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Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

January 2022
Macroheterocycles 15(4):207-304

January 2022
15(4):207-304

DOI:10.6060/mhc224870k

Authors:

Oskar I. Koifman

Ivanovo State University of Chemistry and Technology

Vasilii Otvagin

Nizhny Novgorod State University

Show all 56 authorsHide

Classification of the generations of PSs developed so far.

…

Combining chemotherapy with PDT in conjugated system.

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Approaches toward cationic phthalocyanines and tetra(pyrido/pyrazino)porphyrazines.

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Accumulation kinetics of compounds 2a (A) and 3 (B). The content of compounds in the samples was determined in vivo by local electron spectroscopy on sarcoma S37 tumor-bearing mice. The ratio of areas under the curves of fluorescence and laser reflection peaks was estimated.

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The growth dynamics of the primary tumor node in irradiation modes of 300 J/cm 2 (A) and 150 J/cm 2 (B) and at a photosensitizer dose of 5 mg/kg. The animals were monitored for 15 days after the PDT procedure. The anti-tumor effect was estimated from the dynamics of primary tumor growth in the experimental and control groups.

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Macroheterocycles

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Макрогетероциклы

http://mhc-isuct.ru

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Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 © ISUCT Publishing 207

DOI: 10.6060/mhc224870k

Synthesis Strategy of Tetrapyrrolic Photosensitizers

for Their Practical Application in Photodynamic Therapy

Oskar I. Koifman,a,b Tatyana A. Ageeva,a Natalia S. Kuzmina,c Vasilii F. Otvagin,c

Alexander V. Nyuchev,c Alexey Yu. Fedorov,c Dmitriy V. Belykh,d

Nataliya Sh. Lebedeva,b Elena S. Yurina,b Sergey A. Syrbu,a,b Mikhail O. Koifman,a

Yury A. Gubarev,b Dmitry A. Bunin,e Yulia G. Gorbunova,e,f Alexander G. Martynov,e

Aslan Yu. Tsivadze,e,f Semyon V. Dudkin,g Alexey V. Lyubimtsev,a Larissa A. Maiorova,a,h

Maria B. Kishalova,a Maria V. Petrova,a Vladimir B. Sheinin,b Vladimir S. Tyurin,e

Ilya A. Zamilatskov,e Eduard I. Zenkevich,i Philipp K. Morshnev,a,b Dmitry B. Berezin,a

Eduard A. Drondel,a Andrey V. Kustov,a,b Viktor A. Pogorilyy,k Alexey N. Noev,k,l

Elizaveta A. Eshtukova-Shcheglova,k Ekaterina A. Plotnikova,l Anna D. Plyutinskaya,l

Natalia B. Morozova,l Andrei A. Pankratov,l Mikhail A. Grin,k Olga B. Abramova,m

Ekaterina A. Kozlovtseva,m Valentina V. Drozhzhina,m Elena V. Filonenko,l

Andrey D. Kaprin,l Anastasiya V. Ryabova,n,o Daria V. Pominova,n,o Igor D. Romanishkin,n

Vladimir I.Makarov,n,o Victor B. Loschenov,n,o Kseniya A. Zhdanova,k

Anastasia V. Ivantsova,k Yulia S. Bortnevskaya,k Natal’ya A. Bragina,k Anna B. Solovieva,p

Anastasya S. Kuryanova,p and Petr S. Timashevp,q

aResearch Institute of Macroheterocyclic Compounds, Ivanovo State University of Chemistry and Technology, 153000

Ivanovo, Russia

bG.A. Krestov Institute of Solution Chemistry of the Russian Academy of Sciences, 153045 Ivanovo, Russia

cN.I. Lobachevsky State University of Nizhny Novgorod, 603950 Nizhny Novgorod, Russia

dInstitute of Chemistry of Komi Republic Scientific Centre of the Ural Branch of Russian Academy of Sciences, 167000

Syktyvkar, Russia

eA.N. Frumkin Institute of Physical Chemistry and Electrochemistry, 119991 Moscow, Russia

fN.S. Kurnakov Institute of General and Inorganic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russia

gA.N. Nesmeyanov Institute of Organoelement Compounds of the Russian Academy of Sciences (INEOS RAS), 119334

Moscow, Russia

hInstitute of Pharmacoinformatics, Federal Research Center “Computer Science and Control” of Russian Academy of

Sciences, Moscow, Russia

iBelarussian National Technical University, 220013 Minsk, Belarus

kMIREA-Russian Technological University, 119454 Moscow, Russia

lP. Hertsen Moscow Oncology Research Institute—Branch of the National Medical Research Radiological Centre of the

Ministry of Health of the Russian Federation, 125284 Moscow, Russia

mLaboratory of Experimental Photodynamic Therapy, A.F. Tsyb Medical Radiological Research Center – Branch of the

National Medical Research Radiological Center of the Ministry of Health of Russian Federation, 249034 Obninsk, Russia

nProkhorov General Physics Institute, Russian Academy of Sciences, 119991 Moscow, Russia

oDepartment of Laser Micro-, Nano- and Biotechnologies, National Research Nuclear University MEPhI, 115409 Moscow,

Russia

pN.N. Semenov Federal Research Center for Chemical Physics of the Russian Academy of Sciences, 119991 Moscow, Russia

qInstitute for Regenerative Medicine, Sechenov University, 119991 Moscow, Russia

This review presents a wide range of tetrapyrrole photosensitizers used for photodynamic therapy (PDT), antimicro-

bial photodynamic therapy, photoinactivation of pathogens. Methods of synthesis and design of new photosensitizers

with greater selectivity of accumulation in tumor tissue and increased photoinduced antitumor activity are consid-

ered. The issues of studying the properties of new photosensitizers, their photoactivity, the ability to generate singlet

oxygen, and the possibility of using targeted photodynamic therapy in clinical practice are discussed. The review ex-

amines the work on PDT by national and foreign researchers.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

208 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

Keywords: Photodynamic therapy, antimicrobial photodynamic therapy, tetrapyrrolic macroheterocyclic compounds,

porphyrins, phthalocyanines, chlorins, bacteriochlorins, synthesis, photosensitizers, optical properties, proteins, SARS-

CoV-2, photoinactivation, optochemical diagnostics, self-assembling, nanostructures; Langmuir-Schefer films, extra-

ligation, quantum dots, singlet oxygen generation, amphiphilic polymers, diagnostics, clinical application of PDT.

Стратегия синтеза тетрапиррольных фотосенсибилизаторов

для их практического применения в фотодинамической терапии

О. И. Койфман,a,b T. A. Aгеева,a Н. С. Кузьмина,c В. Ф. Отвагин,c A. В. Нючев,c

A. Ю. Федоров,c Д. В. Белых,d Н. Ш. Лебедева,b Е. С. Юрина,b С. А. Сырбу,a,b

M. O. Koйфман,a Ю. А. Губарев,b Д. А.Бунин,e Ю. Г. Горбунова,e,f A. Г. Мартынов,e

А. Ю. Цивадзе,e,f С. В. Дудкин,g А. В. Любимцев,a Л. А. Майорова,a,h

М. Б. Кишалова,a М. В. Петрова,a В. Б. Шейнин,b В. С. Тюрин,e И. А. Замилацков,e

Э. И. Зенькевич,i Ф. К. Моршнев,a,b Д. Б. Березин,a Э. А. Дрондель,a А. В. Кустов,a,b

В. А. Погорилый,k А. Н. Ноев,k,l E. A. Ештукова-Щеглова,k E. A. Плотникова,l

A. Д. Плютинская,l Н. Б. Морозова,l A. A. Панкратов,l M. A. Грин,k O. Б. Абрамова,m

E. A. Koзловцева,m В. В. Дрожжина,m E. В. Филоненко,n A. Д. Каприн,l

A. В. Рябова,n,o Д. В. Поминова,n,o И. Д. Романишкин,n В. И. Макаров,n,o

В. Б. Лощенов,n,o К. А. Жданова,k А. В. Иванцова,k Ю. С. Бортневская,k

Н. А. Брагина,k A. Б. Соловьева,p A. С. Курьянова,p П. С. Тимашевp,q

Elena V. Guseva,a@ Albina V. Potapova,b and Elena V. Fesikc

aИнститут макрогетероциклических соединений, Ивановский государственный химико-технологический университет,

153000 Иваново, Россия

bИнститут химии растворов им. Г.А. Крестова РАН, 153045 Иваново, Россия

cНижегородский государственный университет им. Н.И. Лобачевского, 603950 Нижний Новгород, Россия

dИнститут химии Коми научного центра Уральского отделения РАН, 167000 Сыктывкар, Россия

eИнститут физической химии и электрохимии им. А.Н. Фрумкина РАН, 119071 Москва, Россия

fИнститут общей и неорганической химии им. Н.С. Курнакова РАН, 119991 Москва, Россия

gИнститут элементоорганических соединений им. А.Н. Несмеянова РАН (ИНЭОС РАН), 119334 Москва, Россия

hИнститут фармакоинформатики Федерального исследовательского центра «И нформатика и управление»

Российской академии наук, Москва, Россия

iБелорусский национальный технический университет, 220013 Минск, Беларусь

kМИРЭА - Российский технологический университет, 119454 Москва, Россия

lМНИОИ им. П.А. Герцена - филиал ФГБУ «НМИЦ радиологии» Минздрава России, 125284 Москва, Россия

mМедицинский радиологический научный центр им. А.Ф. Цыба – филиал ФГБУ «НМИЦ радиологии» Минздрава

России, 249036 Обнинск, Россия

nИнститут общей физики им. А.М. Прохорова РАН, 119991 Москва, Россия

oНациональный исследовательский ядерный университет «МИФИ», 115409 Москва, Россия

pФедеральный исследовательский центр химической физики им. Н.Н. Семенова РАН, 119991 Москва, Россия

qИнститут регенеративной медицины Московского государственного медицинского университета

им. М.М. Сеченова, Москва, Россия

В обзоре представлен широкий спектр тетрапиррольных фотосенсибилизаторов, применяемых для фото-

динамической терапии (ФДТ), антимикробной фотодинамической терапии, фотоинактивации патогенов.

Рассмотрены методы синтеза и дизайн новых фотосенсибилизаторов, обладающих большей селективно-

стью накопления в опухолевой ткани и повышенной фотоиндуцированной противоопухолевой активностью.

Обсуждаются вопросы исследования свойств новых фотосенсибилизаторов, их фотоактивность, способность

генерировать синглетный кислород, возможности применения таргетной фотодинамической терапии в кли-

нической практике. В обзоре рассмотрены работы по ФДТ отечественных и зарубежных исследователей.

Ключевые слова: Фотодинамическая терапия, антимикробная фотодинамическая терапия, тетрапиррольные

макрогетероциклические соединения, порфирины, фталоцианины, хлорины, бактериохлорины, синтез, фото-

сенсибилизаторы, оптические свойства, белки, SARS-CoV-2, фотоинактивация, оптохимическая диагностика,

наноматериалы, самоорганизация, наноструктуры, пленки Ленгмюра-Шеффера, экстракоординация, кванто-

вые точки, генерация синглетного кислорода, амфифильные полимеры, диагностика, клиническое примене-

ние ФДТ.

Macroheterocycles

Review

Макрогетероциклы

http://mhc-isuct.ru

Обзор

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 209

Introduction

The idea of this review was born as a result of a de-

tailed discussion of the problems of photodynamic therapy

at the “PDT Day” section at the XIV International Confer-

ence "Synthesis and Application of Porphyrins and Their

Analogues" (ICPC-14), held at Ivanovo State University of

Chemistry and Technology from 29th June to 4th July,

2022. The unique ability of porphyrins and related com-

pounds to generate reactive oxygen species under the influ-

ence of light necessitates the synthesis of new photosensi-

tizers with the required properties (such as selectivity, viru-

lence, bacteriocide properties, etc.). Such properties are a

crucial part of effective use of photosensitizers in photody-

namic therapy, clinical laboratory diagnostics and photody-

namic inactivation of bacteria and viruses that can be harm-

ful to humans. Therefore, the review focuses on the synthet-

ic aspects of tetrapyrrole macroheterocycles and their modi-

fication for the purposes of targeting photodynamic therapy.

Leading specialists in the photosensitizers’ synthesis and

research, as well as PDT practitioners were involved in

writing this article.

List of Abbreviations:

1O2 – singlet oxygen

aPDT – antibacterial photodynamic therapy

BC – bacteriochlorin (7,8,17,18-tetrahydroporphyrin)

BSA – bovine serum albumin

CMPI – 2-Chloro-1-methylpyridinium iodide

DAMPs – damage-associated molecular patterns

DCC – dicyclohexylcarbodiimide

EDAC – 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

FLI – fluorescence imaging

FRET – fluorescence resonance energy transfer

FRET – foerster resonance energy transfer

GFC – gel filtration chromatography

GSH – glutathione

HCP1 – heme carrier protein 1

HDACi – histone deacetylase inhibitor

HDACs – histone deacetylases

HDL – high-density lipoproteins

HLB – hydrophilic-lipophilic balance

iBC – isobacteriochlorin (2,3,7,8-tetrahydroporphyrin)

LDL – low density lipoproteins

MAM – metastasis-associated macrophages

MDR – multiple drug resistance

MHC – macroheterocyclic compounds

MRI – magnetic resonance imaging procedure

NIR – near infrared

PBS – phosphate buffered saline

Pcs – phthalocyanines

PDT – photodynamic therapy

PL – photoluminescence

PSs – photosensitizers

QD – quantum dots

RBD – receptor-binding domain

ROS – reactive oxygen species

TAM – tumor associated macrophages

TFA – trifluoroacetic acid

TKI – tyrosine kinases inhibitors

TLC – thin-layer chromatography

TNTR – the tumor-to-normal tissue ratio

TOPO – tri-n-octyl phosphine oxide

1. The Key Role of Tetrapyrrole Macroheterocyclic

Compounds in Photodynamic Therapy

Photodynamic therapy (PDT) is a perspective socially

significant method of treatment successfully applied both

for tumor neoplasms and microbial infections caused by

poly-resistant pathogens.[1-9] This method requires the pres-

ence of three components: irradiation (including NIR radia-

tion), photosensitizer (PSs, phototherapeutic agent) and

molecular oxygen. Photosensitizers, substances selectively

localized in tumor or microbial cells, produce reactive oxy-

gen species (ROS)[4,10-13] when exposed to the red light in

the presence of dioxygen.

In recent three decades, photodynamic therapy has at-

tracted much attention due to its non-invasive features, low

side effects and low systemic toxicity.[14-16] The photosensi-

tizers are activated on exposure to light and become photo-

sensitizers’ triplet, which, react with molecular oxygen to

produce reactive oxygen species (Figure 1).[17,18] The hy-

droxyl radical is another reason which leads to the reaction

between the photosensitizer and molecular oxygen. These

cytotoxic molecules induce a series of biological reactions

that ultimately lead to cell death. The outcomes of PDT

depend on the nature of the cells, as well as the on the prop-

erties and localization of photosensitizer and the illumina-

tion conditions. Its obvious advantage is that cause negligi-

ble damage to the surrounding normal tissues and has little

systemic effects.

Correspondingly, PSs is one of the key factors of

PDT, and its physico-chemical properties (e.g. interaction

with molecular oxygen, efficiency of singlet oxygen gen-

eration, solubility, successful targeting and delivery, etc.)

largely determine the outcome of PDT. In this respect, it

was evidently shown that tetrapyrrolic macrocycles (por-

phyrins, chlorins, bacteriochlorins, phthalocyanines) are

potentially translatable PSs with exceptional properties,

multifunctional uses and excellent biocompatibility for

PDT, thus their special affinity to cancer cells makes them

the ligands par excellence for anticancer drugs.[19-28] One of

the prominent advantages of most porphyrins is that they

have special affinity for cancer cells, which enables them to

selectively remain in cancer cells, providing feasibility for

the research of anti-tumor targeted drugs.[29–32]

Figure 1. Schematic diagram of photodynamic therapy.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

210 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

These tetrapyrrolic macroheterocyclic compounds are

the focus of research because of their stability under a wide

range of environmental conditions, absorption in the visible

and near infra-red ranges of the electromagnetic spectrum

(400–1000 nm), many derivatives possess long lived triplet

excited states that sensitize singlet oxygen (1O2) formation

to potentiate phototoxicity as anticancer and antibiotic

agents. These macrocycles can chelate with nearly every

metal in the periodic table which results in variable func-

tional properties such as tuneable excited state lifetimes,

redox potentials, catalytic activities, molar absorption coef-

ficients, and molecular dynamics. Functional groups on the

periphery of structurally rigid porphyrinoids provide topo-

logical diversity, making these pigments well suited for the

engineering of supramolecular materials.[33-36]

Porphyrins and their analogues are organic heterocy-

clic macrocycles with photophysical properties well-suited

for clinical phototherapy and cancer imaging. However,

their wider application in the clinical management of dis-

ease is barred by poor aqueous solubility, bioavailability,

tumour accumulation and skin phototoxicity. These limita-

tions require the search of new photoactive systems devoid

of these disadvantages

Chemists devoted many efforts to improving the effi-

ciency and selectivity of the photosensitizers to prevent the

side-effects of PDT over the last few decades. The perfect

photosensitizers should meet several criteria: (1) pure and

stable molecule, (2) no cytotoxicity in the dark, (3) optimal

absorption, distribution, metabolism and excretion proper-

ties, (4) high molar absorption coefficient in the long wave-

length region (650–800 nm) for maximum light penetration

through tissue, (5) high 1O2 quantum yield, (6) tumor selec-

tivity and (7) ease of synthesis and a scalable process.[37-41]

Today, numerous tetrapyrrole macroheterocyclic compounds

that have been investigated as promising photosensitizers

for use in PDT meet several necessary criteria, such as

strong absorbing properties in the range from 650 to 700 nm,

high quantum yield in the triplet state, high photostability

and often minimal toxicity. In the world clinical practice,

many tetrapyrrol photosensitizers have already found their

application for the treatment of various forms of cancer.

As the PDT method was developing, all photosensitiz-

ers used in the clinical practice were grouped into several

generations, mainly based on their spectral and photophysi-

cal properties.[17,23,42] The first generation of PS includes

porphyrin derivatives, for example, hematoporphyrin iso-

lated from blood (Figure 2). Based on this compound, the

first domestic photosensitizer "Photogem"[43] was developed

by Prof. A.F. Mironov, the founder of our scientific team.

The second generation combines natural and synthetic

compounds, including chlorins, bacteriochlorins, phthalo-

cyanines, benzoporphyrins, and others. Finally, the third

generation includes PS with enhanced spectral properties

that absorb in the near-infrared range of the spectrum and

show targeting against tumors of various genesis.

A huge number of reviews have been published on

the chemistry, photochemical characteristics and clinical

applications of PDT photosensitizers.[44–61] The number of

publications devoted to tetrapyrrol photosensitizers, which

are at different stages of development and clinical trials, is

growing rapidly.

Figure 2. Classification of the generations of PSs developed so

far.

The history of the development of PDT using

tetrapyrrole photosensitizers dates back almost a hundred

years. The starting date was an observation made in 1924

by A. Policard who revealed accumulation in animal tumors

of endogenous porphyrins able to fluoresce under UV irra-

diation.[62] In 1942, H. Auler and G. Banzer recorded red

fluorescence in the primary tumor and metastases in rats

subjected to subcutaneous and intramuscular administration

of hematoporphyrin.[63]

The modern stage of development of fluorescence di-

agnosis and photodynamic therapy began in the 1960s,

when R. Lipson et al. (the United States) revealed the pos-

sibility to record the fluorescence of porphyrin in tumors of

cancer patients who were administered the hematoporphy-

rin derivative prepared by acetylation and reduction of a

porphyrin mixture enriched in hydrophobic oligomers.[64] It

is commonlythat wide clinical use of photodynamic therapy

of cancer began in 1978, when T. Dougherty et al. reported

the results from application of this method for treatment of

25 patients with 113 primary, recurrent, and metastatic skin

tumors.[65]

In Russia, photodynamic therapy of tumors has been

the subject of experimental research for many years, but it

was not until 1992 that it received development in clinical

practice, when the first dosage form of the domestic photo-

sensitizer Photohem from a group of hematoporphyrin de-

rivatives was created. Successful clinical trials were con-

ducted at Gertsen Moscow Research Oncological Institute

and the State Scientific Center of Laser Medicine. After two

years (in 1994), clinical trials of second generation photo-

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 211

sensitizer Photosens, on the base sulfonated aluminum

phthalocyanine, developed at the SSC Research Institute of

Organic Intermediates and Dyes, (Corresponding Member

of RAS Prof. G.N. Vorozhtsov, Prof. E.A. Luk’yanets)

were started. In 1999, clinical application of Alasens, a 5-

aminolevulinic acid-based drug (GHTs NIOPIK, Corre-

sponding Member of RAS Prof. G.N. Vorozhtsov, Prof.

E.A. Luk’yanets) and in 2002 and 2004, that of drugs pre-

pared from e6 chlorin, Radachlorin [Radafarma, Limited

Liability Company, A.V. Reshetnikov], and Photoditazine

(VETA–GRAND, Limited Liability Company, Prof. G.V.

Ponomarev), respectively, were begun. Three decades of

the history of the creation of tetrapyrrol photosensitizers

that have received clinical use in Russia are reflected in

Figure 3. The introduction of new treatment methods in

Russia was facilitated by the development of appropriate

domestic diagnostic and therapeutic equipment. The availa-

ble instrumental base and domestic photosensitizers make

photodynamic therapy not only a highly effective but also

an economically feasible method.[66]

Although a few porphyrin/chlorin based PSs have

been approved for clinical use, PDT is still limited by the

low stability and poor tumor targeting capacity of the large

majority of these macrocycles in vivo. The main obstacles

in effective delivery of PSs are associated with these intrin-

sic properties. At present, two possible approaches to over-

come this problem seem to be of interest in this direction.

One way is the supramolecular organic chemis-

try/photochemistry, a highly interdisciplinary field of sci-

ence covering the chemical, physical, and biological fea-

tures of chemical species held together and organized by

means of intermolecular binding interactions of covalent

and non-covalent nature. The resulting crossover has pro-

vided novel principles and concepts in physico-chemistry

such as molecular recognition, self-organization, regulation,

cooperativity, replication. A significant interest of numer-

ous scientific groups in this direction has been devoted to

the design and investigation of tetrapyrrole compounds that

fold or assemble predictably in order to form multicompo-

nent well-defined arrays which may be perspective in nano-

biomedicine.[72-76]

On the other hand, small molecular PSs (including

tetrapyrrolic macrocycles of various structure and physico-

chemical properties) can be attached to functional organic

or inorganic nanomaterials to enhance the PS stability and

tumor targeted delivery, and, in addition, some functional-

ized nanocarriers themselves can be directly used as

PSs.[51,77,78] To date, nanostructures of different types and

morphology (such as metal nanoparticles, semiconductor

quantum dots, graphene-based nanomaterials, liposomes,

ROS-sensitive nanocarriers and supramolecular nano-

materials)[79-88] have been tested as possible nanoplatforms

for tetrapyrrolic PSs. Various nanomaterial systems have

shown great potentials in improving PDT.[51,78,89]

The application of PDT is not only limited to oncolo-

gy, but is also being explored for the treatment of cardio-

vascular, dermatological, ophthalmic and infectious diseas-

es.[48] In accordance with the achievements of PDT today,

five main areas of application can be distinguished. These

are: (i) the anti-cancer agents for photodynamic therapy

(PDT), (ii) the anti-cancer agents for photoimmunotherapy

(PIT), (iii) the agents for photodynamic therapy (PDT) in

ophthalmology, (iv) the agents for photodynamic therapy

(PDT) in dermatology, and (v) the anti-microbial agents for

photodynamic inactivation (PDI).

How to enhance the selective uptake and uniform dis-

tribution of photosensitizers in tumor tissues is still an ur-

gent conundrum to be solved, especially the specific target-

ing of different cells and tumor tissues, to improve the effi-

cacy and reduce their toxic and side effects on healthy tis-

sues and organs. This may be a promising research direc-

tion, introducing functionally active groups into porphyrin

photosensitizers to improve their targeting and anti-tumor

activity, and then selectively delivering them to tumor tis-

sues with transport carriers such as liposomes, magnetic

nanoparticles and metal–organic frameworks. The ad-

vantages are as follows: photosensitizers bond with bio-

active groups, which can exert synergistic therapeutic

effects and improve the efficacy. Also, combining with a

transport carrier having a bio-recognition function is

conducive to improving the ability of the photosensitiz-

ers to penetrate tissues, enhancing the targeting of tumor

tissues and increasing the drug-level concentration at the

target site.

Further improvement of the photodynamic therapy

technique requires finding new photosensitizers having

higher photoactivity, possessing better tumor-tropic properties,

and excitable in the near-infrared spectral region, as well as

designing highly sensitive and reliable diagnostic and ther-

apeutic equipment. Clinical experience with photodynamic

therapy allows rating this method among the most

promising directions in modern clinical oncology.

 = 630 nm

 = 676 нм

 = 662 nm

 = 661-662 nm

 = 662 nm

 = 747 nm

Photogem[44]

Photosens[67]

Radachlorin[68]

Photoditazine[69]

Photoran E6[70]

Bacteriosens [71]

Figure 3. The Russian tetrapyrrolic photosensitizers that have received clinical use for PDT.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

212 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

2. Conjugation of photosensitizers with cytotoxic molecules

– an emerging trend in combination therapy

One of notorious problems associated with cancer dis-

eases is a lack of the universal treatment protocol for pa-

tients diagnosed with a cancer. Due to genetic uniqueness

of cancer cells, it is almost impossible to propose the treat-

ment, whose outcome is completely predictable. In this

regard, the combined therapy aimed to multiple cellular

targets has many advantages over conventional chemother-

apy.[90] Thus, combining various anticancer drugs it is pos-

sible to reverse drug resistance and fight metastatic can-

cers,[91] what is difficult for singular modalities. Moreover,

combination therapy works in a synergistic manner, and

therefore a lower therapeutic dosage of each individual drug

is required. Among possible drug combinations the utiliza-

tion of photodynamic therapy and chemotherapy together

stands out as an emerging trend.[92,93] The successful out-

come of PDT is highly dependent on a proper irradiation of

a full area where cancer tissues are located. Combined

treatment with a chemotherapeutic drug lowers this risk and

inhibits pathogenic cells survived PDT.[94] Another notable

advantage is the enhanced delivery of chemotherapeutic

drugs after conjugation with porphyrinoid photosenstitizers.

Due to binding with low density lipoproteins (LDL),[95]

heme carrier protein 1 (HCP1)[96] or peripheral benzodiaze-

pine receptor[97] the photosensitizing part can increase con-

centration of the chemotherapeutic counterpart in tumor

cells. Therefore, along with therapeutic properties PS

demonstrates a targeting delivery as well. Thus, such syner-

gistic behavior can lead to reduce of a drug dose and sys-

temic toxicity.[98,99] Another outstanding advantage of com-

bining chemotherapy with PDT is the unique ability of PDT

to induce an immune response to treatment.[100] Since the

result of photodynamic action may be an inflammatory re-

action, the resulting damage-associated molecular patterns

(DAMPs) can be recognized by the corresponding receptors

of neutrophils, which ultimately leads to the activation of

the immune system in response to PDT.

Generally, there are many possible ways to connect PS

with a chemotherapeutic molecule. The first way is admin-

istration of two or more drugs independently during one

course of a treatment. To achieve a proper combinational

effect in this case, a dosage has to be carefully chosen.[101]

Another option is the incorporation of drugs in targeted

systems - liposomes or nanoparticles.[91,102] One of the main

problems associated with such approach is unpredictable

properties arising during manufacturing of these delivery

platforms. Thus, the procedures for surface modification

may strongly modify the size, charge, shape, stability, drug

loading, and releasing ability of the nanovectors,[103] what

can cause undesirable side effects. In recent decades,

covalent binding of a photosensitizer and a cytostatic

agent demonstrated exceptional utility and efficiency

(Figure 4).[92,104–107] Such platform operates with homoge-

neous, chemically pure and stable compounds, which satis-

fies the requirements for an ideal photosensitizer.[92,108] In

addition, the structure of covalent conjugates can be easily

varied over a wide range, thereby changing their biological

activity. In order to create chemical bonding between active

parts specific linkers are used, which provide sufficient

separation in the space of the active fragments.

Figure 4. Combining chemotherapy with PDT in conjugated system.

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 213

Scheme 1.

Interestingly, in some cases the chemotherapeutic part

can also demonstrate binding to key proteins in cells and as

a consequence improves delivery of PS.[106,109–111] Thus,

tyrosine kinases inhibitors (TKI) have been proposed as a

perspective cytostatic vector for the design of conjugates.

One of the widely utilized TKI for construction of conju-

gates is erlotinib, which binds to the epidermal growth fac-

tor receptor (EGFR) known for its overexpression in many

types of tumors.[103] Erlotinib provides targeted binding of

the conjugate with EGFR and inhibits its activity what leads

to a death of cancer cells.

One of the recent examples of photoactive conjugates

designed with utilization of erlotinib is conjugate 1

(Scheme 1).[112] In this work Huang with colleagues sug-

gested a conjugate 1, where the copper(II) porphyrin moiety

plays a dual role both as a photosensitizer and a sensor for

glutathione-dependent apoptotic processes. The decrease of

glutathione (GSH) level is an important benchmark during

apoptotic processes.[113] Interestingly, paramagnetic copper

being bound to NH fragments is capable to coordinate GSH

which leads to fluorescence intensification.[114] However,

when the concentration of glutathione is low, the copper(II)

porphyrin exists in a fluorescence-quenching aggregation

state.[114] This phenomenon was used to visualize apoptotic

cells and monitor the therapeutic effect during PDT.[112]

Copper tetraarylporphyrin and erlotinib were conjugated

with the tetraethyleneglycol linker. The authors showed that

the presence of erlotinib increases the selectivity of con-

jugate accumulation in murine breast cancer cells 4T1

(IC50 (1) = 3.4 μM against IC50 (Cu-porphyrin without er-

lotinib) = 25 μM) with an elevated level of EGFR compared

to the non-tumor human hepatic LO2 line (IC50 (1) = 42.8 μM

against IC50 (Cu-porphyrin without erlotinib) = 31.7 μM)

with a low level of EGFR expression (LED, λ=540 nm).

Furthermore, conjugate 1 could respond to intracellular

glutathione fluctuations. Upon treatment of HepG2 cells

with 1, its fluorescence was restored, which can timely es-

timate the therapeutic efficiency of bimodal chemo- and

photodynamic therapy.

Scheme 2.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

214 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

The research group under supervision of Prof. A.Yu.

Fedorov is also interested in creation of various multifunc-

tional conjugates with improved pharmacological parame-

ters. Thus, the vandetanib derivative, another inhibitor of

EGFR and VEGFR (vascular endothelial growth factor)

tyrosine kinases, as part of a conjugated system based on

the chlorin e6 zinc complex 2 was used (Scheme 2).[110]

Vanetanib was supposed to play the role of a dual agent of

selective delivery due to preferential binding to VEGFR

and EGFR receptors overexpressed by many types of tu-

mors, as well as cytotoxic action due to inhibition of the

activity of the mentioned tyronis kinases.[115] To increase

water solubility, chlorin derivative was modified by the

introduction of three ammonium groups in the form of

quaternized salts. The conjugate 2 showed a selective ac-

cumulation in A431 cells (EGFR-positive, human epider-

moid carcinoma), which was greater than in non-tumor

CHO cells (EGFR-negative, chinese hamster ovary cells)

and HeLa cells (human epidermoid carcinoma) with a low

level of EGFR expression. Under the action of light (LED,

λ= 615-635 nm), the conjugate 2 inhibited the growth of all

cell lines at low micromolar concentrations, while without

irradiation, the values of the half-inhibition index (IC50)

were approximately an order of magnitude higher. This

indicates that when bound to chlorin-e6, Vandetanib deriva-

tive is unable to inhibit cell growth. It should be noted that

chlorin-e6 without Vandetanib 3 had 2-3 times lower toxici-

ty in all cell lines. In experiments performed on CT-26 tu-

mor-grafted mice, the conjugate 2 also demonstrated selec-

tive accumulation in the tumor.

Then, it was decided to change the type of the linkage

between vandetanib and chlorin-e6 from an ether bond to an

ester bond (Scheme 2). It is known that ester bonds in the

body are easily destroyed by the action of esterase,[116]

therefore, the modified conjugate may be capable of releas-

ing vandetanib, which means that the synergistic effect of

PDT and chemotherapy can be observed. The second

change was to replace the tetramethylammonium groups

with maltose-based carbohydrate moieties. Having hydro-

philic properties, carbohydrates also selectively bind to

their respective receptors (galectins and GLUT transporters)

in tumor cells by the Warbug effect.[117-119] Although there

was almost no selectivity for A431 cells compared to CHO,

the conjugate 4 generally accumulated better in these cells

than the conjugate with ammonium fragments 2, and also

showed less pronounced cytotoxicity in the dark compared

to 2. Under light irradiation (LED, λ = 655−675 nm) in

A431 cells, the conjugate 4 was 2 times more active than in

the CHO cell line and 2–5 times more active than unconju-

gated chlorin-e6 5. An in vivo study of activity in mice with

grafted A431 tumors showed a moderate selectivity for

tumor-normal cells, as well as slight differences in the ac-

tivity of the conjugate in the light and in the dark. This fact

implies that the conjugate 4 enters the cells and releases

vandetanib by esterase-mediated ester cleavage. Thus, the

obtained results indicate different behavior of vandetanib-

chlorin-e6 conjugates depending on the type of bond be-

tween the key fragments.

Scheme 3.

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 215

Scheme 4.

Metal-containing drugs are broadly known for their

significant antitumor activity.[120] The conjugation of such

metal complexes with PSs can modulate their hydrophilic

properties. Due to the interaction with DNA, metal com-

plexes[121] may serve as a vector to deliver PS to the cell

nuclei. Organotin complexes are an attractive alternative to

the known platinum-containing cytostatics because of low-

ered systemic toxicity.[122] Importantly, such complexes can

be effective in cell lines possessing cisplatin resistance.[123,124]

In 2021, two conjugates of bacteriochlorin derivative and a

tin complex with amino acid ligands (p-aminobenzoic acid

and L-lysine) 5-6 was obtained by Prof. Grin with col-

leagues (Scheme 3).[125]

The dark cytotoxicity of conjugates 5 and 6 in various

cell lines (prostate gland adenocarcinoma (PC-3); breast

adenocarcinoma (MCF-7); lung carcinoma (A549); cervix

adenocarcinoma (Hela) and mouse sarcoma (S-37)) was 3-5

times higher than that of the corresponding organotin com-

plexes. Based on studies in vitro, the authors concluded that

the conjugation of tin-containing cytostatics with bacterio-

chlorin moieties promotes better internalization and accu-

mulation of conjugates in tumor cells and therefore in-

creased cytotoxicity. Among synthesized conjugates, the

molecule 6 demonstrated exceptional photodynamic effi-

ciency (2-3 fold increased IC50 under light) compared to

referenced bacteriochlorin derivative 7 (halogen lamp,

λ≥720 nm). The nature of the intracellular distribution of

the obtained agents showed a diffuse granular distribution

with predominant accumulation in the near nuclear region,

but it was not possible to observe the localization of conju-

gates in the nuclei themselves. Thus, the authors conclude

that the tin complexes act as target fragments for the deliv-

ery of bacteriochlorins into the cells.

Histone deacetylases (HDACs) are directly involved

in carcinogenesis and therefore represent an attractive tar-

gets for anticancer therapy.[90,126] Inhibition of HDACs is

shown to suppress growth of cancer cells, promote cell

death, modulate immune responses, and inhibit formation

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

216 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

of new blood vessels.[127] Mainly approved for hematologi-

cal malignancies, histone deacetylase inhibitor (HDACi)

has been clinically tested for solid tumors and has shown

better results when combined with another chemotherapy

drug.[128] It is also known that, HDAC6 and HDAC8 are

highly expressed in breast cancer cells, therefore their

downregulation may be beneficial.[129] In 2020 Aru with

colleagues reported the 3-hydroxypyridin-2-thione (3-HPT)

substituted silicon phthalocyanine derivative 8 and evaluat-

ed its anti-cancer properties on two different breast cancer

cell lines (non-invasive MCF-7 and invasive MDA-MB-

231 cells) (LED, λ=700 nm). It was shown, that conjugate 8

activated programmed cell death pathways and induced cell

cycle arrest which was accompanied by decreasing levels of

HDAC6 and HDAC8 (Scheme 4).[130]

Among various breast cancers, its triple negative vari-

ant doesn’t respond to classical hormone therapy and al-

ways associated with poor prognosis.[131] In this regard,

promising results obtained after treatment of cell line

MDA-MB-231 (triple negative breast cancer) with 8 can

become a starting point for a novel treatment of such dis-

ease. Thus, the authors demonstrated pronounced inhibition

of MDA-MB-231 cells with the highest late apoptotic cell

population. Inspired by the obtained results, the authors

evaluated how the substitution in α- or β-position of a

phthalocyanine with HDACi inhibitor affects antitumor

behavior of suggested conjugates.[132] Additionally, differ-

ent metals were chosen for complexation with a phthalocy-

anine core. The influence of the central ion on the internal-

ization of conjugates was different for zinc and indium de-

rivatives. While for α-substituted indium conjugates 9b and

10b no differences were found in their cell uptake, β-

substituted Zn-conjugate 10a in demonstrated enhanced

internalization and, therefore, a more pronounced antitumor

effect compared to its α-analogue-conjugate ZnPc 9a (LED

lamp, red light). It was also noted that although all conju-

gates generated ROS in the mitochondrial network, only

indium compounds 9b and 10b directly targeted mitochon-

dria. Treatment of cells with obtained conjugates also led to

cell cycle arrest in different phases of cell cycle.

Camptothecin 11 is a potent antitumor antibiotic

whose action mechanism involves the inhibition of DNA

relaxation by the stabilization of a covalent binary complex

formed between topoisomerase I and DNA.[133] In 2022

Zhang and colleagues proposed the concept of a prodrug 12

based on the camptothecin derivative (Scheme 5).[134] As a

photosensitizing part a BODIPY-based compound was cho-

sen. At the same time camptothecin derivative was attached

to the BODIPY through a labile phenyl benzoate group.

The phenyl benzoate group is prone to cleavage by cell

esterases what was exploited for the release of camptothe-

cin 11. Importantly, camptothecin 12 attached to the BOD-

IPY photosensitizer can’t inhibit tumor proliferation while

its release could lead to pronounced cytotoxicity due to

DNA binding. In addition, the fluorescence of conjugated

camptothecin was dramatically reduced through the intra-

molecular fluorescence resonance energy transfer (FRET)

process from camptothecin to the BODIPY photosensitizer.

Monitoring the fluorescence level, the authors were able to

observe the release of camptothecin 11. Studies of esterase-

mediated release of camptothecin 11 from conjugate 12

proved its liberation under action of exogenous carboxyles-

terase in phosphate-buffered saline and under action of en-

dogenous carboxylesterase overexpressed in cancer cells.

The authors showed a significant decrease in the IC50 val-

ues of the resulting conjugate against HepG2 human hepa-

tocellular carcinoma and HeLa human cervical carcinoma

cells upon irradiation (LED, λ=660 nm) compared with

camptothecin and BODIPY as controls. The combined anti-

tumor effects of the prodrug 12 were also observed in the

mice bearing H22 murine hepatocarcinoma tumors.

Scheme 5.

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 217

Scheme 6.

Combretastatin A-4 13 (CA4) is a strong antimitotic

agent that can bind to the colchicine-binding site and inhibit

tubulin polymerization[135] (Scheme 6). CA-4 in Z-form

exhibits cytotoxicity at the nanomolar level in various hu-

man tumor cell lines, (MCF-7-IC50 = 9 nM) including mul-

ti-drug resistant (MDR) tumor cells.[136] CA4 contributes to

the blockage of tumor vessels, therefore serves as a vascular

disrupting agent. In 2013, Bio with colleagues developed

“photo-unclick chemistry”, a novel chemical tool involving

the cleavage of aminoacrylate linker by singlet oxygen

(1O2). The researchers applied it to visible light-activatable

prodrugs. The proposed delivery system includes a photo-

sensitizer connected by an aminoacrylate linker to an anti-

tumor drug (combretastatin A4) 14[137] (Scheme 6).

The resulting prodrug 14 was activated by far-red light

(diode laser, λ=690 nm) due to the destruction of the ami-

noacrylate linker by singlet oxygen formed upon irradiation

of the dithioporphyrin photosensitizer. Thus, singlet oxygen

acts not only as a cytotoxic agent, but also as a tool for acti-

vating the chemotherapy drug, resulting in synergistic dual

chemo- and photodynamic therapy. In 2020, Bio with col-

leagues improved the above mentioned system by adding a

2,4-dinitrobenzenesulfonate (DNBS) quencher and biotin as

a tumor-targeting ligand to it [138]. Due to the presence of

the DNBS substituting group, zinc phthalocyanine quench-

ing was achieved for 1O2 production and fluorescence emis-

sion. When interacting with glutathione (GSH) in the cell

environment, DNBS group had removed and prodrug had

recovered photoactivity. In cells HepG2 (biotin receptor

positive) conjugate 15a demonstrated elevated uptake than

that for HCT-116 cells (control, human colorectal carcino-

ma), therefore proving its ability to target biotin receptors.

After GSH-OEt pre-treatment the fluorescence intensity

increased. Under conditions of light irradiation (300 W hal-

ogen lamp, λ=610 nm) of HepG2 cells containing the con-

jugate 15a, their growth was inhibited at a nanomolar con-

centration (Hep2G: IC50 light = 48 nM), while in the dark it

was inactive at a concentration of up to 2 μM. At the same

time, the control conjugate without aminoacrylate linker

15b had a much lower activity both with and without irra-

diation, confirming the proposed mechanism of activation

of the conjugate system by singlet oxygen-induced destruc-

tion of the aminoacrylate linker in conjugate 15a. The au-

thors calculated the combination index (CI)[139] for 15a

(CI~0.25), which indicated strong synergism between pho-

tosensitizing and chemotherapeutic parts. Thus, the new

delivery system is favorably distinguished by its dual action

(PDT and chemotherapy) and high controllability.

Our research group decided to take advantage of com-

bretastatin A-4's photoisomerization from a 1000-fold less

active trans-isomer 17 (IC50 = 10-6M) to a clinically active

cis-isomer 13 (IC50 = 10-9M)[140,141] (Scheme 7A). Combin-

ing trans-combretastatin with a porphyrin photosensitizer

using a photoremovable o-nitrobenzyl linker,[142] we as-

sumed that the action of one- or two-photon irradiation

could trigger a cascade of processes: 1) destruction of the o-

nitrobenzyl linker with simultaneous release of trans-

combretastatin; 2) its photoisomerization into toxic cis-

isomer 13; 3) photoactivation of porphyrin and its genera-

tion of singlet oxygen.[143] As a result, we would be able to

control the toxicity of the conjugates 16a-b using light and

enhance the therapeutic effect due to the simultaneous ac-

tion of PDT and chemotherapy. To increase hydrophilicity,

the porphyrin macrocycle was decorated with carbohydrate

fragments (galactose and maltose, respectively). However,

when using various light sources (UV-A, λ=365 nm, UV-B,

λ=311 nm, and UV-C, λ= 254 nm, LED, λ = 450 nm), it

was not possible to observe the desired release of com-

bretastatin A-4. To determine the cause of the intactness of

the conjugates 16a-b, irradiation of the trans-combretastatin-

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

218 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

linker 18 model system without porphyrin was carried out.

It turned out that this system is capable of releasing a thera-

peutic agent, which is immediately not only isomerized to

active cis-combreatstatin, but further cyclized to a phenan-

threne-type derivative 19 (Scheme 7B). TD-DFT calcula-

tions were also performed to identify the best type of bind-

ing between the porphyrin and the o-nitrobenzyl linker. It

has been shown that the excitation of three orbitals (LUMO,

LUMO+1, and LUMO+2) is possible when the conjugates

are irradiated with 365 nm light, in which the maximum

electron density is shifted towards the porphyrin fragment,

which possibly complicates the photocleavage process. At

the same time, if there are no additional functional groups

between the porphyrin fragment and the o-nitrobenzyl link-

er, then on the LUMO + 2 orbital, which at the same time

has an energy higher than for all considered types of bind-

ing, the electron density shifts towards the linker, which

increases the probability of photodegradation. Our result is

consistent with the experimentally photocleaved porphyrin-

o-nitrobenzyl linker-fluorouracil conjugate by Lin,[144] for

which we also calculated the corresponding orbitals. Thus,

further research will focus on an updated design that elimi-

nates linkers between the photosensitizer moiety and the o-

nitrobenzyl group, and the replacement of combretastatin

A-4 with azo analogs that do not undergo cyclization.[145].

Thus, the substantial progress made in the develop-

ment of multifunctional drugs opens broad possibilities for

the creation of efficient antitumor agents. Even though

there are no approved conjugated drugs so far on the mar-

ket, their further investigation will undoubtedly lead to new

treatment options for people suffering from oncological

diseases. Among main beneficials hiding in this concept the

one can point out: 1) broad variability arising from unlim-

ited tools for conjugation including cytostatics, cleavable

linkers, quenchers etc.; 2) predictability of cellular targets

for main active parts of conjugate; 3) possibility to exploit

multiple mechanisms leading to the death of cancer cells,

including MDR-cells; 4) selective delivery to tumors

through decoration with specific oncovectors or targeted

TKI utilization.

Scheme 7.

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 219

3. Formation of Cationic Groups at the Natural

Chlorins Macrocycle Periphery in the Synthesis

of Potential Antitumor and Antibacterial

Photosensitizers

It is known that the introduction of cationic groups to

the periphery of the chlorin PS macrocycle not only in-

creases hydrophilicity, but also promotes interaction with

the cell membrane of gram-negative bacteria.[42,47,92,146-161]

Chlorophyll a derivatives have spectral properties suitable

for use as antitumor and antibacterial PSs and relatively low

toxicity; therefore, they are widely used as a basis for the

synthesis of new PSs for medical purposes.[42,47,92,146-161]

One of the possible directions of chlorophyll a and its de-

rivatives chemical modification is the introduction of cati-

onic substituents to the macrocycle periphery. It should be

noted that a positive charge can arise both in the protona-

tion of nitrogen atoms (as a rule, nitrogen atoms of periph-

eral substituents[47,150,153,162-164]) and in the presence of pH-

independent cationic groups on the macrocycle periph-

ery.[47,146,148–153,159,165] The dependence on pH of the charge

presence at the macrocycle periphery can reduce the effec-

tiveness of the PS, so the formation of cationic substituents

with a constant charge is more preferable. This review con-

siders methods for the formation of cationic groups at the

periphery of the macrocycle of chlorophyll a derivatives. It

should be noted that not all cationic chlorins, which will be

discussed below, have been studied as potential PS, howev-

er, the methods used in their synthesis can be applied in the

synthesis of potential PS, and these chlorins themselves are

potential PS. Only cationic derivatives with a quaternary

nitrogen atom have been described for a-series chlorins.

The key step in the formation of a cationic group is the in-

troduction of an amino group substituted to varying degrees

(usually it is tertiary amino group). Quaternization of this

group allows the formation of a charged group and is usual-

ly not difficult. Next, will be considered the main ways of

introducing the previous nitrogen-containing groups and

their subsequent transformation into cationic groups.

The Introduction of Cationic Substituents by Forming an

Ester Bond

To introduce cationic groups by forming an ester bond

the action of an alcohol with a tertiary nitrogen atom on the

activated carboxyl groups of chlorine is used, followed by

quaternization of the nitrogen atom. When chlorin e6 is

treated with 2-(N,N-dimethylamino)ethanol (choline) or 3-

hydroxymethylpyridine, followed by alkylation with methyl

iodide, the corresponding chlorins with three cationic sub-

stituents are obtained. Because when using dicyclohexylcar-

bodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl)car-

bodiimide (EDAC) the products are usually a mixture of

mono-, di-, and tri-substituted chlorin e6, mostly mono- and

di-substituted chlorin e6, probably because the structure of

intermediates is too bulky to esterify all of the three car-

boxyl groups, the carboxyl groups “activation” is carried

out with oxalyl chloride. The corresponding acid chloride is

forms, which makes it possible to carry out the reaction at

all three carboxyl groups of chlorin e6[166] (Scheme 8).

Insertion of Cationic Substituents through the Formation of

an Amide Bond

The formation of an amide bond upon the introduction

of a tertiary amino group to the periphery of the chlorine

macrocycle is carried out both using the usual reactions of

“activated” carboxyl groups and using the reactions of chlo-

rophyll a derivatives associated with the features of their

chemical structure. When, for the introduction of the

N,N(dimethyl)ethylenediamine fragment in reactions with

various 17-carboxy derivatives of chlorophyll a, in most

cases, carbodiimide “activation” (DCC, EDC) is used

(Schemes 9-12). The quaternization of the tertiary nitrogen

atom is carried out by the action of CH3I, if it is necessary,

the exchange of the counterion is carried out on an ion ex-

change resin. So, for the synthesis of the cationic derivative

of pheophorbide a, the DCC-activated carboxyl group of the

substrate is treated with (N,N-dimethyl)ethylenediamine[167]

(Scheme 9).

A similar reaction of pyropheophorbide a[168] (Scheme 10),

pyropheophorbide d[169] (Scheme 11), and phorbin, which is

the active substance of Photochlor PS[170] (Scheme 12), is

carried out with EDC “activation”.

If it is necessary to carry out some additional modifi-

cations of chlorin (for example, the synthesis of a complex

or the reduction of the aldehyde group,[169] Scheme 11),

quatrainization is carried out at the final stage, since it is not

so convenient to work with a compound with a cationic

group due to its increased hydrophilicity and also the possi-

bility of uncontrolled exchange of the counterion and its

participation in side reactions. As will be seen from what

follows, this technique is quite universal and is used regard-

less of the method of introducing a substituent with a ter-

tiary nitrogen atom.

Scheme 8.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

220 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

Scheme 9.

Scheme 10.

Scheme 11.

Scheme 12.

The interaction of amines with esters to form the cor-

responding amides is a well-known reaction that is possible

for compounds of various classes, including natural chlo-

rins. Amidation of chlorophyll a derivatives ester groups in

most cases, is carried out after the formation chlorin e6 13-

amide derivatives after the exocycle opening (see below).

However, amidation of the phytylpropionate substituent of

pheophytin a by the action of N,N,N,N,N,N-hexapropyl-

penta(aminoethyl)amine (amine oligoamine with five ter-

tiary nitrogen atoms) is possible before exocycle open-

ing[161] (Scheme 13). The reaction is carried out under the

action of a small molar excess of amine in the presence of

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 221

about 10-fold molar excess of TFA. It is known,[161] that the

same amine can open the pheophytin a exocycle with the

formation of the corresponding 13-amide derivative (see

below), but this reaction does not occur under amidation

conditions. The selectivity of amidation can be provided not

only by a small excess of amine, but also by the presence of

TFA. The role of TFA the authors of[161] do not comment

on. Possibly, the presence of a small amount of TPA pro-

vides an "acidic" catalysis of amidation, which, along with

a slight excess of amine, results in the selectivity of the

process. The target compound in this synthesis was the cat-

ionic zinc complex, as in the cases described above, quater-

nization is carried out at the last stage.

A similar reaction of the amide’s formation occurs

upon the action of amines on cyclic lactones formed during

the oxidation of chlorines with a carboxyl group in position

17.[171,172] As a result of the reaction, the lactone exocycle

opens and the corresponding hydroxychlorines are forms.

This reaction can be used to insert the N-methylpyridyl

group[173] (Scheme 14).

It is known that the exocycle of pheophytin a, methyl

pheophorbide a, pheophorbide a and a number of other

similar natural chlorins is relatively easily opened under the

action of nucleophilic agents such as amines. This reaction

can be used to form cationic groups if the opening is per-

formed with an amine containing tertiary nitrogen atoms or

additional amino groups that can be alkylated. The exocycle

opening under the action of (N,N-dimethyl)ethylenediamine

allows the introduction of a tertiary amino group, the

quaternization of which gives a cationic substituent

(Scheme 15). This pathway was implemented for pheophyt-

in a, methylpheophorbide a and a number of its derivatives

with fragments of biomolecules to increase hydrophilici-

ty.[110,118,174-178] Alkylation of the dimethylamino group is

usually carried out with CH3I.[110,174-178] However, other

alkylating agents, such as C2H5Br, are also possible[118]

(Scheme 15).

Scheme 13.

Scheme 14.

Scheme 15.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

222 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

Scheme 16.

Scheme 17.

Scheme 18.

The use of (N,N-dimethyl)ethylenediamine analogs

with a large number of methylene units between the prima-

ry and tertiary amino groups makes it possible to synthesize

chlorin e6 derivatives with a cationic group remote from the

macrocycle to varying degrees. For example, the reaction

with 1-(N,N-dimethylamino)-3-aminopropane[179] makes it

possible to lengthen the spacer by one methylene group

(Scheme 16).

The exocycle opening with 4-aminomethylpyridine can

be used to introduce the pyridinium fragment[180] (Scheme 16),

but the authors did not perform further alkylation. The cati-

onic group formation is also possible from a primary amino

group. For example, chlorin with a cationic group is synthe-

sized by the action of a large excess of CH3I on the 13-

amide derivative of chlorin e6, obtained by the action of

ethylenediamine on methyl pheophoride a[181] (Scheme 17).

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 223

Scheme 19.

Scheme 20.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

224 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

The interaction of purpurin 18 and its analogs with

amines giving cycloimide derivatives not only significantly

improves the spectral characteristics of chlorin, but also

introduces an additional functional group.[182] This reaction

can be used both for the directly insertion of a substituent

with a tertiary nitrogen fragment[173] and for the formation

of a reaction center, which can then be used to synthesize a

derivative with a tertiary nitrogen fragment. The purpurin

methyl ester 18 cycloimide derivative is obtained from pur-

purin 18 by the action of 4-aminomethylpyridine in three

stages performed “in one flask”, subsequent alkylation of

the pyridine substituent with CH3I gives the target cationic

derivative[173] (Scheme 18). Starting from purpurin methyl

ester 18, a similar derivative can be obtained in one step

by refluxing in toluene with 4-aminomethylpyridine[180]

(Scheme 18). In addition to the direct introduction of a sub-

stituent with a tertiary nitrogen atom, the formation of cy-

cloimide can be used to introduce an additional reaction

center. The action of hydrazine on bacteriopurpurin methyl

ester 18 gives a cycloimide derivative with a reactive amino

group, acylation of which with pyridinecarboxylic acids

leads to derivatives with a pyridinium fragment[183-185]

(Scheme 19). Quaternization of pyridinium fragments by

the action of CH3I gives the corresponding cationic de-

rivatives.

Chlorins with a seven-membered exocycle closed by

an imide bond can be synthesized by the action of CMPI on

13-amd-15-carboxy derivatives of chlorin e6, provided that

the amide group at position 13 is secondary or primary.[186]

In the case when there is a dimethylaminomethyl group at

the amide nitrogen atom, cyclization results in the for-

mation of chlorin, the action of which with CH3I gives the

corresponding cationic derivative[186] (Scheme 20).

Formation of C-N(amine) Bonds in the Synthesis of Cationic

Chlorins

The formation of C-N(amine) in a number of cases is used

to introduce substituents with a tertiary nitrogen atom, the

subsequent quaternization of which gives a cationic substit-

uent. Epoxidation of the vinyl group of chlorin with meta-

chloroperoxybenzoic acid followed by treatment of the in

situ formed polyamine epoxide gives chlorin with a poly-

amine fragment in position 3, a precursor of the pentacation

substituent[161] (Scheme 21). Quaternization of tertiary ni-

trogen atoms in the molecule by the action of CH3I is car-

ried out at the last stage.

Cationic derivatives with a pyridinium group were

synthesized by the action of heterocyclic compounds with a

pyridinium fragment, I2 and AgPF6, on vinylchlorins; the

cationic group is immediately formed in the course of

chemical oxidative pyridylation[152,187] (Scheme 22). Using

the reaction involving pyridine as an example, it was

shown[187] that more accessible silver salts, for example,

halides, can be used instead of AgPF6.

A similar reaction can also be carried out in a series of

bactriochlorophyll a derivatives after the formation of a

vinyl group in position 3[187] (Scheme 23).

In the absence of a vinyl group, the insertion of a cati-

onic substituent during chemical oxidative pyridylation

occurs in meso-positions[188] (Scheme 24). As a result, a

mixture of regioisomers with comparable isolated yields is

formed, which significantly complicates the use of this re-

action for the synthesis of cationic PSs.

A similar reaction can also be carried out in a series of

bactriochlorophyll a derivatives after the formation of a

vinyl group in position 3[187] (Scheme 23).

Scheme 21.

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 225

Scheme 22.

Scheme 23.

Scheme 24.

Scheme 25.

Scheme 26.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

226 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

In the absence of a vinyl group, the insertion of a cati-

onic substituent during chemical oxidative pyridylation

occurs in meso-positions[188] (Scheme 24). As a result, a

mixture of regioisomers with comparable isolated yields is

formed, which significantly complicates the use of this re-

action for the synthesis of cationic PSs.

C-C and C=C Bonds Formation in the Synthesis

of Cationic Chlorins

The formation of a carbon-carbon bond is considered

to be the most preferred when introducing a substituent

with a tertiary nitrogen atom. In the chemical modification

of chlorins in this direction, both some "classical" reactions

(variants of the Mannich reaction, other condensations) and

relatively recently discovered cross-coupling reactions are

used. Under the action of Eschenmoser's reagent on zinc

porphyrinates obtained from methyl pheophorbide a, me-

thyl pyropheophorbide a, and chlorin e6 trimethyl ester,

stereoselective aminomethylation of the vinyl group oc-

curs[189-193] (Scheme 25, methyl pyropheophorbide a as an

example). As a result of the reaction, trans-isomers are

formed, subsequent demetallation with trifluoroacetic acid

and alkylation of the dimethylaminomethyl group of the

obtained derivatives with CH3I gives the corresponding

metal-free monocationic chlorins.

It was shown[194] that, in contrast to Eschenmoser's re-

agent, the action of bis(N,N-dimethylamino)methane on

metal-free 13-amide derivatives of chlorin e6 by refluxing

in acetic acid results in the nonstereoselective insertion of

two dimethylaminomethyl substituents into the vinyl group

(Scheme 26).

A similar reaction occurs at room temperature with the

participation of zinc[195] and nickel[196]) complexes of a-

series vinyl-chlorins.

The vinyl group aminomethylation reaction[194-196] is

used to synthesize dicationic chlorins containing additional

hydrophilic and hydrophobic fragments[197] (Scheme 27), as

well as dicationic derivatives of chlorin e6 with membrano-

tropic substituents (myristic acid fragments[198] (Scheme 28)

and phytol[176,177] (Scheme 29). In addition to aminomethyl-

ation, the introduction of a cationic group can be carried out

using palladium-catalyzed cross-coupling reaction. This

reaction was used to insert a pyridine moiety at position 20

of mesomethylpyropheophorbide a, which was then quaternized

by the action of iodo triethylene oxide[199-201] (Scheme 30).

Condensation of the methylpyropheophorbide d alde-

hyde group with methyl groups of the corresponding heter-

ocycles is used to introduce a heterocyclic fragment with a

pyridyl nitrogen atom. The heterocyclic fragment of the

substituted vinyl group formed as a result of condensation

is quaternized by the action of CH3I[202] (Scheme 31).

Scheme 27.

Scheme 28.

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 227

Scheme 29.

Scheme 30.

Scheme 31.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

228 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

Combination of Reaction Centers of Various Chlorophyll a

Derivatives in the Synthesis of Cationic Chlorins

It often becomes necessary to synthesize chlorins with

different natures, amounts, and positions of cationic sub-

stituents to reveal structure-activity relationships. Combina-

tions of modifications using reactions of various reaction

centers allow not only to increase the number of introduced

cationic groups, but also to synthesize chlorins with differ-

ent arrangement of cationic groups on the periphery of the

macrocycle, which is widely used in the synthesis of cation-

ic PSs based on chlorophyll a derivatives. The chlorin e6

diamide derivative with ten cationic groups was synthe-

sized by combining the amidation of the pheophytin a

phytylpropionate substituent with the action of

N,N,N,N,N,N-hexapropyl-penta(aminoethyl)amine leads to

the opening of the exocycle of the 17-amide derivative con-

taining a fragment of the same amine[161] (Scheme 32). As

in all previous cases, the cationic groups are formed by the

action of CH3I in the last step.

Opening of the cyclic lactone in combination with the

formation of cycloimide makes it possible to synthesize the

purpurin 18 cycloimide derivative with two cationic groups[173]

(Scheme 33).

Scheme 32.

Scheme 33.

Scheme 34.

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 229

Scheme 35.

Reaction conditions: i: CH3NH2-H2O/THF, r.t., 3 h; ii: [(CH3)2N=СH2]+I-, CH2Cl2, r.t., 12 h; iii: H2NCH2CH2N(CH3)2, CH2Cl2, r.t.; iv:

CH2(N(CH3)2)2, AcOH-THF, boiling for 30 min; v:[92] CH3I, CH2Cl2, r.t.

Scheme 36.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

230 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

Scheme 37.

The combination of exocycle opening with ami-

nomethylation of the vinyl group by the action of bis(N,N-

dimethylamino)methane makes it possible to synthesize

tricationic chlorin with a membranotropic phytol frag-

ment[176] (Scheme 34). The same reactions were used for the

synthesis of tricationic chlorin with 4-arylaminoquinazoline

moiety[110] (Scheme 35).

The combination of different variants of the vinyl group

aminomethylation with the exocycle opening makes it pos-

sible to synthesize chlorins with a different number of cati-

onic groups and their arrangement in the macrocycle[178]

(Scheme 36).

Alkylation of amino groups in chlorin e6 di- and triamide

derivatives allows the formation of two and three cationic

groups, respectively[181] (Scheme 37).

Thus, chlorophyll a and its derivatives, having a num-

ber of convenient reaction centers in the molecule, are a

good basis for the synthesis of cationic photosensitizers for

medical purposes.

4. Water-Soluble Cationic Porphyrins

for Photoinactivation of Pathogens

The ability of porphyrins and related compounds to

generate reactive oxygen species under the action of light

necessitates the synthesis of new photosensitizers with the

required properties (selectivity, virucidality, bactericidality,

etc.) for their effective use in photodynamic therapy, clini-

cal laboratory diagnostics, pharmacopoeia,[203] antimicrobi-

al water and air purification,[204] photodynamic inactivation

of bacteria and viruses dangerous to humans and plants.[205]

Photodynamic inactivation of bacteria and viruses is

considered the most promising treatment for drug-resistant

bacteria[206] and zoonotic viruses.[207] The method of pho-

toinactivation is based on the reactions of biomolecules,

biosubstrates, and pathogens with ROS, which are generat-

ed with the participation of PS. It should be noted that, in

addition to tetrapyrrole macroheterocyclic compounds, oth-

er classes of compounds are also considered as PS for pho-

toinactivation of pathogens.[208] For example, the well-

known cationic methylene blue or anionic rose bengal also

show an antibacterial photoelectric effect, but their effec-

tiveness is much lower than that of porphyrin photosensi-

tizers. To date, it is reliably known that cationic tetrapyrrole

MHCs more effectively damage pathogens than anionic and

neutral PSs. The positive charges of PS promote the elec-

trostatic binding of porphyrin to negatively charged sites on

the outer membrane of bacteria and induce damage that

enhances the penetration of the photosensitizer into the

cell.[209,210]

The structure of the outer shell of the pathogen is no

less significant. In general, gram-positive bacteria show a

higher susceptibility to photoinactivation than gram-

negative ones. This is probably the result of differences in

the structure of the cell membranes of these two microbial

groups. gram-positive and gram-negative bacteria have a

cytoplasmic membrane. The membrane of gram-positive

bacteria is more permeable; peptidoglycan structure is po-

rous, filled with teichoic and teichuronic acids. The main

structural difference between gram-positive and gram-

negative bacteria is the thickness of the peptidoglycan and

the presence of an outer membrane. The outer membrane is

present only in gram-negative species. It is a very effective

barrier to the penetration of exogenous substances. This

membrane is very dense, the porins present in it ensure the

passage of only small hydrophilic substances.[211,212] In ad-

dition, due to the presence of lipopolysaccharides, the outer

membrane has a strong negative charge and a high surface

potential.[204]

In order to damage a bacterium, PS must reach the cy-

toplasmic membrane after passing through the outer mem-

brane.[213] Differences in the structure of the cell membrane

of gram-negative and gram-positive bacteria determine the

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 231

possibility of photoinactivation of the latter with the help of

not only cationic, but also anionic and neutral PS, while

gram-negative bacteria are more resistant to photoinactivation.

Photoinactivation of viruses by MHC is a less ex-

plored topic compared to photoinactivation of bacteria, but

as with bacteria, singlet oxygen generated (type II photoox-

idation) and radicals (type I photooxidation)[214, 215] play a

key role in virus inactivation. Potential targets for photo-

sensitizer binding are the protein capsid, nucleic acids, and

virion envelopes.

It is a priori clear that the intracellular localiza-

tion/binding site of a cationic PS is highly dependent on the

structure and intramolecular charge distribution of the por-

phyrin compound and is an important factor in photody-

namic antimicrobial chemotherapy. Therefore, the purpose

of this work was to summarize the literature data and our

own data obtained recently and reflecting the interaction of

cationic porphyrins with biosubstrates in the context of

photoinactivation of the corresponding pathogens.

Very interesting data were obtained in a study[ 217] that

evaluated the efficiency of photoinactivation of bacterio-

phage T4 using tetra-, tri-, and dicationic porphyrins. The

efficiency of photoinactivation of this virus depends on the

charge of the MHC: the greater the charge, the greater the

virucidality of the MHC. The authors attribute the revealed

pattern to the fact that the T4 phage capsid in aqueous solu-

tions with a pH of about 7 is negatively charged; therefore,

tetracationic porphyrins interact electrostatically with the

surface of the virus, and partially oxidize it upon photoirra-

diation. Tricationic porphyrins are also able to interact with

the bacteriophage capsid, in contrast to dicationic MHCs.

The significance of the positive charge of MHC for pho-

toinactivation can also be traced on the example of trisubsti-

tuted porphyrins: 5-(4-methoxycarbonylphenyl)-10,15,20-

tris(N-methylpyridinium-4-yl)porphyrin tri-iodide and 5-(4-

carbonylphenyl)-10,15,20-tris(N-methylpyridinium-4-yl)por-

phyrin tri-iodide. These porphyrins differ only in the nature

of the substituent in one of the phenyl rings. The carboxyl

group of 5-(4-carbonylphenyl)-10,15,20-tris(N-methylpyri-

dinium-4-yl)porphyrin tri-iodide dissociates -CO2H  -CO2-+

+ H+[218] in an aqueous medium, unlike -CO2CH3 group,

which is part of 5-(4-methoxycarbonylphenyl)-10,15,20-

tris(N-methylpyridinium-4-yl)porphyrin tri-iodide and is

incapable of dissociation. As a result, the total formal

charge of a porphyrin with a carboxyl group and virucidal

activity are lower than those of a porphyrin with a -CO2CH3

group.

Results for 5-(4-methoxycarbonylphenyl)-10,15,20-

tris(N-methylpyridinium-4-yl)porphyrin triiodide and 5-

(pentafluorophenyl)-10,15,20-tris(N-methylpyridinium-

4-yl)porphyrin triiodide demonstrate that the presence of a

lipophilic aryl group in one of the meso positions of the

tetrapyrrole macrocycle contributes to phage inactiva-

tion.[219] This conclusion is consistent with the results of the

study of photoinactivation of hepatitis A virus and bacteri-

ophage MS2 with PS differing in the length of the side al-

kyl chain (tetrakis(N-[n-butyl]-4-pyridiniumyl) porphyrin,

tetrakis(N-[n-hexadecyl]-4-pyridinium tetrayl)porphyrin,

tetrakis(N-methyl-4-pyridiniumyl)porphyrin, tetrakis(N-[n-

octyl]-4-pyridiniumyl) porphyrin).[203] At the same concen-

tration of PS and identical conditions for complete photoin-

activation of the hepatitis A virus using tetrakis(N-[n-

butyl]-4-pyridiniumyl) porphyrin, irradiation was required

for 30 min, while when using tetrakis(N-[n-octyl]-4-

pyridiniumyl)porphyrin took 1 min to achieve the same

effect.[203]

The introduction of lipophilic fragments has a positive

effect on the bactericidal activity of PS. Photodynamic ef-

fect of meso-substituted cationic porphyrins, 5-[4-

(trimethylammonium)phenyl]-10,15,20-tris(2,4,6-trimethoxyp-

henyl)porphyrin iodide, 5,10-di(4-methylphenyl)- 15,20-

di(4-trimethylammoniumphenyl)porphyrin iodide and 5-(4-

trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)-

porphyrin iodide have been studied in vitro in gram-

negative Escherichia coli bacteria.[220] It turned out that

after the washing step, only 5-(4-trifluorophenyl)-10,15,20-

tris(4-trimethylammoniumphenyl)porphyrin iodide had bacte-

ricidal activity.

The authors,[221] while studying the antibacterial prop-

erties of meso-substituted tetracationic pyridylporphyrins

quaternized with hydroxyalkyl and haloalkyl residues

against gram-negative and gram-positive microorganisms,

hypothesized that the main factor of photoinactivation is the

presence of positively charged groups in the PS molecule,

which have electrostatic interaction with negative groups on

the surface of bacterial cell membrane. The final binding of

PS to cells is regulated by the lipophilicity of functional

groups, which contributes to the strong association of PS

with the cytoplasmic membrane.

Figure 5. The structure of cell membrane.[216]

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

232 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

When designing a PS, it is also necessary to take into

account the fact that the introduction of a lipophilic substit-

uent can lead to a shift in aggregation equilibria in porphy-

rin solutions towards self-association, which will adversely

affect antibacterial activity. It is this phenomenon that was

discovered by the authors[222] when studying the photophys-

ical properties and antibacterial activity of a number of deriva-

tives of 5,10,15,20-tetrakis-(4-N-methylpyridyl)porphine, in

which one N-methyl group is replaced by a hydrocarbon

chain in the range from C6 to C22. It turned out that the

antibacterial photoactivity of porphyrins increased with an

increase in the length of the N-alkyl chain to C14, which

led to complete inhibition of the growth of E. coli even at

doses of 0.8 μM. A further increase in the size of the sub-

stituent to C18 and C22 was accompanied by a decrease in

the photosensitizing efficiency.

It is known that an asymmetric charge distribution in

the peripheral position of a porphyrin leads to an increase in

the amphiphilic nature of MHC, which can contribute to a

better accumulation in cells during PDT,[223] as well as inac-

tivation of bacteria. For example, 5-(4-trifluorophenyl)-

10,15,20-tris(4-trimethylammoniumphenyl)porphyrin io-

dide,[224] which has a lipophilic trifluoromethyl group in its

composition, exhibits the ability to bind with high affinity

to E. coli (gram-negative) and provides effective photoinac-

tivation. At the same time, 5,10-di(4-methylphenyl)-15,20-

di(4-trimethylammoniumphenyl)porphyrin iodide is able to

cause photodamage of E. coli in a lesser extent, and after

washing out the PS, it does not inhibit the growth of bacte-

ria at all. This fact suggests that the dicationic porphyrin

does not enter the cell.

The inactivation of gram-positive and gram-negative

bacteria with 5,10,15-tris(1-methylpyridinium-4-yl)-20-

(pentafluorophenyl)porphyrin tri-iodide is reported in the

work,[225] which also emphasizes the importance of the lip-

ophilic substituent in the PS molecule for photoinactivation

of gram-negative bacteria.

Quite interesting data were obtained by the authors[209]

in the study of photoinactivation of gram-negative bacteria

Vibrio anguillarum and E. coli by porphyrins. Tetra(4-

sulfonatophenyl)porphine, tetra(4N-methyl-pyridyl)porphine

tetraiodide and the tetra(4-N,N,N,-trimethyl-anilinium)por-

phine were evaluated as PS. All three porphyrins show a

similar pattern of subcellular distribution, localizing mainly

in protoplasts. However, only cationic porphyrins have pho-

toinactivating ability.

It is known that the mechanism of photooxidation with

the participation of PS can proceed in two types: type I,

with the participation of radical forms, and type II, with the

participation of singlet oxygen. As the data show,[226] pho-

toinactivation of gram-negative bacteria of the genus Sal-

monella is carried out to a greater extent due to the genera-

tion of superoxide anion and hydroxyl radical upon irradia-

tion of tetracationic porphyrin containing [Ru(bpy)2Cl]+ -

peripheral groups. The results obtained are credible, since

the formation of radical forms was confirmed by the EPR

method.

As noted above, the cell membrane of gram-negative

bacteria is characterized by low permeability for large mol-

ecules. Nevertheless, the authors[227] succeeded in obtaining

an effective PS for the gram-negative bacterium E. coli

based on a conjugate of tricationic porphyrin with cy-

clodextrin, which, judging by the information presented by

the authors, penetrates into the bacterial cell. The work[228]

emphasizing the significance of the influence of the charged

group position and the bridging heteroatom in porphyrin

conjugates with cyclodextrin on the efficiency of E. coli

photoinactivation is quite indicative. The authors of this

publication found that the replacement of thiopyridine by

methoxypyridine units in the conjugates leads to a signifi-

cant decrease in the antibacterial effect.

Figure 6. Atomic force microscopy for a mycobacterium in the initial state (a), after light exposure (b), after treatment with tetracationic

ZnTMPyP (c), after treatment with tetracationic ZnTMPyP and light exposure (d).

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 233

Figure 7. Effect of photosensitization by the exogenous porphyrins

on the ultrastructure of C. albicans cells. TEM images of C. albicans

cells treated with 3.5 μM TMPyP illuminated with 12 J·cm-2

(TMPyP+Light). C. albicans cells illuminated with 12 J·cm-2 without

an exogenous porphyrin (Light Control) served as control.[234]

Establishing the localization and accumulation sites of

PS in virions and most bacterial cells is a complex and un-

resolved issue in most cases. For bacterial cells, it is ex-

tremely difficult to analyze individual structural elements of

the cell, for example, by flow cytometry methods, where

the bacterial cells themselves are often at the limit of the

resolution of the method[216]. Obtaining information about

the role or behavior of individual cell structures in the pro-

cess of photoinactivation of bacteria requires the use of

complex methods. In some cases, it is possible to establish

the localization of a PS by the presence of its own fluores-

cence[229]. Recently, atomic force microscopy[230-232] or

transmission electron microscopy[323] has been used to

prove the localization of PS in the bacterial cell wall. Figure

6 shows, as an example, atomic force microscopy data for a

mycobacterium in the initial state (a), after light exposure

(b), after treatment with tetracationic zinc(II) 5,10,15,20-

tetra-[4-N-methylpyridyl]porphine (ZnTMPyP) (c), after

treatment with tetracationic ZnTMPyP and light exposure

(d), as well as TEM results for C. albicans with 5,10,15,20-

tetra-[4-N-methylpyridyl]porphine (TMPyP) porphyrin.[230]

TEM makes it possible to detect ultrastructural chang-

es in the cell, so in the presented photo (Figure 7), after

treatment with PS and light, disorganization of the cyto-

plasm is visible, there is a lack of a nucleus and the appear-

ance of white spots, a massive accumulation of wall-like

material in the cytoplasm and damage of cells with a rup-

ture of the membrane, which reflects the leakage of intra-

cellular components.[234]

However, few such data have been obtained so far,

which does not allow us to analyze the dependence of the

PS structure and its predominant localization in the patho-

gen. Nevertheless, the conducted mini-review of the litera-

ture data allows us to conclude that for the effective interac-

tion of meso-substituted porphyrins with pathogens and

their subsequent inactivation, three cationic groups in the

photosensitizer composition are sufficient, and the fourth

peripheral substituent must have a lipophilic character and

can provide the required selectivity. Based on this conclu-

sion, we attempted to design a cationic porphyrin capable to

binding with the SARS-CoV-2 spike protein in the recep-

tor-binding domain (RBD).[235,236] The ultimate goal was to

obtain a porphyrin capable of causing photodestruction of

the RBD S-protein, which would make it impossible for the

SARS-CoV-2 virion to bind to the surface enzymes of hu-

man cells. As a result of theoretical studies,[235-239] the some

porphyrins were designed, which, according to the results

of molecular docking with the S-protein, are localized be-

tween the RBDs of three S-protein polypeptide chains. Us-

ing the C-H-activation method,[240] a targeted synthesis of

porphyrins was carried out: 5-[4′-(1″,3″-benzothiazol-2″-

yl)phenyl]-10,15,20-tris(N-methylpyridinium-3′-yl)porphyrin

triiodide (S-Por), 5- [4′-(1″,3″-benzoxazol-2″-yl)phenyl]-

10,15,20-tris(N-methylpyridinium-3′-yl)porphyrin triiodide

(O-Por), 5-[4-(N-Methyl-1″,3″-benzimidazol-2″-yl)phenyl]-

10,15,20-tris(N-methylpyridinium-3′-yl)-porphyrin triio-

dide (N-Por). For the compounds obtained, the quantum

yields of singlet oxygen were determined, which amounted

to 0.61-0.86.[235,241] In monohetaryl substituted porphyrins,

the replacement of a heteroatom has almost no effect on

both the quantum yield of fluorescence (0.17–0.21) and sin-

glet oxygen, and the lifetime (8.3–9.8 ms) in the triplet state.

To confirm the results of molecular docking, a spectral

study of S-protein binding to the synthesized monoheteryl

substituted porphyrins was performed. Quantitative charac-

teristics reflecting the affinity[242,243] of the S-protein to he-

taryl porphyrins were obtained by spectrophotometric titra-

tion of the S-protein with asymmetrically monosubstituted

hetaryl porphyrins (Table 1).

Table 1. The affinity parameters of S-protein with hetaryl porphy-

rins according to fluorescent titration data.

System

Affinity

The number of

binding sites

S-protein·N-por

5.94·105

1.18·105

3.6

4.7

S-protein·O-por

1.2·106

2.4

S-protein·S-por

7.96·105

2.6

S-protein·ACE2·N-por

1.56·105

1.25·105

2.2

2.4

Figure 8. Structure of the ACE2-RBD (6vw1) complex with N-

por according to X-Ray data. Yellow ACE2, blue RBD. Orange

indicates the RBD region responsible for binding to the N-por.[244]

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

234 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

The results of competitive titration[243] of the S-

protein·ACE2 complex with N-por (Figure 8) clearly demon-

strated that N-por leads to the destruction of the S-protein

complex with ACE2. The competitive influence of N-por and

ACE2 for binding to the S-protein confirms that the binding of

porphyrin occurs precisely in the RBD site of the S-protein.

Determination and evaluation of the bactericidal activ-

ity of porphyrins was carried out on the surface of a dense

nutrient medium (Columbian Agar with sheep's blood) con-

taminated with museum strains of microorganisms -

S.aureus ATCC 29213 and S.epidermidis ATCC 14990. It

was found that the synthesized compounds upon photoacti-

vation exhibit antibacterial activity against staphylococci

(Figure 9, Table 2). Thus, the conducted microbiological

study showed that with photoirradiation for 15', monoheter-

yl substituted porphyrins containing benzothiazole and N-

methylbezimidazole residues provide complete lysis of

S.aureus and S.epidermidis strains. In the case of a hetaryl

substituted porphyrin with a benzooxazole residue, com-

plete lysis was observed in the S.aureus strain and incom-

plete lysis was observed in the S.epidermidis test strain.

Increasing the exposure time to 30' ensured complete lysis

in the S.epidermidis strain.

This review of literature data showed that tri- and tet-

ra-cationic porphyrins are effective photosensitizers; reac-

tive oxygen species generated by them initiate oxidative

destructive reactions in biomolecules and biostructures of

pathogens. Variation of the lipophilic nature of the PS

makes it possible to adjust the bactericidal and virucidal

activity of MHC. To increase the efficiency of photoinacti-

vation of pathogens, further studies are needed to study the

influence of the nature of photosensitizers, their interaction

with cells and subcellular structures, biomolecules, and the

assessment of photoinduced damage.

Por-

phyrin

Exposure time, min

Control in dark

15'

30'

60'

S-por

О-por

N-por

Figure 9. The results of the bactericidal effect of porphyrins in the dark and upon activation by light on the S. aureus ATCC 29213 test

culture.

Table 2. Evaluation of lytic activity on a five-point scale (“-” no lytic activity; “+” low activity; “++” formation of a lysis zone with a

large number of colonies of secondary growth of the bacterium; “+++” lysis zone with single colonies of secondary growth ; "++++"

transparent zone of lysis without secondary growth colonies).

Porphyrin

S-por

O-por

N-por

Exposure time

15'

30'

60'

15'

30'

60'

15'

30'

60'

S.aureus

++++

+++

++++

S.epidermidis

++++

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 235

5. Approaches toward Cationic Photosensitizers

Based on Phthalocyanines

One of the most attractive photosensitizers in PDT are

phthalocyanines (Pcs) and their metal complexes.[10,98, 245–248]

The singlet oxygen mediated process is predominant for

Pcs that possess strong absorbance in the visible region

between 600 nm and 700 nm (referred to as the Q-bands)

resulting from the HOMO → LUMO (π→π*)

excitations.[249,250] This part of spectrum favours the optimal

penetration of the light through a tissue. Moreover, Pcs,

compared with porphyrins, have low absorbance at wave-

lengths between 400 nm and 500 nm, where daylight inten-

sity is highest. The presence of a heavy atom in the phthal-

ocyanine leads to the enhancement of singlet oxygen gener-

ation due to an increase in spin-orbit coupling owing to so

called “heavy atom effect”, and consequently, the transition

of the photosensitizer from the excited singlet state to the

excited triplet state. Outstanding photophysical properties

of phthalocyanines are tailorable through variation of the

central cation, axial ligands, nature of substituents and their

position in the Pc aromatic ring.[98,251–253] Introduction of

cationic substituents into phthalocyanine’s rings may pro-

vide better solubility in water as well as decrease their ten-

dency to aggregation [254,255]. In addition, many cationic Pcs

show a higher photodynamic efficiency than anionic or

uncharged hydrophilic Pcs [255] due to better binding to

DNA molecules.

Cationic function in Pc can be formed via different

ways, such as: quaternization of the nitrogen atom in ali-

phatic and aromatic substituents,[256–258] quaternization of pyri-

do(pyrazo)porphyrazines [255] and exocyclic metalation,[259]

introducing of pnictogenes(V) in the cavity of phthalocya-

nine ring,[260–264] or via meso-N-protonation of phthalocya-

nines bearing alkoxy-groups in non-peripheral positions[265]

(Figure 10).

The synthesis and photophysical properties of some

cationic Pcs bearing quaternized nitrogen-containing groups

have been summarized in two comprehensive reviews.[266,267]

This chapter of the review is an attempt to illustrate novel

examples of Pcs with quaternized nitrogen-containing

groups and other pathways to others types of cationic Pcs.

N-Centered Cationic Charges

Intriguing examples of cationic phthalocyanines with

four and eight extremely bulky 2,6-di(N-methylpyridine-3-

yl)phenoxy substituents were reported by P. Zimcik group

(Figure 11).[257] These Pcs exist as monomers in water and

phosphate buffered saline (PBS) and exhibit moderate fluo-

rescence quantum yields (0.17 and 0.16) both in DMF and

PBS that may be used for bioimaging. Also, high values of

phototherapeutic indices were obtained. The ratio of dark

toxicity to light toxicity (TC50/EC50) for HeLa cell line was

found to be 1409 and 5691 for tetra- and octa-substituted

complexes respectively.

Introduction of imidazolium units into Pcs’s macro-

heterocycles was accomplished by Lioret et al. through SN2

reaction of 4,5-dichlorophthalonitrile and imidazole in the

presence of K2CO3 at 50 °C yielding 75 % of the diimidaz-

olyl derivative. The further template cyclotetramerization of

the resulting phthalonitrile with Zn(OAc)2 lead to octa-

imidazolyl Pc derivative (Figure 12).[258] After quaterni-

zation with methyliodide, the target zinc octa(N-

methylimidazolium)phthalocyaninate was obtained in al-

most quantitively yield. For latter complex photocytotoxici-

ty studies were carried out on murine melanoma B16F10

cancer cells and IC50 = 5.379 mM was determined.

Figure 10. Approaches toward cationic phthalocyanines and tet-

ra(pyrido/pyrazino)porphyrazines.

Figure 11. Synthesis of quaternized zinc complexes with tetra-[257] and octa-substituted[268] [2,6-di(3-pyridyl)phenoxy]phthalocyanines.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

236 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

Figure 12. Synthesis of zinc octa(N-methylimidazolium) phthalocyaninate.[258]

Figure 13. Robust route toward cationic phthalocyanines through reductive amination.[256]

Recently Bunin et al. developed robust route toward

cationic phthalocyanines through reductive amination.[256]

In the synthesis of cationic Pcs reductive amination was

used for the first time. Thus, reaction of 3- and 4-

(formylphenoxy)phthalonitriles with diethylamine and so-

dium tris-acetoxyborohydride in THF resulted in corre-

sponding diethylamino-derivatives of phthalonitriles isolat-

ed in high yields (Figure 13). New Zn(II), Mg(II) and metal-

free phthalocyanines containing four or eight cationic

groups in non-peripheral (-) or peripheral (-) positions

were synthesized starting from the aminated phthalonitrile

derivatives followed by quaternization with CH3I. All novel

cationic Pcs exhibited strong absorption in the photothera-

peutical range from 670 to 700 nm and high quantum yields

of singlet oxygen generation in DMSO where all phthalo-

cyanines existed in monomeric forms. Also, cationic com-

plexes showed moderate solubility in water with high ten-

dency to aggregation, however -substituted Zn(II) and

Mg(II) complexes existed in water in predominantly mon-

omeric states. Further monomerization of tetra--

substituted Zn(II) complex in aqueous solution was

achieved by interaction either with non-ionic surfactant

Tween-80 or bovine serum albumin (BSA). The advantage

of these results was not limited to new convenient synthetic

approach to the cationic phthalocyanines but it also consist-

ed in preparation of new promising phototherapeutic agents

with good photophysical and photochemical properties.

Cationic Charges Centered on Other Atoms

Schneider et al. report the first example of exocyclic

metalation of tetra(pyrido)porphyrazine (Figure 14).[259] In this

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 237

intriguing work, zinc complex with tetra(pyrido)porphyrazine

(as a mixture of regioisomers) was coupled with transplatine

fragments via reaction with fivefold excess of tetrakis-

[trans-Pt(NH3)2Cl]NO3, derived in situ from transplatine

(tetrakis-(trans-Pt(NH3)2Cl2)) and AgNO3 in DMF. Mass-

spectrometry in combination with 195Pt-NMR spectrum for

platinated compound indicates the formation of the tet-

raplatinated derivative as a sole product. This exo-

platinated Pc demonstrate red shift of Q-band in UV-Vis

spectra up to 682-692 nm in comparison with 663-674 nm

for its non-platinated precursor. Interesting, platinated com-

plex of demonstrated low absolute quantum yield of fluo-

rescence (0.01 in DMF) expectedly due to large percentage

of inter system crossing (ISC) that provides «heavy atom

effect» of Pt-atoms, but no generation of singlet oxygen

was also indicated (by direct method in which luminescence

of 1O2 was registered at 1270 nm in DMF-d7). However, in

phototoxicity experiments with HeLa cell line this complex

exhibited IC50 value at 1.6 M at the light and 12 M in the

dark. These results could be rationalized by production of

other forms of reactive oxygen species (ROS) rather than

singlet oxygen. Notably, for zinc complex of tet-

ra(pyrido)porphyrazine light and dark toxicities IC50 were

28 and 34 M respectively. Difference in dark toxicity of

precursor and platinated complex was explained by DNA-

binding ability of transplatine fragments, which was shown

in this work; after irradiation difference in toxicity was ex-

plained by «double-punch» effect (photoxicity in combina-

tion with DNA-binding ability) provided by platinated

complex. Thus, authors proposed further investigation of this

novel exocyclically platinated tetra(pyrido)porphyrazine as a

photosensitizer.

Also, positive charges can be obtained by introduction

of quaternized phosphonium moieties (Figure 15).[269–271]

For example, such complexes were synthesized with the

intention to be used for enhanced mitochondria penetration

owing to four triphenylphosphonium units, they demon-

strated both photodynamic and sonodynamic efficacy against

MCF-7 and HeLa cell lines (IC50 = 12.7 – 45.0 M).

Figure 14. Synthesis of phthalocyaninate modified by four transplatine units.[259]

Figure 15. Synthesis of phthalocyanines with trisphenylphosphonium moieties.[269–271]

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

238 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

Figure 16. Reversible nucleophilic addition to phosphorus(V)

phthalocyaninate (Nu- = OH- or MeO).[264]

Recently Kolomeychuk et al. performed detailed in-

vestigation of phthalocyaninate where the cationic phospho-

rus(V) centre was placed into the cavity of the

macrocycle[264] in order to compare its photophysical prop-

erties with corresponding porphyrin derivatives.[272,273] Dur-

ing this study the authors discovered the unprecedent

switching of aromaticity (Figure 16),[264] which could be

"turned off" by reversible aromatic nucleophilic addition of

OH- or OMe- to the –pyrrole C-atom affording the nonflu-

orescent adduct. Aromaticity and fluorescence could be

restored by acidic treatment of the adduct. Structure of the

adduct was confirmed by single crystal X-ray analysis.

Kobayashi et al. demonstrated the prospective of cati-

onic phthalocyanines with endocyclic pnictogene atoms.

They synthesized non-peripherally substituted octa(2,6-

dimethylthiophenoxy) phthalocyaninates of P(V), As(V)

and Sb(V) and thoroughly investigated their optical proper-

ties (Figure 17).[260–262] Absorption maxima of these com-

plexes (Q-bands) were found at 1018, 1032 and 1056 nm,

respectively, whereas the analogous zinc complex had a Q-

band located at 790 nm (in CH2Cl2). Such strong shifts of

Q-bans into near-IR region for P(V), As(V) and Sb(V)

complexes occurred due to decrease in HOMO–LUMO gap

that was confirmed by DFT calculations. In addition, the

insertion of electron-donating S-atoms in non-peripheral

positions also contributed to the narrowing of the HOMO–

LUMO gap.

Figure 17. Complexes of P(V), As(V) and Sb(V) with non-peripherally 2,6-dimethylthiophenoxy-substituted phthalocyanine.[261]

(a)

(b)

(c)

Figure 18. (a) – Zinc(II) complex with crown-substituted oxanthrenocyanine ZnOc; (b) reversible formation of bifurcate proton bonds

upon acidic treatment; (c) UV-vis spectra of ZnOc and its protonated forms in CHCl3.[265] Adapted with permission from ref. [265] Copy-

right 2016, American Chemical Society.

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 239

Apart from strong bathochromic shifts of Q-bands

non-peripheral substitution provides phthalocyanines with

enhanced basicity of their meso-nitrogen atoms.[274,275] Pro-

tonation has profound effect on optical properties of phthal-

ocyanines resulting in strong bathochromic shifts of their

Q-bands and quenching of fluorescence.[276] For example,

Safonova et al. developed approach toward tetra-15-crown-

5-annelated zinc oxanthrenocyaninate ZnOc (Figure 18a,b)

and demonstrated consecutive formation of four proto-

nated forms having absorption maxima at 798, 867, 974

and 1028 nm respectively whereas non-protonated form

had maximum of absorbance at 732 nm (Figure 18c).[265]

Notably, this protonation is reversable and neutral form can

be recovered under addition of amines. Such proton-

susceptible compound can serve as a molecular switcher for

optoelectronic materials, sensors as well as switchable pho-

tosensitizers.

To conclude, cationization of phthalocyanines is an ef-

fective way to complexes, that are soluble in water and ex-

ist in aqueous solutions predominantly as monomeric spe-

cies. Also, some cationic phthalocyanines and their analogs

have absorbance beyond 1000 nm due to complexation with

pnictogens or formation of protonated species. These prop-

erties of cationic Pcs in conjunction with excellent photo-

dynamic efficiency may be useful in photomedicine and

also, cationic Pcs might be suitable NIR dyes, molecular

switchers and sensors.[277]

6. Effective Near-Infrared Photosensitizers for

PDT and aPDT Based

on meso-Tetrakis(3-pyridyl)bacteriochlorin

The reduced derivatives of porphyrins such as, chlo-

rins and bacteriochlorins, are used as photosensitizers (PSs)

for photodynamic therapy (PDT) of cancer due to their

high-intensity optical absorption and luminescence in the

red and near-IR range.[46,148,278-281] Thus, well-known photo-

sensitizers based on hydrogenated porphyrin derivatives

such as Foscan® (Temoporfin, meso-tetrakis(3-hydroxyp-

henyl)porphyrin),[282,283] Visudyne® (Verteporfin),[46,284] and

Tookad[285, 286] have been designed (Scheme 38).

A porphine molecule has a multiple-contour conjugat-

ed system; its interior chromophore contains 18 π electrons.

Two peripheral Сβ=Сβʹ bonds are quasi-isolated, and their

hydrogenation does not disrupt the main macrocyclic aro-

matic π-conjugated system (Scheme 39).[287–290]

Reduction of the double bond in one of the pyrrolyc

moieties of porphine gives chlorin (2,3-dihydroporphyrin);

reduction of double bonds in two opposite pyrrole moieties

give rise to bacteriochlorin (7,8,17,18-tetrahydroporphyrin,

ВС), while hydrogenation of two adjacent pyrrole moieties

yields isobacteriochlorin (2,3,7,8-tetrahydroporphyrin, iBC)

(Scheme 40).[291]

Scheme 38.

Scheme 39.

Scheme 40.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

240 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

Scheme 41.

Scheme 42.

Scheme 43.

The main data on preparation of synthetic chlorins and

bacteriochlorins have been summarized in reviews.[292-294]

The simplest and most available method for preparation

reduced porphyrin derivatives is Whitlock reduction (reduc-

tion by diimide generated in situ from p-toluenesulfonyl

hydrazide, TsNHNH2) (Scheme 41).[295]

Solubility in bodily fluids, and water in particular, is

one of the key requirements posed on PSs, along with the

high quantum yield generation of singlet oxygen and tropic-

ity to tumor cells [14,296-299]. Therefore, meso-tetrakis(3-

pyridyl)bacteriochlorin is a promising platform for design-

ing novel photosensitizers: on the one hand, this compound

is characterized by strong light absorption in the long-

wavelength region (λmax = 747 nm; ε ~ 112,000 mol–1 L cm–1;

chloroform [300]), while on the other hand, quaternary am-

monium salts can be produced by quaternization of pyridyl

nitrogen atoms upon exposure to various alkylating agents,

giving rise to water-soluble species.

The initial meso-tetra(3-pyridyl)bacteriochlorin

(BC 1) was synthesized using the conventional method: by

Whitlock reduction of meso-tetrakis(3-pyridyl)porphyrin

(Scheme 41, R = 3-Py), and its two water-soluble salts

(tetraiodide (BC 2) and tetratosylate (BC 3)) were immedi-

ately produced by boiling of BC 1 with excess amount of

methyl iodide or methyl-p-toluenesulfonate, respectively

(Scheme 42).[301]

Later on, it was shown that other water-soluble qua-

ternary salts can be produced by boiling BC 1 with excess

amount of 1,4-dibromobutane (BC 4) or ethyl chloroacetate

(ВС 5), respectively, in nitromethane under an inert atmos-

phere (Scheme 42).[300] Aqueous solutions of tetracationic

salts are stable under dark conditions for up to 6 months.[302]

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 241

The presence of bromine atoms in the alkyl chain of

ВС 4 made it possible to conduct further quaternization and

increase the number of cationic sites to n = 8. Boiling BC 4

with excess amount of dry pyridine or dimethylaminoethanol

(DMAE) in methanol under an inert atmosphere for 4.5 h

yielded octabromide (BC 6) and octabromide (ВС 7), re-

spectively (Scheme 43).[300]

Hermann et al.[303] found that tetratosylate ВС 3 gen-

erates singlet oxygen via type I photosensitization. During

type I photosensitization reactions, an electron is detached

from the substrate by a photoexcited photosensitizer mole-

cule, which is accompanied by formation of hydroxyl radi-

cals, superoxide anion radicals, and hydrogen perox-

ide;[304,305] in turn, these species can easily penetrate biolog-

ical membranes, are highly reactive in oxidation processes,

and can further generate radical particles, thus ensuring an

oxidation cascade.

Tetratosylate ВС 3 exhibited high photodynamic ac-

tivity (> 86% dead cells) with respect to bile duct cancer

(BDC, GBC) cell lines in in vitro and in vivo experiments;

furthermore, it possesses low systemic toxicity and derma-

tological toxicity.[306] Later, in vivo experiments showed

that BC 3 is rapidly absorbed by human choroidal melano-

ma cells and has low dark toxicity after 24 h incubation.[307]

The studies of pharmacokinetics and biodistribution of ВС

3 in vivo (in live mice) and ex vivo (in resected organs) in

Balb/c colon-26 carcinoma tumor-bearing mice showed that

ВС 3 is predominantly accumulated in the tumor compared

to most normal tissues except for the kidneys: the tumor-to-

normal tissue ratio (TNTR) for the liver, colon, muscle, and

spleen tissues ranged from 8 to 50.[307]

Physicochemical properties, biodistribution in animal

tissues and photoinduced antitumor efficacy of ВС 4 and

ВС 6 were studied using epidermoid carcinoma of the lar-

ynx (Hep2) cell culture and the Lewis lung carcinoma

(LLC) animal tumor model. In vitro studies showed that

photosensitizers remained stable for a long time and exhib-

ited a high photoinduced activity with respect to НЕр2 cell

culture (IC50 varied from 0.34±0.07 to 0.71±0.09 μМ)[308].

These compounds were shown to be rapidly accumulated in

tumor tissue in mice (the tumor-to-normal tissue ratio being

2.3–3.3), exhibit a high photoinduced activity against LLC

tumor cells – for BC 4 and BC 6 in the in vitro (IC50 is

0.34±0.03 and 0.37±0.08 μМ, respectively) and in vivo sys-

tem (inhibition of tumor growth and response rate are

100%).[308]

Along with being used as conventional photosensitiz-

ers for PDT, BC 1 and its water-soluble derivatives exhibit

antibacterial activity against various pathogens. Thus, ВС 3

was used against Gram-positive S.aureus strains (MSSA

and MRSA) and Gram-negative E.coli and P. aeruginosa

strains.[309,310] BC 3 was found to strongly bind to the cell

wall of both Gram-positive and Gram-negative bacteria and

exhibit no inhibitory activity against S.aureus in the dark,

while irradiation causes almost complete inactivation of the

pathogen.[309] Incubation of the S. aureus MRSA strain in

phosphate-buffered saline (PBS) in the presence of ВС 3

for 30 min followed by irradiation causes almost complete

death of the pathogen.[310]

Along with quaternary ammonium salts, lyophilisate

for solution for infusion based on BC 1 and containing a

non-ionogenic surfactant, Bacteriosens drug, can be used as

a water-soluble photosensitizer.[311] This lyophilisate is sta-

ble during storage under dry conditions for 6 months. Bac-

teriosens exhibits a moderate in vitro hemolytic activity,

does not possess dark cytotoxicity against cultured human

and mouse cancer cells, and exhibits high specific activity

when exposed to light.[312,313]

Loschenov et al. described the application of ВС 1 as

a photobactericidal coating for a hydroxyapatite implant

and demonstrated that this coating was resistant to washout

of photosensitizer particles over time.[314]

The Intrachlorin composition based on meso-

tetrakis(3-pyridyl)bacteriochlorin and being a mixture of

water-soluble meso-tetra(3-pyridyl)bacteriochlorin and me-

so-tetra(3-pyridyl)chlorin (88 : 12, wt. %) was designed.[315,316]

The mixture was prepared by Whitlock reduction of meso-

tetrakis(3-pyridyl)porphyrin, and pyridyl nitrogen atoms

were subsequently quaternized using methyliodide.[316] The

resulting composition is characterized by stability, high-

intensity long-wavelength optical absorption, and high

quantum yields of singlet oxygen generation (~

0.45±5).[317] The Intrachlorin composition was active when

used at a dose of 5–15 mg per 1 g of tumor against model

Lewis tumors, spontaneous pituitary cancer, and breast can-

cer.[318] Rzhevskii et al. reported using the Intrachlorin

composition for photodynamic therapy of C26 colon carci-

noma subcutaneously grafted to Balb/c mice.[319] The recur-

rence-free efficacy during 6 months was 70 %. A systemic

toxicity study of the Intrachlorin composition in Wistar

mice was conducted.[319] The 10 mg/kg dose of the In-

trachlorin composition was found to be safe when adminis-

tered daily during 7 days, both intravenously and subcuta-

neously; the dose > 20 mg/kg repeatedly administered in-

travenously and the dose > 50 mg/kg repeatedly adminis-

tered subcutaneously were found to induce toxic effects.[319]

7. Spectral Properties of Photosensitizers Based

on Tetra(pyridin-3-yl)porphine and Its Reduced

Forms in Solutions and Thin Films

There are numerous tetrapyrrolic compounds that are

used as PSs in PDT, whereas porphyrins and phthalocya-

nines are the most frequently used PSs in antibacterial pho-

todynamic therapy (aPDT). Cationic porphyrins like TMPyP

(5,10,15,20-tetrakis(1-methyl-4-pyridinium)porphyrin tetra(p-

toluenesulfonate)) have fourfold positive charge. Collins et

al. used TMPyP against Pseudomonas aeruginosa biofilms

both wild and mutant strains.[320,321] It was postulated that

this effect might be due to the large molecular structure of

TMPyP or strong electrostatic interaction between the four-

fold positive charge of cationic porphyrin and negative

charge of extracellular polymeric substance molecules.[32 2]

Nowadays cationic antimicrobial peptides or cell pen-

etrating peptides are conjugated to porphyrins to improve

their efficiency. These conjugated porphyrins show a great

cell inactivation during aPDT. Recent studies strongly up-

hold the hypothesis that aPDT can be a satisfactory alterna-

tive since there is a substantial difference in the mode of

action of PSs than that of antibiotics. The key benefits of

aPDT can be outlined as follows: a broad spectrum of ac-

tion compared to antibiotics since PS can act on diverse

organisms such as bacteria, protozoa, fungi; bactericidal

effects independent of antibiotic resistance pattern; more

limited adverse effect profile and damage to the host tissue;

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

242 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

no resistance following multiple sessions of therapy. The

synthetic photosensitizers of the porphyrin series,[323,324]

conjugates of porphyrins with amino acids[325] and saccha-

rides[326,327] are the most studied. A special place in this

range of compounds is occupied by reduced forms of por-

phyrins - chlorins and bacteriochlorins, which can compete

with existing photosensitizers used in clinical practice.

During the last decade, nanotechnology has had a

great impact on PDT. Most of these studies have used na-

noparticles to improve the efficacy of classical PDT while a

few of them have been done on the antimicrobial as-

pects.[328] The results with nanoparticles were more satisfac-

tory than with the PS alone. Furthermore, PS bound to a

nanoparticle penetrates through the membrane better than

free PS. Nanoparticles themselves can act as PS.[329] To date,

low-dimensional porphyrin-based nanostructures with

promising properties have been obtained in three-

dimensional (3D) environments, on solid surfaces using

two-dimensional (2D) templates, exploiting covalent bond-

ing[330,331] or various non-covalent intermolecular interac-

tions.[332,333] Self-assembly is a key player in materials

nanoarchitectonics.[334–336] The nanostructures formed in

this way arise from molecules carrying side groups that

promote the process. Supramolecular polymers were creat-

ed using diverse self-assembly strategies.[337,338] Formation

of different kinds of supramolecular assemblies from some

compounds at the water surface,[339] during electrochemical

deposition on electrodes[340] and in metal-organic frame-

works[341] was reported.

Previously, we demonstrated the possibility of supra-

molecular design at the air-water interface by controlled

self-assembly of porphyrins into 2D and 3D nanoaggre-

gates.[342–349] The concept of nanostructuring of organic

compounds at the air-water interface and a model of a float-

ing layer, the structural units of which can be both individ-

ual molecules (Langmuir's approach, special case) and their

major nanoaggregates (so-called M-nanoaggregates, gen-

eral case), were presented.[342,350] The formation of the por-

phyrin nanoparticles from magnesium porphine, a function-

al element of chlorophyll, was reported.[342] The formation

of nanoparticles of biological and bioactive compounds is a

non-trivial, but very important and urgent task. Recently,

we have established the possibility of formation of nanopar-

ticles by a hydrophobic derivative of vitamin B12 (porphy-

rin-type compound) whose properties differ from the prop-

erties of the parent molecules. They were formed at the

water-air interface. This was important for pharmacology

because this derivative is a neuroprotective agent.[351] Mi-

cro- and nanocapsulation technologies occupy an important

place in the development of means for targeted delivery of

hydrophilic and hydrophobic drugs and modulation of their

biological properties.[352-354] It was found that vitamin B12

itself can also form nanosized ensembles in delivery sys-

tems.[354]

However, nanostructures of photosensitizers based on

3-pyridylporphine, which is parent compound of such im-

portant photosensitizers as chlorin and bacteriochlorin, have

not yet been obtained.

The aim of this work is to synthesize and characterize

the 5,10,15,20-tetra(pyridine-3-yl)chlorin (T3PyCh) and

5,10,15,20-tetra(pyridine-3-yl) bacteriochlorin (T3PyBCh),

which are the products of 5,10,15,20-tetra(pyridine-3-

yl)porphine (T3PyP)[355] reduction according to the proce-

dure of Whitlock

†

[295] (Figure 19). The possibility of form-

ing T3PyP nanostructures by self-assembly at the water-air

interface and in thin films on solid supports was studied.

‡

Figure 19. Structural formulae of 5,10,15,20-tetra(pyridine-3-

yl)porphine (T3PyP); 5,10,15,20-tetra(pyridine-3-yl)chlorin

(T3PyCh); and 5,10,15,20-tetra(pyridine-3-yl) bacteriochlorin

(T3PyBCh).

†

T3PyP in pyridine was heated at 100-105 °C in the presence of

potassium carbonate with p-toluenesulfonylhydrazide (pTSH); the latter

was added gradually. The reaction was carried out until the maximum ratio

of optical densities at 745 and 653 nm (absorption maxima of bacteriochlo-

rin and chlorin, respectively) was reached. It was shown that this ratio is

inconstant and reaches a maximum (8-10) between 12 h and 16 h.

‡

The layers at the water-air interface were formed on a Langmuir

setup (NT-MDT, Russia). A solution of T3PyP in dichloromethane with C

= 0.88·10-4 М was applied to the surface of bidistilled water using a sy-

ringe with a graduation value of a 1 µL (Hamilton, Sweden) at (20±1) ºC.

The solution was taken in such a volume that the initial degree of surface

coverage, assuming perpendicularity of the planes of molecules and the

water surface (ISCD cedge, the ratio of the area occupied by the molecules

of the substance to the surface area of water) was between 25 and 51%.

Also the ISCD cface value for the parallel arrangement of the molecular

planes and the surface was calculated. Surface pressure was measured

using Wilhelmi sensor with an accuracy of 0.02 mN/m. In 15 minutes after

the application of the solution, the formed layer was compressed at a rate

of v=2.3 cm2·min-1.

Langmuir-Schefer (LS) films were prepared at (17±1) ºC. For this

purpose, the barriers compressing the layer on the water surface were

stopped at the selected pressure value, and then the layers were successive-

ly transferred onto the quartz substrate by the horizontal lift method. To

obtain optimal optical density, the number K of times the substrate was in

contact with the layer was varied from 20 to 60. The electronic absorption

spectra of LS films and solutions were recorded on a Shimadzu-Uv-1800

spectrophotometer (wavelength resolution ±1 nm), fluorescence spectra

were measured with the Solar spectrophotometer.

The structure of the layers at water–air interface was analyzed by

using quantitative analysis of compression isotherms of a nanostructured

M-monolayer, based on the concept of a layer as a real two-dimensional

gas with structural elements representing two-dimensional nanoaggregates

5–20 nm in diameter (M-nanoaggregates).[342] The method allows determi-

nation of the critical characteristics of floating layers: the area per mole-

cule in a nanoaggregate, the number of molecules in nanoaggregates, etc.

The stable state of the layer is described by equation π(A–Amol ) = kT/n (1)

where π is the surface pressure, A is the area per molecule in the layer,

Amol is the area per molecule in the aggregate, n is the number of mole-

cules in the aggregate, k is the Boltzmann constant and T is the absolute

temperature. When plotted in πA–π axes, a compression isotherm of the

floating layer has both linear (corresponding to the single-phase states of

the layer) and nonlinear regions. The Amol and n are the main characteris-

tics of a floating monolayer; these values may be determined by approxi-

mating the πA–π plot using a linear function (the least squares method was

used and the πA error did not exceed 3%).

Geometric parameters of the molecule, determined using the Hyper-

chemistry software, are as follows: а = 1.5 nm, b = 1.4 nm, с = 0.7 nm; the

projection areas Aproj(face) = 1.55 nm2, Aproj(edge) = 0.7 nm2; the areas

of circumscribed rectangles Arec(face) = 2.2 nm2 and Arec(edge) = 1.2 nm2.

The areas per molecule in the most densely packed monolayer are

Apack(face) = 2 nm2 and Apack(edge) =1 nm2.

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 243

Although the Whitlock procedure is now the main

method for obtaining chlorins from porphyrins, its course

has not been systematically studied. In turn, understanding

the principles of this interaction would be useful for identi-

fying reaction conditions in which the target product is ei-

ther T3PyCh or T3PyBCh. To this end, we quantitatively

monitored the diimide reduction of T3PyP in the course of

the Whitlock reaction by measuring the optical density of

the reaction mass at 653 and 745 nm after adding each por-

tion of the reducing agent (pTSH).

The reaction can be divided into two steps: the reduc-

tion of porphyrin to chlorin and the formation of bacterio-

chlorin. In the first stage, chlorin is predominantly formed,

which is accompanied by an increase in absorbance at 653 nm

(Figure 20a). In the second stage, chlorin is converted to

bacteriochlorin; absorbance decreases at 653 nm and in-

creases at 745 nm (Figure 20b).

The concentration of chlorin in the mixture initially

increases, reaches a maximum after 7.5 h, and then decreas-

es. On the other hand, the concentration of bacteriochlorin

increases insignificantly in the beginning, but starts to in-

crease sharply after 6 hours. Therefore, if the target product

of the reaction is chlorin, the reaction should be stopped at

the moment when bacteriochlorin absorption starts to grow

rapidly. Maximum conversion to bacteriochlorin is expected

when the maximum ratio of optical densities at 745 nm and

653 nm is reached.

Taking this data into account, we individually isolated

the reduced products. The hydrogenated products,

T3PyCh and T3PyBCh, were isolated by column chroma-

tography.[356] Chromatographic separation was performed

on aluminum oxide, and for the first time on silica gel using

chloroform or dichloromethane and their mixtures (up to

5%) with methanol. In general, chromatography on both

sorbents leads to almost identical results, however, separa-

tion on silica gel is more efficient in terms of product yield.

Figure 21 shows the absorption and fluorescence spec-

tra of the initial porphyrin T3PyP and its reduced forms,

T3PyCh and T3PyBCh.

As can be seen, the transition from porphyrin

T3PyP to chlorin T3PyCh results in significant changes in

the electronic spectra of their solutions. In particular, the

Soret band decreases significantly and broadens with the

formation of the "shoulder", with a simultaneous increase in

the intensity and a slight bathochromic shift of the last band

in the Q-region. Changes in the spectrum of bacteriochlorin

T3PyBCh solution are even more noticeable: intensive

absorption bands appear in the short-wave region at 355 nm

and 378 nm and in the long-wave region at 745 nm.

Figure 20. Electronic absorption spectra (DCM) of the reaction mixture during the first 6 h (а) and the last 6 h (b) of the reaction.

300 400 500 600 700 800

100000

200000

300000

400000

500000

450 500 550 600 650 700 750 800

20000

40000

60000

80000

100000

120000

140000

, нм



, нм

T3PyP

T3PyCh

T3PyBCh



Figure 21. UV-Vis absorbance spectra (DCM) (a) and emission spectra (DCM) (b) at 520 nm of T3PyP, T3PyCh and T3PyBCh.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

244 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

a b

Figure 22. (a) The π-А isotherm of tetra(pyridine-3-yl)porphine. Point A shows the conditions of layer transfer onto the solid support. (b)

UV-Vis absorption spectra of solution in dichloromethane (C=1·10-6 М, dotted line) and of the LS film formed from nanostructured layers

(solid line).

As part of the search for a solution to the problem of

formation of nanoarchitectures of bioactive porphyrin-type

compounds, studies have begun on the possibility of form-

ing nanoparticles of these compounds. The compression

isotherm for the layer formed at cedge=51% is presented in

Figure 22a. The structure of the layers was analyzed within

the framework of the author's model of a nanostructured M-

monolayer using the quantitative method of isotherm analy-

sis [342]. It was found that T3PyP forms nanostructured lay-

ers at the water-air interface. The conditions for the for-

mation and the main characteristics of monomolecular lay-

ers obtained at small values of the ISCD are determined. In

particular, at low surface pressures (less than 1 mN/m) the

number of molecules (n) of T3PyP in the M-nanoaggregate

is 270-330; aggregate diameter (Daggr) is 2-28 nm; the area

per molecule in the nanoaggreagate (Аmol) is 1.1 nm2; the

pressure range of existence of a stable monolayer is 0.1-

0.5 mN/m; water content in the nanoaggregate (when mole-

cules are arranged vertically in stacks) 25%; the degree of

surface coverage by aggregates at the beginning of the re-

gion of stable monolayer is 83 %.

Three-dimensional tetralayer nanoaggregates formed

on the water surface were transferred to a quartz plate by

the Langmuir-Schaefer method at the surface pressure of

50 mN/m (point A in Figure 22a). The positions of the

main bands in the absorption spectrum of T3PyP film

(Figure 22b, solid line) are shifted batochromically com-

pared to the spectrum of the monomer solution (dash-and-

dot line in Figure 22b): the Soret band by 16 nm and Q

bands by 8-12 nm. The half-width of the Soret band in the

film spectrum is twice as large as in the monomer solution.

The ratio of intensities of Q bands has also changed. These

results indicate intermolecular interactions stronger than the

van der Waals ones in the nanostructures formed within the

films of tetra(pyridine-3-yl)porphine.

Thus, 5,10,15,20-tetra(pyridine-3-yl)chlorin (T3PyCh)

and 5,10,15,20-tetra(pyridine-3-yl) bacteriochlorin

(T3PyBCh), the products of 5,10,15,20-tetra(pyridine-3-

yl)porphine reduction, were synthesized and fully charac-

terized, and their photophysical properties were studied.

The first stable T3PyP nanostructures, whose properties

differ from properties of original substance were obtained

by its controlled self-organization within layers at the air-

water interface and films on solid supports.

8. Derivatives of meso-Formylporphyrins:

Synthesis and Application as Components

of Optical Sensors and Photosensitizers

The production of porphyrin materials with target spe-

cific structures is based on various methodologies, differing

in that natural or synthetic porphyrins are used. Synthetic

porphyrins such as



-octaethylporphyrin (OEP) and meso-

tetraphenylporphyrin (TPP) are considered as a good alter-

native to the natural porphyrins due to their availability by

simple synthesis. The porphyrin core, which is easily ob-

tained by tetrapyrrole condensation,[357] then needs to be

functionalized to impart the required properties to the por-

phyrin molecule.[358-360] The free meso-positions of



octaethylporphyrin and meso-arylporphyrins can be func-

tionalized by a variety of methods, among which the

formylation is especially prolific.[361] The advantage of this

functionalization methodology is based on that the aldehyde

group is rich in the possibilities of further transformations

leading to the addition of functional fragments. There are

reported examples of formylporphyrins utilization in a wide

variety of reactions including, but not limited to the Wit-

tig,[362-364] Grignard,[364-366] McMurry,[367] cycloaddition,[368]

Knoevenagel[369] reactions and Schiff bases preparation.[370]

The products of the formylporphyrins transformations are

potential optical sensors[371] and photosensitizers.[46, 372,373]

meso-Functionalization of porphyrins by electrophilic

formylation of tetrapyrrole macrocycle

Although a formyl group can be inserted into a

tetrapyrrole macrocycle by various ways, the “old-

fashioned’’ classical aromatic electrophilic substitution

reaction, namely the Vilsmeier-Haack formylation reaction

stands out, as it allows the formyl group to be inserted into

the porphyrin core with simple procedure and affordable

reagents (DMF/POCl3 (Scheme 44).[361,374] The Vilsmeier

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 245

reagent is an electrophilic agent, the iminium cation,

formed by the interaction of POCl3 with DMF. To promote

the reaction of an electrophilic Vilsmeier reagent with a

nucleophilic porphyrin substrate, the tetrapyrrole aromatic

system is activated by the formation of metal complexes in

which the porphyrin core has a formal charge of -2, and

thus it is enriched with electrons. Porphyrin complexes with

Ni(II), Cu(II), Pd(II), Pt(II) resistant to HCl released during

the reaction, are usually formylated.[361]

meso-Formylporphyrins formed during the formyla-

tion of



-substituted porphyrins differ significantly in phys-

ical and chemical properties from β-formylporphyrins -

products of the formylation of β-unsubstituted porphy-

rins.[374] This is explained by the fact that the aldehyde

group in β-formyl derivatives is conjugated with the

tetrapyrrole macrocycle and exhibits all the properties of

arylaldehydes, while the aldehyde group in meso-

formylporphyrin is largely out of conjugation with the mac-

rocycle due to steric hindrances caused by neighboring pyr-

role substituents.[361] There is an additional wide long-

wavelength band in the region of 650-670 nm in the UV-

Vis spectra of meso-formylporphyrins.

In the process of formylation with a Vilsmeier reagent,

prepared from 3-(dimethylamino)acrolein instead of DMF,

the corresponding acrolein derivatives of porphyrins were

formed. These derivatives were easily cyclized to form an-

nelated six-membered cycles, when heated in an acidic

medium. Smith et al. obtained Ni(II) meso-acroleinyl-β-

octaethylporphyrin 1, which was cyclized by the action of

sulfuric acid at room temperature for two hours into benzo-

chlorin 2 (Scheme 45).[375]

The absorption spectrum of such annulled porphyrins

is significantly bathochromically shifted, which makes

them promising photosensitizers for photodynamic therapy

(PDT).[46,372,373,376] Thus, the product of the double cycliza-

tion of meso-bis-acroleinyl-β-octaethylporphyrin 3 – diben-

zobacteriochlorin 5 [375] possesses a strong absorption band

in the region of 752 nm, which fully corresponds to the

tissue transparency window (Scheme 46).[373,378] The conju-

gates of the similar benzochlorin with carbohydrates were

screened using the galectin-binding-ability assay and exhib-

ited an enhancement of about 300-400-fold compared to

lactose. All conjugates were also shown to possess good

photosensitizing efficacy with fibrosarcoma tumor cells.[379]

The McMurry reaction

The McMurry reaction, first described in 1974, is a re-

action of an organometallic derivative of aldehydes and

ketones to form alkenes, which is based on the use of low-

valent titanium. Peripherally carbonyl-substituted metal

porphyrins were subjected to this reaction with the for-

mation of the variety of bis-porphyrins and their homo- and

hetero-bimetallic complexes.[367] Cu(II) meso-formyl-



octaethylporphyrin (CuOEP-CHO) was dimerized under the

action of TiCl3 and Zn/Cu to form of the copper complex of

bisporphyrin 6 bound by the ethene bridge with a 64%yield in

the form of a mixture of cis and trans isomers (Scheme 47).[375]

A similar reductive dimerization reaction was carried

out with acrolein derivatives, resulting in the formation of

the dimer with a hexatriene bridge. The dimerization of the

benzochlorin acrolein derivative 4 led to the dimer 7, for

which a significant bathochromic shift of the Q band up to

706 nm was observed.

Scheme 44. The Vilsmeier-Haack formylation reaction.

Scheme 45. Transformation of Ni(II) β-octaethylporphyrin (NiOEP)

into Ni(II) benzochlorin 2.

Scheme 46. Preparation of dibenzobacteriochlorin.

Scheme 47. McMurry reaction of Ni(II) and Cu(II) meso-

formylporphyrins.[375]

Scheme 48. McMurry reaction of Ni(II) meso-acroleinylbenzochlorin.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

246 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

Grignard and Wittig reactions

Johnson and Arnold[366] performed the Grignard reac-

tion between Ni(II) meso-formyl-



-octaethylporphyrin

(NiOEP-CHO) and MeMgI, obtaining, as expected, Ni(II)

5-(1-hydroxyethyl)-



-octaethylporphyrin 8, by eliminating

water from which Ni(II) 5-vinyl-



-octaethylporphyrin 9

was obtained (Scheme 49). Smith et al. carried out a similar

reaction with a free base and a zinc complex of meso-

formyl-



-octaethylporphyrin, which resulted in 15-

alkylated products (10), and the formyl group remained

intact (Scheme 50). A regioselective attack of the C-

nucleophile to the 15-carbon of the macrocycle of Zn(II) 5-

formyl-



-octaethylporphyrin was also observed in the case

of the methyllithium reaction, while with the free base



octaethylporphyrin, a product of the attack to the carbonyl

group 5-(l-hydroxyethyl)-



-octaethylporphyrin was ob-

tained. The Wittig reaction is more reliable in this regard,

with the help of which the meso-formyl group of porphyrins

is easily transformed into a meso-vinyl (Scheme 49), 2-

(ethoxycarbonyl)ethenyl, 2-cyanoethenyl, and others.[361,364]

The meso-vinyl group can further be functionalized

with the conjugation extension. The direct C-H borylation

of the meso-vinyl group in 9 was performed with Cu(II)

complex as a catalyst, yielding the meso-(2-

pinacolboronylethenyl)porphyrin 11, which was shown to

act as nucleophilic partner in the Suzuki cross-coupling

leading to porphyrin derivatives with an extended π-

conjugation through the carbon-carbon double bond.[380]

The oxidative homocoupling of the borylporphyrin 11 pro-

duced the dimer 13 (Scheme 51).[381] Thus, this strategy of

the meso-formyl group transformations allows to attach

various aromatic chromophores through the ethene and bu-

tadiene bridges. The products of couplings possess batho-

chromic shift of absorption bands.

Scheme 49. The Grignard and Wittig reaction of Ni(II) meso-formyl-



-octaethylporphyrin.

Scheme 50. The Grignard reaction of Zn(II) meso-formyl-



octaethylporphyrin.

Scheme 51. The borylation of the vinyl-porphyrin with the subsequent

coupling reactions.

Scheme 52. Preparation of an exocyclic derivative of the dimethyl

mesoporphyrin IX.

Lugtenburg et al. obtained a 2-cyanoethenyl derivative

12 from the dimethyl meso-formyl-mesoporphyrin IX (11),

which was then cyclized when heated in an acidic medium

to a quinoline-annelated product 13 (Scheme 52).[382] The

product 13 strongly absorbed light at 685 nm and it was

tested as a photosensitizer in the PDT of the tumor cells of

the ovary of the Chinese hamster (Cricetulus griseus),

providing a good antitumor activity.

Scheme 53. Preparation of copropurpurin I from coproporphyrin I.

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 247

The formyl group in NiOEP-CHO and Ni(II) tetrame-

thyl ester of meso-formyl-coproporphyrin I (14) was trans-

formed by Wittig reaction with ethoxycarbonylmethylenetri-

phenylphosphoran into 2-(ethoxycarbonyl)ethenyl group.[383]

After demetallation in sulfuric acid, the obtained products

were heated for 24 hours in glacial acetic acid in an argon

atmosphere, causing the cyclization with the formation

of etiopurpurin I and copropurpurin I (16), respectively

(Scheme 53).

The authors of the work conducted a study on rats

with the bladder cancer induced. The obtained etiopurpurin

I and tetramethyl copropurpurin I photosensitizers were

emulsified and injected intravenously to the rats. The PDT

of the cancer cells proceeded efficiently with both com-

pounds. However, it was noted that the copropurpurin I has

a lower cytotoxic effect, and selective accumulation of eti-

opurpurin I in tumor cells was also observed.

The Knoevenagel reactions

The Knoevenagel reaction, like the Wittig reaction, is

used to transform formylporphyrins into the corresponding

derivatives of acrylic acid. Meso-formyl-triarylporphyrin

derivatives were usually synthesized under standard condi-

tions of basic catalysis. The meso-cyanoacrylate derivative

of Zn(II) meso-formyl-tri(p-tolyl)porphyrin was obtained

by boiling in piperidine with methanol for 16 hrs.[384] meso-

Formyl-diarylporphyrin reacted with nitromethane, dime-

thylmalonate and malononitrile in a mixture of piperidine,

acetic acid and toluene.[385] The product of the reaction of

meso-formyldiarylporphyrin with malononitrile containing

meso-dicyanovinyl group was shown to act as a fluores-

cence ‘‘turn-on’’ cyanide probe.[386] meso-Nitroethylene

derivative was utilized as fluorescence turn-on probes for

biothiols as it exhibited fast fluorescence enhancement and

high selectivity towards thiols based on the Michael addi-

tion mechanism.[385] It was also successfully applied to fluo-

rescent cell imaging in the NIR wavelength range.

meso-Formyl-



-octaethylporphyrin (NiOEP-CHO) is

less reactive and also easily degraded under basic condi-

tions, therefore it requires a special approach. Activation of

the carbonyl group with TiCl4 in pyridine promoted the

reaction.[369] Using the Knoevenagel reaction with NiOEP-

CHO, heterocyclic derivatives of porphyrins were ob-

tained.[387] First, upon condensation with malonic ester, the

corresponding malonate derivative (17) was obtained,

which reacted with hydrazine in the presence of sodium

ethoxide, resulting in formation of pyrazolidine-3,5-dione

linked to the Ni(II)



-octaethylporphyrin with ethane bridge

(18) (Scheme 54).

Direct synthesis of the porphyrin conjugates with thi-

ohydantoin, and thiobarbituric acid (Scheme 55) was also

performed via the Knoevenagel reaction.

The UV-Vis spectra of the heterocyclic conjugates

contain new bands arose from the interaction of the conju-

gated chromophores as well as bathochromically shifted

original absorption bands. Particularly dramatic changes

were observed in the UV-Vis spectrum of the thiobarbituric

acid conjugate, which exhibited substantial increase of ab-

sorption in green and red spectral region. Such combina-

tions of porphyrin dyes with such heterocyclic chromo-

phores and sensing fragments can be of interest as potential

promising photosensitizers and sensor dyes.

Synthesis and properties of Schiff bases derivatives

of meso-formylporphyrins

Synthesis of Schiff bases from formylporphyrins is a

promising direction of functionalization, since a huge num-

ber of various tetrapyrrole compounds with desired proper-

ties can be obtained from azomethine derivatives, including

photosensitizers and sensor dyes with the required photo-

physical characteristics.[388] There are two main ap-

proaches to the synthesis of Schiff bases: 1) interaction

of formylporphyrins with amines; 2) interaction of amines

with the so called "phosphorus complex" formed during the

Vilsmeier-Haack reaction.[370] The classical reaction of the

preparation of Schiff bases by the reaction of amines with

the aldehyde group of porphyrin was first investigated in

the synthesis of oximes, which were formed within a few

hours from the meso-formylporphyrin with hydroxylamine

hydrochloride in boiling pyridine.[366,389] Oximes are of in-

terest as precursors for the production of cyanoporphy-

rins,[390] which can be consequently transformed to carboxyl

derivatives. Intramolecular cyclizations of meso-substituted

porphyrins often leads to porphyrinoids with fused exocy-

cles like purpurins and benzochlorins. Treatment of nickel

complex of meso-oxime 21 with lead tetraacetate (1.2 eq.)

in methylene chloride in the presence of an excess of tri-

ethylamine at room temperature for 5 min gives the product

22 with fused 1,2-oxazin ring with an overall yield of

77%.[391] When a solution of Zn(II) complex of meso-oxime

23 was stirred in methylene chloride with a small amount of

water, after a few hours the starting complex was trans-

formed to hydroxy-1,2-oxazinochlorin 24 (Scheme 56).[392]

Oxidative hydroxylation of the intermediate 1,2-

oxazinochlorin possibly occurred via a peroxide mechanism

leading to the stable hydroxychlorin. Ni(II) complex of the

oxime vinylog meso-(trans-formylvinyl)-



-octaethylporphyrin

25 underwent similar intramolecular cyclization producing

a condensed eight-membered exocycle 26 (Scheme 57).[393]

Such annulated products are of interest as photosensitizers

due to their strong absorption in red region. The electronic

absorption spectrum of 1,2-oxazocine fused porphyrinoids

26 shows a significant bathochromic shift by 50 nm of all

absorption bands compared to the spectra of the corre-

sponding chlorins.

Scheme 54. Synthesis of the conjugate of Ni(II)



-octaethylporphyrin with pyrazolidine-3,5-dione.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

248 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

Scheme 55. Synthesis of the conjugate of Ni(II)



-octaethylporphyrin with thiohydantoin and thiobarbituric acid.

Scheme 56. Synthesis of the porphyrinoids with fused 1,2-oxazin ring via oxime cyclization.

Scheme 57. Synthesis of the porphyrinoid with fused 1,2-oxazocine ring 26 via vinylogous oxime cyclization.

Scheme 58. Preparation of azomethine derivatives of octaethylporphyrin from the phosphorus complex.

Despite the large number of examples of the utiliza-

tion of the interaction of formylporphyrins with an amino

group for preparation of Schiff bases, the reaction of the

"phosphorus complex" with amines has become more wide-

spread. Ponomarev et al. was the first to isolate phosphorus

complexes of various porphyrins.[370] Azomethine deriva-

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 249

Scheme 59. Preparation of cyclopentane-annelated derivatives.

Scheme 60. Treatment of the N-methylimine derivative of the Ni(II) -octaethylporphyrin (29) with a strong base.

tives of nickel and palladium complexes of various porphy-

rinoids including



-octaethylporphyrin, tetraalkyl esters of

coproporphyrins I and II, mesoporphyrin IX and mesochlo-

rin e6 were obtained by direct interaction of "phosphorus

complexes" with amines (Scheme 58).[388, 389,395]

The metal complexes of azomethine derivatives, ob-

tained by this way, were investigated as potential photosen-

sitizers for photodynamic therapy (PDT).[388] A number of

Schiff bases, in particular, Pt(II) and Pd(II) complexes of



octaethylporphyrin and tetramethyl coproporphyrin I, were

investigated as sensor dyes for determination of oxygen

concentration and acidity.[396,397] An optochemical probe for

cellular diagnostics using phosphorescent determination of

oxygen and pH in living cells was developed based on the

Pt(II) complex of meso-(N-methylimino)-



-octaethylporp-

hyrin.[398]

Upon studying the thermolysis of azomethine deriva-

tives of porphyrins, it was found that the cyclization occurs

with the formation of cyclopentane-fused derivatives

(Scheme 59).[399-401] During thermolysis of metal complexes

of meso-imines of tetraalkyl ester of coproporphyrin I the

formation of both cyclopentane and cyclopentane-lactam

bicycle was observed.[395]

Treatment the Ni(II) complexes of the azomethine deriva-

tives of



-octaethylporphyrin with t-BuOK led to the for

mation of the corresponding meso-nitrile (30), meso-amide

(31) and meso-hydroxy (32) derivatives (Scheme 60). The

latter was demetalated with sulfuric acid (Scheme 61), re-

sulting in the phlorin 33 with strong light absorption in the

region of 700 nm which could be useful for the photosensi-

tizing applications.[395]

meso-Hydrazone derivatives were obtained by reac-

tion of meso-formyl derivatives of



-octaethylporphyrin

and coproporphyrin I with hydrazine and N-substituted hy-

drazines catalyzed by trifluoroacetic acid (Scheme 62).[402]

Hydrazones 34, 35 were formed as a mixture of E- and Z-

isomers.

Scheme 61. Demetalation of the Ni complex of meso-hydroxy-



octaethylporphyrin (32).

Scheme 62. Synthesis of hydrazones from formyl porphyrins.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

250 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

Scheme 63. Preparation of cyclopentane fused porphyrins by thermolysis of meso-tosylhydrazones.

Scheme 64. Synthesis of the porphyrin dyads linked with the imino group.

Thermolysis of N-tosylhydrazones in the presence of a

base produced meso-carbenes that were cyclized leading to

the corresponding cyclopentane fused porphyrinoids via

intramolecular carbene C–H insertion.[4 03] The cyclopentane

fused products 27, 37 obtained were the same as in the

thermolysis of azomethines, however, the second product in

the N-tosylhydrazone of the coproporphyrin I thermolysis

was 6-member ring fused 38 instead of bicyclic lactam

28,[395] and yields of the products in the carbene based reac-

tion were higher (Scheme 63).

By the interaction of metal complexes of meso-

formyltriarylporphyrin 39 and meso-aminotriarylporphyrin

40, dyads 41, linked via an imino group, were obtained

(Scheme 64).[404] However, due to the low activity of the

meso-amino group and its degradation under sufficiently

harsh reaction conditions, yields were low. A satisfactory

yield of 57% was achieved for the zinc complex by cataly-

sis with a tenfold excess of ZnBr2. Such dyads can be used

in the two-photon excited PDT, which provides a highly

targeted treatment.[405]

Unsubstituted meso-hydrazones of



-octaethylporphyrin

and



-octaethylchlorin were used in the preparation of az-

ines 42 by reaction with various aldehydes (Scheme 65).[406]

Scheme 65. Preparation of azines from the Ni(II) complex of the me-

so-hydrazone of



-octaethylporphyrin.

In using chlorins containing an aldehyde group (me-

thyl pyropheophorbide-a (PPPa) and methyl pyropheophor-

bide-d (PPPd)) as aldehyde components in the reaction with

Ni(II) meso-hydrazone of



-octaethylporphyrin 34a, por-

phyrinoid dyads 43, 44 were obtained (Scheme 66).[406] In

the excited state of the dyads the energy was efficiently

transferred from the OEP moiety to the pyropheophorbide

chromophore.

One-pot formylation, hydrazone and azine formation

was realized for the meso-amino substituted octaethylpor-

phyrin.[407] Amino-group was preliminarily protected with

trifluoroacyl group. It was found that under the condi-

tions of formylation of meso-(trifluoroacetamido)-β-

octaethylporphyrin (45), the amide fragment was oxidized

to form hydroxamic acid 49 (Scheme 67). Combination of

trifluoroacetamide and arylazine groups in the products 47-

49 led to the strongly increased absorption near 500 nm and

considerably red-shifted Q-bands up to 650 nm.

Further expansion of the -electron system of the az-

ine functionalized disubstituted



-octaethylporphyrin was

achieved through the conjugation with another tetrapyrrole.

Interaction of the hydrazone 46a with methyl pyrophe-

ophorbide-d (PPPd), containing formyl group at



-position

of the tetrapyrrole ring resulted in formation of the azine

bridged porphyrin-chlorin conjugate 50 (Scheme 68). The

resulting compound features the substantial growth of the

Q-band intensity as well as red-shifting Soret band. The

azine bridged dyad could be of interest as potential photo-

sensitizers, sensor dyes and biologically active compounds.

Porphyrins with meso-fused heterocycles

Interaction of the aldehyde group with 1,2-dithiol is

often used to protect it by the formation of the 1,3-

dithiolane. A family of push–pull quinoidal porphyrin was

obtained from a meso-formyl porphyrin through the at-

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 251

tachment of 1,3-dithiolane (benzo-1,3-dithiolane) and

malononitrile fragments at the opposite meso-positions of

the 5,15-diarylporphyrin (Scheme 69).[408]

Porphyrins with meso-fused 2-imidazolyl heterocycles

53 were obtained from the meso-formylporphyrins as car-

bonyl components in the heterocyclic condensation. 5-

formyl-10,20-diarylporphyrins as well as their Cu(II) com-

plexes reacted with phenanthrene- or phenanthroline-5,6-

dione and ammonium acetate leading to the correspond-

ing imidazolyl-porphyrin conjugates with high yields

(Scheme 70).[409] Ruthenium complex of the free base por-

phyrin-imidazo[4,5‐f]phenanthroline conjugate 56 showed

good binding ability to DNA, which facilitated DNA pho-

tocleavage.[410] Thus, it could be a potential photosensitizer

for PDT.

Scheme 66. Synthesis of porphyrin-chlorin dyads linked by the azine bridge.

Scheme 67. Synthesis of trifluoroacetamide and arylazine oppositely substituted β-octaethylporphyrin.

Scheme 68. Synthesis of the azine bridged dyad 50.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

252 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

Scheme 69. Synthesis of the porphyrins with meso-fused 2-imidazolyl heterocycles.

Scheme 70. Synthesis of the porphyrins with meso-fused 2-imidazolyl heterocycles.

Scheme 71. Synthesis of the meso-linked porphyrin – fullerene conjugate 58.

Scheme 72. Synthesis of the porphyrin-corrole dyads 60.

meso-Formylporphyrins can form azomethine ylide af-

ter interaction with N-methylglycine. 1,3-dipolar cycloaddi-

tion of the intermediate azomethine ylide to the double

bond leads to the porphyrins with meso-fused heterocycles.

The porphyrin – fullerene conjugate 58 was obtained by

this way from meso-formyltriarylporphyrin 57, N-

methylglycine and C60 fullerene (Scheme 71).[368] The ex-

citation of the dyad led to the extremely fast formation of

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 253

the exciplex due to the strong interaction between the por-

phyrin and C60 moieties placed at close proximity.

Directly linked porphyrin-corrole dyads 60 were ob-

tained by the condensation of the meso-formyltriarylporphyrin

59 and dipyrromethane (Scheme 72).[411] The strong exciton

coupling between porphyrin and corrole and reversible en-

ergy transfer were shown to exist in the dyads. When 5,15-

bisformylporphyrin was used in the condensation, the cor-

role–porphyrin–corrole triad was formed.[412] Similar direct-

ly meso-meso linked porphyrin dimers and oligomers were

obtained using condensation of meso-formylated porphyrins

with pyrrole.[413] Such porphyrin dimers and oligomers

were shown to act as prospective photosensitizers.[4 14]

To sum up, meso-functionalization of porphyrins with

formyl group provides powerful tool for development of the

diverse porphyrin derivatives possessing valuable proper-

ties. In particular, promising photosensitizers with strong

red-shifted absorption bands, including NIR bands, were

obtained from the meso-formyl porphyrins via formation of

the annulated cycles such as benzochlorins and dibenzobac-

teriochlorins. meso-Imino derivatives were applied as sen-

sor dyes in the multi-modal, multi-analyte optochemical

sensing platform for cell diagnostics. Easily formed with

the help of the formyl group porphyrin conjugates with het-

erocycles can be used as biologically active compounds and

in sensing applications. Imino- and azino- bridges represent

two alternatives for bonding porphyrins into dyads, utiliz-

ing various pathways for energy transfer between the chro-

mophores. Currently, the post-derivatization of the meso-

formylporphyrins is under intense development.

9. Interaction of Multiporphyrin Complexes and

Nanoassemblies with Molecular Oxygen in

Solutions: Mechanisms and Some Specific Effects

Together with the formation of supramolecular com-

plexes of tetrapyrrole photosensitizers, which have already

found application in biomedicine,[72-76] another approach to

the formation of possible platforms of tetrapyrrole photo-

sensitizers of the third generation can be implemented by

combining tetrapyrroles of various structures with function-

al nanocarriers of organic or inorganic nature. Both ap-

proaches are promising for increasing the stability of PSs

and their targeted delivery to the tumor.

From the photophysical background it should be noted

that in both approaches, the first necessary step is connected

with the quantitative experimental analysis of the influence

of molecular oxygen O2 on deactivation of excited singlet

and triplet states of the given PS which is included in mul-

timolecular complex or attached to nanocarrier. Keeping in

mind this idea, within German-Belarus scientific collabora-

tion such comparative study was carried out for various

multicomponent nanoassemblies containing tetrapyrrolic

macrocycles.[74,75,79,80,88] Herein, we aim to review main

results of this study and provide a comprehensive under-

standing of O2 interaction with multicomponent species in

liquid solutions at ambient temperature. At the first step,

excited S1- and T1-states quenching by molecular oxygen

was studied for a set of Zn-porphyrin and Zn-chlorin chem-

ical dimers in which -conjugated macrocycles are cova-

lently linked via spacers of various nature and flexibility

(-CH2-CH2- bond or phenyl ring in mesoposition, cyclodi-

mers) in non-polar toluene with and without pyridine ad-

mixture. At the second step, self-organized multiporphyrin

triads and pentads were formed via non-covalent binding

interactions of Zn-porphyrin or Zn-chlorin chemical dimers

with di- and tetrapyridyl containing porphyrin or chlorin

extra-ligands, and the main peculiarities of the interaction

of O2with the triads and pentads of various and controlled

geometry were evaluated. Then, we consider perspectives

of semiconductor quantum dots (QD) as well as their nano-

assemblies with porphyrin molecules as possible PDT pho-

tosensitizers, and provide quantitative comparative results

on efficiencies of the singlet oxygen generation by QD-

porphyrin nanoassemblies. Finally, as far as in many exper-

iments the intensity of the singlet oxygen (1О2 or 1Δg) emis-

sion at max = 1.27  is measured in various solvents, some

specific reasons (described in literature) are discussed

which may change the rate constant of the radiative transi-

tion 1∆g → 3Σ-g in 1О2 molecule.

All objects under study (including monomeric porphy-

rins, chemical dimers, triads, pentads as well as QD-

porphyrin nanoassemblies) were synthesized, prepared and

characterized according to methods comprehensively de-

scribed in our earlier publications,[74,75,79,80,88,416 and references

herein] the details of experimental setup, steady-state and

time-resolved spectral measurements, direct detection of

intensity and kinetics for singlet oxygen IR-emission as

well as theoretical calculations one may find in these publi-

cations also. Below some necessary information in this re-

spect as well as the corresponding structures will be pre-

sented directly in appropriate places of the text.

Interaction of Zn-porphyrin and Zn-chlorin chemical

dimers with molecular oxygen

Typically, in degassed liquid solutions for monomeric

Zn-porphyrins and Zn-chlorins as well as for chemical di-

mers on their basis the deactivation of excited S1-states

takes place within S0=1.2-1.6 ns,[416,417] and, thus these ex-

cited states are hardly influenced by the presence of oxy-

gen at normal atmospheric pressure at 293 K (see data in

Table 3). It is not the case with long-lived excited T1-states,

and correspondingly, Table 3 collects experimental T val-

ues and bimolecular rate constants kT showing the specifici-

ty of T1-states quenching by molecular oxygen for the stud-

ied dimers and their complexes with pyridine.

For all dimers collected in Table 3, it was shown[416,417]

that at 77-293 K fluorescence and intersystem crossing

quantum yields as well as the rate constants of the internal

conversion S1S0 are hardly dependent on the interactions

between coupled macrocycles. In addition, it follows from

Table 3 that coordination of pyridine to the central Zn ion

in the dimers moiety has not a substantial effect on fluores-

cence quantum yields and lifetimes. It means that additional

non-radiative deactivation processes of S1-states are not

enhanced in pyridinated dimers. In contrast, there are some

noticeable features for triplet states for pyridinated dimers:

(i) phosphorescence lifetimes and quantum yields exhibit a

decrease by 1.5 - 2.5 times compared to uncomplexed di-

mers;[416,417] (ii) an additional T1-state quenching is found

for pyridinated complexes which diminishes upon the tri-

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

254 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

plet level lowering for a set of the investigated dimers,

(ZnOEP)2Ph  Zn-31,31-cyclodimer  (ZnOEP)2 

 (ZnHTPP)2  (ZnOEChl)2. In the result, the basic con-

clusion is as follows. The dimers (ZnOEP)2Ph, Zn-31,31-

cyclodimer and (ZnOEP)2 with high-energy triplet states

and relatively long phosphorescence decays Ph are sensi-

tive to the coordination with pyridine leading to enhancing

non-radiative deactivation of T1-states. This quenching is

attributed to several reasons: accepting role of the extra-

ligand vibrations, the enhanced spin-orbit coupling due to

the singlet ,* states energy lowering and the out-of-plane

distortion of porphyrin macrocycle. However, for the di-

mers (ZnHTPP)2 and (ZnOEChl)2 with low-energy triplet

states and relatively short lifetimes Ph the rate constants of

intrinsic non-radiative deactivation processes T1S0 (due

to Frank-Condon factor presumably) are so large that addi-

tional channels of the non-radiative deactivation of T1-

states caused by the extra-ligation seem in comparison not

very effective.

According to[417,418] the process of T1-states quenching

by O2 is realized via non-radiative transitions between elec-

tronic states in an intermediate short-living weak collision

complex formed as a result of a diffusional contact of a

given excited organic molecule with an oxygen molecule.

From this point of view, the quenching of T1-state of the

organic molecule (3M1) may take place via different ways

depending on the spin state of the collision complex:

 



 

dis

diff

3kΔMΔO...M~~~O...MOM    

 



, (I)

   

dis

diff

3kOMO...M~~~O...MOM    

 



. (II)

Table 3. Lifetimes of S1- and T1-states and bimolecular rate constants for quenching of excited T1-states by molecular oxygen O2 found

for monomeric precursors and chemical dimers in toluene (TOL) and toluene+pyridine (TOL+PYR) at 293 К. For clarity, the structures of

the compounds under study are presented also, central Zn ions are not shown.

Compound

Structure

Solvent

S, ns

T,

kT, a)

109 M-1s-1

kT(lig)/ kT b)

ZnOEP-CH3

TOL

TOL+PYR

1.6

1.45

330

285

1.65

1.95

1.18

(ZnOEP)2

TOL

TOL+PYR

1.3

1.2

440

205

1.2

1.9

1.6

ZnOEP-Ph

TOL

TOL+PYR

1.5

1.4

335

370

1.6

1.5

0.94

(ZnOEP)2Ph

TOL

TOL+PYR

1.2

1.1

495

490

1.1

1.0

ZnHTPP

TOL

TOL+PYR

1.6

1.5

570

530

1.0

1.05

(ZnHTPP)2

TOL

TOL+PYR

1.5

1.4

590

500

0.95

1.05

1.10

ZnOEChl-CH3

TOL

TOL+PYR

1.3

1.25

290

265

1.9

2.1

1.10

(ZnOEChl)2

TOL

TOL+PYR

1.2

350

285

1.6

2.0

1.25

ZnOEP-

cycle=CH2

TOL

TOL+PYR

2.6

2.5

310

275

1.9

2.0

1.05

Zn-31,51-cyclo-

dimer

TOL

TOL+PYR

2.5

355

295

1.6

1.9

1.2

a) The corresponding values of bimolecular rate constants of the excited S1-and T1-states quenching by molecular oxygen were calculated

using oxygen solubility in methylcohexane and toluene (TOL) at 293 K and the following expression kTS = [(T,S)-1 - [(0T,S)-1]/[O2], where

T and S are the corresponding decays of T1- and S1-states of the given compound in the presence of molecular oxygen, 0T and 0S are

decays in degassed solutions, [O2] is the concentration of dissolved molecular oxygen at 293 K.

b) kT(lig) is the rate constant of triplet state quenching for extra-ligated compound in toluene+pyridine (TOL+PYR) solution.



O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 255

Here, kdiff is a diffusion rate constant, kST is a non-

radiative intersystem crossing rate constant, and k is a the

singlet oxygen generation rate constant. g1=1/9 and g3=1/3

are spin-statistical factors reflecting the rate constants of

the formation of the complex states 1[3M1...3O2] and

3[3M1...3O2], correspondingly; kdis is the rate constant of the

complex dissociation. In the case of porphyrins and chlorins

kdiskS where kS is the bimolecular rate constant of S1-state

quenching by O2.[418] From the considerations presented

in[417,418] it follows that at the same quenching mechanism

(exchange and donor-acceptor interactions) the quenching

scheme may differ. Scheme (I) with generation of singlet

oxygen (1O2 or 1g) corresponds to the case when the ratio

 = kT/kS = 1/9, while scheme (II) without 1O2 generation is

operative at  = kT/kS = 1/3.

It follows from the data collected in Table 3 that in

toluene at 293 K, all non-pyridinated dimers exhibit a no-

ticeable decrease (by 15-30%) of bimolecular rate constants

kT compared to with those known for the corresponding

precursor monomers with the same side substituents. The

reason of that is connected with the fact[74,415] that in chemi-

cal dimers containing Zn-porphyrin subunits coupled by the

phenyl spacer, the non-radiative exchange triplet-triplet

energy transfer realizes at distances RDA=12.6-17.0 Å with

rate constants of kET5106 s-1, while rate constants of T1-

state quenching by dissolved O2 for porphyrin monomers

are of kq= kT[O2]  3106 s-1 in these conditions. Corre-

spondingly, the T-T energy transfer between porphyrin

halves in the dimers competes with quenching of T1-excited

macrocycle by molecular oxygen leading to the relative

decrease of the experimental bimolecular rate constants kT

for the dimers with respect to those found for monomeric

precursors at the same conditions.

It is seen also from Table 3 that kT values for the di-

mers increase sequentially in the following set: (ZnOEP)2Ph,

(ZnHTPP)2 (ZnOEP)2  Zn-31,31-cyclodimer, (ZnOEChl)2.

It is known [415,416,418] that T1-state quenching by O2 is an

electron donor-acceptor by nature [M+…O2] and thus cor-

relates with one electron oxidation potentials (Eox1/2) of por-

phyrins and chlorins. According to[419] Eox1/2 values (meas-

ured vs SCE) for Zn-meso-phenylporphyrins are higher

than Eox1/2 =0.63 V for ZnOEP, and values Eox1/2 =0.20 V for

ZnOEChl and Zn-cyclopentaneporphyrins are lower than

that for ZnOEP. Therefore, the observed sequential increase

of kT values in the presented set of the dimers reflects quali-

tatively the corresponding Eox1/2 values lowering. Finally,

the most pronounced oxygen-induced T1-state quenching

(by 60%, Table 3) is found for pyridinated dimer (ZnOEP)2

with a flexible -CH2-CH2- bridge. There are two factors that

come into play here. Firstly, electron-donating ethane

bridge in the dimer may strengthen the pyridine action in

oxygen-induced T1-state quenching (effect of Eox1/2 de-

crease). Secondly, it is known[415,416] that pyridine-

coordinated dimer (ZnOEP)2 is in the opened fully stag-

gered conformation. It means that the cross-section of the

dimer-oxygen interaction becomes larger thus leading to the

increase of the experimentally detected quenching.

Finally, based on results obtained in[415,416] one may

conclude that quantum efficiencies  of singlet oxygen 1Δg

generation for both monomeric precursors and chemical

dimers of Zn porphyrins coincide practically (within exper-

imental errors) with the corresponding values of quantum

efficiencies γТ of the non-radiative intersystem crossing

S1>T1. It means that for Zn-porphyrin dimers, T1-states

quenching by oxygen O2 is realized according to scheme (I)

followed by the formation of singlet oxygen 1Δg molecule.

Interaction of porphyrin triads and pentad with molecular

oxygen

The formation of self-assembling porphyrin triads and

pentads (based on a “key-hole” principle) was realized via

two-fold extra-ligation of both central Zn ions of Zn-

porphyrin chemical dimers with two nitrogens of meso-

pyridyl rings in the corresponding di- and tetra-pyridyl-

containing porphyrin free bases.[74,420-422] The structures of

monomeric and dimeric precursors as well as optimized

geometries of multiporphyrin arrays under study are pre-

sented in Figure 23. For these multiporphyrin arrays with

known morphology, the main pathways and mechanisms of

the primary photoinduced relaxation processes were evalu-

ated using steady-state and time-resolved lumines-

cence/absorption measurements and theoretical analysis,

and are properly described in our earlier publications.[420-422]

Based on this results, photophysical events leading to the

formation of the final excited states of the triad or pentad

subunits are the following. The fast non-radiative relaxation

of the dimer S1-state (within 1.6 ps) is caused by two com-

peting processes: (i) the resonance energy S-S transfer di-

merextra-ligand, and (ii) the photoinduced electron trans-

fer from the dimer to the extra-ligand leading to the singlet

radical ion pair state formation. It means that the popula-

tion of the dimer T1-state via intersystem crossing

S1(1dimer*)T1(3dimer*) with a rate constant of kISC(5-

7)107s-1 is low probable. In its turn, because of charge

transfer events, the extra-ligand T1-state in triads and pen-

tads cannot be populated via simple S1T1 process in

ligand subunit. The population of the locally excited T1-

state of porphyrin extra-ligands in triads and pentads

(3H2P*) takes place from the upper-lying triplet radical ion

pair state 3(dimer+...H2P-) or directly from the singlet radical

ion pair state 1(dimer+...H2P-). Concluding, after any excita-

tion of the triad or pentad the final lowest locally excited

state is the extra-ligand T1-state. Namely this state should

be taken into account upon examination of the interaction

of triads and pentad with O2 in liquid solutions.

The obtained experimental data are collected in Table 4.

It is seen that at 293 K in degassed solutions, fluorescence

quantum yields F0 and lifetimes S0 do practically not differ

for all pyridyl substituted porphyrin free base derivatives

(exta-ligands). In these conditions T1-state decays are prac-

tically the same (T0= 1.2-1.4 ms) for individual extra-

ligands, triads and pentad. It was shown also[423] that in tri-

ads and pentads containing dimers (ZnHTPP)2 and

(ZnTPP)2, the locally excited T1-state (ET=1.44 eV) of ex-

tra-ligands lies essentially lower than the CT state. There-

fore, in these multiporphyrin arrays the upper lying CT-

state does not influence the deactivation of the locally ex-

cited T1-state of the extra-ligand.

In the presence of oxygen, both S1- and T1-states

quenching is observed for individual pyridyl substituted

porphyrin free bases and those including in triads and pen-

tad. Notably, Table 4 shows that for all extra-ligands and

multiporphyrin arrays under consideration the ratio

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

256 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

= kT/kS is within 0.11-0.15. It corresponds to the spin-

statistical factor of g=1/9 reflecting the situation[417,418]

where triplet states of the compounds are quenched by tri-

plet molecular oxygen, 3O2 (see Scheme 73).

The role of structural organisation of triads and pen-

tads is most readily understood upon their interaction with

molecular oxygen in liquid solutions at 293 K. The com-

parative analysis of data summarised in Table 4 shows that

both kS and kT values for extra-ligands in the triads and

pentad are smaller than those obtained for the correspond-

ing individual pyridyl substituted porphyrins. Therefore, it

is reasonable to connect the relative decrease of kS and kT

values upon transition from monomeric extra-ligands to

those including in the triads and pentad with screening

effects depending on the mutual arrangement of subunits

in multiporphyrin complexes. In fact, the Zn-porphyrin

dimer in the triad (without any populated excited states)

plays a screening role limiting the access of oxygen mole-

cule to the excited extra-ligand. For the “opened” triad

(ZnHTPP)2H2P-(p^Pyr)2 (see Figure 23) this screening

effect is minimal, and the measured decrease of kT value

for this triads is minimal with respect to that obtained for

individual H2P-(p^Pyr)2. In contrast, for “closed” triads,

(ZnHTPP)2H2P-(m^Pyr)2 and (ZnHTPP)2H2P-(m-Pyr)2,

the access of oxygen molecule to the excited extra-ligand

becomes more limited thus resulting in more pronounced

decrease of the observed kS and kT values in comparison

with those found for the corresponding monomers. Con-

sistent with this interpretation is the fact that for pentad

2(ZnOEP)2PhH2P-(m-Pyr)4 where screening effects due

two dimers (ZnOEP)2 are higher with respect to those in

triads, the relative decrease of kT values is maximal in com-

parison with the triads at the same experimental conditions

(see Table 4).

Finally, quantum yields of singlet oxygen generation

by triad (= 0.80) and pentad (=0.70) are high in toluene

and comparable with those known for monomeric free ba-

ses (= 0.68 - 0.73[418]). In fact, for triads or pentads it

means that the quenching of extra-ligands excited states by

O2 takes place with the same efficiency practically like for

monomers, but with smaller bimolecular constants because

of screening effects. In fact, the observation of singlet oxy-

gen generation by triads and pentads in liquid solutions at

room temperature serves the independent direct proof of the

existence of locally excited triplet states in these arrays.

Figure 23. A: Structures of chemical dimers with phenyl spacer, (ZnOEP)2Ph, (ZnHTPP)2, and pyridyl-containing monomeric porphy-

rinsH2P(m^Pyr)2-(iso-PrPh)2, H2P(p^Pyr)2(Ph)2 and H2P(m-Pyr)4 used as extra-ligands upon formation of triads and pentad. The corres-

ponding extra-ligands were used: i) porphyrin, H2P containing two meso-phenyls and two meso-pyridyl rings with different positions of

both pyridyl rings (adjacent) and pyridyl nitrogens N (meta and para): i) adjacent meta-pyridyl rings - H2P(m^Pyr)2-(iso-PrPh)2 and adja-

cent para-pyridyl rings - H2P(p^Pyr)2(Ph)2; ii) porphyrin, H2P containing four meso-pyridyl rings with meta-nitrogens. B: Mutual ar-

rangement of the dimers, (ZnHTPP)2, (ZnOEP)2Ph and various extra-ligands in triads and pentad (optimized geometry, HyperChem soft-

ware package, release 4, semiempirical methods AM1 and PM3). For simplicity, mesophenyl rings in (ZnHTPP)2 are omitted. Mark 

denotes what porphyrin subunits are included in self-assembled triads and pentad.

H2P-(m^Pyr)2: R1,R2= m-Pyr, R3,R4=iso-PrPh H2P-(p^Pyr)2 : R1,R2= p-Pyr, R3,R4=Ph H2P-(m-Pyr)4 : R1,R2,R3,R4= m-Pyr

Zn Zn

R' R'

H2P-(m^Pyr)2

H2P-(p^Pyr)2

H2P-(m-Pyr)4

(ZnOEP)2Ph R=C2H5

(ZnHTPP)2 R´ = HexPh:

(ZnHTPP)2 Н2Р(m^Pyr)2 (ZnHTPP)2 Н2Р(p^Pyr)2 2(ZnOEP)2PhН2Р(m-Pyr)4

(ZnOEP)2Ph  Н2Р(m^Pyr)2

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 257

Table 4. Photophysical parameters and bimolecular rate constants of excited S1- and T1-states quenching by molecular oxygen for

pyridyl-substituted porphyrin free bases, triads and pentad in methylcyclohexane (MCH) and toluene (TOL) at 293K

Compound

Solvent

S, ns

S0, ns

kS, 10-9

M-1s-1

F

F0

T, ns

kT,10-9

M-1s-1

=kT/kS

T (77K),

H2P-(m^Pyr)2

MCH

8.9

11.4

9.3

0.070

0.104

275

1.40

0.14

4.2

H2P-(p^Pyr)2

MCH

9.1

12.8

12.0

0.064

0.098

280

1.37

0.11

4.2

H2P-(m-Pyr)4

MCH

8.4

10.5

9.3

0.071

0.098

260

1.45

0.15

4.1

(ZnHTPP)2 H2P-(p^Pyr)2

MCH

8.0

10.4

11.3

340

1.15

0.10

4.1

(ZnHTPP)2 H2P-(m^Pyr)2

MCH

8.2

10.3

9.6

365

1.05

0.12

3.1

H2P-(m^Pyr)2

TOL

9.5

0.075

380

1.45

(ZnOEP)2Ph H2P-(m^Pyr)2

TOL

7.7

0.05

550

1.0

2(ZnOEP)2Ph H2P-(m-

Pyr)4

TOL

0.04

680

0.80

S, F and S0, F0 are fluorescence decays and quantum yields in the presence and absence of O2 in solution, correspondingly; kS and kT

are bimolecular rate constants of the excited S1-and T1-states quenching by molecular oxygen (for definition see Table 3); T (77K) is T1-

states decay in rigid solutions at 77 K. Mark  denotes which porphyrin subunits are included in self-assembled triads and pentad.

Figure 24. Structural properties of nanoassemblies based on semiconductor CdSe/ZnS QDs capped with n-trioctylphosphine oxide

(TOPO)and H2P(m-Pyr)4 molecules. A: Schematic presentation of QD-porphyrin nanoassemblies. B: An optimized geometry for Cd33Se33

+ H2P(m^Pyr)2 (B, optimization by HyperChem 7.0; simulations by ab initio density functional theory, DFT, with the VASP code[424]). C:

the scales of CdSe core, ZnS shell, porphyrin and TOPO molecules corresponding to relative sizes of the main components of the arrays.

Interaction of “Quantum Dot – Porphyrin” nanoassemblies

with molecular oxygen

Like for multiporphyrin complexes described in the

previous section, a controllable formation of nanoassem-

blies based on semiconductor quantum dots (QD) and por-

phyrins was realized using also a “key-hole” principle upon

quantitative titration experiments in toluene at ambient

conditions.[74,75,79,80] In this case, “QD-Porphyrin” nanoas-

semblies with various porphyrin/QD ratio were successfully

prepared upon two-point coordination interactions of two

nitrogens of meso-pyridyl rings in the tetra-pyridyl-

containing H2P(m-Pyr)4 or H2P(p-Pyr)4 porphyrin free ba-

ses, and surface Zn ions of colloidal core/shell CdSe/ZnS

QDs passivated by tri-n-octyl phosphine oxide (TOPO)

(Figure 24).

From the photochemical point of view, the generation

of singlet oxygen by “QD-porphyrin” nanoassemblies

should involve two steps. First, after excitation of QD the

energy transfer QDporphyrin should be organized result-

ing in S1-state formation followed by the intersystem cross-

ing into porphyrin T1-state via the intersystem crossing

S1T1 pathway. Second, for porphyrin acting as PS,

the diffusional controlled triplet-triplet energy transfer

PS(T1)oxygen(3O2) should take place resulting in 1O2

generation in the surrounding media. This section provides

a description of the obtained basic results and a discussion

of all necessary steps what should be realized upon quanti-

tative analysis of QD PL quenching processes in “QD-

porphyrin” nanoassemblies and efficiencies of the singlet

oxygen generation.

Typically, the formation and “QD-porphyrin” nanoas-

semblies manifests itself in a pronounced quenching of QD

photoluminescence (PL).[74,79,80,424] Correspondingly, for

nanoassemblies consisting of TOPO-capped CdSe/ZnS

QDs (dCdSe = 3.0 nm, 2 ZnS monolayers, CQD = 410−7 M)

and H2P(m-Pyr)4 molecules in toluene, the direct quantita-

tive comparison of QD PL quenching results and sensitiza-

tion data for porphyrin fluorescence was carried out using a

complete set of titration points. Our results showed[79,80,424]

that in “QD-porphyrin” nanoassemblies under consideration,

Foerster resonance energy transfer (FRET) QDporphyrin

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

258 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

occurs with low quantum efficiency of FRET =0.10-0.15,

while the main part of the observed QD PL quenching is

due to non-FRET nature (non-FRET =0.85-0.90). We quanti-

tatively argued for the first time that a mechanism clearly

distinct from the photoinduced charge transfer and FRET

may be responsible for the QD PL quenching in QD-

porphyrin nanocomposites upon the coordinative attach-

ment of the extra-ligand on QD surface.[74,425] This process

is correlated with the extension of the exciton wave func-

tion beyond the interface between the QD and the attached

porphyrin molecule. Based on comparison of experimental

data and quantum mechanical calculations it follows that

the specificity of the exciton non-radiative decay in QD-

porphyrin nanoassemblies (that is QD PL non-FRET

quenching) is connected with the charge tunneling through

ZnS barrier in the conditions of quantum confinement.

Keeping in mind these results, a comparative study-

ing the quantum yields  of the singlet oxygen generation

by alone QDs and “CdSe/ZnS QD-porphyrin” nanoassem-

blies was performed using the direct spectral-kinetic meas-

urements of 1O2 near-IR emission.[79,80,424] It follows from

these experiments (Figure 25B) that at ambient temperature

most of alone QDs are hardly perspective to the direct

generation of singlet oxygen (the quantum efficiency

γΔ =1.5%), and without attached organic PSs cannot be

used in clinical practice.

Figure 25 evidently shows that upon laser excitation at

the same experimental conditions and concentrations in air-

saturated toluene, “QD-porphyrin” nanoassemblies do in-

deed produce singlet oxygen with essentially higher effi-

ciency in comparison with alone QDs or even with alone

H2P(m-Pyr)4 molecules. The radiative relaxation of 1О2 de-

tected at reg=1270 nm is characterized by the same decay

value of (1g) = 30-31 s that is typical for 1О2 emission in

non-polar liquid solutions.[415,416] It seems to be reasonable

to connect this findings with FRET QDporphyrin in

nanoassemblies. Thus, for clear understanding the role of

FRET namely in the increase of the quantum yields  of 1O2

generation by nanoassemblies one should take into account the

role of the excitation conditions and then carry out the quanti

tative analysis of the whole experimental data.

Typically, photoluminescence measurements are car-

ried out at lower excitation energies with respect to experi-

ments with essentially higher laser pulse energies being

used for 1О2 generation, and this thing was taken into ac-

count. From Fig. 25C it is evidently seen that the PL inten-

sity for QDs in “QD-porphyrin” nanoassemblies (curve 1,

em=558 nm) and for alone CdSe/ZnS QDs is not a linear

function of the laser pulse energy. In the case of alone por-

phyrin solutions, the above dependence is linear practically

(not shown), while in contrast, this dependence for porphy-

rin counterpart in the “QD-porphyrin” nanoassembly is

non-linear (curve 2, em=720 nm). The linear dependence

for alone porphyrin solutions in the whole range of nano-

second laser energies is explained by the minor role of the

singlet-singlet and triplet-triplet annihilation processes at

low solute concentrations[426] (C=10-5-10-6 M in the given

case). Non-linear dependence obtained for alone QDs and

QDs in “QD-porphyrin” nanoassemblies reflects the well-

documented pump-intensity dependence of the amplitudes

of the single-exciton caused by non-radiative intraband Au-

ger processes mediated by Coulomb electron-electron inter-

actions in the conditions of the spatial confinement.[427] In

this respect, the non-linear dependence of fluorescence meas-

ured for porphyrin being attached to QD surface (Figure 25C,

curve 2, em=720 nm) presents itself a direct proof of the

realization of namely FRET process QDporphyrin com-

peting with both radiative electron-hole recombination and

non-radiative Auger process in the conditions of powerful

excitation.

The above results evidently show that the relative in-

crease of the efficiency of 1О2 generation by “QD-

porphyrin” nanoassemblies with respect to that for alone

QDs coincides with the observed sensitized increase of the

fluorescence intensity for the attached porphyrins caused by

FRET. Therefore, understanding and ultimately control-

ling the energy of laser excitation we have carried out

the quantitative estimation of FRET efficiency in the

nanoassemblies based on singlet oxygen generation ex-

periments presumably.[79]

Figure 25. Singlet oxygen detection upon laser excitation of ZnS/CdSe QDs and “QD-porphyrin” (1:4) nanoassemblies in air-saturated

toluene. A: 3D presentation of spectrum of singlet oxygen 1О2 emission. B: Comparative presentation of singlet oxygen 1О2 luminescence

decay sensitized by individual CdSe/ZnS QDs (1) with respect to that measured for nanoassemblies (2) at the same molar concentrations

of QDs and laser excitation conditions (exc=532 nm, pulse duration t1/2= 0.7 ns, reg=1270 nm). C: Dependencies of the normalized

emission intensities on the energy of laser excitation for CdSe/ZnS QDs (1, em=558 nm) and H2P(m-Pyr)4 molecules (2, em=720 nm)

being coupled in nanoassemblies.

050000 100000 150000

1000

2000

3000

4000

5000

1O2 Emission Intensity, rel. units

Time, ns

0 5 10 15 20

0,0

0,2

0,4

0,6

0,8

1,0 em = 720 nm

C em = 558 nm

Energy/pulse, J

Normal counts

0 5 10 15 20

0,0

0,2

0,4

0,6

0,8

1,0

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 259

Scheme 73.

Our findings have shown that within the experimental

accuracy the for these nanoassemblies, the values of FRET

efficiencies obtained via the direct 1О2 emission measure-

ments at low laser excitation (FRET = 0.12  0.03), are in

a good agreement with FRET efficiencies found from the

direct sensitization data for porphyrin fluorescence (FRET =

= 0.14  0.02, discussed above). Such quantitative analysis

was done for the first time and shows that namely FRET

process QDporphyrin is a reason of singlet oxygen gen-

eration in the given nanoassemblies according to the fol-

lowing Scheme 73.

To our knowledge, such an approach is a first example

showing quantitatively the role of FRET effects in the sin-

glet oxygen generation by QD – PS nanoassemblies. The

obtained results may be considered as a direct proof of the

realization of namely FRET QDporphyrin process fol-

lowed by the singlet oxygen generation via porphyrin triplet

states. This conclusion has been proven also for the same

nanoassemblies using two-photon excitation.[428] All these

facts raise the conclusion that “CdSe/ZnS QD-porphyrin”

nanoassemblies may be considered as PSs of a novel type

for PDT. However, nanoassemblies based on TOPO capped

CdSe/ZnS QDs and porphyrins are not soluble in water and

contain toxic elements, what limits their biological applica-

tions. Recently, we examined water-soluble glutathione

capped AgInS/ZnS QDs with electrostatically attached Zn-

porphyrins and showed that upon UV excitation, these

nanoassemblies are capable to generate 1g (via FRET

QDporphyrin in ps time scale).[88] These preliminary re-

sults together with a specific dependence of spectral-kinetic

parameters of AgInS /ZnS QDs on pH and local polarity,

studied by us also,[429] make these nanoassemblies more

perspective in various biomedical applications (drug delivery

carriers, the distant testing the local pH, PDT treatment, etc.).

Finally, taking into account that in a lot of investiga-

tions the direct experimental measurements of the singlet

oxygen (1О2 or 1Δg) emission at max = 1.27  are realized in

various solvents, one should pay attention to some specific

effects which may change the rate constant of the radiative

transition 1∆g → 3Σ-g in 1О2 molecule. Luminescence of sin-

glet oxygen 1Δg in solvents is difficult to observe[430,431]

because of fast quenching of 1О2 molecules by solvent vi-

brations, and the rate constant of the radiative transition

1∆g → 3Σ-g in 1О2 molecule is strongly dependent on the

solvent properties (e.g. refractive index and polarizabil-

ity)[432-434] including interface boundaries[435] and biological

dielectrical nanoobjects.[436] Correspondingly, it is im-

portant to know all physico-chemical factors determining

the singlet oxygen 1Δg lifetime (or the rate constant of the

radiative transition 1∆g → 3Σ-g in 1О2 molecule) in various

environments including systems in vivo. In[437] radiative rate

constants kr of the radiative transition 1∆g → 3Σ-g have been

determined for the photoluminescence of singlet oxygen

1O2 in solutions with regularly changed refractive indexes

using laser kinetic spectroscopy. The observed changes in

the radiative rate constants kr are shown to be caused both

by inherent properties of the emitting 1O2 molecule and by

characteristics of an external environment, which defines

the local field factor and the density of photon states of the

field. In addition, the dielectric dipole moment of the transi-

tion 1∆g → 3Σ-g increases significantly as a result of the

contact of 1O2 molecule with solvent molecules.

Nevertheless, in spite of numerous experimental stud-

ies in this direction during last decade, some peculiarities of

electronic mechanisms of 1О2 luminescence decay remain

still under the question.[438] Recently,[439] the molecular

modeling of the influence of 10 solvents on the radiative

rate constant kr was carried out. The data obtained indicate

that the correlation of kr values with molecular polarizabil-

ity that occurs in a number of cases is associated, on the one

hand, with its effect on the strength of dispersion interac-

tions in the complex, and, on the other hand, with the fact

that, to certain extent, it reflects the position of the upper

occupied orbitals of the solvent molecule. Both factors af-

fect the degree of mixing of -orbitals of 1O2 molecule,

which promotes the activation of the radiative transition

1∆g → 3Σ-g . Only taking into account all the above factors

one may give a consistent explanation what happens with

the intensity of 1O2 in various solvents.

Conclusions

The interaction of molecular oxygen O2 with various

multicomponent complexes and nanoassemblies containing

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

260 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

tetrapyrrolic compounds was quantitatively analyzed using

set of steady-state and time-resolved experimental data ob-

tained in liquid solutions at ambient temperature. It was

found that for Zn-porphyrin and Zn-chlorin chemical di-

mers, the extra-ligation of central Zn ions of the porphyrin

macrocycles by pyridine leads to the enhancement of the

non-radiative T1S0 intersystem crossing rate constant

(the most pronounced for the dimers with higher lying T1-

states). This effect is attributed to several reasons: accepting

role of the extra-ligand vibrations, the enhanced spin-orbit

coupling due to the singlet ,* states energy lowering and

the out-of-plane distortion of porphyrin macrocycle. For

pyridinated dimers, their T1-states quenching by molecular

oxygen depends on the spacer flexibility and donor-

acceptor interactions with pyridine.

In the case of self-assembled triads and pentads con-

taining Zn-porphyrin dimers and pyridyl substituted por-

phyrin free bases (extra-ligand), the cooperative existence

of energy and electron transfer processes results in the pop-

ulation of the final locally excited S1- and T1-states of por-

phyrin extra-ligands. The quenching of these states in triads

and pentads by O2 is noticeably weakened with respect to

that for individual pyridyl substituted porphyrins, and is

caused by the screening action of a strongly quenched Zn-

porphyrin dimer subunit in triads and pentads limiting the

access of oxygen molecule to the excited extra-ligand.

These facts have to be taken into account upon the analysis

of natural dark and photoinduced reactions of porphyrin

supramolecular systems with molecular oxygen (oxygena-

tion and deoxygenation in haemoglobin, singlet oxygen

generation in tissues and PDT of cancer).

For inorganic-organic nanoassemblies based on

TOPO-capped semiconductor quantum dots CdSe/ZnS and

tetra-pyridyl porphyrins and formed via a two-fold coordi-

nation Zn….N-pyr “key-hole” principle in toluene at ambi-

ent conditions, the photoluminescence quenching for QD

with superficially attached porphyrin molecules is caused

by two main competitive reasons: the electron tunneling in

the conditions of quantum confinement (the efficiency of

0.85-0.90) as well FRET QDporphyrin (the efficiency of

0.10-0.15). On the basis of near-IR photoluminescence de-

tection of singlet oxygen 1O2 emission, it was proven that

the experimental efficiencies  of 1O2 generation by “QD-

porphyrin” nanoassemblies are essentially higher than

those obtained for alone QDs. It was found that in “QD-

porphyrin” nanoassemblies (1:4), FRET efficiencies

FRET = 0.12  0.03 obtained via the direct 1О2 emission

measurements at low laser excitation, are in a good coinci-

dence with FRET efficiencies FRET = 0.14  0.02 evaluat-

ed from the direct sensitization data for porphyrin fluores-

cence in nanoassemblies. It means that namely FRET pro-

cess QDporphyrin is a reason of singlet oxygen genera-

tion by QD-porphyrin nanoassemblies. Taking into account

experimental data and structure of excitonic states for QDs,

it was concluded that at ambient temperature most of alone

QDs are hardly perspective to the direct generation of sin-

glet oxygen. On the other hand, “QD-porphyrin” or “QD-

chlorin” nanoassemblies may be considered as good candi-

dates for ‘‘see and treat’’ PDT or for oxygen sensing over

physiological oxygen ranges.

Concluding, dealing with the direct quantitative meas-

urements of the singlet oxygen 1Δg emission (max = 1.27 )

in various solvents, one should take into account changes in

the rate constants kr of the radiative transition 1g  3-g in

1Δg molecule which may be caused as discussed in[417-419]

by inherent properties of the emitting singlet oxygen mole-

cule and by characteristics of an external environment (na-

ture of solvent).

10. Hydrophilic-Lipophilic Balance

and Interaction of Chlorin Type Photosensitizers

with a Transport Proteins of Blood

The trend towards the search of new effective photo-

sensitizers, best meeting the requirements applied to drugs

is of current interest for decades and up to the present

time.[6,10,440–448] The ability to generate ROS is one of the

most important characteristics of potential PS designed for

PDT. However, the singlet oxygen as a highly reactive oxi-

dative form has a very short lifetime (≤ 40 ns) limiting its abil-

ity to diffuse inside biological tissues (20~40 nm).[6,12,448] Ac-

cordingly, pharmacodynamics of PS and, namely, mecha-

nisms of its biodistribution in the human body including

intracellular localization, play a crucial part in of PDT effi-

cacy increase.[6,448-451] These biological features are fairly

determined by the structure and hydrophilic-lipophilic bal-

ance (HLB) of the PS molecule, or, in fact, by the pigment

affinity to a cell membrane and ability to penetrate it.[444, 452-

454] In this regard, design of a new antitumor drug for PDT

should involve an estimation of the affinity and accumula-

tion selectivity of PS in malignized or pathogenic cells, as

well as the control of specificity of passive transport of

the drug in vivo and its localization on the target cell

sites.[84,455,456]

Our scientific group is involved in extensive and con-

tinuing series of multidisciplinary studies on the devel-

opment and investigation of new and already known po-

tential sensitizers for antitumor and antimicrobial

PDT[6,9,11,13,75,444,451,453,457-463] based on the porphyrin, chlorin

and some other macroheterocyclic platforms. Our experi-

mental efforts in this field are aimed at finding the ways to

improve efficacy of PDT in terms of pharmacodynamics

and selective biodistribution of potential PSs. Current work

is a brief of literature and own recently obtained results,

related to the influence of the structural features and am-

phiphilicity of the chlorin photosensitizers on the mecha-

nisms of these drugs distribution and transport by proteins

of human blood.

The main emphasis on PSs of chlorophyll-like nature

is made here due to several factors. Naturally derived chlo-

rins have an intense absorption (lgε = 4.5-4.6) in a long-

wavelength part of visible spectrum (~665 nm), a sufficient

quantum yield of singlet oxygen,[6,10-13,373,448] a good am-

phiphilicity[453,454] due to asymmetric location of polar and

non-polar substituents in the molecule and a susceptibility

of macrocycles to chemical modification.[6,373,464,465] Some

chlorin-based drugs like Foscan, Talaporfin, Radachlorin,

Photoditazine or Photoran e6 are currently used in clinical

oncology as antitumor PSs.[6,9,10]

Hydrophilic-lipophilic balance of naturally derived chlorin

photosensitizers

The balance of hydrophilic and lipophilic properties of

PS plays an important part both at the stage of drug admin-

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 261

istration in its dosage form when the covalent or non-

covalent binding of PS to a different type of nanocarriers is

often used[84,451,455-460] and during its delivery to the surface

of the target cell.[6,448,465] The interaction of photosensitizer

with a cellular constituents starts from the main primary

target – membrane of the atypical cell.[466] The generation

of ROS by irradiated cell-localized PSs results in oxidation

of membrane lipids and proteins. The important chemical

targets for photoactivated PS are double bonds of mem-

brane unsaturated phospholipids.[10,467] The accumulation of

lipid hydroperoxides and oxidased forms of cholesterol in

membrane is a trigger for the lateral phase separation of

lipids, perturbation of their functions and, as a result, a de-

crease of membrane permeability and cell death.[468-470] Alt-

hough the mechanism of oxidative transformation of lipid

membranes during PDT remains still poorly established[466]

the most of authors are inclined to state a key role of 1O2,

formed at the photochemical reaction of PS with molecular

oxygen, in the oxidation of unsaturated membrane phos-

pholipids.[467] However, regardless of the type of photooxi-

dation mechanism,[10,75,373,447] the efficacy of a sensitizer in

the deactivation of a malignant or pathogenic cells depends

on the proximity of molecular contact between the PS

and the membrane lipid.[469] Therefore optimized PS mol-

ecule have to possess a certain HLB, to be well soluble in

water or aqueous solutions of carriers,[84,455-457] but not to be

too hydrophilic and not weaken its affinity and transfer

through lipid membranes of cells.[84,444,454]

The human body can be represented as a set of lipid-

like membrane barriers divided by water-like environ-

ment.[26,28,30] The P coefficient[3,452-454,469-475] reflecting parti-

tion of the substance between two immiscible phases is the

main parameter for the comparative quantitative estimation

of the hydrophilic-lipophilic balance (HLB) of any potential

drug.[146,452,471,472] P value of the later give an important in-

formation about its ability to interact and penetrate cell

membranes and, furthermore, about drug bioavailability and

biodistribution pathways. The results of experimental de-

termination of P values for tetrapyrrole photosensitizers and

their theoretical evaluation obtained by various computa-

tional methods are widely presented in the literature.[3,473,474]

Usually the hydrophobic core of lipid membrane is

modeled by 1-octanol (OctOH) and phosphate buffered

saline (PBS, pH 7.4) is used to mimic water-like media for

simulating transfer through the surface of lipid membranes

and experimental evaluation of the hydrophilic-lipophilic

balance of the drug.[452,454,461,462] To calculate partition coef-

ficients (P) the isothermal saturation method is normally

applied. This quantity is determined from the ratio of the

equilibrium concentrations of PS in the lipid-like phase and

in the PBS according to equation (1):

, (1)

where Cm OctOH and Cm aq are equilibrium solute molal con-

centrations in 1-octanol and aqueous phase, respectively.

Our experiments to determine P values were carried

out in thermostatically controlled 50 ml glass cells filled

with a PS solution with the initial concentration within 20 -

90 µmol/kg in a phosphate saline buffer or OctOH. Then

the second component of the immiscible solvent system

was added in a volume ratio of OctOH and PBS equal

40:60. After 36 hours of magnetic stirring at 298 K the so-

lution was kept until complete phase separation before the

probe was taken from the lipid-like layer. The equilibrium

concentration of PS was analyzed by the spectrophotomet-

ric method using previously obtained calibration graphs.

The solute concentration in the aqueous phase was estimat-

ed as the difference between the initial and lipid-like phase

equilibrium concentration of the pigment. Values of the

partition coefficients calculated according to the eq. (1) are

presented in the Table 5.

All the chlorin PSs investigated (compounds 2-9) were

synthesized by chemical modification of methylpheophor-

bide a (1).[451,454,476-478] Methylpheophorbide a and clinically

used photosensitizing agents as “Fotoditazin” (8) and

“Fotoran e6” were purchased from companies “CHLORIN”,

“VETA-GRANT LLC” and “RANFARMA”, respectively.

Trisodium salt of chlorin e6 (9) was obtained by precipita-

tion of “Fotoran” from aqueous solution of HCl (рН ~ 6)

followed by solid powder dissolution in diluted NaOH solu-

tion (рН ~ 8) and further complete water evaporation.[451]

All the compounds were identified using 1H NMR spectros-

copy and mass spectrometry technics.

The analysis of the previously measured at 298 K and

newly obtained partition coefficients of chlorin PSs at the

interface of OctOH and PBS clearly demonstrate these

quantities differentiate strongly depending on the substitu-

tion pattern of the macroheterocyclic molecule. Thus, a

metal-free chlorophyll a appeared to be the most hydropho-

bic natural chlorin studied (P = 326.0[453]) with a highly

pronounced affinity to the lipid surrounding. Transesterifi-

cation of this PS with the replacement of a hydrophobic

phytyl tail with a methyl group (methylpheophorbide a, 1)

leads to a rather significant decrease in the P value down to

210.1.[444,454,476] An additional structural factor contributing

to the chlorin hydrophilic properties is the opening of the

cyclopentanone ring accompanied by the transformation of

phorbine (1) to chlorin (2-9) molecular structure.

Table 5. Partition coefficients (P, 298K) for compounds 1–9 in the 1-octanol/phosphate saline buffer biphasic system and PSs distribution

between transport proteins of human blood (%).

Photosensitizers

Partition in OctOH – PBS system

210.1±

6.0 [444]

29.3 ± 0.9

[454]

20.1 ± 0.9

[451]

8.7 ± 0.2

[476]

1.04 ± 0.2

[476]

0.97 ± 0.3

[476]

19.0 [56]

2.03 ±

0.21 [9]

1.88 ±

0.09 [9]

Proteins

Serum proteins binding, %

LDL

52 [479]

38 [479]

HDL

31 [479]

44 [479]

Albumin

17 [479]

18 [479]

NB – no binding

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

262 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

Figure 26. Molecular structures of PSs studied: 1 — Pheophor-

bide a 17(3)-methyl ester, 2 — Chlorin e6 13(1)-N-methylamide-

17(3)-(1’-(2’-hydrohymethyl-3’,8’-dioxy-quinoxalinyl)methyl

ester)-15(2)-methyl ester, 3 — Chlorin e6 13(1)-N-methylamide-

15(2)-methyl ester-17(3)-O-6'-galactopyranosyl ester, 4 — Chlo-

rin e6 13(1)-N-(2-N’,N’,N’- trimethylammonioethyl iodide) amide-

15(2),17(3)-dimethyl ester, 5 — Chlorin e6 3(1),3(2)-bis(N,N,N-

trimethylaminomethyl iodide)-13(1)-N’-methylamide-15(2),17(3)-

dimethyl ester, 6 — Chlorin e6 3(1),3(2)-bis(N,N,N-trimethylamino-

methyl iodide)-13(1)-N′-(2-N″,N″,N″-trimethylammonioethyl io-

dide) amide-15(2),17(3)-dimethyl ester, 7 — Chlorin e6

13(1),15(2)-dimethyl ester, 8 — Chlorin e6 dimeglumine salt, 9 —

Chlorin e6 trisodium salt.

For instance, at the disclosure of phorbine exo-cycle in

the molecule of macrocyclic PS conjugate with diethylene

glycol an intense decrease of the partition coefficient from

77.7 to 32.6 is observed at 298K.[444]

Any conjugation of one or higher number of hydro-

philic groups to a chlorin macroheterocycle decreases P

parameter (Table 5). At the same time, the incorparation of

a single non-ionic or zwitterionic substituent, such as rese-

dues of glycol[444] or carbohydrate,[451] N-oxide[454] or amino

acid[451] into the PS structure promotes an amphiphilicity of

the molecule without essential increase in water solubility.

Usually, the naturally derived chlorins carrying one of the

substituents listed above (Table 5, compounds 2, 3),

demonstrate partition coefficients ranged from 15 to 30.

Meantime, chlorin molecules containing one or more ionic

groups (4-6, 8, 9) and characterized by P values less than

10 acquire solubility in water to achieve therapeutic con-

centrations of PS.[6,7,9,10] So, the solubility of dicationic

chlorin (5) reaches to 0.04 mol/kg at 298K. [477] Comparison

of partition coefficients of cationic (5, 6) and anionic (8, 9)

type PSs with two and three charged groups in the OctOH –

PBS system in both cases demonstrates the proximity of the

P values within pairs of PSs with a positively and negative-

ly charged groups (Table 5). At the same time, these quanti-

ties are slightly lower for cationic PS 5 and 6 revealing

more hydrophilic character of trimethylammonia groups

(P = 0.97-1.04)[476] as compared to carboxylic ones (P = 1.9-

2.0).[9] The HLB of the monocationic chlorin (4, P = 8.6)

differs significantly from di- and three charged deriva-

tives.[457] Meantime, having moderate amphiphilicity, comp.

4 retains a sufficiently high solubility in water. The mono-

carbaxylate derivative of chlorin e6 (7) is significantly less

hydrophilic (P = 19.0[478]), localized mainly in a lipid-like

phase and almost insoluble in water. An increase in the

number of charged groups in the PS molecule provides the

pronounced enhancement of macrocycle affinity towards a

water-like pool. Nevertheless, even tricationic chlorin e6 (6)

is still equally distributed between lipid-like octanol and

water-like PBS phase[476] due to the bulky non-polar macro-

cyclic part of the molecule.

Hydrophilic chlorin PSs[476] in contrast to more hydro-

phobic ones[444,451,454] demonstrate only modest Arrhenius

dependence already at P < 30. So, for such a compounds

partition coefficients measured at standard temperatures can

be attributed to physiological conditions.[476] It should also

be taken into account the P parameter of chlorin PSs with

an acidic groups depends on pH significantly.[475] In par-

ticular, it is found, that an affinity to a water-like compart-

ment in the case of anionic carboxylic derivatives of chlorin

e6 decreases with pH.[475] This information could be im-

portant for tumor-localized anionic PSs.

Thus, the P value is a reliable quantitative characteris-

tics of the hydrophilic-lipophilic balance of porphyrin and

chlorin-type macroheterocyclic PSs. Knowledge about the

HLB of photosensitizing agents is critically important for

understanding mechanisms of their delivery and pharmaco-

dynamics.[95,449,450,4 9,32] Partition coefficients provide not

only the information about PS affinity towards a water-like

or lipid-like surrounding but also an ability to bind to a dif-

ferent kind of blood proteins determining the mechanisms

of drug biodistribution in organism and, finally, the efficacy

of PDT. That is why in the next section of the paper the

effect of the structure and HLB of chlorin PS on the mech-

anisms of their transport by blood proteins, as well as on

their affinity to the membranes of atypical cells will be ana-

lyzed.

Equilibrium distribution of chlorin PSs between transport

proteins of blood

Extensive material devoted to the study of the interac-

tion of PSs of various structure with blood proteins using a

wide range of spectroscopic, chromatographic and quantum

chemical methods [17,58-63] are accumulated in the literature.

Entering biological tissues the drug participates in the pro-

cess of equilibrium redistribution on the molecules of bio-

environment, primarily on the blood transport proteins –

“light” albumins (M = 66.5 kDa) or “heavy” lipoproteins of

low and high densities (LDL and HDL, M = >230 kDa and

~180, respectively).[478] Redistribution occurs regardless of

whether the PS is taken as a pure compound or as molecular

complex of the drug with any non-covalently bound deliv-

ery vehicle.[17,31-33,64]

It is known that the selectivity of PS for tumor cells is

several times higher compared to healthy mammalian

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 263

ones.[6,448] The most of photosensitizers are mainly hydro-

phobic molecules, therefore, in human circulatory system

they bind predominantly to the lipophilic protein fraction

like low-density lipoproteins (LDL).[6,296,483,486,487] Lipopro-

teins play an important role in supplying cells with choles-

terol, which is used to form membranes during cell divi-

sion. Since tumor cells have an abnormal growth rate, they

require a large excess of cholesterol and the number of LDL

receptors in them per surface unit increases significantly

compared to healthy cells.[488] Thus, the binding of PS to

LDL in the bloodstream is one of the main reasons of the

pigments tumor selectivity due to their targeted delivery.[486]

High-density lipoproteins (HDL) and albumins are al-

so used as a transport proteins of human blood.[449] HDL are

able to deliver the drug to the tumor tissue, but, compared

to LDL, they are accumulated in tumor macrophages, which

can negatively influence the efficacy of the same PS.[487] In

turn, albumins have a less pronounced affinity for malig-

nant cells, so PS associated with this type of transport pro-

teins will be accumulated both in target tumor and healthy

mammalian cells, providing less selectivity.

PS molecules of various structures have different

pharmacodynamics and bind to transport proteins in indi-

vidual ways. It can be expected that essentially hydrophilic

PS bind mainly to albumins, the amphiphilic ones interact

with both HDL and albumins and hydrophobic molecules

make a complex selectively with LDL.[449,451,478,489] This

hypothesis is in a good agreement both with a previously

published results and our data (Table 5).[451,479] Thus, three

types of transport proteins of the circulatory system com-

pete for binding with PS molecule, therefore, and the effi-

cacy of the antitumor agent mainly depends on its hydro-

philic-lipophilic balance (Table 5).

There are a number of ways to determine the nature of

PS binding to various types of proteins, including transport

ones. One of the most common is the titration of the PS

solution with a specific protein using spectral methods to

obtain both equilibrium constants and thermodynamic pa-

rameters of the process, as well as structural characteristics

such as binding sites and geometrical features of the com-

plex.[75,490,491] However, in relation to the study of PS inter-

action with transport proteins of blood the spectral approach

has some disadvantages. Albumins are commercially avail-

able and chemically stable proteins, therefore titration using

spectral measurements is the most preferable in this

case.[296,480,481] At the same time, low- and high-density

blood lipoproteins are hardly separable from each other and

because of the low stability require immediate use after the

sample preparation. In addition, the titration using spectro-

scopic methods in the case of LDL and HDL is also of little

use because it requires separate experiments for different

types of proteins and does not allow to work with a mixture

to assess the competitive distribution of PS between

transport proteins. At the same time, information about the

competitive interaction of potential PS with proteins is im-

portant for understanding the real picture of their redistribu-

tion and transport in vivo.

To solve the above problems, the gel filtration chro-

matography (GFC) capable to separate complexes of

transport human serum proteins with PS divided into frac-

tions according to the size of macromolecules is used.

[478,479,482,483,485,489,492] (Figure 27) Spectrophotometric or

fluorescence analysis of fractions gives the gel-filtration

chromatogram or separation curve which is the dependence

of the relative optical density (A/Amax) measured on the

absorption maxima of tryptophan (280 nm, available in all

types of transport proteins), and naturally derived chlorin

PS, absorbing at ~665 nm, from the volume of the solution

(V, mL) eluting from the chromatographic column. Figure 28

demonstrates an example of gel chromatographic separation

on the column (1.670 cm) filled with polyacrylamide gel

Acrilex P-200. Three pronounced peaks of blood proteins

are observed usually on the gel-chromatogram (curves 1,

Figure 28), where the first one corresponds to LDL (with an

admixture of HDL), while the second and third represent

HDL and albumins, respectively. This finding is con-

firmed by the biochemical analysis of the obtained frac-

tions (Figure 27).[478,489] The area under the PS curve on the

gel chromatogram, measured for individual peaks corre-

sponding to specific types of blood proteins, allows to cal-

culate the percentage of pigment in each fraction (Table 5).

25 30 35 40 45 50 55 60 65 70 75 80 85

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

A/Amax

V, ml

Figure 27. Detection of protein components of human blood by biochemical analyzer “COBAS” (left, 1 – blood protein sample, 2 – low-

density lipoproteins, 3- albumins) and the principle of gel-filtration chromatographic separation of proteins depending on the size of the

molecule (right).

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

264 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

25 30 35 40 45 50 55 60 65 70 75

0.0

0.2

0.4

0.6

0.8

1.0

A/Amax

V, ml

30 35 40 45 50 55 60 65 70 75

0.0

0.2

0.4

0.6

0.8

1.0

A/Amax

V, ml

Figure 28. Gel filtration chromatograms of 70% blood serum (1 mL) with addition of 9∙10-5 mol/kg of PSs 4 (a) or 8 (b): 1 – main protein

(absorption at 280 nm), 2 - analyzed PS (665 nm). Column elution rate is 0.2 mL/min, the volume of each collected fraction is 2.5 mL.

As mentioned above, the degree of amphiphilicity af-

fects significantly the biodistribution of drugs, including

PS, using blood transport system.[478,489] As can be seen

from the literature and our data[9,475,478,479] (Table 5), hydro-

philic chlorin molecules 8 and 9 containing two and three

anionic groups, respectively, are accumulated to a greater

extent in the albumin structure (94%). An increase in the

partition coefficients P and affinity of PS to a lipid-like

compartments, on the contrary, leads to preferential binding

of drug molecule to a less polar lipoprotein fractions. So,

more hydrophobic compound 7 (P = 19.0 56), containing the

only carboxyl group is about equally distributed between

both LDL and HDL (Table 5) with a total binding to lipo-

proteins of 82%. Amphiphilic conjugates of chlorin e6 with

an antimicrobial drug “Dioxidine” (2) and D-galactose (3)

behave in approximately the same way due to a relatively

similar HLB (P = 20 – 30). Binding of the compound 3 to

lipoproteins is overwhelming and reaches 98% (Table 5).

Based on close P values and HLB of di- and tri-

anionic (8, 9) and di- and tricationic PSs (5, 6) an identical

picture of blood proteins binding could be expected for

them. However, it was found that molecules 5 and 6 carry-

ing two and three cationic trialkylammonia groups, respec-

tively, do not demonstrate interaction to any types of

transport proteins during GFC. The reason of that is the

most important characteristic affecting the equilibrium dis-

tribution of PS across transport proteins is not only the

number, but also the sign of the charged substituents pre-

sented in the molecule. As known from the literature[478] the

at the pigment interaction with albumin, the nonpolar mac-

rocyclic core is embedded into the hydrophobic pocket of

the transport protein surrounded by positively charged

groups.[478] Therefore, anionic PSs strongly bind to albu-

mins, whereas cationic pigments such as compounds 4, 5 or

6, are repulsed electrostatically from these transport mole-

cules. At the same time, the moderate hydrophobicity of

monocationic chlorin 4 (P = 8.3) allows it to form stable

complex with blood lipoproteins (in total 93%, see Table 5).

Mainly hydrophilic (P ~ 1) PSs 5 and 6 are not able to be

transported by lipid transport proteins due to its high affini-

ty for the water-like phase.

Thus, based on the fact that LDL is the most preferred

PS transport agent, it can be assumed that cationic pigments

with an appropriate HLB estimated by partition coefficient

of about 10, such as the compound 4, should demonstrate a

good efficacy as photosensitizing agents in antitumor PDT.

In addition, due to the amphiphilic structure and the pres-

ence of a cationic group in the molecule PS 4 is more versa-

tile compared to neutral or anionic chlorins, since it can be

used both in antitumor and antimicrobial PDT. [3,5,6,75,447,476]

Amphiphilic PSs with non-ionic or zwitterionic func-

tional groups (2, 3) interact effectively with LDL and HDL

(Table 5), however, provide negligible solubility in water

and pronounced aggregation in aqueous solutions. The later

factor affects negatively their ability to generate ROS and

reduces PS bioavailability.[10-13,373,447] The application of

micellar, polymer and other types of nanocarriers for PS

binding and solubilization is an effective way to achieve its

therapeutic concentrations in aqueous solutions.[84,471]

The results presented above allow us to draw the fol-

lowing important conclusions:

1. The structural features of photosensitizing mole-

cules designed for PDT determine their polarity, am-

phiphilic properties and significantly affect the biodistribu-

tion of pigments, mechanisms of their transport in the sys-

tem bloodstream and the selectivity of accumulation in the

membranes of malignazed or pathogenic cells.

2. PS distribution coefficients reflecting the state of

its hydrophilic-lipophilic balance are correlated with the

type of their distribution between blood transport proteins.

Thus, highly hydrophilic anionic-type chlorins give a stable

complex with blood albumins, whereas a decrease in the

affinity of PS to the aqueous phase leads to the predominant

binding to lipoprotein fractions of the blood. Cationic PSs

carrying two or more charged groups do not interact with

any type of transport proteins.

3. Gel filtration chromatography is a reliable method

of primary evaluation of the mechanism of PS biodistribu-

tion between proteins of the circulatory system.

Chlorin-type monocationic pigments with moderate

HLB (P ~10) are the most promising photosensitizing

agents, which, on the one hand, maintain the solubility in

water, and, on the other hand, are transported mainly by

lipoproteins in the system bloodstream of human. Thus,

due to the number and sign of the charges PS 4 demon-

strates higher versatility compared to neutral or anionic

chlorins and can be applied both in antitumor and antimi-

crobial PDT.

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 265

11. Sulfur-Containing Derivatives

of Natural Bacteriochlorophyll a as Promising

Photosensitizers for PDT of Cancer

The development of antitumor drugs is one of the

most important areas in the modern medicinal chemistry.[1]

The World Health Organization (WHO) defines cancer as a

broad group of pathological processes that can begin in

almost any organ or tissue and can be accompanied by un-

controlled division of abnormal cells. Despite the variety of

existing therapeutic agents, all of them have a number of

drawbacks, including non-selectivity of action, high toxici-

ty, and resistance of tumors to these agents. Since oncologi-

cal diseases continue to threaten the people’s health and

quality of life around the world, there is a need for innova-

tive targeted therapeutics aiming at specific targets in tumor

cells in order to implement a personalized approach in on-

cology.[2]

The development of sulfur-containing pharmaceutical

substances plays an important role in the development of

medicinal chemistry. There are more than 300 sulfur-

containing drugs approved by the US Food and Drug Ad-

ministration (FDA), the most popular of which include sul-

fonamides, thioethers, sulfones, penicillin, etc.[493] Photo-

sensitizers that acquire new useful properties upon incorpo-

ration of sulfur-containing groups or individual sulfur at-

oms into their structure also attract attention.

One of the most desirable groups of PS includes natu-

ral bacteriochlorins with absorption maxima in the near-

infrared spectral range, which expands the capabilities of

treating deep-seated tumors.[494] Moreover, they have a rela-

tively high yield of singlet oxygen and low toxicity along

with increased tropism to tumor tissues.[106,495]

The glutathione system is one of the key protective

cell systems affecting the efficiency of its photoinduced

death.[496] This system is vitally important for the existence

of mammalian cells, including tumor ones. It is known that

the level of enzymes in this system is elevated in various

types of malignant neoplasms.[497] The thiol-containing

tripeptide glutathione (γ-glutamylcysteinylglycine, GSH)

plays an important role in the detoxification of xenobiotics,

free radical control, maintenance of the cell redox balance,

formation of multiple drug resistance (MDR), etc.[498] This

makes it promising to aim pharmacological compounds at

the glutathione system as a biological target in order to

damage the enzymes of this system, which would favor the

vulnerability of tumor cells to oxidative stress.[499]

Two main enzymes are responsible for the biosynthe-

sis of glutathione in a cell: first, γ-glutamylcysteine ligase

(GCL, EC 6.3.2.2), which catalyzes the reaction between

glutamate and cysteine, followed by the addition of a gly-

cine residue to the resulting dipeptide with involvement of

glutathione synthetase (GSS, EC 6.3.2.3).[500,501] Cellular

glutathione participates in binding to various xenobiotics

with involvement of enzymes of the glutathione S-

transferase family, being an important regulator of intracel-

lular metabolism.[502] Glutathione binding to reactive oxy-

gen species (ROS), which is necessary for the protection

against oxidative stress and regulation of the cell redox bal-

ance, is catalyzed by GST or by specific enzymes of the

glutathione peroxidase family (GPx, EC 1.11.1.9).[503] Upon

interaction with ROS, glutathione is converted to an oxi-

dized dimeric form (GSSG), which, under the action of

glutathione reductase (GR, EC 1.8.1.7), is reduced into the

GSH monomer, thus closing the redox cycle.[504,505]

An important metabolic pathway that uses glutathione

for detoxification of metabolites is the glyoxalase system

consisting of glyoxalase I (GloI, EC 4.4.1.5) and glyoxalase

II enzymes (GloII, EC 3.1.2.6).[506-508] This system catalyzes

the conversion of methylglyoxal, a highly toxic glycolysis

product, to D-lactate via the S-D-lactoylglutathione inter-

mediate. The energy metabolism of the majority of tumor

cells is based on the Warburg effect, i.e., aerobic glycolysis

where glucose is converted to lactate in the presence of

oxygen. This process in tumor cells is characterized by

much higher activity (up to 200-fold) in comparison with

normal cells.[509,510] Such a rate of glycolytic transfor-

mations results in a higher formation rate of glycolysis side

products, including methylglyoxal (MG), a cytotoxic me-

tabolite. Therefore, tumor cells are more sensitive to MG

compared to healthy cells, and the glyoxalase system is a

promising target for anticancer agents.

A number of inhibitors of glutathione system enzymes

are currently known that make it possible to control its

function at different stages of the metabolic pathway. GCL

is the most promising target for the disruption of glutathi-

one biosynthesis in the course of anti-tumor therapy by hin-

dering and disruption of oncogenesis.[500] The best known

inhibitors of γ-glutamylcysteine ligase include methionine

and buthionine sulfoximines (MSO and BSO, respectively).

In vitro and in vivo studies have shown the prospects of

using MSO and BSO in combination with the known

chemotherapeutic agents.[511] Moreover, clinical studies

showed the safety of BSO administered intravenously.[512]

The inhibition of the glyoxalase system mainly in-

volves the inhibition of the rate-limiting enzyme GloI.

The best known GloI inhibitors include S-conjugated

glutathione derivatives such as S-alkylglutathiones, S-p-

bromobenzylglutathione, etc.[513] It was shown in in vitro

studies that S-p-bromobenzylglutathione made it possible to

overcome multiple drug resistance (MDR) and recover the

sensitivity of tumor cells to therapy.[514] Subsequent studies

have shown that tumor cell lines with high GloI expression

are resistant to current clinical antitumor drugs but sensitive

to GloI inhibitors.[515,516]

One of the promising ways for increasing the PDT ef-

ficiency is to create combined-action drugs comprising a

photosensitizer and a chemotherapeutic agent.[92,517] Exam-

ples are known where the chemotherapeutic agent in a

combined drug manifests pro-oxidant action that includes

the inhibition of the tumor cell defense system against oxi-

dative stress.[518,519]

The purpose of this work was to synthesize and study

the photoinduced cytotoxicity of photosensitizers based on

natural bacteriochlorins with sulfur-containing molecules

capable of affecting the glutathione and glyoxalase systems

for photodynamic efficiency improvement. All experi-

mental details are presented in Supporting Information.

The sulfur-containing proteinogenic amino acids cys-

teine and cystine are among the key compounds involved in

the redox processes in malignant cells. They undergo re-

versible enzymatic reactions with glutathione, thus weaken-

ing the antioxidant defense of tumor cells. The use of sul-

fur-containing amino acids in the development of antitumor

drugs may shift the redox equilibrium in a tumor cell to-

ward the generation of oxidative stress, which increases the

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

266 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

PDT efficiency.[524,525] Unlike cysteine and cystine, methio-

nine has a methylated thiol group that cannot directly inter-

act with the molecules of the cell’s glutathione antioxidant

system. However, some tumor cell lines are characterized

by enhanced consumption of this amino acid, a phenome-

non called “methionine dependence”.[626] Therefore, creat-

ing a conjugate with this amino acid would increase the

tropism of the entire molecule to tumor tissues. Moreover,

methionine derivatives, such as L-methionine- and L-

butyonine-sulfoximine, are selective inhibitors of the gluta-

thione antioxidant system, including γ-glutamylcysteine

synthase. Based on all of the above, it is obvious that creat-

ing bacteriochlorin conjugates with sulfur-containing amino

acids (cysteine, cystine, methionine and its derivatives) may

enhance the photoinduced effect of antitumor therapy due

to the action on the redox system of malignancies and en-

hanced tropism to the latter.

Previously we reported a synthesis of derivatives of

O-propyloxime-N-propoxybacteriopurinimide with cysteine

(2a), cystine (2b), methionine (3), methioninesulfoximine

(4), and buthioninesulfoximine (5),[520,521] that involved

addition of the above aminoacids and their derivatives to

the carboxyl group of the propionic residue at macrocycle

position 17 (Scheme 74).

At the first stage of this work, we optimized the condi-

tions for the preparation of DPBP conjugates with esters of

sulfur-containing amino acids presented in Scheme 74. The

reaction yield was taken as the optimization criterion. To

increase it, various agents activating the carboxyl group

were studied (Table 6).

Table 6. Reaction conditions in the synthesis of DPBP conjugates

with sulfur-containing derivatives (all in inert argon atmosphere

during 15 min).

Compound

Reaction conditions, i

Yield, %

DPBP-Cys (2a)

EEDQ, CH2Cl2, 25oC

NHS, CH2Cl2, 0oC

DCC, CH2Cl2, 0oC

EDC, CH2Cl2, 0oC

DCC, NHS, CH2Cl2, 0oC

EDC, NHS, CH2Cl2, 0oC

DPBP-Cys-Cys-

DPBP (2b)

EEDQ, CH2Cl2, 25oC

EDC, CH2Cl2, 0oC

EDC, NHS, CH2Cl2, 0oC

DPBP-Met (3)

EEDQ, CH2Cl2, 25oC

EDC, CH2Cl2, 0oC

EDC, NHS, CH2Cl2, 0oC

DPBP-MSO (4)

EEDQ, CH2Cl2, 25oC

EDC, CH2Cl2, 0oC

EDC, NHS, CH2Cl2, 0oC

DPBP-BSO (5)

EEDQ, CH2Cl2, 25oC

EDC, CH2Cl2, 0oC

EDC, NHS, CH2Cl2, 0oC

Scheme 74. Reagents and conditions: i: provided in the Table 6; ii: NH2-R, CH2Cl2, inert argon atmosphere, 25oC, 48 h.

Figure 29. A study photoinduced activity of compounds 1, 2a,b, 3 in vitro on HeLa tumor cell line. All compounds were used as Kolliphor ELP

4% micellar solutions: A. Exposure in the presence of PS in the incubation medium. The time of incubation with photosensitizers before exposure

to light was 4 h. The power density was 21.0 ± 1.0 mW/cm2 and the calculated light dose was 10 J/cm2; B. Exposure without PS in the incubation

medium. The time of incubation with photosensitizers before exposure to light was 4 h. The power density was 21.0 ± 1.0 mW/cm2 and the calcu-

lated light dose was 10 J/cm2; C. Dark cytotoxicity.

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 267

The combination of 1-ethyl-3-(3-dimethylaminop-

ropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)

as the activating agents was found to be the best. They were

considerably superior to N-еthoxycarbonyl-2-ethoxy-1,2-

dihydroquinoline (EEDQ) and 1,3-dicyclohexylcarbodiimide

(DCC) and allowed the reaction yield to be increased by a

factor of 1.5-2. Due to the high solubility of EDC in water,

multiple extractions can be avoided, while addition of NHS

produces a stable activated derivative of O-propyloxime-N-

propoxybacteriopurpurinimide with N-succinimide.

In the previous model experiments, we observed a re-

versible transition of the free thiol group to the oxidized

disulfide form.[520] Therefore, in this work we were the first

to estimate the in vitro antitumor photodynamic efficacy of

DPBP-Cys (2a), DPBP-Cys-Cys-DPBP (2b), and DPBP-

Met (3) derivatives that can affect the glutathione system of

tumor cells.

Study of photo-induced antitumor activity of DPBP-Cys (2a),

DPBP-Cys-Cys-DPBP (2b), and DPBP-Met (3) in vitro

The HeLa cell line with elevated levels of intracellular

glutathione was selected for in vitro biological tests.[497] The

time of incubation with compounds before light exposure

was 4 h, while the incubation time of cells after irradiation

was 24 h. The cells were irradiated with a standard light

dose of 10 J/cm2 with an exposure time of 13 min. The

starting compound, O-propyloxime-N-propoxybacteriopur-

purinimide (1), was chosen as the reference compound. The

survival of tumor cells was assessed both visually using an

inverted microscope and by the MTT test. The results of the

latter are shown in Figure 29.

The half-maximum inhibition concentration (IC50) of

compounds (2a) and (3) after 4 hours of cell incubation was

2-3 times smaller than that of the reference compound O-

propyloxime-N-propoxybacteriopurinimide (1), both under

exposure to light in the presence of PS in the incubation

medium (Figure 29A) and under exposure to light after PS

removal from the incubation medium (Figure 29B). The

IC50 of the dimeric compound (2b) was slightly larger and

comparable to that of the original compound (1). The dark

cytotoxicity of compounds (2a) and (2b) was slightly high-

er than that of derivative (3) (Figure 29C).

Since the photoinduced IC50 of DPBP-Cys-Cys-DPBP

(2b) was higher than that of cysteine (2a) and methionine

(3), only DPBP-Cys (2a) and DPBP-Met (3) were selected

for in vivo biological studies.

Study of the photoinduced antitumor activity of DPBP-Cys

(2a), DPBP-Met (3) in vivo

The accumulation kinetics of DPBP-Cys (2a) and

DPBP-Met (3) in a tumor compared to healthy tissues was

studied on a tumor model of mouse sarcoma S37 using lo-

cal electron spectroscopy. After intravenous injection, both

drugs showed accumulation of the compounds of interest

in the tumor with the maximum accumulation at 180 and

270 min, respectively (Figure 30). The fluorescence con-

trast in the accumulation maxima was 3.09 and 5.86 for

DPBP-Cys (2a) and DPBP-Met (3), respectively.

The data obtained indicate that accumulation of

DPBP-Cys (2a) and DPBP-Met (3) in the tissues of malig-

nant neoplasms occurs, which, in combination with the re-

stricted area of light exposure, provides an efficient tool for

tumor targeting. Moreover, the maximum accumulation

times of the compounds lie in the range convenient for the

photodynamic therapy procedure.

At the next step, the photoinduced antitumor activity

of DPBP-Cys (2a) and DPBP-Met (3) was studied in vivo

using various irradiation modes. The dose of both photo-

sensitizers was 5 mg/kg; DPBP (1) whose accumulation

kinetics was studied previously was taken as the reference

drug.[527] To select the optimal light dose, two irradiation

modes with light doses of 150 J/cm2 and 300 J/cm2 at a ra-

diation power density of 0.2 W/cm2 were used. The PDT

procedure was performed at the time points when the max-

imum accumulation of drugs took place (15 min, 180 min,

and 270 min after intravenous injection of DPBP (1),

DPBP-Cys (2a), and DPBP-Met (3), respectively).

Analysis of the growth dynamics of the primary tumor

node showed that the highest antitumor effect was shown

by the DPBP-Met compound 3 (Figure 31). The optimal

PDT conditions for this compound are: a photosensitizer

dose of 5 mg/kg, an interval of 270 min between its injec-

tion and the beginning of irradiation, and a light dose of

300 J/cm2.

Figure 30. Accumulation kinetics of compounds 2a (A) and 3 (B). The content of compounds in the samples was determined in vivo by

local electron spectroscopy on sarcoma S37 tumor-bearing mice. The ratio of areas under the curves of fluorescence and laser reflection

peaks was estimated.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

268 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

Figure 31. The growth dynamics of the primary tumor node in irradiation modes of 300 J/cm2 (A) and 150 J/cm2 (B) and at a photosensi-

tizer dose of 5 mg/kg. The animals were monitored for 15 days after the PDT procedure. The anti-tumor effect was estimated from the

dynamics of primary tumor growth in the experimental and control groups.

Figure 32. Tumor growth inhibition in experimental and comparison groups in irradiation modes of 300 J/cm2 (A) and 150 J/cm2 (B) and

at a photosensitizer dose of 5 mg/kg. The animals were monitored for 15 days after the PDT procedure.

Using this PDT mode with DPBP-Met (3), complete

resorption of the tumor node was achieved on day 4 after

the treatment. The figure shows the tumor growth inhibi-

tion (TGI) data in two irradiation modes, 150 J/cm2 (A)

and 300 J/cm2 (B) (Figure 32).

Thus, the in vivo studies on the biological properties

of DPBP-Cys (2a) and DPBP-Met (3) indicated that the

methionine methyl ester derivative (DPBP-Met (3)) has a

two times higher selectivity of accumulation in the tumor

tissue than the cysteine derivative 2a. Moreover, the pho-

toinduced antitumor activity of the DPBP-Met derivative

(3) at a light dose of 300 J/cm2 was higher than that of

DPBP (1) and DPBP-Cys (2a).

Synthesis of DPBP conjugates with sulfur-containing inhib-

itors of enzymes of the glutathione and glyoxalase systems

Inhibitors of γ-glutamylcysteine synthase and glyoxa-

lase I were used as sulfur-containing molecules capable of

affecting the glutathione and glyoxalase systems and pos-

sessing antitumor activity. Inhibition of these enzymes

should increase the efficiency of photoinduced antitumor

therapy and reduce dark toxicity through the intracellular

targeted impact on tumor cells.

Methionine and buthionine sulfoximines were chosen

as potent γ-glutamylcysteine synthase inhibitors. Their con-

jugation with DPBP (1) was performed as described in our

previously studies and resulted in conjugates DPBP-MSO

(4) and DPBP-BSO (5).

S-Hexylglutathione dimethyl ester (8) was chosen as the

glyoxalase I inhibitor. The esters of this class of compounds

are known to have greater antitumor activity than the ana-

logues with free carboxyl groups.[528] Initial attempts to

obtain S-hexylglutathione dimethyl ether (8) by a conven-

tional technique in acid medium failed to give the expected

result, which seems to be due to the formation of a side

product of S-hexylglutathione cyclization. Therefore, dime-

thyl ether (8) was obtained using diazomethane (Scheme 75).

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 269

Scheme 75. Synthesis of S-hexylglutathione dimethyl ether (8). Reagents and conditions: i: 1-iodohexane, 1M aqueous NaOH, EtOH, inert argon

atmosphere, 25°C; ii: CH2N2, Et2O.

Scheme 76. Reagents and conditions: i: compound 8, EDC, NHS,

CH2Cl2, inert argon atmosphere, 25oC.

At the next step, S-hexylglutathione dimethyl ether

was added to the free carboxyl group of O-propyloxime-N-

propoxybacteriopurinimide (1) using the optimized reaction

conditions shown in Table 6 (Scheme 76).

The spectral characteristics of the pigment thus ob-

tained did not differ from those of the original DPBP (1).

The absorption maximum in the long-wave spectrum region

for all the sulfur-containing conjugates (2-5, 9) was 800 nm.

Study of the photoinduced antitumor activity of DPBP-GS-

Hex, DPBP-MSO and DPBP-BSO in vitro

At the final stage of this work, the photoinduced cyto-

toxicity of all the resulting conjugates of O-propyloxime-N-

propoxybacteriopurinimide with sulfur-containing amino

acids (2-5) and with S-hexylglutathione dimethyl ether (9)

was studied in vitro on the Hela tumor cell line. The time of

incubation with compounds before light exposure was 4 h,

while the incubation time after irradiation was 24 h. Cells

were irradiated with a standard light dose of 10 J/cm2, the

exposure time was 13 min. The starting compound, O-

propyloxime-N-propoxybacteriopurpurinimide (1), was

chosen for comparison. Tumor cell survival was estimated

both visually using an inverted microscope and by the MTT

test whose results are presented in Table 7.

The highest photoinduced cytotoxicity in vitro was

observed for compounds DPBP-MSO (4), DPBP-BSO (5),

and DPBP-GS-Hex (9) than have 5-6 times lower IC50 than

O-propyloxime-N-propoxybacteriopurinimide (1) and 2-

3 times lower IC50 than the conjugates with cysteine (2a)

and methionine (3), both upon exposure to light in the pres-

ence of PS in the incubation medium and upon exposure to

light followed by removal of PS from the incubation medi-

um. The lowest cytotoxicity without light exposure (dark

cytotoxicity) was shown by DPBP-GS-Hex (9).

In conclusion, DPBP derivatives with sulfur-

containing amino acids were obtained previously in our

works.[520,521] In this work, we have optimized the reac-

tion conditions for the amidation of O-propyloxime-N-

propoxybacteriopurinimide with sulfur-containing amino

acids and modified glutathione, which allowed us to increase

the yield of the target derivatives by a factor of 1.5-2.

Table 7. Photoinduced cytotoxicity in vitro of sulfur-containing compounds on HeLa tumor cells.

Compound

Exposure in the presence of PS

in the incubation medium

Exposure with subsequent

removing PS from in the incu-

bation medium

Dark cytotoxicity

IC50 value, nM

DPBP (1)

111 ± 8

129 ± 24

2141 ± 763

DPBP-Cys (2a)

50 ± 2

62 ± 6

1260 ± 72

DPBP-Cys-Cys-DPBP (2b)

104 ± 10

117 ± 8

1355 ± 136

DPBP-Met (3)

44 ± 0.4

46 ± 2

785 ± 63

DPBP-MSO (4)[]

16 ± 1

20 ± 5

232±27

DPBP-BSO (5)[]

20 ± 0.7

21 ± 2

485±63

DPBP-GS-Hex (9)

24 ± 4

27 ± 3

2042±981

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

270 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

The in vivo biological studies of solubilized DPBP

conjugates with methyl esters of cysteine 2a and methio-

nine 3 on tumor-bearing mice with subcutaneously inocu-

lated sarcoma S37 were performed for the first time. Methi-

onine derivative 3 was found to have a 1.9-fold higher se-

lectivity of accumulation in tumor tissue than cysteine de-

rivative 2a. Moreover, the photoinduced antitumor activity

of compound 3 under the conditions used (5 mg/kg, light

dose 300 J/cm2) was found to be higher than that of the refer-

ence compound DPBP 1 and cysteine-based pigment 2a.

A new derivative 9 of natural bacteriochlorophyll a

with an inhibitor of the glutathione-dependent enzyme gly-

oxalase I, was obtained.

All the sulfur-containing bacteriochlorin conjugates

obtained were studied in vitro on Hela tumor cells, and their

photoinduced cytotoxicity was shown to increase in com-

parison with the parent compound 1. This pattern shows a

beneficial role of sulfur-containing compounds in the mani-

festation of photodynamic activity and suggests that the

development of such PS for antitumor PDT is a promising

approach.

12. Antitumour Efficacy of Photodynamic Therapy

of Experimental Laboratory Animal Tumours

with a New Photosensitizer of Chlorin Series

Most photosensitizers used for photodynamic therapy

in clinical practice are derivatives of chlorin e6. To date,

research aimed at increasing the anti-tumour efficiency of

PS continues. It is known that only high-purity chlorin e6

has the most pronounced photodynamic properties, and the

presence of related chlorine and the other ingredients in the

dosage form significantly impairs the properties of the pho-

tosensitizer. Concomitant chlorines have a much lower

quantum output of singlet oxygen and less accumulation in

tumour tissues. This requires the application of a higher

light dose and leads to blurring of the clear boundary during

the PDT session and, as a result, significant damage to

healthy tissues adjacent to the tumour node.[529] The pres-

ence of polyvinylpyrrolidone in the composition of the me-

dicinal form of the photosensitizer leads to increased vis-

cosity of the injectable solutions and makes it impossible to

apply a rapid and convenient method of jet administration,

replacing it with only drip intravenous administration. Thus

the presence of impurities reduces the effectiveness of chlo-

rine PSs. As a result, the improvement of PS is now on

course to achieve the highest possible purity of chlorin e6 in

a sustainable form.

The new photosensitizer Heliochlorin, developed by

Professor G.V. Ponomarev and co-authors,[70] is available in

a lyophilically dried form, including trismeglumin salt of

chlorin e6 and meglumin as a cryostabilizer. Heliochlorin is

a PS based on highly purified chlorin e6 from methyl pheo-

pharbide a, with a high core content of 93-98%.

The study of the antitumor efficacy of PDT with the

Heliochlorin on the models of murine Erlich carcinoma and

rat sarcoma M-1 showed that treatment at the optimal time

after intravenous administration of PS with certain parame-

ters of laser exposure allowed to achieve complete regres-

sion of tumour nodes and no recurrence within 90 days after

therapy in 100% of animals.

The animal experiments were conducted in a strict ac-

cordance with the Guidelines for the Care and Use of Labora-

tory Animals of the National Medical Research Radiologi-

cal Centre of the Ministry of Health of the Russian Federa-

tion and in accordance with the rules and requirements of

the European Convention ETS/STE No. 123 and interna-

tional standard GLP (OECD Guide 1:1998).

Table 8. Experiment Design.

Tumour

Test system

Group

PS dose, mg/kg

Mode of

administration

Laser action parameters

E, J/cm2

Ps, W/cm2

Ehrlich’s

carcinoma

mice

1 group-PDT

2.5

intravenously

150

0.48

1 control

2 group-PDT

5.0

intravenously

150

0.48

2 control

Sarcoma M-1

rats

3 group-PDT

2.5

intravenously

150

0.48

3 control

4 group-PDT

5.0

intravenously

150

0.48

4 control

This study was performed using sarcoma M-1 bearing

outbred female rats (totally 32 animals) at three months of

age weighting on average 180-200 g and 48 female outbred

mice two months old, 19-20 g weight with implanted Erlich

carcinoma. The animals were housed in T-3, T-4 cages un-

der the natural light conditions with the forced ventilation

of 16 times/h, at a room temperature and relative humidity

of 50–70%. The animals had free access to water and PK-

120-1 feed for rodents (Laboratorsnab Ltd., Moscow, Rus-

sia). The tumour strains was obtained from the tumor bank

of the N.N. Blokhin National Medical Research Center of

Oncology of the Ministry of Health of Russian Federation.

Sarcoma M-1 was implanted subcutaneously as a 1.0 mm3

piece of donor tumour into the outer side of the left thigh.

The experiment was started 8-9 days after transplantation,

when the largest diameter of the tumour nodes reached 0.8-

1.0 cm. Erlich’s ascitic carcinoma liquid with 2.5 × 106 tumour

cells from the donor mice was injected subcutaneously into

the area of the lateral surface of the left thigh of mice. The

experiment was started 4 days after transplantation, when

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 271

the largest diameter of the tumour nodes reached 0.4-0.6 cm.

The experimental and control groups containing eight rats

and twelve mice each. As the series with the different

treatment regimens were not performed simultaneously,

each series had its own control. As a control, we studied

tumour-bearing animals without any exposure. The session

of photodynamic therapy was performed, as described pre-

viously.[530] The animals of the experimental groups re-

ceived the PS in doses of 2.5 and 5.0 mg/kg into the caudal

vein. The tumours were exposed to the laser irradiation

using an Atkus-2 semiconductor device (Atkus, Russia;

λ = 662 nm, diameter of the light spot = 1 cm for mice and

1.5 cm for rats). The following doses of laser irradiation

were tested: E = 150 J/cm2; Ps = 0.48 W/cm2 (Table 8). In

our preliminary studies the optimal time of beginning of

laser action after administration of the drug (drug-light in-

terval) was determined: 45-60 min after intravenous admin-

istration of PS to mice and 60-75 min to rats.[531] The results

were analyzed in the seventh, fourteenth, twenty-first and

ninetieth days and compiled in Table 9. The antitumor effi-

cacy of the PDT was assessed according to the criteria de-

scribed earlier.[532,533]

After the PDT, there was a pronounced swelling of

soft tissue in the irradiation zone, which is a natural reac-

tion to photodynamic tumor damage with any known PS.

The swelling was accompanied by hyperemia of the radia-

tion zone and surrounding soft tissue. Tissue alteration was

observed and, depending on the size of the surface to be

irradiated, a thick scab of dark color was formed for 2-4 days

after treatment.

Up to 21 days after photodynamic exposure (with a PS

dose of 5.0 mg/kg and laser exposure parameters:

E=150 J/cm2; Ps=0.48 W/cm2) an inhibition effect was ob-

served on murine Erlich carcinoma in all experimental ani-

mals, and remained up to 3 months in 100% of animals.

When the PS dose was reduced to 2.5 mg/kg at the same

light dose, the complete tumour regression was observed to

21 days. Later on, some animals relapsed into tumour for

90 days after therapy, however, the percentage of cured

animals was 66.7% (Table 9) and 33.3% of mice with re-

lapse tumors increased their life expectancy by 45% com-

pared to control (Table 10). All the control groups men-

tioned above demonstrate continuous tumor growth (see

Table 9).

After PDT of rat sarcoma M-1 with the following pa-

rameters: the PS doses 2.5 and 5.0 mg/kg and dose of light:

E=150 J/cm2; Ps=0.48 W/cm2 up to 90 days after therapy total

regression of tumor in 100% of animals was observed (Table. 9).

Table 9. Anti-tumour efficacy of murine Erlich carcinoma and rat sarcoma M-1 PDT with intravenous administration of Heliochlorin

(laser action parameters E = 150 J/cm2; Ps = 0.48 W/cm2).

No. gr.

PS dose,

mg/kg

Observation period, days of

Erlich’s carcinoma of mice

2.5

(2 and 3) 100%

(4) 67%

5.0

(2 and 3) 100%

(4) 100%

Control 1+2

(1) 0.09±0,02

(1) 0.94±0,16

(1) 4.05±2.43

Sarcoma M-1 rats

2.5

(2 and 3) 100%

(4) 100%

5.0

(2 and 3) 100%

(4) 100%

Control 3+4

(1) 2.7±0.5

(1) 10.2±2.1

(1) 17.1±4.2

Note: (1) Tumor volume (V, cm3); (2) Tumor growth inhibition (TGI, %); (3) Percentage of animals with full tumor regression (R); (4)

Percentage of completely cured animals.

Table 10. The life expectancy of animals (days) and an increase in

the mean life span of animals (%) after PDT compared to control.

PS dose, mg/kg

Life expectancy

Increase in the mean life

span of animals, %

Mice

2.5

86.0±4.0

5.0

>90

>100*

control

59.3±3.0

Rats

2.5

>90

>100*

5.0

>90

>100*

control

37.3±2.2

*Significant increase in the mean life span of animals compared to

control;

>90 days and > 100% of animals with full tumour regression, no

recurrence until 90 days after PDT, are removed from the experi-

ment with the help of essential anesthesia.

Thus, the new chlorin e6 derivative drug - Heliochlorin

is an effective photosensitizer. Photodynamic therapy of

experimental malignant tumours: murine Erlich carcinoma

and rat sarcoma M-1 showed high photodynamic activity of

the drug. Treatment involving intravenous administration of

Heliochlorin at doses of 5.0 mg/kg (murine Erlich carcino-

ma) and 2.5 mg/kg (rat sarcoma M-1) at optimal time be-

tween administration of the drug and laser treatment with

energy density 150 J/cm2 and power density 0.48 W/cm2

allowed to achieve the maximum therapeutic effect - com-

plete tumor regression in 100% of animals and no recur-

rence of neoplasms up to 90 days after treatment. It has thus

been shown that Heliochlorin has a pronounced antitumor

activity for photodynamic therapy when administered at

lower doses and at lower laser effects than other known

chlorin drugs, clinical.

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

272 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

13. Clinical Application of Photodynamic Therapy

To date, in clinical practice, new methods of treatment

based on the photochemistry achievements, photobiology

and quantum physics are increasingly spreading. Photody-

namic therapy (PDT) is a completely new technology used

in the treatment of various diseases.[534-536]

Photodynamic therapy is highly effective and side ef-

fects and complications are minimal at the same time. This

method is based on the selective accumulation of a photo-

sensitizing substance in the tissue of a malignant tumor,

followed by the chemical reaction development that causes

the destruction of sensitized substances under the influence

of a laser beam of a certain wavelength and power. PDT has

no negative effect on nearby tissues and organs. Healing

occurs according to the type of natural reparative processes;

therefore, the method is organ-preserving, well-tolerated

and permissible with repeated use.[537,538]

In the last decade, an increasing number of specialists

have turned to studying the possibilities of this method in

various fields of medicine.

Photodynamic therapy is widely and successfully used

in purulent surgery; there is a pronounced antibacterial and

anti-inflammatory effect. Resistance to PDT in the treat-

ment of chronic infectious processes in pathogenic micro-

organisms does not develop. Recently, PDT of purulent

wounds has been carried out using photosensitizers of the

chlorin series and has a positive effect on the course of the

wound process which is manifested by a pronounced anti-

bacterial effect accelerating the cleansing of wounds from

purulent-necrotic detritus and shortening the healing time of

wound defects.[539]

A.V. Heinz and co. A clinical study was conducted on

the treatment of 120 patients with purulent and long-term

non-healing soft tissue wounds of various etiologies and

localization using the photoditazin photosensitizer (VETA-

GRAND LLC, Russia, registration certificate No. LS

001246 of 18.05.2012) in the form of a gel. According to

the results, it was concluded that this method of treatment

contributes to reducing the time of wound cleansing from

purulent-necrotic detritus, the appearance of granulations

and reducing the timing of the epithelialization onset by

1.5–2 times. It also reduces the time of complete healing of

purulent wounds by 5-7 days compared with traditional

treatment.

It is important to note that the developed PDT method

of purulent wounds using photosensitizers of the chlorin

series provides a reduction in the time of granulation and

complete healing of purulent wounds.

The effectiveness of PDT does not depend on the

spectrum of sensitivity of pathogenic microorganisms to

antibiotics and has a detrimental effect on antibiotic-

resistant strains of Staphylococcus aureus, Pseudomonas

aeruginosa and other bacteria. Photodynamic damage is

caused by the cytotoxic effect of singlet oxygen and free

radicals, therefore, the development of resistance of micro-

organisms to PDT is minimal. It should be noted that with

prolonged treatment of local infectious processes, the anti-

microbial effect of PDT does not weaken, and the bacteri-

cidal effect is local in nature and does not have a systemic

detrimental effect on the saprophytic flora of the body.

In the field of endocrinology, the results of treatment

of 72 patients with trophic ulcers who underwent PDT with

photoditazine in the form of a gel, both in an independent

version and in combination with NO-plasma therapy were

analyzed. It was concluded that PDT accelerates the cleans-

ing of wounds from devitalized tissues, normalizes micro-

circulatory disorders and stimulates fibroblast proliferation

and granulation tissue maturation. It also enhances phago-

cytosis promotes the epithelization of ulcerative defect on

the 12th-15th day.[540]

Of no small importance is the use of PDT in arthros-

copy for the arthrosis treatment and other inflammatory

processes in large human joints. The results of treatment of

11 patients with deforming arthrosis, synovitis and bursitis

who underwent PDT with the drug photoditazine have been

published. The drug was administered intravenously at the

rate of 0.05 mg/kg of the patient's body weight. After PDT,

the swelling in the projection of the lesion decreased; the

intensity of the severity of the pain syndrome and the local

temperature reaction of the skin was normalized.[543] Treat-

ment results analysis of 86 patients aged 4 to 17 years with

a clinical diagnosis of rheumatoid arthritis, who underwent

PDT with the drug photoditazine in the form of a gel,

showed that this method eliminates the main clinical symp-

toms and is highly effective in the treatment of inflammato-

ry diseases of the joints.

Clinical trials have been successfully launched in

many countries of the world to study the effectiveness of

PDT in the treatment of ophthalmic diseases, in particular,

such as neovascularization.

The results of PDT treatment effectiveness study in

Russia with photosens (FSUE "SSC "NIOPIC", Russia,

registration certificate RN000199/02 dated 04.03.2010) as

monotherapy and combination of PDT with anti-VEGF in

38 patients with age-related macular degeneration have

been published. According to the angiographic picture of

the fundus and the average thickness of the retina in the

foveolus, patients have increased visual acuity, the activity

of the subretinal neovascular membrane has decreased after

treatment.[541]

These guides indicate the effectiveness of the PDT

method with the drug photosens in the treatment of patients

with subretinal neovascular membrane against the back-

ground of age-related macular degeneration.

PDT is used in otorhinolaryngology to improve the

treatment effectiveness of patients with ear throat and nose

purulent-inflammatory diseases, in conditions of reduced

immunity and increased resistance to antibiotics. The re-

sults of PDT in the treatment of purulent sinusitis in 41 pa-

tients with the photosensitizer radachlorin (LLC "RADA-

PHARMA", Russia, registration certificate No.LS-001868

dated 12/16/2011) have been published. Treatment results

analysis showed high efficiency of PDT. 38 patients had a

complete cure; in 3 patients, a significant decrease in the

titers of the detected pathogen was noted in the absence of

clinical signs of the disease.[542]

Data from a number of studies confirm that the use of

PDT in the toxic-allergic form allows to achieve stable re-

mission and in simple forms of chronic tonsillitis to com-

plete cure of the disease.

For many years, specialists have been investigating

the problem of respiratory papillomatosis. Laryngeal papil-

loma is a benign tumor but is prone to rapid growth and

frequent recurrence. Etiotropic therapy of this disease does

not exist to this day. Photodynamic therapy is actively used

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 273

as an alternative method of treating this disease. Russian

authors report on the results of treatment of 19 patients di-

agnosed with respiratory papillomatosis by PDT with rad-

achlorin. After PDT, therapeutic effect was registered in all

patients; complete eradication of PVI was 77%. The ap-

pearance of scars in the larynx and trachea or the aggra-

vation of the existing scarring process has not been rec-

orded.[543]

Currently, the main role in the pathogenesis of the

precancerous and tumor pathology of the cervix develop-

ment belongs to HPV of high oncogenic risk, namely 16,

18, 31, 33, 45 and 52 types. Russian authors report on the

treatment results of 104 patients diagnosed with dysplasia

of the II-III stage and cancer in situ of the cervix associated

with highly oncogenic HPV genotypes with the drug pho-

togem. After PDT, complete eradication of HPV DNA was

achieved in 94% of cases and partial eradication of HPV

was diagnosed in 6% of cases. This is the evidence of a

pronounced antiviral effect of treatment.[544]

The incidence of epithelial malignant neoplasms of the

skin is increasing annually. Basal cell skin cancer (BCC)

accounts for a large proportion of them. One of the most

effective, minimally invasive and organ-preserving methods

of skin epithelial malignant neoplasms treatment is PDT.

Photodynamic damage effectively destroys the tumor and

pathological tissues preserving the surrounding healthy

ones as much as possible.[545]

The positive effect in the treatment of skin cancer by

PDT varies between 70-100% and depends on the localiza-

tion of the tumor process and the stage, the dose of the pho-

tosensitizer and the chemical structure as well as the pa-

rameters of laser irradiation. Complete regression of tumors

is registered in 75-80% of patients, and the duration of the

relapse-free period ranges from 2 months to 5 years.[546,547]

The PDT method is successfully used for inconvenient

localization of tumors with the absence of cosmetic defects

in the form of gross deformation. PDT is indicated for tu-

mors resistant to previously conducted methods of tradi-

tional therapy.

Along with primary and recurrent malignant tumors, a

special place is occupied by metastatic lesions of the skin.

According to the literature, the frequency of metastases of

cancer of internal organs to the skin ranges from 0.29 to

3.3%.[548] Of the metastatic malignant skin tumors, the larg-

est group is breast cancer.

A group of authors performed PDT with domestic

photosensitizers in 36 patients with intradermal metastases.

When using prolonged PDT in patients with intradermal

metastases of breast cancer and melanoma, complete re-

gression of tumors was obtained in 39.3% and 38%, respec-

tively, partial – in 46% and 52.4%. Against the background

of previously conducted traditional therapy, tumor re-

sistance to traditional treatment was noted in all pa-

tients.[549]

Kaposi's sarcoma is a rare angioproliferative disease

that is associated with human type 8 herpesvirus. PDT is

the most promising method of its treatment. According to

the results of treatment of 15 patients with intra-tumor ad-

ministration of domestic photosensitizers using PDT, a pos-

itive clinical effect was noted by improving the general

condition of patients and reducing the area of tumor lesion,

which in turn leads to an improvement in the patients life

quality.

Lung cancer occupies the first place in the structure of

morbidity and mortality from malignant neoplasms. The

surgical method of treatment is used for early central lung

cancer. At the same time, about 20-50% of patients are in-

operable. Photodynamic therapy can be used in combina-

tion with classical methods; therefore, it is more promis-

ing.[550] Some authors argue that the effectiveness of PDT in

early central lung cancer (CRL) depends on the form of

growth and size of primary bronchial cancer. Thus, with a

superficial type of tumor (up to 5 mm), complete regression

with PDT is achieved in 91% of cases, from 5 to 10 mm —

in 89.4%. In nodular and polypoid types of tumors with a

diameter of up to 5 mm, complete regression was noted in

93.7% of cases, and in tumors from 5 to 10 mm — in

62.0%.[551] A number of other authors after PDT in 99 pa-

tients with early CRL in art. IA with tumors up to 1 cm in

diameter, complete regression was observed in 95% of pa-

tients, with tumors of 2 cm or more — in 46%. An excel-

lent PDT result was indicated when the tumor size was up

to 1 cm when the distal border of the tumor was clearly

visible during bronchoscopy. In this case, full regression

was achieved in 98% of the observations. Tumor recurrence

was diagnosed in 13% of patients.[552] Experts from the UK

noted that 517 patients had a 5-year survival rate of 70%

with complete regression of the tumor. In the USA, in one

of the PDT clinics, complete tumor regression was achieved

in 69.5% of cases. A new focus of metachronous lung can-

cer was detected in 24% during further follow-up.[553]

A number of Russian researchers report the results of

PDT of early CRL in 37 patients with domestic photosensi-

tizers (photogem, radachlorin, photosens). In 87% of pa-

tients, complete regression of the tumor was noted.

According to the data of many authors, PDT CRL is a

highly effective method and has no equal analogues, espe-

cially in patients with primary multiple bronchial lesions

as well as with a high risk of complications of surgical

treatment.

PDT is an alternative method for the stenosing malig-

nant neoplasms treatment of the respiratory tract, such as

lung atelectasis and pneumonia. There is information about

PDT with domestic photosensitizers in 55 patients with

stenosing malignant lung tumors of central localization.

This group of patients had a positive clinical effect after

treatment.

Recently, intrapleural prolonged PDT with the drug

photosens has been used to treat tumor pleurisy in primary

and metastatic lesions of the pleura. According to some

authors, conducting such therapy in patients with mesothe-

lioma and metastatic pleural lesion makes it possible to

achieve a stable cessation of intrapleural exudation in 92%

of patients with a follow-up period of up to 3.5 years.

To date, serious experience has been accumulated in

the use of PDT in the early cancer treatment of the esopha-

gus and stomach. This method allows to be applied repeat-

edly in the form of multi-course treatment with an interval

of several months. Conducting a multi-course PDT is pos-

sible for many years.

In 116 patients (121 tumor foci), photodynamic thera-

py was performed for initial cancer of the esophagus and

stomach with intravenous administration of domestic pho-

tosensitizers. Complete regression was achieved in 90

(74.4%) foci and partial regression was achieved in 31

(25.6%) tumor foci.[554]

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

274 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

Based on the above, it can be concluded that with the

use of PDT, high oncological results can be achieved in the

treatment of initial forms of esophageal and stomach cancer.

Tumor lesion of the digestive tract aggravates the

condition of patients and reduces the quality of life. PDT is

an alternative method of palliative care in inoperable pa-

tients with stenosing cancer of the upper digestive tract. The

only method of eliminating tumor stricture is photodynamic

therapy for recurrent dysphagia after stenting due to tumor

germination through the walls of the prosthesis or tumor

growth above or below the stent. The recanalization effect

lasts on average 3 months. Repeated PDT also has a benefi-

cial effect on the recurrence of dysphagia. To achieve the

highest quality of life in patients of this group, a multi-

course PDT is recommended.[555,556]

Stomach cancer ranks second in the structure of can-

cer mortality. At the time of diagnosis, in 70% of patients,

the tumor process is locally widespread and is the cause of

high mortality in the first year after diagnosis. A search is

underway for new methods of specialized therapeutic ef-

fects on the area of the surgical field and peritoneum. In-

traoperative PDT is one of such methods. The researchers

note that the use of intraoperative PDT with domestic pho-

tosensitizers in patients with peritoneal dissemination in

disseminated gastric cancer in combination with palliative

surgical treatment to increase the duration of the relapse-

free period and the overall survival of patients in this group

is effective and safe.

Among all malignant neoplasms of the genitourinary

system, bladder cancer accounts for 70%. Surgical treat-

ment in the volume of transurethral resection within healthy

tissues to the muscle layer is the main method of treatment.

But at the same time, the percentage of relapses does not

decrease and is 40-90% in the first year of follow-up. Cur-

rently, PDT is one of the most promising areas of relapse

prevention.

A number of experts indicate that when using PDT

with 5-aminolevulinic acid as adjuvant therapy, the re-

lapse-free survival was more than 2.3 years in every sec-

ond patient.[557]

Some authors report surgical treatment (transurethral

resection of the bladder) followed by adjuvant PDT with

domestic photosensitizers and intravesical chemotherapy in

35 patients with non-muscularly invasive bladder cancer.

During the observation period, no signs of progression or

recurrence of the tumor process were found.[558]

Therefore, as an adjuvant treatment, PDT in combina-

tion with chemotherapy or bladder TUR is a promising

method that allows achieving high oncological results in the

treatment of initial forms of bladder cancer.

The high frequency of precancerous diseases and early

cervical cancer in the structure of gynecological pathology

in young women significantly disrupts reproductive func-

tion and is an urgent problem. The human papilloma-

virus (HPV) plays a major role in the carcinogenesis of

the cervix.

One of the newest approaches to the treatment of cer-

vical pathology, combining optimal therapeutic effect and

the absence of undesirable complications, is PDT. This

method has both antitumor and antiviral effects aimed at

both the lesion site and the source of permanent HPV infec-

tion of the epithelial layers.

In the study of B. Monk, PDT of dysplastic changes in

the cervical epithelium and preinvasive cancer was per-

formed with local application of photophrine with an expo-

sure time of 24 h and an energy density of 100-140 J/cm2.

Out the 11 patients (73%), 8 showed complete regression of

pathological changes.

Russian researchers conducted a comparative analysis

of PDT results in 195 women with dysplastic changes and

early cervical cancer with intravenous administration of

domestic photosensitizers. All patients were diagnosed with

HPV types 16/18. Antitumor and antiviral effects were

evaluated after PDT. A complete regression of the tumor

was diagnosed in 178 people, partial – in 6, stabilization -

in 11 people. Complete eradication of the virus was diag-

nosed in 182 people, partial – in 8 people and no effect

in 5 patients.

It is important to note that PDT for precancerous and

early cervical cancer is an effective organ-sparing treatment

method that allows the patient to be cured, as well as con-

tributes to her medical and social rehabilitation.

In addition, PDT can be considered as a method of

secondary prevention of cervical cancer in viruspositive

women and used as an independent method of treatment in

this contingent of patients. PDT is an alternative method of

treatment of precancerous and initial tumor pathology of the

cervix with preservation of anatomical and functional integ-

rity of the organ, which is important for the realization of

reproductive function in women.

An urgent problem of modern clinical oncogynecolo-

gy is the treatment of dystrophic diseases, intraepithelial

neoplasia and initial vulvar cancer. Vulvar cancer among

malignant tumors of the female genital organs is 5-8%.

Vulvar cancer is most often preceded by a variety of back-

ground and precancerous diseases. Surgical intervention is

complicated by the fact that the tumor is often localized in

close proximity to important anatomical structures (urethra,

vagina, rectum) or passes to them. The use of photodynam-

ic therapy has expanded the possibilities of therapy for can-

cer and precancerous vulva.

In one of the works of Russian authors, the results of

patients treatment with dystrophic diseases of the vulva,

who underwent PDT using domestic photosensitizers, were

analyzed. Treatment was carried out in 30 patients with

vulvar lesions. By the nature of the revealed pathology of

the vulva, 20 patients (66.7%) had verified sclerotic lichen

of the vulva, 8 (26.6%) had squamous cell hyperplasia of

the vulva, 2 patients (6.7%) had mixed dystrophy. The age

of the patients ranged from 33 to 80 years, the average age

was 56.5 years. Complete clinical remission in the vulvar

pathology group was noted in 27 (90%) of 30 patients. In 3

(10%) patients with vulvar lichen sclerosis, a second PDT

session was required, after which a clinical cure was diag-

nosed. In all cases, a good cosmetic effect was recorded which

is especially important for women of reproductive age.[559]

According to another clinical study, the results of PDT

in 67 patients were analyzed. In 36 patients (53.7%), vulvar

lichen sclerosis was verified, in 16 (23.9%) – squamous cell

hyperplasia of the vulva and in 15 patients (22.4%) – in-

taepithelial neoplasia of the vulva I-III art. Complete clini-

cal remission was noted in 59 (88.1%) of 67 patients. 8

(11.9%) patients had partial remission, which required a

repeat PDT session. After that clinical cure was diagnosed.

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 275

PDT of the affected vulva area attacked by the dys-

trophic process prevented the progression of the disease and

achieved clinical recovery in 92.5% of patients.[560]

The analysis of the obtained PDT results using domes-

tic photosensitizers in the treatment of dystrophic diseases

and intraepithelial neoplasia of the vulva showed high ther-

apeutic efficacy with minimal side effects and no complica-

tions after the treatment. The PDT method allows perform-

ing organ-preserving treatment in patients with vulvar dis-

eases without deterioration of their quality of life. All this

indicates the high efficiency of this method in the treatment

of patients with vulvar diseases and the prospects for fur-

ther development of this direction in oncogynecology.

14. PDT for Activation

of Antitumor Immune Response

Cancer is one of the leading causes of death world-

wide, being the cause of death for nearly 10 million people

in 2020, or nearly one in six deaths.[561] The global cancer

burden is expected to be 28.4 million cases in 2040, a 47%

increase from 2020.[562] Despite significant scientific and

financial efforts to improve five-year survival rates, statis-

tics for many types of cancer are improving only slightly.

This includes the bronchial, lung, esophageal, stomach,

pancreas, liver, bile ducts, and brain cancers.[563] Other

types of cancer have better survival parameters, which ex-

perts explain either by a good response to chemotherapy or

by late activation of metastasis. This makes it possible to

identify the tumor with instrumental methods before the

metastatic stage and surgically remove it. According to the

American authors, the minimum tumor size diagnosed with

nuclear medicine techniques is 15 mm on average in U.S.

clinics.[564] Impossibility of early detection is a major reason

for treatment failures of tumors that begin to metastasize at

smaller sizes.

In light of this, an important scientific challenge is

finding sensitive methods and tools to diagnose minimally

sized tumors. It is important for early-stage tumors diagno-

sis. Another major scientific problem is the development of

minimally invasive method for evaluating the direction of

the tumor process evolution and the possibility of changing

this direction towards to the destruction of the tumor and

metastases. This is especially important for actively metas-

tasizing tumors, which include most of the types of tumors

with a low five-year survival.

Increasingly popular optical diagnostic techniques us-

ing absorptive and fluorescent dyes have significantly ad-

vanced 5-year survival rates for several types of can-

cer.[565,566] Tissue spectral and video fluorescence analysis

devices, with fluorescence excitation in the red and near-

infrared ranges of the spectrum, have significantly ad-

vanced the diagnostic capabilities of optical methods.[567,568]

For example, in neurosurgical intraoperative navigation

during tumor removal, small bleeding completely blocks

the possibility of fluorescence registration with excitation in

the blue range, whereas with red range radiation, fluores-

cence registration is possible even under a layer of blood up

to 1 mm.[569] The most commonly used near-infrared fluor-

ophores in clinical practice today are protoporphyrin IX

induced by 5-aminolevulinic acid,[570] methylene

blue,[571,572] fluorescein[573,574] and indocyanine green.[575]

One of the main directions of PDT development is the crea-

tion of photosensitizers that allow using infrared laser radia-

tion for excitation, which has a greater depth of penetration

into biological tissues. Currently promising infrared photo-

sensitizers are products of bacteriochlorin series, the ab-

sorption maximums of which lie in the range of 760–

820 nm,[57 6–57 8] products based on nanostructured forms of

phthalocyanine derivatives.[579,580] Indocyanine green with

an absorption maximum around 780 nm, widely used for

fluorescent blood flow studies and identification of diseased

lymph nodes,[581] also has photodynamic effects, as demon-

strated in experimental models of skin cancer[582,583] and

infectious pneumonia.[584]

Tumor stroma

It is known that any primary tumor consists not only

of neoplastic cells, but also of a supporting stroma, the

main elements of which are cells of the hematopoietic sys-

tem (in particular neutrophils and macrophages).[585] A

change in the phenotype of cells in the tumor microenvi-

ronment gives the tumor the opportunity to grow and in-

vade. That is why the study of the transformed cells interac-

tions with the stroma seems relevant and clinically im-

portant. Despite the emergence of various targeted drugs on

the market, which have made a big breakthrough in the

treatment of certain types of oncology, but such diseases as

cancer of the stomach, liver, bile ducts, brain and spinal

cord are still in the shadow of scientific progress and can

not be treated with most drugs. The same applies to the skin

melanoma. To date, particularly relevant is the search for

new approaches to therapy, namely, new specific methods

of influencing tumor stromal cells.[586]

Over the past decade, it has been shown that the stro-

ma is an active and decisive component of tumor growth

and its progression. Stromal cells, such as immune, mesen-

chymal and endothelial cells, can account for more than

half of the cellular contents of the tumor. Their activation

and function are the determining factor for regression or,

conversely, growth of tumors. Further research should pro-

vide a better understanding of the interaction between tu-

mors and the immune system, and this knowledge should

be used to optimize cancer treatments.[587,588]

By secreting immunosuppressive cytokines or other

pro-tumor molecules, tumor cells can create a microenvi-

ronment that inhibits antitumor immunity and promotes

malignancy. Among the cells recruited by the tumor, mac-

rophages are particularly numerous and are present on all

stages of tumor progression. In the primary tumor, macro-

phages (so-called tumor-associated macrophages or TAM)

can stimulate angiogenesis and enhance the invasion of

tumor cells, their mobility and the ability to penetrate into

the vessels.[589] During metastasis, metastasis-associated

macrophages (MAM) contribute to extravasation of tumor

cells, their survival and sustained growth.[590] Tumor-

associated macrophages also suppress immunity by pre-

venting the attack by natural killers and T-cells on tumor

cells during tumor progression and after recovery from

chemo- or immunotherapy. Thus, macrophages are attrac-

tive targets for cancer treatment.

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276 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

Selective accumulation of PS by macrophages

According to literature, the amount of tumor-

associated macrophages in the tumor microenvironment is

20 to 80% of the total number of cells,[591] while the accu-

mulation of a photosensitizer in them can be up to 9 times

higher (for porphyrin series) than in tumor cells.[592] This is

due to the fact that during intravenous administration the

photosensitizers bind to low-density lipoproteins, on the

capture of which macrophages are oriented.

The scientific community already have developments

on the selective accumulation of photosensitizers in tumor-

associated macrophages. A group led Professor Hamblin

conducted studies of targeted photodynamic therapy aimed

at tumor-associated macrophages. A photosensitizer was

developed based on chlorin e6 bound to albumin, which

selectively accumulated in TAM with intracutaneous ad-

ministration and provided high PDT efficiency.[593,594] An-

other group of Japanese researchers suggested using chlorin

conjugated with mannose for selective accumulation in

TAM (M-chlorin), selectivity of the accumulation was

demonstrated on cells in vitro.[595] Particularly successful in

terms of accumulation in macrophage cells are nanoparti-

cles. As such, it was shown that the use of photosensitizers

in the nanoform allows selective accumulation in tumor-

associated macrophages and tumor cells and the effective-

ness of photodynamic therapy in melanoma B16F10 was

demonstrated.[596]

In recent years, the possibility of using photosensitiz-

ers in the form of molecular nanocrystals for fluorescent

diagnosis and photodynamic therapy has been investigat-

ed.[597] When such molecular aluminum phthalocyanine

nanocrystals came in contact with the surrounding biologi-

cal cells, the effect of fluorescence "kindling" and singlet

oxygen generation was recorded.[598] he manifestation of the

photoactive properties of mTHPC nanoparticle colloid, as

well as the ability to change cellular functions, has been

demonstrated in interaction with polarized macrophages.[599]

PDT-induced immune response

Although the researchers initially focused their efforts

on increasing the direct cytotoxicity for tumor cells subject-

ed to photodynamic effects (including increasing the gener-

ation efficiency of singlet oxygen by photosensitizers),

there has recently been growing interest in developing

methods and approaches for further studying and enhancing

the immunostimulatory properties of PDT. During PDT,

apoptosis and / or necrosis of tumor cells, destruction of the

tumor-associated vasculature, as well as inflammation that

can induce an immune response in the patient, which subse-

quently also leads to the death of tumor cells due to their de-

struction by their own immune cells, arises directly.[600,601]

PDT induced immune response not only increases the

effectiveness of the therapy performed by systemically

eliminating residual tumor lesions and thus controlling the

primary growth of the tumor, but also has a certain potential

to fight cancer cells at the metastatic stage and relapse of

the disease.[602] Phototoxic damage to tumor cells and ves-

sels induces the release of mediators provoking a local in-

flammatory reaction, resulting in the activation of innate

immunity and the recruitment of phagocytes (macrophages,

neutrophils, dendritic and mast cells), and natural killer

(NK) that capture damaged and / or dying tumor

cells.[603,604] Mediators of inflammation are perceived by

immune cells as danger signals and can be recognized and

neutralized by phagocytes, which leads to induced cytotoxic

activity of immune cells against tumor cells.[605] In the

event that a disrupting tumor cell is captured by an antigen -

presenting cell (for example, dendritic cell), an effective

antitumor immune response is triggered by activating a

specific T-cell response: converting naïve T-cells into cyto-

toxic tumor-specific T-lymphocytes.[606]

From the point of view of fundamental medicine,

when approaching anti-tumor therapy as the management of

the immune response, many questions arise: how the PDT

affects the polarization of macrophages, which populations

of macrophages die first during PDT and whether the sur-

viving populations of cells change their functions, whether

anti-tumor immunity is acquired as a result of PDT, is it

possible to evaluate non-invasively the quantitative ratio of

macrophages of different polarizations before and after the

PDT is performed directly in the tumor in vivo, how to neu-

tralize the microglia of the brain activated as a result of

PDT into the proinflammatory phenotype after tumor re-

sorption, etc.

Vascular effect of PDT, oxygenation, effect of hypoxia on

immune antitumor response

As a result of the vascular mechanism of PDT and the

destruction of the tumor-associated vascular network, the

nutrition of the tumor ceases, which contributes to its re-

gression.[607] However, since in practice, during the treat-

ment, an evaluation of the state of the vessels is not carried

out, their destruction is often incomplete. An important tool

for evaluating the vascular effect of PDT should be the

measurement of tissue oxygenation during and after thera-

py, with the possibility of evaluating deep-seated tumor

layers.[608] At the moment, separate studies are under way to

monitor the oxygenation of tissues during PDT. The studies

on experimental animals demonstrated the possibility of

monitoring changes in the vascular bed and saturation with

oxygen using photoacoustic microscopy during PDT with

Verteporfin at a resolution of 60 μm.[609] Similar studies are

conducted using optical coherent angiography.[610] In the

past few years, there have been reports of laser speckle vis-

ualization in real time, a method developed for non-

invasive monitoring of in vivo blood flow dynamics and

vascular structure with high spatial and temporal resolu-

tion.[611] The possibility of conducting classical PDT in

combination with the use of specific PET radiopharmaceu-

ticals to evaluate the effect on cells (apoptosis, necrosis,

proliferation, metabolism) or vascular damage was demon-

strated.[612] However, to be widespread clinical practice, the

method of monitoring blood flow parameters should be

simple in management, inexpensive and allow assessing not

only the dynamics of blood vessels of the tumor, but also

their oxygenation which is relevant for PDT with non-

vascular drugs.

Premise of PDT activation of immunity in brain tumors

Fluorescent diagnosis of brain tumors received wide

development in the clinical practice of the Russian Federa-

tion, especially to conduct intraoperative navigation.[613,614]

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 277

Towards PDT methods Russian and Western doctors exhib-

it a more cautious attitude. This is due to the fact that mi-

croglia cells, acting as phagocytic macrophages in the acti-

vated state, can stay in this state for a long time and phago-

cytize healthy cells.[615] Microglia cells are activated by

mechanical, radiation, or photodynamic effects on brain

tissue.[616] One of the first photosensitizers, methylene blue,

found neuroprotective effects on the central nervous system

in traumatic brain injury by increasing autophagy, reducing

brain edema and inhibiting microglia activation.[617,618] It

should be noted that the photodynamic properties of meth-

ylene blue (and it has a rather significant photodynamic

effect[619]) have not been used or discussed. Another point

of caution in the use of PDT for brain tumors is that, in ac-

cordance with the effect mechanism, the irradiation zone

will be destroyed and this will facilitate the migration of

non-killed cancer cells to other areas for continued growth.

In fact, PDT appears to be an assistant to tumor-associated

microglial cells that express matrix metalloproteinases

(MMPs) to destroy the matrix in the microenvironment of

tumor cells, facilitating their migration and, consequently,

metastasis. To mitigate these effects, it is necessary to eval-

uate the accumulation of a photosensitizer in microglia cells

of different polarization and to select the light doses neces-

sary for their deactivation. When the blood-brain barrier is

compromised, which often occurs with malignant growth,

monocytes enter the tumor stroma, which may polarize into

pro-tumor or pro-inflammatory phenotypes upon extravasa-

tion, which also requires a separate study of their interac-

tion with photosensitizers.[620]

Thus, PDT, depending on the time after the introduc-

tion of the photosensitizer and its redistribution between the

cells of the stroma and the tumor, as well as the irradiation

mode, stimulates a cascade of response reactions, the

strength of which can already be controlled at the irradia-

tion stage. For brain tumors, especially for stage 3,4 glio-

mas, precise and long-term monitoring of the tumor bed

condition is necessary not only during surgery, but also

during remission. The most promising for this purpose are

fiber-optic implants, installed for a long time in the projec-

tion of the tumor bed.[621]

15. Multifunctional Agents Based on Tetrapyrroles

for Magnetic Resonance Imaging Guided

Photodynamic Therapy

Despite significant progress in the study of the cancer

biochemical mechanisms and the development of antitumor

drugs, cancer remains one of the leading causes of morbidi-

ty and mortality worldwide.[622] Traditional methods – sur-

gery, chemotherapy and radiotherapy are widely used to

treat oncological diseases, but these methods don’t always

allow the tumors to be completely removed and have seri-

ous side effects. There is an extensive search for safe and

cost-effective approaches to improve the effectiveness of

traditional cancer therapies and to find new alternative ap-

proaches to its treatment.[623-625] Early diagnosis and detec-

tion of the pathological mass are the key to successful can-

cer therapy. Diagnostic imaging implies selective detection

of pathological processes at the cellular and molecular lev-

els, as well as their real-time monitoring with high sensi-

tivity.[627] Over the years, cancer imaging techniques have

progressed significantly from traditional methods providing

structural information to more advanced and relatively new

functional and molecular imaging technologies. In this re-

gard, the concept of theranostics assuming application of

the advantages of molecular imaging agents and tumor

therapy with a single drug is a promising strategy.[628, 629]

Integration of optical imaging and phototherapy meth-

ods have recently initiated phototheranostics.[629-631] Thus,

the combination of optical fluorescence imaging (FLI) in

the near infrared (NIR) (650-1700 nm) window of "optical

tissue permeability", and PS action could be a promising

approach in clinical practice. The combination of PDT and

the magnetic resonance imaging procedure (MRI), which is

widely used in clinical diagnostics due to its high three-

dimensional spatial resolution and ability to penetrate into

deep tissues, is of particular interest.[632,633] The relevance of

MRI is due to its non-invasiveness, non-ionizing, and non-

radiating modality, its ability to provide detailed infor-

mation on tissue anatomy, function, and metabolism in vivo

with excellent anatomical resolution.[634] Non-specific con-

trast agents based on paramagnetic metals (Gd(III), Mn(II),

Fe(III)) are effective for improving sensitivity in clinical

diagnostics, they account for approximately 25-30% of all

MRI scans.[635-637] However, the application of such drugs is

associated with the danger of increased nephrotoxicity due

to the release of metal ion from the chelating complex,

therefore, one of the most important tasks of the creation of

contrast agents is to achieve stable metal chelation. In con-

nection with the above, the design of multifunctional diag-

nostic systems for the combined diagnosis and therapy of

cancer using PDT and MRI methods is a promising ap-

proach in clinical practice.

Figure 33. The chemical structure of theranostic agents for MRI

guided PDT.

The introduction of contrast agent and PS as individu-

al objects leads to limitations in their pharmacodynamics

and pharmacokinetics, and, as a consequence, to differences

in imaging based on their biodistribution. The inclusion of

MRI contrast agent and PS in one theranostic agent has

been shown to overcome this limitation, when the contrast

agent and PS have the same biodistribution patterns, thus

PDT treatment is further optimized.[638] Furthermore, this

strategy of bimodal agents combining within a single mo-

lecular object provides the additional benefits of enhanced

PS hydrophilicity in biological fluids and improved proton

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278 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

relaxation for MRI efficiency due to the increased molecu-

lar weight.[639] In addition, the growing interest in the de-

velopment of effective PS-based contrast agents is related

to the fact that no commercially available contrast agent is

effective enough for accurate detection of malignant neo-

plastic tissue.

In the past few years, agents for phototheranostics

based on tetrapyrrole structures that could combine MRI

diagnosis and PDT have been actively developed.[631,640-643]

The limited cavity size of tetrapyrrole macrocycles cannot

ensure stable binding to the large radius metal ions used in

MRI, it was proposed to introduce an external chelating

fragment into the PS molecule to create the drug-

theranostics (Figure 33).[644-646]

This review is devoted to recent advances in the appli-

cation of tetrapyrrole compounds for MRI guided PDT. It

emphasizes that the introduction of an external chelating

fragment into the structure of tetrapyrroles leads to genera-

tion of promising agents for phototheranostics. Data on the

available contrast agents as well as PS based on

tetrapyrroles containing Gd(III) for MR diagnostics are

presented, methods of inclusion of metal atoms into such

systems are analyzed.

Types of contrast probes used in MRI diagnosis

The principle of MRI is based on the phenomenon of

nuclear magnetic resonance and proton spin relaxation in

the magnetic field divided into 2 types: 1) longitudinal re-

laxation called T1 relaxation or spin-lattice relaxation aris-

ing from the dissipation of absorbed radiofrequency pulse

energy in the surrounding tissue; 2) transverse relaxation,

called T2 relaxation or spin-spin relaxation arising from the

loss of phase coherence during spin precession. Longitudi-

nal and transverse relaxations are two synchronous magnet-

ic processes, each of which provides its own type of con-

trast: T1 recovery and T2 attenuation have brightening

(positive contrast) and darkening (negative contrast) effects,

respectively.[639,647]

The amount of intrinsic contrast between tissues pro-

duced on an MR image depends on the differences in T1

and/or T2 relaxation times of the observed tissues. In some

cases, the difference in signals between diseased and

healthy tissue is difficult to detect on the MR image. Patho-

logical tissues may have no significant difference in T1 or

T2 signals from the surrounding normal tissues. This signal

distinction can be increased by introduction of an exoge-

nous contrast agent (CA contrast agent).[632] The exogenous

contrast agents improve MR images by locally decreasing

T1 and T2 proton relaxation times.

Two main types of magnetic resonance probes are

paramagnetic and superparamagnetic agents with unpaired

electrons, and the corresponding magnetic moments engage

in dipolar and scalar interactions with nearby water pro-

tons.[639] Then the local water protons relax and quickly

exchange with other unrelaxed water protons reducing the

average relaxation time. The amplification of the signal

contrast can be achieved in two ways: either by higher tis-

sue vascularization, which, for example, is typical for tu-

mors, or by increased affinity of CA to the target area.

The contrast agent (CA) should have high relaxation

ability, stability, specific biodistribution, rapid clearance,

low osmolality and viscosity, as well as low toxicity. All

these contrast agents have their advantages and draw-

backs.[636] The typical CA is a chelated metal ion with a

constant magnetic moment.[632,636, 639,643] The acute and

chronic toxic side effects caused by the metal ion as well as

the chelating agent are significantly reduced due to com-

plexation.

T1 contrast agents are called positive CAs, and they

are mostly compounds with paramagnetic metals. The in-

vestigated tissues absorbing such contrast agents become

bright on T1-weighted images and can be distinguished

from other pathological and normal physiological states,

T1-weighted images are usually more preferable to T2 con-

trast agents. T1-type agents contain paramagnetic ions

(Gd3+ and Mn2+), which have high electron spin and con-

stant magnetic moment due to seven and five unpaired elec-

trons, respectively. T2 contrast agents are classified as neg-

ative CAs, they are derived from iron(III) compounds in

most cases. The negative contrast agents reduce T2 signals

by shortening the T2 relaxation time, which leads to a

strong decrease in the MR signal and thus to the negative

contrast in vivo.[29,632,636,639,647]

Gadolinium-based contrast agents

Gadolinium-based CAs are the basis of clinically used

MRI contrast agents due to its properties: the high magnetic

moment and the long electron spin relaxation time.[648-649]

The contrast agents containing gadolinium reduce the T1

and T2 relaxation times of neighboring water protons,

which increases the signal intensity of T1-weighted images

and decreases the signal intensity of T2-weighted images.

The contrast agents containing gadolinium reduce the T1

and T2 relaxation times of adjoining water protons, that

increases the signal intensity of T1-weighted images and

decreases the signal intensity of T2-weighted images. How-

ever, there are some physical restrictions, for example, the

short half-life complicates the differentiation of benign and

malignant tumors. In addition, gadolinium binds weakly to

serum proteins, its salts are usually hydrolyzed and con-

verted to hydroxides, which are absorbed by the reticuloen-

dothelial system (RES) and accumulate in the body, espe-

cially in the liver, spleen and bones, thereby imparting po-

tential toxicity to the drug. To partially overcome these

problems, Gd is introduced in the chelated forms. Currently

nine Gd contrast compounds with multidentate chelating

ligands have been approved for the clinical practice. Gener-

ally, the cyclic ligand is more stable than the linear ligand.

The ionic compounds are slightly more stable than non-

ionic compounds, but they have a higher osmolality at the

same time. The ionic and hydrophilic complexes comprise

gadolinium (III) diethylentriamine pentaacetate (Gd-

DTPA, referred to as gadopentetate dimeglumine),

Gd(III) 1,4,7,10-tetrazacyclododecane-1,4,7,10-tetraacetate

(Gd-DOTA, gadoterate) and polyaspartate Gd(III). The

nonionic hydrophilic gadolinium (III) chelates are Gd(III)-

diethylene-triamine pentaacetate-bis(methylamide) (Gd-

DTPA-BMA, gadodiamide) and macrocyclic chelate Gd-

DOTA, in which the acetic acid functional group is substi-

tuted with 2-propanol radical (Gd-HP-DO3A, gadoteridol).

The ionic and lipophilic gadolinium complexes comprise

Gd-benzyl-oxymethyl derivative of dimethylglucamine salt

of diethyltriaminepentaacetate (Gd-BOPTA, gadobenate

O. I. Koifman et al.

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dimeglumine) and Gd-ethoxybenzyl diethylentriaminepent-

aacetate (Gd-EOB-DTPA, gadoxetate) (Figure 34).[648]

Although clinically used Gd chelates are relatively

safe and generally tolerated by patients well, there have

been some recent concerns about nephrogenic systemic

fibrosis and nephrogenic fibrosing dermopathy in some

patients, as well as retention of small quantities of Gd in

bone and brain tissue has been found. These side effects

have been identified in patients with impaired renal func-

tion and patients, who have been exposed to MRI scans

with contrast repeatedly. However, there is no evidence that

the application of approved gadolinium-based contrast

agents can lead to pathological changes in the body.

Manganese-based contrast agents

Great attention has been recently paid to the develop-

ment of the contrast agents based on Mn, which is an en-

dogenous metal and has less toxicity. Mn(II) forms a

Mn(II) high-spin d5-cation (S = 5/2) and Mn(III) d4-cation

(S = 2). Mn(II) can act as a strong paramagnetic ion gener-

ating T1 signal amplification in vivo.[650,651] Manganese

dipyridoxyldiphosphate (Mn-DPDP, Teslascan) and man-

ganese(II) chloride tetrahydrate (LumenHance) are the only

clinically approved Mn-based contrast agents.[652] Manga-

nese in ionic form and manganese chelates have a short

half-life. Manganese compounds are excreted primarily

through the gastrointestinal tract and the biliary tract unlike

gadolinium contrast agents. However, high concentrations

of manganese have neurotoxic effects similar to Parkinson's

disease. Mn-based contrast agent, manganese-N-picolyl-

N,N',N'-trans-1,2-cyclohexenediamin triacetate (Mn-Py-

C3A), has been recently proposed, and it provides high spin

relaxation and resistance to Mn dissociation simultaneous-

ly. The application of Mn-Py-C3A exhibited the tumor con-

trast enhancement comparable to gadoterate meglumine in

an animal model of breast cancer, and its partial hepatobili-

ary elimination allowed visualization of liver tumors in a

model of the metastasis (Figure 35).[653] However, despite

the rather high efficiency of the proposed Mn(II)-based

MRI contrast agents, the problems of thermodynamic sta-

bility and kinetic inertness of these complexes remain unre-

solved, that calls into question their clinical application.

Iron-based contrast agents

The iron-based preparations were proposed as a poten-

tial alternative to gadolinium contrast compounds more

than thirty years ago. It has led to extensive preclinical and

clinical studies, but today the only commercially available

contrast agent for MRI is Combidex® (iron oxide nanopar-

ticles), approved exclusively in the Netherlands. Magnetic

nanoparticles of iron (III) oxide are T2 contrast agent, and

also Fe3+ ion has a significant T1 contrast enhancement ef-

fect.[639] The magnetic properties of Fe3+ are due to external

unpaired electrons unlike the Gd3+ ion, so they depend

strongly on the coordination method of the metal with the

ligand. Iron is a widespread and endogenous element in the

human body, so high-spin Fe(III) complexes are very prom-

ising as contrast agents for MRI, they have the best bio-

compatibility and safety profiles as compared to other metal

complexes.[654]

Figure 34. Gadolinium-based contrast agents approved for clinical application.[648]

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Figure 35. Liver imaging of an orthotopic mouse model of colorectal liver metastasis with complex Mn-Py-C3A at 4.7 T. (A and B) T1-

weighted axial images of the liver at the level of the tumor (arrow) prior to and during the hepatocellular phase after injection of Mn-Py-

C3A, respectively. (C) Liver parenchyma vs. tumor CNR prior to and following treatment with Mn-Py-C3A. Reprinted with permission

Figure 36. Potential MRI contrast agents based on Fe(III) complexes.[657]

There are two types of iron oxide-based contrast

agents: superparamagnetic iron oxide (SPIO) and ultra-

small superparamagnetic iron oxide (USPIO), which are

colloidal suspensions of iron oxide nanoparticles. SPIO and

USPIO have been successfully used to diagnose liver tu-

mors in some cases. The superparamagnetic iron oxide par-

ticles or their ultra-small forms are removed from the blood

by the reticuloendothelial system of the liver, spleen and

lymph nodes after intravenous injection. Due to this specif-

ic biodistribution, these preparations can be used for the

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organ imaging and the evaluation of metastasis spread, the

macrophage labeling in the vulnerable atherosclerotic

plaques, and the detection of inflammatory lesions after

transplantations. This explains the recent increased interest

in the application of iron-based preparations for MRI.[653] In

terms of the development of new contrast agents, Schellen-

berger et al. have shown that low molecular weight Fe(III)

complexes such as Fe-CDTA (Magnevist®) provide signif-

icant contrast imaging in vivo and exhibit enhancement

kinetics similar to the data for the clinically used agent Gd-

DTPA (Figure 36).[656,657]

The main problem of iron nanoparticles application in

medical practice is related to the fact that nanoparticles tend

to accumulate in vessels, causing their embolization and

blocking the blood flow [654, 657].

Design of tetrapyrroles conjugates with Gd(III)

for phototheranostics

Tetrapyrrole compounds can become a versatile plat-

form for creating such agents due to their structure, func-

tionalization ability, photophysical properties, and affinity

for the tumor. The conjugation of tetrapyrrole PS with

MRI-contrasts seems to be a particularly promising ap-

proach for the theranostics purposes.[658] Since unsubstitut-

ed tetrapyrroles are poorly soluble in aqueous media, the

presence of hydrophilic complexes of Gd(III) and other

paramagnetic metals makes PS soluble under physiological

conditions and improves tissue distribution and excretion in

vivo. In this regard, the molecular design of the therapeutic

molecule, in particular, the selection of the optimal number

and type of paramagnetic metal complexes within the con-

jugate to fulfill all the requirements for a drug and diagnos-

tic drug is a critical point. PS for PDT, in turn, should have

an amphiphilic structure, be non-toxic and have a strong

absorption in the window of biological transparency to

achieve the required depth of the tissue penetration and

accumulation in the tumor.[659] In addition, the growing

interest in the development of effective PS-based contrast

agents is related to the fact that, to date, no commercially

available contrast agent is sufficiently effective for the ac-

curate detection of malignant neoplastic tissues.

Despite the well-known coordination abilities of

tetrapyrroles, there are limitations in obtaining stable com-

plexes with large radius paramagnetic metals, such as

Gd(III) ions. The limited cavity size of the porphyrin mac-

rocycle makes it very difficult to obtain stable compounds

with metals used in MRI. Therefore, the introduction of an

external chelating fragment into the structure of the mole-

cule became necessary to realize the porphyrin-based

theranostics concept, and most of the publications involve

the conjugation of the tetrapyrrole cycle with an external

chelating agent.

Previously, bifunctional agent 1 (Gd2(DTPA)4TPP)

based on meso-5,10,15,20-tetrakis(4-aminophenyl)porphyrin

bound to 4 DTPA complexes, two of which were metallated

with Gd(III) ions, was first proposed in 1993.[660] It was

found that the relaxation value for the obtained conjugate

with Gd(III) was twice as high as for GdDTPA at 20 MHz,

and a significant enhancement of the contrast image of the

tumor compared to the adjacent normal tissues in mice was

indicative of the affinity of the compound to the tumor tis-

sue (Figure 37). Photoinduced toxicity studies performed

Figure 37. Structure of a new bimodal agent.[660]

Figure 38. Chemical structure of the leader theranostic

compound 2.[661]

on two cell lines (HT29 and L1210) under multi-

wavelength laser irradiation with λ=488 nm and 514 nm

showed phototoxicity of the Gd2(DTPA)4TPP conjugate

comparable to values for the commercially available PS

hematoporphyrin. This Gd2(DTPA)4TPP conjugate was the

first bimodal agent prototype developed for the MRI-

controlled PDT.

More than ten years later Pandey et al. developed and

investigated several theranostic agents based on the natural

porphyrin pyropheoforbide with substituents of lipophilic

or hydrophilic nature.[661] These porphyrins were conjugat-

ed to one, two, three, or six GdDTPA complexes. The con-

jugate design included the creation of a chelating agent

bond by an additional branched linker away from the mac-

rocycle, and the stability of the GdDTPA core was main-

tained by chelation with five anionic carboxyl groups.

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Figure 39. Magnetic resonance (MR) images of rat (Fischer) with Ward's colon tumors before (left) and 24 h after (right) injection of

Theranostics containing one or two Gd complexes required

the use of a liposomal composition to impart water solubili-

ty. With three (compound 2) and six GdDTPA fragments in

the conjugate molecule, water solubility was significantly

improved. Compound 2 (HPPH-3GdDTPA) with three che-

lating fragments (Figure 38), was found to be the best can-

didate with respect to imaging and treatment results. This

conjugate showed a marked enhancement of MR contrast in

tumors in mice 24 h after injection at a dose 10 times lower

than the clinical dose used with Magnevist® (Figure 39).

Fluorescence imaging obtained from the emission of

light by the HPPH derivative also showed maximum inten-

sity 24 h after injection. Also, this compound showed an

effective PDT effect after a single irradiation at 665 nm

(70 J/cm2) 24 h after injection.

In the following work, conjugates of tetra- and mono-

substituted meso-arylporphyrins 3 with GdDTPA residues

were obtained (Figure 40).[662] Measurement of the longitu-

dinal relaxation capacity of the products showed 154% and

251% enhancement over the commonly used Gd-DTPA for

the mono- and tetrasubstituted derivatives, respectively, and

their relaxation times were r1 7.82 mM-1s-1 and 14.8 mM-1s-1.

Research [663] describes the production of potential

theranostic drugs based on zinc phthalocyanine complexes

conjugated with one molecule of the GdDO3A complex

(Figure 41). To reduce the degree of aggregation in aqueous

media and increase the solubility, the PS macrocycles were

modified with peripheral hydrophilic substituents. The

ZnPht-1Gd conjugate 4 showed a lower proton relaxation

value (1.43 mM-1s-1 at 128 MHz) compared to the commer-

cial preparation Omniscan™ (3.23 mM-1s-1 at 128 MHz).

This phenomenon may be due to steric hindrances that

make it difficult for water molecules to access the metal

center. At the same time, the ZnPht-1Gd compound pro-

duced cytotoxic singlet oxygen quite efficiently in DMSO

medium when irradiated with a quantum yield of 0.67.[663]

Figure 40. Structure of the new monosubstituted contrast agent 3.[662]

O. I. Koifman et al.

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Figure 41. Chemical structure of 4 (ZnPht-1Gd) [663] and 5

(ZnPz-1Gd).[664]

Figure 42. T1 weighted MR image of WI-38 VA13 cells

incubated with 0.50, and 100 μM of Zn-Pz-1Gd(III) for 24 h at 4.7

The zinc-based porphyrazine complex PS was bound

to one, four, and eight GdDO3A complexes to form bifunc-

tional conjugates (ZnPz-Gd) (Figure 41). These compounds

showed a strong increase in relaxation (up to 12.8 mM-1s-1

for ZnPz-8Gd at 60 MHz and 37 °C) with an increase in the

number of Gd-complexes attached to the PS (Figure 42).

Cellular uptake was observed only for compounds contain-

ing a single Gd complex (ZnPz-1Gd). A noticeable photo-

toxic effect (with 50% cell destruction) was observed after

10-min white light irradiation.[664]

Porphyrin derivatives incorporating the Gd(III) ion in-

to their cavity are an attractive agents due to their simpli-

fied structure and lower synthetic costs. However, numer-

ous studies in vivo demonstrate problems associated with

the reduced stability of these compounds caused by the dis-

sociation of gadolinium ions from the porphyrin cavity [665].

Nevertheless, two compounds based on substituted porphy-

razines 6-7 (GdPz1 and GdPz2), differing in the nature of

the peripheral groups and containing a Gd(III) ion in the

macrocycle cavity were obtained (Figure 43)[665]. Significant

accumulation of the agent in the tumor in vivo was con-

firmed by fluorescence and MRI methods for both com-

pounds. A good relaxation value under very strong magnet-

ic field conditions (4.67 mM-1 s-1 at 9.4 Tesla) was obtained

for compound GdPz1, and cellular uptake, photodynamic

activity and fluorescence in vivo were also studied. PDT

activity was evaluated on CT26 cell line when irradiated

with 615-635 nm light (10-20 J/cm2, incubation concentra-

tion from 10-7 to 10-4 M). Moderate tumor death was ob-

served, so further optimization of various parameters such

as drug dose and light application was necessary.

The use of the multifunctionalized PS for the treat-

ment of tumors with PDT and chemotherapy, as well as for

MRI imaging, has also been reported.[49] Functionalized

meso-arylporphyrin including gadolinium ion in the macro-

cycle cavity was covalently bound to chemotoxic plati-

num(II) complexes. The Gd/Pt-P1 compound 8 was ob-

tained from 5,10,15,20-tetra(4-pyridyl)porphyrin (P1) coor-

dinated with four Pt(II) complexes (Pt-P1) and one Gd(III)

ion (Figure 44). The new polyfunctional agent showed al-

most doubled relaxation capacity at 3 Tesla compared to

commercial GdDTPA. The phototoxic effect was studied

on the C6 cell line after a 10-minute irradiation at 630 nm.

A synergistic chemophotodynamic antitumor effect was

observed in cells from C6 tumor-bearing mice.

Figure 43. Molecular structure of GdPz1 and GdPz2.[665]

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Figure 44. Molecular structure of 8.[666]

Figure 45. Chemical structure of the theranostic compound 9

developed by Chen et al.[667]

Increasing the number of coordinated water molecules

near Gd(III) contrast agents is particularly attractive strate-

gy to receive agents with high relaxation in a strong mag-

netic field. In this case, careful design of the structure of

hepta- or hexadentate ligands is necessary in order to obtain

Gd(III) complexes with good thermodynamic stability and

kinetic inertness and to avoid the formation of ternary com-

plexes with endogenous molecules. Chen et al. proposed a

new structure of potential theranostics 9 (Figure 45) con-

sisting of a central core-tetraphenylporphyrin bound to four

GdDTTA complexes.[667]

This compound showed a high relaxation value

(14.1 mM-1s-1 at 0.55 Tesla), which doubled in the presence

of human serum albumin, indicating a high affinity of the

obtained conjugate for this blood transport protein (Figure 46).

Uptake of H1299 cell line was confirmed by confocal mi-

croscopy. Effective formation of singlet oxygen was ob-

served under irradiation at 650 nm. The results demonstrat-

ed high potential of this compound as a contrast agent for

multimodal (MR and luminescence) imaging and as a PS

for PDT.[667]

In the following work, a number of theranostics based

on zinc porphyrin complexes with an extended π-system

with the external chelate ligand GdDOTA 10-12 were ob-

tained, and the properties of the synthesized single- and

two-photon activated PS as imaging probes were studied

(Figure 47).[668,669] The attachment of GdDOTA to PS was

realized using the commercial ligand DOTAGA (1,4,7,10-

tetraazacyclodododecane-1-glutar-4,7,10-triacetic acid),

which provided local flexibility while maintaining the sta-

bility of the GdDOTA fragment.

High relaxation r1 values were obtained for 10-12,

which were 19.32, 19.94, and 14.33 mM-1-s-1 at 20 MHz,

respectively, which is 4-5 times higher for known contrast

agents. A 20% increase in relaxation was also observed in

the presence of BSA. Compound 10 had a strong single-

photon absorption capacity (εmax = 41000 M-1 cm-1 at

667 nm in water).

Figure 46. (A) T1-weighted phantom MR images for complex 9 with a concentration of 0−50.0 μM. (B) T1-weighted phantom MR imag-

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 285

Figure 47. Chemical structure of theranostics 10-12 developed by Heitz et al.[668,669]

Figure 48. Structure of complexes with Gd(III), Fe(III), Mn(III).

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The high PDT effect evaluated in HeLa cells was ob-

served with single-photon excitation at 660 nm (1 h, 1 μM

incubation), and a moderate two-photon PDT effect was

achieved at 930 nm (300 scans, 1 μM incubation). High

PDT effect was observed in HeLa cells after single-photon

irradiation at 740 nm (30 min, incubation concentration

of 1 μM). The effect of two-photon PDT was observed at

910 nm (300 scans, incubation concentration 2 μM) as a

function of light power, and 100% cell death was observed

at an average power of 108 mW.

In our work, terpyridine derivatives were used as an

external chelating agent to obtain conjugates of meso-

arylporphyrins with Gd(III), Fe(III), and Mn(III).[670] Ter-

pyridine belongs to the type of linear pincer (claw-shaped)

ligands and forms coordination chelates of the tridentate

type with various metal cations in an almost flat geometry

due to the presence of three conjugated nitrogen atoms in

the molecule.[671] We have developed synthetic approaches

for the preparation of conjugates based on porphyrins of

A2B2 and A3B type with an active functional groups (NH2,

COOH, OH, Py) (Figure 48).

The highest yields were achieved when the amide

bond between the porphyrin and terpyridine fragments was

created. Thus, the yields of conjugates 15-17 were 90-95%.

The conditions for obtaining conjugates 13-17 with para-

magnetic ions Gd(III), Fe(III), Mn(III) for the subsequent

creation of MRI-agents were established. Thus, it appeared

that conjugate complexes with Fe(III) and Mn(III) salts

were formed under mild conditions in an inert atmosphere

at room temperature with the addition of sodium acetate as

a catalyst, while the introduction of gadolinium under mild

conditions was not effective enough and required harsher

reaction conditions. To obtain Gd(III) complexes, the reac-

tion was carried out in an inert box in an autoclave when

heated well above the boiling point of the solvents (80°C).

To assess the safety of the obtained conjugates, it was

important to evaluate the effect of the newly synthesized

compounds on the ability to disrupt the integrity of intercel-

lular contacts (test for inhibition of metabolic cooperation

or for promoter activity). The study demonstrated that com-

pounds 13-17 do not exhibit promoter activity in the given

concentration ranges, i.e., they do not disrupt intercellular

contacts. Thus, when exposed to compound 14 at concen-

trations of 4.5 μM; 2.25 μM; 1.125 μM and 0.56 μM, the

degree of cell cooperation was 109%, 105%, 101%, 111%

relative to untreated cells (Figure 49). At the same time, the

degree of cell cooperation in the samples treated with TPA

at a final concentration of 5 μg/ml was 15% to 20%, which

is statistically significantly different from the control

(p<0.01). Figure 49 shows microphotographs of the cell

monolayer in the scratch region from the control and exper-

imental samples.

The dark and light-induced toxicity of the obtained

conjugates was evaluated in vitro on the Hep-2 cell line

with and without irradiation for 1.5 and 24 h. The absence

of dark and light-induced toxicity of the obtained com-

pounds after 1.5 h was shown. However, an increase in

dark-induced toxicity was observed with prolonged incuba-

tion. Moreover, for the conjugates linked by an amide bond,

the toxicity was lower. Thus, for conjugate 13 after 24 h of

incubation the IC50 was 2.47±0.233 μM (irradiation dose

8.073 J/cm2) and 2.55±0.28 μM (no irradiation). For conju-

gate 15, the IC50 was 1.55±0.15 μM (irradiation dose 8.073

J/cm2) and 4.17±0.251 μM (no irradiation) (Figure 50).

Such an effect may be related to the better biocompatibility

(lower toxicity) of the amide bond for cells.

Figure 49. Micrographs of cell monolayer in the scratch area of control and experimental samples. A. Negative control (dH2O). B. Posi-

tive control (TPA, 5 μg/ml). Compound 12: 4.5 μM (C); 2.25 μM (D); 1.125 μM (E) and 0.56 μM (F). LV propagation length in 4 minutes.

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 287

Figure 50. Effect of compounds on the viability of Hep2 cell line. Cells were irradiated for 90 min using the Medical Therapy Philips TL

20W/52 lamp (irradiation dose of 8.073 J/cm2). Incubation of cells with the compound without irradiation in the dark for 90 min. * statisti-

cally significant differences in cell survival relative to values with zero concentration of the compound (p<0.01) were noted. A) conjugate

13; B) conjugate 15.

In conclusion, increasing interest in the development

of effective contrast agents for MRI diagnostics based on

tetrapyrrole PS is related to the fact that, to date, no com-

mercially available contrast agent is effective enough for

accurate detection of malignant neoplastic tissues. Analysis

of the literature has shown that the development of multi-

functional theranostic systems for combined diagnosis and

therapy of cancer using PDT and MRI methods is a promis-

ing approach in clinical practice. Tetrapyrrole PSs are a

suitable chemical platform for the design of multimodal

agents for PDT. The strategy of combining bimodal agents

in the structure of a single molecular object provides addi-

tional advantages of increased hydrophilicity of the PS in

biological fluids and improved proton relaxation for MRI

efficiency due to the increased molecular weight.

16. Effect of Amphiphilic Polymers

and Polysaccharides on the Photosensitizing

Activity of Porphyrin and Non-Porphyrin Dyes

One of the most important priorities of the Strategy for

Scientific and Technological Development of the Russian

Federation is the "Transition to personalized medicine,

high-tech healthcare and health-saving technologies, includ-

ing through the rational use of drugs (primarily antibacterial

drugs)". Antibacterial photodynamic therapy (aPDT) can be

considered not only in connection with the problem of the

growth of resistance of pathogenic microorganisms to anti-

microbial drugs, but also as a certain niche in antibiotic

therapy, the relevance of which is associated with the loss

of antibiotic effectiveness, as a result of which “common

infections and minor injuries that have been curable for

decades can now kill again” (opinion of WHO Assistant

Director-General for Health Security Dr. Keiji Fukuda). It

is precisely because of this circumstance that researchers

from scientific and medical centers in Japan, the USA,

South Korea, France, Australia, etc., begin to develop ideas

about the use of aPDT instead of antibiotic therapy in the

treatment of dental and some other types of chronic in-

flammatory processes. Approaches to the use of aPDT in

the fight against wound infections, trophic ulcers and bed-

sores are also being developed. This is evidenced by the

growing number of publications and relevant international

symposiums. There is also a growing awareness that photo-

dynamic therapy (PDT), including aPDT, should be under-

stood not only as photoinduced effects on pathological tis-

sues with necrosis or apoptosis of affected cells, but also as

a targeted initiation of the effective dynamics of subsequent

regenerative processes of tissue granulation and epitheliali-

sation.[448,673] Such processes are initiated, among other

things, by the body's immune system in the tissues adjacent

to the pathologically developing tissues of the affected ob-

jects. And the task of PDT should be to choose the optimal

regimes of photodynamic effects on pathological tissues so

that the regenerative systems of the body can show their

activity to the full extent. It is for this reason that the level

of energy effects during PDT of affected objects should be

minimized to a certain level in order to initiate, but not sup-

press, the possibilities of the body's regenerative systems.

First of all, a controlled decrease in the level of light expo-

sure during aPDT can be associated with a certain decrease

in the concentration of photosensitizers (PS) introduced into

the corresponding drugs, which is extremely important for

reducing the phototoxicity of such dosage forms. As it was

shown earlier,[674] the solution of such problems can be

achieved by using PS with amphiphilic polymers (AP), with

fragments of which PS molecules can form conformational

complexes. In this case, the specific activity of the PS in the

composition of such complexes with AP can exceed the

specific activity of the initial PS molecules by an order of

magnitude or more (“polymer effect”). Since photoinduced

effects on pathological tissues during PDT are associated

with photosensitized (with the participation of PS mole-

cules, usually porphyrins, their chlorine derivatives, phthal-

ocyanines) generation of singlet oxygen and its other reac-

tive forms (ROS), conclusions about the "polymer effect"

were made preliminary on the basis of the values of effec-

tive rate constant keff of photooxidation of a model sub-

strate, tryptophan, and confirmed by aPDT of infected

wounds in laboratory animals.[674,675] The idea of using PS

together with polymeric components in the preparation of

medicinal drugs for aPDT lies in the development of an

integrated approach to the creation of effective photosensi-

tizing systems. This is, first of all, the search for new com-

ponents for the developed photosensitizing compositions

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

288 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

PS-AP, where PS is a photosensitizer; AP is an amphiphilic

polymer. At the same time, it is possible to use not only

traditional PS, such as phthalocyanine, porphyrin and chlo-

rine derivatives, but also non-porphyrin compounds - dyes

that not only show a high activity in the photogeneration of

reactive oxygen species (ROS), primarily singlet oxygen

1О2 with a quantum yield ФΔ =0.75-0.76, but also exhibit-

ing their own bactericidal activity (which is traditionally

used in medical practice).[320,676,677] This, in particular, ap-

plies to methylene blue (MB) and rose bengal (RB).[678–680]

These PS are currently used in aPDT of bacterial, viral and

fungal infections, and their cost is significantly less than the

cost of porphyrin compounds. It was shown in [673] that MB

and RB dyes, as well as porphyrin compounds, can become

the basis for the search for effective polymer-containing PS

systems for aPDT. Methylene blue (Figure 51,a) is an or-

ganic dye of the thiazine dyes group, which has long been

known as an effective photogenerator of singlet oxygen.

Recently, it has attracted attention as a promising photosen-

sitizer for PDT of cancer and infectious diseases.[681,682]

Methylene blue is used as a PS for the destruction of My-

cobacterium tuberculosis by аPDT.[683] However, unlike

porphyrin derivatives, which require a long incubation peri-

od for photosensitization, phenothiazine derivatives, such as

MB, do not require such a long pretreatment. Rose bengal

(Figure 51,b) is a dianionic xanthene dye, also used in pho-

todynamic therapy of certain types of cancer and fungal

diseases.[684,685] The structure of MB and RB is represented

by polycyclic aromatic compounds, which tend to form

stable aggregates with low PS activity in aqueous solutions.

In addition, due to its anionic nature, RB hardly penetrates

through the cell walls of bacteria.

Studies have shown that ternary block copolymers of

oxyethylene and oxypropylene, pluronics F127 and F108,

polyvinylpyrrolidone, polyethylene glycol (PEG), and pol-

yvinyl alcohol, may be of interest as AP for PS systems for

aPDT. Naturally, the necessary (preliminary) condition for

the use of such AP in PS systems is their promoting role in

the model process of tryptophan photooxidation, which

occurs during the photosensitized energy transfer of triplet

excited PS molecules to molecular oxygen with the for-

mation of singlet oxygen in aqueous media. The kinetic

regularities of tryptophan photooxidation catalyzed by

methylene blue and rose bengal in the presence (and ab-

sence) of the indicated AP, as well as an assessment of the

effect of AP on the absorption and fluorescence spectra of

the dyes, are presented below.

As shown earlier, amphiphilic polymers can initiate

the deaggregation of porphyrins or dyes in an aqueous me-

dium, thereby increasing the proportion of PS molecules

that are effectively involved in the process of photosensitiz-

ing oxidation of substrate molecules. It is known that the

aggregation of porphyrins in aqueous media, which reduces

their photosensitizing activity due to the internal dissipation

of photoexcitation energy, is due to hydrophobic interac-

tions of their macrocycles, while the aggregation of these

dyes is associated with their substituted anthracene struc-

ture. The magnitude of the effect of AP on the state of PS

associates in all cases was analyzed by the value of the ef-

fective rate constant keff of the rate of tryptophan photooxi-

dation in the presence of these PS and AP. At the same

time, the measure of the ability of amphiphilic polymers to

deaggregate the associates of these dyes when interacting

with MB and RB, a kind of “activity series” of AP in the

process of deaggregation, does not coincide with the similar

series of AP activity during the destruction of aggregates of

water-soluble porphyrin PS.[686 -688]

Thus, according to the ability to cause deaggregation

of porphyrin water-soluble PS, in particular, photoditazine

and dimegin, that is, to increase the value of keff, am-

phiphilic polymers are arranged as follows: PVP > F127 >

> F108 > PEG > PVA. A similar pattern for MB and RB

looks somewhat different: PVA > F108 > F127 > PEG >

> PVP. These differences in the nature of the interaction of

amphiphilic polymers with porphyrin PS and dyes are ap-

parently related to the difference in the structures of

macrocyclic PS and anthracene derivatives, which have

small sizes.

The main interactions that determine the supramolecu-

lar structural organization of macrocyclic porphyrin com-

pounds (including photoditazine and dimegine) in aqueous

solutions are associated with hydrophobic, hydrogen, and

donor-acceptor bonds. At the same time, the nature of in-

termolecular interactions of anthracene derivatives should

be greatly influenced by the presence of positively charged

(MB) or electronegative (RB) groups. Indeed, anionic RB,

depending on the pH of the medium, can exist in cationic,

neutral (lactone), and anionic forms (Figure 52). In a neu-

tral photoinactive form, rose bengal exists in a slightly acid-

ic environment (pH < 5) (Figure 52,II). In a neutral and

weakly alkaline medium (pH 7.2–7.6), RB has a character-

istic absorption spectrum with two bands (550 and 515 nm),

corresponding to the dianionic form of RB (Figure 52,III).

Thus, RB works as a PS only in a neutral and weakly alka-

line medium (рН 7.2-7.6).[689,690] Another issue that is usu-

ally discussed in connection with the development of effec-

tive PS systems for aPDT is associated with the simultane-

ous use of biologically active molecules - proteins, proteo-

lytic enzymes, polysaccharides with their own bactericidal

activity during aPDT procedures. However, such biopoly-

mers can reduce and even suppress the activity of dyes in

1О2 photogeneration. It is shown below that one of the pos-

sible ways to solve these problems, specifically, when using

natural polysaccharides with wound healing ability - chi-

tosan (CHT) and sodium alginate (SA) in аPDT procedures

- is the formation of conjugates of PS (in this case, MB and

RB) with the indicated polysaccharides. Such conjugates

provide better penetration of PS through bacterial cell

walls, the structure of which includes polysaccharides and

their complexes with proteins,[691] directly into the cell.

Figure 51. Structural formula of methylene blue (a) and rose

bengal (b).

O. I. Koifman et al.

Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304 289

Cation

Lactone

Dianion

III

Figure 52. Tautomeric forms of rose Bengal.

Figure 53. Schematic representation of the formation of the RB conjugate - a fragment of CHT.

Among microbial pathogens, gram-negative bacteria

have the highest antibiotic resistance, and therefore the dis-

eases caused by such microflora are extremely difficult to

treat with antimicrobial therapy. They are also considered

to be sufficiently resistant to photodynamic effects,[692]

which is associated with the low permeability of their outer

membrane for PS. It is known that cationic PS can most

effectively interact with gram-negative bacteria.[210] Cur-

rently, PS covalently bound to polymeric polycations (natu-

ral and synthetic) are also used in aPDT. Thus, it was

shown in [693] that the treatment of deuteroporphyrin with

the polycationic peptide polymyxin B promotes the penetra-

tion of the formed complex through the membrane of gram-

negative bacteria. The use of conjugates of anionic dyes

(phthalocyanines) with cationic polylysine leads to an in-

crease in their photobactericidal activity.[694] It was also

shown that the binding of photosensitizers used in medical

practice, in particular, photohem and photoditazine, with

chitosan gel reduced the toxicity of the photosensitizer and

significantly reduced hemorrhagic phenomena in the

wound.[695-697] In this case, the interaction of polycationic

chitosan and porphyrin PS significantly reduces their activi-

ty in the processes of generation of singlet oxygen.[698] It

was shown that such a decrease is due to the aggregation of

porphyrin molecules near the protonated amino groups of

chitosan.[699,700] Moreover, as we demonstrated earlier, the

activity of PS-chitosan systems in 1О2 photogeneration in

the aqueous phase can be increased by adding AP, specifi-

cally, polyvinylpyrrolidone or Pluronic F127.[701,702] It was

shown by the proton NMR method that in the presence of

the indicated AP, the photosensitizer is coordinated with the

molecules of the amphiphilic polymer (formation due to

hydrophobic and hydrogen bonds), which promotes PS dis-

aggregation and, obviously, prevents its interaction with

chitosan molecules.[686]

At the same time, the anionic dye RB interacts with

protonated amino groups of chitosan macromolecules to

form ionically bound conjugates (Figure 53), which leads to

a decrease in the value of the effective photooxidation rate

constant keff by 2.5–3 times.

In addition, it was shown that RB molecules that do

not interact with chitosan macromolecules retain their pho-

tocatalytic activity. It should be emphasized that this equi-

librium can exist only in a buffer solution, where the pH for

the RB-CHT system cannot be lower than 4.5–4.0. When

this reaction is carried out in an aqueous solution, the intro-

duction of chitosan almost completely inhibits the process

of photocatalytic oxidation of tryptophan. Since chitosan

becomes soluble only in a slightly acidic medium (0.2–

0.5% acetic acid, pH 4.5), it is also necessary to establish

the role of acetic acid in the observed effect of chitosan on

the PS activity of porphyrin and nonporphyrin photosensi-

tizers, as well as the effect of acetic acid on the structure of

the amphiphilic polymer, which determines the activity of

PS-AP systems under such conditions. It turned out that in

the case of rose bengal in an acetic acid medium, a slight

Synthesis Strategy of Tetrapyrrolic Photosensitizers for Their Practical Application in Photodynamic Therapy

290 Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304

decrease in the value of keff (by approximately 1.3 times) is

observed, i.e. the main role in reducing the photocatalytic

activity of RB belongs to chitosan. At the same time, in the

presence of PVP, the photocatalytic activity of RB is practi-

cally restored to its original value. A similar effect on the

value of keff in the presence of chitosan is observed when

MB is used as a PS. In this case, the drop in keff is 25-35%.

A different picture is observed for PS of porphyrin and

chlorine nature. It is shown that in a solution of 0.2% acetic

acid (pH~4) both the photocatalytic activity of PPS and

their complexes with AP and the spectral properties of

dimegin and chlorin e6 change. Under these conditions, PS

is protonated to form a monoprotonated form. The photo-

catalytic activity of the monoprotonated form of DMG in-

creases by ~1.4 times, while the photoactivity of protonated

Се6 decreases by ~1.2 times.[701] Due to the decrease of the

keff value of tryptophan photooxidation catalyzed by MB

and RB in the presence of chitosan, experiments on the use

of sodium alginate as a polysaccharide (a biopolymer with

wound healing properties) were carried out; Pluronic F127,

PVA, CHT and PVP were used as amphiphilic polymers.

The obtained results are shown in Table 11.

Table 11. Values of keff for MB, RB systems in the presence of

polymers. The concentrations of polymers indicated in parentheses

are given in mass fractions. [PS] = 5·10-6 M, [Trp] = 1·10-4 M,

Trp-tryptophan.

As follows from the data in Table 11, sodium alginate,

as well as CHT, can significantly reduce the photocatalytic

activity of MB, while the addition of amphiphilic polymers

to the CHT-PS mixture can almost completely neutralize

the effect of polysaccharides. The most active in these sys-

tems are PVP, F127, and PVA. It is interesting to note that

sodium alginate in the case of RB practically does not re-

duce the value of keff. Possibly, this is due to the absence

of interaction between the SA polyanion and the anionic

RB. It should also be noted that in PS systems based on

porphyrin photosensitizers, polyvinyl alcohol is completely

inactive. Thus, in order to obtain effective PS polymer

compositions containing polysaccharides and amphiphilic

polymers, it is necessary, starting from the choice of PS, to

carry out a targeted selection of AP and polysaccharide

components.

Acknowledgements. The review was financially supported

by a number of foundations, programs and grants: Section 1

(Koifman O.I. et al.) – by Ministry of Science and Higher

Education of the Russian Federation (grant agreement No

FZZW-2020-0008); Section 2 (Fedorov A.Yu. et al.) – by

the Russian Science Foundation under Grant No. 21-73-

10230 (https://rscf.ru/en/project/21-73-10230/) and by State

assignment via the Research scientific laboratory of

“Chemistry of natural products and their synthetic ana-

logues” of Scientific Educational Centre “Technoplatform

2035” (FSWR-2021-014); Section 3 (Belykh D.V.) – The

work was carried out within the framework of the state

tasks of the Institute of Chemistry of the Komi Research

Center of the Ural Branch of the Russian Academy of Sci-

ences (Syktyvkar) No. 122040600073-3; Section 4 (Leb-

edeva N.Sh. et al.) – by the Russian Science Foundation

(grant no. 21-73-20140); Section 5 (Gorbunova Yu.G. et

al.) – by the Russian Science Foundation (project no. 19-

13-00410-P); Section 6 (Dudkin S.V.) – by the Ministry of

Science and Higher Education of the Russian Federation

(Contract/agreement No. 075-00697-22-00); Section 7

(Lyubimtsev A.V., Maiorova L.A. et al.) – by the grant of

the Russian Science Foundation (20-12-00175), and Minis-

try of Science and Higher Education of the Russian Federa-

tion (FZZW-2020-0008, synthesis of the compounds); Sec-

tion 8 (Tyurin V.S. et al.) – by the Russian Science Founda-

tion, grant 22-23-00903; Section 9 (Zenkevich E.I.) – by

the BSPSR program “Photonics and Electronics for Innova-

tions (2021-2025, Belarus)”, Volkswagen Foundation (Pro-

ject “New Functionalities of Semiconductor Nanocrystals

by Controllable Coupling to Molecules”), and Visiting

Scholar Program of TU Chemnitz, Germany (E.Z., 2020-

2021); Section 10 (Berezin D.B. et al.) – by Russian Foun-

dation for Basic Research, project number 20-03-00153.

Authors are grateful to the Centers for Shared Use of Scien-

tific Equipment of ISUСT (support of the Ministry of Sci-

ence and Higher Education of Russia, project number 075-

15-2021-671) and ISC RAS. The authors are grateful to Dr.

I.I. Hludeev and Dr. V.P. Zorin (Belorussian State University)

for their assistance in realization of gel-chromatographic ex-

periments. Section 11 (Grin M.A. et al.) – Chemical syn-

thesis and article preparation was performed as a part of the

project “Radiopharmaceuticals” during realization of the

RTU MIREA university development program (Federal

academic leadership program Priority 2030). Biological

studies were supported by the Ministry of Science and

Higher Education of the Russian Federation (project №

0706-2020-0019). NMR spectra were performed using the

equipment of the Shared Science and Training Center for

Collective Use RTU MIREA and supported by the Ministry

of Science and Higher Education of the Russian Federation.

Section 14 (Loschenov V. B. et al.) – by the Ministry of

Education and Science of the Russian Federation, grant for

the creation and development of world-class research cen-

ters No. 075-15-2022- 315 “Photonics” and by the Russian

Foundation for Basic Research, grant 20-02-00928. Section

15 (Zhdanova K.A. et al.) – by the Russian Science Founda-

tion (project no. 22-73-10176); Section 16 (Solovieva A.N. et

al.) – by state order No. 122040400099-5 of the N.N. Se-

menov Federal Research Center for Chemical Physics RAS.

The composition of the system in the

order of mixing

keff, L/(mol·s)

1500

MB, SA (0.001)

400

MB, SA (0.0001)

900

MB, PVA (0.04), SA (0.001)

800

MB, PVP (0.04), SA (0.001)

500

MB, F127 (1.30) SA (0.001)

800

MB, F127 (1.30) SA (0.0001)

1450

MB, CHT (0.01)

200

MB, F127 (1.30) CHT (0.01)

700

1600

RB, SA (0.01)

1550

RB, SA, PVA

1800

RB, CHT (0.01)

RB, PVP (1.30), CHT (0.01)

1400

O. I. Koifman et al. 
Макрогетероциклы / Macroheterocycles 2022 15(4) 207-304  291 
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Accepted 07.11.2022 

Opening of forbinexocycle by weak O-nucleophiles. Study and optimisation of free chlorine e6 synthesis methods

Article

Dec 2023

D. R. Karimov

The article considers issues concerning the reaction of exocycle opening in phorbin molecules (on the example of methylpheophorbide a) under the O nucleophiles - water and alcohols action. According to the author, under certain conditions relatively weak nucleophiles as water and alcohols can cause the opening of the exocycle in the molecule of methylpheophorbide a with the formation of chlorine derivatives e6. The study of this reaction, in addition, reveals new synthetic methods to obtain chlorine e6 esters of different degrees of substitution, and eventually - free chlorine e6 in the form of triacids. Free chlorine is a valuable photosensitiser for photodynamic therapy (PDT), and a starting compound for the preparation of other photosensitizers. The article also discusses possible synthetic approaches to obtain free chlorine e6.

Advantages of Long-Wavelength Photosensitizer meso-Tetra(3-pyridyl) Bacteriochlorin in the Therapy of Bulky Tumors

Article

Full-text available

Dec 2023

This research presents a novel synthetic photosensitizer for the photodynamic therapy (PDT) of malignant tumors: meso-tetra(3-pyridyl) bacteriochlorin, which absorbs at 747 nm (in the long-wavelength region of the spectrum) and is stable when stored in the dark. H2Py4BC demonstrates pronounced photoinduced activity in vitro against tumor cells of various geneses (IC50 varies from 21 to 68 nM for HEp2, EJ, S37, CT26, and LLC cultured cells) and in vivo provides pronounced antitumor efficacy in the treatment of mice bearing small or large S37, Colo26, or LLC metastatic tumors, as well as in the treatment of rats bearing RS-1 liver cholangioma. As a result, total regression of primary tumor nodules and cure of 40 to 100% of the animals was proven by the experiment criteria, MRI, and histological analysis. Meso-tetra(3-pyridyl) bacteriochlorin quickly penetrates and accumulates in the tumor tissue and internal organs of mice, and after 24 h, 80% of the dye is excreted from the skin in addition to 87–92% from the liver, kidneys, and spleen.

Iodinated lutetium (III) phthalocyanine: Synthesis, photochemical and nonlinear optical properties

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May 2024

Molecular brushes with long polyfluorene backbones and polymethacrylic acid side chains: Luminescent properties and self‐organization in solutions

Article

Feb 2024

A series of molecular brushes with a polyfluorene (PF) backbone and polymethacrylic acid side chains of varying lengths were prepared by atom transfer radical polymerization. The structure and composition of the synthesized compounds were confirmed by ¹ H NMR and IR spectroscopy. Effect of the length of the backbone on spectral and conformational parameters of the macromolecules in solutions was analyzed. The grafting density of side chains was about 90%. Spectral methods have been used to determine the dependence of side chain grafting on the luminescent properties of polymer solutions, including quantum yields. It was shown that an optimal length of polymethacrylic acid side chains provides solubility of the polymer brushes. Solutions of PF‐ graft ‐polymethacrylic acid complexes with the model substance curcumin were investigated. It was established that the molecular brushes containing curcumin form monomolecular micelles. Molecular brushes with zinc phthalocyanine, potential systems for photodynamic, and photothermal therapy, were studied.

Opening of forbinexocycle by weak O-nucleophiles. Study and optimisation of free chlorine e6 synthesis methods

Article

Dec 2023

D. R. Karimov

Interaction of Cobalt Tetrasulfophthalocyanine with ORF8 Accessory Protein of SARS-CoV-2

Article

Oct 2023

The interaction of cobalt(II) tetrasulfophthalocyanine (CoPc) with the ORF8 accessory protein of SARS-CoV-2 was studied by spectroscopy and calorimetry. The protein was found to shift the aggregation equilibrium in cobalt tetrasulfophthalocyanine solutions towards dimerization. Most probably, the CoPc dimer binds to ORF8 on the greater β-sheet side, thus causing fluorescence quenching. The protein affinity constant to CoPc dimer is 1.5 × 105. Differential scanning calorimetry data indicate that ORF8 undergoes thermally induced denaturation in the temperature range of 38–67°C. Melting of ORF8 includes two stages, which partly overlap. The complex formation of ORF8 with CoPc leads to thermal stabilization of the protein, thus preventing the second stage of protein unfolding. Denaturation of the complex proceeds between 40 and 77°C as two temperature-separated stages. According to gel electrophoresis and immunoblotting data, visible light photoirradiation of the ORF8 complex with CoPc does not induce photooxidation of the protein. It was shown that water-soluble cobalt sulfo-substituted phthalocyanine can be considered as a potential drug inhibiting the ORF8 accessory protein.

A first-in-class β-glucuronidase responsive conjugate for selective dual targeted and photodynamic therapy of bladder cancer

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Mar 2024

Photosensitizing effects and physicochemical properties of chlorophyll a derivatives with hydrophilic oligoethylene glycol fragments at the macrocycle periphery

Article

Feb 2024
PHOTOCH PHOTOBIO SCI

In this work, screening studies of the cytotoxic effect of chlorins with fragments of di-, tri-, and pentaethylene glycol at the macrocycle periphery in relation to HeLa, A549, and HT29 cells were performed. It is shown that, despite different hydrophobicity, all the compounds studied have a comparable photodynamic effect. The conjugate of chlorin e6 with pentaethylene glycol, which has the lowest tendency to association among the studied compounds with tropism for low density lipoproteins and the best characteristics of the formation of molecular complexes with Tween 80, has a significant difference in dark and photoinduced toxicity (ratio IC50(dark)/IC50(photo) approximately 2 orders of magnitude for all cell lines), which allows to hope for a sufficiently large "therapeutic window". A study of the interaction of this compound with HeLa cells shows that the substance penetrates the cell and, after red light irradiation induces ROS appearance inside the cell, associated, apparently, with the photogeneration of singlet oxygen. These data indicate that photoinduced toxic effects are caused by damage to intracellular structures as a result of oxidative stress. Programmed type of cell death characterized with caspase-3 induction is prevailing. So, the conjugate of chlorin e6 with pentaethylene glycol is a promising antitumor PS that can be successfully solubilized with Tween 80, which makes it suitable for further in vivo studies.

Cationic Porphyrins as Antimicrobial and Antiviral Agents in Photodynamic Therapy

Article

Full-text available

Dec 2023
CURR ISSUES MOL BIOL

Antimicrobial photodynamic therapy (APDT) has received a great deal of attention due to its unique ability to kill all currently known classes of microorganisms. To date, infectious diseases caused by bacteria and viruses are one of the main sources of high mortality, mass epidemics and global pandemics among humans. Every year, the emergence of three to four previously unknown species of viruses dangerous to humans is recorded, totaling more than 2/3 of all newly discovered human pathogens. The emergence of bacteria with multidrug resistance leads to the rapid obsolescence of antibiotics and the need to create new types of antibiotics. From this point of view, photodynamic inactivation of viruses and bacteria is of particular interest. This review summarizes the most relevant mechanisms of antiviral and antibacterial action of APDT, molecular targets and correlation between the structure of cationic porphyrins and their photodynamic activity.

Novel cationic conjugates of chlorin e6 with galactose fragments: synthesis and evaluation of photodynamic activity

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