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Received: 4 February 2020
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Revised: 14 June 2020
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Accepted: 3 July 2020
DOI: 10.1002/med.21713
REVIEW ARTICLE
Progress in the development of human carbonic
anhydrase inhibitors and their pharmacological
applications: Where are we today?
Chandra B. Mishra
1,2
|Manisha Tiwari
1
|Claudiu T. Supuran
3
1
Department of Bioorganic Chemistry, Dr.
B. R. Ambedkar Center for Biomedical
Research, University of Delhi, Delhi, India
2
Department of Pharmaceutical Chemistry,
College of Pharmacy, Sookmyung Women's
University, Seoul, South Korea
3
Dipartimento Neurofarba, Sezione di
Scienze Farmaceutiche e Nutraceutiche,
Universitàdegli Studi di Firenze,
Florence, Italy
Correspondence
Manisha Tiwari, Department of Bioorganic
Chemistry, Dr. B. R. Ambedkar Center for
Biomedical Research, University of Delhi,
Delhi 110007, India.
Email: mtiwari07@gmail.com
Claudiu T. Supuran, Dipartimento
Neurofarba, Sezione di Scienze
Farmaceutiche e Nutraceutiche, Università
degli Studi di Firenze, 50019 Florence, Italy.
Email: claudiu.supuran@unifi.it
Abstract
Carbonic anhydrases (CAs, EC 4.2.1.1) are widely distributed
metalloenzymes in both prokaryotes and eukaryotes. They
efficiently catalyze the reversible hydration of carbon diox-
ide to bicarbonate and H
+
ions and play a crucial role in
regulating many physiological processes. CAs are well‐
studied drug target for various disorders such as glaucoma,
epilepsy, sleep apnea, and high altitude sickness. In the past
decades, a large category of diverse families of CA inhibitors
(CAIs) have been developed and many of them showed ef-
fective inhibition toward specific isoforms, and effectiveness
in pathological conditions in preclinical and clinical settings.
The discovery of isoform‐selective CAIs in the last decade
led to diminished side effects associated with off‐target
isoforms inhibition. The many new classes of such com-
pounds will be discussed in the review, together with stra-
tegies for their development. Pharmacological advances of
the newly emerged CAIs in diseases not usually associated
with CA inhibition (neuropathic pain, arthritis, cerebral
ischemia, and cancer) will also be discussed.
KEYWORDS
arthritis, cancer, carbonic anhydrase, coumarin, epilepsy,
glaucoma, inhibitor, neuropathic pain, obesity, selectivity,
SLC‐0111, sulfamate, sulfonamide
Med Res Rev. 2020;1–81. wileyonlinelibrary.com/journal/med © 2020 Wiley Periodicals LLC
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1
1|INTRODUCTION
The carbonic anhydrases (CAs) are ubiquitous metalloenzymes, acting as an efficient catalyst for the reversible
hydrationofcarbondioxidetobicarbonate.Invertebrates, CAs exist in at least 16 different isoforms (CA I‐XV)
belonging to the α‐class, which can be broadly classified into four groups based on the localization (cytosolic,
mitochondrial, secreted, and membrane associated). In other organisms CAs are encoded by eight distinct
genetic families α‐,β‐,γ‐,δ‐,ζ‐,η‐,θ‐,andι‐CAs, differ in their preference for metal ions used within the active
site for performing the catalysis.
1,2
All human CAs (hCAs) belongs to the α‐class which contains a Zn(II) ion at
the active site, coordinated by three histidine residues and a water molecule/hydroxide ion.
3
These isoforms
are known to differ by molecular features, cellular localization, distribution in organs and tissues, expression
levels and response to different classes of inhibitors. hCA I−III, hCA VII, and hCA XIII are cytosolic, hCA IV,
hCA IX, hCA XII, and hCA XIV are categorized as membrane bound, and hCA VA and hCA VB are mitochondrial
isoforms (Table 1). Three acatalytic forms are also known, CA VIII, X, and XI.
4,5
These enzymes are actively
involved to catalyze the reversible conversion of carbon dioxide and water into a bicarbonate ion and a
proton (CO
2
+H
2
O⇆HCO
3
−
+H
+
) by ensuing a two‐step reaction process (except the three acatalytic
isoforms). Additionally, CAs also catalyze numerous other reactions, such as conversion of cyanate to carbamic
acid, cyanamide to urea, sulfonyl chlorides to sulfonic acids, aldehydes to alcohols. They are also esterases with
esters of carboxylic, sulfonic and phosphoric acid derivatives.
6
Most of them were poorly investigated up until
now (Figure 1).
6,7
The widespread expression of these CAs is observed in diverse human organs and tissue,
TABLE 1 Distribution, localization, and catalytic activity of human carbonic anhydrase (CA) isoforms
4,5
CA isoforms
Catalytic activity (CO
2
hydration) Subcellular localization Organ/tissue distribution
CA I Low Cytosol Erythrocytes, gastrointestinal tract, and eye
CA II High Cytosol Erythrocytes, eye, gastrointestinal tract, bone
osteoclasts, kidney, lung, testis, and brain
CA III Very low Cytosol Skeletal muscle and adipocytes
CA IV Medium Membrane‐bound Kidney, lung, pancreas, brain capillaries, colon,
heart muscle, and eye
CA VA Low Mitochondria Liver
CA VB High Mitochondria Heart and skeletal muscle, pancreas, kidney,
spinal cord, and gastrointestinal tract
CA VI Low Saliva and milk secretion Salivary and mammary gland
CA VII High Cytosol Central nervous system (CNS)
CA VIII Acatalytic cytosol CNS
CA IX High Transmembrane Tumors and gastrointestinal mucosa
CA X Acatalytic Cytosol CNS
CA XI Acatalytic Cytosol CNS
CA XII Low Transmembrane kidney, intestine, reproductive epithelia, eye,
tumors, and CNS
CA XIII Low Cytosol Kidney, brain, lung, gut, and reproductive tract
CA XIV Low Transmembrane Kidney, brain, liver, and eye
2
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MISHRA ET AL.
which is indicative of their crucial physiological roles. The CA‐catalyzed reaction is involved in numerous
physiological and pathological processes, including respiration and transport of CO
2
and bicarbonate between
metabolizing tissues and lungs; pH and CO
2
homeostasis; electrolyte secretion in various tissues and organs;
biosynthetic reactions (such as gluconeogenesis, lipogenesis, and ureagenesis); bone resorption; calcification;
tumorigenicity and virulence of various pathogenspathogens.
8–11
FIGURE 1 Reactions catalyzed by α‐carbonic anhydrase [Color figure can be viewed at wileyonlinelibrary.com]
FIGURE 2 hCA Isoforms as drug targets for various pathologies. CA, carbonic anhydrase; hCA, human carbonic
anhydrase [Color figure can be viewed at wileyonlinelibrary.com]
MISHRA ET AL.
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3
Research on CAs has exhaustively proved their significant contribution in diseases such as glaucoma,
obesity, osteoporosis, cancer, high altitude sickness, epilepsy, neuropathic pain, and sleep apnea. As thus, many
ofthehCAsisoformsareimportanttargetsforthedesign of inhibitors with clinical applications (Figure 2).
12–14
CA I is widely present in several tissues and involved in some pathological conditions such as retinal and
cerebral edema, although its precise physiological function is largely a mistery.
15
CA II is also abundantly found
in numerous tissue and plays an important role in many disease conditions such as glaucoma, epilepsy, edema,
high altitude sickness, and renal disorders.
16,17
CA III is linked to the oxidative stress and is associated with
various inflammatory diseases (e. g., myasthenia gravis and rheumatoid arthritis [RA]).
18–20
IsoformCAIVis
well studied as a drug target for glaucoma, stroke, and retinitis pigmentosa.
21,22
The role of the mitochondrial
isoforms (CA VA and CA VB) is well documented for obesity.
23
Although the role of CA VI is less studied, this
isoform is involved in the genesis of caries.
24
CA VII is a well‐studied target for epilepsy and this isoform may
act together with CA II and XIV within the brain in cognition‐related processes.
25,26
CA VIII has been studied
for its involvement in neurodegenerative disorders as well as mental retardation.
27
However, few information
is available regarding the acatalytic isoforms CA VIII, X, and XI regarding their real physiological functions.
27
CA IX as well as CA XII are well‐studied drug targets for hypoxic tumors/metastases, and their inhibition
actively controls tumor growth, progression, and metastasis. CA IX is considered as a valuable marker of
disease development in numerous types of hypoxic tumors. However, CA XII is less studied as compared
with CA IX as an anticancer drug target, but several reports also evoked its role in cancer progression
(Figure 2).
28–31
Some reports also mention its role in epilepsy and other central nervous system (CNS) dis-
orders.
32
CA XIII has been studied for its role in the sperm motility and inhibition of this isoform may be used
to develop contraceptive agents.
33
Furthermore, isoform CA XIV also appeared to be a target for epilepsy and
some types of retinopathies.
34,35
Despite the enormous steps that have been made over the last decades to understand the molecular
machinery associated with CAs, the development of selective CA inhibitors (CAIs) is still an on‐going process.
For the past decades, various research groups from all over the world continuously worked to design and
synthesize novel CAIs effective against various pathological conditions in which the activity of these enzymes
is dysregulated.
36–38
It is rather well‐known that none of the clinically used CAIs appears to be a selective inhibitor for a specific
isozyme, due to the fact that they were discovered in the 1950s and 1970s (except the topically‐acting sulfona-
mides). Clinical and preclinical research outcomes indicated that several CA isoforms are upregulated in various
pathological conditions, therefore, it is necessary to selectively inhibit such an isoform to control disease pro-
gression, without inhibiting other offtarget isoforms.
39
Another drawback of existing nonselective inhibitors cre-
ates elusion to decide the specific role of particular isoform in specific pathological condition.
40
Therefore,
development of highly selective CAIs may open new avenues in the CA research area, and be highly advantageous
to treat CA‐related disorders without exerting severe undesirable side‐effects.
Many CAI classes have been discovered which include sulfonamides, sulfamates, sulfamides, coumarins,
carboxylates, hydroxamates, urea, phenols, thiols, seelenols, boronic acids, benzoxaboroles, and so forth. They act
as effective CAIs and many researchers reviewed CAs along with their modulators in recent periods.
41–44
How-
ever, the detailed structure–activity relationship (SAR) investigation and critical discussion of selective CAIs are
still pending, which is very much necessary for medicinal chemists who are working on CA‐based drug develop-
ment field. A detailed critical analysis of the pharmacological advancement of the newly emerged CAI classes also
appears as a major lacuna in this study field. To fulfill these significant and urgently required issues, is the main goal
of this review article. In this regard, we have categorized isoform‐selective inhibitors, which were discovered in the
past decades and their inhibitory action as well as selectivity over other isoforms are discussed in detail. This
review article will provide exhaustive discussion in the field of drug discovery and development targeting all
physiologically and pathologically important hCAs, where special attention will be focused on selective CAIs
developed in the last 10 years.
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MISHRA ET AL.
2|CASASATHERAPEUTICTARGETSFORCONTROLLING
PATHOLOGICAL CONDITIONS
2.1 |Cancer and metastasis
Tumor progression is the result of continuous division of cells, which generates a hypoxic (low oxygen levels)
environment. These hypoxic conditions are produced by the lack of sufficient blood supply in proliferating tumor
cells, which creates a low oxygen concentration within tumors.
45,46
Hypoxic environments provoke extracellular
acidosis due to anaerobic glycolysis in the tumor cell.
47
This metabolic pathway produces lactic acid and the drop
of pH within the surrounding tumor microenvironment, which further promotes tumor progression. CAs are a main
player of CO
2
metabolism by controlling acid–base balance to maintain a suitable pH.
48
In fact, two CA isoforms,
CA IX and CA XII are reasonably designated as cancer‐associated CA isoforms, as CA IX is overexpressed in almost
all hypoxic tumors and CA XII is associated with many tumor types too.
38,49,50
During the past decade, several
reports demonstrated that CA IX and CA XII are therapeutic targets for cancer management. Several studies have
shown ectopic overexpression of CA IX in various human malignant tumors, including cervix, brain, head and neck,
lungs, colon, breast, and bladder cancer while, whereas CA XII is overexpressed in a more limited number of tumors
which include cervical, breast and renal cell carcinoma (RCC).
51–53
CA IX activity sustains the intracellular pH of
aggressive tumor cells for survival whereas the acidic extracellular pH is maintained for promoting tumor growth
and metastasis.
54
Studies also indicate a role of CA IX in cell proliferation as well as cell–cell interaction. Hypoxia‐
inducible factor 1α(HIF‐1α) modulates the expression of CA IX in response to the oxygen level and the Von
Hippel Lindau (VHL) tumor suppressor protein downregulates its expression by proline hydroxylation, leading
to proteasome degradation of the transcription factor. It was observed that hypoxia malignant tumors contain
increased levels of HIF‐1αand its downstream targets, such as CA IX and XII.
55,56
CA IX is 414 amino acids dimeric protein and consists of several domains which include a short intracytosolic
(IC) tail, a transmembrane domain (TM), a catalytic CA domain, the proteoglycan domain (PG), and signal peptide
(SP). The bearing of the PG domain makes it a distinctive isoform and is supposed to participate in cell adhesion as
well as preservation of catalytic activity in an acidic microenvironment of the hypoxic tumors.
57,58
Pastorekova's group demonstrated the role of CA IX in extracellular acidification of hypoxic tumors and the
fact that its inhibition with sulfonamide CAIs reverses this action. Additionally, it was found that fluorescent CA IX/
XII inhibitors only bind in hypoxic cells, whereas they failed to bind CA IX in normoxic conditions.
59,60
It was also
observed that hypoxia plays a crucial role to open the active site of CA IX, making it accessible for substrates/
inhibitors to bind. Such a characteristic makes CA IX as a perfect target to prevent hypoxic tumor progression,
where less chance of side effect as compared with conventional chemotherapeutic agents might occur.
59,61
Messenger RNA (mRNA) level study indicated that clear‐cell renal adenocarcinoma displayed strong mRNA signals,
while no CA IX mRNA was observed in normal renal parenchyma.
62,63
CA IX overexpression is highly associated
with a high tumor grade, treatment upshot and poor prognosis in lung, breast, brain, esophageal, gastric, and cervix
cancers.
51,52
Investigations indicate that CA IX expression correlates with other prognostic factors, such as EGFR,
MUC‐1, p53, and p300. Association of CA IX with these oncogenic and regulatory pathways components advocates
their possible cross‐talks, which still remain to be clarified.
64,65
CA XII was first identified in a human RCC through a serological screening process.
66
It is a homodimeric
transmembrane glycoprotein that consists of an extracellular catalytic domain. CA XII is shorter (354 aa) in length
as compared with CA IX and consists of four distinctive domains, including a signal peptide, extracellular CA
domain, TM domain, and IC domain. It does not contain the PG domain which is present only in CA IX.
67
Similar to CA IX, overexpression of CA XII has been seen in various types of tumors. Significant expression was
visualized in non‐small cell lung cancer, colorectal cancer, brain tumors, pancreatic tumors as well as cervical
cancer.
68,69
Additionally, 75% of breast carcinomas which were estrogen receptor‐positive and epidermal growth
factor receptor‐negative have shown remarkable CA XII expression.
70
CA XII is normally expressed in the colon
MISHRA ET AL.
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epithelium and overexpression is associated with dysplasia as well as invasive tumor development stage. Studies
have indicated that CA XII is less strongly regulated by hypoxia when compared with CA IX.
71,72
Various cancers
such as colorectal, renal and kidney are correlated with poor prognosis.
72
Riganti's group investigated the role of
CA XII as a therapeutic target for chemoresistance of tumors and observed that CA XII was overexpressed on the
surface of chemoresistant cells together with the efflux pump PGP.
73
In the past decade, numerous sulfonamide, sulfamate and coumarin derivatives with potent CA IX/CA XII
inhibitors have been tested against various cancers in‐vivo models and these compounds successfully stop tumor
progression and their metastasis, which forcefully establishes CA IX/CA XII as a promising target for cancer
management.
74–76
Moreover, a potent CA IX/CA XII inhibitor SLC‐0111 is running in the Phase II clinical trial for
hypoxic, metastatic cancer therapy.
77
Thus, CA IX/CA XII emerged as a valuable therapeutic target for the man-
agement of aggressive tumors and their metastases.
2.2 |Glaucoma and associated eye disorders
Glaucoma is a chronic and progressive degenerative eye disease characterized by high intraocular pressure (IOP)
that causes distinctive alteration of the optic nerve head leading to visual field loss.
78,79
Progressive damage of the
optic nerve ultimately leads to blindness.
80
Glaucoma is considered as the second cause of vision loss worldwide,
and almost 70 million individuals are affected by this disease.
81
Research in the past decades established CA
isoforms as a suitable and effective target to control glaucoma by lowering IOP.
82,83
Studies have shown that
sodium bicarbonate is the main constituent of aqueous humor and several CA isoforms are involved in its secretion
within the ciliary processes of the eye.
84
Several studies revealed that isoforms CA I, CA II, and CA IV are most
widely diffused in the human eye. They are found in the eye lens, whereas only CA I and CA II are expressed in the
corneal endothelium. However, only CA II is expressed in both retina as well as the ciliary processes. Isoform CA IV
is present in choriocapillaris and the retinal pigment epithelium. These isoforms play important role in aqueous
humor secretion which is the key controller of IOP in the eye.
85–87
Liao et al.
88
found overexpression of CA XII in
the eye of glaucoma patients and suggested its role in the pathophysiology of glaucoma. Moreover, it has been
found that the slow cytosolic isozyme CA I is involved in hemorrhagic retinal and cerebral vascular permeability
leakage via prekallikrein activation and production of the active serine protease factor XIIa.
89
These events play a
key role in the pathogenesis of proliferative diabetic retinopathy as well as diabetic macular edema, both of which
are leading causes of vision loss, with few therapeutic options available up until now.
88
Several potent CAIs such as acetazolamide (AAZ), methazolamide, dichlorphenamide, and ethoxzolamide are
widely clinically used drugs to treat glaucoma. Among all, AAZ has been well studied as an anti‐glaucoma drug
which prominently reduced IOP with minimal toxicity.
90,91
However, systemic administration of these sulfona-
mides CAIs displays nonspecific CA inhibition which leads to unwanted side effects such as, metallic taste, malaise,
depression, weight loss, metabolic acidosis, and renal calculi.
92
Additionally, water‐soluble CAIs such as dorzola-
mide and brinzolamide appeared to be potent anti‐glaucoma drugs via the topical administration route, which
effectively reduced IOP with less side effects as compared with the systemically used CAIs.
93
These compelling
evidence ascertain CAs as a therapeutic target to treat glaucoma, retinopathies‐, and macular degeneration.
2.3 |High altitude sickness
High altitude sickness is a common problem for high altitudes travelers and it is symptoms include headache,
anorexia, dizziness, insomnia, nausea, vomiting, dyspnea, peripheral edema, and retinal hemorrhage.
94,95
Ad-
ditionally, pulmonary or cerebral edema was also observed in severe cases.
95
The main reason of high altitude
sickness is associated with reduced oxygen supply at high altitudes, resulting in the development of hypoxemia.
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MISHRA ET AL.
Studies have shown that CA inhibition appeared to be an useful approach for the management of high altitude
sickness.
96
Some studies indicated that inhibition of CAs induces diuresis and bicarbonate excretion leads to a mild
metabolic acidosis.
97
Inhibition of CA IV provokes ventilation that improves arterial oxygenation as well as ven-
tilatory control. In addition to this, inhibition of CA in the peripheral chemoreceptors (PCRs) mediates ventilatory
optimization by receding hypoxic and hypercapnic sensitivity.
98
Thus, diminution in pulmonary vasoconstriction as
well as an alteration in cerebral blood flow may also control cerebral oxygenation, resulting in the control of acute
mountain sickness symptoms.
99
Investigations have also shown the possible effect of the CAI AAZ on central
chemoreceptors (CCRs).
98
It has been reported that cerebral intraventricular administration of AAZ exterminates
enhancement of [HCO
3
−
]
CSF
and interrupts pH of CSF during respiratory acidosis in experimental animals.
100,101
Parati et al. found that systemic resting diastolic and mean arterial blood pressure was increased after 6 h and
2 days of advent at high altitude as compared with sea level. The CAI treatment (AAZ, 250 mg b.i.d) barred this
enhancement, possibly due to lower sympathetic stimulation and elevated NO production.
102
Recently, Burtscher
et al.
103
also showed that low‐dose AAZ pretreatment on the day of ascent to high altitude reduces systemic blood
pressure. Some studies also displayed the beneficial effect of AAZ against periodic breathing during sleep at high
altitude. AAZ significantly attenuated apnea‐associated hypoxemia and improved quality of sleep, which may
contribute to reducing periodic breathing during sleep.
104
Thus, AAZ is the most popular and well‐studied drug for the management of high altitude illness. Dose‐
dependent action study showed that 250 mg/day dose significantly reduces symptoms of high altitude sickness. It
has also found that AAZ in combination with dexamethasone noticeably diminished these symptoms.
105,106
However, other potent clinically used CAIs were not yet investigated in detail against high altitude sickness.
2.4 |RA and osteoporosis
RA affects the lining of joints primarily and progressively causes attrition of the cartilage, which leads to joint
distortion at the later stages.
107
RA is considered as a chronic inflammatory disease which is the result of an
autoimmune response.
108
The Rochester Epidemiology project report indicated that 4.1% of the American
population were diagnosed with RA each year.
107
Although several therapeutic approaches are employed to treat
RA, including surgical procedures, unfortunatly there is no effective and permanent treatment available to date.
108
Several studies observed abnormal expressions of the CAs I, III, and IV in a sample of RA patients, although
only antibodies of these isoforms were detected.
109,110
CA III exhibits low carbon dioxide hydratase activity and its
expression was observed to be high in the skeletal muscle, where free radical production is increased due to
physical exercise.
111
It was hypothesized that this isoform may play a role to control free radicals and thereby
shielding cells against oxidative damage.
112
Recently, overexpression of isoforms IX and XII has been also detected
in patients affected by juvenile idiopathic arthritis (JIA), which is considered as the most common form of RA in
pediatric patients.
113
In vitro studies revealed that CA I significantly promote CaCO
3
formation besides enhancing
hydration reaction. Calcium salt precipitation is a key step of bone formation and CA I play an important role in
this key step. Hence, the enhanced CA I expression in the synovium of RA patients may lead to inappropriate
mineralization through promoting calcium salts deposition.
114,115
Chang et al. demonstrated that enhanced ex-
pression of CA I provokes precipitation and ossification in osteosarcoma cell lines. This study group also found that
overexpression of CA I exacerbates joint inflammation and destruction in a transgenic mice model.
116
It has been
shown that pH plays a vital role to control inflammation and related pain symptoms in RA affected patients. CAs
overexpression elevates ionic concentrations which leads to a local extracellular acidosis. Additionally, the
investigation also showed that tissue acidosis affects humoral as well as cellular immunity processes.
117,118
CAs have also been studied as therapeutic targets for osteoporosis, a disease characterized by depleted bone
mass as well as microarchitectural alterations, which result in bone fragility and an enhanced risk of fractures. It
has been documented that 1.5 million fractures occur in the United States due to osteoporosis. Mortality related to
MISHRA ET AL.
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osteoporotic fractures ranges from 15% to 30% and many of the patients are compelled to live a poor quality of
life.
119
Studies indicate that the highly active CA II is abundantly expressed in osteoclasts and involved to provide
hydrogen ions for mobilization of calcium from the bone by an ATP‐dependent proton pump.
120
Riihonen et al.
showed that membrane‐bound isoenzymes CA IV and CA XIV are expressed in osteoclasts in vivo and in vitro and
these expressions were observed at both mRNA as well as protein levels. By using a membrane‐impermeable CAI,
the authors provided new evidence regarding the involvement of transport metabolons to regulate pH in osteo-
clasts. Additionally, this inhibitor increased osteoclast number and bone resorption activity in rat osteoclast
cultures at low concentration.
121
Thus, these evidence clearly evoke the potential role of CAs as a drug target for
the management of RA and osteoporosis.
2.5 |Obesity
Obesity is a multifactorial disorder defined by the deposition of excess adipose tissue. It emerged as a critical
medical problem all over the world in recent years. It is now considered among the top 10 global health problems
and only in the United States, 65% of the population is affected by this disorder.
122,123
In 2003, WHO anticipated
that more than one billion adults on the planet were overweight and approximately 300 million of them were
obese. Surprisingly, WHO data in 2006 indicated that one‐third of the adult population was overweight and among
them 10% were obese. Obesity is linked with various other diseases such as cardiovascular, diabetes, muscu-
loskeletal disorders, cancer, as well as psychological impairment.
122,124
Although, several molecular targets such as
neuropeptides, endogenous hormones, Glucagon‐like peptide‐1, 5‐HT2C and 11β‐hydroxysteroid dehydrogenase
type1 enzyme were studied for therapy of obesity, no effective drugs are so far available. CAs also emerged as a
potent therapeutic target for the treatment of obesity. The story started from the treatment of epileptic patients
with topiramate (TPM), where this anticonvulsant CAI drug markedly reduced the body weight of obese patients.
Later, the effect of TPM was also observed in Zucker obese rats. TPM is an effective anticonvulsant drug and
displayed good inhibitory potential against CA II (K
i
= 10 nM), CA VA (K
i
= 63 nM), and CA VB (K
i
= 30 nM).
125
The isoform CA VA and CA VB are exclusively expressed in the mitochondria and actively involved in several
metabolic processes such as ureagenesis, gluconeogenesis, and lipogenesis. These and other CA isoforms play a
crucial role in fatty acid biosynthesis: CA VA or/and CA VB within mitochondria and CA II within the cytosol.
126
Mitochondrial pyruvate carboxylase (PC) is required for the efflux of the acetyl group from mitochondria to cytosol
(fatty acid biosynthesis site). In this process, pyruvate is converted into oxaloacetate in the presence of a PC as well
as bicarbonate. The bicarbonate used in this step is produced under the catalytic influence of the mitochondrial
isoforms CA VA and CA VB. Furthermore, oxaloacetate is converted into citrate and translocated in the cytoplasm,
where this citrate is converted into acetyl‐CoA. Acetyl‐CoA is converted into malonyl‐CoA in the presence of
acetyl‐coenzyme A carboxylase (ACC) and bicarbonate. The bicarbonate required for this process is supplied by
the highly active CA II isoform.
127,128
Thus, CA II, CA VA, and CA VB play important roles in lipogenesis and their
inhibition may control excess lipogenesis in obese patients.
129
Several studies confirmed that another potent CAI,
zonisamide, which effectively inhibits CA II, CA VA, and CA VB has therapeutic potential against obesity.
130
Additionally, the CAI trifluoromethansulfonamide (TFM) has significantly decreased lipogenesis in adipocytes, in
cell cultures.
131
Thus, inhibition of mitochondrial isoenzyme CA VA and CA VB along with the cytosolic isoform CA
II may constitute novel targets for the management of obesity by controlling lipogenesis.
2.6 |Epilepsy
Epilepsy is considered as one of the chronic neurological disorder, as it affects 50 million people worldwide. It
is characterized by spontaneous and recurrent seizures in the epileptic patients.
132
Seizure appears due to a
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MISHRA ET AL.
hyper‐synchronous neuronal firing and neuronal hyperexcitability in various regions of the brain. It is a complex
and multifactorial disorder in which numerous receptors, ion channels, and enzymes are involved in the generation
of epileptic seizures. Rapid alterations in ionic composition and pH shifts also play an important role in seizure
initiation and progression, and an increase in the extracellular potassium concentration is identified to enhance
neuronal excitability, causing epileptiform activity.
133,134
There are many CA isoforms present in the mammalian
brain, including CA II, CA IV, CA VA, CA VII, CA XII, and CA XIV. CA II is usually expressed in oligodendrocytes,
choroid plexus, astrocytes as well as myelinated tracts and is involved in many pathophysiologies of the brain,
including epileptogenesis.
135
It was observed that CA II expression increased in the CA1 cells after 3−12 h of
exposure to kainic acid, a model of status epilepticus.
136
Additionally, it is also documented that CA II knockout
mice are more resistant to seizures.
137
Isoform CA VII is also widely expressed in the cortex, hippocampus as well
as the thalamus and endorses depolarizing and excitatory GABAergic transmission by HCO
3
−
currents. Several
reports advocated that CA VII is involved in seizure generation by GABAergic excitation.
138
Recent studies also
showed a key role of CA VII in the initiation of febrile seizures by GABA
A
receptors activation. Intraneuronal
HCO
3
−
influences activity of the GABA receptor, which is possibly involved in hippocampal epileptiform activity.
139
CA isoform XII is also expressed in the brain and high levels of CA XII mRNA were observed in the medial amygdala
as well as dentate granule cells. In addition, the kainic acid treatment also induced CA XII and was linked with
seizure, as the anticonvulsant drug TPM strongly inhibits CA XII too.
136
N‐methyl‐D‐aspartate (NMDA) receptors
induced seizure activity and such an action was suppressed by extracellular protons. The effect of extracellular
protons on the NMDA receptor may provide a remarkable explanation for the antiseizure effects ensued after CA
inhibition.
140
Alteration of intracellular and extracellular pH plays a crucial role in the regulation of neuronal
excitability, with small changes in intracellular and extracellular pH significantly influencing the function of ligand‐
gated and voltage‐gated channels which actively participate in seizure activity. It has been shown that acidification
of the intracellular environment terminates seizure activity in the dentate gyrus. Thus, CAs are main players in this
disease, by actively controlling brain pH.
141
Several studies revealed the role of CA VA in seizure. CA VA expression has been found in astrocytes, where
controls the normal function of PC by providing HCO
3
−
. It was found that patients who have PC deficiency are
more prone to develop severe neurological disorders including convulsions, compared with person who express
this enyme normally.
142
It is also known for decades that some potent CAIs such as AAZ and MZA are used
clinically for the management of epilepsy. AAZ was approved in 1953 as diuretic, and thereafter also for the
treatment of epilepsy. It is still used in myoclonic, partial, absence, and primary generalized tonic‐clonic seizures in
refractory epilepsies. Additionally, other potent CAIs such as zonisamide, methazolamide, and TPM are utilized for
the treatment of epilepsy in more recent periods.
143,144
2.7 |Sleep apnea
Obstructive sleep apnea (OSA) is a common breathing disorder in the adults and is characterized by recurring
occlusion of the pharyngeal airway during sleep. It affects approximately 2–4% of middle‐aged adults. Airway
obstruction increases extra respiratory efforts to maintain normal breathing. OSA compels people to live with poor
sleep, which may contribute to the development of several other disorders such as cognition dysfunction, de-
pression, metabolic disorders, and cardiovascular disease.
145,146
Conventional therapy such as continuous positive
airway pressure (CPAP) and surgery is employed to treat OSA patients. However, these therapeutic strategies are
costly and not feasible for all patients.
147
Several studies revealed CA isoenzymes as a potential therapeutic target
for sleep apnea and the potent CAI AAZ is successfully improved for the management of sleep apnea at high
altitude.
148
Many CA isoforms are expressed in kidneys, red blood cells, and in cerebral areas associated with
central and peripheral chemosensors. Altered levels of CA isoenzymes were found in numerous physiological
stresses such as high altitude associated hypoxia, physical exercise, and hyperventilation.
149–151
Pharmacological
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9
inhibition of CA significantly reduced apnea‐hypopnea index (AHI) and intensified nocturnal oxygenation in OSA
patients.
152
A critical mechanism by which CA inhibition can endorse ventilation is considered to be through the
renal system. CA inhibition in proximal and distal tubules produces a deficit of bicarbonate in urine with metabolic
acidosis which favors ventilation through chemosensory mechanisms.
153
Additionally, the inhibition of CA in red
blood cells and vascular endothelium also promotes ventilation.
154
Hedner's group conducted a randomized placebo‐controlled study with OSA patients and showed that the CAI
zonisamide significantly reduced sleep disorders in OSA patients.
155
This study group studied the relationship
between arterial bicarbonate, apnea severity and hypertension in such patients. Elevated arterial standard
bicarbonate level in OSA patients, which indicated high CA activity was proposed to be due to nocturnal hypoxia
and ubiquitous occurrence of hypertension in OSA.
156
Recently, Hedner research group disclosed that AAZ de-
creased AHI by 42% in OSA patients by decreasing bicarbonate concentrations. Additionally, AAZ alone or
combination with CPAP markedly decreased blood pressure as well as vascular stiffness in OSA patients.
157
It has
been also shown that CA inhibition improves ventilatory stability by decreasing loop gain in OSA patients.
157
Javaheri et al.
158
showed that a single dose of AAZ treatment before sleep significantly improves central sleep
apnea in heart failure patients. These findings clearly indicated a significant role of CAs in blood pressure reg-
ulation and sleep‐disordered breathing in OSA patients.
2.8 |Neuropathic pain
The neuropathic pain is associated with CNS as well as peripherals nervous system (PNS) and is characterized by
paresthesia, tingling, pins and needles sensations. It is caused by damage of the somatosensory nervous system and
is not controlled by classical analgesic therapies.
159,160
The pathogenesis of this disease is complex and associated
with multiple mechanisms. Peripheral neuropathic pain is the result of mutilation within PNS such as radiculopathy,
postherpetic neuralgia, and complex regional pain syndrome, whereas central neuropathic pain is the outcome of
any type of CNS insults including, encephalitis, ischemia, stroke, brain or spinal cord trauma, syrinx formation in the
brainstem or spinal cord, as well as neoplastic disorders.
161
For the treatment of neuropathic pain several types of
medicines are recommended which include antidepressants (tricyclic antidepressants, serotonin reuptake in-
hibitors, and norepinephrine), anticonvulsants (gabapentin, pregabalin, and TPM), opioids, tramadol, capsaicin, and
zonisamide. However, these drugs failed to treat the majority of patients and exert numerous side effects.
162
Price's and Kaila's groups demonstrated the role of CA inhibition for the management of neuropathic pain.
Several CA isoenzymes efficiently generate HCO
3
−
and protons (H
+
)byCO
2
hydration within the brain.
163
The
anion plays a crucial role in the physiology of various neurotransmitters such as γ‐aminobutyric acid (GABA). It
has been shown that peripheral nerve injury (PNI) negatively persuades spinal GABA‐ergic networks through a
diminution in the neuron‐specific potassium‐chloride (K
+
‐Cl
−
) cotransporter (KCC2) and this process has been
connected to the emergence of neuropathic allodynia.
164
Price's group showed that when KCC2 is pharma-
cologically blocked, PNI injury creates a loss of analgesic effect for neurosteroid GABAA allosteric modulators
in naïve mice. Interestingly, CA inhibition by intrathecal AAZ rapidly restored an analgesic effect of KCC2
blockers, demonstrating the imperative function of CA activity in regulating GABAA influenced analgesia after
PNI.
164
Additionally, the spinal administration of AAZ leads to an intense reduction in the mouse formalin pain
test which indicates that inhibition of CA generates analgesia when primary afferent activity is motivated by
chemical mediators. This study group studied that administration of AAZ systemically to rats with PNI gen-
erates the anti‐allodynia effect by itself and an enrichment of the peak analgesic effect with a change in the
dose–response curve shape of a benzodiazepine derivative. The same group found that spinal coadministration
of AAZ and midazolam worked synergistically to diminish neuropathic allodynia after PNI. This finding points
out that the use of CAIs and benzodiazepines collectively may appear effective clinical management for
neuropathic pain.
163,164
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2.9 |Cerebral ischemia and cardiac dysfunction
Brain ischemia is a global health problem that causes deaths or long‐term disability. It is estimated that more than
5 million deaths occur every year worldwide due to ischemic stroke.
165
It is caused by an interruption in cerebral
blood flow due to thrombosis, focal hypoperfusion, and embolism. Insufficient blood flow to the brain leads to poor
oxygen delivery or cerebral hypoxia, which may initiate brain tissue damage or ischemic stroke. Cerebral ischemia
causes alteration in brain metabolism, metabolic rate reduction, and an energy crisis. Ischemic patients encounter
with symptoms such as unconsciousness, coordination deficit, blindness, dysphasia, and body weakness.
166
In focal
brain ischemia, reduction of blood flow occurred in a specific area of the brain which may induce neurons damage
of that particular area whereas in global brain ischemia, blood flow to the brain is arrested or drastically reduced
which may affect large areas of the brain including permanent damage to neurons.
167
Thrombolysis by adminis-
trating tissue‐plasminogen activator (t‐PA) is employed as the most common medication in acute cerebral ischemia.
However, permanent treatment of ischemic patients is not available to date, especially for global cerebral ische-
mia.
167
Several investigations revealed the role of CA isoenzyme in ischemic brain and such studies supported a
potential therapeutic role of CA inhibition in cerebral ischemia.
168,169
Several studies have shown that some CA
isoforms are present in the human brain and participate in its crucial neurophysiological functions by catalyzing the
reversible hydration of carbon dioxide to bicarbonate.
169
Guo et al.
170
found that inhibition of CA by AAZ reduces
brain edema, neuronal death, and neurological deficits after intracerebral hemorrhage, which is suggestive of a
protective role of CA inhibition in brain injury after intracerebral hemorrhage. Recently, Amani's research group
studied the effect of AAZ on stroke patients and the results indicated that AAZ treatment (750 mg/day) effectively
managed hemorrhagic stroke, decreasing Rankin scale and mortality rate in treated patients.
171
Inhibition of CAs
by AAZ enhanced cerebral blood flow in cerebral ischemia patients. Xenon‐133 inhalation technique revealed that
AAZ increased the interhemispheric asymmetry of cerebral blood flow in cerebral ischemia patient with unilateral
occlusion of major cerebral arteries; whereas no significant side‐to‐side asymmetry was observed in patients which
had minor arterial lesions.
171
Swenson's group
172
showed that CA inhibition, mainly with benzolamide and
ethoxzolamide, reduces cardiac dysfunction in rats, being hypothesized that a membrane‐bound CA isoform may
be responsible for such effects. However, detailed mechanistic studies are still missing for understanding which CA
isoforms are involved and the mechanisms by which the drugs exert their effects.
172
In contrast, Mannelli et al. carried out an in vivo evaluation study of sulfonamide‐and coumarin‐based CAIs
against cerebral ischemia. They found that several CAIs at the dose of 1 mg/kg were able to enhance the neu-
rological score by 40%, and reduce the volume of hemisphere infarction in permanent middle cerebral artery
occlusion rat model of cerebral ischemia.
169
2.10 |Miscellaneous conditions
CAs as a target for the treatment of peptic ulcer were well studied starting in 1970.
173
A study has displayed that
AAZ and other sulfonamide CAIs are effective for the treatment of peptic ulcer, but the inhibition of CA isoforms in
other organs exerts numerous side effects which made this approach a poor attraction for the clinician. Subse-
quently, it was found that CAIs control peptic ulcers by inhibiting CA isoform present in the bacterial patho-
gen Helicobacter pylori which are responsible for peptic ulcer, as these CA isoforms are essential for the survival of
the pathogen.
171
CA isoenzymes also studied as a possible drug target for hydrocephalus, being found that inhibition of choroid
plexus CA significantly reduces CSF secretion. AAZ with a combination of cytostatics effectively reduced elevated
intracranial pressure.
174
It is understood that treatment with AAZ is superior as compared with surgical treatment
in children with progressive hydrocephalus. Thus, these studies may be clinically exploited in the management of
elevated intracranial pressure, such as hydrocephalus in children or Wegener's granulomatosis.
175
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11
The diuretic function of CAIs has been well studied and widely used to treat various life‐threatening disorders
such as congestive heart failure, renal disorder, hepatic failure associated edema and diabetes insipidus.
176
CAIs
such as AAZ, ethoxzolamide, dichlorphenamide, and methazolamide are still used for the management of con-
gestive heart failure induced edema as well as drug‐induced edema. CAs present in the renal system play a vital
role in various physiological functions such as acid–base balance homeostasis, bicarbonate reabsorption, and NH
4
+
output.
176
CA II, as well as CA IV inhibition are involved in the diuretic action of CAIs. CA inhibition by AAZ has
also been adopted for the treatment of numerous neurological/neuromuscular disorders such as familial hemiplegic
migraine and ataxia, hypo‐and hyperkalaemic periodic paralysis as well as tardive dyskinesia. These investigations
proved that the therapeutic effects are probably due to the metabolic acidosis ensued after CA inhibition
177
CA
inhibition has been proposed to be used for the management of Parkinson's disease‐associated tremor.
178
CAIs
such as AAZ were found effective alone or in combination with levodopa, benzodiazepines and other agents
against Parkinson's disease. However, limited data are available currently related to the role of CA inhibition
against Parkinson's disease.
178
3|STRUCTURAL ARCHITECTURE OF hCA ISOFORMS
Human associated α‐CAs are found in different subcellular localization; CA I, II, III, VII, and XIII are present in
cytosol, CA IV, IX, XII, and XIV are anchored to the cell membrane (being extracellular proteins), CA VA and VB are
located in mitochondria, whereas CA VI is secreted in saliva and milk.
179,180
To date, 15 isoforms of CA have been
discovered in humans and among them, 12 isoforms (CA I–IV, CA VA–VB, CA, VI–VII, CA IX, and CA XII–XIV)
contains zinc in the active site and are catalytically active. Isoforms CA VIII, X, and XI lack the zinc ion from the
active site, being known as CA‐related proteins (CA‐RPs).
180,181
In the past decades, a huge progress has been made regarding the structural elucidation of most hCA isoforms.
However, to date, the three‐dimensional structure of all isoforms is not available, as for CA VB the structure was
not resolved, whereas for CA VA only the structure of a truncated murine enzyme is available (Figure 3).
182–184
It
has been found that the active site architecture of all isoforms is rather similar to each other. The investigation of
structures revealed that these isoenzymes, independent of their subcellular localization, show high sequence
homology, presenting a conserved structure, typified by a central twisted β‐sheet encircled by helical connections
and additional β‐strands. The active site of CAs is a large, conical cavity, of around 12 Å wide and 13 Å deep and
extents from the protein surface to the center of the molecule. The catalytic zinc ion is situated at the bottom of
the active site cavity, in a tetrahedral coordination with three conserved His residues (His94, His96, and His119) as
well as a water molecule as ligands (Figure 3).
185,186
The CAs contain a bipolar active site architecture. Half of the
active site contains mainly hydrophobic residues and the other half consists of hydrophilic residues. Several studies
have shown that these two halves of the active site are actively involved in the rapid catalytic cycling of CO
2
to
bicarbonate. The hydrophobic region is essential for requisitioning the CO
2
substrate and orientating the carbon
atom for nucleophilic attack by the zinc‐bound hydroxide (Figure 3). A well‐ordered hydrogen‐bond solvent net-
work is present in the hydrophilic site, which is essential to permit the proton transfer reaction from the zinc‐
bound water molecule to the bulk solvent which generates the catalytcally active, zinc hydroxide species of the
enzyme.
181,187–189
All CA isoforms exist in monomeric form except the membrane‐bound isoforms CA IX and CA
XII and the secreted CA VI, which are dimeric in structure. However, the dimeric structure of these isoforms does
not influence their catalytic properties. It has been observed that the crucial differences among CA isoforms in
amino acid sequences are present mainly in the middle and external site, on the rim of the cavity (Figure 3).
190,191
The crystal structure of murine CA VA revealed that it contains a Tyr residue at position 64 instead of histidine,
which is present in other isoforms.
192
X‐ray crystal structures of many CAs with various inhibitors indicated four inhibition mechanisms
193,194
: (i) by
direct coordination of the inhibitor to the Zn(II) ion, replacing the zinc‐bound water/hydroxide ion; (ii) Inhibitors
12
|
MISHRA ET AL.
FIGURE 3 Human catalytically active carbonic anhydrase (CA) isoform structures, as determined by X‐ray
crystallography: (A) CA I, (B) CA II, (C) CA III, (D) CA IV, (E) CA VI, (F) CA VII, (G) CA IX, (H) CA XII,(I) CA XIII, and (J)
CA XIV [Color figure can be viewed at wileyonlinelibrary.com]
MISHRA ET AL.
|
13
which anchor the Zn(II)‐bound water or hydroxide ion. (iii) Occlusion of the CA active site entrance by inhibitors
such as coumarins, lacosamide, and fullerenes, (iv) inhibitors which bind outside the active site.
As mentioned above, the most variable sequence of the amino acid residues is found at entrance of the CA
active site, as compared with the bottom of the active site which is conserved between the various isoforms.
193,194
Therefore, designing of inhibitors which interact with active site entrance may bestow selective CAIs. These drug
design strategies will be discussed in the next sections of the article.
4|DRUG DESIGN AND DEVELOPMENT STRATEGIES FOR
SELECTIVE CAIS
4.1 |CA I‐and CA II‐selective inhibitors
CA I and CA II are the major cytosolic CA isoforms and their inhibitors are widely used to treat glaucoma and
epilepsy for more than five decades. To date, several compounds have been developed to inhibit CA I and CA II
selectively. These compounds comprise several classes such as anions, thioureas, nitro compounds, uracil deri-
vatives, phenols, sulfamates, and sulfonamides. Some of them selectively inhibit CA I/CA II or both over other
isoforms of CAs.
195–233
It should be mentioned however, that although CA I is one of the most abundant isoforms
in many tissues, its precise physiological role is still unknown.
194
4.1.1 |CA I‐selective inhibitors
Various carboxylic acids and their derivatives such as ester, amide, and metal complexes have been tested for their
inhibitory action against isoforms CA I, II, IX, and XII.
195
Inhibition assay result revealed that some of the com-
pounds selectively inhibited CA I. Among all indole‐pyrazole carboxylic 1and hydroxyoxoindolinylidene carbox-
ylate 2showed satisfactory inhibitory action against CA I and also displayed reasonable selectivity over other
isoforms. Compound 1showed a K
i
value of 0.042 µM for CA I, being 43 333‐, 185‐, 185‐fold selective over CA II,
CA IX, and CA XII, respectively. While, compound 2displayed a K
i
value of 0.049 µM and showed 84‐, 164‐, and
102‐fold selectivity over CA II, CA IX, and CA XII, respectively. It was assumed that two nitrogen atoms in the
pyrazole ring of compound 1can modulate the interaction with the zinc ions in active site, while in compound 2
hydroxyl as well as oxo functionalities make selective binding environment toward CA I. However, a bulkier
aromatic group‐containing indole derivatives failed to produce satisfactory inhibition and selectivity. It may be due
to steric hindrance produced by the bulkier group between heteroaryl backbone and amino acid residues present
in the entrance of the active site (Figure 4).
195
Carreyre et al. developed some dodoneine‐based CAIs and assessed
their CA inhibitory action against a panel of CA isoforms. The result indicated that most of the synthesized
compounds displayed selectivity inhibition toward CAI in the micromolar range. SAR investigation revealed that
the hybridization of dodoneine with bicyclic lactone failed to produce inhibition against CA I. However, most of
these lactones showed selective inhibition for hCA III and mCA XIII. Compounds 3,4, and 5have shown
satisfactory inhibitory action toward CA I with a K
i
value of 0.38, 0.76, and 0.13 µM, respectively. These com-
pounds also showed adequate selectivity over hCA II, hCA III, hCA IV, hCA Va, hCA Vb, hCA VI, hCA VII, hCA IX,
hCA XII, mCA XIII, and hCA XIV(Figure 4).
196
A novel series of chiral thiourea derivatives have been synthesized by
Korkmaz et al.
197
and have been evaluated for their CA inhibition action against CA I and CA II isoform.
Investigation revealed that compounds 6–8showed effective inhibitory action toward CA I with K
i
in the range of
3.4–7.6 µM and selectivity in the range of 2–3‐fold over CA II. It was hypothesized that the hydroxyl group in these
compounds might be responsible for effective inhibitory action (Figure 4).
197
A series of sulfonamide derivatives
containing substituted chalcone moieties were synthesized and examined for their CA inhibitory action.
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|
MISHRA ET AL.
Although all derivatives selectively inhibited CA I isoform with nanomolar range inhibitory action, some of the
derivatives such as compounds 9and 10 appeared as a most selective in the entire series over CA II. p‐methoxy
phenyl moiety containing derivative 9showed a K
i
value of 13.05 nM for hCA I and almost 4‐fold selectivity over
CA II, while trimethoxy phenyl holding compound 10 displayed a K
i
value of 21.88 nM for hCA I and appeared to be
almost 2‐fold selective over CA II. The selectivity of these compounds over CA II was not much higher as seen in
results, however, these nanomolar range CAI may prove good lead molecule to develop more selective CAIs.
198
Novel 4‐(2‐(3,4‐dimethoxybenzyl)cyclopentyl)‐1, 2‐dimethoxybenzene derivatives have been developed by Artunc
et al. and their CA inhibition study was performed using esterase assay. Results indicated that these cyclopentyl‐
based derivatives showed medium nanomolar range activity for CA I and CA II. Most of the derivatives did not
display selective inhibition toward CA I. However, some compounds like 11 and 12 showed 2.7‐and 3.4‐fold
selectivity for hCA I, respectively, over CA II. Compound 11 showed a K
i
value of 655.77 nM and compound 12
displayed a K
i
value of 573.18 nM against hCA I (Figures 4and 5).
199
Gul et al. synthesized 4‐(2‐substituted
FIGURE 4 Chemical structure of selective hCA I inhibitors 1–11. hCA, human carbonic anhydrase
MISHRA ET AL.
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15
hydrazinyl)benzenesulfonamides by microwave irradiation method and these derivatives were assessed against CA
I and CA II to study their inhibitory potential. These hydrazinyl benzenesulfonamides compounds have shown low
nanomolar range inhibitory activity for CA I and CA II. Compounds containingp‐bromophenyl 13 and thiophene 14
as a tail bestowed almost 6‐and 4‐fold selectivity over CA II. Compounds 13 and 14 also showed good inhibitory
action against CA I by showing a K
i
value of 1.97 and 2.73 nM, respectively (Figure 4).
200
The CA inhibitory potential of uracil derivatives was also assessed by esterase assay. Tested uracil derivative
showed low to higher micromolar range activity toward CA I and CA II. Inhibition data indicate that methyl
(compound 15) and hydroxyl uracil (compound 16) derivatives showed medium micromolar range activity with a K
i
value of 57.6 and 49.51 µM for hCA I and were not effective against CA II. However, other compounds such as
Orotic acid, Isoorotic acid and 6‐Amino 1,3‐dimethyluracil displayed good micromolar range activity, being 2–3‐
folds selective over CA II isoform. The study provided some uracil‐based good lead molecules as CA I and CA II
inhibitors and may prove useful to develop more potent and selective CA I or CA II inhibitors (Figure 5).
201
FIGURE 5 Chemical structure of selective hCA I inhibitors 12–20. hCA, human carbonic anhydrase
16
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MISHRA ET AL.
A new series of benzenesulphamide derivative has been designed and synthesized, containing benzhydrylpiperizine
as tail and β‐alanyl or nipecotyl as a spacer between the head and tail region. Synthesized derivatives were tested
for their CAs isoforms inhibitory action and most of the compound selectively inhibited CA I isoform with na-
nomolar range affinity. SAR study indicated that β‐alanyl and nipecotyl spacer bestowed some potent and selective
CA I inhibitors. Nipecotyl spacer containing compounds 17 and 18 appeared to be potent CAIs with satisfactory
selectivity over CA II, CA IV, and CA IX. Compound 17 showed a K
i
value of 45.8 nM for CA I and displayed 16.4‐,
30.17‐, and 6.4‐fold selectivity over CA II, CA IV, and CA IX, respectively. The compound containing m‐sulphamide
group 18 has a K
i
value of 71.4 nM against CA I as well as 12.75‐, 22.62‐, and 37.5‐fold selectivity over CA II, CA IV,
and CA IX, respectively. Thus, these benzenesulfonamides represented a suitable lead molecule for further de-
velopment of potent and selective CA I inhibitors (Figure 5).
202
Very recently, Awadallah et al. synthesized
N
1
‐substituted secondary sulfonamides derivatives holding thiazolinone or imidazolone‐indole tails. These deri-
vatives were screened for CA inhibition activity against hCA I, II, IV, and IX. The Inhibition study indicated that
indolo‐benzenesulfonamide 19 and compound containing dihydro imidazole ring 20 appeared to be potent and
selective inhibitors for CA I. Compound 19 showed a K
i
value of 88.5 nM for CA I and 6.5‐,31‐, and 3.6‐fold
selectivity over CA II, CA IV, and CA IX, although, compound 20 displayed a K
i
value of 93.7 nM with 6.4‐,29‐, and
3.5‐fold selectivity over CA II, CA IV, and CA IX. However, the selectivity of these compounds was not up to mark
over cancer‐associated isoform CA IX (Figure 5).
203
4.1.2 |CA II‐selective inhibitors
A library of glycoconjugate benzene sulfonamides has been synthesized by Wilkinson et al. and these derivatives
were screened for their ability to inhibit CA isoforms. Authors have linked sugar tail with zinc‐binding group
benzenesulfonamide by triazole linker. SAR study indicated that compounds containing protected sugar, such as 21
as well as 22 and deprotected sugar holding compound 23 selectively inhibited CA II with low nanomolar range
affinity (Figure 6). Some other derivatives also strongly inhibited CA II but did not appear as selective CA II
inhibitors as like compounds 21,22 and 23. Compound 21 showed a K
i
value of 9.1 nM for hCA II along with 483‐
and 14‐fold selectivity over CA I and CA IX, respectively. Compound 22 displayed a K
i
value of 8.7 nM and was
∼494‐and 12‐fold more potent than at CA I and CA IX, respectively. Remarkably, deprotected ribose analogue 23
showed 573‐and 16‐fold selectivity over hCA I and hCA IX, respectively, with displaying effective inhibition
against hCA II (K
i
= 7.5 nM).
204
Further, same research group synthesized sulfonamide linked neoglycoconjugates
and evaluated their inhibitory potential against hCA I, hCA II, hCA IX, and hCA XII. Most of the conjugates
displayed effective inhibition toward hCA II and hCA XII. N‐4‐(Aminosulfonyl)phenethyl‐S‐(1‐thio‐β‐maltosyl)
sulfonamide (compound 24) and N‐4‐(Aminosulfonyl)phenethyl‐S‐(1‐thio‐β‐lactosyl)sulfonamide (compound 25)
appeared to be the most potent CA II inhibitors which displayed a K
i
value of 4.7 and 4.6 nM (Figure 6). Compound
24 was 20.6‐, 20.4‐, and 2‐fold selective over hCA I, hCA IX, and hCA XII, respectively, while compound 25 showed
17‐, 21.7‐, and 2‐fold selectivity over hCA I, hCA IX, and hCA XII, respectively.
205
Some other 1,5‐disubstituted‐
1,2,3‐triazole benzenesulfonamide glycoconjugates were also synthesized by this study group and examined for
their inhibitory potential against hCA I, hCA II, and hCA IX. The result of this investigation provided some potent
hCA II inhibitors, which have shown a nanomolar range of inhibition. Specially, 1,5‐disubstituted‐1,2,3‐triazole
benzenesulfonamide glycoconjugate 26 which have shown excellent inhibition toward hCA II and also exhibited
satisfactory selectivity over hCA I and hCA IX. This glycoconjugate had a K
i
value of 2.9 nM and showed 1513‐and
23‐fold selectivity over hCA I and hCA IX, respectively. Thus, this glycoconjugate represents a good lead molecule
as a potent and selective hCA II inhibitor.
206
A novel approach has been adopted to design potent CAs inhibitors
where a linear aliphatic chain was tethered with one or two ZBGs (sulfamates) to develop potent and selective CAs
inhibitors. These sulfamates were tested against a panel of CA isoforms and all derivatives effectively inhibited
hCA II isoform. A single sulfamate group‐containing compounds 27 and 28 were the most potent hCA II inhibitors,
MISHRA ET AL.
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17
which have also shown satisfactory selectivity over other isoforms (Figure 6). Hence, these sulfamates derivatives
bring new insight into design potent and selective CAIs, although, they are very simple chemical entities.
207
A series of organic nitrates were tested for their inhibitory action against hCA I and hCA II. These derivatives
displayed millimolar range inhibitory activity for hCA I and micromolar range activity for hCA II. Compounds (2S
(R),7R(S))‐7‐Hydroxybicyclo[2.2.1] heptan‐2‐yl nitrate 29 and (2R(S),7R(S))‐7‐Hydroxybicyclo[2.2.1] heptan‐2‐yl
nitrate 30 showed effective hCA II inhibitory action by showing a K
i
value of 132.7 and 121.4 µM, respectively
(Figure 6). Compound 29 displayed 129‐fold and compound 30 showed 112‐fold selectivity over hCA I. These
finding revealed a new class of CAIs besides well‐known class, such as sulfonamides, sulfamates, and sulfamides,
although the mechanism which governs the CA inhibition action of these nitrates remain unclear.
208
Maresca et al.
reported a series of coumarin derivatives as potent CAIs and these derivatives effectively inhibited various CA
FIGURE 6 Chemical structure of compounds 21–34 displaying selective hCA II inhibitory activity. hCA,
human carbonic anhydrase
18
|
MISHRA ET AL.
isoforms. Some of the coumarin derivatives effectively and selectively inhibited hCA II isoform such as com-
pounds 31,32, and 33 (Figure 6). Coumarins 31,32, and 33 showed a K
i
value of 0.059, 0.066, and 0.099 µM,
respectively, being selective over other isoforms. SAR study indicated that substitution on the coumarin ring plays
an important role in generating potent and selective CAIs. For example, methoxy substitution on coumarin
(compound 32) bestowed potent and selective hCA II inhibitor as compared with ethoxy substituted coumarin
(compound 34). These compounds may constitute a suitable lead for the development of a more potent and
selective hCA II inhibitor because they appeared to be selective over 12 other CA isoforms. Studies have shown
that coumarins represent a different mechanism of binding within the active site of CA as compared with classical
sulfonamide derivatives. The coumarins bind in the hydrolyzed form at the entrance of the active site and do not
intermingle with the metal ion.
209
Pala et al. identified a lead molecule as a potent hCA II inhibitor by virtual
screening protocol. Identified dihydroxy phenyl derivative 35 was evaluated for its CA inhibitory action against
hCA I and hCA II (Figure 7). This molecule showed effective inhibitory potential against hCA II and displayed
45‐fold selectively over hCA I. Molecular docking study of this compound with CA II revealed that it nicely fitted in
the active site and well interacted with the crucial amino acid in the active site. Thus, this compound may be
utilized as a suitable lead for further development of potent and selective hCA II inhibitor.
210
Some phenolic
compounds were also tested against hCA I, hCA II, hCA VI and dCA. The inhibitory profile of these molecules
revealed that they showed micromolar range inhibitory action for all tested isoforms. 3, 4‐Dihydroxybenzoic
acid 36 appeared to be the most active one against hCA II and displayed a K
i
value of 0.47 µM. It showed
2‐,10‐, and 7‐fold selective over hCA I, hCA VI, and dCA (Figure 7).
211
Phenol derivatives were tested as CAIs by Durdagi et al. In this report, hydroxy/methoxy‐substituted benzoic
acids, as well as di/tri‐methoxy benzenes, were submicromolar to low micromolar range hCA inhibitors. Most of the
derivatives were nonselective inhibitors for all four isoforms except 3‐(2‐hydroxyphenyl) acrylic acid (compound
37). Compound 37 had a K
i
value of 9.2 µM and it was >1000‐fold selective over hCA IX and hCA XII. However, CA
II isoform was effectively inhibited by 1,2‐dimethoxybenzene 38 with a K
i
value of 0.50 µM. Compound 38 showed
to be 20.8‐,17‐, and 16‐fold selective over hCA I, hCA IX, and hCA XII, respectively (Figure 7).
212
Interestingly,
Carta et al. synthesized a series of dithiocarbamates (DTCs) and tested against hCA I, II, IX, and XII. Most of the
derivatives showed mixed type inhibitory action for hCA I, II, IX, and XII. However, some of the derivatives
displayed excellent inhibition profile against hCA II with sub‐nanomolar range inhibition constant. DTCs bearing
benzyl 39 and iso‐butyl 40 had sub‐nanomolar range inhibitory action (Figure 7). Compound 40 was an equipotent
inhibitor for hCA I as well as hCA II. Noticeably, benzyl derivative 39 appeared to be a most potent hCA II inhibitor
and also displayed 5.8‐, 27.4‐, and 16.4‐fold selectivity over hCA I, hCA IX, and hCA XII. Thus, these DTCs
represent a new category of potent CAIs and may be explored to generate various selective CAIs in the future. The
CS
2
−
moiety of DTCs presents a novel zinc‐binding function and it is directly bound to the Zn (II) ion in the enzyme
active site. It also contributes to an interaction with the OH moiety of Thr199, which is taken as an essential for the
binding of numerous classes of CAIs.
213
hCA inhibition profile of tosylated aromatic amine derivatives was in-
vestigated against hCA I, II and VI. Most of the derivatives have shown potent and selective inhibition toward hCA
II in the micromolar range (K
i
s = 0.27–14.3 µM). Pyrimidine containing benzenesulfonamide derivative 41 and
pyridine bearing derivative 42 inhibited hCA II strongly with a K
i
value of 0.34 and 0.27 µM, respectively (Figure 7).
Compound 41 was 76‐and 34‐fold more potent for hCA II as compared with hCA I and hCA VI, respectively.
Additionally, Compound 42 has exhibited 102‐and 56‐fold selectivity over hCA I and hCA VI. Thus, these findings
point out that these two potent derivatives may be used as potent lead compounds for further development of
more potent and selective hCA II inhibitors.
214
A small series of tricyclic sulfonamides was reported by Marini et al.
as potent and selective hCA II inhibitors. Designing of these compounds was carried out by inspiring two well‐
known COX inhibitors celecoxib and valdecoxib which also inhibit hCAs effectively. In this study, zinc‐binding
moiety benzenesulfonamide was connected to a pyrazole moiety to explore their activity and isoform selectivity.
Inhibition experiment with these compounds revealed that tricyclic sulfonamides 43–47 have shown the low
nanomolar range inhibitory action against hCA II along with satisfactory selectivity over 11 hCAs and three
MISHRA ET AL.
|
19
mycobacterial β‐CAs (Figure 7). Compounds 43, 44, 45, 46, and 47 showed a K
i
value of 16, 29, 210, 72, and 49 nM,
respectively, against hCA II. Thusly, this category of compounds constitutes highly interesting CAIs which, selec-
tively inhibit hCA II among the 12 catalytically active CA isoforms.
215
A new series of sulfamides incorporating
dopamine scaffold has been designed and synthesized to evaluate their inhibitory potential against hCAs.
These dopamine‐based sulfonamide derivatives displayed low nanomolar range inhibitory activity for hCA II
and micromolar range activity for other isoforms such as hCA I, hCA VA, hCA IX, hCA XII, and hCA XIV.
These derivatives 48–52 had shown effective inhibitory activity for hCA II with a K
i
in the range of 1.47–2.95 nM
and also shown satisfactory selectivity over the above said isoforms (Figure 7). Therefore, these dopamine
derivatives denote a novel type of selective hCA II inhibitor, which may be explored further to investigate their
clinical relevance.
216
FIGURE 7 Chemical structure of compounds 35–54 showing selective hCA II inhibition. hCA, human carbonic
anhydrase
20
|
MISHRA ET AL.
Continuing this study line, 2‐thiophene‐sulfonamides containing 1‐substituted aryl‐1,2,3‐triazolyl moieties were also
developed as potent and selective hCA II inhibitors. These compounds were tested for their inhibition profile against hCA
I, hCA II, hCA IX as well as hCA XII. Most of the derivatives inhibited hCA II very effectively by showing the low
nanomolar range K
i
value. For example, a compound containing trifluoromethyl benzene 53 and trifluoromethyl benzo-
nitrile 54 have displayed selective inhibition toward hCA II (Figure 7). Compound 53 had shown a K
i
value of 2.9 nM and
exhibited 1053‐,307‐,and37‐fold selectivity over hCA I, hCA IX, and hCA XII isoform, respectively. Although, compound
54 showed a K
i
value of 2.2 nM along with 3429‐,368‐,and108‐fold selectivity over hCA I, hCA IX, and hCA XII. Thus,
this investigation provided some potent and selective hCA II inhibitors as lead molecules. These obtained data may be
utilized to achieve more potent hCA II inhibitors, with potential use as diuretics or as anti‐glaucoma agents.
217
A series of 7‐substituted sulfocoumarins and 3,4‐dihydrosulfocoumarins were developed as highly selective
hCA II inhibitors. Contrasting to 6‐substituted sulfocoumarins which appeared to be potent hCA IX and XII
inhibitors and ineffective hCA I and II inhibitors, these sulfocoumarins derivatives showed low nanomolar range
hCA II inhibitory properties. All synthesized compounds of this series were ineffective toward hCA I, hCA IX, and
hCA XII isoforms. These derivatives (compounds 55–70) effectively inhibited hCA II by exhibiting low nanomolar
TABLE 2 hCAs inhibitory action of sulfocoumarins and their derivatives (55–70)
218
K
i
(nM)
Compound R hCA I hCA II hCA VA hCA IX hCA XII
55 >10 000 2.3 591 >10 000 >10 000
56 H >10 000 1.6 5100 >10 000 >10 000
57 >10 000 2 2150 >10 000 >10 000
59 H >10 000 7.6 327 >10 000 >10 000
59 >10 000 1.8 517 >10 000 >10 000
60 >10 000 2.4 91 >10 000 >10 000
61 >10 000 1.5 1675 >10 000 >10 000
62 >10 000 2.2 206 >10 000 >10 000
63 >10 000 3.4 141 >10 000 >10 000
64 >10 000 2.5 835 >10 000 >10 000
65 >10 000 2.1 5090 >10 000 >10 000
66 H >10 000 8.4 788 >10 000 >10 000
67 >10 000 1.5 9960 >10 000 >10 000
68 >10 000 2.2 716 >10 000 >10 000
69 >10 000 2.6 3910 >10 000 >10 000
70 >10 000 1.9 7060 >10 000 >10 000
Abbreviations: CA, carbonic anhydrase; hCA, human carbonic anhydrase.
MISHRA ET AL.
|
21
range inhibition constant (K
i
s = 1.5–8.4 nM). These compounds displayed medium to high nanomolar range in-
hibition constant (K
i
s=64–9960 nM) against hCA VA (Table 2). Hence, these sulfocoumarins derivatives emerged
as highly selective hCA II inhibitor with effective inhibitory action against this isoform. These sulfocoumarins may
serve as potent lead molecules to develop more potent and selective hCA II inhibitors in the future. These hCA II
isoform‐selective inhibitors may be also considered of interest for numerous biomedical applications.
218
Carta et al. synthesized xanthates (RO‐CS
2
−
M
+
) and thioxanthates (organic trithiocarbonates, RS‐CS
2
−
M
+
)
compounds to investigate their hCAs inhibitory action. These compounds are considered as structurally related to
well‐studied class DTCs. Synthesized compounds were screened against hCA I, hCA II, and cancer associated
isoform hCA IX as well as hCA XII. Compound containing phenyl ethyl (compound 71), Adamantyl ethyl (compound
FIGURE 8 Chemical structure of compounds 71–86 displaying selective hCA II inhibition. hCA, human carbonic
anhydrase
22
|
MISHRA ET AL.
72) and tri‐cyclodec‐9‐yl (compound 73) moiety displayed effective hCA II inhibitory action and showed a K
i
value
of 5.4, 6.4, and 6.6 nM, respectively (Figure 8). These derivatives also showed reasonable selectivity over hCA I,
hCA IX, and hCA XII isoforms. The molecular docking study revealed that these xanthates coordinate mono‐
dentately to the Zn (II) in the active site of the enzyme similar to DTCs for which the X‐ray crystal structure was
reported previously. The organic moiety of xanthates showed extended conformation within the active site similar
to DTCs. So, these xanthates appeared to be potent and selective hCA II inhibitors which have shown similar
binding mode as DTCs and some of them have also displayed effective anti‐glaucoma activity in vivo.
219
A small
series of ureido substituted benzenesulfonamide bearing a GABA moiety has been designed and synthesized.
These hybrid molecules 74–79 were tested against thirteen isoforms of hCA and results showed that they
effectively as well as selectively inhibited hCA II (Figure 8). All hybrids displayed low nanomolar range inhibitory
action and showed a range of K
i
value 4.9–41 nM. SAR investigation indicated that substitution on ureido moiety
did not much influence hCA II inhibitory action. It was also noticed that these derivatives were completely inactive
toward hCA III. So, these GABA conjugated benzenesulfonamide derivatives appeared to be a novel class of CAIs,
which have shown admirable inhibitory action as well as selectivity toward hCA II.
220
SitaRam et al. reported a series of 6‐sulfamoyl‐benzothiazoles containing pyrazole moiety as a potent hCA inhibitors.
Some of the derivatives displayed potent inhibitory action toward hCA II and low nanomolar range K
i
values were
observed. The derivative containing COOH group along with 4‐chlorophenyl substitution on the pyrazole ring (compound
80) showed effective and selective inhibition against hCA II. This derivative showed a K
i
value of 2.7 nM and exhibited
255‐, 311‐,and24‐fold selectivity over hCA I, hCA IX, and hCA XII. Also, compound bearing formyl group as well as
4‐fluoro phenyl substituted pyrazole (compound 81) ring effectively inhibited hCA II (K
i
=10.4nM)andappearedtobe
1508‐,98‐, and >961‐fold selective over hCA I, hCA IX, and hCA XII. Thus, this report provided some highly selective and
effective hCA II inhibitors which may be considered as a suitable lead molecule for the development of more potent and
selective hCA II inhibitors (Figure 8).
221
Aseriesof4and5nitro‐1,3‐dioxoisoindolin‐2‐yl benzenesulfonamide derivatives
were synthesized to evaluate their inhibitory potential against hCAs. The result of this report indicated that some of 1,
3‐dioxoisoindolin‐benzenesulfonamide derivatives strongly inhibited hCA II by showing satisfactory selectivity over
other isoforms. Among all derivatives, compound 4‐(5‐nitro‐1,3‐dioxoisoindolin‐2yl)benzenesulfonamide 82,3‐(4‐nitro‐
1,3‐dioxoisoindolin‐2yl)benzenesulfonamide 83,and2‐(4‐nitro‐1,3‐dioxoisoindolin‐2yl)benzenesulfonamide 84 displayed
promising inhibitory action as well as selectivity for hCA II isoform (Figure 8). These compounds inhibited hCA II with low
nanomolar range inhibitory potential (K
i
s=1.7–4.3 nM) and also showed remarkable selectivity over hCA I, hCA IX, and
hCA XII isoforms.
222
Benzenesulfonamides incorporating aroylhydrazone, sulfone, piperidinyl, [1,2,4]triazolo[3,4‐b][1,3,4]
thiadiazinyl‐or 2‐(cyanophenyl‐methylene)‐1,3,4‐thiadiazol‐3(2H)‐yl moieties were also explored as inhibitors of hCA I,
hCA II, hCA IX and hCA XII. Inhibition data evoked that all derivatives excellently inhibited physiologically dominant
isoform hCA II. Indeed, derivatives possessing thienyl moiety (compounds 85 and 86) as tail demonstrated selective
inhibition againsthCAII(Figure8). Compound 85 had a K
i
value of 0.94 nM, while compound 86 showed a K
i
value of
0.89 nM, which indicates their strong inhibitory action against hCA II isoform. In terms of selectivity, compound 85
showed 206‐,376‐, and 220‐fold selectivity over hCA I, hCA IX, and hCA XII, respectively. Even though, another
derivative 86 demonstrated 102‐, 621‐, and 422‐fold selectivity over hCA I, hCA IX, and hCA XII, respectively
(Figure 8).
223
Bozdag et al. synthesized various sulfonamides bearing bulky tails such as benzamide, quinazoline and
4‐oxoquinazoline to investigate their hCA inhibitory action. Results indicated that most of the derivatives very effectively
inhibited hCA II with sub‐nanomolar to low nanomolar range inhibitory potential. SAR study indicated that compounds
containing quinazoline tail displayed excellent inhibitory activity as compared with others toward hCA II. Indeed,
4‐(quinazolin‐4‐ylamino)benzenesulfonamide 87 and 3‐(quinazolin‐4‐ylamino)benzenesulfonamide 88 appeared to be the
most potent inhibitors for hCA II, showed a K
i
value of 0.06 and 0.08 nM, respectively (Figure 9). Additionally, compound
87 showed 158‐, 135‐,21‐fold selectivity over hCA I, hCA IX, and hCA XII, respectively. Interestingly, compound 88 has
also shown 693‐, 1900‐,and12‐fold selectivity over hCA I, hCA IX, and hCA XII. Hence, quinazoline tail played a crucial
role to generate very effective as well as selective hCA inhibitors and these compounds may be used in various
biomedical applications associated with selective hCA II inhibition.
224
MISHRA ET AL.
|
23
Benzenesulfonamides incorporating various substituted (hetero)aryl rings in the para‐position were synthe-
sized and their CA inhibitory action was assessed. The obtained result showed that most of the derivatives
effectively inhibited hCA II, although they were not very selective hCA II inhibitors. However, benzensulfonamide
coupled with p‐cyanophenyl (compound 89) demonstrated selective inhibition toward hCA II, along with low
nanomolar range inhibition (Figure 9). This compound has displayed a K
i
value of 1.2 nM and exhibited 112‐,
190‐, and 11.8‐fold selectivity over hCA I, hCA IX, and hCA XII.
225
Ombouma et al. developed a series of gly-
coinhibitor against hCAs and tested their efficacy against hCA I, hCA II, hCA IX, and hCA XII. Majority of deri-
vatives appeared to be an effective inhibitor of isoform hCA II and displayed K
i
in the range of 1–12 nM. Some
derivatives such as compounds 90 and 91 selectively inhibited hCA II (Figure 9). Compound 90 which had a K
i
value
of 1 nM was 300, 77, and 556 times more potent inhibitor for hCA II as compared with hCA I, hCA IX, and XII,
FIGURE 9 Chemical structure of compounds 87–102 showing hCA II‐selective inhibitory action. hCA,
human carbonic anhydrase
24
|
MISHRA ET AL.
respectively. Derivative 91 displayed 30‐,30‐, and 13‐fold selectivity over hCA I, hCA IX, and hCA XII, along with a
K
i
value of 3 nM.
226
X‐ray crystallography inspired the development of potent and selective hCA II inhibitors have
been also developed. In due course, authors have synthesized a series of 1‐N‐substituted 6‐sulfamoylsaccharins as
well as their hydrolyzed products. Inhibition study against hCAs of these derivatives revealed that hydrolyzed
products, containing free acid group displayed very effective and selective inhibition against hCA II as compared
with close compounds. SAR investigation indicated that compounds containing n‐propyl (compound 92), butyne
(compound 93), and 4‐bromobenzyl (compound 94) side chain showed promising inhibitory action against hCA II
and also appeared to be a selective inhibitor for hCA II (Figure 9). Compound 92 has shown a K
i
value of 0.2 nM
against hCA II and 407‐, 447‐, and 318‐fold selectivity over hCA I, hCA IX, and hCA XII, respectively. While,
compound 93 had displayed 178‐, 111‐, and 96‐fold selectivity over hCA I, hCA IX, and hCA XII, respectively with a
K
i
value of 0.7 nM. While, another compound 94 was looked 129‐, 235‐, and 90‐fold selective over hCA I, hCA IX,
and hCA XII along with showing a K
i
value of 0.3 nM for hCA II. The crystallographic experiment with hCA II
displayed that the R moiety of these compounds may espouse a variety of orientations within the active site of CA
II, which may enlighten their very high affinity and selectivity for this isoform.
227
Schiff bases of sulfanilamide
semicarbazone have been also synthesized and screened for their inhibitory action against hCA I, hCA II, hCA IX,
and hCA XII. The result showed that these Schiff bases displayed promising inhibition toward hCA II with K
i
s in the
range of 4.0–51.6 nM. Some of the derivatives displayed a medium range of selectivity for hCA II isoform. How-
ever, derivative containing 2‐chlorophenyl substitution (compound 95) showed 18‐,50‐, and 10‐fold selectivity
over hCA I, hCA IX, and hCA XII, respectively, along with a K
i
value 4.2 nM against hCA II (Figure 9).
228
A novel
class of CAIs was also designed and synthesized by appending dual tail groups on well‐known CAI AAZ like
compound 96. Additionally, few single tail containing AAZ derivatives (e.g., compound 97) were also synthesized to
compare inhibition property of dual tail derivatives versus single tail. Although these dual tail bearing derivatives
appeared to be less effective hCA II (K
i
s=83–201 nM) inhibitors as compared with AAZ (Figure 9). However, dual
tail containing AAZ derivative showed more selectivity over hCA I as compared with AAZ. Interestingly, single tail
containing derivatives have shown more effective hCA II inhibitory action as compared with AAZ.
229
Krasavin et al.
designed and synthesized oxazole‐based highly effective hCA II inhibitors and some of them have displayed
picomolar range inhibitory action. It was noticed that oxazole derivatives (compound 98) which displayed pico-
molar range inhibitory action against hCA II also very effectively inhibited hCA I as well as hCA IX. Compound 98
displayed a K
i
value 0.01, 0.008, 1.6, and 6.9 nM against hCA I, hCA II, hCA IX, and hCA XII, respectively. However,
some of the oxazole derivatives, possessing methoxy substituted benzenesulfonamide at one end and pyrrolidine
(compound 99) as well as morpholine (compound 100) at other end bestowed effective and selective hCA II
inhibitors (Figure 9). Compound 99 very effectively inhibited hCA II with a K
i
value of 0.01 nM and exhibited
>1 000 000‐, 410‐, and 857 000‐fold selectivity over hCA I, hCA IX, hCA XII, respectively. Whereas, compound 100
also strongly inhibited hCA II by showing a K
i
value of 0.05 nM and demonstrated >2 000 000‐,90‐, and
>2 000 000‐fold selectivity over hCA I, hCA IX, and hCA XII. Hence, these oxazole derivatives provided highly
potent and selective hCA inhibitors and these compounds may appear useful in various biomedical application.
230
Congiu et al. synthesized a series of benzenesulfonamides incorporating ureido moieties as potent hCA II in-
hibitors. The novel compounds, consisting 4‐N‐substituted piperazine fragment tethered with benzenesulfonamide
by ureido linker were examined as inhibitors of hCA I, hCA II, hCA IX, and hCA XII. Inhibition data revealed that the
majority of synthesized compounds inhibited hCA II more effectively as compared with other isoforms such as hCA
I, hCA IX, and hCA XII. Certainly, di‐chlorophenyl (compound 101) and pyridine (compound 102) substituted
benzenesulfonamide derivatives displayed satisfactory selectivity along with effective inhibition toward hCA II
(Figure 9). Compound 101 showed a K
i
value of 5.4 nM and found 1860‐,49‐, and 23‐fold selectivity over hCA I,
hCA IX, and hCA XII. Although, pyridine containing compound 102 displayed sub‐nanomolar range inhibitory action
(K
i
= 0.75 nM) against hCA II and exhibited 35‐, 649‐, and 192‐fold selectivity over hCA I, hCA IX, and hCA XII.
231
Benzensulfonamide containing 8‐chloro‐2‐mercapto‐4‐oxoquinazoline 103 has been also found an effective
hCA II inhibitor with sub‐nanomolar range K
i
value (Figure 10). This novel derivative has exhibited a K
i
value of
MISHRA ET AL.
|
25
0.31 nM with 261‐, 141‐, and 17‐fold selectivity over hCA I, hCA IX, and hCA XII, respectively.
232
Thiazole
derivative 104 which incorporates benzenesulfonamide moieties at both ends has also emerged as a potent hCA II
inhibitor and this compound has shown selective inhibition for hCA II over hCA I as well as hCA IX (Figure 10). This
compound had a K
i
value of 0.41 nM and showed 15.8‐as well as 17.8‐fold selectivity over hCA I and hCA
IX, respectively.
233
A series of benzenesulfonamides linked with aromatic tail by urea linker was synthesized and
tested for their inhibitory potential against four isoforms (hCA I, hCA II, hCA VII, and hCA XII). Most of the
derivatives were not effective against hCA II, however, some of the derivatives like compound 105 (Figure 10)
displayed medium micromolar range (K
i
= 87.8 µM) activity against hCA II, whereas >100 µM inhibition constant
(K
i
) was observed against hCA I, hCA VII as well as hCA XII.
234
Ulus et al. disclosed a series of acridine–AAZ
conjugates which have shown potent and selective inhibition toward hCA II. All investigated isoforms such as hCA
I, hCA II, hCA VII, and hCA IV were inhibited in the low micromolar and the nanomolar range by these conjugates.
However, hCA II was effectively inhibited by most of the derivatives in the low nanomolar range and also selective
FIGURE 10 Chemical structure of selective hCA II inhibitors 103–115. hCA, human carbonic anhydrase
26
|
MISHRA ET AL.
inhibition toward hCA II was also detected. These derivatives inhibited hCA I, hCA II, hCA IV, and hCA VII with K
i
s
in the range of 6.7–335.2, 0.5–55.4, 29.7–708.8, and 1.3–90.7 nM, respectively. SAR study specified that con-
jugates containing 4‐cyanophenyl (compound 106) and 4‐chlorophenyl (compound 107) showed selective inhibition
toward hCA II as compared with other derivatives (Figure 10). Compound 106 has shown 20.8‐, 208‐, and 26.6‐fold
selectivity for hCA II over hCA I, hCA IV, and hCA VII, respectively, along with a K
i
value of 3.4 nM against hCA II.
Additionally, Compound 107 promisingly inhibited hCA II with a K
i
value 0.5 nM and displayed 57‐, 262‐, and
21‐fold selectivity over hCA I, hCA IV, and hCA VII.
235
Some N‐glycosylsulfonamides possessing the phenol
derivative were synthesized and investigated as hCAs inhibitors. However, these derivatives were less effective
hCA II inhibitors as compared with standard inhibitor AAZ. Although, these derivatives selectively inhibited hCA II
and they were more selective as compared with AAZ for hCA II. In this small series, compound 108 displayed a fair
selectivity for hCA II, showing a K
i
value of 137 nM (Figure 10). Likewise, this compound showed a K
i
value of
3840, >50 000, and >50 000 nM against hCA I, hCA IX, and hCA XII.
236
Benzenesulfonamides derivatives, holding
the substituted piperazine tail as well as urea liker also emerged as potent and selective hCA II inhibitors. hCA II
isoform was very effectively inhibited by these sulfonamide derivatives with a K
i
in the range of 0.69–50.7 nM.
Whereas, these derivatives displayed a K
i
ranges of 83.8 to >50 000 and 58.5 to >50 000 nM against hCA I as well
hCA VII, respectively. However, these derivatives were completely inactive against cancer associated hCA XII,
showing K
i
values > 50 000 against this isoform. SAR studies designated derivatives possessing phenyl (compound
109) as well as diphenyl piperazine (compound 110) tails were most selective hCA II inhibitor in the entire series,
being effective inhibitor for hCA II with low nanomolar range inhibitory potential (Figure 10). Compound 109 has
shown a K
i
value of 0.6 nM against hCA II and appeared to be 5057‐fold selective over hCA I and >83 333‐fold
selective over hCA VII as well as hCA XII. Yet, another derivative 110 displayed a K
i
value of 8.7 nM toward hCA II
along with 379‐fold selectivity over hCA I and >5747‐fold selectivity over hCA VII as well as hCA IX. Hence, both
derivatives, specially, 109 emerged as highly selective and potent hCA II inhibitors which may prove as a
suitable lead molecule to generate more potent and selective hCA II inhibitors.
237
Carta et al. reported design and
synthesis of pritelivir (antiviral agent) related compounds and evaluated for their CA Inhibitory action against six
physiologically and pharmacologically relevant hCA (EC 4.2.1.1) isoforms. Pritelivir, N‐[5‐(aminosulfonyl)‐4‐methyl‐
1,3‐thiazol‐2‐yl]‐N‐methyl‐2‐[4‐(2‐pyridinyl)phenyl]acetamide, is a helicase‐primase inhibitor used for the treat-
ment of herpes simplex virus infections. The result of this study indicated that most of the derivatives along with
pritelivir effectively inhibited hCA II in the nanomolar range. Compound containing ethyl substitution on thiazole
ring along with pyridine moiety (compound 111) showed auspicious inhibitory action against hCA II with a K
i
value 1.0 nM (Figure 10). This compound also showed 48‐,60‐,23‐,28‐and 48‐fold selective over hCA I, hCA VA,
hCA VB, hCA IX, and hCA XII.
238
hCA II was effectively and selectively inhibited by carbamates derivatives of
4‐aminobenzenesulfonamide and branched VPA derivatives. These carbamates inhibited hCA II with low nano-
molar range inhibitory potential and displayed a K
i
in the range of 0.6–19.6 nM. However, these compounds
showed a K
i
ranges of 5.9–484.7, 182.6 to >10 000, and 24.5 to >10 000 nM for hCA I, hCA IV, and hCA VII
isoform. Indeed, in the entire series, 2‐ethylhexyl (4‐sulfamoylphenyl)carbamate 112 was the most selective hCA II
inhibitor over hCA I, hCA IV, and hCA VII (Figure 10). This compound possessed 48‐fold selectivity over hCA I and
>1149‐fold selectivity over hCA IV as well as hCA VII. Moreover, these carbamates exhibited effective antic-
onvulsant activity and also appear non‐teratogenic as well as safer AEDs.
239
Angapelly et al. synthesized various
novel sulfonamides incorporating phenylacrylamido functionality and studied their inhibitory action against four
hCA isoforms; hCA I, hCA II, hCA IX as well as hCA XII. Inhibition study of these compounds revealed that most of
derivatives effectively inhibited hCA II, hCA IX and hCA XII. However, some derivatives like 113 which contains
3,4‐dimethoxy phenyl showed effective as well as selective inhibition toward hCA II (Figure 10). This derivative
showed an effective K
i
value of 0.5 nM for hCA II as well as 421‐,18‐, and 17.8‐fold selective over hCA I, hCA IX,
and hCA XII.
240
A similar trend was also observed with curcumin fragment containing sulfonamide derivatives.
These derivatives showed effective inhibition toward hCA II as well as hCA IX. These compounds were found more
selective over hCA I as compared with other isoforms tested. Although some compounds displayed medium‐range
MISHRA ET AL.
|
27
selective inhibition for hCA II. Curcumin derivative containing 2,5 dimethoxy 114 as well as 3,4 dimethoxy moiety
115 exhibited somewhat better selectivity for hCA II as compared with other derivatives (Figure 10). Both deri-
vatives also demonstrated sub‐nanomolar range inhibitory action against hCA II, compound 114 showed a K
i
value
of 0.89 nM, while compound 115 have shown a K
i
value of 0.75 nM for hCA II.
241
Various aromatic primary
sulfonamides possessing diversely substituted 1,2,4‐oxadiazole periphery groups have been also explored as
potent and selective hCA II inhibitors. These compounds inhibited hCA II in the sub‐nanomolar to the low
nanomolar range, while hCA IX was inhibited with low nanomolar range inhibitory action. Some derivatives with
fair selectivity toward hCA II were also noticed. For example, compound containing 2‐chlorophenyl substituted
oxadiazole 116 (Figure 11) illustrated 1306‐, 11 675‐, and 23‐fold selectivity toward hCA II over hCA I, hCA IV, and
hCA IX, respectively, along with a K
i
value of 0.48 nM against hCA II.
242
FIGURE 11 Chemical structure of compounds 116–131 representing selective hCA II inhibition. hCA,
human carbonic anhydrase
28
|
MISHRA ET AL.
Żołnowska et al. synthesized a series of 2‐arylmethylthio‐4‐chloro‐5‐methylbenzenesulfonyl)‐1‐(6‐substituted‐
4‐chloro‐1,3,5‐triazin‐2‐ylamino)guanidine and explored as hCAs inhibitors against hCA I, hCA II, hCA IX and
hCA XII. SAR study revealed that 3‐fluoromethylphenyl substitution bestowed some potent and selective hCA II
inhibitors. Derivatives bearing 3‐fluoromethylphenyl along with 4‐(H
2
NSO
2
)C
6
H
4
CH
2
(compound 117)or
3‐(H
2
NSO
2
)C
6
H
4
(compound 118) were the most selective hCA II inhibitors within the series, being effective hCA
II inhibitors (Figure 11). Compounds 117 and 118 had a K
i
value of 5.4 and 9.3 nM, respectively against hCA II.
Compound 117 displayed 40‐,8‐, and 47‐fold selectivity over hCA I, hCA IX and hCA XII, respectively. Noticeably,
compound 118 has shown 36‐, 359‐, and 24‐fold selective over hCA I, hCA IX, and hCA XII, respectively.
243
Recently, a series of iminothiazolidinone‐sulfonamide hybrids have been synthesized and evaluated for their
inhibitory action against hCAs. Most of the derivatives have shown effective inhibition against hCA II along with
fair selectivity over hCA I, hCA IX, and hCA XII. These derivatives have shown a K
i
in the range of 67.2–664.4 nM
against hCA I, 0.41–37.8 nM against hCA II, 140.1–5887.9 nM against hCA IV and 24.3–368.6 nM against hCA IX.
Thus, these inhibitors selectively inhibited hCA II in the sub‐nanomolar to the low nanomolar range. Additionally,
the off‐target hCA I, and the membrane‐bound hCA IV were weakly inhibited by these hybrid compounds. It was
observed that iminothiazolidinone‐sulfonamide hybrid with 3‐fluoro phenyl substitution (compound 119) provided
more selective hCA II inhibitor as compared with other substitutions (Figure 11). Compound 119 has effectively
inhibited hCA II isoform with a K
i
of value 0.46 nM and showed 161‐, 3680‐as well as 84‐fold selectivity over hCA
I, hCA IV, and hCA IX.
244
Akocak et al. reported a series of N,N'‐diaryl cyanoguanidines which have shown
promising inhibitory action as well as selectivity for hCA II. The authors used guanidine functionality for bioi-
sosteric replacement of urea or thiourea which is present in many potent hCA inhibitors including SLC‐0111. This
replacement strategy conferred numerous potent and selective hCA inhibitors, which may be used for various
biomedical applications where selective hCA II inhibition will be needed. These compounds inhibited hCA II with
low nanomolar range inhibition constant and displayed high selectivity over hCA I, hCA IV, and hCA IX. Cyano-
guanidines derivatives have shown of K
i
s values in the range of 2.8–25.5 nM against hCA II and of 50.5–442.2 nM
against hCA I. hCA IV as well as hCA IX were poorly inhibited by these derivatives, which demonstrated K
i
sof
414–7649.5 nM for hCA IV and of 965.5 to >10 000 nM for hCA IX. Although, all derivatives displayed potent and
selective hCA II inhibition, only the compound containing benzoic acid as tail moiety 120 (Figure 11) showed
promising inhibitory action as well as selectivity against hCA II. Compound 120 exhibited a K
i
value of 3.2 nM for
hCA II and 27.7‐, 1496‐, and >3125‐fold selectivity over hCA I, hCA IV, and hCA IX, respectively.
245
Furthermore,
the same research group developed 1,3‐diaryltriazene‐substituted sulfonamides as potent and selective hCA II
inhibitors. In this study, authors replaced ureido linker of SLC‐0111 (potent hCA inhibitor) with open triazene
linker which leads to various 1,3‐diaryltriazene‐substituted sulfonamide derivatives. It was thought that the in-
troduction of the triazene linker provides more flexible molecules as compared with SLC‐0111. The inhibition
study demonstrated that synthesized derivatives effectively inhibited hCA II and also emerged as a selective hCA II
inhibitors. hCA II was inhibited by these compounds in the sub‐nanomolar range to the low nanomolar range and
displayed K
i
in the range of 0.2–21.5 nM. These compounds moderately inhibited hCA I and weakly inhibited hCA
IX. Thus, triazine linker between benzenesulfonamide head and aromatic tail have donated potent and selective
hCA II inhibitors such as 121, 122 ,and 123 which contain 4‐butoxy, 4‐methoxy and 4,5 dimethylphenyl as tails
respectively (Figure 11). Compounds 121,122, and 123 showed selectivity in the range of 44–1298‐,23–336‐,
57–10693‐fold, respectively, for hCA II over hCA I, hCA VII, and hCA IX
246
correspondingly. Nocentini et al.
prepared a small series of aromatic sulfamates as potent and selective hCA II inhibitors. The inhibitors were
designed by considering selective COX‐2 inhibitors celecoxib and valdecoxib as lead compounds which have also
shown effective inhibition against hCAs. Synthesized derivatives were very weak inhibitors for hCA I and hCA IV as
well as hCA IX. However, these sulfamates appeared to be effective as well as selective inhibitors for hCA II such as
compounds 124 and 125 (Figure 11). Compound 124 inhibited hCA II with a K
i
value of 0.8 nM and exhibited
3427‐,30‐, and 36‐fold selectivity for hCA II over hCA I, hCA IV, and hCA IX, respectively. Whereas, compound
125 showed a K
i
value of 0.4 nM as well as 7857‐,51‐, and 62.5‐fold selectivity over hCA I, hCA IV, and hCA
MISHRA ET AL.
|
29
IX, respectively.
247
Two series of novel benzenesulfonamide derivatives as a potent hCAs inhibitors have been
developed which contain N‐methylacetamide as well as N‐methylpropanamide linker to tie aromatic tail with the
zinc‐binding domain. Synthesized derivatives were tested for their inhibitory action against hCA I, hCA II, hCA VII,
and hCA IX. Most of the derivatives displayed effective inhibition against hCA II as compared with other isoforms
hCA I, hCA VII, and hCA IX. It was also detected that some derivatives such as compounds 126 and 127 (Figure 11)
which contain piperonylpiperazine tail with N‐methylacetamide and N‐methylpropanamide linker, respectively,
were effective hCA II inhibitors. Compounds 126 and 127 have inhibited hCA II with nanomolar range inhibitory
action by exhibiting a K
i
value of 75.5 and 33.2 nM, respectively. Compound 126 has shown 5‐,7‐, and 21‐fold
selectivity for hCA II over hCA I, hCA VII, and hCA IX, respectively, while derivative 127 was 24‐,10‐, and 3‐fold
selective for hCA II over hCA I, hCA VII, and hCA IX, respectively.
248
Novel hybrid compounds consisting of
nonsteroidal‐anti‐inflammatory drugs (NSAIDs) and CAI fragment benzenesulfonamide type have been also tested
against five hCA isoforms, hCA I, hCA II, hCA IV, hCA IX, and hCA XII. Results of inhibition study indicated that
most of the compounds displayed a highly potent inhibition against all five isoforms. Few derivatives showed
moderate selectivity for hCA II along with effective inhibition, such as compounds 128 and 129 which incorporate
flurbiprofen and indomethacin moieties, respectively with a propenamide linker (Figure 11). Hybrid 128 inhibited
hCA II with a K
i
value of 7.0 nM, being 33‐,50‐,35‐,10‐fold selective over hCA I, hCA IV, hCA IX, and hCA
XII, respectively. Indomethacin containing hybrid 129 showed a K
i
value of 4.2 nM for hCA II along with 89‐, 380‐,
42‐,10‐fold selectivity over hCA I, hCA IV, hCA IX, and hCA XII, respectively.
249
[1,4]oxazepine‐based benzenesulfonamide derivatives were also identified as potent hCA II inhibitors. These novel
derivatives were prepared by reacting 4‐chloro‐3‐nitrobenzenesulfonamide with a range of bis‐electrophilic phenols
containing an NH‐acidic functionality at ortho‐position. Synthesized analogous promisingly inhibited hCA II with K
i
sin
the range of 0.52–29.5 nM. Isoforms hCA IV as well as hCA IX were moderately inhibited by most of the derivatives,
while hCA I was weakly inhibited. However, compounds having ethylmorpholine and methyl morpholine substitution on
11‐oxo‐10,11‐dihydrodibenzo[b,f] [1,4]oxazepane (compound 130) and dibenzo[b,f]pyrazolo[1,5‐d][1,4]oxazepane (com-
pound 131), respectively, demonstrated sub‐nanomolar range inhibitory potential (compound 130 showed a K
i
value of
0.59 nM and 131 showed a K
i
value 0.52 nM) along with fair selectivity for hCA II (Figure 11). Moreover, compound 130
was 4975‐,10‐,and403‐fold selective for hCA II over hCA I, hCA IV, and hCA IX, respectively. Indeed, analogue 131 was
found to be 7443‐,9‐, and 489‐fold selective for hCA II over hCA I, hCA IV, and hCA IX, respectively.
250
Recently, Angapelly et al. synthesized a series of sulfamoylphenyl derivatives, which have demonstrated very
selective inhibition for hCA II (Table 3). The synthesized derivatives 132–146 effectively inhibited hCA II and
showed K
i
s in the range of 5.2–92.1 nM, being less effective against other hCA isoforms. Sulfamoylphenyl deri-
vatives 134–137,141, and 144–146 inhibited hCA II very effectively, with K
i
s in the range of 5.1–8.6 nM, being
superior to AAZ (K
i
= 12.1 nM). Other compounds, such as 132,133,138,140,142, and 143 showed slightly
weaker activity than AAZ against hCA II. These analogous showed medium to high nanomolar range inhibitory
potential with K
i
ranges between 45.5 and 5997.1, 470–8756, and 191.3–4365 nM against hCA I, hCA IV, and hCA
IX, respectively. Thus, these sulfamoylphenyl derivatives have been developed as potent leads which further
structural modifications could be beneficial for the development of more effective hCA inhibitors used for the
treatment associated with selective hCA II inhibition (Table 3).
251
4.2 |CA IV‐selective inhibitors
The most distinctive feature of the active site of this enzyme is related to the presence of the four cysteine
residues, which requires building two disulfide bonds, located at the entrance inside the pit.
181
These residues
engross almost the same region of the active site as the histidine cluster found in hCA II.Additionally,iozyme
CA IV consists only one histidine residue His 64 inside active site similar to hCA II that plays a vital role in
catalysis.
187,188
It is well studied that hCA IV imparts a crucial role in many pathological conditions such as
30
|
MISHRA ET AL.
epilepsy, RA, renal disorders, as well as eye disorders.
186
Therefore, the development of selective hCA IV
inhibitors may appear very useful to manage these disorders without exerting severesideeffectsassociated
with other isoform inhibition. However, a very small number of highly selective hCA IV inhibitors have been
developed to date.
252–254
Chiaramonte et al. synthesized 2‐benzylpiperazine derivatives, bearing benzenesulfonamide as zinc‐binding
group, and evaluated their inhibitory performance against hCAs. Among all, some benzylpiperazine derivatives
such as 147 and 148, possessing benzyl and benzoyl moiety showed some selectivity for hCA IV isoform, although
most of the compounds of this series displayed effective hCA IV inhibition (Figure 12). 147 and 148 inhibited hCA
IV in the low nanomolar range and exhibited a K
i
value of 2.3 as well as 4 nM, respectively. Additionally, against this
isoform, compound 147 showed activity 2.0‐and 72.2‐fold higher than on II and IX, respectively. Similarly, 148
displayed 13‐fold selectivity for hCA IV over hCA I as well as hCA II and 414‐fold versus hCA IX.
252
Hybrid
molecules, consisting of 6‐and 7‐substituted coumarins (CAIs) and NSAIDs (indomethacin, ketoprofen, ibuprofen,
diclofenac, sulindac, and ketorolac, etc.) displayed effective and selective inhibition for hCA IV. These hybrids
inhibited hCA II with low nanomolar range K
i
and between 0.44 and 9.8 nM, being more potent than standard AAZ
(K
i
= 74.2 nM). Interestingly, these compounds were found less effective against hCA I, hCA II, and hCA VII and
displayed a K
i
value > 100 nM. However, hCA IX and hCA XII inhibited by most of the derivatives with a medium
nanomolar range K
i
s value. Hybrids such as 149–154 (Figure 12) were selective hCA IV inhibitors, which displayed
reasonable selectivity over other isoforms, especially over hCA I, hCA II, and hCA VII.
253
Some polyamines such as
TABLE 3 Inhibitory action of compounds 132–146 against hCAs isoforms
251
K
i
(nM)
Compounds R R
1
hCA I hCA II hCA IV hCA IX
132 H Morpholine 195.9 17.1 6453.2 1957.5
133 Hcis‐2,6 dimethylmorpholine 646.4 37.5 5006.2 1690.0
134 H Piperidine 88.0 8.6 4890.6 431.5
135 H Pyrrolidine 323.5 5.3 5051.0 3458.5
136 CH
3
Morpholine 82.7 8.3 5253.8 3096.0
137 CH
3
cis‐2,6‐dimethylmorpholine 280.2 6.3 4198.3 3553.5
138 CH
3
Piperidine 820.9 49.3 768.4 3361.0
139 CH
3
Pyrrolidine 943.6 92.1 3891.6 4314.0
140 CH
3
Thiomorpholine 421.4 13.5 3327.1 4092.5
141 Cl Morpholine 87.2 6.5 539.9 4024.5
142 Cl cis‐2,6 dimethylmorpholine 529.8 47.8 470.0 3561.0
143 Cl Pyrrolidine 645.8 23.8 538.7 4365.0
144 F Morpholine 677.5 7.9 840.2 3817.0
145 Fcis‐2,6 dimethylmorpholine 45.5 5.2 8072.3 296.
146 F Piperidine 434.2 6.3 8756.0 191.3
Abbreviation: hCA, human carbonic anhydrase.
MISHRA ET AL.
|
31
spermidine 155, hexamethyl trien 156,1‐naphthyl‐acetamido‐spermine 157, and CF
3
CO containing spermine
derivative 158 also displayed moderate selectivity for hCA IV over other 12 hCA isoforms (Figure 12). Although,
these polyamines showed micromolar range inhibition for hCA IV, but they were selective over other isoforms,
which made it suitable leads for further development. These compounds displayed K
i
s in the range of
0.018–0.11 µM against hCA IV isoforms.
254
Benzenesulfonamide derivatives, containing aryl, carboxylic acid, hydroxymethyl, carboxylic acid hydrazide,
and carboxamide substituted triazole ring showed effective inhibitory action against hCA IV. Most of the deri-
vatives displayed better inhibitory potential as compared with standard AAZ against hCA IV and displayed a K
i
s
ranging from 35.7 to 66.2 nM (AAZ showed a K
i
value of 74 nM). Nevertheless, some derivatives showed moderate
selectivity for hCA IV over hCA I, hCA II, and hCA IX. For example, triazoles substituted with carboxylic acid along
with 4‐chlorophenyl (compound 159), 4‐bromophenyl (compound 160), and 2‐thianyl (compound 161) showed
FIGURE 12 Chemical structure of selective hCA IV inhibitors 147–161. hCA, human carbonic anhydrase
32
|
MISHRA ET AL.
selective inhibition toward hCA IV (Figure 12). These compounds exhibited 8–71‐,6–12‐,16–22‐fold selectivity for
hCA IV versus hCA I, hCA II, and hCA IX respectively. Thus, these compounds also appeared to be suitable leads for
further development of more potent and selective hCA IV inhibitors.
255
4.3 |CA V‐selective inhibitors
Out of 16 mammalian CA isoforms, CA VA and VB are mitochondrial isoforms and they were shown to be involved in
numerous biosynthetic processes, such as ureagenesis, lipogenesis, and gluconeogenesis, in both vertebrates and
invertebrates.
23,24
Mitochondrial CAs are well studied for management of obesity, a serious medical problem associated
with life style.
23
The X‐ray structure of CA VA illustrated that the active site consists of three histidine residues His94,
His96, and His119 as well as a water molecule/hydroxide ion similar to other isoforms, which acts as a nucleophile in
the hydration of carbon dioxide to bicarbonate and a proton. hCA VB is widely distributed as compared with hCA VA,
signifying different physiological roles for hCA VA and hCA VB.
192
It was found that CA VB is catalytically more active
as compared with CA VA and that this isoform could be a drug target.
192
Sulfonamides and sulfamates are the most
effective CAIs studied up to now against all the CA isozymes, including CA VA and VB. Several research groups have
tried to develop potent and selective inhibitors for mitochondrial isoforms CA VA and CA VB. However, very few
inhibitors showed selective inhibition toward CA VA and CA VB over other isoforms.
256–258
Supuran's group tested 34
sulfonamides/sulfamates against these two mitochondrial isoforms. The 10 clinically used drugs such as AAZ, ethox-
zolamide, methazolamide, brinzolamide, dichlorphenamide, TPM, indisulam, dorzolamide, benzolamide, and sulpiride,
displayed effective hCA VB inhibitory action, with K
i
s in the range of 18–62 nM. Interestingly, these inhibitors have
shown better inhibitory potential against hCA VB as compared with hCA VA, however, most of the inhibitors did not
show selective inhibition over cytosolic isoform hCA II. Among them, sulpiride and ethoxzolamide have shown almost
two times more potent inhibitors for mitochondrial isoform over the cytosolic.
256
Aromatic and heterocyclic sulfona-
mides comprising R‐and S‐camphorsulfonyl moieties have unveiled effective and selective inhibition to-
ward mitochondrial isoforms. SAR investigation illustrated that mostly the R‐enantiomers have shown more effective
inhibitory action against hCA VA as compared with their S‐enantiomers. All synthesized derivatives 162–171 have
shown effective low nanomolar range inhibition against hCA VA as well as hCA VB (Figure 13). These compounds have
exhibited K
i
sintherangeof5.9–68.6 nM against hCA VA, while, K
i
sintherangeof7.3–67.3 nM were measured against
hCA VB for these compounds. Derivative 170 (Figure 13) was most active derivative showing a K
i
value of 5.9 nM
against hCA VA, 889‐and 300‐fold selectivity over hCA I and hCA II, respectively.
257
Apigenin 172 and eriocitrin 173
were identified as effective hCA VA inhibitors by a structure‐based virtual screening (Figure 13). In vitro inhibition
study divulged that Apigenin 172 and eriocitrin 173 have demonstrated low micromolar range inhibition against hCA
VA as these compounds exhibited a K
i
value of 0.3 and of 0.15 µM, respectively. Thus, these natural compounds may
serve as an excellent lead to develop more potent and selective hCA VA inhibitors.
258
4.4 |CA VII‐selective inhibitors
As discussed above, CA VII is widely expressed in several regions of the brain and validated as a promising target
for epilepsy.
136
Additionally, Parkkila's group recently observed elevated expression of this isoform in brain tumor
suggested CA VII as a valuable marker for brain tumor.
135
Development of highly selective hCA VII inhibitors is
highly desirable to avoid inhibition of other isoforms present in other parts of the body including CNS. Indeed, our
group is continuously putting effort to develop selective hCA VII and due course several classes of molecules have
been synthesized and tested against this isoform.
234,237,259–266
Villalba et al. reported N,N’‐disubstituted sulfamides and sodium cyclamate as very active and selective
inhibitors for hCA VII. These compounds offered sub‐nanomolar to low nanomolar range inhibition against hCA VII
MISHRA ET AL.
|
33
(K
i
s = 0.3–2.2 nM). Of interest, these derivatives were inactive against off‐target isoforms hCA I (K
i
s > 10 000 nM)
as well as hCA II (K
i
s 4957 to >10 000 nM). Isoforms hCA IX, hCA XII, and hCA IV were inhibited with low
nanomolar range to medium nanomolar range potency by these tested compounds. In this series, sulfamide 174
(Figure 14) showed promising selectivity for hCA VII, being a potent hCA VII inhibitor (K
i
= 0.39 nM). This com-
pound has shown >256 41‐, 25 641‐, 490‐,86‐, and 20‐fold selectivity for hCA VII over hCA I, hCA II, hCA IX, hCA
XII, and hCA XIV, respectively.
259
Mishra et al. have also designed and synthesized benzenesulfonamide‐piperazine
hybrid molecules as potent hCAs inhibitors and some of them like piperonyl containing benzenesulfonamide
(compound 175, Figure 14) indicated low nanomolar range inhibitory action (K
i
= 4 nM) against hCA VII, showing
promising selectivity over hCA I, hCA II, and hCA XII. This compound has shown a K
i
value of 8550, 36, and
>50 000 for hCA I, hCA II, and hCA XII isoforms, respectively.
237
Further, this lead has been modified by providing
more flexible linker between benzenesulfonamide and piperazine tail that bestowed some selective hCA VII
inhibitors like compound 176 (Figure 14) which has shown a K
i
Value of 27.1 nM for hCA VII. This derivative also
inhibited hCA I, hCA II, and hCA IX with a K
i
value of 93.3, 80.4, and 1627.9 nM, respectively.
248
Furthermore,
optimization is extended and recently reported benzenesulfonamides containing substituted piperazine tail as
effective as well as selective hCA VII inhibitors. Compounds having furoyl (compound 177) and dimethylphenyl
(compound 178) substituted piperazine tail showed low nanomolar range inhibitory action against hCA VII,
FIGURE 13 Chemical structure of compounds 162–173 displaying selective inhibition for hCA VA or hCA
VB. hCA, human carbonic anhydrase
34
|
MISHRA ET AL.
displaying a K
i
value of 6.2 and 17.8 nM, respectively (Figure 14). Compound 177 was 10.4‐, 4.9‐, and 288.4‐fold
selective for hCA VII over hCA I, hCA II, and hCA IX. While, 561.7‐, 527.2‐, and 93‐fold selectivity was measured
for compound 178 against hCA VII over hCA I, hCA II, and hCA IX.
260
(R,S)4‐(1‐aryl‐3,4‐dihydro‐1H‐isoquinolin‐2‐
carbonyl)‐benzenesulfonamides have been also examined as powerful and selective hCA VII inhibitors. Most of the
derivatives were sub‐nanomolar range or low nanomolar range inhibitors of hCA VII except derivative 179 (K
i
=
84.4 nM) and showed K
i
s in the range of 0.2–9.2 nM. Derivative 180 was the most the effective inhibitor of hCA VII
in the entire series and demonstrated a K
i
value of 0.2 nM for hCA VII (Figure 14). This derivative has also
presented a K
i
value of 9.3, 3.5, 36.9, 4.7, and 3.3 nM against hCA I, hCA II, hCA IX, hCA XII, and hCA XIV.
Crystallographic as well as docking studies indicated the key interactions of these inhibitors into the CA catalytic
site, signifying the relevant role of nonpolar contacts for this class of inhibitors.
261
A series of potent and selective
imidazoline‐based inhibitors of hCA VII was obtained by simply switching the position of the benzenesulfonamide
moiety from the N1 position (reported earlier, compound 181, Figure 14) to the C2 on the imidazole ring of the
scaffold. This modification bestowed highly selective hCA VII inhibitors, which demonstrated selectivity greater
than 100‐fold over off‐target isoforms hCA I, hCA II, and hCA IV. Most of the synthesized derivatives exhibited
sub‐nanomolar range inhibitory action against hCA VII (K
i
s = 0.83–0.97 nM). Markedly, 3,4‐dimethoxy phenyl
substituted derivative 182 was the most selective inhibitor for hCA VII, which demonstrated 947‐831‐, and 9543‐
fold selectivity over hCA I, hCA II, and hCA IV, respectively. The molecular docking study revealed that these
derivatives participated in an additional hydrogen bonding with the nonconserved Q69 residue in the active site of
hCA VII which may be responsible to generate such type of fabulous selectivity for hCA VII.
262
Selenides bearing
benzenesulfonamide moieties also emerged as influential and selective inhibitors for hCA VII. These selenides
showed sub‐nanomolar to nanomolar range inhibitory action against hCA VII. However, a number of selenides in
this series failed to inhibit hCA VII very selectively, although they showed effective inhibition for hCA VII.
However, some selective and potent hCA VII inhibitors such as 183 and 184 (Figure 14) were also identified which
showed a K
i
value of 0.35 and 0.38 nM. From the selectivity point of perspective, these derivatives showed
6.8–2517‐fold selectivity over hCA I, hCA II, hCA IV, and hCA IX. Thus, these findings exposed the potential of
selenides as effective and selective hCA VII inhibitors, which may serve as a potent lead for further develop-
ment.
263,264
El‐Azab et al. synthesized a library of carboxylates and sulfonamides consisting of phthalic anhydride
and phthalimide moieties which were found to be potent and highly selective inhibitors for hCA VII. These
derivatives inhibited hCA VII with sub‐nanomolar to low nanomolar range inhibitory potential and showed K
i
sin
the range of 0.31–14.5 nM. hCA I and hCA II were poorly inhibited by these derivatives with K
i
s in the range of 12
to >10 000 nM. Carboxylate holding phthalic anhydride 185 (K
i
value of 3.3 nM) and phthalimide 186 (K
i
value of
4.3 nM) arose as very selective hCA VII inhibitors (Figure 14). Both derivatives were ineffective against hCA I, hCA
II as well as hCA IV (K
i
s value of >10 000 nM) and showed moderate inhibitory potential against cancer associated
isoform hCA XII (K
i
value of 57.4 nM for compound 185 and of 51.9 nM for compound 186).
265
Various natural polyphenols such as flavones, flavanones, flavanols, isoflavones, and depsides were also
screened for their inhibitory action against hCA VII. Most of the tested polyphenols showed low nanomolar to
medium nanomolar range inhibitory potential against hCA VII (K
i
s in the range of 3.3–493.1 nM) and appeared to
be highly selective over hCA I as well as hCA II (K
i
s in the range of 168 to >10 000 nM). Interestingly, Hesperitin
187 (K
i
value of 3.3 nM) and Tiliroside 188 (K
i
value of 4.6 nM) showed effective inhibitory action against hCA VII,
being selective over hCA I, hCA II, hCA IV, and hCA XII (Figure 14).
266
4.5 |CA IX‐and CA XII‐selective inhibitors
CA IX and CA XII have been well studied for their role in tumorigenesis, cancer progression and metastasis, as
discussed in earlier section.
267,268
These enzymes belong to the transmembrane class of proteins, with their
extracellular domain comprising the CA active site, being thus involved in regulation of tumor microenvironment
MISHRA ET AL.
|
35
pH.
269,270
It has been demonstrated that reducing the activity of either CA IX/XII affects the pH of the tumor
microenvironment leading to a reduction of tumor cell survival and proliferation. Such features make CA IX/XII
attractive as anticancer drug targets.
271–273
Due to the established role of CA IX/CA XII in cancer, many classes of
potent and selective CA IX/CA XII inhibitors have been developed which have shown promising activity in in vitro
as well as in vivo models. Many researchers have put their effort to develop potent CA IX/CA XII inhibitors, which
selectively inhibit CA IX/CA XII or both over the cytosolic CA isoforms.
269–316
Maresca et al. synthesized 7,8‐as well as 6,7‐disubstituted coumarins and evaluated their CA inhibitory action
against four CA isoforms. These coumarins showed submicromolar inhibition against CA IX and XII, while weakly
inhibited off‐target isoforms CA I and II. Among all series hydroxy (compound 189) as well as methylene chloride
(compound 190) substituted coumarins showed better hCA IX/hCA XII inhibitory action as compared with
other derivatives along with displaying satisfactory selectivity over hCA I as well as hCA II (Figure 15).
FIGURE 14 Chemical structure of hCA VII‐selective inhibitors 174–188. hCA, human carbonic anhydrase
36
|
MISHRA ET AL.
Compound 189 has displayed a K
i
value of 0.19 and 0.68 µM for hCA IX and hCA XII, respectively, whereas
compound 190 have shown a K
i
value of 0.35 and 0.73 µM against hCA IX and hCA XII, respectively.
267
Further,
Maresca et al. reported 7,8‐disubstituted coumarins 191–199 as potent and selective hCA IX/hCA XII inhibitors
(Figure 15). These coumarins exhibited nanomolar/sub‐nanomolar inhibition against CA IX/XII, whereas, micro-
molar range (>100 µM) inhibition was observed against hCA I/hCA II, which indicated the highly selective nature of
these coumarins toward hCA IX/XII. These derivatives have shown K
i
s value in the range of 37.8–78.3 nM for hCA
IX and of 0.98–60.9 nM for hCA XII. Interestingly, 6,7‐substituted coumarins were ineffective toward CA IX and
XII, thus, 7,8 substitution on coumarins highly influences hCA IX/hCA XII inhibitory action.
268
Diaminopteridine‐benzenesulfonamide/benzenesulfonate conjugates were also reported as potent and se-
lective hCA IX inhibitors. The inhibition study showed that diaminopteridine‐benzenesulfonamide conjugates
appeared to be an effective hCA IX inhibitors, whereas diaminopteridine‐benzenesulfonate conjugates failed to
FIGURE 15 Chemical structure of developed hCA IX‐/hCA XII‐selective inhibitors 189–209. hCA,
human carbonic anhydrase
MISHRA ET AL.
|
37
produce effective inhibition against hCA IX. All reported diaminopteridine‐benzenesulfonamide conjugates dis-
played low nanomolar range inhibition toward hCA IX while isoforms hCA I and hCA II were inhibited with high
nanomolar range by these compounds. Diaminopteridine‐benzenesulfonamide conjugates, containing NHCH
2
CH
2
(compound 200), as well as compound 201, and compound 202, showed selective inhibition for hCA IX over hCA I
and hCA II (Figure 15). Conjugates 200,201, and 202 showed a K
i
value of 4.7, 2.5, and 3.7 nM, respectively,
against hCA IX. These potent hCA IX inhibitors have also shown selectivity between 9.6‐and 369‐fold over hCA II
and between 348‐and 5829‐fold over hCA I. Thus, these conjugates emerged as appropriate leads for the de-
velopment of more potent and selective hCA IX inhibitors.
269
Several ureido‐substituted benzenesulfonamides
with low nanomolar CA IX/XII inhibitory action have been discovered, also, displayed good selectivity for the
transmembrane isoforms (CA IX/XII) over the cytosolic isoforms. Although, most of the derivatives in this series
effectively inhibited CA IX/XII, however, derivatives 203–209 showed promising inhibitory action against CA IX/
XII (Figure 15). These derivatives have shown a K
i
in the range between 0.3 and 6.2 nM against hCA IX and
between 2.3 and 5.7 nM toward hCA XII. Additionally, derivative 205 holding 4‐acetylphenyl moiety exhibited
promising selectivity for CA IX/XII over hCA I/hCA II. This compound showed a selectivity ratio of 71.8 for
inhibiting CA IX over CA I and of 196.3 for inhibiting CA IX over CA II. Moreover, a selectivity ratio of 84.3 for
inhibiting CA XII over CA I and of 230.4 for inhibiting CA XII over CA II were also found for this derivative.
270
Some acetylated N‐β‐glycosyl sulfamides 210–213 were also reported as potent and selective hCA IX/hCA XII
inhibitors (Figure 16). These derivatives showed a narrow range of K
i
s, from 5.0 to 7.7 nM for hCA IX and from 5.4
to 6.5 nM for hCA XII. A fair selectivity pattern for hCA IX/hCA XII over hCA I/hCA II was also observed for these
derivatives. These compounds showed a K
i
in the range of 76–85 nM against hCA II and of 3714–9703 nM against
hCA I. Therefore, these sulfamides have shown a very weak affinity toward hCA I.
271
Sulfonamides incorporating 1,3,5‐triazine moieties also inhibited hCA IX/hCA XII very effectively with a K
i
in the
range of 0.15–138 nM against hCA IX and of 0.35–248 nM against hCA XII. It was observed that benzenesulfonamides
coupled with dichloro triazine 214, alanine substituted triazine 215, and ethanolamine substituted triazine 216 dis-
played excellent inhibitory action against hCA IX/hCA XII along with good selectivity over hCA I/hCA II (Figure 16).
Compounds 214,215,and216 have shown a K
i
value of 0.15, 0.96, and 0.75 nM, respectively, against hCA IX, while
displaying a K
i
value of 0.35, 0.58, and 1.6 nM against hCA XII. Additionally, these compounds showed 800–1464‐fold
selectivity for hCA IX over hCA I and appeared to be 28–686‐fold selective for hCA XII over hCA I. However, these
derivatives have shown 49–706‐fold and 23–302‐fold selectivity for hCA IX and hCA XII, respectively, over hCA II.
272
Lopez et al. synthesized a library of carbohydrate‐based CAIs and evaluated for their inhibitory action against hCA IX
and hCA XII. Results demonstrated that most of derivatives effectively inhibited hCA IX/hCA XII with low nanomolar to
medium nanomolar range K
i
s value. Additionally, some of the derivatives behaved as a selective inhibitor for hCA IX/
hCA XII over hCA I as well as hCA II. For example, compound 217, which has shown 673.8‐and 7.8‐fold selectivity for
hCA IX over hCA I and hCA II, respectively, with a K
i
value of 8.4 for hCA IX. This derivative displayed a K
i
value of
7.6 nM against hCA XII, which appeared to be 744.7‐and 8.6‐fold selective over hCA I and hCA II, respectively
(Figure 16). Likewise, derivative 218 (K
i
value of 8.6 nM against hCA IX) also exhibited selective inhibition for hCA IX/
hCA XII, displayed 137‐and 9.5‐fold selectivity for hCA IX over hCA I and hCA II, respectively (Figure 16). Also, this
derivative was more selective toward hCA XII as compared with hCA IX over hCA I/hCA II and showed an impressive K
i
value of 7.3 nM against hCA XII.
273
Glycosyl coumarins have been reported by Touisni et al. as potent and very
selective hCA IX/hCA XII inhibitors. These coumarins showed low to medium nanomolar range inhibition for hCA IX/
hCA XII, however, micromolar range inhibition was observed against hCA I/hCA II. Among all, mannose containing
coumarin 219 and galactose bearing coumarin 220 showed effective and selective inhibition against hCA IX and hCA
XII, respectively (Figure 16). Coumarin 219 has shown low nanomolar inhibition against hCA IX with a K
i
value of
9.2 nM, while a medium nanomolar range K
i
value of 43 nM was detected against hCA XII. This derivative has displayed
aK
i
value of >100 µM for hCA I as well as hCA II, indicating auspicious selectivity toward hCA IX/hCA XII over hCA I/
hCA II. Additionally, hCA XII was strongly inhibited by galactose containing coumarin 220 (Figure 16)withaK
i
value of
8.5 nM, whereas this compound showed a K
i
value of 93 nM against hCA IX. This derivative also appeared to be a
38
|
MISHRA ET AL.
selective hCA IX/hCA XII inhibitor and showed a K
i
value of 0.59 and 77 µM for hCA I and hCA II, respectively.
274
Metallocene‐based compounds were also shown potent hCA IX/hCA XII inhibitory action and displayed low to medium
nanomolar range inhibition against these isoforms. However, these derivatives strongly inhibited hCA XII as compared
with hCA IX isoform, whereas most of the compounds were less effective against hCA I and hCA II. Some of derivatives
such as 221 and 222 showed selective hCA XII inhibitory action as compared with other derivatives of this series
(Figure 16). Compounds 221 and 222 showed a K
i
value of 8.7 and of 26.2 nM against hCA XII, respectively. hCA IX
isoform was inhibited with a K
i
value of 75.5 and 136 nM by derivatives 221 and 222, respectively. Moreover,
compound 221 displayed 296‐and 47‐fold selectivity for hCA XII over hCA I and hCA II, respectively, while compound
222 showed 140‐and 179‐fold selectivity toward hCA XII over hCA I and hCA II, respectively.
275
Gieling et al. developed a series of sulfamate derivatives which have shown high specificity at nanomolar levels
against CA IX/XII isoforms. These ureidobenzene sulfamates have exhibited promising selectivity for hCA IX/XII
FIGURE 16 Chemical structure of compounds 210–224 showing selective hCA IX/hCA XII selective inhibitory
action. hCA, human carbonic anhydrase
MISHRA ET AL.
|
39
and showed K
i
s in the ranges of 6–27 nM and of 1–19 nM against hCA IX and hCA XII, respectively. A range of K
i
s
from 1230 to 5600 nM and from 145 to 546 nM were displayed against hCA I and hCA II, respectively, by these
sulfamates. Interestingly, perfluorophenyl 223 and 3, 5 dimethylphenyl derivative 224 showed promising se-
lectivity for hCA IX/hCA XII over hCA I/hCA II (Figure 16). Compound 223 (showing a K
i
value of 6 nM against hCA
IX and of 1 nM against hCA XII) has shown 530‐and 24.2‐fold selectivity for hCA IX over hCA I and hCA II,
respectively, 3180‐and 145‐fold selectivity for hCA XII over hCA I and hCA II, respectively. Derivative 224 which
showed a K
i
value of 7 nM against hCA IX and of 2 nM against hCA XII, demonstrated 800‐and 78‐fold selectivity
for hCA IX over hCA I and hCA II, respectively, being 2800‐and 273‐fold selective for hCA XII over hCA I and hCA
II, respectively.
276
Phenylethynylbenzenesulfonamide derivatives, which were reported by Knaus et al. also
showed selective inhibition toward hCA IX/hCA XII isoforms over hCA I/hCA II. Methoxy substituted deriva-
tive 225 (Figure 17) showed low nanomolar potency against hCA IX as well as hCA XII and displayed a K
i
of value
6.5 and 1.2 nM for hCA IX and hCA XII, respectively. Compound 225 confirmed 12‐and 65‐fold selectivity for hCA
IX and hCA XII, respectively, over hCA I, 752.3‐and 4075‐fold for hCA IX and hCA XII, respectively, over hCA II.
Interestingly, methyl containing derivative 226 (Figure 17) appeared to be selective inhibitor for hCA IX and
showed 92.7‐, 1554‐, and 92.7‐fold selective over hCA I, hCA II, and hCA XII, respectively.
277
hCA IX was also
selectively inhibited by diarylpyrazole‐benzenesulfonamides. These derivatives showed effective nanomolar range
inhibition toward hCA IX, and some of derivatives such as compounds 227 and 228 (Figure 17) showed selective
inhibition for hCA IX over hCA I as well hCA II. Compound 227 had a K
i
value of 27 nM along with 148‐and 15‐fold
selectivity over hCA I and hCA II, respectively. Compound 228, containing 4‐bromophenyl substitution illustrated a
K
i
value of 15 nM against hCA IX and appeared to be 1272‐and 7.8‐fold selective over hCA I and hCA II, re-
spectively.
278
A library of benzenesulfonamides incorporating cyanoacrylamide moieties has been reported as
potent and selective hCA IX/hCA XII. Isoforms hCA IX and hCA XII were strongly inhibited by most of the
derivatives, generally in the low nanomolar to the subnanomolar range. Inhibitory data revealed that majority of
derivatives showed selective inhibition toward hCA IX/hCA XII over hCA I/hCA II. For example, compounds 229
and 230 (Figure 17) illustrated imperative selectivity toward hCA IX/hCA XII by displaying sub‐nanomolar range
inhibition. Derivative 229 effectively inhibited hCA IX and hCA XII with a K
i
value of 0.86 and 0.57 nM, respec-
tively. This derivative has a selectivity ratio of 46 and 10.4 for inhibiting hCA IX over hCA I and hCA II, respec-
tively. However, compound 229 demonstrated 69.4‐and 15.7‐fold selectivity for hCA XII over hCA I and hCA II.
Likewise, compound 230 have shown 87.9‐and 50.4‐fold selectivity for hCA IX over hCA I and hCA II, respectively,
being an effective inhibitor of hCA IX (K
i
= 0.67 nM). This derivative inhibited hCA XII with a K
i
value of 0.65 nM
and showed selectivity ratio 90.6 and 52 for hCA XII over hCA I and hCA II, respectively.
279
Tertiary (fluorinated)
benzenesulfonamide derivatives have also effectively inhibited hCA IX/hCA XII. Although, it seems that hCA IX was
inhibited more effectively as compared with hCA XII by most of the derivatives. Indeed, some derivatives such
as 231 and 232 showed potent and selective inhibition against hCA IX (Figure 17). Compound 231 has a K
i
value of
9.1 nM for hCA IX and was 8.5‐and 10.9‐fold selective over hCA I and hCA XII, respectively. Compound 232 have
displayed 9.3‐and 8.7‐fold selectivity for hCA IX and hCA XII, respectively, being a potent hCA IX inhibitor
(K
i
= 9.6 nM). Remarkably, most of the derivatives, including compounds 231 and 232 appeared to be ineffective
against the most abundant off‐target hCA II.
280
Saada et al. synthesized a series of sulfonamide‐based potent and
selective hCA IX/hCA XII inhibitors by applying thiol‐ene click chemistry. These derivatives displayed sub‐
nanomolar to low nanomolar range inhibition against hCA IX/hCA II. Indeed, satisfactory selectivity for hCA IX/
hCA XII over hCA I/hCA II was also observed. For example, derivatives 233,234,and 235 (Figure 17) showed a K
i
value of 0.82, 0.92, and 0.75 nM, respectively, against hCA IX, while against hCA XII compounds 233,234, and 235
demonstrated a K
i
value of 0.66, 0.80, and 0.63 nM, respectively. These derivatives illustrated 95.6–1060‐fold and
12.5–80.6‐fold selectivity for hCA IX over hCA I and hCA II, respectively. For hCA XII these compounds showed
110–1318‐fold and 15–100‐fold selectivity over hCA I and hCA II, respectively.
281
Some sulfonamides, containing
4,5,6,7‐tetrabromophthalimide moiety also selectively inhibited hCA IX/hCA XII with low nanomolar inhibition.
Benzenesulfonamide possessing tetrabromophthalimide (compound 236) and chlorobenzene sulfonamide
40
|
MISHRA ET AL.
containing tetrabromophthalimide (compound 237) presented a K
i
value of 9.1 and 8.5 nM, respectively, for
hCA IX, against hCA XII compounds 236 and 237 showed a K
i
value of 6.1 and 5.9 nM, respectively (Figure 17).
In terms of selectivity, compound 236 demonstrated 47.4‐, 7.4‐, and 5.9‐fold selectivity toward hCA IX over hCA I,
hCA II, and hCA VII, respectively. This derivative showed 70.8‐,11‐, and 8.8‐fold selectivity for hCA XII over hCA I,
hCA II, and hCA VII, respectively. However, compound 237 indicated 8.4–24.7‐and 9.3–53.2‐fold selectivity
for hCA IX and hCA XII correspondingly over hCA I, hCA II, and hCA VII.
282
Additionally, hCA IX/hCA XII were strongly and specifically inhibited by 6‐triazolyl‐substituted sulfocoumarins
238–258 which have been prepared by employing click chemistry approach (Table 4). Interestingly, most of the syn-
thesized derivatives strongly inhibited hCA IX/hCA XII, being ineffective inhibitors for hCA I/hCA II. hCA IX was effec-
tively inhibited by most of the triazolyl substituted sulfocoumarins with low nanomolar range inhibition except derivatives
241,244,245,and250. These sulfocoumarins demonstrated K
i
s in the range of 7.2–875 nM against hCA IX and of
FIGURE 17 Chemical structure of selective hCA IX/hCA XII inhibitors 225–237. hCA, human carbonic anhydrase
MISHRA ET AL.
|
41
TABLE 4 hCA I, hCA II, hCA IX, and hCA XII inhibitory action of compounds 238–258
K
i
(nM)
Compound R hCA I hCA II hCA IX hCA XII
238 >10 000 >10 000 60.6 5.9
239 >10 000 >10 000 10.5 5.5
240 >10 000 >10 000 62.7 5.7
241 >10 000 >10 000 93.1 7.6
242 >10 000 >10 000 7.8 17.7
243 >10 000 >10 000 7.9 6.3
244 >10 000 >10 000 875 9.2
245 >10 000 >10 000 136 5.5
246 >10 000 >10 000 9.0 6.9
247 >10 000 >10 000 531 6.7
248 >10 000 >10 000 67.1 9.5
249 >10 000 >10 000 9.5 9.2
250 >10 000 >10 000 102 42.9
251 >10 000 >10 000 9.1 7.6
252 >10 000 >10 000 8.3 44.0
253 >10 000 >10 000 9.6 8.9
254 >10 000 >10 000 7.2 30.1
255 >10 000 >10 000 7.4 7.2
42
|
MISHRA ET AL.
5.5–42.9 nM against hCA XII. Thus, hCA XII was inhibited more effectively as compared with hCA IX by these novel
sulfocoumarins. Noticeably, all derivatives were ineffectivetowardhCAIaswellashCAIIanddisplayaK
i
s value of
>100 000 nM. These highly selective hCA IX/hCA XII inhibitors may appear highly useful for the development of potential
anticancer agents which will be free from side effects associated with hCA I/hCA II inhibition.
283
Moreover, 4‐N,N‐disubstituted sulfanilamides which incorporated 4,4,4‐trifluoro‐3‐oxo‐but‐1‐enyl, phena-
cylthiourea and imidazol‐2(3H)‐one/thione showed selective inhibition toward hCA IX/hCA XII. These sulfanila-
mides displayed a range of K
i
s from 9.5 to 39 nM against hCA IX and from 12 to 36 nM against hCA IX. These
derivatives inhibited hCA I/hCA II with low potency and showed K
i
s in the range of 302–593 nM and 274–510 nM
for hCA I and hCA II, respectively. Benzoyl moiety bearing derivative 259 (Figure 18) effectively inhibited hCA IX
with a K
i
value of 9.5 nM and possessed reasonable selectivity over hCA I/hCA II.
284
A series of 30 cyclic imides
was investigated as hCAs inhibitors by Abdel‐Aziz et al. Most of derivatives showed low nanomolar to sub‐
nanomolar inhibition against hCA XII. Indeed, these derivatives also showed effective inhibition against hCA IX as
well as hCA II. However, some derivatives such as compounds 260 and 261 (Figure 18) showed selective inhibition
toward hCA IX/hCA XII. Compound 260 showed >45‐fold selectivity for hCA IX over hCA I/hCA II and >909‐fold
selective for hCA XII over hCA I/hCA II, being a potent hCA IX (K
i
= 22.1 nM) and hCA XII (K
i
= 1.1 nM) inhibitor.
Another compound 261 has a K
i
value of 5.9 and 1.2 nM against hCA IX and hCA XII, respectively. This derivative
exhibited 169‐and 833‐fold selectivity for hCA IX and hCA XII correspondingly over hCA I/hCA II.
285
Hydro-
xysulfamide glycosides have been prepared from glycals and tested for their hCA IX/hCA XII inhibitory action.
Most of the derivatives have shown effective inhibition against hCA IX as well as hCA XII. Derivatives such as 262
and 263, were the most selective tumor‐associated CAs inhibitors, with selectivity ratios of >100 for inhibiting hCA
IX/XII over hCA I/II (Figure 18).
286
De Monte et al. reported a series of cyclic tertiary sulfamates as very selective
hCA IX/hCA XII inhibitors. Most of derivatives showed low nanomolar range to medium nanomolar range inhibition
against hCA IX and hCA XII. Some derivatives such as 264 (Figure 18)
,
which contains benzoyl moiety showed
effective inhibition against hCA IX as well as hCA XII, being highly selective hCA IX/hCA XII inhibitor. This
derivative demonstrated a K
i
value of 5.7 and 16.5 nM against hCA IX and hCA XII, respectively. Noticeably, this
derivative was a weak inhibitor for hCA I/hCA II, and exhibited a K
i
value of >20 000 nM for these both iso-
forms.
287
The tetrazole‐substituted sulfocoumarins also acted as potent and selective hCA IX/hCA XII inhibitors.
Various alkyl and substituted aryl moieties were substituted at the 1‐position of the tetrazole ring to get a
conclusive SAR. These tetrazole‐sulfocoumarins efficiently inhibited hCA IX and hCA XII with K
i
s ranging from 6.5
to 68.6 nM and between 4.3 and 59.8 nM, respectively, although, off‐target cytosolic isoforms hCA I/hCA II was not
inhibited by these derivatives. Hence, these compounds very selectively inhibited hCA IX/hCA XII over cytosolic
isoforms hCA I/hCA II. All derivatives effectively inhibited hCA IX/hCA XII, however, derivatives 265,266, and 267
strongly inhibited hCA IX/hCA XII with low nanomolar range inhibition (Figure 18). Compounds 265,266, and 267
showed a K
i
value of 8.5, 8.7, and 6.5 nM, respectively against hCA IX, and also demonstrated a K
i
value of 7.1, 6.3,
TABLE 4 (Continued)
K
i
(nM)
Compound R hCA I hCA II hCA IX hCA XII
256 >10 000 >10 000 8.3 7.8
257 >10 000 >10 000 8.8 9.4
258 >10 000 >10 000 9.2 31.2
Abbreviation: hCA, human carbonic anhydrase.
MISHRA ET AL.
|
43
and 5.7 nM, respectively, against hCA XII. All derivatives, including these three showed a K
i
s value > 10 000 nM
against hCA I as well as hCA II, which indicated the inactivity of these derivatives toward hCA I/hCA II.
288
Moeker
et al. synthesized a small series of saccharin derivatives by installing hydrophobic or hydrophilic substituents on
the benzene ring of saccharin to study their inhibitory action against hCA IX. Indeed, some of the derivatives
showed more effective inhibitory action against hCA IX as compared with saccharin. However, hydrophilic gly-
coconjugate derivative 268 (Figure 18) displayed better inhibition against CA IX (K
i
= 49.5 nM) and enormously
weak inhibition against off‐target CAs; hCA I/hCA II (K
i
s > 50 000 nM) compared with saccharin. Thus, this in-
vestigation highlighted the effectiveness of cyclic secondary sulfonamides, which may be explored for the dis-
covery of effective and selective hCA IX inhibitors.
289
Sulfonamides as well as coumarins consisting
arylsulfonylureido tails were also evaluated for their hCA IX/hCA XII inhibitory action. Although, most of the
derivatives showed nonselective/less selective inhibition against all tested isoforms. However, few derivatives
showed hCA IX/hCA XII selective inhibition over hCA I/hCA II. As for example, compound 269 containing 4‐fluoro
phenyl substitution showed 432.9‐and 1298.7‐fold selective inhibition for hCA IX and hCA XII, respectively, over
hCA I as well as hCA II (Figure 18). This derivative has shown a K
i
value of 23.1 and 7.7 nM against hCA IX and hCA
XII, respectively.
290
Sulfamates possessing carbohydrate tails also showed effective and specific inhibition for hCA
IX and hCA XII. Various linkers has been installed between sulfamate and carbohydrate tail to achieve a decisive
SAR. Most of the synthesized derivatives inhibited hCA IX with low nanomolar range inhibition by displaying a K
i
s
FIGURE 18 Chemical structure of synthesized compounds 259–272 showing selective inhibitory action
toward hCA IX/hCA XII. hCA, human carbonic anhydrase
44
|
MISHRA ET AL.
in the range of 2–225 nM. These derivatives also appeared to be a selective hCA IX inhibitors over cytosolic off‐
target isoforms hCA I/hCA II. Among all, derivative 270 showed excellent selectivity for hCA IX over hCA I as well
as hCA II (Figure 18). Compound 270 displayed >1818‐fold selectivity for hCA IX over hCA I and hCA II, by
showing an effective K
i
value of 11 nM against hCA IX.
291
Sildenafil analogues have been also reported as very
selective hCA IX/hCA XII inhibitors. New sildenafil analogues were developed by the introduction of a nitrogen
atom into the pyridine ring in place of carbonyl group of sildenafil. These new analogues showed effective na-
nomolar range inhibition for hCA IX as well as hCA XII. These derivatives exhibited K
i
s in the range of
15.4–42.4 nM for hCA IX and of 3.8–667 nM for hCA IX. Noticeably, these sildenafil analogues demonstrated very
weak inhibition against cytosolic hCA I/hCA II isoforms and showed a K
i
value of >10 000 nM for both hCA I and
hCA II isoforms. Indeed, analogues 271 and 272 were very effective and selective inhibitors for hCA IX and hCA
XII, respectively (Figure 18). Compound 271 has a K
i
value of 15.4 nM against hCA IX and >649‐fold selectivity
over hCA I as well as hCA II. While compound 272 has displayed a K
i
value of 3.8 nM and >2631.5‐fold selectivity
for hCA XII over hCA I/hCA II. Thus, these sildenafil analogues have emerged as a new structural class of potent
and selective hCA IX/hCA XII inhibitors over traditional pharmacophore such as sulfonamide or coumarin.
292
Furthermore, the modification on the sildenafil scaffold was carried out by the same research group by
introducing various substituted phenyl derivatives on the pyrazole ring of sildenafil. However, this modification
could not produce more potent hCA IX/hCA XII inhibitors as compared with previous derivatives, although se-
lectivity for hCA IX/hCA XII over hCA II was significantly enhanced. Noticeably, the inhibitory potential of these
derivatives against off‐target isoform hCA I has been increased as compared with previous sildenafil derivatives. In
this series, Compound 273 showed promising hCA IX inhibitory action with a K
i
value of 13.8 nM (Figure 19). This
derivative had a K
i
value of 82.8, >50 000, and 810 nM against hCA XII, hCA II and hCA I, respectively.
293
Carradori
et al. synthesized amide derivatives of probenecid which has demonstrated effective and selective inhibition
against hCA IX/hCA XII. These probenecids did not display inhibition against hCA I and hCA II (K
i
s > 10 000 nM).
Cancer‐associated isoforms hCA IX and hCA XII were effectively inhibited by these derivatives in low to medium
nanomolar range inhibitory potential. In this series, compound 274 (3‐pyridyl substitution) and compound 275 (2‐
chlorophenyl substitution) showed reasonable inhibitory action against hCA IX and hCA XII, respectively. Com-
pounds 274 and 275 have shown a K
i
value of 9.9 and 23.6 nM for hCA XII and hCA IX, respectively (Figure 19).
294
7‐Amino‐3,4‐dihydro‐1H‐quinolin‐2‐one (compound 276, Figure 19) was also recognized as potent and selective
hCA IX/hCA XII inhibitor. Interestingly, this compound was found to be a selective hCA IX/hCA XII inhibitor, which
has shown reasonable selectivity over hCA I, hCA II, hCA III, hCA IV, hCA VA, hCA VI, hCA VII, hCA IX, hCA XII,
hCA XIII, and hCA XIV. This compound has exhibited a K
i
value of 16.1 and of 124 nM against hCA XII and hCA IX,
respectively. Additionally, it was found that the lactam ring of this compound is not hydrolyzed and intact bicyclic
amino‐quinolinone core is responsible to produce inhibitory action.
295
6‐Substituted sulfocoumarins consisting
trimethylammonium carboxamide, methoxy and cyano moieties, with effective hCA IX and hCA XII inhibitory
action were reported by Grandane et al. Inhibition study specified that tert‐butylcarboxamido (compound 277),
phenylcarboxamido (compound 278), and 4‐pyridylcarboxamido (compound 279) moieties comprising sulfocou-
marins conferred the strong inhibition against hCA IX and hCA XII isoforms with K
i
s between 2.1 and 8.1 nM
(Figure 19). Additionally, all synthesized derivatives, including these three did not display inhibition against off‐
target isoforms hCA I as well as hCA II.
296
Fascinatingly, Arylation on the benzene ring of sulfocoumarins also
bestowed potent and selective hCA IX as well as hCA XII inhibitors. These derivatives also did bot display inhibition
against hCA I and hCA XII (K
i
= > 10 0000 nM). However, all these derivatives were effective hCA IX/hCA XII
inhibitors and have shown K
i
s in the range of 9–95.3 nM against hCA IX and of 3.7–14.2 nM against hCA XII.
Dichlorophenyl substituted sulfocoumarin 280 (Figure 19) was the most potent inhibitor and displayed a K
i
value
of 9.4 and of 5.7 nM for hCA IX and hCA XII, respectively.
297
A small library of 7‐substituted sulfocoumarins
keeping the aryl‐triazolyl moieties tethered through an oxymethylene linker has been developed as effective and
very selective hCA IX/hCA XII inhibitors. Most of the derivatives inhibited hCA XII with low nanomolar range
inhibitory potential and showed K
i
s in the range of 4.3–19.1 nM. Whereas, hCA IX (K
i
s=19–36.5 nM) was inhibited
MISHRA ET AL.
|
45
with slightly lower nanomolar range inhibitory potential as compared with hCA XII. Compound bearing 4‐carboxy
phenyl triazole ring 281 (Figure 19) showed effective inhibition for hCA IX (K
i
= 19.2 nM) as well as hCA XII
(K
i
= 4.6nM). Similar to other sulfocoumarins, these derivatives were also ineffective against hCA I as well as hCA
II.
298
A series of 7‐substituted coumarins possessing aryl‐triazole moieties were also synthesized by click chemistry
and tested against cancer‐associated CA isoforms hCA IX and hCA XII. Most of the synthesized coumarin deri-
vatives were weak inhibitors for hCA I and II, but showed low nanomolar inhibitory action against hCA IX (K
i
sin
the range of 14.3–34.4 nM) as well as hCA XII (K
i
s in the range of 4.7–37.8 nM). Coumarins containing phenyl
triazole (compound 282) and 3‐chlorophenyl triazole ring (compound 283) showed satisfactory selectivity for hCA
IX/hCA XII over hCA I/hCA II, by exhibiting effective inhibition against hCA IX/hCA XII (Figure 19). Coumarin 282
FIGURE 19 Chemical structure of inhibitors 273–288 displaying selective hCA IX/hCA XII inhibition. hCA,
human carbonic anhydrase
46
|
MISHRA ET AL.
was 408‐and 2083‐fold selective for hCA IX and hCA XII, respectively, over hCA I as well as hCA XII. Although,
derivative 283 was 320.5‐and 1818.1‐fold selective for hCA IX and hCA XII over hCA I/hCA II.
299
De Luca et al.
also reported various substituted coumarins derivatives, which have demonstrated very selective inhibition for
hCA IX/hCA XII over hCA I/hCA II. A majority of substituted coumarins showed low nanomolar range inhibition
against hCA IX as well as hCA XII. These compounds were completely inactive against cytosolic hCA I and hCA II
(K
i
s > 10 000). 4‐Aminobenzene substituted coumarin 284 (Figure 19) was found to be the most potent and
selective inhibitor for hCA XII and presented a K
i
value of 5.5 nM for this isoform. Moreover, coumarin 284
inhibited hCA IX with a K
i
value of 24.2 nM, and it was totally inactive against hCA I as well as hCA II.
300
Benzoylamino‐benzamide pendant coumarins were developed as highly selective hCA IX and hCA XII inhibitors
with low nanomolar range inhibitory potential. These coumarins were completely inactive inhibitors for the off‐
target isoforms hCA I as well as hCA II and exhibited K
i
s value of >500 000 nM. Remarkably, most of the deri-
vatives such as 285–288 (Figure 19) showed effective inhibition toward hCA IX (K
i
s in the range of 10.9–21.6 nM)
as well as hCA XII (K
i
s in the range of 6.7–10.2 nM).
301
Coumaryl‐carboxamide derivatives were also bestowed selective inhibition against hCA IX and hCA XII, though,
these derivatives have demonstrated high nanomolar range inhibition for hCA IX/hCA XII. 2‐Oxo‐N‐((2‐(pyrrolidin‐1‐yl)
ethyl)carbamothioyl)‐2H‐chromene‐3‐carboxamide 289 was the most effective inhibitor which inhibited hCA IX with a
K
i
value of 107.9 nM (Figure 20). All derivatives of this series were ineffective against hCA I, hCA II, and hCA VII and
presented K
i
s value of >10 000 nM for all these three isoforms.
302
Schiff base derivatives of sulfanilamide were tested
for their hCA IX and hCA XII inhibitory potential. Indeed, these derivatives were low nanomolar range inhibitor for hCA
XII, however, most of the derivatives did not display effective inhibition against hCA IX. Among all, compound 290
(Figure 20) was a very selective inhibitor for hCA XII with a K
i
of value 9.7 nM. This derivative has shown a K
i
value of
36 nM against hCA IX, and a K
i
value of >50 000 nM against hCA I as well as hCA II. Further, the Schiff base was
reduced and converted into sulfamoylphenyl benzylamine derivatives. Although, these derivatives inhibited hCA IX
more effectively as compared with Schiff bases. However, these derivatives also inhibited hCA I and hCA II effectively
with low nanomolar range inhibitory action.
303
AlibraryofN‐substituted saccharin, as well as N‐/O‐substituted ace-
sulfame derivatives, were prepared as potential hCA IX/hCA XII inhibitors. The result indicated that these compounds
prominently inhibited hCA XII isoform with low nanomolar range inhibition constant and hCA IX was inhibited with low
to medium nanomolar range inhibition constant (K
i
s ranging between 19 and 2482 nM). Off‐target isoforms hCA I and
hCA II were poorly inhibited and a range of K
i
s > 10 µM for hCA II and between 318 nM and 50 µM hCA I were
observed. In saccharin series, derivative 291 and in acesulfame series, derivative 292 was the most active and selective
hCA IX/hCA XII inhibitors (Figure 20). Compound 291 has exhibited a K
i
value of 22 and 4.3 nM against hCA IX and
hCA XII, respectively, while compound 292 have shown a K
i
value of 26 and 4 nM for hCA IX and hCA XII, respectively.
These compounds were ineffective against off‐target cytosolic isoforms hCA I as well as hCA II, and displayed a K
i
value
of >100 000 nM for both isoforms.
304
3‐Hydroxy‐1H‐quinazoline‐2,4‐dione moiety was also used to develop strong and
selective inhibitors for hCA IX and hCA XII. A library of derivatives possessing different substituents such as Cl, NH
2
,
NO
2
,CF
3
, Amido, ureido, and heterocycles on the fused benzo ring were synthesized. Several of them presented
nanomolar activity against hCA IX and XII isoforms, though they were completely inactive to inhibit the cytosolic
enzymes hCAs I and II. In this series, most of the derivatives like nitro (compound 293), chloro (compound 294), CF
3
(compound 295) substituted derivatives and 5,7‐dichloro‐3‐hydroxyquinazoline 296 showed low nanomolar range
inhibition against hCA IX as well as hCA XII (Figure 20). These derivatives inhibited hCA IX with K
i
sintherangeof
4.6–8.2 nM and hCA XII was inhibited with a range of K
i
s from 5.8 to 7.5 nM. These compounds were unable to inhibit
cytosolicisoformshCAIaswellashCAIIanddisplayedK
i
s > 10 000 for both isoforms.
305
3‐nitrobenzoic acid deri-
vatives are developed as selective inhibitors for hCA IX as well as hCA XII, and most of the derivatives exhibited high
selectivity over hCA I, hCA II, hCA IV, and hCA VII by displaying moderate selectivity over hCA VA. The most potent
inhibitor for hCA IX and CA XII was derivative 297 (Figure 20) which showed a K
i
value of 16 and 82.1 nM for hCA IX
and hCA XII, respectively.
306
Ivanova et al. investigated N‐substituted saccharins bearing aryl, alkyl as well as alkynyl
moieties, and some ring‐openedderivativesaseffectivehCAIX/hCAXIIinhibition. Indeed, hCA XII was inhibited more
MISHRA ET AL.
|
47
effectivelybythesederivativesascomparedwithhCAXIIisoformsandshowedK
i
s in the range of 22.1–481 nM for
hCA IX and of 3.9–245 nM for hCA XII. These inhibitors did not inhibit cytosolic isoforms hCA I as well as hCA II and
showed K
i
s > 10 000 µM. Saccharin substituted with Phenyl ethyl (compound 298,Figure20) was the most powerful in
the entire series and showed a K
i
value of 76.1 and 10.4 nM against hCA IX and hCA XII, respectively.
307
Open
saccharin derivatives have been also reported as selective hCA IX and hCA XII inhibitors with nanomolar range
inhibitory action. Derivatives having 3‐methyl phenyl (compound 299)and3‐CF3 phenyl (compound 300)moietyhave
exhibited effective low nanomolar inhibitory potential against hCA XII (Figure 20). Compounds 299 and 300 showed
high nanomolar range inhibition for another cancer‐related isoform hCA IX (299;K
i
= 223 nM, 300;K
i
= 238 nM).
Cytosolic isoforms hCA I and hCA II were not inhibited by these open saccharin derivatives (K
i
s > 10 000).
308
Acridine
orange‐sulfonamide derivative (compound 301) was indeed an effective and selective hCA IX as well as hCA XII
FIGURE 20 Chemical structure of selective hCA IX/hCA XII inhibitors 289–303. hCA, human carbonic
anhydrase
48
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MISHRA ET AL.
inhibitor. This acridineorange‐sulfonamide compound displayed a K
i
value of 9.1 and 4.9 nM against hCA IX and hCA XII
isoforms. Compound 301 (Figure 20) was 843.9‐and 181.3‐fold selective for hCA IX over hCA I and hCA II, respec-
tively. This compound showed 1567.3‐and 336.7‐fold selectivity for hCA XII over hCA I and hCA II, respectively.
309
N’‐phenyl‐N‐hydroxyureas were found to be selective inhibitors for tumor‐associated isoforms hCA IX and hCA II.
These hydroxyurea derivatives appeared to be ineffective for off‐target isoforms hCA I as well as hCA II. Certainly,
most of the compounds showed high nanomolar range inhibitory action against hCA IX as well as hCA XII. However,
some derivatives like 302 (K
i
= 7.2 nM) and 303 (K
i
= 6.9 nM) showed low nanomolar range inhibition against hCA XII
(Figure 20). Moreover, compounds 302 and 303 exhibited a K
i
value of 78.9 and 72.8 nM against hCA IX. Hence, the
investigation provided some selective low nanomolar range hCA XII inhibitors and may be considered to be a suitable
lead for further development as more potent hCA XII inhibitors.
310
Primary benzenesulfonamides tethered to bi/tricyclic scaffolds were disclosed as sub‐nanomolar/low nanomolar
range inhibitors for hCA IX. Most of the derivatives have revealed moderate selectivity over hCA I, hCA II, and hCA
IV. However, benzenesulfonamide linked to the tricyclic scaffold (compound 304,Figure21) was the most selective
hCA IX inhibitor, which has also shown low nanomolar inhibition constant for hCA IX (K
i
= 7.3 nM). It was >1370‐,
577‐, and 990‐fold selective for hCA IX over hCA I, hCA II, and hCA IV. The molecular docking study indicated that
pyrimidine ring nitrogen of compound 304 actively participated to establish H‐bond interaction with Q71 and Q92
side chains, which may be provided favorable binding with hCA IX led to achieving high affinity for hCA IX.
311
Bozdag
et al. developed 2‐aminophenol‐4‐sulfonamide and its urea analogues by modifying lead molecule SLC‐0111as potent
hCA IX/hCA XII inhibitors. Inhibition data showed that compounds 305 (4‐Fluoro substation) and 306 (2‐nitro
substitution) were the most potent inhibitors against hCA IX and hCA XII (Figure 21). Compound 305 showed a K
i
value of 2.6 and 7.6 nM for hCA IX and hCA XII, respectively, while compound 306 has demonstrated a K
i
value of
2.6 nM for hCA IX and of 7.8 nM for hCA XII. Addition to this, compounds 305 and 306 have shown low affinity for
hCA I as well as hCA II and displayed K
i
s in the range of 441–>10 000 nM.
312
A new scaffold psoralen carboxylic
acids and their corresponding benzenesulfonamide derivatives were also developed as potent and selective hCA IX
and hCA XII inhibitors. Indeed, psoralen carboxylic acids derivatives were more selective hCA IX/hCA XII inhibitors
as compared with their corresponding benzenesulfonamide derivatives. In the psoralen carboxylic acids series,
derivative 307 (Figure 21) was the most effective and selective derivative which displayed a K
i
value of 17.7 and
7.4 nM for hCA IX and hCA XII, respectively, a K
i
value of >10000 nM was shown by this derivative for hCA I as well
as hCA II. In psoralen‐benzenesulfonamide series compound 308 (Figure 21) was the most active inhibitor for hCA
IX/hCA XII. It demonstrated a K
i
value of 17.8, 2.4, 55.1, and 6829.7 nM for hCA IX, hCA XII, hCA II, and hCA
I, respectively.
313
Bonardi et al. designed and synthesized 3‐carboxy coumarin derivatives, chromeno[4,3‐c]pyrazol‐4‐
ones, and pyrano[4,3‐c]pyrazol‐4‐ones which have shown highly selective inhibition toward hCA IX and hCA XII. All
these derivatives were not effective against off‐target cytosolic isoforms hCA I as well as hCA II. In entire series
derivatives 309,310, and 311 (Figure 21) have demonstrated the most effective inhibition toward hCA IX (K
i
value of
8.2, 9.6, 8.5nM, respectively) and hCA XII (K
i
value of 5.6, 5.8, and 7.1 nM, respectively).
314
Carboxamide derivatives
of 6‐and 7‐substituted coumarins were also prepared and evaluated to find out potent and selective hCA IX and hCA
XII inhibitors. Indeed, these derivatives were highly selective inhibitors for hCA IX, although, most of the derivatives
showed high nanomolar range inhibitory action. These coumarins did not show effective inhibition against hCA I, hCA
II, and hCA IV (K
i
s > 10 000 nM). In the whole series, coumarin bearing phenethyl‐acetamide moiety 312 and
phenethyl‐acetamide moiety 313 were the most potent hCA IX inhibitors, which indicated a K
i
value of 30.5 and
30.2 nM, respectively (Figure 21).
315
Sulfonamide containing chromone derivatives also strongly and selectively
inhibited hCA IX/hCA XII with low nanomolar range inhibition. Majority of derivatives showed very effective in-
hibition toward hCA IX as well as hCA XII. These chromones poorly inhibited hCA I and hCA II by displaying a high
nanomolar range affinity toward these isoforms. Indeed, some derivatives appeared to be very selective hCA IX as
well as hCA IX inhibitors such as thiazole (compound 314) and isoxazole (compound 315) substituted chromones
(Figure 21). Derivatives 314 and 315 were completely inactive toward hCA I as well as hCA II and displayed a K
i
value of >10000 nM against both isoforms. Additionally, compounds 314 and 315 effectively inhibited hCA IX with a
MISHRA ET AL.
|
49
K
i
value of 19.91 and 32.09 nM, respectively. Although, against hCA XII, 314 and 315 have shown a K
i
value of 22.8
and 10.7 nM, respectively
316
Carradori et al. synthesized Salen and tetrahydrosalen derivatives with micromolar
range inhibitory action against hCA XII. Two chelating groups from salen/tetrahydrosalen were tethered by nu-
merous aliphatic as well as aromatic linkers to optimize the inhibitory activity of these molecules. Some of the
derivatives showed selective inhibition against hCA XII, displaying low micromolar range inhibition. Methoxy phenyl
containing compound 316 was a selective hCA XII inhibitor and displayed a K
i
value of 1.49 µM against hCA XII. This
derivative feebly inhibited hCA I, hCA II, and hCA IX, showing a K
i
value of >100µM against all these isoforms.
Further, the authors studied the interaction of these molecules with hCA XII using molecular modeling and docking
FIGURE 21 Chemical structure of compounds 304–317 with selective hCA IX/hCA XII inhibitory action. hCA,
human carbonic anhydrase
50
|
MISHRA ET AL.
studies. It was noticed that compounds nicely interact with Zn
2+
ion in the active site by displacing zinc‐bound water
molecule.
317
Very recently, this study group developed saccharin/isoxazole and saccharin/isoxazoline hybrid mole-
cules as potent and selective inhibitors for hCA IX as well as hCA XII. Indeed, these hybrid molecules were highly
selective hCA hCA IX/hCA XII inhibitors over hCA I/hCA II isoforms. hCA hCA IX and hCA XII were effectively
inhibited by these molecules with low nanomolar range to medium nanomolar range inhibition. Noticeably, com-
pound 317,containingelectron‐donating group methoxy was very effective inhibitor for hCA IX (K
i
= 22.1 nM) as well
as hCA XII (K
i
= 8.0 nM). Compound 317 was also highly selective over hCA I and hCA II, demonstrating a K
i
value of
>10 000 nM for both isoforms. Additionally, this molecule behaves as chemosensitizer as well as good coadjuvant
with doxorubicin in the MCF7 cell line, being nontoxic in normal cell line primary human fibroblasts.
318
Nguyen et al.
reported perfluoroalkyl substances (PFASs) as a novel type of hCA IX/hCA XII inhibitors. In this investigation, 15
PFASs were investigated for their inhibitory action against hCAs. Some of them effectively inhibited hCA IX and hCA
XII isoforms with low micromolar range inhibition. This novel investigation recognized PFASs as effective hCA IX/
hCA XII inhibitors, and may also open a new door for the development of biocompatible materials as effective
therapeutic agents.
319
5|PHARMACOLOGICAL ADVANCES WITH NEW CA‐SELECTIVE
INHIBITORS: PRECLINICAL STUDIES
5.1 |CAIs as anti‐glaucoma agents
As discussed in the earlier part, CA inhibition especially, hCA I, hCA II, hCA IV, and hCA XII are validated as a useful
strategy for the treatment of glaucoma.
85–87
In this regards, various potent hCA inhibitors have been tested in
animal model of glaucoma proving to possess a potent anti‐glaucoma action.
320–323
Poly(amidoamine) (PAMAM)
dendrimers containing benzenesulfonamide moieties have shown potent hCA inhibitory action (CA II and XII) and
effective anti‐glaucoma action in animal models of glaucoma. It was noticed that dendrimer G2 was the most
potent IOP lowering agent among the tested CAIs and showed effective lowering effect (6 mmHg) just after 2h of
drug administration, it was 33% better than dorzolamide (DRZ; 4 mmHg). Other dendrimers such as G1 and G3
were less potent as compared with G2, but they were quite effective in chronic treatment. Potent hCA II (K
i
value
of 13.2 nM) and hCA XII (K
i
value of 0.76 nM) inhibitor 318 (a dithiocarbamate derivative; DTC) appeared to be an
effective anti‐glaucoma agent and has lowered IOP promisingly in an animal model of glaucoma (Figure 22). The
result of this study indicated that DTC 318 displayed greater than double IOP lowering efficacy (of 8.6 mmHg) as
compared with standard anti‐glaucoma drug DZA at 1 h and it was maintained up to 2 h of drug postadministration.
Remarkably, after 3 h of compound 318 treatment, it was found that this compound had a superior effect on
lowering IOP as compared with DZA. Thus, DTC 318 emerged as potent anti‐glaucoma agents which has greater
efficacy than DZA in terms of maximal IOP lowering as well as the duration of action.
320
Two new sulfonamide
derivatives 319 and 320 (Figure 22) which had shown potent inhibitory action against glaucoma‐associated hCA
isoforms were also evaluated for their anti‐glaucoma potential by complexing with cyclodextrin (γcyclodextrin,
hydroxypropyl‐γcyclodextrin, hydroxypropyl‐βcyclodextrin, and hydroxyethyl‐βcyclodextrin). The study provided
very interesting result and gamma‐cyclodextrin was the best complexing agent for both sulfonamides and its
complexes showed promising IOP lowering action against the animal model of glaucoma. IOP lowering was
36–37 mmHg (Peak value) after 1 h postadministration. Fascinatingly, these complex of sulfonamide maintained a
low IOP pressure (of around 35 mmHg) for the next 24 h, which was not noticed earlier with any other IOP
lowering drugs. Indeed, this study provided potent anti‐glaucoma agents with long duration of action which may
appear useful for therapy of glaucoma in future.
321
Effective hCA inhibiting 2‐benzylpiperazines deriva-
tives 321 and 322 have been also evaluated for their anti‐glaucoma action in transient as well as a stable animal
model of glaucoma (Figure 22). In the transient model, both derivatives effectively reduced IOP level after 60 min
MISHRA ET AL.
|
51
of postadministration and reached the maximum activity after 120 min of postadministration, lowering the IOP of
about 10 mmHg. These compounds were found to be potent similar to standard reference drug DRZ. Compound
322 has been also evaluated against a stable model of glaucoma and significantly reduced the IOP at 24, 48, 72,
and 96 has compared with control. The result indicated that Compound 322 has shown two‐fold higher IOP
lowering capacity as compared with DRZ at the same dose.
252
IOP lowering activity of some monothiocarbamates
(MTCs) such as 323–326 (Figure 22) have been assessed, which were potent hCA II inhibitors. The experimental
outcome indicated that all tested MTCs significantly lowered IOP up to 120 min. MTC 325 appeared to be a very
effective IOP lowering agent and showed effect (3.8 mmHg ΔIOP) at 60 min after injection and after 120min of
injection it displayed maximum IOP lowering activity (ΔIOP = 4.9 mmHg). Compound 326 bestowed an IOP re-
ducing result comparable with DRZ up to 120 min. Remarkably, 323 as well as 326 sustained their IOP reduction
capability up to 240 min as compared with standard drug DRZ. However, compound 324 was unable to produce
FIGURE 22 hCA inhibitors 318–330 displaying anti‐glaucoma activity. hCA, human carbonic anhydrase
52
|
MISHRA ET AL.
significant IOP lowering activity and showed a maximum at 120 min. Thus, these MTCs have been exposed as
potent IOP lowering agents which may appear a good lead molecule for further development as suitable anti‐
glaucoma agents in future.
322
Multitargeted compounds as β‐adrenergic receptor (AR) blocker and a CA (EC
4.2.1.1) inhibitor have been also tested for their anti‐glaucoma action against hypertonic saline‐induced ocular
hypertension in rabbits. Most potent dual‐acting compounds such as 327,328,and 329 dropped IOP more ef-
fectively as compared with standard drug DRZ and timolol (Figure 22). Compounds 327,328, and 329 decreased
IOP of 8.25, 10.75, and 6.75 mmHg at 60 min postadministration, respectively. Compound 329 investigated as the
most effective one, with almost 2‐fold enhanced effectiveness as compared with DRZ and timolol. Interestingly,
compounds 327,328, and 329 were also found effective after 2 h postadministration while compound 329 was
most effective among all to produce an IOP drop of 8.0 mmHg. Markedly, it was observed that 1% eye drops of
dual acting derivatives were more effective as compared with the combination of CAI and β‐blocker which was
used in the ratio 1% + 0.25%. Compound 330 (Figure 22) which has shown a unique CA inhibitory action was also
evaluated for its anti‐glaucoma action and this derivative also showed effective anti‐glaucoma activity in the rabbit
model. Additionally, It was also observed that dual targeting agents 325,328, and 329 significantly enhanced IOP
lowering efficacy as compared with 328 (uniquely inhibited the CAs) at 60 min of postadministration, whereas the
activity was observed weak after 2 h of drug administration.
323
Potent hCA II and XII inhibitors; dithiocarbamates (DTCs) 331 as well as 332 (Figure 23) were also possess
effective anti‐glaucoma activity and displayed promising IOP lowering action against carbomer‐induced rabbits
model of glaucoma. Compounds 331 and 332 were formulated as 2% eye drop solution at neutral pH and standard
drug DZA was formulated at pH 5.5, as a hydrochloride salt. Result evoked that these two derivatives effectively
reduced elevated IOP in a time‐dependent manner for a long duration. After 2 h of administration, compounds
showed a maximal effect (6–10 mmHg) and lasted for up to 4–8 h, almost double as compared with DZA (of
4–5 mmHg). Compound 331 was somewhat more promising than compound 332 as an IOP lowering agent.
213
Xanthates 333–335,71, and 72 (potent hCA II inhibitors) have been developed as potent anti‐glaucoma agents and
FIGURE 23 Chemical structure of potent hCAs 331–337 with effective anti‐glaucoma action. hCA,
human carbonic anhydrase
MISHRA ET AL.
|
53
these xanthates effectively lowered elevated IOP in the normotensive rabbit model as well as a hypertensive
rabbit model of glaucoma (Figure 23). These derivatives significantly decreased IOP (of 1.0–3.5 mmHg) upon direct
administration into the eye. In a hypertensive rabbit model, these xanthates showed strong IOP lowering activity
(4.0−12.0 mmHg). After 1h postadministration these xanthates were two to three times more effective than DZA.
The peak effect to lower IOP was detected around 50−100 min postadministration and activity was also observed
up to 200−250 min postadministration. Among all, compound 71 (K
i
value of 5.4 nM against hCA II) was the best
IOP lowering agent which showed an IOP lowering of 12 mmHg after 100 min postadministration, which was three
times higher at the same concentration of DZA.
219
Some of the potent hCAs inhibitors such as 336 and 337 (K
i
value of 21 and 19.1 nM against hCA II and of 6.8
and 0.67 nM against hCA XII, respectively) belonging to the pyridine sulfonamide class were also evaluated for
their action as anti‐glaucoma agents (Figure 23). Compound 336 (1% concentration) and 337 (2% concentration)
showed effective IOP lowering action in an animal model of glaucoma. The peak effect of the IOP drop was
detected at 1 h postadministration and it was higher than standard drug. These compounds have shown a longer
effect as compared with dorzolamide after 2 h of postadministration and drop of IOP was noticed 16–16.5 mmHg
for both drugs. Indeed, these compounds also maintained efficacy at 4 h of postadministration.
290
5.2 |CAIs with anticancer activity
hCA IX and hCA XII have been validated as an imperative target for therapy of hypoxic tumors and metas-
tasis.
324–326
Several potent and selective hCA IX and hCA XII inhibitors have been tested for their anticancer
activity in various in vitro and in vivo models. Studies revealed that numerous potent hCA IX and hCA XII inhibitors
have shown promising anticancer activity in preclinical investigation.
327–332
In this line, Winum et al. evaluated
anticancer effect of potent and selective hCA IX as well as hCA XII inhibitors belonging to sulfamate class. The
most potent inhibitors 339–342 (Figure 24) exerted a significant cytotoxic effect against four breast cancer cell
lines (SKBR3, MCF10A, ZR75/1, and MDA‐MB‐361) at the concentration of 100 µM. Additionally, these com-
pounds have also induced a dose‐dependent cytotoxic effect in hypoxic MCF7 cells, where hCA IX expression is
highly induced.
324
4‐(thiazol‐2‐ylamino)‐benzenesulfonamides possessing good hCA IX inhibitory action were
tested for their anticancer activity in vitro by using the MCF cell line. Compounds 342–345 have shown IC
50
in the
range of 2.79–11.90 µM against MCF‐7 cells (Figure 24). However, compound 342 was the most active (IC
50
=
2.79 µM) cytotoxic agent against MCF‐7 and it was found four times more active as compared with standard drug
fluorouracil.
233
A series of pyrrole and pyrrolopyrimidine scaffold containing sulfonamides were developed as
potent hCAs inhibitors, which have also shown promising anticancer effect in vitro. Most of the synthesized
compounds showed an effective cytotoxic effect against MCF‐7 cells. Interestingly, Compounds 346 and 347
(Figure 24) were the most potent cytotoxic agents which displayed an IC
50
value of 6.46 µM against MCF‐7 cell
line.
325
Sulfonamides incorporating chromone moiety have been also evaluated for their anticancer activity. These
sulfonamides were potent and highly selective hCA IX as well as hCA XII inhibitors. These derivatives exerted
promising cytotoxic effects against MCF‐7 and A‐549 cell lines. Compound 348 (Figure 25) was the best cytotoxic
agent which displayed an IC
50
value of 0.72 µM against MCF‐7 and of 0.50 µM against A‐549 cells. The apoptotic
potential of this compound was evaluated by Annexin V‐FITC/PI staining using a flow cytometer. The result
indicated that compound 348 significantly induced apoptosis in MCF‐7 cells and A549 cells where 94.12% and
74.77% early apoptotic cells were observed in MCF‐7 cells as well as A549 cells, respectively.
316
A series of sildenafil analogues as potent hCA IX/hCA XII inhibitors were also examined for their anticancer
activity in vitro. These derivatives showed a moderate to good cytotoxic effect against MCF‐7 and MDA‐MB‐231
cell lines. Compound 349 (Figure 25) was the most potent cytotoxic agent among the entire series and this
derivative showed an IC
50
value of 99 and 102 µM against MDA‐MB‐231 and MCF‐7. The cytotoxic potential of
this compound was slightly less as compared with standard chlorambucil which showed an IC
50
value of 93 and
54
|
MISHRA ET AL.
97 µM against MDA‐MB‐231 and MCF‐7 cells. The effect of this compound on DNA synthesis in both cell lines was
also studied and compound 349 has shown an IC
50
value of 80 and 87 µM against MDA‐MB‐231 and MCF‐7 cells,
being slightly less potent than chlorambucil.
326
Benzenesulfonamides bearing pyrrole, pyrrolopyrimidine and fused
pyrrolopyrimidine moieties were selective and very effective hCA IX as well as hCA XII inhibitors also showed
promising cytotoxic effect against MCF‐7 cells. Indeed, most of the derivatives have shown a better cytotoxic
effect (IC
50
in the rage of 7.29–7.84 µM) than doxorubicin (IC
50
value of 8.02 µM) against MCF‐7 cells. However,
compounds 350 and 351 were most effective and displayed an IC
50
value of 7.29 µM against MCF‐7 cells
(Figure 25).
327
Arylsulfonehydrazone benzenesulfonamides 352 and 353 (Figure 25), selective and potent hCA IX
inhibitors also bestowed effective cytotoxic effect against MCF‐7 and MDA‐MB‐231 cancer cell lines under
hypoxic conditions. Compound 352 was more effective as compared with 353 and showed an IC
50
value of 5.14
and 2.33 µM against MDA‐MB‐231 and MCF‐7, respectively. However, Compound 353 displayed an IC
50
value of
FIGURE 24 Chemical structure of hCAs inhibitors 338–347 displaying potent anticancer activity. hCA,
human carbonic anhydrase
MISHRA ET AL.
|
55
18.22 and 3.9 µM against MDA‐MB‐231 and MCF‐7, respectively.
328
Grandane et al. evaluated in vitro anticancer
activity of 6‐Substituted sulfocoumarins which have exhibited very effective and selective inhibition for hCA IX as
well as hCA XII. Sulfocoumarins 278 (Figure 19) and 354 (Figure 25) demonstrated effective cytotoxicity in both
normoxia and hypoxia conditions after 72 h of treatment. In normoxia condition, reduced cell viability was iden-
tified for sulfocoumarin 278 after 16 h incubation at the concentration of 30 μM. With increasing incubation time,
its efficacy enhanced at the same concentration and reached at maximum after 72 h of incubation by showing 43%
cell viability. Incubation time and concentration did not significantly influence the cytotoxic effect of 278.
Remarkably, a hypoxic condition also did not influence the cytotoxic potential of compound 278 and a maximum of
35% cell viability was observed after 72 h of incubation at 100μM concentration. Likewise, another derivative 354
(Figure 25) also showed a maximum cytotoxic effect with 100 μM concentration after 72 h incubation in both
normoxia as well as hypoxia conditions. It was assumed that these compounds bestowed cytotoxic effect by
FIGURE 25 hCAs inhibitors 348–358 possessing potent anticancer action. hCA, human carbonic anhydrase
56
|
MISHRA ET AL.
governing multiple mechanisms of action.
296
CAIs bis‐sulfonamide bearing ethylene glycol oligomeric/polymeric
diamines linkers appeared to be potent anticancer agents. These compounds were evaluated for their anticancer
activity in 2D and 3D (tumor spheroids) models of cancer by using HT‐29, MDA‐Mb231, and SKOV3 cell lines. Bis‐
sulfonanides 355–358 showed significant cytotoxicity against these cell lines by decreasing cell viability
(Figure 25). Bis‐sulfonamide 358 was the most effective CAI, which displayed a propitious cytotoxic effect against
all three cell lines under both conditions; normoxia and hypoxia. It demonstrated excellent cell‐killing effect by
showing cell viabilities 60–70% at 10 μM, 50% at 100 μM and 30–40% at 1 mM. In 3D cultures, compound 358
showed an excellent cell‐killing effect against tumor spheroids of HT‐29 and SKOV‐3 cells, being less effective
FIGURE 26 Chemical structure of hCAs inhibitors 359–373 with potent anticancer action. hCA,
human carbonic anhydrase
MISHRA ET AL.
|
57
against MDA‐MB231 cells. This compound showed a cell viability of 80% in SKOV‐3, 70% in HT‐29, and 90% in
MDA‐MB231 at 100 μM.
329
Coviello et al. reported anticancer activity of 1,2‐benzisothiazole derivatives 359 and 360 as potent hCA IX
and hCA XII inhibitors (Figure 26). Both compounds significantly inhibited proliferation of HT‐29 concentration
dependently with an IC
50
value of 44.10 and 50.97 μM, respectively under hypoxia condition. Additionally, com-
pound 359 displayed robust synergistic action in combination with well‐known standard drugs, SN‐38 and 5‐FU.
After 72 h incubation with SN‐38 and 5‐FU, under hypoxic condition, this compound showed an IC
50
of
0.029 ± 0.012 and 10.95 ± 2.73 μM, respectively, against HT‐29 cells. Thus, these derivatives showed promising
antiproliferative action against HT‐29 cells alone as well as a combination with SN‐38 and 5‐FU, which evokes its
strong synergistic action.
330
Falsini et al. examined the antiproliferative activity of highly selective and potent hCA
IX as well as hCA XII inhibitors 3‐hydroxyquinazoline‐2,4‐dione derivatives against HT‐29 colon cancer cell line.
The result indicated that compounds 361–364 showed significant cytotoxicity by inducing about 50% cell death in
HT‐29 cell (Figure 26). Strangely, no significant difference was detected between normoxia and hypoxia conditions
toward the cytotoxic effect of these compounds. Additionally, a significant cytotoxic effect of compounds 361–364
was highlighted at 30 μM concentration after 72 h incubation.
305
Purine/pyrimidine moieties containing benze-
nesulfonamide derivatives also displayed effective anticancer activity in vitro by inhibiting hCA IX. The cytotoxic
activity of potent hCA IX inhibitors 365–368 has been assessed against HT‐29 cells under normoxia as well as
hypoxia conditions (Figure 26). In normoxic conditions, compounds 366,367, and 368 showed moderate cytotoxic
effect by inducing cell mortality by about 10−15% at the concentration of 300 μM. Interestingly, hypoxia condition
improved cytotoxic effect of derivatives 365,366, and 368 by decreasing 25–30% cell viability after 48 h in-
cubation. In a hypoxic environment, compound 366 inimitably preserved cytotoxic effect after 16 h incubation,
although, compounds 367 and 368 enhanced cytotoxic profile after 48 h of incubation, it may be due to robust
upregulation of hCA IX. Remarkably, the cytotoxic effect of compound 365 was increased with prolonged hypoxia
condition. Thus, adenine analogues 365 and 368 showed a remarkable gain in cytotoxic action after prolong
hypoxia, which evokes their antiproliferative activity due to inhibition of overexpressed hCA IX (Figure 26).
331
Bonardi et al. investigated antiproliferative action of coumarin derivatives which, have emerged as potent and
selective hCA IX as well as hCA XII inhibitors. Antiproliferative action of selected coumarin derivatives was
examined against HT‐29 cells in time and concentration dependent manner under normoxia as well as hypoxia
condition. The result of the cytotoxic evaluation points out that some derivatives such as 309,369, and 370
(Figures 21 and 26) were potent cytotoxic agents and showed a concentration as well as time dependent activity.
Compounds 309 and 370 reduced cell viability up to 35% and 50%, respectively, at the concentration of 100 µM
after 48 h incubation. In hypoxic condition, cytotoxic efficacy was increased and after 48 h of incubation coumarins
analogues 309,310 (Figure 21) and 371 (Figure 26) showed effective cytotoxicity against HT‐29 cells. Coumarin
371 demonstrated excellent cytotoxic action and reduced cell viability up to 70%.
314
Heterocyclic 4‐substituted
pyridine‐3‐sulfonamides emerged as potent hCA IX and hCA XII inhibitors and their anticancer activity were
assessed against a panel of 60 human tumor cell lines at a single concentration 10 µM. Among all, compound 372
containing 4‐(3,4,‐dichlorophenyl)piperazine moiety displayed broad‐spectrum cytotoxicity against 26 cell lines and
exhibited IGP in the range of 25–89% (Figure 26). Particularly, compound 372 showed IGP 72%, 65%, 89%, and
65% against T‐47, OVCAR‐4, SK‐MEL‐5, and K562 cell lines.
332
Bisphosphonates as dual inhibitors of metalloproteinase (MMP) and hCA (hCA IX and hCA XII) also showed
antiproliferation action against the J774 cell line. Five most potent compounds of this series were tested for their
cytotoxic effect against J774 cells and these derivatives showed effective cytotoxic effect in J774 cells. However,
compound 373 was the most effective cytotoxic agent and displayed an IC
50
value of 1.7 µM against J774 cells
(Figure 26).
333
Morris et al. reported antiproliferative action of carbohydrate liked benzene sulfonamides, which
were potent hCA IX and hCA XII inhibitors. In vitro anticancer activity of these derivatives was assessed against
two fibroblast cell lines (one lacking endogenous expression of CA IX and another one over‐expressing CA IX).
Investigation identified compounds 374 and 375 as effective cytotoxic agents that selectively blocked CA IX‐
58
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MISHRA ET AL.
induced cell survival (Figure 27). Derivative 374 was also evaluated against human colon carcinoma cells, LS174Tr
and this compound reduced mRNA level and CA IX protein in colon carcinoma cells under normoxia and hypoxia
conditions Moreover, after 3 days of incubation, compound 374 displayed a 60% decrease in cell viability in colon
carcinoma cells and also decreased 16% cell viability in ca9/ca12 double‐silenced cells.
334
In vivo anticancer
potential of potent and highly selective hCA IX (Ki; 9.3 nM) as well as hCA XII (Ki; 43 nM) inhibitor 7‐glycosylated
4‐methyl coumarin 219 has been evaluated by Touisni et al. In vivo model has been developed by implanting 4T1
cells into the mammary fat pad of BALB/c mice (Figure 16). The result of this study evoked that the treatment of
compound 219 significantly inhibits tumor growth of mice. Compound 219 also significantly reduced tumor volume
as compared with untreated animals. Remarkably, this compound did not produce any noticeable toxicity to the
animals during the whole study period. Thus, selective CA IX inhibitor 219 emerged as a potent anticancer agent
which may be applied alone or combination with conventional chemotherapy or radiation to target the hypoxic
FIGURE 27 Chemical structure of hCAs inhibitors 374–382 with potent anticancer action. hCA,
human carbonic anhydrase
MISHRA ET AL.
|
59
tumor where expression of hCA IX is always high.
274
Benzene sulfamates bearing urea functionality strongly
inhibited cancer cell migration. In vitro study point out that compounds 376,377, and 378 inhibited cell migration
in both normoxia and anoxia (Figure 27). CA IX inhibitors 377 (Figure 27), 223, and 224 (Figure 16) showed
inhibition of eGFP‐MDA‐MB‐231 cell migration by 49%, 32%, and 50%, respectively in anoxia. It was noticed that
these inhibitors significantly inhibited eGFP‐MDA‐MB‐231 cell migration at the concentration of 33 μM. Com-
pound 223 also inhibited cell migration in WRO and FTC‐133 thyroid carcinoma cell lines, which were well studied
CA IX positive. Additionally, derivative 224 inhibited proliferation of MDA‐MB‐231 cells dose dependently and
showed an IC
50
value of 481 μM. This compound also exerted cytotoxic effect in HCT116 and HT29 cells with an
IC
50
value of >1000 and 20 μM, respectively. In an in vivo study, treatment with compound 224 significantly
abridged the metastatic tumor load in the lungs of mice bearing orthotopic eGFP‐MDA‐MB‐231 tumors. Moreover,
treatment with compound 224 did not reduce the average body weight of animals and was found similar to the
vehicle treated animals.
276
Nitroimidazole incorporating sulfamide group (compound 380, Figure 27) was a potent
hCA IX/hCA XII inhibitor and examined for its anticancer activity against HT‐29 tumor bearing mice. Compound
380 significantly inhibited extracellular tumor acidification in colorectal HT‐29 and the cervical HeLa carcinoma
cells overexpressing CA IX. Treatment with compound 380 significantly reduced tumor growth in HT‐29 tumor
bearing mice, with an average time to reach 4× starting volume (T4×SV) of 25.43 and 23.39 days for compound
380 and standard drug doxorubicin. Additionally, compound 380 treatments did not produce any significant
toxicity to mice and no reduction on average body weight was observed during the whole treatment schedule.
Moreover, this compound also sensitizes tumors toward radiotherapy and doxorubicin‐based chemotherapy,
signifying the usefulness of this compound as potent anticancer agent.
335
2‐[4‐Chloro‐5‐methyl‐2‐(naphthalen‐1‐
ylmethylthio)benzenesulfonyl]‐1‐[4‐chloro‐6(4 sulfamoylphenylamino)‐1, 3, 5‐triazin‐2 ylamino]guanidine,
compound 381 was also explored as potent hCA IX/hCA XII inhibitor and antiproliferative agent (Figure 27).
Compound 381 demonstrated effective cytotoxic effect against HeLa cells with an IC
50
value of 17 ± 1 µM. Ad-
ditionally, this compound showed comparatively low cytotoxicity in normal cells HaCaT (IC
50
value of 61 ± 8 µM),
indicative selective cytotoxicity toward cancer cells.
336
Koyuncu et al. studied the anticancer activity of benze-
nesulfonamide Schiff base 382 (Figure 27) by performing various in vitro assays. Compound 382 showed a pro-
mising cytotoxic effect in HeLa cells with an IC
50
value of 11.3 µM at 72 h. This compound also showed low toxicity
in normal cells, such as HEK‐293 and PNT‐1A cells. Compound 382 significantly induced apoptosis in HeLa cells by
inducing extrinsic, intrinsic pathways and an autophagic pathway. Treatment of this compound significantly in-
fluenced Bcl‐2, Bax, caspase‐3, 8, 9, and 12, Beclin and LC3 expression to induce apoptosis in HeLa cells. Moreover,
compound 382 also induced ROS production and arrested the Sub‐G1 phase cell cycle in HeLa cells. Overall, these
extensive studies indicated promising anticancer activity of compound 380 which may appear beneficial to develop
this molecule as a potent chemotherapeutic agent in future.
337
5.3 |CAIs with anticonvulsant action
As discussed, some isoforms of CAs such as hCA II, hCA IV, hCA VII, and hCA XII, were well studied as a promising
target for the management of seizure. Several potent CAIs such as AAZ and TPM have shown effective antic-
onvulsant effect and successfully being used clinically.
237
To the search of potent anticonvulsant agents by tar-
geting hCA isoforms several novel chemical entities have been developed and these compounds were shown
effective anticonvulsant action in various in vivo models of epilepsy.
234,237,248
Our research group has designed and synthesized benzenesulfonamide‐based potent and selective hCA II/hCA
VII inhibitors and their anticonvulsant activity were assessed by using Maximal electroshock (MES‐test) and
pentylenetetrazol (sc‐PTZ test). The result of this investigation exposed that compounds 383,384, and 385 (Potent
hCA II/hCA VII in inhibitors) showed excellent anticonvulsant activity in MES as well as sc‐PTZ test (Figure 28).
Compounds 383,384, and 385 have shown 87%, 100%, and 87% protection, respectively, at 0.5 h time interval,
60
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MISHRA ET AL.
while 75%, 75%, and 87% protection was noticed, respectively, at 3 h time interval against MES induced seizure. In
sc‐PTZ test, these compounds also showed excellent protection against sc‐PTZ induced seizures with showing
protection in the range of 50–87%. These derivatives also appeared to be long duration acting anticonvulsant
agents and have shown 62–75% protection after 6 h of drug administration. These derivatives were also able to
control MES induced seizure upon oral administration and demonstrated 25–75% seizure protection after oral
dosing to Wistar rats. Remarkably, these three potent anticonvulsant agents did not exert any significant toxicity
to the experimental animals in sub‐acute toxicity study.
237
Further, we have altered this lead molecule and
developed two novel series of compounds containing N‐methylacetamide as well as N‐methylpropanamide linker.
Potent and selective hCA II/hCA VII inhibitors such as 386,176, and 387 have been evaluated against MES as well
as sc‐PTZ induced seizure (Figures 13 and 28). These derivatives showed promising protection against both tests
and displayed 50–75% seizure protection in MES test and 25–75% protection in sc‐PTZ test. Compound 387
FIGURE 28 Chemical structure of hCAs inhibitors 383–394 as potent anticonvulsant agents. hCA,
human carbonic anhydrase
MISHRA ET AL.
|
61
emerged as a long duration acting anticonvulsant and was active up to 6 h of postadministration. This derivative
was also orally active and significantly opposed MES induced seizure in rats upon oral administration. To evaluate
the safety parameter of this novel compound, a sub‐acute toxicity study was performed and the compound was
found to be a safe molecule without showing remarkable toxicity to rats.
248
Furthermore, the introduction of a
rigid linker between benzenesulfonamide head and substituted piperazine tail region, led to two series of novel
compounds bearing benzylidenehydrazine/benzylidenehydrazine carbonyl linkers. Some potent hCA II/hCA VII
inhibitors, such as 388 and 178 (Figures 13 and 28), have been appraised for their anticonvulsant action. These
derivatives inhibited MES as well as sc‐PTZ induced seizures in Swiss albino mice. Compound 178 was the most
potent anticonvulsant agent which demonstrated a long duration action and protected 60% of animals from MES
induced seizure after 6 h postadministration. Compound 178 also inhibited MES induced seizure upon oral ad-
ministration to rats, being nontoxic in sub‐acute toxicity study.
260
Masereel et al. reported aromatic/heterocyclic
sulfonamides incorporating valproyl moieties as potent hCAs inhibitors and evaluated their anticonvulsant action
by the MES test. The most effective anticonvulsants in the investigated series were 389,390, and 391 which
exhibited a rate of protection in the range of 25–44% at 0.5 h, and 87–100% at 3 h of postadministration
(Figure 28). 5‐valproylamido‐1,3,4‐thiadiazole‐2‐sulfonamide, 389 was one of the best hCAs inhibitor, which
showed promising anticonvulsant activity in the MES test in mice. Compound 389 also endowed with long duration
action and it showed 63% protection after 6 h of drug administration.
338
A series of 4‐benzenesulfonamide‐
carbamates have been also tested as potential hCAs inhibitors and anticonvulsant agents. These carbamates were
potent hCA II and hCA VII inhibitors and some of them also showed promising anticonvulsant activity in MES as
well as 6 Hz tests in rodents. Carbamates 392,393, and 394 have shown an ED
50
value of 136, 31, and 14 mg/kg in
MES test and 74, 53, and 80 mg/kg in 6Hz, respectively (Figure 28). In the mouse‐corneal‐kindling test, compound
394 had an ED
50
of 59 mg/kg, and this compound also showed an MES‐ED
50
value of 13 mg/kg in rats. These three
potent compounds showed some signal of teratogenicity at the dose of 1.8 mmol/kg dose, but lacked a teratogenic
potential at the dose of 0.9 mmol/kg. The fabulous anticonvulsant action of these compounds established them as a
potential candidate for further extensive evaluation and development of potent and safe new antiepileptic drug.
339
5.4 |CAIs as antiarthritis agents
Evidence from literature revealed a significant role of several CA isoforms in RA. It has been shown that CA
isoforms IX and XII are overexpressed in the inflamed synovium of JIA.
249
The role of other CA isoforms such as
hCAs I, III, and IV in RA was also studied.
253
In this regard, various potent CAIs were developed, which effectively
controlled RA in animal models.
249,253
A series of hybrid compounds integrating 6‐and 7‐substituted coumarins
(CAIs) derivatized with NSAIDs (indomethacin, ketoprofen, diclofenac, ibuprofen, sulindac, and ketorolac, etc.) for
the management of RA. Most of these hybrid molecules were potent hCA IV, hCA IX, and hCA XII inhibitors and
evaluated for their effectiveness at the dose of 10 mg/kg against CFA induced rat model of arthritis. The effects of
compounds were compared with classical NSAID ibuprofen at the dose of 100 mg/kg. The result indicated that
7‐coumarin substituted ibuprofen derivative 395 (Figure 29) was most efficacious among all and demonstrated
effective anti‐hyperalgesic effect at the dose of 10 mg/kg and activity was sustained for 60 min after adminis-
tration. This hybrid molecule was capable to significantly intensify the weight tolerated on the ipsilateral paw at
15 min (58.7 ± 1.3 g) and maximum effect was observed between 30 and 45 min (63.8 ± 1.3 and 61.3 ± 1.3 g,
respectively) after administration.
253
Recently, we have also developed NSAIDs and benzenesulfonamide‐based
hybrid molecules to study their anti‐hyperalgesic activity. These molecules were effective inhibitors for hCA I, hCA
II, hCA IX, and hCA XII. Some of the potent inhibitors were evaluated for their anti‐hyperalgesic action against in
an in vivo RA model by conducting paw‐pressure and incapacitance tests. Hybrid molecules 396 and 397
(Figure 29) were the most active and showed promising antinociceptive effects, which last up to 60 min after
administration. Both derivatives displayed dose dependent antihypersensitive effect and 10 mg/kg dose fully
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MISHRA ET AL.
responded the CFA‐induced hypersensitivity of the ipsilateral paw the effect lasted up to 75 min after adminis-
tration. Thus, hybrids, 396 and 397 were the best performing anti‐hyperalgesic agents with long lasting efficacy.
249
5.5 |Miscellaneous pharmacological actions of CAIs
The pain modulating effect of CAIs was also studied by Carta et al. In this study, authors developed various
sulfonamide‐based CAIs with effective hCA II, hCA VII, hCA IX and hCA XII inhibitory action. Potent CAI 398
(Figure 30) was assessed for its neuropathic pain modulating effect in comparison with AAZ against an oxaliplatin
FIGURE 29 Chemical structure of hCAs inhibitors 395–397 with potent antiarthritisaction. hCA,
human carbonic anhydrase
FIGURE 30 Chemical structure of hCAs inhibitors 398–401 with miscellaneous pharmacological action. hCA,
human carbonic anhydrase
MISHRA ET AL.
|
63
induced mouse model of neuropathic pain. The result showed that treatment with compound 398 relieved neu-
ropathic pain in a dose dependent (10–50 mg/kg) manner. The higher dose of this compound promisingly restores
the pain threshold up to the value of the control group. Compound 398 has a better potential to relieve pain as
compared with AAZ. Indeed, 100 mg/kg dose of AAZ did not completely reduce pain and it was active only for
15 min. Thus, this was the first report about the neuropathic pain modulation action of sulfonamides CAIs.
340
Some
potent CAIs belonging to sulfonamide and coumarin class were also examined against cerebral ischemia animal
model (permanent middle cerebral artery occlusion; pMCAO). The neurological score of pMCAO rats was thea-
trically abridged 24 h after occlusion. Repeated subcutaneous injections of the CAIs 399 and 400 significantly
increased the neurological score by 40% at the dose of 1 mg/kg (Figure 30). Additionally, derivative 400 displayed
the propensity to reduce the volume of hemisphere infarction. Remarkably, the standard CAI AAZ was ineffective
to improve the neurological score as compared with the negative control. However, a detailed investigation
regarding the involvement of a particular CA isoform in cerebral ischemia is needed.
341
Rodrigues et al. studied
anti‐trypanosomal activity of Trypanosoma cruzi (TcCA) associated CAIs, which belong to hydroxamate class.
Derivative 401 was studied extensively and showed promising anti‐trypanosomal activity (Figure 30). At low
concentration this derivative effectively inhibited growth of all three developmental forms of the parasite with an
IC
50
values in the range of 7.0 to <1 μM. The compound showed a selectivity index (SI) of 6.7, without showing
toxicity to macrophage cells. Preliminary in vivo data indicated that compound 401 significantly reduced blood-
stream parasites and interestingly, all treated mice survived and it appeared to be a more potent than the standard
drug benznidazole.
342
6|CLINICAL STAGE DEVELOPMENT STATUS OF CAIS
6.1 |Clinically approved CAIs and their therapeutic potential
Several CAIs such as AAZ, methazolamide, ethoxzolamide, and dichlorphenamide are successfully used clinically
for the last 45 years against various disorders. Recently, some other CAIs such as dorzolamide and brinzolamide
have also found imperative therapeutic applications as anti‐glaucoma drugs. AAZ, methazolamide, ethoxzolamide,
and dichlorphenamide were widely employed in the therapy of glaucoma.
6,342
However, AAZrepresents the best‐
studied drug and has been commonly used for years because it reduces IOP very effectively. In the long‐term
treatment, AAZ is provided in doses of 250 mg for each 6 h, whereas, methazolamide is administered in doses of
25–100 mg t.i.d.
6,343
However, such a way of treatment exerts undesired side effects due to CA inhibition in organs
other than the eye. The most common side effects are numbness and tingling of extremities, depression, metallic
taste, fatigue, weight loss, malaise, gastrointestinal irritation, and metabolic acidosis.
344
Therefore, topical
administration of the sulfonamide inhibitors directly into the eye was introduced by Becker.
345
Unfortunately,
clinically used these drugs presented negative results and ultimately these CAIs are effective for glaucoma
treatment only via the systemic route. Dorzolamide and brinzolamide are potent CAIs with potent anti‐glaucoma
action which were developed by Merck & Co. and Alcon Laboratories, respectively.
346
Both drugs possess a good
water solubility and are sufficiently liposoluble to penetrate through the cornea, and may be directly administered
into the eye, as a 2% water solution, several times a day. Clinically, these two drugs are very effective in reducing
IOP and displayed fewer side effects as compared with the systemically applied drugs.
344,347
AAZ is also very
effective in the therapy of macular edema when provided systemically. Dorzolamide and brinzolamide were also
found effective in this disease condition with topical administration.
348
Additionally, AAZ (375 mg/day) is also
found to be effective for the management of serious retinal detachment.
349
AAZ, methazolamide, and TPM are widely used as anti‐epileptics. The anticonvulsant effects of these CAIs are
possibly due to CO
2
retention secondary to inhibition of the red cell and brain enzyme. But other mechanisms
have been also hypothesized that are blockade of sodium channels, kainate/AMPA receptors, and intensification of
64
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MISHRA ET AL.
GABA‐ergic transmission.
234,237,350,351
AAZ alone or in combination with dexamethasone was shown to be effective in
the preclusion and management of mountain sickness and high altitude associated cerebral edema, due to the enhanced
arterial oxygen concentrations after red blood cell/brain enzyme inhibition by AAZ.
352
Use of AAZ has been also
noticed in the treatment of hydrocephalus by inhibition of CAs present in choroid plexus. It is found that treatment with
AAZ is superior to surgical treatment in kids with a slowly progressive hydrocephalus.
174
AAZ also appeared to be
effective to decrease gastric secretion and gastric ulcer healing, but a high dose (3–4 g/day for 2 weeks) of AAZ is
required to decrease gastric acid secretion. This much higher dose of AAZ may produce a severe unwanted side effect,
therefore, it is not recommended for the treatment of gastric ulcer clinically.
353
AAZ is also an effective diuretic and
was the first nonmercurial diuretic to be used clinically in 1956. AAZ promptly increases the urine volume and changes
normally acidic pH of urine into alkaline. Nowadays, use of AAZ is limited due to its side effect and availability of better
drug molecules. However, AAZ played an important role to understand renal physiology and pharmacology. It also
provided a suitable base for the development of many other effective high ceiling diuretics.
177
6.2 |CAIs in clinical trials
The small molecule CAI SLC‐0111 with potent hCA IX/hCA XII inhibitory action has shown promising anti-
proliferative effect in vitro as well as in vivo models. This compound was developed by the SignalChem Lifesciences
Corporation (SLC, British Columbia, Canada). SLC‐0111has successfully completed phase I clinical trial in Vancouver,
Canada for the treatment of CA IXoverexpressing solid tumors, being now in phase II trials.
354
Another potent CAI is
E7070/indisulam which have also shown anticancer activity against hypoxic tumors, and entered the clinical trials in
2005. This molecule showed very effective antitumor action in phase I clinical trial and currently running in phase II
clinical trial in the United States and Europe. Apart from CA inhibition, indisulam also inhibits cell cycle‐associated
cyclin‐dependent kinases (CDKs) and arrest G1/S phase cell cycle to halt cancer cell proliferation.
355
The monoclonal antibody targeting hCA IX girentuximab is also in Phase III (NCT00087022) clinical trials for
the management of RCCs. This antibody, particularly binds to CA IX expressed tumor cells and activates the
antigen‐dependent cellular cytotoxicity (ADCC) immune response which leads to induce tumor cell death.
356,357
124
I‐Chimeric monoclonal antibody G250, a monoclonal antibody fused with radionuclides provokes antibody‐
dependent cytotoxicity and targeted delivery of the radioactive materials as well as immunodetection. Another
monoclonal antibody radionuclide conjugate in which cG250 was labeled with I
131
has successfully completed Phase
II clinical trials for the treatment of patients with advanced renal carcinoma.
358,359
This conjugate is being in-
vestigated for radio‐immunodetection as well as radioimmunotherapy by targeting hCA IX (Table 5).
360
7|PROSPECTIVES OF CAIS IN LIFE SCIENCES
It is well studied that CAs actively participate in various pathophysiological processes in humans and established as
an imperative target for many disease conditions. Inhibition of CA isoforms by potent CAIs seemed to be very
beneficial to control various diseases like glaucoma, cancer, epilepsy, osteoporosis, RA, high altitude sickness, and
renal disorders, and so forth.
346–353
Some potent CAIs like AAZ, methazolamide, ethoxzolamide, and di-
chlorphenamide are used clinically for the treatment of glaucoma, many others also showed promising IOP low-
ering effect in preclinical study.
346,348–350
In anticancer drug development, CAIs are also playing an important role
and many of hCA IX/hCA XII inhibitor have shown excellent antiproliferative action including SLC‐0111, which is
currently ready to enter in phase II clinical trial for the treatment of metastatic, hypoxic solid tumors. Thus, the
impairment of cancer cell proliferation by targeting hCA IX/hCA XII could be highly beneficial for the treatment of
various types of cancer. This approach may also prove helpful to fight with problems connected to existing cancer
drug resistance.
357–360
MISHRA ET AL.
|
65
The development of CAIs as diagnostic tools could be considered as a good contribution to the health industry.
These investigations consist in the development of CAIs as MRI as well as PET agents.
358,359
These imaging agents have
shown effectiveness in various in vitro and in vivo models, and some of them are running in various stages of clinical
trials. The resistance of existing anti‐biotics is considered as a big challenge in infection management.
359
Studies have
shown that CAs are widely available in many eubacteria as well as Archaea and its inhibition by potent CAIs may kill
these pathogenic bacteria, with auspicious result in the use of ethoxzolamide for the management of meningitis.
361,362
Additionally, α‐,β‐and γ‐CAs have been purified in various species of bacteria such as Escherichia coli, H. pylori, Neisseria,
Synechocystis, Anabaena variabilis, Acetobacterium woodi,andRhodospirillum rubrum.
363
However, very few studies have
been performed to discover potent antibacterial agents by targeting CA isoform of bacteria. Searching of a potent anti‐
infective agent that targets bacterial CA will be very beneficial and demanding work for this era because nowadays
resistance of existing antibiotics is a major obstacle in infection management. Therefore, CA isoforms and their inhibitor
are indeed valuable for the biomedical field, and may offer fascinating opportunities for the development of various
novel drugs as well as diagnostic tools for the management of numerous pathological conditions.
8|CONCLUSIONS
CAs are considered as key enzymes for various physiological process and their catalytic action play a very crucial
role to maintain body function in humans. In past decades, several significant achievements have been made in the
field of CA research. Several selective inhibitors have been documented for hCA I, hCA II, hCA VII, hCA IX, and
TABLE 5 Small molecules/antibodies/imaging agents running in clinical trial by targeting hCA IX
Clinical trial Class CTID Treatment Status
SLC‐0111 Small molecule NCT02215850 Advanced solid tumors Phase I
(Completed)
E7070 Small molecule NCT00003891 Solid tumors Phase I
(Completed)
NCT00080197 Metastatic breast cancer Phase II
(Completed)
NCT01692197 Relapsed AML and High‐Risk
Myelodysplastic Syndrome
Phase II
(Completed)
Girentuximab(cG250) Monoclonal antibody NCT00087022 Patients undergoing non‐
metatstatic kidney cancer
Phase III
(Completed)
BAY 79‐4620 Antibody–Drug
Conjugate
NCT01028755 Advance stage tumor Phase I
(Completed)
124
I‐cG250 PET tracer NCT00003102 Kidney Cancer Phase I
(Completed)
Zr
89
‐gerentuximab Imaging agent NCT02883153 Renal cell Carcinoma Phase III
(Completed)
In
111
‐DOTA‐
gerentuximab‐
IRDye800CW
Imaging agent NCT02497599 Renal cell Carcinoma Recruiting
124
I‐cG250 Imaging agent NCT00606632 Renal cell Carcinoma Phase III
(Completed)
Note: Information acquired from clinical Trials.gov.
Abbreviation: hCA, human carbonic anhydrase.
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MISHRA ET AL.
hCA XII which are mostly belonging to sulfonamide, sulfamate and coumarin class. However, very few selective
inhibitors were reported for isoforms CA IV, CA VA, CA VB, and CA IV up to date. Additionally, numerous potent
CAs inhibitors have shown excellent therapeutic potential for the management of various disorders such as
glaucoma, cancer, epilepsy, arthritis, and obesity in preclinical as well as clinical trial study. Thus, inhibition of CAs
by potent inhibitors has provided a new way for the treatment of various disorders. In this review article, a detailed
debate has been carried out about strategies for the development of selective inhibition mechanisms, and selective
inhibitors developed in past decades have been categorized on the basis of their inhibition against particular
isoforms. Hopefully, this article will help to provide an appropriate way for the discovery and development of
various selective CAIs with effective pharmacological action, which is highly demanding in CA research field.
ACKNOWLEDGMENTS
This study was supported by DHR Young scientist research grant. CBM is thankful to Department of Health
Research (DHR) for Young Scientist Fellowship, MT is grateful to University of Delhi for financial support. CTS is
grateful to a grant from the Italian Ministry for Research and University (MIUR), PRIN: rot. 2017XYBP2R.
ORCID
Claudiu T. Supuran http://orcid.org/0000-0003-4262-0323
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AUTHOR BIOGRAPHIES
Chandra B. Mishra is currently working as a Scientist at College of Pharmacy, Sookmyung Women's University,
Seoul, South Korea. He received BSc degree from Magadh University Bodh Gaya, Bihar, India in 2005. He got
his master degree in 2005 in biomedical science from Bundelkhand University Jhansi, U.P, India. He obtained
PhD degree in 2013 in medicinal chemistry from University of Delhi, Delhi, India, and his research project
during doctoral study was design and synthesis of novel adenosine A
2A
receptor antagonist as potential anti‐
Parkinsonian agents. From 2014 to 2017 he carried out his Dr. D. S. Kothari Post‐doctoral research program
and involved in development of potent anti‐epileptic, anti‐Alzheimer's, and anticancer agents. Currently as a
Young Scientist, he is involved in development of selective carbonic anhydrase inhibitors, anti‐epileptic agents,
anti‐Parkinsonian agents and anticancer agents. He has published his research work in more than 30 papers
and he is also an inventor of four granted patents.
Manisha Tiwari is an assistant professor at Dr. B. R. Ambedkar Center for Biomedical Research, University of
Delhi. She got her master degree in chemistry in 1994 from Indian Institute of Technology, Kanpur, India. She
received her PhD degree in 1998 in chemistry from Department of Chemistry, University of Delhi, Delhi, India.
Her Research interest is drug development against various disorders such as Epilepsy, Alzheimer's, and Cancer.
She has published more than 47 research articles in reputed journal and she is an inventor of two granted
patents
Claudiu T. Supuran is working as a Full Professor at Dipartimento Neurofarba, Universita` degli Studi di
Firenze, Sezione di Scienze Farmaceutiche e Nutraceutiche, Florence, Italy. He received his BSc in chemistry
from the Polytechnic University of Bucharest, Romania (1987), and PhD in chemistry at the same university in
1991. His research interests focus on drug development against Carbonic anhydrases and other enzymes. He
has published more than 1700 research publication on carbonic anhydrase enzyme research and in these fields
being one of the most cited medicinal chemists worldwide. He is Editor‐in‐Chief of Expert Opinion on
Therapeutic Patents,Journal of Enzyme Inhibition and Medicinal Chemistry,Current Enzyme Inhibition, and so forth.
How to cite this article: Mishra CB, Tiwari M, Supuran CT. Progress in the development of human carbonic
anhydrase inhibitors and their pharmacological applications: Where are we today? Med Res Rev. 2020;1‐81.
https://doi.org/10.1002/med.21713
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