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Iranian Journal of Basic Medical Sciences
ijbms.mums.ac.ir
Synthesis and antiplasmodial activity of novel phenanthroline
derivatives: An in vivo study
Azar Tahghighi 1*, Safoura Karimi 1, 2 , Arezoo Rafie Parhizgar 1, 2, Sedigheh Zakeri 1
1 Malaria and Vector Research Group (MVRG), Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran
2 Department of Medicinal Chemistry, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran
A R T I C L E I N F O
A B S T R A C T
Article type:
Original article
Objective(s): Due to the rapid increased drug resistance to Plasmodium parasites, an urgent need to
achieve new antiplasmodial drugs is felt. Therefore, in this study, the new synthetic phenanthroline
derivatives were synthesized with antiplasmodial activity.
Materials and Methods: A series of 1,10-phenanthroline derivatives containing amino-alcohol and
amino-ether substituents were synthesized via facile procedures, starting with 5,6-epoxy-1,10-
phenanthroline. Their antiplasmodial activity was then evaluated using Peter's 4-day suppressive test
against Plasmodium berghei-infected mice (ANKA strain). Furthermore, the mean survival time of the
mice treated with synthetic compounds was compared with the negative control group.
Results: The results demonstrated that the compounds 6-(3-(dibutylamino)propylamino)-5,6-dihydro-
1,10-phenanthroline-5-ol (7b) at the dose of 150 mg/kg/day and 4-(1,10-phenanthroline-5-yloxy)-N,N-
dipropylbutan-1-amine (8b) at the dose of 15 mg/kg/day have 90.58% and 88.32% suppression,
respectively. All synthetic compounds prolonged the mean survival time of treated mice in comparison
with negative control groups, indicating the in vivo antiplasmodial activity of these new compounds.
Conclusion: The present study is the first attempt to achieve new, effective synthetic compounds based
on phenanthroline scaffold with the antiplasmodial activity. However, more research is needed to
optimize their antimalarial activity.
Article history:
Received: Jun 26, 2017
Accepted: Sep 28, 2017
Keywords:
Antiplasmodial activity
Malaria
Plasmodium berghei
Peter's test
1,10-Phenanthroline
Quinoline
►
Please cite this article as:
Tahghighi A, Karimi S, Rafie Parhizgar A, Zakeri S. Synthesis and antiplasmodial activity of novel phenanthroline derivatives: An in vivo
study. Iran J Basic Med Sci 2018; 21:202-211.
Introduction
Malaria is one of the most important parasitic
diseases worldwide, which is transmitted by female
anopheles mosquitoes. Based on WHO reports in
2015, 95 countries had ongoing malaria transmission
with an estimated 3.2 billion people at the risk of
malaria, especially Plasmodium falciparum, as the
most deadly malaria parasite in the world (1).
Furthermore, there were an estimated 214 million
new cases of malaria and 438,000 deaths annually,
which are mostly children. Despite many efforts to
control, eliminate, and eventually eradicate this
infection, malaria still remains the greatest global
health problem. However, for malaria control, there
are various methods such as personal protection,
mosquito control using insect repellents and
insecticides, malaria prophylaxis, and treatment with
antiplasmodial drugs. In fact, the initial detection and
treatment of the disease by itself are sufficient for the
control of this epidemic infection, at least at its early
stages. By these preventive actions, the parasite load
in the community is decreased, thereby reducing the
transmission of the disease.
Drug therapy is one of the main methods of
malaria control. There are some drugs that affect
different stages (exoerythrocytic, erythrocytic, and
sexual) of the parasite’s life cycle. For instance,
chloroquine (CQ), mefloquine (MQ), amodiaquine
(AQ), and halofantrine (HAL) are effective drugs in
parasite’s erythrocytic stage that interfere with
detoxification mechanism of the parasite (Figure 1)
(2). These drugs belong to the family of quinoline
analogs. Actually, CQ and AQ are 4-aminoquinoline
derivatives, whereas MQ and HAL are aryl-amino
alcohols derivatives. All these drugs have already been
used in malaria control, elimination, and eradication
programs because of their easy usage, affordable
synthesis, or great clinical efficacy. Some of them are
also safe for children and pregnant women.
Nevertheless, in recent years, the value of these drugs
for the prevention and treatment of malaria has
decreased after development and the spread of drug
resistance, especially against quinoline analogs (3, 4).
Indeed, the re-emerging of malaria in many endemic
areas of the world is attributed to the rapid increase
of resistance to available antiplasmodial drugs and the
*Corresponding author: Azar Tahghighi. Malaria and Vector Research Group (MVRG), Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran,
Iran. Fax: +98-21-66480749; email: atahghighi2009@gmail.com
Phenanthroline derivatives have antimalarial activity Tahghighi et al.
Iran J Basic Med Sci, Vol. 21, No.2, Feb 2018
203
HO
N
HO
CF3
CF3
N
H
Mefloquine
N
N
Cl
NH
N
N
Cl
NH
Chloroquine
OH
Amodiaquine
F3C
N
Cl
Cl
Halofantrine
N N+
X-NN+
Br-
O
NN+
SO42-
NNNN
HO H
NONPr2
NR2
8a: n=3
8b: n= 4
n
3
7a: R= Et
7b: R= Bu
1-N-benzyl-1,10-phenanthrolinium
bromide (1: X=Br-)
1-N-benzyl-1,10-phenanthrolinium
iodide (2: X=I-)
1-N-(4-methoxy-benzyl)-1,10-
phenanthrolinium bromide (3)
1-N-methyl-1,10-phenanthrolinium
sulfate (4)
2
NN+
Br-
O5
O
Quinine
N
HO
O
N
1-N-(3,4-dimethoxy-benzyl)-1,10-
phenanthrolinium bromide (5)
NN
(N-Benzoyl-N',N'-di(2-hydroxyethylthioureato)-S,O)(4-
methyl-1,10-phenanthroline)platinum(II) Chloride (6)
O
N
S
Pt
N
OH
OH
+
Figure 1. Antimalarial drugs (quinine, chloroquine, mefloquine, amodiaquine, and halofantrine), synthetic compounds with phenanthroline
scaffold (1-N-benzyl-1,10-phenanthrolinium bromide (1); 1-N-benzyl-1,10-phenanthrolinium iodide (2); 1-N-(4-methoxy-benzyl)-1,10-
phenanthrolinium bromide (3); 1-N-methyl-1,10-phenanthrolinium sulfate (4); 1-N-(3,4-dimethoxy-benzyl)-1,10-phenanthrolinium
bromide (5); (N-Benzoyl-N',N'-di(2-hydroxyethylthioureato)-S,O)(4-methyl-1,10-phenanthroline)platinum(II) chloride (6), and designed
compounds (amino-alcohol and amino-ether phenanthroline derivatives 7a-7b and 8a-8b)
resistance of vectors to insecticides. As an example, P.
falciparum is extremely resistant to CQ and MQ in the
areas where these drugs are used widely (5). In addition,
AQ resistance has been reported in South America, Asia,
and East Africa (6). It is noticeable that there is cross-
resistance between these quinoline drugs due to the
similarity of their chemical structures (7).
Considering the resistance problem to CQ and its
quinoline analogs, a new drug with different scaffolds,
known as HAL, was discovered. HAL was primarily
purposed for healthy people, to protect them from
malaria (8). This aryl-amino alcohol derivative with
phenanthrene scaffold is effective against CQ and
multi-drug-resistant P. falciparum malaria. But, its
use is limited to malaria treatment due to the risk of
toxicity and unreliable absorption. On the other hand,
development of MQ resistance resulted in cross-
resistance to HAL, thus reducing its usage (9).
Artemisinins, as the best antimalarial drugs in the
current situation, showed very rapid parasite clearance
Tahghighi et al. Phenanthroline derivatives have antimalarial activity
Iran J Basic Med Sci, Vol. 21, No.2, Feb 2018
204
times. Since artemisinins have a short half-life and are
fast acting, artemisinin-based combination therapy
(ACT), especially with a different class of long-lasting
antimalarial drugs, has been recommended for treating
P. falciparum malaria (10). Recently, resistance to ACTs
has been reported in Asian countries, which can be the
start of a catastrophic incidence in the world (11). It is
remarkable that drug resistance can lead to malaria
prophylaxis and treatment failure in the absence of an
alternative, tolerable and safe drug, particularly for
children and pregnant women. Therefore, pharma-
ceutical companies and academic researchers have
focused on the development of novel antiplasmodial
drugs. In this light, these groups considered two main
features for drug discovery: first, discovery of new
natural products with antimalarial activity and second,
the achievement of new synthetic medicines with
activity against the strains of the parasite, which is a
powerful tool for malaria control (12).
Drug development based on synthetic methods
plays a vital role in modern drug discovery, and in this
concern, the identification of lead compound is very
important. For instance, chloroquine was designed
and synthesized based on quinine, as an identified
natural product, for the purpose of decreasing
quinine side effects (3). Other quinoline analogs (such
as AQ, MQ, and HAL) were also prepared with the
replacement of the side chain or aromatic ring to
overcome drug resistance and to enhance desired
physiochemical or biological properties.
Due to the importance of aromatic or heteroaromatic
scaffolds in medicinal chemistry, other new compounds
with different scaffolds were synthesized and evaluated
in antiplasmodial tests (13). The 1,10-phenanthroline is
one of these heteroaromatic scaffolds that is considered
as diaza-analog of phenanthrene with two nitrogen
atoms at C-1 and C-10 positions and quinoline analog
with a fused pyridine ring. Therefore, considering the
side effects, high cost, and unreliable absorption of HAL,
the researchers synthesized its diaza-analogs by the
replacement of phenanthrene with 1,10-phenanthroline
and evaluated their antiplasmodial activities in both
in vitro and in vivo tests (Figure 1) (14-20).
In the present study, with regard to the spread of
resistance to quinoline antiplasmodial drugs, their
disadvantages, and the great potential of 1,10-
phenanthroline (14-19), four new phenanthroline
derivatives were synthesized and evaluated for the first
time against Plasmodium berghei (ANKA strain). Similar
to the available antiplasmodial drugs, these derivatives
were composed of aliphatic side chain containing
tertiary amine. They were synthesized from 5,6-epoxy-
1,10-phenanthroline as a starting agent. As shown in
Figure 1, the phenanthroline derivatives are divided into two
groups, amino-alcohol, and amino-ether phenanthroline
compounds. The antiplasmodial activity of the synthetic
compounds was also assessed by Peter's test in mice
inoculated with
P. berghei
. Furthermore, the mean survival
time of the mice treated with synthetic compounds was
compared with the negative control groups.
Materials and Methods
Chemistry
All chemical reagents and materials were purchased
from Sigma-Aldrich Company (USA). Solvents were
procured from Sumchun Company (South Korea).
The key intermediates 5,6-epoxy-1,10-phenanthroline
(9) and 5-hydroxy-1,10- phenanthroline (10) were
prepared based on the methods described in literatures
(21, 22). Uncorrected melting points were determined
on a Kofler hot stage apparatus. The IR spectra
were obtained on a Shimadzu 470 spectrophotometer
(potassium bromide dicks). 1H-NMR and 13C-NMR
spectra were recorded on a Varian Unity 500
spectrometer, and chemical shifts (δ) were reported in
parts per million (ppm) relative to tetramethylsilane, as
an internal standard. The mass spectra were run on a
Finigan TSQ-70 spectrometer (Finigan, USA) at 70 eV.
Elemental analyses were carried out on the CHN rapid
elemental analyzer (GmbH, Germany) for C, H, and N,
and the results were within 0.4% of the theoretical
values. Merck silica gel 60 F254 plates were used for
analytical TLC. The logP of compounds were performed
using ACD/ChemSketch Freeware version.
Table 1. The in vivo activities of four synthetic compounds (7a-b and 8a-b) against Plasmodium berghei
SD: Standard Deviation; * shows the most potent compounds
P-value
Mean survival
rate (day)
% Suppression of
parasitemia
Average %
parasitemia ± SD
Dose
(mg/kg)
logP
Compounds
Groups
< 0.0001
22.00
53.47
4.98 ± 0.43
150
1.73
7a
1
16.75
27.08
7.80 ± 1.04
100
17.75
18.50
8.72 ± 1.49
50
< 0.0001
22.25
90.58
1.01 ± 0.94
150
3.18
7b*
2
18.50
74.63
2.71 ± 1.14
100
17.50
22.25
8.32 ± 1.53
50
0.009
22.00
52.98
5.03 ± 1.15
30
3.83
8a
3
20.75
30.89
7.39 ± 1.49
20
19.50
13.22
9.28 ± 1.05
10
< 0.0001
21.67
88.32
1.25 ± 1.24
15
4.28
8b*
4
21.00
47.94
5.57 ± 1.13
12.5
18.75
17.17
8.86 ± 1.34
10
15.50
0
10.47± 2.42
-
-
PBS
5
16.00
0
10.93 ± 2.075
20 %
-
DMSO
6
-
100
-
25
-
CQ
7
Phenanthroline derivatives have antimalarial activity Tahghighi et al.
Iran J Basic Med Sci, Vol. 21, No.2, Feb 2018
205
Synthesis of 6-(3-(diethylamino)propylamino)-5,6-
dihydro-1,10-phenanthroline-5-ol (7a)
A mixture of 0.2 g (1.02 mmol) 5,6-epoxy
phenanthroline (9) and 1.6 ml (10.15 mmol) 3-
(diethylamino)propylamine in absolute ethanol was
refluxed at 80 °C for 24 hr. The completion of the
reaction was detected by TLC, and the solvent was
removed under reduced pressure to obtain a brown
solid. The solid was dissolved in dichloromethane
(100 ml) and washed with aqueous NaOH 10%. Then
the organic layer was separated and washed with brine
(30 ml) and was dried using sodium sulfate. The filtrated
organic layer was concentrated by a rotary evaporator.
The final product was purified by silica gel column
chromatography (dichloromethane/ethanol) for obtain-
ing a cream solid.
Synthesis of 6-(3-(dibutylamino)propylamino)-5,6-
dihydro-1,10-phenanthroline-5-ol (7b)
A mixture of 0.2 g (1.02 mmol) 5,6-epoxy phenan-
throline (9) and 2.4 ml (10.65 mmol) 3-(dibutylamino)
propylamine in absolute ethanol was stirred at room
temperature for eight days. The completion of the
reaction was detected by TLC and the solvent was
removed under reduced pressure to obtain a brown
grassy solid. The solid was dissolved in dichloromethane
(100×2 ml) and washed with aqueous NaOH 10%. The
organic layer was then separated and washed with brine
(30 ml) and dried using sodium sulfate. The filtrated
organic layer was concentrated by a rotary evaporator.
The final product was purified by silica gel column
chromatography (dichloromethane/ethanol) to obtain a
cream solid.
General procedure for the synthesis of intermediates
11a-b
A mixture of 0.11 g (4.58 mmol) sodium hydride and
0.2 g (1.02 mmol) 5-hydroxy-1,10-phenanthroline (10)
in 15 ml of ethanol was stirred vigorously at room
temperature for 30 min. The mixture was then added to
the solution of dibromo alkyl (5.96 mmol) in dry THF
dropwise; this mixture was refluxed for ~3-4 hr. The
completion of the reaction was detected by TLC. After
filtration, the solvents were removed under reduced
pressure to obtain a yellow viscose solid. Excess of
dibromo alkyl was removed by hot petroleum ether, and
finally, a cream solid was obtained.
General procedure for the synthesis of compounds 8a-b
A volume of 0.65 mmol bromoalkoxy-1,10-phenan-
throline (11a-b), 2.5 mmol dipropylamine, and 3 g
potassium carbonate were mixed in 20 ml of absolute
ethanol, and the mixture was then refluxed for ~24-28
hr. The completion of the reaction was detected by TLC,
and the solvent was removed under reduced pressure to
obtain a dark yellow viscose solid. The residue was
decanted with H2O and chloroform. The organic phase
was separated, and the solvent was removed under
reduced pressure to obtain a yellow solid. The solid was
purified by silica gel column chromatography (ethyl
acetate/petroleum ether) until a cream solid was
obtained.
Evaluation of antiplasmodial activity (Peter's test)
The experimental female BALB/c mice (6-8
weeks) were purchased from Pasture Institute of Iran
(Tehran) and were kept under standard conditions
for ten days to adapt to the laboratory animal housing
facilities. The synthetic compounds, 7a-b and 8a-b,
were administered intraperitoneally to three female
BALB/c mice for 5 days with the concentrations of 10
to 150 mg/kg/day. The signs of mortality in each
group were monitored daily. The optimum dose of
compounds 7a-b was 150 mg/kg/day, while for
compounds 8a and 8b were 30 and 15 mg/kg/day,
respectively. The antiplasmodial (schizontocidal)
activity of synthetic compounds (7a-b and 8a-b) was
evaluated using the 4-day suppressive test against P.
berghei infection in mice (23). The 19-22 g mice were
weighed and randomized into seven groups and again
weighted after the experiment. The stock of CQ-sensitive
P. berghei (ANKA) parasite (500 µl containing 25% P.
berghei) was defrosted and injected into two female
BALB/c mice. Next, five animals were selected and
infected with P. berghei through passaging. Each animal
was inoculated IP with 2×107-infected erythrocytes of P.
berghei in PBS (200 μl) on the first day (D0) of the
experiment. The compounds were solubilized in 20%
DMSO and prediluted in PBS to make appropriate
concentrations. The first treatment was accomplished
three hours after the mice were infected (D0) and
treated daily for four consecutive days (D4). Groups 1
and 2 were treated with compounds 7a and 7b (50, 100,
and 150 mg/kg/day) by IP injection for four days,
whereas groups 3 and 4 were treated with compounds
8a (10, 20, and 30 mg/kg/day) and 8b (10, 12.5, and 15
mg/kg/day) (Table 1). Mice groups 5 and 6 received PBS
and 20% DMSO as negative controls, and mice group 7
was treated by CQ (25 mg/kg/day), as a positive control,
for four days (Table 1). On day four, tail blood smears
were taken, stained with 10% Giemsa stain in phosphate
buffer (pH 7.2) for 20 min and then visualized under a
microscope at 100 magnifications to determine the
parasitemia level. The parasitized red blood cells on
at least 2,000 red blood cells were counted to calculate
the percentage of parasitemia (%parasitemia = the
number of infected RBC/the total number of RBC 100).
The percentage of parasitemia suppression for each
group was evaluated by comparing the percentage of
parasitemia in negative controls with that in the treated
group (%suppression = parasitemia in negative control
- parasitemia in treated group/parasitemia in negative
control ×100). During the treatment, all mice were
weighed on days 0 and 4. Also, the dissection of the
internal organs (spleen, liver, and kidney) was done on
Tahghighi et al. Phenanthroline derivatives have antimalarial activity
Iran J Basic Med Sci, Vol. 21, No.2, Feb 2018
206
the seventh day of treatment. The kidneys of the treated
groups did not show any change. Furthermore, the
mortality of mice was monitored daily during
experiment up to 24 days post-infection, and the mean
survival rate of each group was calculated.
Statistical analysis
Control and test data were analyzed using SPSS
(version 22.0, 2012). One-way ANOVA was used to
test the statistical differences for three doses within a
group, followed by LSD and Tukey’s test for pairwise
comparisons. P≤0.05 was considered statistically as
significant.
Results
Chemistry
The pathway for the synthesis of compounds 7a-b
is shown in Scheme 1. The intermediate of 5,6-epoxy-
1,10-phenanthroline (9) was obtained from the
reaction of 1,10-phenanthroline with aqueous sodium
hypochlorite (21, 24; Scheme 1). The reaction of 5,6-
epoxy-1,10-phenanthroline (9) with alkyl diamines in
absolute ethanol gave compounds 7a-b in good yields.
Indeed, epoxide is reactive due to the ring strain and can
easily react with alkyl diamines through nucleophilic
attack. It is remarkable that the epoxide ring opening is
stereospecific, and nucleophilic attack with inversion
gives trans product. The compound 7a, the hydrogens of
the phenanthroline nucleus, indicated a triplet at 8.62
ppm (H1 and H8 in phenanthroline), doublet at 7.90 ppm
(H3 in phenanthroline), and multiplet at 7.40 ppm (H2
and H7 in phenanthroline). The compound 7a had a
trans format, which was confirmed by a doublet at
4.74 ppm (H4 in phenanthroline) and a doublet at 3.80
ppm (H5 in phenanthroline) with coupling constant
~9.5 Hz.
The spectral data confirmed the structure of the
derivatives. In 1HNMR spectra of compound 7b,
the hydrogens of the phenanthroline nucleus showed
a broad singlet at 8.72 ppm (H1 and H8 in
phenanthroline), two sets of doublet at 8.03 ppm
(H3 in phenanthroline) and 7.88 ppm (H6 in
phenanthroline), and a multiplet at 7.31 ppm (H2 and
H7 in phenanthroline). Also, a doublet at~4.84 ppm
(H4 in phenanthroline) and a doublet at~3.92 ppm (H5
in phenanthroline) were detected with coupling
constant~10.5 Hz, which confirms the formation
of trans product. In the 1HNMR spectra of compounds
7a-b, the aliphatic hydrogens in the side chain on the
phenanthroline nucleus were recognizable regarding
their spin-spin splitting patterns.
The synthesis of target compounds 8a and 8b is
outlined in Scheme 1. As shown in the Scheme, the
intermediate of 5-hydroxy-1,10-phenanthroline (10)
was obtained from 5,6-epoxy-1,10-phenanthroline
based on the method reported previously (22). The
intermediates of bromoalkoxy-1,10-phenanthroline
(11a-b) were obtained from the reaction of
compound 10 in the presence of sodium hydride, as a
strong and a solid base in dry ethanol, which can
deprotonate the hydroxyl group. This mixture was
then added to excess dibromo alkyl in dry tetrahydrofuran
and refluxed. The reaction of intermediates 11a-b in
absolute ethanol with dipropylamine in the presence of
excess potassium carbonate afforded the final compounds
(8a-b).
The 1HNMR spectra of compound 8a, the hydrogens of
the phenanthroline nucleus in DMSO-d6 showed a
doublet at 9.12 ppm (H1 in phenanthroline), a broad
singlet at 8.91 ppm (H7 in phenanthroline), a doublet-
doublet at 8.67 ppm (H3 in phenanthroline), a triplet at
8.33 ppm (H5 in phenanthroline), two sets of multiplet at
7.79 and 7.67 ppm (H2 and H6 in phenanthroline), and a
singlet at 7.37 ppm (H4 in phenanthroline). The
compound 8b, the hydrogens of the phenanthroline
nucleus, indicated two sets of triplet at 9.19 and 9.00 ppm
(H1 and H7 in phenanthroline), a doublet-doublet at 8.68
ppm (H3 in phenanthroline), a doublet at 8.07 ppm (H5 in
phenanthroline), two sets of multiplet at 7.63 and 7.54
ppm (H2 and H6 in phenanthroline), and a singlet at 6.92
ppm (H4 in phenanthroline) in its 1HNMR spectra in CDCl3.
The aliphatic hydrogens in the side chain on the
phenanthroline nucleus of compounds 8a-b were
recognizable with regards to their spin-spin splitting
patterns in 1HNMR spectra. Finally, the formation of all
synthetic compounds was confirmed by different analysis
methods, including 13CNMR, Mass, and CHN analysis.
6-(3-(diethylamino)propylamino)-5,6-dihydro-1,10-
phenanthroline-5-ol (7a)
Yield: 78%, m.p. > 300 °C. 1HNMR(CDCl3, 500 MHz) δ:
8.62 (t, 2H, J= 8 Hz, phen), 7.90 (d, 2H, J= 8 Hz, phen),
7.40 (m, 2H, phen), 5.02 (br s, 1H, -OH), 4.74 (d, 1H, J=
9.5 Hz, phen), 3.80 (d, 1H, J= 9.5 Hz, phen), 2.80 (br s,
1H, -NH), 2.60 (t, 2H, J= 7 Hz, -CH2-), 2.38 (m, 4H, J= 7
Hz, -CH2-), 1.52 (t, 2H, J= 7 Hz, -CH2-), 0.93 (t, 6H, J=7
Hz, -CH3). Anal.Calcd for C19H26N4O: C, 69.91; H, 8.03;
N, 17.16. Found: C, 69.73; H, 7.71; N, 17.09.
6-(3-(dibutylamino)propylamino)-5,6-dihydro-1,10-
phenanthroline-5-ol (7b)
Yield: 58%, m.p. > 300 °C. 1HNMR(CDCl3, 500 MHz) δ:
8.72 (br s, 2H, phen), 8.03 (d,1H, J= 8 Hz, phen), 7.88
(d,1H, J= 8 Hz, phen), 7.31 (m, 2H, phen), 4.84 (d, 1H,
J= 10.5 Hz, phen), 3.92 (d, 1H, J= 10.5 Hz, phen), 2.95
(t, 2H, J= 6 Hz, -CH2-), 2.56 (t, 2H, J= 6.5 Hz, -CH2-), 2.42
(m, 4H, J= 7 Hz, -CH2-), 1.74 (m, 2H, J= 6.5 Hz, -CH2-),
1.42 (m, 4H, J= 7 Hz, -CH2-), 1.30 (m, 4H, J= 7.5 Hz, -
CH2-), 0.91 (t, 6H, J= 7.5 Hz, -CH3). MS (m/z, %) = 383.4
[M+, 31], 339.4 (2), 240.2 (6), 210.2 (6), 181.2 (53),
142.2 (100), 100.2 (73), 70.1 (6), 41.1 (33). Anal.Calcd
for C23H34N4O: C, 72.21; H, 8.96; N, 14.65. Found: C,
72.37; H, 8.76; N, 14.86.
Phenanthroline derivatives have antimalarial activity Tahghighi et al.
Iran J Basic Med Sci, Vol. 21, No.2, Feb 2018
207
NN
O
NN
HO H
NNR2
3
H2N-(CH2)3 -NR2
N N C2H5OH
NaClO
HCl , 18 oC
NaOH
97a; R= Et
7b; R= Bu
NN
O
N N
HO
H2SO4
NaOH NPr2
n
NaH
C2H5OH
reflux
reflux
NN
OBr
n
11a; n=3
11b; n= 4
NHPr2
C2H5OH
reflux
10
Br(CH2) nBr
8a; n=3
8b; n= 4
THF
1,10-phenanthroline
Scheme 1. Synthetic route for the preparation of compounds 7a-b and 8a-b
Figure 2. The effect of synthetic compounds (7a-b and 8a-b) intraperitoneally in different doses on the percentage of parasitemia of
Plasmodium berghei-infected mice (ANKA strain) on days 5 and 10 using the Peter's 4-day suppressive test
-5
0
5
10
15
20
25
30
35
40
45
% Parasitemia
Compounds (dose mg/kg/day)
Day 5
Day 10
Tahghighi et al. Phenanthroline derivatives have antimalarial activity
Iran J Basic Med Sci, Vol. 21, No.2, Feb 2018
208
Figure 3. Toxicity assay of treated mice with different doses of drugs (7a-b and 8a-b), including (A) body weight on days 1 and 5, (B)
hepatomegaly on day 7, (C) splenomegaly on day 7, and (D) the survival rate up to 24 days post infection
5-(3-bromopropoxy)-1,10-phenanthroline (11a)
Yield: 85%, m.p. > 300 °C. 1HNMR(DMSO-d6, 500
MHz) δ: 9.15 (d, 1H, J= 4.5 Hz, phen), 8.98 (d, 1H, J=
4.5 Hz, phen), 8.74 (dd, 1H, J= 6.5 & J=1.5 Hz, phen),
8.38 (dd, 1H, J= 6.5 and J =1.5 Hz, phen), 7.81 (m, 1H,
J= 4.5 Hz, phen), 7.71 (m, 1H, J= 4.5 Hz, phen), 7.40 (s,
1H, phen), 4.42 (t, 2H, J= 6.5 Hz, -CH2-), 3.84 (t, 2H, J=
6.5 Hz, -CH2-), 2.49 (m, 2H, J= 6.5 Hz, -CH2-
).13CNMR(DMSO-d6, 500 MHz) δ: 153.54, 153.50,
150.36, 147.49, 147.40, 147.36, 135.01, 130.61,
129.12, 123.44, 123.05, 102.33, 66.19, 31.64, 31.30.
5-(4-bromobutoxy)-1,10-phenanthroline (11b)
Yield: 93%, m.p. > 300 °C. 1HNMR(CDCl3, 500 MHz)
δ: 9.15 (d, 1H, J= 4 Hz, phen), 8.96 (d, 1H, J= 4 Hz,
phen), 8.60 (dd, 1H, J= 6.5 and J= 1.5 Hz, phen), 8.02
(dd, 1H, J= 6.5 and J=1.5 Hz, phen),7.60 (q, 1H, J= 6.5
Hz, phen), 7.49 (q, 1H, J= 6.5 Hz, phen), 6.85 (s, 1H,
phen), 4.21 (br s, 2H, -CH2-), 3.49 (br s, 2H, -CH2-), 2.05
(br s, 2H, -CH2-), 1.22 (br s, 2H, -CH2-).
3-(1,10-phenanthroline-5-yloxy)-N,N-
dipropylpropan-1-amine (8a)
Yield: 65%, m.p. > 300 °C. 1HNMR (DMSO-d6, 500
MHz) δ: 9.12 (d, 1H, J= 3.5 Hz, phen), 8.91 (brs, 1H,
phen), 8.67 (dd, 1H, J= 8 and J= 2.5 Hz, phen), 8.33 (t,
1H, J= 3.5 Hz, phen), 7.79 (m, 1H, J= 4 Hz, phen),
7.67 (m, 1H, J= 4 Hz, phen), 7.37 (s, 1H, phen), 4.41 (t,
2H, J= 6 Hz, -CH2-), 2.89 (t, 4H, J= 7.5 Hz, -CH2-),
2.08 (br s, 2H), 1.55 (t, 4H, J= 7.5 Hz,
-CH2-), 0.89 (t, 6H, J= 7.5 Hz, -CH3). 13CNMR (DMSO-d6,
500 MHz) δ: 153.55, 153.50, 150.26, 147.49, 147.40,
147.36, 135.01, 130.62, 129.11, 123.34, 123.01,
102.31, 66.82, 66.15, 46.43, 31.66, 27.42, 19.15, 11.61.
MS (m/z, %) = 337.4 [M+, 3], 308.3 (6), 268.1(54),
236.2 (44), 196.2 (43), 167.2 (72), 140.1 (22), 114.1
(14), 73.2 (29), 45.1 (100). Anal.Calcd for C21H27N3O:
C, 74.74; H, 8.06; N, 12.45. Found: C, 74.36; H, 8.31; N,
11.87.
4-(1,10-phenanthroline-5-yloxy)-N,N-dipropylbutan-
1-amine (8b)
Yield: 54%, m.p. > 300 °C. 1HNMR(CDCl3, 500 MHz)
δ: 9.19 (t, 1H, J= 2.5 Hz, phen), 9.00 (t, 1H, J= 2.5 Hz,
phen), 8.68 (dd, 1H, J= 8 and J= 2.5 Hz, phen), 8.07 (d,
1H, J= 8 Hz, phen), 7.63 (m, 1H, J= 2.5 Hz, phen), 7.54
(m, 1H, J= 2.5 Hz, phen), 6.92 (s, 1H, phen), 4.27 (t, 2H,
J= 6.5 Hz, -CH2-), 2.62 (t, 2H, J= 7 Hz, -CH2-), 2.53 (t, 4H,
J= 7 Hz, -CH2-), 2.07 (m, 2H, J= 6.5 Hz, -CH2-), 1.90 (m,
2H, J= 7 Hz, -CH2-), 1.47 (m, 4H, J= 7 Hz, -CH2-), 0.89 (t,
6H, J= 6.5 Hz, -CH3). MS (m/z, %) = 351.3 [M+, 25],
322.2 (55), 282.1 (40), 250.1 (37.5), 221.1 (6.25),
196.1 (92.5), 167.1 (65), 140.1 (45), 114.2 (52.5), 87.2
(100), 45.2 (75). Anal.Calcd for C22H29N3O: C, 75.18; H,
8.32; N, 11.96. Found: C, 74.89; H, 8.01; N, 12.15.
In vivo antiplasmodial activity
The compound 8b, as the amino-ether derivative
of 1,10-phenanthroline, showed 88.32% in vivo
suppression of parasitemia at the low dosage of
15 mg/kg/day by IP route using Peter's 4-day
suppressive test against infected P. berghei (Table
1 and Figure 2) (23). However, 90.58% suppression
was observed for the compound 7b, as the amino-
Phenanthroline derivatives have antimalarial activity Tahghighi et al.
Iran J Basic Med Sci, Vol. 21, No.2, Feb 2018
209
Figure 4. Dissection of the internal organs of mice (spleen, liver,
and kidney) after treatment with compounds 7a-b and 8a-b on day
7. Kidneys of the treated groups did not show any change
alcohol derivative of 1,10-phenanthroline, at the high
dose of 150 mg/kg/day. During the treatment, all
mice were weighed on days 0 and 4 (Figure 3A). All
treated mice had weight reduction, which can be
related to the lack of 100% reduction of parasitemia
after treatment with synthetic compounds. Seven
days after treatment, one of the mice in each group
was randomly selected and dissected. The dissection
of the internal organs (spleen, liver, and kidney)
presented a mild enlargement of the spleen and liver
in the treated groups with compounds 7a-7b and 8a-
8b (Figures 3B and 3C, and 4) compared with the
control groups. The kidneys of the treated groups did
not show any change. The mortality of mice after IP
administration of the synthetic compounds was also
investigated, and all of the treated mice had a survival
rate higher than the negative control groups (Table 1
and Figure 3D).
The result of statistical analysis between the groups
demonstrated that the compound 7a in the high dose
(150 mg/kg/day) had a significant difference in
comparison to other doses (P<0.05) but did not show any
difference between the doses of 100 and 50 mg/kg
(P>0.05). The compound also indicated that difference
between the treated groups and the control groups was
statistically significant (P<0.05). The compound 7b
showed a significant difference not only in its three
doses but also in the control groups (P<0.05). On the
other hand, no difference was found for the
compound 8a between doses of 10 and 20 mg/kg as
well as between doses of 20 and 30 mg/kg (P>0.05)
among its groups. However, there was a difference
between the low concentration (10 mg/kg) and the
control groups (P<0.05). The comparison among the
three study groups of compound 8b as well as
between these groups and the control group indicated
no significant difference (P<0.05).
Discussion
Previous studies have shown that phenanthroline
derivatives have antiplasmodial activity (14-20). For
instance, the derivatives of N-benzyl-1,10-phenan-
throline (1 and 2) have been demonstrated to have
good activity against FCR-3 strain with the IC50 values
of 0.1 and 0.18 µM, respectively after 72-hr incubation
(Figure 1) (16). Indeed, the 1,10-phenanthroline ring
has metalloprotease inhibitory activity by chelating
metal ions. However, Sholikhah et al. (16) have
obtained contradictory result when synthesized the
compounds with N-aryl and N-alkyl substitution on
1,10-phenanthroline for blockage of the chelating site.
Their results confirmed that the antiplasmodial
activity of these compounds did not relate to the
chelating capacity. The compounds 1 and 2 are
nonpolar because of benzyl substituent and can easily
penetrate through the cell membrane. Sholikhah
et al. study has shown that the activity of N-benzyl-
1,10-phenanthroline derivatives was higher than that
of N-alkyl-1,10-phenanthroline derivatives. These
compounds have also been evaluated by the classical
4-day suppressive test against P. berghei (18). The
most potent compound was (1)-N-benzyl-1,10-
phenanthrolinium iodide (2) (LD50= 121.42 mg/kg
and ED50= 2.08 mg/kg). Investigations have again
revealed that the benzyl group is the most important
moiety for antiplasmodial activity. The compound
with soft anion conjugate (I-) has more effective
interaction with the cell membrane of the parasite,
hence giving a better antiplasmodial activity.
Modification of drug structure is a usual procedure to
achieve superior activity and less toxicity. Therefore, the
researchers designed and synthesized other 1,10-
phenanthrolinium derivatives. The antiplasmodial
activity of (1)-N-(4-methoxybenzyl)-1,10-phenanthro-
linium bromide (3) against two strains of P. falciparum,
FCR-3, and D10, have been indicated to have the IC50
values of 0.82 and 1.21 µM, respectively (15). The
suppression of parasitemia was never complete (100%
inhibition of parasite growth), and it had lower activity
compared to compounds 1 and 2. A previous study has
presented antiplasmodial activity of (1)-N-methyl-1,10-
phenanthrolinium sulfate (4) with the IC50 value of 260
nM and also showed that chloroquine diphosphate
was more potent than N-alkyl and N-benzyl-1,10
phenanthroline derivatives (17). Furthermore, the
modified fixed-ratio isobologram method has displayed
an in vitro additive interaction between the compound 4
and CQ. The compound (1)-N-(3,4-dimethoxybenzyl)-1,
10-phenanthrolinium bromide (5) was synthesized, and
the result of heme polymerization inhibitory activity
assay revealed that the IC50 value of 3.63 mM had more
antiplasmodial activity than CQ (19). The compound 5
has two nitrogens; the positively charged nitrogen
interacts with the electronegative oxygen at ferriproto-
porphyrin IX, and the other nitrogen (base) reacts with
the carboxylic acid group at ferriprotoporphyrin IX.
Tahghighi et al. Phenanthroline derivatives have antimalarial activity
Iran J Basic Med Sci, Vol. 21, No.2, Feb 2018
210
Thus, the heme polymerization process can be
prevented. A complex of 1,10-phenanthroline platinum
(II) benzoyl thiourea (6) presented a suitable activity
against K1 and D10 strains of P. falciparum with the IC50
values of 488 and 282 nM, respectively (20). The
complex showed a strong in vitro interaction with
ferriprotoporphyrin IX and inhibited β-hematin
formation. The strong interaction of the phenanthroline
complex with ferriprotoporphyrin IX is attributed to the
extended planar structure of phenanthroline ring with
delocalized electrons in all of these complexes.
In vitro studies may lead to outcomes that do not
relate to the situation occurring around a living
organism. Therefore, in vivo studies is often apply
more than in vitro because it is suitable for observing
the overall effects of an experiment on a living
microorganism.
In vivo evaluation of antimalarial compounds
typically begins with the use of rodent malaria
parasites, especially in drug discovery. In an
extensively studied model of murine malaria, mice
are infected with P. berghei, which is considered as a
strong tool for biological studies in the field of
malaria. In fact, P. berghei is genetically similar to P.
falciparum and morphologically to P. vivax; therefore,
it could be a good template for the study of malaria
interventions.
In the present work, new amino-alcohol and amino-
ether phenanthroline derivatives were synthesized and
represented satisfactory results in inhibiting the
parasitemia of P. berghei infection in BALB/c mice, though
the reduction of parasitemia was never completed. Table
1 illustrates the mean percentage of parasitemia and the
percentage of suppression for each group at four
days. The best antiplasmodial compounds, 7b and 8b,
showed a significant activity (P≤ 0.05) and a high
mean survival rate of about 22 days for mice (Table 1,
Figure 3D). More important, compounds 1 and 2
showed 63.71 and 82.27% growth inhibition in a dose
of 12.8 mg/kg, whereas compound 4 presented
92.82% at a dose of 25.6 mg/kg. Antiplasmodial
activity of these compounds was evaluated using
Peter's 4-day suppressive test against inoculated
mouse with 1107 P. berghei-infected erythrocytes.
The compound 8b indicated 88.32% in vivo
suppression of parasitemia at the low dosage of 15
mg/kg/day by IP route against inoculated mouse with
2107 P. berghei-infected erythrocytes. Therefore, we
can draw the conclusion that the compound 8a is a
better candidate than the previously reported
compounds.
Lipophilicity plays an important role in biological
activity. In the current study, the amino-alcohol
compound 7b with logP= 3.18 showed 90.58%
suppression in the high dose (150 mg/kg/day) in
comparison to its analog (7a, logP= 1.73) that indicated
53.47% suppression in the same dose (Table 1).
However, amino-ether compound 8b with high
lipophilicity (logP= 4.28) was toxic in the concentration
higher than 20 mg/kg/day. On the other hand, this
compound showed a high suppressive effect in the
concentration of 15 mg/kg/day, as compared to its
analog 8a (logP= 3.83; Table 1). Both compounds 7b
with N,N-(dibutylamino)propylamino moiety and
compound 8b with N,N-dipropylbutan-1-amine moiety
presented a high antiplasmodial activity in their groups.
Mechanistic studies have shown that CQ and its
analogs interfere with the mechanism of heme
polymerization by malaria parasite (25-27). Indeed, in
the P. falciparum food vacuole (FV) is changed heme to
hemozoin, which is a safe pigment for the parasite. This
process is essential for the survival of the malaria
parasite (26), whereas the antiplasmodial drugs
(quinoline analogs) inhibit heme polymerization, which
results in accumulation of toxic-free heme in FV and also
leads to parasite’s death. Therefore, the inhibition of
hemozoin formation is an excellent drug target for the
development of antimalarial drugs (28). It is assumed
that our synthetic compounds can also accumulate in FV
and trap in its acidic (protonated) form. As a result, the
new compounds have the ability to inhibit the formation
of hemozoin and to increase the intracellular heme,
which is toxic to the parasite. On the other hand, these
compounds with new substitutions at position 4 of
phenanthroline ring can have metalloprotease
inhibitory activity because of free nitrogen atoms. These
mechanistic studies can be evaluated in the next projects
of our research group.
Conclusion
The present study illustrates the synthesis of new
antiplasmodial compounds with phenanthroline
scaffold. The results of this investigation revealed that
the best compounds against P. berghei were
derivatives of amino-alcohol phenanthroline 7b and
amino-ether phenanthroline 8b. Although the
decrease in the percentage of parasitemia was less
than the reference drug in infected mice, with the
spread of CQ resistance in different regions of the
world, the necessity for a new, safe, well-tolerated and
an affordable alternative drug is highly felt. Moreover,
further research is required to be carried out on these
compounds to optimize their antiplasmodial activities
such as formulation strategies, co-formulation with
other antimalarial drugs, and drug delivery systems.
Acknowledgment
The authors are grateful to Dr H Baseri
(Department of Medical Entomology, School of Public
Health, Tehran University, Tehran, Iran) for providing
P. berghei (ANKA). This project (no. 740) has received
a financial support from Pasteur Institute of Iran (PII),
Tehran, Iran.
Phenanthroline derivatives have antimalarial activity Tahghighi et al.
Iran J Basic Med Sci, Vol. 21, No.2, Feb 2018
211
Compliance with ethical standards
All applicable and acceptable guidelines for the
care and use of animals were considered.
Conflicts of interest
The authors declare no conflicts of interest.
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