Synthesis, anti-HIV-1 and antiproliferative evaluation of novel 4-nitroimidazole derivatives combined with 5-hydroxy-4-pyridinone moiety

Article abstract of DOI:10.1016/j.molstruc.2019.127344

In an effort to synthesize more effective non-nucleoside reverse transcriptase inhibitors (NNRTIs) against the HIV-1 infection, a new series of novel 4-nitroimidazole derivatives combined with 5-hydroxy-4-pyridinone moiety were designed by molecular docking studies, prepared and characterized by spectroscopic techniques. All the synthesized compounds were in vitro evaluated for their inhibitory effect against the HIV-1 replication in the MT-4 cells. Results showed that none of these synthesized compounds displayed any specific anti HIV-1 activity. Surprisingly, these compounds showed high cytotoxicity against MT-4 cells with low selectivity index (<1), therefore could be new potential antiproliferative agents. Therefore, their antiproliferative property were evaluated against MOLM-13 and K562 (leukemia) cell lines. Compound 14g showed notable antitumor activity toward both studied cell lines (EC₅₀ = 1.3 μM and EC₅₀ = 1.8 μM respectively).

Full text of DOI:10.1016/j.molstruc.2019.127344

Journal Pre-proof
Synthesis, anti-HIV-1 and antiproliferative evaluation of novel 4-nitroimidazole
derivatives combined with 5-hydroxy-4-pyridinone moiety
Pouria Shirvani, Afshin Fassihi, Lotfollah Saghaie, Siska Van Belle, Zeger Debyser,
Frauke Christ
PII:
S0022-2860(19)31453-X
DOI:
https://doi.org/10.1016/j.molstruc.2019.127344
MOLSTR 127344
Reference:
To appear in: Journal of Molecular Structure
Received Date: 8 September 2019
Revised Date: 31 October 2019
Accepted Date: 1 November 2019
Please cite this article as: P. Shirvani, A. Fassihi, L. Saghaie, S. Van Belle, Z. Debyser, F. Christ,
Synthesis, anti-HIV-1 and antiproliferative evaluation of novel 4-nitroimidazole derivatives combined
with 5-hydroxy-4-pyridinone moiety, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2019.127344.
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Graphical abstract
A series of novel 2-((5-substituted-4-nitro-1H-imidazol-1-yl)methyl)-5-hydroxy-1-methylpyridin-4(1H)-one were prepared and their
anti-HIV-1 activity was evaluated. Because of high cytotoxicity against MT-4 cells, none of them were active toward HIV-1.
However, they displayed promising antiproliferative activity against leukemia cell lines MOLM13 and K562.
MT-4
EC90
MOLM13
K562
Compd.
EC50
(µM)
CC50
(µM)
EC50
(µM)
EC50
(µM)
SI
(µM)
>5.5
>7.3
>3.6
>5.5
5.5
7.3
3.6
<1 4.27±0.89 8.01±0.72
<1 1.3±0.39 1.8±0.44
<1 3.65±0.64 5.79±0.53
14f
14g
14j
>7.3
>3.6

Synthesis, anti-HIV-1 and antiproliferative evaluation of
novel 4-nitroimidazole derivatives combined with
5-hydroxy-4-pyridinone moiety
Pouria Shirvani a, Afshin Fassihi a, b, *, Lotfollah Saghaie a, Siska Van Belle c, Zeger Debyser c,
Frauke Christ c, *
a
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Science, Isfahan University of
Medical Science, Hezar Jerib, 817416-73461, Isfahan, Iran
b Bioinformatics Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical
Sciences, 81746-73461 Isfahan, Iran
c
Department of Pharmacological and Pharmaceutical Sciences, Laboratory of Molecular Virology and Gene
Therapy, KU Leuven, Belgium
*
Corresponding author. Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Science,
Isfahan University of Medical Science, Hezar Jerib, 817416-73461, Isfahan, Iran. Email: Fassihi@pharm.mui.ac.ir
*
Corresponding author. Department of Pharmacological and Pharmaceutical Sciences, Laboratory of Molecular
Virology and Gene Therapy, KU Leuven, Belgium. Email: frauke.christ@kuleuven.be
1

Abstract
In an effort to synthesize more effective non-nucleoside reverse transcriptase inhibitors
(NNRTIs) against the HIV-1 infection, a new series of novel 4-nitroimidazole derivatives
combined with 5-hydroxy-4-pyridinone moiety were designed by molecular docking studies,
prepared and characterized by spectroscopic techniques. All the synthesized compounds were in
vitro evaluated for their inhibitory effect against the HIV-1 replication in the MT-4 cells. Results
showed that none of these synthesized compounds displayed any specific anti HIV-1 activity.
Surprisingly, these compounds showed high cytotoxicity against MT-4 cells with low selectivity
index (<1), therefore could be new potential antiproliferative agents. Therefore, their
antiproliferative property were evaluated against MOLM-13 and K562 (leukemia) cell lines.
Compound 14g showed notable antitumor activity toward both studied cell lines (EC50= 1.3 ꢀM
and EC50= 1.8 ꢀM respectively).
Keywords: 4-nitro-imidazole, anti-HIV activity, 5-hydroxy-4-pyridinone, antiproliferative,
NNRTI
2

1. Introduction
Combination antiretroviral therapy (cART), in which two or more antiretroviral agents are
combined has become an inevitable mean of taking the control of HIV-1 infection [1]. Due to
potent antiviral activity, desirable pharmacokinetic profile, moderate cytotoxicity and high
selectivity, None-nucleoside reverse transcriptase inhibitors (NNRTIs) play indispensable role in
cART regimens [2-4]. They interact in a noncompetitive manner with an allosteric hydrophobic
pocket distal to polymerase active site, called none-nucleoside inhibitor binding pocket (NNIBP)
and change the conformation of polymerase active site [5, 6]. Currently, there are six NNRTIs
which have been approved by the US food and drug administration (FDA) for anti-HIV-1
therapy, namely Nevirapiene (NVP), Delaverdine (DLV), Efavirenze (EFV), Etravirine (TMC-
125), Rilpivirine (TMC-278) and Doravirine (MK-1439) [7, 8]. Unfortunately, the effectiveness
of the first generation of NNRTIs (NVP, DLV and EFV) has been seriously decreased by the
rapid emergence of mutant HIV-1 strains [9, 10]. The chemical structures of these compounds
are provided in Figure 1. Moreover, poor pharmacokinetic and other drug toxicity have been
reported with the second generation of NNRTIs (TMC-125 and TMC-278) [11, 12].
Consequently, looking for novel chemical skeletons with high potency and different resistant
profile is still needed.
Figure 1. Structures of NNRTIs drugs approved by the US FDA
3

Malignant tumors have always been a major health problem with a high mortality rate.
Despite significant progress in the diagnosis and treatment of cancer diseases, many kinds of
tumors survival for a long time. Anti-cancer research is still a challenging and promising field in
the life sciences. Surgery, chemotherapy and radiotherapy are the most common ways in the
treatment of cancer today. However, these traditional methods and specially chemotherapy and
radiotherapy, are associated with disadvantages of poor selectivity, plentiful side effects and the
emergence of drug resistance [13].
By the recent years a significant progress has been achieved toward elucidating the
molecular mechanism of cancer disease, including key cancer cell signal transduction pathways,
apoptosis induction, regulator molecules of cell-cycle, angiogenesis and cell-extracellular matrix
interactions [14]. These findings have brought a new era to cancer treatment called “targeted
therapy”. Therefore, enzymes with crucial roles in signal transduction pathways associated with
cancer cell differentiation and proliferation have attracted attention in developing new anticancer
drugs acting on specific targets. Most of the protein tyrosine kinases enzymes (PTKs) associated
with cell proliferation, survival and migration [15]. thus the small molecule tyrosine kinase
inhibitors (TKIs) are regarded some of the most effective agents for targeted cancer therapy [16].
The imidazole ring as an interesting scaffold could interact with various cations and
anions as well as biological molecules in human body. Imidazole-based compounds have
demonstrated tremendous potential in medicinal chemistry. In fact, several imidazole-based
compounds have been employed as clinical agents such as antifungal (Ketoconazole and
Clotrimazole), antihistamine (Cimetidine), antiparasitic (Benznidazole and Metronidazole) and
anticancer (Azathioprine, Tipifarnib and Dacarbonize) for treatment of diverse types of diseases.
Therefore, there have been increasing research effort for utilizing imidazole-based compounds as
nitric oxide synthase inhibitors [17, 18], DNA alkylating agents [19, 20], kinase inhibitors [21-
23], anti-inflammatory agents [24, 25] and Histamine H3 agonists and antagonists [26, 27].
Moreover, different laboratories engaged in the development of novel NNRTIs using imidazole-
based compounds [28-32]. Capravirine (AG-1549), was the first imidazole-based NNRTI which
reached clinical trials, but its development was discontinued in phase II because of a lack of
significant advantages over other second generation NNRTIs (Figure 2).
4

Figure 2. Chemical structures of Capravirine and Deferiprone
3-Hydroxy-4-pyridinones has been the subject of a great interest since the discovery of
Deferiprone as a strong iron chelator used in the treatment of iron overload, a problem with
transfusion therapy in thalassemia syndrome (Figure 2). This six-membered ring scaffold has
also been conjugated with other molecular moieties to increase the interactions with a particular
binding site or to make multi-target compounds. Moreover, 3-hydroxy-4-pyridinones displayed
anti-viral activity against the HIV-1 replication as well as herpes simplex (HSV-1 and 2),
probably through metal chelating mechanism [33-35]. Today, 3-hydroxy-4-pyridinones are rising
as possible candidates in designing novel multi-target antiretroviral agents.
Based on these results and in continuation of our strategy in employing 3-hydroxy-4-
pyridinones as anti-HIV-1 agents, herein we report the design of new 4-nitroimidazole
derivatives combined with 5-hydroxy-4-pyridinone moiety via structure based drug design
principle. As depicted in Figure 3, the isopropyl moiety in Capravirine was replaced with a nitro
group to enhance the hydrogen bonding of molecules with active site residues. Furthermore, a
hydroxypyridinone motif was employed to establish essential hydrogen bonds as the carbamate
group of Capravirine did. These newly designed 4-nitroimidazole compounds were synthesized
via a facile synthetic root and their anti-HIV-1 activities were determined against wild type
HIV-1. Structural characterization and anti-HIV-1 activities of the prepared compounds will be
discussed in detail [36, 37].
Figure 3. Structure based design of novel 4-nitroimidazoles
5

2. Experimental
2.1. Materials and methods
Chemicals and solvents were obtained from Merck, Sigma-Aldrich, Alfa Aesar and Samchun
chemical companies. Solvents were dried and purified by standard procedures. Melting points
(mp) were determined in Thermo Fisher scientific IA9200 apparatus. FTIR spectra were
measured on Shimadzu FTIR-8300 spectrophotometer with potassium bromide (KBr) as diluent.
13
1H-NMR and C-NMR spectra were recorded on a Bruker (Avance DRX-500) spectrometer in
DMSO-d6 solvent with tetramethylsilane (TMS) as internal standard. Chemical shifts δ were
reported in ppm downfield to TMS and coupling constants values (J) are estimated in Hertz
(HZ). The mass spectra were recorded on Agilent 5975c-inert MSD with Triple-Axis Detector
spectrometer. Elemental analyses were run on a Thermo Finnigan Flash EA-1112 series.
Reactions were monitored using thin layer chromatography (TLC) on pre-coated silica gel 60F254
TLC plates obtained from Merck. Column chromatography was carried out by silica gel 60
Merck (230-270).
The detailed spectral characterizations of the prepared compounds are provided below.
For the ease of NMR assignment, all atoms of the scaffold are numbered by simple, prime and
double prime symbols. It should be clarified that this numbering system is different from IUPAC
numbering method. The numbered scaffold is provided in Figure 4.
O
3²
4
5
OH
2²
3
2
N
4²
O
N1²
O2N
3¢
3¢
4
5
5
6
O
5²
O2N 4¢
6
3
2
1 N
2¢
4¢ N
N
4
2¢
X
1¢
CH3
2¢
O2N
5¢
N
R
3
6
3¢
4¢
1 N
CH3
5¢
1¢
N
H 1¢
N
1
6¢
Br
2
H
R
5¢
Figure 4. The numbered scaffolds of the synthesized compounds
2.1.1. Synthesis of 5-(benzyloxy)-2-(hydroxymethyl)-4H-pyran-4-one (2)
To a solution of kojic acid 1 (2.84 g, 20 mmol) in methanol (10 mL) was added sodium
hydroxide (0.88 g, 22 mmol) dissolved in water (5 mL). The reaction mixture was heated up to
reflux temperature before benzyl chloride (2.53 mL, 22 mmol) was added slowly to the reaction
flask. Then the resulting mixture was refluxed overnight. After removing the solvent by rotary
evaporator, the solid was washed by water and diethyl ether, and then recrystallized in ethanol to
give 2 as colorless needles (3.9 g, 84% yield); mp: 129-131 °C (lit. mp 132-133 °C) [39].
6

2.1.2. Synthesis of 5-(benzyloxy)-2-(hydroxymethyl)-1-methylpyridin-4(1H)-one (3)
4.13 mL of 40% methylamine (50 mmol) was added to a mixture of 2 (5.8 g, 25mmol) in ethanol
(10 mL) and heated at 70 °C for 3 hours. The mixture was concentrated by rotary evaporator, the
precipitate was filtered and washed by water to obtain 3 as off-white solid (5.5 g, 88% yield);
mp: 214-214 °C (lit. mp 215.5-217.5 °C) [39].
2.1.3. Synthesis of 5-(benzyloxy)-2-(chloromethyl)-1-methylpyridin-4(1H)-one (4)
To 12 mL of distilled thionyl chloride at 0 °C was added 5-(benzyloxy)-2-(hydroxymethyl)-1-
methylpyridin-4(1H)-one 3 (5 g, 20 mmol). This mixture was then stirred at room temperature
for 1 hour after which time pale yellow crystals formed. The product was washed by petroleum
ether and a small amount of acetone to afford amorphous white compound 4 (5.1 g, 96% yield);
mp: 166-167.5 °C (lit. mp 165.5-166 °C) [39].
2.1.4. Synthesis of 4-nitro-1H-imidazole (6)
A solution of imidazole 5 (20.4 g, 0.3 mol) in 95% sulfuric acid (60 mL), was heated up to 70 °C
and 60 mL of 65% nitric acid was added dropwise thereto. The temperature of the resulting
mixture was then elevated to 100 °C and refluxed for 5 hours. The reaction solution was poured
onto 100 g crushed ice and neutralized with 25% ammonia solution. Filtration, washing and
drying were further performed to give 6 as a white solid (23 g, 67% yield); mp: 311-312 °C
(lit. mp 310-311 °C) [40].
2.1.5. Synthesis of 4,5-dinitro-1H-imidazole (7)
4-Nitroimidazole 6 (5.6 g, 50 mmol) was dissolved in a minimum amount of concentrated
sulfuric acid and a mixture of fuming nitric acid (20 mL) and concentrated sulfuric acid (25 mL)
was added to it. The resulting solution was refluxed for 5 hours and after cooling poured onto
crushed ice and the mixture was adjusted to pH = 2 with sodium bicarbonate. The product was
extracted by ethyl acetate (3×50 mL). Concentration the extract yielded 7 as yellow crystals (3.5
g, 22 mmol);
mp: 171-173 °C (lit. mp 166-169 °C) [41].
2.1.6. General procedure for the synthesis of 4-Nitro-5-substituted-imidazoles 8a-h
To a solution of aniline derivatives (13 mmol) in ethanol (5 mL) was added 4,5-dinitroimidazole
7 (0.5 g, 3.2 mmol) and the mixture was refluxed at 75 °C for 20-28 hours. The progress of the
reaction was monitored by TLC (eluent: CH2Cl2/MeOH, 9:1), after completion, the reaction
7

mixture was cooled to room temperature and solvent was evaporated in vacuum. The resulting
precipitate was filtered, washed with ethanol and then purified by recrystallization in ethanol to
provide compounds 8a-h.
2.1.6.1. N-(4-bromophenyl)-4-nitro-1H-imidazol-5-amine (8a)
Orange powder; yield: 62%; mp: 272 °C (decomposed); FTIR (KBr, cm-1): 3417, 3309 (C1-NH),
3155, 1643, 1542 (NO), 1450, 1350 (NO), 1203, 1080, 948, 817, 748, 624; 1H-NMR (500 MHz,
DMSO-d6) δ (ppm): 13.72 (br s, 1H, N1ʹ-H), 9.36 (s, 1H, C2ʹ-H), 7.82 (s, 1H, C1-NH), 7.71 (s,
2H, Ar-H2 and Ar-H6), 7.49 (d, J = 8.4 Hz, 2H, Ar-H3 and Ar-H5).
2.1.6.2. N-(4-chlorophenyl)-4-nitro-1H-imidazol-5-amine (8b)
Orange powder; yield: 58%; mp: 258 °C (decomposed); IR (KBr, cm-1): 3309 (C1-NH), 3186,
1
1643, 1542 (NO2), 1458, 1350 (NO2), 1203, 1080, 918, 825, 748, 624; H-NMR (500 MHz,
DMSO-d6) δ (ppm): 13.76 (br s, 1H, N1ʹ-H), 9.37 (s, 1H, C2ʹ-H), 7.79 (s, 3H, Ar-H3, Ar-H5 and
C1-NH), 7.37 (d, J = 8.4 Hz, 2H, Ar-H2 and Ar-H6).
2.1.6.3. 4-Nitro-N-(p-tolyl)-1H-imidazol-5-amine (8c)
Orange powder; yield: 52%; mp: 223 °C (decomposed); IR (KBr, cm-1): 3271 (C1-NH), 3139,
1
2777, 1635, 1542 (NO2), 1460, 1350 (NO2), 1257, 1080, 941, 856, 810, 748, 624; H-NMR
(500 MHz, DMSO-d6) δ (ppm): 13.40 (br s, 1H, N1ʹ-H), 9.27 (s, 1H, C2ʹ-H), 7.71 (s, 1H, C1-
NH), 7.54 (d, 2H, J = 7.9 Hz, Ar-H2 and Ar-H6), 7.15 (d, J = 8.0 Hz, 2H, Ar-H3 and Ar-H5), 2.93
(s, 3H, CH3).
2.1.6.4. 4-((4-Nitro-1H-imidazol-5-yl)amino)benzonitrile (8d)
Orange powder; yield: 32%; mp: 266 °C (decomposed); IR (KBr, cm-1): 3325 (C1-NH), 3124,
1
2229 (CN), 1595 (NO2), 1481, 1427, 1373 (NO2), 1280, 1172, 1095, 833, 640; H-NMR
(500 MHz, DMSO-d6) δ (ppm): 13.99 (br s, 1H, N1ʹ-H), 9.59 (s, 1H, C2ʹ-H), 7.96 (d, 2H,
J = 8.5 Hz, Ar-H3 and Ar-H5), 7.87 (s, 1H, C1-NH), 7.74 (d, J = 8.8 Hz, 2H, Ar-H2 and Ar-H6).
2.1.6.5. N-(4-methoxyphenyl)-4-nitro-1H-imidazol-5-amine (8e)
Orange powder; yield: 32%; mp: 224 °C (decomposed); IR (KBr, cm-1): 3417, 3301 (C1-NH),
1
3193, 1643, 1512 (NO2), 1450, 1357 (NO2), 1249, 1203, 1080, 1033, 817, 612; H-NMR
(500 MHz, DMSO-d6) δ (ppm): 13.18 (br s, 1H, N1ʹ-H), 9.28 (s, 1H, C2ʹ-H), 7.63 (s, 1H, C1-
NH), 7.55 (d, 2H, J = 8.5 Hz, Ar-H2 and Ar-H6), 6.93 (d, J = 9.0 Hz, 2H, Ar-H3 and Ar-H5), 3.74
(s, 3H, O-CH3).
8

2.1.6.6. N-(3-chlorophenyl)-4-nitro-1H-imidazol-5-amine (8f)
Olive powder; yield: 64%; mp: 217 °C (decomposed); IR (KBr, cm-1): 3271 (C1-NH), 3139,
2777, 1620, 1535 (NO2), 1450, 1350 (NO2), 1242, 1087, 894, 810, 756, 671, 617; 1H-NMR (500
MHz, DMSO-d6) δ (ppm): 13.88 (br s, 1H, N1ʹ-H), 9.38 (s, 1H, C2ʹ-H), 7.66 (s, 1H, C1-NH), 7.33
(t, J = 8.1 Hz, 1H, Ar-H5), 7.22 (s, 1H, Ar-H2), 7.09 (d, J = 5.7 Hz, 1H, Ar-H), 6.96 (d, J = 8.0
Hz, 1H, Ar-H).
2.1.6.7. N-(3,5-dichlorophenyl)-4-nitro-1H-imidazol-5-amine (8g)
Orange powder; yield: 38%; mp: 276 °C (decomposed); IR (KBr, cm-1): 3332, 3263 (C1-NH),
3193, 3085, 2808, 1635, 1589 (NO2), 1419, 1350 (NO2), 1203, 1087, 918, 840, 810, 748, 663,
624; 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 14.01 (br s, 1H, N1ʹ-H), 9.48 (s, 1H, C2ʹ-H), 7.96
(s, 1H, Ar-H), 7.91 (s, 1H, Ar-H), 7.18 (s, 1H, C1-NH).
2.1.6.8. 4-Nitro-N-phenyl-1H-imidazol-5-amine (8h)
Olive powder; yield: 49%; mp: 227 °C (decomposed); IR (KBr, cm-1): 3301 (C1-NH), 3170,
1
2777, 1635, 1535 (NO2), 1427, 1350 (NO2), 1257, 1180, 1080, 910, 856, 748, 686, 632; H-
NMR (500 MHz, DMSO-d6) δ (ppm): 13.44 (br s, 1H, N1ʹ-H), 9.31(s, 1H, C2ʹ-H), 7.69 (s, 1H,
C1-NH), 7.34
(t, J = 7.8 Hz, 2H, Ar-H), 7.07 (t, J = 7.4 Hz, 2H, Ar-H), 6.94 (s, 1H, Ar-H).
2.1.7. Preparation of 2,4,5-tribromo-1H-imidazole (9)
To a solution of imidazole 5 (2 g, 30 mmol) and sodium acetate (22 g) in 50 mL glacial acetic
acid, was added a solution of Br2 (4.6 mL, 90 mmol) in acetic acid (10 mL) within 30 minutes.
The reaction mixture was stirred at room temperature for 2 hours and then poured into 50 mL ice
water. The resulting precipitate was filtered and washed with water to afford 9 as white solid (5.4
g, 59% yield); mp: 221-222 °C (lit. mp 218-221 °C) [42].
2.1.8. Preparation of 4-bromo-1H-imidazole (10)
To a reaction flask were added 2,4,5-tribromo-1H-imidazole 9 (3 g, 10 mmol), sodium sulfite
(6.3 g, 50 mmol) and 30 mL water and then heated at 110 °C for 6 hours. After cooling to room
temperature, the product precipitated as a yellowish solid was filtered and the aqueous phase was
extracted by ethyl acetate (3×30 mL). The combined organic extracts were washed with water
(3×30 mL) and dried over anhydrous magnesium sulfate. Ethyl acetate was removed by rotary
evaporator and yellowish solid 10 was obtained (1.25 g, 85% yield); mp: 131-132.5 °C
(lit. mp 130-131 °C) [43].
9

2.1.9. Preparation of 5-bromo-4-nitro-1H-imidazole (11)
4-Bromo-1H-imidazole 10 (1 g, 7 mmol) in 10 mL sulfuric acid was treated with 70% nitric acid
(0.5 mL, 7.7 mmol) and heated at 110 °C for 1 hour. The reaction was cooled to room
temperature and poured intro ice water. The target compound 11 deposited as off-white solid was
collected by filtration and dried (0.9 g, 67% yield); mp: 273-275 °C (lit. mp 271-272 °C) [52].
2.1.10. Preparation of 5-(benzyloxy)-2-((5-bromo-4-nitro-1H-imidazol-1-yl)methyl)-1-
methylpyridin-4(1H)-one (12)
A mixture of 4 (2.63 g, 10 mmol), K2CO3 (1.38 g, 10 mmol), KI (0.1 g) and 11 (1.9 g, 10 mmol),
in acetone (20 mL) was heated at 40 °C overnight. After completion of the reaction indicated by
TLC (eluent CH2Cl2/MeOH, 15:1), the mixture was cooled down to room temperature and the
obtained yellowish solid 12 was filtered and dried (2.9 g, 93% yield); mp: 242.5 °C
(decomposed); IR (KBr, cm-1): 2931, 1627 (C=O), 1581, 1535 (NO2), 1496, 1350 (NO2), 1296,
1
1126, 1010, 825, 732, 617; H-NMR (500 MHz, DMSO-d6, mixture of two isomers) δ (ppm):
8.24 (s, 0.39H,
C2ʹ-H), 8.15 (s, 0.56H, C2ʹ-H), 7.73 (s, 0.64H, C6-H), 7.72 (s, 0.36H, C6-H),
7.41-7.30 (m, 5H, Ar-H), 5,62 (s, 0.74H, N1ʹ-CH2), 5.45 (s, 1.22H, N1ʹ-CH2), 5.40 (s, 0.38H,
C3-H), 5.34 (s, 0.61H, C3-H), 4.97 (s, 2H, O-CH2), 3.70 (s, 3H, N1-CH3).
2.1.11. General procedure for the preparation of 4-Nitroimidazole derivatives 13a-h
A mixture of 4 (0.52 g, 2 mmol), K2CO3 (0.27 g, 2 mmol), KI (20 mg), and corresponding
imidazole derivatives 8a-h (2 mmol) in 10 mL acetone was heated at 55 °C overnight. After the
reactants were disappeared (monitored by TLC), the resulting mixture was cooled to room
temperature and the target compound was collected by filtration.
2.1.11.1.
5-(benzyloxy)-2-((5-((4-bromophenyl)amino)-4-nitro-1H-imidazol-1-yl)methyl)-1-
methylpyridin-4(1H)-one (13a)
Yellowish solid; yield: 92%; mp: 293.5 °C (decomposed); IR (KBr, cm-1): 3440, 3309 (NH),
3093, 3053, 2939, 1620 (C=O), 1573 (NO2), 1466, 1365 (NO2), 1296, 1218, 1126, 979, 817,
740, 624; 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 8.15 (s, 1H, C2ʺ-H), 7.82 (d, J = 8.9 Hz, 2H,
C3ʹ-H and C5ʹ-H), 7.70 (s, 1H, C6-H), 7.53 (d, J = 8.7 Hz, 2H, C2ʹ-H and C6ʹ-H), 7.41-7.30 (m,
5H,
OCH2-phenyl-H), 5.57 (s, 2H, N1ʺ-CH2), 5.40 (s, 1H, C3-H), 4.96 (s, 2H, O-CH2),
3.71 (s, 3H, N1-CH3).
10

2.1.11.2.
5-(benzyloxy)-2-((5-((4-chlorophenyl)amino)-4-nitro-1H-imidazol-1-yl)methyl)-1-
methylpyridin-4(1H)-one (13b)
Yellowish solid; yield: 87%; mp: 294 °C (decomposed); IR (KBr, cm-1): 3433, 3309 (NH), 3039,
2916, 1620 (C=O), 1558 (NO2), 1458, 1373 (NO2), 1296, 1234, 1118, 1010, 825, 748, 640;
1H-NMR (500 MHz, DMSO-d6) δ (ppm): 8.66 (s, 1H, C2ʺ-H), 8.23 (s, 1H, C6-H), 7.88
(d, J = 8.4 Hz, 2H, C2ʹ-H and C6ʹ-H), 7.46 (s, 2H, C3ʹ-H and C5ʹ-H), 7.45-7.37 (m, 5H,
OCH2-phenyl-H), 6.60 (s, 1H, C3-H), 5.84 (s, 2H, N1ʺ-CH2), 5.18 (s, 2H, O-CH2), 4.12 (s, 3H,
N1-CH3).
2.1.11.3.. 5-(benzyloxy)-1-methyl-2-((4-nitro-5-(p-tolylamino)-1H-imidazol-1-yl)methyl)pyridin-
4(1H)-one (13c)
Yellowish solid; yield: 90%; mp: 283.5 °C (decomposed); IR (KBr, cm-1): 3440, 3328 (NH),
3055, 2939, 1627 (C=O), 1573 (NO2), 1488, 1365 (NO2), 1288, 1218, 1134, 1087, 1010, 887,
732, 694, 755; 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 8.17 (s, 1H, C2ʺ-H), 7.59 (s, 1H, C6-H),
7.41-7.35
(m, 5H, OCH2-phenyl-H), 7.14 (d, J = 8.0 Hz, 2H, C2ʹ-H and C6ʹ-H), 7.07 (d, J =
C3ʹ-H and C5ʹ-H), 5.38 (s, 2H, N1ʺ-CH2), 5.26 (s, 1H, C3-H), 4.93 (s, 2H, O-CH2),
8.1 Hz, 2H,
3.61 (s, 3H, N1-CH3), 2.20 (s, 3H, C4ʹ-H).
2.1.11.4.
4-((1-((5-(Benzyloxy)-1-methyl-4-oxo-1,4-dihydropyridin-2-yl)methyl)-4-nitro-1H-
imidazol-5-yl)amino)benzonitrile (13d)
Yellowish solid; yield: 81%; mp: 293 °C (decomposed); IR (KBr, cm-1): 3394, 3325 (NH), 3101,
2931, 2221 (CN), 1604 (C=O), 1566 (NO2), 1465, 1365 (NO2), 1296, 1234, 1134, 1010, 840,
740, 702, 624; 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 8.17 (s, 1H, C2ʺ-H), 7.99 (d, J = 8.5 Hz,
2H, C3ʹ-H and C5ʹ-H), 7.76 (d, J = 8.6 Hz, 2H, C2ʹ-H and C6ʹ-H), 7.70 (s, 1H, C6-H), 7.41-7.35
(m, 5H, OCH2-phenyl-H), 5.58 (s, 2H, N1ʺ-CH2), 5.43 (s, 1H, C3-H), 4.96 (s, 2H, O-CH2), 3.71
(s, 3H, N1-CH3).
2.1.11.5.
5-(Benzyloxy)-2-((5-((4-methoxyphenyl)amino)-4-nitro-1H-imidazol-1-yl)methyl)-1-
methylpyridin-4(1H)-one (13e)
Orange solid; yield: 94%; mp: 265 °C (decomposed); IR (KBr, cm-1): 3440, 3301 (NH), 3093,
3055, 2931, 1627 (C=O), 1581 (NO2), 1458, 1378 (NO2), 1288, 1218, 1118, 1026, 825, 740,
640, 601; 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 8.11 (s, 1H, C2ʺ-H), 7.72 (d, J = 8.0 Hz, 2H,
C2ʹ-H and C6ʹ-H), 7.69 (s, 1H, C6-H), 7.41-7.35 (m, 5H, OCH2-phenyl-H), 6.93 (d, J = 8.9 Hz,
11

2H,
C3ʹ-H and C5ʹ-H), 5.55 (s, 2H, N1ʺ-CH2), 5.40 (s, 1H,
C3-H), 4.96 (s, 2H, O-CH2),
3.74 (s, 3H, O-CH3), 3.71 (s, 3H, N1-CH3).
2.1.11.6.
5-(Benzyloxy)-2-((5-((3-chlorophenyl)amino)-4-nitro-1H-imidazol-1-yl)methyl)-1-
methylpyridin-4(1H)-one (13f)
Mustard solid; yield: 74%; mp: 280 °C (decomposed); IR (KBr, cm-1): 3425, 3317 (NH), 3093,
3083, 1612 (C=O), 1581 (NO2), 1465, 1427, 1380 (NO2), 1288, 1218, 1126, 995, 902, 848, 740,
1
609; H-NMR (500 MHz, DMSO-d6) δ (ppm): 8.17 (s, 1H, C2ʺ-H), 8.07 (s, 1H, C2ʹ-H), 7.75
(dd, J = 8.1, J =2.1 Hz, 1H, C4ʹ-H), 7.72 (s, 1H, C6-H), 7.46, (m, 1H, C5ʹ-H), 7.41-7.35 (m, 5H,
OCH2-phenyl-H), 7.11 (dd, J = 7.9, J =2.0 Hz, 1H, C6ʹ-H), 5.58 (s, 2H, N1ʺ-CH2), 5.43 (s, 1H,
C3-H), 4.96 (s, 2H, O-CH2), 3.72 (s, 3H, N1-CH3).
2.1.11.7. 5-(Benzyloxy)-2-((5-((3,5-dichlorophenyl)amino)-4-nitro-1H-imidazol-1-yl)methyl)-1-
methylpyridin-4(1H)-one (13g)
Orange solid; yield: 22%; mp: 301 °C (decomposed); IR (KBr, cm-1): 3409 (NH), 3101, 1620
(C=O), 1581 (NO2), 1473, 1427, 1365 (NO2), 1308, 1226, 1126, 987, 933, 840, 802, 763, 702,
624; 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 8.19 (s, 1H, C2ʺ-H), 8.03 (s, 2H, C2ʹ-H and C6ʹ-
H), 7.71 (s, 1H, C6-H), 7.41-7.30 (m, 5H, OCH2-phenyl-H), 7.24 (s, 1H, C4ʹ-H), 5.59 (s, 2H,
N1ʺ-CH2), 5.43 (s, 1H, C3-H), 4.96 (s, 2H, O-CH2), 3.72 (s, 3H, N1-CH3).
2.1.11.8. 5-(Benzyloxy)-1-methyl-2-((4-nitro-5-(phenylamino)-1H-imidazol-1-yl)methyl)pyridin-
4(1H)-one (13h)
Mustard solid; yield: 94%; mp: 273 °C (decomposed); IR (KBr, cm-1): 3417, 3309 (NH), 3062,
3016, 2931, 1620 (C=O), 1581 (NO2), 1441, 1380 (NO2), 1288, 1218, 1118, 1010, 910, 732,
694, 601; 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 8.15 (s, 1H, C2ʺ-H), 7.81 (d, J = 8.0 Hz, 2H,
C2ʹ-H and C6ʹ-H), 7.71 (s, 1H, C6-H), 7.41-7.33 (m, 7H, OCH2-phenyl-H, C3ʹ-H and C5ʹ-H), 7.08
(t, J = 7.4 Hz, C4ʹ-H), 5.57 (s, 2H, N1ʺ-CH2), 5.42 (s, 1H, C3-H), 4.97 (s, 2H, O-CH2), 3.72 (s,
3H, N1-CH3).
2.1.12. General procedure for the preparation of 4-Nitroimidazole derivatives 13i-k
Substituted thiophenol (1 mmol) was added to a stirred suspension of 5-(benzyloxy)-2-((5-
bromo-4-nitro-1H-imidazol-1-yl)methyl)-1-methylpyridin-4(1H)-one 12 (0.419 g, 1 mmol) in
ethanol, containing potassium hydroxide ( 0.056 g, 1 mmol). The mixture was refluxed for 24-48
12

hours and then the precipitate that had formed was filtered off and purified by column
chromatography to give the products 13i-k.
2.1.12.1.
5-(Benzyloxy)-1-methyl-2-((4-nitro-5-(phenylthio)-1H-imidazol-1-yl)methyl)pyridin-
4(1H)-one (13i)
Yellow powder; yield: 59%; mp: 290 °C (decomposed); IR (KBr, cm-1): 3417, 1627 (C=O), 1581
1
(NO2), 1481, 1365 (NO2), 1257, 1226, 1141, 1087, 995, 756, 702; H-NMR (500 MHz,
DMSO-d6) δ (ppm): 8.14 (s, 1H, C2ʺ-H), 7.72 (s, 1H, C6-H), 7.68-7.65 (m, 2H, C3ʹ-H and C5ʹ-H),
7.49-7.46 (m, 3H, C2ʹ-H, C4ʹ-H and C6ʹ-H), 7.41-7.36 (m, 5H, OCH2-phenyl-H), 5.61 (s, 2H,
N1ʺ-CH2), 5.34 (s, 1H, C3-H), 4.96 (s, 2H, O-CH2), 3.70 (s, 3H, N1-CH3).
2.1.12.2.
5-(Benzyloxy)-2-((5-((4-chlorophenyl)thio)-4-nitro-1H-imidazol-1-yl)methyl)-1-
methylpyridin-4(1H)-one (13j)
Yellow powder; yield: 45%; mp: 284 °C (decomposed); IR (KBr, cm-1): 3417, 3093, 2923, 1627
1
(C=O), 1581 (NO2), 1481, 1365 (NO2), 1257, 1218, 1087, 1010, 825, 740; H-NMR (500 MHz,
DMSO-d6) δ (ppm): 8.12 (s, 1H, C2ʺ-H), 7.69 (s, 1H, C6-H), 7.57 (d, J = 9.0 Hz, 2H, C2ʹ-H and
C6ʹ-H), 7.41-7.38 (m, 5H, OCH2-phenyl-H), 7.32 (d, J = 8.7 Hz, 2H, C3ʹ-H and C5ʹ-H), 5.58
(s, 2H, N1ʺ-CH2), 5.36 (s, 1H, C3-H), 4.96 (s, 2H, O-CH2), 3.69 (s, 3H, N1-CH3).
2.1.12.3.
5-(Benzyloxy)-1-methyl-2-((4-nitro-5-(p-tolylthio)-1H-imidazol-1-yl)methyl)pyridin-
4(1H)-one (13K)
Yellow powder; yield: 52%; mp: 295 °C (decomposed); IR (KBr, cm-1): 3440, 3101, 3055, 2923,
1627 (C=O), 1573 (NO2), 1535, 1488, 1365 (NO2), 1288, 1218, 1134, 1087, 1010, 887, 833,
1
732, 655; H-NMR (500 MHz, DMSO-d6) δ (ppm): 8.17 (s, 1H, C2ʺ-H), 7.59 (s, 1H, C6-H),
7.41-7.34
(m, 5H, OCH2-phenyl-H), 7.14 (d, J = 8.0 Hz, 2H, C2ʹ-H and C6ʹ-H), 7.07 (d, J =
C3ʹ-H and C5ʹ-H), 5.38 (s, 2H, N1ʺ-CH2), 5.26 (s, 1H, C3-H), 4.93 (s, 2H, O-CH2),
8.1 Hz, 2H,
3.61 (s, 3H, N1-CH3), 2.20 (s, 3H, C4ʹ-CH3).
2.1.13. General procedure for the synthesis of target compounds 4-Nitroimidazole
derivatives 14a-k
To a suspension of compounds 13a-k (0.5 mmol) in dry dichloromethane (25 mL) at 0 °C, boron
trichloride (1M in CH2Cl2, 2 mL) was added under nitrogen and the reaction was stirred for 30
minutes and allowed to stir at room temperature for 5 hours. The reaction mixture was quenched
slowly by methanol (5 mL) to remove the excess BCl3 and left to stir another 30 minutes. The
13

solvent was evaporated in vacuum and the residue was purified by recrystallization from
methanol to afford 14a-k.
2.1.13.1.
2-((5-((4-Bromophenyl)amino)-4-nitro-1H-imidazol-1-yl)methyl)-5-hydroxy-1-
methylpyridin-4(1H)-one (14a)
Yellow powder; yield: 77%; mp 260 °C (decomposed); IR (KBr, cm-1): 3417, 3332 (NH), 3093,
1
1612 (C=O), 1566 (NO2), 1465, 1373 (NO2), 1288, 1211, 1118, 825, 601; H-NMR (500 MHz,
DMSO-d6) δ (ppm): 11.23 (br s, 1H, OH), 9.56 (s, 1H, NH), 8.37 (s, 1H, C2ʺ-H), 8.27 (s, 1H,
C6-H), 7.84 (d, J = 8.6 Hz, 2H, C3ʹ-H and C5ʹ-H), 7.54 (d, J = 8.6 Hz, 2H, C2ʹ-H and C6ʹ-H), 6.82
(s, 1H, C3-H), 5.86 (s, 2H, N1ʺ-CH2), 4.11 (s, 3H, N1-CH3); 13C-NMR (125 MHz, DMSO-d6)
δ (ppm): 160.59 (C=O), 147.59, 144.72, 143.86, 142.87, 137.85, 132.00, 130.96, 121.58, 120.56,
114.51, 109.85, 46.20 (N1ʺ-CH2), 42.65 (N1-CH3); EIMS m/z: 420.9 (M+); Anal. calcd for
C16H14BrN5O4: C, 45.73; H, 3.36; N, 16.67. Found: C, 45.49; H, 3.67; N, 16.51.
2.1.13.2.
2-((5-((4-Chlorophenyl)amino)-4-nitro-1H-imidazol-1-yl)methyl)-5-hydroxy-1-
methylpyridin-4(1H)-one (14b)
Yellow powder; yield: 90%; mp 243 °C (decomposed); IR (KBr, cm-1): 3417, 3317 (NH), 3047,
2368, 1620 (C=O), 1559 (NO2), 1465, 1373 (NO2), 1288, 1211, 1126, 879, 833, 740, 632;
1H-NMR (500 MHz, DMSO-d6) δ (ppm): 11.26 (br s, 1H, OH), 9.56 (s, 1H, NH), 8.36 (s, 1H,
C2ʺ-H), 8.21 (s, 1H, C6-H), 7.35 (d, J = 8.5 Hz, 2H, C2ʹ-H and C6ʹ-H), 7.28 (d, J = 8.6 Hz, 2H,
C3ʹ-H and C5ʹ-H), 6.57 (s, 1H, C3-H), 5.70 (s, 2H, N1ʺ-CH2), 4.12 (s, 3H, N1-CH3); 13C-NMR
(125 MHz, DMSO-d6) δ (ppm): 160.52 (C=O), 149.43, 145.95, 143.13, 139.63, 136.29, 130.96,
129.74, 129.30, 129.19, 121.23, 110.57, 45.25 (N1ʺ-CH2), 43.13 (N1-CH3); EIMS m/z: 375.1
(M+); Anal. calcd for C16H14ClN5O4: C, 51.14; H, 3.76; N, 18.64. Found: C, 51.32; H, 3.98; N,
18.41.
2.1.13.3.
5-Hydroxy-1-methyl-2-((4-nitro-5-(p-tolylamino)-1H-imidazol-1-yl)methyl)pyridin-
4(1H)-one (14c)
yellowish powder; yield: 89%; mp 244 °C (decomposed); IR (KBr, cm-1): 3440, 3326 (NH),
1
2923, 1627 (C=O), 1542 (NO2), 1488, 1380 (NO2), 1326, 1126, 833, 756; H-NMR (500 MHz,
DMSO-d6) δ (ppm): 11.15 (br s, 1H, OH), 9.49 (s, 1H, NH), 8.36 (s, 1H, C2ʺ-H), 8.26 (s, 1H,
C6-H), 7.71 (d, J = 8.2 Hz, 2H, C2ʹ-H and C6ʹ-H), 7.18 (d, J = 8.2 Hz, 2H, C3ʹ-H and C5ʹ-H), 6.78
(s, 1H, C3-H), 5.84 (s, 2H, N1ʺ-CH2), 4.10 (s, 3H, N1-CH3), 2.29 (s, 3H, C4ʹ-CH3); 13C-NMR
14

(125 MHz, DMSO-d6) δ (ppm): 160.74 (C=O), 148.35, 144.84, 143.89, 143.41, 135.70, 132.07,
128.70, 120.24, 119.70, 109.77, 46.11 (N1ʺ-CH2), 42.58 (N1-CH3), 19.91 (C4ʹ-CH3); EIMS m/z:
355.10 (M+); Anal. calcd for C17H17N5O4: C, 57.46; H, 4.82; N, 19.71. Found: C, 57.38; H, 5.01;
N, 19.55.
2.1.13.4.
4-((1-((5-Hydroxy-1-methyl-4-oxo-1,4-dihydropyridin-2-yl)methyl)-4-nitro-1H-
imidazol-5-yl)amino)benzonitrile (14d)
Yellow powder; yield: 85%; mp 270 °C (decomposed); IR (KBr, cm-1): 3425, 3294 (NH), 3062,
1
1620 (C=O), 1559 (NO2), 1465, 1373 (NO2), 1288, 1211, 1126, 879, 833, 740, 632; H-NMR
(500 MHz, DMSO-d6) δ (ppm): 9.56 (s, 1H, NH), 8.37 (s, 1H, C2ʺ-H), 8.27 (s, 1H, C6-H), 7.83
(d, J = 8.6 Hz, C3ʹ-H and C5ʹ-H), 7.54 (d, J = 8.6 Hz, 2H, C2ʹ-H and C6ʹ-H), 6.82 (s, 1H, C3-H),
13
5.86 (s, 2H, N1ʺ-CH2), 4.11 (s, 3H, N1-CH3); C-NMR (125 MHz, DMSO-d6) δ (ppm): 160.59
(C=O), 147.59, 144.72, 143.86, 142.87, 137.85, 132.00, 130.96, 121.58, 120.56, 114.51, 109.85,
46.20 (N1ʺ-CH2), 42.65 (N1-CH3); EIMS m/z: 366.0 (M+); Anal. calcd for C17H14N6O4: C, 55.74;
H, 3.85; N, 22.94. Found: C, 55.59; H, 3.96; N, 23.11.
2.1.13.5.
5-Hydroxy-2-((5-((4-methoxyphenyl)amino)-4-nitro-1H-imidazol-1-yl)methyl)-1-
methylpyridin-4(1H)-one (14e)
Yellowish powder; yield: 82%; mp 214 °C (decomposed); IR (KBr, cm-1): 3440, 3309 (NH),
3093, 1620 (C=O), 1512 (NO2), 1465, 1380 (NO2), 1288, 1242, 1110, 1026, 871, 825, 740, 594;
1H-NMR (500 MHz, DMSO-d6) δ (ppm): 11.12 (br s, 1H, C5-OH), 9.51 (s, 1H, NH), 8.32 (s,
1H, C2ʺ-H), 8.23 (s, 1H, C6-H), 7.73 (d, J = 8.6 Hz, 2H, C2ʹ-H and C6ʹ-H), 6.95 (d, J = 8.7 Hz,
2H, C3ʹ-H and C5ʹ-H), 6.72 (s, 1H, C3-H), 5.81 (s, 2H, N1ʺ-CH2), 4.08 (s, 3H, N1-CH3), 3.76 (s,
3H, O-CH3); 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 161.09 (C=O), 155.18, 148.65, 144.87,
143.96, 143.58, 131.52, 131.23, 121.59, 120.01, 113.44, 109.70, 54.73 (O-CH3), 46.06 (N1ʺ-
CH2), 42.46 (N1-CH3); EIMS m/z: 371.1 (M+); Anal. calcd for C17H17N5O5: C, 54.98; H, 4.61; N,
18.86. Found: C, 55.82; H, 4.82; N, 19.06.
2.1.13.6.
2-((5-((3-Chlorophenyl)amino)-4-nitro-1H-imidazol-1-yl)methyl)-5-hydroxy-1-
methylpyridin-4(1H)-one (14f)
Olive powder; yield: 69%; mp 254.5 °C (decomposed); IR (KBr, cm-1): 3317 (NH), 3093, 2599,
1
1620 (C=O), 1566 (NO2), 1465, 1373 (NO2), 1311, 1226, 1118, 894, 779, 678, 601; H-NMR
(500 MHz, DMSO-d6) δ (ppm): 11.21 (br s, 1H, OH), 9.62 (s, 1H, NH), 8.37 (s, 1H, C2ʺ-H), 8.28
15

(s, 1H, C6-H), 8.08 (s, 1H, C2ʹ-H), 7.78 (d, J = 8.2 Hz, 1H, C4ʹ-H), 7.38 (s, 1H, C5ʹ-H), 7.13
(d, J = 7.9 Hz, 1H, C6ʹ-H), 6.81 (s, 1H, C3-H), 5.85 (s, 2H, N1ʺ-CH2), 4.11 (s, 3H, N1-CH3);
13C-NMR (125 MHz, DMSO-d6) δ (ppm): 160.60 (C=O), 147.38, 144.68, 143.87, 142.72,
140.00, 132.57, 131.94, 129.81, 122.26, 120.65, 118.87, 118.07, 109.87, 46.23 (N1ʺ-CH2), 42.62
(N1-CH3); EIMS m/z: 375.10 (M+); Anal. calcd for C16H14ClN5O4: C, 51.14; H, 3.76; N, 18.64.
Found: C, 51.32; H, 3.58; N, 18.51.
2.1.13.7.
2-((5-((3,5-Dichlorophenyl)amino)-4-nitro-1H-imidazol-1-yl)methyl)-5-hydroxy-1-
methylpyridin-4(1H)-one (14g)
Yellowish powder; yield: 61%; mp 282.5 °C (decomposed); IR (KBr, cm-1): 3425, 3317 (NH),
3093, 1620 (C=O), 1566 (NO2), 1473,1427, 1357 (NO2), 1311, 1211, 1118, 933, 840, 671, 609;
1H-NMR (500 MHz, DMSO-d6) δ (ppm): 11.28 (br s, 1H, OH), 9.70 (s, 1H, NH), 8.37 (s, 1H,
C2ʺ-H), 8.30 (s, 1H, C6-H), 8.05 (s, 2H, C2ʹ-H and C6ʹ-H), 7.26 (s, 1H, C4ʹ-H ), 6.83 (s, 1H, C3-
H), 5.87 (s, 2H, N1ʺ-CH2), 4.11 (s, 3H, N1-CH3); 13C-NMR (125 MHz, DMSO-d6) δ (ppm):
160.61 (C=O), 146.61, 144.57, 143.86, 142.27, 141.14, 133.45, 132.01, 121.39, 120.94, 117.48,
109.92, 46.30 (N1ʺ-CH2), 42.64 (N1-CH3); EIMS m/z: 409.0 (M+); Anal. calcd for
C16H13Cl2N5O4:
C, 46.85; H, 3.19; N, 17.07. Found: C, 46.59; H, 3.31; N, 16.84.
2.1.13.8. 5-Hydroxy-1-methyl-2-((4-nitro-5-(phenylamino)-1H-imidazol-1-yl)methyl)pyridin-
4(1H)-one (14h)
Mustard powder; yield: 78%; mp 240 °C (decomposed); IR (KBr, cm-1): 3386 (NH), 3116, 2923,
1
1635 (C=O), 1550 (NO2), 1404, 1352 (NO2), 1319, 1218, 1118, 987, 763; H-NMR (500 MHz,
DMSO-d6) δ (ppm): 9.05 (s, 1H, NH), 8.22 (s, 1H, C2ʺ-H), 8.14 (s, 1H, C6-H), 7.16-7.47 (m, 5H,
Ar-H), 6.35 (s, 1H, C3-H), 4.85 (s, 2H, N1ʺ-CH2), 3.99 (s, 3H, N1-CH3); 13C-NMR (125 MHz,
DMSO-d6) δ (ppm): 160.02 (C=O), 145.43, 144.32, 142.87, 142.20, 141.77, 141.13, 133.92,
122.09, 121.55, 118.18, 110.14, 46.54 (N1ʺ-CH2), 42.41 (N1-CH3); EIMS m/z: 341.1 (M+);
Anal. calcd for C16H15N5O4: C, 56.30; H, 4.43; N, 20.52. Found: C, 56.46; H, 4.28; N, 20.62.
2.1.13.9.
5-Hydroxy-1-methyl-2-((4-nitro-5-(phenylthio)-1H-imidazol-1-yl)methyl)pyridin-
4(1H)-one (14i)
yellowish powder; yield: 83%; mp 243 °C (decomposed); IR (KBr, cm-1): 3301, 3224, 3116,
3055, 2923, 2476, 1635 (C=O), 1558 (NO2), 1488, 1434, 1357 (NO2), 1296, 1257, 1114, 1010,
879, 748, 702; 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 8.54 (s, 1H, C2ʺ-H), 8.23 (s, 1H, C6-H),
16

7.67
(m, 2H, C3ʹ-H and C5ʹ-H), 7.49 (m, 3H, C2ʹ-H, C4ʹ-H and C6ʹ-H), 6.74 (s, 1H, C3-H),
13
5.87 (s, 2H, N1ʺ-CH2), 4.08 (s, 3H, N1-CH3); C-NMR (125 MHz, DMSO-d6) δ (ppm): 160.80
(C=O), 146.27, 145.07, 143.98, 142.80, 139.07, 134.16, 131.93, 129.84, 128.76, 127.55, 110.04,
46.36 (N1ʺ-CH2), 42.57 (N1-CH3); EIMS m/z: 358.0 (M+); Anal. calcd for C16H14N4O4S: C,
53.62; H, 3.94; N, 15.63. Found: C, 53.51; H, 4.87; N, 15.86.
2.1.13.10.
2-((5-((4-Chlorophenyl)thio)-4-nitro-1H-imidazol-1-yl)methyl)-5-hydroxy-1-
methylpyridin-4(1H)-one (14j)
Yellowish powder; yield: 84%; mp 241 °C (decomposed); IR (KBr, cm-1): 3371, 3070, 2923,
2599, 1635 (C=O), 1550 (NO2), 1473, 1357 (NO2), 1288, 1118, 1010, 879, 833, 748, 740, 640;
1H-NMR (500 MHz, DMSO-d6) δ (ppm): 11.20 (br s, 1H, OH), 8.36 (s, 1H, C2ʺ-H), 8.23 (s, 1H,
C6-H), 7.30 (d, J = 8.7 Hz, 2H, C2ʹ-H and C6ʹ-H), 7.23 (d, J = 8.7 Hz, 2H, C3ʹ-H and C5ʹ-H), 6.52
(s, 1H,
C3-H), 5.74 (s, 2H, N1ʺ-CH2), 4.05 (s, 3H, N1-CH3); 13C-NMR (125 MHz, DMSO-
d6) δ (ppm): 160.09 (C=O), 145.52, 144.01, 142.70, 139.20, 135.86, 132.16, 130.53, 129.31,
128.86, 120.80, 111.07, 46.36 (N1ʺ-CH2), 42.70 (N1-CH3); EIMS m/z: 392.0 (M+); Anal. calcd
for C16H13N4O4S: C, 48.92; H, 3.34; N, 14.26. Found: C, 48.71; H, 3.56; N, 14.13.
2.1.13.11.
5-Hydroxy-1-methyl-2-((4-nitro-5-(p-tolylthio)-1H-imidazol-1-yl)methyl)pyridin-
4(1H)-one (14k)
Off-white powder; yield: 62%; mp 235.5 °C (decomposed); IR (KBr, cm-1): 3217, 3132, 2638,
1
1620 (C=O), 1542 (NO2), 1473, 1380 (NO2), 1288, 1226, 1118, 1018, 833, 802, 648; H-NMR
(500 MHz, DMSO-d6) δ (ppm): 11.17 (br s, 1H, OH), 8.30 (s, 1H, C2ʺ-H), 8.21 (s, 1H, C6-H),
7.13
(d, J = 8.9 Hz, 2H, C2ʹ-H and C6ʹ-H), 7.01 (d, J = 8.9 Hz, 2H, C3ʹ-H and C5ʹ-H),
C3-H), 5.72 (s, 2H, N1ʺ-CH2), 4.04 (s, 3H, N1-CH3), 2.20 (s, 1H, C4ʹ-CH3); 13C-
6.28 (s, 1H,
NMR (125 MHz, DMSO-d6) δ (ppm): 159.95 (C=O), 148.59, 143.88, 142.68, 138.88, 137.26,
131.99, 129.66, 128.83, 127.35, 122.28, 110.68, 44.71 (N1ʺ-CH2), 42.59 (N1-CH3), 19.94 (C4ʹ-
CH3); EIMS m/z:
372.1 (M+); Anal. calcd for C17H16N4O4S: C, 54.83; H, 4.33; N, 15.05.
Found: C, 54.69; H, 4.57; N, 14.92.
2.2. In vitro anti-HIV-1 assay
The inhibitory effect of antiviral drugs on the HIV-induced CPE in MT-4 cell culture was
determined by the MTT-assay [53]. This assay is based on the reduction of the yellow colored
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by mitochondrial
17

dehydrogenase of metabolically active cells to a blue formazan derivative, which can be
measured spectrophotometrically. The 50% cell culture infective dose of the HIV strains was
determined by titration of the virus stock using MT-4 cells. For the drug susceptibility assays,
MT-4 cells were infected with 100 to 300 50% cell culture infective doses of the HIV strains in
the presence of fivefold serial dilutions of the antiviral drugs. The concentration of the compound
achieving 50% protection against the CPE of HIV, which is defined as the 50% effective
concentration (IC50), was determined. The concentration of the compound destroying 50% of the
MT-4 cells, which is defined as the 50% cytotoxic concentration (CC50), was determined as well.
2.3. In vitro antiproliferative assay
Human leukemic cells from MOLM-13 and K562 cell lines were plated in a 24-well plate at a
density of 2x106 cells/mL and treated with compound (between 0.5µM and 10µM) or control
(DMSO). Cells were cultured in RPMI supplemented with 10% FBS and 50µL/mL gentamicin at
37°C. All conditions were exposed to the same amount of DMSO (0.4%). At day four, cells were
seeded at their initial density and provided with fresh compound or DMSO. Three days later,
100µL of cell suspension was transferred to a 96-well plate in addition of 20µL MTT (7.5 mg/L).
After one hour of incubation, the cells were re-suspended in 100µL isopropanol, supplemented
with 6% Triton X-100 and 0.4% methanesulfonic acid and absorbance was measured at 540 nm
using EnVision Multimode Plate Reader.
2.4. Molecular modeling
The crystallographic structure of HIV-1 RT complex with Capravirine (PDB cod: 1EP4) was
retrieved from the protein data bank. The protein structure was modified as follows: (1) Cognate
ligands, co-crystalized ions and solvent molecules were removed. (2) All hydrogens were added,
after computing Kollman charges the nonpolar hydrogens were merged and the result was saved
as pdbqt extension. Structures of the designed compounds were drawn and minimized by MM+
force field and PM3 semi-empirical techniques, respectively in Hyperchem8 software. Then in
Autodock tools, the Gasteiger-Marsili charge was assigned and rotatable bonds were defined for
each minimized molecule, saving the resulting file in pdbqt format. Autodock grids were
generated using a grid box of 60 × 60 × 60 grid points in xyz directions with grid spacing of
0.375 Å, centered on cognate ligand in RT allosteric binding pocket. Autogrid 4.0 was used to
calculate energy grid maps for all atom types in the molecules. The Lamarckian genetic
algorithm was selected for docking simulation of compounds. For the LGA method, an initial
18

population of random individuals with a population size of 150 individuals, a maximum number
of 2.5 × 106 energy evaluations, a maximum number of generations of 27,000, mutation and
crossover rates of 0.02 and 0.8, respectively, were used. Top scoring pose was shown by the
Pymol software, version 1.8.7. The secondary structure of RT was shown in cartoons and only
the key residues of allosteric binding site were shown in yellow sticks. The H-bonds were
displayed in yellow dashed lines.
3. Results and Discussion
3.1. Chemistry
The 5-benzyloxy-4-pyridinone 4 was prepared in three steps from kojic acid 1 according to
previously reported procedures, as depicted in Scheme 1 [38, 39]. Kojic acid 1 was treated with
benzyl chloride under basic condition to yield benzylated compound 2 which was then converted
to compound 3 by reaction with methylamine solution. Finally, the reaction of compound 3 with
thionyl chloride at room temperature (r.t) provided 5-(benzyloxy)-2-(chloromethyl)-1-
methylpyridin-4(1H)-one (4). The FTIR spectrum of 4 was characterized by disappearance of
broad absorption bands of 2-hydroxymethyl and 5-hydroxyl groups in 3000-3400 cm-1 region,
meanwhile the shift of carbonyl group from 1667 to 1620 cm-1.
Scheme 1. Preparation of 5-benzyloxy-2-chloromethyl-4-pyridinone. Reagent and conditions: (a) benzyl chloride,
NaOH, MeOH/H2O, reflux, overnight; (b) MeNH2, H2O, 70 °C, 2h; (c) Thionyl chloride, r.t, 2h.
The synthetic route employed for the imidazole derivatives 14a-k is summarized in
Scheme 2. 4-Nitroimidazole 6 was obtained by nitration of commercially available imidazole 5,
which was then converted to 4,5-dinitroimidazole 7 with fuming nitric acid and sulfuric acid, by
the described earlier procedures [40, 41]. The reaction of 7 with a range of substituted anilines in
ethanol provided 4-nitro-5-phenylaminomidazole derivatives 8a-h. Condensation of 8a-h with 4
1
in the presence of K2CO3 and KI afforded 4-nitroimidazole derivatives 13a-h. In the H-NMR
spectra of 8a-h the imidazole NH proton appeared as a broad singlet between δ 13.21-14.12 ppm
which was disappeared in 13a-h.
19

Treatment of imidazole 5 with bromine in the presence of acetic acid and sodium acetate
yielded 2,4,5-tribromo imidazole 9 [42]. Reaction of 9 with sodium sulfite resulted in
4-bromoimidazole 10 which on treatment with nitric acid and sulfuric acid furnished
4-nitro-5-bromo-imidazole 11, according to the literature methods [43, 44]. It is noteworthy that
previously reported compounds 2-7 and 9-11 have been fully characterized in some papers
[38-44].
13, 14
R
a
b
c
d
e
f
g
h
i
j
k
4-Br 4-Cl 4-CH3 4-CN 4-OCH3 3-Cl 3,5-Cl2
H
H
4-Cl 4-CH3
Scheme 2. (a) H2SO4, HNO3, reflux, 5h; (b) H2SO4, HNO3 (100%), reflux, 5h; (c) Ar-NH2, EtOH, 70 °C, 20h; (d) 4,
K2CO3, KI, acetone, 55 °C, overnight; (e) NaOAc, ACOH, Br2, r.t, 2h; (f) Na2SO3, H2O, 110 °C, 6h; (g) H2SO4,
HNO3, 110 °C, 1h; (h) 4, K2CO3, KI, acetone, 40 °C, overnight; (i) Ar-SH, KOH, EtOH, reflux, 24h; (j) dry CH2Cl2,
N2, BCl3 (1 M), 0 °C, 1h.
Attempts to achieve compound 12 using compounds 4 and 11 yielded two imidazole
derivatives, the 5-bromo-4-nitroimidazole isomer 12a and the 4-bromo-5-nitroimidazole isomer
12b (Scheme 3), whose structures were confirmed by 1H-NMR spectrum. Thin layer
chromatography of 12 showed only one spot, thus it was impossible to separate these two
isomers by column chromatography. Interestingly, 4-halo-5-nitroimidazoles have been reported
to be resistant to nucleophilic displacement of the halo substituent [45, 46].
In this study, the 5-bromo-4-nitroimidazole isomer (12a) readily underwent reaction at
the site of the bromo substituent, whereas the 4-bromo-5-nitroimidazole isomer (12b) was
20

resistant to reaction at the bromo position under the same conditions. Replacement of the 2-
bromo of 12a with the corresponding thiophenols using potassium hydroxide as a base in the
Ethanol, yielded the desired intermediates 13i-k. Thereafter, in the next step, compounds 13i-k
were purified by column chromatography, (eluent CH2Cl2/MeOH, 97:3). Finally, the target
compounds 14a-k were obtained by deprotection of compounds 13a-k using BCl3 in dry CH2Cl2
[39].
Scheme 3. Two isomers of 12a and 12b
1
The structures of the final products 14a-k were verified by their FTIR, H-NMR, 13C-
NMR and mass spectrometry spectra as well as elemental analyses. In the FTIR spectra, the
secondary aromatic NH group showed a signal at 3294-3440 cm-1. Moreover, the absorption
band in
1612-1635 cm-1 was attributed to the carbonyl functionality. In 1H-NMR spectra a
singlet appeared at δ 3.99-4.12 ppm region that was assigned to N-CH3 protons. Two protons of
pyridinone ring had resonance at two different regions δ 6.28-6.83 and 8.14-8.30 ppm. A singlet
signal between
δ 4.85-5.87 ppm was assigned to the N-CH2 protons. In the 13C-NMR
spectra, the low field resonances in the regions δ 42.41-43.13 and 44.71-46.36 ppm were
attributed to the N-CH3 and N-CH2 carbons, respectively. The resonance of the carbonyl group
was observed between
δ 159.95-161.09 ppm. Additionally, the pyridinone and the
aromatic carbons appeared in the regions δ 109.70-155.18 ppm.
3.2. Molecular modeling
In the purpose of understanding the binding mode of the designed compounds as well as
comparing their binding modes with the lead compound, capravirine, molecular modeling studies
were carried out. Compound 14j and capravirine, were docked separately into the non-nucleoside
binding pocket (NNBP) of wild type HIV-1 (PDB cod: 1EP4) by means of Autodock tools
21

(ADT) package version 1.5.6rc3 [47]. The docking results were analyzed by pymol and their
binding modes inside NNBP are depicted in Figure 5.
Figure 5. Predicted binding mode of compounds 14j (left) and Capravirine (right) inside the NNIBP of wild type
HIV-1. H-bonds are depicted as yellow dashed lines.
It is apparent that compound 14j binds to NNIBP in a manner almost similar to
Capravirine. Interestingly, the hydroxypyridinone motif formed H-bonds with both Lys103 and
Pro236 which play crucial roles in anti-HIV-1 activity of Capravirine. Moreover, the 4-
chlorophenyl moiety of 14j is oriented towards the Tyr181 and Tyr188 residues, similar to the
3,5-dichlorophenyl moiety of Capravirine, which established pi-pi interactions.
3.3. In vitro anti-HIV-1 activity
The newly synthesized compounds 14a-k were evaluated for their antiviral activities against WT
HIV-1 strain IIIB in human T-lymphocyte (MT-4) cells based on the MTT assay. The
cytotoxicity of these compounds against T-lymphocyte (MT-4) cells was determined in parallel.
The biological results expressed as EC50, EC90 (anti-HIV-1 activity), CC50 (cytotoxicity) and SI
(selectivity index) are represented in table 1. The FDA approved drugs, ralpivirine (RPV),
azidothymidine (AZT) and ritonavir were included for comparison purpose.
Table 1. In vitro anti-HIV-1 activities of compounds 14a-k.
Compd.
14a
EC50 a (µM)
>6.8
EC90 b (µM)
>6.8
CC50 c (µM)
6.8
SId
<1
×1
>250.0
>250.0
>250.0
14b
22

>25.4
>250.0
>250.0
>5.5
>25.4
>250.0
>250.0
>5.5
25.4
>250.0
>250.0
5.5
<1
×1
×1
<1
<1
×1
<1
<1
<1
-
14c
14d
14e
14f
>7.3
>7.3
7.3
14g
>250.0
>23.6
>3.6
>250.0
>23.6
>3.6
>250.0
23.6
3.6
14h
14i
14j
>29.8
0.0021
0.0170
0.0042
>29.8
0.0049
0.0967
0.4088
29.8
-
14k
RPV
AZT
ritonavir
-
-
-
-
a EC50 and EC90: the effective concentration required to achieve 50% and 90% protection, respectively of MT-4 cells
against viral cytopacthicity
b CC50: the cytotoxic concentration of the compounds that reduce the viability of mock-infected MT-4 cells by 50%
c SI: selectivity index, the ratio of EC50/CC50
As shown in table 1, none of the studied compounds were active against HIV-1
replication in cell culture. In fact, all were toxic for the host cell and four of them, 14a, 14f, 14g
and 14j were the most potent ones thus, it is not possible to judge about the anti-HIV-1 activity
of them. Due to their toxicity toward MT-4 cells infected by HIV-1, the study of the enzymatic
inhibition of the prepared compounds was regarded meaningless. However, based on the SAR
analysis, compounds with halogen substituents on the benzene ring demonstrated higher
cytotoxicity than those with other substituents at the same ring. Furthermore, comparing 14b
with 14j and 14h with 14i revealed that replacing the NH linker between imidazole and benzene
moieties with sulfur, increased the cytotoxicity.
3.4. In vitro antiproliferative activity
23

In order to explore the antitumor activity of the prepared compounds, the three most cytotoxic
compounds 14f, 14g and 14j were selected and tested against acute myeloid leukemia (AML),
MOLM-13, as well as chronic myeloid leukemia (CML), K562, cell lines using MTT method.
As depicted in table 2, all of them were active with EC50 values in the range from 1.3 to 8.01 ꢀM
and 14g was the most cytotoxic compound toward both cell lines.
Table 2 The antiproliferative activities of compounds 14f, 14g and 14j.
EC50 (µM)
Compd.
MOLM-13
4.27±0.89
1.3±0.39
K562
8.01±0.72
1.8±0.44
5.79±0.53
14f
14g
14j
3.65±0.64
It is noteworthy that myeloid leukemia are regularly related to mutation and translocation
in tyrosine kinase genes. Then, most common type of small molecules that have been
investigated in target therapy of AML and CML are tyrosine kinase inhibitors [48, 49].
Furthermore, there are structural similarities between compounds prepared in this paper and
previous reported tyrosine kinase inhibitors against myeloid leukemia (Figure 6) [50, 51]. Hence,
compounds prepared may exert their antiproliferative activity through tyrosine kinase inhibition.
Figure 6. Structures of (a and b) previously reported tyrosine kinase inhibitors and (c) synthesized compounds in
this manuscript.
4. Conclusion
In the current study, a series of 4-nitroimidazole derivatives combined with 5-hydroxy-4-
pyridinones 14a-k were prepared through a multi-step reaction. All the new analogues 14a-k
were characterized by their NMR and mass spectral data as well as elemental analysis. The
designed molecules displayed good binding energies in molecular docking studies, even better
24

than Capravirine. However, in anti-HIV-1 assay of the synthesized derivatives, no activity was
observed. In fact, all of them were cytotoxic against the human T-lymphocyte (MT-4) cells, thus
it was not possible to determine the anti-HIV-1 activity of the compounds. Nevertheless, inspired
by high cytotoxicity of compounds 14f, 14g and 14j toward MT-4 cells, they have been selected
for antiproliferative assay. They exhibited remarkable antitumor activity against leukemia
MOLM-13 as well as K562 cell lines at low micromolar concentrations. Presumably, these
newly synthesized compounds could inhibit tyrosine kinase enzymes, they are worthy of future
studies with the aim of developing novel promising tyrosine kinase inhibitors.
Acknowledgements
The auteurs are grateful to Isfahan University of Medical Science for financial support.
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