Molecular modeling, synthesis and biological evaluation of N-heteroaryl compounds as reverse transcriptase inhibitors against HIV-1

Article abstract of DOI:10.1111/cbdd.12397

Different N-heteroaryl compounds bearing pyrimidine and benzimidazole moieties have been designed in silico using Discovery studio 2.5 software, synthesized and evaluated for their inhibitory activity as reverse transcriptase inhibitors against HIV-1 replication using laboratory adapted strains HIV-1_IIIB (X4, subtype B) and HIV-1_Ada5 (R5, subtype B), and the primary isolates HIV-1_UG070 (X4, subtype D) and HIV-1_VB59 (R5, subtype C). Cell-based assay showed that compounds were active at 1.394 μM concentrations (Selectivity Index: 1.29-38.39). The studies on structure-activity relationship clearly suggested anti-HIV activity of pyrimidine and benzimidazole derivatives and these findings were consistent with the in vitro cell-based experimental data. The results of molecular modeling and docking confirmed that all compounds assumed a butterfly-like conformation and showed H-bond, 'π-π' and 'π-+' and hydrophobic interactions within flexible non-nucleoside inhibitor binding pocket of HIV-1 reverse transcriptase, similar to known non-nucleoside reverse transcriptase inhibitors, such as nevirapine. In view of the results obtained, it can be said that the chemical skeletons of N, N′-bis-(pyridin-2-yl)-succinamide (14 and 15) and 1, 4-bis-benzoimidazol-1-yl-butane-1, 4-dione (16 and 17) may be used for developing potent inhibitors of HIV-1 replication, with suitable structure/pharmacophore modifications.

Full text of DOI:10.1111/cbdd.12397

Received Date : 02-Jan-2014
Revised Date : 08-Jul-2014
Accepted Date : 12-Jul-2014
Article type
: Research Article
Molecular Modeling, Synthesis and Biological Evaluation of N-Heteroaryl Compounds
as Reverse Transcriptase Inhibitors against HIV-1
Anuradha Singh1, Dipti Yadav1, Madhu Yadav1, Ashwini Dhamanage2, Smita Kulkarni2, $ and Ramendra
K. Singh1, $,*
1
Nucleic Acids and Antiviral Research Laboratory, Department of Chemistry, University of Allahabad,
Allahabad-211002, India
2National AIDS Research Institute, Pune, India
$Equal contribution
*Corresponding author: Ramendra K. Singh, rksinghsrk@gmail.com, Telefax. No. +91-532-2461005
Abstract
Different N-heteroaryl compounds bearing pyrimidine and benzimidazole moieties have been designed in silico
using Discovery studio 2.5 software, synthesized and evaluated for their inhibitory activity as reverse
transcriptase inhibitors against HIV-1 replication using laboratory adapted strains HIV-1IIIB (X4, subtype B) and
HIV-1Ada5 (R5, subtype B), and the primary isolates HIV-1UG070 (X4, subtype D) and HIV-1VB59 (R5, subtype C).
Cell based assay showed that compounds were active at 1.394 µM concentrations (Selectivity Index: 1.29-
38.39). The studies on structure–activity relationship clearly suggested anti-HIV activity of pyrimidine and
benzimidazole derivatives and these findings were consistent with the in vitro cell based experimental data. The
results of molecular modeling and docking confirmed that all compounds assumed a butterfly like conformation
and showed H-bond, ‘π-π’ and ‘π- +’ and hydrophobic interactions within flexible non-nucleoside inhibitor
binding pocket of HIV-1 reverse transcriptase, similar to known non-nucleoside reverse transcriptase
inhibitors, such as nevirapine. In view of the results obtained, it can be said that the chemical skeletons of N,
N’-bis-(pyridin-2-yl)-succinamide (14 and 15) and 1, 4-bis-benzoimidazol-1-yl-butane-1, 4-dione (16 and 17)
may be used for developing potent inhibitors of HIV-1 replication, with suitable structure/pharmacophore
modifications.
Key Words: HIV-1 reverse transcriptase, anti-HIV activity, docking, TZM-bl cell lines
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
an 'Accepted Article', doi: 10.1111/cbdd.12397
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Introduction
Acquired immunodeficiency syndrome (AIDS) continues to be a threat to human life since 1983 and presently
around 35.3 million people are living with HIV, its etiological agent (1). Significant progress has been made in
developing therapeutic agents to control HIV replication and introduction of combination therapy, highly active
antiretroviral therapy (HAART) has delayed the onset of AIDS and slowed down its global epidemic and
drastically prolonged the life expectancy of HIV-1 positive patients. However, the imminent development of
drug resistance and the toxic side effects associated with HAART prompt continuous efforts toward the
discovery of new therapeutics with unique mechanism of action. Though current combination therapy has
provided an improved quality of life, researchers are pursuing development of new anti-HIV drugs because of
the problems associated with current anti-retroviral therapy (2-4).
Currently, five classes of FDA approved antiretroviral agents exist, viz. reverse transcriptase inhibitors (RTIs),
protease inhibitors (PIs), integrase inhibitors (IIs), fusion inhibitors (FIs) and chemokine receptor antagonists
(CRAs). The target enzyme reverse transcriptase (RT), focused in present study, plays a crucial role in the life-
cycle of HIV. It has three enzymatic activities - RNA dependent DNA polymerase activity, DNA dependent
DNA polymerase activity and RNase-H activity, and is responsible for transformation of HIV genomic ssRNA
into dsDNA- the proviral DNA. Thus, HIV-RT has been recognized as a prime target for drug development and
several RT inhibitors, like nucleoside reverse transcriptase inhibitors (NRTIs), nucleotide reverse transcriptase
inhibitors (NtRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been developed as
potential drug molecules against HIV (5).
The NRTIs act as chain terminators and bind at the catalytic site as DNA base mimics (6), while NNRTIs, being
small and somewhat nonpolar molecules, exert their biological action on the HIV-RT through interaction within
an allosteric non-substrate site, located about 10 Å away from the catalytic site. The interactions within
hydrophobic and relatively small cleft known as the non-nucleoside inhibitor binding pocket (NNIBP), cause
short- and long range conformational changes (allosteric effect) impending the HIV-RT DNA polymerase
activity. The NNRTIs cross the blood brain barrier (BBB) and thus, NNRTIs not only reduce the viral loads
across the BBB but also prevent complications, like the onset of AIDS associated dementia (7).
The NNRTIs include a variety of conformationally restrained two and three ring structures with varying degree
of flexibility and are specific only for HIV-1 RT because of the presence of a flexible hydrophobic pocket in
which NNRTIs can fit. Favourable pharmacokinetic properties, unique antiviral activity, high specificity and
wide range of chemically diverse structures make NNRTIs a highly striking ingredient of HAART. The amino
acids within the NNRTI binding pocket are prone to mutation, leading to drug resistance and development of
HIV variants. Therefore, there is an urgent need to develop novel, highly potent NNRTIs with broad spectrum
of antiviral activities.
In the light of the need for new anti-HIV agents and the significant role played by NRTIs and NNRTIs, we
focused on developing novel NNRTIs utilizing benzimidazole and pyrimidine moieties having flexible
connecting linkages for better adaptability, unlike known NNRTIs having rigid fused systems, to match the
hydrophobic nature of the allosteric pocket in HIV RT (8-13). Thus, considering the importance of the
benzimidazole and pyrimidine moieties, novel molecules 5-17 with requisite structural features- hydrophilic site
flanked by hydrophobic sites, were designed using the DS 2.5 software, synthesized and screened against HIV-1
using in vitro cell based model. Molecular docking studies with HIV-1 RT enzyme were used to ascertain
behavior of these molecules.
Experimental Procedures
Chemicals were obtained from E. Merck India Ltd, India and Sigma-Aldrich Chemical Company, USA. All
solvents were reagent grade, purified and dried before use. Melting points of compounds determined using
electro thermal apparatus are uncorrected. Thin layer chromatographic analysis (TLC) was done to monitor the
completion of reactions as well as for identification of compounds. The spots were visualized by exposure to
iodine vapor and UV light. UV measurements were carried out on Lambda 25 spectrophotometer. 1H NMR and
13C NMR spectra were recorded on DRX 300 MHz instrument using CDCl3 as solvent and TMS as an internal
standard. Mass spectra were obtained using a Thermofinnigan TRACE-DSQ Electro Spray Ionization (ESI)
mass spectrometer. Elemental analyses were carried out on a Perkin-Elmer 240-C analyzer.
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Chemistry
General procedure for synthesis of compounds 5-13
To compound 1a/2a (1mMol) dissolved in dry acetonitrile and a small amount of pyridine, oxalyl chloride (1.2
mmol) was added drop wise in cold and anhydrous condition. Reaction mixture was stirred at 25°C for 1 h and
the solvent removed in vacuo. The residual oil dissolved in dry pyridine was used directly in the next step
without further purification. To a stirred solution of residual oil in dry pyridine (3 mL) maintained in an ice
bath, added drop wise appropriate amine (1 mmol) dissolved in dry acetonitrile (4 mL) and pyridine (1 mL),
and stirred the reaction mixture at room temperature. After completion of the reaction, as determined by TLC,
the solvent was evaporated under pressure and the residual mass dissolved in ethyl acetate (30 mL). This
organic fraction was washed consecutively with 5% NaHCO3 solution (20 mL), brine (20 mL), H2O (20 mL),
dried over anhydrous Na2SO4, filtered and evaporated under vacuum (40-50°C) to furnish a solid residue, which
was purified by silica-gel column chromatography using hexane–ethyl acetate as eluent.
2-(2, 4-Dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-N-pyridin-2-yl-acetamide (5)
1
White crystals; yield: 68%; Rf = 0.81; m.p. 200 -202°C ; H NMR: δ (ppm) : 8.49 (d, J = 11.0 Hz, 1H, C6-H
uracil), 6.07 (d, J = 10.8 Hz, 1H, C5-H uracil), 5.45 (s, 1H, -CH2), 7.71 (s, 1H, -CONH-), 8.36 (d, J = 5.0, 1.2
Hz, 1H, C6-H pyridine), 7.15 (ddd, J = 8.2, 5.1, 1.2 Hz, 1H, C5-H pyridine), 7.99 (dd, J = 8.2, 1.1 Hz, 1H, C4-H
pyridine), 7.86 (td, J = 8.0, 1.3 Hz, 1H, C3-H pyridine); 13C NMR: δ (ppm) 167 (-CONH-), [165 (C4), 153(C2),
143 (C6), 101(C5) uracil], [153(C2), 148 (C6), 135(C4), 120(C5), 114(C3) pyridine], 53 (-CH2);UV/Vis (EtOH):
λmax = 245 nm; MS (EI, 70 eV): m/z (%): 247.08 (12.5), 246.00 (100) [M+H]+; HRMS-FAB: m/z [M+H]+
calcd for C11H10N4O3: 246.08, found: 246.10; Anal. calcd for C11H10N4O3: C 53.66, H 4.09, N 22.75, found: C
53.68, H 4.08, N 22.78.
N-(5-Bromo-pyridin-2-yl)-2-(2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-acetamide (6)
1
Orange solid; yield: 74%; Rf =0.83; m.p. 218-219 °C ; H NMR : δ(ppm) : 7.89 (d, J = 11.0 Hz, 1H, C6-H
uracil), 5.92 (d, J = 10.8 Hz, 1H, C5-H uracil), 7.71 (s, 1H, -CONH-), 4.42 (s, 2H, -CH2), 9.26 (d, J = 1.3 Hz,
1H, C6-H pyridine), 8.77 (dd, J = 7.9, 1.2 Hz, 1H, C4-H pyridine), 8.49 (d, J = 8.1 Hz,1H, C3-H pyridine); 13C
NMR: δ (ppm) 169 (-CONH-), [165(C4), 153(C2), 143 (C6), 101(C5) uracil], [152(C2), 150 (C6), 142(C4),
117.5(C5), 117(C3) pyridine], 53 (-CH2); UV/Vis (EtOH): λmax = 248 nm; MS (EI, 70 eV): m/z (%): 325.98
(97.3), 323.98 (100) [M+H]+; HRMS-FAB: m/z [M+H]+ calcd for C11H9BrN4O3: 323.99, found: 323.98; Anal.
calcd for C11H9BrN4O3: C 40.64, H 2.79, Br 24.58, N 17.23, found: C 40.62, H 2.78, Br 24.56, N 17.24.
2-(2, 4-Dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-N-(5-nitro-pyridin-2-yl)-acetamide (7)
1
Brown solid; yield: 77%; Rf = 0.88; m.p. 178-182°C ; H NMR : δ(ppm) : 7.92 (d, J = 10.8 Hz, 1H, C6-H
uracil), 5.92 (d, J = 10.8 Hz, 1H, C5-H uracil), 7.71 (s, 1H, -CONH-), 4.42 (s, 2H, -CH2), 9.26 (d, J = 1.3 Hz,
1H, C6-H pyridine), 8.77 (dd, J = 7.9, 1.2 Hz, 1H, C4-H pyridine), 8.49 (d, J = 8.1 Hz, 1H, C3-H pyridine); 13
C
NMR: δ (ppm) 168.1(-CONH-), [165(C4), 153(C2), 143(C6), 100(C5) uracil],[159(C2), 143(C6), 140(C5),
134(C4), 116(C3) pyridine], 53(-CH2),UV/Vis (EtOH): λmax = 315 nm, 256 nm; MS (EI, 70 eV): m/z (%):
292.07 (14.3), 291.06 (100) [M+H]+; HRMS-FAB: m/z [M+H]+ calcd for C11H9N5O5: 291.06, found: 291.08;
Anal. calcd for C11H9N5O5: C 45.37, H 3.11, N 24.05, found: C 45.35, H 3.10, N 24.02.
2-(5-Methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-(N-pyridin-2-yl)-acetamide (8)
1
White solid; yield: 70%; Rf =0.77; m.p. 221°C ; H NMR : δ(ppm) : 8.36 (dd, J = 5.0, 1.2 Hz, 1H, C6-H
pyridine), 7.99 (dd, J = 8.2, 1.1 Hz, 1H, C3-H pyridine), 7.86 (td, J = 8.0, 1.3 Hz, 1H, C4-H pyridine), 7.15
(ddd, J = 8.2, 5.1, 1.2 Hz, 1H, C5-H pyridine), 7.71 (s, 1H, -CONH-), 7.62 (q, J = 1.0 Hz, 1H, C6-H thymine),
1.78 (s, 3H, –CH3 thymine), 4.42 (s, 2H,-CH2); 13C NMR: δ (ppm) 167.9(-CONH-), [164(C4), 153(C2), 138(C6),
109(C5), 13.5(–CH3) thymine], 151(C2), 148(C6), 141(C4), 118(C5), 113(C3) pyridine], 53(-CH2); UV/Vis
(EtOH): λmax = 319 nm; MS (EI, 70 eV): m/z (%): 261.09 (14.8), 260.09 (100) [M+H]+; HRMS-FAB: m/z
[M+H]+ calcd for C12H12N4O3: 260.09, found: 260.20; Anal. calcd for C12H12N4O3: C 55.38, H 4.65, N 21,23,
found: C 55.36, H 4.64, N 21.22.
N-(5-Bromo-pyridin-2-yl)-2-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-acetamide (9)
1
Yellow crystals; yield: 75%; Rf = 0.81; m.p. 229-230 °C ; H NMR: δ(ppm) : 8.22 (d, J = 1.3 Hz, 1H, C6-H
pyridine), 7.97 (dd, J = 7.9, 1.2 Hz, 1H, C4-H pyridine), 8.23 (d, J = 8.1 Hz, 1H, C3-H pyridine), 7.71 (s, 1H, -
CONH-), 7.62 (q, J = 1.0 Hz, 1H, C6-H thymine), 1.76 (s, 3H, –CH3 thymine),4.42 (s, 2H, -CH2); 13C NMR: δ
(ppm) 167.6(-CONH-), [165(C4), 154(C2), 135.3(C6), 109(C5), 12.5(–CH3) thymine], [151(C2), 147.8(C6),
142(C4), 115.8(C5), 115(C3) pyridine], 53(-CH2); UV/Vis (EtOH): λmax = 325 nm; MS (EI, 70 eV): m/z (%):
340.00 (97.5), 338.00 (100) [M+H]+; HRMS-FAB: m/z [M+H]+ calcd for C12H11BrN4O3: 338.00, found:
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338.10; Anal. calcd for C12H11BrN4O3: C 42.50, H 3.27, Br 23.56, N 16.52, found: C 42.49, H 3.25, Br 23.57,
N 16.53.
2-(5-Methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-N-(5-nitro-pyridin-2-yl)-acetamide (10)
Dark brown solid; yield: 80%; Rf = 0.88; m.p. 179-180 °C ; 1H NMR : δ (ppm) : 9.26 (d, J = 1.3 Hz, 1H, C6-H
pyridine), 8.77 (dd, J = 7.9, 1.2 Hz, 1H, C4-H pyridine), 8.49 (d, J = 8.1 Hz, 1H, C3-H pyridine), 7.71 (s, 1H, -
CONH-), 7.62 (q, J = 1.0 Hz, 1H, C6-H thymine), 1.89 (s, 3H, –CH3 thymine),4.42 (s, 2H, -CH2); 13C NMR: δ
(ppm) 167.8(-CONH-), [165(C4), 152(C2), 133(C6), 109, (C5) 13(–CH3) thymine], [153.4(C2), 143.2(C6),
140(C5), 135(C4), 112.2(C3) pyridine], 53(-CH2);UV/Vis (EtOH): λmax = 315 nm, 256 nm; MS (EI, 70 eV): m/z
(%): 306.08 (13.7), 305.09 (100) [M+H]+; HRMS-FAB: m/z [M+H]+ calcd for C12H11N5O5: 305.09, found:
305.12; Anal. calcd for C12H11N5O5: C 47.22, H 3.63, N 22.94, found: C 47.20, H 3.61, N 22.92.
2-Benzoimidazol-1-yl-N-pyridin-2-yl-acetamide (11)
White solid; yield: 78%; Rf = 0.48 ; m.p. 130 °C ; H NMR : δ(ppm) : 8.36 (dd, J = 5.0, 1.2 Hz, 1H, C6-H
1
pyridine), 7.99 (dd, J = 8.2, 1.1 Hz, 1H, C3-H pyridine), 7.86 (td, J = 8.0, 1.3 Hz, 1H, C4-H pyridine), 7.12 (dd, J
= 8.2, 1.1 Hz, 1H, C5-H pyridine),7.71 (s, 1H, -CONH-), 7.64 (dd, J = 7.4, 1.6 Hz, 2H, C4,7 -H benzimidazole),
8.17 (s, 1H, C2-H benzimidazole), 7.32 – 7.25 (m, 1H, C5,6-H benzimidazole), 5.22 (s, 2H, -CH2); 13C NMR: δ
(ppm) 167.7(-CONH-), [141.3(C2), 137.6(C8, C9), 123.5(C5, C6), 114.8(C4, C7) benzimidazole], [152.2 (C2),
145.4(C6), 140.5(C4), 120.5(C5), 115(C3) pyridine], 54.5(-CH2); UV/Vis (EtOH): λmax = 213 nm; MS (EI, 70
eV): m/z (%): 253.09 (17.1), 252.11 (100) [M+H]+; HRMS-FAB: m/z [M+H]+ calcd for C14H12N4O: 252.11,
found: 252.16; Anal. calcd for C14H12N4O: C 66.65, H 4.79, N 22.21, found: C 66.64, H 4.77, N 22.19.
2-Benzoimidazol-1-yl-N-(5-bromo-pyridin-2-yl) – acetamide (12)
1
Light brown solid; yield : 78%; Rf =0.48 ; m.p. 247°C ; H NMR : δ(ppm) : 8.26 – 8.20 (m, 2H, C3, -H
6
pyridine), 7.97 (dd, J = 7.9, 1.3 Hz, 1H, C4-H pyridine), 8.17 (s, 1H, C2-H benzimidazole), 7.64 (dd, J = 7.4, 1.6
Hz, 2H, C4,7 -H benzimidazole), 7.25 (dd, 1H, C6 -H benzimidazole), 7.20 (td, J = 7.5, 1.6 Hz, 1H, C5 -H
benzimidazole), 7.71 (s, 1H,-CONH-), 5.22 (s, 2H,-CH2); 13C NMR: δ (ppm) 169(-CONH-), [153.5(C2),
149(C6), 142(C4), 117(C5), 115.9(C3) pyridine], [142(C2), 136.6(C8, C9), 123.8(C5, C6), 114.2(C4, C7)
benzimidazole], 54.8(-CH2); UV/Vis (EtOH): λmax = 224 nm; MS (EI, 70 eV): m/z (%): 332.00 (98.9), 330.10
(100) [M+H]+; HRMS-FAB: m/z [M+H]+ calcd for C14H11BrN4O: 330.10, found: 330.12; Anal. calcd for
C14H11BrN4O: C 50.77, H 3.35, Br 24.13, N 16.92, found: C 50.75, H 3.34, N 16.90.
2- Benzoimidazol-1-yl-N-(5-nitro-pyridin-2-yl)-acetamide (13)
Dark yellow solid ; yield : 56%; Rf = 0.73; m.p. 138-139 °C ; 1H NMR : δ (ppm) : 9.26 (d, J = 1.3 Hz, 1H, C6-
H pyridine), 8.77 (dd, J = 7.9, 1.2 Hz, 1H, C4-H pyridine), 8.49 (d, J = 8.1 Hz, 1H, C3-H pyridine), 7.71 (s, 1H, -
CONH-), 8.17 (s, 1H, C2-H benzimidazole), 7.64 (dd, J = 7.4, 1.6 Hz, 2H, C4,7 -H benzimidazole), 7.25 (m, 1H,
C6 -H benzimidazole), 7.20 (td, J = 7.5, 1.6 Hz, 1H, C5 -H benzimidazole), 5.22 (s, 2H, -CH2); 13C NMR: δ
(ppm) 169(-CONH-), [157(C2), 143(C6), 140(C5), 134(C4), 114(C3) pyridine],[ 141.3(C2), 136.9(C8, C9),
123.2(C5, C6), 115.2(C4, C7) benzimidazole], 54.8(-CH2); UV/Vis (EtOH): λmax = 210 nm; MS (EI, 70 eV): m/z
(%): 298.11 (17.7), 297.10 (100) [M+H]+; HRMS-FAB: m/z [M+H]+ calcd for C14H11N5O3 297.10, found:
297.12; Anal. calcd for C14H11N5O3: C 56.56, H 3.73, N 23.56, found: C 56.54, H 3.72, N 23.55.
General procedure for synthesis of compounds 14-17
3a (2.5 mmol, in the case of compounds 14-15)/ 4a (2.5 mmol, in the case of compounds 16-17) was suspended
in dry pyridine (4 mL) in a 100 mL round bottomed flask and added succinoyl chloride (1 mmol) and stirred the
reaction mixture for 4 h. The solvent was removed in vacuo and the resulting material was partitioned between
aqueous NaHCO3 solution and DCM. The organic layer was dried over anhydrous Na2SO4, filtered,
concentrated in vacuo and the product was crystallized using a mixture of ethyl acetate and hexane.
N,N’-Bis-(5-bromo-pyridin-2-yl)-succinamide (14)
Light brown crystals; yield: 75%; Rf = 0.81; m.p. 137°C; 1H NMR : δ (ppm): 2.45 (s, 2H,-CH2), 8.26 – 8.20 (m,
2H, C3-6 -H pyridine), 7.97 (dd, J = 7.9, 1.3 Hz, 1H, C4-H pyridine); 13C NMR: δ (ppm) 172.4(-CONH-), [152.2
(C2), 149.4(C6), 140.5(C4), 115.5(C5), 115(C3) pyridine], 30.8(-CH2); UV/Vis (EtOH): λmax = 280 nm; MS (EI,
70 eV): m/z (%): 429.80 (50.0), 427.80 (100) [M+H]+; HRMS-FAB: m/z [M+H]+ calcd for C14H12Br2N4O2:
427.80, found: 427.82; Anal. calcd for C14H12Br2N4O2: C 39.28, H 2.83, Br 37.33, N 13.09, found: C 39.26, H
2.80, Br 37.31, N 13.08.
N,N’-Bis-(5-nitro-pyridin-2-yl)-succinamide (15)
1
white crystals; yield: 85%; Rf = 0.67; m.p. 147°C; H NMR : δ (ppm): 2.45 (s, 2H,-CH2), 9.26 (d, J = 1.3 Hz,
1H, C6 -H pyridine), 8.77 (dd, J = 7.9, 1.2 Hz, 1H, C4-H pyridine), 8.49 (d, J = 8.1 Hz, 1H, C3-H pyridine); 13
C
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NMR: δ (ppm) 172.4(-CONH-), [158.2 (C2), 142.4(C6), 140.5(C5), 132.5(C5), 115(C3) pyridine], 30.2(-CH2);
UV/Vis (EtOH): λmax = 294 nm; MS (EI, 70 eV): m/z (%): 361.10 (18.2), 360.10 (100) [M+H]+; HRMS-FAB:
m/z [M+H]+ calcd for C14H12N6O6: 360.10, found: 360.12; Anal. calcd for C14H12N6O6: C 46.67, H 3.36, N
23.33, found: C 46.65, H 3.34, N 23.30.
1, 4-Bis-benzoimidazol-1-yl-butane-1, 4-dione (16)
Brown solid; yield: 80%; Rf = 0.66; m.p. 145-146 °C; H NMR : δ (ppm): 8.08 (s, 1H, C2-H benzimidazole),
1
7.56 (ddd, J = 7.0, 4.9, 1.7 Hz, 2H, C4,7-H benzimidazole), 7.29 – 7.17 (m, 2H, C5,6-H benzimidazole), 2.73 (s,
2H, -CH2); 13C NMR: δ (ppm) 202(-CONH-), [141.3(C2), 137.6(C8, C9), 123.5(C5, C6), 114.8(C4, C7)
benzimidazole], 26.8(-CH2); UV/Vis (EtOH): λmax = 267 nm; MS (EI, 70 eV): m/z (%): 319.10 (21.8), 318.12
(100) [M+H]+; HRMS-FAB: m/z [M+H]+ calcd for C18H14N4O2: 318.12, found: 318.14; Anal. calcd for
C18H14N4O2: C 67.91, H 4.43, N 17.60, found: C 67.89, H 4.41, N 17.58.
1-Bis-(4-nitro-benzoimidazol-1-yl)-butane-1, 4-dione (17)
1
Yellow solid; yield: 60%; Rf = 0.58; m.p. 154-156 °C; H NMR : δ (ppm): 8.14 (dd, J = 7.5, 1.5 Hz, 1H, C7-H
benzimidazole), 8.08 (s, 1H, C2-H benzimidazole), 7.95 (dd, J = 7.5, 1.5 Hz, 1H, C5-H benzimidazole), 7.49 (t, J
= 7.4 Hz, 1H, C6-H benzimidazole); 13C NMR: δ (ppm) 13C NMR: δ (ppm) 202(-CONH-), [142.3(C2),
138.6(C8), 134.8(C4), 132.7(C9), 122.9(C6), 120.8(C7), 118.5(C5) benzimidazole], 26.9(-CH2); UV/Vis (EtOH):
λmax = 252 nm; MS (EI, 70 eV): m/z (%): 409.10 (22.7), 408.10 (100) [M+H]+; HRMS-FAB: m/z [M+H]+
calcd for C18H12N6O6: 408.10, found: 408.12; Anal. calcd for C18H12N6O6: C 52.95, H 2.96, N 20.58, found: C
52.94, H 2.94, N 20.56.
In vitro Anti-HIV-1 assay
In vitro anti-HIV assay was carried out using published procedure (28).
Cell lines
TZM-bl cell line was used for primary screening of all compounds. TZM-bl cells were obtained from NIH
AIDS Research and Reference Reagent Programme (NIH ARRRP), USA. These are genetically engineered
HeLa cells, which have been modified to express different receptors, like CD4, CXCR4, and CCR5 and contain
Tat-responsive reporter genes for firefly luciferase (Luc) and Escherichia coli β-galactosidase under regulatory
control of an HIV-1 LTR. The cells were maintained in Dulbecco’s Modified Eagle’s Medium DMEM (Gibco,
USA) containing 10% heat inactivated fetal bovine serum (Morgate, Australia), HEPES (Gibco, USA),
penicillin, streptomycin (Gibco, USA) and gentamicin (Sigma, USA) and the cultures were incubated at 370C in
a humidified 5% CO2 atmosphere.
Viral Stocks
The laboratory adapted strains HIV-1IIIB (X4, subtype B), HIV-1Ada5 (R5, subtype B) and the primary isolates
HIV-1UG070 (X4, subtype D) were obtained from NIH ARRRP, whereas R5 tropic isolate HIV-1VB59 (subtype C)
was an Indian isolate from the National AIDS Research Institute, Pune. The viruses were grown in PHA-P
activated (Sigma Aldrich, USA) peripheral blood mononuclear cells (PBMC) derived from healthy donors. The
virus production was quantified in cell culture supernatants by HIV-1 p24 antigen detection kit (Vironostika
HIV-1 Antigen, Biomerieux, Netherlands). Aliquots of cell-free culture viral supernatants, stored at -70oC were
used as the viral stocks and the 50% tissue culture infectivity dose (TCID50) of each isolate was determined in
the appropriate cell line by using the Spearman Karber formula.
Cytotoxicity Assay
The TZM-bl cells (1×104/well/100µL medium) were seeded and the plate was incubated overnight at 37oC with
5% CO2. On the next day, the medium was replaced with 100 µL of two fold dilution series of each compound
and was incubated further. After 48 h, the cell viability was determined using MTT assay (3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma, USA). In this assay, the measured absorbance is
proportional to the viable cell number and inversely to the degree of cytotoxicity. Three independent assays
using quadruplicate measurements were carried out for each compound and the mean CC50 value was calculated.
Cell associated Anti-HIV-1 assay
The TZM-bl cells (1×104/well/100µL media) were seeded in a 96-well plate and incubated overnight at 37oC
with 5% CO2. On the subsequent day, the cells were infected with 400 TCID50 HIV-1 stock and incubated for 2
h at 37oC with 5% CO2. Serial dilutions of each compound showing more than 50% viability were then added in
duplicate onto the cells. The anti-HIV activity was determined after 48 h of incubation by measuring the relative
luminescence units (RLU) using the Britelite Plus substrate (Perkin Elmer). In this assay, RLU is directly
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proportional to the number of virus particles and inversely to the percent of inhibition. The concentration
showing 50% inhibition (IC50) was determined using Luc software. The results were interpreted as mean of two
independent assays. Nevirapine was used as a positive control.
Molecular modeling
The computational studies were carried out with the X-ray crystal structure of HIV-1 RT complexed with a
ligand (PDB: ID 3E01). All computational studies were carried out using Discovery Studio 2.5 (DS 2.5;
Accelrys Ltd., USA) obtained through Apsara Innovations Pvt Ltd, Bangalore, India) on an Intel Pentium 2.99
GHz processor, 1.99 GB RAM with Windows XP professional version 2002 operating system. Receptor from
the target protein (PDB: ID 3E01) and ligands were prepared according to the published procedure (8).
Receptor set up
The target protein [PDB: ID 3E01] was taken, the ligand extracted, the missing hydrogens added, and their
positions optimized using the all-atom CHARMm forcefield and the Adopted Basis set Newton Raphson
(ABNR) method available in DS 2.5 protocol until the root mean square (r.m.s.) gradient was less than 0.05
kcal/mol/Å. The minimized protein was defined as the ‘receptor’ using the binding site module of DS 2.5. The
binding site was defined from the volume of ligand method, which was modified to accommodate all important
interacting residues in the active site of HIV-1 RT. The Input Site Sphere was defined over the binding site, with
a radius of 5Å from the center of the binding site. The protein, thus characterized, was taken as the target
receptor for the docking procedure. Minimization was performed to relax these newly added hydrogen atoms by
fixing all other non-hydrogen atoms. The minimized structure of the protein was used in docking simulations.
Ligand set up
Using the built-and-edit module of DS 2.5, various ligands were built; all-atom CHARMm forcefield
parameterization was assigned and then minimized using the ABNR method. A conformational search of the
ligand was carried out using a stimulated annealing molecular dynamics (MD) approach. The ligand was heated
to a temperature of 700 K and then annealed to 200 K. Thirty such cycles were carried out. The transformation
obtained at the end of each cycle was further subjected to local energy minimization, using the ABNR method.
The 30 energy-minimized structures were then superimposed and the lowest energy conformation occurring in
the major cluster was taken to be the most probable conformation.
Docking and scoring
Molecular docking is a computational method used to predict the binding of the ligand to the receptor binding
site by varying position and conformation of the ligand. Binding mode of inhibitors can be explored by using the
LigandFit docking protocol (14, 15). LigandFit algorithm performed a shape comparison filter with a Monte
Carlo conformational search (bond lengths and bond angles fixed and only the rotatable bonds were allowed to
rotate freely) to generate docked poses parallel to the shape of the binding site. Dreiding forcefield, grid-based
calculated interaction energy, is used to refine these poses by rigid body minimization. The receptor protein was
fixed during docking. The docked poses were further minimized by using all-atom CHARMm forcefield and
smart minimizer method until the r.m.s gradient for potential energy was less than 0.001 kcal mol−1 Å−1 and
evaluated with a set of scoring functions (Table 2). The atoms of ligand and the side chains of the residues of
the receptor within 5 Å from the center of the binding site were kept flexible during minimization. The LUDI III
score was used to score the refined poses. The ligand pose, which corresponded to the highest LUDI III score
was taken as the best docked pose Furthermore, determination of binding energy to assess the binding affinity
of ligands for receptor was calculated by employing highest stable ligand-receptor complex through the protocol
'Calculate Binding Energies' within DS 2.5 using the default settings [16-20].
Validation of the docking methodology
Docking was first tested on known inhibitor - nevirapine, which was docked in the allosteric binding site of
HIV-1 RT, after extracting the ligand from the crystal structure. A deviation was observed in the conformation
of the docked ligand on superimposition with nevirapine. Table 1 showed the docked inhibitors with the HIV-1
RT. This is in good agreement with the observed values for nevirapine. The root mean square deviation (RMSD)
of the best docked poses of the molecules reported herein was less than 2 Å, which was supposed to be an
appropriate condition for developing NNRTIs. Two structures overlapped very well with positional RMSD of
1.2. The docked pose having the highest LUDI III score gave the least RMSD with respect to the crystal
conformation for both the ligands. The analysis of the results showed a sound validation of the docking and
scoring methodology used in this study and the ligand as a whole moved into a more stable position with a much
lower docked energy.
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Calculation of H-acceptors, H-donors, TPSA and logP values
H-acceptors, H-donors and TPSA were calculated using Molinspiration software and logP values using
ChemDraw software.
Results and Discussion
Chemistry
The newly designed N-heteroaryl compounds 5-13 were synthesized via different routes detailed in Scheme 1.
The starting intermediates 1a (uracil N (1)-acetic acid), 1b (thymine N (1)-acetic acid) and 1c (benzimidazole N
(1)-acetic acid) were prepared according to the published procedure (21). To a solution of compound 1a or (1b
or 1c) in dry acetonitrile containing pyridine as a catalyst, was added oxalyl chloride drop wise in cold and
anhydrous condition. The reaction mixture was stirred for 1h and the extra oxalyl chloride removed under
pressure. The residual oil dissolved in dry pyridine was used directly in the next step without further
purification. To a stirred solution of residual oil in dry pyridine maintained in an ice bath, added drop wise
appropriate amine dissolved in dry acetonitrile and pyridine, and stirred the reaction mixture at room
temperature. The derivatives 14-15 were synthesized using 2-amino-5-substituted-pyridine and succinoyl
chloride (Scheme 2). Similarly, other bases, viz. benzimidazole and 4-nitro-benzimidazole were also reacted
with succinoyl chloride to get the products 16 and 17. The reaction mixture was treated with sodium bicarbonate
solution and extracted with dichloromethane. Organic extract was dried over anhydrous sodium sulfate and
concentrated under reduced pressure. Finally, the pure product was separated by silica-gel column
chromatography using ethyl acetate and hexane (22-23).
Molecular docking analysis
We have examined sixty N-heteroaryl compounds to develop some potent and selective inhibitors of HIV-1 RT
by using in silico structure-based approach. On the basis of preliminary in silico investigations, we selected
thirteen promising molecules for further studies. All compounds have amide linkages. The presence of a C=O
dipole and a small N-C dipole allowed these molecules to function as H-bond acceptors as well as H-bond
donors. Since these molecules have both H-donor and H-acceptor sites, they can ionize at an appropriate pH to
further enhance their solubility. Thus, these molecules have potential to internalize better in cells. Some
physically significant descriptors and pharmaceutically relevant properties of designed molecules, like
molecular weight, H-bond donors, H-bond acceptors, logP (octanol/water) have been analysed on the basis of
Lipinski’s rule of five and the results are summarized in Table 1. Molecules showing more than one type of
violations are supposed to have problems with bioavailability, hence rejected. In the present study (Table 1), all
ligands showed the allowed values for the properties analysed and exhibited drug-like characteristics based on
Lipinski’s rule of five.
Molecular docking is frequently used tool in computer-assisted drug discovery. It is used to predict the
preferred orientation, affinity and activity of small molecules into the active site of target protein through a
process involving a series of steps (24). Docking results have been discussed using the parameters, like
hydrogen bond, non-bonded ‘π-π’ and ‘π-+’ interactions as they help stabilize and strengthen receptor-ligand
complexes. The ‘π-π’ and ‘π- +’ interactions are non-covalent interactions and play crucial role in protein-ligand
recognition. According to Gallivan and Dougherty, the effective distance for ‘π- +’ interactions necessary for
receptor-ligand recognition should fall below 6Å (25) and values observed for all compounds (5-17) are well
below this range.
Docking of molecules 5-17 using DS 2.5 into the allosteric site of target protein [PDB: ID 3E01] generated the
plausible binding conformations with the corresponding Ludi_3 values. The cluster analysis revealed a
predominant orientation of the ligands within the binding pocket of active site and the conformation with the
highest Ludi_3 score for each molecule was chosen for further analysis. In order to derive maximum
information about interactive forces for receptor-ligand interaction, the entire set of molecules were rigorously
docked into the allosteric site of HIV-1 RT, using the same protocol and analysed through above mentioned
parameters and results presented in Table 2
Visual inspection of the minimized complexes of inhibitors with HIV-1 RT showed the requisite electronic and
hydrophobic interactions, suitable for providing stability of a certain level to the protein-ligand complex,
between the enzyme and the ligands. These derivatives, like the other NNRTIs, adopted a common "butterfly"
conformation. (Figure 1) However, there are important differences in their conformations and specific
positioning within the NNIBP. The molecules formed one to three H-bonds with various amino acids (Lys 101,
Lys 103, His 235, Pro 236 and Tyr 318) present in the NNRTI binding site of HIV-1 RT.
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Notably, the NNRTI binding pocket is largely hydrophobic and few H-bonds are accommodated. For the
pyrimidine based RTIs, the H-bond between the N(3)H of the inhibitor and the backbone carbonyl group of
Lys101 represented the major hydrophilic interaction. To maximize hydrophobic interactions throughout the
binding pocket, this H-bond not only contributed to the binding affinity but also served as an anchor for the
inhibitor to adopt optimized conformation (26).
In compound 5, uracil ring pointed towards aromatic amino acid residue within NNBP showed π interaction
with Tyr 181 (4.4 Å) and heteroaryl ring showed π interaction with Tyr 318 (4.5 Å), but did not form any H-
bond. In the case of compounds 6 and 7, the C=O portion of the uracil ring formed H-bonds with Lys 103 and
Val 106 and the pyridine ring containing bromo and nitro group did not participate in any interaction and no H-
bond formation occurred. In compound 8, the C=O portion of the thymine ring formed H-bond with Lys 103
(2.8 Å) and the ring showed π interaction with Phe 227 (5.2 Å). In compound 9, C=O portion of the thymine
ring formed one H-bond with Val 106 (3.2 Å) and showed π interaction with Phe 227 (5.4 Å) and the amide
linkage formed H-bonds with Tyr 318 (2.9 Å). In compound 10, the thymine ring formed two H-bonds, the first
between Lys 103 and C=O portion of thymine ring and the second bond (3.1 Å) between Lys 103 and –NH
group of ring . Further, the -NO2 group formed another H-bond with side chain of Tyr 181(3.1 Å). In compound
11, the benzimidazole nucleus showed two π interactions with Tyr 181 (4.4 Å and 5.3 Å). In compounds 12 and
13, the amide linkage formed one H-bond with Lys 103 (2.8 Å each) (Figure 2 and Figure 3). In compound 14,
the amide linkage formed one H-bond with Lys 103. Similarly, in compound 15, the amide linkage formed one
H-bond with Lys 103 (3.1 Å) and the NO2 group showed two π- cation interactions with Trp 229 (6 Å and 5 Å).
Compounds 16 and 17 did not show any interaction with HIV RT. However, surprisingly, their total interaction
energy was comparable or a bit higher than the reference and other compounds (Table 2). This indicated a
unique structure–activity relationship that depended on LogP and total polar surface area (TPSA) of the
compounds and, in turn, on hydrophobicity (Figure 2). Compounds 16 and 17 had higher LogP values and
showed more hydrophobic character. The hydrophobic effect arises due to the interaction of non polar atoms on
surrounding water molecules and the magnitude of the hydrophobic effect is proportional to the TPSA of the
non polar atoms that the molecule contains, as shown by Table 2 (27). Thus, it can be inferred that in the
absence of H-bond, ‘π-π’ and ‘π- +’ interactions with key amino acid residue of NNBP, it is the hydrophobic
interaction that stabilizes the HIVRT-ligand, which depends on LogP and TPSA values for that compound.
Different groups at the C-5 of the heteroaryl moieties also influenced the inhibitory activity of the compounds.
Analogs having nitro group at the termini exhibited better anti-HIV activity (compounds 7, 10, 15, 16 and 17)
however, the compound having Br atom at the terminus (compound 14) showed the highest activity.
Anti-HIV evaluation
The compounds 5–13 and 14-17 presented in this study were evaluated for their anti-HIV-1 activity using cell
based assays. The results, expressed as EC50, CC50 and the selectivity index (SI), are summarized in Table 3. In
the cell based assay, the compounds 14, 15 and 17 were the most potent inhibitors of HIV-1 replication against
HIV-1IIIB, the EC50 ranged from 15 to 25 µM (SI value 10 to 28) and against HIV-1Ada 5, the EC50 ranged from
15 to 64 µM (SI value 7 to 15.08). The other compounds 6, 7, 10 and 16, also showed reasonable anti-HIV-1
potency: the EC50 ranged from 1-65 µM and 1-40 µM against HIV-1IIIB and HIV-1Ada5 strains, respectively. The
compounds having activity against HIV-1IIIB and/or HIV-1Ada5 strains were taken for further anti-HIV-1
screening against the primary HIV-1 isolates from Uganda (HIV-1UG070) and Indian (HIV-1VB59) origin. The
compounds 14, 16 and 17 were found most active against primary isolates (EC50: 5 to 35 µM; SI: 6.28 to 38.39
against HIV-1UG070 strain, EC50: 25 to 98 µM; SI 4.97 to 8.06 against HIV-1VB59 strain) as shown in Table 3.
Thus, in the cell based assay, amongst the 13 compounds the highest inhibitory activity was shown by
compounds 14, 15, 16 and 17 with SI ranging between 4.96-28.04 against lab adapted strains, and with SI 2.31–
38.39 against primary isolates. While the other compounds were moderate inhibitors of HIV-1 replication
against HIV-1IIIB and HIV-1Ada5 strains.
In view of the results obtained against HIV-1 using the strains HIV-1IIIB, HIV-1Ada5 and primary isolates HIV-
1UG070 and HIV-1VB59 from Uganda and Indian, respectively, under in vitro condition, we can conclude that the
molecules acted as HIV-1 reverse transcriptase inhibitors and that the chemical skeletons of compounds 14, 15,
16 and 17 may be developed as lead HIV-1 RT inhibitors with suitable structure/ pharmacophore modifications.
Further results in this direction will be described as separate studies.
Conflict of Interest: The authors declare no conflict of interest.
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Legends
Table 1 N-Heteroaryl Compounds 5-17 in the eyes of Lipinski’s Rule
Table 2 Physical parameters, ligand score and stabilization energy of HIV-1 RT- ligand complexes of 5-
17
Table 3 Results of anti-HIV-1 activity, cytotoxicity and selectivity index in TZM-bl cells against HIV-1
lab
Scheme1 Synthesis of compounds 5-13
Reagents and conditions: i) H2O, aq. KOH, 40 °C, 30 min; ii) CH3CN / Py ( v/v 3:1), oxalyl
adapted strains and primary isolates
chloride, rt, 1h; iii) Py, Et3N, 16 h
Scheme 2 Synthesis of compounds 14-17
Reagents and conditions: i) CH3CN / Py (v/v 3:1), Et3N, rt, 12 h
Figure 1 (A) ‘Butterfly conformation’ of compound 17 showing hydrophobic character (shown by blue dots)
(B)
Binding mode of compound 17 with HIV-1 RT: Stabilization of HIV-1 RT-ligand complex through
hydrophobic interaction (surface view)
Figure 2 Hydrophobic interaction of compound 14-17 within HIV-1 RTwt NNIBP
Figure 3 Binding of compound 5-12 into allosteric site of HIV-1 RTwt (only surrounding residues are shown
in
grey, hydrogen bond shown by green dashed line and pi interaction shown by solid orange line)
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Table 1
N-Heteroaryl Compounds 5-17 in the eyes of Lipinski’s Rule
Compound
MW1
246
H-A2
7
H-D3
2
LogP4
-1.029
-0.05
-0.9
TPSA5
96.85
96.85
142.68
96.85
96.85
142.68
59.81
59.82
105.64
83.98
175.62
69.79
161.44
63.58
120.65
violations
0
0
0
0
0
0
0
0
0
0
1
0
1
1
1
5
325
7
2
6
291
10
7
2
7
260
2
-0.33
0.649
-0.201
1.401
2.38
8
339
7
2
9
305
10
5
2
10
252
1
11
331
5
1
12
297
8
1
1.53
13
428
6
2
2.62
14
15
360
12
6
2
0.92
318
0
2.97
16
408
12
5
0
2.80
17
266.306
435.28
1
1.380
5.027
Nevirapine
Etravirine
7
3
1. Molecular weight
2. number of H bond acceptors
3. number of H bond donors
4. Octanol-water partition coefficient
5. Total polar surface area
Table 2
Physical parameters, ligand score and stabilization energy of HIV-1 RT- ligand complexes of 5-17
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Comp logP1 TP
ound
I.E3
Lu ∆G Lu
No. and
Dista
No., type and
amino acid in
Pi bond
Dista
nce
(Aº)
P
SA2
5
6
di_
di_ EC50 amino acid nce
pred
24
34
in
(Aº)
(kc
al⁄
(μM)
hydrogen
bond
mol
)
-
96.
-40
-41
28
4
-
4.0
2
53
8
4.16
0
--
2 ‘π- π’
(Tyr181,Tyr3 4.5
18)
4.4,
5
1.029 85
×10-
06
-0.05 96.
85
40
9
-
5.8
0
42
0
6.30
2 (Lys103) 3.1,
3.0
--
--
--
6
×10-
05
-0.9
142 -43
.68
33
5
-
4.7
5
46
3
2.34
2 (Lys103) 2.6,
2.7
--
7
×10-
05
-0.33 96.
85
-41
-44
38
9
-
5.5
1
47
0
1.99
1 (Lys103) 2.8
1 ‘π- π’
5.2
--
8
×10-
(Phe227)
05
0.649 96.
85
42
6
-
6.0
4
48
8
1.31
2 (Val106,
Tyr318)
3.2,
2.9
--
--
9
×10-
05
-
142 -44
34
2
-
4.8
5
40
7
8.51
3 (Lys103,
Tyr181)
2.8,
3.1,3.
1
--
10
11
12
13
14
15
16
0.201 .68
×10-
05
1.401 59.
81
-38
-43
35
4
-
5.0
2
75
1
3.0×
10-08
--
--
2 ‘π- π’
(Tyr181)
4.4,
5.3
2.38
1.53
2.62
0.92
2.97
59.
82
40
7
-
5.7
7
42
3
5.88
1 (Lys103) 2.8
1 (Lys103) 2.8
--
--
--
--
--
--
--
--
--
--
×10-
05
105 -44
.64
37
0
-
6.0
4
42
6
5.49
×10-
05
83.
98
-48
45
1
-
6.3
9
46
0
2.51
--
--
--
--
--
--
×10-
05
175 -52
.62
40
1
-
5.6
8
42
0
6.30
×10-
05
69.
79
-46
42
7
-
6.0
5
69
4
1.14
×10-
07
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2.80
161 -53
.44
32
8
-
4.6
5
40
9
8.12
--
--
--
--
--
--
17
×10-
05
1.380 63.
58
-45
39
3
-
5.5
7
55
7
0.27
2 ‘π- π’
(Tyr181,Tyr1 5.1
88)
6.0,
Nevir
apine
×10-
05
1. Octanol-water partition coefficient
2. Total polar surface area
3. Total interaction energy
4. Ludi_2 and Ludi_3, empirical scoring functions derived from the Ludi algorithm
5. Predicted binding free energy
6. Predicted 50% effective concentration required to inhibit HIV1 replication
Table 3 Results of anti-HIV-1 activity, cytotoxicity and selectivity index in TZM-bl cells against HIV-1 lab
adapted strains and primary isolates
Number
of
Anti-HIV-1 activity1 agains t HIV-1 lab
adapted strains
Anti-HIV-1 activity1 against primary
isolates
compou
nd
3
3
EC50 2 ( µM)
CC50
SI4
SI4 EC50 2 (µM)
CC50
SI4
SI4
(µM )
(µM)
HIV-1
IIIB
HIV- TZM- HIV HIV-1 HIV-1 HIV- TZM HIV- HIV-1
1
bl
-1
ADA
UG07
1
-bl
1
VB59
ADA cells
5
IIIB
5
0
VB5 cells UG0
9
70
243
65
178
602
215
6.32
49
2.47 3.38
3.30
113
394
602
215
6.32
49
5.32
1.52
1.6
5
43
5
70
134
3.07
2.63
1.65
1.43
2.4
6
1
1.3
26
6.32 4.86
2.13 1.88
3.32 1.43
4.47 2.56
1.42 NA
2.40
29.6
39.43
32.68
389
2.68
2.35
2.1
7
23
23.2
6
43.5
7
42.1
3
ND
8
17
39.43 56.50
30.65 78.75
56.5
0
78.7
5
1.29
1.86
ND
ND
ND
4.97
9
17.6
381
NA
6.7
28.03
10
11
12
13
14
NA
76
541.9
8
118
542
1.39
ND
2.1
ND
2.7
1.5
ND
NA
NA
ND
9.59
18.14
1.89
8.65
35.04
18.1
4
487.
6
63.08 487.6
17.3 7.73
9
98.1
3
13.9
1
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15.7
40
58.33 442.5
28
7.59
110.5
5.28
191.
6
25.1
5
31.8
6
442.
5
202.
8
184.
7
4
2.31
8.06
5.8
15
16
17
40
202.8
4.96 4.96
38.3
9
6.28
17.1
15.4
184.7
7
10.7 15.08
7
29.41
1. Represents mean of two independent assay
2. EC50 is the 50% effective concentration required to inhibit HIV1 replication for strains IIIB, ADA5,
UG070 and
VB59 in the TZMbl cell line
3. CC50 is the 50% cytotoxic concentration of the respective compound to the TZMbl cells
4. Selectivity index ratio CC50/ EC50
(A)
(B)
(C)
Figure 1 (A) ‘Butterfly conformation’ of compound 17 showing hydrophobic character (shown by blue dots)
(B) Binding mode of compound 17 with HIV-1 RT: Stabilization of HIV-1 RT-ligand complex through
hydrophobic interaction (surface view) (C) Docking results of 17 into NNIBP of HIV-1 RTwt
14
15
16
Figure 2 Hydrophobic interaction of compound 14-17 within NNIBP of HIV-1 RTwt
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5
6
7
8
9
10
11
12
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Figure 3 Binding of compound 5-12 into allosteric site of HIV-1 RTwt (only surrounding residues are shown in grey,
hydrogen bond shown by green dashed line and pi interaction shown by solid orange line)
(A)
O
O
O
R1
R1
NH
O
R2
R1
O
NH
NH
i
ii
iii
Br
O
N
O
N
R2
OH
NH2
N
O
O
NH
OH
N
NH
5-10
1
1 a
(B)
R3
R3
R2
R3
N
N
N
O
i
ii
iii
Br
N
H
N
NH2
N
N
HN
O
OH
OH
R2
OH
N
2
2 a
11-13
5
6
R1 - H - H
7
-H
8
9
10
-CH3 -CH3 -CH3
-H -Br -NO2
11
R2 - H - H
R3 - H -Br
12
13
-H
-NO2
R2 - H -Br -NO2
Scheme 1 Synthesis of compounds 5-13
Reagents and conditions: i) H2O, aq. KOH, 40 °C, 30 min; ii) CH3CN / Py ( v/v 3:1), oxalyl chloride, rt, 1h; iii)
Py, Et3N, 16 h
R1
(C)
R1
R1
N
N
O
i
Cl
NH2
N
Cl
NH
O
HN
C
C
O
3a
O
14 15
-Br -NO2
14-15
R1
(D)
Hydrophobic sites
R2
R2
R2
N
O
N
N
Cl
i
N
Cl
N
N
H
O
C
C
O
O
4a
Hydrophilic sites
16 17
R2 -H -NO2
16-17
Scheme 2 Synthesis of compounds 14-17
Reagents and conditions: i) CH3CN / Py (v/v 3:1), Et3N, rt, 12 h
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