N-hydroxy-substituted 2-aryl acetamide analogs: A novel class of HIV-1 integrase inhibitors

Article abstract of DOI:10.1111/cbdd.12974

An in silico method has been used to discover N-hydroxy-substituted 2-aryl acetamide analogs as a new class of HIV-1 integrase inhibitors. Based on the molecular requirements of the binding pocket of catalytic active site, two molecules (compounds 2 and 4

Full text of DOI:10.1111/cbdd.12974

DR. SETURAM B KATTI (Orcid ID : 0000-0001-9776-2391)
Received Date : 01-Oct-2016
Revised Date : 16-Feb-2017
Accepted Date : 19-Feb-2017
Article type : Research Article
N-hydroxy substituted 2-aryl acetamide analogues: a novel class
of HIV-1 integrase inhibitors
Utsab Debnath1, Prachi Kumar2, Aakanksha Agarwal2, Ajay Kesharwani2, Satish K. Gupta2
and Seturam B. Katti1*
1Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute,
Lucknow 226031, India.
bReproductive Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali
Marg, New Delhi 110 067, India.
Abstract
An in silico method has been used to discover N-hydroxy substituted 2-aryl acetamide
analogues as a new class of HIV-1 integrase inhibitors. Based on the molecular requirements
of the binding pocket of catalytic active site, two molecules (compounds 2 and 4b) were
designed as fragments. These were further synthesized and biologically evaluated. In vitro
potency along with docking studies highlighted compound 4b as an active fragment which
was further used to synthesize new leads as HIV-1 integrase inhibitors. Finally, six promising
compounds (compounds 5b, 5c, 5e, 6-2c, 6-3b and 6-5b) were identified by integrase
inhibition assay (>50% inhibition). Based on in-vitro anti-HIV-1 activity in a reporter gene
based cell assay system, compounds 5d, 6s and 6k were found as novel HIV-1 integrase
inhibitors due to its better selectivity index. Additionally, docking study revealed the
importance of H-bond as well as hydrophobic interactions with Asn155, Lys156 and Lys159
which were required for their anti-HIV-1 activity.
Key words: AIDS, HIV-1 integrase, ZINC database, Molecular modeling
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
doi: 10.1111/cbdd.12974
This article is protected by copyright. All rights reserved.

Corresponding author: Tel: +91-522-2772454, Fax; +91-522-2771941 Email address:
setu_katti@yahoo.com, sb_katti@cdri.res.in (S. B. Katti)
Introduction
Worldwide, more than 36.7 million people are infected with human immunodeficiency virus
(HIV) and approximately 2.1 million new cases are reported each year (UNAIDS Fact sheet,
2016, www.unaids.org/en/resources/fact-sheet). Highly active antiretroviral therapy
(HAART) mainly consisting of HIV protease and reverse transcriptase inhibitors, introduced
in the mid-to-late 1990, has largely improved patient quality of life as well as longevity [1,
2]. However, drug resistance and toxicity are the main problems during HIV treatment [3].
Integrase enzyme is one of the widely explored targets for designing diverse molecules
against HIV-1[4]. This enzyme acts as an intermediate to transfer the viral DNA into the host
genome [5]. Mg+2 and Mn+2 are the two important co-factors in the catalytic site of integrase
enzyme [5]. Moreover, Mg+2 is a preferred divalent co-factor for integration [6]. This co-
factor is used for the 3'-end processing and strand transfer steps. Integrase inhibitor can play
an essential role by the process of inhibition of Mg+2 chelation in the catalytic site of the
enzyme to induce the functional impairment of integrase activity [7].
It has been seen that diverse natural and synthetic molecules are being explored as integrase
inhibitors to stop the viral progression [8-10]. Most of the drugs like Raltegravir,
Elvetegravir and Dolutegravir make a co-ordinate bond with divalent ions (e.g. Mg+2) to stop
the 3'-end processing followed by strand transfer inhibition [11]. Historical structure-activity
relationship (SAR) of all integrase inhibitors indicate that two structural segments are crucial
for their activity: (1) a hydrophobic benzyl moiety that can bind to a hydrophobic site at one
end of the integrase catalytic active site and (2) a planner chelating triad for binding with
Mg+2 ion in hydrophilic zone of the enzyme active site. Generally all active molecules
reported so far contain at least one of these features, while diketo and catechol based
inhibitors contain both hydrophobic and magnesium binding features [6, 8]. Based on this
development, a multitude of different scaffolds have been utilized to develop new inhibitors
for this enzyme. However, due to mutations in the loop region (amino acid residues 140 to
149) of the catalytic site, most of the current drugs like Raltegravir and other related
molecules are facing serious drug resistance problem [11,12]. Therefore, there is an urgent
requirement to develop new active molecules/scaffolds as integrase inhibitors. The central
theme of this study is the de novo design, synthesis and biological evaluation of new scaffold
as HIV-1 integrase inhibitors.
In the present study, we have used an in silico approach to identify a new fragment namely,
N-hydroxyl-2-arylacetimidamide which contains all the required features for integrase
inhibition (vide supra) which was further exploited to prepare in silico ZINC database. The
prepared database is used to identify new N-hydroxy substituted 2-arylacetamide as an anti-
HIV-1 integrase inhibitor. Synthesis and biological evaluation of the identified best docked
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molecule and its analogues thereof to confirm the potential anti-HIV-1 activity of the scaffold
has been described. Furthermore, molecular docking studies were performed for all
synthesized molecules to explain the observed structure-activity relationship. Finally, the lead
is identified after consideration of its good anti HIV-1 activity profile along with its good
selectivity index (SI).
Materials and Methods
In silico screening
An in silico screening method was used to identify a new class of molecules based on binding
scores. To do that, de novo designed fragments were prepared which were used for getting a
ZINC database containing 290 compounds. This database [13] was advanced to a primary
filtration of the 290 compounds through a molecular docking method using SYBYLX 1.3
[14]. For docking experiments, a co-crystal of HIV-1 integrase protein was selected (PDB ID:
1QS4) [15] and prepared by adding hydrogen atoms, fixing backbone amides and applying
Gastregial Huckle charges. Next, the energy of the protein was minimized by the Powell
method using the Tripos force field with a distance-dependent dielectric constant of 1.0 and a
non-bonding interaction cut-off of 8.0 Å and iterations up to 1000 (convergence criteria 0.001
kcal/mol Å). Using ligand-based option procedures, the binding pocket was generated in the
Protomol module of SYBYLX 1.3. The energy-minimized standard inhibitors and
synthesized compounds were docked using the Surflex-Dock-Geom X docking mode into the
pockets of selected target. The best docking poses of the compounds were selected based on
the total, crash and polar scores. The best docked conformation was used for site-directed
residue interaction analysis and visualization in Pymol [16] and MOE [17].
General synthesis procedures
Syntheses were done through general procedures as shown in Schemes 1 and 2 [18, 19].
(i) Synthesis of Compound 2: 4-chlorophenyl acetohydroxamic acid (2) was prepared in a
single-step reaction (Scheme 1) [18]. 4-chlorophenylacetic acid was treated with cyanuric
chloride (TCT) (0.3 equiv) and N-methylmorpholine (NMM) (1.0 equiv) in dichloromethane
(DCM). Additionally, dimethylamino pyridine (DMAP) was added as a catalyst (0.1 equiv),
followed by addition of hydroxylamine hydrochloride (1.1 equiv). Stirring was continued at
room temperature for 6-8 h, until 4-chloro phenylacetic acid was completely consumed as
monitored by TLC. The reaction mixture was filtered and washed with 0.1 (N) HCl and brine.
The desired product was recovered simply by removing the solvent (DCM) at reduced
pressure. The pure form of the product was obtained with high yield value. The triazine by-
products were easily removed by simple aqueous workup using 0.1N HCl solution and brine.
Finally, spectroscopic analyses were done to confirm the structures.
(ii) Synthesis of Compounds 5a-5e & 6-(1a-5c): The synthesis of 5a-e and 6-(1a-5c) was
performed using the corresponding N-hydroxy2-arylacetimidamide derivatives (4a-e) as
starting material, and according to previously reported method [19](Scheme 2). Initially,
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compounds 4a-e were synthesized by condensation of different substituted benzene
acetonitriles (1 mmol), with hydroxylamine hydrochloride (1.1 mmol) and sodium methoxide
(1.1 mmol) in methanol at reflux temperature for 12 h. Afterwards, the compound was
purified by the silica gel column chromatography using appropriate solvent system (1:10: :
methanol:chloroform).
Then, reaction of compounds 4a-e (1mmol) with 3-nitrobenzoyl chloride (1.2 mmol) in
dichloromethane (30 ml) using 1.2 mmol of triethayl amine as base at 0 °C led to the
formation of corresponding N-(3-nitrobenzoyloxy)-2-aryl acetimidamide derivatives (5a-e).
In the same condition of reaction, condensation of 4a-e with different aryl sulphonylchloride
gave the N-arylsulphonyloxy 2-aryl acetamide derivatives 6-(1a-5c). In both cases, reactions
were stirred for 1-2 h. The reaction was monitored by TLC (40% AcOEt/hexane). After
completion of reaction, the organic layer was washed with brine solution and treated with
anhydrous sodium sulphate (Na2SO4). After separating the organic layer, was evaporated
under reduced pressure to get (5a-5e) and 6-(1a-5c) with good yield values (65-85%). The
crude product was purified by silica gel column chromatography using methanol/chloroform
gradient. Spectroscopic analyses were carried out to characterize all the compounds.
In vitro assay for HIV-1 integrase inhibition
The HIV-1 integrase inhibitory activity of the compounds was determined by the commercial
kit (XpressBio, Life Science Products, MD, USA) as per the manufacturer’s instructions.
Raltegravir (2 μM; Santa Cruz Biotechnology, Dallas, Texas, USA) was used as positive
reference control [20].
In vitro anti-HIV-1 activity in reporter gene cell-based assay
TZM-bl cells [HeLa cell line expressing high levels of CD4, HIV-1 co-receptors CCR5 and
CXCR4 with β-galactosidase and luciferase as reporter genes under HIV-1 LTR promoter]
were maintained in Dulbecco's modified Eagle’s medium (DMEM; Sigma-Aldrich Inc., St.
Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries,
Kibbutz beit Haemek, Israel) and an antibiotic-antimycotic cocktail [Penicillin (100 units/ml),
Streptomycin (100 μg/ml) and Amphotericin B (250 ng/ml); Pen-Strep-Ampho sol,
Biological Industries]. TZM-bl cells (5.0 × 104/well) were seeded in 24-well cell culture plate
(Greiner Bio-One, GmbH, Frickenhausen, Germany) and cultured overnight at 37°C in
presence of 75% relative humidity and 5% CO2. In separate vials, HIV-1 NL4.3 (CXCR4
using virus) at a multiplicity of infection (MOI) of 0.05 was treated with various synthetic
compounds or solvent for 1 h at 37ºC [21]. Subsequently, pretreated viruses were added in
duplicates to TZM-bl cells and cultured for 4 h. Nevirapine (Sigma Aldrich Inc.) was used as
a positive reference control whereas negative control comprised of cells without HIV
infection. After incubation, the cells were washed once with cold 50 mM PBS, pH 7.4 to
remove the cell-free virus followed by addition of fresh culture medium with or without the
synthetic compounds. Cells were further incubated for 48 h, washed twice with PBS and
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lysed with 1X lysis buffer (Promega Corporation, Madison, USA) by freeze-thaw. The
supernatant was analyzed for luciferase activity by BrightGlo Luciferase Assay kit (Promega
Corporation) in white opti-plate and luminescence was read using Fluorimeter (BMG
Labtech GmbH, Offenberg, Germany) at a spectral range of 240 nm to740 nm. The results
were expressed as percentage inhibition, calculated by taking the luminescence in
experimental group divided by the luminescence in infected cells in absence of test
compound multiplied by hundred. Percent inhibition was calculated by subtracting the above
value from hundred.
Cytotoxicity assay
The cytotoxicity of the synthetic compounds on TZM-bl cells was assessed by MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma Aldrich Inc.] assay [22]. In
brief, TZM-bl cells were seeded (8 x 103/well) in 96-well cell culture plates (Greiner Bio-
One) and grown overnight at 37°C in presence of 75% relative humidity and 5% CO2. After
24 h, synthetic compounds were added in increasing concentrations followed by further
incubation for 48 h. Negative control included cells treated with solvent/medium. After
incubation, cell viability was assessed by adding 20 μl MTT (5 mg/ml in PBS) per well and
incubated at 37°C for 3 h followed by addition of MTT solvent (100 μl/well; absolute
isopropanol, 0.04 N HCl). The absorbance (OD) was read at 540 nm with reference filter at
630 nm. Experiments were performed in triplicates and percent viability was calculated by
dividing the OD obtained in treatment group by OD of the untreated cell control multiplied
by hundred.
Result and Discussion
Virtual Screening analysis
To increase the available drug repertoire for ‘HAART’ therapy, integrase enzyme is explored
as a crucial target for small molecule inhibitors. In the catalytic domain of the integrase
enzyme, loop regions are used to allow conformational changes that are required
for 3´processing of the viral DNA and strand transfer (STF) reactions, which are the two key
steps of the integration process. Due to the presence of loop regions (connecting area of α
helix and β sheets) in the catalytic core domain (CCD), diverse flexible molecules can be
examined to explore the binding site. To identify new molecules for this target, we employed
an in silico screening method by using online ZINC database. The ZINC database was
prepared using a de novo designed active fragment, compound 4b, due to its better binding
score (total score 4.38, Figure 1) compared to compound 2 (total score 3.81, Figure 1). The
ZINC database containing 290 molecules was further filtered by molecular docking method
to identify the most potent molecules from the database. Based on the total score, crash score,
polar score and C score ten molecules were selected (Table 1, Figure 2) from the database.
Most of them were showing good H-bond interactions (ZINC 02935679, ZINC 08829370,
ZINC 08683725) with important amino acids such as Asn155, Lys156 and Lys159 present in
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the active site of integrase (Table 1, Figure 2). Additionally, ZINC 08829370 and ZINC
02974425 were showing CH-π interactions respectively with Phe121 and Phe139 (Supporting
Information, Figure S1). This led to improved binding scores (ZINC 08829370; total score
8.26, ZINC 02974425; total score 8.61) but also reduce the crash score value near to zero
which indicated correct penetration of these molecules into the binding pocket of integrase
enzyme. From this docking analysis, it was clear that ZINC 08829370 provided the most
promising binding scores (total score 8.26; crash score -0.31; polar score 7.04 and C score 5).
It also displayed the important H-bond interactions with the important amino acids Glu152,
Lys156 and Lys159. Therefore, we synthesized this molecule and its analogues to explore
their structure-activity relationships as HIV-1 integrase inhibitor.
Synthesis and biological evaluations
Newly designed compounds (2 and 4a-4e) were synthesized by two different approaches
(Schemes 1 and 2). Both compounds were obtained through single-step one-pot multi
components reactions with high yields (>90%). Reactions were completed in 6-12 hrs. Again,
compound 4c was used to synthesize ZINC 08829370 (compound 5c) and its different
derivatives by base-catalysed (TEA) elimination reaction. The desired products were
obtained in pure form by silica gel column chromatography.
Synthesized compounds (2 and 4b) were tested for in vitro integrase inhibitory activity to
find out the active one. Here, only compound 4b was considered for in vitro assay since it
showed good docking score as well as maximum structural similarity with compound 2.
Percentage inhibition data at 50 µg/ml concentration confirmed compound 4b to be a potent
integrase inhibitor (Table 2). After identifying compound 4b as a hit molecule, other related
compounds were identified based on in silico screening. Top score based screened molecule
(ZINC 08829370) was synthesized (compound 5c) to examine its inhibitory activity against
integrase enzyme as well as infection by HIV-1 using in-vitro gene reporter cell based assay.
The percent inhibition data pointed towards a good integrase inhibition (Table 2) along with
anti HIV-1 activity (Table 3). This data provide a new scaffold that was used to explore the
activity of other analogues and to scrutinize their ability as an integrase inhibitor.
All synthesized analogues of ZINC 08829370 (5c) were screened for integrase inhibition
activity. Molecules with greater than 50% integrase inhibitions were tested further in an in-
vitro reporter gene cell (TZM-bl) based assay for anti HIV-1 activity. Furthermore, the
cytotoxicity assay was also carried out to estimate the selectivity index of the molecules. It
has been observed that compounds with halogen substitutions (compounds 5a, 5b, 6-1a, 6-5b
etc.) at R position showed moderate to good inhibition against integrase enzyme (Table 2)
whereas para-hydroxyl group substituted compounds at 4th position of a benzyl moiety
resulted in poor inhibitions (compounds 6-4a, 6-4b and 6-4c, Table 2). R1 substitution at
meta and para position of benzene ring gave promising activity against integrase as well as
HIV-1(compounds 5c, 5b, 5e, 6-2c and 6-5b, Tables 2, 3, Figure 3). In most of the cases,
bulky groups like acetamide and methoxy substituted aryl groups were showing less to
moderate activities against integrase (compounds 6-1d, 6-1e, 6-3c, 6-5c etc.). More bulky
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groups like 1,3,5-triisopropylbenzene (compound 6-1c) were less favourable at R1 position.
In vitro percent inhibition data further indicated that hydrophobic substitution on the aryl
moiety at R1 position decreased the inhibitory activities (compounds 6-1c, 6-2a, 6-3a, 6-4a
etc). The biological data also highlighted the importance of the linker of all synthesized
molecules. It may be inferred from the integrase inhibitory activity data that imine (=NH)
group at Y position plays an important role for biological activity (compound 4b, Table 2).
Similarly, linker at X position of all synthesized compounds influenced the activity.
Substitution with both carbonyl (C=O) and sulphonyl groups (SO2) exhibit reasonable anti
HIV-1 integrase activity (compounds 5b, 5c, 6-1a, 6-2c, 6-5b etc.) compared to Raltegravir.
Furthermore, the improved cytotoxicity profile of some compounds (6-2c, 6-5b, Tables 2, 3)
offered safe selectivity index.
Docking analysis of synthesized compounds
After biological evaluations the docking studies was further utilized to explain the SAR by
docking the synthesized compounds in the integrase binding pocket (PDB ID: 1QS4).
Docking analysis indicated that total, crash and polar scores play a significant role for
compound's binding affinity towards integrase enzyme. C score pointed out the overall rank
of the docked molecules. In most of the cases it has been observed that less active compounds
showed crash score values far away from zero (compounds 2, 6-1c, 6-1f, 6-2a and 6-4b),
which represents their inadequate penetration quality into the binding domain. Therefore,
these molecules provided poor binding scores (Supporting information, Table S1). Similarly,
some of the active molecules gave crash score values near to zero, which verified their well
penetrating characteristic into the binding site of integrase. Thus, better crash score value
might improve the total binding scores (Supporting information, Table S1).
Interestingly, molecular substitution with hydrophobic and halo groups at R1 position
(compounds 6-1c, 6-1f, 6-2a) were found to exhibit poor crash and total scores, which were
reflected in their decreased activity against integrase. At the same time, polar groups
containing molecules (compounds 5b, 5e, 6-2c, 6-5b etc.) influenced the total and crash
scores, which were signified through good anti HIV-1 integrase activity (Supporting
information, Table S1). The present results highlight the importance of polar interactions
instead of hydrophobic interactions due to the polar nature of the binding site of the integrase
enzyme. Furthermore, Figure 4 revealed that strong H-bond interactions with important
amino acids (Glu152, Asn155, Lys156 and Lys159) led to the improved activity of the
molecules (compounds 4b, 6-2c, 6-5b etc.). Moreover, H-bond interactions with other amino
acids such as Thr 66, Glu 148 etc. were not so significant for anti-integrase activity
(compounds 2, 6-1c etc.).
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Conclusion
In the present study, in silico based new N-hydroxy substituted 2-aryl acetamide analogues
were designed, synthesized, and evaluated for activity, which exhibited moderate to good
inhibitory activity against HIV-1 integrase enzyme. Selected molecules, having better
inhibitory activities against integrase (>50%) were tested for their anti-HIV-1 activity in
TZM-bl cell based assay. Cytotoxicity profiles of selected molecules were estimated to
identify the potent lead molecule. Among the tested compounds 5c, 5e, 6-2c and 6-5b
showed potent anti-HIV-1 activity with an IC50 value of 5.94, 14.36, 8.17 & 5.87 µg/ml in
TZM-bl cell line respectively. Furthermore, molecular docking study revealed that polar
interaction of the molecules with Asn155, Lys156, and Lys159 were significant for their
inhibitory activity against HIV-integrase. These docking analyses showed a similar binding
mode of 6-5b and Raltegravir with HIV-1 integrase enzyme. Therefore, compound 6-5b may
serves as conceptually new lead to design and prepare novel analogues to improve potency of
this new class of integrase strand transfer inhibitors for the treatment of HIV-1 infection.
Acknowledgements
The author (UD) is thankful to SAIF Division for spectral data and facilities. UD is also
thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi for the
financial support in the form of fellowships. Other authors (PK, AA, AK and SKG) are
thankful to the Department of Biotechnology (DBT), Government of India and the Indian
Council of Medical Research (ICMR), Government of India for financial support. The
authors thank NIH AIDS Research & Reference Reagent program, Division of AIDS,
NIAID, NIH for providing us the molecular clone of HIV-1NL4.3. CDRI communication no. is
336/2015/SBK.
Conflict of interest
The authors declare that no competing interests exist.
Supporting Information
Supporting information includes (1) all experimental data of synthesized compounds (2)
docking poses of selected top ten molecules from ZINC database (Figure S1) and (3)
Docking scores of all synthesized compounds along with their H-bond interaction sites (Table
S1).
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Scheme 1: Synthesis of hydroxamic acid analogue.
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Scheme 2: General scheme for synthesis of N-hydroxy substituted 2-aryl acetamide analogues.
Figure 1: Two dimension figures of docked 2 and 4b compounds with their binding residues of HIV-
1 integrase enzyme. Green and pink colour of the binding residues indicates the H-bond acceptor and
H-bond donor sites respectively. Violet colour near to the aromatic ring of the molecules illustrates
hydrophobic interactions with surrounding amino acids of the binding domain.
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Figure 2: Structures of selected top ten molecules from ZINC database.
Figure 3: Left hand panel shows anti-HIV-1 activity of compounds 5c, 5e, 6-2c, 6-3b and 6-5b
whereas right hand panel shows anti-HIV-1 activity of compound 5b at the tested concentrations of
the compounds.
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Figure 4: Binding mode of compound 6-5b (active) and 6-1c (inactive) with HIV-1 integrase (PDB
ID: 1QS4). Panels (A)/(B) and (D)/(E) showed 3D and 2D conformations of compound 6-5b and 6-1c
along with their interacting nearby important binding residues. Panels (C) and (F) showed
superimpositions of compound 6-5b (white) and 6-1c (white) with standard drug Raltegravir (green)
respectively.
Table 1: Binding scores of top ten ZINC molecules along with H-bond interaction sites.
Total
Score
7.00
Crash
Score
-0.52
-0.63
Polar
Score
3.80
C
Score
4
Sl. No
ZINC ID
H-bond interaction
1
2
ZINC 59512961
ZINC 59512904
Asn155, Lys156, Lys159
Asn155, Lys156, Lys159
Asp64, Asn155, Lys156
Lys159
7.67
4.47
4
3
ZINC 02974425
8.61
-0.58
6.78
4
4
5
ZINC 08829370
ZINC 05068971
8.26
8.16
-0.31
-0.82
7.04
6.58
5
4
Glu152, Lys156, Lys159
Asp64, Lys65, Asn155
Glu152, Asn155, Lys156
Lys159
Glu152, Lys156, Lys159
Asp64, Cys65,Thr66,
Glu152, Lys156, Lys159
Asp64, Lys65, Asn155
Lys156
6
7
8
ZINC 08683725
ZINC 02935679
ZINC 79838265
7.20
8.81
9.27
-0.96
-0.99
-3.61
3.32
7.43
6.06
4
4
5
9
ZINC 02324432
ZINC 80132816
8.00
8.12
-1.23
-1.72
4.26
3.36
5
5
10
Lys65, Thr66, Asn155
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Table 2: In vitro anti HIV-1 integrase activity of all synthesized compounds.
Code
R
R1
X
Y
%
inhibit
ion
Code
R
R1
X
Y
%
inhibit
ion
of
of
integr
ase
integr
ase
(50
(50
g/mL
g/mL
)
)
4-fluro
phenyl
4-iodo
SO2 NH
SO2 NH
SO2 NH
4-Cl
4-Cl
-
-
-
O
NS
3,5-diCl
3,5-diCl
3,5-diCl
23.3
2
6-1f
6-1g
6-1h
NH
NH
31.3
37.8
NSa
NSa
4b
5a
-
phenyl
4-bromo
phenyl
3,5-
diCl
3-nitro
phenyl
3-nitro
phenyl
CO
4-methyl
phenyl
4-Cl
CO
56.1
4-Cl
4-Cl
NSa
29.57
79.92
26.1
NH
SO2 NH
SO2 NH
SO2 NH
SO2 NH
SO2 NH
5b
5c
5d
5e
6-2a
6-2b
6-2c
6-3a
3-nitro
phenyl
4-nitro
phenyl
4-
CO NH 52.10
OCH3
3-nitro
phenyl
4-methoxy
phenyl
4-OH
CO
CO
SO2
NSa
4-Cl
NH
3-nitro
phenyl
4-methyl
phenyl
NH
NH
4-F
56.8
4-OCH3
4-nitro
phenyl
4-nitro
phenyl
3,5-
diCl
37.9
21.7
4-OCH3
4-OCH3
56.4
NSa
6-1a
6-1b
6-3b
6-3c
4-methyl SO2 NH
4-methoxy SO2 NH
3,5-
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diCl
phenyl
2,4,6-
phenyl
4-methyl
phenyl
3,5-
diCl
NSa
4-OH
4-OH
NSa
NSa
SO2 NH
SO2 NH
SO2 NH
SO2 NH
tributyl
phenyl
4-phenyl
acetamid
e
6-1c
6-1d
6-4a
6-4b
4-nitro
phenyl
3,5-
diCl
16.40
4-
4-methoxy
3,5-
diCl
NSa
4-OH
4-F
NSa
methoxy SO2 NH
phenyl
phenyl SO2 NH
6-1e
6-4c
6-5c
4-methyl
4-methoxy
SO2 NH
NSa
24.5
100
SO2 NH
4-F
4-F
6-5a
6-5b
phenyl
phenyl
4-nitro
SO2 NH
Raltegravir (reference)
88.01
phenyl
NSa : Not significant.
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Table 3: Anti-HIV-1 activity, inhibition of HIV-1 integrase activity and cytotoxicity of
selected synthetic compounds along with their docking scores.
Code
IC50 value of in vitro
In vitro cytotoxicity and anti-HIV-1
Total
anti HIV-1 integrase activity of synthetic compounds in TZM- Score
activity of synthetic
compoundsa
( g/mL)
bl cells based assay
IC50 value for
CC50 value by
MTT assay on
TZM-bl cellsc
( g/mL)
S.I.d
anti-HIV-1
activityb
( g/mL)
18.68
21.7
36.34 12.19
251.39 26.34
11.26
4.76
7.94
8.12
7.75
7.33
7.28
8.68
9.43
5b
5c
5e
6-2c
6-3b
6-5b
5.94 0.39
14.36 3.51
8.17 0.28
22.52 13.95
5.87 1.87
0.22
28.32 9.47
104.04 9.16
339.58 53.33
271.63 22.78
172.25 11.82
379.96
17.93
11.35
37.03
12.06
29.34
1727
1.59
13.50
1.55
Raltegravir
a
IC50: Concentration of the compounds resulting in 50% inhibition in the HIV-1 integrase enzyme activity
determined using a commercial kit
b
IC50: The concentration of the compounds that resulted in 50% inhibition in HIV infection. All data presented
are averages of three independent experiments performed in duplicate
c
CC50: The cytotoxic concentration of the compounds that resulted in the reduction of viable cells by 50%. All
data presented are averages of three independent experiments performed in duplicate
d
S.I.: Selectivity Index is CC50/IC50
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