Synthesis, Biological Evaluation, and Molecular Docking Studies of Novel 4-[4-Arylpyridin-1(4H)-yl]benzoic Acid Derivatives as Anti-HIV-1 Agents

Article abstract of DOI:10.1002/cbdv.201700295

The structural similarities between N1 substituted 1,4-dihydropyridines and the known gp41 inhibitors, NB-2 and NB-64, were considered in the current research for the design of some novel anti-HIV-1 agents. A series of novel 4-[4-arylpyridin-1(4H)-yl]benzoic acid derivatives were synthesized and after a comprehensive structural elucidation were screened for in vitro anti-HIV-1 activity. Most of the tested compounds displayed moderate to good inhibitory activity against HIV-1 growth and were evaluated for in vitro cytotoxic activity using XTT assay at the concentration of 100 μm. Among the tested compounds, 1c, 1d and 1e showed potent anti-HIV-1 activity against P24 expression at 100 μm with inhibition percentage of 84.00%, 76.42% and 80.50%, respectively. All the studied compounds possessed no significant cytotoxicity on MT-2 cell line. The binding modes of these compounds to gp41 binding site were determined through molecular docking study. Docking studies proved 1a as the most potent compound and binding maps exhibited that the activities might be attributed to the electrostatic and hydrophobic interactions and additional H-bonds with the gp41 binding site. The Lipinski's ‘rule of five’ and drug-likeness criteria were also calculated for the studied compounds. All derivatives obeyed the Lipinski's ‘rule of five’ and had drug-like features. The findings of this study suggest that novel 4-[4-arylpyridin-1(4H)-yl]benzoic acid might be a promising scaffold for the discovery and development of novel anti-HIV-1 agents.

Full text of DOI:10.1002/cbdv.201700295

Article Type: Full Paper
Synthesis, Biological Evaluation and Molecular Docking Studies of
Novel 4-Arylpyridin-1(4H)-yl) benzoic acid Derivatives as Anti-
HIV-1 Agents
Saghi Sepehri,a,b Sepehr Soleymani,cRezvan Zabihollahi,cMohammad R. Aghasadeghi,c Mehdi
Sadat,c Lotfollah Saghaie,a Afshin Fassihi a,*
aDepartment of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, 81746-
73461, Isfahan, Iran
E-mail: fassihi@pharm.mui.ac.ir (A. Fassihi)
bDepartment of Medicinal Chemistry, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, 5618953141, Iran
cDepartment of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
The structural similarities between N1 substituted 1,4-dihydropyridines and the known gp41 inhibitors, NB-2 and NB-64, were
considered in the current research for the design of some novel anti-HIV-1 agents. A series of novel 4-arylpyridin-1(4H)-yl) benzoic acid
derivatives were synthesized and after a comprehensive structural elucidation were screened for in vitro anti-HIV-1 activity. Most of the
tested compounds displayed moderate to good inhibitory activity against HIV-1 growth and were evaluated for in vitro cytotoxic
activity using XTT assay at the concentration of 100 μM. Among the tested compounds, 1c, 1d and 1e showed potent anti-HIV-1 activity
against P24 expression at 100 μM with inhibition percentage of 84.00, 76.42 and 80.50 %, respectively. All the studied compounds
possessed no significant cytotoxicity on MT-2 cell line. The binding modes of these compounds to gp41 binding site were determined
through molecular docking study. Docking studies proved 1a as the most potent compound and binding maps exhibited that the
activities might be attributed to the electrostatic and hydrophobic interactions and additional H-bonds with the gp41 binding site. The
Lipinski’s ‘‘rule of five’’ and drug-likeness criteria were also calculated for the studied compounds. All derivatives obeyed the Lipinski’s
‘‘rule of five’’ and had drug-like features. The findings of this study suggest that novel 4-arylpyridin-1(4H)-yl) benzoic acid might be a
promising scaffold for the discovery and development of novel anti-HIV-1 agents.
Keywords: Anti-HIV-1• N1 substituted 1,4-dihydropyridines• Gp41• Docking simulation
Introduction
Despite many developments in the prevention and treatment, HIV-1 still remains a universal health challenge due to the continued
evolution of resistance to medications presently provided to combat it. The first anti-retroviral drug was presented in the mid-1980s. It
was a nucleotide mimic inhibiting HIV-1 reverse transcriptase enzyme. Today, 25 anti-HIV drugs acting on different viral targets have
been approved for the treatment of AIDS. A drug protocol called highly-active antiretroviral therapy (HAART) applies a combination of
anti-retroviral drugs, generally including two different nucleoside reverse transcriptase inhibitors (NRTIs) and an non-nucleoside reverse
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doi: 10.1002/cbdv.201700295
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transcriptase inhibitor (NNRTI) or a protease inhibitor.[1] Because of the serious adverse effects, drug resistance and increased number of
patients of HIV-1 infection who fail to respond to HAART, development of novel anti-HIV-1 drugs is necessitated.[2],[3]
Viral entry, the first step of HIV-1 infection, is a promising step for anti HIV-1 drug design.[4],[5] It consists of the attachment of the
viral and target cell membranes, a procedure mediated by a series of molecular events involving viral envelope proteins, gp120 and
gp41.[6] The trans-membrane subunit of glycoproteins 41 (gp41) includes extracellular and trans-membrane domains and an intra-
cytoplasmic tail. The trans-membrane domain contains a hydrophobic N-terminal fusion peptide followed by a polar region, an N-
terminal heptad repeat (NHR), a disulfide-bridged loop region, a C-terminal heptad repeat (CHR), and a membrane-proximal external
region.[7],[8] During fusion, binding of gp120 to CD4 and chemokine receptors (CXCR4 or CCR5) on the target cell results in
conformational changes that disrupt the interactions between gp41 and gp120, upon this the buried trans-membrane protein gp41 will
be exposed. After exposure of gp41, the NHR region (trimer) zips around a preformed trimer of CHR helix and packs in an anti-parallel
manner to form a six-helix bundle (6-HB).[7],[9],[10]
The first entry inhibitor approved for clinical use, enfuvirtide (also referred to as T20), is a peptide that inhibits fusion.[8] Clinical
application of the enfuvirtide and other peptides are limited due to their high production cost, poor pharmacokinetic profile and
induction of drug resistance.[9] Two non-peptide pyrrole derivatives, NB-2 and NB-64 (Fig.1), were presented by high-throughput
screening technique as new HIV-1 fusion inhibitors in 2004. These compounds bind to the gp41 protein via ionic and hydrophobic
interactions.[10],[11] There is a hydrophobic pocket inside the gp41 binding site and the interaction of the six helix bundle (6-HB) with the
residues of this pocket is critical for the stability of the helix. This pocket is considered as a hotspot for small molecule drug design. It can
accommodate compounds with a molecular mass of 500 to 600 Da. Small-molecule compounds that target this pocket have been
identified in several reports.[12]
Figure 1. Some small-molecule HIV-1 fusion inhibitors
Molecular docking analysis of NB-2 and NB-64 indicates that each of them occupy only a part of the deep hydrophobic pocket
on the gp41 NHR trimer.[13] These compounds are small and do not fill the pocket. Compounds with increased hydrophobicity and
additional H-bonding functionalities provide the necessary affinity required for a small molecule inhibitor (compounds 1, 2 and 3;
Fig.1).[14],[15] These compounds provide an expanding linear binding conformation thus occupy more space in the binding pocket.[13],[14]
Some structural similarities can be observed between NB-2 and NB-64 as recognized gp41 inhibitors and 1,4-dihydropyridine
derivatives for anti-HIV-1 activity. Most of the gp41 inhibitors such as NB-2 and NB-64 have a -COOH moiety in their scaffold able to
form salt bridges with positively charged residues (Lys574 or Arg579) located in the gp41 binding site. Aromatic rings in the structures
of gp41 inhibitors as hydrophobic moieties have shown good interactions with the hydrophobic pocket of the gp41 binding site
enhancing the potency.[13-15] Based on this, the anti-HIV-1 activity of 1,4-dihydropyridines containing a phenyl ring substituted on the
N1 and a (het)aryl moiety located on the C4 position of 1,4-DHP scaffold was considered in our research group. Our preliminary
molecular docking studies showed that a -COOH functionality on the para position of the phenyl ring significantly influences the
orientation of the compound in the gp41 binding site. It has been reported that carboxylic acid moiety in most of the gp41 inhibitors
binds electrostatically to the Lys574 or Arg579 residues located on the surface of the groove pocket in gp41 structure. It is verified that
removing this group will reduce the anti-HIV-1 potency of the compound. Another key structural feature of NB-2 and NB-64 which is
involved in the interaction with hydrophobic binding surface of the gp41 is the two intersecting rings, pyrrol and the substituted
phenyl rings. This system of NB structures has an analogue in 1,4-DHP compounds: the substituted phenyl ring attached to the N1 atom
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of the 1,4-DHP scaffold. The substituted aromatic ring attached to the nitrogen atom of 1,4-DHP ring increases the hydrophobicity and
molecular size. This scaffold is supposed to occupy the hydrophobic pocket inside the gp41 binding site efficiently which favors the
anti-HIV-1 activity according to previous researches.
There are few reports on the anti-HIV-1 activity of 1,4-dihydropyridine derivatives. Hilgeroth and coworkers have reported N-
substituted cage dimeric 4-aryl-1,4-dihydropyridines as a novel class of HIV-1 protease inhibitors in 1999. The compounds they
investigated exerted 6-32% inhibition at 25 and 50 μM on the activity of HIV-1 protease.[16] In another work, the same researchers
prepared a series of novel N-H and N-alkyl-substituted cage dimeric 4-aryl-1,4-dihydropyridines. As HIV-1 protease inhibitors their IC50s
were between 16.1 to 262 μM.[17]
1,4-dihydropyridine derivatives have been reported as anti-HIV-1 agents working by moderate inhibition of HIV-1 protease.[18]
There is not any report for the inhibition of HIV-1 gp41 by 1,4-dihydropyridine scaffold.
In this paper we report the design, synthesis, and in vitro HIV-1 inhibitory activity of some novel N1 substituted 1,4-
dihydropyridine compounds:
4-(3,5-bis(alkoxycarbonyl)-2,6-dimethyl-4-arylpyridin-1(4H)-yl)benzoic acid derivatives. The structural
similarities with known gp41 inhibitors were considered in the design of these compounds. Intermolecular interactions of the studied
compounds with the putative viral receptor, gp41, were also investigated using molecular docking simulations.
Results and Discussion
As it was mentioned in the introduction, the most relevant compounds which were previously reported as anti-HIV-1 compounds were
dimeric dihydropyridine derivatives. They were evaluated as inhibitors of HIV-1 protease enzyme. They were different from the
compounds in the present study in both the chemical structure and the biological target. In the following sections the in vitro and in
silico results of anti-HIV-1 studies of the prepared compounds are discussed in detail.
Chemistry
Symmetric N1 substituted 1,4-dihydropyridine derivatives (1a-h) were synthesized as described in Scheme 1. The core 1,4-DHP ring
was constructed according to the Hantzsch multi-component cyclocondensation reaction.[19] In brief, a mixture of the proper aldehyde,
alkyl acetoacetate and para-amino benzoic acid was refluxed in absolute ethanol to provide 4-[3,5-bis(alkoxycarbonyl)-2,6-dimethyl-4-
arylpyridin-1(4H)-yl] benzoic acid derivatives (1a-h). For compounds 1b and 1h the reaction was complete when HCl was used as a
catalyst.
O
Ar
O
O
Ar
N
O
O
H
R
R
EtOH
O
O
O
O
O
CH3
H3C
CH3
H3C
O
Reflux, 24-28h
NH2
HO
O
HO
O
H3C
O
O
=
=
=
=
=
N
H3C
S
S
O2N
O
=
=
=
Scheme 1. Synthesis of 4-[3,5-bis(alkoxycarbonyl)-2,6-dimethyl-4-arylpyridin-1(4H)-yl] benzoic acids (1a-h).
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Structures of compounds 1a-h were unambiguously established on the basis of their spectral (1H-NMR, FT-IR and LC-Mass) and
elemental analysis data. FT-IR spectra of the final compounds exhibited the characteristic broad absorption peaks of carboxylic acid O-H
group around 2500-3500 cm-1. Stretch absorption bands of carboxylic acid C-O were also observed in 1072-1315 cm-1. The presence of
the ester and carboxylic acid C=O groups was confirmed by strong absorption peaks at 1686-1705 cm-1 and 1609-1677 cm-1,
respectively. 1H-NMR spectra revealed broad singlets at 10.63-13.27 ppm demonstrating the presence of carboxylic acid group in the
final compounds. Singlet peaks in 4.83-5.91 ppm belonging to the C-4 proton of the DHP ring confirmed that this ring is formed
successfully. Other 1H-NMR spectral signals were in accordance with the proposed structures.
Biological assay
Anti-HIV activities of all the synthesized derivatives were evaluated in terms of inhibitory activity against HIV-1 replication in HEK cells
cultures. All the synthesized compounds were also tested for their cytotoxicity on MT-2 cell line by XTT assay. Evaluated compounds did
not exhibit significant cytotoxicity at concentrations below 100 μM. HIV-1 replication inhibitory activities at 100 μM from 27 to 84%
were recorded for the studied compounds. The biological results are summarized in Table 1.
Relationships between the structure and the anti-HIV-1 activity of the synthesized derivatives were as follows:
1) Compounds 1c and 1d showed higher activity when compared with other derivatives. It can be postulated that hetero
aromatic groups at C4 position might be responsible for the higher activity of these compounds.These compounds possess 2-furyl
heterocyclic substituent instead of substituted phenyl ring at C4 of the 1,4-DHP scaffold. One possible reason may be related to
additional H-bonding ability of 2-furyl moiety. This is also reflected in the TPSA of these compounds which is 106.29 Å2. The same
postulation has been made by other researchers.[14] Ligand-receptor interaction plots showed that oxygen atom of 2-furan hetero-
aromatic ring at C4 position of 1,4-DHP is involved in hydrogen bonding with indole NH of Trp571. Replacement of the furyl substituent
in compound 1c with thienyl (compound 1e) lowered the activity. According to the 2D ligand-receptor interaction plots, sulfur atom is
not involved in hydrogen bonding with any of the gp41 binding site residues; instead, it has additional hydrophobic interactions with
the target.
Table 1. Anti-HIV-1 activity of the prepared compounds
100 μM
Compound
(%) inhibition rate of P24 expression
% cell viability
1a
1b
1c
30.57 4.08
86.63 1.52
47.50 2.50
84.00 0.17
80.50 1.63
76.42 5.69
50.12 7.50
30.00 5.00
27.00 7.96
100
93.37 4.15
82.12 2.04
98.15 2.20
75.16 5.09
96.30 2.99
80.55 5.57
65.72 2.86
100
1d
1e
1f
1g
1h
BMS-806
a F=22.66303 > F Crit=3.500464, p-value=0.000113
b F=9.703489 > F Crit=3.500464, p-value=0.002322
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2) An explanation for the lower activity of compounds 1a, 1b and 1h might be the inappropriate orientation of styryl and phenyl
derivatives at C4 position of 1,4-dihydropyridine scaffold in the gp41 binding site. This can be seen in Fig.2 by superimposing 1a and 1c
in the gp41 binding site.
Figure 2. Superimposition of 1a (green sticks) and 1c (blue sticks) from docking results.
3) Compound 1g with 5-nitro-2-furyl heterocyclic ring at C4 of the 1,4-DHP ring showed weak antiviral activity which might be
attributed to the decreased lipophilicity of the compound due to the presence of nitro group at C5 position of the 2-furyl ring.
Literature reports have shown that the presence of more than one -COOH group in the compound leads to a decreased inhibitory
activity.[15] On the basis of such rationalization, the presence of both nitro and carboxylic acid groups in compound 1g might be
inappropriate for inhibitory activity.
4) The lower activity of compound 1f (bearing ethoxy carbonyl moiety at C3 and C5 positions of the core scaffold) in comparison with
compound 1e (bearing methoxy carbonyl at the same positions) was thought to be due to the difference in the lipophilicity of the
compounds.
In silico studies
In silico calculation of physicochemical parameters and toxicity studies
As a part of the present investigation, a computational study was performed for estimating the drug-likeness and bioavailability of
compounds. One of the key goals in drug design is to identify potent molecules which possess a rational absorption, distribution,
metabolism and excretion (ADME) profile, i.e., drug-like molecules. The Lipinski’s rule of five measures of drug-likeness which is critical
for rational drug design was applied in determining drug-likeness. This rule states that most molecules with good membrane
permeability should have calculative partition coefficient values (CLogP) in the octanol-water system ≤ 5, molecular weight (MW) ≤ 500,
number of hydrogen bond acceptors ≤ 10 and number of hydrogen bond donors ≤ 5. Compounds that satisfy this rule are considered
as drug-like ones. Thus, compounds with the number of violations not more than 1 display good bioavailability. In addition to these
four criteria, some more measures are nowadays considered to determine the drug-likeness of the molecules. Topological polar surface
area (TPSA), logarithm of aqueous solubility in terms of mol/L (LogS), number of rotatable bonds, and seven more criteria were also
determined in this study as measures of drug-likeness. Numerical values for all of these criteria are given in Tables 2 and 3. On the basis
of in silico drug-likeness study, all compounds obeyed Lipinski’s rule of five and compounds 1c and 1e did not have any violations from
the rule of five.
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Table 2. Calculated pharmacokinetic parameters
Compound
TPSA
CLogP
n-ROTB
HBA
Volume
Lipinski’s violations
93.1
96.4
6.14
5.59
4.27
5.02
4.91
5.67
4.35
5.49
8
8
7
9
7
9
8
7
7
8
405.6
424.1
359.8
393.4
368.9
402.5
383.1
378.2
1
1
0
1
0
1
1
1
1a
1b
1c
1d
1e
1f
106.3
106.3
93.1
8
8
7
93.1
7
152.1
93.1
11
7
1g
1h
TPSA (•2), topological polar surface area; CLogP, logarithm of compound partition coefficient between n-octanol and water; n-ROTB, number
of rotatable bonds; HBA, number of hydrogen bond acceptors. Number of hydrogen bond donors (HBD) for the studied compounds was 1.
Table 3. Prediction of anti-HIV-1 compounds as drug-like molecules
Compound
LogSa
Drug-likeness
Drug-scoreb
Mc
Td
IRe
REf
1a
1b
1c
1d
1e
1f
-4.74
-4.30
-3.95
-4.55
-4.28
-4.88
-4.99
-4.27
-1.85
-1.53
-0.76
-2.20
0.80
0.32
0.13
0.47
0.33
0.54
0.35
0.14
0.46
NO
NO
NO
NO
NO
NO
YES
NO
NO
YES
NO
NO
NO
NO
YES
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
YES
NO
NO
NO
NO
NO
NO
-0.56
-0.82
-0.27
1g
1h
a More than 80% of the drugs on the market have an estimated logS (logarithm of aqueous solubility measured in M) value greater than -4; b
c
d
e
f
drug score combines drug likeness, CLogP, logS, molecular weight and toxicity risks; Mutagenicity; Tumorigenicity; Irritant effect;
Reproductive effect.
Molecular flexibility measured by the number of rotatable bonds (optimally below 8) is another important predictor of good oral
bioavailability. According to data in Table 2, all of the synthesized derivatives had an optimum number of rotatable bonds (<8) except
1d and 1f (number of rotatable bonds = 9) as they obtain a moderate degree of structural rigidity.
Toxicity risk (mutagenicity, tumorogenicity, irritation, and reproduction) are signals alarming that the calculated compounds may
be harmful concerning the risk category specified. Most of the compounds did not show any potential of toxicity in toxicity risk
assessment (Table 3).
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In Osiris calculations the drug-likeness prediction is a fragment based approach. A positive drug-likeness value states that a
molecule contains mainly fragments which are often present in commercial drugs. Osiris study displayed that compound 1c had a high
positive drug-likeness value, and the fragments of this compound had a contribution for drug-like activity.
The studied compounds revealed moderate to good drug score introducing them as potentially safe lead compounds. Drug-
likeness and drug-score values of the designed compounds are presented in Table 3.
Molecular docking study
Molecular docking as an important methods in structure-based computer-assisted drug design predicts the main binding mode(s) of a
ligand with a protein of known three-dimensional structure.[20] From another aspect of view, docking simulation could be applied to
find the binding mode and mechanism of more active derivatives. Molecular docking simulation of the interaction with gp41 was
carried out to investigate the posible inhibition of this protein with the prepared compounds. 4-(3,5-Bis(alkoxycarbonyl)- 2,6-dimethyl-
4-arylpyridin-1(4H)-yl)benzoic acids (1a-h) were all docked into the gp41. Docking results are summarized in Tables 5 and 6.
Docking results showed that evaluated structures possessed similar binding modes in gp41. Observed binding modes were
associated with different docking scores. Previous SAR studies on other chemical series revealed that three major contact motifs
recognizable within the gp41 binding pocket are as follows:
a) Flexible and positively-charged Lys574 and Arg579 residues which could form a strong salt bridge with the negatively charged
carboxylic group of the ligands
b) Hydrophilic amino acids which could form hydrogen bonds (e.g. Gln567 and Gln575)
c) Hydrophobic amino acids providing favorable hydrophobic interactions.
These three key contact motifs may lead to the inhibition of gp41 6-helix bundle formation. NB-2 and NB-64 have been
identified as drug-like compounds during the screening of small-molecule libraries in the recent years.[21] At the first step of docking, we
focused on exploring the binding mode of both NB-2 and NB-64 within the entire protein via a blind-docking procedure to validate
the applied docking protocol. The obtained results showed that the docked compounds were located in a groove containing Trp571,
Gln575, Leu581, Gln577, Lys574, Leu568 and Gln567 residues of gp41. An electrostatic interaction between the carboxylate group of
each ligand and Lys574 as well as a hydrogen bond between the hydroxyl group of NB-2 and Gln567 in gp41 was observed.[11] These
blind-docking results were in accordance with the previous reports.[10,22] Subsequent to the validation of the docking protocol, the 3D
structures of 4-(3,5-bis(alkoxycarbonyl)-2,6-dimethyl-4-arylpyridin-1(4H)-yl)benzoic acid derivatives (1a-h) were all docked into gp41
protein. The following ligand-receptor interaction features were derived from the obtained docking results:
1) Previous researches suggested that compounds with improved anti-HIV-1 potency could be obtained by increasing the molecular
size and hydrophobicity since they can occupy the hydrophobic pocket efficiently.[23],[24] In the present study, the substituted aromatic
ring attached to the nitrogen atom of 1,4-DHP ring increases the hydrophobicity and molecular size which favors the anti-HIV-1 activity
(Table 3).
2) Electrostatic interaction with the positively-charged amino acids of gp41 (Lys574 and/or Arg579) which is crucial for the receptor
inhibition is provided by para-carboxylic acid substitution on the phenyl ring at N1 position of 1,4-DHP. para-Carboxylic acid
substitution on all 1,4-DHP derivatives contributed to form a strong salt bridge with Lys574 and/or Arg579.
3) In almost all the docked 1,4-dihydropyridine structures, hydrogen bond with Gln567 or Gln575 can be detected.
4) It was observed that most of the studied compounds had almost similar binding orientation in gp41 binding site. However, they had
slightly different binding modes. The in silico binding mode of the two most active compounds (1c and 1d) against gp41 can be seen in
Fig.3.
5) Compounds 1a, 1b and 1g showed the highest binding energy. It seemed that the styryl and 4-dimethylamino phenyl substituent
on the C-4 position of 1,4-DHP ring of 1a and 1b might be responsible for additional hydrophobic interactions with the gp41 binding
site. In the case of 1g, the high binding free energy was for the increased electrostatic interactions due to the presence of the nitro
group. As mentioned earlier, the improper permeability as a result of high TPSA made this compound less active in anti-HIV-1
evaluations.
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6) Nitro group substituted on furyl ring at C4 position of 1,4-DHP ring (1g) could be responsible for additional
electrostatic interaction with NH of Lys574 through oxygen atom of a nitro substituent (Fig.4).
Table 4. Interactions between the docked 1,4-dihydropyridine derivatives and gp41 binding site residues.
Hydrogen bond
Distance
(Å)
Compoun
d
Amino acids in electrostatic
Amino acids in non-bonded contacts
contacts
Amino acid
Angle (°)
Lys574,
Gln567
Lys574,
Gln567
Lys574,
Gln567,
Trp571
1.77
1.95
1.74
2.15
1.94
1.90
2.98
133.81
26.42
146.01
34.58
37.82
30.59
66.83
Lys574, Gln567, Trp571, Leu568, Val570,
Trp631, Glu630,Glu634
Lys574, Gln567, Trp571, Leu568, Val570,
Trp631, Glu630,Glu634
1a
1b
Lys574
Lys574
Lys574, Gln567, Trp571, Leu568, Val570,
Trp631, Glu630,Glu634
1c
Arg579
Lys574,
Arg579,
Gln575
1.93
3.09, 1.88
2.12
149.49
112.69,134,84
28.08
Arg579, Lys574, Gln575, Ala578, Gln577,
Trp571
1d
Arg579
Lys574,
Gln567,
Trp571
Lys574,
Arg579,
Gln575
1.81
2.12
2.39
2.37
1.84
2.10
130.22
5.81
41.35
141.01
135.92
26.38
Lys574, Gln567, Trp571, Leu568, Val570,
Trp631
1e*
1f
Lys574
Arg579
Arg579, Lys574, Gln575, Ala578, Gln577,
Trp571
2.15
3.06
1.84
2.22
1.71
2.13
1.89
2.98
121.22
112.98
161.99
11.09
132.20
43.23
Lys574,
Arg579,
Gln575
Arg579, Lys574, Gln575, Ala578, Gln577,
Trp571
1g
Lys574, Arg579
Lys574,
Gln567
Lys574,
Trp571
Lys574, Gln567, Trp571, Leu568, Val570,
Trp631, Glu634
Lys574, Gln567, Trp571, Leu568, Glu634,
Trp631
1h
Lys574
-
11.42
64.82
BMS-806
*1e had cation-π interaction with Lys574 residue inside the active site.
Table 5. Docking results of 1,4-dihydropyridine derivatives docked into fusion HIV-1 virus target (gp41, PDB ID: 1AIK)
Intermol
energy
Electrostatic
energy
a
ΔGbinding
Compound
Vdw_Hbond_desolv energy (kal/mol)
(kcal/mol)
(kcal/mol)
(kcal/mol)
1a
1b
-5.82
-5.75
-5.33
-5.29
-4.25
-4.96
-5.76
-5.56
-6.42
-8.51
-8.44
-7.46
-8.28
-6.64
-7.94
-8.44
-7.94
-7.61
-1.20
-1.12
-1.22
-1.37
-0.97
-1.12
-2.44
-1.21
-0.19
-7.31
-7.32
-6.23
-6.91
-5.67
-6.82
-6.01
-6.73
-7.42
1c
1d
1e
1f
1g
1h
BMS-806
a
F=0.152896 < F Crit=2.069832, p-value= 0.99598
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Figure 3. Compounds 1c (A) and 1d (B) in gp41 binding site.
Figure 4. Compound 1g at binding site of gp41
7) Carboxylic acid groups of 1c, 1d, 1e and 1g showeed electrostatic interaction with Arg579, while in other compounds Lys574 was
involved in such an interaction.
8) Oxygen atom of the 2-furyl and sulfur atom of the 2-thiophenyl hetero-aromatic substituents at the C4 position of 1,4-DHP
(compounds 1c, 1d and 1e, 1f) had a determinant role in H-bond interactions with Lys574.
9) Different H-bond patterns were observed for the sttudied compounds. For 1a, 1b, 1c, 1e and 1h hydrogen bond was formed with
Gln567, while compounds 1d, 1f and 1g participated in hydrogen bonding to Gln575.
10) Oxygen atom of methyl esters at C3 and C5 of the 1,4-DHP ring in 1e and furyl ring at C4 position of 1c participated in H-bond
interaction with Trp571.
11) All the 1,4-DHP structures participated in non-bonded interactions with Trp571, Leu568, and Val570 residues. Compounds 1d, 1f
and 1g also showed additional hydrophobic contacts wwith Ala578.
12) Docking results showed that Trp631, Glu630 and Glu634 contributed to non-bonded contacts with 1a, 1b, 1c and 1h (Table 4) while
they did not show any hydrophobic contacts with 1d, 1f and 1g.
13) Compounds 1d, 1f and 1g formed hydrophobic intteractions with Gln577 located inside the binding pocket.
14) Compound 1e was found to take part in a cation-π stacking interaction with Lys574, while, other compounds did not show such an
interaction.

15) Compound BMS-806 was docked in gp41 binding site and showed interactions almost similar to NB-2 and synthesized derivatives.
This compound forms H-bond with Lys574 and Trp571. It was not able to interact electrostatically with the receptor since it lacks the
proper functionalities (Fig.5).
As illustrated in Scheme 2, compounds 1c-e showed HIV-1 inhibitory activity up to 70%. The inhibitory activity of the reference
compound (BMS-806) was 100% at the same concentration. Other compounds displayed moderate activity (Scheme 2). This suggests
that novel N1-substituted 1,4-DHP derivatives are good candidates for further HIV-1 inhibition studies. In addition, results of Scheme 3
showed that all of the compounds exhibited docking results comparable to NB-2. Finally, it was found that compound 1c (2-furyl ring at
C4 position of 1,4-DHP and methyl ester moieties at C3 and C5 positions of 1,4-DHP) was the most potent compound in HIV-1 inhibition
assay. Compound 1a (styryl group at C4 position of 1,4-DHP) showed the highest ΔGbinding in molecular docking study.
Figure 5. A. Chemical structure of BMS-806, B. Compound BMS-806 at gp41 binding site
100
80
60
40
20
0
Compounds
Scheme 2. Inhibitory activity for 1,4-DHP derivatives at 100 μM

8
7
6
5
4
3
2
1
0
Compounds
Scheme 3. Docking binding energy (ΔGbinding) for 1,4-DHP derivatives in comparison with BMS-806
Conclusions
In the present study, 4-arylpyridin-1(4H)-yl) benzoic acid was introduced as a novel anti-HIV-1 scaffold. Eight different derivatives of 4-
(3,5-bis(alkoxycarbonyl)-2,6-dimethyl-4-arylpyridin-1(4H)-yl)benzoic acid were designed and synthesized as anti-HIV-1 agents. After
structural elucidation by FT-IR, 1H-NMR, LC-Mass and elemental analysis, the anti-HIV-1 activity of all compounds was evaluated. Most of
the studied compounds were moderate to good anti-HIV-1 agents lacking considerable cytotoxicity in cell cultures. The most potent
compounds (1c, 1d and 1e) exhibited 84, 80.5 and 76.42% HIV-1 inhibitory activity at 100 μM, respectively. Structure-activity
relationships analysis revealed that the presence of 2-furyl and 2- thienyl hetero-aromatic substituents at the C4 position of 1,4-DHP
ring enhanced the anti-HIV-1 activity and lowered the cytotoxic effect on MT-2 cell line. All of the compounds (1a-h) obeyed the
Lipinski’s "rule of five" and showed drug-likeness in terms of their ADMET profile.
Molecular docking simulation of the interaction with gp41 was carried out to investigate the posible inhibition of this protein
with the prepared compounds. Docking simulation studies have revealed that 4-(3,5-bis(alkoxycarbonyl)-2,6-dimethyl-4-arylpyridin-
1(4H)-yl)benzoic acids are bound mainly within gp41 binding site and the change in free energy of binding (ΔGbinding) for most of the
compounds was similar to that of NB-2. Autodock 4.2 estimated ΔGbindings were correlated with the experimental in vitro anti-HIV-1
activities. Docking study indicated that all of the derivatives form electrostatic interactions and at least one H-bond with the gp41
binding site residues. These interactions were presumably associated with improved binding energies. Based on the obtained results,
1,4-dihydropyridine scaffold seems to be a promising candidate for the development of new HIV-1 inhibitors and 1c and 1e might be
appropriate compounds for further modification towards more potent and selective anti HIV-1 agents.
Experimental Section
Chemistry
All chemicals, reagent and solvents were obtained from commercial suppliers and were freshly used after purification by standard
procedures. Melting points were determined on the Electrothermal 9200 Melting Point apparatus and were uncorrected. 1H-NMR
spectra were recorded in DMSO-d6 on a Bruker-Ultrashield 400MHz spectrometer (Germany). All the chemical shifts were reported as (δ)
values (ppm) against tetramethylsilane as an internal standard. Electrospray mass spectra (ESI-Mass) were obtained in negative and
positive ion mode on a SHIMADZU LCMS-2010 EV spectrometer using methanol as solvent, a capillary voltage of 4500 V and a cone
voltage of 10 V. FT-IR (KBr) spectra were recorded on a JASCO 6300 (Japan). Elemental analysis was performed using CHNS-923 LECO
analyzer (USA) and the results were within 0.4% of the theoretical values. All the experiments were monitored by analytical thin layer
chromatography (TLC) on pre-coated silica gel 60 F254 aluminum plates (Merck, Germany). Preparative column chromatographies were
carried out on silica gel (230–400 mesh, G60 F254 Merck, Art. 5715). Preparative thin-layer chromatography was done with prepared
glass-backed plates (20×20 cm2, 500 μm) using silica gel (Merck Kieselgel 60 HF254, Art. 7739). Suitable names for all the novel
compounds were given with the aid of ChemOffice 2010 software. All final products were prepared in 25-58% yield. Their purity was
determined by thin layer chromatography using several solvent systems of different polarities and 1H-NMR and CHNOS analysis results.

They were all stable in refrigerator under 5 ºC and in the darkness. General structure and structural details of the prepared compounds
are summarized in Table 6.
Table 6. General structure and structural features of the designed compounds
Compound
(het)Aryl
R
Mol. formula
Mol. weight
1a
1b
1c
1d
1e
1f
Styryl
4-Dimethylaminophenyl
2-Furyl
CH3
CH3
CH3
C2H5
CH3
C2H5
CH3
CH3
C26H25NO6
C26H28N2O6
C22H21NO7
C24H25NO7
C22H21NO6S
C24H25NO6S
C22H20N2O9
C24H23NO6
447.48
464.51
411.40
439.46
427.47
455.52
456.40
421.44
2-Furyl
2-Thienyl
2-Thienyl
1g
1h
5-Nitro-2-furyl
Phenyl
General procedure for the synthesis of 4-(3,5-bis(alkoxycarbonyl)-2,6-dimethyl-4-arylpyridin-1(4H)-yl)benzoic acid (1a-h)
A mixture of corresponding aldehyde (3.0 mmol), alkyl acetoacetate (6.0 mmol) and para-aminobenzoic acid (3.0 mmol) in ethanol (4
mL) was heated and stirred under reflux. The progress of the reaction was monitored by TLC. After completion of the reaction, the
reaction mixture was cooled to room temperature then ethanol was removed under reduced pressure and the product was left
overnight in freezer. The obtained solid was filtered off, washed with small portions of cold ethanol and crystallized from ethanol to
give compounds 1a-h. For compounds 1c and 1d, the process of purification was different: after cooling the mixture, ethanol was
removed under reduced pressure and the product was purified by column chromatography over silica gel using chloroform-methanol.
In the case of compounds 1b and 1h, one milliliter of HCl was used as catalyst in the reaction progress. After the completion of the
reaction, the reaction mixture was cooled to room temperature, 5 mL of water was added, and the resulting mixture was extracted with
chloroform (3×10 mL). The organic layers were combined and dried over anhydrous MgSO4. After filtering the solid off, the solvent was
removed under reduced pressure and the residue was purified by preparative TLC using chloroform-methanol as eluent. All the
obtained products were completely dried in a vacuum oven.
The spectral features of the prepared molecules are provided in detail as follows.
(E)-4-(3,5-Bis(methoxycarbonyl)-2,6-dimethyl-4-styrylpyridin-1(4H)-yl)benzoic acid (1a)
°
Yellow crystals. Yield: 25%.M.p.194-195 C. FT-IR (KBr) ν (cm-1): 2500-3500 (O-H, acid), 2956.34 (C-H, aromatic), 2832.92 (C-H, aliphatic),
1686.44 (C=O, ester), 1654.32 (C=O, acid), 1626.66 (C=C, alkene), 1600.63 & 1571.70 (C=C, aromatic), 1273.75 (C-O, ester), 1166.72 (C-O,
acid). 1H-NMR (DMSO-d6) δ (ppm): 10.63 (1H, s: COOH), 7.95 (2H, d, J=8.4 Hz, C3-H, C5-H: -C6H4COOH), 7.67 (2H, d, J=8.4 Hz, C2-H, C6-H: -
C6H4COOH), 7.31-7.34 (5H, m, C2-H, C3-H, C4-H, C5-H, C6-H: -CH=CHC6H5), 6.59-6.61 (2H, m, Ca-H, Cb-H: -HCa=CbHC6H5), 4.89 (1H, s, C4-
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H: DHP), 3.67 (6H, s:
2×-COOCH3), 2.21 (6H, s, C2-CH3, C6-CH3: DHP). Mass m/z (%): 446.30 (M-1). Anal. Calcd. for C26H25NO6 (447.48):
C, 69.67; H, 5.67; N, 3.21.
C, 69.79; H, 5.63; N, 3.13; found:
4-(4-(4-(Dimethylamino)phenyl)-3,5-bis(methoxycarbonyl)-2,6-dimethylpyridin-1(4H)-yl)benzoic acid (1b)
°
Yellow precipitates, Yield 58%.m.p.: 259-260 C. FT-IR (KBr) ν (cm-1): 2500-3200 (O-H, acid), 2882.70 & 2829.06 (C-H, aliphatic), 1686.44
(C=O, ester), 1658.55 (C=O, acid), 1627.63 & 1599.66 & 1422.24 (C=C, aromatic), 1272.79 (C-O, ester), 1165.76 (C-O, acid). 1H-NMR (DMSO-
d6) δ (ppm): 12.76 (1H, brs: COOH), 7.94 (2H, d, J=8.4 Hz, C3-H, C5-H: -C6H4COOH), 7.76 (2H, d, J=9.2 Hz, C2-H, C6-H: -C6H4COOH), 7.24 (2H,
d, J=8.4 Hz, C2-H, C6-H: -C6H4N(CH3)2), 6.79 (2H, d, J=8.8 Hz, C3-H, C5-H: -C6H4N(CH3)2), 5.31 (1H, s, C4-H: DHP), 3.68 (6H, s: 2×-COOCH3),
3.02 (6H, s: -C6H4N(CH3)2), 2.03 (6H, s, C2-CH3, C6-CH3: DHP). Mass m/z (%): 463.15 (M-1). Anal. Calcd. (%) for C26H28N2O6 (464.51): C, 67.23;
H, 6.08; N, 6.03. Found: C, 67.38; H, 6.09; N, 6.07.
4-(4-(Furan-2-yl)-3,5-bis(methoxycarbonyl)-2,6-dimethylpyridin-1(4H)-yl)benzoic acid (1c)
°
Pale yellow precipitates, Yield 51% m.p.: 248-249 C. FT-IR (KBr) ν (cm-1): 2500-3200 (O-H, acid), 3067.23 (C-H, aromatic), 2925.48 &
2867.02 (C-H, aliphatic), 1704.76 (C=O, ester), 1677.77 (C=O, acid), 1601.59 &1521.56 (C=C, aromatic), 1291.11 (C-O, ester), 1171.64 (C-O,
acid). 1H-NMR (DMSO-d6) δ (ppm): 11.97 (1H, s: COOH), 7.89 (2H, d, J=8.8 Hz, C3-H ,C5-H: -C6H4COOH), 7.77 (2H, d, J=8.8 Hz, C2-H, C6-H: -
C6H4COOH), 7.38 (1H, d, J=8.8 Hz, C3-H: furan), 6.74 (1H, d, J=8.8 Hz, C4-H: furan), 6.59 (1H, d, J=8.8 Hz, C5-H: furan), 5.91 (1H, brs, C4-H:
DHP), 3.71 (6H, s: 2×-COOCH3), 2.06 (6H, s, C2-CH3, C6-CH3: DHP). Mass m/z (%): 410.15 (M-1). Anal. Calcd. (%) for C22H21NO7 (411.40): C,
64.23; H, 5.14; N, 3.40. Found: C, 64.59; H, 4.81; N, 3.68.
4-(3,5-Bis(ethoxycarbonyl)-4-(furan-2-yl)-2,6-dimethylpyridin-1(4H)-yl)benzoic acid (1d)
°
Pale yellow precipitates, Yield 51%.m.p.: 248-249 C. FT-IR (KBr) ν (cm-1): 2500-3200 (O-H, acid), 3064.33 (C-H, aromatic), 2899.45 (C-H,
aliphatic), 1739.49 (C=O, ester), 1709.59 (C=O, acid), 1672.95 & 1604.48 (C=C, aromatic), 1284.36 (C-O, ester), 1174.44 (C-O, acid). 1H-NMR
(DMSO-d6) δ (ppm): 12.32 (1H, s: COOH), 7.84 (2H, d, J=8.8 Hz, C3-H ,C5-H: -C6H4COOH), 7.72 (2H, d, J=8.8 Hz, C2-H, C6-H: -C6H4COOH),
7.37 (1H, d, J=8.8 Hz, C3-H: furan), 6.96 (1H, d, J=2.8 Hz, C4-H: furan), 6.82 (1H, d, J=7.6 Hz, C5-H: furan), 5.89 (1H, brs, C4-H: DHP), 4.80 (4H,
q, J=4.4 Hz: COOCH2CH3),
2.89-2.90 (6H, m: COOCH2CH3), 2.10 (6H, s, C2-CH3, C6-CH3: DHP). Mass m/z (%): 438.20 (M-1). Anal. Calcd.
(%) for C24H25NO7 (439.46): C, 65.59; H, 5.73; N, 3.19. Found: C, 65.38; H, 5.45; N, 2.91.
4-(3,5-Bis(methoxycarbonyl)-2,6-dimethyl-4-(thiophen-2-yl)pyridin-1(4H)-yl)benzoic acid (1e)
White precipitates, Yield 46%.m.p.: 246-247 °C. FT-IR (KBr) ν (cm-1): 2500-3200 (O-H, acid), 2999.73 (C-H, aromatic), 2949.59 & 2843.52 (C-
H, aliphatic), 1698.98 (C=O, ester), 1639.20 (C=O, acid), 1598.70 & 1585.20 & 1429.96 (C=C, aromatic), 1275.68 (C-O, ester), 1205.29 (C-O,
acid). 1H-NMR (DMSO-d6) δ (ppm): 13.27 (1H, s: COOH), 8.07 (2H, d, J=8.4 Hz, C3-H ,C5-H: -C6H4COOH), 7.41 (2H, d, J=8.4 Hz, C2-H, C6-H: -
C6H4COOH), 7.30 (1H, d, J=5.2 Hz, C3-H: thiophen), 6.94 (1H, dd, J=4.0 Hz & J=2.0 Hz, C4-H: thiophen), 6.85 (1H, d, J=3.2 Hz, C5-H:
thiophen), 5.31 (1H, s, C4-H: DHP), 3.67 (6H, s: 2×-COOCH3), 2.00 (6H, s, C2-CH3, C6-CH3: DHP). Mass m/z (%): 426.15 (M-1). Anal. Calcd. (%)
for C22H21NO6S (427.47): C, 61.81; H, 4.95; N, 3.28; S, 7.50. Found: C, 62.15; H, 5.12; N, 3.29; S, 7.82.
4-(3,5-Bis(ethoxycarbonyl)-2,6-dimethyl-4-(thiophen-2-yl)pyridin-1(4H)-yl)benzoic acid (1f)
White precipitates, Yield 41%. m.p.: 242-243 °C. FT-IR (KBr) ν (cm-1): 2500-3200 (O-H, acid), 2956.34 (C-H, aromatic), 2832.92 (C-H,
aliphatic), 1704.76 (C=O, ester), 1676.80 (C=O, acid), 1600.63 & 1524.45 & 1421.28 (C=C, aromatic), 1293.04 (C-O, ester), 1175.40 (C-O,
acid). 1H-NMR (DMSO-d6)
δ (ppm): 12.93 (1H, brs: COOH), 8.04 (2H, d, J=8.4 Hz, C3-H ,C5-H: -C6H4COOH), 7.39 (2H, d, J=8.4 Hz, C2-H,
C6-H: -C6H4COOH), 7.29 (1H, dd, J=1.2 Hz, J=4.0 Hz, C3-H: thiophen), 6.93 (1H, dd, J=4.0 Hz & J=2.0 Hz , C4-H: thiophen), 6.83 (1H, d, J=3.2
Hz, C5-H: thiophen), 5.08 (1H, s, C4-H: DHP), 3.99 (4H, q, J=2.8 Hz: COOCH2CH3), 2.50-2.51 (6H, m: COOCH2CH3), 2.03 (6H, s, C2-CH3, C6-
CH3: DHP). Mass m/z (%): 454.15 (M-1). Anal. Calcd. (%) for C24H25NO6S (455.52): C, 63.28; H, 5.53; N, 3.07; S, 7.04. Found: C, 62.93; H, 5.25;
N, 3.38; S, 7.41.
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4-(3,5-Bis(methoxycarbonyl)-2,6-dimethyl-4-(5-nitrofuran-2-yl)pyridin-1(4H)-yl)benzoic acid (1g)
Pale yellow precipitates, Yield 35%.m.p.: 191-192 °C. FT-IR (KBr) ν (cm-1): 2800-3400 (O-H, acid), 3000.69 (C-H, aromatic), 2955.38 &
2832.92 (C-H, aliphatic), 1687.41 (C=O, ester), 1658.55 (C=O, acid), 1628.66 & 1599.66 & 1420.32 (C=C, aromatic), 1385.60 (N-O, Nitro),
1271.82 (C-O, ester), 1164.79 (C-O, acid). 1H-NMR (DMSO-d6) δ (ppm): 12.79 (1H, s: COOH), 10.58 (1H, brs, C4-H: furan), 8.86 (1H, brs, C5-H:
furan), 7.89 (2H, d, J=8.4 Hz,
C3-H ,C5-H: -C6H4COOH), 7.27 (2H, d, J=8.8 Hz, C2-H, C6-H: -C6H4COOH), 4.83 (1H, s, C4-H: DHP), 3.61 (6H,
s: 2×-COOCH3), 2.15 (6H, s, C2-CH3, C6-CH3: DHP). Mass m/z (%): 455.15 (M-1). Anal. Calcd. (%) for C22H20N2O9 (456.40): C, 57.90; H, 4.42; N,
6.14. Found: C, 58.21; H, 4.72; N, 6.23.
4-(3,5-Bis(methoxycarbonyl)-2,6-dimethyl-4-phenylpyridin-1(4H)-yl)benzoic acid (1h)
°
White crystals, Yield 31%. m.p.: 213-215 C. FT-IR (KBr) ν (cm-1): 2500-3200 (O-H, acid), 3046.98 (C-H, aromatic), 2905.24 (C-H, aliphatic),
1705.73 (C=O, ester), 1609.31 (C=O, acid), 1576.52 & 1505.17 (C=C, aromatic), 1246.75 (C-O, ester), 1114.65 (C-O, acid). 1H-NMR (DMSO-
d6) δ (ppm): 11.97 (1H, brs, COOH), 7.63 (2H, d, J=2.0 Hz, C3-H ,C5-H: -C6H4COOH), 7.23-7.27 (3H, m, C3-H, C4-H, C5-H: phenyl), 7.17 (2H, d,
J=6.4 Hz,C2-H, C6-H: phenyl), 6.54 (2H, d, J=8.4 Hz, C2-H, C6-H: -C6H4COOH), 5.88 (1H, s, C4-H: DHP), 3.68 (6H, s, 2×COOCH3), 2.17 (6H, s,
C2-CH3, C6-CH3: DHP). Mass m/z (%): 420.15 (M-1). Anal. Calcd. (%) for C24H23NO6 (421.44): C, 68.40; H, 5.50; N, 3.32. Found: C, 68.69; H,
5.41; N, 3.39.
Biological assay
Inhibition of the HIV-1 entry by the prepared compounds was investigated. BMS-806 as the control HIV-1 surface glycoprotein inhibitor
was used in the experiments.[25] Synthesized compounds were dissolved in dimethyl sulfoxide (DMSO) at different concentrations.
DMSO with the final concentration of 1% V/V (1 mL DMSO in 100 mL H2O) was considered as the negative solvent control. DMSO plus
MT-2 (human lymphocyte) cells and virus were used to normalize the solvent effect.[26] BMS-806 (200 mM) was dissolved in DMSO and
kept in -20°C. All tests were performed in triplicate.
Inhibition of HIV-1 single-cycle infection
Human embryonic kidney (HEK) and MT-2 cells were obtained from National Cell Bank of Iran. MT-2 cells were produced by co-culturing
normal human cord leukocytes with leukemic T-cells from a patient with adult T-cell leukemia. To measure the inhibition effect, HEK
293T cells were cultured in RPMI 1640 and DMEM (Dulbecco’s modified Eagle’s medium) containing 15% L-FBS (Fetal Bovine Serum),
100 U/mL of penicillin, and 100 μg/mL of streptomycin.[27] In the step of transfection into HEK cells, HEPES (4-(2-hydroxyethyl)-1-
piperazine ethane sulfonic acid) was added and finally the cells were incubated at 37 °C under 5% CO2.
To produce the SCR HIV-1 virions, pMD2G, pmzNL4-3 and pSPAX2 plasmids were used. These plasmids were co-transfected into
HEK 293T cells in 6-well plates (5×105 cells/well) in certain proportion using PolyFect reagent (Qiagen) according to the suggested
method by Qiagen. Supernatants of the transfected cells were harvested after 24, 48 and 72 h after infection and fresh medium was
added to the culture wells. Mediums containing viruses were mixed together and stored at -70 °C after filtration with 0.22 μm filters.[28]
°
Final centrifugation on the filtrate at 60,000 g was performed for 2 h at 4 C. The supernatant was taken out and the virions pellet was
shaken gently overnight in 1/30 volume of RPMI 1640 at 4 °C. SCR HIV-1 virions were produced in 24-well plates to generate viruses for
measuring the rate of production and maturation. 70×103 HEK 293T cells were used for producing SCR HIV-1 virions by 400 ng of
plasmid. Transfection was carried out in the presence of 4 μL of PolyFect reagent. Volume of transfection was 300 μL and the volume of
the added DNA complex was 120 μL. When transfection was complete, 800 μL of the complete medium containing the synthesized
compounds was added to the cells. The culture medium containing the virus was collected 48 h after transfection and the amount of
the purified virus was measured using p24 ELISA assay kit (Cell Biolabs).[29]
Cytotoxicity assay
The toxicity of compounds for target cell (Hela) was assayed using XTT (sodium 3-[1 (phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-
methoxy-6-nitro)benzene sulfonic acid) (Roche, Germany) as described previously. The replication assay plates were directly subjected
to XTT assay after analysis of supernatant for P24 load. Before addition of XTT, the medium was aspirated and replaced with phosphate-
buffered saline (PBS). The XTT solution was prepared and added to the wells according to the user manual. Cell was then incubated for
an additional 2 h to allow the formazan production. The optical densities were measured with ELISA reader at test and reference wave
lengths of 492 nm and 690 nm, respectively.
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In Silico studies
In silico calculation of physicochemical parameters and ADMET prediction
Lipinski’s rule of five which is essential to ensure a drug-like pharmacokinetics profile and the physicochemical properties of the
designed derivatives were assessed using online Osiris property explorer[30] and Molinspiration WebME Editor1.16.[31] These
physicochemical properties were: calculated logarithm of partition coefficient (CLogP), logarithm of solubility (LogS), molecular weight
(MW), drug-likeness, number of hydrogen bond donors (HBD), number of hydrogen bond acceptors (HBA), number of rotatable bonds
(nROTB), drug score, topological polar surface area (TPSA). Absorption, distribution, metabolism and excretion (ADME) of the designed
molecules were some more physicochemical properties calculated in silico.
Molecular docking studies
Molecular docking studies were carried out by AutoDock version 4.2.[32] The three-dimensional (3D) protein structure of gp41 (PDB ID:
1AIK), determined by X-ray crystallography, was retrieved from the RCSB Protein Databank.[33] The protein was then corrected by
removing the extra crystallized water molecules using Accelrys discovery studio visualizer 4.0[34] and adding polar hydrogens and
charges to the molecule byAutoDockTools version 1.5.6rc3.[21] Protonation step corrects the ionization and tautomeric states of residues
and thus modifies the total Kollman charges on the protein structure. All
two-dimensional (2D) structures of the ligands were
drawn by ChemDraw program.[35] Each structure was energy minimized by MM+ force field and PM3 semi-empirical techniques in
HyperChem8 software. Then, partial charges of atoms were calculated using Gasteiger-Marsili procedure. Non-polar hydrogens of
compounds were merged, rotatable bonds were assigned and finally the molecules were saved as pdbqt format file. The resulting
protein structure was used as an input for the AutoGrid program. AutoGrid performs pre-calculated atomic affinity grid maps for each
atom type in the ligand, plus an electrostatics map and a separate desolvation map presented in the substrate molecule. All maps were
calculated with 0.375 Å spacing between grid points. Grid center was centered on the Cα atom of Lys574 residue with x=24.00, y=21.82
and z=23.82 coordinates obtained using Acceryls discovery studio visualizer 4.0. The location and size of the grid were set in a way to
contain not only the active site but also considerable portions of the surrounding surface. For this purpose, box was set at 60×60×60 Å.
Each docking was performed by 150 runs of the AutoDock search by the Lamarckian genetic algorithm (LGA). The factors for LGA were
defined as follows: a maximum number of 5,000,000 energy evaluations; a maximum number of generations of 27,000; mutation and
crossover rates of 0.02 and 0.8, respectively. Lastly, the conformation of the lowest predicted binding free energy of the most occurring
binding modes in the gp41 binding site was selected. Graphic manipulations and visualizations were done by Accelrys discovery studio
visualizer 4.0 software.
Statistical methods
Analysis of variance (ANOVA) was used as a technique for analyzing experimental data in which one or more response variables are
measured under numerous conditions. In an analysis of variance, the difference in the response is divided into variation attributable to
differences between the response variables and variation attributable to random error. A usual aim in ANOVA is to compare means of
the response variable for various combinations of the classification variables. ANOVA table consists of sum-of-squares, degrees of
freedom, mean square, F ratio and p values less than 0.05 which are considered statistically significant. ANOVA was done on the data to
determine the significant variables of the docking and biological assay procedures.
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