Design, synthesis, and evaluation of HIV-1 entry inhibitors based on broadly neutralizing antibody 447-52D and gp120 V3loop interactions

Article abstract of DOI:10.1016/j.bioorg.2021.105313

The third variable loop region (V3 loop) on gp120 plays an important role in cellular entry of HIV-1. Its interaction with the cellular CD4 and coreceptors is an important hallmark in facilitating the bridging by gp41 and subsequent fusion of membranes for transfer of viral genetic material. Further, the virus phenotype determines the cell tropism via respective co– receptor binding. Thus, coreceptor binding motif of envelope is considered to be a potent anti-viral drug target for viral entry inhibition. However, its high variability in sequence is the major hurdle for developing inhibitors targeting the region. In this study, we have used an in silico Virtual Screening and “Fragment-based” method to design small molecules based on the gp120 V3 loop interactions with a potent broadly neutralizing human monoclonal antibody, 447-52D. From the in silico analysis a potent scaffold, 1,3,5-triazine was identified for further development. Derivatives of 1,3,5-triazine with specific functional groups were designed and synthesized keeping the interaction with co-receptor intact. Finally, preliminary evaluation of molecules for HIV-1 inhibition on two different virus strains (clade C, clade B) yielded IC50 < 5.0 μM. The approach used to design molecules based on broadly neutralizing antibody, was useful for development of target specific potent antiviral agents to prevent HIV entry. The study reported promising inhibitors that could be further developed and studied.

Full text of DOI:10.1016/j.bioorg.2021.105313

Bioorganic Chemistry 116 (2021) 105313
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis, and evaluation of HIV-1 entry inhibitors based on
broadly neutralizing antibody 447-52D and gp120 V3loop interactions
,
,
,*
Jagadeesh Senapathia 1, Akhila Bommakantia 1, Srinivas Vangarab, Anand K. Kondapia
a Dept. of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, India
b School of Chemistry, University of Hyderabad, Hyderabad 500046, India
A R T I C L E I N F O
A B S T R A C T
Keyword:
The third variable loop region (V3 loop) on gp120 plays an important role in cellular entry of HIV-1. Its inter-
action with the cellular CD4 and coreceptors is an important hallmark in facilitating the bridging by gp41 and
subsequent fusion of membranes for transfer of viral genetic material. Further, the virus phenotype determines
the cell tropism via respective co– receptor binding. Thus, coreceptor binding motif of envelope is considered to
be a potent anti-viral drug target for viral entry inhibition. However, its high variability in sequence is the major
hurdle for developing inhibitors targeting the region. In this study, we have used an in silico Virtual Screening
and “Fragment-based” method to design small molecules based on the gp120 V3 loop interactions with a potent
broadly neutralizing human monoclonal antibody, 447-52D. From the in silico analysis a potent scaffold, 1,3,5-
triazine was identified for further development. Derivatives of 1,3,5-triazine with specific functional groups were
designed and synthesized keeping the interaction with co-receptor intact. Finally, preliminary evaluation of
HIV-1
V3 loop
CCR5
Entry inhibitors
Drug design
Potent broadly neutralizing human monoclonal
antibody
447-52D
1,3,5-triazine
Sulphonic acid
Isoniazid
molecules for HIV-1 inhibition on two different virus strains (clade C, clade B) yielded IC50 < 5.0 μM. The
Quinazolinone
approach used to design molecules based on broadly neutralizing antibody, was useful for development of target
specific potent antiviral agents to prevent HIV entry. The study reported promising inhibitors that could be
further developed and studied.
1. Introduction
the core of gp120 with disulfide bridges followed by the highly flexible
stem and crown that extends outside of gp120 core when the loop is
HIV-1 Entry is facilitated by the interactions between the viral en-
velope protein (gp120) and host cell receptors and co-receptors
involving conserved (C1-C5) and variable (V1-V5) regions of envelope
[1]. The molecular analysis of interactions revealed that some of these
regions were sufficiently exposed from the viral spike to generate
neutralizing antibodies and participate in the interaction with small
molecule inhibitors. The glycosylation of envelope protein (gp120 and
gp41) varies in different virus strains and may contribute to the gener-
ation of strain-specific antibodies in the serum of HIV-1 infected in-
dividuals [2]. Most of these antibodies recognize the third hypervariable
(V3) loop of gp120, especially T-cell line adapted strains of HIV-1 [3].
The loop contains 35 residues with an overall positive charge in the
range of +2 to +10, which participates in the interactions with negative
charge tyrosine sulphonate (-SO3ꢀ ) group of CCR5 N-terminal [4]. V3
loop is further classified structurally into three areas: a base, stem, and
crown [5]. The base part contains the most conserved residues located at
conformationally open [6]. The conserved residues in the crown region
are immunogenic and also provide interactions with the CCR5-ECL2
loop [7]. High immunogenicity of the V3 loop has been widely exploi-
ted to develop synthetic mimics to act as an efficient antigen to produce
antibodies in the immunization process [8]. X-ray crystallography
studies disclosed that the crown part of the V3 loop adopts β- hairpin
conformation in both V3 loop-containing gp120 core and antibody-V3
loop complex. This is assumed to be because of a conserved region in
the centre of the crown consisting of GPXR epitope (GPGR in subtype B
and GPGQ in subtypes A, C, and D), which preserves the structural el-
ements in the V3 loop irrespective of sequence variability in other
portions of V3 loop [9]. Although most anti V3 -loop antibodies
neutralize only a few strains, some of them neutralize diverse strains of
the virus. The V3 loop native conformation can elicit anti-V3 broadly
neutralizing antibodies like 447-52D and 2219, interacting with other
immunodominant regions on gp120 [10]. This study explores the
* Corresponding author.
E-mail address: akondapi@uohyd.ac.in (A.K. Kondapi).
1
Authors have contributed equally to the work presented in this paper.
https://doi.org/10.1016/j.bioorg.2021.105313
Received 4 July 2021; Received in revised form 25 August 2021; Accepted 27 August 2021
Available online 30 August 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

J. Senapathi et al.
Bioorganic Chemistry 116 (2021) 105313
antibody 447-52D to derive entry inhibitors based on its interaction with
gp120 V3 loop. Previous studies of 447-52D show contact with both
stem and tip of the V3 loop to neutralize the subtype B virus (Fig. 1).
From the crystal structure analysis, 447-52D mAb CDRH3 C-terminal
interacts with V3 crown N -terminal via main chain interactions and side
chains interactions at V3 tip residues (Fig. 1) [11].
2. Results and discussion
2.1. In silico design and development of 447-52D mimetics
Pep:MMs:MIMIC server was used to obtain hits based on the 447-52D
and V3 loop interaction as described. The interacting interface and V3
loop sequence submitted to pep:MMs:MIMIC server led to the develop-
ment of a pharmacophore used to screen the MMsINC database of small
molecules to return hits. Two hundred small molecules were reported
and scored, based on the match to the pharmacophore. Top 30 mole-
cules are presented along with scores in (Table S1). The molecules re-
ported by pep:MMs:MIMIC were docked with the V3 loop structure (PDB
Id:1Q1J) using GOLD by the protocol described to build a ranked hit
library to obtain molecules with high scores and better interactions. The
top 20 molecules (Table S2a) from the hit library were docked to whole
structure of gp120 (PDB ID:2B4C). The amino acid residues, Arg306 and
Lys307, were seen to be involved in the interaction with most molecules.
These residues were also crucial in the interaction interface of 447-52D
and V3 loop. Apart from these residues, Cys297 and Cys331 were also
important for the interaction. Based on the number, type, other pa-
rameters of interactions and the docking score, ten molecules
(Table S2b) were selected for further development. The selected mol-
ecules are given in Fig. 2. These molecules led to identifying a core
structure, s-triazane or 1,3,5-triazine, the sub-structure reported to be
present molecules with broad spectrum activities including antimicro-
bial, anticancer, tumour growth inhibition and estrogen receptor mod-
ulators [15]. This sub-structure was further developed, molecules were
synthesized, and validated using activity studies viz. inhibition of HIV-1
entry and HIV-1 replication.
Interaction stability was explained by:
i. The anionic (AspH95 of the Ab) and cationic (V3 loop Arg315)
residues formed the Salt bridge.
ii. The antibody aromatic residues (TyrH100J and TrpH33 of the
447-52D heavy chain) formed
(Arg315 of V3 loop).
π-cation stacking with cationic
iii. Van der Waals interaction between V3 loop residues (Pro313 of
V3 loop - TrpL91 and V3 loop -TrpL96).
iv. Water-mediated hydrogen bond network at the antigen-binding
site [12].
These interactions were used as a basis for design of small molecules
that can mimic the binding of the antibody to gp120 and subsequently
blocking viral entry. The neutralizing antibody interactions provide
knowledge about valid target site recognition and the compounds
designed to target these sites are expected to modulate the protein–-
protein interactions [13]. The mimicking chemosuperiors are consid-
ering as “hits” for lead molecule development with various
modifications. The small molecule design is based on the structural
consideration of drug-like molecules based on pharmacological stability,
conformational changes, and reduced metabolism [14]. In this paper,
we report the design, synthesis, characterization, and evaluation of
promising molecules those mimic the antibody-V3 loop interaction and
block HIV-1 entry.
2.2. Chemistry
Development of molecules from the selected hits was based mainly
on the significant pharmacophore features of the parent structure.
Molecule UHLMTA-A9 type, has been previously reported (see Fig. 3) to
inhibit HIV-1 infection [16] and hence, this molecule was considered for
Fig. 1. The interaction interface between 447-52D and HIV-1 gp120 V3 loop.
2

J. Senapathi et al.
Bioorganic Chemistry 116 (2021) 105313
Fig. 2. Top nine molecules selected from the virtual screening protocol for 447-52D mimics.
quinazolinone, isoniazid, and other functional groups. The 1,3,5-
triazine scaffolds were synthesized by substitution of less expensive
cyanuric chloride with different functional groups. The synthesis was
carried out mainly through nucleophilic substitutions and coupling re-
action mechanisms in alkali conditions. The sequential ring substitution
was majorly depending on reaction time and temperature [19]. The
cyanuric chloride reaction with different aromatic amines to replace
chlorides on 1,3,5-triazine ring with amino groups to form mono, di, tri
substituted - s-triazines (Schemes 2–4). The substitution of the chlorine
atom depends on the reaction conditions indicated therein. The
substituted groups on 1,3,5-triazine create structural advantage for
strong interactions at target site.
The target compounds 7a-c were prepared as per the procedure in
Scheme 2. The 6a-c arylamines were used for substitution of single
chloride on s-triazine to form corresponding molecules (7a-c). The
◦
whole reaction was controlled under low temperature < 5 C and the
slow addition of aq. NaOH, which was acting as neutralizing agent for
released HCl during reaction [20].
For the synthesis of UHLMTJ-257a-c, the second chloride of mon-
ochloride substituted 7a-c molecules replaced with isoniazid, in the
presence of aq. NaOH between pH 7.5 to 8.0 under room temperature
(RT) in Scheme 3 [21]. However, UHLMTJ-259a-c were synthesized
through nucleophilic substitution of chloride with 2-(4-aminophenyl)-
2,3-dihydro-1H-quinazolin-4-one (4) which is prepared as per the
Scheme 1 [22,23]. The reaction was carried out at overnight under inert
conditions in the presence of K2CO3 that was shown in Scheme 4.
The synthetic route for the preparation of trichloro substituted 1,3,5-
triazine molecules (UHLMTJ-258a-c, 260a-c, 261a-c) was explained in
Schemes 3 and 4. The third chloride replacement was difficult due to less
availability of chlorides on 1,3,5-triazine. So, the reaction was carried in
excess equivalent of corresponding arylamines at a high temperature
(110 ◦C). In that, the excess amine itself is acting as a base to neutralize
the reaction. While excess P-toluidine was treated with UHLMTJ-257a-c
and UHLMTJ-259a-c to form UHLMTJ-258a-c, and UHLMTJ-260a-c,
respectively. Compounds UHLMTJ-261a-c were prepared as per the
Scheme 4 and excess of isoniazid was treated with UHLMTJ-259a-c to
get desired molecules [24].
Fig. 3. Chemical structures of HIV entry inhibitor targeting gp41.
further development. The 1,3,5-triazine skeleton of the UHLMT-A9
molecule is mainly substituted with different functional groups to
design new derivatives (Table 1) with synthetic ease as described by the
below methodology. The functional groups that could mimic in-
teractions of the V3 loop with co-receptor CCR5 were considered for
substitution to facilitate competitive binding with the loop, conferring
blocking interaction with the co-receptor [17]. The V3 loop-CCR5 par-
ticipates principally through ionic interactions involving the positively
charged V3 region and negatively charged sulfate group of CCR5.
However, the sulphonate group mediated interactions alone may not be
sufficient to inhibit entry inhibition [18]. We developed molecules
containing electron abundant negative charge moieties for participation
in hydrophilic, hydrophobic, and cation -π stacking type interactions
with V3 loop residues. The designed analogues of UHLMT-A9 contains
negative charge sulfonate groups, substituted benzene ring,
3

J. Senapathi et al.
Bioorganic Chemistry 116 (2021) 105313
Table 1
The newly designed and synthesized analogues of 1,3,5-triazine.
R1
R2
Compound Code
-Cl
UHLMTJ-257a
UHLMTJ-258a
UHLMTJ-257b
UHLMTJ-257c
UHLMTJ-258c
UHLMTJ-258b
UHLMTJ-259b
-Cl
UHLMTJ-259c
UHLMTJ-259a
UHLMTJ-260b
UHLMTJ-261b
UHLMTJ-260c
UHLMTJ-261c
UHLMTJ-260a
UHLMTJ-261a
Scheme 1.
Scheme 2.
The experimental observation concluded that following order
essential for chloride atom substitution. As per that first electron-
withdrawing groups contained amines and then bulky size amines, fol-
lowed by small, and electron-rich amines. All substituted compounds
were further purified by washing, recrystallized, and characterized by
NMR, IR, and HRMS. The yield and melting points of molecules are
reported with spectral data. The yield of sulfonic acid derivates was
significantly low compared to others. Here, we prepared sodium salt of
sulfonic acid by dissolving sulfonic acid groups contained molecules in
PBS buffer at pH 8.0. The hydrogen ion was replaced by sodium ion to
form the sodium salt of sulfonic acid confirmed by molecular solubility.
4

J. Senapathi et al.
Bioorganic Chemistry 116 (2021) 105313
Scheme 3.
Scheme 4.
2.4. In silico binding studies of 1,3,5-traizine derivatives
2.3. Structural dynamics of gp120-V3 loop
Number of studies have reported that the recognition of gp120 bind-
ing regions by small molecules is dependent on the V3 structural dy-
namics as some molecules recognize CD4 bound open loop gp120
structure while some recognize the native closed structures better [25].
To understand this, the selected molecules were docked with the open,
and closed structures of gp120 and the binding energies were analysed to
understand the mode of action of the compounds. The structure of the
gp120, complexed with CD4 and X5 antibody, retrieved from RCSB PDB
(PDB ID: 2B4C) has an open loop structure which was used for docking.
To obtain a closed loop structure of gp120, the crystal structure was
subjected to MD simulation for 20 ns (Fig. S1). The Carbon backbone of
the structure before and after simulation was overlapped to monitor the
structural changes (Fig. S2). Major structural change was seen in the V3
loop of the gp120 structure, where an open structure seen when com-
plexed with CD4 was changed to a closed structure when subjected to the
molecular dynamics protocol. This change in conformation between CD4
bound and unbound gp120 has been reported in several studies [26].
Analysis of the molecular docking scores of 1,3,5-traizine derivatives
between the open and closed loop conformation of gp120 showed better
affinity with closed structures than open structures in the mimetics
(Table 1). From the docking scores and the interacting residues, it was
observed that the binding region for 447-52D mimetics was similar to
the antibody interacting interface. Electrostatic interactions, Hydrogen
bonding and cation-π interactions were observed in the binding of the
compounds, while electrostatic interactions were dominant. The com-
pounds designed to possess an overall negative charge tend to interact
with greater affinity due to the dominant positive charge of the V3 loop.
It was observed that Trichloro substituted molecules showed better
binding, than dichloro substituted molecules. Further, moieties with
aromatic and anionic properties such as quinazolinone, sulphonate,
sulphanilamide and isoniazid substituted molecules have shown better
binding energies computation from Autodock. Moieties such as quina-
–
– –
zolinone ring
(
NH
C
O), sulfonate
(
SO3ꢀ ), safinamide
–
–
–
–
(
SO2NH2), and ester groups ( COOC2H5) interact with side chains of
5

J. Senapathi et al.
Bioorganic Chemistry 116 (2021) 105313
the crown (HIS308, ILE309, GLY312, PRO313, TYR318), stem (THR
297, PRO299, ARG304) regions to form hydrogen bonds. Along with
those interactions, the quinazolinone, isoniazid and s-triazine aromatic
with IC50 of <2.0 µM on both clades of virus. This difference could be
attributed to the differences in interaction and affinity with V3 loop as
seen in the results of the in silico analysis. These molecules showed
similar anti-HIV-1 activity against both clade C and clade B type of virus,
while relatively better activity exhibited against clade B.
rings were also involved in cation -π stacking at the base part of the V3
loop (Fig. 4). The binding region also included amino acids from the
base of the V3 loop in closed structures, most prominent being Asn332,
Thr297 and Asn295. This was also reflected in preference of 447-52D
mimetics to choose closed structure where the stem, crown, and base
of the V3 loop and other surrounding regions of gp120 were in prox-
imity, due to the folded V3 conformation. Among various residues
outside V3 loop involved in the binding were Arg444, Thr413, Val292,
Lys337 in the closed loop conformation.
2.7. Inhibition of gp120-mediated entry using dye transfer assay
Inhibition of gp120 mediated viral entry was tested by monitoring a
cell-mediated fusion of envelope expressing HL2/3 and SupT1 cells. The
compound UHLMTJ-261c, was tested for inhibition by dye redistribu-
tion assay as described. T-20 (Enfuvirtide) was used as a control and the
compound were tested at respective IC80 concentrations in pre-
incubation experiment, where the compounds were incubated with
HL2/3 cells before co-culturing them with SupT1 at 1:1 ratio. The
compound showed better blocking of entry, as seen in Fig. 7, with fewer
fused cells (cyan coloured cells) in comparison to control cells.
2.5. Cell cytotoxicity (MTT Assay) of selected molecules
The cytotoxicity of the newly designed and synthesized drug-like
molecules on human cell line Sup-T1 cells was conducted in the pres-
ence of increasing concentrations, 50, 100, 250, 500 and 750 μM, and
the cell viability was evaluated using MTT assasy. The results of analysis
of 50% cell survival concentrations (CC50) showed that the molecules
containing sulfonic groups (UHLMTJ-257c, 258c, 259c, 260c, 261c)
exhibited significant toxicity than the molecules devoid of sulfonic
group (UHLMTJ-257a, b etc.,). Along with quinazolinone, isoniazid
combination with sulphnamide and sulfonic group molecules, UHLMTJ-
3. Conclusion
We elaborate the design and development of small-molecule mimics
based on interaction of broadly neutralizing antibody 447-52D with
gp120 V3 loop. The 1,3,5-triazine scaffold of UHLMT-A9 molecules
derivatives were designed and synthesized with different functional
groups. The interaction studies of the derivatives disclosed that most of
260c, 261a, 261c, were more toxic and CC50 was <200.0
μM. The
remaing molecules were modarately toxic at concertation > 300
μM, the
the interactions were hydrophilic along with hydrophobic and
π -π
dose-dependent cytotoxicity is depicted in Fig. 5 and CC50 values are
stacking. Tri-substituted molecules interact effectively with better
docking scores. The antiviral activity results suggested that all molecules
presented in Table 2.
have IC50 activity below 5.0 μM. This study has identified a promising
molecular scaffold that can be further explored to obtain potent HIV-1
inhibitors targeting viral entry.
2.6. Anti-HIV-1 activity (p24 Assay) of molecules
The antiviral activity of the molecules was analysed against HIV-1
replication in SupT1 cells. SupT1 cells were challenged with HIV-1
using 93IN101 (Clade C) and NL4-3 (Clade B) virus in the presence of
increasing concentrations (0.5–10 µM) of the compounds. The virus
replicated was estimated by measuring the p24 capsid protein in the
infected cells using p24 antigen capture assay, the results are presented
in Fig. 6 and IC50 values are presented in Table 2. Tri chloro substituted
compounds showed better anti-HIV-1 activity than di chloro substitution
of corresponding molecules. Among these quinazolinone, isoniazid
combination with sulfonic and sulphonamide contained molecules
UHLMTJ-258c, 259c, 260a, 260c, 261a, 261b, 261c, were highly active
4. Experimental
4.1. Computational design of compounds from 447-52D-V3 loop
interacting complex
The structure 1Q1J solved by Stanfield et al. with a resolution of 2.5
Å was the closest crystal structure record found, for the 447-52D-V3 loop
complex. Pep:MMs:MIMIC server was used to screen the MMs database
for molecules with desired physical properties of the 447-52D-V3 loop
interaction interface. The V3 residues I309, I307, H308, R315 etc. were
Fig. 4. A) Depicts the binding region of all molecules with gp120. The molecules can be seen bound in the interface of the folded V3 loop stem (green) and the base of
the loop and surrounding residues (grey). B) Depicts the interface of the interaction of molecule UHLMTJ ꢀ 261b with gp120.
6

J. Senapathi et al.
Bioorganic Chemistry 116 (2021) 105313
MTT Assay
80
70
60
50
40
30
20
10
0
Compounds
50uM 100uM 250uM 500uM 750uM
Fig. 5. Cytotoxicity of the compounds tested on SupT1 cell line. The toxicity was measured by MTT assay at increasing concentration of the compounds and the %
cytotoxicity was plotted against the concentration. Mean ± SD was plotted. P value was ≤0.05.
Table 2
Binding energy (kcal/mol) in interaction with V3 loop, CC50, IC50 and therapeutic index (TI) of newly synthesized compounds. (ND: Not determined).
S.
Compound
Code
Binding Energy (Kcal/
mol)
CC50 (µM)
IC50 ± SD (µM) (93IN101, Clade
IC50 ± SD (µM) (NL4-3, Clade
TI
TI (NL4-3)
No
(SupT1)
C)
B)
(93IN101)
1
T20
ND
>100
0.035
0.028
>2000
>3000
>100.0
>90.90
123.85
>142.85
>78.94
>300.0
>200.0
>120.0
>250.0
190.25
102.80
251.45
252.81
135.67
373.72
2
UHLMTJ-257a
UHLMTJ-257b
UHLMTJ-257c
UHLMTJ-258a
UHLMTJ-258b
UHLMTJ-258c
UHLMTJ-259a
UHLMTJ-259b
UHLMTJ-259c
UHLMTJ-260a
UHLMTJ-260b
UHLMTJ-260c
UHLMTJ-261a
UHLMTJ-261b
UHLMTJ-261c
ꢀ 7.3
ꢀ 6.8
ꢀ 7.7
ꢀ 8.0
ꢀ 7.2
ꢀ 8.7
ꢀ 8.7
ꢀ 8.3
ꢀ 8.8
ꢀ 9.5
ꢀ 8.5
ꢀ 9.4
ꢀ 10.5
ꢀ 9.0
ꢀ 9.6
>300
4.03 ± 0.15
4.50 ± 0.18
3.25 ± 0.12
2.86 ± 0.15
3.60 ± 0.17
1.14 ± 0.09
2.16 ± 0.19
2.83 ± 0.12
1.54 ± 0.15
1.74 ± 0.12
2.31 ± 0.16
0.94 ± 0.08
0.76 ± 0.05
1.93 ± 0.12
0.52 ± 0.04
3 ± 0.14
>74.44
>66.66
83.83
3
>300
3.3 ± 0.15
2.2 ± 0.18
2.1 ± 0.12
3.8 ± 0.19
1.0 ± 0.11
1.5 ± 0.2
2.5 ± 0.19
1.2 ± 0.13
1.1 ± 0.15
2.6 ± 0.12
0.7 ± 0.04
0.6 ± 0.03
1.8 ± 0.1
0.5 ± 0.06
4
272.47
>300
5
>104.89
>83.33
>263.15
>138.88
>106.00
>194.80
120.27
115.70
187.25
199.59
126.53
359.34
6
>300
7
>300
8
>300
9
>300
10
11
12
13
14
15
16
>300
209.28
267.29
176.02
151.69
244.22
186.86
given as the interacting residues. The scoring was based on both phar-
macophoric (60%) and shape (40%) parameters. The molecules reported
by Pep:MMs: MIMIC were further screened to obtain hits with good
binding affinity to V3 loop. GOLD docking suite was used for screening
of the reported molecules. The processed 1Q1J file was used as the re-
ceptor, and the docking was performed in batch. The binding site residue
was specified as Arg315 and 5 Å was set as the radius of the cavity.
Genetic algorithm was selected as the default program for exploring the
conformational space for ligand binding. The parameters for GA run
were set to default as described; 100 was set as the population size, the
selection pressure was set to 1.1, the number of operations was given as
10,000, and one island and niche size of 2 was set per run. Mutation and
cross over rates were set at 100. The GOLD score was used as a metric to
score the molecules. The output was set to 10 solutions per molecule.
Further, Autodock Vina v1.2 was used to dock the selected, rede-
signed, synthesized, and tested molecules with X-Ray structure of gp120
obtained from PDB record (2B4C) and the minimized and processed
structure of gp120.
4.2. Structural dynamics of gp120-V3 loop
The processed PDB structure of gp120 was subjected to molecular
dynamics simulation using the GROMOS96 43a1 force field. The simu-
lation was carried in water using the SPC/E water model and a cubic box
was defined to build the system (protein in water). The protein was
placed in the centre of the box, 1 nm from the edge. Thirteen water
molecules in the solvent were replaced by negatively charged Chloride
ions to balance the charge and neutralize the system. Before proceeding
with the simulations, the structure was subjected to energy minimiza-
tion using the Steepest Descent Algorithm with 50,000 iteration steps
and a step size of 0.01 KCal/mol. The maximum force on the system was
set to 1000 KJ/mol/nm, and the minimization would stop if the force fell
below the level. The minimized system was equilibrated at constant
pressure and constant volume for 200 ps each. The equilibrated system
was subjected to final dynamic simulation for a 20 ns run. The simulated
structure was evaluated by RMSD and radius of gyration. GROMACS 5.0
was used for setting up and running the MD simulation. The final
structure obtained was used for further docking studies.
7

J. Senapathi et al.
Bioorganic Chemistry 116 (2021) 105313
p24 Assay
5.00
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
Compounds
subtype-C subtype-B
Fig. 6. Graphical representation of action of synthesized molecules against replication of HIV-1 subtype B (NL4-3) subtype C (93IN101) in IC50 value. Each data
point is an average of three independent experiments and presented as Mean ± SD. T20 was used as a positive control.
Fig. 7. Dye transfer assay to monitor inhibition of cell fusion in presence of compound UHLMTJ-261c. The images were captured by Leica Fluorescent Microscope as
different regions of coverslip. Fusion in absence of compounds was considered as control and in presence of T-20 is considered positive control. Fused cells exhibit
cyan color and are indicated by yellow flash for visualization.
4.3. Chemistry
synthesized molecules was confirmed by NMR (600 MHz), ESI- HRMS,
FTIR and melting point apparat. The chemical shift value of compound
peaks reported in ppm (δ scale) and coupling constant (J) vlaues were
given in Hz and DMSO‑d6 were used as a standared solvent. The peaks
multiplicity was explained in singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m) and broad (br). The low solubility of compounds
4.3.1. General experimental information
All reagents and solvents were of analytical grade and were used
without further purification. High precision instruments were used for
molecular characterization. The structural characterization of
8

J. Senapathi et al.
Bioorganic Chemistry 116 (2021) 105313
restricted the 13C NMR of the compounds. It was interesting to note,
2H, Ph H). 13C NMR (DMSO‑d6, 150.91 MHz) δ: 170.21, 169.30,
164.30, 144.72, 137.73, 126.74, 121.20 ppm. IR (neat): νmax 3469,
3270, 1618, 1557, 1498, 1423, 1384, 1319, 1225, 1170, 1127, 1035,
1007, 962, 876, 846, 828 cm-1. HRMS (ESI): m/z [M+H]+ Calcd for
C9H6Cl2N4O3S, 320.9616; Found, 320.9605.
–
1
– –
N
H proton exchange in H NMR of some compounds. Insilco studies
was carried out by using Pep:MMs:MIMIC server, GOLD, GROMACS 5.0,
Autodock Vina V1.2. The biological experiment were conducted by CO2
Incubator, ELISA Plate reader, Fluorescent Microscope (Carl-Zeiss) and
Bruker ImageJ Java 1.8.0_112. Graphpad Prism 6.0 was used for all
statistical analysis.
4.3.2.3. General procedure for synthesis of the target compound (UHLMTJ-
257 a-c). The monochloride substituted triazene 7a-c (0.01 mol), and
the corresponding arylamine (0.01 mol) were dissolved in 1,4- dioxane
at RT. During the reaction, the released HCl was neutralized with
dropwise added 4% of NaOH (Alternatively, dry DMF as a solvent and
K2CO3 as a neutralizing agent also used) and continue reaction up to 3 h
and maintained reaction mixture at pH 7.5–8.0. After completing the
reaction, the total blend was neutralized, washed with excess water,
filtered to get the recrystallized product in dry DMF.
4.3.2. Experimental procedures
4.3.2.1. Synthesis of 2-(4-aminophenyl)-2,3-dihydro-1H-quinazolin-4-one
(4). The anthranilamide (0.73 mmol) was dissolved in 2 ml of aceto-
nitrile, then mixed with 4-nitrobenzaldehyde (0.73 mmol) and cyanuric
chloride (0.135 mmol, 10 mol %) at RT. The reaction mixture was
◦
reflexed at 70 C for 0.5 h, and after the reaction was completed, the
access solvent was evaporated and washed with cooled water to get a
yellow solid (2.8) with a 95 % yield. The solid nitro, compound 2.8
(0.01 mol), was dissolved in a mixture of ethanol (10 ml), water (0.05
ml), and added few drops of a con. HCl. The solution was gently stirred
for 10 min at room temperature and then added iron metal powder
(0.03 mol) and NH4Cl (0.03 mol). Finally, the total mixture was refluxed
for 0.5 h at 60 ◦C. TLC was used for monitoring the completion of the
reaction. After completing the reaction, the reaction mixture was cooled
at RT and then neutralized with the aq.solution of 10% NaHCO3, poured
into cooled water, and filtered to get the solid supernatant cake, which
was washed with hot ethanol and chloroform in 1: 1 ratio. To obtain the
final compound, evaporate the solvent, and got the pure amino com-
pound (2.9) with a 40% yield; no further purification was required.
Yield 40%. mp 202–204 ◦C. 1H NMR (600 MHz, DMSO‑d6) δ 8.0 (brs,
4.3.2.3.1. 4-((4-chloro-6-(2-isonicotinoylhydrazinyl)-1,3,5-triazin-2-
yl)amino)benzenesulphonamide (UHLMTJ-257a). Yield 85%. mp Infus-
1
–
– – –
ible. H NMR (600 MHz, DMSO‑d6): δ11.02 (d, 1H, NH NH
C O),
–
–
– – –
10.65 (brs, 1H, NH NH
–
C O), 10.34 (brs, 1H, NH), 8.85–8.76
–
–
–
–
(m, 4H, Ph H, Py H), 7.82–7.77 (m, 2H, Py H), 7.76–7.09 (m, 2H,
–
–
–
–
Ph H), 7.22 (brs, 2H, NH -S O). IR (neat): νmax 3206, 1555, 1514,
2
1408, 1327, 1189, 1154, 1100, 1034, 986, 903, 833 cmꢀ 1. HRMS (ESI):
m/z [M+H]+ Calcd for C15H13ClN8O3S, 421.0598; Found, 421.0584.
4.3.2.3.2. Ethyl4-((4-chloro-6-(2-isonicotinoylhydrazinyl)-1,3,5-tri-
◦
azin-2 -yl) amino)benzoate (UHLMTJ-257b). Yield 83%. mp > 300 C.
1
–
– –– –
NH C O), 10.64 (brs,
–
H NMR (DMSO‑d6): δ 10.99 (d, 1H, NH
–
– – –
1H, NH NH
–
C O), 10.34 (brs, 1H, NH), 8.82–8.76 (m, 2H,
–
–
–
–
–
Ph H), 7.90–7.77 (m, 4H, Py H, Ph H), 7.65 (d, J = 8.4 Hz, 2H,
–
–
–
Py H), 4.20–4.16 (q, 2H, OCH2 CH3), 1.26 (t, J = 7.2, 3H, CH3).
13C NMR (DMSO‑d6, 150.91 MHz) δ: 169.01, 167.63, 165.59, 164.96,
151.08, 143.43, 139.74, 130.52, 130.04, 124.48, 121.89, 120.01, 60.95,
14.61 ppm. IR (neat): νmax 3281, 1688, 1605, 1539, 1508, 1411, 1366,
1276, 1227, 1176, 1107, 1017, 982, 901, 856 cmꢀ 1. HRMS (ESI): m/z
[M+H]+ Calcd for C18H16ClN7O3, 414.1081; Found, 414.1078.
4.3.2.3.3. 4-((4-chloro-6-(2-isonicotinoylhydrazinyl)-1,3,5-triazin-2-
–
– –
–
C O), 7.58 (d, J = 6.6 Hz 1H, Ph H), 7.18 (d, J = 7.2 Hz,
–
1H, NH
–
–
–
1H, Ph H), 7.13 (d, J = 8.4 Hz, 1H, Ph H) 6.84 (s, 1H, NH),
–
–
6.72–6.60 (m, 3H, Ph H), 6.54 (d, J = 7.8 Hz, 2H, Ph H), 5.72 (d, J =
9.6 Hz, 1H, NH CH NH), 5.55 (s, 1H, NH2). 13C NMR (DMSO‑d6,
150.91 MHz) δ:164.46, 149.36, 148.87, 133.67, 128.76, 128.41,
127.88, 117.47, 115.48, 114.90, 114.03, 67.56 ppm. IR
(neat):νmax3287, 1607, 1557, 1487, 1408, 1329, 1176, 1126, 1033,
1007, 832, 800 cmꢀ 1. HRMS (ESI): m/z [M+H]+ Calcd for C14H11N3O
238.0980;Found, 238.0971.
– – –
–
yl)amino)benzenesulfonic acid (UHLMTJ-257c). Yield 72%. mp 300 ◦C.
1
–
– – –
H NMR (600 MHz, DMSO‑d6) δ 11.16 (brs, 1H, NH NH
C O),
–
–
– – –
10.32 (d, 1H, NH NH
–
C O), 9.98 (brs, 1H, NH), 9.0–8.88 (m,
–
–
–
–
2H, Ph H) 8.10–8.02 (m, 2H, Py H), 7.53 (d, J = 8.4 Hz, 2H, Py H),
–
–
7.40 (d, J = 8.4 Hz, 2H, Ph H), 7.16 (brs, 1H, OH). IR (neat): νmax
3205, 1615, 1568, 1515, 1491, 1317, 1277, 1165, 1030, 984, 908, 834
cmꢀ 1. HRMS (ESI): m/z [M+H]+ Calcd for C15H12ClN7O4S 422.0438,
Found: 422.0431.
4.3.2.2. General procedure for synthesis of the target compound (7a-c).
The mixture of 2,4,6-trichloro -1,3,5-triazene (0.01 mol) and arylamine
(0.01 mol) was taken in acetone and stirring at 0–5 ◦C. After 1 h, 4% of
NaOH solution was added dropwise with rapid stirring and then
continue the reaction up to 2 h. After completing the reaction, the
mixture was poured into crushed ice and then neutralized with 2 M HCl
under continuous stirring. The reaction mixture was filtered, washed
with ice water, dried, and recrystallized in acetone.
4.3.2.4. General procedure for synthesis of the target compound (UHLMTJ-
258 a-c). To the solution of Dichloro substituted triazene UHLMTJ-
257a-c (0.01 mol) and corresponding arylamine (0.02 mol) in dry acetic
acid under N2 conditions were reflexed at 110 ◦C for 1 h. After relaxa-
tion, the reaction mixture was cooled and into ice-cooled water, then
was filtered to get a solid product. Finally, the compound was washed
with hot water and hexane to get a required product, which was further
crystallized with 1,4-dioxane.
4.3.2.2.1. 4-((4,6-dichloro-1,3,5-triazin-2-yl) amino) benzene sulpho-
namide (7a). Yield 92%. mp Infusible. 1H NMR (600 MHz, DMSO‑d6) δ
–
–
11.39 (s, 1H, NH), 7.79 (d, J = 6.0 Hz, 2H, Ph H), 7.72 (d, J = 6.0 Hz,
2H, Ph H), 7.73 (brs, 2H, NH2). 13CNMR (DMSO‑d6, 150.91 MHz)
δ:170.30, 169.52, 164.50, 140.51, 127.17, 121.63, 120.83 ppm. IR
(neat): νmax 3293, 3208, 1619, 1552, 1516, 1493, 1425, 1379, 1328,
1295, 1226, 1162, 1022, 900, 875, 845, 831 cmꢀ 1. HRMS (ESI): m/z
[M+H]+ Calcd for C9H7Cl2N5O2S, 319.9776; Found, 319.9766.
4.3.2.2.2. Ethyl 4-((4,6-dichloro-1,3,5-triazin-2-yl) amino) benzoate
(7b). Yield 93%. mp > 300 ◦C. 1H NMR (DMSO‑d6) δ 10.89 (s, 1H,
–
–
4.3.2.4.1. 4-((4-(2-isonicotinoylhydrazinyl)-6-(p-tolylamino)-1,3,5-
triazin-2-yl)amino)benzenesulfonamide (UHLMTJ-258a). Yield 80%. mp
Infusible. 1H NMR (600 MHz, DMSO‑d6):
δ 10.74 (d, 1H,
–
– – –
– –
C O), 9.27 (brs, 1H, NH), 8.80–8.74 (br, 2H, NH-
–
NH NH
–
–
– – –
C O, NH), 8.0–7.62 (m, 8H, Ph H, Py H), 7.50 (brs, 2H,
–
NH
–
–
–
–
–
–
– –
NH -S O), 7.25–7.07 (m, 4H, Ph H), 3.41 (H2O), 24 (s, 3H, CH3).
–
2
NH), 7.87 (d, 2H, Ph H), 7.70 (d, 2H, Ph H) 4.20 (q, 2H, CH2),
1.22 (t,3H, CH3).13CNMR (DMSO‑d6, 150.91 MHz) δ: 165.79, 154.64,
142.27, 130.73, 125.48, 120.54, 61.08, 14.73 ppm. IR (neat): νmax 3281,
3201, 1687, 1607, 1541, 1504, 1431, 1387, 1337, 1318, 1282, 1253,
13C NMR (DMSO‑d6, 150.91 MHz) δ:167.94, 167.82, 164.81, 164.57,
150.98, 150.72, 140.31, 139.88, 137.10, 129.40, 126.84, 122.26,
121.97, 120.20, 119.55, 20.95 ppm. IR (neat): νmax 3263, 1561, 1487,
1405, 1321, 1154, 1034, 1007, 985, 905, 833 cmꢀ 1. HRMS (ESI): m/z
[M+H]+Calcd for C22H21N9O3S, 492.1566; Found, 492.1557.
–
1226, 1186, 1165, 1124, 1014, 960, 878, 836, 814 cmꢀ 1
.
4.3.2.2.3. 4-((4,6-dichloro-1,3,5-triazin-2-yl) amino) benzenesulfonic
acid (7c). Yield 73%. mp Infusible. 1H NMR (600 MHz, DMSO‑d6) δ
4.3.2.4.2. Ethyl-4-((4-(2-isonicotinoylhydrazinyl)-6-(p-tolylamino)-
1,3,5-triazin-2-yl) amino) benzoate (UHLMTJ-258b). Yield 87%. mp
–
–
11.16 (s, 1H, NH), 7.58 (d, J = 8.4 Hz, 2H, Ph H), 7.52 (d, J = 8.4 Hz,
9

J. Senapathi et al.
Bioorganic Chemistry 116 (2021) 105313
238–240 ◦C. 1H NMR (600 MHz, DMSO‑d6): δ10.75 (d, 1H,
was further crystallized with 1,4-dioxane.
–
–
–
– – –
NH NH
– – – –
O), 9.27–9.21 (br, 2H, NH, NH NH C
C
O), 8.80
4.3.2.6.1. 4-((4-((4-(4-oxo-3-hydroquinazolin-2-yl) phenyl) amino)-
6-(p-tolylamino)-1,3,5-triazin-2-yl) amino)benzenesulfonamide (UHLMTJ-
260a). Yield 74%. mp 300 ◦C. 1H NMR (600 MHz, DMSO‑d6): δ
–
–
–
–
(brs, 1H, NH), 8.0–7.48 (m, 8H, Ph H, Py H), 7.12 (d, J = 6.6 Hz,
–
–
2H, Ph H), 7.07 (d, J = 8.4 Hz, 2H, Ph H), 4.26–4.22 (q, 2H,
–
–
–
–
– – – –
9.66–9.22 (brs, 4H, all NH, NH C O), 8.16–7.95 (m, 4H, Ph H),
–
CH2–CH3), 3.37 (H2O), 2.24 (s, 3H, CH3), 1.26 (t, 3H, CH2–CH3).
–
– – –
7.80–7.43 (m, 8H, Ph H), 7.25–7.10 (m, 6H, NH -S O, Ph H), 3.34
–
2
IR (neat): νmax 3267, 1571, 1486, 1407, 1364, 1273, 1173, 1104, 1033,
1008, 805 cmꢀ 1. HRMS (ESI): m/z [M+H]+ Calcd for C25H24N8O3,
485.2050; Found, 485.2047.
(H2O), 2.23 (brs, 3H,
CH3). 13C NMR (DMSO‑d6, 150.91 MHz)
–
δ:164.61, 164.52, 164.33, 162.98, 152.57, 149.44, 143.71, 137.38,
135.08, 132.10, 129.60, 129.46, 129.36, 128.81, 127.85, 126.91,
126.68, 126.40, 126.14, 121.43, 121.24, 119.87, 119.67, 21.02 ppm. IR
(neat): νmax 3272, 2324, 205, 1914, 1603, 1566, 1538, 1486, 1400,
1320, 1247, 1151, 1095, 987, 833, 802 cmꢀ 1. HRMS (ESI): m/z [M+H]+
Calcd for C30H25N9O3S, 592.1879; Found, 592.1885.
4.3.2.4.3. 4-((4-(2-isonicotinoylhydrazinyl)-6-(p-tolylamino)-1,3,5-
triazin-2-yl) amino) benzenesulfonic acid (UHLMTJ-258c). Yield 68%.
1
◦
–
mp > 300 C. H NMR (600 MHz, DMSO‑d6): δ11.01 (brs, 1H, NH),
–
–
–
– – –
9.50 (d, 1H, NH NH
– – –
C
C
O), 9.40 (d, 1H, NH NH
O), 8.96
–
–
–
– –
(m, 4H, Py H), 8.09 (m, 4H, Ph H), 7.70–7.45 (m, 3H, Ph H, NH),
–
–
–
7.08 (d, J = 8.4 Hz, 2H, Ph H), 2.23 (s, 3H, CH3). OH protan Ex-
4.3.2.6.2. Ethyl-4-((4-((4-(4-oxo-3-hydroquinazolin-2-yl)phenyl)
change. IR (neat): νmax 3291, 1556, 1488, 1407, 1325, 1171, 1123,
amino)-6-(p-tolylamino)-1,3,5-triazin-2-yl)amino)benzoate
(UHLMTJ-
1032, 1006, 833 cmꢀ 1
.
HRMS (ESI): m/z [M+H]+ Calcd for
260b). Yield 78%. mp 258–260 ◦C. 1H NMR (600 MHz, DMSO‑d6):
–
– – – –
δ10.1–9.0 (brs, 4H, all NH, NH C O), 8.20–7.39 (m, 14H, Ph H),
–
C22H20N8O4S, 493.1406; Found, 493.1409.
–
–
7.20–7.05 (m, 2H, Ph H), 4.25–4.18 (q, 3H, CH2), 2.25 (brs, 3H,
–
–
CH3)0.1.22 (t, 3H, CH3). IR (neat):νmax3272, 2350, 2285, 2112,
4.3.2.5. General procedure for synthesis of the target compound (UHLMTJ-
259 a-c). The monochloride substituted triazene, 7a-c (0.01 mol), and
the corresponding arylamine (0.01 mol) were dissolved in 1,4-dioxane
at RT. During the reaction, the released HCl was neutralized with
dropwise added 4% of NaOH (Alternatively, dry DMF as a solvent and
K2CO3 as a neutralizing agent also used) and continue reaction up to 3 h
and maintained reaction mixture at pH 7.5–8.0. After completing the
reaction, the total blend was neutralized, washed with excess water,
filtered to get the recrystallized product in dry DMF.
1981, 1916, 1681, 1614, 1567, 1556, 1514, 1487, 1455, 1403, 1214,
1169, 1120, 1031, 1006, 870, 836, 803 cmꢀ 1. HRMS (ESI): m/z [M+H]+
Calcd for C33H28N8O3, 585.2364; Found, 585.2367.
4.3.2.6.3. 4-((4-((4-(4-oxo-3-hydroquinazolin-2-yl) phenyl) amino)-
6-(p-tolylamino)-1,3,5-triazin-2-yl)
amino)
benzenesulfonic
acid
(UHLMTJ-260c). Yield 65%. mp Infusible,1H NMR (600 MHz,
–
– – –
DMSO‑d6):δ 9.9–9.3 (brs, 4H, all NH, NH
C O) 8.30–8.08 (m, 2H,
–
–
–
–
Ph H), 7.82 (d, J = 8.4 Hz, 1H, Ph H), 7.74–7.43 (m, 12H, Ph H),
–
–
–
7.11 (d, J = 8.4 Hz, 1H,-Ph H)6.8 (brs, 1H, OH), 2.30 (s, 3H, CH3).
IR (neat): νmax 3291, 1557, 1486, 1405, 1324, 1164, 1121, 1031, 1005,
830 cmꢀ 1. HRMS (ESI): m/z [M+H]+ Calcd for C30H25N8O4S, 593.1719;
Found, 593.1724.
4.3.2.5.1. 4-((4-chloro-6-((4-(4-oxo-3-hydroquinazolin-2-yl)phenyl)
amino)-1,3,5-triazin-2-yl)amino)benzene sulfonamide (UHLMTJ-259a).
Yield 74%. mp Infusible. 1H NMR (600 MHz, DMSO‑d6) δ 8.30 (brs, 1H,
–
–
NH = CO), 8.16 (d, J = 8.4 Hz, 2H,-Ph H), 7.80–7.70 (m, 4H,
–
–
–
4.3.2.6.4. 4-((4-(2-isonicotinoylhydrazinyl)-6-((4-(4-oxo-3-hydro-
quinazolin-2-yl)phenyl)amino)-1,3,5-triazin-2-yl)amino)benzene sulfon-
Ph H), 7.59 (d, J = 8.4 Hz, 2H, Ph H), 7.50–7.41 (m, 2H, Ph H),
–
–
–
–
7.27 (s, 2H, NH -S O), 7.23–7.20 (m, 2H, Ph H), 6.73 (d, J = 8.4
2
1
–
amide (UHLMTJ-261a). Yield 79%. mp Infusible. H NMR (600 MHz,
Hz, 1H, Ph- H), 6.67–6.63 (m, 1H, Ph H), 5.71 (s, 1H,- NH), 2.86
–
– – –
NH NH C O), 8.77 (brs, 2H,
–
–
–
DMSO‑d6): δ 10.84 (brs, 1H,
(DMF H), One NH protan Exchange. IR (neat): νmax 3281, 1560,
1490, 1407, 1331, 1237, 1155, 1129, 1035, 1007, 988, 900, 832 cmꢀ 1
.
–
– –
– –
NH
– – –
C O, NH), 8.15 (m, 2H, Ph H), 7.9–7.68 (m, 12H, Ph H,
–
HRMS (ESI) m/z [M+H]+: Calcd for C23H17ClN8O3S, 521.0911; Found,
–
–
–
Py H, NH -S O), 7.45 (d, J = 6.6 Hz, 1H, Ph H), 7.3–7.17 (m, 3H,
2
–
–
Ph H), two NH protans are Exchange. IR (neat): νmax 3287, 1556,
1486, 1407, 1327, 1228, 1155, 1035, 1008, 832 cmꢀ 1. HRMS (ESI): m/z
[M+H]+ Calcd for C29H23N11O4S, 622.1733; Found, 622.1738.
4.3.2.6.5. Ethyl-4-((4-(2-isonicotinoylhydrazinyl)-6-((4-(4-oxo-3-
hydroquinazolin-2-yl) phenyl) amino)-1,3,5-triazin-2-yl) amino) benzoate
(UHLMTJ-261b). Yield 81%. mp 260–262 ◦C.1H NMR (600 MHz,
521.0909.
4.3.2.5.2. Ethyl
4-((4-chloro-6-((4-(4-oxo-3-hydroquinazolin-2-yl)
phenyl)amino)-1,3,5-triazin-2-yl)amino)benzoate (UHLMTJ-259b). Yield
1
◦
81%. mp 240–241 C. H NMR (600 MHz, DMSO‑d6) δ 8.26 (brs, 1H,
–
–
NH = CO), 7.95–7.80 (m, 8H, Ph H), 7.58 (d, J = 6.6 Hz, 1H,
–
–
–
Ph H), 7.44 (d, 1H, Ph H), 7.46–7.20 (m, 1H, Ph H), 7.20 (d, J = 6.6
–
– – –
DMSO‑d6): δ 8.75 (brs, 2H, NH C O, NH), 8.16 (d, J = 8.4 Hz, 1H,
–
–
–
–
Hz, 1H, Ph H), 6.62 (d, J = 6.6 Hz, 1H, Ph H), 5.70 (brs, 1H, NH),
– – –
–
–
–
Py H), 8.10 (d, J = 8.4 Hz, 1H, Py H), 8.0–7.82 (m, 12H, Ph H,
4.24 (q, J = 7.2 Hz, 2H, OCH2 CH3), 2.86 (DMF H), 1.26 (t, J = 7.2
– – –
Py H), 7.80–7.63 (m, 2H, Ph H), 7.44 (brs, 1H, NH), 4.25–4.22 (q,
–
–
Hz, 3H, CH3), One NH protan Exchange. IR (neat): νmax 3287, 1566,
1487, 1409, 1365, 1309, 1274, 1240, 1175, 1107, 1017 cmꢀ 1. HRMS
(ESI): m/z [M+H]+ Calcd for C26H21ClN7O3, 514.1394; Found,
514.1395.
–
–
J = 8.4 Hz, 2H, CH2), 1.27 (t, J = 8.4 Hz, 3H, –CH3) two NH protans
are Exchange. IR (neat): νmax IR (neat): νmax 3205, 1650, 1554, 1484,
1404, 1284, 1177, 1127, 1035, 981, 832, 800 cmꢀ 1. HRMS (ESI): m/z
[M+H]+ Calcd for C32H26N10O4 , 615.2217; Found, 615.2220.
4.3.2.6.6. 4-((4-(2-isonicotinoylhydrazinyl)-6-((4-(4-oxo-3-hydro-
quinazolin-2-yl)phenyl)amino)-1,3,5-triazin-2-yl)amino)benzenesulfonic
acid (UHLMTJ-261c). Yield 73%. mp Infusible. 1H NMR (600 MHz,
4.3.2.5.3. 4-((4-chloro-6-((4-(4-oxo-3-hydroquinazolin-2-yl)phenyl)
amino)-1,3,5-triazin-2 yl)amino)benzenesulfonic acid (UHLMTJ-259c).
1
◦
Yield 68%. mp > 300 C, H NMR (600 MHz, DMSO‑d6) δ 8.14–8.09
–
–
–
–
(brs, 2H, NH = CO, NH), 7.90–7.76 (m, 5H, Ph H), 7.70–7.51 (m,
–
– – –
NH NH C O), 8.8 (brs, 2H,
–
–
–
DMSO‑d6):δ 10.92 (brs, 1H,
6H, Ph- H), 7.22–7.16 (m, 1H, Ph H), 7.11 (s, 1H, OH), One NH
protan Exchange. IR (neat): νmax 3270, 1556, 1489, 1405, 1322, 1162,
1121, 1031, 1006, 986, 832 cmꢀ 1. HRMS (ESI): m/z [M+H]+ Calcd for
–
–
–
– – –
C O, NH), 8.15–7.95 (m, 6H, Ph H, Py H), 7.88–7.37 (m,
–
NH
–
–
–
10H, Ph H, Py H), 6.72 (brs, 1H, OH), two NH are Exchange. IR
(neat): νmax 3291, 1556, 1488, 1407, 1325, 1171, 1123, 1032, 1006,
833 cmꢀ 1. HRMS (ESI): m/z [M+3H]+ Calcd for C29H23N10O5S,
625.1730; Found, 625.1711.
C
23H16ClN7O4S, 522.0751;Found, 522.0748.
4.3.2.6. General procedure for synthesis of the target compound (UHLMTJ-
260 a-c, UHLMTJ-261 a-c). To the solution of dichloro substituted tri-
azene UHLMTJ-259 a-c (0.01 mol) and corresponding arylamine (0.02
mol) in dry acetic acid under N2 conditions were reflexed at 110 ◦C for 1
h. After relaxation, the reaction mixture was cooled and into ice-cooled
water, then was filtered to get a solid product. Finally, the compound
was washed with hot water and hexane to get a required product, which
5. Cell cytotoxicity by MTT assay
Cytotoxicity in SUP-T1 cells was measured by quantifying 3-(4,5-
dimethylthiozol-2-yl) color change -2,5-diphenyltetrazolium bromide
(MTT, Sigma) in the presence of different concentrations of the syn-
thesized compounds and nanoparticles. According to the procedure, the
10

J. Senapathi et al.
Bioorganic Chemistry 116 (2021) 105313
96-well plate was the seed with SUP-T1 cells at a density of 0.2x106
7.2. SupT1 labelling
◦
cells/well and incubated at 37 C in a 5% CO2 incubator for 4 h. The
incubated cells were treated with different concentrations of the syn-
thesized compounds and nanoparticles. After treatment, the cells were
again incubated for 16 h in the same incubation conditions. The cells
were pelleted at 1200 rpm for 7 min and resuspended in complete me-
dium. Now, 20 µl of 5 mg/ml pre-dissolved MTT was added to each well
and incubated for another 4 h. After the incubation, the cells were pel-
leted at 1200 rpm for 10 min, discarded the media from wells, and
formed MTT-formazan crystals were dissolved in DMSO by adding 100
SupT1 cells were incubated with fluorescent dye Hoechst (20 μM) for
1 h at 37 ◦C. After incubation, the cells were pelleted at 350xg, and the
pellet was resuspended in fresh medium and incubated another 30 min.
7.3. Dye redistribution assay
The fluorescent dye-labeled, Env protein-expressing cell line (HL2/
3) and CD4+, CCR5+, CXCR4+ contained cell line (SupT1) were mixed
co-cultured at a ratio of 1:1 and incubated at 37 ◦C for 2 h. The fusion
was monitored at IC80 concentration of the compounds. The fusion in the
presence of 50 nM of T20 was considered positive control and cells in
absence of compounds is considered as negative control. After incuba-
tion, the extent of fused cells was observed using a Leica Fluorescent
microscope or Leica Confocal microscope.
μ
L into each well. Finally, the plate was incubated in the dark for 5 min,
and the color change was recorded in an ELISA reader at 595 nm. The
experiments were conducted in triplicate, and the average with standard
deviation was plotted to represent the cell survival.
6. HIV-1 antiviral activity quantified by p24-ELISA assay
6.1. HIV-1 antiviral assay
Funding
106 SupT1 cells were suspended in RPMI 1640 supplemented with
0.1% FBS were seeded per well and followed by treatment with different
concentrations of the synthesized compounds and nanoparticles. The
compounds were added to the cells, followed by infection with 1 ng/ml
p24 equivalent of 93IN101 and NL43 virus strains of clade C and clade B
were used to study the inhibition by the molecules. The cells were
incubated for 4 h at 37 ◦C in a 5% CO2 incubator. After incubation, the
cells were pelleted at 1200 rpm for 7 min, and the supernatant was
discarded. The cells pellets were washed with fresh medium (RPMI 1640
supplemented with 10% FBS), resuspended in medium, and incubated
for 96 h. After the incubation, the cells were pelleted down, and su-
pernatants were collected for p24 antigen estimation as per the below
described protocol. p24 levels in the absence of compounds were
considered 0% inhibition and were taken as a negative control, and in
the presence of known drugs T20 (Enfuvirtide) were considered positive
control. Each experiment is conducted in triplicates; the data is pre-
sented as Mean ± Standard Deviation.
Research work is funded under DBT project BT/PR24076/Med/29/
1210/2017. JS received funding through the University Grants Com-
mission for doctoral fellowship and support. AB received funding
through Indian Council of Medical Research for doctoral fellowship.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
The authors thank DBT, DST FIST, UGC SAP, UG UPE-II, DBT Builder
program for providing central facilities. The authors thank ACRHEM,
University of Hyderabad for support in spectroscopy experiments.
Appendix A. Supplementary material
6.2. p24-ELISA assay
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105313.
The HIV-1 core p24 antigen was collected from cell supernatent and
quantified by p24 Antigen ELISA Kit after 96 h of incubation. The
infection of the virus was quantified as on detected levels of viral antigen
p24. The p24 quantification was followed as per the manufacturer’s
guidelines. The collected supernatants were diluted to 10-fold with
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