Molecular docking guided structure based design of symmetrical N,N′-disubstituted urea/thiourea as HIV-1 gp120-CD4 binding inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.05.038

Induced fit molecular docking studies were performed on BMS-806 derivatives reported as small molecule inhibitors of HIV-1 gp120-CD4 binding. Comprehensive study of protein-ligand interactions guided in identification and design of novel symmetrical N,N′-disubstituted urea and thiourea as HIV-1 gp120-CD4 binding inhibitors. These molecules were synthesized in aqueous medium using microwave irradiation. Synthesized molecules were screened for their inhibitory ability by HIV-1 gp120-CD4 capture enzyme-linked immunosorbent assay (ELISA). Designed compounds were found to inhibit HIV-1 gp120-CD4 binding in micromolar (0.013-0.247 μM) concentrations.

Full text of DOI:10.1016/j.bmc.2013.05.038

Bioorganic & Medicinal Chemistry 21 (2013) 4591–4599
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Molecular docking guided structure based design of symmetrical
N,N0-disubstituted urea/thiourea as HIV-1 gp120–CD4 binding
inhibitors
⇑
Sree Kanth Sivan, Radhika Vangala, Vijjulatha Manga
Molecular Modeling and Medicinal Chemistry Group, Department of Chemistry, Nizam College, Hyderabad 500001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Induced fit molecular docking studies were performed on BMS-806 derivatives reported as small mole-
cule inhibitors of HIV-1 gp120–CD4 binding. Comprehensive study of protein–ligand interactions guided
in identification and design of novel symmetrical N,N0-disubstituted urea and thiourea as HIV-1 gp120–
CD4 binding inhibitors. These molecules were synthesized in aqueous medium using microwave irradi-
ation. Synthesized molecules were screened for their inhibitory ability by HIV-1 gp120–CD4 capture
enzyme-linked immunosorbent assay (ELISA). Designed compounds were found to inhibit HIV-1
Received 14 February 2013
Revised 16 May 2013
Accepted 17 May 2013
Available online 1 June 2013
Keywords:
HIV-1 gp120
Symmetrical N,N0-disubtituted urea
Symmetrical N,N0-disubtituted thiourea
Molecular docking
gp120–CD4 binding in micromolar (0.013–0.247
l
M) concentrations.
Ó 2013 Elsevier Ltd. All rights reserved.
Induced fit docking (IFD)
Enzyme-linked immunosorbent assay
(ELISA)
1. Introduction
within functional spike, the CD4-binding site (CD4bs) is a recessed
pocket and the co-receptor-binding site (or CD4-induced region) is
either not formed or not exposed until HIV-1 gp120 engages CD4
on target cells.7 During this highly coordinated process, conserved
hidden epitopes in Env are exposed to the surface. Targeting these
highly conserved transiently exposed epitopes as well as confor-
mational epitopes generated by the CD4–HIV-1 gp120–CCR5/
CXCR4 complex by antivirals, may limit significantly viral escape
and broaden the antiviral activity of inhibitory molecules to effec-
tively neutralize a wide range of HIV-1 clades.
HIV-1 cell entry is mediated by sequential interactions of enve-
lope protein gp120 with the receptor CD4 and a co-receptor, usu-
ally CCR5 or CXCR4, depending on the individual virion. Entry of
primate immunodeficiency viruses into the host cell involves bind-
ing of HIV-1 gp120–CD4 glycoprotein, which serves as the primary
receptor.1 This binding creates the necessary exposure of an inter-
acting surface for the CCR5 or CXCR4 host-cell chemokine receptor.
This chain of events triggers the release of HIV-1 gp41 which, ulti-
mately, undergoes a large conformational change responsible for
the viral and cellular membranes apposition, thereby allowing en-
try of viral genetic material into the cytosol.2,3 This series of HIV-1
Env–receptor interactions are the major focus of research aimed at
developing broadly neutralizing antibodies to interrupt the entry
process. Extensive Env glycosylation and conformational masking
of the functional spike (i.e., epitope inaccessibility) make this gly-
coprotein a difficult target for broadly neutralizing antibodies.4–6
Receptor-binding structures of HIV-1 gp120 are conserved among
diverse viral isolates and represent functionally constrained re-
gions that might serve as targets of broadly neutralizing antibod-
ies. However, evidence from structural studies suggest that,
Most of the potent HIV-1 gp120–CD4 inhibitors identified till
date are proteins or peptides.8 However recent publications and
patent disclosures indicate that there has been an increased effort
to identify small-molecule HIV-1 gp120 inhibitors that can block
HIV-1 gp120–CD4 interactions.9 BMS-378806 (BMS-806) and
BMS-4880434 discovered through a cell-based screening assay
(HIV-1 envelope-mediated fusion assay using two populations of
HeLa cells),10 are potent small-molecule inhibitors that are re-
ported to prevent the binding of HIV-1 gp120–CD4 receptors in
nanomolar range.10–12 NBD-556 and NBD-557 identified by a HIV
syncytium formation assay on a drug-like small-molecule chemical
library of 33,000 compounds have been reported to exhibit single
digit micromolar potency against selected HIV-1 laboratory strains
with minimal cytotoxicity.13
⇑
Corresponding author. Tel.: +91 9866845408.
E-mail addresses: sivan.sreekanth@gmail.com (S.K. Sivan), vangalaradhika@
Tamamur and co-workers14–16 have designed and synthesised
small molecule CD4 mimics (NBD-556 derivatives) that inhibit
yahoo.com (R. Vangala), vijjulathamanga@gmail.com (V. Manga).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.038

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S. K. Sivan et al. / Bioorg. Med. Chem. 21 (2013) 4591–4599
gp120–CD4 interactions. These molecules posses substituted phe-
nyl groups attached to oxalamide group at one end and piperidinyl
group at the other end. Molecular docking studies illustrated a
hydrophobic interaction between phenyl group and hydrophobic
residues in the binding site of gp120. The piperidinyl moiety has
an electrostatic interaction with Asp368 and a hydrophobic inter-
action with Val430. Similar interactions were reported by Bewley
et al.18 for batzelladine analogues, and these were further opti-
mized by Kazuo Nagasawa et al.17
In present work novel symmetrical substituted urea and thio-
urea having benzimidazole moieties were designed based on
molecular docking studies, these were synthesized and analyzed
for their inhibitory activity against gp120–CD4 interaction.
Scatter plot of Glide Emodel vs pIC50
9
8
7
6
5
4
3
2
1
0
-30
-35
-40
-45
-50
-55
-60
-65
Glide Emodel (kcal/mol)
2. Results and discussion
Figure 2. Scatter plot of Glide Emodel (kcal/mol) versus experimental pIC50
.
HIV-1 gp120 core is composed of inner and outer domains,
which reflects the likely orientation of HIV-1 gp120 in the assem-
bled trimer, and a bridging sheet. Components of both domains
and the bridging sheet contribute to CD4 binding. CD4 binds in a
recessed pocket on HIV-1 gp120 to a surface that is larger than that
occluded by a typical antibody–protein interaction. The interface
displays several unusual features, including a shallow, water-filled
cavity that is thought to function in immune evasion. A second
interfacial cavity penetrates into the hydrophobic interior of HIV-
1 gp120; it is bounded by conserved interior HIV-1 gp120 residues
derived from all three domains and by Phe43 of CD4 (Fig. 1). Muta-
genesis, conservation and structural analysis all indicate that this
‘‘Phe43 cavity’’ and its surrounding structures are critically impor-
tant for CD4 binding. Phe43 cavity thus constitutes a conserved,
spatially localized feature in a large, otherwise relatively variable
HIV-1 gp120–CD4 interface. Consequently, the CD4–gp120 cavity
has been suggested to be a potential target for drug design.19,20
On the basis of the consideration that Phe43 cavity is highly
conserved and has been hypothesized to be a site less prone to
Table 1
Experimental pIC50, Glide Score, Emodel and predicted activity of BMS derivatives
Compound pIC50
Glide Score (XP)
(kcal/mol)
Emodel (kcal/
mol)
Predicted
activity
1
2
3
4
5
6
7
8
8.097 ꢀ7.166
6.284 ꢀ7.541
ꢀ48.269
ꢀ49.560
ꢀ52.988
ꢀ62.656
ꢀ59.619
ꢀ47.683
ꢀ54.007
ꢀ55.084
ꢀ42.554
ꢀ43.450
ꢀ40.937
ꢀ49.651
ꢀ42.636
ꢀ43.510
ꢀ42.076
ꢀ35.740
ꢀ42.413
ꢀ47.147
ꢀ45.392
ꢀ46.540
ꢀ49.274
ꢀ39.607
ꢀ37.102
ꢀ56.719
ꢀ49.364
ꢀ54.866
ꢀ56.747
ꢀ56.850
ꢀ51.035
ꢀ58.173
6.184
6.321
6.683
7.706
7.385
6.122
6.791
6.905
5.579
5.674
5.408
6.330
5.588
5.681
5.529
4.859
5.565
6.065
5.879
6.001
6.290
5.268
5.003
7.078
6.300
6.882
7.081
7.092
6.477
7.232
7.523 ꢀ7.403
7.588 ꢀ8.305
8.301 ꢀ7.552
6.066 ꢀ7.537
6.377 ꢀ6.099
6.284 ꢀ5.560
5.721 ꢀ6.308
5.638 ꢀ6.484
5.620 ꢀ7.233
5.523 ꢀ8.008
5.481 ꢀ6.758
5.041 ꢀ7.212
4.657 ꢀ7.360
4.796 ꢀ5.797
4.886 ꢀ6.924
6.367 ꢀ7.630
6.699 ꢀ6.769
6.432 ꢀ6.308
5.328 ꢀ6.768
5.372 ꢀ7.132
5.444 ꢀ6.784
6.824 ꢀ5.791
6.699 ꢀ6.339
6.301 ꢀ7.226
7.097 ꢀ7.306
6.824 ꢀ7.290
6.208 ꢀ6.802
7.444 ꢀ7.792
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Figure 1. (a) Crystal structure of HIV-1 gp120 with soluble CD4 retrieved from the
PDB (id: 1RZJ), showing Phe43 of CD4 binding with HIV-1 gp120. (b) Focused view
of Phe43 binding cavity of HIV-1 gp120, the hydrophobic region is represent in pink,
Phe43 residue of CD4 is shown in red, it shows hydrogen bond interactions with
Asp368 of HIV-1 gp120.

S. K. Sivan et al. / Bioorg. Med. Chem. 21 (2013) 4591–4599
4593
Figure 3. Structures of BMS derivatives.
resistance-conferring mutations.21 Phe43 cavity was chosen as tar-
2.1. Molecular docking
get for computational simulations, a computational protocol based
on molecular docking (induced fit docking), was applied to identify
ligands targeting Phe43 cavity.
Glide Score 5.6 XP22–25 is a harder function that exacts severe
penalties for poses that violate established physical chemistry

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S. K. Sivan et al. / Bioorg. Med. Chem. 21 (2013) 4591–4599
Fig. 3 (continued)
principles such as, charged and strongly polar groups should be
adequately exposed to solvent. This minimizes false positives and
is especially useful in lead optimization or other studies in which
only a limited number of compounds will be considered experi-
mentally and each computationally identified compound needs
to be as high in quality as possible. A combination of Glide Score,
the ligand receptor molecular mechanics interaction energy, and
the ligand strain energy is used to select the correctly docked pose.

S. K. Sivan et al. / Bioorg. Med. Chem. 21 (2013) 4591–4599
4595
Table 2
This composite scoring function, is called as Emodel, it is much bet-
ter at selecting the correct pose than, either the molecular mechan-
ics energy or Glide Score alone. A regression analysis of biological
activity (pIC50) and Glide Emodel for known inhibitor was carried
out. Correlation coefficient (r) of 0.768 and standard error of the
estimate (s) of 0.62, model equation obtained from the regression
is given below (Eq. 1), a scatter plot is shown in Figure 2. Glide
Score, Emodel and predicted activity of the molecules are given
in Table 1:
Glide Score, Emodel and predicted activity of symmetrical N,N0-disubstituted urea
and thiourea
Compound Glide Score (XP) (kcal/
mol)
Emodel (kcal/
mol)
Predicted
activity
SU1
SU2
SU3
SU4
SU5
SU6
SU7
SU8
SU9
SU10
SU11
SU12
ꢀ7.458
ꢀ6.027
ꢀ6.667
ꢀ8.992
ꢀ6.356
ꢀ7.260
ꢀ8.868
ꢀ8.163
ꢀ8.157
ꢀ8.130
ꢀ7.411
ꢀ8.698
ꢀ59.855
ꢀ50.756
ꢀ70.425
ꢀ71.903
ꢀ70.226
ꢀ71.038
ꢀ63.446
ꢀ51.133
ꢀ61.415
ꢀ61.528
ꢀ65.303
ꢀ74.100
7.409
6.447
8.528
8.685
8.507
8.593
7.789
6.487
7.575
7.587
7.986
8.917
pIC50 ¼ ðꢀ0:1058 ꢁ EmodelÞ þ 1:0773
ð1Þ
Analyses of protein–ligand interaction for the docked molecules
display a similar binding mode as that of the Phe43 of CD4. In gen-
eral, aromatic ring of benzoyl moiety linked to piperazine ring
occupies the hydrophobic cavity of Phe43. Apart from these, mol-
ecules like 11, 12 and 13 (molecules shown in Fig. 3) having pyra-
zole, benzofuran and pyridine groups were observed to occupy the
hydrophobic cavity. To have better understanding of protein–
ligand interaction and to incorporate protein flexibility during
docking run, an induced fit docking of BMS-806 (molecule 1),
BMS-043 (molecule 4) and high potent derivative of BMS-806
(molecule 5) was carried out. Schrödinger’s induced fit docking
(IFD)26–28 protocol accounts for both small backbone relaxations
in the receptor structure as well as significant side-chain confor-
mational changes. This IFD protocol has been validated on a large
set of pharmaceutically relevant examples with good results.27,28
Dock pose of BMS derivatives into the Phe43 cavity showed a
surprising match between the compound moieties and CD4 resi-
dues Ser42–Phe43–Leu44. Aromatic ring of benzoyl moiety linked
to piperazine is seated deep into the CD4 Phe43 cavity than the
phenyl ring of the CD4 Phe43. Binding pose of most potent deriv-
ative of BMS-806 (molecule 5) is shown in Figure 4, it clearly char-
acterizes that the hydrophobic interaction at this cavity plays a
major role in binding of inhibitors to gp120 receptor. A hydrogen
bond interaction is seen between NH of the indole ring and residue
Trp427. Based on these interactions it was summarized that mole-
cules with hydrophobic groups that can be accommodated within
the hydrophobic cavity and having some hydrogen bond donor
groups that can interact with the hydrophilic region at the entry
of the cavity comprising of residues Trp427, Asp368 would act as
potential inhibitors.
benzothiazole substitutions reported as antibacterial29,30 were
docked by both the docking protocols (XP and Induced Fit). Their
dock scores, Emodel and predicted activities are given in Table 2
and structures are shown in Figure 5. The dock poses obtained
for these molecules were also similar to molecule 5, the benzimid-
azole ring was seating deep into the Phe43 cavity, and the NH of
one of the benzimidazole had a hydrogen bond interaction with
Asp368. Figure 6 represents the dock pose of SU1. SU3 having a
2-phenylbenzimidazole substitution, allows it to occupy an addi-
tional outer hydrophobic cavity, that increases its binding affinity,
which is evident from its Emodel value of ꢀ70.425 kcal/mol. SU4 a
thiourea compound having 2-phenylbenzimidazole, also showed a
similar binding mode having a high Emodel value of ꢀ71.903 kcal/
mol.
This encouraged us to design new symmetrical N,N0-disubsti-
tuted urea and thioureas having different fused heterocyclic group
linked with urea or thiourea by methylene, phenyl and benzyl
linker groups (SU4–SU12), preferably that can provide flexibility
to the molecule to occupy hydrophobic cavity and also have hydro-
gen bond interaction with residues Trp427 and Asp368.
The Emodel, Glide Score, and predicted activity of these mole-
cules are given in Table 2. Newly designed molecule SU12 showed
the highest Emodel value, other molecules showed comparable
values with that of existing inhibitors. Analysis of SU12 dock pose
revealed that the heterocyclic group is occupying the hydrophobic
To validate the assumption, a set of symmetrical N,N0-disubsti-
tuted ureas and thioureas (SU1–SU3) having benzimidazole and
Figure 4. Dock pose of molecule five in the active site of HIV-1 gp120. The benzoyl group is occupying the hydrophobic cavity. The molecule shows a hydrogen bond
interaction with residue Trp427 of HIV-1 gp120.

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S. K. Sivan et al. / Bioorg. Med. Chem. 21 (2013) 4591–4599
Figure 5. Structures of symmetrical N,N0-disubstituted urea and thiourea derivatives.
cavity inside the gp120 binding site of CD4, as well as it is having a
favorable hydrophobic interaction with the residues at the entry
site of the cavity. The molecule also has hydrogen bond interaction
with residue Asp368 of HIV-1 gp120, which allows it to bind
strongly to the receptor that is evident from its Emodel and Glide-
Score values of ꢀ74.10 and ꢀ8.969 kcal/mol, respectively. These
symmetrical N,N0-disubstituted urea and thiourea molecules were
synthesized using a novel microwave assisted synthetic procedure
reported by us elsewhere and screened for their HIV-1 gp120 CD4
binding inhibitory activity.
were prepared by the literature procedure,31 where ortho-phenyl-
enediamine was condensed with aminocarboxylic acid in 5.5 M
hydrochloric acid, refluxing for 12 h–3 days. Amine hydrochloride
thus obtained was dried and reacted with urea or thiourea to yield
symmetrical substituted urea/thiourea, and their final yields are
provided in Table 3.
2.3. HIV-1 gp120–CD4 binding inhibition
Synthesized N,N0-symmetrical disubstituted urea and thio-
ureas were screened for their HIV-1 gp120–CD4 binding inhibi-
tion ability by HIV-1 gp120–CD4 capture Enzyme-linked
immunosorbent assay (ELISA). All the screened compounds were
found to inhibit HIV-1 gp120–CD4 binding in micromolar
2.2. Chemistry
The method applied for synthesis of symmetrical substituted
urea/thiourea is summarized in Scheme 1. Amine hydrochlorides
were reacted with urea or thiourea in 1:2 ratio using water med-
ium under microwave irradiation for 4 min. Amine hydrochlorides
(0.013–0.247
ues are listed in Table 4.
l
M) concentrations.32 Inhibitory activity (IC50) val-

S. K. Sivan et al. / Bioorg. Med. Chem. 21 (2013) 4591–4599
4597
5.6 was used for ligand preparation, protein preparation and in-
duced fit docking. The neutralizing antibody and CD4 chains were
deleted, except for the Ser42–Phe43–Leu44 chain that binds to the
HIV-1 gp120 cavity. Protein was prepared using protein prepara-
tion module applying the default parameters; considering Ser42–
Phe43–Leu44 as ligand moiety. A grid was generated around these
residues of CD4 with receptor van der Waals scaling for non-polar
atoms as 0.9. A set of 30 known HIV-1 gp120 inhibitors having var-
ied range of inhibition concentrations (IC50) were selected from the
literature34 these were built using Maestro build panel and pre-
pared by LigPrep application in Schrödinger 2010 suite. Molecular
docking of 30 molecules into the generated grid was performed by
using the extra precision (XP) docking mode.
Induced fit docking (IFD) protocol was run from the graphical
user interface accessible within Maestro 9.0. It was carried out
on prepared HIV-1 gp120 receptor with BMS-806, BMS-043 and
high potent derivative of BMS-806 as test ligands (molecules 1, 4
and 5, respectively). The overall procedure has four stages: Briefly,
during Stage 1 initial softened-potential Glide docking is per-
formed on a van der Waals scaled-down rigid-receptor, a scaling
of 0.7/0.5 was set for receptor/ligand van der Waals radii, respec-
tively. The top 20 poses for each test ligand was retained. In Stage
2, receptor sampling and refinement was performed on residues
within 5.0 Å of each ligand for each of the 20 ligand–protein com-
plexes, followed by Prime35,36 side-chain sampling and prediction.
The side-chains, as well as the backbone and ligand, undergo sub-
sequent energy minimizations.
Figure 6. Dock pose of SU1 in the active site of HIV-1 gp120, the molecule is
occupying the hydrophobic cavity in a similar manner as BMS derivative. It shows a
hydrogen bonding interaction with residue Asp368 of HIV-1 gp120.
3. Conclusion
A total of 20 induced fit receptor conformations were generated
for each of the test ligands. Stage 3 involved re-docking the test li-
gands into their respective 20 structures that are within 30.0 kcal/
mol of their lowest energy structure. Finally, the ligand poses were
scored in Stage 4 using a combination of Prime and Glide Score
scoring functions in which the top ranked pose for each ligand
was chosen as the final result. The XP scoring function was used
in all docking stages.
Highly improvised docking protocol was employed that can
incorporate the protein flexibility during docking calculation. The
dock pose analysis of the existing inhibitors of HIV-1 gp120 re-
vealed important binding requirements, these include the need
of a hydrophobic group that can interact with the hydrophobic cav-
ity of CD4 Phe43 and also have hydrogen bond donor group that
can interact with residues Trp427 and Asp368. These assumptions
were validated by docking of symmetrically substituted urea and
resulted in design of novel symmetrically substituted urea and
thiourea derivatives that have potentials to act as CD4 mimic and
inhibit the binding of HIV-1 gp120–CD4. These molecules were
synthesized in aqueous medium using microwave irradiation.
Study of inhibitory activity of these synthesized molecules has
been carried out to prove its experimental validity and to further
optimize the inhibitors.
4.2. General procedure for the preparation of heterocyclic
amine hydrochlorides
To a mixture of o-phenylenediamine (0.01 mol) and aminocar-
boxylic acid (0.015 mol), 20 ml of 5.5 M hydrochloric acid was
added and the reaction mixture was refluxed for 12 h–3 days at
150 °C. The reaction mixture was cooled for 12–18 h in
a
refrigerator to obtain the crystals of corresponding amine hydro-
chloride which were filtered, washed with ethanol and recrystal-
lized from methanol.
4. Experimental
4.1. Docking studies
4.3. General procedure for the synthesis of symmetrical
substituted urea
X-ray crystal structure of HIV-1 gp120 envelope glycoprotein
complexed with CD4 and induced neutralizing antibody 17b with
2.2 Å resolution was downloaded from RCSB Protein Data Bank
(http://www.rcsb.org/pdb/home/home.do) (PDB id: 1RZJ).33 GLIDE
Amine hydrochlorides (0.02 mol) were thoroughly grinded with
urea or thiourea (0.01 mol) in a borosil vessel, a paste of reaction
NH . 2HCl
2
N
O
NH2
5.5m HCl
R
NH
2
N
H
+
HO
R
Reflux
NH2
12h - 3 days
X
X
H2O, MW
R
R
N
N
NH2. 2HCl
N
NH
NH
360W (100 - 150 oC)
R
2
NH
NH
N
H
H2N
NH2
4 min
+
X = O or S
Scheme 1.

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S. K. Sivan et al. / Bioorg. Med. Chem. 21 (2013) 4591–4599
Table 3
Aminocarboxylic acids, amine hydrochlorides used for preparation of substituted urea/thiourea and obtained yields of disubstituted urea/thiourea
Compd
SU1
Aminocarboxylic acid
Amine hydrochloride
Yielda (%)
70
N
CH2NH2
2HCL
NH2
.
HOOCCH2NH2
N
H
N
NH2
NH2
HOOC
HOOC
2HCl
2HCl
.
.
SU3
SU4
SU7
85
76
80
N
H
N
NH2
N
H
N
CH2NH2
2HCL
NH2
.
HOOCCH2NH2
N
H
HOOCH2C
NH2
NH2
2HCl
2HCl
.
.
NH
SU9
60
50
N
N
HOOCH2C
NH2
NH
SU10
a
Yields refer to isolated and chromatographically pure products.
Table 4
d 7.74–7.77 (dd, 4H, J1 = J2 = 2.8 Hz), d 10.62 (s, 2H). IR (KBr) band
at 3298, 1629 cmꢀ1 corresponding to NH and C@O stretching of
amide group. LC–MS: m/z (%) = 445 (M+1, 100).
HIV-1 gp120–CD4 binding inhibition (IC50 values) for synthesized substituted urea
and thiourea
S. No.
Compound
IC50
lM
SD
4.3.3. N,N0-Bis[4-(1H-benzimidazol-2-yl)phenyl]thiourea (SU4)
1H NMR (DMSO-d6, 400 MHz) d 7.09–7.15 (complex peak), d
12.50 (s, 2H); 13C NMR (DMSO-d6, 125 MHz) d = 109.42, 121.93,
132.07, 167.70 IR (KBr) band at 3153 and 1514 cmꢀ1 correspond-
ing to NH and C@S stretching of thiocarbamide group. LC–MS: m/
z (%) = 461 (M+1, 100).
1
2
3
4
5
6
SU1
SU3
SU4
SU7
SU9
SU10
0.247 0.048
0.188 0.036
0.126 0.12
0.013 0.1
0.197 0.0032
0.149 0.0073
4.3.4. N,N0-Bis(1H-benzimidazol-2-ylmethyl)thiourea (SU7)
1H NMR (DMSO-d6, 400 MHz) d 5.51–5.52 (d, 4H, J = 4.8 Hz), d
8.27–8.30 (t, 2H, J1 = 6 Hz, J2 = 5.2 Hz), d 8.46–8.51 (m, 4H), d
8.58–8.60 (d, 4H, J = 8.4 Hz). IR (KBr) band at 3319 cmꢀ1 Corre-
sponding to NH stretching and 1531 cmꢀ1 corresponding to C@S
stretching, LC–MS: m/z (%) = 337 (M+1, 100), HRMS (ESI), m/z calcd
mixture was made by adding few drops of water. Then the vessel
was placed in synthetic microwave oven (Catalyst Cata 2R model)
at 360 W power (100–150 °C) until the mixture was dry. Product
was washed with water to remove any unreacted urea or amine
hydrochloride. Solid was recrystallised from 1:1 methanol–water
purified by column chromatography. Structures were confirmed
by 1H NMR, 13C NMR, IR, LC–MS and HRMS. 1H NMR spectra were
recorded in DMSO-d6 on Bruker (Bio-spin) Ultrashield Arance-III
Nano Bay 400 MHz NMR spectrometer and 13C NMR spectra were
recorded in DMSO-d6 on Avance 500 (125 MHz) spectrometer
using SiMe4 as internal standard. IR spectra were obtained on Ten-
sor-27 (Bruker-optics) FTIR spectrophotometer using KBr disc. LC–
MS were obtained on SHIMADZU 2010A. HRMS were obtained on
Thermo Scientific LTQ Orbitrap.
for
C17H16N6S (M+55(Na–CH3OH adduct)) 391.1622, found
391.1657.
4.3.5. N,N0-Bis[4-(1H-benzimidazol-2-ylmethyl)phenyl]urea
(SU9)
1H NMR (DMSO-d6, 400 MHz) d 4.10 (s, 4H), d 7.12–7.13 (d, 4H,
J = 3.2 Hz), d 7.22–7.24 (d, 4H, J = 8 Hz), d 7.37–7.39 (d, 4H,
J = 7.6 Hz), d 7.46–7.47 (d, 4H, J = 3.2 Hz), d 8.64 (s, 2H). 13C NMR
(DMSO-d6, 125 MHz) d = 33.55, 117.66, 120.84, 128.52, 130,
137.66, 151.88, 153.18. IR (KBr) band at 3601 and 1646 cmꢀ1
corresponding to NH and C@O stretching of amide group. LC–MS:
m/z (%) = 473 (M+1, 100), HRMS (ESI), m/z calcd for C29H24N6O
(MH+) 473.2092, found 473.2071.
4.3.1. N,N0-Bis(1H-benzimidazol-2-ylmethyl)urea (SU1)
1H NMR (DMSO-d6, 400 MHz) d 4.59–4.61 (d, 4H, J = 4.4 Hz), d
7.25 (s, 2H), d 7.33–7.34 (d, 4H), d 7.64–7.65 (d, 4H). IR (KBr) band
at 3361 and 1646 cmꢀ1 corresponding to NH and C@O stretching of
amide group.
4.3.6. N,N0-Bis[4-(1H-benzimidazol-2-ylmethyl)phenyl]thiourea
(SU10)
1H NMR (DMSO-d6, 400 MHz) d 4.38 (s, 4H), d 7.35–7.37 (d, 4H,
J = 8 Hz), d 7.38–7.41 (q, 4H, J = 4 Hz), d 7.52–7.54 (d, 4H, J = 8 Hz), d
7.66–7.69 (q, 4H, J = 4 Hz), d 10.19 (s, 2H). 13C NMR (DMSO-d6,
4.3.2. N,N0-Bis[4-(1H-benzimidazol-2-yl)phenyl]urea (SU3)
1H NMR (DMSO-d6, 400 MHz) d 6.60–6.61 (d, 4H, J = 2.4 Hz), d
7.45–7.49 (q, 4H, J1 = J2 = 6 Hz), d 7. 69–7.72 (q, 4H, J1 = J2 = 6 Hz),

S. K. Sivan et al. / Bioorg. Med. Chem. 21 (2013) 4591–4599
4599
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We gratefully acknowledge support for this research from DST
(SR/FT/CS-040/2009), CSIR (01/(2436)/10/EMR-II) and UGC, New
Delhi, India. We are thankful to Department of chemistry, Nizam
College, Hyderabad, India where the research was carried out.
We are grateful to Dr. M. Rupalatha, Duke University Medical Cen-
tre, North Carolina, for her timely help for carrying out HIV-1
gp120–CD4 binding assay for our compounds. We also acknowl-
edge Schrödinger Inc. for GLIDE software, Tripos Inc. for SYBYLX-
1.2. We are thankful to CFRD, Osmania University and IICT, Hyder-
abad for providing spectral data. We do not have any conflict of
interest to declare.
33. Huanga, C. C.; Venturi, M.; Majeed, S.; Moore, M. J.; Phogat, S.; Zhang, M.
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Supplementary data
Supplementary data associated with this article can be found, in
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