Design and synthesis of caffeoyl-anilides as portmanteau inhibitors of HIV-1 integrase and CCR5

Article abstract of DOI:10.1016/j.bmc.2010.12.031

Designing multi-functional ligands is a recent strategy by which multiple targets can be inhibited by a single entity. A series of caffeoyl-anilide compounds based on structures of various integrase and CCR-5 inhibitors have been designed and synthesized

Full text of DOI:10.1016/j.bmc.2010.12.031

Bioorganic & Medicinal Chemistry 19 (2011) 1256–1263
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of caffeoyl-anilides as portmanteau inhibitors
of HIV-1 integrase and CCR5
Hardik S. Bodiwala a, , Sudeep Sabde b, , Pawan Gupta c, Ruchira Mukherjee b, Rajender Kumar d,
a,
Prabha Garg d, Kamlesh Kumar Bhutani a, Debashis Mitra b, , Inder Pal Singh
⇑
⇑
a Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar, Punjab 160 062, India
b National Center for Cell Science (NCCS), Pune University Campus, Ganeshkhind, Pune 411 007, Maharashtra, India
c Center for Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar, Punjab 160 062, India
d Computer Center, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar, Punjab 160 062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Designing multi-functional ligands is a recent strategy by which multiple targets can be inhibited by a
single entity. A series of caffeoyl-anilide compounds based on structures of various integrase and CCR-
5 inhibitors have been designed and synthesized as anti-HIV agents in the present study. Most of the
compounds exhibited potent anti-HIV activity at micromolar concentration in CEM-GFP CD4+ T cells
infected with HIV-1NL4.3 virus. Compound 14 showed a lower EC50 and better TI as compared to AZT.
Mechanism based studies suggest that these compounds inhibit either one or in some cases, both the tar-
gets. The experimental data and the docking results showed that these compounds are potential inhibi-
tors for both HIV-1 IN and CCR5.
Received 23 September 2010
Revised 26 November 2010
Accepted 13 December 2010
Available online 21 December 2010
Keywords:
Caffeoyl-anilide
Anti-HIV
Integrase inhibitor
CCR5 inhibitor
Portmanteau inhibitor
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
multiple targets can be inhibited or blocked using a single entity.
This improves patient compliance and reduces chances of drug
Human immunodeficiency virus-1 (HIV-1) is the causative
agent of the acquired immunodeficiency syndrome (AIDS). Glob-
ally, an estimated 33 million people were living with HIV in
2008.1 Almost all FDA approved drugs are either reverse transcrip-
tase (RT) or protease (PR) inhibitors. Recently approved drugs for
anti-HIV therapy are Maraviroc (entry inhibitor) and Raltegravir
(integrase (IN) inhibitor).2 Currently, highly active antiretroviral
therapy (HAART) comprising of a HIV RT with PR or IN inhibitors
successfully suppresses HIV viral load to an undetectable level.
However, the effect of this therapy is compromised by emergence
of resistant HIV strains.
There has been a recent trend of formulations whereby two or
more drugs are combined in a single tablet or capsule to make dos-
ing regimen simpler, thereby improving patient compliance. Cer-
tain physico-chemical risks like drug-drug and drug-excipient
compatibility are associated with these formulations. Designing
multiple ligand is an upcoming drug discovery strategy, where
resistance. The exploration of such multifunctional ligands has
proven valuable for anti-HIV lead discovery.3 Earlier, Vince et al.
have proposed the term portmanteau inhibitors for molecules that
have two scaffolds merged into one entity and act on different sites
or phases of viral life cycle.4
In an ongoing effort to develop novel anti-HIV molecules,5,6 we
planned to synthesize portmanteau inhibitors of HIV-1 integrase
and CCR5. The purpose of such design was to block the virus at
two different stages of life cycle. HIV-1 pol gene encodes three
essential enzymes viz. RT, IN and PR. The integrase, which is an
intracellular target for HIV, mediates two critical reactions during
viral replication. It removes two nucleotides from 30 end of both
cDNA strands. The host genomic DNA is cleaved and processed
cDNA strands are joined with host DNA. FDA approved integrase
inhibitors block this strand transfer reaction.7 The CCR5 is an extra-
cellular target on the host cell surface and an important mediator
of R5 virus entry. Thus, CCR5 is the target at the entry level, when
virus attaches to the host cell surface and the integrase, when viral
DNA gets integrated into the genome of host cell.
⇑
Corresponding authors. Tel.: +91 20 25708151 (D.M.); tel.: +91 172
Catechol moiety is a significant structural component in many
potent HIV integrase inhibitors such as caffeic acid phenylethyl es-
ter (CAPE, 1) and chicoric acid (CA, 2).8 CA exhibited IC50 values of
2214683x2144; fax: +91 172 2214692 (I.P.S.).
E-mail addresses: dmitra@nccs.res.in (D. Mitra), ipsingh@niper.ac.in, ipsingh67
@yahoo.com (I.P. Singh).
0.1–0.5
0.1–0.2
l
l
M for the end-processing/strand transfer reactions and
Both H.S.B. and S.S. contributed equally to the work. NIPER Communication No.
476.
M for the disintegration reactions.9 Catechol containing
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.031

H. S. Bodiwala et al. / Bioorg. Med. Chem. 19 (2011) 1256–1263
1257
inhibitors were proven toxic, perhaps because of nonspecific bind-
O
O
O
NH2
ing to other targets or due to the formation of oxidized species,
such as semiquinones or orthoquinones.10 Although it was shown
in 2000 that chicoric acid inhibits HIV replication by non-specific
gp120 interactions,11 however, Ho et al. have reported in 2005 that
CAPE and its analogues acted through distinctly different mecha-
nism. The compounds were considered safe as no mortality was
observed in experimental mice. It was also concluded that CAPE
like compounds are worth studying further as potential chemo-
therapeutic agents in HIV infection.12 Many other recent papers
indicate that CAPE and CA derivatives act through inhibition of
HIV integrase.13–15 Previous studies on SAR of CA indicated that
at least one carboxylic acid moiety and bisphenol are necessary
for anti-HIV activity. The expected cleavage of its ester linkage
by hydrolytic enzymes and the relatively large number of rotatable
bonds are limiting steps in its oral bioavailability. It was suggested
that optimization of the lead molecule may be achieved by substi-
tuting the ester groups by more robust linkages, like amide or ether
and preventing free rotation around single bonds by synthesizing
conformationally constrained analogs.9
i
HO
HO
5
6
NH2
7
HO
HO
CHO
ii
HO
HO
O
N
H
R
Scheme 1. Reagents and conditions: (i) CH3SO3H, neat, rt, 5 min; (ii) RCOCl, THF, rt,
4 h.
(ꢀ10%). By using 3 equiv of acid the desired product 7 was ob-
tained in a yield of 45–48%.
Various methods for synthesis of chalcones reported in the liter-
ature involve protection and deprotection of hydroxyl groups.23,24
Further, the acid catalyzed reaction for 7 required refluxing for 8 h.
Therefore, in order to improve the reaction conditions, we modified
the acid catalyzed coupling using methane sulfonic acid (MSA),
which is a clear, colorless liquid with high acid strength (pKa = ꢁ1.9).
It is less aggressive than H2SO4, easy to use and often recyclable. It is
used widely as a catalyst in several reactions (esterification, alkyl-
ation) or as catalyst/solvent for condensation or rearrangement
reactions.25
Higher yield of chalcone 7 was obtained, when reaction was
performed in MSA at RT for a short time of 5 min. Reaction per-
formed in various organic solvents such as, MeOH, EtOH, ACN,
THF etc. did not give the desired product in good yield. The present
method is advantageous over existing acid catalyzed methods for
synthesis of chalcones as (i) it does not require protection and
deprotection of phenolic hydroxyl groups, (ii) uses cheap, easy to
handle and commercially available catalyst (solvent), (iii) room
temperature and non-anhydrous reaction conditions, and (iv)
shorter reaction times. Further, 7 was dissolved in THF and various
acid chlorides were added to the reaction mixture to obtain the fi-
nal products as shown in Figure 2.
TAK-779 (3) was reported as the first small molecule CCR5
antagonist with an IC50 of 1.4 nM.16 Thereafter, various groups
have exploited anilides as CCR5 antagonists as reported compre-
hensively by Kazmierski et al.17 SAR study of 3 suggested that a
proper shape of molecule, suitable bulkiness and location of qua-
ternary ammonium moiety is essential for optimal activity.16 In
further studies, 3 exhibited poor oral absorption because of quater-
nary ammonium moiety.18 Smithkline Beecham Corp. disclosed
various benzanilides as antagonists of receptor. The benzanilides
(4 as a representative) have been claimed for treatment of various
diseases including HIV by CCR5 antagonism with an IC50 value
ranging from 0.0001 to 100 l
M.19–21
Based on these reports, we proposed to synthesize and evaluate
caffeoyl-anilide compounds as anti-HIV agents (Fig. 1) incorporat-
ing caffeoyl moiety of 1 and 2 and anilide moiety of 3 and 4 in the
present study. We synthesized molecules in which caffeoyl unit
was kept intact and anilide unit was modified with different aro-
matic and aliphatic substituents to study the changes in anti-HIV
activity. In this paper, we describe synthesis, mechanism based
(CCR5 and IN) anti-HIV activity and molecular modeling study of
newly designed caffeoyl-anilide molecules.
Intermediate (7) and all synthesized compounds (8–21) were
identified by Mass, IR, 1H, and 13C NMR spectral data. All com-
pounds showed a typical 1H-1H coupling constant of 15 Hz show-
ing trans stereochemistry of double bond.
2. Results and discussion
2.1. Chemistry
2.2. Biology
The target compounds were synthesized as shown in Scheme 1.
Commercially available 4-aminoacetophenone (5) was reacted
with 3,4-dihydroxybenzaldehyde (6) in presence of catalytic
amount of H2SO4 in MeOH under reflux for 8 h.22 However, the
yield of the desired product under these conditions was low
To measure the effect of synthesized caffeoyl-anilide com-
pounds, these were tested against HIV-1 in a cell based assay
where virus infected cells were incubated for 7 days with com-
pounds. The anti-HIV activity assay of compounds was carried
out in CEM-GFP T cells infected with HIV-1NL4.3 virus. CEM-GFP
O
HO
O
N
O
HO
1
O
O
Cl
N
H
HO
HO
HO
HO
O
3
O
O
O
N
H
R
O
O
CO2H
CO2H
N
O
N
H
Caffeoyl-Anilides
O
HO
HO
4
2
Figure 1. Design of portmanteau inhibitors based on IN inhibitors (1 and 2) and CCR5 antagonists (3 and 4).

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H. S. Bodiwala et al. / Bioorg. Med. Chem. 19 (2011) 1256–1263
Table 1
O
Anti-HIV activity of caffeoyl-anilide compounds in HIV-1NL4.3 infected CEM-GFP T
cells
HO
HO
O
N
H
R
F
Compound Concda
(l
M) % Inhibitionb EC50
(l
M) CC50 (l
M) TIe
c
d
R =
7
8
9
3.9
1.7
1.5
1.4
6.0
2.9
2.7
2.6
2.8
2.4
6.4
6.3
2.5
2.5
2.2
5.0
0
0
0
0
ND
ND
ND
ND
4.2 0.1
ND
ND
ND
ND
ND
13.5 0.7
ND
ND
ND
ND
ND
3.2
ND
8
9
16
17
18
-CH3
-CH(CH3)2
10
11
12
13
14
15
16
17
18
19
20
21
AZT
10 -CH2CH(CH3)2
86.1 1.2
24.2 0.9
0
71.6 1.4
73.0 2.1
0
92.4 2.7
16.0 1.1
77.6 3.2
83.2 2.1
10.9 0.8
89.8 7.1
O
11
ND
ND
ND
0.9 0.0
1.6 0.0
ND
1.9 0.0
ND
1.6 0.0
1.8 0.0
ND
51.4 0.2
20.3 0.3
ND
32.3 0.5
ND
15.7 0.5
18.6 0.1
ND
59.1
13.0
ND
17.4
ND
9.8
10.6
ND
O
12
F
F
19
20
S
13
F
F
S
14
F
F
1.1 0.0
24.1 0.6
22.9
21
15
ND = Not determined.
a
Highest noncytotoxic concentration.
% Decrease in p24 antigen at highest noncytotoxic concentration.
EC50 = concentration of compound required to achieve 50% inhibition of virus
b
c
CF3
Figure 2. Structures of caffeoyl-anilides synthesized for anti-HIV activity.
production from HIV-1 infected cells (n = 3).
d
CC50 = concentration of compound required for 50% cytotoxicity against unin-
fected cells (n = 3).
is a human CD4+ reporter T cell line, which expresses GFP upon
HIV infection due to transactivation by Tat protein of stably inte-
grated long terminal repeat regulated GFP gene.26 This cell line is
widely used for determination of anti-HIV activity due to easy
visualization of infected cells.27 HIV-1 NL4.3 is available as a
molecular clone from NIH AIDS Repository and is routinely used
as a representative isolate of HIV-1. Although it is known to be
X4 virus, it has been reported to infect monocytic cell lines like
THP1 and U937. Because NL4.3 was used in screening studies, for
identification of the mechanism of entry, we used a different X4
isolate and thus IIIB was used as a X4 isolate and IndieC was used
as a R5 isolate.
All synthesized compounds were tested for their cytotoxicity in
MTT assay for 48 h before testing for cell based anti-HIV activity.
Based on the results of MTT assay, initially all the synthesized
derivatives were tested for anti-HIV activity at highest non-toxic
concentration. Six out of the 15 compounds showed more than
70% inhibition of HIV-1 replication in CEM-GFP cells at less then
e
TI = (CC50/EC50).
Table 2
HIV-1 integrase inhibition activity using in vitro integrase assay
Compound
Concd (
l
g/mL)
% Inhibition
IC50 (lM)
11
14
15
17
19
20
20
20
20
20
20
20
5%
67.7
66.5
56.0
66.8
74.0
69.7
98.8
36.8 2.0
44.6 1.4
50.2 1.7
25.2 1.9
28.5 0.9
39.3 1.0
1.2 0.07%
Sodium azide
Table 3
Inhibition of HIV-1 entry through CCR5 or CXCR4 co-receptor as analyzed in TZM-bl
cells using R5 and X4 tropic virus isolates
Compound Concda
M)
% Inhibition with R5
tropic virus
% Inhibition with X4
tropic virus
6 lM concentration. These six compounds were then analyzed fur-
(l
ther for determination of EC50 and CC50
.
28 The results of activity are
11
14
15
17
19
20
6.0
2.6
2.8
5.1
2.5
2.5
59.6 0.4
52.9 4.7
58.6 3.6
55.8 10.8
71.5 4.4
73.4 0.3
0
0
shown in Table 1.
The six active compounds were further tested in vitro against
HIV-1 integrase and for HIV-1 entry inhibitory activity in TZM-bl
cells. For the entry inhibition assay, one CCR5 tropic (IndieC) and
one CXCR4 tropic (IIIB) virus was used to identify the mechanism
of inhibition. All active compounds showed potent integrase inhib-
itory activity (Table 2). The entry inhibition assay in TZM-bl cells
clearly indicated that the active compounds potently inhibited
the entry of CCR5 utilizing virus isolate but did not inhibit the en-
try of CXCR4 utilizing virus isolate (Table 3). None of the com-
pound was active against HIV-1 RT (data not shown) suggesting
that the designed compounds have specificity towards the desig-
nated targets.
27.8 0.6
0
0
44.7 0.8
a
Highest noncytotoxic concentration.
11 having bulkier adamantyl substitution was active in the anti
HIV assay. It showed moderate anti HIV IN activity with an IC50
of 36.8 lM and inhibited virus entry through CCR5.
Out of five-membered hetroaromatic anilides with O and S as
heteroatom (12–14), compound 13 having 2-thiophene moiety
was inactive. Interestingly compound 14 having thiophene-2-acyl
moiety was found to be the most active compound in cell based as-
2.3. SAR
To study the structure–activity relationship, small aliphatic
groups like acetyl, isobutyryl, isovaleryl, bulkier aliphatic group
like adamantyl, five-member hetroaromatic groups with O and S
as hetroatoms, naphthyl, phenyl and mono- and di-substituted
phenyl groups were introduced.
say with IC50 of 0.9
length might be important for anti HIV activity. It showed 53%
inhibition of virus entry at 2.6 M concentration.
Three benzanilides (15–17) similar to 4 were synthesized. As
expected, caffeoyl-benzanilide derivatives 15 and 17 were found
lM and TI of 59.1. This suggested that chain
l
Compounds 8–10 which were devoid of aromatic benzanilide
moiety were found to be inactive for anti HIV activity. Compound
to show good anti HIV activity with IC50 of 1.6 and 1.9
tively. In the IN assay, 17 was found be more active (IC50 of
lM, respec-

H. S. Bodiwala et al. / Bioorg. Med. Chem. 19 (2011) 1256–1263
1259
25.2
lM) than 15 (IC50 of 50.2 lM). However, in CCR5 assay, 15
was more active than 17.
In order to explore the effect of substitution pattern on the phe-
nyl ring, electron withdrawing groups like F and CF3 were intro-
duced (18–21). Compounds having 2,4-disubstitution (18 and 21)
were inactive. Compounds having 3,4- and 3,5-disubstitution (19
and 20) were found to be active as anti HIV agents with IC50 of
1.6 and 1.8
pounds inhibited entry of R5 tropic virus at a concentration of
2.53 M. Compound 19 was more active than 20 in HIV-1 IN assay
(IC50 of 28.5 M Vs 39.3 M). The above observations suggest that
lM and TI of 9.8 and 10.6, respectively. Both these com-
l
l
l
substitution at 3, 4 and 5-positions of phenyl ring of anilide part
might be important for anti HIV activity. Compounds with ada-
mantyl group and phenyl group showed better activity than the
others.
2.4. Molecular modeling
The main objective of docking studies is to explore the binding
mode of these compounds against active site of HIV-1 IN and CCR5
proteins. Several computational docking studies using the crystal
structure of the IN core domain and inhibitors have been re-
ported.29–35 The docking study of raltegravir (marketed drug)
was performed in to the active site of HIV-1 IN. It was found that
raltegravir docked near to Mg2+ ion (Fig. 3A) and keto-enol moie-
ties were coordinated by Mg2+ ion (Table S1 in Supplementary data
for coordination distance). This coordination is required for chela-
tion of the metal ion.30,36,37 The H-bonding interaction and cation–
G interaction exhibited with Asn155 and Mg2+, respectively. This
docking result well supported by published docking results of ral-
tegravir.38 Similarly, synthesized compounds were also docked in
to the same active site of HIV-1 IN. All HIV-1 IN active compounds
also occupied same space near to Mg2+ ion as raltegravir (Fig. 3B for
best bound conformation of inhibitors into the active site of HIV-1
IN). Similarly, the keto group of caffeoyl moiety was also coordi-
nated by Mg2+ ion (Table S1 in Supplementary data for coordina-
tion distance). The reported data also showed similar type of
interaction between metal ion of HIV-1 IN and highly electronega-
tive atoms of most of the HIV-1 IN inhibitors.29,36,39,40 The catechol
moieties of all compounds were placed near to Lys159. This residue
Figure 3B. Docking pose of compounds 11 (magenta), 14 (cyan), 15 (yellow), 17
(orange), 19 (blue) and 20 (purple) into the active site residues of HIV-1 IN. Green
sticks represent active site residues around the inhibitors, magenta ball-magnesium
ion.
established cation-G and H-bonding interactions with all the
compounds. Additionally, ring B of compound 14 also exhibited
cation–G interaction with Mg2+ (Table S1 and Figs. S1–S6 in
Supplementary data). The interactions with Lys159 have prime
importance in binding of inhibitors into the active site of HIV-1
IN.34 All the compounds consistently exhibited H-bonding with
residues Thr66, His67, Gln148, and Lys159 (Table S1 and Figs.
S1–S6). The ring A of inhibitors is placed in a hydrophobic cavity
formed by active site residues Cys65, Thr66, His67, Asn155,
Lys156 and Lys159, ring C was found near to residues Gln62,
Leu63, His114, Thr115, Gln148, and Ile151 that exhibited hydro-
phobic interactions. Series of interactions in docking studied
strengthen the binding of inhibitors to the active site of HIV-1 IN.
Docking studies were also performed on CCR5 modelled protein
using AUTODOCK 4.2 program. The putative binding modes of differ-
ent compounds were published in last few years using modelled
CCR5 protein. Based on published results, the active site residues
were considered during docking studies.41–44 The binding site of
CCR5 is hydrophobic in nature due to multiple aromatic residues
present in pocket. This property imparted tight binding to the
inhibitors.45 Figure 4 shows best docked conformation of inhibitors
into the active site of CCR5. Most of the compounds consistently
exhibited H - bonding interaction with backbone carbonyl of
Ser160 and amino hydrogen of Lys197 (Table S1). The interaction
with Lys197 was supported by literature study on site directed
mutagenesis.42 Interestingly, the aromatic side chain of Phe245
interacted via G–G stacking with aromatic rings (see Table S1
and Figs. S7–S12 for G–G interaction in Supplementary data) of
all the inhibitors except compound 11 which only exhibited G–G
stacking with Phe109 and Trp248. Additionally, other compounds
17 and 20 also exhibited G–G stacking with aromatic side chain
of Phe109 and Trp248. The ring B of all inhibitors interacted with
Lys197 (–NH3+) via cation–G interaction (Table S1). Altogether,
hydrophobic nature of CCR5 active site and multiple ring system
of inhibitors imparted hydrophobic interactions with active site
residues (Phe109, Phe112, Gly163, Thr167, Ser169, Ser180,
Phe182, Lys197, Ile200, Leu203, Leu207, Phe245, Leu246, Trp248,
Ala249, Tyr251 and Asn252). The compound 11, unlike other com-
pounds did not superimpose into the active site. (Fig. 4 for com-
pound 11 in magenta color). Low inhibitory activity of compound
Figure 3A. Docking pose of Raltegravir into the active site residues of HIV-1 IN.
Green sticks represent active site residues around the inhibitor, magenta ball-
magnesium ion.

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H. S. Bodiwala et al. / Bioorg. Med. Chem. 19 (2011) 1256–1263
reactions and extractions involved the use of a rotatory evaporator
(Buchi, Switzerland) operating at a reduced pressure. HPLC analy-
ses were performed using Shimadzu HPLC system with a PDA
detector on Phenomenex Luna C18 column (250 ꢂ 4.6 mm, 5
l)
with a mixture of solvents A and B (condition 1: A/B = water/aceto-
nitrile 30:70, flow rate 1.0 mL/min; condition 2: A/B = 0.2% acetic
acid in water/acetonitrile 50:50, flow rate 1.0 mL/min). All
compounds showed >95% purity by HPLC.
4.1.2. Procedure for synthesis of (E)-3-(4-aminophenyl)-1-(3,4-
dihydroxyphenyl)prop-2-en-1-one (7)
4-Aminoacetophenone (5, 500 mg, 3.70 mmol) was allowed to
stir with 3,4-dihydroxybenzaldehyde (6, 391 mg, 2.84 mmol) in
MSA (1.42 mL, 14.85 mmol) for 5 min at room temperature. The
reaction mixture was diluted with water and extracted with EtOAc.
The organic layer was washed with brine and dried over anhydrous
sodium sulfate. Solvent was removed under vacuum and crude
product was purified by column chromatography to yield 7. Yield:
60%; yellow solid; mp 184–185 °C; IR (Neat) mmax 3328, 2922,
Figure 4. Docking pose of compounds 11 (magenta), 14 (cyan), 15 (yellow), 17
(orange), 19 (blue) and 20 (purple) into the active site of modelled CCR5. Green
sticks represent active site residues around the inhibitors.
; d 7.88 (d, 2H,
1646, 1598, 1552 cmꢁ1 1H NMR (CD3OD)
J = 8.2 Hz), 7.60 (d, 1H, J = 15.4 Hz), 7.48 (d, 1H, J = 15.4 Hz), 7.16
(d, 1H, J = 2.1 Hz), 7.07 (d, 1H, J = 8.2 Hz), 6.81 (d, 1H, J = 8.1 Hz),
6.68 (d, 2H, J = 8.6 Hz); 13C NMR (CD3OD) d 190.8, 155.7, 149.9,
147.2, 145.6, 132.7, 129.1, 128.2, 123.6, 120.2, 117.0, 116.0,
114.7; MS (ESI) m/z 256 [M+1]+; HRMS (ESI) m/z calcd for
11 is reinforced by different orientation into active site of CCR5
with low binding energy. All the compounds followed the same
trend in docking studies with good binding score that was sup-
ported by experimental inhibitory activity (Table S1).
C
15H13NNaO3 [M+Na]+: 278.0793, found: 278.0794.
3. Conclusion
4.1.3. General procedure for synthesis of 8–21
To the solution of 7 in THF, various acid chlorides (1.0 equiv)
were added. Reaction mixture was allowed to stir for 4 h at room
temperature. Solvent was evaporated under vacuum and crude
product was washed with n-hexane to yield 8–21 in 74–97% yield.
In conclusion, we have synthesized caffeoyl-anilides as port-
manteau inhibitors of HIV-1 IN and cellular CCR5. The intermediate
4-amino-30,40-dihydroxychalcone was prepared by MSA in good
yield in very short time. Compounds 11, 14, 15, 17, 19 and 20 were
found to be active in cell based anti-HIV assay with EC50 values of
4.1.3.1.(E)-N-(4-(3-(3,4-Dihydroxyphenyl)acryloyl)phenyl)-acet-
amide (8). 77%; Olive green solid; mp >280 °C; IR (Neat) mmax
4.2, 0.9, 1.6, 1.8, 1.6 and 1.8 lM, respectively. The mechanism
based study suggested that synthesized compounds inhibited the
entry of CCR5 tropic viruses and also the integration step of the
virus life cycle. Thus, the anti-HIV activity of these inhibitors might
be due to specificity towards both targets. The molecular modeling
studies suggested that predicted conformations and especially
their predominant interactions with (HIV-1 IN; Thr66, His67,
3441, 3357, 1653, 1598, 1529, 1441, 1277 cmꢁ1 1H NMR (CD3OD)
;
d 7.94 (d, 2H, J = 8.4 Hz), 7.64 (d, 2H, J = 8.4 Hz), 7.57 (d, 1H,
J = 15.4 Hz), 7.41 (d, 1H, J = 15.4 Hz), 7.10 (s, 1H), 7.01 (d, 1H,
J = 8.1 Hz), 6.72 (d, 2H, J = 8.6 Hz), 2.0 (s, 3H); 13C NMR (CD3OD) d
189.8, 170.6, 148.6, 145.5, 145.4, 143.0, 133.4, 129.3, 127.0,
122.2, 118.8, 118.7, 118.0, 115.2, 114.3, 22.6; MS (APCI) m/z 298
[M+1]+; HRMS (ESI) m/z calcd for C17H15NNaO4 [M+Na]+:
320.0899, found: 320.0901.
Gln148, Lys159 and Mg2+ CCR5; Ser160, Lys197 and Phe245) ac-
,
tive site amino acid residues have explained the inhibitory profile
of these compounds for both HIV-1 IN and CCR5.
The major significance of this work as compared to our earlier
reports5,6 is that the present compounds inhibit the HIV-1 virus
by two distinct mechanisms, integrase and entry inhibition as
compared to only rt inhibition in case of previous reports. Because
of this, it is expected that generation of drug resistant viruses will
be reduced with these new dual inhibitors.
4.1.3.2. (E)-N-(4-(3-(3,4-Dihydroxyphenyl)acryloyl)phenyl)-iso-
butyramide (9). 75%; Dark brown oil; IR (KBr disc) mmax 3323,
1646, 1593, 1521 cmꢁ1
; d 8.03 (d, 2H,
1H NMR (CD3OD)
J = 7.2 Hz), 7.76 (d, 2H, J = 6.9 Hz), 7.67 (d, 1H, J = 15.4 Hz), 7.50
(d, 1H, J = 15.2 Hz), 7.18 (s, 1H), 7.10 (d, 1H, J = 6.4 Hz), 6.82 (d,
1H, J = 6.5 Hz), 2.70-2.63 (m, 1H), 1.21 (d, 6H, J = 5.8 Hz); 13C
NMR (CD3OD) d 191.2, 179.0, 150.0, 146.9, 146.9, 146.7, 144.7,
134.8, 131.6, 130.8, 128.8, 128.4, 123.7, 123.0, 120.4, 119.4,
119.2, 116.7, 115.8, 37.1, 19.8; MS (APCI) m/z 326 [M+1]+; HRMS
4. Experimental
4.1. Chemistry
(ESI) m/z calcd for
C
19H19NNaO4 [M+Na]+: 348.1212, found:
348.1214.
4.1.1. General
1H and 13C NMR spectra were recorded on 400 MHz Bruker
FT-NMR (Avance II 400) spectrometer using tetramethylsilane as
an internal standard and the chemical shifts are reported in d units.
Mass spectra were recorded on either CIMS (LtQ, Thermo, USA) or
MALDI MS (Bruker, USA). HRMS were recorded on Bruker Maxis.
Merck silica gel 60 F254 plates were used for TLC. Developed plates
were visualized by UV light. All chemicals were purchased from
Sigma-Aldrich. Solvents used for the chemical synthesis purchased
from commercial sources were of analytical grade and were used
without further purification. Concentration of solutions after
4.1.3.3.
methylbutanamide (10). 78%; Dark brown oil; IR (Neat) mmax
3516, 3309, 1665, 1592, 1564, 1524 cmꢁ1 1H NMR (CD3OD) d
8.05 (d, 2H, J = 8.4 Hz), 7.76 (d, 2H, J = 8.4 Hz), 7.68 (d, 1H,
J = 15.0 Hz), 7.52 (d, 1H, J = 15.4 Hz), 7.20 (s, 1H), 7.11 (d, 1H,
J = 8.2 Hz), 6.83 (d, 1H, J = 8.2 Hz), 2.29 (d, 2H, J = 7.1 Hz), 2.18
(sept, 1H, J = 6.7 Hz), 1.02 (d, 6H, J = 6.4 Hz); 13C NMR (CD3OD) d
189.8, 173.0, 148.6, 145.5, 145.5, 143.0, 133.5, 129.4, 127.0,
122.3, 119.0, 118.0, 115.2, 114.3, 45.9, 26.1, 21.3; MS (APCI) m/z
(E)-N-(4-(3-(3,4-Dihydroxyphenyl)acryloyl)phenyl)-3-
;

H. S. Bodiwala et al. / Bioorg. Med. Chem. 19 (2011) 1256–1263
1261
341 [M+1]+; HRMS (ESI) m/z calcd for C20H21NNaO4 [M+Na]+:
362.1368, found: 362.1362.
7.18 (d, 1H, J = 8.0 Hz), 6.81 (d, 1H, J = 8.0 Hz); 13C NMR (DMSO-
d6) d 187.9, 166.5, 149.1, 146.0, 144.8, 143.9, 134.8, 133.5, 132.4,
132.3, 130.0, 129.5, 128.7, 128.6, 126.2, 127.4, 126.8, 124.9,
120.0, 118.8, 116.2, 116.0; MS (ESI) m/z 432 [M+Na]+; HRMS (ESI)
m/z calcd for C26H19NNaO4 [M+Na]+: 432.1212, found: 432.1200.
4.1.3.4.
adamantylamide (11). 88%; Brown solid; mp 186–188 °C; IR
(KBr disc) mmax 3428, 1646, 1593, 1516 cmꢁ1 1H NMR (CD3OD) d
(E)-N-(4-(3-(3,4-Dihydroxyphenyl)acryloyl)phenyl)-1-
;
8.03 (d, 2H, J = 8.4 Hz), 7.77 (d, 2H, J = 8.8 Hz), 7.67 (d, 1H,
J = 15.2 Hz), 7.49 (d, 1H, J = 15.6 Hz), 7.18 (d, 1H, J = 1.6 Hz), 7.09
(d, 1H, J = 8.4 Hz), 6.81 (d, 1H, J = 8.0 Hz), 1.69–2.10 (15H, protons
of adamantyl); 13C NMR (CD3OD) d 189.8, 178.0, 148.6, 145.5,
145.4, 143.1, 133.6, 129.1, 127.0, 122.3, 120.0, 118.1, 115.2,
114.3, 41.5, 38.6, 36.2, 28.0; MS (ESI) m/z 418 [M+1]+; HRMS
4.1.3.10. (E)-N-(4-(3-(3,4-Dihydroxyphenyl)acryloyl)phenyl)-4-
methoxybenzamide (17). 89%; Buff white solid; mp 212–216 °C;
IR (Neat) mmax 3489, 1684, 1602, 1515 cmꢁ1 1H NMR (CD3OD) d
;
8.06 (d, 2H, J = 8.1 Hz), 7.93 (d, 2H, J = 8.5 Hz), 7.89 (d, 2H,
J = 8.0 Hz), 7.68 (d, 1H, J = 15.4 Hz), 7.51 (d, 1H, J = 15.4 Hz), 7.19
(s, 1H), 7.11 (d, 1H, J = 8.0 Hz), 7.03 (d, 1H, J = 8.0 Hz), 6.82 (d,
1H, J = 8.0 Hz), 3.90 (s, 3H); 13C NMR (CD3OD) d 191.2, 168.5,
164.4, 150.0, 147.0, 146.9, 144.7, 135.0, 132.8, 130.8, 130.7,
128.4, 127.9, 123.7, 121.2, 119.5, 116.7, 115.8, 114.9, 114.7, 56.0;
MS (ESI) m/z 390 [M+1]+; HRMS (ESI) m/z calcd for C23H19NNaO5
[M+Na]+: 412.1161, found: 412.1158.
(ESI) m/z calcd for
440.1830.
C
26H27NNaO4 [M+Na]+: 440.1838, found:
4.1.3.5.
furan-2-carboxamide (12). 94%; Reddish brown solid; mp 268–
270 °C; IR (Neat) mmax 3516, 3266, 1646, 1600, 1575, 1525 cmꢁ1
(E)-N-(4-(3-(3,4-Dihydroxyphenyl)acryloyl)-phenyl)-
;
1H NMR (CD3OD) d 7.99 (d, 2H, J = 8.1 Hz), 7.82 (d, 2H, J = 8.1 Hz),
7.67 (s, 1H), 7.59 (d, 1H, J = 15.5 Hz), 7.44 (d, 1H, J = 15.4 Hz),
7.22 (s, 1H), 7.10 (s, 1H), 7.02 (d, 1H, J = 8.0 Hz), 6.73 (d, 1H,
J = 8.1 Hz), 6.56 (s, 1H); 13C NMR (CD3OD) d 189.8, 157.5, 147.4,
145.6, 145.5, 142.5, 133.9, 129.3, 126.9, 122.3, 119.8, 118.0,
115.3, 115.2, 114.3, 112.0; MS (ESI) m/z 350 [M+1]+; HRMS (ESI)
m/z calcd for C20H15NNaO5 [M+Na]+: 372.0848, found: 372.0841.
4.1.3.11.
2,4-difluorobenzamide (18). 95%; Buff white solid; mp 202–
206 °C; IR (Neat) mmax 3482, 3436, 1687, 1659, 1595, 1524 cmꢁ1
(E)-N-(4-(3-(3,4-Dihydroxyphenyl)acryloyl)phenyl)-
;
1H NMR (CD3OD) d 8.08 (d, 2H, J = 8.4 Hz), 7.87 (d, 2H, J = 8.4 Hz),
7.80 (dd, 1H, J = 7.2, 8.4 Hz), 7.68 (d, 1H, J = 15.6 Hz), 7.52 (d, 1H,
J = 15.2 Hz), 7.20 (s, 1H), 7.11 (m, 3H), 6.82 (d, 1H, J = 8.0 Hz); 13C
NMR (CD3OD) d 189.7, 163.2, 148.6, 145.7, 145.5, 142.6, 134.1,
131.8, 131.7, 129.3, 126.9, 122.3, 119.5, 118.0, 115.2, 114.3, 111.7,
111.5, 104.4, 104.1 103.9; MS (ESI) m/z 396 [M+1]+; HRMS (ESI) m/
z calcd for C22H15F2NNaO4 [M+Na]+: 418.0867, found: 418.0856.
4.1.3.6. (E)-N-(4-(3-(3,4-Dihydroxyphenyl)acryloyl)phenyl)-thi-
ophene-2-carboxamide (13). 97%; Brown solid; mp 189–191 °C;
IR (Neat) mmax 3465, 3339, 1643, 1591, 1523 cmꢁ1 1H NMR
;
(CD3OD) d 8.07 (d, 2H, J = 8.4 Hz), 7.94 (d, 1H, J = 3.2 Hz), 7.88 (d,
2H, J = 8.4 Hz), 7.74 (d, 1H, J = 4.8 Hz), 7.68 (d, 1H, J = 15.6 Hz),
7.52 (d, 1H, J = 15.6 Hz), 7.20 (s, 2H), 7.11 (d, 1H, J = 8.0 Hz), 6.82
(d, 2H, J = 8.4 Hz); 13C NMR (CD3OD) d 189.8, 161.4, 148.6, 145.6,
145.5, 142.9, 139.2, 133.8, 131.7, 129.3, 129.1, 127.6, 126.9,
122.3, 119.7, 118.0, 115.2, 114.3; MS (ESI) m/z 366 [M+1]+; HRMS
(ESI) m/z calcd for C20H15NNaO4S [M+Na]+: 388.0619, found:
388.0616.
4.1.3.12.
(E)-N-(4-(3-(3,4-Dihydroxyphenyl)acryloyl)phenyl)-
3,4-difluorobenzamide (19). 80%; Yellowish green solid; mp
254–257 °C; IR (Neat) mmax 3539, 3347, 3142, 1652, 1594, 1567,
1520 cmꢁ1 1H NMR (CD3OD) d 8.09 (d, 2H, J = 8.0 Hz), 7.90 (d,
;
3H, J = 8.4 Hz), 7.83 (brs, 1H), 7.69 (d, 1H, J = 15.6 Hz), 7.53 (d,
1H, J = 15.6 Hz), 7.41 (dd, 1H, J = 8.0, 16.8 Hz), 7.19 (s, 1H), 7.12
(d, 1H, J = 8.0 Hz), 6.82 (d, 1H, J = 8.0 Hz); 13C NMR (CD3OD) d
191.6, 166.7, 150.4, 147.5, 147.2, 144.7, 135.8, 133.7, 131.1,
128.7, 126.4, 124.1, 121.7, 119.7, 119.2, 118.9, 118.6, 117.0,
116.1; MS (ESI) m/z 396 [M+1]+; HRMS (ESI) m/z calcd for
4.1.3.7.
(thiophen-2-yl)acetamide (14). 90%; Brown solid; mp 226–
229 °C; IR (Neat) mmax 3504, 3270, 1661, 1590, 1560, 1523 cmꢁ1
(E)-N-(4-(3-(3,4-Dihydroxyphenyl)acryloyl)phenyl)-2-
C
22H15F2NNaO4 [M+Na]+: 418.0867, found: 418.0863.
;
1H NMR (CD3OD) d 8.05 (d, 2H, J = 7.6 Hz), 7.76 (d, 2H, J = 7.6 Hz),
7.67 (d, 1H, J = 15.4 Hz), 7.51 (d, 1H, J = 15.4 Hz), 7.29 (d, 1H,
J = 3.8 Hz), 7.19 (s, 1H), 7.11 (d, 1H, J = 8.0 Hz), 7.01–6.98 (m, 2H),
6.82 (d, 1H, J = 8.0 Hz), 3.94 (s, 2H); 13C NMR (CD3OD) d 189.8,
169.9, 148.6, 145.6, 145.5, 142.9, 136.1, 133.7, 129.4, 127.0,
126.4, 126.3, 124.5, 122.3, 119.0, 118.0, 115.2, 114.4, 37.5; MS
(ESI) m/z 380 [M+1]+; HRMS (ESI) m/z calcd for C21H17NNaO4S
[M+Na]+: 402.0776, found: 402.0771.
4.1.3.13. (E)-N-(4-(3-(3,4-Dihydroxyphenyl)acryloyl)phenyl)-
3,5-difluorobenzamide (20). 79%; Yellow brown solid; mp
>280 °C; IR (Neat) mmax 3555, 3476, 3356, 1658, 1594,
1518 cmꢁ1 1H NMR (CD3OD) d 8.08 (d, 2H, J = 8.0 Hz), 7.91 (d,
;
2H, J = 8.0 Hz), 7.68 (d, 1H, J = 15.6 Hz), 7.59 (d, 2H, J = 6.8 Hz),
7.52 (d, 1H, J = 15.6 Hz), 7.24–7.19 (m, 2H), 7.11 (d, 1H,
J = 8.4 Hz), 6.82 (d, 1H, J = 8. 4 Hz); 13C NMR (CD3OD) d 189.8,
164.5, 164.3, 164.2, 161.8, 161.7, 148.7, 145.7, 145.5, 142.7,
138.1, 134.1, 129.3, 126.9, 122.3, 119.9, 118.0, 115.2, 114.4,
112.2, 112.0, 110.7, 110.6, 110.5, 106.9, 106.7, 106.4; MS (ESI) m/
z 396 [M+1]+; HRMS (ESI) m/z calcd for C22H15F2NNaO4 [M+Na]+:
418.0867, found: 418.0857.
4.1.3.8.
benzamide (15). 91%; Light brown solid; mp 198–201 °C; IR
(Neat) mmax 3490, 3295, 1645, 1596, 1557, 1513 cmꢁ1 1H NMR
(E)-N-(4-(3-(3,4-Dihydroxyphenyl)acryloyl)phenyl)-
;
(CD3OD) d 8.09 (d, 2H, J = 8.1 Hz), 7.95 (d, 2H, J = 8.2 Hz), 7.92 (d,
2H, J = 9.2 Hz), 7.68 (d, 1H, J = 15.4 Hz), 7.59 (d, 1H, J = 7.2 Hz), 7.53
(m, 3H), 7.20 (s, 1H), 7.12 (d, 1H, J = 8.1 Hz), 6.82 (d, 1H,
J = 8.1 Hz); 13C NMR (CD3OD) d 189.8, 167.6, 148.6, 145.5, 145.5,
143.1, 134.7, 133.8, 131.7, 129.3, 128.3, 127.3, 127.0, 126.9, 122.3,
119.9, 118.0, 115.2, 114.3; MS (APCI) m/z 360 [M+1]+; HRMS (ESI)
m/z calcd for C22H17NNaO4 [M+Na]+: 382.1055, found: 382.1053.
4.1.3.14. (E)-N-(4-(3-(3,4-Dihydroxyphenyl)acryloyl)phenyl)-2-
fluoro-4-(trifluoromethyl)benzamide (21). 74%; Yellow brown
solid; mp >280 °C; IR (Neat) mmax 3548, 3507, 3352, 3263, 1669,
1647, 1595, 1567, 1521 cmꢁ1 1H NMR (CD3OD) d 8.09 (d, 2H,
;
J = 8.4 Hz), 7.91–7.87 (m, 3H), 7.70–7.54 (m, 4H), 7.20 (s, 1H),
7.11 (d, 1H, J = 7.6 Hz), 6.82 (d, 1H, J = 8.0 Hz); 13C NMR (CD3OD)
d 189.7, 162.7, 160.5, 158.0, 148.7, 145.7, 145.5, 142.4, 143.3,
134.0, 133.0, 131.1 131.0, 129.4, 127.9, 127.7, 126.9, 124.3, 122.3,
121.6, 121.7, 120.6, 119.5, 118.0, 115.2, 114.4, 114.1, 113.9,
(113.6, 113.6, 113.4, 113.3) (CF3, q); MS (ESI) m/z 446 [M+1]+;
HRMS (ESI) m/z calcd for C23H15F4NNaO4 [M+Na]+: 468.0835,
found: 468.0828.
4.1.3.9.
naphthamide (16). 80%; Buff white solid; mp 187–189 °C; IR
(Neat) mmax 3383, 3302, 1655, 1584, 1523 cmꢁ1 1H NMR (DMSO-
(E)-N-(4-(3-(3,4-Dihydroxyphenyl)acryloyl)phenyl)-2-
;
d6) d 10.75 (s, 1H), 9.71 (s, 1H), 9.13 (s, 1H), 8.62 (s, 1H), 8.17 (d,
2H, J = 8.4 Hz), 8.10–7.99 (m, 6H), 7.68–7.57 (m, 4H), 7.26 (s, 1H),

1262
H. S. Bodiwala et al. / Bioorg. Med. Chem. 19 (2011) 1256–1263
4.2. Biology
and Luciferase assay was performed using SteadyliteÒ luciferase
assay kit (Perkin–Elmer Life Science, USA) according to manufac-
turer’s protocol. Relative Luciferase units were compared between
control infected cells and treated infected cells controls to calculate
percent inhibition of virus infection.
4.2.1. Cell cytotoxicity assay using MTT
Cytotoxicity of potential candidates was assayed using MTT Kit
(Roche) in the CEM-GFP cell line according to the manufacturer’s
protocol. Briefly, 2 ꢂ 104 cells/well were seeded in 96-well plate;
samples were then added into the wells at different concentrations
keeping untreated and vehicle treated wells as controls. After 48 h
4.3. Molecular modeling
The six active compounds were selected for docking studies
against HIV-1 IN (PBD ID: 1QS4)47 and CCR5 proteins. 1QS4 is used
for binding mode analysis of HIV-1 IN inhibitory activity. However,
no X-ray crystal structure is available in PDB for CCR5 protein. So,
hypothetical model of CCR5 was generated using MODELLER 9v7 pro-
gram.48 Protein sequence of CCR5 was retrieved from Swissprot ID
P51681. The first step was to search for a number of related se-
quences to find a related three dimensional protein structure as a
template by the PSI-BLAST program. Bovine rhodopsin (PDB ID:
1F88)49 was mostly selected as a template molecule for CCR5 mod-
el development.41,43,45 The sequence alignment was performed
using Align2D module in Modeller program and also alignment
was manually checked for error (Fig. S13 in Supplementary data
shows sequence alignment between CCR5 and template protein).
Protein models were built and minimized using CHARMm to
incubation, 10 lL of MTT Reagent (5 mg/mL) were added in to the
wells to allow the reaction. Formazan crystals produced during the
reaction were solubilized and color development was read at
540 nm. The highest noncytotoxic concentration was determined
by first measuring % viability at three different concentrations,
and then making further dilutions to determine the concentration
that gave 95% viability of the cells.
4.2.2. Anti-HIV screening in CEM-GFP cells
Human CD4+ T cell line, CEM-GFP cells were infected with HIV-
1NL4.3 virus at a multiplicity of infection (MOI) of 0.05 using the
standard protocol previously published from the lab.27,28 The infec-
tion was monitored by GFP visualization under the microscope.
The cells were then incubated with samples for up to 7 days post
infection. Preliminary screening was performed by GFP quantita-
tion by microfluorometry. Virus production was assayed in the cul-
ture supernatant on day-7 post infection by p24 antigen capture
ELISA (Perkin–Elmer Life Science, USA). AZT was used as a positive
control. The activity is reported as% decrease in p24 antigen in the
culture supernatant of HIV-1 infected cells treated with compound
as compared to culture supernatant of untreated HIV-1 infected
cells.
obtain stable structures. Protein models were validated using
50
PROCHECK
and finally one model that fits best in Ramachandran
plot was used for docking studies (Fig. S14 in Supplementary data
shows Ramachandran plot of CCR5 model and Fig. S15 shows super
imposition of model with template protein). All the compounds
were built using Sybyl7.151 and subsequently minimized using
Powell gradient method52 using Gasteiger Huckel charges.53 The
protein structures were prepared for docking using AUTODOCK Tool.
The docking was performed using AUTODOCK 4.2 program.54 Co-
crystallized ligand and all water molecules were removed from
crystal protein (1QS4) while a magnesium ion (Mg2+) at the active
site of HIV-1 IN (1QS4) was maintained. Polar hydrogens were
added and non polar hydrogens were merged, finally Kallman uni-
ted atom charge and atom type parameter was added to both 1QS4
and modelled CCR5 proteins. Grid map dimensions (for HIV-1 IN
56 ꢂ 68 ꢂ 46; for CCR5 64 ꢂ 56 ꢂ 60) were set surrounding active
site. Lamarckian genetic search algorithm54 was employed and
docking run was set to 30.
4.2.3. HIV-1 integrase inhibition assay
In vitro integrase assay was performed to analyze the HIV-1 IN
inhibition by the compounds using HIV-1 Integrase Assay kit
(Xpressbio Life Science Products, USA) according to manufacturer’s
protocol. Briefly, Streptavidin coated 96-well plates were coated
with a double-stranded HIV-1 LTR U5 donor substrate (DS) oligo-
nucleotide containing an end-labeled biotin. Full-length recombi-
nant HIV-1 integrase protein was then loaded onto this oligo
substrate. Compounds were added to the reaction and then a
different double-stranded target substrate (TS) oligo containing
30-end modifications was added to the plate. The HIV-1 integrase
cleaves the terminal two bases from the exposed 30-end of the
HIV-1 LTR DS and then catalyzes a strand-transfer reaction to inte-
grate the DS into the TS. The products of the reaction were detected
colorimetrically using an HRP-labeled antibody directed against
the TS 30-end modification. Percent inhibition in the integrase as-
Acknowledgments
Authors are thankful to Department of Biotechnology, Govern-
ment of India for the financial support to this project (Grant No.
BT/PR7020/Med/14/930/2005 Dt. 23/05/2006). CEM-GFP and
TZM-bl cells were obtained from the NIH AIDS Research and Refer-
ence Reagent Program, Division of AIDS, NIAID, NIH.
say was first measured at 20
more than 50% inhibition, the IC50 values were determined in
mL and then converted to M. Sodium azide was used as a positive
control in this experiment.
l
g/mL, as all the compounds showed
lg/
l
Supplementary data
4.2.4. Entry inhibition assay
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.12.031.
TZM-bl cells are reporter cells expressing CD4, CCR5 and CXCR4
along with reporter proteins luciferase and b-galactosidase under
the control of HIV-1 long terminal repeat (LTR) promoter46
TZM-bl cells (104) were seeded in each well of a 96 well plate. Cells
were incubated overnight at 37 °C in a CO2 incubator. After
16–20 h, medium was removed from the wells and were fed with
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