Anti-HIV activity of semisynthetic derivatives of andrographolide and computational study of HIV-1 gp120 protein binding

Article abstract of DOI:10.1016/j.ejmech.2012.07.030

Andrographolide, a diterpene lactone of the Andrographis paniculata, displays anti-HIV activity in vitro. A series of andrographolide derivatives have been synthesized and evaluated for their anti-HIV activity in a cell-free virus infectivity assay using TZM-bl cells. As compared to andrographolide, 3-nitrobenzylidene derivative 6 showed higher in vitro anti-HIV activity, whereas 2′,6′-dichloro-nicotinoyl ester derivative 9 showed higher Therapeutic Index. The andrographolide and its derivatives, 6 and 9, inhibited gp120-mediated cell fusion of HL2/3 cells (expressing gp120 on its surface) with TZM-bl cells (expressing CD4 and co-receptors CCR5 & CXCR4). Further, computational studies revealed that these molecules bind to the important residues of V3 loop of gp120. These results suggest that andrographolide derivatives may be promising candidates for prevention of HIV infection.

Full text of DOI:10.1016/j.ejmech.2012.07.030

European Journal of Medicinal Chemistry 56 (2012) 368e374
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Anti-HIV activity of semisynthetic derivatives of andrographolide and
computational study of HIV-1 gp120 protein binding
Mayur M. Uttekar a, Tiyasa Das b, Rohan S. Pawar a, Beena Bhandari b, Vidya Menon a, Nutan b,
**
,
a,
*
Satish K. Gupta b , Sujata V. Bhat
a Laboratory for Advanced Research in Natural and Synthetic Chemistry, V. G. Vaze College, Mumbai University, Mithagar Road, Mulund (East), Mumbai 400 081, India
b National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Andrographolide, a diterpene lactone of the Andrographis paniculata, displays anti-HIV activity in vitro. A
series of andrographolide derivatives have been synthesized and evaluated for their anti-HIV activity in
a cell-free virus infectivity assay using TZM-bl cells. As compared to andrographolide, 3-nitrobenzylidene
derivative 6 showed higher in vitro anti-HIV activity, whereas 20,60-dichloro-nicotinoyl ester derivative 9
showed higher Therapeutic Index. The andrographolide and its derivatives, 6 and 9, inhibited gp120-
mediated cell fusion of HL2/3 cells (expressing gp120 on its surface) with TZM-bl cells (expressing
CD4 and co-receptors CCR5 & CXCR4). Further, computational studies revealed that these molecules bind
to the important residues of V3 loop of gp120. These results suggest that andrographolide derivatives
may be promising candidates for prevention of HIV infection.
Received 17 November 2011
Received in revised form
15 July 2012
Accepted 17 July 2012
Available online 24 July 2012
Keywords:
Andrographolide derivatives
Cytotoxicity
Ó 2012 Elsevier Masson SAS. All rights reserved.
Anti-HIV activity
Viral envelope protein gp120 binding
1. Introduction
[6,7]. The compound has multiple pharmacological properties such
as antihepatotoxic, immunostimulant, anticancer and hypogly-
Acquired immune deficiency syndrome (AIDS) caused by the
infection with the human immunodeficiency virus (HIV) represents
a major health problem worldwide, especially in the developing
countries. Highly active antiretroviral combination therapy
(HAART), mainly consisting of HIV protease and reverse transcrip-
tase inhibitors, introduced in the late 1990s, has largely improved
patients’ lives in the developed countries. However, drug resistance
and toxicity are the main problems during HIV treatment. There-
fore, there is a continuous need for new drugs effective against
HIV [1,2].
cemic activities [8e14]. Recent studies indicated that Andro was
also a potential AIDS therapeutic agent [15]. Some derivatives of
Andro such as 14-deoxy-11,12-didehydroandrographolide and the
corresponding oxime of 3-keto derivative, amide derivatives of
andrographolic acid and 12-ester of 12-hydroxy-14-deoxy-13,14-
dehydroandrographolide were reported to exert comparable or
slightly improved anti-HIV effect [16e18], suggesting Andro could
be regarded as an excellent lead compound to develop new
molecules for anti-HIV treatment.
Andrographis paniculata Nees (Fam. Acanthaceae), commonly
known as ‘king of bitters’, is a well-known medicinal herb in
Southeast Asia, India and China. It has been used for the treatment of
a large variety of diseases, which include meningitis, acute hepatitis,
common cold and many other inflammatory conditions [3e5].
Andrographolide 1 (Andro) is a major bioactive component of
the plant. Andro is a bicyclic ent-labdane type diterpene lactone
Abbreviations: AZT, azidothymidine; gp120, glycoprotein 120; Mp, melting
point; THF, tetrahydrofuran; Andro, andrographolide.
1. Andrographolide
* Corresponding author. Tel.: þ91 022 21631421x113; fax: þ91 022 21634262.
** Corresponding author. Tel.: þ91 11 26741249; fax: þ91 11 26742125.
E-mail addresses: skgupta@nii.ac.in (S.K. Gupta), sujata8b@gmail.com (S.V. Bhat).
HIV entry in the host cell is initiated when HIV surface glyco-
protein gp120 binds to host cellular receptor CD4 and subsequently
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.030

M.M. Uttekar et al. / European Journal of Medicinal Chemistry 56 (2012) 368e374
369
to its co-receptor usually either CCR5 or CXCR4. These initial events
trigger conformational changes in HIV-1 envelope proteins and
expose fusion peptide gp41 to initiate membrane fusion. In this
process, the interaction of V3 loop of gp120 with the tyrosine-
sulfated N-terminus of CCR5 is critical [19]. Thus, inhibition of
any of these events is an important target for the design of new
pharmaceuticals for the multidrug therapy of AIDS. Important
examples of this class include triterpene betulinic acid derivatives
such as IC9564 and A43-D that exhibited anti-HIV activity through
binding to V3 loop region of gp120 [20,21].
obtained by selective controlled esterification of C14eOH with 2,6-
dichloronicotinoyl chloride and salicyloyl chloride respectively in
the presence of triethyl amine under N2 atmosphere. In the 1H
spectrum of esters 9 and 10, the peak at d 5.1 due to proton under C-
14 hydroxy group was absent and a new peak at
to proton under C-14 ester group.
d 6.2 appeared due
4.2. In vitro cytotoxicity and anti-HIV activity of Andro and its
derivatives
We report herein, the in vitro anti-HIV activity of 3,19-
benzylidene and 14-ester derivatives of Andro in a cell-free virus
infectivity assay using TZM-bl cells [22]. Further, we have evaluated
these compounds for their efficacy to inhibit HIV-1 gp120-
mediated cell fusion, reverse transcriptase (RT) and protease
enzyme activities. Additionally, computational studies for their
binding to V3 loop of gp120 have been performed.
The observed anti-HIV activity of the compounds may be
sometime due to their toxicity and consequently might result in
erroneous conclusion. Keeping this in view, Andro as well as its
derivatives 2e10, were first evaluated for cytotoxicity on TZM-bl
cells by MTT assay [24]. Out of the ten compounds evaluated
using MTT assay, six compounds (1, 2, 6, 8, 9 and 10) showed CC50
values in the range of 745e10,345
mM (Table 1). Interestingly,
derivatives 8 and 9 showed lower cytotoxicity as compared to
Andro, whereas, cytotoxicity of derivative 2 was comparable to the
parent compound (Table 1). The derivative 7 showed an interme-
2. Chemistry
Andro is a 3,14,19-trihydroxy-diterpene containing
g-lactone
diate cytotoxicity with CC50 value of 440
mM. The semi-synthetic
connected to a decalin ring via an unsaturated C-2 moiety. In the
present study nine derivatives (2e10) were synthesized from
Andro by selective reactions at C3eOH, C19eOH and/or C14eOH
groups (Scheme 1). The structures of these derivatives were sup-
ported by their spectral properties, LCMS and microanalyses.
derivatives 3, 4 and 5 showed high cytotoxicity (CC50 < 179
m
M).
Subsequently, Andro as well as its derivatives 2e10, were eval-
uated at varying concentrations for their anti-HIV activity using
cell-free virus infectivity assay in TZM-bl cells (Table 1). These
investigations revealed that derivatives 6 and 9, including Andro
have IC50 values less than 1
showed lower IC50 value as compared to Andro (0.51 vs 0.59
Derivatives 2, 4, 5 and 8 have IC50 values in the range of 1e3
Remaining derivatives 3, 7 and 10 showed IC50 values greater
than 6 M. The natural products and their derivatives which can be
considered as promising candidates against HIV infection should be
well tolerated; preferably with IC50 values ꢀ1 M and known
m
M (Table 1). Interestingly, derivative 6
M).
M.
3. Biology
m
m
The cytotoxicity of Andro (1) and its derivatives 2e10 was
evaluated using MTT assay in TZM-bl cells [a recombinant HeLa cell
line expressing high levels of CD4 receptors, HIV-1 co-receptors
m
CCR5 and CXCR4, with
b-galactosidase and luciferase as reporter
m
genes under the control of HIV-1 long terminal repeat (LTR)]. These
compounds were evaluated for their in vitro anti-HIV activity using
reporter gene based cell assay. In order to delineate the mechanism
of action, derivatives of Andro (2e10) along with Andro were
evaluated for their ability to inhibit HIV-1 protease and RT activi-
ties. Keeping in view of the recent observations that 14-acetyl
analog of Andro inhibit in vitro entry of Herpes simplex Virus
(HSV) [23], the Andro and its two derivatives (6, 9) showing IC50
mechanism of action. The therapeutic index (TI) of a drug is the
ratio between the toxic dose and the therapeutic dose and is used
as a measure of the relative safety of drug for a particular treatment
i.e. TI > 10 is generally considered as safe. From this perspective,
Andro and all derivatives have TI values either equal to or more
than 10. However, derivative 9 [14-(20,60-dichloronicotinoyl) ester
of Andro] has higher TI value than Andro (12,474 vs 2875; Table 1).
The derivative
6
[3,19-(30-nitrobenzylidene)-andrographolide],
values less than 1
m
M in HIV infectivity assay, were also evaluated
which had a higher anti-HIV activity than Andro has however,
lower TI as compared to the parent compound (Table 1). Consid-
ering the significant problem of drug resistance during anti-HIV
therapy, it is imperative to find new agents, which are safe and
have potent anti-HIV activity. The derivative 9 is a promising
candidate for further evaluation.
for their efficacy to inhibit gp120-mediated cell-based fusion using
HL2/3 HeLa derived cells (expressing HIV-1HxB2/3 Env on the cell
surface in addition to Tat, Gag, Rev and Nef proteins in the cyto-
plasm) and TZM-bl cells. Further, computational modeling studies
suggest that these compounds bind to V3 loop region of HIV-1
envelope protein, gp120.
4.3. Andro and its derivatives 6 and 9 inhibit gp120-mediated cell
4. Results and discussion
fusion
4.1. Synthesis of Andro derivatives
It has been proposed, though not proven, that anti-HIV activity
of Andro and its derivatives may be due to their ability to prevent
HIV entry. Recently, 14-acetyl analog of Andro have been shown to
inhibit in vitro entry of HSV [23]. Hence, we have evaluated the
potential of Andro and its derivatives 6 and 9 to inhibit gp120-
mediated cell fusion [25]. We have employed HL2/3 cells that
express gp120 on its surface and produce Tat, Gag, Rev, & Nef and
TZM-bl cells that express CD4 and chemokine receptors, CCR5 and
CXCR4. Upon co-culture of these two cell types, cellecell fusion
The hydroxyl groups of Andro at C-3 and C-19 positions are
secondary and primary respectively. The condensation of these
hydroxyl groups was carried out by reacting Andro with differently
substituted aromatic aldehydes in the presence of Amberlyst-15Ò
under N2 atmosphere to yield 3,19-benzylidene derivatives 2e7.
The stereoisomers of benzylidene derivatives, 2e7 due to newly
introduced chiral center had identical properties in column chro-
matography over silica gel and in 1H NMR spectra except benzyli-
dene protons, which showed overlapping signals.
occurs, resulting in the formation of syncytia and activation of b-
galactosidase as well as luciferase that are under the control of Tat
regulated LTR. Pre-incubation of HL2/3 cells with Andro and its
derivatives 6 and 9 showed a dose dependent inhibition of lucif-
erase activity, in the cell-based fusion assay (Table 2). The derivative
The C19eOH of Andro could be selectively converted to give C-19
tosylaminocarbonyl derivative 8 by the treatment with p-toluene-
sulfonyl-isocyanate. Similarly, the C-14 esters 9 and 10 were

370
M.M. Uttekar et al. / European Journal of Medicinal Chemistry 56 (2012) 368e374
Scheme 1. Synthesis of andrographolide derivatives.
9 showed slightly lower IC50 value (1.1
(1.7 mM) and derivative 6 (1.6 mM). In addition, these compounds
m
M) as compared to Andro
protease as well as RT enzyme activities (data not shown). These
observations suggest that Andro and its derivatives 6 and 9 inter-
fered with early events in the entry of HIV into target cells by
interacting with the gp120.
also inhibited syncytia formation (data not shown). Further, Bicy-
clam, which inhibits the entry of HIV-1 into CD4þ T cells via
selective blockade of chemokine receptor (CXCR4) [26] failed to
show any inhibition in fusion, if HL2/3 cells were pre-treated with
it. However, pretreatment of TZM-bl cells with Andro and its
derivatives 6 and 9 did not exhibit any inhibition in fusion of these
two cells, whereas Bicyclam significantly inhibited fusion mediated
luciferase expression (Table 2). Andro and its derivatives when
4.4. Molecular modeling
All Andro derivatives obeyed Lipinski’s rule (except molecular
weights of compounds 8 and 9, which are >500, Table 3). Lipinski’s
evaluated at 50
mM failed to significantly inhibit HIV-1 specific
Table 2
Inhibition of gp120-mediated cell fusion by Andro and
Table 1
its derivatives 6 and 9.
Toxicity and in vitro anti-HIV activity of Andro and its derivatives.
Compounds
IC50 (m
m)a
Comp.
Mol. Formula
CC50
(
m
M)a
IC50
(m
M)b
TIc
HL2/3 cells pre-treated with compounds incubated
with untreated TZM-bl cells
1
6
9
1
2
3
4
5
6
7
8
9
C20H30O5
C28H34O7
C28H36O7
C27H34O6
C27H34O7
C27H33NO7
C27H33ClO5
C28H37NO8S
1696
1647
59
0.59
2.75
6.09
2.10
1.09
0.51
9.94
2.08
0.83
84.24
2875
599
10
1.7
1.6
1.1
NS
50
24
179
745
440
2035
10,354
1082
164
1460
44
978
12,474
12
Bicyclam
TZM-bl cells pre-treated with compounds
incubated with untreated HL2/3 cells
1
6
9
NSb
NS
NS
1.6
C26H31Cl2NO6
10
C27H34O7
Bicyclam
a
CC50: The cytotoxic concentration of the compound leading to 50% reduction in
a
cell viability.
IC50: Inhibitory concentration of the compound
leading to 50% inhibition of luciferase expression.
b
IC50: Inhibitory concentration of the compound leading to 50% inhibition in HIV
infection.
b
NS: No significant inhibition of luciferase
c
TI: Therapeutic Index, (CC50 O IC50).
expression.

M.M. Uttekar et al. / European Journal of Medicinal Chemistry 56 (2012) 368e374
371
Table 3
however, has higher TI as compared to Andro due to lower cyto-
toxicity. These derivatives inhibit HIV-1 infection by blocking
gp120-CD4/CXCR4/CCR5 interaction. Further, molecular docking
studies revealed that these compounds may bind to V3 loop of HIV-
1 envelope protein, gp120. These studies demonstrate the potential
of semi-synthetic derivatives of Andro as anti-HIV agents.
Andro and its derivatives in the eyes of Lipinski’s Rule.a
Comp. Formula
Mol. Wt. H-accept. H-donors log P TPSA
1
2
3
4
5
6
7
8
9
10
C
20H30O5
350.45
482.50
484.59
454.56
470.56
483.56
473.01
547.67
5
7
7
6
7
8
5
9
7
7
3
1
2
2
3
1
1
3
2
3
1.05
3.61
3.05
3.24
3.15
3.65
4.39
2.90
3.89
3.50
86.99
83.47
94.46
C28H34O7
C28H36O7
C27H34O6
C27H34O7
85.23
105.46
110.83
65.00
139.24
105.96
113.29
6. Experimental section
C27H33 NO7
C27H33 Cl O5
C28H37NO8S
C26H31Cl2N O6 524.44
C27H34O7 470.56
6.1. General
IR spectra were recorded on PerkineElmer spectrum one spec-
trometer. 1H NMR spectra were recorded on a Varian either 300,
400 or Bruker 500 MHz spectrometers with tetramethylsilane as an
a
H-acceptor, H-donors, log P and TPSA values were calculated using Molinspi-
ration software.
internal standard and chemical shifts are reported in
Microanalyses were performed in Carlo Erba Model 1106
elemental analyzer (Milano, Italy). LCeMS analysis was done on
MS-Varian 500 instrument, with direct injection (10 L) in meth-
d units.
rule has been used to evaluate drug likeness or determine if
a chemical compound with a certain biological activity has prop-
erties that would make it a likely orally active drug in humans [27].
The molecules 1e10 were docked into V3 loop region (residues
296e330) of gp120 HIV-1 envelope protein to study molecular
interactions. The natural stereoisomer of Andro (1) was considered
for docking in the V3 loop of gp120. The docking results (Table 4)
were compared with well-known betulinic acid derivative IC9564,
which has been reported to be a HIV-1 entry inhibitor through
binding into V3 loop region of gp120 [20,21].
Docking studies with V3 loop of gp120, revealed that Andro (1)
(Fig. 1) formed total four hydrogen bonds including one hydrogen
bond between C19eOH and Gly 321, two hydrogen bonds between
lactone carbonyl (C16) and Gln 328 and Arg 298; the fourth hydrogen
bond was formed between lactone oxygen and Gln 328. The nitro
group of derivative 6 formed three hydrogen bonds with Ser 306,
whereas derivative 9 made three hydrogen bonds with C14-ester
carbonyl bonded to Arg 298 and C19eOH bonded to Ser 57 and Asn
58. On similar docking studies with betulinic acid derivative IC9564
showed four hydrogen bonds including C28 and side chain amide
carbonyls formed bonds with Arg 298 and Arg 304, respectively. The
side chain hydroxyl and carboxylic acid groups formed hydrogen
bonds with Tyr 318 and Ser 306 respectively. Interestingly, the
binding scores of all compounds were close to the observed pIC50
values (Table 4). Thus, molecular modeling studies indicate that
Andro and its derivatives bind to V3 loop of gp120.
a
m
anol at 300 ꢁC. Silica gel (100e200 mesh), which was used for
column chromatography was activated by heating at 120 ꢁC for 4 h.
All chemicals were procured from Sigma Aldrich Inc. (St. Louis, MO,
USA) and the various solvents used for chemical synthesis were of
analytical grade. Products were obtained as colorless to pale yellow
solids. TZM-bl cells were maintained in Dulbecco’s modified Eagle’s
medium (DMEM; SigmaeAldrich Inc.), supplemented with 10%
fetal bovine serum (FBS; Biological Industries, Kibbutz Beit Hae-
mek, Israel) and an antibioticeantimycotic cocktail [Penicillin
(100 units/mL), Streptomycin (100
mg/mL) and Amphotericin B
(0.25 g/mL); Pen-Strep-Ampho sol, Biological Industries] under 5%
m
CO2 humidified atmosphere at 37 ꢁC. HeLa-derived HL2/3 cells
(AIDS Research and Reference Reagent Program, Division of AIDS,
NIAID, NIH, USA), which express HIV-1HxB2 Env and other viral
proteins (obtained from Prof Anand K. Kondapi, Department of
Biochemistry, School of Life Sciences, University of Hyderabad,
Hyderabad 500 046, India), were also cultured as described above
for TZM-bl cells except that medium was additionally supple-
mented with 500 mg/mL of G418 (Gibco, Grand Island, NY, USA). The
Andro and derivatives (2e10) were evaluated for anti-HIV activity
at the Bio-safety Laboratory, National Institute of Immunology, New
Delhi after due approval from the Institutional Bio-safety
Committee and following its guidelines.
Presently, most regimens for treatment of HIV infection employ
drugs that primarily inhibit HIV specific RT and/or protease
enzymes. The synthetic Andro derivatives described in the present
study, which inhibit viral entry, may have potential as anti-HIV
agents and need further exploration.
6.2. Chemistry
6.2.1. General procedure for synthesis of 3,19-benzylidene
derivatives of andrographolide (2e7)
A mixture of Andro (100 mg, 0.285 mmol), substituted benzal-
dehyde (0.855 mmol) and activated Amberlyst-15Ò (10 mg) in dry
1,4-dioxane (4 mL) was refluxed for 5 h under N2 atmosphere. The
reaction mixture was cooled and filtered to remove the catalyst.
The organic layer was evaporated in vacuo and the residue was
subjected to the column chromatography on silica gel and elution
with petroleum ether (60e80 ꢁC)/ethyl acetate (80:20) gave 3,19-
benzylidine derivatives 2e7.
5. Conclusion
In summary, these studies revealed that the derivative 6 showed
higher anti-HIV activity as compared to Andro. The derivative 9
Table 4
Docking scores of compounds 1e10 and IC9564.a
Comp.
IC50
0.51
pIC50
Docking score
6
1
9
5
8
4
2
3
6.29
6.23
6.08
5.96
5.68
5.68
5.56
5.22
5.00
4.07
7.00
6.27
6.43
6.22
6.14
5.65
5.47
5.20
5.10
5.58
4.28
6.94
6.2.1.1. 3,19-(30,40-Methylenedioxybenzylidene)-andrographolide
(2). Yield: 104 mg (76%), colorless solid, Mp 238e240 ꢁC (dec.); IR
(KBr): 3439, 2962,1753,1681,1100,1051, 903 cmꢂ1; 1H NMR (CDCl3,
0.59
0.83
1.09
2.08
2.10
2.75
6.09
9.94
84.24
0.10
300 MHz): d 0.99 (s, 3H),1.25 (s, 3H),1.62e1.74 (m, 4H),1.76e1.93 (m,
5H), 2.42 (bt, J ¼ 6.8 Hz, 2H), 3.45e3.62 (m, 2H), 4.08 (d, J ¼ 11.1 Hz,
2H), 4.31 (dd, J ¼ 11.1, 6.8 Hz, 2H), 4.76 (s, 2H), 4.82 (bs,1H), 5.06 (m,
1H), 5.67 (bs, 1H), 5.93 (s, 2H), 6.65 (d, J ¼ 7.8 Hz, 1H), 6.70 (d,
J ¼ 7.8 Hz, 1H), 6.92e6.93 (m, 1H), 7.26 (s, 1H). 13C NMR (CDCl3,
7
10
IC9564
125 MHz): d 172.5,148.0,147.5,143.0, 137.4,133.1,129.1,129.0,122.0,
a
With V3 loop of GP120 protein.
121.4, 120.0, 108.0, 107.0, 101.0, 95.0, 81.5, 69.5, 60.2, 50.0, 41.0, 37.3,

372
M.M. Uttekar et al. / European Journal of Medicinal Chemistry 56 (2012) 368e374
Fig. 1. Binding of compounds 1, 6, 9 and IC9564 in the V3 loop region of HIV-1 gp120 (only main surrounding residues are shown).
36.2, 36.1, 26.0, 22.3, 21.4, 21.2, 16.0; Anal. Calcd for C28H34O7: C,
3H), 1.17 (s, 3H), 1.31e1.48 (m, 4H), 1.72e1.94 (m, 4H), 2.65 (t,
J ¼ 6.5 Hz, 2H), 3.09e3.14 (m, 4H), 4.32 (d, J ¼ 10.5 Hz, 2H), 4.57 (dd,
J ¼ 10.5, 6.5 Hz, 2H), 4.79 (bs, 1H), 4.88 (bs,1H), 5.15 (bs,1H), 5.69 (s,
1H), 6.90 (m, 1H), 7.60 (t, J ¼ 7.5 Hz, 1H), 7.89 (d, J ¼ 7.5 Hz, 1H), 8.25
69.69; H, 7.10. Found: C, 69.57; H, 7.38. LC/MS: m/z 482.5 (Mþ).
6.2.1.2. 3,19-(40-Hydroxy-30-methoxybenzylidene)-andrographolide
(3). Yield: 96 mg (70%), colorless solid, Mp 204e205 ꢁC; IR (CHCl3):
(d, J ¼ 7.5 Hz, 1H), 8.42 (s, 1H). 13C NMR (CDCl3, 125 MHz):
d 170.1,
3409, 2946, 1725, 1101, 1025, 891 cmꢂ1
;
1H NMR (CDCl3, 500 MHz):
149.0, 141.0, 136.0, 132.4, 130.0, 129.2, 127.3, 124.0, 122.0, 93.5, 81.0,
74.3, 69.5, 66.2, 54.0, 51.0, 50.0, 38.1, 36.1, 34.0, 33.5, 28.0, 26.0, 22.2,
19.5, 15.2; Anal. Calcd for C27H33NO7: C, 67.06; H, 6.88; N, 2.89.
Found: C, 67.26; H, 6.67; N, 2.68. LC/MS: m/z 483.5 (Mþ).
d
0.87 (s, 3H),1.49 (s, 3H),1.58e1.88 (m, 5H), 1.92e2.10 (m, 3H), 2.40 (bt,
J ¼ 6.5 Hz, 2H), 2.54e2.62 (m, 2H), 3.64 (m,1H), 3.92 (s, 3H), 4.23e4.26
(m, 1H), 4.29 (d, J ¼ 10.2 Hz, 2H), 4.45 (dd, J ¼ 10.2, 6.5 Hz, 2H), 4.62 (s,
1H), 4.92 (s, 1H), 5.04 (bs, 1H), 5.40 (s, 1H), 5.86 (bs, 1H), 6.89 (d,
J ¼ 8.1 Hz, 1H), 6.95 (d, J ¼ 8.1 Hz, 1H), 6.98e7.00 (m, 1H), 7.04 (m, 1H);
6.2.1.6. 3,19-(40-Chlorobenzylidene)-andrographolide
75 mg (56%), colorless solid, Mp 223e225 ꢁC; IR (KBr): 3347, 2951,
1755,1670,1114, 899 cmꢂ1; 1H NMR (CDCl3, 400 MHz):
0.87 (s, 3H),
(7). Yield:
13C NMR(CDCl3,125MHz):
d170.1,147.0,146.5,143.4,143.1,128.5,127.0,
121.5, 114.5, 110.0, 109.1, 95.0, 92.0, 77.5, 74.3, 68.0, 65.1, 58.1, 56.0, 55.0,
43.2, 40.0, 36.4, 29.3, 27.0, 24.0, 23.0, 16.0; Anal. Calcd for C28H36O7: C,
69.40; H, 7.49. Found: C, 69.24; H, 7.30. LC/MS: m/z 484.5 (Mþ).
d
1.25e1.28 (m, 3H), 1.46 (s, 3H), 1.59e2.02 (m, 5H), 2.44 (t, J ¼ 6.8 Hz,
2H), 2.55e2.57 (m, 1H), 3.58e3.66 (m, 3H), 4.24 (d, J ¼ 10.8 Hz, 2H),
4.47 (dd, J ¼ 10.8, 6.8 Hz, 2H), 4.62 (s,1H), 4.92 (s,1H), 5.08 (s,1H), 5.74
(s,1H), 6.98 (m,1H), 7.33 (d, J ¼ 8.4 Hz, 2H), 7.42 (d, J ¼ 8.4 Hz, 2H); 13C
6.2.1.3. 3,19-(40-Hydroxybenzylidene)-andrographolide (4). Yield:
95 mg (74%), pale yellow solid, Mp 158e160 ꢁC; IR (CHCl3): 3397,
NMR (CDCl3, 125 MHz): d 172.2, 144.1, 137.5, 131.0, 129.2, 129.0, 128.4,
2962, 1738, 1216, 1097, 756 cm1; 1H NMR (CDCl3, 300 MHz):
d
0.83
128.1, 128.0, 126.3, 94.4, 85.0, 70.5, 69.5, 65.5, 56.0, 50.4, 41.3, 39.0,
38.0, 34.2, 30.0, 26.0, 23.0, 21.0, 19.1, 14.0; Anal. Calcd for C27H33ClO5:
C, 68.56; H, 7.03. Found: C, 68.34; H, 7.39. LC/MS: m/z 473.0 (Mþ).
(s, 3H), 1.25 (s, 3H), 1.53e1.77 (m, 4H), 1.89e2.24 (m 4H), 2.55 (bt,
J ¼ 6.9 Hz, 2H), 3.43e3.60 (m, 4H), 4.24 (dd, J ¼ 10.5, 6.9 Hz, 2H),
4.54 (d, J ¼ 10.5 Hz, 2H), 4.82 (bs, 1H), 4.93 (bs,1H), 5.01 (s, 1H), 5.67
(s, 1H), 6.79 (d, J ¼ 8.1 Hz, 2H), 6.92 (m, 1H), 7.33 (d, J ¼ 8.1 Hz, 2H),
9.80 (s, 1H); Anal. Calcd for C27H34O6: C, 71.34; H, 7.54. Found: C,
71.74; H, 7.81. LC/MS: m/z 454.5 (Mþ).
6.2.2. Synthesis of 19-(N-tosylaminocarbonyl)-andrographolide (8)
To a suspension of NaH (30 mg, 1.25 mmol) in dry THF (20 mL),
Andro (50 mg, 0.142 mmol) was added at 0 ꢁC under N2 atmosphere
and the mixture was stirred for 30 min. To the reaction mixture, p-
toluenesulfonyl isocyanate (0.024 mL) in dry THF (5 mL) was added
and the mixture was stirred for 2 h at 0 ꢁC. The reaction mixture
was subjected to the usual work up to give the derivative 8; Yield:
34 mg (44%), colorless solid, Mp 198e199 ꢁC; IR (KBr): 3620, 3240
(b), 2960, 1750, 1597 cmꢂ1; 1H NMR (CDCl3 þ DMSO-d6, 400 MHz):
6.2.1.4. 3,19-(20,40-Dihydroxybenzylidene)-andrographolide (5). Yield:
107 mg (79%), pale yellow solid, Mp 208e209 ꢁC; IR (CHCl3): 3422,
2932, 1751, 1216, 1023, 668 cmꢂ1; 1H NMR (CDCl3, 500 MHz):
d 0.87
(s, 3H), 1.31 (s, 3H), 1.63e1.92 (m, 4H), 2.04e2.34 (m, 4H), 2.62 (t,
J ¼ 6.5 Hz, 2H), 3.39e3.58 (m, 2H), 4.10 (d, J ¼ 10.0 Hz, 2H), 4.32 (dd,
J ¼ 10, 6.5 Hz, 2H), 4.52 (m, 2H), 4.83 (bs, 1H), 4.88 (bs, 1H), 5.10 (bs,
1H), 5.78 (s, 1H), 6.74 (m, 1H), 6.54 (d, J ¼ 8.5 Hz, 1H), 7.02 (m, 1H),
7.47 (d, J ¼ 8.5 Hz, 1H), 9.75 (s, 1H), 11.48 (s, 1H); Anal. Calcd for.
C27H34O7: C, 68.92; H, 7.28. Found: C, 69.24; H, 7.60. LC/MS: m/z
470.5 (Mþ).
d
0.70 (s, 3H), 1.15 (s, 3H), 1.22e1.38 (m, 4H), 1.62e1.78 (m, 5H),
1.82e1.88 (m, 3H), 2.10 (s, 3H), 2.34 (t, J ¼ 6.9 Hz, 2H), 3.93 (m, 2H),
4.01 (d, J ¼ 9.6 Hz, 2H), 4.29 (dd, J ¼ 9.6, 6.9 Hz, 2H), 4.45 (m, 1H),
4.89 (s, 1H), 5.05 (s, 1H), 6.98 (m, 1H), 7.39 (d, J ¼ 7.3 Hz, 2H), 7.85 (d,
J ¼ 7.3 Hz, 2H); 13C NMR (CDCl3, 125 MHz):
d 172.2, 152.0, 151.3,
144.3, 144.0, 137.2, 136.1, 129.3, 129.1, 128.0, 127.5, 126.0, 83.0, 75.3,
65.0, 61.0, 55.0, 42.0, 38.5, 38.0, 36.1, 29.5, 23.0, 22.4, 21.5, 21.4, 21.0,
15.0; Anal. Calcd for C28H37NO8S: C, 61.41; H, 6.81; N, 2.56; S, 5.85.
Found: C, 61.20; H, 6.61; N, 2.24; S, 5.64. LC/MS: m/z 547.6 (Mþ).
6.2.1.5. 3,19-(30-Nitrobenzylidene)-andrographolide
(6). Yield:
88 mg (64%), colorless solid, Mp 161e163 ꢁC; IR (neat): 3388, 2940,
1727,1677,1120,1021, 905 cmꢂ1; 1H NMR (CDCl3, 500 MHz): 0.94 (s,

M.M. Uttekar et al. / European Journal of Medicinal Chemistry 56 (2012) 368e374
373
6.2.3. General procedure for synthesis of C-14 ester of
andrographolide (9e10)
was treated with either test compound or the solvent control for 1 h
at 37 ꢁC. AZT (SigmaeAldrich Inc.) was used as positive control,
whereas negative control comprised of cells with untreated HIV.
Subsequently, pre-treated virus were added in duplicate to TZM-bl
cells and incubated for 4 h at 37 ꢁC in humidified atmosphere of
5% CO2 followed by washing with 50 mM PBS to remove cell free
virus. Cells were treated with fresh culture medium supplemented
with compounds 1e10. After 48 h incubation, the cells were washed
twice with PBS. For quantitation of luciferase activity, cells were
A suspension of Andro (125 mg, 0.3 mmol) in dichloroethane
(2 mL) was cooled to 0 ꢁC under N2 atmosphere and triethyl amine
(60 mg, 0.6 mmol) was added. 2,6-Dichloronicotinoyl chloride
(83 mg, 0.4 mmol) or Salicyloyl chloride (62 mg, 0.4 mM) was
dissolved in dichloroethane (1 mL) and added dropwise to the
reaction mixture. After addition of the acid chloride, the reaction
mixture was allowed to warm slowly to ambient temperature and
stirred for 4 h. The reaction mixture was subjected to the usual
work up followed by silica gel column chromatography and elution
with petroleum ether (60e80 ꢁC)/ethyl acetate (60:40) to give the
ester derivatives 9 and 10 respectively.
lysed in 150 mL of 1X Promega cell culture lysis buffer (Promega
Corporation, Madison, Wisconsin, USA) and lysate was centrifuged
at 14,000ꢃ g for 10 min at 4 ꢁC. Luciferase activity in the cell lysate
was measured by the addition of 60 mL of cell lysate to 30 mL of
luciferase substrate (Promega Corporation) and chemiluminescence
was measured using Luminometer (FLUostar Optima, BMG Labtech,
Offenburg, Germany). The results are expressed as percent inhibi-
tion in HIV infection, calculated by dividing the luciferase activity in
experimental group (i.e. in presence of test compounds/AZT) by
luciferase activity in infected cells in absence of test compounds/AZT
multiplied by hundred. Percent inhibition was calculated by sub-
tracting the above value from hundred. Statistical analyses of
doseeresponse inhibition were done using nonlinear curve-fitting
program Prism to obtain IC50 values (concentration eliciting a half-
maximal inhibitory response).
6.2.3.1. 14-(20,60-Dichloronicotinoyl) ester of andrographolide
(9). Yield: 110 mg (59%), colorless solid, Mp 196e198 ꢁC; IR (KBr):
3326, 2920, 1726, 1278, 1071, 890 cmꢂ1; 1H NMR (CDCl3, 300 MHz):
d
0.61 (s, 3H), 1.23 (s, 3H), 1.61e1.82 (m, 4H), 1.92e2.10 (m, 4H), 2.34
(bt, J ¼ 6.5 Hz, 2H), 3.21e3.54 (m, 5H), 4.14 (dd, J ¼ 11.7, 6.5 Hz, 2H),
4.42 (d, J ¼ 11.7 Hz, 2H), 4.62 (bs, 1H), 4.70 (bs, 1H), 6.02 (m, 1H),
7.01 (m, 1H), 7.40 (d, J ¼ 8.1 Hz, 1H), 8.15 (d, J ¼ 8.1 Hz, 1H); Anal.
Calcd for C26H31Cl2N O6. C, 59.55; H, 5.96; N, 2.67. Found: C, 59.84;
H, 5.67; N, 2.98. LC/MS: m/z 524.4 (Mþ).
6.2.3.2. 14-Salicylate ester of andrographolide (10). Yield: 113 mg
(68%), colorless solid, Mp 209e211 ꢁC; IR (CHCl3): 3408, 2932, 1747,
6.3.3. Cell-based fusion assay
1257, 1051, 752 cmꢂ1; 1H NMR (CDCl3, 300 MHz):
d
0.85 (s, 3H), 1.14
Either HL2/3 or TZM-bl (1.0 ꢃ 105) cells were pre-incubated with
increasing amount of Andro and its derivatives 6 and 9 for 1 h at
37 ꢁC in humidified atmosphere of 5% CO2. Cells incubated with
appropriate volume of solvent used to dissolve various compounds
and Bicyclam (AIDS Research and Reference Reagent Program,
Division of AIDS, NIAID, NIH, USA) were used as respective negative
and positive controls. Subsequently, cells were washed to remove
the unbound compounds and treated HL2/3 cells were co-cultured
with untreated TZM-bl cells and vice versa at 1:1 cell density ratio
(1.0 ꢃ 105 cells/well) in a 12-well culture plate (Greiner Bio One) for
48 h at 37 ꢁC in humidified atmosphere of 5% CO2. Additional
controls comprised of incubation of HL2/3 and TZM-bl cells, sepa-
rately. Following incubation, luciferase activity was determined as
described above. The results were expressed as percent inhibition
in luciferase expression, calculated by dividing luciferase activity in
experimental group (i.e. in presence of test compounds/Bicyclam)
by luciferase activity in cells in absence of test compounds/Bicy-
clam multiplied by hundred. Percent inhibition was calculated by
subtracting the above value from hundred. Statistical analyses of
doseeresponse inhibition were done using nonlinear curve-fitting
program Prism to obtain IC50 values.
(s, 3H), 1.38e1.69 (m, 4H), 1.75e1.87 (m, 4H), 2.02 (t, J ¼ 6.2 Hz, 2H),
3.34e3.47 (m, 4H), 3.51 (s, 1H), 4.34 (dd, J ¼ 11.7, 6.2 Hz, 2H), 4.54
(d, J ¼ 11.7 Hz, 2H), 4.78 (bs, 1H), 4.87 (bs, 1H), 6.06 (m, 1H), 6.88 (m,
1H), 7.08 (d, J ¼ 7.9 Hz, 1H), 7.32 (d, J ¼ 7.9 Hz, 1H) 7.47 (t, J ¼ 7.4 Hz,
1H), 7.95 (t, J ¼ 7.4 Hz, 1H), 8.21 (s, 1H). Anal. Calcd for C27H34O7; C,
68.92; H, 7.28. Found: C, 69.24; H, 7.59. LC/MS: m/z 470.5 (Mþ).
6.3. Biology
6.3.1. Cytotoxicity assay using MTT
To assess cell viability in the presence of Andro and derivatives,
MTT assay was performed. Briefly, 6.0 ꢃ 103 TZM-bl cells were
seeded per well in 96-well culture plate (Greiner Bio One GmbH,
Frickenhausen, Germany) and incubated in humidified atmosphere
for 24 h at 37 ꢁC in presence of 5% CO2. Following incubation, cells
were treated with varying concentrations of Andro and derivatives
(4, 20,100 and 500
mM) or solvent (used to prepare the compounds)
for 48 h. After incubation, 20
mL of methyl thiazolyl diphenyl-
tetrazolium bromide (MTT reagent, 5 mg/mL in 50 mM PBS) was
added to the cells and further incubated for 4 h at 37 ꢁC. After
incubation, cells were centrifuged at 14,000ꢃ g for 15 min and
supernatant was discarded. Following that 200
mL of MTT solvent
6.3.4. HIV-1 RT and protease inhibition assay
[20% sodium dodecyl sulfate (SDS) and 50% dimethyl formamide
(DMF) in 50 mM PBS] was added to the wells and culture plate was
left for overnight incubation at 37 ꢁC. Absorbance was read on ELISA
reader (BioTek Instruments Inc, Highland Park, VT, USA) at 570 nm
with 690 nm reference filter. Percent viability was calculated by
dividing absorbance of test sample treated cells by absorbance of
untreated cells, multiplied by hundred. The CC50 values (cytotoxic
concentration of the compound leading to reduction in cell viability
by 50%) of Andro and derivatives were calculated using the
nonlinear curve-fitting software Prism (GraphPad-Prism).
Andro and derivatives (2e10) were assessed for HIV-1 RT
inhibitory activity by using the commercially available ELISA kit
(Roche Diagnostics GmbH, Roche Applied Science, Mannheim,
Germany) according to manufacturer’s protocol. Nevirapine (1 mM)
was used as the positive control in the assay. These were also
assessed for inhibition of HIV-1 protease activity by the commer-
cially available protease assay kit (AnaSpec, Fremont, CA) following
the manufacturer’s protocol. Saquinavir (1 mM) was used as positive
control in this assay.
6.4. Molecular modeling
6.3.2. Anti-HIV assay in TZM-bl cells
TZM-bl cells (4.0 ꢃ 104/well) were seeded in 24-well plate
(Greiner Bio One) and kept for overnight incubation. In separate vial,
CXCR4-tropic HIV-1 NL4.3 virus at aconcentrationequivalent to0.05
multiplicity of infection (MOI) with respect to seeded TZM-bl cells
6.4.1. General
All computational studies were done using SYBYL-X 1.2 (Tripos
Ltd., St. Louis, MO, USA). The docking study for all compounds (1e10)
was performed with the Surflex-Dock program. All parameters used

374
M.M. Uttekar et al. / European Journal of Medicinal Chemistry 56 (2012) 368e374
in docking were default except for explained. These studies were
carried out with the X-ray crystal structure of HIV-1 envelope
protein gp120 at V3 loop region and were compared with well-
known betulinic acid derivative IC9564 [20,21].
the Department of Chemistry and Sophisticated Analytical Instru-
mentation Facility, Indian Institute of Technology, Bombay and Dr.
Sudha Srivastava of Tata Institute of Fundamental Research,
Mumbai for NMR spectral data. We would also like to thank Dr.
Debashis Mitra, National Centre for Cell Science, Pune for making
available TZM-bl cell line and NIH AIDS Research and Reference
Reagent Program, Division of AIDS, NIAID, NIH for providing us the
molecular clone of HIV (NL4.3).
6.4.2. Receptor setup
The target protein gp120 at V3 loop region (PDB ID: 2B4C) was
taken, water molecules and subunits were removed, and
AMBER7FF99 charges were added to the protein. Hydrogens were
added and their positions were optimized by staged minimization
using Tripos forcefield and Powell method. Initial optimization was
kept as Simplex and Termination was set as Gradient, which was
less than 0.05 kcal/(mol Å). Maximum displacement was set at 0.01,
minimum energy change was kept at 0.05, Simplex threshold was
kept at 1000.0 and 1000 iterations were performed. The minimized
protein was defined as the receptor using binding site module of
SYBYL-X 1.2. The binding site was defined and then modified to
accommodate all important interacting residues in V3 loop region
of gp120 protein [28]. This prepared protein was taken as the target
receptor for the docking procedure [29,30].
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6.4.5. Validation of docking methodology
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loop region of gp120 (Fig. 1). The docking score was found to be
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