Synthesis and evaluation of anti-HIV activity of isatin β-thiosemicarbazone derivatives

Article abstract of DOI:10.1016/j.bmcl.2005.07.046

On the basis of pharmacophoric modelling studies of existing NNRTIs, a series of isatin β-thiosemicarbazone derivatives was synthesized and evaluated for their anti-HIV activity in HTLV-III_B strain in the CEM cell line. Three compounds showed significant anti-HIV activity, whereupon compound 6 was found to be the most active compound with an EC₅₀ value of 2.62 μM and a selectivity index of 17.41, while not being cytotoxic to the cell line at a CC₅₀ value of 44.90 μM. Other tested compounds exhibited marked activity below their toxicity threshold.

Full text of DOI:10.1016/j.bmcl.2005.07.046

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4451–4455
Synthesis and evaluation of anti-HIV activity of isatin
b-thiosemicarbazone derivatives
Tanushree Ratan Bal, Balasubramani Anand,
Perumal Yogeeswari and Dharmarajan Sriram*
Medicinal Chemistry Research Laboratory, Pharmacy group, Birla Institute of Technology and Science, Pilani 333031, India
Received 7 May 2005; revised 7 July 2005; accepted 14 July 2005
Available online 22 August 2005
Abstract—On the basis of pharmacophoric modelling studies of existing NNRTIs, a series of isatin b-thiosemicarbazone derivatives
was synthesized and evaluated for their anti-HIV activity in HTLV-IIIB strain in the CEM cell line. Three compounds showed
significant anti-HIV activity, whereupon compound 6 was found to be the most active compound with an EC50 value of
2.62 lM and a selectivity index of 17.41, while not being cytotoxic to the cell line at a CC50 value of 44.90 lM. Other tested
compounds exhibited marked activity below their toxicity threshold.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
2. Chemistry
The human immunodeficiency (HIV) epidemic contin-
ues to have enormous human health consequences. Dur-
ing 2004, around five million adults and children became
infected with HIV and by the end of the year, an esti-
mated 39.4 million people worldwide were living with
HIV/AIDS. Non-nucleoside reverse transcriptase inhib-
itors (NNRTIs) have gained a definitive place in the
treatment of HIV-1 infections,1 in addition to the nucle-
oside reverse transcriptase inhibitors (NRTIs) and
protease inhibitors (PIs), since they do not function
as chain terminators and do not bind to the dNTP
site,2–4 making them less likely to interfere with the
normal function of other DNA polymerases and there-
fore less toxic for the treatment of HIV-infected
patients. In this respect, isatin b-thiosemicarbazone
derivatives were found to demonstrate a range of antivi-
ral activities (Moloney leukaemia virus, vaccinia virus),
as reported by earlier studies.5–8 The present study was
an attempt to develop new anti-HIV drugs based on
various structural modifications of thiosemicarbazone
derivatives and 3D-pharmacophoric mapping of the
existing NNRTIs.
2.1. Design
Earlier studies have shown that the overall structure of a
widely diverging class of NNRTIs on a closer scrutiny
exhibits several common features that are reminiscent of
a butterfly with a hydrophilic centre as a ÔbodyÕ and two
hydrophobic moieties representing the Ôwings.Õ9,10 The
Ôbutterfly-likeÕ conformation has also been proven by a
crystallographic analysis of nevirapine.11 A 3D-pharma-
cophore model has been derived taking into account five
well-known NNRTIs, as shown in Figure 1, to identify
a set of pharmacophoric elements required for biological
activity from structurally diverse ligands, as well as to
guide in the design of new and more potent compounds.
The selected ligands were geometrically optimized based
on an internal strain energy and the essential structural
components, such as atoms, centroids of collection of
atoms, electron lone pair positions, etc., were matched
in the three-dimensional space of the energetically acces-
sible conformations of the ligands, to arrive at a 3-point
pharmacophore model proposed in Figure 2.
The proposed isatin thiosemicarbazone analogue was
designed based on the derived pharmacophoric model
with the thiosemicarbazo moiety (@N–NH–CS–N—)
constituting the ÔbodyÕ and the aryl ring of isatin and
bulky diethyl moiety constituting the ÔwingsÕ and was
found to fulfil the specification of the pharmacophoric
distance map by complying within the defined range,
Keywords: Anti-HIV; Isatin; Thiosemicarbazones.
*
Corresponding author. Tel.: +91 1596 244684; fax: +91 1596
244183; e-mail: dsriram@bits-pilani.ac.in
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.046

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T. R. Bal et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4451–4455
C
H
N
O
O
CF3
C
O
O
O
S
Cl
S
N
N
N
N
N
N
H
N
H
N
H
O
S
BHAP (U-90152)
Delavirdine
Efavirenz
Benzothiadiazine-1-oxide
N
H
N
S
C2H5
C2H5
N
C
N
O
O
Br
S
NH
Cl
NH2
O
N
N
H
S
N
N
H
O
CH2 R'
Trovirdine
Lead Compound
Indole Carboxamide
Figure 1. Schematic representation of a butterfly-like conformation of the existing NNRTIs and the lead compound.
Figure 2. Schematic representation of a butterfly-like configuration of NNRTIS and the pharmacophoric distance map.
as shown in Table 1. Molecular superposition technique,
namely least-squares superimposition study has shown
that the RMS fit value of 0.187 shows a good correlation
between selected points in the lead compound structure
and corresponding points in the reference molecule, that
is, Efavirenz (Fig. 3).
for 1 h to form a potassium salt of dithiocarbamate. To
the stirred mixture was added hydrazine hydrate
(0.01 mol) and the stirring was continued at 60 °C for
1 h to obtain N,N-diethyl thiosemicarbazide. This mix-
ture was condensed with isatin in the presence of glacial
acetic acid to form 1H-indole-2,3-dione-3-(N,N-diethyl
thiosemicarbazone) (Schiff base). The N-Mannich bases
were synthesized further by condensing the acidic imino
group of isatin derivatives with formaldehyde and vari-
ous secondary amines. The purity of the synthesized
compounds was checked by TLC and elemental analy-
ses; and the compounds of this study were identified
by spectral data. In general, IR spectra14 showed a
C@N (azomethine) peak at 1640 cmꢀ1 and, CH2
2.2. Synthesis
The synthesis of thiosemicarbazone derivatives was car-
ried out in three steps, as shown in Scheme 1. First, to a
solution of diethylamine (0.01 mol) in THF (10 ml) were
added potassium hydroxide (0.01 mol) and carbon disul-
fide (0.75 ml),12 and the mixture was stirred at 15–20 °C

T. R. Bal et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4451–4455
4453
Table 1. The pharmacophoric distance model of bioactive NNRTIs and lead compound by molecular mechanics (MM3) force fields
˚
AB (A)
˚
BC (A)
˚
Compound
CA (A)
Upper limit
Lower limit
Upper limit
Lower limit
Upper limit
Lower limit
Delavirdine
Trovirdine
4.328 0.04
4.235 0.01
4.359 0.01
4.425 0.05
4.512 0.02
6.705 0.15
6.635 0.02
6.705 0.20
6.689 0.16
6.459 0.03
4.256 0.19
4.289 0.08
4.562 0.14
4.247 0.23
4.269 0.14
7.542 0.35
7.269 0.16
7.478 0.07
7.211 0.21
7.545 0.01
9.156 0.04
9.168 0.04
9.125 0.04
9.145 0.04
9.129 0.04
9.382 0.04
9.426 0.04
9.434 0.04
9.440 0.04
9.406 0.04
Indole carboxamide
Efavirenz
Benzothiadiazine-1-oxide
Range
4.22–6.70
4.26 0.18
4.24–7.54
4.27 0.09
9.12–9.44
9.16 0.01 9.33 0.02
Lead compound
6.57 0.12
6.76 0.15
Efavirenz
Lead Compound
Figure 3. Superimposition and RMS fit of the proposed lead compound and Efavirenz (RMS value = 0.187).
O
S
C
C2H5
C2H5
C2H5
C2H5
KOH / CS
2
HN
H2N
N
H
N
+
O
N H .H O
2
4
2
N
H
S
C2H5
N
C N
R1
R2
N
Glacial Acetic acid
Schiff Reaction
HN
HCHO
C2H5
H
O
,
N
H
Mannich Reaction
S
C2H5
N
C N
N
C2H5
H
O
R1
N
CH2
N
R2
1-9
Scheme 1. Protocol for synthesis.
(Mannich methylene) peak at 2860 and 2840 cmꢀ1. In
the 1H NMR spectra, the signals of the respective
protons of the prepared compounds were verified on
the basis of their chemical shifts, multiplicities and
coupling constants. The spectra showed a singlet at d
4.8–5.1 ppm corresponding to the –NCH2N– group.
The elemental analysis results were within 0.4% of
the theoretical values.

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T. R. Bal et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4451–4455
2.3. Anti-HIV activity
anti-HIV activity with an EC50 value in the range of
2.62–3.40 lM. Compound 6 exhibited the highest activ-
ity with an EC50 value of 2.62 lM and a selectivity index
of more than 17, while not being cytotoxic to the cell line
with a CC50 value of 44.90. Other compounds did not
show any marked anti-HIV activity below their toxicity
threshold. All the synthesized compounds were found to
be less active than the standard drug Efavirenz.
The synthesized compounds were screened for anti-HIV
activity on the replication of HIV-1 (HTLV-IIIB) in the
CEM cell line (Table 2).13 The synthesized thiosemicar-
bazone derivatives inhibited the cytopathic effect of
HIV-1 (IIIB), with EC50 values ranging from 2.62 lM
to >14.50 lM. Compounds 3, 6 and 9 showed significant
Table 2. Physical constants and biological activity of compounds (1–9) against HIV-1 (HTLV-IIIB) in the CEM cell line
S
C2H5
NH
O
N
N
C2H5
N
R'
Compound
R0
Molecular weight
Melting point
% yield
Anti-HIV-1 activity (lM)
SIc
a
b
EC50
CC50
CH2 C6H5
N
1
2
485
333
52
66
72
74
>8.29
>2.86
8.29
2.86
<1
CH2 C6H5
CH3
CH3
N
<1
3
4
5
6
7
8
CH2
464
468
495
518
452
401
78
58
92
64
62
85
69
67
70
71
75
70
3.40
>14.50
>11.90
2.62
14.00
14.50
11.90
44.90
12.80
13.30
4.12
N
N
N
N
C6H5
F
<1
N
N
N
NO2
<1
N
17.14
<1
CF3
N
N
N
N
>12.80
>13.30
CH3
N
O
<1
CH3
O
F
COOH
9
607
198
76
3.12
0.78
37.50
12.02
N
N
N
C2H5
Efavirenz
—
>200
>256
a 50% effective concentration or concentration required to inhibit HIV-1 induced cytopathicity in cell lines by 50%.
b 50% cytotoxic concentration or concentration required to reduce the viability of mock-infected cell lines by 50%.
c Selectivity index or ratio of CC50 to EC50
.

T. R. Bal et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4451–4455
4455
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3. Conclusion
On the basis of the 3D-pharmacophoric-distance map
based on the structures of existing NNRTIs, the thio-
semicarbazone derivatives were found to comply to
be within the defined range for anti-HIV activity.
Least-squares superimposition technique also estab-
lished a good correlation between thiosemicarbazone
and Efavirenz with a RMS fit value in the agreeable
range. Further investigation on structure–activity rela-
tionships and appropriate modification among the syn-
thesized thiosemicarbazone derivatives is likely to
provide more effective HIV-1 inhibitors with improved
efficacy.
Acknowledgments
One of the authors, Ms. Tanushree Bal, deeply acknowl-
edges the Council of Scientific and Industrial Research
for providing Senior Research Fellowship. The authors
also thank Dr. Robert H. Shoemaker, National Cancer
Institute, USA, for his support in biological testing.
12. Pandeya, S. N.; Sriram, D.; Nath, G.; Clercd, E. De.
Indian J. Pharm. Sci. 1999, 61, 358.
13. Sriram, D.; Bal, T. R.; Yogeeswari, P. Bioorg. Med. Chem.
2004, 12, 5865.
14. Spectral data for compound 6. IR (KBr): 3200, 2860,
2840, 1640, 1620, 1580, 1015 cmꢀ1 1H NMR (CDCl3)
;
References and notes
d (ppm): 1.0 (t, 6H, 2CH3), 2.59 (t, 4H, CH2NCH2), 3.4
(t, 4H, CH2NCH2), 3.49 (q, 4H, 2CH2), 4.80 (s, 2H,
NCH2N), 7.01–7.58 (m, 8H, Ar-H), 11.26 (s, 1H, –NH,
D2O exchangeable).
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