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- 2.1. Design

ues to have enormous human health consequences. Dur-
ing 2004, around five million adults and children became Earlier studies have shown that the overall structure of a
infected with HIV and by the end of the year, an esti- widely diverging class of NNRTIs on a closer scrutiny
mated 39.4 million people worldwide were living with exhibits several common features that are reminiscent of
HIV/AIDS. Non-nucleoside reverse transcriptase inhib- a butterfly with a hydrophilic centre as a ÔbodyÕ and two
itors (NNRTIs) have gained a definitive place in the hydrophobic moieties representing the Ôwings.Õ9,10 The
treatment of HIV-1 infections,1 in addition to the nucle- Ôbutterfly-likeÕ conformation has also been proven by a
oside reverse transcriptase inhibitors (NRTIs) and crystallographic analysis of nevirapine.11 A 3D-pharma-
protease inhibitors (PIs), since they do not function cophore model has been derived taking into account five
as chain terminators and do not bind to the dNTP well-known NNRTIs, as shown in Figure 1, to identify
site,2–4 making them less likely to interfere with the a set of pharmacophoric elements required for biological
normal function of other DNA polymerases and there- activity from structurally diverse ligands, as well as to
fore less toxic for the treatment of HIV-infected guide in the design of new and more potent compounds.
patients. In this respect, isatin b-thiosemicarbazone The selected ligands were geometrically optimized based
derivatives were found to demonstrate a range of antivi- on an internal strain energy and the essential structural
ral activities (Moloney leukaemia virus, vaccinia virus), components, such as atoms, centroids of collection of
as reported by earlier studies.5–8 The present study was atoms, electron lone pair positions, etc., were matched
an attempt to develop new anti-HIV drugs based on in the three-dimensional space of the energetically acces-
various structural modifications of thiosemicarbazone sible conformations of the ligands, to arrive at a 3-point
derivatives and 3D-pharmacophoric mapping of the pharmacophore model proposed in Figure 2.
existing NNRTIs.
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
Keywords: Anti-HIV; Isatin; Thiosemicarbazones. bulky diethyl moiety constituting the ÔwingsÕ and was
* Corresponding author. Tel.: +91 1596 244684; fax: +91 1596 found to fulfil the specification of the pharmacophoric
244183; e-mail: [email protected] distance map by complying within the defined range,

0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.046
4452 T. R. Bal et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4451–4455

H C
O N
O O CF3 C
S S Cl O
N N N N
N
N S N O N O
H H H

Benzothiadiazine-1-oxide BHAP (U-90152) Efavirenz

Delavirdine

H S C2H5
O S O N N C N Br
C2H5 NH
Cl NH2
O
N N S
N O N
H
H CH2 R'

Indole Carboxamide Lead Compound Trovirdine

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, for 1 h to form a potassium salt of dithiocarbamate. To
namely least-squares superimposition study has shown the stirred mixture was added hydrazine hydrate
that the RMS fit value of 0.187 shows a good correlation (0.01 mol) and the stirring was continued at 60 °C for
between selected points in the lead compound structure 1 h to obtain N,N-diethyl thiosemicarbazide. This mix-
and corresponding points in the reference molecule, that ture was condensed with isatin in the presence of glacial
is, Efavirenz (Fig. 3). acetic acid to form 1H-indole-2,3-dione-3-(N,N-diethyl
thiosemicarbazone) (Schiff base). The N-Mannich bases
2.2. Synthesis were synthesized further by condensing the acidic imino
group of isatin derivatives with formaldehyde and vari-
The synthesis of thiosemicarbazone derivatives was car- ous secondary amines. The purity of the synthesized
ried out in three steps, as shown in Scheme 1. First, to a compounds was checked by TLC and elemental analy-
solution of diethylamine (0.01 mol) in THF (10 ml) were ses; and the compounds of this study were identified
added potassium hydroxide (0.01 mol) and carbon disul- by spectral data. In general, IR spectra14 showed a
fide (0.75 ml),12 and the mixture was stirred at 15–20 °C C@N (azomethine) peak at 1640 cm
1 and, CH2
 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
Compound AB (Å) BC (Å) CA (Å)
Lower limit Upper limit Lower limit Upper limit Lower limit Upper limit
Delavirdine 4.328 ± 0.04 6.705 ± 0.15 4.256 ± 0.19 7.542 ± 0.35 9.156 ± 0.04 9.382 ± 0.04
Trovirdine 4.235 ± 0.01 6.635 ± 0.02 4.289 ± 0.08 7.269 ± 0.16 9.168 ± 0.04 9.426 ± 0.04
Indole carboxamide 4.359 ± 0.01 6.705 ± 0.20 4.562 ± 0.14 7.478 ± 0.07 9.125 ± 0.04 9.434 ± 0.04
Efavirenz 4.425 ± 0.05 6.689 ± 0.16 4.247 ± 0.23 7.211 ± 0.21 9.145 ± 0.04 9.440 ± 0.04
Benzothiadiazine-1-oxide 4.512 ± 0.02 6.459 ± 0.03 4.269 ± 0.14 7.545 ± 0.01 9.129 ± 0.04 9.406 ± 0.04
Range 4.22–6.70 4.24–7.54 9.12–9.44
Lead compound 4.26 ± 0.18 6.57 ± 0.12 4.27 ± 0.09 6.76 ± 0.15 9.16 ± 0.01 9.33 ± 0.02

Efavirenz

Lead Compound

Figure 3. Superimposition and RMS fit of the proposed lead compound and Efavirenz (RMS value = 0.187).

S O
C2H5 KOH / CS 2 C2H5
HN H2N N C N +
N2H4.H2O O
C2H5 H C 2H5
N
S H
C2H5
1
Glacial Acetic acid N N C N R
H C2H5 HN HCHO
2
O R ,
Schiff Reaction
N
Mannich Reaction
H
S C2H5
N N C N
H C2H5
O
N R
1

CH2 N
2
R
1-9

Scheme 1. Protocol for synthesis.

(Mannich methylene) peak at 2860 and 2840 cm
1. In coupling constants. The spectra showed a singlet at d
the 1H NMR spectra, the signals of the respective 4.8–5.1 ppm corresponding to the –NCH2N– group.
protons of the prepared compounds were verified on The elemental analysis results were within ±0.4% of
the basis of their chemical shifts, multiplicities and the theoretical values.
4454 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-
The synthesized compounds were screened for anti-HIV ity with an EC50 value of 2.62 lM and a selectivity index
activity on the replication of HIV-1 (HTLV-IIIB) in the of more than 17, while not being cytotoxic to the cell line
CEM cell line (Table 2).13 The synthesized thiosemicar- with a CC50 value of 44.90. Other compounds did not
bazone derivatives inhibited the cytopathic effect of show any marked anti-HIV activity below their toxicity
HIV-1 (IIIB), with EC50 values ranging from 2.62 lM threshold. All the synthesized compounds were found to
to >14.50 lM. Compounds 3, 6 and 9 showed significant be less active than the standard drug Efavirenz.

Table 2. Physical constants and biological activity of compounds (1–9) against HIV-1 (HTLV-IIIB) in the CEM cell line
S
C 2H 5
N NH N
C2 H5

O
N

R'

0
Compound R Molecular weight Melting point % yield Anti-HIV-1 activity (lM)
EC50a CC50b SIc

CH2 C6H5
1 N 485 52 72 >8.29 8.29 <1
CH2 C6H5

CH3
2 N 333 66 74 >2.86 2.86 <1
CH3

3 N N CH2 C6H5 464 78 69 3.40 14.00 4.12

4 N N F 468 58 67 >14.50 14.50 <1

5 N N NO2 495 92 70 >11.90 11.90 <1

N N
6 518 64 71 2.62 44.90 17.14
CF3

7 N N 452 62 75 >12.80 12.80 <1

N

CH3

8 N O 401 85 70 >13.30 13.30 <1

CH 3

O
F COOH
9 607 198 76 3.12 37.50 12.02
N N
N
C 2 H5

Efavirenz — 0.78 >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

3. Conclusion 3. Kopp, E. B.; Miglietta, J. J.; Shrutkowski, A. G.; Shih, C.

K.; Grob, P. M.; Skoog, M. T. Nucleic Acids Res. 1991, 19,
On the basis of the 3D-pharmacophoric-distance map 3035.
based on the structures of existing NNRTIs, the thio- 4. Romero, D. L.; Busso, M.; Tan, C. K.; Reusser, F.;
Palmer, J. R.; Poppe, S. M.; Aristoff, P. A.; Downey, K.
semicarbazone derivatives were found to comply to
M.; So, A. G.; Resnick, L. Proc. Natl. Acad. Sci. U.S.A.
be within the defined range for anti-HIV activity. 1991, 88, 8806.
Least-squares superimposition technique also estab- 5. Teitz, Y.; Barko, N.; Abramoff, M.; Ronen, D. Chemo-
lished a good correlation between thiosemicarbazone therapy 1994, 40, 195.
and Efavirenz with a RMS fit value in the agreeable 6. Ronen, D.; Teitz, Y. Antimicrob. Agents Chemother. 1984,
range. Further investigation on structure–activity rela- 26, 913.
tionships and appropriate modification among the syn- 7. Teitz, Y.; Ronen, D.; Vansover, A.; Stematsky, T.; Riggs,
thesized thiosemicarbazone derivatives is likely to J. L. Antiviral Res. 1994, 24, 305.
provide more effective HIV-1 inhibitors with improved 8. Sherman, L.; Edelstein, F.; Shtacher, G.; Avramoff, M.;
efficacy. Teitz, Y. J. Gen. Virol. 1980, 46, 195.
9. Kroeger Smith, M. B.; Rouzer, C. A.; Taneyhill, L. A.;
Smith, N. A.; Hughes, S. H.; Boyer, P. L.; Janssen, P. A.;
Moereels, H.; Koymans, L.; Arnold, E. Protein Sci. 1995,
Acknowledgments 4, 2203.
10. Ren, J.; Esnouf, R.; Hopkins, A.; Ross, C.; Jones, Y.;
Stammers, D.; Stuart, D. Structure 1995, 3, 915.
One of the authors, Ms. Tanushree Bal, deeply acknowl- 11. Mui, P. W.; Jacober, S. P.; Hargrave, K. D.; Adams, J.
edges the Council of Scientific and Industrial Research J. Med. Chem. 1992, 35, 201.
for providing Senior Research Fellowship. The authors 12. Pandeya, S. N.; Sriram, D.; Nath, G.; Clercd, E. De.
also thank Dr. Robert H. Shoemaker, National Cancer Indian J. Pharm. Sci. 1999, 61, 358.
Institute, USA, for his support in biological testing. 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 cm
1; 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,
1. De Clercq, E. Antiviral Res. 1998, 38, 153. NCH2N), 7.01–7.58 (m, 8H, Ar-H), 11.26 (s, 1H, –NH,
2. De Clercq, E. Rev. Med. Virol. 2000, 10, 255. D2O exchangeable).