Synthesis of thiocarbohydrazide and carbohydrazide derivatives as possible biologically active agents

Article abstract of DOI:10.1007/s00044-013-0684-3

A series of new β-isatin aldehyde-N,N′-thiocarbohydrazone, bis-β-isatin thiocarbohydrazones, bis-β-isatin carbohydrazones was synthesized by condensation of 5-substituted isatin with thiocarbohydrazide or carbohydrazide. The chemical structures of the newly synthesized compounds were confirmed by FT-IR, ¹H NMR, and mass spectral analysis. The synthesized compounds were evaluated for in vitro antiviral activity against various strains of DNA and RNA viruses, but exhibited moderate antiviral activity compared with the reference compounds. Among all the compounds 6c exhibited the highest chemoprevention activity in a two-stage mouse-skin carcinogenesis test. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-013-0684-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0684-3
ORIGINAL RESEARCH
Synthesis of thiocarbohydrazide and carbohydrazide derivatives
as possible biologically active agents
•
Kiran Gangarapu Sarangapani Manda Anvesh Jallapally
•
•
•
Sreekanth Thota Subhas S. Karki Jan Balzarini
•
•
•
Erik De Clercq Harukuni Tokuda
Received: 23 March 2013 / Accepted: 25 July 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of new b-isatin aldehyde-N,N0-thio-
carbohydrazone, bis-b-isatin thiocarbohydrazones, bis-b-
isatin carbohydrazones was synthesized by condensation of
5-substituted isatin with thiocarbohydrazide or carbohyd-
razide. The chemical structures of the newly synthesized
Introduction
Isatin is a versatile lead molecule for designing potential
antiviral agents. Isatin derivatives display broad spectrum
pharmacological properties, including cytotoxic (Reddy
et al., 2012; Alcaide et al., 2012), antibacterial (Akhaja and
Raval, 2011), antifungal (Reddy et al., 2011), antiviral
(Kang et al., 2011; Bacani et al., 2011; Sin et al., 2009),
anti-HIV (Bal et al., 2005), and antimalarial (Hans et al.,
2011) activities. The first clinically approved antiviral
agent, N-methylisatin-b-thiosemicarbazone (methisazone)
was active against smallpox and vaccinia viruses (Bauer,
1985). N,N0-disubstituted thiosemicarbazone derivatives of
isatin were inhibitory to HIV-1 replication (Teitz and
Ronen, 1984). N-methylisatin-b-thiosemicarbazone is an
inhibitor of Japanese encephalitis virus infection (Sebastain
et al., 2008). The general configuration of the isatin mol-
ecule resembles that of the purines and also tryptophan and
1
compounds were confirmed by FT-IR, H NMR, and mass
spectral analysis. The synthesized compounds were eval-
uated for in vitro antiviral activity against various strains of
DNA and RNA viruses, but exhibited moderate antiviral
activity compared with the reference compounds. Among
all the compounds 6c exhibited the highest chemopreven-
tion activity in a two-stage mouse-skin carcinogenesis test.
Keywords Isatin ꢀ Thiocarbohydrazide ꢀ
Carbohydrazide ꢀ Antiviral ꢀ Anti-influenza ꢀ
Carcinogenesis
K. Gangarapu (&)
S. Thota
Department of Chemistry, Colorado State University,
Fort Collins 80523, CO, USA
Department of Pharmaceutical Chemistry, Kakatiya Institute
of Pharmaceutical Sciences, Pembarthy (V), Hasanparthy (M),
Warangal 506 371, AP, India
e-mail: gangakiran1905@gmail.com
S. S. Karki
Department of Pharmaceutical Chemistry, KLE University’s
College of Pharmacy, Rajajinagar, Bangalore, KN, India
K. Gangarapu
Center for Pharmaceutical Sciences, IST, JNTU, Kukatpally,
Hyderabad 500085, India
J. Balzarini ꢀ E. De Clercq
Rega Institute for Medical Research, KU Leuven, Leuven,
Belgium
S. Manda
Department of Pharmaceutical Chemistry, University
College of Pharmaceutical Sciences, Kakatiya University,
Warangal 506 009, AP, India
H. Tokuda
Department of Complementary and Alternative Medicine,
Clinical R&D, Graduate School of Medical Science Kanazawa
University, Kanazawa, Japan
A. Jallapally
Department of Pharmaceutical Chemistry, Acharya and BM
Reddy College of Pharmacy, Soldevanahalli, Bangalore, KN,
India
123

Med Chem Res
its derivatives, but there is no evidence available as to
whether isatin-b-thiosemicarbazone acts as an antimetab-
olite to these compounds (Bauer and Sadler, 1960). Recent
studies have shown that isatin could inhibit the protease,
caspase, MAO and COX-2 enzymes (Medvedev et al.,
2007).
requirements necessary for the retention of antiviral
activity by heterocyclic thiosemicarbazone in neurovacci-
nia infections in mice. Viral infections primarily affect the
respiratory and gastrointestinal system; epithelium, mucous
membranes and endothelium of skin, mouth and genitals;
lymphoid tissue, liver and other organs; and the central
nervous system (Bruce et al., 2007).
Viral pathogens are responsible for many human dis-
eases and examples of diseases caused by viruses include
the common cold, smallpox, AIDS, cold sores, human
herpes virus, papilloma virus, etc. (Prado-Prado et al.,
2009). The worldwide market for antiviral drugs is esti-
mated to be approximately 20 billion US dollars and is
expected to grow rapidly as new therapies become avail-
able. The need for effective antiviral drugs is further
emphasized by the lack of vaccines for most respiratory
tract virus infections [adenovirus, rhinovirus, parainfluenza
virus and respiratory syncytial virus (RSV), the widely
occurring human papilloma viruses, and herpes simplex
virus (HSV), varicella-zoster virus (VZV), Epstein–Barr
virus, cytomegalovirus (CMV), and the vast array of
hemorrhagic fever viruses (De Clercq, 2002)]. Millions of
people worldwide are affected by infectious diseases
caused by viruses. Further, widespread viral resistance has
renewed the interest in the quest for new antiviral agents.
Earlier work could clearly define the structural
Recently, we have reported that b-isatin-N,N0-thiocar-
bohydrazone derivatives could have antimicrobial and
antioxidant activity (Kiran et al., 2012a). Thiosemicarba-
zones were the first antiviral compounds recognized to
possess broad-spectrum antiviral activity against a range of
DNA and RNA viruses [Castro et al., 2011]. Earlier reports
have described antiviral activity of isatin-b-thiosemicar-
bazone and isatin-b-semicarbohydrazone against different
viral strains [Quenelle et al., 2006; Pandeya et al., 2005].
However, the biological activities of isatin-b-thiocarbo-
hydrazone and isatin-b-carbohydrazone have not been
evaluated against DNA and RNA viruses. In connection
with our ongoing work on isatin (Kiran et al., 2012b), we
are now reporting a simple and efficient method for the
synthesis of some b-isatin-N,N0-thiocarbohydrazone (4a–
g), bis-b-isatin thiocarbohydrazone, and bis-b-isatin thio-
carbohydrazone derivatives (6a–e) as depicted in Schemes
1 and 2. These compounds were screened for in vitro
Scheme 1 Schematic steps of
b-isatin aldehyde-N,N0-
thiocarbohydrazones derivatives
S
S
H
EtOH
Ar
C
H
N
NH
C
NH NH2
Ar CHO
H2N N C NH NH2
+
gl. CH3COOH
synthesis
2
1
O
O
N
R1
3
S
H
N
NH
N
C
N
Ar
C
H
O
N
R1
4
N
O2N
Ar =
S
NH
4c
4a
4b
4d
Cl
H3CO
R1= C6H5CH2-
4e
OCH
3 4f
4g
123

Med Chem Res
X
C
H
H
O
N N
N N
X
R
2
R
R
H
EtOH
H2N N C NH NH2
O
+
O
O
gl. CH3COOH
N
N
N
H
X= O,S
H
H
5
6
6a: R= H; 6b:R=Cl; 6c: R= CH3; 6d:R= NO2; 6e=H, X=S
Scheme 2 Schematic steps of Bis-isatin derivatives synthesis
antiviral activity and also in a two stage mouse-skin car-
cinogenesis test.
compound are comparable and show m.p 170–172 °C
similar to that reported in the literature (Zhou et al., 2010).
Synthesis of monothiocarbohydrazones (2a–g)
Experimental
An equimolar mixture of thiocarbohydrazide (0.01 mol),
substituted aldehyde (0.01 mol), and a few drops of glacial
acetic acid were taken in 30 ml of ethanol in 250 ml
conical flask. The resulting mixture was heated under
reflux for 1 h. Then the reaction mixture was cooled
overnight and the precipitate was collected by filtration.
Further it was purified by recrystallization with ethanol.
The synthesized compounds were comparable with the
reported literature (Mohamed et al., 1991).
Materials and methods
Melting points (m.p.) were determined in open capillaries,
using the Toshniwal melting point apparatus, expressed
in °C and were uncorrected. FT-IR spectra (KBr) were
recorded on a thermo Nicolet Nexus 670S series. NMR
spectra were recorded on an Avance-300 MHz in DMSO-
d6 solvent using TMS as an internal standard (chemical
shifts in d, ppm). Mass spectra were obtained on a LC-
MSD-Trap-SL. Reactions were monitored using thin layer
chromatography (TLC) on aluminum-backed precoated
silica gel 60 F254 plates (E Merck). The purity of the
compounds was checked on silica gel-coated aluminum
sheets by TLC. Column chromatography was performed by
using Qualigen’s silica gel (60–120 mesh). All the solvents
were AR grade and distilled before use. 5-Substituted
isatins were purchased from Himedia (Mumbai, India).
Indole-3-carboxaldehyde was obtained from E. Merck
(India). All substituted aldehydes were purchased from Sd.
Fine Chemicals.
Synthesis of b-isatin aldehyde-N,N0-thiocarbohydrazones
(4a–g)
To a solution of monothiocarbohydrazone (2a–g, 0.01 mol)
and substituted isatin (3, 0.01 mol) in ethanol, a few drops
of glacial acetic acid were added to initiate the reaction.
The progress of the reaction was monitored by TLC. The
residue was cooled, kept overnight and the precipitate was
collected by filtration. The solid was purified by column
chromatography, and subsequently by recrystallization
with ethanol.
General procedure
Synthesis of bis-isatin derivatives (6a–e)
Synthesis of isatin derivatives
A mixture of thiocarbohydrazide or carbohydrazide
(0.01 mol) was mixed with appropriate substituted isatin
derivatives (0.02 mol) in ethanol–acetic acid (1:1). The
reaction mixture was refluxed for 2 h. Then the reaction
mixture was cooled, kept overnight and the precipitate was
collected by filtration. The resulting solid was purified by
column chromatography and subsequently by recrystalli-
zation with ethanol.
The synthesis of isatin derivatives was carried out using a
modified Sandmeyer methodology (Henry and Blatt, 1964).
N-benzyl isatin is prepared by a microwave method as
reported in the literature (Shmidt et al., 2008). All the isatin
derivatives exhibit same physical properties as reported in
the literature.
Synthesis of thiocarbohydrazide (1)
Spectral data of the synthesized compounds
The synthesis of thiocarbohydrazide was carried out by the
Taguchi method. The physical data of the synthesized
3-[(1H-indol-3-ylmethylidene)amino]-1-{[(3Z)-2-oxo-2,
3-dihydro-1H-indol-3-ylidene]amino}thiourea (4a) IR
123

Med Chem Res
(m cm-1): 3226 (br, N–H, lactam), 3138, 3070 (w, C–H,
Ar), 1705 (s, C=O, lactam), 1337 (s, C=S), 1538 (s, C=N),
(m cm-1): 3345 (N–H), 3050 (C–H, Ar), 1630 (C=O, lac-
tam), 900–690 (Ar–H), 785 (C–Cl); 1H-NMR (DMSO- d6)
d ppm; 14.7 (s, 1H), 13.6 (s, 1H), d 11.2 (s, 1H), 7.93 (s,
1H), 7.85–7.79 (m, 2H), 7.68 (dd, J = 7.5, 1.6 Hz, 1H),
7.60 (dd, J = 7.4, 1.6 Hz, 1H), 7.48 (td, J = 7.4, 1.5 Hz,
3H), 7.27–7.20 (m, 1H); mass (ESI–MS): m/z 357 (M?,
100 %), 359 (M?2, 38 %). Elemental analysis for
C16H12N6O3S: calculated (%) C: 53.71; H: 3.38; N: 19.57;
found: C: 53.68; H: 3.42; N: 19.76.
1
1463 (s, C=C). H-NMR (DMSO-d6) d ppm: 14.1 (s, 1H,
NH), 12.2 (s, 1H, NH), 11.7 (s, 1H, NH), 11.3 (s, 1H, NH),
8.67 (1H, =CH–), 8.47 (1H, N=CH), 7.87 (dd, J = 7.4,
1.6 Hz, 1H), 7.64–7.52 (m, 3H), 7.49–7.40 (m, 2H), 7.34
(td, J = 7.4, 1.5 Hz, 1H), 6.59 (d, J = 7.5 Hz, 1H). 13C
NMR (DMSO-d6) d ppm: 173.9 (C=S), 162 (C=O), 143
(C=N), 110–137 (Ar–C); mass (ESI–MS): m/z 363 (M?1,
100 %), 385 (M?Na, 80 %). Elemental analysis for
C18H14N6OS: calculated (%) C: 59.65; H: 3.89; N: 23.19;
found: C: 59.29; H: 3.24; N: 23.45.
1-{[(3,4-Dimethoxyphenyl)methylidene]amino}-3-{[(3Z)-
2-oxo-2,3-dihydro-1H-indol-3-ylidene]amino}thiourea
(4f) IR (m cm-1): 3348 (N–H), 3020 (C–H, Ar), 1648
1
3-{[(3Z)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]amino}-1-
[thiophen-2-ylmethylidene)amino]thiourea (4b) IR (m
cm-1): 3316 (N–H), 3026 (w, C–H, Ar), 1708 (s, C=O,
lactam), 1368 (s, C=S), 1395 (s, C=N), 1528 (s, CH=N).
1H-NMR (DMSO-d6) d ppm: 14.7 (s, 1H, NH), 13.3 (s, 1H,
–NH), 11.9 (s, 1H, –NH), 8.90 (s, 1H), 7.65–7.59 (m, 2H),
7.57–7.45 (m, 2H), 7.31 (dd, J = 7.5, 2.8 Hz, 1H),
7.27–7.20 (m, 1H), 6.96 (dd, J = 7.5, 1.5 Hz, 1H); mass
(ESI–MS): m/z 330 (M?1, 100 %). Elemental analysis for
C14H11N5OS2: calculated (%) C: 51.05; H: 3.37; N: 21.26;
found: C: 51.02; H: 3.34; N: 21.18.
(C=O), 1313 (C=S); H-NMR (DMSO-d6) d ppm; 14.7 (s,
1H), 13.1 (s, 1H), d 10.9 (s, 1H), 7.93 (s, 1H), 7.71–7.57 (m,
4H), 7.48 (td, J = 7.5, 1.5 Hz, 1H), 7.14–7.08 (m, 2H), 3.79
(s, 6H); mass (ESI–MS): m/z 384 (M?1, 100 %). Elemental
analysis for C24H21N5OS: calculated (%) C: 56.38; H, 4.47;
N, 18.27; found: C: 56.34; H: 4.46; N: 18.23.
3-{[(3Z)-1-benzyl-2-oxo-2,3-dihydro-1H-indol-3-ylidene]
amino}-1-[(phenylmethylidene)amino]thiourea (4g) IR
(m cm-1): 3360 (N–H), 3050 (C–H, Ar), 1630 (C=O, lac-
tam), 900–690 (Ar, oop); 1H-NMR (DMSO-d6) d ppm;
14.7 (s, 1H), 13.6 (s, 1H), d 11.2 (s, 1H), 7.93 (s, 1H),
6.8–7.5 (Ar–H), 3.2 (s, –CH2, 2H); mass (ESI–MS): m/
z 428 (M?, 100 %), 450 (M?Na, 38 %). Elemental anal-
ysis for C18H17N5O3S: calculated (%) C: 66.81; H: 4.63; N:
16.94; found: C: 66.78; H: 4.58; N: 16.78.
1-({[4-(dimethylamino)phenyl]methylidene}amino)-3-
{[(3Z)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]amino}
thiourea (4c) IR (m cm-1): 3218 (–NH), 3016 (w, C–H,
Ar), 2700 (C–H, str), 1702 (s, C=O, lactam), 1358 (s, C=S),
1
1528 (s, C=N), 1528 (s, CH=N). H-NMR (DMSO- d6) d
ppm: 14.9 (s, 1H, –NH), 14.5 (s, 1H, –NH), 13.0 (s, 1H,
–NH), 8.06 (s, 1H), 7.76 (dd, J = 7.5, 1.5 Hz, 1H), 7.61
(dd, J = 7.5, 1.7 Hz, 1H), 7.53–7.46 (m, 3H), 7.28–7.20
(m, 1H), 6.85–6.79 (m, 2H), 3.02 (s, 6H); mass (ESI–MS):
m/z 367 (M?1, 100 %), 389 (M?Na, 40 %). Elemental
analysis for C18H18N6OS: calculated (%) C: 59.00; H:
4.95; N: 22.93; found: C: 59.02; H: 4.86; N: 22.78.
1,3-Bis({[(3Z)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]ami-
no})urea (6a) IR (m cm-1): 3350–3050 (NH, NH), 3070
(C–H, Ar), 1742 (s, C=O, lactam), 1608, 1472 (C=C, Ar),
1
1650 (C=O), 1610 (C=N). H-NMR (DMSO-d6) ppm; d
13.2 (s, 2H), 7.93 (s, 2H), 7.15–7.79 (m, 6H); mass (ESI–
MS): m/z 349 (M?1, 100 %), 371 (M?Na, 52 %). Ele-
mental analysis for C17H12N6O3: calculated (%) C:58.62;
H:3.47; N:24.13; found: C: 58.58; H: 3.46; N: 24.20.
1-{[(4-Nitrophenyl)methylidene]amino}-3-{[(3Z)-2-oxo-
2,3-dihydro-1H-indol-3-ylidene]amino}thiourea (4d) IR
(m cm-1): 3120 (–NH), 3006 (w, C–H, Ar), 1690 (s, C=O,
lactam), 1358 (s, C=S), 1528 (s, C=N), 1528 (s, CH=N). 1H-
NMR (DMSO-d6) d ppm: 14.2 (s, 1H,–NH), 13.6 (s, 1H,
–NH), 12.0 (s, 1H, –NH), 8.06 (s, 1H), 7.64 (dd, J = 7.5,
1.5 Hz, 1H), 7.64 (dd, J = 7.5, 1.7 Hz, 1H), 7.42–7.26 (m,
3H), 7.28–7.20 (m, 1H), 6.85–6.79 (m, 2H); mass (ESI–
MS): m/z 369 (M?1, 100 %), 391 (M?Na, 30 %). Ele-
mental analysis for C16H12N6O3S: calculated (%) C: 52.17;
H: 3.28; N: 22.81 found: C: 52.12; H: 3.24; N: 22.76.
1,3-Bis({[(3Z)-5-chloro-2-oxo-2,3-dihydro-1H-indol-3-yli-
dene]amino})urea (6b) IR (m cm-1): 3250–3050 (NH,
NH), 3010 (C–H, Ar), 1642 (s, C=O, lactam), 1608, 1472
1
(C=C, Ar), 1650 (C=O), 1610 (C=N). H-NMR (DMSO-d6)
ppm; d 13.2 (s, 2H), 7.93 (s, 2H), 7.15–7.79 (m, 6H); mass
(ESI–MS): m/z 417 (M?1, 100 %), 439 (M?Na,
46 %).Elemental analysis for C17H10Cl2N6O3: calculated (%)
C:48.94; H:2.42;N: 20.14; found: C: 48.89; H: 2.46; N: 20.20.
1,3-Bis({[(3Z)-5-methyl-2-oxo-2,3-dihydro-1H-indol-3-yli-
dene]amino})urea (6c) IR (m cm-1) 3198 (NH), 3010 (C–
H, Ar), 2980 (-CH str), 1730 (C=O), 1680 (s, C=O, lac-
tam), 1608, 1472 (C=C, Ar), 1620 (C=N). 1H-NMR
1-{[(4-Chlorophenyl)methylidene]amino}-3-{[(3Z)-2-oxo-
2,3-dihydro-1H-indol-3-ylidene]amino}thiourea (4e) IR
123

Med Chem Res
(DMSO-d6) ppm; d 11.2 (s, 2H), 10.2 (s, 2H), 7.15–7.79
(m, 6H) 2.2 (s, –CH3); mass (ESI–MS): m/z 377 (M?1,
100 %), 386 (M?Na, 51 %). Elemental analysis for
C19H16 N6O3: calculated (%) C: 60.63; H: 4.28; N: 22.33;
found: C: 60.69; H: 4.26; N: 22.32.
Clercq (1981, 1986). The compounds were evaluated for
antiviral activity by using the following viruses: human
cytomegalovirus (HCMV) strains AD-169 and Davis, herpes
simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-
deficient (TK-), HSV-1 KOS strain resistant to ACV (ACVr),
herpes simplex virus type 2 (HSV-2) strain G, vaccinia virus,
vesicular stomatitis virus (VSV), VZV strain Oka, TK- VZV
strain 07-1, RSV strain Long, VSV, coxsackie B4, parain-
fluenza 3, Reovirus-1, Sindbis, Punta Toro, feline coronavirus
(FIPV: feline infectious peritonitis virus), influenza A virus
subtypes H1N1 and H3N2, and influenza B virus. Antiviral
assays were carried out in Crandell–Rees feline kidney cells
(feline corona virus and feline herpes virus), HeLa cell cul-
tures (vesicular stomatitis virus, coxsackie virus B4, and
RSV), human embryonic lung (HEL) cell cultures [HSV-1
(KOS), HSV-2 (G), vaccinia virus, vesicular stomatitis virus
and HSV-1 TK- KOS ACVr], Madin Darby canine kidney
(MDCK) cells (influenza A H1N1 subtype, influenza A H3N2
subtype, and influenza B) and Vero cell cultures (parainflu-
enza-3 virus, reovirus-1, sindbis virus, coxsackie virus B4,
and Punta Toro virus). The HEL, Vero, and HeLa cell lines
used in this study were monitored for mycoplasma contam-
ination and were found to be mycoplasma-free.
1,3-Bis({[(3Z)-5-nitro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]
amino})urea (6d) IR (m cm-1): 3097 (NH), 3050 (C–H,
Ar), 1702 (C=O), 1618 (s, C=O, lactam), 1608, 1472 (C=C,
Ar), 1626 (C=N) 1355 (–NO2). 1H-NMR (DMSO-d6) ppm;
d 13.5 (s, 2H), 11.5 (s, 2H), 7.15–7.79 (m, 6H); mass (ESI–
MS): m/z 439 (M?1, 100 %). Elemental analysis for
C17H10 N8O7: calculated (%) C: 46.58; H: 2.30; N: 25.56;
found: C: 46.53; H: 2.26; N: 25.54.
1,3-Bis({[(3Z)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]amino})
thiourea (6e) IR (m cm-1): 3150, 3010 (NH, NH) 1650
(C=O), 1610 (C=N), 1350 (NCSN). H-NMR (DMSO-d6)
1
d ppm; d 13.5 (s, 2H), 7.93 (s, 2H), 7.15–7.79 (m, 6H);
mass (ESI–MS): m/z 365 (M?1, 100 %). Elemental anal-
ysis for C17H12 N6O2S: calculated (%) C: 56.04; H: 3.32;
N: 23.06; found: C: 56.03; H: 3.36; N: 23.04.
Biological activities
Screening for inhibition of virus-induced cytopathic effect
in vitro
Antiviral activity assays
The synthesized compounds were evaluated for antiviral
activity according to well-established procedures by De
Confluent cell cultures in microtiter 96-well plates were
inoculated with 100 CCID50 of virus (1 CCID50 being the
Fig. 1 Inhibitory effects of
compounds 6b and 6c on
mouse-skin carcinogenesis
induced by DMBA and TPA.
a Positive control [DMBA
(390 nmol) ? TPA (1.7 nmol)];
b TPA ? 85 nmol of 6b;
c TPA ? 85 nmol of 6c
123

Med Chem Res
virus dose to infect 50 % of the cell cultures) or with 20
plaque forming units (PFU) (for VZV) and the cell cultures
were incubated in the presence of varying concentrations of
the test compounds. Viral cytopathicity or plaque forma-
tion (VZV) was recorded as soon as it reached completion
in the control virus-infected cell cultures that were not
treated with the test compounds. Antiviral activity was
expressed as the EC50 or compound concentration required
to reduce virus-induced cytopathicity or viral plaque for-
mation by 50 %.
counter. The cytostatic concentration was calculated as
CC50 (the compound concentration required to reduce cell
growth by 50 % relative to the number of cells in the
untreated controls). CC50 values were estimated from
graphic plots of the number of cells (percentage of control)
as a function of the concentration of the test compounds.
Cytotoxicity was expressed as minimum cytotoxic con-
centration (MCC) or the compound concentration that
causes a microscopically detectable alteration of cell
morphology.
Cytotoxicity assays
Anti-influenza virus activity
Cytostatic activity were based on the inhibition of HEL cell
growth. HEL cells were seeded at 5 9 103 cells/well into
96-well microtiter plates and allowed to proliferate for
24 h. Then, medium containing different concentrations of
the test compounds was added. After 3 days of incubation
at 37 °C, the cell number was determined with a Coulter
The synthesized compounds were evaluated for their anti-
viral activity against three influenza virus subtypes [A/
Puerto Rico/8/34 (H1N1); A/Hong Kong/7/87 (H3N2), and
B/Hong Kong/5/72]. Antiviral activity was estimated from
the inhibitory effect on virus-induced cytopathic effect, as
determined by microscopical examination and/or the
Table 1 Inhibitory activity of synthesized isatin derivatives against DNA viruses
S. Compounds Human embryonic cell culture (HEL) ECa50 (lM)
no.
Crandell–Rees Human embryonic
feline kidney
cell culture
(CRFK)
cell culture (HEL)
Cytomegalovirus
Herpes
simplex simplex virus
Herpes Vaccinia Vesicular Herpes
stomatitis simplex
virus
Feline Feline Varicella-zoster
corona herpes virus (VZV)
virus-1
(KOS)
virus-2
(G)
virus-1TK- virus
KOS ACVr
virus
AD-169
strain
Davis strain
TK ? VZV TK-
strain
VZV
strain
1
2
3
4
5
6
7
8
9
4a
4b
4c
4d
4e
4f
12 3.5 15 0.4
[20
[20
[20
[100
[100
[100
[20
[100
[100
[100
[100
[100
[100
0.01
1
[20
[20
[100
[100
[100
[20
[100
[100
[100
[100
[100
[100
[100
22
[20
[20
[20
[20
[20
[20
[4
7.4
16 3.7 11.32 0.08 [20
[20
[20
[100
[100
[100
[20
[100
[100
[100
[100
[100
[100
4
[20
[20
[100
[20
[20
[20
[20
[100
[20
[100
[20
[20
–
[20
[20
[20
[20
[20
[20
[20
[100
[100
[20
[20
[100
[100
[100
[20
[100
[100
[100
[100
[100
[100
0.02
2
[100
[100
[100
[20
[100 [100 [100
[100 [100 [20
[100 [100 [20
[100 [100 [20
[100 [100 [100
[100 [100 [20
[20
[20
[20
4g
6a
6b
[100
[100
[20
[100
[100
[100
[100
[100
[100
[100
[250
[250
[250
–
[100
[100
19.7
55.1
[20
10 6c
11 6d
12 6e
[100
[100
[20
[100
[100 [100 [20
[100 [100 [20
13 Ganciclovir 8.1 0.2 5.5 2.36
[100
1.4
–
–
14 Cidofovir
15 Brivudin
16 Acyclovir
1.0 0.05 0.78 0.49
0.9
–
–
–
–
–
–
–
–
–
0.04
0.4
112
4
50
–
–
2.1
0.019
–
143
0.2
[250
–
22
–
–
104.5
–
17 HHA
–
–
–
4.2
11.0
(lg ml-1
)
18 UDA
–
–
–
–
–
–
–
1.6
1.2
–
–
(lg ml-1
)
‘‘–’’ not performed
a
Required to reduce virus-induced cytopathogenicity by 50 %
123

Med Chem Res
formazan-based MTS cell viability test. The EC50 (50 %
Effective concentration), or concentration producing 50 %
inhibition of virus-induced cytopathic effect, was deter-
mined by visual scoring of the CPE, or by measuring the
cell viability with the colorimetric formazan-based MTS
assay. Cytotoxicity of the test compounds was expressed as
the compound concentration causing minimal changes in
cell morphology (MCC), or the concentration causing
50 % cytotoxicity (CC50), as determined by the MTS
assay.
the skin (Fig. 1). One hour before each treatment with TPA,
the mice were treated with the samples (85 nmol) in ace-
tone (0.1 ml). The incidence of papillomas was examined
weekly over a period of 20 weeks (Tanaka et al., 2001), the
percentages of mice bearing papillomas (Fig. 2a) and the
average number of papillomas per mouse (Fig. 2b) were
recorded.
Results and discussion
Two-stage mouse-skin carcinogenesis test
A series of new b-isatin aldehyde-N,N0-thiocarbohydraz-
one, bis-b-isatin thiocarbohydrazones, and bis-b-isatin
carbohydrazones derivatives was synthesized by micro-
wave-oriented reaction by two-step procedure as depicted
in Schemes 1 and 2. The synthesized compounds were
purified by column chromatography. All of the derivatives
Specific pathogen-free female ICR mice (6 weeks old,
body weight approx. 30 g) were obtained from Japan SLC
Inc., Shizuoka, Japan, and the animals were housed, five
per polycarbonate cage, in a temperature-controlled room
at 24 2 °C and given food and water ad libitum
throughout the experiment. Animals were divided into
three experimental groups containing 15 mice each. The
back (2 9 8 cm2) of each mouse was shaved with surgical
clippers, and the mice were topically treated with 7,12-
dimethyl benz[a]anthracene (DMBA) (100 lg, 390 nmol)
in acetone (0.1 ml) an initiating treatment. One week after
the initiation, papilloma formation was promoted twice
weekly by the application of 12-O-tetradecanoylphorbol-
13-acetate (TPA) (1 lg, 1.7 nmol) in acetone (0.1 ml) to
1
were characterized by FT-IR, H NMR, and mass spectral
data. The FT-IR spectra of the compounds revealed
absorption bands in the region 1,509–1,610 cm-1 corre-
sponding to C=N stretching bands. Absence of carbonyl
(C=O) peak around 1,715 cm-1 revealed the formation of
1
Schiff bases. The H NMR spectrum of the synthesized
compounds showed only one set of signals for the aromatic
protons around 6.73–7.80 ppm, while the –NH of the isatin
and –NH of hydrazones gave rise to two different signals in
the 11–14 ppm range.
Table 2 Inhibitory activity of synthesized isatin derivatives against RNA Viruses
S.
no.
Compound
Antiviral activity ECa50 (lM)
HeLa cell cultures
Vero cell cultures
Vesicular
stomatitis virus
Coxsackie
virus B4
Respiratory
syncytial virus
Para-influenza- Reovirus- Sindbis
3 virus
Coxsackie
virus B4
Punta Toro
virus
1
virus
1
2
3
4
5
6
7
8
9
4a
4b
4c
4d
4e
4f
[20
[4
[20
[4
[20
[4
[20
[20
[20
[20
[100
[20
[100
[20
[100
[20
[20
[20
[100
[20
100
[20
[20
[20
[20
[20
[20
[100
[20
[100
[100
[100
[100
[20
[100
[100
[100
[100
[20
[100
[100
[100
[100
[20
[100
[20
[100
[20
[100
[20
[100
[20
[100
[20
[100
[20
[100
[20
4g
6a
6b
[100
[20
[100
[20
[100
[20
[100
[20
[100
[100
[100
[100
[100
1
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
0.8
[20
[20
[20
[20
10 6c
[20
[20
[20
[20
11 6d
[100
[20
[100
[20
[100
[20
[100
[20
12 6e
13 DS—5,000
[100
[100
[100
[100
(lg ml-1
14 (S)-DHPA
15 Ribavirin
a
)
[250
[250
112
[250
[250
85
[250
146
[250
[250
[250
250
[250
25
4
4
Required to reduce virus-induced cytopathogenicity by 50 %
123

Med Chem Res
Antiviral activity
twelve isatin derivatives using a cell-protection assay. The
data are recorded in Tables 1, 2, 3, and 4.
The novel b-isatin aldehyde-N,N0-thiocarbohydrazones
(4a–g) and bis-isatin (6a–e) were evaluated against various
strains of DNA viruses (i.e., human cytomegalovirus
(HCMV), vaccinia virus (VACV), HSV type 1 (HSV-1)
and type 2 (HSV-2), acyclovir-resistant, ACVr), VZV
(Table 1). The test compounds were also evaluated against
several RNA viruses (i.e., VSV, coxsackie B4 virus, RSV,
parainfluenza virus type 3, reovirus-1, sindbis virus and
Punta Toro virus (Table 2). The EC50 was calculated as the
effective concentration of compound that decreased the
percentage of formazan production in compound treated,
virus-infected cells to 50 % of that produced by com-
pound-free uninfected cells. The 50 % cytostatic concen-
tration (CC50) was calculated as the concentration required
to reduce cell growth by 50 %. MCC is the minimum
cytotoxic concentration that causes a microscopically
detectable alteration of cell morphology. The HEL, Vero,
and HeLa cell lines used in this study were regularly
monitored for mycoplasma contamination and found to be
mycoplasma-free. The antiviral activity was measured for
There was only moderate activity of the 4a and 4b
compounds against both strains of HCMV. Although at
concentrations close to their toxicity levels, no specific
antiviral effects were noted with the evaluated compounds
against HSV-1, (HSV-1 strain KOS), HSV-2 (HSV-2 strain
G), HSV-1 (ACV resistant KOS strain), vaccinia virus,
vesicular stomatitis virus, coxsackie virus B4, respiratory
syncytial virus, parainfluenza-3 virus, reovirus-1, sindbis
virus, and Punta Toro virus. None of the compounds
exhibited specific antiviral effects (i.e., minimal antiviral
effective concentration C fivefold lower than minimal
cytotoxic concentration) against any of the viruses
evaluated.
Anti-influenza virus activity
The anti-influenza virus activities of a series of isatin
derivatives were determined by measuring their inhibitory
effects on virus replication in MDCK cells using three
Table 3 Anti-influenza virus activity and cytotoxicity of compounds in MDCK cell cultures
Compound
Cytotoxicity
Antiviral ECc50 (lM)
CCa50(lM) Minimum cytotoxic
Influenza A H1N1
subtype
Influenza A H3N2 subtype Influenza B
concentrationb
Visual CPE MTS
score
Visual CPE MTS
score
Visual CPE MTS
score
4a
4b
4c
4d
4e
4f
51
20
20
[4
[4
[4
[4
[4
[4
46
[4
[4
[4
[4
[4
[4
[100
[100
[100
[100
[100
[100
[100
46
[100
[100
[100
[100
[100
[100
[100
C20
[100
[100
[100
39 5.5
[100
[100
[100
[20
[100
[100
[100
37 25
[100
[100
[100
[20
[100
[100
[100
27 7.0
[100
[100
[100
[20
[100
[100
[100
27 7.3
[100
[100
[100
[20
[100
[100
[100
45 0.0
[100
[100
[100
[20
[100
[100
[100
37 8.75
[100
4g
6a
6b
6c
6d
6e
[100
[100
[20
[100
[100
[100
[100
[100
[100
47 2.5
72 27
0.4 0.0
55 26
61 32
72 27
72 27
33 1.7
34 9.5
47 2.5
39 5.5
38 1.3
48 31
2.5 0.7
Oseltamivir
carboxylate
0.35 0.2 10.25 9.7 5.45 4.05 6.7 5.3
Ribavirin
[100
[200
[200
[100
[200
[200
2.5 0.5
5
2.8 0.25 3 1.0
2
0.2
2.4 1.6
[200
2.45 0.25
[200
Amantadine
Rimantadine
3.2
0.9
0.4
0.8
0.6
[200
[200
[200
[200
MDCK cells Madin Darby canine kidney cells
a
50 % Cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay
Minimum compound concentration that causes a microscopically detectable alteration of normal cell morphology
b
c
50 % Effective concentration, or concentration producing 50 % inhibition of virus-induced cytopathic effect, as determined by visual scoring
of the CPE, or by measuring the cell viability with the colorimetric formazan-based MTS assay
123

Med Chem Res
Table 4 Cytotoxicity for synthesized isatin derivatives in different cell cultures
S.no
Compound
Human embryonic lung
cell cultures
Crandell-Rees feline
kidney cells
HeLa cell cultures
Vero cell cultures
Minimum cytotoxic
concentrationa (lM)
Minimum cytotoxic
concentrationa (lM)
Minimum cytotoxic
concentrationa (lM)
Minimum cytotoxic
concentrationa (lM)
1
4a
60
86
C20
20
100
100
[100
100
[100
C20
[100
100
100
100
[100
100
–
2
4b
100
63
3
4c
C100
100
[100
[100
[100
100
C100
[100
[100
[100
[100
[100
–
[100
[100
[100
[100
100
4
4d
5
4e
100
6
4f
100
7
4g
C100
C100
100
8
6a
[100
[100
[100
[100
[100
–
9
6b
10
11
12
13
14
15
16
17
18
19
a
6c
[100
C100
100
6d
6e
Acyclovir
Brivudin
Ganciclovir
Cidofovir
DS-5000 (lg ml)
(S)-DHPA
Ribavirin
[400
[300
[350
[300
–
–
–
–
[100
–
–
–
–
–
–
[100
[250
[250
[100
[250
[250
–
–
–
–
Minimum cytotoxic concentration that causes a microscopically detectable alteration of normal cell morphology
Fig. 2 Inhibition effects of compounds 6b and 6c on DMBA–TPA-
induced carcinogenesis. Tumor formation in all mice was initiated
with DMBA (390 nmol) and promoted with TPA (1.7 nmol) twice
weekly, 1 week after initiation. a Percentage of mice with papillomas.
b Average number of papillomas/mouse. Circle control [DMBA
(390 nmol) ? TPA (1.7 nmol)]; filled square TPA ? 85 nmol of 6b;
triangle TPA ? 85 nmol of 6c
strains of the influenza subtype. The antiviral data obtained
by microscopic evaluation of the virus-induced CPE were
confirmed by the MTS cell viability assay and were
expressed as the EC50. The cytotoxicities of the compounds
were expressed as the MCC (the compound concentration
causing minimal alterations in cell morphology as esti-
mated by microscopy) or the CC50 (the compound
concentration causing 50 % reduction in cell viability
based on the MTS assay).
None of the twelve analogs were able to inhibit the
cytopathic effects of three influenza virus subtypes i.e.,
H1N1, H3N2, and influenza B (Table 3), except 4f, 6d, and
6e that showed some activity below the non-toxic con-
centration of 100 lM.
123

Med Chem Res
Two-stage mouse-skin carcinogenesis test
compound 6b. Thus these results suggest that isatin lead
molecules could also be useful as cancer chemopreventive
agents.
In the present study, we selected two compounds 6b and 6c
among the ten compounds to examine their effects on
in vivo two-stage carcinogenesis using mouse-skin papil-
lomas induced by DMBA as an initiator and TPA as a
promoter. During the test, no significant toxic effects, such
as inflammation and lesional damage (edema, erosion, and
ulcer), were observed on the areas of mouse skin topically
treated with test compounds. In our experiment, the body
weight of the animals was not affected by the treatment
with test compounds. As shown in Fig. 2a, the positive
control (DMBA and TPA) group in papilloma-bearing mice
appears rapidly from week 6, and reached 100 % within
10 weeks of promotion. On the other hand, treatment with
compound 6c (85 nmol) along with DMBA/TPA reduced
the percentage of papilloma-bearing mice to 40–70 %
during weeks 10–15, and thereafter 90 % during week 18
and reached 100 % at week 20. As shown in Fig. 2b, in the
positive control group, the number of papillomas per mouse
formed increased rapidly after week 6 and reached 7.9
papillomas/mouse at week 20. On the other hand, mice
treated with compound 6c bore only 5.1 papillomas/mouse
over the period of week 20. A moderate protection is
observed for the compound 6b. The percentage of papillo-
mas increased rapidly from week 7 and reached 100 % after
week 17, and in this group only 1.2, 3.9, and 6.1 papillomas
were formed per mouse at 10, 15, and 20 weeks of pro-
motion. From these results, the inhibitory effects of com-
pound 6c on mouse two-stage carcinogenesis induced by
DMBA and TPA were apparently more potent than those of
compound 6b. Thus, as demonstrated above, the in vivo
anti-tumor promoting activity of compound 6c showed
stronger chemoprevention activity than that of compound
6b. These results suggest that isatin lead molecules may be
useful as cancer chemopreventive agents.
Acknowledgments The authors are very much thankful to M.
Sadalaxmi, President, Kakatiya Institute of Pharmaceutical Sciences,
for providing the laboratory facilities. The antiviral research was
performed by grants of the KU Leuven (GOA 10/14). The authors
thank Leentje Persoons, Frieda De Meyer, Anita Camps, Lies Van
den Heurck and Steven Carmans for excellent technical assistance.
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