Synthesis, cytotoxic and phytotoxic effects of some new N-4aryl substituted isatin-3-thiosemicarbazones

Article abstract of DOI:10.2174/157018011795514159

A series of N⁴-aryl substituted isatin-3-thiosemicarbazones was prepared by the reaction of isatin with an appropriate thiosemicarbazide in ethanol containing a few drops of acetic acid. The newly synthesized compounds were characterized by mea

Full text of DOI:10.2174/157018011795514159

452
Letters in Drug Design & Discovery, 2011, 8, 452-458
Synthesis, Cytotoxic and Phytotoxic Effects of Some New N4-Aryl
Substituted Isatin-3-thiosemicarbazones
Humayun Pervez*,1, Muhammad Ramzan1, Muhammad Yaqub1, and Khalid Mohammed Khan*,2
1Department of Chemistry, Bahauddin Zakariya University, Multan 60800, Pakistan
2Research Institute of Chemistry, International Center for Chemical and Biological Sciences, (H.E.J.) University of
Karachi, Karachi-75270, Pakistan
Received October 27, 2010: Revised March 08, 2011: Accepted March 11, 2011
Abstract: A series of N4-aryl substituted isatin-3-thiosemicarbazones was prepared by the reaction of isatin with an
appropriate thiosemicarbazide in ethanol containing a few drops of acetic acid. The newly synthesized compounds were
characterized by means of their analytical (CHN) and spectral (IR, 1H-NMR, EIMS) data, and evaluated for their
cytotoxicity and phytotoxicity potential. Eleven out of thirteen compounds tested proved to be active in the brine-shrimp
lethality bioassay exhibiting significant cytotoxic activity with LD50 values ranging from 1.75x10-5M to 1.91x10-4M. In
phytotoxicity assay, all the synthesized compounds, regardless of the nature of aryl substituents, demonstrated weak to
moderate (5-30%) plant growth inhibition at the highest tested concentration (500 μg/mL).
Keywords: Isatin, Isatin Derivatives, Thiosemicarbazones, Isatin-3-thiosemicarbazones, Cytotoxicity, Phytotoxicity.
INTRODUCTION
CHEMISTRY
Isatin and its derivatives are important heterocyclic
molecules, which possess numerous types of biological
properties including sedative, anticonvulsant, anxiogenic,
anthelmintic, antimicrobial, antineoplastic, antiviral,
antiplasmodial, antitubercular and enzymatic inhibition [1-
9]. In recent years, isatins-thiosemicarbazones are reported to
display broad spectrum of medicinal properties such as
antimicrobial, antineoplastic, antitubercular, antiulcer,
antiviral and enzymatic inhibition [1-3,10-16]. Tempted by
this and in continuation of our earlier efforts [17-20] in
search of potent organic molecules exhibiting antibacterial,
antifungal, cytotoxic, phytotoxic and urease inhibitory
activities, we have synthesized a new series of thirteen title
thiosemicarbazones by condensation of isatin with
The synthesis of isatin-thiosemicarbazones was simple
and straightforward. A mixture of isatin, appropriate
thiosemicarbazide and ethanol containing a few drops of
glacial acetic acid was refluxed for 2 h (Scheme 1). The
crystalline solid formed during refluxing in each case was
filtered hot. Thorough washing with hot ethanol followed by
ether furnished the required compounds 2a-2m in moderate
to excellent yields (54-92%).
The structures of the synthesized thiosemicarbazones 2a-
2m were established through analytical (CHN) and spectral
1
(IR, H-NMR, EIMS) data. Satisfactory elemental analysis
(± 0.4% of calculated values) was obtained for all
compounds except where noted otherwise. The IR spectra of
compounds 2a-2m showed two separate bands resulting
from NH stretchings of indole and thioamide functions in the
3316-3200 and 3185-3135 cm-1 regions, respectively. The
lactam C=O, azomethine C=N and thioamide C=S
stretchings were observed in the 1699-1680, 1602-1570 and
appropriate 4-substituted 3-thiosemicarbazides
1
and
screened them for their cytotoxic and phytotoxic effects.
RESULTS AND DISCUSSION
1
1207-1161 cm-1 regions, respectively [10,21-23]. The H-
The present work describes the synthesis and in vitro
determination of the cytotoxic and phytotoxic effects of
thirteen new N4-aryl substituted isatin-3-thiosemicarbazones
2a-2m. It also describes in vitro determination of the
NMR spectra of 2a-2m exhibited three singlets at ꢀ 8.15-
10.97,
ꢀ
9.38-11.29 and
ꢀ
12.74-12.94 for the
thiosemicarbazone N4-H, indole NH and thiosemicarbazone
N2-H, respectively [10,21,24,25]. The indole C7-H appeared
as a doublet at ꢀ 6.71-6.95, while the indole C5-H and C6-H
appeared at ꢀ 7.00-7.37 and ꢀ 7.24-7.63, respectively, as a
triplet or a doublet of double doublet. Indole C4-H,
experiencing a deshielding effect caused by inductively
electron-withdrawing C=N function, resonated as a doublet
at ꢀ 7.61-7.99 [10,25-27]. In certain cases, however,
overlapping of two signals corresponding to indole C5-H and
C6-H was observed as multiplets due to combination with
different aromatic protons of N4-substituents. The EI mass
spectra of the thiosemicarbazones showed molecular ions of
different intensity, which confirmed their molecular weights.
The major fragmentation pathways involved rupture of the
exocyclic N-N, NH-CS and endocyclic NH-CO bonds [10].
phytotoxicity potential of
a
previously reported
thiosemicarbazone i.e. 2-(2-oxo- 1,2- dihydro- 3H-indole- 3-
ylidine)- N-phenyl-1-hydrazinecarbothioamide [17].
*Address correspondence to these authors at the Department of Chemistry,
Bahauddin Zakariya University, Multan 60800, Pakistan; Tel: (92-61)
9210083, 9210071-5 (Ext. 2402); Fax: (92-61) 9210138;
E-mail: pdhpervez@hotmail.com
Research Institute of Chemistry, International Center for Chemical and
Biological Sciences, (H.E.J.) University of Karachi, Karachi-75270,
Pakistan; Tel: (92-21) 34824910; Fax: (92-21) 9243190, 9243191;
E-mail: hassaan2@super.net.pk
1570-1808/11 $58.00+.00
© 2011 Bentham Science Publishers Ltd.

N4-Aryl Substituted Isatin-3-thiosemicarbazones
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5 453
O
O
NNHCSNHR
O
N
H
RNHCSNHNH2
N
H
EtOH-AcOH
Reflux, 2h
1
2a-2m
C2H5
H3CO
OCH3
2a R =
2b R =
2h R =
H3CO
C2H5
I
2i R =
2j R =
OCH3
2c R =
I
2d R =
2e R =
Cl
Cl
Cl
Cl
2k R =
2l R =
I
Cl
Cl
CN
2f R =
2g R =
Cl
Cl
CN
2m R =
Scheme 1. Synthesis of title compounds 2a-2m.
Compounds 2c and 2j-2l did not show molecular ion peaks
in their spectra. However, the fragments corresponding to
thiosemicarbazone moiety, formed by N-N and NH-CS bond
cleavage, confirmed their structures. The proposed
fragmentation pattern of 2a is depicted in Fig. (1). X-ray
structures of two representative examples 2a and 2d were
determined in order to confirm the assigned structures and
displayed promising cytotoxicity (LD50 = 1.75 x 10-5M - 1.91
x 10-4M) against Artemia salina in the present assay (Table
1). Compounds 2h and 2i with methoxy substituents at
positions- 2, 4 and -2, 5 gave a value of LD50 >2.80 x10-4M
and, therefore, can be considered to be almost inactive. It
may be noted that compound 2m with chloro substituents at
positions-3 and -5 of the phenyl ring showed maximum
activity (LD50 = 1.75 x 10-5M) and, therefore, proved to be
the most potent compound in this series. The other chloro-
substituted compounds 2j-2l having substituents at positions-
2,3, -2,4 and -2,5, respectively, were found to be less potent
giving LD50 values of 1.02 x 10-4M, 1.20 x 10-4M and 1.66 x
10-4M. Interestingly, substitution of two chloro substituents
at the phenyl ring was found to cause induction of cytotoxic
activity, as the respective monochloro-substituted
compounds gave a value of LD50 > 3.02 x 10-4M in the
earlier assay [18] and were thus considered to be almost
inactive. To the contrary, the monoiodo-substituted
compounds 2c-2e having substituent at positions-2, -3 and -
establish
conformations
of
the
synthesized
thiosemicarbazones 2a-2m. Relevant crystal data and details
of the structure elucidation have been reported elsewhere
[28,29].
BIOLOGICAL TESTING
In Vitro Cytotoxicity
The synthesized thiosemicarbazones 2a-2m were
evaluated for their cytotoxic effects by the brine-shrimp
lethality bioassay. All these compounds except 2h and 2i

454 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5
Pervez et al.
C2H5
N
NCS
O
NNHCSNH
O
NNHCSNH
H2N
-CO
C2H5
C2H5
NH
N
N
H
H
m/z 324 (M+)
m/z 121
m/z 203
m/z 296
H
-C2H6
S
NNH2
NH2
C2H5
C2H5
HN
SCN
N
NH
O
O
N
N
S
H
H
-C2H5
H
m/z 91
m/z 178
N
NH
m/z 161
m/z 163
m/z 147
-HNCO
m/z 150
-HNCO
-C2H5
-NH
H
NH2
SCN
NH2
-CO
NH2
NNH2
O
m/z 118
m/z 104
m/z 133
m/z 105
m/z 134
Fig. (1). The proposed fragmentation pattern of 2a.
4, respectively, exhibited LD50 values of 1.59x10-4M , 1.40 x
10-4M and 1.26 x 10-4M. Similarly, the cyano compounds
having substituents at positions-3 and -4 of the phenyl ring
gave LD50 values of 1.91 x 10-4 M and 1.51 x 10-4M,
respectively. Further, as compared to compound, 2-(2-oxo-
1,2-dihydro-3H-indole-3-ylidine)-N-phenyl-1-hydrazinecarbo-
thioamide [17] having no substituent at the phenyl ring
attached to N4 of the thiosemicarbazone moiety and giving a
value of LD50 > 3.38 x 10-4M in the earlier assay [18],
substitution of ethyl group at position-2 or -4 of its phenyl
ring caused induction of activity (1.74 x 10-4M and 5.10 x
10-5M, respectively). However, compared with the respective
methyl-substituted compounds [18], compound 2b with an
ethyl substituent at position-4 of the phenyl ring exhibited
induced activity (LD50 > 3.22 x 10-4M ꢀ 5.10 x 10-5M),
whereas compound 2a having such a substituent at position-
2 of the phenyl ring displayed reduced activity (LD50 = 8.10
significant (5-30 %) inhibition at the highest tested
concentration (500 ꢁg/ mL) as compared to the reference
point, 2-(2-oxo-1,2-dihydro-3H-indole-3-ylidine)-N-phenyl-
Table 1. Brine-Shrimp Bioassay of Compounds 2a-2m
Compounds
LD50 (M)
2a
2b
2c
2d
2e
2f
1.74 x 10-4
5.10 x 10-5
1.59 x 10-4
1.40 x 10-4
1.26 x 10-4
1.91 x 10-4
1.51 x 10-4
> 2.80 x 10-4
>2.80 x 10-4
1.02 x 10-4
1.20 x 10-4
1.66 x 10-4
1.75 x 10-5
>3.38 x 10-4
2g
2h
2i
x
10-5M 10-4M). These structure-activity
ꢀ 1.74 x
relationships (SAR) may serve as a basis for chemical
modifications aimed at the development of certain cytotoxic
agents of clinical interest.
2j
2k
2l
In Vitro Phytotoxicity
All the synthesized thiosemicarbazones 2a-2m having
one or two substituents about the phenyl ring at N4 of the
thiosemicarbazone moiety were also evaluated for their
phytotoxic effects at three different concentrations i.e. 500,
50 and 5 ꢁg / mL. These compounds displayed weak or non-
2m
2-(2-Oxo-1,2-dihydro-3H-indole-3-
ylidine)-N-phenyl-1-hydrazine-
carbothioamide‡
‡[17,18].

N4-Aryl Substituted Isatin-3-thiosemicarbazones
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5 455
Table 2. Percent Growth Inhibition of Lemna aequinocitalis by Compounds 2a-2m at Different Concentrations*
Compounds
500 (ꢀg/mL) (% G.I. †)
50 (ꢀg/mL) (% G.I. †)
5 (ꢀg/mL) (% G.I. †)
2a
2b
2c
2d
2e
2f
15
15
25
10
30
05
25
20
30
10
20
20
10
40
15
10
20
20
20
05
20
20
25
10
20
15
10
25
25
05
10
05
20
05
10
10
05
10
20
05
05
15
2g
2h
2i
2j
2k
2l
2m
2-(2-Oxo-1,2-dihydro-3H-indole-3-ylidine)-N-
phenyl-1-hydrazinecarbothioamide‡
*The reference compound paraquat shows 100 % growth inhibition at a concentration of 0.015ꢀg/mL; †G.I.: growth inhibition; ‡[17].
1-hydrazinecarbothioamide [17], which demonstrated
moderate (40%) inhibition at the same concentration (Table
2). Similarly, all the synthesized compounds including
reference point exhibited weak or non-significant (5-25%)
plant growth inhibition at the lowest tested concentration (5
ꢀg/ mL). From the results obtained in this bioassay, it may
be concluded that our test compounds showed non-
significant inhibition at much higher levels of concentration
compared with the standard paraquat, which displayed 100
% inhibition at a concentration of 0.015 ꢀg / mL.
Furthermore, the percent growth inhibition values revealed
that the type, number and position of the substituents about
the phenyl ring at N4 of the thiosemicarbazone moiety did
not significantly affect the phytotoxicity potential of the
synthesized compounds at both the highest as well as lowest
tested concentrations (500 ꢀg / mL and 5 ꢀg / mL).
the spots were visualized under ultraviolet light at 254 and
366 nm and / or spraying with iodine vapours. In vitro
biological evaluation of the synthesized compounds was
done at Dr. Panjwani Center for Molecular Medicine and
Drug Research, and H. E. J. Research Institute of Chemistry,
International Center for Chemical and Biological Sciences,
University of Karachi, Pakistan.
Synthesis
General Procedure for the Preparation of Isatin-
thiosemicarbazones (2a-2m)
To a hot solution of isatin (5 mmol) in ethanol (10 mL)
containing a few drops of glacial acetic acid was added the
appropriate thiosemicarbazide (5 mmol) dissolved in ethanol
(10 mL) under stirring. The reaction mixture was then heated
under reflux for 2 h. The crystalline solid formed during
heating was collected by suction filtration. Thorough
washing with hot ethanol followed by ether gave the
required compounds 2a-2m in pure form.
MATERIALS AND METHODS
General
All reagents and solvents were used as obtained from the
suppliers or recrystallized / redistilled as necessary. Melting
points were taken on a Fisher-Johns melting point apparatus
and are uncorrected. Elemental analyses were performed on
a Leco CHNS-9320 elemental analyzer. Infrared spectra
(KBr disks) were run on Shimadzu Prestige-21 FT-IR
N-(2-Ethylphenyl)-2-(2-oxo-1,2-dihydro-3H-indol-3-
ylidene)-1-hydrazinecarbothioamide (2a)
°
Yield 88% as yellow crystals; m.p. >212 C (decomp.);
(Found: C, 63.15; H, 4.95; N, 17.28. Calcd for C17H16N4OS:
C, 62.96; H, 4.94; N, 17.28%); IR (KBr, cm-1): 3316, 3184,
3165, 3145 (NH stretching), 1695 (C = O), 1593 (C = N),
1
spectrometer. The H-NMR spectra were recorded in CDCl3
1
1541 (NH bending), 1161 (C = S); H-NMR (CDCl3): ꢁ
and DMSO-d6 on Bruker (Rhenistetten-Forchheim,
Germany) AM 300 and AM 400 spectrometers operating at
300 MHz and 400 MHz, respectively, using TMS as an
1.31(t, J = 7.5 Hz, 3H, CH3), 2.74 (q, J = 7.5 Hz, 2H, CH2),
6.95 (d, J = 7.8 Hz, 1H, indole C7-H), 7.15 (t, J = 7.5 Hz,
1H, indole C5-H), 7.30-7.41 (m, 3H, indole C6-H, phenyl C3-
H, C4-H), 7.61 (d, J = 7.5 Hz, 1H, indole C4-H), 7.77-7.83
(m, 2H, phenyl C5-H, C6-H), 8.35 (s, 1H, CS-NH), 9.38 (s,
1H, indole NH), 12.88 (s, 1H, N-NH); EI MS (70 eV) m/z
(%): 326 (M+ +2, 2), 325 (M+ +1, 4), 324 (M+,11), 296
(100), 203 (9), 178 (48), 163 (55), 161 (49), 150 (16), 147
1
internal standard. H chemical shifts are reported in ꢁ (ppm)
and coupling constants in Hz. The electron impact mass
spectra (EI MS) were determined with a Finnigan MAT-312
and a JEOL MSRoute mass spectrometer. The progress of
the reaction and the purity of the products were checked on
TLC plates pre-coated with Merck silica gel 60 GF254 and

456 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5
Pervez et al.
(17), 134 (4), 133(14), 132 (20), 121 (17), 118 (34), 105(12),
104 (31), 91 (18), 77 (35).
8.5 Hz, 1H, indole C4-H), 8.50 (s,1H, CS-NH), 9.42 (s, 1H,
indole NH), 12.85 (s, 1H, N-NH); EI MS (70 eV) m/z (%):
424 (M+ +2, 1), 423 (M+ +1, 4), 422 (M+, 17), 394 (80), 276
(1), 261(63), 244 (35), 219 (100), 203 (30), 161 (29),
150(11), 147 (3), 144 (26), 132 (6), 134 (19), 104 (14), 92
(27), 76 (13), 65 (19).
N-(4-Ethylphenyl)-2-(2-oxo-1,2-dihydro-3H-indol-3-
ylidene)-1-hydrazinecarbothioamide (2b)
º
Yield 75% as yellow crystals; m.p. >238 C (decomp.);
(Found: C, 63.16; H, 4.96; N, 17.27. Calcd for C17H16N4OS:
C, 62.96; H, 4.94; N, 17.28%); IR (KBr, cm-1): 3305, 3200,
3165 (NH stretching), 1695 (C = O), 1593 (C = N), 1541
N-(3-Cyanophenyl)-2-(2-oxo-1,2-dihydro-3H-indol-3-
ylidene)-1-hydrazinecarbothioamide (2f)
1
(NH bending), 1202 (C = S); H-NMR (CDCl3): ꢀ 1.20 (t, J
Yield 54% as bright yellow crystals; m.p. >282 ºC
(decomp.); (Found: C, 59.94; H, 3.44; N, 21.82. Calcd for
C16H11N5OS: C, 59.81; H, 3.43; N, 21.81%); IR (KBr, cm-1):
3300, 3251 (NH stretching), 1689 (C = O), 1587 (C = N),
= 7.6 Hz, 3H, CH3), 2.60 (q, J = 7.6 Hz, 2H, CH2), 6.88 (d, J
= 7.8 Hz, 1H, indole C7-H), 6.97- 7.10 (m, 2H, phenyl C2-H,
C6-H), 7.13-7.20 (m, 3H, indole C5-H and phenyl C3-H, C5-
H), 7.28-7.39 (m, 1H, indole C6-H), 7.63 (d, J = 7.6 Hz, 1H,
indole C4-H), 8.15 (s, 1H, CS-NH), 9.42 (s, 1H, indole NH),
12.80 (s, 1H, N-NH); EI MS (70 eV) m/z (%): 325 (M+ +1,
3), 324 (M+, 20), 296 (96), 203 (6), 163 (16), 161 (20), 150
(37), 147 (21), 134 (3), 132 (22), 118 (29), 104 (86), 91 (35),
77 (100), 51 (41).
1
1521 (NH bending), 1170 (C = S); H-NMR (DMSO-d6) ꢀ
6.95 (d, J = 7.8 Hz, 1H, indole C7-H), 7.12 (t, J = 7.5 Hz,
1H, indole C5-H), 7.39 (t, J = 7.8 Hz, 1H, phenyl C5-H), 7.63
(t, J = 7.8 Hz, 1H, indole C6-H), 7.73-7.77 (m, 2H, phenyl
C4-H, C6-H), 7.99 (d, J = 8.1 Hz, 1H, indole C4-H), 8.13 (s,
1H, phenyl C2-H), 10.97 (s, 1H, CS-NH), 11.29 (s, 1H,
indole NH), 12.90 (s, 1H, N-NH); EI MS (70 eV) m/z
(%):322 (M+ +1, 3), 321 (M+, 20), 293 (77), 203 (20), 161
(43), 160 (45), 150 (16), 147 (3), 145 (26), 132 (25), 118
(100), 104 (44), 91 (39), 77 (24), 65 (13), 51 (34).
N-(2-Iodophenyl)-2-(2-oxo-1,2-dihydro-3H-indol-3-
ylidene)-1- hydrazinecarbothioamide (2c)
º
Yield 92% as yellow crystals; m.p. >270 C (decomp.);
(Found: C, 42.81; H, 2.62; N, 13.27. Calcd for C15H11IN4OS:
C, 42.65; H, 2.61; N, 13.27%); IR (KBr, cm-1): 3226, 3184
(NH stretching), 1685 (C = O), 1590 (C = N), 1544 (NH
bending), 1165 (C = S); 1H-NMR (DMSO-d6): ꢀ 6.95 (d, J =
7.8 Hz, 1H, indole C7-H), 7.08-7.15 (m, 2H, phenyl C4-H,
C5-H), 7.37 (ddd, J = 7.8, 1.2 Hz, 1H, indole C5-H), 7.47
(ddd, J = 8.1, 1.2 Hz, 1H, indole C6-H), 7.53 (dd, J = 8.1, 1.8
Hz,1H, phenyl C6-H), 7.73 (d, J = 7.5 Hz, 1H, indole C4-H),
7.96 (dd, J = 8.1, 0.9 Hz, 1H, phenyl C3-H), 10.79 (s, 1H,
CS-NH), 11.28 (s, 1H, indole NH), 12.81 (s, 1H, N-NH); EI
MS (70 eV) m/z (%): 394 (2), 294 (49), 266 (100), 219 (9),
161 (2), 149 (21), 144 (5), 118 (21), 104 (4), 90 (7), 77 (4).
N-(4-Cyanophenyl)-2-(2-oxo-1,2-dihydro-3H-indol-3-
ylidene)-1-hydrazinecarbothioamide(2g)
º
Yield 71% as orange yellow crystals; m.p. >278 C
(decomp.); (Found: C, 59.96; H, 3.44; N, 21.81. Calcd for
C16H11N5OS: C, 59.81; H, 3.43; N, 21.81%); IR (KBr, cm-1):
3300, 3265, 3244 (NH stretching), 1699 (C = O), 1587 (C =
1
N), 1529 (NH bending), 1195 (C = S); H-NMR (CDCl3): ꢀ
6.92 (d, J = 8.3 Hz, 1H, indole C7-H), 7.07-7.15 (m, 3H,
indole C5-H and phenyl C2-H, C6-H), 7.32-7.39 (m, 1H,
indole C6-H), 7.62-7.74 (m, 2H, phenyl C3-H, C5-H), 7.98 (d,
J = 8.1 Hz, 1H, indole C4-H), 9.80 (s, 1H, indole NH), 12.94
(s, 1H, N-NH); EI MS (70 eV) m/z (%): 322 (M+ +1, 4),
321(M+, 22), 293 (95), 203 (19), 161 (51), 160 (56), 150
(24), 147 (4), 145 (25), 132 (29), 118 (100), 104 (49), 91
(36), 77 (27), 65 (11), 51 (29).
N-(3-Iodophenyl)-2-(2-oxo-1,2-dihydro-3H-indol-3-
ylidene)-1-hydrazinecarbothioamide (2d)
Yield 84% as dark yellow crystals; m.p. >230 ºC
(decomp.); (Found: C, 42.79; H, 2.62; N, 13.28. Calcd for
C15H11IN4OS: C, 42.65; H, 2.61; N, 13.27%); IR (KBr, cm-
1): 3280, 3166 (NH stretching), 1689 (C = O), 1570 (C = N),
1523 (NH bending), 1163 (C = S); 1H-NMR (CDCl3): ꢀ 6.92
(d, J = 7.9 Hz, 1H, indole C7-H), 7.00-7.15 (m, 2H, indole
C5-H and phenyl C6-H), 7.27-7.41 (m, 2H, indole C6-H and
phenyl C5-H), 7.57-7.74 (m, 2H, phenyl C2-H, C4-H), 7.79
(d, J = 7.8 Hz, 1H, indole C4-H), 8.50 (s, 1H, CS-NH), 9.42
(s, 1H, indole NH), 12.85 (s, 1H, N-NH); EI MS (70 eV) m/z
(%): 423 (M+ +1, 2), 422 (M+, 12), 394 (77), 276 (2),
261(58), 244 (18), 219 (100), 203 (43), 161 (21), 145 (25),
134 (24), 104 (11), 92 (65), 78 (61), 63 (62).
N-(2, 4-Dimethoxyphenyl)-2-(2-oxo-1,2-dihydro-3H-indol-
3-ylidene)-1-hydrazinecarbothioamide (2h)
Yield 76% as deep yellow crystals; m.p. >256 ºC
(decomp.); (Found: C, 57.46; H, 4.50; N, 15.74. Calcd for
C17H16N4O3S: C, 57.30; H, 4.49; N, 15.73 %); IR (KBr, cm-
1): 3265, 3185, 3135 (NH stretching), 1689 (C = O), 1585 (C
1
= N), 1541 (NH bending), 1161 (C = S); H-NMR (DMSO-
d6): ꢀ 3.80 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 6.57 (dd, J =
8.7, 2.4 Hz, 1H, phenyl C5-H), 6.68 (d, J = 2.7 Hz, 1H,
phenyl C3-H), 6.94 (d, J = 7.8 Hz, 1H, indole C7-H), 7.10 (t,
J = 7.8 Hz, 1H, indole C5-H), 7.37 (ddd, J = 7.8, 1.2 Hz, 1H,
indole C6-H), 7.51 (d, J = 8.4 Hz, 1H, phenyl C6-H), 7.70 (d,
J = 7.2 Hz, 1H, indole C4-H), 10.30 (s, 1H, CS-NH), 11.24
(s, 1H, indole NH), 12.74 (s, 1H, N-NH); EI MS (70 eV) m/z
(%): 358 (M+ +2, 5), 357 (M+ +1, 14), 356 (M+, 63), 328
(100), 295 (2), 195 (43), 153 (79), 150 (8), 147 (4), 138 (85),
132 (8), 118 (23), 104 (11), 91 (8), 77 (8), 65 (5).
N-(4-Iodophenyl)-2-(2-oxo-1,2-dihydro-3H-indol-3-
ylidene)-1-hydrazinecarbothioamide (2e)
º
Yield 81% as yellow crystals; m.p. >250 C (decomp.);
(Found: C, 42.80; H, 2.62; N, 13.26. Calcd for C15H11IN4OS:
C, 42.65; H, 2.61; N, 13.27%); IR (KBr, cm-1): 3324, 3226
(NH stretching), 1680 (C = O), 1602 (C = N), 1555 (NH
bending), 1163 (C = S); 1H-NMR (CDCl3): ꢀ 6.71 (d, J = 8.5
Hz, 1H, indole C7-H), 6.87-6.97 (m, 2H, phenyl C2-H, C6-
H), 7.03-7.17 (m, 1H, indole C5-H), 7.29-7.37 (m, 1H, indole
C6-H), 7.50-7.67 (m, 2H, phenyl C3-H, C5-H), 7.70 (d, J =
N-(2, 5-Dimethoxyphenyl)-2-(2-oxo-1,2-dihydro-3H-indol-
3-ylidene)-1-hydrazinecarbothioamide (2i)
º
Yield 78% as yellow crystals; m.p. >258 C (decomp.);
(Found: C, 57.45; H, 4.50; N, 15.73. Calcd for C17H16N4O3S:

N4-Aryl Substituted Isatin-3-thiosemicarbazones
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5 457
C, 57.30; H, 4.49; N, 15.73 %); IR (KBr, cm-1): 3282, 3246,
3210 (NH stretching), 1691 (C = O), 1590 (C = N), 1544
(NH bending), 1163 (C = S); H-NMR (DMSO-d6): ꢀ 3.72
CS-NH), 11.27 (s, 1H, indole NH), 12.89 (s, 1H, N-NH); EI
MS (70 eV) m/z (%): 338 (12), 337 (3), 329 (100), 301 (3),
203 (17), 185 (32), 161 (28), 160 (6), 150 (21), 147 (27), 145
(34), 132 (25), 118 (23), 104 (41), 91 (12), 77 (22), 65 (6),
51 (18).
1
(s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.83 (dd, J = 9.0, 3.0 Hz,
1H, phenyl C4-H), 6.95 (d, J = 7.8 Hz, 1H, indole C7-H),
7.07 (d, J = 9.0 Hz, 1H, phenyl C3-H), 7.12 (t, J = 7.5 Hz,
1H, indole C5-H), 7.38 (t, J = 7.5 Hz , 1H, indole C6-H), 7.66
(d, J = 7.5 Hz, 1H, indole C4-H), 7.74 (d, J = 2.7 Hz, 1H,
phenyl C6-H), 10.43 (s, 1H, CS-NH), 11.27 (s, 1H, indole
NH), 12.81 (s, 1H, N-NH); EI MS (70 eV) m/z (%): 357 (M+
+1 , 7), 356 (M+ ,33), 328 (100), 297 (22), 195 (27), 181
(70), 161 (17), 150 (18), 147 (9), 145 (28), 138 (82), 132
(22), 118 (27), 104 (50), 91 (15), 77 (41), 65 (21), 51 (38).
N-(3, 5-Dichlorophenyl)-2-(2-oxo-1,2-dihydro-3H-indol-3-
ylidene)-1-hydrazinecarbothioamide (2m)
Yield 80% as deep yellow crystals; m.p. >270 ºC
(decomp.); (Found: C, 49.43; H, 2.73; N, 15.36. Calcd for
C15H10Cl2N4OS: C, 49.32; H, 2.74; N, 15.34 %); IR (KBr,
cm-1): 3315, 3244, 3221 (NH stretching), 1695 (C = O), 1587
(C = N), 1541 (NH bending), 1172 (C = S); 1H-NMR
(DMSO-d6): ꢀ 6.94 (d, J = 8.1 Hz, 1H, indole C7-H), 7.12 (t,
J = 7.5 Hz, 1H, indole C5-H), 7.38 (t, J = 7.5 Hz, 1H, indole
C6-H), 7.50 (t, J =1.8 Hz, 1H, phenyl C4-H), 7.76 (d, J = 7.2
Hz, 1H, indole C4-H), 7.87 (d, J = 1.8 Hz, 2H, phenyl C2-
H,C6-H), 10.90 (s, 1H, CS-NH), 11.29 (s, 1H, indole NH),
12.91 (s, 1H, N-NH); EI MS (70 eV) m/z (%): 366 (M+ +1,
14), 338 (59), 203 (43), 186 (15), 161 (77), 160 (14), 150
(54), 147 (29), 145 (61), 132 (60), 118 (57), 104 (100), 91
(30), 77 (59), 65 (16), 51 (45).
N-(2, 3-Dichlorophenyl)-2-(2-oxo-1,2-dihydro-3H-indol-3-
ylidene)-1-hydrazinecarbothioamide (2j)
º
Yield 85% as yellow crystals; m.p. >290 C (decomp.);
(Found: C, 49.45; H, 2.75; N, 15.34. Calcd for
C15H10Cl2N4OS: C, 49.32; H, 2.74; N, 15.34 %); IR (KBr,
cm-1): 3244, 3230 (NH stretching), 1695 (C = O), 1585 (C =
N), 1527 (NH bending), 1207 (C = S); 1H-NMR (DMSO-d6):
ꢀ 6.94 (d, J = 7.8 Hz, 1H, indole C7-H), 7.10 (t, J = 7.5 Hz,
1H, indole C5-H), 7.37 (ddd, J = 7.68, 0.86 Hz, 1H, indole
C6-H), 7.44 (t, J = 9.0 Hz, 1H, phenyl C5-H), 7.55 (dd, J =
7.8, 1.5 Hz, 1H, phenyl C4-H), 7.65 (dd, J = 8.7, 1.5 Hz, 1H,
phenyl C6-H), 7.69 (d, J = 7.5 Hz, 1H, indole C4-H), 10.88
(s, 1H, CS-NH), 11.26 (s, 1H, indole NH), 12.88 (s, 1H, N-
NH); EI MS (70 eV) m/z (%): 338 (12), 329 (100), 301 (3),
203 (29), 185 (35), 161 (44), 160 (7), 150 (24), 147 (23), 145
(43), 132 (31), 118 (39), 104 (59), 91 (23), 77 (39), 65 (9),
51 (34).
BIOLOGICAL TESTING
In Vitro Cytotoxicity
Brine-shrimp (Artemia salina Leach) eggs were hatched
in a shallow rectangular plastic dish (22x32 cm) filled with
artificial sea water, which was prepared with a commercial
salt mixture (Instant Ocean, Aquarium System, Inc., Mentor,
Ohio, USA) and double-distilled water. An unequal partition
was made in the plastic dish with the help of a perforated
device. Approximately 50 mg of eggs were sprinkled into the
large compartment, which was darkened, while the smaller
compartment was opened to ordinary light. After two days,
nauplii were collected by a pipette from the lighted side. A
sample of the test compound was prepared by dissolving 2
mg of each compound in 2 mL of methanol. From this stock
solution, 500, 50 and 5 ꢁL were transferred to 9 vials, three
for each dilution, and one vial was kept as control having 2
mL of methanol. The solvent was allowed to evaporate
overnight. After two days, when shrimp larvae were ready, 1
mL of sea water and 10 shrimps were added to each vial (30
shrimps/ dilution) and the volume was adjusted with sea
water to 5 mL per vial. After 24 h, the number of survivors
was counted [30,31]. Data were analyzed by a Finney
computer program to determine the LD50 values [32].
N-(2, 4-Dichlorophenyl)-2-(2-oxo-1,2-dihydro-3H-indol-3-
ylidene)-1-hydrazinecarbothioamide (2k)
Yield 87% as deep yellow crystals; m.p. >272 ºC
(decomp.); (Found: C, 49.46; H, 2.74; N, 15.33. Calcd for
C15H10Cl2N4OS: C, 49.32; H, 2.74; N, 15.34 %); IR (KBr,
cm-1): 3300, 3261 (NH stretching), 1695 (C = O), 1579(C =
N), 1533 (NH bending), 1165 (C = S); 1H-NMR (DMSO-d6):
ꢀ 6.94 (d, J = 7.8 Hz, 1H, indole C7-H), 7.10 (t, J = 7.5 Hz
1H, indole C5-H), 7.37 (t, J = 7.6 Hz, 1H, indole C6-H), 7.50
(dd, J = 8.8, 1.5 Hz, 1H, phenyl C5-H), 7.60 (d, J = 8.5 Hz,
1H, phenyl C6-H), 7.69 (d, J = 7.5 Hz, 1H, indole C4-H),
7.76 (d, J = 2.23 Hz, 1H, phenyl C3-H), 10.75 (s, 1H, CS-
NH), 11.24 (s, 1H, indole NH), 12.88 (s, 1H, N-NH); EI MS
(70 eV) m/z (%): 340 (4), 338 (21), 337 (5), 329 (100), 301
(3), 203 (22), 185 (36), 161 (42), 160 (10), 150 (27), 147
(21), 145 (37), 132 (32), 118 (31), 104 (54), 91 (16), 77 (29),
65 (7), 51 (21).
Phytotoxicity Bioassay
N-(2, 5-Dichlorophenyl)-2-(2-oxo-1,2-dihydro-3H-indol-3-
ylidene)-1-hydrazinecarbothioamide (2l)
This test was performed according to the modified
literature protocol [30]. The test compounds were
incorporated into sterilized E-medium at different
concentrations i.e. 5, 50, and 500 ꢁg/mL in methanol.
Sterilized conical flasks were inoculated with compounds of
the desired concentrations prepared from the stock solution
and allowed to evaporate overnight. Each flask was
inoculated with 20 ml of sterilized E-medium and then ten
Lemna aequinocitalis Welv, each containing a rosette of
three fronds were placed on media. Other flasks were
supplemented with methanol serving as negative control and
reference inhibitor i.e. parquet serving as positive control.
º
Yield 83% as yellow crystals; m.p. >270 C (decomp.);
(Found: C, 49.44; H, 2.75; N, 15.34. Calcd for
C15H10Cl2N4OS: C, 49.32; H, 2.74; N, 15.34 %); IR (KBr,
cm-1): 3244, 3200 (NH stretching), 1699 (C = O), 1589 (C =
N), 1533 (NH bending), 1200 (C = S); 1H-NMR (DMSO-d6):
ꢀ 6.94 (d, J = 7.8 Hz, 1H, indole C7-H), 7.10 (t, J = 7.2 Hz,
1H, indole C5-H), 7.37 (ddd, J = 7.5, 1.1 Hz, 1H, indole C6-
H), 7.46 (dd, J = 8.66, 2.53 Hz,1H, phenyl C4-H), 7.63 (d, J
= 8.6 Hz, 1H, phenyl C3-H), 7.68 (d, J = 7.4 Hz, 1H, indole
C4-H), 7.74 (d, J = 2.5 Hz, 1H, phenyl C6-H), 10.81 (s, 1H,

458 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 5
Pervez et al.
thiosemicarbazone derivatives against cruzain falcipain-2 and
rhodesain. Bioorg. Med. Chem. Lett., 2003, 13, 3527-3530.
Bal, T. R.; Anand, B.; Yogeeswari, P.; Sriram, D. Synthesis and
evaluation of anti-HIV activity of isatin ꢀ-thiosemicarbazone
derivatives. Bioorg. Med. Chem. Lett., 2005, 15, 4451- 4455.
Terzioglu, N.; Karali, N.; Gursoy, A.; Pannecouque, C.; Leysen, P.;
Paeshuyse, J.; Neyts, J.; De Clercq, E. Synthesis and primary
antiviral activity evaluation of 3-hydrazono-5-nitro-2-indolinone
derivatives. Arkivoc, 2006, 109-118.
Quenelle, D. C.; Keith, K. A.; Kern, E. R. In vitro and in vivo
evaluation of isatin-ꢀ-thiosemicarbazone and marboran against
vaccinia and cowpox virus infections. Antiviral Res., 2006, 71, 24-
30.
Karali, N.; Gursoy A.; Kandemirli, F.; Shvets, N.; Kaynak, F. B.;
Ozbey, S.; Kovalishyn, V.; Dimoglo, A. Synthesis and structure-
antituberculosis activity relationship of 1H-indole-2,3-dione
derivatives. Bioorg. Med. Chem., 2007, 15, 5888-5904.
Guzel, O.; Karali, N.; Salman, A. Synthesis and antituberculosis
activity of 5-methyl/trifluoromethoxy-1H-indole-2,3-dione 3-
thiosemicarbazone derivatives. Bioorg. Med. Chem., 2008, 16,
8976-8987.
Treatments were replicated three times and the flasks
incubated at 30 C in a Fisons Fi-Totron 600 H growth
°
[12]
[13]
cabinet for seven days, 9000 lux light intensity, 56+10
relative humidity, and 12 h day length. Growth of L.
aequinocitalis in flasks containing the compounds was
determined by counting the number of fronds per dose and
growth inhibition was calculated with reference to negative
control.
[14]
[15]
[16]
CONCLUSIONS
We have demonstrated the potential of N4-aryl
substituted isatin-3- thiosemicarbazones to exhibit cytotoxic
and phytotoxic activities. Further investigation on structure-
activity relationships (SAR) and appropriate chemical
modifications amongst the synthesized thiosemicarbazones
is likely to furnish more effective cytotoxic and phytotoxic
agents.
[17]
[18]
Pervez, H.; Iqbal, M.S.; Tahir, M. Y.; Choudhary, M. I.; Khan, K.
M. Synthesis of some N4-substituted isatin-3-thiosemicarbazones.
Nat. Prod. Res., 2007, 21, 1178-1186.
Pervez, H.; Iqbal, M. S.; Tahir, M. Y.; Nasim, F. H.; Choudhary,
M. I.; Khan, K. M. In vitro cytotoxic, antibacterial, antifungal and
urease inhibitory activities of some N4-substituted isatin-3-
thiosemicarbazones. J. Enz. Inhib. Med. Chem., 2008, 23, 848-854.
Pervez, H.; Chohan, Z. H.; Ramzan, M.; Nasim, F. H.; Khan, K. M.
Synthesis and biological evaluation of some new N4-substituted
isatin-3-thiosemicarbazones. J. Enz. Inhib. Med. Chem., 2009, 24,
437-446.
Pervez, H.; Manzoor, N.; Yaqub, M.; Khan, A.; Khan, K. M.;
Nasim, F. H.; Choudhary, M. I. Synthesis and urease inhibitory
properties of some new N4-substituted 5-nitroisatin-3-
thiosemicarbazones. Lett. Drug. Des. Discov., 2010, 7, 102-108.
Omar, A. M. E.; Eshba, N. H.; Salama, H. M. Synthesis of some
ACKNOWLEDGEMENTS
The authors (HP, MR, MY) thank the Ministry of
Science & Technology (MoST), Government of Pakistan for
the partial financial assistance under “Projects for the
Strengthening of S&T Education in Universities” (Project
No. P&D/ S&T/2001/231).
[19]
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