Synthesis and biological evaluation of some N4-substituted 5-nitroisatin-3-thiosemicarbazones

Article abstract of DOI:10.1007/s00044-011-9745-7

A series of 5-nitroisatin-3-thiosemicarbazones 2a-2l was synthesised and evaluated for selected biological activities. The brine shrimp lethality bioassay was carried out to study their in vitro cytotoxicity potential and besides, their antifungal, phytotoxic and urease inhibitory effects were also investigated. Only compound 2j proved to be active in the brine shrimp assay exhibiting LD₅₀ value 1.16 × 10^-3 M. Compounds 2a and 2d displayed moderate antifungal activity (50 and 40%, respectively) against M. canis. Similarly, compound 2l exhibited moderate activity (40%) against the fungal strain, A. flavus. In phytotoxicity assay, all the synthesised compounds including the reference point 2m showed weak to moderate (20-60%) activity at the highest tested concentrations (1,000 μg and 500 μg/ml, respectively). In urease inhibition assay, compounds 2a, 2i and 2k proved to be potent inhibitors demonstrating pronounced inhibition with IC₅₀ values 0.440, 0.901 and 27.880 μM, respectively. These compounds may act as leads for further studies. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9745-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2251–2262
DOI 10.1007/s00044-011-9745-7
ORIGINAL RESEARCH
Synthesis and biological evaluation of some N4-substituted
5-nitroisatin-3-thiosemicarbazones
•
Humayun Pervez Nazia Manzoor
•
•
•
Muhammad Yaqub Faiz-ul-Hassan Nasim
Khalid M. Khan
Received: 15 December 2010 / Accepted: 8 July 2011 / Published online: 22 July 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A series of 5-nitroisatin-3-thiosemicarbazones
2a–2l was synthesised and evaluated for selected biological
activities. The brine shrimp lethality bioassay was carried
out to study their in vitro cytotoxicity potential and besides,
their antifungal, phytotoxic and urease inhibitory effects
were also investigated. Only compound 2j proved to be
active in the brine shrimp assay exhibiting LD50 value
1.16 9 10-3 M. Compounds 2a and 2d displayed moder-
ate antifungal activity (50 and 40%, respectively) against
M. canis. Similarly, compound 2l exhibited moderate
activity (40%) against the fungal strain, A. flavus. In phy-
totoxicity assay, all the synthesised compounds including
the reference point 2m showed weak to moderate
(20–60%) activity at the highest tested concentrations
(1,000 lg and 500 lg/ml, respectively). In urease inhibi-
tion assay, compounds 2a, 2i and 2k proved to be potent
inhibitors demonstrating pronounced inhibition with IC50
values 0.440, 0.901 and 27.880 lM, respectively. These
compounds may act as leads for further studies.
Keywords 5-Nitroisatin Á Thiosemicarbazones Á
Antifungal Á Cytotoxicity Á Phytotoxicity Á
Urease inhibition
Introduction
Isatin is a naturally occurring product found in many plants
including those of the genus Isatis. In humans, it is found
as a metabolic derivative of adrenaline (Da Silva et al.,
2001). The biological properties of isatin and its derivatives
have been known since long. Isatin itself displayed a
number of biological activities including anticonvulsant,
anxiogenic and sedative (Pandeya et al., 2005). Similarly,
isatin derivatives are reported to exhibit broad spectrum
chemotherapeutic properties including antiviral, antibacte-
rial, anticonvulsant, cysticidal, herbicidal, anthelmintic,
antifungal, antineoplastic, hypotensive and enzymatic
inhibition (Aboul-Fadl and Bin-Jubair, 2010; Bal et al.,
2005; Beauchard et al., 2006; Chen et al., 2005; Chiyanzu
et al., 2005, 2003; Da Silva et al., 2001; Guzel et al., 2008;
Hall et al., 2009; Hyatt et al., 2007; Karali, 2002; Karali
et al., 2007, 2002; Khan et al., 2008; Pandeya et al., 2005;
Quenelle et al., 2006; Ravichandran et al., 2007; Singh
et al., 2010; Terzioglu et al., 2006). Amongst these, isatins-
based synthetic thiosemicarbazones have increased a great
deal of interest (Bal et al., 2005; Chiyanzu et al., 2005,
2003; Da Silva et al., 2001; Guzel et al., 2008; Karali 2002;
Karali, et al., 2007, 2002; Pandeya et al., 2005; Quenelle
et al., 2006; Terzioglu et al., 2006). Incited by this and as a
part of our work on isatin derivatives, we recently reported
the synthesis of some N4-aryl-substituted isatins-3-thiose-
micarbazones as antiurease compounds (Pervez et al.,
2009a, 2007, 2008, 2010). Investigation of the structure–
activity relationship (SAR) studies revealed that, in
H. Pervez (&) Á N. Manzoor Á M. Yaqub
Department of Chemistry, Bahauddin Zakariya University,
Multan 60800, Pakistan
e-mail: pdhpervez@hotmail.com
Faiz-ul-Hassan Nasim
Department of Chemistry, The Islamia University
of Bahawalpur, Bahawalpur, Pakistan
e-mail: faiznasim@hotmail.com
K. M. Khan (&)
H.E.J. Research Institute of Chemistry, International Center
for Chemical and Biological Sciences, University of Karachi,
Karachi 75270, Pakistan
e-mail: khalid.khan@iccs.edu
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Med Chem Res (2012) 21:2251–2262
general, the type and position of the substituents about the
phenyl ring at N4 of the thiosemicarbazone moiety as well
as introduction of a nitro function at position-5 of the isatin
scaffold played an important role in causing rise in the
urease inhibitory potential of these compounds (Pervez
et al., 2008, 2009a, 2010). Encouraged by these findings
and in order to obtain antiurease compounds with improved
efficacy, the study of the attachment of some other groups
(alkyl, alkenyl and aryl having one or two substituents
about the phenyl ring) to N4 of the thiosemicarbazone
moiety was considered worth pursuing. The work reported
herein, therefore, primarily deals with the preparation
and description of urease inhibition effects of twelve
N4-substituted 5-nitroisatin-3-thiosemicarbazones. It also
describes the effects of the nature of functional groups
(alkyl, alkenyl and aryl) attached to N4 of the thiosemi-
carbazone moiety on the cytotoxicity, antifungal and phy-
totoxicity potential of these compounds.
(30 ml). After addition of 3–4 drops of glacial acetic acid,
the mixture was heated under reflux for 2 h. The product
formed during heating was filtered hot and washed thor-
oughly with hot aqueous ethanol to give the required
thiosemicarbazones 2a–2l in pure form.
N-Hexyl-2-(5-nitro-2-oxo-1,2-dihydro-3H-indol-3-
ylidene)-1-hydrazinecarbothioamide (2a)
Yield 82% as yellow crystals; m.p. 240°C (dec.); IR (KBr,
cm-1): 3346, 3275 (NH stretching), 1700 (C=O), 1640
1
(C=N), 1150 (C=S); H-NMR (DMSO-d6, d, ppm): 0.87
(t, J = 6.9 Hz, 3H, (CH2)5CH3), 1.30–1.31 (m, 6H,
CH2(CH2)3CH2CH3), 1.16–1.64 (m, 2H, (CH2)4CH2CH3),
3.61 (q, J = 7.8 Hz, 2H, CH2(CH2)4CH3), 7.11 (d,
J = 8.7 Hz, indole C7-H), 8.26 (dd, J = 8.7, 2.4 Hz, 1H,
indole C6-H), 8.56 (d, J = 2.4 Hz, 1H, indole C4-H), 9.58
(t, J = 5.7 Hz, 1H, CS-NH), 11.80 (s, 1H, indole NH),
12.32 (s, 1H, N–NH); EI MS (70 eV) m/z (%): 349 (M?,
11), 321 (19), 273 (3), 206 (16), 192 (94), 190 (10), 189 (2),
178 (9), 159 (22), 144 (25), 117 (14), 115 (18), 103 (22),
100 (43), 56 (100); Anal. calcd. for C15H19N5O3S: C: 51.56,
H: 5.48, N: 20.04; found: C: 51.52, H: 5.48, N: 20.11%.
Materials and methods
General
All chemicals and solvents were purchased from Aldrich,
Fluka and Merck-Schuchardt. Melting points were deter-
mined on cover slips by using a Fisher-Johns melting point
apparatus and are uncorrected. Micro (elemental) analyses
were performed using a Leco CHNS-9320 (USA) instru-
ment and were within 0.4% of theoretical limits except
where noted otherwise. Infrared (IR) spectra were mea-
sured as solids (KBr pellets) on a Shimadzu 8400 or a
Shimadzu Prestige-21 FT-IR spectrophotometer. Proton
nuclear magnetic resonance (1H-NMR) spectra were
recorded in DMSO-d6 on a 300 MHz Bruker FT-NMR
spectrometer using TMS as an internal standard. The
chemical shifts (d) are given in parts per million (ppm)
relative to TMS (d = 0.00) and coupling constants in Hz.
Mass spectra were recorded by means of a Finnigan MAT-
312 or a JEOL MSRoute mass spectrometer at 70 eV. The
homogeneity of the compounds was monitored by thin-
layer chromatography (TLC) using Merck silica gel 60
GF254 coated glass plates, visualised by iodine vapour or
under UV light at 254/366 nm.
N-Allyl-2-(5-nitro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)-
1-hydrazinecarbothioamide (2b)
Yield 73% as orange crystals; m.p. 230°C (dec.) (lit. (Ka-
rali, 2002; Hall et al., 2009) m.p. 235–236°C, 226–230°C
(dec.)); IR (KBr, cm-1): 3345, 3220 (NH stretching), 1695
1
(C=O), 1610 (C=N), 1163 (C=S); H-NMR (DMSO-d6, d,
ppm): 4.27 (t, J = 5.1 Hz, 2H, CH2CH=CH2), 5.14–5.23
(m, 2H, CH2CH=CH2), 5.86–5.98 (m, 1H, CH2CH=CH2),
7.11 (d, J = 8.7 Hz, 1H, indole C7-H), 8.25 (dd, J = 8.7,
2.4 Hz, 1H, indole C6-H), 8.55 (d, J = 2.1 Hz, 1H, indole
C4-H), 9.75 (t, J = 6.0 Hz, 1H, CS-NH), 11.81 (s, 1H,
indole NH), 12.36 (s, 1H, N–NH); EI MS (70 eV) m/z (%):
305 (M?, 6), 277 (5), 206 (6), 192 (64), 190 (3), 144 (8), 117
(12), 115 (76), 103 (15), 99 (6), 81 (37), 56 (100); Anal.
calcd. for C12H11N5O3S: C: 47.21, H: 3.63, N: 22.94; found:
C: 47.15, H: 3.65, N: 22.98%.
N-(3-Methylphenyl)-2-(5-nitro-2-oxo-1,2-dihydro-3H-
indol-3-ylidene)-1-ydrazinecarbothioamide (2c)
Synthesis
Yield 83% as yellow amorphous solid; m.p. 232°C (dec.);
General method for the synthesis of 5-nitroisatin-
thiosemicarbazones 2a–2l
IR (KBr, cm-1): 3300, 3175 (NH stretching), 1680 (C=O),
1
1610 (C=N), 1151(C=S); H-NMR (DMSO-d6, d, ppm):
2.36 (s, 3H, CH3), 7.11 (d, J = 8.7 Hz, 1H, indole C7-H),
7.12 (d, J = 7.2 Hz,1H, phenyl C4-H), 7.32 (t, J = 7.8 Hz,
1H, phenyl C5-H), 7.39-7.42 (m, 2H, phenyl C2-H, C6-H),
A solution of appropriate N-substituted thiosemicarbazide
(2.5 mmol) in ethanol (10 ml) was added to a hot solution
of 5-nitroisatin (2.5 mmol) in 50% aqueous ethanol
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2253
8.25 (dd, J = 8.7, 2.4 Hz, 1H, indole C6-H), 8.68 (d,
J = 2.1 Hz, 1H, indole C4-H), 11.01 (s, 1H, CS-NH),
11.84 (s, 1H, indole NH), 12.51 (s, 1H, N–NH); EI MS
(70 eV) m/z (%): 355 (M?, 2), 327 (13), 248 (8), 206 (33),
190 (8), 189 (14), 178 (5), 164 (2), 149 (77), 148 (13), 144
(10), 117 (12), 115 (17), 107 (100), 106 (93), 103 (9), 91
(60), 65 (27); Anal. calcd. for C16H13N5O3S: C: 54.08, H:
3.69, N: 19.71; found: C: 54.20, H: 3.64, N: 19.65%.
C6-H), 7.63 (t, J = 7.5 Hz, 1H, phenyl C4-H), 7.80 (t,
J = 7.2 Hz, 1H, phenyl C5-H), 7.86 (d, J = 8.1 Hz, phenyl
C3-H), 8.28 (dd, J = 8.7, 2.4 Hz, 1H, indole C6-H), 8.64
(d, J = 2.4 Hz, 1H, indole C4-H), 11.08 (s, 1H, CS-NH),
11.85 (s, 1H, indole NH), 12.60 (s, 1H, N–NH); EI MS
(70 eV) m/z (%): 409 (M?, 30), 381 (100), 340 (2), 312 (2),
283 (8), 248 (1), 224 (12), 218 (2), 206 (47), 203 (75), 190
(6), 189 (3), 184 (99.7), 178 (21), 176 (8), 163 (5), 161
(31), 149 (29), 148 (5), 145 (45), 144 (15), 117 (8), 115
(41), 103 (31); Anal. calcd. for C16H10F3N5O3S: C: 46.95,
H: 2.46, N: 17.11; found: C: 46.87, H: 2.42, N: 17.16%.
N-(4-Methylphenyl)-2-(5-nitro-2-oxo-1,2-dihydro-3H-
indol-3-ylidene)-1-hydrazinecarbothioamide (2d)
Yield 91% as orange yellow crystals; m.p. 238°C (dec.)
(lit. (Karali, 2002) m.p. 251–253°C); IR (KBr, cm-1):
3300, 3170 (NH stretching), 1707 (C=O), 1620 (C=N),
N-(4-Fluorophenyl)-2-(5-nitro-2-oxo-1,2-dihydro-3H-
indol-3-ylidene)-1-hydrazinecarbothioamide (2g)
1
1157 (C=S); H-NMR (DMSO-d6, d, ppm): 2.34 (s, 3H,
Yield 86% as orange crystals; m.p. 276°C (lit. (Karali,
2002) m.p. 275°C); IR (KBr, cm-1): 3295, 3150 (NH
stretching), 1700 (C=O), 1650 (C=N), 1140(C=S); 1H-
NMR (DMSO-d6, d, ppm): 7.12 (d, J = 8.7 Hz, 1H, indole
C7-H), 7.28 (t, J = 7.8 Hz, 2H, phenyl C2-H, C6-H),
7.57–7.61 (m, 2H, phenyl C3-H, C5-H), 8.26 (dd, J = 8.7,
2.4 Hz, 1H, indole C6-H), 8.65(d, J = 2.4 Hz, 1H, indole
C4-H), 11.06 (s, 1H, CS-NH), 11.85 (s, 1H, indole NH),
12.54(s, 1H, N–NH); EI MS (70 eV) m/z (%): 359 (M?,
13), 331 (59), 301 (3), 248 (3), 206 (18), 190 (5), 189 (3),
178(11), 163 (3), 153 (55), 149 (20), 148 (4), 144 (14), 117
(8), 115 (14), 111 (54), 103 (29), 95 (100), 76 (35), 75 (70);
Anal. calcd. for C15H10FN5O3S: C: 50.14, H: 2.81, N:
19.49; found: C: 50.21, H: 2.77, N: 19.45%.
CH3), 7.12 (d, J = 8.7 Hz, 1H, indole C7-H), 7.25 (d,
J = 8.4 Hz, 2H, phenyl C3-H, C5-H), 7.45 (d, J = 8.4 Hz,
2H, phenyl C2-H, C6-H), 8.26 (dd, J = 8.7, 2.4 Hz, 1H,
indole C6-H), 8.69 (d, J = 2.1 Hz, 1H, indole C4-H), 11.02
(s, 1H, CS-NH), 11.84 (s, 1H, indole NH), 12.52 (s, 1H, N–
NH); EI MS (70 eV) m/z (%): 355 (M?, 1), 327 (7), 248
(3), 206 (29), 190 (3), 189 (8), 178 (3), 149 (66), 148 (24),
144 (6), 117 (7), 115 (13), 107 (70), 106 (100), 103 (8), 91
(71), 65 (28); Anal. calcd. for C16H13N5O3S: C: 54.08, H:
3.69, N: 19.71; found: C: 54.01, H: 3.67, N: 19.75%.
N-(4-Methoxyphenyl)-2-(5-nitro-2-oxo-1,2-dihydro-3H-
indol-3-ylidene)-1-hydrazinecarbothioamide (2e)
Yield 87% as dirty golden amorphous solid; m.p. 233°C
(lit. (Hall et al., 2009) m.p. 220–248°C (dec.)); IR (KBr,
cm-1): 3305, 3180 (NH stretching), 1705 (C=O), 1660
(C=N), 1150(C=S); 1H-NMR (DMSO-d6, d, ppm): 3.79 (s,
3H, OCH3), 7.00 (dd, J = 6.9, 2.1 Hz, 2H, phenyl C3-H,
C5-H), 7.12 (d, J = 8.7 Hz, 1H, indole C7-H), 7.44 (dd,
J = 9.0, 2.1 Hz, 2H, phenyl C2-H, C6-H), 8.26 (dd,
J = 8.7, 2.4 Hz, 1H, indole C6-H), 8.69 (d, J = 2.4 Hz,
1H, indole C4-H), 11.00 (s, 1H, CS-NH), 11.84 (s, 1H,
indole NH), 12.50 (s, 1H, N–NH); EI MS (70 eV) m/z (%):
371 (M?, 8), 343 (9), 248 (9), 206 (9), 190 (16), 189 (8),
165 (34), 163 (4), 150 (29), 148 (12), 144 (22), 133 (14),
123 (53), 117 (7), 115 (18), 108 (100), 103 (12), 80 (83);
Anal. calcd. for C16H13N5O4S: C: 51.75, H: 3.53, N: 18.86;
found: C: 51.82, H: 3.49, N: 18.82%.
N-(4-Bromophenyl)-2-(5-nitro-2-oxo-1,2-dihydro-3H-
indol-3-ylidene)-1-hydrazinecarbothioamide (2h)
Yield 88% as orange yellow amorphous solid; m.p. 262°C
(lit. (Karali, 2002) m.p. 235–236°C); IR (KBr, cm-1):
3300, 3150 (NH stretching), 1680 (C=O), 1600 (C=N),
1159 (C=S); 1H-NMR (DMSO-d6, d, ppm): 7.12 (d,
J = 8.7 Hz, 1H, indole C7-H), 7.57-7.65 (m, 4H, phenyl
C2-H, C3-H, C5-H, C6-H), 8.26 (dd, J = 8.7, 2.4 Hz, 1H,
indole C6-H), 8.66 (d, J = 2.4 Hz, 1H, indole C4-H), 11.07
(s, 1H, CS-NH), 11.86 (s, 1H, indole NH), 12.58 (s, 1H, N–
NH); EI MS (70 eV) m/z (%): 393 (3), 391 (3), 248 (6), 215
(75), 213 (71), 206 (48), 190 (9), 189 (17), 173 (82), 171
(86), 163 (3), 149 (16), 148 (2), 144 (11) 134 (29), 117 (9),
115 (31), 103 (19), 92 (46), 91 (20), 65 (100); Anal. calcd.
for C15H10BrN5O3S: C: 42.87, H: 2.40, N: 16.67; found: C:
42.79, H: 2.39, N: 16.73%.
2-(5-Nitro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)-N-[2-
(trifluoromethyl)phenyl]-1-hydrazinecarbothioamide (2f)
N-(4-Iodophenyl)-2-(5-nitro-2-oxo-1,2-dihydro-3H-indol-
3-ylidene)-1-hydrazinecarbothioamide (2i)
Yield 85% as yellow crystals; m.p. 242°C; IR (KBr, cm-1):
3300, 3210 (NH stretching), 1690 (C=O), 1602 (C=N),
1
1161(C=S); H-NMR (DMSO-d6, d, ppm): 7.14 (d, J =
8.7 Hz, 1H, indole C7-H), 7.56 (d, J = 7.8 Hz, 1H, phenyl
Yield 89% as dirty yellow amorphous solid; m.p. 270°C
(dec.); IR (KBr, cm-1): 3304, 3160 (NH stretching), 1697
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Med Chem Res (2012) 21:2251–2262
1
N-(2,5-Dimethoxyphenyl)-2-(5-nitro-2-oxo-1,2-dihydro-
3H-indol-3-ylidene)-1-hydrazinecarbothioamide (2l)
(C=O), 1622 (C=N), 1159 (C=S); H-NMR (DMSO-d6, d,
ppm): 7.11 (d, J = 8.7 Hz, 1H, indole C7-H), 7.45 (d,
J = 8.7 Hz, 2H, phenyl C2-H, C6-H), 7.79 (dd, J = 8.7,
1.8 Hz, 2H, phenyl C3-H, C5-H), 8.26 (dd, J = 8.7, 2.4 Hz,
1H, indole C6-H), 8.66 (d, J = 2.4 Hz, 1H, indole C4-H),
11.05 (s, 1H, CS-NH), 11.85 (s, 1H, indole NH), 12.57 (s,
1H, N–NH); EI MS (70 eV) m/z (%): 467 (M?, 5), 439
(21), 261 (52), 248 (23), 219 (95), 206 (19), 190 (21), 189
(14), 178 (6), 176 (5), 163 (5), 159 (14), 150 (4), 148 (2),
144 (25), 134 (32), 117 (8), 115 (25), 103 (13), 92 (86), 65
(100); Anal. calcd. for C15H10IN5O3S: C: 38.56, H: 2.16,
N: 14.99; found: C: 38.62, H: 2.13, N: 14.93%.
Yield 82% as light orange red amorphous solid; m.p.
276°C; IR (KBr, cm-1): 3300, 3230 (NH stretching), 1700
1
(C=O), 1610 (C=N), 1109 (C=S); H-NMR (DMSO-d6, d,
ppm): 3.72 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 6.89 (dd,
J = 9.0, 3.0 Hz, 1H, phenyl C4-H), 7.07 (d, J = 9 Hz, 1H,
phenyl C3-H), 7.14 (d, J = 8.7 Hz, 1H, indole C7-H), 7.20
(d, J = 3 Hz, 1H, phenyl C6-H), 8.28 (dd, J = 8.7, 2.4 Hz,
1H, indole C6-H), 8.59 (d, J = 2.4 Hz, 1H, indole C4-H),
10.74 (s, 1H, CS-NH), 12.32 (s, 1H, indole NH), 12.48 (s,
1H, N–NH); EI MS (70 eV) m/z (%): 401 (M?, 3), 373 (4),
206 (21), 195 (38), 190 (2), 189 (9), 180 (49), 176 (1), 153
(41), 148 (2), 144 (7), 138 (100), 115 (20), 107 (5), 103
(10), 95 (34), 76 (14); Anal. calcd. for C17H15N5O5S: C:
50.87, H: 3.77, N: 17.45; found: C: 50.92, H: 3.73, N:
17.40%.
N-(3-Cyanophenyl)-2-(5-nitro-2-oxo-1,2-dihydro-3H-
indol-3-ylidene)-1-hydrazinecarbothioamide (2j)
Yield 80% as yellow amorphous solid; m.p. 275–280°C
(dec.); IR (KBr, cm-1): 3285, 3170 (NH stretching), 1701
1
(C=O), 1624 (C=N), 1157 (C=S); H-NMR (DMSO-d6, d,
Biological testing
ppm): 7.12 (d, J = 8.7 Hz, 1H, indole C7-H), 7.66 (t,
J = 7.8 Hz, 1H, phenyl C5-H), 7.77 (dt, J = 7.8, 1.2 Hz,
1H, phenyl C4-H), 7.98 (ddd, J = 8.1, 2.1, 1.2 Hz, 1H,
phenyl C6-H) 8.11 (t, J = 1.5 Hz, 1H, phenyl C2-H), 8.27
(dd, J = 8.7, 2.4 Hz, 1H, indole C6-H), 8.63 (d,
J = 2.4 Hz, 1H, indole C4-H), 11.17 (s, 1H, CS-NH),
11.88 (s, 1H, indole NH), 12.62 (s, 1H, N–NH); EI MS
(70 eV) m/z (%): 366 (M?, 2), 352 (1), 338 (3), 306 (1),
293 (1), 248 (17), 206 (50), 190 (10), 189 (24), 178 (8), 175
(2), 163 (4), 160 (100), 149 (7), 144 (9), 118 (73), 115 (24),
103 (8), 102 (31), 91 (17), 76 (10), 64 (12); Anal. calcd. for
C16H10N6O3S: C: 52.46, H: 2.75, N: 22.94; found: C:
52.53, H: 2.72, N: 22.89%.
Cytotoxicity in vitro
A hatching tray was filled with sea water (38 g sea salt/l
water, pH 7.4). Brine shrimp (Artemia salina Leach) eggs
were sprinkled in one half of the tray and that part was
covered with a lid. Other half of the tray was left open
under light for 1–2 days at 30 3°C. The eggs hatched
and larvae moved from the dark to enlightened area
through the pores made between the two halves of the tray.
Brine shrimp larvae were collected with the help of Pasteur
pipette. Test samples were dissolved in DMF and different
volumes of the prepared solutions were disposed off in
three test tubes, each corresponding to a different con-
centration (500, 100 and 20 lg). The solvent was evapo-
rated to dryness overnight. After ensuring the complete
evaporation of solvent, sea water was added into the test
tubes to a total volume of 5 ml and 2–4 days old larvae
were transferred from hatching tray to the test tubes con-
taining samples. Incubation was done at 30 3°C for 24 h
under illumination. The number of survivors were then
counted and recorded (Rahman et al., 1999). The test tubes
supplemented with DMF and etoposide (the anticancer
drug) served as negative and positive controls, respec-
tively. The controls were also processed in the same way as
the test samples were. Data were analysed by EZ-Fit 5
software from Perrella Scientific, Inc.
N-(2,4-Dimethoxyphenyl)-2-(5-nitro-2-oxo-1,2-dihydro-
3H-indol-3-ylidene)-1-hydrazinecarbothioamide (2k)
Yield 85% as orange red crystals; m.p. 260°C (dec.); IR
(KBr, cm-1): 3280, 3160 (NH stretching), 1680 (C=O),
1
1610 (C=N), 1143 (C=S); H-NMR (DMSO-d6, d, ppm):
3.80 (s, 6H, OCH3), 6.58 (dd, J = 8.7, 2.7 Hz, 1H, phenyl
C5-H), 6.69 (d, J = 2.7 Hz, 1H, phenyl C3-H), 7.14 (d,
J = 8.7 Hz, 1H, indole C7-H), 7.28 (d, J = 8.4 Hz, 1H,
phenyl C6-H), 8.27 (dd, J = 8.7, 2.4 Hz, 1H, indole C6-H),
8.67 (d, J = 2.4 Hz, 1H, indole C4-H), 10.65 (s, 1H, CS-
NH), 11.82 (s, 1H, indole NH), 12.47 (s, 1H, N–NH); EI
MS (70 eV) m/z (%): 401 (M?, 24), 373 (36), 358 (1), 342
(1), 248 (31), 206 (29), 195 (58), 190 (21), 189 (29), 178
(19), 153 (76), 149 (7), 144 (19), 138 (100), 131 (11), 117
(6), 115 (30), 110 (34), 103 (8), 95 (28) 78 (8), 52 (12);
Anal. calcd. for C17H15N5O5S: C: 50.87, H: 3.77, N: 17.45;
found: C: 50.79, H: 3.75, N: 17.53%.
Antifungal activity in vitro
Antifungal activities of all the compounds were studied
against five fungal cultures. Sabouraud dextrose agar (Ox-
oid, Hampshire, England) was seeded with 105 (cfu) ml-1
123

Med Chem Res (2012) 21:2251–2262
2255
O
fungal spore suspensions and transferred to petri plates.
Discs soaked with 20 ll (200 lg/ml in DMSO) of all the
compounds were placed at different positions on the agar
surface. The plates were incubated at 27–29°C for 7 days.
The results were recorded (Hussain et al., 2003) as zone of
inhibition (mm) and compared with standard drugs, mico-
nazole and amphotericin B.
O2N
O
NNHCSNHR
O
N
O2N
H
RNHCSNHNH2
N
50% EtOH(aq.)-AcOH
Reflux, 2h
H
1
2a-2l
2a R = n-C6H13
2g R = 4-FC6H4
2h R = 4-BrC6H4
2i R = 4-IC6H4
2j R = 3-CNC6H4
2k R = 2,4-(CH3O)2C6H3
2l R = 2,5-(CH3O)2C6H3
2b R = CH2=CH-CH2
2c R = 3-CH3C6H4
2d R = 4-CH3C6H4
2e R = 4-CH3OC6H4
2f R = 2-CF3C6H4
Phytotoxicity in vitro
Phytotoxic activity of all the synthesised compounds was
determined according to the modified literature protocol
(McLaughlin et al., 1991) using MeOH and paraquat as
negative and positive controls, respectively.
Scheme 1 Synthesis of title compounds 2a–2l
The crystalline or amorphous solid formed during heating
in each case was filtered hot. Thorough washing with 50%
hot aqueous ethanol afforded the target thiosemicarbazones
2a–2l in good to excellent yields (73–91%).
Urease inhibitory activity in vitro
The modified Berthelot (phenol-hypochlorite) method
(Weatherburn, 1967) was used for the determination of
antiurease activity of the synthesised compounds 2a–2l.
Briefly, 200 ll of phosphate buffer (K2H2PO4 ? KOH, pH
7) containing 1 unit of Human urease (Gesellschaft fu¨r
Biochemica und Diagonistica mbH, Germany) enzyme was
mixed with 230 ll of phosphate buffer and 20 ll of the test
and thiourea (reference inhibitor of urease) solutions in
DMF containing 0.01–100 lM concentrations (three test
tubes for each concentration). DMF (20 ll) tested alone as
a control did not have any inhibitory effect on the activity
of the enzyme. The reaction mixture was then incubated at
25°C for 5 min. After the incubation period, 400 ll of urea
stock solution (20 mM) was added. Calibration mixture
was prepared with no urea solution. All the test tubes were
further incubated for 10 min at 40°C to accomplish urea
hydrolysis. This was followed by the addition of 1,150 ll
phenol-hypochlorite reagent (freshly prepared by mixing
450 ll phenol reagent (phenol ? sodium nitroprusside)
and 700 ll alkali reagent (NaOH ? NaOCl)). The tubes
were again incubated at 56°C for 25 min. After 5 min of
cooling, absorbance of the blue coloured complex thus
formed was noted at 625 nm and the percentage inhibition
was calculated as the difference of absorbance values with
and without the test compounds (Nagata et al., 1992). IC50
values were calculated using EZ-Fit 5 software from Per-
rella Scientific, Inc.
The structures of all the synthesised compounds 2a–2l
1
were determined by analytical and spectral (IR, H-NMR,
EIMS) methods. The infrared (IR) spectra of 2a–2l dis-
played absorption bands in the 3346–3210 and 3180–3150,
1707–1680, 1660–1600 and 1163–1109 cm-1 regions
attributed to NH, C=O, C=N and C=S functions, respec-
tively (Karali, 2002; Naumov and Anastasova, 2001; Omar
et al., 1984; Petrov et al., 1986). The proton nuclear
magnetic resonance (1H-NMR) spectra of compounds 2c–
2l demonstrated the N4-H and N2-H protons of the thio-
semicarbazone moiety (d 10.65–11.17 and d 12.47–12.62,
respectively) and the indole NH proton (d 11.82–12.32) as
three separate singlets (Karali, 2002; Laatsch et al., 1984;
Nizamuddin et al., 1999; Omar et al.,1984). Similarly, the
1H-NMR spectra of 2a and 2b exhibited two separate
singlets at d 11.80–11.81 and d 12.32–12.36, respectively,
for the indole NH and thiosemicarbazone N2-H. However,
the thiosemicarbazone N4-H resonance in these cases was
observed as a triplet at d 9.58–9.75 due to CH2 coupling.
The indole C7-H resonated as a doublet at d 7.11–7.14,
while the indole C6-H, being deshielded due to electron-
attracting inductive effect of the nitro group at position-5,
appeared at d 8.25–8.28 as a double doublet. Indole C4-H
experienced a deshielding effect due to inductively elec-
tron-withdrawing nitro and C=N functions and resonated
further downfield as a doublet at d 8.55–8.69 (Baron
et al.,1990; Eshba et al., 1987; Karali, 2002; Laatsch et al.,
1984). The electron impact mass spectra (EIMS) of all the
synthesised compounds (except 2h) displayed molecular
ions with different intensities. The structure of compound
2h was, however, confirmed by the presence of the frag-
ments corresponding to thiosemicarbazone moiety, formed
by cleavage of N–N and NH–CS bonds. The proposed
fragmentation pattern of 2f is illustrated in Fig. 1. X-ray
structure of 2a was determined in order to confirm the
Results and discussion
Chemistry
5-Nitroisatin, appropriate thiosemicarbazide and 50%
aqueous ethanol containing a catalytic amount of glacial
acetic acid were heated under reflux for 2 h (Scheme 1).
123

2256
Med Chem Res (2012) 21:2251–2262
Fig. 1 The proposed
fragmentation pattern of
compound 2f
-CO
NNHCSNH
O2N
C(SH)N´
NH
NNHCSNH
O
O2N
-CF3
O2N
.
H
NH
m/z 312
-H2N2
N
m/z 283
H
m/z 340
.
+
.
+
NNCS
O
CF3
O2N
.
+
H2N
CF3
+
NNHCSNH
N
O2N
H
m/z 161
m/z 248
NH
m/z 381
-CO
.
+
CF3
NNHCSNH
O
O2N
O2N
NH2
.
+
O
S
.
+
N
N
CF3
H
H
m/z 178
HN N
m/z 409 (M )
CF3
.
H
-N2
m/z 218
.
.
+
+
CF2
CF3
NNH2
m/z 145
O2N
SCN
SCN
F
O
N
+
O2N
N
H
-NH3
O2N
.
+
m/z 184
m/z 206
CN
m/z 203
O
N
.
HNCO
O2N
-HNCO
H
m/z 190
H
NCO
O2N
.
+
.
+
m/z 189
NH
NNH2
.
NH
H
.
m/z 148
m/z 163
-CO
NO2
H
.
NO2
NO2
H
.
.
+
+
NH
O
CN
O2N
.
+
NNH
NH
NCO
m/z 144
m/z 176
m/z 103
m/z 117
assigned structures and establish conformations of the
synthesised thiosemicarbazones 2a–2l. Relevant crystal
data and details of the structure elucidation have been
reported elsewhere (Pervez et al., 2009b).
compounds of this series except 2j gave LD50 values
greater than 1.07 9 10-3–1.63 9 10-3 M against Artemia
salina in this assay and, therefore, can be considered to be
almost inactive. Compound 2j with a cyano group at
position-3 of the phenyl ring, however, proved to be an
active cytotoxic agent in the present series exhibiting LD50
value 1.16 9 10-3 M.
Biology
Cytotoxicity in vitro
Antifungal activity in vitro
The synthesised compounds 2a–2l were subjected to
cytotoxicity evaluation by the brine shrimp lethality bio-
assay. Compound 2m i.e. 1-(5-nitro-2-oxoindolin-3-yli-
dene)-4-phenylthiosemicarbazide, the synthesis of which
has been reported elsewhere (Pervez et al., 2010), served as
a reference point to evaluate the effects of different sub-
stituents (alkyl, alkenyl and aryl having one or two func-
tions about the phenyl ring) at N4 of the thiosemicarbazone
moiety on cytotoxicity potential of these compounds. From
the data recorded in Table 1, it is evident that all the
Antifungal activity of the synthesised compounds 2a–2l
was determined against five fungal cultures i.e. Candida
albicans, Aspergillus flavus, Microsporum canis, Fusarium
solani and Candida glabrata (Table 2) at 200 lg/ml in
DMSO. Here too, compound 2m served as a reference
point to assess the effects of different substituents attached
to N4 of the thiosemicarbazone moiety on antifungal
potential of these compounds. All the compounds except 2i
were found to be active against one or more pathogens
123

Med Chem Res (2012) 21:2251–2262
2257
Table 1 Brine shrimp bioassay
for compounds 2a–2m
position of the phenyl ring, however, showed 20% inhibi-
tion against A. flavus only. Similarly, compound 2f having
trifluoromethyl function at ortho position of the phenyl ring
exhibited 20% inhibition against M. canis only. Amongst
the halogenated compounds 2g–2i, compounds 2g and 2h
with fluoro and bromo groups at para position were active
against M. canis and F. solani, and A. flavus, respectively,
demonstrating 20% inhibition, whereas compound 2i hav-
ing iodo substituent at the same position was inactive
against all the fungi. Also, similar to compound 2d, com-
pound 2j with cyano substituent at mata position of the
phenyl ring exhibited relatively better activity profile (20,
20 and 15%, respectively) against the same fungal strains
i.e. A. flavus, M. canis and F. solani. Of the remainder
N4-aryl-substituted compounds 2k and 2l having methoxy
substituents at ortho, para (2, 4) and ortho, meta (2, 5)
positions of the phenyl ring, respectively, compound 2l was
found to show relatively more inhibition against A. flavus
(40 vs. 20%). Interestingly, the reference compound 2m
having no substituent about its phenyl ring also displayed
20% inhibition of A. flavus, thus clearly indicating that the
electronic effects of different substituents about the phenyl
ring attached to N4 of the thiosemicarbazone moiety did
not play a significant role in enhancing the capability of
certain active compounds to exhibit added inhibition
against this fungal strain. On the contrary, both 2k and 2l
demonstrated 20% inhibition of M. canis, whereas as
mentioned earlier, 2m was found to be completely inactive
against this fungal culture.
Compounds
LD50 (M)
2a
2b
2c
2d
2e
2f
[1.43 9 10-3
[1.63 9 10-3
[1.40 9 10-3
[1.40 9 10-3
[1.34 9 10-3
[1.22 9 10-3
[1.39 9 10-3
[1.19 9 10-3
[1.07 9 10-3
1.16 9 10-3
2g
2h
2i
2j
2k
[1.24 9 10-3
[1.24 9 10-3
[1.47 9 10-3
2l
2ma
a
Pervez et al., 2010
Table 2 Antifungal activity (in vitro) of compounds 2a–2m*
(% inhibition)
Compounds Microbial species
C. albicans A. flavus M. canis F. solani C. glabrata
2a
2b
2c
2d
2e
2f
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
30
20
00
00
20
00
20
20
40
20
50
20
00
40
00
20
20
00
00
20
20
20
00
00
00
30
30
00
00
20
00
00
15
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
Notably, some fungi, especially C. albicans and
C. glabrata were resistant to all the tested compounds,
whereas the others i.e. A. flavus and F. solani showed
resistance to most of the tested compounds. None of the
compounds of the present series displayed significant
antifungal activity; however, the negative findings or dis-
play of weak or moderate activities do not preclude from
further investigations of these compounds against other
fungal strains.
2g
2h
2i
2j
2k
2l
2m
*Concentration used 200 lg/ml of DMSO
Phytotoxicity in vitro
exhibiting varied inhibition (15–50%). Compounds 2a and
2b having n-hexyl and allyl substituents at N4 of the thi-
osemicarbazone moiety were found to display 50 and 20%
inhibition of M. canis, respectively, whereas 2m with
phenyl group at the same position displayed no activity
against the said fungal strain. Amongst the aryl-substituted
compounds 2c and 2d having methyl substituents at dif-
ferent positions of the phenyl ring, compound 2c with
meta-substituent demonstrated 30% inhibition against
F. solani, whereas the para-substituted compound 2d dis-
played relatively much better activity profile (30, 40 and
30%) against A. flavus, M. canis and F. solani, respec-
tively. Compound 2e having methoxy substituent at para
The synthesised 5-nitroisatin-3-thiosemicarbazones 2a–2l
were also tested for their phytotoxicity potential at 1000,
100 and 10 lg/ml concentrations. The results presented in
Table 3 revealed that all the compounds except 2a and 2b
exhibited weak or non-significant (20–35%) plant growth
inhibition at the highest tested concentration (1,000 lg/ml).
Compounds 2a and 2b having n-hexyl and allyl substitu-
ents at N4 of the thiosemicarbazone moiety, however,
displayed moderate inhibition (60 and 50%, respectively)
at the same tested concentration. Out of ten compounds
possessing different substituents about the phenyl ring
attached to N4 of the thiosemicarbazone moiety, eight i.e.
123

2258
Med Chem Res (2012) 21:2251–2262
From the results obtained in this assay, it may be con-
cluded that compared with the standard drug, paraquat,
which displayed 100% inhibition at a concentration of
0.015 lg/ml, our test compounds exhibited weak or non-
significant inhibition at much higher concentration. Fur-
thermore, the percent growth inhibition values displayed by
our trial compounds revealed that the nature or type of the
substituents at N4 of the thiosemicarbazone moiety did not
significantly affect their phytotoxicity potential at both the
highest as well as lowest tested concentrations (1,000 and
10 lg/ml).
Table 3 Percent growth inhibition of Lemna aequinocitalis by
compounds 2a–2m at different concentrations
Compounds
1000 (lg/ml)
(% G.I.)
100 (lg/ml)
(% G.I.)
10 (lg/ml)
(% G.I.)
2a
2b
2c
2d
2e
2f
60
50
25
30
25
25
20
20
25
25
30
35
20b
35
35
20
20
15
15
15
15
10
15
25
30
10b
25
25
10
15
10
10
05
10
05
05
15
25
05b
2g
2h
2i
Urease inhibition in vitro
2j
Urease inhibitory effects of the synthesised thiosemicar-
bazones 2a–2l were determined according to the protocol
of the Department of Chemistry, The Islamia University of
Bahawalpur, Pakistan. The results given in Table 4 dem-
onstrated that all the compounds tested in this assay except
2g exhibited enzymatic activity at almost all the tested
concentrations (100, 10, 1, 0.1, 0.01 lM). Compound 2d
and the reference point 2m, however, did not show
inhibitory activity at 0.1 and 10 lM concentrations,
respectively, whereas compound 2g showed an anomalous
behaviour displaying increment in the enzymatic activity
at all the tested concentrations. The results collected in
Table 4 also revealed that as compared to compound 2m
with a phenyl group at N4 of the thiosemicarbazone
moiety, 2a and 2b having n-hexyl and allyl functions at
the same position exhibited enhanced enzyme inhibitory
activity at the highest tested concentration (100 lM).
2k
2l
2ma
The reference compound paraquat shows 100% growth inhibition at a
concentration of 0.015 lg/ml
G.I. growth inhibition
a
b
Tested at 500, 50 and 5 lg/ml concentrations; corresponding G.I.
values
2c–2f and 2i–2l were found to exhibit relatively greater
inhibition of plant growth (25–35%), when compared with
the reference point 2m (having un-substituted phenyl ring),
which displayed 20% plant growth inhibition. On the other
hand, all the synthesised compounds including 2m dem-
onstrated weak or non-significant (5–25%) plant growth
inhibition at the lowest tested concentration (10 lg/ml).
Table 4 Inhibition of human urease by compounds 2a–2m
Compounds Inhibition at 100 lM
(% SD)
Inhibition at 10 lM
(% SD)
Inhibition at 1 lM
(% SD)
Inhibition at 0.1 lM Inhibition at 0.01 lM IC50
(% SD)
(% SD)
(lM)
2a
40.9 2.571
15.9 0.950
34 3.000
27 1.000
37 1.527
12 1.000
13.9 0.854
58 1.616
15 0.577
5.8 1.026
NA
60 1.327
21.6 1.184
6.5 2.157
0.440
2b
8
1.001
22 2.640
0.416
2c
2d
7
0.577
4
4
1.386
8
1.517
2e
46.6 1.527
21 1.184
:
38.6 0.529
20.9 0.173
:
33.6 1.527
30.9 1.001
:
44 1.000
23 1.154
:
47.6 0.577
21.6 0.635
:
2f
2g
2h
4
0.896
8
0.577
6.6 0.550
52 1.157
1.8 1.205
61 3.605
15.6 0.692
0.65 0.224
20.92 0.501
4.5 0.814
51 2.200
16 2.804
66.8 2.362
25.9 0.173
5.8 0.763
14.54 1.504
18.9 1.000
57 0.577
20.7 1.123
68 1.258
34.5 2.590
8.8 1.616
8.16 0.501
2i
50 3.055
8.9 1.792
37.9 2.475
11 1.500
47 1.442
12.6 2.281
53.5 2.200
17.8 1.069
NA
0.901
2j
2k
27.880
2l
2m
Thioureaa
6
0.320
94.33 1.003
8.09 2.006
50.105
: Increment in enzymatic activity, NA no inhibitory activity
a
Reference urease inhibitor
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Med Chem Res (2012) 21:2251–2262
2259
Much pronounced enhancement was observed in the
case of 2a (6.0% ? 40.9% with IC50 value 0.440 lM).
Similarly, compared with 2m, all the synthesised N4-aryl-
substituted thiosemicarbazones except 2d and 2h dis-
played increased inhibitory activity at the same tested
concentration. Relatively, much increment was observed
in the cases of 2c, 2e, 2i and 2k (34.0, 46.6, 50.0% with
IC50 value 0.901 lM and 37.9% with IC50 value
27.880 lM, respectively). To the contrary, compound 2h
was found to exhibit slight decrement in the inhibitory
activity (6% ? 4%), whereas 2d displayed almost the
same activity as of the reference point 2m (7 vs. 6%).
Overall, out of 12 compounds tested for their urease
inhibitory potential, 11 i.e. 2a–2f and 2h–2l exhibited
enzymatic inhibition. Of these, three i.e. 2a, 2i and 2k were
found to be potent inhibitors of urease activity. These
compounds may act as leads and could be the potential
candidates for further studies.
of bulky attachment makes it easier for the urease inhibi-
tors to enter the substrate-binding pocket as well as avoid
unfavourable steric interactions with the amino acid resi-
dues in their vicinity (Amtul et al., 2002). The results that
we have observed in the cases of potent thiosemicarba-
zones 2a, 2i and 2k are consistent with these hypotheses.
The SAR studies demonstrated that the differential inhib-
itory potential, as exhibited by these potent compounds,
depended primarily on the electronic and/or steric effects
of the alkyl or aryl substituents attached to N4 of the thi-
osemicarbazone moiety. For example, compared with the
reference point 2m having un-substituted phenyl group at
N4, compound 2a possessing an electron-donating n-hexyl
substituent at the same position displayed much pro-
nounced inhibitory potential (IC50 value 0.440 lM). This
might be attributed to the promotion of electron-avail-
ability on N4 by the n-hexyl function. The unshared elec-
tron-pair on N4 is fully available for delocalization over the
thiocarbonyl sulphur atom, so as to enhance its electron-
affinity for coordination with the electrophilic nickel ions
present at the active site of the enzyme. In the case of 2m,
reduction in electron-availability on N4 occurs by the
partial delocalization of unshared electron-pair over the
benzene ring-carbon atoms via their p orbitals. This caused
reduction in the electron-affinity of the thiocarbonyl sul-
phur atom, which took part in coordination/chelation with
the nickel ions of the enzyme’s active site, making a rel-
atively less stable enzyme-inhibitor chelate and thus
resulting into a much pronounced decrement in inhibitory
activity. To the contrary, compound 2i having iodo sub-
stituent at para position of the phenyl ring exhibited a high
degree of inhibitory potential with IC50 value 0.901 lM.
Iodo group is both electron-donating by mesomeric effect
and electron-attracting by inductive effect. However, the
larger positive mesomeric effect overweighs the smaller
negative inductive effect. The IC50 value indicates that by
comparison, the electron-donating mesomeric effect of the
iodo substituent increases the inhibitory potential of the
compound because of much reduction in delocalization of
the N4 unshared electron-pair over the benzene ring-carbon
atoms. The unshared electron-pair on N4 is now more
available for delocalization over the adjacent electron-
withdrawing thiocarbonyl group, thus increasing the elec-
tron-affinity of the sulphur atom and making a relatively
more stable enzyme-ligand complex. In view of this, it was
expected that substitution of two strong electron-donating
groups at ortho and para positions of the phenyl ring would
have given a more potent urease inhibitor with IC50 value
lower than that of compound 2i. However, quite interest-
ingly, compound 2k with methoxy substituents at ortho and
para positions of the phenyl ring attached to N4 of the
thiosemicarbazone moiety displayed much higher IC50
value (27.880 lM). This effect can be attributed to the
All ureases, regardless of their origin, possess besides
two coordinated Ni(II) ions (Benini et al., 1998, 2000,
2001, 2004; Ciurli et al.,1999; Krajewska and Zaborska,
2007a, b), one, two or three closely related protein subunits
(Mobley et al., 1995). Thus, a urease inactivator inter-
mingles with the enzyme activity by contacting either with
the Ni(II) ions or the protein constituent. b-Mercap-
toethanol (BME), hydroxamic acids (HXAs) and phosp-
horodiamidates (PPDs), for example, are the synthetic
inhibitors, which were reported to interact with the enzyme
activity by binding to the Ni(II) ions present at its active
site (Ciurli et al., 1999; Kuhler et al., 1995; Nagata et al.,
1993; Zaborska et al., 2007). In contrast, sulphenamide,
quinones and heavy metal ions have been found to influ-
ence enzymatic activity by intermingling with the thiol
(S–H) functions present in its protein component (Krajewska
2008; Krajewska and Zaborska, 2007a, b; Krajewska et al.,
2004; Kuhler et al., 1995; Nagata et al., 1993; Zaborska
et al., 2007).
The actual mechanism of urease inhibition by our trial
compounds 2a–2l is not documented. These compounds
seem to be mechanism-based urease inhibitors and inhibit
the enzyme through a chelate interaction, which binds to its
active site in a normal substrate-like mode. This process
appears to be similar to charge-transfer transitions, which
occur between thiolate of b-mercaptoethanol and Ni (II) of
the enzyme, suggesting that the thiolate binds directly to
the nickel ion(s). Different non-covalent interactions
including hydrogen bonds and hydrophobic contacts may
stabilise such enzyme-inhibitors chelate interactions and
thus contribute towards their inhibitory potential. Addi-
tionally, inhibitory potency may also depend upon elec-
tron-affinity of the coordinating atoms. Furthermore, bulky
groups attached to the pharmacophore (the actual ligand-
chelator) may reduce activity of the inhibitors, as the lack
123

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Med Chem Res (2012) 21:2251–2262
steric bulk introduced by methoxy group at ortho position
of the phenyl ring, which overweighed the positive meso-
meric effects of the substituents in part and thus played an
important role in decreasing the inhibitory potential of the
compound. Expectedly, because of steric hindrance of the
substituent at ortho position, the side chains of the enzyme
may not be able to efficiently access the compound/ligand
for binding. This conclusion is supported by the much
smaller IC50 value given by compound 2i having a bulky
iodo substituent at para position of the phenyl ring, which
exhibited either no or much less steric hindrance. Fur-
thermore, contrary to compound 2k, the corresponding
mono-substituted compound possessing a methoxy sub-
stituent at ortho position of the phenyl ring did not show
any inhibitory activity in our earlier assay (Pervez et al.,
2010). However, compared with the reference compound
2m, the corresponding mono-substituted compound 2e
with a methoxy substituent at para position of the phenyl
ring was found to display much enhanced inhibitory
activity in the present assay. Similarly, as compared to 2m,
the respective di-substituted compound 2l having methoxy
substituents at ortho and meta (2,5) positions of the phenyl
ring exhibited enhanced inhibitory activity at all the tested
concentrations in the present assay, but with percent
inhibitory values lower than that of compound 2e. This
lessening in inhibitory potential is attributed to the fact that
the mesomeric effects are similar at ortho and para posi-
tions but different for the substituents at meta position.
These results strongly suggest that mesomeric effects of the
substituents at ortho and para positions of the phenyl ring
play a pivotal role in the inhibition of urease. Nonetheless,
extensive studies are required to elucidate the mechanism
by which isatins-derived thiosemicarbazones exhibit the
urease inhibitory activity.
significant phytotoxic influences at the highest tested con-
centration, invited attention to their effectiveness as potent
inactivators of soil ureases, as they could be combined with
fertilizers in small amounts to increase the overall effi-
ciency of nitrogen utilisation. The SAR studies revealed
that the urease inhibitory potential of the test compounds
depended mainly upon electronic effects of the substituents
attached to N4 of the thiosemicarbazone moiety. Further-
more, these studies may serve as a basis for chemical
modifications aimed at the development of potential bio-
active compounds of agricultural/medicinal interest.
Acknowledgments We acknowledge the partial funding of this
research work and the award of indigenous PhD scholarship to NM by
Higher Education Commission, Pakistan.
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