Synthesis, X-ray molecular structure, biological evaluation and molecular docking studies of some N4-benzyl substituted 5-nitroisatin-3-thiosemicarbazones

Article abstract of DOI:10.1016/j.bmc.2016.12.012

A series of fifteen N⁴-benzyl substituted 5-nitroisatin-3-thiosemicarbazones 5a–o was synthesized and evaluated for urease inhibitory, phytotoxic and cytotoxic influences. All the compounds proved to be highly potent inhibitors of the enzyme, s

Full text of DOI:10.1016/j.bmc.2016.12.012

Accepted Manuscript
Synthesis, X-ray molecular structure, biological evaluation and molecular dock-
ing studies of some N4-benzyl substituted 5-nitroisatin-3-thiosemicarbazones
Humayun Pervez, Nazia Khan, Sumera Zaib, Muhammad Yaqub, Muhammad
Moazzam Naseer, Muhammad Nawaz Tahir, Jamshed Iqbal
PII:
S0968-0896(16)31048-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.12.012
BMC 13439
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 October 2016
7 December 2016
8 December 2016
Please cite this article as: Pervez, H., Khan, N., Zaib, S., Yaqub, M., Naseer, M.M., Tahir, M.N., Iqbal, J., Synthesis,
X-ray molecular structure, biological evaluation and molecular docking studies of some N4-benzyl substituted 5-
nitroisatin-3-thiosemicarbazones, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.
2016.12.012
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Synthesis, X-ray molecular structure, biological evaluation and molecular docking studies of
some N4-benzyl substituted 5-nitroisatin-3-thiosemicarbazones
Humayun Pervez,*,a Nazia Khan,a Sumera Zaib,b Muhammad Yaqub,a Muhammad Moazzam
Naseer,c Muhammad Nawaz Tahir,d Jamshed Iqbal*,b
aInstitute of Chemical Sciences, Organic Chemistry Division, Bahauddin Zakariya University,
Multan 60800, Pakistan
bCentre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad
22060, Pakistan
cDepartment of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
dDepartment of Physics, University of Sargodha, Sargodha 45320, Pakistan
Correspondence:
E-mail addresses: pdhpervez@hotmail.com (H. Pervez); drjamshed@ciit.net.pk (J. Iqbal)
Abstract
A series of fifteen N4-benzyl substituted 5-nitroisatin-3-thiosemicarbazones 5a-o was synthesized
and evaluated for urease inhibitory, phytotoxic and cytotoxic influences. All the compounds proved
to be highly potent inhibitors of the enzyme, showing inhibitory activity (IC50 = 0.87 ± 0.25 – 8.09 ±
0.23 μM) much better than the reference inhibitor, thiourea (IC50 = 22.3 ± 1.12 μM) and may thus act
as persuasive leads for further studies. In phytotoxicity assay, twelve out of fifteen
thiosemicarbazones tested i.e. 5a-e, 5g, 5i and 5k-o appeared to be active, exhibiting weak or non-
significant (5-35%) growth inhibition at the highest tested concentrations (1000 or 500 µg/mL). In
contrast, only one compound i.e. 5i was active in the brine shrimp (Artemia salina) lethality
bioassay, demonstrating cytotoxic activity with LD50 value 2.55 × 10-5 M. Molecular docking studies
of compounds 5a-o were also performed to identify their probable binding modes in the active site of
the enzyme.
Keywords: Cytotoxicity, 5-Nitroisatin, Phytotoxicity, Thiosemicarbazones, Urease inhibition,
Urease inhibitors
1. Introduction

Isatin and its derivatives are reported to exhibit a broad spectrum of chemotherapeutic properties.1-25
Among isatin derivatives, isatin-thiosemicarbazones have been found to demonstrate diverse
biological activities such as analgesic and antiinflammatory, anticancer, antimicrobial,
antitubercular, antiulcer, antiviral and enzymatic inhibition.1-5,7,11,12,17,21-25 In view of these facts and
as a part of our synthetic work on potential biologically active isatin derivatives, we have recently
synthesized a number of N4-aryl substituted isatin-3-thiosemicabazones as antibacterial, antifungal,
antileishmanial, cytotoxic, phytotoxic and more importantly antiurease compounds.26-35 Investigation
of the structure-activity relationships (SAR) revealed that in some cases, the nature and position of
the substituents about the phenyl ring attached to N4 of the thiosemicarbazone moiety and/or the
presence of inductively electron-withdrawing/lipophilic groups (nitro, trifluoromethoxy, fluoro,
chloro) at position-5 of the isatin scaffold played an important role in the inducement and/or
increment of certain activities. Furthermore, during a preliminary screening of some isatin-
thiosemicarbazones prepared in our laboratory, oneviz.N-benzyl-2-(5-nitro-2-oxo-2,3-dihydro-1H-
indol-3-ylidene)-1-hydrazinecarbothioamidedrew our attention, as it showed about four fold more
urease inhibitory activity than the reference inhibitor, thiourea, used in the assay. In view of this, it
was envisaged that the attachment of variously substituted benzyl groups (having one or two
inductively electron-attracting /-donating substituents about the phenyl ring) to N4 of the
thiosemicarbazone moiety would cause an increase in the urease inhibitory potential of the
molecules. Thus, the present work to synthesize a series of fifteen title thiosemicarbazones and
screen them mainly for their urease inhibitory as well as phytotoxicity and cytotoxicity potential was
carried out.
This paper is one of the first to report on N4-benzyl substituted isatin-thiosemicarbazones with urease
inhibitory potential. In it, we confine our discussion to those compounds, which are having attached
concurrently strong inductively electron-attracting/lipophilic nitro group at position-5 of the isatin
scaffold and the benzyl substituents (modified by substituting one inductively electron-donating and

one or two electron-attracting substituents about the phenyl ring) to N4 of the thiosemicarbazone
moiety.
2. Experimental
2.1. General
Melting points (uncorrected) were determined on a Fisher-Johns melting point apparatus. Elemental
analyses were performed by using a Leco CHNS-9320 (USA) elemental analyzer. FT-IR spectra
1
(KBr disks) were run on Shimadzu Prestige-21 FT-IR spectrophotometer. H-NMR spectra were
recorded in DMSO-d6 on Bruker Avance 300 and Bruker Spectrospin 400 spectrometers, operating at
300 and 400 MHz, respectively, and using TMS as an internal standard. The chemical shifts are
reported in δ/ ppm and coupling constants in Hz.13C-NMR spectra were recorded in DMSO-d6 on
Bruker Avance 300, operating at 75 MHz with the same internal standard. Electron impact (EI) mass
spectra were obtained on MAT-312 and JEOL MSRoute mass spectrometers. X-ray crystallographic
data were taken on Bruker Kappa APEXII CCD. All the chemicals and solvents used in the present
study were procured from Merck-Schuchatdt, Fluka and Sigma-Aldrich.
2.2. Synthesis of N-substituted thiosemicarbazides 3
The N-substituted thiosemicarbazides used in this study were synthesized according to the reported
synthetic route.36
2.3. Synthesis of isatin-thiosemicarbazones 5a-o
An appropriate thiosemicarbazide 3 (5 mmol) dissolved in ethanol (10 mL) was added to a hot
solution of 5-nitroisatin 4 (5 mmol) in 50% aqueous ethanol (15 mL) under stirring and the reaction
mixture was then heated to reflux for 2 h. The crystalline solid formed during heating in each case
was collected by suction filtration and washed with hot aqueous ethanol to afford the desired
compounds 5a-o.
2.3.1. N-Benzyl-2-(5-nitro-2-oxo-2,3-dihydro-1H-indol-3-ylidene)-1-hydrazinecarbothioamide
(5a)

Yellow crystals (82%): mp 262-264 °C; IR (KBr, cm-1): 3344, 3118 (NH), 1693 (C=O), 1604 (C=N),
1157 (C=S); 1H-NMR (DMSO-d6, 400 MHz, δ, ppm): 4.91 (d, J = 6.0 Hz, 2H, benzyl CH2), 7.12 (d,
J = 8.8 Hz, 1H, indole C7-H), 7.25-7.39 (m, 5H, benzyl C2-H, C3-H, C4-H, C5-H, C6-H), 8.26 (dd, J =
8.8, 2.4 Hz, 1H, indole C6-H), 8.55 (d, J = 2.0 Hz, 1H, indole C4-H), 10.13 (t, J = 7.5 Hz, 1H,
CSNH), 11.83 (s, 1H, indole NH), 12.44 (s, 1H, NNH); 13C-NMR (DMSO-d6, δ, ppm): 47.24 (CH2),
111.19 (CH), 116.20 (CH), 120.94, 126.89 (CH), 127.03 (CH), 127.19 (CH), 128.29 (CH), 130.10,
138.14, 142.67, 147.39, 162.95, 177.70; EIMS (70eV) m/z (%): 355( M+, 2), 327 (2), 296 (1), 279
(3), 220 (4), 192 (20), 164 (9), 131 (20), 106 (44), 91 (100), 77 (17), 65 (27), 51 (15). Anal. Calcd for
C16H13N5O3S (355): C, 54.08; H, 3.66; N, 19.72. Found: C, 53.89; H, 3.62; N, 19.66.
2.3.2. N-(2-Methylbenzyl)-2-[5-nitro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-1-
hydrazinecarbothioamide (5b)
Yellow fluffy crystals (82%): mp 240-242 °C; IR (KBr, cm-1): 3319, 3246 (NH), 1701 (C=O), 1601
(C=N), 1163 (C=S); 1H-NMR (DMSO-d6, 400 MHz, δ, ppm): 2.35 (s, 3H, CH3), 4.87 (d, J = 6.0 Hz,
2H, benzyl CH2), 7.12 (d, J = 9.0 Hz, 1H, indole C7-H), 7.19 (s, 4H, benzyl C3-H, C4-H, C5-H, C6-
H), 8.26 (dd, J = 9.0, 3.0 Hz, 1H, indole C6-H), 8.56 (d, J = 3.0 Hz, 1H, indole C4-H), 10.05 (t, J =
7.5 Hz, 1H, CSNH), 11.83 (s, 1H, indole NH), 12.47 (s, 1H, NNH); 13C-NMR (DMSO-d6, δ, ppm):
55.27 (CH2), 112.95 (CH), 113.59 (CH), 114.13 (CH), 122.24, 123.65 (CH), 127.20 (CH), 130.62,
131.06, 133.23 (CH), 141.38, 144.07 (CH), 157.43, 162.26, 176.45; EIMS (70eV) m/z (%): 369 (M+,
2), 341(2), 293(1), 220 (3), 192 (23), 178 (17), 163 (12), 145 (25), 120 (38), 105 (100), 91 (38), 79
(52), 77 (65), 65 (20). Anal.Calcd for C17H15N5O3S (369): C, 55.28; H, 4.07; N, 18.97. Found: C,
55.12; H, 4.04; N, 18.93.
2.3.3. N-(3-Methylbenzyl))-2-[5-nitro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-1-
hydrazinecarbothioamide (5c)
Yellow crystals (72%): mp 210-212 °C; IR (KBr, cm-1): 3323, 3069 (NH), 1699 (C=O), 1610 (C=N),
1
1159 (C=S); H-NMR (DMSO-d6, 400 MHz, δ, ppm): 2.56 (DMSO, CH3), 3.35 (DMSO, benzyl
CH2), 6.96 (d, J = 7.1 Hz, 1H, indole C7-H), 7.35-7.33 (m, 1H, benzyl C4-H), 7.39-7.50 (m, 4H,

benzyl C2-H, C5-H, C6-H, indole C6-H), 7.80 (d, J = 2.0 Hz, 1H, indole C4-H), 10.82 (s, 1H, CSNH),
11.36 (s, 1H, indole NH), 12.71 (s, 1H, NNH); 13C-NMR (DMSO-d6, δ, ppm): 112.64 (CH), 116.96
(CH), 120.83 (CH), 121.67, 126.55, 130.80 (CH), 131.75, 141.33, 162.36, 177.89; EIMS (70eV) m/z
(%): 369 (M+, 3), 341 (2), 293 (2), 248 (2), 220 (8), 206 (6), 192 (46), 178 (20), 163 (17), 145 (46),
144 (11), 121 (65), 105 (100), 90 (18), 91 (55), 79 (19), 77 (73), 65 (34). Anal.Calcd for
C17H15N5O3S (369): C, 55.28; H, 4.07; N, 18.97. Found: C, 54.99; H, 4.02; N, 18.94.
2.3.4. N-(4-Methylbenzyl)-2-[5-nitro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-1-
hydrazinecarbothioamide (5d)
Yellow crystals (89%): mp 208-210 °C; IR (KBr, cm-1): 3316, 3163 (NH), 1678 (C=O), 1600 (C=N),
1
1159 (C=S); H-NMR (DMSO-d6, 400 MHz, δ, ppm): 2.29 (s, 3H, CH3), 4.86 (d, J = 5.6 Hz, 2H,
benzyl CH2), 6.84 (d, J = 9.0 Hz, 1H, indole C7-H), 7.07-7.34 (m, 4H, benzyl C2-H, C3-H, C5-H, C6-
H), 8.27 (d, J = 9.0 Hz, 1H, indole C6-H), 8.55 (s, 1H, indole C4-H), 10.08 (t, J = 7.5 Hz, 1H,
CSNH), 11.83 (s, 1H, indole NH), 12.43 (s, 1H, NNH); 13C-NMR (DMSO-d6, δ, ppm): 20.67 (CH3),
47.02 (CH2), 111.19 (CH), 113.44 (CH), 116.20 (CH), 120.95, 126.88 (CH), 127.21 (CH), 128.51
(CH), 128.83 (CH), 130.11, 135.08, 142.66, 147.37, 162.96, 177.54; EIMS (70eV) m/z (%): 369
(M+, 19), 341 (3), 293 (7), 220 (13), 206 (76), 192 (44), 178 (47), 163 (34), 145 (44), 136 (27), 121
(96), 105 (100), 91 (22), 79 (17), 77 (28), 65 (9). Anal.Calcd for C17H15N5O3S (369): C, 55.28; H,
4.07; N, 18.97. Found: C, 54.91; H, 4.05; N, 18.91.
2.3.5. N-(2-Methoxybenzyl)-2-[5-nitro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-1-
hydrazinecarbothioamide (5e)
Yellow crystals (89%): mp 290-292 °C; IR (KBr, cm-1): 3356, 3093 (NH), 1737 (C=O), 1606 (C=N),
1
1137 (C=S); H-NMR (DMSO-d6, 400 MHz, δ, ppm): 3.85 (s, 3H, CH3), 4.41 (d, J = 5.0 Hz, 2H,
benzyl CH2), 6.91-6.99 (m, 2H, benzyl C3-H, C5-H), 7.11 (d, J = 9.0 Hz, 1H, indole C7-H), 7.21-7.29
(m, 2H, benzyl C4-H, C6-H), 8.24 (d, J = 9.0 Hz, 1H, indole C6-H), 8.42-8.49 (m, 2H, indole C4-H,
NH), 11.73 (s, 2H, CSNH, NNH); 13C-NMR (DMSO-d6, δ, ppm): 37.75 (CH3), 55.34 (CH2), 110.30
(CH), 110.97 (CH), 115.43, 115.58 (CH), 120.17 (CH), 120.69 (CH), 126.87 (CH), 127.75 (CH),

142.59, 146.60, 148.38, 153.75, 156.37, 162.92, 165.04; EIMS (70eV) m/z (%): 369 (3), 339 (3), 206
(100), 178 (80), 163 (93), 149 (20), 136 (51), 121 (97), 104 (26), 91 (91), 77 (26), 65 (17). Anal.
Calcd for C17H15N5O4S (385): C, 52.99; H, 3.90; N, 18.18. Found: C, 52.74; H, 3.85; N, 18.12.
2.3.6. N-(3-Methoxybenzyl)-2-[5-nitro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-1-
hydrazinecarbothioamide (5f)
Yellow crystals (81%): mp 260-262 °C; IR (KBr,cm-1): 3302, 3221 (NH), 1714 (C=O), 1597 (C=N),
1
1147 (C=S); H-NMR (DMSO-d6, 300 MHz, δ, ppm): 3.74 (s, 3H, CH3), 4.87 (d, J = 6.3 Hz, 2H,
benzyl CH2), 6.84 (dd, J = 8.4, 2.4 Hz, 1H, benzyl C4-H), 6.92-6.95 (m, 2H, benzyl C2-H, C6-H),
7.11 (d, J = 8.7 Hz, 1H, indole C7-H), 7.27 (t, J = 8.1Hz, 1H, benzyl C5-H), 8.26 (dd, J = 8.7, 2.4 Hz,
1H, indole C6-H), 8.54 (d, J = 2.4 Hz, 1H, indole C4-H), 10.11 (t, J = 6.3 Hz, 1H, CSNH), 11.83 (s,
13
1H, indole NH), 12.44 (s,1H, NNH); C- NMR (DMSO-d6, δ, ppm): 113.03 (CH), 114.17, 120.74,
122.05, 123.75 (CH), 124.44 (CH), 127.86 (CH), 128.79 (CH), 130.24 (CH), 131.27, 133.50 (CH),
139.82, 141.58, 162.27, 176.19; EIMS (70eV) m/z (%): 385 (M+, 5), 309 (2), 220 (12), 193 (23), 161
(29), 136 (49), 121 (100), 109 (31), 104 (19), 91 (92), 77 (84), 65 (65). Anal.Calcd for C17H15N5O4S
(385): C, 52.99; H, 3.90; N, 18.18. Found: C, 52.85; H, 3.88; N, 18.10.
2.3.7. N-(4-Methoxybenzyl)-2-[5-nitro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-1-
hydrazinecarbothioamide (5g)
Orange crystals (87%): mp 274-276 °C; IR (KBr, cm-1): 3369, 3267 (NH), 1687 (C=O), 1620 (C=N),
1
1164 (C=S); H-NMR (DMSO-d6, 400 MHz, δ, ppm): 3.30 (DMSO, CH3, benzylCH2), 6.90 (d, J =
8.3 Hz, indole C7-H), 7.11 (t, J = 9.0 Hz, benzyl C5-H), 7.49-7.50 (m, 4H, benzyl C2-H, C3-H, C6-H,
indole C6-H), 7.99 (d, J = 2.4 Hz, 1H, indole C4-H), 10.92 (s, 1H, CSNH), 11.37 (s, 1H, indole NH),
12.67 (s, 1H, NNH); 13C- NMR (DMSO-d6, δ, ppm): 112.15 (CH), 112.48 (CH), 112.65 (CH),
113.03 (CH), 114.17, 121.34 (CH), 122.06, 123.78 (CH), 129.87 (CH), 129.99 (CH), 131.23, 133.49
(CH), 139.99, 141.57, 159.96, 162.28, 163.18, 176.14; EIMS (70eV) m/z (%): 385 (M+, 2), 309 (2),
248 (2), 206 (13), 194 (22), 161(57), 136 (34), 121 (100), 109 (22), 104 (11), 91 (38), 77 (74), 65

(28). Anal.Calcd for C17H15N5O4S (385): C, 52.99; H, 3.90; N, 18.18. Found: C, 52.88; H, 3.87; N,
18.14.
2.3.8. N-(2-Fluorobenzyl)-2-[5-nitro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-1-
hydrazinecarbothioamide (5h)
Yellow crystals (82%): mp 264-266 °C; IR (KBr, cm-1): 3352, 3052 (NH), 1701 (C=O), 1607 (C=N),
1172 (C=S); 1H-NMR (DMSO-d6, 400 MHz, δ, ppm): 3.85 (s, 2H, benzyl CH2), 6.90 (d, J = 8.3 Hz,
1H, indole C7-H), 7.01 (t, J = 7.5 Hz, 1H, benzyl C4-H), 7.14 (d, J = 8.0 Hz, 1H, benzyl C6-H), 7.29
(td, J = 8.0, 1.2 Hz, 1H, benzyl C5-H), 7.51-7.54 (m, 2H, benzyl C3-H, indole C6-H), 7.93 (d, J = 2.4
Hz, 1H, indole C4-H), 10.56 (s, 1H, CSNH), 11.34 (s, 1H, indole NH), 12.59 (s, 1H, NNH); 13C-
NMR (DMSO-d6, δ, ppm): 55.65 (CH2), 111.89 (CH), 112.98 (CH), 114.13, 120.06 (CH), 122.21,
123.52 (CH), 126.88, 127.97 (CH), 128.10 (CH), 130.73, 133.28 (CH), 141.46, 153.85, 162.24,
176.90; EIMS (70eV) m/z (%): 373 (M+, 4), 345 (2), 297 (2), 220 (4), 192 (26), 149 (47), 124 (53),
109 (100), 90 (12), 83 (55), 77 (21). Anal.Calcd forC16H12FN5O3S (373): C, 51.47; H, 3.22; N,
18.77. Found: C, 51.39; H, 3.19; N, 18.69.
2.3.9. N-(3-Fluorobenzyl)-2-[5-nitro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-1-
hydrazinecarbothioamide (5i)
Yellow crystals (86%): mp 256-258 °C; IR (KBr, cm-1): 3359, 3201 (NH), 1697 (C=O), 1606 (C=N),
1161 (C=S); 1H-NMR (DMSO-d6, 400 MHz, δ, ppm): 4.92 (d, J = 5.0 Hz, 2H, benzyl CH2), 7.07-
7.23 (m, 4H, benzyl C2-H, C5-H, C6-H, indole C7- H), 7.37-7.46 (m, 1H, benzyl C4-H), 8.27 (d, J =
10 Hz, 1H, indole C6-H), 8.54 (d, J = 2.5 Hz, 1H, indole C4-H), 10.13 (t, J = 7.5Hz, 1H, CSNH),
11.84 ( s, 1H, indole NH), 12.47 (s, 1H, NNH); 13C-NMR (DMSO-d6, δ, ppm): 46.71 (CH2), 111.21
(CH), 113.68 (CH), 116.20 (CH), 120.90, 123.16 (CH), 126.96 (CH), 130.24, 130.29, 130.37 (CH),
141.07, 142.67, 147.42, 162.95, 177.89; EIMS (70eV) m/z (%): 373 (M+, 4), 345 (2), 297 (2), 191
(24), 149 (20), 125 (52), 111 (74), 97 (90), 83 (73), 71 (85), 57 (100). Anal.Calcd forC16H12FN5O3S
(373): C, 51.47; H, 3.22; N, 18.77. Found: C, 51.38; H, 3.20; N, 18.75.
2.3.10. N-(4-Fluorobenzyl)-2-[5-nitro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-1-
hydrazinecarbothioamide (5j)

Yellow fluffy crystals (89%): mp 276-278 °C; IR (KBr, cm-1): 3342, 3130 (NH), 1722 (C=O), 1605
(C=N), 1161 (C=S) ); 1H-NMR (DMSO-d6, 400 MHz, δ, ppm): 3.30 (DMSO, benzyl CH2), 6.91 (d,
J = 8.0 Hz, 1H, indole C7-H), 7.39-7.72 (m, 5H, benzyl C2-H, C3-H, C5-H, C6-H, indole C6-H), 7.83
(d, J = 2.5 Hz, 1H, indole C4-H), 10.90 (s, 1H, CSNH), 11.35 (s, 1H, indole NH), 12.64 (s, 1H,
13
NNH); C- NMR (DMSO-d6, δ, ppm): 113.04 (CH), 114.15, 122.16, 123.65 (CH), 127.60 (CH),
129.02 (CH), 129.61 (CH), 130.61, 131.19, 131.57, 133.41 (CH), 135.94, 141.41, 162.22, 177.68;
EIMS (70eV) m/z (%): 373 (M+, 2), 345 (2), 297 (2), 192 (24), 182 (9), 149 (25), 124 (40), 109
(100), 83 (70), 57 (100). Anal.Calcd forC16H12FN5O3S (373): C, 51.47; H, 3.22; N, 18.77. Found: C,
51.39; H. 3.19; N, 18.68.
2.3.11. N-(2-Chlorobenzyl)-2-[5-nitro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-1-
hydrazinecarbothioamide (5k)
Yellow crystals (91%): mp 268-270 °C; IR (KBr, cm-1): 3348, 3157 (NH), 1692 (C=O), 1616 (C=N),
1188 (C=S); 1H-NMR (DMSO-d6, 400 MHz, δ, ppm): 4.96 (d, J = 5.0 Hz, 2H, benzyl CH2), 7.13 (d,
J = 9.0 Hz, 1H, indole C7-H), 7.27-7.37 (m, 3H, benzyl C4-H, C5-H, C6-H), 7.49 (d, J = 7.5 Hz, 1H,
benzyl C3-H), 8.28 (d, J = 9.0 Hz, 1H, indole C6-H), 8.55 (s, 1H, indole C4-H), 10.12 (t, J = 6.0 Hz,
13
1H, CSNH), 11.85 (s, 1H, indole NH), 12.50 (s, 1H, NNH); C-NMR (DMSO-d6, δ, ppm): 45.05
(CH2), 111.24 (CH), 116.24 (CH), 120.89, 126.99 (CH), 127.01 (CH), 127.79, 128.65, 129.00 (CH),
130.00, 131.00, 135.01, 142.68, 147.45, 162.95, 178.19; EIMS (70eV) m/z (%): 354 (71), 325 (5),
268 (7), 206 (62), 192 (18), 183 (18), 178 (12), 163 (13), 140 (25), 125 (100), 106 (17), 89 (14), 77
(10), 63 (7). Anal.Calcd for C16H12ClN5O3S (389.5): C, 49.29; H, 3.08, N, 17.97. Found: C, 49.38;
H, 3.07; N, 17.95.
2.3.12. N-(3-Chlorobenzyl)-2-[5-nitro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-1-
hydrazinecarbothioamide (5l)
Orange crystals (72%): mp 258-260 °C; IR (KBr,cm-1): 3348, 3064 (NH), 1701 (C=O), 1605 (C=N),
1163 (C=S); 1H-NMR (DMSO-d6, 300 MHz, δ, ppm): 4.90 (d, J = 6.0 Hz, 2H, benzyl CH2), 7.12 (d,
J = 9.0 Hz, 1H, indole C7-H), 7.32-7.43 (m, 4H, benzyl C2-H, C4-H, C5-H, C6-H), 8.28 (dd, J = 8.7,

2.4 Hz, 1H, indole C6-H), 8.54 (d, J = 2.4 Hz, 1H, indole C4-H), 10.14 (t, J = 6.0 Hz, 1H, CSNH),
11.84 (s, 1H, indole NH), 12.47 (s, 1H, NNH); 13C-NMR (DMSO-d6, δ, ppm): 47.14 (CH2), 111.74
(CH), 116.70 (CH), 121.41, 126.43 (CH), 127.43 (CH), 127.50 (CH), 130.76 (CH), 130.91, 133.44,
141.25, 143.16, 147.97, 163.48, 178.34; EIMS (70eV) m/z (%): 389 (M+, 3), 313 (2), 220 (6), 192
(50), 178 (4), 165 (16), 140 (49), 125 (100), 89 (80), 77 (58), 64 (25). Anal.Calcd for C16H12ClN5O3S
(389.5): C, 49.29; H, 3.08, N, 17.97. Found: C, 49.46; H, 3.09; N, 17.99.
2.3.13. N-(4-Chlorobenzyl)-2-[5-nitro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-1-
hydrazinecarbothioamide (5m)
Orange crystals (89%): mp 238-240 °C; IR (KBr,cm-1): 3336, 3188 (NH), 1697 (C=O), 1604 (C=N),
1166 (C=S); 1H-NMR (DMSO-d6, 400 MHz, δ, ppm): 4.89 (d, J = 9.0 Hz, 2H, benzyl CH2), 7.13 (d,
J = 7.5 Hz, 1H, indole, C7-H), 7.37-7.48 (m, 4H, benzyl C2-H, C3-H, C5-H, C6-H), 8.27 (d, J = 8.0
Hz, 1H, indole C6-H), 8.54 (s, 1H, indole C4-H), 10.14 (t, J = 9.0 Hz, 1H, CSNH), 11.84 (s, 1H,
indole NH), 12.45 (s, 1H, NNH); 13C-NMR ((DMSO-d6, δ, ppm): 46.56 (CH2), 111.22 (CH), 116.07
(CH), 120.83, 126.77 (CH), 127.92 (CH), 129.09 (CH), 129.95, 131.52, 137.02, 142.30, 147.48,
162.35, 177.75; EIMS (70eV) m/z (%): 391/389 (M+, 2/7), 361/359 (3/2), 220 (4), 206 (46), 192
(25), 178 (7), 163 (7), 140 (25), 125 (100), 89 (13), 77 (8), 65 (16). Anal.Calcd for C16H12ClN5O3S
(389.5): C, 49.29; H, 3.08, N, 17.97. Found: C, 49.36; H, 3.05; N, 17.93.
2.3.14. N-(2,4-Dichlorobenzyl)-2-[5-nitro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-1-
hydrazinecarbothioamide (5n)
Orange fluffy crystals (80%): mp 260-262 °C; IR (KBr,cm-1): 3336, 3130 (NH), 1681 (C=O), 1606
(C=N), 1159 (C=S); 1H-NMR (DMSO-d6, 300 MHz, δ, ppm): 4.91 (d, J = 6.0 Hz, 2H, benzyl CH2),
7.13 (d, J = 8.4 Hz, 1H, indole C7-H), 7.28 (d, J = 9.0 Hz, 1H, benzyl C6-H), 7.46 (dd, J = 8.1, 2.1
Hz, 1H, benzyl C5-H), 7.66 (d, J = 2.1 Hz, 1H, benzyl C3-H), 8.28 (dd, J = 8.7, 2.4 Hz, 1H, indole
C6-H), 8.55 (d, J = 2.4 Hz, 1H, indole C4-H), 10.13 (t, J = 6.0 Hz, 1H, CSNH), 11.86 (s, 1H, indole
13
NH), 12.51 (s, 1H, NNH); C-NMR (DMSO-d6, δ, ppm): 45.18 (CH2), 111.77 (CH), 116.73 (CH),
121.34 (CH), 127.55 (CH), 127.85 (CH), 129.15 (CH), 129.74, 130.96, 132.76, 132.95, 134.79,

143.17, 147.99, 163.45, 178.70;EIMS (70eV) m/z (%): 390/388 (M+-HCl, 10/13), 208/206 (8/2), 192
(35), 176 (22), 159 (100), 140 (20), 123 (28), 103 (22), 89 (40), 75 (37), 63 (48), 51 (22). Anal.Calcd
for C16H11Cl2N5O3S (424): C, 45.28; H, 2.59; N, 16.51. Found: C, 45.22; H, 2.58; N, 16.49.
2.3.15. N-(3,4-Dichlorobenzyl)-2-[5-nitro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]-1-
hydrazinecarbothioamide (5o)
Orange crystals (79%): mp 270-272 °C; IR (KBr,cm-1): 3336, 3228 (NH), 1693 (C=O), 1612 (C=N),
1166 (C=S); 1H-NMR (DMSO-d6, 300 MHz, δ, ppm): 4.87 (d, J = 6.0 Hz, 2H, benzyl CH2), 7.12 (d,
J = 8.7 Hz, 1H, indole C7-H), 7.35 (dd, J = 8.4, 2.1 Hz, 1H, benzyl C6-H), 7.60-7.64 (m, 2H, benzyl
C2-H, C5-H), 8.26 (dd, J = 8.7, 2.4 Hz, 1H, indole C6-H), 8.52 (d, J = 2.1 Hz, 1H, indole C4-H),
10.12 (t, J = 6.0 Hz, 1H, CSNH), 11.84 (s, 1H, indole NH), 12.47 (s, 1H, NNH); 13C-NMR (DMSO-
d6, δ, ppm):): 46.64 (CH2), 111.69 (CH), 116.64 (CH), 121.30 (CH), 127.46 (CH), 128.10 (CH),
129.65 (CH), 130.06, 130.86, 131.01, 131.36, 139.89, 143.13, 147.91, 163.41, 178.75;EIMS (70eV)
m/z (%): 425/423 (M+, 2/2), 395 (4), 347 (3), 234/232 (4/5), 219/217 (17/21), 206 (84), 192 (29),
176/174 (27/36), 161/159 (94/100). 149/147 (91/61), 140 (39), 125/123 (15/7), 105/103 (15/7), 91/89
(10/10). Anal.Calcd for C16H11Cl2N5O3S (424): C, 45.28; H, 2.59; N, 16.51. Found: C, 45.25; H,
2.54; N, 16.47.
2.4. X-ray structure determination
2.4.1. Crystallographic data collection and structural refinement
Specimen of 5g of good quality and size was mounted on a thin glass fiber at room temperature and
the reflection data were collected on a Bruker Kappa APE XII CCD diffractometer equipped with
graphite mono-chromated Mo Kα radiation (λ = 0.71073 Å). The data were corrected to Lorentz and
polarization effect. The structure was solved using SHELXS-97.37 Final refinement on F2 was carried
out by full-matrix least-squares techniques using SHELXL-9737
2.5. Biological assays
In vitro urease inhibitory, phytotoxicity and cytotoxicity potential was determined in accordance
with the literature protocols given in our published research articles.38,34,31

2.6. Molecular docking studies
Molecular docking studies as well as HYDE assessment were carried out according to the literature
method provided in our very recently published research paper.39 In order to perform docking
studies, the crystallographic structure of Jack bean urease (PDB ID: 3LA4) was obtained from the
RCSB PDB database.40
3. Results and discussion
This study describes the synthesis and in vitro evaluation of antiurease, phytotoxic and cytotoxic
potential of fourteen presently and one previously reported41 N4-benzyl substituted 5-nitroisatin-
thiosemicarbazones 5b-o and 5a, respectively.
3.1. Chemistry of compounds 5a-o
Appropriate N-substituted thiosemicarbazides 3 were treated with 5-nitroisatin 4 in 50% ethanol (aq.)
to afford the respective isatin-3-thiosemicarbazones 5a-o (Scheme 1) in good to excellent yields (72-
91%).
The structures of the synthesized isatin-thiosemicarbazones 5a-o were deduced by analytical and
1
spectral (IR, H-NMR, 13C-NMR, EIMS) as well as single crystal X-ray diffraction analyses.
Satisfactory elemental (CHN) data were obtained in all the cases. Also, the spectral data were in tune
with the respective literature.22,24,42,43 The IR spectra of 5a-o showed NH stretchings of indole and
thioamide functions in the 3359-3302 and 3250-3052 cm-1 regions. The lactam C = O, azomethine C
= N and thioamide C = S stretchings were observed in the 1722-1678, 1616-1597 and 1188-1147 cm-
1
1
regions, respectively.22,42,43 The H-NMR spectra exhibited two separate singlets at δ 11.34-11.86
and 12.43-12.71 for indole NH and thiosemicarbazone N2-H, respectively, in all the cases except 5e,
wherein the indole NH resonance was observed as a multiplet at δ 8.42-8.49 due to overlapping of
indole C4-Hsignal, while the thiosemicarbazone N2-H resonated as a singlet at δ 11.73 together with
thiosemicarbazone N4-H. Similarly, the thiosemicarbazone N4-H appeared as a triplet at δ 10.05-
10.14 in all the cases22,24 except 5c, 5g, 5h and 5j, wherein it resonated as a singlet at δ 10.82, 10.92,

10.56 and 10.90, respectively. Furthermore, the benzyl CH2 protons were observed as a doublet at δ
4.41-4.96 in all the cases22,24 except 5c, 5g, 5h and 5j; in the case of 5h, they appeared as a singlet at
δ 3.85, whereas in 5c, 5g and 5j, they resonated together with the CH3 group protons and/or the
residual protons of DMSO. The 13C-NMR spectra of compounds 5a-o supported the IR and 1H-NMR
findings. Also, the electron impact (EI) mass spectra of all the compounds except 5e, 5k and 5n
demonstrated molecular ions of varied intensity, which validated their molecular weights. The
structures of 5e, 5k and 5n were, however, made certain by the existence of the fragments related to
thiosemicarbazone moieties, formed by the breakage of N-N and NH-CS bonds. The major
fragmentation pathway involved the breakage of endocyclic NH-CO and exocyclic N-N, NH-CS
bonds.43 The proposed fragmentation pattern of 5c is depicted in Figure S1 (see the SI). X-ray
structure of a representative compound 5g was determined to substantiate the assigned structures of
the synthesized thiosemicarbazones 5a-o.
o
Scheme 1. Reagents and conditions: (i) Et3N, MeOH, CS2, stir, 30 C (internal), 75 min., r.t., 1 h (ii) CH3I,
MeOH, stir, -10 oC, 20 min., r.t., 2 h (iii) NH2NH2.H2O, EtOH, reflux, 2 h (iv) 50% EtOH (aq.), reflux, 2 h.
3.1.1. X-ray analysis of compound 5g

For the X-ray structure determinations, X-ray quality single crystals of 5g (Table 1) were obtained
from its DMSO solution by slow evaporation at room temperature. The X-ray analysis demonstrates
that it crystallizes in triclinic crystal lattice with the P¯1 space group. The molecular structure
(ORTEP diagram) of 5g along with crystallographic numbering scheme is depicted in Figure 1.The
central N-iminothiourea (thiosemicarbazone) moiety is almost planar adopting preferably the S-cis/S-
trans conformation, most probably due to the formation of intramolecular hydrogen bond [N(5)-
H(4)···N(3) 2.259 Å].44-46 The dihedral angles between S(1)-C(9)-N(4)-H(3), S(1)-C(9)-N(5)-H(4)
and C(9)-N(4)-N(3)-C(7) are -1.61°, 176.07° and 173.08°, respectively. This planarity around the
central N-iminothiourea moiety can be ascribed to extensive delocalization of N lone pairs onto the
thiocarbonyl (C=S) function, which is clearly shown by the shorter N-C bond lengths [N(4)-C(9)
1.376(3) Å and N(5)-C(9) 1.325(3) Å], suggestive of the partial double bond character of N-C bonds.
The slightly longer bond length in the case of N-C bond, directly connected to the imino (-C=N-)
function of the N-iminothiourea moiety, indicates less delocalization of the N lone pair onto the
thiocarbonyl (C=S) group, presumably due to the presence of inductively electron-attracting sp2-
hybridized N atom in its vicinity. Notably, it is the planarity and the mentioned intramolecular
hydrogen bonding due to which the central N-iminothiourea moiety in such molecules adopts S-
cis/S-trans conformation, thus exposing the thioamide function to form centrosymmetric
supramolecular dimers in their solid state packing.44-46 However, compound 5g has an additional
amide moiety, offering a competing environment for the appearance of otherwise two reliable
centrosymmetric dimer synthons [
engineering.44-47
{···H–N–C=S}2 and
{···H–N–C=O}2] in crystal
Table 1. Crystallographic data of 5g
Crystal data
5g
CCDC
1005939
Chemical formula
C19H21N5O5S2

Mr
463.53
Triclinic, P¯1
296
Crystal system, space group
Temperature (K)
a, b, c (Å)
8.6754 (4), 11.9082 (6),
12.2545 (10)
112.750 (3), 98.066 (3),
106.130 (2)
, ,  (°)
V (Å3)
Z
1076.79 (12)
2
Mo K
Radiation type
 (mm-1)
0.29
Crystal size (mm)
0.38 × 0.23 × 0.20
Data collection
Diffractometer
Bruker Kappa APEXII CCD
Multi-scan
Absorption correction
(SADABS; Bruker, 2005)
Tmin, Tmax
0.898, 0.944
No. of measured,
independent and
observed [I> 2(I)]
reflections
17062, 5051, 3780
Rint
0.022
0.658
(sin /)max (Å-1)
Refinement
R[F2> 2(F2)], wR(F2), S
0.045, 0.150, 1.07
No. of reflections
5051
283
No. of parameters
H-atom treatment
max, min (e Å-3)
H-atom parameters constrained
0.85, -0.41

Figure 1. The molecular structure (ORTEP diagram) of 5g.
3.2. Biology of compounds 5a-o
3.2.1. Urease inhibition (in vitro)
The synthesized N4-benzyl substituted 5-nitroisatin-thiosemicarbazones 5a-o were tested for their
urease inhibitory potential against Jack bean urease. Thiourea served as a reference inhibitor in this
assay. All the compounds proved to be highly potent inhibitors of the enzyme, demonstrating
inhibitory activity (IC50 = 0.87 ± 0.25 – 8.09 ± 0.23 μM) much better than the reference inhibitor,
thiourea (IC50 = 22.3 ± 1.12 μM) (Table 2) and may thus act as convincing leads for further studies.
The structure-activity relationship (SAR) studies in the isatin-thiosemicarbazones 5a-o revealed that
in comparison to compound 5a having no substituent about the phenyl ring of the benzyl group
attached to N4 of the thiosemicarbazone moiety, all the other compounds except 5k and 5m,
irrespective of the type, number and place of the functional groups present on the phenyl ring,
showed increased enzymatic activity (IC50 values 0.87 ± 0.25 – 2.54 ± 0.18 μM vs. 5.92 ± 0.25 μM).
Compound 5g possessing methoxy substituent at position-4 of the phenyl ring was found to be the

most potent urease inhibitor of the series in the present assay, exhibiting about seven and twenty six
fold more activity than compound 5a and the reference inhibitor, thiourea (IC50 = 0.87 ± 0.25 μM vs.
5.92 ± 0.25 and 22.3 ± 1.12 μM, respectively). Next most potent urease inhibitor was compound 5c
with methyl substituent at position-3 of the phenyl ring. This compound displayed somewhat less
inhibitory activity than the most potent inhibitor 5g but much more than the reference inhibitor,
thiourea (IC50 value 1.27 ± 0.19 μM vs.0.87 ± 0.25 and 22.3 ± 1.12 μM, respectively). The third most
potent urease inhibitor was compound 5j having a fluoro substituent at position-4 of the phenyl ring.
This compound, regardless of the nature and position of the substituents about the phenyl ring,
exhibited almost the same activity as that of compound 5c (IC50 value 1.29 ± 0.27 vs.1.27 ± 0.19
μM). However, like compound 5c, it was found to be somewhat less active than the most potent
urease inhibitor of the series i.e. compound 5g (IC50 value 1.29 ± 0.27 vs. 0.87 ± 0.25 μM) but about
five and seventeen times more active than compound 5a and the reference inhibitor, thiourea (IC50
value 1.29 ± 0.27 vs. 5.92 ± 0.25 and 22.3 ± 1.12 μM, respectively). The remaining relatively more
potent urease inhibitors were the mono- and dichloro-substituted derivatives 5l and 5n. Among these,
5l having the substituent at position-3 of the phenyl ring was found to be a little bit less active than
the corresponding methyl-substituted derivative 5c but about four and fourteen times more active
than compound 5a and the reference inhibitor, thiourea (IC50 value 1.63 ± 0.18 μM vs. 1.27 ± 0.19,
5.92 ± 0.25 and 22.3 ± 1.106 μM, respectively). In contrast, the dichloro-substituted derivative 5n
with the substituents at positions-2,4 (ortho, para) of the phenyl ring was four times more active than
the monochloro-substituted derivative 5k bearing the substituent at position-2 of the phenyl ring
(IC50 value 1.94 ± 0.13 vs. 8.09 ± 0.23 μM). This indicated that compound 5n with an additional
inductively electron-withdrawing chloro function at position-4 of the phenyl ring meddled with the
enzyme in a different manner, giving rise to an extensive increase in its inhibitory activity.
The above results showed that the attachment of variously substituted benzyl groups (having one or
two inductively electron-donating or –withdrawing functionalities about the phenyl ring) to N4 of the

thiosemicarbazone moieties caused the molecules to interfere with the enzyme differently and in
some instances much more competently.
Table 2. Inhibition of urease by compounds 5a-o
NNHCSNHCH2R
O2N
O
N
H
Compound
R
IC50 ± SEM*
(µM)
C6H5
5.92 ± 0.25
1.39 ± 0.13
1.27 ± 0.19
1.52 ± 0.11
1.33 ± 0.12
1.34 ± 0.17
0.87 ± 0.25
1.58 ± 0.16
2.54 ± 0.18
1.29 ± 0.27
8.09 ± 0.23
1.63 ± 0.18
6.34 ± 0.18
1.94 ± 0.13
2.11 ± 0.07
22.3 ± 1.06
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
2-CH3C6H4
3-CH3C6H4
4-CH3C6H4
2-CH3OC6H4
3-CH3OC6H4
4-CH3OC6H4
2-FC6H4
3-FC6H4
4-FC6H4
2-ClC6H4
3-ClC6H4
4-ClC6H4
2,4-(Cl)2C6H3
3,4-(Cl)2C6H3
Thiourea**
* Standard error of the mean; ** standard inhibitor for urease.
3.2.2. Phytotoxicity (in vitro)
The synthesized 5-nitroisatin-thiosemicarbazones 5a-o were further tested for their phytotoxicity
potential at 1000, 100, 10 or 500, 50, 5 µg / mL concentrations. All the compounds of this series
except 5f, 5h and 5j appeared to be active in the present assay, displaying weak or non-significant
(5-35%) plant growth inhibition at the highest tested concentrations (1000 or 500 µg/mL) (Table 3).
Table 3. Growth inhibition of Lemna aequinocitalis by compounds 5a-o* at different
concentrations

NNHCSNHCH2R
O
O2N
N
H
1000
100
10
500
50
5
Compound
R
µg/mL
(% G.I)
µg/mL
(% G.I)
µg/mL
(% G.I)
µg/mL
(% G.I)
µg/mL
(% G.I)
µg/mL
(% G.I)
C6H5
−
−
−
10
−
−
−
−
−
−
−
−
−
−
−
−
30
10
00
−
−
−
−
−
−
−
−
−
−
−
−
00
00
00
−
−
−
−
−
−
−
−
−
−
−
−
00
00
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
2-CH3C6H4
3-CH3C6H4
4-CH3C6H4
2-CH3OC6H4
3-CH3OC6H4
4-CH3OC6H4
2-FC6H4
3-FC6H4
4-FC6H4
2-ClC6H4
3-ClC6H4
15
20
05
35
00
10
00
10
00
05
05
05
−
05
05
00
15
00
06
00
00
00
00
00
00
−
00
00
00
00
00
00
00
00
00
00
00
00
−
4-ClC6H4
2,4-(Cl)2C6H3
3,4-(Cl)2C6H3
−
−
−
*The standard drug, paraquat, shows 100% growth inhibition at a concentration of 0.015 µg/mL; G.I:
growth inhibition.
3.2.3. Cytotoxicity (in vitro)
The synthesized thiosemicarbazones 5a-o were also evaluated for their cytotoxicity potential by a
brine shrimp lethality bioassay, using etoposide (a standard anticancer drug) as a reference point.
From the data presented in Table 4, it is evident that all the compounds of this series except 5i gave
LD50 values >2.36 x 10-4 – 2.82 x 10-4 M against Artemia salina in the present assay and, therefore,
can be considered to be almost inactive. Compound 5i bearing 3-fluorobenzyl function at N4 of the
thiosemicarbazone moiety, however, proved to be active, exhibiting cytotoxic activity with LD50
value 2.55 × 10-5 M.
Table 4. Brine shrimp (Artemia salina) lethality bioassay for compounds 5a-o

NNHCSNHCH2R
O
O2N
N
H
Compound
R
LD50
(M)
C6H5
>2.82 x 10-4
5a
5b
5c
5d
5e
5f
2-CH3C6H4
3-CH3C6H4
4-CH3C6H4
2-CH3OC6H4
3-CH3OC6H4
4-CH3OC6H4
2-FC6H4
>2.71 x 10-4
>2.71 x 10-4
>2.71 x 10-4
>2.60 x 10-4
>2.60 x 10-4
>2.60 x 10-4
>2.68 x 10-4
2.55 x 10-5
5g
5h
5i
3.3. Molecular
docking
3.3.1. Molecular
of compounds
docking studies
5a-o
3-FC6H4
>2.68 x 10-4
>2.57 x 10-4
>2.57 x 10-4
>2.57 x 10-4
>2.36 x 10-4
>2.36 x 10-4
To
rationalize
the obtained in
results,
4-FC6H4
5j
vitro biological
2-ClC6H4
5k
5l
molecular
docking studies
inhibitors 5a-o
3-ClC6H4
of
all
the
4-ClC6H4
5m
5n
5o
were performed
bean urease PDB
rationalized that
compounds were
against
Jack
2,4-(Cl)2C6H3
3,4-(Cl)2C6H3
ID: 3LA4. It was
most of the
found to bind in
*Tested at 100, 10, 1 µg/mL concentrations.
the same region of the active site. Before docking the most active inhibitor 5g in the active site of the
urease enzyme, molecular docking studies of the co-crystallized ligand KCX (Lysine NZ-carboxylic
acid) was carried out. The ligand was reproduced successfully with an RMSD value of 1.5 Å.
Molecular docking studies of all the test compounds 5a-o were carried out to investigate the

interactions inside the active site. The most potent inhibitor 5g was successfully oriented inside the
active site of the target enzyme. The putative binding mode of 5g is shown in Figure 2. After
reproducing the co-crystallized reference ligand into the active site of the receptor, all the derivatives
were docked inside the active site. The docking studies supported the experimental results that most
of the inhibitors inhibit the catalytic site of the enzyme. As described previously,38,39 the most
important interactions i.e., hydrogen bonding were seen between the oxygen and hydrogen atoms of
the inhibitors and the amino acid residues Arg609, Asp494, Ala440 and His593. Similarly, as
reported earlier by us,48 stable hydrogen bonding interactions within the active pocket were noticed
with the amino acid residues His492, His593 and His594. The 4-methoxy benzyl substituent was
oriented towards bottom of the active site, while the isatin scaffold located towards opening of the
active site and the mid-gorge area. Moreover, the thiosemicarbazone part was found to interact with
Arg439 and Ala636 in the middle of the active site. Similarly, weak interactions were shown by all
the compounds with Ni842 in the catalytic site of the urease enzyme. Furthermore, the benzyl
substituent also showed π-π interaction with the imidazole ring of His594 and π-charge interactions
were identified by the thiosemicarbazone moieties of the inhibitors. The putative binding pose of
compound 5g was selected after HYDE assessment and visualization of top 30 poses and is
represented in Figure 2.

Figure 2. A possible binding mode of compound 5g is shown. Carbon atoms of 5g are colored blue, while that of
protein light green; oxygen, sulfur and nitrogen atoms are colored red, yellow and blue, respectively. The two nickel
ions in the active site are represented as small dark brown spheres.
3.3.2. HYDE assessment of compounds 5a-o
The HYDE affinity assessment was done for the first 20 top ranking docked conformations. The
binding free energy ΔG, FlexX docking score and the most favorable poses for all the derivatives 5a-
o are given in Table 5. Most of the compounds bind to the receptor with a very high binding affinity.
Table 5. Docking scores and their corresponding ranks by HYDE affinity assessment
Compound
FlexX score of the top
ranking pose
-20.82
Poser rank
Binding free energy ΔG
(kJ mol-1)
10
8
-12
-5
5a
5b
5c
5d
5e
-20.78
-20.45
17
12
15
-5
-20.75
-14
-5
-18.60

-22.62
-19.62
-20.88
-22.25
-20.78
-20.30
-21.10
-20.97
-20.33
-19.94
2
19
9
-2
-13
-5
5f
5g
5h
5i
8
-2
12
16
8
-13
-11
-4
5j
5k
5l
7
-12
-12
-7
5m
5n
5o
9
15
4. Conclusions
In this study, a series of fifteen N4-benzyl substituted 5-nitroisatin-thiosemicarbazones 5a-o have
been synthesized and evaluated for in vitro urease inhibitory, phytotoxic and cytotoxic activities. All
the synthesized thiosemicarbazones proved to be highly potent inhibitors of the enzyme, exhibiting
inhibitory activity even better than the reference inhibitor (i.e. thiourea) used in the assay. These
compounds, by demonstrating no or weak/non-significant phytotoxicity, attract much attention to
their utility as valuable soil ureases inhibitors because they could be mixed with fertilizers in small
quantities to enhance the overall effectiveness of nitrogen utilization. Also, being non toxic, they
could prove to be credible candidates for orally effective remedial agents used for the treatment of
certain clinical conditions induced by microbial ureases.
This study presents the first case of display of urease inhibition by N4-benzyl substituted 5-
nitroisatin-thiosemicarbazones and provides a tangible basis for further studies on such compounds
to develop more potent and safe urease inhibitors of agricultural/ medicinal interest.
Acknowledgements
H. Pervez acknowledges the Higher Education Commission (HEC), Pakistan for funding under
National Research Support Program for Universities (Project No. 20-873/R&D/07/452). J. Iqbal is
thankful to the Organization for the Prohibition of Chemical Weapons (OPCW), The Hague, The

Netherlands and the Higher Education Commission of Pakistan for the financial support through
Project No. 20-3733/NRPU/R&D/14/520.
References
1. Da Silva, J. F. M.; Garden, S. J.; Pinto, A. C. J. Braz. Chem. Soc. 2001, 12, 273.
2. Pandeya, S. N.; Smitha, S.; Jyoti, M.; Sridhar, S. K. Acta Pharm. 2005, 55, 27.
3. Cerchiaro, G.; Ferreira, A. M. C. J. Braz. Chem. Soc. 2006, 17, 1473.
4. Vine, K. L.; Matesic, L.; Locke, J. M.; Ranson, M.; Skropeta, D. Anti-Cancer Agents Med.
Chem. 2009, 9, 397.
5. Bhrigu, B.; Pathak, D.; Siddiqui, N.; Alam, M. S.; Ahsan, W. Int. J. Pharm. Drug Res. 2010, 2,
229.
6. Aboul-Fadl, T.; Bin-Jubair, F. A. S. Int. J. Res. Pharm. Sci. 2010, 1, 113.
7. Pal, M.; Sharma, N. K.; Priyanka, J. K.; Jha, K. K. J. Adv. Sci. Res.2011, 2, 35.
8. Raj, V. Int. J. Curr. Pharm. Res. 2012, 4, 1.
9. Pakravan, P.; Kashanian, S.; Khodaei, M. M.; Harding, F. J. Pharmacol. Rep. 2013, 65, 313.
10. Khan, K. M.; Khan, M.; Ali, M.; Qadir, M. I.; Perveen, S.; Karim, A.; Choudhary, M. I. J. Chem.
Soc. Pak. 2013, 35, 987.
11. Vine, K. L.; Matesic, L.; Locke, J. M.; Scropeta, D. In Advances in anticancer agents in
medicinal chemistry, ed. M. Prudhomme, Bentham Science Publishers, Sharjah (UAE), 2013,
Recent highlights in the development of isatin-based anticancer agents, 254.
12. Grewal, A. S. Int. J. Pharm. Res. 2014, 6, 1.
13. Mathur, G.; Nain, S. Med. Chem. 2014, 4, 417.
14. Adhikari, S.; Bari, S. B.; Samanta, A. J. Appl. Chem. Res. 2014, 8, 31.
15. Phogat, P.; Singh, P. Cent. Nerv. Syst. Agents Med. Chem. 2015, 15, 28.
16. Hussain, A. Z.; Meeran, M. N. Res. J. Pharm. Biol. Chem. Sci. 2015, 6, 1598.
17. Rahim, F.; Malik, F.; Ullah, H.; Wadood, A.; Khan, F.; Javid, M. T.; Taha, M.; Rehman, W.;
Rehman, A. Ur.; Khan, K. M. Bioorg. Chem. 2015, 60, 42.
18. Khan, M.; Khan, K. M.; Rahim, F.; Samreen, Perveen, S.; Karim, A.; Imtiazuddin, Choudhary,
M. I. J. Chem. Soc. Pak. 2015, 37, 520.
19. Corona, A.; Meleddu, R.; Esposito, F.; Distinto, S.; Bianco, G.; Masaoka, T.; Maccioni, E.;
Menéndez-Arias, L.; Alcaro, S.; Le Grice, S. F. J.; Tramontano, E. PLoS One 2016,11, 1.
20. Ziarani, G. M.; Moradi, R.; Lashgarib, N. ARKIVOC 2016, i, 1.
21. Karali, N.; Terzioglu, N.; Gursoy, A. Arch. Pharm. (Weinheim) 2002, 335, 374.
22. Ermut, G.; Karalı, N.; Cetin, I.; Topcul, M.; Birteksoz, S. Marmara Pharm. J. 2013, 17, 147.

23. Karali, N.; Gursoy, A.; Kandemirli, F.; Shvets, N.; Kaynak, F. B.; Ozbey, S.; Kovalishyn, V.;
Dimoglo, A. Bioorg. Med. Chem. 2007, 15, 5888.
24. Guzel, O.; Karali, N.; Salman, A. Bioorg. Med. Chem. 2008, 16, 8976.
25. Banerjee, D.; Yogeeswari, P.; Bhat, P.; Thomas, A.; Srividya, M.; Sriram, D. Eur. J. Med. Chem.
2011, 46, 106.
26. Pervez, H.; Iqbal, M. S.; Tahir, M.Y.; Choudhary, M. I.; Khan, K. M. Nat. Prod. Res. 2007, 21,
1178.
27. Pervez, H.; Iqbal, M. S.; Tahir, M. Y.; Nasim, F. H.; Choudhary, M. I.; Khan, K. M. J. Enzyme
Inhib. Med. Chem. 2008, 23, 848.
28. Pervez, H.; Chohan, Z. H.; Ramzan, M.; Nasim, F. H.; Khan, K. M. J. Enzyme Inhib. Med. Chem.
2009, 24, 437.
29. Pervez, H.; Manzoor, N.; Yaqub, M.; Khan, A.; Khan, K. M.; Nasim, F. H.; Choudhary, M. I.
Lett. Drug. Des. Discov. 2010, 7, 102.
30. Pervez, H.; Ramzan, M.; Yaqub, M.; Khan, K. M. Lett. Drug. Des. Discov. 2011, 8, 452.
31. Pervez, H.; Saira, N.; Iqbal, M. S.; Yaqub M.; Khan, K. M. Molecules 2011, 16, 6408.
32. Pervez, H.; Manzoor, N.; Yaqub, M.; Khan, A.; Nasim, F. H.; Khan, K. M. Med. Chem. Res.
2012, 21, 2251.
33. Pervez, H.; Ramzan, M.; Yaqub, M.; Nasim, F. H.; Khan, K. M. Med. Chem. 2012, 8, 505.
34. Pervez, H.; Saira, N.; Iqbal, M. S.; Yaqub M.; Khan, K. M. Med. Chem. Res. 2013, 22, 5878.
35. Pervez, H.; Manzoor, N.; Yaqub, M.; Khan, K. M. J. Enzyme Inhib. Med. Chem. 2014, 29, 628.
36. Ashton, W. T.; Cantone, C. L.; Chang, L. L.; Hutchins, S. M.; Strelitz, R. A.; MacCoss, M.;
Chang, R. S. L.; Lotti, V. J.; Faust, K. A.; Chen, T-. B.; Bunting, P.; Schorn, T. W.; Kivlighn, S.
D.; Siegl, P. K. S. J. Med. Chem. 1993, 36, 591.
37. Sheldrick, G. M. Acta Crystallogr. A 2008, 64, 112.
38. Rauf, M. K.; Talib, A.; Badshah, A.; Zaib, S.; Shoaib, K.; Shahid, M.; Flörke, U.; Din, I. U.; Iqbal,
J. Eur. J. Med. Chem. 2013, 70, 487.
39. Rauf, M. K.; Zaib, S.; Talib, A.; Ebihara, M.; Badshah, A.; Bolte, M.; Iqbal, J. Bioorg. Med. Chem.
2016, 24, 4452.
40. Balasubramanian, A.; Ponnuraj, K. J. Mol. Biol. 2010, 400, 274.
41. Stone, M. D.; Monteen, K.; Mathis, T.; Ford, K.; Huxford, R.; Lisic, E. C. J. Undergrad. Chem.
Res. 2008, 7, 59.
42. Omar, A. -M. M. E.; Eshba, N. H.; Salama, H. M. Arch. Pharm. 1984, 317, 701.
43. Karali, N. Eur. J. Med. Chem. 2002, 37, 909.

44. Jawaria, R.; Hussain, M.; Shafiq, Z.; Ahmad, H. B.; Tahir, M. N.; Shad, H. A.; Naseer, M. M.
Crystengcomm. 2015, 17, 2553.
45. Hameed, A.; Shafiq, Z.; Yaqub, M.; Hussain, M.; Ahmad, H. B.; Tahir, M. N.; Naseer, M. M.
New J. Chem. 2015, 39, 6052.
46. Pervez, H.; Khan, N.; Iqbal, M. S.; Yaqub, M.; Tahir, M. N. Acta Crystallogr. 2012, E68, o2731.
47. Ahmad, M.; Pervez, H.; Ben Hadda, T.; Toupet, L.; Naseer, M. M. Tetrahedron Lett. 2014, 55,
5400.
48. Ahmad, M.; Pervez, H.; Zaib, S.; Yaqub, M.; Naseer, M. M.; Khan, S. U.; Iqbal, J. RSC Adv.
2016, 6, 60826.

Graphical abstract
Synthesis, X-ray molecular structure, biological evaluation and molecular docking studies of
some N4-benzyl substituted 5-nitroisatin-3-thiosemicarbazones
Humayun Pervez, Nazia Khan, Sumera Zaib, Muhammad Yaqub,a Muhammad Moazzam Naseer,
Muhammad Nawaz Tahir, Jamshed Iqbal*
A possible binding mode of compound 5g bound to active pocket of Jack bean urease