Synthesis, spectroscopic, crystal structure and in vitro cytotoxicity studies of N-thiophenoyl-N′-substituted phenyl thiocarbamide derivatives

Article abstract of DOI:10.1016/j.molstruc.2018.12.011

A series of eight biologically active N, N′-disubstituted thiocarbamide compounds (1–8) have been prepared from thiophene-2-carbonyl isothiocyanate and various substituted aromatic primary amines (2,4-dichlorophenyl aniline, 4-chloro-3-nitrophenyl aniline

Full text of DOI:10.1016/j.molstruc.2018.12.011

Accepted Manuscript
Synthesis, spectroscopic, crystal structure and in vitro cytotoxicity studies of N-
thiophenoyl-N′-substituted phenyl thiocarbamide derivatives
Sunil K. Pandey, Seema Pratap, Gaetano Marverti, Manpreet Kaur, Jerry P. Jasinski
PII:
S0022-2860(18)31432-7
DOI:
https://doi.org/10.1016/j.molstruc.2018.12.011
MOLSTR 25948
Reference:
To appear in: Journal of Molecular Structure
Received Date: 10 October 2018
Revised Date: 24 November 2018
Accepted Date: 4 December 2018
Please cite this article as: S.K. Pandey, S. Pratap, G. Marverti, M. Kaur, J.P. Jasinski, Synthesis,
spectroscopic, crystal structure and in vitro cytotoxicity studies of N-thiophenoyl-N′-substituted
phenyl thiocarbamide derivatives, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2018.12.011.
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Graphical Abstract
Synthesis, spectroscopic, crystal structure and in vitro cytotoxicity studies of
N-thiophenoyl-N'-substituted phenyl thiocarbamide derivatives
Sunil K. Pandey a, Seema Pratap a*, Gaetano Marverti b, Manpreet Kaur c Jerry P.
Jasinski c
a Department of Chemistry (M.M.V), Banaras Hindu University, Varanasi−221005,
UP, India
b Department of Biomedical, Metabolic and Neural Sciences, University of
Modena and Reggio Emilia, 41125 Modena, Italy
c Department of Chemistry, Keene State College, 229 Main Street, Keene, NH
03435-2001, USA.
In the present study, a series of biologically active N, N'–disubstituted thiocarbamide compounds
(1−8) have been synthesized and characterized by some spectroscopic (FT-IR, 1H and 13C NMR,
UV-Visible) techniques. The molecular structure of compound 1 was determined by single
crystal X-ray diffraction analysis. All the synthesized compounds (1−8) and five more (9−13)
were screened for their in vitro cytotoxicity activities.

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Synthesis, spectroscopic, crystal structure and in vitro cytotoxicity studies of
N-thiophenoyl-N'-substituted phenyl thiocarbamide derivatives
a
a*
b
Sunil K. Pandey , Seema Pratap , Gaetano Marverti , Manpreet Kaur,c Jerry P.
Jasinski c
aDepartment of Chemistry (M.M.V), Banaras Hindu University, Varanasi−221005, UP, India
bDepartment of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio
Emilia, 41125 Modena, Italy
cDepartment of Chemistry, Keene State College, 229 Main Street, Keene, NH 03435-2001, USA.
Abstract
A series of eight biologically active N, N'-disubstituted thiocarbamide compounds (1–8) have
been prepared from thiophene-2-carbonyl isothiocyanate and various substituted aromatic
primary
amines
(2,4-dichlorophenyl
aniline,
4-chloro-3-nitrophenyl
aniline,
4-
methoxycarbonylphenyl aniline, 3-methoxycarbonylphenyl aniline, 2-methoxycarbonylphenyl
aniline, 4-methoxyphenyl aniline, 2-methoxyphenyl aniline and 2-nitrophenyl aniline). Their
1
structures were confirmed by elemental analyses, various spectroscopic techniques ((FT–IR, H
13
and C NMR) and single crystal X-ray analysis of compound (1). In the molecular structure of
compound (1) twisted confirmation of the carbonyl and thiocarbonyl group across C–N bond of
thiocarbamide moiety and an offset face-to-face π–π stacking between two thiophene and two
benzene ring of two molecules is observed. In vitro cytotoxicity assay of all the above
compounds and five more (9−13) were carried out using seven human cancer cell lines; cervical
(2008 and C13*), colorectal (HT29 and HCT116) and ovarian carcinoma (A2780, A2780/CP and
IGROV-1). The results revealed that compounds 1, 11, 12 and 13 displayed promising inhibitory
activity against all the cell lines tested.
* Email: drseemapratap@gmail.com
Keywords: Thiocarbamide; X-ray crystal structure; In vitro Cytotoxicity; Spectroscopic
techniques; π–π stacking.

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1. Introduction
N, N'-disubstituted thiocarbamides represent one of the most useful class of compounds with a
wide range of applications in medicinal, analytical and other fields [1−5]. The structural motif of
these compounds [-C(O)NHC(S)NH-] is composed of nitrogen, oxygen and sulfur heteroatoms
which facilitate their hydrogen bonding abilities with different molecules and multitude of
coordination behaviour with a variety of heavy trantion metal ions[6−9].The presence of
thiocarbonyl moiety influences the lipophilicity/hydrophilicity and the electronic properties of
the compounds [10, 11]. The medicinal applications of these compounds include their
antibacterial, -fungal, anti-tubercular, anti-thyroid, anti-helmintic, rodenticidal, insecticidal, and
herbicidal and plant growth regulator activities [12−17]. They also exhibit significant anticancer
properties against various leukemia and solid tumors, however, the reports are very limited [18,
19]. Their inhibitory activity against protein tyrosine kinases (PTKs) [20, 21], human sirtuin type
proteins, topoisomerase II and DNA repair synthesis has been reported [22].
These compounds have also been successfully used in liquid-liquid extraction of precious metals
(Pt, Pd, Au Ag.), as anion and cation sensors [23, 24], liquid crystal materials [25, 26], as
catalyst in chemical reactions [6, 27], as precursors or intermediates towards the synthesis of a
variety of heterocyclic compounds. Due to non-linear optical properties single crystal of
substituted thiocarbamides are being extensively employed in the electronic industry as
polarization filters, electronic light shutters, electronic modulators, and as components in
electrooptic and electro-acoustic devices, etc [28, 29].
We have reported anticancer properties of few N, N'-substituted thiocarbamide derivatives in our
previous publications [12, 13, 30, 31]. Since, optimizing compound structure is a usual strategy
to design and construct new antitumor agents, in the present study; we report the synthesis and
anticancer activity of a series of novel thiophene containing substituted thiocarbamides.

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2. Experimental Details
2.1. Chemicals, Instruments and Methods
Thiophene-2-carbonyl chloride, substituted aromatic primary amines (2, 4 – dichlorophenyl
aniline, 4-chloro-3-nitrophenyl aniline, 4-methoxycarbonyl) phenyl aniline, 3-methoxycarbonyl
phenyl aniline, 2-methoxycarbonyl phenyl aniline, 4-methoxyphenyl aniline, 2-methoxyphenyl
aniline and 2-nitrophenyl aniline) and ammonium thiocyanate were purchased from Merck
(Germany). Analytical grade acetone, acetonitrile, and dichloromethane were purchased from
Rankem. The acetone was dried and freshly distilled prior to use. Melting points was measured
on a X–4 digital melting-point apparatus and were uncorrected. Elemental analyses were
performed on a CE-440 Exeter Analytical CHN analyzer. The infrared spectra of the title
compounds as KBr pellets (4000−400 cm-1) were recorded on a Varian 3100 FT-IR Excalibur
13
series spectrophotometer.1H and C NMR spectra were recorded in CDCl3 by using TMS as an
internal standard on a JEOL FT-NMR AL 500 spectrometer. The splitting of proton resonances
1
in the reported H NMR spectra were remarked as s = singlet, d = doublet, t = triplet, dd =
doublet of doublets, and m = multiplet; coupling constants are reported in Hz. UV-Visible
spectra were recorded on a Shimadzu (-UV-) 1700 Pharma Spec spectrophotometer, USA. A
rectangular quartz cell with optical path length of 1cm was used.
2.2. Crystal structure determination
Data collection was performed using CrysAlisPRO on an Oxford Diffraction Xcaliber Ruby
Gemini CCD differactometer using graphite- monochromatic CuKa (k = 1.54178 Å) at 123 K.
The structure was solved by direct methods and refined by full-matrix least-square on F2 using
SHELXL-97 [32]. The non-hydrogen atoms were refined with anisotropic thermal parameters.
All hydrogen atoms were geometrically fixed and allowed to refine using the riding model. The
details of the single crystal X-ray data collection, structure solution and structure refinement
parameters are given in Table 1. Selected bond lenghths and bond angles are listed in Table 2.
2.3. General procedure for synthesis of compounds (1−8)
A solution of thiophene -2-carbonyl chloride (1.06 mmol, 10 mmol) in dry acetone (30 mL) was
mixed to a suspension of ammonium thiocyanate (0.78g, 10 mmol) in dry acetone (30 mL) and
was heated (60 °C) under reflux for 1 h. The resultant thiophene-2-carbonyl isothiocyanate
solution was treated with equimolar quantity of appropriate substituted aromatic primary amine
in acetone (Scheme 1) and further refluxed for 2 h at 27 °C [30, 31, 33−35]. The precipitated

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ammonium chloride was filtered off and the yellow colour solution was evaporated in vacuum to
dryness to obtain crude crystalline product. Yellow crystals of the compound 1 were grown by
the slow evaporation of its solution in acetone/dichloromethane (1:1) in one week at 20°C.
2.3.1. N-(2, 4 – dichlorophenyl) - N'- (thiophene-2-carbonyl)-thiocarbamide (1)
Light yellow solid; (2, 4-dichloroaniline, 1.630g, 10 mmol); Yield: 80%, m.p. 175−176°C.
FT−IR (KBr, cm-1): 3411, 3117, ν (N−H); 3029, ν (Ar, C−H); 1667, ν (C═O); 1273, ν (C═S);
1157, ν(NCN); 776,(C−S); 661, νas(C−Cl); 478, νs(C−Cl). 1H NMR (500 MHz, CDCl3, 25°C): δ
(ppm) 12.54 (s, 1H, −CSNH), 9.10 (s, 1H, −CONH), 8.37 (d, 1H, J = 8.5 Hz, Thiophene-H),
7.76 (dd, 1H, J1 = 8.5 Hz, J2 = 3.2 Hz, Thiophene-H), 7.74 (d, 1H, J = 5.1 Hz, Thiophene-H),
13
7.47-7.30 (m, 2H, Ar-H), 7.29-7.18 (m, 2H, Ar-H). C NMR (125 MHz CDCl3, 25°C): δ(ppm)
178.5 (C═S), 161.1 (C═O), 135.7, 134.7, 133.8, 132.5(Thiophene, Cs), 131.1, 129.5, 128.7,
128.6, 127.2, 126.9 (Ar, Cs). Anal. Calcd. for C12H8N2OS2Cl2 (331.00): C, 43.51; H, 2.43; N,
8.46, Found: C, 43.47; H, 2.39; N, 8.38 %.
2.2.2. N-(4-chloro-3-nitrophenyl) - N'- (thiophene-2-carbonyl)-thiocarbamide (2)
Yellow solid; (4-chloro-3-nitroaniline, 1.731g, 10 mmol); Yield: 91%, m.p. 100−101°C. FT−IR
(KBr, cm-1): 3263, 3105, ν(N−H); 3072, ν(Ar, C−H); 1659, ν(C═O); 1556, νas(−NO2);
1
1356,νs(−NO2); 1272, ν(C═S); 1163,ν(NCN); 766, ν(C−S); 724, νas(C−Cl); 486, νs(C−Cl). H
NMR (500 MHz, CDCl3, 25°C): δ (ppm) 12.71 (s, 1H, −CSNH), 9.03 (s, 1H, −CONH), 8.51 (d,
1H, J = 7.5 Hz, Thiophene-H), 7.85 (dd, 1H, J1 = 6.0 Hz, J2 = 3.5 Hz, Thiophene-H), 7.76 (d,
1H, J = 5.0 Hz, Thiophene-H), 7.70-7.54 (m, 2H, Ar-H), 7.24-7.15 (m, 2H, Ar-H). 13C NMR
(125 MHz CDCl3, 25°C): δ (ppm) 182.1 (C═S), 161.5 (C═O), 137.1, 135.1, 134.5,
132.1(Thiophene, Cs), 131.3, 128.8, 128.5, 128.1, 124.4, 120.6 (Ar, Cs). Anal. Calcd. for
C12H8N3O3S2Cl (341.50): C, 42.17; H, 2.36; N, 12.29, Found: C, 42.09; H, 2.33; N, 12.19 %.
2.2.3. N-(4-methoxycarbonyl) phenyl)-N'-(thiophene-2-carbonyl)-thiocarbamide (3)
Light yellow solid; (4-methoxycarbonylphenyl aniline, 1.520g, 10 mmol); Yield: 88%, m.p.
170−171°C. FT−IR (KBr, cm-1): 3296, 3147, ν(N−H); 3097, ν(Ar, C−H); 1721, ν(−COOR);
1698, ν(C═O); 1270, ν(C═S); 1143, ν(NCN); 769,ν(C−S). 1H NMR (500 MHz, CDCl3, 25°C): δ
(ppm) 12.65 (s, 1H, −CSNH), 8.94 (s, 1H, −CONH), 8.08 (d, 1H, J = 8.5 Hz, Thiophene-H), 7.87
(dd, 1H, J1 = 8.5 Hz, J2 = 3.2 Hz, Thiophene-H), 7.74 (d, 1H, J = 5.1 Hz, Thiophene-H),
7.24−7.21 (m, 2H, Ar-H), 7.20−7.19(m, 2H, Ar-H), 3.91(s, 3H, −COOCH3). 13C NMR (125
MHz CDCl3, 25°C): δ (ppm) 177.4 (C═S), 161.1 (C═O), 139.1, 137.1, 134.5, 132.1(Thiophene,

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Cs), 131.3, 130.7, 130.2, 128.3, 122.6, 121.4 (Ar, Cs), 58.1 (C, –COOCH3). Anal. Calcd. for
C14H12N2O3S2 (320.38): C, 52.48; H, 3.78; N, 8.74, Found: C, 52.27; H, 3.72; N, 8.68 %.
2.2.4. N-(3-methoxycarbonyl) phenyl)-N'-(thiophene-2-carbonyl)-thiocarbamide (4)
Light yellow solid; (3-methoxycarbonylphenyl aniline, 1.520g, 10 mmol); Yield: 92%, m.p.
95−96°C. FT−IR (KBr, cm-1): 3332, 3140, ν(N−H); 3094, ν(Ar, C−H); 1709,ν(−COOR); 1666,
ν(C═O); 1263, ν(C═S); 1137,ν(NCN); 736,ν(C−S). 1H NMR (500 MHz, CDCl3, 25°C): δ (ppm)
13.27 (s, 1H, −CSNH), 10.01 (s, 1H, −CONH), 8.37 (d, 1H, J = 8.0 Hz, Thiophene-H), 8.04 (dd,
1H, J1 = 8.0 Hz, J2 = 3.4 Hz, Thiophene-H), 7.99 (d, 1H, J = 4.1 Hz, Thiophene-H), 7.37−7.34
13
(m, 2H, Ar-H), 7.33−7.20(m, 2H, Ar-H), 3.94(s, 3H, −COOCH3). C NMR (125 MHz CDCl3,
25°C): δ (ppm) 177.9 (C═S), 166.4 (C═O), 138.4, 136.1, 134.2, 132.3(Thiophene, Cs), 131.4,
130.7, 130.8, 128.4, 127.0, 126.2 (Ar, Cs), 57.8 (C, –COOCH3). Anal. Calcd. for C14H12N2O3S2
(320.38): C, 52.48; H, 3.78; N, 8.74, Found: C, 52.39; H, 3.76; N, 8.66 %.
2.2.5. N-(2-methoxycarbonyl) phenyl)-N'-(thiophene-2-carbonyl)-thiocarbamide (5)
Light yellow solid; (2-methoxycarbonylphenyl aniline, 1.520g, 10 mmol); Yield: 85%, m.p.
97−98°C. FT−IR (KBr, cm-1): 3332, 3300, ν(N−H); 3088,ν(Ar, C−H); 1715,ν(−COOR); 1690,
ν(C═O);1262,ν(C═S); 1163,ν(NCN); 758,ν(C−S).1H NMR (500 MHz, CDCl3, 25°C): δ (ppm)
11.93 (s, 1H, −CSNH), 9.80 (s, 1H, −CONH), 8.71 (d, 1H, J = 9.0 Hz, Thiophene-H), 8.28 (dd,
1H, J1 = 8.5 Hz, J2 = 2.4 Hz, Thiophene-H), 7.99 (d, 1H, J = 4.1 Hz, Thiophene-H), 7.97−7.92
(m, 2H, Ar-H), 7.87−7.86(m, 2H, Ar-H), 3.87 (s, 3H, −COOCH3). 13C NMR (125 MHz CDCl3,
25°C): δ (ppm) 178.8 (C═S), 160.1 (C═O), 139.9, 138.2, 134.6, 132.1(Thiophene, Cs), 131.1,
130.7, 130.7, 128.4, 127.8, 126.7 (Ar, Cs), 57.6 (C, –COOCH3). Anal. Calcd. for C14H12N2O3S2
(320.38): C, 52.48; H, 3.78; N, 8.74, Found: C, 52.38; H, 3.75; N, 8.67 %.
2.2.6. N-(4-methoxyphenyl) - N'- (thiophene-2-carbonyl)-thiocarbamide (6)
Yellow solid; (4-methoxyphenyl aniline, 1.231g, 10 mmol); Yield: 78%, m.p. 120−121°C.
FT−IR (KBr, cm-1): 3458, 3207, ν (N−H); 3086, ν (Ar, C−H); 1658, ν(C═O); 1285,ν(C═S);
1180, ν(NCN); 776, ν(C−S). 1H NMR (500 MHz, CDCl3, 25°C): δ(ppm) 13.27 (s, 1H, −CSNH),
8.97 (s, 1H, −CONH), 8.48 (d, 1H, J = 7.5 Hz, Thiophene-H), 8.05 (dd, 1H, J1 = 6.5 Hz, J2 = 2.1
Hz, Thiophene-H), 7.99 (d, 1H, J = 4.0 Hz, Thiophene-H), 7.59−7.57 (m, 2H, Ar-H), 7.33−7.31
(m, 2H, Ar-H), 3.95 (s, 3H, −OCH3). 13C NMR (125 MHz CDCl3, 25°C): δ (ppm) 178.4 (C═S),
160.0 (C═O), 138.6, 136.2, 134.1, 132.5(Thiophene, Cs), 131.1, 130.9, 128.4, 126.5, 126.2,

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122.6 (Ar, Cs), 52.6 (C, –OCH3). Anal. Calcd. for C13H12N2O2S2 (292.37): C, 53.40; H, 4.14; N,
9.58, Found: C, 53.24; H, 4.09; N, 9.47 %.
2.2.7. N-(2-methoxyphenyl) - N'- (thiophene-2-carbonyl)-thiocarbamide (7)
Yellow solid; (2-methoxyphenyl aniline, 1.231g, 10 mmol); Yield: 75%, m.p. 115−116°C.
FT−IR (KBr, cm-1): 3468, 3262, ν(N−H); 3105,ν(Ar, C−H); 1673, ν(C═O); 1270, ν(C═S);
1
1119,ν(NCN); 762,ν(C−S). H NMR (500 MHz, CDCl3, 25°C): δ (ppm) 12.40 (s, 1H, −CSNH),
9.03 (s, 1H, −CONH), 7.74 (d, 1H, J = 10.0 Hz, Thiophene-H), 7.65 (dd, 1H, J1 = 9.5 Hz, J2 =
4.1 Hz, Thiophene-H), 7.99 (d, 1H, J = 4.0 Hz, Thiophene-H), 7.59−7.57 (m, 2H, Ar-H),
7.33−7.31 (m, 2H, Ar-H), 3.95 (s, 3H, −OCH3). 13C NMR (125 MHz CDCl3, 25°C): δ (ppm)
178.2 (C═S), 161.3 (C═O), 138.3, 136.1, 135.8, 134.6(Thiophene, Cs), 132.3, 131.0, 130.1,
129.1, 128.6, 125.4 (Ar, Cs), 58.5 (C, –OCH3). Anal. Calcd. for Calc. for C13H12N2O2S2
(292.73): C, 53.40; H, 4.14; N, 9.58, Found: C, 53.27; H, 4.11; N, 9.51 %
2.2.8. N-(2-nitrophenyl) - N'- (thiophene-2-carbonyl)-thiocarbamide (8)
Dark yellow solid; (2-nitroaniline aniline, 1.381g, 10 mmol); Yield: 91%, m.p. 155−156°C.
FT−IR (KBr, cm-1): 3318, 3101, ν(N−H); 3005,ν(Ar, C−H); 1661,ν(C═O); 1564, νas(−NO2),
1
1319, νs(−NO2); 1264, ν(C═S); 1154,ν(NCN); 743,ν(C−S). H NMR (500 MHz, CDCl3, 25°C):
δ (ppm) 12.88 (s, 1H, −CSNH), 9.01 (s, 1H, −CONH), 8.28 (d, 1H, J = 8.5 Hz, Thiophene-H),
7.78 (dd, 1H, J1 = 6.0 Hz, J2 = 3.1 Hz, Thiophene-H), 7.75 (d, 1H, J = 5.5 Hz, Thiophene-H),
7.24−7.21 (m, 2H, Ar-H), 7.20−7.19(m, 2H, Ar-H). 13C NMR (125 MHz CDCl3, 25°C): δ (ppm)
178.1 (C═S), 161.5 (C═O), 145.2, 143.3, 135.5, 135.0(Thiophene, Cs), 131.1, 128.8, 124.6,
123.2, 121.1 (Ar, Cs).Anal. Calcd. For C12H9N3O3S2 (307.40): C, 46.89; H, 2.95; N, 13.67,
Found: C, 46.78; H, 2.92; N, 13.58 %
3. Biological assays
3.1. Cell lines
Seven human cancer cell lines namely, cervical (2008 and C13*), colorectal (HT29 and
HCT116) and ovarian carcinoma (IGROV-1, A2780 and A2780/CP) were used. Among these,
C13* and A2780/CP are cisplatin (ccDDP)-resistant cells [36, 37]. Cells were grown as
monolayers in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum and 50
µg/ml gentamycin sulfate. All cell media and serum were purchased from Lonza (Verviers,
Belgium). Cultures were equilibrated with humidified 5% CO2 in air at 38 ºC. All studies were

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performed in Mycoplasma negative cells, as routinely determined with the MycoAlert
Mycoplasma detection kit (Lonza, Walkersville, MD, USA).
3.2 Cytotoxicity screening
In vitro cytotoxicity of compounds used in the present study was determined by MTT assay [38,
39]. The cells were seeded into 96-well plates and cultured overnight .Various concentrations of
the test compounds dissolved in DMSO solvents were then added and incubated for 72 h. After
incubation, the medium was removed and added fresh culture medium 100 µl containing 0.5 mg
mL-1 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide; Sigma) and then
incubated at 38ºC for 4 h. The medium was removed and 100 µL DMSO solvent was added to
dissolve the dark blue crystals. After incubation for 30 min at room temperature, to ensure that
all crystals were dissolved, absorbance was measured using an ELISA plate reader at 570 nm
with reference wavelength of 650 nm.

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4. Results and Discussion
4.1 Synthesis and Characterization
Two step synthesis of compounds 1−8 were accomplished by the reaction of thiophene-2-
carbonyl chloride with ammonium thiocyanate and refluxing the resultant thiophene-2-carbonyl
isothiocyanate with appropriate substituted aromatic primary amines (Scheme 1). All the
compounds were obtained in good yield. The slow evaporation at room temperature of acetone
solution of compound (1) afforded single crystals suitable for X-ray diffraction.
Scheme 1 Synthetic route to new thiophene-2-carbonyl-thiocarbamides (1 – 8)
4.2. FT-IR study
In the IR spectra of compounds a sharp and a broad band observed at ~3400 cm-1 and ~3140
cm-1 could be assigned to N1−H and N2ꢀꢀꢀH stretching modes, respectively [40, 41]. The
appearance of N2−H stretching vibration at a lower wave number is due to the presence of
intramolecular hydrogen bonding N2−HꢀꢀꢀO═C whereas the first band may be attributed to free
ν(N1−H) [13]. Characteristic C−H stretching vibrations of the aromatic groups appear as
medium intensity band at ~3050 cm-1. A strong band at ~1660 cm-1 corresponds to ν(C=O) mode

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which resembles in frequency to the hydrogen bonded C=O group (N−HꢀꢀꢀO═C) observed in
many similar compounds. A strong and a medium intensity band at ~1265 cm-1 and ~760 cm-1
could be assigned to C═S and C−S stretching modes, respectively [12, 13].
4.3. NMR study
1H NMR spectra of the compounds (1−8) in CDCl3 exhibited two singlets at 9.00–10.00 and
12.00–13.31 ppm which could be assigned to free N1−H and hydrogen bonded N2−H,
respectively [12, 13]. Aromatic protons appeared as multiplets between 7.20–8.50 ppm. The
chemical shift value of free N−H suffers a downfield shift of ~3.0 ppm in DMSO-d6. Such shifts
could be ascribed to the probable hydrogen bonding between the NH and sulfoxide (S═O)
moiety [14].
13
The C NMR spectra of compounds (1–8) depicted all the signals due to magnetically distinct
carbons present in them. The peaks related to the carbons in CONH and CSNH groups of the
thiocarbamide resonate around 160–170 and 172–182 ppm, respectively [12-14]. The downfield
resonance of C═S in comparison to C=O carbon may be related to steric and electronic factors
[42].
4.4 Electronic study
Electronic spectra of compounds 1–8 recorded in dichloromethane over the scan range of 200–
800 nm show identical trends (Figure S1). A representative UV-Vis spectrum of compound 2 is
shown in Figure 1. A weak band centered at ~261 nm and a strong band at ~296 nm in the
absorption spectra of compounds can be ascribed to π → π* transitions of thiocarbamide moiety
[43].

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Fig. 1 UV−Vis spectrum of compound 2 in dichloromethane
4.5 Description of crystal structure
The ORTEP view, unit cell diagram and packing pattern of compound (1) are depicted in Figures
2(a), 2(b) and 2(c), respectively. The crystallographic and refinement details are shown in Table
1. Selected bond lengths, bond angles and hydrogen bond interaction are listed in Table 2 and
Table S1. In molecular structure thiocarbonyl (C═S) and carbonyl (C═O) groups are opposite to
each other and are almost coplanar with torsion angles S(1)−C(6)−N(1)−C(5) = 177.6(3)º and
N(2)–C(6)−N(1)−C(5) = -3.3(5)°, respectively. The phenyl ring, thiophene and thiocarbamide
moiety lie in a plane, which is evidenced by the torsion angle values N(1)−C(6)−N(2)−C(7)
=173.8(3)º, C(6)–N(1)−C(5)−C(1)= -169.6(3)º, respectively [14,15]. The molecular structure
shows an intramolecular N2–HꢀꢀꢀO=C hydrogen bond stabilizing the (–C(O)NHC(S)NH–)
geometry, forming a pseudo six membered ring (Table S1). The C−N, C═S and other bond
distances (Table 2) exhibit conventional bond parameters similar to other substituted
thiocarbamides [44, 45]. Additionally, an offset face to face π−π stacking interaction is also
observed in compound (1) between thiophene rings and substituted benzene rings of two
molecules [13, 46]. Very weak intermolecular interactions C−HꢀꢀꢀCl, C−SꢀꢀꢀH and Cπ−Cπ
stabilize the crystal packing and lead to an infinite chain length along the b-axis Figure 2(c)

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(a)
(b)

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(c)
Fig. 2 (a) ORTEP diagram of 1 showing the thermal ellipsoids at 50% probability. The
intramolecular hydrogen bonds as shown by dashed lines. (b) Unit cell diagram viewed down the
b axis for compound 1. Intermolecular face-to-face π−π stacking interaction is also observed in
the compound 1. (c) The packing diagram of compound 1. Weak intermolecular interactions
(C−HꢀꢀꢀCl, C−SꢀꢀꢀH and Cπ−Cπ) lead to an infinite chain length along the b-axis.

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Table 1. Crystal data and structure refinement for compound 1
Compound
CCDC No
1
1561945
Empirical formula
Formula weight
Temperature/K
Crystal system
Space group
C12H8Cl2N2OS2
331.22
173(2)
orthorhombic
P212121
a (Å)
3.9537(2)
b (Å)
10.9953(6)
c (Å)
30.0239(16)
α,β, γ (º)
90
Volume/Å3
1305.20(12)
Z
4
ρcalcg/cm3
1.686
m/mm-1
7.402
F(000)
672.0
Crystal size/mm3
Radiation
0.48 × 0.14 × 0.08
CuKα (λ = 1.54184)
8.564 to 142.506
-4 ≤ h ≤ 4, -12 ≤ k ≤ 13, -26 ≤ l ≤ 36
8346
2Θ range for data collection/°
Index ranges
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
Final R indexes [I>=2σ (I)]
Final R indexes [all data]
Largest diff. peak/hole / eÅ-3
Flack parameter
2501 [Rint = 0.0366, Rsigma = 0.0316]
2501/0/172
1.029
R1 = 0.0340, wR2 = 0.0867
R1 = 0.0351, wR2 = 0.0877
0.33/-0.36
-0.008(13)
aR = P||Fo| − |Fc||/P |Fo|
bRw = [Pw(Fo2− Fc2)2/Pw(Fo4)]1/2.

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Table 2. Selected bond lengths (Å) and bond angles (º) for compound 1
Bond Lengths (Å)
N1−C5
N1−C6
N2−C6
N2−C7
O1−C5
C6−S1
1.372(4)
1.394(4)
1.346(4)
1.419(4)
1.229(4)
1.660(3)
S2−C1
1.730(3)
1.706(3)
1.388(5)
1.388(4)
1.738(3)
1.733(3)
S2−C4
C10−C11
C12−C12
C8−Cl1
C10−Cl2
Bond Angles (º)
C4−S2−C1
91.58(16)
122.2(3)
118.3(2)
115.1(3)
118.9(3)
O1−C5−N1
N1−C5−C1
N2−C6−S1
N2−C7−C8
C5−C1−C2
123.4(3)
114.4(3)
126.6(2)
120.3(2)
130.6(3)
O1−C5−C1
N1−C6−S1
N2−C6−N1
N2−C7−C12

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4.6 In vitro cytotoxicity screening
To evaluate the anti-cancer activity of synthesized substituted thiocarbamides (1−8) and five
other (9−13), all the thirteen compounds (1−13) were tested for their in vitro cytotoxicity
activities against a panel of seven human cancer cell lines, cervical (2008 and C13*), colorectal
(HT29 and HCT116) and ovarian carcinoma (A2780, A2780/CP and IGROV-1) using 5-
Fluorouracil (5-FU, a clinically used drug) as a positive control [47−49]. The result in the form
of their IC50 values has been presented in Table 3. The comparative cytotoxicity of the
compounds is depicted in Figure 3. The structural analysis of compounds 9−13
(N−4−Sulfonamide phenyl-N'–2–thiophenoyl thiourea, N–3–Nitrophenyl–N'–2–thiophenoyl
thiourea, N–2–Chloro–4–nitrophenyl–N'–2–thiophenoyl thiourea, N–2–Methoxy–4–nitrophenyl-
N'–2–thiophenoyl thiourea and N–4–Chloro–2–nitrophenyl–N'–2–thiophenoyl thiourea) along
with their other biological properties have already been published by us [50]. The tested
compounds displayed potent inhibitory activity against all the cell lines having IC50 values in
different ranges (Table 3). The strongest inhibitory activity was displayed by compounds 1, 11,
12 and 13 against all the cell lines. All the compounds except 5, 9 and 10 displayed stronger
inhibitory activity than the standard drug (5-FU) against HT29 cell line. In comparison to the
standard drug (5-FU) better inhibitory properties were obtained for compounds 1, 3, 4, 8, 11, 12
and 13 against HCT116 and for compounds 5 and 8 against A2780/CP cancer cell lines,
respectively. The present study demonstrates that nature and number of functional groups
attached to the phenyl ring of the thiocarbamide moiety exert a decisive influence on the
cytotoxicity of this class of compounds. The most promising compounds 1, 11, 12 and 13
contained a nitro and/or chloro electron withdrawing group at ortho and para positions [51, 52]
In general, all the above compounds exhibited better inhibitory properties than those reported in
our previous papers [12, 13, 30, 31]. Previous studies have shown that the presence of
electronegative atom/group at ortho and or/ para position of the aromatic ring increases the
lipophilicity of molecules and is responsible for enhanced cytotoxicity in MTT model [53].
Based on the above results, potential anticancer candidates can be considered for structural
modification and pharmacological evaluation.

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Table 3. IC50 Values (µM) for the compounds (1−13) against seven human cancer cell lines;
(2008 and C13*) cervical, (HT29 and HCT116) colorectal and (A2780, A2780/CP and
IGROV-1) ovarian carcinoma.
Compounds
Cell lines
HT29 HCT116 A2780
2008
C13*
A2780/CP IGROV-
1
7.4±0.3
11.9±0.4 13.4±0.1 11.6±2
21.4±5
19.6±3
19.6±5
23.4±6
15.9±1
1.
11.2±0.4 12.1±0.3 12.9±0.3 13.5±4
17.5±0.4
20.4±0.9
19.8±10
12.9±1
2.
10.5±0.6 11.8±0.2 11.9±0.6 12.5±0.4 26.2±0.7 24.3±1
10.1±0.1 11.2±0.3 11.9±0.3 11.5±0.2 24.6±0.3 27.4±0.2
3.
4.
9.8±0.4
9.75±0.3 22.9±0.5 25.8±0.2 11.4±0.8 10.4±0.5
5.
22.5±0.5 19.6±0.1 15.4±0.3 19.6±0.4 21.4±0.4 22.5±2
21.8±0.2 18.9±0.2 14.8±0.6 18.4±0.2 20.6±0.3 21.3±5
23.3±0.6
21.8±0.7
21.3±0.7
29.6±0.3
31.1±0.7
13.2±0.4
14.8±1
6.
7.
10.2±0.1 11.1±0.3 10.9±0.2 11.5±0.1 29.3±4
10.6±0.5
8.
22.3±0.5 28.6±0.8 21.3±0.6 33.1±0.1 27.4±0.8 35.5±0.9
9.
20.7±0.5 22.6±0.6 24.7±0.7 22.8±0.3 27.6±2
31.9±0.6
14.9±3
10.
11.
12.
13.
5-FU
5.4±0.1
6.2±0.2
5.1±0.2
4.1±0.3
11.2±0.2 12.4±0.2 11.9±0.7 15.2±1
12.3±0.6 13.2±0.2 12.9±0.3 18.5±3
10.6±0.5 11.6±0.2 11.2±0.7 15.1±1
8.5±0.5 15.2±0.1 13.5±0.1 5.5±0.3
16.4±3
14.4±2
11.8±1
12.8±0.5
5.1±0.2

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(A)

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(B)

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(C)
Fig. 3 Comparative in vitro cytotoxicity of compounds 1−13 for a panel of seven human cancer
cell lines; (A). Cervical (2008 and C13*) (B). Colorectal (HT29 and HCT116) (C). Ovarian
carcinoma (A2780, A2780/CP and IGROV-1).

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5. Conclusions
A facile synthesis of a series of N, N'-disubstituted thiocarbamides were performed using the
prescribed method in this paper. All these compounds were structurally characterized by using
various spectroscopic and single crystal X-ray diffraction techniques. The crystal structure of
compound 1 revealed the planar confirmation of thiocarbamide unit which may be due to the
formation of intramolecular hydrogen bond N2−HꢀꢀꢀO═C. An offset face-to-face π –π stacking is
also present between aromatic rings of compound 1.The cytotoxic effects of these compounds
were evaluated against seven human cancer cell lines using the standard MTT assay. The IC50
values indicated all of them to be potent anticancer agents against all the cell lines. Most of the
tested compounds conferred better inhibitory activity than the standard drug (5-FU) against
HT29 and HCT116 cell lines and are worthy of further structural modification and
pharmacological evaluation.
Acknowledgements
The author, SKP is grateful to Banaras Hindu University, Varanasi, India for the financial
assistance. I am thankful to Prof. G. Marverti, Department of Biomedical Sciences, Metabolic
and neurosciences, University of Modena, 41125 Modena, Italy for cytotoxicity studies. I am
also very thankful to Prof. Jerry P. Jasinski, Department of Chemistry, Keene State College, and
229 Main Street, Keene, NH 03435-2001, USA for single crystal X-ray diffraction.
Supporting Information Summary
1
UV−Vis spectra of compounds (1−8) in dichloromethane Figure S1. Scan copies of HNMR
Fig.(S2−S9) and 13CNMR Fig.(S10−S17). X-ray crystallographic hydrogen bonding Table S1 of
compound (1) have been provided in supporting information. Crystallographic data (excluding
structure factors) for the structures in this paper have been deposited with the Cambridge
Crystallographic Data Centre, CCDC, 12 Union Road, Cambridge
CB21EZ, UK. Copies of the data can be obtained free of charge on quoting the depository
numbers CCDC 1561945 for compound (1) (Fax: +44-1223-336-033; Email:
deposit@ccdc.cam.ac.uk, http://www.cam.ac.uk)

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References
[1] A. Saeed, U. Flörke, M. F. Erben, A review on the chemistry, coordination, structure and
biological properties of 1-(acyl/aroyl)-3-(substituted) thioureas, J. Sulfur Chem. 35 (2014)
318−355.
[2] A. A. Aly, E. K. Ahmed, K. M. El-Mokadem, M. El-Amir, F. Hegazy, Update survey on
aroyl substituted thioureas and their applications, J. Sulfur Chem. 28 (2007) 73−93.
[3] K. R. Koch, New chemistry with old ligands: N-alkyl- and N, N-dialkyl-N_-acyl (aroyl)
thioureas in co-ordination, analytical and process chemistry of the platinum group metals, Coord.
Chem. Rev. 216–217 (2001) 473−488.
[4] S. Saeed, N. Rashid, P. G. Jones, M. Ali, R. Hussain, Synthesis, characterization and
biological evaluation of some thiourea derivatives bearing benzothiazole moiety as potential
antimicrobial and anticancer agents, Eur. J. Med. Chem. 45 (2010) 1323−1331.
[5] O. E. Hernández, J. Duque, E. Reguera, Structural features of 1-furoylthioureas 3-
monosubstituted and 3, 3-disubstituted: coordination to cadmium and analytical applications, J.
Sulfur Chem. 32 (2011) 213−222.
[6] N. Gunasekaran, P. Ramesh, M. N. G. Ponnuswamy, R. Karvembu, Monodentate
coordination of N-[di(phenyl/ethyl)carbamothioyl]benzamide ligands: synthesis, crystal structure
and catalytic oxidation property of Cu(I) complexes, Dalton Trans. 40 (2011) 12519−12526.
[7] M. M. Sheeba, M. M. Tamizh, L. J. Farrugia, A. Endo, R. Karvembu, Chiral
(η6 p Cymene)ruthenium(II) Complexes Containing Monodentate Acylthiourea Ligands for
Efficient Asymmetric Transfer Hydrogenation of Ketones, Organometallics.33 (2014) 540−550.
[8] S. Saeed, R. Hussain, Preparation and characterization of copper sulfide nanoparticles from
symmetrical [(Bu)2NC(S)NC(O)C6H3(3,5-NO2)2]2Cu(II) and [(Bu)2NC(S)NC(O)C6H4(4-
NO2)]2Cu(II) complexes by thermolysis, J. Coord.Chem. 67 (2014) 2942−2953.
[9] S. S. Hassan, M. M. Shoukry, R. N. Shallan, R. van Eldik, Synthesis, characterization,
speciation and biological studies on metal chelates of 1- benzoyl(1,2,4-triazol-3-yl)thiourea, J.
Coord. Chem.70 (2017) 1761−1775.
[10] D. Mahendiran, N. Pravin, N. S. P. Bhuvanesh, R.S. Kumar, V. Viswanathan, D.
Velmurugan, A. K. Rahiman, Bis(thiosemicarbazone)copper(I) Complexes as Prospective
Therapeutic Agents: Interaction with DNA/BSA Molecules, and In Vitro and In Vivo Anti-
Proliferative Activities, ChemistrySelect 3 (2018) 7100−7111.

ACCEPTED MANUSCRIPT
[11] M. J. McKeage, L. Maharaj, S. J. B. Price, Mechanisms of cytotoxicity and antitumor
activity of gold (I) phosphine complexes: the possible role of mitochondria, Coord. Chem. Rev.
232 (2002) 127−135.
[12] S. K. Pandey, D. P. Singh, G. Marverti, R. J. Butcher, S. Pratap, Monodentate Coordination
of N, N’-Disubstituted Thiocarbamide Ligands: Syntheses, Structural Analyses, In Vitro
Cytotoxicity and DNA Damage Studies of Cu(I) Complexes, ChemistrySelect 3 (2018)
3675−3679.
[13] S. K. Pandey, S. Pratap, G. Gozzi, G. Marverti, R. J. Butcher, Synthesis, molecular structure
exploration and in vitro cytotoxicity screening of five novel N, N′-disubstituted thiocarbamide
derivatives, Phosphorus, Sulfur, and Silicon Relat. Elem.193 (2018) 507−514.
[14] M. K. Rauf, S. Zaib, A. Talib, M. Ebihara, A. Badshah, M. Bolte, J. Iqbal, Solution-phase
microwave assisted parallel synthesis, biological evaluation and in silico docking studies of
N,N'-disubstituted thioureas derived from 3-chlorobenzoic acid, Bioorg. Med. Chem. 24 (2016)
4452−4463.
[15] J. Haribabu, G. R. Subhashree, S. Saranya, K. Gomathi, R. Karvembu, D. Gayathri,
Synthesis, crystal structure, and in vitro and in silico molecular docking of novel acyl thiourea
derivatives, J. Mol. Struct. 1094 (2015) 281−291.
[16] M. Mahdavi, M. S. Shirazi, R. Taherkhani, M. Saeedi, E. Alipour, F. H. Moghadam, A.
Moradi, H. Nadri, S. Emami, L. Firoozpour, A. Shafiee, A. Foroumadi, Synthesis, biological
evaluation and docking study of 3-aroyl-1-(4-sulfamoylphenyl)thiourea derivatives as 15-
lipoxygenase inhibitors, Eur. J. Med. Chem. 82 (2014) 308−313.
[17] L. Qiao, J. Huang, W. Hu, Y. Zhang, J. Guo, W. Cao, K. Miao, B. Qin, J. Song, Synthesis,
characterization, and in vitro evaluation and in silico molecular docking of thiourea derivatives
incorporating 4-(trifluoromethyl)phenyl moiety, J. Mol. Struct. 1139 (2017) 149−159.
[18] S. G. Kucukguzel, E. E. Oruc¸ S. Rollas, F. Sahin, A. Ozbek, Synthesis, characterisation and
biological activity of novel 4-thiazolidinones, 1,3,4-oxadiazoles and some related compounds,
Eur. J. Med. Chem. 37 (2002) 197−206.
[19] V. S. Palekar, A. J. Damle, S.R. Shukla, Synthesis and antibacterial activity of some novel
bis-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles and bis-4-thiazolidinone derivatives from terephthalic
dihydrazide, Eur. J. Med. Chem. 44 (2009) 5112−5116.

ACCEPTED MANUSCRIPT
[20] F. A. Larik, A. Saeed, P. A. Channar, U. Muqadar, Q. Abbas, M. Hassan, S.Y. Seo, M.
Bolte, Design, synthesis, kinetic mechanism and molecular docking studies of novel 1-
pentanoyl-3-arylthioureas as inhibitors of mushroom tyrosinase and free radical scavengers, Eur.
J. Med. Chem. 141 (2017) 273−281
[21] M. L. Barreca, A. Chimirri, L. D. Luca, A. M. Monforte, P. Monforte, A. Rao, M. Zappala,
J Balzarini, E. D. Clercq, C. Pannecouquec, M. Witvr, Discovery of 2,3-Diaryl-1,3-thiazolidin-4-
ones as Potent Anti-HIV-1 Agents, Bioorg. Med. Chem. Lett. 11 (2001) 1793−1796.
[22] M. Mushtaque, F. Avecilla, M. S. Khan, Z. B. Hafeez, M. M. A. Rezvi, A. Srivastava,
Synthesis, characterization, cytotoxicity, cell cycle analysis of 3-(4-methoxyphenyl)-1-(pyridin-
2-ylmethyl)thiourea and quantum chemical analyses, J. Mol. Struct. 1141 (2017) 119−132.
[23] M. M. Habtu, S. A. Bourne, K. R. Koch, R. C. Luckay, Competitive bulk liquid membrane
transport and solvent extraction of some transition and post-transition metal ions using
acylthiourea ligands as ionophores, New J. Chem. 30 (2006) 1155−1162.
[24] F. Oton, A. Espinosa, A. Tarraga, I. Ratera, K. Wurst, J. Veciana, P. Molina, Mononuclear
Ferrocenophane Structural Motifs with Two Thiourea Arms Acting as a Dual Binding Site for
Anions and Cations, Inorg. Chem. 48 (2009) 1566−1576.
[25] V. Krishnakumar, C. Ramachandraraja, R.S. Sundararajan, Crystal growth and vibrational
spectroscopic studies of the semiorganic non-linear optical crystal-bisthiourea magnesium
sulphate, Spectrochim. Acta Part A. 68 (2007) 113−116.
[26] G. Sharma, D. Singh, R. L. Gardas, Effect of Fluorinated Anion on the Physicochemical,
Rheological and Solvatochromic Properties of Protic and Aprotic Ionic Liquids: Experimental
and Computational Study, ChemistrySelect 2 (2017) 11653−11658.
[27] S. Khanam, S. K. Pandey, S. K. Rai, D. Verma, J. P. Jasinski, S. Pratap, A. K. Tewari,
Synthesis of N, N-Bis-Sulfonylated and N-Alkyl-N-Sulfonylated G1 Dendrimers via Click
Reaction: Application of Thiocarbamide based Cu(I) Catalysts, ChemistrySelect 2 (2017)
6370−6374.
[28] C. Puzzarini, Molecular Structure of Thiourea, J. Phys. Chem. A 116 (2012) 4381−4387.
[29] F. Karipcin, M. Atis, B. Sariboga, H. Celik, M. Tas, Structural, spectral, optical and
antimicrobial properties of synthesized 1-benzoyl-3-furan-2-ylmethyl-thiourea, J. Mol. Struct.
1048 (2013) 69−77.

ACCEPTED MANUSCRIPT
[30] D. P. Singh, S. Pratap, S. K. Pandey, R. J. Butcher, G. Marverti, N-(naphthyl)-N
ꢀ-
(methoxy carbonyl) thiocarbamide and its Cu (I) complex: synthesis, spectroscopic, X-ray, DFT
and in vitro cytotoxicity study, J. Coord. Chem. 68 (2015) 261−276.
[31] D. P. Singh, S. Pratap, M. Shukla, Solvent induced geometry transformation of trigonal
planar Cu(I) complexes of N-((2/4-methyoxy carbonyl) phenyl)-N'-(ethoxy/methoxy carbonyl)
thiocarbamides to square-planar Cu(II) complexes: Synthesis, spectral, single crystal, DFT and in
vitro cytotoxic study, Inorg. Chim.Acta. 423 (2014) 386−396.
[32] L.J. Farrugia, WinGX and ORTEP for windows: an update, J. Appl.Cryst. 45 (2012) 849-
854.
[33] Z. Weiqun, K. Leng, Y. Zhang, Lu Lude, Structural and spectral studies of N-(4-chloro)
benzoyl-N'-2- Tolylthiourea, J. Mol. Struct. 657 (2003) 215−223.
[34] X. Shen, X. Shi, B. Kang, Y. Liu, Y. Tong, L. Gu, Q. Liu, Y. Huang, Preparation and
crystal structure of a new Cu(II) complex derived from the desulfurization of N-(p-nitrophenyl)
N'- ethoxycarbonyl thiourea, Polyhedron 18 (1999) 33−37.
[35] S. K. Pandey, S. Pratap, M. K. Tiwari, G. Marverti, J. P. Jasinski, Experimental and
theoretical exploration of molecular structure and anticancer properties of two N, N'-
disubstituted thiocarbamide derivatives, J. Mol. Struct. 1175 (2019) 963−970.
[36] C. Korch, M. A. Spillman, T.A. Jackson, B. M. Jacobsen, S. K. Murphy, B. A. Lessey, V.
C.Jordan, A. P. Bradford, DNA profiling analysis of endometrial and ovarian cell lines reveals
misidentification, redundancy and contamination, Gynecol Oncol. 127 (2012) 241−248.
[37] A. P. Andrews, A. J. Jones, Characterization of Binding Proteins from Ovarian Carcinoma
and Kidney Tubule Cells that are Specific for Cisplatin Modified DNA, Cancer Commun. 3
(1991) 93−102.
[38] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: Application to
proliferation and cytotoxicity assays, J. Immunol. Methods. 65 (1983) 55−63.
[39] A. A. Stepanenko, V.V. Dmitrenko, Pitfalls of the MTT assay: Direct and off-target effects
of inhibitors can result in over/underestimation of cell viability, Gene 574 (2015) 193−203.
[40] M. Atis, F. Karipcin, B. Sarıboga, M. Tas, H, Celik, Structural, antimicrobial and
computational
characterization
of
1-benzoyl-3-(5-chloro-2-hydroxyphenyl)
thiourea,
Spectrochim. Acta Part A. 98 (2012) 290−301.

ACCEPTED MANUSCRIPT
[41] O. E. Hernandez, E. O. Sanchez, J.L. H. H. de Cisneros, I. N. Rodrıguez, E. Reguera, A
Raman and infrared study of 1-furoyl-3-monosubstituted and 3,3-disubstituted thioureas,
Spectrochim. Acta Part A. 62 (2005) 964−971.
[42] R. M. Silverstein, X. F. Webster, J. D. Kiemle, Spectrometric Identification of Organic
Compounds, Seventh Edition; Wiley: New York. ISBN0-471−39362-2.
[43] A. Saeed, S. Ashraf, J. M. White, D. B. Soria, C. A. Franca, M. F. Erben,′Synthesis, X-ray
crystal structure, thermal behavior and spectroscopic analysis of 1-(1-naphthoyl)-3-(halo-
phenyl)-thioureas complemented with quantum chemical calculations, Spectrochim. Acta Part A.
150 (2015) 409–418.
′
[44] A. Saeed, A. Khurshid, J. P. Jasinski, C. G. Pozzi, A. C. Fantoni, M. F. Erben,′Competing
intramolecular N-H…O=C hydrogen bonds and extended intermolecular network in 1-(4-
chlorobenzoyl)-3-(2-methyl-4-oxopentan-2-yl) thiourea analyzed by experimental and theoretical
Methods, J. Chem. Phys. 431–432 (2014) 39−46.
[45] M. S. M. Yusof, R. H. Jusoh, W. M. Khairul, B. M. Yamin, Synthesis and characterisation a
series of N-(3,4-dichlorophenyl)-N'-(2,3 and 4-methylbenzoyl)thiourea derivatives, J. Mol.
Struct. 975 (2010) 280−284.
[46] A. Saeed, Z. Ashraf, M. F. Erben, J. Simpson, Vibrational spectra and molecular structure of
isomeric 1-(adamantan-1-ylcarbonyl)-3-(dichlorophenyl)thioureas, J. Mol. Struct. 1129 (2017)
283−291.
[47] A. A. Ramos, C. P. Wilsonb, A. R. Collins, Protective effects of Ursolic acid and Luteolin
against oxidative DNA damage include enhancement of DNA repair in Caco-2 cells, Mutat. Res.
692 (2010) 6−11.
[48] K. K. Gnanasekaran, D. M. Benbrook, B. Nammalwar, E. Thavathiru, R. A. Bunce, K. D.
Berlin, Synthesis and evaluation of second generation Flex-Het scaffolds against the human
ovarian cancer A2780 cell line, Eur. J. Med. Chem. 96 (2015) 209−217.
[49] K. M. Khan, F. Naz, M. Taha, A. Khan, S. Perveen, M. I. Choudhary, W. Voelter, Synthesis
and in vitro urease inhibitory activity of N,N'-disubstituted Thioureas, Eur. J. Med. Chem. 74
(2014) 314−323.
[50] D. P. Singh, M. Gangwar, D. Kumar, G. Nath, S. Pratap, Synthesis, Spectroscopic
Characterization, Crystal structure, Antimicrobial and In Vitro Hemolytic Studies of Some Novel
Substituted Thiourea Derivatives, J. Chem. Crystallogr. 43 (2013) 610−621.

ACCEPTED MANUSCRIPT
[51] H. N. Pati, U. Das, J. W. Quail, M. Kawase, H. Sakagami, J. R. Dimmock, Cytotoxic 3,5-
bis(benzylidene) piperidin-4-ones and N-acyl analogs displaying selective toxicity for malignant
cells, Eur. J. Med. Chem. 43 (2008) 1−7.
[52]
structural characterization and in vitro cytotoxicity and anti-bacterial activity of some copper(I)
complexes with N,N'-disubstituted thioureas, J. Inorg. Biochem. 103 (2009) 1135–1144.
′M. K. Rauf, I. Din, A. Badshah, M. Gielen, M. Ebihara, D. deVos, S. Ahmed,′Synthesis,
′
[53] S. Saeed, N. Rashid, P. G. Jones, M. Ali, R. Hussain, Synthesis, characterization and
biological evaluation of some thiourea derivatives bearing benzothiazole moiety as potential
antimicrobial and anticancer agents, Eur. J. Med. Chem. 45 (2010) 1323–1331.

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Highlights:
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Single crystal X-ray of compound 1 and various spectroscopic techniques
(FT-IR, UV-Vis, H and C NMR) were used to establish the structure of
the compounds.
1
13
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The presence of intramolecular hydrogen bond (N─H⋯O═C) compels
planarity of the molecule and trans orientation of C=O and C=S group.
The compounds were screened for their In vitro cytotoxic activity against
seven human cancer cell lines.
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The compounds screened for their cytotoxic activity in seven human cell
line carcinomas exhibited promising anticancer activity
All the compounds except 5, 9 and 10 displayed stronger inhibitory activity
than the standard drug (5-FU) against HT29 cell line.