Synthesis and Anticancer Activity of Novel Urea and Thiourea Bearing Thiophene-2-carboxalate Derivatives

Article abstract of DOI:10.1134/S1070363220070221

Abstract: A new series of urea and thiourea bearing thiophene-2-carboxalate derivatives has been designed against protein tyrosine phosphatase 1B (PTP1B) active site, synthesized and charecterized by ¹H and ¹³C NMR, and mass spectra. The compounds have been evaluated for in vitro anticancer activity against different cancer cell lines using the MTT colorimetric assay and doxorubicin as a standard drug. Among the tested compounds, methyl 3-methoxy-4-{4-[3-(4-methoxyphenyl)thioureido]phenyl}thiophene-2-carboxylate demonstrates the highest inhibitory activity against MCF-7, K562, HepG2, MDA-MB-231, and HeLa cell lines. The new molecular structures and their interactions with PNP1B have been evaluated by docking studies.

Full text of DOI:10.1134/S1070363220070221

ISSN 1070-3632, Russian Journal of General Chemistry, 2020, Vol. 90, No. 7, pp. 1336–1344. © Pleiades Publishing, Ltd., 2020.
Synthesis and Anticancer Activity of Novel Urea
and Thiourea Bearing Thiophene-2-carboxalate Derivatives
K. Ch. Gulipallia, P. Ravulab, S. Bodigea, S. Endooria, P. Koteswara Rao Cherukumallia,
J. N. Narendra Sharath Chandrac, and N. Seelama,*
a Department of Chemistry, Koneru Lakshmaiah Education Foundation, Green Fields, Vaddeswaram,
Guntur, 522502 India
b Department of Pharmaceutical Chemistry, Gurunanak Institutions Technical Campus, School of Pharmacy,
Hyderabad, 501506 India
c Department of Pharmaceutical Chemistry, Gurukrupa Institute of Pharmacy, Maharashtra, 431129 India
*e-mail: nareshvarma.klu@gmail.com
Received May 7, 2020; revised June 27, 2020; accepted July 7, 2020
Abstract—A new series of urea and thiourea bearing thiophene-2-carboxalate derivatives has been designed
against protein tyrosine phosphatase 1B (PTP1B) active site, synthesized and charecterized by 1H and 13C NMR,
and mass spectra. The compounds have been evaluated for in vitro anticancer activity against different cancer cell
lines using the MTT colorimetric assay and doxorubicin as a standard drug. Among the tested compounds, methyl
3-methoxy-4-{4-[3-(4-methoxyphenyl)thioureido]phenyl}thiophene-2-carboxylate demonstrates the highest inhibi-
tory activity against MCF-7, K562, HepG2, MDA-MB-231, and HeLa cell lines. The new molecular structures
and their interactions with PNP1B have been evaluated by docking studies.
Keywords: synthesis, thiophene-2-carboxalate, urea, thiourea, anticancer activity, molecular docking, protein
tyrosine phosphatase inhibitor
DOI: 10.1134/S1070363220070221
INTRODUCTION
for in vitro cytotoxic activity against HeLa, MCF-7,
K562, MDA-MB-231, HepG2, and HEK293 cell lines.
Recently protein tyrosine phosphatase 1B (PTP1B)
has been recognized as an attractive target for anticancer
studies, especially for breast cancer [1–3]. Thiophene
and its derivatives exhibit various biological activities
including BACE1 inhibiting [4], anti-inflammatory [5],
anti-HIV [6], and anti-cancer [7–10]. Urea and thiourea
derivatives have received close attention as building
blocks of molecules characterized as anticancer active due
to their significant inhibitory action on protein tyrosine
kinases (PTKs), Raf kinase, NADH oxidase, and DNA
topoisomerase [11–13]. Thus, more small molecules
bearing urea and thiourea building blocks need to be
synthesized for developing new anticancer compounds.
Recently we reported urea and thiourea derivatives of
thieno[3,2-d]pyrimidine that displayed excellent to good
anti cancer activity [14].
RESULTS AND DISCUSSION
As presented in Scheme 1, the key intermediate
amine 4 was prepared from methyl 4-bromo-3-hydroxy-
thiophene-2-carboxylate (1) using our previously
reported synthetic approach [15]. The amine 4 reaction
with 1,1′-thiocarbonyldiimidazole gave methyl 4-(4-iso-
thiocyanatophenyl)-3-methoxythiophene-2-carboxylate
(5), which reacted with different amines in a microwave
reactor (P = 150 W, t = 85 °C) to afford the corresponding
thioureaderivatives6a–6h. Ontheotherhand, intermediate
4 reacted with different aryl isocyanates in a microwave
reactor leading to the target compounds 7a–7g.
Initially, we attempted to make thiourea derivatives
by the reaction of amine 4 with the corresponding
isothiocyanate (Scheme 2). p-Tolylisothiocyante was
used as one of the precursors. The conventional reaction
in different solvents (CH2Cl2 and THF) and bases (Et3N
and DIPEA) at different temperatures (Table 1) led to
In the present study, we designed a series of urea and
thiourea derivatives bearing thiophene moiety based on
molecular docking against PTP1B, and evaluated those
1336

SYNTHESIS AND ANTICANCER ACTIVITY OF NOVEL UREA
1337
Scheme 1. Synthesis of title compounds.
formation of symmetrical thiourea 8 detected exclusively
by LC-MS. The reaction without a base led to formation
of the desired product with the yield ca 15% (Table 1,
entry 7), but overall conversion was ca 30%. The following
optimization of the reaction was extended to non-
conventional approach that involved ultrasonic irradiation
(entry 8, Table 1) and microwave irradiation (Table 1,
entry 9). The MW initiated process resulted in 80% of
conversion and 30% formation of the desired compound
6a, and upon purification yield was lower than 19%.
For improving the process yield methyl 4-(4-isothio-
cyanatophenyl)-3-methoxythiophene-2-carboxylate (5)
was introduced in the reaction with 4-methylaniline
(Scheme 3) to give the desired thiourea derivatives 6a–6h
in excellent to good yields.
Following the same line, urea derivatives were
synthesised by the reaction of amine compound 4 with
p-tolyl isocyanate (Scheme 4) upon MW irradiation. The
reaction proceeded readily with high yield of compounds
7a–7g.
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GULIPALLI et al.
Table 1. Optimization of the reaction conditions for the synthesis of compound 6a from amine 4a
Entry no.
Solvent
DCM
Base
Et3N
T, °C
Time, h
Conversionb, % Yield, %b (6a : 8)
1
2
3
4
5
6
7
8
9
40
50
25
65
40
65
65
65
80
16.0
16.0
16.0
4.0
40
42
50
70
45
65
30
35
80
0 : 100
0 : 100
0 : 100
0 : 100
0 : 100
0 : 100
15 : 85
20 : 80c
30 : 70d,e
Chloroform
THF
Et3N
Et3N
Et3N
DIPEA
DIPEA
–
THF
DCM
THF
16.0
4.0
THF
4.0
THF
–
2.0
THF
–
0.5
a All reactions were performed using amine 4 (1.00 mmol), p-tolylisothiocyanate (1.20 mmol), base (2.00 mmol).
b Conversion and yield estimated by LC-MS analysis.
c Ultrasonic irradiation.
d MW irradiation.
e Yield 19% upon purification.
Anticancer activity. Anticancer activity of the
synthesized compounds 6a–6h and 7a–7g was evaluated
against MCF-7, K562,HepG2, MDA-MB-231, HeLa, and
HEK293 cell lines using the MTT colorimetric assay and
doxorubicin as a standard drug (Table 2). The products
exhibited moderate to potent anticancer activity. Thiourea
derivatives 6a–6h were determined to be more active than
the urea derivatives 7a–7g.
activity, and replacement of the above substituent with
3-methoxyphenylhenyl in 6b led to some decrease in
activity. The product with 4-methylphenyl group 6a was
characterized by even lower anticancer activity. The
product 6e bearing 4-bromophenyl ring displayed more
inhibitory activity against all cancer cell lines than the
compound 6f with 4-fluorophenyl ring.
Among the urea series 7a–7g, 4-bromophenyl group
containing compound 7e demonstrated activity against
Among the tested compounds, the product 6c with
4-methoxtphenyl ring demonstrated potent inhibitory
Scheme 2. Reaction of amine 4 with p-tolyl isothiocyante.
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SYNTHESIS AND ANTICANCER ACTIVITY OF NOVEL UREA
1339
Scheme 3. Synthesis of thio urea derivatives.
Scheme 4. Reaction of amine 4 with p-tolyl isocyante.
MCF-7 and K562 cancer cell lines.All products exhibited
no significant toxic effect.
with PTP1B inhibitors. The representative interactions
are presented graphically for compound 6b (Fig. 1).
According to docking procedure carried out for 7b, the
urea NH group could participate in polar interactions with
ASP 48 of amino acid residues with 40.0% binding at a
distance of 1.59 Å, the other urea NH group interacted
withASP48 of 61.1% binding at 1.69 Å. The carboxylate
C=O group interacted withARG 221 of 25.3% binding at
a distance of 2.59 Å, and phenyl ring exhibited stacking
Molecular docking. The target molecules were
docked on the PTP1B active site for considering their
binding interactions against PTP 1B. According to
docking results, it was evident that the synthesized
compounds 6a–6h and 7a–7g interacted with key amino
acid residues of PTB1B such as ASP 48, TYR 46, ARG
24, and GLN 262 that are also known for their interactions
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GULIPALLI et al.
Table 2. In vitro anticancer activity of the synthesized compounds
IC50, μM
MDA-MB 231
> 50
Compound
MCF-7
8.84
5.26
2.16
8.32
10.28
12.56
22.35
33.69
13.83
19.82
24.17
18.52
14.23
12.35
32.26
0.41
K562
9.12
7.30
3.27
10.32
9.24
14.23
19.86
29.33
18.54
> 50
22.58
19.28
11.69
18.53
30.26
0.07
HepG2
13.34
9.43
HeLa
14.23
8.29
HEK293
Not determined
Not determined
Not determined
48.25
Not determined
> 50
Not determined
Not determined
> 50
Not determined
48.23
> 50
48.58
Not determined
Not determined
6a
6b
6c
6d
6e
6f
6g
6h
7a
7b
7c
7d
7e
7f
7g
5.33
1.35
9.63
6.23
Not determined
15.33
9.63
15.23
18.63
22.35
35.63
16.38
17.59
25.36
34.81
24.43
32.55
36.76
45.22
0.37
12.54
16.56
21.24
19.58
25.38
29.12
37.34
23.18
16.34
19.56
26.88
0.60
18.12
24.31
18.36
19.26
22.19
23.21
15.89
17.62
22.33
22.53
5.00
Doxorubicin
interactions with TYR 46. The compound molecule could
be surrounded by ARG 47, GLN 267 and PHE 182.
purification. Melting points were uncorrected and
determined in one end open capillary tubes on a Guna
Digital Melting Point apparatus. Elemental analysis was
EXPERIMENTAL
1
performed on a Perkin-Elmer 240 CHN analyzer. H
and 13C NMR spectra were measured on a Bruker AMX
300 spectrometer operating at 400 MHz (1H NMR) and
100 MHz (13C NMR) using TMS as the internal
reference and CDCl3 or DMSO-d6 as the solvents. Mass
spectra were measured on an Agilent Technologies mass
spectrometer.
All chemicals were purchased from Sigma–Aldrich,
Merck and Combi blocks and used without further
Methyl 4-(4-isothiocyanophenyl)-3-methoxy-
thiophene-2-carboxylate (5). To a solution of methyl
4-(4-aminophenyl)-3-methoxythiophene-2-carboxylate
(2.50 g, 9.50 mmol) in CH2Cl2 (25 mL) was added
1,1′-thiocarbonyldiimidazole (2.03 g, 11.4 mmol) and
the mixture was stirred at room temperature for 16 h.
The solvent was evaporated, and the residue was purified
by flash column chromatography (ethyl acetate–hexane)
to afford pure compound 5 as an off-white solid. Yield
62%, mp 118–120°C. 1H NMR spectrum, δ, ppm: 3.81 s
(3H, ArOCH3), 3.82 s (3H, C=OOCH3), 7.51 d (2H,
J = 8.8 Hz, HAr), 7.69 d (2H, J = 8.4 Hz, HAr), 8.09 s
(1H, HAr). 13C NMR spectrum, δC, ppm: 52.00, 62.07,
116.5, 126.2, 128.7, 129.0, 129.2, 133.8, 134.5, 159.4,
160.6. Found, %: C 55.15; H 3.75; N4.48. C14H11NO3S2.
Calculated, %: C 55.06; H 3.63; N 4.59.
Fig. 1. (a) Two-dimensional representation of the interaction
mode of compound 6b with PTP1B enzyme and (b) three-
dimensional structural model of 6b on PTP1B enzyme.
Synthesis of compounds 6a–6h. To a solution of
methyl 4-(4-isothiocyanatophenyl)-3-methoxythiophene-
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SYNTHESIS AND ANTICANCER ACTIVITY OF NOVEL UREA
1341
2-carboxylate (0.50 mmol) in THF (1.00 mL) was added
the desired substituted aniline (0.60 mmol) at room
temperature, and the mixture was subjected to MW
irradiation upon stirring at 85°C and 150 W for 20 min.
The reaction mixture was concentrated under reduced
pressure. The obtained crude product was purified by
column chromatography (ethyl acetate–hexane) to afford
the corresponding pure compound.
spectrum, δ, ppm: 3.82 (6H, 2OCH3), 7.14 d (2H, J =
8.4 Hz, HAr), 7.35 d (2H, J = 8.0 Hz, HAr), 7.57 s (4H,
HAr), 7.97 s (1H, HAr), 9.80 s (1H, C=SNH), 9.81 s (1H,
C=SNH). 13C NMR spectrum, δC, ppm: 51.91, 61.84,
116.3, 118.3, 119.4, 121.7, 127.1, 127.3, 135.8, 138.9,
139.1, 142.6, 152.4, 159.6, 160.8. Found, %: C 52.38; H
3.45; N5.70. C21H17F3N2O4S2. Calculated, %: C 52.27;
H 3.55; N5.81. MS: m/z: 483.1 [М + Н]+.
Methyl 3-methoxy-4-[4-(3-p-tolylthioureido)-
phenyl]thiophene-2-carboxylate (6a). Off white solid,
yield 90%, mp 180–184°C. 1H NMR spectrum, δ, ppm:
2.28 s (3H, ArCH3), 3.81 s (3H, ArOCH3), 3.82 s (3H,
C=OOCH3), 7.14 d (2H, J = 8.0 Hz, HAr), 7.35 d (2H,
J = 8.4 Hz, HAr), 7.57 s (4H, HAr), 7.96 s (1H, HAr),
9.78 s (1H, C=SNH), 9.79 s (1H, C=SNH). 13C NMR
spectrum, δC, ppm: 20.50, 51.93, 61.94, 116.3, 123.2,
123.8, 127.4, 127.9, 128.9, 129.1, 133.7, 135.6, 136.7,
139.1, 159.6, 160.8, 179.4. Found, %: C 61.24; H 4.77;
N6.68. C21H20N2O3S2. Calculated, %: C61.14; H 4.89;
N6.79. MS: m/z: 413.2 [М + Н]+.
Methyl 4-{4-[3-(3-fluorophenyl)thioureido]-
phenyl}-3-methoxythiophene-2-carboxylate (6e).
1
Orange solid, yield 84%, mp 184–189°C. H NMR
spectrum, δ, ppm: 3.82 s (3H, ArOCH3), 3.83 s (3H,
C=OOCH3), 6.93–6.97 m (1H, HAr), 7.26–7.28 m (1H,
HAr), 7.33–7.39 m (1H, HAr), 7.54–7.60 s (5H, HAr), 7.98 s
(1H, HAr), 10.0 br. s (2H, C=SNH). 13C NMR spectrum,
δC, ppm: 51.94, 61.97, 109.8, 110.0, 110.6, 110.8, 116.3,
118.9, 123.4, 127.5, 128.0, 129.5, 129.9, 130.0, 135.6,
138.8, 141.2, 141.3, 159.6, 160.8, 162.9, 179.3. Found,
%: C 57.58; H, 4.21; N, 6.62. C20H17FN2O3S2. Calculated,
%: C 57.68; H, 4.11; N, 6.73. MS: m/z: 417.0 [М + Н]+.
Methyl 3-methoxy-4-{4-[3-(3-methoxyphenyl)-
thioureido]phenyl}thiophene-2-carboxylate (6b).
Light yellow solid, yield 81%, mp 175–179°C. 1H NMR
spectrum, δ, ppm: 3.74 s (3H, C=OOCH3), 3.82 s (6H,
2OCH3), 6.71 dd (1H, J = 8.0 Hz, J = 2.0 Hz, HAr), 7.03 d
(1H, J = 8.4 Hz, HAr), 7.18 s (1H, HAr), 7.24 t (1H, J =
8.0 Hz, HAr), 7.57 s (4H, HAr), 7.97 s (1H, HAr), 9.89 s
(1H, C=SNH), 9.90 s (1H, C=SNH).13C NMR spectrum,
δC, ppm: 51.04, 51.93, 61.94, 109.1, 109.7, 115.4, 116.2,
123.3, 127.4, 127.9, 129.2, 135.6, 139.0, 140.4, 159.2,
159.6, 160.7, 179.1. Found, %: C 58.96; H 4.79; N6.43.
C21H20N2O4S2. Calculated, %: C58.86; H 4.70; N6.54.
MS: m/z: 429.1 [М + Н]+.
Methyl 4-{4-[3-(4-fluorophenyl)thioureido]-
phenyl}-3-methoxythiophene-2-carboxylate (6f).
Brown solid, yield 83%, mp 176–180°C. H NMR
1
spectrum, δ, ppm: 3.82 s (3H, ArOCH3), 7.18 t (2H, J =
8.8 Hz, HAr), 7.47–7.51 m (2H, HAr), 7.58 s (4H, HAr),
7.97 s (1H, HAr), 3.83 s (3H, C=OOCH3), 9.24 s (1H,
C=SNH), 9.86 s (1H, C=SNH). 13C NMR spectrum, δC,
ppm: 51.91, 61.93, 114.9, 115.1, 116.2, 123.3, 126.1,
127.5, 127.9, 129.3, 135.6, 135.7, 138.9, 157.9, 159.6,
160.3, 160.7, 179.7. Found, %: C 57.78; H 4.19; N 6.62.
C20H17FN2O3S2. Calculated, %: C 57.68; H 4.11; N6.73.
MS: m/z: 417.0 [М + Н]+.
Methyl 4-{4-[3-(3,5-difluorophenyl)thioureido]-
Methyl 3-methoxy-4-{4-[3-(4-methoxyphenyl)-
thioureido]phenyl}thiophene-2-carboxylate (6c).
Yellow solid, yield 85%, mp 140–144°C. H NMR
phenyl}-3-methoxythiophene-2-carboxylate (6g).
Orange solid, yield 87%, mp 168–171°C. H NMR
1
1
spectrum, δ, ppm: 3.79 s (3H, ArOCH3), 3.81 s (3H,
C=OOCH3), 7.03–7.07 m (1H, HAr), 7.28–7.34 m (1H,
HAr), 7.50–7.56 m (4H, HAr), 7.92 s (1H, HAr), 8.06–8.12 m
(1H, HAr), 8.54 s (1H, C=SNH), 9.12 s (1H, C=SNH).
13C NMR spectrum, δC, ppm: 51.87, 61.79, 103.7, 110.9,
111.1, 116.2, 118.0, 121.9, 123.9, 124.0, 127.1, 127.3,
127.9, 135.7, 139.0, 152.1, 153.4, 159.6, 160.8. Found,
%: C 55.19; H 3.82; N6.35. C20H16F2N2O3S2. Calculated,
%: C 55.29; H 3.71; N6.45. MS: m/z: 435.2 [М + Н]+.
spectrum, δ, ppm: 3.78 s (3H, C=OOCH3), 3.81 s (6H,
2OCH3), 7.14 d (2H, J = 8.4 Hz, HAr), 7.34 d (2H, J =
8.4 Hz, HAr), 7.56 s (4H, HAr), 7.96 s (1H, HAr), 9.79 s
(1H, C=SNH), 9.80 s (1H, C=SNH).13C NMR spectrum,
δC, ppm: 51.89, 60.14, 61.91, 116.2, 123.2, 123.8,
127.4, 127.8, 128.8, 129.1, 133.7, 135.6, 136.7, 139.1,
159.6, 160.7, 179.4. Found, %: C 58.96; H 4.79; N6.43.
C21H20N2O4S2. Calculated, %: C 58.86; H 4.70; N6.54.
MS: m/z: 429.1 [М + Н]+.
Methyl 4-{4-[3-(5-fluoropyridin-3-yl)thioureido]-
Methyl 3-methoxy-4-{4-[3-(4-(trifluoromethoxy)-
phenyl)thioureido]phenyl}thiophene-2-carboxylate
(6d). Brown solid, yield 78%, mp 174–178°C. 1H NMR
phenyl}-3-methoxythiophene-2-carboxylate (6h).
Off white solid, yield 76%, mp 135–139°C. H NMR
spectrum, δ, ppm: 3.82 s (3H, ArOCH3), 3.83 s (3H,
1
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GULIPALLI et al.
C=OOCH3), 7.55–7.62 m (4H, HAr), 7.99 s (1H, HAr),
8.07–8.11 m (1H, HAr), 8.33 d (1H, J = 2.4 Hz, HAr), 8.49 s
(1H, HAr), 10.1 s (1H, C=SNH), 10.2 s (1H, C=SNH).
13C NMR spectrum, δC, ppm: 51.94, 62.00, 116.3, 117.5,
117.7, 123.6, 127.6, 128.1, 129.8, 132.5, 132.7, 135.5,
137.6, 138.4, 141.0, 156.9, 159.5, 159.6, 160.7, 179.9.
Found, %: C 54.75; H 3.76; N10.17. C19H16FN3O3S2.
Calculated, %: C 54.66; H 3.86; N10.07. MS: m/z: 418.0
[М + Н]+.
(2H, C=ONH). 13C NMR spectrum, δC, ppm: 51.87,
61.90, 109.7, 109.9, 110.5, 110.7, 116.2, 118.8, 123.3,
127.4, 127.9, 129.4, 129.8, 129.9, 135.5, 138.7, 141.1,
141.2, 159.5, 160.7, 162.9, 179.2. Found, %: C 59.89; H
4.17; N7.10. C20H17FN2O4S. Calculated, %: C 59.99; H
4.28; N 7.00. MS: m/z: 401.1 [М + Н]+.
Methyl 4-{4-[3-(4-fluorophenyl)ureido]phenyl}-3-
methoxythiophene-2-carboxylate (7d). Brown solid,
yield 71%, mp 171–175°C. 1H NMR spectrum, δ, ppm:
3.81 s (6H, 2OCH3), 7.29 s (2H, HAr), 7.54 s (6H, HAr),
7.92 s (1H, HAr), 8.83 s (1H, C=ONH), 8.91 s (1H,
C=ONH). 13C NMR spectrum, δC, ppm: 51.90, 61.83,
116.3, 118.3, 119.4, 121.7, 127.1, 127.3, 127.9, 135.8,
138.9, 139.1, 142.6, 152.4, 159.6, 160.8. Found, %: C
59.88; H 4.18; N7.11. C20H17FN2O4S. Calculated, %: C
59.99; H 4.28; N7.00. MS: m/z: 401.1 [М + Н]+.
Synthesis of compounds 7a–7g. To a solution of
methyl 4-(4-aminophenyl)-3-methoxythiophene-2-
carboxylate (0.38 mmol) in THF (1.00 mL), was added a
desired isocyanate (0.45mmol) at room temperature, and
the mixture was subjected to MW irradiation upon stirring
at 85°C and 150 W for 20 min. The reaction mixture was
concentrated under reduced pressure. The obtained crude
product was purified by column chromatography (ethyl
acetate–hexane) to afford the corresponding pure product.
Methyl 4-{4-[3-(4-bromophenyl)ureido]phenyl}-
3-methoxythiophene-2-carboxylate (7e). Brown solid,
yield 77%, mp 153–156°C. 1H NMR spectrum, δ, ppm:
3.80 s (3H, ArOCH3), 3.81 s (3H, C=OOCH3), 7.15 d
(1H, J = 8.0 Hz, HAr), 7.24 t (1H, J = 8.0 Hz, HAr), 7.32 d
(1H, J = 8.0 Hz, HAr), 7.54 s (4H, HAr), 7.87 s (1H,
HAr), 7.92 s (1H, HAr), 8.87 s (1H, C=ONH), 8.92 s (1H,
C=ONH). 13C NMR spectrum, δC, ppm: 51.89, 61.82,
116.3, 117.0, 117.2, 118.4, 120.4, 120.6, 121.7, 124.3,
124.6, 127.1, 127.4, 127.9, 130.6, 135.8, 139.0, 141.3,
152.2, 159.6, 160.8. Found, %: C 52.17; H, 3.79; N, 6.16.
C20H17BrN2O4S. Calculated, %: C 52.07; H, 3.71; N,
6.07. MS: m/z: 462.9 [М + Н]+.
Methyl 3-methoxy-4-[4-(3-m-tolylureido)phenyl]-
thiophene-2-carboxylate (7a). Off white solid, yield
1
71%, mp 182–186°C. H NMR spectrum, δ, ppm:
2.29 s (3H, ArCH3), 3.81 s (3H, ArOCH3), 3.82 s (3H,
C=OOCH3), 6.70 dd (1H, J = 8.4 Hz, J = 2.4 Hz, HAr),
7.03 d (1H, J = 8.4 Hz, HAr), 7.18 s (1H, HAr), 7.23 t (1H,
J = 8.0 Hz, HAr), 7.56 s (4H, HAr), 7.97 s (1H, HAr), 9.88 s
(1H, C=ONH), 9.89 s (1H, C=ONH).13C NMR spectrum,
δC, ppm: 20.51, 55.02, 61.92, 109.0, 109.7, 115.4, 116.2,
123.3, 123.8, 127.4, 127.9, 129.1, 129.2, 135.6, 139.0,
140.4, 159.2, 159.6, 160.7, 179.1. Found, %: C 63.72; H
5.17; N7.17. C21H20N2O4S. Calculated, %: C 63.62; H
5.08; N 7.07. MS: m/z: 397.1 [М + Н]+.
Methyl 3-methoxy-4-{4-[3-(4-(trifluoromethoxy)-
phenyl)ureido]phenyl}thiophene-2-carboxylate (7f).
1
Methyl 3-methoxy-4-[4-(3-p-tolylureido)phenyl]-
thiophene-2-carboxylate (7b). Orange solid, yield 76%,
mp 174–178°C. 1H NMR spectrum, δ, ppm: 2.28 s (3H,
ArCH3), 3.80 s (3H, ArOCH3), 3.81 s (3H, C=OOCH3),
7.28–7.30 m (3H, HAr), 7.53–7.57 m (5H, HAr), 7.92 s
(1H, HAr), 8.38 s (1H, C=ONH), 8.91 s (1H, C=ONH).
13C NMR spectrum, δC, ppm: 20.50, 51.93, 61.95, 116.3,
123.2, 123.8, 123.8, 127.4, 127.9, 128.9, 129.1, 133.7,
135.6, 136.7, 139.1, 159.6, 160.8, 179.4. Found, %: C
63.73; H 5.18; N7.18. C21H20N2O4S. Calculated, %: C
63.62; H 5.08; N 7.07. MS: m/z: 397.1 [М + Н]+.
Off white solid, yield 68%, mp 162–166°C. H NMR
spectrum, δ, ppm: 3.80 s (3H, ArOCH3), 3.82 s (3H,
C=OOCH3), 7.29 d (2H, J = 8.4 Hz, HAr), 7.54–7.59 m
(6H, HAr), 7.92 s (1H, HAr), 8.85 s (1H, C=ONH), 8.93 s
(1H, NHC=ONH). 13C NMR spectrum, δC, ppm: 51.87,
61.80, 116.3, 118.2, 119.4, 121.6, 127.1, 127.3, 127.9,
135.8, 138.9, 139.1, 142.6, 152.3, 159.6, 160.8. Found,
%: C 54.18; H 3.55; N6.12. C21H17F3N2O5S. Calculated,
%: C 54.08; H 3.67; N 6.0. MS: m/z: 467.1 [М + Н]+.
Methyl 4-{4-[3-(3,5-difluorophenyl)ureido]phenyl}-
3-methoxythiophene-2-carboxylate (7g). Brown solid,
yield 79%, mp 168–172°C. 1H NMR spectrum, δ, ppm:
3.80s(3H,ArOCH3), 3.81s(3H, C=OOCH3), 7.03–7.08m
(1H, HAr), 7.28–7.34 m (1H, HAr), 7.51–7.56 m (4H,
HAr), 7.92 s (1H, HAr), 8.07–8.13 m (1H, HAr), 8.54 s
(1H, C=ONH), 9.13 s (1H, C=ONH).13C NMR spectrum,
δC, ppm: 51.89, 61.82, 116.3, 118.0, 122.0, 123.9, 124.0,
Methyl 4-{4-[3-(3-fluorophenyl)ureido]phenyl}-3-
methoxythiophene-2-carboxylate (7c). Off white solid,
yield 85%, mp 181–185°C. 1H NMR spectrum, δ, ppm:
3.82s(3H,ArOCH3), 3.83s(3H, C=OOCH3), 6.92–6.97m
(1H, HAr), 7.27 d (2H, J = 8.4 Hz, HAr), 7.33–7.39 m (1H,
HAr), 7.53–7.60 m (5H, HAr), 7.98 s (1H, HAr), 10.0 br. s
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 90 No. 7 2020

SYNTHESIS AND ANTICANCER ACTIVITY OF NOVEL UREA
1343
127.1, 127.3, 127.9, 135.7, 139.0, 152.2, 159.6, 160.8.
CONCLUSIONS
Found, %: C 57.31; H 3.74; N 6.79. C20H16F2N2O4S.
Calculated, %: C 57.41; H 3.85; N 6.70. MS: m/z: 418.1
[М + Н]+.
A new series of urea and thiourea bearing thiophene-
2-carboxamide derivatives has been designed, synthesized
and evaluated for in vitro anticancer activity against
different cancer cell lines using the MTT colorimetric
assay. Among the testeded compounds, methyl 3-meth-
oxy-4-{4-[3-(4-methoxyphenyl)thioureido]phenyl}-
thiophene-2-carboxylate (6c) demonstrates the highest
inhibitory activity against MCF-7, K562, HepG2, MDA-
MB-231, and HeLa cell lines. The tested compounds show
key interactions as those of known PTP1B inhibitors in
docking studies.
Anticancer activity. Synthesized molecules were
tested for anticancer activity by the MTT colorimetric
assay against human cancer cell lines including breast
cancer (MDA-MB-231), cervix carcinoma (HeLa), breast
adeno carcinoma (MCF-7), hepato cellular carcinoma
(HepG2), chronic myeloid leukemia (K562), and hepato
cellular carcinoma (HEK293). All tumour cells were
grown in DMEM media supplemented with 10% heat
inactivated foetal bovine serum (FBS), 100 mg/mL
streptomycin, 100 IU/mLpenicillin and 2 mM-glutamine.
All cells were maintained at temperature of 37°C under
humidified atmosphere. Approximately 5×103 cells
were allowed to adhere for 24 h in 96-well culture plate.
Compounds dissolved in the DMSO (10 to 100 μg/mL)
were added to the culture wells in triplicate to the 96 well
plate and incubated under 5% CO2 atmosphere at 37°C
for 48 h. Afterwards, the cell culture was stained with
20 μLof MTT (5 mg/mLin PBS) for 3 h, and then DMSO
(100 μL) was added to each 96-well plate. Absorbance
of the test solution was measured at 540 nm using
Benesphera plate reader. The results were expressed in
IC50 and calculated by using origin software [16, 17].
ACKNOWLEDGMENTS
The authors would like to thank the management of AMRI
Hyderabad research centre for giving an opportunity to carry
out this research. The authors are also thankful to Sai Innotech
Labs Pvt.Ltd.for providing synthesis facility.
CONFLICT OF INTEREST
No conflict of interest was declared by the authors.
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