Antiproliferative activity of novel thiophene and thienopyrimidine derivatives

Article abstract of DOI:10.1055/s-0033-1358679

A novel series of newly synthesized thiophene derivatives, ethyl-4,5-dimethyl-2-(3-(3,4,5-trimethoxyphenyl)thioureido)thiophene-3- carboxylate 3, ethyl-2-[(2-(dimethylamino)ethoxy)mercapto)methyleneamino)]-4,5- dimethyl-thiophene-3-carboxylate 9, thienopyrimidines 4, 7, 10-20, triazolothienopyrimidines 5, 6 were prepared and tested for their antiproliferative activity. The structures of the synthesized compounds were confirmed on the basis of elemental analysis, IR, ¹H-NMR, ¹³C-NMR and mass spectral data. The results showed that the synthesized compounds were more active on breast cancer than on colon cancer cell lines and the most potent compounds in this study are compounds 3 and 13 which exerted remarkable activity against MDA-MB-231 (breast cancer) and HT-29 (colon cancer) cell lines with IC₅₀ values (40.68, 49.22 μM) for compound 3 and (34.04, 45.62 μM) for compound 13. Also, compounds 4-6, 9 showed a moderate activity against breast cancer cell line, while compounds 15, 19 and 20 showed no activity. Georg Thieme Verlag KG Stuttgart New York.

Full text of DOI:10.1055/s-0033-1358679

Original Article 313
Antiproliferative Activity of Novel Thiophene and
Thienopyrimidine Derivatives
Authors
M. M. Ghorab1, A. Al-Dhfyan2, M. S. Al-Dosari1, M. G. El-Gazzar3, M. S. AlSaid1
Affiliations
1 Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Kingdom of Saudi Arabia
2 Stem Cell Therapy Program, King Faisal Specialized Hospital and Research Center, Riyadh, Saudi Arabia
3 Department of Drug Radiation Research, National Center for Radiation Research and Technology,
Atomic Energy Authority, Cairo, Egypt
Key words
Abstract
▼
data. The results showed that the synthesized
compounds were more active on breast cancer
than on colon cancer cell lines and the most
potent compounds in this study are compounds
▶
thiophenes
●
▶
●
thienopyrimidines
antiproliferative activity
A novel series of newly synthesized thiophene
derivatives, ethyl-4,5-dimethyl-2-(3-(3,4,5-tri-
methoxyphenyl)thioureido)thiophene-3-car-
boxylate 3, ethyl-2-[(2-(dimethylamino)ethoxy)
mercapto)methyleneamino)]-4,5-dimethyl-
thiophene-3-carboxylate 9, thienopyrimidines
4, 7, 10–20, triazolothienopyrimidines 5, 6 were
prepared and tested for their antiproliferative
activity. The structures of the synthesized com-
pounds were confirmed on the basis of elemental
analysis, IR, 1H-NMR, 13C-NMR and mass spectral
▶
●
3
and 13 which exerted remarkable activ-
ity against MDA-MB-231 (breast cancer) and
HT-29 (colon cancer) cell lines with IC50 values
(40.68, 49.22μM) for compound 3 and (34.04,
45.62μM) for compound 13. Also, compounds
4–6, 9 showed a moderate activity against breast
cancer cell line, while compounds 15, 19 and 20
showed no activity.
Introduction
improved antimicrobial [17] and anticancer [18]
activities. Consequently, the thieno[2,3-d]pyri-
midine ring system constitutes an attractive tar-
get for the design of new anticancer drugs
through wide structure variations. In view of the
above findings and in continuation of our
research program [19–22] concerned with struc-
tural modification of certain biologically active
heterocyclic nuclei with the purpose of enhanc-
ing their biological activity, we report here the
synthesis of several novel thieno[2,3-d]pyrimi-
dines which is either substituted on the pyrimi-
dine with different biologically active moieties or
fused to triazole ring hoping that they could have
chemical and biological interest as anticancer
agents.
▼
Cancer is a disease of striking significance in the
world today as it remains a major cause of death
worldwide [1,2]. Although chemotherapy is the
mainstay of cancer therapy, the use of available
chemotherapeutics is often limited mainly due to
undesirable side effects and a limited choice of
available anticancer drugs [3]. This clearly under-
lines the urgent need for developing novel chem-
otherapeutic agents with more potent anticancer
activities. Many anticancer agents that act as
tyrosine kinase inhibitors comprise the pyrimi-
dine nucleus as a core moiety. This could be
exemplified by different quinazoline derivatives
such as Gefitinib (IressaTM) [4] and Tandutinib
(MLN518) (phase II clinical trials) [5]. On the
other hand, thieno[2,3-d]pyrimidines and their
fused heterocyclic ring systems are potential bio-
active molecules as they bear structural analogs
and isoelectronic relations to purines [6]. A large
number of fused thieno[2,3-d]pyrimidine deriva-
tives exhibited biological activities as potential
antimicrobial [7,8], antiviral [9], analgesic [10],
anti inflammatory [10] and anticancer [11–16]
activities. Moreover, addition of a triazolyl moi-
ety to thieno[2,3-d]pyrimidine nucleus was
reported to yield compounds that showed
received 30.08.2013
accepted 08.10.2013
Bibliography
DOI http://dx.doi.org/
10.1055/s-0033-1358679
Published online:
November 13, 2013
Drug Res 2014;
64: 313–320
© Georg Thieme Verlag KG
Stuttgart · New York
ISSN 2194-9379
Correspondence
M. M. Ghorab
Materials and Method
Professor of Applied Organic
Chemistry
Department of Pharmacognosy
College of Pharmacy
King Saud University
P.O. Box 2457
▼
General
Reagents were obtained from commercial suppli-
ers and were used without purification. Melting
points were determined in open capillary tubes
using Thermo system FP800 Mettler FP80 central
processor supplied with FP81 MBC cell appara-
tus, and were uncorrected. Elemental analyses
(C, H, N) were performed on a Perkin-Elmer 2400
Riyadh 11451
Kingdom of Saudi Arabia
Tel.: +966/53/4292 860
Fax: +966/01/4670 560
mmsghorab@yahoo.com
Ghorab MM et al. Novel Thiophene as Antiproliferatives… Drug Res 2014; 64: 313–320

314 Original Article
Instrument (USA). All compounds were within ±0.4% of the the-
(DMSO-d6) δ: 1.9 [s, 3H, CH3 triazole] 2.2, 2.3 [2s, 6H, 2CH3],
3.76, 3.78 [2s, 9H, 3OCH3], 6.5–7.2 [m, 2H, Ar-H]. 13C-NMR in
(DMSO-d6): 12.4, 12.6, 22.8, 56.1 (2), 59.9, 93.8 (2), 121.8, 129.7,
137.2, 137.8, 138.4, 153.1, 153.7 (2), 160.0, 161.2, 164.8. Anal.
Calcd. For C19H20N4O4S (400.45): C, 56.99; H, 5.03; N, 13.99.
Found: C, 56.76; H, 4.78; N, 14.28.
oretical values. Infrared (IR) spectra (KBr disc) were recorded on
FT-IR spectrophotometer (Perkin Elmer) at the Research Center,
College of Pharmacy, King Saud University, Saudi Arabia. 1H and
13C NMR spectra were recorded on a Ultra Shield Plus 500MHz
(Bruker) spectrometer operating at 500MHz for proton and
125MHz for carbon, respectively. The chemical shift values are
reported in δ (ppm) relative to the residual solvent peak, the
coupling constants (J) are reported in Hertz (Hz). Mass spectra
were run using a HP Model MS-5988 (Hewlett Packard).
5,6-Dimethyl-2-thioxo-3-(3,4,5-trimethoxyphenyl)-2,3-
dihydrothieno-[2,3-d]-pyrimidin-4-(1H)-one (7)
A mixture of 2 ( 4.41g, 0.01mol) and 3,4,5- trimethoxyaniline
(1.83g, 0.01mol) in dimethylformamide (20ml) containing tri-
ethylamine 3 drops was refluxed for 18h. The reaction mixture
was cooled and poured onto ice/water. The obtained solid was
recrystallized from dioxane to give 7. Yield % 58; m.p. 139.8℃;
IR (KBr, cm−1): 3386 (NH), 3066 (CH arom.), 2928, 2836 (CH
Ethyl-4,5-dimethyl-2-(3-(3,4,5-trimethoxyphenyl)
thioureido)thiophene-3-carboxylate (3)
A mixture of ethyl 2-isothiocyanato-4,5-dimethylthiophene-3-
carboxylate 2 (2.41g, 0.01mol) and 3,4,5- trimethoxyaniline
(1.83g, 0.01mol) in dimethylformamide (20ml) was stirred at
room temperature for 3h. The reaction mixture was poured onto
ice/water and the obtained solid was recrystallized from ethanol
to give 3. Yield % 93; m.p. 189.2℃; IR (KBr, cm−1): 3123 (NH),
3091 (CH arom.), 2964, 2899 (CH aliph.), 1655 (C=O), 1227
(C=S). 1H-NMR in (DMSO-d6) δ: 1.2 [t, 3H, CH3 ester], 2.17, 2.21
[2s, 6H, 2CH3 thiophene], 3.68, 3.78, 3.82 [3s, 9H, 3OCH3], 4.2 [q,
2H, CH2 ester], 6.7 [s, 2H, Ar-H], 10.7, 11.9 [2s, 2H, 2NH, D2O
exchangeable]. 13C-NMR in (DMSO-d6): 11.9, 13.9, 14.1, 55.8 (2),
60.0, 60.3, 102.4 (2), 112.9, 122.3, 128.0, 133.0, 135.6, 148.7 (2),
153.0, 165.6, 175.1. Anal. Calcd. For C19H24N2O5S2 (424.53): C,
53.75; H, 5.70; N, 6.60. Found: C, 53.48; H, 5.42; N, 6.29.
1
aliph.), 1654 (C=O), 1237 (C=S). H-NMR in (DMSO-d6) δ: 2.1,
2.2 [2s, 6H, 2CH3], 3.72, 3.78 [2s, 9H, 3OCH3], 6.9–7.1 [m, 2H,
Ar-H], 11.9 [s, 1H, NH, D2O exchangeable]. 13C-NMR in (DMSO-
d6): 11.8 (2), 55.6 (2), 55.8, 95.9 (2), 113.1, 123.0, 127.8, 128.6,
143.0, 152.8(2), 164.2, 166.7, 175.3. Anal. Calcd. ForC17H18N2O4S2
(378.47): C, 53.95; H, 4.79; N, 7.40. Found: C, 54.26; H, 4.98; N,
7.11.
Ethyl-2-[(2-(dimethylamino)ethoxy)mercapto)
methyleneamino)]-4,5-dimethyl-thiophene-3-
carboxylate (9)
A solution of 2 (4.41g, 0.01mol) in 2-(dimethylamino)ethanol
(5ml) was refluxed for 22h. The reaction mixture was concen-
trated and the obtained solid was recrystallized from dioxane to
give 9. Yield % 88; m.p. 110.6℃; IR (KBr, cm−1): 3100 (CH arom.),
2981, 2862 (CH aliph.), 1661(C=O). 1H-NMR in (DMSO-d6) δ:
1.2 [ t, 3H, CH3, ester], 2.0 [ s, 6H, N(CH3)2] 2.11, 2.14 [ m, 4H, 2
CH2 ], 2.51, 2.52 [2s, 6H, 2CH3], 4.1 [ q, 2H, CH2, ester], 7.1 [s, 1H,
SH, D2O exchangeable]. 13C-NMR in (DMSO-d6): 11.9, 14.2, 14.5,
40.0 (2), 44.8, 58.6, 59.0, 111.8, 129.0, 158.0, 162.1, 164.9. Anal.
Calcd. For C14H22N2O3S2 (330.47): C, 50.88; H, 6.71; N, 8.48.
Found: C, 50.66; H, 6.47; N, 8.16.
3-Amino-5,6-dimethyl-2-(3,4,5-trimethoxypenylamino)
thieno[2,3-d]pyrimidin-4- (3H)-one (4)
A mixture of 3 (4.42g, 0.01mol) and hydrazine hydrate (0.5g,
0.01mol) in absolute ethanol (30ml) was refluxed for 8h. The
reaction mixture was poured onto ice/water and the solid
obtained was recrystallized from ethanol to give 4. Yield % 90;
m.p. 221.3℃; IR (KBr, cm−1): 3403, 3391, 3186 (NH, NH2), 3074
(CH arom.), 2934, 2835 (CH aliph.), 1684 (C=O), 1599 (C=N).
1H-NMR in (DMSO-d6) δ: 2.2, 2.3 [2s, 6H, 2CH3], 3.75, 3.78, 3.82
[2s, 9H, 3OCH3], 6.6 [s, 2H, NH2, D2O exchangeable], 7.1–7.4 [m,
2H, Ar-H], 9.2 [s, 1H, NH, D2O exchangeable]. 13C-NMR in (DMSO-
d6): 12.2, 12.7, 56.0 (2), 59.9, 98.6 (2), 114.5, 127.8, 128.1, 129.6,
137.9, 152.5 (2), 153.7, 158.7, 163.9. MS (m/z): 376 [M+] (23.3),
76(100). Anal. Calcd. For C17H20N4O4S (376.43): C, 54.24; H,
5.36; N, 14.88. Found: C, 54.56; H, 5.12; N, 14.55.
2-Benzyl-5,6-dimethylthieno[2,3-d]pyrimidin-4-(3H)-one
(10)
A mixture of ethyl 2- amino-4,5- dimethylthiopene-3-carboxy-
late 1 [19,20] (1.99g, 0.01mol), triethylorthoformate (10ml)
and benzylamine (1.07g, 0.01mol) in decaline (5ml) was
refluxed for 18h. The reaction mixture was cooled and filtered.
The obtained solid was recrystallized from dioxane to give 10.
Yield % 76; m.p. 112.8℃; IR (KBr, cm−1): 3042 (CH arom.), 2928,
2861 (CH aliph.), 1661(C=O), 1568 (C=N). 1H-NMR in (DMSO-
d6) δ 2.1, 2.2 [2s, 6H, 2CH3], 4.9 [q, 2H, CH2], 7.1–7.9 [ m, 6H,
Ar- H+CH pyrimidine]. 13C-NMR in (DMSO-d6): 13.5, 13.8,
116.3, 125.8, 126.1, 127.8, 129.6 (2), 135.4, 137.1, 143.9, 149.2,
154.6, 163.0. MS (m/z): 270 [M+] (78.91), 91 (100). Anal. Calcd.
For C15H14N2OS (270.35): C, 66.64; H, 5.22; N, 10.36. Found: C,
66.39; H, 5.51; N, 10.14.
6,7-Dimethyl-5-oxo-1-(1,2,3-trimethoxy-5-yl)[1,2,4]
triazolo[5,1:2,3]thieno-[2,3-d]pyrimidine (5), 6,7-Dimethyl-
5-oxo-1-(1,2,3-trimethoxy-5-yl)-2-methyl[1,2,4]-
triazolo-[5,1:2,3]thieno[2,3-d]pyrimidine (6).
A solution of 4 (3.76g, 0.01mol) in formic acid and/or acetic
anhydride (20ml) was refluxed for 7h. The reaction mixture was
evaporated under vacuum and the obtained solid was recrystal-
lized from dioxane to give 5 and 6, respectively.
5: Yield % 78; m.p. 287.3℃; IR (KBr, cm−1): 3100 (CH arom.),
2940, 2835 (CH aliph.), 1684 (C=O), 1588 (C=N). 1H-NMR in
(DMSO-d6) δ: 2.1, 2.3 [2s, 6H, 2CH3], 3.7, 3.8 [2s, 9H, 3OCH3],
6.8–7.2 [m, 2H, Ar-H], 9.2 [s, 1H, CH. triazole]. 13C-NMR in
(DMSO-d6): 12.5, 12.7, 57.6 (2), 59.1, 95.9 (2), 119.2, 130.1,
136.7, 138.2, 138.8, 143.9, 152.5 (2), 157.8, 163.2, 164.0. Anal.
Calcd. For C18H18N4O4S (386.42): C, 55.95; H, 4.70; N, 14.50.
Found: C, 55.76; H, 4.98; N, 14.18.
Ethyl-2-(5,6-dimethyl-4-oxothieno[2,3-d]pyrimidin-
3(4H)-yl)acetate (12)
A mixture of 11 [20] (1.8g, 0.01mol), ethychloroacetate (1.23g,
0.01mol) in dry acetone (50ml) containing anhydrous potas-
sium carbonate (1g) was refluxed for 24h. The reaction mixture
was filtered while hot and the product that obtained was recrys-
tallized from ethanol to give 12. Yield % 91; m.p. 133.6℃; IR
(KBr, cm−1): 3065 (CH arom.), 2967, 2840 (CH aliph.), 1732
6: Yield % 68; m.p. 311.7℃; IR (KBr, cm−1): 384 (CH arom.),
2937, 2830 (CH aliph.), 1675 (C=O), 1579 (C=N). 1H-NMR in
Ghorab MM et al. Novel Thiophene as Antiproliferatives… Drug Res 2014; 64: 313–320

Original Article 315
1681 (2C=O), 1589 (C=N). 1H-NMR in (DMSO-d6) δ: 1.2 [t, 3H,
CH3 ester], 2.37, 2.38 [2s, 6H, 2CH3 thiophene], 4.1 [q, 2H, CH2
ester], 4.8 [s, 2H, CH2CO], 8.3 [s, H, CH pyrimidine]. 13C-NMR in
(DMSO-d6): 12.6, 12.7, 13.9, 46.8, 61.2, 122.3, 128.6, 130.4,
147.3, 156.9, 160.8, 167.8. Anal. Calcd. For C12H14N2O3S (266.32):
C, 54.12; H, 5.30; N, 10.52. Found: C, 54.48; H, 5.62; N, 10.19.
methanol to give 16. Yield % 89; m.p. 156.8℃; IR (KBr, cm−1):
3386, 3295 (NH2), 3100 (CH arom.), 2971, 2836 (CH aliph.),
1
1726, 1700, 1678 (3C=O), 1581 (C=N). H-NMR in (DMSO-d6)
δ: 1.2 [t, 3H, CH3 ester], 2.3, 2.4 [2s, 6H, 2CH3 thiophene], 4.2 [q,
2H, CH2 ester], 5.5 [s, 2H, CH2CO], 7.3 [s, 2H, NH2, D2O exchange-
able], 7.8 [s, 1H, CH, pyrazole], 8.3 [s, 1H, CH pyrimidine]. 13C-
NMR in (DMSO-d6): 12.9, 13.4, 15.5, 42.8, 62.0, 109.1, 118.6,
136.4, 137.0, 138.8, 146.1, 154.8, 156.3, 163.9, 167.4, 203.5. MS
(m/z): 375 [M+] (16.3), 103 (100). Anal. Calcd. For C16H17N5O4S
(375.40): C, 51.19; H, 4.56; N, 18.66. Found: C, 51.43; H, 4.25; N,
18.37.
2-(5,6-Dimethyl-4-oxothieno[2,3-d]pyrimidin-3(4H)-yl)
acetohydrazide (13)
A solution of 12 (2.66g, 0.01mol) in ethanol (50ml), hydrazine
hydrate (0.5g, 0.01mol) was added and the reaction mixture
was refluxed for 1h. The obtained solid was separated and
recrystallized from ethanol to give 13. Yield % 85; m.p. 253.7℃;
IR (KBr, cm−1): 3310, 3280, 3268 (NH, NH2), 3075 (CH arom.),
2938, 2876 (CH aliph.), 1691, 1678 (2C=O), 1592 (C=N). 1H-
NMR in (DMSO-d6) δ: 2.3, 2.4 [2s, 6H, 2CH3 thiophene], 4.5 [s,
2H, CH2], 4.9 [s, 2H, NH2, D2O exchangeable], 8.2 [s, 1H, CH pyri-
midine], 9.3 [s,1H, NH, D2O exchangeable]. 13C-NMR in (DMSO-
d6): 13.0, 13.6, 52.0, 118.6, 132.8, 135.7, 150.0, 158.6, 163.2,
173.5. MS (m/z): 252 [M+] (6.7), 193 (100). Anal. Calcd. For
C10H12N4O2S (252.29): C, 47.61; H, 4.79; N, 22.21. Found: C,
47.29; H, 4.48; N, 22.52.
Ethyl-2-(2-(5,6-dimethyl-4-oxothieno[2,3-d]pyrimidin-3-
(4H)-yl)acetyl)hydrazide-carboxylate (17)
A mixture of 13 (2.52g, 0.01mol) and ethylchloroformate (1.09g,
0.01mol) in dimethylformamide (20ml) containing anhydrous
K2CO3 (0.5g) was refluxed for 15min, cooled and acidified with
dilute HCl. The obtained solid was recrystallized from ethanol to
give 17. Yield % 77; m.p. 240.6℃; IR (KBr, cm−1): 3356, 3216
(NH), 3076 (CH arom.), 2976, 2835 (CH aliph.), 1716, 1691,
1656 (3C=O), 1590 (C=N). 1H-NMR in (DMSO-d6) δ: 1.1 [t, 3H,
CH3 ester], 2.36, 2.37 [2s, 6H, 2CH3 thiophene], 4.0 [q, 2H, CH2
ester], 4.7 [s, 2H, CH2CO], 8.2 [s, 1H, CH pyrimidine], 9.2, 10.1 [2s,
2H, 2NH, D2O exchangeable]. 13C-NMR in (DMSO-d6): 12.6, 12.8,
14.4, 46.2, 61.1, 122.3, 128.6, 130.1, 147.8, 155.9, 156.9, 166.6,
170.4. MS (m/z): 324 [M+] (1.7), 58 (100). Anal. Calcd. For
C13H16N4O4S (324.09): C, 48.14; H, 4.97; N, 17.27. Found: C,
48.49; H, 4.68; N, 12.55.
3-[(5-mercapto-4H-1,2,4-triazol-3-yl)methyl)-5,6-
dimethylthieno[2,3-d]pyrimidin-4(3H)-one (14)
Equimolecular amounts of the hydrazide 13 (2.52g, 0.01mol)
and thiourea (0.76g, 0.01mol) was fused at 250℃ in an oil bath
for 15min. The reaction mixture was cooled and the separated
solid was recrystallized from acetic acid to give 14. Yield % 65;
m.p.>350℃; IR (KBr, cm−1): 3278 (NH), 3081(CH arom.), 2946,
2836 (CH aliph.), 1652 (2C=O), 1628 (C=N). 1H-NMR in (DMSO-
d6) δ: 2.1, 2.2 [2s, 6H, 2CH3 thiophene], 4.4 [s, 2H, CH2], 8.4 [s,
1H, CH pyrimidine], 9.1 [s, 1H, NH, D2O exchangeable], 12.0
[s,1H, SH, D2O exchangeable]. 13C-NMR in (DMSO-d6): 11.3, 11.8,
42.7, 119.6, 131.8, 134.7, 146.6, 154.5, 159.9, 161.8, 164.2. MS
(m/z): 293 [M+] (9.80), 180 (100). Anal. Calcd. For C11H11N5OS2
(293.37): C, 45.03; H, 3.87; N, 23.21. Found: C, 45.29; H, 3.52; N,
23.46.
2-(2-(5,6-Dimethyl-4-oxothieno[2,3-d]pyrimidin-3(4H)-
yl)acetyl)hydrazide-carbothioamide (18)
A mixture of 13 (2.52g, 0.01mol) and ammonium thiocyanate
(0.76g, 0.01mol) in dioxane (25ml) was refluxing for 6h. The
reaction mixture was cooled and recrystallized from dioxane to
give 18. Yield % 71; m.p.>350℃; IR (KBr, cm−1): 3405, 3386,
3272 (NH, NH2 ), 3100 (CH arom.), 2971, 2881 (CH aliph.),
1691, 1659 (2C=O), 1586 (C=N), 1281 (C=S). 1H-NMR in
(DMSO-d6) δ: 2.32, 2.33 [2s, 6H, 2CH3 thiophene], 4.8 [s, 2H,
CH2CO], 8.3 [s, 1H, CH pyrimidine], 10.8 [s, 2H, NH2, D2O
exchangeable], 11.6 [s, 2H, 2 NH, D2O exchangeable]. 13C-NMR in
(DMSO-d6): 12.7, 12.8, 46.4, 122.3, 128.5, 130.3, 147.7, 157.0,
160.9, 166.1, 185.6. MS (m/z): 311 [M+] (100). Anal. Calcd. For
C11H13N5O2S2 (311.38): C, 42.43; H, 4.21; N, 22.49. Found: C,
42.12; H, 4.50; N, 22.19.
5,6-Dimethyl-3-(5-oxo-1,2,5,6-tetrahydro-1,2,4-triazole-
3-yl)methyl)-thieno-[2,3-d]-pyrimidin-4-(3H)-one (15)
An equimolecular amount of 13 (2.52g, 0.01mol) and chloroa-
cetamide (0.94g, 0.01mol) in dimethylformamide (20ml) was
heated under reflux for 12h. The reaction mixture was filtered
while hot and the obtained solid was recrystallized from diox-
ane to give 15. Yield % 79; m.p. 320.3℃; IR (KBr, cm−1): 3428,
3125 (NH), 3066 (CH arom.), 2924, 2854 (CH aliph.), 1689,
1670 (2C=O), 1595 (C=N). 1H-NMR in (DMSO-d6) δ: 2.3, 2.4 [2s,
6H, 2CH3 thiophene], 4.0 [s, 2H, CH2], 4.5 [s, 2H, CH2CO], 8.2 [s,
1H, CH, pyrimidine], 11.2 [s, 2H, 2NH, D2O exchangeable]. 13C-
NMR in (DMSO-d6): 13.2, 13.7, 47.4, 56.1, 116.6, 132,7, 138.8,
145.9, 158.6, 162.8, 169.7, 204.6. MS (m/z): 291 [M+] (8.07), 192
(100). Anal. Calcd. For C12H13N5O2S (291.33): C, 49.47; H, 4.50;
N, 24.04. Found: C, 49.10; H, 4.15; N, 24.36.
N-allyl-2-(2-(5,6-dimethyl-4-oxothieno[2,3-d]pyrimidin-
3(4H)-yl)acetyl)-hydrazine-carbothioamide (19)
A mixture of 13 (2.52g, 0.01mol) and allyl isothiocyanate (0.99g,
0.01mol) in dioxane (30ml) was refluxed for 4h. The excess sol-
vent was evaporated under vaccum and the obtained solid was
recrystallized from dioxane to give 19. Yield % 66; m.p. 208.7℃;
IR (KBr, cm−1): 3488, 3378, 3284 (NH), 3076 (CH arom.), 2924,
2854 (CH aliph.), 1689, 1672 (2C=O), 1578 (C=N), 1282 (C=S).
1H-NMR in (DMSO-d6) δ: 2.37, 2.38 [2s, 6H, 2CH3 thiophene], 4.1
[s, 2H, CH2CO], 4.7 [d, 2H, CH2NH, J=7.4Hz], 5.1 [s, 1H, NHCH2,
D2O exchangeable], 5.3, 5.4[d, 2H, CH=CH2, J=7.4Hz], 5.9[m, 1H,
CH=CH2], 8.2 [s, 1H, CH pyrimidine], 9.5, 10.3 [2s, 2H, 2NH, D2O
exchangeable]. 13C-NMR in (DMSO-d6): 12.6, 12.7, 45.9, 47.2,
115.3, 122.3, 128.7, 130.4, 134.5, 147.6, 157.0, 160.7, 170.4,
181.6. MS (m/z): 351 [M+] (1.3), 48(100). Anal. Calcd. For
C14H17N5O2S2 (351.45): C, 47.84; H, 4.88; N, 19.93. Found: C,
47.56; H, 4.57; N, 19.69.
Ethyl-5-amino-1-(2-(5,6-dimethyl-4-oxothieno[2,3-d]
pyrimidin-3(4H)-yl)-acetyl)-1H- pyrazole-4-carboxylate
(16)
A mixture of 13 (2.52g, 0.01mol), and 2-cyano-3-ethoxyacrylate
(1.69g, 0.01mol) in methanol (30ml) containing 3 drops of gla-
cial acetic acid was refluxed for 6h. The solvent was evaporated
under vacuum and the obtained solid was recrystallized from
Ghorab MM et al. Novel Thiophene as Antiproliferatives… Drug Res 2014; 64: 313–320

316 Original Article
Fig. 1 Synthetic pathways for compounds 2–6.
O
O
O
O
CSCl2
N
C
S
NH2
S
S
(1)
O
(2)
NH2
DMF
r.t.
O
O
O
O
NH2
O
O
O
N
N
N2H4- H2O
EtOH
- H2S
HN
O
NH
N
S
S
H
S
(3)
(4)
O
O
O
O
N
R
N
N
i) HCOOH
ii) Ac2O
N
S
O
5; R= H
6; R= CH3
O
O
N-acetyl-2-(5,6-dimethyl-4-oxothieno[2,3-d]pyrimidin-
3(4H)-yl)acetohydrazide (20)
Results and Discussion
▼
A solution of 13 (2.52g, 0.01mol) in glacial acetic acid (20ml)
containing anhydrous sodium acetate (1g) was refluxed for 12h.
The reaction mixture was cooled and poured onto ice/water. The
obtained solid was recrystallized from acetic acid to give 20.
Yield % 81; m.p.>350℃; IR (KBr, cm−1): 3256, 3201 (NH), 3078
(CH arom.), 2972, 2861 (CH aliph.), 1700, 1690, 1679 (3C=O),
1610 (C=N). 1H-NMR in (DMSO-d6) δ: 2.1, 2.2 [2s, 6H, 2CH3 thi-
ophene], 2.6 [s, 3H, COCH3], 4.1 [s, 2H, CH2CO], 8.1 [s, 1H, CH
pyrimidine], 11.3 [s, 2H, 2NH, D2O exchangeable]. 13C-NMR in
(DMSO-d6): 11.4, 12.0, 26.3, 52.6, 118.9, 131.8, 136.7, 149.2,
154.6, 159.9, 168.4, 173.0. MS (m/z): 294 [M+] (132), 221 (100).
Anal. Calcd. For C12H14N4O3S (294.33): C, 48.97; H, 4.79; N,
19.04. Found: C, 48.47; H, 4.50; N, 19.29.
Synthesis
The compounds were designed in the aim of exploring their
antiproliferative activity. The sequence of reaction followed in
the synthesis of the target compounds is illustrated in
▶
(
●
Fig. 1–4). As a part of a program aimed at the synthesis of
novel theinopyrimidine derivatives bearing a biologically active
3,4,5-trimethoxyphenyl moiety, which could be useful for
biological screening, we have investigated the possible utility of
2- isothiocyanatothiophene 2 [19] to react with 3,4,5-trimeth-
oxyaniline in dimethylformamide, at room temperature, to give
thioureido derivative 3 in high yield ( Fig. 1). Structure of com-
pound 3 was established based on the elemental and spectral
data. IR spectrum of 3 showed the disappearance of N=C=S
group and presence of (NH) at 3123cm−1, (C=S) at 1227cm−1
▶
●
.
In vitro antiproliferative activity
WST-1 cell proliferative assay
1H-NMR revealed signals at 3.68, 3.78, 3.82 ppm due to 3 meth-
oxy groups and 10.7, 11.9 ppm assigned to 2 (NH) groups. Com-
pound 3 allowed a fruitful, one step synthesis of several
heterocyclic rings via their reaction with hydrazine hydrate. The
formation of N-amino derivatives 4 proceeded via the elimina-
tion of one molecule of H2S, followed by intramolecular cycliza-
tion. IR spectrum exhibited the presence of characteristic bands
for N-amino group. 1H NMR spectrum revealed the presence of a
singlet at 6.6 ppm, due to N-amino group. Mass spectrum of
compound 4 revealed a molecular ion peak m/z at 376 with a
base peak m/z at 76. Triazolotheinopyrimidines 5 and 6 were
synthesized by a reaction of N-amino derivatives 4 with one car-
bon cyclising reagents, namely formic acid and/or acetic anhy-
MDA-MB-231 breast cancer and HT-29 colon cancer cell lines
were purchased from the American Type Culture Collection.
Cells were maintained in RPMI 1640 (Sigma), supplemented
with 10% FBS (Lonza), 100 IU/mL penicillin, 100mg/mL strepto-
mycin and 2mmol/L L-glutamine (Sigma). Cells were seeded
into 96-well plates at 0.4*104/well and incubated overnight. The
medium was replaced with fresh one containing the desired
concentrations of the synthesized compounds. After 48h, 10μl
of the WST-1 reagent was added to each well and the plates were
reincubated for 4h at 370℃. The amount of formazan was quan-
tified using ELISA reader at 450nm [23–25].
Ghorab MM et al. Novel Thiophene as Antiproliferatives… Drug Res 2014; 64: 313–320

Original Article 317
Fig. 2 Synthetic pathways for compounds 7–9.
O
O
N
O
S
N
OH
N
H
(2)
S
(8)
NH2
DMF
TEA
O
O
O
O
O
O
O
O
O
N
O
N
N
S
S
SH
N
S
H
(7)
(9)
Fig. 3 Synthetic pathways for compounds 10–15.
O
O
N
N
NH
NH2
CH(OC2H5)3
HCOOH
O
(1)
N
S
S
(11)
(10)
Dry Acetone
Anhd. K2CO3
Cl
O
NH2
NH
O
O
O
O
N
N
O
N2H4-H2O
EtOH
N
N
S
S
(13)
(12)
S
NH2
DMF
Fusion
O
Cl
H2N
NH2
O
O
H
N
SH
N
O
N
N
N
N
N
HN NH
N
(15)
S
S
(14)
▶
dride ( Fig. 1). On the other hand, when compound 2 was
●
was confirmed on the basis of elemental analysis, IR which
revealed the absence of N=C=S group and presence of charac-
teristic bands (NH) at 3386cm−1, (C=O) at 1654cm−1, (C=S) at
.
1237cm−1 1H-NMR exhibited singlet at 11.9 ppm assigned to
reacted with 3,4,5- trimethoxyaniline in DMF containing tri-
ethylamine as catalyst the corresponding cyclic theinopyrimi-
dine derivative 7 was obtained. The structure of compound 7
Ghorab MM et al. Novel Thiophene as Antiproliferatives… Drug Res 2014; 64: 313–320

318 Original Article
Fig. 4 Synthetic pathways for compounds 16–20.
O
O
HN
N
HN
NH
N
NaOAC
AcOH
O
N
O
N
S
N
(21)
S
O
O
(20)
N
N
O
O
O
O
HN
NH
O
H
O
O
ClCOOC2H5
N
N
O
OH
EtOH
(13)
N
O
S
N
S
(16)
(17)
S
S
NH2
NH
HN
NH
HN
NH
O
O
CH2=CHCH2NCS
NH4SCN
N
O
N
O
N
N
S
S
(19)
(18)
NH group. Ethyl 2-[(2-(dimethylamino)hoxy) mercapto) meth-
ylamino)]-4,5-dimethylthiophene-3-carboxylate 9 was obtained
in good yield via reaction of compound 2 with 2-(dimethyl-
amino)ethanol. IR spectrum exhibited the absence of N=C=S
band and presence of bands at 3100cm−1 (CH arom.), 1661cm−1
(C=O). 1H-NMR revealed signals at 2.0 ppm for N-dimethyl
group, 7.1 ppm corresponding to SH group. Refluxing compound
1 [19,20] with benzyl amine containing triethylorthoformate
afforded the benzyl derivative 10. 1H-NMR spectrum showed
singlet at 4.9 ppm indicated the presence of CH2 group. The
present paper describes the synthesis of the ester derivative 12
via reaction of thienopyrimidine 11 [20] with ethyl chloroace-
tate in dry acetone in the presence of anhydrous K2CO3. The
structure of 12 was confirmed by elemental analysis, IR, 1H-
NMR and 13C-NMR spectra. The IR spectrum of 12 revealed the
absence of (NH) functional group and presence of absorption
bands at 1750, 1690cm−1 assigned to (2C=O) groups. Also, 1H-
NMR spectrum exhibited the presence of a quartet and triplet
attributed to the ester pattern. The hydrazide derivative 13 was
obtained in excellent yield through reaction of ester 12 with
hydrazine hydrate in refluxing ethanol. IR spectrum showed
bands at 3310, 3280, 3268cm-1 (NH, NH2), 1691, 1678cm−1
(2C=O). 1H-NMR spectrum revealed signals at 4.5 ppm related to
CH2 group, 4.9 ppm corresponding to NH2 group and 9.3 ppm
assigned to (NH) group. The study was extended to the synthesis
of a new thieno[2,3-d]pyrimidines containing 1,2,4- triazole
nucleus having thiole moiety with potential biological activity.
Thus, the reaction of 13 with thiourea under condition of fusion
furnished the corresponding 1,2,4-triazole derivative 14. IR
spectrum exhibited the absence of carbonyl group of the
hydrazide and presence of carbonyl group of the corresponding
thienopyrimidine. 1H-NMR spectrum revealed singlet at 12.0
ppm assigned to SH group. Mass spectrum of 14 showed m/z
293[M+; 9.80%] with a base peak at 180(100%; base peak).
Interaction of 13 with chloroacetamide in dimethylformamide
gave the triazino derivative 15. The structure of compound 15
was supported on the basis elemental analysis and spectral data.
IR spectrum revealed bands at 1689, 1670cm−1 (2C=O). 1H-
NMR spectrum revealed signals at 4.0 ppm assigned to CH2
group, 4.5 ppm due to CH2CO group, 11.2 ppm for 2NH groups.
When compound 13 was reacted with ethyl 2-cyano-3-ethoxy-
acrylate in methanol containing 3 drops of glacial acetic acid
afforded pyrazole derivative 16. IR spectrum showed bands at
1726, 1700, 1678cm−1 (3C=O). 1H-NMR spectrum exhibited
the triplet quartet pattern at 1.2 and 4.2 ppm of the ester group,
7.8 ppm for CH pyrazole. In addition interaction of 13 with ethyl
chloroformate yielded the ethoxycarbonyl acetohydrazide deriv-
1
ative 17. H-NMR spectrum exhibited a triplet for CH3, quartet
for CH2 ester. The hydrazinecarbothioamide derivative 18 was
obtained via reaction of compound 13 with ammonium thiocy-
anate in dry toluene. 1H-NMR spectrum revealed signals at 10.8
ppm assigned to NH2 group, 11.6 ppm due to 2 NH groups. 13C-
NMR showed signals at 12.7, 12.8 for 2 CH3, 185.6 ppm corre-
sponding to (C=S) group. Mass spectrum exhibited m/z 311
[M+, 10.6%], with a base peak appeared at 221(100%, base
peak). Interaction of compound 13 with allylisothiocyanate in
dioxane furnished the corresponding N-allyl derivative 19. IR
spectrum showed bands at 1689, 1672cm−1 (2C=O), 1282cm−1
(C=S). 1H-NMR spectrum revealed singlet at 4.1 for CH2CO
group, duplet at 4.7 ppm assigned to CH2 group, 5.8 ppm due to
CH group. 13C-NMR spectrum showed signal at 181.6 ppm for
(C=S). Finally, the reaction of the hydrazide derivative 13 with
acetic acid in the presence of fused sodium acetate was carried
out in a trail for obtaining 1,2,4-triazine derivative 21, but
▶
instead, the mono acetyl derivative 20 was obtained ( Fig. 4).
●
IR spectrum revealed bands at 1700, 1690, 1679cm−1 for
1
(3C=O). H-NMR spectrum exhibited singlet at 2.6 ppm attrib-
uted to COCH3 group. Mass spectrum of 20 revealed m/z at 294
[M+, 1.32%], with a base peak appeared at 221 (100%, base peak).
In vitro antiproliferative activity
In-vitro antiproliferative activity was measured by the cell
growth inhibition assay. Cytotoxicity activity was determined at
the Stem Cell Therapy Program, King Faisal Specialized Hospital
and Research Center. The general in vitro antitumor evaluation
Ghorab MM et al. Novel Thiophene as Antiproliferatives… Drug Res 2014; 64: 313–320

Original Article 319
ety 9 with IC50 value (56.12μM) and they all showed higher
Table 1 Antiproliferative activity of the newly synthesized compounds
activity than the reference drug doxorubicin (71.8μM). Gener-
ally, the tested compounds are more active on the breast cancer
cell line and the most potent compounds showed higher activity
than the reference drug, while, concerning the colon cancer cell
line, only 4 compounds showed activity and they are of low
potency compared to the reference drug vinblastine (2.3μM).
3–20.
Compound No.
IC50 (μM)a MDA-MB 231
IC50 (μM)a HT-29
3
40.68±0.06
49.79±0.02
51.19±0.01
57.22±0.019
371.05±0.07
56.12±0.015
242.35±0.06
34.04±0.002
266.1±0.02
N A
49.22±0.02
4
109.94±0.01
5
NA
6
NA
7
NA
9
96.61±0.085
Conclusion
▼
12
NA
13
45.62±0.04
In this work, we report the synthesis of novel series of newly synthe-
sized thioureido thiophene-3-carboxylate 3, mercapto-methylene-
amino-4,5-dimethylthiophene-3-carboxylate 9, thienopyrimidines
4, 7, 10–20, triazolothienopyrimidines 5, 6 and tested for their
antiproliferative activity against MDA-MB-231 (breast cancer)
and HT-29 (colon cancer) cell lines. We can conclude that the
tested compounds are more active on the breast cancer cell line
and the most potent compounds showed higher activity than
the reference drug doxorubicin, while, concerning the colon can-
cer cell line, only 4 compounds showed activity and they are of
low potency compared to the reference drug vinblastine.
14
NA
1 5
N A
16
193.24±0.04
100.67±0.03
184.82±0.01
N A
NA
17
NA
18
NA
1 9
N A
2 0
N A
N A
Doxorubicin
Vinblastine
71.8±0.01
–
–
3.2±0.05
a IC50: Concentration of the compound (μM) producing 50% cell growth inhibition
after 48h of compound exposure, as determined by the WST-1 assay. Each experi-
ment was run at least 3 times, and the results are presented as average values±
standard deviation
NA: in active within 400μM concentration range
Acknowledgment
▼
of the newly synthesized compounds was conducted by use
WST-1 reagent for determination of IC50 for each compound
The authors would like to extend their sincere appreciation to
the Deanship of Scientific Research at King Saud University for
its funding of this research through the Research Group Project
no. RGP-VPP-302.
▶
(
Table 1). All the newly synthesized compounds were evalu-
●
ated for their in vitro cytotoxic activity against MDA-MB-231
breast cancer cell line and HT-29 colon cancer cell lines com-
pared to doxorubicin or vinblastine as reference drugs. The rela-
tionship between surviving fraction and drug concentration was
plotted to obtain survival curve of breast and colon cancer cell
lines. The response parameter calculated was IC50 value, which
corresponds to the concentration required for 50% inhibition of
cell viability.
Conflict of Interest
▼
The authors declare that they have no conflicts of interest to dis-
close.
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