Synthesis and Cytotoxicity of Some Thieno[2,3-d]pyrimidine Derivatives

Article abstract of DOI:10.1002/jccs.201600279

A series of compounds 5-amino-2-ethylmercapto-4-phenyl-6-subistitutedthieno[2,3-d]pyrimidines (8a–d), 4-chloro-7-ethylmercapto-9-phenylpyrimido[5′,4′:4,5]thieno[3,2-d]triazine (9), and 2-ethylmercapto-8-oxo-4-phenyl-7-(4-chlorophenyl)pyrimido [4′,5′:4,5]thieno[2,3-d]pyrimidine (10) were synthesized and their structures were confirmed by ¹H NMR, ¹³C NMR, and MS. All compounds were evaluated for their IC₅₀ values against three cancer cell lines (MCF-7, HUH-7 and BHK) and WISH cells. The IC₅₀ of compound (8d) was calculated for each cell line. Interestingly, the IC₅₀ for the normal human amnion WISH cell line was much higher (723 μg/mL) than those found for the tumor cell lines BHK (17 μg/mL), HUH-7 (5.8 μg/mL), and MCF-7 (8.3 μg/mL). The proliferation inhibition of normal (WISH) and tumor (BHK, HUH-7, and MCF-7) cells by compound (8d) was investigated using MTT assay, and the IC₅₀ was calculated after 48 h of treatment for each cell line.

Full text of DOI:10.1002/jccs.201600279

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Article
Synthesis and Cytotoxicity of Some Thieno[2,3-d]pyrimidine Derivatives
a
a
b
a
*
Abdelreheem A. Saddik, Adel M. Kamal El-Dean, Gamal H. El-Sokary, Khairy M. Hassan,
Mohamed S. Abbady,a Ismail A. Ismailb and Saber H. Saberb
aChemistry Department, Faculty of Science, Assiut University, Assiut 71516, Egypt
bZoology Department, Faculty of Science, Assiut University, Assiut 71516, Egypt
(Received: July 27, 2016; Accepted: October 25, 2016; DOI: 10.1002/jccs.201600279)
A series of compounds 5-amino-2-ethylmercapto-4-phenyl-6-subistitutedthieno[2,3-d]pyrimidines
(8a–d), 4-chloro-7-ethylmercapto-9-phenylpyrimido[50,40:4,5]thieno[3,2-d]triazine (9), and 2-ethyl-
mercapto-8-oxo-4-phenyl-7-(4-chlorophenyl)pyrimido [40,50:4,5]thieno[2,3-d]pyrimidine (10) were
1
synthesized and their structures were confirmed by H NMR, 13C NMR, and MS. All compounds
were evaluated for their IC50 values against three cancer cell lines (MCF-7, HUH-7 and BHK)
and WISH cells. The IC50 of compound (8d) was calculated for each cell line. Interestingly, the
IC50 for the normal human amnion WISH cell line was much higher (723 μg/mL) than those
found for the tumor cell lines BHK (17 μg/mL), HUH-7 (5.8 μg/mL), and MCF-7 (8.3 μg/mL).
The proliferation inhibition of normal (WISH) and tumor (BHK, HUH-7, and MCF-7) cells by
compound (8d) was investigated using MTT assay, and the IC50 was calculated after 48 h of treat-
ment for each cell line.
Keywords: Thienopyrimidine; Pyrimidothienotriazine; Pyrimidothienopyrimidine; Cytotoxicity.
INTRODUCTION
Thienopyrimidines are an important class of
activities on HEPG2 and MCF7 cell lines using
doxorubicin as reference. Compound E showed very
good anticancer activity against HEPG2 cell line
and compound F against MCF7 cell line.7 Mavrova
et al. reported the synthesis of compounds G and
H; these compounds showed very good cytotoxicity
against MDA-MB-231, Lep-3, and HT-29 cell
lines.8
In view of the cytotoxicity of thienopyrimi-
dines, and in continuation of our work on the syn-
thesis of thienopyrimidines,9–27 here we report the
synthesis of some thienopyrimidines and evaluate
their cytotoxicity.
heterocyclic compounds because of their therapeutic
activities. These compounds have been reported to
have anticancer activities.1–4 Thienopyrimidines have
been identified as potent inhibitors of VEGFR-2
kinase,2 and some pyrimidine derivatives were shown
to exert potent cytotoxic effects on leukemia cell lines
including P388 cells and J774 cells (compound A).4
Thienopyrimidine derivatives have been identified as
cyclin-dependent kinase 4 (CDK4) inhibitors.5 Al-
Taisan et al. have reported the synthesis of some thie-
nopyrimidine derivatives (B, C, and D) and studied
their in vitro cytotoxic activities on HEPG2, HELA,
MCF7, HCT116, and HEP2 cell lines using doxorubi-
cin as reference. Compound B showed very good
anticancer activity against HEPG2, MCF7, and HEP2
cell lines, compound C against HELA cell line, and
compound D against HCT116.6 On the other hand,
Shaaban et al. prepared thienotriazolopyrimidine deri-
vatives E and F and studied their in vitro cytotoxic
RESULT AND DISCUSSION
Chemistry of the compounds
5-Cyano-4-oxo-6-phenyl-2-thioxo-1,2,3,4-tetrahyd-
ropyrimidine (4)28 was synthesized via multicomponent
reaction (MCR) by one-pot condensation of benzalde-
hyde (1), ethyl cyanoacetate (2), and thiourea (3) in
refluxing ethanol in the presence of potassium
*Corresponding author. Email: abdelraheemseddik@gmail.com
J. Chin. Chem. Soc. 2016
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Article
Saddik et al.
carbonate. Refluxing of (4) with sodium acetate in etha-
nol gave the sodium salt of (4),29 which was then
reacted with ethyl iodide to give 2-ethylylmercapto-4-
oxo-6-phenyl-pyrimidine-5-carbonitriles (5).
Compound 5 was converted into 2-(ethylmer-
capto)-4-chloro-6-phenylpyrimidine-5-carbonitrile
(6)
via reaction with POCl3. The chlorine underwent nucle-
ophilic displacement with the mercapto group using thi-
ourea in refluxed ethanol to give 2-(ethylmercapto)-4-
mercapto-6-phenylpyrimidine-5-carbonitrile (7).27
When compound 7 was allowed to react with
α-halogenated the compounds chloroacetamide, chlor-
oacetonitrile, ethyl chloroacetate, and N-p-chlorophenyl
chloroacetamide in ethanol and in the presence of
potassium carbonate, compounds 8a–d were obtained.30
IR spectra of compounds 8a–d showed absorption
bands from 3475 to 3331 cm−1 corresponding to the
1
NH2 group. H NMR spectra of compounds 8a–d in
(CDCl3) showed signals at δ 4.58 due to the NH2
group.
Compound 8d was cyclized with trietyl ortho-
formate in the presence of a catalytic amount of ace-
tic acid to give the pyrimidothienopyrimidine
derivative 9.30 Compound 8b when treated with
sodium nitrite in HCl afforded pyrimidothienotria-
zine 10.30 Formation of compound 9 was confirmed
1
by IR, H NMR, and mass spectrometry. The mass
spectrum of compound 9 showed a peak at m/z
450 as a molecular ion peak and base peak. IR spec-
trum of compound 9 showed a band at 1689 cm−1
,
characteristic of CO. 1H NMR spectrum of com-
pound 9 in (CDCl3) showed a multiplet at δ 7.3–7.85
for aromatic protons and a signal at 7.96 characteris-
tic of CH pyrimidine. The characteristic signals for
NH and NH2 groups were absent in the spectrum of
compound 8d. The structure of compound 10 was
confirmed on the basis of spectral analysis. The IR
spectrum for compound 10 did not show bands char-
acteristics of the NH2 and CN groups in the case of
Scheme 1. 8a: R CONH2, 8b: R CN, 8c:
R CO2Et, 8d: R CONHPh-Cl-p.
(a) Ethyl iodide, AcONa, EtOH, reflux,
3 h: (b) POCl3, 3 h: (c) NH2CSNH2,
EtOH, reflux, 8 h: (d) RCH2Cl, EtOH,
K2CO3, reflux, 3 h: (e) CH(OEt)3, gla-
cial AcOH, reflux, 2 h: (f ) AcOH, HCl,
NaNO2 (10%), 0–5ꢀC.
1
compound 8b. The H NMR spectrum for compound
10 (CDCl3) showed a signal at δ 1.45 for the CH3
group, 3.3 for the CH2 group, and 7.5–8 for the ben-
zene ring. The signal characteristic of the NH2 group
was absent in compound 8b. The mass spectrum of
compound 10 showed a molecular ion peak at m/z
359.5 (Scheme 1).
Cytotoxicity
The effect of the starting compound (4) and its
derivatives (8a–d, 9, and 10) on the cell viability of sev-
eral cell lines was investigated using MTT cell viability
2
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Synthesis and Cytotoxicity of Some Thieno[2,3-d]pyrimidine Derivatives
assay after 48 h of treatment. All compounds inhibited
HUH-7 cell proliferation, and the IC50 for each com-
pound was calculated after 48 h of treatment (Table 1).
Interestingly, all the derivatives (i.e., 8a–d, 9, and 10)
showed more effective proliferation inhibition of HUH-
7 cells than the starting compound (4) (Figure 1). Com-
pound 8d was the most potent derivative in inhibiting
HUH-7 cell proliferation with IC50 = 5.8 (Figure 1 and
Table 2).
Next, we investigated the effect of 8d as the most
potent cell proliferative inhibitor derivative on several
cell lines (WISH, BHK, HUH-7, and MCF-7)
(Table 3). The IC50 of 8d (Table 4) was calculated for
each cell line as indicated in Figure 2. Interestingly, the
IC50 for the normal human amnion WISH cell line was
much higher (723 μg/mL) than those for the tumor cell
lines BHK (17 μg/mL), HUH-7 (5.8 μg/mL), and
MCF-7 (8.3 μg/mL) (Figure 2).
apparatus. Preparative and analytical thin-layer chro-
matography (TLC) were carried out on silica gel plates
(Fluka 70643-50EA. Sigma-Aldrich, Schwein Furt,
Germany) using UV light. All reactions were carried
out under air atmosphere. MTT (3-[4,5-dimethyl-2-thia-
zolyl]-2,5-diphenyl-2H-tetrazolium bromide) was pur-
chased from Sigma (St. Louis, MO, USA), while
DMEM, fetal bovine serum (FBS), and penicillin/strep-
tomycin antibiotics were from Gibco (Invitrogen Cor-
poration, CA, Carlsbad, California, USA). All other
chemicals and reagents were obtained from standard
commercial sources. Human MCF-7 breast cancer,
human HUH-7 hepatocarcinoma, human WISH nor-
mal amnion, and baby hamster kidney (BHK) fibro-
blast cells were obtained from Vacsera—Cell Culture
Unit, Cairo, Egypt. 5-Cyano-4-oxo-6-phenyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine (4) was synthesized via
MCR by a one-pot condensation reaction according to
the literature.28
These different antiproliferation activities of 8d on
normal (WISH) and tumor (BHK, HUH-7, and MCF-
7) cells was confirmed also in a dose-dependent study
(Figure 3). There was a clear, remarkable differential
inhibitory action of 8d in normal and cancer cells.
These data suggest 8d as a potent anticancer drug
candidate.
Chemistry
2-(Ethylmercapto)-4-hydroxy-6-phenylpyrimidine-5-carbonitrile
(5).
Prepared according literature procedure:29
white crystals, 85% yield, M.p. 238–240ꢀC, (246ꢀC) ,
IR (cm−1 KBr): 3150 (NH), 3030 (CH aromatic), 2219
(CN), 1655 (C O), MS m/z: 257.5 [M]+.
EXPERIMENTAL
2-(Ethylmercapto)-4-chloro-6-phenylpyrimidine-5-carbonitrile
All melting points are uncorrected and measured
on a Fisher-John apparatus, Hampton, New Hamp-
shire, USA. IR spectra were recorded on a Thermo
Nicolet 6700 USA spectrophotometer using the KBr
wafer technique. 1H NMR spectra were obtained on
Bruker, Bremen, Germany 400 MHz spectrometer in
CDCl3 and DMSO-d6 using Me4Si as internal standard.
Chemical shifts are expressed as ppm unless otherwise
stated. Mass spectra were obtained on JEOL JMS-600
(6)27.
Compound 5 (20 gm) in POCl3 (60 mL) was
refluxed for 3 h, then allowed to cool, poured into
crushed ice (600 g), and neutralized with sodium carbo-
nate solution (10%) to just alkaline. The solid precipi-
tate formed was collected by filtration and washed
several times with water and dried in air and recrystal-
lized from ethanol. White crystals, 75% yield.
M.p. 64–66ꢀC, IR (cm−1 KBr): 3047 (CH aromatic),
Table 1. Effect of compounds (8a–d to 10) on HUH-7 cell line
HUH-7 cells
μg/mL
4
8a
100
65.45748
50.14107
39.98388
37.36397
31.15679
8b
100
57.67836
40.91092
37.04152
34.4216
8c
100
49.41556
44.33696
36.9206
8d
100
40.15735
34.10296
26.06465
23.49923
21.41269
9
10
100
67.13534
44.34242
37.68532
34.99761
14.45242
Conc.
0.1
1
10
100
1000
100
100
83.19226
65.01411
50.48368
48.0653
45.8283
43.24869
38.89561
28.37565
19.02459
29.74607
21.76542
37.36397
27.65014
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Article
Saddik et al.
yield. M.p. 178–180 ºC. IR (cm−1 KBr): 3047
(CH aromatic), 2984 (CH aliphatic), 2216 (CN),
+
1255 (C S). MS: m/z 273.5 [M]
.
5-Amino-2-ethylmercapto-4-phenyl-6-subistituted thieno[2,3-d]
pyrimidine (8a–d).
General procedure
Compound 7 (0.01 mol), potassium carbonate
(0.02 mole), and the α-halocarbonyl compounds chlor-
oacetamide, chloloacetonitrile, ethyl chloroacetate, and
p-chloro chloroacetanilide (0.011 mole) in 50 mL etha-
nol were heated under reflux for 3 h. The solid product
was filtered off, washed with water and ethanol, and
dried in air.
5-Amino-2-ethylmercapto-4-phenylthieno[2,3-d]pyrimidine-6-
carbamide (8a).
Pale yellow crystals, 62% yield.
M.p. 174–176ꢀC, IR (cm−1 KBr): 3475, 3429 (NH2),
3327, 3208 (NH2), 3047 (CH aromatic), 2965
(CH aliphatic), 1667(C O). 1H NMR (400 MHz,
CDCl3) δ(ppm) 1.4 (t, J = 26.78 Hz, 3H, CH3), 3.2 (q,
J = 13.15 Hz, 2H, CH2), 5.35 (s, 2H, NH2), 6 (s,2H,
NH2), 7.4–7.6 (t, J = 32.35 Hz, 5Haromatic ); 13C NMR
(400 MHz, CDCl3): 170.19, 168.04, 167.13, 163.07,
146.46, 136.36, 130.58, 128.98, 128.76, 117.31, 94.32,
25.66, 14.44. MS: m/z 329.8 [M]+.
Fig. 1. Effect of the seven compounds on HUH-7
cell proliferations investigated using MTT
cell viability assay. The IC50 of compound
was calculated after 48 h as indicated above.
2925 (CH aliphatic), 2227 (CN), 1640 (C O), 694 (C–
Cl). MS m/z: 275.6 [M]+.
2-(Ethylmercapto)-4-mercapto-6-phenylpyrimidine-5-
5-Amino-2-ethylmercapto-4-phenyl-thieno[2,3-d]pyrimidine-6-
carbonitrile (7)27.
Compound 6 (0.01 mole) and thi-
carbonitrile (8b).
Pale yellow crystals, 65% yield.
ourea (0.02 mole) in 100 mL ethanol was refluxed
for 6 h and then allowed to cool. The yellow precipi-
tate formed was filtered off, washed with ethanol,
treated with NaOH (10%), and acidified with dilute
HCl. The crude product was collected, washed sev-
eral times with water, dried, and recrystalized from
an ethanol/dioxane mixture. Yellow crystals, 63%
M.p. 198–200ꢀC. IR (cm−1 KBr): 3464, 3331 (NH2),
3227 (NH tautomer), 3047 (CH aromatic), 2978
(CH aliphatic), 2202 (CN); 1H NMR (400 MHz,
CDCl3) δ (ppm) 1.36 (t, J = 36.11 Hz, 3H, CH3), 3.17
(q, J = 13.31 Hz, 2H, CH2), 4.58 (s, 2H, NH2), 7.52 (s,
5Haromatic). 13C NMR (400 MHz, CDCl3): 171.16,
169.82, 162.75, 148.49, 136.00, 130.90, 129.17, 128.72,
114.93, 114.74, 25.70, 14.36. MS: m/z 312 [M]+.
Table 2. Most potent derivative in inhibiting HUH-7 cell
proliferation
Ethyl 5-amino-2-ethylmercapto-4-phenyl-thieno[2,3-d]pyrimi-
HUH-7 cells
dine-6-carboxylate (8c).
Pale yellow crystals, 70%
yield. M.p. 168–170ꢀC. IR (cm−1 KBr): 3498, 3365
IC50
SD
(NH2), 3050 (CH aromatic), 2974 (CH aliphatic), 1671
4
28.2
6.6
7.1
7.7
5.8
7.3
8.4
1.5
0.8
0.5
0.42
0.25
0.9
1
(C O). H NMR (400 MHz, CDCl3) δ(ppm) 1.27–1.37
8a
8b
8c
8d
9
(2 t, J = 39.1 Hz, 6H,2CH3), 3.15 (q, J = 10.86 Hz,
2H, CH2), 4.25 (q, J = 11.12 Hz, 2H, CH2), 5.74 (s,
2H, NH2), 7.52 (d, J = 28.39 Hz, 5Haromatic); 13C
NMR (400 MHz, CDCl3) 170.24, 169.78, 165.25,
163.00, 146.83, 136.51, 130.53, 129.01, 128.70, 116.71,
94.73, 25.64, 14.45. MS: m/z 359[M]+.
10
0.71
SD: standard deviation.
4
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Synthesis and Cytotoxicity of Some Thieno[2,3-d]pyrimidine Derivatives
Table 3. Effect of compound 8d on MCF-7, HUH-7, BHK, and WISH cell lines
Cell line
WISH
MCF-7
HUH-7
BHK
Conc.
0.1 M
01 M
10 M
100 M
1000 M
100 ꢁ 3.2
95 ꢁ 2.4
100 ꢁ 3.8
90.86 ꢁ 1.9
59.19 ꢁ 3.5
45.93 ꢁ 1.7
29.65 ꢁ 2.5
18.31 ꢁ 1.3
100 ꢁ 3.8
83.25 ꢁ 2.1
59.5 ꢁ 1.6
40.5 ꢁ 2.4
22.5 ꢁ 1.7
16.5 ꢁ 1.9
100 ꢁ 2.9
82.5 ꢁ 19
68.45 ꢁ 2.5
56.5 ꢁ 1.8
36.5 ꢁ 2.1
22.8 ꢁ 0.8
79 ꢁ 5.1
65 ꢁ 2.5
59.05 ꢁ 3.4
43.25 ꢁ 18
5-Amino-2-ethylmercapto–4-phenyl-N-(p-chlorophenyl)thieno
[2,3-d]-pyrimidine-6-carbamide (8d).
129.97, 128.14, 127.74, 121.23, 118.93, 25.81, 14.46.
MS: m/z 450[M]+.
Orange crystals,
50% yield. M.p. 188–190ꢀC, IR (cm−1 KBr): 3485, 3460
(NH2), 3292 (NH), 3046 (CH aromatic), 2924
(CH aliphatic), 1636 (C O). 1H NMR (400 MHz,
CDCl3) δ(ppm) 1.35(t, J = 24.36 Hz, 3H, CH3), 3.2 (q,
J = 10.49 Hz, 2H, CH2), 6.0 (s, 2H, NH2), 7.22–7.24
(2d, J = 10.45 Hz, 10.35 Hz, 4H, Ar-H); 7.40–7.49 (m,
J = 56 Hz, 6H, Ar-H, NH). 13C NMR (400 MHz,
CDCl3) 170.46, 163.52, 163.09, 146.80, 136.20, 130.69,
129.54, 129.07, 128.76, 121.86, 117.40, 94.76, 25.71,
14.43. MS: m/z 440[M]+.
4-Chloro-7-ethylmercapto-9-phenylpyrimido[50,40:4,5]thieno
[3,2-d]triazine (10).
Compound 8b (1.3 g, 0.005 mol)
was mixed with acetic acid (10 mL) and concentrated
hydrochloric acid (7 mL), and a sodium nitrite solution
10% (4 mL, 0.006 mol) was added with stirring during
5 min. The stirring was continued at 5ꢀC for 3 h. The
formed precipitate was collected and crystallized from
ethanol to give white plates in 62.5% yield.
M.p. 180–181ꢀC; IR (cm−1 KBr): 3093 (CH aromatic),
2950 (CH aliphatic); 1H NMR (400 MHz, CDCl3)
δ(ppm) 1.45 (t, J = 34.56 Hz, 3H, CH3), 3.3 (q,
J = 18.21 Hz, 2H,CH2), 7.51–7.56 (q, J = 26.4 Hz, 3H,
Ar-H), 7.98–8.00 (d, J = 20.8 Hz, 2H, Ar-H). 13C-
NMR (400 MHz, CDCl3) 175.26, 172.00, 165.87,
2-Ethylmercapto-8-oxo-4-phenyl-7(4-chlorophenyl)-pyrimido
[40,50:4,5]thieno[2,3-d]pyrimidine (9).
Compound 8d
(0.01 mol), 20 mL of triethyl orthoformate, and a few
drops of glacial acetic acid were refluxed for 2 h. The
white crystals were formed while hot. After cooling, the
crystals were filtrated and washed with ethanol, and
dried in air. White crystals, 68% yield. M.p. 220–222ꢀC,
IR (cm−1 KBr): 3075 (CH aromatic), 2929
(CH aliphatic), 1689 (C O); 1H NMR (400 MHz,
CDCl3) δ(ppm) 1.4 (t, J = 24.11 Hz, 3H, CH3), 3.25 (q,
J = 12.42, 2H, CH2), 7.27–7.29 (d, J = 18.1 Hz, 2H,
Ar-H), 7.42–7.48 (q, J = 27.2 Hz, 5H, Ar-H), 7.84–
7.85 (d, J = 18.1 Hz, 2H, Ar-H), 7.96 (s, 1H, CH); 13C
NMR (400 MHz, CDCl3) 172.31, 171.49, 164.67,
156.66, 150.12, 147.11, 136.05, 135.72, 134.93, 130.79,
Table 4. IC50 compound 8d on (MCF-7, HUH-7, BHK and
WISH) cell lines
The IC50 of 8d in different cell lines
Cell line
IC50 (μg/mL)
Fig. 2. Proliferation inhibition of normal (WISH)
and tumor (BHK, HUH-7, and MCF-7)
cells by 8d investigated using MTT assay.
IC50 was calculated after 48 h of treatment
for each cell line.
MCF-7
HUH-7
BHK
8.3 ꢁ 0.3
5.8 ꢁ 0.2
17 ꢁ 1.0
723 ꢁ 8.4
WISH
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Article
Saddik et al.
synthesized compounds displayed excellent cytotoxic-
ity. The most promising compound 8d exhibited
strong cytotoxicity against HUH-7, MCF-7, and
BHK cell lines, with IC50 values of 5.8 ꢁ 0.2,
8.3 ꢁ 0.3, and 17 ꢁ 1.0 μg/mL, and against normal
WISH cell line with IC50 value of 723 ꢁ 8.4.
ACKNOWLEDGMENT
We thank Prof. Dr. Ahmed Abdo Geies, President
and Professor of Organic Chemistry, Assiut University
for support.
Fig. 3. Effect of 8d on normal (WISH) and cancer
Supporting Information
(MCF-7, HUH-7, and BHK) cell lines inves-
tigated using MTT cell viability assay after
48 h. 8d strongly inhibited tumor cell growth
and proliferation when compared to its effect
on normal cells.
Supporting information for this article is avail-
able on the www under http://dx.doi.org/10.1002/
jccs.201600279.
REFERENCES
153.05, 151.48, 135.27, 132.01, 130.51, 128.78, 128.31,
1. J. A. Ubersax, E. L. Woodbury, P. N. Quang, M. Paraz,
J. D. Blethrow, K. K. M. Shah, D. O. Shokat, Nature
2003, 425, 859.
2. M. J. Munchhof, J. S. Beebe, J. M. Casavant,
B. A. Cooper, J. L. Doty, R. C. Higdon,
S. M. Hillerman, C. I. Soderstrom, E. A. Knauth,
M. A. Marx, A. M. K. Rossi, S. B. Sobolov, J. Sun,
Bioorg. Med. Chem. Lett. 2004, 14, 21.
3. W. Glen, H. Li, J. Jack, Ortwine, Fred D. PCT Int. Appl.
Wo 02 64,598. C.A. 2002, 137, 1,855,04f.
4. J. A. Katada, M. Muramatsu, M. Takami, E. Yasuda,
M. Hayashi, M. Hattori, Y. Hayashi, Bioorg. Med.
Chem. Lett. 1999, 9, 797.
5. T. Horiuchi, J. Chiba, K. Uoto, T. Soga, Bioorg. Med.
Chem. Lett. 2009, 19, 305.
6. K. M. Al-Taisan, H. M. A. Al-Hazimi, S. S. Al-Shihry,
Molecules 2010, 15, 3932.
7. M. A. Shaaban, M. M. Ghorab, H. I. Heiba,
M. M. Kamel, N. H. Zaher, M. I. Mostafa, Arch. Pharm.
Chem. Life Sci. 2010, 343, 404.
8. A. T. Mavrova, S. Dimov, D. Yancheva, M. Rangelov,
D. Wesselinova, J. A. Tsenov, Eur. Med. Chem. 2016,
123, 69.
115.56, 26.12, 14.27. MS: m/z 359.5 [M]+.
Cytotoxicity
The effects of thieno[2.3-d]pyrimidine and its deri-
vatives (Compounds 8a–d to 10) were investigated for
the proliferation of MCF-7, HUH-7, WISH, and BHK
cells using MTT assay as previously described. Briefly,
the cells were cultured in 100 μL medium in 96-well
plates (5 × 103/well). On the following day, the experi-
mental media containing different concentrations of
thieno[2,3-d]pyrimidines and its derivatives (0.0, 0.0001,
0.001, 0.01, 0.1, and 1.0 mg/mL) were added, and then
incubated for 48 h. Fifty microliters of MTT solution
(2 mg/mL in PBS) was added to each well and incu-
bated for 4 h. After careful removal of the media,
150 μL of DMSO was added to each well, and then
after shaking, the absorbance was read at 570 nm using
an ELISA microplate reader (Molecular Devices, Sun-
nyvale, CA, USA).
CONCLUSION
9. K. M. Hassan, A. M. Kamal El-Dean, M. S. K. Youssef,
F. M. Atta, M. S. Abbady, Phosphorus Sulfur Silicon
Relat. Elem. 1990, 47, 283.
10. M. S. K. Youssef, F. M. Atta, A. M. Kamal El-Dean,
M. S. Abbady, K. M. Hassan, Bull. Fac. Sci. Assiut Univ.
1991, 20, 31.
11. H. S. El-Kashef, A. A. Geies, A. M. Kamal El-Dean,
A. A. Abdel-Hafez, J. Chem. Technol. Biotechnol. 1993, 57, 15.
12. A. A. Abdel-Hafez, A. M. Kamal El-Dean,
A. A. Hassan, H. S. El-Kashef, Bull. Fac. Sci. Assiut
Univ. 1993, 23, 93.
In summary, a series of compounds 5-amino-2-
ethylmercapto-4-phenyl-6-subistitutedthieno[2,3-d]pyri
midine (8a–d), 4-Chloro-7-ethylmercapto-9-phenyl
pyrimido[50,40:4,5]thieno[3,2-d]triazine (9), and 2-ethyl-
mercapto-8-oxo-4-phenyl-7-(4-chlorophenyl)pyrimido
[40,50:4,5]thieno[2,3-d]pyrimidine (10) were designed,
synthesized, and evaluated for cytotoxicity against
three cancer cell lines (HUH-7, MCF-7 and BHK)
in vitro. The pharmacological results indicated that all
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JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Synthesis and Cytotoxicity of Some Thieno[2,3-d]pyrimidine Derivatives
13. A. M. Kamal El-Dean, Phosphorus Sulfur Silicon Relat.
Elem. 1994, 90, 85.
22. A. M. Kamal El-Dean, A. S. N. Al-Kamali, Afinidad
2009, 66, 70.
14. A. M. Kamal El-Dean, J. Chem. Res. 1996, 6, 260.
15. A. A. Geies, A. M. Kamal El-Dean, Bull. Polish Acad.
Sci. 1997, 45, 831.
16. A. M. Kamal El-Dean, Monatsh. Chem. 1998, 129, 523.
17. S. M. Radwan, A. M. Kamal El-Dean, Phosphorus Sulfur
Silicon Relat. Elem. 2000, 164, 299.
23. A. M. Kamal El-Dean, S. M. Radwan, R. M. Zaki, Eur.
J. Med. Chem. 2011, 46, 567.
24. R. M. Zaki, S. M. Radwan, A. M. Kamal El-Dean,
J. Chin. Chem. Soc. 2011, 58, 544.
25. R. M. Zaki, A. M. Kamal El-Dean, S. M. Radwan, Afini-
dad 2012, 68, 424.
18. A. M. Kamal El-Dean, R. Shaker, A. Abo El-Hassan,
F. F. Abdel Latief, J. Chin. Chem. Soc. 2004, 51, 335.
19. A. M. Kamal El-Dean, M. S. A. El-Gaby, A. M. Gaber,
H. A. Eyada, A. S. N. Al-Kamali, Phosphorus Sulfur Sili-
con Relat. Elem. 2005, 180, 413.
26. A. M. Kamal El-Dean, R. M. Zaki, A. Y. Abdulrazzaq,
Russ. J. Bioorgan. Chem. 2015, 41, 112.
27. A. A. Saddik, K. M. Hassan, A. M. Kamal El-Dean,
M. S. Abbady, Eur. Chem. Bull. 2015, 4, 436.
28. S. Kambe, K. Saito, H. Kishi, Synthesis 1979, 4, 287.
29. Z. H. Khalil, A. Abdel Hafez, A. A. Ahmed, Phosphorus
Sulfur Silicon Relat. Elem. 1989, 45, 81.
20. A. M. Kamal El-Dean, S. M. Radwan, Y. A. Elosealy,
J. Chem. Res. 2008, 11, 622.
21. A. M. Kamal El-Dean, S. M. Radwan, R. M. Zaki,
J. Chin. Chem. Soc. 2008, 55, 290.
30. A. M. Kamal El-Dean, S. M. Radwan, R. M. Zaki,
J. Chin. Chem. Soc. 2008, 55, 1290.
J. Chin. Chem. Soc. 2016
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