Synthesis and biological evaluation of novel series of thieno[2,3-d] pyrimidine derivatives as anticancer and antimicrobial agents

Article abstract of DOI:10.1007/s00044-012-0324-3

The present study is concerned with the synthesis, anticancer and antimicrobial screening of several new 3-substituted or 2,3-disubstituted-5- methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamide derivatives. Three compounds (4b, 8c, and 11b) were selected by the National Cancer Institute and were first evaluated at a single-dose primary anticancer assay against 60 human cancer cell lines for their in vitro anticancer activity. Compound 8c which passed the criteria for activity in this assay was evaluated against the full panel of 60 human cancer cell lines at five concentrations at tenfold dilutions where it showed non-selective broad-spectrum activity against all cancer cell lines. Furthermore, compounds 4b, 6, 8c, 8d, and 16 showed pronounced antibacterial activities comparable to ampicillin against P. aeruginosa. Therefore, it was concluded that compound 8c may serve as a useful lead compound in search for powerful dual anticancer-antimicrobial agents.

Full text of DOI:10.1007/s00044-012-0324-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0324-3
ORIGINAL RESEARCH
Synthesis and biological evaluation of novel series of thieno[2,3-d]
pyrimidine derivatives as anticancer and antimicrobial agents
•
Nargues S. Habib Raafat Soliman
•
•
•
Alaa A. El-Tombary Soad A. El-Hawash
Omaima G. Shaaban
Received: 2 November 2011 / Accepted: 6 November 2012
Ó Springer Science+Business Media New York 2012
Abstract The present study is concerned with the syn-
thesis, anticancer and antimicrobial screening of several
new 3-substituted or 2,3-disubstituted-5-methyl-4-oxo-3,4-
biological activities which includes antimicrobial (Eissa
and Moneer, 2004; Omar et al., 2005), antiviral (Nasr and
Gineinah, 2002; Shehata et al., 1996), and anticancer
(Shehata et al., 1996; Jennings et al., 2005) activities.
Some thienopyrimidine derivatives have been reported to
display good activity as phosphodiesterase (Chakraborti
et al., 2003), dihydrofolate reductase (DHFR) (Donkor
et al., 2003), in addition to vascular endothelial growth
factor (VEGF) kinase inhibitors (Munchhof et al., 2004).
The increasing number of neoplastic diseases together
with the accompanied high mortality rates (Eckhardt, 2002)
has stimulated the search for new structure leads that might
be of use in designing novel anticancer drugs.
dihydrothieno[2,3-d]pyrimidine-6-carboxamide
deriva-
tives. Three compounds (4b, 8c, and 11b) were selected by
the National Cancer Institute and were first evaluated at a
single-dose primary anticancer assay against 60 human
cancer cell lines for their in vitro anticancer activity.
Compound 8c which passed the criteria for activity in this
assay was evaluated against the full panel of 60 human
cancer cell lines at five concentrations at tenfold dilutions
where it showed non-selective broad-spectrum activity
against all cancer cell lines. Furthermore, compounds 4b,
6, 8c, 8d, and 16 showed pronounced antibacterial activi-
ties comparable to ampicillin against P. aeruginosa.
Therefore, it was concluded that compound 8c may serve
as a useful lead compound in search for powerful dual
anticancer-antimicrobial agents.
Chemotherapy medicines usually target cells that
quickly divide. Therefore, the most common side effects of
chemotherapy are linked to their effects on other fast
growing cells including those found in the bone marrow,
hair follicles and the lining of the gastrointestinal tract.
Patients suffering from neoplastic disorders and receiv-
ing chemotherapeutic treatment are more likely to get
microbial infections due to subsequent depression of the
immune system (Roche, 2008).
Keywords Thieno[2,3-d]pyrimidine ꢀ Synthesis ꢀ
Anticancer ꢀ Antimicrobial
Multi-drug treatment of neoplastic conditions associated
with microbial infections might inflect some added health
problems especially in patients with impaired liver and/or
kidney functions (Williams, 2008; Ritter et al., 2008).
Hence, mono-therapy by a single drug having both
anticancer and antimicrobial activities with minimum
adverse effects and high safety margin might be advanta-
geous from both therapeutic and cost-effective standpoints.
Interest in this field has been intensified after the dis-
covery of the natural pyrazole C-glycoside pyrazofurin;
4-hydroxy-3-b-D-ribofuranosyl-1H-pyrazole-5 carboxam-
ide. This antibiotic was reported to possess a broad spec-
trum of antimicrobial and antiviral activities in addition to
Introduction
Thienopyrimidines, being bioisostere and structural ana-
logs of the natural purines, occupy a special position
among condensed pyrimidines. Thienopyrimidine deriva-
tives are characterized by a very broad spectrum of
N. S. Habib ꢀ R. Soliman ꢀ A. A. El-Tombary (&) ꢀ
S. A. El-Hawash ꢀ O. G. Shaaban
Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Alexandria University, Alexandria 21521, Egypt
e-mail: alaaeltombary@yahoo.com
123

Med Chem Res
being active against several tumor cell lines (Comber et al.,
1991).
spectrometer (300 MHz) at the microanalytical unit, Faculty
of Science, Cairo University, or on Jeol (500 MHz) at the
microanalytical unit, Faculty of Science, Alexandria Uni-
versity, using DMSO-d6 as a solvent and TMS as internal
standard. The chemical shifts are given in ppm d values (s,
singlet; d, doublet; t, triplet and m, multiplet). 13C-NMR
spectra were determined on Jeol (125 MHz), Faculty of
Science, Alexandria University, using TMS as internal
standard. Mass spectra were run on a Finnigan mass spec-
trometer model SSQ/7000 (70 eV), Faculty of Science,
Cairo University. Microanalyses were performed at the
microanalytical unit, Faculty of Science, Cairo University
and the microanalytical unit, Faculty of Pharmacy, Alexan-
dria University. The results of the microanalyses were
within 0.4 % of the calculated values. Follow-up of the
reactions and checking the homogeneity of the compounds
were made by ascending TLC run on silica gel G (Merck 60)
coated glass plates. The spots were visualized by exposure to
iodine vapor or UV lamp at k 254 nm for few seconds.
Compound 1 was prepared according to the reported
method (Gewald et al., 1966) using cyanoacetamide, eth-
ylcyanoacetate in the presence of sulfur and morpholine.
Since the combination of two pharmacophores on the
same scaffold is a well-established approach to the synthesis
of more potent drugs (Pillai et al., 2003; Venkatachalam
et al., 2006), and based on the above observations in addition
to our new finding on tetrahydrobenzothieno[2,3-d]pyrimi-
dines as antimicrobial agents (Soliman et al., 2009) and in
continuation of our research program on the synthesis of
novel bioactive heterocyclic compounds (El-Hawash et al.,
1999; Habib and El-Hawash, 2005; Soliman et al., 2003;
Habib et al., 2003; Bekhit et al., 2004; El-Hawash et al.,
2006), the present work is concerned with the synthesis of
several 3-substituted or 2,3-disubstituted-5-methyl-4-oxo-
3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamide deriva-
tives hoping to obtain compounds with dual anticancer
and antimicrobial activities. Substituents used in these
compounds include alkyl, aralkyl, phenyl, amino, sulfanyl,
thioether such as phenyl or 4-substituted phenyl carbamoylm-
ethylthio, 2-oxopropylthio, ethyloxycarbonylmethylthio, alkyl/
aralkylthio, 2,3-epoxypropylthio, 3-substituted-2-hydro-
xypropylthio, in addition to sulfones and thiouredo moieties.
It is well documented that these pharmacophoric groups
impart various electronic and lipophilic properties to the
original compounds, thus may contribute to the enhancement
of the anticancer and/or antimicrobial activities of the
thienopyrimidine nucleus. In particular, incorporation of
thioether (Zhao et al., 2009; Patil et al., 2010), 2,3-epoxy-
propylthio (Raic-Malic et al., 1999; Al-Smadi and Al-Mo-
mani, 2008), 3-substituted-2-hydroxypropylthio (Raic-Malic
et al., 1999; Du et al., 2005), sulfones (Patil et al., 2010;
Haruna et al., 2006) and thiouredo (Hallur et al., 2006;
Cunha et al., 2007) moieties into different heterocyclic
templates have been reported to produce compounds with
anticancer and antimicrobial activities. Furthermore, various
thiazoles and thiazolidinones possess anticancer (Kutschy
et al., 2009; Ottana et al., 2005) and antimicrobial activities
(Omar et al., 2005; Bondock et al., 2007). This encouraged
us to incorporate dihydrothiazole and thiazolidinone moie-
ties at the 3-position of thienopyrimidine nucleus hoping to
go a step forward in the field of anticancer and antimicrobial
agents.
5-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-
carboxamide (2)
A mixture of1 (2.28 g, 10 mmol) and formamide(10 ml) was
refluxed for 3 h. The reaction mixture was set aside overnight
at room temperature. The separated solid was filtered, washed
with water, and crystallized from ethanol/water to yield 1.6 g
(76 %) as a white solid, mp 298–299 °C; IR (KBr, t, cm-1):
3419, 3200, 3108 (NH), 1630 (C=O), 1575, 1494 (C=N, C=C,
d NH), 1400, 1342 (C–N lactam), 1242,1027 (C–S–C); 1H-
NMR (300 MHz, DMSO-d6) d (ppm): 2.80 (s, 3H, CH3), 7.60
(s, 2H, NH2, D2O exchangeable), 8.13 (s, 1H, thienopyrimi-
dine C2–H), 12.47 (s, 1H, NH, D2O exchangeable); EI-MS
m/z (relative abundance %): 211 [M??2] (5), 210 [M??1]
(13), 209 [M?] (84), 208 (94), 195 (5), 194 (11), 193 (87), 192
(100), 191 (33), 181 (6), 165 (21), 164 (29), 163 (23), 138 (10),
137 (12), 136 (10), 111 (10), 110 (20), 109 (23), 108 (15), 94
(20), 93 (19), 92 (10), 84 (7), 83 (15), 82 (20), 81 (16), 71 (13),
70 (16), 69 (22), 68 (24). Anal. Calcd. for C8H7N3O2S
(209.23): C, 45.92; H, 3.37; N, 20.08. Found: C, 45.77; H,
3.36; N, 20.04.
Materials and methods
5-Methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-
carboxamide potassium salt (3)
Chemistry
Melting points were determined in open glass capillaries on a
Gallen-Kamp melting point apparatus and were uncorrected.
IR spectra were recorded, for potassium bromide discs, t
(cm-1), on Perkin Elmer 1430 spectrophotometer. 1H-NMR
spectra were determined either on a Bruker Avance
Compound 2 (2.09 g, 10 mmol) was suspended in absolute
ethanol (30 ml) and treated with alcoholic potassium
hydroxide (0.56 g, 10 mmol in absolute ethanol (10 ml)).
The reaction mixture was stirred for 24 h at room tem-
perature. The potassium salt formed was filtered, washed
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Med Chem Res
with absolute ethanol, and dried to yield 2.09 g (85 %) as a
yellowish-white solid, mp [300 °C. The obtained product
was used directly in the next reaction.
(C=N), 1600, 1552 (C=C), 1573 (d NH), 1436, 1357 (C–N
lactam), 1265, 1043 (C–S–C); 1H-NMR (300 MHz,
DMSO-d6) d (ppm): 2.73 (s, 3H, CH3), 3.64 (q, 2H,
J = 5.4 Hz, CH2CH2OH), 4.02 (t, 2H, J = 5.4 Hz,
CH2CH2OH), 4.93 (t, 1H, J = 5.4 Hz, OH, D2O
exchangeable), 7.62 (s, 2H, NH2, D2O exchangeable), 8.31
(s, 1H, thienopyrimidine C2-H). Anal. Calcd. for
C10H11N3O3S (253.28): C, 47.42; H, 4.37; N, 16.59.
Found: C, 47.22; H, 4.36; N, 16.53.
3-Benzyl-5-methyl-4-oxo-3,4-dihydrothieno[2,3-
d]pyrimidine-6-carboxamide (4a)
Method A: A suspension of the potassium salt 3 (2.48 g,
10 mmol) in dry DMF (10 ml) was treated dropwise with
stirring for 15 min with benzyl chloride (10 mmol). The
reaction mixture was refluxed for 3 h, poured on crushed ice.
The obtained solid was filtered, dried, and crystallized from
ethanol to give pale yellow clusters in 65 % yield, mp
216–218 °C; IR (KBr, t, cm-1): 3330, 3174 (NH), 1685
(C=O), 1635 (C=N, C=C), 1571 (d NH), 1407, 1369 (C–N
lactam), 1259, 1072 (C–S–C); 1H-NMR (300 MHz, DMSO-
d6) d (ppm): 2.67 (s, 3H, CH3), 5.14 (s, 2H, CH2), 7.24–7.26
(m, 2H, C6H5 C2,6-H), 7.27–7.32 (m, 3H, C6H5 C3,4,5-H), 7.65
(s, 2H, NH2, D2O exchangeable), 8.65 (s, 1H, thienopyrimi-
dineC2-H).Anal. Calcd. forC15H13N3O2S(299.35):C,60.19;
H, 4.38; N, 14.04. Found: C, 60.15; H, 4.38; N, 13.99.
5-Methyl-4-oxo-3-propyl-3,4-dihydrothieno[2,3-
d]pyrimidine-6-carboxamide (4c)
Yellowish white crystals, yield 82 % (ethanol), mp
237–239 °C; IR (KBr, t, cm-1): 3427, 3379 (NH), 1676
(C=O), 1645 (C=N), 1573 (d NH), 1500 (C=C), 1406, 1367
1
(C–N lactam), 1265, 1099 (C–S–C); H-NMR (500 MHz,
DMSO-d6) d (ppm): 0.84 (t, 3H, J = 7.6 Hz, CH2CH2CH3),
1.64 (sextet, 2H, J = 7.6 Hz, CH2CH2CH3), 2.69 (s, 3H, C5–
CH3), 3.88 (t, 2H, J = 7.6 Hz, CH2CH2CH3), 7.64 (s, 2H,
NH2, D2O exchangeable), 8.44 (s, 1H, thienopyrimidine C2-
H). Anal. Calcd. for C11H13N3O2S (251.30): C, 52.57; H,
5.21; N, 16.72. Found: C, 52.39; H, 5.21; N, 16.68.
Method B: A mixture of 1 (0.23 g, 1 mmol) and tri-
ethylorthoformate (5 ml) was heated for 3 h under reflux.
The reaction mixture was concentrated under vacuum.
Benzyl amine (2 ml) was added, and the mixture was
stirred for 3 h at 40 °C. Aqueous ethanol (1:1) was added
to the reaction mixture, and the precipitated crystals were
filtered, dried and recrystallized from ethanol to give pale
yellow clusters in 83 % yield, mp 216–219 °C.
3-Cyclohexyl-5-methyl-4-oxo-3,4-dihydrothieno[2,3-
d]pyrimidine-6-carboxamide (4d)
White needles, yield 79 % (ethanol), mp 260–262 °C; IR
(KBr, t, cm-1): 3434, 3275 (NH), 1644 (C=O), 1583, 1480
(C=N, C=C), 1531 (d NH), 1407, 1325 (C–N lactam), 1295,
The products obtained from both methods were identical
1
as revealed by mp, mixed mp and superimposability of IR
1
and H NMR spectra. However, method B was superior to
1088 (C–S–C); H-NMR (500 MHz, DMSO-d6) d (ppm):
1.01–1.38 (m, 6H, cyclohexyl C3,4,5-H), 1.61–1.81 (m, 4H,
cyclohexyl C2,6-H), 2.68 (s, 3H, C5-CH3), 4.56–4.61 (m, 1H,
cyclohexyl C1-H), 7.65 (s, 2H, NH2, D2O exchangeable), 8.49
(s, 1H, thienopyrimidine C2-H); 13C-NMR (normal/DEPT-
135)(125 MHz, DMSO-d6) d (ppm): 15.47 (?ve, CH3), 25.17
(-ve, cyclohexyl C4), 26.14 (-ve, cyclohexyl C3,5), 32.05
(-ve, cyclohexyl C2,6), 53.22 (?ve, cyclohexyl C1), 122.83
(ab, C4a), 129.34 (ab, C6), 136.97 (ab, C5), 147.93 (?ve, C2),
157.80(ab,C4),163.05(ab,CONH2),164.14(ab,C7a);EI-MS
m/z (relative abundance %): 292 [M??1](23), 291 [M?] (40),
290 [M?-1] (11), 211 (16), 210 (66), 209 (99), 208 (18), 195
(8), 194 (17), 193 (100), 192 (60), 191 (11), 166 (8), 165 (12),
164 (13), 153 (7), 137 (8), 136 (7), 110 (7), 109 (12), 83 (8), 81
(11), 79 (11), 69 (8), 58 (17), 55 (53), 54 (27), 53 (16). Anal.
Calcd.forC14H17N3O2S(291.37):C,57.71;H, 5.88;N,14.42.
Found: C, 57.72; H, 5.89; N, 14.47.
method A concerning the yield of the prepared compound.
General procedure for the synthesis of 3-substituted-5-
methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-
carboxamides (4b–e)
Compounds 4b–e were synthesized following the above
procedure under method (B) for preparation of 4a, by
heating a mixture of 1 (0.23 g, 1 mmol) and triethylor-
thoformate (5 ml) under reflux for 3 h. The reaction mix-
ture was concentrated under vacuum. The appropriate
amine (2 ml) was added, and the mixture was stirred for
3 h at 40 °C. Aqueous ethanol (1:1) was added to the
reaction mixture and the precipitated crystals were filtered,
dried, and recrystallized from the appropriate solvent.
3-(2-Hydroxyethyl)-5-methyl-4-oxo-3,4-dihydrothieno-
[2,3-d]pyrimidine-6-carboxamide (4b)
5-Methyl-4-oxo-3-(pyridin-2-yl)-3,4-dihydrothieno[2,3-
d]pyrimidine-6-carboxamide (4e)
White clusters, yield 85 % (ethanol), mp 233–234 °C; IR
(KBr, t, cm–1): 3427, 3379 (OH, NH), 1670 (C=O), 1618
Yellowish white crystals, yield 80 % (DMF/ethanol), mp
240–241 °C; IR (KBr, t, cm-1): 3433, 3159 (NH), 1691
123

Med Chem Res
General procedure for the synthesis of 2,5-dimethyl-3-N-
substituted thiocarbamoylamino-4-oxo-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxamides (7a, b)
(C=O), 1611, 1517 (C=N, C=C), 1562 (d NH), 1438, 1347
(C–N lactam), 1282, 1096 (C–S–C); H-NMR (500 MHz,
1
DMSO-d6) d (ppm): 2.70 (s, 3H, CH3), 7.56 (t, 1H,
J = 6.85 Hz, pyridine C5-H), 7.72 (s, 2H, NH2, D2O
exchangeable), 7.75 (d, 1H, J = 8.4 Hz, pyridine C3-H),
8.03 (t, 1H, J = 7.6 Hz, pyridine C4-H), 8.57 (s, 1H, thi-
enopyrimidine C2-H), 8.62 (d, 1H, J = 4.6 Hz, pyridine
C6-H). Anal. Calcd. for C13H10N4O2S (286.31): C, 54.53;
H, 3.52; N, 19.56. Found: C, 54.36; H, 3.51; N, 19.51.
A solution of the aminopyrimidine derivative 6 (2.38 g,
10 mmol) in absolute ethanol (25 ml) was treated with the
selected isothiocyanate derivative (10 mmol) and heated
under reflux for 7–10 h. The reaction mixture was con-
centrated under vacuum and left to cool to room temper-
ature. The deposited product was filtered and crystallized
from ethanol.
Ethyl 2-acetamido-5-carbamoyl-4-methylthiophene-3-
carboxylate (5)
2,5-Dimethyl-3-N-phenylthiocarbamoylamino-4-oxo-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxamide (7a)
A mixture of 1 (0.23 g, 1 mmol) and acetic anhydride
(5 ml) was heated under reflux for 3 h. The reaction mix-
ture was cooled, poured onto ice, and the separated solid
was filtered and crystallized from ethanol to yield 0.21 g
(77 %) as a white needles, mp 196–198 °C; IR (KBr, t,
cm-1): 3253 (NH), 1683 br (C=O), 1544, 1505 (C=N,
C=C, d NH), 1410, 1305 (C–N lactam), 1244, 1110, 1028
(C–S–C) and (C–O–C); 1H-NMR (500 MHz, DMSO-d6) d
(ppm): 1.41 (t, 3H, J = 6.9 Hz, CH2–CH3), 1.6 (s, 1H, NH,
D2O exchangeable), 2.31 (s, 3H, CO–CH3), 2.56 (s, 3H,
CH3), 4.39 (q, 2H, J = 6.9 Hz, CH2–CH3), 11.57 (s, 2H,
NH2, D2O exchangeable); EI-MS m/z (relative abundance
%): 270 [M?] (3), 252 (19), 251 (23), 229 (8), 228 (10),
211 (9), 210 (56), 209 (60), 184 (5), 183 (15), 182 (16), 181
(5), 166 (8), 165 (18), 164 (81), 163 (100), 154 (5), 153 (5),
139 (8), 138 (9), 137 (11), 136 (13), 110 (9), 109 (16), 108
(6), 94 (5), 82 (6), 71 (5), 70 (15), 69 (14), 68 (7), 67 (13),
66 (43), 65 (40), 64 (40), 63 (5), 59 (5), 58 (5). Anal. Calcd.
for C11H14N2O4S (270.30): C, 48.88; H, 5.22; N, 10.36.
Found: C, 48.69; H, 5.22; N, 10.33.
Yellowish-brown microcrystalline powder, yield 83 %, mp
190–192 °C; IR (KBr, t, cm-1): 3393, 3294, 3259 (NH),
1674 (C=O), 1599, 1481 (C=N, C=C), 1527 (d NH), 1428,
1328 (C–N lactam), 1599, 1263, 1180, 1023 (NCS amide I,
II, III and IV mixed vibrational bands, respectively), 1066
1
(C–S–C); H-NMR (500 MHz, DMSO-d6) d (ppm): 2.33
(s, 3H, thienopyrimidine C2-CH3), 2.47 (s, 3H, thienopyr-
imidine C5-CH3), 6.96 (t, 1H, J = 7.6 Hz, C6H5 C4-H),
7.39 (t, 2H, J = 7.6 Hz, C6H5 C3,5-H), 7.56 (d, 2H,
J = 7.6 Hz, C6H5 C2,6-H), 8.11 (s, 2H, NH2, D2O
exchangeable), 10.5 (s, 1H, NH-C6H5, D2O exchangeable),
10.75 (s, ‘ H, thienopyrimidine-NH, D2O exchangeable),
11.04 (s, ‘ H, SH, D2O exchangeable); EI-MS m/z (rela-
tive abundance %): 373 [M?] (absent), 232 (88), 121 (49),
111 (29), 110 (46), 105 (21), 103 (9), 102 (36), 92 (31), 77
(100), 76 (49), 75 (22), 70 (32), 69 (35), 66 (48), 65 (48).
Anal. Calcd. for C16H15N5O2S2 (373.45): C, 51.46; H,
4.05; N, 18.75. Found: C, 51.37; H, 4.04; N, 18.69.
3-N-Benzylthiocarbamoylamino-2,5-dimethyl-4-oxo-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxamide (7b)
3-Amino-2,5-dimethyl-4-oxo-3,4-dihydrothieno[2,3-
d]pyrimidine-6-carboxamide (6)
Yellow needles, yield 82 %, mp 220–222 °C; IR (KBr, t,
cm-1): 3273 br (NH), 1640 (C=O), 1586, 1470 br (C=N,
C=C, d NH), 1526, 1222, 1171, and 961 (NCS amide I, II,
III, and IV mixed vibrational bands, respectively), 1335
(C–N lactam), 1070 (C–S–C); 1H-NMR (300 MHz,
DMSO-d6) d (ppm): 2.50 (s, 3H, thienopyrimidine C2-
CH3), 2.51 (s, 3H, thienopyrimidine C5-CH3), 4.73 (d, 2H,
J = 5.7 Hz, CH2), 7.20–7.23 (m, 5H, CH2-C6H5), 7.77 (s,
2H, NH2, D2O exchangeable), 8.44 (s, 1H, NH-CH2-C6H5,
D2O exchangeable), 9.27 (s, 1H, thienopyrimidine-NH,
D2O exchangeable). Anal. Calcd. for C17H17N5O2S2
(387.48) : C, 52.69; H, 4.42; N, 18.07. Found: C, 52.47; H,
4.42; N, 18.03.
A mixture of 5 (2.7 g, 10 mmol) and hydrazine hydrate
99 % (15 ml, 30 mmol) was dissolved in absolute ethanol
(5 ml). The mixture was refluxed for 6-8 h. The product
obtained after cooling was filtered, washed with water, and
crystallized from ethanol to yield 1.9 g (79 %) as a yellow
needles, mp 172–174 °C; IR (KBr, t, cm-1): 3393, 3293,
3259 (NH), 1674 (C=O), 1599, 1480 (C=N, C=C), 1526 (d
NH), 1428, 1328 (C–N lactam), 1262, 1179, 1097 (C–S–
1
C); H-NMR (300 MHz, DMSO-d6) d (ppm): 2.37 (s, 6H,
2 9 CH3), 2.5 (s, 2H, 3-NH2, D2O exchangeable), 8.11 (s,
2H, CONH2, D2O exchangeable). Anal. Calcd. for
C9H10N4O2S (238.27): C, 45.37; H, 4.23; N, 23.51. Found:
C, 45.21; H, 4.22, N, 23.42.
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Med Chem Res
General procedure for the synthesis of 3-(3-aryl/aralkyl-4-
substituted phenylthiazol-2(3H)-ylideneamino)-2,5-
dimethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-
carboxamides (8a–e)
(C=N, C=C), 1529 (d NH), 1405 (C–N lactam), 1254,
1175, 1072 (C–S–C), 761 (C–Br); 1H-NMR (300 MHz,
DMSO-d6) d (ppm): 2.15 (s, 3H, thienopyrimidine C2-
CH3), 2.50 (s, 3H, thienopyrimidine C5-CH3), 6.53 (s, 1H,
thiazoline C5-H), 7.00 (d, 2H, J = 8.1 Hz, 4-Br-C6H4 C2,6-
H), 7.12 (t, 1H, J = 8.4 Hz, N-C6H5 C4-H), 7.38 (t, 2H,
J = 8.4 Hz, N-C6H5 C3,5-H), 7.48 (d, 2H, J = 8.4 Hz,
A solution of the appropriate thiourea derivative 7a or
b (10 mmol) in absolute ethanol (20 ml) was treated with
equivalent amount of the selected phenacyl bromide. The
reaction mixture was heated under reflux for 6–8 h and left to
cool to room temperature. The deposited product was fil-
tered, washed with aqueous ethanol, and crystallized from
ethanol.
N-C6H5 C2,6-H), 7.67 (d, 2H, J = 8.1 Hz, 4-Br-C6H4 C3,5
H), 7.81 (s, 2H, NH2, D2O exchangeable). Anal. Calcd. for
C24H18BrN5O2S2 (552.47): N, 12.68. Found: N, 12.65.
-
3-(3-Benzyl-4-phenylthiazol-2(3H)-ylideneamino)-2,5-
dimethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-
carboxamide (8c)
3-(4-(4-Chlorophenyl)-3-phenylthiazol-2(3H)-
ylideneamino)-2,5-dimethyl-4-oxo-3,4-dihydrothieno-
[2,3-d]pyrimidine-6-carboxamide (8a)
White needles, yield 80 %, mp 164–165 °C; IR (KBr, t,
cm-1): 3325, 3280 (NH), 1645 (C=O), 1583, 1418 (C=N,
C=C), 1530 (d NH), 1403, 1326 (C–N lactam), 1245, 1081
(C–S–C); 1H-NMR (300 MHz, DMSO-d6) d (ppm): 2.44 (s,
3H, thienopyrimidine C2-CH3), 2.51 (s, 3H, thienopyrimi-
dine C5-CH3), 5.00 (s, 2H, CH2-C6H5), 6.30 (s, 1H, thiazo-
line C5-H), 7.04 (d, 2H, J = 7.2 Hz, CH2-C6H5 C2,6-H),
7.22–7.42 (m, 8H, Ar–H), 9.8 (s, 2H, NH2, D2O exchange-
able). Anal. Calcd. for C25H21N5O2S2 (487.60): C, 61.58; H,
4.34; N, 14.36. Found: C, 61.44; H, 4.33; N, 14.41.
Dark yellow microcrystalline powder, yield 78 %, mp
216–218 °C; IR (KBr, t, cm-1): 3435, 3278 (NH), 1640
(C=O), 1583, 1480 (C=N, C=C), 1550 (d NH), 1326 (C–N
lactam), 1295, 1088 (C–S–C), 829 (C–Cl); 1H-NMR
(500 MHz, DMSO-d6) d (ppm): 2.11 (s, 3H, thienopyrimi-
dine C2-CH3), 2.46 (s, 3H, thienopyrimidine C5-CH3), 6.55
(s, 1H, thiazoline C5-H), 6.95 (d, 2H, J = 7.6 Hz, N-C6H5
C2,6-H), 7.04 (t, 1H, J = 7.6 Hz, N-C6H5 C4-H), 7.14 (d, 2H,
J = 8.4 Hz, 4-Cl-C6H4 C2,6-H), 7.28 (d, 2H, J = 8.4 Hz,
4-Cl-C6H4 C3,5-H), 7.32 (t, 2H, J = 7.6 Hz, N-C6H5 C3,5
-
3-(3-Benzyl-4-(4-chlorophenyl)thiazol-2(3H)-
ylideneamino)-2,5-dimethyl-4-oxo-3,4-dihydrothieno-[2,3-
d]pyrimidine-6-carboxamide (8d)
H), 7.87 (s, 2H, NH2, D2O exchangeable); 13C-NMR (nor-
mal/DEPT-135)(125 MHz, DMSO-d6) d (ppm): 15.83
(?ve, C2-CH3), 16.86 (?ve, C5-CH3), 95.60 (?ve, thiazo-
line C5), 115.94 (ab, C4a), 117.38 (?ve, C6H5 C2,6), 121.38
(?ve, 4-Cl-C6H4 C2,6), 124.06 (?ve, C6H5 C4), 129.09 (?ve,
4-Cl-C6H4 C3,5), 130.17 (?ve, C6H5 C3,5), 134.42 (ab, 4-Cl-
C6H4 C1), 138.77 (ab, 4-Cl-C6H4 C4), 139.22 (ab, C6),
148.39 (ab, C6H5 C1), 149.27 (ab, C5), 149.92 (ab, thiazoline
C4), 155.65 (ab, thiazoline C2), 158.13 (ab, C4), 161.38 (ab,
CONH2), 163.89 (ab, C2), 164.16 (ab, C7a); EI-MS m/z (rel-
ative abundance %): 510 [M??2], 508 [M?] (absent), 467
(17), 466 (10), 465 (33), 373 (13), 303 (19), 302 (11), 301
(51), 300 (10), 289 (14), 288 (40), 287 (38), 286 (100), 285
(48), 250 (7), 210 (10), 179 (33), 170 (17), 169 (10), 168 (29),
165 (39), 151 (18), 150 (25), 149 (18), 140 (9), 138 (19), 137
(14), 136 (18), 135 (11), 134 (19), 133 (27), 132 (10), 125
(19), 124 (14), 123 (10), 113 (10), 111 (14), 110 (21), 91 (10),
89 (33), 77 (52). Anal. Calcd. for C24H18ClN5O2S2.H2O
(526.03): C, 54.79; H, 3.83. Found: C, 54.77; H, 3.82.
Yellow clusters, yield 76 %, mp 148–150 °C; IR (KBr, t,
cm-1): 3289, 3132 (NH), 1630 (C=O), 1558 br (C=N,
C=C, d NH), 1404, 1321 (C–N lactam), 1260, 1088 (C–S–
1
C), 819 (C–Cl); H-NMR (500 MHz, DMSO-d6) d (ppm):
2.04 (s, 3H, thienopyrimidine C2-CH3), 2.46 (s, 3H, thie-
nopyrimidine C5-CH3), 4.97 (s, 2H, CH2C6H5), 6.36 (s, 1H,
thiazoline C5-H), 7.01 (d, 2H, J = 6.9 Hz, CH2-C6H5 C2,6
-
H), 7.17 (t, 1H, J = 6.9 Hz, CH2-C6H5 C4-H), 7.22 (t, 2H,
J = 6.9 Hz, CH2-C6H5 C3,5-H), 7.29 (d, 2H, J = 8.4 Hz,
4-Cl-C6H4 C2,6-H), 7.42 (d, 2H, J = 8.4 Hz, 4-Cl-C6H4
C3,5-H), 7.49 (s, 2H, NH2, D2O exchangeable). Anal.
Calcd. for C25H20ClN5O2S2 (522.04): C, 57.52; H, 3.86; N,
13.42. Found: C, 57.46; H, 3.86; N, 13.37.
3-(3-Benzyl-4-(4-bromophenyl)thiazol-2(3H)-
ylideneamino)-2,5-dimethyl-4-oxo-3,4-dihydrothieno-[2,3-
d]pyrimidine-6-carboxamide (8e)
3-(4-(4-Bromophenyl)-3-phenylthiazol-2(3H)-
ylideneamino)-2,5-dimethyl-4-oxo-3,4-dihydrothieno
[2,3-d]pyrimidine-6-carboxamide (8b)
Yellow microcrystalline powder, yield 70 %, mp
172–174 °C; IR (KBr, t, cm-1): 3419, 3284 (NH), 1648
(C=O), 1560, 1504 (C=N, C=C, d NH), 1455, 1316 (C–N
lactam), 1247, 1063 (C–S–C), 726 (C–Br); 1H-NMR
Pale yellow crystals, yield 71 %, mp 146–148 °C; IR (KBr,
t, cm-1): 3330, 3289, 3107 (NH), 1640 (C=O), 1581, 1481
(500 MHz, DMSO-d6)
d
(ppm): 2.41 (s, 3H,
123

Med Chem Res
thienopyrimidine C2-CH3), 2.46 (s, 3H, thienopyrimidine
C5-CH3), 4.97 (s, 2H, CH2-C6H5), 6.35 (s, 1H, thiazoline C5-
H), 7.01 (d. dist., 2H, CH2-C6H5 C2,6-H), 7.17 (t, 1H,
J = 6.8 Hz, CH2-C6H5 C4-H), 7.21–7.24 (m, 4H, CH2-C6H5
C3,5-H ? 4-Br-C6H4 C2,6-H), 7.56 (d, 2H, J = 8.4 Hz,
4-Br-C6H4 C3,5-H), 7.49 (s, 2H, NH2, D2O exchangeable).
Anal. Calcd. for C25H20BrN5O2S2 (566.49): C, 53.01; H,
3.56; N, 12.36. Found: C, 53.20; H, 3.56; N, 12.38.
DMSO-d6) d (ppm): 2.39 (s, 3H, thienopyrimidine C2-CH3),
2.51 (s, 3H, thienopyrimidine C5-CH3), 4.14 (s, 2H, CH2-
C6H5), 4.87 (s, 2H, thiazolidinone C5-H), 7.23–7.39 (m, 6H,
Ar–H and thiazolidinone C5-H enolic), 7.56 (s, 2H, NH2,
D2O exchangeable), 10.00 (s, 1H, enolic OH, D2O
exchangeable). Anal. Calcd. for C19H17N5O3S2 (427.50): C,
53.38; H, 4.01. Found: C, 53.19; H, 4.00.
2-Mercapto-5-methyl-4-oxo-3-phenyl-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxamide (10)
General procedure for the synthesis of 3-(4-oxo-3-
substituted thiazolidin-2-ylideneamino)-2,5-dimethyl-4-
oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamides
(9a, b)
A mixture of 1 (2.28 g, 10 mmol) and phenylisothiocyanate
(1.35 g, 1.19 ml, 10 mmol) in acetonitrile (30 ml) was
heated under reflux for 15 h in the presence of anhydrous
potassium carbonate (1.4 g, 10 mmol). The reaction mixture
was filtered. The residue obtained was dissolved in water,
and neutralized with acetic acid; the obtained crude product
was filtered, washed with water, dried, and crystallized from
ethanol to yield 2.5 g (78 %) as a yellow amorphous powder,
mp [300 °C; IR (KBr, t, cm-1): 3421, 3347, 3323, 3141
(NH), 2700 (SH), 1698, 1653 (C=O), 1569, 1496 (C=N,
C=C), 1537 (d NH), 1412 (C–N lactam), 1224, 1090 (C–S–
To a solution of the appropriate thiourea derivative 7a or
b (10 mmol) in absolute ethanol (20 ml), ethyl bromoacetate
(1.8 g, 1.1 ml, 10 mmol) was added. The reaction mixture
was heated under reflux for 6 h and left to cool to deposit the
product which was filtered and crystallized from ethanol.
3-(4-Oxo-3-phenylthiazolidin-2-ylideneamino)-2,5-
dimethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-
carboxamide (9a)
1
C); H-NMR (500 MHz, DMSO-d6) d (ppm): 3.55 (s, 3H,
CH3), 7.19 (d, 2H, J = 7.6 Hz, C6H5 C2,6-H), 7.36 (t, 1H,
J = 7.6 Hz, C6H5 C4-H), 7.43 (t, 2H, J = 7.6 Hz, C6H5
C3,5-H), 7.60 (br s, 2H, NH2, D2O exchangeable), 7.90 (s,
1H, OH, imidol, D2O exchangeable), 13.00 (s, 1H, SH, D2O
exchangeable); 13C-NMR (normal/DEPT-135)(125 MHz,
DMSO-d6) d (ppm): 14.83 (?ve, CH3), 117.69 (ab, C4a),
125.66 (ab, C6H5 C1), 128.68 (?ve, C6H5 C4), 129.48 (?ve,
C6H5 C2,6), 129.52 (?ve, C6H5 C3,5), 137.67 (ab, C6), 139.67
(ab, C5), 152.01 (ab, C4), 157.98 (ab, CONH2), 163.95 (ab,
C2), 175.97 (ab, C7a). Anal. Calcd. for C14H11N3O2S2
(317.39): C, 52.98; H, 3.49. Found: C, 53.01; H, 3.50.
Brownish clusters yield 89 %, mp 260–262 °C; IR (KBr, t,
cm-1): 3388, 3281 (NH), 1740, 1637 (C=O), 1601, 1481 (C=N,
C=C), 1559 (d NH), 1445, 1356 (C–N lactam), 1245, 1033 (C–
S–C); 1H-NMR (500 MHz, DMSO-d6) d (ppm): 2.44 (s, 3H,
thienopyrimidine C2-CH3), 2.46 (s, 3H, thienopyrimidine C5-
CH3), 4.09 (s, 2H, thiazolidinone C5-H), 4.21 (s, 1 H, thiazo-
lidinone C5-H, enol form), 6.96 (t, 1H, J = 7.6 Hz, C6H5 C4-
H), 7.32 (t, 2H, J = 7.6 Hz, C6H5 C3,5-H), 7.55 (d, 2H,
J = 7.6 Hz, C6H5 C2,6-H), 8.01 (s, 2H, NH2, D2O exchange-
able), 10.53 (s, 1 H, enolic OH, D2O exchangeable); 13C-NMR
(normal/DEPT-135)(125 MHz, DMSO-d6) d (ppm): 16.78
(?ve, C2-CH3), 16.84 (?ve, C5-CH3), 60.10 (-ve, thiazolidi-
none C5), 85.64 (?ve, thiazolidinone C5, enol form), 99.72 (ab,
thiazolidinone C4, enol form), 116.07 (ab, C4a), 117.38 (?ve,
C6H5 C2,6), 122.30 (?ve, C6H5 C4), 129.67 (?ve, C6H5 C3,5),
139.20 (ab, C6), 148.40 (ab, C6H5 C1), 153.11 (ab, C5), 155.65
(ab, thiazolidinone C2), 157.37 (ab, CONH2), 158.37 (ab, C4),
161.39 (ab, C2), 163.10 (ab, C7a), 173.02 (ab, thiazolidinone
C4). Anal. Calcd. for C18H15N5O3S2 (413.47): C, 52.29; H,
3.66. Found: C, 52.15; H, 3.65.
General procedure for the synthesis of 2-(phenyl or 4-
substituted phenylcarbamoylmethylthio)-5-methyl-4-
oxo-3-phenyl-3,4-dihydrothieno[2,3-d]pyrimidine-6-
carboxamides (11a–c)
A mixture of 10 (0.32 g, 1 mmol) and the appropriate
chloroacetanilide (1 mmol) was refluxed in dry acetone
(20 ml) for 3 h in the presence of anhydrous potassium
carbonate (0.14 g, 1 mmol). The mixture was cooled,
poured into ice-cold water, filtered, and the solid obtained
was dried and crystallized from ethanol.
3-(3-Benzyl-4-oxothiazolidin-2-ylideneamino)-2,5-
dimethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-
carboxamide (9b)
2-(Phenylcarbamoylmethylthio)-5-methyl-4-oxo-3-phenyl-
3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamide (11a)
Pale yellow microcrystalline powder, yield 85 %, mp
244–245 °C; IR (KBr, t, cm-1): 3400, 3350 (NH), 1707,
1680 (C=O), 1620 (C=N), 1585 (d NH), 1500 (C=C), 1323
Yellow clusters, yield 83 %, mp 284–285 °C; IR (KBr, t,
cm-1): 3404, 3274, 3205 (NH), 1662 (C=O), 1600, 1510 (C=N,
C=C), 1544 (d NH), 1433 (C–N lactam), 1228, 1078 (C–S–C);
1
(C–N lactam), 1263, 1074 (C–S–C); H-NMR (300 MHz,
123

Med Chem Res
1H-NMR (300 MHz, DMSO-d6) d (ppm): 2.68 (s, 3H, CH3),
4.10 (s, 2H, CH2), 7.05 (t, 1H, J = 7.5 Hz, NH–C6H5 C4-H),
7.33 (t, 2H, J = 7.5 Hz, NH–C6H5 C3,5-H), 7.30–7.64 (m, 7H,
Ar–H), 7.54 (s, 2H, NH2, D2O exchangeable), 10.3 (s, 1H, NH,
D2O exchangeable). Anal. Calcd. for C22H18N4O3S2 (450.53):
C, 58.65; H, 4.02; N, 12.43. Found: C, 58.48; H, 4.02; N, 12.38.
carbonate (0.21 g, 1.5 mmol) in dry acetone (10 ml) was
heated under reflux for 10 h. The reaction mixture was
cooled, poured into ice-cold water; the precipitate formed
was dried and crystallized from ethanol.
2-(2-Oxopropylthio)-5-methyl-4-oxo-3-phenyl-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxamide (12)
2-(4-Chlorophenylcarbamoylmethylthio)-5-methyl-4-
oxo-3-phenyl-3,4-dihydrothieno[2,3-d]pyrimidine-6-
carboxamide (11b)
Pale yellow crystals, yield 78 %, mp 240–242 °C; IR (KBr,
t, cm-1): 3458, 3312, 3165 (NH), 1732, 1693, 1646
(C=O), 1592, 1509 (C=N, C=C, d NH), 1455, 1304 (C–N
White needles, yield 78 %, mp 292–293 °C; IR (KBr, t,
cm-1): 3364, 3322, 3174 (NH), 1694, 1650 (C=O), 1596,
1500 (C=N, C=C), 1536 (d NH), 1400, 1322 (C–N lactam),
1220, 1158, 1091 (C–S–C), 829 (C–Cl); 1H-NMR (500 MHz,
DMSO-d6) d (ppm): 2.62 (s, 3H, C5-CH3), 4.04 (s, 2H, S–
CH2), 7.33 (d, 2H, J = 9.2 Hz, 4-Cl-C6H4 C2,6-H), 7.42–7.44
1
lactam), 1229, 1171, 1023 (C–S–C); H-NMR (300 MHz,
DMSO-d6) d (ppm): 2.25 (s, 3H, COCH3), 2.66 (s, 3H,
thienopyrimidine C5-CH3), 4.07 (s, 2H, S–CH2), 7.43-7.45
(m, 2H, C6H5 C2,6-H), 7.58–7.61 (m, 3H, C6H5 C3,4,5-H).
Anal. Calcd. for C17H15N3O3S2 (373.45): C, 54.67; H,
4.05; N, 11.25. Found: C, 54.45; H, 4.05; N, 11.22.
(m, 3H, C6H5 C3,4,5-H), 7.54 (d, 2H, J = 8.4 Hz, C6H5 C2,6
-
H), 7.58 (d, 2H, J = 9.2 Hz, 4-Cl-C6H4 C3,5-H), 7.60 (s, 2H,
NH2, D2O exchangeable), 10.46 (s, 1H, NH, D2O exchange-
able). Anal. Calcd. for C22H17ClN4O3S2 (484.98): C, 54.48;
H, 3.53; N, 11.55. Found: C, 54.51; H, 3.53; N, 11.58.
Ethyl 2-(6-carbamoyl-5-methyl-4-oxo-3-phenyl-3,4-
dihydrothieno[2,3-d]pyrimidin- 2-ylthio)acetate (13)
Yellow clusters, yield 87 %, mp 194–196 °C; IR (KBr, t,
cm-1): 3450, 3314, 3163 (NH), 1700, 1692, 1649 (C=O),
1594, 1504 (C=N, C=C, d NH), 1455 (C–N lactam), 1295,
1228, 1152 (C–S–C), 1228, 1046 (C–O–C); 1H-NMR
(500 MHz, DMSO-d6) d (ppm): 1.18 (t, 3H, J = 6.85 Hz,
CH2–CH3), 2.64 (s, 3H, thienopyrimidine C5-CH3), 4.08 (s,
2H, S–CH2), 4.10 (q, 2H, J = 6.85 Hz, CH2–CH3), 7.41 (d,
2H, J = 7.65 Hz, C6H5 C2,6-H), 7.55–7.62 (m, 3H, C6H5
C3,4,5-H), 7.62 (s, 2H, NH2, D2O exchangeable); 13C-NMR
(normal/DEPT-135)(125 MHz, DMSO-d6) d (ppm): 14.74
(?ve, C5-CH3), 15.24 (?ve, CH2CH3) 35.06 (-ve, S–
CH2), 61.68 (-ve, CH2CH3), 120.15 (ab, C4a), 127.77 (ab,
C6H5 C1), 129.79 (?ve, C6H5 C2,6), 130.25 (?ve, C6H5
C3,5), 130.76 (?ve, C6H5 C4), 135.86 (ab, C6), 137.24 (ab,
C5), 158.40 (ab, C4), 160.41 (ab, H2N-CO), 163.07 (ab,
C2), 164.10 (ab, C7a), 168.64 (ab, O–C=O). Anal. Calcd.
for C18H17N3O4S2 (403.48): C, 53.58; H, 4.25; N, 10.41.
Found: C, 53.74; H, 4.26; N, 10.38.
2-(p-Tolylcarbamoylmethylthio)-5-methyl-4-oxo-3-phenyl-
3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamide (11c)
Pale yellow needles, yield 85 %, mp 242–244 °C; IR (KBr,
t, cm-1): 3325, 3181 (NH), 1694, 1650 (C=O), 1599, 1507
(C=N, C=C, d NH), 1410, 1327 (C–N lactam), 1224, 1160,
1
1091 (C–S–C); H-NMR (500 MHz, DMSO-d6) d (ppm):
2.20 (s, 3H, 4-CH3-C6H4), 2.63 (s, 3H, C5-CH3), 4.04 (s, 2H,
S–CH2), 7.06 (d, 2H, J = 7.6 Hz, 4-CH3-C6H4 C3,5-H), 7.39
(t, 1H, J = 8.4 Hz, C6H5 C4-H), 7.43 (d, 2H, J = 7.6 Hz,
4-CH3-C6H4 C2,6-H), 7.53–7.59 (m, 4H, C6H5 C2,3,5,6-H),
7.61 (s, 2H, NH2, D2O exchangeable), 10.23 (s, 1H, NH, D2O
exchangeable); 13C-NMR (normal/DEPT-135)(125 MHz,
DMSO-d6) d (ppm): 15.27 (?ve, C5-CH3), 20.99 (?ve,
4-CH3-C6H4), 38.15 (-ve, S–CH2), 119.65 (?ve, 4-CH3-
C6H5 C2,6), 120.18 (ab, C4a), 127.72 (ab, C6H5 C1), 129.72
(?ve, C6H5 C2,6), 129.84 (?ve, C6H5 C4), 130.20 (?ve,
C6H5 C3,5), 130.67 (?ve, 4-CH3-C6H5 C3,5), 132.99 (ab,
4-CH3-C6H5 C4), 135.99 (ab, 4-CH3-C6H5 C1), 136.82 (ab,
C6), 137.19 (ab, C5), 158.50 (ab, C4), 160.84 (ab, H2N-CO),
163.26 (ab, C2), 164.16 (ab, C7a), 165.51 (ab, CO–NH).
Anal. Calcd. for C23H20N4O3S2 (464.56): C, 59.46; H, 4.34;
N, 12.06. Found: C, 59.41; H, 4.33; N, 12.04.
General procedure for the synthesis of 2-substituted
thio-5-methyl-4-oxo-3-phenyl-3,4-dihydrothieno-[2,3-d]
pyrimidine-6-carboxamides (14a–c)
A solution of 10 (0.32 g, 1 mmol) in 10 % sodium
hydroxide solution (10 ml) was treated gradually with
alkyl or aralkyl halide (CH3I, C2H5I or C6H5–CH2Cl)
(1.5 mmol) over 15 min. The reaction mixture was stirred
for 5 h at ambient temperature. The precipitate formed was
filtered, washed with water, dried, and crystallized from
ethanol.
General procedure for the synthesis of 2-substituted
thio-5-methyl-4-oxo-3-phenyl-3,4-dihydrothieno-
[2,3-d]pyrimidine-6-carboxamides (12, 13)
A mixture of 10 (0.32 g, 1 mmol) and either chloroacetone
or ethyl bromoacetate (1 mmol) and anhydrous potassium
123

Med Chem Res
5-Methyl-2-(methylthio)-4-oxo-3-phenyl-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxamide (14a)
disappeared. The white precipitate formed was filtered, washed
with water, air dried, and crystallized from ethanol.
White needles, yield 85 %, mp 230–232 °C; IR (KBr, t,
cm-1): 3394 (NH), 1680 (C=O), 1635 (C=N), 1564 (d NH),
1509 (C=C), 1402, 1300 (C–N lactam), 1227, 1159, 1080
5-Methyl-2-(methylsulfonyl)-4-oxo-3-phenyl-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxamide (15a)
1
(C–S–C); H-NMR (500 MHz, DMSO-d6) d (ppm): 2.05
Pale yellow needles, yield 63 %, mp 256–258 °C; IR (KBr,
t, cm-1): 3440, 3275, 3187 (NH), 1696, 1655 (C=O), 1598
(C=N, C=C), 1543 (d NH), 1402, 1319 (C–N lactam),
1359, 1149 (SO2), 1269, 1081 (C–S–C); 1H-NMR
(500 MHz, DMSO-d6) d (ppm): 2.46 (s, 3H, SO2-CH3),
2.65 (s, 3H, C5-CH3), 7.40 (t, 1H, J = 7.6 Hz, C6H5 C4),
7.47 (t, 2H, J = 7.6 Hz, C6H5 C3,5-H), 7.52 (d, 2H,
J = 7.6 Hz, C6H5 C2,6-H), 7.83 (s, 2H, NH2, D2O
exchangeable); 13C-NMR (normal/DEPT-135)(125 MHz,
DMSO-d6) d (ppm): 15.20 (?ve, C5-CH3), 41.82 (?ve,
SO2-CH3), 124.64 (ab, C4a), 129.01 (?ve, C6H5 C2,6),
130.33 (?ve, C6H5 C4), 130.55 (?ve, C6H5 C3,5), 132.99
(ab, C6H5 C1), 133.59 (ab, C6), 137.09 (ab, C5), 153.35 (ab,
C4), 158.27 (ab, H2N-CO), 159.58 (ab, C2), 163.64 (ab,
C7a). Anal. Calcd. for C15H13N3O4S2 (363.41): C, 49.57;
H, 3.61; N, 11.56. Found: C, 49.72; H, 3.62; N, 11.61.
(s, 3H, S–CH3), 2.65 (s, 3H, C5-CH3), 7.40 (dd, 2H,
J = 7.2, 2.3 Hz, C6H5 C2,6-H), 7.53–7.54 (m, 3H, C6H5
C3,4,5-H), 7.62 (s, 2H, NH2, D2O exchangeable). Anal.
Calcd. for C15H13N3O2S2 (331.41): C, 54.36; H, 3.95; N,
12.68. Found: C, 54.13; H, 3.94; N, 12.66.
2-(Ethylthio)-5-methyl-4-oxo-3-phenyl-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxamide (14b)
Pale yellow needles, yield 72 %, mp 216–217 °C; IR (KBr,
t, cm-1): 3327, 3267 (NH), 1687 (C=O), 1647 (C=N),
1591, 1504 (C=C, d NH), 1460, 1301 (C–N lactam), 1222,
1
1159, 1095 (C–S–C); H-NMR (500 MHz, DMSO-d6) d
(ppm): 1.19 (t, 3H, J = 7.6 Hz, CH2CH3), 2.63 (s, 3H, C5-
CH3), 3.02 (q, 2H, J = 7.6 Hz, CH2CH3), 7.38 (dd, 2H,
J = 7.6, 1.5 Hz, C6H5 C2,6-H), 7.48-7.55 (m, 3H, C6H5
C3,4,5-H), 7.61 (s, 2H, NH2, D2O exchangeable). Anal.
Calcd. for C16H15N3O2S2 (345.44): C, 55.63; H, 4.38; N,
12.16. Found: C, 55.46; H, 4.37; N, 12.13.
2-(Ethylsulfonyl)-5-methyl-4-oxo-3-phenyl-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxamide (15b)
White needles, yield 65 %, mp 254–255 °C; IR (KBr, t,
cm-1): 3465, 3369 (NH), 1670 (C=O), 1605, 1493 (C=N,
C=C), 1539 (d NH), 1388, 1165 (SO2), 1321 (C–N lactam),
2-(Benzylthio)-5-methyl-4-oxo-3-phenyl-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxamide (14c)
1
1272, 1080 (C–S–C); H-NMR (500 MHz, DMSO-d6) d
(ppm): 1.22 (t, 3H, J = 7.6 Hz, CH2CH3), 2.66 (s, 3H, C5-
CH3), 3.59 (q, 2H, J = 7.6 Hz, CH2CH3), 7.40–7.42 (m,
3H, C6H5 C3,4,5-H), 7.47 (dd, 2H, J = 6.8, 1.5 Hz, C6H5
C2,6-H), 7.82 (s, 2H, NH2, D2O exchangeable). Anal.
Calcd. for C16H15N3O4S2 (377.44): C, 50.91; H, 4.01; N,
11.13. Found: C, 50.97; H, 4.01; N, 11.09.
White shiny crystals, yield 79 %, mp 218–220 °C; IR
(KBr, t, cm-1): 3440, 3247, 3166 (NH), 1693 (C=O), 1606
(C=N), 1566, 1550 (d NH), 1506 (C=C), 1423, 1371 (C–N
lactam), 1271, 1045 (C–S–C); 1H-NMR (300 MHz,
DMSO-d6) d (ppm): 2.68 (s, 3H, C5-CH3), 4.35 (s, 2H,
CH2), 7.23-7.55 (m, 10H, Ar–H), 7.61 (s, 2H, NH2, D2O
exchangeable). Anal. Calcd. for C21H17N3O2S2 (407.51):
C, 61.89; H, 4.20; N, 10.31. Found: C, 61.73; H, 4.10; N,
10.28.
2-(Benzylsulfonyl)-5-methyl-4-oxo-3-phenyl-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxamide (15c)
White crystals, yield 60 %, mp 204–206 °C; IR (KBr, t,
cm-1): 3433, 3266, 3197 (NH), 1685, 1664 (C=O), 1595,
1499 (C=N, C=C), 1539 (d NH), 1411, 1334 (C–N lactam),
1370, 1136 (SO2), 1270, 1078 (C–S–C); 1H-NMR
(500 MHz, DMSO-d6) d (ppm): 2.57 (s, 3H, C5-CH3), 4.44
General procedure for the synthesis of 2-substituted
sulfonyl-5-methyl-4-oxo-3-phenyl-3,4-dihydrothieno[2,3-
d]pyrimidine-6-carboxamides (15a–c)
An aqueous solution of 4 % potassium permanganate was
added dropwise to a stirred solution of the appropriate 14a–c
(0.5 mmol) in glacial acetic acid (5 ml). The addition of
potassium permanganate solution was continued until the
purple color persisted for 30 min. Stirring was maintained at
room temperature for further 2 h. The reaction mixture was left
overnight, then cooled to 5 °C, and saturated sodium sulfite
solution was gradually added with stirring until the brown color
(s, 2H, S–CH2), 7.22 (d, 2H, J = 7.6 Hz, CH2-C6H5 C2,6
-
H), 7.26–7.44 (m, 8H, Ar–H), 7.50 (s, 2H, NH2, D2O
exchangeable); EI-MS m/z (relative abundance %): 440
[M??1] (4), 439 [M?] (9), 376 (9), 375 (21), 374 (58), 301
(18), 298 (8), 286 (7), 285 (20), 284 (81), 283 (5), 212 (5),
182 (12), 167 (36), 119 (5), 110 (18), 109 (6), 103 (7), 92
(15), 91 (100), 90 (10), 89 (5), 82 (5), 77 (20), 66 (16), 65
(27), 64 (9), 63 (7), 52 (6), 51 (15), 50 (8). Anal. Calcd. for
123

Med Chem Res
C21H17N3O4S2 (439.51): C, 57.39; H, 3.90; N, 9.56. Found:
C, 57.17; H, 3.89; N, 9.53.
DMSO-d6) d (ppm): 2.59 (s, 3H, CH3), 2.69, 2.85 (two
singlets, each 1H, SCH2), 3.78 (t, 4H, J = 8.4 Hz, mor-
pholine C3,5-H), 3.79 (t, 4H, J = 8.4 Hz, morpholine C2,6
-
2-[(Oxiran-2-yl)methylthio]-5-methyl-4-oxo-3-phenyl-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxamide (16)
H), 4.13 (dd, 2H, J = 5.8, 5 Hz, N–CH2), 4.41–4.43 (m,
1H, CH), 5.76 (d, 1H, J = 5.35 Hz, OH, D2O exchange-
able), 7.21 (d, 2H, J = 7.60 Hz, C6H5 C2,6-H), 7.39 (t, 1H,
J = 7.60 Hz, C6H5 C4-H), 7.44 (t, 2H, J = 7.60 Hz, C6H5
C3,5-H), 7.59 (br s, 2H, NH2, D2O exchangeable); EI-MS
m/z (relative abundance %): 460 [M?] (absent), 450 (9),
357 (34), 344 (20), 343 (13), 341 (45), 340 (21), 317 (10),
315 (17), 303 (22), 302 (24), 301 (78), 300 (22), 299 (10),
296 (12), 286 (10), 285 (21), 284 (13), 283 (21), 258 (13),
240 (10), 222 (15), 221 (52), 215 (12), 209 (26), 197 (11),
196 (19), 195 (37), 192 (15), 183 (11), 182 (100), 181 (19),
177 (24), 166 (40), 165 (31), 164 (44), 152 (20), 150 (16),
146 (13), 139 (32), 137 (26), 129 (13), 126 (14), 117 (22),
113 (25), 112 (10), 111 (20), 109 (8), 97 (12), 91 (24), 85
(2), 83 (11), 77 (29). Anal. Calcd. for C21H24N4O4S2.H2O
(478.58): C, 52.70; H, 5.48; N, 11.71. Found: C, 52.57; H,
5.48; N, 11.76.
To a stirred solution of the thiol 10 (0.64 g, 2 mmol) and
potassium hydroxide (0.22 g, 4 mmol) in water (50 ml),
epichlorohydrin (0.19 g, 0.16 ml, 2 mmol) was added
dropwise over 10 min. The reaction mixture was stirred at
room temperature for 10 h and the precipitated product was
filtered, washed with water and crystallized from ethanol to
yield 0.6 g (81 %) as a pale yellow amorphous powder, mp
146–148 °C; IR (KBr, t, cm-1): 3369, 3220 (NH), 1681,
1674 (C=O), 1633 (C=N), 1558, 1506, 1488 (C=C), 1539
(d NH), 1434, 1311 (C–N lactam), 1224, 1045, 1026, 771
(C–O–C), 1172, 1072 (C–S–C); 1H-NMR (500 MHz,
DMSO-d6) d (ppm): 2.76 (dd, 1H, J = 15.25, 6.1 Hz,
epoxy CH2-cis H), 2.63 (s, 3H, CH3), 3.51-3.58 (m, 1H,
epoxy CH2-trans H), 4.04–4.09 (m, 2H, S–CH2), 5.09 (dist.
quintet, 1H, epoxy CH), 7.25 (t, 1H, J = 7.65 Hz, C6H5
C4-H), 7.38 (t, 2H, J = 7.65 Hz, C6H5 C3,5-H), 7.52 (s, 2H,
NH2, D2O exchangeable), 7.61 (d, 2H, J = 7.65 Hz, C6H5
C2,6-H); 13C-NMR (normal/DEPT-135)(125 MHz, DMSO-
d6) d (ppm): 15.22 (?ve, C5-CH3), 33.13 (-ve, S–CH2),
33.80 (–ve, epoxy CH2), 42.87 (?ve, epoxy CH), 120.37
(ab, C4a), 128.81 (?ve, C6H5 C4), 129.50 (?ve, C6H5 C2),
129.76 (?ve, C6H5 C6), 130.18 (?ve, C6H5 C3), 130.58
(?ve, C6H5 C5), 130.64 (ab, C6H5 C1), 135.97 (ab, C6),
137.25 (ab, C5), 158.62 (ab, C4), 159.81 (ab, H2N-CO),
163.20 (ab, C2), 164.16 (ab, C7a). Anal. Calcd. for
C17H15N3O3S2.11/2H2O (400.47): C, 50.98; H, 4.53; N,
10.49. Found: C, 50.79; H, 4.51; N, 10.46.
2-(2-Hydroxy-3-(piperidin-1-yl)propylthio)-5-methyl-4-
oxo-3-phenyl-3,4-dihydrothieno[2,3-d]pyrimidine-6-
carboxamide (17b)
Yellow microcrystalline powder, yield 56 % (DMF/water),
mp 202–204 °C; IR (KBr, t, cm-1): 3480 (OH), 3263,
3165 (NH), 1706, 1654 (C=O), 1591, 1575, 1508, 1473
(C=N, C=C), 1558 (d NH), 1419, 1334 (C–N lactam),
1
1271, 1176, 1091 (C–S–C); H-NMR (500 MHz, DMSO-
d6) d (ppm): 1.22-1.60 (m, 6H, piperidine C3,4,5-H),
2.21–2.30 (m, 4H, piperidine C2,6-H), 2.63 (s, 3H, CH3),
3.83 (dd, 2H, J = 14.5, 8.3 Hz, SCH2), 4.16 (dd, 2H,
J = 14.5, 4.05 Hz, N–CH2), 4.44–4.47 (m, 1H, CH), 5.77
(d, 1H, J = 5.1 Hz, OH, D2O exchangeable), 7.23–7.26
(m, 2H, C6H5 C2,6-H), 7.38– 7.50 (m, 3H, C6H5 C3,4,5-H),
7.54 (s, 2H, NH2, D2O exchangeable); 13C-NMR (normal/
DEPT-135)(125 MHz, DMSO-d6) d (ppm): 15.19 (?ve,
C5-CH3), 53.53 (-ve, S–CH2 and piperidine C3,4,5), 54.85
(-ve, piperidine-CH2 and piperidine C2,6), 65.90 (?ve,
CH-OH), 114.76 (ab, C4a), 124.86 (?ve, C6H5 C4), 128.80
(ab, C6H5 C1), 129.46 (?ve, C6H5 C2,3,5,6), 136.38 (ab, C6),
138.20 (ab, C5), 150.52 (ab, C4), 156.53 (H2N-CO), 159.31
(ab, C2), 163.93 (ab, C7a); EI-MS m/z (relative abundance
%): 458 [M?] (absent), 448 (2), 413 (3), 374 (5), 373 (6),
357 (5), 344 (5), 342 (13), 341 (18), 340 (7), 325 (5), 318
(12), 317 (42), 316 (12), 302 (16), 301 (65), 300 (12), 299
(13), 285 (9), 284 (29), 283 (11), 258 (9), 221 (11), 209
(18), 195 (21), 193 (15), 183 (13), 182 (100), 165 (22), 164
(23), 154 (10), 139 (15), 137 (16), 119 (11), 113 (12), 111
(11), 110 (16), 109 (11), 95 (6), 84 (6), 77 (29), 72 (11), 70
(11). Anal. Calcd. for C22H26N4O3S2 (458.60): N, 12.22;
Found: N, 12.18.
General procedure for the synthesis of 2-(3-substituted
2-hydroxypropylthio)-5-methyl-4-oxo-3-phenyl-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxamides (17a, b)
A mixture of 16 (0.37 g, 1 mmol) and the appropriate
amine (1 mmol) in absolute ethanol (25 ml) was heated
under reflux for 10 h, concentrated and left to cool in ice
bath. The separated product was filtered and crystallized
from the appropriate solvent.
2-(2-Hydroxy-3-morpholinopropylthio)-5-methyl-4-oxo-3-
phenyl-3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamide
(17a)
Pale yellow crystals, yield 52 % (ethanol/water), mp
220–222 °C; IR (KBr, t, cm-1): 3413 (OH), 3265, 3130
(NH), 1681,1674 (C=O), 1635 (C=N), 1507, 1485 (C=C),
1535 (d NH), 1432, 1315 (C–N lactam), 1275, 1172, 1075
(C–S–C), 1233, 1027 (C–O–C); 1H-NMR (500 MHz,
123

Med Chem Res
Biological evaluation
which is the drug concentration resulting in total growth
inhibition which signifies the cytostatic effect of the test
agent. LC50 value which is the compounds’ concentration
leading to 50 % net cell death at the end of the incubation
period (48 h) and signifies the cytotoxic effect of the test
agent.
In vitro anticancer screening
Out of the newly synthesized thieno[2,3-d]pyrimidine
derivatives in the present work, compounds 4b (NSC
746752), 8c (NSC 746754), and 11b (NSC 746751) were
selected by the National Cancer Institute (NCI) Develop-
ment Therapeutic Program (DTP) (www.dtp.nci.nih.gov)
for the in vitro disease-oriented human cells screening
panel assay to investigate their in vitro anticancer activity.
Selectivity ratio
Subpanel and full panel mean-graph midpoint values
(MG-MID GI50) are the average of individual real and
default GI50 values of all cell lines in the subpanel or the
full panel. The ratio obtained by dividing the compound
full panel mean-graph midpoint (MG-MID GI50) (lM) by
its individual subpanel MG-MID GI50 (lM) is considered
as a measure of compound selectivity (Boyd and Paull,
1995). Ratio between 3 and 6 refer to moderate selec-
tivity, ratios greater than 6 indicate high selectivity
toward the corresponding cell line, while compounds
meeting neither of these criteria are rated non-selective
(Table 3).
In vitro one-dose anticancer assay
A primary in vitro one-dose anticancer assay was per-
formed using the full panel of about 60 human tumor cell
lines derived from nine neoplastic diseases including leu-
kemia, melanoma, lung, colon, CNS, ovarian, renal, pros-
tate, and breast cancers, in accordance with the current
protocol of the Drug Evaluation Branch, NCI, Bethesda,
MD, USA (Monks et al., 1991; Boyd, 1997; Shoemaker,
2006).
In the screening protocol, each cell line was inoculated
and preincubated for 24–48 h on a microtiter plate. Tested
compounds were added to the culture at a single concen-
tration (10-4 mol) and the cultures were incubated for
48 h. End point determinations were made with a protein
binding dye, sulforhodamine B (SRB). The percentage
growth was evaluated spectrophotometrically versus con-
trols not treated with test agents.
In vitro antimicrobial screening
The newly synthesized compounds were evaluated for their
in vitro antibacterial activity against Staphyloccus aureus
(ATCC6538P) as a representative of gram-positive bacte-
ria, Escherichia coli (ATCC8739), and Pseudomonas
aeruginosa (ATCC9027) as representatives of gram-nega-
tive bacteria and for their in vitro antifungal activity
against Candida albicans (ATCC2091) using the cup dif-
fusion technique (Jain and Kar, 1971). Compounds show-
ing reasonable inhibition zones (C13 mm) were subjected
to determine their minimal inhibitory concentration (MIC)
values using the two fold serial dilution method (Scott,
1989). Ampicillin was used as standard antibacterial agent,
while Clotrimazole was used as standard antifungal agent.
Dimethylformamide (DMF) was used as a blank showed no
inhibition zones.
Results for each tested compound were reported as a
mean graph of the percent growth of the treated cells when
compared to the untreated control cells (Table 1). This
allows detection of both growth inhibition (GI %) (values
between 0 and 100) and lethality (values less than 0).
In vitro 5-dose anticancer assay
The cytotoxic and/or growth inhibitory effects of the most
active selected compounds, which satisfy pre-determined
threshold inhibition criteria designed to efficiently capture
compounds with antiproliferative activity and based on
careful analysis of historical DTP screening data, were
tested in vitro against the full NCI 60 cell panel at tenfold
dilutions of five concentrations ranging from 10-4 to
10-8 mol. A 48-h continuous drug exposure protocol was
followed and an SRB protein assay was used to estimate
cell viability or growth.
Inhibition zone measurement
The agar cup diffusion technique (Jain and Kar, 1971) was
adopted to determine the growth inhibition zones. Sterile
nutrient agar was inoculated with the test organisms (each
100 ml of molten agar (at 45° C) received 1 ml of 6 h broth
culture), and then seeded agar was poured into sterile petri
dishes. Cups (8 mm in diameter) were cut in the agar, and
each cup received 0.1 ml of the test compound solution in
DMF (1 mg/ml). The plates were prepared in triplicate and
then incubated at 37 °C for 24 h for bacteria and 48 h for
fungi. The resulting inhibition zones were measured in mm
diameter (Table 4).
Three dose response parameters (GI50, TGI and LC50)
were calculated for each cell line (Table 2). The GI50
corresponds for the compounds’ concentration causing
50 % decrease in net cell growth which signifies the
growth inhibitory power of the test agent. The TGI value
123

Med Chem Res
Table 1 In vitro mean growth percentage, delta values, and the percentage growth inhibition (GI %) caused by the test compounds against the
most sensitive tumor cell lines at the single-dose assay
Comp. NSC- Mean
no.
Panel
Subpanel tumor cell lines (% growth inhibitory activity)
no.
growth (%)
4b
746752 116.73
746754 19.24
Renal cancer
CNS cancer
TK-10 (47.22)
SF-295 (43.78)
8c
Non-small cell
lung cancer
A549/ATCC (100.00), EKVX (106.97), HOP-62 (75.81), HOP-92 (119.09),
NCI-H226 (69.12), NCI-H23 (79.54), NCI-H322 M (64.60), NCI-H460 (92.54), NCI-H522
(75.09)
Colon cancer
Breast cancer
Ovarian cancer
Leukemia
HCC-2998 (42.89), HCT-116 (70.87), HCT-15 (85.10), HT 29 (20.50), KM12 (86.43), SW-
620 (87.48)
BT-549 (106.70), HS578T (114.57), MCF7 (82.68), MDA-MB-231/ATCC (56.97), MDM-
MB-435 (87.80), NCI/ADR-RES (81.12), T-47D (65.33)
IGROV1 (89.09), OVCAR-3 (95.68), OVCAR-4 (66.53), OVCAR-5 (52.15), OVCAR-8
(85.29)
CCRF-CEM (112.17), HL-60(TB) (135.80), K-562 (91.58), MOLT-4(87.91), RPMI-8226
(110.32), SR (87.20)
Renal cancer
Melanoma
786-0 (49.31), A498 (99.29), ACHN (92.73), CAKI-1 (140.65), RXF393 (105.46), SN12C
(52.27), TK-10 (97.11), UO-31 (95.87)
LOX IMVI (79.44), M 14 (63.64), SK-MEL-2 (89.96), SK-MEL-28 (51.79), SK-MEL-5
(92.56), UACC-257 (95.15)
Prostate cancer
CNS cancer
Ovarian cancer
Renal cancer
Melanoma
DU-145 (57.85), PC-3 (98.03)
SF-268 (78.68), SF-539 (77.83), SNB-19 (70.56), SNB-75 (45.25), U251 (92.24)
11b
746751 109.13
IGROV1 (79.38)
TK-10 (47.22)
SK-MEL-2 (116.09)
The data obtained from NCI’s in vitro disease-oriented human tumor cell screen at 10 lM conc
Minimal inhibitory concentration measurement
carboxylate 1 was prepared by reacting acetoacetamide and
ethyl cyanoacetate in the presence of sulfur and morpholine,
according to the procedure previously reported (Gewald et al.,
1966). The preparation of 3-Benzyl-5-methyl-4-oxo-3,4-di-
hydrothieno[2,3-d]pyrimidine-6-carboxamide derivative 4a
(Scheme 1) was achieved through a three steps procedure by
adopting the previously established reaction conditions that
involved refluxing the amino ester 1 with excess formamide
(El-Baih et al., 2006) to give 5-methyl-4-oxo-3,4-dihydro-
thieno[2,3-d]pyrimidine-6-carboxamide 2. The 1H-NMR
spectrum of 2 showed two singlets at 2.80 and 8.13 ppm
assigned for thienopyrimidine C5-CH3 protons and thieno-
pyrimidine C2-proton, respectively; in addition to two deute-
rium exchangeable singlets at 7.60 and 12.47 ppm assigned
for NH2 and NH protons, respectively. Compound 2 was
converted to the corresponding potassium salt 3 by treatment
with alcoholic potassium hydroxide followed by heating with
benzyl chloride in dry DMF under reflux to afford the required
thienopyrimidine derivative 4a in 65 % yield. Alternatively,
compound 4a was prepared in 83 % yield by reacting the
amino ester 1 with triethylorthoformate and benzyl amine in
accordance with the previously reported reaction conditions
(Nomoto et al., 1991). Accordingly, compounds 4b–e were
synthesized via the one pot reaction conditions in 79–85 %
yields by treating the amino ester 1 with triethylorthoformate
Using the two fold serial dilution method (Scott, 1989), the
test organisms were grown in their suitable broth at 37 °C
for 24 h for bacteria and 48 h for fungi. Two fold serial
dilutions of the test compounds and the proper reference
drugs solutions were prepared using the proper nutrient
broth to obtain concentrations 500, 250, 125, 62.5, 31.25,
15.63, 7.82, and 3.91 lg/ml with the concentration of DMF
not exceeding 2.5 %. The tubes were then inoculated with
the test organisms (each 5 ml received 0.1 ml of the above
inoculum) and were incubated at 37 °C for 48 h. Subse-
quently, the tubes were observed for the presence or
absence of microbial growth. The lowest concentration
showing no growth was taken as the MIC. The MIC values
of the prepared compounds are listed in Table 4.
Results and discussion
Chemistry
The reaction sequences employed for synthesis of target
compounds are depicted in Schemes 1 and 2. The starting
compoundethyl2-amino-5-carbamoyl-4-methylthiophene-3-
123

Med Chem Res
Table 2 In vitro cytotoxic activity of the compound 8c (NSC-No.:
746754) in the five-dose screen (10-4 to 10-8 M) toward 60 cancer
cell lines
Table 2 continued
Panel/cell line
GI50 (lM)
TGI (lM)
LC50 (lM)
Renal cancer
786-0
Panel/cell line
GI50 (lM)
TGI (lM)
LC50 (lM)
3.82
2.53
2.86
3.35
3.43
3.84
3.78
1.66
[100
8.97
[100
[100
[100
[100
[100
[100
[100
[100
Leukemia
CCRF-CEM
HL-60(TB)
K-562
A498
34.2
6.68
8.89
24.6
13.9
5.74
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
ACHN
[100
[100
[100
[100
[100
7.00
CAKI-1
RXF393
MOLT-4
SN12C
RPMI-8226
SR
TK-10
UO-31
Non-small cell lung cancer
A549/ATCC
HOP-62
Prostate cancer
PC-3
3.79
2.82
1.34
3.72
3.12
3.71
2.65
1.73
[100
9.47
[100
[100
[100
[100
[100
[100
[100
[100
2.15
3.90
8.31
[100
[100
DU-145
[100
HOP-92
5.33
Breast cancer
MCF7
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO205
HCC-2998
HCT-116
HCT-15
67.9
2.92
2.90
3.28
3.85
2.89
2.41
3.25
2.65
[100
29.2
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
7.34
NCI/ADR-RES
MDA-MB-231/ATCC
HS578T
8.41
[100
12.5
7.23
MDA-MB-435
BT-549
8.89
2.39
3.05
2.13
2.64
3.04
3.37
3.16
7.32
[100
[100
[100
[100
[100
[100
[100
T-47D
[100
[100
[100
10.03
[100
[100
[100
[100
MDA-MB-468
The data obtained from NCI’s in vitro disease-oriented human tumor
cell screen
HT29
KM12
SW-620
Table 3 Median growth inhibitory concentrations (GI50, lM) of
in vitro subpanel tumor cell lines, GI50 (lM) full panel mean-graph
mid-points (MG-MID) and selectivity ratio of compound 8c toward
the nine tumor cell lines
CNS cancer
SF-268
4.00
2.93
4.02
4.80
2.80
3.31
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
SF-295
Subpanel tumor cell lines MG-MID GI50 (lM) Selectivity ratios
SF-539
SNB-19
Leukemia
15.67
2.86
2.83
3.64
3.34
3.09
3.15
3.03
3.02
4.39a
0.28
1.53
1.55
1.21
1.31
1.42
1.39
1.45
1.45
–
SNB-75
Non-small cell lung cancer
Colon cancer
CNS cancer
U251
Melanoma
LOX IMVI
MALME-3M
M14
3.54
2.18
3.88
3.89
3.56
1.81
3.56
4.26
[100
[100
84.5
[100
[100
[100
[100
[100
7.75
Melanoma
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
Full panel MG-MID
a
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
86.6
[100
3.75
24.3
[100
[100
GI50 (lM) full panel mean-graph mid-point (MG-MID) = the
average sensitivity of all cell lines toward the test agent (8c)
83.2
1.43
2.28
2.50
4.33
3.49
4.50
6.31
[100
[100
[100
[100
[100
[100
1
and the appropriate amine. The H-NMR spectra of com-
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
8.58
pounds 4a–e lacked the deuterium exchangeable singlet for
the NH proton at position 3 in the precursor 2 and showed
signals for the protons of the alkyl and aralkyl substituents at
the 3-position of the thienopyrimidine nucleus beside signals
characteristic for the other protons at their expected chemical
[100
[100
[100
[100
123

Med Chem Res
Table 4 In vitro antimicrobial activity of the synthesized compounds. Inhibition zones (IZ) in mm diameter and MIC in lg/ml
Comp. no.
S. aureus (ATCC6538P)
E. coli (ATCC8739)
P. aeruginosa (ATCC9027)
C. albicans (ATCC2091)
IZ
MIC
IZ
MIC
IZ
MIC
IZ
MIC
2
10
10
10
10
10
10
10
10
10
12
10
10
10
10
11
14
14
12
10
10
10
13
10
10
10
10
11
10
10
10
10
10
30
–
–
13
15
15
17
17
13
16
15
15
13
16
15
15
15
15
15
15
15
15
15
15
15
13
13
16
15
16
13
15
15
13
15
26
–
–
12
16
17
12
12
12
12
17
12
12
12
12
17
17
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
17
12
12
18
–
–
15
15
15
15
15
15
15
15
15
15
15
15
17
15
15
16
16
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
–
–
4a
–
–
–
–
4b
–
–
125
–
–
4c
–
125
125
–
–
4d
–
–
–
4e
–
–
–
5
–
125
–
–
–
6
–
125
–
–
7a
–
–
–
7b
–
–
–
–
8a
–
125
–
–
–
8b
–
–
–
8c
–
–
125
125
–
125
–
8d
–
–
8e
–
–
–
9a
250
250
–
–
–
–
9b
–
–
–
10
–
–
–
11a
–
–
–
–
11b
11c
–
–
–
–
–
–
–
–
12
250
–
–
–
–
13
–
–
–
14a
–
–
–
–
14b
14c
–
–
–
–
–
–
–
–
15a
–
–
–
–
15b
15c
–
–
–
–
–
–
–
–
16
–
–
125
–
–
17a
–
–
–
17b
Ampicillin
Clotrimazole
–
–
–
–
3.91
–
7.81
–
125
–
–
28
7.81
shifts. 13C-NMR spectrum of 4d displayed five highly shiel-
ded signalsduetothienopyrimidine-C5 CH3 at15.47 ppm, the
five methylene carbons resonated as three signals at 25.17,
26.14, and 32.05 ppm corresponding to cyclohexyl C4,
cyclohexylC3 andC5, andcyclohexylC2 andC6, respectively,
and the methine carbon of the cyclohexyl C1 at 53.22 ppm. In
addition, the spectrum showed another deshielded methine
carbon of thienopyrimidine C2 at 147.93 ppm and six de-
shielded signals at 122.83, 129.34, 136.97, 157.80, 163.05,
and 164.14 ppm corresponding to six quaternary carbons of
the thienopyrimidine C4a, C6, C5, C4, CONH2, and C7a,
respectively. The peaks due to quaternary carbon atoms of the
structure disappeared on DEPT experimentation.
Furthermore, refluxing the amino ester 1 in acetic
anhydride gave the N-acetylated derivative 5. The 1H-
NMR spectrum of compound 5 displayed a triplet and a
quartet corresponding to the ethyl ester moiety and two
deuterium exchangeable singlets at 1.60 and 11.57 ppm
assigned for the NH and C5-carbamoyl NH2 protons,
respectively. It also showed the N-acetyl and C4-methyl
protons as two singlets at 2.31 and 2.56 ppm,
respectively.
123

Med Chem Res
Scheme 1 Synthetic route for the preparation of target compounds 2–9
Cyclization of the N-acetylated amino ester 5 following
the previously reported reaction conditions (El-Ansary and
Omar, 2001) by heating under reflux with hydrazine
hydrate in ethanol gave 3-Amino-2,5-dimethyl-4-oxo-3,4-
dihydrothieno[2,3-d]-pyrimidine-6-carboxamide 6. 1H-
NMR spectrum of 6 revealed a singlet at 2.37 ppm inte-
grated for six protons assigned for the two methyl groups in
addition to two deuterium exchangeable singlets at 2.5 and
8.11 ppm due to two NH2 groups. Treatment of 6 with
equimolar amounts of the selected aryl or aralkyl
isothiocyanate derivative in boiling ethanol afforded 3-N-
substituted thiocarbamoylamino derivatives 7a, b. IR
spectra of compounds 7a, b were characterized by the
appearance of absorption bands at 1599–1526, 1263–1222,
1180–1171, and 1023–961 cm-1 attributed to N–C=S
1
function (Omar and AboulWafa, 1984). H-NMR spectra
of 7a, b showed two deuterium exchangeable singlets due
to the protons of the amide-NH2 and the phenyl or benzyl-
NH at 8.11 and 10.50 ppm in case of compound 7a and at
7.77 and 8.44 ppm in case of compound 7b, respectively.
123

Med Chem Res
Scheme 2 Synthetic route for the preparation of target compounds 8–17
1
7a, b. H-NMR spectra of the products 8a–e and 9a, b
The spectra also showed another D2O exchangeable singlet
integrated either for one proton resonated at 9.27 ppm in
case of compound 7b or half proton resonated at
10.75 ppm in case of compound 7a assigned for thieno-
pyrimidine-NH proton. Compound 7a showed as well a
D2O exchangeable singlet at 11.04 ppm integrated for half
proton assigned for SH (thiol form) besides other signals
appeared at their expected chemical shifts.
lacked the NH protons present in the starting material and
showed the singlet for the thiazoline C5-proton (8a–e) at
6.30–6.55 ppm and that for the thiazolidinone C5-protons
(9a, b) at 4.09–4.87 ppm. The spectra also showed other
signals at their expected chemical shifts. Furthermore, the
1H-NMR spectra of the thiazolidinone derivative 9a
showed a singlet at 4.21 ppm integrated for one proton
assigned for the enol form of thiazolidinone C5-H while the
corresponding signal in case of compound 9b was included
within the five aromatic protons between 7.23 and
7.39 ppm. In addition, the spectra showed a D2O
exchangeable singlet between 10.00 and 10.53 ppm inte-
grated for one proton assigned for enolic OH pointing out
that these two compounds exhibited a typical keto-enol
tautomerism. 13C-NMR spectrum of the thiazoline 8a
revealed two high field signals at 15.83 and 16.86 ppm for
the two methyl carbons at 2- and 5-positions of the
Cyclocondensation of the thiourea derivatives 7a, b with
equimolar amount of the appropriate phenacyl bromide or
ethyl bromoacetate in boiling absolute ethanol, as reported
for the preparation of related compounds (El-Feky, 1993),
afforded the corresponding 3-(3-aryl/aralkyl-4-substituted
phenylthiazol-2(3H)-ylideneamino) derivatives 8a–e or
3-(4-oxo-3-substituted thiazolidin-2-ylideneamino) deriva-
tives 9a, b, respectively in good yields (Scheme 1). IR
spectra of 8a–e and 9a, b lacked the mixed vibrational
bands due to N–C=S function present in their precursors
123

Med Chem Res
thienopyrimidine nucleus, respectively. It also showed six
signals due to ten methine carbons of the thiazole C5, the
equivalent phenyl C2 and C6, p-chlorophenyl C2 and C6,
phenyl C4, p-chlorophenyl C3 and C5, and phenyl C3 and
C5 at 95.60, 117.38, 121.38, 124.06, 129.09, and
130.17 ppm, respectively, and the signals characteristic for
the five deshielded quaternary carbons, p-chlorophenyl C1,
p-chlorophenyl C4, phenyl C1, thiazole C4, and thiazole C2
at 134.42, 138.77, 148.39, 149.92, and 155.65 ppm,
respectively. In addition, the spectrum showed the signals
due to thienopyrimidine and CONH2 carbons at their
expected chemical shifts. 13C-NMR spectrum of 9a showed
in addition to the signals corresponding to 2,5-dimethyl-4-
oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamide car-
bons present almost at the same chemical shifts as in
compound 8a, a highly shielded signal due to the methy-
lene carbon of thiazolidinone C5 at 60.10 ppm, four signals
due to six methine carbons of the thiazolidinone C5 enol
form, the equivalent phenyl C2 and C6, phenyl C4 and the
equivalent phenyl C3 and C5 at 85.64, 117.38, 122.30, and
129.67 ppm, respectively and the signals characteristic for
the quaternary carbons, thiazolidinone C4 enol form, phe-
nyl C1, and thiazolidinone C2 at 99.72, 148.40, and
155.65 ppm, respectively.
absorption bands at 1700 cm-1 due to C=O and at 1228
and 1046 cm-1 due to C–O-C of the ester moiety. The 1H-
NMR spectra of 11a–c, 12 and 13 displayed a singlet
between 4.04 and 4.10 ppm corresponding to S–CH2 pro-
1
tons. The H-NMR spectra of 11a–c showed a deuterium
exchangeable singlet between 10.23 and 10.46 ppm
assigned for NH proton. The 1H-NMR spectrum of 12
showed a singlet at 2.25 ppm due to COCH3 protons while
the 1H-NMR spectrum of 13 showed a triplet and a quartet
assigned for the ethyl ester group in addition to the other
signals at their expected chemical shifts. 13C-NMR spec-
trum of 11c showed two high field signals for 4-tolyl-CH3
and S–CH2 at 20.99 and 38.15 ppm, respectively and two
signals at 119.65 and 130.67 ppm due to four methine
carbons of the 4-tolyl moiety. The spectrum also showed
the two 4-tolyl quaternary carbons C4 and C1 signals at
132.99 and 135.99 ppm, respectively while the most de-
shielded signal at 165.51 ppm was characterized for CO–
NH, besides other signals at their expected chemical shifts.
13C-NMR spectrum of 13 showed two high field signals for
the ethyl group at 15.24 and 61.68 ppm and a signal at
35.06 ppm due to S–CH2. The most deshielded signal at
168.64 ppm was characterized for the quaternary O–C=O
carbon. The spectrum also showed the signals corre-
sponding to other carbons at their expected chemical shifts.
Furthermore, reacting the 2-mercapto derivative 10 with
methyl iodide, ethyl iodide, or benzyl chloride in aqueous
sodium hydroxide solution gave the corresponding S-alkyl-
On the other hand, heating the amino ester 1 under reflux
with equimolar amount of phenyl isothiocyanate in aceto-
nitrile in the presence of anhydrous potassium carbonate in
accordance to the previously reported reaction conditions
(Badawey et al., 1993) gave 2-mercapto-5-methyl-4-oxo-3-
phenyl-3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamide
10 (Scheme 2). IR spectrum of 10 displayed a stretching
1
ated derivatives 14a–c. The H-NMR spectra of 14a, 14b,
and 14c displayed a singlet at 2.05 ppm due to S–CH3 pro-
tons, a triplet and a quartet at 1.19 and 3.02 ppm due to S–
CH2CH3 protons and a singlet at 4.35 ppm due to benzyl-
CH2 protons, respectively. In addition, the spectra showed
the signals for other protons at their expected chemical shifts.
Oxidation of the alkylthio compounds 14a–c with potas-
sium permanganate following the previously reported reac-
tion conditions (El-Emam and Ibrahim, 1991) afforded their
corresponding sulfones 15a–c. IR spectra of compounds
15a–c characterized by the appearance of absorption bands at
1388–1359 and 1165–1136 cm-1 attributed to SO2. The 1H-
NMR spectra of compounds 15a, 15b, and 15c showed the
singlet at 2.46 ppm due to SO2-CH3, the triplet and quartet at
1.22 and 3.59 ppm characteristic for SO2-CH2CH3 and the
singlet at 4.44 ppm due to benzyl-CH2 protons, respectively,
in addition to the signals for other protons at their expected
chemical shifts. The 13C-NMR spectrum of 15a showed a
highly shielded signal at 41.82 ppm due to SO2-CH3 carbon,
besides other signals at their expected chemical shifts.
1
absorption band due to SH group at 2,700 cm-1. H-NMR
spectra of 10 displayed a D2O exchangeable singlet at
13.00 ppm assigned for SH proton in addition to the other
protons appeared at their expected chemical shifts. 13C-
NMR spectrum of 10 revealed one shielded signal due to
CH3 at 14.83 ppm and three signals at 128.68, 129.48, and
129.52 ppm due to five methine carbons of the phenyl C4, the
equivalent phenyl C2 and C6, and phenyl C3 and C5,
respectively. In addition, the spectrum showed the eight
quaternary carbons C4a, phenyl C1, C6, C5, C4, CONH2, C2,
and C7a resonated at their expected chemical shifts.
S-alkylation of 10 with chloroacetanilides, chloroace-
tone, or ethyl bromoacetate by heating under reflux in the
presence of anhydrous potassium carbonate in dry acetone
gave the corresponding 2-(phenyl or 4-substituted phe-
nylcarbamoylmethylthio) derivatives 11a–c, 2-(2-oxopro-
pylthio) derivative 12, or 2-(ethoxycarbonylmethylthio)
derivative 13, respectively. The IR spectra of 11a–c, 12,
and 13 lacked the absorption band due to SH function
present in the precursor. The IR spectrum of 12 showed an
additional absorption band at 1732 cm-1 due to CH3-C=O
moiety. The IR spectrum of 13 showed additional
Treatment of an aqueous solution of the potassium salt
of 10 with equimolar amount of epichlorohydrin at room
temperature according to the reported procedure (El-
Meligie et al., 2001) gave 2-[(oxiran-2-yl)methylthio]
derivative 16. IR spectrum of 16 showed the characteristic
123

Med Chem Res
absorption bands of the epoxy C–O–C function at 1224,
1
11b showed mean growth of 116.73 and 109.13 %,
respectively.
1045, 1026, and 771 cm-1. The H-NMR spectrum of 16
showed a doublet of doublet at 2.76 ppm assigned for one
Compound 8c, satisfied the pre-determined threshold
inhibition criteria, has been further evaluated at five con-
centrations ranging from 10-4 to 10-8 mol against the 60
cell lines of above-mentioned types of human cancers.
Three dose response parameters, GI50, TGI, and LC50 were
calculated for each cell line (Table 2).
cis hydrogen of the epoxy CH2,
a multiplet at
3.51–3.58 ppm assigned for the trans proton of the epoxy
CH2, a multiplet at 4.04–4.09 ppm assigned for the S–CH2
protons and a quintet at 5.09 ppm assigned for the CH
epoxy proton in addition to the signals for other protons at
their expected chemical shifts. 13C-NMR spectrum of 16
displayed four high field signals at 15.22, 33.13, 33.80, and
42.87 ppm due to the methyl carbon at C5, S–CH2, epoxy
CH2 and the epoxy CH methine carbon, respectively. The
signals characteristic for 4-oxo-3-phenyl-3,4-dihydrothie-
no[2,3-d]pyrimidine-6-carboxamide carbons resonated at
their expected chemical shifts.
From Table 2, compound 8c showed broad-spectrum
antitumor activity against the 60 human tumor cell line
tested with GI50 values ranging from 1.34 to 8.89 lM with
the exception of leukemia cell lines CCRF-CEM, MOLT-
4, and RPMI-8226 with GI50 values 34.2, 24.6, and 13.9
lM, respectively. 8c showed the most promising activity
toward non-small cell lung cancer HOP-92 and NCI-H522,
melanoma SK-MEL-5, ovarian cancer IGROV1, and renal
cancer UO-31 with GI50 values 1.34, 1.73, 1.81, 1.43, and
1.66 lM, respectively, and TGI values 5.33, 7.23, 3.75,
6.31, and 7.00 lM, respectively.
In addition, it showed moderate activity against colon
cancer COLO205, HCT-116, melanoma MALME-3 M,
ovarian cancer OVCAR-3, OVCAR-4, renal cancer A498,
prostate cancer PC-3 and breast cancer BT-549 with GI50
values 2.39, 2.13, 2.18, 2.28, 2.50, 2.53, 2.15, and 2.41 lM,
respectively while its TGI value against colon cancer
COLO205, HCT-116, ovarian cancer OVCAR-3, renal
cancer A498, prostate cancer PC-3, and breast cancer BT-
549 was 7.32, 10.03, 8.58, 8.97, 8.31, and 8.89 lM,
respectively.
Refluxing the epoxide 16 with the appropriate amine in
the presence of absolute ethanol according to the reported
procedure (El-Meligie et al., 2001), yielded the corre-
sponding 2-(3-substituted 2-hydroxypropylthio) derivatives
1
17a, b. The H-NMR spectrum of compound 17a is char-
acterized by the presence of two singlets at 2.69 and
2.85 ppm attributed to S–CH2 protons, two triplets at 3.78
and 3.79 ppm corresponding to the morpholine C3,5 and
C
2,6 protons, respectively, a doublet of doublet at 4.13 ppm
1
attributed to N–CH2 protons while the H-NMR spectrum
of compound 17b showed two multiplets at 1.22–1.60 and
2.21–2.30 ppm assigned for the piperidine C3,4,5 and C2,6
protons, respectively, and two doublets of doublets at 3.83
and 4.16 ppm assigned for the S–CH2 protons and N–CH2
1
protons, respectively. In addition, the H-NMR spectra of
Furthermore, compound 8c showed minimal cytotoxic-
ity against almost all the cell lines tested with LC50 values
being generally more than 100 lM.
However, although compound 8c showed broad-spec-
trum antitumor activity against the nine tumor subpanels
tested, and demonstrated significant activity in the in vitro
antitumor screening expressed by the full panel MG-MID
GI50 value of 4.39 lM, it was non-selective toward any
specific subpanel as indicated from Table 3 where its
selectivity ratio occurred between 0.28 and 1.55.
17a and 17b showed a multiplet between 4.41 and
4.47 ppm attributed to C–H proton and a deuterium
exchangeable doublet at 5.76 and 5.77 ppm corresponding
to OH proton besides the signals for other protons at their
expected chemical shifts. The 13C-NMR spectrum of 17b
showed seven methylene carbons resonated as two signals
at 53.53 ppm due to S–CH2 and piperidine C3,4,5 and at
54.85 ppm due to piperidine-CH2 and piperidine C2,6. It
also showed six methine carbons appeared as three signals
at 65.90, 124.86, and 129.46 ppm due to CH–OH, phenyl
C4, and phenyl C2,3,5,6, respectively, in addition to the
signal for the phenyl C1 quaternary carbon at 128.80 ppm,
besides other signals at their expected chemical shifts.
In vitro antimicrobial screening
In general, investigation of antimicrobial screening data
(Table 4) revealed that all the newly synthesized com-
pounds displayed variable activities against the four tested
microorganisms. Compounds 9a, 9b, and 12 showed weak
antimicrobial activity against S. aureus (IZ = 13–14 mm),
while compounds 4c, 4d, 5, 8a, 14b, and 15a, showed mild
antimicrobial activity against E. coli (IZ = 16–17 mm).
On the other hand, compounds 4b, 6, 8c, 8d, and 16 were
equipotent and exhibited good antimicrobial activity
against P. aeruginosa (IZ = 17 mm; MIC = 125 lg/ml)
comparable to that of ampicillin (IZ = 18 mm;
Biological evaluation
In vitro anticancer screening
The data obtained from the primary one-dose anticancer
assay (Table 1) revealed that compound 8c exhibited the
highest anticancer activity among the tested compounds
with mean growth of 19.24 % while compounds 4b and
123

Med Chem Res
MIC = 125 lg/ml). In addition, compound 8c showed
mild antifungal activity against C. albicans (IZ = 17 mm).
SAR studies indicated that, the monocyclic thiophene
derivative 5 showed mild antibacterial activity against E. coli
(IZ = 16 mm; MIC = 125 lg/ml) and weak antifungal
activity against C. albicans (IZ = 15 mm). Cyclization of 5
into the 3-Amino-2,5-dimethyl-4-oxo-3,4-dihydrothie-
no[2,3-d]pyrimidine-6-carboxamide 6 improved the anti-
microbial activity that was equipotent to ampicillin against
P. aeruginosa (IZ = 17 mm; MIC = 125 lg/ml) without
any activity against the other microorganisms.
mechanism of anticancer activity of this class of compounds.
Furthermore, compound 8c showed pronounced antibacte-
rial activities comparable to ampicillin against P. aeruginosa
with mild antifungal activity against C. albicans. Therefore,
compound 8c could be considered as useful template for
future development of a new class of dual anticancer-anti-
microbial agents. Moreover, further derivatization or modi-
fication might help to obtain more potent and selective
agents.
Acknowledgments The authors thank the members of the Devel-
opmental Therapeutics Program, Division of Cancer Treatment and
Diagnosis, National Cancer Institute, Bethesda, Maryland, U.S.A. for
carrying out the in vitro anticancer screening and members of the
Department of Microbiology, Faculty of Pharmacy, University of
Alexandria, for performing the in vitro antimicrobial screening.
Converting the 3-NH2 group in 6 into the open chain
N-substituted thiocarbamoylamino derivatives 7a and 7b
having N-phenyl and N-benzyl functions, respectively
abolished the antimicrobial activity. Cyclization of these
thiourea into a rigid 5-membered heterocyclic ring struc-
tures linked through an imino function afforded 8a–e and
9a, b derivatives. Among the thiazoline derivatives 8a–e,
compound 8c (3-benzyl, 4-phenyl) and compound 8d
(3-benzyl, 4-p-chlorophenyl) were equipotent and dis-
played the same antimicrobial activity as ampicillin against
P. aeruginosa (IZ = 17 mm; MIC = 125 lg/ml). Fur-
thermore, compound 8c showed mild activity against
C. albicans (IZ = 17 mm; MIC = 125 lg/ml). On the
other hand, the thiazolidinone derivatives 9a (3-phenyl)
and 9b (3-benzyl) displayed weak antimicrobial activity
against S. aureus (IZ = 14 mm; MIC = 250 lg/ml) and
C. albicans (IZ = 16 mm).
Conflict of interest The authors have declared no conflict of
interest.
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