Synthesis of thiophene-linked pyrimidopyrimidines as pharmaceutical leads

Article abstract of DOI:10.1007/s12039-014-0614-z

Thiophene-substituted chalcones were cyclised with guanidine in the presence of potassium hydroxide to get 4-substituted-6-thiophenopyrimidines 2a-e which were then refluxed with acetylacetone to obtain pyrimidopyrimidines 3a-e. Compounds 2a-e were also refluxed with ethylacetoacetate to afford pyirmidopyirimidines 4a-e which on refluxing with POCl₃ in presence of DMF produced compounds 5a-e. Nucleophilic substitution reactions on 5a-e were carried out with aniline to obtain 6a-e. The structures of the newly synthesised compounds have been confirmed by elemental analysis and spectral studies. Some selected compounds have been screened for antibacterial and analgesic activities.

Full text of DOI:10.1007/s12039-014-0614-z

c
J. Chem. Sci. Vol. 126, No. 3, May 2014, pp. 821–826. ꢀ Indian Academy of Sciences.
Synthesis of thiophene-linked pyrimidopyrimidines as pharmaceutical
leads
M B SIDDESH, BASAVARAJ PADMASHALI∗, K S THRIVENI and C SANDEEP
Department of Chemistry, Sahyadri Science College (Autonomous), Kuvempu University,
Shimoga 577 203, India
e-mail: basavarajpadmashali@yahoo.com
MS received 14 June 2013; revised 3 December 2013; accepted 10 December 2013
Abstract. Thiophene-substituted chalcones were cyclised with guanidine in the presence of potassium
hydroxide to get 4-substituted-6-thiophenopyrimidines 2a–e which were then refluxed with acetylacetone to
obtain pyrimidopyrimidines 3a–e. Compounds 2a–e were also refluxed with ethylacetoacetate to afford pyirmi-
dopyirimidines 4a–e which on refluxing with POCl3 in presence of DMF produced compounds 5a–e. Nucleo-
philic substitution reactions on 5a-e were carried out with aniline to obtain 6a-e. The structures of the
newly synthesised compounds have been confirmed by elemental analysis and spectral studies. Some selected
compounds have been screened for antibacterial and analgesic activities.
Keywords. 2-Acetylthiophene; chalcones; guanidine hydrochloride; pyrimidopyrimidines.
1. Introduction
of heterocycles related to 8-alkylpterins (N8-alkyl-2-
aminopteridin-4(8H)-ones), which have been shown to
be novel substrates for DHFR.
Although various procedures for synthesis of pyrimi-
dine derivatives have been developed, it is convenient
to synthesize substituted pyrimidines by the reaction
of amidine or guanidine derivatives with a variety of
1, 3-dielectrophilic three-carbon units such as α, β-
unsaturated carbonyl compounds.11
Pyrimidines have a long and distinguished history
extending from the days of their discovery as impor-
tant constituents of nucleic acids to their current use
in chemotherapy of AIDS. Pyrimidine nucleus occurs
in biologically important products such as nucleic
acids, vitamins, coenzymes and pharmacologically
useful natural products of plant origin. Pyrimidines
are an important class of heterocyclic compounds,
which possess a wide range of biological activities
such as anticancer,1,2 antibacterial,3 anti-inflammatory,4
antiviral,5 antitubercular,6 antihypertensive7 and anti-
convulsant8 activities.
The pyrimidopyrimidine moiety represents a core
structure that is a useful template for the design of
a variety of tyrosine kinase inhibitors.9 From high
throughput screening, a pyrimidopyrimidine analogue
was identified as a dual inhibitor of the growth factor
receptors. This analogue was a significantly less potent
inhibitor of the other tested kinases.
2. Experimental section
All the melting points were determined in an open capi-
llary and were uncorrected. IR spectra were recorded on
Bruker alpha FT IR spectrophotometer, 1H NMR spec-
tra were measured on Bruker AV 400MHZ using CDCl3
and DMSO as solvent. Chemical shifts are expressed in
δ ppm. Mass spectra were performed on a Joel JMS-D
300 mass spectrometer operating at 70 eV. All the reac-
tions were followed and checked by TLC, and further
purification was done by column chromatography. All
the reagents used were of AR grade and they were again
purified by distillation.
In a programme to design and develop mechanism-
based compounds active as substrates and inhibitors
of dihydrofolate reductase (DHFR), Gebauer et al.10
have reported the synthesis and physical properties
of the 6-methyl, 8-methyl and 8-ethyl derivatives of
the parent 2-aminopyrimido[4,5-d]pyrimidin-4-(3H)-
one. These compounds are the first members of a class
2.1 Preparation of (2E)-3-(4-methoxyphenyl)-1-
(thiophen-2-yl) prop-2-en-1-one (1a)
A mixture of 2-acetylthiophene (1.26 g, 0.01 mol) and
anisaldehyde (1.36 g, 0.01 mol) were stirred in ethanol
(15 mL), then an aqueous solution of 40% potassium
∗For correspondence
821

822
M B Siddesh et al.
hydroxide (10 mL) was added and stirring was conti- using ethanol. Similarly, the compounds 3b–e were
nued for 2 h. The mixture was kept overnight at room prepared.
temperature, reaction mixture was poured into crushed
ice and then acidified with dilute hydrochloric acid.
The solid separated was filtered and recrystalized from
2.3a 2-(4-Methoxyphenyl)-6,8-dimethyl-4-(thiophen-
2-yl)-4H-pyrimido[1,2-a]pyrimidine (3a): IR (KBr)
ethyl acetate, ethanol mixture (8:2). Similarly, the com-
ν (cm−1): 1247 (C-O-C); 1607 (C=N), 817 (C-S-C);
pounds 1b–e were prepared.
1H NMR (400 MHZ, CDCl3)δ (ppm): 2.03-2.17 (s, 6H,
2CH3), 8.09 (s, 1H Ar H), 7.90 (m, 3H, Ar H), 7.58
2.1a 2E)-3-(4-Methoxyphenyl)-1-(thiophen-2-yl)prop
-2-en-1-one (1a): IR (KBr) (νmax cm−1): 1614
(CH=CH), 1644 (C=O); 1H NMR (400 MHZ,
CDCl3) δ (ppm): 8.05 (m, 2H Ar H), 7.70 (d, 2H,
Ar H), 7.25 (m,1H, Ar H), 6.99 (d, 2H, Ar H), 6.93
(dd, 2H, CH=CH), 3.87 (s, 3H, OCH3); MS: m/z
245.Calcd. (%) for C14H12O2S: C, 68.57; H, 4.89;
Found: C, 68.53; H, 4.85.
(m,2H, Ar H), 6.85 (m, 3H, Ar H), 3.88 (s, 3H, OCH3);
MS m/z: 350. Calcd. (%) for C20H19N3OS: C, 68.57;
H, 5.42; N, 12.00; Found: C, 68.51; H, 5.35; N, 11.94.
2.4 Preparation of 2-(4-methoxyphenyl)-8-methyl-4-
(thiophen-2-yl)-4H-pyrimido[1,2-a]pyrimidin-6-ol (4a)
4-(4-Methoxyphenyl)-6-(thiophen-2-yl)pyrimidine-2-
amine (2a) (2.84 g, 0.01 mol) and ethylacetoacetate
(0.13 g, 0.01 mol) in catalytic amount of acetic acid
were taken in a round bottomed flask and the reac-
tion mixture was refluxed for 6 h. The reaction was
monitored by TLC. The mixture was then poured into
crushed ice. The solid separated was filtered, dried
and recrystalized using ethanol, ehylacetate mixture.
Similarly, the compounds 4b–e were prepared.
2.2 Preparation of 4-(4-methoxyphenyl)-6-(thiophen-
2-yl) pyrimidine-2-amine (2a)
A mixture of chalcone (2E)-3-(4-methoxyphenyl)-1-
(thiophen-2-yl)prop-2-en-1-one 1a (2.44 g, 0.01 mol)
and guanidine hydrochloride (0.96 g, 0.01 mol) in 1,4-
dioxane (15 mL) was refluxed on water bath for 5 h.12
The solvent was completely evaporated and the residue
was poured into ice cold water. The precipitated solid
was collected by filtration, purified on silica gel column
using ethyl acetate and petroleum ether mixture (2:8)
solvent system. Similarly, the compounds 2b–e were
prepared.
Table 1. Characterisation data of synthesised compounds.
Sl. No.
Comp.
Nature
Yield (%)
mp (◦C)
1
2
3
4
5
6
7
8
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
Crystalline
83
71
75
89
72
74
62
49
60
52
55
68
60
63
51
58
48
57
43
49
62
55
50
61
60
232–235
246–232
254–261
264–270
239–244
233–240
242–247
257–260
228–235
222–225
266–272
255–261
259–265
270–278
277–282
241–248
234–239
265–270
270–277
278–280
275–280
263–268
271–276
254–260
266–271
2.2a 4-(4-Methoxyphenyl)-6-(thiophen-2-yl) pyrimi-
dine-2-amine (2a): IR (KBr) (νmax cm−1): 3330 (N-
H), 1297-1176 (C-O-C); 1H NMR (400 MHZ, CDCl3)δ
(ppm): 5.09 (s, 2H, NH2), 8.03 (s, 1H Ar H), 7.90 (d,
2H, Ar H), 7.55 (m,2H, Ar H), 6.99 (m, 3H, Ar H),
3.88 (s, 3H, OCH3)7; MS m/z: 284. Calcd. (%) for
C15H13N3OS: C, 63.38; H, 4.57; N, 14.78; Found: C,
63.27; H, 4.55; N, 14.86.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
2.3 Preparation of 2-(4-methoxyphenyl)-6,8-
dimethyl-4-(thiophen-2-yl)-4H-pyrimido
[1,2-a]pyrimidine (3a)
4-(4-Methoxyphenyl)-6-(thiophen-2-yl)pyrimidine-
2-amine (2a) (2.84 g, 0.01 mol) and acetyl acetone
(0.1 g, 0.01 mol) in catalytic amount of acetic acid13
were taken in a round bottomed flask and refluxed for
10 h. The reaction was monitored by TLC, the reaction
mixture was then poured into crushed ice. The product
obtained was filtered, washed, dried and recrystalized

Synthesis of pyrimidopyrimidines as pharmaceutical leads
823
2.4a 2-(4-Methoxyphenyl)-8-methyl-4-(thiophen-2- (4.5 g, 0.03 mol) in dimethyl formamide (10 mL) were
yl)-4H-pyrimido[1,2-a]pyrimidin-6-ol (4a): IR (KBr) refluxed on a heating mantle for 5 h. Then the reaction
1
(νmax cm−1): 3323 (OH), 1607 (C=N); H NMR (400 mixture was cooled and poured into crushed ice. The
MHZ, CDCl3) δ (ppm): 1.21 (s, 3H, CH3), 9.48 (s, solid separated was filtered, dried and recrystalized
1H, OH), 8.02 (s, 1H Ar H), 7.93 (m, 3H, Ar H), using chloroform–hexane. Similarly, the compounds
7.59 (m,2H, Ar H), 6.90 (m, 3H, Ar H), 3.88 (s, 3H, 5b–e were prepared.
OCH3); MS m/z: 351. Calcd. (%) for C19H17N3O2S: C,
64.95; H, 4.84; N, 11.96; Found: C, 64.90; H, 4.78; N,
11.99.
2.5a 6-Chloro-2-(4-methoxyphenyl)-8-methyl-4-
(thiophen-2-yl)-4H-pyrimido[1,2-a] pyrimidine (5a):
IR (KBr) (νmax cm−1): 724 (C-Cl), 830 (C-S-C); 1650
2.5 Preparation of 6-chloro-2-(4-methoxyphenyl)-8-
methyl-4-(thiophen-2-yl)-4H-pyrimido[1,2-
a]pyrimidine (5a)
1
(C=N); H NMR (400 MHZ, CDCl3) δ (ppm): 1.26
(s, 3H, CH3), 8.14 (s, 1H Ar H), 7.99 (m, 3H, Ar H),
7.69 (m,2H, Ar H), 7.25 (m, 3H, Ar H), 3.80 (s, 3H,
A mixture of 2-(4-methoxyphenyl)-8-methyl-4-(thio- OCH3); MS m/z: 370. Calcd. (%) for C19H16ClN3OS:
phen-2-yl)-1,9a-dihydro-4H-pyrimido[1,2-a]pyrimidin- C, 61.62; H, 4.32; N, 11.35; Found: C, 61.58; H, 4.31;
6-ol (4a) (3.51 g, 0.01 mol) and phosphoryl chloride N, 11.29.
R-CHO
CH
+
S
3
O
KOH/Ethanol
S
R
1a-e
O
NH
KOH
1,4-Dioxane
H N
2
NH .HCl
2
R
R
R
S
S
S
Acetyl acetone
Acetic acid
EAA/Acetic acid
H C
3
N
N
N
N
N
N
HO
N
3
N
3
NH
2
4a-e
2a-e
3a-e
CH
CH
POCl /DMF
3
R
R
NH
S
2
S
N
N
NH
N
N
Cl
Ethanol
N
N
3
CH
CH
6a-e
3
5a-e
Scheme 1. General synthetic procedure for 4-substituted-4-(thiophene-2-yl)-pyrimido
[1,2-a] pyrimidines 3a–e and 4-substituted-6-(thiophene-2-yl)-pyrimido[1,2-a]pyrimidin-4-
amine 6a–e.

824
M B Siddesh et al.
2.6 Preparation of 8-(4-methoxyphenyl)-2-methyl-N-
phenyl-6-(thiophen-2-yl)-6H-pyrimido[1,2-
a]pyrimidin-4-amine (6a)
showed that a molecular ion peak at m/z 350 in its mass
spectrum is in agreement with the structure.
Compounds 2a–e on refluxed with ethylacetoacetate
in presence of catalytic amount of acetic acid produced
2-substituted-4-thiopheno-8-methyl-pyrimido-[1,2-a]
pyrimidine-6-ol derivatives 4a–e. The reactions were
monitored by TLC. In confirmation, the IR spectrum of
4a showed a absorption band at 3323 cm−1 due to OH
A mixture of 6-chloro-2-(4-methoxyphenyl)-8-methyl-
4-(thiophen-2-yl)-1,9a-dihydro-4H pyrimido[1,2-a]py-
rimidine 5a (3.7 g, 0.01 mol) and aniline (0.93 g,
0.01 mol) in ethanol (15 mL) media was refluxed for
2 h. The reaction mixture was cooled and poured into
crushed ice. The solid obtained was filtered and recrys-
talized using ethanol. Similarly, the compounds 6b–e
were prepared.
1
stretching. The H NMR spectrum showed a singlet at
δ 3.88 due to three protons of OCH3 group and a singlet
at δ 9.48 due to OH group. Its mass spectra showed a
molecular ion peak at m/z 351 which is in agreement
with the structure.
Chlorination of reactive hydroxyl group of com-
pounds 4a–e to yield 5a–e was done by refluxing 4a–e
with POCl3 in presence of DMF. Formation of 5a–e was
confirmed by the presence of chlorine in elemental ana-
lysis. Nucleophilic substitution reactions on com-
pounds 5a–e to replace reactive chlorine were per-
formed by treatment with aniline which resulted in the
formation of 6a–e.
Physical properties of synthesised compounds are
show in table 1 and some selected compounds
were screened for antibacterial and analgesic activity
(scheme 1).
2.6a 8-(4-Methoxyphenyl)-2-methyl-N-phenyl-6-
(thiophen-2-yl)-6H-pyrimido[1,2-a]pyrimidin-4-amine
(6a): IR (KBr) (νmax cm−1): 3300 (NH), 1638 (C=N);
1H NMR (400 MHZ, CDCl3) δ (ppm): 1.58 (s, 3H,
CH3), 5.13 (s, 2H, NH); 8.24 (m, 3H Ar H), 7.99
(m, 4H, Ar H), 7.62 (m,3H, Ar H), 7.15 (m, 4H, Ar
H), 3.80(s, 3H, OCH3); MS m/z: 427. Calcd. (%) for
C25H22N4OS: C, 59.40; H, 4.15; N, 11.08; Found: C,
59.30; H, 4.11; N, 11.17.
3. Results and discussion
Compound
R
In this article, we report the synthesis and biological
properties of some thiophene-linked pyrimidopyrimi-
dine derivatives. The chalcones 1a–e used as precur-
sors to synthesise various pyrimidine derivatives have
been prepared by refluxing 2-acetylthiophene with aro-
matic aldehydes in presence of potassium hydroxide in
ethanol medium.
OCH
a
3
b
c
Cl
Cl
N
The chalcones 1a–e were refluxed with guani-
dine hydrochloride in presence of KOH in 1,4-
dioxane solvent to afford 4-substituted-2-amino-6-
thiophenopyrimidines 2a–e in good yield. The forma-
tion of 2a–e were monitored by TLC. In confirmation,
2a exhibited a absorption band at 3330 cm−1 corre-
d
e
O
1
sponding to NH stretching in its IR spectrum. The H
NMR spectrum showed a singlet at δ 3.88 due to three
protons of OCH3 group and a broad singlet at δ 5.09 due
to two protons of NH2 group. Further, a molecular ion
peak at m/z 284 in its mass spectrum is in agreement
with the structure.
3.1 Antibacterial activity
Some selected compounds were screened for their
Pyrimidines 3a–e were prepared by the treatment of antibacterial activity against Staphylococcus aureus,
2a–e with acetyl acetone in acetic acid medium. The IR Escherichia coli, S. paratyphi-A and Bacillus subtilis.
spectrum of compound 3a exhibited a absorption band The activity was carried out using cupplate agar
1
at 1607 cm−1 due to C=N group. The H NMR spec- method.14 The zone of inhibition was measured in mil-
trum of compound 3a showed a singlet at δ 3.88 due to limetres. DMF is used as a vehicle. Chlorampheni-
three protons of OCH3 group and two singlets at δ 2.03 col and streptomycin were used as standard drugs for
and δ 2.17 for six protons of two CH3 groups. Further, it comparison. The compounds were tested at 40 μg/mL

Synthesis of pyrimidopyrimidines as pharmaceutical leads
Table 2. Antibacterial activity of synthesised compounds.
825
Diameter of zone of inhibition (mm)
Compound
Staphylococcus aureus Escherichia coli S. paratyphi-A Bacillus subtilis
3a
3c
3d
3e
4a
4c
4d
4e
5a
5c
5d
5e
6a
6c
6d
6e
13
12
15
13
18
14
12
14
15
10
14
15
18
13
11
15
00
20
14
15
12
09
17
13
13
12
11
12
15
13
12
08
10
13
00
16
15
10
12
10
17
14
14
12
16
12
17
08
18
11
14
10
00
21
12
13
10
11
18
18
14
10
17
18
11
10
17
14
16
13
00
22
DMF
Chloramphenicol
concentration. All the synthesised compounds were number of abdominal constrictions for 20 min start-
found to be moderate to poorly active against bacteria. ing 3 min after the injection of acetic acid solution.
Details of zone of inhibition are presented in table 2.
Group 1–10 received the suspension of test compounds
(100 mg/kg dose), respectively, and 11 received the
standard drug suspension (Ibuprofen) at the dosage of
100 mg/kg. After 1 h, acetic acid solution was admin-
istered intraperitonially and number of abdominal con-
strictions was recorded for 20 min starting 3 min after
the injection of acetic acid solution. Analgesic activity
was calculated as the percentage of maximum possi-
ble effect (%MPE) and the results are given in table 3.
Compounds 3a, 3d, 6b and 6d exhibited significant
analgesic activity.
3.2 Analgesic activity
Albino mice of either sex (20–30 g) were subjected
to acetic-acid-induced writhing test for analgesic
activity.15 Acetic acid solution (0.6%, 10 mL/kg) was
used to induce writhing in mice. The mice were
divided into 11 groups, each consisting of six ani-
mals. Analgesic response was assessed by counting the
Table 3. Analgesic activity of synthesised compounds.
Mean number of abdominal constrictions
occurred between 3 and 20 min
Compound
Dose (mg/kg)
Before drug
After drug
% MPE
3a
3b
3c
3d
3e
6a
6b
6c
100
100
100
100
100
100
100
100
100
100
100
25.8 1.2
38.6 2.1
39.1 2.0
24.8 1.2
40.1 2.0
34.1 2.0
23.1 1.5
26.4 1.6
24.8 1.2
34.1 2.0
47.1 2.5
7.4 0.92
15.8 1.43
16.2 1.40
8.4 0.92
15.2 1.40
15.1 0.40
8.9 0.82
10.4 1.3
8.5 1.92
15.1 0.40
11.8 1.27
66.1∗
61.7∗
60.1∗
66.1∗
60.1∗
60.1∗
66.4∗
62.2∗
68.5∗
60.1∗
75.1∗
6d
6e
Ibuprofen
Analgesic activity, *P < 0.001 vs. control; student’s t-test, n = 6

826
M B Siddesh et al.
2. Yamakawa T, Kagechika H, Kawachi E, Hashimoto Y
4. Conclusion
and Shedo K R 1990 J. Med.Chem. 33(5) 1430
3. Isida S, Matsuda A, Kawamura Y and Yamanaka K 1960
Chromatography (Tokyo) 8 146
New pyrimidopyrimidines prepared are additions to
the molecular library. Some compounds have exhibited
significant biological activity.
4. Hogale M B, Dhore N P, Shelar A R and Pawar P K
1986 J. Chem. 2 55
5. Ahluwalia V K, Nayal L, Kalia N, Bala S and Tahim A K
1987 Indian J. Chem. 26B(4) 384
6. Bhat A K, Bhamana R P, Patel M R, Bellare R A and
Deliwala C V 1972 Indian J. Chem. 10(7) 694
7. Ishitsuka H, Ninomiya Y T, Ohsawa C, Fujiu M and
Suhara Y 1982 Antimicrob. Agents Chem-Other. 22(4)
617
Supplementary information
Structural data (NMR, IR, mass and elemental analysis)
of the rest of the compounds are provided in the sup-
plementary information file. The electronic supporting
information can be seen at www.ias.ac.in/chemsci.
8. Ninomiya Y, Shimma N and Ishitsuka H. 1990 Antiviral
Res. 13(2) 61
9. Rossman P, Luk K, Cai J, Chen Y, Dermatakis A,
Flynn T, Garofalo L, Gillespie P, Goodnow R, Graves B,
Harris, W, Huby N, Jackson N, Kabat M, Konzelmann
F, Li S, Liu J, Liu W, Lukacs C, Michoud A, Perrotta
A and Portland L 2004 Proc. Am. Assoc. Cancer Res.
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11. Rahaman A, Rajendra P, Pahani K and Bahrat K 2009
Saudi Pharm. J. 17 259
Acknowledgements
Authors are thankful to the Principal, Sahyadri Sci-
ence College (Autonomous), Shimoga and the Chair-
man, Department of Chemistry, Sahyadri Science Col-
lege (Autonomous), Shimoga, for providing necessary
laboratory facilities. Authors are also thankful to the
Principal, SCS College of Pharmacy, Harapanahalli, for
providing facilities to conduct biological activities.
12. Naik T A and Chikhalia K H 2007 E-J. Chem. 4 60
13. Vishnu J R, Kushwaha D S and Mishra L 1989 Indian
J .Chem. 28B 242
14. Barry A L 1976 The antimicrobial susceptibility
test: Principle and practice; Illuslea and Febiger,
Philadelphia, USA; p. 180
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