Regioselective N1-alkylation of 3,4-dihydropyrimidine-2(1H)-ones: Screening of their biological activities against Ca2+-ATPase

Article abstract of DOI:10.1016/j.ejmech.2012.04.043

A regioselective N1-alkylation of 3,4-dihydropyrimidin-2(1H)-ones using a very efficient mild base Cs₂CO₃ and alkyl halides at room temperature has been reported. The selectivity of this methodology is excellent and the yields of the alkylated products are very good. Furthermore inhibitory action of both the 3,4-dihydropyrimidin-2(1H)-ones and the N1-alkylated derivatives were tested on Ca²⁺-ATPase, which revealed that the parent compounds can act as Ca²⁺-ATPase inhibitors whereas the N1-alkylated derivatives are inefficient for this purpose.

Full text of DOI:10.1016/j.ejmech.2012.04.043

European Journal of Medicinal Chemistry 54 (2012) 223e231
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Regioselective N1-alkylation of 3,4-dihydropyrimidine-2(1H)-ones: Screening
of their biological activities against Ca2þ-ATPase
Salil Putatunda a, Srabasti Chakraborty b, Swatilekha Ghosh c, Pinki Nandi c, Supriya Chakraborty c,
,
a,
*
Parimal C. Sen c , Arijit Chakraborty
*
a Department of Chemistry, Maulana Azad College, 8 Rafi Ahmed Kidwai Road, Kolkata 13, India
b Department of Chemistry, Behala College, Kolkata 60, India
c Division of Molecular Medicine, Bose Institute, Kolkata 54, India
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
< A Regioselective N1-Alkylation of 3, 4-dihydropyrimidine-2(1H)-ones has been done.
< Alkylation was done by using Cs2CO3 and alkyl halides at room temperature.
< Regioselectivity is excellent and the yields of the alkylated products are very good.
< Inhibitory action of 3, 4-DHPMs and their derivatives were tested on Ca2þ-ATPase.
a r t i c l e i n f o
a b s t r a c t
Article history:
A regioselective N1-alkylation of 3,4-dihydropyrimidin-2(1H)-ones using a very efficient mild base
Cs2CO3 and alkyl halides at room temperature has been reported. The selectivity of this methodology is
excellent and the yields of the alkylated products are very good. Furthermore inhibitory action of both
the 3,4-dihydropyrimidin-2(1H)-ones and the N1-alkylated derivatives were tested on Ca2þ-ATPase,
which revealed that the parent compounds can act as Ca2þ-ATPase inhibitors whereas the N1-alkylated
derivatives are inefficient for this purpose.
Received 6 March 2012
Received in revised form
28 April 2012
Accepted 30 April 2012
Available online 16 May 2012
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
N1-Alkylated dihydropyrimidones
Regioselective synthesis
Ca2þ-ATPase
1. Introduction
Dihydropyrimidones (DHPMs) have a wide range of synthetic
importance due to the six diversity points around the heterocyclic
core. DHPMs have been reported to possess diverse biological
* Corresponding authors. Tel.: þ91 9830365972.
E-mail address: arijit_chak2002@yahoo.com (A. Chakraborty).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.043

224
S. Putatunda et al. / European Journal of Medicinal Chemistry 54 (2012) 223e231
R1
of DHPM scaffold using Mitsunobu-type conditions [21], but the
R1
reagents used are reasonably expensive and hazardous. Another
direct alkylation method had been demonstrated by Singh et al.
[22], where strong aqueous alkali was used. The reaction condition
is not suitable for most of the alkyl halides and a huge excess of
alkyl halide is required. Thus a facile and good yielding method still
has its importance.
In the present study not only a simple inexpensive and high
yielding method for the synthesis of DHPM derivatives are
described, their biological importance has also been studied.
Cs2CO3
,
R2X
EtOOC
Me
EtOOC
Me
NH
NH
DMF , r.t.
N
H
O
N
R2
O
Scheme 1.
activities such as antiviral, antibacterial and antitumor effects and
more recently DHPMs have emerged as the integral backbone of
several calcium channel blockers [1e7]. In this report we have
demonstrated the role of some synthetic compounds belonging to
the DHPM group as inhibitors of the Ca2þ-ATPase.
2. Synthetic methodology and discussion
We have found that highly regioselective N1-alkylation of
DHPMs could be achieved by using Cs2CO3 and alkyl halides (in
some cases Bu4NI is required for better yield) in DMF at room
temperature (Scheme 1). The various non-alkylated DHPM moie-
ties (1e6) used in this report are synthesized through Biginelli one
pot condensation reaction following the reported method [23e25].
Cesium salts are well known for their unique physical and
chemical properties, commonly called “cesium effect” [26e28]. In
this transformation Cs2CO3 was chosen due to the following
reasons-
Closely related to the calcium channels are the Ca2þ-ATPases -
the conventional Mg2þ-dependent Ca2þ-ATPase, present in most
mammalian tissues along with Mg2þ-independent Ca2þ-ATPase,
enriched in the reproductive tissue [8e10]. They control Ca2þ
-
homeostasis in cells and maintain a resting cytoplasmic level of
Ca2þ, necessary for the normal functioning of a cell. Additionally,
they have been implicated in regulation of sperm motility and
hence fertility [11,12].
Modification of DHPM moiety for the preparation of novel
bioactive compounds encouraged us to develop an efficient
methodology of N1-alkylation as it is least attempted, most prob-
ably due to its demands for regioselectivity. The achievement of
regioselectivity is difficult because of the small difference in acidity
of N1 and N3 hydrogens. There are only a few literature available
describing N1-alkylation of DHPMs and they have their limitations
in terms of yield, reagent, reaction conditions and selectivity.
Kappe and coworkers introduced several methods for the
preparation of N1-monoalkyl derivatives through Biginelli cyclo-
condensation reaction by using N-monoalkylated urea [13e20].
The yields are not satisfactory in most of the cases and the
commercial availability of substituted ureas are limited, thereby
making variation in the alkyl group is often troublesome. Latter on
Kappe et al. developed a good yielding method for monoalkylation
i) Cs2CO3 is a moderately strong base which is found effective to
capture
a proton selectively from the N1 position in
dihydropyrimidones.
ii) It is appreciably soluble in many organic solvents unlike other
alkali metal carbonates.
Different mild bases like CsF, LiOH.H2O, K2CO3 were tested
(Table 1) but Cs2CO3 was found to be the most efficient in terms of
yield and selectivity for different alkyl halides. Reaction using
LiOH.H2O was very fast but it produced the dialkyl product from the
very beginning of the reaction. It must be mentioned that DMSO is
also a suitable solvent for this reaction whereas acetonitrile and
acetone were not at all effective (Table 2).
The present method demonstrates
a highly regioselective
synthesis of N1-alkyl DHPMs by using 1.3 equiv. of Cs2CO3 and 1.5
Table 1
Optimization of the reaction conditions.
Entry
Electrophile R2X
Mole Equivalent
Base (1.3 mol eq)
Additive
Reaction time (h)
Yield (%)
Mono alkyl (1.1)
Di alkyl (1.2)
1
2
3
4
5
6
7
8
PhCH2Br
PhCH2Br
PhCH2Br
PhCH2Br
n-C8H17Br
n-C8H17Br
n-C8H17Br
n-C8H17Br
1.5
1.5
1.5
1.5
2
2
2
2
Cs2CO3
CsF
e
5
5
5
72
65
60
e
e
10
22
e
LiOH.H2O
K2CO3
Cs2CO3
Cs2CO3
Cs2CO3
CsF
e
e
5
Trace
56
e
24
24
24
24
e
e
60a
77
e
Bu4NIb
e
Bu4NIb
30
e
a
Under elevated temperature (60 ꢀC).
1eq. Bu4NI was added before the addition of alkyl halide.
b

S. Putatunda et al. / European Journal of Medicinal Chemistry 54 (2012) 223e231
225
Table 2
DHPMs. Reactions with allyl bromide were very fast but a small
amount of dialkyl derivatives (less than 8%) were formed for all the
three cases. Cinnamyl chloride showed similar reactivity as benzyl
bromide but it required longer time. Other two electrophiles
PhCH2CH2Br and PhCH2CH2CH2Br were also used in the reaction
with DHPM 3, the yields were 30% and 62% respectively.
Results of the alkylation of compound 1 in various solvents.
Entry Electrophile Solvent
R2X
Base
Reaction Yield (%)
time (h)
1
2
3
4
CH3I
CH3I
CH3I
CH3I
Anhydrous DMF
Anhydrous DMSO
Anhydrous Acetone
Cs2CO3 17
Cs2CO3 17
Cs2CO3 17
94
93
Trace
Trace
All the N1-alkylated DHPMs were characterized by 1H NMR, 13
C
Anhydrous Acetonitrile Cs2CO3 17
NMR, CHN elemental analysis and Mass spectroscopy. The position
of alkylation was confirmed from the 1H NMR splitting pattern of C-
4 proton. In the alkylated product the C-4 proton appeared as
doublet on coupling with adjacent N3-proton with coupling
constant 3 Hz. Finally the structure of the compound 1.1a was
confirmed from X-ray crystallography analysis (Fig. 1). In the
process of single crystal formation from the racemic mixture of the
compound 1.1a only the R-isomer was crystallized out
(CCDC 865427). This phenomenon was reported by different
workers as “chiral amnesia” [29,30].
equiv. of alkyl halide in anhydrous DMF at room temperature. Most
of the alkyl halides furnished N1-alkylated DHPMs with excellent
yields under this condition until and unless mentioned (Table 3).
DHPM 1, 2 and 3 produced respectively 94%, 92% and 90% N-methyl
derivative, no dialkylated or N2-alkylated product was isolated. For
n-butyl bromide the yields were 83%, 73% and 73% respectively.
Both Benzyl bromide and benzyl tosylate were found to be equally
efficient electrophile for this reaction, 72% and 75% respective
yields for compound 1.1f were obtained.
3. Biological results and discussion
Long chain alkyl halides like n-octyl, n-decyl and n-hexadecyl
bromides were less reactive under the reaction condition and fur-
nished N1-alkylated product with poor yield. A modified method
was developed on using tetrabutylammonium iodide as additive,
which leads to very good yield with selectivity for all the three
The inhibitory activities of the unsubstituted DHPMs and N1-
alkyl DHPMs were examined on Mg2þ-dependent and indepen-
dent Ca2þ-ATPase according to the previously established proce-
dures [31]. The percentage of inhibition at different concentration
of the inhibitors indicates the efficiency of the DHPMs (Fig. 2). The
pre-incubation of the DHPM derivatives with the enzyme was done
for about 5 min. However prolonging the incubation time did not
significantly affect the percentage of inhibition. Increase in the
concentration of the enzyme also did not produce any notable
change in the percentage of inhibition. The I50 values of the DHPM
derivatives were determined by measuring the enzyme activities at
different concentrations of the compounds at a fixed concentration
of the enzyme following the same above mentioned assay
procedure.
Table 3
Preparation of N1-alkylated DHPMs with different alkyl halides.
Among all DHPM derivatives, compound 1 to 6 were found to be
most potent in their inhibitory action. They differ only in the
aromatic nucleus present at C-4 position of the heterocyclic core.
The I50 values are listed in the Table 4. This is pertinent to mention
that the N1-alkylated derivatives were found to be less potent or
not at all effective when compared with their parent non-alkylated
compounds (data not shown). These DHPM derivatives also
showed an additive inhibitory effect in the presence of Verapamil,
another potent Ca2þ-ATPase inhibitor (Fig. 3), clearly indicating
that they possess different binding sites in the enzyme. These
Entry DHPM
R2X
Reaction Additive N1-alkyl Yield
time (h)
DHPM
(%)
1
1 (R1]H)
CH3I
17
20
24
24
24
5
4
e
1.1a
1.1b
1.1c
1.1d
1.1e
1.1f
1.1f
1.1g
1.1h
2.1a
2.1b
2.1c
2.1d
2.1e
2.1f
2.1f
2.1g
2.1h
3.1a
3.1b
3.1c
3.1d
3.1e
3.1f
3.1f
3.1g
3.1h
3.1i
94
83
77
78
74
72
75
82a
73
92
73
73
76
73
69
70
78a
69
90
73
72
71
68
68
70
76a
68
30
62
2
3
4
5
6
7
8
n-C4H9Br
n-C8H17Br
n-C10H21Br
n-C16H33Br
PhCH2Br
PhCH2OTs
CH2]CHCH2Br
PhCH]CHCH2Cl 17
e
Bu4NI
Bu4NI
Bu4NI
e
e
20 min
e
9
e
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
2 (R1 ¼ p-Cl)
CH3I
17
20
24
24
24
5
4
e
n-C4H9Br
n-C8H17Br
n-C10H21Br
n-C16H33Br
PhCH2Br
PhCH2OTs
CH2]CHCH2Br
PhCH]CHCH2Cl 17
e
Bu4NI
Bu4NI
Bu4NI
e
e
20 min
e
e
3 (R1]m-NO2) CH3I
n-C4H9Br
17
20
24
24
24
5
4
e
e
n-C8H17Br
n-C10H21Br
n-C16H33Br
PhCH2Br
PhCH2OTs
CH2]CHCH2Br
PhCH]CHCH2Cl 17
PhCH2CH2Br 24
PhCH2CH2CH2Br 24
Bu4NI
Bu4NI
Bu4NI
e
e
20 min
e
e
e
e
3.1j
a
Only 1.1 eq of Cs2CO3 was used and 5e8% dialkylated product was obtained.
Fig. 1. Single crystal X-ray structure of compound 1.1a (R-isomer).

226
S. Putatunda et al. / European Journal of Medicinal Chemistry 54 (2012) 223e231
Fig. 2. Inhibitory activity of DHPMs on (A) Mg2þ-dependent Ca2þ-ATPase and (B) Mg2þ-independent Ca2þ-ATPase.
DHPM derivatives did not exhibit any significant effect on Naþ, Kþ-
ATPase.
mass spectrometer. CHN analysis was done on Perkin Elmer 2400
Series II CHN analyzer. Infrared spectra were recorded on Perkin
Elmer Spectrum 100 FT-IR spectrometer in KBr pellets.
4. Conclusion
5.1. Typical N1-alkylation procedure for the synthesis of ethyl 1,6-
dimethyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (1.1a) [21,22]
It is worth remarking that by using this methodology it is
possible to prepare variously substituted N1-alkyl DHPMs. In
general the alkylation is highly efficient in terms of yield and
selectivity. The reaction procedure is very simple, the reagents are
not very costly and are easily available. Various alkyl halides can be
used without any special precaution, which indicates the generality
of this reaction. Moreover the study of inhibitory action reveals
good potentiality of DHPM derivatives on both Mg2þ-dependent
and Mg2þ-independent Ca2þ-ATPase activity which might be
further investigated so as to develop compounds/drugs for a wide
number of diseases. The novel N1-alkylated products are not good
inhibitors and they have solubility problems in the assay condition.
In a solution of compound 1 (250 mg, 0.96 mmol) in 3 ml
anhydrous DMF, Cs2CO3 (407.28 mg, 1.25 mmol) was added. The
mixture was stirred at room temperature for 1 h under anhydrous
condition. Then methyl iodide (0.09 ml, 1.44 mmol) was added to
the reaction mixture slowly and stirred at r.t. for 17 h. After
completion of the reaction as indicated by TLC, saturated NaCl
solution was added. The reaction mixture was extracted with ethyl
acetate (3 ꢁ 25 ml). The combined organic layer was washed with
water (2 ꢁ 50 ml), followed by brine solution (1 ꢁ 50 ml). The
organic layer was dried over anhydrous Na2SO4 and the solvent was
removed under reduced pressure. The crude mass was subjected to
column chromatography using 100e200 mesh silica gel when the
desired product was obtained by eluting the column with 30%
EtOAc/60e80 ꢀC Petrolium ether as solvent system. (94% yield).
White solid mp 176e177 ꢀC (Ethyl acetate/n-Hexane).
5. Experimental
All reagents used were of analytical grades and obtained from
recognized commercial suppliers and were used without further
purification unless otherwise stated. Column chromatography was
performed on SRL 100e200 mesh silica gel. Thin Layer chroma-
tography was performed on Merck Silica gel 60 F254 plates. Melting
points were determine on a LabX India, Digital Melting Point
apparatus and are uncorrected. 1H NMR and 13C NMR spectra were
recorded at ambient temperature using Bruker 300 MHz (Bruker
AVANCE 300 or Bruker DPX-300) and Bruker 500 MHz (Bruker
Ultrashield Plus 500), FT NMR spectrometers (300 MHz and
500 MHz respectively, for 1H and 75 MHz and 125 MHz respectively
for 13C). HRMS were performed on Waters Micromass Q-tof Micro
1H NMR (300 MHz, CDCl3):
d 7.32e7.22 (m, 5H, ArH), 5.43 (brs,
1H, NH), 5.38 (d, 1H, J ¼ 3 Hz, CH), 4.10 (q, 2H, J ¼ 7.2 Hz, ester-CH2),
3.24 (s, 3H, NeCH3), 2.51 (s, 3H, CH3), 1.18 (t, 3H, J ¼ 7.2 Hz, ester-
CH3).
13C NMR (75 MHz, CDCl3):
d 166.0, 154.1, 149.3, 143.3, 128.6,
127.6, 126.1, 104.1, 60.1, 53.7, 30.2, 16.5, 14.1.
IR (KBr, cmꢂ1): 3231, 3101, 2946, 1689, 1623, 1445, 1395, 1349,
1246, 1185, 1149, 1082, 1051.
Anal. Calcd for C15H18N2O3: C, 65.68; H, 6.61; N, 10.21. Found: C,
65.59; H, 6.65; N, 10.26.
HRMS (m/z) for C15H18N2O3Na [M þ Na]þ : Calculated 297.1215,
found 297.1214.
Table 4
aI50 values of different DHPM derivatives.
Entry
DHPM derivatives
I50 mM
5.2. Ethyl 1-butyl-6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyri-
midine-5-carboxylate (1.1b) [22]
R1
Compound no.
Mg2þ-dependent
Mg2þ-independent
1.
2.
3.
4.
5.
6.
H
1
2
3
4
5
6
222
143
132
242
268
289
226
185
282
258
247
203
p-Cl
m-NO2
m-Cl
o-Cl
White solid (83% yield).
mp 106e107 ꢀC (Ethyl acetate/n-Hexane).
1H NMR (300 MHz, CDCl3):
d 7.31e7.22 (m, 5H, ArH), 5.44 (brs,
p-OH
1H, NH), 5.35 (d, 1H, J ¼ 2.7 Hz, CH), 4.08 (q, 2H, J ¼ 7.2 Hz, ester-
CH2), 3.95e3.85 (m,1H, one H of NeCH2e), 3.62e3.52 (m,1H, one H
of NeCH2e), 2.51 (s, 3H, CH3), 1.62e1.23 (m, 4H, two eCH2- of n-
a
I50 is the concentration (in
tory effect is achieved.
mM) of inhibitors at which half the maximal inhibi-

S. Putatunda et al. / European Journal of Medicinal Chemistry 54 (2012) 223e231
227
Fig. 3. Effect of verapamil on the ATPase activity in the absence and presence of the DHPM derivatives; (A) Mg2þ-dependent Ca2þ-ATPase and (B) Mg2þ-independent Ca2þ-ATPase.
butyl), 1.16 (t, 3H, J ¼ 7.2 Hz, ester-CH3), 0.97e0.88 (m, 3H, eCH3 of
5.5. Ethyl 1-hexadecyl-6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydro-
pyrimidine-5-carboxylate (1.1e)
n-butyl).
13C NMR (75 MHz, CDCl3):
d 166.2, 153.6, 148.8, 143.5, 128.5,
127.63, 126.2, 104.4, 60.1, 53.8, 42.4, 31.8, 19.9, 16.0, 14.1, 13.7.
IR (KBr, cmꢂ1): 3218, 3099, 2960, 1686, 1610, 1466, 1396, 1280,
1206, 1156, 1094.
White solid (74% yield).
mp 75e76 ꢀC (Ethyl acetate/n-Hexane).
1H NMR (300 MHz, CDCl3):
d 7.25e7.17 (m, 5H, ArH), 5.52 (brs,
Anal. Calcd for C18H24N2O3: C, 68.33; H, 7.65; N, 8.85. Found: C,
68.26; H, 7.62; N, 8.91.
1H, NH), 5.31 (s, 1H, CH), 4.03 (q, 2H, J ¼ 7.1 Hz, ester-CH2),
3.99e3.78 (m, 1H, one H of NeCH2e), 3.55e3.46 (m, 1H, one H of
NeCH2e), 2.45 (s, 3H, CH3), 1.93 (m, 4H, two eCH2- of n-hexadecyl),
1.54e1.44 (m, 2H, one eCH2- of n-hexadecyl), 1.19e1.13 (m, 22H,
eleven eCH2- of n-hexadecyl), 1.10 (t, 3H, J ¼ 7.1 Hz, ester-CH3),
0.83e0.79 (m, 3H, eCH3 of n-hexadecyl).
5.3. Ethyl 6-methyl-1-octyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyri-
midine-5-carboxylate (1.1c)
13C NMR (125 MHz, CDCl3):
d 166.3, 153.6, 148.9, 143.7, 128.8,
127.9, 126.4, 104.6, 60.3, 54.3, 42.9, 32.1, 30.0, 29.8, 29.8, 29.7, 29.5,
27.0, 22.8, 16.3, 14.3, 14.2.
White solid (77% yield).
mp 82e84 ꢀC (Ethyl acetate/n-Hexane).
IR (KBr, cmꢂ1): 3220, 3100, 2921, 2849, 1690, 1612, 1466, 1398,
1279, 1194, 1155, 1095.
1H NMR (300 MHz, CDCl3):
d 7.32e7.24 (m, 5H, ArH), 5.60 (brs,
1H, NH), 5.37 (s, 1H, CH), 4.09 (q, 2H, J ¼ 7.1 Hz, ester-CH2),
3.93e3.85 (m, 1H, one H of NeCH2e), 3.62e3.54 (m, 1H, one H of
NeCH2e), 2.52 (s, 3H, CH3), 1.60e1.50 (m, 2H, one eCH2- of n-
octyl), 1.25e1.20 (m, 10H, five eCH2- of n-octyl), 1.17 (t, 3H,
J ¼ 7.1 Hz, ester-CH3), 0.90e0.86 (m, 3H, eCH3 of n-octyl).
Anal. Calcd for C30H48N2O3: C, 74.34; H, 9.98; N, 5.78. Found: C,
74.29; H, 9.85; N, 5.89.
5.6. Ethyl 1-benzyl-6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyri-
midine-5-carboxylate (1.1f) [16,21]
13C NMR (75 MHz, CDCl3):
d 166.2, 153.6, 148.8, 143.5, 128.5,
127.6, 126.2, 104.4, 60.1, 53.8, 42.6, 31.7, 29.8, 29.2, 29.2, 26.7, 22.6,
16.0, 14.1, 14.0.
White solid (72% yield).
mp 156e157 ꢀC (Ethyl acetate/n-Hexane).
IR (KBr, cmꢂ1): 3220, 3102, 2920, 2853, 1686, 1612, 1468, 1411,
1192, 1158, 1098.
1H NMR (300 MHz, CDCl3):
d 7.30e7.23 (m, 8H, ArH), 7.14e7.12
(m, 2H, ArH), 5.64 (brs, 1H, NH), 5.44 (d, 1H, J ¼ 3 Hz, CH), 5.19
(d, 1H, J ¼ 16.5 Hz, one H of NeCH2e), 4.89 (d, 1H, J ¼ 16.5 Hz, one H
of NeCH2e), 4.09 (q, 2H, J ¼ 7.2 Hz, ester-CH2), 2.44 (s, 3H, CH3),1.15
(t, 3H, J ¼ 7.2 Hz, ester-CH3).
Anal. Calcd for C22H32N2O3: C, 70.94; H, 8.66; N, 7.52. Found: C,
70.98; H, 8.60; N, 7.66.
13C NMR (75 MHz, CDCl3):
d 166.0, 154.1, 149.0, 143.2, 137.9,
128.6, 128.6, 127.7, 127.1, 126.4, 126.3, 104.8, 60.2, 53.7, 45.9, 16.4,
14.1.
5.4. Ethyl 1-decyl-6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrim-
idine-5-carboxylate (1.1d) [22]
IR (KBr, cmꢂ1): 3216, 3099, 2930, 1691, 1614, 1445, 1382, 1306,
1201, 1164, 1109.
White solid (78% yield).
Anal. Calcd for C21H22N2O3: C, 71.98; H, 6.33; N, 7.99. Found: C,
71.90; H, 6.21; N, 7.93.
mp 104e105 ꢀC (Ethyl acetate/n-Hexane).
1H NMR (300 MHz, CDCl3):
d 7.29e7.23 (m, 5H, ArH), 5.37e5.36
(m, 2H, NH & CH), 4.09 (q, 2H, J ¼ 7.2 Hz, ester-CH2), 3.95e3.85 (m,
1H, one H of NeCH2e), 3.62e3.52 (m, 1H, one H of NeCH2e), 2.52
(s, 3H, CH3), 1.60e1.53 (m, 2H, one eCH2- of n-decyl), 1.25e1.19 (m,
14H, seven eCH2- of n-decyl), 1.17 (t, 3H, J ¼ 7.2 Hz, ester-CH3),
0.90e0.86 (t, 3H, J ¼ 6 Hz, eCH3 of n-decyl).
5.7. Ethyl 1-allyl-6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyri-
midine-5-carboxylate (1.1g) [22]
Pale yellow solid (82% yield).
13C NMR (75 MHz, CDCl3):
d
166.2, 153.6, 148.8, 143.5, 128.5,
mp 128e129 ꢀC (Ethyl acetate/n-Hexane).
127.6, 126.2, 104.4, 60.1, 53.8, 42.6, 31.8, 29.8, 29.5, 29.3, 29.2, 26.7,
22.6, 16.0, 14.1, 14.1.
1H NMR (300 MHz, CDCl3):
d 7.32e7.23 (m, 5H, ArH), 5.90e5.81
(m, 1H, one H of NeCH2eCH]), 5.70 (brs, 1H, NH), 5.41 (d, 1H,
J ¼ 1.9 Hz, CH), 5.18e5.08 (m, 2H, two H of NeCH2eCH]CH2), 4.47
(dd, 1H, J1 ¼ 4.7 Hz, J2 ¼ 17.1 Hz, one H of NeCH2e), 4.35 (dd, 1H,
J1 ¼ 4.5 Hz, J2 ¼ 17.1 Hz, one H of NeCH2e), 4.09 (q, 2H, J ¼ 7.1 Hz,
ester-CH2), 2.51 (s, 3H, CH3), 1.17 (t, 3H, J ¼ 7.1 Hz, ester-CH3).
IR (KBr, cmꢂ1): 3220, 3099, 2923, 2854, 1690, 1611, 1466, 1409,
1357, 1279, 1195, 1160, 1095.
Anal. Calcd for C24H36N2O3: C, 71.96; H, 9.06; N, 6.99. Found: C,
72.04; H, 9.13; N, 6.91.

228
S. Putatunda et al. / European Journal of Medicinal Chemistry 54 (2012) 223e231
13C NMR (75 MHz, CDCl3):
d
166.1, 153.5, 148.9, 143.3, 133.9,
5.11. Ethyl 4-(4-chlorophenyl)-6-methyl-1-octyl-2-oxo-1,2,3,4-
128.5, 127.6, 126.2, 115.9, 104.2, 60.1, 53.8, 44.7, 15.9, 14.0.
IR (KBr, cmꢂ1): 3210, 3084, 2987, 2938, 1687, 1386, 1270, 1214,
1088.
Anal. Calcd for C17H20N2O3: C, 67.98; H, 6.71; N, 9.33. Found: C,
68.09; H, 6.78; N, 9.20.
tetrahydropyrimidine-5-carboxylate (2.1c)
White solid (73% yield).
mp 132e133 ꢀC (Ethyl acetate/n-Hexane).
1H NMR (500 MHz, CDCl3):
d
7.27 (d, 2H, J ¼ 8.5 Hz, ArH), 7.20 (d,
2H, J ¼ 8.5 Hz, ArH), 6.10 (brs, 1H, NH), 5.36 (d, 1H, J ¼ 3.5 Hz, CH),
4.12 (q, 2H, J ¼ 7 Hz, ester-CH2), 3.95e3.89 (m, 1H, one H of
NeCH2e), 3.59e3.53 (m, 1H, one H of NeCH2e), 2.54 (s, 3H, CH3),
1.76e1.47 (m, 2H, one eCH2- of n-octyl), 1.32e1.23 (m, 10H, five
eCH2- of n-octyl), 1.21 (t, 3H, J ¼ 7 Hz, ester-CH3), 0.90 (t, 3H,
J ¼ 7 Hz, eCH3 of n-octyl).
5.8. Ethyl 1-cinnamyl-6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydr-
opyrimidine-5-carboxylate (1.1h)
Yellow solid (73% yield).
13C NMR (125 MHz, CDCl3):
d 166.1, 153.6, 149.3, 142.2, 133.6,
mp 122e123 ꢀC (Ethyl acetate/n-Hexane).
1H NMR (300 MHz, CDCl3):
d 7.31e7.23 (m, 10H, ArH), 6.43 (d,
128.9, 127.8, 104.3, 60.4, 53.4, 42.8, 31.9, 30.0, 29.4, 29.4, 26.9, 22.8,
16.3, 14.3, 14.2.
1H, J ¼ 16.2 Hz, one H of NeCH2eCH]CH-), 6.24e6.16 (m, 1H, one
H of NeCH2eCH]), 5.57 (brs, 1H, NH), 5.43 (d, 1H, J ¼ 2.7 Hz, CH),
4.68 (dd, 1H, J1 ¼ 4.2 Hz, J2 ¼ 17.1 Hz, one H of NeCH2e), 4.48 (dd,
1H, J1 ¼ 4.2 Hz, J2 ¼ 16.8 Hz, one H of NeCH2e), 4.10 (q, 2H,
J ¼ 6.9 Hz, ester-CH2), 2.56 (s, 3H, CH3), 1.18 (t, 3H, J ¼ 6.9 Hz, ester-
CH3).
IR (KBr, cmꢂ1): 3352, 3211, 3094, 2928, 1687, 1613, 1479, 1390,
1196, 1158, 1096.
Anal. Calcd for C22H31ClN2O3: C, 64.93; H, 7.68; N, 6.88. Found: C,
64.98; H, 7.61; N, 6.85.
13C NMR (75 MHz, CDCl3):
d
166.1, 153.6, 148.8, 143.3, 136.4,
5.12. Ethyl 4-(4-chlorophenyl)-1-decyl-6-methyl-2-oxo-1,2,3,4-
131.6, 128.7, 128.5, 127.8, 127.7, 126.4, 126.3, 125.3, 118.1, 104.8, 60.2,
54.0, 53.4, 44.4, 16.2, 14.1.
tetrahydropyrimidine-5-carboxylate (2.1d)
IR (KBr, cmꢂ1): 3225, 3111, 2972, 1689, 1620, 1410, 1308, 1261,
1205, 1176, 1110.
White solid (76% yield).
mp 103e104 ꢀC (Ethyl acetate/n-Hexane).
Anal. Calcd for C23H24N2O3: C, 73.38; H, 6.43; N, 7.44. Found: C,
73.43; H, 6.49; N, 7.31.
1H NMR (300 MHz, CDCl3):
d
7.27 (d, 2H, J ¼ 8.7 Hz, ArH), 7.18 (d,
2H, J ¼ 8.4 Hz, ArH), 5.62 (brs, 1H, NH), 5.34 (d, 1H, J ¼ 2.7 Hz, CH),
4.10 (q, 2H, J ¼ 7.2 Hz, ester-CH2), 3.95e3.85 (m, 1H, one H of
NeCH2e), 3.60e3.51 (m, 1H, one H of NeCH2e), 2.52 (s, 3H, CH3),
1.62e1.21 (m, 16H, eight eCH2- of n-decyl), 1.19 (t, 3H, J ¼ 7.2 Hz,
ester-CH3), 0.88 (t, 3H, J ¼ 6 Hz, eCH3 of n-decyl).
5.9. Ethyl 4-(4-chlorophenyl)-1,6-dimethyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (2.1a)
13C NMR (125 MHz, CDCl3):
d 166.1, 153.6, 149.3, 142.2, 133.7,
128.9, 127.8, 104.4, 60.4, 53.5, 42.9, 32.0, 30.0, 29.7, 29.7, 29.5, 29.4,
26.9, 22.8, 16.3, 14.3, 14.2.
White solid (92% yield).
mp 130e131 ꢀC (Ethyl acetate/n-Hexane).
IR (KBr, cmꢂ1): 3216, 3095, 2922, 2854, 1693, 1614, 1475, 1390,
1194, 1156, 1094.
1H NMR (300 MHz, CDCl3):
d
7.33 (d, 2H, J ¼ 8.7 Hz, ArH), 7.25 (d,
2H, J ¼ 8.4 Hz, ArH), 5.65 (brs, 1H, NH), 5.43 (d, 1H, J ¼ 3 Hz, CH),
4.17 (q, 2H, J ¼ 6.9 Hz, ester-CH2), 3.29 (s, 3H, NeCH3), 2.58 (s, 3H,
CH3), 1.25 (t, 3H, J ¼ 6.9 Hz, ester-CH3).
Anal. Calcd for C24H35ClN2O3: C, 66.27; H, 8.11; N, 6.44. Found: C,
66.21; H, 8.19; N, 6.49.
13C NMR (75 MHz, CDCl3):
d 166.0, 154.0, 149.7, 142.1, 133.6,
128.9, 127.8, 104.0, 60.4, 53.4, 30.5, 16.7, 14.3, 14.3.
IR (KBr, cmꢂ1): 3220, 3095, 2940, 1710, 1683, 1627, 1490, 1441,
1385, 1302, 1269, 1251, 1198, 1184, 1092, 1050.
Anal. Calcd for C15H17ClN2O3: C, 58.35; H, 5.55; N, 9.07. Found: C,
58.42; H, 5.48; N, 9.17.
5.13. Ethyl 4-(4-chlorophenyl)-1-hexadecyl-6-methyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (2.1e)
White solid (73% yield).
mp 110e111 ꢀC (Ethyl acetate/n-Hexane).
1H NMR (300 MHz, CDCl3):
d
7.25 (d, 2H, J ¼ 8.4 Hz, ArH), 7.16 (d,
2H, 8.4 Hz, ArH), 5.51 (d, 1H, J ¼ 3 Hz, NH), 5.32 (d, 1H, J ¼ 3 Hz, CH),
4.08 (q, 2H, J ¼ 7.2 Hz, ester-CH2), 3.98e3.82 (m, 1H, one H of
NeCH2e), 3.64e3.48 (m, 1H, one H of NeCH2e), 2.50 (s, 3H, CH3),
1.60e1.44 (m, 4H, two eCH2- of n-hexadecyl), 1.44e1.19 (m, 24H,
twelve eCH2- of n-hexadecyl), 1.17 (t, 3H, J ¼ 7.2 Hz, ester-CH3),
0.88e0.84 (m, 3H, eCH3 of n-hexadecyl).
5.10. Ethyl 1-butyl-4-(4-chlorophenyl)-6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (2.1b)
White solid (73% yield).
mp 123e124 ꢀC (Ethyl acetate/n-Hexane).
13C NMR (75 MHz, CDCl3):
d 166.1, 153.5, 149.3, 142.2, 133.7,
1H NMR (300 MHz, CDCl3):
d
7.20 (d, 2H, J ¼ 9 Hz, ArH), 7.12 (d,
128.9, 127.8, 104.3, 60.4, 53.5, 42.9, 32.1, 30.0, 29.8, 29.7, 29.5, 26.9,
22.8, 16.3, 14.3.
2H, J ¼ 9 Hz, ArH), 5.50 (brs, 1H, NH), 5.27 (d, 1H, J ¼ 3 Hz, CH), 4.03
(q, 2H, J ¼ 7.2 Hz, ester-CH2), 3.89e3.79 (m, 1H, one H of NeCH2e),
3.55e3.45 (m, 1H, one H of NeCH2e), 2.45 (s, 3H, CH3), 1.54e1.38
(m, 2H, one eCH2- of n-butyl), 1.30e1.17 (m, 2H, one eCH2- of n-
butyl),1.15 (t, 3H, J ¼ 7.2 Hz, ester-CH3), 0.88 (t, 3H, J ¼ 6 Hz, eCH3 of
n-butyl).
IR (KBr, cmꢂ1): 3212, 3092, 2922, 2850, 1693, 1614, 1474, 1423,
1391, 1195, 1156, 1095.
Anal. Calcd for C30H47ClN2O3: C, 69.40; H, 9.12; N, 5.40. Found: C,
69.51; H, 9.17; N, 5.37.
13C NMR (75 MHz, CDCl3):
d 166.0, 153.7, 149.1, 142.1, 133.3,
128.6, 127.6, 104.1, 60.2, 53.0, 42.3, 31.8, 19.9, 16.0, 14.1, 13.7.
IR (KBr, cmꢂ1): 3344, 3223, 3102, 2963, 1686, 1610, 1476, 1391,
1277, 1205, 1156, 1094.
5.14. Ethyl 1-benzyl-4-(4-chlorophenyl)-6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (2.1f)
Anal. Calcd for C18H23ClN2O3: C, 61.62; H, 6.61; N, 7.98. Found: C,
61.69; H, 6.72; N, 7.85.
White solid (69% yield).
mp 160e161 ꢀC (Ethyl acetate/n-Hexane).

S. Putatunda et al. / European Journal of Medicinal Chemistry 54 (2012) 223e231
229
1H NMR (300 MHz, CDCl3):
d
7.23e7.01 (m, 9H, ArH), 5.60 (brs,
HRMS (m/z) for C15H17N3O5Na [M þ Na]þ : Calculated 342.1066,
1H, NH), 5.31 (d, 1H, J ¼ 3 Hz, CH), 5.10 (d, 1H, J ¼ 16.8 Hz, one H of
NeCH2e), 4.78 (d, 1H, J ¼ 16.2 Hz, one H of NeCH2e), 3.99 (q, 2H,
J ¼ 7.2 Hz, ester-CH2), 2.35 (s, 3H, CH3), 1.07 (t, 3H, J ¼ 7.2 Hz, ester-
CH3).
found 342.1065.
5.18. Ethyl 1-butyl-6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (3.1b)
13C NMR (75 MHz, CDCl3):
d
165.9,154.1,149.3,141.7,137.8,133.4,
128.7, 127.8, 127.3, 126.4, 104.5, 60.3, 53.0, 45.8, 16.4, 14.1.
IR (KBr, cmꢂ1): 3216, 3096, 2982, 2940, 1692, 1618, 1420, 1390,
1318, 1205, 1163, 1107.
Anal. Calcd for C21H21ClN2O3: C, 65.54; H, 5.50; N, 7.28. Found: C,
65.47; H, 5.45; N, 7.39.
Pale yellow solid (73% yield).
mp 76e77 ꢀC (Ethyl acetate/n-Hexane).
1H NMR (300 MHz, CDCl3):
d 8.14e8.11 (m, 2H, ArH), 7.60 (d, 1H,
J ¼ 7.5 Hz, ArH), 7.48 (t, 1H, J ¼ 7.8 Hz, ArH), 6.01 (brs, 1H, NH), 5.47
(d,1H, J ¼ 3 Hz, CH), 4.12 (q, 2H, J ¼ 7.2 Hz, ester-CH2), 4.01e3.91 (m,
1H, one H of NeCH2e), 3.63e3.53 (m, 1H, one H of NeCH2e), 2.56
(s, 3H, CH3), 1.64e1.41 (m, 2H, one eCH2- of n-butyl), 1.36e1.26 (m,
2H, one eCH2- of n-butyl), 1.20 (t, 3H, J ¼ 7.2 Hz, ester-CH3), 0.92 (t,
3H, J ¼ 7.5 Hz, eCH3 of n-butyl).
5.15. Ethyl 1-allyl-4-(4-chlorophenyl)-6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (2.1g)
White solid (78% yield).
mp 124e125 ꢀC (Ethyl acetate/n-Hexane).
13C NMR (75 MHz, CDCl3):
d 165.6, 153.6, 150.1, 148.3, 145.7,
1H NMR (300 MHz, CDCl3):
d
7.20 (d, 2H, J ¼ 8.7 Hz, ArH), 7.13 (d,
132.3, 129.5, 122.6, 121.5, 103.4, 60.4, 53.1, 42.4, 31.8, 19.9, 16.1, 14.1,
2H, J ¼ 8.7 Hz, ArH), 5.84e5.71 (m, 2H, NH & one H of
NeCH2eCH]), 5.32 (d,1H, J ¼ 3 Hz, CH), 5.13e5.00 (m, 2H, two H of
NeCH2eCH]CH2), 4.40 (dd, 1H, J1 ¼ 6.3 Hz, J2 ¼ 18 Hz, one H of
NeCH2e), 4.27 (dd, 1H, J1 ¼ 6.3 Hz, J2 ¼ 16.2 Hz, one H of NeCH2e),
4.04 (q, 2H, J ¼ 7.2 Hz, ester-CH2), 2.44 (s, 3H, CH3), 1.11 (t, 3H,
J ¼ 7.2 Hz, ester-CH3).
13.7.
IR (KBr, cmꢂ1): 3223, 3105, 2930, 1688, 1608, 1527, 1351, 1207,
1153, 1092.
Anal. Calcd for C18H23N3O5: C, 59.82; H, 6.41; N, 11.63. Found: C,
59.87; H, 6.45; N, 11.72.
13C NMR (75 MHz, CDCl3):
d 165.9, 153.5, 149.2, 141.9, 133.8,
133.3, 128.6, 127.7, 116.1, 103.8, 60.2, 53.1, 44.7, 16.0, 14.1.
IR (KBr, cmꢂ1): 3341, 3212, 3093, 2942, 1691, 1621, 1420, 1321,
1204, 1109.
5.19. Ethyl 6-methyl-4-(3-nitrophenyl)-1-octyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (3.1c)
Anal. Calcd for C17H19ClN2O3: C, 60.99; H, 5.72; N, 8.37. Found: C,
60.87; H, 5.79; N, 8.43.
White solid (72% yield).
mp 98e99 ꢀC (Ethyl acetate/n-Hexane).
1H NMR (500 MHz, CDCl3):
d 8.17e8.14 (m, 2H, ArH), 7.62 (d, 1H,
5.16. Ethyl 4-(4-chlorophenyl)-1-cinnamyl-6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (2.1h)
J ¼ 8 Hz, ArH), 7.50 (t, 1H, J ¼ 8 Hz, ArH), 5.80 (brs, 1H, NH), 5.49 (d,
1H, J ¼ 3 Hz, CH), 4.17e4.11 (m, 2H, ester-CH2), 3.99e3.94 (m, 1H,
one H of NeCH2e), 3.62e3.56 (m, 1H, one H of NeCH2e), 2.58 (s,
3H, CH3), 1.58e1.54 (m, 2H, one eCH2- of n-octyl), 1.29e1.26 (m,
10H, five eCH2- of n-octyl), 1.23 (t, 3H, J ¼ 7.5 Hz, ester-CH3), 0.89 (t,
3H, J ¼ 7 Hz, eCH3 of n-octyl).
Pale yellow solid (69% yield).
mp 116e117 ꢀC (Ethyl acetate/n-Hexane).
1H NMR (300 MHz, CDCl3):
d 7.44e7.24 (m, 9H, ArH), 6.42 (d, 1H,
J ¼ 15.9 Hz, one H of NeCH2eCH]CH-), 6.25e6.16 (m, 1H, one H of
NeCH2eCH]), 5.52 (brs, 1H, NH), 5.43 (d, 1H, J ¼ 3 Hz, CH), 4.69
(dd, 1H, J1 ¼ 4.5 Hz, J2 ¼ 16.5 Hz, one H of NeCH2e), 4.50 (dd, 1H,
J1 ¼ 4.5 Hz, J2 ¼ 16.2 Hz, one H of NeCH2e), 4.14 (q, 2H, J ¼ 7.2 Hz,
ester-CH2), 2.58 (s, 3H, CH3), 1.21 (t, 3H, J ¼ 7.2 Hz, ester-CH3).
13C NMR (75 MHz, CDCl3):
d 165.8, 153.5, 150.3, 148.5, 145.9,
132.5, 129.7,122.9,121.7,103.6, 60.6, 53.5, 42.9, 31.9, 30.0, 29.4, 26.9,
22.7, 16.3, 14.3, 14.2.
IR (KBr, cmꢂ1): 3216, 3093, 2925, 2855, 1691, 1617, 1530, 1344,
1158, 1098.
13C NMR (75 MHz, CDCl3):
d
165.9, 153.7, 149.2, 141.8, 136.1,
Anal. Calcd for C22H31N3O5: C, 63.29; H, 7.48; N, 10.06. Found: C,
63.22; H, 7.41; N, 9.94.
133.4, 131.5, 128.7, 128.6, 127.8, 127.7, 126.3, 125.0, 104.4, 60.3, 53.0,
44.3, 16.1, 14.1.
IR (KBr, cmꢂ1): 3221, 3102, 2980, 1690, 1619, 1399, 1310, 1267,
1203, 1179, 1104.
5.20. Ethyl 1-decyl-6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-
Anal. Calcd for C23H23ClN2O3: C, 67.23; H, 5.64; N, 6.82. Found: C,
67.31; H, 5.69; N, 6.79.
tetrahydropyrimidine-5-carboxylate (3.1d)
White solid (71% yield).
5.17. Ethyl 1,6-dimethyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (3.1a) [21]
mp 84e85 ꢀC (Ethyl acetate/n-Hexane).
1H NMR (300 MHz, CDCl3):
d 8.15e8.11(m, 2H, ArH), 7.61 (m, 1H,
ArH), 7.51e7.49 (m, 1H, ArH), 5.81 (brs, 1H, NH), 5.48 (s, 1H, CH),
4.13 (q, 2H, J ¼ 7.1 Hz, ester-CH2), 3.95e3.93 (m, 1H, one H of
NeCH2e), 3.59e3.58 (m, 1H, one H of NeCH2e), 2.56 (s, 3H, CH3),
1.76 (m, 2H, one eCH2- of n-decyl), 1.58 (m, 2H, one eCH2- of n-
decyl),1.25e1.08 (m, 15H, six eCH2- of n-decyl & ester-CH3), 0.88
(t, 3H, J ¼ 6.9 Hz, eCH3 of n-decyl).
White solid (90% yield).
mp 136e137 ꢀC (Ethyl acetate/n-Hexane).
1H NMR (300 MHz, CDCl3):
d 8.13e8.10 (m, 2H, ArH), 7.59 (d, 1H,
J ¼ 7.5 Hz, ArH), 7.48 (t, 1H, J ¼ 7.5 Hz, ArH), 5.97 (brs, 1H, NH), 5.50
(d, 1H, J ¼ 3.3 Hz, CH), 4.13 (q, 2H, J ¼ 7.2 Hz, ester-CH2), 3.26 (s, 3H,
NeCH3), 2.56 (s, 3H, CH3), 1.21 (t, 3H, J ¼ 7.2 Hz, ester-CH3).
13C NMR (125 MHz, CDCl3):
d 165.8, 153.4, 150.2, 148.6, 145.9,
13C NMR (75 MHz, CDCl3):
d
165.6, 154.2, 150.8, 148.2, 145.4,
132.5,129.8,122.9,121.7,103.6, 60.6, 53.6, 43.0, 32.0, 30.0, 29.7, 29.7,
29.4, 26.9, 22.8, 16.3, 14.3, 14.2.
132.1, 129.5, 122.6, 121.6, 102.8, 60.4, 52.8, 30.4, 16.5, 14.1.
IR (KBr, cmꢂ1): 3236, 3104, 2982, 2936, 1683, 1620, 1527, 1385,
1347, 1305, 1172, 1075.
IR (KBr, cmꢂ1): 3222, 3095, 2923, 2853, 1690, 1622, 1532, 1345,
1161, 1099.
Anal. Calcd for C15H17N3O5: C, 56.42; H, 5.37; N, 13.16. Found: C,
56.47; H, 5.40; N, 13.15.
Anal. Calcd for C30H47N3O5: C, 68.02; H, 8.94; N, 7.93. Found: C,
68.11; H, 8.82; N, 7.88.

230
S. Putatunda et al. / European Journal of Medicinal Chemistry 54 (2012) 223e231
5.21. Ethyl 1-hexadecyl-6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-
5.24. Ethyl 1-cinnamyl-6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (3.1e)
tetrahydropyrimidine-5-carboxylate (3.1h)
White solid (68% yield).
White solid (68% yield).
mp 86e87 ꢀC (Ethyl acetate/n-Hexane).
mp 125e126 ꢀC (Ethyl acetate/n-Hexane).
1H NMR (500 MHz, CDCl3):
d
8.14e8.10 (m, 2H, ArH), 7.59 (d, 1H,
1H NMR (300 MHz, CDCl3):
d 8.19e8.11 (m, 2H, ArH), 7.64 (d, 1H,
J ¼ 7.5 Hz, ArH), 7.47 (t,1H, J ¼ 8 Hz, ArH), 6.24 (brs, 1H, NH), 5.47 (d,
1H, J ¼ 2.5 Hz, CH), 4.12 (q, 2H, J ¼ 7.5 Hz, ester-CH2), 3.97e3.91 (m,
1H, one H of NeCH2e), 3.59e3.53 (m, 1H, one H of NeCH2e), 2.55
(s, 3H, CH3),1.80e1.50 (m, 2H, one eCH2- of n-hexadecyl),1.31e1.25
(m, 26H, thirteen eCH2- of n-hexadecyl), 1.19 (t, 3H, J ¼ 7.5 Hz,
ester-CH3), 0.87 (t, 3H, J ¼ 7 Hz, eCH3 of n-hexadecyl).
J ¼ 7.8 Hz, ArH), 7.46 (t, 1H, J ¼ 7.8 Hz, ArH), 7.33e7.26 (m, 5H, ArH),
6.47 (d, 1H, J ¼ 16.2 Hz, one H of NeCH2eCH]CH-), 6.27e6.19 (m,
1H, NeCH2eCH]), 5.87 (d, 1H, J ¼ 2.4 Hz, NH), 5.53 (d, 1H,
J ¼ 3.3 Hz, CH), 4.70 (dd, 1H, J1 ¼ 6 Hz, J2 ¼ 16.8 Hz, one H of
NeCH2e), 4.52 (dd, 1H, J1 ¼ 5.7 Hz, J2 ¼ 16.8 Hz, one H of NeCH2e),
4.13 (q, 2H, J ¼ 7.2 Hz, ester-CH2), 2.61 (s, 3H, CH3), 1.21 (t, 3H,
J ¼ 7.2 Hz, ester-CH3).
13C NMR (125 MHz, CDCl3):
d 165.8, 153.4, 150.2, 148.6, 145.9,
132.5, 129.8, 122.9, 121.7,103.7, 60.6, 53.6, 43.0, 32.1, 30.0, 29.8, 29.8,
29.7, 29.5, 29.4, 27.0, 22.8, 16.3, 14.3, 14.2.
IR (KBr, cmꢂ1): 3216, 3093, 2923, 2851, 1691, 1621, 1535, 1471,
1344, 1278, 1184, 1100.
13C NMR (75 MHz, CDCl3):
d 165.6, 153.5, 150.1, 148.4, 145.6,
136.1, 132.3, 132.0, 129.6, 128.6, 127.9, 126.3, 124.9, 122.7, 121.6,
103.6, 60.5, 53.3, 44.7, 16.3, 14.1.
IR (KBr, cmꢂ1): 3222, 3105, 2981, 1690, 1617, 1527, 1344, 1207,
1171, 1109.
Anal. Calcd for C30H47N3O5: C, 68.02; H, 8.94; N, 7.93. Found: C,
68.09; H, 8.90; N, 7.91.
Anal. Calcd for C23H23N3O5: C, 65.55; H, 5.50; N, 9.97. Found: C,
65.60; H, 5.53; N, 9.92.
HRMS (m/z) for C30H47N3O5Na [M þ H]þ : Calculated 530.3594,
found 530.3584.
HRMS (m/z) for C23H23N3O5 [M]þ : Calculated 421.1638, found
421.1639.
5.22. Ethyl 1-benzyl-6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-
5.25. Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo-1-phenethyl-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (3.1f)
tetrahydropyrimidine-5-carboxylate (3.1i)
Pale yellow solid (68% yield).
White solid (30% yield).
mp 138e139 ꢀC (Ethyl acetate/n-Hexane).
mp 122e123 ꢀC (Ethyl acetate/n-Hexane).
1H NMR (300 MHz, CDCl3):
d
8.04e8.00 (m, 2H, ArH), 7.47 (d,1H,
1H NMR (300 MHz, CDCl3):
d 8.17e8.13 (m, 2H, ArH), 7.58 (d, 1H,
J ¼ 7.5 Hz, ArH), 7.34 (t, 1H, J ¼ 7.8 Hz, ArH), 7.24e7.13 (m, 3H, ArH),
7.06e7.03 (m, 2H, ArH), 6.23 (d, 1H, J ¼ 3 Hz, NH), 5.44 (d, 1H,
J ¼ 3 Hz, CH), 5.13 (d, 1H, J ¼ 16.5 Hz, one H of NeCH2e), 4.79 (d, 1H,
J ¼ 16.5 Hz, one H of NeCH2e), 4.00 (q, 2H, J ¼ 7.2 Hz, ester-CH2),
2.39 (s, 3H, CH3), 1.08 (t, 3H, J ¼ 7.2 Hz, ester-CH3).
J ¼ 7.5 Hz, ArH), 7.49 (t, 1H, J ¼ 7.8 Hz, ArH), 7.32e7.20 (m, 5H, ArH),
5.80 (brs, 1H, NH), 5.48 (d, 1H, J ¼ 2.7 Hz, CH), 4.22e4.09 (m, 3H,
ester-CH2 & one H of NeCH2e), 3.91e3.83 (m, 1H, one H of
NeCH2e), 2.95e2.88 (m, 2H, benzylic CH2), 2.55 (s, 3H, CH3), 1.21 (t,
3H, J ¼ 7.2 Hz, ester-CH3).
13C NMR (75 MHz, CDCl3):
d
165.5, 153.9, 150.3, 148.3, 145.5,
13C NMR (75 MHz, CDCl3):
d 165.6, 153.3, 149.7, 148.3, 145.7,
137.6, 132.5, 129.5, 128.8, 127.3, 126.2, 122.7, 121.6, 103.5, 60.5, 53.2,
46.0, 16.5, 14.1.
138.0, 132.3, 129.6, 128.6, 128.6, 126.6, 122.7, 121.6, 103.3, 60.4, 53.3,
44.2, 35.9, 16.1, 14.1.
IR (KBr, cmꢂ1): 3223, 3107, 2945, 1688, 1628, 1531, 1390, 1351,
1276, 1207, 1175, 1115.
IR (KBr, cmꢂ1): 3265, 3099, 2975, 2933, 1684, 1610, 1527, 1351,
1157, 1098.
Anal. Calcd for C21H21N3O5: C, 63.79; H, 5.35; N, 10.63. Found: C,
63.72; H, 5.38; N, 10.66.
Anal. Calcd for C22H23N3O5: C, 64.54; H, 5.66; N, 10.26. Found: C,
64.58; H, 5.61; N, 10.30.
HRMS (m/z) for C21H21N3O5Na [M þ Na]þ : Calculated 418.1379,
found 418.1377.
HRMS (m/z) for C22H23N3O5Na [M þ Na]þ : Calculated 432.1535,
found 432.1534.
5.26. Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo-1-(3-phenylpropyl)-
5.23. Ethyl 1-allyl-6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (3.1j)
tetrahydropyrimidine-5-carboxylate (3.1g)
White solid (62% yield).
White solid (76% yield).
mp 110e112 ꢀC (Ethyl acetate/n-Hexane).
mp 96e97 ꢀC (Ethyl acetate/n-Hexane).
1H NMR (300 MHz, CDCl3):
d 8.15e8.09 (m, 2H, ArH), 7.59 (d, 1H,
1H NMR (300 MHz, CDCl3):
d
8.17e8.11 (m, 2H, ArH), 7.61 (d, 1H,
J ¼ 7.8 Hz, ArH), 7.45 (t, 1H, J ¼ 7.8 Hz, ArH), 7.31e7.14 (m, 5H, ArH),
6.20 (brs, 1H, NH), 5.47 (d, 1H, J ¼ 2.4 Hz, CH), 4.12 (q, 2H, J ¼ 6.9 Hz,
ester-CH2), 3.96e3.91 (m, 1H, one H of NeCH2e), 3.65e3.60 (m, 1H,
one H of NeCH2e), 2.62 (t, 2H, J ¼ 7.5 Hz, benzylic CH2), 2.44 (s, 3H,
CH3), 1.99e1.87 (m, 2H, two H of NeCH2eCH2-), 1.20 (t, 3H,
J ¼ 6.9 Hz, ester-CH3).
J ¼ 7.5 Hz, ArH), 7.48 (t, 1H, J ¼ 7.8 Hz, ArH), 6.12 (brs, 1H, NH),
5.94e5.81 (m, 1H, one H of NeCH2eCH]), 5.52 (d, 1H, J ¼ 3.3 Hz,
CH), 5.23e5.10 (m, 2H, two H of NeCH2eCH]CH2), 4.52 (dd, 1H,
J1 ¼ 4.8 Hz, J2 ¼ 17.1 Hz, one H of NeCH2e), 4.36 (dd, 1H, J1 ¼ 4.8 Hz,
J2 ¼ 17.1 Hz, one H of NeCH2e), 4.12 (q, 2H, J ¼ 7.2 Hz, ester-CH2),
2.56 (s, 3H, CH3), 1.20 (t, 3H, J ¼ 7.2 Hz, ester-CH3).
13C NMR (75 MHz, CDCl3):
d 165.6, 153.6, 149.9, 148.3, 145.6,
13C NMR (75 MHz, CDCl3):
d
165.6, 153.4, 150.3, 148.2, 145.5, 133.6,
140.8, 132.2, 129.6, 128.4, 128.2, 126.1, 122.6, 121.5, 103.4, 60.4, 53.1,
42.2, 32.8, 31.0, 16.0, 14.1.
132.3, 129.5, 122.6, 121.6, 116.3, 103.0, 60.4, 53.1, 44.9, 16.0, 14.1.
IR (KBr, cmꢂ1): 3237, 3103, 2985, 2917, 1689, 1627, 1529, 1403,
1348, 1187, 1111.
IR (KBr, cmꢂ1): 3229, 3099, 2922, 1688, 1615, 1526, 1341, 1166,
1102.
Anal. Calcd for C17H19N3O5: C, 59.12; H, 5.55; N, 12.17. Found: C,
59.08; H, 5.53; N, 12.20.
Anal. Calcd for C23H25N3O5: C, 65.24; H, 5.95; N, 9.92. Found: C,
65.18; H, 5.99; N, 9.95.
HRMS (m/z) for C17H19N3O5Na [M þ H]þ : Calculated 346.1403,
found 346.1394.
HRMS (m/z) for C23H25N3O5Na [M þ Na]þ : Calculated 446.1692,
found 446.1692.

S. Putatunda et al. / European Journal of Medicinal Chemistry 54 (2012) 223e231
231
5.27. Purification of Ca2þ-ATPase from microsomal membranes of
goat spermatozoa
JRF fellowship to SP), Bose Institute to PN and SC and Council of
Scientific and Industrial Research to SG. We are grateful to Dr. Rina
Ghosh (Jadavpur University), Dr. Dilip Kumar Maiti (University of
Calcutta) and Mr. Saikat Bhattacharya for their various help in
different stages of work.
Ca2þ-ATPase was purified from the enzyme enriched microsomal
membrane of goat spermatozoa as per previously reported literature
[9]. The spermatozoawere collected from the caudal regionof thegoat
epididymis in a buffer containing 25 mM imidazole, 0.25 mM sucrose,
1 mM EDTA and 1 mM 2-mercaptoethanol (pH-7.5). The sperm
suspension was then homogenized in a Teflon homogenizer and spun
at 12,000 rpm for 20 min at 4 ꢀC. The supernatant was collected and
centrifuged at 100,000g for 1 h. The pellet was re-suspended in the
Appendix A. Supplementary data
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2012.04.043.
above mentioned buffer and was found to be enriched with Ca2þ
ATPase.
-
5.28. Assay of Mg2þ-dependent and independent Ca2þ-ATPase
activities
References
For Mg2þ-dependent Ca2þ-ATPase activity assay, the assay
mixture contained in a final volume of 0.25 ml, 50 mM histidine
(pH 7.4) buffer in 25 mM sucrose, 0.5 mM EDTA, 1 mM 2-mer-
[1] K. Kumsars, A. Velena, G. Duburs, J. Uldrikis, A. Zidermane, Biokhimiya 36
(1971) 1201e1209.
[2] M. Ashok, B.S. Holla, N.S. Kumari, Eur. J. Med. Chem. 42 (2007) 380e385.
[3] J.M. Blacquiere, O. Sicora, C.M. Vogels, M. Cuperlovic-Culf, A. decken,
R.J. Ouellette, S.A. Westcott, Can. J. Chem. 83 (2005) 2052e2059.
[4] R. Kumar, S. Malik, R. Chandra, J. Chem. 48B (2009) 718e724.
[5] C.O. Kappe, Acc. Chem. Res. 33 (2000) 879e888.
[6] C.O. Kappe, Eur. J. Med. Chem. 35 (2000) 1043e1052.
[7] K. Singh, D. Arora, K. Singh, S. Singh, Mini-Reviews Med. Chem. 9 (2009)
95e106.
[8] E. Carafoli, M. Crompton, Curr. Top. Membr. Trans. 10 (1978) 151e216.
[9] R. Sikdar, U. Ganguly, P. Pal, B. Mazumder, P.C. Sen, Mol. Cell. Biochem. 103
(1991) 121e130.
[10] S. Vijayasarathy, S. Shivaji, P. Balaram, FEBS Lett. 114 (1980) 45e47.
[11] H. Breitbart, S. Rubinstein, L. Nass-Arden, J. Biol. Chem. 260 (1985)
11548e11553.
captoethanol, 1 mM MgCl2, 3 mM CaCl2, 3 mM ATP, 5 mg enzyme,
in absence and presence of the DHPM derivatives. The reaction
mixture was pre-incubated for 5 min at 37 ꢀC, followed by
addition of ATP and incubation was continued for further 30 min.
The reaction was then terminated with 6% cold TCA. The liberated
inorganic phosphate (Pi) was estimated colorimetrically to
determine the amount of enzymatically released Pi by the addi-
tion of a solution containing 1.75% ammonium molybdate and 2%
ascorbic acid (6:1). The blue colour developed was measured at
820 nm using
a spectrophotometer [28]. The values were
[12] H.T. Zeng, D.R.P. Tulsiani, J. Cell Sci. 116 (2003) 1981e1989.
[13] C.O. Kappe, G. Uray, P. Roschger, W. Linder, C. Kartky, W. Keller, Tetrahedron
48 (1992) 5473.
expressed as the difference in activity in the presence of
Mg2þ þ Ca2þ, and Mg2þ alone. DHPMs inhibitors are soluble in
DMSO. Same concentration of DMSO was used as a positive
control for all the above experiments and the calculations have
been done accordingly.
[14] C.O. Kappe, Tetrahedron 23 (1993) 360.
[15] P. Wipf, A. Cunningham, Tetrahedron Lett. 36 (1995) 7819.
[16] A. Stadler, C.O. Kappe, J. Comb. Chem. 3 (2001) 624e630.
[17] C.O. Kappe, Bioorg. Med. Chem. Lett. 10 (2000) 49.
[18] C.O. Kappe, QSAR -Comb. Sci. 22 (2003) 630e645.
[19] A. Dondoni, A. Massi, Mol. Divers. 6 (2003) 261e270.
[20] G. Aridoss, Y.T. Jeong, Bull. Korean Chem. Soc. 31 (2010) 863.
[21] D. Dallinger, C.O. Kappe, Synlett (2002) 1901e1903.
[22] K. Singh, D. Arora, E. Poremsky, J. Lowery, R.S. Moreland, Eur. J. Med. Chem. 44
(2009) 1997e2001.
[23] B.C. Ranu, A. Hajra, U. Jana, J. Org. Chem. 65 (2000) 6270.
[24] J. Lu, Y. Bai, Synthesis 4 (2002) 466e470.
[25] F.S. Falsone, C.O. Kappe, Arkivoc (2001) 122e134.
[26] A. Steinmetz, The Broad Scope of Cesium Salts in Organic Chemistry,
Catalysts Cesium from Acros Organics, pp 3e11 and the references cited in
the article.
For Mg2þ-independent Ca2þ-ATPase activity assay, above
procedure was used except that the reaction was done in imidazole
buffer and the ATPase activity was determined as the difference in
activity in presence and absence of Ca2þ and no Mg2þ was present
in the mixture.
To determine the effect of verapamil on Mg2þ-dependent and
independent Ca2þ-ATPase activity assay in the absence and presence
of the DHPM derivatives, the I50 concentration of the DHPM deriv-
atives were used at a fixed concentration (300 mM) of verapamil.
[27] G. Dijkstra, W.H. Kruizinga, R.M. Kellogg, J. Org. Chem. 52 (1987)
4230e4234.
[28] R.N. Salvatore, A.S. Nagle, K.W. Jung, J. Org. Chem. 67 (2002) 674e683.
[29] C. Viedma, Astrobiology 7 (2007) 312e319.
[30] D.G. Blackmond, Chem.eEur. J. 13 (2007) 3290e3295.
[31] S. Ghoshal, T. Sengupta, P.C. Sen, BioFactors 26 (2006) 259e271.
Acknowledgements
We gratefully acknowledge financial supports from University
Grants Commission (UGC MRP No. PSW-053/10-11 to AC and UGC-