Synthesis and pharmacological investigation of 3-(substituted 1-phenylethanone)-4-(substituted phenyl)-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylates

Article abstract of DOI:10.1016/j.ejmech.2009.02.021

Fifteen new ethyl 6-methyl-2-methoxy-3-(substituted 1-phenylethanone)-4-(substituted phenyl)-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylates (6a-o) have been synthesized in a two step reaction. In first step ethyl acetoacetate, s-methylisourea and appropri

Full text of DOI:10.1016/j.ejmech.2009.02.021

European Journal of Medicinal Chemistry 44 (2009) 3645–3653
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and pharmacological investigation of 3-(substituted
1-phenylethanone)-4-(substituted phenyl)-1, 2, 3,
4-tetrahydropyrimidine-5-carboxylates
*
R.V. Chikhale , R.P. Bhole, P.B. Khedekar, K.P. Bhusari
Department of Medicinal Chemistry, Sharad Pawar College of Pharmacy, Wanadongri, Hingna Road, Nagpur 441 110, Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Fifteen new ethyl 6-methyl-2-methoxy-3-(substituted 1-phenylethanone)-4-(substituted phenyl)-1, 2, 3,
4-tetrahydropyrimidine-5-carboxylates (6a–o) have been synthesized in a two step reaction. In first step
ethyl acetoacetate, s-methylisourea and appropriate benzaldehydes reacted in a single step reaction to
obtain ethyl 6-methyl-2-methoxy-4-(substituted phenyl)-1, 4-dihydropyrimidine-5-carboxylates (4a–e).
Second step involves synthesis of reaction between substituted phenacyl bromides and 1-4-dihy-
dropyrimidine-5-carboxylates (6a–o). Their structures are confirmed by IR, 1H NMR, mass and elemental
analyses. The compounds were tested for antihypertensive activity by non-invasive tail-cuff, and eval-
uated by carotid artery cannulation method for determining the diastolic blood pressure. Hypertension
was induced by DOCA-salt. Anti-inflammatory activity was carried out by carrageenan induced rat-paw
oedema method. Test compounds 6b, 6c, 6e, 6f, 6j, 6h, 6k, 6l, 6m, 6n and 6o exerted comparative
antihypertensive activity at 10 mg/kg dose level compared to nifedipine. Compounds 6j, 6m and 6o
showed excellent results on evaluation by direct method. Test compounds 6a–6h, 6l, 6m, 6n and 6o
exerted moderate to comparative anti-inflammatory activity at the 100 mg/kg dose level compared to
indomethacin. Their further investigation for analgesic activity and acute ulcerogenesis was carried out,
compounds 6m, 6f, 6k, 6o showed excellent to good analgesic activity and low ulcerogenic activity.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 10 November 2008
Received in revised form
2 February 2009
Accepted 17 February 2009
Available online 27 February 2009
Keywords:
Biginelli reaction
DOCA-salt hypertension
Non-invasive tail-cuff method
Carotid artery cannulation
Antihypertensive activity
Anti-inflammatory activity
Analgesic activity
Ulcerogenic activity
1. Introduction
mixture was identified correctly by Biginelli as 3, 4-dihydropyr-
imidine-2(1H)-one [1].
Similar groups/structures often exhibit similar biological activ-
ities. However, they usually exhibit different potency. The tradi-
tional structure–activity relationship (SAR) is a useful tool in the
search for new drugs. However, SAR is usually determined by
making minor changes to the structure of the existing compound
and assessing the effect on its biological activity. Similarly, struc-
tural analogy has played vital role in designing compounds with
higher potency. One of such structural analogy is seen between 4-
aryl-1, 4-dihydropyridines (DHPs) of the nifedipine type and
dihydropyrimidines (DHPMs). In 1893 Italian chemist Pietro Bigi-
nelli reported on the acid-catalyzed cyclocondensation reaction of
ethyl acetoacetate, benzaldehyde and urea. The reaction was
carried out simply by heating a mixture of three components dis-
solved in ethanol with a catalytic amount of hydrochloric acid at
reflux temperature. The product of this novel one pot, three-
component synthesis that precipitated on cooling of the reaction
The synthetic potential of this new heterocyclic synthesis
remained unexplored for quite some time. In the 1970s and 1980s
interest slowly increased, and the scope of the original cyclo-
condensation reaction was gradually extended by variation of all
three building blocks, allowing access to a large number of multi-
functionalized dihydropyrimidines [2].
In the past decades, a broad range of biological effects, including
antiviral [3], antitumor [4], antibacterial [5] and anti-inflammatory
[6] activities have been ascribed to these partly reduced pyrimidine
derivatives. More recently, DHPMs have emerged as, for e.g., orally
active antihypertensive agents [7]. A very recent highlight in this
context been the identification of the structurally rather simple
DHPM monastrol as a mitotic kinesin motor protein inhibitor and
potential new lead for the development of anticancer drugs [8].
Appropriately functionalized DHPM derivatives have emerged as
potent calcium channel modulators [9]. Apart from synthetic
DHPM derivatives several marine natural products with interesting
biological activities containing the dihydropyrimidine-5-carbox-
ylate core have recently been isolated. Most among these are the
batzelladine alkaloids A and B which inhibit the binding of HIV
* Corresponding author.
E-mail address: rupeshchikhale7@gmail.com (R.V. Chikhale).
0223-5234/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ejmech.2009.02.021

3646
R.V. Chikhale et al. / European Journal of Medicinal Chemistry 44 (2009) 3645–3653
envelop protein gp-120 to human CD4 cells and therefore, are
potential new leads for AIDS therapy [10].
R
NH
O
H5C2
2. Chemistry
+
O
+
H2N
OCH3
O
H3C
The chemistry of pyrimidine-5-carboxylates has been of great
interest. 4-Aryl-1, 4-dihydropyridines of the nifedipine type (I, II)
are most studied class of organic calcium channel modulators, since
their introduction. They have become drugs of immense impor-
tance for the treatment of hypertension, cardiac arrhythmias, etc. In
recent years, interest has also been focused on aza-analogues such
as dihydropyrimidines of types III and IV which show similar
pharmacological profile to classical dihydropyridine calcium
channel modulators, the reported lead compounds show superi-
ority in potency and duration of activity.
O
H
1
2
3
Dimethyl formamide
Sodium bicarbonate
Reflux
3
4
R
R
H
a
b
c
a
b
c
Cl
NO2
O
NO2
OH
CH3
OCH3
H
N
H5C2
d
e
d
e
O
O
O
CH3
N
O
O
H3C
H3C
CH3
O
O
CH3
H3C
N
H
OCH3
O
O
H3C
N
H
CH3
H3C
N
H
CH3
4 a-e
I
II
Scheme 1.
pressure were performed using Power Lab/4SP with ML135 Dual
Bio Amp computerized BP monitor automatic cardiovascular
system (AD instruments Pvt. Ltd., Australia). Anti-inflammatory
activity of synthesized compounds was carried out by carrageenan
induced rat-paw oedema [22] method using UGO BASTILE Ple-
thysmometer 7140. Analgesic activity was carried out by acetic acid
induced writhing method [23]. Acute ulcerogenesis test was done
according to Cioli et al. [24].
CH3
O2N
O
O
S
O
O
O
H3C
NH2
CH3
O
N
H
N
O
N
H
CH3
H3C
N
H
4. Results and discussion
All the compounds synthesized are novel. These derivatives
were obtained from the two step synthesis, their structures were
confirmed by IR, NMR and elemental analyses. We have shown that
2-hetero-1, 4-dihydropyrimidines can be synthesized with selec-
tive substitution of the para-substituted electrophiles at N3 posi-
tion. This selectivity is believed to be due to electron density at N3
and N1. The former being richer in electron density is more reactive
and produces products of exclusive functionalization at N3. Fifteen
derivatives were synthesized, antihypertensive activity was carried
out initially for all the test compounds, those compounds which
were found out to show significant activity by non-invasive
(Tail-cuff method) technique were further evaluated for antihy-
pertensive activity by direct cannulation method. Anti-inflamma-
tory activity was carried out followed by analgesic and acute
ulcerogenesis studies.
III
IV
The pyrimidine-5-carboxylate substituted at third position, i.e.,
on nitrogen in the pyrimidine ring gives antihypertensive activity
similar to nifedipine-type calcium channel modulator, substitution
at the third nitrogen is possible and the resultants can be strong
contenders of anti-inflammatory, antihypertensive activities
[11–18]. It involves the application of Biginelli reaction and its
modifications.
In the first step three-component reaction involving o-methyl-
isourea hydrogensulfate, ethyl acetoacetate and substituted benz-
aldehydes (3a–e) reacted in the presence of sodium bicarbonate
and dimethylformamide to form the substituted ring nucleus
compounds (4a–e) (Scheme 1).
On reaction with various substituted phenacyl bromides 5a–c,
they undergo nucleophilic substitution reaction in the presence of
a base such as pyridine, to form their respective derivatives (6a–o)
(Scheme 2).
4.1. Antihypertensive activity
Antihypertensive activity carried out by the non-invasive
method gave the systolic blood pressure (SBP), from which the
observations are summarized in Table 1. For structure–activity
studies we choose the aromatic substitutions that are commonly
employed in dihydropyridines. 4-Methoxy derivative 6n has
remarkable antihypertensive activity. 3, 4-Disubstituted methoxy
derivative 6o has shown good antihypertensive activity at 10 mg/
kg. Although 3-methoxy 4-hydroxy derivative (6i) is less potent
than the corresponding 6n and 6o. 3, 4-Dichloro analogues are
moderately potent than 6n and 6o. Data are presented as
3. Pharmacology
Antihypertensive activity of synthesized compounds was
carried out by model or method of Deoxycorticosterone Acetate salt
(DOCA-salt) induced hypertension in rats [19–21]. The non-invasive
method to determine systolic blood pressure (SBP), invasive
method to determine diastolic blood pressure (DBP) and mean
arterial blood pressure (MAP) for determining the changes in blood

R.V. Chikhale et al. / European Journal of Medicinal Chemistry 44 (2009) 3645–3653
3647
R
R
O
R1
R1
O
H
O
H
Br
Substituted phenacyl bromides
5a-c
N
H5C2O
H3C
N
H5C2O
O
Pyridine
OCH3
N
H
H3C
OCH3
N
H
5
a
b
c
R1
4a-e
6a-o
H
Cl
OCH3
Comp. 6a 6b
6c
6d 6e
Cl Cl OCH3 OH OH
Cl Cl
6f
6g
6h
6i
6j
6k
6l
CH3 OCH3 OCH3 OCH3
OCH3 Cl OCH3
6m
6n
6o
H
H
H
H
OH
CH3 CH3
Cl
R
Cl OCH3
H
H
Cl OCH3
H
H
R1
Scheme 2.
means ꢀ S.E.M., a repeated measures analysis of variance was used
to obtain the statistical significance between and within groups.
Differences were considered statistically significant at a P level
lower than 0.05 and F value for all compounds are F: 22.33 ꢀ 0.5.
Their results for percentage inhibition are as shown in Table 2 and
Fig. 1 respectively.
From the ANOVA (Analysis of Variance) and % inhibition studies,
it was concluded that except compounds 6a, 6d, 6g and 6i all other
compounds showed significant antihypertensive activity. Those
drugs that showed significant and encouraging results were tested
at lower dose levels of 5 and 2.5 mg/kg respectively.
Those compounds that showed significant activity by tail-cuff
method were furtherevaluated for theirantihypertensive activity by
direct cannulation of the carotid artery. Antihypertensive activity
carried out by cannulation method gave the diastolic blood pressure
(DBP), from which the observations are given in Table 3 Data are
presented as means ꢀ S.E.M., a repeated measures analysis of vari-
ance was used to obtain the statistical significance between and
within groups. Differences were considered statistically significant
at a P level lower than 0.05 and F value for all compounds are F: ꢀ0.5.
Their results are as shown in Table 4 and Fig. 2 respectively.
4.2. Anti-inflammatory activity
Anti-inflammatory activity data is summarized in Table 5. The
effect of structural modification on potency in vitro, and anti-
inflammatory activity with a series of dihydropyrimidines is
summarized in Table 6. The structural activity data suggest that
potency in vitro for anti-inflammatory was optimized in 6m.
Aromatic substitution having methyl 6j and hydroxyl 6g
analogues being considerably less potent then corresponding 6m.
Other compounds show moderate to low activity. Data are pre-
sented as means ꢀ S.E.M., a repeated measures analysis of vari-
ance was used to obtain the statistical significance between and
within groups. Differences were considered statistically significant
at a P level lower than 0.05 and F value for all compounds are
F: 34.61 ꢀ 0.5, their results are as shown in Tables 5 and 6, and
Fig. 3 respectively.
P < 0.0001 and the F value for all compounds are also statisti-
cally (99.99%) significant (F: 34.61 ꢀ 0.5). The readings obtained
from rat-paw oedema method are given in Table 5.
From the ANOVA and % inhibition studies, it was concluded that
compounds 6i and 6j show low anti-inflammatory activity,
Table 1
Antihypertensive activity data obtained by tail-cuff method at 10 mg/kg dose.
Compound (10 mg/kg)
Average systolic blood pressure (mmHg) at time (min)
0
15
30
60
120
180
240
300
360
400
460
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
225 ꢀ 4
226 ꢀ 8
226 ꢀ 8
225 ꢀ 4
225 ꢀ 6
226 ꢀ 3
224 ꢀ 4
226 ꢀ 2
225 ꢀ 4
226 ꢀ 4
226 ꢀ 8
225 ꢀ 8
222 ꢀ 3
228 ꢀ 3
226 ꢀ 3
225 ꢀ 2
225 ꢀ 1
222 ꢀ 8
224 ꢀ 5
224 ꢀ 5
222 ꢀ 8
223 ꢀ 5
224 ꢀ 6
222 ꢀ 8
224 ꢀ 3
222 ꢀ 8
222 ꢀ 8
220 ꢀ 5
220 ꢀ 0
220 ꢀ 6
220 ꢀ 6
220 ꢀ 6
224 ꢀ 1
221 ꢀ 2
221 ꢀ 9
210 ꢀ 5
210 ꢀ 5
220 ꢀ 9
210 ꢀ 6
210 ꢀ 3
220 ꢀ 9
210 ꢀ 8
221 ꢀ 9
220 ꢀ 9
200 ꢀ 5
200 ꢀ 5
210 ꢀ 3
210 ꢀ 3
210 ꢀ 3
225 ꢀ 1
215 ꢀ 1
195 ꢀ 2
193 ꢀ 4
193 ꢀ 4
190 ꢀ 2
195 ꢀ 8
193 ꢀ 5
190 ꢀ 2
190 ꢀ 4
195 ꢀ 2
193 ꢀ 2
193 ꢀ 4
191 ꢀ 4
193 ꢀ 5
183 ꢀ 5
190 ꢀ 5
224 ꢀ 3
195 ꢀ 3
180 ꢀ 4
178 ꢀ 5
178 ꢀ 5
178 ꢀ 4
180 ꢀ 5
178 ꢀ 7
175 ꢀ 4
172 ꢀ 5
180 ꢀ 4
185 ꢀ 4
170 ꢀ 5
170 ꢀ 0
178 ꢀ 7
170 ꢀ 7
175 ꢀ 7
224 ꢀ 4
180 ꢀ 2
175 ꢀ 8
161 ꢀ 4
161 ꢀ 4
172 ꢀ 8
160 ꢀ 8
161 ꢀ 2
172 ꢀ 8
160 ꢀ 4
175 ꢀ 8
175 ꢀ 8
161 ꢀ 4
161 ꢀ 7
161 ꢀ 2
161 ꢀ 2
160 ꢀ 2
224 ꢀ 1
168 ꢀ 1
172 ꢀ 6
142 ꢀ 4
142 ꢀ 4
167 ꢀ 6
145 ꢀ 3
142 ꢀ 5
162 ꢀ 6
142 ꢀ 4
172 ꢀ 6
170 ꢀ 6
142 ꢀ 4
142 ꢀ 2
140 ꢀ 5
140 ꢀ 5
140 ꢀ 5
225 ꢀ 1
145 ꢀ 2
165 ꢀ 4
138 ꢀ 6
138 ꢀ 6
160 ꢀ 4
140 ꢀ 6
134 ꢀ 6
160 ꢀ 4
135 ꢀ 6
165 ꢀ 4
165 ꢀ 4
138 ꢀ 6
135 ꢀ 6
131 ꢀ 6
131 ꢀ 6
128 ꢀ 6
224 ꢀ 4
125 ꢀ 2
160 ꢀ 6
131 ꢀ 7
131 ꢀ 7
158 ꢀ 6
135 ꢀ 7
130 ꢀ 7
154 ꢀ 6
130 ꢀ 7
160 ꢀ 6
161 ꢀ 6
130 ꢀ 7
130 ꢀ 7
128 ꢀ 7
128 ꢀ 7
125 ꢀ 7
225 ꢀ 2
125 ꢀ 1
158 ꢀ 3
125 ꢀ 5
125 ꢀ 5
152 ꢀ 3
125 ꢀ 5
125 ꢀ 5
150 ꢀ 3
125 ꢀ 5
158 ꢀ 3
155 ꢀ 3
125 ꢀ 5
125 ꢀ 0
123 ꢀ 5
113 ꢀ 5
120 ꢀ 5
224 ꢀ 3
122 ꢀ 2
155 ꢀ 3
122 ꢀ 8
122 ꢀ 8
145 ꢀ 3
125 ꢀ 7
121 ꢀ 8
145 ꢀ 3
122 ꢀ 8
155 ꢀ 3
150 ꢀ 3
122 ꢀ 8
122 ꢀ 2
121 ꢀ 8
110 ꢀ 5
115 ꢀ 8
225 ꢀ 2
120 ꢀ 3
Control
Nifedipine

3648
R.V. Chikhale et al. / European Journal of Medicinal Chemistry 44 (2009) 3645–3653
Table 2
Comparative study of inhibition (%) for antihypertensive activity by tail-cuff method.
Compound
Inhibition (%)
0
15
30
1.43
60
120
180
240
300
360
420
480
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
0.8
0.71
0.1
0.8
0.1
0.1
0.36
0.9
0.8
0.5
0.1
0.1
0.9
0.9
0.9
–
0.59
0.7
1.0
0.91
0.27
0.1
0.59
1.0
0.27
0.59
1.61
1.82
1.57
1.57
1.57
–
12.98
13.78
13.78
15.21
12.71
13.74
15.21
15.12
12.71
13.74
13.78
14.67
13.74
18.19
15.06
–
19.61
20.46
20.46
20.5
19.57
20.37
21.84
23.13
19.61
17.38
24.02
24.0
21.56
27.98
27.98
22.9
28.25
28.07
23.0
28.43
21.56
27.98
28.0
28.1
23.33
36.74
36.80
25.62
35.46
36.76
27.77
36.73
23.33
36.80
36.80
36.90
36.0
26.3
28.69
41.52
41.60
29.7
39.75
42.0
31.35
41.97
28.0
41.97
41.97
41.97
42.85
42.85
41.85
–
29.43
45.0
45.1
31.04
46.48
46.48
35.57
44.19
46.06
35.48
45.48
31.04
45.48
45.74
46.30
45.91
51.90
48.57
–
6.49
6.49
1.87
6.45
6.58
1.87
6.36
1.43
1.87
11.09
11.09
6.58
6.58
6.58
–
38.24
38.30
28.35
37.35
40.02
28.53
39.58
26.3
38.30
39.50
39.58
41.35
41.35
40.1
32.1
44.05
44.05
33.0
44.05
29.43
44.05
44.0
44.05
44.98
49.39
46.51
–
20.36
23.82
21.59
–
28.0
28.0
27.8
36.0
36.0
–
Control
Nifedipine
–
–
0.4
1.29
4.44
12.92
19.69
24.98
35.49
44.20
44.51
45.51
46.58
whereas, compounds 6a–6h, 6l, 6m, 6n and 6o showed moderate
to comparative activity of indomethacin, which was used as stan-
dard. Furthermore, those compounds which were found to be
significant were screened at lower dose level of 50 and 25 mg/kg
respectively.
are presented in Table 7. Structural activity data for analgesic and
ulcerogenic activity shows that both the activity were favored by 4-
methoxy aromatic substitution 6o, 4-hydroxy, 4-methyl substitu-
tions being considerably less active compound with standard
(ibuprofen). Other compounds show moderate to low activity.
4.3. Analgesic activity
5. Experimental section
The analgesic activity of the synthesized compounds 6a–6h and
6k–6o was evaluated by acetic acid induced writhing test. The
activity showed that compound 6m exhibited maximum analgesic
activity (59.76%) and its activity was comparable with the standard
drug ibuprofen (62.55%) at a dose of 20 mg/kg p.o. Compounds 6f,
6k, and 6o showed good activity (51.33–53.17%). Results are pre-
sented in Table 7. Analysis of result showed that substitution of
chlorine, methyl and methoxyl group in the ring structure resulted
in increased activity.
5.1. Chemistry: general procedures
Chemicals were obtained from Fluka Chemical Co. (Germany).
Melting points (m.p.) were detected with open capillaries using
Thermonik Precision Melting point cum Boiling point apparatus
(C-PMB-2, Mumbai, India) and are uncorrected. IR spectra (KBr)
were recorded on FTIR-8400s spectrophotometer (Shimadzu,
Japan). 1H NMR was obtained using a Varian EM 390 Spectropho-
tometer (Shimadzu, Japan) using CDCl3. All chemical shift values
were recorded as d (ppm). The purity of compounds was controlled
4.4. Acute ulcerogenesis
by thin layer chromatography (Merck, silica gel, HF254–361, type 60,
0.25 mm, Darmstadt, Germany). The elementary analysis was per-
formed at RTM Nagpur University, India. Elementary analyses for C,
H, N were within ꢀ0.4% of theoretical values.
The compounds, which showed significant anti-inflammatory
activity, were screened for their ulcerogenic activity. The test was
performed according to Cioli et al. The tested compounds showed
ulcerogenic activity ranging from 0.16 to 0.76, whereas the standard
drug ibuprofen showed high severity index of 1.97. The results
indicate that compounds are devoid of ulcerogenic action. Results
5.1.1. General procedure for preparation of compounds 4a–e
A mixture of o-methylisourea hydrogensulfate (60 mmol), ethyl
acetoacetate (55 mmol), and substituted benzaldehydes (50 mmol)
6a
6b
Table 3
60
Antihypertensive activity data obtained by direct cannulation of carotid artery
method at 10 mg/kg dose.
6c
6d
50
6e
Compound Average diastolic blood pressure (mmHg) at time (min)
(10 mg/kg)
6f
0
15
30
60
120
180
240
40
6g
6b
6c
6e
6f
6h
6j
6k
6l
6m
6n
6o
195 ꢀ 8 190 ꢀ 5 176 ꢀ 5 150 ꢀ 4 136 ꢀ 5 121 ꢀ 4 102 ꢀ 4
198 ꢀ 8 191 ꢀ 5 178 ꢀ 5 149 ꢀ 4 136 ꢀ 5 121 ꢀ 4 102 ꢀ 4
196 ꢀ 6 191 ꢀ 5 178 ꢀ 6 149 ꢀ 8 135 ꢀ 5 120 ꢀ 8 105 ꢀ 3
195 ꢀ 3 189 ꢀ 6 174 ꢀ 3 150 ꢀ 5 137 ꢀ 7 121 ꢀ 2 102 ꢀ 5
197 ꢀ 2 192 ꢀ 3 175 ꢀ 8 151 ꢀ 4 136 ꢀ 5 120 ꢀ 4 102 ꢀ 4
197 ꢀ 4 190 ꢀ 8 176 ꢀ 9 150 ꢀ 2 137 ꢀ 4 125 ꢀ 8 100 ꢀ 6
196 ꢀ 8 190 ꢀ 5 175 ꢀ 5 151 ꢀ 4 135 ꢀ 5 121 ꢀ 4 102 ꢀ 4
196 ꢀ 8 189 ꢀ 0 173 ꢀ 5 149 ꢀ 4 136 ꢀ 0 121 ꢀ 7 102 ꢀ 2
195 ꢀ 3 190 ꢀ 6 175 ꢀ 3 151 ꢀ 5 136 ꢀ 7 121 ꢀ 2 101 ꢀ 5
196 ꢀ 3 191 ꢀ 6 176 ꢀ 3 148 ꢀ 5 137 ꢀ 7 121 ꢀ 2 102 ꢀ 5
196 ꢀ 3 188 ꢀ 6 172 ꢀ 3 148 ꢀ 5 134 ꢀ 7 118 ꢀ 2 100 ꢀ 5
195 ꢀ 2 195 ꢀ 1 196 ꢀ 1 195 ꢀ 3 195 ꢀ 4 195 ꢀ 1 196 ꢀ 1
6h
30
6i
6j
20
6k
6l
10
6m
6n
0
6o
0
15 30 60 120 180 240 300 360 420 480
Time (min.)
Std.
Control
Nifedipine 197 ꢀ 1 190 ꢀ 2 173 ꢀ 1 149 ꢀ 3 135 ꢀ 2 118 ꢀ 1 101 ꢀ 2
Fig. 1. Graph showing (%) decrease in systolic blood pressure (SBP).

R.V. Chikhale et al. / European Journal of Medicinal Chemistry 44 (2009) 3645–3653
3649
Table 4
Table 5
Comparative study of inhibition (%) for antihypertensive activity by carotid artery
cannulation method.
Anti-inflammatory activity data at 10 mg/kg dose.
Compound (10 mg/kg)
Average paw volume
Compound
Inhibition (%)
0 h
1 h
3 h
5 h
0
15
30
9.99
8.97
8.92
11.11
10.35
9.79
10.50
11.52
10.62
10.09
12.13
–
60
120
180
240
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
1.15 ꢀ 0.01
1.20 ꢀ 0.02
1.20 ꢀ 0.01
1.15 ꢀ 0.02
1.14 ꢀ 0.01
1.21 ꢀ 0.02
1.14 ꢀ 0.01
1.20 ꢀ 0.01
1.18 ꢀ 0.01
1.17 ꢀ 0.01
1.21 ꢀ 0.02
1.16 ꢀ 0.02
1.18 ꢀ 0.01
1.21 ꢀ 0.02
1.21 ꢀ 0.01
1.86 ꢀ 0.02
1.11 ꢀ 0.01
1.12 ꢀ 0.02
1.18 ꢀ 0.02
1.17 ꢀ 0.01
1.12 ꢀ 0.02
1.12 ꢀ 0.02
1.17 ꢀ 0.02
1.12 ꢀ 0.02
1.18 ꢀ 0.02
1.16 ꢀ 0.02
1.16 ꢀ 0.01
1.17 ꢀ 0.02
1.14 ꢀ 0.02
1.14 ꢀ 0.02
1.16 ꢀ 0.02
1.16 ꢀ 0.01
1.86 ꢀ 0.01
1.04 ꢀ 0.01
1.10 ꢀ 0.02
1.15 ꢀ 0.02
1.15 ꢀ 0.01
1.10 ꢀ 0.02
1.10 ꢀ 0.02
1.14 ꢀ 0.02
1.10 ꢀ 0.02
1.14 ꢀ 0.02
1.15 ꢀ 0.02
1.16 ꢀ 0.01
1.14 ꢀ 0.02
1.12 ꢀ 0.02
1.11 ꢀ 0.01
1.13 ꢀ 0.02
1.12 ꢀ 0.01
1.86 ꢀ 0.02
0.91 ꢀ 0.02
1.10 ꢀ 0.01
1.10 ꢀ 0.02
1.10 ꢀ 0.01
1.10 ꢀ 0.01
1.10 ꢀ 0.01
1.0 ꢀ 0.02
1.10 ꢀ 0.01
1.10 ꢀ 0.01
1.14 ꢀ 0.01
1.16 ꢀ 0.01
1.0 ꢀ 0.02
1.10 ꢀ 0.01
0.9
6b
6c
6e
6f
6h
6j
6k
6l
6m
6n
6o
Control
Nifedipine
0.3
1.8
2.35
1.84
1.84
2.81
1.43
2.20
2.35
3.12
2.30
1.79
3.33
–
22.99
23.50
23.29
22.93
22.47
23.09
22.47
22.52
22.89
23.52
23.52
–
30.14
30.14
30.65
29.52
30.14
30.65
30.39
30.04
30.87
30.12
31.06
–
37.77
37.77
38.08
37.87
38.28
35.52
37.77
37.62
37.87
37.87
39.41
–
47.78
47.78
46.30
47.73
47.78
48.69
47.78
47.88
48.24
47.73
48.75
–
0.7
0.05
1.02
1.12
0.8
0.8
0.05
0.5
0.5
–
0.9
2.51
11.72
23.55
30.80
39.41
48.39
1.10 ꢀ 0.02
1.0 ꢀ 0.01
1.86 ꢀ 0.01
0.93 ꢀ 0.01
Control
Indomethacin
was mixed together with sodium bicarbonate (200 mmol) and
100 mL of dimethylformamide, reaction mixture was heated at
70 ꢁC for 12 h. After cooling to room temperature, the mixture was
diluted with 150 mL of brine and extracted with ether (2 ꢂ 150 mL),
dried over magnesium sulfate and excess of solvent was removed
under reduced pressure. After complete drying the product was
collected and recrystallized from ethanol. The structure of
compounds was confirmed by IR, 1H NMR, mass and element
analyses.
(t, J ¼ 7.38 Hz, 3H, ethyl ester). ESMS: m/z (MHþ) 289. Anal.
(C15H18N2O4) C(62.06/62.09), H(6.25/6.22), N(9.65/9.69).
5.1.1.3. Ethyl 6-methyl-2-methoxy-4-(4-methylphenyl)-1, 4-dihy-
dropyrimidine-5-carboxylate (4c). Yield: 76%, m.p. 144–147 ꢁC. Rf:
0.78 (chloroform–benzene 20:80). IR (KBr)
n
¼ 1687.6 cmꢃ1 (C]O),
1392.5, 3176 (N–H), 1292.22 (C–N). 1H NMR (CDCl3)
d 8.34 (s, 1H,
N1–H), 8.15 (d, J ¼ 7.9 Hz, 1H, aromatic), 8.17 (s, 1H, aromatic), 7.74
(d, J ¼ 7.9 Hz,1H, aromatic), 7.51 (t, J ¼ 7.9 Hz,1H, aromatic), 7.32 (m,
5H, aromatic), 5.98, 4.18 (d, J ¼ 5.29 Hz, 2H, benzylic), 6.00 (s, 1H,
methine), 4.10 (m, 2H, ethyl ester), 2.59 (s, 3H, methyl) and 1.18 (t,
J ¼ 7.38 Hz, 3H, ethyl ester). ESMS: m/z (MHþ) 288. Anal.
(C16H20N2O3) C(66.65/66.68), H(6.99/6.96), N(9.72/9.70).
5.1.1.1. Ethyl 6-methyl-2-methoxy-4-(phenyl)-1, 4-dihydropyrimidine-
5-carboxylate (4a). Yield: 64.47%, m.p. 202–204 ꢁC. Rf: 0.56 (chlo-
roform–benzene 50:50). IR (KBr)
n
¼ 1687.6 cmꢃ1 (C]O), 1392.5,
3176 (N–H), 1292.22 (C–N). 1H NMR (CDCl3)
d 8.32 (s, 1H, N1–H), 8.15
(d, J ¼ 7.9 Hz, 1H, aromatic), 8.11 (s, 1H, aromatic), 7.64 (d, J ¼ 7.9 Hz,
1H, aromatic), 7.51 (t, J ¼ 7.9 Hz, 1H, aromatic), 7.32 (m, 5H,
aromatic), 5.98, 4.18 (d, J ¼ 5.29 Hz, 2H, benzylic), 6.00 (s, 1H,
methine), 4.19 (m, 2H, ethyl ester), 2.39 (s, 3H, methyl) and 1.40–1.70
(d, J ¼ 7.38 Hz, 2H, ethyl ester). ESMS: m/z (MHþ) 274. Anal.
(C15H18N2O3) C(65.68/65.66), H(6.61/6.50), N(10.21/10.24).
5.1.1.4. Ethyl 6-methyl-2-methoxy-4-(4-methoxyphenyl)-1, 4-dihy-
dropyrimidine-5-carboxylate (4d). Yield: 75%, m.p. 157–159 ꢁC. Rf:
0.64 (chloroform–benzene 20:80). IR (KBr)
n
¼ 1687.6 cmꢃ1 (C]O),
1392.5, 3176 (N–H), 1292.22 (C–N). 1H NMR (CDCl3)
d 8.33 (s, 1H,
N1–H), 8.14 (d, J ¼ 7.9 Hz, 1H, aromatic), 8.16 (s, 1H, aromatic), 7.66
(d, J ¼ 7.9 Hz, 1H, aromatic), 7.54 (t, J ¼ 7.9 Hz, 1H, aromatic), 7.33
(m, 5H, aromatic), 5.96, 4.18 (d, J ¼ 5.29 Hz, 2H, benzylic), 6.00 (s,
1H, methine), 4.11 (m, 2H, ethyl ester), 2.342 (s, 3H, methyl) and
1.14 (t, J ¼ 7.38 Hz, 3H, ethyl ester). ESMS: m/z (MHþ) 305. Anal.
(C16H20N2O4) C(63.14/63.16), H(6.62/6.63), N(9.20/9.21)
5.1.1.2. Ethyl 6-methyl-2-methoxy-4-(4-hydroxyphenyl)-1, 4-dihy-
dropyrimidine-5-carboxylate (4b). Yield: 64%, m.p. 216–218 ꢁC. Rf:
0.71 (chloroform–benzene 20:80). IR (KBr)
n
¼ 1687.6 cmꢃ1 (C]O),
1392.5, 3176 (N–H), 1292.22 (C–N). 1H NMR (CDCl3)
d 8.32 (s, 1H,
N1–H), 8.15 (d, J ¼ 7.9 Hz, 1H, aromatic), 8.13 (s, 1H, aromatic), 7.64
(d, J ¼ 7.9 Hz,1H, aromatic), 7.51 (t, J ¼ 7.9 Hz,1H, aromatic), 7.42 (m,
5H, aromatic), 5.90, 4.18 (d, J ¼ 5.29 Hz, 2H, benzylic), 6.00 (s, 1H,
methine), 4.11 (m, 2H, ethyl ester), 2.49 (s, 3H, methyl) and 1.18
Table 6
Comparative study results for inhibition (%) at 10 mg/kg dose for anti-inflammatory
activity with indomethacin as standard.
Compound
% Inhibition [1 ꢃ (Dt/Dc) ꢂ 100]
0 h
1 h
3 h
5 h
6b
60
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
38.09
35.57
35.62
38.25
38.82
35.83
38.84
35.62
36.62
39.09
35.83
37.15
36.62
35.83
35.80
–
39.83
36.62
37.09
39.83
39.83
37.15
39.83
36.62
37.63
37.78
37.15
38.82
38.82
37.78
37.70
–
41.03
38.36
38.41
41.03
41.03
38.76
40.91
38.76
38.25
37.85
38.76
39.83
40.55
39.48
39.83
–
42.05
42.0
6c
6e
50
42.65
42.05
42.05
47.26
42.05
42.05
39.94
38.89
47.26
42.05
52.63
42.0
6f
40
6h
6j
30
6k
6l
20
6m
10
6n
6o
0
std
0
15
30
60
Time (min.)
120
180
240
47.2
–
52.63
Control
Indomethacin
Fig. 2. Graph showing (%) decrease in diastolic blood pressure (DBP) by carotid artery
cannulation method.
41.0
46.38
51.84

3650
R.V. Chikhale et al. / European Journal of Medicinal Chemistry 44 (2009) 3645–3653
n
¼ 1687.6 cmꢃ1 (C]O), 1392.51, 3203 (N–H), 1325.01 (C–Cl)
Anti inflammatory activity
1292.22 (C–N). 1H NMR (CDCl3)
d
7.76 (d, J ¼ 7 Hz, 1H), 7.72 (t,
60
50
40
30
20
10
0
J ¼ 7.0 Hz, 1H), 7.56 (t, J ¼ 7 Hz, 1H), 7.39 (d, J ¼ 7.0 Hz, 1H), 7.05 (d,
J ¼ 10 Hz, 2H), 6.57 (d, J ¼ 10 Hz, 2H), 5.89 (s, 1H), 4.9–4.7 (m, 1H),
3.93 (q, J ¼ 6 Hz, 2H), 3.8 (s, 3H), 2.45 (s, 3H), 0.98 (t, J ¼ 6 Hz, 3H).
ESIMS: m/z (MHþ) 429. Anal. (C23H25 ClN2O4) C(64.41/64.44),
H(5.88/5.90), N(6.5/6.2).
Activity at 5h
5.1.2.3. Ethyl 6-methyl-2-methoxy-3-[1-(4-methoxyphenyl) etha-
none]-4-(phenyl)-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylate
(6c). Yield: 64%, m.p. 108–110 ꢁC. Rf: 0.67 (ethanol–benzene 40:60).
6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 6o std
compound
IR (KBr)
n
¼ 1691.6 cmꢃ1 (C]O), 1390.58, 3245 (N–H), 1355.86 (O–
Fig. 3. Graph showing decrease in inflammation compared with Std. (indomethacin).
CH3), 1292.22 (C–N). 1H NMR (CDCl3)
d
7.76 (d, J ¼ 7 Hz, 1H), 7.64 (t,
J ¼ 7.0 Hz, 1H), 7.55 (t, J ¼ 7 Hz, 1H), 7.39 (d, J ¼ 7.0 Hz, 1H), 7.04 (d,
J ¼ 10 Hz, 2H), 6.57 (d, J ¼ 10 Hz, 2H), 5.94 (s, 1H), 4.8–4.6 (m, 1H),
3.93 (q, J ¼ 6 Hz, 2H), 3.9 (s, 3H), 2.45 (s, 3H), 0.96 (t, J ¼ 6 Hz, 3H).
ESIMS: m/z (MHþ) 424. Anal. (C24H28N2O5) C(67.91/67.93), H(6.65/
6.68), N(6.60/6.63).
5.1.1.5. Ethyl 4-(4-chlorophenyl)-6-methyl-2-methoxy-1, 2, 3, 4-tet-
rahydropyrimidine-5-carboxylate (4e). Yield: 64%, m.p. 240–245 ꢁC.
Rf: 0.74 (chloroform–benzene 20:80). IR (KBr)
n
¼ 1687.6 cmꢃ1
(C]O),1392.5, 3176 (N–H),1292.22 (C–N). 1H NMR (CDCl3)
d 8.31 (s,
1H, N1–H), 8.17 (d, J ¼ 7.9 Hz, 1H, aromatic), 8.14 (s, 1H, aromatic),
7.66 (d, J ¼ 7.9 Hz, 1H, aromatic), 7.54 (t, J ¼ 7.9 Hz, 1H, aromatic),
7.30 (m, 5H, aromatic), 5.96, 4.18 (d, J ¼ 5.29 Hz, 2H, benzylic), 6.00
(s, 1H, methine), 4.11 (m, 2H, ethyl ester), 2.49 (s, 3H, methyl) and
1.18 (t, J ¼ 7.38 Hz, 3H, ethyl ester). ESMS: m/z (MHþ) 307. Anal.
(C15H17ClN2O3) C(58.35/58.40), H(5.55/5.51), N(9.07/9.09).
5.1.2.4. Ethyl 6-methyl-2-methoxy-3-(1-phenylethanone)-4-(4-chlor-
ophenyl)-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylate (6d). Yield:
60%, m.p. 115–120 ꢁC. Rf: 0.67 (ethanol–benzene 40:60). IR (KBr)
n
¼ 1690.6 cmꢃ1 (C]O), 1390.58, 3245 (N–H), 1355.86 (O–CH3),
1325.01 (C–Cl), 1292.22 (C–N). 1H NMR (CDCl3)
d
7.77 (d, J ¼ 7 Hz,
1H), 7.62 (t, J ¼ 7.0 Hz, 1H), 7.58 (t, J ¼ 7 Hz, 1H), 7.35 (d, J ¼ 7.0 Hz,
1H), 7.03 (d, J ¼ 10 Hz, 2H), 6.52 (d, J ¼ 10 Hz, 2H), 5.91 (s,1H), 4.8–4.6
(m,1H), 3.93 (q, J ¼ 6 Hz, 2H), 3.3 (s, 3H), 2.47 (s, 3H), 0.95 (t, J ¼ 6 Hz,
3H). ESIMS: m/z (MHþ) 429. Anal. (C23H25 ClN2O4) C(64.41/64.38),
H(5.88/5.89), N(6.53/6.50).
5.1.2. General procedure for preparation of compounds 6a–o
A mixture of ethyl 6-methyl-2-methoxy-4-(substituted phenyl)-
1, 4-dihydropyrimidine-5-carboxylates 4a–e (0.01 mmol) and
substituted phenacyl bromides 5a–c (0.01 mmol) were taken in
dichloromethane (20 mL) as solvent and pyridine (1.5 mL) as
a catalyst. The resultant mixture was refluxed for 7 h. Then it was
cooled to room temperature and poured on to crushed ice. It was
kept overnight and filtered to obtain solid, which was dried and
recrystallized from ethanol.
5.1.2.5. Ethyl 6-methyl-2-methoxy-3-[1-(4-chlorophenyl) ethanone]-
4-(4-chlorophenyl)-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylate
(6e). Yield: 55%, m.p. 110–115 ꢁC. Rf: 0.67 (ethanol–benzene 40:60).
IR (KBr)
n
¼ 1690.6 cmꢃ1 (C]O), 1390.58, 3245 (N–H), 1355.86 (O–
CH3), 1330, 1325.01 (C–Cl), 1292.22 (C–N). 1H NMR (CDCl3)
d 7.75 (d,
5.1.2.1. Ethyl 6-methyl-2-methoxy-3-(1-phenylethanone)-4-(phenyl)-
J ¼ 7 Hz, 1H), 7.64 (t, J ¼ 7.0 Hz, 1H), 7.45 (t, J ¼ 7 Hz, 1H), 7.38 (d,
J ¼ 7.0 Hz, 1H), 7.04 (d, J ¼ 10 Hz, 2H), 6.67 (d, J ¼ 10 Hz, 2H), 5.95 (s,
1H), 4.8–4.6 (m, 1H), 3.93 (q, J ¼ 6 Hz, 2H), 3.9 (s, 3 H), 2.44 (s, 3H),
0.99 (t, J ¼ 6 Hz, 3H). ESIMS: m/z (MHþ) 464. Anal. (C23H24Cl2N2O4)
C(59.62/59.60), H(5.22/5.27), N(6.05/6.08).
1, 2, 3, 4-tetrahydropyrimidine-5-carboxylate (6a). Yield: 80.88%, m.p.
96–99 ꢁC. Rf: 0.67 (ethanol–benzene 40:60). IR (KBr)
n
¼ 1687.6 cmꢃ1
(C]O), 1392.5, 3176 (N–H), 1292.22 (C–N). 1H NMR (CDCl3)
d 7.78
(d, J ¼ 7 Hz, 1H), 7.64 (t, J ¼ 7.0 Hz, 1H), 7.55 (t, J ¼ 7 Hz, 1 H), 7.37
(d, J ¼ 7.0 Hz,1H), 7.04 (d, J ¼ 10 Hz, 2H), 6.57 (d, J ¼ 10 Hz, 2H), 5.11 (s,
1H), 4.8–4.6 (m,1H), 3.42 (q, J ¼ 6 Hz, 2H), 3.7 (s, 3H), 2.46 (s, 3H), 0.96
(t, J ¼ 6 Hz, 3H). ESIMS: m/z (MHþ) 394. Anal. (C23H26N2O4) C(70.03/
70.06), H(6.64/6.66), N(7.10/7.09).
5.1.2.6. Ethyl 6-methyl-2-methoxy-3-[1-(4-chlorophenyl) ethanone]-
4-(4-methoxyphenyl)-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylate
(6f). Yield: 65%, m.p. 115–120 ꢁC. Rf: 0.67 (ethanol–benzene 40:60).
IR (KBr)
n
¼ 1690.6 cmꢃ1 (C]O), 1390.58, 3245 (N–H), 1355.86
5.1.2.2. Ethyl 6-methyl-2-methoxy-3-[1-(4-chlorophenyl) ethanone]-
4-(phenyl)-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylate (6b). Yield:
45%, m.p. 158–161 ꢁC. Rf: 0.63 (ethanol–benzene 40:60). IR (KBr)
(O–CH3), 1325.01 (C–Cl), 1292.22 (C–N). 1H NMR (CDCl3)
d 7.76 (d,
J ¼ 7 Hz, 1H), 7.62 (t, J ¼ 7.0 Hz, 1H), 7.55 (t, J ¼ 7 Hz, 1H), 7.34
(d, J ¼ 7.0 Hz, 1H), 7.04 (d, J ¼ 10 Hz, 2H), 6.57 (d, J ¼ 10 Hz, 2H), 5.91
(s,1H), 4.8–4.6 (m,1H), 3.93 (q, J ¼ 6 Hz, 2H), 3.7 (s, 3H), 2.45 (s, 3H),
0.96 (t, J ¼ 6 Hz, 3H). ESIMS: m/z (MHþ) 459. Anal. (C24H27ClN2O5)
C(62.81/62.85), H(5.93/5.90), N(6.10/6.15).
Table 7
Analgesic and ulcerogenic activity of compounds 6a–6h and 6k–6o.
Compound
(50 mg/kg)
Analgesic activity
(% protection)
Ulcerogenic activity
(severity index)
5.1.2.7. Ethyl
6-methyl-2-methoxy-3-(1-phenylethanone)-4-(4-
6a
6b
6c
6d
6e
6f
6g
6h
6k
6l
6m
6n
6o
40.14
41.66
45.80
37.87
37.33
51.33
40.33
45.66
53.17
43.33
59.76
49.56
51.33
–
0.16
0.45
0.74
0.45
0.20
0.20
0.18
0.45
0.76
0.56
0.75
0.45
0.20
0.00
1.97
hydroxyphenyl)-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylate
(6g). Yield: 78%, m.p.131–134 ꢁC. Rf: 0.57 (ethanol–benzene 40:60).
IR (KBr)
n
¼ 1691.6 cmꢃ1 (C]O), 1390.51, 3176 (N–H), 1296.22 (C–
N). 1H NMR (CDCl3)
d
7.74 (d, J ¼ 7 Hz, 1H), 7.62 (t, J ¼ 7.0 Hz, 1H),
7.55 (t, J ¼ 7 Hz, 1 H), 7.44 (d, J ¼ 7.0 Hz, 1H), 7.06 (d, J ¼ 10 Hz, 2H),
6.57 (d, J ¼ 10 Hz, 2H), 5.91 (s, 1H), 4.8–4.4 (m, 1H), 3.97 (q, J ¼ 6 Hz,
2H), 3.7 (s, 3H), 2.47 (s, 3H), 0.95 (t, J ¼ 6 Hz, 3H). ESIMS: m/z (MHþ)
411. Anal. (C23H26N2O5) C(67.30/67.35), H(6.38/6.35), N(6.82/6.85).
5.1.2.8. Ethyl 6-methyl-2-methoxy-3-[1-(4-chlorophenyl) ethanone]-
4-(4-hydroxyphenyl)-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylate
(6h). Yield: 57%, m.p. 136–138 ꢁC. Rf: 0.57 (ethanol–benzene
Control
Ibuprofen
62.55

R.V. Chikhale et al. / European Journal of Medicinal Chemistry 44 (2009) 3645–3653
3651
40:60). IR (KBr)
n
¼ 1687.6 cmꢃ1 (C]O), 1390.51, 3203 (N–H),
IR (KBr)
n
¼ 1689.8 cmꢃ1 (C]O),1402.15, 3204 (N–H),1346.01 (C–Cl),
1342.01 (C–Cl), 1296.22 (C–N). 1H NMR (CDCl3)
d
7.76 (d, J ¼ 7 Hz,
1402 (CH3–O–C),1296.22 (C–N).1H NMR (CDCl3)
d 9.55 (s,1H), 8.4 (br
1H), 7.68 (t, J ¼ 7.0 Hz, 1H), 7.56 (t, J ¼ 7 Hz, 1H), 7.35 (d, J ¼ 7.0 Hz,
1H), 7.04 (d, J ¼ 10 Hz, 2H), 6.57 (d, J ¼ 10 Hz, 2H), 5.91 (s, 1H), 4.8–
4.6 (m, 1H), 3.93 (q, J ¼ 6 Hz, 2H), 3.7 (s, 3H), 2.45 (s, 3H), 0.99 (t,
J ¼ 6 Hz, 3H). ESIMS: m/z (MHþ) 444. Anal. (C23H25ClN2O5) C(62.09/
62.11), H(5.66/5.65), N(6.30/6.34).
s,1H), 8.3 (s,1H), 8.10 (d, J ¼ 8.4 Hz,1H), 7.72 (d, J ¼ 7.4 Hz,1H), 7.40 (t,
J ¼ 7.91 Hz,1H), 6.5 (s,1H), 6.3 (br s,1H), 5.10 (t, J ¼ 6.3 Hz,1H), 2.4 (s,
3H),1.20 (d, J ¼ 5.8 Hz, 3H), 1.18 (d, J ¼ 6.3 Hz, 3H). ESIMS: m/z (MHþ)
459. Anal. (C24H27ClN2O5) C(62.81/62.85), H(5.93/5.95), N(6.10/6.11).
5.1.2.15. Ethyl 6-methyl-2-methoxy-3-[1-(4-methoxyphenyl) etha-
none]-4-(4-methoxyphenyl)-1, 2, 3, 4-tetrahydropyrimidine-5-carbox-
ylate (6o). Yield: 74%, m.p. 108–111 ꢁC. Rf: 0.64 (ethanol–benzene
5.1.2.9. Ethyl 6-methyl-2-methoxy-3-[1-(4-methoxyphenyl) etha-
none]-4-(4-hydroxyphenyl)-1, 2, 3, 4-tetrahydropyrimidine-5-carbox-
ylate (6i). Yield: 60%, m.p. 106–108 ꢁC. Rf: 0.57 (ethanol–benzene
40:60). IR (KBr)
n
¼ 1690.8 cmꢃ1 (C]O), 1402.15, 3240 (N–H), 1402
40:60). IR (KBr)
n
¼ 1691.6 cmꢃ1 (C]O), 1390.51, 3245 (N–H),
(CH3–O–C),1402 (CH3–O–C),1296.22 (C–N). 1H NMR (CDCl3)
d 9.78 (s,
2914.24 (O–H), 1296.22 (C–N). 1H NMR (CDCl3)
d
8.76 (d, J ¼ 7 Hz,
1H), 8.7 (br s,1H), 8.2 (s,1H), 8.10 (d, J ¼ 8.4 Hz, 1H), 7.72 (d, J ¼ 7.4 Hz,
1H), 7.52 (t, J ¼ 7.91 Hz, 1H), 6.7 (s, 1H), 6.4 (br, s, 1H), 5.00 (t,
J ¼ 6.3 Hz, 1H), 2.2 (s, 3H), 1.25 (d, J ¼ 5.8 Hz, 3H), 1.26 (d, J ¼ 6.3 Hz,
3H). ESIMS: m/z (MHþ) 455. Anal. (C25H30N2O6) C(66.06/66.06),
H(6.65/6.69), N(6.16/6.18).
1H), 7.62 (t, J ¼ 7.0 Hz, 1H), 7.55 (t, J ¼ 7 Hz, 1H), 7.34 (d, J ¼ 7.0 Hz,
1H), 7.04 (d, J ¼ 10 Hz, 2H), 6.57 (d, J ¼ 10 Hz, 2H), 6.0 (s, 1H), 4.8–4.6
(m,1H), 3.93 (q, J ¼ 6 Hz, 2H), 3.9 (s, 3H), 2.45 (s, 3H), 0.99 (t, J ¼ 6 Hz,
3H). ESIMS: m/z (MHþ) 441. Anal. (C24H28N2O6) C(65.44/65.47),
H(6.41/6.45), N(6.36/6.32).
5.2. Pharmacological study
5.1.2.10. Ethyl 6-methyl-2-methoxy-3-(1-phenylethanone)-4-(4-meth-
ylphenyl)-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylate (6j). Yield:
62.5%, m.p. 116–118 ꢁC. Rf: 0.76 (ethanol–benzene 40:60). IR (KBr)
5.2.1. Antihypertensive activity
5.2.1.1. Non-invasive tail-cuff method. The newly synthesized
compounds 6a–o were used for antihypertensive activity studies.
Norwegian strain of inbred albino rats (male) weighing 200–250 g
were used in experiment. Nifedipine was used as standard drug.
All rats were housed in a temperature and humidity controlled
room with 12-hour light/dark cycle. All rats were allowed free
access to regular food and tap water. The drinking water was
replaced by 1% w/v sodium chloride aqueous solution for rats
used in DOCA experiments. All experimental work was carried out
in accordance with the guidelines provided by the Committee for
the Purpose of Control and Supervision of Experiments in Animal
(CPCSEA), India.
n
¼ 1691.6 cmꢃ1 (C]O), 1390.51, 3176 (N–H), 1296.22 (C–N). 1H NMR
(CDCl3)
d
9.75 (s, 1H), 8.5 (br s, 1H), 8.2 (s, 1H), 8.10 (d, J ¼ 8.4 Hz, 1H),
7.72 (d, J ¼ 7.4 Hz, 1H), 7.48 (t, J ¼ 7.91 Hz, 1H), 6.7 (s, 1H), 6.4 (br, s,
1H), 5.05 (t, J ¼ 6.3 Hz, 1H), 2.4 (s, 3H), 1.29 (d, J ¼ 5.8 Hz, 3H), 1.16 (d,
J ¼ 6.3 Hz, 3H). ESIMS: m/z (MHþ) 409. Anal. (C24H28N2O4) C(70.57/
70.55), H(6.91/6.96), N(6.86/6.85).
5.1.2.11. Ethyl 6-methyl-2-methoxy-3-[1-(4-chlorophenyl) etha-
none]-4-(4-methylphenyl)-1, 2, 3, 4-tetrahydropyrimidine-5-carbox-
ylate (6k). Yield: 45.5%, m.p. 104–107 ꢁC. Rf: 0.68 (ethanol–benzene
40:60). IR (KBr)
n
¼ 1687.6 cmꢃ1 (C]O), 1390.51, 3203 (N–H),
1342.01 (C–Cl) 1296.22 (C–N). 1H NMR (CDCl3)
d 9.75 (s, 1H), 8.5 (br
s, 1H), 8.2 (s, 1H), 8.10 (d, J ¼ 8.4 Hz, 1H), 7.72 (d, J ¼ 7.4 Hz, 1H), 7.48
(t, J ¼ 7.91 Hz, 1H), 6.7 (s, 1H), 6.4 (br s, 1H), 5.05 (t, J ¼ 6.3 Hz, 1H),
2.4 (s, 3H), 1.29 (d, J ¼ 5.8 Hz, 3H),1.16 (d, J ¼ 6.3 Hz, 3H). ESIMS: m/z
(MHþ) 443. Anal. (C24H27 ClN2O5) C(65.08/65.06), H(6.14/6.11),
N(6.32/6.29).
5.2.1.2. DOCA-salt hypertension. Rats were anesthetized by
injecting pentobarbital injection administered intraperitoneally in
a dose of 50 mg/kg body wt. It was placed on a heated surgical
surface maintained at 37 ꢁC. A flank incision was made to expose
the left kidney, which was ligated and removed. This procedure of
removing either of the kidneys is called as uninephrectomy. The
incision was sutured. One week after uninephrectomy, rats were
administered subcutaneously with injection of DOCA (30–50 mg/
kg/week) and drinking water was replaced by 1% w/v sodium
chloride aqueous solution. Control group of rats were unin-
ephrectomized, injection of DOCA-salt was not administered to
them and received vehicle injections and tap water [25].
5.1.2.12. Ethyl 6-methyl-2-methoxy-3-[1-(4-methoxyphenyl) etha-
none]-4-(4-methylphenyl)-1, 2, 3, 4-tetrahydropyrimidine-5-carbox-
ylate (6l). Yield: 66%, m.p. 157–159 ꢁC. Rf: 0.68 (ethanol–benzene
40:60). IR (KBr)
n
¼ 1693.6 cmꢃ1 (C]O), 1390.51, 3245 (N–H), 1402
(CH3–O–C), 1296.22 (C–N). 1H NMR (CDCl3)
d 9.71 (s, 1H), 8.4 (br s,
1H), 8.2 (s, 1H), 8.1 (d, J ¼ 8.4 Hz, 1H), 7.72 (d, J ¼ 7.4 Hz, 1H), 7.48 (t,
J ¼ 7.91 Hz, 1H), 6.8 (s, 1H), 6.4 (br s, 1H), 5.05 (t, J ¼ 6.3 Hz, 1H), 2.6
(s, 3H), 1.29 (d, J ¼ 5.8 Hz, 3H), 1.18 (d, J ¼ 6.3 Hz, 3H). ESIMS: m/z
(MHþ) 439. Anal. (C25H30N2O5) C(68.47/68.50), H(6.90/6.92),
N(6.35/6.38).
5.2.1.2.1. Non-invasive
blood
pressure
(NIBP)
measur-
ements. Indirect blood pressure (BP) was determined with a Power
Lab/4SP with ML135 Dual Bio Amp and computerized BP monitor
(AD instruments Pvt. Ltd., Australia). This system measures systolic
blood pressure (SBP) by recording the cuff pressure at which the
interrupted blood flow returns to the tail. Training the rats for tail-
cuff blood pressure measurements was necessary to reduce the
stress associated with the BP measurements and hence reduces the
variability of BP with successive measurements. Training consisted
of six sessions over 3 days. On day one, rats were introduced into
plastic restrainer for 5 min per session. The tail-cuff was inflated
five times in quick succession. By day three, the training was
extended to 10 min per session. The effect of training was to reduce
the standard deviation around mean BP. At the end of session rats
were ready for BP recording. They were restrained by being placed
into cylindrical restrainer. For better detection of tail pulse, the tail
artery was dilated by placement of restrained mouse into ther-
mostatically controlled Lucite box, heated at 33–34 ꢁC, for 2–5 min
before BP measurement was started [26].
5.1.2.13. Ethyl
6-methyl-2-methoxy-3-(1-phenylethanone)-4-(4-
methoxyphenyl)-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylate
(6m). Yield: 81%, m.p. 134–137 ꢁC. Rf: 0.58 (ethanol–benzene
40:60). IR (KBr)
n
¼ 1687.9 cmꢃ1 (C]O), 1390.51, 3176 (N–H), 1402
(CH3–O–C), 1296.22 (C–N). 1H NMR (CDCl3)
d 9.77 (s, 1H), 8.5 (br s,
1H), 8.4 (s, 1H), 8.10 (d, J ¼ 8.4 Hz, 1H), 7.6 (d, J ¼ 7.4 Hz, 1H), 7.48 (t,
J ¼ 7.91 Hz, 1H), 6.9 (s, 1H), 6.4 (br s, 1H), 5.15 (t, J ¼ 6.3 Hz, 1H), 2.4
(s, 3H), 1.29 (d, J ¼ 5.8 Hz, 3H), 1.17 (d, J ¼ 6.3 Hz, 3H). ESIMS: m/z
(MHþ) 425. Anal. (C24H28N2O5) C(67.91/67.94), H(6.65/6.64),
N(6.60/64).
5.1.2.14. Ethyl 6-methyl-2-methoxy-3-[1-(4-chlorophenyl) ethanone]-
4-(4-methoxyphenyl)-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylate
(6n). Yield: 74%, m.p. 155–158 ꢁC. Rf: 0.78 (ethanol–benzene 40:60).

3652
R.V. Chikhale et al. / European Journal of Medicinal Chemistry 44 (2009) 3645–3653
Tail pulse was detected by passage of tail through a narrow
the sub-plantar tissue of the left hind paw of each rat. Out of this,
one group was treated with indomethacin as standard (100 mg/kg).
The initial volume of paw was measured within 30 s after carra-
geenan injection. Later on paw volume was measured after 1–5 h
respectively. The relative increase in the paw volume was calcu-
lated in the individual animal of the control, test, and standard
groups respectively. The % inhibition of oedema was calculated as
follows:
tail-cuff sensor attached to the amplifier. BP measurements were
started by automatic inflation of tail-cuff to greater than 200 mmHg
and release of pressure. The results were recorded in form of graph.
The computer provides two tracings that start and stop at the same
time. The lower trace channel plots cuff pressure, which is cali-
brated at 500 mmHg at full scale. The tracing sharply rises when
applied to the tail-cuff and falls off gradually during the 15–20 s of
the test. The upper trace channel monitors pulse, with fluctuations
about the centre line suddenly appearing at the onset of pulsations
(Fig. 3). The first onset of pulse is taken as the systolic blood pres-
sure. Initiation of pulse pressure was determined when the baseline
amplitude increased in accordance to the set maximal inflated cuff
pressures, maximal inflation was set at 200 mmHg.
Blood pressure recording was considered to be successful if the
mouse did not move and a clear initial pulse could be seen. Ten tail-
cuff measurements were made in a session. The BP for the session
was accepted as the average of four BP readings that were within
5 mmHg or the average of 10 readings that were within 8 mmHg. BP
measurements were done thrice per week for two weeks.
Twelve groups of six rats weighing between 200 and 250 g were
made. Animals were operated and hypertension was induced as
detailed above. Rats were trained for the experiment. On the first day
of experiment the test compounds were administered by oral feeding
using an oral feeding needle. Compounds 6a–o as test compounds
were prepared in 0.5% carboxymethyl cellulose and dosed 10 mg/kg
orally. One group of animal was treated with standard drug nifedi-
pine. One of the twelve groups was treated with vehicle.
Anti ꢃ inflammatory activity ð%inhibitionÞ [ ½1LðDt=DcÞ3100ꢄ
where Dt is mean relative change in a paw volume in test group and
Dc is mean relative change in paw volume in control group.
The experiment was repeated at two different dose levels (25
and 50 mg/kg) for compounds which showed significant statistical
differences between the test and the control group. ANOVA was
employed as the statistical method.
5.2.3. Analgesic activity: writhing test method
Analgesic activity was carried out by acetic acid induced writhing
method [23] in Swiss albino mice (25–30 g). A 0.6% aqueous acetic
acidsolutionwasinjected intraperitoneally (i.p.) to avolume of 0.1 mL
used as writhing inducing agent. In each group six mice were kept.
Mice were kept individually in test cage, before acetic acid injection
and habituated for 30 min. Screening of analgesic activity was
performed after p.o. administration of test compounds at a dose of
20 mg/kg. The compounds, which exhibited good anti-inflammatory
activity comparable to that of indomethacin, were screened for
analgesic activity. All compounds were dissolved in 1% CMC.
Ibuprofenwasusedasreferencedrug. After 1hofdrugadministration
0.10 mL of 1% acetic acid solution was given to mice intraperitoneally.
Stretching movements consisting of arching of the back, elongation of
bodyand extension of hind limbs were counted for 5–15 min of acetic
acid injection. The analgesic activity was expressed in terms of %
inhibition. % Analgesic activity was calculated as follows:
Prior to dosing the animals, initial graph reading was taken to
record the BP before administration of drug. After 1 h of dosing
recordings were taken as mentioned above. Each compound was
evaluated three times in one week, and average of the recordings
was recorded accordingly.
The experiment was repeated at two different dose levels (5 and
2.5 mg/kg) after animal washing at interval of two weeks respec-
tively, for those compounds that showed significant statistical
differences between the test and control group. Average readings
were calculated by employing ANOVA method.
Analgesic activity ð%inhibitionÞ [ ðnLn0=nÞ3100
where n ¼ mean number of writhes of control group and n0 ¼ mean
number of writhes of test group.
5.2.1.3. Determination of blood pressure by cannulation. Direct
blood pressure measurements were performed to study the dia-
stolic blood pressure (DBP) and mean arterial blood pressure (MAP)
by cannulation technique employing Power Lab/4SP with ML135
Dual Bio Amp and computerized BP monitor (AD instruments Pvt.
Ltd., Australia). Compounds 6b, 6c, 6e, 6f, 6h, 6j, 6k, 6l, 6m, 6n and
6o were tested by direct cannulation method.
Animals were anesthetized with sodium pentobarbital (35 mg/
kg i.p.), and the left carotid artery was exposed, cannulated and
exteriorized between the scapulas. Blood pressure was measured
directly from cannula using transducer attachments of the above
instrument. After animals recovered from surgery and a baseline
blood pressure was established, they were dosed orally with test
compounds via feeding needle. Acute effects were determined by
monitoring blood pressure at 15, 30, 60, 120 and 180 min after oral
dosing. Average readings were calculated by employing ANOVA
method.
Statistical analysis was done using Student’s t-test. The percent
protection in mice brought about by administration of the drug is
shown in table.
5.2.4. Acute ulcerogenesis
Acute ulcerogenesis test was done according to Cioli et al. [24].
Albino rats (150–200 g) were divided into different groups con-
sisting of six animals in each group. Ulcerogenic activity was
evaluated after p.o. administration of test compounds or ibuprofen
at the dose of 50 mg/kg. Control rats received p.o. administration of
vehicle (suspension of 1% methyl cellulose). Food but not water was
removed 24 h before administration of the test compounds. After
the drug treatment, the rats were fed normal diet for 17 h and then
sacrificed. The stomach was removed and opened along the greater
curvature, washed with distilled water and opened along the
greater curvature, washed with distilled water and cleaned gently
by dipping in saline. The gastric mucosa of the rats was examined
by means of a 4ꢂ binocular magnifier. The lesions were counted
and divided into large (greater than 2 mm in diameter), small
(1–2 mm) and punctiform (less than 1 mm). For each stomach the
severity of mucosal damage was assessed according to the
following scoring system: 0dno lesions or up to five punctiform
lesions; 1dmore than five punctiform lesions; 2done to five small
ulcers; 3dmore than five small ulcers or one large ulcer; 4dmore
than one large ulcer.
5.2.2. Anti-inflammatory activity: carrageenan induced
rat-paw oedema method
The method of Winter et al. [22] was employed with some
modifications. All test samples were administered to animals at
a 100 mg/kg dosage, as suspension in 0.5% carboxymethyl cellulose
and administered orally. After 60 min of drug dose, the injection of
0.1 mL of solution of carrageenan (0.5 mg/25 mL) was injected into

R.V. Chikhale et al. / European Journal of Medicinal Chemistry 44 (2009) 3645–3653
3653
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Prof. Jean Jacques Vanden Eynde, University of Mons-Hainaut,
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