The synthesis, anti-inflammatory, and anti-microbial activity evaluation of new series of 4-(3-arylureido)phenyl-1,4-dihydropyridine urea derivatives

Article abstract of DOI:10.1007/s00044-012-0109-8

A new series of 4-(3-arylureido)phenyl-1,4-dihydropyridine urea derivatives were synthesized using simple three component condensation, reduction, and nucleophilic addition sequence in moderate to good yields. All the synthesized compounds 6a-j were evaluated for their anti-inflammatory [against the pro-inflammatory cytokines (tumor necrosis factor-alpha, TNF-α and interleukin-6, IL-6)] and anti-microbial activity (anti-bacterial and anti-fungal). Among all the compound screened, the compound 6b, 6f, and 6j were found to have promising anti-inflammatory activity, 74-83 % TNF-α and 91-96 % IL-6 inhibitory activity, respectively as compared to the standard dexamethasone (71 and 86 % inhibition) but at the MIC of 10 μM/ml. The compounds 6d-e and 6h exhibited relatively lower TNF-α and IL-6 inhibitory activity and found to be moderately potent anti-inflammatory agents. The compounds 6c-e, 6g, and 6i were found to be promising anti-bacterial and anti-fungal agents and remarkably some of the new compounds, viz. 6d and 6i were found be more potent than the standard ciprofloxacin or miconazole. It is to be noted that this is the first report on the anti-inflammatory activity evaluation of novel 1,4-dihydropyridine urea derivatives against the important molecular target, TNF-α, and IL-6.

Full text of DOI:10.1007/s00044-012-0109-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0109-8
ORIGINAL RESEARCH
The synthesis, anti-inflammatory, and anti-microbial activity
evaluation of new series of 4-(3-arylureido)phenyl-1,4-
dihydropyridine urea derivatives
•
Rajesh H. Tale Atish H. Rodge Girish D. Hatnapure
•
•
•
Ashish P. Keche Kalpana M. Patil Rajendra P. Pawar
•
Received: 4 December 2011 / Accepted: 22 May 2012
Ó Springer Science+Business Media, LLC 2012
Abstract A new series of 4-(3-arylureido)phenyl-1,4-
dihydropyridine urea derivatives were synthesized using
simple three component condensation, reduction, and nucle-
ophilic addition sequence in moderate to good yields. All the
synthesized compounds 6a–j were evaluated for their anti-
inflammatory [against the pro-inflammatory cytokines (tumor
necrosis factor-alpha, TNF-a and interleukin-6, IL-6)] and
anti-microbial activity (anti-bacterial and anti-fungal).
Among all the compound screened, the compound 6b, 6f, and
6j were found to have promising anti-inflammatory activity,
74–83 % TNF-a and 91–96 % IL-6 inhibitory activity,
respectively as compared to the standard dexamethasone (71
and 86 % inhibition) but at the MIC of 10 lM/ml. The com-
pounds 6d–e and 6h exhibited relatively lower TNF-a and
IL-6 inhibitory activity and found to be moderately potent
anti-inflammatory agents. The compounds 6c–e, 6g, and 6i
were found to be promising anti-bacterial and anti-fungal
agents and remarkably some of the new compounds, viz. 6d
and 6i were found be more potent than the standard
ciprofloxacin or miconazole. It is to be noted that this is the
first report on the anti-inflammatory activity evaluation of
novel 1,4-dihydropyridine urea derivatives against the
important molecular target, TNF-a, and IL-6.
Keywords 1,4-Dihyropyridine Á Urea derivatives Á
Anti-inflammatory Á Anti-bacterial Á Anti-fungal
Introduction
1,4-Dihydropyridine (1,4-DHP) derivatives is an important
class of bioactive molecules in the pharmaceutical field (Stout
and Meyers, 1982). They possess anti-inflammatory, anti-
microbial (Surendra Kumar et al., 2011), anti-oxidant, and
anti-ulcer activities (Swarnalatha et al., 2011). DHPs are
commercially used as calcium channel blocker for the treat-
ment of cardiovascular diseases, including hypertension
(Gaudio et al., 1994). Recently, the synthesis of DHPs with
respect to multidrug resistance reversal in tumor cell gave a
new dimension to their application (Tanabe et al., 1998;
Tasaka et al., 2001). In addition, 1,4-DHPs are excellent
starting synthons for development of anti-tubercular agent
(Eharkar et al., 2002; Desai et al., 2001). Oxidative aromati-
zation reactions of DHPs are taking place in biological sys-
tems in the presence of certain enzymes. The nitrogen
heterocycles thus prepared by Hantzsch method are of great
importance because of their role in biological system. They
have been served as model compounds for the NAD-NAPH
biological redox systems (Vijesha et al., 2011; Schellenberg and
Weheimer, 1965; Kirschbaum, 1968; Norcross et al., 1962).
Though, anti-microbial activity of 1,4-dihydropyridines
derivatives has been studied and well established in the
literature (Vijesha et al., 2011; Sirisha et al., 2011).
However, there are no reports on the anti-inflammatory
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-012-0109-8) contains supplementary
material, which is available to authorized users.
R. H. Tale (&) Á A. H. Rodge
Department of Chemistry, School of Chemical Sciences,
Swam Ramanand Trith University (SRTM), Nanded,
Maharashtra, India
e-mail: atish.rodge@gmail.com
G. D. Hatnapure Á A. P. Keche
Department of Chemistry, Udaygiri Mahavidyalaya,
Udgir, Maharashtra, India
e-mail: Ashish.keche@gmail.com
K. M. Patil Á R. P. Pawar
P. G. Research Centre, Deogiri College, Station Road,
Aurangabad, Maharashtra, India
123

Med Chem Res
activity of the 1,4-dihydropyridines and most importantly,
the potential of 1,4-dihydropyridines nucleus as to their
anti-inflammatory activity against the pro-inflammatory
cytokines (tumor necrosis factor-alpha, TNF-a and inter-
leukin-6, IL-6) hitherto remained untested.
4-amionophenyl-1,4-DHP 5 with different arylisocyanates
were so rapid that the reactions were to be stopped shortly
(within2 min)failingwhichtheformationofthesideproducts
and decrease in yield was observed. Thus, ten different 4-(3-
arylureido)phenyl-1,4-dihydropyridinurea derivatives 6a–j
were obtained in moderate to good yields using simple three
component condensation, reduction, and nucleophilic addi-
tion reaction sequence and straightforward starting material
under mild conditions. The purity of the newly synthesized
compounds was checked by TLC and HPLC. The 1H NMR
and mass spectral data was found to be consistent with
structures of the newly synthesized 1,4-DHP analogs.
Non-steroidal anti-inflammatory drugs (NSAIDs) are
therapeutically important in the treatment of rheumatic
arthritis and in various types of inflammatory conditions,
but their therapeutic utility has been limited due to their
frequently observed gastrointestinal side effects. Thus,
there is an urgent need for new targets that are required for
the design and development of novel anti-inflammatory
agents as an alternative to NSAIDs (Shishoo et al., 1999).
TNF-a and IL-6, the two important multifunctional pro-
inflammatory cytokines that are involved in the pathogen-
esis of autoimmune, inflammatory, cardiovascular, neuro-
degenerative, and cancer diseases through a series of
cytokine signaling pathways (Krishnamoorthy and Honn,
2006; Dominic and Raj, 2009). IL-6 contributes to the
initiation and extension of the inflammatory process and
considered as a central mediator in a range of inflammatory
diseases but has not received the desired attention in drug
discovery. TNF-a and IL-6 are thus pharmaceutically
important molecular targets for the treatment of the above-
mentioned diseases.
Anti-inflammatory activity
Having secured a series of structurally diverse 4-(3-ary-
lureido)phenyl-1,4-dihydropyridine derivatives, next their
anti-inflammatory and anti-microbial activity was evalu-
ated. The results of the anti-inflammatory, anti-bacterial,
and anti-fungal activity are collected in Tables 1, 2, and 3
respectively. As shown in Table 1, among all the com-
pounds screened, compound 6b, 6f, 6j exhibited promising
TNF-a and IL-6 inhibitory activity ranging from 74–83 and
93–96 %, respectively at MIC of 10 lM. The other com-
pounds, 6d–e and 6h exhibited comparable or little less
anti-inflammatory activity to that of standard dexametha-
sone. It is noteworthy that the compounds 6b (83 and
91 %) and 6j (78 and 96 %) exhibited even higher TNF-a
and IL-6 inhibitory activity than the standard dexametha-
sone (71 and 80 % TNF-a and IL-6 inhibition, respec-
tively) but at MIC of 10 lM/ml. Thus, the two compounds
3b and 3j were found to be the potent anti-inflammatory
agents among the series of compounds 6a–j. The remaining
compounds of this series found to have no activity at all or
very low activity against TNF-a and IL-6.
We have previously reported the anti-inflammatory and
anti-microbial activity of novel 3,4-dihydropyrimidin-
2(1H)-ones urea derivatives (Tale et al., 2011). Encouraged
by the results of our previous work, and to further expand
the scope of 1,4-dihydropyridines derivatives as privileged
medicinal scaffold, herein, we disclose our results on the
synthesis, anti-inflammatory, and anti-microbial activity
evaluation of novel 1,4-dihydropyridines urea derivatives.
Results and discussion
Chemistry
Anti-microbial activity
Our synthetic strategy for novel 4-(3-arylureido)phenyl-1,4-
dihydropyridinureaderivativesisillustrated in Scheme 1. The
key intermediate, 4-nitrophenyl-2,6-dimethyl-1,4-dihydro-
pyridine-3,5-diethylcarboxylate 4 was synthesized by the
three component condensation of 4-nitrobenzaldehyde 1,
ethyl acetoacetate 2, and ammonium acetate 3 using PTSA as
a catalyst in acetonitrile under reflux in high yield. The
reduction of 4 with SnCl2 in ethyl acetate at 40 °C readily
afforded the corresponding amino derivative 5 in almost
quantitative yield. Then, the treatment of the 5 with different
arylisocyanate in THF at room temperature afforded the cor-
responding 4-(3-arylureido)phenyl-2,6-dimethyl-1,4-dihy-
dropyridine-3,5-diethylcarboxylate derivatives 6a–j in
moderate to good yields. It is to be noted that the reactions of
Regarding anti-bacterial and anti-fungal activity (Tables 2,
3) compounds 6c–d and 6i exhibited broad spectrum anti-
bacterial activity against all the bacteria, whereas the
compounds 6c–e, 6g, and 6i found to be the potent anti-
fungal agents against all the fungi at the MIC ranging from
10 to 35 lM/ml. Remarkably, while the compounds 6e and
6i exhibited almost twofold more anti-bacterial activity that
the standard ciprofloxacin almost against all the bacterial
stain tested, the compounds 6d and 6i exhibited much
higher (50 % higher) anti-fungal activity than the standard
miconazole against the selected fungi. Thus, the com-
pounds 6d–e and 6i found to be even more potent anti-
bacterial or anti-fungal agents than standard drugs at the
same concentration level.
123

Med Chem Res
Scheme 1 Reagents and
conditions: (a) PTSA,
NO2
acetonitrile, reflux, 3 h:
(b) SnCl2, EtOAc, 40 °C, 4 h:
(c) different substituted
isocyanates, THF, rt, 2 min
CHO
O
O
O
O
O
a
+
NH4
O
O-
O
O
NO2
N
H
3
1
2
4
R
b
NH
NH2
O
O
NH
c
O
O
O
O
O
O
O
N
H
N
H
R = 6a) 2-Cl,5-CF3
6b) 4-Cl, 2-CF3
6c) 2,4-Cl2
6d) 2,5-Cl2
6e) 2,4-F
6a-j
5
6f) 4-CF3
6g) 4-Cl
6h) 4-F
6i) 4-CN
6j) 2,5-F2
Table 1 Antibacterial activity of thioanalogs of 4-(3-Arylureido)
Phenyl-1, 4-Dihydropyridine urea derivatives (minimum inhibitory
concentrationa values lg/ml)
Table 2 Antifungal activity of thioanalogs of 4-(3-Arylureido) Phe-
nyl-1, 4-Dihydropyridine urea derivatives (minimum inhibitory con-
centrationa values lg/ml)
Compounds Gram-positive
Staphylococcus
aureus
Bacillus Gram- Salmonella
subtilis negative typhimurium
E. coli
Compounds
Candida
albicans
Aspergillus
niger
Fusarium
solani
Aspergillus
flavus
4
–
–
90
90
90
60
25
10
20
90
20
85
25
–
–
4
90
60
80
55
20
15
45
60
65
40
10
–
–
90
40
65
45
10
15
25
90
20
65
15
70
20
90
45
90
60
20
15
25
90
35
65
10
90
25
5
90
65
50
15
10
35
45
30
65
20
85
25
–
90
90
80
20
20
35
70
20
85
20
–
5
85
–
6a
45
85
10
30
20
55
20
45
10
90
20
6a
6b
6c
6d
6e
6f
6g
6h
6i
6b
70
35
25
50
85
80
80
10
–
6c
6d
6e
6f
6g
6h
6i
6j
6j
Miconazole
15
20
Ciprofloxacin 20
25
No activity was observed up to 200 lg/ml
a
No activity was observed up to 200 lg/ml
Values are the average of three reading
a
Values are the average of three reading
effect to the desired target. Thus, the prominent role of
ureido moiety of this novel 1,4-DHP scaffold to impart the
various activities evident by the fact that neither parent
4-nitrophenyl-1,4 DHP, 4 nor the corresponding amino
We believe that the incorporation of arylureido moiety
onto the 4-substituted DHP core might have provided new
chemicals space which resulted into favorable biological
123

Med Chem Res
analogs suggested that the compound 6b, 6f, and 6j
exhibits the promising TNF-a and IL-6 inhibitory activity,
while the compounds 6c–e, 6g, and 6i found to be the
potent anti-bacterial and anti-fungal agents.
Table 3 Anti-inflammatory activity data
Compounds
% Inhibition at 10 lM
TNF-a
IL-6
4
0
0
0
0
5
Experimental
6a
0
12
91
24
51
54
86
18
68
8
6b
83
11
36
28
74
0
General techniques
6c
6d
All reagent used were of synthesis grade (Thomas Baker).
1H NMR spectra were recorded on Bruker Advance spec-
trometer (300 or 500 MHz) using tetramethylsilane as
internal standard. Chemical shifts are reported in ppm (d)
relative to the solvent peak, mass spectra were recorded on
either GCMS (focus GC with TSQ II mass analyzer and
thermoelectro) with auto sampler/direct injection (EI/CI) or
LCMS (APCI/ESI; Bruker Daltanoics Micro TOFQ).
HPLC purity was checked using Water Alliances or Dionex
Ultima 3000 HPLC system. All purifications were done by
Silica gel column chromatography (100–200#). Ethyl
acetate and petroleum ether were used as mobile phase for
TLC (Merck Kiesel 60 F254, 0.2 mm thickness sheet).
6e
6f
6g
6h
42
0
6i
6j
78
71
96
80
Dexamethasone (1 lM)
analog 5 (Scheme 1) exhibited any TNF-a or IL-6 inhibi-
tory activity nor the significant anti-microbial activity.
Regarding structure–activity relationship, biological
activity data suggested the substituents on terminal ben-
zene ring of ureido moiety has remarkable effect on the
anti-inflammatory and anti-microbial activity. Thus, the
compounds 6b, bearing 2-CF3, 4-Cl; and 6j, bearing 2,5-F2,
the most potent anti-inflammatory agents from the series
were found to be completely inactive as anti-bacterial or
anti-fungal agents. However, the compounds with Cl, F,
and CN etc. at one or more than one positions found to be
suitable for high anti-bacterial and anti-fungal activity. The
positions 2,4 or 2,5 were to found to be most suitable for
the favorable effect on these activities but this seems not to
be the general trend. This is evident by the fact that while
compound 6d with 2,5-Cl2 found to be potent anti-micro-
bial agent, 6e bering 2,4-F2 (Tables 2, 3, entry 7) exhibited
moderate anti-bacterial activity and high anti-fungal
activity, compound 6j bearing 2,5-F2 (Tables 2, 3, entry
12) on the other hand found to be completely ineffective as
anti-bacterial and anti-fungal agent. Also, very high
potency of the compound 6i possessing 4-CN group,
implicates that the presence of electron withdrawing sub-
stituent is favorable for high anti-microbial activity.
Chemistry
Synthesis of diethyl 2,6-dimethyl-4-(4-nitrophenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (4)
In a solution of ethyl acetoacetate (1) (6.62 mmol),
4-nitrobenzaldehyde (2) (6.62 mmol), and ammonium
acetate (3) (6.62 mmol), in acetonitrile (30 ml), PTSA
(0.66 mmol) was added then the resultant reaction mixture
heated at 80 °C for a period of 12 h. After completion of
reaction the reaction mixture was poured into ice cold
water and extracted with ethyl acetate (50 9 3), organic
layer concentrated under vacuum to get crude compound
which was purified by silica gel column chromatography
1
(100–200#). Yellow solid. Yield 68 %, H NMR (DMSO,
300 MHz): 1.22 (t, J = 7.6 Hz, 6H), 2.28 (s, 6H), 4.12 (q,
J = 7.6 Hz, 4H), 5.54 (s, 1H), 7.48–7.44 (m, 2H),
8.11–8.07 (m, 2H). MS (APCI); m/z 375.2 [M?1]?.
Synthesis of diethyl 4-(4-(3-(2-chloro-5-
(trifluoromethyl)phenyl)ureido)phenyl)-2,6-dimethyl-1,4-
dihydropyridine-3,5-dicarboxylate (6a)
Conclusion
In conclusion, we have developed the novel approach for
the synthesis of structurally diverse 4-(3-arylurei-
do)phenyl-1,4-dihydropyridine urea derivatives using sim-
ple reaction sequence under mild conditions and their anti-
inflammatory and anti-microbial activity was evaluated.
The biological activity data of newly synthesized 1,4-DHP
In a solution of diethyl 4-(4-aminophenyl)-2,6-dimethyl-
1,4-dihydropyridine-3,5-dicarboxylate (4) (0.29 mmol) in
THF (5 ml), 2-chloro-5-trifluoromethylphenylisocyanate
(0.29 mmol) was added then the resultant reaction mixture
was stirred at room temperature for a period of 2.5 min.
After completion of reaction, the reaction mixture was
123

Med Chem Res
poured into ice cold water and extracted with ethyl acetate
(20 9 3), organic layer concentrated under vacuum to get
crude compound which was purified by silica gel column
chromatography (100–200#). Pale yellow solid. Yield
300 MHz): 1.14 (t, J = 7.2 Hz, 6H), 2.25 (s, 6H), 3.99 (q,
J = 7.2 Hz, 4H), 4.80 (s, 1H), 7.06 (d, J = 8.4 Hz,
2H),7.27 (d, J = 8.4 Hz, 2H), 7.36 (dd, J = 8.8, 2.4 Hz,
1H), 7.60 (d, J = 2.4 Hz, 1H), 8.19 (d, J = 9.2 Hz, 2H),
8.34 (s, 1H), 8.77 (s, 1H), 9.31 (s, 1H). MS (APCI); m/
z 533.1 [M?1]?.
1
19 %, mp 214–216; H NMR (DMSO, 300 MHz): 1.12 (t,
J = 7.2 Hz, 6H), 2.25 (s, 6H), 3.98 (q, J = 7.2 Hz, 4H),
4.80 (s, 1H), 7.08 (d, J = 8.8 Hz, 2H), 7.29 (d, J = 8.4 Hz,
2H), 7.35 (dd, J = 8.4, 2 Hz, 1H), 7.70 (d, J = 8.4 Hz,
1H), 8.57–8.63 (m, 2H), 8.79 (s, 1H), 9.44 (s, 1H). MS
(APCI); m/z 566.3 [M?1]?.
Synthesis of diethyl 2,6-dimethyl-4-(4-(3-(4-
(trifluoromethyl) phenyl) ureido) phenyl)-1,4-
dihydropyridine-3,5-dicarboxylate (6f)
Synthesis of diethyl 4-(4-(3-(4-chloro-3-(trifluoromethyl)
phenyl) ureido) phenyl)-2,6-dimethyl-1,4-dihydropyridine-
3,5-dicarboxylate (6b)
The synthetic method followed the same as for 6a. Pale
1
yellow solid. Yield 21 %, mp 208–210; H NMR (DMSO,
300 MHz): 1.12 (t, J = 7.2 Hz, 6H), 2.24 (s, 6H), 3.99 (q,
J = 7.6 Hz, 4H), 4.78 (s, 1H), 7.04 (d, J = 8.4 Hz, 2H),
7.26–7.23 (m, 2H), 7.33–7.31 (m, 2H), 7.48–7.44 (m, 1H),
8.53 (s, 1H), 8.77–8.75 (m, 2H), 8.85 (s, 1H). MS (APCI);
m/z 532.1 [M?1]?.
The synthetic method followed the same as for 6a. Pale
1
yellow solid. Yield 33 %, mp 222–226; H NMR (DMSO,
300 MHz): 1.12 (t, J = 6.8 Hz, 6H), 2.25 (s, 6H), 3.98 (q,
J = 6.8 Hz, 4H), 4.79 (s, 1H), 7.06 (d, J = 8.8 Hz, 2H),
7.27 (d, J = 8.4 Hz, 2H), 7.71–7.69 (m, 2H), 7.99 (d,
J = 8.8 Hz, 1H), 8.10 (s, 1H), 8.77 (s, 1H), 9.29 (s, 1H).
MS (APCI); m/z 566.7 [M?1]?.
Synthesis of diethyl 4-(4-(3-(4-chlorophenyl) ureido)
phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (6g)
Synthesis of diethyl 4-(4-(3-(3,4-dichlorophenyl) ureido)
phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (6c)
The synthetic method followed the same as for 6a. Pale
1
yellow solid. Yield 18 %, mp 213–215; H NMR (DMSO,
300 MHz): 1.12 (t, J = 7.2 Hz, 6H), 2.25 (s, 6H), 3.99 (q,
J = 6.8 Hz, 4H), 4.79 (s, 1H), 7.04 (d, J = 8.4 Hz, 2H),
7.26–7.23 (m, 2H), 7.33–7.31 (m, 2H), 7.48–7.44 (m, 1H),
8.54 (s, 1H), 8.77–8.75 (m, 2H), 8.86 (s, 1H). MS (APCI);
m/z 498.1 [M?1]?.
The synthetic method followed the same as for 6a. Off white
solid. Yield 29 %, mp 216–218; 1H NMR (DMSO,
300 MHz): 1.14 (t, J = 7.2 Hz, 6H), 2.25 (s, 6H), 3.99 (q,
J = 6.8 Hz, 4H), 4.80 (s, 1H), 7.06 (d, J = 8.4 Hz, 2H), 7.27
(d, J = 8.4 Hz, 2H), 7.36 (dd, J = 8.8, 2.4 Hz, 1H), 7.60 (d,
J = 2.4 Hz, 1H), 8.19 (d, J = 9.2 Hz, 1H), 8.34 (s, 1H), 8.77
(s, 1H), 9.31 (s, 1H). MS (APCI); m/z 533.1 [M?1]?.
Synthesis of diethyl 4-(4-(3-(4-fluorophenyl) ureido)
phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (6h)
Synthesis of diethyl 4-(4-(3-(2,5-dichlorophenyl) ureido)
phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (6d)
The synthetic method followed the same as for 6a. Off
1
white solid. Yield 18 %, mp 228–223; H NMR (DMSO,
300 MHz): 1.12 (t, J = 7.2 Hz, 6H), 2.25 (s, 6H), 3.99 (q,
J = 7.2 Hz, 4H), 4.79 (s, 1H), 7.12–7.03 (m, 4H), 7.24 (d,
J = 8.8 Hz, 2H), 7.45–7.41 (m, 2H), 8.52 (s, 1H),
8.75–8.65 (m, 2H). MS (APCI); m/z 482.1 [M?1]?.
The synthetic method followed the same as for 6a. Off
1
white solid. Yield 41 %, mp 220–223; H NMR (DMSO,
300 MHz): 1.12 (t, J = 7.2 Hz, 6H), 2.25 (s, 6H), 3.99 (q,
J = 7.2 Hz, 4H), 4.80 (s, 1H), 7.08 (d, J = 8.8 Hz,
2H),7.29 (d, J = 8.4 Hz, 2H), 7.35 (dd, J = 8.4, 2 Hz, 1H),
7.70 (d, J = 8.4 Hz, 1H), 8.57–8.64 (m, 2H), 8.78 (s, 1H),
9.43 (s, 1H). MS (APCI); m/z 533.1 [M?1]?.
Synthesis of diethyl 4-(4-(3-(4-cyanophenyl) ureido)
phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (6i)
Synthesis of diethyl 4-(4-(3-(2,4-dichlorophenyl) ureido)
phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (6e)
The synthetic method followed the same as for 6a. Off
1
white solid. Yield 18 %, mp 228–231; H NMR (DMSO,
300 MHz): 1.12 (t, J = 7.2 Hz, 6H), 2.25 (s, 6H), 3.99 (q,
J = 7.2 Hz, 4H), 4.75 (s, 1H), 7.04 (d, J = 8.4 Hz, 2H),
7.24 (d, J = 8.4 Hz, 2H), 8.51 (s, 1H), 8.63 (s, 1H), 8.74 (s,
1H). MS (APCI); m/z 489.1 [M?1]?.
The synthetic method followed the same as for 6a. Off
1
white solid. Yield 32 %, mp 217–219; H NMR (DMSO,
123

Med Chem Res
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Synthesis of diethyl 4-(4-(3-(2,5-difluorophenyl) ureido)
phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (6j)
The synthetic method followed the same as for 6a. Off
1
white solid. Yield 18 %, mp 219–222; H NMR (DMSO,
300 MHz): 1.12 (t, J = 7.6 Hz, 6H), 2.25 (s, 6H), 3.99 (q,
J = 7.6 Hz, 4H), 4.80 (s, 1H), 7.08 (d, J = 8.8 Hz, 2H),
7.30 (d, J = 8.4 Hz, 2H), 7.35 (dd, J = 8.4, 2 Hz, 1H),
7.70 (d, J = 8.4 Hz, 1H), 8.57 (s, 1H), 8.64 (s, 1H), 8.78 (s,
1H), 9.43 (s, 1H). MS (APCI); m/z 500.1 [M?1]?.
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Anti-inflammatory assay
Pro-inflammatory cytokine production by lipopolysaccha-
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method described by Hwang et al. (1993). During assay,
THP-1 cells were cultured in RPMI 1640 culture medium
(Gibco BRL, Pasley, UK) containing 100 U/ml penicillin
and 100 mg/ml streptomycin, 10 % fetal bovine serum
(JRH). Cells were differentiated with phorbol myristate
acetate (Sigma). Following cell plating, the test compounds
in 0.5 % DMSO were added to each well separately and the
plate was incubated for 30 min at 37 °C. Finally, LPS
(Escherichia coli 0127: B8, Sigma Chemical Co., St.
Louis, MO) was added, at a final concentration of 1ug/ml
in each well. Plates were further incubated at 37 °C for
24 h in 5 % CO2. After incubation, supernatants were
harvested, and assayed for TNF-a and IL-6 by ELISA as
described by the manufacturer (BD Biosciences).
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Acknowledgments The authors would like to thank Mr. R. B. Ingle
Department of Microbiology, Shri Shivaji College, Akola for bio-
logical screening studies of the newly synthesized compounds. We
are very grateful to the editor and an anonymous reviewer for their
invaluable comments and suggestions on our study.
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