Novel anti-inflammatory agents based on pyrazole based dimeric compounds; design, synthesis, docking and in vivo activity

Article abstract of DOI:10.1248/cpb.58.634

Series of pyrazole ester prodrugs analogues have been synthesized and found to contain highly potent inhibitors of the cyclooxygenase-2 (COX-2) enzyme. The paper describes synthesis of the target pyrazole analogues. The structure of the synthesized mutual

Full text of DOI:10.1248/cpb.58.634

634
Regular Article
Chem. Pharm. Bull. 58(5) 634—638 (2010)
Vol. 58, No. 5
Novel Anti-inflammatory Agents Based on Pyrazole Based Dimeric
Compounds; Design, Synthesis, Docking and in Vivo Activity
Ashish Kumar TEWARI,*,a Priyanka SRIVASTAVA,a Ved Prakash SINGH,a Amit SINGH,b Raj Kumar GOEL,b
c
and Chethampadi Gopi MOHAN
a Department of Chemistry, Faculty of Science, Banaras Hindu University; b Department of Pharmacology, IMS, Banaras
Hindu University; Varanasi 221 005, India: and c Centre for Pharmacoinformatics, National Institute of Pharmaceutical
Education and Research (NIPER); S.A.S. Nagar-160 062, Punjab, India.
Received October 28, 2009; accepted January 28, 2010; published online February 15, 2010
Series of pyrazole ester prodrugs analogues have been synthesized and found to contain highly potent
inhibitors of the cyclooxygenase-2 (COX-2) enzyme. The paper describes synthesis of the target pyrazole
1
analogues. The structure of the synthesized mutual ester prodrugs (6—8c) were confirmed by H-, 13C-NMR
mass spectroscopy (MS) and their purity were ascertained by TLC and elemental analyses. The biological in vivo
evaluation of these compounds in experimental models (carrageenan-induced oedema) proved the presence of
anti-inflammatory activity. Docking studies into the catalytic site of COX-2 were used to identify potential anti-
inflammatory lead compounds. One lead derivative was chosen endowed with good binding energies.
Key words prodrug; pyrazole; cyclooxygenase; docking; NMR
Various inflammatory diseases are currently treated ous adverse effects (such as bone marrow depression, water
with steroidal and non-steroidal anti inflammatory drugs and salt retention and carcinogenesis), the use of pyrazole
(NSAIDs).1) NSAIDs are drug with analgesic, antipyretic and derivative is limited. This limitation has led to the inves-
i
n higher doses anti inflammatory effects. They inhibit tigation of new pyrazole derivatives with that more potent
synthesis of inflammation mediators.2,3) Most NSAIDs act activity and less toxicity.
as non-selective inhibitors of the enzyme cyclooxygenase
Motivated by the aforementioned findings, it was designed
(COX), by inhibiting the metabolism of arachidonic acid.2) to synthesize novel series of pyrazole derivatives that would
COX catalyses the formation of prostaglandins (PGs) and act as anti inflammatory agents. The substitution pattern of
thromboxane from arachidonic acid. PGs act as messenger the pyrazole ring was rationalized so as to be correlated to
molecules in the process of inflammation. Conventional the diaryl heterocycles template of compounds that are
NSAIDs that non-selectively inhibit major cyclooxygenase known to act selectively as COX-2 inhibitors, such as cele-
isoforms,4—7) (COX-1 and COX-2), are widely used to treat the coxib. Compared with coxibs, new drugs were synthesized
signs and symptoms of inflammation, particularly arthritic by keeping the original pyrazole moiety same in new drugs
pain. COX-1 is the constitutive isoform and is mainly containing heterocycle (like phthalimide, N-pyridone and O-
responsible for the synthesis of cytoprotective PGs in gas- pyridone) linked ethylene linker attached to oxygen adjacent
trointestinal (GI) tract whereas COX-2 is inducible and plays to phenyl group containing nitrogen of pyrazole which pro-
a major role in PG biosynthesis in inflammatory cells.8—10) It vides flexibility to the molecule so that it will become easy to
is believed that the inhibition of COX-1 causes unfavorable fit into the secondary pocket of COX-2 active site. The drugs
GI side effects.11) NSAIDs vary in their potency, duration of were synthesized as acid derivatives (containing ester group),
action and the way in which they are eliminated from the initiating the easy uptake of drug (in salt form). Presence of a
body. Though selective COX-2 inhibitors (coxibs) with better small hydrophobic group, methyl group in pyrazole nucleus
safety profile have been marketed as a new generation of at position 3 can exert important influence on the activity and
NSAIDs,12,13) but careful prospective examination of coxibs selectivity of related drug.
has revealed unexpected adverse effects. The two main ad-
Chemistry All compounds were synthesized via a gen-
verse drug reactions (ADRs) associated with NSAIDs relate eral substitution reaction at room temperature. The starting
to GI effects14,15) and renal effects (kidney failure) of the material in the synthesis of substituted pyrazoles had been
agents. Also, they have shown unexpected cardiovascular ad- synthesized from reaction of ethyl acetoacetate 1 with phenyl
verse effects.16) Therefore, development of novel compounds hydrazine at refluxing temperature22) to synthesize 5-methyl-
having anti inflammatory activity with an improved safety 2-phenyl-2,4-dihydro-pyrazol-3-one 2 (Chart 1). Reaction of
profile is still a necessity. Literature survey revealed that 2 with carbon disulfide23,24) followed by methylation gave 4-
many pyrazole17) derivatives have found their clinical appli- (bis-methyl sulfanyl-methylene)-5-methyl-2-phenyl-2,4-dihy-
cation as NSAIDs. Among the highly marketed COX-2 in-
hibitors that comprise the pyrazole nucleus, celecoxib is the
one which is treated as a safe anti inflammatory and anal-
gesic agent. It is considered as a typical model of the diaryl
heterocycle template that is known to selectively inhibit the
COX-2 enzyme. Some other examples of pyrazole deriva-
tives as NSAIDs are felcobutazone, mefobutazone, mora-
zone, famprofazone and ramifenazone.18—21) But due to seri-
Chart 1
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: tashish2002@yahoo.com

May 2010
635
Chart 2
Chart 3
Chart 4
dro-pyrazol-3-one 3 (in 80—90% yield), the key intermedi-
ate in the synthesis of 4-substituted pyrazoles. Base catalysed lent doses of the target derivatives (6—8C). After 15 min
alcoholysis of 3 followed by acidification gave pyrazole-ester control animals were similarly treated with aqueous solution
4 in 80% yields. The mass spectrum and elemental analysis of carboxymethyl cellulose (CMC, 1% w/v). One percent
1
are in accordance with the assigned structure. The H-NMR carrageenin suspension was prepared as a homogeneous so-
spectrum of 4a had a singlet at d 2.406 for methyl protons lution in distilled water. A volume of 0.1 ml of carrageenin
and a singlet at d 3.920 for methoxy protons and a broad sin- solution was injected through a 26 gauge needle into the
glet at d 9.992 for hydroxyl proton. The mass spectrum of 4a plantar surface of the left hind paw below the plantar aponeu-
1
had the base peak at MS (m/z) Mꢀ 233. Similarly the H- rosis. The volume of paw was measured before and at differ-
NMR and mass spectra of 4b were found in accordance to ent intervals for 3 h after injection of carrageenin. The differ-
the assigned structures. Treatment of 4a with excess of 1,2- ence in paw volume before and after administration of the
dibromoethane (5 eq) in presence of a weak base (anhydrous phlogistic agent was taken as the measure of pedal edema.
K2CO3) and N,N-dimethylmethanamide (DMF) afforded syn-
Measurement of Paw Volume: The volume of hind paw of
thesis of 5a with quantitative yield and high purity. Similarly, the rats up to the ankle joint was measured plathysmographi-
5b was obtained by condensation of 4a with excess of 1,2- cally by the mercury displacement method. The ankle joint
dibromoethane in basic medium. All products were obtained of the rats was marked with a skin marking pencil and the
in quantitative yield with high purity via SiO2 column chro- paw was dipped in the mercury, so that the mark on the paw
matography with 5% ethyl acetate in chloroform as eluant coincides with a prefixed line kept constant on the syringe.
mixture. Reaction of 4a with 1,2-dibromoethane (0.5 eq) in The level of the mercury was every time brought to the level
presence of K2CO3 and DMF afforded synthesis of 6 with of this line by adjusting the height of the displaced mercury.
42.78% yield. The base catalyzed nucleophilic substitution The difference in the paw volume before and after injection
of 5a with 1 eq of phthalimide in DMF as solvent at room of the phlogistic agents was taken as a measure of pedal
temperature gave 7a (Chart 4), in good yield. The pure prod- edema. The change in paw volume was expressed in “ml” of
uct was obtained by crystallization with 2% ethyl acetate in mercury displaced.
hexane. Similarly, 5b reacted with phthalimide to afford syn-
Statistical Analysis: The difference in the means between
thesis of 8a. The base catalyzed nucleophilic substitution of each experimental group was first analyzed by Analysis of
5a and 5b separately with 1 eq of pyridone and DMF at r.t. Variance (ANOVA). When the overall ANOVA was signifi-
afforded the synthesis of 7b, 7c, 8b and 8c, respectively.
cant (pꢂ0.05), the data was further subjected to statistical
Materials and Methods Animals: All experiments have analysis by the student’s t-test.
been conducted on adult Wistar strain albino rats of either
Molecular Docking analysis on COX-2 Inhibitor: A bind-
sex, weighing between 150—200 g. The animals were ob- ing study of COX-2 inhibitor was performed using ligand–
tained from the Central Animal House of the Institute of receptor interaction module of MOE software (Macrovision
Medical Sciences, B.H.U. They were kept in colony cages Europe Ltd.), while docking analysis was conducted using
under identical housing conditions at an ambient temperature FlexXnet software (Schrodinger). In all the studies, in-
of 25ꢁ2 °C and 45—55% relative humidity with a 12 h light– domethacine was used as a reference inhibitor in COX-2
dark cycle in the departmental animal room and fed on stan- crystal structure (PDB code: 4COX), considering that size
dard diet. Animals were acclimatized for a week before use.
and shape complementarity of indomethacine was similar to
Experimental Model of Inflammation: Carrageenin in- the studied compound.
duced paw edema25) was used throughout the investigation.
Result and Observations In all experiments, carrageenin
Carrageenin Induced Paw Edema in Rats: Target com- was administered into the left hind paw and the paw volume
pounds were suspended in aqueous solution of carboxymethyl was measured before and at intervals of 30 min, 90 min and
cellulose (CMC, 1% w/v) and administered orally at a dose 180 min, after carrageenin injection (Table 1).
level of 10 mg/kg of NSAI drugs nimusilide and their equiva-
It was observed that maximum percentage of paw edema

636
Vol. 58, No. 5
Table 1. Paw Edema at Different Time Interval (ml/Rat) (MeanꢁS.E.M.)
Group
0 min
30 min
90 min
180 min
Control
Nimusilide
6
7a
7b
7c
8a
8b
8c
0.99ꢁ0.067
1.18ꢁ0.064
1.16ꢁ0.044
1.28ꢁ0.064*
0.84ꢁ0.056
0.82ꢁ0.038
0.76ꢁ0.033
1.15ꢁ0.050
0.97ꢁ0.086
1.27ꢁ0.043
1.29ꢁ0.041
1.32ꢁ0.029
1.33ꢁ0.061
0.94ꢁ0.044***
0.87ꢁ0.028***
1.02ꢁ0.052**
1.45ꢁ0.028
1.25ꢁ0.034
1.36ꢁ0.070
1.25ꢁ0.024
1.43ꢁ0.025
1.36ꢁ0.063
1.03ꢁ0.057***
0.97ꢁ0.025***
1.11ꢁ0.034**
1.44ꢁ0.012
1.15ꢁ0.050
1.21ꢁ0.072
1.21ꢁ0.021
1.33ꢁ0.046
1.28ꢁ0.093*
0.99ꢁ0.043*
0.99ꢁ0.028*
1.01ꢁ0.052
1.34ꢁ0.045
1.05ꢁ0.022
Results are (meanꢁS.E.M.) of 6 rats in each group. ∗ pꢂ0.05; ∗∗ pꢂ0.01; ∗∗∗ pꢂ0.001.
Table 2. Percentage Edema Growth Relative to Control at Different Time Intervals (MeanꢁS.E.M.)
Group
0 (min)
100ꢁ0
100ꢁ0
100ꢁ0
100ꢁ0
100ꢁ0
100ꢁ0
100ꢁ0
100ꢁ0
100ꢁ0
30 (min)
90 (min)
180 (min)
Control
130.1ꢁ6.54
(30.1)
110.8ꢁ2.58
(10.8)
114.1ꢁ2.88
(14.1)
103.9ꢁ2.59*
(3.9)
115.1ꢁ10.02
(15.1)
106.5ꢁ3.67**
(6.5)
133.9ꢁ3.56
(33.9)
126.8ꢁ5.60
(26.8)
138.7ꢁ4.47
(38.7)
107.90ꢁ3.14
(7.90)
123.4ꢁ3.37*
(23.4)
106.2ꢁ3.98*
(6.2)
123.3ꢁ3.83***
(23.3)
118.4ꢁ3.86
(18.4)
146.5ꢁ6.54
(46.5)
125.6ꢁ4.86
(25.6)
122.3ꢁ5.17
(22.3)
102.54ꢁ2.52
(2.54)
114.9ꢁ4.25
(14.9)
99.6ꢁ2.54**
(0.4)
120.6ꢁ8.34
(20.6)
121.4ꢁ4.55
(21.4)
133.6ꢁ7.58
(33.6)
117.1ꢁ5.86
(17.1)
Nimusilide
6
7a
7b
7c
8a
8b
8c
129.0ꢁ4.53
(29.0)
118.4ꢁ3.98*
(18.4)
108.7ꢁ4.75
(8.7)
nꢄ6ꢄnumber of rats in each group. Results in parentheses indicate percentage change from respective control group. ∗ pꢂ0.05; ∗∗ pꢂ0.01; ∗∗∗ pꢂ0.001.
growth in control group at 90 min was 38.7% which was
found to decrease upto 6.2% in the group of rats treated with
7a and least effect was shown by 8a drug (Table 2).
Discussion and Conclusion The results of present study
shown that compound 7a had shown to possess maximum in-
hibitory effect when compared to control group (Fig. 1). It
was observed that maximum percentage of paw edema
growth in control group at 90 min was 38.7% which was
found to decrease upto 6.2% in the group of rats treated with
7a (Table 2). Also, the values were found statistically very
significant. 8c have also been found to possess very good anti
inflammatory property as the percentage paw edema growth
was shown to be only 18.4% when compared to that of con-
trol group (where it was 38.7% at 90 min). Other drugs i.e.,
6, 7b and 8b have shown moderate to intermediate effects on
Fig. 1. Effect of Different Drugs on Carrageenin Induced Rat Paw Edema
Vertical lines indicate S.E.; ∗ pꢂ0.05; ∗∗ pꢂ0.01; ∗∗∗ pꢂ0.001.
inhibitory properties. But 8a has shown no effect as anti in-
flammatory agent. Also, in case of 7c, the drug was shown to activity) were almost same except that in 7a ester moiety
possess good inhibitory property at 90 min when compared contained methyl group and in 8a ester moiety contained
to control group as the value dropped from 38.7 to 18.4% but ethyl group which being bulkier than methyl group may
at 180 min, the drug has shown almost negligible effect on affect the activity of the particular drug.
inflammation inhibition and the percentage inhibition in-
The ability of compound 7a to interact with the COX-2
creased up to 21.4%. Therefore, the results indicated that 7a was further assessed by in silico studies (Table 3). Figure 2
is potent inhibitor of inflammation and the anti inflammatory depicted the compound docked at the active site, using FlexX
effect of 7a on inflammagen induced edema may depend on software for which the total score obtained was ꢃ8.72 hydro-
inhibition of the formation of several inflammation media- gen bonding interactions are proposed to occur near the
tors. The structures of 7a (drug with maximum anti inflam- active site of COX-2 (i) carbonyl group of dione 7a form
matory activity) and 8a (drug with no anti inflammatory hydrogen bond with the hydroxyl group of Ser 530 and its

May 2010
637
Table 3. Compounds Docking Scores and Compared with indomethacine
Compound
Docking score
Indomethacine
ꢃ12.09
ꢃ6.88
ꢃ8.72
ꢃ7.54
ꢃ8.27
ꢃ7.78
ꢃ6.82
ꢃ6.67
6
7a
7b
7c
8a
8b
8c
distance is 2.227 Å, this active site is thought to reflect
inhibitory binding mode where no viable products can be
produced26) and (ii) nitrogen of the pyrazole ring of 7a form
hydrogen bond with the hydroxyl group of Tyr 355 and its
distance is 2.070 Å, bound in the top channel, similar to
arachidonic acid.27) This conformation of product, in which
the PGG2/PGH2 species hydrogen-bonds with the constric-
tion site residues and bends in an L-shaped fashion at Tyr-
385 and its distance is 2.596 Å, suggests that arachidonic
acid was positioned properly for catalysis presented in Fig. 2.
According to these computational studies, 7a is able to
dock into the enzyme active site in the same way that has
been reported for indomethacine.28)
Fig. 2. Docking Analysis of the Compound (7a) Shown in Capped Stick
Model
Amino acids are in wireframe model, amino acids form hydrogen bonding shown
with broken lines and amino acid residues are indicated using three-letters code.
mixture. mpꢄ68—70 °C. Yieldꢄ0.33 g (55.2%).
1H-NMR (CDCl3, 300 MHz) d in ppm: 2.42 (s, 3H, CH3), 3.79 (s, 3H,
OCH3), 3.99 (s, 2H, CH2), 4.50 (s, 2H, CH2), 7.04—7.77 (m, 9H, Ar-H).
13C-NMR (CDCl3) d in ppm: 15.18 (CH3), 37.57 (CH2), 51.03 (OCH3),
71.58 (CH2), 99.46 (C), 123.14 (CH), 127.24 (CH), 128.68 (CH), 131.86
(CH), 133.83 (CH), 137.06 (C), 150.86 (CH), 154.33 (C), 163.19 (C),
167.79 (C). Element Analysis: (i). Calculated: %Nꢄ10.37, %Cꢄ65.18,
%Hꢄ4.69. (ii). Found: %Nꢄ10.17, %Cꢄ65.77, %Hꢄ4.82. MS: Mꢀ at m/z
406.
Experimental
5-(2-Bromo-ethoxy)-3-methyl-1-phenyl-1H-pyrazole-4-carboxylic
Acid Methyl Ester 5a Compound 4a23) (2.60 g, 11.2 mmol), anhydrous
K2CO3 (2.31 g, 16.8 mmol) and 1,2-dibromoethane (4.8 ml, 56 mmol) were
dissolved in 15 ml DMF and stirred at r.t. for 48 h. TLC was checked and
reaction mixture was worked up. DMF was removed under pressure through
rotary evaporator and crude product was extracted with CHCl3/H2O (100/
50ꢅ2 ml), dried (anhydrous Na2SO4), filtered and evaporated. 5a was ob-
tained via column chromatography. Eluant used was 5% ethyl acetate in
chloroform. mpꢄ48—50 °C. Yieldꢄ3.00 g (78.9 %).
5-[2-(3-Cyano-4,6-dimethyl-2-oxo-2H-pyridin-1-yl)-ethoxy]-3-methyl-
1-phenyl-1H-pyrazole-4-carboxylic Acid Methyl Ester 7b and 5-[2-
(3-Cyano-4,6-dimethyl-pyridin-2-yloxy)-ethoxy]-3-methyl-1-phenyl-1H-
pyrazole-4-carboxylic Acid Methyl Ester 7c 4,6-Dimethyl-2-oxo-1,2-
dihydro-pyridine-3-carbonitrile (0.35 g, 2.3 mmol), anhydrous K2CO3
(0.65 g, 4.7 mmol) and 5a (0.8 g, 2.3 mmol) were stirred in 15 ml DMF for
48 h at r.t. Completion of reaction was confirmed via TLC. Reaction was
worked up. DMF was removed through rota vapour and the product mixture
was extracted with CHCl3/H2O (50/50ꢅ2 ml). The CHCl3 layer was dried
(Na2SO4), filtered and evaporated and pure 7b and 7c were obtained through
crystallization with ethyl acetate in hexane mixture. [7b was crystallized
with ethyl acetate and 7c was crystallized with 1% ethyl acetate in hexane
solution.]
1H-NMR (CDCl3, 300 MHz) d in ppm: 2.46 (s, 3H, CH3), 3.46—3.50 (t,
2H, CH2), 3.87 (s, 3H, OCH3), 4.48—4.52 (t, 2H, CH2), 7.26—7.67 (m, 5H,
Ar-H).
5-(2-Bromo-ethoxy)-3-methyl-1-phenyl-1H-pyrazole-4-carboxylic
Acid Ethyl Ester 5b Compound 4b23) (3 g, 12.1 mmol), anhydrous K2CO3
(1.68 g, 12.1 mmol) and 1,2-dibromoethane (5.25 ml, 60.9 mmol) were dis-
solved in 20 ml DMF and stirred at r.t. for 24 h and TLC checked. Reaction
mixture was worked up as above and pure 5b was obtained as oily liquid via
column loaded with silica in chloroform. Eluant used was 5% ethyl acetate
in chloroform. Yieldꢄ3.32 g (77.4 %).
7b: mpꢄ174—176 °C. Yieldꢄ0.50 g (52.2%).
1H-NMR (CDCl3, 300 MHz) d in ppm: 1.36—1.41 (t, 3H, OCH2CH3), 2.47
(s, 3H, CH3), 3.45—3.50 (t, 2H, OCH2), 4.29—4.36 (q, 2H, OCH2CH3),
4.48—4.53 (t, 2H, CH2), 7.26—7.67 (m, 5H, Ar-H).
1H-NMR (CDCl3, 300 MHz) d in ppm: 2.38 (s, 3H, CH3), 2.42—2.43 (d,
6H, CH3ꢅ2), 3.84 (s, 3H, OCH3), 4.34—4.37 (t, 2H, CH2), 4.49—4.53 (t,
2H, CH2), 5.93 (s, 1H, CH), 7.26—7.33 (m, 5H, Ar-H). 13C-NMR (CDCl3) d
in ppm: 15.25 (CH3), 20.82 (CH3), 21.46 (CH3), 44.98 (CH2), 51.13 (OCH3),
71.98 (CH2), 99.63 (C), 101.39 (C), 109.53 (CH), 115.14 (C), 123.42 (CH),
127.83 (CH), 129.00 (CH), 136.99 (C), 150.72 (C), 151.41 (C), 154.34 (C),
158.24 (C), 160.66 (C), 163.25 (C). Element Analysis: (i). Calculated: %Nꢄ
13.79, %Cꢄ65.02, %Hꢄ5.41. (ii). Found: %Nꢄ13.29, %Cꢄ64.98, %Hꢄ5
.32. MS: Mꢀ at m/z 407.
Preparation of 1-[5-[2-(4-Acetyl-5-methyl-2-phenyl-2H-pyrazol-
3-yloxy)-ethoxy]-3-methyl-1-phenyl-1H-pyrazol-4-yl]-ethanone 6 Com-
pound 4a (2 g, 8.6 mmol), anhydrous K2CO3 (1.18 g, 8.6 mmol) and 1,2-
dibromoethane (0.37 ml, 4.31 mmol) were dissolved in 20 ml DMF and
stirred for 24 h at r.t. TLC checked and reaction mixture was worked up. 6
was obtained in pure form through column loaded with silica in chloroform.
Eluant used was chloroform. mpꢄ88—90 °C. Yieldꢄ1.81 g (42.8%).
1H-NMR (CDCl3, 300 MHz) d in ppm: 2.45 (s, 6H, CH3ꢅ2), 3.80 (s, 6H,
OCH3ꢅ2), 4.50 (s, 4H, CH2ꢅ2), 7.26—7.57 (m, 10H, Ar-H). 13C-NMR
(CDCl3, 300 MHz) d in ppm: 15.36 (CH3), 51.08 (OCH3), 74.00 (CH2),
99.21 (C), 123.07 (CH), 127.43 (CH), 128.91 (CH), 137.38 (C), 150.84 (C),
154.78 (C), 163.33 (C). MS: Mꢀ at m/z 491.
5-[2-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-ethoxy]-3-methyl-1-phenyl-
1H-pyrazole-4-carboxylic Acid Methyl Ester 7a Phthalimide (0.21 g,
1.4 mmol), anhydrous K2CO3 (0.19 g, 1.4 mmol) and 5a (0.5 g, 1.4 mmol)
were taken in 20 ml DMF and stirred at r.t. for 24 h. TLC was checked and
the reaction mixture was worked up. DMF was removed under pressure and
compound was extracted with CHCl3/H2O (20/20ꢅ2 ml). The CHCl3 layer
was dried (anhydrous Na2SO4) and filtered and the solvent was removed.
Pure 7a was obtained by crystallization with 2% ethyl acetate in hexane
7c: mpꢄ98—100 °C. Yieldꢄ0.32 g (33.4%).
1H-NMR (CDCl3, 300 MHz) d in ppm: 2.37 (s, 3H, CH3), 2.42—2.46 (d,
6H, CH3ꢅ2), 3.87 (s, 3H, OCH3), 4.57—4.64 (double-d, 4H, OCH2ꢅ2),
6.66 (s, 1H, CH), 7.17—7.66 (m, 5H, Ar-H). 13C-NMR (CDCl3) d in ppm:
15.27 (CH3), 19.91 (CH3), 24.31 (CH3), 51.06 (OCH3), 65.05 (CH2), 73.42
(CH2), 93.89 (C), 99.29 (C), 114.41 (C), 117.68 (CH), 122.77 (CH), 126.96
(CH), 128.79 (CH), 137.36 (C), 150.85 (C), 154.32 (C), 154.85 (C), 160.21
(C), 162.95 (C), 163.38 (C). Element Analysis: (i). Calculated: %Nꢄ13.79,
%Cꢄ65.02, %Hꢄ5.41. (ii). Found: %Nꢄ13.29, %Cꢄ64.63, %Hꢄ5.49. MS:
Mꢀ at m/z 407.
5-[2-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-ethoxy]-3-methyl-1-phenyl-
1H-pyrazole-4-carboxylic Acid Ethyl Ester 8a Phthalimide (0.20 g, 1.4
mmol), anhydrous K2CO3 (0.19 g, 1.4 mmol) and 5b (0.5 g, 1.4 mmol) were
taken in 10 ml DMF and stirred at r.t. for 24 h and the completion of reaction

638
Vol. 58, No. 5
was confirmed by TLC. The reaction was worked up. DMF was removed edges to CSIR for SRF-CSIR fellowship from CSIR, New Delhi, India.
under pressure and the product was extracted with CHCl3/H2O (30/30ꢅ2
ml). The organic layer was dried (anhydrous Na2SO4), filtered and evapo- References
rated. Pure 8a was obtained from 2% ethyl acetate in hexane mixture. mpꢄ
86—88 °C. Yieldꢄ0.44 g (74.1%).
1) Moeser P. J., Postgrad. Med., 90, 178—182 (1991).
2) Insel P. A., “Analgesic, Antipyretic and Anti-inflammatory Agents and
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1H-NMR (CDCl3, 300 MHz) d in ppm: 1.31—1.36 (t, 3H, OCH2CH3),
2.42 (s, 3H, CH3), 3.97—4.00 (t, 2H, CH2), 4.23—4.31 (q, 2H, OCH2CH3),
4.50—4.54 (t, 2H, CH2), 7.02—7.79 (m, 9H, Ar-H). 13C-NMR (CDCl3) d in
ppm: 14.31 (CH3), 15.27 (CH3), 37.59 (CH2), 59.93 (CH2), 71.58 (CH2),
99.72 (CH), 123.21 (CH), 127.18 (CH), 128.65 (CH), 131.88 (C), 133.81
(CH), 137.09 (C), 150.85 (C), 154.33 (C), 162.81 (C), 167.79 (C). Element
Analysis: (i). Calculated: %Nꢄ10.02, %Cꢄ65.87, %Hꢄ5.01. (ii). Found:
%Nꢄ9.87, %Cꢄ65.80, %Hꢄ4.97. MS: Mꢀ at m/z 420.
5-[2-(3-Cyano-4,6-dimethyl-2-oxo-2H-pyridin-1-yl)-ethoxy]-3-methyl-
1-phenyl-1H-pyrazole-4-carboxylic Acid Ethyl Ester 8b and 5-[2-(3-
Cyano-4,6-dimethyl-pyridin-2-yloxy)-ethoxy]-3-methyl-1-phenyl-1H-
pyrazole-4-carboxylic Acid Ethyl Ester 8c 4,6-Dimethyl-2-oxo-1,2-dihy-
dro-pyridine-3-carbonitrile (0.20 g, 1.4 mmol) and anhydrous K2CO3 (0.39 g,
2.8 mmol) were dissolved in 15 ml DMF for 30 min and 5b (0.50 g, 1.4
mmol) was added slowly with stirring. The reaction was stirred at r.t. for 48
h and TLC checked. Reaction was worked up. Solvent was removed via ro- 11) Mitchell J. A., Akarasereenont P., Thiemeromann C., Flower R. J.,
tary evaporator and the product mixture was extracted with CHCl3/H2O (50/
50ꢅ2 ml). The CHCl3 layer was dried (anhydrous Na2SO4), filtered and
Vane J. R., Proc. Natl. Acad. Sci. U.S.A., 90, 11693—11697 (1993).
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evaporated. The product mixture was crystallized with 5% ethyl acetate in 13) Tally J. J., Bertenshaw R. S., Brown D. L., Carter J. S., Graneto M. J.,
hexane and recrystallized with 2% ethyl acetate in hexane to obtain pure 8b
and 8c.
Kellogg M. S., Kobolt C. M., Yuan J., Zhang Y. Y., Siebert K., J. Med.
Chem., 43, 1661—1663 (2000).
8b: mpꢄ154—156 °C. Yieldꢄ0.12 g (20.2%).
14) Kulkarni S. K., Varghese N. P., Indian Drugs, 35, 245—260 (1998).
15) Kulkarni S. K., Jain N. K., Singh A., Method Find. Exp. Clin. Pharma-
col., 22, 291—298 (2000).
1H-NMR (CDCl3, 300 MHz) d in ppm: 1.35—1.39 (t, 3H, OCH2CH3),
2.38 (s, 3H, CH3), 2.41—2.43 (d, 6H, CH3ꢅ2), 4.29—4.37 (double-t, 4H,
CH2, OCH2CH3), 4.50—4.52 (d, 2H, CH2), 5.92 (s, 1H, CH), 7.25—7.323 16) Donge J. M., Supuran C. T., Pratico D., J. Med. Chem., 48, 2251—
2257 (2005).
(m, 5H, Ar-H). 13C-NMR (CDCl3) d in ppm: 14.38 (CH3), 15.36 (CH3),
20.85 (CH3), 21.51 (CH3), 45.02 (CH2), 60.07 (CH2), 72.00 (CH2), 109.52 17) Tewari A. K., Mishra A., Bioorg. Med. Chem., 9, 715—718 (2001).
(CH), 123.46 (CH), 127.83 (CH), 129.02 (CH), 137.06 (C), 150.75 (C), 18) “The Extra Pharmacopia,” 30th ed., ed. by Reynold J. E. F., Martin-
151.39 (C), 158.23 (C), 160.69 (C), 162.91 (C). Element Analysis: (i). Cal-
dale, Pharmaceutical Press, London, 1993, p. 1.
culated: %Nꢄ13.30, %Cꢄ65.71, %Hꢄ5.71. (ii). Found: %Nꢄ13.03, %Cꢄ 19) Amir M., Kumar S., Indian J. Chem., 443, 2532—2537 (2005).
65.33, %Hꢄ5.69. MS: Mꢀ at m/z 421.
20) Gursoy A., Demirayak S., Capan G., Erol K., Vural K., Eur. J. Med.
Chem., 35, 359—364 (2000).
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Saxena K. K., Lata S., Gupta B., Srivastava V. K., Indian J. Chem., 4,
1979—1984 (2003).
8c: mpꢄ86—88 °C. Yieldꢄ0.10 g (16.8%).
1H-NMR (CDCl3, 300 MHz) d in ppm: 1.37—1.41 (t, 3H, OCH2CH3),
2.36 (s, 3H, CH3), 2.42—2.47 (d, 6H, CH3ꢅ2), 4.30—4.37 (q, 2H,
OCH2CH3), 4.56—4.59 (t, 2H, OCH2), 4.64—4.67 (t, 2H, OCH2), 6.65 (s,
1H, CH), 7.18—7.66 (m, 5H, Ar-H). 13C-NMR (CDCl3) d in ppm: 14.38 22) “Textbook of Practical Organic Chemistry,” 5th ed., Vogel, Pearson
(CH3), 15.39 (CH3), 19.96 (CH3), 24.34 (CH3), 59.94 (CH2), 65.09 (CH2),
Education Singapore, Singapore, 2004, p. 1150.
73.43 (CH2), 93.98 (C), 99.54 (C), 114.44 (C), 117.69 (CH), 122.84 (CH), 23) Apparao S., Ila H., Junjappa H., Synthesis, 1, 65—66 (1981).
126.94 (CH), 128.81 (CH), 137.46 (C) 150.92 (C), 154.36 (C), 154.87 (C), 24) Chauhan S. M. S., Junjappa H., Tetrahederon, 32, 1779—1787 (1976).
160.24 (C), 163.02 (C). Element Analysis: (i). Calculated: %Nꢄ13.30,
%Cꢄ65.71, %Hꢄ5.71. (ii). Found: %Nꢄ13.21, %Cꢄ65.23, %Hꢄ5.32.
MS: Mꢀ at m/z 421.
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Acknowledgement Author is gracefully acknowledged to Department
of Science and Technology, New Delhi for financial assistance in Young Sci-
entist chart and Department of Pharmacology, IMS, Banaras Hindu Univer-
sity, Varanasi for providing assistance in conducting anti inflammatory
27) Kiefer J. R., Pawlitz J. L., Moreland K. T., Stegeman R. A., Hood W.
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97—101 (2000).
experiments and Department of Pharmaceutical Chemistry, IT, Banaras 28) Kurumbail R. G., Stevens A. M., Gierse J. K., McDonald J. J., Stege-
Hindu University, Varanasi, for providing docking analysis, Department of
Chemistry, Faculty of Science, Banaras Hindu University, Varanasi, INDIA is
acknowledged for departmental facilities. CGM also acknowledges support
from Department of Biotechnology, New Delhi, India. VPS also acknowl-
man R. A., Pak J. Y., Gildehaus D., Miyashiro J. M., Penning T. D.,
Seibert K., Isakson P. C., Stallings W. C., Nature (London), 384, 644—
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