Synthesis and pharmacological investigation of 5-substituted-3- methylsulfanyl-1 H-pyrazole-4-carboxylic acid ethyl esters as new analgesic and anti-inflammatory agents

Article abstract of DOI:10.1055/s-0032-1321830

Purpose: To synthesize a new series of 5-substituted-3-methylsulfanyl-1H- pyrazole-4-carboxylic acid ethyl esters for their analgesic and anti-inflammatory activity. Methods: The title compound synthesized by reacting the amino group of 5-amino-3-methylsu

Full text of DOI:10.1055/s-0032-1321830

Original Article 457
Synthesis and Pharmacological Investigation of
5-Substituted-3-methylsulfanyl-1H-pyrazole-4-
carboxylic Acid Ethyl Esters as New Analgesic and
Anti-inflammatory Agents
Authors
P. D. Gokulan1, B. Jayakar1, V. Alagarsamy2, V. Raja Solomon3
Affiliations
1 Department of Pharmaceutical Chemistry, Vinayaka Mission's College of Pharmacy, Sankari Main Road, Ariyanoor, Salem,
India
2 Department of Pharmaceutical Chemistry, MNR College of Pharmacy, Gr. Hyderabad, India
3 Medicinal & Process Chemistry Division, Central Drug Research Institute, Lucknow, India
Key words
Abstract
▼
compounds were investigated for analgesic, anti-
inflammatory and ulcerogenic behaviour.
Results: The compound 5-benzoylamino-3-
methylsulfanyl-1-phenyl-1H-pyrazole-4-carbox-
ylic acid ethyl ester (4c) emerged as the most
active compound and exhibiting imperative anal-
gesic and anti-inflammatory activities. Interest-
ingly the test compounds showed only mild
ulcerogenic potential when compared to
indomethacin.
▶
pyrazole derivatives
analgesic
●
▶
●
Purpose: To synthesize a new series of 5-sub-
stituted-3-methylsulfanyl-1H-pyrazole-4-car-
boxylic acid ethyl esters for their analgesic and
anti-inflammatory activity.
▶
anti-inflammatory activity
ulcerogenic behaviour
●
▶
●
Methods: The title compound synthesized by
reacting the amino group of 5-amino-3-methyl-
sulfanyl-1H-pyrazole-4-carboxylic acid ethyl
ester with acid anhydrides, acid chlorides and
phenyl dithiocarbamates. The synthesized com-
pounds were characterized by IR, 1H-NMR and
mass spectral data; the purity of the compounds
was determined by elemental analysis. The title
Conclusion: The compound (4c) could serve as
a lead molecule for further modification to obtain
a clinically useful novel class of analgesic and
anti-inflammatory agents.
Introduction
▼
anti-inflammatory activities. Therefore, it was
thought worthwhile to explore analgesic and
anti-inflammatory activity of tetra substituted
pyrazole derivatives. Accordingly the present
work is concerned with the synthesis of tetra
substituted pyrazole derivatives with the objec-
tive of discovering novel and potent analgesic
and anti-inflammatory agents that might be
devoid of gastrointestinal side effects. The syn-
thesized compounds were tested for their anal-
gesic, anti-inflammatory and ulcerogenic index
behaviour.
Nonsteroidal anti-inflammatory drugs (NSAIDs)
are widely used for the choice treatment in vari-
ous inflammatory diseases such as arthritis,
rheumatisms and relieve body aches and pain of
everyday life [1–4]. However, long-term use of
the NSAIDs has been associated with gastrointes-
tinal ulceration, bleeding and nephrotoxicity
[1–4]. Therefore the discovery of new safer anti-
inflammatory drugs represents a challenging
goal for such a research area. The currently avail-
able NSAIDs belong to different chemical classes
[5]. The pyrazole and substituted pyrazole deriv-
atives have occupied a prominent place in medic-
inal chemistry because of their significant
therapeutic properties in clinical applications
[5–7]. In particular, it has been reported for
diverse range of pharmacological activities
including antihistamin, anti-arrhythmic, anti-
ulcer, leishmanicidal, HIV-RT inhibitor, anti-can-
cer, antimalarial, anti-microbial, and cytotoxicity
[7–13]. In addition to this several pyrazole ana-
logs were also reported to exhibit analgesic and
anti-inflammatory activity [7,14–16].
received 18.03.2012
accepted 02.07.2012
Bibliography
DOI http://dx.doi.org/
10.1055/s-0032-1321830
Published online:
August 29, 2012
Arzneimittelforschung 2012;
62: 457–462
© Georg Thieme Verlag KG
Stuttgart · New York
ISSN 0004-4172
Materials and Methods
Correspondence
P. D. Gokulan
Department of Pharmaceutical
Chemistry
Vinayaka Mission’s College of
Pharmacy
Sankari Main Road
Ariyanoor
Salem - 636 308
India
Tel.: +91/427/247 7903
Fax: +91/427/247 7903
drvalagarsamy@gmail.com
gokulakila@yahoo.co.in
▼
Chemistry
Melting points (mp) were taken in open capillar-
ies on Thomas Hoover melting point apparatus
(Thomas Hoover, Philadelphia, USA) and are
uncorrected. The IR spectra were recorded in film
or in potassium bromide disks on a Perkin-Elmer
398 spectrometer (Bio Engineering, Wald, Swit-
zerland). The 1H NMR spectra were recorded on a
DPX-300MHz Bruker FT-NMR spectrometer
(Pacific Northwest, Richland, Washington, USA).
The chemical shifts were reported as parts per
However, tetra substituted pyrazole derivatives
were not been studied towards analgesic and
Gokulan PD et al. Synthesis of novel pyrazole analogs… Arzneimittelforschung 2012; 62: 457–462

458 Original Article
million (δ ppm) tetramethylsilane (TMS) as an internal standard.
s, 2H, NH2), 7.49–7.56 (m, 5H, Ar-H); MS (m/z) 277 (M+); Anal.
Calcd for C13H15O2S: C, 56.41; H, 5.37; N, 13.16; Found C, 56.38;
H, 5.35; N, 13.14.
Mass spectra were obtained on a JEOL-SX-102 instrument
(maspec, Tokyo, Japan) using fast atom bombardment (FAB posi-
tive). Elemental analysis was performed on a Perkin-Elmer 2400
CHN analyzer and values were within the acceptable limits of
the calculated values (±0.4%). Spectral data (IR, NMR and mass
spectra) confirmed the structures of the synthesized com-
pounds; the purity of these compounds was ascertained by
microanalysis. The progress of the reaction was monitored on
readymade silica gel plates (Merck Whitehouse Station, NJ, USA)
using chloroform/methanol (9:1) as a solvent system. Iodine was
used as a developing agent. All chemicals and reagents used in
the synthesis were obtained from Aldrich (Sigma-Aldrich,
Spruce St. St. Louis, MO 63103), Lancaster (Alfa Aesar, Johnson
Matthey Company, Ward Hill, MA 01835, USA) or Spectrochem
Pvt.Ltd (Mumbai, India) and were used without further purifica-
tion.
5-Acetylamino-3-methylsulfanyl-1H-pyrazole-4-
carboxylic acid ethyl ester (3a)
5-Amino-3-methylsulfanyl-1H-pyrazole-4-carboxylic acid ethyl
ester (3) (2.01g, 0.01mol) and acetic anhydride (0.05mol) was
placed in a round bottom flask and refluxed for 3h. The reaction
mixture was cooled and poured into ice-water and filtered. The
precipitated product was filtered and washed with water and
dried; the precipitated was recrystallized from ethanol water
(75:25) mixture. Yield 80%; m. p. 150–152°C; Rf 0.39 (CHCl3:
CH3OH, 9: 1); IR (KBr, cm−1): 3344 (Pyrazole NH Str), 3269
(Amide NH Str), 1710 (Ester C=O Str), 1670 (Amide C=O Str),
597 (C=N Str), 1292 (Ester C-O Str), 705 (C-S-C Str); 1H NMR
(500MHz, CDCl3): δ 1.38–1.41 (t, 3H, CH2CH3), 2.26 (s, 3H,
NHCOCH3), 2.53 (s, 3H, SCH3), 4.32–4.36 (q, 2H, CH2CH3), 9.67
(br s, 1H, NH), 11.86 (br s, 1H, NH); MS (m/z) 242 (M+); Anal.
Calcd for C9H13N3O3S: C, 44.43; H, 5.39; N, 17.27; Found C, 44.4;
H, 5.36; N, 17.24.
Ethyl 2-cyano-3,3-bis(methylthio)acrylate (2)
To a solution of dimethylformamide (30mL) and 1.13g (0.01mol)
of ethyl cyanoacetate (1), and sodium hydroxide (0. 8g, 0.02mol)
in a minimum amount of water at 0°C, was added. The reaction
mixtures were treated drop wise 0. 6mL of carbon disulphide for
1h at 0°C with continues stirring. This mixture was further
stirred for 1h with 2. 52g (0.02mol, 1.9mL) of dimethyl sul-
phate, which was added drop wise at 10–15°C. The stirring was
continued for further 3h and the reaction mixture was poured
into ice-water. The precipitated product was filtered and dried;
the precipitated was recrystallized from ethanol. Yield 93%;
m.p.65–66°C; Rf 0.78 (CHCl3: CH3OH, 9: 1); IR (KBr, cm−1): 1710
(Ester C=O Str), 1245 (Ester C-O Str), 767 (CN deformation), 686
(C-S-C Str); 1H NMR (500MHz, CDCl3): δ 1.33–1.36 (t, 3H,
CH2CH3), 2.60 (s, 3H, SCH3), 2.75 (s, 3H, SCH3), 4.26–4.30 (q, 2H,
CH2CH3); MS (m/z) 217 (M+); Anal. Calcd for C8H11NO2S2: C,
44.22; H, 5.1; N, 6.45; Found: C, 44.25; H, 5.08, N, 6.43.
5-Chloroacetylamino-3-methylsulfanyl-1H-pyrazole-4-
carboxylic acid ethyl ester (3b)
5-Amino-3-methylsulfanyl-1H-pyrazole-4-carboxylic acid ethyl
ester (3) (2.01g, 0.01mol) was dissolved in 0.02 mol of glacial
acetic acid, to this chloroacetylchloride (1.12g, 0.01mol) was
added and reaction mixture was refluxed for 1h. The remaining
procedure was the same as for compound 3a.Yield 82% m. p.
115–117°C; Rf 0.37 (CHCl3: CH3OH, 9: 1); IR (KBr, cm−1): 3315
(Pyrazole NH Str), 3279 (Amide NH Str), 1690 (Ester C=O Str),
1664 (Amide C=O Str), 1585 (C=N Str), 1298 (Ester C-O Str),
781 (C-Cl Str); 686 (C-S-C Str); 1H NMR (500MHz, CDCl3):
δ 1.38–1.40 (t, 3H, CH2CH3), 2.27 (s, 3H, SCH3), 4.22 (s, 2H, ClCH2
CONH), 4.34–4.39 (q, 2H, CH2CH3), 9.68 (br s, 1H, NH), 10.66 (br
s, 1H, NH).; MS (m/z) 278 (M+); Anal. Calcd for C9H12ClN3O3S: C,
38.92; H,4.36; N,15.13; Found C,38.89; H,4.33; N,15.11.
5-Amino-3-methylsulfanyl-1H-pyrazole-4-carboxylic
acid ethyl ester (3)
A mixture of 2.17g (0.01mol) of ethyl 2-cyano-3,3-bis(methylthio)
acrylate (2) and 0.5g (0.01mol) of (99%) hydrazine hydrate in
25mL of ethanol was refluxed for 2h. The reaction mixture was
cooled and poured into ice-water and filtered. The precipitated
product was filtered and washed with water and dried; the pre-
cipitated was recrystallized from ethanol water (75:25) mixture.
Yield 81%; m. p. 140–142°C; Rf 0.36 (CHCl3: CH3OH, 9: 1); IR
(KBr, cm−1): 3389–3332 (NH2 Str), 1653 (Ester C=O Str), 1626
(C=N Str), 1287 (Ester C-O Str ), 659 (C-S-C); 1H NMR (500MHz,
CDCl3): δ 1.36–1.39 (t, J=7.0Hz, 3H, CH2CH3), 2.48 (s, 3H,
SCH3),4.28–4.32 (q, 2H, CH2CH3), 6.13 (br s, 2H, NH2), 12.87 (br s,
1H, NH); MS (m/z) 202 (M+1); Anal. Calcd for C7H11N3O2S: C,
41.78; H, 5.51; N, 20.88; Found C, 41.74; H, 5.48; N, 20.85.
5-Benzoylamino-3-methylsulfanyl-1H-pyrazole-4-
carboxylic acid ethyl ester (3c)
5-Amino-3-methylsulfanyl-1H-pyrazole-4-carboxylic acid ethyl
ester (3) (2.01g, 0.01mol) was dissolved in 5mL of pyridine to
this was added benzoyl chloride (0.013mol) drop wise with stir-
ring. After the completion of addition, the mixture was stirred
for further 1h and then the pyridine was removed by high vac-
uum rotavapor and the reaction mixture was dried to obtain the
precipitated product. It was crystallized with chloroform-etha-
nol mixture. Yield 74%; m. p. 123–125°C; Rf 0.71 (CHCl3: CH3OH,
9: 1); IR (KBr, cm−1): 3230 (Pyrazole NH Str), 3190 (Amide NH
Str), 1680 (Ester C=O Str), 1602 (C=N Str), 1550 (Amide C=O
Str), 1250 (Ester C-O Str), 660 (C-S-C); 1H NMR (500MHz,
CDCl3): δ 1.38–1.41 (t, 3H, CH2CH3), 2.53 (s, 3H, SCH3), 4.30–4.35
(q, 2H, CH2CH3), 7.48–7.54 (m, 5H, Ar-H), 8.47 (br s, 1H, NH),
9.41 (br s, 1H, NH); MS (m/z) 306 (M+); Anal. Calcd for
C14H15N3O3S: C, 55.07; H, 4.95; N, 13.76; Found C, 55.04; H, 4.93;
N, 13.73.
5-Amino-3-methylsulfanyl-1-phenyl-1H-pyrazole-4-
carboxylic acid ethyl ester (4)
A mixture of 2.17g (0.01mol) of ethyl 2-cyano-3,3-bis(methylthio)
acrylate (2) and 1.08g (0.01mol) of phenyl hydrazine in 30mL
ethanol was refluxed for 2h. The remaining procedure was the
same as for compound 3. Yield 85%; m. p. 95–97°C; Rf 0.39
(CHCl3: CH3OH, 9: 1); IR (KBr, cm−1): 3331–3314 (NH2 Str),
1669 (Ester C=O Str), 1608 (C=N Str), 1277 (Ester C-O Str), 687
(C-S-C Str); 1H NMR (500MHz, CDCl3): δ 1.39–1.42 (t, 3H,
CH2CH3), 2.54 (s, 3H, SCH3), 4.32–4.36 (q, 2H, CH2CH3), 5.37 (br
3-Methylsulfanyl-5-(3-phenyl-thioureido)-1H-pyrazole-
4-carboxylic acid ethyl ester (3d)
A mixture of compound 5-amino-3-methylsulfanyl-1H-pyra-
zole-4-carboxylic acid ethyl ester (3) (0.01mol) and methyl-(N-
phenyl) dithiocarbamate (0.01mol) was dissolved in ethanol
Gokulan PD et al. Synthesis of novel pyrazole analogs… Arzneimittelforschung 2012; 62: 457–462

Original Article 459
and a pinch potassium carbonate was added and refluxed for 9h.
The remaining procedure was the same as for compound 3a.
Yield 81%; m. p. 232–233°C; Rf 0.39 (CHCl3: CH3OH, 9: 1); IR
(KBr, cm−1):3431 (NH Str), 3323 (Amide NH Str), 1713 (Ester
C=O Str), 1668 (Amide C=O Str), 1608 (C=N Str), 1278 (Ester
C-O Str), 688 (C-S-C Str); 1H NMR (500MHz, CDCl3): δ 1.30–1.33
(t, 3H, CH2CH3), 2.51 (s, 3H, SCH3), 4.62–4.66 (q, 2H, CH2CH3),
7.28–7.39 (m, 5H, Ar-H), 8.12 (br s, 1H, NH), 8.81 (br s, 1H, NH),
9.46 (br s, 1H, NH); MS (m/z) 336 (M+); Anal. Calcd for
C14H16N4O2S2: C, 49.98; H, 4.79; N, 16.65; Found C, 49.95; H,
4.75; N, 16.63.
5-Benzoylamino-3-methylsulfanyl-1-phenyl-1H-
pyrazole-4-carboxylic acid ethyl ester (4c)
A mixture of 5-amino-3-methylsulfanyl-1-phenyl-1H-pyrazole-
4-carboxylic acid ethyl ester (4) (2.77g, 0.01mol) was dissolved
in 5mL of pyridine to this was added benzoyl chloride (0.013mol)
drop wise with stirring. After the completion of addition, the
mixture was stirred for further 1h and then the pyridine was
removed by suction and the reaction mixture was dried to
obtain the precipitated product. It was crystallized with chloro-
form-ethanol mixture. Yield 79%; m. p. 152–155°C; Rf 0.65
(CHCl3: CH3OH, 9: 1); IR (KBr, cm−1): 3290.82 (Amide NH Str),
1713 (Ester C=O Str), 1701 (Amide C=O Str), 1589 (C=N Str),
1236 (Ester C-O Str), 692 (C-S-C Str); 1H NMR (500MHz, CDCl3):
δ 1.22–1.25 (t, 3H, CH2CH3), 2.53 (s, 3H, SCH3) 4.27–4.31 (q, 2H,
CH2CH3), 7.25–7.46 (m, 9H, Ar-H), 7.80 (br s, 1H, CONH); MS
(m/z) 382(M+) Anal. Calcd for C20H19N3O3S: C,62.97; H,5.02;
N,11.02; Found C,62.94; H,4.98; N,10.99.
3-Methylsulfanyl-5-(3-(4-methyl phenyl)-thioureido)-
1H-pyrazole-4-carboxylic acid ethyl ester (3e)
A mixture of compound 5-amino-3-methylsulfanyl-1H-pyra-
zole-4-carboxylic acid ethyl ester (3) (0.01mol) and methyl-(N-
(4-methyl phenyl)) dithiocarbamate (0.01mol) was dissolved in
ethanol and a pinch potassium carbonate was added and
refluxed for 12h. The remaining procedure was the same as for
compound 3a. Yield 77%; m. p. 143–145°C; Rf 0.41 (CHCl3:
CH3OH, 9: 1); IR (KBr, cm−1): 3362 (NH Str), 3296 (Amide NH
Str), 1716 (Ester C=O Str), 1685 (Amide C=O Str), 1639 (C=N
Str), 1226 (Ester C-O Str), 632 (C-S-C Str); 1H NMR (500MHz,
CDCl3): δ 1.32–1.34 (t, 3H, CH2CH3), 2.34 (s, 3H, SCH3), 2.46 (s,
3H, CH3), 4.24–4.27 (q, 2H, CH2CH3), 7.15–7.17 (d, 2H, Ar-H),
7.31–7.32 (d, 2H, Ar-H), 7.39 (br s, 1H, NH), 8.44 (br s, 1H, NH),
9.17 (br s, 1H, NH); MS (m/z) 350 (M+); Anal. Calcd for
C15H18N4O2S2: C, 51.41; H, 5.18; N, 15.99; Found C, 51.37; H,
5.15; N, 15.96.
3-Methylsulfanyl-1-phenyl-5-(3-phenyl-thioureido)-1H-
pyrazole-4-carboxylic acid ethyl ester (4d)
A mixture of compound 5-amino-3-methylsulfanyl-1-phenyl-
1H-pyrazole-4-carboxylic acid ethyl ester (4) (0.01mol) and
methyl-(N-phenyl) dithiocarbamate (0.01mol) was dissolved in
ethanol and a pinch potassium carbonate was added and
refluxed for 10h. The remaining procedure was the same as for
compound 3a. Yield 78%; m. p. 188–190°C; Rf 0.45 (CHCl3:
CH3OH, 9: 1); IR (KBr, cm−1): 3342 (NH Str), 3273 (NH Str), 1700
(Ester C=O Str) 1671 (Amide C=O Str), 1598 (C=N Str) 1278
(Ester C-O Str), 633 (C-S-C Str); 1H NMR (500MHz, CDCl3):
δ 1.26–1.29 (t, 3H, CH2CH3), 2.59 (s, 3H, SCH3), 4.29–4.33 (q, 2H,
CH2CH3), 6.83 (br s, 1H, NH), 7.31–7.40m, (5H, Ar-H), 7.51–7.62
(m, 5H, Ar-H), 8.02 (br s, 1H, NH); MS (m/z) 412 (M+); Anal.
Calcd for C20H20N4O2S2: C,58.23; H,4.89; N,13.58; Found. C,58.2;
H,4.85; N,13.55.
5-Acetylamino-3-methylsulfanyl-1-phenyl-1H-pyrazole-
4-carboxylic acid ethyl ester (4a)
A mixture of 5-amino-3-methylsulfanyl-1-phenyl-1H-pyrazole-
4-carboxylic acid ethyl ester (4) (2.77g, 0.01mol) and acetic
anhydride (0.05mol) was placed in a RB flask and refluxed for
3h. The remaining procedure was the same as for compound 3a.
Yield 79%; m. p. 171–173ºC; Rf 0.48 (CHCl3: CH3OH, 9: 1); IR
(KBr, cm−1): 3388 (Amide NH Str), 1758 (Ester C=O Str), 1734
(Amide C=O Str), 1599 (C=N Str), 1239 (Ester C-O Str), 688
(C-S-C Str); 1H NMR (500MHz, CDCl3): δ 1.40–1–43 (t, 3H,
CH2CH3), 2.03 (s, 3H, NHCOCH3) 2.61 (s, 3H, SCH3), 4.31–4.34 (q,
2H, CH2CH3), 7.51–7.59 (m, 5H, Ar-H), 9.61(br s, 1H, CONH); MS
(m/z) 320 (M+); Anal. Calcd for C15H17N3O3S: C, 56.41; H,5.37;
N,13.16; Found C, 56.38; H,5.35; N13.14.
3-Methylsulfanyl-1-phenyl-5-(3-(4-methyl phenyl)-
thioureido)-1H-pyrazole-4-carboxylic acid ethyl ester
(4e)
A mixture of compound 5-amino-3-methylsulfanyl-1-phenyl-
1H-pyrazole-4-carboxylic acid ethyl ester (4) (0.01mol) and
methyl-(N-(4-methyl phenyl)) dithiocarbamate (0.01mol) was
dissolved in 20mL of ethanol and a pinch potassium carbonate
was added and refluxed for 13h. The remaining procedure was
the same as for compound 2. Yield 81%; m. p. 211–212°C; Rf
0.71 (CHCl3: CH3OH, 9: 1); IR (KBr, cm−1): 3283 (NH Str), 3149
(NH Str), 1693 (Ester C=O Str), 1593 (C=N Str), 1246 (Ester C-O
Str), 1138 (Thioamide C=S Str), 680 (C-S-C Str); 1H NMR
(500MHz, CDCl3): δ 1.39–1.41 (t, 3H, CH2CH3), 2.32 (s, 3H, CH3),
2.54(s, 3H, SCH3), 4.32–4.36 (q, 2H, CH2CH3), 4.64(br s, 1H, NH),
7.20–7.28 (m, 5H, Ar-H), 7.49–7.56 (m, 4H, Ar-H), 7.90 (br s, 1H,
NH); MS (m/z) 427(M+); Anal. Calcd for C21H22N4O2S2: C,59.13;
H,5.2; N,13.13; Found C,59.11; H,5.18; N,13.1
5-Chloroacetylamino-3-methylsulfanyl-1-phenyl-1H-
pyrazole-4-carboxylic acid ethyl ester (4b)
A mixture of 5-amino-3-methylsulfanyl-1-phenyl-1H-pyrazole-
4-carboxylic acid ethyl ester (4) (2.77g, 0.01mol) was dissolved
in glacial acetic acid (0.02mol), to this chloro acetylchloride
(1.12g, 0.01mol) was added and the reaction mixture was
refluxed for 1h. The remaining procedure was the same as for
compound 3a. Yield 89%; m. p. 161–163°C; Rf 0.45 (CHCl3:
CH3OH, 9: 1); IR (KBr, cm−1): 3220 (Amide NH Str), 1706 (Ester
C=O Str), 1669 (Amide C=O Str), 1559 (C=N Str), 1234 (Ester
C-O Str), 774 (C-Cl Str), 657 (C-S-C Str); 1H NMR (500MHz,
CDCl3): δ 1.39–1.40 (t, 3H, CH2CH3), 2.57 (s, 3H, SCH3), 4.04 (s,
2H, CH2ClCONH), 4.31–4.36 (q, 2H, CH2CH3), 7.34–7.52 (m, 5H,
Ar-H), 8.35 (br s, 1H, CONH); MS (m/z) 353 (M+); Anal. Calcd for
C15H16ClN3O3S: C, 50.92; H,4.56; N,11.88; Found. C, 50.89;
H,4.54; N,11.85;
Pharmacology
▼
The synthesized compounds were evaluated for analgesic, anti-
inflammatory and ulcerogenic index. The test compounds and
the standard drugs were administered in the form of a suspen-
sion (using 1% carboxymethylcellulose as a vehicle) by oral route
of administration for analgesic and anti-inflammatory. For ulcer-
ogenicity studies the drug were administrated by intraperito-
Gokulan PD et al. Synthesis of novel pyrazole analogs… Arzneimittelforschung 2012; 62: 457–462

460 Original Article
neally as suspension in 10% V/V Tween 80. Each group consisted
ment technique with the help of a plethysmograph immediately
of six animals. The animals were procured from the Tetrex Bio-
logical Center, Madurai, India, and were maintained in colony
cages at 25±2°C, relative humidity 45–55%, under a 12h light
and dark cycle; they were fed standard animal feed. All the ani-
mals were acclimatized for a week before use. The Institutional
Animal Ethics committee has approved the protocol adopted for
the experimentation of animals.
before and 1, 2 and 3h after carrageenan injection. The percent-
▶
age inhibition of paw oedema was calculated using ( Table 2)
●
the following formula
Percent inhibition I=100[1−(a−x)/(b−y)]
Where x is the mean paw volume of rats before the administra-
tion of carrageenan and test compounds or reference compound
(test group), a is the mean paw volume of rats after the adminis-
tration of carrageenan in the test group (drug treated), b is the
mean paw volume of rats after the administration of carra-
geenan in the control group and y is the mean paw volume of
rats before the administration of carrageenan in the control
group.
Analgesic activity
Test for analgesic activity was performed by tail-flick technique
using Wistar albino mice (25–35g) of either sex selected by a
random sampling technique [17–19]. The test compounds and
pentazocine at a dose level of 5mg kg−1 was administered orally.
The reaction time was recorded at 30min and 1, 2 and 3h after
the treatment, and the cut-off time was 10s. The percentage
▶
analgesic activity ( Table 1) (PAA) was calculated by the follow-
Evaluation of ulcerogenicity index
●
ing formula,
Ulceration in rats was induced as described by reported protocol
[21]. Albino rats of the Wistar strain weighing 150–200g of
either sex were divided into various groups each of 6 animals.
The control groups of animals were administered only 10% V/V
Tween 80 suspension intraperitoneally. One group were adminis-
tered with indomethacin intraperitoneally at a dose of 5mg kg−1
once daily for 3 days. The remaining group of animals was
administered with test compounds intraperitoneally at a dose of
5mg kg−1. On the fourth day, pylorus was ligated as per the
method of Shay et al. [22]. Animals were fasted for 36h before
the pylorus ligation procedure. 4h after the ligation, animals
were sacrificed. The stomach was removed and opened along with
ª
«
¬
º
»
¼
T2 ꢀ T1
10 ꢀ T1
PAA
u100
where T1 is the reaction time (s) before treatment, and T2 is the
reaction time (s) after treatment.
Anti-inflammatory activity
Anti-inflammatory activity was evaluated by carrageenan-
induced paw oedema test in rats [20]. The test compounds and
Diclofenac sodium at a dose level of 5mg kg−1 was administered.
The paw volumes were measured using the mercury displace-
Fig. 1 Synthesis of pyrazole derivatives 3a–e and
4a–e.
NC
SCH3
Na
S
a
b
NC
NC
COOC2H5
Na
COOC2H5
SCH3
COOC2H5
(2)
S
(1)
c
C2H5OOC
H2N
SCH3
C2H5OOC
SCH3
d
R1
N
N
N
N
N
R
H
R
(3) R = H
(4) R = Phenyl
(a) R 1 = -COCH3
(3a-d) R = H
(4a-d) R = Phenyl
(b) R 1 = -COCH2Cl
(c) R 1 = -COC6H5
S
(d) R1
=
=
C
NH
NH
S
R1
C
CH3
(e)
Reagents and conditions: (a) NaOH, CS2 , DMF, 0 °C for 1 h, 30 min; (b) (CH3)2SO4,
10-15 °C, 3 h; (c) R-NH-NH2, ethanol reflux; (d) For compounds 3a and 4a (CH3CO)2O
reflux; For compounds 3b and 4b; gla CH3COOH, Cl-CH2COCl reflux; For compounds
3c and 4c pyridine, benzoyl chloride reflux; For compounds 3d, 3e, 4d and 4e RCSNHR
K2CO3, ethanol reflux.
Gokulan PD et al. Synthesis of novel pyrazole analogs… Arzneimittelforschung 2012; 62: 457–462

Original Article 461
Compound
Dose (mg/kg)
Percent analgesic activity
2h
Table 1 Analgesic activity of syn-
thesized compounds (3a–e and
4a–e) by tail-flick technique.
30min
1h
3h
3a
5
5
5
5
5
5
5
5
5
5
5
–
23.61±0.65d
20.24±0.66e
33.00±0.80c
14.88±0.50e
13.89±1.34 c
30.62±1.11d
27.68±0.94d
47.62±0.87c
20.24±1.12e
23.31±0.96e
39.25±0.79c
1.46±0.69
38.00±0. 60c
31.55±1.01c
39.32±0.99c
27.67±0.50c
28.77±0.91d
57.61±1.26c
46.73±0.53c
69.35±1.10c
36.31±0.65c
42.16±1.16c
66.47±0.73c
2.27±0.97
57.24±0.21c
47.62±0.58c
58.43±0.97c
38.10±0.68c
39.25±0.73c
67.00±1.12c
63.99±0.87c
77.68±1.23c
50.00±0.64c
58.93±1.23c
77.25±0.43c
3.02±0.51
16.07±0.01c
13.39±0.80 c
22.52±0.78d
12.80±0.79 d
12.33±0.71 d
28.84±1.11d
21.13±0.60d
38.99±0.71c
17.56±1.26c
18.75±1.18
65.09±0.71c
2.09±0.67
3b
3c
3d
3e
4a
4b
4c
4d
4e
Pentazocine
Control
Each value represents the mean±SD (n=6). Significant difference relative to control at same time points and comparison were
measured: c p<0.001, d p<0.01, e p<0.05,
1-substituted-1H-pyrazole-4-carboxylic acid ethyl ester (3 and
4) by 1,3-dipolar cyclo addition. The desired pyrazole derivatives
(3a–e and 4a–e) were obtained by the reaction of 5-amino-3-
Table 2 Anti-inflammatory activity of synthesized compounds (3a–e and
4a–e) determined by Carrageenan induced paw oedema test in rats.
Compound
Dose
Percent Protection
2h
methylsulfanyl-1-substituted-1H-pyrazole-4-carboxylicacid
(mg/kg)
1h
3h
▶
ethyl ester with different reagents and conditions ( Fig. 1). The
●
3a
5
5
5
5
5
5
5
5
5
5
5
28.57±1.1c
21.77±0.8c
31.98±1.0c
21.77±0.8c
25.17±1.6c
31.98±0.5c
25.17±0.7d
38.78±0.9c
21.77±0.3c
21.77±0.8c
62.58±0.2c
54.07±0.6c
48.82±0.7c
56.69±0.4c
39.63±0.9c
44.88±0.5c
56.69±0.3c
52.75±0.5c
58.00±0.7c
42.26±0.9c
46.19±0.6c
73.75±0.4c
37.65±0.6c
36.45±0.5c
41.58±0.9c
29.26±0.5c
30.46±0.6c
40.05±0.4c
37.35±0.6c
45.18±0.6c
35.25±0.3c
37.65±0.9c
70.02±0.4c
1
synthesized compounds were characterized by IR, H-NMR, and
3b
mass spectral data. Elemental analysis satisfactorily confirmed
elemental composition and purity of the synthesized compounds.
3c
3d
3e
Pharmacology
4a
Test for analgesic activity was performed by tail-flick technique
using Wistar albino mice [17–19]. The results of analgesic test-
ing indicate that the test compounds exhibited moderate analge-
sic activity after 30min of reaction time and an increase in
4b
4c
4d
4e
Diclofenac
sodium
Control
activity after 1h which reached a peak level after 2h. Decline in
▶
activity was observed after 3h ( Table 1). According to the
●
–
1.17±0.08
1.95±0.05
2.07±0.08
structure–activity relationship (SAR) studies, all the compounds
have shown moderate analgesic activity when compared with
reference drug pentazocine. Further results indicated that differ-
ent substituents on the 5th position and N-1 pyrazole ring,
exerted varied biological activity. Among the various amide
(acetamide (3a), chloroacetamide (3b) and benzoyl amide (3c))
substitution on the 5th position, benzoyl amide substituted
group (3c) showed significant activity. Compounds having
1-methyl-3-phenylthiourea (3d) and 1-methyl-3-p-tolylthiou-
rea substitution (3e) on the 5th position of pyrazole ring system
leads to abolish the analgesic activity. The phenyl substitution
on N-1 pyrazole ring system led to significant increases in anal-
gesic activity when compared to unsubstituted compounds.
Among the series, 5-benzoylamino-3-methylsulfanyl-1-phenyl-
Each value represents the mean±SD (n=6). Significant difference relative to control
at same time points and comparison were measured: cp<0.001, dp<0.01, ep<0.05,
the greater curvature. Ulcer index was determined by the method
▶
of Ganguly and Bhatnagar [23] and is recorded in Table 3.
●
Statistical analysis
Statistical analysis of the biological activity of the synthesized
compounds on animals was evaluated using a one-way analysis
of variance (ANOVA). In all cases, post-hoc comparisons of the
means of individual groups were performed using Tukey's test. A
significance level of p<0.05 denoted significance in all cases. All
values are expressed as mean±SD (standard deviations). For sta-
tistical analysis we have used GraphPad Prism version 3.0.
(GraphPad Software, Inc. San Diego, CA 92130 USA).
1H-pyrazole-4-carboxylic acid ethyl ester (4c) is found to equi-
▶
potent with the reference drug pentazocine ( Table 1) .
●
A n t i - i n flammatory activity was evaluated by carrageenan
induced paw oedema test in rats [20]. The anti-inflammatory
▶
Results and Discussion
activity data ( Table 2) indicated that all the test compounds
●
protected rats from carrageenan-induced inflammation moder-
ately after 30min of reaction time with increased activity after
1h that reached a peak level after 2h. Decline in activity was
observed after 3h. The SAR studies indicated that different sub-
stituents on the 5th position and N−1 pyrazole ring, exerted var-
ied protection effect from carrageenan-induced inflammation.
The compound 5-benzoylamino-3-methylsulfanyl-1-phenyl-
1H-pyrazole-4-carboxylic acid ethyl ester (4c) showed moderate
anti-inflammatory activity when compared to the reference
standard diclofenac sodium.
▼
Chemistry
The target compounds were synthesized according to steps
outlined in
▶
●
Fig. 1. The precursor of ethyl 2-cyano-3,3-
bis(methylthio)acrylate (2) was easily synthesized by treatment
of ethyl cyanoacetate (1) with carbon disulphide and sodium
hydroxide in DMF to give sodium dithiocarbamate, which was
methylated with dimethyl sulphate to afford the dithiocarbamic
acid methyl ester (2). The compound (2) reacted with hydrazine
and substituted hydrazine that gives 5-amino-3-methylsulfanyl-
Gokulan PD et al. Synthesis of novel pyrazole analogs… Arzneimittelforschung 2012; 62: 457–462

462 Original Article
Table 3 Evaluation of ulcerogenic behavior.
Compound
Dose (mg/Kg)
Volume of gastric juice (mL/4h)
pH
Total acid out put (mEq/L)
Free acid (mEq/L)
Ulcer index
3c
5
5
5
–
4.34±0.2a
4.53±0.4a
10.32±0.4
2.12±0.3
2.15±0.3a
2.13±0.2a
2.04±0.3
3.84±0.5
29.54±0.3a
29.65±0.3a
60.33±0.5
13.32±0.4
11.76±0.5a
11.78±0.5a
24.90±0.4
5.85±0.2
1.84±0.5 a
1.68±0.4a
3.79±0.3
0.06±0.01
4c
Indomethacin
Control
Each value represents the mean±SD (n=6). Significant difference relative to control at same time points and comparison were measured: cp<0.001, dp<0.01, ep<0.05
5 Juby PF. In: Antiinflammatory Agents. Scerrer RA, Whitehouse MW.
Two selected compounds (3c and 4c) of the series were evalu-
(Eds.), vol. I:Academic Press, New York: 1984; 91–127
▶
ated for their ulcerogenic behaviour ( Table 3). The ulcer index
●
6 Daidone G, Plescia S, Raffa D et al. Researches on antiinflammatory
agents. Studies on some new 3-(pyrazol-5-yl)-1,2,3-benzotriazin-
4(3H)-ones and -quinazolin-4(3H)-ones. Farmaco 1990; 45: 391–398
7 Menozzi G, Mosti L, Bruno O et al. Synthesis and biological evalua-
tion of [alpha-(1,5-disubstituted 1H-pyrazol-4-yl)benzyl]azoles, ana-
logues of bifonazole. Farmaco 1999; 54: 416–422
8 Naito H, Ohsuki S, Sugimori M et al. Synthesis and antitumor activity of
novel pyrimidinyl pyrazole derivatives. II. Optimization of the phenyl-
piperazine moiety of 1-[5-methyl-1-(2-pyrimidinyl)-4-pyrazolyl]-3-
phenylpiperazinyl-1-trans-propenes. Chem Pharm Bull (Tokyo) 2002;
50: 453–462
9 Park HJ, Lee K, Park SJ et al. Identification of antitumor activity of
pyrazole oxime ethers. Bioorg Med Chem Lett 2005; 15: 3307–3312
10 Smith RA, Fathi Z, Brown SE et al. Constrained analogs of CB-1 antag-
onists: 1,5,6,7-Tetrahydro-4H-pyrrolo[3,2-c]pyridine-4-one deriva-
tives. Bioorg Med Chem Lett 2007; 17: 673–678
of the test compounds showed negligible ulcer index, when
compared to reference standard indomethacin. The indometh-
acin induced ulcer model animals had ulcers and hemorrhagic
streaks, where as in animals treated with the pyrazole deriva-
tives compounds of (3c and 4c) at 5mg/kg dose level, there was
a significant reduction in ulcer index. Furthermore the pyrazole
derivatives (3c and 4c) significantly reduces the total volume of
the gastric juice, free and total acidity of gastric secretion and
also has higher activity against gastric ulcers in rats when it was
compared with indomethacin.
11 Jorand-Lebrun C, Brondyk B, Lin J et al. Identification, synthesis, and
biological evaluation of novel pyrazoles as low molecular weight lutei-
nizing hormone receptor agonists. Bioorg Med Chem Lett 2007; 17:
2080–2085
12 Sakya SM, Hou X, Minich ML et al. 5-Heteroatom substituted pyrazoles
as canine COX-2 inhibitors. Part III: molecular modeling studies on
binding contribution of 1-(5-methylsulfonyl)pyrid-2-yl and 4-nitrile.
Bioorg Med Chem Lett 2007; 17: 1067–1072
13 Diana P, Carbone A, Barraja P et al. 3,5-bis(3′-indolyl)pyrazoles, ana-
logues of marine alkaloid nortopsentin: synthesis and antitumor
properties. Bioorg Med Chem Lett 2007; 17: 6134–6137
14 Farag AM, Mayhoub AS, Barakat SE et al. Synthesis of new N-phe-
nylpyrazole derivatives with potent antimicrobial activity. Bioorg
Med Chem 2008; 16: 4569–4578
15 Penning TD, Talley JJ, Bertenshaw SR et al. Synthesis and biological
evaluation of the 1,5-diarylpyrazole class of cyclooxygenase-2 inhibi-
tors: identification of 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-
pyrazol-1-yl]benze nesulfonamide (SC-58635, celecoxib). J Med Chem
1997; 40: 1347–1365
Conclusion
▼
In the present study, synthesis of a new series of 5-substituted-
3-methylsulfanyl-1H-pyrazole-4-carboxylic acid ethyl esters
(3a–e and 4a–e) has been described. The results of the analgesic
and anti-inflammatory activities of the 5-substituted-3-methyl-
sulfanyl-1H(substituted)-pyrazole-4-carboxylic acid ethyl esters
series showed that moderate enhancement of activity. The com-
pound 5-benzoylamino-3-methylsulfanyl-1-phenyl-1H-pyra-
zole-4-carboxylic acid ethyl ester (4c) emerged as the most
active compound. Hence this series could be developed as a
novel class of analgesic and anti-inflammatory agents. Further
structural modification is planned to obtain compounds with
increased analgesic and anti-inflammatory activities with mini-
mal ulcerogenic behaviour.
16 Srivastava BK, Soni R, Patel JZ et al. Hair growth stimulator property of
thienyl substituted pyrazole carboxamide derivatives as a CB1 recep-
tor antagonist with in vivo antiobesity effect. Bioorg Med Chem Lett
2009; 19: 2546–2550
Acknowledgements
17 Amour RE, Smith DL. A method for determinating loss of pain sensa-
tion. J Pharm Exp Therap 1941; 72: 74–78
18 Kulkarni SK. Heat and other physiological stress-induced analgesia:
Catecholamine mediated and naloxone reversible response. Life Sci-
ences 1980; 27: 185–188
▼
The authors thank the Head of RSIC, Indian Institute of Technol-
ogy, Chennai for the mass spectral data and the head, sophisti-
cated analytical instrument facilities, Central Drug Research
Institute, Lucknow for NMR spectral data.
19 Eaton M. Common animal models for spasticity and pain. J Rehabil
Res Dev 2003; 40: 41–54
20 Winter CA, Risely EA, Nuss GW. Carregeenin induced oedema in hind
paw of the rat as assay for Antiinflammatory drugs. Exp Biol Med
1962; 111: 544–547
Conflict of Interest Statement
21 Goyal RK, Chakrabarti A, Sanyal AK. T h e e ffect of biological variables
on the anti-ulcerogenic effect of vegetable plantain banana. Planta
medica 1985; 29: 85–88
22 Shay M, Komarov SA, Fels D et al. A simple method for the uniform pro-
duction of gastric ulceration in the rats. Gastroenterol 1945; 5: 43–61
23 Ganguly AK, Bhatnagar OP. E ffect of bilateral adrenalectomy on pro-
duction of restraint ulcers in the stomach of albino rats. Can J Physilo
Pharmacol 1973; 51: 748–750
▼
The authors report no conflicts of interest to declare in connec-
tion with the contents of this manuscript.
References
1 Van Ryn J, Botting RM. New insights into the mode of action of anti-
inflammatory drugs. Inflamm Res 1995; 44: 1–10
2 Van Ryn J, Trummlitz G, Pairet M. COX-2 selectivity and inflammatory
processes. Curr Med Chem 2000; 7: 1145–1161
3 Van Ryn J. Inhibition of prostaglandin synthesis as a mechanism of
action for aspirin-like drugs. Nature: New Biology 1971; 231: 232–235
4 Beuck M. Nonsteroidal antiinflammatory drugs: A new generation of
cyclooxygenase inhibitors. Angew Chem Int Ed 1999; 38: 631–633
Gokulan PD et al. Synthesis of novel pyrazole analogs… Arzneimittelforschung 2012; 62: 457–462