Synthesis and evaluation of analgesic, anti-inflammatory, and anticancer activities of new pyrazole-3(5)-carboxylic acid derivatives

Article abstract of DOI:10.1007/s00044-012-0072-4

In this article, we synthesized a series of novel 1-benzyl-5(3)-p-tolyl-1H- pyrazole-3(5)-carboxylic acid derivatives and characterized by IR, ¹H NMR, and mass spectroscopy. Compounds were evaluated for their in vivo analgesic and anti-inflammatory activity using the p-benzoquinone-induced writhing test and the carrageenan-induced paw edema model, respectively. Out of 14 compounds tested, 7a, 7c, 7e, 7f, 7i, 8a-b, and 8f-g exhibited potent analgesic and/or anti-inflammatory activity as compared to reference drugs aspirin and indomethacin. Anticancer activity of these compounds was assessed against five cancer cell lines with the MTT assay (HL-60, human promyelocytic leukemia cells; HeLa, human cervical cancer cells; Raji, human B lymphocyte cell line; MCF7, human breast adenocarcinoma cell line; MDA-MB-231, estrogen-independent human breast cancer cell line). Compounds 7a, 8a, and 8b with high anti-inflammatory activity, and also 7d and 7j with mild anti-inflammatory activity exhibited promising anticancer activity against some selected cell lines.

Full text of DOI:10.1007/s00044-012-0072-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:782–793
DOI 10.1007/s00044-012-0072-4
ORIGINAL RESEARCH
Synthesis and evaluation of analgesic, anti-inflammatory,
and anticancer activities of new pyrazole-3(5)-carboxylic acid
derivatives
˙
•
•
•
Burcu C¸ alıs¸kan Akın Yılmaz Ilker Evren
•
Sevda Menevs¸e Orhan Uludag Erden Banoglu
•
Received: 28 December 2011 / Accepted: 19 April 2012 / Published online: 6 May 2012
Ó Springer Science+Business Media, LLC 2012
Abstract In this article, we synthesized a series of novel
1-benzyl-5(3)-p-tolyl-1H-pyrazole-3(5)-carboxylic acid deriv-
1
atives and characterized by IR, H NMR, and mass spec-
Introduction
Fast and effective relief of pain and inflammation in human
beings is an ongoing challenge for medical researchers.
Non-steroidal anti-inflammatory drugs (NSAIDs) represent
an important therapeutic class for the alleviation of pain and
inflammation associated with a number of pathological
conditions including arthritis, Alzheimer’s disease, athero-
sclerosis, and cancer (Barthel and Axford-Gatley, 2010;
Bhalke and Chavan, 2011; Charo and Taub, 2011; Fischer
et al., 2011; Heneka et al., 2011). Recent epidemiological
studies have shown a link between inflammation and cancer
suggesting that patients with chronic inflammatory diseases
could be more prone to suffering from various types of
cancers such as bladder, cervical, gastric, skin, intestinal,
and prostate cancers (Grossman et al., 2000; Mantovani
et al., 2008; Muller-Decker et al., 1998). An increasing body
of evidence has demonstrated that certain tumors and cancer
lines overexpress arachidonic acid (AA) metabolizing
enzymes, i.e., 5-lipoxygenase (5-LO) and cyclooxygenases
(COX-1 and COX-2) (Grossman et al., 2000; Muller-Decker
et al., 1998; Radmark and Samuelsson, 2010; Sheng et al.,
1997; Wang and Dubois, 2010) and their pro-inflammatory
metabolic products, i.e., leukotrienes (LT) and prostaglan-
dins (PG). Pharmacological inhibition of 5-LO has been
shown to potently suppress tumor cell growth by inducing
cell cycle arrest and triggering cell death via an intrinsic
apoptotic pathway (Ghosh and Myers 1997, 1998). There is
also a strong evidence for COX-2 being the major form in
cancer cells for production of PGE2 which promotes cancer
cell growth and survival by several mechanisms includ-
ing increased proliferation and inhibition of apoptosis
(Greenhough et al., 2009; Grossman et al., 2000; Muller-
Decker et al., 1998; Sheng et al., 1997). In addition, micro-
somal prostaglandin E synthase-1 (mPGES-1), a downstream
troscopy. Compounds were evaluated for their in vivo
analgesic and anti-inflammatory activity using the p-benzo-
quinone-induced writhing test and the carrageenan-induced
paw edema model, respectively. Out of 14 compounds tested,
7a, 7c, 7e, 7f, 7i, 8a–b, and 8f–g exhibited potent analgesic
and/or anti-inflammatory activity as compared to reference
drugs aspirin and indomethacin. Anticancer activity of these
compounds was assessed against five cancer cell lines with
the MTT assay (HL-60, human promyelocytic leukemia cells;
HeLa, human cervical cancer cells; Raji, human B lympho-
cyte cell line; MCF7, human breast adenocarcinoma cell line;
MDA-MB-231, estrogen-independent human breast cancer
cell line). Compounds 7a, 8a, and 8b with high anti-inflam-
matory activity, and also 7d and 7j with mild anti-inflam-
matory activity exhibited promising anticancer activity
against some selected cell lines.
Keywords Carrageenan ꢀ Hind paw edema ꢀ
Benzoquinone ꢀ Writhings ꢀ Cell lines
B. C¸ alıs¸kan ꢀ E. Banoglu (&)
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Gazi University, Etiler, 06330 Yenimahalle, Ankara, Turkey
e-mail: banoglu@gazi.edu.tr
A. Yılmaz ꢀ S. Menevs¸e
Department of Medical Biology and Genetics, Faculty of
Medicine, Gazi University, Etiler, 06100 Besevler, Ankara,
Turkey
˙
I. Evren ꢀ O. Uludag
Department of Pharmacology, Faculty of Pharmacy, Gazi
University, Etiler, 06330 Yenimahalle, Ankara, Turkey
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Med Chem Res (2013) 22:782–793
783
enzyme for PGE2 formation, has been linked to development
of various types of cancers including nonsmall cell lung,
colorectal, gastric, and pancreatic cancers (Nakanishi et al.,
2010; Radmark and Samuelsson, 2010). Although our
understanding of the complicated roles of AA-derived eico-
sanoids in tumor microenvironment is continuously improv-
ing, there is a strong chance that the development of novel
anti-inflammatory agents clearly may lead to more effective
cancer chemopreventive and/or therapeutic agents.
yields, respectively. The benzylation process predominantly
occured at a position to yield 1-benzyl-3-phenylpyrazole-5-
carboxylate isomer in a ratio of 5:1. The difference between the
absolute chemical shift valuesof methylene protonsin 1HNMR
was a clear indication of distinguishing between both regioi-
somers as pointed out previously for similar derivatives (Xia
et al., 2007, 2008). Thus, comparison of the 1H NMR spectra of
both isomers showed that while the two methylene protons in
1-benzyl-3-phenyl isomer (5) appeared at relatively downfield
around 5.8 ppm, the same protons in 1-benzyl-5-phenyl isomer
(6) resonated at relatively upfield at about 5.4 ppm, therefore
clearly distinguishing between the regioisomers. After hydro-
lysis of the esters to carboxylic acids (7, 8), the title amide and
ester derivatives were obtained by the reaction of appropriate
amines and phenols using EDC as the carboxylate activator.
Compounds were purified by automated flash chromatography
and checked for purity with UPLC before being tested in bio-
logical experiments (purity was[97 %). The structures of all
compounds were elucidated by high resolution mass spec-
trometry, IR, and 1H NMR spectral data.
Pyrazoles are widely used as core motifs for a large
number of compounds for development of new therapeutics
for inflammation and cancer (Bekhit et al., 2010; Keter and
Darkwa, 2011). In the last decade, we and others have
reported a large series of pyrazole derivatives having
promising anti-inflammatory (Banoglu et al., 2005, 2004,
2003; Caliskan et al., 2011; Ergun et al., 2010; Nagarapu
et al., 2011) and anticancer activities (Ding et al., 2009;
Farag et al., 2010; Lian et al., 2009; Nitulescu et al., 2010;
Xia et al., 2007, 2008; Zheng et al., 2010, 2009), indicating
the use of pyrazole motif as a powerful tool for novel
medicines. Among the reported studies, pyrazole-5-car-
boxylate (Wei et al., 2006; Zheng et al., 2010), pyrazole-5-
carboxamide (Ding et al., 2009), and pyrazole-5-carbohy-
drazide (Lian et al., 2009; Xia et al., 2007; 2008; Zheng
et al., 2009) derivatives were shown to have a significant
anticancer activity against A549 lung cancer cell lines by
inducing apoptosis or autophagy. Therefore, with the aim
of obtaining new compounds with anti-inflammatory and
also with anticancer activity, we synthesized a series of
novel 1-benzyl-3(5)-p-tolyl-1H-pyrazole-5(3)-carboxylic
acid amides and esters and evaluated the effects of these
compounds on carrageenan-induced hind paw edema and
benzoquinone-induced writhing test as well as for their
anticancer activity against five different cancer cell lines
with the MTT assay.
Pharmacology
Our previous findings with certain pyrazole-based com-
pounds resulted in derivatives with potent in vivo anti-
inflammatory and analgesic activity (Banoglu et al., 2005,
2004, 2003). Our ongoing work also demonstrated that
transformation of 1,5-diarylpyrazole-3-propanoic acid
scaffold into lipophilic amides and esters acted as sup-
pressors of LT biosynthesis in intact PMNL, and some
compounds showed a potent inhibition of COX-1 along
with inhibition of LT biosynthesis providing a potent anti-
inflammatory potential (Caliskan et al., 2011; Ergun et al.,
2010). Especially, amidation with t-butylbenzylpiperazine
and ethyl piperidinecarboxylate, and esterification with
i-propyl- or t-butylphenol highly contributed to the effi-
ciency of compounds for inhibition of LT formation in
intact cells (Caliskan et al., 2011). In addition, combination
of N1-phenyl and C5-p-methylphenyl substitution at a
central pyrazole for amides and esters even increased the
potency leading to dual inhibition of platelet COX-1
activity and LT biosynthesis for potential anti-inflamma-
tory activity. Based on aforementioned observations and
also the previous findings indicating pyrazole-5-carboxylic
acid nucleus as suitable for anti-cancer activity (Wei et al.,
2006; Xia et al., 2007, 2008; Zheng et al., 2010, 2009), we
prepared a novel series of amide and ester derivatives of
1-benzyl-3-(p-tolyl)-1H-pyrazole-5-carboxylic acid (7a–j)
and also the regioisomeric counterparts bearing 1-benzyl-
5-(p-tolyl)-1H-pyrazole-3-carboxylic acid core as 8a–
b and 8f–g. Since we previously reported the positive
effects of C5-p-methylphenyl substitution about a central
pyrazole and amidation with piperazines, we kept the
Results and discussion
Chemistry
The synthesis of the resulting amide and ester derivatives is
outlined in Scheme 1. Methyl 2,4-dioxo-4-(p-tolyl)butanoate
(3) was prepared by Claisen condensation of 4-methylaceto-
phenone (1) with dimethyl oxalate (2) in the presence of sodium
metoxide. Then, the starting intermediate methyl 3-(p-tolyl)-
1H-pyrazole-5-carboxylate (4) was readily obtained by the
reaction of 3 with hydrazine in acetic acid. After benzylation of
4 with benzyl chloride in the presence of K2CO3 in acetonitrile,
the regioisomers formed were successfully separated by
automated flash chromatograpy to yield methyl 1-benzyl-3-
(p-tolyl)-1H-pyrazole-5-carboxylate (5) and methyl 1-benzyl-
5-(p-tolyl)-1H-pyrazole-3-carboxylate (6) in 77 and 15 %
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Med Chem Res (2013) 22:782–793
Scheme 1 Synthesis of the
amide and ester derivatives of
1-benzyl-5(3)-p-tolyl-1H-
pyrazole-3(5)-carboxylic acids
(Reagents and conditions:
a (i) sodium methoxide, MeOH
(ii) H?; b NH2NH2ꢀH2O,
AcOH; c Benzyl chloride,
K2CO3, AcCN,
chromatographic separation of
regioisomers 5 and 6; d LiOH,
THF, H2O; e amine/phenol,
EDC, DMAP, CH2Cl2)
p-methylphenyl substitute pyrazole core structure constant
and studied the effect of different amide and ester side
chains on their analgesic, anti-inflammatory, and antican-
cer activities.
quinone derivatives that were used as a chemical inducer of
algesia exhibited more effective and potent activity as of
acetic acid which is an another commonly used method.
These models induce a characteristic and quantifiable overt
pain-like behavior described as writhing response or
abdominal contortions. It was demonstrated that cytokines
such as TNFa (tumor necrosis factor a), IL (interleukin)-
1b, and chemokines act in synergy to induce the writhing
response. In fact, cyclooxygenase products and cysteinyl
Analgesic activity of title compounds was screened in
vivo in mice using the p-benzoquinone-induced writhing
test (Okun et al., 1963). p-Benzoquinone test commonly
used as a fast screening model to access the activity of
novel candidates as potential analgesics. In addition,
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785
Table 1 Analgesic and anti-inflammatory effects of the test compounds on p-benzoquinone (PBQ)-induced abdominal constriction test and
carrageenan (CG)-induced hind paw edema model in mice, respectively
Test compounds Number of writhings SEM Swelling in thickness (910-2 mm) SEM (inhibition of edema, %)b
(analgesic activity, %)a
90 min
180 min
270 min
360 min
Control
7a
28.2 2.8
38.0 2.0
51.0 3.0
75.0 3.0
77.0 3.0
6.5 2.1 (77.0%)***
29.8 4.3
28.0 4.0 (26.7%) 34.0 5.0 (33.3%) 34.0 4.0 (54.4%)*** 33.0 5.0 (57.6%)***
44.2 5.4 49.0 4.0 (4.1%) 55.0 3.0 (26.7%)* 53.0 2.0 (30.4%)*
31.0 7.0 (17.8%) 28.0 5.0 (44.7%)* 19.0 5.0 (74.4%)*** 36.0 5.0 (53.3%)***
36.0 7.0 (3.7%) 43.0 5.0 (16.5%) 57.0 5.0 (24.4%)* 57.0 8.0 (25.3%)*
29.0 6.0 (23.0%) 39.0 8.0 (24.1%) 42.0 7.0 (44.4%)*** 44.0 6.0 (42.8%)***
7b
7c
13.3 2.7 (53.0%)*
30.3 4.6
7d
7e
14.4 3.5 (48.9%)*
25.1 3.6 (10.9%)
18.7 5.8 (33.0%)
27.4 4.4
7f
40.0 6.1
38.3 4.0
42.5 5.3
40.0 5.8
33.0 4.0 (35.0%) 38.0 4.0 (50.0%)*** 29.0 3.0 (62.0%)***
7g
39.0 4.0 (24.0%) 53.0 5.0 (29.0%)*
47.0 5.0 (9.0%) 68.0 6.0 (10.0%)
43.0 5.0 (17.0%) 58.0 4.0 (22.2%)
53.0 6.0 (31.5%)*
80.0 6.2
7h
7i
12.2 4.2 (56.9%)*
24.0 3.4 (14.9%)
4.8 1.7 (83.2%)***
11.5 3.6 (59.2%)*
7.5 3.5 (73.4%)***
15.3 1.8 (45.7%)
8.8 2.9 (68.9%)***
66.0 4.0 (14.1%)
62.0 7.0 (18.8%)
7j
35.0 5.0 (6.6%) 43.0 4.0 (15.4%) 61.6 5.0 (19.2%)
40.0 4.3
8a
44.0 4.0 (13.9%) 32.0 3.0 (57.8%)*** 31.0 4.0 (59.8%)***
8b
34.0 4.0 (8.9%) 28.0 8.0 (46.3%)* 31.0 5.0 (58.9%)*** 28.0 5.0 (63.4%)***
28.0 4.0 (26.0%) 37.0 6.0 (28.5%) 34.0 6.0 (54.0%)*** 32.0 7.0 (58.7%)***
27.0 5.0 (27.4%) 42.0 8.0 (18.7%) 37.0 5.0 (50.4%)*** 40.0 3.0 (47.8%)***
8f
8g
ASA
INDO
32.0 4.0 (15.5%) 35.0 4.0 (31.7%) 41.0 3.0 (45.6%)*** 42.0 4.0 (45.7%)***
Data obtained from animal experiments were expressed as means SEM. ASA aspirin, 100 mg/kg (body weight); INDO indomethacin, 10 mg/
kg (body weight); and all test drugs, 100 mg/kg (body weight) were s.c. administered to mice (n = 6–12) for a PBQ-induced writhing and b CG-
induced paw edema tests, respectively. * Statistically significance were evaluated from the control by One-way ANOVA post hoc Dunnett’s test
(* p \ 0.05, ** p \ 0.01, *** p \ 0.001)
leukotrienes also contribute to nociception in this model.
The PBQ but not acetic acid writhing also depends on
cytokines such as IL-18 and interferon (IFN)-c, and
endothelin-1. It was also shown that PBQ-induced writhing
response in mice depends on spinal activation of ERK,
JNK, p38, and PI3K, and microglia (Pavao-de-Souza et al.,
2012; Verri et al., 2008). Meanwhile, a hotplate latency
tests are reported to quantify antinociception mediated
primarily by spinal and supraspinal mechanisms, and
generally used to evaluate the analgesic activity of cen-
trally acting substances such as narcotics (Baker et al.,
2002). As it is very common in the literature and also our
experience in our laboratory also confirms this fact in our
various demonstrations, we employed p-Benzoquinone-
induced writhing test to evaluate the analgesic activity of
title compounds.
failed to induce analgesic activity. In addition, trimethox-
ybenzylpiperazine amide (7c) and the carbohydrazide
derivative (7e) of pyrazole-5-carboxylic acid nucleus
resulted in comparable activity with respect to reference
aspirin with a small decrease in the inhibitory potential of
writhing movements. In the case of ester analogs, while the
t-butylphenyl ester of 1H-pyrazole-3-carboxylate core (8f)
demonstrated very potent analgesic activity (73 %), the
regioisomeric counterpart (7f) lacked the similar potency
(11 %). The replacement of t-butyl in 8f with i-propyl (8g)
in the ester part caused a decrease in analgesic potency
(45 %). Moreover, while the 7h with a larger heptyloxy-
phenyl group in the ester portion was ineffective, tri-
fluoromethylphenyloxyphenyl group in the ester portion
(7i) restored the analgesic efficiency (57 %).
Anti-inflammatory activities of the compounds were
also in good correlation with the analgesic activity results,
tested by using the carrageenan-induced hind paw edema
model (Table 1) (Kasahara et al., 1985). As shown, the
same amide derivatives (7a, 7c, 7e, 8a, and 8b) as well
as ester derivatives (7f, 8f, and 8g) resulted in potent
anti-inflammatory activity which was comparable to indo-
methacin. It is claimed that an edema produced by carra-
geenan is a biphasic event and the inhibitory effects of
compounds which act on the first stage are attributable to
The results shown in Table 1 indicated that the prepa-
ration of the amide derivatives of both regioisomeric
1-benzyl-3(5)-p-tolyl-1H-pyrazole-5(3)-carboxylic acids
with ethyl piperidinecarboxylate resulted in compounds
with potent analgesic activity (7a, 77 % and 8a, 83 %)
which was higher than aspirin at the same dose of 100 mg/
kg. In the case of t-butylbenzylpiperazine amides (7b, 8b),
there was a discrepancy in favor of 1-benzyl-5-(p-tolyl)-
1H-pyrazole-3-carboxamide core (8b, 59 %) since 7b
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Med Chem Res (2013) 22:782–793
the inhibition of the release of chemical mediators such as
histamine, serotonin, and bradykinin (Vinegar et al., 1969).
However, the second phase of edema might be related to
the release of AA metabolites since it is potently sup-
pressed by aspirin, indomethacin, and other COX inhibitor
drugs (Vinegar et al., 1969). As shown in Table 1, the most
active derivatives showed a similar pattern with indomet-
acin with an absolute gradual increase in the second phase
(270 min), therefore suggesting that these compounds
might exert their anti-inflammatory activities through the
mechanisms involving the inhibition of the formation of
AA metabolites. It should be noted that the amidation of
the carboxylic acid with lipophilic amines such as ethyl
piperidinecarboxylate (7a–8a), t-butylbenzylpiperazine
(8b), and trimethoxybenzylpiperazine (7c) had a significant
impact for rising the anti-inflammatory potential of either
regioisomeric pyrazole-3(5)-carboxylic acids. In the case
of ester derivatives, only the incorporation of small lipo-
philic ester parts such as t-butylphenyl (7f–8f) or i-pro-
pylphenyl (8g) was able to contribute to an increase in the
anti-inflammatory activity. Taken together, we can con-
clude that 1-benzyl-3(5)-(p-tolyl)-1H-pyrazole-5(3)-car-
boxylic acid cores can be modified to a potent analgesic
and anti-inflammatory compounds by preparing lipophilic
amide (i.e. 4-t-butylbenzylpiperazine and ethyl piperidin-
ecarboxylate) and ester derivatives (i.e. 4-t-butylphenyl)
with potential for novel treatments of inflammation-related
disorders as was in good agreement with our previous
findings for 1,5-diarylpyrazole-3-propanoic acid amides
(Caliskan et al., 2011).
above cell lines which provides a suitable cellular model
system to investigate the anti-cancer potential of these
derivatives.
The results for each tested compounds at a 50 lM
screening dose are reported as growth percentages com-
pared with the untreated control cells after 24 and 48 h of
drug exposure (Table 2). Owing to the solubility problems,
compounds 7c and 7e were not evaluated in the MTT
assay. The compound 8b bearing t-butylbenzylpiperazine
group in the amide part of pyrazole-3-carboxamide core
displayed significant growth inhibitory activity against all
cell lines (85 % for HL-60, 60 % for MDA-MB-231, 65 %
for HeLA, 50 % for MCF7, and 70 % for Raji cells at
48 h). For estrogen-independent breast cancer cells (MDA-
MB-231), all compounds demonstrated mild to moderate
inhibitory activity on cell viability at 48 h (25–75 %
inhibition), as the compound 7j, an imidazolylphenyl ester
of pyrazole-5-carboxylate, being the most efficient deriv-
ative (75 %). The efficiency of compounds against HeLa
cervical cancer cells was also ranged between 20 and 70 %
with 7j (66 %) and 8b (65 %) showing equipotent inhibi-
tory activity which was followed by 8a (52 %) and 7a
(50 %) having the most considerable growth inhibition at
both end time points. The sensitivity of 8a (60 %) and 8b
(70 %) was found higher to B lymphocyte cell line (Raji)
among all other derivatives. For human breast adenocar-
cinoma MCF7 cell line, only 7j (58 %) and 8b (54 %)
showed inhibitory potential on cell growth while others
found mostly inefficient. Lastly, for HL-60 leukemia cell
line, 8b (88 %) followed by 7d (70 %, 3-pyridinylmethyl
amide derivative) displayed significant antitumor activity,
especially at 24 h. HL-60 leukemia cells were insensitive
to all other derivatives. Compounds 7j and 8b were chosen
for concentration–response studies at five-concentrations
on selected cell lines where their inhibitory potential was
more pronounced. IC50 values for 8b for HeLa and HL-60,
and for 7j for HeLa cell lines were calculated as 30.5, 24.4,
and 30.4 lM, respectively.
Since lipophilicity is an important factor for molecules
to cross cell membranes and the partition coefficient log
P is a parameter describing this behavior of a drug mole-
cule, we calculated the predicted lipophilicity (log P) val-
ues of the synthesized compounds using ACD/Labs 6.0
software. While the lipophilicity (log P) values of tested
compounds were in the range of 3.15–8.62, our results
indicated that compounds possessing the lipophilicity with
log P values in the range of 3.15–5.31 (7a, 7d, 7j, 8a, and
8b) demonstrated better inhibitory effects on the growth of
selected cancer cells. Our results showed good correlation
with previously published anticancer activity of similar
pyrazole derivatives in which they showed growth inhibi-
tory effect on A549 lung cancer cells with log P values in
the range of 3.12–4.94 (Xia et al., 2007, 2008).
Since the formation of pro-inflammatory eicosanoids
(i.e. PGs and LTs) are evidenced to be a visible key reg-
ulators of cell proliferation and neo-angiogenesis, the
inhibitors of the pathways for their formation are being
investigated as potential anti-cancer drugs (Claria and
Romano, 2005; Romano and Claria, 2003). Hormone-
dependent MCF-7 cells have a basal level of COX-1 and a
barely detectable and transient COX-2 inducible expres-
sion, and hormone-independent MDA-MB-231 breast
cancer cell lines show a low expression of COX-1 but a
constitutive level of COX-2 (Liu and Rose, 1996). More-
over, overexpression of COX-2 has been reported in
hematological cancer models such as Raji (Burkitt’s lym-
phoma) (Sobolewski et al., 2010; Wun et al., 2004). COX-
2 and 5-LO also differentially expressed in the HeLa
(human cervical cancer cells) and HL-60 (a human pro-
myelocytic leukemia) cell lines and their growth was found
sensitive to NSAID treatment (Agrawal and Fentiman
2008; Chao et al., 2005; Liu et al., 2011; Pereg and Lishner
2005; Sobolewski et al., 2010; Totzke et al., 2003; Werz
et al., 1997). Considering that our compounds displayed
promising in vivo anti-inflammatory activities presumably
by inhibiting the formation of AA metabolites, we selected
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Med Chem Res (2013) 22:782–793
Conclusion
obtained using a Perkin Elmer Spectrum 400 FTIR/FTNIR
spectrometer equipped with a Universal ATR Sampling
Accessory. Flash chromatography was performed with a
CombiflashÒRf automated flash chromatography system
with RediSep columns (Teledyne-Isco, Lincoln, NE, USA)
using hexane–ethyl acetate or dichloromethane–methanol
solvent gradients. The purity of the final compounds was
determined to be[97 % by UPLC with UV detector. 4-(4-
methylphenyl)-2,4-dioxobutanoate (3) was synthesized by
the reaction of 40-methylacetophenone with dimethyloxa-
late (Maurin et al., 2004). 2-Hydrazinoquinolin was syn-
thesized by the reaction of 2-chloroquinoline with
hydrazine hydrate (Gupta et al., 2007). p-Benzoquinone
(PBQ), k-Carrageenan (type IV) and other biochemical’s
used in this study were purchased from Sigma-Aldrich
Chemical Company (St Louis, MO, USA).
Our results clearly show that derivatization of 1-benzyl-
3(5)-(p-tolyl)-1H-pyrazole-5(3)-carboxylic acid scaffolds
into lipophilic amides and esters act as in vivo potent
analgesic and anti-inflammatory compounds as compared
to reference drugs aspirin and indomethacin, exemplified
by 7a, 7c, 7e–f, 8a–b, and 8f–g. Comparison between the
regiosiomeric main pyrazole scaffolds, amide (8a–b), and
ester (8f–g) derivatives of 1-benzyl-5-(p-tolyl)-1H-pyra-
zole-3-carboxylic acid were more preferred over the same
amides (7a–b)/esters (8f–g) of 1-benzyl-3-(p-tolyl)-1H-
pyrazole-5-carboxylic acid. Meanwhile, none of the other
ester derivatives of pyrazole-5-carboxylic acid regioisomer
were found active indicating that amide derivatization was
more useful approach for this scaffold.
The results of the anti-cancer assays revealed an activity
at the tested lM range linked particularly five compounds
(7a, 7d, 7j, 8a, and 8b) in which three of them (7a,
8a–b) endowed with potent in vivo anti-inflammatory
activity in the second phase of carrageenan-induced hind
paw edema. Interestingly, among these compounds, the only
ester derivative 7j with considerable anti-cancer activity for
HeLa and MDA-MB-231 cells lacked the potency as an
analgesic or anti-inflammatory agent. Although induction of
cytotoxicity toward selected cancer-specific cells does not
preclude the probable cytotoxicity against normal cells, the
promising results obtained with the compounds bearing
Methyl 3-(p-tolyl)-1H-pyrazole-5-carboxylate (4)
To a mixture of 4-(p-tolyl)-2,4-dioxobutanoate (1) (12 mmol)
in acetic acid (20 ml), hydrazine hydrate (13.2 mmol) was
added and heated at reflux temperature for 40 min. The
resulting solution was cooled, the precipitate was collected by
filtration and washed with water. The product was used for the
next step without further purification.Yield 96 %. HRMS
(m/z): [M ? H]? calcd for C12H13N2O2, 217.0977; found,
217.0974.
1-benzyl-3(5)-(p-tolyl)-1H-pyrazole-5(3)-carboxylic
acid
Methyl 1-benzyl-3-(4-methylphenyl)-1H-pyrazole-5-
carboxylate (5) and methyl 1-benzyl-5-(4-methylphenyl)-
1H-pyrazole-3-carboxylate (6)
scaffolds encourage us to develop new antitumor substances
with good selectivity toward cancer cells and studies for
determining the selectivity of new series of compounds are
under ongoing work in our laboratory.
To a mixture of 4 (10 mmol) and K2CO3 (12 mmol) in
acetonitrile (40 ml), benzyl chloride (15 mmol) was added
and refluxed for 8 h. The reaction mixture was cooled to
room temperature, filtered, and solvent was evaporated
under reduced pressure and the regioisomers were sepa-
rated by automated flash chromatography. The product 5
was obtained as white solid in 77 % yield and 6 was
obtained as white solid in 15 % yield. For 5; HRMS (m/z):
[M ? H]? calcd for C19H19N2O2, 307.1447; found,
307.1444. For 6; HRMS (m/z): [M ? H]? calcd for
C19H19N2O2, 307.1447; found, 307.1452.
Materials and methods
Chemistry
1H NMR spectra were recorded in CDCl3 or DMSO-d6 on a
Varian Mercury 400 MHz High Performance Digital FT-
NMR spectrometer using tetramethylsilane as the internal
standard at the NMR facility of Faculty of Pharmacy,
Ankara University. All chemical shifts were recorded as d
(ppm). High resolution mass spectra data (HRMS) were
collected in-house using a Waters LCT Premier XE Mass
Spectrometer (high sensitivity orthogonal acceleration
time-of-flight instrument) operating in ESI (?) method,
also coupled to an AQUITY Ultra Performance Liquid
Chromatography system (Waters Corporation, Milford,
MA, USA). Melting points were determined with an SMP-
II Digital Melting Point Apparatus and are uncorrected
(Schorpp Geaetetechnik, Germany). IR spectra were
1-Benzyl-3-(4-methylphenyl)-1H-pyrazole-5-carboxylic
acid (7) and 1-benzyl-5-(4-methylphenyl)-1H-pyrazole-3-
carboxylic acid (8)
Compounds 5 and 6 (5 mmol) were hydrolyzed to corre-
sponding acids, in THF/water (1:1) with LiOHꢀH2O
(15 mmol) by heating at 70 °C for 2 h. The reaction mixture
was poured into water and acidified with 6 N HCl, precipitate
was collected by filtration and washed with water, and used in
123

Med Chem Res (2013) 22:782–793
789
the synthesis of amide and ester derivatives. For 7; Yield
1
3.28 (s, 2H), 3.36 (s, 2H), 3.63 (bs, 2H), 3.85 (s, 3H), 3.87 (s,
6H), 5.52 (s, 2H), 6.52 (s, 1H), 6.62 (d, 1H, J = 8.0 Hz), 6.90
(d, 1H, J = 8.0 Hz), 7.21 (d, 2H, J = 8.0 Hz), 7.24–7.28 (m,
5H), 7.69 (d, 2H, J = 8.0 Hz); HRMS (m/z): [M ? H]?
calcd for C32H37N4O4, 541.2815; found, 541.2795.
96 %; Mp 190–191 °C; IR (ATR): 1,686 cm-1; H NMR
(CDCl3): d 2.38 (s, 3H), 5.80 (s, 2H), 7.22–7.29 (m, 8H), 7.73
(d, 2H, J = 8.0 Hz); HRMS (m/z): [M ? H]? calcd for
C18H17N2O2, 293.1290; found, 293.1290. For 8; yield 98 %;
Mp 120–121°C; IR (ATR): 1,675 cm-1; 1H NMR (CDCl3): d
2.39 (s, 3H), 5.40 (s, 2H), 6.91 (s, 1H), 7.03–7.06 (m, 2H),
7.17–7.28 (m, 7H); HRMS (m/z): [M ? H]? calcd for
C18H17N2O2, 293.1290; found, 293.1281.
1-Benzyl-3-(4-methylphenyl)-N-(pyridin-3-ylmethyl)-1H-
pyrazole-5-carboxamide (7d)
Prepared from pyridin-3-ylmethylamine. Yield 55.7 %; Mp
172–173 °C; IR (ATR): 3035, 2935, 1647, 1522, 1428,
1281, 715 cm-1; 1H NMR (CDCl3): d 2.36 (s, 3H), 4.56 (d,
2H, J = 6.4 Hz), 5.82 (s, 2H), 6.37 (t, 1H), 6.79 (s, 1H),
7.19–7.29 (m, 8H), 7.52 (m, 1H), 7.67 (d, 2H, J = 8.4 Hz),
8.52–8.54 (m, 2H); HRMS (m/z): [M ? H]? calcd for
C24H23N4O, 383.1872; found, 383.1854.
General synthesis of amide and ester derivatives
To a mixture of the appropriate carboxylic acid derivative 7 or
8 (1 mmol) in 10 ml CH2Cl2, the appropriate amine or phenol
derivative (1.1 mmol), 4-dimethylaminopyridine (DMAP)
(0.2 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbo-
diimide (EDC) (1.1 mmol) were added, and stirred at room
temperature overnight. The reaction mixture was diluted with
CH2Cl2 and washed with 1 N HCl (3 9 20 ml), 5 %
NaHCO3 (3 9 20 ml), and brine. After drying over Na2SO4,
solvent was evaporated under reduced pressure and the crude
product was purified by flash chromatography.
1-Benzyl-3-(4-methylphenyl)-N0-quinolin-2-yl-1H-
pyrazole-5-carbohydrazide (7e)
Prepared from 2-hydrazinoquinoline. Yield 25 %; Mp
231–232 °C; IR (ATR): 3252, 2909, 1654, 1618, 1509,
1
1429, 759, 718, 699 cm-1; H NMR (CDCl3): d 2.36 (s,
Ethyl 1-{[1-benzyl-3-(4-methylphenyl)-1H-pyrazol-5-yl]-
carbonyl}piperidine-4-carboxylate (7a)
3H), 5.72 (s, 2H), 6.80 (d, 1H, J = 8.8 Hz), 7.12 (s, 1H),
7.17–7.33 (m, 8H), 7.51–7.54 (m, 1H), 7.62–7.68 (m, 4H),
7.86 (d, 1H, J = 8.4 Hz); HRMS (m/z): [M ? H]? calcd
for C27H24N5O, 434.1981; found, 434.1970.
Prepared from ethyl piperidine-4-carboxylate. Yield 63 %;
Mp 96–97 °C; IR (ATR): 2976, 1717, 1624, 1440, 1043,
1
702 cm-1; H NMR (DMSO-d6): d 1.06 (bs, 1H), 1.18 (t,
4-tert-Butylphenyl 1-benzyl-3-(4-methylphenyl)-1H-
pyrazole-5-carboxylate (7f)
3H, J = 7.2 Hz), 1.40 (bs, 1H), 1.60 (bs, 1H), 1.84 (bs,
1H), 2.32 (s, 3H), 2.52–2.58 (m, 1H), 2.89–3.02 (m, 2H),
3.70 (bs, 1H), 4.07 (q, 2H, J = 7.2 Hz), 4.26 (bs, 1H), 5.42
(s, 2H), 6.93 (s, 1H), 7.18–7.32 (m, 7H), 7.72 (d, 2H,
J = 8.0 Hz); HRMS (m/z): [M ? H]? calcd for
C26H30N3O3, 432.2287; found, 432.2271.
Prepared from 4-t-butylphenol. Yield 78 %; Mp
127–127.6 °C; IR (ATR): 3134, 2958, 1725, 1439, 1247, 1201,
1066, 817 cm-1;1H NMR (CDCl3): d1.33 (s, 9H), 2.39 (s, 3H),
5.82 (s, 2H), 7.05 (d, 2H, J = 8.8 Hz), 7.23–7.34 (m, 7H), 7.42
(d, 2H, J = 8.8 Hz), 7.77 (d, 2H, J = 8.0 Hz); HRMS (m/z):
[M ? H]? calcd for C28H29N2O2, 425.2229; found, 425.2209.
1-{[1-Benzyl-3-(4-methylphenyl)-1H-pyrazol-5-yl]-
carbonyl}-4-(4-tert-butylbenzyl) piperazine (7b)
4-Isopropylphenyl 1-benzyl-3-(4-methylphenyl)-1H-
pyrazole-5-carboxylate (7g)
Prepared from 1-(4-t-butylbenzyl)piperazine. Yield 56 %;
Mp 145.2–145.5 °C; IR (ATR): 2950, 1626, 1442, 832,
1
749, 706 cm-1; H NMR (CDCl3): d 1.31 (s, 9H), 1.93 (s,
Prepared from 4-i-propylphenol. Yield 79 %; Mp 96–97 °C;
IR (ATR): 3127, 2949, 1735, 1504, 1241, 1194, 1046,
817 cm-1; 1H NMR (CDCl3): d 1.25 (d, 6H, J = 6.8 Hz), 2.39
(s, 3H), 2.93 (m, 1H), 5.82 (s, 2H), 7.05 (d, 2H, J = 8.8 Hz),
7.23–7.35 (m, 10H), 7.77 (d, 2H, J = 8.0 Hz); HRMS (m/z):
[M ? H]? calcd for C22H27N2O2, 411.2073; found, 411.2059.
2H), 2.34 (s, 2H), 2.37 (s, 3H), 3.29 (s, 2H), 3.36 (s, 2H),
3.64 (s, 2H), 5.53 (s, 2H), 6.52 (s, 1H), 7.16–7.34 (m, 11H),
7.68 (d, 2H, J = 8.0 Hz); HRMS (m/z): [M ? H]? calcd
for C33H39N4O, 507.3124; found, 507.3127.
1-{[1-Benzyl-3-(4-methylphenyl)-1H-pyrazol-5-yl]-
carbonyl}-4-(2,3,4-trimethoxybenzyl)piperazine (7c)
4-(Heptyloxy)phenyl 1-benzyl-3-(4-methylphenyl)-1H-
pyrazole-5-carboxylate (7h)
Prepared from 1-(2,3,4-trimethoxylbenzyl)piperazine. Yield
50 %; Mp 134–134.5 °C; IR (ATR): 2917, 1628, 1458, 821,
748 cm-1; 1H NMR (CDCl3): d 1.98 (bs, 2H), 2.37 (m, 5H),
Prepared from 4-heptyloxyphenol. Yield 78 %; Mp
91.5–92 °C; IR (ATR): 3062, 2947, 1734, 1504, 1246,
123

790
Med Chem Res (2013) 22:782–793
1
1197, 1060, 818 cm-1; H NMR (CDCl3): d 0.89 (t, 3H,
J = 6.8 Hz), 1.31–1.47 (m, 8H), 1.77 (m, 2H), 2.39 (s,
3H), 3.94 (t, 2H, J = 6.8 Hz), 5.81 (s, 2H), 6.90 (d, 2H,
J = 8.8 Hz), 7.03 (d, 2H, J = 8.8 Hz), 7.23–7.35 (m, 7H),
7.76 (d, 2H, J = 8.0 Hz); HRMS (m/z): [M ? H]? calcd
for C31H35N2O3, 483.2648; found, 483.2632.
4-tert-Butylphenyl 1-benzyl-5-(4-methylphenyl)-1H-
pyrazole-3-carboxylate (8f)
Prepared from 4-t-butylphenol. Yield 67 %; Mp
119.5–120 °C; IR (ATR): 2946, 1732, 1462, 1204, 1196,
1
993, 807, 716 cm-1; H NMR (CDCl3): d 1.33 (s, 9H),
2.39 (s, 3H), 5.46 (s, 2H), 6.99 (s, 1H), 7.06–7.08 (m, 2H),
7.17 (d, 2H, J = 8.8 Hz), 7.19–7.29 (m, 7H), 7.42 (d, 2H,
J = 8.8 Hz); HRMS (m/z): [M ? H]? calcd for
C28H29N2O2, 425.2229; found, 425.2215.
4-[4-(Trifluoromethyl)phenoxy]phenyl 1-benzyl-3-(4-
methylphenyl)-1H-pyrazole-5-carboxylate (7i)
Prepared from
5 4-[4-(trifluoromethyl)phenoxy]phenol.
4-Isopropylphenyl 1-benzyl-5-(4-methylphenyl)-1H-
pyrazole-3-carboxylate (8g)
Yield 68 %; Mp 109–110 °C; IR (ATR): 3037, 1734, 1495,
1318, 1054, 818, 723 cm-1; 1H NMR (CDCl3): d 2.39(s, 3H),
5.83 (s, 2H), 7.06–7.10 (m, 4H), 7.16 (d, 2H, J = 8.8 Hz),
7.24–7.32 (m, 7H), 7.35 (s, 1H), 7.59 (d, 2H, J = 8.8 Hz),
7.77 (d, 2H, J = 8.0 Hz); HRMS (m/z): [M ? H]? calcd for
C31H24F3N2O3, 529.1739; found, 529.1746.
Prepared from 4-i-propylphenol. Yield 82 %; Mp
129–130 °C; IR (ATR): 3141, 2957, 1732, 1455, 1167,
1
990, 873, 734 cm-1; H NMR (CDCl3): d 1.26 (d, 6H,
J = 6.8 Hz), 2.39 (s, 3H), 2.93 (m, 1H), 5.46 (s, 2H), 6.99
(s, 1H), 7.06–7.08 (m, 2H), 7.16 (d, 2H, J = 8.8 Hz),
7.20–7.30 (m, 9H); HRMS (m/z): [M ? H]? calcd for
C27H27N2O2, 411.2073; found, 411.2054.
4-(1H-Imidazol-1-yl)phenyl 1-benzyl-3-(4-methylphenyl)-
1H-pyrazole-5-carboxylate (7j)
Prepared from 4-(1H-imidazol-1-yl)phenol. Yield 64 %;
Mp 167.5–168 °C; IR (ATR): 3133, 2945, 1736, 1518,
1
1508, 1199, 1051, 808, 726 cm-1; H NMR (CDCl3): d
Pharmacological screening
2.40 (s, 3H), 5.82 (s, 2H), 7.22–7.35 (m, 11H), 7.37 (s, 1H),
7.44 (d, 2H, J = 8.4 Hz), 7.78 (d, 2H, J = 8.0 Hz), 7.84 (s,
1H); HRMS (m/z): [M ? H]? calcd for C27H23N4O2,
435.1821; found, 435.1820.
Animals
Male, Swiss albino mice (25–30 g) were purchased from the
animal breeding laboratories of Refik Saydam Central
Institute of Health (Ankara, Turkey). They were housed in a
room with controlled temperature (22 1 °C), humidity
(55 10 %), and photoperiod (12:12 h) light–dark cycle
for at least a week before being used. They were maintained
on standard pellet diet and water ad libitum throughout the
experiment. A minimum of six animals were used in each
group. Throughout the experiments, animals were processed
according to the suggested international ethical guidelines
for the care of laboratory animals under the audit of Gazi
University Commission of Animal Ethics (Permission no:
G.U.ET-11.091/188-17453).
Ethyl 1-{[1-benzyl-5-(4-methylphenyl)-1H-pyrazol-3-yl]-
carbonyl}piperidine-4-carboxylate (8a)
Prepared from ethyl piperidine-4-carboxylate. Yield 79 %;
Mp 98–99 °C; IR (ATR): 3131, 2923, 1720, 1624, 1486,
1
1164, 1043, 987, 734 cm-1; H NMR (CDCl3): d 1.26 (t,
3H, J = 7.0 Hz), 1.73–1.83 (m, 2H), 1.92 (m, 1H), 2.02
(m, 1H), 2.38 (s, 3H), 2.59 (m, 1H), 3.00 (t, 1H), 3.30 (t,
1H), 4.15 (q, 2H, J = 7.0 Hz), 4.57 (m, 1H), 4.68 (m, 1H),
5.32 (s, 2H), 6.69 (s, 1H), 7.04–7.06 (m, 2H), 7.23 (s, 4H),
7.24–7.30 (m, 3H); HRMS (m/z): [M ? H]? calcd for
C26H30N3O3, 432.2287; found, 432.2273.
Analgesic activity
1-{[1-benzyl-5-(4-methylphenyl)-1H-pyrazol-3-yl]-
carbonyl}-4-(4-tert-butylbenzyl) piperazine (8b)
Analgesic activity was evaluated using the p-Benzoqui-
none (PBQ)-induced writhing test in mice (Okun et al.,
1963). Thirty minutes after the subcutaneous administra-
tion of a test sample in dimethyl sulfoxide (DMSO)
(100 mg/kg body weight), the mice were intraperitoneally
injected with 0.1 ml/10 g body weight of 2.5 % (w/v) PBQ
solution in distilled water. Control animals received an
appropriate volume of dosing vehicle (DMSO). The mice
were then kept individually for the observation and the
total number of the abdominal contractions (writhing
Prepared from 1-(4-t-butylbenzyl)piperazine. Yield 49 %;
Mp 125–126 °C; IR (ATR): 3022, 2958, 1621, 1483, 1255,
1
963, 815, 729 cm-1; H NMR (CDCl3): d 1.32 (s, 9H),
2.39 (s, 3H), 2.80 (bs, 2H), 3.40 (bs, 2H), 3.71 (s, 1H), 4.13
(m, 3H), 4.80 (bs, 1H), 5.28 (s, 3H), 6.77 (s, 1H), 7.00–7.02
(m, 2H), 7.18–7.29 (m, 7H), 7.46 (d, 2H, J = 8.0 Hz), 7.53
(d, 2H, J = 8.0 Hz); HRMS (m/z): [M ? H]? calcd for
C33H39N4O, 507.3124; found, 507.3114.
123

Med Chem Res (2013) 22:782–793
791
movements) was counted for the following 15 min, starting
5 min after the PBQ injection. The data represent the
average of the total number of writhes observed. Analgesic
activity was then expressed as the percentage change from
writhing controls.
USA) at a wavelength of 570 nm. Cell treated with 10 lM
cisplatin were used as a positive control.
Statistical analysis
The data were expressed as means SEM or SD. The
significance of differences between the treatment and the
control group of animals were determined by one-way
ANOVA with Barlett test following post hoc Student–
Newman–Keuls multiple comparisons test for analgesic
activity and two-way ANOVA following post hoc Bon-
ferroni test for anti-inflammatory activity. Data for anti-
cancer activity were analyzed by one-way ANOVA.
P value \0.05 was considered statistically significant.
Anti-inflammatory activity
Carrageenan-induced hind paw edema model was utilized
for evaluation of anti-inflammatory activity (Kupeli et al.,
2007). Each experimental group contained seven animals
minimally. Thirty minutes after the subcutaneous admin-
istration of a test sample in DMSO (100 mg/kg body
weight) or dosing vehicle, each mouse was injected with
freshly prepared suspension of carrageenan (0.5 mg/25 ll)
in physiological saline into sub-plantar tissue of the right
hind paw. As a control group, 25 ll saline was injected into
the left hind paw. Paw edema was measured in 90, 180,
270, and 360 min after carrageenan injections. The dif-
ference in hind paw thickness was measured by a caliber
compasses (Ozaki Co., Tokyo, Japan). Mean values of each
treated groups were compared with control group and
analyzed by using statistical methods.
Acknowledgments This investigation was supported by a Scientific
Research Grant 02/2010-20 awarded by Gazi University BAP. The
authors greatly acknowledge D. A. Alkan (Department of Pharma-
cology) for her assistance for performing in vivo biological experi-
ments. The authors report no conflicts of interest.
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