Synthesis of 1H-Pyrazole-1-carboxamide derivatives and their antiproliferative activity against melanoma cell line

Article abstract of DOI:10.1002/ardp.201000057

Synthesis of a new series of 1H-pyrazole-1-carboxamide derivatives is described. Their antiproliferative activity against A375 human melanoma cell line was tested and the effect of substituents on the diarylpyrazole scaffold was investigated. The pharmacological results indicated that most of the newly synthesized compounds showed moderate activity against A375, compared with sorafenib. Among all of these derivatives, compound IIe which has N-methylpiperazinyl and phenolic moieties showed the most potent antiproliferative activity against A375 human melanoma cell line.

Full text of DOI:10.1002/ardp.201000057

Arch. Pharm. Chem. Life Sci. 2011, 11, 197–204
197
Full Paper
Synthesis of 1H-Pyrazole-1-carboxamide Derivatives and
Their Antiproliferative Activity against Melanoma Cell Line
Mohammed I. El-Gamal1,2, Tae Bo Sim1, Jun Hee Hong3, Jung-Hyuck Cho1, Kyung Ho Yoo1, and
Chang-Hyun Oh1,2
1 Biomaterials Center, Korea Institute of Science and Technology, Cheongryang, Seoul, Republic of Korea
2 Department of Biomolecular Science, University of Science and Technology, Daejeon, Republic of Korea
3 College of Pharmacy, Chosun University, Gwangju, Republic of Korea
Synthesis of a new series of 1H-pyrazole-1-carboxamide derivatives is described. Their antiproliferative
activity against A375 human melanoma cell line was tested and the effect of substituents on the
diarylpyrazole scaffold was investigated. The pharmacological results indicated that most of the
newly synthesized compounds showed moderate activity against A375, compared with sorafenib.
Among all of these derivatives, compound IIe which has N-methylpiperazinyl and phenolic moieties
showed the most potent antiproliferative activity against A375 human melanoma cell line.
Keywords: A375 / Antiproliferative activity / 3,4-Diarylpyrazole / Melanoma / 1H-Pyrazole-1-carboxamide
Received: February 23, 2010; Revised: April 26, 2010; Accepted: April 26, 2010
DOI 10.1002/ardp.201000057
Introduction
After reviewing the literature, we found many promising,
potent, and selective antiproliferative agents for treatment of
melanoma. The most famous one of these agents is sorafenib
[10–15]. Sorafenib is an oral multikinase inhibitor that tar-
gets 2 classes of kinases which are known to be involved in
both tumor proliferation and angiogenesis [16]. It inhibits
RAF kinases (RAF-1 and B-RAF), as well as proangiogenic
receptor tyrosine kinases of the PDGFR and VEGFR family
[10]. The antiproliferative activity of sorafenib against mela-
noma is assumed to be due to B-RAF inhibition and induction
of apoptosis in a caspase-independent manner [17]. Sorafenib
demonstrated high antiproliferative activity against differ-
ent melanoma xenografts and cell lines [17], but not in case of
advanced metastatic melanoma (stage IV) [16]. In addition,
sorafenib has been implicated in the development of revers-
ible posterior leukoencephalopathy syndrome and secondary
erythrocytosis [18]. The poor efficiency of sorafenib in case
advanced metastatic melanoma and its side effects encourage
the search for new antiproliferative compounds for treat-
ment of melanoma.
Melanoma is the most aggressive form of skin cancer and is
the fastest growing cancer in the United States [1, 2]. Early
stage melanoma can be cured surgically. However, mela-
noma metastasizing to major organs (stage IV) is virtually
incurable [2]. Patients with advanced melanoma have a
median survival time of less than one year, and the estimated
5-year survival time is less than 15% [2, 3]. With the incidence
of melanoma rapidly rising in the United States and other
developed countries, there is an urgent need to develop more
effective drugs [4–6].
The current treatments involve surgical removal of the
tumor, immunotherapy, radiotherapy, chemotherapy, vari-
ous combinations, or the use of new treatments in clinical
trials. As for immunotherapy, interferon alfa-2b (intron-A) [7]
has been approved by both the FDA and EMEA for adjuvant
treatment of melanoma patients, and aldesleukin (proleukin)
[8, 9] is also approved for the treatment of metastatic
melanoma in the USA.
Moreover, antiproliferative agents with 3,4-diarylpyrazole
scaffold targeting B-RAF kinase and can be efficient for treat-
ment of melanoma have been identified [19, 20]. Among these
derivatives, compound A [19] (Fig. 1) was considered as the
lead compound in order to design our target compounds. In
our efforts in order to develop new antiproliferative agents
Correspondence: Chang-Hyun Oh, Biomaterials Center, Korea Institute
of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650,
Republic of Korea,
E-mail: choh@kist.re.kr
Fax: þ82-2-958-5189.
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198
M. I. El-Gamal et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 197–204
OR1
OH
R
O
CF3
HN
N
N
H3C
O
Cl
Cl
Cl
N
H
O
N
N
N
O
N
N
H
H
N
N
N
Sorafenib
R = dialkylamino, heterocycloalkyls
R1 = H, CH3
A
Figure 1. Structures of sorafenib, the lead compound A, and target compounds.
for treatment of melanoma, we designed and synthesized 18 Results and discussion
new 3,4-diarylpyrazole derivatives possessing an amide
moiety at position 1 of the pyrazole ring. The newly synthe-
sized compounds Ia–i and IIa–i were designed by isosteric
replacement of the pyrimidine ring of the lead compound A
with a pyridine ring and introduction of different amide tails
at position 1 of the pyrazole ring instead of the ethyl group.
The phenolic hydroxy group was retained in compounds
IIa–i and modified into methoxy group in compounds Ia–i
in order to study its effect on the activity (Fig. 1). Herein, we
report the synthesis and antiproliferative activity against
A375 human melanoma cell line of these compounds.
Chemistry
3,4-Diarylpyrazole derivatives Ia–i and IIa–i with amide
moiety at position 1 of the pyrazole ring were prepared
according to the sequence of reactions shown in Scheme 1.
Heating 3,5-dichlorobenzoic acid (1) with three molecular
equivalents of sodium methoxide in hexamethylphosphora-
mide (HMPA) followed by acidification with HCl gave 3-
chloro-5-methoxybenzoic acid (2), which upon esterification
with methanol in the presence of acetyl chloride afforded the
corresponding methyl ester 3 [21]. The pyridyl derivative 4
OCH3
Cl
OCH3
OCH3
a
c
b
OH
OH
O
Cl
Cl
Cl
CH3
Cl
O
O
O
O
N
1
2
3
4
OCH3
OCH3
OCH3
COOPh
e
f
d
N
N
N
Cl
R
Cl
Cl
N
NH
N COOPh
+
N
N
N
5
7
6
OCH3
OH
OCH3
O
NH
R
R
HN
HN
O
N
N
N
Cl
Cl
Cl
g
N
N
N
+
O
N
N
N
8
Ia-i
IIa-i
Reagents and conditions: (a) sodium methoxide, HMPA, 115–120°C, 15 h; (b) acetyl chloride, MeOH; (c) 4-picoline, LHMDS,
THF; (d) (i) DMF-DMA, (ii) hydrazine monohydrate, EtOH; (e) phenyl chloroformate, TEA, THF; (f) substituted ethanamine,
K2CO3, CH2Cl2; (g) BF3 Me2S, CH2Cl2.
Scheme 1. Synthesis of the target compounds Ia–i and IIa–i.
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1H-Pyrazole-1-carboxamides
199
Table 1. Antiproliferative activity of methoxy compounds Ia–i
against A375P cell line
was obtained by treatment of 3 with 4-picoline in THF in the
presence of lithium bis(trimethylsilyl)amide (LHMDS).
Cyclization to the pyrazole compound 5 was carried out by
treatment of 4 with dimethylformamide dimethyl acetal
(DMF-DMA), and subsequent treatment with hydrazine mono-
hydrate [19]. Interaction of 5 with phenyl chloroformate in
the presence of TEA in dry THF gave a mixture of pyrazole-2-
carboxylate derivative 6 and pyrazole-1-carboxylate 7 in an
approximate ratio of 1:4. The mixture was then reacted with
different ethanamines to furnish the target methoxy com-
pounds Ia–i in combination with their regioisomers 8.
Compounds Ia–i with lower Rf values on TLC were obtained
in the pure form after purification by flash column chroma-
tography. Demethylation of the methoxy group of Ia–i using
boron trifluoride–methyl sulfide complex afforded the cor-
responding hydroxy derivatives IIa–i.
Structure
Comp. No.
Ia
R
IC50 (mM)
CH3
N
9.6
CH3
CH3
Ib
Ic
16.3
N
CH3
>20
N
O
CH3
O
Id
N
>20
Antiproliferative activity and discussion
OCH3
The antiproliferative activity of the newly synthesized com-
pounds against A375 human melanoma cell line was tested.
The ability of the 1H-pyrazole-1-carboxamide derivatives to
inhibit the growth of A375 cell line is summarized in Tables 1
and 2. The results are expressed as IC50 values. Sorafenib was
selected as the reference standard, because it has been exten-
sively used in clinical trials for melanoma [4, 22].
CH3
R
Cl
HN
O
N
N
Ie
If
6.9
N
N CH3
N
O
>20
N
N
As listed in Tables 1 and 2, most of the compounds showed
moderate activity, while compounds Ie, IIa, IIb, IIe, and IIh
having IC50 values ranging from 4.2 to 6.9 mM exhibited
similar activity to that of Sorafenib (IC50 ¼ 5.6 mM).
Compound Ie possesses a methoxy group on the benzene
ring while compounds IIa, IIb, IIe, and IIh possess a hydroxy
group. In addition, the R moiety of compounds Ie and IIe is N-
methylpiperazinyl moiety while the R moieties of com-
pounds IIa, IIb, and IIh are dimethylamino, diethylamino,
and 2-methylpiperidinyl, respectively.
CH3
Ig
Ih
>20
>20
N
N
H3C
The compounds in Table 2 were more potent than those in
Table 1, which suggests that the hydroxy group on position 3
of the benzene ring is optimal for the activity. This may be
attributed to hydrogen bond formation at the receptor site.
The effect of the terminal substituents of the tail at posi-
tion 1 of the pyrazole ring was also investigated. Compounds
Ii and IIi having a 5-membered ring, pyrrolidine ring, showed
diminished activity. We can conclude that this moiety is
unfavorable for antiproliferative activity against melanoma
compared with dialkylamino and 6-membered rings. By com-
paring compounds Ia and Ib, we find that smaller alkyl
groups, two methyl groups, are more optimal for activity
than the slightly longer groups, ethyl groups. Upon compar-
ing the activity of the piperazinyl derivatives, it was found
that the N-methyl derivatives (Ie and IIe) were more potent
than that of N-acetyl derivatives (If and IIf). These results may
be rationalized by the steric and/or electronic effects of the
Ii
>20
N
Sorafenib
5.6
acetyl group, compared with the methyl group. Introduction
of a methyl group on the piperidine ring (compound IIh)
enhanced the activity compared with non-methylated piper-
idine ring (compound IIg). On the other hand, introduction
of two methyl groups on the morpholine ring (compounds
Id and IId) did not alter the activity, compared with deriva-
tives with non-methylated morpholine ring (compounds Ic
and IIc).
In conclusion, a new series of 3,4-diaryl-1H-pyrazole-1-car-
boxamide derivatives was synthesized based on our previous
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Arch. Pharm. Chem. Life Sci. 2011, 11, 197–204
Table 2. Antiproliferative activity of hydroxy compounds IIa–i
against A375P cell line
Experimental
Chemistry
Structure
Comp. No.
IIa
R
IC50 (mM)
All melting points were obtained on a Walden Precision
Apparatus Electrothermal 9300 apparatus and are uncorrected.
Mass spectra (MS) were taken in ESI mode on a Waters 3100 Mass
Detecter (Waters, Milford, MA, USA). Nuclear magnetic resonance
(NMR) spectroscopy was performed using a Bruker ARX-300,
300 MHz spectrometers (Bruker Bioscience, Billerica, MA, USA)
with TMS as an internal standard. IR spectra (KBr disks) were
recorded with a Bruker FT-IR instrument (Bruker Bioscience,
Billerica, MA, USA). Unless otherwise noted, all solvents and
reagents were commercially available and used without further
purification.
CH3
N
6.6
CH3
CH3
IIb
IIc
6.8
N
CH3
10.8
N
O
3-Chloro-5-methoxybenzoic acid 2
A mixture of 3,5-dichlorobenzoic acid (1, 2.0 g, 10.4 mmol) and
NaOMe (6.74 mL, 25 wt-% solution in methanol, 31.2 mmol) in
HMPA (40 mL) was heated at 1208C for 15 h. The mixture was
poured into ice-water and acidified with conc. HCl to give 3-
chloro-5-methoxybenzoic acid (2, 1.56 g, 80%) as a precipitate
which was collected by filtration and used in the next step
without purification. 1H-NMR (DMSO-d6) d [ppm]: 3.82 (s, 3H),
7.30 (t, 1H, J ¼ 2.0 Hz), 7.38 (q, 1H, J ¼ 1.3 Hz), 7.47 (t, 1H,
J ¼ 1.6 Hz).
CH3
O
IId
IIe
N
10.8
4.2
OH
CH3
R
Cl
HN
O
N
N
N
N CH3
N
Methyl 3-chloro-5-methoxybenzoate 3
Acetyl chloride (1.9 mL, 28.1 mmol) was added dropwise to a
solution of 2 (1.0 g, 5.4 mmol) in MeOH (40 mL) at 08C and the
reaction mixture was then stirred at room temperature for 15 h.
After evaporation of the organic solvent, the residue was purified
by flash column chromatography (silica gel, hexane/methylene
chloride 5:1 v/v then switching to hexane/methylene chloride 1:1
v/v) to give 3 (0.7 g, 65.1%) as an oil. 1H-NMR (CDCl3) d [ppm]:
3.85 (s, 3H), 3.92 (s, 3H), 7.09 (t, 1H, J ¼ 2.2 Hz), 7.45 (q, 1H,
J ¼ 1.3 Hz), 7.61 (t, 1H, J ¼ 1.6 Hz).
O
N
N
IIf
IIg
IIh
13.0
17.2
6.8
CH3
N
N
1-(3-Chloro-5-methoxyphenyl)-2-(pyridin-4-yl)ethanone 4
To a mixture of compound 3 (1.0 g, 5.0 mmol) and 4-picoline
(0.5 mL, 5.6 mmol) in THF (5 mL) in a cooled bath at ꢀ258C,
LHMDS (3.7 mL, 1.0 M solution in THF, 19.9 mmol) was slowly
added to maintain the temperature at ꢀ258C. The resulting
mixture was stirred overnight at room temperature. The mixture
was quenched with saturated aqueous NH4Cl. Ethyl acetate was
added and the organic layer was separated. The aqueous layer
was extracted with ethyl acetate (3 ꢁ 10 mL). The combined
organic layer extracts were washed with brine and dried over
anhydrous Na2SO4. The organic solvent was evaporated under
reduced pressure and the residue was purified by flash column
chromatography (silica gel, hexane/ethyl acetate 1:1 v/v then
switching to hexane/ethyl acetate 1:5 v/v) to yield compound 4
(0.52 g, 40%). 1H-NMR (CDCl3) d [ppm]: 3.85 (s, 3H), 4.25 (s, 2H),
7.12 (t, 1H, J ¼ 2.0 Hz), 7.19 (d, 2H, J ¼ 5.7 Hz), 7.39 (q, 1H,
J ¼ 1.4 Hz), 7.54 (t, 1H, J ¼ 1.5 Hz), 8.58 (d, 2H, J ¼ 5.7 Hz).
H3C
IIi
>20
N
Sorafenib
5.6
literature studies. Among all of these derivatives, compound
IIe having m-hydroxyphenyl substituted N-methylpiperazinyl
moieties showed the most potent antiproliferative activity
against A375 human melanoma cell line. One can conclude
that these moieties are optimal for antiproliferative activity
of this series of compounds. Further modification of these
compounds in order to improve their potency is currently in
progress. Our ultimate goal is to identify several compounds
that are highly potent against melanoma cells.
4-[3-(3-Chloro-5-methoxyphenyl)-1H-pyrazol-4-yl]
pyridine 5
Compound 4 (1.0 g, 3.8 mmol) was added to DMF-DMA (5.14 mL,
38.2 mmol) and the mixture was stirred at room temperature for
18 h. The resulting solution was concentrated to dryness to
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1H-Pyrazole-1-carboxamides
201
furnish an oil which was used in the next step without purifi-
cation. To a portion of the oil from the previous step (0.137 g,
0.457 mmol) in EtOH (3 mL) was added hydrazine monohydrate
(0.04 mL, 0.76 mmol) and the reaction mixture was stirred over-
night at room temperature. Water (5 mL) was added to the
reaction mixture and the organics were extracted with ethyl
acetate (3 ꢁ 5 mL). The combined organic layer extracts were
washed with brine and dried over anhydrous Na2SO4. After
evaporation of the organic solvent, the residue was purified
by column chromatography (silica gel, hexane/ethyl acetate
1:1 v/v then switching to hexane/ethyl acetate 1:5 v/v) to yield
compound 5 (0.11 g, 81%). 1H-NMR (DMSO-d6) d [ppm]: 3.77 (s, 3H),
6.85 (brs, 1H), 6.94 (brs, 1H), 7.06 (brs, 1H), 7.23 (d, 2H, J ¼ 5.4 Hz),
7.83 (s, 1H), 8.55 (d, 2H, J ¼ 5.3 Hz).
6H, J ¼ 6.3 Hz), 2.47 (q, 4H, J ¼ 6.3 Hz), 2.62 (t, 2H, J ¼ 5.8 Hz),
3.15 (t, 2H, J ¼ 5.2 Hz), 3.72 (s, 3H), 6.92 (dd, 1H,
J ¼ 2.0 Hz, J ¼ 2.2 Hz), 7.13 (dd, 1H, J ¼ 2.0 Hz, J ¼ 2.2 Hz),
7.33 (dd, 1H, J ¼ 1.9 Hz, J ¼ 1.8 Hz), 7.36 (d, 2H, J ¼ 4.3 Hz),
8.50 (brs, 1H), 8.54 (d, 2H, J ¼ 4.2 Hz), 8.75 (s, 1H); 13C-NMR
(CD3OD) d [ppm]: 162.6, 152.4, 149.0, 143.0, 138.5, 135.8,
135.4, 134.8, 132.5, 120.4, 114.4, 112.6, 109.1, 54.7, 51.2, 48.4,
39.7, 10.2; MS m/z: 429.1 [M þ Hþ].
3-(3-Chloro-5-methoxyphenyl)-N-(2-morpholinoethyl)-4-
(pyridin-4-yl)-1H-pyrazole-1-carboxamide Ic
It was purified by flash column chromatography (silica gel, ethyl
acetate then switching to ethyl acetate/methanol 4:1 v/v). 45%
Yield; m.p.: 103–1058C; IR (KBr) [cmꢀ1]: 3252.1, 2964.2, 2943.2,
1727.0, 1609.0, 1578.4, 1509.5, 1411.6, 1296.0, 1139.5, 1115.4,
1042.0; 1H-NMR (DMSO-d6) d [ppm]: 2.44-2.52 (m, 6H), 3.16 (t, 2H,
J ¼ 5.0 Hz), 3.58 (t, 4H, J ¼ 4.1 Hz), 3.73 (s, 3H), 6.94 (dd, 1H,
J ¼ 1.9 Hz, J ¼ 2.0 Hz), 7.13 (dd, 1H, J ¼ 2.0 Hz, J ¼ 2.0 Hz), 7.36
(dd, 1H, J ¼ 2.0 Hz, J ¼ 2.1 Hz), 7.53 (d, 2H, J ¼ 6.0 Hz), 8.56 (d,
2H, J ¼ 6.0 Hz), 8.61 (brs, 1H), 8.80 (s, 1H); 13C-NMR (DMSO-d6) d
[ppm]: 160.6, 151.3, 149.7, 140.9, 134.8, 134.2, 129.3, 128.9, 121.4,
120.4, 114.4, 112.7, 109.2, 66.4, 57.6, 54.8, 53.3, 37.3; MS m/z:
443.0 [M þ Hþ].
Phenyl 3-(3-chloro-5-methoxyphenyl)-4-(pyridin-4-yl)-1H-
pyrazole-1-carboxylate 7
To a solution of compound 5 (0.1 g, 0.35 mmol) in anhydrous
THF (10 mL), triethylamine (0.112 g, 1.1 mmol) was slowly added
at 08C. Phenyl chloroformate (0.165 g, 1.05 mmol) was slowly
added to the above solution at 08C. The reaction mixture was
stirred at the same temperature for 2 h. The mixture was diluted
with H2O (10 mL) and CH2Cl2 (15 mL). The organic layer was
separated, washed with brine, and dried over anhydrous MgSO4.
The organic solvent was evaporated under reduced pressure and
the residue (a mixture of compounds 6 and 7) was used in the
next step without further purification.
3-(3-Chloro-5-methoxyphenyl)-N-(2-(2,6-
dimethylmorpholino)ethyl)-4-(pyridin-4-yl)-1H-pyrazole-1-
carboxamide Id
It was purified by flash column chromatography (silica gel, ethyl
acetate/hexane 2:1 v/v then switching to ethyl acetate). 54%
Yield; m.p.: 80–828C; 1H-NMR (CDCl3) d [ppm]: 1.18 (d, 6H,
J ¼ 6.3 Hz), 2.59–2.80 (m, 6H), 3.44 (t, 2H, J ¼ 4.9 Hz), 3.71–
3.77 (m, 5H), 6.94 (dd, 1H, J ¼ 2.0 Hz, J ¼ 2.0 Hz), 7.13 (dd, 1H,
J ¼ 1.9 Hz, J ¼ 2.0 Hz), 7.26 (dd, 1H, J ¼ 2.0 Hz, J ¼ 2.0 Hz), 7.63
(d, 2H, J ¼ 5.1 Hz), 8.30 (brs, 1H), 8.42 (d, 2H, J ¼ 5.0 Hz), 8.58 (s,
1H); MS m/z: 471.0 [M þ Hþ].
General procedure for preparation of compounds Ia–i
To a solution of the crude product of the last step (0.1 g,
0.246 mmol) in dry CH2Cl2 (3 mL), a solution of the appropriate
ethanamine derivative (0.738 mmol) in dry CH2Cl2 (2 mL), and
anhydrous K2CO3 (68 mg, 0.492 mmol) were added. The reaction
mixture was stirred at room temperature for 1 h. Water (5 mL) was
added to the reaction mixture and the organic layer was separated.
The aqueous layer was extracted with CH2Cl2 (2 ꢁ 3 mL) and the
combined organic layer extracts were washed with brine and dried
over anhydrous MgSO4. The organic solvent was evaporated under
reduced pressure and the residue was obtained.
3-(3-Chloro-5-methoxyphenyl)-N-(2-(4-methylpiperazin-1-
yl)ethyl)-4-(pyridin-4-yl)-1H-pyrazole-1-carboxamide Ie
It was purified by flash column chromatography (silica gel, ethyl
acetate/hexane 1:1 v/v then switching to ethyl acetate). 66%
3-(3-Chloro-5-methoxyphenyl)-N-(2-
(dimethylamino)ethyl)-4-(pyridin-4-yl)-1H-pyrazole-1-
carboxamide Ia
1
Yield; m.p.: 112–1158C; H-NMR (DMSO-d6) d [ppm]: 2.28 (s, 3H),
2.37–2.58 (m, 8H), 2.62 (t, 2H, J ¼ 6.0 Hz), 2.96 (t, 2H, J ¼ 5.9 Hz),
3.69 (s, 3H), 6.98 (dd, 1H, J ¼ 1.9 Hz, J ¼ 2.0 Hz), 7.04 (dd, 1H,
J ¼ 2.0 Hz, J ¼ 2.1 Hz), 7.24 (dd, 1H, J ¼ 2.0 Hz, J ¼ 1.9 Hz), 7.35
(d, 2H, J ¼ 6.1 Hz), 7.90 (brs, 1H), 8.21 (s, 1H), 8.45 (d, 2H,
J ¼ 6.2 Hz); 13C-NMR (CD3OD) d [ppm]: 160.6, 150.1, 149.7,
142.9, 140.9, 135.5, 134.8, 134.2, 121.4, 120.4, 114.4, 112.7,
109.4, 57.2, 54.8, 53.3, 53.2, 46.8, 37.3; MS m/z: 455.9 [M þ Hþ].
It was purified by flash column chromatography (silica gel, ethyl
acetate then switching to ethyl acetate/methanol 4:1 v/v). 58%
Yield; oil; 1H-NMR (CD3OD) d [ppm]: 2.37 (s, 6H), 2.66 (t, 2H,
J ¼ 6.7 Hz), 3.58 (t, 2H, J ¼ 6.5 Hz), 3.75 (s, 3H), 6.96 (dd, 1H,
J ¼ 1.7 Hz, J ¼ 2.0 Hz), 7.02 (dd, 1H, J ¼ 2.0 Hz, J ¼ 2.2 Hz), 7.14
(dd, 1H, J ¼ 1.6 Hz, J ¼ 2.0 Hz), 7.39 (d, 2H, J ¼ 6.2 Hz), 8.51 (d,
2H, J ¼ 6.1 Hz), 8.62 (s, 1H); 13C-NMR (CD3OD) d [ppm]: 160.6,
150.2, 149.8, 141.9, 141.0, 134.8, 134.3, 129.3, 120.4, 120.3, 114.8,
112.7, 112.6, 57.9, 54.7, 44.1, 37.5; MS m/z: 400.9 [M þ Hþ].
N-(2-(4-Acetylpiperazin-1-yl)ethyl)-3-(3-chloro-5-
methoxyphenyl)-4-(pyridin-4-yl)-1H-pyrazole-1-
carboxamide If
It was purified by flash column chromatography (silica gel, ethyl
acetate then switching to ethyl acetate/methanol 4:1 v/v). 45%
Yield; m.p.: 187–1908C; 1H-NMR (CD3OD) d [ppm]: 2.11 (s, 3H),
2.49–2.62 (m, 6H), 3.33 (t, 2H, J ¼ 4.9 Hz), 3.53 (t, 4H, J ¼ 4.0 Hz),
3.75 (s, 3H), 7.09 (dd, 1H, J ¼ 2.0 Hz, J ¼ 2.1 Hz), 7.14 (dd, 1H,
J ¼ 2.1 Hz, J ¼ 1.9 Hz), 7.26 (dd, 1H, J ¼ 2.0 Hz, J ¼ 2.1 Hz), 7.35
(d, 2H, J ¼ 6.2 Hz), 8.50 (d, 2H, J ¼ 6.1 Hz), 8.61 (s, 1H); MS m/z:
484.0 [M þ Hþ].
3-(3-Chloro-5-methoxyphenyl)-N-(2-(diethylamino)ethyl)-
4-(pyridin-4-yl)-1H-pyrazole-1-carboxamide Ib
It was purified by flash column chromatography (silica gel, ethyl
acetate then switching to ethyl acetate-methanol 6:1 v/v). 70%
Yield; m.p.: 104–1078C; IR (KBr) [cmꢀ1]: 3318.3, 2967.8, 2935.2,
1737.3, 1610.8, 1578.4, 1550.5, 1509.9, 1471.5, 1409.7, 1266.3,
1210.5, 1173.9, 1138.6, 1043.5; 1H-NMR (DMSO-d6) d [ppm]: 0.98 (t,
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J ¼ 2.1 Hz, J ¼ 1.8 Hz), 6.85 (dd, 1H, J ¼ 1.9 Hz, J ¼ 2.0 Hz),
7.05 (dd, 1H, J ¼ 2.0 Hz, J ¼ 2.1 Hz), 7.41 (d, 2H, J ¼ 5.9 Hz),
8.51 (d, 2H, J ¼ 6.0 Hz), 8.63 (s, 1H); 13C-NMR (CD3OD) d [ppm]:
158.5, 150.5, 149.9, 142.7, 141.0, 138.5, 134.6, 134.2, 123.3, 120.3,
119.1, 115.6, 114.0, 57.8, 46.7, 37.3; MS m/z: 386.95 [M þ Hþ].
3-(3-Chloro-5-methoxyphenyl)-N-(2-(piperidin-1-yl)ethyl)-
4-(pyridin-4-yl)-1H-pyrazole-1-carboxamide Ig
It was purified by flash column chromatography (silica gel, ethyl
acetate/hexane 1:1 v/v then switching to ethyl acetate). 41%
Yield; m.p.: 39–418C; IR (KBr) [cmꢀ1]: 3317.1, 2967.8, 1737.3,
1610.8, 1578.4, 1509.9, 1409.6, 1266.3, 1173.9, 1138.6, 1072.2,
1043.5; 1H-NMR (CDCl3) d [ppm]: 1.39–1.63 (m, 6H), 2.27 (t, 4H,
J ¼ 7.6 Hz), 2.72 (t, 2H, J ¼ 5.9 Hz), 3.42 (t, 2H, J ¼ 5.6 Hz), 3.80 (s,
3H), 6.88 (dd, 1H, J ¼ 2.0 Hz, J ¼ 2.1 Hz), 7.07 (dd, 1H,
J ¼ 1.9 Hz, J ¼ 2.0 Hz), 7.22 (dd, 1H, J ¼ 2.1 Hz, J ¼ 2.0 Hz),
7.35 (d, 2H, J ¼ 5.1 Hz), 7.79 (s, 1H), 8.51 (d, 2H, J ¼ 4.9 Hz);
13C-NMR (CDCl3) d [ppm]: 160.3, 150.0, 149.8, 143.0, 138.6,
136.6, 135.2, 132.4, 122.7, 120.7, 114.5, 112.6, 98.3, 57.1, 55.6,
53.9, 36.8, 24.3; MS m/z: 440.95 [M þ Hþ].
3-(3-Chloro-5-hydroxyphenyl)-N-(2-(diethylamino)ethyl)-
4-(pyridin-4-yl)-1H-pyrazole-1-carboxamide IIb
It was purified by flash column chromatography (silica gel, ethyl
acetate then switching to ethyl acetate/methanol 6:1 v/v). 48%
Yield; m.p.: 118–1208C; IR (KBr) [cmꢀ1]: 3392.3, 3127.0, 2970.7,
1729.7, 1610.4, 1593.5, 1512.0, 1417.6, 1345.1, 1293.0, 1205.6,
1153.7, 1003.4; 1H-NMR (CD3OD) d [ppm]: 1.16 (t, 6H, J ¼ 7.1 Hz),
2.78 (q, 4H, J ¼ 7.1 Hz), 2.87 (t, 2H, J ¼ 6.9 Hz), 3.57 (t, 2H,
J ¼ 6.9 Hz), 6.78 (dd, 1H, J ¼ 1.4 Hz, J ¼ 1.9 Hz), 6.85 (dd, 1H,
J ¼ 1.8 Hz, J ¼ 2.0 Hz), 7.04 (dd, 1H, J ¼ 1.3 Hz, J ¼ 1.9 Hz), 7.40
(d, 2H, J ¼ 4.8 Hz), 8.51 (d, 2H, J ¼ 4.9 Hz), 8.63 (s, 1H); 13C-NMR
(CD3OD) d [ppm]: 158.6, 150.5, 149.9, 141.0, 135.9, 134.6, 134.2,
129.2, 123.3, 120.4, 119.1, 115.7, 114.0, 51.1, 48.4, 37.0, 9.8; MS
m/z: 414.96 [M þ Hþ].
3-(3-Chloro-5-methoxyphenyl)-N-(2-(2-methylpiperidin-1-
yl)ethyl)-4-(pyridin-4-yl)-1H-pyrazole-1-carboxamide Ih
It was purified by flash column chromatography (silica gel, ethyl
acetate/hexane 1:1 v/v then switching to ethyl acetate). 40%
Yield; m.p.: 73–758C; 1H-NMR (CD3OD) d [ppm]: 1.15 (d, 3H,
J ¼ 5.3 Hz), 1.38–1.66 (m, 6H), 2.17–2.63 (m, 5H), 3.31 (t, 2H,
J ¼ 5.0 Hz), 3.75 (s, 3H), 6.96 (dd, 1H, J ¼ 1.9 Hz, J ¼ 2.0 Hz),
7.03 (dd, 1H, J ¼ 2.0 Hz, J ¼ 2.0 Hz), 7.15 (dd, 1H,
J ¼ 1.9 Hz, J ¼ 2.0 Hz), 7.40 (d, 2H, J ¼ 5.9 Hz), 8.52 (d, 2H,
J ¼ 6.1 Hz), 8.62 (s, 1H); MS m/z: 455.07 [M þ Hþ].
3-(3-Chloro-5-hydroxyphenyl)-N-(2-morpholinoethyl)-4-
(pyridin-4-yl)-1H-pyrazole-1-carboxamide IIc
It was purified by flash column chromatography (silica gel, ethyl
acetate then switching to ethyl acetate/methanol 6:1 v/v). 55%
Yield; m.p.: 247–2508C; IR (KBr) [cmꢀ1]: 3400.0, 3124.7, 2973.7,
1732.8, 1610.6, 1593.5, 1510.3, 1404.7, 1324.0, 1293.7, 1154.0,
1094.1, 1002.1; 1H-NMR (CD3OD) d [ppm]: 2.50 (t, 4H, J ¼ 6.9 Hz),
2.61 (t, 2H, J ¼ 6.6 Hz), 3.30–3.72 (m, 6H), 6.78 (dd, 1H,
J ¼ 1.5 Hz, J ¼ 1.9 Hz), 6.87 (dd, 1H, J ¼ 1.8 Hz, J ¼ 2.0 Hz),
7.09 (dd, 1H, J ¼ 1.9 Hz, J ¼ 2.0 Hz), 7.41 (d, 2H, J ¼ 5.9 Hz),
8.45 (s, 1H), 8.62 (d, 2H, J ¼ 6.0 Hz); MS m/z: 428.92 [M þ Hþ].
3-(3-Chloro-5-methoxyphenyl)-4-(pyridin-4-yl)-N-(2-
(pyrrolidin-1-yl)ethyl)-1H-pyrazole-1-carboxamide Ii
It was purified by flash column chromatography (silica gel, ethyl
acetate then switching to ethyl acetate/methanol 3:1 v/v). 46%
Yield; oil; 1H-NMR (CDCl3) d [ppm]: 1.83 (t, 4H, J ¼ 5.3 Hz), 2.61 (t,
4H, J ¼ 5.1 Hz), 2.77 (t, 2H, J ¼ 6.3 Hz), 3.32 (t, 2H, J ¼ 5.9 Hz),
3.73 (s, 3H), 6.83 (dd, 1H, J ¼ 1.9 Hz, J ¼ 2.0 Hz), 7.11 (dd, 1H,
J ¼ 1.9 Hz, J ¼ 2.1 Hz), 7.22 (dd, 1H, J ¼ 1.6 Hz, J ¼ 1.8 Hz), 7.27
(d, 2H, J ¼ 5.8 Hz), 7.59 (brs, 1H), 8.43 (s, 1H), 8.58 (d, 2H,
J ¼ 6.0 Hz); 13C-NMR (CDCl3) d [ppm]: 160.3, 150.3, 149.3,
143.0, 139.7, 135.9, 135.3, 133.9, 121.7, 120.9, 115.4, 115.0,
112.8, 55.7, 55.6, 53.9, 39.4, 23.5; MS m/z: 426.93 [M þ Hþ].
3-(3-Chloro-5-hydroxyphenyl)-N-(2-(2,6-
dimethylmorpholino)ethyl)-4-(pyridin-4-yl)-1H-pyrazole-1-
carboxamide IId
It was purified by flash column chromatography (silica gel, ethyl
acetate then switching to ethyl acetate/methanol 10:1 v/v). 60%
Yield; m.p.: 175–1788C; IR (KBr) [cmꢀ1]: 3399.4, 3124.7, 2936.2,
1732.8, 1610.8, 1593.6, 1510.2, 1404.8, 1324.2, 1293.7, 1153.9,
1094.1, 1002.1, 1H-NMR (CD3OD) d [ppm]: 1.20 (d, 6H, J ¼ 6.3 Hz),
2.48–2.70 (m, 6H), 3.36 (t, 2H, J ¼ 4.8 Hz), 3.74 (m, 2H), 6.80 (dd,
1H, J ¼ 2.0 Hz, J ¼ 2.1 Hz), 6.93 (dd, 1H, J ¼ 1.9 Hz, J ¼ 1.8 Hz),
7.15 (dd, 1H, J ¼ 1.9 Hz, J ¼ 2.0 Hz), 7.54 (d, 2H, J ¼ 5.0 Hz), 8.42
(d, 2H, J ¼ 4.9 Hz), 8.55 (s, 1H); MS m/z: 456.97 [M þ Hþ].
General procedure for preparation of compounds IIa–i
To a solution of compound Ia–i (0.1 mmol) in methylene
chloride (3 mL), BF3 Me2S (0.13 g, 1 mmol) was added dropwise
at room temperature under N2 and the reaction mixture was
stirred at the same temperature for 48 h. The mixture was
quenched with saturated aqueous NaHCO3. Ethyl acetate
(3 mL) was added and the organic layer was separated. The
aqueous layer was extracted with ethyl acetate (3 ꢁ 2 mL).
The combined organic layer extracts were washed with brine
and dried over anhydrous Na2SO4. The organic solvent was evapo-
rated under reduced pressure and the residue was obtained.
3-(3-Chloro-5-hydroxyphenyl)-N-(2-(4-methylpiperazin-1-
yl)ethyl)-4-(pyridin-4-yl)-1H-pyrazole-1-carboxamide IIe
It was purified by flash column chromatography (silica gel, ethyl
acetate). 63% Yield; m.p.: 120–1228C; IR (KBr) [cmꢀ1]: 3399.2,
3124.8, 2973.8, 1732.7, 1610.6, 1593.3, 1510.2, 1404.8, 1356.2,
1293.9, 1154.0, 1094.2, 1002.2; 1H-NMR (DMSO-d6) d [ppm]: 2.25 (s,
3H), 2.45–2.59 (m, 8H), 2.60 (t, 2H, J ¼ 6.1 Hz), 3.02 (t, 2H,
J ¼ 5.9 Hz), 6.78 (dd, 1H, J ¼ 1.8 Hz, J ¼ 2.1 Hz), 6.84 (dd, 1H,
J ¼ 2.0 Hz, J ¼ 2.0 Hz), 7.14 (dd, 1H, J ¼ 2.0 Hz, J ¼ 1.8 Hz), 7.53
(d, 2H, J ¼ 6.2 Hz), 8.10 (brs, 1H), 8.43 (d, 2H, J ¼ 6.2 Hz), 8.54 (s,
1H); 13C-NMR (DMSO-d6) d [ppm]: 159.8, 149.3, 142.6, 138.2, 135.2,
134.8, 134.1, 121.1, 119.9, 114.6, 113.0, 109.1, 57.2, 55.3, 54.7,
52.2, 42.8, 37.9; MS m/z: 441.95 [M þ Hþ].
3-(3-Chloro-5-hydroxyphenyl)-N-(2-(dimethylamino)ethyl)-
4-(pyridin-4-yl)-1H-pyrazole-1-carboxamide IIa
It was purified by flash column chromatography (silica gel, ethyl
acetate then switching to ethyl acetate/methanol 4:1 v/v). 41%
Yield; m.p.: 179–1818C; IR (KBr) [cmꢀ1]: 3391.6, 3126.9, 2970.7,
2924.2, 1730.0, 1610.5, 1593.1, 1512.0, 1417.5, 1345.1, 1293.0,
1205.5, 1153.7, 1003.4; 1H-NMR (CD3OD) d [ppm]: 2.39 (s, 6H), 2.69
(t, 2H, J ¼ 6.7 Hz), 3.59 (t, 2H, J ¼ 6.5 Hz), 6.76 (dd, 1H,
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1H-Pyrazole-1-carboxamides
203
CO2 at 378C. A375P cells were taken from culture substrate with
0.05% trypsin-0.02% EDTA and plated at a density of 5 ꢁ 103 cells/
well in 96 well plates and then incubated at 378C for 24 h in a
humidified atmosphere with 5% CO2 prior to treatment with
various concentrations (3-fold serial dilution, 12 points) of test
compounds. The cells were incubated for 48 h after treatment
with the test compounds. The A357P cell viability was assessed by
the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) reduction assay. MTT assays were carried
out with CellTiter 961 (Promega) according to the manufac-
turer’s instructions. The absorbance at 590 nm was recorded
using EnVision 2103 (Perkin Elmer; Boston, MA, USA). The IC50
was calculated using GraphPad Prism 4.0 software.
N-(2-(4-Acetylpiperazin-1-yl)ethyl)-3-(3-chloro-5-
hydroxyphenyl)-4-(pyridin-4-yl)-1H-pyrazole-1-
carboxamide IIf
It was purified by flash column chromatography (silica gel, ethyl
acetate then switching to ethyl acetate/methanol 4:1 v/v). 50%
Yield; m.p.: 197–1998C; IR (KBr) [cmꢀ1]: 3346.6, 3126.5, 2930.4,
1731.5, 1610.7, 1510.1, 1433.5, 1345.6, 1154.5, 1001.9; 1H-NMR
(CD3OD) d [ppm]: 2.04 (s, 3H), 2.53–2.72 (m, 6H), 2.93 (t, 2H,
J ¼ 4.9 Hz), 3.42 (t, 4H, J ¼ 5.0 Hz), 6.87 (dd, 1H,
J ¼ 1.7 Hz, J ¼ 1.9 Hz), 6.94 (dd, 1H, J ¼ 1.6 Hz, J ¼ 2.0 Hz),
7.10 (dd, 1H, J ¼ 1.7 Hz, J ¼ 2.1 Hz), 7.38 (d, 2H, J ¼ 4.6 Hz),
8.10 (s, 1H), 8.59 (d, 2H, J ¼ 4.7 Hz); MS m/z: 470.0 [M þ Hþ].
3-(3-Chloro-5-hydroxyphenyl)-N-(2-(piperidin-1-yl)ethyl)-
4-(pyridin-4-yl)-1H-pyrazole-1-carboxamide IIg
The authors have declared no conflict of interest.
It was purified by flash column chromatography (silica gel, ethyl
acetate). 50% Yield; m.p.: 205–2088C; IR (KBr) [cmꢀ1]: 3415.1,
2932.2, 1731.5, 1610.7, 1510.0, 1433.7, 1345.2, 1154.7, 1001.8;
1H-NMR (CD3OD) d [ppm]: 1.37–1.59 (m, 6H), 2.23 (t, 4H,
J ¼ 7.0 Hz), 2.68 (t, 2H, J ¼ 6.2 Hz), 3.28 (t, 2H, J ¼ 6.1 Hz),
6.77 (dd, 1H, J ¼ 1.9 Hz, J ¼ 2.1 Hz), 6.91 (dd, 1H,
J ¼ 1.9 Hz, J ¼ 1.8 Hz), 7.11 (dd, 1H, J ¼ 1.8 Hz, J ¼ 2.0 Hz),
7.58 (d, 2H, J ¼ 5.0 Hz), 8.02 (s, 1H), 8.51 (d, 2H, J ¼ 5.1 Hz);
MS m/z: 426.95 [M þ Hþ].
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