Chemoselective synthesis, antiproliferative activities, and SAR study of 1H-pyrazol-5-yl-N,N-dimethylformamidines and pyrazolyl-2-Azadienes

Article abstract of DOI:10.1007/s00044-011-9872-1

Chemoselective microwave-Assisted amidination was successfully developed to synthesize 1H-pyrazol-5-yl-N,N-dimethylformamidines and pyrazolyl-2-Azadienes. All of the starting materials and resulting products were tested against NCI-H226, NPC-TW01, and Jurkat cancer cells to evaluate their antiproliferative activities. 1H-Pyrazol-5-yl-N,N-dimethylformamidines 2b, 2c, and 2d were most potent with IC50 values in low micromolar range. The formyl group at C-4 position and the grafted amidinyl group in the main core of pyrazolic molecule were necessary for the inhibitory activity. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9872-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3920–3928
DOI 10.1007/s00044-011-9872-1
ORIGINAL RESEARCH
Chemoselective synthesis, antiproliferative activities, and SAR
study of 1H-pyrazol-5-yl-N,N-dimethylformamidines
and pyrazolyl-2-azadienes
•
Kau-Shan Wen Hui-Yi Lin Yu-Ying Huang
•
•
•
Kimiyoshi Kaneko Hiroyuki Takayama
•
•
Masayuki Kimura Shin-Hun Juang Fung Fuh Wong
•
Received: 26 May 2011 / Accepted: 1 November 2011 / Published online: 20 December 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Chemoselective microwave-assisted amidin-
ation was successfully developed to synthesize 1H-pyrazol-
5-yl-N,N-dimethylformamidines and pyrazolyl-2-azadi-
enes. All of the starting materials and resulting products
were tested against NCI-H226, NPC-TW01, and Jurkat
cancer cells to evaluate their antiproliferative activities.
1H-Pyrazol-5-yl-N,N-dimethylformamidines 2b, 2c, and
2d were most potent with IC50 values in low micromolar
range. The formyl group at C-4 position and the grafted
amidinyl group in the main core of pyrazolic molecule
were necessary for the inhibitory activity.
Introduction
Pyrazoles attract attentions due to their wide range of
pharmacological properties (Haddad et al., 2004; Ramesh
Kakarla and Gerritz, 2007). The bioactivities of function-
alized N-arylpyrazoles are extensively studied (Elguero,
1984, 1996; Huang and Katzenellenbogen, 2000; Kost and
Grandberg, 1966; Lee et al., 2003) and the C-5 substituted
pyrazoles are also explored in the design of pharmaceuti-
cals and agrochemical agents (Sakya and Rast, 2003). As a
result, we focus to develop the new methodology for the
synthesis a series of N-arylpyrzoles containing aminidyl
group derivatives by microwave irradiation. These com-
pounds have represented an attractive target in view of
versatile biological activity (Cheng et al., 2010).
Keywords Amidination ꢀ Pyrazoles ꢀ
Antiproliferative activity ꢀ Microwave-assisted reaction ꢀ
Methnimidamide
Amidinyl groups are intensively studied since they
contribute to the activities of many biologically important
compounds (Bielawska et al., 2004; Collins et al., 1998;
Fastier et al., 1962; Grout, 1975; Kreutzberger, 1968;
Panico et al., 2002; Sielecki et al., 2001; Sienkiewich et al.,
2005; Stephens et al., 2001). Except for acting as valuable
pharmacophore, amidines are also important building
blocks for the preparation of various heterocyclic com-
pounds (Boyd, 1991; Croce et al., 1997; Mason et al.,
2001a, b; Palacios et al., 2006, 2009), protecting groups for
primary amines (Pocha, 1986; Rudyk et al., 2003), support
linkers in solid phase synthesis (Furth et al., 1997a, b), and
auxiliaries in asymmetric synthesis (Matulenko and
Meyers, 1996; Meyers et al., 1991; Meyers, 1992; Meyers
and Elworthy, 1992; Meyers and Hutchings, 1993, 1996).
The introduction of an amidinyl group into a known bio-
logical molecule is consequently of interest in the field of
medicinal chemistry and demonstrates good results in
several models (Lund and Tybring, 1972; Oszczapowicz
et al., 1997). Herein, we reported a new chemoselective
Kau-Shan Wen and Hui-Yi Lin have contributed equally to this work.
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-011-9872-1) contains supplementary
material, which is available to authorized users.
K.-S. Wen ꢀ Y.-Y. Huang ꢀ S.-H. Juang (&) ꢀ F. F. Wong (&)
Graduate Institute of Pharmaceutical Chemistry, China Medical
University, No. 91, Hsueh-Shih Rd., Taichung 40402,
Taiwan, ROC
e-mail: paul@mail.cmu.edu.tw
F. F. Wong
e-mail: wongfungfuh@yahoo.com.tw; ffwong@mail.cmu.edu.tw
H.-Y. Lin
School of Pharmacy, China Medical University, No. 91,
Hsueh-Shih Rd., Taichung 40402, Taiwan, ROC
K. Kaneko ꢀ H. Takayama ꢀ M. Kimura
Department of Medico Pharmaceutical Science,
Nihon Pharmaceutical University, 10281, Komuro,
Inamachi, Kita-Adachigun, Saitama, Japan
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Med Chem Res (2012) 21:3920–3928
3921
microwave-assisted amidination method to synthesize 1H-
pyrazol-5-yl-N,N-dimethylformamidines and pyrazolyl-2-
azadienes by using the suitable amount of basic pyridine as
the basic catalyst. The reactivity and bioactivity for the
different skeletal of methnimidamides and starting material
5-amino-1,3-disubstituted pyrazole were also explored.
model. Initially, we chose 5-amino-1,3-diphenylpyrazole
1a as modeling case and treated 1a with different inorganic
or organic basic agents to quench the excess amount of
active imineniun species or neutralize hydrochloride for
diminishing the formation of the formylated methnimida-
mide product 2a. The bases included sodium hydroxide
(NaOH), potassium carbonate (K2CO3), cesium carbonate
(CsCO3), triethylamine (NEt3), dimethylaminopyridine
(DMAP), and pyridine. Firstly, the amidination reaction
was performed on 5-amino-1-3-diphenylpyrazole 1a with-
out basic catalytic agent as the blank study. The reaction
only provided the formylated methnimidamide 2a in 94%
yield. When the reaction was treated with 2.0 equivalent of
inorganic base including sodium hydroxide (NaOH),
potassium carbonate (K2CO3), and cesium carbonate
(CsCO3), the methnimidamide product 3a without the
formyl group was provided in poor yields, except for using
cesium carbonate (see entries 2–4 of Table 2). For cesium
carbonate, the methnimidamide product 3a was obtained in
78% isolated yield with the recovery of a small amount of
the starting materials 1a. On the other hand, the starting
materials 1a and the small amount of the formylated
methnimidamide compound 2a were simultaneously
obtained in NaOH as basic catalytic agent.
Result and discussion
Chemistry
Scheme 1 illustrates a newly developed chemoselective
microwave-assisted amidination (Oszczapowicz et al., 1997)
of 5-amino-1,3-disubstituted pyrazoles 1 to give the corre-
sponding 1H-pyrazol-5-yl-N,N-dimethylformamidines 2 and
pyrazolyl-2-azadienes 3. A series of 1H-pyrazol-5-yl-N,N-
dimethylformamidines 2b–2e were designed based on our
previous study (Oszczapowicz et al., 1997) with enhanced
antiproliferative activity. A model procedure involved the
treatment of 5-amino-1,3-disubstituted pyrazoles 1a–1e with
POCl3 (*1.2 equivalent) in DMF at 30–40°C with 100 W of
microwave energy within 15–20 min. After work-up and
purification by column chromatography on silica gel, the
corresponding 1H-pyrazol-5-yl-N,N-dimethylformamidines
2a–2e were readily obtained in 81–94% yields (see Table 1;
Scheme 1). In addition to grafting the amidinyl group on the
main core of 5-amino pyrazole, the formyl group was also
introduced at the C-4 position of pyrazolic ring (Besan et al.,
1980). Compounds 2a–2e were fully characterized by spec-
troscopic methods.
When the same condition was applied to the commer-
cially available organic bases including triethylamine
(NEt3), dimethylaminopyridine (DMAP), or pyridine, the
methnimidamide product 3a without formyl group was
obtained in 34–82% yields as the major product (see entries
5–7 of Table 2). Particularly, the best chemoselective result
was achieved by using pyridine as the basic catalyst. The use
of various equivalent of pyridine was also studied from 1.0
equiv to 4.0 equiv. We found that the use 3.0 equivalent of
pyridine can give pyrazolyl-2-azadiene product 3a in the
best yield (97% yield, see entry 9 of Table 2). Furthermore,
the newly chemoselective methodology can be applicable to
For the investigation of the effect of formyl group on
activity, we evaluated the chemoselective microwave-
assisted amidination methodology to prepare a series of
pyrazolyl-2-azadienes 3a–3e without introducing a formyl
group at C-4 position on pyrazolic ring as the comparing
H
X
X
N
NMe2
NH2
CHO
DMF without pyridine
NaOH
MeOH
N
N
CHO
N
N
POCl3, MW
X
Y
Y
NH2
2a-2e
4a-4e
N
N
H
Y
X
N
NMe2
DMF with pyridine
POCl3, MW
1a-1e
N
N
1a. 2a. 3a. 4a. X = H, Y = H
Y
1b. 2b. 3b. 4b. X = m-Cl, Y = Me, 1c. 2c. 3c. 4c. X = m-Cl, Y = Cl
1d. 2d. 3d. 4d. X = p-Br, Y = Me, 1e. 2e. 3e. 4e. X = p-Br, Y = Cl
3a-3e
Scheme 1 Synthesis of methnimidamides 2a–2e, pyrazolyl-2-azadienes 3a–3e, and 5-amino-4-formylpyrazoles 4a–4e
123

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Med Chem Res (2012) 21:3920–3928
Table 1 The results of synthesis of methnimidamides 2a–2e and
5-amino-4-formylpyrazoles 4a–4e
Table 3 The results of chemoselective amidination reaction for
preparation of pyrazolyl-2-azadiene products 3a–3e
5-Amino-1,3-N,N-
disubstituted pyrazoles
Methnimidamides
(2a–2e)
5-Amino-4-
formylpyrazoles
(4a–4e)
5-Amino-1,3-N,N-disubstituted
pyrazoles
Pyrazolyl-2-azadienes
(3a–3e)
S.M. (1a–1e)
X
Y
Products
Yields (%)
S.M.
(1a–1e)
X
Y
Products Yields Products Yields
(%)
1a
1b
1c
1d
1e
H
H
3a
3b
3c
3d
3e
97
91
98
93
78
(%)
m-Cl
m-Cl
p-Br
p-Br
Me
Cl
1a
1b
1c
1d
1e
H
H
2a
2b
2c
2d
2e
94
82
81
90
92
4a
4b
4c
4d
4e
92
85
87
83
96
m-Cl
m-Cl
p-Br
p-Br
Me
Cl
Me
Cl
Me
Cl
compounds 4a–4e were sequentially prepared as the com-
parison model for the further structure–activity relationship
study. When we searched the previous reported literature
about de-amidination, only one method was found by using
HCl aqueous solution (Mason et al., 2001). However, the
purification procedure was troublesome, especially in
neutralization procedure. Consequently we investigated a
newly basic condition by using NaOH in MeOH solution.
The reliable procedure involved the treatment of methni-
midamides 2a–2e with two equivalent of NaOH at reflux in
MeOH solution within 2–3 h. After the extraction work-up
and simple purification through the short column chroma-
tography on silica gel, the corresponding de-amidination
5-amino-4-formylpyrazole products 4a–4e were obtained
in 83–96% yields (see Table 1; Scheme 1).
Table 2 The basic catalyzed study for preparation of pyrazolyl-2-
azadienes 3a without formyl group in the chemoselective microwave-
assisted amidination
Entry Basic agents
Catalyst
Yield (%)
Equiva Formylated
Pyrazolyl-
methnimidamides 2-azadienes
(2a)
(3a)
b
1
2
3
4
5
6
Without catalyst
NaOH
–
2
2
2
2
2
94
27
–
b,c
–
b,c
–
K2CO3
4
78
34
50
b,c
–
CsCO3
b,c
–
Triethylamine (NEt3)
b,c
–
Dimethylaminopyridine
(DMAP)
7
Pyridine
Pyridine
Pyridine
Pyridine
2
1
3
4
18
15
82
75
97
53
Biological evaluations
8
b
9
–
The growth inhibitory activity of all amidine compounds is
evaluated against a panel of human cancer cell lines,
including lung carcinoma (NCI-H226), nasopharyngeal
(NPC-TW01), and T cell leukemia (Jurkat) cells. The GI50
value is the concentration that results in a 50% decrease in
the cell growth relative to an untreated control. All of
starting materials 1a–1e were selected and used as the
comparison model for the inhibitory activity study. Among
of starting substrates, only compound 1d possessed the
negligible inhibitory activity against three cell lines (the
GI50 values of 1d are 54.3 lM (NCI-H226), 80.2 lM
(NPC-TW01), and 45.0 lM (Jurkat), see Table 4).
Formylated methnimidamide 2a was also used as the
comparison model for other analogs 2b–2e against
the cancer cell lines. Compounds 2b and 2c containing the
same m-Cl–Ph substituted group on N-1 position and either
p-Cl–Ph or p-Me–Ph groups on C-3 position in pyrazolic
ring displayed the better inhibitory activity against the
three cancer cell lines with GI50 values between 7.2 and 9.2
lM (see Table 4). The results also showed that they were
more active against NPC-TW01 and Jurkat than NCI-
H226. For compounds 2d and 2e with p-Br–Ph on N-1
10
44
a
Based on the weight of 5-amino-1-3-diphenylpyrazole (1a)
Not detectable
b
c
Starting material 1a was recovery
compounds 1a–1e to provide the corresponding pyrazolyl-2-
azadiene products 3a–3e without formyl group in 78–98%
yields (see Table 3). The reliable chemoselective procedure
involved the treatment of 5-amino-1,3-disubstituted pyra-
zoles 1a–1e with *1.2 equivalent of POCl3 and 3.0
equivalent of pyridine in DMF at 30–40°C with 100 W of
microwave energy within 15–20 min. After work-up and
purification were performed, the desired pyrazolyl-2-azadi-
ene products 3a–3e without formyl group were obtained in
78–98% isolated yields (see Table 3). Following the afore-
mentioned studies, the chemoselective amidination reaction
seemed determinate to the suitable amount of pyridine basic
agent.
To identify the essentiality of amidinyl group for the
inhibitory activity study, a series of de-amidination
123

Med Chem Res (2012) 21:3920–3928
3923
Table 4 Antiproliferative activity of the pyrazole derivatives
H
H
X
X
X
X
NH2
N
NMe2
N
NMe2
NH2
N
N
CHO
N
CHO
N
N
N
N
N
Y
Y
Y
Y
2a-2e
3a-3e
1a-1e
4a-4e
Compounds
Pyrazoles (1a–1e, 2a–2e, 3a–3e, and 4a–4e)
GI50 (lM)a,b
X (N-1)
Y (C-3)
NCI-H226
NPC-TW01
Jurkat
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
a
H
H
72.2
63.5
75.1
54.3
58.7
31.4
8.9
[100
[100
[100
80.2
83.0
56.6
[100
45.0
61.3
23.5
7.8
m-Cl
m-Cl
p-Br
p-Br
H
Me
Cl
Me
Cl
64.4
H
9.3
m-Cl
m-Cl
p-Br
p-Br
H
Me
Cl
7.2
9.2
7.4
7.7
Me
Cl
8.2
6.0
6.7
62.9
[100
80.9
75.6
73.9
59.8
[100
71.8
79.7
8.5
[100
[100
[100
92.7
38.9
[100
[100
74.5
[100
84.9
87.3
86.3
78.9
34.4
90.7
H
m-Cl
m-Cl
p-Br
p-Br
H
Me
Cl
Me
Cl
[100
60.7
H
[100
[100
[100
28.2
m-Cl
m-Cl
p-Br
p-Br
Me
Cl
Me
Cl
49.1
59.7
NCI-H226: human lung carcinoma; NPC-TW01: human nasopharyngeal carcinoma; Jurkat: human T cell leukemia
b
All tested compounds were dissolved in 100% DMSO at a concentration of 20 mM as the stock solution. Cells were cultured without or in the
presence of the methnimidamide derivatives at different concentrations for 72 h. Cell survival was determined by MTT assay. Drug molar
concentration causing 50% cell growth inhibition (GI50) was calculated. Each value represents the mean SD of three independent experiments
position and either p-Cl–Ph or p-Me–Ph groups at C-3
position on pyrazolic ring, compound 2d showed the better
inhibitory activity against the three cancer cell lines with
GI50 values between 6.0 and 8.2 lM. Due to the bulky
p-Br-Ph group and p-Cl-Ph groups on the N-1 and C-3
position of pyrazole not favoring to reach the blocking
side, the poor result of bioactivity was observed in com-
pound 2e. Following the structure–activity relationship
study results, compounds 2b–2d possessed the better
activity than 2a and 2e. On the other hand, the antiprolif-
erative activity data was consistent with our design
approach and compound 2b–2d can be considered as the
potency lead drugs.
compounds 4a–4e were evaluated against three cancer cell
lines as the comparison study. Following the antiprolifer-
ative activity result, the data indicated that compounds 3a–
3e [GI50:[59.8 lM (NCI-H226),[60.7 lM (NPC-TW01),
and[74.5 lM (Jurkat)] and 4a–4e [GI50:[8.5 lM (NCI-
H226), [28.2 lM (NPC-TW01), and[34.4 lM (Jurkat)]
were less potent than compounds 2a–2d. The experimental
result in Table 4 demonstrated the formyl group at C-4
position and grating the amidinyl group toward amino
moiety at C-5 in pyrazolic ring are essential for the pro-
motion of inhibitory activity. Furthermore, the data indi-
cated that tendency for sensitivity is nasopharyngeal
(NPC-TW01) [ T cell leukemia (Jurkat) cell [ lung
carcinoma (NCI-H266) for methnimidamide compounds
2a–2e.
For the further the structure–activity relationship investi-
gation, pyrazolyl-2-azadienes 3a–3e and de-amidination
123

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Med Chem Res (2012) 21:3920–3928
Conclusion
center of the CDCl3 triplet (d 77.0 ppm). Multiplicities are
recorded by the following abbreviations: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; J, coupling
constant (Hz). Microwave irradiation instrument was pur-
chased from CEM Discover. The microwave irradiation
condition was set in 100 W at 30–40°C within 10–20 min.
ESI–MS spectra were obtained from an Applied Biosys-
tems API 300 mass spectrometer. High-resolution mass
spectra were obtained by means of a JEOL JMS-HX110
mass spectrometer. Elemental analyses were carried out on
a Heraeus CHN–O RAPID element analyzer.
We have successfully developed a new chemoselective
microwave-assisted amidination method to prepare
1H-pyrazol-5-yl-N,N-dimethylformamidines 2a–2e with
the formyl group and pyrazolyl-2-azadienes 3a–3e without
formylation by using pyridine as the basic agent. Further-
more, we have also evaluated the new de-amidination
methodology to prepare the 5-amino-4-formylpyrazoles
4a–4e as the compared study. Following the structure–
activity relationship study, we have demonstrated that
introducing formyl group at C-4 position and grafting
amidinyl group in the pyrazole core molecule are necessary
for the improved bioactivity. Based on the growth inhibi-
tory activity data, compounds 2b, 2c, and 2d with m-Cl-Ph
and p-Br-Ph groups at N-1 position and p-Me-Ph and
p-Cl-Ph groups at C-3 position in pyrazolic ring possessed
the most potent activity.
Standard procedure for the synthesis of methnimidamide
compounds (2a–2e)
A solution of pyrazol-5-amine derivatives (1a–1e, 1.0
equiv) and POCl3 (1.2 equiv) in DMF solution (3 ml) at
30–40°C was treated with 100 W of microwave energy
within 10–20 min. When the reaction was completed, the
reaction mixture was concentrated, added to water (10 ml),
and extracted with CH2Cl2 (4 9 20 ml). The organic
extracts were washed with saturated NaHCO3, dried over
MgSO4, filtered, and concentrated under reduced pressure.
The residue solution was purified by column chromatog-
raphy on silica gel to give the corresponding methnimi-
damide products (2a–2e) in 81–94% yields.
Experimental
Chemistry
All chemicals were reagent grade and use as purchased. All
reactions were carried out under argon or nitrogen atmo-
sphere and monitored by TLC. Flash column chromatog-
raphy was carried out on silica gel (230–400 mesh). Ethyl
acetate and hexanes, purchased from Mallinckrodt Chem-
ical Co., were dried and distilled from CaH2. Toluene
(reagent grade, from Merck Chemical Co.) was dried by
distillation from CaH2 under nitrogen. 4-Methylbenzoyl-
acetonitrile, phenylhydrazine was purchased from Acros
Chemical Co. 4-Bromophenylhydrazine hydrochloride,
4-chlorobenzoylacetonitrile, and 3-chlorophenylhydrazine
hydrochloride were purchased from Alfa Aesar Chemical
Company. Benzoylacetonitrile was purchased from TCI.
N,N-Dimethylformamide and pyridine were purchased
from Scharlau Chemical Co. Phosphorylchloride was pur-
chased from FERAK Chemical Co.
N0-[4-Formyl-1,3-diphenyl-1H-pyrazol-5-yl-N,N-dimethyl-
methanimidamide (2a) Mp (purified by column chroma-
tography on silica gel) 120–122°C; 1H NMR (CDCl3,
200 MHz) d 3.01 (s, 3 H, CH3), 3.12 (s, 3 H, CH3),
7.26–7.47 (m, 6 H, ArH), 7.65–7.70 (m, 2 H, ArH),
7.84–7.89 (m, 2 H, ArH), 8.68 (s, 1H, N=C–H), 9.68 (s,
1H, aldehyde); 13C NMR (50 MHz, CDCl3) d 34.3 (CH3),
40.7 (CH3), 108.4, 124.3 (2 9 CH), 126.8, 128.4
(2 9 CH), 128.5 (2 9 CH), 128.8, 129.4 (2 9 CH), 132.3,
139.2, 154.1, 155.8, 159.0, 185.2; IR (KBr) 3059 (m), 2920
(m), 2800 (w), 2742 (w), 1670 (s), 1597 (m), 1508 (m),
1381 (m), 1257 (m), 1134 (m), 1095 (m), 975 (m), 767 (m),
694 (m) cm-1; EIMS m/z (relative intensity) 318 (100), 317
(M?, 42), 303 (17), 289 (9), 274 (19), 248 (8), 186 (14),
159 (7), 77 (24), 51 (5); Anal. Calcd for C19H18N4O; C:
71.68; H: 5.70; N: 17.60, Found: C: 71.72; H: 5.71; N:
17.58.
Analytical thin-layer chromatography (TLC) was per-
formed on precoated plates (silica gel 60 F-254) purchased
from Merck Inc. Mixtures of ethyl acetate and hexanes
were used as eluants. Infrared (IR) spectra were measured
on a Bomem Michelson Series FT-IR spectrometer. The
wavenumbers reported are referenced to the polystyrene
absorption at 1601 cm-1. Absorption intensities are
recorded by the following abbreviations: s, strong; m,
medium; w, weak. Proton NMR spectra were obtained on a
Bruker (200 or 400 MHz) spectrometer by use of CDCl3 as
solvent. Carbon-13 NMR spectra were obtained on a
Bruker (75 or 100 MHz) spectrometer by used of CDCl3 as
solvent. Carbon-13 chemical shifts are referenced to the
N0-[1-(2-Chlorophenyl)-4-formyl-3-(4-methylphenyl)-1H-
pyrazol-5-yl]-N,N-dimethyl-methanimidamide (2b) Mp
(purified by column chromatography on silica gel)
1
166–168°C; H NMR (CDCl3, 200 MHz) d 2.40 (s, 3 H,
CH3), 3.01 (s, 3 H, CH3), 3.11 (s, 3 H, CH3), 7.17–7.34 (m, 4
H, ArH), 7.51–7.55 (m, 2 H, ArH), 7.51–7.55 (m, 2 H, ArH),
7.79–7.85 (m, 2 H, ArH), 8.01–8.03 (m, 2 H, ArH), 8.69 (s, 1
H, N=C–H), 9.64 (s, 1H, aldehyde); 13C NMR (50 MHz,
123

Med Chem Res (2012) 21:3920–3928
3925
CDCl3) d 21.4 (CH3), 34.4 (CH3), 40.8 (CH3), 108.5, 122.0,
124.2, 126.5, 129.2 (6 9 CH), 133.9, 138.9, 140.3, 154.2,
156.1, 159.1, 185.2; IR (KBr) 2920 (m), 1666 (s), 1627 (m),
1589 (m), 1489(m), 1384 (m), 1261 (m), 1134 (m), 1099 (m),
1072 (m), 987 (m), 825 (m), 825 (m), 781 (m), 740 (m), 682
(m) cm-1; EIMS m/z (relative intensity) 366 (100), 365 (M?,
20), 337 (8), 322 (14), 220 (11), 185 (7), 111 (11), 91 (7), 83
(7), 75 (4); Anal. Calcd for C20H19ClN4O; C: 65.48; H: 5.22;
N: 15.27, Found: C: 65.50; H: 5.19; N: 15.23.
(2 9 CH), 120.3, 125.6 (2 9 CH), 128.7 (2 9 CH), 130.5
(2 9 CH), 131.5 (2 9 CH), 135.0, 138.1, 154.5 (2 9 CH),
158.9, 184.4; IR (KBr) 2364 (m), 2333 (m), 1666 (m), 1624
(m), 1516 (m), 1485 (m), 1381 (m), 1261 (m), 1138 (m),
1076 (m), 1010 (m), 813 (m), 740 (m), 578 (m), 547 (m),
505 (m) cm-1; EIMS m/z (relative intensity) 430 (73), 429
(M?, 20), 388 (13), 374 (8), 266 (14), 232 (8), 205 (9), 155
(11), 111 (4), 83 (10); Anal. Calcd for C19H16ClN4O; C:
52.86; H: 3.74; N: 12.98, Found: C: 52.88; H: 3.71; N:
13.01.
N0-[4-Formyl-1-(2-chlorophenyl)-3-(4-chlorophenyl)-1H-
pyrazol-5-yl]-N,N-dimethyl-methanimidamide (2c) Mp
(purified by column chromatography on silica gel)
Standard procedure for the synthesis of pyrazolyl-2-
azadiene compounds (3a–3e)
1
162–164°C; H NMR (CDCl3, 200 MHz) d 3.03 (s, 3 H,
CH3), 3.13 (s, 3 H, CH3), 7.23–7.43 (m, 4 H, ArH),
7.58–7.64 (m, 2 H, ArH), 7.77–7.82 (m, 1 H, ArH),
7.99–8.01 (m, 1 H, ArH), 8.63 (s, 1 H, N=C–H), 9.60 (s, 1
H, aldehyde); 13C NMR (50 MHz, CDCl3) d 34.5 (CH3),
40.8 (CH3), 108.5, 121.9, 124.1, 126.6, 128.7 (2 9 CH),
129.4, 130.5 (3 9 CH), 134.0, 135.0, 140.1, 154.5, 154.5,
158.9, 184.4; IR (KBr) 2924 (m), 2360 (m), 1666 (s), 1624
(m), 1585 (m), 1481 (m), 1384 (m), 1095 (m), 837 (m), 783
(m), 736 (m) cm-1; EIMS m/z (relative intensity) 386
(100), 385 (M?, 19), 371 (16), 357 (9), 342 (14), 330 (9),
316 (8), 220 (16), 111 (18), 83 (9); Anal. Calcd for
C19H16ClN4O; C: 58.93; H: 4.16; N: 14.47, Found: C:
58.89; H: 4.17; N: 14.46.
A solution of pyrazol-5-amine derivatives (1a–1b, 1.0
equiv), POCl3 (1.2 equiv), and pyridine (3.0 equiv) in DMF
solution (3 ml) at 30–40°C was treated with 100 W of
microwave energy within 10–20 min. When the reaction
was completed, the reaction mixture was concentrated,
added to water (10 ml), and extracted with CH2Cl2
(4 9 20 ml). The organic extracts were washed with sat-
urated NaHCO3, dried over MgSO4, filtered, and concen-
trated under reduced pressure. The residue solution was
purified by column chromatography on silica gel to give
the corresponding methnimidamide products (3a–3e) in
78–98% yields.
N0-(4-Formyl-1,3-diphenyl-1H-pyrazol-5-yl)-N,N-dimethyl-
methanimidamide (3a) Mp (purified by column chroma-
tography on silica gel) 113–115°C; 1H NMR (CDCl3,
200 MHz) d 2.29 (s, 3 H, CH3), 3.01 (s, 3 H, CH3), 6.15
(s, 1 H), 7.17–7.43 (m, 6 H, ArH), 7.78 (s, 1 H), 7.83–7.97
(m, 3 H, ArH); 13C NMR (50 MHz, CDCl3) d 34.5 (CH3),
40.2 (CH3), 88.4, 123.5 (2 9 CH), 125.5, 127.6, 128.3
(2 9 CH), 128.5 (3 9 CH), 134.0, 140.3, 150.8, 152.6,
154.4; IR (KBr) 3059 (m), 2920 (m), 1635 (s), 1593 (m),
1543 (m), 1496 (m), 1392 (m), 1361 (m), 1257 (m), 1103
(m), 948 (m), 759 (m), 694 (m) cm-1; EIMS m/z (relative
intensity) 290 (100), 298 (M?, 10), 246 (29), 219 (7), 198
(8), 186 (14), 171 (15), 145 (10), 83 (9), 77 (20); Anal.
Calcd for C19H19ClN4; C: 67.35; H: 5.65; N: 16.54, Found:
C: 74.43; H: 6.28; N: 19.27.
N0-[1-(4-Bromophenyl)-4-formyl-3-(4-methylphenyl)-1H-
pyrazol-5-yl]-N,N-dimethyl-methanimidamide (2d) Mp
(purified by column chromatography on silica gel)
1
198–200°C; H NMR (CDCl3, 200 MHz) d 2.36 (s, 3 H,
CH3), 2.98 (s, 3 H, CH3), 3.09 (s, 3 H, CH3), 7.21–7.23 (m,
2 H, ArH), 7.47–7.55 (m, 4 H, ArH), 7.77–7.81 (m, 2 H,
ArH), 8.68 (s, 1 H, N=C–H), 9.64 (s, 1 H, aldehyde); 13C
NMR (50 MHz, CDCl3) d 21.4 (CH3), 34.4 (CH3), 40.7
(CH3), 108.5, 120.1, 125.6 (2 9 CH), 129.21 (5 9 CH),
131.4 (2 9 CH), 128.3, 138.9, 154.1, 156.0, 159.1, 185.1;
IR (KBr) 2920 (m), 1662 (s), 1624 (m), 1489 (s), 1381 (m),
1265 (m), 1134 (m), 1091 (m), ‘1010 (m), 975(m), 829 (m),
740 (m), 501 (m) cm-1; EIMS m/z (relative intensity) 410
(100), 409 (M?, 26), 395 (12), 366 (15), 266 (10), 185 (10),
155 (6), 83 (7), 58 (5); Anal. Calcd for C20H19BrN4O; C:
58.40; H: 4.66; N: 13.62, Found: C: 58.44; H: 4.69; N:
13.58.
N0-[1-(2-Chlorophenyl)-4-formyl-3-(4-methylphenyl)-1H-pyr-
azol-5-yl]-N,N-dimethyl-methanimidamide (3b) Mp (puri-
fied by column chromatography on silica gel) 109–115°C;
1H NMR (CDCl3, 200 MHz) d 2.38 (s, 3 H, CH3), 2.94
(s, 3 H, CH3), 2.95 (s, 3 H, CH3), 6.10 (s, 1 H), 7.15–7.35
(m, 5 H, ArH), 7.70 (s 1 H, ArH), 7.75–7.80 (m, 2 H, ArH),
7.94–8.00 (m, 1 H, ArH), 8.18–8.20 (m, 1 H, ArH); 13C
NMR (50 MHz, CDCl3) d 21.1 (CH3), 34.3 (CH3), 40.0
(CH3), 88.2, 120.6, 122.3, 124.9, 125.3 (2 9 CH), 129.0
(2 9 CH), 130.8, 133.6, 137.4, 141.4, 151.0, 152.8, 154.2;
N0-[1-(4-Bromophenyl)-4-formyl-3-(4-chlorophenyl)-1H-pyr-
azol-5-yl]-N,N-dimethyl-methanimidamide (2e) Mp (puri-
fied by column chromatography on silica gel) 195–197°C;
1H NMR (CDCl3, 200 MHz) d 3.01 (s, 3 H, CH3), 3.13
(s, 3H, CH3), 7.38–7.63 (m, 6 H, ArH), 7.73–7.79 (m, 2 H,
ArH), 8.63 (s, 1 H, N=C–H), 9.61 (s, 1 H, aldehyde); 13C
NMR (50 MHz, CDCl3) d 34.5 (CH3), 40.8 (CH3), 108.5
123

3926
Med Chem Res (2012) 21:3920–3928
IR (KBr) 3109 (m), 2920 (s), 2808 (m), 1647 (s), 1585 (m),
1546 (m), 1523 (m), 1489 (m), 1388 (m), 1354 (m), 1261
(m), 1149 (m), 1103 (s), 1072 (m), 1037 (m), 948 (m), 875
(2 9 CH), 132.4, 133.3, 139.4, 149.9, 153.0, 154.5; IR
(KBr) 2920 (m), 1635 (s), 1539 (m), 1489 (m), 1357 (m),
1099 (m), 1010 (m), 948 (m), 829 (m), 759 (m), 497
(m) cm-1; EIMS m/z (relative intensity) 402 (89), 401
(M?, 4), 360 (18), 279 (9), 266 (11), 205 (15), 155 (7), 115
(4), 83 (9), 57 (4); Anal. Calcd for C18H16BrClN4; C:
53.55; H: 3.99; N: 13.88, Found: C: 53.51; H: 4.02; N:
13.91.
(m), 825 (s), 783 (m), 756 (m), 678 (m), 513 (m) cm-1
;
EIMS m/z (relative intensity) 338 (100), 317 (M?, 10), 294
(15), 279 (6), 220 (10), 185 (18), 151 (3), 111 (7), 91 (4),
83 (9); Anal. Calcd for C19H19ClN4; C: 67.35; H: 5.65; N:
16.54, Found: C: 67.36; H: 5.62; N: 16.51.
N0-[4-Formyl-1-(2-chlorophenyl)-3-(4-chlorophenyl)-1H-pyr-
azol-5-yl]-N,N-dimethyl-methanimidamide (3c) Mp (puri-
fied by column chromatography on silica gel) 108–110°C;
1H NMR (CDCl3, 200 MHz) d 2.93 (s, 3 H, CH3), 3.02
(s, 3 H, CH3), 6.03 (s, 1 H), 7.18–7.35 (m, 3 H, ArH), 7.65
(s, 1 H), 7.73–7.78 (m, 2 H, ArH), 7.77–7.82 (m, 1 H,
ArH), 7.90–7.95 (m, 1 H, ArH), 8.13–8.15 (m, 1 H, ArH);
13C NMR (50 MHz, CDCl3) d 34.5 (CH3), 40.2 (CH3),
88.3, 120.8, 122.9, 125.3, 126.8 (2 9 CH), 128.6
(2 9 CH), 129.3, 132.3, 133.3, 133.8, 141.3, 149.9, 153.1,
154.4; IR (KBr) 2920 (m), 1643 (s), 1585 (m), 1543 (m),
1504 (m), 1485 (m), 1354 (m), 1261 (m), 1153 (m), 1107
(m), 1014 (m), 948 (m), 879 (m), 837 (s), 783 (m), 756 (m),
678 (m) cm-1; EIMS m/z (relative intensity) 358 (100), 357
(M?, 7), 316 (15), 299 (6), 220 (12), 205 (13), 179 (7), 111
(12), 96 (2), 83 (10); Anal. Calcd for C18H16Cl2N4; C:
60.18; H: 4.49; N: 15.60, Found: C: 60.21; H: 4.53; N:
15.57.
Standard procedure for the synthesis of 5-amino-4-
formylpyrazoles (4a–4e)
A solution of methnimidamide derivatives (2a–2e, 1.0
equiv) and NaOH (2.0 equiv) in MeOH solution (15 ml) at
reflux within 2–3 h. When the reaction was completed, the
reaction mixture was concentrated to remove solvent,
added to water (10 ml) and extracted with CH2Cl2
(3 9 20 ml). The organic extracts were washed with sat-
urated NaHCO3, dried over MgSO4, filtered, and concen-
trated under reduced pressure. The residue solution was
purified by short column chromatography on silica gel to
give the corresponding 5-amino-4-formylpyrazole products
(4a–4e) in 83–96% yields.
5-Amino-1,3-diphenyl-1H-pyrazole-4-carbaldehyde (4a) Mp
(purified by column chromatography on silica gel)
1
154–155°C; H NMR (CDCl3, 200 MHz) d 6.13 (s, 2 H,
NH2), 7.39–7.72 (m, 10 H, ArH), 9.81 (s, 1 H, CHO); 13C
NMR (50 MHz, CDCl3) d 104, 124.0 (2 9 CH), 128.4,
128.6 (2 9 CH), 128.8 (2 9 CH), 129.2, 129.9 (2 9 CH),
131.6, 136.9, 150.1, 153.4, 185.4 (CHO); IR (KBr) 3425
(m), 3309 (m), 2827 (m), 2353 (m), 1647 (s), 1508 (m),
1253 (m), 1165 (m), 979 (m), 914 (m), 844 (m), 755
(m) cm-1; EIMS m/z (relative intensity) 263 (M?, 100);
Anal. Calcd for C16H13N3O; C: 72.99; H: 4.98; N: 15.96,
Found: C: 73.02; H: 5.01; N: 15.93.
N0-[1-(4-Bromophenyl)-4-formyl-3-(4-methylphenyl)-1H-pyr-
azol-5-yl]-N,N-dimethyl-methanimidamide (3d) Mp (puri-
fied by column chromatography on silica gel) 115–117°C;
1H NMR (CDCl3, 200 MHz) d 2.35 (s, 3 H, CH3), 2.97
(s, 3 H, CH3), 3.00 (s, 3 H, CH3), 6.11 (s, 1 H), 7.16–7.20
(m, 2 H, ArH), 7.71–7.75 (m, 3 H, ArH), 7.85–7.92 (m, 2
H, ArH); 13C NMR (50 MHz, CDCl3) d 21.3 (CH3), 34.5
(CH3), 40.3 (CH3), 88.3, 118.7, 124.7 (2 9 CH), 125.4
(2 9 CH), 129.2 (2 9 CH), 130.9, 131.3 (2 9 CH), 137.5,
139.4, 151.2, 152.7, 154.5; IR (KBr) 2920 (m), 1631 (s),
1489(m), 1388 (m), 1103 (m), 829 (m), 759 (m), 497
(m) cm-1; EIMS m/z (relative intensity) 382 (99), 381
(M?, 5), 340 (17), 326 (5), 259 (10), 185 (21), 155 (4), 115
(7), 91 (4), 83 (7); Anal. Calcd for C18H19BrN4; C: 59.54;
H: 5.00; N: 14.62, Found: C: 59.57; H: 5.02; N: 14.58.
5-Amino-1-(2-chlorophenyl)-3-(4-methylphenyl)-1H-pyra-
zole-4-carbaldehyde (4b) Mp (purified by column chro-
1
matography on silica gel) 147–148°C; H NMR (CDCl3,
200 MHz) d 2.40 (s, 3 H, CH3), 6.01 (s, 2 H, NH2),
7.25–7.27 (m, 2 H, ArH), 7.37–7.39 (m, 1 H, ArH),
7.43–7.48 (m, 2 H, ArH), 7.57–7.58 (m, 2 H, ArH), 7.64 (s,
1 H, ArH), 9.84 (s, 1 H, CHO); 13C NMR (50 MHz,
CDCl3) d 21.4 (CH3), 104.9, 121.6, 124.2, 128.5, 129.5
(4 9 CH), 130.9 (2 9 CH), 135.8, 138.1, 139.3, 150.0,
153.9, 185.7 (CHO); IR (KBr) 3406 (m), 3298 (m), 2368
(m), 1624 (s), 1512 (m), 1226 (m), 1168 (m), 1087 (m),
1033 (m), 829 (m), 744 (m) cm-1; EIMS m/z (relative
intensity) 313 (M ? 2, 32), 311 (M?, 100); Anal. Calcd for
C17H14ClN3O; C: 65.49; H: 4.53; N: 13.48, Found: C:
45.47; H: 4.56; N: 13.47.
N0-[1-(4-Bromophenyl)-4-formyl-3-(4-chlorophenyl)-1H-pyr-
azol-5-yl]-N,N-dimethyl-methanimidamide (3e) Mp (puri-
fied by column chromatography on silica gel) 152–154°C;
1H NMR (CDCl3, 200 MHz) d 2.86 (s, 3 H, CH3), 2.92
(s, 3H, CH3), 6.04 (s, 1 H), 7.30–7.34 (m, 2 H, ArH),
7.45–7.53 (m, 2 H, ArH), 7.64 (s, 1 H), 7.72–7.77 (m, 2 H,
ArH), 7.85–7.92 (m, 2 H, ArH); 13C NMR (50 MHz,
CDCl3) d 34.5 (CH3), 40.3 (CH3), 88.3, 118.9, 124.6
(2 9 CH), 126.8 (2 9 CH), 128.6 (2 9 CH), 131.3
123

Med Chem Res (2012) 21:3920–3928
3927
5-Amino-1-(4-chlorophenyl)-3-(4-chlorophenyl)-1H-pyr-
azole-4-carbaldehyde (4c) Mp (purified by column
chromatography on silica gel) 144–145°C; 1H NMR
(CDCl3, 200 MHz) d 6.06 (s, 2 H, NH2), 7.37–7.48 (m, 5
Research Center (BCRC, Taiwan). All the tumor cell lines
were maintained in either RPMI-1640 or Modified essential
medium (MEM) supplied with 10% fetal bovine serum at
37°C in a humidified atmosphere of 5% CO2/95% air in the
present of penicillin and streptomycin.
H, ArH), 7.61–7.62 (m, 3 H, ArH), 9.80 (s, 1 H, CHO); 13
C
NMR (50 MHz, CDCl3) d 104.7, 121.6, 124.2 128.6, 129.0
(2 9 CH), 129.7 (2 9 CH), 129.8, 130.9, 135.4, 135.8,
137.9, 150.1, 152.5, 185.0 (CHO); IR (KBr) 3406 (m),
3298 (m), 2924 (m), 2850 (m), 2368 (m), 1624 (s), 1512
(m), 1359 (m), 1222 (m), 1168 (m), 1095 (m), 829 (m), 744
(m) cm-1; EIMS m/z (relative intensity) 333 (M ? 2, 64),
331 (M?, 100); Anal. Calcd for C16H11Cl2N3O; C: 57.85;
H: 3.34; N: 12.65, Found: C: 57.88; H: 3.32; N: 12.69.
Growth inhibition assay
Logarithmic phase cells were seeded in a 96-well plate and
incubated overnight prior to addition of the designated
compounds. After incubation with different concentrations
of the tested compounds for 72 h, cells were incubated with
MEM containing 0.4 mg/ml MTT for 2 h. The conversion
of MTT to formazan by metabolically viable cells was
measured by the absorbance at 570 nm in a 96-well
microtiter plate reader. The percentage conversion by
mock-treated control cells was used to evaluate the effect
of the chemicals on cell growth and to determine the
concentration that inhibited 50% of growth (GI50).
5-Amino-1-(4-bromophenyl)-3-(4-methylphenyl)-1H-pyra-
zole-4-carbaldehyde (4d) Mp (purified by column chro-
matography on silica gel) 87–88°C; 1H NMR (CDCl3,
200 MHz) d 6.10 (s, 2 H, NH2), 7.24–7.25 (m, 2 H, ArH),
7.41–7.42 (m, 2 H, ArH), 7.53–7.57 (m, 4 H, ArH), 9.75 (s,
1 H, CHO); 13C NMR (50 MHz, CDCl3) d 21.2 (CH3),
104.7, 121.7, 125.1 (2 9 CH), 128.3 (3 9 CH), 129.4
(2 9 CH), 132.8 (2 9 CH), 135.9, 139.1, 149.9, 153.6,
185.3 (CHO); IR (KBr) 3290 (m), 2924 (m), 2850 (m),
2368 (m), 1643 (s), 1519 (m), 1373 (m), 1249 (m), 1165
(m), 1072 (m), 983 (m), 825 (m), 740 (m) cm-1; EIMS m/z
(relative intensity) 357 (M ? 2, 98), 355 (M?, 100); Anal.
Calcd for C17H14BrN3O; C: 57.32; H: 3.96; N: 11.80,
Found: C: 57.28; H: 3.94; N: 11.81.
Acknowledgments We are grateful to the China Medical Univer-
sity (CMU100-S-TS-13) and the National Science Council of
Republic of China for financial support (NSC 99-2320-B-039-014-
MY3).
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