Synthesis and discovery of novel pyrazole carboxamide derivatives as potential osteogenesis inducers

Article abstract of DOI:10.1002/ardp.201200180

A series of novel N-aryl-3-aryl-1-arylmethyl-1H-pyrazole-5-carboxamide derivatives 4a-l was synthesized by the reaction of 3-aryl-1-arylmethyl-1H- pyrazole-5-carbonyl chloride with substituted aniline in good to excellent yields. Structures of the compoun

Full text of DOI:10.1002/ardp.201200180

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
1
Full Paper
Synthesis and Discovery of Novel Pyrazole Carboxamide
Derivatives as Potential Osteogenesis Inducers
Hong-Shui Lv1*, Qian-Qian Han2,3*, Xiao-Ling Ding4, Ji-Liang Zhou1, Pi-Shan Yang2,3
Jun-Ying Miao5, and Bao-Xiang Zhao1
,
1 Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University,
Jinan, P. R. China
2 Department of Periodontology, School of Stomatology, Shandong University, Jinan, P. R. China
3 Key Lab of Oral Biomedicine of Shandong Province, School of Stomatology, Shandong University, Jinan,
P. R. China
4 College of Advanced Professional Technology, Qingdao University, Qingdao, P. R. China
5 School of Life Science, Shandong University, Jinan, P. R. China
A series of novel N-aryl-3-aryl-1-arylmethyl-1H-pyrazole-5-carboxamide derivatives 4a–l was synthesized by
the reaction of 3-aryl-1-arylmethyl-1H-pyrazole-5-carbonyl chloride with substituted aniline in good to
excellent yields. Structures of the compounds were determined by IR, 1H NMR, and HR-MS spectroscopy.
The molecular structure was confirmed by the X-ray crystal analysis of one compound (4j) that was prone
to crystallization. These compounds were used to induce mouse osteoblast precursors MC3T3-E1 into
osteoblasts and the induction was assessed by alkaline phosphatase (ALP) activity and the gene expression
of bone sialoprotein (BSP). The results showed that the compounds 4a–d, 4g, 4h, and 4k could increase the
ALP activity in comparison with the negative control group and compound 4h was the most effective
one which could induce osteogenesis. Furthermore, mRNA expression of BSP which is a marker of
osteogenesis was up-regulated by the compound 4h.
Keywords: ALP activity / MC3T3-E1 / Osteogenesis / Pyrazole-5-carboxamide / Synthesis / X-ray
Received: April 27, 2012; Revised: June 20, 2012; Accepted: June 27, 2012
DOI 10.1002/ardp.201200180
Introduction
current treatment of periodontal disease, which consists of
three parts: the source of seed cells for tissue engineering,
suitable scaffold materials, and effective cytokines. As for
cytokines, present research has focused on peptide growth
factors such as BMP-2, NGF, VEGF, of which recombinant
human bone morphogenetic protein-2 (BMP-2) activity was
approved by the FDA in 2002 and is being used clinically as a
bone graft substitute to achieve solid bony fusion and reduce
the morbidity of iliac bone harvest in patients who require
spine fusion surgery due to spinal instability [9, 10]. However,
the application of growth factors in vivo is limited due to
disadvantages such as the short half life, susceptibility to
degradation, large side-effects, and high price [11, 12].
Small synthetic compounds are easier to manufacture than
recombinant proteins and are more likely to scale-up for its
Bone is a dynamic tissue that constantly undergoes a remod-
eling process where bone resorption and bone deposition are
balanced. Dysregulation of this coupled remodeling can lead
to various diseases such as osteoporosis [1, 2], rheumatoid
arthritis (RA) [3], periodontal disease [4], and other metabolic
bone diseases [5–7]. Periodontal disease is a chronic infectious
disease characterized by destruction of periodontal support-
ing tissue. It can cause significant destruction of alveolar
bone, periodontal ligament (PDL), and gingiva [8]. The ulti-
mate goal of periodontal therapy is the reproduction and
reconstitution of the periodontium to restore the function of
the lost periodontium. Tissue engineering is one choice of
Correspondence: Bao-Xiang Zhao, Institute of Organic Chemistry,
School of Chemistry and Chemical Engineering, Shandong University,
Jinan 250100, P. R. China.
Additional correspondence: Prof. Pi-Shan Yang.
Email: yangps@sdu.edu.cn
E-mail: bxzhao@sdu.edu.cn
Fax: þ86 531 8856 4464
Fax: þ86 531 82950194
*These authors contributed equally to this work.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
low cost. Small molecule compounds have a variety of bio-
logical functions and serve as cell signaling molecules, tools
in molecular biology, or therapeutic drugs. Small molecule
BMP stimulators that are able to bypass the need for high
doses of BMP and induce bone formation have also been
reported [13–15]. Therefore, the discovery of novel natural
and synthetic small molecule regulators of the differen-
tiation and maturation of osteoblasts is expected to extend
clinical application of tissue engineering.
Many pyrazole derivatives are known to exhibit a wide
range of biological properties such as CB1 cannabinoid recep-
tor–ligand [16–19], inhibitor of CDK2, with good activity
against a range of human tumor cell lines [20], EP1 receptor
antagonists [21], trypsin-like serine protease factor Xa inhibi-
tors [22, 23], HIV-1 integrase inhibitors [24], non-steroidal
selective glucocorticoid receptor (GR) agonists and potent
PPARa activators [25, 26].
As part of ongoing research directed toward the develop-
ment of pyrazole-based compounds with structure diversity
[27–34], we here report the synthesis of novel pyrazole car-
boxamides and screening aimed to discover osteogenesis
inducers that promote bone regeneration instead of polypep-
tide growth factors or reduce the amount of polypeptide
growth factors.
Figure 1. The molecule structure of 4j.
ate 1. 3-Aryl-1-arylmethyl-1H-pyrazole-5-carbonyl chloride 3
was synthesized by the reaction of carboxylic acid 2 with oxalyl
chloride in CH2Cl2 under reflux. Then N-aryl-3-aryl-1-aryl-
methyl-1H-pyrazole-5-carboxamides 4 were obtained in 66.0–
97.4% yield by the reaction of 3-aryl-1-arylmethyl-1H-pyrazole-
5-carbonyl chloride 3 with substituted aniline in the presence
of triethylamine in CH2Cl2 at room temperature. Structures
of the compounds 4a–l were determined by IR, 1H NMR, and
HR-MS spectroscopy.
Results and discussion
Chemistry
The synthesis of N-aryl-3-aryl-1-arylmethyl-1H-pyrazole-5-
carboxamide derivatives 4 was performed as outlined in
Scheme 1 starting from ethyl 3-aryl-1-arylmethyl-1H-pyr-
azole-5-carboxylate 1 that can be synthesized as described
in our previous paper [35]. Briefly, 3-aryl-1-arylmethyl-1H-pyr-
azole-5-carboxylic acid 2 was firstly obtained by alkali
hydrolysis of ethyl 3-aryl-1-arylmethyl-1H-pyrazole-5-carboxyl-
Crystal structure of 4c has been reported in our previous
work [36]. Here we report the crystal structure of compound
4j as shown in Fig. 1. A summary of crystallographic data
collection parameters and refinement parameters are com-
piled in Table 1, meanwhile important bond lengths and
Scheme 1. Synthesis of N-aryl-3-aryl-1-
arylmethyl-1H-pyrazole-5-carboxamide deri-
vatives 4a–l. Reagents and conditions:
(i) KOH/EtOH, reflux, 4 h, then HCl aq;
(ii) (COCl)2, CH2Cl2, reflux, 3 h; (iii) CH2Cl2,
Et3N, ArNH2, rt, 2–3 h, 66.0–97.4% overall
yields.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Synthesis and Discovery of Novel Pyrazole Carboxamide Derivatives
3
Table 1. Summary of crystal data and structure refinement
4j
Empirical formula
Formula weight
Temperature
C27H25BrClN3O
522.86
273(2) K
˚
Wavelength
0.71073 A
Monoclinic
P21/c
Crystal system
Space group
Unit cell dimensions
˚
a ¼ 13.673(2) A, a ¼ 908
˚
b ¼ 20.520(3) A, b ¼ 104.739(3)8
˚
c ¼ 9.4083(15) A, g ¼ 908
Volume
Z
Calculated density
Absorption coefficient
Max. and min. transmission
F(000)
Crystal size
u range for data collection
Limiting indices
2552.7(7) A3
4
1.360 Mg/m3
1.739 mmꢀ1
0.8453 and 0.7224
1072
Figure 2. A packing diagram of the crystal structure of 4j.
0.20 ꢂ 0.10 ꢂ 0.10 mm3
1.54–26.408
ꢀ17 ꢃ h ꢃ 16, ꢀ24 ꢃ k ꢃ 25,
ꢀ11 ꢃ l ꢃ 10
Reflections collected/unique
Completeness to theta ¼ 25.05
Absorption correction
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
13962/5201 [R(int) ¼ 0.0309]
˚
99.4%
Multi-scan
Table 3. Hydrogen bond geometry (A, 8)
Full-matrix least-squares on F2
5201/0/358
1.024
D–Hꢁ ꢁ ꢁA
D–H
Hꢁ ꢁ ꢁA
Dꢁ ꢁ ꢁA
D–Hꢁ ꢁ ꢁA
N1–H1ꢁ ꢁ ꢁO1i
0.81
0.97
1.05
1.05
0.80
0.92
2.16
2.49
2.71
2.50
2.60
2.45
2.937(3)
2.961(4)
3.478(3)
3.304(3)
2.868(4)
3.082(4)
160
110
130
133
102
126
Final R indices [I > 2s(I)]
R indices (all data)
Largest diff. peak and hole
CCDC no.
R1 ¼ 0.0424, wR2 ¼ 0.1134
Intra C3–H3ꢁ ꢁ ꢁO1
C5–H5ꢁ ꢁ ꢁCl2ii
R1 ¼ 0.0785, wR2 ¼ 0.1416
3
˚
0.293 and ꢀ0.356 e/A
C5–H5ꢁ ꢁ ꢁO1i
878298
Intra C12–H12ꢁ ꢁ ꢁN3
Intra C17–H17Aꢁ ꢁ ꢁO1
Symmetry code: (i) x, (1/2) S y, (1/2) þ z; (ii) S1 þ x, (1/2) S y,
(3/2) S z.
D and A are the hydrogen-bond donor and acceptor, respectively.
angles are depicted in Table 2. The pyrazole ring makes
dihedral angles of 59.52(11), 7.51(23), and 82.56(12)8 with
the 4-bromophenyl, 4-chlorophenyl, and 4-tert-butylphenyl
rings, respectively. The orientation of the 4-bromophenyl
moiety is defined by the torsion angle of C(4)-N(1)-C(7)-C(8)
ꢀ169.7(3)8. The packing diagram of 4j is shown in Fig. 2. The
molecules of 4j are stabilized by intramolecular C–Hꢁ ꢁ ꢁO
and C–Hꢁ ꢁ ꢁN bonds, meanwhile linked by intermolecular
C–Hꢁ ꢁ ꢁO, C–Hꢁ ꢁ ꢁCl, and N–Hꢁ ꢁ ꢁO bonds (shown in Table 3).
Effects of compounds 4a–l on cellular alkaline
phosphatase (ALP) activity
Mouse osteoblast precursor MC3T3-E1 is a widely accepted
cell line that can be used to study the role of bone formation
in vitro. ALP is widely considered to be the marker of the
differentiation and function of the osteoblast [37]. ALP
activity is an early osteogenic marker and the testing
methods are relatively simple. When ST2 cells were treated
with BMP-2 at concentrations over 10 ng/mL for 6 days, ALP
activity was significantly increased [38]. ST2 is a mouse bone
marrow stromal stem cell line and MC3T3-E1 is a mouse
osteoblast precursor. Both cells have the potential to differ-
entiate into osteoblasts. In our present research, we chose
MC3T3-E1 cells as cell model and detected the ALP activity
after exposing to compounds 4a–l for 7 days. As shown in
Fig. 3, compounds 4a–d, 4g, 4h, and 4k could increase the ALP
activity in comparison with the negative control group.
Among them, compound 4h exhibited higher ALP activity
than other groups. The results showed that compound 4h
might have a relatively strong osteoinduction effect.
˚
Table 2. Selected bond lengths (A), bond angles (8), and torsion
angles (8) of compound 4j
C(10)–C(11)
1.467(4)
C(7)–C(8)
1.476(4)
C(9)–C(10)
N(2)–C(8)
N(3)–C(10)
C(18)–C(17)
N(1)–C(4)
C(10)–N(3)–N(2)
N(1)–C(7)–C(8)
N(2)–C(17)–C(18)
C(4)–N(1)–C(7)–C(8)
1.403(4)
1.361(4)
1.341(3)
1.504(5)
1.411(3)
105.4(2)
113.1(2)
110.9(2)
C(9)–C(8)
N(2)–N(3)
N(2)–C(17)
N(1)–C(7)
O(1)–C(7)
N(3)–N(2)–C(8)
C(7)–N(1)–C(4)
O(1)–C(7)–C(8)
1.368(4)
1.348(3)
1.480(4)
1.356(4)
1.238(3)
111.7(2)
127.3(2)
123.2(2)
80.7(4)
ꢀ169.7(3) C(8)–N(2)–C(17)–C(18)
C(19)–C(18)–C(17)–N(2) ꢀ104.8(4) C(23)–C(18)–C(17)–N(2) 75.2(4)
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ation but promote cell differentiation [39, 40]. Therefore, we
use MTT to detect the effect of compound 4h on cell prolifer-
ation in vitro. MTT results (Fig. 5) showed that cell viability
depended on the concentration of compound 4h signifi-
cantly. When treated with 1 and 4 mM of compound 4h
for 24 h, the cell viability of MC3T3-E1 was 90.9 ꢄ 1.50
and 87.9 ꢄ 5.63%, respectively, which was not changed obvi-
ously. Moreover, when cells were treated with 1 and 4 mM of
compound 4h for 48 h, the cell viability of MC3T3-E1 was
89.5 ꢄ 2.69 and 86.9 ꢄ 2.58%, respectively, which was also
not changed notably. However, when cells were treated with
16 and 40 mM of compound 4h for 24 h, the cell viability of
MC3T3-E1 (80.3 ꢄ 6.10, 67.1 ꢄ 6.20%, respectively) was
decreased significantly. When cells were treated for 48 h,
the cell viability decreased more significantly (57.9 ꢄ 3.80,
55.7 ꢄ 1.81%, respectively). Therefore, we believed that the
selected 4 mM of compound 4h was suitable for a further
study.
Figure 3. Effect of pyrazole carboxamide derivatives 4a–l on ALP
activity of MC3T3-E1, an early marker of osteogenic differentiation,
after culturing cells for 7 days in growth medium. Controlꢀ,
medium with 4 mM DMSO; 4a–l, medium supplemented with com-
pound 4a–l; Controlþ, osteogenetic medium. ꢅp < 0.05, ꢅꢅp < 0.01,
ꢅꢅꢅp < 0.001.
Effect of compound 4h on mRNA expression level of
BSP
Effects of compound 4h on cellular alkaline
phosphatase (ALP) activity and cell viability
Bone sialoprotein (BSP) is a highly post-translationally
modified protein which is an abundant non-collagenous
component of the bone matrix and it is a marker of osteo-
genesis. BSP expression was up-regulated when human den-
tal pulp stem cells (DPSCs) were induced into osteoblasts [41],
and ALP activity and BSP level were down-regulated when
compounds were used to suppress osteoblastic differen-
tiation and mineralized nodule formation [42]. To further
determine the effect of compound 4h on osteoinduction
activity of MC3T3-E1 cells, total RNA was isolated, and RT-
PCR was performed to measure gene expression levels of BSP.
As shown in Fig. 6, we found that in the compound 4h
induced group, the mRNA expression levels of BSP showed
a 1.69-fold increase compared with that in the negative con-
trol group after 4 days induction. The increase was similar to
that of the positive control group which was a 1.73-fold
increase compared with the negative control group.
Next, the effect of the compound 4h at different concen-
trations on ALP activity was assessed. As shown in Fig. 4,
when cells were treated with various concentrations of com-
pound 4h for 7 days, cellular alkaline phosphatase (ALP)
activity was affected and the result showed that compound
4h at the concentration of 4 mmol/L exhibited the highest
ALP activity.
Cell proliferation and differentiation are two relatively
independent processes. Several studies confirmed that a
variety of cytokines or compounds could inhibit cell prolifer-
Conclusion
A series of novel N-aryl-3-aryl-1-arylmethyl-1H-pyrazole-5-
carboxamide derivatives 4a–4l was synthesized and charac-
terized by IR, 1H NMR, and HRMS spectroscopy. The molecu-
lar structure was confirmed by X-ray crystal analysis of
one compound (4j). These compounds were used to induce
differentiation of mouse osteoblast precursors MC3T3-E1 into
osteoblasts and the induction was assessed by ALP activity
and mRNA expression of BSP. The results showed that the
compounds 4a–d, 4g, 4h, and 4k could increase the ALP activity
in comparison with the negative control group and compound
4h was the most effective one. Furthermore, the mRNA
Figure 4. Effect of compound 4h at different concentrations on
ALP activity of MC3T3-E1 after culturing confluent cells for 7 days
in medium containing different concentrations of compound 4h.
Controlꢀ, medium with 4 mmol/L DMSO; ꢅp < 0.01, ꢅꢅp < 0.001.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Synthesis and Discovery of Novel Pyrazole Carboxamide Derivatives
5
Figure 5. Effects of compound 4h at different
concentrations (1, 4, 16, and 40 mM) on viability
of MC3T3-E1 cells treated for 24 h (A) and 48 h
(B). Controlꢀ, medium. ꢅp < 0.05, ꢅꢅp < 0.01.
paper [35]. To a solution of 1 (10 mmol) in ethanol (50 mL)
was added KOH (30 mmol). The mixture was refluxed for 2 h.
After cooling, ethanol was removed under reduced pressure. The
residue was dissolved in 50 mL water. The solution was added
hydrochloric acid (3 M) until pH ¼ 4–5. The precipitate was
filtered and dried to give 3-aryl-1-arylmethyl-1H-pyrazole-5-
carboxylic acid (2) as pure product in 100% yield. A solution
of 2 (1 mmol), oxalyl chloride (0.5 mL), and two drops of DMF
in CH2Cl2 (10 mL) was refluxed for 4 h. After cooling, the
solvent was removed to give 3, which was used without further
purification.
expression of BSP which is a marker of osteogenesis was
up-regulated by compound 4h. Therefore, compound 4h
may have the osteoinduction activity for MC3T3-E1 cells.
Currently, investigations are underway to elucidate the
osteoinduction activity and the mechanism by which com-
pound 4h promotes osteogenetic differentiation on MC3T3-
E1 and the results will be reported in due course.
Experimental
All reagents were of analytical grade or chemically pure.
Analytical TLC was performed on silica gel 60 F254 plates
(Merck KGaA). m.p. was determined on an XD-4 digital micromelt-
ing point apparatus. 1H NMR spectra were recorded on a Bruker
Avance 400 spectrometer, using CDCl3 as solvent and TMS as
internal standard. IR spectra were recorded with an IR spectro-
photometer Avtar 370 FT-IR (Termo Nicolet). HRMS spectra were
recorded on a LTQ Orbitrap Hybrid mass spectrometer.
N-Aryl-3-aryl-1-arylmethyl-1H-pyrazole-5-carboxamide 4
To a solution of substituted aniline (1 mmol) in CH2Cl2 (10 mL)
containing Et3N (0.5 mL), 3-aryl-1-arylmethyl-1H-pyrazole-5-
carbonyl chloride (3) (1 mmol) dissolved in CH2Cl2 (5 mL) was
added dropwise at room temperature. The mixture was stirred
overnight. The end of reaction was detected by TLC. After which
the solution was washed by water (15 mL ꢂ 2), the water phase
was extracted with CH2Cl2 (15 mL ꢂ 1). The combined organic
phase was dried over MgSO4. After filtration, the solvent was
removed under reduced pressure. The residue was purified by
column chromatography on silica gel using PE/EtOAc as an
eluent to afford title compound 4 in 66.0–97.4% (overall yields).
3-Aryl-1-arylmethyl-1H-pyrazole-5-carbonyl chloride 3
The starting material ethyl 3-aryl-1-arylmethyl-1H-pyrazole-5-
carboxylate (1) was synthesized as described in our previous
N-(4-Chlorophenyl)-1-((6-chloropyridin-3-yl)methyl)-
3-phenyl-1H-pyrazole-5-carboxamide 4a
White solid, yield: 66.0%, m.p.: 214–2168C, IR: nmax cmꢀ1 3412
1
(N–H), 1674 (C O). H NMR (CDCl , 400 MHz): 5.82 (s, 2H, –CH ),
–
–
3
2
7.06 (s, 1H, pyrazole–H), 7.26 (d, 1H, J ¼ 8.1 Hz, pyridine–H), 7.34
(d, 2H, J ¼ 8.7 Hz, Ar–H), 7.36–7.47 (m, 3H, Ar–H), 7.56 (d, 2H,
J ¼ 8.7 Hz, Ar–H), 7.74 (dd, 1H, J1 ¼ 8.1 Hz, J2 ¼ 2.2 Hz,
pyridine–H), 7.81 (d, 2H, J ¼ 7.4 Hz, Ar–H), 7.93 (s, 1H, N–H),
8.47 (d, 1H, J ¼ 2.2 Hz, pyridine–H); HRMS calcd for
[MþH]þ C22H17Cl2N4O: 423.0779, found 423.0769.
N-(4-Bromophenyl)-1-((6-chloropyridin-3-yl)methyl)-
3-phenyl-1H-pyrazole-5-carboxamide 4b
White solid, yield: 72.8%, m.p.: 194–1988C, IR: nmax cmꢀ1 3412
1
(N–H), 1675 (C O). H NMR (CDCl , 400 MHz): 5.81 (s, 2H, –CH ),
–
–
3
2
6.96 (s, 1H, pyrazole–H), 7.26 (d, 1H, J ¼ 8.2 Hz, pyridine–H),
7.33–7.54 (m, 7H, Ar–H), 7.69 (s, 1H, N–H), 7.73 (dd, 1H,
J1 ¼ 8.2 Hz, J2 ¼ 2.3 Hz, pyridine–H), 7.80 (d, 2H, J ¼ 7.1 Hz,
Ar–H), 8.48 (d, 1H, J ¼ 2.2 Hz, pyridine–H); HRMS calcd for
[MþH]þ C22H17BrClN4O: 467.0274, found 467.0261.
Figure 6. Effects of compound 4h on BSPexpression in MC3T3-E1
osteoblastic cells analyzed by RT-PCR. 4h, medium with 4 mM
compound 4h; Controlꢀ, medium with 4 mM DMSO; Controlþ,
osteogenetic medium. ꢅp < 0.01.
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H.-S. Lv et al.
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7.82 (d, 2H, J ¼ 7.6 Hz, Ar–H); HRMS calcd for [MþH]þ
1-((6-Chloropyridin-3-yl)methyl)-N-(4-ethoxyphenyl)-
3-phenyl-1H-pyrazole-5-carboxamide 4c
C28H30N3O2: 440.2338, found 440.2338.
Yellow solid, yield: 77.7%, m.p.: 164–1658C, IR: nmax cmꢀ1 3339
(N–H), 1663 (C O). 1H NMR (CDCl3, 400 MHz): 1.42 (t, 3H,
–
–
1-(4-(Tert-butyl)benzyl)-N,3-bis(4-chlorophenyl)-1H-
pyrazole-5-carboxamide 4i
J ¼ 7.0 Hz, –CH3), 4.03 (q, 2H, J ¼ 7.0 Hz, –OCH2), 5.82 (s, 2H,
–CH2), 6.90 (d, 2H, J ¼ 8.9 Hz, Ar–H), 6.93 (s, 1H, pyrazole–H), 7.24
(d, 1H, J ¼ 8.2 Hz, pyridine–H), 7.32–7.39 (m, 1H, Ar–H), 7.39–
7.48 (m, 4H, Ar–H), 7.63 (s, 1H, N–H), 7.74 (dd, 1H, J1 ¼ 8.2 Hz,
J2 ¼ 2.3 Hz, pyridine–H), 7.79 (d, 2H, J ¼ 7.2 Hz, Ar–H), 8.48
(d, 1H, J ¼ 2.3 Hz, pyridine–H); HRMS calcd for [MþH]þ
C24H22ClN4O2: 433.1431, found 433.1425.
White solid, yield: 68.6%, m.p.: 213–2158C, IR: nmax cmꢀ1 3283
(N–H), 1658 (C O). 1H NMR (CDCl3, 400 MHz): 1.26 (s, 9H,
–
–
–C(CH3)3), 5.78 (s, 2H, –CH2), 6.88 (s, 1H, pyrazole–H), 7.28–7.42
(m, 6H, Ar–H), 7.38 (d, 2H, J ¼ 8.1 Hz, Ar–H), 7.50 (d, 2H,
J ¼ 8.6 Hz, Ar–H), 7.62 (s, 1H, N–H), 7.75 (d, 2H, J ¼ 8.1 Hz,
Ar–H); HRMS calcd for [MþH]þ C27H26Cl2N3O: 478.1453, found
478.1450.
1-((6-Chloropyridin-3-yl)methyl)-N-(4-methoxyphenyl)-
3-phenyl-1H-pyrazole-5-carboxamide 4d
N-(4-Bromophenyl)-1-(4-(tert-butyl)benzyl)-3-
(4-chlorophenyl)-1H-pyrazole-5-carboxamide 4j
Yellow solid, yield: 75.9%, m.p.: 118–1218C, IR: nmax cmꢀ1 3359
1
(N–H), 1666 (C O). H NMR (CDCl , 400 MHz): 3.82 (s, 3H, –OCH ),
–
–
Yellow solid, yield: 97.4%, m.p.: 202–2068C, IR: nmax cmꢀ1 3283
3
3
5.82 (s, 2H, –CH2), 6.91 (d, 2H, J ¼ 8.9 Hz, Ar–H), 6.94 (s, 1H,
pyrazole–H), 7.24 (d, 1H, J ¼ 8.2 Hz, pyridine–H), 7.33–7.39
(m, 1H, Ar–H), 7.39–7.50 (m, 4H, Ar–H), 7.64 (s, 1H, N–H), 7.74
(dd, 1H, J1 ¼ 8.2 Hz, J2 ¼ 2.3 Hz, pyridine–H), 7.80 (d, 2H,
J ¼ 7.2 Hz, Ar–H), 8.48 (d, 1H, J ¼ 2.3 Hz, pyridine–H); HRMS
calcd for [MþH]þ C23H20ClN4O2: 419.1275, found 419.1276.
(N–H), 1659 (C O). 1H NMR (CDCl3, 400 MHz): 1.26 (s, 9H,
–
–
–C(CH3)3), 5.77 (s, 2H, –CH2), 6.88 (s, 1H, pyrazole–H), 7.27–7.34
(m, 4H, Ar–H), 7.38 (d, 2H, J ¼ 8.5 Hz, Ar–H), 7.42–7.50 (m, 4H,
Ar–H), 7.63 (s, 1H, N–H), 7.74 (d, 2H, J ¼ 8.5 Hz, Ar–H); HRMS
calcd for [MþH]þ C27H26BrClN3O: 522.0948, found 522.0935.
1-(4-(Tert-butyl)benzyl)-3-(4-chlorophenyl)-N-
(4-ethoxyphenyl)-1H-pyrazole-5-carboxamide 4k
1-(4-(Tert-butyl)benzyl)-N-(4-chlorophenyl)-3-phenyl-1H-
pyrazole-5-carboxamide 4e
Yellow solid, yield: 84.6%, m.p.: 185–1878C, IR: nmax cmꢀ1 3275
Yellow solid, yield: 73.6%, m.p.: 182–1838C, IR: nmax cmꢀ1 3372
(N–H), 1649 (C O). 1H NMR (CDCl3, 400 MHz): 1.27 (s, 9H,
–
(N–H), 1654 (C O). 1H NMR (CDCl3, 400 MHz): 1.26 (s, 9H,
–
–
–
–C(CH3)3), 1.41 (t, 3H, J ¼ 7.0 Hz, –CH3), 4.03 (q, 2H, J ¼ 7.0 Hz,
–OCH2), 5.79 (s, 2H, –CH2), 6.87 (s, 1H, pyrazole–H), 6.89 (d, 2H,
J ¼ 9.0 Hz, Ar–H), 7.28–7.34 (m, 4H, Ar–H), 7.38 (d, 2H,
J ¼ 8.5 Hz, Ar–H), 7.42 (d, 2H, J ¼ 8.5 Hz, Ar–H), 7.53 (s, 1H,
N–H), 7.75 (d, 2H, J ¼ 8.5 Hz, Ar–H); HRMS calcd for
[MþH]þ C29H31ClN3O2: 488.2105, found 488.2107.
–C(CH3)3), 5.78 (s, 2H, –CH2), 6.90 (s, 1H, pyrazole–H), 7.26–7.37
(m, 7H, Ar–H), 7.37–7.45 (m, 2H, Ar–H), 7.49 (d, 2H, J ¼ 8.7 Hz,
Ar–H), 7.65 (s, 1H, N–H), 7.81 (d, 2H, J ¼ 7.2 Hz, Ar–H); HRMS
calcd for [MþH]þ C27H27ClN3O: 444.1843, found 444.1848.
N-(4-Bromophenyl)-1-(4-(tert-butyl)benzyl)-3-phenyl-1H-
pyrazole-5-carboxamide 4f
1-(4-(Tert-butyl)benzyl)-3-(4-chlorophenyl)-N-
(4-methoxyphenyl)-1H-pyrazole-5-carboxamide 4l
Yellow solid, yield: 81.0%, m.p.: 175–1788C, IR: nmax cmꢀ1 3276
(N–H), 1656 (C O). 1H NMR (CDCl3, 400 MHz): 1.26 (s, 9H,
–
–
Yellow solid, yield: 89.6%, m.p.: 201–2038C, IR: nmax cmꢀ1 3283
–C(CH3)3), 5.78 (s, 2H, –CH2), 6.91 (s, 1H, pyrazole–H), 7.27–7.32
(m, 4H, Ar–H), 7.32–7.37 (m, 1H, Ar–H), 7.38–7.48 (m, 6H, Ar–H),
7.65 (s, 1H, N–H), 7.81 (d, 2H, J ¼ 7.8 Hz, Ar–H); HRMS calcd for
[MþH]þ C27H27BrN3O: 488.1338, found 488.1329.
(N–H), 1653 (C O). 1H NMR (CDCl3, 400 MHz): 1.27 (s, 9H,
–
–
–C(CH3)3), 3.81 (s, 3H, –OCH3), 5.78 (s, 2H, –CH2), 6.87 (s, 1H,
pyrazole–H), 6.89 (d, 2H, J ¼ 8.5 Hz, Ar–H), 7.28–7.34 (m, 4H,
Ar–H), 7.38 (d, 2H, J ¼ 8.5 Hz, Ar–H), 7.44 (d, 2H, J ¼ 8.6 Hz,
Ar–H), 7.55 (s, 1H, N–H), 7.75 (d, 2H, J ¼ 8.5 Hz, Ar–H); HRMS
calcd for [MþH]þ C28H29ClN3O2: 474.1948, found 474.1952.
1-(4-(Tert-butyl)benzyl)-N-(4-ethoxyphenyl)-3-phenyl-1H-
pyrazole-5-carboxamide 4g
Yellow solid, yield: 74.2%, m.p.: 149–1518C, IR: nmax cmꢀ1 3267
Crystal structure determination of compound 4j
(N–H), 1645 (C O). 1H NMR (CDCl3, 400 MHz): 1.26 (s, 9H,
–
–
Suitable single crystals of 4j for X-ray structural analysis were
obtained by recrystallization from dichloromethane/ethyl acetate.
The diffraction data was collected with a Bruker SMART CCD
diffractometer using a graphite monochromated Mo Ka radiation
–C(CH3)3), 1.40 (t, 3H, J ¼ 7.0 Hz, –CH3), 4.01 (q, 2H, J ¼ 7.0 Hz,
–OCH2), 5.78 (s, 2H, –CH2), 6.83–6.93 (m, 3H, pyrazole–H þ Ar–H),
7.27–7.36 (m, 5H, Ar–H), 7.36–7.47 (m, 4H, Ar–H), 7.65 (s, 1H,
N–H), 7.81 (d, 2H, J ¼ 7.2 Hz, Ar–H); HRMS calcd for
[MþH]þ C29H32N3O2: 454.2495, found 454.2497.
˚
(l ¼ 0.71073 A) at 273(2) K. The structures were solved by direct
methods with the SHELXS-97 program and refinements on F2
were performed with the SHELXL-97 [43] program by full-matrix
least-squares techniques with anisotropic thermal parameters
for the non-hydrogen atoms. All H atoms were initially located
in a difference Fourier map. The methyl H atoms were then
1-(4-(Tert-butyl)benzyl)-N-(4-methoxyphenyl)-3-phenyl-
1H-pyrazole-5-carboxamide 4h
White solid, yield: 75.6%, m.p.: 164–1678C, IR: nmax cmꢀ1 3261
(N–H), 1646 (C O). 1H NMR (CDCl3, 400 MHz): 1.26 (s, 9H,
–
˚
constrained to an ideal geometry, with C–H ¼ 0.96 A and
–
Uiso(H) ¼ 1.5 Ueq(C). All other H atoms were placed in geometri-
–C(CH3)3), 3.81 (s, 3H, –OCH3), 5.80 (s, 2H, –CH2), 6.87–6.93
(m, 3H, pyrazole–H þ Ar–H), 7.28–7.32 (m, 4H, Ar–H), 7.32–
7.36 (m, 1H, Ar–H), 7.38–7.47 (m, 4H, Ar–H), 7.57 (s, 1H, N–H),
cally idealized positions and constrained to ride on their parent
˚
atoms, with C–H ¼ 0.93 A and Uiso(H) ¼ 1.2 Ueq(C).
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Synthesis and Discovery of Novel Pyrazole Carboxamide Derivatives
7
Table 4. The sequences of the primers for amplication of mouse
BSP and GAPDH
Cell culture and ALP activity of compounds 4a–l
MC3T3-E1 cells were cultured in a-MEM (Hyclone, USA) supple-
mented with 10% FBS (Gibco, USA), 100 units/mL penicillin, and
100 mg/mL streptomycin. When reaching 80% confluence, the
cells were seeded at a density of 2 ꢂ 104 cells/well on 24-well
plates and allowed to grow to confluence. Then cells were main-
tained in compounds-stimulated media [a-MEM with 10% v/v
FBS, supplemented with different kinds of compounds 4a–l
(4 mmol/L)]. Cells cultured on basic media supplemented with
4 mmol/L of DMSO were the negative control and cells cultured
in osteogenic media (a-MEM supplemented with 10ꢀ8 mol/L
dexamethasone, 50 mg/L ascorbic acid, and 10 mmol/L b-glycero-
phosphate) were the positive control. Media were changed every
2 days. After 7 days of induction, cells were washed with
0.01 mol/L PBS and scraped into 100 mL of 0.2% TritonX-100.
Then cells were sonicated and cell lysates were centrifuged at
14 000 g for 10 min at 48C. ALP activity in the supernatant was
assayed according to the instructions of the manufacturer
(Nanjing Jiancheng, China). Results were adjusted according
to protein content detected by BCA standard (KeyGEN
BioTECH, China).
Primer
GAPDH
BSP
Sequence
Forward: 50-AGGTCGGTGTGAACGGATTTG-30
Reverse: 50-TGTAGACCATGTAGTTGAGGTCA-30
Forward: 50-CAGGGAGGCAGTGACTCTTC-30
Reverse: 50-AGTGTGGAAAGTGTGGCGTT-30
Statistical analysis
Results were presented as the means ꢄ SD of three to five rep-
licates for each experiment. The statistical significance of the
differences between different groups was assessed by one-way
ANOVA. p < 0.05 was considered as statistically significant.
This work was supported by National Natural Science Foundation of China
(20972088, 90813022, and 81070835) and the Independent Innovation
Foundation of Shandong University (2009JC007).
ALP activity of compound 4h at different
concentrations
The authors have declared no conflict of interest.
MC3T3-E1 were cultured in a-MEM supplemented with 10% FBS.
When reaching 80% confluence, cells were seeded in 6-well plates
at a density of 1 ꢂ l05 cells/well. Then the media were replaced
with fresh a-MEM containing different concentrations of com-
pound 4h (0, 1, 4, 16, and 40 mmol/L) for 7 days. Media were
changed every 2 days. After 7 days of induction, ALP activity was
detected.
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