Synthesis and discovery of a novel pyrazole derivative as an inhibitor of apoptosis through modulating integrin β4, ROS, and p53 levels in vascular endothelial cells

Article abstract of DOI:10.1016/j.bmc.2008.03.011

Recently, pyrazole derivatives as high affinity and selective A2A adenosine receptor antagonists have been reported. But, so far, there are no reports about the inhibitory effects of multi-substituted pyrazole derivatives on apoptosis of vascular endothelial cells (VECs). In this study, we synthesized six pyrazole derivatives and characterized the structures of the compounds by IR, ¹H NMR, mass spectroscopy, and element analysis. The biology assay showed that a novel pyrazole derivative, ethyl 3-(o-chlorophenyl)-5-methyl-1-phenyl-1H-pyrazole-4-carboxylate (MPD) at low concentration (25 μM) increased VECs viability and inhibited VECs apoptosis induced by deprivation of serum and FGF-2. During this process, the levels of integrin β4, reactive oxygen species (ROS), and p53 were depressed obviously. The data suggested that MPD was a potential inhibitor of apoptosis associated with the signal pathway mediated by integrin β4, ROS, and p53 in VECs.

Full text of DOI:10.1016/j.bmc.2008.03.011

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5171–5180
Synthesis and discovery of a novel pyrazole derivative as
an inhibitor of apoptosis through modulating integrin b4, ROS,
and p53 levels in vascular endothelial cells
Bao-Xiang Zhao,a,* Lu Zhang,b,c Xing-Shang Zhu,b,c Mao-Sheng Wan,d Jing Zhao,b,c
Yun Zhang,c Shang-Li Zhangb,c and Jun-Ying Miaob,c,
*
aInstitute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China
bInstitute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, China
cThe Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education
and Chinese Ministry of Health, Shandong University Qilu Hospital, Jinan 250100, China
dShandong Drug and Food Vocational College, Weihai 264210, China
Received 30 January 2008; revised 2 March 2008; accepted 4 March 2008
Available online 6 March 2008
Abstract—Recently, pyrazole derivatives as high affinity and selective A2A adenosine receptor antagonists have been reported. But,
so far, there are no reports about the inhibitory effects of multi-substituted pyrazole derivatives on apoptosis of vascular endothelial
1
cells (VECs). In this study, we synthesized six pyrazole derivatives and characterized the structures of the compounds by IR, H
NMR, mass spectroscopy, and element analysis. The biology assay showed that a novel pyrazole derivative, ethyl 3-(o-chloro-
phenyl)-5-methyl-1-phenyl-1H-pyrazole-4-carboxylate (MPD) at low concentration (25 lM) increased VECs viability and inhibited
VECs apoptosis induced by deprivation of serum and FGF-2. During this process, the levels of integrin b4, reactive oxygen species
(ROS), and p53 were depressed obviously. The data suggested that MPD was a potential inhibitor of apoptosis associated with the
signal pathway mediated by integrin b4, ROS, and p53 in VECs.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
way, the inflammatory infiltrate within lesions perpetu-
ates and amplifies the increased apoptosis within lesions.
Apoptosis of VECs leads to increased risk for thrombo-
sis. During apoptosis VECs lose anticoagulant mem-
brane components and become procoagulant by an
increased exposure of phosphatidylserine on their sur-
face. In the presence of coagulation factors V and VII,
phosphatidylserine can promote thrombin generation.5
It is presumed that the inhibition of VEC apoptosis
can be a clue to the development of new strategies in
atherosclerosis therapy.
Vascular endothelial cells (VEC) form the inner lining of
all blood vessels and function to maintain vascular tone
and anticoagulant properties of blood vessels. VEC
apoptosis is important in many disease states, including
atherosclerosis. Traditional hypotheses of atherogenesis
have suggested that the injury of VECs is critical for the
development of atherosclerosis.1 The sites where plaques
develop may be associated with increased VEC turnover
rate, suggesting a mechanical link with susceptibility to
atherosclerosis, perhaps as an increase in apoptosis.2
In addition, membrane vesicles from apoptotic cells con-
tain biologically active oxidized phospholipids that can
induce monocyte adhesion to the endothelium.3,4 In this
The use of small molecules to affect biological phenom-
ena, also known as chemical genetics, has made a signif-
icant impact in diverse areas of biology.6–8 The design of
the library is the first and a very crucial step in the forward
chemical genetics process, and this step determines the
success of the library.9 In addition to natural products li-
brary and known drug-like library, the design and synthe-
sis of novel small molecules library with valuable
chemical diversity has been shown to be challenging.10
Keywords: Pyrazole derivative; Apoptosis; Vascular endothelial cell;
Integrin b4; ROS; p53.
Corresponding authors. Tel.: +86 531 88366425; fax: +86 531
88564464; e-mail addresses: bxzhao@sdu.edu.cn; miaojy@sdu.
edu.cn
*
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.011

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The pyrazole unit is one of the core structures in a num-
ber of natural products. Many pyrazole derivatives are
known to exhibit a wide range of biological properties
and have been attracted attention in the widely field of
biology. Extensive studies have been devoted to arylpy-
razole derivatives such as Celecoxib, a well-known
cyclooxygenase-2 inhibitor.11–14 Recently, pyrazole
derivatives as high affinity and selective A2B adenosine
receptor antagonists have been reported.15 But, so far,
there are no reports about the effects of pyrazole deriv-
atives, especially multi-substituted pyrazole derivatives,
on the apoptosis of vascular endothelial cells. In our
previous papers, we described the synthesis and preli-
minary biological evaluation of novel 1-substituted-3-
aryl-1H-pyrazole-5-carboxylate derivatives and novel
5-alkyl-2-ferrocenyl-6,7-dihydropyrazolo[1,5-a]pyrazin-
4(5H)-one derivatives.16–19 In an ongoing study in our
laboratory on the discovery and the development of
apoptosis inducers or inhibitor as potential new athero-
sclerosis therapy,20–22 we are interested in synthesizing
novel multi-substituted pyrazole derivatives and screen-
ing potential small molecules for the investigation of
molecular mechanism.
tures of compounds 4 were characterized by IR, 1H
NMR, mass spectroscopy, and element analysis.
Interestingly, when 3a–3c were treated with hydrazine
hydrate, respectively, and 3a was treated with phenyl
hydrazine in ethanol, 4a–4d were obtained in good
yields; however, the reaction of 3b with phenyl hydra-
zine and the reaction of 3d with hydrazine hydrate gave
4e and 4f. The structures of compounds 4a–4f were as-
1
signed with H NMR, IR, and mass spectrum. Further-
more, intermediates 30 and 3 exist in tautomerism. It was
reported that the melting point of intermediate 3c0 was
55–56 °C,23 however, in the present study, we observed
that the melting point of 3c was 128–131 °C. It is not
surprising there is the difference because intermediates
30 and 3 exist in tautomerism and can be isolated in suit-
able condition.
2.2. Effects of the compounds 4 on VEC apoptosis
In order to evaluate the effects of the compounds on
VEC apoptosis, we used the given model of VEC apop-
tosis induced by deprivation of serum and FGF-2. The
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bro-
mide (MTT) cell proliferation assay is widely accepted as
a reliable way to measure the cell proliferation rate, and
conversely when metabolic events lead to apoptosis or
necrosis. The results showed that compounds 4a, 4b, 4e,
and 4f had no obvious effects on VEC apoptosis at the
concentrations of 25–100 lM (data not shown), mean-
while it was interesting that ethyl 3-(o-chlorophenyl)-5-
methyl-1-phenyl-1H-pyrazole-4-carboxylate 4d (MPD)
suppressed the apoptosis at 25 lM and promoted the
apoptosis at the concentrations of 50 and 100 lM
(p < 0.01) (Fig. 2). This finding is similar to the phenom-
ena observed in our previous study with other small
molecules.20,24
Herein, we would like to report the synthesis of novel
multi-substituted pyrazole derivatives and the findings
of their biological activities in VEC apoptosis, espe-
cially, the modulation of ethyl 3-(o-chlorophenyl)-5-
methyl-1-phenyl-1H-pyrazole-4-carboxylate (MPD) on
the levels of integrin b4, reactive oxygen species
(ROS), and p53 which play important roles in VECs
apoptotic signaling.
2. Results and discussion
2.1. Chemistry
Multi-substituted pyrazole derivatives 4 were synthe-
sized as outlined in Figure 1. The precursors 3 to the
multi-substituted pyrazole derivatives were easily syn-
thesized by the treatment of ethyl acetoacetate with
the appropriate aryl acid chlorides in the mixture of
aqueous solution of sodium hydroxide and petroleum
ether. The desired pyrazole derivatives 4 were obtained
by the reaction of 3 with hydrazine hydrate and phenyl
hydrazine in ethanol at refluxing condition. The struc-
2.3. Effects of compound MPD on the morphological
changes of VECs
When VECs were deprived of FGF-2 and serum, cell
growth was inhibited (p < 0.01), and the cells gradually
detached from the dish and apoptosis occurred
(Fig. 2B and E). After treatment with MPD from 5 to
100 lM for 24 and 48 h, we examined the changes of
morphology and viability of the cells. The results
R2
O
O
O
O
OH
O
O
N
N
O
O
NaOH/H2O/PE
R2NHNH2
Cl
OEt
OEt
R3
+
H3C
OEt
R4
EtOH/reflux
R1
H3C
R1
H3C
R1
R1
3
1
3'
4
2
4a: R1 = o-Cl, R2 = H, R3 = CH3, R4 = CO2Et
4b: R1 = o-NO2, R2 = H, R3 = CH3, R4 = CO2Et
4c: R1 = p-NO2, R2 = H, R3 = CH3, R4 = CO2Et
4d: R1 = o-Cl, R2 = Ph, R3 = CH3, R4 = CO2Et
4e: R1 = o-NO2, R2 = Ph, R3 = OH, R4 = H
4f: R1 = p-CH3, R2 = H, R3 = OH, R4 = H
3a: R1 = o-Cl
3b: R1 = o-NO2
3c: R1 = p-NO2
3d: R1 = p-CH3
Figure 1. Scheme for the synthesis of multi-substituted pyrazole derivatives.

B.-X. Zhao et al. / Bioorg. Med. Chem. 16 (2008) 5171–5180
5173
Figure 2. Effects of MPD on cell morphology and viability of VECs. (A and D) Cells cultured in M199 medium with serum and FGF-2 for 24 and
48 h, respectively. (B and E) Cells cultured in basal M199 for 24 and 48 h, respectively. (C and F) Cells treated with MPD at 25 lM for 24 and 48 h,
respectively. (G) Effects of MPD on viability of VECs. Normal, cells cultured in M199 medium with serum and FGF-2. Control, cells cultured in
basal M199 medium (without serum and FGF-2). 5, 10, 20, 25, 50 and 100, cells treated with MPD at 5, 10, 20, 25, 50 and 100 lM, respectively.
(*p < 0.05, **p < 0.01 vs control group, n = 3).
Figure 3. Effects of MPD on nuclear DNA fragmentation. (A) Nuclei of normal cells cultured in M199 medium with serum and FGF-2. (B) Nuclei of
control cells deprived of serum and FGF-2 for 24 h. (C) Nuclei of cells treated with MPD at 25 lM for 24 h.
showed that MPD most effectively increased the cell via-
bility at 25 lM. The morphological changes associated
with apoptosis were impaired compared with the control
(Fig. 2C, F, and G). Thus, 25 lM concentration of
MPD was used in the next experiments.
2.4. Effects of the compound MPD on nuclear DNA
condensation and fragmentation in VECs
In order to confirm the anti-apoptotic effect of the com-
pound MPD, its effects on nuclear DNA condensation

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B.-X. Zhao et al. / Bioorg. Med. Chem. 16 (2008) 5171–5180
and fragmentation in VECs were analyzed by acridine
orange (AO) staining and TUNEL assay. The results
showed that nuclear DNA condensation and fragmenta-
tion were inhibited obviously when the cells were ex-
posed to MPD at 25 lM for 24 h (Fig. 3). When the
cells were deprived of serum and FGF-2 for 24 h, the
proportion of apoptotic cells was 41.38 3.74%, which
was much higher than that in the normal group
(p < 0.01) (Fig. 4A, B, and D). After VECs were treated
with MPD at 25 lM for 24 h, the proportion of apopto-
tic cells was reduced to 19.16 5.53% (p < 0.01) (Fig. 4C
and D). The result showed that MPD could effectively
inhibit VEC apoptosis induced by deprivation of serum
and FGF-2.
in MPD-treated VECs. The immunofluorescence results
showed that the relative level of integrin b4 in the cells
deprived of serum and FGF-2 was much higher than
that in the normal cells (#p < 0.05) (Fig. 5A, B, D, E,
G, H, and J). The fluorescent intensity of the cells trea-
ted with MPD for 12, 24, and 48 h was 6.307 1.225,
9.083 1.010, 13.14 1.667, respectively. The fluores-
cent intensity of control group cells (12, 24 and 48 h)
was 11.384 1.962, 11.725 1.404, 16.331 0.858,
respectively. In the cells treated with MPD for 12 and
48 h, the relative level of integrin b4 was depressed obvi-
ously (*p < 0.05) (Fig. 5C, I, and J).
2.6. The compound MPD depressed the level of intracel-
lular ROS
Taken together, these findings encouraged us to investi-
gate the mechanism of MPD action and its modulation
on the levels of integrin b4, ROS, and p53 which play
important roles in VEC apoptotic signaling.25–27
It was shown that integrin b4 participated in regulation
of ROS level in VECs.30,31 Therefore, we checked the
effects of MPD on ROS level in VECs. In the cells of
control group, the relative fluorescent intensity of
20,70-dichlorofluorescin (DCF) was much higher than
that in the normal cells (p < 0.01) (Fig. 6A, B, D, E,
G, H, and J). But when the cells deprived of serum
and FGF-2 were treated with MPD at 25 lM, the fluo-
rescent intensity decreased significantly (**p < 0.01 and
*p < 0.05) (Fig. 6C, F, I, and J). Our results showed that
MPD could depress intracellular ROS levels induced by
deprivation of serum and FGF-2.
2.5. The compound MPD depressed the level of integrin
b4
Integrin b4 is found in hemidesmosomes, providing firm
mechanical links between the basal lamina and the inter-
mediate filament cytoskeleton system.28 It is a key mem-
brane protein in cellular signal transduction and
involved in apoptotic cell death of VECs.26,27 Thus, we
further explored the effects of MPD on integrin b4 level
to understand the mechanism of MPD action. In our
previous studies, it has been shown that integrin b4 is
a key factor involved VEC apoptotic signaling.29 In or-
der to know whether MPD can modulate this integrin
subunit, we determined the changes of integrin b4 level
2.7. The compound MPD depressed the level of p53
Integrin b4, ROS, and p53 were involved in the signal
pathway of VEC apoptosis induced by deprivation of
serum and FGF-2. The levels of p53 protein in the cells
Figure 4. Quantification of apoptotic cells by TUNEL assay. Fluorescent micrographs show the TUNEL staining of VECs. (A) Normal, cells
cultured in M199 medium with serum and FGF-2 for 24 h. (B) Control, cells cultured in basal M199 medium for 24 h. (C) Cells treated with MPD at
25 lM for 24 h. (D) The quantity of apoptotic cells (##p < 0.01 vs normal group, **p < 0.01 vs control group, n = 3).

B.-X. Zhao et al. / Bioorg. Med. Chem. 16 (2008) 5171–5180
5175
Figure 5. Effects of MPD on the level of integrin b4 in VECs. (A–I) Fluorescent micrographs show the relative intensity of integrin b4. (A, D and G)
Normal, cells cultured in M199 medium with serum and FGF-2 for 12, 24 and 48 h, respectively. (B, E and H) Control, cells deprived of serum and
FGF-2 for 12, 24 and 48 h, respectively. (C, F and I) Cells treated with MPD at 25 lM for 12, 24 and 48 h, respectively. (J) The relative quantity of
#
*
integrin b4 level in VECs ( p < 0.05 vs normal group, p < 0.05 vs control group, n = 3).
deprived of serum and FGF-2 were much higher than
that in the normal cells (p < 0.01) (Fig. 7A, B, D, E,
G, H, and J). But in the MPD-treated cells, the levels
of p53 were depressed significantly (p < 0.01) (Fig. 7C,
F, I, and J). This result showed that the increase of
p53 level induced by deprivation of serum and FGF-2
could be inhibited by MPD.
inhibition of endothelial cell apoptosis is of great impor-
tance in various vascular diseases. In the previous re-
searches, we found several kinds of chemical
compounds that can promote or inhibit VEC apoptosis.
These small molecules are very helpful tools for under-
standing the mechanisms of VEC apoptosis.20,32 Fur-
thermore, they could modulate the level of integrin b4
that is a very important integrin subunit in cellular sig-
naling. Our recent report showed that 3-benzyl-5-((2-
nitrophenoxy)methyl)-dihydrofuran-2(3H)-one (3BDO)
could inhibit VEC apoptosis and suppress integrin b4
VEC apoptosis is an important mechanism of vascular
injury, resulting in vascular leak, inflammation, coagula-
tion and atherosclerotic lesion formation. Therefore,

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B.-X. Zhao et al. / Bioorg. Med. Chem. 16 (2008) 5171–5180
Figure 6. Effects of MPD on the level of ROS in VECs. (A–I) Fluorescent micrographs show the relative intensity of ROS. (A, D and G) Normal,
cells cultured in M199 medium with serum and FGF-2 for 12, 24 and 48 h, respectively. (B, E and H) Control, cells deprived of serum and FGF-2. (C,
F and I) Cells treated with MPD at 25 lM for 12, 24 and 48 h, respectively. (D) The relative quantity of ROS level in VECs (#p < 0.01 vs normal
group, *p < 0.05 vs control group, **p < 0.01 vs control group, n = 3).
expression, but it could not depress the ROS level in-
duced by deprivation of serum and FGF-2.20 In the
present study, we found that MPD more effectively
inhibited VEC apoptosis at low concentrations than
3BDO. Moreover, it not only depressed the level of inte-
grin b4, but also suppressed the increase of ROS in-
duced by deprivation of serum and FGF-2. The data
suggested that MPD would provide much information
about the relationship between integrin b4 and ROS in
regulation of VEC apoptosis, and it was a more poten-
tial inhibitor of VEC apoptosis than 3BDO.
In the past decades, considerable progress has been made
in understanding how integrin b4 regulated cell function,
and most researches were focused on carcinoma cells.
However, the function of this integrin in normal VECs
is not well known. In our previous studies, we found that
integrin b4 was involved in VEC apoptosis and the re-
sults showed that integrin b4 mediated VEC apoptosis
through modulating the levels of ROS and p53, suggest-
ing that integrin b4, ROS, and p53 regulated VEC apop-
tosis in a cooperative manner.20,25,30 The data of the
present study support our previous reports.

B.-X. Zhao et al. / Bioorg. Med. Chem. 16 (2008) 5171–5180
5177
Figure 7. Effects of MPD on the level of p53 in VECs. (A–I) Fluorescent micrographs show the relative intensity of p53. (A, D and G) Normal, cells
cultured in M199 medium with serum and FGF-2 for 12, 24 and 48 h, respectively. (B, E and H) Control, cells deprived of serum and FGF-2 for 12,
24 and 48 h, respectively. (C, F and I) Cells treated with MPD at 25 lM for 12, 24 and 48 h, respectively. (J) The relative quantity of p53 level in
VECs (**p < 0.01 vs control group, ##p < 0.01 vs normal group, n = 3).
3. Conclusion
nism of VEC apoptosis, and MPD might be useful in the
development of new strategies in vascular disease
therapy.
In summary, multi-substituted pyrazole derivatives were
synthesized and preliminary biological evaluation in
VEC apoptosis were determined. By screening, we dis-
covered ethyl 3-(o-chlorophenyl)-5-methyl-1-phenyl-
1H-pyrazole-4-carboxylate (MPD) should be a potential
inhibitor of VEC apoptosis induced by deprivation of
serum and FGF-2. It inhibited the apoptosis through
down-regulating the levels of integrin b4, p53, and
ROS. Cell-permeable MPD with anti-apoptotic effects
in VECs offered us an exciting tool to study the mecha-
4. Experimental
4.1. General methods
Thin-layer chromatography (TLC) was conducted on
silica gel 60 F254 plates (Merck KgaA). 1H NMR spectra
were recorded on JEOL-FX-90Q instrument, using

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B.-X. Zhao et al. / Bioorg. Med. Chem. 16 (2008) 5171–5180
CDCl3 or DMSO as solvent. Melting points were deter-
mined on an XD-4 digital micro melting point appara-
tus. IR spectra were recorded with an IR
spectrophotometer Avtar 370 FT-IR (Termo Nicolet).
MS spectra were recorded on a Trace DSQ mass spec-
trograph (Thermo Finnigan).
4.3. Synthesis of multi-substituted pyrazole derivatives 4
(4a for a typical procedure)
4.3.1. Ethyl 3-(o-chlorophenyl)-5-methyl-1H-pyrazole-4-
carboxylate 4a. To a stirred solution of the Z-ethyl 2-
(hydroxy(o-chlorophenyl)methylene)-3-oxobutanoate
(5.37 g, 0.02 mol) in ethanol (250 mL), hydrazine
(0.06 mol, 80%) was added dropwise at room tempera-
ture. The reaction mixture was stirred at refluxing for
19 h. Then the mixture was concentrated under reduced
pressure, and the residue was dissolved in ethyl acetate,
washed with water, organic phase was dried over
MgSO4. After concentration, the residue was chroma-
tographied on silica gel by elution of petroleum and
ethyl acetate (1:1 V/V) to give 4a (4.3 g, yield 79%); IR
(KBr) v: 3190, 2981, 1708, 1492, 1294, 1184, 1104, 989,
4.2. General procedure for the synthesis of Z-ethyl 2-
(hydroxy(aryl)methylene)-3-oxobutanoate 3a–3d (for a
typical 3a)
To the mixture of ethyl acetoacetate (13 g, 0.1 mol) and
petroleum ether (17 mL) was added 33% (w/w) of solu-
tion of sodium hydroxide (5 mL), and the mixture was
stirred for 30 min at 0 °C. Then, 2-chlorobenzoyl chlo-
ride (13.6 mL, 0.105 mol) and 33% (w/w) of solution
of sodium hydroxide (18 mL) were added dropwise from
two funnel, respectively. After stirring for 1 h at 0 °C,
the mixture was heated to 35 °C and stirred for 1 h.
Then cooled overnight, precipitate was filtered, washed
with water and petroleum ether, dried, to afford Z-ethyl
2-(hydroxy(o-chlorophenyl)methylene)-3-oxobutanoate
3a 16.4 g (yield 61%).
763 cmꢀ1 1H NMR (CDCl3) d: 11.60 (s, 1 H, NH),
;
7.22–7.35 (m, 4H, ArH), 4.01 (q, J = 7.3 Hz, 2H,
CH2CH3), 2.01 (s, 3H, CH3), 0.96 (t, J = 7.3 Hz, 3H,
CH2CH3); MS m/z (%): 229 (M+ꢀCl, 86), 201 (100),
200 (18), 127 (24), 126 (16), 75 (16). Anal. Calcd for
C13H13ClN2O2: C, 58.99; H, 4.95; N, 10.58. Found: C,
58.96; H, 4.98; N, 10.60.
4.2.1. Z-Ethyl 2-(hydroxy(o-chlorophenyl)methylene)-3-
oxobutanoate 3a. White solid, mp 229–232 °C; IR (KBr)
v: 2980, 1681, 1620, 1600, 1409, 1390, 1342, 1282, 1086,
4.3.2. Ethyl 5-methyl-3-(o-nitrophenyl)-1H-pyrazole-4-
carboxylate 4b. Pale gray solid, yield 71%, mp 137–
141 °C; IR (KBr) v: 3194, 1685, 1606, 1527, 1350,
766 cmꢀ1
;
1H NMR (DMSO) d: 7.01–7.21 (m, 4H,
750 cmꢀ1 1H NMR (CDCl3) d: 10.70 (s, 1H, NH),
;
ArH), 3.48 (q, J = 7.2 Hz, 2H, CH2CH3), 2.07 (s, 3H,
CH3), 0.54 (t, J = 7.2 Hz, 3H, CH2CH3). Anal. Calcd
for C13H13ClO4: C, 58.11; H, 4.88. Found: C, 58.14;
H, 4.85.
7.46–8.05 (m, 4 H, ArH), 3.99 (q, J = 7.3 Hz, 2H,
CH2CH3), 2.20 (s, 3H, CH3), 1.00 (t, J = 7.3 Hz, 3H,
CH2CH3); MS m/z (%): 230 (M+ꢀOEt, 20), 229 (62),
201 (100), 184 (16), 129 (12), 103 (20), 76 (16), 42 (18).
Anal. Calcd for C13H13N3O4: C, 56.72; H, 4.76; N,
15.27. Found: C, 56.69; H, 4.79; N, 15.30.
4.2.2. Z-Ethyl 2-(hydroxy(o-nitrophenyl)methylene)-3-
oxobutanoate 3b. Pale yellow solid, yield 64%, mp
123–124 °C; IR (KBr) v: 2987, 1678, 1649, 1600,
4.3.3. Ethyl 5-methyl-3-(p-nitrophenyl)-1H-pyrazole-4-
carboxylate 4c. Yellow solid, yield 42%, mp 156–
157 °C; IR (KBr) v: 3343, 3221, 1699, 1677, 1518,
1567, 1526, 1389, 1343, 1088, 863, 706 cmꢀ1
;
1H
NMR (DMSO) d: 7.88–7.97 (m, 1H, ArH), 7.39–7.58
(m, 2H, ArH), 7.13–7.20 (m, 1H, ArH), 3.62 (q,
J = 7.3 Hz, 2H, CH2CH3), 2.17 (s, 3H, CH3), 0.64 (t,
J = 7.3 Hz, 3H, CH2CH3). Anal. Calcd for
C13H13NO6: C, 55.91; H, 4.69; N, 5.02. Found: C,
55.89; H, 4.72; N, 5.04.
1351, 854, 788 cmꢀ1 1H NMR (CDCl3) d: 10.36 (s,
;
1H, N–H), 8.25 (d, J = 8.2 Hz, 2H, ArH), 7.83 (d,
J = 8.2 Hz, 2H, ArH), 4.25 (q, J = 7.0 Hz, 2H,
CH2CH3), 2.56 (s, 3H, CH3), 1.26 (t, J = 7.0 Hz, 3H,
CH2CH3); MS m/z (%): 275 (M+, 50), 247 (30), 230
(100), 229 (24), 200 (10), 184 (45), 128 (20), 115 (13),
77 (14), 42 (9). Anal. Calcd for C13H13N3O4: C, 56.72;
H, 4.76; N, 15.27. Found: C, 56.75; H, 4.78; N, 15.25.
4.2.3. Z-Ethyl 2-(hydroxy(p-nitrophenyl)methylene)-3-
oxobutanoate 3c. Pale yellow solid, yield 95%, mp 128–
131 °C; IR (KBr) v: 3537, 3394, 2986, 1658, 1646,
1
1583, 1520, 1383, 1346, 1078, 981, 840, 712 cmꢀ1; H
4.3.4. Ethyl 3-(o-chlorophenyl)-5-methyl-1-phenyl-1H-
pyrazole-4-carboxylate 4d. White solid, yield 76%, mp
153–157 °C; IR (KBr) v: 3064, 2980, 1709, 1597, 1504,
NMR (DMSO) d: 8.08 (d, J = 8.5 Hz, 2H, ArH), 7.40
(d, J = 8.5 Hz, 2H, ArH), 3.53 (q, J = 7.3 Hz, 2H,
CH2CH3), 2.07 (s, 3H, CH3), 0.56 (t, J = 7.3 Hz, 3H,
CH2CH3). Anal. Calcd for C13H13NO6: C, 55.91; H,
4.69; N, 5.02. Found: C, 55.95; H, 4.75; N, 4.98.
1
1244, 1105, 758, 692 cmꢀ1; H NMR (CDCl3) d: 7.13–
7.30 (m, 9H, ArH), 3.95 (q, J = 7.3 Hz, 2H, CH2CH3),
2.54 (s, 3H, CH3), 0.97 (t, J = 7.3 Hz, 3H, CH2CH3);
MS m/z (%): 340 (M+, 20), 339 (76), 311 (100), 277
(9), 190 (6), 126 (5), 77 (38), 51 (9). Anal. Calcd for
C19H17ClN2O2: C, 66.96; H, 5.03; N, 8.22. Found: C,
66.98; H, 5.00; N, 8.20.
4.2.4. Z-Ethyl 2-(hydroxy(p-tolyl)methylene)-3-oxobut-
anoate 3d. White solid, yield 85%, mp 221–223 °C; IR
(KBr) v: 2977, 1696, 1657, 1598, 1410, 1234, 1067, 903,
782, 773 cmꢀ1
;
1H NMR (DMSO) d: 7.23 (d,
J = 8.3 Hz, 2H, ArH), 7.00 (d, J = 8.3 Hz, 2H, ArH),
3.50 (q, J = 7.2 Hz, 2H, CH2CH3), 2.23 (s, 3H, CH3 in
CH3C6H4), 2.01 (s, 3H, C=OCH3), 0.50 (t, J = 7.2 Hz,
3H, CH2CH3). Anal. Calcd for C14H16O4: C, 67.73; H,
6.50. Found: C, 67.69; H, 6.52.
4.3.5. 3-(o-Nitrophenyl)-1-phenyl-1H-pyrazol-5-ol 4e.
Pale yellow solid, yield 69%, mp 139–140 °C; IR (KBr)
v: 3384, 3339, 1675, 1539, 1497, 1347, 859, 767 cmꢀ1
;
1H NMR (DMSO) d: 10.36 (s, 1H, OH), 7.21–8.09 (m,
5H, ArH), 6.59–7.12 (m, 5H, ArH and pyrazole); MS

B.-X. Zhao et al. / Bioorg. Med. Chem. 16 (2008) 5171–5180
5179
m/z (%): 281 (M+, 8), 223 (12), 196 (15), 195 (100), 167
(83), 166 (45), 139 (20), 91 (18), 77 (38), 43 (32). Anal.
Calcd for C15H11N3O3: C, 64.05; H, 3.94; N, 14.94.
Found: C, 64.02; H, 3.97; N, 14.90.
iated dUTP nick-end labeling technique was used to de-
tect in situ nuclear DNA fragmentation and count
apoptosis ratio.35 The cells treated as above-mentioned
three ways for 24 h, then DNA fragmentation was de-
tected by the DeadEndTM Fluorometric TUNEL System
(Promega, USA) according to the manufacturer’s proto-
col. Samples were evaluated under the laser scanning
confocal microscope (Leica, Germany). The apoptosis
rate was quantified according to the TUNEL-positive
rate.
4.3.6. 3-p-Tolyl-1H-pyrazol-5-ol 4f. White solid, yield
85%, mp 223–226 °C; IR (KBr) v: 3123, 2585, 1627,
1
1605, 1513, 930, 826, 775 cmꢀ1; H NMR (DMSO) d:
10.60 (s, 2H, NH and OH), 7.50 (d, J = 8.2 Hz, 2H,
ArH), 7.14 (d, J = 8.2 Hz, 2H, ArH), 5.78 (s, 1H), 2.25
(s, 3H, CH3); MS m/z (%): 174 (M+, 100), 145 (5), 131
(10), 117 (45), 115 (25), 91 (15), 73 (8), 58 (15), 43
(33). Anal. Calcd for C10H10N2O: C, 68.95; H, 5.79;
N, 16.08. Found: C, 68.91; H, 5.81; N, 16.11.
4.4.6. Immunofluorescence. Immunofluorescence was per-
formed as the method of Zhao et al.36 After adding the
primary (rabbit anti-human p53, integrin b4) and second
antibodies (FITC-IgG) (Santa Cruz Biotechnology),
samples were evaluated with laser scanning confocal
microscopy (Leica, Germany). The immunofluorescence
techniques allow semiquantitative evaluation of protein
expression.37
4.4. Biological assay
4.4.1. Cell culture. Human umbilical vein endothelial
cells (HUVECs) were obtained as described previ-
ously.33 The cells were cultured on gelatin-coated plastic
dishes with M199 medium (Gibco, USA) with 20% fetal
bovine serum (FBS, Hyclone Lab Inc., USA) and
70 ng/mL FGF-2, at 37 °C in 5% CO2 and 95% air (nor-
mal group). All experiments were performed on cells
from 10 to 20 passages.
4.4.7. ROS assay. The levels of intracellular ROS were
detected in cells treated with or without MPD using a
fluorescent
probe,
20,70-dichlorodihydrofluorescin
(DCHF, Sigma, USA), which can be oxidized into fluo-
rescent 20,70-dichlorofluorescin (DCF) by intracellular
ROS. This assay is a reliable method for measuring the
intracellular ROS.38 The fluorescence was monitored
with laser scanning confocal microscopy (Leica, Ger-
many). The amount of ROS was quantified as the relative
fluorescence intensity of DCF per cell in the scanned area.
4.4.2. Exposure of VEC to compounds 4. When the cul-
tures of VEC reached sub-confluence, the culture med-
ium was replaced with basal M199 culture medium
(without FGF-2 and FBS) after once wash with the
same medium. Then the cells were treated in three ways.
(a) Cells were cultured in M199 medium with serum and
FGF-2 (normal group). (b) Cells were cultured in basal
M199 medium (control group). (c) Cells were cultured in
basal M199 medium with compounds 4 at 25–100 lM,
particularly with compound 4d at 5–100 lM (MPD
group). Compounds 4 were dissolved in dimethyl sulfox-
ide (DMSO) and applied to cells such that DMSO at the
final concentration did not affect the viability of the
cells. The morphological changes of cells were observed
with phase contrast microscope (Nikon, Japan) at 24
and 48 h, respectively.
4.4.8. Statistics analysis. The results are expressed as
means SE. Statistical analysis was done by t test,
and differences at p < 0.05 were considered statistically
significant.
Acknowledgments
This work was financially supported by the National 973
Research Project (No. 2006CB503803) and by Natural
Science Foundation of Shandong Province (Y2005B12
and Z2006D02).
4.4.3. Cell growth assay. When the cells cultured on 96-
well cell culture plate reached sub-confluence (the cells
did not contact with each other), the cells were washed
once with M199 medium and then, were cultured in
the medium without serum and FGF-2 (control group).
The cells were treated with compound 4 at 25–100 lM,
particularly with compound 4d, at 5, 10, 20, 25, 50,
and 100 lM, respectively. At 24 and 48 h, cell viability
was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-di-
phenyl-tetrazolium (MTT) assay following the proce-
dure described by Price and McMillan.34
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