Synthesis, crystal structure and biological evaluation of novel 2-(5-(hydroxymethyl)-3-phenyl-1H-pyrazol-1-yl)-1-phenylethanol derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.09.041

A series of novel 2-(5-(hydroxymethyl)-3-phenyl-1H-pyrazol-1-yl)-1- phenylethanol derivatives (4) was synthesized from ethyl 1-(2-oxo-2-phenylethyl) -3-phenyl-1H-pyrazole-5-carboxylate derivatives (3) and characterized by means of IR, ¹H NMR, HRMS and X-ray crystal diffraction. Structures of 4a, 4d, 4e and 4f were also determined by ¹³C NMR. Isomeric intermediates, 3a and 5a, were unambiguously confirmed by X-ray crystal structure analysis and successfully differentiated with ¹H NMR chemical shifts of methylene bonded to pyrazole ring. Preliminary biological evaluation showed that compounds 4d and 4e could suppress A549 lung cancer cell growth through cell cycle arrest and autophagy.

Full text of DOI:10.1016/j.ejmech.2010.09.041

European Journal of Medicinal Chemistry 45 (2010) 5792e5799
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, crystal structure and biological evaluation of novel
2-(5-(hydroxymethyl)-3-phenyl-1H-pyrazol-1-yl)-1-phenylethanol derivatives
,
,
,
b
a
b,
Liang-Wen Zheng a 1, Jian Zhu b 1, Bao-Xiang Zhao a , Yao-Hui Huang , Jun Ding , Jun-Ying Miao
*
*
a Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China
b Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 1 October 2010
A series of novel 2-(5-(hydroxymethyl)-3-phenyl-1H-pyrazol-1-yl)-1-phenylethanol derivatives (4) was
synthesized from ethyl 1-(2-oxo-2-phenylethyl)-3-phenyl-1H-pyrazole-5-carboxylate derivatives (3) and
characterized by means of IR, 1H NMR, HRMS and X-ray crystal diffraction. Structures of 4a, 4d, 4e and 4f
were also determined by 13C NMR. Isomeric intermediates, 3a and 5a, were unambiguously confirmed by
X-ray crystal structure analysis and successfully differentiated with 1H NMR chemical shifts of methylene
bonded to pyrazole ring. Preliminary biological evaluation showed that compounds 4d and 4e could
suppress A549 lung cancer cell growth through cell cycle arrest and autophagy.
Keywords:
Synthesis
Pyrazole
Crystal
A549 cell
Autophagy
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
these compounds can inhibit A549 lung cancer cell growth.
Encouraged by these findings, we struggle to modify the structure
Lung cancer is one of the most common malignant human
tumors and is also one of the leading causes of death worldwide [1].
A549 cells are the adenocarcinoma cells, so they are the best model
to study lung cancer. A key feature of cancer cells is their uncon-
trolled proliferation, thus, inhibition of proliferative pathways is
believed to be an effective strategy to fight cancer and much
attention has recently been paid to the discovery and development
of new, more selective anticancer agents [2e4].
of pyrazole derivatives in order to expand structure diversity and
screen anticancer compounds. Herein, we would like to report the
synthesis and structure characterization of 2-(5-(hydroxymethyl)-
3-phenyl-1H-pyrazol-1-yl)-1-phenylethanol derivatives as well as
preliminary biological evaluation.
2. Chemistry
The design and synthesis of new pyrazole derivatives has been
a subject of consistent interest. Pyrazole derivatives have occupied
a unique position in the drug discovery due to their broad range of
biological activities, including antiinflammatory, antiangiogenic,
antibacterial, antimicrobial, anticancer, antidepressant, antioxi-
dant, antiinfluenza and analgetic activities [5e13]. In our effort to
discover and develop apoptosis or autophagy inducers as potential
new anticancer agents, we reported a series of novel pyrazole
derivatives such as ethyl 1-(20-hydroxy-30-aroxypropyl)-3-aryl-1H-
pyrazole-5-carboxylate derivatives, 1-arylmethyl-3-aryl-1H-pyr-
azole-5-carbohydrazide derivatives, 1-arylmethyl-3-aryl-1H-pyr-
azole-5-carbohydrazide hydrazone derivatives and 3-aryl-1-(2-
(hydroxyimino)-2-arylethyl)-1H-pyrazole-5-carboxylate deriva-
tives [14e17]. The evaluation of biological activity showed that
2.1. Synthesis
The synthesis route for novel 2-(5-(hydroxymethyl)-3-phenyl-
1H-pyrazol-1-yl)-1-phenylethanol derivatives 4 is summarized in
Scheme 1. The starting material ethyl 3-phenyl-1H-pyrazole-5-
carboxylate analogue 1 was prepared following the procedure
described in our previous paper [17]. The reaction of compound 1
with 2-bromo-1-phenylethanones 2 in the presence of potassium
carbonate as the base in acetonitrile gave the mixture of isomeric
intermediates 3 and 5 (Scheme 2). After column chromatography on
silica gel, the isomers of ethyl 1-(2-oxo-2-phenylethyl)-3-phenyl-
1H-pyrazole-5-carboxylate derivatives 3 and ethyl 1-(2-oxo-2-
phenylethyl)-5-phenyl-1H-pyrazole-3-carboxylate derivatives
5
were easily isolated. The 2-(5-(hydroxymethyl)-3-phenyl-1H-pyr-
azol-1-yl)-1-phenylethanols 4 were prepared by the reduction of 3
with sodium borohydride in ethanol.
In chemistry, owning to the existence of annular tautomerism of
the 1H-pyrazole, alkylation and acylation at NeH position of
* Corresponding authors. Tel.: þ86 531 88366425; fax: þ86 531 88564464.
E-mail addresses: bxzhao@sdu.edu.cn (B.-X. Zhao), miaojy@sdu.edu.cn(J.-Y. Miao).
Both the authors contributed equally.
1
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.041

L.-W. Zheng et al. / European Journal of Medicinal Chemistry 45 (2010) 5792e5799
5793
Scheme 1. Synthetic route of compound 4.
pyrazole often results in two isomers. It should be noted that it was
difficult to distinguish structures of two isomers, though many
convenient treatments were conducted on the separation of the
two isomers. In general, it is because there is lack of specific
spectroscopic data in the literature. To our continuous interests,
isomeric intermediates of 3a and 5a can be distinguished by
comparing the chemical shifts, especially the signals of methylene
bonded to nitrogen of pyrazole moiety as described in our previous
works [18]. Thus, for example in 3a, two protons in methylene
of compound 4awas found inagreement with theassigned molecular
structure confirmed by its consistent 13C NMR spectra.
2.2. Single-crystal structure
Single crystals of 3a, 5a and 4b were all grown via slow evap-
oration of ethyl acetate solutions. The molecular views of 3a, 5a and
4b are shown in Fig.1 and Fig. 2, respectively. Details of crystal data,
data collection and refinement parameters are compiled in Table 1.
The molecular structure of 4b is shown in Fig. 2 with the atome
numbering scheme. In the crystal structure of 4b, the N2eC11 bond
has antiperiplanar orientationwith respect to the C12eC13 bond (the
N2eC11eC12eC13 torsion angle is ꢀ177.6(2)ꢁ). C11eC12 bond has
bonded to nitrogen showed single peak at
corresponding protons in 5a appeared at
¼ 5.65 ppm. Moreover, it
is clearly found protons at 4-position of pyrazole in 3a and 5a show
different chemical shifts at
d
¼ 6.07 ppm, whereas
d
d
¼ 7.25 and 6.94 ppm, respectively.
The structures of novel pyrazole derivatives 4 were determined by
IR, 1H NMR and HRMS spectroscopy. For example, in the IR spectra of
compound 4a, only hydroxyl group absorption was clearly found at
the region of 3288 cmꢀ1. In the 1H NMR spectra of 4a, two ortho-
aromatic protons signals in 3-phenyl moiety appeared at the range of
a
perpendicular orientation to the core pyrazole ring (the
N1eN2eC11eC12 torsion angle is ꢀ95.8(2)ꢁ). All the aromatic rings
are approximately planar within 0.008(2)ꢁ deviation. Moreover, the
pyrazole plane and the substituted benzene ring are almost coplanar
with a 2.14(7)ꢁ dihedral angle. Instead of coplanar, the pyrazole and
the unsubstituted benzene ring are paralleled to each other with
a dihedral angle 8.03(7)ꢁ.
d
¼ 7.39 and 7.78 ppm as triplet and doublet peaks (J ¼ 7.4 Hz),
respectively. Two hydroxyl groups show as doublet and triplet at
¼ 5.73 ppm (J ¼ 4.5 Hz) and 5.26 ppm (J ¼ 5.5 Hz), respectively. In
addition,singletsignalofpyrazoleprotonappearedat
d
Molecules of 4b feature a S(8) hydrogen bond between the
hydroxyl H2 atom and the O2 atom of the adjacent hydroxyl group
[19]. Two racemic molecules are linked by an intermolecular
O2eH1/O1 hydrogen bond between the hydroxyl groups on one
and the other. Hydroxyl atom O2 thus serves as an acceptor for
intramolecular hydrogen bond and as a donor for intermolecular
hydrogen bonds. From the packing diagram (Fig. 3), it is observed
that molecules linked into infinite chains by the O2eH1/O1
d
¼ 6.57ppm. In
the HRMS, in accordance with the molecular structure of C18H18N2O2,
compound 4a gave a [M þ H]-ion peak at m/z 295.1446. The structure
intermolecular hydrogen bond and C10eH10A/p interactions
(Table 2).
3. Pharmacology
Screening of synthesized substances was carried out using lung
cancer A549 cell line. Proliferation percentage was determined by
the SRB assay [20,21]. Cells were incubated with substances at 15,
30 and 60 mM for 48 h and the cell proliferation/viability deter-
mination using the survival percentage obtained with the cell
treated only with the solvent (DMSO at 0.1%) as reference. The
results are expressed as the average of triplicate assays. Effect
factors related to antiproliferation such as apoptosis, autophagy
and cell cycle distribution were determined.
Scheme 2. Annular tautomerism of pyrazole leads the reaction forming two isomeric
intermediates.

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L.-W. Zheng et al. / European Journal of Medicinal Chemistry 45 (2010) 5792e5799
Fig. 1. Molecular structures of 3a and 5a, with the atomenumbering scheme. Thermal ellipsoids for non-hydrogen atoms are drawn at the 30% probability level.
4. Results and discussion
group and the cells treated with the compounds 4def at 60 mM for
48 h (Data not shown). The results indicated that the compounds at
the test range of concentration did not cause necrosis in A549 cells.
Taken together, the compounds 4def did not induce apoptosis and
necrosis in A549 lung cancer cells.
4.1. Effects of the compounds on the viability of A549 lung cancer
cells
At first, we observed the morphological image of the cells
treated with compounds 4aei under a phase contrast microscope.
We found that there were no morphological changes of the cells,
4.3. Compounds 4def induced autophagic cell death in A549 cells
even if the cells treated with compounds at 60 mM for 48 h, but we
Based on the cell morphology and Hoechst assay, we observed
that the compounds 4def might not induce apoptosis of A549 lung
cancer cell. The compounds 4def might inhibit cell proliferation
through other pathways. The understanding of the mechanisms of
cell death execution and the role that they play in different diseases
opens new therapeutic strategies. Currently, increasing evidence
indicates that autophagy is a frequent cell death mechanism and
several autophagy inducers have been used as anticancer agents.
Since vacuolization, a sign of autophagy, was first observed in A549
found that the quantities of the cells treated with compounds 4def
were decreased. In order to evaluate the inhibitory effects on the
growth of A549 cells we carried out the SRB assay with the
compounds at 48 h. The data obtained by SRB assay showed that
compounds 4def had obviously inhibitory effects on the growth of
A549 cells (Fig. 4). Other compounds did not affect the growth of
A549 cells (Date not shown).
cells treated with 60 mM of 4def, we speculated that the inhibitory
4.2. Compounds 4def do not induce apoptosis and necrosis in A549
effects of these compounds on cell growth were due to autophagic
cell death. It is acceptable that LC3 is the credible marker of the
autophagosome in mammalian cells. Enhancement of the conver-
sion of LC3-I to LC3-II and upregulation of LC3 expression occurs
when autophagy is induced. Thus, induction or suppression of
autophagy can be easily monitored by examining the levels of LC3-
II by immunoblot [22]. The results showed an elevated protein level
of LC3-II in the A549 cells treated with these compounds at 48 h
(p < 0.05) (Fig. 6). Taken together, these results indicated that 4def
might inhibit A549 cell proliferation via inducing autophagic cell
death.
lung cancer cells
To detect whether compounds 4def resulted in apoptosis or
necrosis of A549 cells, we performed Hoechst 33258 staining and
LDH assay. It is well known that DNA fragmentation, chromatin
condensation, cell shrinkage, and membrane blebbing are the
characteristics of apoptotic cells. The chromatin condensation and
DNA fragmentation in the cells were determined by Hoechst 33258
staining under a fluorescence microscope. The results showed that
nuclear DNA fragmentation and chromatin condensation did not
occur in the cells treated with 60 mM of compounds 4def for 48 h
(Fig. 5). In order to determine if the growth inhibition of cells were
due to necrosis, we measured the LDH activity in cell culture
medium and the results showed that there was no significant
difference (p > 0.05) in LDH release between the cells of control
4.4. Flow cytometry analysis of cell cycle distribution
To further analyze the antiproliferative effects of compounds
4def, their effects on the cell cycle progression was next investi-
gated. It is well known that cell cycle dysregulation is a hallmark of
tumor cells. The eukaryotic cell cycle consists of alternating rounds
of DNA replication (S phase) and cell division (M phase) separated
by the gap phases G0, G1 and G2. Regulation of proteins that
mediate critical events of the cell cycle may be a useful anti-tumor
target [23,24] In order to get a more precise insight of growth
inhibition, flow cytometry analysis was taken to detect whether
compounds 4def induce cell cycle arrest. The results showed that
compounds 4d and 4e treatment with 60
phase arrest efficaciously (Fig. 7). After 48 h of treatment with
60 M 4d, 4e, the G1 population was noticeably enhanced by 24%
mM at 48 h induced G1-
m
and 22% compared with control, respectively. The increase in the
G1-phase cell population was accompanied by a decrease in the S
and G2-phase cell populations. However, compound 4f did not
Fig. 2. Molecular structure of 4b, with the atomenumbering scheme. Thermal ellip-
soids for non-hydrogen atoms are drawn at the 30% probability level.

L.-W. Zheng et al. / European Journal of Medicinal Chemistry 45 (2010) 5792e5799
5795
Table 1
Crystal data and structure refinement for compounds 3a, 5a and 4b.
Compound
3a
5a
4b
Empirical formula
Formula weight
Temperature (K)
Crystal system
Space group
a (Å)
C20H18N2O3
334.36
295(2)
Triclinic
Pe1
7.9619(8)
9.4634(9)
12.8068(13)
92.029(2)
107.9700(10)
106.004(2)
2
C20H18N2O3
334.36
295(2)
Triclinic
Pe1
9.081(4)
10.309(4)
11.253(4)
112.433(6)
91.880(7)
109.375(6)
2
C18H17ClN2O2
328.79
298(2)
Monoclinic
P21/c
10.4320(10)
18.2429(17)
8.5917(8)
90
94.249(2)
90
4
1.339
0.245
b (Å)
c (Å)
a
b
g
Z
(ꢁ)
(ꢁ)
(ꢁ)
Dx (g/cm3)
1.270
0.086
352
1.230
0.084
352
m
(mmꢀ1
F(000)
Crystal size (mm)
)
688
0.30 ꢂ 0.20 ꢂ 0.20
0.20 ꢂ 0.10 ꢂ 0.10
0.20 ꢂ 0.10 ꢂ 0.10
q
Range for data
collection (ꢁ)
2.26e26.20
1.99e25.69
2.23e27.00
Ranges of indices h, k, l
ꢀ9 ꢃ h ꢃ 9
ꢀ11 ꢃ k ꢃ 9
ꢀ15 ꢃ l ꢃ 15
98.5%
ꢀ11 ꢃ h ꢃ 10
ꢀ11 ꢃ k ꢃ 12
ꢀ13 ꢃ l ꢃ 8
98.7%
ꢀ13 ꢃ h ꢃ 12
ꢀ23 ꢃ k ꢃ 20
ꢀ10 ꢃ l ꢃ 10
99.1%
Completeness to
q
Reflections collected/unique
Data/restraints/parameters
R(int)
4793/3443
3443/0/299
0.0126
4785/3396
3396/1/226
0.0169
9245/3531
3531/0/276
0.0255
Final R indices [I > 2
s
(I)]
R1 ¼ 0.0387
wR2 ¼ 0.1133
R1 ¼ 0.0471
wR2 ¼ 0.1218
1.029
R1 ¼ 0.0538
wR2 ¼ 0.1393
R1 ¼ 0.0863
wR2 ¼ 0.1614
1.028
R1 ¼ 0.0444
wR2 ¼ 0.1168
R1 ¼ 0.0787
wR2 ¼ 0.1367
1.045
R indices (all data)
Goodness of fit on F2
Dr (max./min) [e Åꢀ3
]
0.182; ꢀ0.164
0.319; ꢀ0.214
0.248; ꢀ0.245
affect the cell cycle distribution. The fact that compounds 4d and 4e
treatment induced a strong G1-phase arrest is concomitant with
the growth inhibitory effect.
6. Experimental
6.1. General
All reagents were of analytical grade or chemically pure.
Analytical TLC was performed on silica gel 60 F254 plates (Merck
KGaA). Melting points were determined on a XDe4 digital micro
melting point apparatus and not corrected. 1H NMR spectra were
recorded on a Bruker Avance 400 (400 MHz) spectrometer, using
CDCl3 or DMSO as solvent and tetramethylsilane (TMS) as internal
standard. IR spectra were recorded with an IR spectrophotometer
Avtar 370 FT-IR (Termo Nicolet). HRMS spectra were recorded on
a LTQ Orbitrap Hybrid mass spectrograph. X-ray diffraction data
were recorded on a Bruker Smart CCD diffractometer.
5. Conclusion
In summary, a series of novel pyrazole derivatives has been
successfully synthesized. During the reaction, ester groups in the
reactants were interestingly reduced to hydroxyl groups in the
presence of sodium borohydride. Structures of all products were
determined by IR, 1H NMR and HRMS. Single crystal structures of
two isomeric intermediates 3a, 5a and product 4b were reported in
this work. Upon a series of biological evaluations, it is found that
compounds 4d and 4e suppressed A549 lung cancer cell growth
through cell cycle arrest and autophagy.
6.2. Synthesis of 2-(5-(hydroxymethyl)-3-phenyl-1H-pyrazol-1-yl)-
1-phenylethanols (4)
Into a solution of compound 3 (3.0 mmol) in ethanol (40 ml),
sodium borohydride (3.3 mmol, 126 mg) was added. The reaction
mixture was stirred and refluxed for 0.5e2 h and the reaction was
monitored by TLC on silica gel (petroleum ether and ethyl acetate,
v/v ¼ 2:1). After this time, solvent was removed under reduced
pressure. The residue was dissolved in water (40 ml) and the
mixture was neutralized using 1 M HCl (aq). The solution was
extracted with EtOAc (20 ml ꢂ 3). The organic phase was dried over
Table 2
Hydrogen bond geometries for 4b.
Compound DeH/A
DeH (Å) H/A (Å) D/A (Å) :DeH/A (ꢁ)
4b
O2eH1/O1a 0.85(3)
O1eH2/O2 1.02(3)
1.86(3)
1.72(3)
2.708(2)
2.747(2)
175(3)
176(3)
Fig. 3. Packing diagram of 4b. Hydrogen bonds and CeH/
p interaction are showed
with blue and red dashed lines, respectively (for interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article).
a
Symmetry codes: x, 1/2 ꢀ y, 1/2 þ z.

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L.-W. Zheng et al. / European Journal of Medicinal Chemistry 45 (2010) 5792e5799
MgSO4 and concentrated under reduced pressure to afford crude
product 4. Recrystallization from absolute ethanol gave pure
product 4 in 50e74% yield.
6.2.1. 2-(5-(Hydroxymethyl)-3-phenyl-1H-pyrazol-1-yl)-1-
phenylethanol (4a)
White solid, yield 74%, mp 136e139 ꢁC. IR (KBr)
n
: 3288
: 4.20 (dd, J ¼ 14.1, 4.8 Hz,
1H, CH2), 4.29 (dd, J ¼ 14.1, 8.3 Hz, 1H, CH2), 4.32 (dd, J ¼ 13.6,
(OH) cmꢀ1. 1H NMR (DMSO, 400 MHz)
d
Fig. 4. Effectsofthecompounds4defonA549cellviabilityattheconcentrationof15, 30or
60
mM or left untreated (control) for 48 h (*P < 0.05 vs control, **P < 0.01 vs control, n ¼ 3).
Fig. 5. Compounds 4d, 4e and 4f do not induces apoptosis in A549 cell at 48 h significantly. Fluorescent micrographs of Hoechst 33258 staining (200ꢂ) were taken under
a fluorescent microscope (Nikon).

L.-W. Zheng et al. / European Journal of Medicinal Chemistry 45 (2010) 5792e5799
5797
(dd, J ¼ 13.9, 4.8 Hz, 1H, CH2), 4.27 (dd, J ¼ 13.9, 8.1 Hz, 1H, CH2),
4.31 (dd, J ¼ 13.3, 5.5 Hz, 1H, CH2), 4.40 (dd, J ¼ 13.3, 5.5 Hz, 1H,
CH2), 4.98e5.02 (m, 1H, CH), 5.17 (t, J ¼ 5.5 Hz, 1H, OH), 5.65 (d,
J ¼ 4.5 Hz, 1H, OH), 6.46 (s, 1H, 4-H), 6.95 (d, J ¼ 8.7 Hz, 2H, ArH),
7.26e7.29 (m, 1H, ArH), 7.34 (s, 2H, ArH), 7.35 (s, 2H, ArH), 7.69 (d,
J ¼ 8.7 Hz, 2H, ArH). HRMS calcd for C19H21N2O3 [M þ H]þ:
325.1552, found: 325.1557.
6.2.4. 1-(4-Chlorophenyl)-2-(5-(hydroxymethyl)-3-phenyl-1H-
pyrazol-1-yl)ethanol (4d)
White solid, yield 56%, mp 126e127 ꢁC. IR (KBr)
n
: 3298
(OH) cmꢀ1. 1H NMR (DMSO, 400 MHz)
d
: 4.19 (dd, J ¼ 13.9, 5.0 Hz,
1H, CH2), 4.29 (dd, J ¼ 13.9, 8.0 Hz, 1H, CH2), 4.34 (dd, J ¼ 13.5,
5.5 Hz, 1H, CH2), 4.44 (dd, J ¼ 13.5, 5.5 Hz, 1H, CH2), 5.00e5.36 (m,
1H, CH), 5.28 (t, J ¼ 5.5 Hz, 1H, OH), 5.82 (d, J ¼ 4.6 Hz, 1H, OH), 6.57
(s, 1H, 4-H), 7.28 (t, J ¼ 7.3 Hz, 1H, ArH), 7.36 (t, J ¼ 7.3 Hz, 2H, ArH),
7.37 (d, J ¼ 7.3 Hz, 2H, ArH), 7.40 (d, J ¼ 8.4 Hz, 2H, ArH), 7.76 (d,
J ¼ 8.4 Hz, 2H, ArH). 13C NMR (CDCl3, 100 MHz)
d: 151.2,143.6, 139.2,
133.9, 132.8, 128.8 (2C), 128.7 (2C), 128.0, 127.4 (2C), 125.6 (2C),
103.2, 73.0, 56.0, 55.1. HRMS calcd for C18H18ClN2O2 [M þ H]þ:
329.1057, found: 329.1057.
Fig. 6. Compounds 4def induce autophagy in A549 cells. (A) Western blot analysis of
the protein level of LC3-II and -actin as a normalization control. A549 cells were
treated with 60 M 4d, 4e and 4f for 48 h. (B) Quantification of LC3-II protein level.
b
m
6.2.5. 1-(4-Chlorophenyl)-2-(3-(4-chlorophenyl)-5-
(*P < 0.05 vs control, n ¼ 3).
(hydroxymethyl)-1H-pyrazol-1-yl)ethanol (4e)
White solid, yield 62%, mp 160e162 ꢁC. IR (KBr)
n
: 3281
(OH) cmꢀ1. 1H NMR (DMSO, 400 MHz)
d
: 4.19 (dd, J ¼ 13.9, 4.9 Hz,
5.5 Hz, 1H, CH2), 4.42 (dd, J ¼ 13.6, 5.5 Hz, 1H, CH2), 4.98e5.03 (m,
1H, CH), 5.26 (t, J ¼ 5.5 Hz, 1H, OH), 5.73 (d, J ¼ 4.5 Hz, 1H, OH), 6.57
(s, 1H, 4-H), 7.26e7.30 (m, 2H, ArH), 7.34 (s, 2H, ArH), 7.35 (s, 2H,
ArH), 7.39 (t, J ¼ 7.4 Hz, 2H, ArH), 7.78 (d, J ¼ 7.4 Hz, 2H, ArH). 13C
1H, CH2), 4.28 (dd, J ¼ 13.9, 8.1 Hz, 1H, CH2), 4.35 (dd, J ¼ 13.5,
5.5 Hz, 1H, CH2), 4.44 (dd, J ¼ 13.5, 5.5 Hz, 1H, CH2), 4.99e5.03 (m,
1H, CH), 5.29 (t, J ¼ 5.5 Hz, 1H, OH), 5.82 (d, J ¼ 4.6 Hz, 1H, OH), 6.60
(s,1H, 4-H), 7.34 (d, J ¼ 8.5 Hz, 2H, ArH), 7.39 (d, J ¼ 8.5 Hz, 2H, ArH),
7.44 (d, J ¼ 8.5 Hz, 2H, ArH), 7.78 (d, J ¼ 8.5 Hz, 2H, ArH). 13C NMR
NMR (CDCl3, 100 MHz) d: 151.2, 143.7, 140.7, 133.0, 128.7 (4C), 128.2,
127.9, 126.0 (2C), 125.7 (2C), 103.2, 73.7, 56.2, 55.0. HRMS calcd for
C18H19N2O2 [M þ H]þ: 295.1447, found: 295.1446.
(CDCl3, 100 MHz) d: 150.1,143.8, 139.1, 134.0, 133.8, 131.4, 128.9 (2C),
128.8 (2C), 127.3 (2C), 126.9 (2C), 103.2, 73.1, 56.1, 55.1. HRMS calcd
for C18H17Cl2N2O2 [M þ H]þ: 363.0667, found: 363.0662.
6.2.2. 2-(3-(4-Chlorophenyl)-5-(hydroxymethyl)-1H-pyrazol-1-yl)-
1-phenylethanol (4b)
White solid, yield 50%, mp 161e162 ꢁC. IR (KBr)
n
: 3254
6.2.6. 1-(4-Chlorophenyl)-2-(5-(hydroxymethyl)-3-(4-
(OH) cmꢀ1. 1H NMR (DMSO, 400 MHz)
d: 4.19 (dd, J ¼ 13.9, 4.7 Hz,
methoxyphenyl)-1H-pyrazol-1-yl)ethanol (4f)
White solid, yield 52%, mp 147e148 ꢁC. IR (KBr)
n
: 3495
1H, CH2), 4.29 (dd, J ¼ 13.9, 8.4 Hz, 1H, CH2), 4.32 (dd, J ¼ 13.5,
5.5 Hz, 1H, CH2), 4.42 (dd, J ¼ 13.5, 5.5 Hz, 1H, CH2), 4.98e5.02 (m,
1H, CH), 5.26 (t, J ¼ 5.5 Hz, 1H, OH), 5.71 (d, J ¼ 4.5 Hz, 1H, OH), 6.60
(s, 1H, 4-H), 7.27e7.30 (m, 1H, ArH), 7.34 (s, 2H, ArH), 7.35 (s, 2H,
ArH), 7.45 (d, J ¼ 8.5 Hz, 2H, ArH), 7.80 (d, J ¼ 8.5 Hz, 2H, ArH).
HRMS calcd for C18H18ClN2O2 [M þ H]þ: 329.1057, found: 329.1065.
(OH) cmꢀ1 1H NMR (DMSO, 400 MHz)
. d: 3.77 (s, 3H, OCH3), 4.16
(dd, J ¼ 13.9, 5.0 Hz, 1H, CH2), 4.26 (dd, J ¼ 13.9, 8.0 Hz, 1H, CH2),
4.32 (dd, J ¼ 13.4, 5.5 Hz, 1H, CH2), 4.41 (dd, J ¼ 13.4, 5.5 Hz, 1H,
CH2), 4.98e5.03 (m, 1H, CH), 5.25 (t, J ¼ 5.5 Hz, 1H, OH), 5.80 (d,
J ¼ 4.6 Hz, 1H, OH), 6.47 (s, 1H, 4-H), 6.95 (d, J ¼ 8.7 Hz, 2H, ArH),
7.34 (d, J ¼ 8.5 Hz, 2H, ArH), 7.39 (d, J ¼ 8.5 Hz, 2H, ArH), 7.68 (d,
J ¼ 8.7 Hz, 2H, ArH). 13C NMR (CDCl3, 100 MHz)
d: 159.6, 151.0, 143.5,
6.2.3. 2-(5-(Hydroxymethyl)-3-(4-methoxyphenyl)-1H-pyrazol-1-
yl)-1-phenylethanol (4c)
139.2, 133.9, 128.8 (2C), 127.3 (2C), 126.9 (2C), 125.6, 114.1 (2C),
102.8, 73.0, 55.9, 55.3, 55.1. HRMS calcd for C19H20ClN2O3 [M þ H]þ:
359.1162, found: 359.1168.
White solid, yield 55%, mp 156e159 ꢁC. IR (KBr)
n
: 3244
(OH) cmꢀ1 1H NMR (DMSO, 400 MHz)
. d: 3.78 (s, 3H, OCH3), 4.17
Fig. 7. Effects of compounds 4d, 4e and 4f on cell cycle distribution of lung cancer A549 cells. Cells were exposed to 4d, 4e, 4f at 60 mM and incubated for 48 h, and then was stained
with PI. Values are expressed as percentage of the cell population in the G1, S, and G2-phase of cell cycle.

5798
L.-W. Zheng et al. / European Journal of Medicinal Chemistry 45 (2010) 5792e5799
6.2.7. 2-(5-(Hydroxymethyl)-3-phenyl-1H-pyrazol-1-yl)-1-
(4-methoxyphenyl)ethanol (4g)
the SRB assay. Cells were incubated with compounds at 15e60 mM
for 48 h and the cell proliferation/viability determination using the
survival percentage obtained with the cell treated only with the
solvent (DMSO at 0.1%) as reference. The results are expressed as
the average of triplicate assays.
White solid, yield 65%, mp 113e114 ꢁC. IR (KBr)
n: 3359
(OH) cmꢀ1 1H NMR (DMSO, 400 MHz)
. d: 3.74 (s, 3H, OCH3), 4.15
(dd, J ¼ 13.9, 4.8 Hz, 1H, CH2), 4.27 (dd, J ¼ 13.9, 8.3 Hz, 1H, CH2),
4.33 (dd, J ¼ 13.4, 5.5 Hz, 1H, CH2), 4.42 (dd, J ¼ 13.4, 5.5 Hz, 1H,
CH2), 4.92e4.97 (m, 1H, CH), 5.25 (t, J ¼ 5.5 Hz, 1H, OH), 5.62 (d,
J ¼ 4.4 Hz, 1H, OH), 6.56 (s, 1H, 4-H), 6.90 (d, J ¼ 8.5 Hz, 2H, ArH),
7.25 (d, J ¼ 8.6 Hz, 2H, ArH), 7.29 (t, J ¼ 8.6 Hz, 1H, ArH), 7.39 (t,
J ¼ 8.6 Hz, 2H, ArH), 7.78 (d, J ¼ 8.5 Hz, 2H, ArH). HRMS calcd for
C19H21N2O3 [M þ H]þ: 325.1552, found: 325.1555.
6.4.3. Flow cytometry analysis of cell cycle distribution
Cells were harvested and then fixed with 70% ethanol, stained
with 50 mg/mL propidium iodide (PI) containing 10 mg/mL RNase A
at 4 ꢁC for 1 h. The stained cells were analyzed using FACSCalibur
flow cytometer (BD Bioscience, USA). Cell cycle distribution was
analyzed by ModiFit software (BD Bioscience, USA).
6.2.8. 2-(3-(4-Chlorophenyl)-5-(hydroxymethyl)-1H-pyrazol-1-yl)-
1-(4-methoxyphenyl)ethanol (4h)
6.4.4. LDH assay
White solid, yield 70%, mp 119e120 ꢁC. IR (KBr)
n
: 3318
Cell culture medium was collected after 48 h treatment with
0.1% DMSO (as control) or the compound. LDH assay was performed
using Lactate Dehydrogenase (LDH) kit (Nanjing Jiancheng Co.,
China) according to the manufacturer’s instructions.
(OH) cmꢀ1. 1H NMR (CDCl3, 400 MHz)
d: 3.82 (s, 3H, OCH3), 4.26 (dd,
J ¼ 14.3, 8.8 Hz, 1H, CH2), 4.37 (dd, J ¼ 14.3, 2.8 Hz, 1H, CH2), 4.56 (d,
J ¼ 13.4 Hz, 1H, CH2), 4.61 (d, J ¼ 13.4 Hz, 1H, CH2), 5.15 (dd, J ¼ 8.8,
2.8 Hz, 1H, CH), 6.50 (s, 1H, 4-H), 6.90 (d, J ¼ 8.6 Hz, 2H, ArH), 7.32
(d, J ¼ 8.5 Hz, 2H, ArH), 7.36 (d, J ¼ 8.5 Hz, 2H, ArH), 7.71 (d,
J ¼ 8.6 Hz, 2H, ArH). HRMS calcd for C19H20ClN2O3 [M þ H]þ:
359.1162, found: 359.1176.
6.4.5. Western blot analysis
Cells were washed twice with ice cold PBS and were then lysed
in protein lysis buffer (0.5% SDS in 25 mM TriseHCl, pH 7.5, 4 mM
EDTA, 100 mM NaCl, 1 mM PMSF, 10 mg/mL leupeptin and 10 mg/mL
6.2.9. 2-(5-(Hydroxymethyl)-3-(4-methoxyphenyl)-1H-pyrazol-1-
soybean trypsin inhibitor). The protein concentration of the cells
was determined by the Bradford method [29]. After adding loading
buffer and boiling, equal amount of protein was loaded onto a 15%
SDS polyacrylamide gel. After electrophoresis, the resolved protein
was electrophoretically transferred to a Polyvinylidene Fluoride
(PVDF) membrane (Millipore, MA, USA). The membrane was
blocked with 5% non-fat milk in PBST (PBS containing 0.05% Tween
20) for 1 h at room temperature. Subsequently the membrane was
probed with Rabbit anti-human LC3b antibody, anti-p62 mouse
yl)-1-(4-methoxyphenyl)ethanol (4i)
White solid, yield 69%, mp 119e120 ꢁC. IR (KBr)
n
: 3228
(OH) cmꢀ1. 1H NMR (CDCl3, 400 MHz)
d: 3.81 (s, 3H, OCH3), 3.84 (s,
3H, OCH3), 4.23 (dd, J ¼ 14.2, 8.7 Hz, 1H, CH2), 4.38 (dd, J ¼ 14.2,
2.6 Hz, 1H, CH2), 4.53 (d, J ¼ 13.3 Hz, 1H, CH2), 4.57 (d, J ¼ 13.3 Hz,
1H, CH2), 5.14 (dd, J ¼ 8.7, 2.6 Hz, 1H, CH), 6.44 (s, 1H, 4-H), 6.89 (d,
J ¼ 8.7 Hz, 2H, ArH), 6.93 (d, J ¼ 8.6 Hz, 2H, ArH), 7.31 (d, J ¼ 8.6 Hz,
2H, ArH), 7.71 (d, J ¼ 8.7 Hz, 2H, ArH). HRMS calcd for C20H23N2O4
[M þ H]þ: 355.1658, found: 355.1648.
monoclonal antibody or anti-b-actin mouse monoclonal antibody
overnight at 4 ꢁC, and was then washed twice with PBST, each time
for 5 min. The membrane was subsequently incubated with HRP-
conjugated goat anti-rabbit IgG or polyclonal rabbit anti-mouse
immunoglobulins/HRP for 1 h at room temperature and then
washed 3 times with PBST. Then the membrane was incubated with
HRP substrate for 5 min and the fluorescence signals were detected
with X-ray films. Intensity of the protein bands was quantified
using Quantity-One software (Bio-Rad).
6.3. X-ray crystal structure determination
The X-ray data were measured on a Bruker Smart CCD diffrac-
tometer using MoK
a
(
l
¼ 0.71073 Å) at room temperature. The
collected data were integrated using SAINT software. The structures
were solved by direct methods and refined by full matrix least-
squares on F2 for all data using SHELXL97 and SHELXS97 [25].
Structure analysis was used by the PLATON [26] program. The
diagrams were generated by using ORTEP [27] and DIAMOND
version 3.2 [28].
6.4.6. Statistical analysis
Data were presented as means ꢄ SE from at least three inde-
pendent experiments and analyzed by Student’s t-test. Differences
at p < 0.05 were considered statistically significant.
Details of the X-ray data collection and final refinement
parameters including anisotropic thermal parameters and full list
of the bond lengths and angles have been deposited with the
Cambridge Crystallographic Data Center in the CIF format as
supplementary publication No.: CCDC 742567 (3a), 742566 (5a)
and 787255 (4b). Copies of these information can be obtained free
of charge from the Director, CCDC, 12 Union Road, Cambridge, CB2
1EZ, UK (Fax: þ44 1223 336033; email: deposit@ccdc.cam.ac.uk).
Acknowledgments
Thanks are due to the Science and Technology Developmental
Project of Shandong Province (2008GG10002034) and National
Natural Science Foundation of China (20972088 and 90813022) for
financial support.
6.4. Preliminary biological evaluation
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well plates or other appropriate dishes containing the medium at
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