Synthesis and discovery of pyrazole-5-carbohydrazide N-glycosides as inducer of autophagy in A549 lung cancer cells

Article abstract of DOI:10.1016/j.bmc.2009.09.004

A series of novel 3-aryl-1-arylmethyl-1H-pyrazole-5-carbohydrazide N-β-glycoside derivatives was synthesized by the reaction of substituted 1H-pyrazole-5-carbohydrazide with d-sugar and the effects of all the compounds on A549 cell growth were investigate

Full text of DOI:10.1016/j.bmc.2009.09.004

Bioorganic & Medicinal Chemistry 17 (2009) 7085–7092
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and discovery of pyrazole-5-carbohydrazide N-glycosides
as inducer of autophagy in A549 lung cancer cells
a
a
b,
*
Song Lian a, , Hua Su b, , Bao-Xiang Zhao a, , Wei-Yong Liu , Liang-Wen Zheng , 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:
Received 23 July 2009
Revised 2 September 2009
Accepted 3 September 2009
Available online 6 September 2009
A series of novel 3-aryl-1-arylmethyl-1H-pyrazole-5-carbohydrazide N-b-glycoside derivatives was syn-
thesized by the reaction of substituted 1H-pyrazole-5-carbohydrazide with -sugar and the effects of all
the compounds on A549 cell growth were investigated. The results showed that all compounds had
inhibitory effects on the growth of A549 lung cancer cells and compound 3d possessed the highest
growth inhibitory effect and induced autophagy of A549 lung cancer cells.
Ó 2009 Elsevier Ltd. All rights reserved.
D
Keywords:
Pyrazole
N-Glycoside
Synthesis
Growth inhibitory
Autophagy
1. Introduction
avoid toxic or other disadvantageous side-effects and thus, obtain
molecules with better chances for pharmacological applications. Su-
Lung cancer is one of the leading causes of death worldwide.1
Our understanding of the biology of cancer has undoubtedly im-
proved in the last decade. One characteristic of cancer cells is their
highly proliferative nature. Consequently, inhibition of prolifera-
tive pathways is considered to be an effective strategy to fight can-
cer and much attention has recently been paid to the discovery and
development of new, more selective anticancer agents.2–4
The understanding of the mechanisms of cell-death execution
and the role that they play in different diseases opens new therapeu-
tic strategies. Induction of apoptosis by anticancer agents has been
shown to correlate with tumor response. However, increasing evi-
dence is emerging for the influence of another process that regulates
the life and death of cancer cells—autophagy, an evolutionarily con-
served process that involves cellular self-eating. The fact that
autophagy can have both tumor suppressive and tumor promoting
functions makes it an interesting topic in cancer research.5–9 It is
proposed that the role and regulation of autophagy in cancer is
apparently quite complex, making autophagy a challenging, but
potentially very important, target for cancer prevention and
treatment.10
gar moieties are known to influence the pharmacokinetic properties
of the respective compounds such as absorption, distribution,
metabolism, and elimination. The incorporation of sugar into pro-
spective pharmaceutical candidates is a major strategy to obtain
activity and safety advantages.11,12 A large number of bioactive nat-
ural compounds possess b-glycosidic structures such as C-glyco-
sides,13,14 O-glycosides,15–22 and N-glycosides,23–25 frequently
containing one or more
-lyxose units.
Pyrazole derivatives have long been well known for their bio-
D-glucose, D and L-arabinose, D-xylose, and
D
logical activities including such as antitumoral,26 anti-inflamma-
tory,27 antinociceptive activity,28 antimicrobial activity.29 The
synthesis of pyrazole derivatives with sugar moiety has also re-
ceived considerable attention due to their broad spectrum of bio-
logical activities.30–40 However, a search of the literature revealed
that pyrazole-5-carbohydrazide derivatives with a carbohydrate
moiety have not been described; therefore their synthesis and bio-
logical study seemed to be an attractive task.
Our research group is interested in the synthesis and screen of
pyrazole derivatives as potential anticancer agents.41–46 Recently,
we reported the synthesis and structure–activity relationships of
novel 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide deriva-
tives as potential agents against A549 lung cancer cells. The results
showed that the carbohydrazide derivatives had more significant
growth inhibitory activity against A549 cell than the carboxylate
derivatives.47–49 Thus, it is important to extend the modification
of pyrazole derivatives and identify the interaction mechanism of
It is generally accepted that incorporation of potential biologi-
cally active moieties into body-friendly type compounds should
* Corresponding authors. Tel.: +86 531 88366425; fax: +86 531 88564464.
E-mail addresses: bxzhao@sdu.edu.cn (B.-X. Zhao), miaojy@sdu.edu.cn (J.-Y.
Miao).
Equal contribution.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.004

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S. Lian et al. / Bioorg. Med. Chem. 17 (2009) 7085–7092
small molecules with targets. Herein, we would like to report the
synthesis of 3-aryl-1-arylmethyl-1H-pyrazole-5-carbohydrazide
with sugar moiety and the findings of their biological activities in
suppressing the growth of A549 lung cancer cells by inducing
autophagy.
ety appear at 4.98 (J = 5.0 Hz), 5.06 (J = 4.5 Hz), 5.13 (J = 4.4 Hz),
respectively. Five multiplet peaks attributable to 5 protons on
the sugar ring were observed. The doublet of double signals ap-
peared at d = 5.71 with coupling constant J = 14.6 Hz and 22.5 are
consistent with methylene protons in 4-tert-butylbenzyl moiety.
Moreover, two ortho-aromatic protons signals in 4-tert-butylben-
zyl moiety appeared at the range of d = 7.19 and 7.34 ppm as dou-
blet peaks (J = 8.2 Hz). Two ortho-aromatic protons signals in 4-
chlorobenzene moiety appeared at the range of d = 7.50 and
7.76 ppm as doublet peaks (J = 8.4 Hz). A singlet signal appeared
at d = 7.35 ppm are consistent with the proton in pyrazole moiety.
2. Results and discussion
2.1. Synthesis of 3-aryl-1-arylmethyl-1H-pyrazole-5-carbohyd-
razide b-glycoside
The synthesis of 3-aryl-1-arylmethyl-1H-pyrazole-5-carbohyd-
razide b-glycoside derivatives has been accomplished as outlined
in Scheme 1 starting from 3-aryl-1-arylmethyl-1H-pyrazole-5-car-
bohydrazide (1) that can be synthesized as described in our previ-
2.2. Effects of the compounds on the morphology of A549 cells
In order to evaluate the biological effects of the compounds 3a–
h on cancer cells, we used the compounds to treat A549 lung car-
ous paper46 and
D-glucose (2a) or D-xylose (2b) in the mixture of
cinoma cells at the concentrations of 10, 20 or 40
48 h. Vacuolization were observed with A549 cells treated with
40 M 3a, 3b, 3e or 20 M 3d at 24 h, and cell number greatly de-
lM for 24 or
ethanol and acetic acid over a 2–4 h reflux period in 81.2–90.5%
yields.
l
l
The structures of obtained compounds were determined by IR,
1H NMR and HRMS mass spectroscopy. Thus, for example 1-(4-
tert-butylbenzyl)-3-(4-chlorophenyl)-N0-(3,4,5-trihydroxytetrahy-
dro-2H-pyran-2-yl)-1H-pyrazole-5-carbohydrazide (3d), obtained
as white crystal, gave a [M+H]-ion peak at m/z 515.2051 in the
HRESI-MS, in accord with the molecular formula C26H32ClN4O5.
In the IR spectra, the carbonyl group absorption in hydrazide moi-
ety was observed in the 1664 cmꢀ1 and NH bands in CONH and OH
absorptions were displayed in 3321–2220 cmꢀ1 region, respec-
tively. The 1H NMR spectra indicated the chemical shift of the
NH proton in the CONH at d = 10.27 ppm in the form of doublet
peak with coupling constant J = 5.3 Hz. Another NH proton bonded
to sugar moiety appeared at 5.93 ppm as triplet peaks with cou-
pling constant J = 4.0 Hz. The anomeric hydrogen of sugar moiety
displayed at 3.84 ppm as double doublet with coupling constant
J1 = 3.1 Hz and J2 = 8.4 Hz that is in the range resulting from an ax-
ial–axial configuration of H1 and H2 and therefore, for these sugar,
there was a b-N-glycosidic linkage. The preference for formation of
the b isomer can be rationalized in terms of the competition be-
tween substitution at the sterically least hindered position (b) ver-
sus substitution at a position which incurs stabilization from the
creased in the mean time with these cells (Fig. 1). The decrease in
cell number was even more obvious at 48 h for these cells. More-
over, vacuolization was observed with cells treated with 40 lM
3f, 3g or 3h at 48 h as well (Fig. 2).
2.3. Inhibitory effects of compounds 3a–h on the proliferation
of A549 lung cancer cells
Cell viability was analyzed by SRB assay51,52 and the results
showed that the compounds 3a–h exhibited an inhibitory effect
on the proliferation of A549 cells in a time and dose-dependent
manner (Fig. 3). Among them, 3d showed a most potent inhibitory
effect on A549 cells growth, followed by 3a, 3b, 3e and 3g accord-
ing to the IC50 values (Table 1).
2.4. Structure–activity relationships
Low IC50 values obtained for compounds 3a–g indicated that
these compounds had pronounced anti-proliferative effects on
lung cancer A549 cells. From these results we could propose that
the sugar moiety play more important role in molecule. For
example, the IC50 values of compounds 3a, 3d and 3f (15.17,
13.90 and 35.00, respectively) are lower than that of correspond-
anomeric effect (a). The substitution is driven predominantly by
sterics and the reaction is stereoselective for the b configuration.50
Three doublet signals of protons in hydroxy group on xylose moi-
Scheme 1.

S. Lian et al. / Bioorg. Med. Chem. 17 (2009) 7085–7092
7087
Figure 1. Effects of the compounds on A549 cell morphology at 24 h. A549 cells were treated with the 8 compounds (B–I: 3a–h, respectively) or left untreated (A) for 24 h at a
concentration of 40 M, except for 3d, which induced necrosis at 40 M. A concentration of 20 M was used for 3d for all the experiments except the viability assay, for at this
l
l
l
concentration no necrosis was detected by LDH assay. Microscopic photographs (400ꢁ) were taken under an inverted phase contrast microscope (Nikon).
Figure 2. Effects of the compounds on A549 cell morphology at 48 h. A549 cells were treated with the 8 compounds (B–I: 3a–h, respectively) or left untreated (A) for 48 h at a
concentration of 40
microscope (Nikon).
lM, except for 3d, for which a concentration of 20 lM was used. Microscopic photographs (400ꢁ) were taken under an inverted phase contrast
ing compounds without sugar moiety (26.55, 18.52 and 67.71,
respectively).46 In addition, it is interesting that compounds with
xylose moiety 3a, 3d and 3g alter the antitumor effect signifi-
cantly than that with glucose moiety 3b, 3e and 3h. Also,
replacement of the hydrogen at the 4-position of 3-aryl ring
with electron-donating methoxy group and chlorine altered the
cytotoxicities against the cancer cells tested, for example, com-
pound 3d with chlorine had lower IC50 values (IC50 = 13.90) than
that of compound 3g with methoxy group ((IC50 = 24.88). In con-
sistent with previous observation, the substituent at the 4-posi-
tion of 1-aryl ring can affect significantly activity. The
compounds with a bulkier tert-butyl group at the 4-position of
1-aryl ring resulted in a significant activity increasing. Taken to-
gether, the cytotoxic potency was highly dependent, as expected,
on the substitution types and patterns on the aryl ring as well as
the kind of sugar.

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S. Lian et al. / Bioorg. Med. Chem. 17 (2009) 7085–7092
Figure 3. Effects of the compounds on A549 cell viability. A549 cells were treated with the 8 compounds or left untreated (control) for 24 h (upper) or 48 h (lower) at
concentrations of 10, 20 or 40 l
M. Cell viability was analyzed by SRB assay and illustrated in column figures. Results were presented as mean SE; n = 3. *P <0.05 versus
control; **P <0.01 versus control.
Table 1
3e or 20 lM of 3d, we speculated that the inhibitory effects of
Growth inhibitory properties IC50
(
l
M) for the compounds at 48 h in A549 cells
3d 3e 3f 3g 3h
15.17 21.57 31.83 13.90 26.63 35.00 24.88 38.87
these compounds on cell growth were due to autophagic cell
death. It is acceptable that LC3 is the credible marker of the auto-
phagosome in mammalian cells. Enhancement of the conversion of
LC3-I to LC3-II and up-regulation 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.55 An alternative method for detecting the autophagic
flux is measuring p62 (SQSTM1/sequestosome 1) degradation,56
therefore we also detected the protein level of p62 in the cells.
The results showed an elevated protein level of LC3-II in the
A549 cells treated with these compounds at 48 h (p <0.05) though
the LC3-II level was not altered at 24 h and p62 protein levels sig-
nificantly (p <0.05) decreased upon drug treatment at both 24 and
48 h (Fig. 5). Taken together, these results indicated that 3a, 3b, 3d
and 3e inhibit A549 cell proliferation via inducing autophagic cell
death.
Compound 3a
IC50 M)
3b
3c
(
l
2.5. Compounds 3a, 3b, 3d and 3e induced autophagic cell
death in A549 cells
In order to determine if the growth inhibitory effects were due
to necrosis that is believed to be an unwanted side effect of cancer-
fighting agents, we performed the LDH assay and the results
showed that 40 lM of 3d at 48 h caused a significant (p <0.01) in-
crease in LDH release, indicative of necrosis induced by 3d at this
concentration. However, no increase of LDH release was detected
for the other 7 compounds, at 40
if 20 M of 3d induced necrosis and the result showed no obvious
increase of LDH release at 48 h (Fig. 4). Therefore, we used 20 M of
3d and 40 M of 3a, b, c, e, f, g and h for the following
experiments.
lM for 48 h. We further tested
l
l
3. Conclusion
l
A dose- and time-dependent inhibition of proliferation was ob-
served in A549 cells following compounds treatment and the
induction of autophagy was demonstrated by up-regulation of
LC3-II and p62 decrease. The study of structure–activity relation-
ships showed that the cytotoxic potency of the compounds was
highly dependent on the substitution types and patterns on the
aryl ring as well as the kind of sugar in which compounds with xy-
lose moiety altered the antitumor effect significantly.
The understanding of the mechanisms of cell-death execution
and the role that they play in different diseases opens new thera-
peutic strategies. Currently, increasing evidence indicates that
autophagy is a frequent cell-death mechanism and several autoph-
agy inducers have been used as anticancer agents. Although com-
plete tumor eradication has not been demonstrated, the antitumor
effect is very promising.53,54 Since vacuolization, a sign of autoph-
agy, was first observed in A549 cells treated with 40 lM of 3a, 3b,

S. Lian et al. / Bioorg. Med. Chem. 17 (2009) 7085–7092
7089
Figure 4. Effects of the compounds on LDH release in A549 cells. A549 cells were treated with the 8 compounds at the concentration of 40
untreated (control) for 48 h. Results were presented as mean SE; n = 3. **P <0.01 versus control.
lM (3a–h) or 3d at 20
lM, or left
Figure 5. Western blot to test the protein level of LC3-II and p62 upon drug treatment. A549 cells were treated with 40 lM 3a, 3b, 3e or 20 lM 3d for 24 or 48 h. The protein
levels of LC3-II and p62 were detected by Western blot analysis (A and B, respectively). The relative levels of LC3-II and p62 were normalized by the level of b-actin, and
represented as percent of control (C and D). (*P <0.05 vs control, n = 3).
monoclonal antibody (Cat. No. 610833) was purchased from BD
Bioscience (USA). Anti-b-actin mouse monoclonal antibody (sc-
47778) and HRP-conjugated goat anti-rabbit IgG (sc-2004) were
obtained from Santa Cruz Biotechnology (USA). Polyclonal rabbit
anti-mouse immunoglobulins/HRP (code No. P 0161) was provided
by DakoCytomation (USA). SuperSignal West Pico Chemilumines-
cent Substrate (HRP substrate, Cat. No. 34080) was purchased from
Thermo Scientific (USA). Sulforhodamine B (SRB) (Cat. No. S-9012)
was provided by Sigma–Aldrich (USA).
4. Experimental
4.1. Reagents and apparatus
Thin-layer chromatography (TLC) was carried out on Silica Gel
60 F254 plates (Merck KGaA). 1H NMR spectra were recorded on a
Bruker Avance 400 (400 MHz) spectrometer or a Bruker Avance
300 (300 MHz) spectrometer, using DMSO as solvent and tetra-
methylsilane (TMS) as internal standard. Melting points were
determined on an XD-4 digital micro melting point apparatus
and are uncorrected. IR spectra were recorded with an IR spectro-
photometer VERTEX 70 FT-IR (Bruker Optics). HRMS spectra were
recorded on a Q-TOF6510 spectrograph.
4.2. General procedure for the synthesis of 3-aryl-1-arylmethyl-
1H-pyrazole-5-carbohydrazide b-glycoside derivatives (3a–h)
To a suspension of glucose (2a) or xylose (2b) (0.5 mmol) in
4 mL of ethanol and one drop of glacial acetic acid was added
hydrazide (1a–e) (0.5 mmol). The reaction mixture was stirred
and heated at reflux for 2–4 h. After cooling to room temperature,
RPMI 1640 was obtained from Gibco BRL Co. (Grand Island,
USA). Bovine calf serum was provided by DingGuo Biotechnology
(Beijing, China). Rabbit anti-human LC3b antibody (Cat. No.
2775S) was purchased from Cell Signaling (USA). Anti-p62 mouse

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S. Lian et al. / Bioorg. Med. Chem. 17 (2009) 7085–7092
the precipitate was filtered to afford the corresponding product
3a–h in 81.2–90.5% yields.
4.2.5. 1-(4-tert-Butylbenzyl)-3-(4-chlorophenyl)-N0-(3,4,5-
trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-1H-
pyrazole-5-carbohydrazide (3e)
4.2.1. 1-(4-tert-Butylbenzyl)-3-phenyl-N0-(3,4,5-trihydroxy-
tetrahydro-2H-pyran-2-yl)-1H-pyrazole-5-carbohydrazide (3a)
Yield: 86.3%, White solid, mp: 205–207 °C; IR (KBr)
m: 3488,
3438, 3286 (–OH, –NH), 1668 (C@O) cmꢀ1 1H-NMR (300 MHz,
;
DMSO) d: 1.23 (s, 9H, C(CH3)3), 2.93–3.07 (m, 2H, CH2), 3.11–3.15
(m, 1H, CH), 3.19–3.27 (m, 1H, CH), 3.41–3.49 (m, 1H, CH), 3.66–
3.71 (m, 1H, CH), 3.87 (dd, 1H, J1 = 8.7 Hz, J2 = 3.0 Hz, CH), 4.34 (t,
1H, J = 5.7 Hz, 6-OH), 4.97 (d, 1H, J = 5.1 Hz, OH), 5.07 (d, 1H,
J = 4.5 Hz, OH), 5.16 (d, 1H, J = 4.2 Hz, OH), 5.70 (dd, 2H,
J1 = 24.6 Hz, J2 = 15.3 Hz, NCH2Ar), 5.95 (t, 1H, J = 3.8 Hz, NH),
7.20 (d, 2H, J = 8.4 Hz, ArH), 7.35 (d, 2H, J = 8.1 Hz, ArH), 7.35 (s,
1H, pyrazolyl-H), 7.50 (d, 2H, J = 8.4 Hz, ArH), 7.77 (d, 2H,
J = 8.4 Hz, ArH), 10.19 (d, 1H, J = 4.8 Hz, NHC@O); HRMS m/z
[M+H]+ calcd 545.2161, found 545.2169.
Yield: 85.8%, White solid, mp: 192–195 °C; IR (KBr)
m: 3435,
3269 (–OH, –NH), 1664 (C@O) cmꢀ1 1H NMR (400 MHz, DMSO)
;
d: 1.24 (s, 9H, C(CH3)3), 3.00–3.07 (m, 2H, CH2), 3.16–3.22 (m,
1H, CH), 3.23–3.29 (m, 1H, CH), 3.72 (dd, 1H, J1 = 11.2 Hz,
J2 = 5.1 Hz, CH), 3.85 (dd, 1H, J1 = 8.4 Hz, J2 = 3.3 Hz, CH), 4.94 (d,
1H, J = 4.8 Hz, OH), 5.01 (d, 1H, J = 4.6 Hz, OH), 5.10 (d, 1H,
J = 4.4 Hz, OH), 5.71 (dd, 2H, J1 = 21.2 Hz, J2 = 14.6 Hz, NCH2Ar),
5.92 (t, 1H, J = 4.1 Hz, NH), 7.19 (d, 2H, J = 8.2 Hz, ArH), 7.33 (d,
4H, J = 7.2 Hz, ArH, pyrazolyl-H), 7.43 (t, 2H, J = 7.5 Hz, ArH), 7.75
(d, 2H, J = 7.5 Hz, ArH), 10.23 (d, 1H, J = 5.3 Hz, NHC@O); HRMS
m/z [M+H]+ calcd 481.2445, found 481.2428.
4.2.6. 3-(4-Chlorophenyl)-1-((6-chloropyridin-3-yl)methyl)-N0-
(3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)-1H-pyrazole-5-carbohydrazide (3f)
4.2.2. 1-(4-tert-Butylbenzyl)-3-phenyl-N0-(3,4,5-trihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-1H-pyrazole-5-
carbohydrazide (3b)
Yield: 90.5%, White solid, mp: 223–224 °C; IR (KBr)
m: 3445,
3381, 3313 (–OH, –NH), 1664 (C@O) cmꢀ1 1H NMR (400 MHz,
;
Yield: 81.2%, White solid, mp: 227–229 °C; IR (KBr)
m
: 3446,
3384, 3345, 3271 (–OH, –NH), 1662 (C@O) cmꢀ1
;
1H NMR
DMSO) d: 3.00–3.08 (m, 2H, CH2), 3.11–3.13 (m, 1H, CH), 3.19–
3.25 (m, 1H, CH), 3.41–3.49 (m, 1H, CH), 3.67–3.71 (m, 1H,
CH), 3.88 (dd, 1H, J1 = 8.5 Hz, J2 = 2.5 Hz, CH), 4.27 (t, 1H,
J = 5.6 Hz, 6-OH), 4.90 (d, 1H, J = 5.2 Hz, OH), 4.96 (d, 1H,
J = 4.6 Hz, OH), 5.08 (d, 1H, J = 4.3 Hz, OH), 5.77 (dd, 2H,
J1 = 20.9 Hz, J2 = 15.0 Hz, NCH2Ar), 5.92 (t, 1H, J = 3.8 Hz, NH),
7.40 (s, 1H, pyrazolyl-H), 7.49 (d, 1H, J = 8.2 Hz, ArH), 7.50 (d,
2H, J = 8.5 Hz, ArH), 7.71 (dd, 1H, J1 = 8.3 Hz, J2 = 2.3 Hz, ArH),
7.77 (d, 2H, J = 8.5 Hz, ArH), 8.36 (d, 1H, J = 2.0 Hz, ArH), 10.21
(d, 1H, J = 5.1 Hz, NHC@O); HRMS m/z [M+H]+ calcd 524.1098,
found 524.1095.
(400 MHz, DMSO) d: 1.23 (s, 9H, C(CH3)3), 2.98–3.07 (m, 2H,
CH2), 3.12–3.15 (m, 1H, CH), 3.21–3.26 (m, 1H, CH), 3.43–3.49
(m, 1H, CH), 3.67–3.71 (m, 1H, CH), 3.88 (dd, 1H, J1 = 8.7 Hz,
J2 = 2.8 Hz, CH), 4.32 (t, 1H, J = 5.6 Hz, 6-OH), 4.96 (d, 1H,
J = 5.3 Hz, OH), 5.05 (d, 1H, J = 4.7 Hz, OH), 5.15 (d, 1H, J = 4.2 Hz,
OH), 5.71 (dd, 2H, J1 = 28.5 Hz, J2 = 14.6 Hz, NCH2Ar), 5.95 (t, 1H,
J = 3.7 Hz, NH), 7.20 (d, 2H, J = 8.2 Hz, ArH), 7.32–7.36 (m, 4H,
ArH, pyrazolyl-H), 7.43 (t, 2H, J = 7.6 Hz, ArH), 7.76 (d, 2H,
J = 7.4 Hz, ArH), 10.17 (d, 1H, J = 5.0 Hz, NHC@O); HRMS m/z
[M+H]+ calcd 511.2551, found 511.2549.
4.2.7. 1-(4-tert-Butylbenzyl)-3-(4-methoxyphenyl)-N0-(3,4,5-
trihydroxytetrahydro-2H-pyran-2-yl)-1H-pyrazole-5-
carbohydrazide (3g)
4.2.3. 1-Benzyl-3-(4-chlorophenyl)-N0-(3,4,5-trihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-1H-pyrazole-5-
carbohydrazide (3c)
Yield: 90.2%, White solid, mp: 200–201 °C; IR (KBr)
m: 3451,
Yield: 90.1%, White solid, mp: 224–228 °C; IR (KBr)
m
: 3444,
3296, 3238 (–OH, –NH), 1665 (C@O) cmꢀ1 1H NMR (300 MHz,
;
3388, 3299, 3266 (–OH, –NH), 1665 (C@O) cmꢀ1
;
1H NMR
DMSO) d: 1.23 (s, 9H, C(CH3)3), 2.98–3.07 (m, 2H, CH2), 3.15–
3.32 (m, 2H, CH), 3.71 (dd, 1H, J1 = 11.0 Hz, J2 = 5.0 Hz, CH),
3.78 (s, 3H, OCH3), 3.84 (dd, 1H, J1 = 8.6 Hz, J2 = 3.2 Hz, CH),
5.01 (d, 1H, J = 4.8 Hz, OH), 5.10 (d, 1H, J = 4.5 Hz, OH), 5.17 (d,
1H, J = 4.2 Hz, OH), 5.69 (dd, 2H, J1 = 19.8 Hz, J2 = 14.7 Hz,
NCH2Ar), 5.94 (t, 1H, J = 4.2 Hz, NH), 6.99 (d, 2H, J = 8.7 Hz,
ArH), 7.18 (d, 2H, J = 8.4 Hz, ArH), 7.26 (s, 1H, pyrazolyl-H),
7.34 (d, 2H, J = 8.1 Hz, ArH), 7.67 (d, 2H, J = 9.0 Hz, ArH), 10.25
(d, 1H, J = 5.1 Hz, NHC@O); HRMS m/z [M+H]+ calcd 511.2551,
found 511.2543.
(400 MHz, DMSO) d: 3.02–3.07 (m, 2H, CH2), 3.11–3.15 (m, 1H,
CH), 3.20–3.26 (m, 1H, CH), 3.43–3.49 (m, 1H, CH), 3.67–3.72 (m,
1H, CH), 3.87 (dd, 1H, J1 = 8.7 Hz, J2 = 2.9 Hz, CH), 4.27 (t, 1H,
J = 5.7 Hz, 6-OH), 4.91 (d, 1H, J = 5.2 Hz, OH), 4.97 (d, 1H,
J = 4.8 Hz, OH), 5.08 (d, 1H, J = 4.3 Hz, OH), 5.75 (dd, 2H,
J1 = 26.4 Hz, J2 = 14.7 Hz, NCH2Ar), 5.92 (t, 1H, J = 3.9 Hz, NH),
7.26 (t, 3H, J = 7.0 Hz, ArH), 7.33 (t, 2H, J = 7.4 Hz, ArH), 7.37 (s,
1H, pyrazolyl-H), 7.50 (d, 2H, J = 8.4 Hz, ArH), 7.78 (d, 2H,
J = 8.6 Hz, ArH), 10.15 (d, 1H, J = 5.0 Hz, NHC@O); HRMS m/z
[M+H]+ calcd 489.1535, found 489.1519.
4.2.4. 1-(4-tert-Butylbenzyl)-3-(4-chlorophenyl)-N0-(3,4,5-tri-
hydroxytetrahydro-2H-pyran-2-yl)-1H-pyrazole-5-carbo-
hydrazide (3d)
4.2.8. 1-(4-tert-Butylbenzyl)-3-(4-methoxyphenyl)-N0-(3,4,5-
trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-1H-
pyrazole-5-carbohydrazide (3h)
Yield: 81.6%, White solid, mp: 192–194 °C; IR (KBr)
m
: 3445,
Yield: 90%, White solid, mp: 215–216 °C; IR (KBr)
m: 3429, 3288
3321, 3280, 3220 (–OH, –NH), 1664 (C@O) cmꢀ1
;
1H NMR
(–OH, –NH), 1667 (C@O) cmꢀ1 1H NMR (400 MHz, DMSO) d: 1.23
;
(400 MHz, DMSO) d: 1.23 (s, 9H, C(CH3)3), 3.00–3.06 (m, 2H,
CH2), 3.16–3.21 (m, 1H, CH), 3.25–3.30 (m, 1H, CH), 3.71 (dd, 1H,
J1 = 11.2 Hz, J2 = 5.1 Hz, CH), 3.84 (dd, 1H, J1 = 8.4 Hz, J2 = 3.1 Hz,
CH), 4.98 (d, 1H, J = 5.0 Hz, OH), 5.06 (d, 1H, J = 4.5 Hz, OH), 5.13
(d, 1H, J = 4.4 Hz, OH), 5.71 (dd, 2H, J1 = 22.5 Hz, J2 = 14.6 Hz,
NCH2Ar), 5.94 (t, 1H, J = 4.1 Hz, NH), 7.19 (d, 2H, J = 8.2 Hz, ArH),
7.34 (d, 2H, J = 8.2 Hz, ArH), 7.35 (s, 1H, pyrazolyl-H), 7.50 (d, 2H,
J = 8.4 Hz, ArH), 7.76 (d, 2H, J = 8.4 Hz, ArH), 10.27 (d, 1H,
J = 5.3 Hz, NHC@O); HRMS m/z [M+H]+ calcd 515.2061, found
515.2051.
(s, 9H, C(CH3)3), 2.98–3.07 (m, 2H, CH2), 3.11–3.15 (m, 1H, CH),
3.20–3.26 (m, 1H, CH), 3.43–3.49 (m, 1H, CH), 3.66–3.71 (m, 1H,
CH), 3.78 (s, 3H, OCH3), 3.87 (dd, 1H, J1 = 8.7 Hz, J2 = 2.9 Hz, CH),
4.26 (t, 1H, J = 5.6 Hz, 6-OH), 4.90 (d, 1H, J = 5.2 Hz, OH), 4.97 (d,
1H, J = 4.8 Hz, OH), 5.10 (d, 1H, J = 4.2 Hz, OH), 5.67 (dd, 2H,
J1 = 26.7 Hz, J2 = 14.6 Hz, NCH2Ar), 5.91 (t, 1H, J = 3.9 Hz, NH),
6.99 (d, 2H, J = 8.8 Hz, ArH), 7.19 (d, 2H, J = 8.4 Hz, ArH), 7.24 (s,
1H, pyrazolyl-H), 7.34 (d, 2H, J = 8.4 Hz, ArH), 7.68 (d, 2H,
J = 8.7 Hz, ArH), 10.09 (d, 1H, J = 5.0 Hz, NHC@O); HRMS m/z
[M+H]+ calcd 541.2657, found 541.2669.

S. Lian et al. / Bioorg. Med. Chem. 17 (2009) 7085–7092
7091
4.3. Cell culture
2007GG20002004) and National Natural Science Foundation of
China (90813022).
A549 lung cancer cells were cultured in RPMI 1640 medium at
37 °C with 5% CO2, and 95% air, supplemented with 10% (v/v) bo-
vine calf serum and 80 U/ml gentamicin. The cells were seeded
onto 96-well plates or other appropriate dishes containing the
medium at the density of 6250/cm2.
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This study was supported by the Science and Technology Devel-
opmental Project of Shandong Province (2008GG10002034 and

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