Synthesis and discovery of autophagy inducers for A549 and H460 lung cancer cells, novel 1-(2′-hydroxy-3′-aroxypropyl)-3-aryl-1H-pyrazole-5-carbohydr azide derivatives

Article abstract of DOI:10.1016/j.bmcl.2008.06.058

A series of novel 1-(2′-hydroxy-3′-aroxypropyl)-3-aryl-1H-pyrazole-5-carbohydr azide derivatives were synthesized, and the effects of the compounds on A549 cell growth were investigated. The results showed that all of the 1-(2′-hydroxy-3′-aroxypropyl)-3-aryl-1H-pyrazole-5-carbohydr azide derivatives 2 could inhibit the growth of A549 cells in dosage- and time-dependent manners. Typically, compound 2a and 2d induced A549 cells to autophagy but did not cause apoptosis and necrosis in the cells, and 2d had the most autophagy inducing effect in H460 cells. More importantly, 2a and 2d did not inhibit the growth of HUVEC cells.

Full text of DOI:10.1016/j.bmcl.2008.06.058

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3860–3864
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and discovery of autophagy inducers for A549 and H460 lung
cancer cells, novel 1-(20-hydroxy-30-aroxypropyl)-3-aryl-1H-pyrazole-5-carbo-
hydrazide derivatives
b
a
c
a,
Chuan-Dong Fan a, Bao-Xiang Zhao b, , Fang Wei , Gai-Hua Zhang , Wen-Liang Dong , Jun-Ying Miao
*
*
a Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, China
b Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China
c Shandong University of Traditional Chinese Medicine, Jinan 250355, China
a r t i c l e i n f o
a b s t r a c t
A series of novel 1-(20-hydroxy-30-aroxypropyl)-3-aryl-1H-pyrazole-5-carbohydrazide derivatives were
synthesized, and the effects of the compounds on A549 cell growth were investigated. The results showed
that all of the 1-(20-hydroxy-30-aroxypropyl)-3-aryl-1H-pyrazole-5-carbohydrazide derivatives 2 could
inhibit the growth of A549 cells in dosage- and time-dependent manners. Typically, compound 2a and
2d induced A549 cells to autophagy but did not cause apoptosis and necrosis in the cells, and 2d had
the most autophagy inducing effect in H460 cells. More importantly, 2a and 2d did not inhibit the growth
of HUVEC cells.
Article history:
Received 25 January 2008
Revised 20 May 2008
Accepted 17 June 2008
Available online 20 June 2008
Keywords:
Pyrazole carbohydrazide
A549 cells
Ó 2008 Elsevier Ltd. All rights reserved.
H460 cells
Autophagy
Cancer is the second leading cause of death in developed
countries, accounting for nearly one in five deaths. Lung cancer
is one of the leading causes of death in the world. Since current
treatment modalities are inadequate, novel therapies are needed
to reduce the effects of the increasing incidence in pulmonary
neoplasm.1,2 An elaborate search of new anticancer agents has
primarily been triggered by the unveiling of new molecular tar-
gets on which they intervene, followed by the discovery of novel
classes of compounds that interact with such targets.3,4 In our
effort to discover and develop apoptosis inducers as potential
new anticancer agents, we have identified several classes of mol-
ecules as novel apoptosis inducers, including safrole oxide, 1-alk-
organelles. Recently, interest in autophagy has been renewed
among oncologists, because different types of cancer cells undergo
autophagy after various anticancer therapies.18–22
Many pyrazole derivatives are known to exhibit a wide range of
biological properties such as anti-hyperglycemic, analgesic, anti-
inflammatory, anti-pyretic, anti-bacterial, hypoglycemic, seda-
tive–hypnotic activity, and anticoagulant activity.23–25 Extensive
studies have been devoted to arylpyrazole derivatives such as
Celecoxib, a well-known cyclooxygenase-2 inhibitor.26–28 In the
previous paper, we investigated the effects of ethyl 1-(20-hydro-
xy-30-aroxypropyl)-3-aryl-1H-pyrazole-5-carboxylate derivatives
on the growth of A549 lung cancer cell and found that these com-
pounds could suppress A549 lung cancer cell growth.29 Recently,
we described the synthesis and preliminary biological evaluation
of ethyl 1-arylmethyl-3-aryl-1H-pyrazole-5-carboxylate deriva-
tives.30 The modification of pyrazole such as basic substituent moi-
ety should provide potential bioactivities. A few of heterocyclic
carbohydrazide derivatives have been reported.31 Recently, we
reported the synthesis and structure–activity relationships of no-
vel 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide derivatives
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.32
oxy-3-(30,40-methylenedioxy)phenyl-2-propanol,
morpholinone
derivatives, and 2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine
derivatives.5–17
Induction of apoptosis by anticancer agents has been shown to
correlate with tumor response; however, non-apoptotic forms of
cell death, such as autophagy and extrinsic senescence, have also
been shown to contribute to the overall tumor response. Non-
apoptotic forms of programmed cell death are targets for novel ap-
proaches in anticancer therapy. Autophagy is a process in which
subcellular membranes undergo dynamic morphological changes
that lead to the degradation of cellular proteins and cytoplasmic
Thus, it is important to expand the modification of pyrazole
derivatives and identify the interaction mechanism of small
molecules with targets. Herein, we would like to report the synthe-
* Corresponding authors. Tel.: +86 531 88364929; fax: +86 531 88564464.
E-mail addresses: bxzhao@sdu.edu.cn, sduzhao@hotmail.com (B.-X. Zhao),
miaojy@sdu.edu.cn (J.-Y. Miao).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.058

C.-D. Fan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3860–3864
3861
sis of 1-(20-hydroxy-30-aroxypropyl)-3-aryl-1H-pyrazole-5-carbo-
hydrazide and the findings of their biological activities in suppress-
ing the growth of A549 lung cancer cells by inducing autophagy.
Chemistry. The synthesis of 1-(20-hydroxy-30-aroxypropyl)-3-
aryl-1H-pyrazole-5-carbohydrazide derivatives has been accom-
plished as outlined in Scheme 1 starting from ethyl 1-(20-hydro-
xy-30-aroxypropyl)-3-aryl-1H-pyrazole-5-carboxylate (1) that can
be synthesized as described in our previous paper.29 For example,
1-(30-(40-chlorophenoxy)-2-hydroxypropyl)-3-(40-chlorophenyl)-
1H-pyrazole-5-carbohydrazide (2d) was synthesized in 86% yield
by the reaction of ethyl 1-(30-(40-chlorophenoxy)-2-hydroxypro-
pyl)-3-(40-chlorophenyl)-1H-pyrazole-5-carboxylate with hydra-
zine hydrate in methanol over a 4 h reflux period. The structures
of 2a–2h were determined by IR, 1H NMR and mass spectroscopy.33
Thus, for example, 2d, obtained as white crystal, gave a [M+H]-ion
peak at m/z 422.4 in the ESI-MS, in accord with the molecular for-
mula C19H18Cl2N4O3. The carbonyl group absorption in hydrazide
moiety was observed in the 1678 cmꢀ1 in IR spectra. The 1H
NMR (DMSO) spectra indicated two ortho-aromatic protons signals
in p-Cl phenol moiety appeared at the range of d = 6.87 and
7.27 ppm as doublet peaks (J = 9.0 Hz). Two ortho-aromatic protons
signals in p-Cl phenyl moiety appeared at the range of d = 7.52 and
7.63 ppm as doublet peaks (J = 8.4 Hz). One proton signal in pyra-
zole moiety appeared at d = 6.85 ppm. Two double doublet signals
appeared at d = 3.92 and 4.03 ppm are consistent with two methy-
lene protons, respectively. Three protons appeared at the range of
4.26–4.37 ppm as multilet peaks. The signals of hydroxyl and NH
appeared at d = 5.55 and 9.98 ppm, respectively. The element anal-
ysis shows satisfactory accord.
influence the normal somatic cell growth and have no toxicity to
normal cells. Interestingly, compound 2d at 10 M promotes HU-
VEC cell growth at 24 h. Taken together, compound 2d was the
most effective compound in suppressing A549 cell growth. The
growth inhibitory properties (IC50) for the compounds 2a–2h are
listed in Table 1.
Effects of the compounds on the morphology of A549 cells. Mor-
phological changes are associated with the physiological and path-
ological processes in A549 cells. After A549 cells were treated with
l
compounds 2a and 2d at 10–80 lM for 24 and 48 h, the cells vac-
uolated gradually as the increase of the concentration and the pro-
longation of the time. Compounds 2b, 2c, and 2e–2h had a similar
but inferior vacuolating effect in A549 cells compared to 2a and 2d
(data not shown). The obviously morphological changes of the cells
treated with compounds 2a and 2d at 80 lM for 24 and 48 h were
observed under a contrast phase microscope, as shown in Figure 2.
Such morphological changes were not apparent in the control cells.
The results showed that the autophagy should take place. It was
confirmed by the analysis of nuclear and acidic compartment mor-
phology as well as LDH activity assay.
Effect of the compounds on nuclear and acidic compartment mor-
phology. DNA fragmentation, chromatin condensation, cell shrink-
age, and membrane blebbing are the characteristics of apoptotic
cells. To detect whether compounds induce apoptosis in A549
and H460 lung cancer cells, we performed acridine orange (AO)
staining under an Olympus BH-2 fluorescence microscope. The
morphology revealed the appearance of large membranous vacu-
oles in the cytoplasm, but the nuclei of these cells appeared normal
with minimum evidence of chromatin condensation. The forma-
tion of these membranous vacuoles increased progressively with
increasing compound exposure time (Fig. 3). The results suggested
that the compounds did not induce apoptosis in A549 and H460
cells. Autophagy is the process of sequestering cytoplasmic pro-
teins into the lytic compartment of the cell, and it is characterized
by the formation of acidic vesicular organelles. In acridine orange-
stained cells, the cytoplasm and nucleolus fluoresce bright green,
whereas acidic compartments fluoresce bright red. Therefore, acri-
dine orange-staining has been accepted as a marker for autoph-
agy.20,34 Thus, the results showed there were increased acidic
vacuoles in the cells treated with compounds 2a and 2d compared
to the untreated cells. Other compounds had a similar but inferior
effect compared to 2a and 2d (data not shown). Such morphologi-
cal changes were not apparent in the control cells. Therefore, the
compounds 2a and 2d might induce autophagy in A549 and
H460 cells.
Effects of the compounds on the viability of A549 lung cancer cells
and HUVEC cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) cell proliferation assay is widely accepted
as a reliable way to measure the cell proliferation rate, and con-
versely when metabolic events lead to apoptosis or necrosis. The
data obtained by MTT assay showed that compounds 2a–2h had
inhibitory effects on the growth of A549 cells in dosage- and
time-dependent manners as indicated by the results shown in
Figure 1. As typically shown in Figure 1, exposure of cells to 2a,
2d, 2e, and 2g at 40 lM for 24 h resulted in cell viability decrease
from 100% to 72–62% (p < 0.05). When the exposure continued on
to 48 h, compared with the control group, the cell viability reduced
more significantly from 100% to 56–40% (p < 0.01). Further, expo-
sure of cells to 2a, 2d, and 2e at 80 lM for 48 and 96 h, the cell via-
bility reduced more significantly from 100% to 29% and 6%,
respectively (p < 0.01). As shown in Figure 1B, 2a and 2d did not in-
hibit the growth of HUVEC (human umbilical vein endothelial
cells), as normal somatic cells, for 24 h and 48 h. They might not
Effect of the compounds on LDH release in A549 cells. To detect
whether compounds resulted in necrosis of A549 cells, LDH activ-
Scheme 1. Synthesis of 1-(20-hydroxy-30-aroxypropyl)-3-aryl-1H-pyrazole-5-carbohydrazide.

3862
C.-D. Fan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3860–3864
Figure 1. Effects of the compounds on the viability of A549 lung cancer and HUVEC cells. (A) Effects of the compounds 2a–2h on the viability of A549 lung cancer cells. The
viability of the cells treated with the compounds at the concentrations indicated for 24, 48, and 96 h, respectively. (B) Effects of compounds 2a and 2d on the viability of
HUVEC cells for 24 and 48 h. Data are means SE from three independent experiments. (*P < 0.05 and **P < 0.01 vs the DMSO group.)
tween the cells of control group and the cells treated with the com-
pounds at 80 M for 24 and 48 h. The results indicated that the
Table 1
Growth inhibitory properties (IC50) for the compounds 2a–h at 48 and 96 h
l
compounds at the test range of concentration did not cause necro-
sis in A549 cells (Fig. 4) and in H460 cells (data not shown). Thus,
our results suggested that the compounds 2a–2h induced A549
and H460 cells to autophagy, among them 2a and 2d had more ef-
fect in A549 cells, and 2d had the most effect in H460 cells.
In summary, all of the 1-(20-hydroxy-30-aroxypropyl)-3-aryl-
Compound
2a
2b
2c
2d
2e
2f
2g
2h
96 h (
48 h (
l
l
M)
M)
30
48
45
72
49
72
27
32
37
39
40
74
35
39
51
70
ity in cell culture medium was measured. As shown in Figure 4,
there was no significant difference (p > 0.05) in LDH release be-
1H-pyrazole-5-carbohydrazide derivatives 2 could inhibit the

C.-D. Fan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3860–3864
3863
Figure 2. Morphology image of A549 cells treated with the compounds 2a and 2d (80
(600ꢁ).
lM) for 24 and 48 h. Control: the cells treated with DMSO 0.1% (v/v) as a vehicle control
Figure 3. Determination of autophagy in A549 (A) and H460 (B) cells by acridine orange-staining. Control: the cells treated with DMSO 0.1% (v/v) as a vehicle control; 2a and
2d: the cells treated with the compounds 2a and 2d (80
M) for 24 and 48 h, respectively (400ꢁ).
l

3864
C.-D. Fan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3860–3864
21. Levine, B. Nature 2007, 446, 745.
22. Karantza-Wadsworth, V.; White, E. Autophagy 2007, 3, 610.
23. Cottineau, B.; Toto, P.; Marot, C.; Pipaud, A.; Chenault, J. Bioorg. Med. Chem. Lett.
2002, 12, 2105.
24. Lee, K. Y.; Kim, J. M.; Kim, J. N. Tetrahedron Lett. 2003, 44, 6737.
25. Jia, Z. J.; Wu, Y.; Huang, W.; Zhang, P.; Song, Y.; Woolfrey, J.; Sinha, U.;
Arfsten, A. E.; Edwards, S. T.; Hutchaleelaha, A.; Hollennbach, S. J.;
Lambing, J. L.; Scarborough, R. M.; Zhu, B.-Y. Bioorg. Med. Chem. Lett. 2004,
14, 1229.
26. Hashimoto, H.; Imamura, K.; Haruta, J.-I.; Wakitani, K. J. Med. Chem. 2002, 45,
1511.
27. Ranatunge, R. R.; Earl, R. A.; Garvey, D. S.; Janero, D. R.; Letts, L. G.; Martino, A.
M.; Murty, M. G.; Richardson, S. K.; Schwalb, D. J.; Young, D. V.; Zemtseva, I. S.
Bioorg. Med. Chem. Lett. 2004, 14, 6049.
28. Sakya, S. M.; DeMello, K. M. L.; Minich, M. L.; Rast, B.; Shavnya, A.; Rafka, R. J.;
Koss, D. A.; Cheng, H.; Li, J.; Jaynes, B. H.; Ziegler, C. B.; Mann, D. W.; Petras, C.
F.; Seibel, S. B.; Silvia, A. M.; George, D. M.; Lund, L. A.; St. Denis, S.; Hickman,
A.; Haven, M. L.; Lynch, M. P. Bioorg. Med. Chem. Lett. 2006, 16, 288.
29. Wei, F.; Zhao, B.-X.; Huang, B.; Zhang, L.; Sun, C.-H.; Dong, W.-L.; Shin, D.-S.;
Miao, J.-Y. Bioorg. Med. Chem. Lett. 2006, 16, 6342.
30. Ding, X-L.; Meng, N.; Xia, Y.; Wang, L.-J.; Zhang, X.-F.; Zhao, B.-X.; Miao, J.-Y.
Chin. J. Org. Chem. 2007, 27, 1542.
31. Elliott, J. M.; Carling, R. M.; Chambers, M.; Chicchi, G. G.; Hutson, P. H.; Jones, A.
B.; MacLeod, R.; Meneses-Lorente, G.; Murray, F.; Rigby, M.; Royo, I.; Russell, M.
G. N.; Sohal, B.; Tsaoc, K.; Williamsa, B. Bioorg. Med. Chem. Lett. 2006, 16, 5748.
32. Xia, Y.; Dong, Z.-W.; Zhao, B.-X.; Ge, X.; Meng, N.; Shin, D.-S.; Miao, J.-Y. Bioorg.
Med. Chem. 2007, 15, 6893.
33. Spectral data of compounds 2a–2h.
1-(30-(40-Chlorophenoxy)-20-hydroxypropyl)-3-phenyl-1H-pyrazole-5-carbo-
hydrazide (2a): Yield 58%, white solid. mp 148–150 °C; IR (KBr)
m: 3476–
3219 (OH, NH, NH2), 1675 (C@O) cmꢀ1 1H NMR (300 MHz, CDCl3) d: 3.80
;
(dd, 1H, J = 6.3, 9.6 Hz), 3.93 (dd, 1H, J = 4.5, 9.6 Hz), 4.08 (br s, 2H, NH2),
4.31 (dd, 1H, J = 7.2, 13.8 Hz), 4.37 (dd, 1H, J = 4.2, 13.8 Hz), 4.43–4.46 (m,
1H), 4.92 (br s, 1H, OH), 6.69 (d, 2H, J = 6.9 Hz), 6.81 (s, 1H), 7.20 (d, 2H,
J = 6.9 Hz), 7.39–7.45 (m, 5H, ArH), 8.75 (br s, 1H, NH); ESI-MS: 387.4
(M+H)+.
Figure 4. Effects of compounds 2a–2h on LDH activities of A549 cells. The culture
medium was collected as samples for LDH assay after 24 and 48 h treatment at the
concentration of 80 lM. (p > 0.05 vs control group, n = 3.)
1-(20-Hydroxy-30-(40-nitrophenoxy)propyl)-3-phenyl-1H-pyrazole-5-carbo-
growth of A549 cells in dosage- and time-dependent manners, typ-
ically compounds 2a and 2d induced A549 cells to autophagy but
did not cause apoptosis and necrosis in the cells, and 2d had the
most autophagy inducing effect in H460 cells. More importantly,
2a and 2d did not inhibit the growth of HUVEC cells.
hydrazide (2b): Yield 56%, white solid. mp 171–174 °C; IR (KBr)
m
: 3450–
3286 (OH, NH, NH2), 1670 (C@O), 1517 and 1254 (NO2) cmꢀ1
;
1H NMR
(300 MHz, CDCl3/DMSO-d6) d: 4.02–4.03 (m, 2H, CH2), 4.30–4.41 (m, 2H,
CH2), 4.43–4.51 (m, 1H, CH), 5.53 (br s, 1H, OH), 6.76 (s, 1H), 6.91 (d, 2H,
J = 9.3 Hz), 7.42–7.46 (m, 5H, ArH), 8.15 (d, 2H, J = 9.3 Hz), 9.18 (br s, 1H,
NH); ESI-MS: 398.4 (M+H)+.
1-(20-Hydroxy-30-(20-methoxyphenoxy)propyl)-3-phenyl-1H-pyrazole-5-
carbohydrazide (2c): Yield 55%, oil. IR (film)
m: 3492–3180 (OH, NH, NH2), 1681
Acknowledgment
(C@O) cmꢀ1 1H NMR (300 MHz, CDCl3) d: 3.77 (s, 3H, OCH3), 3.93–4.04 (m, 2H,
;
CH2), 4.40–4.41 (m, 2H, CH2), 4.46–4.52 (m, 1H, CH), 6.82–7.00 (m, 5H, ArH and
pyrazole moiety), 7.42 (m, 5H, ArH), 9.46 (br s, 1H, NH); ESI-MS: 383.5
(M+H)+.1-(30-(40-Chlorophenoxy)-20-hydroxypropyl)-3-(40-chlorophenyl)-1H-
pyrazole-5-carbo hydrazide (2d): Yield 86%, white solid. mp 166–168 °C; IR
This study was supported by the Natural Science Foundation of
Shandong Province (Z2006D02).
(KBr) m ;
: 3353–3090 (NH, NH2), 1678 (C@O) cmꢀ1 1H NMR (300 MHz, DMSO) d:
References and notes
3.92 (dd, 1H,J = 4.5, 13.9 Hz, CH2), 4.03 (dd, 1H, J = 6.9, 13.9 Hz, CH2), 4.26-4.37
(m, 3H, CH2, CH), 5.55 (d, 1H, J = 4.8 Hz, OH), 6.85 (s, 1H), 6.87 (d, 2H, J = 9.0 Hz,
ArH), 7.27 (d, 2H, J = 9.0 Hz, ArH), 7.52 (d, 2H, J = 8.4 Hz, ArH), 7.63 (d,
2H,J = 8.4 Hz, ArH), 9.98 (br s, 1H, NH); ESI-MS: 422.4 (M+H)+.
1. Raez, L. E.; Lilenbaum, R. Clin. Adv. Hematol. Oncol. 2004, 2, 173.
2. Kelly, K. Clin. Cancer Res. 2005, 11, 5027.
3-(40-Chlorophenyl)-1-(20-hydroxy-30-(40-nitrophenoxy)propyl)-1H-pyrazole-5-
3. Nie, Z.; Perretta, C.; Erickson, P.; Margosiak, S.; Almassy, R.; Lu, J.; Averill, A.;
Yager, K. M.; Chu, S. Bioorg. Med. Chem. Lett. 2007, 17, 4191.
4. Jones, D. A.; Fitzpatrick, F. A. Curr. Opin. Chem. Biol. 1999, 3, 71.
5. Du, A.-Y.; Zhao, B.-X.; Yin, D.-L.; Zhang, S.-L.; Miao, J.-Y. Bioorg. Med. Chem. Lett.
2006, 16, 81.
6. Zhao, J.; Miao, J.-Y.; Zhao, B.-X.; Zhang, S.-L. FEBS Lett. 2005, 579, 5809.
7. Zhao, J.; Miao, J.-Y.; Zhao, B.-X.; Zhang, S.-L.; Yin, D.-L. Vasc. Pharmacol. 2005, 43,
69.
8. Du, A.-Y.; Zhao, B.-X.; Zhang, S.-L.; Miao, J.-Y. Bioorg. Med. Chem. 2005, 13, 4176.
9. Du, A.-Y.; Zhao, B.-X.; Zhang, S.-L.; Miao, J.-Y. Exp. Lung Res. 2004, 30, 1.
10. Zhao, B.-X.; Du, A.-Y.; Miao, J.-Y.; Zhao, K.-W.; Du, C.-Q. Vasc. Pharmacol. 2003,
40, 183.
11. Zhao, B.-X.; Wang, D.-W.; Zuo, H.; Qiang, G.-T.; Miao, J.-Y. Chin. J. Org. Chem.
2003, 23, 1026.
12. Zhao, J.; Zhao, B.-X.; Du, A.-Y.; Zhao, Q.-T.; Miao, J.-Y. Endothelium 2004, 11, 267.
13. Du, A.-Y.; Zhao, B.-X.; Miao, J.-Y.; Yin, D.-L.; Zhang, S.-L. Bioorg. Med. Chem.
2006, 14, 2438.
14. Su, L.; Zhao, J.; Zhao, B.-X.; Miao, J.-Y.; Yin, D.-L.; Zhang, S.-L. BBA-Mol. Cell Res.
2006, 1763, 247.
15. He, Q.-X.; Zhu, X.-S.; Shi, M.; Zhao, B.-X.; Zhao, J.; Zhang, S.-L.; Miao, J.-Y. Bioorg.
Med. Chem. 2007, 15, 3889.
16. Tan, W.; Zhao, B.-X.; Shi, M.; Sha, L.; Miao, J.-Y.; Shin, D.-S. Chin. J. Chem. 2006,
24, 396.
carbohydrazide (2e): Yield 87%, pale yellowish solid. mp 156–159 °C; IR (KBr)
m:
3421–3122 (OH, NH, NH2), 1673 (C@O), 1517 and 1254 (NO2) cmꢀ1 1H NMR
;
(300 MHz, CDCl3) d: 3.90–4.18 (m, 4H, NH2 and CH2), 4.30–4.40 (m, 2H, CH2),
4.45–4.51 (m, 1H, CH), 5.31 (br s, 1H, OH), 6.80 (s, 1H), 6.87 (d, 2H, J = 9.0 Hz),
7.40–7.47 (m, 5H, ArH), 8.18 (d, 2H, J = 9.0 Hz, ArH), 8.80 (br s, 1H, NH);
ESI-MS: 432.5 (M+H)+.
3-(40-Chlorophenyl)-1-(20-hydroxy-30-(20-methoxyphenoxy)propyl)-1H-pyrazole-
5-carbohydrazide (2f): Yield 93%, white solid, mp 150–153 °C; IR (KBr)
m: 3391–
3350 (OH, NH, NH2), 1681 (C@O) cmꢀ1 1H NMR (300 MHz, CDCl3) d: 3.77 (s,
;
3H, CH3), 3.91 (dd, 1H, J = 5.6, 9.8 Hz, CH2), 3.99 (dd, 1H, J = 5.1, 9.8 Hz, CH2),
4.36 (d, 2H, J = 5.6 Hz, CH2), 4.48–4.53 (m, 1H, CH), 6.80–6.91 (m, 4H, ArH),
6.94–6.98 (m, 1H, ArH), 7.33–7.37 (m, 4H, ArH); ESI-MS: 417.5 (M+H)+.
1-(30-(40-Chlorophenoxy)-20-hydroxypropyl)-3-(40-methoxyphenyl)-1H-pyrazole-
5-carbohydrazide (2g): Yield 81%, white solid, mp 180–182 °C; IR (KBr)
m:
3348–2935 (OH, NH, NH2), 1680 (C@O) cmꢀ1 1H NMR (300 MHz, CDCl3) d:
;
3.79 (dd, 1H, J = 5.6, 9.6 Hz, CH2), 3.83 (s, 3H, CH3), 3.90 (dd, 1H, J = 4.4, 9.6 Hz,
CH2), 4.34–4.45 (m, 3H, CH and CH2), 6.67 (d, 2H, J = 7.0 Hz, ArH), 6.88 (d, 2H,
J = 6.0 Hz, ArH), 6.92 (s, 1H, ArH), 7.19 (d, 2H, J = 6.0 Hz, ArH), 7.28 (d, 2H,
J = 7.0 Hz, ArH), 9.39 (br s, 1H, NH); ESI-MS: 417.5 (M+H)+.
1-(20-Hydroxy-30-(20-methoxyphenoxy)propyl)-3-(40-methoxyphenyl)-1H-pyrazole-
5-carbohydrazide (2h): Yield 71%, white oil. IR (film)
m: 3350–2933 (OH, NH,
NH2), 1677 (C@O) cmꢀ1 1H NMR (300 MHz, DMSO/CDCl3) d: 3.71 (s, 3H,
;
CH3),3.80 (s, 3H, CH3), 3.99 (d, 1H, J = 4.8 Hz, NH2), 4.15–4.42 (m, 2H, CH2),
4.43–4.50 (m, 3H, CH and CH2), 5.42 (d, 1H, J = 4.8 Hz, OH), 6.67 (s, 1H, ArH),
6.82-6.96 (m, 4H, ArH), 7.03 (d, 2H, J = 8.7 Hz, ArH), 7.51 (d, 2H, J = 8.7 Hz, ArH),
9.41 (s, 1H, NH); ESI-MS: 413.6 (M+H)+.
17. Jiao, P.-F.; Zhao, B.-X.; Wang, W.-W.; He, Q.-X.; Wan, M.-S.; Shin, D.-S.; Miao,
J.-Y. Bioorg. Med. Chem. Lett. 2006, 16, 2862.
18. Jin, S.; White, E. Autophagy 2007, 3, 28.
19. Mizushima, N. Genes Dev. 2007, 21, 2861.
20. Arthur, C. R.; Gupton, J. T.; Kellogg, G. E.; Yeudall, W. A.; Cabot, M. C.; Newsham,
I.; Gewirtz, D. A. Biochem. Pharmacol. 2007, 74, 981.
34. Paglin, S.; Hollister, T.; Delohery, T.; Hackett, N.; McMahill, M.; Sphicas, E.;
Domingo, D.; Yahalom, J. Cancer Res. 2001, 61, 439.