Novel 2H-pyrazolo[4,3-c]hexahydropyridine derivatives: Synthesis, crystal structure, fluorescence properties and cytotoxicity evaluation against human breast cancer cells

Article abstract of DOI:10.1007/s11426-013-4840-x

A series of novel 2H-pyrazolo[4,3-c]hexahydropyridine derivatives (II) have been designed and synthesized. The target compounds have been identified by elemental analysis and spectral (¹H NMR, IR, and MS) data and the absolute configuration of compound (II ₁ ) was confirmed by single crystal X-ray diffraction. The cytotoxicity of the target compounds have been evaluated in vitro against two human breast cancer cell lines MCF-7 and MDA-MB-231 by MTT assay. Most compounds exhibited good inhibition, and compounds II ₂₁ (IC₅₀ = 4.7 μM for MCF-7 and IC₅₀ = 9.3 μM for MDA-MB-231), II ₃₃ (IC₅₀ = 2.4 μM for MCF-7 and IC₅₀ = 4.2 μM for MDA-MB-231) and II ₄₀ (IC₅₀ = 3.3 μM for MCF-7 and IC₅₀ =8.6 μM for MDA-MB-231) displayed better inhibitory activity than 5-fluorouracil (IC ₅₀ = 4.8 μM for MCF-7 and IC₅₀ = 9.6 μM for MDA-MB-231, respectively). Flow cytometric analysis and DNA fragmentation suggest that II ₃₃ is cytotoxic and able to induce the apoptosis of MCF-7 cells. The fluorescence properties of compounds II ₁, II ₆, II ₁₁, II ₁₆, II ₂₃, II ₂₈, and II ₃₅ were also studied and compound II ₂₈ afforded the highest photoluminescence quantum yield (38%).

Full text of DOI:10.1007/s11426-013-4840-x

SCIENCE CHINA
Chemistry
June 2013 Vol.56 No.6: 702–715
doi: 10.1007/s11426-013-4840-x
• ARTICLES •
Novel 2H-pyrazolo[4,3-c]hexahydropyridine derivatives: Synthesis,
crystal structure, fluorescence properties and cytotoxicity
evaluation against human breast cancer cells
PANG ChunCheng, SUN ChuanWen*, WANG Jing, XIAO Di, DING Li & BU HongFei
College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, China
Received September 13, 2012; accepted November 7, 2012; published online March 13, 2013
A series of novel 2H-pyrazolo[4,3-c]hexahydropyridine derivatives (II) have been designed and synthesized. The target com-
pounds have been identified by elemental analysis and spectral (1H NMR, IR, and MS) data and the absolute configuration of
compound (II1) was confirmed by single crystal X-ray diffraction. The cytotoxicity of the target compounds have been evalu-
ated in vitro against two human breast cancer cell lines MCF-7 and MDA-MB-231 by MTT assay. Most compounds exhibited
good inhibition, and compounds II21 (IC50 = 4.7 μM for MCF-7 and IC50 = 9.3 μM for MDA-MB-231), II33 (IC50 = 2.4 μM for
MCF-7 and IC50 = 4.2 μM for MDA-MB-231) and II40 (IC50 = 3.3 μM for MCF-7 and IC50 =8.6 μM for MDA-MB-231) dis-
played better inhibitory activity than 5-fluorouracil (IC50 = 4.8 μM for MCF-7 and IC50 = 9.6 μM for MDA-MB-231, respec-
tively). Flow cytometric analysis and DNA fragmentation suggest that II33 is cytotoxic and able to induce the apoptosis of
MCF-7 cells. The fluorescence properties of compounds II1, II6, II11, II16, II23, II28, and II35 were also studied and compound
II28 afforded the highest photoluminescence quantum yield (38%).
pyrazolopyridine, synthesis, crystal structure, breast cancer cells, cytotoxicity evaluation, apoptosis, fluorescence prop-
erties
cal role, stimulating cancer cell proliferation [6, 7]. Some
1 Introduction
studies have showed that the human breast cancer cell lines
ER (estrogen receptor)-positive MCF-7 and ER (estrogen
receptor)-negative MDA-MB-231 are considerably more
resistant to apoptosis than a number of other human breast
cancer cell lines [8]. Thus, there is a strong impetus to iden-
tify new anti-breast cancer agents with improved cytotoxi-
city for ER-positive MCF-7 and ER-negative MDA-MB-
231 human breast cancer cell lines.
Five and six-membered nitrogen-containing heterocycles
are abundant in nature and exhibit diverse and important
biological properties [9, 10]. Pyrazolopyridine derivatives
are an important class of heterocyclic compounds, which
exhibit a variety of biological activities including anticancer,
anxiolytic, antiviral, antileishmanial and anti-inflammatory
activities [11–16]. Because of their biological activities
these compounds have distinguished themselves as hetero-
Among diseases cancer is considered as the deadliest and a
substantial number of new antineoplastic agents have been
discovered [1, 2]. Cancer cells differ from their normal
counterparts in a number of biochemical processes, particu-
larly during the control of cell growth and division. One
characteristic of cancer cells, that distinguishes them from
most normal cells, is their high proliferative index [3]. As a
result, targeting of proliferative pathways resulting in cell
death via apoptosis is considered as an effective strategy for
fighting this disease.
Breast cancer is the most prevalent form of cancer diag-
nosed in women, and its incidence is steadily rising [4, 5].
In hormone dependent breast cancer, estrogens play a criti-
*Corresponding author (email: willin112@163.com)
© Science China Press and Springer-Verlag Berlin Heidelberg 2013
chem.scichina.com www.springerlink.com

Pang CC, et al. Sci China Chem June (2013) Vol.56 No.6
703
cycles of profound chemical and biological significance
[17]. As a result, the design of new pyrazolopyridine deriv-
atives has attracted considerable attention.
as anti-breast cancer agents. The structures of all new com-
pounds have been confirmed by H NMR, IR, MS and ele-
1
mental analysis, and the absolute configuration of the com-
pound II1 was confirmed by single crystal X-ray diffraction.
Photoluminescence spectra were studied for compounds II1,
II6, II11, II16, II23, II28, and II35 and these compounds each
gave an intense emission. Biological evaluation indicated
that some of the synthesized compounds exhibited good
inhibition, among them compound II33 was found to be the
most potent compound with IC50 values of 2.4 and 4.2 μM
against MCF-7 and MDA-MB-231, respectively. Moreover,
the compound II33 induced apoptosis of MCF-7 cells, which
was detected by DNA fragmentation and flow cytometric
analysis using annexin V and PI double staining.
In our previous publication we described synthesis and in
vitro antiproliferative activity against leukemia K562 cell
lines of N-(substituted benzyl)-3,5-bis(benzylidene)-4-
piperidones (Scheme 1, I). Significant antiproliferative ac-
tivity of compounds I were found and some of these com-
pounds have better inhibitory activities than the anticancer
drug 5-fluorouracil (5-FU, IC50 = 8.56), however, no an-
ti-breast cancer activity was observed with these com-
pounds [18]. Therefore, further structural modification of I
with the goal of discovering novel compounds with potent
anti-breast cancer activity is necessary.
Pyrazolopyridine derivatives have mostly been synthe-
sized through the reaction of α,β-unsaturated ketones with
substituted hydrazine [17]. Motivated by this method, in the
search for effective anti-breast cancer agents, we have di-
rected our efforts toward the derivatization of N-(substituted
benzyl)-3,5-bis(benzylidene)-4-piperidones (I) to afford
2H-pyrazolo[4,3-c]hexahydropyridine derivatives (II). The
derivatives (II) can be easily obtained by reacting (I) and
phenylhydrazine as raw materials catalyzed by NaOEt in
HOEt.
2 Experimental
2.1 Materials and apparatus
All the chemical reagents purchased were of analytical
grade and used without further purification, except for the
toluene, which was dried by refluxing in the presence of
1
sodium and distilled prior to use. H NMR spectra were
recorded on a Bruker Avance 400 (400 MHz) spectrometer,
using CDCl3 or DMSO-d6 as solvents and tetramethylsilane
(TMS) as internal standard. Melting points were determined
by an RK1 microscopic melting apparatus (uncorrected).
Herein, in a continuation of our research on anti-breast
cancer compounds, we report the synthesis of a series of
2H-pyrazolo[4,3-c]hexahydropyridine derivatives (II1–II41)
Scheme 1 General synthetic route to 2H-pyrazolo[4,3-c]hexahydropyridine derivatives (II). Reagents and conditions: (a) absolute methanol, refluxing; (b)
sodium/absolute methanol, toluene, refluxing; (c) 25% HCl, refluxing; (d) 35% sodium hydroxide, r.t.; (e) aromatic aldehyde, EtOH, 10% NaOH, r.t.; (f)
phenylhydrazine, EtONa, refluxing.

704
Pang CC, et al. Sci China Chem June (2013) Vol.56 No.6
Elemental analysis was performed with a PerkinElmer 2400
instrument. MS spectra were recorded in ESI mode on a
Bruker microTOF-Q II spectrometer. IR spectra were ob-
tained on a Nicolet 5DX FT-IR spectrophotometer in the
region 4000–400 cm–1 using KBr discs. X-ray diffraction
data were recorded on a Bruker Smart CCD diffractometer.
The single-photon fluorescence spectra were collected on a
PerkinElmer LS55 luminescence spectrometer. Visible ab-
sorption spectra were determined on PerkinElmer Lambda
35 spectrophotometer.
H, 7.08; N, 8.48.
5-(4-Methylbenzyl)-7-(4-methoxylbenzylidene)-3,3a,4,5,6,7-
hexahydro-3-(4-methoxylphenyl)-2-phenyl-2H-pyrazolo[4,3-
c]pyridine (II3)
1
Yield, 78%; chartreuse solid; mp 186187 °C; H NMR
(CDCl3, 400 MHz) δ 2.30 (s, 3H), 2.43 (t, J = 10.2 Hz, 1H),
3.163.23 (m, 2H), 3.263.52 (m, 1H), 3.61 (dd, J = 13.1,
38.3 Hz, 2H), 3.81 (s, 3H), 3.82 (s, 3H), 4.03 (d, J = 13.9
Hz, 1H), 4.52 (d, J = 12.3 Hz, 1H), 6.796.85(m,1H),
6.907.30 (m,17H); 13C NMR (CDCl3, 100 MHz) δ 20.19,
52.68, 52.71, 53.74, 54.25, 54.73, 70.58, 112.70, 114.08,
114.24, 114.28, 119.40, 125.08, 125.39, 125.99, 127.65,
127.92, 128.67, 128.88, 132.27, 136.29, 136.32, 137.56,
141.29, 145.81, 150.11, 152.38. IR (KBr, cm–1) 2948, 2831,
1596, 1509, 1497, 1250, 1030, 829; ESI-MS: 552.2
(C35H35N3NaO2, [M+Na]+). Anal. Calcd. for C35H35N3O2: C,
79.32; H, 6.67; N, 7.92. Found: C, 79.37; H,6.66; N, 7.93.
2.2 General procedure for the preparation of com-
pounds II
N-(substituted benzyl)-3,5- bis(ben- zylidene)-4-piperidones
(I) were prepared according to reported procedures [16]. A
mixture of (I) (0.001 mol), phenylhydrazine (0.004 mol)
and Na metal (0.05 g) in ethanol (4 mL) was heated under
reflux for 10–24 h. After this time, the separated solid was
filtered off and recrystallized from CH2Cl2:EtOH = 1:2 to
obtain title compounds II.
7-(4-Fluorobenzylidene)-5-(4-methylbenzyl)-3,3a,4,5,6,7-
hexahydro-3-(4-fluorophenyl)-2-phenyl-2H-pyrazolo[4,3-
c]pyridine (II4)
7-Benzylidene-5-(4-methylbenzyl)-3,3a,4,5,6,7-hexahydro-2,
1
Yield, 70%; chartreuse solid; mp 183185 °C; H NMR
3-diphenyl-2H-pyrazolo[4,3-c]pyridine (II1)
(CDCl3, 400 MHz) δ 2.34 (s, 3H), 2.52(t, J = 10.1 Hz, 1H),
3.143.28 (m, 3H), 3.54 (d, J = 13.0Hz, 1H), 3.65 (d, J =
13.1Hz, 1H), 4.04 (d, J = 13.5Hz, 1H), 4.55 (d, J = 12.6Hz,
1H), 6.81 (t, J = 7.2Hz, 1H), 6.836.88 (m, 8H), 7.037.06
(m, 4H), 7.097.16 (m, 2H), 7.257.27 (m, 3H); 13C NMR
(CDCl3 , 100 MHz) δ 20.21, 53.02, 53.08, 54.18, 57.38,
71.12, 112.69, 114.13, 114.19, 114.39, 115.04, 115.25,
119.52, 124.25, 126.67, 126.76, 127.74, 129.06, 130.06,
130.21, 131.09, 131.13, 136.23, 136.26, 145.50, 150.46,
157.86, 159.80, 162.3. IR (KBr, cm–1) 3022, 2918, 1584,
1485, 1273, 1207, 1092; ESI-MS: 528.3 (C33H29F2N3Na,
[M+Na]+). Anal. Calcd. for C33H29F2N3: C, 78.37; H, 5.79;
N, 8.34. Found: C, 78.39; H, 5.78; N, 8.31.
1
Yield, 87%; chartreuse solid; mp 168169 °C; H NMR
(CDCl3, 400 MHz) δ 2.30 (s, 3H), 2.47 (t, J = 10.4Hz, 1H),
3.143.18 (m, 1H), 3.223.36 (m, 2H), 3.523.56 (m, 2H),
4.02 (d, J = 12.1Hz, 1H), 4.61 (d, J = 12.4Hz, 1H), 6.80 (t, J
= 7.2Hz, 1H), 7.047.37 (m,19H); 13C NMR (CDCl3, 100
MHz) δ 20.11, 53.54, 54.08, 54.46, 56.12, 70.66, 114.09,
114.24, 114.82, 119.62, 122.46, 125.05, 125.51, 125.67,
127.69, 128.09, 128.38, 128.91, 132.09, 135.54, 136.44,
136.49, 137.46, 145.72, 147.79, 148.96, 150.16. IR (KBr,
cm–1) 3001, 2819, 1705, 1581, 1278, 1207, 1090; ESI-MS:
492.3 (C33H31N3Na, [M+Na]+); Anal. Calcd. for C33H31N3:
C, 84.47; H, 6.69; N, 8.94. Found: C, 84.40; H, 6.65; N,
8.95.
7-(4-Chlorobenzylidene)-5-(4-methylbenzyl)-3,3a,4,5,6,7-
hexahydro-3-(4-chlorophenyl)-2-phenyl-2H-pyrazolo[4,3-
c]pyridine (II5)
5-(4-Methylbenzyl)-7-(4-methylbenzylidene)-3,3a,4,5,6,7-
hexahydro-3-(4-methylphenyl)-2-phenyl-2H-pyrazolo[4,3-
c]pyridine (II2)
1
1
Yield, 81%; chartreuse solid; mp 210212 °C; H NMR
Yield, 81%; chartreuse solid; mp 208209 °C; H NMR
(CDCl3, 400 MHz) δ 2.30 (s, 3H), 2.402.85 (m, 1H),
3.023.33 (m, 3H), 3.54(d, J = 12.4Hz, 1H), 3.65(d, J =
12.6 Hz, 1H), 4.04 (d, J = 13.4Hz, 1H), 4.55 (d, J = 12.0Hz,
1H), 6.81 (t, J = 6.8Hz, 1H), 7.087.16 (m, 4H), 7.167.28
(m, 10H), 7.307.37 (m, 3H). 13C NMR (CDCl3 , 100 MHz)
δ 20.30, 52.86, 53.85, 54.21, 58.02, 70.11, 112.71, 114.08,
119.61, 124.17, 126.47, 127.48, 127.78, 128.24, 128.42,
128.79, 129.03, 129.64, 132.21, 132.42, 133.43, 139.04,
145.31, 150.29, 157.87. IR (KBr, cm–1) 3022, 2918, 1592,
1584, 1273, 1107, 1023; ESI-MS: 560.1(C33H29Cl2N3Na,
[M+Na]+). Anal. Calcd. for C33H29Cl2N3: C, 73.62; H, 5.40;
N, 13.19. Found: C, 73.60; H, 5.43; N, 13.17.
(CDCl3, 400 MHz) δ 2.30 (s, 3H), 2.34 (s, 6H), 2.43 (t, J =
10.2Hz, 1H), 3.143.20 (m, 1H), 3.213.26 (m, 1H),
3.283.30 (m, 1H), 3.54 (d, J = 13.1Hz, 1H), 3.65 (d, J =
13.1Hz, 1H), 4.04 (d, J = 13.9Hz, 1H), 4.55 (d, J = 12.4Hz,
1H), 6.81 (t, J = 7.2Hz, 1H), 7.037.27 (m, 17H). 13C NMR
(CDCl3 , 100 MHz) δ 20.10, 20.22, 20.28, 53.31, 54.09,
54.42, 57.37, 70.44, 113.56, 114.05, 114.21, 114.22, 119.26,
125.03, 125.06, 126.29, 127.64, 127.73, 129.32, 129.39,
129.89, 132.32, 132.36, 132.51, 145.87, 150.72, 157.94,
158.07. IR (KBr, cm–1) 3022, 2918, 1596, 1509, 1042, 1030,
997; ESI-MS: 520.6 (C35H35N3Na, [M+ Na]+); Anal. Calcd.
for C35H35N3: C, 84.47; H, 7.09; N, 8.44. Found: C, 84.48;

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7-Benzylidene-5-(4-methoxylbenzyl)-3,3a,4,5,6,7-hexahydro
(400 MHz, CDCl3) δ 2.40 (t, J = 10.8 Hz, 1H), 3.123.34
(m, 3H), 3.563.72 (m, 2H), 3.79 (s, 3H), 4.07 (d, J = 12.8
Hz, 1H), 4.51 (d, J = 11.4 Hz, 1H), 6.81 (t, J = 7.2 Hz, 1H),
6.927.23 (m, 17H); 13C NMR (CDCl3 , 100 MHz) δ 53.01,
53.09, 54.20, 54.28, 54.48, 60.15, 70.11, 112.68, 114.10,
114.17, 114.38, 115.03, 115.25, 119.51, 124.23, 126.67,
126.74, 127.73, 129.05, 130.06, 130.14, 131.09, 131.13,
136.22, 136.25, 145.50, 150.45, 157.85, 159.69, 162.15. IR
(KBr, cm–1) 2985, 2876, 1608, 1554, 1273, 1147, 1068;
ESI-MS: 544.2(C33H29F2N3NaO, [M+Na]+). Anal. Calcd.
for C33H29F2N3O: C, 75.92; H, 5.67; N, 8.02. Found: C,
75.99; H, 5.60; N, 8.06.
-2,3-diphenyl-2H-pyrazolo[4,3-c]pyridine (II6)
1
Yield, 88%; chartreuse solid; mp 180-181 °C; H NMR
(400 MHz, CDCl3) δ 2.382.43 (m, 1H), 3.133.25 (m, 3H),
3.553.64 (m, 2H), 3.76 (s, 3H), 4.04 (d, J = 12.5 Hz, 1H),
4.55 (d, J = 12.8 Hz, 1H), 6.83 (t, J = 7.2 Hz, 1H),
6.92-7.23 (m, 19H); 13C NMR (CDCl3 , 100 MHz) δ 53.05,
54.10, 54.19, 54.48, 60.15, 71.12, 114.08, 114.24, 114.46,
119.66, 122.62, 122.78, 123.06, 124.16, 126.47, 127.23,
127.49, 127.77, 128.20, 128.43, 129.67, 130.32, 132.21,
132.43, 133.38, 139.02, 145.29, 149.94, 159.03, 161.50. IR
(KBr, cm–1) 2972, 2850, 1568, 1552, 1266, 1112, 1042;
ESI-MS: 508.2 (C33H31N3NaO, [M+Na]+). Anal. Calcd. for
C33H31N3O: C, 81.64; H, 6.47; N, 8.62. Found: C, 81.62; H,
6.43; N, 8.65.
7-(4-Chlorobenzylidene)-5-(4-methoxylbenzyl)-3,3a,4,5,6,7-
hexahydro-3-(4-chlorophenyl)-2-phenyl-2H-pyrazolo[4,3-c]
pyridine (II10)
1
Yield, 73%; chartreuse solid; mp 200201 °C; H NMR
5-(4-Methoxylbenzyl)-7-(4-methylbenzylidene)-3,3a,4,5,6,7-
(400 MHz, CDCl3) δ 2.43 (t, J = 10.3 Hz, 1H), 3.123.34
(m, 3H), 3.553.61 (m, 2H), 3.78 (s, 3H), 4.14 (d, J = 11.5
Hz, 1H), 4.52 (d, J = 12.3 Hz, 1H), 6.81 (t, J = 7.2 Hz, 1H),
6.927.23 (m, 17H); 13C NMR (CDCl3 , 100 MHz) δ 52.99,
53.84, 54.20, 54.39, 60.12, 70.11, 112.70, 114.07, 119.61,
124.07, 126.46, 127.47, 127.76, 128.24, 128.41, 128.79,
129.03, 129.64, 132.20, 132.42, 133.42, 139.04, 145.31,
150.28, 157.87. IR (KBr, cm–1) 2982, 2861, 1566, 1554, 1273,
1147, 1082; ESI-MS:576.1 (C33H29Cl2N3NaO, [M+Na]+).
Anal. Calcd. for C33H29Cl2N3O: C, 71.42; H, 5.27; N, 7.52.
Found: C, 71.48; H, 5.27; N, 7.58.
hexahydro-3-(4-methylphenyl)-2-phenyl-2H-pyrazolo[4,3-
c]pyridine (II7)
1
Yield, 83%; chartreuse solid; mp 170172 °C; H NMR
(400 MHz, CDCl3) δ 2.30 (s, 6H), 2.43 (t, J = 10.3 Hz, 1H),
3.123.34 (m, 3H), 3.553.61 (m, 2H), 3.78 (s, 3H), 4.14 (d,
J = 11.5 Hz, 1H), 4.52 (d, J = 12.3 Hz, 1H), 6.81 (t, J = 7.2
Hz, 1H), 6.927.23 (m, 17H); 13C NMR (CDCl3 , 100 MHz)
δ 20.11, 20.29, 52.69, 52.78, 53.75, 54.25, 54.75, 70.57,
112.71, 114.08, 114.23, 114.26, 119.42, 125.06, 125.36,
125.94, 127.63, 127.99, 128.61, 128.83, 132.27, 136.29,
136.32, 137.58, 141.29, 145.81, 150.12, 152.36. IR (KBr,
cm–1) 2972, 2889, 1590, 1513, 1280, 1096, 1043; ESI-MS:
536.2(C35H35N3NaO, [M+Na]+). Anal. Calcd. for C35H35N3O:
C, 81.82; H, 6.87; N, 8.12. Found: C, 81.84; H, 6.87; N,
8.18.
7-Benzylidene-5-(2-fluorobenzyl)-3,3a,4,5,6,7-hexahydro-2,
3-diphenyl-2H-pyrazolo[4,3-c]pyridine (II11)
1
Yield, 88%; chartreuse solid; mp 165167 °C; H NMR
(CDCl3, 400 MHz) δ 2.57 (s, 1H), 2.933.38 (m, 3H),
3.75(s, 2H), 4.08 (d, J = 14.1Hz, 1H), 4.63 (d, J = 12.4Hz,
1H), 6.77 (t, J = 7.2Hz, 1H), 6.957.54 (m, 19H). 13C NMR
(CDCl3 , 100 MHz) δ 50.06, 53.38, 53.98, 54.56, 71.32,
112.68, 113.52, 114.18, 114.37, 119.21, 122.95, 122.99,
124.98, 126.29, 127.62, 127.77, 128.01, 128.09, 129.92,
130.33, 132.54, 145.82, 150.53, 158.02, 159.05, 161.49. IR
(KBr, cm–1) 3447, 1643, 1491, 1420, 1083; ESI-MS:
498.1(C32H28FN3Na, [M+ Na]+). Anal. Calcd. for
C32H28FN3: C, 81.15; H, 5.93; N, 8.86. Found: C, 81.16; H,
5.96; N, 8.87.
5-(4-Methoxylbenzyl)-7-(4-methoxylbenzylidene)-3,3a,4,5,6,
7-hexahydro-3-(4-methoxylphenyl)-2-phenyl-2H-pyrazolo[4,
3-c]pyridine (II8)
1
Yield, 83%; chartreuse solid; mp 161163 °C; H NMR
(400 MHz, CDCl3) δ 2.45 (t, J = 12.6 Hz, 1H), 3.163.34
(m, 3H), 3.563.61 (m, 2H), 3.77(s, 3H), 3.81 (s, 6H), 3.98
(d, J = 13.5 Hz, 1H), 4.52 (d, J = 12.3 Hz, 1H), 6.84 (t, J =
7.1 Hz, 1H), 6.92-7.23 (m, 17H); 13C NMR (CDCl3 , 100
MHz) δ 52.25, 53.29, 54.26, 54.44, 57.38, 60.03, 70.42,
113.54, 114.01, 114.17, 114.22, 119.24, 125.01, 125.02,
126.27, 126.64, 127.72, 129.30, 129.38, 129.87, 132.32,
132.35, 132.51, 145.86, 150.71, 157.93, 158.06. IR (KBr,
cm–1) 2981, 2868, 1546, 1520, 1268, 1162, 1062; ESI
-MS: 568.6(C33H35N3NaO3, [M+Na]+). Anal. Calcd. for
C35H35N3O3: C, 77.02; H, 6.47; N, 7.72. Found: C, 77.04; H,
6.47; N, 7.70.
5-(2-Fluorobenzyl)-7-(4-methylbenzylidene)-3,3a,4,5,6,7-
hexahydro-3-(4-methylphenyl)-2-phenyl-2H-pyrazolo[4,3-
c]pyridine (II12)
1
Yield, 85%; chartreuse solid; mp 193195 °C; H NMR
(CDCl3, 400 MHz) δ 2.06(s,1H), 2.37 (s, 6H), 2.463.33 (m,
3H), 3.74 (td, J = 12.0, 7.0 Hz, 2H), 4.14 (dd, J = 14.3,7.2
Hz,1H), 4.58 (d, J = 11.0Hz, 1H), 6.83 (t, J = 7.3Hz, 1H),
7.077.54 (m,17H). 13C NMR (CDCl3 , 100 MHz) δ 20.11,
20.27, 53.06, 53.33, 54.04, 54.57, 70.63, 114.09, 114.17,
114.39, 119.21, 122.96, 122.99, 125.06, 125.29, 127.64,
127.96, 128.08, 128.43, 128.85, 130.35, 130.41, 132.22,
7-(4-Fluorobenzylidene)-5-(4-methoxylbenzyl)-3,3a,4,5,6,7-
hexahydro-3-(4-fluorophenyl)-2-phenyl-2H-pyrazolo[4,3-c]
pyridine (II9)
1
Yield, 63%; chartreuse solid; mp 183184 °C; H NMR

706
Pang CC, et al. Sci China Chem June (2013) Vol.56 No.6
136.36, 137.57, 145.78, 159.06, 161.50. IR (KBr, cm–1)
3448, 1643, 1485, 1419, 1084, 1022, 1175, 753; ESI-MS:
524.3 (C34H32FN3Na, [M+ Na]+). Anal. Calcd. for
C34H32FN3: C, 81.42; H, 6.41; N, 8.36. Found: C, 81.41; H,
6.43; N, 8.38.
7-Benzylidene-5-(4-fluorobenzyl)-3,3a,4,5,6,7-hexahydro-2,
3-diphenyl-2H-pyrazolo[4,3-c]pyridine (II16)
1
Yield, 78%; chartreuse solid; mp 174175 °C; H NMR
(CDCl3, 400 MHz) δ 2.392.48 (m, 1H), 3.143.33 (m, 3H),
3.54 (d, J = 12.8Hz, 1H), 3.65 (d, J = 13.1Hz, 1H), 4.04 (d,
J = 12.1Hz, 1H), 4.55 (d, J = 13.1Hz, 1H), 6.81 (t, J =
7.2Hz, 1H), 6.867.00 (m, 4H), 7.047.12 (m, 11H),
7.157.21 (m, 4H). 13C NMR (CDCl3 , 100 MHz) δ 50.12,
53.42, 53.98, 54.34, 71.02, 112.48, 113.58, 114.16, 114.38,
119.26, 122.92, 122.99, 124.98, 126.35, 127.68, 127.77,
128.01, 128.09, 129.98, 130.38, 132.53, 145.88, 150.55,
158.01, 159.05, 161.22. IR (KBr, cm–1) 3002, 2819, 1605,
1581, 1278, 1187, 1090; ESI-MS: 498.2(C32H28FN3Na, [M+
Na]+). Anal. Calcd. for C32H28FN3: C, 81.17; H, 5.99; N,
8.84. Found: C, 81.16; H, 5.96; N, 8.87.
5-(2-Fluorobenzyl)-7-(4-methoxylbenzylidene)-3,3a,4,5,6,7-
hexahydro-3-(4-methoxylphenyl)-2-phenyl-2H-pyrazolo [4,3-
c]pyridine (II13)
1
Yield, 73%; chartreuse solid; mp 176178 °C; H NMR
(CDCl3, 400 MHz) δ 2.55 (s,1H), 3.27 (m, 3H), 3.613.68
(m, 2H), 3.74 (s, 3H), 3.75 (s, 3H), 3.93 (d, J = 13.9 Hz,
1H), 4.56 (d, J = 12.4 Hz, 1H), 6.74 (dd, J = 4.2, 10.4 Hz,
1H), 6.807.26 (m, 17H). 13C NMR (CDCl3 , 100 MHz) δ
50.06, 53.36, 53.99, 54.23, 54.53, 70.34, 112.72, 113.53,
114.17, 114.37, 119.20, 122.95, 124.98, 126.29, 127.62,
127.77, 128.01, 128.09, 129.93, 130.32, 132.53, 145.87,
150.52, 157.90, 158.06, 159.04, 161.49. IR (KBr, cm–1)
3438, 1597, 1501, 1424, 1247, 1087, 1028, 759; ESI-MS:
556.1(C34H32FN3NaO2, [M+Na]+). Anal. Calcd. for
C34H32FN3O2: C, 76.52; H, 6.07; N, 7.86. Found: C, 76.51;
H, 6.04; N, 7.87.
5-(4-Fluorobenzyl)-7-(4-methylbenzylidene)-3,3a,4,5,6,7-
hexahydro-3-(4-methylphenyl)-2-phenyl-2H-pyrazolo[4,3-
c]pyridine (II17)
1
Yield, 67%; chartreuse solid; mp 190191 °C; H NMR
(CDCl3, 400 MHz) δ 2.35 (s, 6H), 2.463.33 (m, 4H), 3.58
(dd, J = 28.3, 13.2 Hz, 2H), 4.01 (d, J = 13.9 Hz,1H), 4.52
(d, J = 12.5Hz, 1H), 6.84 (t, J = 7.3Hz, 1H), 6.92 (dd, J =
12.1, 5.3 Hz, 2H), 7.04 (dd, J = 8.7, 1.0 Hz, 2H), 7.117.21
(m, 10H), 7.25 (s, 1H), 7.267.30 (m, 2H). 13C NMR
(CDCl3, 100 MHz) δ 20.10, 20.26, 53.36, 53.58, 54.28,
54.87, 70.03, 114.09, 114.16, 114.62, 119.11, 122.46,
122.87, 125.09, 125.31, 127.68, 127.85, 128.11, 128.46,
128.85, 130.32, 130.46, 132.28, 136.36, 137.57, 148.32,
156.02, 159.54. IR (KBr, cm–1) 2998, 2918, 1588, 1482,
1175, 1111, 998; ESI-MS: 524.2 (C34H32FN3Na, [M+ Na]+).
Anal. Calcd. for C34H32FN3: C, 81.47; H, 6.49; N, 8.34.
Found: C, 81.41; H, 6.43; N, 8.38.
5-(2-Fluorobenzyl)-7-(4-fluorobenzylidene)-3,3a,4,5,6,7-
hexahydro-3-(4-fluorophenyl)-2-phenyl-2H-pyrazolo[4,3-
c]pyridine (II14)
1
Yield, 75%; chartreuse solid; mp 172174 °C; H NMR
(CDCl3, 400 MHz) δ 2.57 (s, 1H), 3.143.41 (m, 3H), 3.76
(s, 2H), 4.19 (d, J = 13.9 Hz, 1H), 4.61 (d, J = 12.3 Hz, 1H),
6.88 (t, J = 7.2 Hz, 1H), 7.057.41 (m, 17H); 13C NMR
(CDCl3 , 100 MHz) δ 53.11, 54.05, 54.46, 60.06, 70.14,
114.11, 114.23, 114.46, 115.03, 115.31, 119.55, 120.11,
124.36, 126.40, 126.69, 126.71, 127.70, 129.34, 130.08,
130.09, 130.43, 131.21, 131.97, 135.45, 136.11, 145.42,
150.10, 159.73, 162.20. IR (KBr, cm–1) 3448, 1598, 1498,
1088, 1027, 835; ESI-MS: 532.2(C32H26F3N3Na,[M+Na]+).
Anal. Calcd. for C32H26F3N3: C,75.41; H, 5.15; N, 8.28.
Found: C, 75.43; H, 5.14; N, 8.25.
5-(4-Fluorobenzyl)-7-(4-isopropylbenzylidene)-3,3a,4,5,6,7-
hexahydro-3-(4-isopropylphenyl)-2-phenyl-2H-pyrazolo
[4,3-c]pyridine (II18)
1
Yield, 75%; chartreuse solid; mp 148150 °C; H NMR
7-(4-Chlorobenzylidene)-5-(2-fluorobenzyl)-3,3a,4,5,6,7-
hexahydro-3-(4-chlorophenyl)-2-phenyl-2H-pyrazolo[4,3-
c]pyridine (II15)
(CDCl3, 400 MHz) δ 1.16 (d, J = 8.2Hz, 12H), 2.47 (t, J =
10.3 Hz, 1H), 2.762.83 (m, 2H), 3.123.34 (m, 3H), 3.61
(dd, J = 13.1, 38.3 Hz, 2H), 4.03 (d, J = 13.9 Hz, 1H), 4.52
(d, J = 12.3 Hz, 1H), 6.81 (t, J = 7.2 Hz, 1H), 6.83 6.92 (m,
4H), 7.05 (t, J = 8.1 Hz, 4H), 7.087.23 (m, 7H), 7.29 (d, J
= 8.7 Hz, 2H); 13C NMR (CDCl3 , 100 MHz) δ 18.26, 19.03,
30.07, 30.12, 50.28, 53.46, 54.22, 59.48, 71.26, 112.52,
112.68, 114.26, 114.38, 119.64, 122.88, 122.94, 126.26,
127.37, 127.68, 128.22, 128.43, 130.23, 130.59, 133.34,
141.82, 146.52, 151.95, 154.02, 159.12, 160.03. IR (KBr,
cm–1) 2996, 2901, 1620, 1579, 1273, 1207, 1004; ESI-MS:
580.3(C38H40FN3Na, [M+Na]+). Anal. Calcd. for C38H40FN3:
C, 81.82; H, 7.27; N, 7.52. Found: C, 81.83; H,7.23; N,
7.53.
1
Yield, 82%; chartreuse solid; mp 187189 °C; H NMR
(CDCl3, 400 MHz) δ 2.56 (s, 1H), 3.123.36 (m, 3H),
3.76(s, 2H), 4.01(d, J = 13.9Hz, 1H), 4.61 (d, J = 12.4Hz,
1H), 6.83 (t, J = 7.3Hz, 1H), 6.997.41 (m, 17H);13C NMR
(CDCl3, 100 MHz) δ 52.78, 53.32, 53.88, 54.46, 70.07,
114.09, 114.24, 114.46, 119.66, 122.61, 122.76, 123.03,
124.16, 126.46, 127.22, 127.49, 127.76, 128.19, 128.43,
129.66, 130.24, 132.28, 132.48, 133.38, 139.01, 145.28,
149.93, 159.03, 161.47. IR (KBr, cm–1) 3449, 1643, 1593,
1488, 1088, 1021, 829; ESI-MS: 564.1 (C32H26Cl2FN3Na,
[M+Na]+). Anal. Calcd. for C32H26Cl2FN3: C, 70.84; H, 4.84;
N, 7.76. Found: C, 70.85; H, 4.83; N, 7.75.

Pang CC, et al. Sci China Chem June (2013) Vol.56 No.6
707
5-(4-Fluorobenzyl)-7-(4-methoxylbenzylidene)-3,3a,4,5,6,7-
hexahydro-3-(4-methoxylphenyl)-2-phenyl-2H-pyrazolo [4,3-
c]pyridine (II19)
7-(4-Chlorobenzylidene)-5-(4-fluorobenzyl)-3,3a,4,5,6,7-
hexahydro-3-(4-chlorophenyl)-2-phenyl-2H-pyrazolo[4,3-
c]pyridine (II22)
1
1
Yield, 73%; chartreuse solid; mp 159161 °C; H NMR
Yield, 69%; chartreuse solid; mp 210212 °C; H NMR
(CDCl3, 400 MHz) δ 2.39 (t, J = 10.5 Hz,1H), 3.013.20 (m,
3H), 3.53 (dd, J = 13.2, 28.0 Hz, 2H), 3.75 (s, 3H), 3.76 (s,
3H), 3.93 (d, J = 13.9 Hz, 1H), 4.46 (d, J = 12.5 Hz, 1H),
6.74 (dd, J = 4.2, 10.4 Hz, 1H), 6.80 (dd, J = 5.4, 9.7 Hz,
3H), 6.896.82 (m, 2H), 6.97 (dd, J = 1.0, 8.8 Hz, 2H),
7.147.26 (m, 10H). 13C NMR (CDCl3 , 100 MHz) δ 50.21,
53.46, 53.92, 54.23, 59.42, 71.30, 112.52, 113.55, 114.23,
114.38, 119.35, 122.88, 124.94, 126.36, 127.48, 127.72,
128.01, 128.46, 129.93, 130.54, 132.31, 148.87, 151.52,
152.90, 156.06, 158.04, 160.42. IR (KBr, cm–1) 2994, 2877,
1683, 1584, 1224, 1207, 1082; ESI-MS: 556.2
(C34H32FN3NaO2, [M+Na]+). Anal. Calcd. for C34H32FN3O2:
C, 76.52; H, 6.07; N, 7.86. Found: C, 76.51; H, 6.04; N,
7.87.
(CDCl3, 400 MHz) δ 2.402.85 (m, 1H), 3.023.33 (m, 3H),
3.54 (d, J = 12.4Hz, 1H), 3.65 (d, J = 12.6Hz, 1H), 4.04 (d,
J = 13.4Hz, 1H), 4.55 (d, J = 12.0Hz, 1H), 6.81 (t, J =
6.82Hz, 1H), 7.087.37 (m, 17H). 13C NMR (CDCl3 , 100
MHz) δ 52.89, 53.38, 53.96, 54.38, 70.11, 114.15, 114.24,
114.86, 119.68, 122.67, 122.78, 123.23, 124.16, 126.46,
127.28, 127.41, 127.78, 128.19, 128.46, 129.68, 130.24,
132.28, 132.48, 133.64, 138.55, 145.20, 149.90, 159.55,
162.49. IR (KBr, cm–1) 3025, 2911, 1610, 1584, 1273, 1201,
1003; ESI-MS: 564.1(C32H26Cl2FN3Na, [M+Na]+). Anal.
Calcd. for C32H26Cl2FN3: C, 70.82; H, 4.85; N, 7.73. Found:
C, 70.85; H, 4.83; N, 7.75.
5-(4-Bromobenzyl)-7-benzylidene-3,3a,4,5,6,7-hexahydro-2,
3-diphenyl-2H-pyrazolo[4,3-c]pyridine (II23)
1
5-(4-Fluorobenzyl)-7-(3-fluorobenzylidene)-3,3a,4,5,6,7-
hexahydro-3-(3-fluorophenyl)-2-phenyl-2H-pyrazolo[4,3-
c]pyridine (II20)
Yield, 78%; chartreuse solid; mp 178179 °C; H NMR
(CDCl3, 400 MHz) δ 2.43 (t, J = 10.9Hz, 1H), 3.143.36 (m,
3H), 3.54 (d, J = 12.4Hz, 1H), 3.65 (d, J = 12.8Hz, 1H),
4.04 (d, J = 12.4Hz, 1H), 4.55 (d, J = 13.1Hz, 1H), 6.89 (t, J
= 7.41Hz, 1H), 6.987.28 (m, 19H). 13C NMR (CDCl3 , 100
MHz) δ 51.16, 53.40, 54.01, 54.34, 70.12, 112.48, 112.58,
114.12, 114.38, 119.28, 122.90, 122.97, 126.08, 126.35,
127.68, 127.72, 128.35, 128.76, 129.23, 131.32, 132.53,
145.80, 150.55, 158.01, 159.05, 160.38. IR (KBr, cm–1)
2998, 2919, 1633, 1581, 1223, 1186, 1065; ESI-MS: 555.1
(C32H28BrN3Na, [M+Na]+). Anal. Calcd. for C32H28BrN3: C,
71.97; H, 5.28; N, 7.84; Found: C, 71.91; H, 5.28; N, 7.86.
1
Yield, 80%; chartreuse solid; mp 153155 °C; H NMR
(CDCl3, 400 MHz) δ 2.40 (t, J = 10.8 Hz, 1H), 3.12-3.26 (m,
3H), 3.51 (d, J =13.2Hz, 1H), 3.69 (d, J =13.2Hz, 1H), 4.02
(d, J =12.5Hz, 1H), 4.61 (d, J =13.6Hz, 1H), 6.80 (t, J
=7.3Hz, 1H), 6.826.88 (m, 8H), 7.037.06 (m, 4H),
7.097.16 (m, 2H), 7.217.25 (m, 3H); 13C NMR (CDCl3 ,
100 MHz) δ 50.11, 53.05, 57.46, 58.06, 71.14, 112.11,
112.23, 113.46, 114.88, 114.91, 119.50, 122.15, 124.56,
126.42, 126.66, 126.70, 127.55, 129.34, 130.34, 130.49,
130.83, 131.21, 131.97, 135.45, 136.11, 141.42, 155.10,
157.23, 161.20. IR (KBr, cm–1) 3020, 2917, 1633, 1577,
1265, 1207, 1012; ESI-MS: 532.2(C32H26F3N3Na, [M+Na]+).
Anal. Calcd. for C32H26F3N3: C,75.42; H, 5.14; N, 8.26.
Found: C, 75.43; H, 5.14; N, 8.25.
5-(4-Bromobenzyl)-7-(4-methylbenzylidene)-3,3a,4,5,6,7-
hexahydro-3-(4-methylphenyl)-2-phenyl-2H-pyrazolo[4,3-
c]pyridine (II24)
1
Yield, 76%; chartreuse solid; mp 200202 °C; H NMR
(CDCl3, 400 MHz) δ 2.33 (s, 6H), 2.46 (t, J = 10.3 Hz, 1H),
3.153.36 (m, 3H), 3.503.54 (m, 2H), 4.03 (d, J = 12.9
Hz,1H), 4.65 (d, J = 12.5Hz, 1H), 6.81 (t, J = 7.3Hz, 1H),
6.927.30 (m, 17H). 13C NMR (CDCl3 , 100 MHz) δ 20.16,
21.09, 53.26, 53.73, 54.18, 54.89, 71.25, 112.09, 112.16,
114.62, 119.13, 122.46, 122.87, 125.09, 125.31, 127.68,
127.85, 128.11, 128.46, 128.89, 130.32, 130.48, 132.18,
137.38, 138.54, 148.63, 158.68, 160.09. IR (KBr, cm–1)
3022, 2918, 1644, 1584, 1273, 1207, 1000; ESI-MS: 584.1
(C34H32BrN3 Na, [M+Na]+). Anal. Calcd. for C34H32BrN3: C,
72.57; H, 5.79; N, 7.44. Found: C, 72.59; H, 5.73; N, 7.47.
5-(4-Fluorobenzyl)-7-(4-fluorobenzylidene)-3,3a,4,5,6,7-
hexahydro-3-(4-fluorophenyl)-2-phenyl-2H-pyrazolo[4,3-
c]pyridine (II21)
1
Yield, 78%; chartreuse solid; mp 196197 °C; H NMR
(CDCl3, 400 MHz) δ 2.44 (t, J = 11.3 Hz, 1H), 3.143.28
(m, 3H), 3.54 (d, J = 13.0Hz, 1H), 3.68 (d, J = 13.4Hz, 1H),
4.04 (d, J = 13.5Hz, 1H), 4.55 (d, J = 12.6Hz, 1H), 6.83 (t, J
= 7.5Hz, 1H), 6.927.33 (m, 17H); 13C NMR (CDCl3 , 100
MHz) δ 53.12, 54.14, 54.46, 60.01, 70.12, 114.20, 114.28,
114.46, 115.03, 115.38, 119.42, 120.11, 124.36, 126.38,
126.69, 126.71, 127.70, 129.39, 131.08, 131.09, 131.43,
135.21, 135.97, 136.45, 138.16, 149.45, 151.13, 157.62,
159.98. IR (KBr, cm–1) 3022, 2918, 1603, 1584, 1273, 1200,
1082; ESI-MS: 532.3(C32H26F3N3Na, [M+Na]+). Anal. Calcd.
for C32H26F3N3: C,75.42; H, 5.14; N, 8.26. Found: C, 75.43;
H, 5.14; N, 8.25.
5-(4-Bromobenzyl)-7-(4-methoxylbenzylidene)-3,3a,4,5,6,7-
hexahydro-3-(4-methoxylphenyl)-2-phenyl-2H-pyrazolo 4,3-
c]pyridine (II25)
1
Yield, 85%; chartreuse solid; mp 203-205 °C; H NMR
(CDCl3, 400 MHz) δ 2.38 (t, J = 10.8 Hz, 1H), 3.14-3.37 (m,

708
Pang CC, et al. Sci China Chem June (2013) Vol.56 No.6
3H), 3.50-3.74 (m, 2H), 3.88(s, 6H), 4.00 (d, J = 13.9
Hz,1H), 4.58 (d, J =12.5Hz, 1H), 6.81 (t, J =7.3Hz, 1H),
6.96-7.38 (m, 17H). 13C NMR (CDCl3 , 100 MHz) δ 53.50,
54.02, 54.23, 54.40, 58.13, 70.48, 112.81, 113.52, 114.15,
119.31, 122.48, 124.61, 125.17, 126.29, 127.61, 127.63,
129.86, 132.01, 132.31, 135.57, 138.47, 145.82, 147.85,
149.01, 150.23, 157.98, 158.18. IR (KBr, cm–1) 3448, 1596,
1501, 1424, 1247, 1087, 1027; ESI-MS: 616.1
125.02, 125.50, 125.66, 127.67, 128.04, 128.38, 128.89,
132.07, 132.58, 133.24, 135.44, 138.85, 145.21, 147.95,
149.09, 149.75. IR (KBr, cm–1) 3444, 1643, 1419, 1082,
1022, 843, 751; ESI-MS: 479.2 (C31H28N4Na, [M+Na]+);
Anal. Calcd. for C31H28N4: C, 81.57; H, 6.19; N, 12.28.
Found: C, 81.55; H, 6.18; N, 12.27.
7-(4-Methylbenzylidene)-5-(3-pyridylmethyl)-3,3a,4,5,6,7-
hexahydro-3-(4-methylphenyl)-2-phenyl-2H-pyrazolo 4,3-
c]pyridine (II29)
(C34H32BrN3NaO2,
[M+Na]+).
Anal.
Calcd.
for
C34H32BrN3O2: C, 68.67; H, 5.49; N, 7.04. Found: C, 68.69;
H, 5.43; N, 7.07.
1
Yield, 79%; chartreuse solid; mp 217219 °C; H NMR
(CDCl3, 400 MHz) δ 2.132.26 (m, 1H), 2.36(s, 6H),
3.173.31 (m, 3H), 3.433.62 (m, 2H), 4.07 (d, J = 14.2
Hz,1H), 4.66 (d, J = 12.6Hz, 1H), 6.76 (t, J = 7.2Hz, 1H),
7.077.52 (m, 15H), 8.41 (d, J = 3.4Hz, 2H). 13C NMR
(CDCl3 , 100 MHz) δ 20.11, 20.26, 53.56, 54.10, 54.43,
56.09, 70.65, 114.09, 119.28, 122.44, 125.02, 125.50,
125.66, 127.67, 128.04, 128.38, 128.89, 132.07, 135.53,
136.28, 136.44, 137.44, 145.71, 147.75, 148.99, 150.15. IR
(KBr, cm–1) 3451, 1643, 1602, 1419, 1084, 1026, 750;
ESI-MS: 507.2(C33H32N4Na, [M+Na]+); Anal. Calcd. for
C33H32N4: C, 81.77; H, 6.69; N, 11.58. Found: C, 81.78; H,
6.66; N, 11.56.
5-(4-Bromobenzyl)-7-(4-fluorobenzylidene)-3,3a,4,5,6,7-
hexahydro-3-(4-fluorophenyl)-2-phenyl-2H-pyrazolo 4,3-
c]pyridine (II26)
1
Yield, 81%; chartreuse solid; mp 197199 °C; H NMR
(CDCl3, 400 MHz) δ 2.46 (t, J = 10.3 Hz, 1H), 3.15-3.36 (m,
3H), 3.503.54 (m, 2H), 4.03 (d, J = 12.9 Hz, 1H), 4.65 (d,
J = 12.8Hz, 1H), 6.81 (t, J = 7.3Hz, 1H), 6.927.30 (m,
17H). 13C NMR (CDCl3 , 100 MHz) δ 53.23, 54.08, 54.47,
60.06, 70.10, 114.12, 114.25, 114.46, 115.09, 115.30,
119.58, 120.14, 124.34, 126.47, 126.64, 126.72, 127.75,
129.35, 130.01, 130.09, 130.45, 130.97, 131.00, 135.55,
136.12, 136.15, 145.42, 150.10, 159.73, 162.20. IR (KBr,
cm–1) 3447, 1597, 1496, 1088, 1027, 835, 753; ESI-MS:
592.1 (C32H26BrF2N3Na, [M+Na]+). Anal. Calcd. for
C32H26BrF2N3: C, 67.37; H, 4.59; N, 7.34. Found: C, 67.37;
H, 4.59; N, 7.37.
7-(4-Isopropylbenzylidene)-5-(3-pyridylmethyl)3,3a,4,5,6,7-
hexahydro-3-(4-isopropylphenyl)-2-phenyl-2H-pyrazolo 4,3-
c]pyridine (II30)
1
Yield, 80%; chartreuse solid; mp 215216 °C; H NMR
(CDCl3, 400 MHz) δ 1.46 (m,12H), 2.522.54 (m, 1H), 3.12
(m, 2H), 3.163.30 (m, 3H), 3.633.71 (m, 2H), 4.04 (d, J =
14.2Hz,1H), 4.66 (d, J = 12.6Hz,1H), 6.87 (t, J = 7.2Hz,1H),
6.977.75 (m,15H), 8.51(d, J = 3.2Hz, 2H). 13C NMR
(CDCl3 , 100 MHz) δ 18.28, 19.42, 30.11, 30.26, 51.55,
53.92, 54.43, 58.59, 70.65, 112.23, 119.26, 122.45, 125.02,
125.58, 125.83, 127.69, 128.14, 128.28, 129.21, 131.27,
133.53, 135.41, 135.82, 138.49, 145.71, 147.75, 148.99,
151.18. IR (KBr, cm–1) 3451, 1644, 1602, 1495, 1419, 1384,
1293, 1084; ESI-MS: 563.3(C37H40NaN4, [M+Na]+); Anal.
Calcd. for C37H40N4: C, 82.17; H, 7.49; N, 10.38. Found: C,
82.18; H, 7.46; N, 10.36.
5-(4-Bromobenzyl)-7-(4-chlorobenzylidene)-3,3a,4,5,6,7-
hexahydro-3-(4-chlorophenyl)-2-phenyl-2H-pyrazolo 4,3-c]
pyridine (II27)
1
Yield, 79%; chartreuse solid; mp 206207 °C; H NMR
(CDCl3, 400 MHz) δ 2.37 (t, J = 11.3 Hz, 1H), 3.173.46
(m, 3H), 3.553.62 (m, 2H), 4.17 (d, J = 12.9 Hz, 1H), 4.75
(d, J = 12.5Hz, 1H), 6.87 (t, J = 7.1Hz, 1H), 6.907.34 (m,
17H). 13C NMR (CDCl3 , 100 MHz) δ 53.37, 53.87, 54.33,
58.12, 70.16, 114.11, 119.77, 122.45, 124.31, 126.43,
126.93, 127.51, 127.70, 128.45, 129.56, 131.84, 132.40,
132.50, 133.50, 135.30, 138.63, 138.81, 145.28, 147.93,
149.07, 149.72. IR (KBr, cm–1) 3449, 1643, 1594, 1487,
1088, 1027, 829; ESI-MS: 624.0 (C32H26BrCl2N3Na, [M+
Na]+). Anal. Calcd. for C32H26BrCl2N3: C, 63.68; H, 4.30; N,
6.94. Found: C, 63.70; H, 4.34; N, 6.96.
7-(4-Methoxylbenzylidene)-5-(3-pyridylmethyl)-3,3a,4,5,6,7-
hexahydro-3-(4-methoxylphenyl)-2-phenyl-2H-pyrazolo[4,3-
c]pyridine (II31)
1
Yield, 77%; chartreuse solid; mp 187189 ℃; H NMR
7-Benzylidene-5-(3-pyridylmethyl)-3,3a,4,5,6,7-hexahydro-
(CDCl3, 400 MHz) δ 2.532.56 (m, 1H), 3.163.30 (m, 3H),
3.563.72 (m, 2H), 3.78 (s, 6H), 3.97 (d, J = 14.2 Hz,1H),
4.56 (d, J = 12.6Hz, 1H), 6.77 (t, J = 7.2Hz, 1H), 6.977.68
(m, 15H), 8.41 (d, J = 3.1Hz, 2H); 13C NMR (CDCl3 , 100
MHz) δ 53.55, 54.03, 54.25, 54.41, 58.12, 70.44, 112.80,
113.59, 114.18, 119.30, 122.45, 124.61, 125.19, 126.27,
127.60, 127.65, 129.88, 132.07, 132.36, 135.51, 145.80,
147.80, 149.02, 150.29, 157.99, 158.12. IR (KBr, cm–1)
2,3-diphenyl-2H-pyrazolo[4,3-c]pyridine (II28)
1
Yield, 71%; chartreuse solid; mp 213215 °C; H NMR
(CDCl3, 400 MHz) δ 2.632.68 (m, 1H), 3.37 (s, 3H),
3.713.83 (m, 2H), 4.06 (d, J = 14.3 Hz,1H), 4.64 (d, J =
12.6 Hz, 1H), 6.86 (t, J = 7.2Hz, 1H), 7.027.45 (m, 17H),
8.50(d, J = 3.3 Hz ,2H). 13C NMR (CDCl3 , 100 MHz) δ
53.52, 54.09, 54.43, 57.21, 70.16, 114.09, 119.29, 122.35,

Pang CC, et al. Sci China Chem June (2013) Vol.56 No.6
709
3452, 1644, 1496, 1419, 1083, 1027, 750; ESI-MS:
539.2(C33H32N4NaO2, [M+Na]+); Anal. Calcd. for
C33H32N4O2: C, 76.77; H, 6.29; N, 10.88. Found: C, 76.72;
H, 6.24; N, 10.84.
7-Benzylidene-5-(2-furfuryl)-3,3a,4,5,6,7-hexahydro-2,3-
diphenyl-2H-pyrazolo[4,3-c]pyridine (II35)
1
Yield, 77%; chartreuse solid; mp 223225 °C; H NMR
(400 MHz, CDCl3) δ 2.52 (s, 1H), 3.213.43 (m, 3H),
3.663.98 (m, 2H), 4.06 (d, J = 14.1Hz, 1H), 4.63 (d, J =
12.4 Hz, 1H), 6.17（s，1H）, 6.31（s，1H）, 6.80 (t, J = 7.2Hz，
7-(3-Fluorobenzylidene)-5-(3-pyridylmethyl)-3,3a,4,5,6,7-
hexahydro-3-(3-fluorophenyl)-2-phenyl-2H-pyrazolo [4,3-
c]pyridine (II32)
1H), 7.077.51 (m, 16H). 13C NMR (CDCl3 , 100 MHz) δ
52.37, 52.81, 53.26, 54.25, 70.59, 107.97, 109.59, 114.28,
119.21, 125.21, 125.47, 125.86, 127.32, 127.87, 128.35,
128.85, 132.55, 136.29, 136.33, 137.63, 141.69, 145.86,
150.18, 150.42. IR (KBr, cm–1) 3444, 1641,1599, 1494,
1436,1081,749; ESI-MS:468.2 (C30H27N3NaO, [M+Na]+);
Anal. Calcd. for C30H27N3O: C, 80.83; H, 6.15; N, 9.49.
Found: C, 80.87; H, 6.11; N, 9.43.
1
Yield, 73%; chartreuse solid; mp 205206 °C; H NMR
(CDCl3, 400 MHz) δ 2.572.59 (m, 1H), 3.173.31 (m, 3H),
3.573.73 (m, 2H), 4.07 (d, J = 14.2 Hz,1H), 4.59 (d, J =
12.6Hz, 1H), 6.85 (t, J = 7.2Hz, 1H), 6.967.61 (m, 15H),
8.45 (d, J = 3.2 Hz, 2H). 13C NMR (CDCl3 , 100 MHz) δ
53.01, 53.38, 54.28, 58.28, 70.11, 112.72, 114.10, 114.17,
114.38, 115.17, 115.25, 119.51, 124.23, 126.67, 126.74,
127.73, 129.05, 130.06, 130.14, 131.09, 131.13, 136.22,
136.25, 145.73, 151.78, 159.69, 162.10. IR (KBr, cm–1)
3451, 1643, 1495, 1419, 1024, 750; ESI-MS: 515.2
(C31H26F2NaN4, [M+Na]+); Anal. Calcd. for C31H26F2N4: C,
75.57; H, 5.33; N, 11.38. Found: C, 75.59; H, 5.32; N,
11.37.
5-(2-Furfuryl)-7-(4-methylbenzylidene)-3,3a,4,5,6,7-
hexahydro-3-(4-methylphenyl)-2-phenyl-2H-pyrazolo[4,3-
c]pyridine (II36)
1
Yield, 79%; chartreuse solid; mp 227228 °C; H NMR
(400 MHz, CDCl3) δ 2.21 (d, J = 13.0 Hz, 1H), 2.38 (s, 6H) ,
3.213.31 (m, 3H), 3.653.75 (m, 2H), 4.05 (d, J = 14.2
Hz,1H), 4.59 (d, J = 11.2 Hz, 1H), 6.14 (s, 1H), 6.32 (s, 1H),
6.83 (t, J = 7.1 Hz, 1H), 7.037.38 (m,14H); 13C NMR
(CDCl3 , 100 MHz) δ 20.11, 20.28, 52.68, 52.76, 53.77,
54.65, 70.57, 107.97, 109.09, 114.07, 119.18, 125.04,
125.33, 125.93, 127.62, 127.97, 128.48, 128.82, 132.26,
136.28, 136.31, 137.57, 141.28, 145.80, 150.12, 150.35. IR
(KBr, cm–1) 3445,2357, 1643, 1495, 1420, 1292, 1082,
1022; ESI-MS: 496.2(C32H31N3NaO, [M+Na]+); Anal.
Calcd. for C32H31N3O: C, 81.17; H, 6.69; N, 8.88. Found: C,
81.15; H, 6.60; N, 8.87.
7-(4-Fluorobenzylidene)-5-(3-pyridylmethyl)-3,3a,4,5,6,7-
hexahydro-3-(4-fluorophenyl)-2-phenyl-2H-pyrazolo [4,3-
c]pyridine (II33)
1
Yield, 79%; chartreuse solid; mp 163164 °C; H NMR
(CDCl3, 400 MHz) δ 2.532.56 (m, 1H), 3.163.30 (m, 3H),
3.563.72 (m, 2H), 4.07 (d, J = 14.2 Hz,1H), 4.56 (d, J =
12.6 Hz, 1H), 6.77 (t, J = 7.2Hz, 1H), 6.977.68 (m, 15H),
8.41 (d, J = 3.1Hz, 2H). 13C NMR (CDCl3 , 100 MHz) δ
52.01, 54.07, 56.11, 57.59, 71.07, 114.35, 119.86, 122.32,
124.64, 126.28, 126.95, 127.58, 128.19, 128.55, 129.44,
131.46, 132.74, 132.98, 133.68, 135.50, 138.83, 146.20,
148.12, 151.36, 159.65. IR (KBr, cm–1) 3453, 1644, 1484,
1417, 1085, 1022, 833; ESI-MS: 515.2(C31H26F2N4Na,
[M+Na]+); Anal. Calcd. for C31H26F2N4: C, 81.57; H, 6.19;
N, 12.28. Found: C, 81.55; H, 6.18; N, 12.27.
5-(2-Furfuryl)-7-(4-isopropylbenzylidene)-3,3a,4,5,6,7-
hexahydro-3-(4-isopropylphenyl)-2-phenyl-2H-pyrazolo
[4,3-c]pyridine (II37)
1
Yield, 71%; chartreuse solid; mp 222223 °C; H NMR
(400 MHz, CDCl3) δ 1.55 (m,12H) , 2.522.54 (m, 1H),
3.12 (m, 2H) 3.163.30 (m, 3H), 3.623.69 (m, 2H), 4.03 (d,
J=14.2 Hz, 1H),4.65 (d, J = 12.6Hz, 1H), 6.13 (s, 1H), 6.34
(s,1H), 6.87 (t, J = 7.2Hz, 1H), 7.117.72 (m, 14H).
13C NMR (CDCl3 , 100 MHz) δ 18.01, 18.48, 30.22, 31.04,
51.08, 52.76, 55.43, 58.86, 70.57, 107.03, 109.21, 112.15,
119.35, 122.43, 122.64, 124.86, 126.42, 127.84, 128.49,
128.96, 131.88, 135.30, 135.78, 138.52, 141.28, 146.80,
152.25, 158.48. IR (KBr, cm–1) 3444, 2356, 1642, 1494,
1421, 1291, 1081, 1032; ESI-MS: 552.3(C36H39N3NaO,
[M+Na]+); Anal. Calcd. for C36H39N3O: C, 81.67; H, 7.49;
N, 7.92. Found: C, 81.63; H, 7.42; N, 7.93.
7-(4-Chlorobenzylidene)-5-(3-pyridylmethyl)-3,3a,4,5,6,7-
hexahydro-3-(4-chlorophenyl)-2-phenyl-2H-pyrazolo[4,3-
c]pyridine (II34)
1
Yield, 86%; chartreuse solid; mp 156157 °C; H NMR
(CDCl3, 400 MHz) δ 2.472.51 (m, 1H), 3.123.29 (m, 3H),
3.533.73 (m, 2H), 4.11 (d, J = 14.2 Hz,1H), 4.56 (d, J =
12.6 Hz, 1H), 6.87 (t, J = 7.2Hz, 1H), 7.017.59 (m, 15H),
8.41(d, J = 3.3Hz, 2H). 13C NMR (CDCl3 , 100 MHz) δ
53.35, 53.90, 54.38, 58.10, 70.12, 114.11, 119.74, 122.44,
124.30, 126.44, 126.93, 127.57, 127.79, 128.49, 129.60,
131.86, 132.41, 132.56, 133.56, 135.34, 138.85, 145.21,
147.96, 149.04, 149.74. IR (KBr, cm–1) 3451, 1643, 1486,
1416, 1085, 1021, 833; ESI-MS: 547.1(C31H26Cl2N4Na,
[M+Na]+); Anal. Calcd. for C31H26Cl2N4: C, 70.83; H, 4.95;
N, 10.69. Found: C, 70.86; H, 4.99; N, 10.66.
5-(2-Furfuryl)-7-(4-methoxylbenzylidene)-3,3a,4,5,6,7-
hexahydro-3-(4-methoxylphenyl)-2-phenyl-2H-pyrazolo[4,3-
c]pyridine (II38)
1
Yield, 81%; chartreuse solid; mp 225226 °C; H NMR
(400 MHz, CDCl3) δ 2.272.34 (m, 1H), 3.123.21 (m, 3H),

710
Pang CC, et al. Sci China Chem June (2013) Vol.56 No.6
129.82, 130.33, 130.46, 132.47, 139.68, 145.33, 149.26,
159.56, 161.49. IR (KBr, cm–1) 3446, 1641, 1601, 1495,
1435, 1082, 889; ESI-MS: 536.1 (C30H25Cl2N3NaO,
[M+Na]+); Anal. Calcd. for C30H25Cl2N3O: C, 70.03; H,
4.95; N, 8.19. Found: C, 70.04; H, 4.90; N, 8.17.
3.633.74 (m, 2H), 3.77 (s, 3H), 3.78 (s, 3H), 4.07 (d, J =
13.9 Hz, 1H), 4.59 (d, J = 11.2 Hz, 1H), 6.18 (s, 1H), 6.37
(s, 1H), 6.81 (t, J = 7.2Hz, 1H), 7.037.47 (m, 14H);
13C NMR (CDCl3 , 100 MHz) δ 50.08, 52.73, 53.97, 54.53,
54.68, 70.35, 107.62, 109.22, 114.82, 119.15, 122.94,
124.33, 125.27, 127.30, 127.47, 128.27, 128.82, 132.53,
136.28, 136.31, 137.57, 141.28, 145.87, 150.52, 158.05. IR
(KBr, cm–1) 3444, 1643, 1594, 1420, 1082, 1021, 811, 548;
ESI-MS: 528.2(C32H31N3NaO3, [M+Na]+); Anal. Calcd. for
C32H31N3O3: C, 76.07; H, 6.19; N, 8.32. Found: C, 76.02; H,
6.18; N, 8.31.
2.3 X-ray crystallography
Compound (II1) was recrystallized by slow evaporation
from a mixed solution of methylene dichloride/ethanol (v:v
= 1:5). A chartreuse crystal of (II1) with dimensions of 0.16
mm x 0.15 mm x 0.10 mm was mounted on a glass fiber for
data collection which was made on a Bruker SMART
APEX 1000 CCD diffractometer equipped with a graph-
ite-monochromatic Mo K radiation (λ = 0.71073 Å) by
using a φ–ω scan mode in the range 2.35  θ  20.55° (–7 
h  7, –11  k  11, –69  l  69) at 298(2) K. A total of
30742 reflections were collected with 6366 unique ones
(Rint = 0.0545), of which 3804 with I > 2σ(I) were consid-
ered as observed and used in the succeeding refinements.
The intensity data were corrected for Lp factors and empir-
ical absorption. All of the non-hydrogen atoms were refined
anisotropically, and hydrogen atoms were located at their
idealized positions. The structure was solved by direct
methods and refined by full-matrix least-squares techniques
on F2 using the SHELXTL program package. All of the
non-hydrogen atoms were refined anisotropically, and hy-
drogen atoms were located at their idealized positions. The
final R = 0.0545, wR = 0.1211 (w = 1/[2(Fo2) + (0.0568P)2
+ 0.2960P], where P = (Fo2 + 2Fc2)/3), S = 1.025, (∆/σ)max
= 0.093, (∆ρ)max = 0.186 and (∆ρ)min = –0.189 e/Å3. The
structural graphics were drawn with the SHELXTL-97
software package. Other details of the structure have been
deposited with the Cambridge Crystallographic Data Centre,
CCDC 845735.
5-(2-Furfuryl)-7-(3-fluorobenzylidene)-3,3a,4,5,6,7-hexahy
dro-3-(3-fluorophenyl)-2-phenyl-2H-pyrazolo[4,3-c]pyridine
(II39)
1
Yield, 82%; chartreuse solid; mp 231232 °C; H NMR
(400 MHz, CDCl3) δ 2.532.56 (m, 1H), 3.173.31 (m, 3H),
3.573.73 (m, 2H), 4.07 (d, J = 14.2 Hz, 1H), 4.59 (d, J =
12.6 Hz, 1H), 6.15 (s, 1H), 6.32 (s, 1H), 6.85 (t, J = 7.2 Hz,
1H), 7.047.59 (m, 14H); 13C NMR (CDCl3 , 100 MHz) δ
53.02, 54.53, 54.97, 60.03, 70.15, 108.12, 109.66, 114.15,
118.96, 120.94, 124.36, 126.46, 127.62, 127.79, 128.47,
128.62, 130.53, 135.28, 135.31, 137.57, 145.08, 150.07,
150.52, 154.05. IR (KBr, cm–1) 3448, 1642, 1599, 1493,
1438, 1083, 749; ESI-MS: 504.1(C30H25F2N3NaO,
[M+Na]+). Anal. Calcd. for C30H25F2N3O: C, 74.81; H, 5.24;
N, 8.74. Found: C, 74.83; H, 5.23; N, 8.73.
5-(2-Furfuryl)-7-(4-fluorobenzylidene)-3,3a,4,5,6,7-hexahy
dro-3-(4-fluorophenyl)-2-phenyl-2H-pyrazolo[4,3-c]pyridine
(II40)
1
Yield, 79%; chartreuse solid; mp 237238 °C; H NMR
(400 MHz, CDCl3) δ 2.432.49 (m, 1H), 3.153.32 (m, 3H),
3.473.63 (m, 2H), 4.07 (d, J = 14.1 Hz, 1H), 4.49 (d, J =
12.7Hz, 1H), 6.11 (s, 1H), ,6.37 (s, 1H), 6.81 (t, J = 7.2Hz,
1H), 7.017.72 (m, 14H); 13C NMR (CDCl3 , 100 MHz) δ
53.01, 53.09, 54.19, 60.13, 71.11, 107.57, 109.04, 112.65,
119.56, 122.24, 124.36, 126.73, 127.52, 127.82, 129.17,
129.82, 132.33, 134.56, 135.31, 138.68, 145.74, 151.26,
152.56, 155.47. IR (KBr, cm–1) 3445, 1640, 1597, 1495,
1436, 1081, 749; ESI-MS: 504.2 (C30H25F2N3NaO,
[M+Na]+); Anal. Calcd. for C30H25F2N3O: C, 74.80; H, 5.23;
N, 8.75. Found: C, 74.83; H, 5.23; N, 8.73.
2.4 Fluorescence measurements
The single-photon fluorescence spectra were collected on a
PerkinElmer LS55 luminescence spectrometer. Visible ab-
sorption spectra were determined on PerkinElmer Lambda
35 spectrophotometer.
The solution concentration of the compounds in CH2Cl2
was 1.0 × 10–5 mol L–1 and the samples were excited at a
wavelength of 360 nm. The photoluminescence quantum
efficiencies of the compounds and their derivatives were
calculated using 9,10-diphenylanthracene as the standard
[19].
5-(2-Furfuryl)-7-(4-chlorobenzylidene)-3,3a,4,5,6,7-hexahy
dro-3-(4-chlorophenyl)-2-phenyl-2H-pyrazolo[4,3-c]pyridi-
ne (II41)
1
Yield, 89%; chartreuse solid; mp 234235 °C; H NMR
(400 MHz, CDCl3) δ 2.232.46 (m, 1H), 3.123.31 (m, 3H),
3.543.71 (m, 2H), 4.03 (d, J = 14.1 Hz, 1H), 4.61 (d, J =
12.6Hz, 1H), 6.25 (s, 1H), 6.42 (s, 1H), 6.95 (t, J = 7.2Hz,
1H), 7.147.74 (m, 14H); 13C NMR (CDCl3 , 100 MHz) δ
52.92, 53.32, 53.88, 58.20, 70.08, 107.60, 109.25, 114.21,
119.56, 122.24, 122.86, 124.13, 126.52, 127.24, 128.43,
2.5 Biology
2.5.1 Cell lines and culture
The anti-breast cancer activity tests of the 2H-pyrazolo
[4,3-c]hexahydropyridine derivatives (II) were evaluated

Pang CC, et al. Sci China Chem June (2013) Vol.56 No.6
711
with human breast cancer MCF-7 and MDA-MB-231 cell
lines by the standard MTT assay in vitro [20]. Human breast
cancer cells MCF-7 and MDA-MB-231 cell lines were cul-
tured in RPMI 1640 medium at 37 °C with 5% CO2, and
95% air, supplemented with 10% (v/v) bovine calf serum
and 80 U mL1 gentamicin. The cells were seeded onto
96-well plates at the density of 6250 cm2. About 10 mg of
the title compounds were dissolved in 100 μL of dimethyl-
sulfoxide (DMSO), then concentrations of 1.0, 10.0, and
100.0 μg mL were adjusted stepwise in each 96-well
plates, and the concentration of DMSO was 1‰. One hun-
dred thousand cells, 20 μL of FBS, RPMI-1640 and differ-
ent concentrations of the title compounds were seeded in
each 100 μL well of a 96-well plate. Each group had eight
wells. The control well of 5-fluorouracil (5-FU) was estab-
lished and the wells (containing cells) were incubated. After
44 h in a humidified atmosphere with 5% CO2 at 37 °C, 10
μL of MTT (5 mg/mL) was added to every well, and 100 μL
of 10% sodium dodecyl sulfate (SDS) solution was added to
stop the reaction after 4 h. After leaving overnight at 37 °C,
the number of absorbency (A) of every well was measured
by enzyme immunity at 570 nm.
firmed by single crystal X-ray diffraction. Figure 1 shows
that the structure is composed of six rings: four benzene
rings, one hexahydropyridine ring and one dihydropyrazolo
ring. The benzene rings are marked A, B, C, and D. The
dihedral angle between the mean planes of the benzene A
and benzene B rings is 60.06 (0.05)°, between benzene C
and benzene D rings is 79.16 (0.06)°. In the structure of
compound II1, the hexahydropyridine ring adopts a skew
boat conformation and the dihedral angle between the ben-
zene A and hexahydropyridine (N1–C9–C11–C12) rings is
86.46 (0.06)°. The carbon–carbon double bonds (C10–C14)
in II1 clearly adopt an (E)-configuration. The steric repul-
sion is reduced by the expansion of the bond angle
C(15)–C(14)–C(10) (132.16o) relative to the normal angle
of 120o.
The relative stereochemistry of the remaining com-
pounds II was determined by NMR analysis. The anti and
syn isomers were identified by the coupling constants (J) of
the vicinal protons adjacent to N–Ph and CH2 groups in
1
their H NMR spectra. The coupling constants (J) of the
1
anti isomer are higher than those of the syn isomer. The H
NMR spectrum of (II1) showed two doubles at δ = 4.02,
4.61 from CH protons, and the corresponding coupling con-
stant is J = 12.4; this indicates that compounds (II) have the
anti configuration. We suggest that the anti configuration of
structure (II1) is the thermodynamically more stable config-
uration because of the exclusion between crowded aryl sys-
tems adjacent to N=C and CH moieties (Figure 2).
2.5.2 Apoptosis detection
Fluorescein isothiocyanate (FITC)-conjugated annexin V
was utilized to detect the externalization of phosphatidyl-
serine that occurs at an early stage of apoptosis. Propidium
iodide (PI) was used as a marker of necrosis due to cell
membrane destruction. To elucidate whether compound-
induced cell death involved apoptosis or necrosis, we per-
formed a flow cytometric analysis using annexin V and PI
double-staining.
3.2 Bioactivity
The anti-breast cancer activity tests in vitro of the 2H-
pyrazolo[4,3-c]hexahydropyridine derivatives (II) along
with 5-fluorouracil for comparison were carried out on hu-
man breast cancer MCF-7 and MDA-MB-231 cell lines.
The screening results expressed as IC50 are summarized in
Table 1. Most of the compounds showed good inhibitory
activity against breast cancer cells, among them, compound
II33 found to be the most potent compound with IC50 values
of 2.4 μM, 4.2 μM against MCF-7 and MDA-MB-231, re-
spectively, an improvement of more than two-fold com-
pared with 5-fluorouracil (IC50 = 4.8 μM for MCF-7 and
IC50 =9.6 μM for MDA-MB-231, respectively) and com-
pounds II21 and II40 also displayed better inhibitory activity
than 5-fluorouracil.
The activity of the tested compounds can be correlated
with the structure modifications. By investigating the tested
compounds with the two cell lines, it was found that differ-
ent A rings led to different anti-breast cancer activity. When
the substituents on B and C rings were constant, compounds
(II28–II41) with a heteroaromatic A ring showed better ac-
tivities than when A was a benzene ring (II1–II27). When A
was a benzene ring, change of substituents on the ring also
affects the activities of the compounds. A fluoro substituent
2.5.3 Electrophoretic analysis of DNA fragmentation
Human breast cancer cells MCF-7 were lysed in 200.0 mL
lysis buffer (10.0 mM EDTA:50.0 mM Tris–HCl, pH 8.0;
0.5% sodium lauryl sulfate; 100.0 mg mL1 proteinase K) at
37 °C for 12 h, then incubated with RNase (50.0 mg mL1)
at 37 °C for an additional 1 h. After incubation, DNA in the
lysate was extracted with an equal volume of phe-
nol/chloroform/isoamyl alcohol (25:24:1), then with chlo-
roform. DNA was precipitated with two volumes of ethanol
in the presence of 0.3 M sodium acetate. After centrifuga-
tion at 12,000 g for 15 min, the DNA pellets were washed
with 70% ethanol, air-dried, and resuspended in 20.0 mL of
TE (10.0 mM Tris–HCl and 1.0 mM EDTA, pH 8.0). DNA
was separated on 1.5% agarose gels containing 0.5 mg mL1
ethidium bromide and photographed by Bio-Rad GD2000
(Bio-Rad, Hercules, CA, USA).
3 Results and discussion
3.1 Crystal structure analysis
The structure of compound (II1) was unambiguously con-

712
Pang CC, et al. Sci China Chem June (2013) Vol.56 No.6
Figure 1 Molecular structure of compound II1 (Number CCDC 845735) with atom-labeling.
compounds with a meta-fluoro substituent. Our results
therefore show that the anti-breast cancer activities of our
compounds were largely influenced by the A ring and the
anti-breast cancer activities of these compounds could be
increased by the introduction of a 3-pyridyl ring or 2-furyl
ring as the A ring.
3.3 Apoptosis in MCF-7 cells and DNA fragmentation
induced by II33
Figure 2 The structures of the anti and syn isomers of II1.
Owing to the notable cytotoxic effect of II33, we performed
further tests to elucidate whether its proliferation inhibitory
of MCF-7 cells was related to apoptosis. MCF-7 cells which
treated with II33 at concentrations of 1, 10 and 100 μg mL1
double stained with annexin V-FITC and propidium iodide
(PI) were analyzed by flow cytometry for 24 h. Annexin V
staining in cells make it possible to detect the expression of
phosphatidylserine on the plasma membrane surface, which
is a characteristic related to apoptosis. As shown in Figure
3(a), the proportion of cells stained with annexin V was
increased with II33 treatment, analyzed by flow cytometry.
These results suggested that II33 induced cytotoxicity of
MCF-7 cells by the induction of apoptosis.
The parameter which was selected to assess the DNA
damage upon treatment with II33 was chromosomal DNA
fragmentation. The chromosomal DNA extracted from the
MCF-7 cells treated with increasing concentrations (10, 50
and 100 μg mL1) of II33 after 72 h, was used for agarose
gel electrophoresis. The results showed fragmentation of the
DNA leading to a smear in the lanes in which cells were
treated with II33 (Figure 3(b)). The observed smear is the
result of DNA breakage at multiple positions across the
on the benzene ring leads to increased activity, and a para-
fluoro substituent on the A ring gave better anticancer activ-
ities than the corresponding compound with an ortho-fluoro
substituent. For example, the potency order decreased in the
order 3-pyridyl (II33) > 2-furyl (II40) > 4- fluorophenyl
(II21) > 2-fluorophenyl (II14) > 4-bromophenyl (II26) > 4-
methoxylphenyl (II9) > 4-methylphenyl (II4).
Regarding the compounds (II28–II34) which all have a
3-pyridyl A ring, compounds with electron-withdrawing
substituents (II32 –II34) in the B and C rings showed more
potent activities than those with electron-donating substitu-
ents (II29 –II31) in these rings. A comparison of the substit-
uents on the
B and C rings demonstrated that electron-withdrawing
groups II32, II33, and II34 gave significantly improved anti-
proliferative activity, whereas Me (II29), CH(CH3)2 (II30)
and OCH3 (II31) substituents gave slightly reduced antipro-
liferative activity compared with II28, which has no substit-
uent. Moreover, compounds with a para-fluoro substituent
on the B and C rings showed better anticancer activity than

Pang CC, et al. Sci China Chem June (2013) Vol.56 No.6
713
Table 1 Inhibition (IC50) of MCF-7 and MDB-MB-231 cells proliferation by compounds (II1 –II41)
IC50 (μM)
IC50 (μM)
MDB-MB-231
Compound
Compound
MCF-7
>50
>50
>50
48.3
>50
>50
>50
>50
42.8
44.2
40.1
40.3
40.3
9.6
MDB-MB-231
>50
MCF-7
14.9
41.8
46.4
48.2
27.6
33.2
10.6
13.8
16.2
21.6
17.6
2.4
II1
II2
II22
II23
II24
II25
II26
II27
II28
II29
II30
II31
II32
II33
II34
II35
II36
II37
II38
II39
II40
II41
5-FU
12.7
>50
>50
>50
33.5
39.2
14.7
19.4
23.4
32.4
26.5
4.2
>50
II3
>50
II4
>50
II5
>50
II6
>50
II7
>50
II8
>50
II9
38.6
41.5
34.4
36.2
38.8
20.3
24.5
36.3
37.2
42.9
44.8
28.6
9.3
II10
II11
II12
II13
II14
II15
II16
II17
II18
8.6
14.2
26.8
29.3
33.4
40.6
21.2
8.6
23.6
29.6
30.5
32.8
16.8
3.3
16.4
32.4
35.4
38.4
38.6
23.5
4.7
II19
II20
II21
7.9
17.5
9.6
4.8
Figure 3 Effects of compound II33 on MCF-7 cells. (a) Flow cytometric analysis of phosphatidylserine externalization and propidium iodide at 1, 10 and
100 μg mL1 for 24 h; (b) DNA ladder assay. Lane 1: DMSO; Lane 2–4: MCF-7 cells treated with 10, 50 and 100 μg mL1, respectively. ‘M’ is a Marker.

714
Pang CC, et al. Sci China Chem June (2013) Vol.56 No.6
chromosomal DNA. The intensity of the smear increased
with the dose. In case of II33, a dose of 50 μg mL1 showed
moderate smearing and a dose of 100 μg mL1 showed
maximum smearing.
and the intensity decreased in the order: 2-F (II11) > 4-F
(II16) > 4-OCH3 (II6)> 4-CH3 (II1) > 4-Br (II23).
The photoluminescence data are summarized in Table 2.
The photoluminescent properties of these compounds may
indicate their great potential for numerous optical applica-
tions and for use as medicinal biomarkers.
3.4 Fluorescence measurements
Photoluminescence spectra were studied for the compounds
II1, II6, II11, II16, II23, II28, and II35 with excitation at 360
nm. The most striking feature was that all six compounds
gave an intense emission and the photoluminescence spectra
of these compounds in dichloromethane are shown in Figure
4. The maximum luminescence intensity was observed at
773 nm for compound II28 with a photoluminescence quan-
tum yield of 39%. When A ring was 3-pyridyl (II28) or
2-furfuryl (II35), this led an increase in the intensity of the
main peaks and a shift to higher emission wavelength.
When A was a benzene ring, different substituents on the
benzene ring lead to different intensities of the main peaks
4 Conclusion
In an attempt to develop more potent anti-breast cancer
agents, a series of previously unreported 2H-pyrazolo
[4,3-c]hexahydropyridine derivatives (II) have been synthe-
sized. The cytotoxicity of the target compounds have been
evaluated in vitro against two human breast cancer cell lines
MCF-7 and MDA-MB-231 by MTT assay. Most com-
pounds exhibited good inhibition, among them compounds
II21 (IC50 = 4.7 μM for MCF-7 and IC50 = 9.3 μM for
MDA-MB-231), II33 (IC50 = 2.4 μM for MCF-7 and IC50
=
4.2 μM for MDA-MB-231) and II40 (IC50 = 3.3 μM for
MCF-7 and IC50 =8.6 μM for MDA-MB-231) displayed
better inhibitory activity than 5-fluorouracil (IC50 = 4.8 μM
for MCF-7 and IC50 =9.6 μM for MDA-MB-231). Structure-
activity relationships further indicated that the anti-breast
cancer activities of our compounds were largely influenced
by the nature of the A ring and the anti-breast cancer activi-
ties could be increased by the introduction of a 3-pyridyl
ring or a 2-furyl ring as the A ring. The fluorescence prop-
erties of compounds II1, II6, II11, II16, II23, II28, and II35
were studied and the photoluminescent properties of these
compounds may indicate their great potential for numerous
optical applications and for use as medicinal biomarkers.
Moreover, the compound II33 induced apoptosis of MCF-7
cells and was studied by DNA fragmentation and flow cy-
tometric analysis, and much more studies of the biological
mechanism of compound II33 are currently ongoing.
Figure 4 Photoluminescence spectra of compounds in CH2Cl2. samples
were excited at 360 nm.
Table 2 Photoluminescence data for selected compounds II
λmax (Ex) (nm) a)
λmax(Em) (nm) c)
φf (%) e)
31
b)
d)
Compound
II1
I(Ex)
500
510
557
557
409
773
676
I(Em)
503
503
576
552
412
765
690
363
362
363
363
362
363
364
494
495
504
498
496
499
502
II6
33
II11
34
II16
33
II23
30
II28
39
II35
37
a) λmax(Ex): maximum excitation wavelength; b) I(Ex): maximum excitation intensity; c) λmax(Em): maximum emission wavelength; d) I(Em): maximum emis-
sion intensity; e) φf: quantum yield.
The bioassay was supported by Zooblast-molecular Biology Laboratory of
Shanghai Normal University. This work was supported by the National
Natural Science Foundation of China (21042010, 21102092 and
30870560), the Key Scientific "Twelfth Five-Year" National Technology
Support Program (2011BAE06B01-17), the Innovation Project of Shanghai
Education Commission (12YZ078), the Key Project of the Science and
Technology Commission of Shanghai 105405503400), the Leading Aca-
demic Discipline Project of Shanghai Normal University (DZL808), and
Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai
Normal University (07dz22303).

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