Synthesis and preliminary biological evaluation of novel pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives as potential agents against A549 lung cancer cells

Article abstract of DOI:10.1016/j.bmc.2008.10.066

A series of novel pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives were synthesized by the reaction of ethyl 3-aryl-1-(2-bromoethyl)-1H-pyrazole-5-carboxylate and amine in the general heating condition and microwave-assisted condition. The structures of the compounds were determined by IR, ¹H NMR and mass spectroscopy, in addition, representative single-crystal structures were characterized by using X-ray diffraction analysis. Preliminary biological evaluation showed that the compounds could inhibit the growth of A549 cells in dosage- and time-dependent manners. The study on structure-activity relationships showed that compounds with 4-chlorophenyl group at pyrazole moiety, such as 5-benzyl-2-(4-chlorophenyl)-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3o) had much more inhibitory effects. Compound 3o was the most effective small molecule in inhibiting A549 cell growth and might perform its action through modulating autophagy.

Full text of DOI:10.1016/j.bmc.2008.10.066

Bioorganic & Medicinal Chemistry 16 (2008) 10165–10171
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and preliminary biological evaluation of novel pyrazolo[1,5-a]
pyrazin-4(5H)-one derivatives as potential agents against A549 lung cancer cells
c
d
Jin-Hua Zhang a, Chuan-Dong Fan b, Bao-Xiang Zhao a, , Dong-Soo Shin , Wen-Liang Dong ,
*
Yong-Sheng Xie a, Jun-Ying Miao b,
*
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
c Department of Chemistry, Changwon National University, Changwon 641-773, South Korea
d Shandong University of Traditional Chinese Medicine, Jinan 250355, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives were synthesized by the reaction of ethyl
3-aryl-1-(2-bromoethyl)-1H-pyrazole-5-carboxylate and amine in the general heating condition and
microwave-assisted condition. The structures of the compounds were determined by IR, 1H NMR and
mass spectroscopy, in addition, representative single-crystal structures were characterized by using X-
ray diffraction analysis. Preliminary biological evaluation showed that the compounds could inhibit
the growth of A549 cells in dosage- and time-dependent manners. The study on structure-activity rela-
tionships showed that compounds with 4-chlorophenyl group at pyrazole moiety, such as 5-benzyl-2-(4-
chlorophenyl)-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3o) had much more inhibitory effects.
Compound 3o was the most effective small molecule in inhibiting A549 cell growth and might perform
its action through modulating autophagy.
Received 19 September 2008
Revised 29 October 2008
Accepted 30 October 2008
Available online 5 November 2008
Keywords:
Pyrazole
Pyrazinone
Microwave-assisted synthesis
Single-crystal
A549 cells
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
including 6-(aroxymethyl)-2-aryl-6,7-dihydropyrazolo[5,1-c][1,4]
oxazin-4-one derivatives and 5-alkyl-2-ferrocenyl-6,7-dihydropy-
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 an effective strategy to fight cancer
and much attention has recently been paid to the discovery and
development of new, more selective anticancer agents.2–4
Many pyrazole derivatives are known to exhibit a wide range of
biological properties such as cannabinoid hCB1 and hCB2 receptor,
anti-inflammatory, inhibitors of p38 Kinase, CB1 receptor antago-
nists, antimicrobial activity.5–9 Extensive studies have been devoted
to arylpyrazole derivativessuch as Celecoxib, a well-knowncycloox-
ygenase-2inhibitor.10–12 Theincorporationof heterocyclicringsinto
prospective pharmaceutical candidates is a major strategy to obtain
activity and safety advantages. As a consequence, much attention
has been paid to the design and synthesis of fused-pyrazole deriva-
tives.13–17 However, a search of the literature revealed very few re-
ports concerning pyrazolo-pyrazinones.18–20 In our previous
papers, we synthesized a series of novel fused-pyrazole derivatives
razolo[1,5-a]pyrazin-4(5H)-one.21,22 The evaluation of biological
activity showed that these compounds can inhibit A549 lung cancer
cell growth. To extend the diversity of fused-pyrazole skeleton and
screen anticancer agents, the modification of structure is needed.
Microwave-assisted chemistry has blossomed into a useful
technique for a variety of applications in organic synthesis. There
are some excellent reviews and reports on the broad use of micro-
wave irradiation in organic synthesis. It has been demonstrated
that the use of microwave heating can dramatically cut down reac-
tion time, increase product purity and yields, and allow precise
control of reaction conditions, all of which make it suited to meet
the increased demands of high throughput chemistry.23–30 How-
ever, reports concerning microwave-assisted rapid synthesis of
pyrazole-fused pyrazinone derivatives has not been reported.
Herein, we would like to report the microwave-assisted synthe-
sis, structural characterization and preliminary biological evalua-
tion of novel pyrazole-fused pyrazinone derivatives.
2. Results and discussion
2.1. Chemistry
* Corresponding authors. Tel.: +86 531 88366425; fax: +86 531 88564464 (B.-X.
Zhao).
The synthesis of pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives
3 has been accomplished as outlined in Scheme 1 starting from
E-mail addresses: bxzhao@sdu.edu.cn (B.-X. Zhao), miaojy@sdu.edu.cn (J.-Y.
Miao).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.066

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J.-H. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 10165–10171
Scheme 1. Synthesis of pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives.
Table 1
ethyl 3-aryl-1-(2-bromoethyl)-1H-pyrazole-5-carboxylate 2 and
amine. Firstly, the N-alkylation reaction of ethyl 3-aryl-1H-pyra-
zole-5-carboxylate 1 with excess 1,2-dibromoethane was achieved
in the presence of potassium carbonate as the base in acetonitrile.
After flash chromatography on silica gel, the ethyl 3-aryl-1-(2-
bromoethyl)-1H-pyrazole-5-carboxylate 2 and the isomer, ethyl
5-aryl-1-(2-bromoethyl)-1H-pyrazole-3-carboxylate were ob-
tained in 85 and 13% yields, respectively. The isomers can be easily
distinguished by comparing the chemical shift in 1H NMR spectra
as described in our previous paper.22 Thus, for example, 5-butyl-
2-phenyl-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3b) was
synthesized in 78.1% yield by the reaction of ethyl 1-(2-bromoeth-
yl)-3-phenyl-1H-pyrazole-5-carboxylate (2a) with butylamine in
acetonitrile over a 5 h reflux period. The structures of pyrazol-
o[1,5-a]pyrazin-4(5H)-one 3 were determined by IR, 1H NMR
and mass spectroscopy. For example, 5-((6-chloropyridin-3-yl)-
methyl)-2-(4-methoxyphenyl)-6,7-dihydropyrazolo[1,5-a]pyra-
zin-4(5H)-one (3k), obtained in 67.3% yield as white crystal, gave a
[M]-ion peak at m/z 368.8 in the ESI-MS, in accord with the molec-
ular formula C19H17ClN4O2. In the IR spectra, the carbonyl group
absorption was observed in the 1659 cmÀ1. The 1H NMR spectra
indicated the chemical shift of the protons in methylene at
d = 4.75 (s, CH2) and methyl at d = 3.84 (s, CH3). Two methylene
protons in the pyrazine moiety appeared at 3.71 and 4.39 as triplet
peaks (J = 6.2 Hz). A proton signal in pyrazole moiety appeared at
7.11 as singlet peak. Two ortho- aromatic protons signals in 4-
methoxybenzene moiety appeared at the range of d = 6.95 and
7.72 ppm as doublet peaks (J = 8.7 Hz), respectively. The signals
of three protons in pyridine appeared at d = 8.38 (d, J = 2.1 Hz),
7.71 (dd, J = 2.1, 8.2 Hz) and 7.34 (d, J = 8.2 Hz), respectively.
We focused our attention on the microwave-assisted synthesis
technique after obtaining compounds 3 by classical heating meth-
od. These reactions are performed in a modified domestic micro-
wave oven due to its low cost and ready availability. In a typical
experiment, ethyl 3-aryl-1-(2-bromoethyl)-1H-pyrazole-5-carbox-
ylate 2 and amine were mixed in acetonitrile and added in a flask
with a condenser, and irradiated under refluxing condition for
0.8–5.7 h. After work-up, desired compounds were obtained. Com-
paring two methods, microwave-assisted synthesis technique dra-
matically cut down reaction time, increase product yields as shown
in Table 1. The yields of compounds 3 were depended on the struc-
ture of reagent amine regardless of classical heating or microwave-
assisted. From the Table 1 we can find that the more the steric hin-
drance of amine was, the lower the yield of compounds 3. For
example, in the case of 3a, 3g and 3m, because the steric hindrance
of isopropylamine is larger than n-butylamine, the yields of 3a, 3g
and 3m are 24.7, 22.8 and 36.6%, respectively, which are less than
the yields of 3b, 3h and 3n (78.1, 84.3 and 64.0%) in the classical
heating condition. In the microwave-assisted condition, we did
not obtained 3a, 3g and 3m in good yields as other compounds.
The yields of compounds 3 in the condition of classical heating and microwave-
assisted.
Entry
Compounds
Classical heating
Time (h) Yields (%)
Microwave-assisted
Time (h)
Yields (%)
1
2
3
4
5
6
7
8
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
7
24.7
78.1
84.2
64.0
43.1
74.3
22.8
84.3
87.1
68.7
67.0
68.2
36.6
64.0
70.2
54.8
55.2
56.6
—
—
5
8
9
2.5
1.3
3.2
5.7
0.8
—
75.8
88.5
54.3
59.1
94.4
—
15
10
7
7
7
2.7
1.2
3.2
4
1.2
—
80.9
84.8
67.6
70.6
71.3
—
9
10
11
12
13
14
15
16
17
18
8
17
4
6
5
2.4
1.7
3
66.7
88.5
83.1
59.1
86.4
8
9
17
3
5
1.2
In the case of the amine with electron-donating group, such as ben-
zylamine (entry 3, 9 and 15) as well as 3,4-dimethoxyphenylethyl-
amine (entry 6, 12 and 18), products 3 could be obtained in shorter
time and satisfactory yields. On the other hand, in the case of
amine with electron-withdrawing group, such as (6-chloropyri-
din-3-yl)methanamine (entry 5, 11 and 17), the reaction time were
longer and the yields were lower in the both classical heating con-
dition and microwave-assisted condition.
2.2. Single-crystal structural characterization by X-ray
The spatial structures of compounds 3k and 3l were determined
by using X-ray diffraction analysis. The single crystals were grown
from ethyl acetate at room temperature. The molecular views of 3k
and 3l are shown in Figures 1 and 2. Crystal data and structure
refinement for 3k and 3l are shown in Table 2.
The molecule of 3k (Fig. 1) consists of four fragments, a planar
pyrazole ring, aryl ring bonded to pyrazole, pyrazinone ring and
arylalkyl group. An optimal electronic overlap of the p-system de-
mands a coplanar arrangement. Indeed, the pyrazole ring and pyr-
azinone ring are approximately coplanar besides C7, which
distance to the plane is 0.575 Å. The coplanar makes dihedral an-
gles of 3.27(8)° and 73.65(8)° with the phenyl and pyridine rings,
respectively, while that between the phenyl and pyridine rings is
76.91(9)°. In compound 3l (Fig. 2), which is a close analogue of
3k, the pyrazole ring and pyrazinone ring are approximately
coplanar besides C12, which distance to the plane is 0.538 Å. The
coplanar makes dihedral angles of 12.85(11)° and 88.23(10)° with

J.-H. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 10165–10171
10167
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one derivatives could inhi-
bit A549 cell growth obviously in the concentrations of 5–
40 lM after 48 h of the treatment and the cytotoxic potency
was highly dependent on the substitution types and patterns on
the pyrazinone ring, for example, replacing the alkyl at the N-po-
sition of pyrazinone ring with a arylalkyl group resulted in a sig-
nificant activity increasing.22 In the present study, we observed
that the nature and the position of substituent on the molecule
improve effectively biological functions. Growth inhibitory prop-
erties (IC50) on the growth of A549 cells for the compounds 3a–
3r are listed in Table 3. The data suggested that compounds with
4-chlorophenyl group are more effective in dosage- and time-
dependent manners (Figs. 3 and 4).
Figure 1. The molecular structure of compound 3k, with displacement ellipsoids
drawn at the 50% probability level.
As typically shown in Figure 4, exposure of cells to compounds
3m and 3o at 32 lM for 12 h resulted in cell viability decrease from
100% to 81.4 and 78.7%, respectively. Continuing the exposure for
24 h resulted in cell viability decrease from 100% to 75.7 and
73.6%, respectively. When the exposure continued on to 48 h, com-
pared with the control group, the cell viability reduced more signif-
icantly from 100% to 50 and 39.2% (p < 0.01). Further, exposure of
cells to compounds 3m and 3o at 64 lM for 48 h, the cell viability re-
duced more significantly from 100% to 34.6 and 26.5%, respectively.
Figure 2. The molecular structure of compound 3l, with displacement ellipsoids
drawn at the 50% probability level.
2.4. Analysis of DNA fragmentation and autophagy detection by
acridine orange (AO) staining
the 4-methoxyphenyl and 3,4-dimethoxyphenyl rings, respec-
tively, while that between the two phenyl rings is 75.84(12)°.
The single structure characterization should be valuable for fur-
ther investigation.
DNA fragmentation, chromatin condensation, cell shrinkage,
and membrane blebbing are the characteristics of apoptotic cells.
The chromatin condensation and DNA fragmentation in the cells
were not observed by acridine orange (AO) staining under a Nikon
fluorescence microscope. The results showed that no obvious nu-
clear DNA fragmentation occurred in the cells treated with
2.3. Effects of the compounds on the viability of A549 lung
cancer cells
16 lM or 32 lM of compound 3o for 24 h and 48 h (Fig. 5). At
the same time, the volume of the cellular acidic compartment, as
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) cell proliferation assay has been widely accepted as
a reliable way to measure the cell proliferation rate, and con-
versely when metabolic events lead to apoptosis or necrosis. In
our previous paper, we reported that 5-alkyl-2-ferrocenyl-6,7-
a marker of autophagy, was visualized by acridine orange stain-
ing.31–33 As shown in Figure 5, compound 3o at 32
lM promoted
autophagy dramatically. The data suggested that the compound
might inhibit A549 cell growth through modulating autophagy.
Table 2
Crystal data and structure refinement for 3k and 3l.
3k
3l
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
space group
C19H17ClN4O2
368.82
273(2) K
0.71073 Å
Monoclinic
P21/n
C23H25N3O4
407.46
273(2) K
0.71073 Å
Triclinic
ꢀ
P1
Unit cell dimensions
a = 7.33070(10) Å,
a
= 90°
a = 7.4342(2) Å,
a = 106.820(2)°
b = 17.1568(2) Å, b = 92.8480(10)°
b = 12.0228(4) Å, b = 99.827(2)°
c = 13.4779(2) Å,
1693.04(4) A3
4
1.447 Mg/m3
0.248 mmÀ1
768
c
= 90°
c = 12.6883(4) Å,
1026.55(5) A3
2
c = 102.451(2)°
Volume
Z
Calculated density
Absorption coefficient
F(000)
1.318 Mg/m3
0.091 mmÀ1
432
Crystal size
h range for data collection
Limiting indices
Reflections collected/unique
Completeness to h = 27.52°
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
0.30 Â 0.25 Â 0.20 mm
0.40 Â 0.10 Â 0.10 mm
1.92–27.53°
1.73–27.52°
À9 6 h 6 9, À22 6 k 6 16, À17 6 l 6 17
12929/3914 [R(int) = 0.0223]
99.9%
À9 6 h 6 7, À15 6 k 6 15, -11 6 l 6 16
9746/4649 [R(int) = 0.0370]
98.2%
None
None
0.9520 and 0.9293
Full-matrix least-squares on F2
3914/0/235
0.9909 and 0.9644
Full-matrix least-squares on F2
4649/0/347
0.935
1.000
Final R indices [I > 2
R indices (all data)
Largest diff. peak and hole
r
(I)]
R1 = 0.0421, wR2 = 0.1148
R1 = 0.0610, wR2 = 0.1294
0.158 and À0.539 e ÅÀ3
R1 = 0.0583, wR2 = 0.1147
R1 = 0.1288, wR2 = 0.1417
0.193 and À0.219 e ÅÀ3

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J.-H. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 10165–10171
Table 3
Growth inhibitory properties for the compounds 3a–3r at 48 h.
Compounds
IC50 M)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
(
l
>64
>64
>64
>64
>64
31.5
>64
62.5
>64
50.2
>64
>64
31
29.8
22.3
>64
39.5
>64
tion of ethyl 3-aryl-1-(2-bromoethyl)-1H-pyrazole-5-carboxylate
with primary amine under the general heating condition and the
microwave-assisted condition. We found that compounds 3f, 3m,
3n, 3o, 3q could suppress A549 lung cancer cell growth. Compound
3o was the most effective small molecule in inhibiting A549 cell
growth and might perform its action through modulating autoph-
agy. The representative single-crystal structural characterization of
the compounds 3k and 3l were studied by X-ray and it should be
valuable for further investigation.
4. Experimental
Thin-layer chromatography (TLC) was conducted on silica gel
60F254 plates (Merck KgaA). 1H NMR spectra were recorded on a
Bruker Avance 400 (400 MHz) spectrometer, using CDCl3 as sol-
vents and tetramethylsilane (TMS) as internal standard. Melting
points were determined on an XD-4 digital micro melting point
apparatus and uncorrected. IR spectra were recorded with an IR
spectrophotometer Avtar 370 FT-IR (Termo Nicolet). MS spectra
were recorded on a Trace DSQ mass spectrograph. X-ray diffraction
data were recorded on a Bruker Smart Apex2CCD diffractometer.
4.1. General procedure for the synthesis of pyrazolo[1,5-
a]pyrazin-4(5H)-one derivatives (3a–3r) by classical heating
technique
The intermediates 2 were prepared according to the previous
paper.22
A solution containing 2, non-aromatic primary amines (10 equiv)
and potassium iodide (0.2 equiv) in acetonitrile was refluxed under
nitrogen for several hours (shown as Table 1). Then the mixture was
cooled, filtered, and the solvent was removed by evaporating under
reduced pressure. The products 3 were obtained by column chroma-
tography on silica gel using ethyl acetate as eluent.
4.2. General procedure for the synthesis of pyrazolo[1,5-
a]pyrazin-4(5H)-one derivatives (3a–3r) by microwave-assisted
technique
Figure 3. Viability of A549 cells treated with compounds 3a–3r. Cells were seeded
A solution containing 2, non-aromatic primary amines (10
equiv) and potassium iodide (0.2 equiv) in acetonitrile was irradi-
ated with high power region (700 W) to reflux under nitrogen for
several hours (shown as Table 1). After work-up as the same with
classical method, the products 3 were obtained.
in 96-well plates at the density of 6250/cm2. Cells were treated with the
compounds at concentrations of 0, 16, 32, 64 lM, for 48 h. The cell viability was
determined by MTT assay. (*p < 0.05 and **p < 0.01 vs control, n = 3).
2.5. Necrosis detection by LDH activity assay
4.3. Spectral data of compounds 3a–3r
To further confirm the mode of cell death induced by compounds
3f, 3j, 3m, 3n, 3o and 3q typically, LDH assay were performedon cells
treated with or without these compounds. As shown in Figure 6,
there were no significant differences in LDH release between the
cells in the control group (normal group) and the compounds treat-
ment group. The results indicated that the compounds at the test
range of concentration did not cause necrosis in the A549 cells. Thus,
these compounds did not induce necrosis.
4.3.1. 5-Isopropyl-2-phenyl-6,7-dihydropyrazolo[1,5-a]pyrazin-
4(5H)-one (3a)
White solid, mp 179–181 °C; IR (KBr) m ;
: 1651 (C@O) cmÀ1 1H
NMR (400 MHz, CDCl3) d: 1.24 (d, J = 7.1 Hz, 6H, CH3), 3.70 (t,
J = 6.1 Hz, 2H, CH2NCO), 4.41 (t, J = 6.1 Hz, 2H, NCH2), 4.99–5.06
(m, 1H, CH), 7.15 (s, 1H, 4-H), 7.34 (t, J = 7.3 Hz, 1H, ArH), 7.42 (t,
J = 7.5 Hz, 2H, ArH), 7.81 (d, J = 7.3 Hz, 2H, ArH); ESI-MS: 255.8 (M)+.
3. Conclusion
4.3.2. 5-Butyl-2-phenyl-6,7-dihydropyrazolo[1,5-a]pyrazin-
4(5H)-one (3b)
White solid, mp 161–163 °C; IR (KBr)
NMR (400 MHz, CDCl3) d: 0.97 (t, J = 7.3 Hz, 3H, CH3), 1.35–1.45
In summary, we have described a facile approach to prepare
pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives 3a–3l by the reac-
m ;
: 1651 (C@O) cmÀ1 1H

J.-H. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 10165–10171
10169
Figure 4. Viability of A549 cells treated with compounds 3f, 3j, 3m and 3o. Cells were treated with 3f, 3j, 3m and 3o at concentrations of 0, 16, 32, 64
48 h. Compounds 3f, 3j, 3m and 3o inhibit the cell growth in dosage- and time-dependent manners. (*p < 0.05 and **p < 0.01 vs control, n = 3).
lM, for 12, 24, and
(m, 2H, CH2), 1.59–1.67 (m, 2H, CH2), 3.58 (t, J = 7.4 Hz, 2H, CH2),
3.79 (t, J = 6.2 Hz, 2H, CH2NCO), 4.44 (t, J = 6.2 Hz, 2H, NCH2),
7.14 (s, 1H, 4-H), 7.32 (t, J = 7.3 Hz, 1H, ArH), 7.42 (t, J = 7.5 Hz,
2H, ArH), 7.81 (d, J = 7.3 Hz, 2H, ArH); ESI-MS: 270.6 (M+H)+.
4.3.3. 5-Benzyl-2-phenyl-6,7-dihydropyrazolo[1,5-a]pyrazin-
4(5H)-one (3c)
White solid, mp 174–175 °C; IR (KBr) m ;
: 1649 (C@O) cmÀ1 1H
NMR (400 MHz, CDCl3) d: 3.70 (t, J = 6.2 Hz, 2H, CH2NCO), 4.40 (t,
J = 6.2 Hz, 2H, NCH2), 4.79 (s, 2H, CH2Ph), 7.21 (s, 1H, 4-H), 7.32–
7.37 (m, 6H, ArH), 7.43 (t, J = 7.4 Hz, 2H, ArH), 7.82 (d, J = 7.8 Hz,
2H, ArH); ESI-MS: 304.4 (M+H)+.
4.3.4. 5-(4-tert-Butylbenzyl)-2-phenyl-6,7-dihydro-
pyrazolo[1,5-a]pyrazin-4(5H)-one (3d)
White solid, mp 185–187 °C; IR (KBr) m ;
: 1644 (C@O) cmÀ1 1H
NMR (400 MHz, CDCl3) d: 1.31 (s, 9H, CH3), 3.70 (t, J = 6.2 Hz, 2H,
CH2NCO), 4.38 (t, J = 6.2 Hz, 2H, NCH2), 4.75 (s, 2H, CH2Ph), 7.20
(s, 1H, 4-H), 7.26 (d, J = 8.3 Hz, 2H, ArH), 7.34–7.44 (m, 5H, ArH),
7.81 (d, J = 7.1 Hz, 2H, ArH); ESI-MS: 360.4 (M+H)+.
Figure 5. DNA fragmentation and autophagy detections by acridine orange (AO)
staining in A549 cells treated with 16 lM or 32 lM of compound 3o for 24 h and
48 h. (n = 4).
4.3.5. 5-((6-Chloropyridin-3-yl)methyl)-2-phenyl-6,7-
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3e)
Yellow solid, mp 226–228 °C; IR (KBr) m ;
: 1649 (C@O) cmÀ1 1H
NMR (400 MHz, CDCl3) d: 3.73 (t, J = 6.2 Hz, 2H, CH2NCO), 4.41 (t,
J = 6.2 Hz, 2H, NCH2), 4.76 (s, 2H, CH2), 7.20 (s, 1H, 4-H), 7.32–
7.36 (m, 2H, ArH, PyH), 7.42 (t, J = 7.2 Hz, 2H, ArH), 7.72 (dd,
J = 2.5, 8.2 Hz, 1H, PyH), 7.80 (d, J = 7.2 Hz, 2H, ArH), 8.38 (d,
J = 2.3 Hz, 1H, PyH); ESI-MS: 339.3 (M+H)+.
4.3.6. 5-(3,4-Dimethoxyphenethyl)-2-phenyl-6,7-
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3f)
White solid, mp 152–154 °C; IR (KBr) m ;
: 1658 (C@O) cmÀ1 1H
NMR (400 MHz, CDCl3) d: 2.93 (t, J = 7.1 Hz, 2H, CH2), 3.56 (t,
J = 6.0 Hz, 2H, CH2NCO), 3.80 (t, J = 7.1 Hz, 2H, CH2), 3.86 (s, 6H,
OCH3), 4.27 (t, J = 6.0 Hz, 2H, NCH2), 6.77–6.82 (m, 3H, ArH), 7.15
(s, 1H, 4-H), 7.33 (t, J = 7.3 Hz, 1H, ArH), 7.42 (t, J = 7.5 Hz, 2H,
ArH), 7.80 (d, J = 7.8 Hz, 2H, ArH); ESI-MS: 378.6 (M+H)+.
Figure 6. Effects of the compounds 3f, 3j, 3m, 3n, 3o and 3q on the release of LDH
from A549 cells. The culture media from the cells treated with the compounds
64 lM for 48 h respectively. Light absorption was analyzed at 340 nm using a
model Cintra 5 UV–vis spectrometer. There was no significant difference in LDH
release among the four groups. (p > 0.05 vs control group, n = 3).

10170
J.-H. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 10165–10171
4.3.7. 5-Isopropyl-2-(4-methoxyphenyl)-6,7-dihydro-
7.10 (s, 1H, 4-H), 7.38 (d, J = 8.3 Hz, 2H, ArH), 7.73 (d, J = 8.3 Hz,
pyrazolo[1,5-a]pyrazin-4(5H)-one (3g)
2H, ArH); ESI-MS: (C16H18ClN3O), 303.9 (M)+.
White solid, mp 157–159 °C; IR (KBr) m ;
: 1650 (C@O) cmÀ1 1H
NMR (400 MHz, CDCl3) d: 1.24 (d, J = 6.8 Hz, 6H, CH3), 3.69 (t,
J = 6.0 Hz, 2H, CH2NCO), 3.85 (s, 3H, OCH3), 4.42 (t, J = 6.0 Hz, 2H,
NCH2), 4.98–5.05 (m, 1H, CH), 6.95 (d, J = 8.6 Hz, 2H, ArH), 7.07
(s, 1H, 4-H), 7.74 (d, J = 8.6 Hz, 2H, ArH); ESI-MS: 286.0 (M+H)+.
4.3.15. 5-Benzyl-2-(4-chlorophenyl)-6,7-dihydropyrazolo[1,5-
a]pyrazin-4(5H)-one (3o)
White solid, mp 142–145 °C; IR (KBr) m ;
: 1647 (C@O) cmÀ1 1H
NMR (400 MHz, CDCl3) d: 3.69 (t, J = 6.1 Hz, 2H, CH2NCO), 4.36 (t,
J = 6.1 Hz, 2H, NCH2), 4.78 (s, 2H, CH2Ph), 7.17 (s, 1H, 4-H), 7.32–
7.39 (m, 7H, ArH), 7.73 (d, J = 8.6 Hz, 2H, ArH); ESI-MS:
(C19H17ClN3O), 338.1 (M+H)+.
4.3.8. 5-Butyl-2-(4-methoxyphenyl)-6,7-dihydropyrazolo[1,5-
a]pyrazin-4(5H)-one (3h)
White solid, mp 160–162 °C; IR (KBr) m ;
: 1652 (C@O) cmÀ1 1H
NMR (400 MHz, CDCl3) d: 0.97 (t, J = 7.3 Hz, 3H, CH3), 1.35–1.44
(m, 2H, CH2), 1.59–1.66 (m, 2H, CH2), 3.58 (t, J = 7.4 Hz, 2H, CH2),
3.79 (t, J = 6.1 Hz, 2H, CH2NCO), 3.85 (s, 3H, OCH3), 4.44 (t, J = 6.1
Hz, 2H, NCH2), 6.95 (d, J = 8.7 Hz, 2H, ArH), 7.07 (s, 1H, 4-H), 7.74
(d, J = 8.7 Hz, 2H, ArH); ESI-MS: 300.5 (M+H)+.
4.3.16. 5-(4-tert-Butylbenzyl)-2-(4-chlorophenyl)-6,7-
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3p)
White solid, mp 212–214 °C; IR (KBr) m ;
: 1649 (C@O) cmÀ1 1H
NMR (400 MHz, CDCl3) d: 1.13 (s, 9H, CH3), 3.69 (t, J = 6.2 Hz, 2H,
CH2NCO), 4.36 (t, J = 6.2 Hz, 2H, NCH2), 4.75 (s, 2H, CH2Ph), 7.16 (s,
1H, 4-H), 7.26 (d, J = 8.2 Hz, 2H, ArH), 7.38 (dd, J = 3.1, 8.4 Hz, 4H,
ArH), 7.73 (d, J = 8.6 Hz, 2H, ArH); ESI-MS: (C23H25ClN3O), 394.3
(M+H)+.
4.3.9. 5-Benzyl-2-(4-methoxyphenyl)-6,7-dihydropyrazolo[1,5-
a]pyrazin-4(5H)-one (3i)
White solid, mp 163–164 °C; IR (KBr) m ;
: 1657 (C@O) cmÀ1 1H
NMR (400 MHz, CDCl3) d: 3.68 (t, J = 6.2 Hz, 2H, CH2NCO), 3.84 (s,
3H, OCH3), 4.35 (t, J = 6.2 Hz, 2H, NCH2), 4.78 (s, 2H, CH2Ph), 6.95
(d, J = 8.8 Hz, 2H, ArH), 7.13 (s, 1H, 4-H), 7.31–7.39 (m, 5H, ArH),
7.73 (d, J = 8.8 Hz, 2H, ArH); ESI-MS: 334.2 (M+H)+.
4.3.17. 2-(4-Chlorophenyl)-5-((6-chloropyridin-3-yl)methyl)-
6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3q)
White solid, mp 192–195 °C; IR (KBr) m ;
: 1652 (C@O) cmÀ1 1H
NMR (400 MHz, CDCl3) d: 3.73 (t, J = 6.2 Hz, 2H, CH2NCO), 4.40 (t,
J = 6.2 Hz, 2H, NCH2), 4.76 (s, 2H, CH2), 7.16 (s, 1H, 4-H), 7.35 (d,
J = 8.2 Hz, 1H, PyH), 7.38 (d, J = 7.6 Hz, 2H, ArH), 7.70 (d, J = 2.4
Hz, 1H, PyH), 7.73 (d, J = 7.6 Hz, 2H, ArH), 8.38 (d, J = 2.3 Hz, 1H,
PyH); ESI-MS: (C18H15Cl2N4O), 373.1 (M+H)+.
4.3.10. 5-(4-tert-Butylbenzyl)-2-(4-methoxyphenyl)-6,7-
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3j)
White solid, mp 147–149 °C; IR (KBr) m ;
: 1659 (C@O) cmÀ1 1H
NMR (400 MHz, CDCl3) d: 1.31 (s, 9H, CH3), 3.69 (t, J = 6.2 Hz, 2H,
CH2NCO), 3.84 (s, 3H, OCH3), 4.35 (t, 2H, J = 6.2 Hz, NCH2), 4.74
(s, 2H, CH2Ph), 6.95 (d, J = 8.8 Hz, 2H, ArH), 7.12 (s, 1H, 4-H), 7.26
(d, J = 8.2 Hz, 2H, ArH), 7.37 (d, J = 8.2 Hz, 2H, ArH), 7.73 (d,
J = 8.8 Hz, 2H, ArH); ESI-MS: 390.0 (M+H)+.
4.3.18. 2-(4-Chlorophenyl)-5-(3,4-dimethoxyphenethyl)-6,7-
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3r)
Yellow solid, mp 175–178 °C; IR (KBr) m ;
: 1656 (C@O) cmÀ1 1H
NMR (400 MHz, CDCl3) d: 2.93 (t, J = 7.1 Hz, 2H, CH2), 3.56 (t,
J = 6.1 Hz, 2H, CH2NCO), 3.80 (t, J = 7.1 Hz, 2H, CH2), 3.86 (s, 6H,
OCH3), 4.24 (t, J = 6.1 Hz, 2H, NCH2), 6.78–6.82 (m, 3H, ArH), 7.10
(s, 1H, 4-H), 7.38 (d, J = 8.5 Hz, 2H, ArH), 7.72 (d, J = 8.5 Hz, 2H,
ArH); ESI-MS: (C22H23ClN3O3), 412.3 (M+H)+.
4.3.11. 5-((6-Chloropyridin-3-yl)methyl)-2-(4-methoxyphenyl)-
6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3k)
White solid, mp 197–199 °C; IR (KBr) m ;
: 1659 (C@O) cmÀ1 1H
NMR (400 MHz, CDCl3) d: 3.71 (t, J = 6.2 Hz, 2H, CH2NCO), 3.84 (s,
3H, OCH3), 4.39 (t, J = 6.2 Hz, 2H, NCH2), 4.75 (s, 2H, CH2), 6.95
(d, J = 8.7 Hz, 2H, ArH), 7.11 (s, 1H, 4-H), 7.34 (d, J = 8.2 Hz, 1H,
PyH), 7.70 (d, J = 2.4 Hz, 1H, PyH), 7.72 (d, J = 7.6 Hz, 2H, ArH),
8.38 (d, J = 2.1 Hz, 1H, PyH); ESI-MS: 368.8 (M)+.
4.4. Cell culture
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.
4.3.12. 5-(3,4-Dimethoxyphenethyl)-2-(4-methoxyphenyl)-6,7-
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3l)
White solid, mp 171–172 °C; IR (KBr) m ;
: 1656 (C@O) cmÀ1 1H
NMR (400 MHz, CDCl3) d: 2.93 (t, J = 6.9 Hz, 2H, CH2), 3.56 (t,
J = 5.7 Hz, 2H, CH2NCO), 3.79 (t, J = 6.9 Hz, 2H, CH2), 4.24 (t,
J = 5.7 Hz, 2H, NCH2), 3.85 (s, 9H, OCH3), 6.78–6.80 (m, 3H, ArH),
6.94 (d, J = 8.6 Hz, 2H, ArH), 7.06 (s, 1H, 4-H), 7.72 (d, J = 8.6 Hz,
2H, ArH); ESI-MS: 408.6 (M+H)+.
4.5. Cell viability assay
As the previous report, cells were seeded onto 96-well plates and
treated with compounds 3a–3r at 8, 16, 32 and 64 lM for 12, 24 and
48 h, respectively. Cell viability was determined by MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay according to
Price et al.34 The light absorption was measured at 570 nm using
Spectra MAX 190 microplate spectrophotometer (GMI Co., USA).
4.3.13. 2-(4-Chlorophenyl)-5-isopropyl-6,7-dihydropyraz-
olo[1,5-a]pyrazin-4(5H)-one (3m)
White solid, mp 188–190 °C; IR (KBr) m ;
: 1654 (C@O) cmÀ1 1H
NMR (400 MHz, CDCl3) d: 1.24 (d, J = 6.8 Hz, 6H, CH3), 3.69 (t,
J = 6.1 Hz, 2H, CH2NCO), 4.39 (t, J = 6.1 Hz, 2H, NCH2), 4.98–5.04
(m, 1H, CH), 7.10 (s, 1H, 4-H), 7.38 (d, J = 8.6 Hz, 2H, ArH), 7.73
(d, J = 8.6 Hz, 2H, ArH); ESI-MS: (C15H16ClN3O), 289.9 (M)+.
4.6. Acridine orange (AO) staining
The cells were incubated with compounds 3o (16, 32
lM) for 24
and 48 h, and stained with 5 g/ml of acridine orange (AO) at room
l
temperature for 1 min. Then the cells were observed and photo-
4.3.14. 5-Butyl-2-(4-chlorophenyl)-6,7-dihydropyrazolo[1,5-
graphed using a Nikon fluorescence microscope.
a]pyrazin-4(5H)-one (3n)
White solid, mp 173–175 °C; IR (KBr)
m
: 1651 (C@O) cmÀ1
;
1H
4.7. LDH assay
NMR (400 MHz, CDCl3) d: 0.97 (t, J = 7.3 Hz, 3H, CH3), 1.37–1.43
(m, 2H, CH2), 1.61–1.65 (m, 2H, CH2), 3.58 (t, J = 7.4 Hz, 2H, CH2),
3.78 (t, J = 6.1 Hz, 2H, CH2NCO), 4.42 (t, J = 6.1 Hz, 2H, NCH2),
Lactate dehydrogenase (LDH) assay was performed on cells
treated with 64 lM compounds 3f, 3j, 3m, 3n, 3o and 3q for 48

J.-H. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 10165–10171
10171
11. Ranatunge, R. R.; Earl, R. A.; Garvey, D. S.; Janero, D. R.; Letts, L. G.; Martino, A.
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Acknowledgments
This study was supported by the Science and Technology Devel-
opmental Project of Shandong Province (2008GG10002034 and
2007GG20002004) and National Natural Science Foundation of
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