Microwave-assisted synthesis, crystal structure of pyrazolo[1,5-a]pyrazin- 4(5H)-ones and their selective effects on lung cancer cells

Article abstract of DOI:10.1016/j.ejmech.2011.03.018

A series of novel pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives with hydrophilic group was synthesized under general heating condition and microwave-assisted condition. The structures of compounds were determined by IR, ¹H NMR and HRMS, moreover

Full text of DOI:10.1016/j.ejmech.2011.03.018

European Journal of Medicinal Chemistry 46 (2011) 2359e2367
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Microwave-assisted synthesis, crystal structure of pyrazolo[1,5-a]pyrazin-4(5H)-
ones and their selective effects on lung cancer cells
,
,
,
a
,
,
Ning Liu a 1, Jin-Hua Zhang b 1, Bao-Xiang Zhao b , Jing Zhao c, Le Su a c, Wen-Liang Dong d,
*
Shang-Li Zhang a c, Jun-Ying Miao a
,
,c,
**
a Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Science, Shandong University, Jinan 250100, PR China
b Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China
c The Key Laboratory of Experimental Teratology, Ministry of Education, Jinan 250012, PR China
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 with hydrophilic group was synthesized
under general heating condition and microwave-assisted condition. The structures of compounds were
determined by IR, 1H NMR and HRMS, moreover, representative crystal structures were characterized by
using X-ray diffraction analysis. Preliminary biological evaluation showed that some compounds could
inhibit the growth of A549, H322 and H1299 cells in dosage dependent manners. The compounds could
inhibit growth of A549, H322 and H1299 cells in different mechanism. Compounds 3eeh inhibited
growth of A549 cells by inducing a strong G1-phase arrest. Whereas these compounds inhibited growth
of H1299 and H322 cells by inducing apoptosis.
Received 24 September 2010
Received in revised form
26 February 2011
Accepted 8 March 2011
Available online 15 March 2011
Keywords:
Pyrazole
Pyrazinone
Ó 2011 Elsevier Masson SAS. All rights reserved.
Microwave-assisted synthesis
Single-crystal
Lung cancer cells
1. Introduction
Many pyrazole derivatives are known to exhibit a wide range of
biological properties and fused-pyrazole derivatives have attracted
Lung cancer has been recognized to be one of the leading causes
of death worldwide. Highly proliferative nature of cancer cells has
undoubtedly been realized and consequently, inhibition of prolif-
erative pathways is considered to be an effective strategy to fight
cancer. Much attention has recently been paid to the discovery and
development of new, more selective anticancer agents [1e3].
Apoptotic cell death regulators are currently considered to have
significant potential as targets for cancer therapeutics although cell
death can occur by mechanisms including necrosis, mitotic catas-
trophe and autophagy [4]. It is now recognized that one hallmark of
cancer cells is their compromised ability to undergo apoptosis.
Targeting critical apoptosis regulators is an attractive strategy for
the development of new classes of therapies for the treatment of
cancer and other human diseases.
considerable attention in the design and synthesis of potential anti-
cancer agents [5e12]. However, a search of the literature revealed
very few reports concerning pyrazolo-pyrazinones [13e15]. In our
previous papers, we synthesized a series of novel fused-pyrazole
derivatives including 6-(aroxymethyl)-2-aryl-6,7-dihydropyrazolo
[5,1-c][1,4] oxazin-4-one derivatives, 5-alkyl-2-aryl-6,7-dihydropyr-
azolo[1,5-a]pyrazin-4(5H)-one and 5-alkyl-2-ferrocenyl-6,7-dihy-
dropyrazolo[1,5-a]pyrazin-4(5H)-one [16e19]. The evaluation of
biological activity showed that these compounds could inhibit A549
lung cancer cell growth. However, because the substituent in pyr-
azinone moiety was generally more hydrophobic, the modification of
structure is needed to extend the diversity of fused-pyrazole skeleton
and screen anticancer agents.
Microwave-assisted chemistry has blossomed into a useful
technique for a variety of applications in organic synthesis. It has
been demonstrated that the use of microwave heating can
dramatically cut down reaction 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 [20e27]. In the previous paper, for example, we reported
the microwave-assisted synthesis of pyrazole-fused pyrazinone
* Corresponding author. Tel.: þ86 531 88366425; fax: þ86 531 88564464.
** Corresponding author. Shandong Provincial Key Laboratory of Animal Cells and
Developmental Biology, School of Life Science, Shandong University, Jinan 250100,
PR China.
E-mail addresses: bxzhao@sdu.edu.cn, sduzhao@hotmail.com (B.-X. Zhao),
miaojy@sdu.edu.cn (J.-Y. Miao).
1
Equal contribution.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.018

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N. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 2359e2367
derivatives, in which it is fast 3e10 fold than general heating
method although it took 1e6 h to finish reaction [18].
Herein, we would like to report the microwave-assisted
synthesis, structural characterization and preliminary biological
evaluation of novel pyrazole-fused pyrazinone derivatives modified
with hydrophilic group, such as hydroxyl group.
low cost and ready availability. In a typical experiment, ethyl 3-aryl-
1-(2-bromoethyl)-1H-pyrazole-5-carboxylate (2) and amine were
mixed in a flask, and irradiated for 1e2 min. After work-up, desired
compounds 3 were obtained. Comparing two methods, microwave-
assisted synthesis technique dramatically cut down reaction time,
increase product yields as shown in Table 1. The yields of
compounds 3 were depended on the structure of amine regardless
of classical heating or microwave-assisted. Microwave-assisted
reaction is fast 45e240 fold than general heating method.
2. Chemistry
2.1. Synthesis
2.2. Single-crystal structural characterization by X-ray
The synthesis of pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives
(3) has been accomplished as outlined in Scheme 1 starting from
ethyl 3-aryl-1-(2-bromoethyl)-1H-pyrazole-5-carboxylate (2) and
amine. Firstly, the N-alkylation reaction of ethyl 3-aryl-1H-pyrazole-
5-carboxylate (1) with excess 1,2-dibromoethane was achieved in
the presence of potassium carbonate in acetonitrile according to our
previous report [17]. After flash chromatography on silica gel, the
ethyl 3-aryl-1-(2-bromoethyl)-1H-pyrazole-5-carboxylate (2) was
obtained. Thus, for example, 5-(2-hydroxyethyl)-2-phenyl-6,7-
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3a) was synthesized in
99% yield by the reaction of ethyl 1-(2-bromoethyl)-3-phenyl-1H-
pyrazole-5-carboxylate (2a) with ethanolamine in acetonitrile over
a 3 h reflux period. The structures of pyrazolo[1,5-a]pyrazin-4(5H)-
one (3) were determined by IR, 1H NMR and HRMS. For example,
2-(4-chlorophenyl)-5-(2-morpholinoethyl)-6,7-dihydropyrazolo
[1,5-a]pyrazin-4(5H)-one (3h), obtained in 58% yield as white
crystal, gave a [M þ H]-ion peak at m/z 361.1435 in the HRESI-MS, in
accord with the molecular formula C18H22ClN4O2. In the IR spectra,
the carbonyl group absorption was observed in the 1653 cmꢀ1. The
1H NMR spectra indicated two ortho- aromatic protons signals in
The spatial structures of compounds 3h and 3j were determined
by using X-ray diffraction analysis. The single crystals were grown
from ethyl acetate solution at room temperature. The molecular
views of 3h and 3j are shown in Figs. 1 and 2.
The molecule of 3h consists of four fragments, a planar pyrazole
ring, aryl ring bonded to pyrazole, pyrazinone ring and 2-mor-
pholinoethyl group. An optimal electronic overlap of the
p-system
demands a coplanar arrangement. Indeed, the pyrazole ring and
pyrazinone ring are approximately coplanar besides C12 with
0.326 Å deviation. The plane makes dihedral angle of 5.79(11)ꢁ with
the 4-chlorophenyl ring. The morpholine ring is in a chair confor-
mation. In compound 3j, which is a close analogue of 3h, the pyr-
azole ring and pyrazinone ring are approximately coplanar besides
C7 with 0.385 Å deviation. The plane makes dihedral angles of
13.37(14)ꢁ with methoxyphenyl ring.
3. Pharmacology
4-chlorobenzene moiety appeared at the range of
d
¼ 7.74 and
7.39 ppm, respectively, as doublet peaks with coupling constant
8.4 Hz. A proton signal in pyrazole moiety appeared at 7.11 as singlet
peak. The chemical shift of the protons in methylenes (7-CH2 and
Screening of synthesized substances was carried out using lung
cancer A549, H322 and H1299 cell lines, which are p53 wild-type,
p53-mutant and p53-null cells, respectively. Proliferation
percentage was determined by the SRB assay [28,29]. Cells were
6-CH2) appeared at
respectively. Other signals are consist with the structure although it
is difficulty to attribute to assigned protons.
We focused our attention on the microwave-assisted synthesis
technique after obtaining compounds 3 by classical heating method
in order to shorten reaction time and free solvent. These reactions
are performed in a modified domestic microwave oven due to its
d
¼ 4.43 and 3.89 as triplet peaks (J ¼ 6.2 Hz),
incubated with substances at 8, 16, 32 and 64 mM for 48 h and the
cell proliferation/viability determination using the survival
percentage obtained with the cell treated only with the solvent
(DMSO at 0.1%) as reference. The results are expressed as the
average of triplicate assays. Effect factors related to antiprolifera-
tion such as apoptosis and cell cycle distribution were determined.
Scheme 1. Synthesis of pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives.

N. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 2359e2367
2361
Table 1
that DNA fragmentation, chromatin condensation, cell shrinkage,
and membrane blebbing are the characteristics of apoptotic cells.
The chromatin condensation and DNA fragmentation in the cells
were determined by Hoechst 33258 staining under a fluorescence
microscope. It was interestingly found that compounds 3eeh could
induce apoptosis in H322 and H1299 cells, which are p53-mutant
and p53-null cells, respectively, as shown in Figs. 7 and 8. However,
from Fig. 9 it can be found that compounds 3eeh did not induce
apoptosis in A549 cells which are p53 wild-type cells. Thus,
mechanism of compounds 3eeh induced apoptosis of H322 and
H1299 cell lines remains unclear. Pathway of apoptosis should be
worthy to be investigated in the future.
Yields of compounds 3ael in the condition of classical heating and microwave-
assisted.
Compound 3
General heating
Time (min)
Microwave
Time (min)
Yield (%)
Yield (%)
a
b
c
d
e
f
g
h
i
180
210
180
120
240
180
180
120
240
150
180
90
98
98
85
88
88
86
78
58
96
87
90
56
1
1
1
1
1
1
1
1
1
1
1
2
94
90
91
99
86
99
89
99
99
97
99
80
j
k
l
4.3. Flow cytometry analysis of cell cycle distribution
In order to analyze effects of compounds 3eeh on proliferation
of A549 cells, the cell cycle progression was next investigated. It is
well known that cell cycle dysregulation is a hallmark of tumor
cells. The eukaryotic cell cycle consists of alternating rounds of DNA
replication (S phase) and cell division (M phase) separated by the
gap phases G0, G1 and G2. Regulation of proteins that mediate
critical events of the cell cycle may be a useful anti-tumor target
[30,31]. In order to get a more precise insight of growth inhibition,
flow cytometry analysis was taken to detect whether compounds
3eeh induce cell cycle arrest. The results showed that compounds
4. Results and discussion
4.1. Effects of the compounds on the viability of A549, H322, H1299
cells and normal human embryo lung cells
Firstly, cell viability of A549 cells treated with compounds 3ael
was analyzed by SRB assay and the results showed that compounds
3eeh exhibited an inhibitory effect on the proliferation of A549 cells
in dose-dependent manner at 48 h (Fig. 3). However, other
compounds did not exhibit effect on the cell viability. Thus,
compounds 3eeh were chosen to examine their effects on H322,
H1299 cells and normal human embryo lung cells. The results
indicated that these compounds had similar effects on the prolifer-
ation of H322 and H1299 cells in dose-dependent manner at 48 h as
shown in Figs. 4 and 5, but had no effect on growth of human embryo
lung cells (Fig. 6). In our previous paper, we reported that 5-alkyl-2-
ferrocenyl-6,7-dihydropyrazolo[1,5-a]pyrazin-4 (5H)-one deriva-
tives could inhibit A549 cell growth obviously in the concentrations
3f and 3h treatment with 64
mM at 48 h induced G1-phase arrest
effectively (Fig. 10). After 48 h treatment with 64
mM, 3f or 3h, the
G1 population was noticeably enhanced by 32% and 27%, respec-
tively, compared with control. In the case of compounds 3e and 3g,
the G1 population was enhanced by 18% and 13%, respectively. The
increase in the G1-phase cell population was accompanied by
a decrease in the S and G2-phase cell populations. The fact that
compounds 3eeh induced a strong G1-phase arrest is concomitant
with the growth inhibitory effect.
of 5e40 mM after 48 h 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-position of
pyrazinone ring with an arylalkyl group resulted in a significant
activity increasing [17,18]. In the present study, we observed that the
nature and the position of substituent on the molecule improved
effectively biological functions. From Fig. 3 it can be found that
compounds 3eeh with 4-chlorophenyl group were moreeffective in
dosage dependent manners, whereas, compounds 3aed with
phenyl and compounds 3i-l with methoxylphenyl group had no
effects on the proliferation of A549 cells.
4.4. Necrosis detection by LDH activity assay
In order to determine if the growth inhibitory effects were due
to necrosis that is believed to be an unwanted side effect of cancer-
fighting agents, LDH assay were performed on cells treated with
compounds 3eeh or 0.1% DMSO (as control). As shown in Fig. 11 for
A549 cells, there were no significant differences in LDH release
between the cells in the control 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 cells.
4.2. Compounds 3eeh induced apoptosis in H322 and H1299 cells
but did not in A549 lung cancer cells
To detect whether compounds 3eeh resulted in apoptosis of the
cells, we performed Hoechst 33258 staining assay. It is well known
Fig. 1. The molecular structure of compound 3h, with displacement ellipsoids drawn
Fig. 2. The molecular structure of compound 3j, with displacement ellipsoids drawn at
at the 50% probability level.
the 50% probability level.

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N. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 2359e2367
Fig. 3. Effects of the compounds on A549 cell viability. A549 cells were treated with compounds 3ael or 0.1% DMSO (control) for 48 h at the concentration of 8, 16, 32 or 64
viability was analyzed by SRB assay. Data are means ꢂ SE from three independent experiments (*p < 0.05 and **p < 0.01 vs. control).
mM. Cell
5. Conclusion
solvent 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.
In summary, we have described a facile approach to prepare
pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives 3ael by the reaction
of ethyl 3-aryl-1-(2-bromoethyl)-1H-pyrazole-5-carboxylate with
amine under the general heating condition and the microwave-
assisted condition. Microwave-assisted reaction is fast 45e240 fold
than general heating method. We also studied the representative
single-crystal structural characterization of the compounds 3h and
3j by X-ray that should be valuable for further investigation. We
found that compounds 3eeh with chlorophenyl group in pyrazole
moiety could suppress A549, H322 and H1299 cells growth. The
compounds might perform their action through inducing a strong
G1-phase arrest rather than apoptosis for A549 cell, whereas by
inducing apoptosis for H322 and H1299 cells. Mechanism of
compounds 3eeh induced apoptosis of H322 and H1299 cell lines
should be worthy to be investigated in the future.
6.2. General procedure for the synthesis of pyrazolo[1,5-a]pyrazin-4
(5H)-one derivatives (3ael) by classical heating technique
The intermediates 2 were prepared according to the previous
paper [17,18]. A mixture of 2 (1 mmol), amine (10 mmol) and
potassium iodide (0.2 mmol) in acetonitrile (30 ml) 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 chromatography on silica gel using ethyl acetate as
eluent.
6. Experimental
6.3. General procedure for the synthesis of pyrazolo[1,5-a]pyrazin-4
6.1. General
(5H)-one derivatives (3ael) by microwave-assisted technique
Thin-layer chromatography (TLC) was conducted on silica gel 60
F254 plates (Merck KGaA). 1H NMR spectra were recorded on
a Bruker Avance 400 (400 MHz) spectrometer, using CDCl3 as
A mixture of 2 (1 mmol), amine (10 mmol) and potassium iodide
(0.2 mmol) was irradiated with high power region (700 W) in free
solvent for 1e2 min (shown as Table 1). After work-up as the same
with classical method, the products 3 were obtained.
Fig. 4. Effects of the compounds on H322 cell viability. H322 cells were treated with
compounds 3eeh or 0.1% DMSO (control) for 48 h at the concentration of 16, 32 and
Fig. 5. Effects of the compounds on H1299 cell viability. H1299 cells were treated with
compounds 3eeh or 0.1% DMSO (control) for 48 h at the concentration of 16, 32 or
64
m
M. Cell viability was analyzed by SRB assay. Data are means ꢂ SE from three
64
m
M. Cell viability was analyzed by SRB assay. Data are means ꢂ SE from three
independent experiments (*p < 0.05 and **p < 0.01 vs. control).
independent experiments (*p < 0.05 and **p < 0.01 vs. control).

N. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 2359e2367
2363
(t, J ¼ 7.5 Hz, 2H, ArH), 7.33 (t, J ¼ 7.4 Hz, 1H, ArH), 7.14 (s, 1H, 3-H),
4.45 (t, J ¼ 6.2 Hz, 2H, CH2), 3.89e3.95 (m, 4H, CH2), 3.75 (t,
J ¼ 5.1 Hz, 2H, CH2); HRMS m/z calcd for C14H16N3O2 [M þ H]þ
258.1243; Found: 258.1239. Calcd for C14H15N3O2Na [M þ Na]þ:
280.1062, Found: 280.1055.
6.4.2. 5-(3-Hydroxypropyl)-2-phenyl-6,7-dihydropyrazolo[1,5-a]
pyrazin-4(5H)-one (3b)
White solid, mp: 179e181 ꢁC; IR (KBr)
n
: 3401 (OH), 1635 (C]O)
cmꢀ1; 1H NMR (400 MHz, CDCl3)
d: 7.79 (d, J ¼ 7.7 Hz, 2H, ArH), 7.40
(t, J ¼ 7.4 Hz, 2H, ArH), 7.33 (t, J ¼ 7.3 Hz, 1H, ArH), 7.19 (s, 1H, 3-H),
4.46 (t, J ¼ 6.2 Hz, 2H, CH2), 3.81 (t, J ¼ 6.2 Hz, 2H, CH2), 3.76 (t,
J ¼ 6.1 Hz, 2H, CH2), 3.65 (t, J ¼ 5.4 Hz, 2H, CH2), 1.83e1.89 (m, 2H,
CH2); HRMS m/z calcd for C15H18N3O2 [M þ H]þ 272.1399; Found:
272.1397. Calcd for C15H17N3O2Na [M þ Na]þ: 294.1219, Found:
294.1220.
Fig. 6. Effects of the compounds on human embryo lung cells viability. Cells were
treated with compounds 3eeh, 5-FU (positive control) or 0.1% DMSO (vehicle control)
for 48 h at the concentration of 32 and 64 mM. Cell viability was analyzed by SRB assay.
Data are means ꢂ SE from three independent experiments (*p < 0.05 and **p < 0.01 vs.
6.4.3. 5-(2-(2-Hydroxyethoxy)ethyl)-2-phenyl-6,7-dihydropyrazolo
[1,5-a]pyrazin-4(5H)-one (3c)
control).
White solid, mp: 97e99 ꢁC; IR (KBr)
n
: 3429 (OH), 1637 (C]O)
cmꢀ1; 1H NMR (400 MHz, CDCl3)
d: 7.79 (d, J ¼ 7.7 Hz, 2H, ArH), 7.40
6.4. Spectral data of compounds 3ael
(t, J ¼ 7.1 Hz, 2H, ArH), 7.33 (t, J ¼ 7.5 Hz, 1H, ArH), 7.13 (s, 1H, 3-H),
4.42 (t, J ¼ 6.2 Hz, 2H, CH2), 3.93 (t, J ¼ 6.2 Hz, 2H, CH2), 3.75e3.81
(m, 6H, CH2), 3.65 (t, J ¼ 4.4 Hz, 2H, CH2); HRMS m/z calcd for
C16H20N3O3 [M þ H]þ 302.1505; Found: 302.1500. Calcd for
C16H19N3O3Na [M þ Na]þ: 324.1324, Found: 324.1325.
6.4.1. 5-(2-Hydroxyethyl)-2-phenyl-6,7-dihydropyrazolo[1,5-a]
pyrazin-4(5H)-one (3a)
White solid, mp: 109e112 ꢁC; IR (KBr)
n
: 3380 (OH), 1638 (C]O)
cmꢀ1; 1H NMR (400 MHz, CDCl3)
d: 7.80 (d, J ¼ 8.0 Hz, 2H, ArH), 7.41
Fig. 7. Compounds 3eeh induced apoptosis in H322 cell. Cells were treated with the compounds or 0.1% DMSO (control) for 48 h at the concentration of 32 or 64
mM, and then were
stained with Hoechst 33258. The apoptosis rate calculated based on Hoechst 33258 staining, at least 500 cells were counted (*p < 0.05 and **p < 0.01 vs. control).

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N. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 2359e2367
Fig. 8. Compounds 3eeh induced apoptosis in H1299 cell. Cells were treated with the compounds or 0.1% DMSO (control) for 48 h at the concentration of 32 or 64
mM, and then
were stained with Hoechst 33258. The apoptosis rate calculated based on Hoechst 33258 staining, at least 500 cells were counted (*p < 0.05 and **p < 0.01 vs. control).
6.4.4. 5-(2-Morpholinoethyl)-2-phenyl-6,7-dihydropyrazolo[1,5-a]
3.82 (t, J ¼ 6.2 Hz, 2H, CH2), 3.75 (t, J ¼ 6.2 Hz, 2H, CH2), 3.62 (t,
J ¼ 5.6 Hz, 2H, CH2), 1.81e1.87 (m, 2H, CH2); HRMS m/z calcd for
C15H17ClN3O2 [M þ H]þ: 306.1009, Found: 306.1005.
pyrazin-4(5H)-one (3d)
White solid, mp: 189e192 ꢁC; IR (KBr)
n
: 1654 (C]O) cmꢀ1; 1H
: 7.80 (d, J ¼ 7.5 Hz, 2H, ArH), 7.42 (t,
NMR (400 MHz, CDCl3)
d
J ¼ 7.1 Hz, 2H, ArH), 7.34 (t, J ¼ 7.3 Hz, 1H, ArH), 7.14 (s, 1H, 3-H),
4.44 (t, J ¼ 6.1 Hz, 2H, CH2), 3.89 (t, J ¼ 6.1 Hz, 2H, CH2), 3.65e3.75
(m, 6H, CH2), 2.60e2.70 (m, 2H, CH2), 2.50e2.60 (m, 4H, CH2);
HRMS m/z calcd for C18H23N4O2 [M þ H]þ: 327.1821, Found:
327.1821.
6.4.7. 2-(4-Chlorophenyl)-5-(2-(2-hydroxyethoxy)ethyl)-6,7-
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3g)
White solid, mp: 90e93 ꢁC; IR (KBr)
n
: 3385 (OH), 1668 (C]O)
cmꢀ1; 1H NMR (400 MHz, CDCl3)
d
: 7.73 (d, J ¼ 6.7 Hz, 2H, ArH), 7.38
(d, J ¼ 6.7 Hz, 2H, ArH), 7.11 (s, 1H, 3-H), 4.42 (t, J ¼ 6.2 Hz, 2H, CH2),
3.94 (t, J ¼ 6.2 Hz, 2H, CH2), 3.73e3.80 (m, 6H, CH2), 3.61 (t,
J ¼ 4.4 Hz, 2H, CH2); HRMS m/z calcd for C16H19ClN3O3 [M þ H]þ:
336.1115; Found: 336.1103. Calcd for C16H18ClN3O3Na [M þ Na]þ:
358.0935, Found: 358.0918.
6.4.5. 2-(4-Chlorophenyl)-5-(2-hydroxyethyl)-6,7-dihydropyrazolo
[1,5-a]pyrazin-4(5H)-one (3e)
White solid, mp: 131e132 ꢁC; IR (KBr)
n: 3394 (OH), 1636 (C]O)
cmꢀ1; 1H NMR (400 MHz, CDCl3)
d
: 7.73 (d, J ¼ 7.7 Hz, 2H, ArH), 7.38
(d, J ¼ 7.7 Hz, 2H, ArH), 7.11 (s, 1H, 3-H), 4.45 (t, J ¼ 6.1 Hz, 2H, CH2),
6.4.8. 2-(4-Chlorophenyl)-5-(2-morpholinoethyl)-6,7-
3.91e3.96 (m, 4H, CH2), 3.76 (t, J ¼ 5.0 Hz, 2H, CH2); 13C NMR
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3h)
(100 MHz, CDCl3)
d
: 46.19, 47.33, 49.73, 61.59, 104.63, 126.89 (2C),
White solid, mp: 191e193 ꢁC; IR (KBr) : 1653 (C]O) cmꢀ1 1H
n ;
128.98 (2C), 131.02, 134.05, 135.65, 150.94, 158.70; HRMS m/z calcd
NMR (400 MHz, CDCl3)
d
: 7.74 (d, J ¼ 8.4 Hz, 2H, ArH), 7.39 (d,
for C14H15ClN3O2 [M þ H]þ: 292.0853, Found: 292.0848.
J ¼ 8.4 Hz, 2H, ArH), 7.11 (s, 1H, 3-H), 4.43 (t, J ¼ 6.2 Hz, 2H, CH2),
3.89 (t, J ¼ 6.2 Hz, 2H, CH2), 3.68e3.73 (m, 6H, CH2), 2.63 (t,
J ¼ 6.2 Hz, 2H, CH2), 2.51e2.57 (m, 4H, CH2); 13C NMR (100 MHz,
6.4.6. 2-(4-Chlorophenyl)-5-(3-hydroxypropyl)-6,7-
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3f)
CDCl3) d: 43.20, 46.19, 46.31, 53.12, 53.72, 56.35, 66.73, 66.84,
White solid, mp: 123e126 ꢁC; IR (KBr)
n
: 3381 (OH), 1646 (C]O)
: 7.73 (d, J ¼ 8.4 Hz, 2H, ArH), 7.39
(d, J ¼ 8.4 Hz, 2H, ArH), 7.12 (s, 1H, 3-H), 4.45 (t, J ¼ 6.2 Hz, 2H, CH2),
104.55, 126.88 (2C), 128.98 (2C), 131.11, 134.01, 135.82, 150.92,
157.75; HRMS m/z calcd for C18H22ClN4O2 [M þ H]þ: 361.1431,
Found: 361.1435.
cmꢀ1; 1H NMR (400 MHz, CDCl3)
d

N. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 2359e2367
2365
Fig. 9. Compounds 3eeh did not induce apoptosis in A549 cell at 48 h significantly. Cells were treated with the compounds or 0.1% DMSO (control) for 48 h at the concentration of
16, 32 or 64
M, and then were stained with Hoechst 33258. Fluorescent micrographs of Hoechst 33258 staining (200ꢃ) were taken under a fluorescent microscope (Nikon).
m
6.4.9. 5-(2-Hydroxyethyl)-2-(4-methoxyphenyl)-6,7-
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3i)
J ¼ 6.3 Hz, 2H, CH2), 3.84 (s, 3H, OCH3), 3.68e3.72 (m, 6H, CH2), 2.62
(t, J ¼ 6.3 Hz, 2H, CH2), 2.53 (t, J ¼ 4.1 Hz, 4H, CH2); HRMS m/z calcd
for C19H25N4O3 [M þ H]þ: 357.1926, Found: 357.1929.
White solid, mp: 145e147 ꢁC; IR (KBr)
n
: 3400 (OH), 1640 (C]O)
cmꢀ1; 1H NMR (400 MHz, CDCl3)
d: 7.72 (d, J ¼ 6.8 Hz, 2H, ArH), 7.08
(s, 1H, 3-H), 6.94 (d, J ¼ 6.8 Hz, 2H, ArH), 4.44 (t, J ¼ 6.2 Hz, 2H, CH2),
3.90e3.94 (m, 4H, CH2), 3.84 (s, 3H, OCH3), 3.76 (t, J ¼ 5.0 Hz, 2H,
CH2); HRMS m/z calcd for C15H18N3O3 [M þ H]þ: 288.1348. Found:
288.1342.
6.5. Cell culture
A549, H322 and H1299 lung cancer cells and human embryo
lung cells were cultured 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 gentamicin. The cells were seeded onto 96-well plates
or other appropriate dishes containing the medium at the density
of 6250/cm2.
6.4.10. 5-(3-Hydroxypropyl)-2-(4-methoxyphenyl)-6,7-
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3j)
White solid, mp: 113e116 ꢁC; IR (KBr)
n
: 3409 (OH), 1632 (C]O)
cmꢀ1; 1H NMR (400 MHz, CDCl3)
d
: 7.73 (d, J ¼ 7.8 Hz, 2H, ArH), 7.08
(s, 1H, 3-H), 6.95 (d, J ¼ 6.8 Hz, 2H, ArH), 4.44 (t, J ¼ 6.2 Hz, 2H, CH2),
3.85 (s, 3H, OCH3), 3.81 (t, J ¼ 6.2 Hz, 2H, CH2), 3.75 (t, J ¼ 6.1 Hz, 2H,
CH2), 3.59e3.64 (m, 2H, CH2), 3.41 (s, 1H, OH), 1.81e1.86 (m, 2H,
CH2); HRMS m/z calcd for C16H20N3O3 [M þ H]þ: 302.1505; Found:
302.1502; Calcd for C16H19N3O3Na [M þ Na]þ: 324.1324, Found:
324.1323.
6.6. Cell viability assay
Cells were seeded onto 96-well plates and treated with
compounds 3ael at 8, 16, 32 and 64 mM for 48 h, respectively. The
cell proliferation/viability determination using the survival
percentage obtained with the cell treated only with the solvent
(DMSO at 0.1%) as reference. The results are expressed as the
average of triplicate assays.
6.4.11. 5-(2-(2-Hydroxyethoxy)ethyl)-2-(4-methoxyphenyl)-6,7-
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3k)
White solid, mp: 103e106 ꢁC; IR (KBr)
n: 3361 (OH), 1634 (C]O)
6.7. LDH assay
cmꢀ1 1H NMR (400 MHz, CDCl3)
; d
: 7.68 (d, J ¼ 8.4 Hz, 2H, ArH),
7.00 (s,1H, 3-H), 6.92 (d, J ¼ 8.4 Hz, 2H, ArH), 4.34 (t, J ¼ 5.96 Hz, 2H,
CH2), 3.82e3.92 (m, 8H, CH2), 3.81 (s, 3H, OCH3), 3.70e3.75 (m, 2H,
CH2); HRMS m/z calcd for C17H22N3O4 [M þ H]þ: 332.1610; Found:
332.1609; Calcd for C17H21N3O4Na [M þ Na]þ: 354.1430, Found:
354.1428.
Lactate dehydrogenase (LDH) assay was performed on cells
treated with 64 mM compounds 3eeh, for 48 h using a LDH kit
(Nanjing Jiancheng, China) according to the manufacturer’s
protocol. Light absorption was measured at 440 nm using a model
Cintra 5 UVevis spectrometer (GBC, Australia).
6.4.12. 2-(4-Methoxyphenyl)-5-(2-morpholinoethyl)-6,7-
6.8. Hoechst 33258 staining to detect apoptosis
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (3l)
White solid, mp: 195e196 ꢁC; IR (KBr)
n
: 1653 (C]O) cmꢀ1; 1H
For A549, the living cells were stained with 10 mg/mL of Hoechst
NMR (400 MHz, CDCl3)
H), 6.95 (d, J ¼ 8.7 Hz, 2H, ArH), 4.41 (t, J ¼ 6.1 Hz, 2H, CH2), 3.86 (t,
d
: 7.73 (d, J ¼ 8.7 Hz, 2H, ArH), 7.06 (s, 1H, 3-
33258 in the medium for 10 min at 37 ꢁC. For H322 and H1299, cell
fixation was done with 4% formaldehyde in PBS for 10 min before

2366
N. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 2359e2367
Fig. 10. Effect of compounds 3eeh on cell cycle distribution of A549 cells. Cells were exposed to the compounds 3eeh at 64 mM and incubated for 48 h. Values are expressed as
percentage of the cell population in the G1, S, and G2 phase of cell cycle. (A) Control; (B) 3e; (C) 3f; (D) 3g; and (E) 3h. The result shown here is one representative experiment from
three independent experiments.
staining with 2
m
g/mL of Hoechst 33258 at 37 ꢁC for 30 min.
6.10. Statistical analyses
Subsequently the cells were gently washed once with PBS, and
were then observed under a fluorescence microscope (Nikon). The
condensed DNA of apoptotic cells was identified by intense local
staining in the nucleus, in contrast to diffused staining of DNA in
normal cells. A minimum of 500 cells was counted, and each
experiment was performed in triplicate.
Data were presented as means ꢂ SE and analyzed by SPSS soft-
ware. Pictures were processed with Photoshop software. Mean
values were derived from at least three independent experiments.
Differences at p < 0.05 were considered statistically significant.
Acknowledgments
6.9. Flow cytometry analysis of cell cycle distribution
This study was supported by the Science and Technology
Developmental Project of Shandong Province (2008GG10002034)
and National Natural Science Foundation of China (90813022 and
20972088).
Cells were harvested and then fixed with 70% cold ethanol,
stained with 50 mg/mL propidium iodide (PI) containing 10 mg/mL
Rnase A at 4 ꢁC for 1 h. The stained cells were analyzed using
FACSCalibur flow cytometer (BD Bioscience, USA). Cell cycle
distribution was analyzed by ModiFit software (BD Bioscience,
USA).
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