Synthesis of 5-benzyl-2-phenylpyrazolo[1,5-a]pyrazin-4,6(5H,7H)-dione derivatives and discovery of an apoptosis inducer for H322 lung cancer cells

Article abstract of DOI:10.1016/j.bmcl.2011.12.049

A series of substituted 5-benzyl-2-phenylpyrazolo[1,5-a]pyrazin-4,6(5H,7H)- dione derivatives was synthesized by one-step reaction of ethyl 3-phenyl-1H-pyrazole-5-carboxylate derivatives and N-arylalkyl-2- chloroacetamide. Structures of the compounds were determined by IR, ¹H NMR and mass spectroscopy. In addition, a representative single-crystal structure was characterized by using X-ray diffraction analysis. The compound 5j could selectively inhibit the growth of H322 lung cancer cells which contain a mutated p53 gene in a dose-dependent manner through inducing apoptosis of cells.

Full text of DOI:10.1016/j.bmcl.2011.12.049

Bioorganic & Medicinal Chemistry Letters 22 (2012) 844–849
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 5-benzyl-2-phenylpyrazolo[1,5-a]pyrazin-4,6(5H,7H)-dione
derivatives and discovery of an apoptosis inducer for H322 lung cancer cells
Hong-Shui Lv a, , Xiang-Qian Kong c, , Qian-Qian Ming a, Xing Jin c, Jun-Ying Miao b, , Bao-Xiang Zhao a,
⇑
⇑
a School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China
b School of Life Science, Shandong University, Jinan 250100, China
c Department of Vascular Surgery, Provincial Hospital Affiliated to Shandong University, Jinan 250021, 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 substituted 5-benzyl-2-phenylpyrazolo[1,5-a]pyrazin-4,6(5H,7H)-dione derivatives was syn-
thesized by one-step reaction of ethyl 3-phenyl-1H-pyrazole-5-carboxylate derivatives and N-arylal-
kyl-2-chloroacetamide. Structures of the compounds were determined by IR, 1H NMR and mass
spectroscopy. In addition, a representative single-crystal structure was characterized by using X-ray dif-
fraction analysis. The compound 5j could selectively inhibit the growth of H322 lung cancer cells which
contain a mutated p53 gene in a dose-dependent manner through inducing apoptosis of cells.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 26 September 2011
Revised 7 December 2011
Accepted 9 December 2011
Available online 16 December 2011
Keywords:
Synthesis
Pyrazole
Pyrazinone
X-ray
H322 cells
Apoptosis
Cancer, being one of the leading causes of death globally, is a
disease of worldwide importance. Among them, carcinoma of the
lung is the most common cancer and remains the leading cause
of cancer mortality worldwide, with non-small cell lung cancer
(NSCLC) constituting about 85% of all new diagnoses. Therefore,
there is an impetus to design, synthesize and develop more potent
therapeutic agents for cancer. Our interest in investigating pyra-
zole derivatives for their potential anticancer effects has recently
encouraged us to examine the influences of fused-pyrazole hetero-
cyclic compounds on lung cancer cells. Fused-pyrazole heterocyclic
compound is an interesting class of widely studied heterocyclic
compounds, as they have been shown to possess excellent biolog-
ical properties such as antibacterial,1–3 antiangiogenic,4 anti-
fungal,5,6 and antitumor7,8 activities. We bear in mind that imide
derivatives are a valuable group of bioactive compounds showing
androgen receptor antagonists,9,10 anti-inflammatory,11 anxio-
lytic,12 antiviral,13–15 antibacterial,16,17 and antitumor18,19 proper-
ties. Thus, pyrazole-fused imide derivatives could be of interest
and potential target compounds. As part of ongoing research direc-
ted toward the development of pyrazole-based compounds with
structure diversity, we have already reported efficient methods
for the construction of pyrazole-fused heterocycles, for example,
pyrazolo[5,1-c][1,4]oxazin-4-one20,21 and pyrazolo[1,5-a]pyrazin-
4(5H)-one.22–25 Herein we provide the first synthesis of pyrazole-
fused imide compounds. The compounds were evaluated for the
cell cytotoxicity against human lung cancer cells A549 and H322.
A compound, 5j, displayed potent effects in the inhibition of
H322 tumor cell proliferation. Hoechst 33258 staining assay
revealed that compound 5j induced apoptosis of H322 cells.
Chemistry: We first expected that imide compounds could be
synthesized from anhydride compounds like literature26–28 and
much effort had been focused on the cyclizing condensation of
dicarboxylic acids to obtain anhydride compounds as shown in
Scheme 1. We performed the approach from starting material ethyl
3-phenyl-1H-pyrazole-5-carboxylate 1a (R1 = H). Thus, in the pres-
ence of potassium carbonate, 1a reacted with ethyl 2-chloroacetate
under refluxing in acetonitrile to easily afford compound 2a in 76%
yield. Hydrolysis of compound 2a in an ethanol solution of potas-
sium hydroxide gave carboxylic acid 3a in 94% yield. Much effort
was attempted to get compound 4a, however, it failed. We have
paid attention to other routes. It was interesting to find that com-
pound 1a reacted with compound 7a to afford desired product 5a
and intermediate 6a, which can be converted to 5a. Encouraged by
this result, we synthesized a series of compounds 5 by one step
strategy as shown in Scheme 2.
⇑
Corresponding authors. Tel.: +86 531 88366425; fax: +86 531 88564464
(B.-X.Z.).
Structures of 5-benzyl-2-phenylpyrazolo[1,5-a]pyrazin-
4,6(5H,7H)-dione derivatives (5) were determined by IR, 1H NMR
and HRMS spectroscopy.29 For example 5a, the IR spectrum
E-mail addresses: miaojy@sdu.edu.cn (J.-Y. Miao), bxzhao@sdu.edu.cn
(B.-X. Zhao).
These authors contributed equally.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.049

H.-S. Lv et al. / Bioorg. Med. Chem. Lett. 22 (2012) 844–849
845
Scheme 1. Retrosynthetic analysis.
Scheme 2. Synthesis of 5-benzyl-2-phenylpyrazolo[1,5-a]pyrazin-4,6(5H,7H)-dione derivatives.
indicated the presence of two C@O bonds due to the existence of
peaks at 1678 and 1723 cmÀ1 arising from C@O in 4 and 6 position
stretching vibrations, respectively. In 1H NMR spectra, signal of
two methylenes in benzyl and pyrazine ring appeared at the range
of d = 5.16 and 5.24 ppm as single peak, respectively. Singlet signal
of pyrazole proton displayed at d = 7.29 ppm. Two protons of
benzene bound to pyrazole ring appeared as doublet peaks at
d = 7.79 ppm (J = 7.1 Hz). Other protons in benzene moiety
appeared in the range from 7.28 to 7.51 ppm. In the HRMS, in
accordance with the molecular structure of C19H15N3O2, compound
5a gave a [M+H]-ion peak at m/z 318.1230.
Crystallography: The single-crystal of 5e was grown from ethyl
acetate solution at room temperature. The molecular view of 5e
is shown in Figure 1. A summary of crystallographic data collection
parameters and refinement parameters for 5e is compiled in Table
1. In the crystal structure of 5e, C13–C14 bond has a perpendicular

846
H.-S. Lv et al. / Bioorg. Med. Chem. Lett. 22 (2012) 844–849
Figure 1. The molecular structure of compound 5e, with displacement ellipsoids drawn at the 30% probability level.
Table 1
Summary of crystallographic data and structure refinement details for 5e
5e
Empirical formula
Formula weight
Temperature
C19H14ClN3O2
351.78
298(2)
Wavelength
Crystal system
space group
0.71073 Å
Monoclinic
P2(1)/n
Unit cell dimensions
a = 11.331(2) Å, a = 90°
b = 8.5694(16) Å, b = 95.632(3)°
c = 16.851(3) Å,
1628.3(5) A3
4
c = 90°
Volume
Z
Calculated density
Absorption coefficient
F(000)
1.435 Mg/m3
0.253 mmÀ1
728
Crystal size
h range for data collection
Limiting indices
Reflections collected/unique
Completeness to theta = 25.00
Absorption correction
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
0.15 Â 0.10 Â 0.10 mm
2.08 to 25.00°
13 6 h 6 11,À10 6 k 6 10, À20 6 l 6 13
7825/2864 [R(int) = 0.0216]
99.9%
None
Full-matrix least-squares on F2
2864/0/282
1.022
Final R indices [I > 2
R indices (all data)
Largest diff. peak and hole
CCDC No.
r
(I)]
R1 = 0.0350, wR2 = 0.0853
R1 = 0.0484, wR2 = 0.0919
0.162 and À0.209 e ÅÀ3
844337
orientation to the core pyrazole ring (the C11–N3–C13–C14 torsion
angle is À90.5(2)°). The atoms of N1, N2, C7, C8, C9, C10, C11, N3
and C12 are coplanar within 0.0088 Å of deviation. The coplanar
makes dihedral angles of 5.77(10)° and 63.69(5)° with 4-chloro-
phenyl and phenyl rings, respectively, while that between two
phenyl rings is 62.73(6)°. The molecular conformation is stabilized
by C3–H3Á Á ÁN1, C13–H13AÁ Á ÁO2 and C13–H13BÁÁÁO1 intramolecu-
lar hydrogen bonds (Fig. 1, Table 2). The packing diagram (Fig. 2)
shows that molecules of 5e are stabilized by the C2–H2Á Á ÁO2 inter-
molecular interaction and linked into infinite chains. The mole-
C14–C19 ring, H10AÁ Á ÁCg4 2.972(18) Å, C10Á Á ÁCg4 3.872(3) Å,
C10–H10AÁ Á ÁCg4 156.3(14)° and symmetry code: 1/2Àx, À1/2+y,
1/2Àz; C18–H18Á Á ÁCg3, Cg3 is the centroid of the C1–C6 ring,
H18Á Á ÁCg3 2.75(2) Å, C18Á Á ÁCg3 3.666(2) Å, C18–H18Á Á ÁCg3
154.6(14)° and symmetry code: 1/2Àx, À1/2+y, 1/2Àz;
C11–O1Á Á ÁCg1, Cg1 is the centroid of the N1, N2, C9, C8, C7 ring,
O1Á Á ÁCg1 3.3751(17) Å, C11Á Á ÁCg1 4.568(2) Å, C11–O1Á Á ÁCg1
168.98(12)° and symmetry code: 1/2Àx, À1/2+y, 1/2Àz;
C11–O1Á Á ÁCg2, Cg2 is the centroid of the N2, C9, C12, N3, C11,
C10 ring, O1Á Á ÁCg2 3.1113(17) Å, C11Á Á ÁCg1 4.220(2) Å,
C11–O1Á Á ÁCg2 152.33(12)° and symmetry code: 1/2Àx, À1/2+y,
1/2Àz.
cules of 5e are further stabilized by C–HÁ Á Á
p
and C–OÁ Á Á
p
hydrogen bonds: C10–H10AÁ Á ÁCg4, Cg4 is the centroid of the
Inhibitory effects of compounds 5a–l on the proliferation of A549
and H322 lung cancer cells: All the compounds synthesized were
evaluated for their cytotoxicity against A549 and H322 lung cancer
cell lines. After incubated with the compounds at the concentra-
Table 2
Hydrogen bond geometries for 5e
tion of 20 or 40 lM for 24 or 48 h, respectively, the cells were trea-
D–HÁ Á ÁA
d(D–H)
d(HÁ Á ÁA)
d(DÁ Á ÁA)
<(DHA)
ted with SRB to measure their growth/viability (% of the untreated
control) using a spectrophotometer as described previously.30–32
The results showed that the compounds displayed almost no
effects on the growth of A549 lung cancer cells which are p53
wild-type cells (data not shown), but some of them inhibited the
growth of H322 lung cancer cells which are p53-mutant, as shown
C(2)–H(2) Á Á ÁO(2)a
0.951(19)
0.932(18)
0.93(2)
2.568(19)
2.559(17)
2.373(18)
2.376(18)
3.363(2)
2.875(2)
2.739(2)
2.729(3)
141.3(14)
100.2(12)
103.0(13)
100.9(12)
C(3)–H(3)Á Á ÁN(1)
C(13)–H(13A)Á Á ÁO(2)
C(13)–H(13B)Á Á ÁO(1)
0.967(17)
a
Symmetry codes: x+1/2, Ày+1/2, zÀ1/2.

H.-S. Lv et al. / Bioorg. Med. Chem. Lett. 22 (2012) 844–849
847
Figure 2. The crystal packing of compound 5e.
Figure 3. Effects of the compounds 5a–l on H322 cells viability. H322 cells were treated with compounds 5a–l at concentration of 20
Cell viability was analyzed by SRB assay and illustrated in column. Results are presented as mean SE; (n = 3. ⁄, P <0.05 vs control).
l
M or left untreated (control) for 24 h.
which anticancer drugs kill tumor cells.33,34 The morphological fea-
tures of apoptosis include chromatin condensation, cell shrinkage,
and nuclear fragmentation.35 In view of the fact that compound 5j
inhibited more growth of H322 cells, we performed furthermore
Hoechst 33258 staining assay to observe whether compound 5j in-
duces apoptosis of H322 cells. As illustrated in Figure 5, compared
with vehicle (0.1% DMSO), cells cultured with compound 5j had
some cells with condensed chromatin. Granulation of the nucleus
appeared as fluorescent blue in the detail of the Hoechst 33258
stained cells (those from which vehicle was absent). Since nucleus
granulation is a feature of the morphological change in apoptosis,
these results suggest that 5j may induce partly apoptosis in
H322 cells.
Figure 4. Effects of the compound 5j on H322 cells viability. H322 cells were
treated with compound 5j at concentrations of 10, 20, 30 or 40 M or left untreated
(control) for 24 h. Cell viability was analyzed by SRB assay and illustrated in
column. Results are presented as mean SE; (n = 3. ⁄, P <0.05 vs control; ⁄⁄, P<0.01
vs control).
To further explore the selectivity of the compound against H322
cells, compound 5j was examined against HUVECs which were de-
rived from normal vascular endothelial cells. Compound 5j, despite
being very potent against H322 cells, were found to be noncytotox-
l
ic toward HUVECs at up to 40 lM (data not shown). These results
indicated that compound 5j has the potential to be developed as
apoptosis inducer because of its high selectivity against H322 cells.
In summary, we have developed synthesis of a series of novel
substituted 5-benzyl-2-phenylpyrazolo[1,5-a]pyrazin-4,6(5H,7H)-
dione derivatives from ethyl 3-phenyl-1H-pyrazole-5-carboxylate
derivatives and N-arylalkyl-2-chloroacetamide by one-step reac-
tion. The biological evaluation of these compounds on A549 and
H322 lung cancer cell growth showed that the compound 5j inhib-
ited selectively much more proliferation of H322 cells. More inter-
estingly, compound 5j might inhibit the H322 cell growth by
induce apoptosis; whereas compound 5j might be noncytotoxic
in Figure 3. Although compound 5i exhibited effects on the growth
of H322 lung cancer cells at 20 lM for 24 h, its solubility is not
good enough to examine IC50. Thus, we selected compound 5j for
further investigation. Compound 5j showed a most potent inhibi-
tory effect on H322 cells growth in a dose-dependent manner with
IC50 = 28.5 lM at 24 h (Fig. 4). The data suggest that the inhibitory
effect of compound 5j on cancer cell growth is selective.
Compound 5j induces apoptosis of H322 cells: Recent advances in
understanding lung cancer biology have suggested novel treatment
strategies. Activation of apoptotic pathway is a key method by

848
H.-S. Lv et al. / Bioorg. Med. Chem. Lett. 22 (2012) 844–849
Figure 5. Compound 5j induces H322 cells apoptosis significantly at 24 h. (A) Fluorescent micrographs of Hoechst 33258 staining (400Â). (B) Ratio of apoptotic cells vs
control. Results are presented as mean SE; (n = 3. ⁄, P <0.05 vs control; ⁄⁄, P <0.01 vs control).
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Acknowledgments
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This research was supported by National Natural Science Foun-
dation of China (90813022 and 20972088) and Natural Science
Foundation of Shandong Province (Z2008B10).
22. Xie, Y.-S.; Zhao, H.-L.; Su, H.; Zhao, B.-X.; Liu, J.-T.; Li, J.-K.; Lv, H.-S.; Wang, B.-
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29. General procedure for the preparation of 5-aralkyl-2-aryl-pyrazolo[1,5-
a]pyrazine-4,6(5H,7H)-dione 5 and spectroscopy data.
A mixture of ethyl 3-aryl-1H-pyrazole-5-carboxylate 1 (2 mmol), N-aralkyl -2-
chloroacetamide 7 (3 mmol), anhydrous potassium carbonate (4 mmol) and
dry acetonitrile (60 ml) was refluxed for 1–5 h, after which the mixture was
cooled to room temperature and filtered. The filtrate was condensed under
reduced pressure. The crude product was purified by chromatographer on
silica gel using PE/EtOAc (3:1) as an eluent to afford the desired compounds 5.
5-Benzyl-2-phenylpyrazolo[1,5-a]pyrazine-4,6(5H,7H)-dione (5a): White solid,
mp 214–216 °C; IR (KBr) m
/cmÀ1: 3121, 3063, 3032, 2961, 2921, 1723, 1678,
1440, 1378, 1347, 1211; 1H NMR (400 MHz, CDCl3) d: 5.16 (s, 2H, CH2), 5.24 (s,
2H, CH2), 7.29 (s, 1H, Pyrazole H), 7.28–7.51 (m, 8H, ArH), 7.79 (d, 2H,
J = 7.1 Hz, ArH); HRMS calcd for [M+H]+ C19H16N3O2: 318.1243; found:
318.1230.
5-Phenethyl-2-phenylpyrazolo[1,5-a]pyrazine-4,6(5H,7H)-dione
(5b):
Light
yellow solid, mp 189–190 °C; IR (KBr)
m
/cmÀ1: 3125, 3061, 2961, 2920, 1729,
1669, 1440, 1354, 1208; 1H NMR (400 MHz, CDCl3) d: 2.95 (t, 2H, J = 7.9 Hz,
CH2), 4.22 (t, 2H, J = 7.9 Hz, CH2), 5.20 (s, 2H, CH2), 7.18–7.48 (m, 9H,
ArH + Pyrazole H), 7.81 (d, 2H, J = 7.2 Hz, ArH); HRMS calcd for [M+H]+
C20H18N3O2: 332.1399; found: 332.1388.
5-(4-Fluorophenethyl)-2-phenylpyrazolo[1,5-a]pyrazine-4,6(5H,7H)-dione (5c):
Yellow solid, mp 222–224 °C; IR (KBr) m
/cmÀ1: 2962, 2936, 1722, 1670, 1511,
1441, 1351, 1262, 1226; 1H NMR (400 MHz, CDCl3) d: 2.92 (t, 2H, J = 7.9 Hz,
CH2), 4.18 (t, 2H, J = 7.9 Hz, CH2), 5.21 (s, 2H, CH2), 6.99 (t, 2H, J = 8.7 Hz, ArH),
7.24 (d, 2H, J = 8.7 Hz, ArH), 7.29 (s, 1H, Pyrazole H), 7.36–7.41(m, 1H, ArH),
7.42–7.48 (m, 2H, ArH), 7.82 (d, 2H, J = 7.7 Hz, ArH); HRMS calcd for [M+H]+
C20H17FN3O2: 350.1305, found: 350.1296.
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52, 1370.

H.-S. Lv et al. / Bioorg. Med. Chem. Lett. 22 (2012) 844–849
849
5-(3-Methoxyphenethyl)-2-phenylpyrazolo[1,5-a]pyrazine-4,6(5H,7H)-dione
2836, 1729, 1674, 1609, 1452, 1344, 1216; 1H NMR (400 MHz, CDCl3) d: 3.85 (s,
3H, OCH3), 5.15 (s, 2H, CH2), 5.22 (s, 2H, CH2), 6.95 (d, 2H, J = 8.8 Hz, ArH), 7.21
(s, 1H, Pyrazole H), 7.28–7.36 (m, 3H, ArH), 7.47 (d, 2H, J = 7.4 Hz, ArH), 7.72 (d,
2H, J = 8.8 Hz, ArH); HRMS calcd for [M+H]+ C20H18N3O3: 348.1348, found:
348.1335.
(5d): White solid, mp 167–169 °C; IR (KBr) m
/cmÀ1: 3132, 2957, 2935, 2835,
1726, 1677, 1437, 1374, 1318, 1249; 1H NMR (400 MHz, CDCl3) d: 2.92 (t, 2H,
J = 8.0 Hz, CH2), 3.80 (s, 3H, OCH3), 4.21 (t, 2H, J = 8.0 Hz, CH2), 5.21 (s, 2H, CH2),
6.78 (dd, 1H, J1 = 7.8 Hz, J2 = 2.2 Hz, ArH), 6.83 (s, 1H, ArH), 6.88 (d, 1H,
J = 7.8 Hz, ArH), 7.23 (t, 1H, J = 7.8 Hz, ArH), 7.29 (s, 1H, Pyrazole H), 7.35–7.41
(m, 1H, ArH), 7.41 (m, 2H, ArH), 7.82 (d, 2H, J = 7.8 Hz, ArH); HRMS calcd for
[M+H]+ C21H20N3O3: 362.1505, found: 362.1493.
2-(4-Methoxyphenyl)-5-phenethylpyrazolo[1,5-a]pyrazine-4,6(5H,7H)-dione (5j):
Light yellow solid, mp 207–209 °C; IR (KBr) m
/cmÀ1: 3029, 2963, 2937, 2837,
1725, 1671, 1547, 1456, 1428, 1357, 1260; 1H NMR (400 MHz, CDCl3) d: 2.95 (t,
2H, J = 7.9 Hz, CH2), 3.85 (s, 3H, OCH3), 4.21 (t, 2H, J = 7.9 Hz, CH2), 5.18 (s, 2H,
CH2), 6.97 (d, 2H, J = 8.8 Hz, ArH), 7.21 (s, 1H, Pyrazole H), 7.22–7.35 (m, 5H,
ArH), 7.74 (d, 2H, J = 8.8 Hz, ArH); HRMS calcd for [M+H]+ C21H20N3O3:
362.1505, found: 362.1495.
5-Benzyl-2-(4-chlorophenyl)pyrazolo[1,5-a]pyrazine-4,6(5H,7H)-dione (5e): Light
yellow solid, mp 193–196 °C; IR (KBr) m
/cmÀ1: 3133, 2960, 2924, 2853, 1723,
1681, 1576, 1440, 1370, 1337, 1210; 1H NMR (400 MHz, CDCl3) d: 5.16 (s, 2H,
CH2), 5.23 (s, 2H, CH2), 7.26 (s, 1H, Pyrazole H), 7.28–7.36 (m, 3H, ArH), 7.40 (d,
2H, J = 8.6, ArH), 7.48 (dd, 2H, J1 = 7.3 Hz, J2 = 1.4 Hz, ArH), 7.73 (d, 2H,
J = 8.6 Hz, ArH); HRMS calcd for [M+H]+ C19H15ClN3O2: 352.0853, found:
352.0862.
5-(4-Fluorophenethyl)-2-(4-methoxyphenyl)pyrazolo[1,5-a]pyrazine-4,6(5H,7H)-
dione (5k): Orange solid, mp 225 °C; IR (KBr) m
/cmÀ1: 3012, 2965, 2939, 2839,
1725, 1672, 1509, 1456, 1427, 1348, 1261; 1H NMR (400 MHz, CDCl3) d: 2.92 (t,
2H, J = 7.9 Hz, CH2), 3.85 (s, 3H, OCH3), 4.18 (t, 2H, J = 7.9 Hz, CH2), 5.18 (s, 2H,
CH2), 6.97 (d, 2H, J = 8.8 Hz, ArH), 6.99 (t, 2H, J = 8.6 Hz, ArH), 7.21 (s, 1H,
Pyrazole H), 7.23 (d, 2H, J = 8.6 Hz, ArH), 7.74 (d, 2H, J = 8.8 Hz, ArH); HRMS
calcd for [M+H]+ C21H19FN3O3: 380.1410, found: 380.1406.
2-(4-Chlorophenyl)-5-phenethylpyrazolo[1,5-a]pyrazine-4,6(5H,7H)-dione (5f):
Orange solid, mp 229–232 °C; IR (KBr)
m : 3121, 3088, 2962, 2927,
/cmÀ1
1729, 1673, 1442, 1356, 1205; 1H NMR (400 MHz, CDCl3) d: 2.95 (t, 2H,
J = 7.9 Hz, CH2), 4.21 (t, 2H, J = 7.9 Hz, CH2), 5.19 (s, 2H, CH2), 7.21–7.35 (m, 6H,
ArH + Pyrazole H), 7.41 (d, 2H, J = 8.5 Hz, ArH), 7.75 (d, 2H, J = 8.5 Hz, ArH);
HRMS calcd for [M+H]+ C20H17ClN3O2: 366.1009, found: 366.1008.
5-(3-Methoxyphenethyl)-2-(4-methoxyphenyl)pyrazolo[1,5-a]pyrazine-
4,6(5H,7H)-dione (5l): Light yellow solid, mp 171–172 °C; IR (KBr)
m :
/cmÀ1
2-(4-Chlorophenyl)-5-(4-fluorophenethyl)pyrazolo[1,5-a]pyrazine-4,6(5H,7H)-
3125, 3017, 2970, 2939, 2840, 1725, 1672, 1613, 1455, 1357, 1253; 1H NMR
(400 MHz, CDCl3) d: 2.92 (t, 2H, J = 8.0 Hz, CH2), 3.80 (s, 3H, OCH3), 3.85 (s, 3H,
OCH3), 4.21 (t, 2H, J = 8.0 Hz, CH2), 5.19 (s, 2H, CH2), 6.78 (dd, 1H, J1 = 7.8 Hz,
J2 = 2.3 Hz, ArH), 6.83 (s, 1H, ArH), 6.87 (d, 1H, J = 7.8 Hz, ArH), 6.97 (d, 2H,
J = 8.7 Hz, ArH), 7.21 (s, 1H, Pyrazole H), 7.23 (t, 1H, J = 7.8 Hz, ArH), 7.74 (d, 2H,
J = 8.7 Hz, ArH); HRMS calcd for [M+H]+ C22H22N3O4: 392.1610, found:
392.1604.
dione (5g): Orange solid, mp 221–222 °C; IR (KBr) m
/cmÀ1: 3123, 3092, 2962,
2931, 1730, 1673, 1511, 1443, 1354, 1207; 1H NMR (400 MHz, CDCl3) d: 2.92 (t,
2H, J = 7.9 Hz, CH2), 4.18 (t, 2H, J = 7.9 Hz, CH2), 5.19 (s, 2H, CH2), 6.99 (t, 2H,
J = 8.7 Hz, ArH), 7.23 (d, 2H, J = 8.7 Hz, ArH), 7.24 (s, 1H, pyrazole H), 7.41 (d,
2H, J = 8.5 Hz, ArH), 7.75 (d, 2H, J = 8.5 Hz, ArH); HRMS calcd for [M+H]+
C20H16ClFN3O2: 384.0915, found: 384.0902.
2-(4-Chlorophenyl)-5-(3-methoxyphenethyl)pyrazolo[1,5-a]pyrazine-4,6(5H,7H)-
30. Lian, S.; Su, H.; Zhao, B.-X.; Liu, W.-Y.; Zheng, L.-W.; Miao, J.-Y. Bioorg. Med.
Chem. 2009, 17, 7085.
31. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;
Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107.
32. Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simon, R. M.; Tosini, S.; Skehan,
P.; Scudiero, D. A.; Monks, A.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1113.
33. Kaufmann, S. H.; Earnshaw, W. C. Exp. Cell Res. 2000, 256, 42.
34. Fulda, S.; Debatin, K. M. Oncogene 2006, 25, 4798.
dione (5h): Light yellow solid, mp 211–212 °C; IR (KBr) m
/cmÀ1: 3132, 2957,
2932, 2829, 1724, 1676, 1448, 1372, 1312, 1247; 1H NMR (400 MHz, CDCl3) d:
2.92 (t, 2H, J = 7.9 Hz, CH2), 3.80 (s, 3H, OCH3), 4.21 (t, 2H, J = 7.9 Hz, CH2), 5.20
(s, 2H, CH2), 6.79 (dd, 1H, J1 = 7.9 Hz, J2 = 2.3 Hz, ArH), 6.83 (s, 1H, ArH), 6.87 (d,
1H, J = 7.9 Hz, ArH), 7.23 (t, 1H, J = 7.9 Hz, ArH), 7.26 (s, 1H, Pyrazole H), 7.41 (d,
2H, J = 8.3 Hz, ArH), 7.75 (d, 2H, J = 8.3 Hz, ArH); HRMS calcd for [M+H]+
C21H19ClN3O3: 396.1115, found: 396.1112.
5-Benzyl-2-(4-methoxyphenyl)pyrazolo[1,5-a]pyrazine-4,6(5H,7H)-dione
(5i):
Light yellow solid, mp 189–190 °C; IR (KBr)
m
/cmÀ1: 3126, 3001, 2956, 2934,
35. Vaux, D. L.; Korsmeyer, S. J. Cell 1999, 96, 245.