One-pot synthesis and antiproliferative evaluation of pyrazolo[3,4-d] pyrimidine derivatives

Article abstract of DOI:10.1016/j.tet.2012.09.054

An efficient one-pot methodology for the synthesis of pyrazolo[3,4-d] pyrimidines was developed by using 5-aminopyrazoles with formamide in presence of PBr₃ as the coupling agent. Among the examples presented in this work, compounds 41 and 54-5

Full text of DOI:10.1016/j.tet.2012.09.054

Tetrahedron 68 (2012) 9658e9664
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
One-pot synthesis and antiproliferative evaluation of pyrazolo[3,4-d]pyrimidine
derivatives
,
Yu-Ying Huang a, Li-Ya Wang b, Chun-Hsi Chang a, Yueh-Hsiung Kuo c d, Kimiyoshi Kaneko e,
,
a,
*
Hiroyuki Takayama e, Masayuki Kimura e, Shin-Hun Juang a , Fung Fuh Wong
*
a Graduate Institute of Pharmaceutical Chemistry, China Medical University, No. 91, Hsueh-Shih Rd., Taichung 40402, Taiwan, ROC
b The Ph.D. Program for Cancer Biology and Drug Discovery, China Medical University, No. 91, Hsueh-Shih Rd., Taichung 40402, Taiwan, ROC
c The Graduate Institute of Chinese Pharmaceutical Sciences, China Medical University, No. 91, Hsueh-Shih Rd., Taichung 40402, Taiwan, ROC
d Tsuzuki Institute for Traditional Medicine, China Medical University, No. 91, Hsueh-Shih Rd., Taichung 40402, Taiwan, ROC
e Department of Medico Pharmaceutical Science, Nihon Pharmaceutical University, 10281, Komuro, Inamachi, Kita-Adachigun, Saitama, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2012
Received in revised form 3 September 2012
Accepted 11 September 2012
Available online 16 September 2012
An efficient one-pot methodology for the synthesis of pyrazolo[3,4-d]pyrimidines was developed by using
5-aminopyrazoles with formamide in presence of PBr3 as the coupling agent. Among the examples
presented in this work, compounds 41 and 54e56 with phenyl or 2-quinolinyl groups at N-1 and p-MeePh,
p-ClePh, or p-OMeePh group at C-3 position in the pyrazole ring possessed better potency against
NCI-H226 and NPC-TW01 cancer cells with GI50 values between 18 mM and 39 mM.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Pyrazolo[3,4-d]pyrimidines
5-Aminopyrazoles
Antiproliferative activity
One-pot synthesis
1. Introduction
5-aminopyrazoles, formamide using PBr3 as the coupling agent.
Furthermore, the cytotoxicities of these pyrazolo[3,4-d]pyrimidine
derivatives were explored for realizing the structureeactivity
relationship and identifying the structural fragments responsible
for the biological activity. Among the compounds, pyrazolo[3,4-d]
pyrimidines 41 and 54e56 possessed better refficacy to inhibit the
growth of NCI-H226 and NPC-TW01 cancer cells with GI50 values of
Pyrazolopyrimidines and related fused heterocycles have been
found to possess significant pharmacological activities,1 such as
antibacterial,2 antifungal,3,4 antitumor,5 herbicidal,6 antivirus,7 and
as the effective inhibitors of inflammatory mediators in intact
cells.8 Owing to the structural similarity of pyrazolo[3,4-d]pyrimi-
dines with purines,9 they are also known to function as CNS
(Central Nervous System) depressants,10 neuroleptic agents,11 and
as tuberculostatic.12 Moreover, some novel pyrazolo[3,4-d]pyrimi-
dine derivatives determine the antioxidant properties to play an
important role in numerous degenerative or chronic diseases, such
as atherosclerosis and cancer.13
18e39 mM.
2. Result and discussion
2.1. Synthesis of pyrazolo[3,4-d]pyrimidine derivatives
Most of synthesis approach of pyrazolopyrimidines were
performed through heterocyclization reaction using 5-amino-4-
formypyrazoles,14 5-amino-1H-4-pyrazolcarbonitriles,2 5-amino-
1H-4-pyrazolcarboxamides2 or other similar chemical reagents
as the starting substrates.15e17 Herein, we developed an efficient
one-pot synthesis of pyrazolo[3,4-d]pyrimidines by treating
Scheme 1 shows the newly developed one-pot methodology
for synthesis of pyrazolo[3,4-d]pyrimidines. Variation of 5-amino-
1,3-disubstituted pyrazole substrates 1e28 were prepared as
the starting materials by following our previous developed
efficient synthesis methodology.18 The reliable model procedure
involved the treatment of 5-aminopyrazoles with 3.0 equiv of
PBr3 in formamide at 60 ꢀC for 0.5e1.0 h. After aqueous worked-
up and purified by column chromatography on silica gel, the
corresponding pyrazolo[3,4-d]pyrimidine products 29e58
was isolated in good to excellent yields (>76%, see Scheme 1
and Table 1).
* Corresponding author. Tel.: þ886 4 2205 3366x5603; fax: þ886 4 2207
8083; e-mail addresses: wongfungfuh@yahoo.com.tw, ffwong@mail.cmu.edu.tw
(F.F. Wong).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.09.054

Y.-Y. Huang et al. / Tetrahedron 68 (2012) 9658e9664
9659
Table 2
O
The inhibitory activity of the pyrazolo[3,4-d]pyrimidine derivatives in NCI-H226,
NPC-TW01, and Jurkat
NH2
N
N
H
NH2
PBr3
X
N
X
N
N
N
N
N
R
R
X = aryl, 2-pyridinyl, 2-quinolinyl
R = Me, t-Bu, Ph, p-Me-Ph, p-Cl-Ph,
p-OMe-Ph
29-56
X
N
1-28
N
R
Scheme 1.
Compounds Pyrazolo[3,4-d]pyrimidines 29e56 GI50
(
m
M)a,b
NCI-H226 NPC-TW01 Jurkat
X (N-1)
R (C-3)
Table 1
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
46
85
44
75
50
60
45
47
46
75
67
35
18
41
95
58
85
79
62
59
42
55
78
54
49
23
41
82
57
72
>100
99
58
45
55
>100
39
The results of the one-pot synthesis of pyrazolo[3,4-d]pyrimidines from
5-aminopyrazoles, formamide, and PBr3
o-MeePh
o-ClePh
m-MeePh
m-ClePh
p-NO2ePh
p-ClePh
p-BrePh
p-OMeePh
Ph
Ph
Ph
Ph
Ph
o-ClePh
p-ClePh
2-Pyridinyl
2-Pyridinyl
2-Pyridinyl
2-Pyridinyl
2-Pyridinyl
2-Pyridinyl
2-Quinolinyl
2-Quinolinyl
2-Quinolinyl
2-Quinolinyl
2-Quinolinyl
2-Quinolinyl
NH2
Pyrazolo[3,4-d]pyrimidine
29e56
X
N
N
R
Ph
Me
t-Bu
p-MeePh
p-ClePh
p-OMeePh
p-ClePh
p-ClePh
Me
t-Bu
Ph
p-MeePh
p-ClePh
p-OMeePh
Me
t-Bu
Ph
p-MeePh
p-ClePh
p-OMeePh
Substrates
X
R
No.
Yields (%)
1
2
3
4
5
6
7
8
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
96
93
91
92
96
87
91
95
89
93
91
93
91
94
93
95
97
93
91
92
94
89
84
86
92
89
87
76
48
36
90
o-MeePh
o-ClePh
m-MeePh
m-ClePh
m-NO2ePh
p-ClePh
p-BrePh
p-OMeePh
Ph
Ph
Ph
Ph
Ph
o-ClePh
p-ClePh
2-Pyridinyl
2-Pyridinyl
2-Pyridinyl
2-Pyridinyl
2-Pyridinyl
2-Pyridinyl
2-Quinolinyl
2-Quinolinyl
2-Quinolinyl
2-Quinolinyl
2-Quinolinyl
2-Quinolinyl
46
83
>100
>100
>100
>100
>100
>100
68
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
30
>100
>100
>100
>100
>100
>100
>100
61
>100
92
>100
54
9
Ph
Me
t-Bu
p-MeePh
p-ClePh
p-OMeePh
p-ClePh
p-ClePh
Me
t-Bu
Ph
p-MeePh
p-ClePh
p-OMeePh
Me
t-Bu
Ph
p-MeePh
p-ClePh
p-OMeePh
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
65
>100
>100
67
29
39
35
36
69
>100
37
a
NCI-H226: human lung carcinoma; NPC-TW01: human nasopharyngeal carci-
noma; Jurkat: human T-cell leukemia.
b
All tested compounds were dissolved in 100% DMSO at a concentration of
20 mM as the stock solution. Cells were cultured without or in the presence of the
methnimidamide derivatives at different concentrations for 72 h. Cell survival was
determined by MTT assay. Drug molar concentration causing 50% cell growth in-
hibition (GI50) was calculated. Each value represents the meanꢁSD of three in-
dependent experiments.
applied to 5-amino-1-(2-pyridinyl)-3-arylpyrazole 17e22, or
5-amino-1-(2-quinolinyl)-3-arylpyrazole 23e28. The correspond-
ing pyrazolo[3,4-d]pyrimidines 45e50, and 51e56 were obtained in
89e94% and 76e92% yields, respectively (see Scheme 1 and Table
1). All of pyrazolo[3,4-d]pyrimidines 29e56 were fully character-
ized by spectroscopic methods. For example, compound 29
To evaluate the one-pot synthesis method for the preparation
of pyrazolo[3,4-d]pyrimidines, 5-amino-1-3-diphenylpyrazole (1)
was used as the model case. Various coupling agents with various
equivalents were used to provide the optimal condition. These
reagents include benzoyl chloride (PhCOCl), oxalyl chloride (ClCO-
COCl), phosphorous tribromide (PBr3), thionyl chloride (SOCl2), and
p-toluenesulfonic acid (TsOH). We found the use of 3.0 equiv of PBr3
can be provide the best yield of desired pyrazolo[3,4-d]pyrimidine
(29, 96% yield). Employing the same reaction condition to
5-aminopyrazole substrates 2e9 bearing N1 aryl group with various
substituents, including o-Me, o-Cl, m-Me, m-Cl, m-NO2, p-Cl, and
p-OMe, the one-pot heterocyclization methodology also smoothly
gave the corresponding pyrazolo[3,4-d]pyrimidines 30e37 in
87e97% yields (see Scheme 1 and Table 2). Furthermore, we
extended this new method toward 5-amino-1-aryl-3-substituted
pyrazoles 10e16, which contain methyl, tert-butyl, p-MeePh,
position p-OMeePh groups at C-3 or Ph, m-ClePh, p-OMeePh
groups at N1-substituted position in pyrazolic ring. The corre-
sponding 38e44 were obtained in 91e95% isolated yields (see
Scheme 1 and Table 1). This same reaction condition was also
presented two singlet peaks at
d 9.02 and 9.31 ppm for pyrimidine
ring in 1H NMR. In 13C NMR spectrum, compound 29 possessed
characterization absorptions at
d
155.0 ppm for pyrimidine carbon
N]13CHeN]C, at
for eC (13CH3)3.
d
34.4 ppm for e13C(CH3)3, and at
d
30.0 ppm
2.2. Antiproliferative activities of pyrazolo[3,4-d]pyrimidine
derivatives
The growth inhibitory activities of all pyrazolo[3,4-d]pyrimi-
dines 29e56 were evaluated against a panel of human cancer cell
lines in vitro, including lung carcinoma (NCI-H226), nasopharyn-
geal (NPC-TW01), and T-cell leukemia (Jurkat) cells. The GI50 value
indicates the concentration of the compound that results in a 50%
decrease in the cell growth relative to the vehicle. The results are
presented in Table 2 and indicated that pyrazolo[3,4-d]pyrimidines

9660
Y.-Y. Huang et al. / Tetrahedron 68 (2012) 9658e9664
29e56 compounds showed the better inhibitory potency against
nasopharyngeal (NPC-TW01) and lung carcinoma (NCI-H226).
Compound 29 was selected as the compared model for the in-
monitored by TLC analysis. Flash column chromatography was
carried out on silica gel (230e400 mesh). Infrared (IR) spectra
were measured on a Bomem Michelson Series FT-IR spectrometer.
The wavenumbers reported are referenced to the polystyrene
1601 cmꢂ1 absorption. Absorption intensities are recorded by the
following abbreviations: s, strong; m, medium; w, weak. Proton
NMR spectra were obtained on a Bruker (200, 400, or 500 MHz)
spectrometer by use of CDCl3 and DMSO-d6 as solvent. Carbon-13
NMR spectra were obtained on a Bruker (50, 100, or 125 MHz)
spectrometer by used of CDCl3 as solvent. Carbon-13 chemical
hibitory activity study. Their GI50 values are 46
mM (NCI-H226),
41 M (NPC-TW01), and 41 M (Jurkat), respectively. In comparison
m
m
with compounds 30e37 containing various substituents, including
H, o-Me, o-Cl, m-Me, m-Cl, p-NO2, p-Cl, p-Br, and p-OMe, at N-1 of
the phenyl ring, the results showed that pyrazolo[3,4-d]pyrimi-
dines 31, 35e37 possessed similar inhibitory activity against
NCI-H226, NPC-TW01 and Jurkat with GI50 values between 42
and 59 M. In particular, compounds 31 and 36 with o-Cl or p-Br at
N-1 of the phenyl ring showed the better inhibitory potency against
NCI-H226 near 44 M and 47 M and compound 36 also presented
better against NPC-TW01 and Jurkat (42 M and 45 M, see Table
mM
m
shifts are referenced to the center of the CDCl3 triplet (d 77.0 ppm).
Multiplicities are recorded by the following abbreviations: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; J, coupling
constant (hertz). Elemental analyses were carried out on a Heraeus
CHNeO RAPID element analyzer. ESI-MS spectra were obtained
from an Applied Biosystems API 300 mass spectrometer. High-
resolution mass spectra were obtained by means of a JEOL
JMS-HX110 mass spectrometer.
m
m
m
m
2). However, compound 31 with m-ClePh at N-1 on pyrazole ring
displayed poor inhibitory activity (see Table 2).
For further structureeactivity relationship study, we modified
pyrazolo[3,4-d]pyrimidine 29 to compounds 38e42 with various
substituents at C-3 position in pyrazole ring, such as methyl (38),
tert-butyl (39), p-MeePh (40), p-ClePh (41), and p-OMeePh (42).
Their antiproliferative activities were presented in Table 2.
Most of the modified pyrazolo[3,4-d]pyrimidine 38e42 exhibited
similar potency. Particularly, compounds 41 showed the best
3.2. Standard procedure for synthesis of pyrazolo[3,4-d]py-
rimidines (29e56)
The reliable procedure involved the treatmentof 5-aminopyrazoles
(1e28, 1.0 equiv) with catalytic amount of PBr3 (w3 equiv) in form-
amide solution (2 mL) at 50e60 ꢀC within 0.5e1 h. When the reaction
was completed, the reaction mixture was added to saturate sodium
bicarbonate (15 mL) and extracted with dichloromethane (15 mL). The
organic extracts were dried over MgSO4, filtered, and concentrated
under reduced pressure. The residue solution was purified by column
chromatographyon silica gel to give the corresponding pyrazolo[3,4-d]
pyrimidine products (29e56) in 76e96% yields.
antiproliferative activity against NCI-H226 (18
mM), NPC-TW01
(23 M), and Jurkat (36 M, see Table 2), which possess p-ClePh
m
m
group at C-3 position on the pyrazole ring.
Based on the results presented in Table 2, we believed that the
o-Cl and p-Cl at N-1 of the phenyl ring (compounds 29 and 33) and
p-ClePh group at C-3 position on pyrazolic ring (compounds 39)
are essential for promoting the inhibitory activity. As a result, we
designed and synthesized compounds 43 and 44 for testing the
growth inhibitory activity. Unfortunately, compounds 43 and 44
were indicated the poor potent activity with GI50 values >100
for three cancer cell lines, which might come from to their poor
solubility property.
m
M
3.2.1. 1,3-Diphenyl-1H-pyrazolo[3,4-d]pyrimidine (29). Mp (puri-
fied by column chromatography on silica gel) 157e158 ꢀC; 1H NMR
(CDCl3, 500 MHz)
7.48e7.56 (m, 4H, ArH), 7.97e8.07 (m, 2H, ArH), 8.25e8.35 (m, 2H,
ArH), 9.09 (s, 1H), 9.45 (s, 1H); 13C NMR (125 MHz, CDCl3)
114.1,
d 7.29e7.36 (m, 1H, ArH), 7.41e7.48 (m, 1H, ArH),
Furthermore, 1-phenyl-1H-pyrazolo[3,4-d]pyrimidines 38e44
were modified at the N-1-aryl groups to provide compounds 45e50
and 51e56 with different methyl, tert-butyl, p-MeePh, p-ClePh,
and p-OMeePh substituents at C-3. Compounds 45e50 displayed
poor potency the cancer cells that might be due to their poor
solubility. However, 1-(quinolin-2-yl)-1H-pyrazolo[3,4-d]pyrimi-
dines 51e56 showed better biological activity than compounds
45e50, especially compounds 54e56 with p-Me, p-Cl and p-OMe at
N-1 phenyl ring. They inhibited the growth of NCI-H226 and
d
121.2 (2ꢃ CH), 126.7, 127.2 (2ꢃ CH), 129.0 (2ꢃ CH), 129.1 (2ꢃ CH),
129.5, 131.4, 138.5, 144.8,152.7, 153.2, 155.5; IR (KBr) 2364 (m), 2331
(m), 1589 (m), 1498 (m), 1417 (m), 1369 (s), 1219 (m), 1093 (m), 950
(m), 759 (m), 690 (m), 590 (m) cmꢂ1; EIMS m/z (relative intensity)
272 (100), 271 (Mþ, 34), 244 (5), 218 (5), 195 (6), 169 (4), 142 (12),
115 (4), 91 (2), 77 (24); HRMS (EI) calcd for C17H12N4 272.1062,
found 272.1059; Anal. Calcd for C17H12N4; C: 74.98; H: 4.44; N:
20.58, found: C: 74.96; H: 4.45; N: 20.61.
NPC-TW01 with GI50 values between 29 mM and 37 mM.
In conclusion, we developed a one-pot method to prepare a series
of pyrazolo[3,4-d]pyrimidines by treating 5-aminopyrazoles in
presence of PBr3 coupling agent in formamide. Following the struc-
tureeactivity relationship study, we found that introducing o-ClePh
or p-BrePh groups at N-1 and p-MeePh or p-ClePh group at C-3
position in the pyrazole are necessary for the improved bioactivity.
Further SAR indicated the better potency of 1-(quinolin-2-yl)-1H-
pyrazolo[3,4-d]pyrimidines than 1-(pyridin-2-yl)-1H-pyrazolo[3,4-
d]pyrimidines, compound 54e56 with p-Me, p-Cl and p-OMe at N-1
phenyl ring were the examples. Their inhibit values of the growth of
3.2.2. 1-(2-Methylphenyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidine
(30). Mp (purified by column chromatography on silica gel)
142e143 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 2.21 (s, 3H, CH3),
7.33e7.43 (m, 3H, ArH), 7.44e7.50 (m, 2H, ArH), 7.51e7.57 (m, 2H,
ArH), 8.02e8.08 (m, 2H, ArH), 9.05 (s, 1H), 9.53 (s, 1H); 13C NMR
(125 MHz, CDCl3)
d
18.2 (CH3), 112.8, 126.7, 127.2 (2ꢃ CH), 127.6,
129.2 (2ꢃ CH), 129.5 (2ꢃ CH), 131.4, 131.7, 135.5, 136, 144.9, 152.8,
154.1, 155.8; IR (KBr) 3053 (w), 2922 (w), 2360 (m), 2339 (m), 1579
(m), 1556 (s), 1498 (m), 1222 (m), 1085 (m), 952 (m), 759 (m), 829
(m), 692 (m) cmꢂ1; EIMS m/z (relative intensity) 286 (100), 285
(Mþ, 34), 244 (5), 218 (5), 195 (6), 169 (4), 142 (12), 115 (4), 91 (2), 77
(23); HRMS (EI) calcd for C18H14N4 286.1218, found 286.1219; Anal.
Calcd for C18H14N4; C: 75.50; H: 4.93; N: 19.57, found: C: 75.53; H:
4.92; N: 19.58.
NCI-H226 and NPC-TW01 two cells between 29 mM and 37 mM.
Based on the antiproliferative evaluation of pyrazolo[3,4-d]pyrimi-
dines, compounds 41 possessed the best efficacy against NCI-H226
(18 mM) and NPC-TW01 (23 mM) cancer cells.
3. Experimental section
3.1. General procedure
3.2.3. 1-(2-Chlorophenyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidine
(31). Mp (purified by column chromatography on silica gel)
142e143 ꢀC; 1H NMR (CDCl3, 500 MHz)
d
7.43e7.49 (m, 3H, ArH),
7.50e7.56 (m, 2H, ArH), 7.57e7.65 (m, 2H, ArH), 8.02e8.08 (m, 2H,
ArH), 9.07 (s, 1H), 9.52 (s, 1H); 13C NMR (125 MHz, CDCl3)
122.9,
All chemicals were reagent grade and used as purchased.
All reactions were carried out under nitrogen atmosphere and
d

Y.-Y. Huang et al. / Tetrahedron 68 (2012) 9658e9664
9661
127.3 (2ꢃ CH), 127.6, 129.2 (2ꢃ CH), 129.6 (2ꢃ CH), 130.7 (2ꢃ CH),
131.4, 132.1, 134.7, 145.6, 152.8, 154.5, 155.9; IR (KBr) 3061 (w), 2924
(w), 2370 (w), 2349 (w), 1581 (m), 1557 (m), 1516 (m), 1497 (m),
calcd for C17H11ClN4 306.0672, found 306.0669; Anal. Calcd
for C17H11ClN4; C: 66.56; H: 3.61; N: 18.26, found: C: 66.53; H: 3.58;
N: 18.23.
1304 (m), 1223 (m), 1088 (m), 955 (m), 760 (m), 684 (m) cmꢂ1
;
EIMS m/z (relative intensity) 308 (Mþ2, 23), 306 (89), 305 (Mþ, 3),
271 (100), 244 (5),195 (15),176 (5),168 (14),141 (4),114 (6), 77 (22);
HRMS (EI) calcd for C17H11ClN4 306.0672, found 306.0674; Anal.
Calcd for C17H11ClN4; C: 66.56; H: 3.61; N: 18.26, found: C: 66.59;
H: 3.58; N: 18.22.
3.2.8. 1-(4-Bromophenyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidine
(36). Mp (purified by column chromatography on silica gel)
176e177 ꢀC; 1H NMR (CDCl3, 500 MHz)
d
7.45e7.57 (m, 3H, ArH),
7.60e7.66 (m, 2H, ArH), 7.98e8.06 (m, 2H, ArH), 8.21e8.29 (m, 2H,
ArH), 9.10 (s, 1H), 9.47 (s, 1H); 13C NMR (125 MHz, CDCl3)
114.3,
d
120.0, 122.4 (2ꢃ CH), 127.3 (2ꢃ CH), 129.2 (2ꢃ CH), 130.0, 131.2,
132.2 (2ꢃ CH), 137.6, 145.2, 152.8, 153.4, 155.6; IR (KBr) 2374 (w),
2347 (w), 1585 (m), 1495 (m), 1398 (m), 1389 (m), 1215 (m), 1072
(m), 951 (m), 822 (m), 760 (m), 689 (m), 588 (m) cmꢂ1; EIMS m/z
(relative intensity) 352 (Mþ2, 98), 350 (100), 349 (Mþ, 11), 298 (3),
270 (13), 243 (4), 217 (3), 194 (11), 168 (6), 140 (4), 114 (11), 90 (6),
77 (43); HRMS (EI) calcd for C17H11BrN4 350.0167, found 350.0166;
Anal. Calcd for C17H11BrN4; C: 58.14; H: 3.16; N: 15.95, found:
C: 58.16; H: 3.18; N: 15.91.
3.2.4. 1-(3-Methylphenyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidine
(32). Mp (purified by column chromatography on silica gel)
75e76 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 2.47 (s, 3H, CH3), 7.12e7.18
(m, 1H, ArH), 7.38e7.42 (m, 1H, ArH), 7.43e7.49 (m, 1H, ArH),
7.50e7.56 (m, 2H, ArH), 8.00e8.06 (m, 2H, ArH), 8.07e8.11 (m, 2H,
ArH), 9.10 (s, 1H), 9.46 (s, 1H); 13C NMR (125 MHz, CDCl3)
d 21.6
(CH3), 114.1, 118.6, 122.0, 127.3 (2ꢃ CH), 127.6 (2ꢃ CH), 129.0, 129
(2ꢃ CH), 129.5, 131.5, 138.3, 139.2, 144.7, 152.7, 153.2, 155.5; IR (KBr)
3053 (w), 2920 (w), 1611 (m), 1582 (m), 1557 (m), 1495 (m), 1386
(m), 1229 (m), 1094 (m), 959 (m), 783 (m), 764 (m), 692 (m) cmꢂ1
;
3.2.9. 1-(4-Methoxylphenyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimi-
EIMS m/z (relative intensity) 286 (100), 285 (Mþ, 22), 271 (4), 232
(3), 209 (4), 156 (6), 128 (2), 91 (5), 77 (7), 65 (4); HRMS (EI) calcd
for C18H14N4 286.1218, found 286.1215; Anal. Calcd for C18H14N4;
C: 75.50; H: 4.93; N: 19.57, found: C: 75.54; H: 4.96; N: 19.53.
dine (37). Mp (purified by column chromatography on silica gel)
149e150 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 3.84 (s, 3H, CH3),
7.00e7.06 (m, 2H, ArH), 7.42e7.48 (m, 1H, ArH), 7.50e7.54 (m, 2H,
ArH), 7.99e8.05 (m, 2H, ArH), 8.09e8.13 (m, 2H, ArH), 9.10 (s, 1H),
9.45 (s, 1H); 13C NMR (125 MHz, CDCl3)
d 55.5 (CH3), 113.8, 114.3
3.2.5. 1-(3-Chlorophenyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidine
(2ꢃ CH), 123.1 (2ꢃ CH), 127.2 (2ꢃ CH), 129.1 (2ꢃ CH), 129.4, 131.5,
131.6, 144.5, 152.7, 152.8, 155.4, 158.4; IR (KBr) 3053 (w), 2836 (w),
2347 (w), 1580 (m), 1555 (m), 1514 (m), 1416 (m), 1366 (m), 1250
(m), 1219 (m), 1177 (m), 1088 (m), 1034 (m) cmꢂ1; EIMS m/z
(relative intensity) 302 (100), 301 (Mþ, 34), 244 (5), 218 (5), 195 (6),
169 (4), 142 (12), 115 (4), 91 (2), 77 (17); HRMS (EI) calcd for
C18H14N4O 302.1168, found 302.1165; Anal. Calcd for C18H14N4O; C:
71.51; H: 4.67; N: 18.53, found: C: 75.49; H: 4.71; N: 18.51.
(33). Mp (purified by column chromatography on silica gel)
188e189 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 7.27e7.33 (m, 1H, ArH),
7.43e7.48 (m, 1H, ArH), 7.48e7.52 (m, 1H, ArH), 7.53e7.59 (m, 2H,
ArH), 8.01e8.07 (m, 2H, ArH), 8.32e8.36 (m, 1H, ArH), 8.42e8.44
(m, 1H, ArH), 9.14 (s, 1H), 9.50 (s, 1H); 13C NMR (125 MHz, CDCl3)
d
114.5,118.9,121.1, 126.6, 127.4 (2ꢃ CH), 129.3 (2ꢃ CH), 129.9, 130.2,
131.2,135.0,139.6,145.4,152.9,153.6,155.8; IR (KBr) 3099 (w), 3062
(w), 2922 (w), 2851 (w),1585 (m),1491 (m),1406 (m),1215 (m), 951
(m), 866 (m), 779 (m), 758 (m), 691 (m) cmꢂ1; EIMS m/z (relative
intensity) 308 (Mþ2, 36), 306 (100), 305 (Mþ, 23), 286 (4), 271 (4),
252 (4), 229 (4), 176 (7), 111 (5), 91 (5), 77 (15); HRMS (EI) calcd for
C17H11ClN4 306.0672, found 306.0671; Anal. Calcd for C17H11ClN4;
C: 66.56; H: 3.61; N: 18.26, found: C: 66.58; H: 3.62; N: 18.29.
3.2.10. 3-Methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (38). Mp
(purified by column chromatography on silica gel) 79e80 ꢀC; 1H
NMR (CDCl3, 500 MHz)
7.42e7.50 (m, 2H, ArH), 8.13e8.19 (m, 2H, ArH), 9.02 (s, 1H), 9.10
(s, 1H); 13C NMR (125 MHz, CDCl3)
12.4 (CH3), 115.6, 120.8
d 2.63 (s, 3H, CH3), 7.23e7.29 (m, 1H, ArH),
d
(2ꢃ CH), 126.3, 129.1 (2ꢃ CH), 138.4, 143.2, 151.6, 152.6, 155.5; IR
(KBr) 3051 (w), 2922 (w), 2852 (w), 1593 (m), 1562 (m), 1508 (m),
1440 (m), 1355 (m), 1211 (m), 1066 (m), 754 (m), 691 (m), 592 (m)
cmꢂ1; EIMS m/z (relative intensity) 210 (100), 209 (Mþ, 27), 195
(13), 168 (4), 142 (16), 115 (4), 91 (6), 77 (24); HRMS (EI) calcd for
C12H10N4 210.0905, found 210.0903; Anal. Calcd for C12H10N4; C:
68.56; H: 4.79; N: 26.65, found: C: 68.58; H: 4.76; N: 26.61.
3.2.6. 1-(3-Nitrophenyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidine
(34). Mp (purified by column chromatography on silica gel)
183e184 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 7.50e7.61 (m, 3H, ArH),
7.67e7.73 (m, 1H, ArH), 8.02e8.10 (m, 2H, ArH), 8.14e8.20 (m, 1H,
ArH), 8.81e8.87 (m, 1H, ArH), 9.18 (s, 1H), 9.31e9.35 (m, 1H, ArH),
9.52 (s, 1H); 13C NMR (125 MHz, CDCl3)
d 114.7, 115.6, 120.8, 126.0,
127.5 (2ꢃ CH), 129.3 (2ꢃ CH), 130.1, 130.1, 130.8, 139.6, 146.0, 148.8,
153.1, 154.0, 156.0; IR (KBr) 2924 (w), 2348 (w), 1531 (m), 1431 (m),
1416 (m), 1343 (m), 1215 (m), 1092 (m), 954 (m), 762 (m), 739 (m)
cmꢂ1; EIMS m/z (relative intensity) 317 (100), 271 (11), 243 (6), 194
(4), 168 (10), 141 (8), 114 (5), 104 (8), 90 (4), 77 (15); HRMS (EI)
calcd for C17H11N5O2 317.0913, found 317.0916; Anal. Calcd for
C17H11N5O2; C: 64.35; H: 3.49; N: 22.07, found: C: 64.37; H: 3.51; N:
22.03.
3.2.11. 3-tert-Butyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine
(39). Mp (purified by column chromatography on silica gel)
48e49 ꢀC; 1H NMR (CDCl3, 500 MHz)
d
1.55 (s, 9H, 3ꢃ CH3),
7.23e7.29 (m, 1H, ArH), 7.44e7.52 (m, 2H, ArH), 8.19e8.12 (m, 2H,
ArH), 9.02 (s, 1H), 9.31 (s, 1H); 13C NMR (125 MHz, CDCl3)
30.3
d
(3ꢃ CH), 34.4, 114.0, 121.0 (2ꢃ CH), 126.2, 129.0 (2ꢃ CH), 138.6,
152.8, 153.1, 154.8, 155.0; IR (KBr) 3049 (w), 2968 (w), 2666 (w),
1599 (m), 1578 (m), 1555 (m), 1508 (m), 1427 (m), 1366 (m), 1261
(m), 1098 (m), 961 (m), 756 (m) cmꢂ1; EIMS m/z (relative intensity)
252 (44), 251 (Mþ, 1), 237 (100), 195 (5), 168 (2), 142 (2), 116 (1), 77
(14); HRMS (EI) calcd for C15H16N4 252.1375, found 252.1377; Anal.
Calcd for C15H16N4; C: 71.40; H: 6.39; N: 22.21, found: C: 71.43; H:
6.35; N: 22.17.
3.2.7. 1-(4-Chlorophenyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidine
(35). Mp (purified by column chromatography on silica gel)
144e145 ꢀC; 1H NMR (CDCl3, 500 MHz)
d
7.46e7.52 (m, 3H, ArH),
7.52e7.58 (m, 2H, ArH), 8.00e8.06 (m, 2H, ArH), 8.29e8.35 (m, 2H,
ArH), 9.12 (s, 1H), 9.49 (s, 1H); 13C NMR (125 MHz, CDCl3)
114.3,
d
122.2 (2ꢃ CH), 127.4 (2ꢃ CH), 129.2 (2ꢃ CH), 129.3 (2ꢃ CH), 129.8,
131.3, 132.1, 137.2, 145.2, 152.9, 153.4, 155.7; IR (KBr) 3051 (w), 2922
(w), 2851 (w), 2384 (w), 2349 (w), 1589 (m), 1554 (m), 1500 (m),
1406 (m), 1367 (m), 1215 (m), 955 (m), 822 (m) cmꢂ1; EIMS m/z
(relative intensity) 308 (Mþ2, 33), 306 (100), 305 (Mþ, 18), 286 (4),
271 (8), 252 (5), 229 (4), 176 (9), 111 (5), 91 (3), 77 (16); HRMS (EI)
3.2.12. 3-(4-Methylphenyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine
(40). Mp (purified by column chromatography on silica gel)
139e140 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 2.43 (s, 3H, CH3),
7.32e7.42 (m, 3H, ArH), 7.51e7.61 (m, 2H, ArH), 7.86e8.02 (m, 2H,
ArH), 8.24e8.38 (m, 2H, ArH), 9.11 (s, 1H), 9.47 (s, 1H); 13C NMR

9662
Y.-Y. Huang et al. / Tetrahedron 68 (2012) 9658e9664
(125 MHz, CDCl3)
d
21.4 (CH3), 114.2, 121.3 (2ꢃ CH), 126.7, 127.2
78e79 ꢀC; 1H NMR (CDCl3, 500 MHz)
(m, 1H, ArH), 7.82e7.86 (m, 1H, ArH), 8.57e8.61 (m, 1H, ArH),
9.08e9.12 (m, 2H, ArH); 13C NMR (125 MHz, CDCl3)
12.5, 29.5,
115.5, 116.1, 121.6, 138.4, 144.5, 148.9, 150.5, 151.7, 153.2, 156.1; IR
(KBr) 2920 (w), 2853 (w), 1730 (w), 1589 (m), 1506 (m), 1317 (m),
1213 (m), 1124 (m), 1067 (m), 779 (m), 738 (m), 690 (m) cmꢂ1; Anal.
Calcd for C11H9N5; C: 62.55; H: 4.29; N: 33.16, found: C: 62.53; H:
4.27; N: 33.17.
d 2.65 (s, 3H, CH3), 7.16e7.22
(2ꢃ CH), 128.7, 129.2 (2ꢃ CH), 129.9 (2ꢃ CH), 138.5, 139.8, 145.0,
152.8, 153.3, 155.5; IR (KBr) 3030 (w), 2920 (w), 2374 (w), 2349 (w),
2313 (w), 1584 (m), 1503 (m), 1430 (m), 1368 (m), 1220 (m), 953
(m), 785 (m), 756 (m) cmꢂ1; EIMS m/z (relative intensity) 286 (100),
285 (Mþ, 34), 244 (5), 218 (5), 195 (6), 169 (4), 142 (12), 115 (4), 91
(2), 77 (18); HRMS (EI) calcd for C18H14N4 286.1218, found 286.1216;
Anal. Calcd for C18H14N4; C: 75.50; H: 4.93; N: 19.57, found: C:
75.49; H: 4.91; N: 19.55.
d
3.2.18. 3-tert-Butyl-1-(pyridin-2-yl)-1H-pyrazolo[3,4-d]pyrimidine
3.2.13. 3-(4-Chlorophenyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine
(46). Mp (purified by column chromatography on silica gel)
(41). Mp (purified by column chromatography on silica gel)
64e65 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 1.50 (s, 9H, CH3), 7.15e7.19
196e197 ꢀC; 1H NMR (CDCl3, 500 MHz)
d
7.34e7.38 (m, 1H, ArH),
7.48e7.58 (m, 4H, ArH), 7.96e8.02 (m, 2H, ArH), 8.24e8.30 (m, 2H,
ArH), 9.12 (s, 1H), 9.46 (s, 1H); 13C NMR (125 MHz, CDCl3)
114.0,
(m, 1H, ArH), 7.77e7.81 (m, 1H, ArH), 8.05e8.07 (m, 1H, ArH),
8.60e8.62 (m, 1H, ArH), 9.07 (s, 1H), 9.28 (s, 1H); 13C NMR
d
(125 MHz, CDCl3) d 29.7, 34.4, 114.2, 116.1, 121.5, 138.2, 148.8, 151.1,
121.5 (2ꢃ CH), 127.0, 128.5 (2ꢃ CH), 129.3 (2ꢃ CH), 129.5 (2ꢃ CH),
130.0, 135.7, 138.4, 143.8, 152.6, 153.3, 155.7; IR (KBr) 3043 (w), 2926
(w), 2848 (w),1585 (m),1555 (m),1503 (m),1368 (m),1219 (m),1093
(m), 954 (m), 831 (m), 789 (m), 745 (m) cmꢂ1; EIMS m/z (relative
intensity) 308 (Mþ2, 36), 306 (100), 305 (Mþ, 21), 286 (5), 271 (7),
252 (4), 229 (3), 176 (9), 111 (6), 91 (3), 77 (18); HRMS (EI) calcd for
C17H11ClN4 306.0672, found 306.0671; Anal. Calcd for C17H11ClN4; C:
66.56; H: 3.61; N: 18.26, found: C: 66.53; H: 3.62; N: 18.29.
152.8, 153.6, 155.7, 155.7; IR (KBr) 2970 (w), 2227 (w), 1595 (m),
1556 (m), 1498 (m), 1450 (m), 1369 (m), 1192 (m), 1112 (m), 9100
(m), 779 (m) cmꢂ1; EIMS m/z (relative intensity) 253 (23), 252
(Mþ, 2), 238 (100), 198 (12), 171 (2), 105 (3), 78 (31), 57 (5), 51 (7);
Anal. Calcd for C14H15N5; C: 66.38; H: 5.97; N: 27.65, found: C:
66.40; H: 5.99; N: 27.63.
3.2.19. 3-Phenyl-1-(pyridin-2-yl)-1H-pyrazolo[3,4-d]pyrimidine
(47). Mp (purified by column chromatography on silica gel)
3.2.14. 3-(4-Methoxylphenyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimi-
206e207 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 7.22e7.30 (m, 1H, ArH),
dine (42). Mp (purified by column chromatography on silica gel)
7.41e7.55 (m, 3H, ArH), 7.83e7.91 (m, 1H, ArH), 7.99e8.05 (m, 2H,
ArH), 8.20e8.26 (m, 1H, ArH), 8.66e8.70 (m, 1H, ArH), 9.19 (s, 1H),
175e176 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 3.87 (s, 3H, CH3),
7.08e7.84 (m, 2H, ArH), 7.32e7.36 (m, 1H, ArH), 7.50e7.56 (m, 2H,
ArH), 7.95e8.01 (m, 2H, ArH), 8.28e8.32 (m, 2H, ArH), 9.10 (s, 1H),
9.47 (s, 1H); 13C NMR (125 MHz, CDCl3)
d
114, 116, 122, 128 (2ꢃ CH),
129 (2ꢃ CH), 130, 131, 139, 146, 149, 151, 153, 154, 156; IR (KBr) 3049
(w), 2920 (w), 2851 (w), 2380 (w), 2349 (w), 1593 (m), 1481 (m),
1452 (m), 1368 (m), 1265 (m), 1219 (m), 1080 (m), 957 (m), 736 (m),
592 (m) cmꢂ1; EIMS m/z (relative intensity) 273 (100), 272 (Mþ, 17),
230 (16), 196 (10),170 (22), 143 (27), 91 (20), 78 (35), 51 (17); Anal.
Calcd for C16H11N5; C: 70.32; H: 4.06; N: 25.63, found: C: 70.28;
H: 4.09; N: 25.66.
9.45 (s, 1H); 13C NMR (125 MHz, CDCl3)
d 55.4 (CH3), 114.2, 114.6
(2ꢃ CH), 121.3 (2ꢃ CH), 124.1, 126.7, 128.7 (2ꢃ CH), 129.2 (2ꢃ CH),
138.6, 144.8, 152.8, 153.2, 155.5, 160.8; IR (KBr) 3047 (w), 2924 (w),
1612 (m), 1582 (m), 1503 (m), 1431 (m), 1356 (m), 1254 (m), 1219
(m), 1092 (m), 962 (m), 835 (m), 758 (m) cmꢂ1; EIMS m/z (relative
intensity) 302 (100), 301 (Mþ, 6), 287 (25), 265 (4), 259 (8), 233 (3),
195 (2), 151 (4), 91 (5), 77 (12); HRMS (EI) calcd for C18H14N4O
302.1168, found 302.1169; Anal. Calcd for C18H14N4O; C: 71.51; H:
4.67; N: 18.53, found: C: 71.48; H: 4.68; N: 18.50.
3.2.20. 3-(4-Methylphenyl)-1-(pyridin-2-yl)-1H-pyrazolo[3,4-d]py-
rimidine (48). Mp (purified by column chromatography on silica gel)
127e128 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 2.42 (s,1H, CH3), 7.28e7.34
3.2.15. 1-(2-Chlorophenyl)-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]
(m, 3H, ArH), 7.89e7.95 (m, 3H, ArH), 8.26e8.28 (m, 1H, ArH),
pyrimidine (43). Mp (purified by column chromatography on silica
8.71e8.72 (m, 1H, ArH), 9.21 (s, 1H), 9.49 (s, 1H); 13C NMR (125 MHz,
gel) 178e179 ꢀC; 1H NMR (CDCl3, 500 MHz)
d
7.34e7.38 (m, 1H,
CDCl3)
d
21.4, 114.7, 116.4, 122.0, 127.5 (2ꢃ CH), 128.2, 129.8 (2ꢃ CH),
ArH), 7.48e7.58 (m, 4H, ArH), 7.96e8.02 (m, 2H, ArH), 8.24e8.30
138.5, 140.1, 146.2,149.0, 151.1, 152.9,153.9,156.2; IR (KBr) 2922 (W),
2853 (W), 1591 (m), 1481 (m), 1445 (m), 1366 (m), 1317 (m), 1221
(m), 1080 (m), 957 (m), 793 (m) cmꢂ1; EIMS m/z (relative intensity)
287 (100), 286 (Mþ, 22), 259 (7),196 (6),170 (18),154 (9),143 (21), 91
(9), 78 (32), 65 (7), 51 (10); Anal. Calcd for C17H13N5; C: 71.06;
H: 4.56; N: 24.37, found: C: 71.08; H: 4.54; N: 24.39.
(m, 2H, ArH), 9.12 (s, 1H), 9.46 (s, 1H); 13C NMR (125 MHz, CDCl3)
d
114.0, 121.5 (2ꢃ CH), 127.0, 128.5 (2ꢃ CH), 129.3 (2ꢃ CH), 129.5
(2ꢃ CH), 130.0, 135.7, 138.4, 143.8, 152.6, 153.3, 155.7; IR (KBr) 3043
(w), 2926 (w), 2848 (w), 1585 (m), 1555 (m), 1503 (m), 1368 (m),
1219 (m),1093 (m), 954 (m), 831 (m), 789 (m), 745 (m) cmꢂ1; HRMS
(EI) calcd for C17H10Cl2N4 340.0283, found 340.0287; Anal. Calcd for
C17H10Cl2N4; C: 59.84; H: 2.95; N: 16.42, found: C: 59.79; H: 2.93;
N: 16.39.
3.2.21. 3-(4-Chlorophenyl)-1-(pyridin-2-yl)-1H-pyrazolo[3,4-d]py-
rimidine (49). Mp (purified by column chromatography on silica
gel) 164e165 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 7.28e7.34 (m, 1H,
3.2.16. 1-(4-Chlorophenyl)-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]
ArH), 7.48e7.52 (m, 2H, ArH), 7.88e8.02 (m, 3H, ArH), 8.25e8.29
pyrimidine (44). Mp (purified by column chromatography on silica
(m, 1H, ArH), 8.70e8.74 (m, 1H, ArH), 9.22 (s, 1H), 9.48 (s, 1H); 13C
gel) 153e154 ꢀC; 1H NMR (CDCl3, 500 MHz)
d
7.34e7.38 (m,1H, ArH),
7.48e7.58 (m, 4H, ArH), 7.96e8.02 (m, 2H, ArH), 8.24e8.30 (m, 2H,
ArH), 9.12 (s, 1H), 9.46 (s, 1H); 13C NMR (125 MHz, CDCl3)
114.0,
NMR (125 MHz, CDCl3)
d
114.5, 116.5, 122.3, 128.5 (2ꢃ CH), 128.8,
129.5 (2ꢃ CH), 136.0, 138.7, 145.0, 150.9, 152.7, 153.9, 156.4; IR (KBr)
2920 (w), 2851 (w), 1587 (m), 1481 (m), 1217 (m), 1094 (m), 957
(m), 833 (m), 770 (m) cmꢂ1; EIMS m/z (relative intensity) 309
(Mþ2, 30), 307 (100), 306 (Mþ, 13), 279 (5),196 (9), 174 (8), 170 (27),
143 (30), 111 (6), 78 (39), 75 (6), 51 (13); Anal. Calcd for C16H10ClN5;
C: 62.45; H: 3.28; N: 22.76, found: C: 62.43; H: 3.30; N: 22.75.
d
121.5 (2ꢃ CH), 127.0, 128.5 (2ꢃ CH), 129.3 (2ꢃ CH), 129.5 (2ꢃ CH),
130.0, 135.7, 138.4, 143.8, 152.6, 153.3, 155.7; IR (KBr) 3043 (w), 2926
(w), 2848 (w),1585 (m),1555 (m),1503 (m),1368 (m),1219 (m),1093
(m), 954 (m), 831 (m), 789 (m), 745 (m) cmꢂ1; HRMS (EI) calcd for
C17H10Cl2N4 341.0283, found 341.0281; Anal. Calcd for C17H10Cl2N4;
C: 59.84; H: 2.95; N: 16.42, found: C: 59.86; H: 2.98; N: 16.38.
3.2.22. 3-(4-Methoxyphenyl)-1-(pyridin-2-yl)-1H-pyrazolo[3,4-d]
pyrimidine (50). Mp (purified by column chromatography on silica
3.2.17. 3-Methyl-1-(pyridin-2-yl)-1H-pyrazolo[3,4-d]pyrimidine
gel) 167e168 ꢀC; 1H NMR (CDCl3, 500 MHz)
d
3.84 (s, 1H, CH3),
(45). Mp (purified by column chromatography on silica gel)
6.99e7.05 (m, 2H, ArH), 7.22e7.28 (m, 1H, ArH), 7.87e7.99

Y.-Y. Huang et al. / Tetrahedron 68 (2012) 9658e9664
9663
(m, 3H, ArH), 8.22e8.26 (m, 1H, ArH), 8.66e8.70 (m, 1H, ArH), 9.18
(s, 1H), 9.44 (s, 1H); 13C NMR (125 MHz, CDCl3)
55.4, 114.0
154.4, 156.4; IR (KBr) 3455 (m), 3051 (w), 2922 (w), 1602 (m), 1581
(m), 1554 (m), 1477 (m), 1431 (m), 1219 (m), 1089 (m) cmꢂ1; EIMS
m/z (relative intensity) 359 (Mþ2, 34), 357 (100), 356 (Mþ, 17), 246
(13), 220 (26), 193 (36), 128 (42), 101 (15), 77 (7); Anal. Calcd for
C20H12ClN5; C: 67.14; H: 3.38; N: 19.57, found: C: 67.12; H: 3.41; N:
19.57.
d
(2ꢃ CH), 114.6, 116.3, 122.0, 123.6, 129.0 (2ꢃ CH), 138.5, 146.0, 149.0,
151.1, 152.9, 153.8, 156.2, 161.0; IR (KBr) 3460 (w), 3057 (w), 2960
(w), 2924 (w), 2850 (w), 1614 (m), 1529 (m), 1367 (m), 1253 (m),
1219 (m), 1178 (m), 1080 (m) cmꢂ1; Anal. Calcd for C17H13N5O; C:
67.32; H: 4.32; N: 23.09, found: C: 67.35; H: 4.30; N: 23.11.
3.2.28. 3-(4-Methoxyphenyl)-1-(quinolin-2-yl)-1H-pyrazolo[3,4-d]
3.2.23. 3-Methyl-1-(quinolin-2-yl)-1H-pyrazolo[3,4-d]pyrimidine
pyrimidine (56). Mp (purified by column chromatography on silica
(51). Mp (purified by column chromatography on silica gel)
gel) 153e154 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 3.86 (s, 3H, CH3),
158e159 ꢀC; 1H NMR (CDCl3, 500 MHz)
d
2.78 (s, 3H, CH3),
7.05e7.07 (m, 2H, ArH), 7.53e7.55 (m, 1H, ArH), 7.72e7.76 (m, 1H,
ArH), 7.83e7.85 (m, 1H, ArH), 8.02e8.04 (m, 2H, ArH), 8.23e8.25
(m, 1H, ArH), 8.32e8.34 (m, 1H, ArH), 8.45e8.47 (m, 1H, ArH), 9.21
7.51e7.59 (m, 1H, ArH), 7.71e7.87 (m, 2H, ArH), 8.22e8.38 (m, 2H,
ArH), 8.58e8.62 (m,1H, ArH), 9.19 (s,1H), 9.21 (s,1H); 13C NMR (125
MHz, CDCl3)
d
29.7, 115.0, 116.5, 126.6, 127.0, 127.5, 129.4, 130.3
(s,1H), 9.49 (s,1H); 13C NMR (125 MHz, CDCl3)
d
55.5,114.7 (2ꢃ CH),
139.0, 145.3, 147.0, 149.4, 151.9, 154.0, 156.3; IR (KBr) 2924 (w), 2855
(w), 2361 (w), 1597 (m), 1504 (m), 1427 (m), 1327 (m), 1211 (m),
1134 (m), 1080 (m) cmꢂ1; Anal. Calcd for C15H11N5; C: 68.95; H:
4.24; N: 26.80, Found: C: 68.98; H: 4.27; N: 26.83.
114.9, 115.5, 123.7, 126.7, 127.1, 127.5, 129.1 (2ꢃ CH), 129.4, 130.4,
139.0, 146.4, 147.0, 149.8, 153.0, 156.3, 161.1,167.8; IR (KBr) 2922 (w),
2853 (w), 1589 (m), 1481 (m), 1447 (m), 1366 (m), 1219 (m), 1080
(m), 957 (m), 791 (m) cmꢂ1; Anal. Calcd for C21H15N5O; C: 71.38; H:
4.28; N: 19.82, found: C: 71.39; H: 4.25; N: 19.81.
3.2.24. 3-tert-Butyl-1-(quinolin-2-yl)-1H-pyrazolo[3,4-d]pyrimidine
(52). Mp (purified by column chromatography on silica gel)
3.3. Cell lines
144e145 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 1.58 (s, 3H, CH3),
7.49e7.53 (m, 1H, ArH), 7.70e7.72 (m, 1H, ArH), 7.80e7.82 (m, 1H,
ArH), 8.18e8.20 (m, 1H, ArH), 8.29e8.31 (m, 1H, ArH), 9.18 (s, 1H),
Human non-small cell lung carcinoma (NCI-H226) was pur-
chased from American Type Culture Collection (ATCC; Rockville,
MD). T-cell leukemia (MT-2) was obtained from Japanese Collection
of Research Bioresources (JCRB) and nasopharyngeal carcinoma
(NPC-TW01) was purchased from Bioresource Collection and
Research Center (BCRC, Taiwan). All the tumor cell lines were
maintained in either RPMI-1640 or Modified essential medium
(MEM) supplied with 10% fetal bovine serum at 37 ꢀC in a humidi-
fied atmosphere of 5% CO2/95% air in the present of penicillin and
streptomycin.
9.38 (s, 1H); 13C NMR (125 MHz, CDCl3)
d 29.8, 34.6, 114.6, 115.5,
126.5, 126.9, 127.4, 129.3, 130.1, 138.8, 146.9, 150.0, 152.9, 154.3,
154.7, 156.2; IR (KBr) 2964 (w), 2922 (w), 2851 (w), 1622 (m), 1600
(m), 1583 (m), 1502 (m), 1433 (m), 1367 (m), 1190 (m), 1047 (m)
cmꢂ1; Anal. Calcd for C18H17N5; C: 71.27; H: 5.65; N: 23.09, found:
C: 71.29; H: 5.62; N: 23.11.
3.2.25. 3-Phenyl-1-(quinolin-2-yl)-1H-pyrazolo[3,4-d]pyrimidine
(53). Mp (purified by column chromatography on silica gel)
194e195 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 7.49e7.59 (m, 4H, ArH),
3.4. Growth inhibition assay
7.75e7.79 (m, 1H, ArH), 7.87e7.89 (m, 1H, ArH), 8.11e8.13 (m, 2H,
ArH), 8.37e8.39 (m, 1H, ArH), 8.47e8.49 (m, 1H, ArH), 9.26 (s, 1H),
Logarithmic phase cells were seeded in a 96-well plate and
incubated overnight prior to addition of the designated com-
pounds. After incubation with different concentrations of the tested
compounds for 72 h, cells were incubated with MEM containing
0.4 mg/mL MTT for 2 h. The conversion of MTT to formazan by
metabolically viable cells was measured by the absorbance at
570 nm in a 96-well microtiter plate reader. The percentage
conversion by mock-treated control cells was used to evaluate the
effect of the chemicals on cell growth and to determine the
concentration that inhibited 50% of growth (GI50).
9.55 (s, 1H); 13C NMR (125 MHz, CDCl3)
d 114.9, 115.6, 126.8, 127.2,
127.5, 127.8 (2ꢃ CH), 129.2 (2ꢃ CH), 129.5, 130.4, 131.2, 139.0, 146.6,
147.0, 149.8, 153.0, 154.4, 156.4; IR (KBr) 3055 (w), 2376 (w), 2347
(w), 1582 (m), 1557 (m), 1502 (m), 1477 (m), 1409 (m), 1368 (m),
1325 (m), 1219 (m), 1090 (m), 962 (m), 825 (m), 761 (m), 694 (m)
cmꢂ1; EIMS m/z (relative intensity) 323 (100), 295 (Mþ, 21) 246
(17), 220 (29), 193 (39), 140 (12), 128 (46), 101 (18), 77 (21); Anal.
Calcd for C20H13N5; C: 74.29; H: 4.05; N: 21.66, found: C: 74.31; H:
4.07; N: 21.69.
3.2.26. 3-(4-Methylphenyl)-1-(quinolin-2-yl)-1H-pyrazolo[3,4-d]py-
Acknowledgements
rimidine (54). Mp (purified by column chromatography on silica
gel) 163e164 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 2.42 (s, 3H, CH3),
We are grateful to the China Medical University (CMU100-
ASIAe17 and CMU-101-S-23), Tsuzuki Institute for Traditional
Medicine, and the National Science Council of Republic of China for
financial support (NSC-99-2320-B-039-014-MY3). This study is also
supported in part by Taiwan Department of Health Clinical Trial and
Research Center of Excellence (DOH100-TD-B-111-004).
7.32e7.36 (m, 2H, ArH), 7.50e7.58 (m, 1H, ArH), 7.70e7.86 (m, 2H,
ArH), 7.96e8.00 (m, 2H, ArH), 8.22e8.48 (m, 3H, ArH), 9.23 (s, 1H),
9.51 (s, 1H); 13C NMR (125 MHz, CDCl3)
d 21.4, 114.8, 115.4, 126.7,
127.0, 127.4, 127.5 (2ꢃ CH), 128.3, 129.4, 129.8 (2ꢃ CH), 130.3, 138.8,
140.1, 146.5, 146.9, 149.7, 152.9, 154.3, 156.3; IR (KBr) 3473 (m), 3048
(w), 2924 (w), 2855 (w), 1557 (m), 1504 (m), 1477 (m), 1431 (m),
1367 (m), 1325 (m), 1217 (m), 1089 (m), 1022 (m) cmꢂ1; Anal. Calcd
for C21H15N5; C: 74.46; H: 4.48; N: 20.76, found: C: 74.48; H: 4.50;
N: 20.73.
Supplementary data
Supplementary data related to this article can be found online at
http://dx.doi.org/10.1016/j.tet.2012.09.054.
3.2.27. 3-(4-Chlorophenyl)-1-(quinolin-2-yl)-1H-pyrazolo[3,4-d]py-
rimidine (55). Mp (purified by column chromatography on silica
References and notes
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