Synthesis and biological evaluation of pyrazolo[4,3-d]pyrimidine analogues

Article abstract of DOI:10.1016/j.ejmech.2013.05.019

A series of pyrazolo[3,4-d]pyrimidine analogues 3, 4 , 5aef, 6aef with various amines and ester groups at C-4 and N-1 were synthesized and evaluated for antitumour activity. They were also evaluated for xanthine oxidase inhibitory activity, with most compounds having no significant impact. Compound 5e had the strongest activity against human hepatoma carcinoma cells 7402 and 7221, with half-maximal inhibitory concentration values of 4.55 and 6.28, respectively. Structureeactivity relationship studies indicate that chlorine atoms in the structure of 4-((4-(substituted amides)phenyl)amino pyrazolo[4,3-d] pyrimidine analogues is crucial for antitumour activity.

Full text of DOI:10.1016/j.ejmech.2013.05.019

Accepted Manuscript
Synthesis and biological evaluation of pyrazolo[4,3-d] pyrimidine analogues
Li Yuan, ChangWei Song, CaiHu Li, Ying Li, Lin Dong, ShuFan Yin
PII:
S0223-5234(13)00328-0
10.1016/j.ejmech.2013.05.019
EJMECH 6197
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 January 2013
Revised Date: 1 May 2013
Accepted Date: 16 May 2013
Please cite this article as: L. Yuan, C. Song, C. Li, Y. Li, L. Dong, S. Yin, Synthesis and biological
evaluation of pyrazolo[4,3-d] pyrimidine analogues, European Journal of Medicinal Chemistry (2013),
doi: 10.1016/j.ejmech.2013.05.019.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Cl
O
NH
HN
N
N
N
N
CH2COOCH2CH3
5e
Compound 5e possessed the best antitumor activity than 17AAG and
allopurinol.

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of pyrazolo[4,3-d]
pyrimidine analogues
Li Yuan, ChangWei Song, CaiHu Li, Ying Li, Lin Dong, ShuFan Yin*
College of Chemistry, Sichuan University, 29 Wangjiang Road, Chengdu 610064, Sichuan
Province, China
ABSTRACT
A series of pyrazolo[3,4-d]pyrimidine analogues 3, 4, 5a–5f, 6a–6f with various
amines and ester groups at C-4 and N-1 were synthesized and evaluated for
antitumour activity. They were also evaluated for xanthine oxidase inhibitory activity,
with most compounds having no significant impact. Compound 5e had the strongest
activity against human hepatoma carcinoma cells 7402 and 7221, with half-maximal
inhibitory concentration values of 4.55 and 6.28, respectively. Structure–activity
relationship studies indicate that chlorine atoms in the structure of 4-((4-(substituted
amides)phenyl)amino pyrazolo[4,3-d]pyrimidine analogues is crucial for antitumour
activity.
Keywords: pyrazolo[3,4-d]pyrimidines; XOD; antitumour; 17AAG; allopurinol.
1. Introduction
The term cancer encompasses a wide range of types such as lung cancer, colon
cancer, and the more obscure acute leukaemia. Malignant cancers are very common
and are the second largest cause of death in the West after cardiovascular disease. It is
one of the major challenges of this century and is a concern for medical communities
all over the world. The diversity of tumour types and their great similarity to normal
cells are the main obstacles that prevent the discovery of a cure [1-6].
In the last decade or so, researchers have reported that purine derivatives of
allopurinol (Fig. 1) have a certain inhibitory activity towards tumour cells [7]; they
can inhibit heat shock protein 90 (Hsp90), which is involved in the degradation of
somatic proteins. In addition, allopurinol is a structural isomer of hypoxanthine (a
naturally occurring purine in the body) and can be used as inhibitor of the
enzyme xanthine oxidase (XOD) [8-12]. Hsp90 is an important target for antitumour
drugs owing to its effect on proteins, which can promote the growth and metastasis of
tumours[13,14]. Purine derivative PU3 (Fig. 1), similar to geldanamycin (GA, Fig. 1),
is the first purine compound discovered that can inhibit Hsp90 biological activity.
Researchers have also found that PU24F-Cl has a stronger affinity to Hsp90 than PU3,
and it has higher solubility than 17-AAG [15,16](Fig. 1). Although they are very
structurally similar; the hydroxyl being replaced by an amino in the pyrimidine ring,
and N-1 and C-2 being replaced by alkyl and 3,4,5-methoxy-1-benzyl in the pyrazole
ring, respectively.
In this study, we carried out chemical modification of purine by introduction of a
* Corresponding author. Tel.: +86 028 85414832; fax: +86 028 85503392.
E-mail address: chuandayouji217@163.com (S.-F. Yin)
1

ACCEPTED MANUSCRIPT
pyrazole ring instead of an imidazole ring to provide a specific binding mode different
from
that
of
allopurinol
analogues
[17-22].
We
synthesized
pyrazolo[3,4-d]pyrimidines analogues 3, 4, 5a–5f, 6a–6f by introducing various
substituent groups at the C-4 and N-1 positions of the pyrazolopyrimidine ring
(Scheme 1). The aim is to produce a potent and selective Hsp90 inhibitor. XOD
inhibitory activity was evaluated, and all these new compounds were also tested for
antitumour activity against the human hepatoma carcinoma cells 7402 and 7221 using
the half-maximal inhibitory concentration (IC50) values.
2. Results and Discussion
2.1. Synthesis
The general synthetic route for preparation of two series of Hsp90 inhibitors (5a–5f
and 6a–6f) is shown in Scheme 1. In the pyrazolopyrimidine ring system, the chloro
substituent, as a leaving group, was introduced at C-4, which was the most reactive
site for nucleophilic attack. Heating commercially available allopurinol (1) with
excess N,N-dimethylaniline and phosphorus oxychloride gave an intermediate product
2, which was treated with ethyl bromoacetate to afford the corresponding ethyl
2-(4-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetate (3). Reacting
3
with
p-phenylenediamine in acetonitrile at room temperature yielded compound 4.
Amination of 4 with sulfonyl chloride or chloride gave target compounds 5a–5f,
Scheme 1.
2.2. Effect on XOD inhibitory activity
Inhibition of the XOD-catalyzed conversion of xanthine to uric acid by all
1-N-alkyl-4-N-substituted amino allopurinol derivative inhibitors was evaluated and
compared with the standard inhibitor allopurinol. Enzyme activity was
spectrophotometrically monitored by measuring uric acid formation at 290 nm. This
was done with a saturated concentration of xanthine (20 ꢀM) as the substrate in 1 mL
of 200 mM phosphate buffer at pH 7.5 and at 25 °C. All samples were tested for XOD
inhibitory activity at different concentrations (5, 10, 15 and 20 ꢀM). The test results
are listed in Table 1.
On observing the data in Table 1, there seems to be no correlation between XOD
inhibitory activity and the substituent size at C-4 and N-1 of the pyrazolopyrimidine
skeleton, as there is no significant difference from the standard allopurinol compound.
Most compounds did not show any significant XOD inhibitory activity. Compounds
5d, 5f, 6e and 6f exhibited potent XOD inhibitory activity against the target
organisms, possibly because of the structural differences i.e. 4-hydroxyl versus amino.
A previous literature report has shown that allopurinol derivatives such as
4-amino-substituted allopurinol have a lower XOD inhibitory activity than
4-hydroxyl-substituted allopurinol [9]. The results of these structure–activity studies
suggest that the structure of purine is crucial for XOD inhibitory activity.
2.3. Effect on antitumour activity
The synthesized compounds (3–6; Scheme 1) were subjected to the human cell
lines screening assay for evaluation of their in vitro antitumour activity. A single high
dose (64 ꢀM) of the test compounds were used in the full NCI two cell lines panel
2

ACCEPTED MANUSCRIPT
assay which includes human hepatoma carcinoma cells 7402 and 7221. The data were
reported as a mean graph of the percent growth of treated cells and presented as IC50
values, Table 2. The obtained results of the tested allopurinol derivatives 3, 4, 5a, 5b,
5c, 5d, 5f, 6a, 6b, 6c, 6d, 6e, 6f showed IC50 values >60 ꢀg/ml, a distinctive potential
pattern of selectivity, as well as broad-spectrum antitumour activity. Compound 5e
showed potency against human hepatoma carcinoma cells 7402 and 7221 lines with
IC50 values of 7.27 and 3.51, respectively. It also has a higher antitumour activity
compared with 17AAG. This difference indicates that 5e may exert its cell growth
inhibitory effect through partial disruption or suppressed dynamics of microtubules or
even through interactions with other cellular targets.
2.4. Structure–activity correlation
Structure–activity correlation, based on the number of cell lines that showed
sensitivity toward each of the synthesized individual compounds, revealed that ethyl
2-(4-((4-(substituted
amides)phenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)
acetate (5) is a more active antitumour agent than 2-(4-((4-(4-substituted amides)
phenyl)amino)-1H-pyrazolo[3,4-d] pyrimidin-1-yl)acetic acid (6). It is possible that
the introduction of an ester group may increase the lipid/water partition coefficient.
As previously reported for PU3 containing an amino group at position 4 and an ethyl
group at position 1 (Fig. 1), amino substitution at position 4 and ester group
substitution at position 1 were also observed in compound 4. The presence of an ester
group at position N1, rather than a carboxylic acid group, proved essential for activity.
Ethyl 2-(4-((4-aminophenyl) amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetate (4) is
a
more
active
antitumour
agent
than
ethyl
2-(4-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetate (3). This suggested that the
presence of a nitrogen atom (aminoanilino group) at position C4 enhances the
antitumour effect. Among the derivatives of 5 (5a–5c), the substituted
benzenesulfonamide pyrazolo[4,3-d]pyrimidine derivative showed more potent
activity than the unsubstituted derivative. Moreover, among the derivatives of 5
(5d–5f), 5e and 5f are small molecules of fatty acid amides. The negative inductive
effect of 5f reduces the density of the electron cloud. Although the presence of a
strong negative halogen atom in 5e exerts a strong electron-withdrawing effect, the
presence of chlorine atoms in 5e can enhance the activity toward the cell lines.
3. Conclusion
Among the compounds that displayed stronger antitumour activity, only 5e
exhibited a reasonable antitumour activity. Pyrazolo[4,3-d]pyrimidines were found to
have a much weaker XOD inhibitory activity than the standard allopurinol. On the
other
hand,
ethyl
2-(4-((4-(2-chloroacetamido)phenyl)amino)-1H-pyrazolo
[3,4-d]pyrimidin-1-yl) acetate (5e) (Fig. 2) exhibited an antitumour activity higher
than 17AAG and allopurinol. Therefore, the unique dual specificity of 5e, which
displays an antitumour activity more potent than conventional drugs, represents an
attractive starting point for further optimization. Further studies on the antitumour
mechanisms of 5e are underway.
4. Experiment
Melting Point were measured with a micromelting point tester and are uncorrected.
3

ACCEPTED MANUSCRIPT
1H and 13C NMR spectra were obtained in DMSO-d6 or CDCl3 solutions on a Bruker
AVII-400 spectrometer operating at 400 and 100 MHz, respectively. High-resolution
mass spectra were obtained on a Waters Q-TOF-Premiter instrument (TOF-MS).
Infrared (IR) spectra were obtained by Perkin-Eliner 16PC-FT infrared spectrometer.
Thin layer chromatography (TLC) was employed to routinely monitor the reaction
samples and confirm the homogeneity of the analytical samples by using Kieselgel
60F254 (0.25 mm) silica gel TLC aluminum sheets. Chromatography was carried out
using Merck silica yel 60, 200-300 mesh. All reagents were commercially available
and were used without further purification unless otherwise indicated. Allopurinol and
xanthine oxidase were purchased from Sigma. Dimethylsulfoximine (DMSO) was
purchased from Amresco.
4.1. Chemistry
4.1.1. 4-chloro-1H-pyrazolo[3,4-d]pyrimidine (2)
A mixture of Allopurinol 1 (2.00 g, 14.69 mmol) and N,N-dimethylaniline (2.00 g,
16.52 mmol) was stirred in POCl3 (25 mL) at 80 °C for 2 h. The reaction mixture was
diluted with water (35 mL), and extracted with ethyl acetate. The organic layer was
washed with water and the organic phase was concentrated to dryness, and the residue
was purified by column chromatography on silica gel using 4:1.5 petroleum ether/
ethyl acetate as the eluent to give 2.
4.1.2. ethyl 2-(4-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetate (3)
To a solution of 4-chloro-1H-pyrazolo[3,4-d]pyrimidine 2 (0.81 g, 5.26 mmol) in
anhydrous DMF (15 mL ) was added Triethylamine (1.78 g, 17.62 mmol). The
mixture was stirred at room temperature for 30min. Then Ethylbromoacetate (1.06g,
6.39mmol) was added and the mixture was stirred at room temperature for 1 h and KI
was added. The reaction mixture was subsequently diluted with water (45 mL),
acidified with HCl and extracted with ethyl acetate. Finally, the organic layer was
washed with water and the organic phase was concentrated to dryness, and the residue
was purified by column chromatography on silica gel using 10:1 petroleum ether/
ethyl acetate as the eluent to give 3: white powder; mp 83-84 ; 1H NMR (400 MHz,
DMSO-d6) δ: 8.91 (s, 1H, CH), 8.58 (s, 1H, CH), 5.45 (s, 2H, CH2), 4.17 (dd, J = 7.10
Hz, J = 14.22 Hz, 2H, CH2), 1.20 (t, J = 7.10 Hz, 3H, CH3); 13C NMR (100 MHz,
DMSO-d6) 167.90, 156.53, 151.47, 151.09, 135.32, 106.29, 61.80, 48.86, 14.36; IR
(KBr, ν, cm-1): 3458, 3116, 2992, 2938, 1734, 1591, 1556, 1486, 1404, 1357, 1250,
1218, 1188, 1134, 1019, 950, 861, 786, 713, 568; HRMS (ESI) calcd for C9H9ClN4O2
[M䟌H]䟌, 240.0414; found, 241.0492.
4.1.3. ethyl 2-(4-((4-aminophenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1- yl)acetate
(4)
To a solution of ethyl 2-(4-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetate 3
(1.73 g, 7.21 mmol) in acetonitrile (5 mL) was added benzene-1,4-diamine (0.78 g,
7.21 mmol). The mixture was stirred at 80
for 3 h. The reaction mixture was
concentrated to dryness, and the residue was purified by recrystallization using ethyl
acetate to give 4: mp 179.8–181.9 ; 1H NMR (400 MHz, DMSO-d6) δ: 9.76 (br, 1H,
4

ACCEPTED MANUSCRIPT
NH), 8.27 (s, 2H, CH), 7.37 (s, 2H, ArH), 6.62 (s, 2H, ArH), 5.02–5.18(m, 4H), 4.14
13
(dd, J = 7.04 Hz, J = 14.16 Hz, 2H, CH2), 1.19 (t, J = 7.08 Hz, 3H, CH3); C NMR
(100 MHz, DMSO-d6) 167.80, 155.73, 154.30, 153.61, 132.52, 127.41, 123.34,
113.94, 100.76, 61.18, 47.80, 13.94; IR (KBr, ν, cm-1): 3476, 3381, 3200, 2940, 1735,
1591, 1514, 1440, 1329, 1291, 1254, 1212, 1019, 915, 795, 740, 706, 603, 516;
HRMS (ESI) calcd for C15H16N6O2 [M䟌H]䟌, 312.1335; found, 313.1439.
4.1.6. ethyl 2-(4-((4-(4-bromophenylsulfonamido)phenyl)amino)-1H-pyrazolo[3,4-d]
pyrimidin-1-yl)acetate (5a)
To
a
solution
of
ethyl
2-(4-((4-aminophenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetate 4 (0.36 g,
1.15 mmol) in THF(10 mL) was added NaH (0.069 g, 2.88 mmol) at 0 °C and stirred
for 30 min. And then 4-bromobenzene-1-sulfonyl chloride (0.34 g, 1.34 mmol ) was
added and stirred at 0 °C for 2 h. The reaction mixture was diluted with water (15
mL), and extracted with dichloromethane. The organic layer was washed with water
and the organic phase was concentrated to dryness, and the residue was purified by
column chromatography on silica gel using 2:1 petroleum ether/ acetone as the eluent
to give 5a: 54%; mp 201.5–203 °C; 1H NMR (400 MHz, DMSO-d6) δ: 10.28 (br, 1H,
NH), 10.09 (br, 1H, NH), 8.38 (s, 1H, CH), 8.22 (s, 1H, CH), 7.79 (d, J = 8.60 Hz, 2H,
ArH), 7.65–7.71 (m, 4H, ArH), 7.10 (d, J = 8.88 Hz, 2H, ArH), 5.23(s, 2H, CH2), 4.15
(dd, J = 7.12 Hz, J = 14.24 Hz, 2H, CH2), 1.19(t, J = 7.10 Hz, 3H, CH3); 13C NMR
(100 MHz, DMSO-d6) 168.20, 155.99, 154.67, 154.05, 139.16, 136.31, 133.24,
132.79, 129.19, 127.22, 122.63, 122.16, 101.32, 61.71, 48.41, 14.44; IR (KBr, ν, cm-1):
3369, 3221, 2927, 2854, 1747, 1622, 1576, 1508, 1435, 1321, 1291, 1209, 1157, 1091,
1026, 917, 793, 742, 655, 606, 549, 419; HRMS (ESI) calcd for C21H19BrN6O4S[M䟌
H]䟌, 530.0372; found, 531.0442.
4.1.7.
ethyl
2-(4-((4-(4-nitrophenylsulfonamido)phenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl
)acetate (5b)
To
a
solution
of
ethyl
2-(4-((4-aminophenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetate 4 (0.36 g,
1.15 mmol) in THF(10mL) was added NaH (0.069 g, 2.88 mmol) at 0 °C and stirred
for 30 min. And then 4-nitrobenzene-1-sulfonyl chloride (0.30 g, 1.34 mmol) was
added and stirred at 0 °C for 2 h. The reaction mixture was diluted with water (15
mL), and extracted with dichloromethane. The organic layer was washed with water
and the organic phase was concentrated to dryness, and the residue was purified by
column chromatography on silica gel using 2:1 petroleum ether/ acetone as the eluent
1
to give 5b: 48%; mp182–183 °C; H NMR (400 MHz, DMSO-d6) δ: 10.53 (br, 1H,
5

ACCEPTED MANUSCRIPT
NH), 10.10 (br, 1H, NH), 8.37–8.41(m, 3H), 8.20 (s, 1H, CH), 8.00 (d, J = 8.88 Hz,
2H, ArH), 7.72 (d, J = 8.72 Hz, 2H, ArH), 7.12(d, J = 8.88 Hz, 2H, ArH), 5.22 (s, 2H,
CH2), 4.15 (dd, J = 7.12 Hz, J = 14.24 Hz, 2H, CH2), 1.19 (t, J = 7.12 Hz, 3H, CH3);
13C NMR (100 MHz, DMSO-d6) 168.18, 155.94, 154.66, 154.03, 150.25, 145.33,
136.66, 132.87, 132.68, 128.80, 125.04, 122.49, 101.31, 61.72, 48.38, 14.41; IR (KBr,
ν, cm-1): 3390, 3253, 3108, 1734, 1620, 1573, 1529, 1481, 1379, 1346, 1213, 1166,
1090, 1015, 919, 857, 792, 737, 607, 547, 464; HRMS (ESI) calcd for C21H19N7O6S
[M䟌H]䟌, 497.1118; found, 498.1199.
4.1.8. ethyl 2-(4-((4-(phenylsulfonamido)phenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin
-1-yl)acetate (5c)
To
a
solution
of
ethyl
2-(4-((4-aminophenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetate 4 (0.36 g,
1.15 mmol) in THF(10 mL) was added NaH(0.069 g, 2.88 mmol) at 0 °C and stirred
for 30min.And then benzenesulfonyl chloride ( 0.24 g, 1.34 mmol ) was added and
stirred at 0 °C for 2 h. The reaction mixture was diluted with water (15 mL), and
extracted with dichloromethane. The organic layer was washed with water and the
organic phase was concentrated to dryness, and the residue was purified by column
chromatography on silica gel using 2:1 petroleum ether/ acetone as the eluent to give
5c: 57%; mp224–226 °C;1H NMR (400 MHz, DMSO-d6) δ: 10.21 (br, 1H, NH),
10.06 (br, 1H, NH), 8.37 (s, 1H, CH), 8.20 (s, 1H, CH), 7.76 (d, J = 7.12 Hz, 2H,
ArH), 7.54–7.67(m, 5H, ArH), 7.11 (d, J = 8.84 Hz, 2H, ArH), 5.22 (s, 2H, CH2), 4.14
13
(dd, J = 7.12 Hz, J = 14.20 Hz, 2H, CH2), 1.19 (t, J = 7.10 Hz, 3H, CH3); C NMR
(100 MHz, DMSO-d6) 167.72, 155.51, 154.23, 153.56, 139.43, 135.46, 133.20,
132.81, 132.38, 129.18, 126.67, 122.32, 121.30, 100.76, 61.21, 47.91, 13.95; IR (KBr,
ν, cm-1): 3345, 3236, 2927, 2854, 1740, 1621, 1573, 1508, 1483, 1441, 1375, 1294,
1212, 1235, 1162, 1093, 1016, 909, 870, 783, 725, 657, 583, 547; HRMS (ESI) calcd
for C21H20N6O4S [M䟌H]䟌, 452.1267; found, 453.1355.
4.1.9.
ethyl
2-(4-((4-benzamidophenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1
-yl)acetate (5d)
To
a
solution of ethyl
2-(4-((4-aminophenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetate 4 (0.50 g, 1.6
mmol) in THF(13 mL) was added Triethylamine (0.50 g, 4.95 mmol) at 0 °C and
stirred for 30min. And then benzoyl chloride ( 0.25 g, 1.79 mmol ) was added and
stirred at 0 °C for 2 h. The reaction mixture was diluted with water (15 mL), and
extracted with dichloromethane. The organic layer was washed with water and the
organic phase was concentrated to dryness, and the residue was purified by column
chromatography on silica gel using 2:1 petroleum ether/ acetone as the eluent to give
1
5d: 59%; mp 233–235 ; H NMR (400 MHz, DMSO-d6) δ: 10.30 (br, 1H, NH),
6

ACCEPTED MANUSCRIPT
10.15 (br, 1H, NH), 8.42 (s, 1H, CH), 8.27 (s, 1H, CH), 7.97 (d, J = 7.72 Hz, 2H,
ArH), 7.78–7.83 (m, 4H, ArH), 7.52–7.62 (m, 3H, ArH), 5.24 (s, 2H, CH2), 4.16 (dd,
13
J = 7.10 Hz, J = 14.22 Hz, 2H, CH2), 1.20 (t, J = 7.08 Hz, 3H, CH3); C NMR (100
MHz, DMSO-d6) 168.25, 165.83, 156.11, 154.88, 154.11, 135.45, 135.12, 132.98,
131.94, 128.82, 128.09, 122.30, 121.25, 101.29, 61.72, 48.41, 14.45; IR (KBr, ν, cm-1):
3328, 3268, 3165, 2995, 2933, 1948, 1893, 1743, 1631, 1587, 1544, 1514, 1488, 1432,
1381, 1317, 1303, 1242, 1214, 1108, 1025, 919, 831, 784, 681, 524; HRMS (ESI)
calcd for C22H20N6O3 [M䟌H]䟌, 416.1597; found, 417.1672.
4.1.10.
pyrimidin-1-yl) acetate (5e)
To
ethyl
2-(4-((4-(2-chloroacetamido)phenyl)amino)-1H-pyrazolo[3,4-d]
a
solution
of
ethyl
2-(4-((4-aminophenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetate 4 (0.50 g, 1.6
mmol) in THF(13 mL) was added Triethylamine (0.50 g, 4.95 mmol) at 0 °C and
stirred for 30min. And then 2-chloroacetyl chloride ( 0.20 g, 1.79 mmol ) was added
and stirred at 0 °C for 2 h. The reaction mixture was diluted with water (15 mL), and
extracted with dichloromethane. The organic layer was washed with water and the
organic phase was concentrated to dryness, and the residue was purified by column
chromatography on silica gel using 2:1 petroleum ether/ acetone as the eluent to give
1
5d: 52%; mp 189.7–191.5 ; H NMR (400 MHz, DMSO-d6) δ: 10.34 (br, 1H, NH),
10.14 (br, 1H, NH), 8.40 (s, 1H, CH), 8.26 (s, 1H, CH), 7.78 (d, J = 8.52 Hz, 2H,
ArH), 7.62 (d, J = 8.96 Hz, 2H, ArH), 5.23 (s, 2H, CH2), 4.26 (s, 2H, CH2), 4.15 (dd,
13
J = 7.10 Hz, J = 14.22 Hz, 2H, CH2), 1.20 (t, J = 7.10 Hz, 3H, CH3); C NMR (100
MHz, DMSO-d6) 168.23, 164.89, 156.00, 154.78, 154.05, 135.26, 134.86, 132.97,
122.40, 120.27, 101.29, 61.72, 48.40, 44.04, 14.43; IR (KBr, ν, cm-1): 3627, 3265,
3197, 3110, 2992, 1742, 1667, 1572, 1513, 1434, 1294, 1209, 1095, 1019, 966, 919,
844, 785, 705, 656, 520; HRMS (ESI) calcd for C17H17ClN6O3 [M䟌H]䟌, 388.1051;
found, 389.1121.
4.1.11.
1-yl)acetate (5f)
ethyl
2-(4-((4-acetamidophenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-
solution of ethyl
To
a
2-(4-((4-aminophenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetate 4 (0.50 g, 1.6
mmol) in THF(13 mL) was added Triethylamine (0.50 g, 4.95 mmol) at 0 °C and
stirred for 30min. And then acetyl chloride ( 0.14 g, 1.79 mmol ) was added and
stirred at 0 °C for 2 h. The reaction mixture was diluted with water (15 mL), and
extracted with dichloromethane. The organic layer was washed with water and the
organic phase was concentrated to dryness, and the residue was purified by column
chromatography on silica gel using 2:1 petroleum ether/ acetone as the eluent to give
5f: 57%; mp 201–203 1H NMR (400 MHz, DMSO-d6) δ: 11.42 (br, 1H, NH),
10.25 (br, 1H, NH), 8.42–8.68 (m, 2H, CH), 7.61–7.72 (m, 4H, ArH), 5.31 (s, 2H,
7

ACCEPTED MANUSCRIPT
CH2), 4.18 (dd, J = 7.00 Hz, J = 14.08 Hz, 2H, CH2), 2.08 (s, 3H, CH3), 1.21 (t, J =
7.04 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) 168.31, 167.45, 152.20, 134.17,
119.58, 100.19, 61.38, 48.32, 23.94, 13.97; IR (KBr, ν, cm-1): 3359, 2921, 2851, 1746,
1665, 1524, 1396, 1295, 1211, 1096, 1022, 850, 791, 692, 596, 553; HRMS (ESI)
calcd for C17H18N6O3 [M䟌H]䟌, 354.1440; found, 355.1451.
4.1.12.
pyrimidin-1-yl)acetic acid (6a)
To
2-(4-((4-(4-bromophenylsulfonamido)phenyl)amino)-1H-pyrazolo[3,4-d]
2-(4-((4-(4-bromophenylsulfonamido)phenyl)amino)-1H-pyrazolo[3,4-d]
a
solution
of
ethyl
pyrimidin-1-yl)acetate 5a (0.30 g, 0.57 mmol) in anhydrous anhydrous ethanol (5 mL )
was added 1 N NaOH aqueous solution (1 mL). The mixture was stirred at room
temperature 3 h. Then the reaction mixture was diluted with water (45 mL), acidified
with HCl to 3 and extracted with ethyl acetate. The organic layer was washed with
water and the organic phase was concentrated to dryness, and the residue was purified
by recrystallization using anhydrous ethanol to give 6a. In accordance with this
1
method to get 6b–6f. 49%; Pale yellow solid; mp 212–213 ; H NMR (400 MHz,
DMSO-d6) δ: 10.02 (br, 1H, NH), 8.28 (s, 1H, CH), 8.07 (s, 1H, CH), 7.65–7.72(m, J
= 8.70 Hz, J = 21.46 Hz, 4H, ArH), 7.59 (s, 2H, ArH), 7.00 (d, J = 8.88 Hz, 2H, ArH),
4.72 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) 175.45, 154.76, 154.19, 152.79,
139.19, 135.63, 133.27, 132.15, 131.03, 128.71, 126.39, 122.02, 121.65, 100.73,
50.62; IR (KBr, ν, cm-1): 3226, 1574, 1511, 1430, 1330, 1287, 1161, 1091, 1067, 1034,
924, 821, 788, 741, 706, 664, 602, 549; HRMS (ESI) calcd for C19H15BrN6O4S [M䟌
K]䟌, 502.0059; found, 540.9611.
4.1.13.
2-(4-((4-(4-nitrophenylsulfonamido)phenyl)amino)-1H-pyrazolo[3,4-d]
pyrimidin-1-yl) acetic acid (6b)
60%; Yellow solid; mp䟟240 ; 1H NMR (400 MHz, DMSO-d6) δ: 9.82 (br, 1H, NH),
8.48 (s, 1H, CH), 8.28 (d, J = 8.80 Hz, 2H, ArH), 8.24 (s, 1H, CH), 7.95 (d, J = 8.84
Hz, 2H, ArH), 7.44 (s, 2H, ArH), 6.95 (d, J = 8.84 Hz, 2H, ArH), 4.71 (s, 2H, CH2);
13C NMR (100 MHz, DMSO-d6) 174.91, 155.24, 154.68, 153.41, 149.81, 146.98,
135.92, 131.69, 128.67, 124.80, 122.31, 101.24, 50.95; IR (KBr, ν, cm-1): 3108, 1614,
1533, 1434, 1349, 1165, 1092, 1034, 926, 855, 789, 740, 664, 613, 551; HRMS (ESI)
calcd for C19H15N7O6S [M䟌K]䟌, 469.0805; found, 508.0348.
4.1.14.
2-(4-((4-(phenylsulfonamido)phenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin
-1-yl)acetic acid (6c)
1
57%; Gray solid; mp 151–152 ; H NMR (400 MHz, DMSO-d6) δ: 10.85 (br, 1H,
8

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COOH), 10.25 (br, 2H, NH), 8.37 (s, 1H, CH), 8.23 (s, 1H, CH), 7.77 (d, J = 7.04 Hz,
2H, ArH), 7.54–7.68 (m, 5H, ArH), 7.12 (d, J = 8.88 Hz, 2H, ArH), 5.12 (s, 2H, CH2);
13C NMR (100 MHz, DMSO-d6) 169.55, 154.93, 154.21, 153.55, 139.92, 135.45,
133.33, 133.23, 129.68, 127.18, 123.20, 121.75, 101.18, 48.61; IR (KBr, ν, cm-1):
3227, 2924, 2854, 1731, 1621, 1576, 1508, 1385, 1332, 1290, 1218, 1160, 1091, 1036,
922, 839, 810, 687, 582; HRMS (ESI) calcd for C19H16N6O4S [M䟌H]䟌, 424.0954;
found, 425.1029.
4.1.15. 2-(4-((4-benzamidophenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetic
acid (6d)
1
61%; White solid; mp䟟240 ; H NMR (400 MHz, DMSO-d6) δ: 13.20 (br, 1H,
COOH), 10.30 (br, 1H, NH), 10.12 (br, 1H, NH), 8.41 (s, 1H, CH), 8.25 (s, 1H, CH),
7.97 (d, J = 7.74 Hz, 2H, ArH), 7.76–7.83 (m, 4H, ArH), 7.52–7.62 (m, 3H, ArH),
5.13 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) 169.70, 165.84, 156.00, 154.88,
154.02, 135.44, 135.16, 133.06, 132.69, 131.96, 128.84, 128.09, 122.28, 121.27,
101.29, 48.50; IR (KBr, ν, cm-1): 3306, 3113, 2922, 1738, 1633, 1530, 1491, 1405,
1300,1266, 1216, 1042, 926, 815, 717, 678, 543, 416. HRMS (ESI) calcd for
C20H16N6O3 [M䟌H]䟌, 388.1284; found, 389.1367.
4.1.16. 2-(4-((4-(2-chloroacetamido)phenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-
yl)acetic acid (6e)
1
64%; Pink solid; mp䟟240 ; H NMR (400 MHz, DMSO-d6) δ: 13.19 (br, 1H,
COOH), 10.35 (br, 1H, NH), 10.13 (br, 1H, NH), 8.40 (s, 1H, CH), 8.24 (s, 1H, CH),
7.78 (d, J = 8.60 Hz, 2H, ArH), 7.63 (d, J = 8.96 Hz, 2H, ArH), 5.12 (s, 2H, CH2),
4.27 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) 169.67, 164.89, 155.89, 154.74,
153.96, 135.29, 134.82, 132.72, 122.48, 120.27, 101.29, 48.49, 44.04; IR (KBr, ν,
cm-1): 3296, 3126, 3072, 2924, 2854, 2507, 1914, 1652, 1607, 1556, 1510, 1464,
1414, 1380, 1275, 1255, 1219, 1144, 1041, 969, 840, 775, 731, 700, 642, 619, 520;
HRMS (ESI) calcd for C15H13ClN6O3 [M䟌H]䟌, 360.0738; found, 361.0823.
4.1.17.
2-(4-((4-acetamidophenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetic
acid (6f)
1
58%; Pink solid; mp䟟240 ; H NMR (400 MHz, DMSO-d6) δ: 13.20 (br, 1H,
COOH), 10.20 (br, 1H, NH), 10.01 (br, 1H, NH), 8.26–8.43 (m, 2H, CH), 7.60–7.72
(m, 4H, ArH), 5.12 (s, 2H, CH2), 2.05 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6)
169.13, 168.06, 155.15, 153.36, 133.76, 132.35, 119.34, 100.69, 48.02, 23.90; IR
(KBr, ν, cm-1): 3296, 2923, 1666, 1613, 1558, 1512, 1382, 1277, 1220, 1093, 1047,
968, 837, 781, 702, 619; HRMS (ESI) calcd for C15H14N6O3 [M䟌H]䟌, 326.1127;
9

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found, 327.1141.
4.2 Determination of XOD inhibitory activity
Measurement of XOD inhibition was carried out by slightly modifying the method
of Cos et al [23-25]. First, 1092 ꢀL of 0.1 unit of XOD in buffer (200 mM sodium
pyrophosphate/HCl, pH 7.5) and 2 ꢀL(5, 10, 15, and 25 ꢀM) of the test extracts or
compounds in DMSO were mixed at 37
for 5 min. The control group did not
contain the test agent. The reaction started by adding 200 ꢀL of 0.6 mM xanthine in
double distilled water to the mixture. The reaction mixture was incubated at ambient
temperature. Finally, absorption increments at 295 nm indicating the formation of uric
acid were determined every minute up to 8 min. Allopurinol was used as the positive
control. Three replicates were made for each test sample. The inhibition ratio (%) was
calculated according to the equation [(rate of control reaction − rate of sample
reaction)/rate of control reaction] × 100.
4.3 Antitumour activity assay
4.3.1. Cells and culture.
The human hepatoma carcinoma cell lines 7402 and 7221 were obtained from the
State Key Laboratory of Biotherapy, Sichuan University. Logarithmically growing
human hepatoma carcinoma cells 7402 and 7221 were incubated with 0.05% trypsin
containing 1 mM EDTA at 37 °C for about 4 min until the cells were nonadherent and
formed a single-cell suspension. Trypsin activity was neutralized by adding a 20-fold
excess of serum-containing medium. The cells were cultured at 37 °C in air with 5%
CO2.
4.3.2. Cell survival assay.
Cell viability was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT). Briefly, human hepatoma carcinoma cells 7402 and 7221 were
plated at a density of 1 × 105 cells/mL into 96-well plates and 7 × 103-well plates,
respectively. After overnight growth, the cells were pretreated with a series of
concentrations of acacetin for 24 h. The final concentrations of dimethyl sulfoxide in
the culture medium were <0.1%. At the end of the treatment, 10 ꢀL of MTT was
added, and the cells were further incubated for 4 h. Cell viability was determined by
scanning with an ELISA reader using a 570 nm filter [26-28].
Acknowledgment
The authors are thankful to the Analytical & Testing Center of Sichuan University,
P. R. China, for providing analytical data and Mr. Yang (State Key Laboratory of
Biotherapy, Sichuan University) for carrying out the antitumour activity tests.
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Tables
Table 1. XOD inhibitory activity of new compounds a
compounds
IC50/ꢀg˙mL−1
allopurinol
1.60
3
inactive
86.65
inactive
>100
4
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
6f
77.74
34.10
40.59
38.79
>100
89.28
61.25
40.32
36.83
33.77
a
The IC50 value (ꢀg·mL−1) of the reducing power assay is the effective concentration at which the
absorbance is 0.5 of the reducing power.
12

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Table 2. In vitro activities of compounds (IC50 in ꢀM) against human hepatoma carcinoma cells 7402
and 7221a
compounds IC50(ꢀg˙mL−1)
72h
7402
7.27
7221
17-AAG
5.77
--
Allopurinol --
3
>100
>100
>64
4
>64
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
6f
>64
>64
>64
>64
>100
>100
7.27
>64
>100
>100
3.51
>64
>64
>64
>100
>100
>100
>100
>64
>100
>100
>100
>100
>64
a IC50 values represent the drug concentrations required to inhibit cancer cell replication by 50%. The
compounds were tested up to a concentration of 64 ꢀM. IC50 values were calculated by probit analysis
(P < 0.05, χ2 test).
13

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Figures
Geldanamycin
Fig. 1 Compound allopurinol, PU3, natural product Hsp90 inhibitor (17AAG) and geldanamycin
14

ACCEPTED MANUSCRIPT
Cl
O
NH
HN
N
N
N
N
CH2COOCH2CH3
5e
Fig. 2 Structures of the active antitumour agents.
15

ACCEPTED MANUSCRIPT
Schemes
Scheme 1. Reagents and conditions: (a) POCl3, (CH3)2NC6H5, 80 °C; (b) BrCH2COOCH2CH3,
TEA, DMF, rt; (c) H2NC6H4NH2, CH3CN, 80 °C; (d) NaH, THF, 0 °C, Sulfonyl chloride or
Chloride; (e) NaOH (1N), CH3CH2OH, rt.
16

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Highlights
►A series of pyrazolo[3,4-d]pyrimidines analogues were designed and
synthesized.
► In-vitro Xanthine Oxidase inhibitory activity and antitumor activity of
the new compounds were evaluated.
►Compound 5e possessed the best antitumor activity than 17AAG and
allopurinol.