Synthesis of N-aryl-5-amino-4-cyanopyrazole derivatives as potent xanthine oxidase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2007.06.002

Some pyrazolo[3,4-d]pyrimidines, structurally related with allopurinol, a well known xanthine oxidase inhibitor, clinically used in the therapy of gout, have also been reported as potent inhibitors of xanthine oxidase and the growth of several human tumour cell lines. Considering the potential interest of this family of compounds, the aim of the present study was to synthesise and provide a full chemical characterization of new N-aryl-5-amino-4-cyanopyrazole derivatives and their corresponding pyrazolo[3,4-d]pyrimidines. Their biological activity pertaining to the xanthine oxidase inhibition and effect on the growth of three tumour cell lines (MCF-7, NCI-H460, and SF-268) are also provided. With only one exception, the synthesised compounds showed no effect on the growth of the three tumour cell lines. However, a strong xanthine oxidase inhibitory activity was observed for almost all pyrazolo[3,4-d]pyrimidines tested, revealing some of them IC₅₀ values below 1 μM. The results of the molecular docking studies of these compounds, against xanthine oxidoreductase are also described, providing an atomistic explanation of the differences in the inhibitory efficiency. MEP calculations were used to explain different inhibitory efficiency of similar inhibitors.

Full text of DOI:10.1016/j.ejmech.2007.06.002

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 771e780
http://www.elsevier.com/locate/ejmech
Original article
Synthesis of N-aryl-5-amino-4-cyanopyrazole derivatives
as potent xanthine oxidase inhibitors
a
a
a
Sanjay Gupta , Lıgia M. Rodrigues , Ana P. Esteves , Ana M.F. Oliveira-Campos
a,
*
´
M. Sao Jose Nascimento , N. Nazareth , Honorina Cidade , Marta P. Neves ,
,
b
b
b
b
´
Eduarda Fernandes , Madalena Pinto , Nuno M.F.S.A. Cerqueira , Natercia Bras
~
c
b
d
d
´
´
a Centro de Qu´ımica, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
b Centro de Estudos de Qu´ımica Orgaˆnica, Fitoqu´ımica e Farmacologia da Universidade do Porto (CEQOFFUP),
Laborato´rios de Microbiologia e Qu´ımica Orgaˆnica da Faculdade de Farma´cia, 4050-047 Porto, Portugal
c REQUIMTE, Departamento de Qu´ımica-F´ısica da Faculdade de Farma´cia da Universidade do Porto, 4050-047 Porto, Portugal
d REQUIMTE, Departamento de Qu´ımica da Faculdade de Cieˆncias da Universidade do Porto, 4169-007 Porto, Portugal
Received 2 March 2006; received in revised form 4 June 2007; accepted 11 June 2007
Available online 5 July 2007
Abstract
Some pyrazolo[3,4-d]pyrimidines, structurally related with allopurinol, a well known xanthine oxidase inhibitor, clinically used in the therapy
of gout, have also been reported as potent inhibitors of xanthine oxidase and the growth of several human tumour cell lines. Considering the
potential interest of this family of compounds, the aim of the present study was to synthesise and provide a full chemical characterization of
new N-aryl-5-amino-4-cyanopyrazole derivatives and their corresponding pyrazolo[3,4-d]pyrimidines. Their biological activity pertaining to
the xanthine oxidase inhibition and effect on the growth of three tumour cell lines (MCF-7, NCI-H460, and SF-268) are also provided. With
only one exception, the synthesised compounds showed no effect on the growth of the three tumour cell lines. However, a strong xanthine oxidase
inhibitory activity was observed for almost all pyrazolo[3,4-d]pyrimidines tested, revealing some of them IC50 values below 1 mM. The results of
the molecular docking studies of these compounds, against xanthine oxidoreductase are also described, providing an atomistic explanation of the
differences in the inhibitory efficiency. MEP calculations were used to explain different inhibitory efficiency of similar inhibitors.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Nitrogen heterocycles; Aminocyanopyrazole derivatives; Pyrazolo[3,4-d]pyrimidines; Xanthine oxidase; Antitumour agents; Cytotoxic; Docking;
Computational studies
1. Introduction
studied pertaining to its mechanism of inhibition and phar-
macokinetic properties [5]. The results suggested that Y-700
will be a promising candidate for the treatment of hyperurice-
mia and other diseases in which xanthine oxidase may be
involved [5].
The formation of pyrazolo[3,4-d]pyrimidines (2) from
pyrazole derivatives with ortho-aminonitriles (3) is well docu-
mented [6e9]. These compounds are of great pharmacological
importance, namely as glycogen synthase kinase-3 (GSK-3)
inhibitors [10a] and antihistaminic [10b]. Some pyrazolo
[3,4-d]pyrimidines, structurally related with allopurinol (4),
a well known xanthine oxidase inhibitor clinically used in the
therapy of gout, have also been reported as potent inhibitors
Pyrazole derivatives constitute an important family of com-
pounds due to their applications as pharmaceuticals, agro-
chemicals and dyestuffs [1e3]. Several 1-phenylpyrazoles
(1) have been designed and tested as xanthine oxidase inhibi-
tors [4]. Among them, Y-700 ([1-[3-cyano-4-(2,2-dimethyl-
propoxy)phenyl]-1H-pyrazole-4-carboxylic acid), was shown
to be a potent xanthine oxidase inhibitor, and was already
* Corresponding author. Tel.: þ351 253 604377; fax: þ351 253 604382.
E-mail address: amcampos@quimica.uminho.pt (A.M.F. Oliveira-Campos).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.06.002

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S. Gupta et al. / European Journal of Medicinal Chemistry 43 (2008) 771e780
of xanthine oxidase [7,11] and the growth of several human
tumour cell lines [6,12e15].
a drug may increase its bioavailability [21]. It was then
decided to bind compounds (5c) and (3c) to glycine or to
a-acetobromoglucose.
R
R
R
Thisworkstartedwithreactionoftherequiredhydrazineswith
ethoxymethylenemalononitrileinboiling ethanol. Whenthe aryl-
hydrazine hydrochloride was the starting material the reaction
proceeded in the presence of triethylamine, and lower yields
were then obtained. The 5-amino-4-cyano-N-phenylpyrazoles
(3) were obtained in yields from 43% (for 3e) to 89% (for 3c)
(Scheme 1). Aminocyanopyrazoles (3) were evidenced on IR
by signals at 2215e2243 cmꢀ1 and in the NMR spectra by a
singlet for proton 3 (e.g. at 7.86 ppm for compound 3d) and
the pair of doublets expected for the para substituted phenyl
ring. Other techniques such as 13C, HMQC and HMBC were
also used.
Pyrazolo[3,4-d]pyrimidines (5) were obtained by reflux of
the corresponding pyrazoles with triethyl orthoformate in ace-
tic anhydride, to give the imidates which were then, in the
crude form, cyclised with ammonia in methanol (Scheme 1).
The yields were moderate to good (24% for 5a and 82% for
5d) except for (5e) where a poor yield was obtained (12%).
The formation of the pyrazolopyrimidine products was
evident from the NMR spectra where two singlets (due to
protons 3 and 6) were observed.
H
N
N
N
N
R'
N
N
N
NH2
CN
N
N
N
N
N
COOH
OH
R'
1
2
3
4
Following previous successful work on the synthesis of ar-
yldicyanoaminopyrazoles [16] and arylaminocyanopyrazoles
containing groups in the meta position of the phenyl ring
[17], it was now decided to obtain similar heterocyclic com-
pounds with substitution in the para position and also build
up a fused pyrimidine ring. The compounds synthesised
were tested for their capacity to inhibit xanthine oxidase activ-
ity and the in vitro growth of three tumour cell lines, MCF-7
(breast cancer), NCI-H460 (non-small cell lung cancer) and
SF-268 (CNS cancer).
2. Results and discussion
The amino group allowed modifications of the molecules
through binding to sugars or aminoacids. The alkylations of
the pyrazole (3c) and pyrazolopyrimidine (5c) on the amino
group with a-acetobromoglucose in DMSO in the presence of
triethylamine were carried out (Schemes 2 and 3). Compounds
(6) and (7) were isolated in 43 and 44% yields, respectively. All
the signals due to the glucose ring and the four acetyl groups
were observed, and it may also be worth mentioning, e.g. for
compound (7), two double doublets at 4.33 (J ¼ 12.6 and
4.5 Hz) and 4.15 (J ¼ 12.6 and 1.8 Hz) for both hydrogen atoms
(sugar-CH2OAc) and the ddd at 3.96 (J ¼ 9.6, 4.5 and 1.8 Hz)
for ring 50 proton.
2.1. Chemistry
In this work a classical method of pyrazole synthesis in-
volving the reaction between hydrazines and b-difunctional
compounds was applied for the preparation of compounds
(3). Aminopyrazoles (3a), (3e) and (3g) are known [6] and
the aminopyrazole (3e) was used for making azo dyes [18],
however, they could not be fully characterized at that time.
Recently, in our group, compounds (3c) and (3e) were pre-
pared, fully characterized and used to make dyes derived
from anilines and b-naphthol [19].
Pyrazolopyrimidines (5) were prepared (Scheme 1) by
a known procedure [20]. Although three of them, (5a), (5e)
and (5g), were reported earlier [6], their NMR data are not
yet available. The 4-amino group and/or the carboxylic group
in these systems confers them great versatility.
Compounds (3c) and (5c) were also reacted with glycine
methyl ester hydrochloride (Schemes 2 and 3), affording the
corresponding (3h) and (5h) in 32 (crude) and 72% yield, re-
spectively, as evidenced by their NMR spectra. For example
Purine derivatives containing aminoacid residues in their
molecules have received some attention in cancer chemo-
therapy [7]. It is also known that a sugar moiety linked to
CONHCH2CO2CH3
1'
CO2H
i)
ii)
4'
N
NH2
CN
N
NH2
CN
R
5
R
N
N
3
H
CN
C
CN
R
3c
3h
iii)
CO2H
2'
C
i) HC(OEt)3, Ac2O
ii) NH3
EtO
N
NH2
CN
N
N
N
N
AcO
OAc
6'
N
NHNH2
1''
O
N
NH
CN
OAc
OAc
5
NH2
5''
N
3
3
5
6
6''
R: a=Me; b=OMe; c=CO2H; d=CN; e=NO2; f=CF3; g=Cl
Scheme 1. Synthesis of compounds 3 and 5.
Scheme 2. Reaction conditions: (i) HBTU, DMF, RT; (ii) GlyOMe, HCl, TEA,
DMF, RT; (iii) DMSO, NEt3, a-acetobromoglucose.

S. Gupta et al. / European Journal of Medicinal Chemistry 43 (2008) 771e780
773
CONHCH2CO2CH3
1'
CO2H
4'
N
i)
ii)
N
N
N
6''
N
N
N
3''
N
CO2H
2''
5c
5h
iii)
NH2
NH2
6''
N
N
6
N
3
N
NH
OAc
OAc
1'
O
5'
6'
7
OAc OAc
Scheme 3. Reaction conditions: (i) HBTU, DMF, RT; (ii) GlyOMe,HCl, TEA, DMF, RT; (iii) DMSO, NEt3, a-acetobromoglucose.
a typical triplet (J ¼ 5.7 Hz) corresponding to NHGly appears
at 9.08 and 9.02 ppm for compounds (3h) and (5h),
respectively.
All the compounds were characterized by spectroscopic
methods and elemental analysis or high resolution mass
spectrometry.
(5ae5h and 7) was examined at the maximum concentration
of 100 mM. Compounds presenting an inhibitory effect higher
than 50%, at this concentration, were further tested at a wide
range of concentrations in order to determine its IC50 values
(Table 1). The tested aminocyanopyrazoles showed no appre-
ciable activity except compounds (3c) and (3h), which ex-
hibited a moderate xanthine oxidase inhibition, with an IC50
of 36.11 ꢁ 1.45 and 37.92 ꢁ 3.03 mM, respectively. However,
a strong inhibitory effect was observed for all of the pyra-
zolo[3,4-d]pyrimidines tested. Only compound (5c) showed
a weak effect (IC50 ¼ 80.97 ꢁ 4.64 mM). Among the pyra-
zolo[3,4-d]pyrimidines, the strongest effects were observed in
2.2. Biological activity
2.2.1. Effects of compounds on xanthine oxidase activity
The inhibition of xanthine oxidase activity by aminocyano-
pyrazoles (3ae3h and 6) and pyrazolo[3,4-d]pyrimidines
Table 1
Effect of compounds on xanthine oxidase and on the growth of human tumour cell lines
Compd
R
XO inhibitory activity IC50 (mM)a
Inhibition of tumour cell lines GI50 (mM)b
3a
3b
3c
3d
3e
3f
CH3
OCH3
>100
>100
>150
>150
ND
>150
>150
ND
>150
>150
ND
COOH
CN
36.11 ꢁ 1.45
>100
>100
>150
64.01 ꢁ 4.03
>150
>150
>150
ND
>150
135.02 ꢁ 9.61
>150
>150
>150
ND
>150
127.51 ꢁ 7.50
>150
>150
>150
ND
NO2
CF3
>100
>100
3g
3h
5a
5b
5c
5d
5e
5f
Cl
CONHCH2COOCH3
37.92 ꢁ 3.03
17.35 ꢁ 1.19
19.58 ꢁ 0.20
80.97 ꢁ 4.64
0.40 ꢁ 0.01
2.20 ꢁ 0.05
0.18 ꢁ 0.02
0.08 ꢁ 0.01
>100
CH3
OCH3
>150
ND
ND
>150
ND
ND
>150
ND
ND
COOH
CN
NO2
CF3
ND
ND
ND
ND
ND
ND
5h
6
CONHCH2COOCH3
>150
ND
>150
>150
ND
>150
>150
ND
>150
e
e
7
18.44 ꢁ 2.59
ND: Not done.
a
Results are expressed as means ꢁ SEM of 3 independent observations performed in triplicate. Allopurinol was used as positive control (% inhibition
(100 mM) ¼ 96.30 ꢁ 0.30, IC50 ¼ 24.40 ꢁ 0.50 mM).
b
Results are mean ꢁ SEM of 1e6 independent experiments performed in duplicate. Doxorubicin was used as positive control: GI50 (MCF-
7) ¼ 42.80 ꢁ 8.20 nM; GI50 (SF-268) ¼ 93.00 ꢁ 7.00 nM; GI50 (NCI-H460) ¼ 94.00 ꢁ 8.70 nM.

774
S. Gupta et al. / European Journal of Medicinal Chemistry 43 (2008) 771e780
35
30
25
20
15
10
5
those possessing a glycine methyl ester (5h), cyano (5d), nitro
(5e) or trifluoromethyl group (5f), exhibiting IC50 values near or
below 2 mM. Compounds (5a), (5b), and (7) showed an interest-
ing activity with IC50 around 20 mM.
Control
3c (40 microM)
3c (10 microM)
Although the number of tested compounds is limited,
some structural features, important to the xanthine oxidase in-
hibitory effect, can be inferred. In general, when comparing
the xanthine oxidase inhibitory effect of aminocyanopyra-
zoles with that of pyrazolo[3,4-d]pyrimidines, it can be con-
cluded that the addition of a pyrimidine ring was responsible
for an increased inhibition. However, comparing the com-
pounds with a carboxylic group (3c and 5c), that differ
only in the presence of a pyrimidine ring, it seems that this
ring was responsible for a two-fold decrease of activity.
Moreover, the introduction of a sugar linked to the amino
group of compound (5c) led to a stronger inhibitory effect
of compound (7). On the contrary, the introduction of the
sugar on the amino group of compound 3c led to a loss of
inhibitory effect. The formation of the amide between com-
pound (5c) and glycine methyl ester was associated with
a strong increase of xanthine oxidase inhibition, but the
same structural modification on compound (3c) did not affect
the inhibitory effect.
0
-0.2
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
0.2
-5
1/S
35
30
25
20
15
10
5
Control
3h (40 microM)
3h (10 microM)
0
-0.2
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
0.2
-5
1/S
In order to evaluate the type of xanthine oxidase inhibition
of the most potent aminocyanopyrazoles and pyrazolo[3,4-d]
pyrimidines studied, their inhibitory effects were tested at dif-
ferent concentrations of the substrate (xanthine), as shown in
Fig. 1. Both the Vmax and Km values obtained in the presence
of the inhibitors are different from the same values obtained in
their absence (Table 2), suggesting a mixed non-competitive
inhibitory effect, that is the inhibitors bind both to the enzyme
and to the xanthine/xanthine oxidase complex, but with greater
affinity for the latter [22].
Previous studies on the effects of the introduction of phenyl
groups in purines and pyrazolopyrimidines [11] on their activ-
ity suggested that there is a hydrophobic bonding region, ad-
jacent to the active site on xanthine oxidase, that can complex
aryl groups attached to those systems. The authors found that,
in general, the introduction of an aryl group in position 1
of the pyrazole ring or in position 6 (pyrimidine ring) in-
creased the inhibitory activity, particularly when an electron-
withdrawing group was present. In our work compounds 5
containing an aryl ring substituted by electron-withdrawing
groups (CF3, NO2, CN) showed enhanced activity. These find-
ings are coherent with the observations of the authors above
referred [11].
35
Control
30
25
20
15
10
5
5h (0.1 microM)
5h (0.025 microM)
0
-0.2
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
0.2
-5
1/S
Fig. 1. LineweavereBurk plots in the absence (control) and in the presence of
inhibitors 3c, 3h, and 5h with xanthine as the substrate. V ¼ DA/min;
S ¼ xanthine (mM).
inhibitory effect that was two-fold higher for MCF-7 than
to the other cell lines. This different cell line response may
reflect a possible tumour type-specific sensitivity of this
compound for breast cancer cell line.
Table 2
Vmax (enzyme activity) and Km (apparent substrate activity) for xanthine oxi-
dase in the absence (control) and in the presence of two different concentra-
tions of inhibitors 3c, 3h, and 5h
2.2.2. Effects of compounds on the growth of human tumour
cell lines
The ability of aminocyanopyrazoles (3ae3h and 6) and pyr-
azolo[3,4-d]pyrimidines (5ae5h and 7) to inhibit the in vitro
growth of MCF-7, NCI-H460 and SF-268 cell lines was also
evaluated and the results, given in concentrations that were
able to cause 50% of cell growth inhibition (GI50), are summa-
rized in Table 1. No activity was observed for the compounds
tested even at maximum concentration (150 mM), except
for aminocyanopyrazole (3e). This compound revealed an
Km (mM)
Vmax (DA/min)
Control (xanthine)
7.16
7.79
0.26
0.12
0.08
0.23
0.09
0.16
0.13
Xanthine þ 3c (10 mM)
Xanthine þ 3c (40 mM)
Xanthine þ 3h (10 mM)
Xanthine þ 3h (40 mM)
Xanthine þ 5h (0.025 mM)
Xanthine þ 5h (0.1 mM)
10.33
10.41
5.77
5.86
7.53

S. Gupta et al. / European Journal of Medicinal Chemistry 43 (2008) 771e780
775
2.3. Computational studies
that in these cases, a tight net of hydrogen bonds with center
1 is generated and a closer interaction with the Mo(IV) center
is achieved, thus increasing the protein ligand affinity.
The known X-ray structures from xanthine oxidoreductase
with bound molecules show a highly specific active site. Its
overall structure remains almost unchanged from the unbound
structure, presenting a narrow tunnel towards the Mo(IV)
complex [5,23].
According to our docking studies, all inhibitors bind within
four phenylalanine residues, Phe649, Phe914, Phe1009 and
Phe1013, what is believed to promote the stabilization of the
binding positions of the aromatic substrate and might be
important for substrate recognition. The presence of these res-
idues makes the region closer to the Mo(IV) complex very
tight, which compromises the binding of larger substituted
rings. This was observed with all docked inhibitors in which
only one phenyl ring was allowed to bind in that region.
(See Fig. 2 for compounds 3c, 5c, 7, 5h and Y-700).
On the other hand compounds of series 3 have higher IC50
values, which can be related to the small size and the global
linear shape of the structures that preclude an efficient binding
with center 1 and center 2 of the active site. Docking results
show two possible main binding orientations, and such com-
petitive behaviour may also be responsible for the lower activ-
ity for these compounds. One exception is compound 3h
where it appears that a good interaction with both centers of
the active site is achieved, improving the ligand binding.
Compounds 6 and 7 have a glucosydic moiety connected to
the pyrazolo or pyrazolopyrimidine rings, respectively. For
compound 6, the introduction of a sugar moiety in the 5-amino
position of the pyrazole ring resulted in complete loss of activity,
as compared with 3c. For pyrazolopyrimidine 7 the position and
the size of the sugar group did not influence its binding inside the
active site cavity and closer interactions with center 1 and 2 are
still observed. Interestingly, the IC50 value for compound 7 is
four times lower than for 5c, although both compounds differ
only in the glucosydic group. The result for compound 7 seems
to be dependent on extra hydrogen bonds that can be established
between the glucosydic moiety and the residues located at the en-
In the active site there are two regions that interact very
closely with the docked inhibitors by hydrogen bonds. One re-
gion involves Arg880, Thr1010 and Glu1261 (center 1) and the
other Asn768, Lys771 and Glu802 (center 2).
Considering the IC50 values of all synthesised compounds
(Table 1), it is clear that the presence of a pyrazolopyrimidine
ring is associated with the substantial increase of the inhibitory
effect. The results of docking and molecular electrostatic poten-
tial (MEP) studies show that, apart from its size, this molecular
moiety can fit inside the active site cavity, interacting very
closely with center 2. Moreover, all ligands of this series occupy
the same region inside the active site and the substituents at the
phenyl ring interact very closely with center 1 pointing towards
the Mo(IV) center. The IC50 values for compounds 5 show that
electron-withdrawing substituents, in general, lead to an in-
crease in the inhibitory effect (5de5h). The results point out
trance of the binding pocket, namely Asn650, Ser744 and Lys771
.
Compounds 3c, 5c, 5h and 7 have structural similarities,
however, the IC50 values are quite different. The calculated
molecular electrostatic potential (MEP) show some tendencies
that allow to explain these differences (Fig. 2). These surfaces
show areas which are susceptible to electrophilic (red colour)
and nucleophilic interactions (blue colour) (for interpretation
of the references to colour in this figure legend, the reader is
referred to the web version of this article).
Fig. 2. Docking results and MEP results obtained for compounds 5c (A), 5h (B), 7 (C), Y-700 (D) and 3c (E1, E2).

776
S. Gupta et al. / European Journal of Medicinal Chemistry 43 (2008) 771e780
Compound 5c has only one region that can establish strong
on a AutoSpec E spectrometer. Elemental analyses were ob-
tained on a Leco CHNS-932 instrument.
interactions with center 1. Compounds 3c and 7 show two well-
defined regions that are able to interact with center 1 and 2. In
addition, compound 7 shows several negative potential regions
around the glucosydic moiety that are able to interact with
Asn650, Ser744 and Lys771 residues. From these results it can
be concluded that these regions are crucial for the inhibitory ef-
fect but, their correct alignment with center 1 and 2 is rather
important, favouring the formation of strong hydrogen bonds,
and therefore enhanced binding.
Thin layer chromatography was carried out on pre-coated
plates (Merck Kieselgel 60F254). Column chromatography
was performed on silica gel with mixtures of light petroleum
and diethyl ether of increasing polarity, unless other conditions
are described. Light petroleum refers to the fraction boiling in
the range 40e60 ꢂC.
4.1.2. Aminocyanopyrazoles (3)
From all the compounds analysed, pyrazolopyrimidine 5h
has the highest inhibitory effect. The larger substituent at the
pyrazolopyrimidine ring did not influence the binding of the
ligand and its high flexibility favours a closer interaction
with center 1 of the active site and with the Mo(IV) center.
The calculated molecular electrostatic potential of this com-
pound shows areas of negative potential similar to those of
Y-700, i.e., three well-defined regions susceptible to electro-
philic attack, that are specifically directed to the regions of
center 1 and 2, where hydrogen bonds occur.
A mixture of arylhydrazine (33 mmol) and ethoxymethyle-
nemalononitrile (33 mmol) was stirred for 30 min and then
ethanol (20 mL) was added. The mixture was stirred at room
temperature for 24 h. The product, which precipitates, was fil-
tered and recrystallised from ethanol or ethanolewater or
other solvent as described. In some cases, as indicated, column
chromatography was used. When the arylhydrazine hydrochlo-
ride was the starting material, triethylamine, equimolar
amount, was added. After stirring the mixture for 5 min, the
method described above was followed.
3. Conclusions
4.1.2.1. 5-Amino-1-p-tolyl-1H-pyrazole-4-carbonitrile (3a). It
was prepared following the general method but the reaction
mixture was refluxed for 4 h. A white solid was obtained after
recrystallisation from chloroformediethyl ether (62%, m.p.
149e150 ꢂC; lit. [6] m.p. 158e159 ꢂC). IR (Nujol mull):
Sixteen compounds of the pyrazole or pyrazolopyrimidine
type (10 of them new) were prepared and fully characterized.
Aminopyrazoles showed no significant activity on xanthine
oxidase, while pyrazolo[3,4-d]pyrimidines showed to be po-
tent inhibitors of this enzyme specially those with cyano
(5d), nitro (5e), trifluoromethyl group (5f) or the derivative
of glycine methyl ester (5h). Concerning the antitumoral activ-
ity, the aminocyanopyrazole (3e) showed an interesting speci-
ficity for the MCF-7 cell line, while the effect of the other
tested compounds on the growth of human tumour cell lines
was negligible. The results of the molecular docking studies
of these compounds against xanthine oxidoreductase provided
some explanations of the differences in inhibitory effect. The
computational studies concerning ligands of series 3 and 5
lead to the conclusion that the compounds of series 5 showed
a better performance for xanthine oxidase inhibition. These
results can contribute for the design and development of
new xanthine oxidase inhibitors related with the N-aryl-5-
amino-4-cyanopyrazole scaffold.
n ¼ 3333, 3298, 3181, 2215 (CN), 1661, 1538 cmꢀ1
.
1H
NMR (DMSO-d6): d ¼ 2.35 (s, 3H, CH3), 6.60 (br s, 2H,
NH2), 7.31 (d, J ¼ 8.5 Hz, 2H, 20-H and 60-H), 7.36 (d,
J ¼ 8.5 Hz, 2H, 30-H and 50-H), 7.75 (s, 1H, 3-H). 13C NMR
(DMSO-d6): d ¼ 20.65 (CH3), 73.23 (C-4), 114.88 (CN),
124.15 (C-30and C-50), 129.89 (C-20 and C-60), 135.00 (C-
10), 137.47 (C-40), 141.49 (C-3), 151.15 (C-5). HRMS: calcd.
for C11H10N4 [M]þ 198.0905; found 198.0904.
4.1.2.2. 5-Amino-1-(40-methoxyphenyl)-1H-pyrazole-4-carbo-
nitrile (3b). A beige solid was obtained after purification by
column chromatography (40% ethyl acetate:hexane) (44%,
m.p. 128e131 ꢂC). IR (neat): n ¼ 3426, 3332, 2927, 2217
1
(CN), 1631, 1563, 1533, 1514 cmꢀ1. H NMR (DMSO-d6):
d ¼ 3.79 (s, 3H, OCH3), 6.54 (br s, 2H, NH2), 7.05 (d,
J ¼ 9.0 Hz, 2H, 30-H and 50-H), 7.37 (d, J ¼ 9.0 Hz, 2H, 20-
H and 60-H), 7.72 (s, 1H, 3-H). 13C NMR (DMSO-d6):
d ¼ 55.48 (CH3), 72.89 (C-4), 114.57 (C-30and C-50), 114.93
(CN), 126.17 (C-20and C-60), 130.24 (C-10), 141.24 (C-3),
151.23 (C-5), 158.80 (C-40). HRMS: calcd. for C11H10N4O
[M]þ 214.0855; found 214.0857.
4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were determined on a Gallenkamp melting
point apparatus and are uncorrected. IR spectra and UV spec-
tra were determined on a Perkin Elmer FTIR-1600 and on a
4.1.2.3. 5-Amino-1-(40-carboxyphenyl)-1H-pyrazole-4-carbo-
nitrile (3c) [19]. It was obtained as an orange solid, recrystal-
1
lised from ethanolewater (89%), m.p. 294e295 ꢂC. H NMR
1
Hitachi U-2000, respectively. H NMR spectra were recorded
(DMSO-d6): d ¼ 6.88 (br s, 2H, NH2), 7.65 (d, J ¼ 8.7 Hz, 2H,
20-H and 60-H), 7.84 (s, 1H, 3-H), 8.06 (d, J ¼ 8.7 Hz, 2H, 30-H
and 50-H), 13.15 (br s, 1H, COOH).
at 300 MHz and 13C NMR spectra were determined at
75.4 MHz both on a Varian Unity Plus Spectrometer. Double
resonance, HMQC and HMBC experiments were carried out
for complete assignment of proton and carbon signals in the
NMR spectra. High resolution mass spectra were obtained
4.1.2.4. 5-Amino-1-(40-cyanophenyl)-1H-pyrazole-4-carboni-
trile (3d). It was obtained as a beige solid (78%, m.p.

S. Gupta et al. / European Journal of Medicinal Chemistry 43 (2008) 771e780
777
226e228 ꢂC; no crystallisation was needed). IR (neat):
40e45 min (the pH of the reaction mixture was 12e13) then
cooled to 0e5 ꢂC and diluted with distilled water (40 mL).
The pH dropped to 10e12. After 30 min stirring at 0e5 ꢂC,
the product was filtered and washed with water (10 mL) and
dried at 50e60 ꢂC. The product was purified by column chro-
matography and/or recrystallisation.
n ¼ 3357, 3183, 2227 (CN), 1666, 1607, 1538 cmꢀ1
.
1H
NMR (DMSO-d6): d ¼ 6.97 (br s, 2H, NH2), 7.74 (d,
J ¼ 8.4 Hz, 2H, 20-H and 60-H), 7.86 (s, 1H, 3-H), 7.98 (d,
J ¼ 8.4 Hz, 2H, 30-H and 50-H). 13C NMR (DMSO-d6):
d ¼ 74.21 (C-4), 109.90 (C-40) 114.47 (CN), 118.35 (CN),
124.22 (C-20and C-60), 133.72 (C-30and C-50), 141.25 (C-10),
142.81 (C-3), 151.78 (C-5). HRMS: calcd. for C11H7N5
[M]þ 209.0701; found 209.0703.
4.1.3.1.
1-p-Tolyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine
(5a). The crude cream coloured solid (86%) obtained was pu-
rified by crystallisation (ethanol) followed by column chroma-
tography (ethyl acetate), affording the pure compound (24%),
m.p. 223.7e224.3 ꢂC (lit.[6] m.p. >300 ꢂC). IR (neat):
4.1.2.5. 5-Amino-1-(40-nitrophenyl)-1H-pyrazole-4-carboni-
trile (3e) [19]. The title compound was obtained as a light yel-
low solid after recrystallisation from acetoneewater (43%),
m.p. 222e224 ꢂC (lit. [6] m.p. 224e225 ꢂC). 1H NMR
(DMSO-d6): d ¼ 7.06 (br s, 2H, NH2), 7.83 (d, J ¼ 9.0 Hz,
2H, 20-H and 60-H), 7.90 (1H, s, 3-H), 8.35 (d, J ¼ 9.3 Hz,
2H, 30-H and 50-H).
n ¼ 3427 (br), 1660, 1588, 1556, 1514 cmꢀ1
.
1H NMR
(acetone-d6): d ¼ 2.41 (s, 3H, Me), 7.25 (br s, 2H, NH2),
7.36 (d, J ¼ 9.3 Hz, 2H, 20-H and 60-H), 8.21 (d, J ¼ 9.3 Hz,
2H, 30-H and 50-H), 8.33 (1H, s, 3-H), 8.37 (1H, s, 6-H). 13C
NMR (acetone-d6): d ¼ 20.78 (CH3), 102.57 (C-3a), 121.46
(C-30and C-50), 130.16 (C-20and C-60), 133.72 (C-3), 136.37
(C-10), 138.15 (C-40), 154.43 (C-4), 157.27 (C-6), 159.42
(C-7a). HRMS: calcd. for C12H11N5 [M]þ 225.1014; found
225.1012.
4.1.2.6. 5-Amino-1-(40-trifluoromethylphenyl)-1H-pyrazole-4-
carbonitrile (3f). It was obtained as a yellowish solid after re-
crystallisation from ethanol (71%, m.p. 170e171 ꢂC). IR
(neat): n ¼ 3457, 3343, 2214 (CN), 1651, 1614, 1564,
1
1531 cmꢀ1. H NMR (DMSO-d6): d ¼ 6.92 (br s, 2H, NH2),
4.1.3.2. (40-Methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl-
amine (5b). After recrystallisation (ethanolewater) it was ob-
tained as a light brown solid. Further purification by column
chromatography (eluted with ethyl acetate) yielded a light yel-
low coloured solid (36%), m.p. 218.2e219.8 ꢂC. IR (neat):
7.76 (d, J ¼ 8.5 Hz, 2H, 20-H and 60-H), 7.85 (s, 1H, 3-H),
7.88 (d, J ¼ 8.5 Hz, 2H, 30-H and 50-H). 13C NMR (DMSO-
d6): d ¼ 74.00 (C-4), 114.57 (CN), 122.19 and 125.80 (right
1
side of q, J ¼ 273 Hz, CF3; it was not possible to assign the
other half of this quartet), 124.41 (C-20and C-60), 126.72 (C-
n ¼ 3418, 1672, 1658, 1596, 1567, 1522 cmꢀ1
.
1H NMR
2
30and C-50), 127.77 (q, J ¼ 32 Hz, C-40), 140.89 (C-10),
(acetone-d6): d ¼ 3.89 (s, 3H, OCH3), 7.12 (d, J ¼ 9.3 Hz,
2H, 30-H and 50-H), 7.30 (br s, 2H, NH2), 8.19 (d,
J ¼ 9.3 Hz, 2H, 20-H and 60-H), 8.31 (s, 1H, 3-H), 8.34 (s,
1H, 6-H). 13C NMR (acetone-d6): d ¼ 55.78 (OCH3), 102.39
(C-3a), 114.82 (C-30and C-50), 123.15 (C-20and C-60), 133.46
(C-3), 133.74 (C-10), 154.15 (C-4), 157.27 (C-6), 158.8þ0
(C-40), 159.41 (C-7a). HRMS: calcd. for C12H11N5O [M]
241.0919; found 241.0972.
142.58 (C-3), 151.74 (C-5). C11H7N4F3 (252.06): calcd. C
52.41, H 2.80, N 22.22; found C 52.44, H 2.96, N 22.14.
HRMS: calcd. for C11H7N4F3 [M]þ 252.0623; found
252.0620.
4.1.2.7. 5-Amino-1-(40-chlorophenyl)-1H-pyrazole-4-carboni-
trile (3g). It was obtained as a light yellow solid (58%) after
recrystallisation from a mixture of chloroformemethanol,
m.p. 166e168 ꢂC (lit. [6] m.p. 167e167.5 ꢂC). IR (Nujol
4.1.3.3. N-(40-Carboxyphenyl)-1H-4-aminopyrazolo[3,4-d]py-
rimidine (5c). The crude product (54%), was purified by
column chromatography (60% methanolechloroform) fol-
lowed by recrystallisation (ethanolewater) affording the title
compound (25%) as a white solid, which does not melt, but de-
composes below 280 ꢂC. IR (neat): n ¼ 3471e3173 (br), 1666,
mull): n ¼ 3299, 3175, 2230 (CN), 1665, 1539 cmꢀ1
.
1H
NMR (DMSO-d6): d ¼ 6.76 (br s, 2H, NH2), 7.52 (d,
J ¼ 8.7 Hz, 2H, 20-H and 60-H), 7.57 (d, J ¼ 8.7 Hz, 2H, 30-
H and 50-H), 7.79 (s, 1H, 3-H). 13C NMR (DMSO-d6):
d ¼ 73.49 (C-4), 114.68 (CN), 126.03 (C-20and C-60), 129.44
(C-30and C-50), 132.24 (C-10), 136.31 (C-40), 142.00 (C-3),
151.44 (C-5). C10H7N4Cl (218.04): calcd. C 54.87, H 3.22,
N 25.60; found C 54.51, H 3.38, N 25.32. HRMS: calcd. for
C10H7N4Cl [M þ 1, 35Cl]þ 219.0437; found 219.0429.
[M þ 1, 37Cl]þ: calcd. 221.0408; found 221.0403.
1
1600, 1556 cmꢀ1. H NMR (DMSO-d6): d ¼ 7.80 (br s, 2H,
NH2), 8.08 (d, J ¼ 8.7 Hz, 2H, 20-H and 60-H), 8.18 (d,
J ¼ 8.7 Hz, 2H, 30-H and 50-H), 8.30 (s, 1H, 6-H), 8.38 (s,
1H, 3-H), OH not observed. 13C NMR (DMSO-d6):
d ¼ 101.52 (C-3a), 119.23 (C-30and C-50), 130.14 (C-20and
C-60), 134.35 (C-3), 135.92 (C-40), 140.02 (C-10), 153.42 (C-
4), 156.80 (C-6), 158.36 (C-7a), 170.02 (CO). HRMS: calcd
for C12H9N5O2 [M]þ 225.0756; found 225.0759.
4.1.3. Pyrazolopyrimidines (5ae5g)
The required pyrazole (1.0 mmol) was treated with triethyl
orthoformate (12.5 mmol) and acetic anhydride (6.0 mmol)
and the mixture was refluxed for 24 h. After cooling, the reac-
tion mixture was concentrated to dryness and then complete
acetic anhydride removal was ensured by evaporation with
60 mL of CCl4. This residue was stirred with aqueous ammo-
nium hydroxide (25%, 3 ꢃ 4 mL, added at different times) for
4.1.3.4. 4-(40-Aminopyrazolo[3,4-d]pyrimidin-1-yl)-benzoni-
trile (5d). Obtained as a light brown solid, (82%) which was
purified by crystallisation from ethanolewater, m.p. 350.9e
351.4 ꢂC. IR (neat): n ¼ 3500e3286 (br), 2239 (CN), 2218
1
(CN), 1679, 1602, 1582, 1518 cmꢀ1. H NMR (DMSO-d6):

778
S. Gupta et al. / European Journal of Medicinal Chemistry 43 (2008) 771e780
d ¼ 7.91 and 8.08 (2 br s, 2H, NH2), 7.97 (d, J ¼ 8.7 Hz, 2H,
2-H and 6-H), 8.33 (s, 1H, 60-H), 8.41 (s, 1H, 30-H), 8.48 (d,
J ¼ 9.0 Hz, 2H, 3-H and 5-H). 13C NMR (DMSO-d6):
d ¼ 101.87 (C-30a), 107.72 (C-4), 118.71 (C-3 and C-5),
120.01 (CN), 133.60 (C-2 and C-6), 135.64 (C-30), 142.47
(C-1), 154.18 (C-40), 157.09 (C-60), 158.35 (C-70a). HRMS:
calcd. for C12H8N6 [M]þ 236.0810; found 236.0811.
J ¼ 5.7 Hz, 1H, NHGly). 13C NMR (DMSO-d6): d ¼ 41.27
(CH2), 51.79 (OCH3), 73.76 (C-4), 114.66 (CN), 123.59
(C-20 and C-60), 128.57 (C-30 and C-60), 132.39 (C-40),
139.98 (C-10), 142.26 (C-3), 151.49 (C-5), 165.66 (CO amide),
170.34 (CO ester). C14H13N5O3 (299.11): calcd. C 56.18, H
4.38, N 23.40; found C 56.03, H 4.44, H 23.62. HRMS: calcd
for C14H13N5O2 [M]þ 299.1018; found 299.1017.
4.1.3.5. 1-(40-Nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl-
amine (5e). It was obtained as a grey to light brown solid
which was recrystallised twice from acetoneewater (12%),
m.p. 319e320 ꢂC (lit. [6] m.p. >300 ꢂC). IR (neat):
4.1.4.2. [40-(400-Aminopyrazolo[3,4-d]pyrimidin-100-yl)-benzoy-
lamino]-acetic acid methyl ester (5h). The crude product
(72%) was purified by column chromatography (eluent: meth-
anol then by ethanol) followed by recrystallisation from chlor-
oformemethanol 1:1 affording the pure compound as a white
solid, m.p. 272e274 ꢂC. IR (Nujol mull): n ¼ 3583, 3338,
n ¼ 3540e3286 (br), 1681, 1596, 1505 cmꢀ1
.
1H NMR
(DMSO-d6): d ¼ 8.00 and 8.10 (2 br s, 2H, NH2), 8.38 (s,
1H, 6-H), 8.43 (d, J ¼ 9.6 Hz, 2H, 20-H and 60-H), 8.47 (s,
1H, 3-H), 8.62 (d, J ¼ 9.3 Hz, 2H, 30-H and 50-H). 13C NMR
(DMSO-d6): d ¼ 101.91 (C-3a), 119.49 (C-30and C-50),
125.21 (C-20and C-60), 136.13 (C-3), 144.09 (C-10), 144.32
(C-40), 154.46 (C-4), 157.31 (C-6), 158.42 (C-7a). HRMS:
calcd. for C11H8N6O2 [M]þ 256.0709; found 256.0719.
3190, 1740, 1677, 1645, 1600, 1510 cmꢀ1
.
1H NMR
(DMSO-d6): d ¼ 3.66 (s, 3H, OCH3), 4.05 (d, J ¼ 6.3 Hz,
2H, CH2), 7.88 (br s, 2H, NH2), 8.04 (d, J ¼ 8.7 Hz, 2H, 30-
H and 50-H), 8.34 (s, 1H, 600-H), 8.38 (d, J ¼ 8.7 Hz, 2H, 20-
H and 60-H), 8.40 (s, 1H, 300-H), 9.02 (t, J ¼ 5.7 Hz, 1H,
NHGly). 13C NMR (DMSO-d6): d ¼ 41.27 (CH2), 51.78
(OCH3), 101.64 (C-300a), 119.56 (C-20and C-60), 128.46 (C-
30and C-50), 130.64 (C-40), 134.86 (C-300), 141.41 (C-10),
153.75 (C-400), 156.95 (C-600), 158.33 (C-700a), 165.94 (CO am-
ide), 170.43 (CO ester). C15H14N6O3 (326.11): calcd. C 55.21,
H 4.32, N 25.76; found C 54.95, H 4.49, N 25.43.
4.1.3.6. 1-(40-Trifluoromethylphenyl)-1H-pyrazolo[3,4-d]pyri-
midin-4-ylamine (5f). It was purified by recrystallisation (eth-
anol) and it was obtained as an off-cream coloured solid
(34%), m.p. 240.9e242.3 ꢂC. IR (neat): n ¼ 3616e3250 (br),
1660, 1597 cmꢀ1
.
1H NMR (DMSO-d6): d ¼ 7.92 (d,
J ¼ 9.0 Hz, 2H, 20-H and 60-H), 8.10 (very br s, 2H, NH2),
8.35 (s, 1H, 6-H), 8.43 (s, 1H, 3-H), 8.52 (d, J ¼ 8.7 Hz, 2H,
30-H and 50-H). 13C NMR (DMSO-d6): d ¼ 101.71 (C-3a),
120.15 (C-30and C-50), 124.20 (q, 1J ¼ 270 Hz, CF3),
4.1.5. Synthesis of compounds (6) and (7)
An anhydrous DMSO (10 mL) solution of pyrazolopyrimi-
dine (5c) or pyrazole (3c) (0.39 mmol) and TEA (1.18 mmol)
was stirred under a nitrogen atmosphere. After 25 min a-
acetobromoglucose (0.39 mmol) was added. The mixture
was stirred at RT for 2 h and water (60 mL) was added. A solid
precipitated and the suspension was cooled between 0 and
5 ꢂC for 20 min and filtered. This solid was recrystallised
from adequate solvent. For the glycosylation of pyrazole the
reaction mixture was stirred for 20 h.
2
125.83(q, J ¼ 32 Hz, C-40), 126.49 and 126.54 (C-20and C-
60), 135.25 (C-3), 142.12 (C-10), 153.98 (C-4), 157.05 (C-6),
158.37 (C-7a). HRMS: calcd. for C12H8N5F3 [M]þ
279.0732; found 279.0737.
4.1.4. Synthesis of compounds (3h) and (5h)
Compound (3c) or (5c) (6.6 mmol) and HBTU [O-(benzo-
triazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophos-
phate] (6.6 mmol) were taken in anhydrous DMF (30 mL)
and the solution was stirred at RT for 25e30 min. A solution
of GlyOMe,HCl in DMF (10 mL), containing triethylamine
(TEA, 6.6 mmol), was added and the mixture stirred at room
temperature for 24 h. Addition of water (300 mL), and stirring
for 2.5 h precipitated the by-products. The aqueous phase was
extracted with diethyl ether (100 mL) and ethyl acetate
(2 ꢃ 100 mL) and the combined organic extracts were dried
(MgSO4) and concentrated. Recrystallisation from the appro-
priate solvent afforded the desired compound.
4.1.5.1. N-(200,300,400,600-Tetra-O-acetyl-b-D-glucopyranosyl)-5-
amino-1-(40-carboxyphenyl)-1H-pyrazole-4-carbonitrile (6).
The product was recrystallised from 1:1:1 ethyl acetate:hexa-
ne:ethyl ether (12 mL). A pale yellow solid was obtained
(43.5% yield), m.p. 190.5e192.3 ꢂC. [a]D20 ¼ ꢀ15.29ꢂ
(c ¼ 2.55 in CHCl3). IR (neat): n ¼ 3337, 2220 (CN), 1748,
1
1637, 1608, 1531 cmꢀ1. H NMR (CDCl3): d ¼ 2.00, 2.05,
2.06 and 2.08 (4 s, 12H, 4 ꢃ OCH3), 3.96 (ddd, J ¼ 9.6, 4.5
and 1.8, 1H, 500-H), 4.15 (dd, J ¼ 12.6 and 1.8 Hz, 1H, 600-
Ha), 4.33 (dd, J ¼ 12.6 and 4.5 Hz, 1H, 600-Hb), 4.82 (br s,
1H, NH), 5.20 (t, J ¼ 9.6 Hz, 1H, 400-H), 5.37e5.34 (m, 2H,
200-H and 300-H), 5.93 (d, J ¼ 6.9 Hz, 1H, 100-H), 7.67 (d,
J ¼ 8.4 Hz, 2H, 30-H and 50-H), 7.68 (s, 1H, 3-H), 8.19 (d,
J ¼ 8.7 Hz, 2H, 20-H and 60-H), OH not observed. 13C NMR
(CDCl3): d ¼ 20.54 and 20.66 (4 ꢃ CH3), 61.38 (C-600),
67.79 (C-400), 70.09 (C-200 or C-300), 72.41 (C-200 or C-300),
72.84 (C-50), 92.59 (C-100), 113.54 (CN), 123.27 (C-30and C-
50), 128.09 (C-4), 131.93 (C-20and C-60), 141.67 (C-10),
142.00 (C-3), 150.03 (C-5), 163.30 (CO acid), 169.36 (CO
acetyl), 169.42 (CO acetyl), 170.03 (CO acetyl), 170.56 (CO
4.1.4.1.
[40-(5-Amino-4-cyanopyrazol-1-yl)-benzoylamino]-
acetic acid methyl ester (3h). A white solid was obtained after
recrystallisation from methanol (32% yield, m.p. 196e
198 ꢂC). IR (neat): n ¼ 3370, 3274, 3138, 2223 (CN), 1751,
1
1661, 1527, 1507 cmꢀ1. H NMR (DMSO-d6): d ¼ 3.66 (s,
3H, OCH3), 4.03 (d, J ¼ 5.7 Hz, 2H, CH2), 6.84 (s, 2H,
NH2), 7.64 (d, J ¼ 8.7 Hz, 2H, 20-H and 60-H), 7.83 (s, 1H,
3-H), 8.01 (d, J ¼ 8.4 Hz, 2H, 30-H and 50-H), 9.08 (t,

S. Gupta et al. / European Journal of Medicinal Chemistry 43 (2008) 771e780
779
acetyl). HRMS: calcd. for C25H26N4O11 [M þ 1]þ 559.1676;
the experimental period, was the same as originally published
[24] and was, respectively, 7.5 ꢃ 104 cells/mL to NCI-H460,
1.5 ꢃ 105 cells/mL to MCF-7 and SF-268. Cells were exposed
for 48 h to five concentrations of compounds starting from
a maximum concentration of 150 mM. Compounds, prepared
in DMSO, were freshly diluted with cell culture medium
just prior the assays. Final concentrations of DMSO
(ꢄ0.25%) did not interfere with the cell lines growth. For
each test compound and for each cell line a dose-response
curve was generated and the growth inhibition of 50%
(GI50), corresponding to the concentration of compound that
inhibits 50% of the net cell growth was determined as
described [24].
found 559.1659.
4.1.5.2. N-(400-Carboxyphenyl)-N-(20,30,40,60-tetra-O-acetyl-b-
D-glucopyranosyl)pyrazolo[3,4-d] pyrimidine (7). Recrystalli-
sation from ethanol yielded an off-white solid (43%), m.p.
212e214 ꢂC. [a]D20 ¼ ꢀ6.25ꢂ (c ¼ 4.0 in CH2Cl2). IR (neat):
n ¼ 3430 (very br), 1746, 1643, 1596 cmꢀ1
.
1H NMR
(acetone-d6): d ¼ 2.02, 2.03, 2.04 and 2.06 (4 s, 12H,
4 ꢃ OCH3), 4.19 (dd, J ¼ 12.3 and 2.1 Hz, 1H, 60-Ha), 4.27e
4.33 (m, 1H, 50-H), 4.35 (dd, J ¼ 12.3 and 4.5 Hz, 1H, 60-
Hb), 5.22 (app t, J ¼ 9.9 and 9.3 Hz, 1H, 40-H), 5.33 (dd,
J ¼ 9.9 and 8.4 Hz, 1H, 20-H), 5.55 (t, J ¼ 9.6 Hz, 1H, 30-H),
6.19 (d, J ¼ 8.4 Hz, 1H, 10-H), 7.46 (br s, 1H, NH), 8.21 (d,
J ¼ 9.0 Hz, 2H, 200-H and 600-H), 8.427 (s, 1H, 3-H), 8.430
(s, 1H, 6-H), 8.67 (d, J ¼ 9.0 Hz, 2H, 300-H and 500-H), OH
not observed. 13C NMR (acetone-d6): d ¼ 29.29, 29.54,
30.06 and 30.32 (4 ꢃ CH3), 62.42 (C-60), 68.97 (C-40), 71.21
(C-20), 73.00 (C-30), 73.31 (C-50), 93.15 (C-10), 102.98 (C-
3a), 120.60 (C-200and C-600), 126.30 (C-400), 131.79 (C-300and
C-500), 135.46 (C-3), 144.91 (C-100), 155.56 (C-4), 157.83
(C-6), 159.43 (C-7a), 164.39 (CO), 169.99 (CO), 170.02
(CO), 170.25 (CO), 170.68 (CO). HRMS: calcd. for
C26H27O11N5 [M]þ 586.1785; found 586.1786.
4.3. Modelling and computational details
The crystal structure of xanthine oxidoreductase was re-
trieved from the PDB database with the corresponding entry
˚
code 1VDV (resolution 1.98 A) [5]. The protein was prepared
by removing all water molecules and adding all hydrogen
atoms using the Insight software [26]. The ligands, once built
in Insight, were docked onto the active site using the molecu-
lar docking software GOLD [27,28].
GOLD is a program for calculating the docking modes of
small molecules into protein-binding sites. A genetic algorithm
is used to search the best binding of a ligand inside the active
site cavity and two different scoring functions can then be
used for analysing the obtained results. In this study we have
used ChemScore [29], a scoring function that is derived from
regression against ligandereceptor binding free energies. The
validation of this score function was performed with two
X-ray structures of the enzyme xanthine oxidoreductase with
bounded inhibitors: 1VDV (inhibitor Y-700) [5] and 1N5X
(inhibitor: TEI-6720) [23]. In the docking process the active
site was defined as a group of residues that are located within
4.2. Biological assays
4.2.1. Xanthine oxidase assay
The reaction mixture containing 50 mM potassium dihy-
drogen phosphate buffer, pH 7.4, xanthine oxidase (0.066 U/
mL), and a solution of test compounds in DMSO was incu-
bated at room temperature, during 15 min. The reaction was
started by addition of xanthine (100 mM) in the presence of
EDTA (1 mM), and uric acid formation was followed by mea-
sure of absorbance at 295 nm during 2 min. Each study corre-
sponds to three experiments, performed in triplicate.
Final concentrations of DMSO (1%) did not interfere with
enzyme activity. Allopurinol was used as positive control.
Additionally, this procedure was repeated for the most po-
tent aminocyanopyrazoles and pyrazolo[3,4-d]pyrimidines,
tested with several concentrations of xanthine (100, 75, 50,
and 25 mM), in order to evaluate the type of inhibition using
the LineweavereBurk plot.
˚
a radius of 15 A from the carbon atom 15 243 of the phenyl
group of the residue Phe1009. For each ligand 10 docking
runs were performed.
Molecular electrostatic potentials (MEPs) of the ligands
were generated at the B3LYP/6-31G* level using Gaussian
[30] . We used MOLEKEL [31] to map the electrostatic poten-
tial onto an electron density surface of 0.002 electrons/bohr3
(generally used, corresponding to about 95% of the electronic
charge) and to draw three-dimensional electrostatic potential
isosurfaces. The latter are used to predict long-range
interactions.
4.2.2. Tumour cell growth assay
The effects of compounds on the growth of tumour cell
lines MCF-7 (breast adenocarcinoma), NCI-H460 (non-small
cell lung cancer) and SF-268 (CNS cancer) were evaluated ac-
cording to the procedure adopted by the National Cancer Insti-
tute (NCI, USA) for the in vitro anticancer drug screening that
use the protein-binding dye sulforhodamine B (SRB) to assess
growth inhibition [24,25]. Cells were routinely maintained as
adherent cell cultures in RPMI-1640 medium supplemented
with 5% heat-inactivated fetal bovine serum, 2 mM glutamine
and 50 mg/mL of gentamicin at 37 ꢂC in a humidified atmo-
sphere containing 5% CO2. The optimal plating density of
each cell line, that ensure exponential growth throughout all
Acknowledgements
~
ˆ
Financial support from Fundac¸ao para a Ciencia e Tecnolo-
gia and FEDER (POCTI-SFA-3-686); (I&D no. 226/94),
POCTI, POCI, and FEDER; PhD grant from FCT to Marta
P. Neves (SFRH/BD/21770/2005) are gratefully acknowl-
´
edged. We also acknowledge MSc. Marılia Silva for the prep-
aration of a-acetobromoglucose and Miss Elisa Pinto for
obtaining the NMR, and elemental analyses data.

780
S. Gupta et al. / European Journal of Medicinal Chemistry 43 (2008) 771e780
[19] S. Gupta, A. Sivasubramanian, L.M. Rodrigues, A.P. Esteves, R. Hrdina,
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