Pyrazolo[3,4-d]pyrimidine derivatives as COX-2 selective inhibitors: Synthesis and molecular modelling studies

Article abstract of DOI:10.1002/ardp.200800140

The pyrazolo[3,4-d]pyrimidine system shows a multitude of interesting pharmacological properties. Owing to the potential anti-inflammatory activity of 5-benzamido-pyrazolo[3,4-d]pyrimidin-4-one derivatives and considering the easy synthesis of this class

Full text of DOI:10.1002/ardp.200800140

Arch. Pharm. Chem. Life Sci. 2009, 342, 321–326
D. Raffa et al.
321
Full Paper
Pyrazolo[3,4-d]pyrimidine Derivatives as COX-2 Selective
Inhibitors: Synthesis and Molecular Modelling Studies
Demetrio Raffa, Benedetta Maggio, Fabiana Plescia, Stella Cascioferro, Maria Valeria Raimondi,
Salvatore Plescia, and Maria Grazia Cusimano
Dipartimento di Chimica e Tecnologie Farmaceutiche, Universitꢀ degli Studi di Palermo, Palermo, Italy
The pyrazolo[3,4-d]pyrimidine system shows a multitude of interesting pharmacological proper-
ties. Owing to the potential anti-inflammatory activity of 5-benzamido-pyrazolo[3,4-d]pyrimidin-
4-one derivatives and considering the easy synthesis of this class of compounds, a set of new 5-
benzamido-1H-pyrazolo[3,4-d]pyrimidin-4-ones has been prepared in 42-80% yields by reacting 5-
aminopyrazole-4(N-benzoyl)carbohydrazide derivatives and the opportune triethylorthoesters.
Compounds 8a, b, 10a–d, and 11a, b revealed a superior inhibitory profile against COX-2, when
compared to that of reference standards NS398 and indomethacin. Molecular modelling studies
confirmed the obtained biological results.
Keywords: COX-2 inhibitors / Docking / Pyrazolo[3,4-d]pyrimidine / 4(3H)-Quinazolinone /
Received: August 1, 2008; accepted: September 29, 2008
DOI 10.1002/ardp.200800140
Introduction
Molecules based on the pyrazolo[3,4-d]pyrimidine ring
system exhibit a multitude of interesting pharmacologi-
cal properties including antimicrobial [1, 2], antiviral
[3, 4], anti-allergic [5], antihypertensive [6], and anti-
inflammatory activities [7].
Figure 1. Structurally related pyrazolo[3,4-d]pyrimidine 1 and
4(3H)-quinazolinone 2 nuclei.
The last activity is very intriguing taking into account
that the pyrazolo[3,4-d]pyrimidine system 1 is structur-
ally related to the 4(3H)-quinazolinone nucleus 2 (Fig. 1).
Our research group has long been interested in the N-het-
erocycle-substituted quinazolinones which are character-
ized by good analgesic and anti-inflammatory activities
and, at the same time, very low ulcerogenicity [8–14].
In the course of our medicinal chemistry researches,
we reacted the pyrazole[3,4-d]-1,3-oxazinones 3a–h with
hydrazine hydrate to obtain compounds 4a–h in 70–
90% yields together with, in the case of 4a, c, f, small
amounts of the isomer compounds 5a, c, f (Scheme 1)
[15].
Moreover, by reacting compound 5a with ethyl ortho-
formate, the 1-phenyl-5-benzamido-1H-pyrazolo[3,4-
d]pyrimidin-4-one 8a was afforded. Compound 8a was
also obtained by benzoylation of the 5-amino-1H-pyra-
zolo[3,4-d]pyrimidin-4-one 6 (Scheme 1).
At this point, owing to the easy synthesis of pyra-
zolo[3,4-d]pyrimidine derivative 8a and considering the
potential anti-inflammatory activity of this class of com-
pounds, we thought it would be of interest to extend this
synthesis to other analogues of 8a.
Moreover, using a docking-simulation approach, we
have tried to address the structural basis of COX-inhibitor
interactions in order to determine differences in the
binding mode of compounds in the COX-1 and COX-2 iso-
forms. Experimental COX-1 and COX-2 activities for com-
Correspondence: Prof. Demetrio Raffa, Dipartimento di Chimica e Tec-
nologie Farmaceutiche, Via Archirafi, 32, I-90123-Palermo, Italy.
E-mail: demraffa@unipa.it
Fax: +39 91 623-6117
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D. Raffa et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 321–326
Scheme 2. Synthetic pathway for the formation of compounds 8,
10, and 11.
Scheme 1. Synthetic pathway for the formation of compound
8a.
Figure 2. The amino NMR signal of anthranoylhydrazide 12.
pound-8a analogues were finally performed with the aim
to confirm the in silico results.
tures of the above mentioned compounds was assigned
on the basis of satisfactory elemental analyses as well as
spectroscopic data. In the case of derivatives 11, the H-
1
NMR spectra indicated hindered internal rotation
around the bond that links the pyrazolo[3,4-d]pyrimidine
nucleus to the benzamido moiety. In fact, the methylene
protons of the ethyl group, bonded at 6-position of the
pyrazolo[3,4-d]pyrimidine ring are diastereotopic and
showed signals that appeared as multiplets in the 2.60 to
2.97 ppm region, due to geminal and vicinal coupling.
The ability of compound 8a–c, 10a-d, and 11a–c to
inhibit the conversion of arachidonic acid to prostaglan-
din H2 (PGH2) was determined using a COX-1 / COX-2
inhibitor screening assay kit (kit n#560101;Cayman
Chemical, Ann Arbor, MI, USA) using N-[2-(cyclohexi-
loxy)4-nitrophenyl]methane sulphonamide (NS398) and
indomethacin (INDO) as reference compounds. Results
are shown in Table 1.
Results and discussion
The
5-benzamido-1H-pyrazolo[3,4-d]pyrimidine-4-one
derivatives 8b–e, 10a–e, and 11a, c, d were synthesized
according to the procedure described in Scheme 2.
In particular, condensation of 5-amino-1H-pyrazole-4-
carbohydrazide 9a, d with the opportune benzoyl chlor-
ide 7a–d in acetonitrile afforded the 5-aminopyrazole-4-
(N-benzoyl)carbohydrazides 5a [15], b, c [15], d, e. We
excluded the possibility to obtain derivatives of type 4
(Scheme 1) on the basis of spectroscopic as well as biblio-
graphic data comparison. In particular, the 1H-NMR spec-
tra of the compounds 5 showed the presence of the pyra-
zole amino group signal at about 6 ppm as reported for
the compounds 5a, c [15] and for the anthranoylhydra-
zide 12 [16] and, the absence of the signal at about
4.38 ppm for the more shielded hydrazide amino group
(Fig. 2) [15, 16].
Crystal structures (6COX [17] and 1Q4G [18]) from the
Brookhaven Protein Data Bank provided Cartesian coor-
dinates for COX-2 in complex with SC558 13 and ovine
COX-1 in complex with a-methyl-4-biphenylacetic acid
14, respectively.
Finally, the reaction of the 5-aminopyrazole-4-(N-ben-
zoyl)carbohydrazides 5 with the opportune triethylor-
thoesters afforded the corresponding pyrazolo[3,4-d]pyri-
midine 8b–e, 10a–e, and 11a, c, d (Scheme 2). The struc-
Insigth II program 2005 (Accelrys Software Inc., San
Diego, CA, USA) was used to check the 6COX and 1Q4G
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Pyrazolo[3,4-d]pyrimidines as COX-2 Selective Inhibitors
323
Table 1. Percent-inhibition of COX-1 and COX-2 at 10 lM con-
centration.
Compound
COX-1
COX-2
8a
8b
8c
19.8
ns
24.9
ns
27.6
ns
29.8
47.2
ns
78.9
75.9
ns
10.2
75.4
62.8
66.0
77.9
29.2
ns
10a
10b
10c
10d
11a
11b
11c
NS398
INDO
36.4
22.8
36.4
54.0
29.2
ns = not significant (%-inhibition a10%).
Figure 3. Structures of ligands: SC558 13 and a-methyl-4-
biphenylacetic acid 14.
structures for missing atoms, bound and contacts. Cer-
ius2 4.10 program (Accelrys Software Inc.,) was used to
add hydrogen to the enzyme structures, to manually
delete ligands and water molecules.
The structures of SC558 13 and a-methyl-4-biphenyl-
acetic acid 14 (Fig. 3) were obtained with Cerius2 4.10 by
selecting and cutting the ligands from the protein-ligand
complex.
Structures of the pyrazolo[3,4-d]pyrimidine 8a–c,
10a–d, and 11a–c were generated by molecular model-
ling with Cerius2 4.10 software by optimizing their geom-
etry using the Dreiding 2.21 force field with Gasteiger
charges.
The active sites of 6COX and 1Q4G were defined by the
“docked-ligand” procedure in the program Cerius2 4.10
according to the shape of the ligands SC558 13 and a-
methyl-4-biphenylacetic acid 14, respectively, as grid
points around the ones which are unoccupied by protein
atoms. A rigid docking experiment was performed for
SC558 13 and a-methyl-4-biphenylacetic acid 14, the
ligand fit predicted conformations with their x-ray crys-
tallographic structures show a very high overlap with a
RMS of 0.37 ꢀ for 13 and 0.18 ꢀ for 14.
Figure 4. Overlay of the docked orientations for: (A) a-methyl-4-
biphenylacetic acid 14 (black) and 5-benzamido-1H-pyra-
zolo[3,4-d]pyrimidine-4-one derivatives 8a, b, 10b (white) in the
active site of COX-1; (B) SC558 13 (black) and 5-benzamido-
1H-pyrazolo[3,4-d]pyrimidine-4-one derivatives 8a, b, 10b
(white) in the active site of COX-2.
All inhibitors were docked into the active sites of 6COX
and 1Q4G to generate the docked conformations using
the flexible docking procedure of “ligand fit” in the pro-
gram Cerius2 4.10.
The cyclooxygenase-binding site in both isoenzymes is
a long, narrow, hydrophobic channel extending from
the membrane-binding region of the protein [19]. A se-
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Table 2. Analytical and spectroscopical data of compounds 5b, d, e.
Comp.
Composition
Yield (%)
75
M.p. (8C)
IR (cm– 1
)
1H-NMR (DMSO-d6) (ppm)
5b
C17H14N6O4 calc.: C, 55.74;
H, 3.85; N, 22.94; found:
C, 55.93; H, 3.78; N, 22.64.
C18H17N5O2: calc.: C, 64.47; 90
H, 5.11; N, 20.88; found:
255
3450-3100 (NH2 and
NH-NH), 1673 (CO).
6.43 (s, 2H, NH2); 7-45-8.40 (a set of sig-
nals, 10H, C6H4, C6H5 and pyrazole H-3),
10.60 (s, 1H, NH); 10.70 (s, 1H, NH).
2.38 (s, 3H, CH3); 6.42 (s, 2H, NH2); 7.31-
5d
5e
230
3400-3100 (NH2 and
NH-NH), 1675 (br, CO). 8.04 (a set of signals, 10H, C6H4, C6H5
and pyrazole H-3); 9.88 (s, 1H, NH);
C, 64.43; H, 5.30; N, 20.62.
10.25 (s, 1H, NH, with D2O).
C12H13N5O2 calc.: C, 55.59;
H, 5.05; N, 27.01; found:
C, 55.53; H, 5.28; N, 26.98.
85
273–275
3400-3100 (NH2 and
NH-NH), 1685-1650
br, CO).
3.57 (s, 3H, CH3); 6.04 (s, 2H, NH2); 7.58-
7.93 (a set of signals, 6H, C6H5 and pyra-
zole H-3); 9.85 (s, 1H, NH); 10.83 (s, 1H,
NH).
Crystallization solvent: ethanol.
Table 3. Analytical and spectroscopical data of compounds 8b–e.
Comp.
Composition
Yields (%)
62
M.p. (8C)
IR (cm– 1
)
1H-NMR (DMSO-d6) (ppm)
8b
C18H12N6O4 calc.: C, 57.45;
H, 3.21; N, 22.33; found:
C, 57.15; H, 3.38; N, 22.64.
304–305
3172 (br, NH),
7.44-8.68 (a set of signals, 11H, C6H5,
1709 (CO), 1686 (CO). C6H4, quinazolinone H-2 and pyrazole
H-3); 12.26 (s, 1H, NH).
8c
C18H12ClN5O2 calc.: C, 59.11; 69
H, 3.31; N, 19.15; found:
C, 59.12; H, 3.50; N, 19.21.
279–280
262–263
167–168
240–241
176–177
284–285
261–262
149–150
172–173
184–185
3180 (br, NH),
7.41-8.59 (a set of signals, 11H, C6H5,
1707 (CO), 1690 (CO). C6H4, quinazolinone H-2 and pyrazole
H-3); 12.06 (s, 1H, NH).
3180 (br, NH),
1693 (CO).
8d
C19H15N5O2 calc.: C, 66.08;
H, 4.38; N, 20.28; found:
C, 65.85; H, 4.06; N, 20.61.
C13H11N5O2 calc.: C, 57.99;
H, 4.12; N, 26.01; found:
C, 57.92; H, 4.27; N, 26.38.
C19H15N5O2 calc.: C, 66.08;
H, 4.38; N, 20.28; found:
C, 66.34; H, 4.30; N, 20.11.
C19H14N6O4 calc.: C, 58.46;
H, 3.61; N, 21.53; found:
C, 58.39; H, 3.52; N, 21.32.
62
75
48
46
2.41 (s, 3H, CH3); 7.40-8.64 (a set of sig-
nals, 11H, C6H5, C6H4, quinazolinone H-
2 and pyrazole H-3); 11.83 (s, 1H, NH).
3.78 (s, 3H, CH3); 7.55-8.08 (a set of sig-
nals, 6H, C6H5 and pyrazole H-3); 10.70
(br s, 1H, NH).
2.54 (s, 3H, CH3); 7.42-8.43 (a set of sig-
nals, 11H, 2 x C6H5, and pyrazole H-3);
11.67 (s, 1H, NH).
8e
3260-3150 (NH),
1660–1630 (26CO).
10a
10b
10c
10d
10e
11a
11c
3215 (br, NH), 1726
(CO), 1677 (CO).
3269 (br, NH),
2.54 (s, 3H, CH3); 7.42-8.47 (a set of sig-
1718 (CO), 1693 (CO). nals, 10H, C6H5, C6H4 and pyrazole H-3);
12.00 (s, 1H, NH).
3180 (br, NH),
C19H14Cl N5O2 calc.: C, 60.09; 53
H, 3.72; N, 18.44; found:
C, 59.85; H, 3.39; N, 18.57.
2.54 (s, 3H, CH3); 7.45-8.43 (a set of sig-
nals, 10H, C6H5, C6H4 and pyrazole H-3);
11.79 (s, 1H, NH).
2.43 (s, 3H, CH3); 2.52 (s, 3H, CH3); 7.40-
8.42 (a set of signals, 10H, C6H5, C6H4
and pyrazole H-3); 11.58 (s, 1H, NH).
2.46 (s, 3H, CH3); 3.77 (s, 3H, CH3); 7.55-
8.04 (a set of signals, 6H, C6H5 and pyra-
zole H-3); 10.63 (s, 1H, NH).
1710–1690 (26CO).
C20H17N5O2 calc.: C, 66.84;
H, 4.77; N, 19.49; found:
C, 67.15; H, 4.71; N, 19.53.
C14H13N5O2 calc.: C, 59.36;
H, 4.63; N, 24.72; found:
C, 59.65; H, 4.44; N, 24.68.
C20H17N5O2 calc.: C, 66.84;
H, 4.77; N, 19.49; found:
C, 66.89; H, 4.58; N, 19.51.
48
80
42
3260 (NH),
1700–1691 (26CO).
3250 (br, NH),
1680–1635 (26CO).
3233 (br, NH),
1.27 (t, 3H, CH3); 2.69-2.97 (m, 2H, CH2)
1726 (CO), 1667 (CO). 7.41-8.43 (a set of signals,11H, 2 x C6H5
and pyrazole H-3); 11.58 (s, 1H, NH).
3393 (br, NH),
C20H16ClN5O2 calc.: C, 60.99; 46
H, 4.09; N, 17.78; found:
1.26 (t, 3H, CH3); 2.66-2.95 (m, 2H, CH2)
1715 (CO), 1691 (CO). 7.44-8.43 (a set of signals, 10H, C6H5,
C, 60.86; H, 3.99; N, 17.62.
C6H4, and pyrazole H-3); 11.72 (s, 1H,
NH).
11d
C21H19N5O2 calc.: C, 67.55;
H, 5.13; N, 18.76; found:
C, 67.71; H, 5.27; N, 18.59.
45
229–230
3250 (NH),
1722–1699 (26CO).
1.26 (t, 3H, CH3); 2.42 (s, 3H, CH3); 2.67-
2.95 (2 m, H, CH2) 7.40-8.43 (a set of sig-
nals, 10H, C6H5, C6H4, and pyrazole H-3);
11.46 (s, 1H, NH).
Crystallization solvent: dioxane for 8b–e, 10a, b, d, and 11a, b; ethyl acetate for 10e; ethanol for 10c and 11d.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 321–326
Pyrazolo[3,4-d]pyrimidines as COX-2 Selective Inhibitors
325
accommodating the 5-benzamido group of the 1H-pyra-
zolo[3,4-d]pyrimidine-4-ones 8a, b, 10b (Figs. 4 and 5).
These results are in agreement with the inhibitory
activity against the purified human COX-2 and ovine
COX-1 reported for compounds 8a–c, 10a–d, and 11a–c
(Table 1). In particular, as predicted by the docking
experiments, the pyrazolo[3,4-d]pyrimidines 8a, b, 10b
showed the best inhibitory activity against COX-2 (75.4 to
78.9% at 10 lM) and a very high COX-2 / COX-1 selectivity.
Analytical and spectroscopical data of compounds 5
and 8b-e are reported in Tables 2 and 3, respectively.
Financial support from MIUR (fund 60%) is gratefully acknowl-
edged.
The authors have declared no conflict of interest.
Experimental
Chemistry
All benzoyl chloride 7a–d were purchased from Aldrich Chemi-
cal Company (Sigma-Aldrich, Italy) and were used as received.
TLC was performed on silica gel plates (Merck 60, F254, 0.2 mm;
Merck, Germany). Melting points were determined on a Bꢁchi
530 capillary melting point apparatus (Bꢁchi Labortechnik, Fla-
wil, Switzerland) and are uncorrected; IR spectra were recorded
with a Jasco IR-810 spectrophotometer (Jasco, Tokyo, Japan) as
nujol mulls; H-NMR spectra were obtained in DMSO-d6 on a
Bruker AC-E 250 MHz spectrometer (TMS as internal standard;
Bruker Bioscience, USA).
Figure 5. Overlay of the docked orientation for compounds 8a,
b, 10b (white) and SC558 13 (black) docked in the COX-2.
cond cavity branched off from the main channel that
lead to the cyclooxygenase active site is observed in COX-
2. A similar pocket exists in COX-1, but it is smaller and
inaccessible because of the bulkier isoleucine at position
523 present in place of the valine in COX-2 [17]. So, the
bulky 5-benzamido-1H-pyrazolo[3,4-d]pyrimidine-4-one
structures are allowed to bind in the active site of COX-2
in a better way than the active site of COX-1 (Fig. 4).
In particular, analysis of the docked structure of the
synthesized pyrazolo[3,4-d]pyrimidines 8a, b, 10b in the
active sites of 6COX and 1Q4G reveals that these com-
pounds are not capable to take up the COX-1 binding site
and their structures are partially outside.
1
General procedure for 5-aminopyrazole-4-(N-
benzoyl)carbohydrazides 5b, d, e
A solution of pyrazole-4-carbohydrazides 9a, b (0.01 mol) in ace-
tonitrile (50 mL) was refluxed with 0.01 mol of the opportune
benzoyl chloride 7b, d, e for 7 h. After this time, the solid that
separated off was collected and recrystallized from ethanol to
give pure 5; yields 85 to 90%.
General procedure for 5-benzamido-1H-pyrazolo[3,4-
d]pyrimidine-4-ones 8, 10, 11
On the contrary, the bigger COX-2 binding site accom-
modates well the compounds 8a, b, and 10b. Comparison
of the different docking results of the most active com-
pounds 8a, b, 10b and SC588 13 shows that, in principle,
they adopt the same binding mode in the COX-2 binding
site (Fig. 5). The ligands are oriented so that their pyra-
zole N-phenyl ring overlapped with the 5-bromophenyl
ring of SC558 13. Furthermore, concerning the pyra-
zolo[3,4-d]pyrimidine nucleus, the pyrimidinone ring
overlappes with the phenylsulphonamide moiety of
SC558 13 and both take up the second cavity branched
off from the main channel which is also capable of
1-R-5-Aminopyrazole-4-(N-benzoyl)carbohydrazides 5 (0.03 mol)
were refluxed with 10 mL of the opportune triethylorthoester
for 4 h. After this time, the solid which separated off was col-
lected and recrystallized from the respective solvent (dioxane
for 8b–e, 10a, b, d, and 11a, b; ethyl acetate for 10e; ethanol for
10c and 11d) to give pure 8, 10, and 11; yields 44 to 75%.
Enzyme assay
The reference compounds indomethacin and NS398 were pur-
chased from Cayman Chemical, Ann Arbor, MI, USA (cat. N8
70270 and 70590, respectively).
The ability of the test compound to inhibit the conversion of
arachidonic acid to prostaglandin H2 (PGH2) was determined
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326
D. Raffa et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 321–326
using a COX1/ COX2 inhibitor screening assay kit (kit n#560101;
Cayman Chemical) according to the manufacturer's protocol.
Cyclooxygenase catalyzes the first step in the biosynthesis of
arachidonic acid to PGH2. The COX (ovine) Inhibitor Screening
Assay directly measures PGF2a produced by SnCl2 reduction, by
enzyme immunoassay (ACETM competitive EIA). This assay is
based on the competition between PG and PG-acetylcholinester-
ase (AChE) conjugate (PG-tracer) for a limited amount of PG
monoclonal antibody.
Since the concentration of PG varies, the amount of PG-tracer
that is able to bind to the PG monoclonal antibody will be inver-
sely proportional to the concentration of PG in the well. This
antibody-PG complex binds to goat polyclonal anti-mouse IgG
that has been attached to the well previously. The plate was
washed to remove any unbound reagents and Ellman's Reagent
(which contains the substrate to acetylcholinesterase) is added
to the well.
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