Exploiting the pyrazolo[3,4-d]pyrimidin-4-one ring system as a useful template to obtain potent adenosine deaminase inhibitors

Article abstract of DOI:10.1021/jm801427r

A number of pyrazolo[3,4-d]pyrimidin-4-ones bearing either alkyl or arylalkyl substituents in position 2 of the nucleus were synthesized and tested for their ability to inhibit adenosine deaminase (ADA) from bovine spleen. The 2-arylalkyl derivatives exhi

Full text of DOI:10.1021/jm801427r

J. Med. Chem. 2009, 52, 1681–1692
1681
Exploiting the Pyrazolo[3,4-d]pyrimidin-4-one Ring System as a Useful Template To Obtain
Potent Adenosine Deaminase Inhibitors
Concettina La Motta,*,† Stefania Sartini,† Laura Mugnaini,† Silvia Salerno,† Francesca Simorini,† Sabrina Taliani,†
Anna Maria Marini,† Federico Da Settimo,† Antonio Lavecchia,§ Ettore Novellino,§ Luca Antonioli,‡ Matteo Fornai,‡
Corrado Blandizzi,‡ and Mario Del Tacca‡
Dipartimento di Scienze Farmaceutiche, UniVersita` di Pisa, Via Bonanno, 6, 56126 Pisa, Italy, Dipartimento di Chimica Farmaceutica e
Tossicologica, UniVersita` di Napoli “Federico II”, Via D. Montesano, 49, 80131 Napoli, Italy, and Centro Interdipartimentale di Ricerche di
Farmacologia Clinica e Terapia Sperimentale, Via Roma 55, 56126 Pisa, Italy
ReceiVed NoVember 13, 2008
A number of pyrazolo[3,4-d]pyrimidin-4-ones bearing either alkyl or arylalkyl substituents in position 2 of
the nucleus were synthesized and tested for their ability to inhibit adenosine deaminase (ADA) from bovine
spleen. The 2-arylalkyl derivatives exhibited excellent inhibitory activity, showing Ki values in the nanomolar/
subnanomolar range. The most active compound, 1-(4-((4-oxo-4,5-dihydropyrazolo[3,4-d]pyrimidin-2-
yl)methyl)phenyl)-3-(4-(trifluoromethyl)phenyl)urea, 14d, was tested in rats with colitis induced by 2,4-
dinitrobenzenesulfonic acid to assess its efficacy to attenuate bowel inflammation. The treatment with 14d
induced a significant amelioration of both systemic and intestinal inflammatory alterations in animals with
experimental colitis. Docking simulations of the synthesized compounds into the ADA catalytic site were
also performed to rationalize the structure-activity relationships observed and to highlight the key
pharmacophoric elements of these products, thus prospectively guiding the design of novel ADA inhibitors.
Introduction
pathological processes involving site- or event-mediated ad-
enosine release. With regard to inflammation, high concentra-
tions of extracellular adenosine are generated as a consequence
of cellular stress, damage, or release of proinflammatory
mediators. Accumulated adenosine is then able to counteract
inflammation via reduction of cytokine biosynthesis and neu-
trophil functions, thus preventing phagocytosis, generation of
toxic oxygen metabolites, and cell adhesion. As the blockade
of ADA activity may increase the concentration of adenosine
at inflamed sites, ADA inhibitors might have therapeutic
relevance as novel anti-inflammatory drugs endowed with
limited adverse effects.14-18
Several compounds have been shown to inhibit ADA with
various degrees of potency. They are generally grouped into
two main classes designated as “transition-state inhibitors”, and
“ground-state inhibitors”. CF, 1,19 and dCF, 2,20 are the most
potent compounds in the first class, while erythro-9-(2-hydroxy-
3-nonyl)adenine21 ((+)-EHNA, 3, Chart 1) belongs to the second
one. Current “transition-state inhibitors” are bound to the
enzyme so tightly that their activity is nearly irreversible, thus
giving rise to serious toxic effects. Moreover, they suffer from
unfavorable pharmacokinetics, which reduces their oral bio-
availability. On the other hand, the rapid metabolism of “ground-
state inhibitors”, such as (+)-EHNA, allows a fast recovery of
the enzyme activity, with poor therapeutic effects. For these
reasons, research efforts in this area are currently focused on
the development of novel ADA inhibitors able to combine
potent, prolonged, and reversible activity with favorable phar-
macokinetic properties.
Adenosine deaminase (adenosine aminohydrolase, ADA,a EC
3.5.4.4) is a 41 kDa zinc protein involved in purine metabolism.
It catalyzes the irreversible hydrolytic deamination of adenosine
and 2′-deoxyadenosine to inosine and 2′-deoxyinosine, respec-
tively, thus regulating the endogenous levels of these nucleo-
sides. In addition, it plays a pivotal role in the development of
the lymphoid system.1-5 The importance of ADA activity for
the maturation and differentiation of immune functions is
supported by the evidence that an inherited deficiency of this
enzyme in human beings triggers a form of severe combined
immunodeficiency disease (SCID), characterized by a serious
lymphoid cell shortage, leading to both a reduced number of T
cells and their altered response to mitogens or antigens.
Therefore, ADA inhibition can induce an immunosuppressive
status, which can be profitably exploited for the treatment of
different types of cancer affecting the immune system, such as
leukemia and lymphoma.6-10 In keeping with this concept, the
antitumor properties of two natural antibiotics, coformycin (CF,
1, Chart 1) and 2′-deoxycoformycin (dCF, 2, Chart 1), have
been associated with their ability to exert a potent inhibition of
ADA.11-13
By preventing the breakdown of endogenous adenosine, ADA
inhibitors may also have a therapeutic potential for managing
* To whom all correspondence should be addressed. Phone:
(+)390502219593. Fax: (+)390502219605. E-mail: lamotta@
farm.unipi.it.
†
Universita` di Pisa.
Universita` “Federico II” di Napoli.
Centro Interdipartimentale di Ricerche di Farmacologia Clinica e
§
In order to discover novel drug candidates for the treatment
of inflammatory bowel diseases (IBD), our group has started a
research program on ADA, disclosing a novel class of inhibitors
derived from the 4-aminopyrazolo[3,4-d]pyrimidine ring system
and designed as (+)-EHNA analogues. The lead compound 4,
(R)-4-amino-2-(2-hydroxy-1-decyl)pyrazolo[3,4-d]pyrimidine,
proved to be a highly potent ADA inhibitor, showing a Ki value
of 53 pM.22 Moreover, when administered intraperitoneally to
‡
Terapia Sperimentale.
a Abbreviations: ADA, adenosine deaminase; SCID, severe combined
immunodeficiency disease; CF, coformycin; dCF, 2′-deoxycoformycin;
EHNA, erythro-9-(2-hydroxy-3-nonyl)adenine; APP, (R)-4-amino-2-(2-
hydroxy-1-decyl)pyrazolo[3,4-d]pyrimidine; IBD, inflammatory bowel dis-
ease; DNBS, 2,4-dinitrobenzenesulfonic acid; TNF-R, tumor necrosis factor-
R; IL-6, interleukin-6; MDA, malondialdehyde; PPOs, pyrazolopyrimidinones,
SARs, structure-activity relationships.
10.1021/jm801427r CCC: $40.75
2009 American Chemical Society
Published on Web 02/18/2009

1682 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Chart 1. Adenosine Deaminase Inhibitors
La Motta et al.
rats with colitis induced by 2,4-dinitrobenzenesulfonic acid
(DNBS), 4 was found to attenuate both systemic and intestinal
inflammatory parameters, improving macroscopic and histologi-
cal features of colonic tissue and reducing levels of inflammatory
mediators such as tumor necrosis factor-R (TNF-R), interleu-
kin-6 (IL-6), and malondialdehyde (MDA).23 In a subsequent
step of our studies, we focused our attention on the novel, potent,
and effective non-nucleoside inhibitors described by Terasaka
and co-workers, 5 and 6 (Chart 1), exploiting the carboxamide
moiety as a key framework for a fruitful and firm anchoring to
the enzyme binding pocket.24-28 Moving from these observa-
tions, we planned to change the 4-aminopyrazolo[3,4-d]pyri-
midine heterocyclic core of our products into the pyrazolo[3,4-
d]pyrimidin-4-one scaffold, thus developing a novel class of
inhibitors as sterically constrained derivatives of 5 and 6.
In the present article, we report the synthesis and the in vitro
functional evaluation of a number of pyrazolopyrimidinones,
PPOs, bearing either alkyl or arylalkyl substituents in position
2 of the nucleus. Compound 14d, found to be the most active
among all derivatives, was investigated in vivo for its anti-
inflammatory activity in a rat model of IBD. Moreover, docking
simulations of PPOs into the ADA catalytic site were carried
out to propose their binding mode in the light of the
structure-activity relationships (SARs).
temperature in the presence of Pd/C, 11 afforded the 2-(4-
aminobenzyl)pyrazolo[3,4-d]pyrimidin-4-one 12. The key in-
termediate 12 led to the desired inhibitors 13a-d,h by reaction
with the appropriate aroyl chloride in the presence of triethyl-
amine and to inhibitors 14a-h by treatment with the appropriate
isocyanate, both protocols being carried out under microwave
irradiation (Scheme 1).
Results and Discussion
Functional Evaluation. On the basis of a close parallel with
the previously developed 4-aminopyrazolo[3,4-d]pyrimidines,
APPs,22 our program started with the synthesis of the inhibitors
9a-c, bearing a n-nonyl, n-decyl, and n-undecyl chains,
respectively, in position 2 of the heterocyclic core. Once tested
for their efficacy against ADA from bovine spleen, these
compounds did not show any appreciable inhibitory activity
(Table 1), at variance with the parent APPs. Therefore,
hypothesizing that the structural modification of the main
scaffold could lead to a different interaction with the active site
of the enzyme, we abandoned the substitution patterns of the
prior series, focusing our attention toward bulkier substituents.
Thus, we developed compounds 13a-d,h, carrying an aroyl-
aminoarylalkyl group in the same position 2 of the nucleus. As
shown in Table 1, all the novel compounds were able to inhibit
the in vitro activity of ADA, exhibiting Ki values in the
nanomolar/subnanomolar range. In this subseries, the insertion
of an electron-donating group in the para position of the distal
phenyl ring did not affect significantly the inhibitory potency.
Indeed, compound 13b, bearing a methoxy function (Ki ) 15.56
nM), was almost as potent as the unsubstituted parent compound
13a (Ki ) 12.65 nM). Conversely, the presence of an electron-
withdrawing substituent in the same position of the pendent ring
gave rise to a remarkable increase in potency. The insertion of
a fluoro atom, as in 13c (Ki ) 0.96 nM), gave a 13-fold increase
in inhibitory potency with respect to 13a, while the presence
of the bulkier trifluoromethyl group resulted in an even more
remarkable increase in efficacy, and compound 13d, with a Ki
Synthesis
The synthesis of the target inhibitors, 9a-c, 13a-d,h, and
14a-h, was performed as outlined in Scheme 1. Alkylation of
the commercially available 3-amino-4-pyrazolecarbonitrile, 7,
with the suitable alkyl bromides in DMF at 100 °C and in the
presence of K2CO3 yielded the N1-alkylpyrazoles 8a-c as the
main reaction products. Cyclization of 8a-c with boiling formic
acid provided the corresponding pyrazolo[3,4-d]pyrimidin-4-
ones 9a-c. Reaction of 7 with p-nitrobenzyl chloride gave the
N1-benzylpyrazole 10, which was then converted to the pyra-
zolopyrimidinone 11 with boiling formic acid. Upon catalytic
hydrogenation, performed under atmospheric pressure and room

Pyrimidin-4-one Ring as Template
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1683
Scheme 1. Synthesis of Pyrazolo[3,4-d]pyrimidin-4-ones 9a-c, 13a-d,h, and 14a-h
value of 0.51 nM, turned out to be the most potent of the whole
subseries, showing a 25-fold gain in activity when compared
to 13a. Finally, an increase in the distance between the phenyl
ring and the amide function through a methylene spacer, as in
13h (Ki ) 47.91 nM), was tolerated, as it led to a moderate
decrease in activity with respect to 13a.
group: it exhibited a 3-fold increase in efficacy with respect to
the amide 13d, proving it to be the most potent among all the
synthesized PPOs. However, it has to be pointed out that, similar
to inhibitors 13, in the subseries 14 the insertion of an electron-
withdrawing group in the para position of the distal phenyl ring
produced an enhancement in inhibitory potency (compounds
14c and 14d with respect to 14a). A further structure-activity
investigation for this class of inhibitors was carried out through
the insertion of a dimethylamino group, as in 14e, a benzyl
group, as in 14f, and a benzyloxy group, as in 14g. Compound
14e (Ki ) 2.01 nM) resulted a potent ADA inhibitor, showing
a Ki value in the nanomolar range, while both derivatives 14f
(Ki ) 186.0 nM) and 14g (Ki ) 108.0 nM) displayed a low
inhibitory activity in the submicromolar range, thus proving that
the substitution pattern, providing an additional aromatic ring,
was detrimental against a favorable interaction with the active
site of the enzyme.
Taking into account the favorable results obtained by
Terasaka and co-workers through a similar modification on ADA
inhibitors,26 we then replaced the amide linker connecting the
two phenyl rings of 13 with an urea residue, thus developing
derivatives 14a-h. This structural change led to opposed
outcomes. While the unsubstituted urea derivative 14a (Ki )
2.42 nM) proved to be a more potent inhibitor than the parent
amide 14a (Ki ) 12.65 nM), compounds 14b,c, bearing either
an electron-donating group, such as methoxy (14b, Ki ) 28.27
nM), or an electron-withdrawing substituent, such as fluoro (14c,
Ki ) 1.15 nM), in the para position of the phenyl ring, were
less effective than the corresponding 13b,c. The same was also
true for the benzyl derivative 14h (Ki ) 85.7 nM), which showed
an almost 2-fold decrease in inhibitory activity when compared
to the parent 13h. In contrast, an opposite trend was shown by
compound 14d (Ki ) 0.16 nM), carrying a trifluoromethyl
Pharmacological Evaluation of Compound 14d. The main
purpose of our research program was to identify potent ADA
inhibitors as novel anti-inflammatory drug candidates. Therefore,
we tested the efficacy of the most potent compound 14d in an

1684 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
La Motta et al.
Table 1. ADA Inhibition Data of Derivatives 9a-c, 13a-e,i, and 14a-i
compd
9a
9b
n
R
Ki (nM)a
8
nab
9
10
0
0
0
0
1
0
0
0
0
0
0
0
1
nab
9c
nab
13a
13b
13c
13d
13h
14a
14b
14c
14d
14e
14f
14g
14h
(+)-EHNA
H
12.65 ( 1.15
15.56 ( 1.52
0.96 ( 0.081
0.51 ( 0.042
47.91 ( 4.13
2.42 ( 0.19
28.27 ( 2.45
1.15 ( 0.10
0.16 ( 0.010
2.01 ( 0.17
186 ( 16.58
108 ( 9.70
OCH3
F
CF3
H
H
OCH3
F
CF3
N(CH3)2
CH2C6H5
OCH2C6H5
H
85.7 ( 6.60
1.14 ( 0.10
a The Ki values are mean values ( SEM. b na: nonactive. Inhibition occurred at a concentration higher than 10 µM.
animal model of experimental colitis, induced in rats through
administration of DNBS, according to a previously reported
protocol.23
The test compound, 14d, was administered ip for 7 days in
a dose regimen ranging from 5 to 45 µmol/kg, starting 1 day
before the induction of colitis. Its effectiveness was evaluated
with respect to dexamethasone, a synthetic glucocorticoid
derivative endowed with potent anti-inflammatory activity. At
day 6 after colitis induction, a significant body weight loss was
recorded: inflamed rats displayed a mean decrease of -20 (
4.5 g in their body weight, while normal animals showed a
weight gain (+25.5 ( 7.5 g) (Figure 1a). A significant reduction
in weight loss was observed in animals treated with 45 µmol/
kg 14d (+2.30 ( 2.80 g), while 5 µmol/kg 14d (-16.50 (
2.10 g), 15 µmol/kg 14d (-10.40 ( 1.80 g), and 0.25 µmol/kg
dexamethasone were without effects (Figure 1a). Measurement
of spleen weight was assumed as an index of systemic
inflammation.29 Treatment with DNBS resulted in a significant
increment of spleen weight (+28 ( 4.50%) (Figure 1b). Such
an increase was dose-dependently reduced by administration
of 14d (+23.80 ( 4%, +18.90 ( 2.9%, and +10.10 ( 3.6%
at 5, 15, 45 µmol/kg, respectively) or dexamethasone (Figure
1b).
Colonic tissues were excised, scored for macroscopic and
processed to assess the ability of 14d to act on parameters related
to intestinal inflammation, in particular MDA and TNF-R. The
distal colon from DNBS-treated rats appeared thickened and
ulcerated with evident areas of transmural inflammation. Adhe-
sions were often present, and the bowel was occasionally dilated,
with a macroscopic damage accounting for 7.3 ( 1.5. Rats
treated with 14d displayed a dose-dependent reduction in
macroscopic damage score (6.90 ( 1.40 at 5 µmol/kg, 5.60 (
0.90 at 15 µmol/kg, and 4.30 ( 0.88 at 45 µmol/kg). Dexa-
methasone-treated animals showed also a significant decrease
in macroscopic damage score (Figure 2). MDA levels in colonic
tissues from control rats were 157 ( 26.2 µmol/mg. In DNBS-
treated animals, a marked increase in MDA concentrations was
observed (576 ( 28.1 µmol/mg). Treatments with increasing
Figure 1. Effects of 14d (5, 15, and 45 µmol/kg) or dexamethasone
(0.25 µmol/kg) on body weight (a) and spleen weight (b) at day 6 after
induction of colitis with DNBS in rats. Each column represents the
mean ( SEM (n ) 6): (*) p < 0.05, significant difference vs control
group; (a) p < 0.05, significant difference vs DNBS group.
doses of 14d or dexamethasone, 0.25 µmol/kg, significantly
attenuated the increase in tissue MDA associated with colitis,
although values did not return to basal levels (Figure 3a).
Moreover, colonic inflammation induced by DNBS was associ-
ated with a significant increase in tissue TNF-R levels (from

Pyrimidin-4-one Ring as Template
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1685
points. Nevertheless, following repeated administration of
compound 14d, we could observe that animals displayed normal
behavior, and favorable variations in body weight and food
intake which, together with a lack of mortality at all doses tested,
suggest a good tolerability and encourage further preclinical
evaluations, including acute and chronic toxicity. Overall, this
novel compound could represent a basis for the development
of novel anti-inflammatory drugs with potential therapeutic
activity against IBDs.
Docking Studies. To better understand the inhibitory potency
of our compounds at a molecolar level and to explain the results
obtained from SARs data, a number of PPOs were docked into
the publicly available X-ray crystal structure of ADA complexed
with the highly potent non-nucleoside inhibitor 6 (PDB code
1O5R).27
X-ray studies, performed by different authors, clearly dem-
onstrated that the active site of ADA can adopt two distinct
conformations: a closed and an open one (Figure 1 in Supporting
Information). In the closed form the active site consists of an
hydrophobic subsite, namely, F0, and an hydrophilic area,
namely, S0. This latter is perfectly enclosed within a structural
gate consisting of the peptide backbone of a ꢀ-strand (L182-
D185) and two leucine side chains (L58 and L62) from an
R-helix (T57-A73). The closed form is usually observed in the
complexes with substrate analogues possessing the adenine
framework.32-34 When the structural gate opens, the active site
turns into the open form which conserves the closed-form area,
consisting of the S0 and F0 subsites, and shows two additional
hydrophobic subsites around the gate, defined as F1 and F2.
Figure 2. Macroscopic damage scores estimated for colon in rats under
normal conditions or following DNBS treatment, either alone or in the
presence of 14d or dexamethasone administration. Each column
represents the mean ( SEM (n ) 6): (*) p < 0.05, significant difference
vs control group; (a) p < 0.05, significant difference vs DNBS group.
Comparing crystal structures of apo-ADA and ligated-ADA
with various inhibitors, Kinoshita et al.35,36 hypothesized that
removal of a specific water molecule binding at the bottom of
the active site might be the trigger of a conformational change
from the open to the closed from. While the apo-ADA adopts
the open form and the “trigger water” molecule exists at the
end of the active site, substrate adenosine or substrate mimic
compounds such as HDPR, binding to the hydrophilic S0
subsite, interfere with the water molecule leading to its removal.
As a result, the side chain of F65, residing at the R-helix
consisting of the active site lid, moves slightly into the active
site pocket to occupy the resulting space. This motion causes
conformation change of ADA from the open to the closed form,
and the interaction between substrate and ADA is increased.
Conversely, the non-nucleoside type inhibitors occupy the
critical water-binding position, thus causing no significant
conformational change of the protein, which remains in the open
form. The same is also true for the semitight-binding inhibitor
EHNA, whose crystal structure in complex with ADA has been
recently published.36 The adenine nucleus of this inhibitor binds
to E217 via two water molecules, thus placing its C8 atom in
the “trigger water” binding position.
So far, different experimental structures of various ligands
that bind to the binding site of ADA have been deposited in
the PDB database37 (PDB IDs: 1KRM,38,39 1NDW, 1NDV,
1NDZ, 1NDY,27 1V7A, 1V78, 1V79,26 1UML, 1QXL, 1O5R,27
2E1W, 1WXZ, 1WXY,28 2Z7G,36 1ADD,32,33 and 1A4M34).
Overlay of the open and closed ADA structures showed that
the shape of the active site in the closed form is entirely
preserved in the open conformation of the enzyme. For this
reason, we chose the open form structure 1O5R,27 whose
cocrystallized ligand (compound 6) has a phenylurea substruc-
ture, which is also present in our ligands.
Figure 3. MDA (a) and TNF-R levels (b) in colonic tissues from
control rats or in animals treated with DNBS, either alone or in
combination with 14d (5, 15, and 45 µmol/kg) or dexamethasone (0.25
µmol/kg). Each column represents the mean ( SEM (n ) 6): (*) p <
0.05, significant difference vs control rats; (a) p < 0.05, significant
difference vs DNBS group.
3.1 ( 0.7 to 9.3 ( 0.85 pg/mg). Such an increment was
significantly counteracted by 45 µmol/kg 14d (5.80 ( 0.70 pg/
mg) and 0.25 µmol/kg dexamethasone (Figure 3b). These results
indicate that treatment with the adenosine deaminase inhibitor
14d significantly improved both systemic and tissue inflamma-
tory parameters in a dose-dependent fashion. Besides this in
vivo anti-inflammatory activity, the tolerability profile of
compound 14d is also a relevant issue, in light of the significant
toxicity of other known adenosine deaminase inhibitors, such
as CF and dCF.30,31 In this respect, the present study was not
specifically designed and powered to assess toxicological end
Docking investigations focused on the most active inhibitors
13a-d, 14a-d, 14f, and 14g, using the automated docking

1686 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
La Motta et al.
Figure 4. Binding mode of compounds 13c (a) and 13d (b) into the ADA binding cavity. Ligands (yellow) and interacting key residues (pink) are
represented as stick models, while the enzyme is represented as a green ribbon model. Zinc atom is represented as a cyan ball. H-bonds are shown
as dashed yellow lines.
program GOLD, version 4.0,40,41 which in several studies has
been shown to yield better performances compared to other
similar programs.42-45
(Figures 4 and 5). Key interactions stabilized the compounds
inside the binding pocket. The pyrazolopyrimidinone CdO
oxygen was coordinated to the zinc ion at a distance of 2.5 Å,
and its adjacent NH moiety donated a H-bond to the E217
carboxylate group. It is interesting to note that the CdO group
on the pyrazolopyrimidinone moiety occupies the same position
of the water molecule ligated to and activated by the zinc atom.
The diphenylamide or urea chain was localized in a deep and
narrow channel at the entrance of the enzyme. Hydrophobic
and π-π stacking interactions were found to stabilize the
inhibitor/enzyme complexes. The first aromatic system of the
urea or amide chain interacted with the lipophilic residues L62
and M155 and, at the same time, formed a face-on-face π-π
stacking contact with the F65 aromatic ring. The distal phenyl
ring of the same chain was accommodated in a pocket framed
by residues L106, P116, W117, H157, and W161, with which
it formed hydrophobic interactions. Moreover, the benzo-fused
ring of W117 appeared to be optimally oriented for a favorable
face-on-face π-π stacking interaction with the distal phenyl
ring of amide or urea chain: the planes of the two rings are
fairly parallel and separated by a distance ranging between 3.5
and 4.3 Å, respectively. Such an interaction would be consistent
with the activity trend of these compounds showing that
lipophilic and electron-withdrawing substituents in the para
position of the distal aromatic ring increase the potency. In fact,
the electron-deficient aromatic ring of 13c, 13d, 14c, and 14d
would more favorably realize the π-stacking charge transfer
interactions with the electron-rich benzo-fused ring of W117.
Kinoshita et al.38,39 reported that the D296 side chain is
flexible in the active site of ADA; that is, it can adjust to keep
the interaction with the respective inhibitor. Accordingly, the
side chain of D296 was allowed to move during the docking
experiments.
The GoldScore-CS docking protocol46 was adopted in this
study. In this protocol, the poses obtained with the original
GoldScore function are rescored and reranked with the GOLD
implementation of the ChemScore function.46,47 To test the
validity of this protocol for the bovine ADA system, the
cocrystallized ligand (compound 6) was first docked back into
its binding site. In this docking run, the 200 poses produced by
GOLD resulted in only one prevailing cluster on the basis of
their conformations: 23 of the poses closely resembled the
cocrystallized conformation with a heavy atom root-mean-square
deviation (rmsd) ranging from 0.6 to 1.8 Å. ChemScore was
able to rank 19 out of the 23 poses from this cluster as the
highest ranked nineteen poses. Thus, this docking protocol was
considered to be suitable for the subsequent docking runs for
compounds 13a-d, 14a-d, 14f, and 14g.
When 13c, 13d, 14c, and 14d were docked within the ADA
active site, about 90% of the conformations generated by GOLD
adopted only one highly conserved orientation. The inhibitors
entirely occupied the S0 subsite and most of the F0 hydrophobic
subsite, but F1 and F2 subsites in the complex remained empty

Pyrimidin-4-one Ring as Template
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1687
Figure 5. Binding mode of compounds 14c (a) and 14d (b) into the ADA binding cavity. Ligands (yellow) and interacting key residues (pink) are
represented as stick models, while the enzyme is represented as a green ribbon model. Zinc atom is represented as a cyan ball. H-bonds are shown
as dashed yellow lines.
Indeed, visual inspection of the inhibitor/ADA complexes
revealed that one CF3 fluorine atom of 13d formed a H-bond
with NH backbone of P116 (Figure 4b), while the same fluorine
atom of 14d established two H-bonds with both NH backbone
and NH2 side chain of N118 (Figure 5b). The H-bonding
capability of the trifluoromethyl group is well-documented in
the literature, and several structural studies highlight the evident
H-bond acceptor capability in protein-ligand complexes, as
assessed by PDB analysis.48 These results are consistent with
the SAR data showing that 13d and 14d are the most potent
ADA inhibitors of the series with Ki values of 0.51 and 0.16
nM, respectively.
In the most frequently occurring and most favorable docking
results, compounds 13b, 14b, and 14e were found to bind in
the known binding pocket in a manner similar to the above-
described compounds, with the pyrazolopyrimidinone CdO
oxygen coordinating the zinc ion and the amide or urea chain
localized in a deep and narrow channel at the entrance of the
enzyme. However, 13b and 14b were weak ADA inhibitors
showing Ki values of 15.56 and 28.27 nM, respectively. The
reduced activity is probably ascribable to both electronic and
steric factors: the p-methoxy group, which produces an electron-
donating (resonance) effect together with a low withdrawing
(inductive) one, could decrease the π-π stacking interaction
between W117 and the distal aromatic ring of both 13b and
14b. Moreover, the p-methoxy group of the longer derivative
14b causes steric hindrance with P114 and I115 CdO backbone,
explaining its reduced ADA inhibitory activity.
In light of these results, compound 14e, which has the
electron-donating p-dimethylamino substituent on the distal
phenyl ring, should be expected to exhibit low inhibitory
activity. However, this compound showed a Ki value of 2.01
nM, which is compatible with the decrease in electron-donating
power of the p-dimethylamino group caused by its protonation
at physiological pH. In fact, the protonation of this moiety (using
the nitrogen electron lone pair) removes the effect of the lone-
pair delocalization.
When the less active 14f and 14g were docked within the
ADA active site, only 20% of the generated conformations
adopted the above-described binding mode, whereas 80% were
in a different orientation. The molecules fully occupied the F1,
F2, and F0 subsites (Figure 6). The pyrazolopyrimidinone ring
and the first aromatic system of the urea chain made hydro-
phobic interactions with both sides of the hydrophobic gate
consisting of the F1 and F2 subsites, while the para-substituted
distal aromatic ring of the urea chain fully occupied the
hydrophobic F0 subsite. The NH pyrazolopyrimidinone of the
inhibitors was involved in a H-bond with the L56 CdO
backbone. Interestingly, the inhibitors were positioned far from
the active center and did not bind to the hydrophilic S0 subsite
at all. These observations plainly explain the loss in the ADA
inhibitory potency for 14f and 14g.

1688 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
La Motta et al.
Figure 6. Binding mode of compounds 14f (a) and 14g (b) into the ADA binding cavity. Ligands (yellow) and interacting key residues (pink) are
represented as stick models, while the enzyme is represented as a green ribbon model. Zinc atom is represented as a cyan ball. H-bonds are shown
as dashed yellow lines.
washed with water, and recrystallized from the appropriate solvent
(Supporting Information, Tables 1 and 2).
In summary, the most active PPOs proved to interact with
the enzyme active site through a direct coordination with the
catalytic zinc ion. The mode of binding here proposed is fully
substantiated by UV spectroscopy measurements, performed to
demonstrate the ability of the pyrazolopyrimidinone ring system
to coordinate this metal ion. UV spectra were recorded using
derivative 9a as representative of the whole class. Upon addition
of zinc(II) ion to a homogeneous solution of the reference
compound, a pronounced decrease in optical density of the
absorption maximum was observed (Figure 2 in Supporting
Information). An understanding of the binding mode of our
inhibitors, fully consistent with the observed SARs, provides a
sound platform for a future structure-guided design of novel
analogues with higher potency.
3-Amino-1-(4-nitrobenzyl)-4-pyrazolecarbonitrile 10. A solution
of 1-(chloromethyl)-4-nitrobenzene (2.05 g, 12.0 mmol) in DMF
was added dropwise to a suspension of 3-amino-4-pyrazolecarbo-
nitrile 7 (1.08 g, 10.0 mmol) and anhydrous potassium carbonate
(1.66 g, 12.0 mmol) in 25 mL of DMF, and the resulting reaction
mixture was stirred at 50 °C for 8 h. After the mixture was cooled,
the inorganic material was filtered off and the solution was
evaporated to dryness under reduced pressure. The residue was then
purified by flash chromatography (eluting system, ethyl acetate/
petroleum ether 60-80 °C 7/3) to obtain 10 as a yellow solid, which
was recrystallized from ethanol. Yield 67%. Mp: 120-122 °C. IR,
1
ν cm-1: 3390, 3226, 2223, 1639, 1560, 1516. H NMR, δ ppm:
5.27 (s, 2H, CH2), 5.66 (s, 2H, NH2, exc), 7.46 (d, 2H, ArH), 8.22
(d, 2H, ArH), 8.31 (s, 1H, H5). Anal. (C11H9N5O2) C, H, N.
2-(4-Nitrobenzyl)-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one 11. A
suspension of 3-amino-1-(4-nitrobenzyl)-4-pyrazolecarbonitrile 10
(10.0 mmol) in 3.0 mL of formic acid was vigorously boiled, under
stirring, for 5 h. The cooled solution was then diluted with
ice-water, and the solid separated was filtered, washed with water,
and recrystallized from ethanol. Yield 40%. Mp: 216-218 °C. IR,
Experimental Section
Chemistry.MeltingpointsweredeterminedusingaReichert-Ko¨fler
hot-stage apparatus and are uncorrected. Infrared spectra were
recorded with a FT-IR spectrometer Nicolet/Avatar in Nujol mulls.
1
Routine H NMR spectra were recorded in DMSO-d6 solution on
1
ν cm-1: 3453, 1692, 1610, 1542. H NMR, δ ppm: 5.68 (s, 2H,
a Varian Gemini 200 spectrometer operating at 200 MHz. Evapora-
tion was performed in vacuo (rotary evaporator). Anhydrous sodium
sulfate was always used as the drying agent. Analytical TLC was
carried out on Merck 0.2 mm precoated silica gel aluminum sheets
(60 F-254), with visualization by irradiation with a UV lamp. Flash
chromatography was performed with Merck silica gel 60 (230-400
mesh ASTM). The microwave-assisted procedures were carried out
in sealed vessels using a CEM/Discover LabMate 220VAC/50 Hz
microwave system. The UV spectra were measured on a Perkin-
Elmer Lambda 25 spectrophotometer at T ) 20 °C using potassium
phosphate buffer, pH 7.2, as solvent and cuvettes with 1 cm path
length. Elemental analyses were performed by our analytical
laboratory and agreed with theoretical values to within (0.4%.
CH2), 7.56 (d, 2H, ArH), 7.96 (s, 1H, H3), 8.25 (d, 2H, ArH), 8.77
(s, 1H, H6), 11.80 (s, 1H, NH, exc). Anal. (C12H9N5O3) C, H, N.
2-(4-Aminobenzyl)-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one 12.
A suspension of 2-(4-nitrobenzyl)-2H-pyrazolo[3,4-d]pyrimidin-
4(5H)-one 11 (2.71 g, 10.0 mmol) and 10% palladium on carbon
(1.00 mmol) in 250 mL of absolute ethanol was hydrogenated at
atmospheric pressure and room temperature until the theoretical
uptake of hydrogen was achieved. After the catalyst was filtered,
the solvent was evaporated to dryness to give a pale-yellow solid
which was collected and recrystallized from ethanol. Yield 95%.
1
Mp: 225-227 °C. IR, ν cm-1: 3409, 3327, 3153, 1700, 1605. H
NMR, δ ppm: 5.17 (s, 2H, NH2, exc), 5.24 (s, 2H, CH2), 6.53 (d,
2H, ArH), 7.07 (d, 2H, ArH), 7.92 (s, 1H, H3), 8.54 (s, 1H, H6),
11.70 (s, 1H, NH, exc). Anal. (C12H11N5O) C, H, N.
The alkyl bromides, the aroyl chlorides, and the isocyanates used
to obtain compounds 8a-c, 13a-d,h, and 14a-h, respectively,
as well as the 1-(chloromethyl)-4-nitrobenzene, were from Sigma-
Aldrich. All other chemicals were of reagent grade.
General Procedure for the Synthesis of N-(4-((4-oxo-4,5-dihy-
dropyrazolo[3,4-d]pyrimidin-2-yl)methyl)phenyl)arylamides 13a-d,h.
2-(4-Aminobenzyl)-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, 12 (2.41
g, 10.0 mmol), the suitable aroyl chloride (12.0 mmol), and
triethylamine (1.67 mL, 12.0 mmol) were mixed thoroughly and
irradiated with microwaves at 140 °C for 10 min. The cooled residue
was then diluted with ice-water and the solid separated was filtered
and purified by recrystallization from the appropriate solvent to
give the target compounds 13a-d,h (Supporting Information,
Tables 1 and 2).
The 5-amino-1-alkyl-4-pyrazolecarbonitriles 8a-c were prepared
in accordance with previously reported procedures.22
General Procedure for the Synthesis of 2-Alkylpyrazolo[3,4-
d]pyrimidin-4-ones 9a-c. A suspension of 1-alkyl-3-amino-4-
pyrazolecarbonitrile 8a-c (10.0 mmol) in 3 mL of formic acid was
vigorously boiled, under stirring, until the disappearance of the
starting material (5-8 h, TLC analysis). The cooled solution was
then diluted with ice-water, and the solid separated was filtered,

Pyrimidin-4-one Ring as Template
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1689
General Procedure for the Synthesis of 1-(4-((4-Oxo-4,5-dihy-
dropyrazolo[3,4-d]pyrimidin-2-yl)methyl)phenyl)-3-arylureas 14a-h.
2-(4-Aminobenzyl)-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one 12 (2.41
g, 10.0 mmol) and the suitable isocyanate (12.0 mmol) were mixed
thoroughly and irradiated with microwaves at 140 °C for 10 min.
The cooled residue was then diluted with toluene and the solid
separated was filtered and purified by recrystallization from the
appropriate solvent to give the target compounds 14a-h (Support-
ing Information, Tables 1 and 2).
performed on specimens taken from a region of inflamed colon
immediately adjacent and distal to the gross necrotic damage.
Assessment of Colitis. At the end of treatments, colonic tissues
were excised, rinsed with saline, and scored for macroscopic injury,
in accordance with the criteria previously reported by Fornai et
al.51 The macroscopic damage was scored on a 0-6 point scale
based on the following criteria: presence of adhesions between
colonic tissue and other organs (0 none, 1 minor, 2 major adhesions)
and consistency of colonic fecal material (0 formed, 1 loose, 2 liquid
stools). All the parameters of macroscopic damage were recorded
and scored for each rat by two observers blinded to the treatment.
At the time of colitis assessment, the weight of spleen was also
measured.
Biology. ADA type IX from bovine spleen (150-200 U/mg)
and adenosine were purchased from Sigma Chemical Co. All other
chemicals were of reagent grade.
Enzymatic Assay. The activity of ADA was determined spec-
trophotometrically by monitoring for 2 min the change in absor-
bance at 262 nm, which is due to the deamination of adenosine
catalyzed by the enzyme. The change in adenosine concentration/
min was determined using a Beckman DU-64 kinetics software
program (Solf Pack TM Module). ADA activity was assayed at 30
°C in a reaction mixture containing 50 µM adenosine, 50 mM
potassium phosphate buffer, pH 7.2, and 0.3 nM enzyme solution
in a total volume of 500 µL. The inhibitory activity of the newly
synthesized compounds was assayed by adding 100 µL of the
inhibitor solution to the reaction mixture described above. All the
inhibitors were dissolved in water, and the solubility was facilitated
by using DMSO, whose concentration never exceeded 4% in the
final reaction mixture. To correct for the nonenzymatic change in
adenosine concentration and the absorption by the test compounds,
a reference blank containing all the above assay components except
the substrate was prepared. The inhibitory effect of the new
derivatives was routinely estimated at a concentration of 10-5 M.
Those compounds found to be active were tested at additional
concentrations between 10-5 and 10-11 M. Each inhibitor concen-
tration was tested in triplicate, and the determination of the IC50
values was performed by linear regression analysis of the log of
the concentration response curve. The Ki values were calculated
from IC50 values by means of the Cheng and Prusoff equation.49
Pharmacology. Albino male Sprague-Dawley rats, 250-300
g body weight, were employed throughout the study. The animals
were fed standard laboratory chow and tap water ad libitum and
were not subjected to experimental procedures for at least 1 week
after their delivery to the laboratory. Their care and handling were
in accordance with the provisions of the European Union Council
Directive 86-609, recognized and adopted by the Italian Govern-
ment. DNBS and dexamethasone were purchased from Sigma
Aldrich (St. Louis, MO). 14d and dexamethasone were dissolved
in sterile dimethyl sulfoxide, and further dilutions were made with
sterile saline. The solutions were frozen into aliquots of 2 mL and
stored at -80 °C until use.
Evaluation of tissue MDA. MDA concentration in colonic
specimens was evaluated to obtain quantitative estimation of
membrane lipid peroxidation. Colonic tissues were weighed, minced
by forceps, homogenized in 2 mL of cold buffer (Tris-HCl, 20
mmol/L, pH 7.4) by a Polytron homogenizer (Cole Palmer
homogenizer), and spun by centrifugation at 1500g for 10 min at
4 °C. Colonic MDA concentrations were determined by means of
a kit for colorimetric assay (Calbiochem-Novabiochem Corporation,
San Diego, CA), and the results were expressed as µmol of MDA
per mg of colonic tissue.
TNF-r Assay. Tissue TNF-R levels were measured using a kit
for enzyme-linked immunosorbent assay (Biosource International,
Camarillo, CA). For this purpose, as described by Marquez et al.,52
tissue samples, previously stored at -80 °C, were weighed, thawed,
and homogenized in 0.3 mL of phosphate buffered saline (pH 7.2)/
100 mg of tissue at 4 °C and centrifuged at 13400g for 20 min.
One hundred microliter aliquots of the supernatants were then used
for assay. Tissue TNF-R levels were expressed as picogram per
milligram of tissue.
Statistical Analysis. The results are given as the mean (
standard error of the mean (SEM). The statistical significance of
data was evaluated by one way analysis of variance (ANOVA)
followed by post hoc analysis by Student-Newman-Keuls test,
and P values lower than 0.05 were considered significant. All
statistical procedures were performed using GraphPad Prism,
version 3.0, software (GraphPad, San Diego, CA).
Computational Chemistry. Molecular modeling and graphic
manipulations were performed using the molecular operating
environment (MOE)53 and UCSF-CHIMERA54 software packages
running on a 2 CPU (PIV 2.0-3.0 GHz) Linux workstation. Energy
minimizations were realized by employing the AMBER, version
9, program,55 selecting the Cornell et al. force field.56
Ligand and Protein Setup. The core structures of compounds
13a-d, 14a-d,f,g were constructed using standard bond lengths
and bond angles of the MOE fragment library. Geometry optimiza-
tions were accomplished with the MMFF94X force field,57-61
available within MOE. The crystal structure of the bovine ADA
complexed with the highly potent non-nucleoside inhibitor 6 (PDB
code 1O5R)27 recovered from Brookhaven Protein Database37 was
used for the docking experiments. Bound ligand and water
molecules were removed. A correct atom assignment for Asn, Gln,
and His residues was done, and hydrogen atoms were added using
standard MOE geometries. Partial atomic charges were computed
by MOE using the Amber99 force field.
Induction of Colitis and Drug Treatments. Colitis was induced
in accordance with the method previously described by Antonioli
et al.23 Briefly, during a short anesthesia with isoflurane (Abbott,
Rome, Italy), an amount of 15 mg of DNBS in 0.25 mL of 50%
ethanol was administered intrarectally via a polyethylene PE-60
catheter inserted 8 cm proximal to the anus. Control rats received
0.25 mL of 50% ethanol. Animals underwent subsequent experi-
mental procedures 6 days after DNBS administration to allow a
full development of histologically evident colonic inflammation.
Docking Simulations. Docking of 13a-d, 14a-d,f,g to ADA
was performed with GOLD, version 4.0,40 which uses a genetic
algorithm for determining the docking modes of ligands and
proteins. An advantage of GOLD over other docking methods is
the program’s ability to account for some rotational protein
flexibility, as well as full ligand flexibility. Specifically, OH groups
of S, T, and Y and amino groups of K are allowed to rotate during
docking to optimize H-bonding to the ligand. GOLD requires a
user-defined binding site. It searches for a cavity within the defined
area and considers all the solvent-accessible atoms in that area as
active-site atoms. The fitness score function that was implemented
in GOLD (GOLDScore) is made up of four components that
account for protein-ligand binding energy: protein-ligand hydro-
gen bond energy (external H-bond), protein-ligand van der Waals
Test drugs were administered intraperitoneally for 7 days, starting
1 day before the induction of colitis. Animals were assigned to the
following treatment groups, each consisting of six rats: 14d (5, 15,
45 µmol/kg) or dexamethasone (0.25 µmol/kg). DNBS-untreated
animals (control group) and DNBS-treated rats (DNBS group)
received drug vehicle to serve as controls. Body weight was
monitored daily starting from the onset of drug treatments. To
evaluate the anti-inflammatory effects of 14d, increasing doses of
this compound were tested on body weight, spleen weight,
macroscopic damage score, tissue TNF-R, and MDA levels in
animals with colitis. Dexamethasone was used as comparator drug,
and the dose was selected on the basis of previous studies per-
formed on rat models of colitis.23,50 The macroscopic score was
evaluated on the whole colon, whereas biochemical assays were

1690 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
La Motta et al.
energy (external vdw), ligand internal vdw energy (internal vdw),
and ligand torsional strain energy (internal torsion). Parameters used
in the fitness function (hydrogen bond energies, atom radii,
polarizabilities, torsion potentials, hydrogen bond directionalities,
and so forth) are taken from the GOLD parameter file. The fitness
score is taken as the negative of the sum of the energy terms, so
larger fitness scores indicated better bindings. The fitness function
has been optimized for the prediction of ligand binding positions
rather than the prediction of binding affinities, although some
correlation with the latter can also be found.46 The protein input
file may be the entire protein structure or a part of it comprising
only the residues that are in the region of the ligand binding site.
In the present study, GOLD was allowed to calculate interaction
energies within a sphere of a 7 Å radius centered on the cocrystal
compound 6 in the ADA structure. The Goldscore-CS docking
protocol46 was adopted in this study. In this protocol, the poses
obtained with the original Goldscore function are rescored and
reranked with the GOLD implementation of the CHEMscore
function.46,47 The mobility of D296 side chain was set up using
the FLEXIBLE SIDECHAINS option in the GOLD front end,
which incorporates the rotamer library reported by Lovell et al.62
To perform a thorough and unbiased search of the conformation
space, each docking run was allowed to produce 200 poses without
the option of early termination, using standard default settings. The
top solution obtained after reranking of the poses with CHEMscore
was selected to generate the ADA/inhibitor complexes.
References
(1) Cristalli, G.; Costanzi, S.; Lambertucci, C.; Lupidi, G.; Vittori, S.;
Volpini, R.; Campioni, E. Adenosine Deaminase: Functional Implica-
tions and Different Classes of Inhibitors. Med. Res. ReV. 2001, 21,
105–128.
(2) Yegutkin, G. G. Nucleotide- and Nucleoside-Converting Ectoenzymes:
Important Modulators of Purinergic Signalling Cascade. Biochim.
Biophys. Acta, Mol. Cell Res. 2008, 1783, 673–694.
(3) Franco, R.; Mallol, J.; Casado, V.; Lluis, C.; Canela, E. I.; Saura, C.;
Blanco, J.; Ciruela, F. Ecto-Adenosine Deaminase: An Ecto-Enzyme
and a Costimulatory Protein Acting on a Variety of Cell Surface
Receptors. Drug DeV. Res. 1998, 45, 261–268.
(4) Franco, R.; Valenzuela, A.; Lluis, C.; Blanco, J. Enzymatic and
Extraenzymatic Role of Ecto-Adenosine Deaminase in Lymphocytes.
Immunol. ReV. 1998, 161, 27–42.
(5) Aldrich, M. B.; Blackburn, M. R.; Kellems, R. E. The Importance of
Adenosine Deaminase for Lymphocyte Development and Function.
Biochem. Biophys. Res. Commun. 2000, 272, 311–315.
(6) Chen, T.; Smyth, T.; Abbott, C. A. CD26. J. Biol. Regul. Homeostatic
Agents 2004, 181, 47–54.
(7) Blackburn, M. R.; Kellems, R. E. Adenosine Deaminase Deficiency:
Metabolic Basis of Immune Deficiency and Pulmonary Inflammation.
AdV. Immunol. 2005, 86, 1–41.
(8) Hershfield, M. S. New Insights into Adenosine-Receptor-Mediated
Immunosuppression and the Role of Adenosine in Causing the
Immunodeficiency Associated with Adenosine Deaminase Deficiency.
Eur. J. Immunol. 2005, 35, 25–30.
(9) Resta, R.; Thompson, L. F. SCID: The Role of Adenosine Deaminase
Deficiency. Immunol. Today 1997, 18, 371–374.
Energy Refinement of the Ligand/Enzyme Complexes. To
eliminate any residual geometric strain, the obtained complexes
were energy minimized for 5000 steps using combined steepest
descent and conjugate gradient methods until a convergence value
of 0.001 kcal/(mol ·Å). The zinc divalent cation in the ADA crystal
structure was replaced by the tetrahedron-shaped zinc divalent cation
that has four cationic dummy atoms surrounding the central zinc
ion,63 without resorting to the use of a covalent bond between zinc
and its coordinate64-66 or to semiempirical calculations.67 This
approach uses four identical dummy atoms tetrahedrically attached
to the zinc ion and transfers all the atomic charge of the zinc divalent
cation evenly to the four dummy atoms. The four peripheral atoms
are “dummy” in that they interact with other atoms electrostatically
but not sterically, thus mimicking zinc’s 4s4p3 vacant orbitals that
accommodate the lone-pair electrons of zinc coordinates. H15, H17,
H214, and D295, which are the first-shell zinc coordinates, were
treated as histidinate (HIN) and aspartate (ASP).63,68-71 D181 and
E260, which form a H-bond with His214 and His15, respectively,
were treated as glutamic acid (GLH). Upon minimization, the
protein backbone atoms were held fixed. Geometry optimizations
were performed using the SANDER module in the AMBER suite
of programs, employing the Cornell et al. force field to assign
parameters for the standard amino acids. General AMBER force
field (GAFF) parameters were assigned to ligands, while the
published force field parameters were used for the tetrahedron-
shaped zinc divalent cation.72 The partial charges were calculated
using the AM1-BCC method as implemented in the ANTECHAM-
BER suite of AMBER.
(10) Hershfield, M. S. Adenosine Deaminase Deficiency: Clinical Expres-
sion, Molecular Basis, and Therapy. Semin Hematol. 1998, 35, 291–
298.
(11) Sauter, C.; Lamanna, N.; Weiss, M. A. Pentostatin in Chronic
Lymphocytic Leukemia. Expert Opin. Drug Metab. Toxicol. 2008, 4,
1217–1222.
(12) Honma, Y. A Novel Therapeutic Strategy Against Monocytic Leu-
kemia with Deoxyadenosine Analogs and Adenosine Deaminase
Inhibitors. Leuk. Lymphoma 2001, 42, 953–962.
(13) Robak, T.; Korycka, A.; Kasznicki, M.; Wrzesien-Kus, A.; Smolewski,
P. Purine Nucleoside Analogues for the Treatment of Hematological
Malignancies: Pharmacology and Clinical Applications. Curr. Cancer
Drug Targets 2005, 5, 421–444.
(14) Ko¨se, K.; Utas¸, S.; Yazici, C.; Akdas¸, A.; Keles¸timur, F. Effect of
Propylthiouracil on Adenosine Deaminase Activity and Thyroid
Function in Patients with Psoriasis. Br. J. Dermatol. 2001, 144, 1121–
1126.
(15) Cronstein, B. N. Adenosine: An Endogenous Anti-Inflammatory Agent.
J. Appl. Physiol. 1994, 76, 5–13.
(16) Ohta, A.; Sitkovsky, M. Role of G-Protein-Coupled Adenosine
Receptors in Down-Regulation of Inflammation and Protection from
Tissue Damage. Nature 2001, 414, 916–920.
(17) Hasko, G.; Deitch, E. A.; Szabo, C.; Nemeth, Z. H.; Vizi, E. S.
Adenosine: A Potential Mediator of Immunosuppression in Multiple
Organ Failure. Curr. Opin. Pharmacol. 2002, 2, 440–444.
(18) Hasko, G.; Cronstein, B. N. Adenosine: An Endogenous Regulator of
Innate Immunity. Trends Immunol. 2004, 25, 33–39.
(19) Cha, S.; Agarwal, R. P.; Parks, R. E., Jr. Thigh-Binding Inhibitors.
II. Non-Steady-State Nature of Inhibition of Milk Xantine Oxidase
by Allopurinol and Alloxanthine and of Human Erythrocytic Adenos-
ine Deaminase by Coformicin. Biochem. Pharmacol. 1975, 24, 2187–
2197.
UV Absorption Spectroscopic Analyses. Stock solutions of
100.0 µM ZnCl2, from Sigma-Aldrich, and of 50.00 µM 9a were
prepared by dissolving the required amount of substance in distilled
water. For 9a the solubility was facilitated by using DMSO. Metal
coordination experiments were carried out by adding from 0 to 2.0
mL of zinc(II) ion solution, in 0.5 mL increments, to 2.0 mL of
50.00 µM 9a and diluting to a total volume of 5.0 mL with
potassium phosphate, pH 7.2. Titration was monitored by UV-vis
spectroscopy measuring the absorbance of each solution, at λ
ranging from 190 to 260 nm and at T ) 20 °C, using a PerkinElmer
Lambda 25 spectrophotometer.
(20) Agarwal, R. P.; Spector, T.; Parks, R. E., Jr. Thigh-Binding Inhibitors.
IV. Inhibition of Adenosine Deaminase by Various Inhibitors. Bio-
chem. Pharmacol. 1977, 26, 359–367.
(21) Schaeffer, H. J.; Schwender, C. F. Enzyme Inhibitors. 26. Bridging
Hydrophobic and Hydrophilic Regions of Adenosine Deaminase
with Some 9-(2-Hydroxy-3-alkyl)adenines. J. Med. Chem. 1974, 17,
6–8.
(22) Da Settimo, F.; Primofiore, G.; La Motta, C.; Taliani, S.; Simorini,
F.; Marini, A. M.; Mugnaini, L.; Lavecchia, A.; Novellino, E.;
Tuscano, D.; Martini, C. Novel, Highly Potent Adenosine Deami-
nase Inhibitors Containing the Pyrazolo[3,4-d]pyrimidine Ring
System. Synthesis, Structure-Activity Relationships, and Molecular
Modeling Studies. J. Med. Chem. 2005, 48, 5162–5174.
(23) Antonioli, L.; Fornai, M.; Colucci, R.; Ghisu, N.; Da Settimo, F.;
Natale, G.; Kastsiuchenka, O.; Duranti, E.; Virdis, A.; Vassalle,
C.; La Motta, C.; Mugnaini, L.; Breschi, M. C.; Blandizzi, C.; Del
Tacca, M. Inhibition of Adenosine Deaminase Attenuates Inflam-
mation in Experimental Colitis. J. Pharmacol. Exp. Ther. 2007, 322,
435–442.
Supporting Information Available: Tables of physical, spectral,
and analytical data of compounds described; figures showing the
closed and open forms of ADA and UV absorption spectra of
compound 9a. This material is available free of charge via the
Internet at http://pubs.acs.org.

Pyrimidin-4-one Ring as Template
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1691
(24) Terasaka, T.; Nakanishi, I.; Nakamura, K.; Eikyu, Y.; Kinoshita,
T.; Nishio, N.; Sato, A.; Kuno, M.; Seki, N.; Sakane, K. Structure-
Based de Novo Design of Non-Nucleoside Adenosine Deaminase
Inhibitors. Bioorg. Med. Chem. Lett. 2003, 13, 1115–1118.
(25) Terasaka, T.; Kinoshita, T.; Kuno, M.; Nakanishi, I. A Highly Potent
Non-Nucleoside Adenosine Deaminase Inhibitor: Efficient Drug
Discovery by Intentional Lead Hybridization. J. Am. Chem. Soc.
2004, 126, 34–35.
(26) Terasaka, T.; Okumura, H.; Tsuji, K.; Kato, T.; Nakanishi, I.;
Kinoshita, T.; Kato, Y.; Kuno, M.; Seki, N.; Naoe, Y.; Inoue, T.;
Tanaka, K.; Nakamura, K. Structure-Based Design and Synthesis
of Non-Nucleoside, Potent, and Orally Bioavailable Adenosine
Deaminase Inhibitors. J. Med. Chem. 2004, 47, 2728–2731.
(27) Terasaka, T.; Kinoshita, T.; Kuno, M.; Seki, N.; Tanaka, K.; Nakanishi,
I. Structure-Based Design, Synthesis, and Structure-Activity Relation-
ship Studies of Novel Non-Nucleoside Adenosine Deaminase Inhibi-
tors. J. Med. Chem. 2004, 47, 3730–3743.
(28) Terasaka, T.; Tsuji, K.; Kato, T.; Nakanishi, I.; Kinoshita, T.; Kato,
Y.; Kuno, M.; Inoue, T.; Tanaka, K.; Nakamura, K. Rational Design
of Non-Nucleoside, Potent, and Orally Bioavailable Adenosine
Deaminase Inhibitors: Predicting Enzyme Conformational Change
and Metabolism. J. Med. Chem. 2005, 48, 4750–4753.
(29) Siegmund, B.; Rieder, F.; Albrich, S.; Wolf, K.; Bidlingmaier, C.;
Firestein, G. S.; Boyle, D.; Lehr, H. A.; Loher, F.; Hartmann, G.;
Endres, S.; Eigler, A. Adenosine Kinase Inhibitor GP515 Improves
Experimental Colitis in Mice. J. Pharmacol. Exp. Ther. 2001, 296,
99–105.
(30) Robak, T. Therapy of Chronic Lymphocytic Leukaemia with Purine
Nucleoside Analogues: Facts and Controversies. Drugs Aging 2005,
22, 983–1012.
(31) Sauter, C.; Lamanna, N.; Weiss, M. A. Pentostatin in Chronic
Lymphocytic Leukemia. Expert Opin. Drug Metab. Toxicol. 2008, 4,
1217–22.
(32) Wilson, D. K.; Rudolph, F. B.; Quiocho, F. A. Atomic Structure of
Adenosine Deaminase Complexed with a Transition State Analog:
Understanding Catalysis and Immunodeficiency Mutation. Science
1991, 252, 1278–1284.
(33) Wilson, D. K.; Quiocho, F. A. A Pre-Transition-State Mimic of an
Enzyme: X-ray Structure of Adenosine Deaminase with Bound
1-Deazaadenosine and Zinc Activated Water. Biochemistry 1993, 32,
1689–1694.
(46) Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.; Taylor,
R. D. Improved Protein-Ligand Docking Using GOLD. Proteins:
Struct., Funct., Genet. 2003, 52, 609–623.
(47) Eldridge, M. D.; Murray, C. W.; Auton, T. R.; Paolini, G. V.; Mee,
R. P. Empirical Scoring Functions: i. The Development of a Fast
Empirical Scoring Function To Estimate the Binding Affinity of
Ligands in Receptor Complexes. J. Comput.-Aided Mol. Des. 1997,
11, 425–445.
(48) Carosati, E.; Sciabola, S.; Cruciani, G. Hydrogen Bonding Interactions
of Covalently Bonded Fluorine Atoms: From Crystallographic Data
to a New Angular Function in the GRID Force Field. J. Med. Chem.
2004, 47, 5114–5125.
(49) Cheng, Y. C.; Prusoff, W. H. Relation Between Inhibition Constant
Ki and the Concentration of Inhibitor Which Causes Fifty Percent
Inhibition (IC50) of an Enzymatic Reaction. Biochem. Pharmacol. 1973,
22, 3099–3108.
(50) Nakase, H.; Okazaki, K.; Tabata, Y.; Uose, S.; Ohana, M.; Uchida,
K.; Nishi, T.; Debreceni, A.; Itoh, T.; Kawanami, C.; Iwano, M.; Ikada,
Y.; Chiba, T. An Oral Drug Delivery System Targeting Immune-
Regulating Cells Ameliorates Mucosal Injury in Trinitrobenzene
Sulfonic Acid-Induced Colitis. J. Pharmacol. Exp. Ther. 2001, 297,
1122–1128.
(51) Fornai, M.; Blandizzi, C.; Antonioli, L.; Colucci, R.; Bernardini, N.;
Segnani, C.; De Ponti, F.; Del Tacca, M. Differential Role of
Cyclooxygenase 1 and 2 Isoforms in the Modulation of Colonic
Neuromuscular Function in Experimental Inflammation. J. Pharmacol.
Exp. Ther. 2006, 317, 938–945.
(52) Marquez, E.; Sanchez-Fidalgo, S.; Calvo, J. R.; de la Lastra, C. A.;
Motilva, V. Acutely Administered Melatonin Is Beneficial While
Chronic Melatonin Treatment Aggravates the Evolution of TNBS-
Induced Colitis. J. Pineal Res. 2006, 40, 48–55.
(53) Molecular Operating EnVironment (MOE), version 2005.06; Chemical
Computing Group, Inc.: Montreal, Canada, 2005.
(54) Huang, C. C.; Couch, G. S.; Pettersen, E. F.; Ferrin, T. E. Chimera:
An Extensible Molecular Modelling Application Constructed Using
Standard Components. Pac. Symp. Biocomput. 1996, 1, 724. (http://
www.cgl.ucsf.edu/chimera).
(55) Case, D. A.; Darden, T. A.; Cheatham, T. E., III; Simmerling, C. L.;
Wang, J.; Duke, R. E.; Luo, R.; Merz, K. M.; Pearlman, D. A.;
Crowley, M.; Walker, R. C.; Zhang, W.; Wang, B.; Hayik, S.;
Roitberg, A.; Seabra, G.; Wong, K. F.; Paesani, F.; Wu, X.; Brozell,
S.; Tsui, V.; Gohlke, H.; Yang, L.; Tan, C.; Mongan, J.; Hornak, V.;
Cui, G.; Beroza, P.; Mathews, D. H.; Schafmeister, C.; Ross, W. S.;
Kollman, P. A. AMBER, version 9; University of California: San
Francisco, CA, 2006.
(56) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M.;
Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman,
P. A. A Second Generation Force Field for the Simulation of Proteins,
Nucleic Acids, and Organic Molecules. J. Am. Chem. Soc. 1995, 117,
5179–5197.
(57) Halgren, T. A. Merck Molecular Force Field. I. Basis, Form, Scope,
Parameterization, and Performance of MMFF94. J. Comput. Chem.
1996, 17, 490–512.
(34) Wang, Z.; Quiocho, F. A. Complexes of Adenosine Deaminase with
Two Potent Inhibitors: X-ray Structures in Four Independent
Molecules at pH of Maximum Activity. Biochemistry 1998, 37,
8314–8324.
(35) Kinoshita, T.; Nakanishi, I.; Terasaka, T.; Kuno, M.; Seki, N.;
Warizaya, M.; Matsumura, H.; Inoue, T.; Takano, K.; Adachi, H.;
Mori, Y.; Fujii, T. Structural Basis of Compound Recognition by
Adenosine Deaminase. Biochemistry 2005, 44, 10562–10569.
(36) Kinoshita, T.; Tada, T.; Nakanishi, I. Conformational Change of
Adenosine Deaminase during Ligand-Exchange in a Crystal. Biochem.
Biophys. Res. Commun. 2008, 373, 53–57.
(37) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.;
Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data Bank.
Nucleic Acids Res. 2000, 28, 235–242.
(58) Halgren, T. A. Merck Molecular Force Field. II. MMFF94 van der
Waals and Electrostatic Parameters for Intermolecular Interactions.
J. Comput. Chem. 1996, 17, 520–552.
(38) Kinoshita, T.; Nishio, N.; Sato, A.; Murata, M. Crystallization and
Preliminary Analysis of Bovine Adenosine Deaminase. Acta Crys-
tallogr. 1999, D55, 2031–2032.
(59) Halgren, T. A. Merck Molecular Force Field. III. Molecular Geometries
and Vibrational Parameters for Intermolecular Interactions. J. Comput.
Chem. 1996, 17, 553–586.
(39) Kinoshita, T.; Nishio, N.; Nakanishi, I.; Sato, A.; Fujii, T. Crystal
Structure of Bovine Adenosine Deaminase Complexed with 6-Hy-
droxyl-1,6-dihydropurine Riboside. Acta Crystallogr. 2003, D59, 299–
303.
(40) GOLD, version 4.0; CCDC Software Limited: Cambridge, U.K., 2008.
(41) Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R.
Development and Validation of a Genetic Algorithm for Flexible
Docking. J. Mol. Biol. 1997, 267, 727–748.
(60) Halgren, T. A. Merck Molecular Force Field. IV. Conformational
Energies and Geometries for MMFF94. J. Comput. Chem. 1996, 17,
587–615.
(61) Halgren, T. A. Merck Molecular Force Field. V. Extension of MMFF94
Using Experimental Data, Additional Computational Data, and
Empirical Rules. J. Comput. Chem. 1996, 17, 616–641.
(62) Lovell, S. C.; Word, J. M.; Richardson, J. S.; Richardson, D. C. The
Penultimate Rotamer Library. Proteins 2000, 40, 389–408.
(63) Pang, Y. P. Novel Zinc Protein Molecular Dynamics Simulations: Steps
Toward Antiangiogenesis for Cancer Treatment. J. Mol. Model. 1999,
5, 196–202.
(42) Schulz-Gasch, T.; Stahl, M. Binding Site Characteristics in Structure-
Based Virtual Screening: Evaluation of Current Docking Tools. J. Mol.
Model. 2003, 9, 47–57.
(43) Wang, R.; Lu, Y.; Wang, S. Comparative Evaluation of 11 Scoring
Functions for Molecular Docking. J. Med. Chem. 2003, 46, 2287–
2303.
(44) Kellenberger, E.; Rodrigo, J.; Muller, P.; Rognan, D. Comparative
Evaluation of Eight Docking Tools for Docking and Virtual
Screening Accuracy. Proteins: Struct., Funct., Bioinf. 2004, 57, 225–
242.
(45) Warre, G. L.; Andrews, C. W.; Capelli, A. M.; Clarke, B.; LaLonde,
J.; Lambert, M. H.; Lindvall, M.; Nevins, N.; Semus, S. F.; Senger,
S.; Tedesco, G.; Wall, I. D.; Woolven, J. M.; Peishoff, C. E.; Head,
M. S. A Critical Assessment of Docking Programs and Scoring
Functions. J. Med. Chem. 2006, 49, 5912–5931.
(64) Hoops, S. C.; Anderson, K. W.; Merz, K. M. J. Force Field Design
for Metalloproteins. J. Am. Chem. Soc. 1991, 113, 8262–8270.
(65) Ryde, U. Molecular Dynamics Simulations of Alcohol Dehydrogenase
with a Four- or Five-Coordinate Catalytic Zinc Ion. Proteins 1995,
21, 40–56.
(66) Lu, D. S.; Voth, G. A. Molecular Dynamics Simulations of Human
Carbonic Anhydrase II: Insight into Experimental Results and the Role
of Solvation. Proteins 1998, 33, 119–134.
(67) Berweger, C. D.; Thiel, W.; van Gunsteren, W. F. Molecular-
Dynamics Simulation of the Beta Domain of Metallothionein with
a Semiempirical Treatment of the Metal Core. Proteins 2000, 41,
299–315.

1692 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
La Motta et al.
(68) Pang, Y. P.; Xu, K.; El Yazal, J.; Prendergast, F. G. Successful
Molecular Dynamics Simulation of the Zinc-Bound Farnesyltrans-
ferase Using the Cationic Dummy Atom Approach. Protein Sci.
2000, 9, 1857–1865.
(69) El Yazal, J.; Pang, Y. P. Ab Initio Calculations of Proton Dissociation
Energies of Zinc Ligands: Hypothesis of Imidazolate as Zinc Ligand
in Proteins. J. Phys. Chem. B 1999, 103, 8773–8779.
(70) El Yazal, J.; Roe, R. R.; Pang, Y. P. Zinc’s Affect on Proton Transfer
between Imidazole and Acetate Predicted by ab Initio Calculations.
J. Phys. Chem. B 2000, 104, 6662–6667.
(71) El Yazal, J.; Pang, Y. P. Comparison of DFT, Moller-Plesset, and
Coupled Cluster Calculations of the Proton Dissociation Energies
of Imidazole and N-Methylacetamide in the Presence of Zinc(II).
J. Mol. Struct.: THEOCHEM 2001, 545, 271–274.
(72) Pang, Y. P. Successful Molecular Dynamics Simulation of Two Zinc
Complexes Bridged by a Hydroxide in Phosphotriesterase Using the
Cationic Dummy Atom Method. Proteins 2001, 45, 183–189.
JM801427R