Novel, highly potent adenosine deaminase inhibitors containing the pyrazolo[3,4-d]pyrimidine ring system. Synthesis, structure-activity relationships, and molecular modeling studies

Article abstract of DOI:10.1021/jm050136d

This study reports the synthesis of a number of 1- and 2-alkyl derivatives of the 4-aminopyrazolo[3,4-d]pyrimidine (APP) nucleus and their evaluation as inhibitors of ADA from bovine spleen. The 2-substituted aminopyrazolopyrimidines proved to be potent inhibitors, most of them exhibiting K_i values in the nanomolar/subnanomolar range. In this series the inhibitory activity is enhanced with the increase in length of the alkyl chain, reaching a maximum with the n-decyl substituent. Insertion of a 2′-hydroxy group in the ra-decyl chain gave 3k, whose (R)-isomer displayed the highest inhibitory potency of the series (K_i 0.053 nM), showing an activity 2 orders of magnitude higher than that of (+)-EHNA (K_i 1.14 nM), which was taken as the reference standard. Docking simulations of aminopyrazolopyrimidines into the ADA binding site were also performed, to rationalize the structure-activity relationships of this class of inhibitors.

Full text of DOI:10.1021/jm050136d

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5162
J. Med. Chem. 2005, 48, 5162-5174
Novel, Highly Potent Adenosine Deaminase Inhibitors Containing the
Pyrazolo[3,4-d]pyrimidine Ring System. Synthesis, Structure-Activity
Relationships, and Molecular Modeling Studies
Federico Da Settimo,† Giampaolo Primofiore,† Concettina La Motta,*,† Sabrina Taliani, Francesca Simorini,
Anna Maria Marini, Laura Mugnaini,† Antonio Lavecchia,*,§ Ettore Novellino,§ Daniela Tuscano,‡ and
Claudia Martini‡
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 Dipartimento
di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy
Received February 11, 2005
This study reports the synthesis of a number of 1- and 2-alkyl derivatives of the 4-amino-
pyrazolo[3,4-d]pyrimidine (APP) nucleus and their evaluation as inhibitors of ADA from bovine
spleen. The 2-substituted aminopyrazolopyrimidines proved to be potent inhibitors, most of
them exhibiting Ki values in the nanomolar/subnanomolar range. In this series the inhibitory
activity is enhanced with the increase in length of the alkyl chain, reaching a maximum with
the n-decyl substituent. Insertion of a 2′-hydroxy group in the n-decyl chain gave 3k, whose
(R)-isomer displayed the highest inhibitory potency of the series (Ki 0.053 nM), showing an
activity 2 orders of magnitude higher than that of (+)-EHNA (Ki 1.14 nM), which was taken
as the reference standard. Docking simulations of aminopyrazolopyrimidines into the ADA
binding site were also performed, to rationalize the structure-activity relationships of this
class of inhibitors.
Chart 1
Introduction
Adenosine deaminase (ADA, EC 3.5.4.4) is a catabolic
enzyme of purine metabolism that catalyzes the ir-
reversible deamination of adenosine and 2′-deoxy-
adenosine to inosine and 2′-deoxyinosine, respectively.
It is a ubiquitous enzyme which regulates the levels of
endogenous adenosine and, in addition, plays a central
role in the differentiation and maturation of the lym-
phoid system.1 ADA has been the object of considerable
interest mainly because its congenital defect in human
beings causes severe combined immunodeficiency
disease (SCID), in which both B-cell and T-cell de-
velopment is impaired. Actually, SCID is caused by a
high concentration of 2′-deoxyadenosine-5′-triphosphate
(dATP), which is lymphotoxic; consequently ADA inhibi-
tion may be useful for the selective treatment of lym-
phoproliferative malignancies.2-5 Indeed, the ability of
the two natural strong ADA inhibitors coformycin6
(CF, I) and 2′-deoxycoformycin7 (dCF, pentostatin, II,
Chart 1) to mimic ADA deficiency has been associated
with their potent antitumor properties, since they
should act as immunosuppressants to control cancers
of the hyperimmune system, such as leukemia and
lymphoma. ADA inhibition may also be used to prevent
deamination and subsequent deactivation of adenine-
containing chemotherapeutic agents such as 8-aza-
adenosine, arabinofuranosyladenine (ara-A), or formycin
A, thus potentiating their therapeutic activity.8,9 Fur-
thermore, as both cerebral and myocardial ischemia
result in the release of adenosine which appears to limit
the extent of degeneration, another potential use of ADA
inhibitors is that of protective agents in these events.10-13
Certainly, inhibition of ADA will inevitably be a
nonselective treatment, as it will stimulate all adenosine
receptor subtypes; thus, to minimize undesiderable side
effects, ADA inhibitors have to be selective for the
enzyme without interfering with adenosine receptors,
which could lead to the alteration of the physiology of
many organ systems. Due to this wide therapeutic
potential, there is a continuous interest in the develop-
ment of new ADA inhibitors.
* To whom all correspondence should be addressed. C.L.M.: Tel:
(+39)0502219547; Fax: (+)390502219605; E-mail: lamotta@
farm.unipi.it. A.L.V.: Tel and Fax: (+39)081678613; E-mail: lavecchi@
unina.it.
† Dipartimento di Scienze Farmaceutiche, Universita` di Pisa.
§ Dipartimento di Chimica Farmaceutica e Tossicologica, Universita`
“Federico II” di Napoli.
Potent ADA inhibitors may be divided into two
classes, which are called ground-state inhibitors if their
‡ Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Bio-
tecnologie, Universita` di Pisa.
10.1021/jm050136d CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/09/2005

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Adenosine Deaminase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5163
Scheme 1
structure is similar to that of the endogenous ADA
substrate adenosine, and transition-state inhibitors
when their structure resembles that of the tetrahedral
transition-state intermediate which forms during the
deamination process catalyzed by the enzyme. Erythro-
9-(2-hydroxy-3-nonyl)adenine14 (EHNA, III, Chart 1)
and 2′-deoxycoformycin (dCF, II, Chart 1) are the most
potent examples of the first and the second classes,
respectively. dCF is so tightly bound to the enzyme (Ki
2.5 × 10-3 nM) that its inhibitory action is nearly
irreversible. The toxicity observed with dCF therapy
seems to be principally due to the prolonged inhibition
of ADA, leading to immunosuppressive effects. On the
other hand, the rapid metabolism of EHNA allows a fast
recovery of the enzyme activity, with poor therapeutic
effects. Thus, it is believed that attractive inhibitors
would be analogues that are 1-2 orders of magnitude
more potent than EHNA.
the highest potency for ADA inhibition among this set
of compounds. The synthesis of both R and S isomers
of 3k was therefore undertaken, to investigate how their
potency is affected by the stereochemistry at the hy-
droxyl-bearing â-carbon, as occurs for the 9-(2-hydroxy-
3-nonyl) side chain of EHNA.
The crystal structure of a murine ADA complexed
with 6-hydroxy-1,6-dihydropurine ribonucleoside23
(HDPR, IV, Chart 1), a tight-binding transition-state
inhibitor, was determined at 2.4 Å resolution. However,
no similar data are available for a semi-tight-binding
inhibitor such as EHNA; neither the conformation nor
its binding site have been fully elucidated yet. Thus,
docking simulations of our very potent inhibitor 3e
together with (R)- and (S)-isomers of 3k, and EHNA,
were performed, both to rationalize the SARs observed
and to gain more information about the ADA catalytic
site.
These observations greatly stimulated our interest
in discovering EHNA analogues in which the adenine
base was substituted by the 4-aminopyrazolo[3,4-d]-
pyrimidine (APP) system bearing alkyl, arylalkyl, or
hydroxyalkyl chains in positions 1 or 2 (compounds
3a-l and 12a-f,h,i, respectively). Actually, the pyr-
azolo[3,4-d]pyrimidine nucleus proved to be a suitable
scaffold for adenosine antagonists and of inhibitors of
another purine metabolism enzyme, namely adenosine
kinase.15-20 Moreover, this scaffold, which offered the
possibility of substituting two different positions (1 and
2), appeared to be particularly versatile to investigate
the structural requirements of the ADA catalytic site.
To better delineate the structure-activity relationships
(SARs) of this new class of ADA inhibitors, the 6-phenyl
derivative 13 of the most potent compound 3e was also
prepared and tested, since it has been reported21 that a
2-phenyl group increases the binding of EHNA toward
ADA. Finally, to verify if also for this class of inhibitors,
as for 9-alkyladenines, substitution at the hexocyclic
amino group was detrimental for inhibitory activity,22
the N4-acetyl derivative 4 was synthesized and tested.
Chemistry
The synthesis of the N2-substituted pyrazolo[3,4-d]-
pyrimidines 3a-l and 4 was performed starting from
the commercially available 3-amino-4-pyrazolecarbo-
nitrile 1, as illustrated in Scheme 1. Alkylation of 1 with
the suitable reactants in DMF at 100 °C, in the presence
of K2CO3, gave the N1-alkylpyrazoles 2a-l as the main
reaction products. In particular, treatment of 1 with the
appropriate alkyl bromides led to compounds 2a-i,
reaction with 1,2-epoxyoctane, 1,2-epoxydecane, and (R)-
(+)- and (S)-(-)-1,2-epoxydecane provided the alcohols
2j, 2k, and (R)-2k and (S)-2k, respectively, and alkyl-
ation with 3-bromononan-2-one24 gave the pyrazole 2l,
which, after reduction with NaBH4 in methanolic solu-
tion at room temperature, afforded the intermediate 2m,
bearing the EHNA side chain.
Cyclization of 2a-k,m with boiling formamide pro-
vided the desired inhibitors 3a-l. The acetylamino
derivative 4 was obtained with a good yield upon
treatment of a pyridine solution of 3e with acetic
anhydride at 100 °C. The (S)-(-)-1,2-epoxydecane used
to obtain enantiomerically pure (S)-3k was prepared
from the commercially available (R)-(+)-1,2-decanediole
5 in the four-step sequence outlined in Scheme 2, which
involved an inversion at C2. Treatment of 5 with tert-
butyldimethylsilyl chloride in anhydrous THF, in the
As several products of this study presented a chiral
center (3j, 3k, 3l, 12f, and 12h), a preliminary biological
evaluation was conducted on their stereoisomeric mix-
tures, to identify the racemate with the best ability to
inhibit the enzyme. Derivative 3k (Ki 0.28 nM) showed

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5164 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Da Settimo et al.
Table 1. Physical Properties of 3-Amino-4-pyrazolecarbonitrile Derivatives 2a-m
no.
R
yield (%)
recryst solv
mp (°C)
formulaa
2a
(CH2)5CH3
(CH2)6CH3
(CH2)7CH3
(CH2)8CH3
(CH2)9CH3
(CH2)10CH3
(CH2)11CH3
CH2C6H5
44
52
54
56
49
41
57
70
80
70
50
60
41
88
45
CHCl3
119-120
96-97
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
10H16N4
11H18N4
12H20N4
13H22N4
14H24N4
15H26N4
16H28N4
11H10N4
12H12N4
12H20N4O
14H24N4O
14H24N4O
14H24N4O
13H20N4O
13H22N4O
2b
CH2Cl2
2c
n-hexane
n-heptane
cyclohexane
cyclohexane
i-PrOH
84-85
2d
88-89
2e
60-62b
148-151
108-111
121-123c
102-103d
133-135
130
2f
2g
2h
AcOEt/cyclohexane
AcOEt/cyclohexane
EtOH
2i
CH2CH2C6H5
2j
CH2CHOH(CH2)5CH3
CH2CHOH(CH2)7CH3
CH2CHOH(CH2)7CH3
CH2CHOH(CH2)7CH3
CHCOCH3(CH2)5CH3
2k
EtOH
e
f
(R)-2k
(S)-2k
2l
EtOH
124-126
124-126
87-89
EtOH
cyclohexane
i-PrOH
2m
CH(CHOHCH3)(CH2)5CH3
108-111
a Elemental analyses for C, H, N were within (0.4% of the calculated values. b Lett.51 mp 69 °C. c Lett.51 mp 132-134 °C. d Lett.52 mp
98-100 °C. e [R]18 +120 (CH3OH). f [R]18 -119 (CH3OH).
Table 2. Physical Properties of Pyrazolo[3,4-d]pyrimidine Derivatives 3a-l, 4, and 8
no.
3a
3b
3c
3d
3e
3f
R1
R2
R3
yield (%)
recryst solv
mp (°C)
formulaa
H
H
H
H
H
H
H
H
H
H
H
H
H
H
(CH2)5CH3
(CH2)6CH3
(CH2)7CH3
(CH2)8CH3
(CH2)9CH3
(CH2)10CH3
(CH2)11CH3
CH2C6H5
H
84
32
38
26
25
30
44
75
78
70
64
40
34
45
73
80
CHCl3
215-217
205-208
208-210
200-202
196-198
192-195
168-170
248-250
188-190
213-216
214-216
206-208
206-208
192-194
90-92
C
11H17N5
12H19N5
13H21N5
14H23N5
15H25N5
16H27N5
17H29N5
12H11N5
13H13N5
13H21N5O
15H25N5O
15H25N5O
15H25N5O
14H23N5O
17H27N5O
15H16N4O
H
AcOEt
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
H
AcOEt
H
AcOEt
H
AcOEt
H
AcOEt
3g
3h
3i
H
AcOEt
H
AcOEt/cyclohexane
AcOEt/cyclohexane
i-PrOH
CH2CH2C6H5
H
3j
CH2CHOH(CH2)5CH3
CH2CHOH(CH2)7CH3
CH2CHOH(CH2)7CH3
CH2CHOH(CH2)7CH3
H
3k
(R)-3k
(S)-3k
3l
H
EtOH
b
c
H
EtOH
H
EtOH
CH(CHOHCH3)(CH2)5CH3
(CH2)9CH3
H
EtOH
4
8
COCH3
H
H
cyclohexane
toluene
(CH2)9CH3
C6H5
133
a Elemental analyses for C, H, N were within (0.4% of the calculated values. b [R]18 +51 (CH3OH). c [R]18 -50 (CH3OH).
Scheme 2
presence of imidazole, gave the C1-protected intermedi-
ate 6, which was converted to mesylate 7 by reaction
with methanesulfonyl chloride in anhydrous pyridine
at 0 °C. Deprotection of 7 with tetrabutylammonium
fluoride afforded the primary alcohol 8, which provided
the desired (S)-(-)-1,2-epoxydecane 9 by reaction with
potassium tert-butoxide in MeOH.
3-bromononan-2-one24 led to the keto derivative 12g,
which was converted to the desired 12h by reduction
with NaBH4 in methanolic solution at room tempera-
ture. Ring closure of 5-amino-4-cyano-1-(â-hydroxy-
ethyl)pyrazole 1026 with boiling formamide provided
compound 12i.
The synthetic route for compound 13 is shown in
Scheme 4. A number of procedures have been described
for the conversion of 3-amino-4-pyrazolecarbonitrile to
6-substituted pyrazolo[3,4-d]pyrimidines.27,28 Since most
of them require harsh conditions and proceed with poor
yields, a new method for the synthesis of the candidate
13 was devised by us, which greatly reduced the reaction
time and enhanced the yield of the product. This
solventless method involved the mixing of 3-amino-1-
decyl-4-pyrazolecarbonitrile 2e and benzonitrile with
The isomeric series of N1-substituted pyrazolo[3,4-d]-
pyrimidines 12a-i were prepared as illustrated in
Scheme 3. 4-Aminopyrazolo[3,4-d]pyrimidine 11 was
obtained from 1 with boiling formamide, following a
previously reported procedure.25 The sodium salt of 11
was then alkylated with the suitable reactant in DMF
solution at 100 °C to give the target inhibitors 12. The
appropriate alkyl bromide gave products 12a-e, 1,2-
epoxyoctane afforded the hydroxy derivative 12f, and

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Adenosine Deaminase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5165
Scheme 3
Table 3. Physical Properties of 4-Amino-1-alkylpyrazolo[3,4-d]pyrimidine Derivatives 7a-i
no.
R
yield (%)
recryst solv
mp (°C)
formulaa
12a
12b
12c
12d
12e
12f
12g
12h
12i
(CH2)9CH3
(CH2)10CH3
(CH2)11CH3
CH2C6H5
25
24
29
30
35
29
40
60
96
AcOEt
AcOEt
AcOEt
i-PrOH
AcOEt
EtOH
-
187-189
159-161
146-148
238b
C
15H25N5
16H27N5
17H29N5
12H11N5
13H13N5
13H21N5O
14H21N5O
14H23N5O
C
C
C
C
C
C
C
CH2CH2C6H5
185
CH2CHOH(CH2)5CH3
CH(COCH3)(CH2)5CH3
CH(CHOHCH3)(CH2)5CH3
CH2CH2OH
146-148
oil
-
oil
toluene
217-219c
C7H9N5O
a Elemental analyses for C, H, N were within (0.4% of the calculated values. b Lett.53 mp 235-236 °C. c Lett.54 mp 223-224 °C.
Scheme 4
nanomolar range. It should be noted that the inhibitory
activity in the 2-substituted aminopyrazolopyrimidine
series is enhanced with the increasing of the length of
the side alkyl chain (3a < 3b < 3c < 3d < 3e ≈ 3f),
reaching its maximum with the n-decyl and the n-
undecyl chains of 3e and 3f showing Ki values of 0.13
nM and 0.47 nM, respectively, 1 order of magnitude
lower than that of (+)-EHNA (1.14 nM). Further in-
crease in the length of the side chain was dramatically
potassium tert-butoxide in an open glass tube, and
irradiation of the reaction mixture in a household
microwave oven for 3 min. The crude product was
readily purified to afford the desired compound 13 with
a high yield (80%).
detrimental for activity (compound 3g, Ki
> 10000 nM).
Insertion of a 2′-hydroxy group into the n-octyl chain
of 3c (Ki 530 nM) led to compound 3j whose inhibitory
activity was 3 orders of magnitude higher than that of
the parent compound. The remarkable beneficial effect
of the 2′-hydroxy group in 3j may be related to the
formation of an additional H-bond with the enzyme.
The â-hydroxy substitution on the n-decyl chain of the
potent inhibitor 3e gave compound 3k, which proved
to be highly potent, like its parent derivative (Ki 0.28
nM). As it is well-known that the ADA inhibitory
potency of EHNA depends on the stereochemistry of its
side chain, the two enantiomers of 3k were prepared
and tested. The (R)-3k isomer (Ki 0.053 nM) proved to
be the most active compound of the whole series, more
Results and Discussion
Biological Evaluation. The Ki values for inhibition
of bovine spleen ADA by aminopyrazolopyrimidine
derivatives 3a-l, 4, 12a-f,h,i, and 13 are presented
in Table 4. (+)-EHNA was used as the reference
standard.
Results showed that the 2-substituted aminopyrazolo-
pyrimidine derivatives 3 were in general more potent
than their 1-substituted analogues 12, with compounds
3d-f,j,k exhibiting Ki values in the nanomolar/sub-