Structure-based design and synthesis of non-nucleoside, potent, and orally bioavailable adenosine deaminase inhibitors

Article abstract of DOI:10.1021/jm0499559

We disclose optimization efforts based on the novel non-nucleoside adenosine deaminase (ADA) inhibitor, 4 (K_i = 680 nM). Structure-based drug design utilizing the crystal structure of the 4/ADA complex led to discovery of 5 (K_i = 11

Full text of DOI:10.1021/jm0499559

2728
J . Med. Chem. 2004, 47, 2728-2731
Str u ctu r e-Ba sed Design a n d Syn th esis of
Non -Nu cleosid e, P oten t, a n d Or a lly
Bioa va ila ble Ad en osin e Dea m in a se
In h ibitor s
Tadashi Terasaka,*,† Hiroyuki Okumura,†
Kiyoshi Tsuji,† Takeshi Kato,† Isao Nakanishi,‡,#
Takayoshi Kinoshita,‡ Yasuko Kato,§ Masako Kuno,§
Nobuo Seki,§ Yoshinori Naoe,§ Takeshi Inoue,§
Kohichiro Tanaka,| and Katsuya Nakamura†
F igu r e 1. Chemical structures of adenosine and known ADA
inhibitors.
Medicinal Chemistry Research Laboratories, Basic Research
Laboratories, Medicinal Biology Research Laboratories, and
Biopharmaceutical and Pharmacokinetic Research
Laboratories, Fujisawa Pharmaceutical Co., Ltd.,
A number of ADA inhibitors have been reported in
the literature. However, almost all are purine nucleoside
or alkyladenine analogues, for example, (+)-erythro-9-
(2-hydroxy-3-nonyl)adenine ((+)-EHNA) (ground-state
inhibitor),11 pentostatin (transition-state inhibitor),12
and various derivatives.13 As a result, they have many
problems, such as poor pharmacokinetics14 and/or sev-
eral toxicities.15 Because of these problems, pentostatin
is the only ADA inhibitor in clinical use; however, it is
limited to the treatment of adult patients with hairy
cell leukemia and is only available via intravenous
administration.16
2-1-6, Kashima, Yodogawa-ku, Osaka 532-8514, J apan
Received J anuary 15, 2004
Abstr a ct: We disclose optimization efforts based on the novel
non-nucleoside adenosine deaminase (ADA) inhibitor, 4 (Ki )
680 nM). Structure-based drug design utilizing the crystal
structure of the 4/ADA complex led to discovery of 5 (Ki ) 11
nM, BA ) 30% in rats). Furthermore, from metabolic consid-
erations, we discovered two inhibitors with improved oral
bioavailability [6 (Ki ) 13 nM, BA ) 44%) and 7 (Ki ) 9.8 nM,
BA ) 42%)]. 6 demonstrated in vivo efficacy in models of
inflammation and lymphoma.
We considered that ADA inhibitors with reduced
toxicity and oral bioavailability would be expected to not
only improve the treatment of leukemia but also have
potential use as anti-inflammatory drugs. We specu-
lated that the problems of the known inhibitors could
be improved by changing the nucleoside framework to
a non-nucleoside framework. Despite the difficulty of
converting a nucleoside to a non-nucleoside, a recent
report from our laboratories described the discovery of
a novel, highly potent non-nucleoside ADA inhibitor 3
(Ki ) 7.7 nM, human ADA) by intentional hybridization
of the structurally distinct lead 1 (Ki ) 5.9 µM) and 2
(Ki ) 1.2 µM) by only two structure-based drug design
(SBDD) iterations (Figure 1).17,18 Further study of 3,
however, revealed its poor solubility and absorption
(Table 1). On the other hand, in the first step of our
hybridization process of 1 and 2, 4 was designed (Figure
2). Compound 4 is still relatively weak in terms of ADA
inhibitory activity (Ki ) 680 nM) but was considered as
a good lead compound for optimization because of its
simple, non-nucleoside framework and low molecular
weight (MW), and moreover, the sum of hydrogen bond
acceptors (Ha) and donors (Hd) is low compared to that
of 3 (MW and Ha/Hd for 3 and 4 are 499.57 and 309.37,
and 10/4 and 5/3, respectively) (Lipinski’s rule19). There-
fore, we attempted to optimize 4 based on a crystal
structure of the 4/ADA complex. In this paper, we
disclose these optimization efforts and the discovery of
a potent and orally bioavailable ADA inhibitor by a
structure-based and metabolism-directed design ap-
proach.
Adenosine deaminase (ADA) (EC. 3.5.4.4) is a key
enzyme in purine metabolism and catalyzes the ir-
reversible deamination of adenosine and 2′-deoxy-
adenosine to inosine and 2′-deoxyinosine, respectively.1
ADA is ubiquitous in almost all human tissues and is
assumed to play a crucial role in the development of the
immune system because genetic ADA deficiency results
in severe combined immunodeficiency disease by im-
pairment of the differentiation and maturation of lym-
phoid cells.2 ADA abnormalities have also been reported
in a variety of other diseases,3 such as rheumatoid
arthritis4 and leukemia.5 In recent years, adenosine has
come to be considered as an important factor in the
attenuation of inflammation,6,7 since it has been re-
ported that the concentration of adenosine is increased
in inflammatory lesions.8 Furthermore, it is also be-
lieved that ecto-ADA has an extra enzymatic function
via binding with CD26 on the surface of activated
lymphocytes9 and could perpetuate chronic inflamma-
tion by metabolizing adenosine released at inflamed
sites that are toxic for lymphocytes.10 Therefore, it is
considered that an ADA inhibitor may prevent adeno-
sine released specifically at inflamed sites from metabo-
lism by ecto-ADA and has great potential as an anti-
inflammatory drug with few side effects.
* To whom correspondence should be addressed. Phone: +81-6-
6390-1286. Fax: +81-6-6304-5435. E-mail: tadashi_terasaka@
po.fujisawa.co.jp.
Compound 4 was originally designed by introducing
a hindered planar naphthyl ring in place of the phenyl
ring of 1 in order to rotate the phenyl face of 1 and fit
the narrow planar hydrophobic space (F1), which is
occupied by the thiazole and thiophene rings of 2.18 The
crystal structure of the 4/ADA complex confirmed our
† Medicinal Chemistry Research Laboratories.
‡ Basic Research Laboratories.
# Current address: Department of Theoretical Drug Design, Gradu-
ate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku,
Kyoto 606-8501, J apan.
§ Medicinal Biology Research Laboratories.
| Biopharmaceutical and Pharmacokinetic Research Laboratories.
10.1021/jm0499559 CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/14/2004

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2729
Ta ble 1. In Vitro Clearance with Liver Microsomes of Rat, Dog, and Human and Pharmacokinetic Parameters of 3 and 5-7 after po
Administration to Rats and Dogs (10 mg/kg, n ) 2-3)
b
in vitro clearance (mL min-1 kg-1
)
pharmacokinetic parameters in ratsb
BA (%)c
solubility
Ki (nM)a
(µg/mL)b
rat
dog
human
Cmax (µg/mL)
t1/2 (h)
AUC0-24h (µg‚h/mL)
rat
dog
3
5
6
7
7.7
11
13
4.2
481
500
80
35
307
111
79
97
30
21
11
123
45
4.5
3.8
<0.1
1.25
1.63
0.97
ndd
0.66
1.66
1.12
<0.1
1.37
2.11
1.85
ndd
30
44
ndd
38
43
9.8
42
91
a
b
Ki values were measured with human ADA. Assays were performed in duplicate. See Supporting Information. c BA ) bioavailability.
d
nd ) not determined.
hypothesized (Figure 2): oxidation of the naphthyl ring
and the benzylic position. Assuming that metabolism
is the major route of clearance, chemical modification
may lead to compounds with improved pharmacokinet-
ics. In consideration of the narrow planar hydrophobic
space available at F1 and protection of the designed
compound from metabolism, a 2,3-dichlorophenyl ring
was introduced in place of the naphthyl ring of 5. The
resulting 6 showed potent inhibitory activity (Ki ) 13
nM) similar to that of 5 (Figure 2). According to the
binding mode of the 1/ADA complex, 1 does not utilize
the F1 space in binding, and moreover, the F1 space
and the phenyl face of 1 are in a perpendicular relation-
ship.18 However, the crystal structure of the 6/ADA
complex confirmed that the 2,3-dichlorophenyl ring was
indeed bound to the F1 space as predicted, and the other
parts showed the same binding mode as 5 (Figure 3e).
This observation suggests that replacement of the
naphthyl ring of 5 with a 2,3-dichlorophenyl ring is
effective in terms of ADA inhibitory potency.
F igu r e 2. Design process for discovery of 5-7.
prediction (Figure 3a) and revealed that the imidazole-
carboxamide group of 4 bound at the bottom of the
catalytic site interacting with ADA through direct
hydrogen bonds with Asp296 and via three water
molecules and the hydroxyl group of 4 formed hydrogen
bonds with His17 and Asp19 (Figure 3b). Furthermore,
although 4 is racemic, only the (R)-stereoisomer was
observed in the complex crystal as predicted by molec-
ular modeling. Therefore, further studies were per-
formed on the basis of the (R)-stereoisomer of 4.
On the other hand, according to the crystal structures
of the 4/ADA and 5/ADA complexes, the naphthyl
moiety is located at the entrance position of the enzyme
and does not utilize the F1 space sufficiently (Figure
3a,e). That is, the hydrophobic space that is occupied
by the thiophene ring of 2 is not utilized to bind these
inhibitors. Therefore, we planned to use this space and
block metabolism of the benzyl position in order to
enhance activity and improve pharmacokinetics. Ether-
linked analogues of 5, which are substituted at the 1-
or 2-position of the naphthyl ring, were considered
(Table 2). Molecular modeling studies with ADA were
performed to determine the best link position for the
ether-linked compounds. Table 2 shows the simulated
interaction and internal energies of the designed com-
pounds. A 2-substituent was shown to be more stable
than a 1-substituent because a 2-substituent has a
higher interaction energy (∆Einteraction ) 1.6 kcal/mol)
but much lower conformational strain energy (∆Estrain
) -6.3 kcal/mol) than a 1-substituent in order to fit to
the active site. Certainly, it appeared from binding
modes with ADA that the naphthyl moiety of a 1-sub-
stituted analogue is strained in order to fit to the active
site without clashes with the enzyme wall, but the
naphthyl ring of a 2-substituted compound utilizes the
inner space of the active site in a good manner (Figure
3f,g). Therefore, we selected 2-substituted ether-linked
7 and only 7 was actually synthesized and evaluated.
Consequently, 7 showed potent inhibitory activity simi-
lar to 5 (Figure 2). The crystal structure of the 7/ADA
complex revealed that although the 2-naphthyloxy
group shifted to the active-site entrance compared to
the simulation, the other parts showed the same binding
mode as 5 (Figure 3h).
On the other hand, EHNA was designed by Schaeffer
on the basis of studies on a large number of 9-substi-
tuted adenines 30 years ago20 and further SAR studies
about chirality and activity of various EHNA isomers
have been reported to date.11 Since the crystal structure
of an EHNA/ADA complex has never been reported, the
binding mode is not clear. However, a molecular model-
ing simulation suggested that the binding positions of
the adenine and the hydroxyl group of EHNA were the
same as those of the imidazolecarboxamide and the
hydroxyl group of 4, respectively. Moreover, (+)-EHNA
has a (S)-methyl group at the R position of the hydroxyl
group. On the basis of the comparison described above,
careful analysis of the interaction of 4 with the active
site of ADA (Figure 3c) led to the prediction that a
hydrophobic cavity exists and suggested introduction of
a (S)-methyl group at the R position of the hydroxyl
group of 4, the same as in EHNA. Therefore, to enhance
the activity of 4, we synthesized the compound with a
(S)-methyl group at the R position to the hydroxyl group,
the (2S,3R)-isomer 5 (Figure 2). As a result, 5 showed
high potency (Ki ) 11 nM) for ADA inhibition and was
60-fold more potent than 4. This observation indicates
that introducing a (S)-methyl group at the R position
to the hydroxyl group is effective to enhance activity.
The crystal structure of the 5/ADA complex verified that
the (S)-methyl group was bound to the small hydropho-
bic cavity as predicted, and the other parts of 5 showed
the same binding mode as 4 (Figure 3a-e).
Further metabolic study of 5 suggested formation of
two principal metabolites that were monooxygenated
derivatives of 5. On the basis of its chemical structure,
the two principal sites of metabolism of 5 could be

2730 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
Letters
F igu r e 3. Binding mode of inhibitors at the ADA active site. Accessible surfaces of the carbon atoms at the active sites are
shown by the mesh. The upper portion of each figure is active site entrance (solvent region), and other nonmeshed black regions
are occupied by protein: (a) binding orientations of 4 and 2 superimposed onto the active site surface of the 4/ADA complex; (b)
hydrogen bond networks around the carboxamide moieties of 4 (hydrogen bonds are shown by dashed lines); (c) view from the
side by 90° of (a); (d) binding orientation of 5 (ball-and-stick)/ADA complex (PDB code, 1V78); (e) binding orientations of 6 and 5
superimposed onto the active site surface of the 5/ADA complex; (f, g) simulated binding mode of ether-linked analogues of 5 to
the active site of 5/ADA complex; (h) binding orientations of 7 and 5 superimposed onto the active site surface of the 5/ADA
complex.
Ta ble 2. Simulated Interaction Energies for Ether-Inserted
Analogues of 5
Ta ble 3. Comparative Antitumor Activity of 6 and 7 and
Pentostatin on U937 Lymphoma Model
drug
control
dose (mg/kg)b
MST (day)
22.0
23.5
34.0
42.5
38.0
30.0
35.5
30.0
28.5
34.0
42.5
AraA
10
6a (po)
10 (b.i.d.)
32 (b.i.d.)
32 (u.i.d.)
10 (b.i.d.)
32 (b.i.d.)
32 (u.i.d)
0.025
Einteraction (kcal/mol)a
Estrain (kcal/mol)b
-93.4
14.0
-91.8
7.7
7 (po)
a
b
Calculated interaction energies. Differential of calculated
internal energies.
pentostatin (ip)
0.25
2.5
Because the primary objective of this study was
identification of ADA inhibitors with superior pharmaco-
kinetics, 5-7 with potency similar to that of 3 were
directly subjected to several pharmacokinetic experi-
ments (Table 1). All three analogues displayed 20- to
120-fold better solubility than 3, and upon oral dosing
of 5 (10 mg/kg), a 1.25 µg/mL maximum plasma con-
centration (Cmax) and a 0.66 h plasma half-life (t1/2) were
achieved (bioavailability BA ) 30%), despite its high in
vitro clearance in rat liver microsomes. In contrast, no
detectable serum levels were observed with 3. Com-
pounds 6 and 7, which were discovered by metabolism-
directed design from 5, displayed improved metabolic
stability, since in vitro clearance was improved com-
pared to 5, especially, in dog and human liver mi-
crosomes. As anticipated from the metabolism study, 6
and 7 also exceeded 5 in most pharmacokinetic param-
eters, as shown in Table 1. Upon dosing rats with 6 (10
mg/kg), a substantial improvement of Cmax (1.63 µg/mL),
AUC (2.11 µg‚h/mL), and t1/2 (1.66 h) were observed (BA
) 44%). Dosing rats with 7 (10 mg/kg) led to a reason-
able Cmax (0.97 µg/mL) and some improvement of AUC
(1.85 µg‚h/mL) and better t1/2 (1.12 h) (BA ) 42%). In
particular, 7 displayed excellent oral bioavailability in
dogs (BA ) 91%).
a
b
HCl salt. b.i.d. ) twice daily dosing. u.i.d. ) once daily
dosing.
inoculated with U937 cells were unhealthy in appear-
ance and had swollen abdomens. The median survival
time (MST) was 22.0 days. Oral administration (po) of
6 at 32 mg/kg u.i.d. and b.i.d. with the antitumor 9-â-
D-arabinofuranosyladenine (AraA) resulted in improved
survival with MST of 38.0 and 42.5 days, respectively.
Mice treated with 7 at 10 and 32 mg/kg survived
significantly longer (MST of 30-35.5 days) than control
mice. Pentostatin prolonged the life of mice bearing
U937 in a dose-dependent manner. The MST of pento-
statin (2.5 mg/kg) with AraA was 42.5 days. Compound
6 had antitumor activity against lymphoma in combina-
tion with AraA because it prolonged the survival time
of SCID mice inoculated with U937 cells and exhibited
oral activity comparable to that observed for intra-
peritoneal administration (ip) of pentostatin.
Furthermore, in the adjuvant arthritis model in rats,
6 also showed an anti-inflammatory effect orally (ED30
) 1.6 mg/kg po). This is the first report of in vivo anti-
inflammatory efficacy for an ADA inhibitor after oral
administration.
The syntheses of 5-7 are outlined in Scheme 1. The
reaction of epoxide 822 with the suitable aryl Grignard
reagent, prepared in situ, and separation of the dia-
Compounds 6 and 7 were next evaluated in in vivo
models of inflammation and lymphoma. The in vivo
antitumor activity of 6, 7, and pentostatin on lymphoma
was evaluated in a SCID mouse model21 (Table 3). Mice

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2731
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displacement by SN2 reaction with 4-imidazolecarbox-
amide in the presence of NaH in DMF afforded 10.
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the corresponding 10.
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in Et2O at 0 °C gave the primary alcohol 12. Removal
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ADA inhibitors by a structure-based and metabolism-
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J M0499559