Synthetic studies on selective adenosine A2A receptor antagonists. Part II: Synthesis and structure-activity relationships of novel benzofuran derivatives

Article abstract of DOI:10.1016/j.bmcl.2010.04.058

Based on the previously reported lead compound, a series of benzofuran derivatives were prepared to study their antagonistic activities to A _2A receptor. The replacement of the phenyl group at the 4-position with a heterocyclic ring improved th

Full text of DOI:10.1016/j.bmcl.2010.04.058

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3768–3771
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Bioorganic & Medicinal Chemistry Letters
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Synthetic studies on selective adenosine A2A receptor antagonists. Part II:
Synthesis and structure–activity relationships of novel benzofuran derivatives
*
Osamu Saku , Mayumi Saki, Masako Kurokawa, Ken Ikeda, Shin-ichi Uchida, Takuya Takizawa,
Noriaki Uesaka
Fuji Research Park, Kyowa Hakko Kirin Co., Ltd, 1188 Shimotogari, Nagaizumi-cho, Suntou-gun, Shizuoka 411-8731, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the previously reported lead compound, a series of benzofuran derivatives were prepared to
study their antagonistic activities to A2A receptor. The replacement of the phenyl group at the 4-position
with a heterocyclic ring improved the PK profile and aqueous solubility. From these studies, we discov-
ered a potent new A2A antagonist, 12a, which has both a good oral bioavailability and in vivo efficacy on
motor disability in MPTP-treated common marmosets.
Received 4 March 2010
Revised 8 April 2010
Accepted 15 April 2010
Available online 18 April 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Adenosine
A2A
A2A Receptor
A2A Antagonist
A2A Receptor antagonist
Parkinson’s disease
Adenosine receptors have been extensively characterized and
are divided into four different subtypes (A1, A2A, A2B, and A3).1
These four receptors play central roles in a variety of biological re-
sponses that could be exploited for a number of clinical applica-
tions. Of the four adenosine receptor subtypes, the A2A receptor
is the focus of intense research within the neurological field, partic-
ularly in relation to the hypoactive and hyperactive movement dis-
orders.2 Our own interest in this area stemmed from the potential
benefit of adenosine A2A antagonists in the central nervous system
(CNS) area and, specifically, for the treatment of basal ganglia dis-
orders such as Parkinson’s disease (PD).3 PD is a very serious neu-
rological disorder which involves the chronic and progressive
degeneration of the brain and impairs motor control, speech, and
Some xanthine and non-xanthine compounds have been found
to have high A2A affinity with varying degrees of A2A versus A1 selec-
tivity.7 We have previously reported the identification of new A2A
antagonists with a benzofuran skeleton.8 In an effort to understand
this novel class of A2A antagonists, we have investigated the effect
of chemically modifying the 2- and 4-positions of the molecule on
compound potency and pharmacokinetic parameters. It became
apparent that reducing the intrinsic lipophilicity of the compounds
was desirable for more potent compounds. In the current study, a
variety of six-membered heterocyclic moieties with one or two
nitrogen atoms in the ring were incorporated to investigate their
contribution to various pharmacokinetic parameters. In most cases,
the introduction of heterocyclic moieties improved the metabolic
stability and aqueous solubility, thus resulting in the discovery of
a potent new A2A antagonist, 12a, which demonstrated good oral
bioavailability and in vivo efficacy on motor disability in MPTP-
treated common marmosets.
Scheme 1 describes the preparation of 4-substituted benzofu-
rans by reactions involving the bromo-functionalized analogue 3.
Stille coupling of 3 with various tributylstannyl pyrazines and pyr-
idines gave the arylated analogues 4a–g. Hydrolysis of the esters
followed by amidation of the resulting carboxylic acids yielded
the desired benzofurans 6a–g.
4-Morpholinobenzofurans were prepared as shown in Scheme
2. The nitration of 2-ethoxycarbonyl-7-methoxybenzofuran 7 with
nitric acid in acetic anhydride gave the isomeric mixture of 8 and
13, which was separated by recrystallization from methanol and
other functions. L-Dopa has long been the gold standard of therapy
for Parkinson’s disease, but its long-term shortcomings are motor
response complications such as dyskinesias, end-of-dose deteriora-
tion or wearing-off, and the fluctuating regulation of motor symp-
toms.4 The selective antagonism of the A2A receptor in a primate
model of PD can ameliorate motor depression.5 Phase III clinical
studies of KW-6002 (Istradefylline) in PD patients with L-dopa-re-
lated motor complications yielded promising results with regard to
motor symptom relief without motor side effects.6 Selective A2A
antagonists should provide a novel nondopaminergic approach to
PD therapy.
* Corresponding author. Tel.: +81 55 989 2014; fax: +81 55 986 7430.
E-mail address: osamu.saku@kyowa-kirin.co.jp (O. Saku).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.058

O. Saku et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3768–3771
3769
Scheme 4. Reagents and conditions: (a) concd HNO3, Ac2O; (b) H2, Pd/C, EtOH;
(c) bis(bromoethyl)ether, iPr2NEt, toluene; (d) LiOHÁH2O, THF, H2O; (e) 4-amino-
pyridine, WSCÁHCl, HOBtÁH2O, Et3N, DMF.
modifications of the amide moiety showed enhanced A2A antago-
nistic activity. The reduction of the intrinsic lipophilicity of the
compounds was performed by an alternative strategy to further
improve the metabolic stability by human liver microsomes. A
study for the initial set of compounds was carried out to determine
the effect of replacing the phenyl group at 4-position with either
pyridine or pyrazine. Changes to the heteroaryl substituents at
the 4-position influenced potency, as demonstrated by the assay
data summarized in Table 1. The 2-pyridine analogue 6a and pyr-
azine analogues 6b and 6c were significantly less potent than the
corresponding previously reported phenyl analogue 19,8 thus sug-
gesting an unfavorable interaction with the nitrogen in the ring.
However, the introduction of 3-pyridyl (6d, 6e) and 4-pyridyl (6f,
6g) groups to the same position provided analogues that were
roughly equipotent to 19. In addition, substitutions of the phenyl
group at the amide moiety with (1H-imidazol-1-yl)methyl group
were beneficial for the antagonistic activity, regardless of the
substituent at 4-position (6d vs 6e and 6f vs 6g). The 4-phen-
ylbenzofuran derivative 19 showed a poor pharmacokinetic profile
and exhibited a high clearance rate. This resulted in low bioavail-
ability, which was possibly due to a high first-pass metabolism.
On the other hand, changing the phenyl group at 4-positon to pyr-
idyl and pyrazyl groups significantly improved the pharmacoki-
netic profile. On the basis of potent in vitro activities at A2A and
good pharmacokinetic profiles, compounds 6d–g were evaluated
in the mouse CGS 21680-induced catalepsy model,9 which is com-
monly used to assess the activity of compounds for anti-parkinso-
nian effects. Despite their improvement in clearance rates over the
previous compound 19, these new compounds still had a moderate
efficiency when dosed at 10 mg/kg in this model, and therefore
that there remains significant room for improvement. Next, we
compared some of the physicochemical properties for the potent
compounds in the series. The data indicated that the aqueous sol-
Scheme 1. Reagents and conditions: (a) Br2, AcOH; (b) ethyl chloroacetate, K2CO3,
KI, DMF; (c) R1SnBu3, Pd(PPh3)4, toluene; (d) LiOHÁH2O, THF, H2O; (e) R2NH2,
WSCÁHCl, HOBtÁH2O, Et3N, DMF.
Scheme 2. Reagents and conditions: (a) concd HNO3, Ac2O; (b) H2, Pd/C, EtOH; (c)
bis(bromoethyl)ether, iPr2NEt, toluene; (d) LiOHÁH2O, THF, H2O; (e) RNH2, WSCÁHCl,
HOBtÁH2O, Et3N, DMF.
chloroform to yield 4-nitrobenzofuran 8. The subsequent hydroge-
nation of the nitro group and cyclization of the amino group of 9
with bis(bromoethyl)ether gave the desired 4-morpholinobenzofu-
ran 10. Compounds 12a–f were obtained by the same procedure
described in Scheme 1.
Similarly, 6-morpholinobenzofuran 15 was obtained from the
6-nitrobenzofuran 13, which was formed as a byproduct in the
nitration of 7 (Scheme 3).
7-Morpholinobenzofuran 18 was prepared from 2-ethoxycar-
bonyl-4-methoxybenzofuran 16 according to the same method de-
scribed for the synthesis of compounds 12a–f (Scheme 4).
The compounds were evaluated for affinity to the three sub-
types of adenosine receptors in binding assays. The assays were
performed with recombinant human receptors using the same
radioligand protocol as described previously.8 Our previous find-
ings on the SAR of benzofuran derivatives indicated that the incor-
poration of a 4-phenyl group improved the PK profile, while
ubility of these compounds was not sufficient (<6 lg/ml) in a solu-
tion such as JP2 fluid (‘2nd fluid’ as described in the disintegration
test of Japanese Pharmacopoeia).
With this information in hand, we prepared a series of 4-mor-
pholinobenzofuran derivatives designed to improve the aqueous
solubility (Table 2). This modification resulted in a dramatic
improvement in both the aqueous solubility and metabolic stabil-
ity while retaining the desired antagonistic activity on A2A recep-
tor. The incorporation of (1H-imidazol-1-yl)methyl group (12b),
which proved to be highly beneficial in previous SAR studies, pro-
duced a slight improvement in potency, albeit the solubility of this
compound in water was very low. Compound 12c, containing an
additional water-solubilizing group at the amide moiety, also dem-
onstrated a slight improvement in potency while retaining the
desirable aqueous solubility. Aliphatic substituents appended to
the amide nitrogen were tolerated and yielded good activities, with
potency increasing as the size of the substituent increased (12e
(R = Et) >12d (R = Me)). This trend did not extend to the larger het-
eroaliphatic ring substituted analogue 12f, however, which exhib-
ited a very low activity. The position of attachment of the
morpholine ring to the benzofuran nucleus was investigated as
shown by the comparison of the 6-subtituted (15) and 7-substi-
tuted (18) analogues. Compound 15 displayed much lower potency
Scheme 3. Reagents and conditions: (a) H2, Pd/C, EtOH; (b) bis(bromoethyl)ether,
iPr2NEt, toluene; (c) LiOHÁH2O, THF, H2O; (d) 4-aminopyridine, WSCÁHCl, HOBtÁH2O,
Et3N, DMF.

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O. Saku et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3768–3771
Table 1
Structure–activity relationships and in vitro clearances for 4-heteroarylbenzofurans
R1
R2
A2A lnhibn. (%) 10À8/10À7 (mol/L)
Human CLint/fm (L/h/kg)
CGS catalepsy lnhibn. (%) 10 mg/kg, po
a
b
Compd
19
29/87
4/27
34.8
2.23
40
6a
NT
6b
24/22
9.21
NT
6c
20/44
27/67
<0.47
3.42
NT
38
6d
6e
6f
46/83
18/72
56/90
3.18
1.97
2.44
60
48
60
6g
a
Average of triplicate measurements.
Compounds were incubated with human liver microsomes for 60 min in the presence of NADPH. CLint was determined by in vitro T1/2 method.10 NT = not tested.
b
Table 2
Structure–activity relationships, in vitro clearances and aqueous solubilities for 4-morpholinobenzofurans
A2A lnhibn. (%) 10À8/10À7 (mol/L)
Marmoset CLint (L/h/kg)
Solubility (
lg/mL) JP2
CGS catalepsy lnhibn. (%) 10 mg/kg, po
76
a
b
Compd
R
12a
30/73
50/89
<0.43
8.0
87
12b
12c
2
NT
50
39/74
NT
47
12d
12e
–Me
–Et
23/58
37/87
1.3
NT
97
NT
NT
44
12f
À2/26
NT
NT
NT
15
18
À16/À5
NT
NT
NT
NT
NT
NT
26/77
a
Average of triplicate measurements.
Compounds were incubated with marmoset liver microsomes for 60 min in the presence of NADPH. CLint was determined by in vitro T1/2 method.10 NT = not tested.
b
as compared to the corresponding compound 12a, while the 7-
morpholino analogue 18, which has the regiochemically opposite
substitution pattern to 12a, exhibited an equipotent antagonistic
activity. Some of the more potent compounds were evaluated for
their anti-parkinsonian effects in vivo. The excellent activity of
compound 12a against the catalepsy induced by the intracerebro-
ventricular administration of CGS 21680 can be explained by the
improvement in aqueous solubility and metabolic stability.
Common marmosets exhibiting parkinsonian symptoms fol-
lowing MPTP treatment appear to be the most suitable pharmaco-
logical models of Parkinson’s disease because their response to
anti-parkinsonian agents mimics that of human patients with
similar side effects, such as vomiting, stereotyped behavior, and
L
-DOPA-induced dyskinesias.11 Compound 12a was selected for
further in vivo testing based on its superior potency in the mouse
CGS 21680-induced catalepsy model as well as the excellent

O. Saku et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3768–3771
3771
Figure 1. Time course of the effect of compound 12a (10 mg/kg, po) on motor disability. Each point represents the mean disability score/10 min ( SEM, n = 8). Open circles
show the vehicle treatment group. Closed circles show the compound 12a treatment group. *P <0.05, **P <0.01 compared with vehicle control: Sign–Wilcoxon test.
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<0.43 L/h/kg). As shown in Figure 1, compound 12a (10 mg/kg,
po) caused a long-lasting (up to 5 h) reduction in motor disabil-
ity,12 and increased locomotor activity (data not shown). In addi-
tion, neither nausea nor vomiting was observed. This result
indicates that compound 12a is a promising candidate for future
pre-clinical evaluations.
In summary, a novel class of adenosine A2A antagonists was
identified by the replacement of the phenyl group at 4-position
of the previously reported benzofuran derivative 19. The improved
aqueous solubility and pharmacokinetic property with a water-sol-
ubilizing group at the 4-position suggested that this strategy of
reducing the intrinsic lipophilicity of the compounds is a promising
approach. Moreover, the SAR studies uncovered some important
factors for the design of potent A2A antagonists in the benzofuran
series, and identified some potent analogues that exhibited an
acceptable in vivo efficacy in mice. This investigation culminated
in the discovery of compound 12a,13 which demonstrated an excel-
lent in vivo efficacy in MPTP-treated common marmosets. Addi-
tional SAR analyses, as well as further evaluations of the in vivo
efficacy of related compounds, will be reported in the future.
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10. Obach, R. S.; Baxter, J. G.; Liston, T. E.; Silber, B. M.; Jones, B. C.; Macintype, F.;
Rance, D. J.; Wastall, P. J. Pharmacol. Exp. Ther. 1997, 283, 46.
11. (a) Loschmann, P. A.; Smith, L. A.; Lange, K. W.; Jenner, P.; Marsden, C. D.
Psychopharmacology 1992, 109, 49; (b) Pearce, R. K. B.; Jackson, M. J.; Smith, L.
A.; Jenner, P.; Marsden, C. D. Mov. Disord. 1995, 10, 731; (c) Smith, L. A.; Gordin,
A.; Jenner, P.; Marsden, C. D. Mov. Disord. 1997, 12, 935.
12. The motor disability score was evaluated using the rating scale as described in
detail in Ref. 10. Trained observers continuously monitored the animals for
scoring periods and scored them every 10 min.
13. Spectroscopic data of 12a: 1H NMR (270 MHz, CDCl3) d: 3.12–3.15 (m, 4H),
3.91–3.94 (m, 4H), 4.01 (s, 3H), 6.68 (d, J = 8.4 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H),
7.68 (dd, J = 1.6, 4.9 Hz, 2H), 7.69 (s, 1H), 8.51 (br, 1H), 8.58 (dd, J = 1.6, 4.9 Hz,
2H). Anal. Calcd for C19H19N3O4Á0.5H2O: C, 62.97; H, 5.56; N, 11.60. Found: C,
62.77; H, 5.42; N, 11.34. MS (APCI): 354 [M+H]+.
References and notes
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