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

Article abstract of DOI:10.1016/j.bmcl.2009.12.028

A series of benzofuran derivatives were prepared to study their antagonistic activities to the A_2A receptor. Replacement of the ester group of the lead compound 1 with phenyl ring improved the PK profile, while modifications of the amide moiety

Full text of DOI:10.1016/j.bmcl.2009.12.028

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1090–1093
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthetic studies on selective adenosine A2A receptor antagonists: Synthesis
and structure–activity relationships of novel benzofuran derivatives
*
Osamu Saku , Mayumi Saki, Masako Kurokawa, Ken Ikeda, 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:
A series of benzofuran derivatives were prepared to study their antagonistic activities to the A2A receptor.
Replacement of the ester group of the lead compound 1 with phenyl ring improved the PK profile, while
modifications of the amide moiety showed enhanced antagonistic activity. From these studies, com-
pounds 13c, 13f, and 24a showed good potency in vitro and were identified as novel A2A receptor antag-
onists suitable for oral activity evaluation in animal models of catalepsy.
Received 18 November 2009
Revised 3 December 2009
Accepted 5 December 2009
Available online 11 December 2009
Ó 2009 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
divided into four different subtypes (A1, A2A, A2B, and A3).1 These
four receptors play central roles in a variety of biological responses
that could be beneficial in a number of clinical applications. The
A2A receptor is abundant in the medium-sized neurons of the cau-
date, putamen, and accumbens nuclei, and stimulates adenylyl cy-
clase.2 Epidemiological studies have shown that consumption of
caffeine, an A2A receptor antagonist, is associated with a decreased
risk of developing Parkinson’s disease (PD).3 PD is a very serious
neurological disorder, a chronic and progressive degenerative dis-
ease of the brain that impairs motor control, speech, and other
functions, and current methods of treatment fail to achieve long-
term control. Selective antagonism of the A2A receptor in a primate
model of PD ameliorated motor depression.4 Phase III clinical stud-
ies of KW-6002 (Istradefylline) in PD patients with L-dopa-related
motor complications yielded promising results with regard to mo-
tor symptom relief without motor side effects.5 Selective A2A
antagonists should provide a novel non-dopaminergic approach
to PD therapy.
OMe
OMe
O
O
F
N
NH
O
HN
R2
S
COOMe
R1
1
2
Figure 1.
Previous non-xanthine A2A adenosine receptor antagonists from
the literature include compound 2.7 These compounds share amide
bonds and 5,6-fused heterocyclic skeletons substituted by a meth-
oxy group as a common structural motif. In an effort to better
understand this class of compounds as A2A antagonists, we have
extensively investigated the effect of modifying the 2- and 4-posi-
tion of the molecule on the potency and pharmacokinetic parame-
ters. Here, we report the structure–activity relationship of a series
of benzofuran derivatives as potent and selective A2A antagonists.
Scheme 1 details the synthesis of benzofuran derivatives with
modifications at the 2- and 4-position. The synthesis started with
2-hydroxy-3-methoxybenzaldehyde (3). Treatment of compound
3 with ethyl bromoacetate afforded benzofuran 4. Subsequent
reaction with dichloromethyl methyl ether in the presence of TiCl4
provided 4-formyl benzofuran derivative 5. Hydrolysis of the ester
followed by amidation of the resulting carboxylic acid gave the de-
sired benzofurans 7. Methyl ester 8 was synthesized from 4-formyl
benzofurans 7.
Some xanthine and non-xanthine compounds have been found
to have high A2A affinity with varying degrees of A2A versus A1
selectivity.6 The search for a new A2A antagonist that is capable
of crossing the blood–brain barrier in order to achieve efficacy in
rodent model of PD started with benzofuran derivative 1, identified
by high-throughput screening of our compound library (Fig. 1).
The synthesis of 4-aryl benzofurans is outlined in Scheme 2.
Biphenyls 10 were synthesized via Suzuki coupling reaction using
* 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 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.028

O. Saku et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1090–1093
OMe OMe
1091
OMe
OH
OMe
OMe
OMe
a
O
O
b
b
a
O
O
O
O
O
COOEt
COOEt
COOEt
OEt
OEt
CHO
COCH2Br
CHO
N
3
4
5
S
4
14
15
R1
OMe
OMe
OMe
O
O
O
c
d
e
O
O
O
OMe
R
R
O
c, d
OH
N
H
N
H
R2
O
N
H
CHO
CHO
COOMe
6
8
7
N
R1
S
16
Scheme 1. Reagents and conditions: (a) ethyl bromoacetate, K2CO3, DMF; (b)
Cl2CHOCH3, TiCl4, CH2Cl2; (c) LiOHÁH2O, THF, H2O; (d) RNH2, WSCÁHCl, HOBtÁH2O,
Et3N, DMF; (e) I2, KOH, MeOH.
Scheme 3. Reagents: (a) BrCOCH2Br, AlCl3, CH2Cl2; (b) R1CSNH2, dioxane; (c)
LiOHÁH2O, THF, H2O; (d) R2NH2, WSCÁHCl, HOBtÁH2O, Et3N, DMF.
OMe
OH
OMe
OH
OMe
OH
b
a
OMe
OH
OMe
OMe
a
b
O
O
CHO
CHO
CN
CN
CHO
Br
R1
CHO
Br
Br
17
3
9
10
9
18
OMe
OMe
OMe
O
O
O
c
d
e
R2
O
O
O
OEt
OH
N
H
R1
R1
R1
O
OMe
OMe
NHPh
O
OMe
c
N
d
e
H
N
O
O
O
11
12
13
N
NH2
O
Scheme 2. Reagents: (a) Br2, AcOH; (b) R1B(OH)2, PdCl2(P(o-tol)3)2, K2CO3, H2O,
EtOH, toluene; (c) ethyl bromoacetate, K2CO3, DMF; (d) LiOHÁH2O, THF, H2O; (e)
R2NH2, WSCÁHCl, HOBtÁH2O, Et3N, DMF.
19
20
21
palladium catalyst from 6-bromo-2-hydroxy-3-methoxybenzalde-
hyde (9). A screening of palladium catalysts and ligands revealed
that PdCl2(P(o-tol)3)2 was a good catalyst for the reaction. The
4-aryl benzofurans 13 were obtained by the same procedure de-
scribed in Scheme 1.
For the preparation of 4-thiazolyl benzofurans, compound 4
was bromoacetylated by Friedel–Crafts reaction, followed by the
cyclization using thioamide to give compound 15. Subsequent
hydrolysis and amidation gave the desired compound 16
(Scheme 3).
Scheme 4. Reagents: (a) bromoacetonitrile, K2CO3, DMF; (b) PhB(OH)2,
PdCl2(P(o-tol)3)2, K2CO3, H2O, EtOH, toluene; (c) aniline, AlCl3, CH2Cl2; (d)
iodobenzene diacetate, toluene; (e) aniline, toluene.
OMe
OMe
OMe
O
O
O
a
b
O
O
O
NH2
OEt
N
H
N3
23
11f
22
The reverse-amide compound was prepared through the route
described in Scheme 4. To avoid the use of 2-aminobenzofuran,
which is difficult to treat because of the instability of the com-
pound, an indirect synthetic method was developed. First, 2-cya-
nobenzofuran 17 was prepared by cyclization of compound 9
with bromoacetonitrile, followed by Suzuki coupling reaction to
give 4-phenylbenzofuran 18. Treatment of compound 18 with ani-
line afforded amidine 19. The reaction of compound 19 with iodo-
benzene diacetate gave the rearranged product 20, which was
further treated with aniline to afford N-(7-methoxy-4-phen-
ylbenzofuran-2-yl)acetamide 21.
OMe
H
N
O
O
R
c
O
24
23
OMe
H
N
H
O
N
d
O
F
Carbamates 24 and urea 25 were prepared starting with the
hydrazination of ester 11f. The reaction of hydrazine with sodium
nitrite provided azide 23, followed by Curtius rearrangement with
alcohols or anilines, providing the desired compounds (24 and 25)
(Scheme 5).
25
Scheme 5. Reagents: (a) N2H4, EtOH; (b) NaNO2, AcOH, H2O; (c) ROH, toluene; (d)
4-fluoroaniline, toluene.
The corresponding benzothiophene analogues were synthesized
as shown in Scheme 6. The starting 3-fluoro-2-formyl-4-methoxy-
biphenyl (26) was cyclized in the presence of methyl 2-mercap-
toacetate and then hydrolyzed to give 2-carboxy substituted
benzothiophene 27. The reaction of the carboxylic acid 27 with
amines afforded the amides 28.
recombinant human receptors using the same radioligand protocol
as described previously.8 A study for the initial set of compounds
aimed at determining the effect of substituents on the 2-position
of the benzofuran ring. The effects of alterations made to the ani-
line ring of compound 1 on the inhibitory activity are summarized
in Table 1. The non-substituted (8a), 4-chloro (8b), and 4-methoxy
(8c) substituted analogues showed equivalent potency to lead
The compounds were evaluated in binding assays to the three
subtypes of adenosine receptors. The assays were performed with

1092
O. Saku et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1090–1093
Table 2
OMe
OMe
OMe
O
c
Structure–activity relationships for 4-phenylbenzofurans
a, b
F
S
S
R
COOH
N
OMe
H
CHO
O
O
R2
N
H
R1
26
27
28
Compound R1
R2
A2A
CGS
a
Scheme 6. Reagents: (a) methyl 2-mercaptoacetate, NaH, DMSO; (b) LiOHÁH2O,
THF, H2O; (c) RNH2, WSCÁHCl, HOBtÁH2O, Et3N, DMF.
catalepsy
lnhibn. (%)
10 mg/kg,
po
lnhibn. (%) 10À8
10À7/10À6
/
(mol/L)
Table 1
Structure–activity relationships for 4-methoxycarbonylbenzofurans
1
–COOMe
14/58/86
48
OMe
O
F
O
R
13a
13b
13c
13d
–Me
57/88/—
18
NT
78
40
N
H
COOMe
F
13/49/67
39/81/103
29/87/100
a
Compound
R
A2A
CGS catalepsy
lnhibn. (%)
10 mg/kg, po
lnhibn. (%) 10À8
/
N
O
N
10À7/10À6 (mol/L)
N
1
14/58/86
48
F
13e
24/64/95
12
N
8a
8b
8c
6/49/86
1/43/77
—/53/91
NT
NT
NT
Cl
Cl
Cl
N
N
13f
83/101/—
18/28/—
74
OMe
N
13g
Cl
Cl
NT
8d
40/80/98
0
N
N
13h
13i
8/33/—
NT
NT
a
N
Average of triplicate measurements; NT = not tested.
COOEt
OMe
—/À2/À8
compound 1. From the structural similarity to Roche’s compounds
2,7 the introduction of (1H-imidazol-1-yl)methyl group was evalu-
ated, and this analogue 8d exhibited increased potency. However,
compound 8d showed no activity in the mouse CGS21680-induced
catalepsy model, which is commonly used to assess the activity of
compounds for antiparkinsonian effects. This result may be due to
the pharmacokinetic instability of the ester group at the 4-position.
Therefore, we turned our attention to the introduction of a vari-
ety of substituents at the 4-position that are capable of replacing
the methoxycarbonyl group. While the introduction of the formyl,
carboxyl, and acyl groups was detrimental to the antagonistic
activity (data not shown), the analogues with phenyl group at
the 4-position exhibited equipotent activities to those of the
4-methoxycarbonyl analogues (Table 2). Substitutions of the amide
with methyl (13a), phenyl (13b), morpholino (13c), 4-pyridyl
(13d), or (1H-imidazol-1-yl)methyl (13e) group were also toler-
ated in 4-phenyl benzofuran derivatives. In particular, 2,6-di-
chloro-4-pyridyl analogue 13f showed the most potent activity.
Oral administration of the compounds at 10 mg/kg was found to
be active in the mouse model CGS-induced catalepsy. The intro-
duction of the chloro (13g, h) or ethoxycarbonyl (13i) group at
the 4-position of the phenyl group showed significantly less
potency, while the 4-methoxy analogue 13j was approximately
equipotent. The analogue 16a–d, with 2-thiazolyl substituent at
the 4-position, significantly diminished the antagonistic activity
(see Table 3).
N
13j
24/63/90
54
N
a
Average of triplicate measurements; NT = not tested.
The amide template was further modified by replacing it with a
reversed amide, carbamate, or urea template, as shown in Table 4.
Reversed amide bond was tolerated, and the analogue containing a
methylcarbamate exhibited good in vivo efficacy. This result indi-
cated that the pharmacological characteristics of compound 24a
are advantageous for penetration of the blood–brain barrier.
Encouraged by this result, we next examined aryl carbamates
and ureas to adjust the physicochemical properties of the com-
pounds in this series. However, these analogues displayed signifi-
cantly lower potencies as compared to the corresponding amide
derivatives.
Most of the benzofuran derivatives showed remarkable selec-
tivity of A2A over A1 and A2B. Specifically, the compounds 13e
and 13j, having an imidazolyl group, showed no activity against
A1 or A2B receptors at 10À5 mol/L, which implies the nature and
size of the substituents at the amide moiety are critical for good
selectivity. The compounds with good inhibitory activity were
selected and their rat liver microsomal stability was evaluated.
The results are shown in Table 5. For a direct comparison of the
in vitro metabolic stability, CLint/fm can be calculated from the
experimental apparent intrinsic clearance, CLint, by correcting for
free fraction of test compounds in the incubations. As discussed
above, the stability of the benzofuran derivative 8d having an ester
group at 4-position was relatively low. On the other hand, 4-phe-
nyl benzofurans were demonstrably better in terms of their
The next set of compounds was prepared to study the influence
of the replacement of the oxygen atom by a sulfur atom in the ben-
zofuran ring. The benzothiophene derivatives 28a, b were found to
be less potent than the corresponding benzofuran derivatives. This
result suggested the superiority of benzofuran skeleton with re-
spect to its affinity for the A2A receptor.

O. Saku et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1090–1093
1093
Table 3
Table 5
Structure–activity relationships for 4-(2-thiazolyl)benzofurans and 4-phenylbenzothio
phenes
Selectivities and in vitro clearances for selected compounds
a
a
Compound
A1
A2B
cLog P
CLint/fm
(L/h/kg)
OMe
O
lnhibn. (%) 10À6
10À5 (mol/L)
/
lnhibn. (%) 10À6
10À5 (mol/L)
/
X
R2
N
H
8a
NT
NT
3.52
3.57
4.50
4.84
4.77
45.5
1.22
34.8
156
NT
R1
13c
13d
13e
13j
37/82
20/77
9/10
À4/À83
81/98
21/58
1/1
À19/4
a
Compound
R1
R2
X
A2A
lnhibn. (%) 10À8
/
10À7/10À6 (mol/L)
Me
N
N
F
16a
16b
O
O
1/15/60
S
S
OMe
an alternative strategy of reducing the intrinsic lipophilicity of
the compounds was desirable to design more potent compounds.
In summary, we have prepared a series of A2A antagonists based
on the lead compound 1. The SAR studies uncovered some impor-
tant factors for the design of potent A2A antagonists in the benzo-
furan series, and identified 13c, 13f, and 24a that exhibited
excellent in vivo efficacy. Further exploration of the SAR and biol-
ogy of the benzofuran series will be reported in due course.
6/11/À19
N
Me
Me
F
16c
O
17/6/4
N
N
S
S
N
N
N
16d
28a
28b
O
S
12/23/20
11/31/—
25/20/—
N
References and notes
N
N
S
1. Fredholm, B. B.; Cunha, R. A.; Svenningsson, P. Curr. Top. Med. Chem. 2003, 3, 413.
2. Schiffmann, S. N.; Libert, F.; Vassart, G.; Vanderhaeghen, J.-J. Neurosci. Lett. 1991,
2, 177.
a
Average of triplicate measurements.
3. Schwarzschild, M. A.; Chen, J.-F.; Ascherio, A. Neurology 2002, 58, 1154–1160.
4. Kase, H.; Mori, A.; Jenner, P. Drug Discovery Today: Ther. Strateg. 2004, 1, 51.
5. LeWitt, P. A.; Guttman, M.; Tetrud, J. W.; Tuite, P. J.; Mori, A.; Chaikin, P.;
Sussman, N. M. Ann. Neurol. 2008, 63, 295.
6. (a) Zhang, X.; Tellew, J. E.; Luo, Z.; Moorjani, M.; Lin, E.; Lanier, M. C.; Chen, Y.;
Williams, J. P.; Saunders, J.; Lechner, S. M.; Markison, S.; Joswig, T.; Petroski, R.;
Piercey, J.; Kargo, W.; Malany, S.; Santos, M.; Gross, R. S.; Wen, J.; Jalali, K.;
O’Brien, Z.; Stotz, C. E.; Crespo, M. I.; Díaz, J.-L.; Slee, D. H. J. Med. Chem. 2008, 51,
7099; (b) Neustadt, B. R.; Liu, H.; Hao, J.; Greenlee, W. J.; Stamford, A. W.; Foster,
C.; Arik, L.; Lachowicz, J.; Zhang, H.; Bertorelli, R.; Fredduzzi, S.; Varty, G.; Cohen-
Williams, M.; Ng, K. Bioorg. Med. Chem. Lett. 2009, 19, 967; (c) Shao, Y.; Cole, A.
G.; Brescia, M.-R.; Qin, L.-Y.; Duo, J.; Stauffer, T. M.; Rokosz, L. L.; McGuinness, B.
F.; Henderson, I. Bioorg. Med. Chem. Lett. 2009, 19, 1399; (d) Gillespie, R. J.;
Bamford, S. J.; Gaur, S.; Jordan, A. M.; Lerpiniere, J.; Mansell, H. L.; Stratton, G. C.
Bioorg. Med. Chem. Lett. 2009, 19, 2664; (e) Vu, C. B. Curr. Opin. Drug Disc. Dev.
2005, 8, 458.
Table 4
Structure–activity relationships for reversed amide, carbamate, and urea derivatives
OMe
H
O
N
R
O
a
Compound
R
A2A
CGS catalepsy
lnhibn. (%)
10 mg/kg, po
lnhibn. (%) 10À8
1CÀ7 (mol/L)
/
7. Alanine, A.; Flohr, A.; Miller, A. K.; Norcross, R. D.; Riemer, C. PCT Int. Appl.
WO2001097786, 2001.
21
24a
24b
–Me
–OMe
–OPh
62/91
28/85
23/65
28
86
34
8. Adenosine A2A binding assay: The human A2A receptor transfectant membrane
(Receptor Biology Inc., Beltsville, MD) was diluted 20-fold with incubation
buffer (50 mmol/L Tris (pH 7.4), 120 mmol/L NaCl, 5 mmol/L KCl, 10 mmol/L
MgCl2, 2 mmol/L CaCl2, and
membrane fraction. The membrane fraction (20
concentration: 75 nmol/L) and the test drug (20
assay and added with the incubation buffer to make the total volume of 200
After incubation at 25 °C for 90 min, the reaction mixture in the tube was
filtrated with 0.5% polyethyleneimine-treated 934 A/H filter (Whatman,
2
U/mL adenosine deaminase) to prepare the
L), [3H]CGS21680 (75
L, final
L) were placed in a tube for
L.
24c
25
O
F
F
11/32
22/37
NT
NT
l
l
l
H
N
l
a
Average of triplicate measurements; NT = not tested.
a
Maidstone, UK) using a cell harvester M-24R (Brandel, Graithersburg, MD). The
filtrated filter was transferred to a scintillation vial and dried. The filter was then
added with 0.5 mL of Ultima gold (Packard, Downers Grove, IL), and the
radioactivity was determined by means of a scintillation counter (LS 6500,
Beckman CO., Ltd, Fullerton, CA). The nonspecific binding was determined as the
metabolic stability. Above all, compound 13c showed the best sta-
bility. The lower lipophilicity of 13c may be partially responsible
for the improvement in the CLint/fm value. It was apparent that
radioactivity measured in the presence of 50 lmol/L NECA.