Selective, high affinity A2B adenosine receptor antagonists: N-1 monosubstituted 8-(pyrazol-4-yl)xanthines

Article abstract of DOI:10.1016/j.bmcl.2008.01.008

A series of N-1 monosubstituted 8-pyrazolyl xanthines have been synthesized and evaluated for their affinity for the adenosine receptors (AdoRs). We have discovered two compounds 18 (CVT-7124) and 28 (CVT-6694) that display good affinity for the A_2B AdoR (K_i = 6 nM and 7 nM, respectively) and greater selectivity for the human A₁, A_2A, and A₃ AdoRs (>1000-, >830-, and >1500-fold; >850-, >700-, and >1280-fold, respectively). CVT-6694 has been shown to block the release of interleukin-6 and monocyte chemotactic protein-1 from bronchial smooth muscle cells (BSMC), a process believed to be promoted by activation of A_2B AdoR.

Full text of DOI:10.1016/j.bmcl.2008.01.008

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1397–1401
Selective, high affinity A2B adenosine receptor antagonists:
N-1 monosubstituted 8-(pyrazol-4-yl)xanthines
Rao V. Kalla,a,* Elfatih Elzein,a Thao Perry,a Xiaofen Li,a Art Gimbel,b Ming Yang,b
Dewan Zengb and Jeff Zablockia
aDepartment of Bioorganic Chemistry, CV Therapeutics Inc., 3172 Porter Drive, Palo Alto, CA 94304, USA
bDepartment of Drug Research and Pharmacological Sciences, CV Therapeutics Inc., 3172 Porter Drive, Palo Alto, CA 94304, USA
Received 28 November 2007; revised 27 December 2007; accepted 2 January 2008
Available online 8 January 2008
Abstract—A series of N-1 monosubstituted 8-pyrazolyl xanthines have been synthesized and evaluated for their affinity for the aden-
osine receptors (AdoRs). We have discovered two compounds 18 (CVT-7124) and 28 (CVT-6694) that display good affinity for the
A2B AdoR (Ki = 6 nM and 7 nM, respectively) and greater selectivity for the human A1, A2A, and A3 AdoRs (>1000-, >830-, and
>1500-fold; >850-, >700-, and >1280-fold, respectively). CVT-6694 has been shown to block the release of interleukin-6 and mono-
cyte chemotactic protein-1 from bronchial smooth muscle cells (BSMC), a process believed to be promoted by activation of A2B
AdoR.
Ó 2008 Elsevier Ltd. All rights reserved.
In our recent publications,1–3 we have reported on the
O
N
O
N
H
N
H
N
discovery of novel 8-pyrazolyl xanthine derivatives that
display high affinity and selectivity for the A2B adeno-
sine receptors (AdoRs). While exploring the SAR for
this series of compounds, we initially focused on sym-
metrical disubstitution at the N-1 and N-3 positions of
the xanthine core. Hayallah and co-workers have shown
that N-1 monosubstituted xanthines that have a phenyl
substitution at the 8-position display higher A2B AdoR
selectivity compared to the 1,3-disubstituted xanthines.4
The goal of the present study is to explore the effect of
N-1 or N-3 monosubstitution of the xanthine core on
A2B AdoR affinity and selectivity for the 8-pyrazolyl
xanthines. For the purpose of this study, we synthesized
the m-F benzyl and m-CF3 benzyl-pyrazol-4-yl groups,
as these substitutions imparted good affinity and selec-
tivity in the 1,3-diethyl and 1,3-dipropyl derivatives
compared with other substitution patterns.1 For com-
parison purposes, the corresponding unsubstituted ben-
zyl derivatives were prepared as well (Fig. 1 and Table
1). The N-1 monosubstituted (5-phenyl oxadiazolyl)-1-
methyl pyrazolyl and (5-phenylisoxazolyl)-1-methyl pyr-
azolyl derivatives were also synthesized as these substi-
tutions on the pyrazole ring displayed good affinity
X
N
N
N
N
N
N
N
N
N
O
O
O
R
2, X = N; 3, X = C;
1a, R= H; 1b, R = F; 1c, R = CF3
Figure 1. Structures of disubstituted 8-pyrazolyl xanthines.
and selectivity in the 1,3-disubstituted xanthines
(Fig. 1 and Table 2).2
The N-1 and N-3 monosubstituted 8-pyrazolyl xanthine
derivatives were synthesized following the synthetic
route illustrated in Scheme 1. The monosubstituted urea
I was treated with cyanoacetic acid in the presence of
freshly prepared sodium ethoxide to afford the 6-amino
N-1 substituted uracil derivative II.5 The 6-amino uracil
V was treated with 1,1,1,3,3,3-hexamethyldisilazane
(HMDS) at high temperature and quenched with the
corresponding halide to provide the N-3 substituted 6-
amino uracil derivatives VI.6
The 6-amino uracils II and VI were converted to the cor-
responding diamines by nitrosation with NaNO2 fol-
lowed by reducing with Na2S2O4.1 The diamines IV
and VIII were selectively acylated at the 5-position by
coupling with the pyrazole acid 4 using 1-[3-(dimethyl-
Keywords: A2B; Adenosine; Antagonist.
*
Corresponding author. Tel.: +1 650 384 8568; fax: +1 650 858
0390; e-mail: rao.kalla@cvt.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.008

1398
R. V. Kalla et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1397–1401
O
N
O
O
Na2S2O4,
15%NH4OH
NaNO2,
CH3COOH/H2O
Ethyl cyano-
acetate
O
NH2
NH2
H
NO
R2
HN
HN
HN
N
NH2 NaOMe
70 °C, 1 h;
70 °C,1 h
O
H
O
N
R2
II
NH2
O
N
NH2
R2
IV
R2
III
I
HMDS,
(NH4)2SO4
125 °C
R1 -Br,
O
O
O
O
Na2S2O4,
15%NH4OH
NaNO2,
CH3COOH/H2O
R1
O
R1
O
H
R1
O
H
NO
NH2
NH2
HN
N
N
N
70 °C, 1 h;
70 °C, 1 h
O
N
H
NH2
N
NH2
N
H
NH2
N
100 °C
H
H
VII
VIII
VI
V
O
O
R1
O
R1
O
i) EDCI,M eOH,
r.t., 16h
H
N
NH2
NH2
N
N
N
N
N
N
+
HOOC
ii) HMDS, (NH4)2SO4
120 °C, 16 h
N
N
N
R2
R2
R1 = H, IV;R2 = H, VIII
4
R1 = H, 5 - 10; R2= H, 11-16
(Table1)
Scheme 1. Synthesis of N-1 and N-3 monosubstituted 8-pyrazolyl xanthines.
O
O
SEM
N
R1
O
R1
H
N
SEM-Cl, K2CO3, DMF
N
N
N
N
N
80 °C, 18 h
N
N
N
N
O
N
SEM
H
IX
11-16
Br
O
O
SEM
R1
Pd(OH)2,
Cyclohexene/EtOH
R1
O
H
N
N
N
NH
N
N
N
N
R
80 °C, 48 h
N
N
i) K2CO3, DMF, 80 °C
ii) 3N HCl, EtOH, 70 °C
N
SEM
O
N
H
R
X
17-26
X
Cl
O
R1
R
H
N
X
O
N
N
N
N
R
N
O
i) K2CO3, DMF, 80 °C
ii) 3N HCl, EtOH, 70 °C
N
O
N
H
27-37
Scheme 2. Synthesis of N-1 monosubstituted 8-pyrazolyl xanthines.
amino)propyl]-3-ethylcarbodiimide
hydrochloride
as previously described.2,8 Compound X was then alkyl-
ated with the corresponding oxadiazoles and isoxazoles,
followed by SEM deprotection with 3 N HCl to afford
the target molecules 27–37.
(EDCI) to furnish the amides that were subsequently
cyclized in the presence of HMDS and ammonium sul-
fate to provide the 8-pyrazolyl derivatives 5–16.7 For
the synthesis of the substituted benzyl derivatives
(Scheme 2) the N-3 and N-7 positions of the N-1 mon-
osubstituted derivatives 11–16 were protected with 2-
(trimethylsilyl)ethoxymethyl chloride (SEM-Cl) using
K2CO3 to provide derivatives IX. Debenzylation was
accomplished by hydrogenolysis (Pearlman’s catalyst)
affording the 8-(N-1H-pyrazol-4-yl) derivative X with
the N-3 and N-7 positions SEM protected. Alkylation
of the derivative X with 3-F and 3-CF3 benzyl groups
followed by SEM deprotection with 3 N HCl furnished
the 8-(1-substituted benzyl-pyrazol-4-yl)-N-1 substituted
xanthine derivatives 17–26 in good yields. The 5-phenyl-
(1,2,4-oxadiazoles) and 5-phenyl-isooxazoles required
for the synthesis of the derivatives 27–37 were prepared
The human A1, A2A, A2B, and A3 adenosine receptor
binding affinities for the monosubstituted derivatives
5–16 and 17–37 were evaluated (Tables 1 and 2). To
identify whether the N-3 or N-1 position of the xanthine
core provides the highest A2B affinity and selectivity we
initially synthesized the benzyl-pyrazolyl derivatives.
Compounds 5–10, that are N-3 monosubstituted xan-
thine derivatives, demonstrated lower affinity toward
the A2B AdoR regardless of the substitution at the N-3
position (Table 1). The N-1 monosubstituted 8-pyrazol-
yl xanthine derivatives 11–16 have displayed good A2B
AdoR affinity and selectivity over the other adenosine
receptor subtypes (Table 1). Compound 12 the N-1 pro-

R. V. Kalla et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1397–1401
1399
Table 1. Binding affinities of disubstituted and monosubstituted analogues for the A1, A2A, A2B, and A3 AdoRs
O
H
R1
N
N
N
N
N
O
N
R2
R
Compound
R1
R2
R
Ki nM#
A2B selectivity
A2A/A2B A3/A2B
a
b
c
d
hA2B
hA1
hA2A
hA3
A1/A2B
1a
1b
1c
5
Propyl
Propyl
Propyl
Propyl
Propyl
Methyl
Ethyl
Propyl
n-Butyl
i-Butyl
Benzyl
H
H
F
11
14
14
76
290
230
400
nd
170
58
7
13
26
18
16
170
160
nd
nd
nd
nd
nd
nd
4
10
Propyl
H
CF3
H
140
nd
nd
nd
nd
nd
nd
12
nd
27
nd
>6000
>6000
>6000
>6000
>6000
>6000
37
nd
nd
nd
nd
nd
nd
40
6
H
H
nd
nd
nd
nd
nd
nd
7
H
H
8
H
H
nd
nd
nd
nd
nd
nd
9
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
H
Ethyl
H
nd
3800
nd
nd
H
5600
1600
1500
120
150
120
150
>460
550
nd
100
>380
100
>380
460
nd
Propyl
n-Butyl
i-Butyl
H
H
13
34
>5000
3400
10
H
H
5100
110
3
90
H
H
13
6
>6000
3290
nd
>5000
2760
nd
1100
180
Cyclopropyl methyl
Benzyl
Ethyl
H
H
30
H
H
>6000
73
nd
nd
30
H
F
>6000
>6000
1800
>5000
>5000
1730
2117
>9000
160
>80
>1000
160
>750
230
>200
>250
>210
42
>70
>830
160
>620
>280
>170
>200
>180
20
Ethyl (CVT-7124)
Propyl
Propyl
H
CF3
F
6
11
>1500
15
80
H
H
CF3
F
8
18
>6000
4200
>5000
>5000
>5000
>5000
>5000
100
700
270
n-Butyl
n-Butyl
H
15
>300
70
H
CF3
F
30
24
>6000
>6000
>6000
210
>9000
1600
>9000
nd
i-Butyl
i-Butyl
H
H
CF3
F
28
5
>320
nd
300
Cyclopropyl methyl
Cyclopropyl methyl
H
H
CF3
3
>6000
>5000
1000
>2000
>1600
a Binding affinity for the A2B AdoR was determined by competition for binding sites labeled by 3H-ZM241385 (14 nM) in membranes prepared from
HEK-A2B cells.
b Binding affinity for the A1 AdoR was determined by competition for binding sites labeled by 3H-CPX (0.5 nM) in membranes prepared from CHO-
A1 cells.
c Binding affinity for the A2A AdoR was determined by competition for binding sites labeled by 3H-ZM241385 (2 nM) in membranes prepared from
HEK-A2A cells.
d Binding affinity for A3 AdoR was determined using CHO-A3 cells with 125I-AB-MECA as the radioligand.
# 95% Confidence intervals are generally within 15% of the mean value.
pyl derivative has A2B affinity of 13 nM that is similar to
the disubstituted derivative 1a (Ki = 11 nM, Table 1) but
displayed higher selectivity compared to 1a. The N-1
ethyl (11) and N-1 butyl (13) derivatives exhibited lower
affinity than the N-1-propyl derivative (12). Branched
alkyl chain N-1-isobutyl derivative 14 showed similar
affinity (Ki = 13 nM) compared to the propyl derivative
and also displayed higher selectivity. Similarly, the
cyclopropyl methyl derivative 15 has good A2B affinity
(Ki = 6 nM) and greater selectivity compared to 12.
Introduction of a bulky benzyl group at the N-1-posi-
tion as in 16 has a significantly lower affinity for the
A2B receptor. These findings clearly demonstrate that
the N-1 monosubstituted 8-(1-benzyl-pyrazol-4-yl) xan-
thine derivatives show good affinity and greater selectiv-
ity related to the N-3-monosubstituted derivatives.
Encouraged by these results, we further explored the ef-
fect of m-F and m-CF3 benzyl substitution on the pyra-
zoles with respect to the N-1 monosubstituted xanthines
(Table 1). In comparison N-1 ethyl analogue 17 with a
3-F substitution has lower affinity than the benzyl ana-
logue 11, but the m-CF3 analogue 18 (CVT-7124) dis-
played good affinity (Ki = 6 nM) for the A2B AdoR
and greater selectivity against the other AdoRs (Table
1). The F (19) and CF3 (20) analogues in the N-1 propyl
series have good affinity for the A2B AdoR (11 and 8 nM),
respectively, and good selectivity over the A1 and A2A
AdoRs but not against the A3 AdoR. The F (21) and
CF3 (22) derivatives in the N-1 butyl series have similar
affinity and selectivity compared to the corresponding
benzyl derivative 13 (Table 1). The isobutyl analogues
23 and 24 have displayed lower A2B AdoR affinity and
lower selectivity than the benzyl analogue 14. Similarly
the cyclopropyl methyl derivative 26 with CF3 substitu-
tion showed good A2B AdoR affinity and selectivity. In
this series of compounds substitution of m-CF3 benzyl
group on the pyrazole ring led to a high affinity and
selective A2B AdoR antagonist 18 (CVT-7124).
The results of the N-1 monosubstituted 8-(1-benzyl-pyr-
azol-4-yl)xanthines persuaded us to further expand our
SAR by replacing the benzyl group with 5-phenyl oxadi-

1400
R. V. Kalla et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1397–1401
Table 2. Binding affinities of N-1 monosubstituted 5-phenyl oxadiazoles and 5-phenyl-isoxazole analogues 33–43
O
H
R1
N
X
N
N
N
R
N
N
O
O
N
R2
Compound
R1
R2
X
R
Ki# (nM)
A2B selectivity
A2A/A2B
a
b
c
d
hA2B
hA1
hA2A
hA3
630
A1/A2B
A3/A2B
2
27
28
29
30
31
32
33
3
Propyl
Propyl
Propyl
H
N
N
N
N
N
N
N
N
C
C
C
C
C
H
21
15
7
1000
3500
1800
50
200
85
30
H
Cl
>5000
>5000
>5000
>5000
>5000
>5000
>5000
420
>9000
>9000
>9000
>9000
>9000
>9000
>9000
1800
>300
>700
>300
>210
>350
>100
>380
30
>600
>1280
>600
>400
>650
>190
>700
130
Propyl (CVT-6694)
Propyl
n-Butyl
H
>6000
1400
>850
90
H
CF3
Cl
15
23
14
48
13
14
22
170
12
15
H
>6000
>6000
>6000
>6000
1500
>260
>400
>120
>460
110
i-Butyl
H
CF3
Cl
Cyclopropyl methyl
Cyclopropyl methyl
Propyl
H
H
CF3
H
Propyl
H
34
35
36
37
Propyl
Propyl
H
Cl
>6000
>6000
>6000
>6000
>5000
>5000
>5000
>5000
>9000
nd
>270
>30
>230
>30
>400
nd
H
i-Butyl
Cyclopropyl methyl
H
H
CF3
>9000
>9000
>500
>400
>410
>300
>750
>600
H
a Binding affinity for the A2B AdoR was determined by competition for binding sites labeled by 3H-ZM241385 (14 nM) in membranes prepared from
HEK-A2B cells.
b Binding affinity for the A1 AdoR was determined by competition for binding sites labeled by 3H-CPX (0.5 nM) in membranes prepared from CHO-
A1 cells.
c Binding affinity for the A2A AdoR was determined by competition for binding sites labeled by 3H-ZM241385 (2 nM) in membranes prepared from
HEK-A2A cells.
d Binding affinity for A3 AdoR was determined using CHO-A3 cells with 125I-AB-MECA as the radioligand.
# 95% confidence intervals are generally within 15% of the mean value.
azoles and 5-phenyl-isoxazoles as these groups in the
1,3-disubstituted series exhibited higher A2B affinity
and selectivity (Table 2).2 The 5-phenyl oxadiazole
derivative with mono N-1 propyl substitution, as in
27, demonstrated good affinity and higher selectivity
for the A2B AdoR compared to the disubstituted deriv-
ative 2. The N-1 propyl analogue with 4-chloro-5-phenyl
oxadiazole 28 (CVT-6694) has displayed high A2B AdoR
affinity (Ki = 7 nM) and excellent selectivity for the hu-
man A1, A2A, and A3 AdoRs compared to the disubsti-
tuted analogue 2. Replacing the chloro group with CF3
as in 29 resulted in a decrease in A2B AdoR affinity and
selectivity compared to the chloro analogue 28. Replac-
ing the propyl group of 28 with a butyl group as in 30
resulted in decrease in A2B AdoR affinity and selectivity.
The N-1 isobutyl analogue 31 with 4-CF3 substitution
on the phenyl group has displayed similar A2B AdoR
affinity and selectivity compared to the propyl analogue
29. In the N-1 cyclopropyl methyl analogues 4-CF3
derivative 33 presented better affinity compared to the
4-chloro analogue 32, and both compounds displayed
very good selectivity. Similarly the N-1 propyl substi-
tuted isoxazole analogue 34 showed better selectivity
while retaining the A2B AdoR affinity compared to the
disubstituted analogue 3 (Table 2). Introduction of a
4-chloro substituent on the phenyl ring of 34 resulted
in 5-fold loss in A2B AdoR affinity. Substitution of iso-
butyl group in place of propyl group as in 36 increased
the A2B AdoR affinity and selectivity compared to 34.
Compound 37 that has a cyclopropyl methyl group at
the N-1 position also displayed very good selectivity
and A2B AdoR affinity. In general, the monosubstituted
5-phenyl oxadiazole and isoxazole analogues displayed
greater selectivity for the A2B AdoR compared to the
disubstituted compounds regardless of the substitution
at the N-1 position of the xanthine.
In conclusion, we have shown that N-1 monosubstituted
8-pyrazolyl xanthine derivatives display higher selectiv-
ity while retaining the A2B AdoR affinity compared to
their corresponding 1,3-disubstituted analogues. We
have also shown that the N-1 substitution not the N-3
substitution on the xanthines provide greater selectivity
for the A2B AdoRs of the 8-pyrazolyl xanthines. In this
process, we have discovered two high affinity and selec-
tive A2B AdoR antagonists [18 (CVT-7124) and 28
(CVT-6694)]. Compound 28 (CVT-6694) has been
shown to block the release of interleukin-6 and monocyte
chemotactic protein-1 from bronchial smooth muscle
cells (BSMC), a process believed to be promoted by the
activation of A2B AdoR.9 The activation of A2B AdoR
in human lung fibroblasts (HLFs) increases the release
of IL-6 and induces the differentiation of fibroblasts into
myofibroblasts that has been shown to be attenuated by
the selective A2B AdoR antagonist 28 (CVT-6694).10
References and notes
1. Kalla, R. V.; Elzein, E.; Perry, T.; Li, X.; Palle, V.;
Varkhedkar, V.; Gimbel, A.; Maa, T.; Zeng, D.; Zablocki,
J. J. Med. Chem. 2006, 49, 3682.

R. V. Kalla et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1397–1401
1401
2. Elzein, E.; Kalla, R.; Li, X.; Perry, T.; Parkhill, E.; Palle,
V.; Varkhedkar, V.; Gimbel, A.; Zeng, D.; Lustig, D.;
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3. Zablocki, J.; Elzein, E.; Kalla, R. Expert Opin. Ther.
Patents 2006, 16, 1347.
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42, 2344.
6. Muller, C. E.; Shi, D.; Manning, M., Jr.; Daly, J. W. J.
Med. Chem. 1993, 36, 3341.
7. Muller, C. E. Synthesis 1993, 125.
8. Zablocki, J.; Kalla, R.; Perry, T.; Palle, V.; Varkhedkar,
V.; Xiao, D.; Piscopio, A.; Maa, T.; Gimbel, A.; Hao, J.;
Chu, N.; Leung, K.; Zeng, D. Bioorg. Med. Chem. Lett.
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9. Zhong, H.; Belardinelli, L.; Maa, T.; Feoktistov, I.;
Biaggioni, I.; Zeng, D. Am. J. Respir. Cell Mol. Biol.
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10. Zhong, H.; Belardinelli, L.; Maa, T.; Zeng, D. Am. J.
Respir. Cell Mol. Biol. 2005, 32, 2.