4-Substituted-7-N-alkyl-N-acetyl 2-aminobenzothiazole amides: Drug-like and non-xanthine based A2B adenosine receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.05.056

7-N-Acetamide-4-methoxy-2-aminobenzothiazole 4-fluorobenzamide (compound 1) was chosen as a drug-like and non-xanthine based starting point for the discovery of A_2B receptor antagonists because of its slight selectivity against A₁ and A_2A receptors and modest A_2B potency. SAR exploration of compound 1 described herein included modifications to the 7-N-acetamide group, substitution of the 4-methoxy group by halogens as well as replacement of the p-flouro-benzamide side chain. This work culminated in the identification of compound 37 with excellent A_2B potency, modest selectivity versus A_2A and A₁ receptors, and good rodent PK properties.

Full text of DOI:10.1016/j.bmcl.2010.05.056

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4140–4146
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4-Substituted-7-N-alkyl-N-acetyl 2-aminobenzothiazole amides: Drug-like
and non-xanthine based A2B adenosine receptor antagonists
a
a
b
a
Adrian Wai-Hing Cheung a, , John Brinkman , Fariborz Firooznia , Alexander Flohr , Joseph Grimsby ,
Mary Lou Gubler a, Kevin Guertin a, Rachid Hamid a, Nicholas Marcopulos a, Roger D. Norcross b, Lida Qi a,
Gwendolyn Ramsey a, Jenny Tan a, Yang Wen a, Ramakanth Sarabu a
*
a Roche Research Center, Hoffmann-La Roche Inc., Nutley, NJ 07110, USA
b F. Hoffmann-La Roche Ltd, Pharma Research, CH-4070 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
7-N-Acetamide-4-methoxy-2-aminobenzothiazole 4-fluorobenzamide (compound 1) was chosen as a
drug-like and non-xanthine based starting point for the discovery of A2B receptor antagonists because
of its slight selectivity against A1 and A2A receptors and modest A2B potency. SAR exploration of com-
pound 1 described herein included modifications to the 7-N-acetamide group, substitution of the 4-meth-
oxy group by halogens as well as replacement of the p-flouro-benzamide side chain. This work
culminated in the identification of compound 37 with excellent A2B potency, modest selectivity versus
A2A and A1 receptors, and good rodent PK properties.
Received 12 April 2010
Revised 13 May 2010
Accepted 14 May 2010
Available online 20 May 2010
Keywords:
Adenosine
GPCR
Ó 2010 Elsevier Ltd. All rights reserved.
Antagonist
Adenosine is an autocoid produced in many tissues, which medi-
ates various functions through four G-protein coupled receptors
(GPCRs), namely A1, A2A, A2B, and A3. The A1 and A3 receptors are cou-
pled to Gi and Go proteins, respectively, while the A2A and A2B recep-
tors are coupled to Gs proteins.1 Due to these differences in receptor
function, adenosine signals an increase in intracellular cAMP levels
via its action through the A2A and A2B receptors, and a decrease in
cAMP levels through the A1 and A3 receptors. In addition, adenosine
increases intracellular calcium ion levels via the A2B receptor
through its coupling to Gq proteins. Adenosine’s agonist potency at
its four receptors individually expressed in Chinese Hamster Ovary
production.8 Based on the SAR of 2-alkynyl-8-aryl-9-methylade-
nines, they identified a series of A2B receptor antagonists that were
efficacious in lowering fasting and fed glucose levels in KK-Ay mice,9
a well recognized modelof type 2 diabetes. Thus, the potential utility
of adenosine A2B receptor antagonists for the treatment of asthma
and type 2 diabetes encouraged us to seek novel antagonists.
The majority of potent A2B receptor antagonists reported to date
are based on xanthine or adenine core structures.10 In our search
for drug-like, non-xanthine based A2B receptor antagonists, we
utilized a series of previously disclosed A2A receptor antagonists,
4-methoxy-7-aryl (I) and 4-methoxy-7-morpholino (II) 2-amin-
obenzothiazoles as the starting point.11 During exploratory studies
with 4-methoxy-7-substituted 2-aminobenzothiazoles, we sys-
tematically modified the 7-substituent to determine whether
(CHO) cells was determined to be, A3 (EC50 = 0.29
l
M) ꢀ A1 (EC50
=
0.31 M) > A2A (EC50 = 0.7
l
lM) ꢁ A2B (EC50 = 24
l
M).2 Based on this
relative order of adenosine agonist potency, it is believed that the
A2B receptor remains silent under normal physiological conditions
and is activated as a consequence of elevated extracellular adeno-
sine levels during chronic, high oxidative stress conditions, such as
hyperglycemia and mast-cell activation. For example, in an asth-
matic lung exposed to an allergen, the increased levels of adenosine
signal through the A2B receptor which mediates its proinflammatory
effects.3,4 CVT-6883 (Fig. 1), a potent and selective A2B receptor
antagonist was found to be efficacious in various animal models of
asthma, COPD and pulmonary fibrosis.5–7 Researchers at Eisai, using
specific agonists and antagonists of adenosine receptors, illustrated
the key role of A2B receptor antagonism in inhibiting hepatic glucose
A2B-selectivity could be achieved within the series but disappoint-
ingly found that almost all the analogs bearing aromatic, heteroar-
omatic and heterocyclic 7-substituents were potent A2A-selective
antagonists (data not shown). We then narrowed our focus on
7-N-acetyl analog 1 which is unique in possessing slight A2B-selec-
tivity over A1 and A2A receptors (Table 1). With the goal of improv-
ing the A2B potency and selectivity of 1, we discuss in this
communication the results of our SAR studies which included
modifications to the 7-N-acetamide group, substitution of the
4-methoxy group by halogens as well as replacement of the p-flo-
uro-benzamide side chain (Fig. 1). This work culminated in the
identification of compound 37, which incorporates excellent A2B
potency, modest selectivity versus A2A and A1 receptors, and good
rodent PK properties.
* Corresponding author. Fax: +1 973 235 7239.
E-mail address: adrian.cheung@roche.com (A. W. -H. Cheung).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.056

A. W. -H. Cheung et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4140–4146
4141
O 4
O
F
F
R
5
6
O
N
N
N
H
N
N
N
F
N
N
S
7
O
I
CVT-6883
O4
O
O
O
4
5
6
R
N
S
F
5
6
N
N
H
N
H
S
7
7
N
O
NH
O
1
II
Figure 1. Structures of CVT-6883, compounds I-II and 1.
Table 1
Functional and binding activities of various 7-N-alkylated 7-N-acetamide-4-methoxy-2-aminobenzothiazole 4-fluorobenzamides (compounds 1, 6–19)
O
O
4
F
N
N
H
S
O
7 N
R1
Compound
R1
=
A2B cAMP (IC50, nM)
A1 Bindinga (Ki, nM)
A2A Bindingb (Ki, nM)
A2B Bindingc (Ki, nM)
1
6
H (7-N-acetyl)
CH3
350
15
1600
66
250
39
130
22
7
8
9
CH2CH3
CH2CH(CH3)2
CH(CH3)2
12
23
19
40
66
22
13
37
3
NTd
NTd
12
NTd
42
NTd
35
10
11
12
13
14
15
16
17
18
19
CH2CF3
CHF2
230
77
42
33
8
6000
60
350
600
30
61
98
29
38
NTd
NTd
NTd
NTd
NTd
NTd
NTd
NTd
NTd
CH2CH2OH
CH2CH2CH2OH
CH2CH2OCH3
CH2CO2CH3
CH2CO2H
CH2CONH2
CH2CONHCH3
CH2CON(CH3)2
450
100
110
23
2600
560
790
1600
5
2200
160
330
480
Binding affinity for the A1 receptor was determined by competition for binding sites labeled by 3H-DPCPX (4.8 nM) in commercial (Euroscreen) A1 membranes.
Binding affinity for the A2A receptor was determined by competition for binding sites labeled by 3H-ZM241385 (5 nM) in commercial (Perkin–Elmer) A2A membranes.
Binding affinity for the A2B receptor was determined by competition for binding sites labeled by 3H-ZM241385 (30 nM) in whole cells (CHO) expressing A2B receptors.
NT: not tested.
a
b
c
d
Various 7-N-alkylated analogs (compounds 6–19) were pre-
and the 7-N-acetamide anion, being more nucleophilic than the
2-aminobenzothiazole benzamide anion, was preferentially alkyl-
ated when treated with 1 equiv of alkylating agent at 0 °C to give
compounds 6–19.
The corresponding 4-Cl analog (compound 20) of compound 7
was made in an analogous manner to that shown in Scheme 1
starting with commercially available N-(3-amino-4-chloro-phe-
nyl)-acetamide.
Various aryl and heteroaryl amides of 7-N-ethyl-acetamide-4-
methoxy-2-aminobenzothiazole (compounds 22–42) were pre-
pared according to Scheme 2. Treatment of compound 7 with 1 N
sodium hydroxide/methanol (1:1) under reflux for 2–3 days gave
N-7-(acetyl-ethyl-amino)-4-methoxy-2-aminobenzothiazole (21).
pared according to Scheme 1. Coupling of commercially available
N-(3-amino-4-methoxy-phenyl)-acetamide with benzoyl isothio-
cyanate12 gave benzoylated thiourea 2. De-benzoylation of com-
pound 2 was achieved using sodium methoxide in methanol13
and the resulting thiourea 3 was cyclized with bromine in acetic
acid14 to give 4-methoxy-7-N-acetamide 2-aminobenzothiazole
(4). The first three steps in the synthetic scheme could be carried
out on a multi-gram scale and did not require chromatography.
The p-fluorobenzoyl group was coupled to the 2-aminobenzothia-
zole core using the corresponding acid chloride to give compound
5. Treatment of compound 5 with 2.5 equiv of sodium hydride in
N,N-dimethylformamide generated the corresponding dianion

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A. W. -H. Cheung et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4140–4146
N
O
O
S
H
N
H
N
O
NaOCH3
NH2
S
O
CH3OH, r.t.,
88% yield
acetone, reflux
69% yield
O
NH
O
NH
2
Cl
HBr
O
O
H
N
F
NH2
N
O
Br2, AcOH
NH2
S
S
40 oC to 90 oC
100% yield
THF, iPr2EtN,
72% yield
O
NH
O
NH
4
3
O
N
O
NaH, DMF
N
S
H
N
N
H
N
R1-Br or R1-I
0 oC to r.t.
F
S
F
O
O
O
R1
O
NH
6-19
5
Scheme 1. Synthesis of various 7-N-alkylated 7-N-acetamide-4-methoxy-2-aminobenzothiazole 4-fluorobenzamides (compounds 6–19).
O
O
Cl
N
R3
R3
N
S
H
N
NH2
1N NaOH
O
S
F
O
N
O
or
O
N
CH3OH, reflux
70% yield
HO
O
7
21
coupling reagent
or
O
N
O
N
S
H
N
N
S
H
HO
R4
O
N
R4
R3
O
O
O
O
N
coupling reagent
37-42
22-36
Scheme 2. Synthesis of various amides of 7-N-ethyl-acetamide-4-methoxy-2-aminobenzothiazole (compounds 22–42).
Various amides (compounds 22–42) were synthesized by reacting
compound 21 with either the corresponding acid chloride or the
corresponding carboxylic acid and an amide coupling reagent
(selected from 1,10-carbonyldiimidazole, benzotriazole-1-yl-oxy-
tris-(dimethylamino)-phosphonium hexafluorophosphate or 1-meth-
ylimidazole/p-toluenesulfonyl chloride15).
As shown in Table 1, introduction of simple alkyl groups such as
methyl (compound 6), ethyl (compound 7), i-butyl (compound 8),
and i-propyl (compound 9) to the 7-N-acetamide moiety of com-
pound 1 led to significant improvements in A2B cAMP potency.
The most A2B potent analog, compound 7 (7-N-ethyl, cAMP
IC50 = 12 nM) is about fivefold selective over A1 (A1, Ki = 66 nM vs
All the new analogs prepared were tested in the A2B cAMP as-
A2B, Ki = 13 nM) and almost equipotent between A2B and A2A (A2A,
say16 and the most interesting analogs were further tested in A1,
A
Ki = 22 nM vs A2B, Ki = 13 nM). Introduction of fluorine atoms such
as trifluoromethyl (compound 10) led to a slight drop in A2B cAMP
potency without any gain in A1 and A2A selectivity, compared to
compound 7. Unexpectedly, replacement of two hydrogens of the
7-N-methyl of compound 6 by fluorines (difluoromethyl group,
2A, and A2B receptor binding assays.17 The data from the above as-
says are summarized in Tables 1–4. The values reported are the
average of at least two separate experiments and it is typical for
duplicate values to be within twofold of each other.

A. W. -H. Cheung et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4140–4146
4143
Table 2
compound 11) led to a 15-fold drop in A2B cAMP potency without
any significant change in A1 and A2A binding affinity, compared to
compound 6.
Functional and binding activities of 4-substituted 7-N-ethyl-acetamide-2-aminoben-
zothiazole 4-fluorobenzamides (compounds 7 and 20)
R2
Introduction of polar groups such as hydroxy and methoxy to
the 7-N-alkyl group (compounds 12–14) led to slight drops (two
to sixfold) in A2B cAMP potency and this approach was not pursued
further. Introduction of a methyl ester to the 7-N-alkyl group gave
compound 15 which is of similar A2B potency in the cAMP assay to
compound 7. In an attempt to improve selectivity against A1 and
A2A receptors and increase the metabolic stability of compound
15, its methyl ester was converted to the corresponding acid (com-
pound 16) and amides (compounds 17–19). Unfortunately, these
analogs all showed significant decreases in potency in the A2B
cAMP assay.
O
4
F
N
N
H
S
O
7 N
Compound R2
=
A2B cAMP
(IC50, nM)
A1 Bindinga A2A
(Ki, nM)
A2B
Bindingb
(Ki, nM)
Bindingc
(Ki, nM)
7
20
OCH3 12
Cl 23
66
30
22
15
13
NTd
Investigation of SAR at the 4-position of the 2-aminobenzothia-
zole core was carried out next. Our previous explorations at the
a,b,c,d
See footnotes to Table 1.
Table 3
Functional and binding activities of various p-substituted benzamides of 4-methoxy-7-N-ethyl-acetamide-2-aminobenzothiazole (compounds 7, 22–36)
O
O
R3
N
N
H
S
O
N
Compound
R3
F
SO2CH3
CN
CO2CH3
NHSO2CH3
=
A2B cAMP (IC50, nM)
A1 Bindinga (Ki, nM)
A2A Bindingb (Ki, nM)
A2B Bindingc (Ki, nM)
7
22
23
24
25
12
36
28
35
37
66
390
71
185
110
22
92
50
170
46
13
19
10
15
22
O
O
26
27
19
28
410
400
71
27
4
N
N
S
O
14
28
29
19
28
150
130
77
80
8
N
N
N
N
N
N
N
N
23
30
31
21
16
78
66
18
4
O
N
300
100
O
N
32
33
21
13
160
180
76
26
7
O
O
N
13
O
(continued on next page)

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A. W. -H. Cheung et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4140–4146
Table 3 (continued)
Compound
R3
=
A2B cAMP (IC50, nM)
A1 Bindinga (Ki, nM)
A2A Bindingb (Ki, nM)
A2B Bindingc (Ki, nM)
O
N
34
35
38
120
89
18
N
O
21
16
185
50
84
68
NTd
NTd
N
O
36
N
a,b,c,d
See footnotes to Table 1.
Table 4
Functional and binding activities of various amides of 7-N-ethyl-acetamide-2-aminobenzothiazole (compounds 37–42)
O
O
R4
N
N
H
S
O
N
Compound
R4
=
A2B, cAMP (IC50, nM)
A1 Bindinga (Ki, nM)
A2A Bindingb (Ki, nM)
A2B Bindingc (Ki, nM)
37
21
100
51
8
N
N
N
38
39
40
7
21
29
35
18
NTd
NTd
19
N
N
NTd
NTd
NTd
41
N
N
F
F
F
41
42
6
6
9
3
3
4
6
N
N
F
N
N
12
a,b,c,d
See footnotes to Table 1.
Table 5
PK parameters of compound 37 after iv and po administration to Han Wistar Rats
Compound
PK Profilea (iv, 2.5 mg/kg)
PK Profilea (po, 50 mg/kg)
AUC extrap. (ng h/mL)
74,400
F (%)
CL (mL/min/kg)
46
Vd (L/kg)
AUC extrap. (ng h/mL)
929
Cmax (ng/mL)
t1/2 (h)
37
3.0
7420
2.1
400
a
A dose of compound 37 was either intravenously (2.5 mg/kg, DMA/PEG 400/40% HPBCD/H2O) injected into the tail vein of male Han Wistar rat (n = 4) or orally (50 mg/kg,
Capmul PG8) administered using an intubation tube (n = 4). Plasma samples were collected up to 24 h after intravenous or oral administration. The plasma concentrations of
compound 37 were determined by LC–MS.
4-position of a structurally simpler 2-aminobenzothiazole tem-
plate (without 7-substitution) showed that 4-substituents such
as –OCH3, –Cl, and –F gave analogs with the best A2B potency (data
not shown). Therefore, the 4-OCH3 moiety of compound 7 was
replaced by 4-Cl and disappointingly, the resulting compound 20
showed a slight drop in A2B cAMP potency and no gain in selectiv-

A. W. -H. Cheung et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4140–4146
4145
ity against A1 or A2A receptors (Table 2). The 4-F analog of com-
pound 7 was also prepared and was less potent than compound
7 in A2B cAMP assay (data not shown). The 4-F series was therefore
not pursued further. Based on the results of our SAR at the 4- and
our work and to Dr. Alexander Alanine, Walter Vifian and Philipp
Schmid for the synthesis of compound 1 and initial analogs. We
would like to thank Drs. Navita Mallalieu and Aruna Railkar for car-
rying out the rodent PK studies. We would also like to thank Roche
Physical Chemistry Department for spectroscopic measurements
and interpretations and Dr. Jefferson Tilley for critical reading of
the manuscript.
7-positions of 2-aminobenzothiazole core, compound
7 with
4-OCH3 and 7-N-ethyl-acetamide substitutions was selected as
having the best combination of A2B potency and selectivity against
A1 and A2A receptors for further optimization.
Thus, the p-fluoro group of the benzamide side chain of
compound 7 was replaced by a diverse set of substituents such as
methylsulfone (22), cyano (23), N-methanesulfonamide (25),
methyl ester (24) and its corresponding amides (31–36), five-mem-
bered heteroaromatics (28–30), together with N-methyl-N-meth-
anesulfonamide (26) and pyrrolidinone (27) both extended by a
methylene spacer. Interestingly, all the above analogs showed
similar potency in the A2B cAMP assay (Table 3, IC50 within 13–
38 nM range) and similar A2B receptor binding affinity (Ki within
4–23 nM range).18 In terms of A1 and A2A selectivity, although most
compounds in Table 3 have moderate A1 selectivity (A1, Ki >100 nM),
only two of them showed A2A Ki equal to or above 100 nM (com-
pounds 24 and 31).
Moving away from a phenyl ring in the amide region, five-
membered heteroaromatic rings were explored and the data for
those analogs are shown in Table 4. Compound 37, incorporating
2-methyl-2H-pyrazole, exhibited good potency in the A2B cAMP
assay and reasonable selectivity against A1 (about 12-fold) and A2A
(about sixfold). Slight variations in the pyrazole ring of compound
37 led to compounds 38–40 which are similar to compound 37 in
A2B potency, but do not offer an advantage in A1 and A2A receptor
selectivity. It was reported at the time of our investigation that
m-F and m-CF3 benzyl-pyrazol-4-yl groups imparted good A2B affin-
ity and selectivity in the 1,3-diethyl and 1,3-dipropyl derivatives in
the xanthine class of compounds.19 We therefore prepared analogs
41 and 42 with similar side chains in our series. Although com-
pounds 41 and 42 are very potent in A2B assays, they did not show
adenosine receptor subtype selectivity, and thus were not profiled
further.
References and notes
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Pharmacol. Rev. 2001, 53, 527.
2. Fredholm, B. B.; Irenius, E.; Kull, B.; Schulte, G. Biochem. Pharmacol. 2001, 61,
443.
3. Wilson, C. N. Br. J. Pharmacol. 2008, 155, 475.
4. Holgate, S. T. Br. J. Pharmacol. 2005, 145, 1009.
5. Kalla, R. V.; Zablocki, J. Purinergic Signalling 2009, 5, 21.
6. Sun, C.-X.; Zhong, H.; Mohsenin, A.; Morschl, E.; Chunn, J. L.; Molina, J. G.;
Belardinelli, L.; Zeng, D.; Blackburn, M. J. Clin. Invest. 2006, 116, 2173.
7. Mustafa, S. J.; Nadeem, A.; Fan, M.; Zhong, H.; Belardinelli, L.; Zeng, D. J.
Pharmacol. Exp. Ther. 2007, 320, 1246.
8. Harada, H.; Asano, O.; Kawata, T.; Inoue, T.; Horizoe, T.; Yasuda, N.; Nagata, K.;
Murakami, M.; Nagaoka, J.; Kobayashi, S.; Tanaka, I.; Abe, S. Bioorg. Med. Chem.
2001, 9, 2709.
9. Harada, H.; Asano, O.; Hoshino, Y.; Yoshikawa, S.; Matsukura, M.; Kabasawa, Y.;
Niijima, J.; Kotake, Y.; Watanabe, N.; Kawata, T.; Inoue, T.; Horizoe, T.; Yasuda,
N.; Minami, H.; Nagata, K.; Murakami, M.; Nagaoka, J.; Kobayashi, S.; Tanaka, I.;
Abe, S. J. Med. Chem. 2001, 44, 170.
10. Kalla, R. V.; Zablocki, J.; Tabrizi, M. A.; Baraldi, P. G. Handb. Exp. Pharmacol.
2009, 193, 99.
11. Flohr, A.; Jakob-Roetne, R.; Norcross, R. D.; Riemer, C. WO 2003043636; CAN
139:6865 and related patents.
12. Sarkis, G. Y.; Faisal, E. D. J. Heterocycl. Chem. 1985, 22, 137.
13. Rajappa, S.; Advani, B. G.; Sreenivasan, R. Indian J. Chem. 1980, 19B, 536.
14. Alanine, A.; Flohr, A.; Miller, A. K.; Norcross, R. D.; Riemer, C. WO 2001097786;
CAN 136:69803.
15. Wakasugi, K.; Iida, A.; Misaki, T.; Nishii, Y.; Tanabe, Y. Adv. Synth. Catal. 2003,
345, 1209.
16. A2B cAMP assay: CHO cells were stably transfected with human A2B receptor
and cultured under 5% CO2/95% O2 atmosphere at 37 °C in DMEM and DMEM/
F-12 (1:1 mixture) medium (Invitrogen) with 10% fetal calf serum (Invitrogen),
100 U/mL penicillin (Invitrogen), 100 U/mL streptomycin (Invitrogen), 1 mg/
mL G418 (Invitrogen) and 0.2 mg/mL Hygromycin B (Invitrogen). Experimental
cultures were grown overnight as a monolayer in 384-well tissue culture
plates (0.06 mL/well–7500 cells/well). Each well was washed once with 0.1 mL
of Krebs buffer. To each well was added 50
lL of Krebs buffer containing
Based on A2B potency and selectivity against A1 and A2A, some of
the analogs described above (compounds 6, 7, 22, 26, 27, 34, and
37) were tested for PK exposure via oral route of administration
(formulation: 2% Klucel/0.1% Tween 80 aqueous suspension) in
C57 mice at 50 mg/kg dose. Of the seven compounds tested, only
compounds 6, 7, and 37 showed plasma drug exposures (Cmax
and AUC, data not shown) that warranted further profiling. Com-
pound 37 was studied more thoroughly in a rat PK study and
was found to have moderate volume of distribution, moderately
high clearance and excellent oral bioavailability (Table 5).20
In summary, 7-N-acetamide-4-methoxy-2-aminobenzothiazole
4-fluorobenzamide (compound 1) was chosen as a drug-like and
non-xanthine based starting point for the discovery of A2B receptor
antagonists because of its slight selectivity against A1 and A2A
receptors and modest A2B potency. SAR exploration of 1 included
modifications to the 7-N-acetamide group, substitution of the
4-methoxy group by halogens, as well as replacement of the
p-flouro-benzamide side chain. This work culminated in the iden-
tification of compound 37 with excellent A2B potency, modest
selectivity versus A2A and A1 receptors, and good rodent PK
properties. In vivo pharmacological evaluation of compound 37
and further attempts to improve its A1 and A2A selectivity will be
described in future publications.
100 M of the phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)-2-
l
imidazolidinone, 100 nM NECA (Sigma–Aldrich), 0.02% BSA Fraction V (Roche
Biochemicals), the test compound (appropriate concentration). The final
concentration of DMSO was 1.1%. After incubation for 20–25 min, the wells
were emptied and blotted on paper towel to remove residual solution. The
HitHunter cAMP Assay Kit from DiscoverX for adherent cells was used for
lysing the cells and measuring cAMP concentrations.
17. Binding assays: (a) Human A1 membrane receptors (Euroscreen) were diluted
in assay buffer (HEPES 50 mM, NaCl 100 mM and MgCl2 1 mM) to yield final
concentration of 10 lg/well. The test compounds (10 lL) and 40 l
L of [3H]-
DPCPX ligand (4.8 nM final conc., Perkin–Elmer), were added to 96-well
polypropylene plates (Becton Dickinson) followed by addition of membranes
(150
Human A2A membrane receptors (Perkin–Elmer) were diluted in assay buffer
(HEPES 50 mM, EDTA 1 mM) to yield final concentration of 8.5 g/well. The
test compounds (10 L) and 40
L of [3H]-ZM241385 ligand (5 nM final) were
added to 96-well polypropylene plates (Becton Dickinson) followed by
addition of membranes (150 L) and incubation at room temperature for 1 h
lL) and incubation at room temperature for 1 h on an orbital shaker. (b)
l
l
l
l
on an orbital shaker. (c) For human A2B receptor, whole cells (CHO cells)
expressing the receptor were used. Confluent (80%) T75 flasks were harvested
mechanically and frozen in aliquots of 1 mL. On the day of assay, a single vial
was suspended in 25 mL of assay buffer. The test compounds (10
ZM241385 ligand 40 L (30 nM final) were added to 96-well polypropylene
plates followed by addition of cell suspension (150 L) and incubation at room
l
L) and [3H]
l
l
temperature for 1 h on an orbital shaker. Reactions were harvested using 96-
well MultiScreen FB plates (0.5% polyethyleneimine-treated) and
MultiScreenHTS vacuum manifold (Millipore). Plates were air dried followed
by addition of scintillation fluid and read on MicroBeta counter (Perkin–Elmer).
(d) Human A3 receptor binding data was not obtained routinely because its
membrane preparations are not commercially available in the US due to patent
restrictions. Only compound 37 from the manuscript was tested in A3 binding
assay and its Ki in A3 was determined to be 80 nM.
Acknowledgments
18. A strong (r2 = 0.64) and linear relationship was observed between the potency
of A2B antagonists for the cAMP (IC50) and binding assay (Ki) across a wide
The authors are grateful to Drs. Claus Riemer and Jean-Luc
Moreau (A2A team) for their generous advices and help throughout
range of potencies (single digit nM–lM cAMP IC50 values) and across multiple
structural subtypes (over 300 compounds were analyzed, data not shown).

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19. Elzein, E.; Li, X.; Kalla, R.; Perry, T.; Palle, V.; Varkhedkar, V; Maa, T.; Nguyen,
M.; Wu, Y.; Maydanik, V.; Lustig, D.; Leung, K.; Zeng, D.; Zablocki, J. Abstracts of
Papers, 227th National Meeting of the American Chemical Society, Anaheim,
CA, Mar 28–Apr 1, 2004; American Chemical Society: Washington, DC, 2004;
MEDI-251.
20. A likely explanation for the greater than 100% oral bioavailability is saturation
of clearance since the oral dose is twenty times higher than the iv dose.
However, we cannot rule out other possibilities such as precipitation at the
injection site or insufficient sampling following iv administration and the
consequent failure to fully capture the distribution phase.