Discovery of benzothiazole-based adenosine A2B receptor antagonists with improved A2A selectivity

Article abstract of DOI:10.1016/j.bmcl.2011.02.053

The highly potent but modestly selective N-(2-amino-4-methoxy-benzothiazol- 7-yl)-N-ethyl-acetamide derivative 2 was selected as the starting point for the design of novel selective A_2B antagonists, due to its excellent potency, and good drug-l

Full text of DOI:10.1016/j.bmcl.2011.02.053

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1933–1936
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of benzothiazole-based adenosine A2B receptor antagonists
with improved A2A selectivity
Fariborz Firooznia, Adrian Wai-Hing Cheung, John Brinkman, Joseph Grimsby, Mary Lou Gubler,
Rachid Hamid, Nicholas Marcopulos, Gwendolyn Ramsey, Jenny Tan, Yang Wen, Ramakanth Sarabu
⇑
Roche Research Center, Hoffmann-La Roche, Inc., Nutley, NJ 07110, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The highly potent but modestly selective N-(2-amino-4-methoxy-benzothiazol-7-yl)-N-ethyl-acetamide
derivative 2 was selected as the starting point for the design of novel selective A2B antagonists, due to its
excellent potency, and good drug-like properties. A series of compounds containing nonaromatic amides
or ureas of five- or six-membered rings, and also bearing an m-trifluoromethyl-phenyl group (shown to
impart superior potency) were prepared and evaluated for their selectivity against the A2A and A1 recep-
tors. This work resulted in the identification of compound 30, with excellent potency and high selectivity
against both A2A and A1 receptors.
Received 4 January 2011
Revised 5 February 2011
Accepted 14 February 2011
Available online 17 February 2011
Keywords:
Adenosine A2B receptor antagonists
Adenosine A2A receptor
Benzothiazoles
Ó 2011 Elsevier Ltd. All rights reserved.
Adenosine is an autocoid that mediates protective effects under
normal physiological conditions through four G-protein coupled
receptors, namely the A1 and A3 receptors (which are coupled to
Gi and Go proteins, respectively), and the A2A and A2B receptors
(which are coupled to Gs proteins).1 Interestingly, adenosine0s ago-
nist potency is determined to be significantly varied at its four
receptors, which were individually expressed in Chinese Hamster
O
O
N
O
N
O
N
O
N
O
N
N
N
N
S
F
N
S
N
S
N
N
H
H
H
HN
CF3
O
O
O
1
2
3
Figure 1. Structures of compounds 1–3.
Ovary (CHO) cells: A3 (EC50 = 0.29
l
M) ꢀ A1 (EC50 = 0.31
lM) >A2A
(EC50 = 0.7 M) >>A2B (EC50 = 24
l
l
M).2 Thus, the low affinity A2B
(Table 1), in contrast to what was observed with the xanthine
series.13
The good in vitro potency of compound 2, as well as its good
rodent PK profile,10 prompted us to further characterize this com-
pound in a number of in vitro selectivity and safety assays. We were
pleased to find that compound 2 did not show any significant activ-
ity (defined as >50% inhibition at 10 lM) against a panel of 63
receptors, enzymes and ion channels tested at MDS Panlabs (Bot-
hell, WA). In addition, compound 2 exhibited virtually no inhibition
receptor is believed to remain silent at normal physiological state,
and become activated when under elevated extracellular adenosine
levels during chronic high oxidative stress conditions, such as
hyperglycemia or mast-cell activation. Antagonists of the A2B recep-
tor have therefore been evaluated as potential thearapeutic agents
in models of diabetes, asthma, COPD, and pulmonary fibrosis.3–9
As discussed in an earlier communication,10 our initial SAR explo-
ration of A2B receptor antagonists, starting with 7-N-acetamide-4-
methoxy-2-aminobenzothiazole 4-fluorobenzamide (compound 1,
Fig. 1), led to the identification of the 7-N-ethyl derivative 2 with
good A2B potency, and modest selectivity versus A2A (ca. six-fold)
and A1 (ca. 12-fold) receptors (Table 1).11,12 Furthermore, incorpora-
tion of a m-trifluoromethyl-benzylsubstitutedpyrazole regioisomer
(which was previously reported to provide a potency boost towards
(IC50 >50
l
M) of CYP450 enzymes (2C9, 2C19, 2D6 and 3A4), as well
M). Fur-
as a low risk of QT interval prolongation (hERG, IC50 >100
l
thermore, compound 2 did not show any genotoxicity liability in
the microsuspension version of the Ames test in the absence and/
or presence of an exogenous metabolic activation system (S9).
In summary, compound 2 possessed all the desired characteris-
tics for a pre-clinical candidate (excellent A2B potency, desirable
PK properties, absence of in vitro toxicological liabilities), but lacked
sufficient selectivity against A1 and A2A receptors. Thus, we focused
our efforts on trying to improve the one remaining shortcoming of
our aminobenzothiazole A2B antagonists, namely sub-optimal
adenosine receptor selectivity, while maintaining their excellent
A
2B in other A2B antagonists)13 resulted in the identification of com-
pound 3, which displayed even greater potency towards A2B, albeit
with a complete loss of selectivity versus the A2A and A1 receptors
⇑
Corresponding author. Fax: +1 973 235 2448.
E-mail address: ramakanth.sarabu@roche.com (R. Sarabu).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.053

1934
F. Firooznia et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1933–1936
Table 1
amides were then removed upon treatment with trifluoroacetic
acid or 1N HCl (step II. Boc-deprotection). Finally, the resulting un-
masked amines were further derivatized (step III. N-capping) by
either (i) acylation with 3-trifluoromethyl-benzoyl chloride (9) or
(ii) sulfonylation with 3-trifluoromethyl-benzenesulfonyl chloride
(10) or (iii) reductive amination with 3-trifluoromethyl-benzalde-
hyde (11), to produce the A2B antagonists 13–27.
Additionally, a few piperidine urea derivatives (compounds
28–30) were also prepared, via the conversion of starting material
4 to the corresponding p-nitrophenyl carbamate via treatment
with 12, followed by the displacement of p-nitrophenol with an
appropriately substituted amine, as shown in Scheme 2.
The new analogs thus prepared were then profiled in the A2B
cAMP assay11 as well as A1 and A2A receptor binding assays (Tables
2–5).12 As there were no commercially available membrane prep-
arations for A2B receptors, only a selected number of the more
interesting compounds were tested in the A2B binding assay14
The data are summarized below.15
Initially, we chose to evaluate analogs bearing five-membered
ring nonaromatic spacers (i.e., spacers derived from 5 or 6,
Table 3), as they were the ‘structurally closest’ analogs to com-
pounds 2 and 3. For synthetic ease, the carbonyl linker was used to
connect the spacers to the m-triflurophenyl group. In the case of
pyrrolidine derivatives, we noted a slight preference for the (R)-
enantiomer (compound 13) over the corresponding (S)-enantiomer
(compound 14) in the A2B cAMP assay. We were especially pleased to
see that the more potent enantiomer 13, while maintaining a similar
potency to compound 2, showed much improved A2A binding selec-
Functional and binding activities of three 7-N-acetamide-4-methoxy-2-aminobenzo-
thiazole amides (compounds 1–3)
Compound A2B cAMP
(IC50, nM)
A1 binding
(Ki, nM)
A2A binding
(Ki, nM)
A2B binding
(Ki, nM)
1
2
3
350
21
6
1600
100
6
250
51
3
130
8
4
potency. We chose to keep the core structure of compounds 2 and 3
[i.e., the N-(2-amino-4-methoxy-benzothiazol-7-yl)-N-ethyl-acet-
amide moiety] intact, while further modifying the right hand
(amide) portion of the molecules. As previously described,10 amides
of simple five- or six-membered aromatic and heteroaromatic rings
did not produce significant breakthroughs in terms of selectivity
against A1 and A2A receptors. Aiming to take advantage of the
m-trifluorophenyl moiety in compound 3 (in order to maintain A2B
potency), we therefore decidedto synthesizeamidesor ureas of non-
aromatic, five- or six-membered rings (compounds 13–30), in hopes
that the added nonplanar structural elements would impart better
selectivity to our A2B antagonists. Thus, all new compounds were de-
signed to contain a nonaromatic spacer (piperidine, pyrrolidine,
aminocyclohexane, or aminocyclopentane), followed by a linker
(carbonyl, sulfonyl, or methylene) group, connecting the core ami-
nobenzothiazole to the m-trifluromethylphenyl moiety required
for potency (Fig. 2).
The amides of nonaromatic five- or six-membered rings (com-
pounds 13–27) were synthesized as shown in Scheme 1, via a three
step sequence starting from intermediate 4 (which was prepared
on a multi-gram scale as previously described).10 The N-Boc-pro-
tected amino acids 5–8 were coupled to starting material 4 (step
I. amide coupling) using either standard coupling procedures
(CDI in THF), or alternatively via activation of the acids with
1-methylimidazole and p-toluenesulfonyl chloride, followed by
treatment with amine 4. The N-Boc protecting groups of the resulting
tivity (Ki >2 lM), without losing its modest A1 selectivity.
Interestingly, when the spacer length was increased by one
atom, the selectivity profiles of the compound were drastically
altered. The chiral cis- and trans-3-N-Boc-amino-cyclopentane ana-
logs 15 and 16 (which retained the same preferred stereochemistry
as 13 at the carbon attached to the benzothiazole core) showed a
O2N
O
O
O
N
Cl
O
N
S
N
S
N
O
O
N
O
N
amine R2H
iPr2NEt
12
N
O
N
O
N
R2
Spacer
N
S
N
S
N
O
N
N
O
Linker
pyridine
CH2Cl2
r.t.
H
H
THF
CF3
CF
3
50 oC
4
28-30
O
O
3
13-30
Scheme 2. Synthesis of various N-(2-amino-4-methoxy-benzothiazol-7-yl)-N-
ethyl-acetamide ureas (compounds 28–30).
Figure 2. Design of compounds 13–30.
II. Boc-
III. N-capping
I. Amide coupling
O
deprotection
N
Boc
F
F
O
F
O
N
5 (R- or S-)
O
N
Cl
N
S
N
S
O
N
O
N
Boc
N
O
R1
9
TFA
O
or
6 (cis- or trans-)
O
O
or 1N HCl
F
F
F
O
4
13-27
N Boc
O
Cl
O
S
7
O
10
or
O
N
F
F
F
O
Boc
H
8 (cis- or trans-)
O
11
Scheme 1. Synthesis of various N-(2-amino-4-methoxy-benzothiazol-7-yl)-N-ethyl-acetamide amides (compounds 13–27).

F. Firooznia et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1933–1936
1935
Table 2
Table 3
Functional and binding activities of various 7-(acetyl-ethyl-amino)-4-methoxy-
Functional and binding activities of various 7-(acetyl-ethyl-amino)-4-methoxy-
benzothiazol-2-yl amides (compounds 2, 13–19)
benzothiazol-2-yl amides (compounds 2, 18–25)
O
O
O
O
R1
N
R1
N
N
N
S
O
S
O
N
N
Compound R1
A2B cAMP
(IC50, nM)
A1
binding
(Ki, nM)
A2A
binding
(Ki, nM)
Compound R1
A2B cAMP
(IC50, nM)
A1 binding
(Ki, nM)
A2A binding
(Ki, nM)
2
21
100
100
51
N
N
2
21
17
100
2
51
N
N
N
F
F
F
O
13
14
2040
F
20
230
N
O
F
F
F
O
F
O
F
N S
F
21
22
23
49
44
28
78
10
7
1040
1000
470
14
15
41
8
120
61
710
81
N
F
F
O
N
F
F
N
F
O
O
F
F
F
F
F
F
N
N
F
F
16
17
8
72
19
31
NTa
220
31
NTa
180
O
F
F
F
F
F
F
F
F
N
24
25a
26
13
30
26
6
67
20
200
280
52
S
O
N
O
F
F
N
18b
N
F
F
F
F
F
F
F
F
F
N
O
19
42
220
600
N
O
S
F
O
F
F
N
a
27
16
46
110
NT: not tested.
Compound was prepared as the trifluoroacetate salt.
S
b
O
O
a
Compound was prepared as the trifluoroacetate salt.
slight improvement (two- to three-fold) in A2B cAMP potency,
when compared with 2 or 13. Unfortunately, this modest gain in
potency was accompanied by significant losses in A2A selectivity
for both compounds.
when compared with 2, they suffered from complete loss of A1
selectivity.
Analogs 23–27, which were synthesized from either trans- or
cis-3-N-Boc-amino-cyclohexanecarboxylic acid (8), were next eval-
uated. All of these derivatives were of similar potency as com-
pound 2 in the A2B cAMP assay, and most offered improved A2A
binding selectivity (with the exception of 26). Unfortunately, the
poor A1 selectivity for these compounds rendered them unsuitable
for further studies.
Finally, we chose to evaluate a limited number of antagonists
which contained nonaromatic, six-membered ring urea spacers in
place of the amide moiety. Three of the most potent ureas are
shown in Table 4. We were pleased to discover that compared to
compound 2, ureas 28–30 are of similar potency in the A2B cAMP
assay, but have significantly improved A2A selectivity. In the case
of sulfone-containing analogs 19, 21, 27, and 30, we have noted a
trend towards a simultaneous loss of affinity to both A1 and A2A
receptors, while maintaining potency at the A2B receptor. Most
importantly, urea 30, which is equipotent to compound 2 in the
A2B cAMP assay, not only displayed significantly improved A2A
We then procceded to prepare the amine analogs 17 and 18 to
investigate the effects of a methylene linker in conjunction with
the five-membered ring spacers. Disappointingly, replacement of
the amide in 13 with the corresponding amine in 17 resulted in
a five-fold loss in A2B cAMP potency. In the case of the one-carbon
extended analogs 15 and 18, going from a carbonyl linker to a
methylene linker resulted in a slight benefit in terms of selectivity
against the A1 receptor, although with an accompanied loss in A2B
cAMP potency. Finally, the sulfonamide analog 19, although of sim-
ilar potency (within two-fold) in the A2B cAMP assay, provided no
additional benefits in terms of selectivity against A1 and and A2A
,
when compared to compound 13.
Next, we turned our attention to A2B antagonists bearing
six-membered ring nonaromatic spacers (Table 4, compounds
20–22), which were prepared from N-Boc-piperidine-4-carboxylic
acid (7). While these three analogs maintained comparable
potency in A2B cAMP assay and significantly improved A2A selectivity

1936
F. Firooznia et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1933–1936
Table 4
Dr. Paul Gillespie for critical reading of the manuscript and helpful
discussions.
Functional and binding activities of various 7-(acetyl-ethyl-amino)-4-methoxy-
benzothiazol-2-yl amide and ureas (compounds 2, 27–32)
References and notes
O
N
O
R2
N
1. Fredholm, B. B.; Ijzerman, A. P.; Jacobson, K. A.; Klotz, K.-N.; Linden, J.
Pharmacol. Rev. 2001, 53, 527.
2. Fredholm, B. B.; Irenius, E.; Kull, B.; Schulte, G. Biochem. Pharmacol. 2001, 61,
443.
N
H
S
O
3. Volpini, R.; Costanzi, S.; Vittori, S.; Cristalli, G.; Klotz, K.-N. Curr. Top. Med. Chem.
2003, 3, 427.
4. Holgate, S. T. Br. J. Pharmacol. 2005, 145, 1009.
5. Kalla, R. V.; Zablocki, J. Purinergic Signalling 2009, 5, 21.
Compound R2
A2B cAMP
(IC50, nM)
A1 binding A2A
6. Sun, C.-X.; Zhong, H.; Mohsenin, A.; Morschl, E.; Chunn, J. L.; Molina, J. G.;
Belardinelli, L.; Zeng, D.; Blackburn, M. R. 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.; Hoshino, Y.; Yoshikawa, S.; Matsukura, M.; Kabasawa, Y.;
Nijima, 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.
(Ki, nM)
binding
(Ki, nM)
2
21
8
100
19
51
N
N
O
F
N
28
29
F
210
240
9. 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.
F
N
N
10. Cheung, A. W.-H.; Brinkman, J.; Firooznia, F.; Flohr, A.; Grimsby, J.; Gubler, M.
L.; Guertin, K.; Hamid, R.; Marcopulos, N.; Norcross, R. D.; Qi, L.; Ramsey, G.;
Tan, J.; Wen, Y.; Sarabu, R. Bioorg. Med. Chem. Lett. 2010, 20, 4140.
11. 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
18
20
5
F
F
F
O O
S
F
30
690
530
F
F
of Krebs buffer. To each well was added 50
lL of Krebs buffer containing
100 M of the phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)-2-
l
Table 5
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.
Functional/binding activities of selected 7-(acetyl-ethyl-amino)-4-methoxy-ben-
zothiazol-2-yl analogs
Compound A2B cAMP
(IC50, nM)
A1 binding
(Ki, nM)
A2A binding
(Ki, nM)
A2B binding
(Ki, nM)
12. 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 a final
2
13
25
28
21
14
30
8
100
100
67
51
2040
280
8
18
18
5
concentration of 10
DPCPX ligand (4.8 nM final conc., Perkin Elmer), were added to 96-well
polypropylene plates (Becton Dickinson) followed by addition of membranes
lg/well. The test compounds (10 lL) and 40 l
L of [3H]-
19
210
(150
Human A2A membrane receptors (Perkin Elmer) were diluted in assay buffer
(HEPES 50 mM, EDTA 1 mM) to yield a 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
selectivity (10-fold improvement over compound 2), but also
exhibited a superior A1 selectivity profile (ca. seven-fold improve-
ment over compound 2).
In summary, starting with compound 2, and through investiga-
tion of nonaromatic amide derivatives of A2B antagonists containing
the N-(2-amino-4-methoxy-benzothiazol-7-yl)-N-ethyl-acetamide
core structure, we have been able to identify several highly potent
A2B antagonists with very good A2A selectivity Table 5. Moreover,
extension of our SAR exploration into similar urea derivatives led
to the identification of compound 30, which displayed excellent
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
temperature for 1 h on an orbital shaker. Reactions were harvested using 96-
well MultiScreen FB plates (0.5% polyethyleneimine-treated) and
l
L) and [3H]-
l
l
a
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.
A2B potency, as well as good A2A and A1 selectivity. Future commu-
nications will describe further attempts in improving the A1 and
A2A selectivities, as well as in vivo studies with selected A2B
antagonists.
13. Elzein, E.; Kalla, R. V.; Li, X.; Perry, T.; Gimbel, A.; Zeng, D.; Lustig, D.; Leung, K.;
Zablocki, J. J. Med. Chem. 2008, 51, 2267. and references cited therein.
14. Over the course of our studies on various classes of A2B antagonists, 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 range of
Acknowledgments
potencies (single digit nM–lM cAMP IC50 values) and across multiple
structural subtypes (data not shown). Thus, we felt comfortable to use the
more readily available cAMP assay as a measure of both A2B potency and for
the assessment of selectivity.
The authors are grateful to Drs. Claus Riemer and Jean-Luc
Moreau (A2A team) for their generous advices and help throughout
our work and to to Dr. Alexander Alanine for the synthesis of
compound 1. We also thank the Roche Physical Chemistry Depart-
ment for spectroscopic measurements and interpretations, and
15. The values reported are the average of at least two separate experiments,
where typically the duplicate values were within two-fold of each other.