Design, synthesis of novel, potent, selective, orally bioavailable adenosine A2A receptor antagonists and their biological evaluation

Article abstract of DOI:10.1021/acs.jmedchem.6b01584

Our initial structure-activity relationship studies on 7-methoxy-4-morpholino-benzothiazole derivatives featured by aryloxy-2-methylpropanamide moieties at the 2-position led to identification of compound 25 as a potent and selective A₂A adenosine receptor (A₂AAdoR) antagonist with reasonable ADME and pharmacokinetic properties. However, poor intrinsic solubility and low to moderate oral bioavailability made this series unsuitable for further development. Further optimization using structure-based drug design approach resulted in discovery of potent and selective adenosine A₂A receptor antagonists bearing substituted 1-methylcyclohexyl-carboxamide groups at position 2 of the benzothiazole scaffold and endowed with better solubility and oral bioavailability. Compounds 41 and 49 demonstrated a number of positive attributes with respect to in vitro ADME properties. Both compounds displayed good pharmacokinetic properties with 63% and 61% oral bioavailability, respectively, in rat. Further, compound 49 displayed oral efficacy in 6-OHDA lesioned rat model of Parkinson diseases.

Full text of DOI:10.1021/acs.jmedchem.6b01584

Subscriber access provided by GAZI UNIV
Article
Design, Synthesis of Novel, Potent, Selective, Orally Bio-available
2A
Adenosine A Receptor Antagonists and Their Biological Evaluation
Sujay Basu, Dinesh A Barawkar, Sachin Thorat, Yogesh D Shejul, Meena Patel, Minakshi
Naykodi, Vaibhav Jain, Yogesh Salve, Vandna Prasad, Sumit Chaudhary, Indraneel Ghosh,
Ganesh Bhat, Azfar Quraishi, Harish Patil, Shariq Ansari, Suraj Menon, Vishal Unadkat,
Rhishikesh Thakare, Madhav S Seervi, Ashwinkumar V Meru, Siddhartha De, Ravi K
Bhamidipati, Sreekanth Rouduri, Venkata P Palle, Anita Chug, and Kasim A Mookhtiar
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01584 • Publication Date (Web): 05 Jan 2017
Downloaded from http://pubs.acs.org on January 5, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Design, Synthesis of Novel, Potent, Selective, Orally Bio-available Adenosine A2A Receptor
Antagonists and Their Biological Evaluation
Sujay Basu*, Dinesh A. Barawkar, Sachin Thorat, Yogesh D. Shejul, Meena Patel, Minakshi
Naykodi, Vaibhav Jain, Yogesh Salve, Vandna Prasad, Sumit Chaudhary, Indraneel Ghosh,
Ganesh Bhat, Azfar Quraishi, Harish Patil, Shariq Ansari, Suraj Menon, Vishal Unadkat,
Rhishikesh Thakare, Madhav S. Seervi, Ashwinkumar V. Meru, Siddhartha De, Ravi K.
Bhamidipati, Sreekanth R. Rouduri, Venkata P. Palle, Anita Chug, Kasim A. Mookhtiar
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Advinus Therapeutics Ltd., Drug Discovery Facility, Quantum Towers, Plotꢀ9, PhaseꢀI, Rajiv
Gandhi Infotech Park, Hinjawadi, Pune 411 057, India.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT:
Our initial structureꢀactivity relationship studies on 7ꢀmethoxyꢀ4ꢀmorpholinoꢀbenzothiazole
derivatives featured by aryloxyꢀ2ꢀmethylpropanamide moieties at the 2ꢀposition lead to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
identification of compound 25 as a potent and selective A2A adenosine receptor (A AdoR)
2A
antagonist with reasonable ADME and pharmacokinetic properties. However, poor intrinsic
solubility and low to moderate oral bioavailability made this series unsuitable for further
development. Further optimization using structureꢀbased drug design approach resulted in
discovery of potent and selective adenosine A2A receptor antagonists bearing substituted 1ꢀ
methylcyclohexylꢀcarboxamide groups at position 2 of the benzothiazole scaffold and endowed
with better solubility and oral bioavailability. Compounds 41 and 49 demonstrated a number of
positive attributes with respect to in vitro ADME properties. Both compounds displayed good
pharmacokinetic properties with 63% and 61% oral bioavailability respectively, in rat. Further,
compound 49 displayed oral efficacy in 6ꢀOHDA lesioned rat model of Parkinson diseases.
INTRODUCTION
Adenosine, an endogenous purine nucleoside, and mediates its effect through four specific cell
membrane receptors called adenosine receptors (AdoRs). These AdoRs, named A , A , A , and
1
2A
2B
1
A in human, belong to the Gꢀproteinꢀcoupled receptors (GPCR) family. AdoRs exhibit high
3
sequence homology but differ in their affinity for adenosine and downstream signaling
2
ꢀ3
mechanism. AdoRs have been implicated in several key physiological processes, and thus
offer broad therapeutic potentials including cell growth, asthma, immune system, inflammatory
4ꢀ6
diseases and central nervous system (CNS) disorders, etc.
ACS Paragon Plus Environment

Page 3 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Adenosine A receptor (A AdoR) is a highly distributed receptor in the CNS. It is expressed at
2
A
2A
high levels in the nigrostriatum (basal ganglia), where it coꢀlocalizes with dopamine D2
7
receptors on striatopallidal output neurons. Several pharmacological studies suggest that
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A AdoR antagonists have potential for use in combination with existing therapies across
2
A
8
multiple therapeutic area. Parkinson’s disease (PD) is a progressive neurodegenerative
movement disorder affecting approximately 1% population over the age of 65. Unfortunately,
current dopamine replacement therapies for PD suffer from poor long term control and
undesirable side effects, mainly dyskinesia (involuntary movements). Antagonism of A AdoR
2
A
9
offers a nonꢀdopaminergic approach to treat PD. Stimulation of A AdoR decreases the binding
2
A
1
0
affinity of dopamine for dopamine D2 receptors and elicits effects, opposite to dopamine D2
11
receptor activation at the level of second messenger system and earlyꢀgene expression.
Another potential disease modifying benefit of A AdoR antagonism is the prevention of
2
A
development of levodopa (LꢀDOPA) induced dyskinesias in PD as demonstrated in rodent and
1
2
monkey studies. A AdoR are expressed on several immune cell typesꢀ T lymphocytes,
2
A
dendritic cells, natural killer cells. A AdoR activation on T cells and NK cells causes
2
A
immunosuppression by reducing their proliferation, cytokine production and tumor killing
1
3ꢀ14
activity.
In recent years, A AdoR has shown exciting progress in the development of
2A
1
5
immunotherapy for the treatment of cancer and multiple deals emphasize commercial potential
of A AdoR antagonists. Numerous research groups have devoted significant effort towards the
2
A
discovery of selective A AdoR antagonists for the treatment of Parkinson's disease. Some
2
A
1
6
representative examples are shown in Figure 1. A AdoR antagonists are classified as xanthine
2A
1
7ꢀ19
and nonꢀxanthine derivatives.
Numerous A AdoR antagonists have reached phase I clinical
2A
trials and beyond. Among xanthine derivatives, 1 (Istradefylline, KWꢀ6002, Kyowa Hakko Kirin
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
16
Co Ltd.), was launched in Japan for PD. Several classes of nonꢀxanthine A AdoR antagonists
2
A
2
0ꢀ21
22ꢀ23
have been reported in literature and they are either monocyclic,
bicyclic
or tricyclic coreꢀ
2
4ꢀ26
based antagonists.
However, the discovery of A AdoR antagonist with acceptable
2
A
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
selectivity, physicochemical, and pharmacological properties has continued to remain
challenging and consequently, majority of research has progressed towards identifying novel
nonꢀxanthine scaffolds. HoffmannꢀLa Roche has identified benzothiazole scaffold based 2
2
7ꢀ28
(SYNꢀ115)
which is a potent, selective A AdoR antagonist with desired ADME properties
2A
namely, and is currently in phase III clinical trials.
We initiated our research with the goal to discover a novel, potent and selective A AdoR
2
A
antagonist with desirable physicochemical, pharmacokinetic and pharmacological properties to
provide therapeutic intervention for PD and cancer immunotherapy. Amongst A AdoR
2
A
antagonists with several structural types, benzothiazole and thiazolo[5,4ꢀc]pyridine scaffold were
chosen as starting point and new analogs were discovered subsequently (Figure 2). Herein, we
describe the discovery and optimization of novel potent A AdoR antagonist based on the
2
A
chemical structure of 2. After analyzing the available A coꢀcrystal structures with antagonists
2
A
and docking pose of 2, we decided to modify the most flexible part on right hand side of 2 with
well tolerated and novel aryloxyꢀ2ꢀmethylpropanamide or cyclohexaneꢀcarboxamide moieties
(
Figure 2). In the present study, a structure–activity relationship (SAR) was established via
synthesis of analogues of 2 having urea linker replacement and various terminal substituents as
shown in schemes 1 to 3.
ACS Paragon Plus Environment

Page 5 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1, Istradefylline
Kyowa-Hakko Kirin
2, Tozadenant (SYN-115)
Roche/UCB/Biotie
3, Heptares
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4, Vipadenant
Biogen/Vernalis
5, Preladenant
Merck
Figure 1. Representative examples of A AdoR antagonists
2
A
Aryloxy-2-methylpropanamide, HIT (12)
Cyclohexane-carboxamide, Lead (49)
Figure 2. Identification of novel aryloxyꢀ2ꢀmethylpropanamide and cyclohexaneꢀcarboxamide.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CHEMISTRY
a
Scheme 1. Synthesis of 12-36
i
ii
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
8
9
7
a: R= p-F; b: R= m-F; c: R= p-CH
d: R= 2,4-diF; e: R= 2,6-diF
f: R= 3,4-diF; g: R= 3,4-diOMe
h: R= m-(2-methoxyethoxy)
i: R= 1,3-dioxole; j: R= p-Cl
3
k: R= p-CF
m: R= p-t-Butyl; n: R= o-F; o: R= m-CF
3
; l: R= p-CN
3
iii
9
1
1
0 (X=C)
1 (X=N)
1
2
2-24 (X=C)
5-36 (X=N)
a
o
Reagents and conditions: (i) K CO , DMF, rt, 16 h; (ii) NaOH, THF/MeOH/H O (3:2:1), 80 C,
2
3
2
16 h; (iii) EDCI, HOBt, NMM, DMF, rt.
In scheme 1, substituted ethyl 2ꢀmethylꢀ2ꢀphenoxyꢀpropanoate derivatives (8) were prepared by
reaction of ethylꢀ2ꢀbromoisobutyrate (6) and phenol (7) in DMF at rt in presence of K CO3.
2
2
9
Hydrolysis of these variously substituted esters 8a-o under aqueous basic conditions yielded
27ꢀ28
30
acid derivatives 9a-o. Amide coupling of 9a-o with 10
2ꢀ36 in overall good yields.
and 11 afforded final compounds
1
ACS Paragon Plus Environment

Page 7 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
Scheme 2. Synthesis of 40-43
O
N
O
1
R1
N
S
R
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
H
O H
O
N
iii
O
O
O
40
O
O
N
S
N
H
O
O H
O
O
1
i
ii
iv
N
S
R
N
H
OH
O
O
O
N
O
37
38
39
OH
OH
41-43
a
Reagents and conditions: (i) LiOH.H O, THF/MeOH/H O (3:2:1), rt, 18 h; (ii) 10, EDCI,
2
2
o
HOBt, NMM, DMF, rt,18 h; (iii) NaBH , EtOH, 0 C ꢀrt, 3 h; (iv) 39, appropriate Grignard
4
o
reagents, dry THF, 0 C ꢀ rt, 1h.
The synthesis of compounds 40ꢀ43 is outlined in scheme 2. Starting material 37, prepared as
31
described in literature and its hydrolysis under aqueous basic conditions yielded 38. Amide
coupling of 38 with 10 afforded compound 39. Reduction of 39 with NaBH yielded final
4
compound 40, with terminal secondary hydroxyl group. Compound 39, when treated with 1.2
molar equivalents of appropriate Grignard reagents gave corresponding tertiary hydroxyl
derivatives as a mixture of isomers 41-43.
Compound 49 was synthesized as illustrated in Scheme 3. Compound 37 upon treatment with
3
2
methanolic ammonia followed by hydrogenation gave corresponding amine 44, which was
further protected by bocꢀanhydride to yielded 45. Hydrolysis of 45, under aqueous basic
conditions yielded 46. Amide coupling of 46 with 10 using standard EDCIꢀHOBt method
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
afforded 47 which on treatment with methanolic HCl provided compound 48. Finally, compound
49 (mixture of cis and trans) was prepared from compound 48 by reductive amination.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 3. Synthesis of 49
i
ii
iii
iv
3
7
44
45
46
47
v
vi
4
8
4
9
a
Reagents and conditions: (i) Methanolic NH , H , 10 % Pd/C, rt, 24 h; (ii) Boc anhydride, Et N,
3
2
3
o
MeOH, rt , 4 h; (iii) LiOH.H O, THF/MeOH/H O (3:2:1), 60 C,18 h; (iv) 10, EDCI, HOBt,
2
2
NMM, DMF , rt, 14h; (v) methanolic HCl, DCM, rt 24 h; (vi) acetaldehyde, NaCNBH , MeOH,
3
NaOAc, rt ,24 h.
RESULTS AND DISCUSSION
Biological data and structure–activity relationships
Initially, a new series of 2ꢀsubstituted benzothiazole and thiazolo[5,4ꢀc]pyridine derivatives were
prepared with the objective of identifying novel, potent and selective A AdoR antagonists with
2
A
ACS Paragon Plus Environment

Page 9 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
acceptable pharmacokinetic properties. For this purpose, urea linkage of 2 was replaced by
aryloxyꢀ2ꢀmethylpropanamide moieties. The compounds were first evaluated for affinity against
A AdoR and only compounds showing A affinity <20 nM were evaluated for binding
2
A
2A
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
selectivy over A , A and A AdoR. The data obtained is listed in Table 1. Most of the aryloxyꢀ
1
2B
3
2ꢀmethylpropanamide derivatives, 12-24 (except 21-24) showed good binding affinity with K in
i
the range of 2.8ꢀ22 nM for human A AdoR (hA ). Compounds 25-36 (thiazolo[5,4ꢀc]pyridine
2
A
2A
core) also exhibited good binding affinity with K in the range of 3.4ꢀ55 nM for human
i
A AdoR. In general, compounds containing small electron withdrawing groups like ꢀF, ꢀdiꢀF or
2
A
electron releasing groups such as ꢀMe, and –OMe on phenyl ring (compound 12-20) showed
good binding affinity with K of <22 nM for A AdoR.
i
2A
However, compounds 21-23, with larger electron withdrawing groups such as –Cl, ꢀCF , ꢀCN,
3
showed modest binding affinity of 51, 76 and 58 nM, respectively, for A AdoR. Compound 24
2
A
with more bulkier and hydrophobic pꢀtꢀbutyl substitution had large negative effect on the
affinity, and reduce binding K of 500 nM for A AdoR. Compounds 34-36 with electron
i
2A
releasing group had moderate A AdoR binding affinity of 42, 55 and 42 nM, respectively.
2
A
Having identified compounds with good binding affinity, potent compounds with binding K < 20
i
nM for A AdoR were tested for adenosine receptor subtype selectivity. Compounds 12-20, 25,
2
A
30, 31 and 33 had moderate to good binding selectivity, while compound 26 showed poor
selectivity and compound 32 showed moderate selectivity over A , A and A AdoR
1
2B
3
The potent and selective compounds, selected on the basis of binding affinity were subsequently
assessed for stability in liver microsomes. Most of the compounds were metabolically stable in
rat liver microsomes (RLM) and human liver microsomes (HLM) (Table 1). Compounds with
very low nanoꢀmolar binding affinity (K <20 nM), good binding selectivity and metabolically
i
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
stable, were further tested for functional potency in cAMP assay using hA ꢀHEK293 cells
2
A
(
Table 1). Compound 12-18, 20 and 25-26 showed excellent functional potency with K in the
i
range of 0.95ꢀ8.3 nM. Compound 30-33 exhibited moderate functional potency with K in the
i
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
range of 13.5ꢀ28 nM and were stable in RLM and HLM. To identify a lead, three potent,
selective and metabolically stable compounds namely 12, 17 and 25, were shortlisted for further
profiling. The data is summarized in Table 2 and 3.
Table 1. Affinity, potency, selectivity and microsomal stability of aryloxyꢀ2ꢀmethylpropanamide
derivatives
X = C: 12-24
X = N: 25-36
.
c
%
Inhibition at 1
µM
MR
Binding
Functional
(nmol/min/mg)
Compound
R
K (nM)
hA2A
K (nM)
i
i
a
b
a
a
a
hA2A
hA1
16
52
7
hA2B
hA3
11
20
5
RLM
0.04
0.1
HLM
0.02
0.08
0.09
0.11
0.16
0.07
0.06
12
13
pꢀF
mꢀF
13
2.8
5.4
7
0
4.2
0.95
8.3
2.6
1.0
2.4
1.8
36
14
0
14
15
16
17
18
pꢀCH
3
0.1
2,4ꢀdiF
2,6ꢀdiF
33
37
38
0
21
10
28
0
0.07
0.09
0.09
0.11
8
19
34
25
3,4ꢀdiF
6.4
19
3,4ꢀdiOMe
ACS Paragon Plus Environment

Page 11 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
mꢀ(2ꢀ
methoxyethoxy)
1
9
0
22
17
34
0
38
0
20
29
nt
nt
nt
nt
mꢀ(1,3ꢀ
benzodioxole
2
2.1
0.05
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
2
2
2
2
2
2
2
2
3
3
3
1
2
3
4
5
6
7
8
9
0
1
2
pꢀCl
51
76
58
500
3.4
14
21
29
24
13
15
12
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
pꢀCF
3
pꢀCN
pꢀtertꢀButyl
pꢀF
nt
nt
46
90
nt
24
42
nt
46
94
nt
6.5
4.7
nt
<0.04 <0.04
<0.02 <0.04
mꢀF
oꢀF
nt
nt
nt
nt
nt
nt
pꢀCl
nt
nt
nt
nt
mꢀCF
nt
nt
nt
nt
3
2,4ꢀdiF
2,6ꢀdiF
3,4ꢀdiF
39
33
50
11
42
42
47
48
47
13.5
26.5
24
<0.01 <0.03
0.05
0.03
0.11
0.05
mꢀ(2ꢀ
methoxyethoxy)
33
14
42
38
nt
23
nt
41
nt
28
nt
0.05
nt
0.1
nt
mꢀ(1,3ꢀ
benzodioxole)
34
35
36
pꢀCH
3
55
42
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
mꢀOCH
3
a
ntꢀ not tested. Binding affinities were determined by using membrane preparations from HEKꢀ293 cells
overexpressing the relevant human AdoR isoform. All data points were evaluated in triplicates For Ki
b
determination, 6 concentrations IC50 curves were plotted. Compounds were evaluated at 12
concentrations, each data point in triplicates ( n =2). The mean K from two independent experiments has
been reported. Metabolic Rate (MR) <0.1 nmol/min/mg is considered metabolically stable. RLM: Rat
liver microsomes, HLM: Human liver microsomes.
i
c
All three compounds (12, 17 and 25) showed high binding as well as functional selectivity over
other AdoRs similar to that of 2 (Table 2). These compounds did not show any CYP liability
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
against major CYPs (data not shown) and hERG liability in binding assay (<5% inhibition at 3,
10 µM for compound 12 and 17; <10 % inhibition at 10, 30 µM for compound 25). Compound
25 was found to be stable in rat hepatocytes. Among these three compounds, 25 showed better
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
pharmacokinetic (PK) profile compared to 12 and 17 with Cmax of 1.1 µM, systemic exposure
AUC(0ꢀt) of 12 µM.h, t of 5.4 h and oral bioavailability of 21% (Table 3).
1/2
Table 2. Binding affinity and functional potency of compounds 12, 17, 25 and 2
Binding K (nM)
Functional K (nM)
i
i
Compound
a
a
a
a
b
b
b
hA2A
hA1
hA2B
hA3
hA2A
hA1
hA2B
1
1
2
2
7
5
13
5000 10000 5000
4.2
2000
nr
nr
6.4
3.4
11
1100 5000
500 1400
500
400
2.4
nr
6.5
nr
2000
2
1700 5000 5000
0.3
nt
nt
a
ntꢀnot tested, nrꢀ no response at highest tested concentration 10 µM.
Binding
affinities
were
determined by using membrane preparations from HEKꢀ293 cells overexpressing the relevant human
AdoR isoform. Compounds were evaluated at 8 concentrations with each data point in triplicates ( n = 2).
b
Compounds were evaluated at 12 concentrations, each data point in triplicates (n = 2). The mean K from
i
two independent experiments has been reported.
a
Table 3. Pharmacokinetic profile of compounds 12, 17 and 25 in male Wistar Rats
Compound
12
17
25
Route of
administration
IV
PO
IV
PO
IV
PO
Dose (mg/kg)
Cmax (µM)
Tmax (h)
3
10
3
10
3
10
NA
NA
0.19 ± 0.0
0.5 ± 0.0
NA
NA
1.33 ± 0.5
0.50 ± 0.0
NA
NA
1.1 ± 0.1
2.75 ± 0.9
12 ± 1.3
AUC0ꢀt
7.6 ± 0.6 1.5 ± 0.25 10.7 ± 2.2 6.2 ± 1.7 16.4 ± 0.6
ACS Paragon Plus Environment

Page 13 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
µM.h)
V (L/Kg)
ss
1.4 ± 0.2
5 ± 1.2
NA
NA
2.1 ± 0.44
10.2 ± 2.5
NA
NA
2.1 ± 0.3
6.6 ± 0.2
NA
NA
CL
1
(mL/min/Kg)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
t (h)
6.3 ± 0.6
NA
NA
6
5.5 ± 0.6
NA
NA
5.4 ± 1.0
NA
NA
1
/2
F (%)
17 ± 4.7
21.7 ± 2.4
a
Values indicate mean for n = 4. NA: Not applicable; Compound 12 : IVꢀNMPꢀ10%, PEG300ꢀ15%,
20 % w/v HPßꢀCD in 0.1 M acetate buffer (pH 4.2) q.s; Compound 17: IVꢀNMPꢀ10%,Cr EL 10%,
.1 M acetate buffer (pH 4.2) q.s; Compound 25: Vehicle: IVꢀNMPꢀ10%, Cr EL ꢀ10%, PEGꢀ300ꢀ 10
, 0.1 M Ammonium acetate buffer q.s (pH 4.2); Compound 12, 17 and 25: PO: Tween 80ꢀ 1%,
.5% w/v NaCMC q.s.; NA: Not applicable
0
%
0
All three compounds from aryloxyꢀ2ꢀmethylpropanamide series however showed poor oral
bioavailability which could be due to poor solubility (aqueous solubility of compounds 12, 17
and 25 are <6 µM at various pH such 2.1, 4 and 7.4). To overcome the issue of bioavailability
and solubility of these compounds, we later assessed a series of cyclohexaneꢀcarboxamide
derivatives, where a more rigid linker was employed and the phenyl ring was replaced by
cyclohexane, thus reducing the potential stacking interaction. Further, compounds 40-43 were
prepared with polar –OH group attached to cyclohexane ring and 49 containing tertiary amine
with the intention to improve solubility. Compounds 40-43 (41-43 are mixture of geometrical
isomers) retained binding affinity (K = 2.1 ꢀ 25 nM) and functional potency (K = 0.2 ꢀ 2.1 nM)
i
i
for A2A AdoR (Table 4). Compounds 40, 41 and 49 showed single digit nanoꢀmolar binding
affinity with K value of 5.9 nM, 8.6 nM and 2.1 nM, respectively. These compounds (40, 41and
i
4
9) also exhibited subꢀnanomolar functional potency with K value of 1.1 nM, 0.25 nM and 0.20
i
nM, respectively. These compounds were further tested for AdoR binding selectivity. Compound
1 (with tert-hydroxy group) and 49 (with tertꢀamine group) showed high selectivity over
4
A AdoR (>300 fold) while no response was seen in A AdoR. Both compounds showed moderate
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
to high selectivity over A AdoR (>50 fold for 41 and >200 fold for 49). Compound 40 however
2
B
showed low binding selectivity over A AdoR (A /A = 15 fold) and A AdoR (A /A = 4 fold)
1
1
2A
2B
2B 2A
but high selectivity over A AdoR. When tested for functional selectivity, compound 40 exhibited
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
low selectivity over A AdoR (A /A = 38 fold) and high selectivity over A AdoR (A /A =
1
1
2A
2B
2B 2A
900 fold). Compounds 41 and 49 were further tested for functional selectivity and found to be
highly selective against human A AdoR and A AdoR (Table 4).
1
2B
ACS Paragon Plus Environment

Page 15 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 4.- Affinity, potency and selectivity of cyclohexaneꢀcarboxamide derivatives
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Functional
K (nM)
i
Binding K (nM)
i
1
Compound
R
a
a
a
a
b
b
b
hA2A hA1 hA2B hA3
hA2A
hA1 hA2B
40
41
42
5.9
8.6
25
90
23
nr
nr
nt
1.1
41.8 1000
2900 500
0.25
nt
nr
nt
2000
nt
nt
nt
nt
nt
43
17
nt
nr
2.1
nt
nr
nt
49
2.1
1200 500
0.20
650
a
ntꢀ not tested; nrꢀ no response at highest tested concentration 30 µM. Binding affinities were
determined by using membrane preparations from HEKꢀ293 cells overexpressing the relevant
human AdoR isoform. Compounds were evaluated at 8 concentrations with each data point in
b
triplicates (n=2). Compounds were evaluated in triplicates at 12 concentrations. The mean Ki
from two independent experiments has been reported (n=2)
.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Based on the overall profile of best compounds 41 and 49 in terms of high binding as well as
functional potency and selectivity, they were progressed for further profiling and the data has
been summarized in Table 5. In general, this series of compounds showed high aqueous
solubility across all pH. Among 41 and 49, the latter compound (49) showed high aqueous
solubility across all pH (Table 5). Both the compounds were metabolically stable in RLM and
HLM as well as in rat hepatocytes. Compound 41 had high permeability towards absorptive
direction (AꢀB) in Cacoꢀ2 cells, while compound 49 showed moderate permeability in the same.
In addition, both compounds did not show any major CYP and hERG liability, and were not
cytototoxic (IC >100 µM) to HepG2 cells. Serum protein binding of the compounds 41 and 49
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
were found to be 87% and 76%, respectively, as determined in vitro in spiked samples of rat.
Compounds 41 and 49 were further evaluated for PK profile before proceeding for in vivo
efficacy studies. A moderate systemic clearance (~60ꢀ65% of hepatic blood flow) was observed
for these compounds, in accordance with low intrinsic clearance in rat hepatocytes (Table 6).
Volume of distribution of compound 41 was found to be ~ 2 fold higher, while volume of
distribution of compound 49 was approximately 8 fold higher than total body water normalized
to body weight of rat. Furthermore, both compounds exhibited high oral bioavailability (F =
63%and 61% for compound 41 and 49, respectively). Compound 49 had much better half life
than compound 41. Compound 49 displayed low brain penetration (brain/plasma AUC Ratio=
0
ꢀt
0.25 ± 0.08; Plasma AUC (µM.hr)= 4.7 ± 1.7; Brain AUC (µM.hr)= 1.1 ± 0.3; 10 mg/kg po,
0
ꢀt
0ꢀt
in Wistar rats, n=4).
ACS Paragon Plus Environment

Page 17 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 5. In vitro drug metabolism and pharmacokinetics (DMPK) of compound 41 and 49
CYP P450
Inhibition
Rat
a
hERG; %
(IC : µM; Binding
MR
(
Aqueous
hepatocyte Cacoꢀ2
t (min); permeability
parent (nm/sec)
remaining (AꢀB)
RLM HLM at 60 min
5
0
nmol/min/mg)
Solubility at
pH 2.1, 4.0
and 7.4(µM)
1
/2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compound
for
C9, 2C19, at 10 and
2D6 and 30 µM
A4)
1A2, Inhibition
%
2
3
1
39, 111,
114
4
1
9
<0.04 <0.04
>60; 88
>60; 89
122
All >25
All >50
NI, 26
13, 18
1
840, 1807,
4
0.01
0.03
37
1586
a
NIꢀ No inhibition; Metabolic Rate (MR) < 0.1 nmol/min/mg is considered metabolically stable. RLM:
Rat liver microsomes, HLM: Human liver microsomes
a
Table 6. Phamacokinetic profile of compounds 41 and 49 in male Wistar Rats
Compound
41
49
Route of
administration
IV
PO
IV
PO
Dose (mg/kg)
Cmax (µM)
Tmax (h)
3
10
3
10
2.02 ± 1.1
0.63
NA
NA
5.4 ± 1.6
0.5 ± 0.0
NA
NA
AUC0ꢀt
3
.7 ± 0.5
7.8 ± 2.4
NA
2.7 ± 0.2
6.1 ± 0.6
37.2 ± 3.1
5.5 ± 0.6
NA
(µM.h)
V (L/Kg)
ss
1.5 ± 0.3
32 ± 4.2
CL
mL/min/Kg)
NA
NA
(
t (h)
0.6 ± 0.07
NA
NA
2.4 ± 0.3
NA
NA
1
/2
F (%)
63 ± 19
61.0 ± 7.0
a
Values indicate mean for n = 4 Compound 41: Vehicle: IVꢀNMPꢀ10%, PEGꢀ300ꢀ 15 %, 0.1 M
Ammonium acetate buffer q.s (pH 4.2); Compound 49: Vehicle: IVꢀNMPꢀ10%, Cr EL ꢀ10%, 0.1 M
Ammonium acetate buffer q.s (pH 4.2); Compound 41, 49: PO: Tween 80ꢀ 1%, 0.5% w/v NaCMC q.s.
NA: Not applicable
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
MOLECULAR MODELING
Molecular modeling studies were carried out to understand the binding poses and intermolecular
interactions of representative active molecules as well as to rationalize the observed SAR
differences among the aryloxyꢀ2ꢀmethylpropanamide series compounds. Molecular docking
analysis of 2 in A AdoR showed that it fits nicely in the adenosine binding site. Based on the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
A
docking model of 2, we identified aryloxyꢀ2ꢀmethylpropanamide and cyclohexaneꢀcarboxamide
as suitable replacement for 4ꢀhydroxyꢀmethyl piperidine moiety as it can be altered easily
without losing the key interactions and activity. Compounds 41-49 were obtained as a mixture
containing both cis and trans geometric isomers. Molecular docking analysis of compounds 41
and 49 isomers revealed that both can fit nicely in the A2A binding site in a similar binding
orientation. However, compounds 41 cis isomer and 49 trans isomer were having better Glide
score and more intermolecular interactions as compared to their other isomeric counterpart
(Figure S1, Supplementary information). Therefore, we have compared only the superior
geometric isomer of the compounds with 2. Docking pose overlay of the representative active
compounds (12, 41 and 49) on 2 showed that compounds binding pose and orientation are
similar to that of 2 (Figure 3). Furthermore, Glide docking score of the compounds 12 (ꢀ7.99
kcal/mol), 41 (ꢀ7.58 kcal/mol) and 49 (ꢀ6.89 kcal/mol) were also comparable to that of 2 (ꢀ7.58
kcal/mol).
In detail, binding model of 2 showed that benzothiazole core was buried in the hydrophobic
pocket; had piꢀpi interaction with Phe168 and hydrophobic interactions with Leu249 and Ile274.
Methoxy group and carbonyl group of the compound formed hydrogen bonds with Asn253 and
Tyr271 side chains, respectively. Morpholine group was surrounded by a few hydrophobic
groups and the top portion 4ꢀhydroxyꢀmethyl piperidine was exposed to solvent front as shown
ACS Paragon Plus Environment

Page 19 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
in Figure 3 and 4. Individual docking poses of compounds 12, 41 and 49 showed that they
retained the intermolecular interactions similar to that of 2 (Figure 4). Interestingly, these
compounds made additional interactions viz. compound 12 dimethyl groups hydrophobically
interacted with Tyr271 and Leu267; hydroxyl group of compound 41 at cyclochexyl position
formed hydrogen bond with Glu169; compound 49, tꢀnitrogen had cationicꢀpi interaction with
His264. The 2D interaction diagram for all the four compounds is provided in supplementary
Figure S1. We also attempted to justify the reason behind low potency of compound 24 using our
docking model, which suggested that pꢀtertꢀbutyl of phenoxy ring could be causing steric clashes
with Lys153. The most plausible reason for modest/low potency of compounds 21-24 with larger
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
electron withdrawing groups such as –Cl, ꢀCF , ꢀCN and –C(CH ) is due to their unfavorable
3
3 3
interactions with the polar bulk solvent.
Figure 3. Overlay of Compound 12 (Cyan), 41 (magenta) and 49 (salmon) on reference
compound 2 (green) in the A receptor (shown in cartoon representation) ligand binding pocket.
2
A
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Docking poses of reference compound 2 and representative active compounds 12, 41
and 49 in the A AdoR. Ligands are shown in magenta color and stick representation, while
2
A
residues are shown in green and line representation. Protein is shown in cartoon rendering in the
background.
ACS Paragon Plus Environment

Page 21 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
IN VIVO EFFICACY STUDY
Compound 49 was tested for efficacy in potentiation of levodopaꢀinduced contralateral rotations
in 6ꢀOHDA (6ꢀhydroxydopamine) lesioned rats. Compound 49, at 30 and 100 mg/kg, p.o, was
tested in Lꢀdopaꢀinduced rotations in 6ꢀOHDA lesioned rats and showed dose dependent increase
in contralateral rotations, post 60 min administration. The potentiation of LꢀDopa induced
contralateral rotations, measured upto 2 h, with compound 49 at 100 mg/kg, p.o. was significant
when compared to LꢀDOPA (4 mg/kg, i.p.) alone. Compound 2 (30 mg/kg, p.o.) also showed
significant effect on potentiation of LꢀDOPA induced contralateral rotations measured up to 2 h
in 6ꢀOHDA lesioned rats as compared with LꢀDOPA (4 mg/kg, i.p.) alone (Figure 5).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. Effect of compound 49 on potentiation of levodopaꢀinduced contralateral rotations in
6ꢀOHDA lesioned rats. The data represent Mean ± SEM (n=6). *Significantly different as
compared to LꢀDOPA, 4 mg/kg, IP alone group (*P < 0.05).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CONCLUSIONS
We have identified aryloxyꢀ2ꢀmethylpropanamide and cyclohexaneꢀcarboxamide based
compounds with good potency for A AdoR and high selectivity against A , A and A AdoR.
2
A
1
2B
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
These compounds showed molecular recognition interactions with A AdoR similar to that of 2.
2
A
Several compounds showed low nanoꢀmolar activity with reasonable selectivity and ADME
properties. Though the initial aryloxyꢀ2ꢀmethylpropanamide series showed low to moderate oralꢀ
bioavailability; cyclohexaneꢀcarboxamides had better oralꢀbioavailability than former.
Compound 49 was identified as a potent and selective A AdoR antagonist with desirable
2A
physicochemical and drugꢀlike properties, including high oral bioavailability. The compound on
further profiling also showed in vivo efficacy in PD animal model where it potentiated the Lꢀ
DOPA induced contralateral rotations significantly at 100 mg/kg, the compound can thus be
considered as a potential lead for further exploration. Data from further optimization and
evaluation of these series of compounds and their in vivo efficacy study will be reported in due
course of time.
ACS Paragon Plus Environment

Page 23 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
EXPERIMENTAL SECTION
General Chemistry - Commercial chemicals and solvents were of reagent grade and were used
without further purification. Anhydrous solvents were used without further drying. The
following abbreviations are used for reagent and solvents: DCMꢀdichloromethane; DMFꢀ
dimethyl formamide; DMSOꢀdimethyl sulfoxide; EtOAcꢀethyl acetate; EtOHꢀEthanol; and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
MeOHꢀmethanol.
EDCIꢀ
1ꢀEthylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide,
HOBtꢀ1ꢀ
Hydroxybenzotriazole, NMMꢀNꢀMethylmorpholine Globe chemie silica gel (100ꢀ200 or 230ꢀ
400 mesh) was used for column chromatography. Analtech thin layer chromatography plates (20
1
x 20 cm, 2000 microns) were used for preparative thin layer chromatography. Proton NMR ( H
NMR) spectra were recorded on a Varian 400 spectrometer (400 MHz). Solutions were typically
prepared in either deuterated dimethyl sulfoxide (DMSOꢀd ), deuterated methanol (CD OD) or
6
3
deuterated chloroform (CDCl ). Chemical shifts are reported in δ units (parts per million)
3
downfield from tetramethylsilane and are assigned as: singlet (s), doublet (d), doublet of doublet
(dd), triplet (t), quartet (q) and multiplets (m), and broad (br). Coupling constants (J) are reported
in Hertz (Hz). Mass spectra (MS) were recorded on Agilent 6110. HPLC were recorded on
Agilent RRLC using Eclipse XOBꢀC18 (250x4.6) mm Su column and 0.05 % fumaric acid (aq.)
and acetonitrile as mobile phase with flow rate of 1ml/min at 30 °C for run time of 17 min and
the HPLC purity is ≥95% unless otherwise stated.
General Procedure for the Synthesis of Compounds 12-36.
To a solution of substituted amines (10 or 11) (1 equiv) and intermediate 9 (1.3 equiv) in DMF
(2 mL) were added 1ꢀ(3ꢀDimethylaminopropyl)ꢀ3ꢀethylcarbodiimide hydrochloride (1.5 equiv),
1ꢀhydroxybenzotriazole (1 equiv) and Nꢀmethylmorpholine (1.5 equiv). The reaction mixture
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was stirred at room temperature for 14 h. The reaction mixture was quenched with water and the
solid obtained was filtered. The crude product was purified by flash chromatography (100ꢀ200
silica gel, ethylacetate in hexane 0/100 to 50/100) to afford the products (12-36).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2-(4-Fluorophenoxy)-N-(4-methoxy-7-morpholino-1,3-benzothiazol-2-yl)-2-methyl-
o
1
propanamide (12) . Off white solid; 23% yield; mp 178ꢀ179 C; H NMR (DMSOꢀd ): δ 12.67
6
(s, 1H), 7.07ꢀ7.12 (m, 2H), 6.91ꢀ6.94 (m, 4H), 3.83 (s, 3H), 3.79 (t, J = 4.4 Hz, 4H), 3.01 (t, J =
1
3
4
.4 Hz, 4H), 1.57 (s, 6H); C NMR (DMSOꢀd ): δ 173.2, 157.7, 156.4, 150.6, 148.2, 140.0,
6
+
1
39.1, 126.7, 121.9, 115.7, 113.0, 108.1, 80.3, 66.5, 55.8, 51.4, 24.2; MS (m/z ) 446.1(M + H) ;
+
HPLC 98%; HRMS (C H FN O S) calulated 446.1544 (M + H) ; found 446.1541
2
2
25
3
4
2-(3-Fluorophenoxy)-N-(4-methoxy-7-morpholino-1,3-benzothiazol-2-yl)-2-methyl-
o
1
propanamide (13). Off white solid; 23% yield; mp 170ꢀ171 C; H NMR (DMSOꢀd ): δ 12.75
6
(bs, 1H), 7.27ꢀ7.33 (m, 1H), 6.91ꢀ6.96 (m, 2H), 6.82ꢀ6.84 (m, 1H), 6.70ꢀ6.76 (m, 2H), 3.85 (s,
+
3H), 3.78 (t, J = 4.4 Hz, 4H), 3.02 (t, J = 4.4 Hz, 4H), 1.62 (s, 6H); MS (m/z ) 446.1 (M + H) ;
HPLC 99%.
N-(4-Methoxy-7-morpholino-1,3-benzothiazol-2-yl)-2-methyl-2-(4-
o
1
methylphenoxy)propanamide (14). Off white solid; 34% yield; mp 152ꢀ153 C; H NMR
(DMSOꢀd ): δ 12.59 (bs, 1H), 7.05 (d, J = 8.4 Hz, 2H), 6.90ꢀ6.95 (m, 2H), 6.77 (d, J = 8.4 Hz,
6
2H), 3.85 (s, 3H), 3.78(t, J = 4.5 Hz, 4H), 3.02 (t, J = 4.4Hz, 4H), 2.20 (s, 3H), 1.56 (s, 6H). MS
+
(
m/z ) 442.1(M + H) ; HPLC 99%.
ACS Paragon Plus Environment

Page 25 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
-(2,4-Difluorophenoxy)-N-(4-methoxy-7-morpholino-1,3-benzothiazol-2-yl)-2-methyl-
o
1
propanamide (15). Off white solid (55mg, 16% ) mp 83ꢀ85 C. H NMR (DMSOꢀd ): δ 12.68
6
(bs, 1H), 7.31ꢀ7.37 (m, 1H), 7.06ꢀ7.12 (m, 1H), 7.00ꢀ7.03 (m, 1H), 6.92ꢀ6.99 (m, 2H), 3.87 (s,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
3
H), 3.78 (t, J = 4.4 Hz, 4H), 3.03 (t, J = 4.4 Hz, 4H), 1.55 (s, 6H); MS (m/z) 464.1 (M + H) ;
HPLC 98%.
-(2,6-Difluorophenoxy)-N-(4-methoxy-7-morpholino-1,3-benzothiazol-2-yl)-2-methyl-
2
o
1
propanamide (16). Off white solid; 16% yield; mp 150ꢀ153 C; H NMR (DMSOꢀd ): δ 12.54
6
(
bs, 1H), 7.13ꢀ7.20 (m, 3H), 6.91ꢀ6.97 (m, 2H), 3.88 (s, 3H), 3.79 (t, J = 4.4 Hz, 4H), 3.03 (t, J =
+
4
.4 Hz, 4H), 1.55 (s, 6H); MS (m/z) 464.1 (M + H) ; HPLC 95.66%.
2-(3,4-Difluorophenoxy)-N-(4-methoxy-7-morpholino-1,3-benzothiazol-2-yl)-2-methyl-
o
1
propanamide (17). Off white solid; 26% yield; mp 158ꢀ159 C; H NMR (DMSOꢀd ): δ 12.69
6
(bs, 1H ), 7.01ꢀ7.10 (m, 1H), 6.87ꢀ6.92 (m, 2H), 6.72ꢀ6.74 (m, 1H), 3.84 (s, 3H), 3.76 (t, J = 4.4
13
Hz, 4H), 3.00 (t, J = 4.4 Hz, 4H), 1.56 (s, 6H); C NMR (DMSOꢀd ): δ 172.8, 150.9, 149.0,
6
148.2, 145.3, 140.0, 139.0, 126.7, 117.3, 116.4, 113.1, 110.3, 108.1, 81.0, 66.5, 55.8, 51.4, 24.1;
+
MS (m/z ) 464.1 (M + H) HPLC 100%.
2
-(3,4-Dimethoxyphenoxy)-N-(4-methoxy-7-morpholino-1,3-benzothiazol-2-yl)-2-methyl-
o
1
propanamide (18). White solid; 12% yield; mp 157ꢀ158 C; H NMR (DMSOꢀd ): δ 12.59 (s,
6
1
H), 6.91ꢀ6.96 (m, 2H), 6.81 (d, J = 8.8 Hz, 1H), 6.67 (d, J = 2 Hz, 1H), 6.36ꢀ6.39 (m, 1H), 3.87
(s, 3H), 3.79 (t, J = 4.4 Hz, 4H), 3.67 (d, J = 3.6 Hz, 6H), 3.03 (t, J = 4.4 Hz, 4H), 1.55 (s, 6H);
+
MS (m/z ) 488.2 (M + H) ; HPLC 99%.
2-[3-(2-Methoxyethoxy)phenoxy]-N-(4-methoxy-7-morpholino-1,3-benzothiazol-2-yl)-2-
o
1
methyl-propanamide (19). Off white solid; 15% yield; mp 130ꢀ132 C; H NMR (DMSOꢀd ):
6
δ 12.72 (s, 1H), 7.12ꢀ7.16 (m, 1H), 6.91ꢀ6.96 (m, 2H), 6.57ꢀ6.59 (m, 1H), 6.42ꢀ6.44 (m, 2H),
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
3
2
.99 (t, J = 4.4 Hz, 2H), 3.85 (s, 3H), 3.79 (t, J = 4.4 Hz, 4H), 3.58 (t, J = 4.8 Hz, 2H), 3.24 (s,
+
H), 3.02 (t, J = 4 Hz, 4H), 1.60 (s, 6H); MS (m/z ) 502.1 (M + H) ; HPLC 100%.
-(1,3-Benzodioxol-5-yloxy)-N-(4-methoxy-7-morpholino-1,3-benzothiazol-2-yl)-2-methyl-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
1
propanamide (20). White solid; 51% yield; mp 182ꢀ184 C; H NMR (DMSOꢀd ): δ 12.59 (s,
6
1H) 6.90ꢀ6.95 (m, 2H), 6.78 (d, J = 8.8 Hz, 1H), 6.61 (d, J = 2.4Hz, 1H), 6.37 (dd, J = 8.8 Hz,
2
6
2
.4 Hz, 1H), 5.95 (s, 2H), 3.85 (s, 3H), 3.77 (t, J = 4.4 Hz, 4H), 3.01 (t, J = 4.4 Hz, 4H), 1.51 (s,
+
H); MS (m/z ) 472.3 (M + H) ; HPLC 100%.
-(4-Chlorophenoxy)-N-(4-methoxy-7-morpholino-1,3-benzothiazol-2-yl)-2-methyl-
o
1
propanamide (21). Off white solid; 26% yield; mp 178ꢀ178 C. H NMR (DMSOꢀd ): δ 12.74
6
(s, 1H), 7.33 (d, J = 9.2 Hz, 2H), 6.89ꢀ6.96 (m, 4H), 3.85 (s, 3H), 3.79 (t, J = 4 Hz, 4H), 3.02 (t,
+
J = 4.4 Hz, 4H), 1.60 (s, 6H); MS (m/z ) 462.1 (M + H) ; HPLC 98.65%.
N-(4-Methoxy-7-morpholino-1,3-benzothiazol-2-yl)-2-methyl-2-[4-
o
1
(trifluoromethyl)phenoxy]propanamide (22). Off white solid; 21% yield; mp 102ꢀ103 C. H
NMR (DMSOꢀd ): δ 13.02 (bs, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.24 (d, J = 8.8 Hz, 2H), 7.14ꢀ7.17
6
(m, 2H), 4.07 (s, 3H), 4.02 (t, J = 4.4 Hz, 4H), 3.93 (t, J = 4.4 Hz, 4H), 1.90 (s, 6H); MS (m/z )
+
496.1(M + H) ; HPLC 100%.
2-(4-Cyanophenoxy)-N-(4-methoxy-7-morpholino-1,3-benzothiazol-2-yl)-2-methyl-
o
1
propanamide (23). Off white solid; 29% yield; mp 157ꢀ159 C; H NMR (DMSOꢀd ): δ 12.84
6
(s, 1H), 7.74 (d, J = 8.8 Hz, 2H), 6.93ꢀ6.98 (m, 4H), 3.83 (s, 3H), 3.78 (bs, 4H), 3.02 (bs, 4H),
+
1.67 (s, 6H); MS (m/z) 453.0 (M + H) ; HPLC 100%.
2
-(4-Tert-butylphenoxy)-N-(4-methoxy-7-morpholino-1,3-benzothiazol-2-yl)-2-methyl-
o
1
propanamide (24). White solid; 23% yield; mp 229ꢀ230 C; H NMR (DMSOꢀd ): δ 12.64 (s,
6
1
H), 7.25 (d, J = 8.8 Hz, 2H), 6.89ꢀ6.94 (m, 2H), 6.76 (d, J = 8.8 Hz, 2H), 3.83 (s, 3H), 3.77 (t, J
ACS Paragon Plus Environment

Page 27 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
+
=
4.4 Hz, 4H), 3.00 (t, J = 4.4 Hz, 4H), 1.56 (s, 6H), 1.19 (s, 9H); MS (m/z) 484.2 (M + H) ;
HPLC 100%.
-(4-Fluorophenoxy)-N-(7-methoxy-4-morpholino-thiazolo[5,4-c]pyridin-2-yl)-2-methyl
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
o
1
propanamide (25) . Off white solid; 38% yield; mp 280ꢀ281 C; H NMR (DMSOꢀd ): δ 13.06
6
(bs, 1H), 7.89 (s, 1H), 7.08ꢀ7.12 (m, 2H), 6.91ꢀ6.95 (m, 2H), 3.90 (s, 3H), 3.76 (t, J = 4.4 Hz,
13
4
H), 3.22 (t, 4H), 1.55(s, 6H); C NMR (DMSOꢀd ): δ 174.2, 157.8, 150.3, 148.7, 143.4, 122.1,
6
+
116.9, 115.8, 80.3, 65.8, 57.0, 48.8, 24.1; MS (m/z) 447.3 (M + H) ; HPLC 99%; HRMS
+
(
C H FN O S) calulated 447.1497 (M + H) ; found 447.1492.
2
1
24
4
4
2
-(3-Fluorophenoxy)-N-(7-methoxy-4-morpholino-thiazolo[5,4-c]pyridin-2-yl)-2-methyl-
o
1
propanamide (26). Off white solid; 62% yield; mp 204ꢀ205 C; H NMR (DMSOꢀd ): δ 13.12
6
(
s, 1H), 7.91 (s, 1H), 7.28ꢀ7.34 (m, 1H), 6.86 (t, J = 8.4 Hz, 1H), 6.70ꢀ6.78 (m, 2H), 3.91 (s, 3H),
+
3.79 (bs, 4H), 3.36 (bs, 4H), 1.63 (s, 6H); MS (m/z) 447.1 (M + H) ; HPLC 99%.
2
-(2-Fluorophenoxy)-N-(7-methoxy-4-morpholino-thiazolo[5,4-c]pyridin-2-yl)-2-methyl-
o
1
propanamide (27). Off white solid; 63% yield; mp 188ꢀ189 C;. H NMR (DMSOꢀd ): δ 10.46
6
(
bs, 1H), 7.87 (s, 1H), 7.08ꢀ7.16 (m, 4H), 4.04 (s, 3H), 3.90 (t, J = 4.4 Hz, 4H), 3.48 (t, J = 4.4
+
Hz, 4H), 1.60 (bs, 6H); MS (m/z) 447.2 (M + H) ; HPLC 99%.
2-(4-Chlorophenoxy)-N-(7-methoxy-4-morpholino-thiazolo[5,4-c]pyridin-2-yl)-2-methyl-
o
1
propanamide (28). Off white solid; 21% yield; mp 179ꢀ180 C; H NMR (DMSOꢀd ): δ 13.30
6
(
bs, 1H), 7.88 (s, 1H), 7.30 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 3.89 (s, 3H), 3.76 (bs,
+
4
H), 3.31 (bs, 4H), 1.58 (s, 6H); MS (m/z) 463.1 (M + H) ; HPLC 97%.
N-(7-methoxy-4-morpholino-thiazolo[5,4-c]pyridin-2-yl)-2-methyl-2-[3-
1
(trifluoromethyl)phenoxy]propanamide (29). Off white solid; 60% yield; mp 210ꢀ212 ºC; H
NMR (DMSOꢀd ): δ 13.33 (bs, 1H), 8.08 (s, 1H), 7.69 (bs, 1H), 7.56 (bs, 1H), 7.45 (bs, 1H),
6
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
7
.35 (bs, 1H), 4.08 (s, 3H), 3.96 (bs, 4H), 3.51 (bs, 4H), 1.81 (s, 6H); MS (m/z) 497.2 (M + H) ;
HPLC 99%.
-(2,4-Difluorophenoxy)-N-(7-methoxy-4-morpholino-thiazolo[5,4-c]pyridin-2-yl)-2-methyl-
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
1
propanamide (30). Off white solid; 50% yield; mp 148ꢀ149 C; H NMR (DMSOꢀd ): δ 10.42
6
(bs, 1H), 7.87 (s, 1H), 7.03ꢀ7.09 (m, 1H), 6.91ꢀ6.96 (m, 1H), 6.83ꢀ6.87 (m, 1H), 4.04 (s, 3H),
+
3
9
2
.90 (t, J = 4.4 Hz, 4H), 3.48 (t, J = 4.4 Hz, 4H), 1.60 (bs, 6H); MS (m/z) 465.2 (M + H) ; HPLC
5%.
-(2,6-Difluorophenoxy)-N-(7-methoxy-4-morpholino-thiazolo[5,4-c]pyridin-2-yl)-2-methyl-
o
1
propanamide (31). off white solid (85 mg, 62%) mp 188ꢀ189 C. H NMR (DMSOꢀd ): δ 10.08
6
(bs, 1H), 7.87 (s, 1H), 6.60ꢀ6.64 (m, 1H), 6.48ꢀ6.51 (m, 2H), 4.03 (s, 3H), 3.91 (t, J = 4.4 Hz,
+
4H), 3.48 (t, J = 4.4 Hz, 4H), 1.67 (s, 6H); MS (m/z) 465.2 (M + H) HPLC 99%.
2
-(3,4-Difluorophenoxy)-N-(7-methoxy-4-morpholino-thiazolo[5,4-c]pyridin-2-yl)-2-methyl-
o
1
propanamide (32). Off white solid; 8% yield; mp 169ꢀ170 C; H NMR (DMSOꢀd ): δ 13.08
6
(
bs, 1H), 7.91 (s, 1H), 7.35 (m, 1H), 7.07ꢀ7.13 (m, 1H), 6.75ꢀ6.77 (m, 1H), 3.92 (s, 3H), 3.78 (t,
+
J = 4.0 Hz, 4H), 1.59 (s, 6H); MS (m/z) 465 (M + H) ; HPLC 99%.
2
-[3-(2-Methoxyethoxy)phenoxy]-N-(7-methoxy-4-morpholino-thiazolo[5,4-c]pyridin-2-yl)-
o
1
2
-methyl-propanamide (33). Off white solid; 49% yield; mp 166ꢀ167 C; H NMR (DMSOꢀ
d6): δ 13.07 (s, 1H), 7.91 (s, 1H), 7.15 (t, J = 8.0 Hz, 1H), 6.60 (d, J = 8.4 Hz, 1H), 6.42ꢀ6.45 (m,
2
H), 4.00 (t, J = 4.4 Hz, 2H), 3.91 (s, 3H), 3.79 (bs, 4H), 3.59 (t, J = 4.4 Hz, 2H), 3.35 (bs, 4H),
+
3.25 (s, 3H), 1.61 (s, 6H); MS (m/z) 503.2 (M + H) ; HPLC 99%.
2-(1,3-Benzodioxol-5-yloxy)-N-(7-methoxy-4-morpholino-thiazolo[5,4-c]pyridin-2-yl)-2-
o
1
methyl-propanamide (34). Off white solid; 45% yield; mp 205ꢀ206 C; H NMR (DMSOꢀd ):
6
δ 12.99 (s, 1H), 7.92 (s, 1H), 6.80 (d, J = 8.4 Hz, 1H), 6.63 (d, J = 2.0 Hz, 1H), 6.39 (dd, J = 2.4
ACS Paragon Plus Environment

Page 29 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Hz, 8.8 Hz, 1H), 5.98 (s, 2H), 3.93 (s, 3H), 3.79 (bs, 4H), 3.35 (bs, 4H), 1.54 (s, 6H); MS (m/z)
+
4
73.2 (M + H) ; HPLC 99%.
N-(7-Methoxy-4-morpholino-thiazolo[5,4-c]pyridin-2-yl)-2-methyl-2-(4-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
o
1
methylphenoxy)propanamide (35). Off white solid; 61% yield; mp 192ꢀ193 C; H NMR
(DMSOꢀd ): δ 13.05 (s, 1H), 7.91 (s, 1H), 7.07 (d, J = 8.0 Hz, 2H), 6.78 (d, J = 8.4 Hz, 2H), 3.91
6
+
(
s, 3H), 3.79 (bs, 4H), 3.35 (bs, 4H), 2.21 (s, 3H), 1.57 (s, 6H); MS (m/z) 443.2 (M + H) ; HPLC
9
9%.
N-(7-Methoxy-4-morpholino-thiazolo[5,4-c]pyridin-2-yl)-2-(3-methoxyphenoxy)-2-methyl-
o
1
propanamide (36). Off white solid; 65% yield; mp 153ꢀ154 C; H NMR (DMSOꢀd ): δ 10.27
6
(bs, 1H), 7.86 (s, 1H), 7.19 (t, J = 8.0 Hz, 1H), 6.69 (d, J = 8.8 Hz, 1H), 6.51ꢀ6.54 (m, 2H), 4.02
(
s, 3H), 3.90 (t, J = 4.0 Hz, 4H), 3.79 (s, 3H), 3.47 (t, J = 4.0 Hz, 4H), 1.60 (s, 6H); MS (m/z)
+
4
59.2 (M + H) ; HPLC 99%.
1-Methyl-4-oxo-cyclohexanecarboxylic acid (38).
To a solution of ethyl 1ꢀmethylꢀ4ꢀoxoꢀcyclohexanecarboxylate (37) (1 g, 5.42 mmol) in 21 ml of
THF: MeOH was added a solution of lithium hydroxide (0.65g, 15.49 mmol) in 4 ml of water.
The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was
concentrated under reduced pressure. The residue was taken in water and washed with hexane.
Aqueous layer was acidified up to pH 1ꢀ2 using 3 N HCl, Extracted with ethyl acetate. The
organic layer dried (Na SO ) and concentrated under reduced pressure to afford the title
2
4
compound 38 (0.7 g, 83 %).
1
H NMR (CDCl ): δ 2.33ꢀ2.54 ( m, 7H), 1.68ꢀ1.76 (m, 2H), 1.38 (s, 3H).
3
ACS Paragon Plus Environment