Amidoalkylindoles as Potent and Selective Cannabinoid Type 2 Receptor Agonists with in Vivo Efficacy in a Mouse Model of Multiple Sclerosis

Article abstract of DOI:10.1021/acs.jmedchem.7b00724

Selective CB₂ agonists represent an attractive therapeutic strategy for the treatment of a variety of diseases without psychiatric side effects mediated by the CB₁ receptor. We carried out a rational optimization of a black market designer drug SDB-001 that led to the identification of potent and selective CB₂ agonists. A 7-methoxy or 7-methylthio substitution at the 3-amidoalkylindoles resulted in potent CB₂ antagonists (27 or 28, IC₅₀ = 16-28 nM). Replacement of the amidoalkyls from 3-position to the 2-position of the indole ring dramatically increased the agonist selectivity on the CB₂ over CB₁ receptor. Particularly, compound 57 displayed a potent agonist activity on the CB₂ receptor (EC₅₀ = 114-142 nM) without observable agonist or antagonist activity on the CB₁ receptor. Furthermore, 57 significantly alleviated the clinical symptoms and protected the murine central nervous system from immune damage in an experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis.

Full text of DOI:10.1021/acs.jmedchem.7b00724

Subscriber access provided by University of Florida | Smathers Libraries
Article
Amidoalkylindoles as Potent and Selective Cannabinoid Type 2 Receptor
Agonists with In Vivo Efficacy in a Mouse Model of Multiple Sclerosis
Ying Shi, Yan-Hui Duan, Yue-Yang Ji, Zhi-Long Wang, Yan-Ran Wu, Hendra Gunosewoyo,
Xiao-Yu Xie, Jian-Zhong Chen, Fan Yang, Jing Li, Jie Tang, Xin Xie, and Li-Fang Yu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00724 • Publication Date (Web): 20 Jul 2017
Downloaded from http://pubs.acs.org on July 20, 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 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
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
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 58
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
Amidoalkylindoles as Potent and Selective Cannabinoid Type 2 Receptor
Agonists with In Vivo Efficacy in a Mouse Model of Multiple Sclerosis
†
, ‡
¶ , ‡
†
§
†
┴
Ying Shi, YanꢀHui Duan,
YueꢀYang Ji, ZhiꢀLong Wang, YanꢀRan Wu, Hendra Gunosewoyo,
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
ǁ
ǁ
†
§
�
*,¶,§
*, †
XiaoꢀYu Xie, JianꢀZhong Chen, Fan Yang, Jing Li, Jie Tang, Xin Xie,
LiꢀFang Yu
†
Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development,
School of Chemistry and Molecular Engineering, East China Normal University, 3663 North
Zhongshan Road, Shanghai 200062, China
¶
Shanghai Key Laboratory of Signaling and Disease Research, Laboratory of Receptorꢀbased Bioꢀ
medicine, Collaborative Innovation Center for Brain Science, School of Life Sciences and
Technology, Tongji University, 1239 Siping Road, Shanghai 200092, China
§
CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, 189 Guo Shou Jing Road, Shanghai 201203, China.
┴
School of Pharmacy, Faculty of Health Sciences, Curtin University, Bentley, Perth, WA 6102,
Australia
ǁ
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China
�
Shanghai Key Laboratory of Green Chemistry and Chemical Process, School of Chemistry and
Molecular Engineering, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062,
China
ACS Paragon Plus Environment

Page 3 of 58
Journal of Medicinal Chemistry
1
2
3
ABSTRACT
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
Selective CB agonists represent an attractive therapeutic strategy for the treatment of a variety of
2
diseases without psychiatric side effects mediated by the CB receptor. We carried out a rational
1
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
optimization of a black market designer drug SDBꢀ001 that led to the identification of potent and
selective CB agonists. A 7ꢀmethoxy or 7ꢀmethylthio substitution at the 3ꢀamidoalkylindoles resulted in
2
potent CB antagonists (27 or 28, IC = 16–28 nM). Replacement of the amidoalkyls from 3ꢀposition to
2
50
the 2ꢀposition of the indole ring dramatically increased the agonist selectivity on the CB over CB
2
1
receptor. Particularly, compound 57 displayed a potent agonist activity on the CB receptor (EC = 114–
2
50
1
42 nM) without observable agonist or antagonist activity on the CB receptor. Furthermore, 57
1
significantly alleviated the clinical symptoms and protected the murine central nervous system from
immune damage in an experimental autoimmune encephalomyelitis (EAE) mouse model of multiple
sclerosis.
INTRODUCTION
The endocannabinoid system consists of at least two cannabinoid (CB) receptors, namely the CB and
1
1
CB receptors. Cloned in the early 1990s, these receptors belong to the class A family of G proteinꢀ
2
2
ꢀ4
coupled receptors (GPCRs). Both receptors are coupled to the G proteins and subsequently inhibit
i/o
the activity of adenylyl cyclase (AC), thus reducing the intracellular cyclic adenosine monophosphate
5
(
cAMP) production. Stimulation of the CB receptor results in blockade of the Nꢀtype and P/Qꢀtype
1
+
6, 7
calcium channels, while activating the inwardly rectifying K channels. The CB receptor consists of
1
4
72 amino acids and is one of the most abundant GPCR in the brain, especially in the hippocampus,
8
cortex, basal ganglia and cerebellum. It is expressed mainly in the presynaptic terminals where it
7
, 9
modulates the release of various neurotransmitters.
Stimulation of the CB receptor leads to
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 58
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
modulation of feeding behavior, implicating an important role for CB receptor in obesity and metabolic
1
1
0, 11
disorders.
Rimonabant (1, SR141716A, SanofiꢀAventis), an inverse agonist/antagonist of the CB1
1
2
receptor, was approved in 2006 in Europe for the treatment of obesity. However, it was withdrawn 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
years later due to its severe central nervous system (CNS)ꢀrelated side effects including depression,
1
3
14
anxiety, and suicidal thoughts. The crystal structure of human CB receptor was very recently
1
resolved in a complex with the analogue of compound 1, 4ꢀ(4ꢀ(1ꢀ(2,4ꢀdichlorophenyl)ꢀ4ꢀmethylꢀ3ꢀ
1
5
(
piperidinꢀ1ꢀylcarbamoyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)phenyl)butꢀ3ꢀynꢀ1ꢀyl nitrate (AM6538), providing crucial
insights into both the mechanistic studies and drug development in the field of CB receptor.
On the other hand, the CB receptor is primarily located in the peripheral immune cells such as
2
1
splenocytes and leukocytes, with significantly lower expression levels in the neurons. The CB receptor
2
signaling typically involves the activation of mitogenꢀactivated protein kinases (MAPK) and JUN Nꢀ
terminal kinases (JNKs), as well as a transient increase in the intracellular calcium levels, resulting in
1
6
complex physiological functions. The human CB receptor consists of 360 amino acids sharing 44%
2
1
homology with CB at the amino acid level and 68% homology within the transmembrane regions. The
1
crystal structure of the human CB receptor has not yet been resolved to date. Of particular interest, the
2
stimulation of CB receptor was found to modulate the chemotactic profile of human monocytes via
2
downregulation of the chemokine receptors and inhibition of interferon γ (IFNγ)ꢀinduced intercellular
17
adhesion moleculeꢀ1 (ICAMꢀ1) expression. The CB receptor is responsible for many aspects of the
2
CBs’ immunomodulatory and antiꢀinflammatory effects, and therefore CB receptor agonists have been
2
suggested as a viable therapeutic option for conditions such as pain, osteoporosis and multiple sclerosis
1
8ꢀ21
(MS).
ACS Paragon Plus Environment

Page 5 of 58
Journal of Medicinal Chemistry
1
2
3
MS is a neuroinflammatory disorder characterized by damages in the myelin sheath which leads to
reduced nerve conductivity and the clinical symptoms of MS. The majority of MS patients initially have
a relapsingꢀremitting MS and eventually progressing to a secondary stage of MS where loss of
neurological function manifests. There is no cure for MS to date and the main goal of currently available
treatment is to relieve the symptoms such as spasticity and pain. In clinical settings, cannabinoids
exhibit beneficial effects on relieving the symptoms of MS. Nabiximols (Sativex, GW Pharmaceuticals)
is a combination of botanical extracts containing approximately 1:1 tetrahydrocannabinol (2,
THC)/cannabidiol (3, CBD) and is marketed in more than 25 countries worldwide for the treatment of
MS. The metabolite of compound 2, (6aR,10aR)ꢀ1ꢀhydroxyꢀ6,6ꢀdimethylꢀ3ꢀ(2ꢀmethyloctanꢀ2ꢀyl)ꢀ
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
6
a,7,10,10aꢀtetrahydroꢀ6Hꢀbenzo[c]chromeneꢀ9ꢀcarboxylic acid (JBTꢀ101, ajulemic acid, Corbus
2
2
Pharmaceuticals) was found to selectively bind to the CB receptor on immune cells and fibroblasts,
2
eventually granted an FDA fastꢀtrack development status for the treatment of systemic sclerosis in 2015.
Recent studies have also shown that potent CB agonists which are devoid of CB activity could offer
2
1
therapeutic benefits for alleviating the symptoms of MS, although the exact mechanisms of action
23, 24
remain to be further established.
In preclinical studies, the beneficial effects of cannabinoids have
been reported in different animal models of MS including experimental autoimmune encephalomyelitis
EAE), chronic relapsing experimental allergic encephalomyelitis (CREAE), and Theiler’s murine
(
2
5
encephalomyelitis virusꢀinduced demyelinating disease (TMEVꢀIDD). Chromenopyrazole (4), a
selective and potent CB receptor agonist, was found to dampen neuroinflammation in the TMEVꢀIDD
2
26
mouse model by reducing microglial activation. Administration of a selective CB receptor agonist
2
quinolineꢀ2,4(1H,3H)ꢀdione (5) reduced the clinical scores in the mouse model of EAE, as shown by the
2
7
decreased leukocyte infiltration and demyelination in the CNS. The bicyclic sesquiterpene (ꢀ)ꢀβꢀ
caryophyllene (6) was previously shown to exhibit antiꢀinflammatory and analgesic effects in mouse
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 58
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
28
models. Very recently, this compound was reported to prevent the progression of clinical symptoms
2
9
and neuroinflammation in the mouse model of EAE.
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
N
NH
OH
OH
N
N
Cl
Cl
O
HO
Cl
SR141716A, 1
9ꢀTHC, 2
Cannabidiol, 3
O
N
HN
O
N
O
O
H
H
O
4
5
(ꢀ)ꢀ ꢀcaryophyllene, 6
I
OH
O
O
OH
O
NO2
N
N
N
O
OH
CP55940, 7
O
N
N
WINꢀ55212ꢀ2, 8
JWHꢀ015, 9
AM1241, 10
O
O
O
NH
N
N
N
SDBꢀ001, 11
ABꢀ001, 12
ABꢀ002, 13
Figure 1. Selected CB receptor ligands.
To date, numerous synthetic ligands for both CB and CB receptors have been investigated both in
1
2
30
academia and pharma industry settings such as the widely used tool compound CP55940 (7). In
particular, indoleꢀcontaining compounds have been recognized as a popular scaffold for the
ACS Paragon Plus Environment

Page 7 of 58
Journal of Medicinal Chemistry
1
2
3
investigation of synthetic CB receptor ligands. WIN55212ꢀ2 (8) represents a nonꢀselective CB agonist
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
1
(
Ki, CB1 = 1.9 nM, Ki, CB2 = 0.3 nM) that has been widely used as pharmacological tool. Selective CB
2
agonists were developed based on an indole scaffold, such as aminoalkylindoles JWHꢀ015 (9) and
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
32ꢀ35
AM1241 (10).
Dual function compounds with CB receptor antagonist and CB receptor agonist
1 2
activities were also developed via the optimization of one common component of K2/spice naphthalenꢀ
36, 37
1
ꢀylꢀ(1ꢀbutylindolꢀ3ꢀyl)methanone (JWHꢀ073).
SDBꢀ001 (11) and ABꢀ001 (12) belong to the 3ꢀ
amidoalkylindole and 3ꢀacylalkylindole class of cannabimimetics, identified in Japan and Ireland as a
3
8, 39
“designer drug” in 2012 and 2010 respectively.
Pharmacological assessments reveal that compounds
1
1–13 were potent CB agonists, with similar EC values for both CB (35–40 nM) and CB (29–89 nM)
50
1
2
4
0
receptors. Compound 11 induced hypothermia and heart rate in rats at 1 mg/kg, while compounds 12
and 13 only produced a weak cannabimimetic activity in rat up to 30 mg/kg. Although the linker moiety
between the adamantane and the indole ring was not important for potent in vitro binding, an amide
linker was crucial for the in vivo activities, as demonstrated by the significantly lower activity of the
4
0
adamantyl ketone 12. Based on these observations, we selected 3ꢀamidoalkylindole 11 as a startingꢀ
point for the development of selective CB receptor ligands with minimal CNSꢀrelated adverse effects.
2
Herein we report our efforts on the investigation of 2ꢀ and 3ꢀamidoalkylindoles that led to the
identification of potent and highly selective CB receptor ligands with beneficial effects in the mouse
2
EAE model of MS.
RESULTS AND DISCUSSION
Chemistry
The 3ꢀamidoalkylindoles 11, 16, 25–36, 39–41 were synthesized starting from commercially available
substituted indoles utilizing the synthetic routes shown in Scheme 1. Indole was first Nꢀalkylated with 4ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 58
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
(
2ꢀbromoethyl)morpholine in dimethylformamide (DMF) using sodium hydride as a base to form
4
1
morpholine indole 14 (route A). Treatment with trifluoroacetic anhydride followed by basic hydrolysis
gave carboxylic acid 15. Subsequent reaction with oxalyl chloride and amantadine afforded the final
product 16. Alternatively, compound 16 could also be synthesized via route B: amidation followed by
Nꢀalkylation. Compounds 11, 25–28, 32–36 were synthesized in a similar manner to compound 16
starting from indole or various substituted indoles with appropriate bromides and amines. The sulfinyl
analog 29 was prepared via oxidation of the thiomethyl compound 28 using metaꢀchloroperoxybenzoic
acid. tertꢀButyldimethylsilyl (TBS) protection of commercially available 4ꢀchlorobutanꢀ1ꢀol and 5ꢀ
chloropentanꢀ1ꢀol yielded silyl ethers 37 and 38 respectively, which were reacted with indole 30 in the
presence of sodium hydride to form Nꢀalkylated 3ꢀamidoalkylindoles. Deprotection of TBS with
tetrabutylammonium fluoride (TBAF) in THF at ambient temperature afforded the final compounds 39
and 40. Compound 40 was further methylated with methyl iodide in the presence of sodium hydride to
afford methyl ether 41.
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 1
ACS Paragon Plus Environment

Page 9 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
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
Reagents and conditions: (a) NaH, DMF, 0 °C; 4ꢀ(2ꢀbromoethyl)morpholine, 100 ºC; (b) i.
(CF CO) O, DMF, 0 °C−rt; ii. NaOH, H O, reflux; (c) (COCl) , DMF (cat.), CH Cl , rt; amantadine,
3
2
2
2
2
2
Et N, CH Cl , rt; (d) TBSCl, imidazole, CH Cl , rt; (e) 30, NaH, DMF, 0 °C−100 ºC; (f) TBAF, THF, rt;
3
2
2
2
2
(g) metaꢀchloroperoxybenzoic acid, CH Cl , ꢀ40 ºC; (h) CH I, NaH, DMF, 0 °C−rt.
2 2 3
The syntheses of 2ꢀamidoalkylindoles 44–52, 54, 56–62, 66–68, and 73–75 were described in Scheme 2.
Commercially available ethyl 1Hꢀindoleꢀ2ꢀcarboxylate (42) was hydrolyzed in aqueous sodium
hydroxide. The resultant carboxylic acid was treated with oxalyl chloride to generate the acid chloride
and subsequently reacted with amantadine to form key intermediate 43. NꢀAlkylation with various
bromides gave the final products 44–47, 49–52. Compound 48 was obtained via basic hydrolysis of
compound
47.
Compound
43
was
reacted
with
1ꢀchloromethylꢀ4ꢀfluoroꢀ1,4ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 58
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
diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (SelectFluor) to afford the fluorinated compound 55.
NꢀAlkylation of compounds 43 and 55 with (3ꢀbromopropoxy)(tertꢀbutyl)dimethylsilane, 37 or 38,
followed by a subsequent TBS deprotection in the presence of TBAF afforded compounds 56–60.
Oxidation of primary alcohol 58 afforded the aldehyde 61, which was converted to a gemꢀdifluoride 62
using diethylaminosulfur trifluoride (DAST). Similarly, compounds 66–68 were prepared by employing
the same strategy starting from indole ester 42. Compounds 71 and 72 were obtained in two steps from
commercially available 4ꢀaminobutanꢀ1ꢀol (69) or 5ꢀaminopentanꢀ1ꢀol (70). Sequential alkylation with
indole 43 and removal of the Boc protecting group under acidic condition afforded the amines 73 and
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
7
4. Amine 74 was next reacted with 1Hꢀpyrazoleꢀ1ꢀcarboxamidine hydrochloride in methanol to afford
guanidine 75.
a
Scheme 2
ACS Paragon Plus Environment

Page 11 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
4
4 R = n-C H
5
11
1
HN
O
HN
O
45 R = (CH2)2CN
a, b
c
1
46 R = (CH ) CN
2
3
N
N
R1
1
4
4
4
5
7 R = (CH ) CN
2 4
H
d
1
8 R = (CH ) CO H
2
4
2
4
3
1
9 R = (CH ) F
O
O
2 5
1
0 R = (CH ) CF
2
3
3
1
N
51 R = 4-methyltetrahydro-2H-pyran
52 R1 = 2-(2-oxooxazolidin-3-yl)ethyl
H
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
2
HN
O
HN
O
a, b
c
N
N
H
53
54
CN
R2
R2
HN
HN
O
HN
O
HN
O
f, g
h
i
N
O
N
58
N
N
H
(CH )n
2
OH
56 n = 1, R2 = H
7 n = 2, R = H
3 R2 = H
5 R2 = F
F
4
5
61
CHO
62
e
F
2
5
5
5
6
2
8 n = 3, R = H
2
9 n = 2, R = F
0 n = 3, R = F
2
O
NHR1
NHR1
a, b
f, g
N
O
N
O
H
N
O
H
42
1
66 R1 = tert-butyl OH
67 R1 = cyclopentyl
68 R1 = cyclohexyl
6
3 R = tert-butyl
64 R1 = cyclopentyl
5 R1 = cyclohexyl
6
HN
O
HO
TSO
HN
j
N
(CH )n
k, l
m
(CH )n
2
2
N
O
74
NH2
NHBoc
(
CH )n
2
NH2
NH
69 n = 2
0 n = 3
71 n = 2
2 n = 3
73 n = 2
74 n = 3
NH2
7
7
75
HN
a
Reagents and conditions: (a) NaOH, H O, reflux; (b) i. (COCl) , DMF (cat.), CH Cl , rt; ii.
2
2
2
2
1
1
adamantanꢀ1ꢀamine or aniline, Et N, CH Cl , rt; (c) NaH, R Br or R Cl, DMF, 0–100 ºC; (d) aqueous
3
2
2
KOH, reflux; (e) SelectFluor, NaHCO , THF, rt; (f) (3ꢀbromopropoxy)(tertꢀbutyl)dimethylsilane, 37 or
3
3
8, NaH, DMF, 100 ºC; (g) TBAF, THF, rt; (h) DessꢀMartin periodinane, CH Cl , rt; (i) DAST, CCl , rt;
2 2 4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 58
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
(
j) i. (Boc) O, CH Cl , rt; ii. TsCl, Et N, CH Cl ; (k) 43, NaH, DMF; (l) HCl in EtOAc, rt; (m) 1Hꢀ
2 2 2 3 2 2
pyrazoleꢀ1ꢀcarboxamidine hydrochloride, MeOH, 40 ºC.
In Vitro Functional Characterization–Calcium Mobilization Studies and ForskolinꢀStimulated
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
cAMP Assay.
The functional profile of all synthesized compounds were characterized using the calcium mobilization
4
2
assay in Chinese hamster ovary (CHO) cells expressing human CB or CB receptors. Compounds
1
2
displaying more than 50% activation or more than 50% inhibition at 10 ꢁM in the primary assay were
evaluated further in the doseꢀresponse studies. Their half maximal effective concentrations (EC ) and
5
0
maximal effect relative to compound 7 (Emax), or half maximal inhibitory concentrations (IC , with 100
50
nM of compound 7 as the agonist) were calculated as listed in Tables 1 and 2.
Table 1. Agonist and Antagonist Activities of 3ꢀAmidoalkylindoles in CHO Cells Expressing Human
a
CB or CB Receptor by Calcium Mobilization Assays.
1
2
Compound
Agonism
Antagonism
CB1
CB2
CB1
CB2
EC , ꢁM
Emax, %
EC , ꢁM
Emax, %
IC , ꢁM
IC , ꢁM
5
0
50
50
50
b
1
1
0.039 ± 0.004 68.3 ± 11.7 0.059 ± 0.020
66.3 ± 4.4
NA
NA
NA
16
3.3 ± 0.1
NA
75.0 ± 7.6
0.059 ± 0.005 56.7 ± 12.0
NA
NA
NA
NA
NA
c
30
25
26
27
ND
0.63 ± 0.24
NA
75.0 ± 2.9
ND
NA
NA
ND
ND
ND
NA
NA
NA
ND
NA
NA
NA
ND
0.016 ±
ACS Paragon Plus Environment

Page 13 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
0
.001
0.028 ±
.005
28
NA
ND
NA
ND
NA
0
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
9
NA
6.7 ± 2.0
NA
ND
63.3 ± 1.7
ND
NA
ND
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
ꢀ
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
ꢀ
32
NA
NA
31
0.50 ± 0.05
0.038 ± 0.006
0.41 ± 0.05
0.18 ± 0.03
0.031 ± 0.003
0.16 ± 0.04
0.062 ± 0.009
0.082 ± 0.016
0.085 ± 0.009
60.0 ± 2.9
76.7 ± 6.0
66.3 ± 1.7
73.3 ± 1.7
70.0 ± 5.0
68.3 ± 1.7
63.3 ± 1.7
70.0 ± 2.9
100 ± 6
33
0.084 ± 0.015
NA
133 ± 12
ND
34
35
36
39
NA
ND
0.63 ± 0.11
13 ± 0.1
4.1 ± 1.3
NA
105 ± 3
58.3 ± 1.7
76.7 ± 9.3
ND
40
41
7
0.022 ± 0.005
100 ± 15
a
See Experimental Section. Data represent mean values ± SEM of eightꢀpoint experiments each
performed in triplicates from three independent experiments. In the agonist mode, compound 7 was used
as a positive control. In the antagonist mode, cells were preincubated with either the test compounds or
b
compound 1 as a positive control for 10 min, followed by addition of agonist 7 at 100 nM. NA: not
c
active, defined as < 50% activation or < 50% inhibitory activity at 10 ꢁM in the primary assay. ND: not
determined, for compounds defined as not active, their maximal effects were not determined.
Compound 11 showed agonist profiles on both CB and CB receptors with similar potency (EC =
50, CB1
1
2
4
0
3
9 nM, EC50, CB2 = 59 nM), which is consistent with previously reported data (EC
= 34 nM, EC50,
50, CB1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 58
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
=
29 nM) in mouse AtT20 neuroblastoma cells using a fluorometric imaging plate reader (FLIPR)
CB2
membrane potential assay. In line with the previous studies demonstrating the importance of the 3ꢀ
4
0
amidoadamantylindole skeleton, we incorporated various Nꢀ1 substituents on the indole ring (16, 25–
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
9, 32–36, 39–41). Deletion of the nꢀC H substituent at this position resulted in compounds that are
5
11
10ꢀfold less potent as CB agonists and devoid of any activity on the CB receptor (30 and 31 vs 11).
2
1
The morpholinoethyl and tetrahydropyranylꢀmethyl groups were previously identified as Nꢀ1
substituents that were beneficial for the selectivity at CB over CB in the SAR studies reported for a
2
1
4
3
series of 3ꢀacylalkylindoles. Applying this within our amidoalkylindole series, replacement of the Nꢀ
pentyl with morpholinylethyl (16), cyanomethyl (34), cyanoethyl (35), cyanopropyl (36), hydroxybutyl
(
39), hydroxypentyl (40) or methoxypentyl (41), generally resulted in the retention of CB partial
2
agonist activities, accompanied with at least over 15ꢀfold reduction at the CB potency compared to
1
compound 11. In particular, the cyanomethyl (34), cyanoethyl (35) and methoxypentyl (41) had no
appreciable agonist activity at the CB receptor. On the other hand, a tetrahydropyranylꢀmethyl (33)
1
substitution maintained functional potencies at both receptors (EC values of 84 nM and 38 nM at CB
1
5
0
and CB receptors, respectively). We then examined the effects of monoꢀsubstitutions at the indole ring
2
on the functional profiles. Introduction of a methoxy group at the 4ꢀ, 5ꢀ, or 7ꢀposition of indole ring (25–
2
7) resulted in a complete loss of agonist activities at both CB and CB receptors. However, for
1 2
compound 27, the 7ꢀmethoxy substitution resulted in a potent and selective CB antagonist with an IC
2
50
value of 16 nM. A similar trend was obtained for the 7ꢀmethylthio analog (28) with an IC value of 28
5
0
nM at the CB receptor. Oxidation or deletion of the sulfur atom of compound 28 resulted in the 7ꢀ
2
methylsulfinyl (29) or 7ꢀmethyl (32) analogs, respectively. Unfortunately, both of these compounds
were found to be completely inactive at the CB receptor, while only the 7ꢀmethyl analog (32) displayed
2
weak, partial agonist activity at the CB receptor (EC value of 6.7 ꢀM).
1
50
ACS Paragon Plus Environment

Page 15 of 58
Journal of Medicinal Chemistry
1
2
3
Table 2. Agonist and Antagonist Activities of 2ꢀAmidoalkylindoles in CHO Cells Expressing human
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
a
CB or CB Receptor by Calcium Mobilization Assays.
1
2
Compound
Agonism
Antagonism
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
CB1
EC , ꢁM
CB2
CB1
CB2
b
Emax , %
EC , ꢁM
Emax, %
IC , ꢁM
IC , ꢁM
50
50
50
50
b
c
4
4
4
4
3
4
5
6
NA
ND
0.98 ± 0.19
66.7 ± 6.0
53.3 ± 4.4
93.3 ± 4.4
61.7 ± 4.4
41.7 ± 1.7
43.3 ± 3.3
53.3 ± 4.4
63.3 ± 3.3
41.7 ± 4.4
66.7 ± 3.3
48.3 ± 4.4
83.3 ± 3.3
86.7 ± 3.3
85.0 ± 2.9
70.0 ± 2.9
46.7 ± 3.3
60.0 ± 2.9
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.72 ± 0.13
0.15 ± 0.01
0.27 ± 0.06
47
0.075 ± 0.004
7.13 ± 1.91
0.14 ± 0.03
0.49 ± 0.04
0.41 ± 0.26
0.22 ± 0.03
0.20 ± 0.06
3.8 ± 1.5
4
4
5
5
8
9
0
1
52
5
5
5
5
3
4
6
7
0.16 ± 0.03
0.12 ± 0.04
0.10 ± 0.03
0.41 ± 0.12
0.61 ± 0.08
58
5
6
9
0
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 58
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
6
1
NA
NA
NA
NA
NA
NA
NA
NA
ND
0.18 ± 0.06
0.092 ± 0.009
0.43 ± 0.07
NA
73.3 ± 4.4
NA
NA
NA
NA
NA
NA
NA
NA
ꢀ
NA
NA
NA
NA
NA
NA
NA
NA
ꢀ
62
ND
ND
ND
ND
ND
ND
ND
60.0 ± 5.0
51.7 ± 1.7
ND
6
6
6
7
6
7
8
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
0.42 ± 0.10
4.7 ± 0.8
50.0 ± 2.9
86.7 ± 1.7
93.3 ± 4.4
ND
74
8.6 ± 3.6
75
NA
7
0.022 ± 0.005 100 ± 15
0.085 ± 0.009
100 ± 6
a
See Experimental Section. Data represent mean values ± SEM of eightꢀpoint experiments each
performed in triplicates from three independent experiments. In the agonist mode, compound 7 was used
as a positive control. In the antagonist mode, cells were preincubated with either the test compounds or
b
compound 1 as a positive control for 10 min, followed by addition of agonist 7 at 100 nM. NA: not
c
active, defined as < 50% activation or < 50% inhibitory activity at 10 ꢁM in the primary assay. ND: not
determined, for compounds defined as not active, their maximal effects were not determined.
Subsequent round of SAR studies were focused on replacing the amidoadamantyl substituent from 3ꢀ
position to the 2ꢀposition of the indole (44–52, 54, 56–62, and 66–68). To our delight, no agonist or
antagonist activity at the CB receptor were observed for all the 25 tested compounds. These 2ꢀ
1
amidoalkylindoles generally behave as CB partial agonists with moderate to high potency (EC values
2
50
between 720 nM and 75 nM), with the exception of compounds 48, 55, 73, and 74 possessing EC50
values over 3 ꢀM and the totally inactive compounds 67 and 75. The polar nature of compounds 48, and
7
3–75 may be an important factor for the observed significant loss of activity. The Nꢀ1 unsubstituted
compound (43) showed a similar functional activity to the nꢀC H substituted compound (44). Various
5
11
ACS Paragon Plus Environment

Page 17 of 58
Journal of Medicinal Chemistry
1
2
3
Nꢀ1 substituents were well tolerated for CB partial agonist activity including the cyanoalkyls (45–47),
2
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
5
2
7
ꢀfluoropentyl (49), 5,5ꢀdifluoropentyl (62), 4,4,4ꢀtrifluoropentyl (50), tetrahydropyranylꢀmethyl (51),
ꢀ(2ꢀoxooxazolidinꢀ3ꢀyl)ethyl (52), hydroxyalkyls (56–58), 5ꢀoxopentyl (61), with EC values between
50
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 and 490 nM. It was noted that the Nꢀ1 tetrahydropyranylꢀmethyl substitution within the 2ꢀ
amidoalkylindole series (51) confers no agonist activity at the CB receptor, unlike the trend previously
1
seen in the 3ꢀamidoalkylindoles (33). However, the carboxyalkyl (48), aminoalkyls (73 and 74), and
guanidinoalkyl (75) moieties were found to be detrimental to the activity compared to the original pentyl
substitution (44). The cyanoalkyl (45 and 47), fluoroalkyl (49 and 62) and hydroxyalkyl (56–58) Nꢀ1
substitutions yielded the most potent CB partial agonists with EC values of less than 160 nM, similar
2
50
to the positive control compound 7 (EC50 = 85 nM, Emax 100%). The hydroxyalkyl substituted
compounds 56–58 generally showed higher Emax (70–90%) compared to the fluoroalkyls (50–60%) and
cyanoalkyls (40–90%). Replacement of the adamantane ring with a benzene resulted in a slight decrease
of potency at the CB receptor (54 vs 47). Among the pentanolꢀsubstituted indoles 66–68, replacement
2
of the adamantane with tertꢀbutyl (66) or cyclohexane (68) groups gave CB partial agonists with EC
2
50
values of around 400 nM, while the cyclopentyl analog 67 was inactive. Substitution of the hydrogen
atom at position 3 of the indole ring with a fluoro group (55, 59, and 60) also resulted in less potent CB2
partial agonists (55 vs 43, 59 vs 57, 60 vs 58).
Compound 57 was selected to be further assessed in a cAMP assay using CHO cells expressing CB . As
2
shown in Figure 2, compound 57 doseꢀdependently inhibits the forskolinꢀstimulated cAMP
accumulation in CHO cells with an EC value of 142 nM. Data were normalized to the amounts of
5
0
cAMP in forskolinꢀstimulated cells (100%).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 58
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
cAMP Assay for CB2
Fsk: 3 uM
1
20
100
8
0
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
0
4
0
2
0
EC50 = 142.2 ± 28.6 nM
0
-20
-12 -11 -10 -9 -8 -7 -6 -5 -4 -3
Compound 57 Log[M]
Figure 2. Compound 57 inhibits forskolinꢀstimulated cAMP formation in CHO cells expressing CB2.
EC50 value represents mean values ± SEM of three independent experiments each performed in
triplicates.
Analyses of ReceptorꢀLigand Interactions.
Relatively small structural modifications of GPCR ligands could lead to a major change in their
4
4
functional profiles. As shown in Figure 3, in the 4ꢀquinoloneꢀ3ꢀcarboxamide series of CB ligands,
replacement of an isopropyl group (76) at the 6ꢀposition with a furanꢀ2ꢀyl (77) alters its CB functional
2
45ꢀ47
profile from an agonist to an inverse agonist in GTPγS assay.
Similarly, for the quinoloneꢀ
2,4(1H,3H)ꢀdiones, C5ꢀ or C8ꢀmethyl (78) substitutions were reported as CB receptor agonists, while
2
2
7
the C6ꢀ or C7ꢀmethyl (79) substituted compounds act as antagonists. We found that a 7ꢀmethoxy or 7ꢀ
methylthio substitution at the 3ꢀamidoalkylindole 16 resulted in potent CB antagonists (27 or 28, IC =
2
50
16–28 nM) without observable agonist activity on CB2.
ACS Paragon Plus Environment

Page 19 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
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 3. Selected examples of small structural modifications of CB receptor ligands leading to a major
2
change in their functional activities.
We next performed flexible docking simulations to predict the receptor
ꢀligand interactions between
48
compound 16 or 27 and CB based on the homology model of CB active state, using the FlexiDock
2
2
method of Sybyl x1.3. As shown in Figure 4, the morpholinylethyl moiety of either 16 or 27 occupies
the deep hydrophobic pocket surrounded by conserved aromatic residues W172, Y190, and W194,
4
9
which have been demonstrated as crucial residues for ligand recognition in the CB receptor. The
2
adamantyl group of 16 or 27 may make contact with a hydrophobic cavity composed of F87, F91, F94,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 58
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
F106, K109, and I110. The carbonyl group of either 16 or 27 may have hydrogen bond interactions with
the side chain guanidine group of residue R177, which was proposed to be an important residue
4
interacting with CP55940 binding to the CB receptor. Our docking simulations also suggested that the
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
agonist 16 would have a different interaction pattern with the antagonist 27 for their binding to the CB2
receptor. In the dockingꢀsimulated structural model of complex CB ꢀ16 (Figure 4. left), the phenyl side
2
chain of F117 may have a nearly parallel πꢀπ interaction with the aromatic side chain of W258.
However, the methoxyl group of the antagonist 27 may induce a steric hindrance resulting in the rotation
of the indole ring of W258 to a weak, Tꢀshaped πꢀπ interaction with the phenyl group of F117 in the
dockingꢀsimulated CB ꢀ27 (Figure 4. right). This docking analysis is in agreement with the previously
2
reported interaction mode calculated between CB and CB ꢀselective antagonist C6ꢀ or C7ꢀsubstituted
2
2
quinoloneꢀ2,4(1H,3H)ꢀdiones, which also have their C6ꢀ or C7ꢀsubstituent to block the movement of the
2
7
indole ring of W258. As reported on the other rhodopsinꢀlike GPCRs, F3.36/W6.48 motif is believed
6
to be the rotamer toggle switch for the receptor activation.
Figure 4. Docking results of CB receptor agonist 16 (left) and CB receptor antagonist 27 (right)
2
2
complexes. The carbon atoms of F117/W258 motif are colored light green, and the Hꢀbonds are shown
as blue dashed lines.
ACS Paragon Plus Environment

Page 21 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
In Vivo Efficacy−Mouse EAE Model for Multiple Sclerosis
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
As one of the most potent and selective CB receptor partial agonists identified in this amidoalkylindoles
2
series, compound 57 was selected for in vivo studies using a mouse EAE model, which is a widely used
animal model of MS. In these animals, the integrity of the bloodꢀbrain barrier (BBB) is damaged and the
pathogenic T cells were allowed to infiltrate the CNS. The consequent accumulation of immune cells
along with the concomitant activation of glia cells eventually results in demyelination, axonal damage,
impaired nerve conduction, and paralysis.
Briefly, mice were administered compound 57 (10 mg/kg or 30 mg/kg, ip) beginning at the
asymptomatic stage of the disease on day 3 post immunization with MOG (myelin oligodendrocyte
glycoprotein) peptide. The clinical EAE scores were recorded from a scale of 0 to 5 and the data are
shown in Figure 5, with 0 as showing no clinical signs and 5 as moribund state or death. Compound 57
significantly reduced the clinical scores of the EAE mice at both the tested doses, with the higher dose
(30 mg/kg) showing superior effects (Figure 5A). Histological examination of spinal cords was
performed on day 19 post immunization. Hematoxylin and eosin (H&E) staining showed multiple
widespread areas of leukocyte infiltration in the white matter region of vehicleꢀtreated EAE mice,
whereas in compound 57 treated mice, infiltration was significantly reduced (Figure 5B and 5D).
Compared to the vehicle group, treatment with compound 57 also significantly reduced the extensive
demyelination in white matter (Figure 5C and 5E). These data from EAE mice suggest that compound
57, a potent and selective partial agonist of CB receptor, may alleviate the clinical symptoms in this
2
model by reducing the extent of infiltration of leukocytes and demyelination in CNS.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 58
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 5. (A) Efficacy of compound 57 in mouse EAE model. EAE mice were treated with 57 (10 or 30
mg/kg, n = 8) or vehicle (n = 10) intraperitoneally once daily from day 3 post immunization until the
end of the study. Data are shown as mean ± SEM of the values obtained in at least 8 animals: ***p <
0
.001 (twoꢀway ANOVA test). (B) Hematoxylin and eosin (H&E) and (C) Luxol fast blue (LFB)
ACS Paragon Plus Environment

Page 23 of 58
Journal of Medicinal Chemistry
1
2
3
staining of paraffin sections of the spinal cords isolated from naive, vehicle, or 57 (10 or 30 mg/kg)
treated EAE mice on day 19 post immunization. Quantitative analysis was performed on the number of
total infiltrates in H&E stained sections (D), and the amount of demyelination in Luxol fast blue stained
sections (E). Three mice from each group were sacrificed, and ≥ 5 sections from each mouse were
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
analyzed. Data are presented as mean ± SEM: *** p < 0.001 vs naive, p < 0.05,
p < 0.001 vs vehicle
###
#
group (Student’s t test).
In Vivo Pharmacokinetics Studies of Compound 57
The preliminary in vivo pharmacokinetic studies of compound 57 were conducted in mice. Compound
7 (10 mg/kg, ip) was administered to C57 mice before collection of plasma samples at the following
5
time points: 0.25, 0.5, 1, and 2 hr (n = 3/group). The concentrations of compound 57 in the plasma
samples were determined by a developed LC−MS/MS method. The plasma halfꢀlives of compound 57
ꢀ1
were found to be 1.04 ± 0.18 hr. The AUC0−t value were 317 hr·ng·mL in the plasma, while the Cmax
value was 350 ng/mL (958 nM), which was about 8ꢀfold higher compared to the in vitro functional EC50
value (118 nM, Table 2) in the calcium mobilization assay. These data suggest that the PK profile is well
in agreement with the in vivo efficacy of compound 57 in the mouse model of EAE.
Table 3. Preliminary Pharmacokinetics Parameters of 57 in C57 mice (10 mg/kg, ip)
PK Parameters
IPꢀ1
0.742
0.934
0.25
365
IPꢀ2
0.748
0.927
0.25
381
IPꢀ3
0.555
1.25
0.25
303
Mean
0.682
1.04
ꢀ
SD
0.110
0.184
ꢀ
RSD (%)
ꢀ1
K , hr
el
16
18
ꢀ
t , hr
1/2
tmax, hr
ꢀ
1
Cmax, ng·mL
350
317
428
41.2
71.9
72.6
12
23
17
ꢀ
1
AUC , hr·ng·mL
312
391
247
0
ꢀt
ꢀ
1
AUC0ꢀinf, hr·ng·mL
410
507
365
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 58
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
ꢀ1
AUMC , hr·hr·ng·mL
237
565
1.38
314
703
1.39
194
643
1.76
248
637
1.51
60.6
69.4
24
11
15
0ꢀt
ꢀ1
AUMC0ꢀinf, hr·hr·ng·mL
MRT, h
0.220
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
Conclusions.
In summary, we report herein the optimization of a promiscuous designer drug 11 that leads to the
identification of potent and selective CB partial agonists that are devoid of CB ꢀmediated psychiatric
2
1
side effects. A total of 42 amidoalkylindoles were synthesized and tested for their functional activities
on both the CB and CB receptors. Within the 3ꢀamidoalkylindoles series, morpholinylethyl (16),
1
2
cyanoalkyls (34–36) and hydroxyalkyls (39 and 40) substituents on the indole nitrogen generally
resulted in retention of the CB receptor partial agonist activity, accompanied with a significant
2
reduction at the CB receptor agonist activity. Monosubstitutions on the benzene ring of the indole
1
influence the functional profiles of these compounds at both CB receptors, particularly with the 7ꢀ
methoxy (27) or 7ꢀmethylthio (28) analogs functionally acting as CB receptor antagonists in the
2
calcium mobilization assay. Furthermore，we found that replacement of the amidoalkyl substituents
from position 3 to the position 2 of the indole leads to highly selective CB receptor partial agonists with
2
no CB receptor agonist activity or antagonism at both receptors. Various Nꢀ1 substituents were also
1
found to be well tolerated in the 2ꢀamidoalkylindoles. A representative analog 57 was selected to be
studied in a mouse EAE model of MS. Administration of this selective CB partial agonist at 10 and 30
2
mg/kg daily ip resulted in a significant drop of the clinical scores in this model. Further histological
examination of spinal cords demonstrated significant reduction in leukocyte infiltration and the extent of
2
6
demyelination, consistent with other reports supporting the efficacy of selective CB agonists in animal
2
models of MS.
ACS Paragon Plus Environment

Page 25 of 58
Journal of Medicinal Chemistry
1
2
3
EXPERIMENTAL SECTION
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
General Chemistry Methods: Starting materials, reagents, and solvents were purchased from
commercial suppliers and used without further purification, unless otherwise stated. Anhydrous THF
and CH Cl were obtained by distillation over sodium wire or CaH , respectively. All nonꢀaqueous
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
2
2
reactions were run under a nitrogen atmosphere with exclusion of moisture from reagents, and all
reaction vessels were ovenꢀdried. The progress of reactions was monitored by TLC on SiO . Spots were
2
visualized by their quenching of the fluorescence of an indicator admixed to the SiO layer, or by
2
dipping into phosphomolybdic acid ethanol solution followed by heating. SiO for flash chromatography
2
was of 200−300 mesh particle size, and an EtOAc/hexane mixture or gradient was used unless stated
1
13
otherwise. H NMR spectra were recorded at a spectrometer frequency of 400 MHz, C NMR spectra at
101 MHz. Chemical shifts are reported in δ (ppm) using the δ 0 signal of tetramethylsilane (TMS) as
internal standard. High resolution mass spectra were performed using a Bruker ESIꢀTOF highꢀresolution
mass spectrometer. Purities of final compounds (> 95%) were established by analytical HPLC, which
was carried out on an Waters e2695 HPLC system with a ZORBAX SBꢀC18 column, with detection at
2
40 nm and 280 nm on a variable wavelength detector 2998 PDA; flow rate = 1.2 mL/min. Gradient I of
20 to 100% methanol in water (both containing 0.05 vol % of TFA) in 30 min; Gradient II of 50 to 95%
acetonitrile in water (both containing 0.05 vol % of TFA) in 21 min.
Nꢀ((3s,5s,7s)ꢀAdamantanꢀ1ꢀyl)ꢀ1ꢀpentylꢀ1Hꢀindoleꢀ3ꢀcarboxamide (11). This compound was
obtained from indole and 1ꢀbromopentane via route A. White solid; yields 94%, 92%, and 67%; purity
1
9
7.4% (Gradient II). H NMR (400 MHz, CDCl ) δ 7.88 (d, J = 7.6 Hz, 1H), 7.64 (s, 1H), 7.37–7.34 (m,
3
1
H), 7.28–7.19 (m, 2H), 5.72 (s, 1H), 4.09 (t, J = 7.2 Hz, 2H), 2.19–2.13 (m, 9H), 1.86–1.78 (m, 2H),
1
3
1.79–1.68 (m, 6H), 1.38–1.24 (m, 4H), 0.87 (t, J = 7.0 Hz, 3H). C NMR (101 MHz, CDCl ) δ 164.5,
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 58
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
1
36.6, 131.3, 125.3, 122.2, 121.1, 120.0, 112.2, 110.3, 52.1, 46.8, 42.3 (3C), 36.6 (3C), 29.7, 29.6 (3C),
+
2
9.0, 22.3, 13.9. HRMS (ESI): Calcd for C H N NaO [M+Na] , 387.2407; found 387.2413.
24 32
2
General procedure of for the preparation of 3ꢀamidoalkylindoles.
Route 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
4
ꢀ(2ꢀ(1HꢀIndolꢀ1ꢀyl)ethyl)morpholine (14). To a stirred solution of indole (2.60 g, 17.37 mmol) in
anhydrous DMF (10 mL) was added NaH (1.60 g, 50% in mineral oil, 34.74 mmol) with ice cooling
under N . After stirring for 15 min at rt 4ꢀ(2ꢀchloroethyl)morpholine (2.03 g, 17.37 mmol) was added.
2
Following stirring for 2 hr at rt, the reaction was quenched with water (15 mL) with ice cooling. The
resulting mixture was extracted with EtOAc (3×20 mL) and the combined organic layers were washed
with water (3×20 mL) and brine (15 mL), dried over Na SO , and concentrated under vacuum. The
2
4
residue was purified by flash chromatography on silica gel, eluting with hexane/EtOAc (5:1) to provide
1
the title compound as light red oil; yield 50%; H NMR (400 MHz, CDCl ) δ 7.59 (d, J = 8.0 Hz, 1H),
3
7
.28 (d, J = 8.0 Hz, 1H), 7.18–7.14 (m, 1H), 7.08–7.04 (m, 2H), 6.45
–6.43 (m, 1H), 4.08 (t, J = 7.0 Hz,
2
H), 3.61 (t, J = 4.6 Hz, 4H), 2.60 (t, J = 6.8 Hz, 2H), 2.34 (t, J = 4.6 Hz, 4H).
1ꢀ(2ꢀMorpholinoethyl)ꢀ1Hꢀindoleꢀ3ꢀcarboxylic acid (15). To a stirred solution of 14 (1.93 g, 8.4
mmol) in anhydrous DMF (10 mL) was added trifluoroacetic anhydride (2.29 g, 11.0 mmol) at 0 ºC.
After stirring for 2 hr at rt, the reaction was quenched with water (15 mL) and extracted with EtOAc
(3×20 mL). The combined organic layers were washed with water (3×20 mL) and saturated sodium
bicarbonate (2×15 mL), and concentrated under vacuum. The residue was then mixed with a solution of
NaOH (1.02 g, 25.2 mmol) in water (10 mL). After refluxing for 3 hr, the reaction was cooled to rt. A
1
0% aqueous hydrochloric acid solution was added to the mixture until pH value dropped to 5–6,
followed by extraction with CH Cl (3×30 mL). The combined organic layers were dried over Na SO ,
2
2
2
4
ACS Paragon Plus Environment

Page 27 of 58
Journal of Medicinal Chemistry
1
2
3
1
and concentrated under vacuum to give the title compound. White solid; yield 83%; H NMR (400 MHz,
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
DMSOꢀd ) δ 8.10 (s, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.26–7.18 (m, 2H), 4.34 (t,
6
1
3
J = 6.4 Hz, 2H), 3.53 (m, J = 4.0 Hz, 4H), 2.68 (t, J = 6.2 Hz, 2H), 2.42 (br s, 4H). C NMR (101 MHz,
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
DMSOꢀd ) δ 165.6, 136.3, 135.7, 126.4, 122.1, 121.2, 120.8, 110.7, 106.3, 66.2, 57.4, 53.2, 43.1.
6
Nꢀ((3s,5s,7s)ꢀAdamantanꢀ1ꢀyl)ꢀ1ꢀ(2ꢀmorpholinoethyl)ꢀ1Hꢀindoleꢀ3ꢀcarboxamide (16). To a solution
of 15 (400 mg, 1.46 mmol) and DMF (0.1 mL) in CH Cl (10 mL) was added oxalyl chloride (0.2 mL,
2
2
2
.2 mmol). After stirring for 2 hr at rt, the mixture was concentrated under vacuum. The residue was
dissolved in CH Cl (10 mL), followed by the additions of amantadine (220 mg, 1.46 mmol) and
2
2
triethylamine (595 mg, 5.84 mmol). The mixture was stirred overnight and filtered off. The filtrate was
concentrated under vacuum and the residue was purified by flash chromatography on silica gel, eluting
with hexane/ EtOAc (10:1) to give the title compound. White solid; yield 62%; purity 98.7% (Gradient
1
I). H NMR (400 MHz, CDCl ) δ 7.91–7.87 (m, 1H), 7.70 (s, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.28–7.20
3
(m, 2H), 5.72 (s, 1H), 4.20 (t, J = 6.8 Hz, 2H), 3.68 (t, J = 4.0 Hz, 4H), 2.72 (t, J = 6.8 Hz, 2H), 2.46 (t,
13
J = 4.0 Hz, 4H), 2.18–2.13 (m, 9H), 1.79–1.68 (m, 6H). C NMR (101 MHz, CDCl ) δ 164.4, 136.5,
3
1
31.5, 125.3, 122.3, 121.2, 120.0, 112.5, 110.0, 66.9, 57.9, 53.8, 52.0, 44.1, 42.2 (3C), 36.5 (3C), 29.6
+
(
3C). HRMS (ESI): Calcd for C H N NaO [M+Na] , 430.2465; found 430.2457.
25 33
3
2
1
4ꢀ(2ꢀ(4ꢀMethoxyꢀ1Hꢀindolꢀ1ꢀyl) ethyl) morpholine (17). Light yellow oil; yield 88%; H NMR (400
MHz, CDCl ) δ 7.10 (t, J = 8.0 Hz, 1H), 6.99 (d, J = 2.8 Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H), 6.57 (d, J =
3
2
.8 Hz, 1H), 6.48 (d, J = 7.6 Hz, 1H), 4.11 (t, J = 7.0 Hz, 2H), 3.90 (s, 3H), 3.64 (t, J = 4.6 Hz, 4H), 2.64
1
3
(t, J = 7.0 Hz, 2H), 2.38 (t, J = 4.4 Hz, 4H). C NMR (101 MHz, CDCl ) δ 153.6, 137.5, 126.6, 122.4,
3
1
19.2, 102.8, 99.3, 98.6, 67.0, 58.2, 55.3, 53.9, 44.2.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 58
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
1
4
ꢀ(2ꢀ(5ꢀMethoxyꢀ1Hꢀindolꢀ1ꢀyl) ethyl) morpholine (18). Light yellow oil; yield 70%; H NMR (400
MHz, CDCl ) δ 7.19 (d, J = 8.8 Hz, 1H), 7.07–7.04 (m, 2H), 6.85 (dd, J = 8.8, 2.4 Hz, 1H), 6.38 (d, J =
3
2
.8 Hz, 1H), 4.11 (t, J = 7.0 Hz, 2H), 3.80 (s, 3H), 3.64 (t, J = 4.6 Hz, 4H), 2.64 (t, J = 6.8 Hz, 2H), 2.39
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
13
(
t, J = 4.4 Hz, 4H). C NMR (101 MHz, CDCl ) δ 154.1, 131.3, 129.0, 128.6, 111.9, 110.0, 102.6,
3
100.9, 67.0, 58.3, 55.9, 53.9, 44.2.
1
4
ꢀ(2ꢀ(7ꢀMethoxyꢀ1Hꢀindolꢀ1ꢀyl) ethyl) morpholine (19). Light yellow oil; yield 99%; H NMR (400
MHz, CDCl ) δ 7.08 (d, J = 8.0 Hz, 1H), 6.87–6.83 (m, 2H), 6.46 (d, J = 7.6 Hz, 1H), 6.29 (d, J = 3.2
3
Hz, 1H), 4.32 (t, J = 7.2 Hz, 2H), 3.75 (s, 3H), 3.55 (t, J = 4.6 Hz, 4H), 2.57 (t, J = 7.2 Hz, 2H), 2.31 (t,
1
3
J = 4.4 Hz, 4H). C NMR (101 MHz, CDCl ) δ 147.5, 131.1, 129.4, 125.6, 119.9, 113.9, 102.3, 101.5,
3
6
7.1, 60.2, 55.2, 54.0, 46.7.
1
4
ꢀ(2ꢀ(7ꢀ(Methylthio)ꢀ1Hꢀindolꢀ1ꢀyl)ethyl)morpholine (20). Light yellow oil; yield 92%; H NMR (400
MHz, CDCl ) δ 7.51 (d, J = 7.6 Hz, 1H), 7.19 (d, J = 7.2 Hz, 1H), 7.10–7.06 (m, 2H), 6.51–6.50 (m,
3
13
1
H), 4.75 (t, J = 7.2 Hz, 2H), 3.73 (t, J = 4.2 Hz, 4H), 2.79 (t, J = 7.2 Hz, 2H), 2.55–2.52 (m, 7H). C
NMR (101 MHz, CDCl ) δ 134.0, 130.6, 130.3, 124.5, 120.1, 119.9, 119.8, 102.0, 67.0, 60.1, 54.0, 46.6,
3
1
9.0.
1
4
ꢀMethoxyꢀ1ꢀ(2ꢀmorpholinoethyl)ꢀ1Hꢀindoleꢀ3ꢀcarboxylic acid (21). White solid; yield 80%; H
NMR (400 MHz, CDCl ) δ 11.69 (brs, 1H), 8.02 (s, 1H), 7.25 (t, J = 7.8 Hz, 1H), 7.11 (d, J = 8.4 Hz,
3
1
2
H), 6.76 (d, J = 7.6 Hz, 1H), 4.24 (t, J = 6.4 Hz, 2H), 4.12 (s, 3H), 3.68 (brs, 4H), 2.76 (t, J = 6.2 Hz,
1
3
H), 2.47 (brs, 4H). C NMR (101 MHz, CDCl ) δ 164.2, 150.9, 138.4, 137.0, 123.8, 114.5, 107.4,
3
105.1, 102.4, 66.8, 57.6, 56.5, 53.8, 44.7.
1
5
ꢀMethoxyꢀ1ꢀ(2ꢀmorpholinoethyl)ꢀ1Hꢀindoleꢀ3ꢀcarboxylic acid (22). White solid; yield 71%; H
NMR (400 MHz, CDCl ) δ 12.10 (brs, 1H), 7.92 (s, 1H), 7.68 (s, 1H), 7.27–7.26 (d, J = 8.4 Hz, 1H),
3
ACS Paragon Plus Environment

Page 29 of 58
Journal of Medicinal Chemistry
1
2
3
6
4
5
.92 (d, J = 8.4 Hz, 1H), 4.22 (brs, 2H), 3.89 (s, 3H), 3.71 (brs, 4H), 2.77 (t, J = 5.8 Hz, 2H), 2.50 (brs,
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
1
3
H). C NMR (101 MHz, CDCl ) δ 170.2, 156.0, 135.7, 131.6, 127.8, 113.4, 110.7, 106.5, 103.2, 66.7,
3
7.7, 55.8, 53.7, 44.5.
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
1
7
ꢀMethoxyꢀ1ꢀ(2ꢀmorpholinoethyl)ꢀ1Hꢀindoleꢀ3ꢀcarboxylic acid (23). White solid; yield 56%; H
NMR (400 MHz, CDCl ) δ 7.83–7.81 (m, 2H), 7.18 (t, J = 7.8 Hz, 1H), 6.71 (d, J = 7.6 Hz, 1H), 4.55 (t,
3
1
3
J = 6.2 Hz, 2H), 3.95 (s, 3H), 3.73 (brs, 4H), 2.80 (t, J = 6.4 Hz, 2H), 2.53 (brs, 4H). C NMR (101
MHz, DMSOꢀd ) δ 166.2, 147.1, 136.2, 129.0, 125.4, 121.7, 113.7, 107.6, 103.4, 66.2, 59.2, 55.4, 53.3,
6
46.1.
1
7ꢀ(Methylthio)ꢀ1ꢀ(2ꢀmorpholinoethyl)ꢀ1Hꢀindoleꢀ3ꢀcarboxylic acid (24). White solid; yield 87%; H
NMR (400 MHz, CD OD) δ 8.07–8.04 (m, 2H), 7.31 (d, J = 7.2 Hz, 1H), 7.20 (t, J = 7.8 Hz, 1H), 5.02
3
1
3
(t, J = 7.2 Hz, 2H), 3.84 (t, J = 4.6 Hz, 4H), 3.60 (s, 2H), 3.02 (brs, 4H), 2.57 (s, 3H). C NMR (101
MHz, CD OD) δ 168.0, 138.9, 135.8, 130.0, 127.1, 123.6, 122.0, 121.4, 108.9, 66.4, 64.3, 59.4, 54.4,
3
1
9.1.
Nꢀ((3s,5s,7s)ꢀAdamantanꢀ1ꢀyl)ꢀ4ꢀmethoxyꢀ1ꢀ(2ꢀmorpholinoethyl)ꢀ1Hꢀindoleꢀ3ꢀcarboxamide (25).
This compound was obtained from 4ꢀmethoxyꢀ1Hꢀindole and 4ꢀ(2ꢀchloroethyl)morpholine via route A.
1
Pale yellow solid; yields 88%, 72%, and 14%; purity 96.4% (Gradient II). H NMR (400 MHz, CDCl )
3
δ 8.65 (s, 1H), 7.91 (s, 1H), 7.16 (t, J = 8.0 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.65 (d, J = 8.0 Hz, 1H),
4
.19 (t, J = 7.0 Hz, 2H), 3.99 (s, 3H), 3.68 (t, J = 4.4 Hz, 4H), 2.73 (t, J = 7.0 Hz, 2H), 2.47 (t, J = 4.4
1
3
Hz, 4H), 2.18–2.13 (m, 9 H), 1.78–1.70 (m, 6H). C NMR (101 MHz, CDCl ) δ 163.3, 152.4, 138.9,
3
134.4, 122.9, 114.1, 113.5, 104.2, 101.7, 66.8, 57.8, 55.2, 53.8, 51.3, 44.3, 41.9 (3C), 36.6 (3C), 29.6
+
(3C). HRMS (ESI): Calcd for C H N O [M+H] , 438.2751; found 438.2793.
2
6
36
3
3
ACS Paragon Plus Environment