Amidoalkylindoles as Potent and Selective Cannabinoid Type 2

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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

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Journal of Medicinal Chemistry

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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,ǁ Jian-Zhong Chen,ǁ Fan Yang, † Jing Li,§ Jie Tang, Xin Xie,*,¶,§ Li-Fang 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 Receptor-based Bio-

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

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ABSTRACT 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. INTRODUCTION The endocannabinoid system consists of at least two cannabinoid (CB) receptors, namely the CB1 and CB2 receptors.1 Cloned in the early 1990s, these receptors belong to the class A family of G proteincoupled receptors (GPCRs).2-4 Both receptors are coupled to the Gi/o proteins and subsequently inhibit the activity of adenylyl cyclase (AC), thus reducing the intracellular cyclic adenosine monophosphate (cAMP) production.5 Stimulation of the CB1 receptor results in blockade of the N-type and P/Q-type calcium channels, while activating the inwardly rectifying K+ channels.6, 7 The CB1 receptor consists of 472 amino acids and is one of the most abundant GPCR in the brain, especially in the hippocampus, cortex, basal ganglia and cerebellum.8 It is expressed mainly in the presynaptic terminals where it modulates the release of various neurotransmitters.7,

9

Stimulation of the CB1 receptor leads to



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modulation of feeding behavior, implicating an important role for CB1 receptor in obesity and metabolic disorders.10, 11 Rimonabant (1, SR141716A, Sanofi-Aventis), an inverse agonist/antagonist of the CB1 receptor, was approved in 2006 in Europe for the treatment of obesity.12 However, it was withdrawn 2 years later due to its severe central nervous system (CNS)-related side effects including depression, anxiety, and suicidal thoughts.13 The crystal structure of human CB1 receptor14 was very recently resolved in a complex with the analogue of compound 1, 4-(4-(1-(2,4-dichlorophenyl)-4-methyl-3(piperidin-1-ylcarbamoyl)-1H-pyrazol-5-yl)phenyl)but-3-yn-1-yl nitrate (AM6538),15 providing crucial insights into both the mechanistic studies and drug development in the field of CB receptor. On the other hand, the CB2 receptor is primarily located in the peripheral immune cells such as splenocytes and leukocytes, with significantly lower expression levels in the neurons.1 The CB2 receptor signaling typically involves the activation of mitogen-activated protein kinases (MAPK) and JUN Nterminal kinases (JNKs), as well as a transient increase in the intracellular calcium levels, resulting in complex physiological functions.16 The human CB2 receptor consists of 360 amino acids sharing 44% homology with CB1 at the amino acid level and 68% homology within the transmembrane regions.1 The crystal structure of the human CB2 receptor has not yet been resolved to date. Of particular interest, the stimulation of CB2 receptor was found to modulate the chemotactic profile of human monocytes via downregulation of the chemokine receptors and inhibition of interferon γ (IFNγ)-induced intercellular adhesion molecule-1 (ICAM-1) expression.17 The CB2 receptor is responsible for many aspects of the CBs’ immunomodulatory and anti-inflammatory effects, and therefore CB2 receptor agonists have been suggested as a viable therapeutic option for conditions such as pain, osteoporosis and multiple sclerosis (MS).18-21


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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 relapsing-remitting 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)-1-hydroxy-6,6-dimethyl-3-(2-methyloctan-2-yl)6a,7,10,10a-tetrahydro-6H-benzo[c]chromene-9-carboxylic acid (JBT-101, ajulemic acid, Corbus Pharmaceuticals) was found to selectively bind to the CB2 receptor on immune cells and fibroblasts,22 eventually granted an FDA fast-track development status for the treatment of systemic sclerosis in 2015. Recent studies have also shown that potent CB2 agonists which are devoid of CB1 activity could offer therapeutic benefits for alleviating the symptoms of MS, although the exact mechanisms of action remain to be further established.23, 24 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 encephalomyelitis virus-induced demyelinating disease (TMEV-IDD).25 Chromenopyrazole (4), a selective and potent CB2 receptor agonist, was found to dampen neuroinflammation in the TMEV-IDD mouse model by reducing microglial activation.26 Administration of a selective CB2 receptor agonist quinoline-2,4(1H,3H)-dione (5) reduced the clinical scores in the mouse model of EAE, as shown by the decreased leukocyte infiltration and demyelination in the CNS.27 The bicyclic sesquiterpene (-)-βcaryophyllene (6) was previously shown to exhibit anti-inflammatory and analgesic effects in mouse



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models.28 Very recently, this compound was reported to prevent the progression of clinical symptoms and neuroinflammation in the mouse model of EAE.29

O

N NH OH

N

OH

N

Cl

Cl O Cl SR141716A, 1

N O

HO

9-THC,

O

O

O

HN

N

O

4

Cannabidiol, 3

2

H

H

(-)- -caryophyllene, 6

5

I

OH OH

O

O

O

NO2 N

N OH

O

CP55940, 7

N

O N

N

WIN-55212-2, 8

JWH-015, 9

AM1241, 10

O O

O

NH N

N

SDB-001, 11

N

AB-001, 12

AB-002, 13

Figure 1. Selected CB receptor ligands. To date, numerous synthetic ligands for both CB1 and CB2 receptors have been investigated both in academia and pharma industry settings such as the widely used tool compound CP55940 (7).30 In particular, indole-containing compounds have been recognized as a popular scaffold for the


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investigation of synthetic CB receptor ligands. WIN55212-2 (8) represents a non-selective CB agonist (Ki, CB1 = 1.9 nM, Ki, CB2 = 0.3 nM) that has been widely used as pharmacological tool.31 Selective CB2 agonists were developed based on an indole scaffold, such as aminoalkylindoles JWH-015 (9) and AM1241 (10).32-35 Dual function compounds with CB1 receptor antagonist and CB2 receptor agonist activities were also developed via the optimization of one common component of K2/spice naphthalen1-yl-(1-butylindol-3-yl)methanone (JWH-073).36, 37 SDB-001 (11) and AB-001 (12) belong to the 3amidoalkylindole and 3-acylalkylindole class of cannabimimetics, identified in Japan and Ireland as a “designer drug” in 2012 and 2010 respectively.38, 39 Pharmacological assessments reveal that compounds 11–13 were potent CB agonists, with similar EC50 values for both CB1 (35–40 nM) and CB2 (29–89 nM) receptors.40 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 adamantyl ketone 12.40 Based on these observations, we selected 3-amidoalkylindole 11 as a startingpoint for the development of selective CB2 receptor ligands with minimal CNS-related adverse effects. Herein we report our efforts on the investigation of 2- and 3-amidoalkylindoles that led to the identification of potent and highly selective CB2 receptor ligands with beneficial effects in the mouse EAE model of MS. RESULTS AND DISCUSSION Chemistry The 3-amidoalkylindoles 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 N-alkylated with 4-



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(2-bromoethyl)morpholine in dimethylformamide (DMF) using sodium hydride as a base to form morpholine indole 14 (route A). Treatment with trifluoroacetic anhydride41 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 N-alkylation. 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 meta-chloroperoxybenzoic acid. tert-Butyldimethylsilyl (TBS) protection of commercially available 4-chlorobutan-1-ol and 5chloropentan-1-ol yielded silyl ethers 37 and 38 respectively, which were reacted with indole 30 in the presence of sodium hydride to form N-alkylated 3-amidoalkylindoles. 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. Scheme 1a


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a


Reagents and conditions: (a) NaH, DMF, 0 °C; 4-(2-bromoethyl)morpholine, 100 ºC; (b) i.

(CF3CO)2O, DMF, 0 °C−rt; ii. NaOH, H2O, reflux; (c) (COCl)2, DMF (cat.), CH2Cl2, rt; amantadine, Et3N, CH2Cl2, rt; (d) TBSCl, imidazole, CH2Cl2, rt; (e) 30, NaH, DMF, 0 °C−100 ºC; (f) TBAF, THF, rt; (g) meta-chloroperoxybenzoic acid, CH2Cl2, -40 ºC; (h) CH3I, NaH, DMF, 0 °C−rt. The syntheses of 2-amidoalkylindoles 44–52, 54, 56–62, 66–68, and 73–75 were described in Scheme 2. Commercially available ethyl 1H-indole-2-carboxylate (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. N-Alkylation 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


1-chloromethyl-4-fluoro-1,4-


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diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (SelectFluor) to afford the fluorinated compound 55. N-Alkylation of compounds 43 and 55 with (3-bromopropoxy)(tert-butyl)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 gem-difluoride 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 4-aminobutan-1-ol (69) or 5-aminopentan-1-ol (70). Sequential alkylation with indole 43 and removal of the Boc protecting group under acidic condition afforded the amines 73 and 74. Amine 74 was next reacted with 1H-pyrazole-1-carboxamidine hydrochloride in methanol to afford guanidine 75. Scheme 2a


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HN c

a, b N H

N R1

O

O d

43

O N H

44 R1 = n-C5H11 45 R1 = (CH2)2CN 46 R1 = (CH2)3CN 47 R1 = (CH2)4CN 48 R1 = (CH2)4CO2H 49 R1 = (CH2)5F 50 R1 = (CH2)3CF3 51 R1 = 4-methyltetrahydro-2 H-pyran 52 R1 = 2-(2-oxooxazolidin-3-yl)ethyl

HN

O

42

HN

a, b N H

HN

c N

O 53

O

54 CN

R2

R2

HN

HN f, g N H

N

O

HN

HN h

58

O

N

i

O

N

O

62

F

(CH2)n OH

43 R2 = H 55 R2 = F

e

56 57 58 59 60

O

NHR1

a, b N H

O

N H

CHO

61

n = 1, R2 = H n = 2, R2 = H n = 3, R2 = H n = 2, R2 = F n = 3, R2 = F

F

NHR1

f, g N

O

O

42 63 R1 = tert-butyl 64 R1 = cyclopentyl 65 R1 = cyclohexyl

66 R1 = tert-butyl OH 67 R1 = cyclopentyl 68 R1 = cyclohexyl

HN HN

TSO

HO (CH2)n

j

(CH2)n NHBoc

NH2

k, l

N

74

O

m

N

(CH2)n NH2

69 n = 2 70 n = 3

a

71 n = 2 72 n = 3

O

73 n = 2 74 n = 3

NH 75

HN

NH2

Reagents and conditions: (a) NaOH, H2O, reflux; (b) i. (COCl)2, DMF (cat.), CH2Cl2, rt; ii.

adamantan-1-amine or aniline, Et3N, CH2Cl2, rt; (c) NaH, R1Br or R1Cl, DMF, 0–100 ºC; (d) aqueous KOH, reflux; (e) SelectFluor, NaHCO3, THF, rt; (f) (3-bromopropoxy)(tert-butyl)dimethylsilane, 37 or 38, NaH, DMF, 100 ºC; (g) TBAF, THF, rt; (h) Dess-Martin periodinane, CH2Cl2, rt; (i) DAST, CCl4, rt;



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(j) i. (Boc)2O, CH2Cl2, rt; ii. TsCl, Et3N, CH2Cl2; (k) 43, NaH, DMF; (l) HCl in EtOAc, rt; (m) 1Hpyrazole-1-carboxamidine hydrochloride, MeOH, 40 ºC. In Vitro Functional Characterization–Calcium Mobilization Studies and Forskolin-Stimulated cAMP Assay. The functional profile of all synthesized compounds were characterized using the calcium mobilization assay42 in Chinese hamster ovary (CHO) cells expressing human CB1 or CB2 receptors. Compounds displaying more than 50% activation or more than 50% inhibition at 10 µM in the primary assay were evaluated further in the dose-response studies. Their half maximal effective concentrations (EC50) and maximal effect relative to compound 7 (Emax), or half maximal inhibitory concentrations (IC50, with 100 nM of compound 7 as the agonist) were calculated as listed in Tables 1 and 2. Table 1. Agonist and Antagonist Activities of 3-Amidoalkylindoles in CHO Cells Expressing Human CB1 or CB2 Receptor by Calcium Mobilization Assays.a Compound

Agonism

Antagonism

CB1

CB2

CB1

CB2

EC50, µM

Emax, %

EC50, µM

Emax, %

IC50, µM

IC50, µM

11

0.039 ± 0.004

68.3 ± 11.7

0.059 ± 0.020

66.3 ± 4.4

NAb

NA

16

3.3 ± 0.1

75.0 ± 7.6

0.059 ± 0.005

56.7 ± 12.0

NA

NA

30

NA

NDc

0.63 ± 0.24

75.0 ± 2.9

NA

NA

25

NA

ND

NA

ND

NA

NA

26

NA

ND

NA

ND

NA

NA

27

NA

ND

NA

ND

NA

0.016 ±


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0.001 0.028 ± 28

NA

ND

NA

ND

NA 0.005

a

29

NA

ND

NA

ND

NA

NA

32

6.7 ± 2.0

63.3 ± 1.7

NA

NA

NA

NA

31

NA

ND

0.50 ± 0.05

60.0 ± 2.9

NA

NA

33

0.084 ± 0.015

133 ± 12

0.038 ± 0.006

76.7 ± 6.0

NA

NA

34

NA

ND

0.41 ± 0.05

66.3 ± 1.7

NA

NA

35

NA

ND

0.18 ± 0.03

73.3 ± 1.7

NA

NA

36

0.63 ± 0.11

105 ± 3

0.031 ± 0.003

70.0 ± 5.0

NA

NA

39

13 ± 0.1

58.3 ± 1.7

0.16 ± 0.04

68.3 ± 1.7

NA

NA

40

4.1 ± 1.3

76.7 ± 9.3

0.062 ± 0.009

63.3 ± 1.7

NA

NA

41

NA

ND

0.082 ± 0.016

70.0 ± 2.9

NA

NA

7

0.022 ± 0.005

100 ± 15

0.085 ± 0.009

100 ± 6

-

-

See Experimental Section. Data represent mean values ± SEM of eight-point 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 compound 1 as a positive control for 10 min, followed by addition of agonist 7 at 100 nM. bNA: not active, defined as < 50% activation or < 50% inhibitory activity at 10 µM in the primary assay. cND: not determined, for compounds defined as not active, their maximal effects were not determined. Compound 11 showed agonist profiles on both CB1 and CB2 receptors with similar potency (EC50, CB1 = 39 nM, EC50, CB2 = 59 nM), which is consistent with previously reported data40 (EC50, CB1 = 34 nM, EC50,



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CB2

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= 29 nM) in mouse AtT20 neuroblastoma cells using a fluorometric imaging plate reader (FLIPR)

membrane potential assay. In line with the previous studies demonstrating the importance of the 3amidoadamantylindole skeleton,40 we incorporated various N-1 substituents on the indole ring (16, 25– 29, 32–36, 39–41). Deletion of the n-C5H11 substituent at this position resulted in compounds that are 10-fold less potent as CB2 agonists and devoid of any activity on the CB1 receptor (30 and 31 vs 11). The morpholinoethyl and tetrahydropyranyl-methyl groups were previously identified as N-1 substituents that were beneficial for the selectivity at CB2 over CB1 in the SAR studies reported for a series of 3-acylalkylindoles.43 Applying this within our amidoalkylindole series, replacement of the Npentyl with morpholinylethyl (16), cyanomethyl (34), cyanoethyl (35), cyanopropyl (36), hydroxybutyl (39), hydroxypentyl (40) or methoxypentyl (41), generally resulted in the retention of CB2 partial agonist activities, accompanied with at least over 15-fold reduction at the CB1 potency compared to compound 11. In particular, the cyanomethyl (34), cyanoethyl (35) and methoxypentyl (41) had no appreciable agonist activity at the CB1 receptor. On the other hand, a tetrahydropyranyl-methyl (33) substitution maintained functional potencies at both receptors (EC50 values of 84 nM and 38 nM at CB1 and CB2 receptors, respectively). We then examined the effects of mono-substitutions at the indole ring on the functional profiles. Introduction of a methoxy group at the 4-, 5-, or 7-position of indole ring (25– 27) resulted in a complete loss of agonist activities at both CB1 and CB2 receptors. However, for compound 27, the 7-methoxy substitution resulted in a potent and selective CB2 antagonist with an IC50 value of 16 nM. A similar trend was obtained for the 7-methylthio analog (28) with an IC50 value of 28 nM at the CB2 receptor. Oxidation or deletion of the sulfur atom of compound 28 resulted in the 7methylsulfinyl (29) or 7-methyl (32) analogs, respectively. Unfortunately, both of these compounds were found to be completely inactive at the CB2 receptor, while only the 7-methyl analog (32) displayed weak, partial agonist activity at the CB1 receptor (EC50 value of 6.7 µM).


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Table 2. Agonist and Antagonist Activities of 2-Amidoalkylindoles in CHO Cells Expressing human CB1 or CB2 Receptor by Calcium Mobilization Assays.a Compound

Agonism CB1

Antagonism CB2

CB1

CB2

EC50, µM

Emaxb, %

EC50, µM

Emax, %

IC50, µM

IC50, µM

43

NAb

NDc

0.98 ± 0.19

66.7 ± 6.0

NA

NA

44

NA

ND

0.72 ± 0.13

53.3 ± 4.4

NA

NA

45

NA

ND

0.15 ± 0.01

93.3 ± 4.4

NA

NA

46

NA

ND

0.27 ± 0.06

61.7 ± 4.4

NA

NA

47

NA

ND

0.075 ± 0.004

41.7 ± 1.7

NA

NA

48

NA

ND

7.13 ± 1.91

43.3 ± 3.3

NA

NA

49

NA

ND

0.14 ± 0.03

53.3 ± 4.4

NA

NA

50

NA

ND

0.49 ± 0.04

63.3 ± 3.3

NA

NA

51

NA

ND

0.41 ± 0.26

41.7 ± 4.4

NA

NA

52

NA

ND

0.22 ± 0.03

66.7 ± 3.3

NA

NA

53

NA

ND

0.20 ± 0.06

48.3 ± 4.4

NA

NA

54

NA

ND

3.8 ± 1.5

83.3 ± 3.3

NA

NA

56

NA

ND

0.16 ± 0.03

86.7 ± 3.3

NA

NA

57

NA

ND

0.12 ± 0.04

85.0 ± 2.9

NA

NA

58

NA

ND

0.10 ± 0.03

70.0 ± 2.9

NA

NA

59

NA

ND

0.41 ± 0.12

46.7 ± 3.3

NA

NA

60

NA

ND

0.61 ± 0.08

60.0 ± 2.9

NA

NA



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a

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61

NA

ND

0.18 ± 0.06

73.3 ± 4.4

NA

NA

62

NA

ND

0.092 ± 0.009

60.0 ± 5.0

NA

NA

66

NA

ND

0.43 ± 0.07

51.7 ± 1.7

NA

NA

67

NA

ND

NA

ND

NA

NA

68

NA

ND

0.42 ± 0.10

50.0 ± 2.9

NA

NA

73

NA

ND

4.7 ± 0.8

86.7 ± 1.7

NA

NA

74

NA

ND

8.6 ± 3.6

93.3 ± 4.4

NA

NA

75

NA

ND

NA

ND

NA

NA

7

0.022 ± 0.005

100 ± 15

0.085 ± 0.009

100 ± 6

-

-

See Experimental Section. Data represent mean values ± SEM of eight-point 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 compound 1 as a positive control for 10 min, followed by addition of agonist 7 at 100 nM. bNA: not active, defined as < 50% activation or < 50% inhibitory activity at 10 µM in the primary assay. cND: 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 3position to the 2-position of the indole (44–52, 54, 56–62, and 66–68). To our delight, no agonist or antagonist activity at the CB1 receptor were observed for all the 25 tested compounds. These 2amidoalkylindoles generally behave as CB2 partial agonists with moderate to high potency (EC50 values between 720 nM and 75 nM), with the exception of compounds 48, 55, 73, and 74 possessing EC50 values over 3 µM and the totally inactive compounds 67 and 75. The polar nature of compounds 48, and 73–75 may be an important factor for the observed significant loss of activity. The N-1 unsubstituted compound (43) showed a similar functional activity to the n-C5H11 substituted compound (44). Various


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N-1 substituents were well tolerated for CB2 partial agonist activity including the cyanoalkyls (45–47), 5-fluoropentyl (49), 5,5-difluoropentyl (62), 4,4,4-trifluoropentyl (50), tetrahydropyranyl-methyl (51), 2-(2-oxooxazolidin-3-yl)ethyl (52), hydroxyalkyls (56–58), 5-oxopentyl (61), with EC50 values between 75 and 490 nM. It was noted that the N-1 tetrahydropyranyl-methyl substitution within the 2amidoalkylindole series (51) confers no agonist activity at the CB1 receptor, unlike the trend previously seen in the 3-amidoalkylindoles (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) N-1 substitutions yielded the most potent CB2 partial agonists with EC50 values of less than 160 nM, similar 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 CB2 receptor (54 vs 47). Among the pentanol-substituted indoles 66–68, replacement of the adamantane with tert-butyl (66) or cyclohexane (68) groups gave CB2 partial agonists with EC50 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 CB2. As shown in Figure 2, compound 57 dose-dependently inhibits the forskolin-stimulated cAMP accumulation in CHO cells with an EC50 value of 142 nM. Data were normalized to the amounts of cAMP in forskolin-stimulated cells (100%).



cAMP Assay for CB2 Fsk: 3 uM 120 100

Response %

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80 60 40 20 0

EC50 = 142.2 ± 28.6 nM

-20 -12 -11 -10

-9

-8

-7

-6

-5

-4

-3

Compound 57 Log[M]

Figure 2. Compound 57 inhibits forskolin-stimulated cAMP formation in CHO cells expressing CB2. EC50 value represents mean values ± SEM of three independent experiments each performed in triplicates. Analyses of Receptor-Ligand Interactions. Relatively small structural modifications of GPCR ligands could lead to a major change in their functional profiles.44 As shown in Figure 3, in the 4-quinolone-3-carboxamide series of CB ligands, replacement of an isopropyl group (76) at the 6-position with a furan-2-yl (77) alters its CB2 functional profile from an agonist to an inverse agonist in GTPγS assay.45-47 Similarly, for the quinolone2,4(1H,3H)-diones, C5- or C8-methyl (78) substitutions were reported as CB2 receptor agonists, while the C6- or C7-methyl (79) substituted compounds act as antagonists.27 We found that a 7-methoxy or 7methylthio substitution at the 3-amidoalkylindole 16 resulted in potent CB2 antagonists (27 or 28, IC50 = 16–28 nM) without observable agonist activity on CB2.


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Figure 3. Selected examples of small structural modifications of CB2 receptor ligands leading to a major change in their functional activities. We next performed flexible docking simulations to predict the receptor-ligand interactions between compound 16 or 27 and CB2 based on the homology model of CB2 active state,48 using the FlexiDock 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, which have been demonstrated as crucial residues for ligand recognition in the CB2 receptor.49 The adamantyl group of 16 or 27 may make contact with a hydrophobic cavity composed of F87, F91, F94,



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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 interacting with CP55940 binding to the CB2 receptor.4 Our docking simulations also suggested that the agonist 16 would have a different interaction pattern with the antagonist 27 for their binding to the CB2 receptor. In the docking-simulated structural model of complex CB2-16 (Figure 4. left), the phenyl side chain of F117 may have a nearly parallel π-π 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, T-shaped π-π interaction with the phenyl group of F117 in the docking-simulated CB2-27 (Figure 4. right). This docking analysis is in agreement with the previously reported interaction mode calculated between CB2 and CB2-selective antagonist C6- or C7-substituted quinolone-2,4(1H,3H)-diones, which also have their C6- or C7-substituent to block the movement of the indole ring of W258.27 As reported on the other rhodopsin-like GPCRs, F3.36/W6.48 motif is believed to be the rotamer toggle switch for the receptor activation.6

Figure 4. Docking results of CB2 receptor agonist 16 (left) and CB2 receptor antagonist 27 (right) complexes. The carbon atoms of F117/W258 motif are colored light green, and the H-bonds are shown as blue dashed lines.


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In Vivo Efficacy−Mouse EAE Model for Multiple Sclerosis As one of the most potent and selective CB2 receptor partial agonists identified in this amidoalkylindoles 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 blood-brain 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 vehicle-treated 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 CB2 receptor, may alleviate the clinical symptoms in this model by reducing the extent of infiltration of leukocytes and demyelination in CNS.



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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

95%) were established by analytical HPLC, which was carried out on an Waters e2695 HPLC system with a ZORBAX SB-C18 column, with detection at 240 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. N-((3s,5s,7s)-Adamantan-1-yl)-1-pentyl-1H-indole-3-carboxamide

(11).

This

compound

was

obtained from indole and 1-bromopentane via route A. White solid; yields 94%, 92%, and 67%; purity 97.4% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 7.6 Hz, 1H), 7.64 (s, 1H), 7.37–7.34 (m, 1H), 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), 1.79–1.68 (m, 6H), 1.38–1.24 (m, 4H), 0.87 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 164.5,



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136.6, 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), 29.0, 22.3, 13.9. HRMS (ESI): Calcd for C24H32N2NaO [M+Na]+, 387.2407; found 387.2413. General procedure of for the preparation of 3-amidoalkylindoles. Route A: 4-(2-(1H-Indol-1-yl)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 N2. After stirring for 15 min at rt 4-(2-chloroethyl)morpholine (2.03 g, 17.37 mmol) was added. 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 Na2SO4, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel, eluting with hexane/EtOAc (5:1) to provide the title compound as light red oil; yield 50%; 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 8.0 Hz, 1H), 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, 2H), 3.61 (t, J = 4.6 Hz, 4H), 2.60 (t, J = 6.8 Hz, 2H), 2.34 (t, J = 4.6 Hz, 4H). 1-(2-Morpholinoethyl)-1H-indole-3-carboxylic 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 10% aqueous hydrochloric acid solution was added to the mixture until pH value dropped to 5–6, followed by extraction with CH2Cl2 (3×30 mL). The combined organic layers were dried over Na2SO4,


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and concentrated under vacuum to give the title compound. White solid; yield 83%; 1H NMR (400 MHz, DMSO-d6) 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, 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). 13C NMR (101 MHz, DMSO-d6) 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. N-((3s,5s,7s)-Adamantan-1-yl)-1-(2-morpholinoethyl)-1H-indole-3-carboxamide (16). To a solution of 15 (400 mg, 1.46 mmol) and DMF (0.1 mL) in CH2Cl2 (10 mL) was added oxalyl chloride (0.2 mL, 2.2 mmol). After stirring for 2 hr at rt, the mixture was concentrated under vacuum. The residue was dissolved in CH2Cl2 (10 mL), followed by the additions of amantadine (220 mg, 1.46 mmol) and 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 I). 1H NMR (400 MHz, CDCl3) δ 7.91–7.87 (m, 1H), 7.70 (s, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.28–7.20 (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, J = 4.0 Hz, 4H), 2.18–2.13 (m, 9H), 1.79–1.68 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 164.4, 136.5, 131.5, 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 C25H33N3NaO2 [M+Na]+, 430.2465; found 430.2457. 4-(2-(4-Methoxy-1H-indol-1-yl) ethyl) morpholine (17). Light yellow oil; yield 88%; 1H NMR (400 MHz, CDCl3) 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 = 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 (t, J = 7.0 Hz, 2H), 2.38 (t, J = 4.4 Hz, 4H). 13C NMR (101 MHz, CDCl3) δ 153.6, 137.5, 126.6, 122.4, 119.2, 102.8, 99.3, 98.6, 67.0, 58.2, 55.3, 53.9, 44.2.



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4-(2-(5-Methoxy-1H-indol-1-yl) ethyl) morpholine (18). Light yellow oil; yield 70%; 1H NMR (400 MHz, CDCl3) 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 = 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 (t, J = 4.4 Hz, 4H). 13C NMR (101 MHz, CDCl3) δ 154.1, 131.3, 129.0, 128.6, 111.9, 110.0, 102.6, 100.9, 67.0, 58.3, 55.9, 53.9, 44.2. 4-(2-(7-Methoxy-1H-indol-1-yl) ethyl) morpholine (19). Light yellow oil; yield 99%; 1H NMR (400 MHz, CDCl3) 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 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, J = 4.4 Hz, 4H). 13C NMR (101 MHz, CDCl3) δ 147.5, 131.1, 129.4, 125.6, 119.9, 113.9, 102.3, 101.5, 67.1, 60.2, 55.2, 54.0, 46.7. 4-(2-(7-(Methylthio)-1H-indol-1-yl)ethyl)morpholine (20). Light yellow oil; yield 92%; 1H NMR (400 MHz, CDCl3) 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, 1H), 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). 13C NMR (101 MHz, CDCl3) 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, 19.0. 4-Methoxy-1-(2-morpholinoethyl)-1H-indole-3-carboxylic acid (21). White solid; yield 80%; 1H NMR (400 MHz, CDCl3) δ 11.69 (brs, 1H), 8.02 (s, 1H), 7.25 (t, J = 7.8 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 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, 2H), 2.47 (brs, 4H). 13C NMR (101 MHz, CDCl3) δ 164.2, 150.9, 138.4, 137.0, 123.8, 114.5, 107.4, 105.1, 102.4, 66.8, 57.6, 56.5, 53.8, 44.7. 5-Methoxy-1-(2-morpholinoethyl)-1H-indole-3-carboxylic acid (22). White solid; yield 71%; 1H NMR (400 MHz, CDCl3) δ 12.10 (brs, 1H), 7.92 (s, 1H), 7.68 (s, 1H), 7.27–7.26 (d, J = 8.4 Hz, 1H),


Page 29 of 58

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6.92 (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, 4H). 13C NMR (101 MHz, CDCl3) δ 170.2, 156.0, 135.7, 131.6, 127.8, 113.4, 110.7, 106.5, 103.2, 66.7, 57.7, 55.8, 53.7, 44.5. 7-Methoxy-1-(2-morpholinoethyl)-1H-indole-3-carboxylic acid (23). White solid; yield 56%; 1H NMR (400 MHz, CDCl3) 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, 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). 13C NMR (101 MHz, DMSO-d6) 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, 46.1. 7-(Methylthio)-1-(2-morpholinoethyl)-1H-indole-3-carboxylic acid (24). White solid; yield 87%; 1H NMR (400 MHz, CD3OD) 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 (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). 13C NMR (101 MHz, CD3OD) 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, 19.1. N-((3s,5s,7s)-Adamantan-1-yl)-4-methoxy-1-(2-morpholinoethyl)-1H-indole-3-carboxamide

(25).

This compound was obtained from 4-methoxy-1H-indole and 4-(2-chloroethyl)morpholine via route A. Pale yellow solid; yields 88%, 72%, and 14%; purity 96.4% (Gradient II). 1H NMR (400 MHz, CDCl3) 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 Hz, 4H), 2.18–2.13 (m, 9 H), 1.78–1.70 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 163.3, 152.4, 138.9, 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 C26H36N3O3 [M+H]+, 438.2751; found 438.2793.



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N-((3s,5s,7s)-Adamantan-1-yl)-5-methoxy-1-(2-morpholinoethyl)-1H-indole-3-carboxamide

Page 30 of 58

(26).

This compound was obtained from 5-methoxy-1H-indole and 4-(2-chloroethyl)morpholine via route A. White solid; yields 70%, 63%, and 42%; purity 99.4% (Gradient I).1H NMR (400 MHz, CDCl3) δ 7.56 (s, 1H), 7.51 (d, J = 2.4 Hz, 1H), 7.23 (d, J = 8.8 Hz, 1H), 6.92–6.89 (m, 1H), 5.58 (s, 1H), 4.17 (t, J = 6.8 Hz, 2H), 3.88 (s, 3H), 3.68 (t, J =4.6 Hz, 4H), 2.71 (t, J = 6.0 Hz, 2H), 2.45 (t, J = 4.6 Hz, 4H), 2.17–2.13 (m, 9H), 1.78–1.70 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 164.7, 155.4, 131.8, 130.6, 126.6, 112.8, 111.9, 110.7, 102.6, 67.0, 58.1, 56.0, 54.0, 52.0, 44.5, 42.3 (3C), 36.5 (3C), 29.7 (3C). HRMS (ESI): Calcd for C26H36N3O3 [M+H]+, 438.2751; found 438.2754. N-((3s,5s,7s)-Adamantan-1-yl)-7-methoxy-1-(2-morpholinoethyl)-1H-indole-3-carboxamide

(27).

This compound was obtained from 7-methoxy-1H-indole and 4-(2-chloroethyl)morpholine via route A. White solid; yields 99%, 56%, and 60%; purity 95.6% (Gradient I). 1H NMR (400 MHz, CDCl3) δ 7.55 (s, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.11 (t, J = 8.0 Hz, 1H), 6.67 (d, J = 8.0 Hz, 1H), 5.68 (s, 1H), 4.49 (t, J = 7.2 Hz 2H), 3.93 (s, 3H), 3.71 (t, J = 4.6 Hz, 4H), 2.74 (t, J = 7.2 Hz, 2H), 2.50 (t, J = 4.6 Hz, 4H), 2.17–2.13 (m, 9H), 1.77–1.69 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 163.4, 146.6, 131.7, 126.6, 125.0, 120.8, 111.5, 111.5, 102.1, 65.9, 58.9, 54.3, 52.8, 51.0, 46.0, 41.2 (3C), 35.5 (3C), 28.6 (3C). HRMS (ESI): Calcd for C26H35N3NaO3 [M+Na]+, 460.2571; found 460.2555. N-((3s,5s,7s)-Adamantan-1-yl)-7-(methylthio)-1-(2-morpholinoethyl)-1H-indole-3-carboxamide (28). This compound was obtained from 7-methylthio-1H-indole and 4-(2-chloroethyl)morpholine via route A. White solid; yields 92%, 56%, and 70%; purity 98.9% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.86–7.84 (m, 1H), 7.56 (s, 1H), 7.21–7.15 (m, 2H), 5.60 (s, 1H), 4.73 (t, J = 7.0 Hz, 2H), 3.70 (t, J = 4.0 Hz, 4H), 2.77 (t, J = 7.2 Hz, 2H), 2.53–2.50 (m, 7H), 2.16–2.13 (m, 9H), 1.79–1.68 (m, 6H). 13

C NMR (101 MHz, CDCl3) δ 164.1, 134.5, 133.3, 127.3, 124.8, 121.8, 121.1, 118.9, 112.8, 67.1, 59.9,


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54.1, 52.2, 47.2, 42.3 (3C), 36.6 (3C), 29.7 (3C), 18.8. HRMS (ESI): Calcd for C26H36N3O2S [M+H]+, 454.2523; found 454.2551. N-((3s,5s,7s)-Adamantan-1-yl)-7-(methylsulfinyl)-1-(2-morpholinoethyl)-1H-indole-3-carboxamide (29). To a stirred solution of 28 (100 mg, 0.22 mmol) in CH2Cl2 (1.5 mL) was added 3chloroperoxybenzoic acid (38 mg, 0.44 mmol) at -40 ºC. After stirring 2 hr, the mixture was concentrated under vacuum and the residue was purified by flash chromatography on silica gel, eluting with CH2Cl2/methanol (20:1) to provide the title compound. White solid; yield 14%; purity 98.6% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 8.18 (d, J = 8.0 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.61 (s, 1H), 7.44 (t, J = 7.8 Hz, 1H), 5.59 (s, 1H), 4.44–4.23 (m, 2H), 3.74–3.61 (m, 4H), 2.84–2.76 (m, 4H), 2.71–2.65 (m, 1H), 2.51–2.43 (m, 4H), 2.17–2.15 (m, 9H), 1.78–1.71 (m, 6H).

13

C NMR (101 MHz,

CD3OD) δ 166.5, 134.9, 132.9, 130.4, 130.0, 126.3, 122.6, 119.4, 113.9, 67.8, 59.5, 55.0, 53.5, 53.3, 44.1, 42.7 (3C), 37.6 (3C), 31.1 (3C). HRMS (ESI): Calcd for C26H36N3O3S [M+H]+, 470.2472; found 470.2505. Route B: N-((3s,5s,7s)-Adamantan-1-yl)-1H-indole-3-carboxamide (30). To a stirred solution of indole (300 mg, 2.56 mmol) in anhydrous DMF (3 mL) was added trifluoroacetic anhydride (1.07 g, 5.12 mmol) at 0 ºC. After stirring for 2 hr at rt, the reaction was quenched with water (10 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 added to a 5 mL aqueous solution of NaOH (307.2 mg, 7.68 mmol). After refluxing for 3 hr, the reaction mixture was cooled to rt. A 10% aq. hydrochloric acid solution was added to the mixture until pH value dropped to 5–6. The resulting mixture was extracted with CH2Cl2 (3×20 mL) and the combined organic layers were dried



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over Na2SO4, and concentrated under vacuum. The residue and DMF (0.1 mL) were dissolved in CH2Cl2 (5 mL). To this solution was added oxalyl chloride (0.44 mL, 5.2 mmol) dropwise under N2. After stirring for 2 hr at rt, the mixture was concentrated under vacuum. The residue was redissolved in CH2Cl2 (5 mL), followed by the addition of amantadine (472 mg, 3.12 mmol) and triethylamine (1.4 mL, 10.4 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 hexanes/EtOAc (5:1) to provide the title compound. White solid; yield 50%; purity 99.9% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 10.50–9.82 (m, 1H), 7.88–7.82 (m, 1H), 7.60–7.56 (m, 1H), 7.44–7.39 (m, 1H), 7.21– 7.19 (m, 2H), 5.84–5.82 (m, 1H), 2.20–2.14 (m, 9H), 1.79–1.68 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 165.5, 136.8, 128.7, 124.3, 122.5, 121.2, 119.1, 112.8, 112.6, 52.3, 42.2 (3C), 36.5 (3C), 29.5 (3C). HRMS (ESI): Calcd for C19H23N2O [M+H]+, 295.1805; found 295.1809. To a stirred solution of 30 (100 mg, 0.34 mmol) in anhydrous DMF (2 mL) was added NaH (32.0 mg, 50% in mineral oil, 0.68 mmol) with ice cooling. After stirring for 15 min, 4-(2-chloroethyl)morpholine (76.3 mg, 0.51 mmol) was added under N2. After stirring for 2 hr at 100 ºC, the reaction was quenched with water (10 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were washed with water (3×10 mL) and brine (10 mL), dried over Na2SO4, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel, eluting with hexane/EtOAc (10:1) to give 16 as white solid; yield 65%. N-((3s,5s,7s)-Adamantan-1-yl)-7-methyl-1H-indole-3-carboxamide

(31).

This

compound

was

obtained from 7-methyl-1H-indole via route B. White solid; yield 76%; purity 99.2% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 9.05 (s, 1H), 7.73 (s, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), 7.04 (d, J = 7.2 Hz, 1H), 5.78 (s, 1H), 2.51 (s, 3H), 2.19–2.14 (m, 9H), 1.78–1.70 (m, 6H). 13C NMR


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(101 MHz, CDCl3) δ 165.2, 136.2, 128.3, 123.9, 123.1, 121.9, 121.5, 116.8, 113.8, 52.2, 42.2 (3C), 36.5 (3C), 29.6 (3C), 16.8. HRMS (ESI): Calcd for C20H24N2NaO [M+Na]+, 331.1781; found 331.1797. N-((3s,5s,7s)-Adamantan-1-yl)-7-methyl-1-(2-morpholinoethyl)-1H-indole-3-carboxamide

(32).

This compound was obtained from 7-methyl-1H-indole and 4-(2-chloroethyl)morpholine via route B. White solid; yields 76% and 65%; purity 99.8% (Gradient I). 1H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 8.0 Hz, 1H), 7.57 (s, 1H), 7.09 (t, J = 8.0 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 5.69 (s, 1H), 4.40 (t, J = 7.0 Hz, 2H), 3.68 (br s, 4H), 2.75–2.65 (m, 5H), 2.44 (br s, 4H), 2.17–2.13 (m, 9H), 1.79–1.68 (m, 6H). 13C NMR (101 MHz, CD3OD) δ 167.4, 136.2, 133.6, 128.8, 126.3, 122.2, 122.0, 120.0, 112.3, 67.6, 60.7, 54.8, 53.0, 47.4, 42.7 (3C), 37.4 (3C), 30.9 (3C), 19.9. HRMS (ESI): Calcd for C26H36N3O2 [M+H]+, 422.2802; found 422.2832. N-((3s,5s,7s)-Adamantan-1-yl)-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-indole-3-carboxamide (33). This compound was obtained from indole and 4-(bromomethyl)tetrahydro-2H-pyran via route A. White solid; yields 95%, 65% and 76%; purity 98.9% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.90–7.84 (m, 1H), 7.62 (s, 1H), 7.39–7.33 (m, 1H), 7.30–7.19 (m, 2H), 5.70 (s, 1H), 4.05–3.89 (m, 4H), 3.37–3.22 (m, 2H), 2.23–2.13 (m, 9H), 2.12–2.06 (m, 1H), 1.80–1.68 (m, 6H), 1.53–1.33 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 164.3, 136.8, 131.7, 125.2, 122.3, 121.2, 119.9, 112.5, 110.3, 67.3, 52.6, 52.1, 42.2 (3C), 36.5 (3C), 36.1, 30.8, 29.6 (3C). HRMS (ESI): Calcd for C25H32N2NaO2 [M+Na]+, 415.2356; found 415.2375. N-((3s,5s,7s)-Adamantan-1-yl)-1-(cyanomethyl)-1H-indole-3-carboxamide (34). This compound was obtained from indole and 2-bromoacetonitrile via route B. White solid; yield 80%; purity 99.8% (Gradient II). 1H NMR (400 MHz, DMSO-d6) δ 8.14–8.11 (m, 2H), 7.60 (d, J = 8.0 Hz, 1H), 7.31–7.18 (m, 3H), 5.59 (s, 2H), 2.13–2.02 (m, 9H), 1.67 (br s, 6H). 13C NMR (101 MHz, DMSO-d6) δ 163.4,



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135.6, 130.4, 126.8, 122.7, 121.6, 121.3, 116.2, 112.8, 110.0, 51.1, 41.2 (3C), 36.1 (3C), 34.0, 28.9 (3C). HRMS (ESI): Calcd for C21H23N3NaO [M+Na]+, 356.1733; Found 356.1716. N-((3s,5s,7s)-Adamantan-1-yl)-1-(2-cyanoethyl)-1H-indole-3-carboxamide (35). This compound was obtained from indole and 2-bromopropanenitrile via route B. White solid; yield 25%; purity 99.1% (Gradient II). 1H NMR (400 MHz, DMSO-d6) δ 8.12–8.09 (m, 2H), 7.60 (d, J = 8.0 Hz, 1H), 7.22–7.12 (m, 2H), 7.05 (s, 1H), 4.50 (t, J = 6.2 Hz, 2H), 3.07 (t, J = 6.2 Hz, 2H), 2.14–2.02 (m, 9H), 1.67 (br s, 6H).

13

C NMR (101 MHz, DMSO-d6) δ 163.7, 135.7, 130.6, 126.6, 122.0, 121.3, 120.7, 118.6, 111.6,

110.2, 51.0, 41.6, 41.3 (3C), 36.1 (3C), 28.9 (3C), 18.4. HRMS (ESI): Calcd for C22H25N3NaO [M+Na]+, 370.1890; Found 370.1872. N-((3s,5s,7s)-Adamantan-1-yl)-1-(3-cyanopropyl)-1H-indole-3-carboxamide (36). This compound was obtained from indole and 2-bromobutanenitrile via route B. White solid; yield 41%; purity 99.8% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.93–7.91 (m, 1H), 7.63 (s, 1H), 7.39–7.37 (m, 1H), 7.31– 7.25 (m, 2H), 5.73 (s, 1H), 4.29 (t, J = 6.0 Hz, 2H), 2.30–2.25 (m, 2H), 2.23–2.11 (m, 11H), 1.78–1.70 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 164.3, 136.6, 130.8, 125.7, 123.1, 121.8, 120.6, 118.7, 113.5, 110.1, 52.4, 44.9, 42.4 (3C), 36.7 (3C), 29.9 (3C), 26.1, 14.8. HRMS (ESI): Calcd for C23H27N3NaO [M+Na]+, 384.2046; Found 384.2054. N-((3s,5s,7s)-Adamantan-1-yl)-1-(4-hydroxybutyl)-1H-indole-3-carboxamide (39). To a solution of 4-chlorobutan-1-ol (2.0 g, 18.4 mmol) in CH2Cl2 (15 mL) was added tert-butyldimethylsilyl chloride (3.04 g, 20.3 mmol) and imidazole (1.40 g, 20.3 mmol) at rt. After stirring for 3 hr, the reaction mixture was filtered off and the filtrate was concentrated to provide crude silyl ether 37 as a colorless oil. To a solution of 30 (40 mg, 0.14 mmol) in anhydrous DMF (1 mL) was added NaH (13 mg, 50% in mineral oil, 0.27 mmol) with ice cooling. After stirring for 15 min, the crude silyl ether 37 (61 mg, 0.27 mmol)


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was added to the reaction mixture. After stirring for 4 hr at 100 ºC, the reaction was quenched with water (10 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were washed with water (3×10 mL) and brine (10 mL) and concentrated under vacuum. The residue was dissolved in a solution of TBAF in THF (2 mL, 0.6 mmol). After stirring overnight at rt, the mixture was concentrated under vacuum and the residue was purified by flash chromatography on silica gel, eluting with hexanes/ethyl acetate (5:1) to provide the title compound. White solid; overall yield 67%; purity 97.8% (Gradient II). 1H NMR (400 MHz, CD3OD) δ 8.02 (d, J = 8.0 Hz, 1H), 7.85 (s, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.24–7.12 (m, 2H), 4.23 (t, J = 7.0 Hz, 2H), 3.55 (t, J = 6.4 Hz, 2H), 2.20–2.11 (m, 9H), 1.97–1.88 (m, 2H), 1.82–1.72 (m, 6H), 1.56–1.47 (m, 2H). 13C NMR (101 MHz, CD3OD) δ 167.6, 137.9, 131.9, 127.9, 123.4, 122.0, 122.0, 112.3, 111.2, 62.4, 53.2, 47.4, 42.9 (3C), 37.6 (3C), 31.1 (3C), 30.8, 27.8. HRMS (ESI): Calcd for C23H30N2NaO2 [M+Na]+, 389.2199; found 389.2188. N-((3s,5s,7s)-Adamantan-1-yl)-1-(5-hydroxypentyl)-1H-indole-3-carboxamide (40). This compound was synthesized from 5-chloropentan-1-ol and 30 according to the methodology described for compound 39. White solid; overall yield 95%; purity 97.5% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.99–7.84 (m, 1H), 7.65 (s, 1H), 7.37–7.35 (m, 1H), 7.28–7.20 (m, 2H), 5.71 (s, 1H), 4.13 (t, J = 7.0 Hz, 2H), 3.61 (t, J = 6.4 Hz, 2H), 2.19–2.13 (m, 9H), 1.92–1.83 (m, 2H), 1.79–1.68 (m, 7H), 1.62–1.53 (m, 2H), 1.44–1.34 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 164.5, 136.5, 131.3, 125.2, 122.2, 121.1, 119.9, 112.2, 110.2, 62.4, 52.0, 46.7, 42.2 (3C), 36.5 (3C), 32.2, 29.8, 29.6 (3C), 23.2. HRMS (ESI): Calcd for C24H32N2NaO2 [M+Na]+, 403.2356; found 403.2345. N-((3s,5s,7s)-Adamantan-1-yl)-1-(5-methoxypentyl)-1H-indole-3-carboxamide (41). To a solution of 40 (72 mg, 0.19 mmol) in anhydrous DMF (1 mL) was added NaH (18 mg, 50% in mineral oil, 0.38 mmol) with ice cooling. After stirring for 15 min, CH3I (54 mg, 0.38 mmol) was added. After stirring for 2 hr at rt, the reaction was quenched with water (10 mL) and extracted with EtOAc (3×10 mL). The



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combined organic layers were washed with water (3×20 mL) and brine (10 mL), dried over Na2SO4, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel, eluting with hexane/EtOAc (5:1) to provide the title compound. White solid; yield 67%; purity 96.9% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.90–7.87 (m, 1H), 7.64 (s, 1H), 7.36–7.34 (m, 1H), 7.27–7.20 (m, 2H), 5.71 (s, 1H), 4.10 (t, J = 7.2 Hz, 2H), 3.33 (t, J = 8.0 Hz, 2H), 3.30 (s, 3H), 2.22–2.10 (m, 9H), 1.89–1.82 (m, 2H), 1.78–1.70 (m, 6H), 1.61–1.54 (m, 2H), 1.42–1.34 (m, 2H).

13

C NMR (101 MHz,

CDCl3) δ 164.6, 136.7, 131.4, 125.5, 122.4, 121.3, 120.2, 112.4, 110.4, 72.5, 58.7, 52.2, 46.8, 42.4 (3C), 36.7 (3C), 30.0, 29.8, 29.4 (3C), 23.8. HRMS (ESI): Calcd for C25H34N2NaO [M+Na]+ 417.2512; Found 417.2504. N-((3s,5s,7s)-Adamantan-1-yl)-1H-indole-2-carboxamide (43). To a solution of NaOH (3.2 g, 79.4 mmol) in water (40 mL) was added 42 (3.0 g, 15.9 mmol). After refluxing for 1 hr, the reaction mixture was cooled to rt. Hydrochloric acid (10% aq.) was added to the mixture until pH value dropped to 3–4 and extracted with CH2Cl2 (3×50 mL). The combined organic layers were dried over Na2SO4, and concentrated under vacuum. The residue and DMF (0.1 mL) were dissolved in CH2Cl2 (50 mL), and to this solution was added oxalyl chloride (2.7 mL, 31.74 mmol) dropwise. After stirring for 3 hr, the mixture was concentrated under vacuum. The residue was redissolved in CH2Cl2 (50 mL) and to the solution was added amantadine (2.9 g, 19.0 mmol) and triethylamine (4.4 mL, 31.7 mmol). After stirring overnight the reaction mixture was filtered off, the filtrate was concentrated under vacuum and the residue was purified by flash chromatography on silica gel, eluting with hexane/EtOAc (5:1) to provide the title compound. White solid; overall yield 76%; purity 99.9% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 10.40 (s, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.26–7.22 (m, 1H), 7.11 (t, J = 7.6 Hz, 1H), 6.77 (s, 1H), 5.96 (s, 1H), 2.19–2.16 (m, 9H), 1.81–1.70 (m, 6H). 13C NMR (101 MHz,


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CDCl3) δ 161.1, 136.6, 131.9, 127.6, 124.0, 121.7, 120.4, 112.3, 101.3, 52.5, 41.9 (3C), 36.4 (3C), 29.5 (3C). HRMS (ESI): Calcd for C19H22N2NaO [M+Na]+, 317.1624; found 317.1636. N-((3s,5s,7s)-Adamantan-1-yl)-1-pentyl-1H-indole-2-carboxamide (44). To a solution of 43 (100 mg, 0.34 mmol) in anhydrous DMF (2 mL) was added NaH (32 mg, 50% in mineral oil, 0.68 mmol) with ice cooling. After stirring for 15 min, 1-bromopentane (76 mg, 0.50 mmol) was added. After stirring for 4 hr at 100 ºC, the reaction was quenched with water (10 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were washed with water (3×20 mL) and brine (15 mL), dried over Na2SO4, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel, eluting with hexane/EtOAc (5:1) to provide the title compound. White solid; yield 73%; purity 96.8% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.30–7.23 (m, 1H), 7.11 (t, J = 8.0 Hz, 1H), 6.73 (s, 1H), 5.90 (s, 1H), 4.51 (t, J = 8.0 Hz, 2H), 2.15–2.13 (m, 9H), 1.81– 1.67 (m, 8H), 1.34–1.23 (m, 4H), 0.87 (t, J = 6.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 162.1, 138.1, 133.4, 126.2, 123.6, 121.7, 120.3, 110.4, 103.2, 52.5, 44.4, 41.8 (3C), 36.4 (3C), 30.4, 29.5 (3C), 29.2, 22.6, 14.2. HRMS (ESI): Calcd for C24H32N2NaO [M+Na]+, 387.2407; found 387.2411. N-((3s,5s,7s)-Adamantan-1-yl)-1-(2-cyanoethyl)-1H-indole-2-carboxamide (45). This compound was synthesized from 43 and 3-bromopropanenitrile according to the methodology described for 44. White solid; yield 76%; purity 99.5% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 8.0 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.36–7.32 (m, 1H), 7.17 (t, J = 8.0 Hz, 1H), 6.81 (s, 1H), 5.94 (s, 1H), 4.79 (t, J = 6.0 Hz, 2H), 2.97 (t, J = 6.0 Hz, 2H), 2.13 (br s, 9H), 1.79–1.68 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 161.5, 137.9, 132.3, 126.3, 124.6, 122.1, 121.2, 117.9, 109.7, 104.6, 52.7, 41.8 (3C), 40.5, 36.3 (3C), 29.5 (3C), 19.2. HRMS (ESI): Calcd for C22H25N3NaO [M+Na]+, 370.1890; found 370.1867.



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N-((3s,5s,7s)-Adamantan-1-yl)-1-(3-cyanopropyl)-1H-indole-2-carboxamide (46). This compound was synthesized from 43 and 4-bromobutanenitrile according to the methodology described for 44. White solid; yield 65%; purity 95.4% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.63 (d, J = 8.0 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.35–7.31 (m, 1H), 7.19–7.13 (m, 1H), 6.79 (s, 1H), 5.92 (s, 1H), 4.64 (t, J = 8.0 Hz, 2H), 2.39–2.35 (m, 2H), 2.30–2.20 (m, 2H), 2.13 (br s, 9H), 1.79–1.68 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 161.7, 138.1, 132.6, 126.1, 124.3, 121.9, 120.9, 119.4, 109.9, 104.0, 52.6, 42.9, 41.8 (3C), 36.3 (3C), 29.5 (3C), 26.6, 14.8. HRMS (ESI): Calcd for C23H27N3NaO [M+Na]+, 384.2046; found 384.2052. N-((3s,5s,7s)-Adamantan-1-yl)-1-(4-cyanobutyl)-1H-indole-2-carboxamide (47). This compound was synthesized from 43 and 5-bromopentanenitrile according to the methodology described for 44. White solid; yield 33%; purity 98.6% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 8.0 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.33–7.27 (m, 1H), 7.17–7.11 (m, 1H), 6.76 (s, 1H), 5.92 (s, 1H), 4.59 (t, J = 7.0 Hz, 2H), 2.34–2.30 (m, 2H), 2.13 (s, 9H), 2.02–1.92 (m, 2H), 1.77–1.70(m, 6H), 1.69–1.63 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 161.9, 138.0, 132.9, 126.2, 124.0, 121.9, 120.6, 119.5, 110.1, 103.6, 52.6, 43.2, 41.8 (3C), 36.3 (3C), 29.7, 29.5 (3C), 22.8, 16.9. HRMS (ESI): Calcd for C24H30N3O [M+H]+, 376.2383; found 376.2384. 5-(2-(((3s,5s,7s)-Adamantan-1-yl)carbamoyl)-1H-indol-1-yl)pentanoic acid (48). To a stirred solution of KOH (151 mg, 10 mmol) in water (5 mL) was added 47 (100 mg, 0.27 mmol). After refluxing for 24 hr, the mixture was cooled to rt. To the reaction mixture was added hydrochloric acid (10% aq.) until pH value dropped to 3–4, followed by extraction with CH2Cl2 (3×10 mL). The combined organic layers were dried over Na2SO4, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel, eluting with CH2Cl2 /methanol (20:1) to provide the title compound. White solid; yield 67%; purity 99.4% (Gradient I). 1H NMR (400 MHz, CDCl3) δ 7.61 (d, J = 8.0 Hz,


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1H), 7.36 (d, J = 8.0 Hz, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.12 (t, J = 8.0 Hz, 1H), 6.74 (s, 1H), 5.91 (s, 1H), 4.54 (t, J = 8.0 Hz, 2H), 2.35 (t, J = 8.0 Hz, 2H), 2.12 (br s, 9H), 1.90–1.83 (m, 2H), 1.77–1.69 (m, 6H), 1.69–1.59 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 178.3, 162.0, 138.0, 133.2, 126.2, 123.8, 121.7, 120.4, 110.2, 103.4, 52.5, 43.8, 41.7 (3C), 36.3 (3C), 33.6, 29.8, 29.5 (3C), 22.1. HRMS (ESI): Calcd for C24H31N2O3 [M+H]+, 395.2329; found 395.2360. N-((3s,5s,7s)-Adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-2-carboxamide (49). This compound was synthesized from 43 and 1-bromo-5-fluoropentane according to the methodology described for 44. White solid; yield 47%; purity 98.0% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.29–7.27 (m, 1H), 7.13–7.10 (m, 1H), 6.73 (s, 1H), 5.89 (s, 1H), 4.54 (t, J = 6.0 Hz, 2H), 4.40 (dt, J = 48.0 Hz, 2H), 2.13 (br s, 9H), 1.88–1.81 (m, 2H), 1.78–1.63 (m, 8H), 1.48–1.38 (m, 2H). 13C NMR (101 MHz, CDCl3) δ162.0, 138.1, 133.3, 126.2, 123.7, 121.7, 120.3, 110.3, 103.2, 83.9 (d, JC-F = 162.6 Hz), 52.4, 44.1, 41.8 (3C), 36.4 (3C), 30.2, 30.1 (d, JC-F = 20.2 Hz), 29.5 (3C), 22.7 (d, JC-F = 5.1 Hz). HRMS (ESI): Calcd for C24H31 FN2NaO [M+Na]+, 405.2313; found 405.2307. N-((3s,5s,7s)-Adamantan-1-yl)-1-(4,4,4-trifluorobutyl)-1H-indole-2-carboxamide

(50).

This

compound was synthesized from 43 and 4-bromo-1,1,1-trifluorobutane according to the methodology described for 44. White solid; yield 72%; purity 99.0% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 8.0 Hz, 1H), 7.36–7.28 (m, 2H), 7.15 (t, J = 7.2 Hz, 1H), 6.77 (s, 1H), 5.91 (s, 1H), 4.61 (t, J = 6.0 Hz, 2H), 2.17–2.06 (m, 13H), 1.80–1.66 (m, 6H).

13

C NMR (101 MHz, CDCl3) δ 160.7, 137.0,

131.8, 126.0 (q, JC-F = 276.7 Hz). 125.2, 123.0, 120.8, 119.6, 108.9, 102.7, 51.5, 41.9, 40.7 (3C), 35.3 (3C), 30.2 (q, JC-F = 29.0 Hz), 28.4 (3C), 22.0 (q, JC-F = 2.6 Hz). HRMS (ESI): Calcd for C23H27N2NaO [M+Na]+, 427.1968; Found 427.1969.



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N-((3s,5s,7s)-Adamantan-1-yl)-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-indole-2-carboxamide (51). This compound was synthesized from 43 and 4-(bromomethyl)tetrahydro-2H-pyran according to the methodology described for 44. White solid; yield 34%; purity 99.8% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.27 (t, J = 7.8 Hz, 1H), 7.12 (t, J = 7.4 Hz, 1H), 6.74 (s, 1H), 5.90 (s, 1H), 4.48 (d, J = 8.0 Hz, 2H), 3.91 (d, J = 10.8 Hz, 2H), 3.30–3.24(m, 2H), 2.16–2.03 (m, 10H), 1.79–1.69 (m, 6H), 1.48–1.37 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 162.1, 138.5, 133.6, 126.1, 123.7, 121.7, 120.4, 110.6, 103.4, 67.63, 52.5, 49.3, 41.8 (3C), 36.7, 36.4 (3C), 30.7, 29.5 (3C). HRMS (ESI): Calcd for C25H32N2NaO2 [M+Na]+, 415.2356; found 415.2374. N-((3s,5s,7s)-Adamantan-1-yl)-1-(2-(2-oxooxazolidin-3-yl)ethyl)-1H-indole-2-carboxamide

(52).

This compound was synthesized from 43 and 3-(2-bromoethyl)oxazolidin-2-one according to the methodology described for 44. White solid; yield 18%; purity 99.2% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.34–7.28 (m, 1H), 7.14 (t, J = 8.0 Hz, 1H), 6.80 (s, 1H), 5.94 (s, 1H), 4.69 (t, J = 7.8 Hz, 2H), 3.97–3.88 (m, 2H), 3.73 (t, J = 6.0 Hz, 2H), 3.11– 3.02 (m, 2H), 2.13 (br s, 9H), 1.79–1.69 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 161.8, 158.5, 138.7, 132.6, 126.0, 124.4, 121.7, 120.9, 110.1, 104.1, 61.9, 52.6, 45.6, 45.1, 42.8, 41.8 (3C), 36.3 (3C), 29.5 (3C). HRMS (ESI): Calcd for C24H30N3O3 [M+H]+, 408.2282; found 408.2295. N-Phenyl-1H-indole-2-carboxamide (53). This compound was synthesized from 42 and aniline according to the methodology described for 43. White solid; yield 64%. 1H NMR (400 MHz, DMSO-d6) δ 11.72 (s, 1H), 10.19 (s, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.68 (d, J = 8.0 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.42 (s, 1H), 7.37 (t, J = 8.0 Hz, 2H), 7.22 (t, J = 7.6 Hz, 1H), 7.11–7.05 (m, 2H). 13C NMR (101 MHz, CD3OD) δ 162.4, 139.8, 138.7, 132.5, 129.9, 129.2, 125.4, 125.4, 123.0, 122.1, 121.3, 113.2, 105.3.


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1-(4-Cyanobutyl)-N-phenyl-1H-indole-2-carboxamide (54). This compound was synthesized from 53 and 5-bromopentanenitrile according to the methodology described for 44. White solid; yield 30%; purity 99.6% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 8.06 (s, 1H), 7.65–7.59 (m, 3H), 7.37–7.31 (m, 4H), 7.18–7.12 (m, 2H), 6.98 (s, 1H), 4.56 (t, J = 7.2 Hz, 2H), 2.27 (t, J = 7.2 Hz, 2H), 2.02–1.90 (m, 2H), 1.70–1.60 (m, 2H).

13

C NMR (101 MHz, CDCl3) δ 159.5, 137.5, 136.6, 130.3, 128.1, 125.1,

123.7, 123.6, 121.2, 119.9, 119.3, 118.5, 109.2, 104.1, 42.6, 28.5, 21.8, 15.8. HRMS (ESI): Calcd for C20H19N3NaO [M+Na]+, 340.1420; found 340.1428. N-((3s,5s,7s)-Adamantan-1-yl)-3-fluoro-1H-indole-2-carboxamide (55). To a solution of 43 (294 mg, 1 mmol) in THF (5 mL) was added saturated NaHCO3 aqueous (2 mL) and 1-chloromethyl-4-fluoro1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (425 mg, 1.2 mmol) at 0 ºC. After stirring overnight at rt, water (10 mL) was added. The mixture was extracted with EtOAc (3×20 mL) and the combined organic layers were dried over Na2SO4, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel, eluting with hexane/EtOAc (20:1) to provide the title compound. White solid; yield 34%; purity 99.8% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 9.79 (s, 1H), 7.63–7.61 (m, 1H), 7.47 (dd, J = 16.0, 4.0 Hz, 1H), 7.31–7.25 (m, 1H), 7.17–7.09 (m, 1H), 6.22 (d, J = 8.0 Hz, 1H), 2.20–2.10 (m, 9H), 1.82–1.70 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 159.4 (d, JC-F = 4.0 Hz), 145.0, 142.5, 132.1 (d, JC-F = 6.0 Hz), 125.1, 120.4, 117.9 (d, JC-F = 3.0 Hz), 115.5 (dd, JC-F = 119.7, 18.3 Hz), 112.5, 52.6, 41.9 (3C), 36.4 (3C), 29.5 (3C). HRMS (ESI): Calcd for C19H21FN2NaO [M+Na]+, 335.1530; found 335.1546. N-((3s,5s,7s)-Adamantan-1-yl)-1-(3-hydroxypropyl)-1H-indole-2-carboxamide (56). This compound was synthesized from 43 and tert-butyl(3-bromopropoxy)dimethylsilane according to the methodology described for 39. White solid; yield 67%; purity 99.7% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 8.0 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 7.14 (t, J = 8.0 Hz, 1H), 6.78



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(s, 1H), 6.02 (s, 1H), 4.68 (s, 1H), 4.65–4.60 (m, 2H), 3.46–3.40 (m, 2H), 2.12 (br s, 9H), 2.09–2.05 (m, 2H), 1.75–1.69 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 162.7, 138.0, 133.3, 126.3, 124.0, 121.9, 120.7, 110.7, 103.6, 57.9, 52.9, 41.6 (3C), 40.8, 36.3 (3C), 32.6, 29.5 (3C). HRMS (ESI): Calcd for C22H28N2NaO2 [M+Na]+, 375.2043; found 375.2034. N-((3s,5s,7s)-Adamantan-1-yl)-1-(4-hydroxybutyl)-1H-indole-2-carboxamide (57). This compound was synthesized from 43 and 37 according to the methodology described for 39. White solid; yield 97%; purity 98.5% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 7.6 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.31–7.23 (m, 1H), 7.11 (t, J = 7.4 Hz, 1H), 6.74 (s, 1H), 5.95 (s, 1H), 4.51 (t, J = 7.6 Hz, 2H), 3.67 (t, J = 6.2 Hz, 2H), 2.45 (s, 1H), 2.12 (br s, 9H), 1.93–1.87 (m, 2H), 1.77–1.68 (m, 6H), 1.63–1.55 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 162.2, 138.1, 132.9, 126.2, 123.8, 121.8, 120.4, 110.3, 103.5, 61.9, 52.6, 43.8, 41.8 (3C), 36.4 (3C), 29.6 (3C), 29.5, 26.7. HRMS (ESI): Calcd for C23H31N2O2 [M+H]+, 367.2380; found 367.2406. N-((3s,5s,7s)-Adamantan-1-yl)-1-(5-hydroxypentyl)-1H-indole-2-carboxamide (58). This compound was synthesized from 43 and 38 according to the methodology described for 39. White solid; yield 93%; purity 99.4% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.29–7.25 (m, 1H), 7.12 (t, J = 8.0 Hz, 1H), 6.74 (s, 1H), 5.92 (s, 1H), 4.51 (t, J = 7.4 Hz, 2H), 3.60 (t, J = 6.4 Hz, 2H), 2.13 (br s, 9H), 1.87–1.79 (m, 2H), 1.78–1.68 (m, 6H), 1.62–1.53 (m, 2H), 1.43–1.35 (m, 2H), 1.27–1.24 (m, 1H). 13C NMR (101 MHz, CDCl3) δ 162.0, 138.0, 133.2, 126.1, 123.6, 121.7, 120.3, 110.3, 103.2, 62.7, 52.4, 44.2, 41.7 (3C), 36.3 (3C), 32.3, 30.2, 29.5 (3C), 23.1. HRMS (ESI): Calcd for C24H33N2O2 [M+H]+, 381.2537; found 381.2562. N-((3s,5s,7s)-Adamantan-1-yl)-3-fluoro-1-(4-hydroxybutyl)-1H-indole-2-carboxamide (59). This compound was synthesized from 55 and 37 according to the methodology described for 39. White solid;


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yield 70%; purity 99.2% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 8.0 Hz, 1H), 7.38– 7.28 (m, 2H), 7.16–7.11 (m, 1H), 6.26 (d, J = 9.6 Hz, 1H), 4.58–4.51 (m, 2H), 3.72 (t, J = 6.0 Hz, 2H), 2.14 (br s, 9H), 1.91–1.84 (m, 2H), 1.77–1.69 (m, 6H), 1.63–1.57(m, 3H). 13C NMR (101 MHz, CDCl3) δ 159.7, 143.8, 133.7 (d, JC-F = 6.1 Hz), 125.1, 120.3, 117.9 (d, JC-F = 2.6 Hz), 114.7 (d, JC-F = 9.4 Hz), 114.6 (d, JC-F = 9.0 Hz), 110.1 (d, JC-F = 1.9 Hz), 61.7, 52.6, 43.5, 41.9 (3C), 36.4 (3C), 29.5 (3C), 29.3, 26.6. HRMS (ESI): Calcd for C23H29FN2NaO2 [M+Na]+, 407.2105; found 407.2123. N-((3s,5s,7s)-Adamantan-1-yl)-3-fluoro-1-(5-hydroxypentyl)-1H-indole-2-carboxamide (60). This compound was synthesized from 55 and 38 according to the methodology described for 39. White solid; yield 95%; purity 97.1% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 8.0 Hz, 1H), 7.35– 7.27 (m, 2H), 7.15–7.10 (m, 1H), 6.21 (d, J = 8.0 Hz, 1H), 4.55 (t, J = 8.0 Hz, 2H), 3.62 (t, J = 6.4 Hz, 2H), 2.14 (br s, 9H), 1.83–1.70 (m, 8H), 1.63–1.55 (m, 3H), 1.43–1.36 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 159.6 (d, JC-F = 4.0 Hz), 146.2, 143.7, 133.7 (d, JC-F = 6.0 Hz), 125.0, 120.2, 117.9 (d, JC-F = 2.0 Hz), 114.9 (dd, JC-F = 41.6, 17.5 Hz), 110.2 (d, JC-F = 1.7 Hz). 62.7, 52.5, 44.2, 41.9 (3C), 36.4 (3C), 32.4, 30.3, 29.5 (3C), 23.0. HRMS (ESI): Calcd for C24H31FN2NaO2 [M+Na]+, 421.2262; found 421.2226. N-((3s,5s,7s)-Adamantan-1-yl)-1-(5-oxopentyl)-1H-indole-2-carboxamide (61). To a solution of 58 (270 mg, 0.71 mmol) in CH2Cl2 (5 mL) was added Dess-Martin periodinane (452 mg, 1.1 mmol) with ice cooling. After stirring for 5 hr at rt, the mixture was filtered off and the filtrate was concentrated under vacuum. The residue was purified by flash chromatography on silica gel, eluting with hexane/EtOAc (5:1) to provide the title compound. White solid; yield 60%; purity 96.8% (Gradient II). 1

H NMR (400 MHz, CDCl3) δ 9.74–9.72 (m, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H),

7.30–7.29 (m, 1H), 7.15–7.10 (m, 1H), 6.75 (s, 1H), 5.90 (s, 1H), 4.59–4.51 (m, 2H), 2.48–2.41 (m, 2H), 2.13 (br s, 9H), 1.90–1.80 (m, 2H), 1.79–1.69 (m, 6H), 1.69–1.60 (m, 2H). 13C NMR (101 MHz, CDCl3)



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δ 202.1, 162.0, 138.1, 133.2, 126.2, 123.8, 121.7, 120.4, 110.2, 103.4, 52.5, 43.9, 43.5, 41.8 (3C), 36.4 (3C), 30.0, 29.5 (3C), 19.4. HRMS (ESI): Calcd for C24H31 N2O2 [M+H]+, 379.2380; found 379.2396. N-((3s,5s,7s)-Adamantan-1-yl)-1-(5,5-difluoropentyl)-1H-indole-2-carboxamide (62). To a solution of 61 (50 mg, 0.13 mmol) in CCl4 (1 mL) was added DAST (48 mg, 0.3 mmol). After stirring for 30 min, the reaction was quenched with water (5 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were dried over Na2SO4, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel, eluting with hexane/EtOAc (15:1) to provide the title compound. White solid; yield 72%; purity 96.7% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.61 (d, J = 7.6 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.30–7.28 (m, 1H), 7.15–7.10 (m, 1H), 6.75 (s, 1H), 5.91–5.61 (m, 1H), 4.55 (t, J = 7.2 Hz, 2H), 2.13 (br s, 9H), 1.90–1.80 (m, 4H), 1.77–1.70 (m, 6H), 1.54–1.44 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 161.9, 138.0, 133.1, 126.2, 123.8, 121.8, 120.4, 117.2 (t, JC-F = 237.5 Hz), 110.2, 103.4, 52.5, 43.9, 41.8 (3C), 36.4 (3C), 33.8 (t, JC-F = 20.5 Hz), 29.9, 29.5 (3C), 19.6 (t, JC-F = 5.6 Hz). HRMS (ESI): Calcd for C24H30 F2N2NaO [M+Na]+, 423.2218; found 423.2242. N-(tert-Butyl)-1H-indole-2-carboxamide (63). White solid; yield 82%; 1H NMR (400 MHz, CDCl3) δ 10.32 (brs, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.26 (t, J = 7.2 Hz, 1H), 7.11 (t, J = 7.6 Hz, 1H), 6.77 (d, J = 1.6 Hz, 1H), 6.09 (s, 1H), 1.54 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 161.5, 136.6, 131.9, 127.7, 124.1, 121.7, 120.4, 112.2, 101.4, 51.9, 29.1. N-Cyclopentyl-1H-indole-2-carboxamide (64). White solid; yield 83%; 1H NMR (400 MHz, DMSOd6) δ 11.52 (s, 1H), 8.27 (d, J = 7.2 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.19– 7.15 (m, 2H), 7.02 (t, J = 7.4 Hz, 1H), 4.31–4.22 (m, 1H), 1.92–1.89 (m, 2H), 1.72–1.69 (m, 2H), 1.59– 1.51 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 160.6, 136.3, 131.9, 127.1, 123.1, 121.4, 119.6, 112.2, 102.5, 50.5, 32.2, 23.6.


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N-Cyclohexyl-1H-indole-2-carboxamide (65). White solid; yield 86%; 1H NMR (400 MHz, CDCl3) δ 9.51 (s, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.43 (dd, J = 8.4, 0.8 Hz, 1H), 7.30–7.28 (m, 1H), 7.13 (t, J = 7.2 Hz 1H), 6.81 (d, J = 1.2 Hz, 1H), 6.06–6.04 (m, 2H), 4.06–3.97 (m, 1H), 2.08–2.04 (m, 2H), 1.81–1.76 (m, 2H), 1.69–1.66 (m, 2H), 1.50–1.39 (m, 2H), 1.33–1.23 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 160.8, 136.2, 131.1, 127.7, 124.4, 121.8, 120.6, 112.0, 101.5, 48.6, 33.3, 25.5, 24.9. N-(tert-Butyl)-1-(5-hydroxypentyl)-1H-indole-2-carboxamide (66). This compound was synthesized from 42 and 2-methylpropan-2-amine according to the methodology described for 58. White solid; overall yield 48%; purity 96.3% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 7.6 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H), 6.72 (s, 1H), 6.09 (s, 1H), 4.50 (t, J = 7.4 Hz, 2H), 3.57 (t, J = 6.4 Hz, 2H), 2.00 (s, 1H), 1.85–1.75 (m, 2H), 1.59–1.51 (m, 2H), 1.46 (s, 9H), 1.38–1.33 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 162.4, 138.1, 133.2, 126.2, 123.7, 121.7, 120.3, 110.3, 103.3, 62.5, 51.7, 44.3, 32.3, 30.2, 28.9, 23.1. HRMS (ESI): Calcd for C18H26N2NaO2 [M+Na]+, 325.1886; found 325.1881. N-Cyclopentyl-1-(5-hydroxypentyl)-1H-indole-2-carboxamide (67). This compound was synthesized from 42 and cyclopentylamine according to the methodology described for 58. White solid; overall yield 24%; purity 98.6% (Gradient I). 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.32–7.24 (m, 1H), 7.12 (t, J = 7.4 Hz, 1H), 6.78 (s, 1H), 6.21 (d, J = 6.8 Hz, 1H), 4.52 (t, J = 7.4 Hz, 2H), 4.42–4.31 (m, 1H), 3.59 (t, J = 6.4 Hz, 2H), 2.12–2.04 (m, 2H), 1.87–1.79 (m, 3H), 1.77– 1.61 (m, 4H), 1.61–1.45 (m, 4H), 1.45–1.34 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 162.3, 138.2, 132.1, 126.2, 123.8, 121.8, 120.4, 110.4, 103.6, 62.6, 51.4, 44.5, 33.2, 32.3, 30.2, 23.8, 23.1. HRMS (ESI): Calcd for C19H26N2NaO2 [M+Na]+, 337.1886; found 337.1902.



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N-Cyclohexyl-1-(5-hydroxypentyl)-1H-indole-2-carboxamide (68). This compound was synthesized from 42 and cyclohexylamine according to the methodology described for 58. White solid; overall yield 67%; purity 99.4% (Gradient II). 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 7.12 (t, J = 8.0 Hz, 1H), 6.79 (s, 1H), 6.13 (d, J = 8.0 Hz, 1H), 4.52 (t, J = 7.4 Hz, 2H), 4.00–3.87 (m, 1H), 3.59 (t, J = 6.4 Hz, 2H), 2.08–1.98 (m, 2H), 1.92–1.70 (m, 5H), 1.70–1.52 (m, 3H), 1.47–1.38 (m, 4H), 1.33–1.14 (m, 3H). 13C NMR (101 MHz, CDCl3) δ 161.8, 138.2, 132.2, 126.2, 123.8, 121.8, 120.4, 110.4, 103.5, 62.6, 48.4, 44.5, 33.2, 32.4, 30.3, 25.6, 24.9, 23.1. HRMS (ESI): Calcd for C20H28N2NaO2 [M+Na]+, 351.2043; found 351.2028. 4-((tert-Butoxycarbonyl)amino)butyl 4-methylbenzenesulfonate (71). To a solution of 4-aminobutan1-ol (69) (1.0 g, 11.22 mmol) in CH2Cl2 (10 mL) was added (Boc)2O (2.7 g, 12.3 mmol). After stirring for 2 hr, to the reaction mixture were added tosyl chloride (3.2 g, 16.8 mmol) and Et3N (4.1 mL, 33.7 mmol). After stirring overnight, the reaction was quenched with water (10 mL) and extracted with EtOAc (3×30 mL). The combined organic layers were dried over Na2SO4, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel, eluting with hexane/EtOAc (5:1) to provide the title compound as colorless oil; yield 55%. 1H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 4.59 (s, 1H), 4.03 (t, J = 6.0 Hz, 2H), 3.08 (t, J = 6.0 Hz, 2H), 2.45 (s, 3H), 1.72–1.62 (m, 2H), 1.54–1.49 (m, 2H), 1.42 (s, 9H). 5-((tert-Butoxycarbonyl)amino)pentyl

4-methylbenzenesulfonate

(72).

This

compound

was

synthesized from 70 according to the methodology described for 71. Colorless oil; yield 46%; 1H NMR (400 MHz, CDCl3) δ 7.70 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 4.56 (s, 1H), 3.93 (t, J = 6.0 Hz, 2H), 2.97 (s, 2H), 2.37 (s, 3H), 1.63–1.53 (m, 2H), 1.35–1.33 (m, 11H), 1.29–1.21 (m, 2H).


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N-((3s,5s,7s)-Adamantan-1-yl)-1-(4-aminobutyl)-1H-indole-2-carboxamide (73). To a solution of 43 (300 mg, 1.02 mmol) in anhydrous DMF (5 mL) was added NaH (72 mg, 50% in mineral oil, 2.04 mmol) with ice cooling. After stirring for 15 min, 71 (700 mg, 2.0 mmol) was added. After stirring for 3 hr at 100 ºC, 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 brine (15 mL), dried over Na2SO4, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel, eluting with hexane/EtOAc (5:1) to provide the intermediate as white solid. The white solid was next dissolved in a saturated solution of HCl and stirred for 3 hr. The mixture was filtered off and the filter cake was dissolved in 20% aqueous NaOH (10 mL) and extracted with CH2Cl2 (3×20 mL), the combined organic layers were dried over Na2SO4, and concentrated under vacuum to provide the title compound. White solid; yield 13% over two steps; purity 96.2% (Gradient I). 1H NMR (400 MHz, CDCl3) δ 7.50 (d, J = 8.0 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 7.23–7.16 (m, 1H), 7.02 (t, J = 7.4 Hz, 1H), 6.66 (s, 1H), 5.94 (s, 1H), 4.38 (t, J = 7.0 Hz, 2H), 2.92 (t, J = 7.0 Hz, 2H), 2.04 (br s, 9H), 1.89–1.80 (m, 2H), 1.78–1.71 (m, 2H), 1.68–1.60 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 161.0, 137.0, 131.7, 125.1, 123.0, 120.7, 119.5, 109.3, 102.7, 51.6, 42.5, 40.7 (3C), 38.3, 35.3 (3C), 28.4 (3C), 26.1, 23.6. HRMS (ESI): Calcd for C23H32N3O [M+H]+, 366.2540; found 366.2546. N-((3s,5s,7s)-Adamantan-1-yl)-1-(5-aminopentyl)-1H-indole-2-carboxamide (74). This compound was synthesized from 72 according to the methodology described for 73. White solid; overall yield 67%; purity 95.6% (Gradient I). 1H NMR (400 MHz, CDCl3) δ 7.58 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.23 (s, 1H), 7.09 (t, J = 7.2 Hz, 1H), 6.72 (s, 1H), 5.97 (s, 1H), 4.49 (t, J = 6.8 Hz, 2H), 4.27 (s, 2H), 2.81 (t, J = 6.8 Hz, 2H), 2.11 (br s, 9H), 1.81–1.60 (m, 10H), 1.37 (s, 2H).

13

C NMR (101 MHz,

CDCl3) δ 162.0, 138.0, 133.0, 126.1, 123.7, 121.7, 120.4, 110.3, 103.5, 52.5, 44.0, 41.8 (3C), 40.3, 36.3



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(3C), 30.0, 29.5 (3C), 29.1, 23.9. HRMS (ESI): Calcd for C24H34N3O [M+H]+, 380.2696; Found 380.2724. N-((3s,5s,7s)-Adamantan-1-yl)-1-(5-guanidinopentyl)-1H-indole-2-carboxamide (75). To a solution of 1H-pyrazole-1-carboxamidine hydrochloride (42.5 mg, 0.29 mmol) was added 74 (100 mg, 0.26 mmol). After stirring for 30 hr at rt, the mixture was concentrated under vacuum. The residue was purified by flash chromatography on silica gel, eluting with CH2Cl2/methanol (10:1) to provide the title compound. White solid; yield 30%; purity 99.2% (Gradient I). 1H NMR (400 MHz, CD3OD) δ 7.58 (d, J = 8.0 Hz, 1H), 7.53 (s, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.24 (t, J = 7.6 Hz, 1H), 7.06 (t, J = 7.4 Hz, 1H), 6.91 (s, 1H), 4.49 (t, J = 7.0 Hz, 2H), 3.12 (t, J = 7.0 Hz, 2H), 2.17–2.10 (m, 9H), 1.80–1.76 (m, 8H), 1.61–1.54 (m, 2H), 1.40–1.30 (m, 2H).13C NMR (101 MHz, CD3OD) δ 164.8, 158.6, 139.5, 134.5, 127.8, 124.7, 122.9, 121.2, 111.3, 106.0, 53.8, 44.8, 42.5 (3C), 42.4, 37.6 (3C), 31.1, 31.0 (3C), 29.5, 25.0. HRMS (ESI): Calcd for C25H36N5O [M+H]+, 422.2914; found 422.2907. Calcium Mobilization Assay.42 Chinese hamster ovarian (CHO) cells stably expressing Gα16 with either CB1 or CB2 receptor were seeded onto 96-well plates and incubated for 24 hr. Cells were loaded with 2 µM fluo-4 AM in Hanks’ balanced salt solution (HBSS, containing 5.4 mM KCl, 0.3 mM Na2HPO4, 0.4 mM KH2PO4, 4.2 mM NaHCO3, 1.3 mM CaCl2, 0.5 mM MgCl2, 0.6 mM MgSO4, 137.0 mM NaCl, 5.6 mM D-glucose and 250 µM sulfinpyrazone, pH 7.4) at 37 ºC for 45 min. In the agonist mode, 50 µL HBSS was added to the dye-loaded cells, and 25 µL of the test compounds with various concentrations, compound 7 (positive control), or DMSO (negative control) was added to a FlexStation microplate reader. Meanwhile, the intracellular calcium change was recorded at an excitation wavelength of 485 nm and an emission wavelength of 525 nm. In the antagonist mode, the excess dye was removed and 50 µL HBSS containing variable concentrations of test compounds, compound 1 (positive control), or DMSO (negative control) was added. After incubation at room temperature 10 min,


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compound 7 (25 µL) was dispensed into the wells using a FlexStation microplate reader, and intracellular calcium change was recorded at an excitation wavelength of 485 nm and an emission wavelength of 525 nm. All experiments were performed in triplicates in three independent experiments. EC50 and IC50 values were analyzed with sigmoidal dose-response curve fitting using GraphPad Prism program. cAMP Assay. CHO Cells were harvested with PBS and pelleted by centrifugation for 5 min at 1100 rounds per minute. The cells were then resuspended in an appropriate volume of assay buffer (PBS containing 500 µmol/L IBMX) to obtain a final cell count of 4×105 cells/mL. The cells were then plated in a 384-well assay plate at 2000 cells/5 µL per well. Another 5 µL of buffer containing the testing compound at various concentrations was added to the assay plate and incubated for 20 min at room temperature. Then 5 µL of buffer containing forskolin was added to reach a final concentration of 3 µmol/L and the assay plate was incubated for another 30 min at room temperature. Intracellular cAMP measurement was performed with a HTRF Dynamic 2 cAMP kit (Cisbio, Cat No 62AM4PEJ) and an EnVision microplate reader according to the manufacturer's instructions. Docking Stimulations. The structures of ligands 16 and 27 were respectively built up using Sybyl X1.3/SKETCH module (Sybyl Molecular Modeling Software Packages, version X1.3; Tripos Associates, Inc.: St. Louis, MO, 2011), and minimized by Tripos force field with Gasteiger-Hückel atomic charge. The generated conformation of either 16 or 27 was manually put into the binding pocket of CB2 receptor predicted by previous modeling simulations based on the homology model of CB2 receptor.27 The flexible docking simulations were then performed to calculate the interaction mode of each ligand binding to CB2 receptor using the FlexiDock method of Sybyl X1.3. A CB2 receptor binding pocket was first defined to cover all residues around 5 Å of the ligand in the original CB2 receptorligand complexes. During the docking simulations, the docking ligand was allowed to move freely



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within such the potential binding pocket, and all the single bonds of the ligand and the side chains of residues around the defined binding pocket were regarded as rotatable bonds. The suitable atoms were marked as the hydrogen bond donors or acceptors for both CB2 receptor and ligand. The atomic charges were recalculated by using the Kollman all-atom approach for the protein and the Gasteiger-Hückel approach for the ligand. The interaction energy was calculated including the terms of van der Waals, electrostatics, and torsion energy defined in the Tripos force field. The structure optimization was performed for 150,000 generations using a genetic algorithm, and the 20 best-scoring ligand-protein complexes, which have very similar 3D structures with little different energies, were screened for further research. Therefore, one best CB2 receptor-ligand complex was selected to analyze the interaction modes. EAE mouse model. All experiments were approved and conducted in accordance with the guidelines of the Animal Care Committee of Tongji University. C57BL/6 mice were purchased from Shanghai Laboratory Animal Center (Shanghai, China). All mice were maintained in pathogen-free conditions with standard laboratory chow and water ad libitum. For EAE induction, female mice (10 weeks age) were immunized s.c. with 200 µg MOG35–55 (MEVGWYRSPFSRVVHLYRNGK; obtained from GL Biochem) in CFA containing heat-killed Mycobacterium tuberculosis (H37Ra strain, 5 mg/mL; BD Diagnostics). The mice then received intraperitoneal (i.p.) injections with 200 ng pertussis toxin on day 0 and 2. For drug treatment, mice received daily i.p. injection of compound 57 (10 and 30 mg/kg in 0.5% CMC; n = 8) or vehicle control (0.5% CMC; n = 10). Disease severity was scored daily on a scale of 0–5 as follow: 0, no clinical signs; 1, paralyzed tail; 2, paresis (weakness, incomplete paralysis of one or two hindlimbs); 3, paraplegia (complete paralysis of both hindlimbs); 4, paraplegia with forelimb weakness or paralysis; and 5, moribund state or death.


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Histopathological analysis. For histopathological analysis, mice (n = 3) were anesthetized and perfused with PBS (pH 7.4) followed by 4% (w/v) paraformaldehyde, spinal cord tissues were isolated and fixed in 4% (w/v) paraformaldehyde overnight. Paraffin-embedded sections of spinal cord were stained with hematoxylin and eosin (H&E) or Luxol fast blue for the analysis of leukocytes infiltration or demyelination by Image Pro, respectively. Statistics. Data were analyzed with GraphPad Prism software. The statistical significance of the EAE clinical scores between treatments was analyzed with a two-way ANOVA test. Histological analysis was assessed by a Student t test. A p value < 0.05 was considered statistically significant. ASSOCIATED CONTENT ABBREVIATIONS USED: Cannabinoid, CB; G-protein coupled receptors, GPCRs; adenylyl cyclase, AC; cyclic adenosine monophosphate, cAMP; central nervous system, CNS; mitogen-activated protein kinases, MAPK; JUN N-terminal kinases, JNKs; interferon γ, IFNγ; intercellular adhesion molecule-1, ICAM-1; multiple sclerosis, MS; experimental autoimmune encephalomyelitis, EAE; chronic relapsing experimental allergic encephalomyelitis, CREAE; Theiler‘s murine encephalomyelitis virus-induced demyelinating

disease,

TMEV-IDD;

tetrahydrocannabinol,

THC;

cannabidiol,

CBD;

dimethylformamide, DMF; tert-butyldimethylsilyl, TBS; tetrabutylammonium fluoride, TBAF; trifluoroacetic

acid,

TFA;

1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane

bis(tetrafluoroborate), SelectFluor; diethylaminosulfur trifluoride, DAST; Chinese hamster ovary, CHO; fluorometric imaging plate reader, FLIPR; blood-brain barrier, BBB; tetramethylsilane, TMS; intraperitoneal, i.p.. AUTHOR INFORMATION Author Contributions



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Ying Shi, †, ‡ Yan-Hui Duan, § , ‡, these authors contributed equally to this work. Corresponding Authors *Xin Xie, Phone: +86-21-50801313; E-mail: [email protected] *Li-Fang Yu, Phone: +86-021-62231385; E-mail: [email protected] ACKNOWLEDGMENTS This work was supported by grants from the Innovation Program of Shanghai Municipal Education Commission (15ZZ027), the Ministry of Science and Technology (2014CB541906), the National Natural Science Foundation of China (81425024), and the Open Program of State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (SIMM1501KF05). Supporting Information. Molecular Formula Strings. REFERENCES 1.

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Amidoalkylindoles as Potent and Selective Cannabinoid Type 2

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