Structure-activity relationship study of indole-2-carboxamides identifies a potent allosteric modulator for the cannabinoid receptor 1 (CB1)

Article abstract of DOI:10.1021/jm4009828

The cannabinoid CB1 receptor is involved in complex physiological functions. The discovery of CB1 allosteric modulators generates new opportunities for drug discovery targeting the pharmacologically important CB1 receptor. 5-Chloro-3-ethyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2- carboxamide (ORG27569; 1) represents a new class of indole-2-carboxamides that exhibit allostery of CB1. To better understand the SAR, a group of indole-2-carboxamide analogues were synthesized and assessed for allostery of the CB1 receptor. We found that within the structure of indole-2-carboxamides, the presence of the indole ring is preferred for maintaining the modulator's high binding affinity for the allosteric site but not for generating allostery on the orthosteric site. However, the C3 substituents of the indole-2-carboxamides significantly impact the allostery of the ligand. A robust CB1 allosteric modulator 5-chloro-N-(4-(dimethylamino)phenethyl)-3-pentyl-1H- indole-2-carboxamide (11j) was identified. It showed an equilibrium dissociation constant (K_B) of 167.3 nM with a markedly high binding cooperativity factor (α = 16.55) and potent antagonism of agonist-induced GTPγS binding.

Full text of DOI:10.1021/jm4009828

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Article
Structure-Activity Relationship Study of Indole-2-carboxamides Identifies
a Potent Allosteric Modulator for the Cannabinoid Receptor 1 (CB1)
Mariam M. Mahmoud, Hamed I Ali, Kwang H. Ahn, Aparna Damaraju, Sushma
Samala, Venkata K. Pulipati, Srikanth Kolluru, Debra A. Kendall, and Dai Lu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm4009828 • Publication Date (Web): 20 Sep 2013
Downloaded from http://pubs.acs.org on September 30, 2013
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StructureꢀActivity Relationship Study of Indoleꢀ2ꢀcarboxamides Identifies
a Potent Allosteric Modulator for the Cannabinoid Receptor 1 (CB1)
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Mariam M. Mahmoud†, Hamed I. Ali§, Kwang H. Ahn††, Aparna Damaraju§, Sushma Samala§, Venkata K.
Pulipati§, Srikanth Kolluru§, Debra A. Kendall††∗, andDai Lu§ ∗
†Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, United
States
††Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut, United States
§
Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, Texas,
United States
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ABSTRACT. The cannabinoid CB1 receptor is involved in complex physiological functions.
The discovery of CB1 allosteric modulators generates new opportunities for drug discovery
targeting the pharmacologically important CB1 receptor. 5ꢀchloroꢀ3ꢀethylꢀNꢀ(4ꢀ(piperidinꢀ1ꢀ
yl)phenethyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (ORG27569; 1) represents a new class of indoleꢀ2ꢀ
carboxamides that exhibit allostery of CB1. To better understand the SAR, a group of indoleꢀ2ꢀ
carboxamide analogs were synthesized and assessed for allostery of the CB1 receptor. We found
that within the structure of indoleꢀ2ꢀcarboxamides, the presence of the indole ring is preferred for
maintaining the modulator’s high binding affinity for the allosteric site, but not for generating
allostery on the orthosteric site. However, the C3 substituents of the indoleꢀ2ꢀcarboxamides
significantly impact the allostery of the ligand. A robust CB1 allosteric modulator 5ꢀchloroꢀNꢀ
(4ꢀ(dimethylamino)phenethyl)ꢀ3ꢀpentylꢀ1Hꢀindoleꢀ2ꢀcarboxamide (11j) was identified. It
showed an equilibrium dissociation constant (KB) of 167.3 nM with a markedly high binding
cooperativity factor (α=16.55) and potent antagonism of agonistꢀinduced GTPγS binding.
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INTRODUCTION
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The endocannabinoid system is composed of at least two Gꢀprotein coupled receptors
(GPCRs), CB1 and CB2, a group of endogenous lipid ligands and several catabolic and anabolic
enzymes that are involved in the biosynthesis and degradation of the endogenous ligands.1 The
CB1 receptors are mainly found on neurons of the central and peripheral nervous system2 where
they attenuate neurotransmitter release. CB1 receptors are also present at much lower
concentrations in some peripheral tissues, including spleen, tonsil, reproductive organs,
endocrine glands, arteries, bone marrow, lungs and heart.3ꢀ5 Consistent with its widespread
distribution, the CB1 receptor regulates a wide variety of physiological functions including
neuronal development, neuromodulatory processes, energy metabolism, cardiovascular,
respiratory as well as reproductive functions.6ꢀ8 In addition, it also modulates cell proliferation,
motility, adhesion and apoptosis.9, 10
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Like many GPCRs, the CB1 receptor displays a multiplicity of intracellular signal
transduction mechanisms. It preferentially couples to Gi/o type G proteins while its interaction
with Gs11 or Gq12 is also possible. Activation of the CB1 receptor primarily leads to the inhibition
of adenylylꢀcyclase thereby reducing cAMP levels in cells. The CB1 receptor also modulates the
activation of mitogenꢀactivated protein kinases (MAPKs), including extracellular signalꢀ
regulated kinaseꢀ1 and ꢀ2 (ERK1/2), p38 MAPK, p42/p44 MAPK and cꢀJun Nꢀterminal kinase
(JNK).13 Additionally, CB1 can negatively couple to Nꢀ and P/Qꢀtype voltageꢀgated Ca2+
channels and positively couple to Aꢀtype and inwardly rectifying K+ channels.14 CB1 also
interacts with nonꢀG protein partners such as βꢀarrestins, adaptor protein APꢀ3, GPCRꢀassociated
sorting protein 1 (GASP1) and the adaptor protein FAN to control receptor signaling or
trafficking.15, 16
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The activation of the CB1 receptor by endogenous and exogenous agonists such as
anandamide and (ꢀ)ꢀꢀ9ꢀtetrahydrocannabinol (ꢀ9ꢀTHC) has been linked to a wide range of
pharmacological effects.17 Growing evidence suggests that the CB1 receptor exists in multiple
active conformations, each of which may display distinct abilities to regulate individual signaling
pathways.18 Thus, the assumption that there is only one active conformation of the CB1 receptor
is overly simplistic and is inadequate to explain all cannabinoidꢀmediated responses. Recently,
several small molecules (Figure 1) have been identified as allosteric modulators of the CB1
receptor. The representative members include ORG27569 (1),19 (1ꢀ(4ꢀchlorophenyl)ꢀ3ꢀ(3ꢀ(6ꢀ
(pyrrolidinꢀ1ꢀyl)pyridinꢀ2ꢀyl)phenyl)urea (2, PSNCBAMꢀ1),20 3ꢀ(4ꢀchlorophenyl)ꢀ5ꢀ(8ꢀmethylꢀ
3ꢀpꢀtolylꢀ8ꢀazabicyclo[3.2.1]octanꢀ2ꢀyl)isoxazole (3, RTIꢀ371)21 and the endogenous ligand
(5S,6R,7E,9E,11Z,13E,15S)ꢀ5,6,15ꢀtrihydroxyicosaꢀ7,9,11,13ꢀtetraenoic acid (4, lipoxin A4).22
The identification of these ligands indicates the existence of allosteric site(s) on the CB1 receptor
and suggests new opportunities for fineꢀtuning the signaling pathways of the CB1 receptor.
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Cl
O
N
Cl
N
N
NH
O
N
N
H
H
N
H
2
(PSNCBAM-1)
1
(ORG27569)
N
O
N
HO OH
Cl
COOH
OH
3
(RTI-371)
4 (Lipoxin A4)
Figure 1. Structures of representative allosteric modulators of the CB1 receptor
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Indoleꢀ2ꢀcarboxamide 1 was the first allosteric modulator identified for the CB1
receptor.19 It increases specific binding of the tritium labeled CB1 agonist 2ꢀ[(1R,2R,5R)ꢀ5ꢀ
hydroxyꢀ2ꢀ(3ꢀhydroxypropyl)cyclohexyl]ꢀ5ꢀ(2ꢀmethyloctanꢀ2ꢀyl)phenol ([3H]CP55,940) but
decreases the binding of the tritium labeled CB1 inverse agonist 5ꢀ(4ꢀchlorophenyl)ꢀ1ꢀ(2,4ꢀ
dichloroꢀphenyl)ꢀ4ꢀmethylꢀNꢀ(piperidinꢀ1ꢀyl)ꢀ1Hꢀpyrazoleꢀ3ꢀcarboxamide ([3H]SR141716A) in
membranes from cells expressing the CB1 receptor. Despite acting as an enhancer of agonist
binding, it acts as an inhibitor of agonistꢀinduced Gαi protein coupling.19, 23 It was further
demonstrated that the 1ꢀinduced conformational change of the CB1 receptor leads to cellular
internalization of the receptor and downstream activation of ERK signaling,23 both of which have
been conventionally recognized as the consequences of CB1 receptor activation. More recent
studies showed that the 1ꢀinduced activation of ERK1/2 signaling is Giꢀindependent24, 25 and
mediated by βꢀarrestins.23 Hence, 1 not only acts as an allosteric agonist which is able to induce
receptor internalization and ERK1/2 phosphorylation on its own, but also functions as an
antagonist of CB1 agonistꢀinduced [35S]GTPγS binding. This supports the notion that allosteric
modulators can selectively regulate the functions of the CB1 receptor. Consequently, more
functionallyꢀdefined pharmacological effects can be achieved for the physiologically and
pharmacologically important CB1 receptors. Along with 1, a few other indoleꢀ2ꢀcarboxamides19,
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24, 26
have also shown allosteric modulation of the CB1 receptor. To study the structureꢀactivity
relationships (SAR) of this class of compounds, we synthesized a series of analogs of 1 with the
general structure shown in Figure 2, and individual structures shown in Schemes 1 and 2.
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Bicyclic aryl side
R1
Amide side
Cl
O
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X
HN R2
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X = NH, O
Figure 2. General structures of indoleꢀ and benzofuranꢀ2ꢀcarboxamide analogs
Previous results from us24 and others26 indicated that the alkyl substituents at the C3
position have a profound influence on the ligand’s allostery. For instance, replacing compound
1’s C3 ethyl group with a nꢀpentyl group led to the enhancement of the allosteric effects, which
is reflected by an improvement in the cooperativity factor α from 6.95 (1) to 17.6 (11a).24 The α
cooperativity factor is a value that quantifies the magnitude by which the affinity of one ligand is
changed by the other ligand when both are bound to the receptor simultaneously to form the
ternary complex. When α is 1.0, the test modulator does not affect the binding of the orthosteric
ligand. If α is less than 1.0, the test modulator reduces orthosteric ligand binding (negative
allosteric modulation on orthosteric ligand binding). If α is greater than 1.0, the modulator
increases orthosteric ligand binding (positive allosteric modulation).27 This prompted us to
continue the investigation of the C3 substituents. Therefore, alkyl groups of different lengths, a
cyclic ring and an aromatic ring were introduced at the C3 position to elucidate the SAR. Since
we previously found that 11a displayed a remarkably enhanced binding cooperativity factor α24,
we chose to use it as a template to vary the amines of the amide side, with the aim of improving
the affinity of the allosteric modulator to its allosteric site, which is denoted by its equilibrium
dissociation constant (KB).27 The above efforts yielded compounds 11bꢀ11k (Scheme 1, Table 1
and Table 2). Next, to elucidate the significance of the indole ring in the indoleꢀ2ꢀcarboxamide
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structure, we replaced the indole ring of 11a with other bicyclic aromatic rings such as the
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benzofuran ring. This effort led to the compounds represented by the structure 13aꢀ13d (Scheme
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2, Table 3).
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CHEMISTRY
The syntheses of the indoleꢀ2ꢀcarboxamide analogs (11) were achieved through the
methods illustrated in Scheme 1.
R
R
O
RCOCl
(6a-f)
Cl
Cl
Cl
COOEt
COOEt
COOEt
(i)
N
N
N
(ii)
H
H
H
5
7a-f
8a-f
R2
R
H2N Y
R2
R1
Cl
Cl
O
10a-f
COOH
N
H
N
HN Y
(iv)
(iii)
H
9a-f
11a-l
6-9
10a-f
a: Y = (CH2)2, R2 = N-piperidinyl
b: Y = CH2, R2 = N-piperidinyl
c: Y = (CH2)2, R2 = N-morpholinyl
d: Y = (CH2)2, R2 = Phenoxyl
e: Y = (CH2)2, R2 = N(CH3)2
a: R1 = n-C5H11, Y = (CH2)2, R2 = N-piperidinyl
b: R1 = Ph, Y = (CH2)2, R2 = N-piperidinyl
a: R= n-C4H9
b: R= Ph
c: R= c-hexyl
d: R= n-C6H13
e: R= n-C8H17
f: R= CH3
c: R1 = CH2Ph, Y = (CH2)2, R2 = N-piperidinyl
d: R1 = CH2-c-hexyl, Y = (CH2)2, R2 = N-piperidinyl
e: R1 = n-C7H15, Y = (CH2)2, R2 = N-piperidinyl
f: R1 = n-C9H19, Y = (CH2)2, R2 = N-piperidinyl
g: R1 = n-C5H11, Y = CH2, R2 = N-piperidinyl
h: R1 = n-C5H11, Y = (CH2)2, R2 = N-morpholinyl
i: R1 = n-C5H11, Y = (CH2)2, R2 = Phenoxyl
j: R1 = n-C5H11, Y = (CH2)2, R2 = N(CH3)2
k: R1 = n-C5H11, Y =CH(CH2OH)CH2, R2 = N(CH3)2
l: R1 = CH2CH3, Y = (CH2)2, R2 = N(CH3)2
f: Y = CH(CH2OH)CH2, R2 = N(CH3)2
Scheme 1. Syntheses of indoleꢀ2ꢀcarboxamide analogs (11aꢀl). Reagents and Conditions: (i)
AlCl3, 1,2ꢀdichloromethane, reflux, 2ꢀ3 h; (ii) (Et)3SiH, CF3COOH, 0 °C ꢀ rt, 4ꢀ12 h; (iii) NaOH,
EtOH, reflux, 2 h; (iv) BOP, DIPEA, DMF, rt, 4ꢀ12 h.
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The C3 substituents were introduced through acylation of the commercially available
ethyl 5ꢀchloroindoleꢀ2ꢀcarboxylate (5) by FriedelꢀCrafts reaction catalyzed by aluminum
chloride. Acylation of 5 with the selected acyl chloride (6) provided the desired 3ꢀacylꢀ5ꢀ
chloroindoleꢀ2ꢀcarboxylates (7). Reduction of the carbonyl group of 7 by triethylsilane generated
the C3 alkylated 5ꢀchloroindoleꢀ2ꢀcarboxylates (8), which were then hydrolyzed in basic
conditions to yield the key intermediate acids (9). The final compounds (11aꢀ11l) were prepared
by coupling commercially available amines (10aꢀf) with the indoleꢀ2ꢀcarboxylic acids (9aꢀf) and
commercially available 5ꢀchloroꢀ3ꢀphenylꢀ2ꢀcarboxylic acid individually in the presence of BOP
and diisopropylethyl amine (DIPEA) in anhydrous DMF at room temperature. Similarly,
benzofuranꢀ2ꢀcarboxamides (13aꢀd) were prepared according to the methods depicted in Scheme
2.
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R1
R1
Cl
Cl
H2N
O
COOH +
R2
(i)
O
O
HN
R2
12
10
a: R2 = N-piperidinyl
13
a: R1 = H; R2 = N-piperidinyl
a: R1 = H
e:
b
R2 = N(CH3)2
b
: R1 = Et
: R1 = Et; R2 = N-piperidinyl
c: R1 = Et; R2 = N(CH3)2
d
: R1 = H: R2 = N(CH3)2
Scheme 2. Syntheses of benzofuranꢀ2ꢀcarboxamide analogs 13aꢀd. Reagents and Conditions: (i)
BOP, DIPEA, DMF, rt, 4ꢀ12 h.
The commercially available 5ꢀchlorobenzofuranꢀ2ꢀcarboxylic acid 12a (AlfaAesar, Ward
Hill, MA) or 5ꢀchloroꢀ3ꢀethylꢀbenzofuranꢀ2ꢀcarboxylic acid 12b (Princeton BioMolecular
Research, Monmouth Junction, NJ) was coupled with selected amines (10a or 10e) in the
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presence of BOP and DIPEA in anhydrous DMF at room temperature to provide the desired
benzofuranꢀ2ꢀcarboxamides (13aꢀ13d). Most of the final compounds of the 11 and 13 series had
acceptable solubility in organic solvents and were therefore purified through chromatography.
However, compounds 11b, 11c and 11d had very poor solubility in most of the common organic
solvents, such as acetone, ethyl acetate, tetrahydrofuran, dichloromethane, diethyl ether, ethanol
and methanol. They were therefore purified using a combination of trituration, recrystallization
and chromatography as detailed in the experimental section.
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RESULTS AND DISCUSSION.
To investigate the allostery of the newly synthesized compounds against the CB1
receptor, we evaluated each compound’s binding cooperativity factor α to quantify the
magnitude of the allosteric effect on orthosteric ligand binding. Some of the compounds that
showed α values comparable to that of 1 were selected for further testing their capability to
modulate the agonist CP55,940ꢀinduced G protein coupling. Additionally, to evaluate the
effectiveness of the modulator binding to the allosteric site, their equilibrium dissociation
constants KB were also determined. The α and KB values were analyzed according to the
allosteric ternary complex model.19 (See equation (1) in experimental section).
The results for compounds with structural variation at the C3 position are shown in Table
1. We found that replacing the C3 alkyl group of 1 with a phenyl ring completely abolished the
allosteric effect of the ligand (11b compared to previously tested 123 and 11a24), whereas a
benzyl group at C3 was somewhat tolerated but with a significantly increased KB value
indicative of lower affinity (11c). Replacing the phenyl ring of the benzyl group in ligand 11c
with a cyclohexyl group (11d) further increased KB although allosteric modulatory effects were
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retained. These data suggest that aliphatic or aromatic rings on the C3 alkyl group significantly
decrease the affinity of the allosteric ligand. Therefore, linear alkyl chains as C3 substituents
were employed for further exploration. The C3ꢀnꢀheptyl (11e) and C3ꢀnꢀnonyl (11f) analogs of 1
were synthesized. Although 11e and 11f displayed appreciable KB values, the α values
substantially decreased relative to 11a, suggesting that a C3ꢀnꢀpentyl is optimal for maximum
allosteric modulatory effects.
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Table 1. Allostery of indoleꢀ2ꢀcarboxamide analogs 11aꢀf
R1
Cl
O
N
HN
H
N
R1
KB (nM)a
αb
217.3 (170.3ꢀ277.2) 6.95
[35S]GTPγS bindingc
Cmpd
1d
Et
nꢀC5H11
Ph
Antagonizing
11ae
11b
11c
11d
11e
11f
469.9 (126.2ꢀ1750)
ND
17.60
1.0
Antagonizing
ꢀ
CH2Ph
1067 (347.3ꢀ3344)
2408 (570.2ꢀ8151)
651.2 (81.51ꢀ5203)
259.7 (87.56ꢀ770)
11.37
9.84
7.38
6.80
ꢀ
CH2ꢀcꢀhexyl
nꢀC7H15
nꢀC9H19
ꢀ
ꢀ
Antagonizing
a KB: equilibrium disassociation constant.
b α: binding cooperativity factor between CP55,940 and the tested allosteric modulators.
Effects on CP55,940ꢀinduced [35S]GTPγS binding in the presence of allosteric
c
modulator.
d Data cited for 1 is from our earlier report and given for comparison.23
e Data cited for 11a is from our earlier report and given for comparison.24
ND: no detectable modulation of [3H]CP55,940 binding using up to 32 ꢁM of test
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Overall, this series of analogs indicates that the type and size of the substituents at the C3
position of indoleꢀ2ꢀcarboxamides have a critical role in the allosteric effects on the CB1
receptor. Our results support the hypothesis of the presence of a hydrophobic subꢀpocket in close
proximity of the C3ꢀposition of 1 when it is bound to the allosteric site. This subꢀsite could only
be occupied by linear alkyl groups but not cyclic structures.
Table 2. Allostery of indoleꢀ2ꢀcarboxamide analogs 11gꢀk
Cl
O
HN R2
N
H
Cmpd
R2
KB (nM)a
αb
[35S]GTPγS bindingc
11ad
469.9 (126.2ꢀ1750) 17.06
Antagonizing
N
N
ND
1.0
ꢀ
11g
11h
508.4 (96.34ꢀ2683) 10.0
2335 (695.3ꢀ7542) 13.15
167.3 (23.39ꢀ1197) 16.55
Antagonizing
N
O
N
O
ꢀ
11i
11j
Antagonizing
OH
1018 (81.51ꢀ5203) 13.27
ꢀ
11k
N
a KB: equilibrium disassociation constant.
bα: binding cooperativity factor between CP55,940 and the tested allosteric modulators.
c Effects on CP55,940ꢀinduced GTPγS binding in the presence of allosteric modulator.
d Data cited for 11a is from our earlier report and given for comparison.24
ND: no detectable modulation of [3H]CP55,940 binding using up to 32 ꢁM of test compound.
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Among the C3ꢀalkyl analogs of 1, the previously tested compound 11a24 exhibited the
most robust allostery (α=17.6) of the CB1 receptor. In an earlier communication, we reported
that 11a promoted the phosphorylation of CB1 downstream effectors ERK1/2 in a Giꢀ
independent but βꢀarrestinꢀ1 mediated manner.24 Although a markedly high α factor was
achieved, 11a exhibited moderately weak affinity for CB1, as reflected by its relatively high KB
value (469.9 nM). Hence, this compound was selected for further modification on the amide side
with the aim of exploring SAR and improving the affinity to the allosteric site on CB1. This
effort led to the synthesis of the compounds presented in Table 2.
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When a methylene group of the ethylene linker between the piperidinylphenyl group and
the amide bond was removed, it resulted in complete elimination of the allostery of the ligand
(11g vs 11a). This indicates that the substituted phenyl ring on the amide side must be separated
from the amide bond by at least a 2ꢀcarbon linkage. This ethylene moiety was therefore retained
for the rest of this series of compounds (11hꢀ11k). When the piperidinyl group was replaced by a
structurally related but more hydrophilic morpholinyl group (11h), the KB value increased and
the cooperativity factor α decreased. This suggests that the hydrophilic substitution on the amino
group decreased the affinity of the ligand for the allosteric site and reduced the allosteric
modulation of the orthosteric site. Replacing the piperidinyl group with a phenoxy group (11i)
resulted in a significant increase in the KB value, but the high binding cooperativity (α) was
retained. Next, swapping the cyclic piperidinyl group with an acyclic dimethyl amino group on
the phenyl ring led to 11j which exhibited the most robust allostery (KB = 167.3 nM; α= 16.55)
reported so far for the CB1 receptor. A low KB value and a high α factor, as achieved in 11j, are
significant and indicate the low concentration of compound 11j that is needed to achieve the
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increase in affinity this allosteric modulator has on CP55,940 binding; i.e. a smaller
concentration of 11j (compared to all CB1 allosteric modulators) is needed to achieve its robust
allosteric effect on agonist binding. This is a key factor that is taken into consideration in
therapeutic drug treatments. Moving forward to introduce a hydroxymethyl group at the α
position of the ethylene linker of 11j reduced its affinity, but a relatively good binding
cooperativity (α value) was maintained (11k). Overall, in this series of compounds, we found
that structural modifications on the amide side generally do not significantly affect the
cooperativity factor α, whereas the influence on the KB of the allosteric modulators is much
more profound.
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To date, the majority of structural modifications on 1 have been focused on the
substitution of the indole ring19, 24 and the variation of the amide side.26 To unveil the importance
of the indole ring on the KB and α values, we synthesized several benzofuranꢀ2ꢀcarboxamides
and compared them with their indoleꢀ2ꢀcarboxamide counterparts. The results are shown in
Table 3.
Table 3. Allostery of indoleꢀ/benzofuranꢀ2ꢀcarboxamide analogs (11l and 13aꢀd)
R1
Cl
O
HN R2
X
Cmpd X R1
1d
NH Et
11le NH Et
R2
KB (nM)a
αb
[35S]GTPγS bindingc
217.3 (170.3ꢀ277.2) 6.95
Antagonizing
N
N
N
N
207.4 (155.9ꢀ2759)
ND
19.68
1.0
ꢀ
O H
O Et
ꢀ
13a
13b
2594 (1005ꢀ6696)
18.04
Antagonizing
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O Et
O H
627.1 (151.5ꢀ2596)
11.59
1.0
ꢀ
ꢀ
13c
13d
N
ND
N
a KB: equilibrium disassociation constant.
b α: binding cooperativity factor between CP55,940 and the tested allosteric modulators.
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c Effects on CP55,940ꢀinduced GTPγS binding in the presence of allosteric modulator.
d Data cited for 1 is from our earlier report and given for comparison.23
Compound 11l was reported earlier26 but was synthesized here and pharmacologically
characterized for comparison with other analogs under the same conditions.
ND: no detectable modulation of [3H]CP55,940 binding using up to 32 ꢁM of test compound.
ꢀ : not performed.
e
We learned that replacing the indole ring of 1 with a benzofuran ring (13b) resulted in a
significant loss of the affinity of the ligand (KB = 2594 nM), however, the binding cooperativity
with the orthosteric ligand was markedly enhanced (13b vs 1). Similarly, replacing the indole
ring of a potent CB1 allosteric modulator reported earlier (11l)26 with a benzofuran ring (13c) led
to a 3ꢀfold increase in KB while the high binding cooperativity (α=11.59) was wellꢀpreserved.
These results suggest that the bicyclic aryl moiety (i.e. the indole ring, Figure 2) significantly
impacts the ligand’s affinity compared to its impact on the binding cooperativity. The indole
ring, therefore, accounts more for the ligand’s capability to bind to the allosteric site, but is less
influential on the ligand’s capability to modulate the orthosteric site. In the benzofuranꢀ2ꢀ
carboxamides, removal of the C3 alkyl group (i.e. 13c vs 13d) eliminated the allosteric effects
completely.
To further characterize the nature of the compounds that show positive allosteric
modulation on the binding of the CB1 orthosteric agonist [3H]CP55,940, compounds 11f, 11h,
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11j and 13b were chosen for their strong allosteric properties (i.e. low KB value or high α value)
as representatives from each series of analogs and further tested for their effects on CP55,940ꢀ
induced and basal G protein coupling using a [35S]GTPγS binding assay. Figure 3 shows that
11f, 11h, 11j and 13b decrease agonistꢀinduced G protein coupling to the CB1 receptor in a
concentrationꢀdependent manner similar to the effects exhibited by the previously reported 1, 2,
11a and 4.19, 20, 22ꢀ24 Remarkably, compound 11j exhibited a robust inhibitory effect in CP55,940ꢀ
induced GTPγS binding (Figure 3C) evident by a progressive decrease in the Emax values with an
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increasing concentration of 11j. For example, at 0.1 ꢁM of 11j, the CP55,940ꢀinduced
[35S]GTPγS binding was substantially reduced (Emax = 53.4 ± 3.1 fmol/mg) compared with that in
the absence of 11j (Emax = 113.0 ± 2.6 fmol/mg) while compounds 11f, 11h and 13b exhibited
comparable levels of reduction at 3.2 ꢁM and higher (Emax = 61.4 ± 2.0, 58.2 ± 1.07 and 59.5 ±
1.4 fmol/mg, respectively; Figure 3A, B and D). Treatment with 1 ꢁM of 11j showed complete
inhibition of CP55,940ꢀinduced [35S]GTPγS binding (Emax = 36.04 ± 2.0 fmol/mg) while at least
10 ꢁM of 11f, 11h and 13b was needed to achieve a similar effect. Because of the robust
allostery that compound 11j displayed, the basal levels of G protein coupling in the absence of
CP55,940 (Figure 3E) were also examined. Compound 11j also antagonized the basal level of
[35S]GTPγS in a concentrationꢀdependent manner. Treatment with 10 ꢁM of 11j resulted in
complete inhibition of this basal activity. While this pattern of G protein coupling is consistent
with an inverse agonist’s profile in its ability to inhibit basal and CP55,940ꢀinduced GTPγS
binding, when collectively taken with its enhancement of the orthosteric agonist CP55,940
binding, it indicates that these compounds are allosteric modulators that are likely able to
selectively regulate the functions of the CB1 receptor. To date, compound 11j is the most
effective allosteric antagonist of CB1 G protein coupling and surpasses both 1 and 11a which we
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also found inhibited G protein coupling at 3.2 ꢁM.23, 24 Moreover, this pattern is consistent with
the robust allostery of compound 11j as reflected by its binding parameters (Table 2).
A recent study of the CB1 receptor explored the mechanism of action of 1 using a siteꢀ
directed fluorescence labeling approach.28 The results showed that the agonist CP55,940 induces
a movement in the cytoplasmic end of TM6, which accompanies G protein coupling. Upon coꢀ
treatment with 1, this agonistꢀinduced movement is blocked. This state of CB1 may explain how
1 and its analogs can elicit differential effects on CB1 agonist affinity and efficacy (i.e. a positive
allosteric modulator of binding but an antagonist of G protein coupling activity).
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Figure 3. Doseꢀresponse curves for CP55,940ꢀinduced [35S]GTPγS binding to human embryonic
kidney (HEK293) cell membranes expressing the CB1 wildꢀtype receptor in the absence and
presence of compounds 11f (A), 11h (B), 11j (C) and 13b (D) at the indicated concentrations.
The basal level of [35S]GTPγS binding was also measured in the absence of any orthosteric
ligand and the inhibition of it was tested at the indicated concentration of compound 11j (E).
Data are presented as specific binding of GTPγS to the membranes. Nonꢀspecific binding was
determined in the presence of 10 ꢁM unlabeled GTPγS. Each data point represents the
mean ± S.E. (error bars) of at least three independent experiments performed in duplicate.
Statistical significance of the differences in each concentration of 11j compared to basal (i.e. no
ligand) in (E) was assessed using oneꢀway analysis of variance and Bonferroni’s postꢀhoc test;
***, p < 0.005. The dotted line in (E) indicates the level of non CB1ꢀmediated GTPγS binding
obtained from [35S]GTPγS binding to the mockꢀtransfected membrane sample.
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CONCLUSION
We synthesized a group of analogs of the CB1 allosteric modulator 1 to understand the
structure activity relationship of indoleꢀ2ꢀcarboxamides in allosteric modulation of the CB1
receptor. Within the structure of indoleꢀ2ꢀcarboxamides, the presence of the indole ring seems to
be more influential on the ligand’s binding affinity to the allosteric site than on the binding
cooperativity (α) between the allosteric site and the orthosteric site. The C3 alkyl groups on the
indoleꢀ2ꢀcarboxides profoundly impacted the allostery of the ligand. Through structural
modification, we identified a robust CB1 allosteric modulator 11j which showed an equilibrium
dissociation constant KB of 167.3 nM with a markedly high binding cooperativity factor
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(α=16.55) and potent inhibition of GTPγS binding. Although a small binding constant KB for the
allosteric modulator is not necessary for maximum binding cooperativity with the CB1
orthosteric ligand, CB1 modulators with high binding affinities to the allosteric site and high
cooperativity towards the orthosteric site are desirable for therapeutic applications.
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In recent years, drug discovery targeting allosteric sites of GPCRs has gained
momentum.29 This approach offers a number of potential advantages over drugs targeting the
orthosteric site, such as improved selectivity across receptor subtypes and the capability to
maintain the spatial and temporal signaling profile of the endogenous ligand. The nature of
biased signaling of some CB1 allosteric modulators such as 123, 25, 11a,24 and 422 suggested a
possibility to selectively regulate specific functions of the CB1 receptor. The activation and
inactivation of the CB1 receptor have been linked to therapeutic implications for many diseases
such as drug addiction, anxiety, depression, obesity, neurodegeneration, cancer and
inflammation. Compound 1 has been the prototype to date for allosteric modulators of the CB1
receptor. Growing evidence indicates that 123, 25 and its analogs24 are functionally positive
allosteric modulators of the CB1 receptor although they antagonize Giꢀprotein coupling activity
to CB1. This class of compounds may selectively transduce the orthosteric agonist signals
through the ERK1/2 phosphorylation pathway. Very recently, Pamplona et al demonstrated using
in vivo experiments that the CB1 positive allosteric modulator 4 is protective against βꢀamyloidꢀ
induced neurotoxicity.22 The therapeutic potential of indoleꢀ2ꢀcarboximides represented by 1 as
neuroprotective agents remains to be explored. Only recently, the explicit search for CB1
allosteric modulators has been initiated. Our work provides new insight into the structural
requirements of CB1 allosteric modulators of the class of indoleꢀ2ꢀcarboxamides. It will aid the
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future design and synthesis of more robust allosteric modulators for the pharmacologically and
physiologically vital CB1 receptor.
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EXPERIMENTAL SECTION
Compounds
Tested compounds (11aꢀ11l and 13aꢀ13d) were synthesized for this study except 1, which was
purchased from Tocris Bioscience (Minneapolis, MN). Compounds 11a19 and 11l26 have
previously been reported and were used in this study for comparison.
CB1 Expression and Membrane Preparation. HEK293 cells were maintained in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal bovine serum and 3.5 mg/ml glucose at
37 °C in 5% CO2. For transient receptor expression, HEK293 cells were plated at 800,000
cells/100ꢀmm dish on the day prior to transfection. The cells were transfected using the calcium
phosphate precipitation method.30 24 h postꢀtransfection, membranes of transfected cells were
prepared as described previously.31
Radioligand Binding Assay. Membranes from HEK293 cells expressing the CB1 wildꢀtype
receptor were prepared as previously described.31 In assays used to determine the cooperativity
between allosteric and orthosteric ligands, approximately 4 ꢁg of membrane was incubated at 30
°C for 60 min with a fixed tracer ([3H]CP55,940; 147.9 Ci/mmol, PerkinElmer Life Sciences
(Boston, MA)) concentration typically at its Kd (2.5 nM) which was determined from a saturation
binding isotherm. Binding assays were performed with at least nine concentrations of unlabeled
allosteric compound (ranging between 100 pM and 100 ꢁM) as described previously.23
Nonspecific binding was determined in the presence of 1 ꢁM unlabeled CP55,940. Reactions
were terminated by adding 250 ꢁL of TME buffer containing 5% BSA followed by filtration
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with a Brandel cell harvester through Whatman GF/C filter paper. Radioactivity was measured
using liquid scintillation counting. The total assay volume and the amount of membrane samples
were adjusted to avoid ligand depletion by keeping the bound ligand less than 10% of the total.
GTPγS Binding Assay. GTPγS binding assays were performed as described previously.23
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Briefly, 7.5 ꢁg of membranes were incubated for 60 min at 30°C in a total volume of 200 ꢁL
GTPγS binding assay buffer (50 mM TrisꢀHCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, and 100
mM NaCl) with unlabeled CP55,940 (at least nine different concentrations were used ranging
between 100 pM and 100 ꢁM), 0.1 nM [35S]GTPγS (1250 Ci/mmol; PerkinElmer Life Sciences,
Boston, MA), 10 ꢁM GDP (Sigma, St. Louis, MO), and 0.1% (w/v) BSA in the absence and
presence of varying concentrations of the allosteric compounds as indicated. The basal GTPγS
binding was measured in the absence of orthosteric ligand and with 5 ꢁM GDP to increase the
window of basal activity. Nonspecific binding was determined with 10 ꢁM unlabeled GTPγS
(Sigma, St. Louis, MO). The reaction was terminated by rapid filtration through Whatman GF/C
filters. The radioactivity trapped in the filters was determined by liquid scintillation counting.
Ligand and GTPγS Binding Data Analysis. All ligand binding assays and GTPγS binding
assays were carried out in duplicate. Data are presented as the mean ± S.E. value or the mean
with the corresponding 95% confidence limits from at least three independent experiments. The
interactions between the orthosteric radiolabeled agonist [3H]CP55,940 and the test modulators
were analyzed using Prism 6.0 (GraphPad Software Inc., San Diego, CA) according to the
following
allosteric
ternary
complex
ꢁ ꢂ
model
of
interaction.19
A
ꢀ =
ꢄ
ꢁ ꢂ⁄ ꢅ
ꢁ ꢂ⁄ ꢅ
ꢃA 1 + B ꢃB
ꢁ ꢂ
A +
ꢄ
1 + ꢆ B ꢃB
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Equation 1.
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Where Y denotes the specific bound orthosteric ligand divided by the total concentration of
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orthosteric ligand [A]. [B] denotes the concentration of the allosteric ligands. KA and KB are
equilibrium dissociation constants for their respective ligands, and α is a cooperativity factor that
quantifies the allosteric interaction in terms of magnitude and direction between the two ligands
when they simultaneously occupy the receptor. When α is 1.0, the tested modulator does not
change orthosteric ligand binding. If α is less than 1.0, the tested modulator reduces orthosteric
ligand binding (negative allosteric modulation on orthosteric ligand binding). If α is greater than
1.0, the modulator increases orthosteric ligand binding (positive allosteric modulation). For this
analysis, the value of [A] was kept constant and was the average of radioactive orthosteric ligand
concentration employed in the binding assays. KA was also fixed as a constant and was
determined from previous CP55, 940 saturation binding assays, whereas values of α and KB were
determined by nonlinear regression.
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Synthesis. All chemical reagents and solvents were purchased from SigmaꢀAldrich Chemical
Company unless specified otherwise and used without further purification. All anhydrous
reactions were performed under a static argon atmosphere in dried glassware using anhydrous
solvents. Organic phases in work up were dried over anhydrous Na2SO4, and removed by
evaporation under reduced pressure. The crude compounds were purified by a Combiflash Rf
chromatography system (Teledyne Technologies, Inc, Thousand Oaks, CA) unless specified
otherwise. Purities of the intermediates were established by Thinꢀlayer Chromatography (TLC),
melting point, 1HNMR, and mass spectrometry. Analytical Thinꢀlayer Chromatography (TLC)
was run on preꢀcoated silica gel TLC aluminum plates (Whatman, UV254, layer thickness 250
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ꢁm), and the chromatograms were visualized under ultraviolet (UV) light. Melting points were
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determined on a capillary Electrothermal melting point apparatus and are uncorrected. HNMR
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spectra of intermediates were recorded on a Bruker Avance DPXꢀ300 spectrometer operating at
300 MHz. 1HNMR spectra of final compounds were recorded on a Bruker AVꢀ500 spectrometer
operating at 500 MHz. All NMR spectra were recorded using CDCl3 or DMSOꢀd6 as solvent
unless otherwise stated and chemical shifts are reported in ppm (parts per million) relative to
tetramethylsilane (TMS) as an internal standard. Multiplicities are indicated as br (broadened), s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), bs (broadened singlet) and coupling
constants (J) are reported in hertz (Hz). Low resolution mass spectra were performed at the
School of Chemical Sciences, University of Illinois at UrbanaꢀChampaign. The purity of each
tested compound was analyzed by combustion elemental analysis and was confirmed to be
greater than 99%. Corresponding combustion elemental analysis was conducted in Roberson
Microlit laboratories (Madison, NJ).
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General procedure A: Preparation of ethyl 3ꢀactylꢀ5ꢀchloroꢀ1Hꢀindoleꢀ2ꢀcarboxylates (7aꢀ
7f). The solution of ethyl 5ꢀchloroꢀ1Hꢀindoleꢀ2ꢀcarboxylate (5; 10 mmol) in anhydrous 1,2ꢀ
dichloroethane (25 mL) was added anhydrous aluminum chloride (10 mmol) powder and the
selected acyl chloride liquid (6; 11.5 mmol) dropwise at room temperature. The reaction mixture
was stirred and refluxed under argon atmosphere for 2ꢀ3 h. The completion of the reaction was
monitored by TLC (25ꢀ33% ethyl acetate in hexane). Upon completion, the reaction mixture was
poured into iceꢀcold water (150 mL) then treated with 4N HCl to pH 2. The mixture was
extracted with ethyl acetate twice. The combined organic phase was washed with water and brine
successively. The organic layer was separated and dried over anhydrous sodium sulfate.
Filtration and removal of solvent in vacuo provided the corresponding crude product, which was
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purified by Combiflash chromatography (0ꢀ40% of ethyl acetate in hexane) and yielded the
desired products 7aꢀ7f.
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General procedure B: Preparation of ethyl 3ꢀalkylꢀ5ꢀchloroꢀ1Hꢀindoleꢀ2ꢀcarboxylates (8aꢀ
8f). Liquid triethylsilane (1.18 mL, 7.40 mmol) was added dropwise to the solution of ethyl 3ꢀ
actylꢀ5ꢀchloroꢀ1Hꢀindoleꢀ2ꢀcarboxylate (7; 3.76 mmol) in trifluoroacetic acid (2.88 mL) at 0 °C.
The reaction mixture was then gradually warmed up to room temperature and stirred for 4ꢀ12 h.
The reaction is monitored by TLC (30% of ethyl acetate in hexane). Upon reaction completion,
the mixture was poured onto ice and treated with saturated sodium carbonate aqueous solution to
pH 7, and was then extracted with ethyl acetate. The combined organic phase was washed with
water and brine, and dried over anhydrous sodium sulfate. Filtration and removal of solvent
provided the crude product, which was purified either by Combiflash silica gel column
chromatography (0ꢀ30% of ethyl acetate in hexane) or recrystallization in the mixture of acetone
and ethyl acetate (1:4) to yield the corresponding products 8aꢀ8f.
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General procedure C: Preparation of 3ꢀalkylꢀ5ꢀchloroꢀ1Hꢀindoleꢀ2ꢀcarboxylic acids (9aꢀ9f).
The solution of ethyl 5ꢀchloroꢀ3ꢀalkylꢀ1Hꢀindoleꢀ2ꢀcarboxylate (8; 1.9 mmol) in anhydrous
ethanol (20 mL) was added 3 N sodium hydroxide aqueous solution (3.8 mL, 11.44 mmol). The
reaction mixture was stirred and refluxed for 2 h. Upon completion of the hydrolysis monitored
by TLC (30% ethyl acetate in hexane), it was cooled to room temperature and treated with 0.1 N
HCl to pH 2. White solid precipitated and was filtered. The cake was washed with a minimal
amount of cold water and hexane (2 mL) and then dried in a vacuum oven over night to provide
the white solid products (9aꢀ9f).
General procedure D: Preparation of 5ꢀchloroindoleꢀ2ꢀcarboxamides (11aꢀ11l) or 5ꢀchloroꢀ
benzofuranꢀ2ꢀcarboxamides (13aꢀ13d). To the solution of 5ꢀchloroindoleꢀ2ꢀcarboxylic acid (9;
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0.68 mmol) or 5ꢀchlorobenzofuranꢀ2ꢀcarboxlic acid (12; 0.68 mmol), BOP (502 mg, 1.02 mmol),
and N,Nꢀdiisopropylethylamine (0.71 mL, 4.08 mmol) in anhydrous DMF (4 mL) were added the
solution of appropriate amine (10; 0.82 mmol) in 1 mL of anhydrous DMF. The reaction mixture
was stirred at room temperature for 4ꢀ12 h. Upon completion of the coupling reaction, which was
monitored by TLC (30% ethyl acetate in hexane), the mixture was poured into cold water (40
mL) and extracted with ethyl acetate twice. The organic layer was separated and washed with
water, brine and dried over anhydrous sodium sulfate. Filtration and removal of solvent in vacuo
provided the crude product, which was then purified either by trituration followed by
recrystallization in the solvent specified in the supporting information or by flash
chromatography to generate the final compounds (11aꢀ11l) or (13aꢀ13d) respectively.
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5ꢀChloroꢀ3ꢀpentylꢀNꢀ(4ꢀ(piperidinꢀ1ꢀyl)phenethyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (11a). The
title compound was reported earlier by us.24 It was resynthesized by the modified methods
reported here to be used as a template for further modifications, and was obtained from 5ꢀchloroꢀ
3ꢀpentylꢀ1Hꢀindoleꢀ2ꢀcarboxylic acid (9a, 214 mg, 0.81 mmol) and commercially available 2ꢀ(4ꢀ
piperidinꢀ1ꢀylꢀphenyl)ꢀethylamine (10a, Oakwood Chemical, 200 mg, 0.98 mmol) according to
the general procedure D. The product was purified by Combiflash chromatography (0ꢀ50% of
°
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ethyl acetate in hexane) and yielded 120 mg (32.8 %) of white solid. mp: 218ꢀ219 C. H NMR
(500 MHz, DMSOꢀd6): δ 11.32 (s, 1H), 7.94 (t, J = 5.5 Hz, 1H), 7.61 (bs, 1H), 7.39 (d, J = 8.6
Hz, 1H), 7.17 (dd, J = 8.6, 1.0 Hz, 1H), 7.09 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.4 Hz, 2H), 3.53
(m, 2H), 3.13 (t, J = 5.6 Hz, 4H), 3.00 (t, J = 7.5 Hz, 2H), 2.75 (t, J = 7.5 Hz, 2H), 1.69ꢀ1.64 (m,
4H), 1.59ꢀ1.54 (m, 4H), 1.36ꢀ1.28 (m, 4H), 0.90 (t, J = 7.5 Hz, 3H). MS (EI): m/z = 451.2 (M+).
Anal. calcd. for (C27H34ClN3O): C, 71.74; H, 7.58; N, 9.30; found: C, 71.78; H, 7.57; N, 9.04.
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5ꢀChloroꢀ3ꢀphenylꢀNꢀ(4ꢀ(piperidinꢀ1ꢀyl)phenethyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (11b). The
title compound was prepared from 5ꢀchloroꢀ3ꢀphenylꢀ1Hꢀindoleꢀ2ꢀcarboxylic acid (9b; 200 mg,
0.74 mmol) and 2ꢀ(4ꢀpiperidinꢀ1ꢀylꢀphenyl)ꢀethylamine (10a, Oakwood Chemical, 180 mg, 0.88
mmol) according to the general procedure D. The crude product (470 mg) was triturated
sequentially with acetone (5 mL) and diethyl ether (5 mL) to yield 200 mg of lightꢀyellow
colored solid which was then purified by Combiflash chromatography (0ꢀ50% of ethyl acetate in
hexane) to yield 57 mg of lightꢀyellow colored solid. The mother solution was concentrated and
purified by Combiflash chromatography (0ꢀ50 % of ethyl acetate in hexane) to yield 86 mg of
lightꢀyellow colored solid. Hence, a total of 143 mg (42.2 %) of solid was collected. mp: 183ꢀ
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185 C. H NMR (500 MHz, Chloroformꢀd): δ 9.54 (bs, 1H), 7.46ꢀ7.42 (m, 3H), 7.40ꢀ7.32 (m,
4H), 7.25 (dd, J = 9.0 Hz, 1.5 Hz, 1H), 6.86 (d, J = 10.0 Hz, 2H), 6.81 (d, J = 10.0 Hz, 2H), 6.02
(t, J = 5.3 Hz, 1H), 3.54 (q, J = 6.4 Hz, 2H), 3.12 (t, J = 6.5 Hz, 4H), 2.63 (t, J = 6.7 Hz, 2H),
1.74ꢀ1.69 (m, 4H), 1.60ꢀ1.55 (m, 2H). MS (EI): m/z = 457.2 (M+). Anal. Calcd. for
(C28H28ClN3Oꢂ½H2O): C, 72.01; H, 6.26; N, 9.00; found: C, 72.27; H, 6.07; N, 8.67.
3ꢀBenzylꢀ5ꢀchloroꢀNꢀ(4ꢀ(piperidinꢀ1ꢀyl)phenethyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (11c) The title
compound was synthesized from 3ꢀbenzylꢀ5ꢀchloroꢀ1Hꢀindoleꢀ2ꢀcarboxylic acid (9c; 200 mg,
0.7 mmol) and commercially available 2ꢀ(4ꢀ(piperidinꢀ1ꢀyl)phenyl)ꢀethylamine (10a, Oakwood
Chemical, 172 mg, 0.84 mmol) according to the general procedure D. The obtained crude
product was triturated with methanol to provide 100 mg of offꢀwhite solid. The mother solution
was then concentrated and yielded a lightꢀred solid, which was then purified by Combiflash
chromatography (0ꢀ40% of ethyl acetate in hexane) and yielded 36 mg of white solid, which was
recrystallized in hot ethyl acetate (6.5 mL) to yield 23 mg of white solid. Therefore, a total of
°
1
123 mg (37.2 %) of the desired product was isolated. mp: 180ꢀ182 C. HNMR (500 MHz,
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DMSOꢀd6): δ 11.4 (s, 1H), 8.04 (t, J = 5.5 Hz, 1H), 7.46 (s, 1H), 7.34 (d, J = 8.5 Hz, 1H), 7.19ꢀ
7.14 (m, 4H), 7.11 (dd, J = 8.7 Hz, 2 Hz, 1H), 7.07ꢀ7.03 (m, 1H), 7.0 (d, J = 8.2 Hz, 2H), 6.76
(d, J = 7.9 Hz, 2H), 4.30 (s, 2H), 3.41(q, J = 6.8 Hz, 2H), 2.98 (t, J = 5.7 Hz, 4H), 2.67 (t, J = 7.3
Hz, 2H), 1.50ꢀ1.55 (m, 4H), 1.41ꢀ1.45 (m, 2H). MS (EI): m/z = 471.2 (M+). Anal. Calcd. for
(C29H30ClN3O): C, 73.79; H, 6.41; N, 8.90; found: C, 73.76; H, 6.46; N, 8.83.
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5ꢀChloroꢀ3ꢀ(cyclohexylmethyl)ꢀNꢀ(4ꢀ(piperidinꢀ1ꢀyl)phenethyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide
(11d). The title compound was synthesized from 5ꢀchloroꢀ3ꢀ(cyclohexylmethyl)ꢀ1Hꢀindoleꢀ2ꢀ
carboxylic acid (9c; 200 mg, 0.69 mmol), and commercially available 2ꢀ(4ꢀPiperidinꢀ1ꢀylꢀ
phenyl)ꢀethylamine (10a, Oakwood Chemical, 168 mg, 0.82 mmol) according to the general
procedure D. The obtained solid crude product was recrystallized in a hot solvent mixture of
ethyl acetate and acetone (6:1) and provided 132 mg of white crystals. The mother solution was
concentrated, and purified by Combiflash chromatography (0ꢀ50% of ethyl acetate in hexane)
and yielded 46 mg of white solid. Thus, a total of 178 mg (54.0 %) of product was isolated. mp:
°
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218ꢀ220 C. HNMR (500 MHz, DMSOꢀd6): δ 11.35 (s, 1H), 7.94 (t, J = 5.5 Hz, 1H), 7.60 (s,
1H), 7.38 (d, J = 8.9 Hz, 1H), 7.17 (dd, J = 8.9 Hz, 2.0 Hz, 1H), 7.10 (d, J = 8.2 Hz, 2H), 6.86 (d,
J = 8.2 Hz, 2H), 3.48 (q, J = 6.8 Hz, 2H), 3.07 (t, J = 5.0 Hz, 4H), 2.83 (d, J = 7 Hz, 2H), 2.75 (t,
J = 7.3 Hz, 2H), 1.62ꢀ1.58 (m, 6H), 1.56ꢀ1.48 (m, 6H), 1.13ꢀ1.02 (m, 3H), 0.99ꢀ0.88 (m, 2H).
MS (EI): m/z = 477.2 (M+). Anal. Calcd. for (C29H36ClN3O) C, 72.86; H, 7.59; N, 8.79; found: C,
72.75; H, 7.56; N, 8.69.
5ꢀChloroꢀ3ꢀheptylꢀNꢀ(4ꢀpiperidinꢀ1ꢀyl)phenethyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (11e). The title
compound was prepared from ethylꢀ5ꢀchloroꢀ3ꢀheptylꢀ1Hꢀindoleꢀ2ꢀcarboxylic acid (9d; 200 mg,
0.62 mmol) and commercially available 2ꢀ(4ꢀpiperidinꢀ1ꢀylphenyl)ꢀethylamine (10a; Oakwood
Chemical, 208 mg, 1.02 mmol) according to the general procedure D. 115 mg (38.6 %) of white
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solid was isolated as product by Combiflash chromatography (0ꢀ40% of ethyl acetate in hexane).
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mp:178ꢀ180 C. HNMR (500 MHz, Chloroformꢀd): δ 9.11 (bs, 1H), 7.52 (d, J = 1.5 Hz, 1H),
7.28 (d, J = 8.5 Hz, 1H), 7.19 (dd, J = 7.5 Hz, 1.5 Hz, 1H), 7.11 (d, J = 8.2 Hz, 2H), 6.89 (d, J =
8.2 Hz, 2H), 6.03 (t, J = 5.3 Hz, 1H), 3.76 (q, J = 6.4 Hz, 2H), 3.11 (t, J = 5.5 Hz, 4H), 2.86 (t, J
= 6.6 Hz, 2H), 2.68 (t, J = 7.8 Hz, 2H), 1.70 (quin, J = 11.0 Hz, 4H), 1.49ꢀ1.41 (m, 2H), 1.30ꢀ
1.16 (m, 10H), 0.87 (t, J = 7.5 Hz, 3H). MS (EI): m/z = 479.2 (M+); Anal. Calcd. for
(C29H38ClN3O): C, 72.55; H, 7.98; N, 8.75; found: C, 72.31; H, 8.01; N, 8.55.
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5ꢀChloroꢀ3ꢀnonylꢀNꢀ(4ꢀpiperidinꢀ1ꢀyl)phenethyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (11f). The title
compound was prepared from ethylꢀ5ꢀchloroꢀ3ꢀnonylꢀ1Hꢀindoleꢀ2ꢀcarboxylic acid (9e; 200 mg,
0.57 mmol) and commercially available 2ꢀ(4ꢀpiperidinꢀ1ꢀylꢀphenyl)ethylamine (10a, Oakwood
Chemical, 190 mg, 0.93 mmol) according to general procedure D. 116 mg (40 %) of white solid
°
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was isolated as product. mp: 148ꢀ150 C. HNMR (500 MHz, Chloroformꢀd): δ 9.09 (bs, 1H),
7.54 (d, J = 1.5 Hz, 1H), 7.30 (d, J = 8.5 Hz, 1H), 7.21 (dd, J = 7.5 Hz, 1.5 Hz, 1H), 7.14 (d, J =
7.9 Hz, 2H), 6.99ꢀ6.91 (m, 2H), 6.03 (bs, 1H), 3.78 (q, J = 6.4 Hz, 2H), 3.20ꢀ3.09 (m, 4H), 2.88
(t, J = 6.6 Hz, 2H), 2.70 (t, J = 7.8 Hz, 2H), 1.80ꢀ1.66 (m, 4H), 1.65ꢀ1.54 (m, 2H), 1.52ꢀ1.42 (m,
2H), 1.32ꢀ1.15 (m, 12H), 0.89 (t, J = 7.5 Hz, 3H). MS (EI): m/z = 507.2 (M+); Anal. Calcd. for
(C31H42ClN3O): C, 73.27; H, 8.33; N, 8.27; found: C, 73.49; H, 8.57; N, 8.09.
5ꢀChloroꢀ3ꢀpentylꢀNꢀ(4ꢀ(piperidinꢀ1ꢀyl)benzyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (11g). The title
compound was prepared from 5ꢀchloroꢀ3ꢀpentylꢀ1Hꢀindoleꢀ2ꢀcarboxylic acid (9a, 232 mg, 0.87
mmol) and commercially available (4ꢀ(piperidinꢀ1ꢀyl)phenyl)methanamine (10b, 200 mg, 1.05
mmol) according to the general procedure D. 105 mg (27.6%) of white solid product was
°
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isolated by Combiflash chromatography (0ꢀ30% ethyl acetate in hexane). mp:196ꢀ198 C; H
NMR (500 MHz, Chloroformꢀd): δ 9.26 (bs, 1H), 7.56 (s, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.24 (d,
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J = 8.2 Hz, 2H), 7.23 (dd, J = 8.9, 2.1 Hz, 1H), 6.94 (d, J = 8.2 Hz, 2H), 6.27 (bs, 1H), 4.59 (d, J
= 5.5 Hz, 2H), 3.16 (t, J = 5.5 Hz, 4H), 2.85 (t, J = 5.5 Hz, 2H), 1.79ꢀ1.67 (m, 4H), 1.65ꢀ1.55 (m,
2H), 1.33ꢀ1.20 (m, 6H), 0.84 (t, J = 5.5 Hz, 3H). MS (EI): m/z = 437.2 (M+). Anal. Calcd. for
(C26H32ClN3O): C, 71.30; H, 7.36; N, 9.59; found: C, 71.06; H, 7.40; N, 9.37.
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5ꢀChloroꢀNꢀ(4ꢀmorpholinophenethyl)ꢀ3ꢀpentylꢀ1Hꢀindoleꢀ2ꢀcarboxamide (11h). The title
compound was synthesized from 5ꢀchloroꢀ3ꢀpentylꢀ1Hꢀindoleꢀ2ꢀcarboxylic acid (9a; 200 mg,
0.75 mmol), and commercially available 2ꢀ(4ꢀmorpholinophenyl)ethanamine (10c, Matrix
Scientific, 186 mg, 0.9 mmol). The product was purified by Combiflash chromatography (0ꢀ60%
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ethyl acetate in hexane) and yielded 110 mg (32.3 %) of white solid. mp: 180ꢀ182 C. H NMR
(500 MHz, DMSOꢀd6): δ 11.25 (s, 1H), 7.87 (t, J = 5.5 Hz, 1H), 7.54 (s, 1H), 7.32 (d, J = 8.5 Hz,
1H), 7.10 (dd, J = 8.6 Hz, 1.6 Hz, 1H), 7.06 (d, J = 8.2 Hz, 2H), 6.81 (d, J = 8.2 Hz, 2H), 3.65 (t,
J = 4.0 Hz, 4H), 3.41 (q, J = 6.9 Hz, 2H), 2.98 (t, J = 4.3 Hz, 4H), 2.87 (t, J = 7.5 Hz, 2H), 2.70
(t, J = 7.3 Hz, 2H), 1.43 (quin, J = 7.3 Hz, 2H), 1.25ꢀ1.12 (m, 4H), 0.77 (t, J = 7.3 Hz, 3H). MS
(EI): m/z = 453.2 (M+). Anal. Calcd. for (C26H32ClN3O2): C, 68.78; H, 7.10; N, 9.26; found: C,
68.79; H, 7.01; N, 9.11.
5ꢀChloroꢀ3ꢀpentylꢀ1Hꢀindoleꢀ2ꢀcarboxylic acid [2ꢀ(4ꢀphenoxyꢀphenyl)ꢀethyl]ꢀamide (11i).
The title compound was synthesized from 5ꢀchloroꢀ3ꢀpentylꢀ1Hꢀindoleꢀ2ꢀcarboxylic acid (9a;
200 mg, 0.75 mmol) and commercially available 2ꢀ(4ꢀphenoxyꢀphenyl)ethylamine (10d, Matrix
Scientific, 219 mg, 1.03 mmol) according to the general procedure D. The product was purified
by Combiflash chromatography (0ꢀ30% ethyl acetate in hexane) and yielded 122 mg (35 %) of
°
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white solid. mp: 143ꢀ145 C. H NMR (500 MHz, Chloroformꢀd): δ 9.06 (bs, 1H), 7.55 (s, 1H),
7.34ꢀ7.29 (m, 2H), 7.38ꢀ7.34 (m, 2H), 7.23 (d, J = 8.2 Hz, 2H), 7.12 (td, J = 7.4, 1.1 Hz, 1H),
7.00 (t, J = 8.2 Hz, 4H), 6.02 (bs, 1H), 3.82 (q, J = 6.3 Hz, 2H), 2.96 (t, J = 6.3 Hz, 2H), 2.73 (t,
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