Design, synthesis, and structure-activity relationship study of conformationally constrained analogs of indole-3-carboxamides as novel CB1 cannabinoid receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2010.06.067

Novel tricyclic indole-3-carboxamides were synthesized as structurally restricted analogs of bicyclic indoles, and found to be potent CB1 cannabinoid receptor agonists. The CB1 agonist activity depended on the absolute configuration of the chiral center of the tricyclic ring. The preferred enantiomer was more potent than the structurally unconstrained lead compound. Structure-activity relationships in the amide side chain of the indole C-3 position were also investigated.

Full text of DOI:10.1016/j.bmcl.2010.06.067

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4918–4921
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and structure–activity relationship study of
conformationally constrained analogs of indole-3-carboxamides as novel
CB1 cannabinoid receptor agonists
Takao Kiyoi a, Mark York a, Stuart Francis a, Darren Edwards a, Glenn Walker b, Andrea K. Houghton b,
Jean E. Cottney b, James Baker b, Julia M. Adam a,
*
a Department of Chemistry, Schering-Plough Research Institute, Newhouse, Lanarkshire ML1 5SH, UK
b Department of Pharmacology, Schering-Plough Research Institute, Newhouse, Lanarkshire ML1 5SH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel tricyclic indole-3-carboxamides were synthesized as structurally restricted analogs of bicyclic
indoles, and found to be potent CB1 cannabinoid receptor agonists. The CB1 agonist activity depended
on the absolute configuration of the chiral center of the tricyclic ring. The preferred enantiomer was more
potent than the structurally unconstrained lead compound. Structure–activity relationships in the amide
side chain of the indole C-3 position were also investigated.
Received 15 April 2010
Revised 10 June 2010
Accepted 11 June 2010
Available online 17 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Cannabinoid
CB1
GPCR agonist
The CB1 cannabinoid receptor is a member of G-protein coupled
receptor (GPCR) superfamily, which is characterized by seven-
transmembrane receptors. The CB1 receptor is located primarily
in the central nervous system but is also expressed on peripheral
neurones. Recently many studies revealed that the CB1 receptor
is a potential therapeutic target against pain and several other dis-
eases including glaucoma, traumatic brain injury, and multiple
sclerosis.1 Several lines of evidence have been reported regarding
the analgesic effects of CB1 agonists in both experimental animal
models and clinical studies. Moreover, a couple of CB1 agonists
bution to the binding of a particular conformation. Stereoselective
synthesis of both enantiomers revealed the structural require-
ments for highly potent CB1 agonist activity. Further structure–
activity relationship studies were also performed to find the opti-
mum amide substituent.
The racemic mixture of tricyclic indole 1, which has the –OCH2–
linkage, could be synthesized using Fischer indole synthesis meth-
odology3 as show in Scheme 1. The key precursor 24 was synthe-
sized efficiently by intramolecular reductive amination using 20
which was obtained from 2-nitrophenol and
a-bromoketone 19.
including
D
9-THC, one of the major bioactive components of can-
The indole ring formation of 24 proceeded in good yield and the
subsequent de-carboxylation and regioselective introduction of a
piperazinylcarbonyl group afforded desired compound 1.
nabis, are used clinically as antiemetics in cancer chemotherapy
or appetite stimulants in AIDS patients. However, the classical
cannabinoids represented by
D
9-THC are highly lipophilic and
The carbon analog 2, which has an ethylene moiety instead of
the –OCH2– of 1, was synthesized in a similar manner using
2-cyclohexyl-1,2,3,4-tetrahydroquinoline 25 as a precursor of in-
dole ring formation reaction, which could be obtained by the reac-
tion4 of quinoline with cyclohexane carboxylic acid followed by the
partial reduction of the quinoline ring.5 The homochiral analogs of
1 could be obtained from commercially available (S)- or (R)-N-Boc-
cyclohexyl-glycine 30 as illustrated in Scheme 2. Mitsunobu reac-
tion of 2-bromophenol with the alcohol 31, which was obtained by
the reduction of 30, afforded aryl ether 32.
the administration methods are still limited. We have previously
described indole-3-carboxamide derivatives as water soluble CB1
agonists which might be suitable for intravenous administration
as peri-operative analgesics.2 We now report a series of conforma-
tionally constrained indole derivatives in which the direction of the
cyclohexyl group is restricted by the formation of a six-membered
ring between indole 1- and 7-position. Generally, unconstrained
agonists exist as a mixture of conformers in solution and we
hypothesized that limiting the number of conformations of these
molecules might improve potency by lowering the entropic contri-
Intramolecular palladium catalyzed amination6 gave 3,4-dihy-
dro-2H-1,4-benzoxazine derivative 33. After removing the Boc
group of 33, the tricyclic core could be constructed in a similar
manner as shown in Figure 1. The subsequent introduction of the
* Corresponding author. Fax: +44 (0) 1698 736187.
E-mail addresses: julia.adam@spcorp.com, julia.adam@merck.com (J.M. Adam).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.067

T. Kiyoi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4918–4921
4919
O
CO2Et
N
R
NO2
O
NH
i, j
N
b
f, g, h
c
28; X = O
29; X = CH2
X
X
X
O
18; R = H
19; R = Br
a
26; X = O
27; X = CH2
24; X = O
25; X = CH2
20
k, l
e
O
NEt
N
HO2C
N
1; X = O
2; X = CH2
d
+
N
N
X
21
22
23
Scheme 1. Reagents and conditions: (a) Br2, quant.; (b) 2-nitrophenol, K2CO3, KI, 62%; (c) H2, Pd–C, quant.; (d) AgNO3, (NH4)2S2O8, TFA, chlorobenzene, water, 140 °C, 13%;
(e) NaBH3CN, AcOH, 59%; (f) NaNO2; (g) LAH, 61% from 24, 68% from 25; (h) ethyl pyruvate, 75%; (i) NaOH; (j) Cu, quinoline, 65% from 26, 48% from 27; (k) oxalyl chloride,
quant.; (l) 1-ethylpiperazine, quant.
NHBoc
NHBoc
NH2
e, f, g
HO
a, b
d
c
NBoc
N
HO2C
Br
NHBoc
O
O
O
30
32
33
31
34
R'
R
O
N
N
CO2Et
h
i, j
N
k or l, m
N
O
O
N
O
36
35
3, 6, 7, 11, 12
l
R
N
O
CO2H
N
O
NH
R
O
N
N
N
p or q
m
n
N
N
N
O
N
O
O
O
37
38; R = Bn
14; R = H
o
p
10
8, 9, 15, 16
13; R = Me
Scheme 2. Reagents and conditions: (a) MeI, NaHCO3, DMF, 90%; (b) NaBH4, CaCl2, THF, MeOH, 85%; (c) 2-bromophenol, DIAD, PPh3, toluene, 0 °C to rt, 46%; (d) Pd(PPh3)4,
t-BuONa, toluene, microwave 120 °C, 67%; (e) HCl, EtOH, 70 °C, quant.; (f) NaNO2, DMF, water, 0 °C, 85%; (g) LAH, THF, 0 °C, 70%; (h) ethyl pyruvate, H2SO4, EtOH, reflux, 85%;
(i) NaOH, EtOH, 70 °C, 92%; (j) Cu, quinoline, 210 °C, 86%; (k) i—oxalyl chloride, 1,1,2,2-tetrachloroethane, 120 °C; ii—amine, Et3N, two steps 44–52%; (l) i—(CF3CO)2O, DMF,
0 °C to rt, 75%; ii—NaOH, 1,4-dioxane, water, reflux, quant.; (m) amine, EDCI, HOBt, DMF, 48%; (n) i—oxalyl chloride, DCM; ii—1-benzyl-cis-3,5-dimethylpiperazine, DIEA,
DCM, two steps 39%; (o) H2, Pd–C, EtOH, 60%; (p) aldehyde, NaBH(OAc)3, EtOH; (q) alkylbromide, CH3CN, microwave, 150 °C.
piperazinylcarbonyl moiety was performed either by direct amide
formation reaction using oxalyl chloride or by stepwise reaction
via carboxylic acid intermediate 37 to afford a variety of substi-
tuted piperazine derivatives. The alternative enantiomer 4 could
be synthesized by the same route.
The prepared compounds were tested for CB1 agonist activity
using CHO cells doubly transfected with human CB1 and a lucifer-
ase reporter gene.7 As shown in Table 1, racemic compounds 1 and
2 were 5- to 6-fold more potent than the non-constrained parent
compound 5 (pEC50 = 7.5, 7.6, and 6.8, respectively). The result
encouraged us to prepare and test both enantiomers of 1. Interest-
ingly, the (R)-isomer 3 retained similar activity to the racemic 1,
though the (S)-isomer 4 completely lost activity. This result indi-
cated that the correct orientation of the cyclohexyl group is essen-
tial to express potent CB1 agonist activity.
The SAR studies on the piperazine moiety of (R)-tricyclic deriv-
atives are summarized in Table 2. The substituent at the 4-position
on the piperazine ring showed an important influence on the CB1

4920
T. Kiyoi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4918–4921
R'
agonist activity, that is, replacement of the ethyl group in com-
pound 3 with methyl group slightly attenuated the activity
(pEC50 = 7.5–7.0), whereas removal of the alkyl group showed con-
siderably reduced potency (6 and 7, respectively). Introduction of
two methyl groups on 3- and 5-position resulted in significantly
improved potency; pEC50 values of cis-4-ethyl-3,5-dimethyl deriv-
ative 8 and cis-3,4,5-trimethyl derivative 9 were 8.4 and 8.2,
respectively. The activity of 3,4-dimethyl derivative 11 was be-
tween that of trimethyl derivative 9 and monomethyl compound
6. The 2,4,6-trimethyl derivative 13, a regioisomer of 9, displayed
similar activity to 4-methylpiperazine 6. Among all of these substi-
tuted piperazines, removal of alkyl group on the 4-position re-
sulted in loss of activity (10, 12, and 14). Interestingly,
introduction of a hydrophilic moiety such as hydroxy or methoxy
group, that could improve water solubility and PK/PD properties
of the molecule, was well tolerated at the piperazine 4-position
(pEC50 = 8.2 for 15 and 8.3 for 16, respectively).
CB1 agonist activity of related unconstrained indole deriva-
tives are summarized in Table 3. Interestingly, the SAR of con-
strained and unconstrained derivatives on the piperazine ring
were quite similar, that is, 4-ethyl-3,5-dimethyl piperazine deriv-
atives showed the highest agonist activity in each series, the
activity of 4-methyl derivatives were slightly lower than the 4-
ethyl derivatives (17b vs 17a and 9 vs 8, respectively), and 3,4-
dimethyl derivatives 17c and 11 displayed better activity than
the corresponding 4-ethyl derivatives (5 and 3). When bearing
the same substituents on the piperazine ring, the constrained
compounds always showed better activity than the uncon-
strained ones. These results strongly support the hypothesis that
limiting the number of conformations of a molecule can improve
its CB1 receptor agonist activity.
O
NR
R"
N
O
NEt
N
N
N
X
*
OMe
X = O or CH2
Figure 1. The structure of the original lead compound and newly designed scaffold.
Table 1
CB1 agonist activities for conformationally constrained compounds 1–4 and non-
constrained compound 5
O
NEt
O
N
NEt
N
N
N
X
OMe
*
1 - 4
X
5
a
Compound
Absolute configuration
pEC50
1
2
3
4
5
O
CH2
O
O
—
Racemic mixture
Racemic mixture
7.5
7.6
7.5
<5
R
S
—
6.8
a
Values are means of three experiments.
Table 2
CB1 agonist activities for (R)-tricyclic indole derivatives
R'
O
NR
N
R"
N
O
a
a
Compound
Piperazine moieties
pEC50
Compound
Piperazine moieties
pEC50
7.8
N
N
N
NEt
NMe
NH
3
7.5
7.0
11
N
N
N
NMe
NH
6
7
12
13
6.3
7.2
5.4
8.4
8.2
6.9
NMe
8
9
N
N
N
NEt
NMe
NH
14
15
16
N
N
N
NH
N
6.1
8.2
8.3
OH
O
10
N
a
Values are means of three experiments.

T. Kiyoi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4918–4921
4921
Table 3
binding to either hCB1 or hCB2 receptors expressed in insect Sf9
membranes.
CB1 agonist activities for unconstrained indole derivatives
R
It is worthwhile to mention that structural constraint did not
negatively influence the aqueous solubility of the molecules. The
aqueous solubility of the HCl salt of the tricyclic derivative 4 was
>4000 mg/L at pH 4.9 and 308 mg/L at pH 7.0, respectively,
whereas solubility of the corresponding bicyclic derivative (5)
was measured as 3398 mg/L at pH 5.0 and 216 mg/L at pH 6.7,
respectively.2 Although substitution of the oxygen atom in the tri-
cyclic ring for a methylene linker (CH2) slightly reduced aqueous
solubility as shown in Table 5 (compound 2 vs compound 4), these
compounds are still sufficiently soluble for intravenous adminis-
tration. As expected, hydrophilic moieties like secondary amines
or hydroxy groups afforded further solubility improvements (data
not shown).
O
NR'
R"
N
N
OMe
a
Compound
R
R0
R00
pEC50
5
17a
17b
17c
H
Et
Et
Me
Me
H
6.8
8.0
7.6
7.6
Me
Me
Me
Me
Me
H
a
The in vitro and in vivo DMPK profiles of compound 11 are sum-
marized in Table 6. The compound was rapidly metabolized by
mouse hepatic microsomes. Mouse brain and plasma levels were
Values are means of three experiments.
determined following intravenous administration of a 0.5 lmol/
kg dose (terminal sampling using CO2). Good CNS penetration
was seen, as expected based on the physico-chemical properties
of the compound. However, compound 11 was rapidly cleared in
vivo, as predicted from the rapid microsomal metabolism.
The antinociceptive activity of compound 11 was determined in
the mouse tail flick test8 after iv administration. The compound
significantly increased the tail flick latency; the ED50 value was
Table 4
Profile of CB1 agonists in in vitro hCB1 and hCB2 binding assays
Compound
CB1 pKi
CB2 pKi
3
11
7.9
8.7
8.4
9.4
0.19 lmol/kg.
Table 5
In summary, a series of conformationally constrained 3-(pipera-
zin-1-ylcabonyl)indole derivatives were synthesized stereoselec-
tively, revealing that one of the stereoisomers was more potent
than the non-constrained compound, while the other enantiomer
was inactive. A systematic SAR study on the piperazine ring re-
vealed a number of highly potent CB1 receptor agonists with drug
like properties. Further studies are in progress to improve meta-
bolic stability within the series by attention to metabolic hot-spots
such as the cyclohexane and alkyl piperazine moieties.
Aqueous solubility of the bicyclic and tricyclic indole derivatives
Compound
MSF solubility (mg/L)
Citrate pH 5 (final pH)
PBS pH 7.4 (final pH)
2
4
5
3509 (4.9)
>4000 (4.9)
3398 (5.0)
114 (7.0)
308 (7.0)
216 (6.7)
Table 6
DMPK profile of CB1 agonist 11
References and notes
Microsomal stability, mouse CLint
(ll/min/mg)
>180
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244047.
PK (ICR mouse, 0.5
Vehicle
lmol/kg, iv)
Saline
51.5
23.7
134
0.23
2.6
Plasma Cmax (ng/ml; t = 0.05 h)
AUCplasma, iv (h ng/ml)
Clearance (ml/min/kg)
T1/2 elimination (h)
Vss (L/kg)
Brain penetration (ICR mouse) same studies as above
Brain Cmax (ng/g)
Brain tmax (h)
Brain AUC0–1h (h ng/g)
Brain:plasma Cmax ratio
76.2
0.17
44.6
1.48
7. Price, M. R.; Baillie, G. L.; Thomas, A.; Stevenson, L. A.; Easson, M.; Goodwin, R.;
McLean, A.; McIntosh, L.; Goodwin, G.; Walker, G.; Westwood, P.; Marrs, J.;
Thomson, F.; Cowley, P.; Christopoulos, A.; Pertwee, R. G.; Ross, R. A. Mol.
Pharmacol. 2005, 68, 1484.
8. Whiteside, G. T.; Gottshall, S. L.; Boulet, J. M.; Chaffer, S. M.; Harrison, J. E.;
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The results of binding assays for both CB1 and CB2 cannabinoid
receptors are listed in Table 4. Compounds 3 and 11 exhibited high
affinity for both CB1 and CB2 cannabinoid receptors, as determined
by radioligand competition binding assays using [3H]CP 55,940