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

Article abstract of DOI:10.1016/j.bmcl.2010.10.061

Bicyclic piperazine derivatives were synthesized as conformationally constrained analogs of N-alkyl piperazines and were found to be potent CB1 receptor agonists. The CB1 receptor agonist activity was dependent upon the absolute configuration of the chiral center of the bicyclic ring system. Although the conformational constraint did not protect the compounds from metabolism by N-dealkylation, several bicyclic analogs were found to be more potent than the unconstrained lead compound. Compound 8b demonstrated potent antinociceptive activity in vivo.

Full text of DOI:10.1016/j.bmcl.2010.10.061

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7327–7330
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and structure–activity relationship study of bicyclic
piperazine analogs of indole-3-carboxamides as novel cannabinoid
CB1 receptor agonists
Elizabeth M. Moir a, Kazuya Yoshiizumi a, Jim Cairns a, Phillip Cowley a, Morag Ferguson a,
Fiona Jeremiah a, Takao Kiyoi a, Richard Morphy a, Jason Tierney a, Grant Wishart a, Mark York a,
James Baker b, Jean E. Cottney b, Andrea K. Houghton b, Petula McPhail b, Andrew Osprey b,
Glenn Walker b, Julia M. Adam a,
⇑
a Department of Chemistry, Merck Research Laboratories, MSD Ltd, Newhouse, Lanarkshire ML1 5SH, UK
b Department of Pharmacology, Merck Research Laboratories, MSD Ltd, 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:
Bicyclic piperazine derivatives were synthesized as conformationally constrained analogs of N-alkyl
piperazines and were found to be potent CB1 receptor agonists. The CB1 receptor agonist activity was
dependent upon the absolute configuration of the chiral center of the bicyclic ring system. Although
the conformational constraint did not protect the compounds from metabolism by N-dealkylation, sev-
eral bicyclic analogs were found to be more potent than the unconstrained lead compound. Compound
8b demonstrated potent antinociceptive activity in vivo.
Received 31 August 2010
Revised 13 October 2010
Accepted 14 October 2010
Available online 20 October 2010
Keywords:
Cannabinoid CB1 receptor
GPCR agonist
Ó 2010 Elsevier Ltd. All rights reserved.
The CB1 cannabinoid receptor is a member of G-protein coupled
receptor (GPCR) superfamily, which is characterized by seven-
transmembrane receptors.1 The CB1 receptor is located primarily
in the central nervous system but is also expressed on peripheral
neurones. Activation of the CB1 receptor has been suggested as a
potential strategy for the treatment of pain and several other dis-
eases including glaucoma, traumatic brain injury, and multiple
sclerosis,2 while inhibition of CB1 receptors has been explored as
a strategy for the treatment of obesity and addiction.3 Several lines
of evidence have been reported regarding the analgesic effects of
CB1 receptor agonists in both experimental animal models and
clinical studies. While limited in clinical utility by their small ther-
apeutic window with respect to psychotropic side effects, a couple
classical cannabinoid agonists.5 Moreover, the classical cannabi-
noid agonists represented by
9-THC are highly lipophilic and
the administration methods are still limited.
D
Previous publications described indole-3-carboxamide deriva-
tives as water soluble CB1 receptor agonists suitable for intrave-
nous administration as potential post-operative analgesics.6,7 This
Letter describes a series of bicyclic piperazine analogs in which
the piperazine N-alkyl substituent is tethered back onto the piper-
azine ring ( Fig. 2). Metabolite identification studies on initial
compounds in the mono-cyclic series indicated piperazine N-deal-
kylation as a major route of metabolism. It was proposed that teth-
ering the N-alkyl group back onto the piperazine ring system
would favorably affect the metabolic stability of these compounds
and, in addition, potentially improve potency within the series. The
impact of stereochemistry and effects of substitution in the bicyclic
piperazine system on CB1 receptor agonist potency and micro-
somal stability were investigated.
Compounds were synthesized as described in Scheme 1. Four-
teen optically pure bicyclic piperazines 6a–n were prepared using
parallel synthesis techniques, applying the previously reported
method for the synthesis (S)-1,4-diazabicyclo[4.3.0]nonane.8 Reac-
tion of amino acid methyl esters 2 and cyclic carboxylic acids 3
afforded amides 4. Boc deprotection and cyclization followed by
reduction of the amide carbonyl afforded bicyclic piperazines
6a–n. Introduction of the piperazinylcarbonyl moiety to N-cyclo-
hexylmethyl-7-methoxyindole 7 was performed by direct amide
of CB1 receptor agonists including
D -
9-tetrahydrocannabinol (D9
THC, Fig. 1), one of the major bioactive components of cannabis,
are used clinically as antiemetics in cancer chemotherapy or appe-
tite stimulants in AIDS patients.4 Sativex™, a medicinal cannabis
extract containing a mixture of
D
9-THC and cannabidiol, has been
recently launched for treatment of multiple sclerosis (MS)- and
cancer-associated neuropathic pain, and for MS-associated spastic-
ity. In addition, several lines of research are being progressed
toward identifying novel cannabinoid related medicines that avoid
or minimize the adverse effects associated with administration of
⇑
Corresponding author. Tel.: +44 1698 736000; fax: +44 1698 736187.
E-mail address: julia.adam@merck.com (J.M. Adam).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.061

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E. M. Moir et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7327–7330
O
OH
OH
OH
N
O
C6H13
C5H11
O
N
O
HO
Δ9-THC
CP 55,940
WIN 55,212-2
Figure 1. Representative naturally occurring and synthetic cannabinoid receptor agonists.
held true for the R1, R2 dimethyl analogs, comparing derivative 8j
R'
O
with 8i. In cases where there was a single substituent at R1 and
R2 = H (examples 8e–h), the combined effect of stereochemistry
at the two chiral centers (* and **) was less clear cut. Replacement
of methyl with the larger isobutyl substituent at R1 was not toler-
ated, illustrated by the reduced potency of compounds 8k and 8l in
comparison with derivatives 8e and 8f, respectively. Addition of a
methyl substituent at the bridgehead position, R3, was detrimental
to activity (comparing analog 8m with 8d). Incorporation of a fur-
ther heteroatom into the bicyclic piperazine system was tolerated.
Indeed for X = S (example 8n), there was an improvement in po-
tency from pEC50 = 6.3 for compound 8g to pEC50 = 7.5 for 8n.
Where tested, different salt forms (e.g., HCl salt/free base) had no
effect on the in vitro potency (data not shown).
O
N
N
N
N
R"
mal
N
N
O
O
A
1
pEC50 6.8
Figure 2. The structure of the original lead compound 1 and newly designed
scaffold A.
Microsomal stability was determined for all compounds in
Table 1 but no improvement in stability over the initial lead, 1,
was observed. Compounds 8a–n all showed a half-life of <5 min
in human and mouse liver microsome preparations.
formation6 using oxalyl chloride in 1,1,2,2-tetrachloroethane at
120 °C for 2 h, followed by addition of bicyclic piperazine 6a–n
and triethylamine, to form the piperazine amides 8a–n.
The prepared compounds were tested for CB1 receptor agonist
activity using CHO cells doubly transfected with human CB1 and
a luciferase reporter gene.9 As shown in Table 1, a number of bicy-
clic piperazine analogs were more potent at CB1 than the uncon-
strained N-ethyl piperazine 1 (Fig. 2), for example, compound 8b
with pEC50 = 7.9 versus pEC50 = 6.8 for compound 1. In general,
In order to investigate whether tethering back the N-alkyl substi-
tuent had perhaps shifted the major site of metabolism to a different
region of the molecule, compound 8b was briefly incubated with hu-
man liver microsomes (5 min) and the profiles of the metabolites
were analyzed by LC–MS–MS (Scheme 2 and Table 2). To our
surprise, the major metabolites were still found to correspond to
metabolism in the bicyclic piperazine portion of the molecule, with
N-dealkylation remaining a significant metabolic pathway as dem-
onstrated by formation of metabolite M2. The enzymes responsible
for this metabolism have not been characterized; however,
**
for analogs with one chiral center ( ) at the bridgehead position,
the (S)-isomer was more potent than the (R)-isomer. This was true
for 6,6- and 6,5-bicyclic piperazine ring systems, comparing deriv-
ative 8b with 8a, and derivative 8d with 8c. This general rule also
X
R3
X
R3
O
n
MeO2C
R1
NH2
R2
a
b
n
N
Boc
+
c
HO2C
N
Boc
MeO2C
R1
NH
R2
2
3
4a-n
R3
X
n
O
N
X
N
X
N
R3
n
R3
HN
O
n
d
R1
N
R2
HN
R1 R2
N
O
R1 R2
O
N
O
6a-n
5a-n
7
8a-n
Scheme 1. Reagents and conditions (reaction yields depicted below were from synthesis of 8b as a typical example): (a) (Me)2N(CH2)3N@C@NEtÁHCl, HOBt, NEt3, DCM, rt,
40 h, 98%; (b) (i) CF3COOH, rt, 2 h, (ii) NEt3, MeOH, reflux, 6 h, two steps 52%; (c) LiAlH4, THF, reflux, 3 h, 68%; (d) oxalyl chloride, 1,1,2,2-tetrachloroethane, 120 °C, 2 h, then
NEt3 and 7, rt, 7 h, 61%.

E. M. Moir et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7327–7330
7329
Table 1
12
l
M, and CYP2D6 13
l
M). It was concluded that relatively high
CB1 receptor agonist activities for bicyclic piperazine compounds 8a–n
lipophilicity (c log P = 5.5 for compound 8b) was likely to be a major
contributing factor to the metabolic vulnerability10 and that more
profound changes to the physico-chemical properties of these mol-
ecules would be required in order to achieve metabolically stable
CB1 receptor agonists.
R3
X
**
n
O
N
N
In view of the unexpectedly high potency of compound 2 and
the earlier observation7 that linking the 1- and 7-positions of the
indole core improved the potency, we decided to combine these
methods. The target compounds 10a and 10b were synthesized
by the previously described methods using the optically pure tricy-
clic indole7 (R)-9 or (S)-9 and the bicyclic amine 6b (Scheme 3).
As depicted in Table 3, the (R,S)-isomer 10a showed comparable
CB1 receptor agonist activity to 8b. The stereochemistry of the tricy-
clic indole core was crucial for the activity; the potency of the
(S,S)-isomer 10b was more than two log units lower than that of
10a. Similar effects of this chiral center on CB1 receptor agonist
activity were observed in the previously reported tricyclic indole
series.7 As expected, compounds 10a and 10b also showed a half-life
of <5 min in human and mouse liver microsome preparations.
Based on their high potency, compounds 8b and 10a were se-
lected for further evaluation. As shown in Table 4, compounds 8b
and 10a exhibited high affinity for both CB1 and CB2 cannabinoid
receptors, as determined by radioligand competition binding as-
says using [3H]CP 55,940 binding to either hCB1 or hCB2 receptors
expressed in insect Sf9 membranes.
*
R1
R2
N
O
8a - n
R1
R2
R3
*
**
pEC50
a
Compd
n
X
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
2
2
1
1
1
1
1
1
1
1
1
1
1
1
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
S
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
iBut
iBut
H
H
H
H
H
H
H
H
H
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
—
—
—
—
R
S
S
R
—
—
R
S
—
S
R
S
R
S
R
S
R
S
R
S
R
S
S
R
7.0b
7.9c
7.2b
7.7c
7.2c
7.0b
6.3c
6.0c
6.2b
7.0b
6.5b
<5.0b
5.9c
7.5c
6.8d
7.7
8m
The antinociceptive activities of compounds 8b and 10a were
determined in the tail flick test6 after iv administration in the
mouse. A beam of radiant heat is focused onto the tail of the animal
and the time recorded for the mouse to flick it’s tail away from the
heat source. The compounds significantly increased the tail flick la-
tency, indicating an antinociceptive response, with ED50 values of
8n
CH3
H
16
CP 55,940
WIN 55,212-2
7.3
a
b
c
Values are means of three experiments.
Data for free base.
Data for HCl salt.
0.58 and 0.42 lmol/kg, respectively (Table 5).
d
Data for maleic acid salt.
The antinociceptive profile of compound 8b was examined
further in the formalin model of inflammatory pain, in which dilute
formalin is injected into one hind paw of the mouse. The resultant
nociceptive behavior is characterized by flinching, licking, lifting,
compound 8b was shown to have inhibitory activity at a number of
human P450s (IC50 values—CYP3A4 6
lM, CYP2C19 10 lM, CYP2C9
H
H
B
O
N
N
O
N
H
NH
human liver
microsomes
+
Other metabolites (M1, M3-M7)
A
N
N
hydroxylation on
regions A and/or B
O
O
Metabolite M2
8b
Scheme 2. Metabolism of compound 8b in human liver microsomes.
Table 2
Metabolites of compound 8b
Compd
Retention time (min)
Apparent [M+H]
Metabolic transformation
Proposed site of metabolism
Relative peak area ratio metabolite/parent
M1
M2
M3
M4
M5
M6
M7
6.43
6.14
4.54
4.87
4.66
5.10
4.37
426
384
442
426
442
442
426
Hydroxylation
Dealkylation
Region B
Region B
At least one OH region A
Region A
At least one OH region A
Unknown
0.6
0.4
0.2
0.1
0.05
0.05
0.05
Di-hydroxylation
Hydroxylation
Di-hydroxylation
Di-hydroxylation
Hydroxylation
Region A
Conditions; column; Develosil RP aqueous 150 Â 4.6 mm, eluents; 0.1% formic acid in MeCN/0.1% formic acid in water, 10/90 to 100/0, detected by Mariner-TOF.

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E. M. Moir et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7327–7330
H
O
N
N
H
N
*
+
O
N
N
*
HN
O
(R)-9
6b
or
(S)-9
10a-b
Scheme 3. Reagents and conditions: oxalyl chloride, 1,1,2,2-tetrachloroethane, 120 °C, 1.5 h, then NEt3 and 6b, rt, 7 h, 76% for 10a and 64% for 10b.
pound 8b (0.03–3.0 l ) demonstrated dose-dependent
mol kgÀ1
Table 3
CB1 receptor agonist activities for tricyclic indoles 10a
and 10b
inhibition of nociceptive behavior in both phases of the formalin
response in the mouse.6 The antinociceptive response was statisti-
cally significant from control at doses equal to, and above,
0.3 l
mol kgÀ1. The effect of 8b in this test was markedly attenu-
H
ated by pretreatment with the CB1 antagonist, SR-141716A, which
indicated that the antinociceptive activity of this compound is
mediated via activation of CB1 receptors. Furthermore, compound
8b showed no genotoxic effects in Vitotox™ assay,11 which can
rapidly assess genotoxicity, at concentrations up to 10À4 M.
In summary, a series of conformationally constrained and opti-
cally pure bicyclic piperazines were synthesized and tested for CB1
receptor agonist activity and metabolic stability. Although the
modifications failed to protect the compounds from metabolism
by N-dealkylation, several analogs were found to have improved
potency compared to the unconstrained N-alkyl piperazines. The
selected compounds demonstrated potent antinociceptive activity
in the tail flick and formalin paw tests in the mouse.
O
N
N
N
O
*
a
Compound
*
pEC50
10a
10b
R
S
8.0b
5.7b
a
Values are means of three experiments.
Data for HCl salt.
b
References and notes
1. Matsuda, L. A.; Lolait, S. J.; Brownstein, M. J.; Young, A. C.; Bonner, T. I. Nature
1990, 346, 561.
Table 4
2. Recent reviews for cannabinoid receptor agonists: (a) Pertwee, R. G. Expert
Opin. Investig. Drugs 2000, 9, 1553; (b) Adam, J.; Cowley, P. M.; Kiyoi, T.;
Morrison, A. J.; Mort, C. J. W. In Progress in Medicinal Chemistry; King, F. D.,
Lawton, G., Eds.; Elsevier, 2006; Vol. 44, pp 207–329; (c)Handbook of
Experimental Pharmacology; Pertwee, R. G., Ed.; Springer, 2005; Vol. 168, (d)
Huffman, J. W.; Padgett, L. W. Curr. Med. Chem. 2005, 12, 1395.
3. (a) Jagerovic, N.; Fernandez-Fernandez, C.; Goya, P. Curr. Top. Med. Chem. 2008,
8, 205; (b) Bermudez-Silva, F. J.; Viveros, M. P.; McPartland, J. M.; Rodriguez de
Fonseca, F. Pharmacol., Biochem. Behav. 2010, 95, 375.
Profiles of CB1 receptor agonists 8b and 10a in in vitro
hCB1 and hCB2 binding assays
Compound
CB1 pKi
CB2 pKi
8b
10a
CP 55,940
WIN 55,212-2
8.6
8.8
9.5
7.9
8.4
9.4
9.5
8.6
4. Mackie, K. Annu. Rev. Pharmacol. Toxicol. 2006, 46, 101.
5. Thakur, G. A.; Tichkule, R.; Bajaj, S.; Makriyannis, A. Expert Opin. Ther. Patents
2009, 19, 1647.
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Edwards, D.; Ferguson, M.; Goodwin, R.; Jeremiah, F.; Kiyoi, T.; Mistry, A.; Moir,
E.; Morphy, R.; Tierney, J.; York, M.; Baker, J.; Cottney, J. E.; Houghton, A. K.;
Westwood, P. J.; Walker, G. Med. Chem. Commun. 2010, 1, 54.
7. Kiyoi, T.; York, M.; Francis, S.; Edwards, D.; Walker, G.; Houghton, A. K.;
Cottney, J. E.; Baker, J.; Adam, J. M. Bioorg. Med. Chem. Lett. 2010, 20, 4918.
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Bowen, W. J. Med. Chem. 1993, 36, 2311.
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McLean, A.; McIntosh, L.; Goodwin, G.; Walker, G.; Westwood, P.; Marrs, J.;
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Table 5
Effects of CB1 receptor agonists following
intravenous dosing in the tail flick test in the
mouse
Compound
ED50 (lmol/kg)
8b
10a
CP 55,940
WIN 55,212-2
0.58
0.42
0.11
1.7
10. Yoon, H. C.; Choi, E.; Park, J. E.; Cho, M.; Seo, J. J.; Oh, S. J.; Kang, J. S.; Kim, H. M.;
Park, S. K.; Lee, K.; Han, G. Bioorg. Med. Chem. Lett. 2010. doi:10.1016/
j.bmcl.2010.08.117.
11. Westerink, W. M.; Stevenson, J. C.; Lauwers, A.; Griffioen, G.; Horbach, G. J.;
Schoonen, W. G. Mutat. Res. 2009, 676, 113.
and biting the affected paw, and occurs in two distinct phases sep-
arated by a short period of quiescence. Iv administration of com-