Synthesis and evaluation of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-aminopropanamide as human cannabinoid-1 receptor (CB1R) inverse agonists

Article abstract of DOI:10.1016/j.bmcl.2009.07.046

Obesity is a chronic medical condition that is affecting large population throughout the world. CB1 as a target for treatment of obesity has been under intensive studies. Taranabant was discovered and then developed by Merck as the 1st generation CB1R inv

Full text of DOI:10.1016/j.bmcl.2009.07.046

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5195–5199
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-
methylpropyl]-2-methyl-2-aminopropanamide as human cannabinoid-1
receptor (CB1R) inverse agonists
a
a
a
b
b
b
Wu Du a, , James P. Jewell , Linus S. Lin , Vincent J. Colandrea , Jing C. Xiao , Julie Lao , Chun-Pyn Shen ,
Thomas J. Bateman c, Vijay B. G. Reddy c, Sookhee N. Ha a, Shrenik K. Shah a,
Tung M. Fong b, Jeffrey J. Hale a, William K. Hagmann a
*
a Department of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, United States
b Department of Metabolic Disorders, Merck Research Laboratories, Rahway, NJ 07065, United States
c Department of Preclinical DMPK, Merck Research Laboratories, Rahway, NJ 07065, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Obesity is a chronic medical condition that is affecting large population throughout the world. CB1 as a
target for treatment of obesity has been under intensive studies. Taranabant was discovered and then
developed by Merck as the 1st generation CB1R inverse agonist. Reported here is part of our effort on
the 2nd generation of CB1R inverse agonist from the acyclic amide scaffold. We replaced the oxygen lin-
ker in taranabant with nitrogen and prepared a series of amino heterocyclic analogs through a divergent
synthesis. Although in general, the amine linker gave reduced binding affinity, potent and selective CB1R
inverse agonist was identified from the amino heterocycle series. Molecular modeling was applied to
study the binding of the amino heterocycle series at CB1 binding site. The in vitro metabolism of repre-
sentative members was studied and only trace glucuronidation was found. Thus, it suggests that the right
hand side of the molecule may not be the appropriate site for glucuronidation.
Received 5 May 2009
Revised 29 June 2009
Accepted 2 July 2009
Available online 24 July 2009
Keywords:
CB1R
Inverse agonist
Obesity
Synthesis
Metabolism
Ó 2009 Elsevier Ltd. All rights reserved.
Characterized by excessive body weight and fat, obesity is a
chronic medical condition that is affecting people throughout the
world. It is often the cause of further serious medical complications
including insulin resistance, Type 2 diabetes, cardiovascular dis-
ease (CVD), stroke, hypertension, cancer and arthritis.1 There are
two FDA-approved drugs for chronic treatment of obesity with
modest efficacy: the sympathomimetic agent, sibutramine, and
the lipase inhibitor, orlistat. More efficacious and diverse treat-
ments for obesity and weight maintenance are highly desired.
It has been established that the cannabinoid system plays a role
in regulating feeding behavior and body weight.2 There are two
types of cannabinoid receptors: cannabinoid-1 receptor (CB1R)
and cannabinoid-2 receptor (CB2R). Among the pharmaceutical
activities of agonists of CB1R are enhanced food intake and in-
creased body weight. Conversely, antagonists/inverse agonists sup-
press food intake and reduce body weight.3 Thus the CB1R has
been an intensively studied target for development of new anti-
obesity agents. Rimonabant (SR141716, 1)4 was developed by
Sanofi-Aventis and had been approved in Europe as a new treat-
ment for obesity, but was withdrawn later due to psychiatric side
effects. After extensive studies on various scaffolds as CB1R inverse
agonists,5 taranabant (MK-0364, 2, Fig. 1)6 was discovered by
Merck scientists and was then advanced into development and
completed Phase III clinic trials.7
Our efforts on second generation CB1R inverse agonists focused
on discovering compounds that maintained a similar CB1R profile
to MK-0364 but exhibited a more balanced metabolism. Thus, it
would be less dependent on CYP3A4 mediated clearance than
MK-0364. By introducing a suitable functional group for direct con-
jugation, for example, we could minimize the potential for
DDI’s.7b,c It has been shown that in addition to carboxylic acids,
some heterocycles can also form glucuronide adducts.8 Thus, one
strategy was to replace the trifluoromethylpyridyloxy moiety in
2 with an amino heterocycle moiety as shown in structure 3. Either
the exo-cyclic nitrogen or the heterocyclic nitrogen may be the site
for glucuronidation. This is depending on the steric and electronic
factors specific to the heterocycle. The exo-cyclic nitrogen is steri-
cally hindered due to the adjacent gem-dimethyl group and also
less nucleophilic due to conjugation with the heterocycle. Thus,
the heterocycle nitrogen may have better chance to be the site
for glucuronidation.
To evaluate the amine linker versus an oxygen linker, com-
pound 3a was synthesized (Scheme 1) for direct comparison with
compound 2. Amino acid 4 was reacted with 2-chloro-5-trifluo-
romethylpyridine 5 in the presence of 2 equiv of DBU to give
* Corresponding author.
E-mail address: wuduchemistry@gmail.com (W. Du).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.046

5196
W. Du et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5195–5199
O
N
N
O
O
N
H
H
N
O
N
NC
Cl
N
H
NC
Cl
N
H
R
N
CF3
Cl
Cl
3
2
1
Cl
MK-0364
SR141716
Figure 1.
tion while its role in weight control is not well understood. It is
desirable to maintain a high selectivity for CB1R when obesity
treatment is concerned to avoid possible complication in future
development. Chloropyridines 3b and 3c are slightly less active
in binding than 3a. Comparing 3a with 3c indicates that o-substi-
tution may result in reduced binding affinity. Moving the CF3 group
from 5-position to 4-position resulted in fivefold improvement to
give highly potent compound 3d (entry 5) that showed an excel-
lent IC50 of 1.6 nM. The CB1/CB2 selectivity is also significantly im-
proved. Apparently, the lipophilic CF3 is important for binding
affinity: as shown in 3e, changing CF3 to a less lipophilic CH3 re-
sulted in an IC50 of 37.7 nM, which is 24-fold less active than 3d.
Further reduction of the local lipophilicity by removing the methyl
group afforded even lower binding activity as shown by 3f. Chang-
ing pyridine to pyrimidine gave no improvement for binding to
CB1 receptor but the CB1/CB2 selectivity was significantly im-
proved as showed by 3g.
Cl
N
a
O
H
N
O
N
CF3
5
HO
NH2
HO
CF3
6
4
N
NC
NH2
HCl
O
H
N
N
7
N
Cl
H
CF3
b
3a
Cl
Scheme 1. Reagents and conditions: (a) DBU, dimethylsulfoxide, 150 °C, 44%; (b) N-
methylmorpholine, PyBop, 59%.
In addition to six-membered ring heterocycles, some five-mem-
bered heterocycles were also tested. As showed in entries 9 and 10,
isoxazoles 3h and 3i gave similar IC50 of 46.1 nM and 47.7 nM,
respectively. Following similar trends observed in the pyridine ser-
ies, an additional methyl group in 3j resulted in a twofold improve-
ment in binding. Increasing local lipophilicity by introducing a t-
butyl group resulted in further improved binding activity: 5-t-bu-
tyl oxazole 3k showed an IC50 of 8.6 nM. 5-Phenylisoxazole 3l,
however, gave a much lower binding activity of 186 nM. This sug-
gests that there may be some steric limitation in this part of bind-
ing pocket. 4-Methylpyrazole 3m and N-methylpyrazole 3n both
gave reduced binding affinity. Two fused heterocycles were also
tested and a similar trend was observed. N-Methylbenzimidazole
3o (entry 16) gave an IC50 of 97 nM, twofold improvement com-
pared to 3n that has a similar N-methyl group. Isoquinoline 3p (en-
try 17) gave an IC50 of 10 nM, more than threefold of improvement
compared to methylpyridine 3e. An amino alkyl group was tested
to replace the heterocycle moiety. As shown in entry 18, compound
3r that contains a dimethylaminopropyl group lost much of the
CB1 activity.
aminopyridine 6 in 44% yield. The acid was then coupled with
amine 7 to give compound 3a, which was less active than 2 but still
quite potent with an IC50 of 7.5 nM against CB1R. Encouraged by
this result, more compounds from the amino heterocycle series
were explored.
To prepare more amino heterocycle analogs, a more efficient
divergent synthesis was then developed as shown in Scheme 2.
Amine 7 was coupled with 2-bromo-2,2-dimethylacetic acid to
yield 2-bromo acetamide 8. This compound was used as a common
intermediate for other amino heterocycle analogs. To validate this
strategy, compound 3a was synthesized by this method. 2-Amino-
5-trifluoromethyl pyridine was first treated with NaH, followed by
compound 8 (conditions A). The reaction, however, gave a reversed
amide product 10a in 26% yield. A mechanism for the formation of
10a is outlined in Scheme 2. In the presence of a strong base, the
amide N–H was first deprotonated. The subsequent intramolecular
N-alkylation gave an aziridine lactam intermediate 9, which can be
opened by a nucleophile through either pathway a or b. Similar azi-
ridine lactam formation has been reported in the literature.9 Usu-
ally a ‘hard’ nucleophile takes pathway a to give the reversed
amide 10 while a ‘soft’ nucleophile takes pathway b to give the de-
sired product 3. A solution of 2-amino-5-trifluoromethylpyridine
and 8 in CH2Cl2 was treated with 50% aqueous NaOH (conditions
B). Upon completion, the reaction afforded the desired isomer 3a
in 37% yield. These conditions B were then applied to other amino-
heterocycles and yielded compounds 3b–3p in 20–70% yields.
The inhibition of the human CB1 and CB2 receptors by com-
pounds 2, 3 and 10 are shown in Table 1. Compounds 3 are still
selective for CB1 receptor with respect to CB2 receptor. However,
in terms of CB1R binding affinity, compound 3a (entry 2) is 25-fold
less active than compound 2 with the simple change from oxygen
to nitrogen. Meanwhile, such replacement resulted in reduced
CB1/CB2 selectivity. CB2R is known to play a role in pain percep-
Interestingly, the structurally distinct reversed amides 10a and
10l also showed binding activity for CB1 receptor and were selec-
tive for CB1 with respect to CB2. Compound 10a gave an IC50 of
132 nM, 17-fold less active than 3a. The isoxazole 10l showed an
IC50 of 136 nM that was slightly more active than its counterpart
3l, and a higher CB1/CB2 selectivity than 3l. On the other hand,
the simple switch of the gem-dimethyl group and the carbonyl
group had a big impact on CYP activity. Compound 3a afforded
an IC50 = 2.03
lM for CYP 3A4 while compound 10a exhibited an
IC50 > 35 M for CYP 3A4. With the advantage of significantly im-
l
proved CYP activity, the reversed amide 10 may be a lead for the
future development of new CB1R agents.
Some of the compounds that showed good binding affinity were
tested for their functional activity by measuring the forskolin in-
duced intra-cellular cAMP level. The results are shown in Table 2.

W. Du et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5195–5199
5197
O
Br
O
HO
Br
NC
Cl
NH2
HCl
NC
Cl
N
H
a
7
8
b
H
N
O
Conditions A
pathway a
NC
N
NC
N
R
a
H
O
b
10
Cl
H2N
9
Cl
R
pathway b
Conditions B
O
H
N
NC
Cl
N
H
R
3
Scheme 2. Reagents and conditions: (a) Et3N, EDCI, 94%; (b) conditions A: NaH, THF; conditions B: 50% aqueous NaOH, Bu4NBr, CH2Cl2, 20–70%.
These four compounds from the amino heterocycle series also be-
haved as inverse agonists and showed functional activities that
were better or comparable to that of taranabant 2. Compound 3a
showed an EC50 of 3.2 nM. Compound 3d that showed the best
binding affinity also gave the best functional activity: an EC50 of
0.9 nM, 2.5-fold better than that of 2. Compounds 3k and 3p gave
EC50 of 3.5 nM and 1.9 nM, respectively. Thus, the amino heterocy-
cle compounds are still potent CB1R inverse agonists.
in the presence of UDPGA and analyzed by LC–MS/MS for the cor-
responding glucuronide.13 The results are shown in Table 3. The
aminopyridine series compounds afforded only trace amount of
glucuronide in human liver microsomes. The aminoxazole com-
pound formed slightly more glucuronide in both rat and human li-
ver microsomes but the amount was still minor based on LC–MS.
Compounds from the pyrimidine series were not tested in the liver
microsomal studies due to their low CB1R binding affinity. In gen-
eral, two criteria have to be met in order for a molecule to form a
glucuronide adduct. First, it must have a functional group that is
chemically capable of forming a glucuronide that is reasonably sta-
ble. Second, this functional group must be in an appropriate posi-
tion in the molecule where allows the formation of the
glucuronide. The latter criteria relates to the compound’s affinity
for the enzymes which catalyze glucuronidation (UGT’s). Pyridine
is known to form glucuronides, but compound 3f and other hetero-
cycles failed to form glucuronides in a meaningful amount. This
suggests that the right hand side of 3 may not be a favorable region
for glucuronidation.
The metabolism of compound 3a was also studied in both rat
and human cryopreserved hepatocytes. Similar to compound 2,
the aminopyridine 3a is still metabolized through oxidative path-
way by formation of metabolite with molecular weight of M+16,
which then forms the glucuronide adduct. The direct glucuronide
adduct of 3a was not observed in either study.
In conclusion, we developed a divergent synthesis of acyclic 2-
N-heterocyclicamino acetamide as human cannabinoid-1 receptor
(CB1R) inverse agonists. This method provided an efficient synthe-
sis of a variety of aminoheterocycle analogs of this family.
Although in general, replacing the oxygen in taranabant (2) with
nitrogen resulted in lower binding affinity, potent CB1R inverse
agonists were still identified from the aminoheterocycle series.
The most active compound from this series is more potent than 2
For further understanding of the binding of the aminoheterocy-
cle series, a modeling study of 3a was pursued. A bound conforma-
tion of 2 was established by overlaying representative conformers
of 2 with rimonabant. The active conformation of compound 3a
overlays well with that of 2. However, 3a requires more energy
(0.54 kcal/mol) than 2 to convert the lowest energy conformation
to active conformation. This is probably because that the NH moi-
ety in 3a tends to be coplanar with the pyridine moiety. Thus, it re-
quires extra energy to break this conjugation to form the active
conformation in which the NH moiety is not coplanar with the pyr-
idine moiety. The computational and docking studies of 2 (MK-
0364) with a CB1R homology model was recently reported.11 Same
homology model was applied here and binding mode is shown in
Figure 2. It was found that amino acid residue Lys192 is close to
the NH moiety of compound 3a. This may destabilize the binding
of 3a due to disfavored positive–positive electrostatic interaction
between the two amine moieties. That the amino moiety of 3a
may have disfavorable interaction on the binding site is also sup-
ported by that the calculated binding energy for compound 3a is
5.9 kcal/mol higher than that of MK-0364. Thus, the decreased
binding affinity of 3a may be due to a less favored effect of the ami-
no moiety on conformation and with the receptor binding site.
To determine whether compounds from the amino heterocycles
series are capable of forming glucuronide adducts, several com-
pounds were incubated with rat and human liver microsomes12

5198
W. Du et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5195–5199
Table 1
Table 1 (continued)
The CB1 and CB2 binding affinity of compounds 2, 3 and 1010
a
a
Entry
16
Compound
R
CB1 IC50
(nM)
CB2 IC50
(nM)
Selectivity
CB2/CB1
a
a
Entry
Compound
R
CB1 IC50
(nM)
CB2 IC50
(nM)
Selectivity
CB2/CB1
1
2
0.3
285
950
N
3o
97
10.1
2982
132
1414
117
15
12
8.7
13
17
N
N
2
3
3a
3b
3c
3d
3e
3f
7.5
322
43
CF3
N
17
18
19
3p
N
12.2
17.9
1.6
699
419
57
23
Cl
Cl
Cl
3r
25890
1721
2312
N
N
4
CF3
N
N
10a
CF3
5
353
221
15
CF3
N
N
O
20
10l
136
6
37.7
49
547
Ph
a
Binding affinity determined by inhibition of binding of [3H]CP-55940 to
recombinant human CB1 or CB2 receptors expressed on Chinese Hasmster Ovary
(CHO) cells. (n = 2).
N
N
7
1830
14020
3863
3213
5158
254
37
Table 2
The CB1 functional activity of 2 and selected compounds 310
a
8
3g
3h
3i
49.2
46.1
47.7
19.5
8.6
286
84
Entry
1
Compound
R
CB1 EC50 (nM)
2.4
N
2
N
2
3
4
3a
3d
3k
3p
3.2
0.9
3.5
1.9
Ο
9
N
CF3
N
N
10
11
12
13
14
15
67
O
O
O
CF3
N
O
N
N
3j
265
30
N
5
3k
3l
a
Functional activity was determined by measuring the cAMP level using aden-
ylyl cyclase activation flash plate assay following the procedure recommended by
the manufacture (n = 2).
N
O
186
301
203
234
1.3
Ph
in functional activity assay. Computational studies of 3a provided
some explanations for the reduced binding affinity caused by the
simple change from oxygen to nitrogen. Some representative
aminoheterocycle compounds were tested in rat and human liver
microsomal incubation, where it was found that glucuronides were
only formed in trace amounts. Thus it suggested that the right
hand side of the molecule may not be an appropriate site to intro-
duce phase II metabolism. Further studies on the 2nd generation of
N
NH
N
3m
3n
6113
13290
20
N
65

W. Du et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5195–5199
5199
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Marsh, D. J.; Stribling, D. S.; Shearman, L. P.; Strack, A. M.; MacIntyre, D. E.;
Van der Ploeg, L. H. T.; Goulet, M. T. Bioorg. Med. Chem. Lett. 2006, 16, 681;
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Shen, C.-P.; Lao, J.; Kumar, S.; Yin, W.; Payack, J. F.; Springfield, S. A.;
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K.; Stoch, A.; Wagner, J. J. Clin. Pharmacol. 2008, 48, 418; (d) Addy, C.;
Wright, H.; Van Laere, K.; Gantz, I.; Erondu, N.; Musser, B. J.; Lu, K.; Yuan, J.;
Sanabria-Bohorquez, S. M.; Stoch, A.; Stevens, C.; Fong, T. M.; De Lepeleire, I.;
Cilissen, C.; Cote, J.; Rosko, K.; Gendrano, I. N., III; Nguyen, A. M.; Gumbiner,
B.; Rothenberg, P.; de Hoon, J.; Bormans, G.; Depre, M.; Eng, W.; Ravussin, E.;
Klein, S.; Blundell, J.; Herman, G. A.; Burns, H. D.; Hargreaves, R. J.; Wagner,
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Fabre, G. Drug Metab. Disp. 2008, 36, 851; (c) Yan, Z.; Caldwell, G. W.; Gauthier,
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Figure 2. The binding mode of 3a in the CB1 homology model.
Table 3
Glucuronide formation studies of 3d, 3f, 3g in rat and human liver microsomes
Entry
Compounds
In rat liver microsome
In human liver microsome
1
2
3
3d
3f
3g
Not observed
Not observed
Trace
Trace
Trace
Trace
CB1R inverse agonist from the acyclic amide scaffold will be
reported in due courses.
Acknowledgment
We thank Dr. Joseph M. Laquidara for providing the intermedi-
ate 7.
10. Shen, C.-P.; Xiao, C. J.; Amstrong, H.; Hagmann, W. K.; Fong, T. M. Eur. J.
Pharmacol 2006, 531, 41.
11. Lin, L. S.; Ha, S.; Ball, R. G.; Tsou, N. N.; Castonguay, L. A.; Doss, G. A.; Fong, T. M.;
Shen, C.-P.; Xiao, J. C.; Goulet, M. T.; Hagmann, W. K. J. Med. Chem. 2008, 51,
2108.
12. Rat and human liver microsomes were prepared in house as outlined in the
literature: Lu, A. Y.; Levin, W. Biochem. Biophys. Res. Commun. 1972, 46, 1334.
13. Incubation of the compound with hepatic microsomes: compound (10
lM)
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was incubated with rat or human liver microsomes (1 mg/mL protein) in
potassium phosphate buffer (100 mM, pH 7.4) containing MgCl2 (10 mM),
alamethicine (10 lg/mg protein, Sigma), D-saccharic acid 1,4-lactone (5 mM,
Sigma) and uridine 50-diphosphoglucuronic acid trisodium salt (UDPGA, 3 mM,
sigma). The incubation was carried out at 37 °C in a shaking water bath for
90 min, terminated using two volumes cold acetonitrile, vortexed, and
centrifuged at 10,000 g for 10 min. The supernatant was analyzed via LC–MS/
MS.