Synthesis and structure-activity relationships of biarylcarboxamide bis-aminopyrrolidine urea derived small-molecule antagonists of the melanin-concentrating hormone receptor-1 (MCH-R1)

Article abstract of DOI:10.1016/j.bmcl.2005.05.015

A novel series of bis-aminopyrrolidine ureas containing either a 4-biphenylcarboxmide or 5-phenyl-2-thiophenecarboxamide group have been identified as potent and functional antagonists of the melanin-concentrating hormone receptor-1. Syntheses and SAR are

Full text of DOI:10.1016/j.bmcl.2005.05.015

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3439–3445
Synthesis and structure–activity relationships
of biarylcarboxamide bis-aminopyrrolidine urea derived
small-molecule antagonists of the melanin-concentrating
hormone receptor-1 (MCH-R1)
Martin W. Rowbottom,a,* Troy D. Vickers,a Brian Dyck,a Junko Tamiya,a
Mingzhu Zhang,a Liren Zhao,a Jonathan Grey,a David Provencal,b David Schwarz,c
Christopher E. Heise,d Monica Mistry,d Andrew Fisher,e Teresa Dong,e Tao Hu,a
John Saundersa and Val S. Goodfellowa
aDepartment of Medicinal Chemistry, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA
bDepartment of Process Chemistry, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA
cDepartment of Molecular Biology, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA
dDepartment of Pharmacology, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA
eDepartment of Preclinical Development, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA
Received 26 February 2005; revised 27 April 2005; accepted 3 May 2005
Available online 9 June 2005
Abstract—A novel series of bis-aminopyrrolidine ureas containing either a 4-biphenylcarboxmide or 5-phenyl-2-thiophenecarbox-
amide group have been identified as potent and functional antagonists of the melanin-concentrating hormone receptor-1. Syntheses
and SAR are described, which led to the discovery of compounds with high binding affinity (Ki = 1 nM) for the receptor. Prelimin-
ary in vitro metabolic stability data are also reported for key compounds.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Mammalian melanin-concentrating hormone (MCH) is
a 19 amino acid cyclic peptide, which was first isolated
from the brain tissue of rats.1 Subsequently, it was dis-
covered that in humans, MCH selectively binds and acti-
vates two G protein-coupled receptors, MCH-R1 and
MCH-R2.2 In the rat CNS, MCH peptide is strongly
expressed within the lateral hypothalamus and the
distribution of MCH-R1 strongly correlates to the
expression of MCH peptide.2,3 It is well known that
the lateral hypothalamus plays a central role in the con-
trol of energy homeostasis, feeding behavior, and body
weight. Indeed, there is mounting evidence suggesting
that in rodents, MCH and the MCH-R1 receptor play
a pivotal role in these processes. In rodents, increased
brain levels of the MCH peptide lead to stimulation of
food intake, obesity, and insulin resistance,4 which is be-
lieved to occur through activation of the MCH-R1
receptor.5 It has also been observed that mice lacking
MCH are both hypophagic and lean.4 Interestingly,
MCH-R1 deficient mice (mchr1À/À) are lean, hyper-
phagic, have altered metabolism, and are resistant to
diet-induced obesity, suggesting that MCH and the
MCH-R1 receptor have a role in controlling metabolic
processes as well as regulation of appetite.4 Therefore,
the development of selective MCH-R1 antagonists
may be useful for the treatment of obesity and related
diseases. Both peptide6 and small-molecule antagonists7
of the MCH-R1 receptor have been reported to suppress
MCH-stimulated food intake in rodents. In addition, re-
cent studies suggest that MCH may be involved in the
regulation of stress behavior,4,8 and it was discovered
that potent and selective small-molecule antagonists of
the MCH-R1 receptor displayed anxiolytic and antide-
pressant properties in animal models.7b,8b It is therefore
not surprising that there is currently great interest in the
development of small-molecule MCH-R1 receptor
Keywords: Melanin-concentrating hormone; MCH; MCH antagonists;
Obesity.
*
Corresponding author. Tel.: + 1 858 617 7799; fax: +1 858 617
7601; e-mail: mrowbottom@neurocrine.com
URL: http://www.neurocrine.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.015

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M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3439–3445
antagonists, which hold the potential to treat both obes-
ity and stress related disorders.7,8b,9
with di-tert-butyl-dicarbonate to give N-Boc protected
(S)-3-(methylamino)pyrrolidine-1-carboxylic acid [(R)-
1-benzylpyrrolidin-3-yl]methylamide 5 in 68% yield
(from 4), as a single diastereoisomer.11 N-Debenzyla-
tion, followed by reductive alkylation with formalde-
hyde, gave N-Boc protected (S)-3-[(N)-methylamino]
pyrrolidine-1-carboxylic acid [(R)-1-methylpyrrolidin-3-
yl]methylamide 6 in 89% yield. N-Boc deprotection (of
6), followed by coupling with either 4-iodobenzoic acid
or 5-bromothiophene-2-carboxylic acid, gave the key
intermediates 4-iodophenylcarboxamide 7 (in 83% yield)
and 5-bromo-2-thiophenecarboxamide 8 (in 77% yield),
respectively. The reaction of 4-iodophenylcarboxamide
7 or 5-bromo-2-thiophenecarboxamide 8 with a variety
of substituted phenylboronic acids (via a Suzuki cou-
pling) gave the desired 4-biphenylcarboxamides 9–11
and 5-phenyl-2-thiophenecarboxamides 12–42, respec-
tively (Scheme 2 and Tables 1–3).12 All biarylcarboxa-
mide bis-aminopyrrolidine ureas described were
assayed for their ability to displace radiolabeled
As part of our efforts to develop novel medicines fo-
cused on the treatment of CNS disorders, we initiated
research toward discovery of orally active small-mole-
cule MCH-R1 antagonists. Previously, we discovered
that bis-aminopyrrolidine urea 1 (Fig. 1), containing
both an N-3-(4-chlorophenyl)propyl chain and a 4-(4-
trifluoromethyl)biphenyl substituted methylcarboxa-
mide, was a potent and functional antagonist of the
MCH-R1 receptor.9i Unfortunately, the in vitro meta-
bolic stability (in human liver microsomes) of bis-
aminopyrrolidine urea 1 proved to be low, suggesting
that it would be rapidly cleared in vivo. This may be
partly due to the molecule having a relatively high logP
(for compound 1, the calculated logP is 6.4).10 Removal
of the large 3-(4-chlorophenyl)propyl side chain would
result in analogs with a reduced log P, in the range of
2–3 log units. We therefore explored a series of bis-
aminopyrrolidine ureas containing an N-methyl substi-
tuent (represented by 2, Fig. 1). These compounds
proved to be potent antagonists of the MCH-R1 recep-
tor with improved drug-like properties. In this letter, we
wish to report the synthesis and SAR of this novel class
of small-molecule MCH-R1 antagonists. In particular,
changes around the biarylcarboxamide portion of the
molecule will be described and the resulting SAR will
be discussed, along with preliminary metabolic stability
data for this series.
[
125I-Tyr13]MCH in a competitive binding assay.13 The
assay was performed using a human MCH-R1 receptor
that is modified for optimal expression in HEK293 cells.
Selected results are summarized in Tables 1–3. The func-
tional antagonism of all compounds with measured KiÕs
less than 50 nM was further confirmed based on their
ability to inhibit, in a dose-dependent manner, MCH
stimulated G protein–[35S]–GTPcS binding in cells
expressing the native human MCH-R1 receptor (as rep-
resented by compound 35, Fig. 2).14 In addition, the
compounds described were shown to be inactive against
human MCH-R215 and optimized compounds exhibited
good selectivity when assayed against a standard panel
of GPCRs, including those of the serotonin, dopamine,
opioid, and muscarinic family of receptors.16
All biarylcarboxamide bis-aminopyrrolidine urea deriv-
atives 2 were synthesized from the Boc-protected core 6,
which was prepared as outlined in Scheme 1. Reaction
of commercially available (R)-(À)-1-benzyl-3-(methyla-
mino)pyrrolidine with 4-nitrophenylchloroformate gave
(R)-1-benzyl-3-[(4-nitrophenoxycarbonyl)methylamino]-
pyrrolidinium hydrochloride 3 in 83% yield. Reaction of
pyrrolidinium salt 3 with commercially available (R)-
(À)-3-pyrrolidinol hydrochloride in the presence of
TEA gave pyrrolidinol urea 4 in 77% yield. The hydrox-
yl group of 4 was activated using 4-nitrophenylsulfonyl
chloride to give the nosylate intermediate, which was
then displaced by methylamine, followed by reaction
Bis-aminopyrrolidine ureas containing a 4-biphenyl-
carboxamide group (see compounds 9–11, Table 1) are
very potent antagonists of the MCH-R1 receptor. It
was observed that the terminal phenyl ring could either
be electron rich or electron deficient (see compounds 9–
11). Not surprisingly, when the central phenyl ring was
replaced with a thiophene ring, potency was maintained
(see compounds 12–15, Table 1). Incorporation of a
methyl substituent ortho to the biaryl bond did not
significantly affect potency in the case of the 5-phenyl-
2-thiophenecarboxamide derivatives (compare com-
pounds 14 and 15), but potency was slightly decreased
in the case of the 4-biphenyl derivatives. The 4-(4-meth-
oxy-2-methylphenyl)phenylcarboxamide derivative 11
had a Ki of 16 nM, which was approximately 3-fold less
potent than the corresponding des-methyl analog 10
(Ki = 5 nM). We also explored the effect of changing
the absolute configuration of the two stereocenters, as
summarized in Table 2. The three other diastereoiso-
mers of 5-[4-(trifluoromethyl)phenyl]-2-thiophenecarb-
oxamide derivative 12 were prepared (see compounds
16–18, Table 2).17 The (S,R) diasteroisomer 12 proved
to be the most potent with a measured Ki of 2 nM.
The (S,S) diasteroisomer 16 was only 7-fold less potent
(Ki = 14 nM), whereas both the (R,S) (compound 17)
and (R, R) (compound 18) diasteroisomers were at least
75-fold less active.
Figure 1. General structures of bis-aminopyrrolidine urea MCH-R1
antagonists.

M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3439–3445
3441
Scheme 1. Reagents and conditions: (a) 4-nitrophenyl chloroformate, THF, 5 ꢁC to rt, 3 h, 83%; (b) (R)-(À)-3-pyrrolidinol hydrochloride, TEA,
DMF, 70 ꢁC, 18 h, 77%; (c) 4-nitrobenzenesulfonyl chloride, TEA, THF, 5 ꢁC to rt, 18 h; (d) methylamine, THF/H2O, 50 ꢁC, 18 h; (e) (Boc)2O, THF,
rt, 30 min, 68% (from 4); (f) H2 (40 psi), Pd(OH)2/C, EtOH, rt, 17 h, quantitative; (g) formaldehyde, NaBH(OAc)3, 1,2-DCE/H2O, rt, 4 h, 89%; (h)
TFA, DCM, rt, 1 h, quantitative; (i) 4-iodobenzoic acid, EDC, HOBT, TEA, DCM, rt, 20 h, 83%; (j) 5-bromothiophene-2-carboxylic acid, EDC,
HOBT, TEA, DCM, rt, 20 h, 77%.
Scheme 2. Reagents and conditions: (a) ArB(OH)2, Pd(PPh3)4, Na2CO3, 1,4-dioxane/H2O, reflux, 12 h.
Using 5-[4-(trifluoromethyl)phenyl]-2-thiophenecarbox-
amide bis-aminopyrrolidine urea 12 as a lead structure,
substitution around the terminal phenyl ring was
explored and selected results are summarized in
Table 3. Moving the trifluoromethyl group to the 3-po-
sition of the phenyl ring led to a dramatic loss in po-
tency. The 3-(trifluoromethyl)phenyl derivative 19 had
a Ki of 1,800 nM and was approximately 900-fold less
potent than the 4-(trifluoromethyl)phenyl analog 12
(Ki = 2 nM; see Table 1). The 2-(trifluoromethyl)phenyl
derivative 20 (Ki of 61 nM) also proved to be less active
than the 4-substituted analog 12. It was generally ob-
served that compounds containing a meta-substituted
phenyl ring were not well tolerated by the receptor.
For instance, both the 3-chlorophenyl 21 (Ki = 320 nM)
and 3-methoxyphenyl 23 (Ki = 900 nM) derivatives were
much less active than their corresponding 4-substituted
analogs 13 (Ki = 2 nM) and 14 (Ki = 4 nM), respectively
(see Table 1). Interestingly, the 2-chlorophenyl deriva-
tive 22 had a Ki of 10 nM, which proved to be 6-fold
more potent than the 2-(trifluoromethyl)phenyl deriva-
tive 20 and over 300-fold more potent than the corre-
sponding 2-methoxyphenyl analog 24 (Ki = 3,400 nM).
In addition, the 2-methylphenyl derivative 26 had a Ki
of 19 nM, whereas the 2,6-dimethylphenyl derivative
28 was 16-fold less potent (Ki of 310 nM). This shows
that compounds containing a 2,6-disubstituted phenyl
are less well tolerated by the receptor. This may be
due to a certain steric interaction or that the phenyl ring
is forced to adopt an unfavorable conformation (relative
to the adjacent thiophene). Importantly, the 5-(2,4-di-
methylphenyl)-2-thiophenecarboxamide derivative 27
had a Ki of 3 nM, this being 6-fold more potent than
the 2-methylphenyl analog 26. Together these results
emphasize the importance of para-substitution (on the
phenyl ring) for optimal receptor binding and a more
detailed study to fully explore the SAR of this key sub-
stituent was carried out.

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M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3439–3445
Table 3. Binding affinities of bis-aminopyrrolidine ureas 19–42 toward
the MCH-R1 receptor
Table 1. Binding affinities of bis-aminopyrrolidine ureas 9–15 toward
the MCH-R1 receptor
Compound
R1
Ki (nM)a
Compound
Ar
Ki (nM)a
9
2
19
1,800
10
11
12
13
14
15
5
16
2
20
21
22
23
24
25
26
27
28
29
61
320
10
2
900
4
3,400
3
3
a Data are average of three or more independent measurements.13
19
Table 2. Binding affinities of bis-aminopyrrolidine ureas 12, 16–18
toward the MCH-R1 receptor
3
310
6,000
Compound
a,b
Ki (nM)a
Optical purity (%)b
12
16
17
18
S,R
S,S
R,S
R,R
2
14
>97
>97
>97
>97
190
150
a Data are average of three or more independent measurements.13
b Experimentally determined.17
(continued on next page)

M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3439–3445
3443
Table 3 (continued)
Compound
Ar
Ki (nM)a
30
31
32
33
34
35
36
37
38
39
40
41
42
2,400
>10,000
Figure 2. Inhibition of MCH (10 nM) stimulated G protein–[35S]–
GTPcS binding, by compound 35. Compound 35 has a measured IC50
of 14 nM (n = 3).14
7
Incorporation of relatively polar or bulky functionality
at the 4-position of the phenyl ring had a detrimental ef-
fect on potency, though the electronic nature of this sub-
stituent was not important, as compounds containing
either an electron rich or electron deficient phenyl ring
were well tolerated by the receptor. The 4-(methylsulfo-
nyl)phenyl 29, 4-cyanophenyl 30, and 4-hydroxyphenyl
31 derivatives (Table 3) had KiÕs of 6000, 2400, and
>10,000 nM, respectively, these substituents being too
polar in nature. Derivatives incorporating more lipo-
philic substituents proved to be much more potent.
For instance, compounds 32, 33, and 35 had KiÕs of 7,
4, and 1 nM, respectively, a level of activity not wit-
nessed with the slightly less lipophilic 4-fluorophenyl
analog 34. Indeed the 4-ethylphenyl derivative 35
proved to be one of the most potent analogs prepared
in this series. With the introduction of the slightly larger
isopropyl group (compound 36, Ki = 5 nM) potency was
maintained. However, introduction of progressively lar-
ger groups led to a noticeable reduction in potency
(compare compound 14 with both compounds 37 and
38). Potency may be improved by addition of a suitable
substituent ortho to the biaryl bond. For instance, the
2-chloro-4-ethoxyphenyl derivative 42 had a Ki of
2 nM and was approximately 30-fold more potent than
the 4-ethoxyphenyl analog 37 (Ki = 63 nM). This sug-
gests that the 2-chloro substituent may help to orient
the phenyl ring into a particular conformation (relative
to the adjacent thiophene), which may be preferred for
optimal receptor binding. In addition, the 2,4-dichlor-
ophenyl 39, 4-chloro-2-methylphenyl 40, and 2-chloro-
4-methylphenyl 41 derivatives had KiÕs of 3, 3, and
1 nM, respectively. Together these data suggest that
the receptor binding pocket in which the phenyl (of
the 5-phenyl-2-thiophenecarboxamide moiety) sits is rel-
atively tight and lipophilic in nature. A 4-substituted or
2,4-disubstituted phenyl is preferred, whereas 3-substitu-
ents are not well tolerated. In addition, the presence of
either a relatively polar or bulky substituent (at the 2-
or 4-position) tends to reduce binding affinity with the
receptor.
4
37
1
5
63
3,300
3
3
1
2
Ultimately our focus was on the discovery of orally ac-
tive small-molecule antagonists of the MCH-R1 recep-
tor, so we were particularly interested in the PK
profile of this novel series of compounds. Incubation
with human liver microsomes (HLM) can be used to
estimate how much of a target compound will be re-
a Data are average of three or more independent measurements.13

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M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3439–3445
6. Shearman, L. P.; Camacho, R. E.; Stribling, D. S.; Zhou,
D.; Bednarek, M. A.; Hreniuk, D. L.; Feighner, S. D.;
Tan, C. P.; Howard, A. D.; Van der Ploeg, L. H. T.;
MacIntyre, D. E.; Hickey, G. J.; Strack, A. M. Eur. J.
Pharmacol. 2003, 475, 37.
7. (a) Takekawa, S.; Asami, A.; Ishihara, Y.; Terauchi, J.;
Kato, K.; Shimomura, Y.; Mori, M.; Murakoshi, H.;
Kato, K.; Suzuki, N.; Nishimura, O.; Fujino, M. Eur. J.
Pharmacol. 2002, 438, 129; (b) Borowsky, B.; Durkin, M.
M.; Ogozalek, K.; Marzabadi, M. R.; DeLeon, J.;
Heurich, R.; Lichtblau, H.; Shaposhnik, Z.; Daniewska,
I.; Blackburn, T. P.; Branchek, T. A.; Gerald, C.; Vaysse,
P. J.; Forray, C. Nat. Med. 2002, 8, 825.
8. (a) Kela, J.; Salmi, P.; Rimondini-Giorgini, R.; Heilig, M.;
Wahlestedt, C. Regul. Pept. 2003, 114, 109; (b) Kennedy,
A. R.; Todd, J. F.; Dhillo, W. S.; Seal, L. J.; Ghatei, M.
A.; OÕToole, C. P.; Jones, M.; Witty, D.; Winborne, K.;
Riley, G.; Hervieu, G.; Wilson, S.; Bloom, S. R. J.
Neuroendocrinol. 2003, 15, 268.
moved by hepatic first-pass metabolism in vivo. For in-
stance in an in vitro HLM assay,18 5-(4-ethylphenyl)-2-
thiophenecarboxamide derivative 35 (Table 3) had an
intrinsic clearance of 95 mL/min/kg, implying that this
compound is being rapidly metabolized. Other examples
proved to be much more stable to metabolism by HLM.
For instance, the 2-chloro-4-methylphenyl 41 and 2-
chloro-4-ethoxyphenyl 42 analogs (Table 3) had intrin-
sic clearance values of 19 and 6 mL/min/kg, respectively.
Inhibition of certain cytochrome P450 (CYP450) metab-
olizing enzymes (particularly CYP2D6 and CYP3A4)
may lead to undesirable drug–drug interactions in vivo.
The compounds described proved to be very weak inhib-
itors of both CYP2D6 and CYP3A4. For instance, in vi-
tro the aforementioned compounds 35, 41, and 42 all
had measured IC50 values >20 lM, against both
CYP2D6 and 3A4.19
9. (a) Carpenter, A. J.; Hertzog, D. L. Expert Opin. Ther.
Patents 2002, 12, 1639; (b) Collins, C. A.; Kym, P. R.
Curr. Opin. Investig. Drugs 2003, 4, 386; (c) Clark, D. E.;
Higgs, C.; Wren, S. P.; Dyke, H. J.; Wong, M.; Norman,
D.; Lockey, P. M.; Roach, A. G. J. Med. Chem. 2004,
47, 3962; (d) Arienzo, R.; Clark, D. E.; Cramp, S.; Daly,
S.; Dyke, H. J.; Lockey, P.; Norman, D.; Roach, A. G.;
Stuttle, K.; Tomlinson, M.; Wong, M.; Wren, S. P.
Bioorg. Med. Chem. Lett. 2004, 14, 4099; (e) Souers, A.
J.; Wodka, D.; Gao, J.; Lewis, J. C.; Vasudevan, A.;
Gentles, R.; Brodjian, S.; Dayton, B.; Ogiela, C. A.; Fry,
D.; Hernandez, L. E.; Marsh, K. C.; Collins, C. A.;
Kym, P. R. Bioorg. Med. Chem. Lett 2004, 14, 4873; (f)
Vasudevan, A.; Wodka, D.; Verzal, M. K.; Souers, A. J.;
Gao, J.; Brodjian, S.; Fry, D.; Dayton, B.; Marsh, K. C.;
Hernandez, L. E.; Ogiela, C. A.; Collins, C. A.; Kym, P.
R. Bioorg. Med. Chem. Lett. 2004, 14, 4879; (g) Souers,
A. J.; Wodka, D.; Gao, J.; Lewis, J. C.; Vasudevan, A.;
Gentles, R.; Brodjian, S.; Dayton, B.; Ogiela, C. A.; Fry,
D.; Hernandez, L. E.; Marsh, K. C.; Collins, C. A.;
Kym, P. R. Bioorg. Med. Chem. Lett. 2004, 14, 4883; (h)
Receveur, J.-M.; Bjurling, E.; Ulven, T.; Little, P. B.;
Nørregaard, P. K.; Ho¨gberg, T. Bioorg. Med. Chem.
Lett. 2004, 14, 5075; (i) Grey, J.; Dyck, B.; Rowbottom,
M. W.; Tamiya, J.; Vickers, T. D.; Zhang, M.; Zhao, L.;
Heise, C. E.; Schwarz, D.; Saunders, J.; Goodfellow, V.
S. Bioorg. Med. Chem. Lett. 2005, 15, 999; (j) Su, J.;
McKittrick, B. A.; Tang, H.; Czarniecki, M.; Greenlee,
W. J.; Hawes, B. E.; OÕNeill, K. Bioorg. Med. Chem.
2005, 13, 1829.
In conclusion, we have discovered a novel series of func-
tional MCH-R1 antagonists based on an N-methylated
bis-aminopyrrolidine urea core, containing either a
4-biphenylcarboxamide or 5-phenyl-2-thiophenecarbox-
amide group. SAR around the 5-phenyl-2-thiophene-
carboxamide group was explored, where it was observed
that either a 4-substituted or 2,4-disubstituted phenyl ring
was preferred for optimal binding with the receptor. In
addition, examples from this series display promising in
vitro metabolic stability profiles, exhibiting good stability
in the HLM assay with no significant inhibition of key
CYP450 metabolizing enzymes. This new series proved
to be significantly more stable (in vitro) than previously
described first-generation bis-aminopyrrolidine urea
MCH-R1 antagonists. Further results, including in vivo
PK and efficacy data in animal models, will be reported
in due course.
Acknowledgments
We are indebted to Mr. John Harman, Mr. Chris De-
vore, and Mr. Shawn Ayube for LC-MS support, Mrs.
Mila Lagman for HPLC support, and Dr. Fabio C. Tuc-
ci and Dr. Warren Wade for technical advice. This work
was partly supported by NIH Grant 2-R44-DK059107-
02.
10. Calculated using ACD/Labs Log P database, version 6.0
(2002), Advanced Chemistry Development Inc., Toronto,
Ontario, Canada (http://www.acdlabs.com).
References and notes
11. The diastereomeric purity of compound 5 was >98%, as
measured by HPLC. This was determined by direct com-
parison to an equal mixture of both possible diastereomeric
products, N-Boc protected (R)- and (S)-3-[(N-)methylami-
no]pyrrolidine-1-carboxylic acid [(R)-1-benzylpyrrolidin-3-
yl]methylamide, which was prepared as described (Scheme
1) using commercially available racemic 3-pyrrolidinol
hydrochloride
12. Analytical data for a typical example, the hydrochloride
salt of 5-(4-trifluoromethylphenyl)-2-thiophenecarbox-
amide bis-aminopyrrolidine urea 12: 1H NMR dH
(300 MHz; CDCl3) 12.83 and 12.40 (2· br m, 1H), 7.73
(d, J = 8.3 Hz, 2H), 7.66 (d, J = 8.3 Hz, 2H), 7.38 (m, 1H),
7.34 (d, J = 3.3 Hz, 1H), 5.04 (m, 1H), 4.76 and 4.17 (2·
m, 1H), 3.82 (m, 1H), 3.41–3.63 (m, 6H), 3.27 (m, 1H),
3.19 (s, 3H), 2.74–2.98 (m, 7H), 2.52 (m, 1H), 2.12–2.33
(m, 2H); MS (CI) m/z: 495.0 (M+H); Anal. Calcd for
1. Vaughan, J. M.; Fischer, W. H.; Hoeger, C.; Rivier, J.;
Vale, W. Endocrinology 1989, 125, 1660.
2. Boutin, J. A.; Suply, T.; Audinot, V.; Rodriguez, M.;
`
Beauverger, P.; Nicolas, J.-P.; Galizzi, J.-P.; Fauchere, J.-
L. Can. J. Physiol. Pharmacol. 2002, 80, 388, and
references cited therein.
3. For binding sites within the human brain, see, Sone, M.;
Takahashi, K.; Murakami, O.; Totsune, K.; Arihara, Z.;
Satoh, F.; Sasano, H.; Ito, H.; Mouri, T. Peptides 2000,
21, 245.
4. Hervieu, G. Expert Opin. Ther. Targets 2003, 7, 495, and
references cited therein.
5. Suply, T.; Della-Zuana, O.; Audinot, V.; Rodriguez, M.;
Beauverger, P.; Duhault, J.; Canet, E.; Galizzi, J.-P.;
Nahon, J.-L.; Levens, N.; Boutin, J. A. J. Pharmacol. Exp.
Ther. 2001, 299, 137.

M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3439–3445
3445
C24H29F3N4O2S–HCl–H2O: C, 52.50; H, 5.87; N, 10.20; S,
5.84. Found: C, 52.39; H, 6.22; N, 10.26; S, 6.23.
16. Cross-reactivity assays were performed by CEREP
(www.cerep.com). For example, compound 12 displayed
>500-fold selectivity when assayed against more than 75
GPCR receptors.
17. Each diastereoisomer was prepared from commercially
available optically pure building blocks. Measured by
reverse-phase chiral HPLC (at 254 nm), the optical purity
of each was determined to be >97% (when directly
compared to a mixture of all four diastereoisomers).
Analytical conditions: ChiralpakꢂADTM-RH column,
dimensions 0.46 · 15 cm. Flow rate of 0.5 mL/min, isocratic
elution with acetonitrile/water (9:1) (containing 0.1%
diethylamine).
18. General experimental details for this assay may be found
in the following reference. Guo, Z.; Zhu, Y.-F.; Gross, T.
D.; Tucci, F. C.; Gao, Y.; Moorjani, M.; Connors, P. J.;
Rowbottom, M. W.; Chen, Y.; Struthers, R. S.; Xie, Q.;
Saunders, J.; Reinhart, G.; Chen, T. K.; Bonneville, A. L.
K.; Chen, C. J. Med. Chem. 2004, 47, 1259.
19. Inhibition assays were carried out using microsomes
isolated from transfected cells expressing only the
relevant CYP450 isoform (2D6 or 3A4) and in the
presence of a fluorescent substrate (AMMC for 2D6 and
BFC for 3A4). Quinidine and ketoconazole were used as
positive controls for the CYP2D6 and 3A4 assays,
respectively.
13. On each assay plate, a standard antagonist of compa-
rable affinity to those being tested was included as a
control for plate-to-plate variability. Overall Ki values
were highly reproducible with an average standard
deviation of <45% for replicate determinations. All
compounds described were assayed in at least three
independent experiments.
14. Assays were performed using membrane preparations of
CHO cells stably expressing the human MCH-R1 recep-
tor. Each experiment was run in the presence of MCH
peptide (10 nM), [c-35S]GTP (0.5 nM), and GDP (10 lM).
On each assay plate, a standard antagonist of comparable
IC50 to those being tested was included as a control for
plate-to-plate variability. Overall IC50 values were highly
reproducible with an average standard deviation of <45%
for replicate determinations. All key compounds
(Ki < 50 nM) were assayed in at least three independent
experiments. In addition, representative compounds from
this series behaved as functional antagonists in a FLIPR
based Ca2+ flux assay, using cells stably expressing the
hMCH-R1 receptor.
15. For example, in an hMCH-R2 binding assay compound
12 had a measured IC50 > 100 lM and was inactive in a
corresponding Ca2+ flux functional assay.