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

Article abstract of DOI:10.1016/j.bmcl.2006.06.045

The design, synthesis, and SAR of a series of retro bis-aminopyrrolidine ureas are described. Compounds from this series exhibited potent binding affinity and functional activity at MCH-R1, and good oral bioavailability in rat.

Full text of DOI:10.1016/j.bmcl.2006.06.045

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4450–4457
Synthesis and structure–activity relationships of retro bis-
aminopyrrolidine urea (rAPU) derived small-molecule antagonists
of the melanin-concentrating hormone receptor-1
(MCH-R1). Part 1
Martin W. Rowbottom,a,* Troy D. Vickers,a Brian Dyck,a Jonathan Grey,a
Junko Tamiya,a Mingzhu Zhang,a Mehrak Kiankarimi,a Dongpei Wu,a
Wesley Dwight,a Warren S. Wade,a David Schwarz,b Christopher E. Heise,c
Ajay Madan,d Andrew Fisher,d Robert Petroskie and Val S. Goodfellowa,*
aDepartment of Medicinal Chemistry, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA
bDepartment of Molecular Biology, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA
cDepartment of Pharmacology, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA
dDepartment of Preclinical Development, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA
eDepartment of Neuroscience, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA
Received 4 May 2006; revised 12 June 2006; accepted 12 June 2006
Available online 30 June 2006
Abstract—The design, synthesis, and SAR of a series of retro bis-aminopyrrolidine ureas are described. Compounds from this series
exhibited potent binding affinity and functional activity at MCH-R1, and good oral bioavailability in rat.
ꢀ 2006 Published by Elsevier Ltd.
Mammalian melanin-concentrating hormone (MCH) is
a 19 amino acid cyclic peptide, first isolated from the
brain tissue of rats.1 It has since been shown that in
mammals, expression of MCH occurs throughout the
brain, though particularly high concentrations are found
in the lateral hypothalamus,2 an area known to be
involved in the control of feeding behavior. MCH selec-
tively binds and activates two 7-transmembrane G pro-
tein-coupled receptors, namely MCH-R1 and R2.2a
Although it is not yet clear what role MCH-R2 plays
in vivo, recent research suggests that MCH-R1 is
involved in the modulation of energy homeostasis, food
intake, and body weight. In rodents, increased brain
levels of MCH leads to increased food intake, obesity,
and insulin resistance.3 Conversely, mice lacking the
MCH peptide (mchꢀ/ꢀ) are lean and hypophagic,
whereas those deficient in MCH-R1 (mch1ꢀ/ꢀ) are
lean, hyperphagic, and resistant to diet-induced obesi-
ty.3 It has also been shown that administration of a po-
tent and selective small-molecule MCH-R1 antagonist
to rats leads to a significant reduction in food intake
and/or weight loss in these animals.4 These and other
data suggest that in man, blockade of MCH-R1 may
lead to a clinical treatment for chronic obesity, and a
number of groups have recently reported on their efforts
toward the development of selective small-molecule
MCH-R1 antagonists.4,5
Keywords: Melanin-concentrating hormone (MCH); Melanin-concen-
trating hormone receptor (MCH-R1); Melanin-concentrating hor-
mone receptor antagonists (MCH-R1 antagonists); Obesity;
Aminopyrrolidine urea; Retro aminopyrrolidine urea.
We recently disclosed the discovery and SAR of a series
of bis-aminopyrrolidine urea (APU) derived small-
molecule MCH-R1 antagonists, exemplified by
1
(Fig. 1).5i,k APU 1 is a potent functional antagonist of
MCH-R1, but exhibits low metabolic stability in an in vi-
tro human liver microsome (HLM) assay and had only
moderate oral exposure in rat.6 Metabolite profiling of
*
Corresponding authors. Tel.: +1 858 617 7600; fax: +1 858 617 7601
(M.W.R.); e-mail addresses: mrowbottom@neurocrine.com;
vgoodfellow@neurocrine.com
URL: http://www.neurocrine.com
0960-894X/$ - see front matter ꢀ 2006 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2006.06.045

M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4450–4457
4451
O
O
N
N
N
3S
Cl
N
Me
Me
3'R
1
F3C
O
O
O
N
Cl
N
N
NH
Me
Me
2a
2b
F3C
O
O
N
N
3
N
*
Me
Me
Me
N
H
3'
*
F3C
Cl
3a (3S ,3'R)
3b (3S, 3'S)
Figure 1. General structures of APU and rAPU MCH-R1 antagonists.
1 in HLM identified compound 2a as the major metab-
olite (Fig. 1), which probably occurs via oxidative deam-
ination of the right-hand pyrrolidine ring of 1 giving
3-pyrrolidinone in the process. We reasoned that rever-
sal of the connectivity of the bis-aminopyrrolidine ring
system to give the retro bis-aminopyrrolidine urea
(rAPU) core (exemplified by 3a and 3b, Fig. 1) might
prevent this metabolic pathway from occurring. This
could yield molecules with increased metabolic stability,
without adversely affecting potency at MCH-R1. In this
letter we report on the synthesis and SAR of this novel
series of small-molecule MCH-R1 antagonists and, in
particular, we describe basic SAR around both the
amine and biarylcarboxamide motifs, by reporting bind-
ing affinity data (Ki) for MCH-R1.
tion of a secondary amine.11 Compound 3b displayed a
very similar in vitro and in vivo profile to that described
for 3a. In light of the above findings we decided to fur-
ther explore the rAPU series.
In order to investigate the SAR around the basic nitro-
gen of the rAPU template, compounds 7–12 (Schemes 1,
2 and Table 1) were prepared incorporating both the
4-(4-trifluoromethylphenyl)phenylcarboxamide group
and the (3S,30R) stereoconfiguration, as employed in
the lead compound 3a. Compounds 8a–l and 10–12 were
obtained from primary amine 7a, whereas compounds
9a–j were obtained from secondary amine 7b. The syn-
thetic route employed for the preparation of both inter-
mediates (7a and 7b) is outlined in Scheme 1. Coupling
of commercially available (S)-1-benzyl-3-(methylami-
no)pyrrolidine12 with 4-(4-trifluoromethylphenyl)benzo-
yl chloride, followed by N-debenzylation, gave
pyrrolidine 4 in good yield. Carbamates 6a and 6b were
obtained directly via reaction of 4-nitrophenyl chloro-
formate with (R)-3-(tert-butoxycarbonyl)aminopyrrol-
idines 5a and 5b, respectively.13 Reaction of pyrrolidine
4 with either carbamate 6a or 6b in the presence of
N,N-4-dimethylaminopyridine in DMF gave, after
N-Boc deprotection, 7a and 7b, respectively. Both
coupled intermediates (N-Boc protected 7a and 7b) were
obtained in only modest yield due to formation of a mix-
ture of unidentified by-products. Subsequent N-alkyl-
ation of 7a and 7b gave compounds 3a, 8a–l, and 9a–j,
respectively.14 Compounds 10–12 were accessed from
compound 3a, as outlined in Scheme 2. All rAPUs
described herein (Tables 1 and 2) were obtained as single
diastereoisomers and were subsequently assayed for
their ability to displace radiolabeled [125I-Tyr13]-MCH
in a competitive binding assay.15 The assay was
performed using human MCH-R1 that is modified for
For the APU series, incorporation of a biarylcarboxa-
mide moiety was favored for optimal receptor binding.
In addition, of the four possible diastereoisomers the
(3S,30R) and (3S, 30S) configurations were preferred
(see 1, Fig. 1).5i,k The rAPU derivative 3a (3S,30R) is a
comparable analog to APU 1, and exhibited a somewhat
similar Ki value of 14 nM (n = 3), with the diastereoiso-
mer 3b (3S, 30S) being equipotent (Ki = 22 nM, n = 3).
The metabolic stability of 3a in human liver microsomes
did indeed show improvement, with a scaled predicted
intrinsic clearance of 19 mL/min/kg versus 60 mL/min/
kg for compound 1.7,8 In addition, compound 3a exhib-
ited greater oral bioavailability in rat, %F = 42,9 howev-
er brain exposure was low and needed to be addressed.
Compound
1
also showed strong inhibition
(IC50 = 170 nM) of the HERG potassium channel, in a
whole cell patch-clamp assay.10 Therefore, we were
pleased to discover that compound 3a showed signifi-
cantly less activity (IC50 = 2600 nM) in the HERG
patch-clamp assay, which may be due to the incorpora-

4452
M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4450–4457
O
NH
a, b
N
N
HN
Me
Me
O
O
N
4
F3C
d, e
N
N
Me
NH
R1
7a: R1 = H
F3C
tBuO
tBuO
c
7b: R1 = Me
O
O
O
NH
N
N
N
NO2
R1
R1
O
f
or
g
6a: R1 = H
6b: R1 = Me
5a: R1 = H
5b: R1 = Me
O
O
N
N
N
Me
R 2
N
R1
F3C
8: R1 = H
9: R1 = Me
Scheme 1. Reagents and conditions: (a) 4-(4-trifluoromethylphenyl)benzoyl chloride, TEA, DCM, rt, 12 h, quantitative; (b) H2 (40 psi), Pd(OH)2/C,
MeOH, rt, 2 h, 63%; (c) 4-nitrophenyl chloroformate, TEA, THF, 0 ꢁC to rt, 2 h, 83% (6a) and 90% (6b); (d) DMAP, TEA, DMF, 100 ꢁC, 12 h; (e)
For 7a: TFA, DCM, rt, 1 h, 38% (from 6a); for 7b: HCl, Et2O, DCM, rt, 12 h, 35% (from 6b); (f) aldehyde or ketone, NaCNBH3 or NaBH4, MeOH,
rt, 12 h, 20–60%; (g) alkyl bromide, NaI, K2CO3, DMF, 80 ꢁC, 3 h, 20–50%.
O
O
O
O
N
N
N
N
N
N
Me
Me
Me
Me
Me
Me
N
R1
N
H
F3C
F3C
Cl
Cl
10: R1 = Me
11: R1 = Et
12: R1 = Ac
3a
Scheme 2. Reagents and conditions: for 10: formaldehyde, NaCNBH3, MeOH, rt, 12 h, 61%; for 11: acetaldehyde, NaBH4, MeOH, rt, 12 h, 59%; for
12: acetyl chloride, TEA, DCM, rt, 12 h, 92%.
optimal expression in HEK293 cells. The functional
antagonism of all compounds with measured Kis less
than 50 nM was further confirmed based on their ability
to inhibit, in a dose-dependent manner, MCH-stimu-
lated G protein-GTPc35S binding in cells expressing na-
tive human MCH-R1.16
tion of a phenyl containing substituent in this region
of the molecule gave potent MCH compounds and
interestingly seemed to significantly reduce CYP2D6
activity. The phenethyl (8f), phenpropyl (8g), and 3-(4-
chlorophenyl)-3-methylbutyl (3a) derivatives had Kis of
17, 10, and 14 nM, respectively, and compounds 8g
and 3a had IC50s at CYP2D6 of 2000 and 3300 nM,
respectively. Saturation of the aryl ring did not adverse-
ly affect potency at the receptor as the cyclohexyl propyl
derivative 8e had a Ki of 22 nM, though incorporation
of relatively polar heteroaryl groups close to the basic
nitrogen, such as (5-methyl-2-furanyl)methyl (8i) or
(2-pyridyl)methyl (8j), was detrimental for receptor
potency.18 Combined, these results suggest that the re-
gion of the receptor surrounding this part of the mole-
cule is relatively open, lipophilic in nature, and prefers
relatively non-polar groups. In addition to secondary
amines, a number of tertiary amines were also prepared.
The N,N-dimethyl derivative (9a, Ki = 34 nM) was
approximately equipotent to the corresponding second-
The unsubstituted amine alone was reasonably potent
(compound 7a, Table 1), having a measured Ki of
51 nM. Addition of a methyl group (7b, Ki = 53 nM)
did not improve potency, though the introduction of
larger lipophilic substituents increased potency up to
5-fold. For instance, the cyclobutylmethyl (compound
8a, Ki = 11 nM), cyclohexylmethyl (8b, Ki = 12 nM),
and 4-methylcyclohexyl (8c, Ki = 13 nM) derivatives
were equipotent. Unfortunately, although molecules
such as 8b and 8c were very potent MCH-R1 antago-
nists, they also exhibited potent inhibition of the metab-
olizing enzyme CYP2D6. For example, compound 8c
inhibited CYP2D6 with an IC50 of 55 nM.17 The addi-

M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4450–4457
Table 1. Binding affinities of rAPUs 3a and 7–12 toward MCH-R1 Table 1 (continued)
4453
Compound
R1
R2
Ki (pKi)a (nM)
O
N
O
9bb
Me
Me
Me
Me
Me
Me
Me
Me
Et
54 (7.27 0.1)
38 (7.42 0.1)
28 (7.55 0.1)
24 (7.62 0.1)
42 (7.38 0.1)
51 (7.29 0.1)
29 (7.54 0.2)
33 (7.48 0.3)
N
2
R
1
N
(S)
N
Me
Me
(R)
9cb
9db
9eb
9fc
R
Me
F3C
Compound
R1
H
R2
Ki (pKi)a (nM)
Cl
3ab,d
14 (7.85 0.05)
Me
Me
7a
H
H
H
H
51 (7.29 0.1)
53 (7.27 0.05)
11 (7.98 0.2)
O
9gb
9hb
9ib
Me
7b
Me
O
8ac
N
Me
8bb
H
12 (7.91 0.2)
9jb
10
Me
Me
26 (7.59 0.1)
14 (7.84 0.05)
Me
O
8cb
8db
H
H
13 (7.88 0.2)
21 (7.68 0.2)
Cl
Cl
Cl
N
Me
Me
Me
11
12
Et
27 (7.57 0.1)
48 (7.32 0.1)
8eb
8fc
H
H
H
H
22 (7.66 0.2)
17 (7.78 0.2)
10 (8.01 0.05)
19 (7.73 0.2)
Me
Me
Me
Me
Ac
8gb
8hc
a Ki (pKi SEM, n = 3 or greater).15
b Prepared via a reductive alkylation as the last step.
c Prepared via a Finkelstein alkylation as the last step.
d Prepared from compound 7a and 3-(4-chlorophenyl)-3-methyl-
butyraldehyde.21
F
O
O
8ib
8jb
H
H
110 (6.96 0.2)
180 (6.74 0.1)
Me
ary amine 7b, and the addition of larger lipophilic
groups such as ethyl (9b, Ki = 54 nM) or isobutyl (9c,
Ki = 38 nM) did not improve potency. In most cases
we discovered that going from a secondary to tertiary
amine resulted in a reduction in binding affinity. For
instance, the N-methyl-(cyclobutylmethyl) derivative 9f
(Ki = 42 nM) was approximately 4-fold less potent than
N
N
8kc
8lc
H
40 (7.40 0.2)
16 (7.79 0.2)
34 (7.47 0.1)
the corresponding secondary amine 8a (with
a
Ki = 11 nM). An exception proved to be compound 10
(Ki = 14 nM), which was equipotent with the corre-
sponding secondary amine 3a. Unfortunately compound
10 proved to be very metabolically unstable, the
scaled predicted intrinsic clearance (in HLM) being
500 mL/min/kg, 25-fold greater than for 3a. Presumably
CF3
H
9ab
Me
Me

4454
M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4450–4457
Table 2. Binding affinities of rAPUs 16 toward MCH-R1
Table 2 (continued)
Compound
Ar
Ki (pKi)a (nM)
O
N
O
N
MeO
N
(S)
16o
16p
1000 (6.00 0.1)
N
Me
(S)
H
N
N
Ar
44 (7.36 0.1)
Compound
Ar
Ki (pKi)a (nM)
MeO
a Ki (pKi SEM, n = 3 or greater).15
16a
16b
H2N
630 (6.20 0.05)
O
N-demethylation occurs rapidly, giving the correspond-
ing secondary amine 3a. Replacement of the N-methyl
group of 10 with either ethyl (11, Ki = 27 nM) or acetyl
(12, Ki = 48 nM) gave compounds with slightly reduced
binding affinity and did very little to improve metabolic
stability. Surprisingly the N-acetyl derivative 12, though
exhibiting moderately potent binding affinity, did not
show appreciable functional activity. This suggests that
for this series at least, the presence of a basic center in
this region of the molecule is important for functional
activity.
>10,000
MeO2S
N
H
O
16c
4000 (5.40 0.1)
Me
N
H
16d
16e
16f
16g
16h
16i
MeO
73 (7.13 0.2)
49 (7.31 0.1)
19 (7.73 0.2)
6 (8.19 0.2)
8 (8.09 0.2)
7 (8.13 0.2)
O
We were also interested in the SAR around the terminal
aryl of the biarylcarboxamide motif. Compounds 16a–p
were prepared as the (3S,30S) diastereomers (which dis-
play comparable potency at MCH-R1; compare 3a,
Ki = 14 nM, with 3b, Ki = 22 nM) and were directly ob-
tained from intermediate 15 as outlined in Scheme 3.
Reaction of commercially available (S)-3-(trifluoroace-
tamido)pyrrolidine hydrochloride with 4-nitrophenyl
chloroformate in the presence of a base gave carbamate
13 in good yield. Subsequent reaction of 13 with
(S)-3-[(tert-butoxycarbonyl)methylamino]pyrroli-
dine19 followed by N-Boc deprotection gave the inter-
mediate methyl amine 14. Coupling of 14 with
4-bromobenzoyl chloride, followed by N-deprotection
then reductive alkylation with hydrocinnamaldehyde,
gave intermediate 15. Subsequent reaction of 15 with a
variety of aryl boronic acids via a Suzuki coupling gave
final compounds 16a–p. We examined the effect of vary-
ing the para substituent on the terminal phenyl ring
(compounds 16a–k) and, not too surprisingly, discov-
ered that the SAR was very similar to that of the origi-
nal APU series.5k The electron-donating/withdrawing
capability of the para substituent (its r value) did not
seem important. Rather, potency was determined by
the substituent’s relative lipophilicity (or p value).20
For example those compounds containing a phenyl
ring substituted by very polar groups such as carboxam-
ide (16a, Ki = 630 nM; p = ꢀ1.5), sulfonamide (16b,
Ki > 10,000 nM; p = ꢀ1.9) and acetamide (16c,
Ki = 4000 nM; p = ꢀ0.8) proved to be poor MCH-R1
binders. The incorporation of more lipophilic function-
ality in this position resulted in a dramatic increase in
potency. The 4-methylcarboxylate (16d; p = 0) and
4-cyano (16e; p = ꢀ0.3) derivatives had measured Kis
of 73 and 49 nM, respectively, whereas the 4-chloro
(16g; p = 0.8), 4-trifluoromethoxy (16i; p = 1.2) and
4-thiomethyl (16j; p = 0.6) had Kis of 6, 7, and 4 nM,
NC
F
Cl
MeO
CF3O
16j
4 (8.42 0.2)
11 (7.96 0.05)
4800 (5.32 0.1)
400 (6.40 0.05)
640 (6.20 0.02)
MeS
F3C
16k
16l
N
N
16m
16n
N
N

M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4450–4457
4455
O
N
O
O
a
N
b, c
O
F3C
F3C
NH
N
O
N
H
N
H
HN
Me
NO2
HCl
O
CF3
N
H
13
14
d-f
O
N
O
N
g
O
N
O
N
N
N
Me
Me
N
N
H
H
Ar
Br
16
15
Scheme 3. Reagents and conditions: (a) 4-nitrophenyl chloroformate, TEA, THF, 0 ꢁC to rt, 2 h, 85%; (b) (S)-3-tert-butoxycarbonyl
(methylamino)pyrrolidine, TEA, DMF, 70 ꢁC, 10 h, 25%; (c) TFA, DCM, rt, 1 h, quantitative; (d) 4-bromobenzoyl chloride, TEA, DCM, rt,
12 h; (e) K2CO3, MeOH, H2O, 75 ꢁC, 12 h; (f) hydrocinnamaldehyde, TEA, NaBH4, MeOH, rt, 12 h, 44% (from 14); (g) aryl boronic acid,
Pd(PPh3)4, K2CO3, 1,4-dioxane, H2O, 90 ꢁC, 12 h, 25–65%.
respectively. Replacing the phenyl with a more polar
aromatic ring, such as pyrimidyl (16l, Ki = 4800 nM),
3-pyridyl (16m, Ki = 400 nM) or 4-pyridyl (16n,
Ki = 640 nM), resulted in a large decrease in potency.
Interestingly, by introducing a methoxy substituent para
to the biaryl bond, potency was regained. The
2-methoxypyrid-5-yl derivative 16p (Ki = 44 nM) was
approximately 10-fold more potent than the
corresponding unsubstituted pyridyl 16m. As observed
previously with the APU series, compounds containing
a meta-substituted aryl ring were much less tolerated
by the receptor. For instance, the 3-methoxypyrid-5-yl
derivative 16o (Ki = 1000 nM) was approximately
20-fold less potent than 16p, suggesting that the
same steric factors may play a role in this series. Overall
these data suggest that the receptor binding pocket
surrounding the biaryl motif is sterically tight and
lipophilic in nature, as only relatively non-polar groups
are tolerated and para substitution is preferred. A
proposed model, based on site-directed mutagenesis, of
the interaction of biphenylcarboxamide containing
antagonists with MCH-R1 supports these general
conclusions.22
Acknowledgments
We are indebted to Dr. John Saunders for stimulating
scientific discussions, Mr. John Harman, Mr. Chris
Devore, and Mr. Shawn Ayube for LC–MS support,
Ms. Monica Mistry for pharmacology support, and
Dr. Yang Sai for metabolite identification support.
This work was partly supported by NIH Grant
2-R44-DK059107-02.
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In conclusion, we have discovered a novel series of
potent and functional MCH-R1 antagonists based
around a rAPU scaffold. SAR around both the basic
nitrogen and biarylcarboxamide groups was explored
and potent MCH-R1 binding was achieved via incor-
poration of both a lipophilic substituted secondary
amine and a lipophilic para substituted biphenylcarb-
oxamide group. Examples from this series exhibited
good oral exposure and metabolic stability, with im-
proved profiles against the HERG channel. Unfortu-
nately, issues with both inhibition of CYP2D6 and
poor brain penetration were identified, and our efforts
toward addressing these problems will be reported in
our next letter.

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Wang, X.-C.; Saunders, J.; Zhu, Y.-F. Bioorg. Med.
Chem. Lett. 2005, 15, 3701; (g) Vasudevan, A.; LaMar-
che, M. J.; Blackburn, C.; Che, J. L.; Luchaco-Cullis, C.
A.; Lai, S.; Marsilje, T. H.; Patane, M. A.; Souers, A. J.;
Wodka, D.; Geddes, B.; Chen, S.; Brodjian, S.; Falls, D.
H.; Dayton, B. D.; Bush, E.; Brune, M.; Shapiro, R. D.;
Marsh, K. C.; Hernandez, L. E.; Sham, H. L.; Collins, C.
A.; Kym, P. R. Bioorg. Med. Chem. Lett. 2005, 15, 4174;
(h) Palani, A.; Shapiro, S.; McBriar, M. D.; Clader, J.
W.; Greenlee, W. J.; Spar, B.; Kowalski, T. J.; Farley, C.;
Cook, J.; Van Heek, M.; Weig, B.; O’Neill, K.; Grazi-
ano, M.; Hawes, B. J. Med. Chem. 2005, 48, 4746; (i)
Kym, P. R.; Iyengar, R.; Souers, A. J.; Lynch, J. K.;
Judd, A. S.; Gao, J.; Freeman, J.; Mulhern, M.; Zhao,
G.; Vasudevan, A.; Wodka, D.; Blackburn, C.; Brown,
J.; Che, J. L.; Cullis, C.; Lai, S. J.; LaMarche, M. J.;
Marsilje, T.; Roses, J.; Sells, T.; Geddes, B.; Govek, E.;
Patane, M.; Fry, D.; Dayton, B. D.; Brodjian, S.; Falls,
D.; Brune, M.; Bush, E.; Shapiro, R.; Knourek-Segel, V.;
Fey, T.; McDowell, C.; Reinhart, G. A.; Preusser, L. C.;
Marsh, K.; Hernandez, L.; Sham, H. L.; Collins, C. A.
J. Med. Chem. 2005, 48, 5888.
6. In rat following a single iv dose of 5 mg/kg, plasma
AUC0–24h, CL, Cmax, t1/2, and Vd were determined to be
4017 ngh/mL, 19.5 mL/min/kg, 27 ng/mL, 4.4 h, and
7.6 L/kg, respectively. Following a single oral dose of
10 mg/kg, plasma AUC0–24h and absolute bioavailability
were determined to be 398 ngh/mL and 5%, respectively.
All data were determined in male Wistar rats (n = 3).
7. 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.
8. Oxidative metabolism of the carbon centeradjacent to the 4-
chlorophenyl ring, of compound 1, was not observed upon
incubation with human liver microsomes (As confirmed by
HPLC–MS-MS). This strongly suggests that the observed
increase in metabolic stability (in HLM) of compound 3a is
not due to the presence of the geminal dimethyl group.
9. In rat following a single iv dose of 5 mg/kg, plasma
AUC0–24h, CL, Cmax, t1/2, and Vd were determined to be
4037 ngh/mL, 21 mL/min/kg, 326 ng/mL, 4.8 h, and 8.8 L/
kg, respectively. Following a single oral dose of 10 mg/kg,
plasma AUC0–24h was determined to be 3396 nghr/mL.
All data were determined in male Wistar rats (n = 3).
10. The HERG potassium current was recorded from a
HERG/HEK cell line using established patch-clamp
methods. The effects of test compounds on the HERG
current were determined at the end of a 5 min application.
Test compound(s) were tested at six concentrations
(0.1 nM, 1 nM, 10 nM, 100 nM, 1 lM, and 10 lM).
Cisapride (30 nM) was used as a positive control.
11. Rowley, M.; Hallett, D. J.; Goodacre, S.; Moyes, C.;
Crawforth, J.; Sparey, T. J.; Patel, S.; Marwood, R.; Patel,
S.; Thomas, S.; Hitzel, L.; O’Connor, D.; Szeto, N.; Castro,
J. L.; Hutson, P. H.; MacLeod, A. M. J. Med. Chem. 2001,
44, 1603.
12. (S)-1-Benzyl-3-(methylamino)pyrrolidine is commercially
available from Tokyo Chemical Industry (TCI) America,
Portland, Oregon. The ee was determined to be >97%.
13. (R)-3-(tert-Butoxycarbonyl)aminopyrrolidine 5a is com-
mercially available from Tokyo Chemical Industry (TCI)
America, Portland, Oregon. (R)-3-[(tert-Butoxycarbon-
yl)methylamino]pyrrolidine 5b is obtained from commer-
cially available (R)-1-benzyl-3-(methylamino)pyrrolidine
(TCI America, ee > 97%) via N-3 Boc protection followed
by N-1 debenzylation.
5. (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.; Hernan-
dez, 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.; Hernan-
dez, 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.;
O’Neill, K. Bioorg. Med. Chem. 2005, 13, 1829; (k)
Rowbottom, M. W.; Vickers, T. V.; Dyck, B.; Tamiya,
J.; Zhang, M.; Zhao, L.; Grey, J.; Provencal, D.; Schwarz,
D.; Heise, C. E.; Mistry, M.; Fisher, A.; Dong, T.; Hu, T.;
Saunders, J.; Goodfellow, V. S. Bioorg. Med. Chem. Lett.
2005, 15, 3439; (l) Kanuma, K.; Omedera, K.; Nishiguchi,
M.; Funakoshi, T.; Chaki, S.; Semple, G.; Tran, T.-A.;
Kramer, B.; Hsu, D.; Casper, M.; Thomsen, B.; Beeley,
N.; Sekiguchi, Y. Bioorg. Med. Chem. Lett. 2005, 15, 2565;
(m) Kanuma, K.; Omedera, K.; Nishiguchi, M.; Funako-
shi, T.; Chaki, S.; Semple, G.; Tran, T.-A.; Kramer, B.;
Hsu, D.; Casper, M.; Thomsen, B.; Sekiguchi, Y. Bioorg.
Med. Chem. Lett. 2005, 15, 3853; (n) Ulven, T.; Frimurer,
14. Typical experimental procedure for preparation of
rAPU’s, via reductive alkylation: Synthesis of 3a. To a
stirred solution of the HCl salt of 7a (601 mg,
1.21 mmol) and TEA (239 mg, 2.37 mmol) in 35 mL
MeOH at ambient temperature, was added a solution of
3-(4-chlorophenyl)-3-methylbutyraldehyde21
(285 mg,
1.45 mmol) in 5 mL MeOH. Stirring was continued for
a further 16 h before the addition of sodium borohydride
(500 mg, 13.16 mmol). After stirring for 30 min, 10 mL
of 1 N aqueous NaOH solution was added. The MeOH
was removed in vacuo and the remaining aqueous phase
extracted with EtOAc (2 · 100 mL). The combined
organic layers were washed with brine then dried
(MgSO4). Concentration in vacuo gave an oil, which
was purified by silica gel chromatography (gradient
elution with 100% EtOAc to 5% MeOH in EtOAc to
9% MeOH/1% TEA in EtOAc) to afford a colorless
foam, which was redissolved in a solution of methane-
sulfonic acid (77 mg, 0.80 mmol, in 20 mL of DCM).
After stirring for 1.5 h at ambient temperature, concen-

M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4450–4457
4457
tration in vacuo afforded the mesylate salt of 3a (565 mg,
58%) as a colorless solid: 1H NMR (DMSO-d6) d 7.94
(d, 2H, J = 6.6 Hz), 7.84 (d, 2H, J = 7.7 Hz), 7.82 (d, 2H,
J = 6.6 Hz), 7.54 (d, 2H, J = 6.2 Hz), 7.37–7.40 (m, 4H),
3.66 (m, 1 H), 3.56 (dd, 1H, J = 9.2, 5.0 Hz), 3.25–3.50
(m, 8H), 2.91 (s, 3H), 2.63–2.66 (m, 2H), 2.34 (s, 3H),
2.05–2.10 (m, 3H), 1.91–1.95 (m, 3H), 1.29 (s, 6H); MS
(m/z) 641.2 (M+H)+. Anal. Calcd for C35H40N4O2Cl-
F3ÆCH3SO3HÆ11/2H2O: C, 56.57; H, 6.20; N, 7.33.
Found C, 56.79; H, 6.59; N, 7.45.
control for plate-to-plate variability and overall IC50
values were highly reproducible. Key compounds (with
Ki <50 nM) were assayed in at least three independent
experiments.
17. The CYP2D6 inhibition assay was carried out in the
presence of the fluorescent substrate, AMMC. Quinidine
was used as positive control.
18. Calculated logPs for 2,5-dimethylfuran and 2-methylpyri-
dine are 2.3 and 1.2, respectively, significantly lower than for
methylcyclohexane with a logP = 3.9. Calculated using
ACD/Labs logP database, Advanced Chemistry Develop-
ment Inc., Toronto, Ontario, Canada (http://www.acdlabs.
com).
15. On each assay plate, a standard antagonist of comparable
affinity to those being tested was included as a control for
plate-to-plate variability. Overall pKi values were highly
reproducible, the standard error of the mean (SEM) being
reported. All compounds described were assayed in at
least three independent experiments.
19. Obtained in two steps from commercially available (S)-1-
benzyl-3-(methylamino)pyrrolidine via N-3 Boc protec-
tion followed by N-1 debenzylation.
16. For example, compound 16j had a measured IC50 of
2.2 1.2 nM (n = 3). Assays were performed using mem-
brane preparations of CHO cells stably expressing human
MCH-R1. Each experiment was run in the presence of
MCH peptide (10 nM), GTPc35S (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
20. Craig, P. N. J. Med. Chem. 1971, 14, 680.
21. 3-(4-Chlorophenyl)-3-methylbutyraldehyde was prepared
from 3-methyl-2-butenal, as described in Goodfellow, V.;
Rowbottom, M.; Dyck, B. P.; Tamiya, J.; Zhang, M.;
Grey, J.; Vickers, T.; Kiankarimi, M.; Wade, W.; Hudson,
S. C. Patent Application WO 2004/080411, 2004.
22. Unpublished results.