The discovery of potent antagonists of NPBWR1 (GPR7)

Article abstract of DOI:10.1016/j.bmcl.2011.11.126

The synthesis and evaluation of small molecule antagonists of the G protein-coupled receptor NPBWR1 (GPR7) are reported for the first time. [4-(5-Chloropyridin-2-yl)piperazin-1-yl][(1S,2S,4R)-4-{[(1R)-1-(4-methoxyphenyl) ethyl]amino}-2-(thiophen-3-yl)cycl

Full text of DOI:10.1016/j.bmcl.2011.11.126

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1014–1018
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery of potent antagonists of NPBWR1 (GPR7)
F. Anthony Romero a, , Nicholas B. Hastings b, Remond Moningka a, Zhiqiang Guo a, Ming Wang a,
⇑
Jerry Di Salvo c, Ying Lei c, Dorina Trusca c, Qiaolin Deng d, Vincent Tong e, Jenna L. Terebetski f,
Richard G. Ball g, Feroze Ujjainwalla a
a Merck & Co., Inc., Department of Medicinal Chemistry, PO Box 2000, Rahway, NJ 07065, USA
b Merck & Co., Inc., Department of Neuroscience and Ophthalmology, Kenilworth, NJ 07033, USA
c Merck & Co., Inc., Department of In Vitro Pharmacology, PO Box 2000, Rahway, NJ 07065, USA
d Merck & Co., Inc., Department of Chemistry Modeling and Informatics , PO Box 2000, Rahway, NJ 07065, USA
e Merck & Co., Inc., Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, PO Box 2000, Rahway, NJ 07065, USA
f Merck & Co., Inc., Department of Basic Pharmaceutical Sciences, PO Box 2000, Rahway, NJ 07065, USA
g Merck & Co., Inc., Department of Analytical Chemistry, PO Box 2000, Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and evaluation of small molecule antagonists of the G protein-coupled receptor NPBWR1
(GPR7) are reported for the first time. [4-(5-Chloropyridin-2-yl)piperazin-1-yl][(1S,2S,4R)-4-{[(1R)-1-
(4-methoxyphenyl)ethyl]amino}-2-(thiophen-3-yl)cyclohexyl]methanone (1) emerged as a hit from a
high-throughput screen. Examination of substituents that focused on replacing the 5-chloropyridine
and 4-methoxybenzylamino groups of 1 led to the identification of compounds that exhibited subnanom-
olar potencies as low as 660 pM (9k) in the functional assay and 200 pM in the binding assay (9i).
Ó 2011 Elsevier Ltd. All rights reserved.
Received 27 October 2011
Revised 29 November 2011
Accepted 30 November 2011
Available online 8 December 2011
Keywords:
NPBWR1
GPR7
Antagonist
Anti-obesity
G protein-coupled receptor
In 2002, G protein-coupled receptor 7 (GPR7) was deorphanized
with the identification of endogenous ligands neuropeptide B (NPB)
and neuropeptide W (NPW).1 Shortly thereafter it was reclassified as
Neuropeptide B/W receptor-1 (NPBWR1). Since the deorphaniza-
tion there have been several studies that have suggested NPBWR1
to be involved in feeding behavior, energy homeostasis, neuroendo-
crine function, and modulating inflammatory pain.2
In humans and rodents, mRNA encoding NPBWR1 and it’s
ligands NPB and NPW have been found to be co-localized in periph-
eral tissues (alimentary tract and endocrine system), but are mainly
concentrated within the central nervous system (CNS). NPBWR1
appears to be abundant in the hippocampus, subcortical limbic tel-
encephalon, hypothalamus, olfactory cortex, midbrain periaqu-
eductal gray and tegmental area.3 These regions correspond well
to the localization of NPB-/NPW-immunoreactive processes to li-
gand binding sites localized using radioiodinated NPB in rat brain
or NPW in mouse brain, as well as regions in which cFos immuno-
reactivity is enhanced following intracerebroventricular (i.c.v.)
administration of NPW to the rat.3–8 These observations suggest
that the NPBWR1 receptor may be involved in cognitive and affec-
tive reactions to stress, arousal, locomotor activity, cardiovascular
and neuroendocrine responses, as well as energy homeostasis.
Among the pharmacological effects of NPB and NPW we were pri-
marily interested in their effects on food intake and body weight.
Studies have shown that i.c.v. injection of NPW in rats caused acute
hyperphagia in male rats while i.c.v. injection of NPB increased feed-
ing.1c,d As such, NPBWR1 has emerged as an interesting new thera-
peutic target. In an effort to probe NPBWR1 as a novel anti-obesity
target we wished to develop a small ’molecule antagonist.
To date no known small molecule antagonists of NPBWR1 have
been reported in the literature. In an effort to validate NPBWR1 as
Cl
OCH
3
H
N
(R)
(S)
(R)
N
N
(S)
N
O
S
1
mouse cAMP / binding IC = 363 / 25 nM
50
human cAMP / binding IC = 1925 / 324 nM
50
⇑
Corresponding author. Tel.: +1 732 594 6040.
E-mail address: anthony_romero@merck.com (F. Anthony Romero).
Figure 1. In vitro potency of lead compound 1 at mouse and human NPBWR1.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.126

F. Anthony Romero et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1014–1018
1015
an anti-obesity target, we started a discovery program aimed at
developing a small molecule antagonist as a pharmacological tool.
In this regard, a high-throughput screen was performed of the Merck
collection. One lead that emerged from this screen was compound 1
(Fig. 1). This compound exhibited moderate potency (IC50 = 363 nM)
in a mouse functional cyclic-AMP (cAMP) assay and was shown in
our mouse binding assay to be relatively potent with an IC50 of
25 nM. While the human in vitro potency was shifted toward weak-
er potency by 13- and 5-fold in the cAMP and binding assays, respec-
tively, our primary interest was in identifying a tool compound that
would be suitable in a mouse in vivo model to probe the mechanism
of NPBWR1. Compound 1 was evaluated in both mouse liver micro-
somes and Pgp efflux studies (LLC-PK1:MDR1 cell line). Compound 1
was found to have poor intrinsic metabolic stability (0% parent
remaining at 30 min) and was also found to be a Pgp substrate.
The limited metabolic stability of 1 coupled with it also being a
Pgp substrate precluded it from further pharmacokinetic studies
and being used as an in vivo tool that could be administered periph-
erally (po or iv). As the initial structure–activity relationships (SAR)
of 1 were being developed, many of these compounds were also
shown to be have limited metabolic stability and were also Pgp sub-
strates (data not shown) thus decreasing the chance of sufficient iv
or po exposure in the CNS. An alternative method for dosing we con-
sidered was through an i.c.v. route since we were targeting NPBWR1
in the CNS and metabolic stability Pgp efflux would not be an issue.
We had decided early onin this program todevelop a compoundthat
could be used as an i.c.v. tool, thus we sought to optimize compound
1 on in vitro potency. For the purposes of this article the potencies
described will be at the mouse receptor unless otherwise indicated.
Here we report the optimization of 1 at mouse NPBWR1, the identi-
fication of the first potent antagonists of NPBWR1 and the identifica-
tion of candidate pharmacological in vivo tool compounds.
regioisomers 3 and 4 in a 3:1 ratio, respectively. Compound 3
was the major product and was easily separable from 4 by silica
gel chromatography. Hydrolysis of the pentafluorophenyl ester
and resolution on a preparative SFC chiral column yielded enantio-
pure intermediate ent-5. Intermediate ent-5 underwent amide cou-
pling with the appropriate aryl substituted piperazine with use of a
resin bound coupling agent to produce ent-6. After filtration of the
resin and evaporation of the solvent, the crude coupling product
was then subjected to reductive amination with the appropriate
amine by enlisting resin bound cyanoborohydride to afford two
diastereomers (3:2 ratio; 7–9 : 10) of which the major diastereo-
mer (7–9) was the one we required. The diastereomers 7–9 and
10 were easily separated by reverse phase HPLC to provide the req-
uisite candidate antagonists (7a–o, 8a–n and 9a–k).10 The absolute
stereochemistry of the final compounds was determined by
obtaining an X-ray crystal structure of compound 8f (Fig. 2; CCDC
854950).11
The structure–activity relationships (SAR) of compound 1 fo-
cused on left-hand aryl ring (Fig. 3) and right-hand benzylamine
(Fig. 4). To explore the left-hand aryl moiety a systematic series
of modifications were made and some key interactions emerged
from this series (Fig. 3, 7a–o). Removal of the pyridine nitrogen
of 1, compound 7a, proved detrimental to activity and resulted in
a ꢀ10-fold loss in potency. This loss of binding potency proves con-
sistent with a loss in binding affinity due to a hydrogen-bond inter-
action within the binding site. While removal of the chlorine atom
of 1, compound 7b, had little effect on the functional potency, the
binding affinity decreased by 12-fold. Substitution of the chlorine
atom of 1 at different positions around the pyridine ring (7c–e) re-
sulted in loss of potency. Replacement of the chlorine atom with
other lipophilic groups such as a methyl (7f) or trifluoromethyl
(7g) maintained potency as compared to 1 whereas hydrophilic
moieties appear to reduce potency significantly. To further explore
the putative hydrogen-bonding of the pyridine nitrogen of 1, we
explored a few heterocycles that would position nitrogen similar
to the pyridine nitrogen of 1. Compound 7j was indistinguishable
in potency from 7b and indicated that replacement of the pyridine
with other heterocycles would be tolerated. Placement of a lipo-
philic substituent on the heterocycle (7k and 7l) increased po-
tency. Attaching a fused phenyl ring to a heterocycle as in 7m–o
led to the largest increase in potency with 7n notably having a
nine-fold increase in binding potency and 20-fold increase in func-
tional activity over lead compound 1.
Key to the divergent synthesis9 of the antagonists was the prep-
aration of intermediate ent-5 from which all the requisite com-
pounds could be synthesized (Scheme 1). A Diels–Alder reaction
between 2-trimethylsilyloxy-1,3-butadiene and cinnamyl ester 2
followed by treatment with HCl produced two cyclohexanone
PFP
O
O
O
O
O
O
a
PFP
PFP
Concurrent with these studies, the SAR of the right-hand
methyl benzylamine was explored (Fig. 4, 8a–n). Removal of the
-methyl substituent resulted in a significant loss in potency.
The stereogenic -methyl was replaced with a gem-dimethyl sub-
a-
O
O
S
S
S
a
2
3
4
PFP = pentafluorophenyl
a
1
stituent (8c) and a two-fold increase in binding potency was ob-
served over 1. The gem-dimethyl substituent may help contribute
towards rigidification of the benzyl side chain whereby it orients
the 4-methoxyphenyl substituent towards a specific interaction
O
R
O
N
HO
O
N
b
c
O
S
S
5
6
ent-
ent-
H
N
H
1
1
R
R
N
2
2
N
R
N
R
d
N
N
O
O
S
S
10
7a o
−
8a−n
9a−k
Scheme 1. Reagents and conditions: (a) 2-trimethylsilyloxy-1,3-butadiene, 150 °C
sealed tube, 24 h then 3 N HCl; (b) LiOH then SFC AD-H column; (c) R1-piperazine,
PS-carbodiimide, HOBT; (d) R2-NH2, MP-cyanoborohydride, AcOH.
Figure 2. X-ray crystallography analysis confirmed the absolute stereochemistry of
compound 8f. C1, C2, C5, C21 are S, S, R, R, respectively.

1016
F. Anthony Romero et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1014–1018
OCH Cl
3
H
N
H
N
R
N
N
R
N
N
N
O
O
S
S
cmpd
1
cAMP IC (nM) binding IC (nM)
50 50
cmpd
1
cAMP IC (nM) binding IC (nM)
R
R
50
50
OCH
Cl
Cl
3
360
25
360
25
N
OCH
3
7a
7b
3865
330
3715
290
8a
8b
8c
3595
981
1525
217
N
Cl
OCH
3
3
3
43
11
56
3.1
7c
2143
N.D.
OCH
OCH
N
8d
8e
523
20
Cl
7d
7e
>32000
7486
N.D.
N.D.
N
N
Br
Cl
8f
2628
2208
134
7f
400
200
74
8g ortho
Br
2196
160
82
N
N
F C
3
8g−i
8h meta 972
8i para 601
7g
18
OCH
3
H CO
3
7h
7303
N.D.
8j
1020
118
N
N
NC
7i
7j
7054
207
1140
194
NO
2
8k
8l
1100
1700
320
100
27
S
Cl
N
N
Br
S
S
7k
7l
55
14
42
F
17
8m
8n
F C
3
115
N
211
16
N
S
7m
7n
7o
24
18
20
4.5
2.8
8.3
Figure 4. Exploration of the
a-methyl substituent and substitution on the benzyl
N
N
N
aryl ring.
O
1 and 500-fold over compound 8a. Additional effects of the substi-
tuent on the benzyl aryl ring were also examined (8f–n). Para sub-
stitution was favored on the phenyl ring as indicated by cyclobutyl
containing compounds 8e and 8g–j. One trend that emerged from
examination of substituents in the para position of a series of gem-
dimethyl containing compounds (8c and 8k–n) was that the bind-
ing potency decreased as the substituent on the phenyl ring be-
came more electronegative. Although generalizations are not
fully defined by this limited data set, it does appear that elec-
tron-donating substituents are more positively impactful on po-
tency than electron-withdrawing substituents. Although the
nature of the interaction of the phenyl group in the binding site
NH
Figure 3. Exploration of the aryl substituent on the piperazine.
in the binding site. This concept was further explored with
substitution of a cyclopropyl (8d) and cyclobutyl (8e) substituent.
Unexpectedly, the substitution of a cyclopropyl substitutent (8d)
led to a five-fold decrease in binding potency compared to 8c.
However, a cyclobutyl substituent (8e) proved quite favorable
and notably increased binding potency eight-fold over compound
is unclear, it does not appear to be involved in a
p–p interaction
because the opposite trend would have been observed.

F. Anthony Romero et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1014–1018
1017
With the discovery of productive modifications on the left- and
right-hand sides of the molecule we sought to combine some of
these better substituents (9a–g) in the hope of providing a further
benefit to potency. Figure 5 summarizes some of these combina-
tions. Gratifyingly, these combinations did indeed provide an
enhancement in both functional and binding potency. Compound
9b exhibited subnanomolar binding affinity and single digit nano-
molar functional potency providing a 36-fold and 61-fold increase
in binding and functional potency, respectively, compared to the
lead compound 1. Exploration of substitution on the benzoxazole
and benzimidazole ring was performed (9h–k). While the addition
of a methyl substituent matched the potency of unsubstituted
benzoxazole 9b, placement of a fluorine atom on the benzoxazole
(9i and 9j) and benzimidazole (9k) rings led to the most potent
compounds of the series. Both compounds 9i and 9k exhibited
subnanomolar binding and functional potency. Most notably, com-
pound 9i resulted in a 420-fold increase in functional potency and
125-fold increase in binding potency over lead compound 1.
Functional and binding potency at the human receptor were
also determined for some of the compounds in Figure 4. As with
our initial lead compound (1), the human potencies were shifted
toward weaker potency between 5- and 43-fold for the binding
potencies and between 4- and 24-fold for the functional potencies.
Since our goal was to develop a compound to probe target engage-
ment in the mouse, the shift in human potency was not a concern
at this stage. Although we had abandoned early on to optimize
these compounds for metabolic stability and Pgp efflux we had
decided to evaluate compounds 9a–k as they were some of the
most potent compounds. Unfortunately, these compounds were
no different as compound 1. All were Pgp substrates and had
<10% parent remaining at 30 min in mouse liver microsomes. In
a future publication we will describe our results with the identifi-
cation of an i.c.v. in vivo tool compound and results on engaging
the target.
In summary, we have prepared and evaluated analogues of
compound 1 for NPBWR1 antagonist activity. Simultaneous explo-
ration of the aryl group on the piperazine as well as exploration of
the benzylamine allowed us to identify groups that were produc-
tive towards increasing potency. Combination of these groups
and further SAR allowed us to identify the first potent small mole-
cule antagonists of NPBWR1. Most notable of this examination was
the substitution of a 5-fluorobenzoxazole for the 5-chloropyridine
of 1 and substitution of a spirocyclobutane for the stereogenic
methyl of 1 which produced compound 9i with picomolar potency
in both the functional and binding assays. More importantly, sev-
eral of these antagonists are potential i.c.v. pharmacological
in vivo tools that may help elucidate the role of NPBWR1 as an
anti-obesity target.
OCH
3
H
N
1
R
N
2
3
R
R
N
O
S
Mouse
Human
a
a
a
a
cAMP binding
IC
cAMP binding
1
2
3
cmpd
9a
R
R , R
IC
IC
IC
50
50
50
50
O
9.2
1.9
110
21
N
O
9b
9c
5.9
4.6
0.69
2.4
40
39
4.2
35
Supplementary data
N
N
NH
Supplementary data (methods and conditions for the binding
and functional assays) associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2011.11.126.
NH
S
9d
9e
3.0
1.8
1.1
11
5.9
References and notes
N
N
1. (a) Brezillon, S.; Lannoy, V.; Franssen, J. D.; Le Poul, E.; Dupriez, V.; Lucchetti, J.;
Detheux, M.; Parmentier, M. J. Biol. Chem. 2003, 278, 776; (b) Fujii, R.; Yoshida,
H.; Fukusumi, S.; Habata, Y.; Hosoya, M.; Kawamata, Y.; Yano, T.; Hinuma, S.;
Kitada, C.; Asami, T.; Mori, M.; Fujisawa, Y.; Fujino, M. J. Biol. Chem. 2002, 277,
34010; (c) Shimomura, Y.; Harada, M.; Goto, M.; Sugo, T.; Matsumoto, Y.; Abe,
M.; Watanabe, T.; Asami, T.; Kitada, C.; Mori, M.; Onda, H.; Fujino, M. J. Biol.
Chem. 2002, 277, 35826; (d) Tanaka, H.; Yoshida, T.; Miyamoto, N.; Motoike, T.;
Kuroso, H.; Shibata, K.; Yamanaka, A.; Williams, S. C.; Richardson, S. J.; Tsujino,
N.; Garry, M. G.; Lerner, M. R.; King, D. S.; O’Dowd, B. F.; Sakurai, T.;
Yanagisawa, M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6251.
2. Hondo, M.; Ishii, M.; Sakurai, T. Results Probl. Cell. Differ. 2008, 46, 239.
3. Jackson, V. R.; Lin, S. H.; Wang, Z.; Nothacker, H. P.; Civelli, O. J. Comp. Neurol.
2006, 497, 367.
4. Kitamura, Y.; Tanaka, H.; Motoike, T.; Ishii, M.; Williams, S. C.; Yanagisawa, M.;
Sakurai, T. Brain Res. 2006, 1093, 123.
5. Schulz, S.; Stumm, R.; Hollt, V. Neurosci. Lett. 2007, 16, 842.
6. Singh, G.; Maguire, J. J.; Kuc, R. E.; Fidock, M.; Davenport, A. P. Brain Res. 2004,
1017, 222.
7. Takenoya, F.; Kitamura, S.; Kageyama, H.; Nonaka, N.; Seki, M.; Itabashi, K.;
Date, Y.; Nakazato, M.; Shioda, S. Regul. Pept. 2008, 145, 116.
8. Levine, A.; Winsky-Sommerer, R.; Huitron-Resendiz, S.; Grace, M.; de Lecea, L.
Am. J. Physiol. Regul. Integr. Comp. Physiol. 2005, 288, R1727.
9. Representative experimental procedure (compound 9c): To a round-bottom
0.33
N.D.
N.D.
F C
3
S
S
9f
14
12
2.2
2.6
342
197
95
18
N
N
N
Br
9g
9h
N
O
O
3.9
0.43
0.20
70
6.0
N
N
N
N
F
9i
0.86
N.D.
N.D.
F
O
N
9j
1.3
0.18
0.42
N.D.
N.D.
N.D.
N.D.
flask
was
added
3-(3-thienyl)acrylic
acid (700 mg, 4.54 mmol),
pentafluorophenol (1.08 g, 5.90 mmol), EDC (4.04 g, 5.45 mmol), DMAP
(55.5 mg, 0.45 mmol) and dichloromethane (23.0 mL) at rt. The resulting
mixture was stirred at rt overnight before quenching with 1 N HCl (20 mL). The
two phases were separated and the organic layer was washed with saturated
aqueous NaHCO3 and brine, and dried over Na2SO4, filtered, and concentrated
under vacuum. The crude mixture was purified by silica gel chromatography
eluting with 0–10% EtOAc/hexanes to give pentafluorophenyl (2E)-3-
F
9k
0.66
a
values in nM
Figure 5. Combination of the some of the most potent substituents and substitu-
tion on the benzoxazole and benzimidazole rings.
(thiophen-3-yl)prop-2-enoate
2 (1.35 g, 93%) as a
white solid. 1H NMR

1018
F. Anthony Romero et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1014–1018
(CDCl3, 500 MHz): d 7.96 (d, J = 15.9 Hz, 1H), 7.68 (dd, J = 2.7, 1.0 Hz, 1H), 7.44
0.55 mmol) and 2-(piperazin-1-yl)-1H-benzimidazole hydrochloride (127 mg,
(dd, J = 5.1, 2.9 Hz, 1H), 7.41 (dd, J = 5.0, 1.2 Hz, 1H), 6.49 (d, J = 15.9 Hz, 1H).
A mixture of pentafluorophenyl (2E)-3-(thiophen-3-yl)prop-2-enoate 2 (1.35 g,
4.22 mmol), 2-(trimethylsiloxy)-1,3-butadiene (2.97 mL, 16.7 mmol) and
0.46 mmol) were dissolved in tetrahydrofuran (7.60 mL), and HOBt (106 mg,
0.69 mmol) was added followed by diisopropyl ethyl amine (120 lL,
0.69 mmol) and PS-carbodimide (1.24 g, 1.38 mmol). The reaction mixture
was put in a shaker at rt overnight. Upon completion, PS-trisamine (674 mg,
2.30 mmol) was added, and the resulting mixture was further shaken at rt for
2 h. Filtration of the resins followed by evaporation of the solvent afforded
hydroquinone (0.02 g, 0.21 mmol) in toluene (2.81 mL) was heated in
a
thick-walled glass tube for 24 h at 170 °C. The reaction mixture was allowed
to cool to rt and concentrated under vacuum. To the crude mixture in methanol
(5 mL) was added 3 N HCl (5 mL) at rt. After 30 min of stirring at rt, the reaction
mixture was concentrated under vacuum and then water was added. The
resulting mixture was extracted with EtOAc (2Â, 50 mL) and the combined
extracts were dried over Na2SO4, filtered and concentrated under vacuum. The
crude residue was purified by silica gel chromatography eluting with 0–95%
EtOAc/hexanes to give in order of elution racemic compound 4 (528 mg, 32%
yield) as a colorless oil 1H NMR (CDCl3, 500 MHz): d 7.32 (dd, J = 4.9, 3.0 Hz,
1H), 7.11–7.13 (m, 1H), 7.04 (d, J = 5.0 Hz, 1H), 3.38 (ddd, J = 15.4, 12.3, 3.3 Hz,
1H), 2.89 (ddd, J = 15.5, 12.1, 3.5 Hz, 1H), 2.35 (dt, J = 13.6, 3.3 Hz, 1H), 2.28 (dq,
J = 13.7, 6.1, 3.0 Hz, 1H), 2.18 (dq, J = 13.2, 7.1, 3.7 Hz, 1H), 1.93–2.03 (m, 1H),
1.51–1.66 (m, 2H) and racemic compound 3 (731 mg, 44% yield) as a white
solid. 1H NMR (CDCl3, 400 MHz): d 7.32 (dd, J = 5.0, 2.9 Hz, 1H), 7.10–7.13 (m,
1H), 7.01 (dd, J = 4.9, 1.2 Hz, 1H), 3.65 (ddd, J = 15.3, 11.1, 4.8 Hz, 1H), 3.31 (ddd,
J = 14.1, 10.3, 3.8 Hz, 1H), 2.75 (ddd, J = 14.8, 4.9, 1.6 Hz, 1H), 2.58–2.68 (m, 2H),
2.50–2.56 (m, 1H), 2.39–2.49 (m, 1H), 2.12–2.25 (m, 1H).
crude
(3S,4S)-4-{[4-(1H-benzimidazol-2-yl)piperazin-1-yl]carbonyl}-3-
(thiophen-3-yl)cyclohexanone, which was used directly in the next step
without further purification.
(3S,4S)-4-{[4-(1H-Benzimidazol-2-yl)piperazin-1-yl]carbonyl}-3-(thiophen-3-
yl)cyclohexanone (30 mg, 0.07 mmol) was dissolved in a mixture of MeOH
(367
(59.2 mg, 0.29 mmol) was added followed by MP-cyanoborohydride (159 mg,
0.367 mmol) and acetic acid (13.0 L, 0.22 mmol) at rt. The reaction mixture
lL) and DCE (367 lL), and 2-(4-methoxyphenyl)propan-2-amine
l
was shaken overnight at 55 °C, cooled to rt, filtered and concentrated under
vacuum. LC–MS indicated a 3:2 ratio of diastereomeric reductive amination
products. The two diastereomers were separated by reverse phase HPLC
(Sunfire prep C18 30 mm  100 mm) eluting with acetonitrile/water + 0.1%
TFA to give [4-(1H-benzimidazol-2-yl)piperazin-1-yl][(1S,2S,4R)-4-{[2-(4-
methoxyphenyl)propan-2-yl]amino}-2-(thiophen-3-yl)cyclohexyl]methanone
TFA salt (9c) as a white solid (only the slower eluting isomer was isolated as the
desired compound). 1H NMR (CD3OD, 400 MHz): d 7.49–7.55 (m, 2H), 7.26 (dd,
J = 5.0, 3.0 Hz, 1H), 7.16–7.22 (m, 1H), 7.19 (dd, J = 5.9, 3.2 Hz, 1H), 7.00–7.03
(m, 1H), 6.91–6.99 (m, 5H), 3.80–3.88 (m, 1H), 3.74 (s, 3H), 3.35–3.53 (m, 5H),
3.17–3.33 (m, 2H), 2.92–3.02 (m, 2H), 2.52–2.63 (m, 1H), 1.83–196 (m, 2H),
1.78 (s, 3H), 1.77 (s, 3H), 1.54–1.72 (m, 4H).
To racemic compound 3 (100 mg, 0.256 mmol) in a mixture of tetrahydrofuran
(1921 lL) and water (640 lL) was added LiOH (18.41 mg, 0.769 mmol) in one
portion at rt. The reaction mixture was stirred at 60 °C for 2 h. After cooling to
rt, the reaction mixture was acidified with 1 N HCl, and poured into water
(10 mL). The resulting mixture was extracted with dichloromethane (2Â,
50 mL). The combined organic layers were dried over Na2SO4, filtered, and
concentrated under vacuum. Resolution on a preparative SFC chiral column
(AD-3 column, faster eluting isomer) gave enantiopure (1S,2S)-4-oxo-2-
(thiophene-3-yl)cyclohexanecarboxylic acid as a brown solid. 1H NMR (CDCl3,
400 MHz): d 7.28 (dd, J = 4.9, 2.9 Hz, 1H), 7.04 (d, J = 1.6 Hz, 1H), 6.96 (dd,
J = 5.0, 1.1 Hz, 1H), 3.53 (ddd, J = 15.1, 10.8, 4.8 Hz, 1H), 2.96 (ddd, J = 13.7, 10.0,
3.8 Hz, 1H), 2.69 (ddd, J = 14.8, 4.8, 1.6 Hz, 1H), 2.50–2.61 (m, 2H), 2.38–2.48
(m, 1H), 2.20–2.30 (m, 1H), 1.98–2.11 (m, 1H).
10. All eight stereoisomers of compound 1 were evaluated that were derived from
the enantiomers of intermediate 5. Only compound 1 as shown in Figure 1
possessing the S, S, R, R configuration was shown to have significant activity.
For the purposes of this paper the SAR surrounding this single enantiomer is
discussed.
11. In order to determine the absolute configuration of the candidate antagonists
we obtained an X-ray structure of compound 8f. Commercially available (R)-4-
bromo-
a-methylbenzylamine was used in order to establish the absolute
(1S,2S)-4-Oxo-2-(thiophene-3-yl)cyclohexanecarboxylic acid
5
(124 mg,
configuration of the stereogenic centers in this molecule.