Discovery of a novel class of bicyclo[3.1.0]hexanylpiperazines as noncompetitive neuropeptide y Y1 antagonists

Article abstract of DOI:10.1021/ml200265m

A novel class of bicyclo[3.1.0]hexanylpiperazine neuropeptide Y (NPY) Y1 antagonists has been designed and synthesized. Scatchard binding analysis showed these compounds to be noncompetitive with [¹²⁵I]PYY binding to the Y1 receptor. The most potent member, 1-((1α,3α,5α,6β)-6- (3-ethoxyphenyl)-3-methylbicyclo[3.1.0]hexan-6-yl)-4-phenylpiperazine (2) had an IC₅₀ = 62 nM and displayed excellent oral bioavailability in rat (% F po = 80), as well as good brain penetration (B/P ratio = 0.61). In a spontaneous nocturnal feeding study with male Sprague-Dawley rats, 2 significantly reduced food intake during a 12 h period.

Full text of DOI:10.1021/ml200265m

Letter
pubs.acs.org/acsmedchemlett
Discovery of a Novel Class of Bicyclo[3.1.0]hexanylpiperazines as
Noncompetitive Neuropeptide Y Y1 Antagonists
Shuanghua Hu,* Yazhong Huang, Milind Deshpande, Guanglin Luo, Marc A. Bruce, Ling Chen,
Gail Mattson, Lawrence G. Iben, Jie Zhang, John W. Russell, Wendy J. Clarke, John B. Hogan,
Astrid Ortiz, Oliver Flint, Andrew Henwood, Qi Gao, Ildiko Antal-Zimanyi, and Graham S. Poindexter
Research & Development, Bristol-Myers Squibb Company, 5 Research Parkway, Wallingford, Connecticut 06492, United States
S
* Supporting Information
ABSTRACT: A novel class of bicyclo[3.1.0]hexanylpiperazine neuropeptide
Y (NPY) Y1 antagonists has been designed and synthesized. Scatchard bind-
ing analysis showed these compounds to be noncompetitive with [125I]PYY
binding to the Y1 receptor. The most potent member, 1-((1α,3α,5α,6β)-
6-(3-ethoxyphenyl)-3-methylbicyclo[3.1.0]hexan-6-yl)-4-phenylpiperazine (2)
had an IC50 = 62 nM and displayed excellent oral bioavailability in rat (% F
po = 80), as well as good brain penetration (B/P ratio = 0.61). In a spontaneous
nocturnal feeding study with male Sprague−Dawley rats, 2 significantly
reduced food intake during a 12 h period.
KEYWORDS: bicyclo[3.1.0]hexanylpiperazines, noncompetitive neuropeptide Y Y1 antagonists, brain penetration
ith obesity posing an ever-increasing threat to public
health, the need to develop effective medicines for its
overlapping site to that of 1229U91.26 Subsequently, Peterson
et al. disclosed a novel class of Y1 antagonists (1) of smaller
molecular weight, and the dihydrochloride salt of one com-
pound from this series was found to have good oral bioavailability
(% F po = 75).27,28
In the research presented here, we report our work on novel
bicyclo[3.1.0]hexanylpiperazine NPY Y1 antagonists (Scheme 1).
A preliminary structure−activity relationship (SAR) investigation
is discussed, and the pharmacokinetic (PK) profile and in vivo
effects of an early lead compound are reviewed.
In considering the N′-aryl-N-benzylpiperazine scaffold present
in 1, we note that it is comparatively flexible; the cyclohexane
ring is able to pseudorotate so that either the phenylpiperazine or
the aryl group could occupy an axial position. We anticipated
that this flexibility might contribute to the reduced potency
observed with this compound class. Correspondingly, we expected
that compounds with more rigid scaffolds would fix the sub-
stituents in either the axial or the equatorial position and may
offer advantages in terms of potency and physiochemical prop-
erties. We concluded that this could be achieved by employing
a bicyclo[3.1.0]hexane scaffold and investigated this hypothesis
as described below.
The stereoselective syntheses of endo bicyclo[3.1.0]-
hexanylpiperidines 5 was achieved using the methodology
shown in Scheme 1. This chemistry was employed as it allowed
the efficient introduction of substituents at the 6-postion of
the bicyclo[3.1.0] hexanylpiperazine and facilitated the rapid
development of an SAR at this sector of the chemotype.
W
prevention and treatment has become urgent. Becuase of the
complex mechanisms involved in the regulation of food intake
and energy expenditure, a number of neurological targets have
been pursued for therapeutic intervention. Of interest to us was
neuropeptide Y (NPY).1−5 This molecule is highly expressed in
the brain and has been shown to be a potent orexigenic agent.6−9
Additionally, NPY and its mRNA are found at substantially higher
levels in the brains of genetically obese rodents relative to their
lean littermates, and concentrations of both increase in normal
rats after fasting.10−12 In other studies, NPY-deficient ob/ob
mice showed less interest in food consumption and were less obese
than WT ob/ob mice.12 Additionally, chronic central nervous
system administration of NPY in rodents results in hyper-
phagia and robust weight gain.13
Five NPY receptors (Y1−Y5) have been cloned and expressed.
Of these, Y1 and Y5 are generally believed to be involved in both
food intake and energy expenditure, although recent literature
suggests the involvement of the Y2 and Y4 subtypes as well.14
The pharmacology of the Y1 receptor has been studied using
potent and selective Y1 receptor antagonists (Figure 1). The
effect of NPY administration in Y5 knockout mice was
completely blocked by coinjection of the peptidic Y1 antagonist,
1229U91.15,16 Other peptidic antagonists17 have been reported,
as well as a number of small molecule Y1 antagonists such as
BIBP3226,18 SR-120819A,19 LY357897,20 BMS-193885,21,22
and a novel carbazole series (structure not shown).23 However,
common characteristics of many of these agents are poor
oral bioavailability and low brain penetration. Contrastingly,
J-10487024,25 showed the first reduced spontaneous food
intake in Zucker fatty rats after oral administration (100 mg/kg).
J-104870 is a Y1 antagonist that binds to a distinct but
Received: November 8, 2011
Accepted: January 4, 2012
Published: January 4, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
investigations; subsequent work provided crystalline material
from which we were able to confirm the assigned stereo-
chemistry; see Figure 2. In the addition to the chloroenamine
Figure 1. Reported NPY Y1 receptor antagonists.
a
Scheme 1. Synthesis of endo Bicyclo[3.1.0]hexanylpiperazines
Figure 2. Crystal structure of 1-((1α,3α,5α,6β)-6-(3-ethoxyphenyl)-
3-methylbicyclo [3.1.0]hexan-6-yl)-4-phenylpiperazine (2). Note the
cis-disposition of methyl and aryl moieties.30
intermediate 4, the Grignard reagents may initiate the cyclization
by attacking the α-imine-carbon from anti-Cl direction and then
simultaneously knocking off the chlorine on the C-6 to form the
endo-bicyclic products.
Subsequently, with this chemistry optimized, we conducted
the synthesis of a 45-membered library using a variety of dif-
ferent Grignard reagents. Compounds from the library were
screened by measuring their ability to inhibit binding of [125I]PYY
(polypeptide YY) to the Y1 receptor in human neuroblastoma
(SK-N-MC) cell membranes, and the IC50 values generated in this
assay for select examples are provided in Table 1.
Table 1. Inhibition of [125I]PYY Binding to NPY Y1
Receptor in Human Neuroblastoma (SK-N-MC) Cell
Membranes
a
a
Reagents and conditions: (a) Cat. TsOH, toluene, reflux. (b) NCS
compd
R
IC50 (μM)
(1 equiv), CH2Cl2, −65 °C to room temperature. (c) Ether, 1.0 equiv
of RMgBr, room temperature; quench with aqueous HCl (1 N,
1 equiv).
2
3-ethoxyphenyl
phenyl
0.062 (n = 10)
6
2.2
7
2-methoxyphenyl
3-methoxyphenyl
3-aminophenyl
3-chlorophenyl
p-tolyl
1.3
8
0.30 (n = 4)
0.19
The synthesis involves condensation of 4-methylcyclohexanone
with 1-phenylpiperazine in the presence of a catalytic amount of
p-tolunesulfonic acid to form an intermediate, 1-(4-methylcyclohex-
1-enyl)-4-phenylpiperazine (3). The latter can then be chlorinated
stereoselectively using N-chlorosuccinimide to afford trans-
1-(6-chloro-4-methylcyclohex-1-enyl)-4-phenylpiperazine (4).
NOEs and the J-coupling constants observed in the NMR
spectra of 4 were consistent with the proposed structural assign-
ment with the chloro substituent anti to the pseudoequatorial
methyl group. Vilsmaier29 has shown that such intermediates can
readily form fused cyclopropyl ring systems on treatment with
nucleophiles, although his investigations were limited to cyanide
and hydride. Here, we extended this reaction to aryl and alkyl
Grignard nucleophiles, which add to the chloroenamine inter-
mediate 4 in a stereoselective fashion to provide the endo bicycl-
[3.1.0]hexanylpiperidines 5 in good yield. The stereochemistry
of the products was again assigned based on extensive NMR
9
10
11
12
13
14
1a
>10
0.46
4-hydroxyphenyl
4-methoxylphenyl
4-fluorophenyl
3-ethoxy
0.17
0.55
0.18
0.12 (n = 3)
a
IC50 values of the library members were obtained from a single
experiment with each point being run in duplicate. BIBP 3226
displayed Ki of 15 nM in this Y1 binding assay.
Early in our SAR investigations, we noted that all compounds
with nonaromatic functionality depicted in Scheme 1 were
devoid of Y1 binding affinity (IC50 > 10 μM), suggesting the
necessity of an aromatic moiety at this position. However, the
simple phenyl derivative 6 displayed moderate activity, IC50
2.2 μM. ortho-Substituents on the phenyl group as exemplified
=
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ACS Medicinal Chemistry Letters
Letter
by 7 seemed to have little impact on binding affinity. Contrastingly,
meta and para substituents as shown in 8, 9, and 11−14
significantly enhanced affinity except in 10 with a meta-chlorine
(IC50 > 10 μM). Electron-donating functionality appeared to
further advance the potency, and of the compounds examined,
the ethoxy derivative, 1-((1α,3α,5α,6β)-6-(3-ethoxyphenyl)-
3-methylbicyclo[3.1.0]hexan-6-yl)-4-phenylpiperazine (2) was
found to be the most active with an IC50 of 62 nM. In the same
treated and buffer-treated membrane. Receptor binding was
identical (data not shown), indicating that 2 is a reversible ligand.
In a functional assay with CHO cells stably expressing the human
neuropeptide Y1 receptor, 2 antagonized NPY-mediated inhibition
of forskolin-stimulated cAMP accumulation; see Figure 4.
[
125I]PYY binding assay, the corresponding member (1a) of
chemical series 1 wherein R is an ethoxy had an IC50 of 120
nM. This result provided evidence that the increased rigidity
indeed had a positive impact on potency. However, because the
cis isomers in chemical series 1 (Me vs arylpiperidines) were
reported to be more potent than the trans isomers,27 it needs to
be investigated in the future whether the exo bicyclo[3.1.0]-
hexanylpiperazines, wherein the methyl is cis to arylpiperidines,
would be more potent than the endo isomers, which are stereo-
selectively synthesized in this work. Compound 2 was further
screened against other NPY receptors and did not exhibit
affinity for the Y2 or Y5 receptor subtypes. With this result
in hand, a more extensive profiling of compound 2 was
undertaken.
Figure 4. Effect of NPY and 2 on forskolin-stimulated (10 μM) cyclic
AMP accumulation in CHO cells stably expressing the human NPY Y1
receptor.
Binding dynamics of 2 with the Y1 receptor were studied in
equilibrium binding experiments (Figure 3). Scatchard analysis
Schild analysis yielded a Kb of 209 nM, which is larger than its
affinity value as a result of the noncompetitive nature of 2.
Subsequently, PK studies of 2 were carried out in rats (Figure 5).
After intra-arterial infusion (4 mg/kg), brain uptake appeared
Figure 5. Concentration of compound 2 in brain and plasma after iv
(4 mg/kg) and oral (10 mg/kg) administration to jugular vein-
cannulated rats.
to be rapid, and the concentration in the brain remained rela-
tively constant over 3 h. Following oral administration (10 mg/kg),
a mean concentration of 2.0 μM (745 ng/mL) in brain was
achieved at 6 h before declining to 0.64 μM (242 ng/mL) by
24 h. After oral administration, a brain to plasma ratio of 0.61
was estimated from the brain and plasma AUC0 → 24 h, and
these results show that 2 had comparable or better brain
penetration than J-104870.24,25 Because the clearance of 2 from
brain is lower than that from plasma, longer dosing intervals to
maintain efficacy might be expected. In a separate PK study
(graphics not shown), 2 exhibited an excellent PK profile with
an oral bioavailability of 80%, half-life of 8.9 h, low clearance
(10.1 mL min/kg), and a Vdss of 1.8 L/kg, which showed the
drug was well distributed extravascularly.
Figure 3. Equilibrium binding experiments and Scatchard analysis of
compound 2 (50 nM) and BMS-193885 (5 nM). Data were analyzed
by LIGAND program.
found that its inhibitory effect on [125I]PYY binding to Y1
receptor in SK-N-MC cell membranes was noncompetitive. At
the concentration of 50 nM, it reduced PYY binding capacity
(Bmax of 1.1 to 0.3 nmol/mg protein) but had little effect on
PYY binding affinity (Kd = 1.1
0.3 vs 1.2
0.2 nM);
meanwhile, a known competitive antagonist, BMS-193885,21,22
at a concentration of 5 nM, did not affect PYY Bmax but did
reduce its binding affinity (Kd = 3.1 0.9 nM).
To prove that 2 is not binding irreversibly to the receptor, we
have preincubated SK-N-MC cell membrane expressing the Y1
receptor with the compound, removed it with subsequent
washes, and performed competitive binding on the compound-
Compound 2 was then evaluated for potential hepatotoxicity
using immortalized human hepatocyte cell lines expressing the
most four relevant human cytochrome isozymes: CYP1A2,
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CYP3A4, CYP2C9, and CYP2D6;31 CYP3A4 is largely res-
ponsible for drug metabolism, and polymorphisms of the other
three isozymes may contribute to hepatotoxicity. Toxicity in
these cell lines was compared with their parent cell line, THLE-
5B-c15, which does not express phase I-metabolizing activity.
End points measured following 48 h of exposure to test com-
pounds included ATP content, an indicator of cell viability. In
these tests (Table 2), 2 was relatively nontoxic in the parent
Table 2. Effect of Compound 2 on Human Hepatocytes
a
IC50 (μg/mL)
cell lines
ATP
30.3
31.4
267.6
25.9
63.7
n. red
61.2
Figure 6. Oral administration of 2 at 10 and 30 mg/kg dose to male
Sprague−Dawley rates (220−280 g) 2−2.5 h before the onset of
darkness reduced 12 h spontaneous nocturnal food intake in rats.
THLE-5B-c15
THLE-5B-1A2
THLE-5B-3A4
THLE-5B-2C9
THLE-5B-2D6
112.4
347.9
55.7
Results are presented as means
SEMs and were analyzed using
ANOVA (F = 6.8, p = 0.004) followed by Bonferroni/Dunn posthoc
test (n = 9−11, *p = 0.01, and **p = 0.001 significantly different from
vehicle). The vehicle used was 95% capmul and 5% water.
290.9
a
Cell lines were exposed to five test concentrations of 0, 5, 50, 100,
250, and 500 μg/mL. IC50 values were the average of four experiments.
ASSOCIATED CONTENT
* Supporting Information
■
S
and all recombinant cell lines, suggesting a low probability of
hepatic necrosis of 2 or its metabolites. It is estimated that
hepatic necrosis accounts for nearly 50% of all drug-related
hepatic damage, more frequent than immune-mediated hepatic
and cholestatic injury.32
Compound 2 was also evaluated for its mutagenicity in an
Ames test with Salmonella typhimurium tester strains TA98 and
TA100 in the absence and presence of induced rat liver S-9
metabolic activation. Compound 2 was not mutagenic in either
tester strain when tested at concentrations up to 5000
μg/plate.33
Compound 2 was last tested in vivo in a spontaneous noc-
turnal feeding model with male Sprague−Dawley rats at
oral dosing of 10 and 30 mg/kg. BMS-193885 was previously
shown to reduce spontaneous nocturnal food intake measured
12−15 h after intraperitoneal administration (10 mg/kg) in
rats. It also blocked centrally administrated NPY-induced food
intake in rats and significantly reduced food intake and body
weight in rats during chronic dosing of BMS-193885 (ip
10 mg/kg) for 44 days.22 However, because BMS-193885 is not
orally bioavailable, fenfluramine (1 and 3 mg/kg either orally
or ip) was used instead, in these experiments, as a positive
control; 2 was administrated 2−2.5 h before the onset of
darkness, and the amount of food consumed during a 12 h
period was recorded. As shown in Figure 6, the effect on food
intake reached statistical significance for both doses tested during
the 12 h period. The vehicle effect was observed up to the first
6 h, but the compound effect was significantly separated from the
vehicle effect beginning 9 h after the administration.
In summary, we have identified a novel series of bioavailable
NPY Y1 antagonists (e.g., 80% with 2). Despite being a non-
competitive Y1 antagonist, 2 showed significant oral efficacy in
reducing food intake in a spontaneous nocturnal feeding model,
thus contributing to the evidence that Y1 antagonists can
potentially inhibit food intake. Lastly, our results indicate that
bicyclo[3.1.0]hexane may be used, in general, as a conforma-
tionally constrained isosteric replacement of the cyclohexane
ring in drug design and discovery.
Procedure for large scale synthesis of 2 and its full charac-
terization; preparation of NPY Y1 expressed human neuro-
blastoma cells, SK-N-MC, and cell membranes; radioligand
binding studies and library screening; in vitro functional
assay; PKs and brain uptake; in vitro toxicity in cultures of
immortalized human hepatocytes; Ames reverse-mutation study
in Salmonella; and spontaneous nocturnal feeding studies upon
oral administration. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 203-677-6314. E-mail: shuanghua.hu@bms.com.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Dr. Robert Gentles for helpful discussions of the
manuscript.
■
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