Identification of potent, selective, CNS-targeted inverse agonists of the ghrelin receptor

Article abstract of DOI:10.1016/j.bmcl.2013.07.044

The optimization for selectivity and central receptor occupancy for a series of spirocyclic azetidine-piperidine inverse agonists of the ghrelin receptor is described. Decreased mAChR muscarinic M2 binding was achieved by use of a chiral indane in place of a substituted benzylic group. Compounds with desirable balance of human in vitro clearance and ex vivo central receptor occupancy were discovered by incorporation of heterocycles. Specifically, heteroaryl rings with nitrogen(s) vicinal to the indane linkage provided the most attractive overall properties.

Full text of DOI:10.1016/j.bmcl.2013.07.044

Bioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of potent, selective, CNS-targeted inverse agonists
of the ghrelin receptor
Kim F. McClure a,b, , Margaret Jackson a,b, Kimberly O. Cameron a,b, Daniel W. Kung a, David A. Perry a
⇑
Suvi T. M. Orr a, Yingxin Zhang a, Jeffrey Kohrt a, Meihua Tu a,b, Hua Gao a, Dilinie Fernando a, Ryan Jones a,
Noe Erasga a, Guoqiang Wang a, Jana Polivkova a, Wenhua Jiao a, Roger Swartz a, Hirokazu Ueno a
Samit K. Bhattacharya a,b, Ingrid A. Stock a, Sam Varma a, Victoria Bagdasarian a, Sylvie Perez a,b
Dawn Kelly-Sullivan a, Ruduan Wang a, Jimmy Kong a,b, Peter Cornelius a, Laura Michael a, Eunsun Lee a
Ann Janssen a, Stefanus J. Steyn a, Kimberly Lapham a, Theunis Goosen a
a Departments of Medicinal Chemistry, Discovery Biology, Drug Metabolism and Pharmaceutical Sciences, Pfizer Worldwide Research and Development, Eastern Point Road, Groton,
CT 06340, United States
b 620 Memorial Drive, Cambridge, MA 02319, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The optimization for selectivity and central receptor occupancy for a series of spirocyclic azetidine–piper-
idine inverse agonists of the ghrelin receptor is described. Decreased mAChR muscarinic M2 binding was
achieved by use of a chiral indane in place of a substituted benzylic group. Compounds with desirable
balance of human in vitro clearance and ex vivo central receptor occupancy were discovered by incorpo-
ration of heterocycles. Specifically, heteroaryl rings with nitrogen(s) vicinal to the indane linkage pro-
vided the most attractive overall properties.
Received 28 May 2013
Revised 14 July 2013
Accepted 22 July 2013
Available online xxxx
Keywords:
Ghrelin
Ó 2013 Elsevier Ltd. All rights reserved.
Antagonist
Inverse agonist
Receptor
Receptor occupancy
Muscarinic
Obesity is an epidemic worldwide. Estimates from the World
Health Organization indicate that obesity is increasing in urban
areas across the world in both developed and developing coun-
tries.1 More than 10% of adults worldwide are now obese. The Cen-
ters for Disease Control (CDC) estimates that over a third of
American adults have a body mass index (BMI) exceeding the ob-
ese criteria.2 Not only is the prevalence of obesity increasing, the
upper boundaries of BMI have spawned new categories such as
‘super obese’ to describe this subset of individuals.3 Fortunately,
obesity is also a preventable disease. Addressing the imbalance in
caloric intake and burn through changes in lifestyle is a major path
to prevention. Pharmacologic treatment, while limited, has re-
cently seen an increase in the available options with the approval
of Qysmia™ and BelviqÒ.4,5 The vast majority of the drug treatment
options are centrally acting compounds that work by decreasing
appetite.
N
N
O
N
N
N
N
N
Cl
O
O
O
N
S
1
2
N
MeO
GHS-R1a pIC50 = 6.67
GHS-R1a pKb = 6.40
(inverse agonist)
ClogP = 4.4
GHS-R1a pKi = 8.26
GHS-R1a pKb = 8.24
(inverse agonist)
ClogP = 3.4
mAChR M2 pKi=6.57
⇑
Corresponding author. Tel.: +1 617 551 3516; fax: +1 617 551 3082.
E-mail address: kim.f.mcclure@pfizer.com (K.F. McClure).
Figure 1. Lead structures.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.07.044
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2
K. F. McClure et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Cl
Cl
O
IMFQSESTYTFVMVSFFWFHRNFY
ISWLDYVSNQLITTAAFWYNVWYY
ISWLDYVSNQFVTTAAFWYNVWYY
ISWLDYVSNQLITTAAFWYNVWYY
GHS-R1a
CO2Me
CO2Me
M1
a
b, c
d
M2
M3
Br
N
N
N
6
7
8
Boc
Boc
Boc
Figure 2. Binding pocket comparison for GHS-R1a and mAChR M1–M3.
9
H2N
Table 1
GHS-R1a and mAChR M2 binding potencies for 3a–3g
R2
R2
Br
O
R3
5
N
N
*
e
f
N
MeO
N
N
N
N
3a-g
N
10
11
5 or 12
N
N
N
Boc
Boc
Compound R3
Configuration⁄ Human GHS-
Human mAChR
O
R1a pKi21
M2 pKi22
X
3a
3b
3c
3d
3e
3f
CH2OMe R/S
5.44 0.13
5.95a
5.15a
Me
Me
Me
i-Pr
i-Pr
i-Pr
S
R
R/S
R/S
S
5.65
4.60a
4.70a
R1
5.77 0.11
5.62 0.46
6.82 0.25
6.04a
>4.52a
>4.68
>4.53
3g
R
7.32a
Unless otherwise noted, all in vitro biology assay data are reported based on n >2 as
the geometric mean plus/minus the standard error in negative log(molar) base-10
format.
Scheme 1. General synthesis for indanes 5 and 12. Reagents and conditions: (a)
LDA (2 equiv), ClCH2I, THF, 66%, (b) LiAlH4, THF, 0 °C, 30 min 96% (c) (COCl)2, DMSO,
Et3N, CH2Cl2, 94%. (d) AcOH, NaCNBH3, MeOH, 70 °C, 20 h, >99%; (e) Pd(dppf)Cl2,
bis(pinacolato)diboron, KOAc, dioxane, 100 °C, 3 h then Pd(dppf)Cl2, Cl-het (R2),
K2CO3 (aq), 100 °C, 18 h, 80–90%; (f) HBTU, DIEA, R1-arylacetic acid, DMF, 63–99%.
a
Less than three independent replicates.
The growth hormone secretagogue receptor 1a (GHS-R1a) has
attracted significant interest for both obesity6 and diabetes7–9
indications. Although the receptor is mainly expressed in the
pituitary, it has a wide tissue distribution including the pancreas
and centers of the brain that are important for feeding and energy
homeostasis. The natural ligand for GHS-R1a is the so-called
hunger hormone ghrelin, a 28 amino acid acylated peptide with
powerful appetite stimulating effects.10,11 The challenge of
competing with this potent hormone in the CNS is made somewhat
more tractable by the observation that the N-terminal amino acids
(5–7) are largely responsible for its binding and pharmacology.12
Nevertheless, there are few reports describing compounds with
the desired pharmacologic profile, good central exposure and
acceptable pharmacokinetics.13,14 Herein we describe the progres-
sion of a series of inverse agonists that were optimized to achieve
desirable central receptor occupancy, selectivity, and predicted
human pharmacokinetic properties.
Previously we described the identification of a series of spiro-
azetidine–piperidine analogues derived from screening of our
corporate file using a ghrelin binding assay.15 The perceived advan-
tage of this chemical series was its consistent inverse agonist
pharmacology and the potential for brain exposure. Optimization
of the lead structures exemplified by 1 led to the more potent
analogue 2 by variation of the substituents at either end of the
Table 2
GHS-R1a and mAChR M2 binding potencies for indane isomers 5a–5i
O
O
5
N
N
Cl
N
N
MeO
R1
4
5a-i
R2
GHS-R1apIC50 = 7.80
mAChR M2 pKi = 5.71
N
N
Compound
R1
R2
Configuration at C5
Human GHS-R1a pKi21
Human mAChR M2 pKi22
5a
5b
5c
5d
5e
5f
5g
5h
5i
4-MeOPh–
4-MeOPh–
4-MeOPh–
3-MeO-pyridine-6-yl
3-MeO-pyridine-6-yl
3-MeO-pyridine-6-yl
3-MeO-pyridine-6-yl
3-MeO-pyridine-6-yl
3-MeO-pyridine-6-yl
Pyrimidine-2-yl
Pyrimidine-2-yl
Pyrimidine-2-yl
Pyrimidine-2-yl
Pyrimidine-2-yl
Pyrimidine-2-yl
Thiazole-2-yl
R/S
S
R
R/S
S
R
R/S
S
R
7.90 0.10a
6.56 0.06
8.20 0.17
7.43 0.00a
5.96 0.21a
7.53 0.45
7.52 0.26a
5.86 1.57
8.40 0.24
6.06 1.11
5.92 0.22
4.85 0.06
5.79 0.23
6.39 0.07
4.84 0.10
6.06 1.37
5.81 0.47
>4.25
Thiazole-2-yl
Thiazole-2-yl
Unless otherwise noted, all in vitro biology assay data are reported based on n >2 as the geometric mean plus/minus the standard error in negative log(molar) base-10 format.
a
pIC50
.
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K. F. McClure et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
3
piperidine–azetidine core (Fig. 1). Similar to the other ghrelin
antagonist efforts16 compounds such as 2 had undesired off-target
aminergic GPCR pharmacology that had to be addressed. In partic-
ular, 2 showed muscarinic acetylcholine receptor (mAChR) M2
pharmacology, which was generally observed with the compounds
that demonstrated more potent inverse agonism at the ghrelin
receptor. The cardiovascular risks associated with M2 pharmacol-
ogy, regardless of functional profile, made a large margin of
selectivity vital, especially considering the obese and/or diabetic
target patient population.
change. As shown in Table 2, the R enantiomer consistently favors
binding to GHS-R1a over mAChR M2.
The preparation of the compounds of general structure 5 and 12
is shown in Scheme 1. The route is shown for synthesis of a single
enantiomer, but initially racemates were synthesized and enantio-
mers of the final targets were separated by chiral chromatography.
As previously described, the synthesis relies on introduction of the
chiral center with the amino-indane as a component in the con-
struction of the azetidine–piperidine spirocyclic ring system.23,24
Chiral 10 is a versatile indane intermediate that allows for late-
stage introduction of heterocyclic R2 groups by Suzuki coupling.25
For triazoles such as 12a, it was necessary to form the triazole-
indane bond prior to azetidine formation (Scheme 2).
A potential approach to address this selectivity issue was to
exploit structural differences in the two receptor families. As
shown in Figure 2, the putative ligand binding region was
modeled,17 revealing an area for differentiation around the
conserved Asp3.32 for the muscarinc receptors and the correspond-
ing adjacent residue on TMIII for GHS-R1a, Glu3.33 (Ballesteros–
Weinstein nomenclature18). The hypothesis was that the same
electrostatic interaction that localizes protonated biogenic amines
to Asp3.32(103) for mAChR M219 would place the charged azetidine
portion of 2 (pKa’s measured at 4.5 and 7.9) in the same region
of GHS-R1a near the acid residue Glu3.33(124). On this basis,
GHS-R1a presented a few additional polar residues as compared
to mAChR M2. This modeling supported an approach focused on
identifying small changes in the azetidine region of the molecule
that were directed toward increasing selectivity for binding to
GHS-R1a over M2. Table 1 shows the impact of adding carbon-
based substituents to the benzylic carbon. Compounds such as 3a
that both decrease the basicity of the azetidine and present a near-
by heteroatom led to nearly equivalent binding to both M2 and
GHS-R1a.20 Similarly, there was no benefit to simple methyl substi-
tution regardless of stereochemical configuration (see 3b–d).
Isopropyl substitution (3e–g), however, provided the first signs of
significant selectivity; the fact that the outcome favored binding
to M2 was disappointing, but it confirmed the potential of this re-
gion of the molecule for pharmacologic discrimination between
the two receptors.
With an expedient stereoselective synthesis of the selectivity-
imparting azetidinyl-indane fragment, our focus turned to opti-
mizing the compounds for low metabolic clearance (human liver
microsomes) and high central receptor occupancy (RO; rat
in vivo assay).26 Since our interest was testing this mechanism in
human, little weight was placed on demonstrating weight loss in
pre-clinical species. Modification of the terminal aromatic rings
to impart physicochemical properties that were consistent with
low clearance and high brain penetration was the overall strategy
in the design of target compounds. Because clearance generally
correlated with lipophilicity, maintaining clogP <3.5 typically gave
acceptable clearance profiles. To enhance the probability of accept-
able brain exposure, compounds were designed to avoid H-bond
donors, to minimize molecular weight, and to maintain polar
surface area (PSA) <70 Å2.27 The preferred R2 groups that met these
structural constraints and imparted useful levels of potency and
selectivity were heteroaryls linked to the indane ring at the 2-posi-
tion relative to a nitrogen atom, such as pyrimidine, thiazole, and
triazole (Table 3). The preferred R1-arylacetamide groups on the
piperidine were substituted phenyl- and pyridyl-acetamides.
To evaluate the potential for brain exposure, compounds were
tested experimentally for P-gp mediated efflux by measuring the
bidirectional permeability properties of the compounds in a Ma-
din–Darby canine kidney (MDCK) cell line expressing the human
MDR-1 gene.28 Compounds with a 2.5-fold or greater efflux ratio
were viewed as having a risk of efflux, although some compounds
above this threshold achieved reasonable brain penetration as
gauged by rat in vivo receptor occupancy (e.g., 5f and 12b). Recep-
tor occupancy of GHS-R1a in rat hypothalamus was measured over
a time period of up to 8 h after a single oral dose of compound.
Several compounds with potent inverse agonist pharmacology
and relatively high receptor occupancy were identified (5c, 12a,
12h). Although we did not identify a sole parameter that was
independently useful for predicting RO, the overall set of physico-
chemical properties and in vitro assays outlined above, provided
reasonable criteria for selecting compounds to test in the RO assay.
From earlier SAR15 around 2, we recognized the importance of
the chloro group to potency, knowledge that initially deterred mak-
ing changes to this part of the molecule. This chloro group was
hypothesized to provide both conformational restriction and bene-
ficial hydrophobic interactions. In contemplating replacements for
the chloro group, the possibility of forming a hydrocarbon ring with
the adjacent benzylic center appeared to be a good way to preserve
the lipophilic interaction, restrict the conformational freedom and
change the environment around the azetidine nitrogen (see com-
pound 4, Table 2). This would also lead to the creation of a chiral
center, a change that ultimately led to an improvement in the bind-
ing selectivity for GHS-R1a over M2. As exemplified by 5a, 5d, and
5g in Table 2, the racemic mixtures mask the true potential of this
N
N
Br
N
N
N
N
a
N
+
+
N
N
H
O
O
O
13
14
15
N
N
N
N
N
c, d
b
N
16
17
HO
H2N
Scheme 2. Synthesis of triazoleindane 17. Reagents and conditions: (a) K2CO3 (1.0 equiv), Fe(acac)3 (10%), CuO (5%), DMF, 80 °C, 16 h, 52% 14 24% 15; (b) R-CBS-catalyst,
BH3ꢀTHF, THF, 0 °C, 2.5 h, 94%, 92% ee (recryst, 98% ee); (c) DPPA, DBU, 2-MeTHF; (d) LiAlH4, 2-MeTHF, 0 °C, 30 min, 96%, 98% ee (2 steps).
Please cite this article in press as: McClure, K. F.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.07.044

4
K. F. McClure et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Table 3
Rat receptor occupancy and in vitro properties
O
N
N
R1
X
R2
5c, f, i & 12a-h
b
Compound R1
R2
X
MW clog P MDR
PSA Human GHS-
R1a pKi21
Human GHS-R1a Human mAChR
pKb
CLint,app,scaled
(lL/min/mg)
ROc
(%)
a,21
efflux ratio
M2 pKi22
5c
5f
5i
12a
12b
MeO Pyrimidine-2-yl
MeO Pyrimidine-2-yl
MeO Thiazole-2-yl
MeO 1,2,3-Triazol-2-yl
MeO 5-
CH 468
2.8
1.8
2.6
3.3
2.9
2.4
3.9
2.3
2.0
4.1
59
72
59
64
72
8.20 0.17
7.76 0.13 (ꢂ39) 4.85 0.06
7.60 0.09 (ꢂ42) 4.84 0.10
7.40 0.29 (ꢂ39) >4.25
8.27 0.06 (ꢂ21) >4.26d
7.37 0.06 (ꢂ29) >4.17
37
14
24
48
20
85
45
35
74
64
N
N
469
474
7.53 0.45
8.40 0.24
8.13 0.21
8.04 0.14
CH 457
N
497
ethylpyrimidin-
2-yl
12c
12d
MeO 1,2,3-Triazol-2-yl
MeO 5-
N
N
458
483
2.3
2.3
2.6
4.5
76
72
8.48 0.28
8.04 0.14
7.57 0.76 (ꢂ42) >4.42
7.07 0.13 (ꢂ40) >4.17
25
18
50
5
methylpyrimidin-
2-yl
12e
12f
12g
12h
Et
Pyrimidine-2-yl
1,2,3-Triazol-2-yl
Pyrimidine-2-yl
1,2,3-Triazol-2-yl
N
N
N
N
467
442
453
456
2.4
2.4
1.9
2.9
3.8
1.7
2.0
1.9
62
67
62
67
7.81 0.16
7.98 0.20
7.56 0.59
7.79 0.16
7.13 0.16 (ꢂ22) 4.55 1.10d
7.23 0.35 (ꢂ40) 4.66 0.21
6.98 0.10 (ꢂ21) >4.56
50
39
22
91
10
23
0.2
74
Me
Me
Et
7.38 0.12 (ꢂ23) >4.59
Unless otherwise noted, all in vitro biology assay data are reported based on n >2 as the geometric mean plus/minus the standard error in negative log(molar) base-10 format.
a
Minimum percent effect in parentheses; a negative value indicates an inverse agonist.
Human microsomal clearance uncorrected for fraction unbound.
Receptor occupancy at 1 h from 10mpk po dose.
Less than three independent replicates.
b
c
d
13. Yu, M.; Lizarzaburu, M.; Beckmann, H.; Connors, R.; Dai, K.; Haller, K.; Li, C.;
In summary, we described the progression of a series of inverse
agonists of GHS-R1a. The initial compounds demonstrated an inad-
Liang, L.; Lindstrom, M.; Ma, J.; Motani, A.; Wanska, M.; Zhang, A.; Li, L.;
Medina, J. C. Bioorg. Med. Chem. Lett. 2010, 20, 1758.
equate selectivity margin versus the muscarinic receptor M2.
Selectivity was increased by exploiting stereochemical effects near
the basic amine center. Within a subset of indane derivatives, opti-
mization of heteroaromatic rings and substituents provided struc-
tures with high central RO and low predicted human clearance.
Clearance was largely driven by lipophilicity, but the central recep-
tor occupancy was multi-factorial and was not well predicted by
any single parameter. Ultimately, several molecules were identi-
fied that possessed a good balance of inverse agonism, central
RO, human microsomal clearance, and selectivity versus M2. How-
ever, the optimized structures were not sufficiently potent to give
us confidence to conduct human trials in the absence of the means
to measure central receptor occupancy. Our inability to develop a
CNS target biomarker, or improve the functional potency profile
enough to proceed without one, led us to terminate our CNS
approach to this target.
14. McCoull, W.; Barton, P.; Broo, A.; Brown, A. J. H.; Clarke, D. S.; Coope, G.; Davies,
R. D. M.; Dossetter, A. G.; Kelly, E. E.; Knerr, L.; MacFaul, P.; Holmes, J. L.;
Martin, N.; Moore, J. E.; Morgan, D.; Newton, C.; Osterlund, K.; Robb, G. R.;
Rosevere, E.; Selmi, N.; Stokes, S.; Svensson, T. S.; Ullah, V. B. K.; Williams, E. J.
Med. Chem. Commun. 2013, 4, 456.
15. Kung, D. W.; Coffey, S. B.; Jones, R. M.; Cabral, S.; Jiao, W.; Fichtner, M.; Carpino,
P. A.; Rose, C. R.; Hank, R. F.; Lopaze, M. G.; Swartz, R.; Chen, H. T.; Hendsch, Z.;
Posner, B.; Wielis, C. F.; Manning, B.; Dubins, J.; Stock, I. A.; Varma, S.;
Campbell, M.; DeBartola, D.; Kosa-Maines, R.; Steyn, S. J.; McClure, K. F. Bioorg.
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16. Pasternak, A.; Goble, S. D.; deJesus, R. K.; Hreniuk, D. L.; Chung, C. C.; Tota, M.
R.; Mazur, P.; Feighner, S. D.; Howard, A. D.; Mills, S. G.; Yang, L. Bioorg. Med.
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17. The binding site residue alignment was conducted with MOE: Chemical
Computing Groups, Inc. MOE 2009.09. 1255 University Street, Montreal,
Quebec, Canada, H3B 2ꢁ3.
18. Ballesteros, J. A.; Weinstein, H. In Methods in Neurosciences; Stuart, C. S., Ed.;
Academic Press, 1995; p 366.
19. Haga, K.; Kruse, A. C.; Asada, H.; Yurugi-Kobayashi, T.; Shiroishi, M.; Zhang, C.;
Weis, W. I.; Okada, T.; Kobilka, B. K.; Haga, T.; Kobayashi, T. Nature 2012, 482, 547.
20. Compounds 3a–g were prepared by reductive amination with the appropriated
ketone in the presence of Ti(iPrO)4 and Na(OAc)3BH as reported in ref.23
21. All GHS-R1a binding and functional data were generated as previously
described in ref.15
References and notes
22. The mAChR M2 binding assay was performed as follows: M2 muscarinic receptor
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lL
containing 50 g human M2 receptor (from Chinese Hamster Ovary (CHO) cells
l
2. Overweight and Obesity Fact Sheet. Centers for Disease Control: Atlanta, GA,
USA, January 2012.
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stably expressing the recombinant human M2 muscarinic receptor) coupled to
0.75 mg scintillation proximity assay (SPA) beads (wheat germ agglutinin
coated, GE Healthcare, RPNQ0060). The membranes and beads were incubated
2 h at 4 °C prior to the addition of 3 nM N-methyl [3H]-scopolamine (Perkin
Elmer Life Sciences, NET636) and varying concentrations of test compound or
vehicle. Controls were included on each assay plate to define 0% effect (ZPE)
where no compound was included in the binding reaction and 100% effect
(HPE) where 5 lM atropine was added to maximally displace the radioligand.
8. Tong, J.; Prigeon, R. L.; Davis, H. W.; Bidlingmaier, M.; Kahn, S. E.; Cummings, D.
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All reagents were diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 0.2%
BSA, EDTA-free protease inhibitors mix, pH 7.4) and reactions were incubated
for 2 h at room temperature to allow binding to reach equilibrium. The amount
of receptor–ligand complex was determined by liquid scintillation counting
using a 1450 Microbeta Trilux (Wallac). Data analysis was performed using a
proprietary software package. Briefly, the percent effect for each compound
concentration (sample) was calculated from raw data as follows:
%effect ¼ 100 ꢂ 100 ꢃ ððsample ꢂ HPEÞ=ðZPE ꢂ HPEÞÞ where HPE and ZPE
values were averages of 16 wells each. The compound% effect values were
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K. F. McClure et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
then plotted versus concentration, and the IC50 was determined using
standard 4-parameter fit algorithm. The Kb value was then calculated using the
Cheng–Prussoff equation: IC50 ¼ Kb ꢃ ð1 þ ð½Lꢄ=KdÞÞ where [L] was the
radioligand concentration in the assay and Kd was the equilibrium
dissociation constant specific for the batch of receptor membrane used in
the experiment.
5
a
(0.32 M sucrose, 5 mM Tris–HCL, pH 7.4) supplemented with 1ꢁ protease
inhibitor cocktail (Roche, Ref# 11873580001). The homogenates was first
centrifuged at 1000ꢁg for 10 min, the pellet was discarded and the
supernatant was centrifuged at 25,000ꢁg for 30 min. The resulting pellet
was resuspended in ice-cold homogenization buffer and immediately used to
determine protein concentration and for binding assay. GHS-R1a binding assay
23. Orr, S. T. M.; Cabral, S.; Fernando, D. P.; Makowski, T. Tetrahedron Lett. 2011, 52,
3618.
with hypothalamus membrane was performed using [His
(Perkin Elmer, Cat# NEX388) as radioligand. The membranes (corresponding
to 100 g protein) were incubated in replicates of six at 4 °C for 60 min, with
150 pM of [His [125I]]-Ghrelin in a final volume of 200
L assay buffer (50 mM
HEPES, 10 mM MgCl2, 0.2% BSA, pH 7.4, 1ꢁ protease inhibitor cocktail). Parallel
incubations where 10 m of compound 2 was also present were used to
[
125I]]-Ghrelin
24. Fernando, D. P.; Jiao, W.; Polivkova, J.; Xiao, J.; Coffey, S. B.; Rose, C.; Londregan,
A.; Saenz, J.; Beveridge, R.; Zhang, Y.; Storer, G. E.; Vrieze, D.; Erasga, N.; Jones,
R.; Khot, V.; Cameron, K. O.; McClure, K. F.; Bhattacharya, S. K.; Orr, S. T. M.
Tetrahedron Lett. 2012, 53, 6351.
l
l
l
25. The absolute stereochemistry for these indanes was assigned based on
anomalous dispersion X-ray experiments with the R isomer 10. The structure
can be obtained from the Cambridge Crystallographic Data Centre (CCDC) with
accession number CCDC 948624.
26. Rat hypothalamus GHS-R1a occupancy study: Male Sprague Dawley rats were
weighted and randomly assigned to different groups (8–10 rats/group). Rats
were orally administered either vehicle (0.5% methyl-cellulose) or GHS-R1a
antagonists at doses of 3, 10, 30, 60 and 100 mg/kg. 0.5, 1, 2, 4, and 8 h after
drug administration, rats were sacrificed by carbon dioxide. Cerebrospinal fluid
(CSF) and brains were rapidly removed. The hypothalamus was dissected on an
ice-cold plastic board, wrapped in aluminum foil and immediately frozen in
determine nonspecific binding. Nonspecific binding was subtracted from total
binding to yield specific binding values. The binding reaction was terminated
by adding ice-cold assay buffer followed by filtration through Glass fiber
Filtermat (Wallac, Turku, Finland, Cat# 1205-401). Filters were washed four
times with ice-cold wash buffer (50 mM HEPES, 10 mM MgCl2, pH 7.4, 0.01%
TritonX-100), and after the filters dried, the radioactivity remaining bound to
the filters was measured by
a liquid scintillation counter (Wallac, 1205
Betaplate). The receptor occupancy rate (%) was calculated and expressed as
follows: RO (%) = 100 ꢁ (1 ꢂ receptor labeling of drug-treated rats/receptor
labeling of vehicle-treated rats).
27. Hitchcock, S. A.; Pennington, L. D. J. Med. Chem. 2006, 49, 7559.
28. Feng, B.; Mills, J. B.; Davidson, R. E.; Mireles, R. J.; Janiszewski, J. S.; Troutman,
M. D.; de Morais, S. M. Drug Metab. Dispos. 2008, 36, 268.
liquid nitrogen. Frozen hypothalami were homogenized with
a power
homogenizer (Polytron PT 1200) for 1 min on ice in homogenization buffer
Please cite this article in press as: McClure, K. F.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.07.044