Identification of piperazine-bisamide GHSR antagonists for the treatment of obesity

Article abstract of DOI:10.1016/j.bmcl.2010.01.043

Piperazine-bisamide analogs were discovered as partial agonists of human growth hormone secretagogue receptor (GHSR) in a high throughput screen. The partial agonists were optimized for potency and converted into antagonists through structure-activity relationship (SAR) studies. The efforts also led to the identification of potent antagonist with favorable PK profile suitable as a tool compound for in vivo studies.

Full text of DOI:10.1016/j.bmcl.2010.01.043

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1758–1762
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of piperazine-bisamide GHSR antagonists for the treatment
of obesity
a
a
a
a
b
a
Ming Yu a, , Mike Lizarzaburu , Holger Beckmann , Richard Connors , Kang Dai , Katrin Haller , Cong Li ,
Lingming Liang a, Michelle Lindstrom a, Ji Ma a, Alykhan Motani a, Malgorzata Wanska a,
Alex Zhang a, Leping Li c, Julio C. Medina a
*
a Amgen Inc. 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
b Syntacoll Gmbh, Donaustr. 24, 93342 Saal/Donau, Germany
c Presidio Pharmaceuticals, Inc. 1700 Owens Street, Suite 585, San Francisco, CA 94158, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Piperazine-bisamide analogs were discovered as partial agonists of human growth hormone secretagogue
receptor (GHSR) in a high throughput screen. The partial agonists were optimized for potency and con-
verted into antagonists through structure–activity relationship (SAR) studies. The efforts also led to the
identification of potent antagonist with favorable PK profile suitable as a tool compound for in vivo
studies.
Received 10 November 2009
Revised 5 January 2010
Accepted 6 January 2010
Available online 20 January 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Piperazine-bisamide
Ghrelin
Growth hormone secretagogue receptor
(GHSR)
Agonist
Antagonist
Inositol phosphate (IP) assay
Pituitary assay
Obesity affects life quality and endangers the general health of a
large population, and it may replace tobacco as the number one
health risk for developed societies.1 It is estimated that about
127 million adults in the United States are overweight or obese,2
and approximately 300,000 deaths per year are directly attribut-
able to obesity, mainly due to associated heart disease, diabetes,
or cancer.3 Several other medical conditions can also be attributed
to obesity, including asthma, sleep apnea, arthritis, reproductive
complications, and psychological disturbances.3Over the years,
many therapeutic interventions have been used for the treatment
of obesity, but most of them are either complicated with safety
issues4 or limited in patient population.5
(GH) secretion, appetite and fat deposition.8 Administration of
ghrelin to rats results in weight gain as a consequence of changes
in energy intake and fuel utilization.9 Moreover, systemic ghrelin
administration in humans stimulates sensations of hunger and in-
duces overeating.10 Based on these findings, ghrelin is believed to
play a crucial role in the regulation of appetite and body weight,
and GHSR antagonists are expected to reduce appetite and food
intake, and may provide treatment of eating disorders and obes-
ity.11 Thus, we and others12 have been interested in identifying
GHSR antagonists for the treatment of obesity. Herein, we report
the discovery and optimization of piperazine-bisamide based
GHSR antagonists.
Ghrelin, a 28 amino acid peptide hormone bearing an octanoyl
side chain at the third amino acid from its N-terminus (serine 3), is
an endogenous ligand for the growth hormone secretagogue recep-
tor (GHSR).6 It is synthesized primarily in the stomach and found in
the circulation of healthy humans. The GHSR was identified to be a
G protein-coupled receptor (GPCR) located predominantly in the
pituitary gland and the hypothalamus.7 Ghrelin levels in plasma
are influenced by nutritional status and regulate growth hormone
High throughput screening of our chemical library was con-
ducted with an aequorin flash luminescence (Aeq) assay using
CHO cells stably expressing human GHSR.13 This assay measured
the inhibition of compounds on the intracellular calcium increases
induced by ghrelin (1.0 lM) and identified the piperazine-bisa-
mide 1a (Fig. 1) as a potent antagonist (IC50 = 100 nM) for the GHSR
receptor. However, further study of 1a in a human inositol phos-
phate (IP) accumulation assay demonstrated an 18% increase in
IP at 10 l
M relative to ghrelin’s maximal response.14,15 Therefore,
efforts to optimize the potency of compounds in the aequrin assay
* Corresponding author.
while reducing their partial agonist properties observed with the IP
E-mail address: yum@amgen.com (M. Yu).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.043

M. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1758–1762
1759
switched the order of the amide bond formation and thus provided
compounds 4a–b. We also designed a synthesis for the middle
B-ring replacements with heterocycles as shown on route C. Pre-
installation of the heterocyclic B-ring onto the biaryl tail of the
molecule through Suzuki coupling of the Br- or Cl-heteroaryl
methyl carboxylates with the corresponding boronic acids IX fol-
lowed by saponification provided intermediate carboxylic acid
XI. Coupling of XI with IV offered the corresponding compounds
9a–b, 10 and 11 in moderate to good yields.
O
H
N
Biaryl tail group
N
N
Head group
O
1a: h-GHSR-IC50 (Aeq) = 100 nM
Figure 1. HTS hit from high throughput screening.
Our initial lead optimization started with the modification of
the indole head group as summarized in Figure 2. Extensive explo-
ration indicated that there was limited tolerance of structural
change in this region of the molecule. All of the modifications to
the indole moiety, including N substitution (1b), linkage alteration
(1c–d) or replacement of the indole with either closely related het-
erocycles (1e–h) or more diverse chemical motifs (1i–k) resulted
assay were pursued. All compounds tested in the human or rat IP
assays were evaluated at 0.1
for purposes of comparing their maximal agonistic response in IP
assays we chose to report the compounds activity at 10 M.
lM, 1.0 lM, and 10 lM. However,
l
The synthetic route to obtain these piperazine-bisamide com-
pounds is outlined in Scheme 1. Following route A, treating com-
mercially available I (tert-butyl piperazine-1-carboxylate:n = 1, or
tert-butyl 1,4-diazepane-1-carboxylate:n = 2) with 4-biphenyl car-
boxylic acid or 30-methoxy-biphenyl-4-carboxylic acid in the pres-
ence of HBTU and Hünig’s base in DMF followed by boc-group
deprotection afforded intermediate II, which upon second amide
coupling with the corresponding aryl-carboxylic acid provided
1b–j, 2, and 5, respectively. Reductive amination of the amine
intermediate II with indole-6-carboxylaldehyde afforded 1k in
54% yield. Starting from III, this synthetic approach provided
access to bromo-substituted intermediate V, which after either
Suzuki or Stille coupling reactions gave compounds 3a–n,
12a,c,e,f, 6, 7, 8a–c, 13 or 12b,d. The intermediate IV involved in
this route also provided convenient access to compound 12g after
amide coupling with the corresponding commercial carboxylic
acids. In contrast to this approach, while route B also shared the
same starting material VI (either the S or R enantiomer), it
in significant loss of potency (IC50 > 10 lM).
While no potency improvement was achieved from the indole
head group modification, we quickly found that 3-methoxy biphe-
nyl as the tail group improved the potency by four folds in com-
pound 2 (Table 1). Using 3-methoxy biphenyl as the tail group,
our optimization was then focused on the middle piperazine core.
Compound 3a with s-CH3 group at R3 position was twice as potent
(IC50 = 12 nM) as compound 2, but all the other methylated piper-
azines (3b, 4a, and 4b) were detrimental toward potency. Enlarg-
ing the piperazine core as in compound 5 also failed to improve
the activity. After this exploration, the methylated piperazine core
in compound 3a was selected as the template for subsequent
modifications.
With the piperazine core optimized, we conducted a detailed
study of modifications to the biphenyl tail group of the molecule.
Route A
O
NH
( )n
a
b
a
R
N
( )n
Ar
R
NH
( )n
N
N
N
Boc
79-95%
79-82%
45-73%
O
O
c
I
II
1b-j, 2 : n = 1
5: n = 2
H
N
N
N
O
54%
1k
R'
R'
O
O
N
O
het
H
N
H
R''
O
H
Br
R''
N
N
NH
N
N
R
a, b
a
N
N
N
HN
Boc
79%
75-89%
O
O
III
IV
V
O
condition d: 3a-n, 12a,c,e, 6, 7, 8a-c, 13 (67-92%)
condition e: 12f (32%)
condition f: 12b,d (18-52%)
H
N
a
N
N
45%
N
O
N
12g
Route B
O
O
H
N
H
N
Br
Br
a
NH
NH
N
a, b
MeO
N
d
N
N
N
*
Boc
*
78%
88%
68%
*
*
O
O
VI
Route C
VIII
VII
O
4a-b
X
O
X
X
H
X
g
h
79-92%
i
63-81%
N
N
het
N
Het
Het
23-79%
B(OH)2
CO2Me
CO2H
IX
X = H; F; Cl or CH3
X
XI
O
9a-b, 10, 11
Scheme 1. Reagents and conditions: (a) ArCO2H, HBTU, iPr2NEt, DMF, rt, 0.5–3.5 h; (b) 20% TFA/CH2Cl2, rt to 50 °C 3.5–5.0 h or 4.0 N HCl/dioxane, rt, 1.5–2.0 h; (c) indole-6-
carboxylaldehyde, NaB(OAc)3H, dichloroethane, 55 °C, 45 min; (d) ArB(OH)2, Pd(PPh3)4, DMF, Cs2CO3, 80–100 °C, 1.0–4.0 h; (e) 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)-1H-pyrazole, [Pd(PPh3)2Cl2]2, DMF, Cs2CO3, 110 °C, 3.5 h, 32%; (f) ArSnBu3, [Pd(PPh3)2Cl2]2, DME, K3PO4, 90–100 °C, 3.0–6.0 h, 18–52%; (g) Br-ArCO2Me or Cl-ArCO2Me,
i
[Pd(PPh3)2Cl2]2, Cs2CO3, DMF, 80–100 °C, 1.5–4.5 h; (h) LiOH, dioxane/H2O (4:1), 1.5–3.0 h; (i) IV, HBTU, Pr2NEt, DMF, rt; 1.0 h.

1760
M. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1758–1762
derivative 3f with the optimized piperazine core was also a very
potent compound.
O
H
R
N
O
N
H
N
N
With potency optimized, we decided to further evaluate the
partial agonist activity. For this purpose, compound 3h was se-
lected as a tool compound partially based on its favorable PK prop-
erties.16 Like our lead compound, 1a, compound 3h continued to
behave as a partial agonist in the IP assay (Fig. 3 and Table 3).
CHO cells expressing the human GHSR receptor treated with
N
O
O
1a: R = H
1b: R = Me
1c
1d
O
O
O
X
H
N
N
Y
N
10 lM, 1.0 lM or 0.1 lM concentration of compound 3h afforded
1e: X = NH, Y = CH
1f: X = NH, Y = N
1g: X = S, Y = CH
1h
a 33%, 31%, and 21% increase in inositol phosphate relative to ghre-
lin’s maximal increase.14 Moreover, compound 3h also acted as a
partial agonist in rat IP assays17 and in an ex vivo primary rat pitu-
itary cell assay,18 wherein it stimulated rat growth hormone (GH)
secretion equivalent to 24% of the maximal ghrelin response at
1i
1k
Figure 2. Early exploration of indole head group replacements.
1.0 lM. In addition, the partial agonist activity seemed rather pre-
Table 1
SAR of piperazine ring modification
valent on this lead series and all the potent compounds from Table
2 (3a, 3f, and 3h–l) demonstrated partial agonist activity in both
the IP and rat ex vivo assays.
O
H
N
R1
N
( )n
In order to decrease the agonist activity, we modified the phe-
nyl B-ring of the biphenyl moiety (Table 4). Because of the good
correlation of the agonist activity readouts across the three assays
in Table 3, the rat IP assay was chosen to guide the lead optimiza-
tion efforts. In short, several B-ring modifications decreased the
agonist activity, such as in compounds 8a–11. But many of these
modifications also led to loss of potency. The fluorine substitution
on the B-ring (8a) well maintained the potency.
N
R3
R2
R3
O
R1
n
R2
h-GHSR, Aeq IC50 (nM)
a,b
Compound
1ª
2
3a
3b
4a
4b
5
H
1
1
1
1
1
1
2
H
H
H
H
H
H
100
25
12
640
280
68
OMe
OMe
OMe
OMe
OMe
H
-(S)-Me
-(R)-Me
-(S)-Me
-(R)-Me
H
H
H
H
The breakthrough of eliminating agonist activity came from fur-
ther exploration on the A-ring of the biphenyl tail (Table 5).
6500
a
Values were the means of three determinations, standard derivation was 630%.
See Ref. 13 for assay protocol.
b
1.2
1.0
Ghrelin
As illustrated in Table 2, with compound 3a as a starting point, we
investigated several other meta substituents for the terminal phe-
nyl A-ring, but found that this meta position was much less toler-
ant of electron withdrawing groups, with 3–11 folds loss of
potency on compounds 3c–e. This was in contrast to the observa-
tion made at the ortho position, wherein almost all the substitu-
tions (3h–k) studied improved the potency as compared to 3a,
except for the 2-methoxy group (3g). Substitutions on the para po-
sition had similar effects as those on the meta positions: the elec-
tron donating methoxy group (3l) was more potent than electron
withdrawing ones (3m–n). It is noteworthy that the unsubstituted
0.8
0.6
0.4
3h
0.2
0.0
a
Substance P
-0.2
-0.4
-6
-4
-2
0
2
Log[Cmpd] µM
Figure 3. Partial agonist activity of compound 3h in human IP assay. aThe reference
compound was chosen as substance P, a peptide inverse agonist of the GHSR;
bvalues were the means of three determinations, p <0.005; csee Ref. 14 for assay
protocol.
Table 2
Substitution SAR of terminal phenyl ring
O
X
H
N
A
N
B
N
Table 3
Agonist activity as %fraction of maximal ghrelin response
O
Compound
In vitro IPa,b,c
Human
Rat ex vivo pituitarya,d,e
a,b
Compound
X
h-GHSR, Aeq IC50 (nM)
Rat
3a
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3-OMe
3-OCF3
3-CF3
3-F
12.0
46.5
77.0
147.0
6.0
26.0
9.1
2.1
4.1
2.9
7.0
136.0
64.0
f
1a
3h
3a
3f
3i
3j
3k
3l
18%
33%
45%
29%
35%
24%
32%
33%
14%
33%
46%
26%
32%
28%
31%
34%
—
24%
40%
f
—
H
24%
21%
18%
2-OMe
2-F
2-Cl
2-CF3
2-Me
4-OMe
4-OCF3
4-F
f
—
a
b
c
d
e
f
Values were the means of three determinations, standard derivation was 630%.
%Fraction of maximal ghrelin response at 10
See Ref. 14 for assay protocol.
%Fraction of maximal ghrelin response at 1.0
See Ref. 18 for assay protocol.
Data not obtained.
l
M.
l
M.
a
Values were the means of three determinations, standard derivation was 630%.
b
See Ref. 13 for assay protocol.

M. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1758–1762
1761
Table 4
detectable agonist activity in the rat IP assay. Other heterocyclic
replacements of phenyl A-ring all reduced the agonist activity,
but not as completely or as potent as 4-pyridyl.
Combination of the optimal features discovered on biphenyl A-
ring and B-ring, respectively provided a set of compounds (8b–c,
12a, and 13, Table 6) that were free of agonist activity on the IP as-
say. Compound 8b19 was the most potent in the rat and human
aequorin assays, therefore, it was chosen for further evaluation.
This compound displayed high in vitro metabolic stability across
species. After 30 min incubation in liver microsomes, the percent-
SAR of middle B-ring to reduce agonism
X
O
A
H
N
N
B
N
O
Compound
X
B-ring
h-GHSR, Aeq
IC50 (nM)
Rat IP
%agonisma,c,d
a,b
3h
3k
F
9.1
2.9
33%
31%
age remaining of 8b (initial concentration: 1.0 lM) was 78% and
CH3
87% for rat and human, respectively. The in vivo pharmacokinetic
properties of 8b were evaluated in mouse. After iv administration
of 8b at dosage of 0.5 mg/kg, the clearance (Cl) was 1.96 L/h/kg, the
MRT was 0.70 h and the Vdss was 1.4 L/kg. This compound also had
decent unbound free drug fraction in both mouse and rat plasma
(fub = 4%, respectively). Although compound 8b showed high affin-
ity toward p-glycoprotein (p-gp) transporter (efflux ratio = 14) and
low brain/plasma AUC ratio (0.16) in FVB mice, this compound was
able to achieve excellent brain uptake in mdr1a knockout mice
(brain/plasma AUC ratio = 2.3),20 which ensured sufficient CNS
exposure for in vivo proof-of-concept studies in this animal. In
addition, compound 8b also exhibited good selectivity over a
screening against our internal receptor panel.21
F
6
7
H
H
17.7
14.3
43%
36%
Me
F
8a
CH3
9.0
16%
N
9a
9b
H
Cl
20.0
13.0
19%
14%
N
10
11
Cl
F
37.2
33.7
18%
14%
The eradication of agonist activity in compound 8b was also
confirmed in the ex vivo growth hormone (GH) release experiment
conducted in isolated primary rat pituitary cells as shown on
Figure 4. Compound 8b did not produce any noticeable GH
S
N
a
b
c
Values were the means of three determinations, standard derivation was 30%.
See Ref. 13 for assay protocol.
Table 6
Summary of top compounds
%Fraction of maximal ghrelin response at 10
See Ref. 14 for assay protocol.
lM.
d
X
O
N
Y
H
Table 5
N
N
SAR of terminal phenyl A-ring to eliminate agonism
N
O
A
H
N
O
a,b
a,c,d
N
Compound
X
Y
Aeq IC50 (nM)
Rat IP
%agonism
N
Human
Rat
O
12a
8b
8c
H
H
F
H
F
F
12.7
12.2
42.0
31.0
20.5
10.3
45.0
49.0
ꢀ2%
ꢀ16%
ꢀ1%
ꢀ2%
Rat IP %agonisma,c,d
a,b
Compound
A-ring
h-GHSR, Aeq IC50 (nM)
F
13
F
H
3h
9.1
33%
a
b
c
Values were the means of three determinations, standard derivation was 630%.
See Ref. 13 for assay protocol.
%Fraction of maximal ghrelin response at 10
See Ref. 14 for assay protocol.
N
12a
12b
12.7
42.2
ꢀ2%
lM.
d
N
12%
N
12c
12d
143.0
65.0
21%
5%
125
100
75
8b
rGhrelin
N
S
N
O
12e
244.6
5%
50
HN
N
12f
123.0
338.7
17%
25
O
c
12g
—
N
0
a
b
c
Values were the means of three determinations, standard derivation was 30%.
See Ref. 13 for assay protocol.
-25
-4
-3
-2
-1
0
1
2
%Fraction of maximal ghrelin response at 10
See Ref. 14 for assay protocol.
lM.
d
Log [CMPD] µM
Figure 4. Agonist activity of ghrelin versus 8b on growth hormone (GH) release in
primary rat pituitary cells. Values were the means of three determinations,
p <0.005; see Ref. 18 for assay protocol.
Employing 4-pyridyl as A-ring provided compound 12a, which
maintained potency in the aequorin assay while showing no

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25
0
-25
-4
-2
0
2
Log [CMPD] µM
Figure 5. Antagonist effect of compound 8b on rghrelin (0.2
hormone (GH) release in primary rat pituitary cells. Values were the means of three
determinations, p <0.005; see Ref. 18 for assay protocol.
l
M) induced growth
secretion at up to 10
the stimulating effect of ghrelin (0.2
pituitary cells (IC50 = 93 nM), as shown in Figure 5.
l
M concentration. It could also antagonize
M) on GH release from rat
l
In summary, we optimized a series of piperazine-bisamide
based GHSR inhibitors for potency and removed the partial agonist
activity seen with the early lead compounds in the IP assays. The
efforts led to the discovery of tool compound 8b, which was fea-
tured with high potency, satisfactory PK profile and sufficient
CNS exposure in mdr1a knockout mice. The compound was also
confirmed to be an antagonist in the ex vivo study of GH release
from isolated primary rat pituitary cells. Compound 8b was proved
to be a useful tool for evaluation in in vivo proof-of-concept studies
in mouse, and the results will be published in due course.
13. This aequorin flash luminescence (Aeq) assay used
a CHO-K1 cell line
expressing the human ghrelin receptor and the aequorin gene (purchased
from Euroscreen, Belgium). The assay was performed as previously described
by An et al. and Bandoh et al.: (a) An, S.; Bleu, T.; Zheng, Y.; Goetzl, E. J. Mol.
Pharm. 1998, 54, 881; (b) Bandoh, K.; Aoki, J.; Hosono, H.; Kobayashi, S.;
Kobayashi, T.; Murakami-Murofushi, K.; Tsujimoto, M.; Arai, H.; Inoue, K. J. Biol.
Chem. 1999, 274, 27776.
14. These assays measured the relative magnitude of inositol phosphate (IP)
accumulation that induced by subject compounds as percentage of the
maximal ghrelin’s response at concentrations of 10, 1.0, and 0.1 lM. The
assay protocol was as the following: The CHO cells stably expressing human or
rat GHSR placed in 96-well TC plates were incubated for 12 h with 3H inositol
(1 lCi/mL in DMEM solution). The cells were then incubated with ghrelin or
Acknowledgments
subject compounds at 37 °C for 1.0 h followed by treatment with 20 mM
formic acid at 4 °C for 4 h. The formic acid solution was extracted to Amersham
SPA beads pre-placed in 96-well plates. After incubation in dark for 12 h,
radioactivity was counted on Top Counting.
We thank Dr. Jiwen Liu and Dr. Songli Wang for constructive
discussions.
15. Compound 1a also behaved as a partial agonist in the IP assay with CHO cells
References and notes
stably expressing rat GHSR. In this assay 1a was evaluated at 10
and 0.1 M and afforded a 14%, 14%, and 24% in IP accumulation relative to
ghrelin’s maximal response, respectively.
lM, 1.0 lM,
l
1. Marshall, E. Science 2004, 304, 804.
2. (a) Ogden, C. L.; Carroll, M. D.; Curtin, L. R. J. Am. Med. Assoc. 2006, 295, 1549; (b)
Melnikova, I.; Wages, D. Nat. Rev. Drug Disc. 2006, 5, 369; (c) Baskin, M. L.; Ard,
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3. Gale, S. M.; Castracane, V. D.; Mantzoros, C. S. J. Nutr. 2004, 134, 295.
4. A representative example is the vascular heart disease associated with the
combined use of fenfluramine and phentermine, see: (a) Connolly, H.; Cray, J.
L.; Mcgoon, M. D.; Hensrud, D. D.; Edwards, B. S.; Edwards, W. D.; Schaff, H. V.
N. Eng. J. Med. 1997, 37, 581–588; For other examples, see: (b) Padwal, R.;
Majumdar, S. The Lancet 2007, 369, 71; (c) Nisoli, E.; Carruba, M. O. Obes. Rev.
2001, 1, 127; (d) Kolanowski, J. Drug Safety 1999, 20, 119.
5. Bray, G. A.; Greenway, F. L. Endocr. Rev. 1999, 20, 805.
6. Kojima, M.; Hosoda, H.; Date, Y.; Nakazato, M.; Matsuo, H.; Kangawa, K. Nature
1999, 402, 656.
7. (a) Jeffery, P. L.; Herington, A. C.; Chopin, L. K. J. Endocrinol. 2002, 172, R7; (b)
Zigman, J. M.; Jones, F. E.; Lee, C. E.; Saper, C. B.; Elmquist, J. K. J. Comp. Neurol.
2006, 294, 528.
8. (a) Cummings, D. E.; Overduin, J.; Foster-Schubert, K. E. Curr. Opin. Endocr.
Diabetes 2005, 12, 72; (b) Camina, J. P.; Carreira, M. C.; El Messari, S.; Llorens-
Cortes, C.; Smith, R. G.; Casanueva, F. F. Endocrinology 2004, 145, 930–940; (c)
Nakazato, M.; Murakami, N.; Date, Y.; Kojima, Y.; Matsuo, H.; Kangawa, K.;
Matsukura, S. Nature 2001, 409, 194; (d) Wren, A. M.; Small, C. L.; Abbott, C. R.;
Dhillo, W. S.; Seal, L. J.; Cohen, M. A.; Batterham, R. L.; Taheri, S. A.; Ghatei, M.
A.; Bloom, S. R. Diabetes. 2001, 50, 2540; (e) Hataya, Y.; Akamizu, T.; Takaya, K.;
Kanamoto, N.; Ariyasu, H.; Saijo, M.; Moriyama, K.; Shimatsu, A.; Kojima, M.;
Kangawa, K.; Nakao, K. J. Clin. Endocrinol. Metab. 2001, 86, 4552; (f) Peino, R.;
Baldelli, R.; Rodriguez-Garcia, J.; Rodriguez-Segade, S.; Kojima, M.; Kangawa,
K.; Arvat, E.; Ghigo, E.; Dieguez, C.; Casanueva, F. F. Eur. J. Endocrinol. 2000, 143,
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16. Compound 3h displayed reasonable rodent PK profile (rat iv @ 2.0 mg/kg:
Cl = 2.1 L/h/kg, MRT = 1.9 h, Vdss = 3.7 L/kg; po @ 0.5 mg/kg: F = 17%), sufficient
brain uptake (brain/plasma AUC ratio = 0.26 in FVB mice), and high potency
across species (IC50 = 2.0 nM in rat aequorin assay). This compound had no
hERG or CYP inhibition liability (IC50 > 10
17. Compound 3h afforded a 33%, 42%, and 41% relative to ghrelin’s maximal
increase in the IP assay using CHO cells stably expressing rat GHSR at 10 M,
1.0 M, and 0.1 M, respectively.
18. The assay protocol was as the following: pituitary cells freshly collected from
6–8 weeks old male SD rats were plated onto 96-well poly- -lysine coated
lM).
l
l
l
D
plates and incubated at 37 °C for 3 days. The cells were then washed with
DMEM assay buffer containing 20 mM Hepes and 0.3% BSA, and incubated at
37 °C for 5 min. For agonist activity experiment, the cells were treated with rat
ghrelin or subject compounds for 20 min. For antagonist activity experiment
(8b), the cells were pretreated with the subject compounds for 10 min before
co-incubation with rat ghrelin and the subject compounds for additional
20 min. The sample was taken for rat growth hormone secretion readout by
RIA assay (LINCO Research).
19. The analytic data for compound 8b: 1H NMR (500 MHz, CDCl3): d ppm 1.24 (br s,
3H), 2.97–3.78 (m, 4H), 4.16–4.80 (m, 3H), 6.53 (br s, 1H), 7.08 (d, J = 8.1 Hz,
1H), 7.22–7.27 (m, 1H), 7.29 (dd, J = 7.7, 7.7, 2 Hz), 7.42–7.57 (m, 4H), 7.61 (d,
J = 8.1 Hz, 1H), 8.69 (m, J = 5.0 Hz, 2H), 9.92 (br s, 1H); LC/MS: MS ESI (pos) m/e:
443.2 (M+H)+.
20. The cell membrane permeability of this compound in the human MDR1–MDCK
cell assay is: PaapB-A/PaapA-B = 1.7.
21. This lead series (e.g., 1a, 3a–n) is highly selective (Ki > 10
5HT2 5HT2 SERT, Adr 2A, D2, D3, DAT, Opioid Opioid
NET, and H2.
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