Combination of a beta adrenoceptor modulator and a norepinephrine-serotonin uptake inhibitor for the treatment of obesity

Article abstract of DOI:10.1021/ml200071k

We report the novel combination of a selective beta adrenoceptor modulator and a norepinephrine-serotonin uptake inhibitor (sibutramine) with potential for the treatment of obesity. The synthesis and characterization of 6-[4-[2-[[(2S)-3-(9H-carbazol-4-ylo

Full text of DOI:10.1021/ml200071k

LETTER
pubs.acs.org/acsmedchemlett
Combination of a Beta Adrenoceptor Modulator and a
Norepinephrine-Serotonin Uptake Inhibitor for the Treatment of Obesity
Cynthia D. Jesudason,* James E. Baker, Robert D. Bryant, Jack W. Fisher, Libbey S. O’Farrell,
Gregory A. Gaich, Minxia M. He, Steven D. Kahl, Aidas V. Kriauciunas, Mark L. Heiman, Mary A. Peters,
†
Christopher J. Rito, Julie H. Satterwhite, Frank C. Tinsley, William G. Trankle, and Anthony J. Shuker
Lilly Research Laboratories, Eli Lilly & Company, Lilly Corporate Center, Indianapolis, Indiana 46285, United States
S Supporting Information
b
ABSTRACT: We report the novel combination of a selective beta adrenoceptor modulator and a
norepinephrine-serotonin uptake inhibitor (sibutramine) with potential for the treatment of
obesity. The synthesis and characterization of 6-[4-[2-[[(2S)-3-(9H-carbazol-4-yloxy)-2-hydro-
xypropyl]amino]-2-methylpropyl]phenoxy]pyridine-3-carboxamide (LY377604), a human β3-
adrenergic receptor agonist and β - and β -adrenergic receptor antagonist with no sympathomi-
1
2
metic activity at the β - and β -adrenergic receptors, is reported. Some in vivo data in both rats and humans is presented.
1
2
KEYWORDS: Selective beta adrenoceptor modulator, β -adrenergic receptor agonist, norepinephrine-serotonin uptake inhibitor,
3
sibutramine, LY377604, obesity
besity has increasingly been cited as a major health issue in
1
O
recent decades. While many industrialized countries have
experienced similar increases, obesity rates in the United States
are among the highest in the world, with thirty-three states
having a prevalence equal to or greater than 25% and nine of
2
these have a prevalence equal to or greater than 30% in 2009.
Three drugs have been marketed for the long-term treatment
of obesity: orlistat, which inhibits pancreatic lipase and conse-
quently fat digestion; sibutramine, which acts centrally to inhibit
norepinephrine and serotonin reuptake; and rimonabant, which
antagonizes the cannabinoid receptor-1 in both the brain and
peripheral tissues. Rimonabant and sibutramine have recently
been withdrawn, owing to concerns about psychiatric side effects
and cardiovascular safety concerns, respectively. Orlistat has had
modest sales, in part because of its side effects and limited
efficacy.
Figure 1. Selected β adrenergic receptor agonists reported in the
literature.
3
The discovery of a third β-adrenergic receptor in the early
1
980s and the finding that stimulation of this receptor by
1
2
selective agonists leads to marked weight loss and glycemic
improvements in rodent models of obesity and diabetes, respec-
tively, have led to intensive research efforts to develop β3
adrenergic agonists for the treatment of obesity and Type 2
diabetes in humans.
markedly increases thermogenesis in rodents, and in fat tissue it
stimulates oxidation of free fatty acids and lipolysis. Early
clinical studies on BRL 35135 and BRL 26830A (1 and 2,
Figure 1) support the potential antihyperglycemic and antiobe-
sity effects of β -adrenergic receptor agonists in humans.
The role of brown adipose tissue (BAT) metabolism in humans
is still debated, and there is disagreement, for example, on the
mRNA. Furthermore, the effects of β -receptor stimulation
3
within BAT have been demonstrated in human subjects or tissues
isolated from humans using the nonselective β-receptor agonist
isoproterenol in the presence of β
blockade or by more selective “atypical” partial β
3
agonists, such as CGP 12177 (3, Figure 1).
treatment with β
preadipocytes to BAT in both rats and humans. These data
suggest that the amount and the metabolic activity of BAT may
be increased with chronic β
studies with our human β -adrenergic receptor agonist LY377604
3
- and β
-adrenergic receptor
-receptor
In addition,
-receptor agonists results in differentiation of
3
1
2
3
À5
Activation of the β -adrenergic receptor
3
13À15
6,7
16
8
À10
17,18
-receptor agonist therapy.
3
Clinical
3
11
amount of BAT that is present in adults. It is clear, however,
that, within brown adipocytes, significant levels of uncoupling
protein mRNA have been detected, along with β -receptor
Received: March 14, 2011
Accepted: May 23, 2011
Published: May 23, 2011
3
r 2011 American Chemical Society
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dx.doi.org/10.1021/ml200071k
|ACS Med. Chem. Lett. 2011, 2, 583–586

ACS Medicinal Chemistry Letters
4) have shown an increase in energy expenditure in healthy male
LETTER
(
ovary (CHO) cells. The compound was tested and found to be a
potent agonist (EC50 = 2.4 nM, n = 14) and to cause a maximal
increase in cyclic adenosine monophosphate (cAMP) levels that
was approximately 74% of the maximal response induced by
isoproterenol. LY377604 did not stimulate cAMP accumulation
1
9
subjects, and this is further corroborated by single dose data
20,21
from L-796,568 (5) in obese subjects.
One dose-related side effect of sibutramine (6, Figure 2)
treatment is an increase in blood pressure and heart rate due to
activation of the sympathetic nervous system. We postulated that
dosing in combination with LY377604 would ameliorate this by
taking advantage of the β and β blocking properties of this
in CHO cells transfected with either the human β
receptor or the human β adrenergic receptor. Interestingly, this
lack of efficacy to stimulate cAMP production was not a result of
poor binding to the human β and β adrenergic receptors.
LY377604 bound with high affinity to membranes expressing
the human β adrenergic receptor and the human β adrenergic
adrenergic
1
2
1
2
compound, in addition to perhaps giving a synergistic increase in
1
2
body weight loss in humans.
The synthesis of LY377604 (4) is outlined in Scheme 1.
22
A
1
2
mixture of 4-hydroxybenzyl alcohol (7) and 2-nitropropane was
heated with potassium tert-butoxide using a DeanÀStark trap
to provide 4-(2-methyl-2-nitropropyl)phenol (8), which was
hydrogenated to yield the corresponding amine (9). The next
receptor. The in vitro data for LY377604 is summarized in Table 1.
step in the synthesis involved a based-promoted S Ar, where-
N
by 4-(2-methyl-2-aminopropyl)phenol (9) was treated with
powdered potassium carbonate and 6-chloronicotinamide to
provide the coupled product (10) in 86% yield. In the final
2
3
step, (S)-4-(oxiranylmethoxy)-9H-carbazole is treated with
equiv of the amine (10) to provide LY377604 (4) as a free
base which is subsequently converted to the HCl salt or
2
2
4
hemisuccinate.
LY377604 was initially examined for its ability to stimulate the
human β adrenergic receptor expressed in Chinese hamster
3
Figure 3. LY377604 decreases the respiratory quotient in diet-induced
obese male LongÀEvans rats for 4 h at both 3 and 10 mg/kg orally (p <
0
.05 ANOVA, SNK). The respiratory quotient was measured by indirect
Figure 2. Structure of sibutramine.
calorimetry.
a
Scheme 1
a
Reagents and conditions: (a) 2-nitropropane, KOtBu, diglyme, 132 °C; (b) 5% Pd/C, H , 50 psi, HOAc, MeOH/EtOH, 50 °C; (c)
2
6
-chloronicotinamide, K CO , DMAC/iso-octane 140 °C; (d) (S)-4-(oxiranylmethoxy)-9H-carbazole, MeOH, 60 °C; (e) HCl, EtOAc; (f) succinic
2
3
acid (0.5 equiv), EtOH.
a
Table 1. Beta Adrenergic Binding and Functional Activities of LY377604 HCl or LY377604 Hemisuccinate
3
3
human β
3
functional
rat β
cAMP accumulation
max (%) EC50 (nM) max (%)
3
functional
human β
cAMP accumulation
EC50 (nM) max (%) EC50 (nM)
2
functional
human β
1
functional
human β
2
radioligand
1
human β radioligand
cAMP accumulation
cAMP accumulation
binding
binding
b
b
EC50 (nM)
E
E
E
Emax (%)
K
i
(nM)
i
K (nM)
2
.4 ( 0.2
n = 14)
73.8 ( 1.0
(n = 14)
5.0 ( 1.2
(n = 12)
55.5 ( 2.0
(n = 12)
>3160
<10
>3160
<10
0.012 ( 0.002
(n = 4)
0.023 ( 0.005
(n = 4)
(
(n = 4)
(n = 4)
(n = 4)
(n = 4)
a
b
Standard deviations are indicated by ( of the geometric mean, and the number of runs is indicated by (n). Data obtained on
LY377604 hemisuccinate.
3
5
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ACS Medicinal Chemistry Letters
LETTER
by a slower terminal elimination phase; the estimated half-life
was 9 h after a 60-mg dose.
In preparation for a phase II obesity study of LY377604 in
combination with sibutramine, it was determined that LY377604
did not alter the pharmacokinetics of sibutramine or its active
metabolites over the dosing range studied (45À300 mg) in
human volunteers. All dose levels of LY377604 blocked the
cardiovascular effects of a 30 mg dose of sibutramine to a similar
extent and demonstrated net decreases from baseline for heart rate
(
À10.5 bpm, Figure 5), systolic blood pressure (À5.5 mmHg), and
diastolic blood pressure (À4.2 mmHg). Results from the phase II
obesity study for this combination will be reported in due course.
’
ASSOCIATED CONTENT
S
Supporting Information. Synthetic procedures and
b
Figure 4. LY377604 increases caloric expenditure in diet-induced obese
male LongÀEvans rats for 4 h at 10 mg/kg orally (p < 0.05 ANOVA,
SNK). Energy expenditure was measured by indirect calorimetry.
characterization data for all compounds, protocols for the human
β adrenergic functional and binding assays, and DIO rat obesity
studies. This material is available free of charge via the Internet at
http://pubs.acs.org.
’
AUTHOR INFORMATION
Corresponding Author
*
Telephone: 317-276-7984. Fax: 317-277-7287. E-mail: jesudason_
cynthia_d@lilly.com.
Notes
In memoriam of Anthony J. Shuker, who passed away on March
†
2
6, 2009.
’
ACKNOWLEDGMENT
We thank Robert D. Boyer, Lawrence A. Goodwin, and
Douglas F. Schmidt for analytical support, Don Bennett for
assay support, and Marlene L. Cohen for advice and mentorship.
’
ABBREVIATIONS
β , beta 1; β , beta 2; β , beta 2; LY377604, 6-[4-[2-[[(2S)-
1
2
3
Figure 5. LY377604 blocks the sibutramine-induced increase in heart
rate produced by a 30 mg dose of sibutramine at all dose levels in healthy
volunteers.
3
-(9H-carbazol-4-yloxy)-2-hydroxypropyl]amino]-2-methylpr-
opyl]phenoxy]pyridine-3-carboxamide; cAMP, cyclic adenosine
monophosphate; ANOVA, analysis of variance; SNK, StudentÀ
NewmanÀKeuls
Administration of LY377604 to male LongÀEvans rats fed a
caloric dense diet resulted in stimulation of lipid utilization. This
was observed as a decrease in respiratory quotient and persisted
for about 4 h before returning to that measured from vehicle-
’
REFERENCES
(1) Kelly, T.; Wang, W.; Chen, C.-S.; Reynolds, K.; He, J. Global burden
25
of obesity in 2005 and projections to 2030. Int. J. Obes. 2008, 32, 1431–1437.
treated rats (Figure 3). The increase in lipid utilization was
dependent on dose. This was accompanied by a concomitant
dose-dependent increase in energy expenditure (Figure 4). The
increased fuel consumption was also maintained for about 4 h.
We have previously reported the clinical confirmation of this
increase in energy expenditure and reduction in respiratory
quotient in a placebo controlled phase I dose escalation study
(
(
2) http://www.cdc.gov/obesity/data/trends.html.
3) de Souza, C. J.; Burkey, B. F. Beta 3-Adrenoceptor Agonists as
Anti-diabetic and Anti-obesity Drugs in Humans. Curr. Pharm. Des.
2001, 7, 1433–1449.
(4) Weyer, C.; de Souza, C. J. Development of β -Adrenoceptor
3
Agonists as Antiobesity and Antidiabetes Drugs in Humans: Current
Status and Future Prospects. Drug Dev. Res. 2000, 51, 80–93.
(5) Arch, J. R. S. The discovery of drugs for obesity, the metabolic
effects of leptin and variable receptor pharmacology: perspectives from
1
9
of LY377604. Single doses of LY377604 also resulted in the
expected β adrenergic antagonist activity as assessed by venous
2
β
2
3
adrenoceptor agonists. Naunyn-Schmiedeberg's Arch. Pharmacol.
008, 378, 225–240.
6) Arch, J. R. S.; Ainsworth, A. T.; Cawthorne, M. A.; Piercy, V.;
tonometry during localized isoproterenol administration in a
1
9
dorsal hand vein.
(
In healthy volunteers, LY377604 was rapidly absorbed, with
maximum plasma concentrations achieved at approximately 2 h.
LY377604 showed a rapid initial decline from plasma, followed
Sennitt, M. V.; Thody, V. E.; Wilson, C.; Wilson, S. Atypical β-adrenoceptor
on brown adipocytes as target for anti-obesity drugs. Nature 1984,
309, 163–165.
5
85
dx.doi.org/10.1021/ml200071k |ACS Med. Chem. Lett. 2011, 2, 583–586

ACS Medicinal Chemistry Letters
7) Arch, J. R. S.; Ainsworth, A, T.; Ellis, R. D. M.; Piercy, V.; Thody,
LETTER
(
V. E.; Thurlby, P. L.; Wilson, C.; Wilson, S.; Young, P. Treatment of
obesity with thermogenic β-adrenoceptor agonists: studies on BRL
26830A in rodents. Int. J. Obes. 1984, 8 (Suppl 1), 1–11.
(8) Connacher, A. A.; Bennet, W. M.; Jung, R. T.; Rennie, M. J.
Metabolic effects of three weeks administration of the beta-adrenoceptor
agonist BRL 26830A. Int. J. Obes. Relat. Metab. Disord. 1992, 16, 685–694.
(9) Mitchell, T. H.; Ellis, R. D.; Smith, S. A.; Robb, G.; Cawthorne,
M. A. Effects of BRL 35135, a beta-adrenoceptor agonist with novel
selectivity, on glucose tolerance and insulin sensitivity in obese subjects.
Int. J. Obes. 1989, 13, 757–766.
(
10) Cawthorne, M. A.; Sennitt, M. V.; Arch, J. R.; Smith, S. A. BRL
5135, a potent and selective atypical beta-adrenoceptor agonist. Am. J.
Clin. Nutr. 1992, 55 (1 Suppl), 252S–257.
11) Nedergaard, J.; Bengtsson, T.; Cannon, B. Unexpected evi-
3
(
dence for active brown adipose tissue in adult humans. Am. J. Physiol.
Endocrinol. Metab. 2007, 293, E444–E452.
(12) Champigny, O.; Ricquier, D. Evidence from in vitro differen-
tiating cells that adrenoceptor agonists can increase uncoupling protein
mRNA level in adipocytes of adult humans: an RT-PCR study. J. Lipid
Res. 1996, 37, 1907–1914.
(13) Lonnqvist, F.; Krief, S.; Strosberg, A. D.; Nyberg, S.; Emorine,
L. J.; Arner, P. Evidence for a functional beta 3-adrenoceptor in man. Br.
J. Pharmacol. 1993, 110, 929–936.
(14) Wheeldon, N. M.; McDevitt, D. G.; Lipworth, B. J. Do beta
3-adrenoceptors mediate metabolic responses to isoprenaline? Q. J. Med.
1
993, 86, 595–600.
(
15) Enocksson, S.; Shimizu, M.; Lonnqvist, F.; Nordenstrom, J.;
Arner, P. Demonstration of an in vivo functional beta 3-adrenoceptor in
man. J. Clin. Invest. 1995, 95, 2239–2245.
(16) Himms-Hagen, J.; Ricquier, D. Brown adipose tissue. In Hand-
book of obesity; Bray, G. A., Bouchard, C., James, W. P. T., Eds.; Marcel
Dekker: New York, pp 415À441.
(17) Holloway, B. R. Reactivation of brown adipose tissue. Proc.
Nutr. Soc. 1989, 48, 225–230.
(18) Kawashita, N. H.; Moura, M. A.; Brito, M. N.; Brito, S. M.;
Garofalo, M. A.; Kettelhut, I. C.; Migliorini, R. H. Relative importance of
sympathetic outflow and insulin in the reactivation of brown adipose
tissue lipogenesis in rats adapted to a high-protein diet. Metabolism
2
002, 51, 343–349.
19) Miller, J. W.; Farid, N. A.; Johnson, R. D.; Smith, B. P.;
Dananberg, J. Stimulation of energy expenditure by LY377604, a β
(
3
adrenergic receptor agonist with β1/2 antagonist properties in healthy
male subjects. Obes. Res. 1999, 7 (Suppl. 1), 121S.
(
20) van Baak, M. A.; Hul, G. B. J.; Toubro, S.; Astrup, A.;
Gottesdiener, K. M.; DeSmet, M.; Saris, W. H. M. Acute effect of
L-796568, a novel β -adrenergic receptor agonist, on energy expenditure
in obese men. Clin. Pharmacol. Ther. 2002, 71, 272–279.
21) Larsen, T. M.; Toubro, S.; van Baak, M. A.; Gottesdiener, K. M.;
Larson, P.; Saris, W. H.; Astrup, A. Effect of a 28-d treatment with
L-796568, a novel β -adrenergic receptor agonist, on energy expenditure
and body composition in obese men. Am. J. Clin. Nutr. 2002, 76, 780–8.
22) Czeskis, B. A.; Wheeler, W. J.; Clodfelter, D. K. Convergent
synthesis of two 14C-labeled β -adrenergic receptor agonists. J. Labelled
Compd. Radiopharm. 2006, 49, 663–673.
23) Czeskis, B. A.; Wheeler, W. J. Synthesis of β3 adrenergic
3
(
3
(
3
(
receptor agonist LY377604 and its metabolite 4-hydroxycarbazole,
labeled with carbon-14 and deuterium. J. Labelled Compd. Radiopharm.
2
005, 48, 407–419.
(
24) Crowell, T. A.; Evrard, D. A.; Jones, C. D.; Muehl, B. S.; Rito
C. J.; Shuker, A. J.; Thorpe, A. J.; Thrasher, K. J.; Czeskis, B. A.; Wheeler,
W. J. EP Patent 0 827 746, 2002.
(
25) Procedure based on method reported in: Chen, Y.; Heiman,
M. L. Chronic leptin administration promotes lipid utilization until fat
mass is greatly reduced and preserves lean mass of normal female rats.
Regul. Pept. 2000, 92, 113–119.
5
86
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