Note: Descriptions are shown in the official language in which they were submitted.
CA 02373035 2001-11-05
WO 00/68197 PCT/US00/10981
3a,4,5,9b-TETRAHYDRO-1H-BENZ~eJINDOL-2-YL AMINE-DERIVED
NEUROPEPTIDE Y RECEPTORS LIGANDS
USEFUL IN THE TREATMENT OF OBESITY AND OTHER DISORDERS
This is a non-provisional of Application Serial No. 60/132,660, filed May 5,
1999.
FIELD OF THE INVENTION
This invention relates to a series of 3a,4,5,9b-tetrahydro-1 H-benz[e]indol-2-
yl
amine derivatives, pharmaceutical compositions containing them and
intermediates
used in their preparation. The compounds of the invention are ligands for the
neuropeptide Y Y5 (NPY5) receptor, a receptor which is associated with a
number
of central nervous system disorders and affective conditions.
BACKGROUND OF THE INVENTION
Regulation and function of the mammalian central nervous system is
governed by a series of interdependent receptors, neurons, neurotransmitters,
and
proteins. The neurons play a vital role in this system for, when externally or
internally stimulated, they react by releasing neurotransmitters that bind to
specific
proteins. Common examples of endogenous small molecule neurotransmitters such
as acetylcholine, adrenaline, norepinephrine, dopamine, serotonin, glutamate,
and
gamma-aminobutyric acid are well known, as are the specific receptors that
recognize these compounds as ligands ("The Biochemical Basis of
Neuropharmacology", Sixth Edition, Cooper, J. R.; Bloom, F. E.; Roth, R. H.
Eds.,
Oxford University Press, New York, NY 1991 ).
In addition to the endogenous small molecule neurotransmitters, there is
increasing evidence that neuropeptides play an integral role in neuronal
operations.
Neuropeptides are now believed to be co-localized with perhaps more than one-
half
of the 100 billion neurons of the human central nervous system. In addition to
humans, neuropeptides have been discovered in a number of animal species. In
some instances the composition of these peptides is remarkably homogenous
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WO 00/68197 PCT/US00/10981
among species. This finding suggests that the function of neuropeptides is
vital and
has been impervious to evolutionary changes. Furthermore, neuropeptides,
unlike
small molecule neurotransmitters, are typically synthesized by the neuronal
ribosome. In some cases, the active neuropeptides are produced as part of a
larger
protein which is enzymatically processed to yield the active substance. Based
upon
these differences, compared to small molecule neurotransmitters, neuropeptide
based strategies may offer novel therapies for CNS diseases and disorders.
Specifically, agents that affect the binding of neuropeptides to their
respective
receptors or ameliorate responses that are mediated by neuropeptides are
potentially useful in therapies for diseases associated with neuropeptides.
There are a number of afflictions that are associated with the complex
interdependent system of receptors and ligands within the central nervous
system;
these include neurodegenerative diseases, affective disorders such as anxiety,
depression, pain and schizophrenia, and affective conditions that include a
metabolic component, namely obesity. Such conditions, disorders and diseases
have been treated with small molecules and peptides which modulate neuronal
responses to endogenous neurotransmitters.
One example of the class of neuropeptides is neuropeptide Y (NPY). NPY
was first isolated from porcine brain (Tatemoto, K. et al. Nature 1982, 296,
659) and
was shown to be structurally similar to other members of the pancreatic
polypeptide
(PP) family such as peptide YY, which is primarily synthesized by endocrine
cells in
the gut, and pancreatic polypeptide, which is synthesized by the pancreas.
Neuropeptide Y is a single peptide protein that consists of thirty-six amino
acids
containing an amidated C-terminus. Like other members of the pancreatic
polypeptide family, NPY has a distinctive conformation that consists of an N-
terminal
polyproline helical region and an amphiphilic oc-helix joined by a
characteristic PP-
fold (Vladimir, S. et. AI. Biochemistry 1990, 20, 4509). Furthermore, NPY
sequences from a number of animal species have been elucidated and all show a
high degree of amino acid homology to the human protein (>94% in rat, dog,
rabbit,
pig, cow, sheep) (see Larhammar, D. in "The Biology of Neuropeptide Y and
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WO 00/68197 PCT/LTS00/10981
Related Peptides", Colmers, W. F. and Wahlestedt, C. Eds., Humana Press,
Totowa, NJ 1993).
Endogenous receptor proteins that bind NPY and related peptides as ligands
have been identified and distinguished, and several such proteins have been
cloned
and expressed. Six different receptor subtypes [Y1, Y2, Y3, Y4(PP), Y5, Y6
(formerly designated as a Y5 receptor)] are recognized today based upon
binding
profile, pharmacology and / or composition if identity is known (Wahlestedt,
C. et. al.
Ann. NY Acad. Sci. 1990, 611, 7; Larhammar, D. et. al. J. Biol. Chem. 1992,
267,
10935; Wahlestedt, C. et. al. Regul. Pept. 1986, 13, 307; Fuhlendorff, J. U.
et. al.
Proc. Natl. Acad. Sci. USA 1990, 87, 182; Grundemar, L. et. al. J. Pharmacol.
Exp.
Ther. 1991, 258, 633; Laburthe, M. et. al. Endocrinology 1986, 118, 1910;
Castan, I.
et. al. Endocrinology 1992, 131, 1970; Gerald, C. et. al. Nature 1996, 382,
168;
Weinberg, D. H. et. al. Journal of Biological Chemistry 1996, 271, 16435;
Gehlert, D.
et. al. Current Pharmaceutical Design 1995, 1, 295; Lundberg, J. M. et. al.
Trends in
Pharmaceutical Sciences 1996, 17, 301 ). Most and perhaps all NPY receptor
proteins belong to the family of so-called G-protein coupled receptors
(GPCRs).
The neuropeptide Y5 receptor, a putative GPCR, is negatively coupled to
cellular
cyclic adenosine monophosphate (CAMP) levels via the action of adenylate
cyclase
(Gerald, C. et. al. Nature 1996, 382, 168; Gerald, C. et. al. PCT WO
96/16542). For
example, NPY inhibits forskolin-stimulated cAMP production / levels in a
neuroblastoma cell line. A Y5 ligand that mimics NPY in this fashion is an
agonist
whereas one that competitively reverses the NPY inhibition of forskolin-
stimulated
cAMP production is an antagonist.
Neuropeptide Y itself is the archetypal substrate for the NPY receptors and
its
binding can elicit a variety of pharmacological and biological effects in
vitro and in
vivo. When administered to the brain of live animals
(intracerebroventricularly (icv)
or into the amygdala), NPY produces anxiolytic effects in established animal
models
of anxiety such as the elevated plus-maze, Vogel punished drinking and Geller-
Seifter's bar-pressing conflict paradigms (Heilig, M. et. al.
Psychopharmacology
1989, 98, 524; Heilig, M. et. al. Reg. Peptides 1992, 41, 61; Heilig, M. et.
al.
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Neuropsycho-pharmacology 1993, 8, 357). Thus compounds that mimic NPY are
postulated to be useful for the treatment of anxiolytic disorders.
The immunoreactivity of neuropeptide Y is notably decreased in the
cerebrospinal fluid of patients with major depression and those of suicide
victims
(Widdowson, P. S. et. al. Journal of Neurochemistry 1992, 59, 73), and rats
treated
with tricyclic antidepressants display significant increases of NPY relative
to a
control group (Heilig, M. et. al. European Journal of Pharmacology 1988, 147,
465).
These findings suggest that an inadequate NPY response may play a role in some
depressive illnesses, and that compounds that regulate the NPY-ergic system
may
be useful for the treatment of depression.
Neuropeptide Y improves memory and performance scores in animal models
of learning (Flood, J. F. et. al. Brain Research 1987, 421, 280) and therefore
may
serve as a cognition enhancer for the treatment of neurodegenerative diseases
such
as Alzheimer's Disease (AD) as well as AIDS-related and senile dementia.
Elevated plasma levels of NPY are present in animals and humans
experiencing episodes of high sympathetic nerve activity such as surgery,
newborn
delivery and hemorrhage (Morris, M. J. et. al. Journal of Autonomic Nervous
System
1986, 17, 143). Thus chemical substances that alter the NPY-ergic system may
be
useful for alleviating the condition of stress.
Neuropeptide Y also mediates endocrine functions such as the release of
luteinizing hormone (LH) in rodents (Kalra, S. P. et. al. Frontiers in
Neuroendrocrinology 1992, 13, 1 ). Since LH is vital for mammalian ovulation,
a
compound that mimics the action of NPY could be useful for the treatment of
infertility, particularly in women with so-called luteal phase defects.
Neuropeptide Y is a powerful stimulant of food intake; as little as one-
billionth
of a gram, when injected directly into the CNS, causes satiated rats to
overeat
(Clark, J. T. et. al. Endocrinology 1984, 115, 427; Levine, A. S. et. al.
Peptides
1984, 5, 1025; Stanley, B. G. et. al. Life Sci. 1984, 35, 2635; Stanley, B. G.
et. al.
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WO 00/68197 PCT/LTS00/10981
Proc. Nat. Acad. Sci. USA 1985, 82, 3940). Thus NPY is orexigenic in rodents
but
not anxiogenic when given intracerebroventricularly and so antagonism of
neuropeptide receptors may be useful for the treatment of eating disorders
such as
obesity, anorexia nervosa and bulimia nervosa.
In recent years, a variety of potent, structurally distinct small molecule Y1
antagonists has been discovered and developed (Hipskind, P. A. et. al. Annu.
Rep.
Med. Chem. 1996, 31, 1-10; Rudolf, K. et. al. Eur. J. Pharmacol. 1994, 271,
R11;
Serradeil-Le Gal, C. et. al. FEBS Lett. 1995, 362, 192; Wright, J. et. al.
Bioorg. Med.
Chem. Lett. 1996, 6, 1809; Poindexter, G. S. et. al. United States Patent
5,668,151;
Peterson, J. M. et, al. W09614307 (1996)). However, despite claims of activity
in
rodent models of feeding, it is unclear if inhibition of a feeding response
can be
solely attributed to antagonism of the Y1 receptor.
Several landmark studies suggest that an "atypical Y1" receptor and / or the
Y5 receptor, rather than the classic Y1 receptor, is responsible for invoking
NPY-
stimulated food consumption in animals. It has been shown that the NPY
fragment
NPY2-3s is a potent inducer of feeding despite poor binding at the classic Y1
receptor (Stanley, B. G. et. al. Peptides 1992, 13, 581 ). Conversely, a
potent and
selective Y1 agonist has been reported to be inactive at stimulating feeding
in
animals (Kirby, D. A. et. al. J. Med. Chem. 1995, 38, 4579). More pertinent to
the
invention described herein, [~-Trp32]NPY, a selective Y5 receptor activator
has been
reported to stimulate food intake when injected into the hypothalamus of rats
(Gerald, C. et. al. Nature 1996, 382, 168). Since [~-Trp32]NPY appears to be a
full
agonist of the Y5 receptor with no appreciable Y1 activity, the Y5 receptor is
hypothesized to be responsible for the feeding response. Accordingly compounds
that antagonize the Y5 receptor should be effective in inhibiting food intake,
particularly that stimulated by NPY.
Certain arylsulfonamides that act as Y5 antagonists are known in the prior
art. In PCT WO 97/19682, aryl sulfonamides and sulfamides derived from
arylalkylamines are described as Y5 antagonists and are reported to reduce
food
consumption in animals. In PCT WO 97/20820, PCT WO 97/20822 and PCT WO
5
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WO 00/68197 PCT/US00/10981
97/20823, sulfonamides containing heterocyclic systems such as quinazolin-2,4-
diazirines, are likewise claimed as Y5 antagonists and reported to reduce
feeding.
In WO 98/35957, various amide derivatives, including those that contain a
benzimidazolinone group are claimed to be neuropeptide Y receptor antagonists.
However, none of these compounds known in the prior art contain the amidine
ring
system present in the compounds of this invention. The cyclic amidino
sulfonamides and amidino benzimidazolinones and amidino arylpiperazines
described in this application are novel molecular entities that may have
binding
motifs that are different from Y5 receptor ligands that have been disclosed in
prior
publications, and yet bind to a similar region of the Y5 receptor. In addition
to
exhibiting an affinity for the neuropeptide Y5 receptor, the compounds of this
invention may also produce pharmacological and biological responses that are,
in
part or wholly, due to activation or antagonism of other Y receptor subtypes
(e.g.,
Y1, Y2, Y4).
SUMMARY OF THE INVENTION
The present invention is related to compounds of formula A
R2
N-Y-L-Z
B2 a \ N
~R~ )n j
in which
R, is independently selected from the group consisting of hydrogen; hydroxy;
halo; C,_$alkyl; C,_8alkoxy; substituted C,_8 alkyl wherein the substituent is
selected from halo, such as chloro, bromo, fluoro and iodo; substituted C,_8
alkoxy wherein the substituent is selected from halo, such as chloro, bromo,
fluoro and iodo; trifluoroalkyl; C,_8alkylthio and substituted C,_8alkylthio
wherein the substituent is selected from halo, such as chloro, bromo, fluoro
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WO 00/68197 PCT/US00/10981
and iodo, trifluoroalkyl and C,_8alkoxy; C3_6cycloalkyl; C3_8cycloalkyloxy;
nitro;
amino; C,_salkylamino; C,_8dialkylamino; C4_8cycloalkylamino; cyano; carboxy;
C,_5alkylcarbonyloxy; C,_5alkoxycarbonyloxy; formyl; carbamoyl; phenyl and
substituted phenyl wherein the substitutent is selected from halo, hydroxyl,
nitro, amino and cyano;
n is 0-2;
BZ is selected from the group consisting of hydrogen; C,_5alkyl; substituted
C,_5alkyl wherein the substituent is halo;
BZ may have either a cis- or traps- stereochemical orientation with respect to
B,; both enantiomers of each diastereomeric set are part of the present
invention;
Y is methylene (-CH2 ) or carbonyl (C=O)
L is selected from the group consisting of
C,_$alkylene; C2_,oalkenylene; Cz_,oalkynylene; C3_,cycloalkylene;
C3_,cycloaIkyIC,~alkylene;
arylC,~alkylene;
(N-methylene)piperid in-4-yl;
~~H2 N
(N-methylene)piperazin-4-yl;
-CH2 N N-
and (N-methylene)piperidin-4,4-diyl;
~~H2 N~
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RZ is independently selected from the group consisting of hydrogen; C,_5alkyl;
substituted C,_Salkyl wherein the substituent is halo;
B, is hydrogen;
B, may have either a cis- or trans- stereochemical orientation with respect to
Bz; both enantiomers of each diastereomeric set are part of this invention.
Z is selected from the group consisting of:
phenyl;
~R4)m
N-sulfonamido;
0
~- ~ -S-R3
H o
N-(aryl)sulfonamido;
H ~ -/ ~R4)m
-N-S
O
2,3-dihydro-2-oxo-1 H-benzimidazol-1-yl;
0
-N~NH
~R4)m
and 1-aryl-2,3-dihydro-4-oxo-imidazol-5,5-diyl;
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WO 00/68197 PCT/US00/10981
O
N
y
~R4)m
R3 is independently selected from the group consisting of C,_$alkyl;
substituted
C,_$alkyl wherein the substituent is selected from C,_8alkoxy and halo;
cycloalkyl; substituted cycloalkyl wherein the substituent is selected from
C,_
8alkoxy and halo; naphthyl; substituted naphthyl wherein the substituent is
selected from halo, nitro, amino and cyano; heteroaryl wherein the heteroaryl
group is selected from pyridyl, pyrimidyl, furyl, thienyl and imidazolyl; and
substituted heteroaryl wherein the substituent is selected from halo, nitro,
amino and cyano;
R4 is independently selected from the group consisting of C,_$alkyl; alkoxy;
hydroxy; halogen; cyano, nitro; amino and alkylamino; substituted C,_8alkyl
wherein the substituent is halo;
m is 0-2;
and enantiomers, diastereomers, and pharmaceutically acceptable salts
thereof,
with the following provisions:
when L is C,_8alkylene, Cz_,oalkenylene, CZ_,oalkynylene, C3_,cycloalkylene,
C3_,cycloaIkyIC,.~alkylene, arylC,~alkylene or (N-methylene)piperidin-4-yl,
then Z is phenyl, N-sulfonamido, N-(aryl)sulfonamido or
2,3-dihydro-2-oxo-1 H-benzimidazol-1-yl;
when L is (N-methylene)piperazin-4-yl,
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then Z is phenyl or naphthyl;
and when L is (N-methylene)piperidin-4,4,-diyl;
then Z is 1-aryl-2,3-dihydro-4-oxo-imidazol-5,5-diyl;
As used herein unless otherwise noted the terms "alkyl" and "alkoxy" whether
used alone or as part of a substituent group, include straight and branched
chains
having 1-8 carbon atoms. For example, alkyl radicals include methyl, ethyl,
propyl,
isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, 2-methyl-3-butyl, 1-
methylbutyl, 2-
methylbutyl, neopentyl, hexyl, 1-methylpentyl, 3-methylpentyl. Alkoxy radicals
are
oxygen ethers formed from the previously described straight or branched chain
alkyl
groups. The term "aryl" is intended to include phenyl and naphthyl. The term
"halo",
unless otherwise indicated, includes bromo, chloro, fluoro and iodo. The term
"cycloalkyl" is intended to include cycloalkyl groups having 3-7 carbon atoms.
With
reference to substituents, the term "independently" means that when more than
one
of such substituent is possible, such substituents may be the same or
different from
each other.
Those compounds of the present invention which contain a basic moiety can
be converted to the corresponding acid addition salts by techniques known to
those
skilled in the art. Suitable acids which can be employed for this purpose
include
hydrochloric, hydrobromic, hydriodic, perchloric, sulfuric, nitric,
phosphoric, acetic,
propionic, glycolic, lactic, pyruvic, oxalic, malonic, succinic, malefic,
fumaric, malic,
tartaric, citric, benzoic, cinnamic, mandelic, methanesulfonic, p-
toluenesulfonic,
cyclohexanesulfamic, salicylic, 2-phenoxybenzoic, 2-acetoxybenzoic, or
saccharin,
and the like. In general, the acid addition salts can be prepared by reacting
the free
base of compounds of formula A with the acid and isolating the salt.
Pharmaceutical compositions containing one or more of the compounds of
the invention described herein as the active ingredient can be prepared by
intimately
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WO 00/68197 PCT/US00/10981
mixing the compound or compounds with a pharmaceutical carrier according to
conventional pharmaceutical compounding techniques. The carrier may take a
wide
variety of forms depending upon the desired route of administration (e.g.,
oral,
parenteral). Thus for liquid oral preparations such as suspensions, elixirs
and
solutions, suitable carriers and additives include water, glycols, oils,
alcohols,
flavoring agents, preservatives, stabilizers, coloring agents and the like;
for solid oral
preparations, such as powders, capsules and tablets, suitable carriers and
additives
include starches, sugars, diluents, granulating agents, lubricants, binders,
disintegrating agents and the like. Solid oral preparations may also be coated
with
substances such as sugars or be enteric-coated so as to modulate the major
site of
absorption. For parenteral administration, the carrier will usually consist of
sterile
water and other ingredients may be added to increase solubility or
preservation.
Injectable suspensions or solutions may also be prepared utilizing aqueous
carriers
along with appropriate additives.
The daily dose of the active ingredient to be administered will depend on the
age of the patient in need of such treatment, the particular condition to be
treated
and the manner of administration. Generally, an approximate daily dose of
about 10
to about 500 mg is to be administered depending upon the mode of
administration
and the weight of the patient being treated. Determination of the optimum
doses
and frequency of administration for a particular disease state or disorder is
within the
experimental capabilities of those knowledgeable of the specific disease or
disorder
being treated.
For the treatment of disorders of the central nervous system, the
pharmaceutical compositions described herein will typically contain from about
1 to
about 1000 mg of the active ingredient per dosage; one or more doses per day
may
be administered. Determination of optimum doses and frequency of dosing for a
particular disease state or disorder is within the experimental capabilities
of those
knowledgeable in the treatment of central nervous system disorders. The
preferred
dose range is from about 1-100 mg/kg.
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As modulators of the NPY5 receptor, the compounds of Formula A are useful
10
for treating feeding disorders such as obesity, anorexia nervosa and bulimia
nervosa, and abnormal conditions such as epilepsy, depression, anxiety,
sleeping
disorders, dyspilipidimia, diabetes, hypertension, migraine, pain and sexual /
reproductive disorders in which modulation of the NPY5 receptor may be useful.
The compounds compete with the endogenous ligand PYY and possibly NPY and
possibly non-endogenous ligands as well, and bind to the NPY5 receptor. In
addition, the compounds demonstrate antagonist activity by antagonizing the
action
of NPY upon binding to the Y5 receptor.
The compounds described herein are ligands of the NPY5 receptor, but are
not necessarily limited solely in their pharmacological or biological action
due to
binding to this or any neuropeptide, neurotransmitter or G-protein coupled
receptor.
For example, the described compounds may also undergo binding to dopamine or
serotonin receptors. The compounds described herein are potentially useful in
the
regulation of metabolic and endocrine functions, particularly those associated
with
feeding, and as such, may be useful for the treatment of obesity. In addition,
the
compounds described herein are potentially useful for modulating other
endocrine
functions, particularly those controlled by the pituitary and hypothalamic
glands, and
therefore may be useful for the treatment of inovulation/infertility due to
insufficient
release of luteinizing hormone (LH).
The present invention comprises pharmaceutical compositions containing
one or more of the compounds of Formula A.
z
wherein R,, R2, B,, B2, Y, n, L and Z are defined as above. In addition, the
present
invention comprises intermediates used in the manufacture of these compounds.
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Examples of preferred compounds of formula A include:
,,,~ o
H-S-CH3
O
,,,~~ ~ ~CH3
N-S-C H
O CH3
S
H 101
,,,, \N-O ~ S
H I I-
O
O O
H ISI
a O
,,,\ O
H S
O N
13
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WO 00/68197 PCT/US00/10981
O
H ISI
O
O
H ISI
O
O
I I
H iSl
O
O
I I
H ISI
O
F
O
H ISI
O
F
O
H ISI
O
02N
,~ O
H-S ~ / CI
O
CI
14
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WO 00/68197 PCT/US00/10981
O
H ISI
O
\\
N
I
H3C0~ v v ,,,\ O
N H ISI
/ O
O
H ISI
O
F
~',, ,,\N-O
H II
O
F
O
H
O
F
O
H
O
F
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WO 00/68197 PCT/US00/10981
F
O
I I
H ISI
O
F
O
v II
N~'~~' H
F
H N
HO ''',,,\ O
N H ISI
O
02N
O
H ISI
O
,,,,,~ O
H ISI
O
O
H ISI
O
H3C0
,,,,,~ O
H ISI
O
02N
16
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WO 00/68197 PCT/US00/10981
,,,~ O
H ISI
O
CI
N
O
H ISI
O
CI
CI
O
I I
H ISI
O
O
\ II
H-S ~ / OCH3
O
OCH3
17
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WO 00/68197 PCT/US00/10981
H
N~N O
~N O
N~N
H
NON O
\N
N~NH
\
N~N O
~N IIO
N~NH
~N O
N~NH
H3
H
N~N O
~N 'I0
N~NH
\
HO
H
NON O
\N
N~NH
\
HO
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N~N O
HO \ N N NH
HO
N~ O
N,
\\
HO \ N N NH
HO
N~ O
N,
~\ I~~//I\\O
F \ N N NH
F
H
NON O
F N ~N~NH
F
H
N~N O
\O
N N NH
\
F
H
NON O
\N
~N~NH
\
F
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WO 00/68197 PCT/US00/10981
O
H
N
N NH
~N O
\
O
H
N
N NH
N
\
N
~N O
N N NH
Br
H3C CH3
H
N~ O
N,
\N
\ N NH
/
Br
H3C CH3
H
N
~N O
~ N IOI
H3C0 \ N NH
/
H3C0
~N O
H3' N~NH
H3
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WO 00/68197 PCT/US00/10981
N O
N
NH
\ N N
O
N
NH
N
N' ~ O
'N
~~ O \NH
\ N N
H3C0
O
N
NH
N
H3
N O
N
NH
\ N N
HO
H
N~ O
N
\NH
\ N N
O
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H
N\ ~ O
'N
\N O NH
\ N
F /
H
N~ O
N
N NH
I \ N-~
F
H
N\ ~ O
'N
F \N O ~ H
I\ N
F /
~N O
NH
N
H
N\ ~ O
'N
~N O ~ H
N
I\
HO /
H
N~ O
N
HO N ~ H
I \ N
HO /
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H
N~N~ OCH3
~N IOI ~N
\ \
I/ I/
H
N
~N~ OCH3
~N ~N
\ \
I / I /
N~N~ OCH3
~N IO' ~N
\ \
I/ I/
H3C0
H
N
~N~ OCH3
~N ~N
\ \
I/ I/
H3C0
H
N\
_N
O N-O _
N
I \ H 101
H
N~
N
~ N I~~//I\\ N-O
I \ H 101
H
N
~N OCH3
HO \ N I \
I/ /
HO
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H
N~N~ OCH3
~ N IOI ~ N \
/ ~ /
F
N
~N~ OCH3
~N ~N
\ \
/ ( /
F
H
N~N~ OCH3
~ N IOI ~ N \
F \
/
F
H
N
~N~ OCH3
F \ N \/N \
/ ~/
F
H
N~N~ OCH3
~N '0I ~N \
HO \
/
HO /
N
~N~ OCH3
HO \ N ~N \
/ ~/
HO
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H
N~N~ OCH3
~N '0I ~N \
H3C0 \
/ ~ /
H3C0
H
N
~N~ OCH3
H3C0 \N ~N \
H3C0 /
H
N~N~ OH
\N '0I ~N \
\
HO /
H
N
~N~ OH
\N ~N \
\
HO /
N~N~ N02
N IOI ~ N \
/ ~ /
HO
H
N
~N~ N02
\N V N \
\
HO
H
N
~N~ F
\N ~N \
~/
HO
CA 02373035 2001-11-05
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DETAILED DESCRIPTION OF THE INVENTION
The cyclic amidines of formula A that comprise this invention are synthesized
via several distinct chemical syntheses which are described in detail in the
Examples set forth below. In general, each synthetic route consists of several
sequential chemical operations which are outlined in Schemes 1-8 and which can
be
generalized as described below:
~ Introduction of the a-cyanomethyl group onto a a-tetralone nucleus.
~ Concomitant reductive amination / cyclization to produce amidine
intermediates.
~ Acylation of cyclic amidine intermediates to afford compounds of formula A
in
which Y = carbonyl (C=O).
~ Reduction of the amide to generate the cyclic amidines of formula A in which
Y =
methylene (-CH2 ).
It is generally preferred that the respective product of each process step be
separated from other components of the reaction mixture and subjected to
purification before its use as a starting material in a subsequent step.
Separation
techniques typically include evaporation, extraction, precipitation and
filtration.
Purification techniques typically include column chromatography (Still, W. C.
et. al.,
J. Org. Chem. 1978, 43, 2921 ), thin-layer chromatography, crystallization and
distillation. The structures of the final products, intermediates and starting
materials
are confirmed by spectroscopic, spectrometric and analytical methods including
nuclear magnetic resonance (NMR), mass spectrometry (MS) and liquid
chromatography (HPLC). In the descriptions for the preparation of compounds of
this invention, ethyl ether, tetrahydrofuran and dioxane are common examples
of an
ethereal solvent; benzene, toluene, hexanes and cyclohexane are typical
hydrocarbon solvents and dichloromethane and dichloroethane are representative
halohydrocarbon solvents. In those cases wherein the product is isolated as
the
acid addition salt, the free base is obtained by techniques known to those
skilled in
the art.
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Specifically, an appropriately substituted ~-tetralone (I) is reacted with a
secondary amine such as pyrrolidine in an inert halohydrocarbon solvent such
as,
for example, dichloromethane or a hydrocarbon solvent such as benzene for
example, under Dean-Stark conditions (removal of water) or in an ethereal
solvent
such as tetrahydrofuran or an alcohol solvent such as methanol, at a
temperature
ranging from ambient temperature to reflux, to afford enamine (II).
Cyanomethylation of enamine (II) is accomplished by reaction with an a-
haloacetonitrile, such as bromoacetonitrile, in an inert solvent such as
acetonitrile, at
a temperature ranging from ambient temperature to reflux, to afford the
iminium salt
(III). The iminium salt is hydrolyzed by treatment with an aqueous acid
solution,
such as hydrochloric or acetic acid, which may contain an organic solvent such
as
an alcohol or dioxane to facilitate dissolution and reaction, to afford the a-
cyanomethyl-~-tetralone (IV). Reductive amination and concomitant cyclization
of
tetralone (IV) is accomplished by reaction with a reducing agent such as, for
example, sodium cyanoborohydride, and an ammonium equivalent such as, for
example, ammonium acetate, in an alcohol solvent such as methanol or in a
halohydrocarbon solvent such as dichloromethane, at a temperature ranging from
ambient temperature to reflux. An organic acid, such as acetic acid for
example,
may be added to facilitate this transformation; cyclization under these
reaction
conditions typically affords the cis-amidine (V) as the major product. Amidine
(V)
may be converted to its acid addition salt upon treatment with organic acids
such as
trifluoroacetic acid, or via treatment with inorganic acids such as
hydrochloric acid,
to afford the corresponding amidine salt (VI) (Scheme 1 ). HX in Scheme 1
represents the hydrochloride salt.
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H
0
U
\ \\
(R~)n i / -.~ (R~)n i
(I)
(II)
N~~C N~~C
Br-CH2-CN ~ H+, HZO O
\ ~+ \
(R~)n i / B~ ~ (R~)n i /
(IV)
(III)
NH NH2
reductive ,
amination ~N H+ \N
i \ ~ ~ \
(borohydride) (R~ )n (R )n
/ 1 ~ / ~HX
(V) (VI)
Scheme 1
The amidine products described above ((V) and (VI)) are acylated via suitable
amidation methods (see Gross and Meienhofer, Eds., "The Peptides", Vols. 1-3,
Academic Press, New York, NY, 1979-1981 ). A carboxylic acid is converted to
an
activated ester via peptide coupling methods known to those skilled in the
art, and
the product of this reaction is subsequently reacted with amidine (V) or (VI)
to afford
the corresponding amide product. For example, traps-4-
(benzenesulfonamido)methylcyclohexane carboxylic acid is reacted with HBTU (2-
(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and
amidine (VI) in the presence of a base such as diisopropylethylamine, in an
inert
solvent such as N,N-dimethylformamide, at a temperature from ambient
temperature
to reflux, to afford sulfonamides (VII) of formula A in which Y = carbonyl and
Z =
(aryl)sulfonamido (Scheme 2). Reaction of amidine (VI) or (V) with alkyl- or
heteroaryl- sulfonyl halides, under similar conditions, affords sulfonamides
(VIII) of
formula A. During these transformations, minor amounts of regiomers (IX) and
(X)
are formed respectively; compounds of this type are considered to be part of
this
invention as well.
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NH NH
O~L-N-O /Ra H 101 -~Ra
\ N H H O I I ~ ~ N L-N-S
o \ ~ II
(R~ )n i / (R~ )n i O O
~HX peptide coupling /
(VI)
(IX) +
H ~ -/Ra
L-N-S
O
(R~
(VII)
O O NH
H II
H O~L-N ~ R3 H O
N L-N-S-R3
(VI) \ ~ O
peptide coupling (R~)n i / O
(e.g. HBTU) / base
(X) + O
(minor) H H II
N~L-N-S-R3
IOI O
N
(R~)n
Scheme 2
(VIII)
Alternatively, a sulfonamido-carboxylic acid is first treated with an amine
base, such as triethylamine, in an inert hydrocarbon, ethereal or
halohydrocarbon
solvent, such as dichloroethane, and subsequently reacted with isobutyl
chloroformate at a temperature from about -20°C to 80°C. This
resulting mixture is
then reacted with amidine (V), in a suitable inert solvent such
dichloromethane at a
temperature from about -20°C to reflux, to afford the sulfonamides
(VII) and (VIII) of
formula A respectively, in which Y = carbonyl and Z = (aryl)sulfonamido or
sulfonamido.
The amidino sulfonamides of formula A in which Y = methylene are prepared
via reduction of amidino amides (VII) and (VIII) by reaction with a suitable
reducing
agent such as borane-tetrahydrofuran complex or lithium aluminum hydride in an
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WO 00/68197 PCT/US00/10981
inert hydrocarbon solvent such as toluene or ethereal solvent such as
tetrahydrofuran, at a temperature from ambient temperature to reflux. The
crude
product is treated with an aqueous acid solution such as hydrochloric acid (3M-
6M)
in order to cleave any boron complexes; neutralization affords sulfonamides
(XI) and
(X11) as corresponding free bases. Preferably, these materials are isolated as
an
acid addition salts upon treatment with a suitable organic acid such as
trifluoroacetic
acid or inorganic acid such as hydrochloric acid (Scheme 3).
H H 101 -/R4 -N-~ -/Ra
N~L-N-S ~ ~ H II
II O
\\ p 1 ) IRI O
N
(Ri )n i 2) H+
(VII) (R'
(XI) H O
H ~ -N-S-Rs
-N-S-R3 1 ) Reduction O
O (e.g., BH3 / Th
(Ri 2) H+ (R~
(VIII)
(XI I )
Scheme 3
Reduction of the regiomeric amides (IX) and (X) by the methods described above
in
Scheme 3, affords amines (X111) and (XIV) (Scheme 4).
NH
H O -/Ra
1 ) [RED] ~ N-CHZ L-N-S
(IX) 2) H~ (R~)n j / O
'HX
(X111)
NH
O
H II
1 ) [RED] ~ N-CH2 L-N-S-R3
(X) _---~ (R~)n i O
2) H+ / HX
(xiv)
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Scheme 4
Compounds of formula A in which Z = 2,3-dihydro-2-oxo-1H-benzimidazol-1-
yl and L = (N-methylene)piperidin-4-yl are prepared from amidines (V) or (VI)
and [4-
(2-keto-1-benzimidazolinyl)piperidin-1-yl]acetic acid. For example, 4-(2-keto-
1-
benzimidazolinyl)piperidine is reacted with a bromoacetic acid ester, such as
ethyl
bromoacetate, in the presence of an amine base, such as diisopropylethylamine,
in
an inert solvent such as acetonitrile, at a temperature ranging from ambient
temperature to reflux, to afford ethyl [4-(2-keto-1-benzimidazolinyl)piperidin-
1-
yl]acetate. This ester is subjected to hydrolysis under basic conditions, for
example,
by treatment with sodium hydroxide in an alcoholic solution such as aqueous
methanol, to yield, upon acidification with an inorganic or organic acid such
as
hydrochloric or acetic acid for example, [4-(2-keto-1-
benzimidazolinyl)piperidin-1-
yl]acetic acid. This carboxylic acid is reacted directly with amidine (V) or
(VI), in the
presence of an amine base, under peptide coupling conditions described above,
to
afford amidino benzimidazolinones (XV) of formula A in which Y = carbonyl and
L =
(N-methylene)piperidin-4-yl (Scheme 5).
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O
O O ~-N H
~-N H \ ~ N
N Br v _OEt O /
/ base / CH CN EtO~N
HNJ \ ~ s
1 ) NaOH / Aq. MeOH
2) H+
NH2 O
~NH
N N
\ + O /
(R~)n j
N
.HX HOJ'~/ \
(VI)
peptide coupling
(e.g. HBTU) / base
H
N
N~ O
~N O
\ N
(R~)n j / \ NH
Scheme 5
Compounds (XVI) of formula A in which Y = methylene and L = (N-
methylene)piperidin-4-yl and Z - 2,3-dihydro-2-oxo-1 H-benzimidazol-1-yl are
prepared by reduction of amides (XV) with a reducing agent such as borane-
tetrahydrofuran complex or lithium aluminum hydride as described above (Scheme
6).
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WO 00/68197 PCT/US00/10981
H
N\
_N O
\N O
N
\ NH
(R~ )n ~ / w
(
1 ) [RED]
(e.g., BH3-THF)
2) H+
H
N~
\ N~ O
N ~
\ N' \
(R~)n ~~ NH
/ 'HX
(xvi)
Scheme 6
Compounds of formula A in which Y = carbonyl, L = (N-methylene)piperazin-
4-yl and Z = phenyl are prepared by reacting a phenylpiperazine with a
haloacetic
acid ester, such as, for example, ethyl bromoacetate, in the presence of an
amine
base, such as diisopropylethylamine, in an inert solvent such as acetonitrile,
at a
temperature ranging from ambient temperature to reflux, to afford ethyl (4-
arylpiperazin-1-yl)acetate. This ester is subjected to hydrolysis under basic
conditions, for example, by treatment with sodium hydroxide in an aqueous
methanol, to yield, upon acidification with an inorganic or organic acid such
as
hydrochloric or acetic acid for example, (4-arylpiperazin-1-yl)acetic acid.
This
carboxylic acid is reacted directly with amidine (V) or (VI) in the presence
of a base,
such as triethylamine for example, under peptide coupling conditions described
above, to afford arylpiperidines (XVII) of formula A in which Y = carbonyl, L
= (N-
methylene)piperazin-4-yl and Z = aryl or substituted aryl (Scheme 7)
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WO 00/68197 PCT/US00/10981
/
\ ~~ Br~OEt O ~N Ra
~N ~N~
HN J Ra base / CH3CN Et0
1 ) NaOH / Aq. MeOH
NH2 2) H+
~N \
\ + O ~ N \R
4
(R~)n i / Et0- v NJ
~HX
(VI)
peptide coupling
(e.g. HBTU) / base
H
N\
'jI( \ N
~ N O \/ N \
\
(R~)n j Ra
/ /
(XV I I )
Scheme 7
Compounds (XVIII) of formula A in which Y - methylene, L - (N-
methylene)piperazin-4-yl and Z = aryl are prepared by reduction of amides
(XVII)
with a reducing agent such as borane-tetrahydrofuran complex or lithium
aluminum
hydride (Scheme 8). Replacement of (4-arylpiperazin-1-yl)acetic acid with a (4-
arylpiperidin-1-yl)acetic acid in Schemes 7 and 8 affords compounds of formula
A in
which L = (N-methylene)piperidin-4-yl and Z = aryl.
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WO 00/68197 PCT/US00/10981
H
\ N~N~ '
N O vN \
1
(R~ )n ~ Ra
/ /
(XV I I )
1 ) [RED]
(e.g., BH3-THF)
2) H+
N
v N \
(R~ ~ / I Ra
(XVIII)
Scheme 8
Compounds of formula A in which Y = carbonyl, L = (N-methylene)piperidin-
4,4-diyl and Z = 1-aryl-2,3-dihydro-4-oxo-imidazol-5,5-diyl are prepared by
reacting
1-aryl-1,3,8-triazaspiro-[4,5]decan-4-one with a haloacetic acid ester, such
as ethyl
bromoacetate, in the presence of an amine base, such as diisopropylethylamine,
in
an inert solvent such as acetonitrile, at a temperature from ambient
temperature to
reflux, to afford ethyl (1-aryl-1,3,8-triazaspiro-[4,5]decan-4-one-8-
yl)acetate. This
ester is subjected to hydrolysis under basic conditions, for example, by
treatment
with sodium hydroxide in an alcoholic solution such as aqueous methanol, to
yield
upon acidification with an inorganic or organic acid such as hydrochloric or
acetic
acid for example, (1-aryl-1,3,8-triazaspiro-[4,5]decan-4-one-8-yl)acetic acid.
This
carboxylic acid is reacted directly with amidine (V) or (VI), in the presence
of a base
such as triethylamine for example, under peptide coupling conditions described
above, to afford amides (XIX) of formula A in which Y = carbonyl, L = (N-
methylene)piperidin-4,4-diyl and Z - 1-aryl-2,3-dihydro-4-oxo-imidazol-5,5-
diyl
(Scheme 9).
CA 02373035 2001-11-05
WO 00/68197 PCT/US00/10981
O
Br~ O
OEt
Hp base / CH3CN Et0' v
Ra
R4 1 ) NaOH / Aq. MeOH
2) H+
NH2
\\
N
i ~ +
(R~ )n ~
~HX
(v1)
Ra
peptide coupling
(e.g. HBTU) / base
R4
N
N'
N/>H
(R~)n
O
(XIX)
Scheme 9
Other compounds of this invention having the formula A can be prepared
using the methods described herein; modifications of the experimental
protocols
described above are known or obvious or within the ability of those skilled in
the art.
For example, a variety of ~3-tetralones are known or readily prepared by
reaction of
phenylacetic acids with ethylene gas in the presence of a Lewis acid (for
example,
Stjernlof, P. et. al. J. Med. Chem. 1995, 38, 2202). Compounds in which the R,
groups) is varied are obtained using this chemistry; in some cases, protecting
group
manipulations are used and these are obvious or known to those skilled in the
art.
Examples include masking an amine group as a carbamate, amide or phthalamide,
and masking an hydroxyl group as an ether or ester. Other R, substituents are
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WO 00/68197 PCT/US00/10981
available through (other) functional group manipulations such as, for example,
reduction of a nitro group to an amine or dehydration of an amide to a
nitrite.
Compounds in which the L group is varied, are derived from amino-carboxylic
acids or piperazines or piperidines; hundreds of such compounds are
commercially
available and many more are known. Compounds of formula A where Z =
sulfonamido or (aryl)sulfonamido, in which either the R3 or the R4 group is
varied,
are accessible by sulfonylation; there are hundreds of sulfonyl halides or
sulfonic
acids that are commercially available and more that are known. Compounds of
formula A where Z = sulfonamido or (aryl)sulfonamido, in which the R3
substituent is
heteroaryl can be prepared by substituting a pyridinyl, thienyl or furyl
sulfonylchloride for a benzenesulfonamide as described in Scheme 2. N-
alkylimidazolylsulfonyl chlorides can be used to prepare sulfonamides of
formula A
in which the R3 substituent is imidazolyl. Similarly, alkylsulfonyl and
cycloalkylsulfonyl halides, alone or in the presence of an activating agent
such as a
Lewis acid, can be used to prepare sulfonamides of formula A in which the R3
substituent is alkyl or cycloalkyl respectively.
Compounds of formula A with L groups other than methylene are prepared by
substituting bromoacetic acid esters with other ~-bromo acid esters in Schemes
5, 7
and 9. There are hundreds of ~-bromo acids and esters that are either
commercially available or known. Compounds of formula A in which the L group
is
alkylene are derived from arylalkylenecarboxylic acids; many compounds of this
structural type are either commercially available or known. Similarly,
arylalkenylene-
, arylalkynylene- and arylcycloalkylene- carboxylic acids are known or
available and
can be used to make compounds of formula A in which L is alkenylene,
alkynylene
or cycloalkylene respectively.
Compounds in which BZ is other than hydrogen are made starting from an
appropriate a-methylated-~i-tetralone and carrying out the chemistry described
in
Scheme 1 and subsequent schemes and examples. Compounds in which R2 is
other than hydrogen are made by reaction of a cyclic amidine with an
alkylation
agent such as methyl iodide.
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EXAMPLES
The following examples describe the invention in greater detail and are
intended to illustrate the invention, but not to limit it. All compounds were
identified
by a variety of methods including nuclear magnetic resonance spectroscopy,
mass
spectrometry and in some cases, infrared spectroscopy and elemental analysis.
Nuclear magnetic resonance (300 MHz NMR) data are reported in parts per
million
downfield from tetramethylsilane. Mass spectra data are reported in
mass/charge
(m/z) units. Unless otherwise noted, the materials used in the examples were
obtained from readily available commercial sources or synthesized by standard
methods known to those skilled in the art.
EXAMPLE 1
traps-4-[[(Phenylsulfonyl)amino]methyl]-N-(cis-3a,4,5,9b-tetrahydro-7-methoxy-
1H-Benz[e]indol-2-yl)cyclohexanecarboxamide (7).
1-(3,4-Dihydro-6-methoxynaphthalen-2-yl)-pyrrolidine (2).
A solution of 6-methoxy-3,4-dihydro-1 H-naphthalen-2-one (1 ) (5.64 g, 32
mmol) in
methanol (60 mL) was treated with pyrrolidine (3.5 mL, 41.6 mmol) and the
resultant
mixture was stirred at ambient temperature for 1.5 h. The product precipitated
from
solution within minutes of the addition of pyrrolidine. The resultant
suspension was
cooled in an ice bath and the enamine product (2) was collected by filtration
as a
white solid (5.6 g, 76 %). NMR (CDC13): 8 1.86-1.94 (m, 4 H), 2.46 (t, 2H),
2.80 (t, 2
H), 3.19-3.25 (m, 4 H), 3.77 (s, 3 H), 5.10 (s, 1 H), 6.59-6.65 (m, 2 H) and
6.78 (d, 1
H).
1-[1-(Cyanomethyl)-3,4-dihydro-6-methoxy-2(11-x-naphthalenylidene)]
pyrrolidinium bromide (3).
A solution of 1-(3,4-dihydro-6-methoxynaphthalen-2-yl)-pyrrolidine (2) (5.6 g,
24.4
mmol) in acetonitrile (60 mL) was treated with bromoacetonitrile (2.21 mL,
31.7
mmol). The resultant solution was stirred at ambient temperature for 1 h. The
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CA 02373035 2001-11-05
WO 00/68197 PCT/US00/10981
pyrrolidinium salt (3), was collected by filtration and washed with diethyl
ether, to
give the pyrrolidinium bromide as a hygroscopic colorless solid which was used
directly in the subsequent reaction. MS 269 (M+).
(1,2,3,4-Tetrahydro-6-methoxy-2-oxo-naphthalen-1-yl)-acetonitrile (4).
A solution of 1-[1-(cyanomethyl)-3,4-dihydro-6-methoxy-2(1H)-
naphthalenylidene)]
pyrrolidinium bromide (3) (24.4 mmol) and acetic acid (5 mL) in
dichloromethane/
methanol/ water (60 mL/ 100 mL/ 50 mL) was stirred at ambient temperature for
18
h. An organic layer was separated and the aqueous layer was extracted with
dichloromethane (100 mL). The combined organics were washed with water, then
washed with a saturated solution of aqueous sodium bicarbonate, and dried over
magnesium sulfate. The solvent was evaporated in vacuo to give the a-cyano-~
tetralone product (4) as a brown oil (3.3 g, 63%, 2 steps). IR(neat): 1715,
1722,
2251 crm'; NMR(CDCI3): 8 2.47-2.58 (m, 1 H), 2.67-2.80 (m, 1 H), 2.88-3.13 (m,
4
H), 3.77 (t, 1 H), 3.77 (s, 3 H), 6.81-6.90 (m, 2 H), 7.19 (d, 1 H).
3a,4,5,9b-Tetrahydro-7-methoxy-1 H-benzo[e]indol-2-yl)-amine (5) and
3a,4,5,9b-tetrahydro-7-methoxy-1 H-benzo[e]indol-2-yl)-amine hydrochloride
(6). A solution of (1,2,3,4-tetrahydro-6-methoxy-2-oxo-naphthalen-1-yl)-
acetonitrile
(4) (3.5 g, 16.2 mmol) and ammonium acetate (18.8 g, 0.24 mol) in methanol (50
mL) was stirred at ambient temperature for 15 min. Sodium cyanoborohydride
(5.11
g, 0.081 mol) was added and the resultant solution was heated at reflux for 1
h. The
solvent was evaporated in vacuo, and the residue was treated with a solution
of
sodium hydroxide (12 g, 0.3 mol) in water (100 mL) at 0°C. A pale gray
solid
precipitated out of solution and was collected by filtration, washed with
water and
triturated in diethyl ether to give the crude cyclic amidine (5) (3.5 g,
100%). This
material (3.0 g, 13.8 mmol) was dissolved in tetrahydrofuran/methanol (~9:1,
75 mL)
and treated with 1 M hydrochloric acid in diethyl ether (40 mL) at 0°C
to induce
precipitation. The resultant precipitate was collected by filtration and
washed with
diethyl ether to give 3a,4,5,9b-tetrahydro-7-methoxy-1 H-benzo[e]indol-2-yl)-
amine
hydrochloride (6) (1.57 g, 45%) as an off white solid. IR(KBr): 1611, 1681,
1703,
2832, 3106 cm-'; NMR(DMSO-ds): 8 1.79-1.91 (m, 2 H), 2.60-2.74 (m, 3 H), 3.33-
3.46 (m, 1 H), 3.63-3.73 (m, 1 H), 3.71 (s, 3 H), 6.80 (d, 1 H), 6.71 (d of d,
1 H), 7.17
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(d, 1 H), 9.08 (br s, 1 H), 9.34 (br s, 1 H) and 10.06 (br s, 1 H); MS 217
(M+H)+.
(Figure 1 ).
H
N
O ~ N
\ \\
H3C0 ~1 ) MeOH H3C0
()
N
C
Br-CH2-CN ~ HOAc
\ +
R.T. BrQ CH2C12,
MeOH,
H3C0
(3) H20 l4l
H2
NH40Ac
NaBH3CN 1 M HCI
MeOH, reflux Et20
HCI
hl (b)
Figure 1
trans-4-[[(Phenylsulfonyl)amino]methyl]-N-(cis-3a,4,5,9b-tetrahydro-7-methoxy-
1H-benz[e]indol-2-yl)cyclohexanecarboxamide (7).
A solution of trans-4-(benzenesulfonamido)methylcyclohexane carboxylic acid
(1.16
g, 4.15 mmol), O-benzotriazol-1-yl-N,N,N;N'-tetramethyluronium
hexafluorophosphate (1.58 g, 4.15 mmol) and N,N-diisopropylethylamine (2.41
mL,
13.8 mmol) in N,N-dimethylformamide (15 mL) was stirred at ambient temperature
for 15 min. After this time, 3a,4,5,9b-tetrahydro-7-methoxy-1 H-benzo[e]indol-
2-yl)-
amine hydrochloride (6) (1.0 g, 3.96 mmol) was added, and the resultant
solution
was heated to 45°C for 1.5 h. The solution was then poured into ice
water and the
product which precipitated was collected by filtration, washed with water and
air
dried. This solid was triturated in diethyl ether to give trans-4-
[[(phenylsulfonyl)amino]methyl]-N-(cis-3a,4,5,9b-tetrahydro-7-methoxy-1 H-
benz[e]indol-2-yl)cyclohexanecarboxamide (7) as a colorless solid (1.87 g,
95%).
NMR (DMSO-d6): s 0.69-0.89 (m, 2 H), 1.10-1.34 (m, 3 H), 1.63-1.88 (m, 5 H),
2.10-
2.27 (m, 1 H), 3.24-3.50 (m, 3 H), 3.70 (s, 3 H), 4.04-4.13 (m, 1 H), 6.63 (d,
1 H),
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6.74 (d of d, 1 H), 7.05 (d, 1 H), 7.54-7.67 (m, 4 H) and 7.74-7.83 (m, 2 H);
MS 496
(M+H)+. (Figure 2).
EXAMPLE 2
N-[[traps-4-[[(cis-3a,4,5,9b-tetrahydro-7-methoxy-1 H-Benz[e]indol-2-yl)
amino]methyl]cyclohexyl]methyl]benzenesulfonamide (8).
traps-4-[[[(Phenylsulfonyl)amino]methyl]-N-(cis-3a,4,5,9b-tetrahydro-7-methoxy-
1 H-
benz[e]indol-2-yl]cyclohexanecarboxamide (7) (1.6 g, 3.22 mmol) was added in
portions, with stirring, to a solution of lithium aluminum hydride (16.1 mmol)
in
tetrahydrofuran (36 mL) at ambient temperature. The resultant solution was
heated
at reflux for 45 min. The solution was then cooled on an ice bath, and then a
solution of water (0.65 mL) in tetrahydrofuran (5 mL) was carefully added,
followed
by the addition of ten percent aqueous sodium hydroxide (0.65 mL) and water
(2.1
mL). The resultant suspension, which formed, was stirred at ambient
temperature
for 30 min and then dried over sodium sulfate. The insoluble inorganic
material was
removed by filtration, and washed generously with tetrahydrofuran. The solvent
was
evaporated in vacuo, the residue was dissolved in a minimum amount of
isopropanol
and this solution was treated with a concentrated solution of hydrogen
chloride in
isopropanol. The solvents were evaporated in vacuo to give crude give N-
[[traps-4-
[[(cis-3a,4,5,9b-tetrahydro-7-methoxy-1 H-benz[e]indol-2-yl)amino]methyl]
cyclohexyl]methyl]benzenesulfonamide hydrochloride salt as a pale pink solid
(1.38
g; estimated purity ~75% by HPLC). A 300 mg. portion of this material was
purified
by preparative HPLC on a C18 reverse phase column (4 cm by 45 cm), eluted with
a
gradient of water/acetonitrile/trifluoroacetic acid from 90/10/0.1 to
10/90/0.1 (v/v)
(flow rate of 40 mL per minute) over 50 minutes. The product obtained was
converted to the hydrochloride salt with ethanolic hydrogen chloride to give
pure give
N-[[traps-4-[[(cis-3a,4,5,9b-tetrahydro-7-methoxy-1 H-benz[e]indol-2-
yl)amino]methyl]
cyclohexyl]methyl]benzenesulfonamide hydrochloride (8) as a colorless solid
(0.15
g). NMR (DMSO-ds): 8 0.70-0.94 (m, 4 H), 1.20-1.50 (m, 2 H), 1.62-1.77 (m, 4
H),
1.80-1.94 (m, 2 H), 2.55-2.73 (m, 5 H), 3.03-3.16 (m, 2 H), 3.31-3.46 (m, 1
H), 3.63-
3.73 (m, 1 H), 3.71 (s, 3 H), 4.24-4.32 (m, 1 H), 6.70 (d, 1 H), 6.79 (d of d,
1 H), 7.14
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(d, 1 H), 7.55-7.67 (m, 4 H), 7.74-7.82 (m, 2 H), 9.66 (br t, 1 H) and 10.09
(br s, 1 H);
MS 482 (M+H)+. (Figure 2).
(dl
2
O
I I
O H N-S
HO ~/
HBTU, iPr2NEt
DMF
O
.'''\ II
1 ) LiAIH4 / THF
2) HCI
O
,.'''\ _1I
HCI
Figure 2
EXAMPLE 3
traps-4-[[(Phenylsulfonyl)amino]methyl]-N-(cis-3a,4,5,9b-tetrahydro-7-hydroxy-
1H-benz[e]indol-2-yl)cyclohexanecarboxamide (9).
A suspension of traps-4-[[(phenylsulfonyl)amino]methyl]-N-(cis-3a,4,5,9b-
tetrahydro-
7-methoxy-1 H-Benz[e]indol-2-yl)cyclohexanecarboxamide (7) (0.200 g, 0.403
mmol)
in dichloromethane (2 mL) was added dropwise with stirring, to a solution of
boron
tribromide (1.6 mmol) in dichloromethane (12 mL) at 0°C. The resultant
suspension
42
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was stirred at 0°C for 30 min. Methanol (--1 mL) was added at which
point the
mixture became a clear yellow solution. The solution was stirred for 30 min at
0°C.
The solvents were evaporated in vacuo, and the residue was purified by
preparative
HPLC on C,8 reverse phase column, using water/acetonitrile/trifluoroacetic
acid
(50:50:0.1 ) as the eluent. The product obtained was dissolved in a minimum
amount
of methanol and converted to the hydrochloride salt by treatment with
ethanolic
hydrogen chloride. The solvents were evaporated in vacuo and the residue was
triturated with diethyl ether to give trans-4-[[(phenylsulfonyl)amino]methyl]-
N-(cis-
3a,4,5,9b-tetrahydro-7-hydroxy-1 H-Benz[e]indol-2-yl)cyclohexanecarboxamide
(9) as
a beige solid (0.093 g, 45%). NMR (DMSO-ds): 8 0.77-0.95 (m, 2 H), 1.20-1.37
(m, 3
H), 1.64-1.80 (m, 2 H), 1.83-1.98 (m, 3 H), 2.43-2.64 (m, 5 H), 2.91 (d of d,
1 H),
3.48-3.74 (m, 4 H), 4.37-4.47 (m, 1 H), 6.54 (d, 1 H), 6.65 (d of d, 1 H),
7.03 (d, 1 H),
7.53-7.83 (m, 6 H), 9.37 (br s, 1 H), 11.56 (br s, 1 H), 13.22 (br s, 1 H); MS
482
(M+H)+ (Figure 3).
0
j o \ /
I
O 'HCI
(7) 1 ) BBr3
~, 2) HCI
H
O
,;
N H IOSI \ /
~N O
'HCI
O
(9)
Figure 3
EXAMPLE 4
N-[[traps-4-[[(cis-3a,4,5,9b-tetrahydro-7-hydroxy-1 H-benz[e]indol-2-yl)
amino]methyl]cyclohexyl]methyl]benzenesulfonamide hydrochloride (10).
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A solution of boron tribromide (3.4 mmol) in dichloromethane (3.4 mL) was
added to
a solution of N-[[trans-4-[[(cis-3a,4,5,9b-tetrahydro-7-methoxy-1 H-
benz[e]indol-2-
yl)amino]methyl]cyclohexyl]methyl]benzenesulfonamide hydrochloride (8) (0.37
g,
0.714 mmol) and triethylamine (0.235 mL, 1.69 mmol) in dichloromethane (20 mL)
at 0°C. The resultant mixture was stirred at 0°C for 1 h, and
stirring was continued at
room temperature for an additional 1 h. The mixture was cooled on an ice bath
and
methanol was added. After stirring for several minutes, the solvents were
evaporated in vacuo. The residue was purified by preparative HPLC on a C18
reverse phase column, eluted with water/acetonitrile/trifluoroacetic acid
(50:50:0.1 ).
The product was dissolved in a minimum amount of methanol and converted to the
hydrochloride salt with ethanolic hydrogen chloride. The solvents were
evaporated
in vacuo to give N-[[trans-4-[[(cis-3a,4,5,9b-tetrahydro-7-hydroxy-1 H-
benz[e]indol-2-
yl)amino]methyl]cyclohexyl]methyl]benzenesulfonamide hydrochloride as a
colorless
solid (0.15 g, 42%). NMR (DMSO-ds): 8 0.67-0.98 (m, 4 H), 1.21-1.50 (m, 2 H),
1.58-
1.97 (m, 6 H), 2.50-2.75 (m, 5 H), 3.03-3.23 (m, 2 H), 3.31-3.46 (m, 1 H),
3.55-3.67
(m, 1 H), 4.17-4.33 (m, 1 H), 6.53 (s, 1 H), 6.64 (d, 1 H), 6.99 (d, 1 H),
7.52-7.83 (m,
6 H), 9.34 (br s, 1 H), 9.76 (br s, 1 H) and 10.24 (br s, 1 H); MS 468 (M+H)+
(Figure
4).
0
0
HCI
1 ) BBr3
2) HCI
($)
'' ,~N-O
H ~ lOl
HCI
H
(10)
Figure 4
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EXAMPLE 5
2-([4-(2,3-Dihydro-2-oxo-1H-benzimidazol-1-yl)-1-piperidinyl]acetyl]-
3a,4,5,9b-
tetrahydro-1 H-benzo[e]indol-2-yl)-amine (11 ).
A solution of 4-(2-keto-1-benzimidazolinyl)piperidine (10.0 g, 46 mmol), ethyl
bromoacetate (5.1 mL, 46 mmol) and N,N-diisopropylethylamine (8.8 mL, 50.6
mmol) in acetonitrile (200 mL) was heated at reflux for 1 hour. The solvent
was
evaporated in vacuo, and the residue was suspended in water 0200 mL). The
suspension was made basic with the addition of a saturated aqueous solution of
sodium bicarbonate. The resultant solid was collected by filtration, washed
with
water and dried in vacuo to give the ethyl (4-(2-keto-1-
benzimidazolinyl)piperidin-1-
yl)acetate as a colorless solid (13.2 g, 94%). MS m/z 304 (MH+); NMR(CDC13): 8
1.32 (t, 3 H), 1.84 (br d, 2 H), 2.40-2.66 (m, 4 H), 3.13 (br d, 2 H), 3.31
(s, 2 H), 4.23
(q, 2 H), 4.45-4.49 (m, 1 H), 6.99-7.10 (m, 2 H), 7.12-7.19 (m, 1 H), 7.27-
7.34 (m, 1
H) and 10.54 (br s, 1 H).
A solution of ethyl (4-(2-keto-1-benzimidazolinyl)piperidin-1-yl)acetate (13.0
g, 42.8
mmol) in methanol (150 mL) was treated with an aqueous solution of sodium
hydroxide (3 N, 30 mL, 90 mmol) and heated at reflux for 2 hours. The solution
was
cooled to room temperature and neutralized with the addition of concentrated
hydrochloric acid (12 N, 7.5 mL). The solvent was evaporated in vacuo, and the
resultant amorphous solid was dried in vacuo with heating (~50°C)
overnight to give
(4-(2-keto-1-benzimidazolinyl)piperidin-1-yl)acetic acid (17.2 g) which was
used in
the subsequent step without purification. MS m/z 304 (MH+); NMR(DMSO-ds): 8
1.74
(br d, 2 H), 2.53-2.67 (m, 2 H), 2.74-2.86 (m, 2 H), 3.33 (s, 2 H), 4.29-4.42
(m, 1 H),
6.97-7.05 (m, 3 H) and 7.38-7.43 (m, 1 H).
A mixture of (4-(2-keto-1-benzimidazolinyl)piperidin-1-yl)acetic acid (2.34 g,
5.83
mmol), 2-(1 H-benzotriazole-1-yl)-1,1,3,3-tetramethyuronium
hexafluorophosphate
(1.87 g, 4.93 mmol) and N,N-diisopropylethylamine (3.1 mL, 17.9 mmol) in N,N-
dimethylformamide (15 mL) was stirred at 45°C for 10 min. After this
time,
3a,4,5,9b-tetrahydro-1H-benzo[e]indol-2-yl)-amine (1.0 g, 4.49 mmol) was added
to
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the mixture, and the resultant solution was stirred at room temperature for an
additional two hours. A white precipitate was collected by filtration and
washed with
water. The product was purified by flash chromatography on silica gel using 5
to
10% methanol in dichloromethane as the eluent. The product was triturated with
diethyl ether and dried in vacuo to give 2-[[4-(2,3-dihydro-2-oxo-1H-
benzimidazol-1-
yl)-1-piperidinyl]acetyl]-3a,4,5,9b-tetrahydro-1 H-benzo[e]indol-2-yl)-amine
(11 ) as a
colorless solid, (0.23 g, 9%). An additional 0.6 g of product was recovered
from the
mother liquor as well as several impure fractions of the chromatography. MS
m/z
444 (MH+); NMR(CDCI3): s 1.78-2.10 (m, 4 H), 2.34-2.83 (m, 6 H), 3.00-3.17 (m,
3
H), 3.20 (s, 2 H), 3.56-3.78 (m, 2 H), 4.27-4.45 (m, 2 H) and 7.00-7.23 (m, 8
H).
(Figure 5).
O
0 O ~NH
~-N H ~ ~ N
N Br~OEt O /
/ base / CH CN Et0' v NJ
HNJ \ ~ s
1 ) NaOH / Aq. MeOH
2) H+
NH2 O
~NH
N N
/ ~/N~ \
~HX HO
N~)
HBTU) / base
H
l.i .~ l
Figure 5
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EXAMPLE 6
2-[[4-(2,3-Dihydro-2-oxo-1H-benzimidazol-1-yl)-1-piperidinyl]ethyl]- 3a,4,5,9b-
tetrahydro-7-methoxy-1H-benzo[e]indol-2-yl)-amine (12).
2-[[4-(2,3-Dihydro-2-oxo-1 H-benzimidazol-1-yl)-1-piperidinyl]acetyl]-
3a,4,5,9b-
tetrahydro-7-methoxy-1 H-benzo[e]indol-2-yl)-amine (0.500 g, 1.13 mmol) was
carefully added in portions, with stirring, to a solution of lithium aluminum
hydride
(4.0 mmol) in tetrahydrofuran (20 mL). Considerable foaming occurred with each
addition. The resultant mixture was heated at reflux for 1.5 hours. The
resultant
solution was cooled on an ice bath, and a solution of water (0.16 mL) in
tetrahydrofuran (5 mL) was carefully added, with stirring, to the solution.
With care,
15% aqueous sodium hydroxide (0.16 mL) was added followed by the addition of
another aliquot of water (0.5 mL). The inorganic salts were removed by
filtration and
washed successively with tetrahydrofuran and dichloromethane. The organic
solutions were combined, and the solvents were evaporated in vacuo. The
residue
was purified by preparative HPLC on a C18 reverse phase column eluted with a
gradient of acetonitrile/water/trifluoroacetic acid from 10/90/0.1 (v/v) to
90/10/0.1 to
give 2-[[4-(2,3-dihydro-2-oxo-1 H-benzimidazol-1-yl)-1-piperidinyl]acetyl]-
3a,4,5,9b
tetrahydro-7-methoxy-1 H-benzo[e]indol-2-yl)-amine (12) as a trifluoroacetic
acid salt,
(0.166 g, 29%). MS m/z 430 (MH+); NMR(DMSO-ds): 8 1.86-2.03 (m, 4 H), 2.58
2.83 (m, 4 H), 3.15-3.87 (m, 11 H), 4.33-4.45 (m, 1 H), 4.47-4.60 (m, 1 H),
6.94-7.04
(m, 3 H), 7.11-7.38 (m, 5 H), 10.03 (br s, 1 H), 10.41 (br s, 1 H) and 10.98
(br s, 1
H). (Figure 6).
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H
II N O
O
N
NH
1 ) LAH
2) H+
H
~N O
N' \
NH
~TFA
(12)
Figure 6
The following compounds of this invention were prepared from appropriately
substituted ~-tetralones as the starting material using the experimental
protocols
described above.
trans-4-[[(Phenylsulfonyl)amino]methyl]-N-(cis-3a,4,5,9b-tetrahydro-1 H-
benz[e]indol-2-yl)cyclohexanecarboxamide (13).
Calculated mass: 465; MS: 466 (M+H)+
N-[[traps-4-[[(cis-3a,4,5,9b-tetrahydro-1 H-Benz[e]indol-2-
yl)amino]methyl]cyclohexyl]methyl]benzenesulfonamide (14).
Calculated mass: 451; MS: 452 (M+H)+
traps-4-[[(Phenylsulfonyl)amino]methyl]-N-(cis-3a,4,5,9b-tetrahydro-7-fluoro-
1H-benz[e]indol-2-yl)cyclohexanecarboxamide (15).
Calculated mass: 483; MS: 484 (M+H)+
N-[[traps-4-[[(cis-3a,4,5,9b-tetrahydro-7-fluoro-1 H-benz[e]indol-2-
yl)amino]methyl]cyclohexyl]methyl]benzenesulfonamide (16).
Calculated mass: 469; MS: 470 (M+H)+
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traps-4-[[(Phenylsulfonyl)amino]methyl]-N-(cis-3a,4,5,9b-tetrahydro-7-chloro-
1H-Benz[e]indol-2-yl)cyclohexanecarboxamide (17).
Calculated mass: 499; MS: 500 (M+H)+
N-[[traps-4-[[(cis-3a,4,5,9b-tetrahydro-7-chloro-1 H-benz[e]indol-2-
yl)amino]methyl]cyclohexyl]methyl]benzenesulfonamide (18).
Calculated mass: 485; MS: 486 (M+H)+
traps-4-[[(Phenylsulfonyl)amino]methyl]-N-(cis-3a,4,5,9b-tetrahydro-7-
methoxy-1H-benz[e]indol-2-yl)cyclohexanecarboxamide (19).
Calculated mass: 513; MS: 514 (M+H)+
2-[[4-(2,3-Dihydro-2-oxo-1 H-benzimidazol-1-yl)-1-piperidinyl]acetyl]-
[3a,4,5,9b-
tetrahydro-7-methoxy-1H-benzo[e]indol-2-yl]-amine (20).
Calculated mass: 473; MS: 474 (M+H)+
2-[[4-(2,3-Dihydro-2-oxo-1 H-benzimidazol-1-yl)-1-piperidinyl]acetyl]-
[3a,4,5,9b-
tetrahydro-7-hydroxy-1H-benzo[e]indol-2-yl]-amine (21).
Calculated mass: 459; MS: 460 (M+H)+
IN VITRO ASSAYS
NPY5 HTS Centrifugation Assay
The compounds described in this invention were evaluated for binding to the
human neuropeptide Y5 receptor.
Stable Transfection
The human NPY5 receptor cDNA (Genbank Accession number U66275) was
inserted into the vector pClneo (Invitrogen) and transfected into human
embryonic
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kidney cells (HEK-293) via calcium phosphate method (Cullen 1987). Stably
transfected cells were selected with G-418 (600 ~,g/mL). Stably transfected
cells
served as the source for the membranes for the NPY5 receptor binding assay.
Membrane Preparation
NPYS-transfected HEK293 cells were grown to confluence in 150 cm2 culture
dishes. Cells were washed once with phosphate-buffered saline (Gibco Cat#
14040-133). Cells were then incubated in phosphate-buffered saline without
calcium and without magnesium, supplemented with 2 mM EDTA: Cells were
incubated for 10 minutes at room temperature and the cells were collected by
repetitive pipeting. Cells were formed into pellets and then frozen at -
80°C until
needed. Frozen pellets were homogenized with a polytron at full speed for 12
seconds in a homogenization buffer (20 mM Tris HCI, 5 mM EDTA, pH 7.4).
Homogenates were centrifuged for 5 minutes at 4 ° C at 200g.
Supernatants were
transferred to corex tubes and centrifuged for 25 minutes at 28,000g. Pellets
were
re-suspended in Binding (20mM HEPES, 10 mM NaCI, 0.22 mM KH2P04, 1.3mM
CaCl2, 0.8 mM MgS04, pH 7.4). Membranes were kept on ice until use.
A competition binding assay, known to those skilled in the art, was used in
which compounds of formula A compete with '251-PYY for binding to cell
membranes. In simple terms, the less '251-PYY bound to the membranes implies
that a compound is a good inhibitor (competitor). Bound '251-PYY is determined
by
centrifugation of membranes, aspirating supernatant, washing away residual
'251
PYY and subsequently counting the bound sample in a g-counter.
Procedure for Radioligand binding assay
Compounds to be tested were prepared as 10x stocks in binding buffer and
added first to assay tubes (RIA vials, Sarstedt). Twenty (20) wL of each 10x
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compound stock is pipeted into vials and 80 ~.L of '251-PYY (NEN catalog
number
NEX240), which has been diluted to a concentration of 200 pM in 0.25 % BSA in
binding buffer, is added to the compound tubes (final concentration of '251-
PYY is 80
pM). To each tube is added 100 ~L of membranes and the mixture is agitated by
pipeting 2 times. Samples are incubated for 1 hr at room temperature. Aluminum
cast plates (Sarstedt) containing the vials are then centrifuged 10 minutes at
3200
rpm in a Sorvall RT6000. Supernatant is then aspirated. To each vial 400 ~L
PBS
is added and this is then aspirated again. Vials are then put in carrier
polypropylene
12x75 tube and counted in gamma counter (Packard). Non-specific binding is
determined in the presence of 300 nM NPY. Percent inhibition of '251-PYY
binding is
calculated by subtracting non-specific binding from the test samples (compound
(I)),
taking these counts and dividing by total binding, and multiplying by 100.
Inhibitory
concentration values (ICSO) of compounds that show appreciable inhibition of
'2sl-
PYY binding are calculated by obtaining percent inhibition of '251-PYY binding
values
at different concentrations of the test compound, and using a graphing program
such as GraphPad Prism (San Diego, CA) to calculate the concentration of test
compound that inhibits fifty-percent of '251-PYY binding (Table 4).
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Binding Affinities of Compounds of Formula A for the Human NPY Y5 Receptor
(expressed as % Inhibition of '251-PYY Binding)
# %Inh %Inh
3 uM 300 nM
7 98 82
8 98 85
9 97 97
97 74
11 85 48
12 53 9
13 100 80
14 93 89
100 89
16 100 76
17 107 86
18 109 90
19 99 80
50 0
21 78 20
Table 1
5
IN VIVO ASSAYS
Rodent Feeding Model:
Measurement of Food Intake in Food-Deprived Rats
Male Long-Evans rats (180-200 grams) are housed individually and are
maintained on a once-a-day feeding schedule (i.e., 10 a.m. until 4 p.m.) for
five days
following quarantine to allow the animals to acclimate to feeding on powdered
chow
(#5002 PMI Certified Rodent Meal) during the allotted time. The chow is made
available in an open jar, anchored in the cage by a wire, with a metal
follower
covering the food to minimize spillage. Water is available ad-libitum.
Animals are fasted for 18 hours prior to testing. At the end of the fasting
period, animals are administered either compounds of the invention or vehicle.
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Vehicle and test compounds are administered either orally (5 mL/kg) 60 minutes
prior to the experiment, or 30 minutes prior when given subcutaneously (1
mL/kg) or
intraperitoneally (1 mL/kg). Compounds of the invention are administered
orally as
a suspension in aqueous 0.5% methylcellulose-0.4% Tween 80, or
intraperitoneally
as a solution or suspension in PEG 200; compound concentrations typically
range
from 1 mg/kg to 100 mg/kg, preferably from 10-30 mg/kg. Food intake is
measured
at 2, 4, and 6 hours after administration by weighing the special jar
containing the
food before the experiment and at the specified times. Upon completion of the
experiment, all animals are given a one-week washout period before retesting.
Percent reduction of food consumption is calculated subtracting the grams of
food consumed by the treated group from the grams of food consumed by the
control group divided by the grams of food consumed by the control group,
multiplied by 100. A negative value indicates a reduction in food consumption
and a
positive value indicates an increase in food consumption.
change = Treatment - Vehicle X 100
Vehicle
Food Consumption (avg. grams)
Compound Dose(mglkg) 0-2 h 0-4 h 0-6 h 2-6h
# (#raty (%ch~) y%chg.J~ ~(%chg.~%cha.)
Vehicle PEG-2000 N=8 10.19 g 13.71 g 21.03 g 10.84 g
10 30 (i.p.) 4.38 g 6.43 g 10.75 g 6.37 g
N=6 (-57%) (-53%) (-49%) (-41 %)
Vehicle PEG-2000 N=8 9.13 g 12.75 g 21.25 g 12.13 g
8 10 (i. p. ) 4.38 g 7.88 g 12.00 g 7.63 g
N=8 (-52%) (-38%) (-43%) (-37%)
53
