GHRELI N -ACYL
The present invention relates to compounds useful for inliibiting ghrelin O-acyi
transferase (GOAT), pharmaceutical compositions and methods for treating diseases
related to GOAT activity.
GOAT belongs to the membrane-bound Q-acyl transferase (MBOAT) family of
enzymes. It converts desacyl-ghrelin (also known as unacylated ghrelin or UAG) to a
biologically active form, a y -ghre tn (AG), by transferring a fatty acid to the Ser3
residue of the desacylghrelin peptide. Acyl-ghrelin has been shown to increase food
intake and increase adiposity in humans and in rodents. Infusion of AG in humans has
also been shown to suppress glucose-induced insulin secretion. Elimination of the ghrelin
gene has been shown to enhance insulin release to prevent or ameliorate glucose
intolerance in high-fat diet fed ob/ob mice.
Small molecule GOAT inhibitors have been reported in the literature. See WO
2013/125732.
However, the prevalence of obesity and diabetes coupled with the variable
effectiveness and responses to current treatments for obesity and diabetes necessitate that
more treatment choices be available to patients. The present invention provides certain
novel compounds that are GOAT inhibitors. Such new compounds could address the
need for potent, effective treatment of obesity it is further believed that a GOAT
inhibitor may also be useful in reducing weight gain or weight regain as an adjunct to diet
and/or exercise, other therapeutic medicinal agents or procedures designed to reducing
weight gain or treat obesity. Similarly, a GOAT inhibitor may be useful in treating type 2
diabetes, singly or in combination with other treatments for type 2 diabetes.
The present invention rovides a compound of formula
wherein R is selected from - - alkyl optionally substituted with -OH; -OC 1-C4 alkyl;
pyrazolyl, oxazolyl, thiazolyl, or thiadiazolyl, wherein pyrazolyl, oxazolyl, or thiazolyl
each may be optionally substituted one to two times with - C ¾; pyridinyl, pyridazinyl, or
pyrazinyl, wherein each may be optionally substituted with -CI; and phenyl optionally
substituted with -OCH3 or a pharmaceutically acceptable salt thereof.
The present invention provides a pharmaceutical composition comprising a
compound of the invention, or a pharmaceutically acceptable salt thereof, with one or
more pharmaceutically acceptable carriers, diluents, or excipients. In a further
embodiment, the composition is used in combination with one or more other therapeutic
agents.
A further aspect of the present invention provides a method of reducing weight
gain or weight regain or treating type 2 diabetes or obesity comprising administering a
compound of the present invention, or a pharmaceutically acceptable salt thereof, to a
patient in need thereof.
The present invention also provides a compound of the invention, or a
pharmaceutically acceptable salt thereof, for use in therapy, in particular for reducing
weight gain or weight regain or treating type 2 diabetes or obesity. Even further, the
present invention provides a compound of the invention, or a pharmaceutically acceptable
salt thereof, for use in reducing weight gain or weight regain or treating type 2 diabetes or
obesity. Furthermore, the present invention provides the use of a compound of the
invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a
medicament for reducing weight gain or weight regain or treating type 2 diabetes or
obesity.
The present invention further provides a method of treating the sequelae of an
ischemic event comprising administering a compound of the present invention, or a
pharmaceutically acceptable salt thereof, to a patient in need thereof. In a further
embodiment, the ischemic event is myocardial ischemia or cardiac ischemia or cerebral
ischemia.
In yet another aspect, the present invention provides a compound of the invention,
or a pharmaceutically acceptable salt thereof, for use in therapy, in particular for treating
the sequelae of an ischemic event. Even further, the present invention provides a
compound of the invention, or a pharmaceutically acceptable salt thereof, for use in
treating the sequelae of an ischemic event. Furthermore, the present invention provides
the use of a compound of the invention, or a pharmaceutically acceptable salt thereof, in
the manufacture of a medicament for treating the sequelae of an ischemic event. In a
further embodiment, the ischemic event is myocardial ischemia or cardiac ischemia or
cerebral ischemia.
The present invention further provides a method of treating addiction disorders
comprising administering a compound of the present invention, or a pharmaceutically
acceptable salt thereof, to a patient in need thereof. In a further embodiment, the
addiction disorder involves consummately behaviors, such as alcohol, smoking,
overeating, or use of illicit drugs.
The present invention provides a method to ameliorate the consequences of stress
that promote addictive behaviors comprising administering a compound of the present
invention, or a phannaceutically acceptable salt thereof, to a patient in need thereof. In a
further embodiment, the addictive behaviors involve consummately behaviors, such as
alcohol, smoking, overeating, or use of illicit drugs.
The present invention also provides a compound of the invention, or a
pharmaceutically acceptable salt thereof, for use in therapy, in particular for treating
addiction disorders. Even further, the present invention provides a compound of the
invention, or a pharmaceutically acceptable salt thereof, for use in treating addiction
disorders. Furthermore, the present invention provides the use of a compound of the
invention, or a phannaceutically acceptable salt thereof, in the manufacture of a
medicament for treating addiction disorders. In a further embodiment, the addiction
disorder involves consummatory behaviors, such as alcohol, smoking, overeating, or use
of illicit drugs.
'The present invention also provides a compound of the invention, or a
pharmaceutically acceptable salt thereof, for use in therapy, in particular for ameliorating
the consequences of stress that promote addictive behaviors. In a further embodiment,
the addiction disorder involves consummatory behaviors, such as alcohol, smoking,
overeating, or use of illicit drugs. Even further, the present invention provides a
compound of the invention, or a pharmaceutically acceptable salt thereof, for use in
ameliorating the consequences of stress that promote addictive behaviors. Furthermore,
the present invention provides the use of a compound of the invention, or a
pharmaceutically acceptable salt thereof, in the manufacture of a medicament for
ameliorating the consequences of stress that promote addictive behaviors. In a further
embodiment, the addictive behaviors involve consummately behaviors, such as alcohol,
smoking, overeating, or use of illicit drugs.
The present invention also encompasses intermediates and processes useful for the
synthesis of a compound of the present invention.
The term "treating" (or "treat" or "treatment") as used herein refers to restraining,
slowing, stopping, or reversing the progression or severity of an existing symptom,
condition or disorder.
As used herein, the term "reducing weight gain" refers to diminishing the increase
in weight of a patient. The term "reducing weight regain" refers to diminishing the
increase in weight of a patient experiencing rebound in weight after weight loss. Weight
regain may be due to a rebound effect following cessation of weight loss achieved via
diet, exercise, behavior modification, or approved therapies. For avoidance of doubt
weight gain or weight regain as used herein refers to weight gain or weight regain
induced by food intake or eating habits and does not refer to non-food related weight gain
such as build up of fluids, weight due to water retention, muscle mass, or inflammation.
An "ischemic event" as used herein refers to an insufficient supply of blood to an
organ or body part. The decrease in blood flow reduces the supply of oxygen to the
affected organ or body part. An ischemic event may also be known as ischemia. One
skilled in the art will know that ischemia can affect different organs or parts of the body,
for example the heart, such as myocardial ischemia or cardiac ischemia, or the brain, such
as cerebral ischemia.
"Addiction disorders" as used herein describes excessive maladaptive behaviors
for which an mdividual exhibits an inability to control despite negative consequences. Of
particular relevance to the present invention are addiction disorders involving
consummately behaviors such as alcohol intake, smoking, overeating, and use of illicit
drugs. This invention normalizes aberrant incentive and reward neural substrates that are
dysregulated in individuals with addictive disorders. Stress is often a precipitating agent
in the etiology and maintenance of addictive disorders; this invention provides a method
to ameliorate the consequences of stress that promote addictive behaviors.
A compound of the present invention may react to form pharmaceutically
acceptable salts. Pharmaceutically acceptable salts and common methodology for
preparing them are well known in the art. See, e.g., P. Stahl, et al. Handbook of
Pharmaceutical Salts: Properties, Selection and Use, 2 Revised Edition (Wiley-VCH,
2011); S.M. Berge, etal, "Pharmaceutical Salts," Journal of Pharmaceutical Sciences,
Vol. 66, No. 1, January 1977
The skilled artisan will appreciate that the compound of the invention, or
pharmaceutically acceptable salt thereof, are comprised of a core that contains at least one
chiral center, represented by * in (!) below:
(I)
Preferred compounds of the invention are represented by (ΪΪ) :
(II)
or pharmaceutically acceptable salts thereof.
The skilled artisan will appreciate that additional chiral centers may be created i
the compounds of the invention by the selection of certain variables. The present
invention contemplates all individual enantiomers or diastereomers, as well as mixtures of
the enantiomers and diastereomers of said compounds including racemates.
The skilled artisan will also appreciate that the Cahn-Ingold-Prelog (R) or (S)
designations for all chiral centers will vary depending upon the substitution patterns of
the particular compound. The single enantiomers or diastereomers may be prepared
beginning with chiral reagents or by stereoselective or stereospecific synthetic techniques.
Alternatively, the single enantiomers or diastereomers may be isolated from mixtures by
standard chiral chromatographic or crystallization techniques at any convenient point in
the synthesis of compounds of the invention. Single enantiomers of compounds of the
invention are a preferred embodiment of the invention.
A compound of the present invention is preferably formulated as pharmaceutical
compositions administered by a variety of routes, such as oral administration. Such
pharmaceutical compositions and processes for preparing the same are well known in the
art. See, e.g., Remington: The Science and Practice of Pharmacy (A. Gennaro, et aL,
eds , 2 1st ed., Mack Publishing Co., 2005). More particularly preferred, is a
pharmaceutical composition comprising a compound of the invention represented by the
formula
wherein R is selected from - -C3 alkyl optionally substituted with -OH; - OC -C alkyl;
pyrazolyl, oxazolyl, thiazolyi, or thiadiazolyi, wherein pyrazolyl, oxazolyl, or thiazoiyl
each may be optionally substttuted one to two times with - ¾; pyridinyl, pyndazinyl, or
pyrazinyl, wherein each may be optionally substituted with -CI; and phenyl optionally
substituted with -OCH or a pharmaceutically acceptable salt thereof and one or more
pharmaceutically acceptable carriers or diluents.
Although all of the exemplified compounds of the invention are GOAT inhibitors,
certain classes of compounds are preferred. The following paragraphs describe such
preferred classes:
a) R is - -C3alkyl optionally substituted with -OH; -OC 1-C4 alkyl; pyrazolyl,
oxazolyl, thiazoiyl, or thiadiazolyi, wherein pyrazolyl, oxazolyl, or thiazolyi
each may be optionally substituted one to two times with -CH 3; pyridinyl,
pyridazinyl, or pyrazinyl, wherein each may be optionally substituted with
-CI; or phenyl optionally substituted with -OCH 3;
R is pyrazolyl, oxazolyl, thiazoiyl, or thiadiazolyi, wherein pyrazolyl,
oxazolyl, or thiazoiyl each may be optionally substituted one to two times with
-CH 3; pyridinyl or pyrazinyl, wherein each may be optionally substituted with
-CI; or phenyl optionally substituted with -OCH 3.
c) R is pyrazolyl, oxazolyl, thiazolyl, or thiadiazolyl, wherein pyrazolyl,
oxazolyl, or thiazolyl each may be optionally substituted one to two times with
-CH 3;
d) R is pyrazolyl, oxazolyl, thiazolyl, or thiadiazolyl, wherein pyrazolyl,
oxazolyl, or thiazolyl each may be optionally substituted one to two times with
CH 3;
e) R is pyridinyl, pyridazinyl, or pyrazinyl, wherein each may be optionally
substituted with -C ;
f) R is phenyl optionally substituted with -QCH ;
g) R is -CH 3 optionally substituted with -OH, -OCH 3, or -OC(G¾)3;
h) R is -CH 3 optionally substituted with -OH;
i) R is -OCH3 or -OC(CH 3)3;
j ) R is pyrazolyl;
k) R is pyrazolyl substituted with -CH ;
1) the compound of the present invention is the free base;
m) the methyl substituent adjacent to -NC(0)R is in the S configuration in the
compound of the present invention;
A preferred embodiment of the present invention relates to compounds of the
formula,
wherein R is selected from - C1-C3 alkyl optionally substituted with -OH; - QC - alkyl;
pyrazolyl, oxazolyl, thiazolyl, or thiadiazolyl, wherein pyrazolyl, oxazolyl, or thiazolyl
each may be optionally substituted one to two times with ~CH : pyridinyl, pyridazinyl, or
pyrazinyl, wherein each may be optionally substituted with -CI; and phenyl optionally
substituted with -OCH 3 or a pharmaceutically acceptable salt thereof.
Another preferred embodiment of the invention relates to compounds of the
following formula
wherein R is selected from -CH3 optionally substituted with -OH; -OCH3 or -OC(C3¾)3;
pyrazolyl, oxazolyl, thiazolyl, or thiadiazolyl, wherein pyrazolyl, oxazolyl, or thiazolyl
each may be optionally substituted one to two times with - C ¾ pyridinyl, pyiidazinyl, or
pyrazinyl, wherein each may be optionally substituted with -CI; and phenyl optionally
substituted with -OCH 3 or a pharmaceutically acceptable salt thereof.
Another preferred embodiment of the invention relates to compounds of the
following formula
wherein R is selected from pyrazolyl, oxazolyl, thiazolyl, or thiadiazolyl, wherein
pyrazolyl, oxazolyl, or thiazolyl each may be optionally substituted one to two times with
-CH 3; pyridinyl, pyridazinyl, or pyrazinyl, wherein each may be optionally substituted
with -CI; and phenyl optionally substituted with -OCH 3; or a pharmaceutically acceptable
salt thereof.
Another preferred embodiment of the invention relates to compounds of the
following formula
wherein R is selected from pyrazolyl, oxazolyl, thiazolyl, or thiadiazolyl, wherein
pyrazolyl, oxazolyl, or thiazolyl each may be optionally substituted one to two times with
-CH 3; pyridinyl or pyrazinyl, wherein each may be optionally substituted with - C ; and
phenyl optionally substituted with -OCH ; or a pharmaceutically acceptable salt thereof.
Another preferred embodiment of the present invention relates to compounds of
the formula,
wherein R is selected from pyrazoly], oxazolyl, thiazoiyl, and thiadiazolyl, wherem
pyrazolyl, oxazolyl, or thiazoiyl each may be optionally substituted one to two times with
-CH 3; or a pharmaceutically acceptable salt thereof.
A further preferred embodiment of the present invention relates to compounds of
the formula:
wherein R is selected from pyridinyl, pyridazinyl, and pyrazinyl, wherein each may be
optionally substituted with -CI; or a pharmaceutically acceptable salt thereof.
Another preferred embodiment of the invention relates to compounds of the
following formula
wherein R is phenyl optionally substituted with - QC or a pharmaceutically acceptable
salt thereof.
Another preferred embodiment of the invention relates to compounds of the
following formula
wherein R is selected from -CH3 optionally substituted with -OH, -OCH3, and
-- C(CH )3; or a pharmaceutically acceptable salt thereof.
Another preferred embodiment of the invention relates to compounds of the
following formula
wherein R is -CH3 optionally substituted with -OH; or a pharmaceutically acceptable
thereof.
Another preferred embodiment of the invention relates to compounds of the
following formula
wherein R is selected from -OCH3 and C(C 3) 3 or a pharmaceutically acceptable salt
thereof.
An especially preferred embodiment of the present invention relates to compounds
of the formula:
or a pharmaceutically acceptable salt thereof.
Another especially preferred embodiment of the present invention relates to the
compound of the formula:
or a pharmaceutically acceptable salt thereof.
A further especially preferred embodiment of the present invention relates to the
compound of formula:
The compound of the present invention is generally effective over a wide dosage
range. For example, dosages per day fall within the range of about 0.03 to about 30
mg g of body weight. In some instances dosage levels below the lower limit of the
aforesaid range may be more than adequate, while in other cases still larger doses may be
employed while maintaining a favorable benefit/risk profile, and therefore the above
dosage range is not intended to limit the scope of the invention in any way. t will be
understood that the amount of the compound actually administered will be determined by
a physician, in the light of the relevant circumstances, including the condition to be
treated, the chosen route of administration, the actual compound or compounds
administered, the age, weight, and response of the individual patient, and the severity of
the patient's symptoms.
It is well known in the art that agents for the treatment of diabetes and/or obesity
may be combined with other agents for the treatment of diabetes and/or obesity. The
compound of the invention, or a pharmaceutically acceptable salt thereof, may be co
administered, simultaneously or sequentially, with other effective treatment(s) for
diabetes or obesity. The compound of the invention, or a pharmaceutically acceptable
salt thereof, alone or in combination with other effective treatement(s) may be
administered, simultaneously or sequentially, following approved medical procedures
such as bariatric surgeries, for example, gastric bypass surgery or adjustable gastric
banding procedures
The compounds of the present invention, or salts thereof, may be prepared by a
variety of procedures known in the art, some of which are illustrated in the Schemes,
Preparations, and Examples below. The specific synthetic steps for each of the routes
described may be combined in different ways, or in conjunction with steps from different
schemes, to prepare compounds or salts of the present invention. The products of each
step in the Schemes below can be recovered by conventional methods well known in the
art, including extraction, evaporation, precipitation, chromatography, filtration,
trituration, and crystallization. In the Schemes below, all substituents unless otherwise
indicated, are as previously defined. The reagents and starting materials are readily
available to one of ordinary skill in the art.
Additionally, certain intermediates described in the following schemes may
contain one or more nitrogen protecting groups. The variable protecting group may be
the same or different in each occurrence depending on the particular reaction conditions
and the particular transformations to be performed. The protection and deprotection
conditions are well known to the skilled artisan and are described in the literature (See for
example "Greene 's Protective Groups in Organic Synthesis", Fourth Edition, by Peter
G.M. Wuts and Theodora W. Greene, John Wiley and Sons, Inc. 2007).
Certain stereochemical centers have been left unspecified and certain substituents
have been eliminated i the following schemes for the sake of clarity and are not intended
to limit the teaching of the schemes in any way. Single enantiomers or diastereomers
may be prepared beginning with chiral reagents or by stereoselective or stereospecific
synthetic techniques. Alternatively, the single enantiomers or racemates may be isolated
from mixtures by standard chiral chromatographic or crystallization techniques at any
convenient point in the synthesis of compounds of the invention by methods such as
selective crystallization techniques or chiral chromatography (See for example, J.
Jacques, et al., "Enantiomers, Racemates, and Resolutions" , John Wiley and Sons, Inc.,
1981, and E . Eliel and S.H. Wilen," Stereochemistry of Organic Compounds", Wiley-
Interscience, 1994).
Some intermediates or compounds of the present invention may have one or more
chiral centers. The present invention contemplates all individual enantiomers or
diastereomers, as well as mixtures of the enantiomers and diastereomers of said
compounds including racemates. It is preferred that compounds of the present inventi o
containing at least one chiral center exist as a single enantiomer or diastereomer. The
single enantiomer or diastereomer may be prepared beginning with chiral reagents or by
stereoselective or stereospecific synthetic techniques. Alternatively, the single
enantiomer or diastereomer may be isolated from mixtures by standard chiral
chromatographic or crystallization techniques. The skilled artisan will appreciate that in
some circumstances the elution order of enantiomers or diastereomers may be different
due to different chromatographic columns and mobile phases.
Certain abbreviations are defined as follows: "AC " refers to acetonitrile; "BSA"
refers to Bovine Serum Albumin; "DCC" refers to 1,3-dicyclohexylcarbodiimide; "DCM"
refers to dichloromethane; "DIC" refers to diisopropylcarbodiimide; "DIPEA" refers to
diisopropylethylamine or N -ethyl -N -isopropyl-propan-2-amine; "DMAP" refers to
dimethylaminopyridine; "DMF" refers to dimethylformamide; "DMSO" refers to
dimethylsulfoxide; "EDCI" refers to l-(3-dimethylaTninopropyl)~3-ethylcarbodiimtde
hydrochloride; "EDTA" refers to ethylenediaminetetraacetic acid; "ee" refers to
enantiomeric excess; "ELISA" refers to enzyme-linked immuno assay; "EtOAc" refers to
ethyl acetate; "EtOH" refers to ethanol or ethyl alcohol; "Ex" refers to example; "FBS"
refers to retal bovine serum; "HATU" refers to (dimethylamino )-N y^-dimethyl(3 H-
[l,2 3]triazo2o[4,5-6 ]pyridin-3-yloxy)methaniminium hexafluorophosphate; "HOAt"
refers to l-hydroxy-7-azobenzotriazole; "FJOBt" refers to 1-hydroxy Ibenzotriazole
hydrate; "HBTIJ" refers to refers to 2-(l H -benzotriazole-l -yl)- l ,1,3,3-
tetramethyluronium hexafluorophosphate; "HPLC" refers to High Performance Liquid
Chromatography; "HRP" refers to horseradish peroxidase; "IC50" refers to the
concentration of an agent that produces 50% of the maximal inhibitory response possible
for that agent; "LC-ES/MS" refers to Liquid Chromatography Electrospray Mass
Spectrometry; "min" refers to minute or minutes; "MeOH" refers to methanol or methyl
alcohol; "MS" refers to Mass Spectrometry; "OAc" refers to acetate; "PBS" refers to
phosphate buffered saline, "PG" refers to protecting group; "Prep" refers to preparation;
"PYBOP ¾" refers to benzotriazol- 1-yloxytripyrrolidino-phosphoriium
hexafluorophosphate; "PYBROP®" refers to bromo-tris-pyrrolidino
phosphoniumhexafluoro phosphate; "RT" refers to room temperature; "SCX" refers to
strong cation exchange; "SFC" refers to supercritical fluid chromatography; "SPE" refers
to solid phase extraction; "TFA" refers to trifluoroacetic acid, "T'MB" refers to 3,3',5,5'-
tetramethylbenzidine; and "T " refers to time of retention.
In the Schemes below, all substituents unless otherwise indicated, are as
previously defined. The reagents and starting materials are readily available to one of
ordinary skill in the art. Others may be made by standard techniques of organic and
heterocyclic chemistry which are analogous to the syntheses of known structurally-similar
compounds and the procedures described in the Preparations and Examples which follow
including any novel procedures.
Scheme 1
(5)
In scheme 1, is an appropriate amine protecting group. Amine protecting
groups are well known and appreciated in the art, and may incl de carbamates and
amides. One skilled in the art will recognize alternative reagents and procedures to add
and remove said protecting groups.
Compound (2) may be prepared by treating compound (1) with a halogenating
agent, such as iodine monochloride, I2, or N-iodosuccinimide. One skilled in the art wil
recognize that there are a number of methods of heteroaromatic haiogenation. In a further
step, compound (4) may be prepared by coupling compound (2) with an alkyne (3), under
standard coupling conditions, utilizing a palladium derived organometallic reagent, such
as Pd(PPh3)2Cl2 Pd(OAc)¾ or Pd2(dba)3, in the presence of a catalyst, such as Cui, and a
base, such as Et3N, DIPEA, K2CO3, or Cs2C0 3. One skilled in the art will recognize that
there are alternative organometallic reagents derived from metals such as Cu or Zn.
Alternatively, the corresponding free amine of compound (3) may be purchased and
protected by an appropriate amine protecting group. Compound (4) is reduced by
catalytic hydrogenation in the presence of a transition metal catalyst such as platinum
oxide. Other hydrogenation catalysts are well known i the art; for example palladium on
carbon or rhodium derivatives are known to reduce alkynes. One skilled in the art will
recognize that there are other methods for alkyne reduction, including treatment with
sodium in ethanol or zinc in acid. The protecting group can then be removed under
conditions well known in the art, such as under acidic or basic conditions to provide
compound (5)
Compound (7) may be synthesized by reacting compound (5) with compound (6),
under standard coupling conditions. One skilled in the art will recognize that there are a
number of methods and reagents for amide formation resulting from the reaction of
carboxylic acids and amines. The coupling of compound (5) with compound (6) can be
effected in the presence of a suitable coupling reagent and a suitable amine base, such as
DIPEA or trtmethyiamtne. Coupling reagents include carbodiimides, such as DCC, DIC,
EDO, and other coupling reagents, such as HOBt and HOAt. Additionally, uronium or
phosphonium salts of non-nucleophilic anions, such as HAITI, HBTU, PYBOP , and
PYBROP® can be used in place of the more traditional coupling reagents. Additives such
as DMAP may be used to enhance the reactions. Alternatively, compound (5) can be
acylated using substituted acyl chloride of compound (6) in the presence of a base, such
as triethylamine or pyridine.
The protecting group, Rx, in intermediate (7) can be removed under conditions
well known in the art, such as acidic or basic conditions. The resulting amine intermediate
can be reacted with compound (8) under standard coupling conditions, including those
previously described n the preparation of compound (7), to give a compound of Formula
(I). The skilled artisan will recognize that there are alternative methods to prepare a
compound of Formula (I) from deprotected compound (7), including reacting with an acid
chloride in the presence of an organic base such as triethylamine or with an anhydride in
the presence of a catalyst such as DMAP.
In an optional step, a pharmaceutically acceptable salt of a compound of Formula
(I) can be formed by reaction of an appropriate free base of Formula (I) with an
appropriate pharmaceutically acceptable acid in a suitable solvent under standard
conditions. Additionally, the formation of such salts can occur simultaneously upon
deprotection of a nitrogen protecting group. The formation of such salts is well known
and appreciated in the art.
Preparations and Examples
The following Preparations and Examples further illustrate the invention and
represent typical synthesis of the compound of the invention. The reagents and starting
materials are readily available or may be readily synthesized by one of ordinary skill in
the art. It should be understood that the Preparations and Examples are set forth by way
of illustration and not limitation, and that various modifications may be made by one of
ordinary skill in the art.
The R or S configuration of the compound of the invention may be determined by
standard techniques such as X-ray analysis and correlation with ehiral-HPLC retention
time. The naming of the following Preparations and Examples is generally performed
using the IUPAC naming feature in MDL ACCELRYS* Draw version 4.1.
LC-ES/MS is performed on an AGILENT ® HP1100 liquid chromatography
system. Electrospray mass spectrometry measurements (acquired in positive mode) are
performed on a Mass Selective Detector quadrupole mass spectrometer interfaced to the
HP1 100 HPLC. LC-MS conditions (low pH): column: PHENOMENEX® GEMINI ®NX
C 8 2.1 50 mm 3.0 m; gradient: 5-100% B in 3 min, then 100% B for 0.75 min column
temperature: 50 °C +/-10 °C; flow rate: 1mL/min; Solvent A: deionized water with
0.1% formic acid; Solvent B: ACN with 0.1% formic acid. Alternate LC-MS conditions
(low pH): column: XTERRA ® MS C18 columns 2.1 50 mm, 3.5 um; gradient: 5%of
solvent A for 0.25 min, gradient from 5% to 100% of solvent B in 3 min and 100% of
solvent B for 0.5 min or 10% to 100% of solvent B in 3 min and at 100% of solvent B for
L7-
0.75 in; column temperature: 50 °C +/-10 °C; flow rate: 1 mL/min; Solvent A: 10 mM
ammonium hydrogencarbonate pH 9; Solvent B: ACN ; wavelength: 214 run.
All preparative reversed phase chromatography is performed on an AGILENT*'
1200 LC MS equipped with a Mass Selective Detector mass spectrometer and a LEAP®
autosampler/fraction collector. High pH methods are run on a 75 X 30 mm
PHENOMENEX ® GEMINI®-NX, 5 particle size column with a 10 X 20 mm guard.
Flow rate of 85 mL/min. Eluent is 10 mM ammonium bicarbonate (pH 10) in
acetonitrile.
A Waters ZQ mass spectrometer and 29998 Diode Array Detector is used for
acquiring mass and UV data during supercritical fluid chiOmatography (SFC). Material
exhibiting the correct mass (electrospray ionization) and UV absorbance is collected.
Preparation 1
5-Iodo-6-methyl-2-(trifluoromemyl)pyrimidin-4-amine
Add a solution of iodine monochloride (4.14 g, 23.37 mmol) in DCM (20.1 mL)
to a flask containing 6-memyl-2-trifluoromemyl-pyrimidin-4-amine (4.14 g, 23.37 mmol)
in MeOH ( .4 mL). Stir the mixture at room temperature for 48 hours. Upon reaction
completion, add a 10% aqueous sodium sulfite solution (200 mL). Extract the resulting
mixture with EtOAc (4 x 100 mL), diy the organic phase over Na2S04, filter and
concentrate under vacuum to obtain the crude title compound as a light yellow solid
(7.0 g, 99%). Use material without additional purification. LC-ES/MS m/'z 303.8 (M+H).
ert-B y l 4-[2-[4-amino-6-methyl-2-(trifluorQm
-carboxyiate
Slurry 5-iodo-6-me1hyl-2-(trifluoromethyl)pyrimidin-4-ariiine (2 05 g, 6.75
mmol), 4-ethynyl-piperidine-l-carboxylic acid -butyl ester (1.41 g, 6.75 mmol),
bis(triphenylphosphine)palladium(II) chloride (239 mg, 0.34 mmol) and copper(I) iodide
(130 mg, 0.68 mmol) in 10 mL DMF in a 20 L microwave vial and bubble nitrogen
through the suspension for 5 min. Add triethylamine (1.88 mL, 13.5 mmol) and continue
to bubble nitrogen through the mixture for 5 additional min. Heat the mixture in a
microwave at 00 °C for 60 min. Cool the mixture to room temperature and pour into
saturated aqueous NaCl (500 mL). Extract with DCM, dry organic layer over MgS0 4,
filter and concentrate under vacuum. Purify the resulting residue via chromatography
over silica gel (5-35% EtOAc:hexanes over 45 min). Concentrate the purified fractions to
dryness to obtai the title compound (1.25 g, 48%) as a light yellow solid. LC-ES/MS
m/z 385.2 (M+H).
rt-Butyl 4~[2-[4-ammo-6-methyl-2-(1xif!uoromethyl)pyrimidm-5-yl]ethyl]piperi
carbox iate
Combine tert-butyl 4-[2-[4-amino-6-methyl~2~(trifluoromethyl)pyrimidin~5-
yl]ethynyl]piperidine-l -carboxyiate (6.28 g, 16.34 mmol) and platinum(IV) oxide (744
mg, 3.27 mmol) in EtOH ( 110 mL). Alternately evacuate and charge the flask with
hydrogen under a hydrogen balloon, fill the system with hydrogen and agitate at room
temperature for 18 hours. Filter the mixture through diatomaceous earth, rinsing with hot
EtOH (30 mL) followed by 2M NH /MeO (20 mL). Concentrate the solution under
reduced pressure to obtain the title compound (6.15 g, 97%) as a white solid. Use without
additional purification. LC-ES/MS m/z 389.2 (M+H).
Preparation 4
6~Methyl~5-[2-(4-piperidyl)ethyl]-2-(tTifluoromethy])p>TiTnidin-4-amtne
Dissolve t rt-butyl 4~[2-[4-ammo-6-methyl-2-(trifluoromethyl)pyrimidin-5-
yl]ethyl]piperidine-l-carboxylate (6.07 g, 15.63 mmol) in DCM (25 mL) and add TFA
(10 mL, 132.25 mmol). Stir the solution for 4 hours at room temperature. Concentrate
the mixture under reduced pressure, dissolve resulting residue in DCM (15 mL) and apply
to an SCX column (50 g), eluting with DCM (100 mL), MeOH (100 mL), and eluting
desired material with 2M I¾/Me H (100 mL). Evaporate methanolic ammonia
fractions to dryness to obtain the title compound (4.4 g, 97%) as an off-white solid. Use
without additional purification. LC-ES/MS m/z 289.2 (M+H).
Preparation 5
6-Methyl-5-[2-(4~piperidyl)ethyl]-2-(trtfluoromethyl)pyrimidin-4-amine dihvdrochloride
Add acetyl chloride (180.1 mL) in a slow steady stream to a 50 C solution of
isopropanol (1.26 L) and stir at 50 °C for 30 inin. Portionwise add tert-butyl 4-[2-[4-
armno-6-methyl-2-(triiluoromethy2)pyrimidin-5-y3]ethyl]piperidine-l-carboxylate (180.1
g, 463.7 mmol) from preparation 3 and continue heating for .5 hours. Cool to T and
add diethyl ether (3.6 L). Collect solid by filtration and wash with diethyl ether (2 x 300
mL). Dry resulting solid in a vacuum oven at 50 °C overnight to obtain the title
compound (174.0 g, 98%) as a white free-flowing powder. Use without additional
purification. LC-ES/MS m/z 289.2 (M+H).
Example 1
teri-Butyl N-/71S)-2-[4~[2~[4~ainino-6-methy
iperidyl]-1-methyl-2-oxo-ethyi] carbamate
Dissolve 6-inethyl~5~[2-(4-piperidyl)ethyl]-2-(1xifiuoromethyl)pyrimtdm-4-amine
(3.9 g, 13.53 mmol) in DMF (20 mL); add (25)-2 -(t rt-butoxycarbonylamino)propanoic
acid (2 8 g, 14.88 mmol), 1-hydroxybenzotriazole (7.46 g, 54.11 mmol), l-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.63 g, 13.53 mmol) and
diisopropylethylamine (7.08 mL, 40.58 mmol). Stir resulting mixture at room
temperature overnight. Pour reaction mixture into saturated aqueous NaHC0 3 (500 mL)
and extract with DCM. Dry organic phase over MgS0 4, filter, concentrate under vacuum
and purify by chromatography over silica gel (10-75% EtOAc:hexanes over 45 min) to
obtain, after solvent removal, the title compound (4.66 g, 75%) as a white foam. LCES/
MS m/z 460.2 (M+H).
Alternative Procedure for Example 1
tert-Butyl N-[(l¾-2-[4-[2-[4-amino-6-methyi-2-(trifluoromethyl)pyrimidin-5-yl]ethyl]-liperi
dyl]-1-methyl-2-oxo-ethyl]carbamate
Add diisopropylethylamine (308.6 mL, 1770 mmol) to a suspension of 6-methyl-
5-[2-(4-piperidyi)ethyl]-2-(trifluoromethyl)pyrimidin-4-amine dihydrochloride (170 g,
442.4 mmol), (25)-2-(t rt-butoxycarbonylamino)propanoic acid (92.1 g, 486.6 mmol) in
DCM (1.6 L) to give a pale yellow suspension. Cool to 0 °C in an ice bath and
portionwise add (dimethylarnino)-N ^-dime1hyl(3 H-[l,2,3]triazolo[4,5-6]pyridin-3-
yloxy)methaniiniiiium hexafluorophosphate (167.3 g, 442.4 mmol). Stir bright yellow
suspension at 0 °C for 30 min and warm to RT with stirring for 2h. Concentrate to ca. 1L
under reduced pressure and dilute reaction mixture with EtOAc ( 1 L) and partition with
saturated aqueous N C 1 (ca. 500 mL); separate organic layer and fiirther extract aqueous
layer with EtOAc (2 x 500 mL). Combine organic phases, wash with saturated aqueous
NH4CI (4 x 400 mL), saturated aqueous NaHCi¾ (400 mL), water (400 mL), saturated
aqueous NaCl (400 mL). Dry over MgSQ4, filter and concentrate under reduced pressure,
azeotroping with z'se-hexanes (750 ml) to obtain the title compound (237 g, 99%) as a
white foam, suitable for use without additional purification. LC-ES/MS m/z 460.3
(M+H). Chiral analysis (SFC MiniGram®, 5% MeOH/CO2/0.2% iso -propylamine, 5
mL/min, 100 bar, 35 °C, 220 run) >98% ee.
Preparation 6
(2$)-2-Amino-l-[4-[2-[4-ammo-6-methy
piperidyljpropan- 1-one
Dissolve rt-butyl N- i'15)-2-[4-[2-[4-amino-6-methyl-2-
(tri uoromethyl)pyrimid
(4.66 g, 10.14 mmol) i DCM (150 mL.) and add trif!uoroacetic acid (7.67 mL, 101.4
mmol). Stir resulting mixture at room temperature overnight. Concentrate solvent under
vacuum, reconstitute residue in DCM (20 mL) and apply to an SCX column (50 g),
eluting with 100 mL. DCM, 100 mL MeOH, and eluting desired material with 2M
NH3/MeOH (100 ml). Evaporate methanolic ammonia fractions to dryness to obtain the
title compound (3.58 g, 98%) as a white foam. Use without additional purification. LCES/
MS m/z 360.2 (M+H).
Example 2
N-[(lS)-2-[4-[2-[4-Ammo-6-memyl-2-(trifluorome&^
piperidyl] -1-methyl-2-oxo-ethyl] -2-methyl-pyrazole-3-carboxamide
Dissolve (25)-2-a.mmo-l~[4~[2-[4-ammo-6-methyl-2-(trifluoromethyl)pyri^
5-yl]ethyl]-l-piperidyl]propan-l-one (560 mg, 1.56 mmol) in DCM (20 mL) containing
DMF (3 mL); add 1-methyl- lH-pyrazole-5-carboxylic acid (216 mg, 1.71 mmol), 1-
hydroxybenzotriazole (969 mg, 6.23 mmol), l-(3-dimethylammopropyl)-3-
ethylcarbodiimide hydrochloride (303 mg, 1.56 mmol) and diisopropylethylaniine (1.36
mL, 7.8 mmol). Stir resulting mixture at room temperature overnight. Pour reaction
mixture into saturated aqueous NaHCC 3 (200 mL) and extract with DCM. Dr organic
phase over MgS0 4, filter, concentrate under vacuum and purify by chromatography over
silica gel (0-10% MeOH:DCM over 30 min) to obtain, after solvent removal, the title
compound (665 mg, 91%) as a white foam. LC-ES/MS m/z 468.0 (M+H).
Prepare the Examples in Table 1 below by essentially following the procedure
described in Example 2, using (25)-2-arnmo-l-[4-[2-[4-amino-6-methyl-2-
(trifluoromemyl)pyrimidin-5-yl]ethy ] -1-piperidyLJpropan- 1-one and the appropriately
substituted carboxylic acid.
Table 1

A¾15)-2-[4-[2~[4~Amino-6-methyl-2-(M
piperidyl] -1-meth l-2-oxo-ethyl] -2-methyl -pyrazole-3 -carbox amide
Add a slow, steady stream of 1-propanephosphonic acid cyclic anhydride (410.6
mL, 683.9 mmol) to a slurry of (25)-2-amino-l-[4-[2-[4-amino-6-methyl-2-
(trifluoromethyl)pyrimidm-5-yl]ethyl]-l-piperidyl]propan-l-one (168,0 g, 342.0 mmol),
l-methyl-l H-pyrazole-5-carboxylic acid (64.7 g, 513.0 mmol) and diisopropvlethylamme
(244.5 mL, 1400 mmol) suspended in DCM (1.5 L) in an ce bath, maintaining the
internal temperature at 5-10 °C. Warm to RT with stirring over 2.5 hours. Concentrate to
ca. 500 mL under reduced pressure and dilute resulting residue with EtOAc (2 L) and
water ( 1 L); separate layers, extract aqueous layer with EtOAc (2 x 400 mL) and wash
combined organic layers with saturated NH4CI (500 mL), saturated aqueous S a CO;; ( 1
L), water (500 mL), saturated aqueous NaCl (500 mL). Dry organic layer over MgS04,
filter, evaporate under reduced pressure. Dissolve resulting residue in isopropyl acetate
( 180 mL), treat with heptanes ( 1 L) and heat to 70 °C for 4 hours to eject a white powder.
Cool to RT, collect solids by filtration, wash with 9 1 heptane: isopropyl acetate (100 mL)
followed by heptanes (2 x 100 mL). Dry in vacuum oven at 45 °C overnight to obtain the
title compound as a white powder ( 1 g, 73%). LC-ES/MS m/z 468.0 (VI— 1).
N- [( 1 -2-[4-[2~ [4-Amino-6-methyl-2-(trifluoromethyl)pyrimtdin-5-yl]ethyl] -1-
piperidyl]-l-methyl-2-oxo-ethyl]acetamide
Prepare the title compound (47.7 g, 67%) by essentially following the procedur
described in Example 1, using (25)-2-amino-l-[4-[2-[4-amino-6-chloro-2-
(trifluoromethyl)pyrimidin-5-yl]ethyl]-l-piperidyl]propan-l-one (70 mg, 0.19 mmol),
acetic anhydride (184.1 , 1.95 mmol) and DMAP (1.2 mg, 0.01 mmol) in DCM (3.9
mL, 0.05 ). ES/LC-MS m/z 402.2 (M+l).
Methyl N- [(15)-2-[4- [2- [4-amino-6-methyl-2-(trifluoromethyl)pyrimidin-5 -yljethyl] -1-
iper dyljj- -methyl-2-oxo-ethyl]carbamate
Dissolve (2 S)-2-amino-l~[4~[2-[4-amino-6-methyl-2-(1xifluoromethy])pyriTrddin-
5-yi]ethyl]-l-piperidyl]propan-l-one (43.8 mg, 0.12 mmol) in DCM (3 mL) and add
dimethyl dicarbonate (22.0 ,uL, 182.8 umol) and pyridine (30 , 365.6 umol). Stir
resulting mixture at RT overnight, pour into saturated aqueous NaCl (100 mL) and extract
with DCM (3 x 30 mL). Wash combined organic layers with 0.1 N HC1 (2 x 100 mL),
water (100 L), saturated aqueous NaCl (100 ml), dry over MgS04, filter and
concentrate under reduced pressure to obtain the title compound as an off-white solid (22
mg, 43%) LC-ES/MS m/z 418.2 (M+H).
Example 21
N-[(1S)~2~ [4~[2- [4-Amino~6~methyl-2-(irifluoromethyl)pyrimidin~5~yl]ethyl] -1-
piperidyl]-l-methyl-2-oxo-ethyl]pyridine-2-cai -boxarriide hydrochloride
Dissolve N-[(15)-2-[4-[2- [4-aniino-6-methyl-2-(trifluoromethyl)pyriiiridin-5 -
yl]ethyl]-l-piperidyl]-l-methyl-2-oxo-ethyl]pyridine-2-carboxamide (69 mg, 0 15 mmol)
in DCM ( 1 L) and add HC1 (IM in dioxane, 500 mL). Stir at room temperature for 10
min, then concentrate under vacuum. Triturate resulting residue with DCM ( 1 mL)
followed by Et2Q (2 mL) Filter and collect resulting light yellow solid to obtain the title
compound (74 mg, 98%). LC-ES/MS m/z 465.2 (M+H).
Assays
GOAT is the principal enzyme that converts UAG to AG. For reviews of the role
of GOAT and ghrelin see: Kristy M. Heppner et al, The ghrelin O-acyltransferaseghrelin
system: a novel regulator of glucose metabolism., Current Opinion in
Endocrinology, Diabetes & Obesity 201 1, 18:50-55; Phillip A Cole et al, Glucose and
Weight Control in Mice with a Designed Ghrelin OAcyltransferase Inhibitor, Science.
2010 December 17; 330(6011): 1689-1692. doi: 10.1 126/science.l 196154, Matthias H.
Tschop et al., Gastric Q-acyl transferase activates hunger signal to the brain, Proc Natl
Acad Sci U S A. 2008 April 29; 105(17): 6213-6214, and Jesus Gutierrez, et al., Ghrelin
octanoylation mediated by an orphan lipid transferase, Proc Natl Acad Sci U S A., 2008
April 29, 105 (17): 6320-6325.
The role of GOAT is supported by the phenotypes observed in mice devoid of
GOAT gene. Therefore, inhibition of GOAT is expected to decrease circulating AG and
raise circulating UAG. Consequently, the ratio of AG to total ghrelin (UAG + AG) is
reduced after GOAT inhibitor treatment.
n vitro ce free GOAT enzymatic assay
Human GOAT gene (Accession number: M 00 1100 16) is subcloned to pAN5 1
baculoviral expression vector. Baculovirus stock is prepared following the Bac-to-Bac
Protocol provided by the vendor, Invitrogen, California, USA. Five mililiters of huma
GOAT baculoviral stock are added to 500 mL Sf cells in HyQ SFX-lnsect M media
(HyClone catalog number SH30278.02) at the density of 1 x 106 cells per milliliter in a 2
L Erlenmeyer flask. The flask with human GOAT gene infected Sf9 cells is put on a
plate shaker at 120 rpm at 28 C for 48 hours. After 48 hours incubation, cells are
centrifuged at LOOOxg for 0 min at 4 °C. The cell pellets are collected and stored at -80
°C in a freezer until ready for further processing.
Preparation of microsomal membrane of GOAT enzyme for the enzymatic assay:
One gram cell pellets are suspended in 9 mL chilled homogenization buffer (50
mM Tris-HCl, 250 mM sucrose, adjusted to pH 7.5 and sterile filtered through 0.2 
Mtllipore filter). The cell suspension is transferred to a Bounce glass homogenizer. Cell
pellets are homogenized with 40 strokes on ice. The homogenate is centrifuged at 3,000
rpm in a Beckman swing bucket rotor at 4 °C for 10 min to remove unbroken cells. The
supernatant is collected and centrifuged at 40,000 xg for 1 hour at 4 °C. The resulting
membrane pellet is suspended in the homogenization buffer using a Dounce glass
homogenizer and stored at -20 °C in the freezer for the assay. For long term storage of
the human GOAT enzyme membrane preparation, the suspended membrane is stored in a
-80 °C freezer.
Human GOAT enzymatic assay protocol:
Prepare test compounds in DMSO to make up a 0.2 mM stock solution. Serially
dilute the stock solution in DMSO to obtain a ten-point dilution curve with final
compound concentrations ranging from 10 to 0.5 nM in a 96-well round-bottom
plate. Prepare enzyme and substrate solutions in assay buffer (0.02% TWEEN™-20 in 50
mM Tris, pH 7.5/250 mM sucrose/1 mg/mL BSA/10 mM EDTA). Add diluted
compound ( 1 ) to each well of row A to N of a corresponding low protein binding 384
well plate. Add human GOAT substrate mix (10 uL), consisting of huma desacylghrelin-
biotin (CPC Scientific Inc., 6.0 final), octanoyl-coenzyme A (CoA) (Sigma,
60 final) and an AG specific antibody (WO 2006/091381)(1.0 g/m final), to the
compounds. Add GOAT-His/sf9 enzyme preparation, that has been prepared in assay
buffer (9 uL), to each well of the plate containing substrate and test compounds res ulting
in a final concentration of 0.01 ,ug''m to initiate the reaction. Incubate the mixture for 1
hour at RT on a gently rotating oscillator. Add 4M guanidine hydrochloride (20 to
all wells, mix, and incubate for 3 hours to stop the reaction.
Prepare ELISA plates (STREPTAVIDIN SPECTRAPLATE™ 384, Perkin Elmer)
by blocking with 2% Heat-Inactivated FBS in PBS (40 ) (Invitrogen) blocking buffer
for 3 hours. Aspirate the blocking buffer from ELISA plate and add blocking buffer (23
) to columns 1-24, rows A-N. Reserve rows O and P for the acylghrelin standard
curve. Add the reaction mix (2 ) to the ELISA plates. Prepare a 10 point standard
curve (biotin-labeled octanoyl-ghrelin) by serial 2X dilution in blocking buffer containing
0.2M Guanidine hydrochloride starting at 2.5 pM. Incubate the reaction mixture or
biotin-labeled AG standard in the ELISA plate overnight at 4 °C. The following day,
wash the plate 3* with wash buffer (0.1% TWEEN™-20/PBS, 100 per well in each
wash cycle). Add AG specific antibody (WO 2006/091381) (25 of 0.5 g/mL in
blocking buffer) to each well and incubate at RT for 1 hour. Wash the plate 3 with the
wash buffer, similarly to the previous step. Add Protein G-HRP (25 ) ( ώ •
Biotech) diluted 3,00Qx in blocking buffer and incubate 1 hour at RT. Wash the late 3
with wash buffer, as in the previous steps. Add TMB reagent (25 ) (Kirkegaard &
Perry Laboratories, Inc.) to each well and let develop for 20 min and stop with 1
phosphoric acid (25 per well). Read plates at 450 nm using an ENVISION®
Multilabei plate reader. AG levels are calculated versus a fitted standard curve and
percent inhibition calculated. The 10-point inhibition curve is plotted and fitted with the
four-parameter logistic equation to obtain IC5o values using ACTWITYBASE* (ver.
7.3.2.1).
Following a protocol essentially as described above, all of the compounds of the
Examples herein were tested and exhibited an IC50 for the in vitro cell free human GOAT
enzymatic assay of lower than 1 uM. The following exemplified compounds of the
invention were tested essentially as described above and exhibited the following activity
as illustrated in Table 2 below.
The data in Table 2 demonstrate that the compounds of Table 2 inhibit purified
GOAT enzyme activity in vitro.
Comparing the change in the ratio of AG to total ghrelin in the compound treated
group and that of the vehicle treated group reflects the degree of GOAT enzyme
inhibition in vivo, due to the dynamic processing of UAG to AG by the GOAT enzyme.
In the in vivo pharmacodynamic studies herein, the levels of AG and UAG in plasma and
stomach in the vehicle and compound treated groups are measured by ELISA specifically
to these two analytes. The total ghrelin level of each sample is computed as the sum of
AG and UAG by these ELISA measurements. The ratio of AG to total ghrelin is defined
by the level of AG in each sample divided by the level of total ghrelin in the same
sample. The levels of AG, UAG and ratio of AG to total ghreli in the vehicle treated
group is computed and set as 00%. The relative change of these parameters in the
compound treated group is then computed to determine the effectiveness of the test
compound.
In vivo dose dependent 3 day BIB study for GOATinhibitor^
Animals and treatment:
Purchase male C57BL/6 mice from Harlan (Indianapolis, IN) at 9 weeks of age.
House the mice individually in a temperature-controlled (24 °C) facility with a 12 hours
light/dark cycle (lights on 2200 h), and allow free access to a standard rodent cho (diet
2014, Harlan) and water. Typically, use the mice when they are 10-13 weeks of age at
the time of the study. On day 0 of the experiment, randomize the mice into treatment
groups (N=7/group) so each group has similar mean body weights. On day 1 and day 2,
treat the animals with vehicle (1% hydroxyethylcellulose, 0.25% TWEEN™ 80, 0.05%
antifoam) or test compound prepared in the vehicle as suspension at various dosages by
oral gavage at 7 am and 7 pm. On day 3, fast the animals, move them into clean cages
and dose with vehicle or the test compound again at 8 am by oral gavage. That same day
at 1 pm, sacrifice the animals by decapitation to collect blood . For details of blood
collection and plasma treatments see Blood collection and Extraction of Ghrelin from
Plasma section below.
Blood collection:
Collect approximately 600 blood into a pre-weighed EDTA tube containing
600 (defined as V r serv freshly -prepared preservative (4 mM PEFABLOC ® [4-(2-
aminoethyl) benzenesulfonvl fluoride hydrochloride], 72 mM NaCl, 58 mM NaF, 0.032
N hydrochloric acid, pH 3.0) and mix immediately. Weigh the tube again and keep on
ice. To accurately determine the exact blood volume of each sample using this blood
collection procedure, the weight of the blood for each mouse is computed using the
following equation:
Weight of Blood = (Weight of the tube containing Blood + preservative) -
(Weight of the tube containing preservative)
Blood volume V ) - (Weight of blood) / 1.06
Note, the density of rodent blood is assumed as 1.06 g/mL.
Within 5 minutes after the blood collection, samples are centrifuged at 5000 rpm
at 4 °C for 8 min. Remove plasma (650 ) to a 5 mL glass tube containing 1 N
hydrochloric acid (65), mix and keep on ice.
Ghrelin extraction by SEP-PAK® column:
AG and UAG are extracted from plasma using SEP-PAK®_C column to remove
interference prior to performing the ELISA. The solid phase extraction of AG and UAG
peptides by SEP-PA Ci columns can be performed on a vacuum manifold (Waters
Corp) or using a peristaltic pump. The sample SEP-PAK*_column extraction procedure is
independently applied to the plasma sample obtained from each individual mouse. The
general extraction protocol is described as follows.
Ail solutions used for the entire protocol of the SEP-PAK® column extraction
should be at ice cold condition. Wet SEP-PAK®_co2umns (WAT054960, Waters Corp,
Mtlford MA) with 99.9% ACN/0.1% TFA ( 1 mL of solution of 100 mL AC /0. mL
TFA). Apply pressure to adjust the flow-rate to about lmL/min to remove liquid from the
column bed but do not allow the column to dry out at any point. Once liquid is removed
from the column, stop the pressure. Equilibrate the columns with 3% ACN/0.1% TFA ( 1
m , of 97 mL water, 3 mL ACN, 0.1 mL TFA). Apply pressure to adjust the flow-rate to
about lmL/min to remove liquid from the column bed, but do not let the column dry out.
Dilute approximately 650 acidified plasma (defined as added to col n to 1.4 mL
ice cold 0.1% TFA. Load all diluted acidified plasma fro the previous step onto the
columns. Apply pressure to adjust the flow-rate to about 0.5mL/min to allow sample
passing through the colum and ghrelin peptides to absorb onto the resin of the column.
Do not let the column dry out. Wash with 3% ACN/0.1% TFA (0.9 mL of 97 mi, water,
3 mL ACN, 0.1 mL TFA). Apply pressure to adjust the flow-rate to about lmL/min to
remove liquid from the column bed but do not let the column dr out.. Repeat the wash
two more times. Elute with 60% ACN/0.1% TFA ( 1 mL of 40 mL water, 60 mL ACN,
0.1 m TFA). Put a collection tube underneath of each column, apply pressure to adjust
the flow-rate to about 0.5 mL/min to push liquid through the column and collect the
eluent into the collection tube. Freeze the samples on dry ice immediately. Lyophilize
the samples in a speed-vac (Model# SCI 10A, Savant) and store at -20 °C until the ELISA
assay is performed
ELISA assay for Ghrelin:
Coat 96-well MULTI-ARRAY ® MSD® plates (Meso Scale Discovery,
Gaithersberg, MD, Catalog # L15XA-3) with 100 i of 1 g/mL of an antibody (WO
2005/026211 AND WO2006/0 19577) that recognizes the mid-domain of both the acyl
and unacylated forms of ghrelin in PBS (Invitrogen). Tap the sides of the plates to ensure
coverage of wells, seal with adhesive plate sealer, and incubate overnight at RT. Discard
the contents and add BLOCKER™ Casein in PBS (25 ) (Thermo Scientific, Rockiord,
IL, Catalog #37528) to each well. Reseal the plates and put on a plate shaker at RT for 1
hour.
Reconstitute the lyophilized preserved plasma samples from the SEP-PAK® C
col mn extraction in BLOCKER™ Casein in PBS (400 L to each sample, this volume is
defined as re to fc ), mix well with a vortex mixer and incubate on ice for 45-60 .
Dtscard the contents from the plates and add reconstituted plasma samples at 25 . to
each well. Prepare acylghrelin and unacylated ghrelin standard curves beginning wit
8000 pg/niL and performing serial 1:4 dilutions for 8 total concentrations. Add the
prepared standards in duplicate to the blocked plates with 25 in each well Seal the
plates and incubate at RT on a plate shaker for 2 h.
Discard the plate contents and wash three times with PBS including 0.1%
TWEEN™ 20 (150 )(). Acylghrelin specific antibody (WO 2006/091381) or
unacylated ghrelin specific antibody (WO 2006/055347) labeled with MSD® SULFOTAG
™(Meso Scale Discovery) are diluted to 0.05 g/mL in 0.2 Blocker Casein
containing 0 05% TWEEN™ 20, named secondary antibody solution. Remove the final
wash and add secondary antibody solution (25 to each well) which specifically
recognizes AG or UAG. The plates are resealed and incubated for 1hour at RT on a plate
shaker before finally washing 3 again with PBS-T (150 ).
Discard the final wash and replace with 1x MSD® Read Buffer ( 50 ).
Read the electrochemiluminescent signal generated by activation of the bound MSD®
SULFO-TAG™ label to the electrodes on the plates using the MSD* SECTOR* Imager
6000 analyzer (Meso Scale Discovery). Calculate concentrations of acylghrelin or
unacylated ghrelin based on the respective standard curve generated by the MSD®
software. Determine the actual plasma concentration for each sample by multiplying the
measured acylghrelin or unacylated ghrelin level by a dilution factor. The dilution factor
for each plasma sample is computed with the following equation.
Di to r =
Results:
Administration of the compound of Example for 3 days decreases plasma AG by
40%, 60%, 56%, 63%, and 63%, and increases UAG by 2.10, 2.22, 3.57, 3.49 and 3.78
fold, respectively at 0.1, 0.3, 1, 3, and 10 g kg (results tablulated below).
Administration at 0.1, 0.3, 1, 3, and 10 mg/kg results in 57, 62, 79, 82 and 82% reduction
respectively in AG to total ghrelin ratio when compared to the vehicle-treated control
animals.
Table 3
The results demonstrate that the compound of Example 2 suppresses AG production and
elevates the UAG in circulation, as shown in the GOAT knock-out mouse, in vivo.
claim:
1. A compound of formula
wherein R is selected from -C1 -C3 alkyl optionally substituted with -OH;
OC -C4 alkyl; pyrazolyl, oxazolyl, thiazolyl, or thiadiazolyl, wherein pyrazolyl,
oxazolyl, or thiazolyl each may be optionally substituted one to two times with
-CH 3; pyridinyl, pyridazinyl, or pyrazinyl, wherei each may be optionally
substituted with -CI; and phenyl optionally substituted with -OCH 3; or a
pharmaceutically acceptable salt thereof
The compound of Claim 1wherein R is selected from -CH3 optionally substituted
with -OH; -OCH3 or -OC(CH 3) ; pyrazolyl, oxazolyl, thiazolyl, or thiadiazolyl,
wherein pyrazolyl, oxazolyl, or thiazolyl each may be optionally substituted one
to two times with -CH 3 pyridinyl, pyridazinyl, or pyrazinyl, wherein each may be
optionally substituted with -CI; and phenyl optionally substituted with -OCH or
a pharmaceutically acceptable salt thereof.
The compound of Claims 1 or 2 wherein R is selected from pyrazolyl, oxazolyl,
thiazolyl, or thiadiazolyl, wherein pyrazolyl, oxazolyl, or thiazolyl each may be
optionally substituted one to two times with -CH 3; pyridinyl or pyrazinyl, wherein
each may be optionally substituted with -CI; and phenyl optionally substituted
with - CH . or a pharmaceutically acceptable salt thereof.
The compound of any of Claims 1 to 3 wherem the configuration of the carbon
atom with the methyl substituent is (S):
or a pharmaceutically acceptable salt thereof.
The compound of any of Claims 1 to 4 of the fonnula
or a p armaceutica y acceptable salt thereof.
6. The compound of any of Claims 1to 5 of the nnula
or a pharmaceutically acceptable salt thereof.
7. The co of any of Claims 1to 6 of the formula
8. A pharmaceutical composition comprising a compound of any of Claims 1to 7, or
a pharmaceutically acceptable salt thereof, with one or more pharmaceutically
acceptable carriers, diluents, or excipients
9. The pharmaceutical composition according to Claim 8 in combination with one or
more other therapeutic agents.
0. A method of reducing weight gain comprising administering a compound
according to any of Claims 1 to 7, or a pharmaceutically acceptable salt thereof, to
a patient in need thereof.
11. A method of reducing weight regain comprising administering a compound
according to any of Claims 1 to 7, or a pharmaceutically acceptable salt thereof, to
a patient in need thereof.
12. A method of treating obesity comprising administering a compound according to
any of Claims 1 to 7, or a pharmaceutically acceptable salt thereof, to a patient in
need thereof.
13. A method of treating type 2 diabetes comprising administering a compound
according to any of Claims 1 to 7, or a pharmaceutically acceptable salt thereof, to
a patient in need thereof.
14. A compound according to any of Claims 1 to 7, or a pharmaceutically acceptable
salt thereof, for use in therapy.
15. A compound according to any of Claims 1 to 7, or a pharmaceutically acceptable
salt thereof, for use in reducing weight gain.
16. A compound according to any of Claims 1 to 7, or a pharmaceutically acceptable
salt thereof, for use in reducing weight regain.
7. A compound according to any of Claims 1 to 7, or a pharmaceutically acceptable
salt thereof, for use in treating obesity.
8. A compound according to any of Claims 1 to 7, or a pharmaceutically acceptable
salt thereof, for use in treating type 2 diabetes.
1 . The use of a compound of any of Claims 1 to 7, or a pharmaceutically acceptable
salt thereof, in the manufacture of a medicament for reducing weight gain.
20. The use of a compound of any of Claims 1 to 7, or a pharmaceutically acceptable
salt thereof, in the manufacture of a medicament for reducing weight regain.
21. The use of a compound of any of Claims 1 to 7, or a pharmaceutically acceptable
salt thereof, in the manufacture of a medicament for treating type 2 diabetes.
22 The use of a compound of any of Claims 1 to 7, or a pharmaceutically acceptable
salt thereof, in the manufacture of a medicament for treating obesity.