A Novel 1,2,3,4-Tetrahydroquinoline Derivative Useful for the Treatment of Diabetes

Diabetes is a serious health care problem facing the developing world. It
would be desirable to provide a safe and effective oral treatment for diabetes. Some
successful commercially available oral treatments for type two diabetes (T2D) are
believed to act through modulation of the peroxisome proliferator-activated receptor
(PPAR) gamma receptor. Administration of these medicines has been associated with
undesired adverse effects that sometimes include hypoglycemia, liver damage,
gastrointestinal disease, weight gain, or other undesired effects that may be associated
with the PPAR gamma activity. New treatment options offering a more desirable
safety profile for managing T2D are desired to effectively treat or prevent diabetes in
more patients. In particular, novel mechanism-based treatment methods that may
minimize or avoid effects that have been associated with PPAR gamma activation are
especially desired.
The G protein-coupled receptor 40 (GPR-40), also known as Free Fatty Acid
Receptor 1 (FFAl or FFARl), is reported as predominately expressed at high levels in
rodent pancreatic beta cells, insulinoma cell lines, and human islets. This receptor is
activated by medium and long-chain fatty acids. The glucose dependency of insulin
secretion is an important feature of activating GPR-40, making this receptor an
excellent target for developing efficacious therapies with a desired safety profile for
use in the treatment of T2D. Compounds that offer efficacy and a more desirable
safety profile compared to existing therapies such as insulin and sulfonylureas can be
especially desirable.
Two recently published patent applications, US201 1009253 1 and
WO201 1066183 disclose compounds possessing a spiro-bicyclic group which exhibit
GPR-40 activity.
This invention provides a compound for the treatment of diabetes particularly
T2D. The compound for this invention is a potent activator of GPR-40. This
invention provides a desired novel treatment option acting through a pharmacological
mechanism that is unique compared to commercially available treatments and further
provides a compound that selectively activates GPR-40 as compared to PPAR gamma.
The pharmacological profile of the compound of this invention, as a selective GPR-40
activator, can be particularly desirable for use in the treatment of T2D. Additionally,
the selective GPR-40 modulation may provide a particularly desirable safety profile
for use in the treatment of T2D by avoiding effects associated with PPAR gamma
modulation.
The present invention provides a compound of the Formula I below:
I
or a pharmaceutically acceptable salt thereof.
The compound of the present invention can have a chiral carbon identified in the
structure above with an asterisk (*). The preferred compound has the configuration
shown above, which by convention is known as the S configuration.
The present invention also provides a pharmaceutical composition comprising a
compound of Formula I as described above or a pharmaceutically acceptable salt thereof
together with one or more pharmaceutically acceptable carriers, diluents or excipients.
The present invention also provides a pharmaceutical composition comprising a
compound of Formula I as described above or a pharmaceutically acceptable salt thereof
together with one or more pharmaceutically acceptable carriers, diluents or excipients,
and optionally one or more therapeutic agents.
The present invention also provides a method for treating diabetes in a mammal.
The method comprises administering to the mammal in need of treatment a compound as
described above for Formula I, or a pharmaceutically acceptable salt thereof. More
preferably the present invention provides a method of treating type two diabetes in a
mammal in need of treatment by administering to the mammal a compound as described
above for Formula I or a pharmaceutically acceptable salt thereof. Preferably the
mammal is a human.
The present invention also provides a method for treating diabetes in a mammal
by administering to the mammal in need of treatment a pharmaceutical composition
comprising a compound as described above for Formula I, or a pharmaceutically
acceptable salt thereof. More preferably the present invention provides a method of
treating type two diabetes in a mammal in need of treatment by administering to the
mammal a pharmaceutical composition comprising a compound as described above for
Formula I, or a pharmaceutically acceptable salt thereof. Preferably the mammal is a
human.
The present invention provides a compound according to Formula I or a
pharmaceutically acceptable salt thereof as described above for use in therapy.
In yet another form, the present invention provides a compound as described
above according to Formula I, a pharmaceutically acceptable salt thereof, or
pharmaceutical composition for use in the treatment of diabetes in a mammal in need
thereof. Preferably the use is for the treatment of type two diabetes and the mammal is a
human.
The present invention provides use of a compound according to Formula I, or a
pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the
treatment of diabetes. Preferably the medicament is for the treatment of type two diabetes
and for treating mammal particularly humans.
In yet another form, the present invention provides an intermediate compound of
the Formula II
II.
wherein R is selected from a C1-4 alkyl, C1-4 haloalkyl, C 3-6 cycloalkyl, C1-4 alkyl-C3-6
cycloalkyl, phenyl, and C1-5 alkylphenyl to provide a compound of Formula I, or a
pharmaceutically acceptable salt thereof. Preferred R groups include C1-2 alkyl, C1-2
haloalkyl, phenyl, and C1-2 alkylphenyl. Particularly preferred R groups include methyl,
ethyl, phenyl, and benzyl.
The present invention also provides a process of preparing (3S)-3-[4-[[5-[(8-
Methoxy-3,4-dihydro-2H-quinolin-l-yl)methyl]-2-thienyl]methoxy]phenyl]hex-4-ynoic
acid described above for Formula I . The method comprises deprotecting or de-esterifying
the intermediate compound according to Formula II to prepare the compound of Formula
1 or a pharmaceutically acceptable salt thereof.
One skilled in the art would readily understand and be able to implement
deprotecting reactions without undue experimentation. It will be recognized by those
skilled in the art that in addition to the carboxylic acid and protected carboxylic acid,
other functional groups that can be readily converted to a carboxylic acid can be used in
place the carboxylic acid or protected acid. Such functional groups, preparations, and
transformations of these groups to carboxylic acids can be found in "Comprehensive
Organic Transformations: A Guide to Functional Group Preparations" by Larock. R.C,
Wiley VCH, 1999 and in "March's Advanced Organic Chemistry, Reactions,
Mechanisms and Structure" Smith, M.B., and March, J., Wiley -Interscience, 6th Ed.
2007.
The compound of the present invention, (3S)-3-[4-[[5-[(8-Methoxy-3,4-dihydro-
2H-quinolin-l-yl)methyl]-2-thienyl]methoxy]phenyl]hex-4-ynoic acid, can be provided
as a pharmaceutically acceptable salt. "Pharmaceutically-acceptable salt" refers to salts
of the compound of the invention considered to be acceptable for clinical and/or
veterinary use. 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, (VCHA/Wiley-VCH, 2002); S.M.
Berge, et al, "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, Vol. 66, No. 1,
January 1977.
The term "pharmaceutically acceptable carrier, diluent, or excipients" means that the
carrier, diluent, and excipients are pharmaceutically compatible with the other ingredients
of the composition.
Certain substituents have been eliminated in the following Schemes for the sake of
clarity and is not intended to limit the teaching of the Schemes in any way. Furthermore,
individual isomers, enantiomers, or diastereomers may be separated at any convenient
point in the synthesis of the compound of Formula I by methods such as chiral
chromatography. Additionally, the intermediates described in the following Schemes and
preparations contain a number of nitrogen, hydroxy, and acid protecting groups such as
esters. 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. e.g., Greene and Wuts, Protective Groups
in Organic Synthesis, (T. Greene and P. Wuts, eds., 2d ed. 1991).
The abbreviations used herein are defined according to Aldrichimica Acta, Vol.
17, No. 1, 1984. Other abbreviations are defined as follows: "ADDP" refers to 1-
(azodicarbonyl)dipiperidine ; "BSA" refers to Bovine Serum Albumin; "DIBAL-H"
refers to diisobutylaluminum hydride; "DIPEA" refers to diisopropylethyl amine;
"DMEM" refers to Dulbecco's Modified Eagle's Medium; "DTT" refers to dithiothreitol;
"ESI" refers to electrospray ionization "EtOAc" refers to ethyl acetate; "EtOH" refers to
ethyl alcohol or ethanol; "F12" refers to Ham's F12 medium; "FBS" refers to Fetal
Bovine Serum; "HEK" refers to human embryonic kidney; "IC50" refers to the
concentration of an agent that produces 50% of the maximal inhibitory response possible
for that agent; "MeOH" refers to methyl alcohol or methanol; "NBS" refers to Nbromosuccinimide;
"PPAR" refers to peroxisome proliferator-activated receptor; "PPRE"
refers to peroxisome proliferator response element; "RFU" refers to relative fluorescence
unit; "RPMI" refers to Roswell Park Memorial Institute; "RT" refers to ambient room
temperature; "THF" refers to tetrahydrofuran; and "TK" refers to thymidine kinase.
The term alkyl as used herein is a straight chain alkyl such as ethyl or n-propyl, or
a branched chain alkyl such as isopropyl or tert-butyl. The term C1-4 haloalkyl refers to
an alkyl group that has 1, 2, 3, or more halo groups attached to the carbons of the alkyl
chain. If there are two or more halogens, the halogens need not be attached to the same
carbon. This term also includes perhalo alkyls where all the hydrogen atoms of the alkyl
group are replaced with a halogen.
In the schemes below, all substituents unless otherwise indicated, are as
previously defined. The reagents and starting materials are generally 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 structurallysimilar
compounds, and the following procedures described in the Preparations and
Examples including any novel procedures.
Scheme 1
(6) (I)
Scheme 2
S ^C0 2H S0 CI , MeOH S C¾C H3 NBS/chloroform S C ¾CH 3 J reflux 4 h J reflux 7 h
B r J
Preparations and Examples
The following Preparations and Examples further illustrate the invention and
represent typical synthesis of the compound of Formula (I). The compounds are named
by IUPACNAME ACDLABS or Symyx Draw 3.2.
Preparation 1
8-Methoxyquinoline
Add potassium hydroxide (435 g, 7.76 mol) to a solution of 8-hydroxy quinoline
(250 g, 1.724 mol) in THF (10 L) at ambient temperature and stir. Add methyl iodide
(435 g, 2.58 mol) dropwise and stir overnight. Filter the reaction mixture and wash the
solid with THF (2 L). Concentrate the solution to dryness; add water; extract with
dichloromethane (2 x 3 L); combine the organic layers; and wash with brine. Collect the
organic layers and dry over sodium sulfate. Remove the solids by filtration. Collect the
filtrate and concentrate under reduced pressure to give a red oil, which solidifies on
standing, to give the title compound (28 1 g, 102%), which can be used without further
purification. ESI (m/z) 160(M+H).
Preparation 2
8-Methoxy- 1,2,3 ,4-tetrahydroquinoline
Add sodium cyanoborohydride (505 g, 8.11 mol) in EtOH ( 1 L) to a solution of 8-
methoxy quinoline (425 g, 2.673 mol) in EtOH (9 L), and stir. Cool the reaction mixture
to an internal temperature of 0°C and add HC1 (35%, 1.12 L, 10.962 mol) dropwise over
60 min so that the internal temperature did not rise above 20 °C. Allow the reaction
mixture to warm to ambient temperature and then heat to reflux for 2.5 hours. Cool to
ambient temperature and stir overnight. Add ammonium hydroxide (25%, 1 L); dilute
with water (15 L); and extract the mixture with dichloromethane (3 x 10 L). Combine
the organic layers and dry over sodium sulfate. Remove the solids by filtration. Collect
the filtrate and concentrate under reduced pressure to give a residue. Purify the residue
by silica gel flash chromatography, eluting with ethyl acetate: hexane (1: 10) to give the
title compound (357 g, 82%). ESI (m/z) 164(M+H).
Preparation 3
Methyl-5-methylthiophene-2-carboxylate
Add thionyl chloride (153 ml, 2.1 mol) dropwise over 20 min to a solution of 5-
methylthiophene-2-carboxylic acid (100 g, 0.703 mol) in MeOH ( 1 L) at 0 °C and stir.
After the addition is complete, heat the reaction mixture to reflux for 3.5 hours. Cool and
concentrate in vacuo to give a thick oil. Dilute the oil with EtOAc (500 ml) and
sequentially wash with water (300 ml) then brine (300 ml). Dry the organic layer over
sodium sulfate. Remove the solids by filtration. Collect the filtrate and concentrate under
reduced pressure to give the title compound (106 g, 97%), which is used without further
purification. ESI (m/z) 156(M+H).
Preparation 4
Methyl 5-(bromomethyl)thiophene-2-carboxylate
Add freshly recrystallised NBS (323.8 g, 1.81 mol) to a solution of methyl-5-
methylthiophene-2-carboxylate (258 g, 1.65 mol) in chloroform (2.6 L) at room
temperature, and stir. Add benzoyl peroxide (3.99 g, 0.016 mol) and heat the reaction
mixture to reflux for 7 hours. Cool the reaction mixture to ambient temperature and filter
through diatomaceous earth. Wash the filter cake with chloroform (250 ml). Collect the
organic layers and remove the solvent to give the title compound (388 g, 100%), which is
used without further purification. ESI (m/z) 236(M+H).
Preparation 5
Methyl-5-[(8-methoxy-3,4-dihydro-2H-quinolin-l-yl)methyl]thiophene-2-carboxylate
Add methyl-5-(bromoethyl)thiophene-2-carboxylate (432.5 g, 1.84 mol) in EtOH
(500 ml) to a solution of 8-methoxy-l,2,3,4-tetrahydroquinoline (300 g 1.84 mol) in
EtOH ( 1 L) and stir. Add DIPEA (641 ml, 3.67 mol) dropwise and stir at room
temperature overnight. After completion of the reaction, remove the EtOH in vacuo, and
add water (5 L). Extract the aqueous with EtOAc (3 x 3 L); combine the organic layers;
and dry over sodium sulfate. Filter the solution and concentrate under reduced pressure
to give a residue. Purify the residue by silica gel flash chromatography eluting with ethyl
acetate: hexane (6:94) to give the title compound (325 g, 56%). ESI (m/z) 318(M+H).
Preparation 6
[5-[(8-Methoxy-3,4-dihydro-2H-quinolin-l-yl)methyl]-2-thienyl]methanol
Add DIBAL-H ( 1 M in toluene 2.7 L, 2.66 mol) slowly via a cannula over a
period of 1.5 h to a stirred solution of methyl-5-(8-methoxy-3,4-dihydroquinolin-l(2H)-
yl)methyl)thiophene-2-carboxylate (281 g, 0.886 mol) in THF (4 L) at -70 °C. Monitor
the reaction via thin layer chromatography (TLC) for completion. After completion of
the reaction, allow the reaction mixture to warm to 20 °C and add a saturated solution of
ammonium chloride. Add a solution of sodium potassium tartrate (1.3 Kg in 5 L of
water), and stir overnight. Separate the organic layer; extract the aqueous phase with
EtOAc (2 x 5 L); then combine the organic layers; and dry the combined organic layers
over sodium sulfate. Remove the solids by filtration. Remove the solvent from the
filtrate under reduced pressure to give the title compound as a white solid (252 g, 98%).
ESI (m/z) 290(M+H).
Preparation 7
Ethyl (3S)-3-[4-[[5-[(8-methoxy-3,4-dihydro-2H-quinolin-l-yl)methyl]-2-
thienyl]methoxy]phenyl]hex-4-ynoate
Add tributylphosphine (50% solution in EtOAc, 543ml, 1.34 mol) to a solution of
ADDP (282.5 g, 1.5 eq) in THF (3 L) and cool the mixture to an internal temperature of 0
°C, then stir for 15 minutes. Add (S)-ethyl 3-(4-hydroxyphenyl)hex-4-ynoate (173. 5g,
0.747 mol) in THF (3 L) dropwise over 15 min; then add 5-((8-methoxy-3,4-
dihydroquinolin-l(2H)-yl)methyl)thiophene-2-yl)methanol (216 g, 0747 mol) in THF (5
L) dropwise. Allow the reaction mixture to warm to ambient temperature and stir
overnight. Filter the reaction mixture through diatomaceous earth and wash the filter
cake with ethyl acetate (2 L). Concentrate the organic filtrate to dryness. Add water (4
L); extract with ethyl acetate (3 x 5 L); combine the organic layers; and dry the combined
organic layers over sodium sulfate. Remove the solids by filtration and concentrate under
reduced pressure to give an oil. Purify the residue by silica gel flash chromatography by
eluting with ethyl acetate: hexane (6:94) to give the title compound (167 g, 44%). ESI
(m/z) 504(M+H).
Example 1
(3S)-3-[4-[[5-[(8-Methoxy-3,4-dihydro-2H-quinolin-l-yl)methyl]-2-
thienyl]methoxy]phenyl]hex-4-ynoic acid
Add a solution of potassium hydroxide (49.76 g, 0.88 mol) in water (372 ml) to a
solution of (S)-ethyl-3-(4-((5-8-methoxy-3,4-dihydroquinolin-l(2H)-yl)methyl)thiophen-
2-yl)methoxy) phenyl)hex-4-ynoate (149 g, 0.296 mol) in EtOH (1.49 L) at room
temperature and stir overnight. Concentrate the reaction mixture to dryness and add
water (1.3 L). Extract the resulting solution with EtOAc (2 x 300 ml) and separate.
Adjust the pH of the aqueous layer to pH = 6 with 2 N HC1. Collect the resulting solids.
Recrystallise the solids from hot MeOH (298 ml, 2 vol) to give the title compound (91 g,
65%). ESI (m/z) 476(M+H).
GPR40; Information
Results of studies using transgenic mice over-expressing the human GPR40 gene
under control of the insulin II promoter recently reported by Nagasumi further support
that GPR40 plays an important role in the regulation of GDIS and plasma glucose levels
in-vivo, especially in rodent models of insulin resistance. Nagasumi K, et. al,
Overexpression of GPR40 in pancreatic b-cells augments glucose-stimulated insulin
secretion and improves glucose tolerance in normal and diabetic mice, Diabetes 58:
1067-1076, 2009. See also, Briscoe CP et al, The orphan Gprotein-coupled receptor
GPR40 is activated by medium and long chain fatty acids, Journal Biological Chemistry
278: 11303 - 1131 1, 2003. These findings further support that the development of new
GPR40 modulator compounds may be particularly desired for use in the treatment of
T2D.
Calcium Flux Primary Assay
The compound of Example 1 is tested essentially as described below and exhibits
an EC50 value for the Calcium Flux Primary assay of lower than 1 mM.
This assay is used to screen compounds by measuring the increase in intracellular
calcium levels that results when a ligand binds and activates GPR40, thus demonstrating
the potency and efficacy of GPR40 agonists. HEK293 cells over expressing the human
GPR40 cDNA maintained in Dulbecco's modified Eagle's medium with F12 medium in
3:1 ratio supplemented with 10% FBS and 800 mg/ml geneticin at 37 °C and 5% CO2 are
employed for the study. Agonist assays are performed using a Calcium 4 Dye assay kit
(Molecular Devices) in the presence of 0.1% fatty acid free BSA in the assay buffer (lx
HBSS (Hank's Balanced Salt Solution) & 20 mM HEPES (4-(2-hydroxyethyl)-lpiperazineethanesulfonic
acid). Receptor activation is measured as an increase in
intracellular calcium using the Fluorometric Imaging Plate Reader (FLIPR). Maximum
change in fluorescence over the base line is used to determine agonist response. An EC50
(effective concentration at half the maximal response) value of the compound is
calculated using Excel Fit software (version 4; IDBS) by plotting concentration vs
relative fluorescence units (RFUs). Percent efficacy is calculated based on the maximal
response exhibited by compound compared to the natural ligand, linoleic acid. The test
compound of Example 1 has an EC50 of 152 +/- 52 nM with 84 +/- 24% efficacy when
examined in this assay. These results further demonstrate the desired potency and
efficacy of this compound as a GPR40 agonist.
Glucose Dependent Insulin Secretion (GDIS) Assays
Because activation of GPR40 is known to result in insulin secretion, which is
dependent on high glucose concentrations, two separate assay systems (an insulinoma cell
line and primary rodent islets) are developed to further characterize compounds that are
known to increase intracellular calcium in the GPR40 primary assay discussed above.
GDIS assays are performed using the mouse insulinoma cell line Min6. The Min6
cells are maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing nonessential
amino acids, 10% FBS, 50 mM 2-mercaptoethanol and 1% penicillin and
streptomycin at 37 °C plus 5% C0 2. On the day of the experiment, the cells are washed
twice with 200 mΐ of pre-warmed Krebs-ringer buffer without glucose. Addition of 200
mΐ of pre-warmed Krebs-ringer buffer containing 2.5 mM glucose is used to starve the
cells followed by the addition of compounds in the presence of a high concentration of
glucose (25 mM). The plate is incubated at 37 °C for 2 hours. At the end of the 2 h
incubation, the supernatant is gently transferred into a Millipore filter plate and spun at
200 g (gravitational force) for 3 minutes. Insulin is assayed using a Mercodia Insulin
estimation kit. Addition of Example 1 at 0.01, 0.1, 1.0, and 10.0 mM plus 25 mM glucose
to the Min6 cells resulted in a dose dependent increase in insulin secretion with a
statistically significant (P < 0.01) increase (2.68 fold over that achieved with 25 mM
glucose) at the 1.0 mM dose.
GDIS assays using primary rodent pancreatic islets of Langerhans are also used to
characterize the exemplified compound. Pancreatic islets are isolated from male SD
(Sprague Dawley) rats by collagenase digestion and Histopaque density gradient
separation. The islets are cultured overnight in RPMI-1640 medium with GlutaMAXn
(stabilized, dipeptide form of L-glutamine (Invitrogen catalog # 61870-010)) to facilitate
recovery from the isolation process. Insulin secretion is determined by a 90 minute
incubation in EBSS (Earle's Balances Salt Solution) buffer in a 48-well plate. Briefly,
islets are first preincubated in EBSS with 2.8 mM glucose for 30 min, and are then
transferred to a 48-well plate (four islets/well) containing 150 mΐ 2.8 mM glucose, and
incubated with 150 mΐ of EBSS with 2.8 or 11.2 mM glucose in the presence or absence
of test compound for 90 minutes. The buffer is removed from the wells at the end of the
incubation, and assayed for insulin levels using the Rat Insulin ELISA kit (Mercodia). In
this assay system, incubation of Example 1 at 1, 3, and 10 mM with rat islets and 11.2
mM glucose results in a statistically significant (P <0.05) increase in insulin at 3.0 uM
(2.1 -fold ) compared to that achieved with 11.2 mM glucose. Thus, the compound of
Example 1 induces insulin production under the conditions of this assay.
Selectivity assays;
Peroxisome Proliferator-Activated Receptor (PPAR) , d, and g Binding and
Functional Assays;
Because GPR40 is known to be activated by ligands to PPARy, the exemplified
compound is examined in PPARa, PPAR8, and PPARy binding and functional assays to
determine the selectivity of the compound of Example 1 for GPR40. The compound of
Example 1 is tested essentially as described below for PPAR binding and it exhibits
binding values greater than 1000 nM with 10 mM concentrations of test compound, and is
thus considered negative for PPAR activity.
Binding affinities of the compound for the PPAR a, d, and g receptors are assessed
using Scintillation Proximity Assay (SPA) technology. Biotinylated oligonucleotide
Direct Repeat 2 (DR2) is used for binding the receptors to Yttrium silicate streptavidincoated
SPA beads. PPAR a, d, g and retinoid X receptor (RXR) a are over expressed in
HEK293 cells, and cell lysates containing the specific receptors are used in the individual
assays. The DR2 is attached to the SPA beads over a 30 minute period in a binding
buffer containing 10 mM HEPES pH 7.8, 80 mM KC1, 0.5 mM MgCl2, 1mM DTT, 0.5%
3[(3-cholamidopropyl)dimethylammonio]-propanesulfonic acid (CHAPS ), and 4.4%
bovine serum. The cell lysates are incubated in each well with one of 11 concentrations
of compound in the presence of a radio-labeled (-0.033.8 m H) PPAR a/d dual agonist
reference compound (butanoic acid, 2-[4-[2-[[[(2,4-difluorophenyl)amino]carbonyl]
heptylamino]ethyl]phenoxy]-2-methyl, see Burris T.P. et al, Molecular Pharmacology
2005, 67, (3) 948-954) for the alpha and delta receptor assays and a radio-labeled
(-0.037.3 m H) PPARy agonist reference compound (propanoic acid, 2-methyl-2-[4-
[3-[propyl[[5-(2-pyridinyl)-2-thienyl]sulfonyl]amino]propyl]phenoxy] see Burris T.P. et
al, Molecular Pharmacology 2005, 67, (3) 948-954) for the gamma receptor assays, 110.3
mg of Yttrium SPA Streptavidin coated beads, 0.126 nM HD Oligo DR2, and either 0.3
mg PPARa with 0.5 mg RXRa, 0.5 mg PPAR5 with 0.5 mg RXRa, or 1.25 mg PPARy with
3.03 mg RXRa in the binding buffer above plus 14% glycerol and 5 mg of sheared salmon
sperm DNA. Non-specific binding is determined in the presence of 10,000 nM of the
unlabeled PPAR a/d dual agonist reference compound for the alpha and delta receptor
assays and the PPARy agonist reference compound for the gamma receptor assay. The
binding reaction (100 mΐ per well in a 96 well [Costar 3632] plate) is incubated for 10 h
and counted disintegration per minutes (dpm) on a Wallac Microbeta. Receptor binding
affinity (IC50) for the compound is determined by fitting an 11 point concentrationresponse
curve with a 4-paramater logistic equation. K is determined from the IC50 using
the Cheng-Prussoff equation and Kd determined by saturation binding. For the
compound of Example 1, no binding is detected in any of the three PPAR binding assays
with concentrations up to 10 mM. Thus, the assays set forth herein support that the
compound of Example 1 selectively activates GPR40 while avoiding the undesired PPAR
activity. The relative ICsos for the exemplified compound when tested up to 30 mM is
greater than 10 mM for the PPAR isoforms, supporting that the exemplified compound
avoids PPAR activity while providing the desired GPR40 activation.
Gal4 PPARa, Gal4 PPAR5, and PPARy reporter functional assays are also used to
monitor the selectivity of the exemplified compound. CV1 cells, which are derived from
the renal tissue of an African green monkey, are transfected with various receptor and
reporter plasmids using Fugene. For the Gal4 PPARa and PPAR5 assays, a reporter
plasmid containing five tandem copies of the yeast transcription protein Gal4 response
element, cloned upstream of a firefly luciferase gene driven by the major late promoter of
adenovirus, is transfected together with a Simian Virus 40 (SV40) driven plasmid
constitutively expressing a hybrid protein containing the Gal4 DNA binding domain
(DBD), and either the PPARa or PPAR5 ligand binding. For the PPARy assay, plasmids
encoding PPARy and RXRa, both driven by a cytomegalovirus (CMV) promoter are
transfected together with a plasmid containing luciferase reporter cDNA driven by the TK
promoter and a receptor response element (2X PPRE). Cells are transfected in T225 cm2
cell culture flasks in DMEM media with 5% charcoal-stripped FBS. After an overnight
incubation, transfected cells are trypsinized; plated in opaque 96 well dishes (15,000
cells/well) in DMEM media containing 5% charcoal-stripped FBS, incubated for 4 h; and
exposed to 0.17 hM to 10 mM of test compound or reference compound in half log
dilutions. After 24 hours incubation with the compound, cells are lysed and luciferase
activity is determined as a measure of receptor activation by luminescence. Data are
fitted to a four parameter-fit logistics model to determine EC50 values. The maximum
percent stimulation is determined versus maximum stimulation obtained with 10 mM of
an appropriate PPAR agonist reference compound. No functional activation of PPARa,
PPAR8, or PPARy is detected with the compound of Example 1when examined up to 10
mM in the specific PPAR co-transfection (CTF) / functional assays described above.
Thus, the assay supports that the exemplified compound avoids PPAR agonist activity, as
desired.
In Vivo Efficacy; Intraperitoneal Glucose Tolerance Test (IPGTT)
To examine the ability of exemplified the compound to activate GPR40 in-vivo
resulting in anti-diabetic efficacy, i.e. reduction in plasma glucose levels, a 4-day
intraperitoneal glucose tolerance test (ipGTT) study is completed, and the data is shown
for the compound tested below.
Male Balb/c (Albino mice) mice (8-9 weeks of age) are single housed, and fed
with normal rodent chow diet and water ad libitum. Animals are weighed; randomized by
body weight; and their daily body weights are recorded. Animals are dosed once per day
orally for three days using a formulation carrying methylcellulose and tween-80. On the
night before Day 4, animals are fasted overnight. On the morning of Day 4, animals are
dosed orally with compound or vehicle alone 60 minutes prior to the glucose tolerance
test (glucose 2 g/kg, i.p.). Blood glucose levels are determined from tail bleeds taken at
0, 3, 7, 15, 30, and 60 min after glucose challenge. The blood glucose excursion profile
from t=0 to t=60 min is used to integrate an area under the curve (AUC) for each
treatment. Percent lowering in glucose is calculated from the AUC data of the compound
with respect to the AUC of vehicle group. The test compound is orally administered at
0.3, 1.0, 3.0, 10, or 30 mg/kg, and a positive control (3-[4-(2-methyl-benzyloxy)-phenyl]-
hex-4-ynoic acid, see WO2005086661.) is administered at 10 mg/kg. Glucose levels are
significantly lowered compared to those achieved with the vehicle control at the 15
minute time points with the 3, 10 and 30 mg/kg doses and at the 30 and 60 minute time
points with the 1.0, 3.0, 10, and 30 mg/kg doses of Example 1. Glucose levels are
lowered at the 15, 30, and 60 minute time points for the positive control. The ED 0 for
this compound based on AUCs for glucose lowering is 1.0 mg/kg. Results from this
study demonstrate that activation of GPR40 by Examples 1 leads to in-vivo anti-diabetic
efficacy.
The exemplified compound of the present invention can be readily formulated into
pharmaceutical compositions in accordance with accepted practices known in the art such
as found in Remington's "Pharmaceutical Sciences", Gennaro, Ed., Mack Publishing Co.
Easton Pa. 1990 such as tablets, solid or gel filled capsules, powders, suspensions, or
solutions. The composition can also include one or more pharmaceutically acceptable
carriers, excipients, and diluents. Non limiting examples of pharmaceutically acceptable
carriers, excipients, and diluents are suitable for such formulations include the following:
starch, sugars, mannitol, and silica derivatives; binding agents such as carboxymethyl
cellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone;
moisturizing agents such as glycerol; disintegrating agents such as calcium carbonate and
sodium bicarbonate; agents for retarding dissolution such as paraffin; resorption
accelerators such as quaternary ammonium compounds; surface active agents such as
cetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite; and
lubricants such as talc, calcium, and magnesium stearate, and solid polyethyl glycols.
Preferred pharmaceutical compositions include formulated as a tablet or capsule
for oral administration. The tablet or capsule can include a compound of the present
invention in an amount effective to treat diabetes particularly type two diabetes.
The pharmaceutical composition is administered to a patient in amounts effective
to treat diabetes, more particularly, type two diabetes. An appropriate amount or dose
effective to treat a patient can be determined by a health care provider.
What is claimed is:
1. A compound which is :
or a pharmaceutically acceptable salt thereof.
2. A pharmaceutical composition comprising a compound according to claim
1 or a pharmaceutically acceptable salt thereof and at least one of a pharmaceutically
acceptable carrier, diluent, or excipient.
3. A pharmaceutical composition according to claim 2 further comprising
one or more additional therapeutic agents.
4. A method of treating diabetes in a mammal in need thereof, comprising
administering to the mammal a compound, or a pharmaceutically acceptable salt thereof
according to claim 1.
5. A method of treating diabetes in a mammal in need thereof, comprising
administering to the mammal a pharmaceutical composition according to claim 2 or 3.
6. A method according to claim 4 or 5 wherein treating diabetes comprises
treating a mammal for type two diabetes.
7. A compound, or a pharmaceutically acceptable salt thereof, according to
claim 1 for use in therapy.
8. A compound, or a pharmaceutically acceptable salt thereof, according to
claim 1 for use in the treatment of diabetes in a mammal.
9. Use of a compound, or a pharmaceutically acceptable salt thereof,
according to claim 1 in the manufacture of a medicament to treat diabetes.
10. A compound according to formula II
wherein R is selected from: C1-4 alky1, C1-4 haloalkyl, C3-6 cycloalkyl, C1-4 alkyl-C3-6
cycloalkyl, phenyl, and C1-5 alkylphenyl.
11. A method of preparing (3S)-3-[4-[[5-[(8-Methoxy-3,4-dihydro-2Hquinolin-
l-yl)methyl]-2-thienyl]methoxy]phenyl]hex-4-ynoic acid or a pharmaceutically
acceptable salt thereof, said method comprising de-esterifying a compound of formula II;
II
Where R is selected from: C1-4 alky 1, C1-4 haloalkyl, C3-6 cycloalkyl, C1-4 alkyl-C3-6
cycloalkyl, phenyl, and C1-5 alkylphenyl to provide a compound of formula I, or a
pharmaceutically acceptable salt thereof
I .