U.S. patent application number 16/483217 was filed with the patent office on 2020-11-05 for trpc ion channel inhibitors for use in therapy.
The applicant listed for this patent is UNIVERSITY OF LEEDS. Invention is credited to David John BEECH, Sin Ying CHEUNG, Richard James FOSTER, Baptiste Michel RODE.
Application Number | 20200345741 16/483217 |
Document ID | / |
Family ID | 1000005032775 |
Filed Date | 2020-11-05 |
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United States Patent
Application |
20200345741 |
Kind Code |
A1 |
BEECH; David John ; et
al. |
November 5, 2020 |
TRPC ION CHANNEL INHIBITORS FOR USE IN THERAPY
Abstract
Described herein are inhibitors Transient Receptor Potential
Canonical (TRPC) ion channels comprising TRPC4 protein and/or TRPC5
protein for use in combating obesity and other medical conditions
including insulin resistance associated with Type II diabetes or
development of Type II diabetes (pre-diabetes), metabolic syndrome,
non-alcoholic fatty liver disease (NAFLD) and non-alcoholic
steatohepatitis (NASH). Also disclosed is the use of the inhibitors
for cosmetic purposes, such as cosmetic weight loss.
Inventors: |
BEECH; David John; (Leeds,
Yorkshire, GB) ; FOSTER; Richard James; (Leeds,
Yorkshire, GB) ; CHEUNG; Sin Ying; (Stanford, CA)
; RODE; Baptiste Michel; (Bordeaux, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF LEEDS |
Leeds |
|
GB |
|
|
Family ID: |
1000005032775 |
Appl. No.: |
16/483217 |
Filed: |
February 9, 2018 |
PCT Filed: |
February 9, 2018 |
PCT NO: |
PCT/GB2018/050369 |
371 Date: |
August 2, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/522 20130101;
A61P 3/04 20180101; A61P 3/10 20180101; A61K 31/713 20130101; A61K
31/4184 20130101 |
International
Class: |
A61K 31/522 20060101
A61K031/522; A61K 31/4184 20060101 A61K031/4184; A61K 31/713
20060101 A61K031/713; A61P 3/10 20060101 A61P003/10; A61P 3/04
20060101 A61P003/04 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 9, 2017 |
GB |
1702160.1 |
Claims
1. An inhibitor which directly targets a Transient Receptor
Potential Canonical (TRPC) ion channel comprising TRPC4 and/or
TRPC5 as present in adipocytes for use in the treatment or
prophylaxis of a condition selected from obesity, insulin
resistance, metabolic syndrome, non-alcoholic fatty liver disease
(NAFLD) and non-alcoholic steatohepatitis (NASH).
2. An inhibitor as claimed in claim 1, wherein the TRPC ion channel
comprises TRPC5.
3. An inhibitor as claimed in claim 1, wherein the TRPC ion channel
comprises TRPC4.
4. An inhibitor as claimed in claim 1, wherein the TRPC ion channel
further comprises TRPC1.
5. An inhibitor as claimed in claim 1 which directly inhibits the
expression or function of TRPC4 and/or TRPC5
6. An inhibitor as claimed in claim 1, wherein said inhibitor is
for use in the treatment or prophylaxis of obesity.
7. An inhibitor as claimed in claim 1 for use in the treatment or
prophylaxis of insulin resistance in a subject.
8. An inhibitor as claimed in claim 7, wherein the subject is
obese.
9. An inhibitor as claimed in claim 7 wherein the insulin
resistance is associated with Type II diabetes or prediabetes.
10. An inhibitor as claimed in claim 1 for use in the treatment or
prophylaxis of metabolic syndrome.
11. An inhibitor as claimed in claim 10, wherein the treatment of
metabolic syndrome comprises reducing abdominal obesity and/or
reducing fasting blood glucose concentration.
12. An inhibitor as claimed in claim 1 wherein the inhibitor is
administered to a subject predetermined to have an elevated level
in adipose tissue relative to control of one or more screening
targets selected from TRPC1 mRNA and/or protein, TRPC4 mRNA and/or
protein and TRPC5 mRNA and/or protein.
13. A method of identifying a subject according to claim 12, which
comprises: a. determining the level of one or more screening
targets selected from TRPC1 protein and/or mRNA, TRPC4 mRNA and/or
protein and or TRPC5 mRNA and/or protein in a sample of adipose
tissue from a subject; and b. selecting said subject for
administration of the inhibitor if the level of said screening
target exceeds a control level.
14. An inhibitor as claimed in claim 1, wherein the inhibitor is an
antibody or an antigen-binding fragment thereof.
15. An inhibitor as claimed in claim 14 wherein said antibody binds
TRPC4 and/or TRPC5.
16. An inhibitor as claimed in claim 1 wherein the inhibitor is a
siRNA or antisense oligonucleotide.
17. An inhibitor as claimed in claim 16 which inhibits expression
of TRPC4 protein, TRPC5 protein or both,
18. An inhibitor as claimed in claim 1, wherein the inhibitor is a
small molecule of 2000 daltons or less.
19. The inhibitor of claim 18, wherein the inhibitor is a compound
of the Formula (II), or a pharmaceutically acceptable salt thereof:
##STR00043## wherein: R.sup.1 is C.sub.1-6 alkyl, C.sub.2-6 alkenyl
or C.sub.2-6 alkynyl, each of which is optionally substituted with
1-4 R.sup.5; R.sup.2 is C.sub.1-6 alkyl, C.sub.1-6 heteroalkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl, halo,
C.sub.1-6 haloalkoxy, hydroxyl, C.sub.1-6 alkoxy, C.sub.3-7
cycloalkyloxy, C.sub.6-10 aryl, C.sub.6-10 aryloxy, C.sub.7-16
arylalkoxy, amino, C.sub.1-6 akylamino, C.sub.2-12 dialkylamino,
--S(O).sub.xR' (wherein x is 0, 1 or 2 and each R' is independently
H or C.sub.1-6 alkyl), heterocycloalkyl, heteroaryl, heteroaryloxy,
sulfonamidyl, amido, urea, sulfonylurea, acyl, nitro, cyano,
wherein each C.sub.1-6 alkyl, C.sub.1-6 heteroalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl,
C.sub.1-6haloalkoxy, hydroxyl, C.sub.1-6 alkoxy, C.sub.3-7
cycloalkyloxy, C.sub.6-10 aryl, C.sub.6-10 aryloxy, C.sub.7-16
arylalkoxy, amino, C.sub.1-6 akylamino, C.sub.2-12 dialkylamino,
--S(O).sub.xR', heterocycloalkyl, heteroaryl, heteroaryloxy,
sulfonamidyl, amido, urea, sulfonylurea, acyl, is optionally
substituted with 1-3 R.sup.6; R.sup.3 is C.sub.1-6 alkyl, C.sub.1-6
heteroalkyl, C.sub.3-7 cycloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.2-6 hydroxyalkyl, or C.sub.1-6 alkoxy, each of which
is optionally substituted with 1-4 R.sup.7; R.sup.4 is C1.6 alkyl,
C1.6 heteroalkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl, each of
which is optionally substituted with 1-4 R.sup.1; R.sup.5, R.sup.6,
R.sup.7, and R.sup.8 are each independently hydrogen, C.sub.1-6
alkyl, C.sub.1-6 heteroalkyl, halo, C.sub.1-6 haloalkyl, C.sub.1-6
haloalkoxy, hydroxyl, C.sub.1-6 alkoxy, amino, C.sub.1-6
alkylamino, C.sub.2-12 dialkylamino, cyano, nitro, amido, C.sub.1-6
alkylamido, C.sub.2-12 dialkylamido, --S(O).sub.xR' (wherein x is
0, 1 or 2), --C(O)OR', --C(O)R', C.sub.3-7 cycloalkyl, C.sub.6-10
aryl, heterocycloalkyl, or heteroaryl, wherein each R' is
independently H or C.sub.1-6 alkyl, wherein each of C.sub.1-6
alkyl, C.sub.1-6 heteroalkyl, C.sub.1-6 haloalkyl, C.sub.1-6
haloalkoxy, hydroxyl, C.sub.1-6 alkoxy, amino, C.sub.1-6
alkylamino, C.sub.2-12 dialkylamino, amido, C.sub.1-6 alkylamido,
C.sub.2-12 dialkylamido, --C(O)O--C.sub.1-6 alkyl, C.sub.3-7
cycloalkyl, C.sub.6-10 aryl, heterocycloalkyl, or heteroaryl is
optionally substituted with 1-3 R.sup.9; and each R.sup.9 is
independently C.sub.1-6 alkyl, C.sub.1-6 heteroalkyl, C.sub.1-6
haloalkyl, C.sub.1-6 haloalkoxy, heterocycloalkyl, C.sub.6-10 aryl,
heteroaryl, C.sub.4-10 cycloalkylalkyl, heterocycloalkyl-C.sub.1-6
alkyl, C.sub.7-16 arylalkyl, heteroaryl-C.sub.1-6 alkyl, halo,
hydroxyl, C.sub.1-6 alkoxy, C.sub.6-10 aryloxy, C.sub.7-16
arylalkoxy, C2-s alkoxyalkoxy, amino, C.sub.1-6 akylamino,
C.sub.2-12 dialkylamino, C.sub.1-6 aryl-amino-C.sub.1-6 alkyl,
C.sub.1-6 alkyl-amino-C.sub.2-12 dialkyl, --S(O).sub.xR' (wherein x
is 0, 1 or 2), sulfonamidyl, amido, urea, sulfonylurea, acyl,
--C(O)--C.sub.1-6 alkyl, --C(O)--C.sub.6-10 aryl,
--NHC(O)--C.sub.1-4 alkyl, --NHC(O)--C.sub.6-10 aryl, --C(O)NR'R',
--C(O)NH--C.sub.6-10 aryl, --C(O)OR', --OC(O)R', acyl, nitro, or
cyano.
20. The inhibitor of claim 19, wherein the inhibitor is:
##STR00044## or pharmaceutically acceptable salt thereof.
21. The inhibitor of claim 19, wherein the inhibitor is:
##STR00045## or a pharmaceutically acceptable salt thereof.
22. The inhibitor of claim 18, wherein the inhibitor is
##STR00046## or a pharmaceutically acceptable salt thereof.
23. A compound of the formula (X), or a pharmaceutically acceptable
salt thereof: ##STR00047## wherein R.sup.1 and R.sup.2 are
independently H or C.sub.1-4 alkyl.
24. A compound of claim 23 of the formula (Xa): ##STR00048## or a
pharmaceutically acceptable salt thereof.
25.-28. (canceled)
Description
FIELD OF THE INVENTION
[0001] The invention relates to direct inhibitors of Transient
Receptor Potential Canonical (TRPC) ion channels comprising TRPC4
protein and/or TRPC5 protein as present in adipocytes for use
especially in combating obesity (reducing obesity or inhibiting
on-set of obesity); this may be for therapeutic purpose or cosmetic
weight loss. More particularly, such an inhibitor may target
expression or function of TRPC4 and/or TRPC 5 so as to affect ion
channel activity or formation. It is envisaged that such use may
also extend to the treatment or prophylaxis of insulin resistance
associated with Type II diabetes or development of Type II diabetes
(pre-diabetes), metabolic syndrome, non-alcoholic fatty liver
disease (NAFLD) and non-alcoholic steatohepatitis (NASH).
BACKGROUND OF THE INVENTION
[0002] Obesity is now a deadly global pandemic. Worldwide obesity
has more than doubled since 1980. In 2014, 39% of adults over 18
were considered overweight and 13% (over 600 million adults) were
obese according to the World Health Organisation. Even a modest
degree of obesity, particularly if the excess fat is located in the
abdomen, increases the risks for Type II diabetes, cardiovascular
diseases, stroke and some forms of cancer. The economic cost of
obesity is estimated to be $2 trillion annually or roughly 2.8
percent of global GDP according to the McKinsey Global Institute.
Methods for managing body weight by dietary restriction and/or by
exercise are largely ineffective as few people stick to dietary
regimens for a long time, and compliance to regular exercise is
equally poor. The result is generally a transient phase of weight
loss (or weight stability) followed by a return on the trajectory
towards obesity. These failures have highlighted the need for safe
anti-obesity therapies.
[0003] In humans and many other mammals, fat is stored in adipose
tissues. Adipose tissues are classified into two types-white
adipose tissue (i.e., "white fat") and brown adipose tissue (i.e.,
"brown fat"). After food consumption, excess calories are stored as
fat in adipocytes of white fat. By contrast, brown fat stores
little fat, instead burning it to produce heat and regulate body
temperature.
[0004] White fat adipocytes have previously been shown to present
ion channels formed by Transient Receptor Potential Canonical
(TRPC) proteins which have been linked to modulation of
adiponectin, an adipokine signalling molecule which has been
implicated in increasing insulin sensitivity, decreasing
inflammation and protecting against atherosclerosis and myocardial
decline. Decreased concentrations of adiponection occur in
obesity-induced insulin resistance and are associated with
endothelial dysfunction, Type II diabetes and hypertension. As
further expanded upon below, more recently TRPC1 and TRPC 5 have
been shown to be up-regulated in murine adipocytes as they mature
leading to constitutively-active Ca.sup.2+--permeable channels.
Moreover, constitutive Ca.sup.2+ influx through TRPC5-comprising
channels has been linked to suppression of adiponectin generation
(Sukumar et al. (2012) Circ. Res. 111, 191-200).
[0005] TRPC channels are a subfamily of the Ca.sup.2+ permeable
channels formed by Transient Receptor Potential (TRP) proteins. TRP
proteins are classified into sub-families based on amino acid
sequence. The canonical (C) sub-family is one such family which
contains six members in humans (TRPC1, 3-7); the additional TRPC 2
protein found in other mammals is not expressed in humans due
correspondence with a pseudo-gene. TRPC1, TRPC4 and TRPC5 are
considered to form a sub-group and TRPC3, TRPC6 and TRPC7 another
sub-group. At the proximal C-terminus of proteins of the TRPC
sub-family is a TRP box motif containing the invariant EWKFAR
sequence and near the N-terminus between 3 and 4 ankyrin repeats.
Each TRP protein is considered to have 6 membrane-spanning segments
and intracellular N- and C-termini. To form trans-membrane ion
channels, TRP proteins assemble together around a central
ion-selectivity filter and gate, most likely as a group of 4 TRP
proteins. All known TRPC channels are non-selective cationic
channels that can enable entry into cells of both Ca.sup.2+ and
Na.sup.+
[0006] The specific TRP proteins that form an individual channel
may be identical (homomers) or different (heteromers). TRPCs are
recognised to be promiscuous in forming heteromers and there is
even evidence that this extends outside the TRPC subfamily to TRPV4
and TRPP2. Much remains to be determined about the compositions of
native TRPC-containing channels and the functional significance of
TRPC heteromerization. TRPC1 has been suggested to be an oddity
amongst TRPC proteins in that it forms ion channels poorly or not
at all when expressed alone in vitro in heterologous systems. In
contrast other TRPCs form plasma membrane channels quite readily
when expressed alone (i.e. they form functional homomers). TRPC1 is
thought probably only to be a component of heteromeric ion channels
with other TRPCs. Although some studies have shown signals when
TRPC1 is expressed alone, the signals have generally been small and
could be explained by TRPC1 forming heteromers with endogeneous
TRPs of the expression system. [Beech (2013) Circ. J. 77, 570-579:
Characteristics of Transient Receptor Potential Canonical Calcium
Permeable Channels and Their relevance to Vascular Physiology and
Disease; Bon and Beech (2013) Brit. J. Pharmacol. 170 459-474,
Review: In pursuit of small molecule chemistry for
calcium-permeable non-selective TRPC channels-mirage or pot of
gold?]. Thus up to now it has been recognised that mature
adipocytes may have functional TRPC5-containing ion channels which
are either homomeric or additionally include TRPC1. Detection of
TRPC4 mRNA by RT-PCR has been reported in immature human adipocytes
(Hu et al. (2009) Characterization of calcium signalling pathways
in human preadipocytes J Cell. Physiol. 220, 765-770) but no role
for TRPC4 in mature adipocytes has previously been recognised. In
the above noted studies of Sukumar et al., no TRPC4 mRNA was
detected in mouse 3T3-L1 cells, which were employed as an
extensively characterised model of in vivo adipocytes. In contrast,
marked up-regulation of TRPC1 mRNA (15.5 times) and TRPC5 mRNA
(almost 40 times) was observed in differentiated mature 3LT3-L1
cells.
[0007] TRPC4 and TRPC5 proteins share about 65% sequence identity.
The proteins were initially identified by their sequence similarity
with the D. melanogaster TRP protein (40% sequence identity), the
founding member of the TRP superfamily of proteins. TRPC4 is
recognised to be expressed in a broad range of tissues, including
brain, peripheral sensory neurones, endothelium, and intestinal
smooth muscle. In smooth muscle cells, TRPC4 channels are gated by
muscarinic acetylcholine receptors and contribute more than 80% to
the muscarinic receptor-induced cation current. In these cells,
TRPC4 channels couple muscarinic receptors to smooth muscle cell
depolarization, voltage-activated Ca.sup.2+ influx, and
contraction, and thereby accelerate small intestinal motility.
TRPC5 has previously been suggested to be predominantly expressed
in the brain. Although channel properties are similar to those of
TRPC4, TRPC5 channels are cold-sensitive and can be activated by a
variety of additional stimuli, including a rise of cytosolic
Ca.sup.2+ [Beck et al. (2013) J. Biol. Chem. 288, 19471-19483].
[0008] The amino acid sequence for human TRPC 4 can be found in
UniProtKB database as entry Q9UBN4.
[0009] The amino acid sequence for human TRPC5 can be found in the
UniProtKB database as entry Q9UL62.
[0010] Based on the observation that antibody inhibition of
TRPC5-containing channels in isolated 3T3-L1 cells led to increased
secretion of adiponectin, Sukumar et al. went on to investigate the
role of TRPC5-containing channels in vivo. For this purpose
transgenic mice were employed capable of expressing a faulty TRPC5
protein (DNT5) which disrupts ion channel function under the
control of a doxycycline-regulated transgene. As predicted, DNT5
expression occurred in adipose tissue of doxycycline-treated double
transgenic mice. Such mice and controls were either fed for 6 weeks
chow diet or a high-fat diet which induced inflammatory indicators
but not obesity. The double transgene mice had as expected
increased circulating adiponectin which was linked to
anti-inflammatory effects but showed no difference in weight or
well-being compared to control mice. Hence the study did not
investigate excess weight gain or its consequences. Furthermore,
Kubota et al. (2007) Cell. Metabolism 6, 55-68 had previously
reported linkage of increased adiponectin to increased food intake
in mice which directly points away from consideration of inhibition
of TRPC5-comprising channels as a means of reducing excess weight
gain.
[0011] Moreover, the same studies of Sukumar et al. also showed no
effect of the DNT5 mutant protein in fat-fed mice on plasma insulin
consistent with the animals not being insulin-resistant. Hence,
nothing could be gleaned from such studies concerning value of
inhibiting TRPC5-containing channels in relation to insulin
resistance associated with Type II diabetes.
[0012] Hu et al. (2009) Mol. Endrocrinol. 23, 689-699 reported
increased TRPC1, TRPC5 and TRPC6 expression in adrenal medulla in
pigs with metabolic syndrome. However, no information is provided
on the functional implications of those observations for treatment
of metabolic syndrome per se. Increases in protein expression can
reflect a role of a mechanism in driving a condition or an attempt
by the system to overcome the condition. It is therefore not
possible to glean from those studies that inhibition of
TRPC5-containing channels would be beneficial, adverse or lacking
in effect in metabolic syndrome.
[0013] As noted above, anti-murine TRPC5 antibodies are known which
will block function of TRPC5-containing ion channels in
differentiated 3T3-L1 cells. Sukumar et al ibid also reported knock
down of TRPC1 and TRPC5 expression in such cells as model
adipocytes using siRNAs. The same disclosure additionally reports
identification of various dietary fatty acids, e.g. linolenic acid,
as inhibitors of function of human TRPC-5 homomeric or TRPC1/5
heteromeric channels as expressed in HEK293 cells. Screening using
HEK239 cells expressing TRPC5 has identified other natural products
as inhibitors of TRPC5 ion channel function, more particularly
galangin (a natural product from ginger), resveratrol (a red wine
component and vitamin C [Naylor et al. (2016) Brit. J. Pharmacol.
173, 562-574; Naylor et al. (2011) J. Biol. Chem. 286, 5078-5086].
However, such studies do not add to the pool of information on
therapeutic value of inhibiting TRPC channels.
[0014] Various small molecule inhibitors of TRPC5-containing
channels which target TRPC5 have also been identified, see for
example WO 2014/143799, WO 2016/023826, WO 2016/023825, WO
2016/023831, WO 2016/023830 and WO 2016/023832 which discloses such
inhibitors as having value as anxiolytic agents. Various agents,
including small molecules, have also been reported as inhibitors of
TRPC4 ion channel function, see for example WO2011/022638 which
teaches various such inhibitors, but only in the context of
treatment of neuropathic pain, including such pain associated with
diabetes. Miller et al. (2011) J. Biol. Chem. 286, 33436-33446
reports identification of a compound ML204 as a relatively
selective TRPC4/C5 antagonist which displays at least 20-fold
higher selectivity for TRPC4 over a collection of other ion
channels including TRPC6 and members of other TRP sub-families, but
only in the context of provision of a further research tool for
investigating functional significance of TRPC4/C5-containing
channels. Westlund et al. (2014) Neuroscience 262, 165-175 reports
that rats with a TRPC4 knock-out mutation or treated with ML204
have higher tolerance to visceral pain. For further information on
inhibitors of TRPC4 and/or TRPC5 reference may also be made to Bon
and Beech (2013) ibid. However, no teaching is provided which would
direct consideration of any such inhibitor in relation to weight
control or insulin resistance.
SUMMARY OF THE INVENTION
[0015] The present inventors have now found that
genetically-modified mice in which the TRPC4 protein or TRPC5
protein is absent, designated as TRPC4 knockout mice (C4.sup.KO)
and TRPC5 knockout mice (C5.sup.KO) respectively, have improved
weight control when on a high fat diet providing excess calorie
intake over an 8 week period compared to control wild-type
littermates on the same diet. The control mice gained excess total
fat and fat pad weights as expected. In contrast, the C4.sup.(KO)
and C5.sup.(KO) mice on the high fat diet were after the same
period strikingly similar to mice on chow diet, showing only normal
weight gain as the mice matured.C4.sup.KO and C5.sup.KO mice on
chow diet showed no significant difference in body weight or fat
pad weight to littermate controls. Evidence as presented herein
suggests that the anti-obesity effect of TRPC4 knockout or TRPC5
knockout may be explained by white adipocytes shifting to a
thermogenic ("energy-burning") phenotype, commonly referred to as
adipocyte beiging.
[0016] A new approach to combating obesity and other conditions
associated with obesity such as insulin resistance is thus proposed
relying on inhibition of TRPC4 and/or TRPC5-containing channels,
more particularly such channels as present in adipocytes. It is
envisaged that such channels may also include heteromeric channels
including TRPC1. The terms TRPC4, TRPC5 and TRPC1 as used herein
will be understood to include any native form of those proteins as
occurs in ion channels present in adipocytes, including adipocytes
of human white adipose tissue.
[0017] Accordingly in one aspect, the present invention provides an
inhibitor which directly targets a TRPC ion channel comprising
TRPC4 and/or TRPC5 as present in adipocytes for use in the
treatment or prophylaxis of a condition selected from obesity,
insulin resistance, metabolic syndrome, non-alcoholic fatty liver
disease (NAFLD) and non-alcoholic steatohepatitis (NASH).
[0018] In a further aspect there is provided cosmetic use of an
inhibitor which directly targets a TRPC ion channel comprising
TRPC4 and/or TRPC5 as present in adipocytes to reduce or inhibit
excess weight gain. Such use will be understood to encompass
non-therapeutic use of a suitable inhibitor e.g. where a suitable
inhibitor is supplied as a non-prescription over-the-counter aid to
weight reduction or prevention of excess weight gain.
[0019] The inhibitor may be any agent which acts directly on a TRPC
ion channel comprising TRPC4 and/or TRPC5 as present in adipocytes,
suitably the inhibitor is a small molecule inhibitor. The inventors
have found that the compound disclosed as Example 31 in WO
2014/143799 (also designated herein as "C31") is a potent TRPC4 and
TRPC5 inhibitor and is therefore suitable for any of the uses
disclosed herein.
[0020] The present invention also provides certain novel compounds.
The novel compounds are suitable for use in inhibiting TRPC4 and/or
TRPC5 ion channels, more particularly the novel compounds may be
for any of the uses described herein, particularly the novel
compounds are inhibitors which directly target a TRPC ion channel
comprising TRPC4 and/or TRPC5 as present in adipocytes for use in
the treatment or prophylaxis of a condition selected from obesity,
insulin resistance, metabolic syndrome, non-alcoholic fatty liver
disease (NAFLD) and non-alcoholic steatohepatitis (NASH). A
specific novel compound of the invention is a compound of the
formula (IXa), or a pharmaceutically acceptable salt thereof (also
designated herein as "DE2").
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Embodiments of the invention are further described
hereinafter with reference to the accompanying drawings, in
which:
[0022] FIG. 1 demonstrates that TRPC4 knockout mice and TRPC5
knockout mice demonstrated normal body weight on a chow diet. (A)
Body weight (mean.+-.SEM, n=6). (B) Subcutaneous white adipose
tissue (scWAT) fat pad weight (mean.+-.SEM, n=6). NS indicates not
statistically significantly different. C4WT, wildtype litter-mate
controls for TRPC4 knockout mice. C5WT, wildtype litter-mate
controls for TRPC5 knockout mice. C4KO, TRPC4 knockout mice. C5KO,
TRPC5 knockout mice.
[0023] FIG. 2 demonstrates that TRPC4 knockout mice and TRPC5
knockout mice were unable to gain excess total body weight on high
fat diet. Summary data for weights of mice during 8 weeks of diet
feeding (mean.+-.SEM, n=7-8). Diets were chow (Chow) or 60%
high-fat (HFD). C4WT, wildtype litter-mate controls for TRPC4
knockout mice. C5WT, wildtype litter-mate controls for TRPC5
knockout mice. C4KO, TRPC4 knockout mice. C5KO, TRPC5 knockout
mice. * indicates statistically significantly different.
[0024] FIG. 3 demonstrates that TRPC4 knockout mice and TRPC5
knockout mice were unable to gain excess fat pad weight on high fat
diet. Summary data for subcutaneous white adipose tissue (scWAT)
fat pad weights of mice during 8 weeks of diet feeding
(mean.+-.SEM, n=7-8). Diets were chow (Chow) or 60% high-fat (HFD).
C4WT, wildtype litter-mate controls for TRPC4 knockout mice. C5WT,
wildtype litter-mate controls for TRPC5 knockout mice. C4KO, TRPC4
knockout mice. C5KO, TRPC5 knockout mice. * indicates statistically
significantly different.
[0025] FIG. 4 demonstrates that TRPC4 knockout mice and TRPC5
knockout mice show protection against hyperglycaemia. Blood glucose
concentrations after bolus intraperitoneal (IP) injection of
glucose (glucose tolerance test, GTT). Mice were on chow or 60%
high fat diet (HFD) (mean.+-.SEM, n=7-8). C4WT, wildtype
litter-mate controls for TRPC4 knockout mice. C5WT, wildtype
litter-mate controls for TRPC5 knockout mice. C4KO, TRPC4 knockout
mice. C5KO, TRPC5 knockout mice. * indicates statistically
significantly different.
[0026] FIG. 5 demonstrates that TRPC4 knockout mice and TRPC5
knockout mice show protection against insulin-resistance. Blood
glucose concentrations after bolus intraperitoneal (IP) injection
of insulin (insulin tolerance test, ITT). Mice were either on chow
or 60% high fat diet (HFD) (mean.+-.SEM, n=7-8). C4WT, wildtype
litter-mate controls for TRPC4 knockout mice. C5WT, wildtype
litter-mate controls for TRPC5 knockout mice. C4KO, TRPC4 knockout
mice. C5KO, TRPC5 knockout mice. * indicates statistically
significantly different.
[0027] FIG. 6 demonstrates that TRPC4 knockout mice and TRPC5
knockout mice show protection against systemic inflammation. Serum
tumour necrosis factor alpha (TNF.alpha.) concentration measured by
ELISA. Mice were either on chow or 60% high fat diet (HFD)
(mean.+-.SEM, n=5). C4WT, wildtype litter-mate controls for TRPC4
knockout mice. C5WT, wildtype litter-mate controls for TRPC5
knockout mice. C4KO, TRPC4 knockout mice. C5.sup.KO, TRPC5 knockout
mice. * indicates statistically significantly different.
[0028] FIG. 7 demonstrates that TRPC4 knockout mice and TRPC5
knockout mice show protection against adipose tissue inflammation.
Relative mRNA analysis for the pro-inflammatory markers TNF.alpha.
and interleukin-6 (IL6) in fat pad. Mice were either on chow or 60%
high fat diet (HFD) (mean.+-.SEM, n=4). C4WT, wildtype litter-mate
controls for TRPC4 knockout mice. C5WT, wildtype litter-mate
controls for TRPC5 knockout mice. C4KO, TRPC4 knockout mice. C5KO,
TRPC5 knockout mice. * indicates statistically significantly
different.
[0029] FIG. 8 demonstrates that TRPC4 knockout mice and TRPC5
knockout mice show protection against ectopic fat in the liver
(steatosis). Total liver weight (upper bar chart, n=7-8) and
percentage liver fat determined by histological analysis (lower bar
chart, n=5). Mice were either on chow or 60% high fat diet (HFD).
Data are mean.+-.SEM. C4WT, wildtype litter-mate controls for TRPC4
knockout mice. C5WT, wildtype litter-mate controls for TRPC5
knockout mice. C4KO, TRPC4 knockout mice. C5KO, TRPC5 knockout
mice. * indicates statistically significantly different.
[0030] FIG. 9 demonstrates that TRPC4 knockout mice and TRPC5
knockout mice show no change in food intake or excretion. Food
intake, fecal excretion and urinary excretion were determined in
mice housed in metabolic cages (mean.+-.SEM, n=6). Mice were either
on chow or 60% high fat diet (HFD). C4WT, wildtype litter-mate
controls for TRPC4 knockout mice. C5WT, wildtype litter-mate
controls for TRPC5 knockout mice. C4KO, TRPC4 knockout mice. C5KO,
TRPC5 knockout mice. * indicates statistically significantly
different.
[0031] FIG. 10 demonstrates that TRPC4 knockout mice and TRPC5
knockout mice show increased expression of markers of
white-to-brown adipocyte phenotypic switch ("beiging"). Summary
data for western blot analysis of UCP1 and Cytochrome C in
subcutaneous white adipose tissue (mean.+-.SEM, n=4). C4WT,
wildtype litter-mate controls for TRPC4 knockout mice. C5WT,
wildtype litter-mate controls for TRPC5 knockout mice. C4KO, TRPC4
knockout mice. C5KO, TRPC5 knockout mice. * indicates statistically
significantly different.
[0032] FIG. 11 demonstrates that TRPC4 knockout mice and TRPC5
knockout mice show increased adipocyte thermogenesis. Oxygen
consumption data for adipocytes from subcutaneous white adipose
tissue of mice. Data are for routine respiration after addition of
sodium pyruvate, mitochondrial leak after addition of oligomycin,
maximum electron transport chain-mediated oxygen consumption after
sequential FCCP addition (ETS), and non-OXPHO respiration after
addition of rotenone and antimycin A (ROX). C4WT, wildtype
litter-mate controls for TRPC4 knockout mice. C5WT, wildtype
litter-mate controls for TRPC5 knockout mice. C4KO, TRPC4 knockout
mice. C5KO, TRPC5 knockout mice. Data are mean.+-.SEM (n=3-4). *
indicates statistically significantly different.
[0033] FIG. 12 demonstrates the inhibition of TRPC4 and TRPC5
channels by DE2 and C31. Representative intracellular Ca.sup.2+
measurements from HEK 293 cells stably expressing inducible human
TRPC4 (upper panel) or human TRPC5 (lower panel). Data are
mean.+-.SEM (3-4 replicates each) and representative of 4
independent experiments each. Channels were activated by the
TRPC4/TRPC5 channel agonist (-)-Englerin A (EA, 100 nM). The
vehicle control was DMSO. Cells were pre-incubated with (30 min)
and maintain in 10 .mu.M DE2 or 100 nM C31 where indicated.
[0034] FIG. 13 demonstrates that small-molecule inhibitors increase
adipocyte thermogenesis. Oxygen consumption data for adipocytes
from subcutaneous white adipose tissue of mice. Data are for
routine respiration after addition of sodium pyruvate,
mitochondrial leak after addition of oligomycin, maximum electron
transport chain-mediated oxygen consumption after sequential FCCP
addition (ETS), and non-OXPHO respiration after addition of
rotenone and antimycin A (ROX). Cells were pre-incubated with (24
hr) and maintain in 10 .mu.M DE2 or 100 nM C31 where indicated. The
vehicle control was DMSO.
[0035] FIG. 14 demonstrates that the small-molecule inhibitors C31
and DE2 increase expression of markers of white-to-brown adipocyte
phenotypic switch ("beiging"). Summary data for mRNA analysis of
UCP1 and Cytochrome C expression in adipocytes from subcutaneous
white adipose tissue (mean.+-.SEM, n=4-5). Cells were pre-incubated
with (24 hr) and maintain in 10 .mu.M DE2 or 100 nM C31 where
indicated. The vehicle control was DMSO. C4WT, wildtype litter-mate
controls for TRPC4 knockout mice. C5WT, wildtype litter-mate
controls for TRPC5 knockout mice. C4KO, TRPC4 knockout mice. C5KO,
TRPC5 knockout mice. * indicates statistically significantly
different.
[0036] FIG. 15 demonstrates that bi-daily oral dosing (BID) with
C31 (designated "Test item" in the figure) significantly reduced
plasma insulin after an oral glucose tolerance test (OGTT) in
HFD-fed wildtype adult male mice (DIN). Data for comparator
established drugs are also shown. N=10 per group.
[0037] FIG. 16 demonstrates that bi-daily oral dosing (BID) with
C31 (designated "Test item" in the figure) significantly reduced
blood glucose after an insulin tolerance test in HFD-fed wildtype
adult male mice (DIN). Data for comparator established drugs are
also shown. N=10 per group.
[0038] FIG. 17 demonstrates that in vivo expression of a dominant
negative TRPC5 ion pore mutant from a transgene (DNTS)
significantly reduced body weight gain in hypercholesterolaemic
(ApoE-/-) adult male mice fed western-style diet. The mutant enters
native TRPC1/4/5 channels to inhibit ion permeation. N=16 DNTS mice
and N=19 control littermates (which lacked DNTS).
[0039] FIG. 18 demonstrates that in vivo expression of a dominant
negative TRPC5 ion pore mutant from a transgene (DNTS)
significantly reduced liver steatosis in hypercholesterolaemic
(ApoE-/-) adult male mice fed western-style diet. N=14 DNTS and
N=11 control littermates (which lacked DNTS).
DETAILED DESCRIPTION
[0040] The present invention relates to an inhibitor which directly
targets a transient receptor potential canonical (TRPC) ion channel
comprising TRPC4 and/or TRPC5 as present in adipocytes for use in
the treatment or prophylaxis of a condition selected from: obesity,
insulin resistance, metabolic syndrome, non-alcoholic fatty liver
disease (NAFLD) and non-alcoholic steatohepatitis (NASH). It is
envisaged that the use of such an inhibitor may extend to the
treatment or prophylaxis of insulin resistance associated with Type
II diabetes or development of Type II diabetes (pre-diabetes).
[0041] The TRPC inhibitors described herein are capable of
inhibiting a TRPC ion channel, as present in adipocytes, comprising
TRCP4 or TRPC5. Optionally, such an inhibitor will directly target
a TRPC ion channel, as present in adipocytes, comprising TRPC4.
Optionally, such an inhibitor will directly target a TRPC ion
channel, as present in adipocytes, comprising TRPC5. Also, it will
be understood that ion channels comprising TRPC4, TRPC5 or both
TRPC4 and TRPC5 may also include other channel proteins, in
particular other TRPC proteins. Accordingly, it will be appreciated
that the TRPC inhibitor may also be capable of inhibiting an ion
channel wherein the TRPC ion channel further comprises another TRPC
protein, for example TRPC1. Optionally, such an inhibitor will
directly target a TRPC ion channel, as present in adipocytes,
wherein the channel comprises TRPC4, TRPC5 and TRPC1.
[0042] A TRPC channel which is directly targeted by the inhibitors
described herein may be a homomeric channel or a heteromeric
channel. When the channel is a homomeric channel it may comprise
four TRPC4 proteins or four TRPC5 proteins. When the ion channel is
a heteromeric channel, the channel comprises (i) at least one TRPC4
and three other TRP proteins, at least one of which is not TRPC4;
or (ii) at least one TRPC5 and three other TRP proteins, at least
one of which is not TRPC5. In embodiments the ion channel may
optionally comprise TRPC4 and TRPC5. Alternatively, the channel may
optionally comprise TRPC4 and TRPC1 or TRPC5 and TRPC1. Optionally
the channel may comprise TRPC4, TRPC5 and TRPC1. Accordingly, the
heteromeric channel is intended to encompass any TRPC ion channel
provided that the channel includes at least one TRPC 4 or one TRPC
5 protein as a component of the ion channel. Representative
examples of heteromeric channels, include, but are not limited to,
for example the ion channel may comprise one TRPC4 and three TRPC1;
two TRPC4 and two TRPC1; three TRPC4 and one TRPC1; one TRPC5 and
three TRPC1; two TRPC5 and two TRPC1; three TRPC5 and one TRPC1;
one TRPC4 and three TRPC5; two TRPC4 and two TRPC5; three TRPC4 and
one TRPC5; one TRPC4 and three other TRP proteins, two TRPC 4 and
two other TRP proteins; three TRPC4 and one other TRP protein; two
TRPC5, one TRPC1 and one other TRP protein; or one TRPC4, one
TRPC5, one TRPC1 and one other TRP protein etc.
[0043] It will be appreciated that an inhibitor operated in
accordance with the invention may inhibit the function or
expression of TRPC4 and/or TRPC5. Preferably, an inhibitor operated
in accordance with the invention may inhibit the function or
expression of TRPC4 and/or TRPC5. TRPC4 is a known protein encoded
by the TRPC4 gene. The amino acid sequence for human TRPC4 may be
found in the UniProtKB database as entry Q9UBN4.TRPC5 is a known
protein encoded by the TRPC5 gene. The amino acid sequence for
human TRPC5 may be found in the UniProtKB database as entry Q9UL62.
Reference to the ion channel "as present in adipocytes" refers to
the form of the ion channel present in adipocytes and includes
natural variants of the channels, including, for example splice
variants and/or naturally occurring variants of the TRPC proteins
forming the ion channel. It is to be understood that the ion
channels comprising TRPC4 and/or TRPC5 are formed in adipocyte
cells. However, such channels may also be present in other tissues.
The inhibitor may therefore act on channels present in adipocytes
or other tissues to provide the therapeutic and cosmetic effects
described herein, for example the treatment of obesity.
[0044] The "TRPC inhibitor" refers to any agent which acts directly
on a TRPC ion channel, as present in adipocytes, comprising TRPC4
and/or TRPC5 to inhibit the function of the ion channel. A TRPC
inhibitor in accordance with the invention may inhibit the function
or expression of TRPC4 and/or TRPC5. Inhibition of the ion channel
by the TRPC inhibitor may include, for example, inhibiting or
preventing the association of the TRP proteins which form the TRPC
ion channel comprising TRPC4 and/or TRPC5. The inhibitor may act to
inhibit the ion channel function. For example, the inhibitor may
bind to the TRPC ion channel such that it blocks completely or
reduces transport of cations (Ca.sup.2+ and Na.sup.+) into or out
of the ion channel. Alternatively it might act allosterically to
modulate the gating and opening probability of the channel. The
inhibitor may be any agent which acts directly on a TRPC ion
channel comprising TRPC4 and/or TRPC5 as present in adipocytes,
suitably the inhibitor is a small molecule inhibitor. Particularly
advantageously, the inventors have found that the compound
disclosed as Example 31 in WO 2014/143799 (also designated herein
"C31") is a potent TRPC4 and TRPC5 inhibitor and is therefore
suitable for any of the uses disclosed herein.
[0045] The present invention has shown that when expression of
TRPC4 or TRPC5 is inhibited, upon exposure to calorific excess in
the form of a high fat diet: excess weight gain may be reduced,
markers of inflammation in adipose tissue may be reduced;
thermogenesis and mitochondrial respirations in adipose tissue may
be increased; a protective effect against insulin resistance and
glucose intolerance may be seen; weight gain in the liver may be
decrease and ectopic fat in the liver may be reduced.
[0046] Advantageously, the inventors have discovered that mutations
in the genes encoding TRPC4 and TRPC5 result in protection against
hyperglycaemia, insulin-resistance, systemic and adipose tissue
inflammation and steatosis associated with excess calorie
intake.
[0047] Accordingly, the invention provides a TRPC inhibitor,
wherein the inhibitor is a siRNA or antisense oligonucleotide.
Where the inhibitor is a siRNA or antisense oligonucleotide, the
inhibitor may inhibit expression of TRPC4 protein, TRPC5 protein or
both.
[0048] Accordingly, the present invention provides an inhibitor
which may be used to eliminate or reduce expression of TRPC4 and/or
TRPC5 in adipocytes, wherein adipocytes are transformed with the
polynucleotide in an antisense orientation such that there is
reduction in, or elimination of, expression or level of native
TRPC4 or TRPC5 polypeptide in such cells compared to untransformed
cells. In accordance with the present invention, antisense
polynucleotides have polynucleotide sequences which hybridize under
stringent conditions to the nucleotide sequences encoding TRPC4 or
TRPC5. Such sequences may be useful in down-regulating expression
of TRPC4 and/or TRPC5. Whilst in certain embodiments, TRPC4 or
TRPC5 may be down-regulated (suppressed) in adipocytes cells, it is
envisaged that it may in certain circumstances be desirable to
down-regulate both TRPC4 and TRPC5.
[0049] Accordingly, it will be understood that a suitable inhibitor
may be capable of specifically reducing or eliminating expression
of TRPC4 and/or TRPC5 in a sequence-specific manner. Such
expression control may, for example, suitably be exerted at either
the transcript or protein level. Accordingly, a TRPC4 or TRPC5
inhibitor may suitably be a small interfering RNA (siRNA) or
antisense oligonucleotide which specifically targets, and inhibits
expression of TRPC4 or TRPC5 respectively. For example such an
inhibitor may operate to reduce or eliminate TRPC4 or TRPC5 mRNA
transcript or protein accumulation in adipocytes. Conveniently,
where it is desired to reduce or eliminate the expression of both
TRPC4 and TRPC5, more than one inhibitor, each specifically
targeting either TRPC4 or TRPC5 may be deployed. In preferred
aspects, the inhibition of, and/or reduction in TRPC4 and/or TRPC5
expression may take place specifically in adipocytes, for example
by using an siRNA construct the expression of which is driven by an
adipocyte-specific promoter e.g. the PdgfR.alpha. or Adiponectin
promoter.
[0050] The invention provides a TRPC inhibitor as defined herein,
wherein the inhibitor is an antibody or an antigen-binding fragment
thereof or a nanobody, affimer or adhiron. The antibody or
antigen-binding fragment thereof may interact directly with the
TRPC4 or TRPC5 proteins to prevent or inhibit the formation of the
TRPC ion channel and/or which acts to directly inhibit the function
of the ion channel for example by binding to the ion channel such
that the transport of cations into or out of the channel is
inhibited or prevented. In preferred embodiments, the inhibitor may
be a monoclonal antibody or the antigen-binding fragment of an
antibody is a fragment of a monoclonal antibody. Monoclonal
antibody (mAb) as used herein refers to a highly-specific antibody
directed against a single antigenic site, obtained from a
population of substantially homogeneous antibodies, i.e., the
individual antibodies comprising the population are identical
except for possible naturally occurring mutations that may be
present in minor amounts. Such monoclonal antibodies may be
synthesised by methods which are well known in the art, for example
by hybridoma culture, or by recombinant DNA methods. Such a
monoclonal antibody may be a chimeric antibody (immunoglobulins) in
which a portion of the heavy and/or light chain is identical with
or homologous to corresponding sequences in antibodies derived from
a particular species or belonging to a particular antibody class or
subclass, while the remainder of the chain(s) is identical with or
homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass,
as well as fragments of such antibodies, so long as they exhibit
the desired biological activity. Preferably, such an antibody binds
TRPC4 and/or TRPC5. Preferably such an antibody is humanised, i.e.
an antibody from a non-human species whereby the protein sequences
have been modified to increase their similarity to antibody
variants produced naturally in humans. An antibody for use in
accordance with the invention may be, for example, an antibody that
targets the E3 loop of a relevant TRPC ion channel forming protein
to suppress channel function as discussed in Beech (2013) Circul.
J. 77, 570-579.
[0051] Such an antibody may also usefully be provided as a
conjugate, i.e. a heterogeneous molecule formed by the covalent
attachment of one or more antibody fragment(s) to one or more
polymer molecule(s).
[0052] In some embodiments the TRPC inhibitor is a small molecule
of 2000 daltons or less. Suitable small molecule inhibitors for the
uses and methods of the invention include, but are not limited to
any of the small molecules set out herein.
[0053] Surprisingly the inventors have discovered that mutations in
the genes encoding TRPC4 and TRPC5 result in an inability to gain
excess total body weight and fat pad weight on high fat diet.
Accordingly, the invention also provides a TRPC inhibitor, wherein
the TRPC inhibitor is for use in the treatment or prophylaxis of
obesity. Optionally, the TRPC inhibitor may be used in treatment or
prevention of abdominal obesity. Typically, the TRPC inhibitor may
be used in the treatment of adiposity. Optionally, TRPC inhibitor
may be used in preventing the accumulation of visceral fat or
reducing the amount of visceral fat in a subject treated with the
inhibitor. Optionally, the TRPC inhibitor may be used in preventing
the accumulation of subcutaneous fat or reducing the amount of
subcutaneous fat in a subject treated with the inhibitor.
[0054] The TRPC inhibitors described herein may also be used in
preventing the accumulation of ectopic fat or reducing the amount
of ectopic fat in a subject. In particular, the TRPC inhibitor
described herein may be used in preventing the accumulation of
ectopic fat or reducing the amount of ectopic fat in the liver of a
subject.
[0055] Optionally, the TRPC inhibitor may be used in the treatment
or prophylaxis of insulin resistance in a subject. Commonly, the
TRPC inhibitor may be used in the treatment or prophylaxis of
insulin resistance in an obese subject. Typically, the insulin
resistance is associated with Type II diabetes or prediabetes.
[0056] Accordingly, said inhibitor may be for use in the treatment
or prophylaxis of metabolic syndrome. Where the inhibitor is for
use for use in the treatment or prophylaxis of metabolic syndrome,
the treatment of metabolic syndrome may comprise reducing abdominal
obesity and/or reducing fasting blood glucose concentration. In
certain embodiments the metabolic syndrome may be a syndrome that
is not associated with the reduction of glucose levels.
[0057] The effect inhibitor may also be beneficial in the treatment
of conditions associated with the conditions controlled by use of
the inhibitor. For example control of obesity, ectopic fat,
insulin-resistance and/or inflammation resulting from the use of
the inhibitor may also provide beneficial effects in diseases and
conditions associated with those conditions. Accordingly the
inhibitor may be for use in the treatment or prevention of a
disease or medical condition associated with obesity, excess
ectopic fat, insulin-resistance and/or inflammatory cytokines
arising from adipocytes. In embodiments the inhibitor is for use in
the treatment or prevention of a condition selected from: coronary
artery disease, cerebral artery disease, peripheral vascular
disease, heart failure, dyslipidaemia, diabetic retinopathy,
diabetic nephropathy diabetic neuropathy and cancer.
[0058] Additionally, it has been discovered that mutations in the
genes encoding TRPC4 and TRPC5 result in greater expression of the
mitochondrial proteins UCP1 and Cytochrome C in white adipose
tissue indicating an increase in thermogenesis and that inhibition
of the proteins encoded by these genes has a similar effect.
[0059] Accordingly, the invention also provides a TRPC inhibitor as
defined herein, wherein the inhibitor reduces expression of
TNF.alpha. or IL6 in adipose tissue in the subject treated with
said inhibitor. Optionally, the inhibitor may reduce the expression
of TNF.alpha. and IL6 in adipose tissue in the subject treated with
said inhibitor.
[0060] Accordingly, the invention also provides a TRPC inhibitor,
wherein the inhibitor increases mitochondrial respiration in
adipose tissue in the subject treated with said inhibitor.
[0061] Accordingly, the invention also provides a TRPC inhibitor,
wherein the inhibitor increases thermogenesis in adipose tissue in
the subject treated with said inhibitor.
[0062] The inventors have previously found that elevated levels of
TRPC1 and TRPC5 are negatively correlated with levels of
adiponectin and in particular that levels of TRPC1 and TRPC5 are
elevated in adipose tissue of subjects which display metabolic
syndrome or major cardiovascular diseases. Consequently,
measurement of TRPC1 levels may be of use in screening for subjects
for which the TRPC inhibitor may be particularly effective.
Accordingly, in any aspect of the invention, optionally, a TRPC
inhibitor as defined herein may usefully be administered to a
subject with elevated levels of TRPC1 in adipose tissue relative to
a control level in order to treating or prevent a condition
selected from; obesity, insulin resistance, metabolic syndrome,
non-alcoholic fatty liver disease (NAFLD) and non-alcoholic
steatohepatitis (NASH). It is envisaged that such use may also
extend to the treatment or prophylaxis of insulin resistance
associated with Type II diabetes or development of Type II diabetes
(pre-diabetes).
[0063] The invention therefore further provides a TRPC inhibitor as
herein before defined, wherein the inhibitor is administered to a
subject predetermined to have an elevated level in adipose tissue
relative to control of one or more screening targets selected from
TRPC1 mRNA and/or protein, TRPC4 mRNA and/or protein and TRPC5 mRNA
and/or protein.
[0064] The invention further provides a method of identifying a
subject having an elevated level in adipose tissue relative to
control of one or more screening targets selected from TRPC1 mRNA
and/or protein, TRPC4 mRNA and/or protein and TRPC5 mRNA and/or
protein. which comprises: [0065] a. determining the level of one or
more screening targets selected from TRPC1 protein and/or mRNA,
TRPC4 mRNA and/or protein and or TRPC5 mRNA and/or protein in a
sample of adipose tissue from a subject; and [0066] b. selecting
said subject for administration of the inhibitor if the level of
said screening target exceeds a control level.
[0067] In certain aspects the method may optionally comprise
determining the level of TRPC4 protein or mRNA in a sample of
adipose tissue from a subject; and/or optionally determining the
level of TRPC5 protein or mRNA in a sample of adipose tissue from a
subject; and administering the inhibitor to the subject if the
level of TRPC1 and TRPC4 exceed control levels, or if the level of
TRPC1 and TRPC5 exceed control levels, or the level of TRPC1, TRPC4
and TRPC5 exceed control levels.
[0068] Specifically determining the level of TRPC1, TRPC4 and/or
TRPC5 protein or mRNA in a sample of adipose tissue from a subject
may conveniently be achieved by methods and devices which are well
known in the art. For example by qPCR, microarray, Northern
Blotting or Next generation sequencing where the target is mRNA, or
e.g. Western Blotting, ELISA or mass spectrometry where the target
is protein. Suitably the method may comprise the use of specific
binding partners which selectively bind to a target molecule
indicative of the presence or expression of a polynucleotide or
polypeptide as hereinbefore defined. Target molecules may suitably
be RNA molecules, DNA molecules, cDNA molecules or alternatively
proteins or polypeptides encoded by a polynucleotide as
hereinbefore defined. A variety of suitable array or chip-based or
liquid-based capture technologies are well known in the art and
suitable for the purpose.
[0069] Suitably the method may comprise using at least one binding
partner selected from the group consisting of: complementary
nucleic acids; aptamers; antibodies or antibody fragments. Suitable
classes of binding partners for any given polynucleotide or protein
will be apparent to the skilled person.
[0070] The method will be adapted to detect and quantify the levels
of said polynucleotides or proteins present in the adipose tissue
sample. This may be with reference to a positive control or
alternatively with reference to an internal standard.
[0071] Preferably, the adipose tissue sample is an extract or
lysate from an adipose cell or tissue. The adipose tissue sample
may suitably be homogenized, processed, buffered and/or purified
prior to quantification of the levels of TRPC1. Suitably the levels
of the target molecules in the biological sample may be detected by
direct assessment of binding between the target molecules and
binding partners.
[0072] Advantageously, the present invention provides a compound as
defined herein for use in therapy. The present invention further
provides that any of the compounds defined herein may be used as a
TRPC inhibitor for use in the treatment or prophylaxis of a
condition selected from: obesity, insulin resistance, metabolic
syndrome, non-alcoholic fatty liver disease (NAFLD) and
non-alcoholic steatohepatitis (NASH). It is envisaged that the use
of such an inhibitor may also extend to the treatment or
prophylaxis of insulin resistance associated with Type II diabetes
or development of Type II diabetes (pre-diabetes).
[0073] The present invention also provides for the cosmetic use of
a TRPC inhibitor as defined herein, which directly targets TRPC ion
channels comprising TRPC4 and/or TRPC5 as present in adipocytes to
reduce or inhibit excess weight gain. Such use will be understood
to encompass non-therapeutic use of a suitable inhibitor e.g. where
a suitable inhibitor is supplied as a non-prescription
over-the-counter aid to weight reduction or prevention of excess
weight gain.
[0074] Such a TRPC inhibitor may therefore appropriately be
formulated for cosmetic use, for example in tablet form. Such
cosmetic compositions may contain the formulation in sufficient
amounts to inhibit expression of TRPC4 and/or TRPC5 in adipocytes
and a pharmaceutically acceptable carrier or excipient.
Particularly, a cosmetic composition containing the formulation can
be used to reduce or inhibit excess weight gain.
[0075] In one embodiment the inhibitor is a 2-aminoquinoline of the
formula (I), or a pharmaceutically acceptable salt thereof:
##STR00001##
wherein R.sup.1 and R.sup.2 are independently H or C.sub.1-4 alkyl,
or R.sup.1 and R.sup.2 together with the nitrogen to which they are
attached form a 4 to 7 membered heterocyclyl, for example
pyrrolidinyl, piperidinyl, piperazine, or homopiperidinyl; R.sup.3
is C.sub.1-4 alkyl; and R.sup.4 is H, halo or C.sub.1-4 alkyl.
[0076] In this embodiment a particular compound of the Formula (I)
is ML204:
##STR00002##
[0077] In another embodiment the inhibitor is a
3,5-bis(trifluoromethyl)pyrazole derivative, for example Pyr2
(BTP2, YM-58483):
##STR00003##
[0078] In another embodiment the inhibitor is a steroid, for
example pregnenolone or pregnanolone.
[0079] In another embodiment the inhibitor is a piperazine or
piperidine derivative, for example BD1063, BD1047 or 4-IBP:
##STR00004##
[0080] In another embodiment the inhibitor is M084:
##STR00005##
[0081] In another embodiment the inhibitor is an
N-phenylanthranilic acid derivative, for example flufenamic acid,
mefenamic acid, niflumic acid, diclofenac or
N-(p-amylcinnamoyl)anthranilic acid.
[0082] In an embodiment the inhibitor is a compound disclosed in WO
2014/143799. In this embodiment the inhibitor may be any one of the
compound disclosed in the Examples of WO 2014/143799, which are
described as TRPC5 inhibitors therein, or a pharmaceutically
acceptable salt thereof. The inhibitor may be any of Compounds 1 to
640 described in Table A of WO 2014/143799, or a pharmaceutically
acceptable salt thereof.
[0083] In embodiments the inhibitor is a compound of the Formula
(II), or a pharmaceutically acceptable salt thereof:
##STR00006##
[0084] wherein: [0085] R.sup.1 is C.sub.1-6 alkyl, C.sub.2-6
alkenyl or C.sub.2-6 alkynyl, each of which is optionally
substituted with 1-4 R.sup.5; [0086] R.sup.2 is C.sub.1-6 alkyl,
C.sub.1-6 heteroalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
C.sub.1-6 haloalkyl, halo, C.sub.1-6 haloalkoxy, hydroxyl,
C.sub.1-6 alkoxy, C.sub.3-7 cycloalkyloxy, C.sub.6-10 aryl,
C.sub.6-10 aryloxy, C.sub.7-16 arylalkoxy, amino, C.sub.1-6
akylamino, C.sub.2-12 dialkylamino, --S(O).sub.xR' (wherein x is 0,
1 or 2 and each R' is independently H or C.sub.1-6 alkyl),
heterocycloalkyl, heteroaryl, heteroaryloxy, sulfonamidyl, amido,
urea, sulfonylurea, acyl, nitro, cyano, [0087] wherein each
C.sub.1-6 alkyl, C.sub.1-6 heteroalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl, C.sub.1-6haloalkoxy,
hydroxyl, C.sub.1-6 alkoxy, C.sub.3-7 cycloalkyloxy, C.sub.6-10
aryl, C.sub.6-10 aryloxy, C.sub.7-16 arylalkoxy, amino, C.sub.1-6
akylamino, C.sub.2-12 dialkylamino, --S(O).sub.xR',
heterocycloalkyl, heteroaryl, heteroaryloxy, sulfonamidyl, amido,
urea, sulfonylurea, acyl, is optionally substituted with 1-3
R.sup.6; [0088] R.sup.3 is C.sub.1-6 alkyl, C.sub.1-6 heteroalkyl,
C.sub.3-7 cycloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
C.sub.2-6 hydroxyalkyl, or C.sub.1-6 alkoxy, each of which is
optionally substituted with 1-4 R.sup.7; [0089] R.sup.4 is
C.sub.1-6 alkyl, C.sub.1-6 heteroalkyl, C.sub.2-6 alkenyl or
C.sub.2-6 alkynyl, each of which is optionally substituted with 1-4
R.sup.8; [0090] R.sup.5, R.sup.6, R.sup.7, and R.sup.8 are each
independently hydrogen, C.sub.1-6 alkyl, C.sub.1-6 heteroalkyl,
halo, C.sub.1-6 haloalkyl, C.sub.1-6 haloalkoxy, hydroxyl,
C.sub.1-6 alkoxy, amino, C.sub.1-6 alkylamino, C.sub.2-12
dialkylamino, cyano, nitro, amido, C.sub.1-6 alkylamido, C.sub.2-12
dialkylamido, --S(O).sub.xR' (wherein x is 0, 1 or 2), --C(O)OR',
--C(O)R', C.sub.3-7 cycloalkyl, C.sub.6-10 aryl, heterocycloalkyl,
or heteroaryl, wherein each R' is independently H or C.sub.1-6
alkyl, [0091] wherein each of C.sub.1-6 alkyl, C.sub.1-6
heteroalkyl, C.sub.1-6 haloalkyl, C.sub.1-6 haloalkoxy, hydroxyl,
C.sub.1-6 alkoxy, amino, C.sub.1-6 alkylamino, C.sub.2-12
dialkylamino, amido, C.sub.1-6 alkylamido, C.sub.2-12 dialkylamido,
--C(O)O--C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, C.sub.6-10 aryl,
heterocycloalkyl, or heteroaryl is optionally substituted with 1-3
R.sup.9; and [0092] each R.sup.9 is independently C.sub.1-6 alkyl,
C.sub.1-6 heteroalkyl, C.sub.1-6 haloalkyl, C.sub.1-6 haloalkoxy,
heterocycloalkyl, C.sub.6-10 aryl, heteroaryl, C.sub.4-10
cycloalkylalkyl, heterocycloalkyl-C.sub.1-6 alkyl, C.sub.7-16
arylalkyl, heteroaryl-C.sub.1-6 alkyl, halo, hydroxyl, C.sub.1-6
alkoxy, C.sub.6-10 aryloxy, C.sub.7-16 arylalkoxy, C.sub.2-8
alkoxyalkoxy, amino, C.sub.1-6 akylamino, C.sub.2-12 dialkylamino,
C.sub.1-6 aryl-amino-C.sub.1-6 alkyl, C.sub.1-6
alkyl-amino-C.sub.2-12 dialkyl, --S(O).sub.xR' (wherein x is 0, 1
or 2), sulfonamidyl, amido, urea, sulfonylurea, acyl,
--C(O)--C.sub.1-6 alkyl, --C(O)--C.sub.6-10 aryl,
--NHC(O)--C.sub.1-4 alkyl, --NHC(O)--C.sub.6-10 aryl, --C(O)NR'R',
--C(O)NH--C.sub.6-10 aryl, --C(O)OR', --OC(O)R', acyl, nitro, or
cyano.
[0093] In embodiments R.sup.1 is C.sub.1-6 alkyl optionally
substituted by R.sup.5.
[0094] In embodiments R.sup.2 is selected from C.sub.1-6 alkyl,
C.sub.1-6 alkoxy, amino, C.sub.1-6 alkylamino, di-C.sub.1-6
alkylamino, C.sub.1-6 alkyl-S(O).sub.2--, C.sub.6-10 aryl,
C.sub.6-10 aryloxy, 5-6 membered heteroaryloxy, wherein any aryl or
heteroaryl is optionally substituted by halo, C.sub.1-4 alkyl,
C.sub.1-4 haloalkyl, C.sub.1-4 alkoxy or C.sub.1-4 haloalkoxy.
[0095] In embodiments R.sup.3 is selected from C.sub.1-6 alkyl,
hydroxyC.sub.2-6 alkyl or C.sub.1-6 alkoxy.
[0096] In embodiments R.sup.4 is C.sub.1-6 alkyl.
[0097] In embodiments R.sup.5 is C.sub.3-6 cycloalkyl, 4-7 membered
heterocyclyl, C.sub.6-10 aryl, C.sub.6-10 aryloxy, 5-6 membered
heteroaryloxy, wherein any cycloalkyl, heterocyclyl, aryl or
heteroaryl is optionally substituted by halo, C.sub.1-4 alkyl,
C.sub.1-4 haloalkyl, C.sub.1-4 alkoxy or C.sub.1-4 haloalkoxy.
[0098] The compound of formula (II) may be a compound of the
formula (III), or a pharmaceutically acceptable salt thereof:
##STR00007##
wherein [0099] R.sup.1A is phenyl or a 5 or 6 membered heteroaryl,
which aryl or heteroaryl is optionally substituted by 1 or 2
substituents independently selected from halo, C.sub.1-6 alkyl,
C.sub.1-6haloalkyl and C.sub.1-6 haloalkoxy; [0100] R.sup.2 is
C.sub.1-6 alkoxy, or C.sub.6-10 aryloxy substituted by 1-3 R.sup.6;
[0101] R.sup.3 is hydroxy-C.sub.1-6 alkyl or C.sub.1-6 heteroalkyl;
[0102] R.sup.4 is C.sub.1-6 alkyl; [0103] each R.sup.6 is
independently selected from halo, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl or C.sub.1-6 haloalkoxy; and [0104] m is 1, 2 or 3.
[0105] It may be that R.sup.1A is selected from phenyl, thiazolyl,
oxazolyl and pyridyl, each of which is optionally substituted by 1
or 2 substituents independently selected from halo, C.sub.1-6 alkyl
and C.sub.1-6 haloalkyl. Preferably, R.sup.1A is phenyl optionally
substituted by 1 or 2 substituents independently selected from
halo, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C.sub.1-4 haloalkyl and
C.sub.1-4 haloalkoxy. Preferably m is 1.
[0106] It may be that the compound of formula (II) is a compound
selected from:
##STR00008## ##STR00009##
or a pharmaceutically acceptable salt thereof.
[0107] In a specific embodiment the compound of formula (II) is
##STR00010##
or pharmaceutically acceptable salt thereof.
[0108] In an embodiment the inhibitor is:
##STR00011##
or pharmaceutically acceptable salt thereof.
[0109] Compound 31 disclosed in WO 2014/143799 is a potent
inhibitor of TRPC4 and TRPC5. Accordingly a preferred inhibitor is
C31, or a pharmaceutically acceptable salt thereof:
##STR00012##
[0110] In another embodiment the inhibitor is a compound described
in WO 2016/023826, WO 2016/023825, WO 2016/023831, WO 2016/023830
or WO 2016/023832.
[0111] The inhibitor may be a compound of the formula (IV) or a
pharmaceutically acceptable salt thereof:
##STR00013##
wherein
[0112] R.sup.1 is C.sub.2-10 hydroxyalkyl, optionally substituted
with 1-3 R.sup.5;
[0113] R.sup.2 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.3-10 cycloalkyl, C.sub.1-6 hydroxyalkyl, or
C.sub.1-6 alkoxy;
[0114] R.sup.3 is C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-6 acyl, C.sub.3-10 cycloalkyl, C.sub.1-6 alkoxy,
C.sub.4-10 cycloalkyloxy, halo, C.sub.1-6 haloalkyl, C.sub.1-6
haloalkoxy, hydroxyl, C.sub.1-6 hydroxyalkyl, C.sub.1-6 alkylthio,
thionyl, sulfonyl, sulfonamidyl, C.sub.6-12 aryl, 5-14-membered
heteroaryl, C.sub.6-12 aryl-C.sub.1-6 alkyl, 5-14-membered
heteroaryl-C.sub.1-6 alkyl, C.sub.6-12 aryloxy, --O--C.sub.6-12
aryl-C.sub.1-6 alkyl, --O--C.sub.1-6 alkyl-C.sub.6-12 aryl,
--C.sub.6-12 aryl-C.sub.1-6 alkyl-OR.sup.5, 5-14-membered
heteroaryloxy, 3-18-membered heterocycloalkyl, amino, C.sub.1-6
alkylamino, C.sub.2-12 dialkylamino, --C(O)NR'R', --NR'C(O)R',
urea, sulfonylurea, nitro, or cyano, wherein each R' is
independently H or C.sub.1-6 alkyl and wherein R.sup.3 is
optionally substituted with 1-5 R.sup.5;
[0115] R.sup.4 is C.sub.1-6 alkyl, C.sub.1-6 acyl, C.sub.3-6
cycloalkyl, C.sub.1-6 alkoxy, C.sub.4-10 cycloalkyloxy, halo,
C.sub.1-6 haloalkyl, C.sub.1-6 haloalkoxy, hydroxyl, C.sub.1-6
hydroxyalkyl, C.sub.1-6 alkylthio, thionyl, sulfonyl, sulfonamidyl,
C.sub.6-12 aryl, 5-14-membered heteroaryl, C.sub.6-12
aryl-C.sub.1-6 alkyl, 5-14-membered heteroaryl-C.sub.1-6 alkyl,
C.sub.6-12 aryloxy, --O--C.sub.6-12 aryl-C.sub.1-6 alkyl,
--O--C.sub.1-6 alkyl-C.sub.6-12 aryl, --C.sub.6-12-aryl-C.sub.1-6
alkyl-OR', 5-14-membered heteroaryloxy, 3-18-membered
heterocycloalkyl, amino, C.sub.1-6 alkylamino, C.sub.2-12
dialkylamino, --C(O)NR'R', --NR'C(O)R', urea, sulfonylurea, nitro,
or cyano, wherein each R' is independently H or C.sub.1-6 alkyl and
wherein R.sup.4 is optionally substituted with 1-5 R.sup.5;
[0116] each R.sup.5 is independently H, C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 acyl, C.sub.1-6 alkoxy,
C.sub.4-10 cycloalkyloxy, halo, C.sub.1-6 haloalkyl, C.sub.1-6
haloalkoxy, hydroxyl, C.sub.1-6 hydroxyalkyl, amino, C.sub.6-12
aryl, 5-14-membered heteroaryl, C.sub.6-12 aryl-C.sub.1-6 alkyl,
C.sub.6-12 aryloxy, --O--C.sub.6-12 aryl-C.sub.1-6 alkyl,
--O--C.sub.1-6 alkyl-C.sub.6-12 aryl, --C.sub.6-12 aryl-C.sub.1-6
alkyl-OR.sup.6, 5-14-membered heteroaryloxy, each of which is
optionally substituted with 1-5 R.sup.6; and
[0117] each R.sup.6 is independently H, C.sub.1-6 alkyl, C.sub.1-6
alkoxy, C.sub.1-6 cycloalkyloxy, halo, C.sub.1-6 haloalkyl,
C.sub.1-6 haloalkoxy, hydroxyl, C.sub.1-6 hydroxyalkyl, amino,
C.sub.1-6 alkylamino, C.sub.2-12 dialkylamino, cyano, nitro,
--C(O)NH.sub.2--C(O)NHC.sub.1-4 alkyl, --C(O)NH(C.sub.1-4
alkyl).sub.2, --NHC(O)C.sub.1-4 alkyl, --N(C.sub.1-4
alkyl)C(O)C.sub.1-4 alkyl, --C(O)OC.sub.1-4 alkyl, --C(O)OH,
--OC(O)C.sub.1-6 alkyl, --C(O)C.sub.1-6 alkyl, nitro, or cyano.
[0118] In embodiments R.sup.1 is C.sub.2-6 hydroxyalkyl, for
example 3-hydroxypropyl.
[0119] In embodiments R2 is C1-4 alkyl, for example methyl
[0120] In embodiments R3 is C1-6 alkoxy, C1-6 haloalkoxy, phenyl or
phenoxy (for example methoxy, ethoxy, propoxy, trifluoromethoxy or
butoxy, phenyl, phenoxy), wherein a phenyl or phenoxy in R3 is
optionally substituted with one or more fluorine, chlorine or
--
[0121] OCF3 group.
[0122] In embodiments R4 is benzyl or isopropyl optionally
substituted with one or more, fluorine, chlorine or --OCF3.
[0123] The compound of formula (IV) may be a compound disclosed in
claim 10 of WO 2016/023826 selected from:
##STR00014## ##STR00015## ##STR00016##
or a pharmaceutically acceptable salt thereof.
[0124] Compounds of the formula (IV) may be prepared as described
in WO 2016/023826.
[0125] In an embodiment the inhibitor is a compound of the formula
(V), or a pharmaceutically acceptable salt thereof:
##STR00017##
wherein
[0126] R.sup.1 is C.sub.2-10 hydroxyalkyl optionally substituted
with 1-3 C.sub.3-10 cycloalkyl groups;
[0127] R.sup.2 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.3-10 cycloalkyl, C.sub.1-10 hydroxyalkyl, or
C.sub.1-6 alkoxy;
[0128] R.sup.3 is C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-6 acyl, C.sub.3-10 cycloalkyl, C.sub.1-6 alkoxy,
C.sub.4-10 cycloalkyloxy, halo, C.sub.1-6 haloalkyl, C.sub.1-6
haloalkoxy, hydroxy, C.sub.1-10 hydroxyalkyl, C.sub.1-6 alkylthio,
thionyl, sulfonyl, sulfonamidyl, C.sub.6-12 aryl, 5-14-membered
heteroaryl, C.sub.6-12aryl-C.sub.1-6 alkyl, 5-14-membered
heteroaryl-C.sub.1-6 alkyl, C.sub.6-12 aryloxy, --O--C.sub.6-12
aryl-C.sub.1-6 alkyl, --O--C.sub.1-6 alkyl-C.sub.6-12aryl,
--C.sub.6-12aryl-C.sub.1-6alkyl-OR', 5-14-membered heteroaryloxy,
3-18-membered heterocycloalkyl, amino, C.sub.1-6 alkylamino,
C.sub.2-12dialkylamino, --C(O)NR'R', --NR'C(O)R', urea,
sulfonylurea, nitro, or cyano, wherein each R' is independently H
or C.sub.1-6 alkyl and wherein R.sup.3 is optionally substituted
with 1-5 R.sup.5;
[0129] R.sup.4 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-6 acyl, C.sub.3-10 cycloalkyl, C.sub.1-6 alkoxy,
C.sub.4-10 cycloalkyloxy, halo, C.sub.1-6 haloalkyl, C.sub.1-6
haloalkoxy, hydroxy, C.sub.1-10 hydroxyalkyl, C.sub.1-6 alkylthio,
thionyl, sulfonyl, sulfonamidyl, C.sub.6-12 aryl, 5-14-membered
heteroaryl, C.sub.6-12 aryl-C.sub.1-6 alkyl, 5-14-membered
heteroaryl-C.sub.1-6 alkyl, C.sub.6-12 aryloxy, --O--C.sub.6-12
aryl-C.sub.1-6 alkyl, --O--C.sub.1-6 alkyl-C.sub.6-12 aryl,
--C.sub.6-12 aryl-C.sub.1-6 alkyl-OR', 5-14-membered heteroaryloxy,
3-18-membered heterocycloalkyl, amino, C.sub.1-6 alkylamino,
C.sub.2-12 dialkylamino, --C(O)NR'R', --NR'C(O)R', urea,
sulfonylurea, nitro, or cyano, wherein each R' is independently H
or C.sub.1-6 alkyl and wherein R.sup.4 is optionally substituted
with 1-5 R.sup.5; and
[0130] each R.sup.5 is independently H, C.sub.1-6 alkyl, halo,
C.sub.1-6 haloalkyl, C.sub.1-6 haloalkoxy, hydroxy, C.sub.1-6
alkoxy, C.sub.6-12aryl, 5-14-membered heteroaryl, C.sub.6-12
aryloxy, --O--C.sub.6-12 aryl-C.sub.1-6 alkyl, --O--C.sub.1-6
alkyl-C.sub.6-12 aryl, --C.sub.6-12 aryl-C.sub.1-6 alkyl-OR',
5-14-membered heteroaryloxy, --C(O)OR', --OC(O)R', --C(O)R', nitro,
or cyano, wherein each R' is independently H or C.sub.1-6
alkyl.
[0131] In embodiments in the compound of formula (V) R.sup.1 is
C.sub.2-6 hydroxyalkyl, for example 3-hydroxypropyl.
[0132] In embodiments in the compound of formula (V) R.sup.2 is
C.sub.1-4 alkyl, for example methyl.
[0133] In embodiments in the compound of formula (V) R.sup.3 is
phenyl, phenylC.sub.1-4alkoxy or phenylC.sub.1-4 alkyl each of
which is optionally substituted with one or more substituent
selected from chloro, C.sub.1-4 alkyl, --CF.sub.3 or --OCF.sub.3.
For example R.sup.3 is phenoxy or phenyl each of which is
optionally substituted with one or more chloro, C.sub.1-4 alkyl or
--OCF.sub.3. Suitably R.sub.3 is isopropyltoluene, chlorophenoxy,
chlorophenyl, or trifluoromethoxyphenyl.
[0134] In embodiments in the compound of formula (V) R.sub.4 is H,
C.sub.1-6 alkyl or phenyl-C.sub.1-4 alkyl, wherein the phenyl is
optionally substituted by one or more halo. For example R.sup.4 is
C.sub.1-6 alkyl or phenyl-C.sub.1-4 alkyl, wherein the phenyl is
optionally substituted by one or more chloro. For example R.sup.4
is benzyl optionally substituted by chloro.
[0135] The compound of formula (V) may be a compound disclosed in
claim 13 of WO2016/023825 selected from:
##STR00018## ##STR00019##
or a pharmaceutically acceptable salt thereof.
[0136] Compounds of the formula (V) may be prepared as described in
WO2016/023825.
[0137] In an embodiment the inhibitor is a compound of the formula
(VI), or a pharmaceutically acceptable salt thereof:
##STR00020##
wherein
[0138] R.sup.1 is C.sub.2-10 hydroxyalkyl, optionally substituted
with 1-3 R.sup.6;
[0139] R.sup.2 is H, C.sub.1-6 alkyl, C.sub.2-6 hydroxyalkyl,
C.sub.3-10 cycloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, or
C.sub.1-6 alkoxy;
[0140] R.sup.3 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-6 alkoxy, C.sub.1-6 acyl, C.sub.3-10 cycloalkyl,
halo, hydroxyl, C.sub.6-12aryl, 5-14-membered heteroaryl,
3-18-membered heterocycloalkyl, amino, C.sub.1-6 alkylamino,
C.sub.2-12 dialkylamino, --C(O)NR'R', --NR'C(O)R', urea,
sulfonylurea, nitro, or cyano, wherein each R' is independently H
or C.sub.1-6 alkyl and wherein each R.sup.3 is optionally
substituted with 1-4 R.sup.6
[0141] R.sup.4 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-6 acyl, C.sub.1-6 alkoxy, C.sub.4-10cycloalkyloxy,
halo, C.sub.1-6 haloalkyl, C.sub.1-6 haloalkoxy, hydroxy, C.sub.1-6
alkylthio, sulfonamidyl, C.sub.6-12 aryl, 5-14-membered heteroaryl,
C.sub.6-12 aryl-C.sub.1-6 alkyl, 5-14-membered heteroaryl-C.sub.1-6
alkyl, C.sub.6-12 aryloxy, --O--C.sub.6-12 aryl-C.sub.1-6 alkyl,
--O--C.sub.1-6 alkyl-C.sub.6-12 aryl, --C.sub.6-12 aryl-C.sub.1-6
alkyl-OR', 5-14-membered heteroaryloxy, 3-18-membered
heterocycloalkyl, amino, C.sub.1-6 alkylamino, C.sub.2-12
dialkylamino, --C(O)NR'R', --NR'C(O)R', urea, sulfonylurea, nitro,
or cyano, wherein each R' is independently H or C.sub.1-6 alkyl and
wherein R.sup.4 is optionally substituted with 1-4 R.sup.7;
[0142] R.sup.5 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-6 acyl, C.sub.1-6 alkoxy, C.sub.4-10
cycloalkyloxy, halo, C.sub.1-6 haloalkyl, C.sub.1-6 haloalkoxy,
hydroxy, C.sub.1-6alkylthio, sulfonamidyl, C.sub.6-12 aryl,
5-14-membered heteroaryl, C.sub.6-12 aryl-C.sub.1-6 alkyl,
5-14-membered heteroaryl C.sub.1-6 alkyl, C.sub.6-12 aryloxy,
--O--C.sub.6-12 aryl-C.sub.1-6 alkyl, --O--C.sub.1-6
alkyl-C.sub.6-12 aryl, --C.sub.6-12 aryl-C.sub.1-6 alkyl-OR',
5-14-membered heteroaryloxy, 3-18-membered heterocycloalkyl, amino,
C.sub.1-6 alkylamino, C.sub.2-12 dialkylamino, --C(O)NR'R',
--NR'C(O)R', urea, sulfonylurea, nitro, or cyano, wherein each R'
is independently H or C.sub.1-6 alkyl and wherein R.sup.5 is
optionally substituted with 1-4 R.sup.7;
[0143] wherein at least two of R.sup.3, R.sup.4 and R.sup.5 are not
H;
[0144] each R.sup.6 is independently H, C.sub.1-3 alkyl, halo,
hydroxy, or amino; and
[0145] each R.sup.7 is independently H, C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 acyl, halo, C.sub.1-6
haloalkyl, C.sub.1-6 haloalkoxy, hydroxy, aryl, heteroaryl,
arylC.sub.1-6alkyl, heteroarylC.sub.1-6alkyl, C.sub.1-6 alkoxy,
C.sub.3-6 cycloalkyloxy, aryloxy, aryl-C.sub.1-6 alkoxy,
heteroaryloxy, amino, C.sub.1-6 alkylamino, C.sub.2-12
dialkylamino, --C(O)NR''R', --NR''C(O)R'', nitro, or cyano; wherein
each R'' is independently H or C.sub.1-4 alkyl.
[0146] In embodiments in the compound of the formula (VI) R.sub.1
is C.sub.2-6 hydroxyalkyl, for example 3-hydroxypropyl.
[0147] In embodiments in the compound of the formula (VI) R.sub.2
is C.sub.1-4 alkyl, for example methyl.
[0148] In embodiments in the compound of the formula (VI) R.sub.3
and R.sub.5 are each independently selected from H, C.sub.1-4
alkyl, phenyl and phenyl substituted with halo, for example
chloro.
[0149] In embodiments in the compound of the formula (VI) R.sub.4
is H, C.sub.1-4 alkyl, phenyl or phenyl substituted with halo or
--OCF.sub.3, for example R.sub.4 is 3-chlorophenyl or
3-trifluoromethoxyphenyl.
[0150] In one embodiment in the compound of the formula (VI)
R.sub.1 is 3-hydroxypropyl; R.sub.2 is methyl; R.sub.3 is H, methyl
or 3-chlorophenyl; R.sub.4 is 3-chlorophenyl or
3-trifluoromethoxyphenyl; and R.sub.5 is H, methyl, or
4-chlorobenzyl.
[0151] The compound of formula (VI) may be a compound disclosed in
claim 12 of WO 2016/023831 selected from:
##STR00021## ##STR00022##
or a pharmaceutically acceptable salt thereof.
[0152] Compounds of formula (VI) may be prepared as described in WO
2016/023831.
[0153] In an embodiment the inhibitor is a compound of the formula
(VII), or a pharmaceutically acceptable salt thereof:
##STR00023##
wherein
[0154] R.sup.1 is C.sub.2-10 hydroxyalkyl, optionally substituted
with 1-3 R.sup.6; R.sup.2 is H, C.sub.1-6 alkyl, C.sub.1-6
hydroxyalkyl, C.sub.3-10 cycloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, or C.sub.1-6 alkoxy;
[0155] R.sup.3 is C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-6 acyl, C.sub.3-10 cycloalkyl, C.sub.1-6 alkoxy,
C.sub.4-10 cycloalkyloxy, halo, C.sub.1-6 haloalkyl, C.sub.1-6
haloalkoxy, hydroxyl, C.sub.1-10 hydroxyalkyl, C.sub.1-6 alkylthio,
thionyl, sulfonyl, sulfonamidyl, C.sub.6-12 aryl, 5-14-membered
heteroaryl, C.sub.6-12 aryl-C.sub.1-6 alkyl, 5-14-membered
heteroaryl-C.sub.1-6 alkyl, C.sub.6-12 aryloxy, --O--C.sub.6-12
aryl-C.sub.1-6 alkyl, --O--C.sub.1-6 alkyl-C.sub.6-12aryl,
--C.sub.6-12 aryl-C.sub.1-6 alkyl-O, 5-14-membered heteroaryloxy,
3-18-membered heterocycloalkyl, amino, C.sub.1-6 alkylamino,
C.sub.2-12 dialkylamino, --C(O)NR'R', --NR'C(O)R', urea,
sulfonylurea, nitro, or cyano, wherein each R' is independently H
or C.sub.1-6 alkyl and wherein R.sup.3 optionally substituted with
1-5 R.sup.5;
[0156] R.sup.4 is C.sub.1-6 alkyl, C.sub.1-6 acyl, C.sub.3-10
cycloalkyl, C.sub.1-6 alkoxy, C.sub.4-10 cycloalkyloxy, halo,
C.sub.1-6 haloalkyl, C.sub.1-6 haloalkoxy, hydroxyl, C.sub.1-6
alkylthio, thionyl, sulfonyl, sulfonamidyl, C.sub.6-12 aryl,
5-14-membered heteroaryl, C.sub.6-12 aryl-C.sub.1-6 alkyl,
5-14-membered heteroaryl-C.sub.1-6 alkyl, C.sub.6-12 aryloxy,
--O--C.sub.6-12 aryl-C.sub.1-6 alkyl, --O--C.sub.1-6
alkyl-C.sub.6-12 aryl, --C.sub.6-12 aryl-C.sub.1-6alkyl-OR',
5-14-membered heteroaryl-C.sub.1-6 alkyl, 5-14-membered
heteroaryloxy, 3-18-membered heterocycloalkyl, amino, C.sub.1-6
alkylamino, C.sub.2-12 dialkylamino, --C(O)NR'R', --NR'C(O)R',
urea, sulfonylurea, nitro, or cyano, wherein each R' is
independently H or C.sub.1-6 alkyl and wherein R.sup.4 is
optionally substituted with 1-5 R.sup.5;
[0157] each R.sup.5 is independently C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 acyl, C.sub.1-6 alkoxy,
C.sub.4-10 cycloalkyloxy, halo, C.sub.1-6 haloalkyl, C.sub.1-6
haloalkoxy, hydroxy, C.sub.1-10 hydroxyalkyl, amino, C.sub.6-12
aryl, 5-14-membered heteroaryl, C.sub.6-12 aryl-C.sub.1-6 alkyl,
C.sub.6-12 aryloxy, --O--C.sub.6-12 aryl-C.sub.1-6 alkyl,
--O--C.sub.1-6 alkyl-C.sub.6-12 aryl, --C.sub.6-12aryl-C.sub.1-6
alkyl-OR', or 5-14-membered heteroaryloxy, wherein each R' is
independently H or C.sub.1-6 alkyl and wherein R.sup.5 is
optionally substituted with 1-5 R.sup.6; and
[0158] each R.sup.6 is independently C.sub.1-6 alkyl, C.sub.1-6
alkoxy, C.sub.3-10 cycloalkyl, halo, C.sub.1-6 haloalkyl, C.sub.1-6
haloalkoxy, hydroxyl, amino, C.sub.1-6 alkylamino, C.sub.2-12
dialkylamino, cyano, nitro, --C(O)NR'R', --NR'C(O)R', --C(O)OR',
--C(O)R', acyl, nitro, or cyano, wherein each R' is independently H
or C.sub.1-6-alkyl.
[0159] In embodiments in the compound of the formula (VII) R.sub.1
is C.sub.2-6 hydroxyalkyl, for example 3-hydroxypropyl.
[0160] In embodiments in the compound of the formula (VII) R.sub.2
is C.sub.1-4 alkyl, for example methyl.
[0161] In embodiments in the compound of the formula (VII) R.sub.3
is C.sub.1-4 alkyl, C.sub.1-4 alkoxy, phenyl-C.sub.1-4 alkoxy-, 5-6
membered heteroaryl-C.sub.1-4 alkyl-, phenyl-C.sub.1-4 alkyl-, 5-6
membered heteroaryl-C.sub.1-4 alkyl-, phenoxy-, 5-6 membered
heteroaryloxy-, phenyl or 5-6 membered heteroaryl, wherein any
phenyl or heteroaryl group in R.sub.3 is optionally substituted by
one or more substituents selected from halo, C.sub.1-4 alkyl,
C.sub.1-4 alkoxy C.sub.1-4 haloalkyl or C.sub.1-4 haloalkoxy.
[0162] In embodiments in the compound of the formula (VII) R.sub.4
is C.sub.1-6 alkyl, 5-6 membered heteroaryl-C.sub.1-6 alkyl-,
phenyl-C.sub.1-6 alkyl- or 5-6 membered heteroaryl-C.sub.1-6
alkyl-, wherein any phenyl or heteroaryl group in R.sub.4 is
optionally substituted by one or more substituents selected from
halo, C.sub.1-4 alkyl, C.sub.1-4 alkoxy C.sub.1-4 haloalkyl or
C.sub.1-4 haloalkoxy.
[0163] The compound of formula (VII) may be a compound disclosed in
claim 13 of WO 2016/023830 selected from:
##STR00024## ##STR00025## ##STR00026## ##STR00027## ##STR00028##
##STR00029##
or a pharmaceutically acceptable salt thereof.
[0164] The compounds of formula (VII) may be prepared as described
in WO 2016/023830.
[0165] The inhibitor may be a compound of the formula (VIII) or a
pharmaceutically acceptable salt thereof:
##STR00030##
wherein
[0166] X is S or O;
[0167] R.sup.1 is C.sub.2-10 hydroxyalkyl, optionally substituted
with 1-3 R.sup.5; R.sup.2 is H, C.sub.1-6 alkyl, C.sub.1-6
hydroxyalkyl, C.sub.3-10 cycloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, or C.sub.1-6 alkoxy;
[0168] R.sup.3 is C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-6 acyl, C.sub.3-10 cycloalkyl, C.sub.1-6 alkoxy,
C.sub.4-10 cycloalkyloxy, halo, C.sub.1-6 haloalkyl, C.sub.1-6
haloalkoxy, hydroxyl, C.sub.1-6 alkylthio, thionyl, sulfonyl,
sulfonamidyl, C.sub.6-12 aryl, 5-14-membered heteroaryl, C.sub.6-12
aryl-C.sub.1-6 alkyl, 5-14-membered heteroaryl-C.sub.1-6 alkyl,
C.sub.6-12 aryloxy, --O--C.sub.6-12 aryl-C.sub.1-6 alkyl,
--O--C.sub.1-6 alkyl-C.sub.6-12 aryl, --C.sub.6-12 aryl-C.sub.1-6
alkyl-OR', 5-14-membered heteroaryloxy, 3-18-membered
heterocycloalkyl, amino, C.sub.1-6 alkylamino, C.sub.2-12
dialkylamino, --C(O)NR'R', --NR'C(O)R', urea, sulfonylurea, nitro,
cyano, wherein each R' is independently H or C.sub.1-6 alkyl and
wherein R.sup.3 is optionally substituted with 1-4 R.sup.5;
[0169] R.sup.4 is C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-6 acyl, C.sub.3-10 cycloalkyl, C.sub.1-6 alkoxy,
C.sub.4-10 cycloalkyloxy, halo, C.sub.1-6 haloalkyl, C.sub.1-6
haloalkoxy, hydroxyl, C.sub.1-6 alkylthio, thionyl, sulfonyl,
sulfonamidyl, C.sub.6-12 aryl, 5-14-membered heteroaryl, C.sub.6-12
aryl-C.sub.1-6 alkyl, 5-14-membered heteroaryl-C.sub.1-6 alkyl,
C.sub.6-12 aryloxy, --O--C.sub.6-12 aryl-C.sub.1-6 alkyl,
--O--C.sub.1-6 alkyl-C.sub.6-12 aryl, --C.sub.6-12 aryl-C.sub.1-6
alkyl-OR', 5-14-membered heteroaryl-C.sub.1-6 alkyl, 5-14-membered
heteroaryloxy, 3-18-membered heterocycloalkyl, amino, C.sub.1-6
alkylamino, C.sub.2-12 dialkylamino, --C(O)NR'R', --NR'C(O)R',
urea, sulfonylurea, nitro, cyano, wherein each R' is independently
H or C.sub.1-6 alkyl and wherein R.sup.4 is optionally substituted
with 1-4 R.sup.5
[0170] each R.sup.5 is independently C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 acyl, C.sub.3-10 cycloalkyl,
C.sub.1-6 alkoxy, C.sub.4-10 cycloalkyloxy, halo, C.sub.1-6
haloalkyl, C.sub.1-6 haloalkoxy, hydroxyl, C.sub.1-6 alkylthio,
thionyl, sulfonyl, sulfonamidyl, amino, C.sub.1-6 alkylamino,
C.sub.2-12 dialkylamino, --C(O)NR'R', --NR'C(O)R', urea,
sulfonylurea, nitro, cyano, wherein each R' is independently H or
C.sub.1-6 alkyl and R.sup.5 is optionally substituted with 1-5
R.sup.6; and
[0171] each R.sup.6 is independently C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 acyl, C.sub.1-6 alkoxy, halo,
C.sub.1-6 haloalkyl, C.sub.1-6 haloalkoxy, hydroxyl, amino,
--C(O)NR'R', --NR'C(O)R', --C(O)R', nitro or cyano.
[0172] In embodiments in the compound of the formula (VIII) R.sub.1
is C.sub.2-6 hydroxyalkyl, for example 3-hydroxypropyl.
[0173] In embodiments in the compound of the formula (VIII) R.sup.2
is C.sub.1-4 hydroxyalkyl or C.sub.1-4 alkyl, for example methyl,
ethyl or 3-hydroxypropyl.
[0174] In embodiments in the compound of the formula (VIII) R.sub.3
is C.sub.1-4 alkyl, C.sub.1-4 alkoxy, aryl-C.sub.1-4 alkoxy-, 5-6
membered heteroaryl-C.sub.1-4 alkyl-, aryl-C.sub.1-4 alkyl-, 5-6
membered heteroaryl-C.sub.1-4 alkyl-, aryloxy-, 5-6 membered
heteroaryloxy-, aryl or 5-6 membered heteroaryl, wherein any aryl
or heteroaryl group in R.sub.3 is optionally substituted by one or
more substituents selected from halo, C.sub.1-4 alkyl, C.sub.1-4
alkoxy C.sub.1-4 haloalkyl, C.sub.1-4 haloalkoxy or phenyl.
Suitably in this embodiment aryl is phenyl or naphthyl, preferably
phenyl. For example R.sub.3 is phenyl or 5-6 membered heteroaryl,
optionally substituted by one or more substituents selected from
halo, C.sub.1-4 alkyl, C.sub.1-4 alkoxy C.sub.1-4 haloalkyl and
C.sub.1-4 haloalkoxy.
[0175] In embodiments in the compound of the formula (VIII) R.sub.4
is C.sub.1-6 alkyl, C.sub.1-6 acyl or benzyl, wherein R.sub.4 is
optionally substituted by one or more substituents selected from
halo, C.sub.1-4 alkyl or OR'', wherein R'' is H or methyl. For
example R.sub.4 is methyl,
[0176] In one embodiment in the compound of the formula (VIII) X is
S.
[0177] In one embodiment in the compound of the formula (VIII) X is
O.
[0178] In one embodiment in the compound of the formula (VIII) X is
O or S; R.sub.1 is 3-hydroxypropyl; R.sub.2 methyl or
3-hydroxypropyl; R.sub.3 is phenyl, naphthyl, pyridyl, phenoxy or
pyridyloxy optionally substituted by one or wo substituents
selected from halo, --CF.sub.3, or --OCF.sub.3; and R.sub.4 is C1-6
alkyl, C.sub.1-6 acyl or benzyl, wherein R.sub.4 is optionally
substituted by one or more substituents selected from halo or
hydroxyl.
[0179] The inhibitor may be a compound of the formula (IX) or a
pharmaceutically acceptable salt thereof:
##STR00031##
wherein:
[0180] A is S or O;
[0181] R.sup.1 is C.sub.2-10 hydroxyalkyl, optionally substituted
with 1-3 R.sup.5;
[0182] R.sup.2 is H, C.sub.1-6 alkyl, C.sub.1-6 hydroxyalkyl,
C.sub.3-10 cycloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, or
C.sub.1-6 alkoxy;
[0183] R.sup.3 is C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-6 acyl, C.sub.3-10 cycloalkyl, C.sub.1-6 alkoxy,
C.sub.4-10 cycloalkyloxy, halo, C.sub.1-6 haloalkyl, C.sub.1-6
haloalkoxy, hydroxyl, C.sub.1-6 alkylthio, thionyl, sulfonyl,
sulfonamidyl, C.sub.6-12 aryl, 5-14-membered heteroaryl, C.sub.6-12
aryl-C.sub.1-6 alkyl, 5-14-membered heteroaryl-C.sub.1-6 alkyl,
C.sub.6-12 aryloxy, --O--C.sub.6-12 aryl-C.sub.1-6 alkyl,
--O--C.sub.1-6 alkyl-C.sub.6-12 aryl, --C.sub.6-12 aryl-C.sub.1-6
alkyl-OH', 5-14-membered heteroaryloxy, 3-18-membered
heterocycloalkyl, amino, C.sub.1-6 alkylamino, C.sub.2-12
dialkylamino, --C(O)NH--, --C(O)N--C.sub.1-6 alkyl-, --NHC(O)--,
--N--C.sub.1-6 alkyl C(O)--, urea, sulfonylurea, nitro, cyano, and
wherein R.sup.3 is optionally substituted with 1-4 R.sup.5;
[0184] R.sup.4 is C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-6 acyl, C.sub.3-10 cycloalkyl, C.sub.1-6 alkoxy,
C.sub.4-10 cycloalkyloxy, halo, C.sub.1-6 haloalkyl, C.sub.1-6
haloalkoxy, hydroxyl, C.sub.1-6 alkylthio, thionyl, sulfonyl,
sulfonamidyl, C.sub.6-12 aryl, 5-14-membered heteroaryl, C.sub.6-12
aryl-C.sub.1-6 alkyl, 5-14-membered heteroaryl-C.sub.1-6 alkyl,
C.sub.6-12 aryloxy, --O--C.sub.6-12 aryl-C.sub.1-6 alkyl,
--O--C.sub.1-6 alkyl-C.sub.6-12 aryl, --C.sub.6-12 aryl-C.sub.1-6
alkyl-OH, 5-14-membered heteroaryl-C.sub.1-6 alkyl, 5-14-membered
heteroaryloxy, 3-18-membered heterocycloalkyl, amino, C.sub.1-6
alkylamino, C.sub.2-12 dialkylamino, --C(O)NH--, --C(O)N--C.sub.1-6
alkyl-, --NHC(O)--, --N--C.sub.1-6 alkyl C(O)--, urea,
sulfonylurea, nitro, cyano, and wherein R.sup.4 is optionally
substituted with 1-4 R.sup.5
[0185] each R.sup.5 is independently C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 acyl, C.sub.3-10 cycloalkyl,
C.sub.1-6 alkoxy, C.sub.4-10 cycloalkyloxy, halo, C.sub.1-6
haloalkyl, C.sub.1-6 haloalkoxy, hydroxyl, C.sub.1-6 alkylthio,
thionyl, sulfonyl, sulfonamidyl, amino, C.sub.1-6 alkylamino,
C.sub.2-12 dialkylamino, keto, C(O)NH--, --C(O)N--C.sub.1-6 alkyl-,
--NHC(O)--, --N--C.sub.1-6 alkyl C(O)--, urea, sulfonylurea, nitro,
cyano, wherein each R' is independently H or C.sub.1-6 alkyl and
R.sup.5 is optionally substituted with 1-5 R.sup.6; and
[0186] each R.sup.6 is independently C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 acyl, C.sub.1-6 alkoxy, halo,
C.sub.1-6 haloalkyl, C.sub.1-6 haloalkoxy, hydroxyl, amino,
--C(O)NH--, --C(O)N--C.sub.1-6 alkyl-, --NHC(O)--, --N--C.sub.1-6
alkyl C(O)--, nitro or cyano.
[0187] The compounds of formula (IX) may be prepared as described
in WO 2016/023832. WO 2016/023832 is hereby incorporated by
reference, including any of the embodiments disclosed therein.
[0188] In one embodiment in the compound of the formula (IX) may be
a compound disclosed in WO 2016/023832 selected from:
##STR00032## ##STR00033## ##STR00034## ##STR00035##
##STR00036##
or a pharmaceutically acceptable salt thereof.
[0189] Compounds of the Formula (IX) may be prepared as disclosed
in WO 2016/023832.
[0190] Clemizole is a potent and selective inhibitor of TRPC5 (Mol
Pharmacol. 2014 November; 86(5):514-21). In an embodiment the
inhibitor is clemizole or a derivative thereof. In one embodiment
the inhibitor is a compound of the formula (X), or a
pharmaceutically acceptable salt thereof:
##STR00037##
[0191] wherein R.sup.1 and R.sup.2 are independently H or C.sub.1-4
alkyl, or R.sup.1 and R.sup.2 together with the nitrogen to which
they are attached form a 4 to 6 membered heterocyclyl.
[0192] R.sup.1 and R.sup.2 may be methyl, ethyl, propyl or
isopropyl.
[0193] In an embodiment R.sup.1 and R.sup.2 together with the
nitrogen to which they are attached form pyrrolidinyl, piperidinyl,
piperazinyl or morpholinyl.
[0194] In one particular embodiment inhibitor is clemizole, or a
pharmaceutically acceptable salt thereof:
##STR00038##
[0195] Certain compounds of the formula (IX) are novel and form a
further aspect of the invention. Accordingly there is provided the
compound of the formula (IX), or a pharmaceutically acceptable salt
thereof, wherein R.sup.1 and R.sup.2 are independently H or
C.sub.1-4 alkyl. Particular compounds of the formula (IX) include
those wherein R.sup.1 and R.sup.2 are both C.sub.1-4 alkyl, for
example R.sup.1 and R.sup.2 are both independently selected from
methyl, ethyl, propyl or isopropyl. A particular novel compound of
the invention is a compound of the formula (IXa):
##STR00039##
or a pharmaceutically acceptable salt thereof.
[0196] The inventors have found the compound of the formula (Xa)
(also described herein as DE2) is a potent inhibitor of TRPC4 and
TRPC5. Accordingly, in embodiments the inhibitor is a compound of
the formula (Xa), or a pharmaceutically acceptable salt
thereof.
[0197] In embodiments the inhibitor is AC1903 or a pharmaceutically
acceptable salt thereof, as disclosed in "A small-molecule
inhibitor of TRPC5 ion channels suppresses progressive kidney
disease in animal models", Zhou et al, Science 358, 13321336.
##STR00040##
[0198] In another embodiment the inhibitor is not a compound
disclosed in Bon et al., British Journal of Pharmacology (2013) 170
459-474. In a further embodiment the inhibitor is not a natural
product, for example inhibitors found in foods, such as fatty
acids. In certain embodiments the inhibitor is not one or more of
the following: [0199] a fatty acid, particularly the inhibitor is
not an omega-3 fatty acid such as .alpha.-linolenic acid,
eicosapentaenoic acid, or docosahexaenoic acid; [0200] a
polyphenol, for example gallic acid, resveratrol or
diethylstillbestrol; [0201] galangin (a natural product obtained
from ginger); [0202] a flavonoid (particularly a flavonoid
described in Naylor, J. et al (2016), Natural and synthetic
flavonoid modulation of TRPC5 channels. British Journal of
Pharmacology 173, 562-74); or [0203] an antioxidant (for example
vitamin C and particularly the anti-oxidants disclosed in Naylor,
J. et al (2011), TRPC5 channel sensitivities to antioxidants and
hydroxylated stilbenes, Journal of Biological Chemistry 286,
5078-5086).
[0204] Suitably the inhibitor is a small molecule inhibitor of
TRPC4 or TRPC5 which inhibits Ca.sup.2+ ion influx in the
Intracellular Ca.sup.2+ assay described in the Examples at a
concentration of 10 .mu.M or less. Suitable the inhibitor is one
which inhibits Ca.sup.2+ ion influx in this assay at a
concentration of 5 .mu.M or less and preferably at a concentration
of 1 .mu.M or less.
Definitions
[0205] Unless otherwise stated, the following terms used in the
specification and claims have the following meanings set out
below.
[0206] It is to be appreciated that references to "treating" or
"treatment" include prophylaxis as well as the alleviation of
established symptoms of a condition. "Treating" or "treatment" of a
state, disorder or condition therefore includes: (1) preventing or
delaying the appearance of clinical symptoms of the state, disorder
or condition developing in a human that may be afflicted with or
predisposed to the state, disorder or condition but does not yet
experience or display clinical or subclinical symptoms of the
state, disorder or condition, (2) inhibiting the state, disorder or
condition, i.e., arresting, reducing or delaying the development of
the disease or a relapse thereof (in case of maintenance treatment)
or at least one clinical or subclinical symptom thereof, or (3)
relieving or attenuating the disease, i.e., causing regression of
the state, disorder or condition or at least one of its clinical or
subclinical symptoms.
[0207] A "therapeutically effective amount" means the amount of a
compound that, when administered to a mammal for treating a
disease, is sufficient to effect such treatment for the disease.
The "therapeutically effective amount" will vary depending on the
compound, the disease and its severity and the age, weight, etc.,
of the mammal to be treated.
[0208] A "subject" or "patient" refers to a living organism
suffering from or prone to a disease or condition that can be
treated by administration of an inhibitor, as provided herein.
Non-limiting examples include humans, other mammals, bovines, rats,
mice, dogs, cats, monkeys, goat, sheep, cows, deer, horses and
other non-mammalian animals. Preferably the patient or subject is
human.
[0209] The term "halo" or "halogen" refers to one of the halogens,
group 17 of the periodic table. In particular the term refers to
fluorine, chlorine, bromine and iodine. Preferably, the term refers
to fluorine or chlorine.
[0210] The term C.sub.m-n refers to a group with m to n carbon
atoms.
[0211] The term "C.sub.1-6 alkyl" refers to a linear or branched
hydrocarbon chain containing 1, 2, 3, 4, 5 or 6 carbon atoms, for
example methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl,
tert-butyl, n-pentyl and n-hexyl. "C.sub.1-4 alkyl" similarly
refers to such groups containing up to 4 carbon atoms. Alkylene
groups are divalent alkyl groups and may likewise be linear or
branched and have two points of attachment to the remainder of the
molecule. Furthermore, an alkylene group may, for example,
correspond to one of those alkyl groups listed in this paragraph.
The alkyl and alkylene groups may be unsubstituted or substituted
by one or more substituents. Possible substituents are described
below. Substituents for the alkyl group may be halogen, e.g.
fluorine, chlorine, bromine and iodine, OH, C.sub.1-C.sub.4alkoxy.
Other substituents for the alkyl group may alternatively be
used.
[0212] The term "C.sub.1-6 haloalkyl", e.g. "C.sub.1-4 haloalkyl",
refers to a hydrocarbon chain substituted with at least one halogen
atom independently chosen at each occurrence, for example fluorine,
chlorine, bromine and iodine. The halogen atom may be present at
any position on the hydrocarbon chain. For example, C.sub.1-6
haloalkyl may refer to chloromethyl, fluoromethyl, trifluoromethyl,
chloroethyl e.g. 1-chloromethyl and 2-chloroethyl, trichloroethyl
e.g. 1,2,2-trichloroethyl, 2,2,2-trichloroethyl, fluoroethyl e.g.
1-fluoromethyl and 2-fluoroethyl, trifluoroethyl e.g.
1,2,2-trifluoroethyl and 2,2,2-trifluoroethyl, chloropropyl,
trichloropropyl, fluoropropyl, trifluoropropyl.
[0213] The term "C.sub.2-6 alkenyl" includes a branched or linear
hydrocarbon chain containing at least one double bond and having 2,
3, 4, 5 or 6 carbon atoms. The double bond(s) may be present as the
E or Z isomer. The double bond may be at any possible position of
the hydrocarbon chain. For example, the "C.sub.2-6 alkenyl" may be
ethenyl, propenyl, butenyl, butadienyl, pentenyl, pentadienyl,
hexenyl and hexadienyl.
[0214] The term "C.sub.2-6 alkynyl" includes a branched or linear
hydrocarbon chain containing at least one triple bond and having 2,
3, 4, 5 or 6 carbon atoms. The triple bond may be at any possible
position of the hydrocarbon chain. For example, the "C.sub.2-6
alkynyl" may be ethynyl, propynyl, butynyl, pentynyl and
hexynyl.
[0215] The term "C.sub.1-6 alkoxy" includes a branched or linear
C.sub.1-6 alkyl-O-- having 1, 2, 3, 4, 5 or 6 carbon atoms. For
example, C.sub.1-6 alkoxy may be methoxy, ethoxy, propyloxy,
isopropyloxy, butyloxy, isobutyloxy, tert-butyloxy, pentyloxy, or
hexyloxy, and the like.
[0216] The term "haloalkoxy" includes a C.sub.1-6 alkoxy
substituted with at least one halogen atom independently chosen at
each occurrence, for example fluorine, chlorine, bromine and
iodine. The halogen atom may be present at any position on the
C.sub.1-6 alkoxy. For example the haloalkoxy may be chloromethoxy,
fluoromethoxy, trifluoromethoxy or chloroethoxy.
[0217] The term "C.sub.3-6 cycloalkyl" includes a saturated
hydrocarbon ring system containing 3, 4, 5 or 6 carbon atoms. For
example, the "C.sub.3-C.sub.6 cycloalkyl" may be cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, bicycle[2.1.1]hexane or
bicycle[1.1.1]pentane.
[0218] The term "amide" or "amido" includes --C(O)NR'R', wherein R'
are independently H or C.sub.1-6 alkyl.
[0219] The term "acyl" includes --C(O)R', wherein R' is H or
C.sub.1-6 alkyl.
[0220] The term "alkyl-amino" includes C.sub.1-6 alkyl-amino (i.e.
C.sub.1-6 alkyl-NH), for example methylamino or ethylamino.
[0221] The term "dialkylamino" includes (C.sub.1-6 alkyl).sub.2N--,
for example dimethylamino, diethylamino or N-methyl,
N-ethylamino.
[0222] The term "hydroxyl-alkyl" includes HO--C.sub.1-6 alkyl-, for
example HO--C.sub.2-6 alkyl-. For example the hydroxyalkyl may be
hydroxymethyl, hydroxyethyl or 2-hydroxybutyl.
[0223] The term "urea" includes --NR'C(O)NR'R', wherein R' is H or
C.sub.1-6 alkyl.
[0224] The term "thionyl" includes --S(O)R', wherein R' is H or
C.sub.1-6 alkyl.
[0225] The term "sulfonyl" includes --S(O).sub.2R', wherein R' is H
or C.sub.1-6 alkyl.
[0226] The term "sulfonylurea" includes --S(O).sub.2NR'C(O)NR'R',
wherein R' is H or C.sub.1-6 alkyl.
[0227] The term "sulfonamidyl" includes --S(O).sub.2NR'R', wherein
R' is H or C.sub.1-6 alkyl.
[0228] The term "aromatic" when applied to a substituent as a whole
includes a single ring or polycyclic ring system with 4n+2
electrons in a conjugated .pi. system within the ring or ring
system where all atoms contributing to the conjugated .pi. system
are in the same plane.
[0229] The term "aryl" includes an aromatic hydrocarbon ring
system. The ring system has 4n+2 electrons in a conjugated .pi.
system within a ring where all atoms contributing to the conjugated
.pi. system are in the same plane. For example, the "aryl" may be
phenyl and naphthyl. The aryl system itself may be substituted with
other groups.
[0230] The term "aryl-C.sub.m-n alkyl-" includes an aryl group
covalently attached to a C.sub.m-n alkylene group, both of which
are defined herein. Examples of aralkyl groups include benzyl.
[0231] The term "aryloxy-" includes an aryl group covalently
attached to --O--. Examples of aryloxy groups include phenoxy.
[0232] The term "heteroaryl" includes an aromatic mono- or bicyclic
ring incorporating one or more (for example 1-4, particularly 1, 2
or 3) heteroatoms selected from nitrogen, oxygen or sulfur. The
ring or ring system has 4n+2 electrons in a conjugated .pi. system
where all atoms contributing to the conjugated .pi. system are in
the same plane.
[0233] Examples of heteroaryl groups are monocyclic and bicyclic
groups containing from five to twelve ring members, and more
usually from five to ten ring members. The heteroaryl group can be,
for example, a 5- or 6-membered monocyclic ring or a 9- or
10-membered bicyclic ring, for example a bicyclic structure formed
from fused five and six membered rings or two fused six membered
rings. Each ring may contain up to about four heteroatoms typically
selected from nitrogen, sulfur and oxygen. Typically the heteroaryl
ring will contain up to 3 heteroatoms, more usually up to 2, for
example a single heteroatom. In one embodiment, the heteroaryl ring
contains at least one ring nitrogen atom. The nitrogen atoms in the
heteroaryl rings can be basic, as in the case of an imidazole or
pyridine, or essentially non-basic as in the case of an indole or
pyrrole nitrogen. In general the number of basic nitrogen atoms
present in the heteroaryl group, including any amino group
substituents of the ring, will be less than five. Examples of
heteroaryl include pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl,
triazinyl, furyl (furanyl), quinolyl, isoquinolyl, thienyl,
imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzo furyl,
benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl,
tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl,
benzothienyl, purinyl, carbazolyl, benzimidazolyl or indolinyl.
[0234] "Heteroaryl-C.sub.m-n alkyl-" includes a heteroaryl group
covalently attached to a C.sub.m-n alkylene group, both of which
are defined herein. Examples of heteroaralkyl groups include
pyridin-3-ylmethyl and the like.
[0235] The term "heteroaryloxy-" includes a heteroaryl group
covalently attached to --O--. Examples of heteroaryloxy groups
include pyridyloxy.
[0236] The term "heterocyclyl", "heterocycloakyl" "heterocyclic" or
"heterocycle" includes a non-aromatic saturated or partially
saturated monocyclic or fused, bridged, or spiro bicyclic
heterocyclic ring system(s). Monocyclic heterocyclic rings may
contain from about 3 to 12 (suitably from 3 to 7) ring atoms, with
from 1 to 5 (suitably 1, 2 or 3) heteroatoms selected from
nitrogen, oxygen or sulfur in the ring. Examples include Examples
of heterocycloalkyl groups include, but are not limited to, groups
such as dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl,
imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl,
morpholinyl, octahydroindolyl, octahydroisoindolyl,
2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl,
oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl,
pyrrolidinyl, pyrazohdinyl, quinuclidinyl, thiazolidinyl,
tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl,
1-oxo-thiomorpholinyl or 1,1-dioxothiomorpholinyl.
[0237] The term "Heterocyclyl-C.sub.m-n alkyl" includes a
heterocyclyl group covalently attached to a C.sub.m-n alkylene
group, both of which are defined herein.
[0238] The term "optionally substituted" includes either groups,
structures, or molecules that are substituted and those that are
not substituted.
[0239] Where optional substituents are chosen from "one or more"
groups it is to be understood that this definition includes all
substituents being chosen from one of the specified groups or the
substituents being chosen from two or more of the specified
groups.
[0240] Where a moiety is substituted, it may be substituted at any
point on the moiety where chemically possible and consistent with
atomic valency requirements. The moiety may be substituted by one
or more substituents, e.g. 1, 2, 3 or 4 substituents; optionally
there are 1 or 2 substituents on a group. Where there are two or
more substituents, the substituents may be the same or
different.
[0241] Substituents are only present at positions where they are
chemically possible, the person skilled in the art being able to
decide (either experimentally or theoretically) without undue
effort which substitutions are chemically possible and which are
not.
[0242] Throughout the description and claims of this specification,
the words "comprise" and "contain" and variations of them mean
"including but not limited to", and they are not intended to (and
do not) exclude other moieties, additives, components, integers or
steps. Throughout the description and claims of this specification,
the singular encompasses the plural unless the context otherwise
requires. In particular, where the indefinite article is used, the
specification is to be understood as contemplating plurality as
well as singularity, unless the context requires otherwise.
[0243] Features, integers, characteristics, compounds, chemical
moieties or groups described in conjunction with a particular
aspect, embodiment or example of the invention are to be understood
to be applicable to any other aspect, embodiment or example
described herein unless incompatible therewith. All of the features
disclosed in this specification (including any accompanying claims,
abstract and drawings), and/or all of the steps of any method or
process so disclosed, may be combined in any combination, except
combinations where at least some of such features and/or steps are
mutually exclusive. The invention is not restricted to the details
of any foregoing embodiments. The invention extends to any novel
one, or any novel combination, of the features disclosed in this
specification (including any accompanying claims, abstract and
drawings), or to any novel one, or any novel combination, of the
steps of any method or process so disclosed.
[0244] The reader's attention is directed to all papers and
documents which are filed concurrently with or previous to this
specification in connection with this application and which are
open to public inspection with this specification, and the contents
of all such papers and documents are incorporated herein by
reference.
[0245] The various functional groups and substituents making up the
compounds of the present invention are typically chosen such that
the molecular weight of the compound does not exceed 1000. More
usually, the molecular weight of the compound will be less than
750, for example less than 700, or less than 650, or less than 600,
or less than 550. More preferably, the molecular weight is less
than 525 and, for example, is 500 or less.
[0246] Suitable or preferred features of any compounds of the
present invention may also be suitable features of any other
aspect.
[0247] The invention contemplates pharmaceutically acceptable salts
of the compounds of the invention. These may include the acid
addition and base salts of the compounds. These may be acid
addition and base salts of the compounds.
[0248] Suitable acid addition salts are formed from acids which
form non-toxic salts. Examples include the acetate, aspartate,
benzoate, besylate, bicarbonate/carbonate, bisulfate/sulfate,
borate, camsylate, citrate, edisylate, esylate, formate, fumarate,
gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate,
hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide,
isethionate, lactate, malate, maleate, malonate, mesylate,
methylsulfate, naphthylate, 1,5-naphthalenedisulfonate,
2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate,
pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate,
saccharate, stearate, succinate, tartrate, tosylate and
trifluoroacetate salts.
[0249] Suitable base salts are formed from bases which form
non-toxic salts. Examples include the aluminium, arginine,
benzathine, calcium, choline, diethylamine, diolamine, glycine,
lysine, magnesium, meglumine, olamine, potassium, sodium,
tromethamine and zinc salts. Hemisalts of acids and bases may also
be formed, for example, hemisulfate and hemicalcium salts. For a
review on suitable salts, see "Handbook of Pharmaceutical Salts:
Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH,
Weinheim, Germany, 2002).
[0250] Pharmaceutically acceptable salts of compounds of the
invention may be prepared by for example, one or more of the
following methods:
[0251] by reacting the compound of the invention with the desired
acid or base;
[0252] by removing an acid- or base-labile protecting group from a
suitable precursor of the compound of the invention or by
ring-opening a suitable cyclic precursor, for example, a lactone or
lactam, using the desired acid or base; or
[0253] by converting one salt of the compound of the invention to
another by reaction with an appropriate acid or base or by means of
a suitable ion exchange column.
[0254] These methods are typically carried out in solution. The
resulting salt may precipitate out and be collected by filtration
or may be recovered by evaporation of the solvent. The degree of
ionisation in the resulting salt may vary from completely ionised
to almost non-ionised.
[0255] Compounds that have the same molecular formula but differ in
the nature or sequence of bonding of their atoms or the arrangement
of their atoms in space are termed "isomers". Isomers that differ
in the arrangement of their atoms in space are termed
"stereoisomers". Stereoisomers that are not mirror images of one
another are termed "diastereomers" and those that are
non-superimposable mirror images of each other are termed
"enantiomers". When a compound has an asymmetric centre, for
example, it is bonded to four different groups, a pair of
enantiomers is possible. An enantiomer can be characterized by the
absolute configuration of its asymmetric centre and is described by
the R- and S-sequencing rules of Cahn and Prelog, or by the manner
in which the molecule rotates the plane of polarized light and
designated as dextrorotatory or levorotatory (i.e., as (+) or
(-)-isomers respectively). A chiral compound can exist as either
individual enantiomer or as a mixture thereof. A mixture containing
equal proportions of the enantiomers is called a "racemic mixture".
Where a compound of the invention has two or more stereo centres
any combination of (R) and (S) stereoisomers is contemplated. The
combination of (R) and (S) stereoisomers may result in a
diastereomeric mixture or a single diastereoisomer. The compounds
of the invention may be present as a single stereoisomer or may be
mixtures of stereoisomers, for example racemic mixtures and other
enantiomeric mixtures, and diasteroemeric mixtures. Where the
mixture is a mixture of enantiomers the enantiomeric excess may be
any of those disclosed above. Where the compound is a single
stereoisomer the compounds may still contain other diasteroisomers
or enantiomers as impurities. Hence a single stereoisomer does not
necessarily have an enantiomeric excess (e.e.) or diastereomeric
excess (d.e.) of 100% but could have an e.e. or d.e. of about at
least 85%
[0256] The compounds described herein may possess one or more
asymmetric centres; such compounds can therefore be produced as
individual (R)- or (S)-stereoisomers or as mixtures thereof. Unless
indicated otherwise, the description or naming of a particular
compound in the specification and claims is intended to include
both individual enantiomers and mixtures, racemic or otherwise,
thereof. The methods for the determination of stereochemistry and
the separation of stereoisomers are well-known in the art (see
discussion in Chapter 4 of "Advanced Organic Chemistry", 4th
edition J. March, John Wiley and Sons, New York, 2001), for example
by synthesis from optically active starting materials or by
resolution of a racemic form. Some of the compounds of the
invention may have geometric isomeric centres (E- and Z-isomers).
It is to be understood that the present invention encompasses all
optical, diastereoisomers and geometric isomers and mixtures
thereof.
[0257] Compounds and salts described in this specification may be
isotopically-labelled (or "radio-labelled"). Accordingly, one or
more atoms are replaced by an atom having an atomic mass or mass
number different from the atomic mass or mass number typically
found in nature. Examples of radionuclides that may be incorporated
include .sup.2H (also written as "D" for deuterium), .sup.3H (also
written as "T" for tritium), .sup.11C, .sup.13C, .sup.14C,
.sup.15O, .sup.17O, .sup.18O, .sup.18F and the like. The
radionuclide that is used will depend on the specific application
of that radio-labelled derivative. For example, for in vitro
competition assays, .sup.3H or .sup.14C are often useful. For
radio-imaging applications, .sup.11C or .sup.18F are often useful.
In some embodiments, the radionuclide is .sup.3H. In some
embodiments, the radionuclide is .sup.14C. In some embodiments, the
radionuclide is .sup.11C. And in some embodiments, the radionuclide
is .sup.18F.
[0258] It is also to be understood that certain compounds described
herein may exist in solvated as well as unsolvated forms such as,
for example, hydrated forms. It is to be understood that the
invention encompasses all such solvated forms.
[0259] It is also to be understood that certain compounds described
herein may exhibit polymorphism, and that the invention encompasses
all such.
[0260] Compounds described herein may exist in a number of
different tautomeric forms and references to compounds of the
invention include all such forms. For the avoidance of doubt, where
a compound can exist in one of several tautomeric forms, and only
one is specifically described or shown, all others are nevertheless
embraced by compounds of the invention. Examples of tautomeric
forms include keto-, enol-, and enolate-forms, as in, for example,
the following tautomeric pairs: keto/enol (illustrated below),
imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime,
thioketone/enethiol, and nitro/aci-nitro.
[0261] Throughout the description and claims of this specification,
the words "comprise" and "contain" and variations of them mean
"including but not limited to", and they are not intended to (and
do not) exclude other moieties, additives, components, integers or
steps.
[0262] Throughout the description and claims of this specification,
the singular encompasses the plural unless the context otherwise
requires. In particular, where the indefinite article is used, the
specification is to be understood as contemplating plurality as
well as singularity, unless the context requires otherwise.
[0263] Features, integers, characteristics, compounds, chemical
moieties or groups described in conjunction with a particular
aspect, embodiment or example of the invention are to be understood
to be applicable to any other aspect, embodiment or example
described herein unless incompatible therewith. All of the features
disclosed in this specification (including any accompanying claims,
abstract and drawings), and/or all of the steps of any method or
process so disclosed, may be combined in any combination, except
combinations where at least some of such features and/or steps are
mutually exclusive. The invention is not restricted to the details
of any foregoing embodiments. The invention extends to any novel
one, or any novel combination, of the features disclosed in this
specification (including any accompanying claims, abstract and
drawings), or to any novel one, or any novel combination, of the
steps of any method or process so disclosed.
[0264] The reader's attention is directed to all papers and
documents which are filed concurrently with or previous to this
specification in connection with this application and which are
open to public inspection with this specification, and the contents
of all such papers and documents are incorporated herein by
reference.
EXAMPLES
Methods
Animals.
[0265] Trpc4 gene-disrupted (knockout) mice on the C57BL/6J
background (B6.129P2-Trpc4.sup.tm1Dgen/H) were generated by
Deltagen Inc. and supplied by the Medical Research Council Harwell,
UK. The sequence spanning base 1272 to base 1330 of the Trpc4 gene
was deleted and inserted with a Lac-Z neo cassette to create a
detectable mutation in the mice. Trpc5 gene-disrupted (knockout)
C57BL/6 mice were generated as part of the International Mouse
Phenotyping Consortium (IMPC) based on Trpc5 gene-targeted ES cells
originally created by the Knockout Mouse Project (KOMP)
(Trpc5tm1b(KOMP)Wtsi) and provided by Riken BRC, Japan. Mice were
intercrossed to generate wild-types and knockouts. Mice were weaned
at 3 weeks of age and 2-5 mice were housed in the same cage with
same-sex littermates under a 12 hr light/dark cycle. Only male mice
were used in this study. Food pellets and water were provided ad
libitum. All procedures were approved by the University of Leeds
Animal Welfare and Ethical Review Body and were conducted under a
moderate protocol on a project license issued by the competent
authority of the United Kingdom.
Glucose and Insulin Tolerance Tests.
[0266] For Glucose Tolerance Test (GTT) mice were fasted overnight
for 16 hr following application of 1 gkg.sup.-1 D-glucose
intraperitoneally. Blood glucose levels were then measured every 0,
30, 60, 90 and 120 min with a glucose meter. For Insulin Tolerance
Test (ITT) mice were fasted for 2 hr followed by administration of
0.75 unitkg.sup.-1 insulin intraperitoneally. Blood glucose
measurements were performed in the same manner as GTT.
Metabolic Measurements
[0267] Individual mice were housed in single metabolic cages where
food powder and water were provided ad libitum. Mice were allowed
to acclimatize to the cage conditions for 72 hr. Food and water are
then replaced with exact amounts of each, followed by measurement
of food and water intake and excretory output after 48 hr. Body
temperatures were measured by an infrared thermometer.
Cell Culture
[0268] Mouse stromal vascular fraction (SVF) cells from
subcutaneous adipose tissue were isolated by mincing of the tissue
followed by enzymatic digestion with 0.3 mgmL.sup.-1 Collagenase
type II (Sigma), 0.3 mgmL.sup.-1 Collagenase type IV (Sigma), 2
mgmL.sup.-1 BSA (Sigma), 0.08 mgmL.sup.-1 DNase I (Sigma) and 0.04
mgmL.sup.-1 Dispase (Thermo Fisher Scientific) in serum free RPMI
1640 media (Gibco) for 45 min at 37.degree. C. in 95% O.sub.2 and
5% CO.sub.2 environment. Isolated cells were centrifuged and
processed through a 100 .mu.m cell strainer. Following
centrifugation, the cell pellet was reconstituted in full RPMI 1640
media containing 15% fetal calf serum and 1%
penicillin-streptomycin (Gibco). Media was changed on the following
day to remove non-adherent cells. Adipocyte differentiation.sup.32
was done at 100% cell confluence in full RPMI 1640 media containing
1 .mu.M dexamethasone (Sigma), 0.5 mM isobitylmethylxanthine
(Sigma), 20 .mu.gmL.sup.-1 insulin (Sigma), 50 nM T3 (Sigma) and 5
.mu.M troglitazone (Sigma) for 48 hr followed by incubation with
full RPMI 1640 media containing 20 .mu.gmL.sup.-1 insulin for 72
hr. Cells are then maintained in full RPMI 1640 media for another 2
days until maturation. HEK 293 cells stably expressing
tetracycline-inducible TRPC4 and TRPC5 were maintained in
DMEM+GlutaMAX-1 (Thermo Fisher Scientific) containing 10% fetal
calf serum, 1% penicillin/streptomycin, 400 .mu.gmL.sup.-1 zeocin
and 5 .mu.gmL.sup.-1 blasticidin S at 37.degree. C. in a 5%
CO.sub.2 incubator. Expression was induced by incubation with 1
.mu.gmL.sup.-1 tetracycline 24 hr prior to experiments.
Immunohistochemistry
[0269] Tissues were fixed in 4% paraformaldehyde, processed in
paraffin and sectioned into 5 .mu.m sections for staining. Standard
Hematoxylin and Eosin and Sirius Red staining protocols were
applied. For UCP1 staining in adipose tissue sections, enzymatic
antigen retrieval was done using 0.1% (v/v) Trypsin, 0.1% (w/v)
CaCl.sub.2) in phosphate buffered saline (PBS), pH 7.8 (NaOH) in a
37.degree. C. humidifier incubator for 1.5 hr. Following primary
antibody incubation, signals were amplified using a biotin
conjugated secondary antibody (Jackson ImmunoResearch) followed by
streptavidin conjugated HRP (Abcam) incubation for 1 hr and
3,3'-diaminobenzidine tetrahydrochloride (DAB) (Thermo Fisher
Scientific) incubation for 15 min. Images were collected using a
standard light microscope. Fiji (ImageJ) (National Institute of
Health) was used to calculate % of staining in IHC images.
Quantitative RT-PCR Analysis
[0270] RNA from cell and tissue samples was obtained using TRIzol
reagent (Thermo Fisher Scientific). 1 .mu.g RNA was
reverse-transcribed using High-Capacity cDNA Reverse Transcription
Kit (Applied Biosystems) and RT-PCR analysis was performed using
SYBR Select Masters Mix (Thermo Fisher Scientific) and
LightCycler0480 (Roche Life Science). The primers used are
tabulated in Supplementary Table 1.sup.34. Relative fold-change in
expression was calculated using the comparative cycle method
(2.sup.-.DELTA..DELTA.Ct. Mouse Gapdh gene was used as the
reference.
Intracellular Ca.sup.2+ Measurement
[0271] HEK 293 cells containing stable incorporation of inducible
TRPC4 or TRPC5 expression were seeded into 96-well clear-bottomed
poly-D-lysine-coated black plates (Corning Life Sciences) at 90%
confluence 24 hr before the experiment. Cells were loaded with 2
.mu.M Fura-2-AM in Standard Bath Solution (SBS) at 37.degree. C.
for 45 min in the presence of 0.01% pluronic acid (Sigma). SBS
contained (in mM): 140 NaCl, 5 KCl, 1.2 MgCl.sub.2, 1.5
CaCl.sub.2), 8 Glucose and 10 HEPES, pH 7.4 (NaOH).sup.35. Cells
were washed three times with SBS prior to Ca.sup.2+ measurements.
In some wells cells were incubated with DE2 or C31 for 30 minutes
prior to the recording. The Fura-2-AM fluorescence was recorded
using a 96-well fluorescence plate reader FlexStation II (Molecular
Devices) at excitation wavelengths of 340 nm and 380 nm, and the
emitted light was collected at 510 nm. Measurements were made at
room temperature (21.+-.3.degree. C.).
[0272] ELISA Mouse TNF.alpha. in mouse serum was measured in
accordance with the manufacturer's guidelines (EMTNFA, Thermo
Fisher Scientific).
Protein Preparation and Immunoblotting
[0273] Proteins were isolated from cells and tissues using
radio-immunoprecipitation assay (RIPA) buffer containing (in mM)
150 NaCl, 20 Tris HCl, 1 EGTA, 1 EDTA, 1% NP-40, 0.1% sodium
dodecyl sulfate (SDS) and 1% sodium deoxylate. Complete.TM.
protease inhibitor (Roche Life Science) was added into RIPA fresh
before use in accordance with the manufacturer's instructions.
Immunoblotting was carried out under standard protocols with
primary antibodies anti-UCP1 (1:500, ab23841; Abcam), Cytochrome C
(1:4000, ab110325; Abcam), Akt (pan) (1:1000, 4691; Cell Signaling)
and Phospho-Akt (Ser473) (1:500, 4060; Cell Signaling). Anti-GAPDH
(1:4000, AM4300; Ambion Life Technologies) was used as the loading
control. Secondary antibodies anti-rabbit-HRP (1:3000, 711-035-152,
Jackson ImmunoResearch) and anti-mouse-HRP (1:3000, 715-035-150,
Jackson ImmunoResearch) were used. Chemiluminescence signals were
visualized using SuperSignal.RTM. West Femto Maximum Sensitivity
Substrate (Thermo Fisher Scientific).
Respirometry
[0274] scWAT was digested with the method stated in Cell culture.
.about.3,000 000 cells from the SVF and adipocyte fractions were
pooled into individual cell culture dishes. Cells were incubated
with either TRPC4/5 inhibitors DE2/C31 or vehicle DMSO in full RPMI
1640 for 24 hrs in a cell culture incubator. Prior to the
experiment cells were harvested from culture dishes, centrifuged
and reconstituted in mitochondrial respiration medium MiR05
containing (in mM): 0.5 EGTA, 3 MgCl.sub.2.6H.sub.2O, 60
Lactobionic acid, 20 Taurine, 10 KH.sub.2PO.sub.4, 20 HEPES, 110
D-Sucrose, added with 1 gL.sup.-1 BSA. The cells were inoculated
into the OROBOROS Oxygraph-2k chambers and oxidative
phosphorylation analyses were performed using the mitochondrial
substrate-uncoupler-inhibitor-titration protocol consisting of:
substrate activation using sodium pyruvate, inhibition of ATP
synthase using oligomycin, uncoupling via sequential addition of
FCCP to maximum oxygen flux, mitochondria complex I and III
inhibition using Rotenone and Antimycin A. Traces were analyzed
using DatLab 6 Software (OROBOROS), measuring for routine state
respiration (routine), mitochondrial leak (leak), maximum
uncoupling capacity of the electron transport system (ETS) and
rotenone and antimycin A-inhibited residual oxygen consumption
(ROX).
Immunocytochemistry
[0275] MitoTracker.RTM. Red FM (M22425, Thermo Fisher Scientific)
was used according to manufacturer's instructions. Adipocytes were
incubated with 400 nM MitoTracker.RTM. Red FM in full RPMI 1640 for
45 min. Cells were then washed 5 times with serum free RPMI 1640
and fixed in 4% PFA for 1 hr at room temperature. Following
fixation, cells were permeabilized with 0.1% Triton-X100 in TBST
containing (in mM) 150.6 NaCl, 12.4 Tris-base, 2.68 KCl and 0.2%
Tween-20, pH 7.1-7.2 (HCl) and incubated with primary antibody UCP1
overnight at 4.degree. C. UCP1 signal was amplified using a
secondary antibody conjugated to AlexFluor-488 (Jackson
ImmunoResearch). Cells were counterstained with DAPI (62248, Thermo
Fisher Scientific) before mounting. For whole mount
immunocytochemistry, adipose tissue was excised and fixed in 4% PFA
overnight. Tissues were dehydrated with ascending concentrations of
methanol in PBS and alternated between room temperature and
-80.degree. C. for antigen retrieval. Following rehydration with
descending concentrations of methanol in TBST, tissues were
incubated in TBST containing 0.1% Triton-X100 for 2 hr. Tissues
were incubated in blocking solution overnight. Tissues were
incubated in primary antibody to caveolin-1 (1:200, 611339, BD
Transduction Laboratories.TM.) and conjugated primary-secondary
antibody F4/80:Pacific Blue.RTM. (1:2000, MCA497PBT, Bio-Rad)
overnight. Caveolin-1 staining was amplified using
AlexaFluor680.RTM. (1:200, cat no. 115-635-174, Jackson
immunoresearch). Images were collected using a LSM880 with Airyscan
(Zeiss).
Chemicals
[0276] (-)-Englerin A (EA) powder (#82530, Phytolab) was
reconstituted in DMSO and the stock solution was 10 mM. Compound 31
(C31) was synthesized as previously described in WO 2014/143799 and
dissolved in DMSO. The stock solution was 5 mM. DE2 was synthesized
as described below. The stock solution was 10 mM in DMSO. Sodium
pyruvate (Sigma) was made fresh at 2 M in ddH.sub.2O. FCCP (Sigma)
was made at 1 mM in EtOH. Rotenone (Sigma) was made at 0.1 mM in
EtOH. Oligomycin was made at 4 mgml.sup.-1 in EtOH. Antimycin A was
made at 5 mM in EtOH.
Synthesis of DE2:
({1-[(4-Chlorophenyl)methyl]-1H-benzimidazol-2-yl}methyl)diethylamine
(1H-Benzimidazol-2-ylmethyl)diethylamine
##STR00041##
[0278] A mixture of diethylamine (220 mg, 3.00 mmol) and anhydrous
potassium carbonate (210 mg, 1.50 mmol) in acetone (5 mL) was
stirred for 5 minutes before the addition of
2-(chloromethyl)-1H-benzimidazole (250 mg, 1.50 mmol). The reaction
mixture was stirred for 8 hr at room temperature and upon
completion, the solvent was removed in vacuo. The crude product was
extracted with EtOAc twice (10 mL.times.2) and the combined organic
extracts were washed with brine (10 mL) and dried (MgSO.sub.4). The
solution was concentrated and purified using flash column by
eluting with 0-25% MeOH in DCM. The selected fractions were
evaporated to give a yellow solid (230 mg, 76%). R.sub.1 0.63 (2:3
MeOH:DCM). LC-MS m/z 204.0 (M+H)+, RT 1.03 min; .delta..sub.H (500
MHz, CD.sub.3OD) 1.05 (6H, t, J=7.3 Hz, H2'), 2.57 (4H, quartet,
J=7.3 Hz, H1'), 3.82 (2H, s, CH.sub.2), 5.36 (1H, br s, NH),
7.06-7.19 (2H, m, H5, H6), 7.53-7.56 (2H, m, H7, H4); .delta..sub.c
(125 MHz, CD.sub.3OD) 12.1 (C2'), 48.5 (C3'), 52.0 (CH.sub.2),
115.7 (C7), 123.3 (C4), 139.6 (C4a), 154.6 (C2); FT-IR (cm.sup.-1)
3046 (C--H, aromatic), 1273 (C--N, aromatic amine); HRMS (ESI): m/z
calcd. for C.sub.12H.sub.18N.sub.3[M+H].sup.+ 204.1497. Found:
204.1495.
({1-[(4-Chlorophenyl)methyl]-1H-benzimidazol-2-yl}methyl)diethylamine
##STR00042##
[0280] Under N.sub.2 gas, (1H-benzimidazol-2-ylmethyl)diethylamine
(430 mg, 2.0 mmol) was dissolved in anhydrous THF (5 mL), then
cooled to 0.degree. C. To this solution was added sodium hydride
(60% suspension in mineral oil) (96 mg, 4.0 mmol) and the mixture
stirred at 0.degree. C. for 5 minutes. Then, 4-chlorobenzyl
chloride (430 mg, 2.10 mmol) was added followed by the addition of
tetrabutylammonium bromide (0.06% eq.). The mixture was stirred at
room temperature overnight, then diluted with a solution of water
(2 drops) in THF (5 mL). The mixture was filtered through Celite,
washed with EtOAc (5 mL) and concentrated to dryness.
[0281] The crude was purified using flash column by eluting with a
0-100% EtOAc in Pet.Ether. The product fractions were combined and
the solvent was evaporated to afford a yellow solid (470 mg, 69%).
R 0.39 (4:1 EtOAc:Pet. Ether). LC-MS m/z 328.0 (M+H).sup.+, RT 1.65
min; .delta.H (500 MHz, CDCl.sub.3) 1.01 (6H, t, J=7.0 Hz, H2'),
2.60 (4H, quartet, J=7.0 Hz, H1'), 3.86 (2H, s,
CH.sub.2-diethylamine), 5.63 (2H, s, CH.sub.2-4-chlorophenyl), 7.01
(2H, d, J=8.0 Hz, H2''), 7.19-7.31 (5H, m, H3'', H7, H6, H5), 7.80
(1H, d, J=8.0 Hz, H4); .delta.c (125 MHz, CDCl.sub.3) 11.3 (C2'),
46.5 (CH.sub.2-diethylamine), 47.0 (C1'), 51.5
(CH.sub.2-4-chlorophenyl), 109.7 (C7), 119.8 (C4), 122.2 (C5),
122.9 (C6), 127.7 (C2''), 129.0 (C3''), 133.4 (C4''), 135.2 (C7a),
135.9 (C1''), 142.4 (C4a), 152.0 (C2); FT-IR (cm.sup.-1) 3057
(C--H, aromatic), 1249 (C--N, aromatic amine); HRMS (ESI): m/z
calcd. for C.sub.1H.sub.23ClN.sub.3 [M+H].sup.+ 328.1584. Found:
328.1575; M.P 63-65.degree. C.
Statistical Analysis
[0282] Data were analyzed using Origin software (version 9.1,
OriginLab, Northampton, Mass., USA). Paired 2-sample t-test and
one-way ANOVA were used to perform tests of significance. n is the
number of independent experiments. In experiments on intracellular
Ca.sup.2+ measurements, n refers to the number of individual
96-well plates used and N is the total number of wells measured.
Statistical significance is indicated by numerical values on
designated figures.
Results
[0283] Studies were performed on genetically-modified mice in which
TRPC4 protein or TRPC5 protein was absent, designated as TRPC4 and
TRPC5 knockout mice (C4.sup.KO and C5KO)
Example 1: Normal Body Weight on Chow Diet
[0284] Body weight and Subcutaneous white adipose tissue (scWAT)
fat pad weight were measured in C4WT (wildtype litter-mate controls
for TRPC4 knockout mice), C5WT (wildtype litter-mate controls for
TRPC5 knockout mice), C4KO (TRPC4 knockout mice) and C5KO (TRPC5
knockout mice) (n=6).
[0285] C4.sup.KO and C5.sup.KO mice on chow diet were apparently
normal, showing no difference in body weight or fat pad weight
compared with litter-mate control mice (FIG. 1).
Example 2: Inability to Gain Excess Total Body Weight on High Fat
Diet
[0286] Mice were next studied on 60% high-fat diet (excess calorie
intake). Bodyweights of mice (C4WT, C5WT, C4KO and C5KO) during 8
weeks of diet feeding were measured (n=7-8). Diets were chow (Chow)
or 60% high-fat (HFD). Control wildtype (WT) litter-mates
(C4.sup.WT and C5.sup.WT) gained excess total weight as expected
during the 8 week experiment period (FIG. 2). C4.sup.KO and
C5.sup.KO mice were, by contrast, strikingly similar to mice on
chow diet, showing only normal weight gain as the mice matured
(FIG. 2).
Example 3: Inability to Gain Excess Fat Pad Weight on High Fat
Diet
[0287] Mice were next studied on 60% high-fat diet (excess calorie
intake). Subcutaneous white adipose tissue (scWAT) fat pad weights
of mice (C4WT, C5WT, C4KO and C5KO) during 8 weeks of diet feeding
were measured (n=7-8). Diets were chow (Chow) or 60% high-fat
(HFD). Control wildtype (WT) litter-mates (C4.sup.WT and C5.sup.WT)
gained excess fat pad weight as expected during the 8 week
experiment period (FIG. 3). C4.sup.KO and C5.sup.KO mice were, by
contrast, strikingly similar to mice on chow diet, showing only
normal weight gain as the mice matured (FIG. 3).
Example 4: Protection Against Hyperglycaemia
[0288] Blood glucose concentrations after bolus intraperitoneal
(IP) injection of glucose (glucose tolerance test, GTT) were also
measured for mice (C4WT, C5WT, C4KO and C5KO) on chow or 60% high
fat diet (HFD) (n=7-8). Wild-type mice on 60% high-fat diet showed
hyperglycemia (FIG. 4), which is an expected additional
characteristic of animals on excess calorie intake. However,
C4.sup.KO and C5.sup.KO mice on this diet were protected against
these effects (FIG. 4).
Example 5: Protection Against Insulin-Resistance
[0289] Blood glucose concentrations after bolus intraperitoneal
(IP) injection of insulin (insulin tolerance test, ITT) were also
measured for mice (C4WT, C5WT, C4KO and C5KO) on chow or 60% high
fat diet (HFD) (n=7-8). Wild-type mice on 60% high-fat diet showed
insulin-resistance (FIG. 5), which is an expected additional
characteristic of animals on excess calorie intake. However,
C4.sup.KO and C5.sup.KO mice on this diet were protected against
these effects (FIG. 5).
Example 6: Protection Against Systemic Inflammation
[0290] Serum tumour necrosis factor alpha (TNF.alpha.)
concentration was measured by ELISA in mice (C4WT, C5WT, C4KO and
C5KO) on chow or 60% high fat diet (HFD) (n=5). Wild-type mice on
60% high-fat diet showed systemic tissue inflammation (FIG. 6),
which is an expected additional characteristic of animals on excess
calorie intake. However, C4.sup.KO and C5.sup.KO mice on this diet
were protected against these effects (FIG. 6).
Example 7: Protection Against Adipose Tissue Inflammation
[0291] Relative mRNA analysis for the pro-inflammatory markers
TNF.alpha. and interleukin-6 (IL6) in fat pad were also measured
for mice (C4WT, C5WT, C4KO and C5KO) on chow or 60% high fat diet
(HFD) (n=4). Wild-type mice on 60% high-fat diet showed adipose
tissue inflammation (FIG. 7), which is an expected additional
characteristic of animals on excess calorie intake. However,
C4.sup.KO and C5.sup.KO mice on this diet were protected against
these effects (FIG. 7).
Example 8: Protection Against Ectopic Fat in the Liver
(Steatosis)
[0292] Total liver weight (FIG. 8 upper bar chart, n=7-8) and
percentage liver fat determined by histological analysis (FIG. 8
lower bar chart, n=5) were also measured for mice (C4WT, C5WT, C4KO
and C5KO) on chow or 60% high fat diet (HFD). Wild-type mice on 60%
high-fat diet showed steatosis (FIG. 8), which is an expected
additional characteristic of animals on excess calorie intake.
However, C4.sup.KO and C5.sup.KO mice on this diet were protected
against these effects (FIG. 8).
Example 9: No Change in Food Intake or Excretion
[0293] In order to determine whether the protection observed in
C4.sup.KO and C5.sup.KO mice could be explained by differences in
food intake or excretion, food intake, fecal excretion and urinary
excretion were determined in mice housed in metabolic cages (n=6).
Mice (C4WT, C5WT, C4KO and C5KO) were either on chow or 60% high
fat diet (HFD). The protection observed in C4.sup.KO and C5.sup.KO
mice was not explained by differences in food intake or excretion
(FIG. 9).
Example 10: Increased Expression of Markers of White-to-Brown
Adipocyte Phenotypic Switch ("Beiging")
[0294] An alternative explanation could be that white adipocytes in
C4.sup.KO and C5.sup.KO mice shift to a thermogenic
("energy-burning") phenotype, often referred to as adipocyte
beiging.
[0295] Western blot analysis of UCP1 and Cytochrome C in
subcutaneous white adipose tissue (n=4) was carried out in C4WT,
C5WT, C4KO and C5KO mice. In support of this idea there was greater
expression of the mitochondrial proteins UCP1 and Cytochrome C in
white adipose tissue of C4.sup.KO and C5.sup.KO mice (FIG. 10).
Example 11: Increased Adipocyte Thermogenesis
[0296] Oxygen consumption was also measured in adipocytes from
subcutaneous white adipose tissue of C4WT, C5WT, C4KO and C5KO mice
(n=3-4). Oxygen consumption was measured for routine respiration
after addition of sodium pyruvate, mitochondrial leak after
addition of oligomycin, maximum electron transport chain-mediated
oxygen consumption after sequential FCCP addition (ETS), and
non-OXPHO respiration after addition of rotenone and antimycin A
(ROX).
[0297] High-resolution respirometry showed increased mitochondrial
respiration in adipocytes of C4.sup.KO and C5.sup.KO mice (FIG.
11), providing further support that white adipocytes in C4.sup.KO
and C5.sup.KO mice shift to a thermogenic ("energy-burning")
phenotype.
Example 12: Inhibition of TRPC4 and TRPC5 Channels by DE2 and
C31
[0298] To examine the effects of the small molecule inhibitors DE2
and C31, intracellular Ca.sup.2+ measurements were made from HEK
293 cells stably expressing inducible human TRPC4 (upper panel) or
human TRPC5 (lower panel). Data presented in FIG. 12 are
mean.+-.SEM (3-4 replicates each) and representative of 4
independent experiments each. Channels were activated by the
TRPC4/TRPC5 channel agonist (-)-Englerin A (EA, 100 nM). The
vehicle control was DMSO. Cells were pre-incubated with (30 min)
and maintained in 10 .mu.M DE2 or 100 nM C31 where indicated. DE2
and C31 demonstrated inhibition of TRPC4 and TRPC5 channels (FIG.
12).
Example 13: Small-Molecule Inhibitors Increase Adipocyte
Thermogenesis
[0299] To examine the effect of DE2 and C31 on thermogenesis in
adipocytes, oxygen consumption was measured in adipocytes from
subcutaneous white adipose tissue of mice. Data are for routine
respiration after addition of sodium pyruvate, mitochondrial leak
after addition of oligomycin, maximum electron transport
chain-mediated oxygen consumption after sequential FCCP addition
(ETS), and non-OXPHO respiration after addition of rotenone and
antimycin A (ROX). Cells were pre-incubated with (24 hr) and
maintained in 10 .mu.M DE2 or 100 nM C31 where indicated. The
vehicle control was DMSO.
[0300] Incubation with both agents (DE2 and C31) caused increases
in adipocyte mitochondrial respiration (FIG. 13). The effects were
quantitatively similar to those caused by TRPC4 and TRPC5
knockout.
Example 14: Small-Molecule Inhibitors Increase Expression of
Markers of White-to-Brown Adipocyte Phenotypic Switch
("Beiging")
[0301] To determine the influence of DE2 and C31 on expression of
markers of white-to-brown adipocyte phenotypic switching,
transcript accumulation of UCP1 and Cytochrome C was measured by
mRNA analysis in adipocytes from subcutaneous white adipose tissue
(n=4-5) of C4WT, C5WT, C4KO, and C5KO mice. Cells were
pre-incubated with (24 hr) and maintained in 10 .mu.M DE2 or 100 nM
C31 where indicated. The vehicle control was DMSO.
[0302] Incubation with both agents (DE2 and C31) caused increases
in adipocyte UCP1/Cytochrome C expression (FIG. 14). The effects
were quantitatively similar to those caused by TRPC4 and TRPC5
knockout (FIG. 14).
Example 15: Small Molecules Significantly Reduced Plasma Insulin
Following Oral Dosing
[0303] C31 (designated "Test item" in FIG. 15) was administered to
HFD-fed wildtype adult male mice (DIN) with a bi-daily oral dosing
(BID) regimen (N=10 per group). In parallel groups comparator,
established drugs were also administered. The HFD-fed wildtype
adult male mice (DIN) administered with C31 had significantly
reduced plasma insulin after an oral glucose tolerance test (OGTT),
see FIG. 15.
[0304] In a similar study, C31 (designated "Test item" in FIG. 16)
was administered to HFD-fed wildtype adult male mice (DIN) with a
bi-daily oral dosing (BID) regimen (N=10 per group). In parallel
groups comparator, established drugs were also administered. The
HFD-fed wildtype adult male mice (DIN) administered with C31 had
significantly reduced blood glucose after an insulin tolerance test
(FIG. 16).
Example 16
[0305] In a study including 16 DNT5 mice and 19 control littermates
(which lacked DNT5) it was observed that in vivo expression of a
dominant negative TRPC5 ion pore mutant from a transgene (DNT5)
significantly reduced body weight gain in hypercholesterolaemic
(ApoE-/-) adult male mice fed western-style diet. The mutant enters
native TRPC1/4/5 channels to inhibit ion permeation (FIG. 17).
[0306] In a similar study including N=14 DNT5 and N=11 control
littermates (which lacked DNT5) it was observed that in vivo
expression of a dominant negative TRPC5 ion pore mutant from a
transgene (DNT5) significantly reduced liver steatosis in
hypercholesterolaemic (ApoE-/-) adult male mice fed western-style
diet (FIG. 18).
* * * * *