U.S. patent application number 15/840429 was filed with the patent office on 2018-04-12 for apoptosis signal-regulating kinase inhibitor.
The applicant listed for this patent is Gilead Sciences, Inc.. Invention is credited to GREGORY NOTTE.
Application Number | 20180099950 15/840429 |
Document ID | / |
Family ID | 47684038 |
Filed Date | 2018-04-12 |
United States Patent
Application |
20180099950 |
Kind Code |
A1 |
NOTTE; GREGORY |
April 12, 2018 |
APOPTOSIS SIGNAL-REGULATING KINASE INHIBITOR
Abstract
The present invention relates to a compound of formula (I):
##STR00001## The compound has apoptosis signal-regulating kinase
("ASK1") inhibitory activity, and is thus useful in the treatment
of diseases such as kidney disease, diabetic nephropathy and kidney
fibrosis.
Inventors: |
NOTTE; GREGORY; (Redwood
City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Gilead Sciences, Inc. |
Foster City |
CA |
US |
|
|
Family ID: |
47684038 |
Appl. No.: |
15/840429 |
Filed: |
December 13, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15491689 |
Apr 19, 2017 |
9873682 |
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15840429 |
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15140204 |
Apr 27, 2016 |
9750730 |
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15491689 |
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14254359 |
Apr 16, 2014 |
9333197 |
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15140204 |
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13748901 |
Jan 24, 2013 |
8742126 |
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14254359 |
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61591710 |
Jan 27, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 31/12 20180101; A61K 31/4439 20130101; C07D 401/04 20130101;
C07D 401/14 20130101; A61P 9/00 20180101; A61P 31/00 20180101; A61K
9/20 20130101; A61P 1/16 20180101; A61K 9/0053 20130101; A61P 13/12
20180101; A61P 19/04 20180101; C07D 233/56 20130101; A61K 45/06
20130101; A61P 3/10 20180101; A61P 11/00 20180101; C07D 213/56
20130101 |
International
Class: |
C07D 401/14 20060101
C07D401/14 |
Claims
1. A compound of formula (I) ##STR00011## Namely,
5-(4-cyclopropyl-1H-imidazol-1-yl)--N-(6-(4-isopropyl-4H-1,2,4-triazol-3--
yl)pyridin-2-yl)-2-fluoro-4-methylbenzamide, or a pharmaceutically
acceptable salt thereof.
2. A pharmaceutical composition comprising a therapeutically
effective amount of a compound or pharmaceutically acceptable salt
of claim 1 and a pharmaceutically acceptable carrier.
3. A method of treating chronic kidney disease comprising
administering a therapeutically effective amount of a compound of
claim 1, or pharmaceutically acceptable salt thereof, to a patient
in need thereof.
4. A method of treating diabetic kidney disease, diabetic
nephropathy, kidney fibrosis, liver fibrosis, or lung fibrosis
comprising administering a therapeutically effective amount of a
compound or pharmaceutically acceptable salt of claim 1, to a
patient in need thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 61/591,710, filed Jan. 27, 2012, the entirety
of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a novel compound for use in
the treatment of ASK1-mediated diseases. The invention also relates
to intermediates for its preparation and to pharmaceutical
compositions containing said novel compound.
BACKGROUND
[0003] Apoptosis signal-regulating kinase 1 (ASK1) is a member of
the mitogen-activated protein kinase kinase kinase ("MAP3K") family
that activates the c-Jun N-terminal protein kinase ("JNK") and p38
MAP kinase (Ichijo, H., Nishida, E., Irie, K., Dijke, P. T.,
Saitoh, M.,
[0004] Moriguchi, T., Matsumoto, K., Miyazono, K., and Gotoh, Y.
(1997) Science, 275, 90-94). ASK1 is activated by a variety of
stimuli including oxidative stress, reactive oxygen species (ROS),
LPS, TNF-.alpha., FasL, ER stress, and increased intracellular
calcium concentrations (Hattori, K., Naguro, I., Runchel, C., and
Ichijo, H. (2009) Cell Comm. Signal. 7:1-10; Takeda, K., Noguchi,
T., Naguro, I., and Ichijo, H. (2007) Annu. Rev. Pharmacol.
Toxicol. 48: 1-8.27; Nagai, H., Noguchi, T., Takeda, K., and
Ichijo, I. (2007) J. Biochem. Mol. Biol. 40:1-6). Phosphorylation
of ASK1 protein can lead to apoptosis or other cellular responses
depending on the cell type. ASK1 activation and signaling have been
reported to play an important role in a broad range of diseases
including neurodegenerative, cardiovascular, inflammatory,
autoimmune, and metabolic disorders. In addition, ASK1 has been
implicated in mediating organ damage following ischemia and
reperfusion of the heart, brain, and kidney (Watanabe et al. (2005)
BBRC 333, 562-567; Zhang et al., (2003) Life Sci 74-37-43; Terada
et al. (2007) BBRC 364: 1043-49).
[0005] ROS are reported be associated with increases of
inflammatory cytokine production, fibrosis, apoptosis, and necrosis
in the kidney. (Singh DK, Winocour P, Farrington K. Oxidative
stress in early diabetic nephropathy: fueling the fire. Nat Rev
Endocrinol 2011 Mar;7(3):176-184; Brownlee M. Biochemistry and
molecular cell biology of diabetic complications. Nature 2001 Dec
13; 414(6865):813-820; Mimura I, Nangaku M. The suffocating kidney:
tubulointerstitial hypoxia in end-stage renal disease. Nat Rev
Nephrol 2010 Nov; 6(11):667-678).
[0006] Moreover, oxidative stress facilitates the formation of
advanced glycation end-products (AGEs) that cause further renal
injury and production of ROS. (Hung K Y, et al.
N-acetylcysteine-mediated antioxidation prevents
hyperglycemia-induced apoptosis and collagen synthesis in rat
mesangial cells. Am J Nephrol 2009;29(3):192-202).
[0007] Tubulointerstitial fibrosis in the kidney is a strong
predictor of progression to renal failure in patients with chronic
kidney diseases (Schainuck L I, et al. Structural-functional
correlations in renal disease. Part II: The correlations. Hum
Pathol 1970; 1: 631-641.). Unilateral ureteral obstruction (UUO) in
rats is a widely used model of tubulointerstitial fibrosis. UUO
causes tubulointerstital inflammation, increased expression of
transforming growth factor beta (TGF-.beta.), and accumulation of
myofibroblasts, which secrete matrix proteins such as collagen and
fibronectin. The UUO model can be used to test for a drug's
potential to treat chronic kidney disease by inhibiting renal
fibrosis (Chevalier et al., Ureteral obstruction as a model of
renal interstitial fibrosis and obstructive nephropathy, Kidney
International (2009) 75, 1145-1152.
[0008] Thus, therapeutic agents that function as inhibitors of ASK1
signaling have the potential to remedy or improve the lives of
patients in need of treatment for diseases or conditions such as
neurodegenerative, cardiovascular, inflammatory, autoimmune, and
metabolic disorders. In particular, ASK1 inhibitors have the
potential to treat cardio-renal diseases, including kidney disease,
diabetic kidney disease, chronic kidney disease, fibrotic diseases
(including lung and kidney fibrosis), respiratory diseases
(including chronic obstructive pulmonary disease (COPD) and acute
lung injury), acute and chronic liver diseases.
[0009] U.S. Publication No. 2007/0276050 describes methods for
identifying ASK1 inhibitors useful for preventing and/or treating
cardiovascular disease and methods for preventing and/or treating
cardiovascular disease in an animal.
WO2009027283 discloses triazolopyridine compounds, methods for
preparation thereof and methods for treating autoimmune disorders,
inflammatory diseases, cardiovascular diseases and
neurodegenerative diseases.
[0010] U.S. Patent Publication No. 2001/00095410A1, published Jan.
13,2011, discloses compounds useful as ASK-1 inhibitors. U.S.
Patent Publication 2001/00095410A1 relates to compounds of Formula
(I):
##STR00002##
wherein:
[0011] R.sup.1 is alkyl, alkenyl, alkynyl, cycloalkyl, aryl,
heteroaryl, or heterocyclyl, all of which are optionally
substituted with 1, 2, or 3 substituents selected from halo, oxo,
alkyl, cycloalkyl, heterocyclyl, aryl, aryloxy, --NO.sub.2,
R.sup.6, --C(O)--R.sup.6, --OC(O)--R.sup.6 --C(O)--O--R.sup.6,
--C(O)--N(R.sup.6)(R.sup.7), --OC(O)--N(R.sup.6)(R.sup.7),
--S--R.sup.6, --S(.dbd.O)--R.sup.6, --S(.dbd.O).sub.2R.sup.6,
--S(.dbd.O).sub.2--N(R.sup.6)(R.sup.7),
--S(.dbd.O).sub.2--O--R.sup.6, --N(R.sup.6)(R.sup.7),
--N(R.sup.6)--C(O)--R.sup.7, --N(R.sup.6)--C(O)--O--R.sup.7,
--N(R.sup.6)--C(O)--N (R.sup.6)(R.sup.7),
--N(R.sup.6)--S(.dbd.O).sub.2--R.sup.6, --CN, and --O--R.sup.6,
[0012] wherein alkyl, cycloalkyl, heterocyclyl, phenyl, and phenoxy
are optionally substituted by 1, 2, or 3 substituents selected from
alkyl, cycloalkyl, alkoxy, hydroxyl, and halo; wherein R.sup.6 and
R.sup.7 are independently selected from the group consisting of
hydrogen, C.sub.1-C.sub.15 alkyl, cycloalkyl, heterocyclyl, aryl,
and heteroaryl, all of which are optionally substituted with 1-3
substituents selected from halo, alkyl, mono- or dialkylamino,
alkyl or aryl or heteroaryl amide, --CN, lower alkoxy, --CF.sub.3,
aryl, and heteroaryl; or R.sup.6 and R.sup.7 when taken together
with the nitrogen to which they are attached form a
heterocycle;
R.sup.2 is hydrogen, halo, cyano, alkoxy, or alkyl optionally
substituted by halo;
[0013] R.sup.3 is aryl, heteroaryl, or heterocyclyl, all of which
are optionally substituted with one or more substituents selected
from alkyl, alkoxy, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl,
heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, halo,
oxo, --NO.sub.2, haloalkyl, haloalkoxy, --CN, --O--R.sup.6,
--O--C(O)--R.sup.6, --O--C(O)--N(R.sup.6)(R.sup.7), --S--R.sup.6,
--N (R.sup.6)(R.sup.7), --S(.dbd.O)--R.sup.6,
--S(.dbd.O).sub.2R.sup.6, --S(.dbd.O).sub.2--N(R.sup.6)(R.sup.7),
--S(.dbd.O).sub.2--O--R.sup.6, --N(R.sup.6)--C(O)--R.sup.7,
--N(R.sup.6)--C(O)--O--R.sup.7,
--N(R.sup.6)--C(O)--N(R.sup.6)(R.sup.7), --C(O)--R.sup.6,
--C(O)--O--R.sup.6, --C(O)--N (R.sup.6)(R.sup.7), and
--N(R.sup.6)--S(.dbd.O).sub.2--R.sup.7, wherein the alkyl, alkoxy,
cycloalkyl, aryl, heteroaryl or heterocyclyl is further optionally
substituted with one or more substituents selected from halo, oxo,
--NO.sub.2, alkyl, haloalkyl, haloalkoxy, --N(R.sup.6)(R.sup.7),
--C(O)--R.sup.6, --C (O)--O--R.sup.6, --C(O)--N(R.sup.6)(R.sup.7),
--CN, --O--R.sup.6, cycloalkyl, aryl, heteroaryl and heterocyclyl;
with the proviso that the heteroaryl or heterocyclyl moiety
includes at least one ring nitrogen atom;
[0014] X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6,
X.sup.7 and X.sup.8 are independently C(R.sup.4) or N, in which
each R.sup.4 is independently hydrogen, alkyl, alkoxy, cycloalkyl,
aryl, heteroaryl, heterocyclyl, halo, --NO.sub.2, haloalkyl,
haloalkoxy, --CN, --O--R.sup.6, --S--R.sup.6,
--N(R.sup.6)(R.sup.7), --S(.dbd.O)--R.sup.6,
--S(.dbd.O).sub.2R.sup.6, --S(.dbd.O).sub.2--N(R.sup.6)(R.sup.7),
--S(.dbd.O).sub.2--O--R.sup.6, --N(R.sup.6)--C(O)--R.sup.7,
--N(R.sup.6)--C(O)--O--R.sup.7, --N(R.sup.6)--C(O)--N
(R.sup.6)(R.sup.7), --C(O)--R.sup.6, --C(O)--O--R.sup.6,
--C(O)--N(R.sup.6)(R.sup.7), or
--N(R.sup.6)--S(.dbd.O).sub.2--R.sup.7, wherein the alkyl,
cycloalkyl, aryl, heteroaryl, and heterocyclyl is further
optionally substituted with one or more substituents selected from
halo, oxo, --NO.sub.2, --CF.sub.3, --O--CF.sub.3,
--N(R.sup.6)(R.sup.7), --C(O)--R.sup.6, --C(O)--O--R.sup.7,
--C(O)--N(R.sup.6)(R.sup.7), --CN, --O--R.sup.6; or
[0015] X.sup.5 and X.sup.6 or X.sup.6 and X.sup.7 are joined to
provide optionally substituted fused aryl or optionally substituted
fused heteroaryl; and
with the proviso that at least one of X.sup.2, X.sup.3, and X.sup.4
is C(R.sup.4); at least two of X.sup.5, X.sup.6, X.sup.7, and
X.sup.8 are C(R.sup.4); and at least one of X.sup.2, X.sup.3,
X.sup.4, X.sup.5, X.sup.6, X.sup.7 and X.sup.8 is N.
[0016] The above disclosures notwithstanding, there is a need for
compounds that are potent and exhibit improved pharmacokinetic
and/or pharmacodynamic profiles for the treatment of diseases
related to ASK1 activation.
[0017] Surprisingly, applicants have discovered a novel compound
within the scope of U.S. patent publication US2011/0009410A
exhibiting good potency, improved pharmacokinetic and/or
pharmacodynamic profiles, on aggregate, compared to compounds
disclosed therein.
SUMMARY OF THE INVENTION
[0018] The present invention relates to a compound of the
formula:
##STR00003##
or a pharmaceutically acceptable salt thereof.
[0019] In one embodiment, the invention relates to the use of a
compound of formula (I) in the treatment of a disease in a patient
in need of treatment with an ASK1 inhibitor.
[0020] In another embodiment, the invention relates to a
pharmaceutical composition comprising a compound of formula (I) or
a pharmaceutically acceptable salt thereof, and one or more
pharmaceutically acceptable carriers.
[0021] In another embodiment, the invention is a method of treating
diabetic nephropathy, or complications of diabetes, comprising
administering a therapeutically effective amount of a compound of
formula (I) or a pharmaceutically acceptable salt thereof, to a
patient in need thereof.
[0022] In another embodiment, the invention relates to a method of
treating kidney disease, or diabetic kidney disease comprising
administering a therapeutically effective amount of a compound of
formula (I) or a pharmaceutically acceptable salt thereof, to a
patient in need thereof.
[0023] In another embodiment, the invention relates to a method of
treating kidney fibrosis, lung fibrosis, or idiopathic pulmonary
fibrosis (IPF) comprising administering a therapeutically effective
amount of a compound of formula (I) or a pharmaceutically
acceptable salt thereof, to a patient in need thereof.
[0024] In another embodiment, the invention relates to a method of
treating diabetic kidney disease, diabetic nephropathy, kidney
fibrosis, liver fibrosis, or lung fibrosis comprising administering
a therapeutically effective amount of a compound or salt of forumia
(I), to a patient in need thereof.
[0025] In another embodiment, the invention relates to
intermediates useful for the synthesis of the compound of formula
(I).
[0026] In another embodiment, the invention relates to the use of a
compound of formula (I) or a pharmaceutically acceptable salt
thereof for the treatment of chronic kidney disease.
[0027] In another embodiment, the invention relates to the use of a
compound of formula (I) or a pharmaceutically acceptable salt
thereof for the treatment of diabetic kidney disease.
[0028] In another embodiment, the invention relates to the use of a
compound of formula (I) or a pharmaceutically acceptable salt
thereof, in the manufacture of a medicament for the treatment of
chronic kidney disease.
[0029] In yet another embodiment, the invention relates to the
compound of formula (I) for use in therapy.
DETAILED DESCRIPTION OF THE INVENTION
FIGURES
[0030] FIG. 1 is a bar graph showing the levels of Collagen IV in
the kidney cortex of rats subjected to seven days of unilateral
ureteral obstruction and treated with either vehicle, or compound
of formula (I) at 1, 3, 10, or 30 mg/kg b.i.d. per day.
[0031] FIG. 2 shows representative images of kidney cortex sections
stained with alpha-smooth muscle actin (a marker of activated
myofibroblasts) from rats subjected to seven days of unilateral
ureteral obstruction and treated with either vehicle, or compound
of formula (I) at 1, 3, 10, or 30 mg/kg b.i.d. per day.
DEFINITIONS AND GENERAL PARAMETERS
[0032] As used herein, the following words and phrases are intended
to have the meanings set forth below, except to the extent that the
context in which they are used indicates otherwise. Where no
indication or definition is given, the ordinary meaning of the word
or phrase as found in a relevant dictionary or in common usage
known to one of skill in the art is implied.
[0033] The term "chronic kidney disease" as used herein refers to
progressive loss of kidney function over time typically months or
even years. Chronic kidney disease (CKD) is diagnosed by a
competent care giver using appropriate information, tests or
markers known to one of skill in the art. Chronic kidney disease
includes by implication kidney disease.
[0034] The term "diabetic kidney disease" as used herein refers to
kidney disease caused by diabetes, exacerbated by diabetes, or
co-presenting with diabetes. It is a form of chronic kidney disease
occurring in approximately 30% of patients with diabetes. It is
defined as diabetes with the presence of albuminuria and/or
impaired renal function (i.e. decreased glomerular filtration rate
(See. de B, I, et al. Temporal trends in the prevalence of diabetic
kidney disease in the United States. JAMA 2011 Jun 22;
305(24):2532-2539).
[0035] The term "pharmaceutically acceptable salt" refers to salts
of pharmaceutical compounds e.g. compound of formula (I) that
retain the biological effectiveness and properties of the
underlying compound, and which are not biologically or otherwise
undesirable. There are acid addition salts and base addition salts.
Pharmaceutically acceptable acid addition salts may be prepared
from inorganic and organic acids.
[0036] Acids and bases useful for reaction with an underlying
compound to form pharmaceutically acceptable salts (acid addition
or base addition salts respectively) are known to one of skill in
the art. Similarly, methods of preparing pharmaceutically
acceptable salts from an underlying compound (upon disclosure) are
known to one of skill in the art and are disclosed in for example,
Berge, at al. Journal of Pharmaceutical Science, January 1977 vol.
66, No.1, and other sources. Salts derived from inorganic acids
include but are not limited to hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid, and the like. Salts
derived from organic acids include but are not limited to maleic
acid, fumaric acid, tartaric acid, p-toluene-sulfonic acid, and the
like. Bases useful for forming base addition salts are known to one
of skill in the art. An example of a pharmaceutically acceptable
salt of the compound of formula (I) is the hydrochloride salt of
the compound of formula (I).
[0037] As used herein, "pharmaceutically acceptable carrier"
includes excipients or agents such as solvents, diluents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents and the like that are not
deleterious to the compound of the invention or use thereof. The
use of such carriers and agents to prepare compositions of
pharmaceutically active substances is well known in the art (see,
e.g., Remington's Pharmaceutical Sciences, Mace Publishing Co.,
Philadelphia, Pa. 17th Ed. (1985); and Modern Pharmaceutics, Marcel
Dekker, Inc. 3rd Ed. (G. S. Banker & C. T. Rhodes, Eds.)
[0038] The term "cardio-renal diseases" as used herein refers to
diseases, related to the function of the kidney, that are caused or
exacerbated by cardiovascular problems such as, for example, high
blood pressure or hypertension. It is believed that hypertension is
a major contributor to kidney disease.
[0039] The term "respiratory diseases" as used herein refers to
diseases including chronic obstructive pulmonary disease (COPD) and
idiopathic pulmonary fibrosis (IPF).
[0040] The term "therapeutically effective amount" refers to an
amount of the compound of formula (I) that is sufficient to effect
treatment as defined below, when administered to a patient
(particularly a human) in need of such treatment in one or more
doses. The therapeutically effective amount will vary, depending
upon the patient, the disease being treated, the weight and/or age
of the patient, the severity of the disease, or the manner of
administration as determined by a qualified prescriber or care
giver.
[0041] The term "treatment" or "treating" means administering a
compound or pharmaceutically acceptable salt of formula (I) for the
purpose of: [0042] (i) delaying the onset of a disease, that is,
causing the clinical symptoms of the disease not to develop or
delaying the development thereof; [0043] (ii) inhibiting the
disease, that is, arresting the development of clinical symptoms;
and/or [0044] (iii) relieving the disease, that is, causing the
regression of clinical symptoms or the severity thereof.
[0045] In a preferred embodiment, the invention relates to the use
of the compound of formula (I) in treating chronic kidney disease
comprising administering a therapeutically effective amount to a
patient in need thereof.
[0046] In another preferred embodiment the invention relates to the
use of the compound of formula (I) in treating diabetic kidney
disease comprising administering a therapeutically effective amount
to a patient in need thereof.
[0047] In another preferred embodiment the invention relates to the
use of the compound of formula (I) in treating lung or kidney
fibrosis comprising administering a therapeutically effective
amount to a patient in need thereof.
[0048] The half maximal inhibitory concentration (IC.sub.50) of a
therapeutic agent is the concentration of a therapeutic agent
necessary to produce 50% of the maximum inhibition against a target
enzyme. It is a desirable goal to discover a therapeutic agent, for
example a compound that inhibits apoptosis signal-regulating kinase
(ASK1) with a low IC.sub.50. In this manner, undesirable side
effects are minimized by the ability to use a lower dose of the
therapeutic agent to inhibit the ASK1 enzyme.
[0049] Similarly, it is a desirable goal to discover a therapeutic
agent that has a low dissociation constant (K.sub.d). K.sub.d is
used to describe the affinity between a ligand (such as a
therapeutic agent) and the corresponding kinase or receptor; i.e. a
measure of how tightly a therapeutic agent binds to a particular
kinase, for example the apoptosis signal-regulating kinase ASK1.
Thus, a lower K.sub.d is generally preferred in drug
development.
[0050] Similarly, it is a desirable goal to discover a compound
having a low EC.sub.50. EC.sub.50 is the concentration of a drug
that achieves 50% maximal efficacy in the cell. The EC.sub.50 value
translates to the concentration of a compound in the assay medium
necessary to achieve 50% of the maximum efficacy. Thus, a lower
EC.sub.50 is generally preferred for drug development. A useful
unit of measure associated with EC.sub.50 is the protein binding
adjusted EC.sub.50 (PB.sub.adj.EC.sub.50 as used herein). This
value measures the amount of a drug e.g. compound of formula (I)
correlated to the fraction of the drug that is unbound to protein
which provides 50% maximal efficacy. This value measures the
efficacy of the drug corrected for or correlated to the amount of
drug that is available at the target site of action.
[0051] Another desirable property is having a compound with a low
cell membrane efflux ratio as determined by CACO cell permeability
studies. An efflux ratio ((B/A)/(A/B)) less than 3.0 is preferred.
A compound with a ratio greater than 3 is expected to undergo
active rapid efflux from the cell and may not have sufficient
duration in the cell to achieve maximal efficacy.
[0052] Another desirable goal is to discover a drug that exhibits
minimal off-target inhibition. That is, a drug that minimally
inhibits the Cyp450 (cytochrome p450) enzymes. More particularly, a
drug that is a weak inhibitor of cyp3A4, the most important of the
P450 enzymes, is desired. A weak inhibitor is a compound that
causes at least 1.25-fold but less than 2-fold increase in the
plasma AUC values, or 20-50% decrease in clearance
(wikipedia.org/wiki/cyp3A4, visited 11/12/11). Generally, a
compound exhibiting a Cyp3A4 IC.sub.50 of greater than 10uM is
considered a weak inhibitor.
A measure useful for comparing cyp3A4 inhibition among drug
candidates is the ratio of Cyp3A4 inhibition and the protein
binding adjusted EC.sub.50. This value gives an indication of the
relative potential for cyp inhibition corrected for the protein
binding adjusted EC.sub.50 which is specific to each drug. A higher
ratio in this measure is preferred as indicative of lower potential
for cyp3A4 inhibition.
[0053] Unexpectedly and advantageously, applicants have discovered
a compound (of formula (I) herein) within the generic scope of U.S.
Patent publication No. 2001/00095410A1 that provides advantages
compared to structurally close compounds (herein designated as
compounds A and B) disclosed in U.S. Patent publication No.
2001/00095410A1
##STR00004##
[0054] Therefore, objects of the present invention include but are
not limited to the provision of a compound of formula (I) or
pharmaceutically acceptable salt thereof, and methods of using the
compound of formula (I) for the treatment of kidney disease,
chronic kidney disease, diabetic kidney disease, diabetic
nephropathy, kidney fibrosis or lung fibrosis.
Combination Therapy
[0055] Patients being treated for cardio-renal diseases such as
chronic kidney disease may benefit from combination drug treatment.
For example the compound of the present invention may be combined
with one or more of angiotensin converting enzyme (ACE) inhibitors
such as enalapril, captopril, ramipril, lisinopril, and quinapril;
or angiontesin II receptor blockers (ARBs) such as losartan,
olmesartan, and irbesartan; or antihypertensive agents such as
amlodipine, nifedipine, and felodipine. The benefit of combination
may be increased efficacy and/or reduced side effects for a
component as the dose of that component may be adjusted down to
reduce its side effects while benefiting from its efficacy
augmented by the efficacy of the compound of formula (I) and/or
other active component(s).
[0056] Patients presenting with chronic kidney disease treatable
with ASKI inhibitors such as compound of formula (I), may also
exhibit conditions that benefit from co-administration (as directed
by a qualified caregiver) of a therapeutic agent or agents that are
antibiotic, analgesic, antidepressant and/or anti-anxiety agents in
combination with compound of formula (I). Combination treatments
may be administered simultaneously or one after the other within
intervals as directed by a qualified caregiver or via a fixed dose
(all active ingredients are combined into the a single dosage form
e.g. tablet) presentation of two or more active agents.
Pharmaceutical Compositions and Administration
[0057] The compound of the present invention may be administered in
the form of a pharmaceutical composition. The present invention
therefore provides pharmaceutical compositions that contain, as the
active ingredient, the compound of formula (I), or a
pharmaceutically acceptable salt thereof, and one or more
pharmaceutically acceptable excipients and/or carriers, including
inert solid diluents and fillers, diluents, including sterile
aqueous solution and various organic solvents, permeation
enhancers, solubilizers and adjuvants. The pharmaceutical
compositions may be administered alone or in combination with other
therapeutic agents. Compositions may be prepared for delivery as
solid tablets, capsules, caplets, ointments, skin patches,
sustained release, fast disintegrating tablets, inhalation
preparations, etc. Typical pharmaceutical compositions are prepared
and/or administered using methods and/or processes well known in
the pharmaceutical art (see, e.g., Remington's Pharmaceutical
Sciences, Mace Publishing Co., Philadelphia, Pa. 17th Ed. (1985);
and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (G. S. Banker
& C. T. Rhodes, Eds.).
[0058] Formulations for combination treatments comprising the
compound of formula (I) may be presented as fixed dose formulations
e.g. tablets, elixirs, liquids, ointments, inhalants, gels, etc.,
using procedures known to one of skill in the art.
[0059] Pharmaceutical compositions of the compound of formula (I)
may be administered in either single or multiple doses by routes
including, for example, rectal, buccal, intranasal and transdermal
routes; by intra-arterial injection, intravenously,
intraperitoneally, parenterally, intramuscularly, subcutaneously,
orally, topically, as an inhalant, or via an impregnated or coated
device such as a stent, for example, or an artery-inserted
cylindrical polymer. Most preferred routes of administration
include oral, parental and intravenous administration.
[0060] The compound of formula (I) may be administered in a
pharmaceutically effective amount. For oral administration, each
dosage unit preferably contains from 1 mg to 500 mg of the compound
of formula (I). A more preferred dose is from 1 mg to 250 mg of the
compound of formula (I). Particularly preferred is a dose of the
compound of formula (I) ranging from about 20 mg twice a day to
about 50 mg twice a day. It will be understood, however, that the
amount of the compound actually administered usually will be
determined by a physician in light of the relevant circumstances
including the condition to be treated, the chosen route of
administration, co-administration compound if applicable, the age,
weight, response of the individual patient, the severity of the
patient's symptoms, and the like. Nomenclature The name of the
compound of the present invention as generated using
ChemBioDraw
[0061] Ultra 11.
##STR00005##
[0062] is
5-(4-cyclopropyl-1H-imidazol-1-yl)--N-(6-(4-isopropyl-4H-1,2,4-t-
riazol-3-yl)pyridin-2-yl)-2-fluoro-4-methylbenzamide also known as
5-((4-cyclopropyl-1H-imdazol-1-yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-tr-
iazole-3-yl) pyridine-2-yl)-4-methylbenzamide.
Synthesis of the Compound of Formula (I)
[0063] The compound of the invention may be prepared using methods
disclosed herein or modifications thereof which will be apparent
given the disclosure herein. The synthesis of the compound of the
invention may be accomplished as described in the following
example. If available, reagents may be purchased commercially, e.g.
from Sigma Aldrich or other chemical suppliers. Alternatively,
reagents may be prepared using reaction schemes and methods known
to one of skill in the art.
Synthetic Reaction Parameters
[0064] The terms "solvent," "inert organic solvent" or "inert
solvent" refer to a solvent inert under the conditions of the
reaction being described in conjunction therewith (including, for
example, benzene, toluene, acetonitrile, tetrahydrofuran (THF),
dimethylformamide (DMF), chloroform, methylene chloride (or
dichloromethane), diethyl ether, petroleum ether (PE), methanol,
pyridine, ethyl acetate (EA) and the like. Unless specified to the
contrary, the solvents used in the reactions of the present
invention are inert organic solvents, and the reactions are carried
out under an inert gas, preferably nitrogen.
[0065] One method of preparing compounds of formula (I) is shown in
Reaction Schemes 1 and 2 below.
##STR00006##
Preparation of Compound A
[0066] To a solution of methyl 6-aminopicolinate (432 g, 2.84 mol)
in MeOH (5 L) was added NH.sub.2NH.sub.2.H.sub.2O (284 g, 5.68 mol,
2.0 eq.). The reaction mixture was heated under reflux for 3 hr and
then cooled to room temperature. The precipitate formed in the
mixture was collected by filtration, washed with EA (2 L.times.2)
and then dried in vacuo to give compound A (405 g, 94% yield) as
white solid.
Preparation of Compound B
[0067] A mixture of compound A (405 g, 2.66 mol) in
dimethylformamide-dimethylacetal (DMF-DMA) (3.54 L) was heated
under reflux for 18 hr, cooled to room temperature and then
concentrated under reduced pressure. The residue was taken up in EA
(700 mL) and heated at 50.degree. C. for 20 min. After being cooled
to room temperature, the solid was collected by filtration and
dried in vacuo to give compound B (572 g, 82% yield) as white
solid.
Preparation of C
[0068] To a solution of compound B (572 g, 2.18 mol) in a mixture
of CH.sub.3CN-AcOH (3.6 L, 4:1) was added propan-2-amine (646 g,
5.0 eq.). The resulting mixture was heated under reflux for 24 hr
and then cooled to room temperature, and the solvent was removed
under reduced pressure. The residue was dissolved in water (2.8 L)
and 1 N aqueous NaOH was added to a pH of 8.0 H. The precipitate
was collected by filtration and the filtrate was extracted with EA
(500 mL.times.3). The combined organic layers were dried over
anhydrous Na.sub.2SO.sub.4, and then concentrated to a volume of
150 mL. To this mixture at 0.degree. C. was slowly added PE (400
mL) and the resulting suspension was filtered. The combined solid
was re-crystallized from EA-PE to give compound C (253 g, 57%
yield) as off-white solid.
[0069] .sup.1H-NMR (400 MHz, CDC13): .delta. 8.24 (s, 1H), 7.52 (m,
2 H), 6.51 (dd, J=1.6, 7.2 Hz, 1H), 5.55 (m, 1H), 4.46 (bs, 2 H),
1.45 (d, J=6.8 Hz, 6 H). MS (ESI+) m/z: 204 (M+1).sup.+. Compound C
is a key intermediate for the synthesis of the compound of formula
(I). Thus, an object of the present invention is also the provision
of the intermediate compound C,
##STR00007##
its salts or protected forms thereof, for the preparation of the
compound of formula (I). An example of a salt of the compound C is
the HCl addition salt. An example of a protected form of compound C
is the carbamate compound such as obtained with Cbz-Cl. Protective
groups, their preparation and uses are taught in Peter G. M. Wuts
and Theodora W. Greene, Protective Groups in Organic Chemistry,
2.sup.nd edition, 1991, Wiley and Sons, Publishers. Preparation of
the Compound of formula (I) continued:
##STR00008##
Compound 6 is a key intermediate for the synthesis of the compound
of formula (I). Thus an object of the present invention is also the
provision of intermediate compound 6,
##STR00009##
salts or protected forms thereof, for the preparation of the
compound of formula (I). An example of a salt of the compound 6 is
the HCl addition salt. An example of a protected form of the
compound 6 is an ester (e.g. methyl, ethyl or benzyl esters) or the
carbamate compound such as obtained with Cbz-Cl. Protective groups,
their preparations and uses are taught in Peter G. M. Wuts and
Theodora W. Greene, Protective Groups in Organic Chemistry,
2.sup.nd edition, 1991, Wiley and Sons, Publishers. Step
1--Preparation of 5-amino-2-fluoro-4-methylbenzonitrile--Compound
(2)
[0070] The starting 5-bromo-4-fluoro-2-methylaniline (1) (20 g, 98
mmol) was dissolved in anhydrous 1-methylpyrrolidinone (100 mL),
and copper (I) cyanide (17.6 g, 196 mmol) was added. The reaction
was heated to 180.degree. C. for 3 hours, cooled to room
temperature, and water (300 mL) and concentrated ammonium hydroxide
(300 mL) added. The mixture was stirred for 30 minutes and
extracted with EA (3.times.200 mL). The combined extracts were
dried over magnesium sulfate, and the solvent was removed under
reduced pressure. The oily residue was washed with hexanes
(2.times.100 mL), and the solid dissolved in dichloromethane and
loaded onto a silica gel column. Eluting with 0 to 25% EA in
hexanes gradient provided 5-amino-2-fluoro-4-methylbenzonitrile
(10.06g, 67.1 mmol). LC/MS (m/z:151 M.sup.+1).
Step 2--Preparation of
5-(2-cyclopropyl-2-oxoethylamino)-2-fluoro-4-methylbenzonitrile--Compound
(3)
[0071] 5-Amino-2-fluoro-4-methylbenzonitrile (12 g, 80 mmol) was
dissolved in anhydrous N,N-dimethylformamide (160 mL) under
nitrogen, and potassium carbonate (13.27 g, 96 mmol) and potassium
iodide (14.61 g, 88 mmol) were added as solids with stirring. The
reaction was stirred for 5 minutes at room temperature and then
bromomethyl cyclopropylketone (20.24 mL, 180 mmol) was added. The
reaction mixture was heated to 60.degree. C. for 3 hours, and then
the solvents removed under reduced pressure. The residue was
dissolved in EA (400 mL) and washed with 400 mL of water. The
organic layer was dried over magnesium sulfate, and solvent was
removed under reduced pressure. The residue was re-dissolved in a
minimum amount of EA, and hexanes were added to bring the solution
to 3:1 hexanes: EA by volume. The product precipitated out of
solution and was collected by filtration to provide
5-(2-cyclopropyl-2-oxoethylamino)-2-fluoro-4-methylbenzonitrile
(14.19 g, 61.2 mmol). LC/MS (m/z : 233, M.sup.+1)
Step 3--Preparation of
5-(4-cyclopropyl-2-mercapto-1H-imidazol-1-yl)-2-fluoro-4-methylbenzonitri-
le--Compound (4)
[0072]
5-(2-Cyclopropyl-2-oxoethylamino)-2-fluoro-4-methylbenzonitrile
(14.19 g, 61.2 mmol) was dissolved in glacial acetic acid (300 mL).
Potassium thiocyanate (11.9 g, 122.4 mmol) was added as a solid
with stirring. The reaction mixture was heated to 110.degree. C.
for 4 hours at which time the solvent was removed under reduced
pressure. The residue was taken up in dichloromethane (200 mL) and
washed with 200 mL water. The aqueous extract was extracted with
(2.times.200 mL) additional dichloromethane, the organic extracts
combined and dried over magnesium sulfate. The solvent was removed
under reduced pressure and the oily residue was re-dissolved in EA
(50 mL) and 150 mL hexanes was added. A dark layer formed and a
stir bar was added to the flask. Vigorous stirring caused the
product to precipitate as a peach colored solid. The product was
collected by filtration, to yield
5-(4-cyclopropyl-2-mercapto-1H-imidazol-1-yl)-2-fluoro-4-methylbenzonitri-
le, (14.26 g, 52.23 mmol). Anal. LC/MS (m/z : 274, M.sup.+1)
Step 4--Preparation of
5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzonitrile--Compoun-
d (5)
[0073] In a 500 mL three neck round bottom flask was placed acetic
acid (96 mL), water (19 mL) and hydrogen peroxide (30%, 7.47 mL,
65.88 mmol). The mixture was heated to 45.degree. C. with stirring
under nitrogen while monitoring the internal temperature.
5-(4--Cyclopropyl-2-mercapto-1H-imidazol-1-yl)-2-fluoro-4-methylbenzonitr-
ile (6.00 g, 21.96 mmol) was then added as a solid in small
portions over 30 minutes while maintaining an internal temperature
below 55.degree. C. When addition of the thioimidazole was complete
the reaction was stirred for 30 minutes at a temperature of
45.degree. C., and then cooled to room temperature, and a solution
of 20% wt/wt sodium sulfite in water (6 mL) was slowly added. The
mixture was stirred for 30 minutes and solvents were removed under
reduced pressure. The residue was suspended in 250 mL of water and
4N aqueous ammonium hydroxide was added to bring the pH to
.about.10. The mixture was extracted with dichloromethane
(3.times.200 ml), the organics combined, dried over magnesium
sulfate, and the solvent was removed under reduced pressure. The
residue was dissolved in 20 mL EA, and 80 mL of hexanes were added
with stirring. The solvents were decanted off and an oily residue
was left behind. This process was repeated and the product,
5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzonitrile
was obtained as a viscous oil (5.14 g, 21.33 mmol) Anal. LC/MS
(m/z: 242, M.sup.+1)
Step 5--Preparation of
5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzoic acid
hydrochloride (6)
[0074]
5-(4-Cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzonitrile
(11.21 g, 46.50 mmol) was placed in a round bottom flask fitted
with a reflux condenser, and suspended in 38% hydrochloric acid
(200 mL). The mixture was heated to 100.degree. C. for 4.5 hours,
and then cooled to room temperature. Solvent was removed under
reduced pressure to give a pink solid, to which was added 100 ml of
EA. The solid product was collected by filtration and washed with
3.times.100 mL EA. To the solid product was added 100 mL 10%
methanol in dichloromethane, the mixture stirred, and the filtrate
collected. This was repeated with 2 more 100 ml portions of 10%
methanol in dichloromethane. The filtrates were combined and
solvent was removed under reduced pressure, to provide crude
5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzoic acid
hydrochloride. No further purification was carried out (11.13 g,
37.54 mmol). Anal. LC/MS (m/z: 261, M.sup.+1)
Step 6--Preparation of
5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro--N-(6-(4-isopropyl-4H-1,2,4-t-
riazol-3-yl) pyridin-2-yl)-4-methylbenzamide--formula (I)
[0075] 5-(4-Cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzoic
acid hydrochloride (1.5 g, 5.07 mmol) was suspended in anhydrous
1,2-dichloromethane (25 mL) at room temperature. Oxalyl chloride
(0.575 ml, 6.59 mmol) was added with stirring under nitrogen,
followed by N,N-dimethylformamide (0.044 ml, 0.507 mmol). The
mixture was stirred for 4 hr at room temperature, and then the
solvent was removed under reduced pressure. The residue was
dissolved in 25 mL anhydrous dichloromethane.
6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (1.13 g, 5.58
mmol) (compound C) and 4-dimethylaminopyridine (0.62 g, 5.07 mmol)
were rapidly added with stirring under nitrogen. The reaction was
stirred for 2 hours at room temperature and aqueous saturated
NaHCO.sub.3 (15 mL) was added. The mixture was stirred for 10
minutes, and the layers were separated, and the aqueous layer was
washed 1.times.20 mL dichloromethane. The combined organics were
dried (MgSO.sub.4), filtered and concentrated. The residue was
dissolved in a minimum amount of CH.sub.3CN and water was slowly
added until solids precipitated from the mixture. The solid was
collected by filtration and dried to give
5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-tr-
iazol-3- yl) pyridin-2-yl)-4-methylbenzamide in .about.96% purity
(1.28 g, 2.88 mmol). Anal. LC/MS (m/z: 446, M.sup.+1). The material
was further purified by RP-HPLC (reverse phase HPLC) to obtain an
analytically pure sample as the HCl salt.
##STR00010##
[0076] C.sub.24H.sub.24FN.sub.7O--HCl. 446.2 (M+1). .sup.1H-NMR
(DMSO): .delta. 11.12 (s, 1H), 9.41 (s, 1H), 9.32 (s, 1H), 8.20 (d,
J=8.4 Hz, 1H), 8.07 (t, J=8.4 Hz, 1H), 7.95 (d, J=6.4 Hz, 1H), 7.92
(d, J=7.6 Hz, 1H), 7.79 (s, 1H), 7.59 (d, J=10.4 Hz, 1H), 5.72
(sept, J=6.8 Hz, 1H), 2.29 (s, 3H), 2.00-2.05 (m, 1H), 1.44 (d,
J=6.8 Hz, 6H), 1.01-1.06 (m, 2H), 0.85-0.89 (m, 2H).
Biological Assays
ASK1 (Apoptosis Signal-Regulating Kinase 1) TR-FRET Kinase Assay
(Biochemical IC.sub.50)
[0077] The ability of compounds to inhibit ASK1 kinase activity was
determined using a time resolved fluorescence resonance energy
transfer [TR-FRET] assay utilizing biotinylated myelin basic
protein [biotin-MBP] as the protein substrate. A Beckman Biomek FX
liquid handling robot was utilized to spot 2 .mu.L/well of
compounds in 2.44% aqueous DMSO into low volume 384-well
polypropylene plates [Nunc, #267460] to give a final concentration
of between 100 .mu.M and 0.5nM compound in the kinase assay. A
Deerac Fluidics Equator was used to dispense 3 .mu.L/well of
0.667ng/.mu.L [Upstate Biotechnologies, #14-606, or the equivalent
protein prepared in-house] and 0.1665 ng/mL biotin-MBP [Upstate
Biotechnologies, #13-111] in buffer (85 mM MOPS, pH 7.0, 8.5 mM
Mg-acetate, 5% glycerol, 0.085% NP-40, 1.7mM DTT and 1.7 mg/mL BSA)
into the plates containing the spotted compounds.
[0078] The enzyme was allowed to pre-incubate with compound for 20
minutes prior to initiating the kinase reaction with the addition
of 5 .mu.L/well 300 .mu.M ATP in buffer (50 mM MOPS, pH 7.0, 5 mM
Mg-acetate, 1 mM DTT, 5% DMSO) using the Deerac Fluidics Equator.
The kinase reactions were allowed to proceed for 20 minutes at
ambient temperature and were subsequently stopped with the addition
of 5 .mu.L/well 25 mM EDTA using the Deerac Fluidics Equator. The
Biomek FX was then used to transfer 1 .mu.L/well of each completed
kinase reaction to the wells of an OptiPlate-1536 white polystyrene
plate [PerkinElmer, #6004299] that contained 5 .mu.L/well detection
reagents (1.11 nM Eu-W1024 labeled anti-phosphothreonine antibody
[PerkinElmer, #AD0094] and 55.56 nM streptavidin allophycocyanin
[PerkinElmer, #CR130-100] in 1.times. LANCE detection buffer
[PerkinElmer, #CR97-100]). The TR-FRET signal was then read on a
Perkin Elmer Envision plate reader after incubating the plates at
ambient temperature for 2 hours.
[0079] The 100% inhibition positive control wells were generated by
switching the order of addition of the EDTA and ATP solutions
described above. These wells and 0% inhibition wells containing
spots of 2.44% DMSO at the beginning of the assay were used in
calculating the % inhibition for the test compounds.
Result
[0080] The compound of formula (I) inhibited ASK1 with an IC.sub.50
of 3.0 nM. This data suggests that the compound of formula (I) is a
potent inhibitor of ASK1 in the presence of the competitive ligand
ATP.
[0081] In an updated version of the assay above, the inhibitory
activity of compound of the invention against ASK1 was examined
using a TR-FRET ASK1 assay which determined the amount of phosphate
transferred to a peptide substrate from ATP.
Materials and Methods
Reagents
[0082] Dephosphorylated recombinant human ASK1 kinase was from
Gilead Sciences. Small molecule kinase inhibitor staurosporine
(Catalogue #S6942) and dithiothreitol (DTT, catalogue #43815-5G)
were obtained from Sigma Chemicals (St. Louis, Mo.). ATP (catalogue
#7724) was from Affymetrix (Santa Clara, Calif.) and the compound
of formula (I) was from Gilead Sciences. HTRF KinEASE.TM.--STK S3
kit was obtained from Cisbio (Bedford, Mass). All other reagents
were of the highest grade commercially available.
Assays
[0083] The assay measures the phosphorylation level of a
biotinylated peptide substrate by the ASK1 kinase using HTRF
detection (6.1). This is a competitive, time-resolved fluorescence
resonance energy transfer (TR-FRET) immunoassay, based on HTRF.RTM.
KinEASE.TM.--STK manual from Cisbio (6.1). Test compound, 1 .mu.M
STK3 peptide substrate, 4 nM of ASK1 kinase are incubated with 10
mM MOP buffer, pH. 7.0 containing 10 mM Mg-acetate, 0.025% NP-40, 1
mM DTT, 0.05% BSA and 1.5% glycerol for 30 minutes then 100
.quadrature.M ATP is added to start the kinase reaction and
incubated for 3 hr. Peptide antibody labeled with 1.times.
Eu.sup.3+ Cryptate buffer containing 10 mM EDTA and 125 nM
Streptavidin XL665 are added to stop the reaction and
phosphorylated peptide substrate is detected using Envision 2103
Multilabeled reader from PerkinElmer. The fluorescence is measured
at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615
nm is calculated for each well. The resulting TR-FRET level (a
ratio of 665 nm/615 nm) is proportional to the phosphorylation
level. Under these assay conditions, the degree of phosphorylation
of peptide substrate was linear with time and concentration for the
enzyme. The assay system yielded consistent results with regard to
K.sub.m and specific activities for the enzyme. For inhibition
experiments (IC.sub.50 values), activities were performed with
constant concentrations of ATP, peptide and several fixed
concentrations of inhibitors. Staurosporine, the nonselective
kinase inhibitor, was used as the positive control. All enzyme
activity data are reported as an average of quadruplicate
determination.
Data Analysis
[0084] The IC.sub.50 values were calculated following equation:
y=Range/{1+(x/IC.sub.50).sup.s}+Background
Where x and y represent the concentration of inhibitors and enzyme
activity, respectively. Enzyme activity is expressed as the amount
of Phosphate incorporated into substrate peptide from ATP. Range is
the maximum y range (no inhibitor, DMSO control) and s is a slope
factor (6.2).
Results
[0085] The compound of formula (I) exhibited an IC50 of 3.2 nM
under this test condition. The data demonstrates that the compound
of formula (I) is a potent inhibitor of the ASK-1 1 receptor.
ASK1 (Apoptosis Signal-Regulating Kinase 1) 293 Cell-Based Assay
(Cellular EC.sub.50)
[0086] The cellular potency of compounds was assayed in cells
stably expressing an AP-1:luciferase reporter construct
(293/AP1-Luc cells - Panomics Inc., 6519 Dumbarton Circle, Fremont,
Calif.). Cells were infected with an adenovirus expressing kinase
active ASK1 (631-1381 of rat ASK1 cDNA), which will activate the
AP-1 transcription factor and increase the expression of
luciferase. Inhibitors of ASK1 will decrease the enzyme activity of
ASK1 and therefore decrease the activity of AP-1 transcription
factor and the expression of luciferase.
1. Materials Required for This Protocol
TABLE-US-00001 [0087] Media and Reagents Source Company Catalog No.
AP-1 Reporter 293 Stable Cell Line Panomics Unknown DMEM (w/high
glucose, w/o L- MediaTech 15-018-CM glutamine, w/pyruvate, w/HEPES
DMEM (w/high glucose, w/o L- Invitrogen 31053-028 glutamine, w/o
pyruvate, w/o HEPES, w/o phenol red HEPES, 1M Invitrogen 15630-080
Sodium Pyruvate, 100 mM Invitrogen 11360-070 Fetal Bovine Serum,
"FBS" Hyclone SH30088.03 Pen-Strep-Glut., "PSG" Invitrogen
10378-016 HygromycinB Calbiochem 400052 Dulbecco's PBS (sterile)
MediaTech 21-030-CM Trypsin-EDTA (0.25%) Invitrogen 25200-056
Steady-Glo Luciferase Assay Promega E2550 System Labware Source
Catalog No. Flasks (poly-D-Lysine coated, 150 BD Biosciences 356538
cm.sup.2, vented cap) Plates (poly-D-Lysine coated, 384- Greiner
(through 781944 well, white/clear, sterile TCT) VWR Scientific)
(82051-354) White Backing Tape PerkinElmer 6005199 Cell Strainers
(40 um nylon, blue VWR Scientific 21008-949 ring, fits 50 mL
conical vials)
2. Reference Materials
[0088] Panomics 293/AP1-Luc stable cell-line product insert.
Promega Steady-Glo Luciferase Assay System product insert.
3. Media Required
Complete Growth Medium, "CGM"
DMEM (MediaTech)
10% FBS
1% PSG
[0089] 100 ug/mL HygromycinB
Assay Medium, "AM"
DMEM (Invitrogen)
25 mM HEPES
1 mM Sodium Pyruvate
1% PSG
4. Methods
Maintenance:
[0090] 293/AP1-Luc Maintain 293/Acells per vendor's instructions;
harvest cells at .about.80% confluence in T150 flasks as
follows:
Aspirate media, wash gently with .about.12 mL sterile D-PBS,
aspirate. Add 5 mL Trypsin-EDTA, tilt gently to coat flask, and
incubate .about.5 min at 37.degree. C. Do not tap flask; add 5 mL
CGM, wash flask 4X with cell suspension, transfer to 50 mL conical
vial, centrifuge 5 min at 1200 rpm. Aspirate media from cell
pellet, add 20 to 30 mL CGM, resuspend pellet by pipeting 6.times.,
pass through cell strainer to disperse clumps (if necessary), and
count cells with hemocytometer.
Assay Day 1:
[0091] Harvest cells as above, except resuspend cell pellet.
[0092] Count cells and dilute to 1.5.times.10.sup.5 cells per mL;
add adenovirus such that there are 5 infectious forming units per
cell.
Prime (20 to 30 mL) and plate cells in Greiner poly-D-Lysine coated
384-well plates at 1.2.times.10.sup.4 cells per well using BioTek
uFill (80 uL per well). Immediately dose plates with 0.4 uL of
compound dose series (in 100% DMSO) incubate 24 hours in humidified
incubator (37.degree. C., 5% CO.sub.2).
Assay Day 2:
[0093] Process plates (per manufacturer's instructions) as
follows:
Set plates in laminar flow hood & uncover for 30 minutes at
room temperature to cool. Remove 60 uL of AM from assay wells Add
20 uL per well Steady-Glo Firefly substrate, let sit for 10-20
minutes at room temperature Cover bottom of assay plates with white
backing tape. Acquire data on a fluorescence plate reader The 100%
inhibition positive control wells were generated by infecting cells
with an adenovirus expressing catalytically inactive ASK1 mutant
with lysine to argine mutation at residue 709.
Result
[0094] The compound of formula (I) exhibits an EC.sub.50 of 2.0
nM.
Determination of Kd
Kinase Assays
[0095] Kinase-tagged T7 phage strains were prepared in an E. coli
host derived from the BL21 strain. E. coli were grown to log-phase
and infected with T7 phage and incubated with shaking at 32.degree.
C. until lysis. The lysates were centrifuged and filtered to remove
cell debris. The remaining kinases were produced in HEK-293 cells
and subsequently tagged with DNA for qPCR detection.
Streptavidin-coated magnetic beads were treated with biotinylated
small molecule ligands for 30 minutes at room temperature to
generate affinity resins for kinase assays.
[0096] The liganded beads were blocked with excess biotin and
washed with blocking buffer (SeaBlock (Pierce), 1% (bovine serum
albumin), 0.05% Tween 20, 1 mM DTT(dithiothreitol)) to remove
unbound ligand and to reduce non-specific binding. Binding
reactions were assembled by combining kinases, liganded affinity
beads, and test compounds in 1.times. binding buffer (20% SeaBlock,
0.17.times.PBS, 0.05% Tween 20, 6 mM DTT). All reactions were
performed in polystyrene 96-well plates in a final volume of 0.135
mL. The assay plates were incubated at room temperature with
shaking for 1 hour and the affinity beads were washed with wash
buffer (1.times.PBS, 0.05% Tween 20). The beads were then
re-suspended in elution buffer (1.times.PBS, 0.05% Tween 20, 0.5
.mu.M non-biotinylated affinity ligand) and incubated at room
temperature with shaking for 30 minutes. The kinase concentration
in the eluates was measured by qPCR.
[0097] Binding constants (Kds) were calculated with a standard
dose-response curve using the Hill equation.
Result
[0098] The compound of formula (I) exhibited a K.sub.d of 0.24 nM.
This data suggests that the compound of formula (I) binds potently
to ASK1 receptor in the absence of ATP.
Determination of Percent of Compound Bound to Plasma
Experimental Design:
[0099] 1 mL Teflon dialysis cells from Harvard Apparatus
(Holliston, Mass., USA) were used in these experiments. Prior to
the study, dialysis membrane was soaked for approximately one hour
in 0.133 M phosphate buffer, pH 7.4. A nominal concentration of 2
.mu.M of compound was spiked into 1 mL of plasma or 1 mL of cell
culture media. The total volume of liquid on each side of the cell
was 1 mL. After 3 hours equilibration in a 37.degree. C. water
bath, samples from each side of the cell were aliquoted into the
appropriate vials containing either 1 mL of human plasma (cell
culture media), or buffer. Sample vials were weighed and recorded.
A 100 .mu.L aliquot was removed and added to 400 .mu.L quenching
solution (50% methanol, 25% acetonitrile, 25% water and internal
standard). Samples were vortexed and centrifuged for 15 minutes at
12000 G. 200 pL of the supernatant was removed and placed into a
new 96 well plate. An additional 200 .mu.L of 1:1 ACN:water was
added. The plate was then vortexed and subjected to LC-MS analysis.
The percent unbound for an analyte in plasma was calculated using
the following equations
% Unbound=100(C.sub.f/C.sub.t)
where C.sub.f and C.sub.t are the post-dialysis buffer and plasma
concentrations, respectively.
Results
[0100] The percent unbound measured in human plasma for the
compound of formula (I) is 11.94%
Determination of CACO-2 Efflux Ratio
Experimental:
[0101] Caco-2 cells were maintained in Dulbecco's Modification of
Eagle's Medium (DMEM) with sodium pyruvate, Glutmax supplemented
with 1% Pen/Strep, 1% NEAA and 10% fetal bovine serum in an
incubator set at 37.degree. C., 90% humidity and 5% CO.sub.2.
Caco-2 cells between passage 62 and 72 were seeded at 2100
cells/well and were grown to confluence over at least 21-days on 24
well PET (polyethylene-terephthalate) plates (BD Biosciences). The
receiver well contained HBSS buffer (10 mM HEPES, 15 mM Glucose
with pH adjusted to pH 6.5) supplemented with 1% BSA pH adjusted to
pH 7.4. After an initial equilibration with transport buffer, TEER
values were read to test membrane integrity. Buffers containing
test compounds were added and 100 .mu.l of solution was taken at 1
and 2 hrs from the receiver compartment. Removed buffer was
replaced with fresh buffer and a correction is applied to all
calculations for the removed material. The experiment was carried
out in replicate. All samples were immediately collected into 400
.mu.l 100% acetonitrile acid to precipitate protein and stabilize
test compounds. Cells were dosed on the apical or basolateral side
to determine forward (A to B) and reverse (B to A) permeability.
Permeability through a cell free trans-well is also determined as a
measure of cellular permeability through the membrane and
non-specific binding. To test for non-specific binding and compound
instability percent recovery is determined. Samples were analyzed
by LC/MS/MS.
[0102] The apparent permeability, P.sub.app, and % recovery were
calculated as follows:
P.sub.app=(dR/dt).times.V.sub.r/(A.times.D.sub.0)
%
Recovery=100.times.((V.sub.r.times.R.sub.120)+(V.sub.d.times.D.sub.120-
))/(V.sub.d.times.D.sub.0)
where, dR/dt is the slope of the cumulative concentration in the
receiver compartment versus time in .mu.M/s based on receiver
concentrations measured at 60 and 120 minutes. V.sub.r and V.sub.d
is the volume in the receiver and donor compartment in cm.sup.3,
respectively. A is the area of the cell monolayer (0.33 cm.sup.2).
D.sub.0 and D.sub.120 is the measured donor concentration at the
beginning and end of the experiment, respectively. R.sub.120 is the
receiver concentration at the end of the experiment (120
minutes).
Absorption and Efflux Classification:
TABLE-US-00002 [0103] P.sub.app (A to B) .gtoreq. 1.0 .times.
10.sup.-6 cm/s High 1.0 .times. 10.sup.-6 cm/s > P.sub.app (A to
B) .gtoreq. Medium 0.5 .times. 10.sup.-6 cm/s P.sub.app (A to B)
< 0.5 .times. 10.sup.-6 cm/s Low P.sub.app (B to A)/P.sub.app (A
to B) .gtoreq. 3 Significant Efflux % recovery < 20% May affect
measured permeability Cell Free P.sub.app < 15 May affect
measured permeability
Result
[0104] The compound of formula (I) was observed to have a CACO
A.fwdarw.B value of 27; and a CACO B.fwdarw.A value of 35 resulting
in a efflux ratio (B.fwdarw.A)/(A.fwdarw.B) of 1.3.
Determination of Metabolic Stability in Hepatic Microsomal
Fraction:
Experimental:
[0105] Metabolic stability was assessed using cofactors for both
oxidative metabolism (NADPH) and conjugation (UDP glucuronic acid
(UDPGA)). Duplicate aliquots of the compound of formula (I) (3
.mu.L of 0.5 mM DMSO stock) or metabolic stability standards
(Buspirone) were added to microsome stock diluted with potassium
phosphate buffer, pH 7.4, to obtain a protein concentration of 1.0
mg/mL and containing alamethicin as a permeabilizing agent.
Metabolic reactions were initiated by the addition of NADPH
regenerating system and UDPGA cofactor. The final composition of
each reaction mixture was: 3 .mu.M test compound, 1 mg microsomal
protein/mL, 5 mM UDPGA, 23.4 .mu.g/mL alamethicin, 1.25 mM NADP,
3.3 mM glucose-6-phosphate, 0.4 U/mL glucose-6-phosphate
dehydrogenase and 3.3 mM MgCl.sub.2 in 50 mM potassium phosphate
buffer, pH 7.4. At 0, 2, 5, 10, 15, 30, 45, and 60 min, 25 .mu.L
aliquots of the reaction mixture were transferred to plates
containing 250 .mu.l of IS/Q (quenching solution containing
internal standard). After quenching, the plates were centrifuged at
3000.times.g for 30 minutes, and 10 .mu.L aliquots of the
supernatant were analyzed using LC/MS to obtain analyte/internal
standard peak area ratios.
[0106] Metabolic stability in microsomal fractions were determined
by measuring the rate of disappearance of the compound of formula
(I). Data (% of parent remaining) were plotted on a semi
logarithmic scale and fitted using an exponential fit:
C.sub.t=C.sub.0e.sup.-Kt and T.sub.1/2=ln2/K where
C.sub.t % of parent remaining at time=t C.sub.0 % of parent
remaining at time=0 t time (hr) K First order elimination rate
constant (hr.sup.-1) T1/2 In vitro half-life (hr) The predicted
hepatic clearance was calculated as follows {reference 1}:
CL.sub.int=KVY.sub.P/P or CL.sub.int=KVY.sub.H/H
CL.sub.h=(CL.sub.intQ.sub.h)/(CL.sub.int+Q.sub.h), where
CL.sub.h Predicted hepatic clearance (L/hr/kg body weight)
CL.sub.int Intrinsic hepatic clearance (L/hr/kg body weight) V
Incubation volume (L) Y.sub.P Microsome protein yield (mg
protein/kg body weight) Y.sub.H Hepatocyte yield (millions of
cells/kg body weight) P Mass of protein in the incubation (mg) H
Number of hepatocytes in the incubation (million) Q.sub.h Hepatic
blood flow (L/hr/kg body weight)
[0107] Predicted hepatic extraction was then calculated by
comparison of predicted hepatic clearance to hepatic blood flow. A
compound was considered stable if the reduction of substrate
concentration was <10% over the course of the incubation
(corresponding to an extrapolated half-life of >395 min in
microsomal fractions and >39.5 hr in hepatocytes).
[0108] Values used for calculation of the predicted hepatic
clearance are shown in the tables below:
TABLE-US-00003 TABLE 1 Values Used for Calculation of the Predicted
Hepatic Clearance from Microsomal Stability Hepatic Microsomes V P
Y Q.sub.h Species (L) (mg) (mg/kg) (L/kg) Rat 0.001 1.0 1520 4.2
Cynomolgus Monkey 0.001 1.0 684 1.6 Rhesus Monkey 0.001 1.0 1170
2.3 Dog 0.001 1.0 1216 1.8 Human 0.001 1.0 977 1.3
Result:
[0109] The predicted hepatic clearance in human as determined from
in vitro experiments in microsomal fractions is 0.1 L/h/kg.
Determination of Rat CL and Vss for Test Compounds
[0110] Pharmacokinetics of Test Compounds following a 1 mg/kg IV
infusion and 5.0 mg/kg PO dose in rats
Test Article and Formulation
[0111] For IV administration the test compound was formulated in
60:40 PEG 400:water with 1 equivalent HCl at 0.5 mg/mL. The
formulation was a solution. For PO (oral) administration, the test
compound was formulated in 5/75/10/10 ethanol/PG/solutol/water at
2.5 mg/mL. The formulation was a solution.
Animals Used
[0112] IV and PO dosing groups each consisted of 3 male SD rats. At
dosing, the animals generally weighed between 0.317 and 0.355 kg.
The animals were fasted overnight prior to dose administration and
up to 4 hr after dosing.
Dosing
[0113] For the IV infusion group, the test compound was
administered by intravenous infusion over 30 minutes. The rate of
infusion was adjusted according to the body weight of each animal
to deliver a dose of 1 mg/kg at 2 mL/kg. For the oral dosing group,
the test article was administered by oral gavage at 2 mL/kg for a
dose of 5.0 mg/kg.
Sample Collection
[0114] Serial venous blood samples (approximately 0.4 mL each) were
taken at specified time points after dosing from each animal. The
blood samples were collected into Vacutainer.TM. tubes
(Becton-Disckinson Corp, N.J., USA) containing EDTA as the
anti-coagulant and were immediately placed on wet ice pending
centrifugation for plasma.
Determination of the Concentrations of the Compound of Formula (I)
in Plasma
[0115] An LC/MS/MS method was used to measure the concentration of
test compound in plasma.
Calculations
[0116] Non-compartmental pharmacokinetic analysis was performed on
the plasma concentration-time data.
Results
[0117] The compound of formula (I) exhibited a CL of 0.09 L/hr/kg;
an oral bioavailability of 75%; t.sub.112 of 5.07 hr and a Vss of
0.55 L/kg in rats.
Cyp Inhibition Assay
Objective
[0118] To assess the potential of the test compound to inhibit the
main cytochrome P450 isoforms, CYP1A, CYP1A2, CYP2B6, CYP2C8,
CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (2 substrates).
Cytochrome P450 Inhibition IC.sub.50 Determination (8 Isoform, 9
Substrates)
Protocol Summary
[0119] Test compound (0.1 .mu.M-25 .mu.M) is incubated with human
liver microsomes and NADPH in the presence of a cytochrome P450
isoform-specific probe substrate. For the CYP2B6, CYP2C8, CYP2C9,
CYP2C19, CYP2D6 and CYP3A4 specific reactions, the metabolites are
monitored by mass spectrometry. CYP1A activity is monitored by
measuring the formation of a fluorescent metabolite. A decrease in
the formation of the metabolite compared to the vehicle control is
used to calculate an IC50 value (test compound concentration which
produces 50% inhibition).
Assay Requirements
[0120] 500 .mu.L of a 10 mM test compound solution in DMSO.
Experimental Procedure
CYP1A Inhibition
[0121] Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25
.mu.M in DMSO; final DMSO concentration=0.3%) are incubated with
human liver microsomes (0.25 mg/mL) and NADPH (1 mM) in the
presence of the probe substrate ethoxyresorufin (0.5 .mu.M) for 5
min at 37.degree. C. The selective CYP1A inhibitor,
alpha-naphthoflavone, is screened alongside the test compounds as a
positive control.
CYP2B6 Inhibition
[0122] Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25
.mu.M in DMSO; final DMSO concentration=0.3%) are incubated with
human liver microsomes (0.1 mg/mL) and NADPH (1 mM) in the presence
of the probe substrate bupropion (110 .mu.M) for 5 min at
37.degree. C. The selective CYP2B6 inhibitor, ticlopidine, is
screened alongside the test compounds as a positive control.
CYP2C8 Inhibition
[0123] Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25
.mu.M in DMSO; final DMSO concentration=0.3%) are incubated with
human liver microsomes (0.25 mg/mL) and NADPH (1 mM) in the
presence of the probe substrate paclitaxel (7.5 .mu.M) for 30 min
at 37.degree. C. The selective CYP2C8 inhibitor, montelukast, is
screened alongside the test compounds as a positive control.
CYP2C9 Inhibition
[0124] Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25
.mu.M in DMSO; final DMSO concentration=0.25%) are incubated with
human liver microsomes (1 mg/mL) and NADPH (1 mM) in the presence
of the probe substrate tolbutamide (120 .mu.M) for 60 min at
37.degree. C. The selective CYP2C9 inhibitor, sulphaphenazole, is
screened alongside the test compounds as a positive control.
CYP2C19 Inhibition
[0125] Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25
.mu.M in DMSO; final DMSO concentration=0.25%) are incubated with
human liver microsomes (0.5 mg/mL) and NADPH (1 mM) in the presence
of the probe substrate mephenytoin (25 .mu.M) for 60 min at
37.degree. C. The selective CYP2C19 inhibitor, tranylcypromine, is
screened alongside the test compounds as a positive control.
CYP2D6 Inhibition
[0126] Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25
.mu.M in DMSO; final DMSO concentration=0.25%) are incubated with
human liver microsomes (0.5 mg/mL) and NADPH (1 mM) in the presence
of the probe substrate dextromethorphan (5 .mu.M) for 5 min at
37.degree. C. The selective CYP2D6 inhibitor, quinidine, is
screened alongside the test compounds as a positive control.
CYP3A4 Inhibition (Midazolam)
[0127] Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25
.mu.M in DMSO; final DMSO concentration=0.26%) are incubated with
human liver microsomes (0.1 mg/mL) and NADPH (1 mM) in the presence
of the probe substrate midazolam (2.5 .mu.M) for 5 min at
37.degree. C. The selective CYP3A4 inhibitor, ketoconazole, is
screened alongside the test compounds as a positive control.
CYP3A4 Inhibition (Testosterone)
[0128] Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25
.mu.M in DMSO; final DMSO concentration=0.275%) are incubated with
human liver microsomes (0.5 mg/mL) and NADPH (1 mM) in the presence
of the probe substrate testosterone (50 .mu.M) for 5 min at
37.degree. C. The selective CYP3A4 inhibitor, ketoconazole, is
screened alongside the test compounds as a positive control.
[0129] For the CYP1A incubations, the reactions are terminated by
methanol, and the formation of the metabolite, resorufin, is
monitored by fluorescence (excitation wavelength=535 nm, emission
wavelength=595 nm). For the CYP2B6, CYP2C9, CYP2C19, CYP2D6, and
CYP3A4 incubations, the reactions are terminated by methanol. The
samples are then centrifuged, and the supernatants are combined,
for the simultaneous analysis of 4-hydroxytolbutamide,
4-hydroxymephenytoin, dextrorphan, and 1-hydroxymidazolam by
LC-MS/MS. Hydroxybupropion, 6a-hydroxypaclitaxel and
6.beta.-hydroxytestosterone are analysed separately by LC-MS/MS.
Formic acid in deionised water (final concentration=0.1%)
containing internal standard is added to the final sample prior to
analysis. A decrease in the formation of the metabolites compared
to vehicle control is used to calculate an IC50 value (test
compound concentration which produces 50% inhibition).
Results
TABLE-US-00004 [0130] CYP450 Calculated Isoform Substrate
Metabolite IC.sub.50 (.mu.M) 1A Ethoxyresorufin Resorufin >25
.mu.M 1A2 Phenacetin Acetaminophen >25 .mu.M 2B6 Bupropion
Hydroxybupropion 19.2 .mu.M 2C8 Paclitaxel
6.alpha.-Hydroxypaclitaxel 21.6 .mu.M 2C9 Tolbutamide
4-Hydroxytolbutamide >25 .mu.M 2C19 S-mephenytoin
4-Hydroxymephenytoin >25 .mu.M 2D6 Dextromethorphan Dextrorphan
17.7 .mu.M 3A4 Midazolam Hydroxymidazolam 2.7 .mu.M 3A4
Testosterone 6 .beta. Hydroxytestosterone 10.5 .mu.M.
General Study Design for the Rat Unilateral Ureter Obstruction
(UUO) Model of Kidney Fibrosis.
[0131] Male Sprague-Dawley rats were fed normal chow, housed under
standard conditions, and allowed to acclimate for at least 7 days
before surgery. At the inception of study, rats were placed into
weight-matched groups, and administered (2 ml/kg p.o. bid) via oral
gavage vehicle, one of four dose levels of compounds (1, 3, 10, or
30 mg/kg). Rats were anesthetized with isoflurane anesthesia on a
nosecone, and laparotomy was performed. Rats underwent complete
obstruction of the right ureter (UUO) using heat sterilized
instruments and aseptic surgical technique. Rats were administered
50 .mu.l Penicillin G (i.m.) immediately post-operatively. Rats
were allowed to recover in a clean, heated cage before being
returned to normal vivarium conditions. Rats were administered
compounds at the dose described above twice daily (at 12 hour
intervals) for the subsequent 7 days. On day 7 following surgery,
rats were anesthetized with isoflurane and serum, plasma, and urine
collected. Animals were then euthanized, the kidneys harvested, and
renal cortical biopsies collected for morphological, histological,
and biochemical analysis. All tissues for biochemical analysis are
flash-frozen in liquid nitrogen and stored at -80.degree. C.,
tissues for histological analysis were fixed in 10% neutral
buffered formalin
[0132] Renal fibrosis was evaluated by measuring the amount of
collagen IV in the kidney by an ELISA method and by examining the
accumulation of alpha-smooth muscle actin positive myofibroblasts
in the kidney by immunohistochemistry. For the former, a small
piece of frozen kidney cortex was transferred homengenized in RIPA
buffer then centrifuged at 14000.times.g for 10 minutes at at
4.degree. C. The supernatant was collected into pre-chilled tubes
and the protein concentration was determined. Equivalent amount of
total protein were subjected to a Col IV ELISA assay (Exocell)
according to the manufacturers instructions.
Formalin fixed and paraffin embedded kidney tissue was stained with
an alpha-smooth muscle actin as previously described (Stambe et
al., The Role of p38 Mitogen-Activated Protein Kinase Activation in
Renal Fibrosis J Am Soc Nephrol 15: 370-379, 2004).
Results:
[0133] The compound of formula (I) was found to significantly
reduce kidney Collagen IV induction (FIG. 1) and accumulation of
alpha-smooth muscle positive myofibroblasts (FIG. 2) at doses of 3
to 30 mg/kg.
Comparative Data for Compound of Formula (I) and Reference
Compounds
[0134] The following table provides comparative results for the
compound of formula (I) and the reference compounds A and B
disclosed in U.S. Patent publication No. 2001/00095410A1, published
Jan. 13, 2011. Applicants note that experiments for which results
are compared below were performed under similar conditions but not
necessarily simultaneously.
TABLE-US-00005 TABLE Compound of formula (I) Compound A Compound B
IC.sub.50 (nM) 3 5 6.5 EC.sub.50 (nM) 2 3.4 18 (9X) PBadj EC.sub.50
(nM) 17 71 (4X) 563 (33X) CACO (A/B, B/A) 27/35 3.1/18.5 0.26/4
Efflux ratio (B/A)/ 1.3 6.0 15.4 (A/B) fu 12 4.8 3.2 Cyp3A4
IC.sub.50 11 1.1 (10X) 4 (2.8X) Testesterone (TST)) (uM) Cyp3A4
IC.sub.50/ 647 15 (43X) 7 (92X) PBAdj.EC.sub.50 Vss (L/Kg) in rats
0.55 0.17 (3.2X) 0.54 CL (L/hr/kg) in rats 0.11 0.30 (2.7X) 0.39
(3.6X) % F in rats 75 11 (6.8X) 50 (1.5X) t1/2 in rats (hr) 5.07
0.59 (8.6X) 1.3 (3.9X) ( ) values in parenthesis represent the
number of times the compound of formula (I) shows an improvement
over the indicated compound for the indicated parameter.
[0135] The following can be deduced from the above comparative
data: The compound of formula (I) has an EC.sub.50 that is
comparable to that of Compound A. The compound of formula (I) has a
functional IC.sub.50 that is comparable to IC.sub.50s for compounds
A and B.
[0136] The compound of formula (I) has a protein binding adjusted
EC.sub.50 that is 4 times lower than that of compound A and 33
times lower than that of compound B.
[0137] The compound of formula (I) is a weaker Cyp3A4 inhibitor
compared to compounds A and B.
[0138] The compound of Formula (I) has a CYP3A4 IC.sub.50/
PBAdj.EC.sub.50 value that is 43 times higher than that for
compound of formula A, and 92 times higher than for the compound of
formula B.
[0139] The compound of formula (I) has a Rat CL value that is 2.7
times lower than that for compound of formula A, and 3.6 times
lower than that for the compound of formula B. The compound of
formula (I) has a percent bioavailability in rats that is 6.8 times
higher than compound A and 1.5 times higher than compound B.
[0140] The compound of formula (I) has a half life in rats that is
8.6 times longer than that of compound A and 3.9 times longer than
that of compound B.
[0141] The above data fairly suggest that the compound of formula
(I) has unexpected and advantageous properties compared to
compounds of formula A and B; and that the compound of formula (I)
is likely a better candidate for further development for the
treatment of chronic kidney disease, lung and/or kidney fibrosis,
and/or cardio-renal diseases.
* * * * *