1,2-BENZOTHIAZOLE COMPOUNDS FOR THE TREATMENT OF
KIDNEY DISORDERS
Renal or kidney disorders in man and animals involve an alteration in the normal
physiology and function of the kidney. Renal disorders can result from a wide range of
acute and chronic conditions and events, including physical, chemical, or biological
injury, insult or trauma, disease, and various inflammatory and autoimmune diseases.
Kidney disorders can lead to reduced kidney function, seriously compromising quality
and duration of life. Regardless of the initial insult or cause, kidney disorders are
characterized by progressive destruction of the renal parenchyma and the loss of
functional nephrons. This progression often leads to chronic kidney disease (CKD) and
end-stage renal disease and failure (ESRD/ESRF).
CKD is characterized by the progressive loss of kidney function. Increased
albuminuria and gradual, progressive loss of renal function are primary manifestations in
CKD. Decreased renal function results in increased blood creatinine and blood urea
nitrogen (BUN). CKD patients experience over time an increase in albuminuria,
proteinuria, serum creatinine, and renal histopathological lesions.
In humans, CKD has been, and continues to be, a considerable social and
economic problem in all industrialized countries. In the USA, 102,567 patients began
dialysis in 2003 (341 patients/year per million), and similar rates were found in
developing countries and in particular ethnic groups (2006, USRDS Am J Kidney Dis
47:1-286; Meguid El Nahas, A., and Bello, A. K. 2005. Chronic kidney disease: the
global challenge. Lancet 365:331-340.). However, these numbers are a small fraction of
the millions of patients who are thought to have some degree of renal impairment. In the
United States, the prevalence of chronically reduced kidney function is estimated to be
around 10% of adults (http://kidney .niddk.nih.gov/kudiseases/pubs/kustats/index.htm,
pages 1-4). Worsening CKD evolves into ESRD for many patients, requiring either
dialysis or kidney transplant. Glomerular filtration rate (GFR) is used to classify the
severity of CKD for patients, with lower GFR corresponding to more severe CKD.
Reducing the rate at which GFR declines in patients is expected to delay or prevent the
development of ESRD. Angiotensin converting enzyme (ACE) inhibitors e.g.,
benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril,
ramipril, and trandolapril; or angiotensin II receptor antagonists or blockers (ARBs) e.g.,
candesartan, eprosartan, irbesartan, telmisartan, valsartan, losartan, and olmesartan; or
combinations thereof, are used as current standard of care to slow the progression of CKD
to ERSD, but these have been shown inadequate to stop the ultimate progression to
dialysis.
The prevalence of renal disorders is also high in cats, whereas chronic renal
failure is considered the most important one. The prevalence of feline CKD has been
reported to reach up to 20%, with 53% of those cats being older than 7 years (Lefebre,
Toutain 2004, J. Vet. Pharm. Therap. 27, 265-281; Wolf, North. Am. Vet Congress
2006). Survival in cats with mild to moderate azotemia and extrarenal clinical signs
(International Renal Interest Society (IRIS) stages 2 and 3) ranges from 1 to 3 years.
Current therapy aims to delay the progression of the disease in cats by improving renal
function. This includes dietary protein restriction, modification of dietary lipid intake,
phosphate restriction, and treatment with ACE inhibitors (P. J. Barber (2004) The Kidney,
in: Chandler E A, Gaskell C J, Gaskell R M, (eds.) Feline Medicine and Therapeutics, 3rd
edition, Blackwell Publishing, Oxford, UK).
There remains a need in the art to provide alternative therapies for treating kidney
disorders in man and animals. Particularly acute needs are alternative therapies for
treating human and feline CKD.
A particularly high risk group for CKD include those with diabetes. Diabetic
nephropathy is chronic kidney disease or damage that results as a complication of
diabetes and is the leading cause of ERSD. Thus, diabetic nephropathy is both a subset of
chronic kidney disease and a complication of diabetes. The overall risk of developing
diabetic nephropathy varies between about 10% of type II diabetics (diabetes of late
onset) to about 30% of type I diabetics (diabetes of early onset). It is believed that
hyperglycemia (uncontrolled high blood sugar) leads to the development of kidney
damage, especially when high blood pressure is also present.
Multiple biochemical pathways have been proposed to explain the adverse effects
of hyperglycemia including activation of the diacylglycerol (DAG)-protein kinase C
(PKC) pathway. PKC is a family of serine/threonine kinases consisting of 12 isoforms:
conventional PKCs (, ΐ , 2, and ) that bind both Ca + and DAG, novel PKCs (, , ,
, and ) that bind DAG, but not Ca , and atypical PKCs (, , and ) that bind neither.
The activation of conventional and novel PKC isoforms requires the correct
phosphorylation of the isoforms and the presence of cofactors such as Ca + and DAG.
When properly phosphorylated, rapid or chronic increases in Ca + or DAG will induce its
translocation to the membranous compartments of the cells to elicit biological actions.
Rapid and short-term increases of DAG and Ca + levels are usually induced by cytokines
via the activation of phospholipase C. Chronic activation of PKCs requires sustained
elevations of DAG, which involves the activation of phospholipase D/C or the de novo
synthesis of DAG. PKC activation directly increases the permeability of albumin and
other macromolecules through barriers formed by endothelial cells. In the hyperglycemic
and diabetic states, all of these pathways probably contribute to the activation of the
DAG-PKC cascade.
PKC inhibitors are already known in the art for the treatment of certain diabetic
complications; see for example, US 5,552,386 and US 5,710,145.
Currently, there is no cure for diabetic nephropathy. Diabetic nephropathy, as
with CKD, is initially treated with medicines that lower blood pressure, such as ACE
inhibitors, ARBs, or combinations thereof. These classes of compounds also appear to
exhibit anti-inflammatory effects. Unfortunately, such treatments only slow disease
progression and are not successful in halting the progression or repairing damage done to
the kidneys. Treatments eventually become more aggressive (dialysis and/or kidney
transplantation) as the kidneys deteriorate towards failure.
Therefore, there exists a need for alternative compounds for diabetic nephropathy.
Preferably such compounds would be more efficacious and could optionally be combined
with an ACE inhibitor, an ARB, or a combination thereof. Preferably such compounds
would not inhibit Akt, a signaling molecule in the insulin signaling pathway, but would
inhibit the activation of conventional and novel PKC isoforms that could provide
treatment for diabetic complications such as atherosclerosis, cardiomyopathy,
retinopathy, nephropathy, and neuropathy.
The present invention provides a compound of the formula:
R i and R2 are each independently hydrogen, fluoro, chloro, methyl, or cyano,
wherein at least one of R i or R is not hydrogen;
Z is
R3, R6, R9, and Rio are each independently hydrogen or methyl;
R4 and R 5 are each independently hydrogen or methyl, or R4 and R 5 taken together
with the carbon to which they are attached form cyclopropyl; and
R7 and R are each independently hydrogen or methyl, or R7 and R taken together
with the carbon to which they are attached form cyclopropyl; wherein at least one of R3,
R4, R R7 R R9, or Rio is not hydrogen. The compound of formula I
may have clinical use in the treatment of kidney disorders, including chronic kidney
disease, and more particularly diabetic nephropathy. Further, a compound of formula I
may have clinical use in the treatment of diabetic complications other than or in addition
to diabetic nephropathy, such as atherosclerosis, cardiomyopathy, retinopathy, and
neuropathy.
In an embodiment of the invention, R i and R2 are each independently hydrogen,
fluoro, chloro, or methyl. In an embodiment of the invention, R i and R are each
independently fluoro, chloro, methyl, or cyano. In an embodiment of the invention, R i
and R are each independently fluoro, chloro, or methyl. In an embodiment of the
invention, R i and R are each independently fluoro or chloro. In an embodiment of the
invention, R i and R are each independently fluoro or methyl. In an embodiment of the
invention, R i and R are each independently chloro or methyl. In an embodiment of the
invention, R i and R are each fluoro. In an embodiment of the invention, R i is hydrogen.
In an embodiment of the invention, R is hydrogen. In an embodiment of the invention,
R 6 is methyl. In an embodiment of the invention, R4 and R5 are each independently
hydrogen or methyl. In an embodiment of the invention, R7 and R are each
independently hydrogen or methyl. In an embodiment of the invention, R3 is hydrogen.
In an embodiment of the invention, R3 is methyl. In an embodiment of the invention, R4
is hydrogen. In an embodiment of the invention, R4 is methyl. In an embodiment of the
invention, R5 is hydrogen. In an embodiment of the invention, R5 is methyl. In an
embodiment of the invention, R7 is hydrogen. In an embodiment of the invention, R7 is
methyl. In an embodiment of the invention, R is hydrogen. In an embodiment of the
invention, R is methyl. In an embodiment of the invention, R9 is hydrogen. In an
embodiment of the invention, R9 is methyl. In an embodiment of the invention, Rio is
hydrogen. In an embodiment of the invention, Rio is methyl. In an embodiment of the
invention, if R4 and R 5 are taken together with the carbon to which they are attached to
form cyclopropyl, then R7 and R are not taken together with the carbon to which they are
attached to form cyclopropyl. In an embodiment of the invention, if R7 and R are taken
together with the carbon to which they are attached to form cyclopropyl, then R4 and R 5
are not taken together with the carbon to which they are attached to form cyclopropyl.
In a preferred embodiment of the invention, A is
In a preferred embodiment of the invention, R2 is fluoro. In a preferred embodiment of
the invention, A is
In a preferred embodiment of the invention, Z is
a preferred embodiment of the invention, Z
In a preferred embodiment of the invention, Z is
The present invention also provides a method of treating a kidney disorder (such
as CKD or diabetic nephropathy) and/or a diabetic complication in a patient comprising
administering to a patient in need of such treatment an effective amount of a compound or
salt thereof of the present invention. The present invention also provides the above
methods further including administering in simultaneous, separate, or sequential
combination an additional active ingredient, such as an ACE inhibitor selected from the
group consisting of benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril,
perindopril, quinapril, ramipril, and trandolapril; an ARB selected from the group
consisting of candesartan, eprosartan, irbesartan, telmisartan, valsartan, losartan, and
olmesartan; or a combination thereof.
This invention also provides pharmaceutical compositions comprising a
compound or salt of the present invention with one or more pharmaceutically acceptable
carriers. In a particular embodiment, the pharmaceutical composition further comprises
one or more other therapeutic agents, for example, an ACE inhibitor selected from the
group consisting of benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril,
perindopril, quinapril, ramipril, and trandolapril; an ARB selected from the group
consisting of candesartan, eprosartan, irbesartan, telmisartan, valsartan, losartan, and
olmesartan; or a combination thereof.
This invention also provides a compound or salt of the present invention for use in
therapy. The invention also provides a compound or salt of the present invention for use
in the treatment of a kidney disorder (such as CKD or diabetic nephropathy) and/or a
diabetic complication. Additionally, this invention provides use of a compound or salt of
the present invention in the manufacture of a medicament for treating a kidney disorder
(such as CKD or diabetic nephropathy) and/or a diabetic complication. In an embodiment
of the invention, the compound or salt of the present invention is for use in simultaneous,
separate, or sequential use of an ACE inhibitor selected from the group consisting of
benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril,
ramipril, and trandolapril; an ARB selected from the group consisting of candesartan,
eprosartan, irbesartan, telmisartan, valsartan, losartan, and olmesartan; or a combination
thereof.
In an embodiment of the invention, provided are methods of decreasing
proteinuria in a patient, comprising administering to a patient in need of such treatment an
effective amount of a compound or salt thereof of the present invention. In an
embodiment of the invention, provided is a compound or salt thereof of the present
invention for use in decreasing proteinuria. In an embodiment of the invention, provided
is the use of a compound or salt thereof of the present invention for the manufacture of a
medicament for decreasing proteinuria.
In an embodiment of the invention, provided are methods of decreasing
albuminuria in a patient, comprising administering to a patient in need of such treatment
an effective amount of a compound or salt thereof of the present invention. In an
embodiment of the invention, provided is a compound or salt thereof of the present
invention for use in decreasing albuminuria. In an embodiment of the invention, provided
is the use of a compound or salt thereof of the present invention for the manufacture of a
medicament for decreasing albuminuria.
In an embodiment of the invention, provided are methods of slowing the rate of
progression to ESRD in a patient comprising administering to a patient in need of such
treatment an effective amount of a compound or salt thereof of the present invention. In
an embodiment of the invention, provided is a compound or salt thereof of the present
invention, for use in slowing the rate of progression to ESRD in a patient. In an
embodiment of the invention, provided is the use of a compound or salt thereof of the
present invention, for the manufacture of a medicament for slowing the rate of
progression to ESRD.
The term "kidney disorder" means any renal disorder, renal disease, or kidney
disease where there is any alteration in normal physiology and function of the kidney.
This can result from a wide range of acute and chronic conditions and events, including
physical, chemical or biological injury, insult, trauma or disease, such as for example
hypertension, diabetes, congestive heart failure, lupus, sickle cell anemia and various
inflammatory, infectious and autoimmune diseases, HIV(or related diseases)-associated
nephropathies etc. This term includes but is not limited to diseases and conditions such as
kidney transplant, nephropathy; chronic kidney disease (CKD); glomerulonephritis;
inherited diseases such as polycystic kidney disease; nephromegaly (extreme hypertrophy
of one or both kidneys); nephrotic syndrome; end stage renal disease (ESRD); acute and
chronic renal failure; interstitial disease; nephritis; sclerosis, an induration or hardening of
tissues and/or vessels resulting from causes that include, for example, inflammation due
to disease or injury; renal fibrosis and scarring; renal-associated proliferative disorders;
and other primary or secondary nephrogenic conditions. Fibrosis associated with dialysis
following kidney failure and catheter placement, e.g., peritoneal and vascular access
fibrosis, is also included.
In some embodiments, the kidney disorder may be generally defined as a
"nephropathy" or "nephropathies". The terms "nephropathy" or "nephropathies"
encompass all clinical-pathological changes in the kidney which may result in kidney
fibrosis and/or glomerular diseases {e.g. glomerulosclerosis, glomerulonephritis) and/or
chronic renal insufficiency, and can cause end stage renal disease and/or renal failure. In
some embodiments, the terms "nephropathy" or "nephropathies" refers specifically to a
disorder or disease where there is either the presence of proteins {i.e. proteinuria) in the
urine of a subject and/or the presence of renal insufficiency.
The term "fibrosis" refers to abnormal processing of fibrous tissue, or fibroid or
fibrous degeneration. Fibrosis can result from various injuries or diseases, and can often
result from chronic transplant rejection relating to the transplantation of various organs.
Fibrosis typically involves the abnormal production, accumulation, or deposition of
extracellular matrix components, including overproduction and increased deposition of,
for example, collagen and fibronectin. As used herein, the terms "kidney fibrosis" or
"renal fibrosis" or "fibrosis of the kidney" refer to diseases or disorders associated with
the overproduction or abnormal deposition of extracellular matrix components,
particularly collagen, leading to the degradation or impairment of kidney function.
A "diabetic complication" includes, but is not limited to atherosclerosis,
cardiomyopathy, retinopathy, nephropathy, and neuropathy.
The term "patient" includes living organisms in which a kidney disorder (such as
chronic kidney disease or diabetic nephropathy), and/or a diabetic complication can
occur, or which are susceptible to such pathologies. The term includes animals, (e.g.,
mammals, e.g., cats, dogs, horses, pigs, cows, goats, sheep, rodents, e.g., mice or rats,
rabbits, squirrels, bears, primates (e.g., chimpanzees, monkeys, gorillas, and humans)), as
well as chickens, ducks, Peking ducks, geese, and transgenic species thereof. Preferably,
the patient is a mammal. More preferably, the patient is a human or a feline.
The terms "treatment," "treat," "treating," and the like, are meant to include
slowing or reversing the progression of a disorder. These terms also include alleviating,
ameliorating, attenuating, eliminating, or reducing one or more symptoms of a disorder or
condition, even if the disorder or condition is not actually eliminated and even if
progression of the disorder or condition is not itself slowed or reversed.
"Pharmaceutically acceptable" as used in this application, for example with
reference to salts and formulation components such as carriers, includes "veterinarily
acceptable", and thus includes both human and non-human animal applications
independently.
The compounds and salts of the present invention are preferably formulated as
pharmaceutical compositions, which include veterinary compositions. The
pharmaceutical compositions may be administered by a variety of routes. Most
preferably, such compositions are for oral or intravenous administration, and include
tablets, capsules, solutions, and suspensions. "Carrier" is used herein to describe any
ingredient other than the active component(s) in a formulation. The choice of carrier will
to a large extent depend on factors such as the particular mode of administration or
application, the effect of the carrier on solubility and stability, and the nature of the
dosage form. Such pharmaceutical compositions and processes for preparing the same
are well known in the art. See, e.g., REMINGTON: THE SCIENCE AND PRACTICE
OF PHARMACY (D. Troy, et al, eds., 2 1st ed., Lippincott Williams & Wilkins, 2005).
The compounds of the present invention are generally effective over a wide
dosage range. "Effective amount" means the amount of the compound for the methods
and uses of the present invention that will elicit the biological or medical response of, or
desired therapeutic effect on, a tissue, system, or patient that is being sought by the
researcher, medical doctor, veterinarian, or other clinician. An effective amount of the
compound may vary according to factors such as the specific disease involved, the
disease state, age, sex, and weight of the patient, the ability of the compound to elicit a
desired response in the patient, the response of the patient, the particular compound
administered, the mode of administration, the bioavailability characteristics of the
preparation administered, the dose regimen selected, and the use of any concomitant
medications. An effective amount is also one in which any toxic or detrimental effect of
the compound is outweighed by the therapeutically beneficial effects. The frequency of
the administration will also be dependent upon several factors, and can be a single or
multiple dose administration.
It will be understood by the skilled reader that the compounds of the present
invention may capable of forming salts, including pharmaceutically acceptable salts. For
example, the compound of Example 1 contains basic amines, and accordingly reacts with
any of a number of inorganic and organic acids to form pharmaceutically acceptable acid
addition salts. Such pharmaceutically acceptable salts and common methodology for
preparing them are well known in the art. See, e.g., P. Stahl, et ah, HANDBOOK OF
PHARMACEUTICAL SALTS: PROPERTIES, SELECTION AND USE, (VCHA/Wiley-
VCH, 2008); S.M. Berge, et ah, "Pharmaceutical Salts", Journal of Pharmaceutical
Sciences, Vol. 66, No. 1, January 1977.
The skilled artisan will appreciate that compounds of the present invention may
contain at least one chiral center. The present invention contemplates all individual
enantiomers or diastereomers, as well as mixtures of the enantiomers and diastereomers
of said compounds including racemates. It is preferred that compounds of the present
invention containing at least one chiral center exist as single enantiomers or
diastereomers. The single enantiomers or diastereomers may be prepared beginning with
chiral reagents or by stereoselective or stereospecific synthetic techniques. Alternatively,
the single enantiomers or diastereomers may be isolated from mixtures by standard chiral
chromatographic or crystallization techniques. For Examples 10 and 3 1 below, Isomer 1
elutes from the column first and isomer 2 elutes second.
The following Preparations and Examples further illustrate the invention and
represent typical synthesis of the compounds of the invention. The reagents and starting
materials are readily available or may be readily synthesized by one of ordinary skill in
the art. It should be understood that the Preparations and Examples are set forth by way
of illustration and not limitation, and that various modifications may be made by one of
ordinary skill in the art. The compounds illustrated herein are named using
IUPACNAME ACDLABS.
As used herein, the following terms have the meanings indicated: "ATCC" refers
to American Type Culture Collection; "ATP" refers to adenosine-5'-triphosphate; " BSA"
refers to bovine serum albumin; "DMF" refers to N,N-dimethylformamide; 'DMSO"
refers to dimethyl sulfoxide; "EDTA" refers to ethylenediaminetetraacetic acid; "EGTA"
refers to ethylene glycol tetraacetic acid; "h" refers to hour or hours; "HATU" refers to
0-(7-azabenzotriazole- 1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate;
"HTRF" refers to homogeneous time resolved fluorescence; "ICR" refers to imprinting
control region; "IgG" refers to immunoglobulin G; "min" refers to minute or minutes;
"OGTT' refers to oral glucose tolerance test; "PBS" refers to phosphate buffered saline;
"PBMC" refers to peripheral blood mononuclear cells; "Pd(dppf)Cl2" refers to 1, -
Bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex;
"PKC" refers to protein kinase C; "PMA" refers to phorbol myristate acetate; "Ppleckstrin"
refers to phosphorylated pleckstrin; "RPMI" refers to Roswell Park Memorial
Institute; "SDS-PAGE" refers to sodium dodecyl sulfate polyacrylamide gel
electrophoresis; "STK" refers to serine/threonine kinase; and "THF" refers to
tetrahydrofuran.
Preparation 1
5-Bromo-2-fluoro-3 -methyl-benzaldehyde
A solution of 4-bromo-l-fluoro-2-methyl-benzene (300 g, 1.59 mol) in THF (1.4
L) is cooled to -65 to -70 °C under nitrogen. Then 2.0 M lithium diisopropylamide in
THF ( 1 L, 2.0 mol, 1.26 eq) is added over 2.5 h, keeping the internal temperature between
-65 to -70 °C. The reaction temperature is maintained for an additional 1 h after the
addition is complete. A solution of DMF (240 mL) and THF (100 mL) are added. The
mixture is stirred at -78 °C for 2 h, then warmed to -10 °C over 16 h. Saturated
ammonium chloride (5 L) is added, and the mixture is extracted with ethyl acetate (2 3
L). The combined extracts are washed with water (5 L), washed with saturated aqueous
sodium chloride (brine) (5 L), and concentrated to give the crude title compound (300 g),
which is used without further purification.
Preparation 2
5-Bromo-2-t rt-butylsulfanyl-3-methyl-benzaldehyde
To a solution of 5-bromo-2-fluoro-3-methyl-benzaldehyde (480 g, 2.534 mol) in
DMF (2.5 L), is added potassium carbonate (600 g, 4.3 mol) followed by 2-
methylpropane-2-thiol (300 mL, 2.7 mol). The resulting mixture is heated to 60 °C for 16
h. Water ( 1.5 L) is added at this temperature, and then the mixture is stirred at 60 °C for
an additional 8 h. The reaction mixture is cooled to about 25 °C, poured into water (10
L), and then extracted with ethyl acetate (7 L). The organic layer is washed with water (7
L), 5% brine (5 L) and concentrated to afford the crude title compound (600 g), which is
used without further purification. LC-ES/MS m/z (7 Br/ 1Br) 230.9/232.9 [Misobutene+
H]+.
Preparation 3
5-Bromo-2-t rt-butylsulfanyl-3-methyl-benzaldehyde oxime
To a solution of 5-bromo-2-t rt-butylsulfanyl-3-methyl-benzaldehyde (600 g,
1.985 mol) in 95% ethanol (3 L) is added hydroxylamine hydrochloride (218 g, 3.1 mol).
Then sodium bicarbonate (280 g, 3.34 mol) is added in portions over 10 min. After the
addition is complete, the reaction mixture is stirred at room temperature for 3 h. The
reaction mixture is diluted with water (3 L) and extracted with ethyl acetate (3 L). The
organic layer is washed with 5% brine and concentrated. The resulting residue is
triturated in petroleum ether (5 L), filtered, and washed with petroleum ether (2 1 L).
The cake is air-dried to afford the title compound as a yellow solid (460 g, 63% yield for
three steps). LC-ES/MS m/z (7 Br/ 1Br) 304.0/306.0 [M+H]+.
Preparation 4
5-Bromo-7-methyl- 1,2-benzothiazole
To a suspension of 5-bromo-2 -t rt-butylsulfanyl-3-methyl-benzaldehyde oxime
(460 g, 1.5 mol) in toluene (3 L) is added 4-methylbenzenesulfonic acid (50 g, 0.26 mol).
The reaction mixture is heated at 80 °C for 2 h, and then is refluxed for 2 h. The reaction
is cooled and diluted with ethyl acetate (3 L). The organic portion is washed with water
(3 L), saturated sodium bicarbonate (3 L) and brine (3 L). The organic portion is
concentrated. The resulting residue is slurred in petroleum ether (3 L) and filtered. The
filter cake is washed with petroleum ether (2 1 L) to afford the title compound as a
brown solid (250 g, 72%). LC-ES/MS m/z (7 Br/ 1Br) 227.9/229.9 [M+H]+. H NMR
(400 MHz, DMSO-i¾ 9.08 (s, 1H), 8.26 (s, 1H), 7.57 (s, 1H), 2.54 (s, 3H).
Preparation 5
5-Bromo-l ,2-benzothiazole-7-carboxylic acid
To a solution of 5-bromo-7-methyl-l,2-benzothiazole (50 g, 0.22 mol) in carbon
tetrachloride (1.5 L) is added N-bromosuccinimide (234.1 g, 1.3 mol) and benzoyl
peroxide (10.6 g, 44 mmol). The mixture is heated at reflux for 16 h, cooled to 25 °C and
filtered. The filter cake is dissolved in water (2.5 L) and ethyl acetate (2 L). The organic
layer and filtrate are combined together and concentrated to give a solid. The residual
solid is dried in the air to afford the crude mixture of 5-bromo-7-(dibromomethyl)-l,2-
benzothiazole (38% purity detected by UV absorption at 214 nm, LC-ES/MS m z
(7 Br/ 1Br) 384/386/388/390 [M+H]+) and 5-bromo-7-(tribromomethyl)-l,2-
benzothiazole (28% purity detected by UV absorption at 214 nm, LC-ES/MS m z
(7 Br/ 1Br) 462/464/466/468/470 [M+H]+) as a solid (80 g). The crude mixture (80 g) is
added to a solution of lithium hydroxide ( 17.4 g, 0.4 15 mol) in water (800 mL) and
dioxane (800 mL) and heated at reflux for 16 h. The solution is cooled to 25 °C and
acidified to pH = 2 to 3 with 2 N HC1. The mixture is extracted with ethyl acetate (3
500 mL) and the combined organic portions are concentrated to give a crude mixture of
5-bromo-l,2-benzothiazole-7-carbaldehyde (50% purity detected by UV absorption at
214 nm, LC-ES/MS m z (7 Br/ 1Br) 242/244 [M+H]+) and 5-bromo-l,2-benzothiazole-7-
carboxylic acid (8% purity detected by UV absorption at 214 nm, LC-ES/MS m z
(7 Br/ 1Br) 258/260 [M+H]+) (100 g). The crude mixture (100 g) is taken up in THF (960
mL), t rt-butyl alcohol (320 mL), and water (320 mL). Sodium chlorite (32.2 g, 0.3 11
mol), sodium dihydrogen phosphate monohydrate (98.3 g, 0.62 mol), and sulfamic acid
(32.2 g, 0.33 mol) are added. The mixture is stirred at 25 °C for 16 h, and then
concentrated. The resulting residue is purified by triturating with dichloromethane and
water (1:1, 400 mL). The slurry is filtered and the cake is dried in air to give the title
compound as a solid (26 g, 46% 3-step yield). LC-ES/MS m z (7 Br/ 1Br) 258/260
[M+H]+. H NMR (400 MHz, DMSO-i¾ 9.18 (d, J = 7.2 Hz, 1 H), 8.28 (d, J = 2.4 Hz,
1 H), 7.12 (d, J = 7.6 Hz, 1 H), 6.714 - 6.719 (m, 1H).
Preparation 6
(5-Bromo-l,2-benzothiazol-7-yl )-[c ,-3,5-dimethylpiperazin-l-yl]methanone
To a mixture of 5-bromo-l,2-benzothiazole-7-carboxylic acid (10 g, 0.039 mol)
and DMF (100 mL) is added cw-2,6-dimethylpiperazine (6.64 g, 0.058 mol) at 28 °C.
The resulting mixture is cooled to 0 °C and HATU (22.1 g, 0.058 mol) is added in
portions (internal temperature is 0 to 2 °C). The mixture is warmed to 25 °C and stirred
at this temperature for 16 h. The mixture is concentrated under vacuum, poured into
water (30 mL) and extracted with ethyl acetate (3 50 mL). The combined organic
portions are washed with saturated sodium bicarbonate solution (30 mL) and brine (30
mL). The organic portion is concentrated and the resulting residue is dissolved in a
mixture of dichloromethane (30 mL) and water (30 mL). 6 N HC1 is added dropwise
until a majority of the solid appears. The solid is collected by filtration. The aqueous
phase of the filtrate is separated and combined with the solid cake, basified with sodium
bicarbonate solution to pH 8, and then extracted with dichloromethane (3 50 mL). The
organic phase is concentrated to give the title compound (10.1 g, 74%). LC-ES/MS m/z
(7 Br/ 1Br) 354/356 [M+H]+.
Preparation 7
[5-(2,6-Difluoro-4-methoxy -phenyl)- 1,2-benzothiazol-7-yl] -[cis-3 ,5-dimethylpiperazin- 1-
l methanone
A mixture of (5-bromo- 1,2-benzothiazol-7-yl)- [cis-3 ,5-dimethylpiperazin- 1-
yl]methanone (2.0 g, 5.65 mmol), 2,6-difluoro-4-methoxyphenyl boronic acid (1.6 g,
2.47 mmol), [l , -bis(diphenylphosphino)ferrocene]dichloropalladium (II) (Pd(dppf)Cl2)
(415 mg, 0.57 mmol), sodium carbonate (1.83 g, 169 mmol), 1,4-dioxane (18 mL), and
water (2 mL) is stirred at 85 °C under a nitrogen atmosphere for 16 h. The mixture is
cooled to 25 °C and filtered. The filtrate is concentrated under vacuum. The residue is
purified by column chromatography on silica gel (eluting with dichloromethane in
methanol = 150:1 to 100:1). The resulting crude product is dissolved in dichloromethane
(20 mL), 4 N HCl in 1,4-dioxane (5 mL, 20 mmol) and a little water is added dropwise.
The resulting solid is filtered off and washed with dichloromethane (2 50 mL). The
solid is then suspended in water (10 mL), basified with saturated sodium carbonate
solution, and extracted with dichloromethane (3 100 mL). The combined organic
portions are dried over sodium sulfate, filtered, and the filtrate concentrated to give the
title compound (1.1 g, 47%). LC-ES/MS m/z 418.1 [M+H]+.
Example 1
4-(7- {[(3R,5S)-3,5-DIMETHYLPIPERAZIN- 1-YL]CARBONYL }-1,2-
BENZOTHIAZOL-5 -YL)-3,5-DIFLUOROPHENOL
To a mixture of 5-(2,6-difluoro-4-methoxy-phenyl)-l,2-benzothiazol-7-yl ] - [ ,-
3,5-dimethylpiperazin-l-yl]methanone (1.0 g, 2.40 mmol) and dichloromethane (30 mL)
is added boron tribromide (2.7 mL, 10.54 mmol) dropwise at -78 °C under a nitrogen
atmosphere. The mixture is allowed to warm to 25 °C and stirred for 2 1 h. The reaction
is quenched with methanol at -40 °C and basified with ammonia to pH = 8. The mixture
is extracted with a solution of dichloromethane and isopropyl alcohol (3 160 mL, 3/1
v/v). The combined organic portions are dried over sodium sulfate, filtered, and
concentrated. The resulting residue is slurried with methanol (5 mL) and filtered. The
filter cake is washed with dichloromethane to give the title compound (0.5 g, 51%). LCES/
MS m z 404.0 [M+H]+.
Example 2
4-(7- {[(3R,5S)-3,5-DIMETHYLPIPERAZIN- 1-YL]CARBONYL}-1,2-
BENZOTHIAZOL-5-YL)-3,5-DIFLUOROPHENOL HYDROCHLORIDE
To a mixture of [5-(2,6-difluoro-4-hydroxy-phenyl)-l,2-benzothiazol-7-yl ]-[cz5'-
3,5-dimethylpiperazin-l-yl]methanone (9.0 g, 22.3 mmol) in methanol (300 mL) and
dichloromethane (300 mL) is added 4 N HCI in 1,4-dioxane (30 mL, 120 mmol). The
mixture is stirred at 25 °C for 16 h. The reaction mixture is concentrated under vacuum
and the residue is washed with methanol (3 50 mL) to give the title compound as a
white solid (8.66 g, 88%). LC-ES/MS m/z 404.0 [M+H]+. H NMR (300 MHz, DMSOd
6) 10.75(s, 1H), 9.72 (d, J = 11.7 Hz, 1H), 9.22 (s, 2H), 8.41 (s, 1H), 7.92 (s, 1H), 6.71
(d, J = 10.2 Hz, 2H), 4.32-4.25 (m, 2H), 3.46-3.55 (m, 3H), 3.13-3.17 (m, 1H), 1.27 (d, J
= 6.3 Hz, 6H).
Example 3
[5-(2,6-DIFLUORO-4-HYDROXYPHENYL)-l,2-BENZOTHIAZOL-7-YL][(3R,5S)-
3,5-DIMETHYLPIPERAZIN- 1-YLJMETHANONE
To a solution of [5-(2,6-difluoro-4-hydroxy-phenyl)-l,2-benzothiazol-7-yl ] - [ ,-
3,5-dimethylpiperazin-l-yl]methanone (967.2 mg, 2.4 mmol) in dichloromethane (8 mL)
and methanol (30 mL) is added 0.2 N methanesulfonic acid aqueous solution (12 mL, 2.4
mmol). The mixture is stirred at 25 °C for 30 min and concentrated under reduced
pressure at 40 °C to give the title compound as a light yellow solid (1.18 g, 98%). ES/MS
m z 404.0 [M+H]+. H NMR (300 MHz, CD3OD) 9.13(s, 1H), 8.45 (s, 1H), 7.92 (s,
1H), 6.65 (d, J = 13.5 Hz, 2H), 4.55-4.60 (m, 2H), 3.55-3.62 (m, 2H), 3.14-3.34 (m, 2H),
2.75 (s, 3H), 1.42(d, J = 6.9 Hz, 6H).
Example 4
ETHANE- 1,2-DISULFONIC ACID - [5-(2,6-DIFLUORO-4-HYDROXYPHENYL)-l,2-
BENZOTHIAZOL-7- YL [(3R,5 S)-3 ,5-DIMETHYLPIPERAZIN- 1-YL] METHANONE
To a solution of [5-(2,6-difluoro-4-hydroxy-phenyl)-l,2-benzothiazol-7-yl ]- [ ,-
3,5-dimethylpiperazin-l-yl]methanone (967.2 mg, 2.4 mmol) in dichloromethane (8 mL)
and methanol (30 mL) is added 0.05 N ethane- 1,2-disulfonic acid aqueous solution (24
mL, 1.2 mmol). The mixture is stirred at 25 °C for 30 min and concentrated under
reduced pressure at 40 °C to give the title compound as a white solid ( 1.18 g, 99%). LCES/
MS m/z 404.0 [M+H]+. H NMR (300 MHz, DMSO-i¾ 10.71 (s, 1H), 9.31 (s, 2H),
8.65 (s, 1H), 7.92 (s, 1H), 6.73 (d, J = 16.5 Hz, 2H), 4.33-4.37 (m, 2H), 3.36-3.48 (m,
2H), 3.02-3.14 (m, 2H), 2.50-2.52 (m, 2H), 1.42(d, J = 6.3 Hz, 6H).
The following compounds may be prepared in an analagous fashion as set forth in
Preparations 1-7 and Examples 1-4.
Table 1
Example Name Physical Data
5 [5-(3-FLUORO-4-HYDROXY-2-
METHYLPHENYL)- 1,2-
BENZOTHIAZOL-7-YL](2,2,5,5- LC-ES/MS m/z 428.2 [M+H]+
TETRAMETHYLPIPERAZIN- 1-
YL)METHANONE
HYDROCHLORIDE
6 [5-(2,6-DIFLUORO-4-
HYDROXYPHENYL)- 1,2-
BENZOTHIAZOL-7-YL] [(2R,3 S,5R)- LC-ES/MS m/z 418.1 [M+H]+
2,3 ,5-TRIMETHYLPIPERAZIN- 1-
YL]METHANONE
HYDROCHLORIDE
7 3-CHLORO-4-(7-{[(3R,5S)-3,5-
DIMETHYLPIPERAZIN- 1-
YL]CARBONYL}-l,2- LC-ES/MS m/z 419.9 [M+H]+
BENZOTHIAZOL-5 -YL)-5-
FLUOROPHENOL
HYDROCHLORIDE
8 ETHANESULFONIC ACID - [5-(2,6-
DIFLUORO-4-HYDROXYPHENYL)-
l,2-BENZOTHIAZOL-7-YL][(3R,5S)- LC-ES/MS m/z 404.1 [M+H]+
3,5-DIMETHYLPIPERAZIN- 1-
YL]METHANONE
9 4-(7-{[(2S,5R)-2,5-
DIMETHYLPIPERAZIN- 1-
YL|CARBONYL}-l,2-
Example Name Physical Data
BENZOTHIAZOL-5 -YL)-3,5- LC-ES/MS m/z 404.2 [M+H]+
DIFLUOROPHENOL
TRIFLUOROACETATE (SALT)
10 [5-(2,6-DIFLUORO-4-
HYDROXYPHENYL)- 1,2-
BENZOTHIAZOL-7-YL](2,3,5,6- LC-ES/MS m/z 432.2 [M+H]+
TETRAMETHYLPIPERAZIN- 1-
YL)METHANONE
HYDROCHLORIDE (Isomer 1)
1 1 5-CHLORO-4-(7-{[(3R,5S)-3,5-
DIMETHYLPIPERAZIN- 1-
YL]CARB0NYL}-1,2- LC-ES/MS m/z 420.0 [M+H]+
BENZOTHIAZOL-5 -YL)-2-
FLUOROPHENOL
HYDROCHLORIDE
12 4-(7-{[(3R,5S)-3,5-
DIMETHYLPIPERAZIN- 1-
YL]CARB0NYL}-1,2- LC-ES/MS m/z 404.2 [M+H]+
BENZOTHIAZOL-5 -YL)-3,5-
DIFLUOROPHENOL
TRIFLUOROACETATE (SALT)
13 [5-(2,6-DIFLUORO-4-
HYDROXYPHENYL)- 1,2-
BENZOTHIAZOL-7-YL] [(2R,3 S,5R)- LC-ES/MS m/z 418.1 [M+H]+
2,3 ,5-TRIMETHYLPIPERAZIN- 1-
YL]METHANONE
HYDROCHLORIDE
14 [5-(2,6-DIFLUORO-4-
HYDROXYPHENYL)- 1,2-
BENZOTHIAZOL-7-YL] [(2R,5R)- LC-ES/MS m/z 404.1 [M+H]+
2,5-DIMETHYLPIPERAZIN- 1-
YL]METHANONE
HYDROCHLORIDE
15 [5-(2,6-DIFLUORO-4-
HYDROXYPHENYL)- 1,2-
BENZOTHIAZOL-7- LC-ES/MS m/z 432.1 [M+H]+
YL][(2R,3S,5R,6S)-2,3,5,6-
TETRAMETHYLPIPERAZIN- 1-
YL]METHANONE
HYDROCHLORIDE
16 [5-(2,6-DIFLUORO-4-
HYDROXYPHENYL)- 1,2-
BENZOTHIAZOL-7-YL] [(5 S)-3,3 ,5- LC-ES/MS m/z 418.1 [M+H]+
TRIMETHYLPIPERAZIN- 1-
Example Name Physical Data
YL]METHANONE
HYDROCHLORIDE
17 3-CHLORO-5-FLUORO-4-(7-{[(3R)-
3-METHYLPIPERAZIN- 1-
YL]CARB0NYL}-1,2- LC-ES/MS m/z 405.9 [M+H]+
BENZOTHIAZOL-5-YL)PHENOL
HYDROCHLORIDE
18 [5-(2,6-DIFLUORO-4-
HYDROXYPHENYL)- 1,2-
BENZOTHIAZOL-7-YL] [(3R,5 S)- LC-ES/MS m/z 418.1 [M+H]+
3,4,5-TRIMETHYLPIPERAZIN-l-
YL]METHANONE
HYDROCHLORIDE
19 3-CHLORO-4-(7-{[(3R,5S)-3,5-
DIMETHYLPIPERAZIN- 1- LC-ES/MS m/z 402.0 [M+H]+
YL]CARB0NYL}-1,2-
BENZOTHIAZOL-5-YL)PHENOL
HYDROCHLORIDE
20 [5-(2-CHLORO-6-FLUORO-4-
HYDROXYPHENYL)- 1,2-
BENZOTHIAZOL-7-YL] [(5 S)-3,3 ,5- LC-ES/MS m/z 433.9 [M+H]+
TRIMETHYLPIPERAZIN- 1-
YL]METHANONE
HYDROCHLORIDE
2 1 [5-(2-CHLORO-5-FLUORO-4-
HYDROXYPHENYL)- 1,2-
BENZOTHIAZOL-7-YL](2,2,5,5- LC-ES/MS m/z 448.1 [M+H]+
TETRAMETHYLPIPERAZIN- 1-
YL)METHANONE
HYDROCHLORIDE
22 4-(7-{[(3R,5R)-3,5-
DIMETHYLPIPERAZIN- 1-
YL]CARB0NYL}-1,2- LC-ES/MS m/z 404.1 [M+H]+
BENZOTHIAZOL-5 -YL)-3,5-
DIFLUOROPHENOL
HYDROCHLORIDE
23 [(3 S,5 S)-3,5-DIMETHYLPIPERAZINl-
YL][5-(2-FLUORO-4-
HYDROXYPHENYL)- 1,2- LC-ES/MS m/z 386.1 [M+H]+
BENZOTHIAZOL-7-
YL]METHANONE
HYDROCHLORIDE
24 3-CHLORO-4-{7-[(3,3-
DIMETHYLPIPERAZIN- 1-
Example Name Physical Data
YL)CARBONYL]-l,2-
BENZOTHIAZOL-5-YL}-5-
FLUOROPHENOL LC-ES/MS m/z 420.1 [M+H]+
HYDROCHLORIDE
25 4-{7-[(3,3-DIMETHYLPIPERAZIN-l-
YL)CARBONYL]-l,2-
BENZOTHIAZOL-5-YL}-3- LC-ES/MS m/z 400.0 [M+H]+
FLUORO-5-METHYLPHENOL
HYDROCHLORIDE
26 2-(7-{[(2S,5R)-2,5-
DIMETHYLPIPERAZIN- 1-
YL]CARB0NYL}-1,2- LC-ES/MS m/z 411.1 [M+H]+
BENZOTHIAZOL-5-YL)-3-
FLUORO-5-
HYDROXYBENZONITRILE
HYDROCHLORIDE
27 [5-(2-CHLORO-6-FLUORO-4-
HYDROXYPHENYL)- 1,2-
BENZOTHIAZOL-7-YL](4,7- LC-ES/MS m/z 418.1 [M+H]+
DIAZASPIRO[2.5]OCT-7-
YL)METHANONE
HYDROCHLORIDE
28 [5-(2,5-DIFLUORO-4-
HYDROXYPHENYL)- 1,2-
BENZOTHIAZOL-7-YL][(3R,5S)-3,5- LC-ES/MS m/z 404.1 [M+H]+
DIMETHYLPIPERAZIN- 1-
YL]METHANONE
HYDROCHLORIDE
29 [5-(5-FLUORO-4-HYDROXY-2-
METHYLPHENYL)- 1,2-
BENZOTHIAZOL-7-YL](2,2,5,5- LC-ES/MS m/z 428.2 [M+H]+
TETRAMETHYLPIPERAZIN- 1-
YL)METHANONE
HYDROCHLORIDE
30 [(3R,5S)-3,5-
DIMETHYLPIPERAZIN-1 -YL] [5-(5-
FLUORO-4-HYDROXY-2- LC-ES/MS m/z 400.0 [M+H]+
METHYLPHENYL)- 1,2-
BENZOTHIAZOL-7-
YL]METHANONE
HYDROCHLORIDE
3 1 [5-(2,6-DIFLUORO-4-
HYDROXYPHENYL)- 1,2-
BENZOTHIAZOL-7-YL](2,3,5,6-
Example Name Physical Data
TETRAMETHYLPIPERAZIN- 1- LC-ES/MS m/z 432.2 [M+H]+
YL)METHANONE
HYDROCHLORIDE (Isomer 2)
32 [5-(2-CHLORO-3-FLUORO-4-
HYDROXYPHENYL)- 1,2-
BENZOTHIAZOL-7-YL](3 ,3- LC-ES/MS m/z 420.1 [M+H]+
DIMETHYLPIPERAZIN- 1-
YL)METHANONE
HYDROCHLORIDE
33 3-CHLORO-4-(7-{[(2S,5R)-2,5-
DIMETHYLPIPERAZIN- 1-
YL]CARB0NYL}-1,2- LC-ES/MS m/z 419.9 [M+H]+
BENZOTHIAZOL-5-YL)-5-
FLUOROPHENOL
HYDROCHLORIDE
34 [5-(2,5-DIFLUORO-4-
HYDROXYPHENYL)- 1,2-
BENZOTHIAZOL-7-YL](2,2,5,5- LC-ES/MS m/z 432.2 [M+H]+
TETRAMETHYLPIPERAZIN- 1-
YL)METHANONE
HYDROCHLORIDE
35 3-CHLORO-4-(7-{[(2S,5R)-2,5-
DIMETHYLPIPERAZIN- 1-
YL]CARB0NYL}-1,2- LC-ES/MS m/z 419.9 [M+H]+
BENZOTHIAZOL-5-YL)-2-
FLUOROPHENOL
HYDROCHLORIDE
36 4-(7-{[(2R,6S)-2,6-
DIMETHYLPIPERAZIN- 1-
YL]CARB0NYL}-1,2- LC-ES/MS m/z 404.2 [M+H]+
BENZOTHIAZOL-5-YL)-3,5-
DIFLUOROPHENOL
HYDROCHLORIDE
37 [(2S,5R)-2,5-
DIMETHYLPIPERAZIN-1 -YL] [5-(3-
FLUORO-4-HYDROXY-2- LC-ES/MS m/z 400.2 [M+H]+
METHYLPHENYL)- 1,2-
BENZOTHIAZOL-7-
YL]METHANONE
HYDROCHLORIDE
38 [5-(2,6-DIFLUORO-4-
HYDROXYPHENYL)- 1,2-
BENZOTHIAZOL-7-YL] [(5R)-3,3,5- LC-ES/MS m/z 418.1 [M+H]+
TRIMETHYLPIPERAZIN- 1-
Example Name Physical Data
YL]METHANONE
HYDROCHLORIDE
39 4-{7-[(3,3-DIMETHYLPIPERAZIN-l-
YL)CARBONYL]-l,2-
BENZOTHIAZOL-5-YL} -3,5- LC-ES/MS m/z 403.9 [M+H]+
DIFLUOROPHENOL
40 3-FLUORO-5-METHYL-4-(7-{[(3R)-
3-METHYLPIPERAZIN- 1-
YL]CARB0NYL}-1,2- LC-ES/MS m/z 386.0 [M+H]+
BENZOTHIAZOL-5-YL)PHENOL
HYDROCHLORIDE
4 1 4-[7-(4,7-DIAZASPIRO[2.5]OCT-7-
YLCARBONYL)-l ,2-
BENZOTHIAZOL-5-YL]-3,5- LC-ES/MS m/z 402 [M+H]+
DIFLUOROPHENOL
TRIFLUOROACETATE (SALT)
42 4-(7-{[(2R,6S)-2,6-
DIMETHYLPIPERAZIN- 1-
YL]CARB0NYL}-1,2- LC-ES/MS m/z 399.9 [M+H]+
BENZOTHIAZOL-5-YL)-3-
FLUORO-5-METHYLPHENOL
HYDROCHLORIDE
Biological Assays
Considerable literature evidence suggests that the intrarenal renin-angiotensin
system plays an important role in diabetic nephropathy. Since PKC is activated by
angiotensin II and initial treatment for diabetic nephropathy is with angiotensinconverting
enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), or
combinations thereof, it is likely that the combination of a PKC inhibitor with an ACE
inhibitor, an ARB, or both may present a more effective treatment option for patients with
diabetic nephropathy.
Furthermore, the effects of ruboxistaurin, reported to be a selective PKC-
inhibitor, administered in combination with an ACE inhibitor, ARB, or both have been
described in the literature. Turtle, K.T. et al., "The Effect of Ruboxistaurin on
Nephropathy in Type 2 Diabetes", Diabetes Care, Volume 28, No. 11: 2686-2690
(November 2005). It was reported there that ruboxistaurin had favorable effects on
albuminuria and renal function in persons with type 2 diabetes and nephropathy.
Therefore, it is believed that a compound that inhibits multiple PKC isoforms, i.e.
conventional (including PKC-) and/or novel isoforms, particularly when used in
combination with an ACE inhibitor, ARB, or both, may provide an effective treatment for
diabetic nephropathy.
The prevalence of diabetic nephropathy has placed it at the forefront of attempts
to discover new therapies for progressive kidney disease. However, non-diabetic kidney
diseases or disorders, principally due to various forms of glomerulopathy, remain a major
contributor to the number of patients requiring dialysis and transplants. While the
aetiologies of these two major categories of kidney disease are clearly different, they
share common clinical manifestations such as hypertension, proteinuria (albumin) and
declining glomerular filtration rate (GFR) as well as major histopathological
characteristics, including glomerulosclerosis, tubulointerstitial fibrosis and macrophage
infiltration. Numerous findings also suggest common pathogenetic mechanisms that link
diabetic and non-diabetic kidney disease, such as the renin-angiotensin system (RAS) and
the elaboration of profibrotic growth factors. Together, these findings raise the possibility
that treatments designed to target the diabetic kidney might also be effective in the nondiabetic
setting. Kelley, D.J. et ah, "Protein Kinase C-Inhibition Attenuates the
Progression of Nephropathy in Non-Diabetic Kidney Disease", Nephrol. Dial.
Transplant, Vol. 24, 1782-1790 (2009). Therefore, it is believed that a compound of the
current invention may provide an effective treatment for kidney disorders, including
chronic kidney disease.
The following assays demonstrate that the exemplified compounds of the present
invention are potent inhibitors of PKC, demonstrate efficacy by reducing albuminuria, are
inhibitors of the conventional and novel isoforms of PKC possibly providing a more
effective treatment for diabetic nephropathy by targeting multiple diabetic complications
simultaneously, and are preferably not potent inhibitors of Akt.
Assay 1: PKC isoforms based HTRF KINEASE - STK jumbo Assay Protocol
The purpose of this assay is to evaluate the inhibitory activity of compounds on
various PKC isoforms. It has been reported that perturbations in vascular cell
homeostasis caused by different PKC isoforms (PKC-a, -/2, and PKC-) are linked to
the development of pathologies affecting large vessel (atherosclerosis, cardiomyopathy)
and small vessel (retinopathy, nephropathy, and neuropathy) complications. Geraldes, P.
and King, G.L., "Activation of Protein Kinase C Isoforms and Its Impact on Diabetic
Complications", Cir. Res., 106: 1319-1331 (2010). So a compound that inhibits multiple
conventional and novel PKC isoforms may provide a more effective treatment option for
diabetic nephropathy by targeting multiple diabetic complications simultaneously.
Effects of a test compound on the kinase activity of PKCa,P2, , , , and
, are determined using a HTRF KINEASE™-STK assay kit (Cisbio) in accordance with
the protocol provided by the vendor. It is a general method for measuring
Serine/Threonine kinase activities using kinase substrates and a universal detection
system. In the current protocol, biotinilated STK1provided in the assay kit is used as the
substrate that can be phosphorylated by any PKC isoform. The HTRF KINEASE™- STK
assay kit format involves the two steps (Enzymatic step and Detection step) described
below. First, the kinase phosphorylates the substrate in the presence of ATP. Second, the
detection reagents provided in the assay kit recognize the phosphorylated substrate.
Detection reagents include the STK-antibody labeled with Eu3+-Cryptate and
streptavidin-XL665. The antibody recognizes only the phosphorylated form of the STK
substrate. Streptavidin binds to biotin attached to the kinase substrate. Cryptate serves as
the energy donor, while XL665 fluorophor associated with streptavidin serves as an
acceptor. Physical proximity between the donor and acceptor results in the fluorescence
resonance energy transfer and associated fluorescent signal. Intensity of the signal is
proportional to the level of substrate phosphorylation.
Staurosporin (Sigma) may be used as a positive control. It is prepared in DMSO
to make up a 10 mM stock solution and then serially diluted (10 - 0.005 ) to
obtain a ten-point dilution curve. Test compounds are prepared in a similar manner with
the top dose at 100 . Each compound is serially diluted to obtain a ten-point dilution
curve.
The reaction system that is used in the Enzymatic step, consists of the reaction
buffer, substrate, and an enzyme. Two kinds of reaction buffers are prepared from the
kinase buffer for different PKC isoforms. 5x Kinase Buffer is provided as a part of the kit
and consists of HEPES 250 mM (pH7.0), NaN3 0.1%, BSA 0.05%, and Orthovanadate
0.5 mM.
For PKCa and PKCs, 1.25x Reaction Buffer consists of 12.5 mM MgCl2, 1.25
mM DTT, 0.125 mM CaCl2, and 1.25x Kinase Buffer. For PKCp2, PKC8, PKCi, and
PKCC, 1.35x Reaction Buffer is comprised of 12.5 mM MgCl2, 1.25 mM DTT, 1.25 mM
lipid activator (Upstate Biotechnology, Inc.), and 1.25x Kinase Buffer. These buffers are
used for preparation of the substrate mix and enzyme mix. Substrate and enzyme mix
differ for various PKC isoforms. For PKCa, the substrate mix consists of 50 ATP
and 2.5 STKl, while the enzyme mix is represented by 3 nM PKCa. For PKCP2, the
substrate mix consists of 25 ATP and 2.5 STKl, while the enzyme is represented
by 3.75 nM of PKCp2. For PKC8, the substrate mix consists of 62.5 ATP and 2.5
STKl, while the enzyme is represented by 3.75 nM PKC8. For PKCs, the substrate
mix consists of 25 ATP and 2.5 STKl, while the enzyme mix is represented by 3
nM PKCs. For PKCi, the substrate mix consists of 87.5 ATP and 2.5 STKl,
while the enzyme is represented by 7.5 nM PKCi. For , the substrate mix consists
of 10 ATP and 2.5 STKl, while the enzyme is represented by 7.5 nM .
For each PKC isoform, to generate 50 of reaction system, \ of diluted
compound is transferred from a dilution plate to an assay plate, using a Thermo Scientific
Matrix. Then 20 of substrate mix is added to the assay plate (using a MULTIDROP®
Combi dispenser, ThermoScientific) and centrifuged at 500 rpm for 1 min. Finally, 20 
of enzyme mix is added to the assay plate (using the MULTIDROP®) to initiate the
reaction. The plate is shaken for 60 sec on a platform shaker and centrifuged at 1000 rpm
for 1 min. The reaction system is incubated at room temperature for various time periods
specific for individual PKC isoforms: 30 min for PKCa, PKCp , and PKCs; 40 min for
PKC8; 70 min for PKC and PKC .
In the Detection step, the detection mix contains 0.5625 of STK-antibody
labeled with Eu3+-Cryptate, 0.1563 uM streptavidin-XL665 and EDTA. After incubation
with the reaction system, the enzymatic reaction is stopped by adding 40 of detection
mix (using the MULTIDROP®). The detection mix is capable of stopping the enzymatic
reaction due to the presence of EDTA. Solutes are mixed (using a platform shaker) for 1
min and centrifuged at 1000 rpm for 1 min. The plates are incubated at room temperature
for 1 h (protected from light), and then the fluorescence is measured at 620 nm (Cryptate)
and 665 nm (XL665) using Victor-3. A ratio is calculated (665/620) for each well.
Specific signal is calculated as a Ratio (Sample) - Ratio (Negative control). The data are
processed using XLFIT ® Software (IDBS).
Following a protocol essentially as described above for human PKCa, 2, , , ,
and enzyme inhibitor assays, the results demonstrate that exemplified compounds of the
invention exhibit selective inhibition of the desired conventional and novel PKC isoforms
, 2, , , as compared to the atypical PKC isoforms and . For example, the
compound of Example 2 has IC50s of about 16.6 nM, 51.0 nM, 68.0 nM, 21.9 nM, >
20,000 nM, and 15,500 nM, respectively. Staurosporin, tested in this assay concurrently
with the compound of Example 2, has IC50s of about 30.5 nM, 76.1 nM, 27.9 nM, 0.8 nM,
69.4 nM, and 141.9 nM, respectively for PKCa, 2, , , , and .
Assay 2: Cell-based Assay of PKC inhibition
The purpose of this assay is to test the inhibitory effects of a test compound on
PKC in a cell-based system by measuring the level of PKC substrate phosphorylation.
Staurosporin may be used as a positive control. It is prepared in DMSO to make
up a 10 stock solution and then serially diluted in plain RPMI 1640 culture medium
(10 - 0.0005 ) to obtain a ten-point dilution curve. Test compounds are prepared
in the same manner.
THP-1 cells (human macrophage cell line) are obtained from ATCC (ATCC TIB-
202 lot 4028542). The cells are cultured in 96-well plates coated with poly-d-lysine
(Becton Dickinson) with seeding density of 4000 cells/well in RPMI 1640 culture
medium (Gibco) with 1% fetal bovine serum (Gibco). Cells are treated with test
compound, dosing at 10 points of 1:3 dilutions across the range of 10 to 0.0005 ,
and with final DMSO concentration at 0.2% for 1.5 h at 37 °C prior to PKC stimulation.
"Stimulation" refers to PKC activation by an exogenous stimulus. PKC is stimulated
with 20 nM phorbol-12-myristate 13-acetate (PMA, Sigma, P1585) for 30 min at 37 °C.
After the incubation, the cells are first fixed in the Prefer fixative (ANATECH
LTD) for 30 min at room temperature. Then the fixative is aspirated. 50 of 0.1%
TRITON® X-100 in PBS without Ca++ and Mg++ is added to permeabilize the cells.
The cells are permeabilized for 15 min at room temperature. The plates are washed twice
with PBS (100 ), and 50 of the primary antibody solution is added. The
cells are incubated with the primary antibody overnight at 4 °C, washed with PBS, and
then incubated with the labeled secondary antibody for one hour at room temperature.
The primary antibody is an anti-phospho-(Serine) PKC substrate IgG (Cell Signaling),
diluted 1:1000 in PBS with 1% BSA. The secondary antibody is ALEXA FLUOR® 488
Goat anti-Rabbit IgG (Molecular Probes) diluted 1:1000 in PBS. The plates are washed
with PBS. The nuclei are counterstained with 50 ΐ \ 5 propidium iodide solution
(Molecular Probes) and 50 g/mL ribonuclease (Sigma) for one hour at room
temperature. Plates are scanned with an ACUMEN EXPLORER™ Laser-scanning
fluorescence microplate cytometer (TTP LABTECH LTD), with 488 nm laser excitation
to detect emission fluorescence at 655 nm-705 nm (emission of DNA bound propidium
iodide) for cell counting/well, and emission of 505 nm-530 nm fluorescence of antibody
binding to phosphorylated PKC substrates in cells. The main assay output is a ratio of
total fluorescence of phosphorylated PKC substrates to total cell number (ratio= total
intensity/total cell number). The ratio is used to calculate the dose response and IC50.
Following a protocol essentially as described above for determining PKC
substrate phosphorylation in human THP-1 cells, exemplified compounds of the invention
demonstrate PKC inhibitory effects in the cell, with most having an absolute IC50 ()
of 1 or less. For example, the compound of Example 2 has an IC50 of about 0.80 .
Staurosporin, tested in this assay has an IC50 of about 0.01 1 (conducted in
conjunction with the compound of Example 2).
Assay 3: PKC in vivo target inhibition assay
The purpose of this assay is to evaluate the effects of in vivo administration of a
test compound on PKC substrate phosphorylation in blood cells.
Pleckstrin is one of the PKC protein substrates and is primarily expressed in
platelets and peripheral blood mononuclear cells (PBMC). Pleckstrin phosphorylation is
proportional to PKC activity. The phosphorylation state of the pleckstrin protein in
purified platelets and PBMC from mouse blood is analyzed via Western blotting using a
primary antibody specific for phosphorylated PKC substrates. This antibody recognizes
multiple phosphorylated PKC substrates that include, but are not limited to p-pleckstrin.
The p-pleckstrin band is identified based on its molecular weight.
Male ICR mice (Charles River) are dosed by oral gavage with a test compound
homogenized in 1% hydroxethylcellulose/0.25% TWEEN® 80, at a concentration range
of 0.3-3 mg/mL, depending on dose; thus dosing volume is 10 ml/kg body weight.
Staurosporine, dissolved in the same vehicle, may be used as a positive control to verify
that the assay is functioning. The mice are sacrificed 2 h after treatment by C0 2
asphyxiation, and blood is collected via cardiac puncture. Blood is treated with EDTA
anti-coagulant as well as protease and phosphatase inhibitors Protease Inhibitor (Roche,
#1836170), Phosphatase Inhibitor I (Sigma, P2850), and Phosphatase Inhibitor II (Sigma,
P5726).
Platelets are prepared from mouse blood by low speed spin at 200 x g for 4 min.
Platelet-rich plasma is removed and transferred to an eppendorf tube which is then spun at
1,400 x g for 5 min. Platelet pellets are suspended in a small volume of lysis buffer (150
mM NaCl, 20 mM Tris (pH 7.5), 1mM EDTA, 1mM EGTA, 1% TRITON X-100®,
Protease Inhibitor (Roche, #1836170), Phosphatase Inhibitor I (Sigma, P2850), and
Phosphatase Inhibitor II (Sigma, P5726)) and frozen until further analysis. The remaining
blood is mixed and applied to BD VACUTAINER® CPT™ tubes (Becton Dickinson) to
purify PBMC via centrifugation at 1,600-1,800 x g for 20-25 minutes. The PBMC band
is recovered with a pipette, diluted with pH 7.5 buffer and spun down at 1,400 x g for 5
min, suspended in a small volume of lysis buffer and frozen until further analysis.
Proteins are obtained by incubation of the cells with the lysis buffer .
10% SDS-PAGE is conducted on the cell lysates (30 g protein loaded per lane),
with 130 V for 90 min. After transfer, nitrocellulose membranes are incubated with the
rabbit polyclonal anti-phospho-PKC substrate antibody (Cell Signaling Technology Inc).
The membranes are washed and incubated with the secondary conjugated fluorescent
anti-rabbit antibody (ALEXA FLUOR® 680 goat anti-rabbit IgG, Invitrogen Molecular
Probes). The membranes are scanned with an ODYSSEY® Infrared Imaging System (LICOR).
The phospho-pleckstrin band in the gel is identified by its relative migration in the
gel relative to molecular weight marker proteins, and (in selected cases) by phosphoproteomic
analysis. Intensity of near-infrared fluorescence is proportional to the level of
PKC substrate phosphorylation. It is quantitated with ODYSSEY® software. For a doseresponse
experiment, the optical density values (y) are averaged and plotted vs dose
values (x) to generate the ID50.
Following a protocol essentially as described above for determining pleckstrin
phosphorylation by PKC in vivo, the compound of Example 2 has an ID50 of about 11.85
mg/kg for the platelets and about 18.03 mg/kg for the PBMCs. This result demonstrates
that Example 2 has inhibitory effects on PKC substrate phosphorylation in vivo.
Assay 4: Efficacy animal model of diabetic nephropathy
The purpose of this assay is to analyze compound efficacy in a mouse model of
diabetic nephropathy. The earliest clinical manifestation of diabetic nephropathy is
albuminuria, leakage of albumin in the urine. Because diabetic nephropathy can be
present without any symptoms, early diagnosis is critical so that treatment can be started.
The key test for early diagnosis of diabetic nephropathy involves checking for the
presence of albuminuria. Thus, if a compound reduces albumin levels in the urine in a
diabetic patient, it would likely indicate efficacy of the compound in the treatment of
diabetic nephropathy.
To model diabetic nephropathy, a combination of genetically driven type 2
diabetes and uninephrectomy is utilized. Six week-old db/db mice (genetic strain: C57
BL KsJ, ChemPartner, Shanghai) receive standard rodent chow and water ad libitum.
The left kidney is removed under anesthesia with 0.6% sodium pentobarbital at 60 mg/kg
body weight (10 / i.p.). Seven days after surgery, urine is collected for 24 h in a
metabolic cage to measure albuminuria levels and blood is collected by tail snip to
determine glucose levels. For compound treatment, the animals are randomized based on
24-h albuminuria, blood glucose, and body weight. A test compound is dissolved in 4%
DMSO (aqueous) and administered twice a day by oral gavage. Control and treatment
groups consisted of 10 mice each. The control group is treated with the vehicle (4%
DMSO) in a similar manner. During the treatment period, albuminuria is analyzed
monthly. After 2 months of treatment, blood is collected by heart puncture under
isofluorane anesthesia, and the mice are euthanized by removing the heart.
Following a protocol essentially as described above for determining reduction of
albuminuria in db/db mice, the compound of Example 2 has an ED50 of about
25.33 mg/kg. Specifically, at the dose of 30 mg/kg, actual mean levels of albumin in the
urine (collected during 24 hours) were 85.0+6.9 g (n=12) while in the urine of vehicletreated
mice they were 135.9+14.3 g (n=12). This result demonstrates that the
compound of Example 2 reduces albumin levels in the urine of mice in an animal model
of diabetic nephropathy.
We Claim:
1. A compound of the formula:
R i and R2 are each independently hydrogen, fluoro, chloro, methyl, or cyano,
wherein at least one of R i or R2 is not hydrogen;
Z is
R3, R6, R9, and Rio are each independently hydrogen or methyl;
R4 and R 5 are each independently hydrogen or methyl, or R4 and R 5 taken together
with the carbon to which they are attached form cyclopropyl; and
R7 and R are each independently hydrogen or methyl, or R7 and R taken together
with the carbon to which they are attached form cyclopropyl; wherein at least one of R3,
R4, R R7 R R9, or Rio is not hydrogen.
2. The compound or salt according to claim 1, wherein A is
The compound or salt according to claim 1 or 2, wherein R2 is fluoro.
The compound or salt according to any of claims 1 to 3, wherein A is
5. The compound or salt according to any of claims 1 to 4, wherein Z is
6. The compound or salt according to claim 5, wherein Z is
7. The compound or salt according to claim 6, wherein Z is
8. A compound or salt accordin to claim 1, which is:
9. The hydrochloride salt, the methane sulfonic acid salt, or the hemi ethane- 1,2-
disulfonic acid salt of the compound according to claim 8.
10. A pharmaceutical composition comprising the compound or salt according to
any one of claims 1 to 9 and one or more pharmaceutically acceptable carriers.
11. The pharmaceutical composition according to claim 10 further comprising
one or more additional therapeutic agents.
12. A method of treating a kidney disorder in a patient in need thereof comprising
administering a compound or salt according to any of claims 1 to 9.
13. The method of claim 12, wherein said kidney disorder is chronic kidney
disease.
14. The method according to claim 12 or 13, wherein the kidney disorder is
diabetic nephropathy.
15. The method according to any of claims 12 to 14, wherein said patient is
human.
16. The method according to any of claims 12 to 14, wherein said patient is
feline.
17. A method of treating a diabetic complication in a patient in need thereof
comprising administering a compound or salt according to any of claims 1 to 9.
18. The method of claim 17, wherein said diabetic complication is selected from
the group consisting of atherosclerosis, cardiomyopathy, retinopathy, nephropathy, and
neuropathy.
19. A compound or salt according to any one of claims 1 to 9, for use in therapy.
20. A compound or salt according to any one of claims 1 to 9, for use in the
treatment of a kidney disorder.
21. The compound or salt for use according to claim 20, wherein the kidney
disorder is chronic kidney disease.
22. The compound or salt for use according to claim 20, wherein the kidney
disorder is diabetic nephropathy.
23. A compound or salt according to any one of claims 1 to 9, for use in the
treatment of a diabetic complication.
24. The compound or salt for use according to claim 23, wherein the diabetic
complication is selected from the group consisting of atherosclerosis, cardiomyopathy,
retinopathy, nephropathy, and neuropathy.