WO2022159566A1 - Benzenesulfonamide agonists of trap1 for treating organ fibrosis - Google Patents

Benzenesulfonamide agonists of trap1 for treating organ fibrosis Download PDF

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WO2022159566A1
WO2022159566A1 PCT/US2022/013106 US2022013106W WO2022159566A1 WO 2022159566 A1 WO2022159566 A1 WO 2022159566A1 US 2022013106 W US2022013106 W US 2022013106W WO 2022159566 A1 WO2022159566 A1 WO 2022159566A1
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alkyl
optionally substituted
fibrosis
hydrogen
halogen
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French (fr)
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Yiannis A. Ioannou
Fannie W. CHEN
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Icahn School Of Medicine At Mount Sinai
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present application relates generally to methods and compositions for treating fibrosis, particularly kidney and liver fibrosis.
  • the methods involve administering a genus of benzenesulfonamides, particularly N-[3- (aminosulfonyl)phenyl]heteroarylamides.
  • oxidative stress is thought to be a key contributor to the pathogenesis of diabetic nephropathy.
  • Pharmacological inhibition of oxidative phosphorylation has been shown to ameliorate chronic kidney injury by reducing oxidative stress.
  • the plethora of evidence for the role of mitochondrial dysfunction in nephropathy has recently been summarized [Saxena, S., Mathur, A. & Kakkar, P. Critical role of mitochondrial dysfunction and impaired mitophagy in diabetic nephropathy. J. Cell. Physiol.
  • Autophagy/mitophagy plays an important protective role in a multitude of kidney cell types, and autophagic flux is compromised under hyperglycemic conditions, which further contributes to the development and progression of diabetic kidney disease.
  • the progressive disorder ultimately leads to organ fibrosis, which culminates in organ failure and a need for transplantation.
  • organ fibrosis which culminates in organ failure and a need for transplantation.
  • NASH non-alcoholic fatty liver disease
  • a number of other organ-specific fibrotic disorders are known, in addition to kidney and liver fibrosis, including cardiac and pulmonary fibrosis. Radiation-induced fibrosis is also treatable with the compounds described hereinbelow. There is a greatunmet need to develop novel approaches to therapy and establish new paradigms for these devastating disorders. A new protein target for drug development that can address various forms of organ fibrosis is described hereinbelow.
  • the invention is directed to pharmaceutical compositions and methods for treating fibrotic diseases and disorders.
  • the present invention provides, in a first aspect, a method of treating a fibrotic disorder comprising administering a compound of formula I
  • R 1 is selected from hydrogen and (Ci-Ce)alkyl
  • R 2 is selected from optionally substituted heteroaryl, meta-substituted phenyl and para-substituted phenyl, wherein substituents are selected from (Ci-Ce)alkyl, halogen, halo(Ci-Ce)alkyl, (Ci-Ce) oxaalkyl, and cyano; or
  • R 1 and R 2 together with the nitrogen to which they are attached, form an optionally substituted and/or fused heterocycle, wherein substituents are selected from (Ci-Ce)alkyl, halogen, halo(Ci-Ce)alkyl, and (Ci-Ce) oxaalkyl;
  • R 3 , R 4 , R 5 and R 6 are independently selected from hydrogen, (Ci-Ce)alkyl, halogen, cyano, hydroxy, halo(Ci-Ce)alkyl, and (Ci-Ce)alkoxy; and
  • Cy is optionally substituted monocyclic heteroaryl, wherein the substituents on the heteroaryl, when present, are selected from (Ci-C?)hydrocarbon, (Ci-Ce)acyl, halo(Ci-Ce)alkyl, (Ci-Cio)oxaalkyl and halogen.
  • organ fibrosis particularly kidney and liver fibrosis.
  • Cy is optionally substituted pyrazole, pyrrole, thiazole, imidazole, oxazole, pyridine, pyridazine, pyrimidine, thiophene, or furan, wherein the substituents on the ring are selected from (Ci-Ce)alkyl, (Ci-Ce)acyl, halo(Ci-Ce)alkyl, (Ci-Cio)oxaalkyl and halogen.
  • R 1 and R 2 taken together do not form a ring.
  • R 1 may be selected from hydrogen and (Ci-Ce)alkyl.
  • R 2 may be pyridine, substituted pyridine, meta- or para-substituted phenyl, and the substituents may be chosen from bromo, cyano and acetyl. It is to be understood that the recitation of ortho- substituted, meta-substituted and (in other contexts) para-substituted means that a substituent will be found at the denominated position. Unless further explicitly restricted, (e.g. “monosubstituted at the ortho position”), it is not meant to imply that no other substituents will be found anywhere else on the ring.
  • R 1 and R 2 together with the nitrogen to which they are attached, form an optionally substituted and/or fused heterocyclic ring.
  • the ring may be a saturated nitrogen heterocycle, for example, a pyrrolidine, piperidine, azepine, morpholine or piperazine, or it may be a fused heterocycle such as tetrahydrobenzoazepine, tetrahydroquinoline, tetrahydroisoquinoline, indoline or isoindoline.
  • R 3 is selected from hydrogen, (Ci- Ce)alkyl, halogen, hydroxy, cyano, halo(Ci-Ce)alkyl, and (Ci-Ce)alkoxy; and R 4 , R 5 and R 6 are independently selected from hydrogen and fluorine.
  • R 3 is selected from hydrogen, methyl, ethyl, methoxy and hydroxy; R 4 and R 6 are hydrogen; and R 5 is hydrogen or fluorine.
  • substituted refers to the replacement of one or more hydrogen atoms in a specified group with a specified radical. For example, substituted aryl, heterocyclyl etc.
  • Oxaalkyl refers to alkyl residues in which one or more carbons (and their associated hydrogens) have been replaced by oxygen. Examples include methoxy, ethoxy, propoxy, methoxypropoxy, 3,6,9-trioxadecyl and the like.
  • the term oxaalkyl is intended as it is understood in the art [see Naming and Indexing of Chemical Substances for Chemical Abstracts, published by the American Chemical Society, 196, but without the restriction of 127(a)], i.e. it refers to compounds in which the oxygen is bonded via a single bond to its adjacent atoms (forming ether bonds); it does not refer to doubly bonded oxygen, as would be found in carbonyl groups.
  • structures depicted herein are also meant to include all stereoisomeric (e.g., enantiomeric, diastereomeric, and cis-trans isomeric) forms of the structure in those cases in which the substituents permit for asymmetry. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13 C- or 14 C-enriched carbon are within the scope of this invention.
  • solvate refers to a compound of Formula I in the solid state, wherein molecules of a suitable solvent are incorporated in the crystal lattice.
  • a suitable solvent for therapeutic administration is physiologically tolerable at the dosage administered.
  • suitable solvents for therapeutic administration are ethanol and water. When water is the solvent, the solvate is referred to as a hydrate.
  • solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions.
  • treatment or “treating,” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit.
  • therapeutic benefit is meant amelioration of the underlying disorder.
  • a benefit is achieved with the amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder.
  • treating kidney fibrosis means alleviating at least one symptom associated with kidney fibrosis; it is not required that the patient no longer exhibit any symptoms of kidney fibrosis or that kidney function be brought to a normal state.
  • the compositions may be administered to a patient at risk of developing fibrosis, or to a patient reporting one or more of the physiological systems of fibrosis, even though a diagnosis of this disease may not have been made.
  • the terms “comprising” and “including” or grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof.
  • This term encompasses the terms “consisting of’ and “consisting essentially of’.
  • the phrase “consisting essentially of’ or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition or method.
  • the formulations related to this invention include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), rectal and topical (including dermal, buccal, sublingual and intraocular) administration.
  • parenteral including subcutaneous, intradermal, intramuscular, intravenous and intraarticular
  • topical including dermal, buccal, sublingual and intraocular
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • Preferred unit dosage formulations are oral unit dosage forms containing an effective dose, or an appropriate fraction thereof, of the active ingredient.
  • Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain anti-oxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient.
  • Formulations for parenteral administration also include aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose of multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example saline, phosphate-buffered saline (PBS) or the like, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • formulations related to this invention may include other agents conventional in the art.
  • those suitable for oral administration may include other therapeutic ingredients, anti-caking agents, preservatives, sweetening agents, colorants, flavors, desiccants, plasticizers, dyes, disintegrants, lubricants and the like.
  • reagents shown above are exemplary and can be replaced by analogous reagents well known in the art for accomplishing the same transformation.
  • CyCOOH can be condensed with the aniline by any of the common reagents used in amide and peptide synthesis.
  • the nitro group can be reduced with hydrogen and catalyst or another metal/acid combination. Exemplary syntheses follow.
  • Step 1 A solution of 1 -methoxy -4-nitrobenzene A (6.2 g, 41 mmol) in CICH2CH2CI (5 mL) was cooled to 0 °C and treated with dropwise addition of chlorosulfonic acid (4 mL, 6 mmol). The reaction was warmed to RT, refluxed for 2 hr, and then cooled. Water was added carefully to quench excess chlorosulfonic acid. Precipitation of solids was observed that dissolved back on addition and stirring of the mixture with chloroform. The organic layer was separated, dried (MgSO 4 ), filtered, and concentrated to provide 2- methoxy-5-nitrobenzene-l -sulfonyl chloride B (1.78 g, 7.07 mmol, 17.5 % yield).
  • Step 2 (1.7 g, 6.8 mmol) was treated with 4 -bromoaniline (1.7 g, 10 mmol) in pyridine (10 mL). The reaction was attached to a reflux condenser and stirred for 16 hr at 90 °C. Most of the pyridine was removed via rotary evaporation; the residue was diluted with EtOAc and then washed with water, and then brine. The organic layer was separated, dried with (MgSO 4 ), filtered, concentrated, and purified to yield 1.7 g (65%) of C.
  • Step 3 C (0.90 g, 2.30 mmol) was dissolved in EtOH (24 mL), treated with tin (II) chloride dihydrate (2.1 g, 9.3 mmol). The reaction was refluxed for 1 hr, cooled, and treated with 1 N NaOH till pH ⁇ 6. A white suspension, presumably consisting of tin salts, was observed. EtOAc was added and the mixture stirred overnight vigorously. The aqueous layer was still a white suspension. The mixture was filtered through celite, the organic layer was separated, dried (MgSO 4 ), filtered, and concentrated.
  • the protein target was unexpectedly suggested to be one of three heat shock chaperones: ERP29, HY0U1, or TRAP1. Further studies confirmed that the target of this compound was the mitochondrial chaperone TRAP1.
  • TRAP1 is the protein target of our leads: We demonstrated direct binding of ML405 to TRAP1, specifically the amino-terminus third of the protein surrounding the ATP binding pocket, with tryptophan fluorescence shift assays using full length and truncated TRAP1 proteins. These results were in agreement with our thermal shift studies, which showed a Tm (-dl/dT max) of 50 degrees upon incubation with ML405, compared to a Tm of 46.5 degrees upon incubation with vehicle.
  • TRAP1 As the target of ML405 via a number of physical and biochemical studies based on known functions of TRAP 1.
  • Treatment of cells with ML405 mimics activation of TRAP1 in: 1) raising cellular ATP levels, 2) reducing mitochondrial oxidative stress, and 3) reducing endoplasmic reticulum stress.
  • mitochondrial chaperone TRAP1 as the protein target of the compounds of formula I.
  • TRAP1 increases SIRT3 protein levels and activity, stimulates autophagy, reduces oxidative stress, and decreases TGF pi -induced fibrotic gene expression.
  • the SIRT3 activation by TRAP1 is particularly exciting based on current knowledge of SIRT3 function and its role in fibrosis.
  • SIRT3 activation suppresses the NLRP3 inflammasome in mitochondria by activating superoxide dismutase 2 (SOD2), which is critical for protecting against oxidative damage. It has been shown that Probucol could reverse the epithelial-mesenchymal transition (EMT) and lung fibrosis by restoring SIRT3 expression.
  • EMT epithelial-mesenchymal transition
  • Recent studies have drawn attention to the protective functions of SIRT3 against kidney disease and fibrosis, and its identification as a TRAP1 client provides a very strong rationale for targeting TRAP1 to modulate SIRT3.
  • Treatment with TRAP1 agonist 1685 Treatment with TRAP1 agonist 1685
  • ML 1685 produced a drug concentration-dependent increase in SIRT3 protein levels.
  • ML405 significantly reduced the mRNA expression of MMP2, TEMPI, TIMP2 at both high (2 pM) and low (0.2 pM) concentrations and PDGFR and TGFpRl at the high concentration. ML405 also decreased the protein levels of pro-fibrotic a-SMA and Coll Al, confirming that ML405 has anti-fibrotic activity in these cells.
  • proximal tubule kidney epithelial HK-2 cells by inactivating their a-galactosidase A gene via CRISPR. These modified proximal tubule kidney epithelial HK-2 cells (which we will call HK-MOD) store large amounts of globotriaosyl ceramide (Gb3). Treatment of HK-MOD cells with 2 pM of ML405 for 48 hours results in significant reduction of Gb3 storage.
  • a mouse knockout model for Fabry disease stores Gb3 but does not develop the renal and cardiac fibrosis seen in human patients.
  • This mouse was crossed with a human Gb3 synthase (Gb3S)-expressing transgenic mouse to produce a transgenic mouse (TGM- Gb3) whose increased Gb3 load recapitulates the human disease.
  • Gb3S human Gb3 synthase
  • TGM- Gb3 transgenic mouse
  • These mice exhibit renal impairment and nephropathy, with fibrosis and inflammation detected by ⁇ 10 weeks, ataxia by ⁇ 20 weeks, and death by ⁇ 36 weeks.
  • Compounds ML405 and 1685 were administered by oral gavage twice weekly to male mice, beginning at 4 weeks of age and continuing until they were 20 weeks old.
  • Compounds were formulated in saline/solutol/DMA as described above. The compounds ameliorate fibrotic progression in these mice.
  • hydroxyproline is one of the few proteins that contains the modified amino acid hydroxyproline
  • the amount of hydroxyproline in a tissue is indicative of its collagen content.
  • Levels of hydroxyproline in kidneys were determined as follows. Briefly, 10-20 mg of kidney is excised and vacuum dried. Because different regions of tissue vary in their collagen content, care was taken to excise samples from the same region of each kidney. Kidneys were then rehydrated for 20hrs at 4°C in buffer (50mM Tris/HCl, pH 7.5, 150mM NaCl + protease inhibitor cocktail) at lOmg/lOOmL. Tissues were hydrolyzed in 6M HC1 at 120°C for 3 hrs and then cooled on ice.
  • Activated charcoal (4mg) was added to the samples, which are mixed and centrifuged at 10,000xg for 3 min. The supernatant was removed and kept on ice. Samples were spotted in 96-well plates and baked in a 60°C oven to dryness. A standard curve of 0-1 pg hydroxyproline was also spotted. Oxidation buffer (60mM chloramine T in acetate-citrate buffer, pH 6.0) was added to each well and incubated for 20 min at room temperature, after which Ehrlich’s reagent (IM DMAB in 30% HCl/70% isopropanol) was added. The plate was incubated at 60°C for 20 minutes and absorbance read at 558nm. There is increased collagen content in the TGM-Gb3 mice. Treatment with ML405 resulted in reduction of hydroxyproline content, with some animals showing greater than 50% reduction, suggesting a notable improvement (decrease) in kidney fibrosis.
  • Oxidation buffer 60mM chloramine T in
  • Mouse blood samples were collected in K2EDTA tubes at 0.167, 0.5, 1, 1.5, 2, 4, 7 and 24 hr after drug administration, and plasma was harvested after centrifugation at 3000 rpm for 10 min. Liver and brain tissues were collected, flash frozen in liquid nitrogen and transferred to 48-well plates. All plasma and tissue samples were stored at -80°C until analysis.
  • the pharmacokinetic parameters were calculated using the non-compartmental approach (Model 200) of the pharmacokinetic software Phoenix WinNonlin, version 6.2 (Certara, St. Louis, MO).
  • the area under the plasma concentration versus time curve (AUC) was calculated using the linear trapezoidal method.
  • the slope of the apparent terminal phase was estimated by log linear regression using at least 3 data points and the terminal rate constant (X) was derived from the slope.
  • AUCo-® was estimated as the sum of the AUCo-t (where t is the time of the last measurable concentration) and Ct/X.
  • the apparent terminal half-life (b/ 2 ) was calculated as 0.693/X.
  • Ultra-performance liquid chromatography -tandem mass spectrometry (UPLC- MS/MS) methods were developed to determine ML405 and ML1685 concentrations in mouse plasma and tissue samples.
  • Mass spectrometric analysis was performed on a Waters Xevo TQ-S triple quadrupole instrument using electrospray ionization in positive mode with the selected reaction monitoring.
  • the separation of test compounds from endogenous components was performed on an Acquity BEH Cl 8 column (50 x 2.1 mm, 1.7 p) using a Waters Acquity UPLC system with 0.6 mL/min flow rate and gradient elution.
  • the mobile phases were 0.1% formic acid in water and 0.1% formic acid in acetonitrile.
  • the calibration standards and quality control samples were prepared in the blank mouse plasma and tissue homogenate. Aliquots of 10 pL samples were mixed with 200 pL internal standard in acetonitrile to precipitate proteins in a 96-well plate. 0.5 pL supernatant was injected for the UPLC-MS/MS analysis. Data were analyzed using MassLynx v4.1 (Waters Corp., Milford, MA). The liver to plasma AUC ratios were about 7 and 14 for ML405 and 1685, respectively. The brain AUC of ML405 and 1685 was ⁇ 4% of plasma AUC values. [0037] These results demonstrate that both compounds exhibit low brain penetration and high liver penetration, suggesting potential utility of compounds of the invention in treating hepatic fibrosis with reduced risk of CNS side-effects.

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Abstract

Methods and compositions for treating organ fibrosis are disclosed. The methods involve administering a genus of benzenesulfonamides, particularly N-[3-(aminosulfonyl)phenyl]-benzamides and heteroarylamides. A genus of suitable compounds is shown in formula (I).

Description

BENZENESULFONAMIDE AGONISTS OF TRAP1 FOR TREATING ORGAN FIBROSIS
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority of US provisional application 63/140,001, filed January 21, 2021, the entire disclosure of which is hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
Technical Field
[0002] The present application relates generally to methods and compositions for treating fibrosis, particularly kidney and liver fibrosis. The methods involve administering a genus of benzenesulfonamides, particularly N-[3- (aminosulfonyl)phenyl]heteroarylamides.
Background Information
[0003] The prevalence of chronic liver and kidney diseases is a worldwide public health concern due to the lack of widely available, effective treatments. In the United States, NASH (non alcoholic steatohepatitis) is expected to be the leading cause for liver transplantation by 2020, whereas chronic kidney disease (CKD) currently affects 10% of the world’s population. Both conditions are characterized by organ fibrosis, which eventually leads to organ failure and a need for transplantation. In recent years, mitochondrial homeostasis has garnered attention as a potential therapeutic target for fibrosis. Supporting evidence comes from studies in which pathways involving mitochondrial function are the most down-regulated in patients with CKD compared to healthy controls. The connection between mitochondrial oxidative stress and diabetic kidney disease is well documented. For example, cells grown under conditions to mimic diabetes exhibit reduced respiration and ATP production, and oxidative stress is thought to be a key contributor to the pathogenesis of diabetic nephropathy. Pharmacological inhibition of oxidative phosphorylation has been shown to ameliorate chronic kidney injury by reducing oxidative stress. The plethora of evidence for the role of mitochondrial dysfunction in nephropathy has recently been summarized [Saxena, S., Mathur, A. & Kakkar, P. Critical role of mitochondrial dysfunction and impaired mitophagy in diabetic nephropathy. J. Cell. Physiol. 27, 458 (2019).] Autophagy/mitophagy plays an important protective role in a multitude of kidney cell types, and autophagic flux is compromised under hyperglycemic conditions, which further contributes to the development and progression of diabetic kidney disease. The progressive disorder ultimately leads to organ fibrosis, which culminates in organ failure and a need for transplantation. There is also large body of evidence showing that hepatic fat accumulation in non-alcoholic fatty liver disease (NAFLD) leads to mitochondrial dysfunction and oxidative stress, which in turn promotes the progression into NASH, fibrosis, and hepatocellular carcinoma.
[0004] A number of other organ-specific fibrotic disorders are known, in addition to kidney and liver fibrosis, including cardiac and pulmonary fibrosis. Radiation-induced fibrosis is also treatable with the compounds described hereinbelow. There is a greatunmet need to develop novel approaches to therapy and establish new paradigms for these devastating disorders. A new protein target for drug development that can address various forms of organ fibrosis is described hereinbelow.
[0005] Many of the compounds described herein as useful for treating fibrosis are disclosed in PCT application WO 2016/130774.
SUMMARY OF THE INVENTION
[0006] The invention is directed to pharmaceutical compositions and methods for treating fibrotic diseases and disorders.
[0007] The present invention provides, in a first aspect, a method of treating a fibrotic disorder comprising administering a compound of formula I
Figure imgf000004_0001
wherein
R1 is selected from hydrogen and (Ci-Ce)alkyl;
R2 is selected from optionally substituted heteroaryl, meta-substituted phenyl and para-substituted phenyl, wherein substituents are selected from (Ci-Ce)alkyl, halogen, halo(Ci-Ce)alkyl, (Ci-Ce) oxaalkyl, and cyano; or
R1 and R2, together with the nitrogen to which they are attached, form an optionally substituted and/or fused heterocycle, wherein substituents are selected from (Ci-Ce)alkyl, halogen, halo(Ci-Ce)alkyl, and (Ci-Ce) oxaalkyl;
R3, R4, R5 and R6 are independently selected from hydrogen, (Ci-Ce)alkyl, halogen, cyano, hydroxy, halo(Ci-Ce)alkyl, and (Ci-Ce)alkoxy; and
Cy is optionally substituted monocyclic heteroaryl, wherein the substituents on the heteroaryl, when present, are selected from (Ci-C?)hydrocarbon, (Ci-Ce)acyl, halo(Ci-Ce)alkyl, (Ci-Cio)oxaalkyl and halogen.
DETAILED DESCRIPTION OF THE INVENTION
[0008] It has been found that compounds of formula I
Figure imgf000005_0001
are useful for treating organ fibrosis, particularly kidney and liver fibrosis.
[0009] In some embodiments of the formula I, Cy is optionally substituted pyrazole, pyrrole, thiazole, imidazole, oxazole, pyridine, pyridazine, pyrimidine, thiophene, or furan, wherein the substituents on the ring are selected from (Ci-Ce)alkyl, (Ci-Ce)acyl, halo(Ci-Ce)alkyl, (Ci-Cio)oxaalkyl and halogen.
[0010] In some embodiments of the formula I, R1 and R2 taken together do not form a ring. In these embodiments, R1 may be selected from hydrogen and (Ci-Ce)alkyl. In some embodiments, R2 may be pyridine, substituted pyridine, meta- or para-substituted phenyl, and the substituents may be chosen from bromo, cyano and acetyl. It is to be understood that the recitation of ortho- substituted, meta-substituted and (in other contexts) para-substituted means that a substituent will be found at the denominated position. Unless further explicitly restricted, (e.g. “monosubstituted at the ortho position”), it is not meant to imply that no other substituents will be found anywhere else on the ring.
[0011] In some embodiments of the formula I, R1 and R2, together with the nitrogen to which they are attached, form an optionally substituted and/or fused heterocyclic ring. In these embodiments, the ring may be a saturated nitrogen heterocycle, for example, a pyrrolidine, piperidine, azepine, morpholine or piperazine, or it may be a fused heterocycle such as tetrahydrobenzoazepine, tetrahydroquinoline, tetrahydroisoquinoline, indoline or isoindoline.
[0012] In some embodiments of the formula I, R3 is selected from hydrogen, (Ci- Ce)alkyl, halogen, hydroxy, cyano, halo(Ci-Ce)alkyl, and (Ci-Ce)alkoxy; and R4, R5 and R6 are independently selected from hydrogen and fluorine. In some embodiments, R3 is selected from hydrogen, methyl, ethyl, methoxy and hydroxy; R4 and R6 are hydrogen; and R5 is hydrogen or fluorine.
[0013] As used herein, the term “optionally substituted” may be used interchangeably with “unsubstituted or substituted”. The term “substituted” refers to the replacement of one or more hydrogen atoms in a specified group with a specified radical. For example, substituted aryl, heterocyclyl etc. refer to aryl or heterocyclyl wherein one or more H atoms in each residue are replaced with halogen, haloalkyl, alkyl, acyl, alkoxyalkyl, hydroxyloweralkyl, carbonyl, phenyl, heteroaryl, benzenesulfonyl, hydroxy, loweralkoxy, haloalkoxy, oxaalkyl, carboxy, alkoxycarbonyl [-C(=O)O-alkyl], carboxamido [-C(=O)NH2], alkylaminocarbonyl [-C(=O)NH-alkyl], cyano, acetoxy, nitro, amino, alkylamino, dialkylamino, dialkylaminoalkyl, dialkylaminoalkoxy, heterocyclylalkoxy, mercapto, alkylthio, sulfoxide, sulfone, sulfonylamino, alkylsulfinyl, alkylsulfonyl, benzyl, heterocyclyl, phenoxy, benzyloxy, heteroaryloxy, aminosulfonyl, amidino, guanidino, and ureido.
[0014] Oxaalkyl refers to alkyl residues in which one or more carbons (and their associated hydrogens) have been replaced by oxygen. Examples include methoxy, ethoxy, propoxy, methoxypropoxy, 3,6,9-trioxadecyl and the like. The term oxaalkyl is intended as it is understood in the art [see Naming and Indexing of Chemical Substances for Chemical Abstracts, published by the American Chemical Society, 196, but without the restriction of 127(a)], i.e. it refers to compounds in which the oxygen is bonded via a single bond to its adjacent atoms (forming ether bonds); it does not refer to doubly bonded oxygen, as would be found in carbonyl groups.
[0015] As used herein, and as would be understood by the person of skill in the art, the recitation of “a compound” - unless expressly further limited - is intended to include salts and solvates of that compound. Thus, for example, the recitation “a compound of formula I” as depicted above, in which Cy is pyridine or imidazole, would include salts in which the pyridine or imidazole is protonated, with the proton arising from a pharmaceutically acceptable acid. Unless otherwise stated or depicted, structures depicted herein are also meant to include all stereoisomeric (e.g., enantiomeric, diastereomeric, and cis-trans isomeric) forms of the structure in those cases in which the substituents permit for asymmetry. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or probes in biological assays. The term "solvate" refers to a compound of Formula I in the solid state, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent for therapeutic administration is physiologically tolerable at the dosage administered. Examples of suitable solvents for therapeutic administration are ethanol and water. When water is the solvent, the solvate is referred to as a hydrate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions.
[0016] As used herein, “treatment” or “treating,” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit. By therapeutic benefit is meant amelioration of the underlying disorder. Also, a benefit is achieved with the amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. For example, “treating kidney fibrosis” means alleviating at least one symptom associated with kidney fibrosis; it is not required that the patient no longer exhibit any symptoms of kidney fibrosis or that kidney function be brought to a normal state. The compositions may be administered to a patient at risk of developing fibrosis, or to a patient reporting one or more of the physiological systems of fibrosis, even though a diagnosis of this disease may not have been made.
[0017] As used herein, the terms “comprising” and “including” or grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. This term encompasses the terms “consisting of’ and “consisting essentially of’. The phrase “consisting essentially of’ or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition or method. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprise” (and any form of comprise, such as “comprises” and “comprising”), “have” (and any form of have, such as “has” and “having”), “include” (and any form of include, such as “includes” and “including”), and “contain” (and any form contain, such as “contains” and “containing”) are open-ended linking verbs. As a result, a method that “comprises”, “has”, “includes” or “contains” one or more steps or elements possesses those one or more steps or elements, but is not limited to possessing only those one or more steps or elements.
[0018] The formulations related to this invention include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), rectal and topical (including dermal, buccal, sublingual and intraocular) administration. The most suitable route may depend upon the decision of a medical professional. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. Preferred unit dosage formulations are oral unit dosage forms containing an effective dose, or an appropriate fraction thereof, of the active ingredient. [0019] Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain anti-oxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient. Formulations for parenteral administration also include aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in unit-dose of multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example saline, phosphate-buffered saline (PBS) or the like, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
[0020] It should be understood that in addition to the ingredients particularly mentioned above, the formulations related to this invention may include other agents conventional in the art. For example, those suitable for oral administration may include other therapeutic ingredients, anti-caking agents, preservatives, sweetening agents, colorants, flavors, desiccants, plasticizers, dyes, disintegrants, lubricants and the like.
[0021] Many of the compounds described herein are known in the literature and may be purchased from commercial sources. Compounds not commercially available may be synthesized by the following route:
Scheme 1
Figure imgf000010_0001
The person of skill will recognize that the reagents shown above are exemplary and can be replaced by analogous reagents well known in the art for accomplishing the same transformation. For example, CyCOOH can be condensed with the aniline by any of the common reagents used in amide and peptide synthesis. Similarly, the nitro group can be reduced with hydrogen and catalyst or another metal/acid combination. Exemplary syntheses follow.
[0022] Preparation of A-(3-(A-(4-bromophenyl)sulfamoy l)-4-methoxyphenyl)-4- m ethylnicotinamide (ML405):
Step 1. A solution of 1 -methoxy -4-nitrobenzene A (6.2 g, 41 mmol) in CICH2CH2CI (5 mL) was cooled to 0 °C and treated with dropwise addition of chlorosulfonic acid (4 mL, 6 mmol). The reaction was warmed to RT, refluxed for 2 hr, and then cooled. Water was added carefully to quench excess chlorosulfonic acid. Precipitation of solids was observed that dissolved back on addition and stirring of the mixture with chloroform. The organic layer was separated, dried (MgSO4), filtered, and concentrated to provide 2- methoxy-5-nitrobenzene-l -sulfonyl chloride B (1.78 g, 7.07 mmol, 17.5 % yield).
Step 2. B (1.7 g, 6.8 mmol) was treated with 4 -bromoaniline (1.7 g, 10 mmol) in pyridine (10 mL). The reaction was attached to a reflux condenser and stirred for 16 hr at 90 °C. Most of the pyridine was removed via rotary evaporation; the residue was diluted with EtOAc and then washed with water, and then brine. The organic layer was separated, dried with (MgSO4), filtered, concentrated, and purified to yield 1.7 g (65%) of C. LC/MS (Agilent system) Retention time ti (short) = 3.50 min, MS (ESI) m/z calculated for C26H22Br2N4NaOioS2 [2M + Na]+ 796.9, found 796.8. 'H NMR (400 MHz, DMSO-t/e) 8 ppm 10.54 (br. s., 1 H), 8.49 (d, J=2.7 Hz, 1 H), 8.45 (dd, J=8.4 Hz, ./=2,8 Hz, 1 H), 7.38 - 7.43 (m, 3 H), 7.09 - 7.02 (m, 2 H), 4.01 (s, 3 H).
Step 3. C (0.90 g, 2.30 mmol) was dissolved in EtOH (24 mL), treated with tin (II) chloride dihydrate (2.1 g, 9.3 mmol). The reaction was refluxed for 1 hr, cooled, and treated with 1 N NaOH till pH~6. A white suspension, presumably consisting of tin salts, was observed. EtOAc was added and the mixture stirred overnight vigorously. The aqueous layer was still a white suspension. The mixture was filtered through celite, the organic layer was separated, dried (MgSO4), filtered, and concentrated. Purification by flash silica gel chromatography with an isocratic 35%EtOAc/hexanes solvent system separated a closely eluting non polar compound to provide pure aniline D (0.56 g, 1.57 mmol, 67 % yield). LC/MS (Agilent system) Retention time ti (short) = 2.78 min, MS (ESI) m/z calculated for Ci3Hi4BrN203S [M + H]+ 357.0, found 356.9. 'H NMR (400 MHz, DMSO-d6) 6 9.96 (s, 4H), 7.44 - 7.33 (m, 8H), 7.09 - 6.97 (m, 12H), 6.87 (d, J=
8.8 Hz, 4H), 6.72 (dd, J= 8.7, 2.8 Hz, 4H), 5.02 (s, 10H), 4.03 (q, J= 7.1 Hz, 1H), 3.71 (s, 12H), 1.99 (s, 1H), 1.17 (t, J= 7.1 Hz, 1H). 'H NMR (400 MHz, DMS0-d6) 6 9.96 (s, 1H), 7.42 - 7.33 (m, 2H), 7.07 - 6.98 (m, 3H), 6.87 (d, J= 8.8 Hz, 1H), 6.72 (dd, J= 8.7,
2.8 Hz, 1H), 5.02 (s, 2H), 3.71 (s, 311).
Step 4. 5-Amino-N-(4-bromophenyl)-2-methoxybenzenesulfonamide D (70 mg, 0.20 mmol) was dissolved in DMF and treated with 4-methylnicotinic acid (81 mg, 0.59 mmol), 2,4,6-tripropyl-l,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (0.37 g, 0.59 mmol) (T3P®), and triethylamine (0.11 mL, 0.78 mmol). The reaction stirred at 60 °C for 48 hr. After cooling the mixture, which had turned into a gel, was diluted with water and extracted with EtOAc. The organic layer was separated, concentrated, and purified by C18 reverse phase column chromatography to yield N-(3-(N-(4-bromophenylsulfamoyl)- 4-methoxyphenyl)-4-methylnicotinamide (ML 405): 32 mg (0.07 mmol, 34%): LC/MS (Agilent system) Retention time ti (long) = 4.13 min, MS (ESI) m/z calculated for C2oHi9BrN3O4S [M + Hl]+ 476.0 found 476.0 ; 'H NMR (400 MHz, DMSO-t/6) 8 10.58 (s, 1H), 10.17 (s, 1H), 8.72 (s, 1H), 8.58 (d, J = 5.1 Hz, 1H), 8.25 (d, J = 2.6 Hz, 1H), 7.88 (dd, J = 9.0, 2.7 Hz, 1H), 7.45 (d, J = 5.2 Hz, 1H), 7.43 — 7.37 (m, 2H), 7.19 (d, J = 9.0 Hz, 1H), 7.10 — 7.03 (m, 2H), 3.86 (s, 3H), 2.44 (s, 311); HRMS (ESI) m/z calculated for C2oHi9BrN3O4S [M + H]+476.0274 found 476.0284.
[0023] Preparation of N-(3-(N-(4-bromophenyl)sulfamoyl)-4-methoxyphenyl)-2- phenyl-lH-imidazole-4-carboxamide (ML 1685):
A mixture of 5-amino-N-(4-bromophenyl)-2-methoxybenzenesulfonamide D (0.5 g, 1.400 mmol), 2-phenyl-lH-imidazole-4-carboxylic acid (0.790 g, 4.20 mmol), HOBt (0.729 g, 4.76 mmol) was treated with DMF/CH2CI2 1 : 1 (Volume: 2.80 ml). To the solution was added DIEA (2.445 ml, 14.00 mmol) followed by EDC (0.805 g, 4.20 mmol). The resulting solution was microwaved at 80°C for 20 minutes then stirred at 50°C overnight. After cooling to room temperature, the reaction solution was diluted with water and extracted with EtOAc. The organic layer was separated, concentrated and purified by C18 reversed phase column chromatography to yield N-(3-(N-(4- bromophenyl)sulfamoyl)-4-methoxyphenyl)-2-phenyl-lH-imidazole-4-carboxamide (1685): lOOmg (0.19 mmol, 14%) LC/MS (Agilent system) Retention time tl (long) = 4.86 min, MS (ESI) m/z calculated for C23Hi9BrN4O4S [M + H]+ 527.4 found 527.0 'H NMR (400 MHz, DMSO-d6) 8 10.22 (s, 1H), 10.11 (s, 1H), 8.33 (d, J= 2.7 Hz, 1H), 8.10 - 8.03 (m, 2H), 8.03 - 7.96 (m, 2H), 7.55 - 7.47 (m, 2H), 7.47 - 7.40 (m, 1H), 7.40 - 7.34 (m, 2H), 7.19 - 7.13 (m, 1H), 7.07 - 7.01 (m, 2H), 3.84 (s, 3H).
[0024] We developed a cell-based reporter assay by which to identify compounds that modify expression from the Rab9 promoter. Using this assay, we identified a group of compounds that showed significant efficacy. The compounds of the invention are shown in Table 1 :
Figure imgf000013_0001
Figure imgf000013_0002
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0002
[0025] Since these compounds were identified using a phenotypic screen, their protein target was not obvious. We used the drug affinity responsive target stability (DARTS) approach to identify the protein target of one of these compounds, which is designated ML405:
Figure imgf000020_0001
ML405
The protein target was unexpectedly suggested to be one of three heat shock chaperones: ERP29, HY0U1, or TRAP1. Further studies confirmed that the target of this compound was the mitochondrial chaperone TRAP1.
[0026] TRAP1 is the protein target of our leads: We demonstrated direct binding of ML405 to TRAP1, specifically the amino-terminus third of the protein surrounding the ATP binding pocket, with tryptophan fluorescence shift assays using full length and truncated TRAP1 proteins. These results were in agreement with our thermal shift studies, which showed a Tm (-dl/dT max) of 50 degrees upon incubation with ML405, compared to a Tm of 46.5 degrees upon incubation with vehicle.
[0027] We validated TRAP1 as the target of ML405 via a number of physical and biochemical studies based on known functions of TRAP 1. Treatment of cells with ML405 mimics activation of TRAP1 in: 1) raising cellular ATP levels, 2) reducing mitochondrial oxidative stress, and 3) reducing endoplasmic reticulum stress. We have now identified the mitochondrial chaperone TRAP1 as the protein target of the compounds of formula I. Furthermore, we have extensively characterized the cellular events following engagement of TRAP 1 by our agonists, such as reduction of oxidative stress in both the mitochondria and endoplasmic reticulum and an increase in cellular ATP levels.
[0028] We have discovered that TRAP1 increases SIRT3 protein levels and activity, stimulates autophagy, reduces oxidative stress, and decreases TGF pi -induced fibrotic gene expression. The SIRT3 activation by TRAP1 is particularly exciting based on current knowledge of SIRT3 function and its role in fibrosis. SIRT3 activation suppresses the NLRP3 inflammasome in mitochondria by activating superoxide dismutase 2 (SOD2), which is critical for protecting against oxidative damage. It has been shown that Probucol could reverse the epithelial-mesenchymal transition (EMT) and lung fibrosis by restoring SIRT3 expression. Recent studies have drawn attention to the protective functions of SIRT3 against kidney disease and fibrosis, and its identification as a TRAP1 client provides a very strong rationale for targeting TRAP1 to modulate SIRT3. Treatment with TRAP1 agonist 1685
Figure imgf000021_0001
ML 1685 produced a drug concentration-dependent increase in SIRT3 protein levels.
[0029] The drug concentration-dependent increase in SIRT3 protein levels effect was TRAP 1 -dependent, since 1685 had no effect on SIRT3 levels in TRAP 1 -null cells. Moreover, this increase in SIRT3 protein levels correlated to increased SIRT3 deacetylase activity: mitochondria isolated from cells treated with 5pM 1685 for 24 hr or 72 hr exhibited 160% and 270% more SIRT3 activity, respectively, than untreated cells. Furthermore, administration of 5mg/kg 1685 three times a week for two weeks in mice increased liver levels of SIRT3, indicating that TRAP 1 -dependent regulation of SIRT3 is relevant in vivo.
[0030] We tested ML405, in the LX2 model for fibrosis [Xu, L. et al. Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis. Gut 54, 142- 151 (2005)]. ML405 significantly reduced the mRNA expression of MMP2, TEMPI, TIMP2 at both high (2 pM) and low (0.2 pM) concentrations and PDGFR and TGFpRl at the high concentration. ML405 also decreased the protein levels of pro-fibrotic a-SMA and Coll Al, confirming that ML405 has anti-fibrotic activity in these cells.
[0031] We engineered proximal tubule kidney epithelial HK-2 cells by inactivating their a-galactosidase A gene via CRISPR. These modified proximal tubule kidney epithelial HK-2 cells (which we will call HK-MOD) store large amounts of globotriaosyl ceramide (Gb3). Treatment of HK-MOD cells with 2 pM of ML405 for 48 hours results in significant reduction of Gb3 storage. In addition, confirming our working hypothesis that TRAP1 upregulation is therapeutically beneficial, expression of TRAP 1 in HK- 2M0D cells results in significant reduction of Gb3 storage similar to the effects of ML405 treatment, validating the use of this cell model for our studies.
[0032] A mouse knockout model for Fabry disease stores Gb3 but does not develop the renal and cardiac fibrosis seen in human patients. This mouse was crossed with a human Gb3 synthase (Gb3S)-expressing transgenic mouse to produce a transgenic mouse (TGM- Gb3) whose increased Gb3 load recapitulates the human disease. These mice exhibit renal impairment and nephropathy, with fibrosis and inflammation detected by ~10 weeks, ataxia by ~20 weeks, and death by ~36 weeks. Compounds ML405 and 1685 were administered by oral gavage twice weekly to male mice, beginning at 4 weeks of age and continuing until they were 20 weeks old. Compounds were formulated in saline/solutol/DMA as described above. The compounds ameliorate fibrotic progression in these mice.
[0033] Since collagen is one of the few proteins that contains the modified amino acid hydroxyproline, the amount of hydroxyproline in a tissue is indicative of its collagen content. Levels of hydroxyproline in kidneys were determined as follows. Briefly, 10-20 mg of kidney is excised and vacuum dried. Because different regions of tissue vary in their collagen content, care was taken to excise samples from the same region of each kidney. Kidneys were then rehydrated for 20hrs at 4°C in buffer (50mM Tris/HCl, pH 7.5, 150mM NaCl + protease inhibitor cocktail) at lOmg/lOOmL. Tissues were hydrolyzed in 6M HC1 at 120°C for 3 hrs and then cooled on ice. Activated charcoal (4mg) was added to the samples, which are mixed and centrifuged at 10,000xg for 3 min. The supernatant was removed and kept on ice. Samples were spotted in 96-well plates and baked in a 60°C oven to dryness. A standard curve of 0-1 pg hydroxyproline was also spotted. Oxidation buffer (60mM chloramine T in acetate-citrate buffer, pH 6.0) was added to each well and incubated for 20 min at room temperature, after which Ehrlich’s reagent (IM DMAB in 30% HCl/70% isopropanol) was added. The plate was incubated at 60°C for 20 minutes and absorbance read at 558nm. There is increased collagen content in the TGM-Gb3 mice. Treatment with ML405 resulted in reduction of hydroxyproline content, with some animals showing greater than 50% reduction, suggesting a notable improvement (decrease) in kidney fibrosis.
[0034] To confirm that compounds of the invention reach the target organs, pharmacokinetic studies of ML405 and ML1685 were carried out in mice. Adult male C57B16 mice (n=3/sampling time point) were obtained from Charles River Laboratories (Wilmington, MA). All experimental procedures were approved by the Animal Care and Use Committee (ACUC) of the NIH Division of Veterinary Resources (DVR). A single dose of 10 and 30 mg/kg was administered through intraperitoneal (IP) route of administration. Dosing solutions were freshly prepared on the day of administration in 50% PEG200/50% water. Mouse blood samples were collected in K2EDTA tubes at 0.167, 0.5, 1, 1.5, 2, 4, 7 and 24 hr after drug administration, and plasma was harvested after centrifugation at 3000 rpm for 10 min. Liver and brain tissues were collected, flash frozen in liquid nitrogen and transferred to 48-well plates. All plasma and tissue samples were stored at -80°C until analysis.
[0035] The pharmacokinetic parameters were calculated using the non-compartmental approach (Model 200) of the pharmacokinetic software Phoenix WinNonlin, version 6.2 (Certara, St. Louis, MO). The area under the plasma concentration versus time curve (AUC) was calculated using the linear trapezoidal method. The slope of the apparent terminal phase was estimated by log linear regression using at least 3 data points and the terminal rate constant (X) was derived from the slope. AUCo-® was estimated as the sum of the AUCo-t (where t is the time of the last measurable concentration) and Ct/X. The apparent terminal half-life (b/2) was calculated as 0.693/X.
[0036] Ultra-performance liquid chromatography -tandem mass spectrometry (UPLC- MS/MS) methods were developed to determine ML405 and ML1685 concentrations in mouse plasma and tissue samples. Mass spectrometric analysis was performed on a Waters Xevo TQ-S triple quadrupole instrument using electrospray ionization in positive mode with the selected reaction monitoring. The separation of test compounds from endogenous components was performed on an Acquity BEH Cl 8 column (50 x 2.1 mm, 1.7 p) using a Waters Acquity UPLC system with 0.6 mL/min flow rate and gradient elution. The mobile phases were 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The calibration standards and quality control samples were prepared in the blank mouse plasma and tissue homogenate. Aliquots of 10 pL samples were mixed with 200 pL internal standard in acetonitrile to precipitate proteins in a 96-well plate. 0.5 pL supernatant was injected for the UPLC-MS/MS analysis. Data were analyzed using MassLynx v4.1 (Waters Corp., Milford, MA). The liver to plasma AUC ratios were about 7 and 14 for ML405 and 1685, respectively. The brain AUC of ML405 and 1685 was ~ 4% of plasma AUC values. [0037] These results demonstrate that both compounds exhibit low brain penetration and high liver penetration, suggesting potential utility of compounds of the invention in treating hepatic fibrosis with reduced risk of CNS side-effects.

Claims

-25-CLAIMS
1. A method of treating organ fibrosis comprising administering to a subject in need of treatment for fibrosis a compound of formula
Figure imgf000026_0001
I wherein
R1 is selected from hydrogen and (Ci-Ce)alkyl;
R2 is selected from optionally substituted heteroaryl, meta-substituted phenyl and para-substituted phenyl, wherein substituents are selected from (Ci-Ce)alkyl, halogen, halo(Ci-Ce)alkyl, (Ci-Ce) oxaalkyl, and cyano; or
R1 and R2, together with the nitrogen to which they are attached, form an optionally substituted and/or fused heterocycle, wherein substituents are selected from (Ci-Ce)alkyl, halogen, halo(Ci-Ce)alkyl, and (Ci-Ce) oxaalkyl;
R3, R4, R5 and R6 are independently selected from hydrogen, (Ci-Ce)alkyl, halogen, cyano, hydroxy, halo(Ci-Ce)alkyl, and (Ci-Ce)alkoxy; and
Cy is optionally substituted monocyclic heteroaryl, wherein the substituents on the heteroaryl, when present, are selected from (Ci-C?)hydrocarbon, (Ci-Ce)acyl, halo(Ci-Ce)alkyl, (Ci-Cio)oxaalkyl and halogen.
2. The method according to claim 1 wherein Cy is optionally substituted monocyclic nitrogenous heteroaryl.
3. The method according to claim 1 wherein Cy is an optionally substituted ring selected from optionally substituted pyrazole, pyrrole, thiazole, imidazole, oxazole, pyridine, pyridazine, pyrimidine, thiophene, and furan.
4. The method according to claim 3 wherein Cy is a ring selected from optionally substituted pyridine and optionally substituted imidazole.
5. The method according to claim 1 wherein R1 is selected from hydrogen and (Ci-C3)alkyl, and R2 is selected from pyridine, substituted pyridine, meta-substituted phenyl and para-substituted phenyl, and said substituents are chosen from fluoro, bromo, chloro, cyano and acetyl.
6. The method according to claim 1 wherein R1 and R2, together with the nitrogen to which they are attached, form an optionally substituted monocyclic or fused bicyclic heterocycle.
7. The method according to claim 6 wherein R1 and R2 form an optionally substituted azepine, benzazepine, piperidine, N-methylpiperazine, morpholine, or pyrrolidine.
8. The method according to claim 1 wherein R3 is selected from hydrogen, (Ci- Ce)alkyl, halogen, hydroxy, cyano, halo(Ci-Ce)alkyl, and (Ci-Ce)alkoxy; and R4, R5 and R6 are independently selected from hydrogen and fluorine.
9. The method according to claim 1 wherein R1 is hydrogen; R2 is para-substituted phenyl, wherein substituents on the phenyl are selected from (Ci-Ce)alkyl, halogen, halo(Ci-Ce)alkyl, and (Ci-Ce)alkoxy; R4, R5 and R6 are hydrogen; and Cy is optionally substituted pyridine or imidazole.
10. The method according to claim 9 wherein R2 is para-substituted phenyl, wherein substituents on the phenyl are selected from halogen and halo(Ci-Ce)alkyl; R3, is methoxy; and Cy is optionally substituted pyridine or imidazole.
11. The method according to claim 1 wherein the compound of formula I is
Figure imgf000028_0001
12. The method of any one of claims 1-11 wherein said organ fibrosis is kidney fibrosis.
13. The method of any one of claims 1-11 wherein said organ fibrosis is liver fibrosis.
14. The method of any one of claims 1-11 wherein said organ fibrosis is lung fibrosis.
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