Classifications

• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
  • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
  • C07D491/04—Ortho-condensed systems
  • C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
  • C07D491/052—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being six-membered
• • 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
  • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/436—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
• • 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
  • 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
  • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

• This invention relates to oxadiazolyl dihydropyrano[2,3-b]pyridine inhibitors of homeodomain interacting protein kinase 2 (HIPK2).
• HIPK2 homeodomain interacting protein kinase 2
• Fibrosis is characterized by excessive production and accumulation of extracellular matrix proteins, which leads to progressive loss of tissue function and eventual organ failure.
• Chronic kidney diseases irrespective of primary insults, are usually accompanied by kidney interstitial fibrosis.
• Therapeutic strategy for chronic kidney disease, in order to halt decline of kidney function requires not only removal of the causal factors, such as hyperglycemia, hypertension, and HIV infection, but also anti-fibrosis therapy to restore the normal kidney structure and function.
• a number of other organ-specific fibrotic disorders are known, in addition to kidney fibrosis, including liver, cardiac and pulmonary fibrosis.
• Renal fibrosis is considered the final convergent pathway for progressive chronic kidney disease, regardless of the original etiologies of the disease. Although much has been learned of the molecular mechanisms underlying renal fibrogenesis, there is still a paucity of success in translating this knowledge to clinical application. It has been demonstrated that HIPK2 is a multifunctional activator of TGF- ⁇ /Smad3, NF- k B, and p53 pathways and that the global knockout of HIPK2 in mice attenuated kidney fibrosis in vivo. United States patent 10,669,266 discloses small molecule inhibitors of HIPK2 that specifically block TGF- ⁇ /Smad3 pathway to attenuate renal fibrosis without causing adverse systemic effects.
• TGF- ⁇ 1 Transforming growth factor- ⁇ 1 (TGF- ⁇ 1) has been identified to be the most important pro-fibrogenic factor for kidney disease.
• TGF- ⁇ 1 binds to type II TGF- ⁇ receptor, allowing its dimerization with type I TGF- ⁇ receptor and leading to phosphorylation of Smad2 and Smad3.
• Phosphorylated Smad3 relocates into nuclei, thereby binds to Smad binding element in promoter and activating the transcription of the target genes including pro-fibrotic genes such as collagen I, fibronectin, and alpha-smooth muscle actin ( ⁇ -SMA).
• pro-fibrotic genes such as collagen I, fibronectin, and alpha-smooth muscle actin ( ⁇ -SMA).
• Smad3 is highly activated in fibrotic kidney and that knockout of Smad3 attenuates kidney fibrosis in animal models of kidney disease. Blockade ofTGF- ⁇ 1/Smad3 pathway therefore provides a therapeutic strategy for kidney fibrosis.
• the invention relates to compounds of general formula I wherein Ar is
• R 4 is chosen from hydrogen, hydroxy, (C 1 -C 3 )alkyl, (C 1 -C 3 )alkoxy, amino, (C 1 - C3)alkylamino, and (C 1 -C 3 )dialkylamino
• R 5 is chosen from hydrogen, hydroxy, (C 1 -C 3 )alkyl, (C 1 -C 3 )alkoxy, amino, (C 1 - C3)alkylamino, and (C 1 -C 3 )dialkylamino;
• R 6 is chosen from hydrogen and (C 1 -C 6 )hydrocarbyl
• R 7 is chosen from hydrogen and (C 1 -C 3 )alkyl; with the proviso that not all of R 4 , R 5 , R 6 , and R 7 are hydrogen.
• the invention relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a compound of formula I.
• the invention relates to methods for inhibiting the interaction of homeodomain interacting protein kinase 2 (HIPK2) with Smad3.
• the method comprises bringing HIPK2 into contact with a compound of formula I.
• the invention in another aspect, relates to methods for inhibiting Smad3 activation.
• the method comprises bringing Smad3 into contact with a compound of formula I.
• the invention relates to a method for treating a fibrotic disease comprising administering a compound of formula I.
• the invention relates to compounds having general formula I as described above:
• Ar is phenyl.
• the phenyl may be para-substituted with a substituent chosen from bromo, chloro, fluoro, fluoromethyl, difluoromethyl, trifluoromethyl, methyl, oxetanyl, and -B(OH)2.
• the phenyl may be substituted with other substituents in addition to the one at the para position.
• Ar is a 5-membered heteroaryl.
• the 5- membered heteroaryl is chosen from oxazole, thiophene, pyrazole, and thiazole, which may be unsubstituted or substituted with a substituent chosen from -(C 1 -C 3 )hydrocarbyl, bromo, chloro, fluoro, fluorom ethyl, difluorom ethyl, and trifluorom ethyl.
• Ar is thiazol-2-yl substituted at 3- or 4-with chloro or methyl.
• R 4 is chosen from hydrogen, hydroxy and amino.
• R 5 is chosen from hydrogen, hydroxy and amino.
• R 6 and R 7 are independently chosen from hydrogen and methyl .
• Ar is chlorophenyl, R 4 is hydrogen, R 5 is hydroxyl and both of R 6 and R 7 are methyl.
• Ar is chlorophenyl, R 4 is hydroxyl, R 5 is hydrogen, and both of R 6 and R 7 are methyl.
• alkyl (or alkylene) is intended to include linear or branched saturated hydrocarbon structures and combinations thereof.
• Alkyl refers to alkyl groups of from 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, s- butyl, t-butyl and the like.
• Cycloalkyl is a subset of hydrocarbon and includes cyclic hydrocarbon groups of from 3 to 8 carbon atoms.
• Examples of cycloalkyl groups include c-propyl, c-butyl, c-pentyl, norbomyl and the like.
• Hydrocarbon or hydrocarbyl includes alkyl, cycloalkyl, poly cycloalkyl, alkenyl, alkynyl, aryl and combinations thereof. Examples include benzyl, phenethyl, cyclohexylmethyl, adamantyl, camphoryl and naphthyl ethyl. Hydrocarbon or hydrocarbyl refer to any substituent comprised of hydrogen and carbon as the only elemental constituents. Aliphatic hydrocarbons or hydrocarbyls are hydrocarbons or hydrocarbyls that are not aromatic; they may be saturated or unsaturated, cyclic, linear or branched.
• aliphatic hydrocarbons or hydrocarbyls examples include isopropyl, 2-butenyl, 2-butynyl, cyclopentyl, norbomyl, etc.
• Aromatic hydrocarbons or hydrocarbyls include benzene (phenyl), naphthalene (naphthyl), anthracene, etc.
• Oxaalkyl refers to alkyl residues in which one or more carbons (and their associated hydrogens) have been replaced by oxygen. Examples include include methoxy, ethoxy, propoxy, isopropoxy, methoxypropoxy, 3,6,9-trioxadecyl and the like.
• 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.
• Alkoxy is a subset of oxaalkyl and includes groups of a straight or branched configuration. Examples include methoxy, ethoxy, propoxy, isopropoxy and the like. Lower-alkoxy refers to groups containing one to four carbons.
• 1, 2 or 3 hydrogen atoms are replaced with a specified radical.
• more than three hydrogen atoms can be replaced by fluorine; indeed, all available hydrogen atoms could be replaced by fluorine.
• Such compounds e.g.perfluoroalkyl fall within the class of “halohydrocarbon” and “haloalkyl”.
• halogen means fluorine, chlorine, bromine or iodine atoms.
• acyl refers to formyl and to groups of 1, 2, 3, 4, 5, 6, 7 and 8 carbon atoms of a straight, branched, cyclic configuration, saturated, unsaturated and aromatic and combinations thereof, attached to the parent structure through a carbonyl functionality. Examples include acetyl, benzoyl, propionyl, isobutyryl and the like. Lower- acyl refers to groups containing one to four carbons.
• the double bonded oxygen, when referred to as a substituent itself is called “oxo”.
• the recitation of “a compound” - unless expressly further limited - is intended to include salts of that compound.
• the recitation “a compound of formula I” as depicted above would include salts of the (3-aryl-l,2,4-oxadiazol-5-yl)-3,4-dihydro-2H-pyrano[2,3- b]pyridine: is a counterion, preferably a pharmaceutically acceptable anion.
• the term “compound of formula I” refers to the compound or a pharmaceutically acceptable salt thereof.
• Suitable pharmaceutically acceptable acids for salts of the compounds of the present invention include, for example, acetic, adipic, alginic, ascorbic, aspartic, benzenesulfonic (besylate), benzoic, boric, butyric, camphoric, camphorsulfonic, carbonic, citric, ethanedisulfonic, ethanesulfonic, ethylenediaminetetraacetic, formic, fumaric, glucoheptonic, gluconic, glutamic, hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic, laurylsulfonic, maleic, malic, mandelic, methanesulfonic, mucic, naphthylenesulfonic, ni
• Suitable pharmaceutically acceptable base addition salts for the compounds of the present invention include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, arginine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine.
• Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium cations and carboxylate, sulfonate and phosphonate anions attached to alkyl having from 1 to 20 carbon atoms.
• a “therapeutically effective amount” means the amount that, when administered to a subject for treating a disorder or condition, is sufficient to effect such treatment.
• the compounds of this invention can exist in radiolabeled form, i.e., the compounds may contain one or more atoms containing an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
• a plurality of molecules of a single structure may include at least one atom that occurs in an isotopic ratio that is different from the isotopic ratio found in nature.
• Radioisotopes of hydrogen, carbon, phosphorous, fluorine, chlorine and iodine include 2 H,
• Radiolabeled compounds of formula I of this invention can generally be prepared by methods well known to those skilled in the art. Conveniently, such radiolabeled compounds can be prepared by carrying out the procedures disclosed in the Examples and Schemes by substituting a readily available radiolabeled reagent for a non-radiolabeled reagent.
• HIPK2 can be localized in tissues, cells and organelles.
• the present invention provides a pharmaceutical composition comprising a compound of formula I, together with one or more pharmaceutically acceptable carriers thereof and optionally one or more other therapeutic ingredients.
• the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
• Formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), rectal and topical (including dermal, buccal, sublingual and intraocular) administration.
• the compounds are preferably administered orally or by injection (intravenous or subcutaneous).
• the precise amount of compound administered to a patient will be the responsibility of the attendant physician. However, the dose employed will depend on a number of factors, including the age and sex of the patient, the precise disorder being treated, and its severity. Also, the route of administration may vary depending on the condition and its severity.
• 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. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
• 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.
• formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
• treatment or “treating,” or “palliating” or “ameliorating” are used interchangeably herein. These terms refers to an approach for obtaining a therapeutic benefit in the form of eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or 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.
• the compositions may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.
• a protecting group refers to a group which is used to mask a functionality during a process step in which it would otherwise react, but in which reaction is undesirable.
• the protecting group prevents reaction at that step, but may be subsequently removed to expose the original functionality. The removal or "deprotection” occurs after the completion of the reaction or reactions in which the functionality would interfere.
• the compounds of the present invention may be prepared by the methods illustrated in the general reaction scheme as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants that are in themselves known, but are not mentioned here.
• the starting materials are either commercially available, synthesized as described in the examples or may be obtained by the methods well known to persons of skill in the art.
• Example 1 The synthesis of ethyl 2,2-dimethyl-2H-pyrano[2,3-b]pyridine-6-carboxylate (0097-1).
• Example 2 The synthesis of ethyl 4-amino-3 -hydroxy -2, 2-dimethyl-3,4-dihydro-2H- pyrano[2,3-b]pyridine-6-carboxylate (0098-2). racemic
• Agilent LCMS 1200-6120 Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 pm); Column Temperature: 40 °C; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] to 0% [water + 10 mM NH 4 HCO 3 ] and 100% [CH 3 CN] in 1.6 min, then under this condition for 1.4 min, finally changed to 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] in 0.1 min and under this condition for 0.7 min. Purity:
• Examples 3 and 4 The synthesis of ethyl (ri)-6-(3-(4-bromophenyl)-1,2,4-oxadiazol-5-yl)-2- m ethyl-3, 4-dihydro-2H-pyrano[2,3-b]pyridine (SMS21032-0130) and ethyl (f?)-6-(3-(4- bromophenyl)-1,2,4-oxadiazol-5-yl)-2-methyl-3,4-dihydro-2H -pyrano[2,3-b]pyridine
• Example 5 The synthesis of 6-(3-(4-bromophenyl)-1,2,4-oxadiazol-5-yl)-2,2-dimethyl-3,4- dihy dro-2H-pyrano[2, 3 -b ]pyridin-4-ol (SMS21032-0143)
• SMS21032-0142 (40 mg, 0.1 mmol) in MeOH (1 mL)/THF (2 mL) was added NaBFL (4 mg, 0.1 mmol) at 5 °C , then the mixture was stirred at 5 °C for 1 h. Concentrated to oil, which was purified by Prep-HPLC to give SMS21032-0143 (8 mg, yield: 20%) as a white solid.
• Agilent HPLC 1200 Column: Waters X-Bridge C18 (150 mm*4.6 mm*3.5 pm); Column Temperature: 40 °C; Flow Rate: 1.0 mL/min; Mobile Phase: from 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] to 0% [water + 10 mM NH 4 HCO 3 ] and 100% [CH 3 CN] in 10 min, then under this condition for 5 min, finally changed to 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] in 0.1 min and under this condition for 5 min.
• Example 6 The synthesis of (R)-3 -hydroxy-2, 2-dimethyl-3,4-dihydro-2H-pyrano[2, 3- b]pyridine-6-carboxylic acid (0097-5a).
• Agilent LCMS 1200-6120 Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 pm); Column Temperature: 40 °C; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] to 0% [water + 10 mM NH 4 HCO 3 ] and 100% [CH 3 CN] in 1.6 min, then under this condition for 1.4 min, finally changed to 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] in 0.1 min and under this condition for 0.7 min. Purity:
• Example 7 The synthesis of (S)-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-pyrano[2,3- b]pyridine-6-carboxylic acid (0097-5b).
• Example 8 The synthesis of (3R,4S)-ethyl 3, 4-dihydroxy -2, 2-dimethyl-3,4-dihydro-2H- pyrano[2, 3 -b ]pyridine-6-carboxylate (0164-2) .
• Agilent LCMS 1200-6120 Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 pm); Column Temperature: 40 °C; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] to 0% [water + 10 mM NH 4 HCO 3 ] and 100% [CH 3 CN] in 1.6 min, then under this condition for 1.4 min, finally changed to 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] in 0.1 min and under this condition for 0.7 min. Purity:
• Example 9 The synthesis of (3S,4R)-ethyl 3, 4-dihydroxy -2, 2-dimethyl-3,4-dihydro-2H- pyrano[2, 3 -b ]pyridine-6-carboxylate (0165-2) .
• Agilent LCMS 1200-6120 Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 pm); Column Temperature: 40 °C; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] to 0% [water + 10 mM NH 4 HCO 3 ] and 100% [CH 3 CN] in 1.6 min, then under this condition for 1.4 min, finally changed to 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] in 0.1 min and under this condition for 0.7 min. Purity:
• Example 10 and 11 The synthesis of (R)-6-(3-(4-bromophenyl)-1,2,4-oxadiazol-5-yl)-2,2- dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridin-4-ol (SMS21032-0171) and (S)-6-(3-(4- bromophenyl)-1,2,4-oxadiazol-5-yl)-2,2-dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridin-4-ol
• SMS21032-0169 Crude
• THF THF
• HC1 2 N, 0.2 mL
• satNaHCCL satNaHCCL
• SMS21032-0171 and SMS21032-0172 were separated by chiral HPLC using a CHIRALCEL OX-10 column (30 x 250 mm, 10 um) with MeOH as the mobile phase.
• the SMS21032- 0171 (10 mg) was the first eluting compound and SMS21032-0172 (10 mg) was the second eluting compound.
• Example 12 The synthesis of (R)-6-(3-(4-chlorophenyl)-1,2,4-oxadiazol-5-yl)-2,2- dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridin-3-ol (SMS21032-0173-01).
• Agilent LCMS 1200-6120 Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 pm); Column Temperature: 40 °C; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] to 0% [water + 10 mM NH 4 HCO 3 ] and 100% [CH 3 CN] in 1.6 min, then under this condition for 1.4 min, finally changed to 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] in 0.1 min and under this condition for 0.7 min. Purity:
• Example 13 The synthesis of (S)-6-(3-(4-chlorophenyl)-1,2,4-oxadiazol-5-yl)-2,2- dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridin-3-ol (SMS21032-0174-01).
• Agilent LCMS 1200-6120 Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 pm); Column Temperature: 40 °C; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] to 0% [water + 10 mM NH 4 HCO 3 ] and 100% [CH 3 CN] in 1.6 min, then under this condition for 1.4 min, finally changed to 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] in 0.1 min and under this condition for 0.7 min. Purity:
• Example 14 The synthesis of (R)-2,2-dimethyl-6-(3-(thiazol-2-yl)-1,2,4-oxadiazol-5-yl)- 3,4-dihydro-2H-pyrano[2,3-b]pyridin-3-ol (SMS21032-0182-01).
• Agilent LCMS 1200-6120 Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 pm); Column Temperature: 40 °C; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] to 0% [water + 10 mM NH 4 HCO 3 ] and 100% [CH 3 CN] in 1.6 min, then under this condition for 1.4 min, finally changed to 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] in 0.1 min and under this condition for 0.7 min. Purity:
• Agilent LCMS 1200-6120 Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 pm); Column Temperature: 40 °C; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] to 0% [water + 10 mM NH 4 HCO 3 ] and 100% [CH 3 CN] in 1.6 min, then under this condition for 1.4 min, finally changed to 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] in 0.1 min and under this condition for 0.7 min. Purity:
• Example 17 The synthesis of (R)-2,2-dimethyl-6-(3-(l-methyl-lH-pyrazol-3-yl)-1,2,4- oxadiazol-5-yl)-3,4-dihydro-2H-pyrano[2,3-b]pyridin-3-ol (SMS21032-0184-01). absolute stereochemistry
• Agilent LCMS 1200-6120 Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 pm); Column Temperature: 40 °C; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] to 0% [water + 10 mM NH 4 HCO 3 ] and 100% [CH 3 CN] in 1.6 min, then under this condition for 1.4 min, finally changed to 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] in 0.1 min and under this condition for 0.7 min. Purity:
• Example 18 The synthesis of (S)-2,2-dimethyl-6-(3-(l-methyl-lH-pyrazol-3-yl)-1,2,4- oxadiazol-5-yl)-3,4-dihydro-2H-pyrano[2,3-b]pyridin-3-ol (SMS21032-0185-01). absolute stereochemistry
• Agilent LCMS 1200-6120 Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 pm); Column Temperature: 40 °C; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] to 0% [water + 10 mM NH 4 HCO 3 ] and 100% [CH 3 CN] in 1.6 min, then under this condition for 1.4 min, finally changed to 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] in 0.1 min and under this condition for 0.7 min. Purity:
• Example 19 The synthesis of (S)-6-(3-(4-chlorophenyl)-1,2,4-oxadiazol-5-yl)-3-methoxy- 2,2-dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridine (SMS21032-0187-01).
• Example 20 The synthesis of (S)-6-(3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl)-2,2-dimethyl- 3 ,4-dihy dro-2H-pyrano[2, 3 -b]pyri din-3 -ol (SMS21032-0190)
• Example 22 The synthesis of (Z)-3,4-dichloro-/V-hydroxybenzimidamide (0192-2).
• LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 pm); Column Temperature: 40 °C; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] to 0% [water + 10 mM NH 4 HCO 3 ] and 100% [CH 3 CN] in 1.6 min, then under this condition for 1.4 min, finally changed to 95% [water +
• Example 24 The synthesis of (S)-2,2-dimethyl-6-(3-(5-methylthiazol-2-yl)-1,2,4-oxadiazol- 5 -yl)-3 ,4-dihy dro-2H-pyrano[2, 3 -b]pyri din-3 -ol (SMS21032-0194)
• SMS21032-0194 (10 mg, yield: 13%) as a white solid.
• LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 pm); Column Temperature: 40 °C; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] to 0% [water + 10 mM NH 4 HCO 3 ] and 100% [CH 3 CN] in 1.6 min, then under this condition for 1.4 min, finally changed to 95% [water +
• Example 25 The synthesis of (R)-6-(3-(5-chlorothiazol-2-yl)-1,2,4-oxadiazol-5-yl)-2,2- dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridin-3-ol (SMS21032-0197)
• LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 pm); Column Temperature: 40 °C; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] to 0% [water + 10 mM NH 4 HCO 3 ] and 100% [CH 3 CN] in 1.6 min, then under this condition for 1.4 min, finally changed to 95% [water +
• Example 27 The synthesis of (R)-6-(3-(4-chlorothiazol-2-yl)-1,2,4-oxadiazol-5-yl)-2,2- dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridin-3-ol (SMS21032-0199)
• 4-chlorothiazole 1.0 g, 8.36 mmol
• THF 15 mL
• n-BuLi 4 mL
• DMF (673 mg, 9.20 mmol) was added, the mixture was stirred at -78 °C for 10 min, then warmed to room temperature and stirred for 2 h.
• LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 pm); Column Temperature: 40 °C; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] to 0% [water + 10 mM NH 4 HCO 3 ] and 100% [CH 3 CN] in 1.6 min, then under this condition for 1.4 min, finally changed to 95% [water +
• Example 28 The synthesis of (S,E)-4-chloro-N'-(3-hydroxy-2,2-dimethyl-3,4-dihydro-2H- pyrano[2,3-b]pyridine-6-carbonyloxy)thiazole-2-carboximidamide (200-1)
• SMS21032-0200 9 mg, yield: 16%) as a white solid.
• LC-MS (Agilent LCMS 1200-6120, Column: Waters X-Bridge C18 (50 mm*4.6 mm*3.5 pm); Column Temperature: 40 °C; Flow Rate: 2.0 mL/min; Mobile Phase: from 95% [water + 10 mM NH 4 HCO 3 ] and 5% [CH 3 CN] to 0% [water + 10 mM NH 4 HCO 3 ] and 100% [CH 3 CN] in 1.6 min, then under this condition for 1.4 min, finally changed to 95% [water +
• Example 29 The synthesis of 1 -ethyl-1H-pyrazole-3-carbonitrile (201-1)
• Example 30 The synthesis of (S)-6-(3-(3-chlorophenyl)-1,2,4-oxadiazol-5-yl)-2,2-dimethyl- 3,4-dihydro-2H-pyrano[2,3-b]pyridin-3-ol (SMS21032-0211-01)
• Example 31 The synthesis of (R)-6-(3-(3-chlorophenyl)-1, 2, 4-oxadiazol-5-yl)-2, 2-dimethyl -3 ,4-dihy dro-2H-pyrano [2,3 -b]pyri din-3 -ol (SMS21032-0212-01)
• the product was purified by Prep HPLC (basic). (3R)-6-[3 -(3 -fluorophenyl)- 1,2, 4-oxadiazol- 5-yl]-2,2-dimethyl-3,4-dihydropyrano[2,3-b]pyridin-3-ol (23 mg, 67.38 umol) as a white solid was obtained.
• Example 40 The synthesis of (R)-6-(3-(3,5-dichlorophenyl)-1,2,4-oxadiazol-5-yl)-2,2- dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridin-3-ol (SMS21032-0261-01)
• HIPK2 homeodomain interacting protein kinase 2
• TGF- ⁇ 1/Smad3 pathway a critical regulator of multiple pro-fibrosis pathways including TGF- ⁇ 1/Smad3 pathway.
• HIPK2 regulates the pathway by physical association with Smad3, which thereby modulates its activity.
• Knockdown of HIPK2 in human primary tubular cells suppresses activation of the TGF- ⁇ 1/Smad3 pathway induced by TGF- ⁇ 1.
• HIPK2 knockout inhibits TGF- ⁇ 1/Smad3 activity and kidney fibrosis in both HIV1 -transgenic mice (Tg26) and mice with unilateral ureteral obstruction (UUO). Suppression of TGF- ⁇ 1/Smad3 pathway through inhibition of HIPK2, therefore provides an approach to anti-fibrosis therapy for kidney disease.
• HIPK2 inhibitors have not been well developed and are not commercially available. Recently Cozza etal [PLoS One, 9: e89176, 2014] described a selective HIPK2 inhibitor that competes for the ATP binding in the kinase domain. However, since HIPK2 regulates multiple signaling pathways, including regulation of p53, there is a concern that broad inhibition of HIPK2 may not be beneficial in all cellular contexts. Here we describe compounds that may inhibit TGF- ⁇ 1/Smad3 pathway through the interruption of HIPK2- Smad3 protein-protein interaction without significant inhibition of HIPK2 kinase activity or inhibition of p53 activation.
• examples of compounds of the invention inhibited the pro-fibrosis pathway in vitro in cultured human renal tubular epithelial cells (RTEC) and in vivo in murine models of kidney fibrosis (Tg26 and UUO mice).
• RTEC human renal tubular epithelial cells
• Tg26 and UUO mice murine models of kidney fibrosis
• HEK 293T (293T) cells are cultured in Dulbecco's Modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (FBS), 0.5% penicillin and streptomycin at 37°C and 5% CO2 humidified environment.
• Human primary tubular cells (PromoCell GmbH, Heidelberg, Germany) are cultured in Renal Epithelial Cell Growth Medium-2 (Promocell GmbH) with supplements according to manufacturer's protocol.
• Human primary renal tubular epithelial cells with ⁇ 5 passages are used for all studies.
• pNL4-3 AG/P -EGFP, a gag/pol-deleted HIV-1 construct that contains EGFP in the gag open reading frame, and pHR -IRES- EGFP, a control EGFP construct, are used to generate the VSV-G pseudotyped virus.
• Cells are infected with HIV pseudotyped virus or control virus for 2 days before the treatment with test compound.
• 4X Smad binding element-driven firefly luciferase (SBE4-Luc) plasmid and Renilla luciferase reporter plasmid (pRL) are available commercially.
• Active domain deleted HIPK2 was previously described by Jin et al. [Nat Med, 18: 580-588], The His 6 -HIPK2 construct is generated by PCR amplification of coding region using plasmid containing human HIPK2 gene (GeneCopoeiaTM) as the template.
• 293T cells seeded in 12-well plate are co-transfected with SBE4- Luc (0.5 ⁇ g) and pRL plasmids (0.2 ⁇ g) using the PolyJet transfection kit according to manufacturer's instructions (SignaGen Laboratories, MD). Forty-eight hours post- transfection cells are treated with assigned concentrations of test compound together with or without lOng/ml TGF- ⁇ 1 for 16 hours. Luciferase activities are measured using the Dual- Luciferase Reporter Assay kit (Promega, #E1910). Data are expressed as the ratio of firefly luciferase activity over renilla luciferase activity.
• pcDNA 3.1 empty vector 0.5 ⁇ g
• HIPK2 KD plasmid 0.5 ⁇ g
• the desired test concentration is 30 mM
• 5 ⁇ L of 660 mM compound is added.
• TGF ⁇ was added to 10 ng/mL.
• the plate was incubated at 37 ° C in a CO2 incubator overnight (18 hours). One hour prior to assay all medium was replaced.
• the luciferase assay was run using ONE-StepTM Luciferase Assay System by adding 100 m ⁇ of ONE-StepTM Luciferase reagent per well, rocking at room temperature for -15 to 30 minutes and measuring luminescence using a luminometer.
• the signals were normalized to percentage inhibition, with non-TGF treated cells as 100% inhibition, and the data were processed by Graphpad Prism.
• Tg26 mice of FVB/N genetic background bearing a defective HIV-1 provirus lacking gag-pol have been described by Feng et al. ⁇ J Am Soc Nephrol 2009; 20: 2138-2146 ].
• Six- week old heterozygous Tg26 mice are used. Wildtype littermates are used as controls.
• Mice in the treatment group receive test compound dissolved in 20% NMP, 60% PEG400, and 20% H2O by oral gavage at a dose of 30, 60, or 90mg/kg body weight per day.
• Mice in the control group receive the same volume of vehicle. The mice are treated for a total of 4 weeks and sacrificed at age of 10 weeks.
• 5- to 6-week old Tg26 mice were treated with Example 13 (90mg/kg daily, PO) or vehicle for 4 weeks: 38% (8/21 mice) died of kidney failure in the control group within the experimental duration; 0% died in the HTPK2i-174 group (0/18 mice).

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