Classifications

• • 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
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
• • 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

Definitions

• the present invention relates to pyrrolidine substituted flavones, represented by the compounds of formula 1 (as described herein) or stereoisomers or tautomers thereof, or pharmaceutically acceptable salts or solvates thereof, for use in the treatment of renal cystic diseases.
• the present invention also relates to pharmaceutical compositions containing the compounds of formula 1 for use in the treatment of a renal cystic disease.
• Renal cystic diseases include diseases such as acquired renal cystic disease (ARCD), dialysis-associated cystic disease, polycystic kidney disease (PKD), congenital multicystic kidney disease (CMKD), multicystic dysplastic kidney disease (MCDKD), end-stage renal disease (ESRD), medullary sponge kidney disease (MSK), nephronophthisis-medullary cystic kidney disease complex (NMCD), nephronophthisis-uremic medullary cystic disease complex, juvenile nephronophthisis, medullary cystic disease, renal cell carcinoma (RCC), tuberous sclerosis (TS) and von Hippel-Lindau syndrome (VHLS).
• ARCD acquired renal cystic disease
• PWD polycystic kidney disease
• CKD congenital multicystic kidney disease
• MCDKD multicystic dysplastic kidney disease
• ESRD end-stage renal disease
• MSK medullary sponge kidney disease
• NMCD ne
• PTD Polycystic kidney disease
• ADPKD autosomal dominant trait
• ARPKD autosomal recessive trait
• ARPKD is the most common heritable cystic renal disease occurring in infancy and childhood. Although the cysts associated with ARPKD are smaller is size than ADPKD, they eventually populate the entire kidney tissue to result in end stage renal failure.
• Polycystic kidney disease (PKD) is the most frequently inherited disease; it affects approximately 600,000 people in the United States and over 12,000,000 worldwide. Most of the patients of PKD particularly suffer from ADPKD. It is the fourth leading cause of kidney failure and causes 10 % of all end-stage renal disease (ESRD) (BMC Med Genet. 201 1 ; 12: 164).
• the first line of treatment for renal diseases involves use of ACE (Angiotensin converting enzyme) inhibitors and ARBs (Angiotensin II receptor blockers) by aggressive management of high blood pressure which helps slow the progression of renal disease.
• Treatment of end-stage kidney disease may include kidney dialysis or kidney transplantation.
• ACE inhibitors and ARBs are often used only to control the associated hypertension, which is allegedly partially mediated by renin.
• US 201 10046164 relates to methods for the treatment of cystic diseases by administering roscovitine, which is a cyclin dependent kinase (CDK) inhibitor, or an analog thereof.
• roscovitine which is a cyclin dependent kinase (CDK) inhibitor, or an analog thereof.
• the present invention relates to a compound of formula 1 (as described herein) or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof; for use in the treatment of a renal cystic disease.
• the present invention relates to pharmaceutical compositions comprising one or more compounds of formula 1 or a pharmaceutically acceptable salt thereof along with a pharmaceutically acceptable carrier or excipient for use in the treatment of a renal cystic disease.
• the present invention relates to pharmaceutical compositions comprising one or more compounds of formula 1 or a pharmaceutically acceptable salt thereof along with a pharmaceutically acceptable carrier or excipient for use in the treatment of polycystic kidney disease.
• the present invention relates to a method for the treatment of a renal cystic disease in a subject comprising administering to the subject a therapeutically effective amount of a compound of formula 1 or a stereoisomer or a tautomer thereof or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.
• the present invention relates to a method for the treatment of polycystic kidney disease in a subject comprising administering to the subject a therapeutically effective amount of a compound of formula 1 or a stereoisomer or a tautomer thereof or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.
• the present invention relates to use of the compound of formula 1 or a stereoisomer or a tautomer thereof or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, for the manufacture of a medicament for the treatment of a renal cystic disease.
• the present invention relates to use of the compound of formula 1 or a stereoisomer or a tautomer thereof or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, for the manufacture of a medicament for the treatment of polycystic kidney disease.
• Figure 1 depicts percentage cyst size of the kidney on treatment with compound A for ift80 morphant.
• Figure 2 depicts percentage cyst size of the kidney on treatment with compound A for pkd2 morphant.
• Figure 3 depicts percentage cyst size of the kidney on treatment with compound A for nek8 morphant.
• Figure 4 depicts percentage cyst size of the kidney on treatment with compound B for ift80 morphant.
• Figure 5 depicts percentage cyst size of the kidney on treatment with compound B for nek8 morphant.
• Figure 6 depicts percentage cyst size of the kidney on treatment with compound B for pkd2 morphant.
• Figure 7 depicts percentage cyst size of the kidney on treatment with compound A for ift80 morphant.
• Figure 8 depicts percentage dye filtered by the kidney on treatment with compound A for pkd2 morphant.
• Figure 9 depicts percentage dye filtered by the kidney on treatment with compound A for nek8 morphant.
• Figure 10 depicts percentage dye filtered by the kidney on treatment with compound A for ift80 morphant.
• Figure 1 1 depicts percentage dye filtered by the kidney on treatment with compound B for nek8 morphant.
• Figure 12 depicts percentage dye filtered by the kidney on treatment with compound B for Ift80 morphant.
• Figure 13 depicts percentage dye filtered by the kidney on treatment with compound B for pkd2 morphant.
• Figure 14 depicts therapeutic window for compound A and compound B.
• substitution or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, as well as represents a stable compound, which does not readily undergo transformation such as rearrangement, cyclization, elimination, etc.
• -C 4 )alkyl refers to the radical of saturated aliphatic groups, including straight or branched-chain containing from 1 to 4 carbon atoms. Examples of alkyl groups include but are not limited to methyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl, te/t-butyl and the like.
• (C-i-C 4 )alkoxy refers to an alkyl group as defined above attached via oxygen linkage to the rest of the molecule.
• alkoxy include, but are not limited to methoxy, ethoxy, propoxy, butoxy, tert-butoxy and the like.
• halogen refers to fluorine, chlorine, bromine and iodine.
• hydroxy or "hydroxyl” as used herein, refers to -OH group.
• PTD polycystic kidney disease
• the cysts are filled with fluid.
• PKD cysts can slowly replace much of the mass of the kidneys, reducing kidney function and leading to kidney failure.
• the signs and symptoms of PKD include, but are not limited to, pain in the back and lower sides, frequent urination, headaches, urinary tract infections, blood in the urine, cysts in the kidneys and other organs.
• subject refers to an animal, preferably a mammal, most preferably a human, who has been the object of the treatment, observation or experiment.
• mammal as used herein is intended to encompass humans, as well as non-human mammals.
• Non-human mammals include but are not limited to domestic animals, such as cows, pigs, horses, dogs, cats, rabbits, rats and mice, and non-domestic animals.
• the terms “compound of formula 1 ", “compounds of formula 1 ", and “compounds of the present invention” include all the, stereoisomeric and tautomeric forms and mixtures thereof in all ratios, and the pharmaceutically acceptable salts and pharmaceutically acceptable solvates thereof.
• the term "pharmaceutically acceptable” means that the carrier, diluent, excipients, and/or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
• the term "pharmaceutical composition” refers to a liquid or solid composition that contains a pharmaceutically active ingredient (e.g. compound of formula 1 ) and at least a carrier, diluent, or excipient, according to known methods of formulation.
• the present invention furthermore includes all solvates of the compounds of the formula 1 , for example hydrates, and the solvates formed with other solvents of crystallization, such as alcohols, ethers, ethyl acetate, dioxane, dimethylformamide or a lower alkyl ketone, such as acetone, or mixtures thereof.
• Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms.
• Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
• renal efficacy refers to functional and morphological improvement in the renal function in the form of a decreased cyst size and improved glomerular filtration rate.
• LOEC refers to the lowest concentration of the compound wherein 50 % of the zebrafish embryos exhibit toxic effect post compound exposure.
• the lethal end point i.e. the median lethal concentration (LC 5 o) refers to the concentration of the compound which resulted in 50 % mortality of the embryos.
• the no effect observed concentration refers to the concentration of the compound which had no effects on the zebrafish larval morphology, growth and development.
• therapeutic window refers to the serum drug concentration at which a desired therapeutic effect occurs in the absence of any toxicity.
• therapeutic window is defined as the range of compound concentrations at which the compound exhibits a therapeutic effect in the absence of any toxicity.
• terapéuticaally effective amount in reference to the treatment of renal cystic disease including the polycystic kidney disease, refers to an amount capable of invoking one or more of the following effects in a subject treated with the compounds of formula 1 : (i) slowing down of the disease; or (ii) complete cure of the disease and/or (iii) relief, to some extent, of one or more symptoms associated with the renal cystic disease including the polycystic kidney disease.
• i t 80 morphants used in the present invention refers to zebrafish larvae with deletion of ift 80 gene by either splice blocking or translation blocking morpholino oligonucleotides. These morphants elucidate the organ forming mechanism and the onset and progression of polycystic kidney disease. This morphant was used to evaluate the potential of the compounds of formula 1 to treat PKD.
• pkd2 morphants used in the present invention refers to zebrafish larvae with deletion of pkd2 gene by either splice blocking or translation blocking morpholino oligonucleotides. These morphants elucidate the organ forming mechanism and the onset and progression of polycystic kidney disease. This morphant was used to evaluate the potential of the compounds of formula 1 to treat PKD.
• nek 8 morphants used in the present invention refers to zebrafish larvae with deletion of nek 8 gene by either splice blocking or translation blocking morpholino oligonucleotides. These morphants elucidate the organ forming mechanism and the onset and progression of polycystic kidney disease. This morphant was used to evaluate the potential of the compounds of formula 1 to treat PKD.
• the compounds for use in the treatment of a renal kidney disease are selected from the compounds represented by the following formula 1 ;
• Ar is a phenyl group which is substituted by 1 or 2 identical or different substituents selected from: halogen, nitro, cyano, (Ci-C 4 )alkyl, trifluoromethyl, hydroxy or (C-i-C 4 )alkoxy; or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, for use in the treatment of a renal cystic disease.
• the compounds of formula 1 are CDK inhibitors and are disclosed in PCT Patent Publication No. WO2004004632 (corresponding to U.S. Patent 7,271 ,193) and PCT Patent Publication No. WO2007148158.
• Ar is a phenyl group, which is substituted by 1 or 2 identical or different substituents selected from halogen, nitro, cyano, (C-
• a compound of formula 1 wherein the phenyl group is substituted by 1 or 2 identical or different substituents selected from: halogen, (CrC 4 )alkyl and trifluoromethyl, for use in the treatment of a renal cystic disease.
• a compound of formula 1 wherein the phenyl group is substituted by 1 or 2 halogens, for use in the treatment of a renal cystic disease.
• a compound of formula 1 wherein the phenyl group is substituted by chlorine, for use in the treatment of a renal cystic disease.
• compound of formula 1 is (+)-frans-2-(2-chloro- phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methyl-pyrrolidin-3-yl)-chromen-4-one or its pharmaceutically acceptable salt, which is provided for use in the treatment of a renal cystic disease.
• the compound of formula 1 is (+)-irans-2-(2- chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methyl-pyrrolidin-3-yl)-chromen- 4-one hydrochloride (referred to herein as "compound A”), which is provided for use in the treatment of a renal cystic disease.
• a compound of formula 1 wherein the phenyl group is substituted by 2 different substituents selected from chloro and trifluoromethyl, for use in the treatment of a renal cystic disease.
• the compound of formula 1 is (+)-irans-2-(2-chloro-4- trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methyl-pyrrolidin-3-yl)- chromen-4-one; or its pharmaceutically acceptable salt, which is provided for use in the treatment of a renal cystic disease.
• the compound of formula 1 is (+)-irans-2-(2- chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methylpyrrolidin- 3-yl)-chromen-4-one hydrochloride (referred to herein as "compound B”), which is provided for use in the treatment of a renal cystic disease.
• the compounds of formula 1 contain at least two chiral centers and hence, exist in the form of two different optical isomers (i.e. (+) or (-) enantiomers). All such enantiomers and mixtures thereof including racemic mixtures are included within the scope of the invention.
• the enantiomers of the compound of formula 1 e.g. the compounds of formula 1 A can be obtained as described above, by methods disclosed in PCT Patent Publication No. WO2004004632, WO2008007169 and WO2007148158 or the enantiomers of the compound of formula 1 can also be obtained by methods well known in the art, such as chiral HPLC and enzymatic resolution.
• enantiomerically pure describes a compound which is present in an enantiomeric excess (ee) of greater than 95 %. In another embodiment, the enantiomeric excess is greater than 97 %. In still another embodiment, the enantiomeric excess is greater than 99 %.
• enantiomeric excess describes the difference between the amount of one enantiomer and the amount of another enantiomer that is present in the product mixture.
• the enantiomers of the compounds of formula 1 e.g. the compounds of formula 1 A can be synthesized by using optically active starting materials.
• all possible stereoisomers and mixtures thereof, of the compounds of formula 1 can be provided for use in the treatment of renal cystic diseases including polycystic kidney disease.
• reference to the compounds of formula 1 shall encompass within its scope the racemic forms and the isolated optical isomers; which can be provided for use in the treatment of renal cystic diseases including polycystic kidney disease.
• the present invention provides a method for the treatment of a renal cystic disease in a subject comprising administering to the subject a therapeutically effective amount of a compound of formula 1 or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula 1 or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, for use in the treatment of a renal cystic disease.
• the present invention provides use of the compound of formula 1 or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, for the manufacture of a medicament for the treatment of a renal cystic disease.
• the renal cystic disease for the treatment of which the compounds of formula 1 are provided is selected from: acquired renal cystic disease (ARCD), dialysis-associated cystic disease, polycystic kidney disease (PKD), congenital multicystic kidney disease (CMKD), multicystic dysplastic kidney disease (MCDKD), end-stage renal disease (ESRD), medullary sponge kidney disease (MSK), nephronophthisis-medullary cystic kidney disease complex (NMCD), nephronophthisis-uremic medullary cystic disease complex, juvenile nephronophthisis, medullary cystic disease, renal cell carcinoma (RCC), tuberous sclerosis (TS) or von Hippel-Lindau syndrome (VHLS).
• ARCD acquired renal cystic disease
• PPD polycystic kidney disease
• CKD congenital multicystic kidney disease
• MCDKD multicystic dysplastic kidney disease
• ESRD end-stage renal disease
• MSK medull
• the renal cystic disease is polycystic kidney disease.
• the present invention relates to a compound of formula 1 or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, for use in the treatment of polycystic kidney disease.
• the present invention relates to a method for the treatment of polycystic kidney disease in a subject comprising administering to the subject a therapeutically effective amount of a compound of formula 1 or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.
• the present invention provides use of the compound of formula 1 or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, for the manufacture of a medicament for the treatment of polycystic kidney disease.
• the compound of formula 1 is selected from the compound A or the compound B.
• the compound of formula 1 provided for use in the treatment of polycystic kidney disease is the compound A.
• the compound of formula 1 provided for use in the treatment of polycystic kidney disease is the compound B.
• polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD) or autosomal recessive polycystic kidney disease (ARPKD).
• ADPKD autosomal dominant polycystic kidney disease
• ARPKD autosomal recessive polycystic kidney disease
• polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD).
• ADPKD autosomal dominant polycystic kidney disease
• the polycystic kidney disease is autosomal recessive polycystic kidney disease (ARPKD).
• ARPKD autosomal recessive polycystic kidney disease
• the pharmaceutical composition may require different routes of administration, because of different physical and chemical characteristics.
• the pharmaceutical compositions comprising compounds of formula 1 may be administered either orally or parenterally.
• the compounds of formula 1 may be administered, for example, in the form of tablets or capsules, powders, dispersible granules or cachets, or as aqueous solutions or suspensions.
• carriers which are commonly used include lactose, corn starch, magnesium carbonate, talc and sugar, and lubricating agents such as magnesium stearate are commonly added.
• useful carriers include lactose, corn starch, magnesium carbonate, talc and sugar.
• sterile solutions of the active ingredient are usually employed, and the pH of the solutions should be suitably adjusted and buffered.
• the sterile solutions of the active ingredient used are prepared in saline or distilled water.
• the compounds of formula 1 may be administered for example in the form of ointments or creams or transdermally, in the form of patches, or in other ways, in the form of aerosols or nasal sprays.
• compositions or preparations provided for use in the treatment of renal cystic diseases including the polycystic kidney disease are prepared in a manner known and familiar to one skilled in the art.
• Pharmaceutically acceptable inert inorganic and/or organic carriers and/or additives can be used in addition to the compound(s) of formula 1 , and/or its (their) pharmaceutically acceptable salt(s).
• Carriers for soft gelatin capsules and suppositories are, for example, fats, waxes, natural or hardened oils, etc.
• Suitable carriers for the production of solutions for example injection solutions, or of emulsions or syrups are, for example, water, physiological sodium chloride solution or alcohols, for example, ethanol, propanol or glycerol, sugar solutions, such as glucose solutions or mannitol solutions, or a mixture of the various solvents thereof.
• the pharmaceutical compositions or preparations normally contain about 1 % to 99 %, for example, about 5 % to 70 %, or from about 10 % to about 30 % by weight of the compound of the formula 1 or its pharmaceutically acceptable salt.
• the amount of the compound of the formula 1 or its pharmaceutically acceptable salt in the pharmaceutical compositions or preparations normally is from about 5 mg to 500 mg.
• the dose of the compounds of formula 1 which is to be administered, can cover a wide range. The dose to be administered daily is to be selected to suit the desired effect.
• a suitable dose can range from about 0.01 mg/kg/day to 100 mg/kg/day of the compound of formula 1 or its pharmaceutically acceptable salt, for example, about 0.1 mg/kg/day to about 50 mg/kg/day of a compound of formula 1 or its pharmaceutically acceptable salt. If required, higher or lower daily doses can also be administered.
• the selected dosage level will depend upon a variety of factors including the activity of the particular compound of formula 1 that is employed, or a pharmaceutically acceptable salt thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compounds employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
• Cystic kidney disorders are chronic in nature progressing over a long period of time to end stage renal disease or chronic renal failure.
• the medical interventions are drugs which have to be taken either repeatedly in cycles or daily.
• a clinically effective dose which is significantly lower than its minimal toxic dose.
• the present invention demonstrates therapeutic index suitable for chronic administration.
• the pharmaceutical compositions or preparations can contain additives such as, for example, fillers, antioxidants, dispersants, emulsifiers, defoamers, flavors, preservatives, solubilizers or colorants. They can also contain two or more compounds of formula 1 or their pharmaceutically acceptable salts.
• the present invention also encompasses within its scope use of a compound of formula 1 or its pharmaceutically acceptable salt in combination, with a further therapeutically active agent for the treatment of polycystic kidney disease.
• the therapeutically active agent used in combination with one or more compounds of formula 1 or its pharmaceutically acceptable salt can be selected from, but not limited to, roscovitine (Institute of Cancer Research and CNRS), pyrimethamine (EXR-101 ; GlaxoSmithKline), lisinopril (ICI-209K, MK-521 ; Merck & Co.), pravastatin sodium (Daiichi Sankyo), rapamycin (Pfizer), telmisartan (Boehringer Ingelheim), tolvaptam (Otsuka pharmaceuticals), bosutinib (Pfizer), EXEL-7647 (Exelixis) or PLX-5568 (Plexxikon).
• the compounds of formula 1 A can be prepared according to the methods disclosed in PCT Patent Publication No. WO2004004632 and PCT Patent Publication No. WO2007148158, which are incorporated herein by reference.
• the method described in the following Scheme I can be used to prepare intermediate of formula VIA.
• the hydroxyl function on the piperidine ring may be converted to a leaving group such as tosyl, mesyl, triflate or halide by treatment with an appropriate reagent such as p-toluenesulfonyl chloride, methanesulfonyl chloride, triflic anhydride or phosphorous pentachloride in the presence of oxygen nucleophiles such as triethylamine, pyridine, potassium carbonate or sodium carbonate , followed by ring contraction in the presence of oxygen nucleophiles such as sodium acetate or potassium acetate in an alcoholic solvent such as isopropanol, ethanol or propanol.
• the ring contraction involved in this step may be effected before flavone formation as depicted in the above scheme or it may be done after building the flavone with the desired substitutions.
• Enantiomerically pure ⁇ -)-trans enantiomer of an intermediate compound of the formula VIA (as described below), is used for the preparation of an enantiomerically pure compound of the formula 1 .
• an intermediate having a high enantiomeric purity as a starting compound in the process, the resultant (+)- trans enantiomer of pyrrolidines substituted with flavone represented by the formula 1 produced by the process has a correspondingly high enantiomeric purity.
• the process for the preparation of an enantiomerically pure ⁇ +)-trans enantiomer of a compound of formula 1 , or a pharmaceutically acceptable salt thereof, from the resolved enantiomerically pure ⁇ -)-trans enantiomer of the intermediate compound of formula VIA comprises the following steps:
• the Lewis acid catalyst utilized in the step (a) above can be selected from: BF 3i Et 2 0, zinc chloride, aluminium chloride and titanium chloride.
• the base utilized in the process step (b) can be selected from triethylamine, pyridine and a DCC-DMAP combination (combination of N, N'-dicyclohexyl carbodiimide and 4-dimethylaminopyridine).
• the base used in the process step (c) can be selected from: lithium hexamethyl disilazide, sodium hexamethyldisilazide, potassium hexamethyldisilazide, sodium hydride and potassium hydride.
• a preferred base is lithium hexamethyl disilazide.
• the dealkylating agent used in process step (e) for the dealkylation of the compound of formula IXA can be selected from: pyridine hydrochloride, boron tribromide, boron trifluoride etherate and aluminium trichloride.
• a preferred dealkylating agent is pyridine hydrochloride.
• the representative compounds of formula 1 namely the compound A and compound B that are used in the pharmacological assays refers to (+)-trans-2-(2- chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methyl-pyrrolidin-3-yl)-chromen- 4-one hydrochloride and (+)-irans-2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy- 8-(2-hydroxymethyl-1 -methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
• nek8 NIMA (never in mitosis gene a) related kinase 8 gene
• the reaction mixture was poured over crushed ice (300 g), acidified with 1 :1 HCI (pH 2) and extracted using EtOAc (2 x 100 ml_).
• the aqueous layer was basified using a saturated Na 2 C0 3 (pH 10) and extracted using CHCI 3 (3 x 200 ml_).
• the organic layer was dried (anhydrous Na 2 S0 4 ) and concentrated. To the residue, cone. HCI (25 ml_) was added and stirred at room temperature for 2 h.
• the reaction mixture was poured over crushed ice (300 g) and made basic using a saturated aqueous Na 2 C0 3 solution.
• the mixture was extracted using CHCI 3 (3 x 200 ml_).
• reaction was allowed to warm to room temperature and stirred for 2.5 h.
• the reaction mixture was acidified with dilute HCI, and basified with 10 % sodium bicarbonate to pH 8 to 9.
• the aqueous layer was extracted with chloroform (3 x 25 mL).
• the organic layer was washed with water (25 mL), brine (25 mL) and dried over anhydrous Na 2 S0 .
• Example 1 The procedures involved herein were carried out with the approval of the Institutional Animal Ethics committee, India (29/1999/CPCSEA). Zebrafish embryos were obtained by wild-type pair matings.
• Example 1 The procedures involved herein were carried out with the approval of the Institutional Animal Ethics committee, India (29/1999/CPCSEA). Zebrafish embryos were obtained by wild-type pair matings.
• Example 1 The procedures involved herein were carried out with the approval of the Institutional Animal Ethics committee, India (29/1999/CPCSEA). Zebrafish embryos were obtained by wild-type pair matings.
• Example 1 Example 1 :
• the zebrafish embryos obtained were injected at the two-cell stage with a solution containing 1 mM ift80 or 0.25 mM pkd2 or 0.5 ⁇ nek8 translation blocking antisense morpholino oligonucleotides (Gene-Tools LLC, USA) in 200 mM KCI, and 0.1 % Phenol Red. Control morpholino oligonucleotides were injected and exhibited no effect on embryonic development. For prophylactic treatment, compound A/ compound B exposure was initiated 6 h post-fertilization while for therapeutic treatment compound A/ compound B exposure was initiated at 24 h post-fertilization (hpf).
• the concentrations for compound A/ compound B were maintained below their LC 5 o concentrations to prevent interference with the disease progression and the results were recorded.
• the solutions for compound A and compound B were prepared by dissolving a known amount (approximately 4-5 mg of compound/mL of 100 % dimethyl sulfoxide (DMSO), Sigma) to prepare a 10 mM stock solution. The compounds were vortexed after addition of DMSO to ensure proper dissolution of the respective compounds. A negative control was used for each plate which contained 0.1 % DMSO. The plates were then incubated at 28 °C in an incubator and were read at 24 h, 48 h and at 96 h post fertilization.
• DMSO dimethyl sulfoxide
• kidney oedema was measured by drawing a polygon around the swelling and calculating the surface area in microns squared using Zeiss Axiolmager 4.5 (Pediatr. Nephrol., 2008, 23, 2095- 2099).
• Treatment with compound A was initiated at 24 h post fertilization (hpf).
• the ift80 morphants were continuously exposed to compound A for 5 days post fertilization (dpf).
• Cyst size regression post treatment with compound A was expressed at the lowest treatment concentration of 5 ⁇ ( Figure 1 ).
• Dose dependent decrease in renal cyst size with increase in compound A concentrations (upto 20 ⁇ ) was observed.
• Maximum decrease (cyst size reduction of > 50 %) in size was observed with 20 ⁇ of compound A.
• Treatment with compound A resulted in stabilization and further regression of the renal cysts indicating activity of the compound in the ift80 morphants.
• Treatment with compound B was initiated at 24 h post fertilization (hpf).
• the ift80 morphants were continuously exposed to compound B for 5 days post fertilization. Cyst size regression post treatment with compound B was expressed at the lowest concentration of 10 ⁇ ( Figure 4). There was a limited decrease (10 to 20 %) in cyst size with increase in compound B concentrations upto (20 ⁇ ), suggestive of stabilization of the disease.
• Treatment with compound B in ift80 morphants was associated with > 25 % decrease in the renal cyst size, followed by stabilization of the disease or non significant dose dependent reduction.
• Ability to limit disease progression and deterioration of renal function is one aspect of this application indicating activity of the compound in the ift80 morphants.
• Treatment with compound B was initiated at 24 h post fertilization (hpf). The nek8 morphants were continuously exposed to compound B for 5 days post fertilization. Cyst size regression post treatment with compound B was expressed at the lowest treatment concentration of 10 ⁇ ( Figure 5). Treatment with compound B in nek8 morphants was associated with > 40 % decrease in the renal cyst size, suggestive of dose dependent regression of the disease. Treatment with compound B resulted in stabilization and further regression of the renal cysts indicating activity of the compound in the nek8 morphants. The cyst size reduction (41 % at 30 ⁇ ) on treatment with compound B was relatively higher than that of roscovitine (39 % at 25 ⁇ ) at the maximal possible concentration for both the compounds.
• Treatment with compound A was initiated at 6 h post fertilization (hpf).
• the ift80 morphants were continuously exposed to compound A for 5 days post fertilization. Cyst size regression post treatment with compound A was expressed at the lowest treatment concentration of 5 ⁇ ( Figure 7). Significant decrease in cyst size was further observed at 10 ⁇ .
• Treatment with compound A was associated with > 20 % decrease in the renal cyst size at 5 ⁇ concentration and with > 40% decrease in the renal cyst size at 10 ⁇ concentration suggestive of dose dependent regression of the disease. Regression in cyst size and improvement in the renal function are features indicating activity of the compound in vivo. Dose dependent regression of the renal cysts indicates prophylactic efficacy and utility of the compound in the ift80 morphants.
• Hentschel et al. Acute renal failure in zebrafish: a novel system to study a complex disease; Am. J. Physiol. Renal Physiol., 2005, 288, F923-F929) was adapted.
• the zebrafish embryos obtained were injected at the two-cell stage with a solution containing 1 mM ift80 or 0.25 mM pkd2 or 0.5 ⁇ nek8 translation blocking antisense morpholino oligonucleotides (Gene- Tools LLC, USA) in 200 mM KCI, and 0.1 % Phenol Red.
• Control morpholino oligonucleotides were injected and exhibited no effect on embryonic development.
• PTU N-phenylthiourea, Sigma Aldrich, St. Louis, MO, USA
• Tricaine Sigma, St. Louis, MO, USA
• the decrease in fluorescent intensity correlates with efficiency of renal filtration as the dye is selectively removed from the blood and excreted by the glomerulus.
• the relative amount of fluorescence remaining in the embryo's hearts after 24 h was calculated by measuring the fluorescent intensity of the hearts using Image J (a public domain Java image processing program inspired by National Institutes of Health, USA). A region 100x100 pixels was drawn around the heart and the average intensity was recorded. An average and standard deviation of intensities was recorded for at least 10 embryos for each treatment.
• the amount of dye excreted was calculated by subtracting the 24 h fluorescence from the initial 3 h fluorescence.
• Increase in the filtration indicates improvement in the renal function and dye clearance due to improved glomerular filtration rate. Any improvement in the dye filtration reflects upon an improvement in glomerular filtration rate and indicative of the activity of the compound in polycystic kidney disease.
• Treatment with compound A was initiated at 24 h post fertilization (hpf).
• the pkd2 morphants were continuously exposed to compound A for 5 days post fertilization.
• the glomerular filtration studies were initiated at 72 h post fertilization. Decrease in the dye filtration is secondary to the inability of the zebrafish pronephros (zebrafish kidneys) to clear the dye from the blood stream due to decrease in the filtering capacity of the kidney.
• the compound A used in this study did not have any effect on the heart rate at the concentrations evaluated. Hence, the retainment in the dye was contributed to by the decrease in the cardiac pumping. Dose dependent increase in the dye excreted was observed on treatment with compound A.
• the dye excretion improved from 9 % in ift80 morphants zebrafish larvae to 59 % in ift80 morphants zebrafish larvae exposed to compound A at highest possible concentration (20 ⁇ ) without causing toxicity ( Figure 10).
• Significant increase in the dye excretion exhibited by exposure to compound A indicates the activity of the compound in polycystic kidney disease.
• Treatment with compound B was initiated at 24 h post fertilization (hpf).
• the nek8 morphants were continuously exposed to compound B for 5 days post fertilization.
• the glomerular filtration studies (used to assess the renal function) were initiated at 72 h post fertilization. Decrease in the dye filtration is secondary to the inability of the zebrafish pronephros (zebrafish kidneys) to clear the dye from the blood stream due to decrease in the filtering capacity of the kidney.
• the compound B used in this study did not have any effect on the heart rate at the concentrations evaluated. Hence, the retainment in the dye was contributed to by the decrease in the cardiac pumping. Dose dependent increase in the dye excreted was observed on treatment with compound B.
• Treatment with compound B was initiated at 24 h post fertilization (hpf).
• the ift80 morphants were continuously exposed to compound B for 5 days post fertilization.
• the glomerular filtration studies (used to assess the renal function) were initiated at 72 h post fertilization. Decrease in the dye filtration is secondary to the inability of the zebrafish pronephros (zebrafish kidneys) to clear the dye from the blood stream due to decrease in the filtering capacity of the kidney.
• the compound B used in this study did not have any effect on the heart rate at the concentrations evaluated. Hence, the retainment in the dye was contributed to by the decrease in the cardiac pumping. Dose dependent increase in the dye excreted was observed on treatment with compound B.
• the dye excretion improved from 13 % in ift80 morphants zebrafish larvae to about 48 % in ift80 morphants zebrafish larvae exposed to compound B ( Figure 12).
• Treatment with compound B was initiated at 24 h post fertilization (hpf).
• the pkd2 morphants were continuously exposed to compound B for 5 days post fertilization.
• the glomerular filtration studies (used to assess the renal function) were initiated at 72 h post fertilization. Decrease in the dye filtration is secondary to the inability of the zebrafish pronephros (zebrafish kidneys) to clear the dye from the blood stream due to decrease in the filtering capacity of the kidney.
• the compounds of interest in this study did not have any effect on the heart rate at the concentrations evaluated. The retainment in the dye hence was contributed to by the decrease in the cardiac pumping. Dose dependent increase in the dye excreted was observed on treatment with compound B.
• the zebrafish embryos obtained after spawning were dispensed in 96 well plates with one embryo per well in the 96 well microtitre plate.
• the compounds were dissolved in minimum volume of DMSO and diluted with water such that the final concentration achieved was 0.1 % DMSO solution.
• the embryos were exposed to varying concentrations of compound A and compound B with 10 embryos per concentration. The studies were repeated in triplicate. A negative control was used for each plate which contained 0.1 % DMSO.
• the plates were then incubated at 28 °C in an incubator and were read at 24, 48 and at 96 h post fertilization. LC 5 o, LOEC and NOEC were recorded.
• LC 5 o or the median lethal concentration was assigned to the concentration of the compound, which resulted in 50 % mortality of the embryos over a period of 48 h.
• Apical endpoints were evaluated in the study and refer to the toxicity features laid down by the OECD guideline 203 for zebrafish.
• the apical endpoints considered for acute toxicity include coagulation of eggs, irregular formation of somites, and absence of tail detachment and lack of the heartbeat. (Nagel et al.; The embryo test with the Zebrafish Danio rerio - a general model in ecotoxicology and toxicology; ALTEX, 2002, 19, Suppl. 1 ). Presence of anyone of these features was taken suggestive of toxic effect of the compound.
• the no effect observed concentration was experimentally derived and related to the compound concentration, which had no effects on the zebrafish larval morphology, growth and development.
• LOEC referred to the test substance concentration, treatment of which led to 50 % of the zebrafish larvae exhibiting abnormal/pathological morphology suggestive of compound associated toxicity.
• the lethal end point i.e. the median lethal concentration (LC 5 o) which corresponds to the concentration of the test compound which resulted in 50 % mortality of the embryos and LOEC was measured using an inverted microscope (Zeiss Axio Observer A.1 ) at the end of 48 h of embryonic development.

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