Compositions and methods of use thereof

Abstract

The present invention relates to compositions and methods of use thereof. Methods are drawn towards treating oxalate production-related disease or disorder by inhibiting the activity of glycolate oxidase (GO) and lactate dehydrogenase (LDHA) while activating degradation of GO and / or LDHA.

Description

COMPOSITIONS AND METHODS OF USE THEREOF

[0001] This application claims priority to U. S. Provisional Application No. 63 / 717,063 filed on November 06, 2024, the entire contents of which are incorporated herein by reference.

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U. S. Patent and Trademark Office patent file or records but otherwise reserves any and all copyright rights.GOVERNMENT INTERESTS

[0004] This invention was made with government support under R01 DK 136685 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION

[0005] The present invention relates to compositions and methods of treating disease.BACKGROUND OF THE INVENTION

[0006] This disclosure comprises small molecule drugs with a pharmacological mechanism combining enzyme inhibition with activation of enzyme degradation pathways.SUMMARY OF THE INVENTION

[0007] Aspects of the invention are drawn towards compound of Formula (I):Formula (I)or a pharmaceutically acceptable salt, prodrug, hydrate, or solvate thereof, wherein A-B is selected from -CH2-CH2-, -CH=CH- or -C=C-; C is selected from -CH2- or -CO-; D is selected from the group consisting of -CH2, -O-, or -NH-; E is selected from the group consisting of -CH2, -O-, or -NH-; F is selected from the group consisting of -O-, -NH-, -CO-NH-, or -NH-CO-; G = is selected from -CH2-, -CO- or -CO-CH2-; H-I is selected from -CH2-CH2- or -CH=CH-; m is 0 or 1; n is 0 or 1; o is 0, 1, 2, or 3; and p is 0, 1, 2, 3, or 4. In embodiments, wherein the compound is:YSL-ad1.In embodiments, the pharmaceutically acceptable salt of the compound described herein or combination of compounds described herein is selected from an inorganic salt or an organic salt. In embodiments, the inorganic salt is selected from an ammonium salt, a sodium salt, a potassium salt, a lithium salt, a calcium salt, a magnesium salt, an aluminum salt, a zinc salt, a ferrous salt, a ferric salt, a hydrochloride salt, a hydrobromide salt, a bisulfate salt, a phosphate salt, or a bicarbonate salt. In embodiments, the organic salt is selected from a mesylate salt, a tosylate salt, a besylate salt, a camphrosulfonate salt, an acetate salt, a formate salt, a maleate salt, a succinate salt, a glutarate salt, a tartrate salt, a citrate salt, a malate salt, or a fumarate salt.

[0008] Aspects of the disclosure are drawn towards a pharmaceutical composition comprising a compound described herein, or a combination thereof, and a pharmaceutically acceptable carrier, excipient, or diluent. In embodiments, the excipient comprises methyl cellulose. In embodiments, the pharmaceutically acceptable carrier further comprising polysorbate 80. In embodiments, the pharmaceutical composition comprises an adjuvant, an additional active agent, or a combination thereof. In embodiments, the adjuvant comprises an amino acid, a polyol, or a combination thereof. In embodiments, the amino acid is selected from the group consisting of L-Alanine, L-Arginine, L-Lysine, L-Histidine, L-Ornitine, L-Serine, L-Valine, L-Leucine, L-Proline, or Glycine. In embodiments, the polyol is selected from N-methyl-D-glucamine, tromethamine, choline, glucosamine, or diethanolamine. In embodiments, the pharmaceutical composition furthercomprising a vitamin B6 derivative. In embodiments, the vitamin B6 derivative is selected from the group consisting of pyridoxinium, pyridoxamine, piridoxal, or nicotinamide.

[0009] Aspects of the disclosure are drawn towards a method of inhibiting the activity of glycolate oxidase (GO) and lactate dehydrogenase (LDHA) while activating degradation of glycolate oxidase (GO) and / or lactate dehydrogenase (LDHA) in a subject. In embodiments, the method comprises administering a therapeutically effective amount of the pharmaceutical composition of described herein. In embodiments, the therapeutically effective amount comprises less than about 0.1 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 120 mg / kg, about 135 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, and greater than about 200 mg / kg. In embodiments, the therapeutically effective amount comprises less than about 1 uM, about 1 uM, about 1.5 uM, about 2 uM, about 2.5 uM, about 3 uM, about 4 uM, about 5 uM, about 6 uM, about 7 uM, about 8 uM, about 9 uM, about 10 uM, about 12.5 uM, about 15 uM, about 20 uM, about 22.5 uM, about 25 uM, about 30 uM, about 35 uM, about 40 uM, about 45 uM, about 50 uM, about 60 uM, about 70 uM, or greater than about 70 uM.

[0010] Aspects of the disclosure are drawn towards a method of treating a subject afflicted with an oxalate production-related disease or disorder. In embodiments, the method comprises: measuring the circulating oxalate levels of a subject; and administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein. In embodiments, the oxalate production-related disease or disorder comprises a cardiometabolic disease or disorder, a cardiovascular disease or disorder, a metabolic disease or disorder, a liver disease or disorder, a renal disease or disorder, or a combination thereof. In embodiments, the oxalate production-related disease or disorder comprises a GO-associated disease or disorder and / or an LDHA-associated disease or disorder. In embodiments, the cardiovascular disease or disorder comprises hypertension, atherosclerotic cardiovascular disease (ASCVD), coronary artery disease (CAD), acute coronary syndrome (ACS), myocardial infarction (MI), ischemic stroke, peripheral artery disease (PAD), carotid atherosclerotic disease, hear failure, myocardial ischemia-reperfusion injury, vascular calcification, arterial stiffening, an aneurysm, or a combination thereof. In embodiments, the renal disease or disorder comprises nephrolithiasis,recurrent kidney stone disease, chronic kidney disease (CKD), end-stage renal disease (ESRD), or a combination thereof. In embodiments, the metabolic disease or disorder comprises diabetes, dyslipidemia, obesity, hyperoxaluria, primary hyperoxaluria (PH), PHI, PH2, PH3, systemic oxalosis, or a combination thereof. In embodiments, the liver disease or disorder comprises cirrhosis, hepatitis, metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction-associated steatohepatitis (MASH), compensated and decompensated cirrhosis secondary to MASLD / MASH, portal hypertension, ascites, hepatocellular carcinoma (HCC), alcohol-associated liver disease (ALD), specific aetiology steatoic liver disease, drug-induced liver injury, a monogenic steatotic liver disease, a cryptogenic steatotic liver disease, or a combination thereof. In embodiments, the therapeutically effective amount of the composition inhibits glycolate oxidase (GO) activity and lactate dehydrogenase (LDHA) activity. In embodiments, the therapeutically effective amount of the composition activates degradation of glycolate oxidase (GO) and / or lactate dehydrogenase (LDHA). In embodiments, measuring the circulating oxalate levels of a subject comprises an enzymatic assay, mass spectrometry, or a combination thereof. In embodiments, the subject’s circulating oxalate level is at least about 1 uM, about 1 uM, about 1.5 uM, about 2 uM, about 2.5 uM, about 3 uM, about 4 uM, about 5 uM, about 6 uM, about 7 uM, about 8 uM, about 9 uM, about 10 uM, or greater than about 10 uM. In embodiments, the therapeutically effective amount comprises less than about 0.1 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 120 mg / kg, about 135 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, and greater than about 200 mg / kg. In embodiments, the therapeutically effective amount comprises less than about 1 uM, about 1 uM, about 1.5 uM, about 2 uM, about 2.5 uM, about 3 uM, about 4 uM, about 5 uM, about 6 uM, about 7 uM, about 8 uM, about 9 uM, about 10 uM, about 12.5 uM, about 15 uM, about 20 uM, about 22.5 uM, about 25 uM, about 30 uM, about 35 uM, about 40 uM, about 45 uM, about 50 uM, about 60 uM, about 70 uM, or greater than about 70 uM.

[0011] Aspects of the disclosure are drawn towards the use of a compound or a pharmaceutical composition described herein for treating a subject afflicted with an oxalate production-related disease or disorder. In embodiments, the use of the pharmaceutical composition is for inhibitingthe activity of glycolate oxidase (GO) and lactate dehydrogenase (LDHA) while activating degradation of glycolate oxidase (GO) and / or lactate dehydrogenase (LDHA) in a subject.

[0012] Aspects of the disclosure are drawn to a compound of Formula (II):(Formula II)or a pharmaceutically acceptable salt, prodrug, hydrate, or solvate thereof, wherein A-B is selected from -CH2-CH2-, -CH=CH- or -C=C-; C is selected from -CH2- or -CO-; D is selected from CH or N; E is selected from -CH2 or -CO-; F is selected from CH or N; and G = is selected from -CH2-, -CO- or -CO-CH2-; and H-I is selected from -CH2-CH2- or -CH=CH-. In embodiments, the compound of claim 2 wherein the compound is:YSL-ad6.In embodiments, the pharmaceutically acceptable salt of the compound described herein or combination of compounds described herein is selected from an inorganic salt or an organic salt. In embodiments, the inorganic salt is selected from an ammonium salt, a sodium salt, a potassium salt, a lithium salt, a calcium salt, a magnesium salt, an aluminum salt, a zinc salt, a ferrous salt, a ferric salt, a hydrochloride salt, a hydrobromide salt, a bisulfate salt, a phosphate salt, or a bicarbonate salt. In embodiments, the organic salt is selected from a mesylate salt, a tosylate salt, a besylate salt, a camphrosulfonate salt, an acetate salt, a formate salt, a maleate salt, a succinate salt, a glutarate salt, a tartrate salt, a citrate salt, a malate salt, or a fumarate salt.

[0013] Aspects of the disclosure are drawn towards a compound of Formula (III):(Formula III)or a pharmaceutically acceptable salt, prodrug, hydrate, or solvate thereof, wherein A-B is selected from -CH2-CH2-, -CH=CH- or -C=C-; C is selected from -CH2- or -CO-; D is selected from the group consisting of -CH2, -O-, or -NH-; E is selected from the group consisting of -CH2,-O-, or -NH-; H-I is selected from -CH2-CH2- or -CH=CH-; m is 0 or 1; n is 0 or 1; o is 0, 1, 2, or 3; and p is 0, 1, 2, 3, or 4; and wherein either D = -O-, E = -O-, or both D = -O- and E = -O-. In embodiments, the compound is:In embodiments, the pharmaceutically acceptable salt of the compound described herein or combination of compounds described herein is selected from an inorganic salt or an organic salt. In embodiments, the inorganic salt is selected from an ammonium salt, a sodium salt, a potassium salt, a lithium salt, a calcium salt, a magnesium salt, an aluminum salt, a zinc salt, a ferrous salt, a ferric salt, a hydrochloride salt, a hydrobromide salt, a bisulfate salt, a phosphate salt, or a bicarbonate salt. In embodiments, the organic salt is selected from a mesylate salt, a tosylate salt, a besylate salt, a camphrosulfonate salt, an acetate salt, a formate salt, a maleate salt, a succinate salt, a glutarate salt, a tartrate salt, a citrate salt, a malate salt, or a fumarate salt.

[0014] Aspects of the disclosure are drawn towards a pharmaceutical composition comprising a compound described herein, or a combination thereof, and a pharmaceutically acceptable carrier, excipient, or diluent. In embodiments, the excipient comprises methyl cellulose. In embodiments, the pharmaceutically acceptable carrier further comprising polysorbate 80. In embodiments, the pharmaceutical composition comprises an adjuvant, an additional active agent, or a combination thereof. In embodiments, the adjuvant comprises an amino acid, a polyol, or a combination thereof. In embodiments, the amino acid is selected from the group consisting of L-Alanine, L-Arginine, L-Lysine, L-Histidine, L-Ornitine, L-Serine, L-Valine, L-Leucine, L-Proline, or Glycine. In embodiments, the polyol is selected from N-methyl-D-glucamine, tromethamine, choline,glucosamine, or diethanolamine. In embodiments, the pharmaceutical composition further comprising a vitamin B6 derivative. In embodiments, the vitamin B6 derivative is selected from the group consisting of pyridoxinium, pyridoxamine, piridoxal, or nicotinamide.

[0015] Aspects of the disclosure are drawn towards a method of inhibiting the activity of glycolate oxidase (GO) and lactate dehydrogenase (LDHA) while activating degradation of glycolate oxidase (GO) and / or lactate dehydrogenase (LDHA) in a subject. In embodiments, the method comprises administering a therapeutically effective amount of the pharmaceutical composition of described herein. In embodiments, the therapeutically effective amount comprises less than about 0.1 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 120 mg / kg, about 135 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, and greater than about 200 mg / kg. In embodiments, the therapeutically effective amount comprises less than about 1 uM, about 1 uM, about 1.5 uM, about 2 uM, about 2.5 uM, about 3 uM, about 4 uM, about 5 uM, about 6 uM, about 7 uM, about 8 uM, about 9 uM, about 10 uM, about 12.5 uM, about 15 uM, about 20 uM, about 22.5 uM, about 25 uM, about 30 uM, about 35 uM, about 40 uM, about 45 uM, about 50 uM, about 60 uM, about 70 uM, or greater than about 70 uM.

[0016] Aspects of the disclosure are drawn towards a method of treating a subject afflicted with an oxalate production-related disease or disorder. In embodiments, the method comprises: measuring the circulating oxalate levels of a subject; and administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein. In embodiments, the oxalate production-related disease or disorder comprises a cardiometabolic disease or disorder, a cardiovascular disease or disorder, a metabolic disease or disorder, a liver disease or disorder, a renal disease or disorder, or a combination thereof. In embodiments, the oxalate production-related disease or disorder comprises a GO-associated disease or disorder and / or an LDHA-associated disease or disorder. In embodiments, the cardiovascular disease or disorder comprises hypertension, atherosclerotic cardiovascular disease (ASCVD), coronary artery disease (CAD), acute coronary syndrome (ACS), myocardial infarction (MI), ischemic stroke, peripheral artery disease (PAD), carotid atherosclerotic disease, hear failure, myocardial ischemia-reperfusion injury, vascular calcification, arterial stiffening, an aneurysm, or acombination thereof. In embodiments, the renal disease or disorder comprises nephrolithiasis, recurrent kidney stone disease, chronic kidney disease (CKD), end-stage renal disease (ESRD), or a combination thereof. In embodiments, the metabolic disease or disorder comprises diabetes, dyslipidemia, obesity, hyperoxaluria, primary hyperoxaluria (PH), PHI, PH2, PH3, systemic oxalosis, or a combination thereof. In embodiments, the liver disease or disorder comprises cirrhosis, hepatitis, metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction-associated steatohepatitis (MASH), compensated and decompensated cirrhosis secondary to MASLD / MASH, portal hypertension, ascites, hepatocellular carcinoma (HCC), alcohol-associated liver disease (ALD), specific aetiology steatoic liver disease, drug-induced liver injury, a monogenic steatotic liver disease, a cryptogenic steatotic liver disease, or a combination thereof. In embodiments, the therapeutically effective amount of the composition inhibits glycolate oxidase (GO) activity and lactate dehydrogenase (LDHA) activity. In embodiments, the therapeutically effective amount of the composition activates degradation of glycolate oxidase (GO) and / or lactate dehydrogenase (LDHA). In embodiments, measuring the circulating oxalate levels of a subject comprises an enzymatic assay, mass spectrometry, or a combination thereof. In embodiments, the subject’s circulating oxalate level is at least about 1 uM, about 1 uM, about 1.5 uM, about 2 uM, about 2.5 uM, about 3 uM, about 4 uM, about 5 uM, about 6 uM, about 7 uM, about 8 uM, about 9 uM, about 10 uM, or greater than about 10 uM. In embodiments, the therapeutically effective amount comprises less than about 0.1 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 120 mg / kg, about 135 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, and greater than about 200 mg / kg. In embodiments, the therapeutically effective amount comprises less than about 1 uM, about 1 uM, about 1.5 uM, about 2 uM, about 2.5 uM, about 3 uM, about 4 uM, about 5 uM, about 6 uM, about 7 uM, about 8 uM, about 9 uM, about 10 uM, about 12.5 uM, about 15 uM, about 20 uM, about 22.5 uM, about 25 uM, about 30 uM, about 35 uM, about 40 uM, about 45 uM, about 50 uM, about 60 uM, about 70 uM, or greater than about 70 uM.

[0017] Aspects of the disclosure are drawn towards the use of a compound or a pharmaceutical composition described herein for treating a subject afflicted with an oxalate production-relateddisease or disorder. In embodiments, the use of the pharmaceutical composition is for inhibiting the activity of glycolate oxidase (GO) and lactate dehydrogenase (LDHA) while activating degradation of glycolate oxidase (GO) and / or lactate dehydrogenase (LDHA) in a subject.

[0018] Other objects and advantages of this invention will become readily apparent from the ensuing description.BRIEF DESCRIPTION OF THE FIGURES

[0019] FIG. 1 shows a non-limiting, exemplary structure of compounds with acyclic linkers.

[0020] FIG. 2 shows a non-limiting, exemplary general structure of compounds containing cyclic fragments.

[0021] FIG. 3 shows non-limiting, exemplary experimental data. Panel A. Decrease of intracellular oxalate in mouse primary hepatocytes (steatosis model) treated with FAB-599 in doses of 10 and 50 pM. Comparison with untreated primary hepatocytes (control DMSO). Panel B. Dose-dependent decrease of LDHA concentration (normalized with P-actin) in primary hepatocytes treated with FAB-599 in doses of 2, 10, 20 and 50 pM. Comparison with untreated primary hepatocytes (control).

[0022] FIG. 4 shows a non-limiting, exemplary 'H-NMR of Methyl 5-(5-formyl-2-furanyl)-2-hydroxybenzoate (MDMG-409E).

[0023] FIG. 5A shows a non-limiting, exemplary 'H-NMR of Methyl (A)-2-hydroxy-5-{5-[3-(4-iodophenyl)-3-oxoprop- 1 -en- 1 -yl]furan-2-yl (benzoate (FAB-545)

[0024] FIG. 5B shows a non-limiting, exemplary 13C-NMR of Methyl (£)-2-hydroxy-5-{5-[3 -(4-iodophenyl)-3 -oxoprop- l-en-l-yl]furan-2-yl (benzoate (FAB-545)

[0025]

[0026] FIG. 6 shows a non-limiting, exemplary 'H-NMR of Methyl (E)-2-hydroxy-5-{ 5-[3-(4-bromophenyl)-3-oxoprop-l-en-l-yl]furan-2-yl(benzoate (FAB-535C).

[0027] FIG. 7A shows a non-limiting, exemplary 'H-NMR of A-(Adamantan-l-ylmethyl)acrylamide (FAB-595).

[0028] FIG. 7B shows a non-limiting, exemplary13C-NMR of / V-(Adamantan-l-ylmethyl)acrylamide (FAB-595).

[0029] FIG. 8A shows a non-limiting, exemplary 'H-NMR of / V-(Adamantan-l-ylmethyl)hex-5-yn-l-amine (FAB -607).

[0030] FIG.8B shows a non-limiting, exemplary13C-NMR of A-(Adamantan-l -ylmethyl)hex-5-yn-l-amine (FAB -607).

[0031] FIG. 9A shows a non-limiting, exemplary 'H-NMR of / c77-Butyl-A-(6-hydroxyhexyl)carbamate (PLN-6).

[0032] FIG. 9B shows a non-limiting, exemplary13C-NMR of / c77-Butyl-A-(6-hydroxyhexyl)carbamate (PLN-6).

[0033] FIG. 10A shows a non-limiting, exemplary 'H-NMR of 5-{5-{(£)-3-{4-[(E)-3-(Adamantan- 1 -y lmethylamino)-3 -oxoprop- 1 -eny l]pheny 1 } -3 -oxoprop- 1 -eny 1 } furan-2-y 1 } -2-hydroxybenzoic acid (FAB-596).

[0034] FIG. 10B shows a non-limiting, exemplary13C-NMR of 5-{5-{(E)-3-{4-[(£)-3-(Adamantan- 1 -ylmethylamino)-3-oxoprop- 1 -enyl]phenyl } -3-oxoprop- 1 -enyl }furan-2-yl } -2-hydroxybenzoic acid (FAB-596).

[0035] FIG. 11 shows a non-limiting, exemplary 'H-NMR of Methyl 5-{5-{(£)-3-{4-[6-(adamantan- 1 -ylmethylamino)hex- 1 -yny l]phenyl } -3 -oxoprop- 1 -enyl } furan-2-yl } -2-hydroxybenzoate (FAB-599e).

[0036] FIG. 12A shows a non-limiting, exemplary 'H-NMR of Methyl 5-{5-{(£)-3-{4-{6-[6-(tert-butoxy carbonylamino)hexyloxy ]hex- 1 -yny 1 } phenyl } -3 -oxoprop- 1 -enyl } furan-2-yl } -2-hydroxybenzoate (PLN-7).

[0037] FIG. 12B shows a non-limiting, exemplary13C-NMR of Methyl 5-{5-{(E)-3-{4-{6-[6-(tert-butoxy carbonylamino)hexyloxy ]hex- 1 -yny 1 } phenyl } -3 -oxoprop- 1 -enyl } furan-2-yl } -2-hydroxybenzoate (PLN-7).

[0038] FIG. 13A shows a non-limiting, exemplary 'H-NMR of 5-{5-{(£)-3-{4-[6-(Adamantan- 1 -ylmethylamino)hex- 1 -ynyl]phenyl } -3 -oxoprop- 1 -enyl }furan-2-yl } -2-hydroxybenzoic acid (FAB-599).

[0039] FIG. 13B shows a non-limiting, exemplary13C-NMR of 5-{ 5-{(£)-3-{4-[6-(Adamantan- 1 -ylmethylamino)hex- 1 -ynyl]phenyl } -3 -oxoprop- 1 -enyl }furan-2-yl } -2-hydroxybenzoic acid (FAB-599).

[0040] FIG. 14A shows a non-limiting, exemplary 'H-NMR of 5-{5-{( )-3-{4-{6-[6-(terA Butoxy carbonylamino)hexyloxy ]hex- 1 -ynyl } phenyl } -3 -oxoprop- 1 -enyl } furan-2-yl } -2-hydroxybenzoic acid (PLN-8).

[0041] FIG. 14B shows a non-limiting, exemplary13C-NMR of 5-{5-{(L)-3-{4-{6-[6-(te / 7-Butoxy carbonylamino)hexyloxy ]hex- 1 -ynyl } phenyl } -3 -oxoprop- 1 -eny 1 } furan-2-yl } -2-hydroxybenzoic acid (PLN-8).

[0042] FIG. 15A shows a non-limiting, exemplary ’H-NMR of 5-{5-{(E')-3-{4-[6-(6-aminohexyloxy)hex- 1 -ynyl]phenyl } -3 -oxoprop- 1 -enyl }furan-2-yl } -2-hydroxybenzoic acid (PLN-9).

[0043] FIG. 15B shows a non-limiting, exemplary13C-NMR of 5-{5-{(E)-3-{4-[6-(6-aminohexyloxy)hex- 1 -ynyl] phenyl } -3 -oxoprop- 1 -enyl }furan-2-yl } -2-hydroxybenzoic acid (PLN-9).

[0044] FIG. 16A shows a non-limiting, exemplary simplified schematic of primary hyperoxaluria type 1 (PHI).

[0045] FIG. 16B shows a non-limiting, exemplary overview of previous therapeutic strategies developed by our research group: furyl salicylic acids (FSAs) and aminomethyl furyl salicylic acids (AMFSAs).

[0046] FIG. 16C shows a non-limiting, exemplary general structure of oxophenylpropenyl furyl salicylic acids (OPPFSAs) and their derivatization into compound 2, a hydrophobic-tag protein degrader (HyT-PD).

[0047] FIG. 16D shows non-limiting, exemplary illustrations of In vitro evaluation of the most potent inhibitors.

[0048] FIG. 16E shows a non-limiting, exemplary illustration of In silico characterization of compound-target interactions and pharmacokinetic properties.

[0049] FIG. 16F shows a non-limiting, exemplary illustration of in vivo translation of the findings.

[0050] FIG. 17 shows non-limiting, exemplary structural analogues of OPPFSAs (pother structures'"

[0051] FIG. 18A shows anon-limiting, exemplary graph of distribution of inhibitors according to the remaining activity of recombinant enzymes after treatment. Comparison with the reference compound 1. large rectangle: compounds that decrease enzyme activity in a greater extent than 1. small rectangle: compounds that produce more than 90% inhibition for both enzymes.

[0052] FIG. 18B shows a non-limiting, exemplary heatmap of enzymatic activity of recombinant hGO and hLDHA following treatment with representative compounds. Activity values areexpressed relative to vehicle treated controls (set at 100%) and represent the mean of four replicates (n = 4).

[0053] FIG. 19 -FIG. 43 show non-limiting, exemplary graphs of embodiments showing nonlinear “log (cone.) vs relative hGO activity” plot (media of four replicates)(left), and non-linear “log (cone.) vs relative hGO activity” plots of the four replicates (using ten concentrations of inhibitor and 180 pM glycolate)(right). Error bars show standard deviation.

[0054] FIG. 44 shows a non-limiting, exemplary image of AGXT protein abundance in livers of Agxt+ / +and Agxt ' mice. Livers from mice Agxt+ / +(n=2) and Agxt ''' (n=2) mice were isolated and AGXT protein expression was assessed.

[0055] FIG. 45 shows non-limiting, exemplary heatmaps of intracellular oxalate levels (fold change) in primary hepatocytes from Agxt- / - mice following treatment with dual inhibitors. Agxt- / - primary hepatocytes were isolated, incubated with glycolic acid (GA, 5 mM) and treated with either vehicle (DMSO) or inhibitors at 10 pM and 50 pM for 24 h (n = 4)(right), and extracellular oxalate levels (fold change) in primary hepatocytes from ^^wZ-Z-mice following treatment with OPPFSAs. Agxt-I- primary hepatocytes were isolated, incubated in the presence or absenceof glycolic acid (GA, 5 mM) and treated with either vehicle (DMSO) or inhibitors at 10 pM and 50 pM for 24 h (n = 4)(left). Statistical analysis: Comparisons between GA control and inhibitor-treated groups were performed using one-way ANOVA followed by Tukey’s post hoc test. Significance levels (*p < 0.05; **p < 0.01; ***p < 0.001). Additional comparisons between compounds that significantly reduced oxalate and reference compound 1, were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Significance levels (®p < 0.05;Y / Y'p < 0.01).

[0056] FIG. 46 shows a non-limiting, exemplary graph of percent cell viability (n = 3) in the evaluation of dual inhibitors on hepatocytes of PHI mice. Agxt'1' primary hepatocytes were isolated, stimulated with GA (5 mM) and treated with either vehicle (DMSO) or inhibitors (50pM) for 24 h.

[0057] FIG. 47 shows a non-limiting, exemplary graph of calculated logP(o / w) and hlogD of OPPFSAs and reference 1. Graph: Bubble size indicates fold change intracellular oxalate (small bubble indicates low intracellular oxalate after treatment 10 pM concentrations of the compound). The dashed blue lines represent a non-limiting, exemplary contemplated range of lipophilicity (hlogD) for a compound to ensure an acceptable permeability / solubility balance.

[0058] FTG. 48 shows non-limiting, exemplary graphs of non-linear concentration vs initial velocity (top) and Lineweaver-Burk (bottom) plots for compound 20 on enzyme AGO (using four concentrations of inhibitor and ten different concentrations of glycolate; measurements every minute during 4 min) (built from data in Table 54). Color code indicates concentration of 20: 2 pM (orange); 0,50 pM (green); 0,25 pM (red); 0 pM (blue).

[0059] FIG. 49 shows non-limiting, exemplary graphs of observed variation on the parameters Vmax (A fluorescence / min) and KM (pM) and the ratio Vmax / KM with increasing concentrations of the inhibitor 20, showing non-competitive mixed type inhibition (a>l) (built from data in Table 55). Top Plot: Curvilinear decrease of Vmax. and curvilinear increase of KM. Bottom Plot: Curvilinear decrease of the ration Vmax / KM.

[0060] FIG. 50 shows non-limiting, exemplary graphs of non-linear concentration vs initial velocity (top) and Lineweaver-Burk (bottom) plots for compound 23 on enzyme AGO (using three concentrations of inhibitor and ten different concentrations of glycolate; measurements every minute during 4 min) (built from data in Table 57). Color code indicates concentration of 23: 0,75 pM (green); 0,5 pM (red); 0 pM (blue).

[0061] FIG. 51 shows non-limiting, exemplary graphs of observed variation on the parameters Vmax (A fluorescence / min) and KM (pM) and the ratio Vmax / KM with increasing concentrations of the inhibitor 23, showing non-competitive mixed type inhibition (a>l) (built from data in Table 58). Top Plot: Curvilinear decrease of Vmax and curvilinear increase of KM. Bottom Plot: Curvilinear decrease of the ration Vmax / KM.

[0062] FIG. 52 shows non-limiting, exemplary graphs of non-linear concentration vs initial velocity (top) and Lineweaver-Burk (bottom) plots for compound 2 on enzyme AGO (using four concentrations of inhibitor and ten different concentrations of glycolate; measurements every minute during 4 min) (built from data in Table 60). Color code indicates concentration of 2: 1,6 pM (orange), 0,8 pM (green); 0,5 pM (red); 0 pM (blue).

[0063] FIG. 53 shows non-limiting, exemplary graphs of observed variation on the parameters Vmax (A fluorescence / min) and KM (pM) and the ratio Vmax / KM with increasing concentrations of the inhibitor 2, showing non-competitive mixed type inhibition (a>l) (built from data in Table 61). Top Plot: Curvilinear decrease of Vmax and curvilinear increase of KM. Bottom Plot: Curvilinear decrease of the ration Vmax / KM.

[0064] FTG. 54 shows non-limiting, exemplary graphs of non-linear concentration vs initial velocity (top) and Lineweaver-Burk (bottom) plots for compound 20 on enzyme hLDHA (using four concentrations of inhibitor and ten different concentrations of pyruvate; measurements every minute during 10 min) (built from data in Table 63). Color code indicates concentration of 20: 0,5 pM (orange); 0,125 pM (green); 0.05 pM (red) and 0 pM (blue).

[0065] FIG. 55 shows non-limiting, exemplary graphs of observed variation on the parameters Vmax (A fluorescence / min) and KM (pM) and the ratio Vmax / KM with increasing concentrations of the inhibitor 20, showing non-competitive inhibition (built from data in Table 64). Top Plot: Curvilinear decrease of Vmax and no increasing or slightly decreasing trend of KM. Bottom Plot: Curvilinear decrease of the ratio Vmax / KM.

[0066] FIG. 56 shows non-limiting, exemplary graphs of non-linear concentration vs initial velocity (top) and Lineweaver-Burk (middle) plots for compound 23 on enzyme / ? LDHA. Colour code indicates concentration of 23: 0,1 pM (purple); 0,075 pM (green); 0.05 pM (red) and 0 pM (blue). Cornish-Bowden (bottom) plot. All plots are prepared using four concentrations of inhibitor and ten different concentrations of pyruvate; measurements every minute during 10 min) (built from data in Table 68).

[0067] FIG. 57 shows non-limiting, exemplary graphs of observed variation on the parameters Vmax (A fluorescence / min) and Ku (pM) and the ratio Vmax / K with increasing concentrations of the inhibitor 23, showing competitive inhibition (built from data in Table 69). Top Plot: No increasing or decreasing trend of Vmax and linear increase of Ku. Bottom Plot: Curvilinear decrease of the ration Vmax / Ku.

[0068] FIG. 58 shows non-limiting, exemplary graphs of non-linear concentration vs initial velocity (top) and Lineweaver-Burk (bottom) plots for compound 2 on enzyme ALDHA (using four concentrations of inhibitor and ten different concentrations of pyruvate; measurements every minute during 10 min) (built from data in Table 71). Color code indicates concentration of 2: 0,12 pM (purple); 0,040 pM (green); 0.010 pM (red) and 0 pM (blue).

[0069] FIG. 59 shows non-limiting, exemplary graphs of observed variation on the parameters Vmax (A fluorescence / min) and Ku (pM) and the ratio Vmax / K with increasing concentrations of the inhibitor 2, showing non-competitive inhibition (built from data in Table 72). Top Plot: Curvilinear decrease of Vmax and no increasing or decreasing trend of Ku. Bottom Plot: Curvilinear decrease of the ratio Vmax / Ku.

[0070] FTG. 60A shows non-limiting, exemplary graphs illustrating Agxt- / - primary hepatocytes isolated and treated with either vehicle (DMSO) or increasing concentrations of 2 (2, 10, 20 and 50 pM) for 24 h. GO protein abundance and quantification relative to P-Actin (n = 4). LDHA protein abundance and quantification relative to P-Actin (n = 4). Haol mRNA expression (n = 4). Ldha mRNA expression (n = 4). Data are expressed as mean ± SEM. Normality was assessed using the Shapiro-Wilk test. Comparisons among multiple groups that passed normality testing were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Groups that did not meet normality assumptions were analyzed using the Kruskal-Wallis test.

[0071] FIG. 60B shows a non-limiting, exemplary schematic of possible LDHA degradation mechanisms and strategy of study.

[0072] FIG. 60C shows non-limiting, exemplary graphs of LDHA protein abundance and quantification relative to P-Actin (n = 3) in the presence or absence of compound 2 (50 pM) and the proteasome inhibitor MG-132 (10 pM)(top). LDHA and ATG5 protein abundance and quantification relative to P-Actin (n = 3) in the presence or absence of 2 (50 pM), siRNA against the autophagy mediator Atg5 or control scramble siRNA(bottom). Data are expressed as mean ± SEM. Normality was assessed using the Shapiro-Wilk test. Comparisons among multiple groups that passed normality testing were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Groups that did not meet normality assumptions were analyzed using the Kruskal-Wallis test.

[0073] FIG.61A shows a non-limiting, exemplary molecular dynamics image of superposition of the representative pose of 2 (blue) within the most populated cluster of AGO (2RDT) (tan) and the initial conformation of 2 (pink) in the 2-AGO ensemble (grey) used to initiate the MD simulation (frame 1). Hydrogen bonds are represented by dashed yellow lines.

[0074] FIG.61B shows a non-limiting, exemplary molecular dynamics image of superposition of the representative pose of 2 (blue) in the most populated cluster of ALDHA (1110) subunit B (tan) and the initial conformation of 2 (pink)-ALDHA dimeric ensemble (grey) (frame 1) that started the MD simulation. Hydrogen bonds are represented by dashed yellow lines.

[0075] FIG. 61C shows non-limiting, exemplary surface representation of the starting frame 1 of 2 that initiated the MD simulation (left), and the most populated clusters of 2 at the end of the MD simulation (right) on AGO. Exposure of the adamantane hydrophobic tag over the protein surface can be observed in ALDHA but not in AGO.

[0076] FTG. 61D shows non-limiting, exemplary Surface representations of the starting frame 1 of 2 that initiated the MD simulation (left), and the most populated clusters of 2 at the end of the MD simulation (right) on LDHA. Exposure of the adamantane hydrophobic tag over the protein surface can be observed in ALDHA but not in hGO.

[0077] FIG. 62A shows a non-limiting, exemplary scatter plot of radius of gyration (Rgyr) versus tridimensional polar surface area (3D-PSA) for all conformers; points are colored by intramolecular hydrogen bonds (IMHB) count (purple= 0, blue = 1, green = 2, yellow = 3). Cross markers denote chloroform conformers; circles denote water conformers.

[0078] FIG. 62B shows a non-limiting, exemplary image of the superposition of the centroid conformers of compound 2 representing the closed (orange), semi-closed (green) and open (cyan) states identified by k-means clustering.

[0079] FIG. 63A shows a non-limiting, exemplary schematic representation of the experimental approach and endpoint analyses. Male Agxt- / - mice were orally administered with either vehicle (0.6% methylcellulose and 0.5% Tween® 80 in water, n = 10) or compound 2 (20 mg / kg body weight, n = 10) daily for 10 days (green triangles indicate the time points at which urine oxalate was measured). Normality was assessed using the Shapiro-Wilk test. Comparisons between two groups were performed using Student’s t-test, as normality was assumed in all cases.

[0080] FIG. 63B shows a non-limiting, exemplary image and data of LDHA protein abundance relative to P-Actin in the liver at the end of the treatment. Data are expressed as mean ± SEM. Normality was assessed using the Shapiro-Wilk test. Comparisons between two groups were performed using Student’s t-test, as normality was assumed in all cases.

[0081] FIG. 63C shows a non-limiting, exemplary image and data of GO protein abundance relative to [3-Actin in the liver at the end of the treatment. Data are expressed as mean ± SEM. Normality was assessed using the Shapiro-Wilk test. Comparisons between two groups were performed using Student’s t-test, as normality was assumed in all cases.

[0082] FIG. 63D shows a non-limiting, exemplary graph of LDH activity measured in the liver at the end of the treatment. Data are expressed as mean ± SEM. Normality was assessed using the Shapiro-Wilk test. Comparisons between two groups were performed using Student’s t-test, as normality was assumed in all cases.

[0083] FIG. 63E shows a non-limiting, exemplary graph of plasma levels of AST at the end of the treatment. Normality was assessed using the Shapiro-Wilk test. Comparisons between two groups were performed using Student’s t-test, as normality was assumed in all cases.

[0084] FIG. 63F shows a non-limiting, exemplary graph plasma levels of AST at the end of the treatment. Normality was assessed using the Shapiro-Wilk test. Comparisons between two groups were performed using Student’s t-test, as normality was assumed in all cases.

[0085] FIG. 63G shows a non-limiting, exemplary images H& E staining of liver sections from mice at the end of the treatment (scale bar 100 pm).

[0086] FIG.64A shows non-limiting, exemplary progression of urinary oxalic acid excretion over treatment represented in absolute values and relative values versus baseline. Male Agxt- / - mice were orally administered with either vehicle (0.6% methylcellulose and 0.5% Tween® 80 in water, n = 10) or 2 (20 mg / kg body weight, n = 10) daily for 10 days. Data are expressed as mean ± SEM. Normality was assessed using the Shapiro-Wilk test. Comparisons among multiple groups that passed normality testing were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Groups that did not meet normality assumptions were analyzed using the Kruskal-Wallis test followed by Dunn’s post hoc test. Comparisons between two groups were performed using the Mann-Whitney test, as normality was not assumed.

[0087] FIG. 64B shows non-limiting, exemplary progression of urinary glycolic acid excretion over treatment represented in absolute values and relative values versus baseline. Male Agxt- / -mice were orally administered with either vehicle (0.6% methylcellulose and 0.5% Tween® 80 in water, n = 10) or 2 (20 mg / kg body weight, n = 10) daily for 10 days. Data are expressed as mean ± SEM. Normality was assessed using the Shapiro-Wilk test. Comparisons among multiple groups that passed normality testing were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Groups that did not meet normality assumptions were analyzed using the Kruskal-Wallis test followed by Dunn’ s post hoc test. Comparisons between two groups were performed using the Mann-Whitney test, as normality was not assumed.

[0088] FIG. 64C shows a non-limiting, exemplary Quantification of calcium-oxalate (Ca-Ox) deposition by % area per image (n=10). Male Agxt- / - mice were orally administered with either vehicle (0.6% methylcellulose and 0.5% Tween® 80 in water, n = 10) or 2 (20 mg / kg body weight, n = 10) daily for 10 days. Data are expressed as mean ± SEM. Normality was assessed using the Shapiro-Wilk test. Comparisons among multiple groups that passed normality testing wereanalyzed using one-way ANOVA followed by Tukey’s post hoc test. Groups that did not meet normality assumptions were analyzed using the Kruskal-Wallis test followed by Dunn’s post hoc test. Comparisons between two groups were performed using the Mann-Whitney test, as normality was not assumed.

[0089] FIG. 64D shows non-limiting, exemplary polarized light microscopy images of kidney tissue stained with H& E at the end of the treatment (scale bar = 200 pm).

[0090] FIG. 64E shows a non-limiting, exemplary graph of total number of Ca-Ox crystals per section. Histopathological analyses were performed by two blinded and one non-blinded pathologist, and results were combined to obtain the overall means for each parameter. Data are presented as mean ± SEM. Normality was assessed using the Shapiro-Wilk test, and group comparisons were performed using the Mann-Whitney test, as data did not meet the assumption of normality.

[0091] FIG. 64F shows a non-limiting, exemplary graph size of the largest Ca-Ox crystal per section. Histopathological analyses were performed by two blinded and one non-blinded pathologist, and results were combined to obtain the overall means for each parameter. Data are presented as mean ± SEM. Normality was assessed using the Shapiro-Wilk test, and group comparisons were performed using the Mann- Whitney test, as data did not meet the assumption of normality.

[0092] FIG. 64G shows a non-limiting, exemplary graph of mean Ca-Ox crystal size per section. Histopathological analyses were performed by two blinded and one non-blinded pathologist, and results were combined to obtain the overall means for each parameter. Data are presented as mean ± SEM. Normality was assessed using the Shapiro-Wilk test, and group comparisons were performed using the Mann-Whitney test, as data did not meet the assumption of normality.

[0093] FIG. 64H shows a non-limiting, exemplary graph of Standard deviation of Ca-Ox crystal distribution. Histopathological analyses were performed by two blinded and one non-blinded pathologist, and results were combined to obtain the overall means for each parameter. Data are presented as mean ± SEM. Normality was assessed using the Shapiro-Wilk test, and group comparisons were performed using the Mann-Whitney test, as data did not meet the assumption of normality.

[0094] FIG. 65 shows a non-limiting, exemplary graph of quantification of ATG5 abundance relative to [3-Actin (n = 3). Agxf'' primary hepatocytes were isolated and transfected with either siScr or siAtg5 (20 nM),

[0095] FIG. 66 shows a non-limiting, exemplary image of superposition of cognate crystal structure (purple) and docked pose (green) of CDST in the hGO (2RDT) binding site (grey). Hydrogen bonds are represented by dashed yellow lines.

[0096] FIG.67 shows a non-limiting, exemplary image of the docking pose of 2 (blue) in the hGO (2RDT) binding site (grey). Hydrogen bonds are represented by dashed yellow lines.

[0097] FIG. 68 shows non-limiting, exemplary images of (left)docking pose of 2 (blue) in the hLDHA (1110) binding site (tan), and close-up(right) of the superposed docking pose of 2 (blue) and crystalized oxamic acic (purple). Hydrogen bonds are represented by dashed yellow lines.

[0098] FIG. 69 shows a non-limiting, exemplary graph of 100 ns MD simulation RMSD of homology-modeled hGO (PDB ID 2RDT) alpha carbons (blue), and standalone ligand 2 (orange) (FAB-599 = 2).

[0099] FIG. 70 shows a non-limiting, exemplary image of Superpositions of the first 10 frames, starting from frame 0 (t=0) to frame 10 (t=10 ns) at 1 ns interval, are presented. The gating loop (loop 4) is highlighted in yellow, while the rest of the protein is depicted in tan. For reference, 2 and cofactor FMN from frame 0 are shown and colored blue.

[0100] FIG. 71A shows a non-limiting, exemplary graph of Distance (A) between Argl67 guanidine group and 2 salicylic carboxylic acid oxygen atoms.

[0101] FIG. 71B shows a non-limiting, exemplary graph of distance (A) between Arg263 guanidine group and 2 salicylic carboxylic acid oxygen atoms.

[0102] FIG. 71C shows a non-limiting, exemplary graph of distance (A) between Leul64 and the phenyl ring of the salicylic ring (blue); TrpllO and 2 phenylketone moiety (orange); Tyrl34 and 2 phenylketone moiety (grey).

[0103] FIG. 72 shows non-limiting, exemplary graphs of 100 ns simulation SASA of hGO on water (left) and 100 ns simulation SASA of ligand 2 on water (right).

[0104] FIG. 73 shows non-limiting, exemplary images of representative pose(left) of 2 (blue) in the most populated cluster of hGO homology model (2RDT) (tan), and the superposition of the representative pose of 2 (blue) in the most populated cluster of hGO (2RDT) (tan) and frame0 2 (pink)-hGO ensemble (grey) that started the MD simulation(right). Hydrogen bonds are represented by dashed yellow lines.

[0105] FIG. 74 shows a non-limiting, exemplary graph of 100 ns simulation RMSD of hLDHA dimeric complex alpha carbons (blue); hLDHA monomer A alpha carbons (orange); hLDHA monomer B alpha carbons (green).

[0106] FIG. 75 shows a non-limiting, exemplary graph of 100 ns simulation SASA of ALDHA dimeric complex on water.

[0107] FIG. 76 shows a non-limiting, exemplary graph of 100 ns simulation RMSD of 2 in the dimeric ALDHA complex.

[0108] FIG. 77A shows a non-limiting, exemplary graph of distance (A) between Argl05 guanidine group and 2 salicylic carboxylic acid.

[0109] FIG. 77B shows a non-limiting, exemplary graph of distance (A) between Argl68 guanidine group and 2 salicylic carboxylic acid.

[0110] FIG. 77C shows a non-limiting, exemplary graph of distance (A) between Val30 methyl group and 2 furane ring.

[0111] FIG. 77D shows a non-limiting, exemplary graph of distance (A) between Arg98 NH and 2 phenyl ring.

[0112] FIG. 78 shows non-limiting, exemplary images of representative pose(left) of 2 (blue) in the most populated cluster of ALDHA (1110) subunit B (tan), and superposition of the representative pose of 2 (blue) in the most populated cluster of / vLDHA (1110) subunit B (tan) and the frame 0 2 (pink)-ALDHA dimeric ensemble (grey) that started the MD simulation(right). Hydrogen bonds are represented by dashed yellow lines.

[0113] FIG. 79A shows a non-limiting, exemplary graph of distribution of 3D polar surface area (3D-PSA) values for conformers in water and chloroform.

[0114] FIG. 79B shows a non-limiting, exemplary graph of distribution of radius of gyration (Rgyr) for conformers in water and chloroform.

[0115] FIG. 79C shows a non-limiting, exemplary graph of distribution of intramolecular hydrogen bonds (IMHBs) per conformer in water (blue) and chloroform (red).

[0116] FIG. 80 shows a non-limiting, exemplary image of superposition of the centroid conformers of compound 2 representing the closed (orange), semi-closed (green) and open (cyan) states identified by k-means clustering.

[0117] FIG. 81 A shows a non-limiting, exemplary graph of plasmatic levels of glucose. Male Agxtl- / - mice were orally administered with either vehicle (0.5% methyl cellulose) (n = 10) or 2 (20mg / kg body weight) (n = 10) daily for 10 days.

[0118] FIG. 81B shows a non-limiting, exemplary graph of body weight. Male Agxtl- / - mice were orally administered with either vehicle (0.5% methyl cellulose) (n = 10) or 2 (20mg / kg body weight) (n = 10) daily for 10 days.

[0119] FIG. 81C shows a non-limiting, exemplary graph of liver weight. Male Agxtl- / - mice were orally administered with either vehicle (0.5% methyl cellulose) (n = 10) or 2 (20mg / kg body weight) (n = 10) daily for 10 days.

[0120] FIG. 81D shows a non-limiting, exemplary graph of liver to body weight ratio. Male Agxtl- / - mice were orally administered with either vehicle (0.5% methyl cellulose) (n = 10) or 2 (20mg / kg body weight) (n = 10) daily for 10 days.

[0121] FIG. 82 - FIG. 105 show non-limiting, exemplary graphs of embodiments showing nonlinear “log (cone.) vs relative ALDHA activity” plot (media of four replicates)(left), and non-linear “log (cone.) vs relative ALDHA activity” plots of the four replicates (using ten concentrations of inhibitor and 180 pM pyruvate)(right). Error bars show standard deviation.

[0122] FIG. 106 shows non-limiting, exemplary Western blots and graphs indicating the proteolytic activity of compound 26. Agxt" primary hepatocytes were isolated and treated with either vehicle (DMSO) or increasing concentrations of compound 26 (2, 10, 20 and 50 pM) for 24 h. Panel A. GO protein abundance and quantification relative to 0-Actin (n = 4). Panel B. LDHA protein abundance and quantification relative to P-Actin (n = 4). Data are expressed as mean ± SEM. Normality was assessed using the Shapiro-Wilk test. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test.

[0123] FIG. 107 shows a schematic of compounds their respective ICso GO and ICso LDHA values.

[0124] FIG. 108 shows non-limiting, exemplary graphs of diet-induced MASH in atheroprone mice. Male (n=10-12 / group) Ldlr- / - mice were fed a standard diet (SD), Western diet (WD), modified choline-deficient high-fat diet (mCDHFD), or modified MASH diet (mMASHD) for 24 weeks. Gross and liver morphology, Liver-to-body weight ratio, Plasma ALT and AST. Data are mean±SEM. One-way ANOVA with Tukey’s post hoc test.

[0125] FIG. 109 shows non-limiting, exemplary graphs of effects of MASH-inducing diets on atherosclerosis in atheroprone mice. En face analysis of lesions along the aortic tree in male Ldlr- / - mice (n=10-12 / group). H& E, Oil Oed O and Van Gieson staining, in aortic sinuses (scale bars, 200 pm).

[0126] FIG. 110 shows non-limiting, exemplary graphs illustrating the effects of FAB-573 in a MASH and atherosclerosis mouse model. Male Ldlr- / - mice (n=13 / group) were fed a WD for 24 weeks. After the first 12 weeks, animals were orally administered with either vehicle (0.5% methyl cellulose) or FAB-573 (lOmg / kg body weight) once daily for the remaining 12 weeks. Parameters measured included body weight, liver weight, liver to body weight, blood glucose, plasma ALT and AST activity, plasma triglycerides and total cholesterol levels, as well as Oil Red O positive area in the aortic sinus, indicative of lipid deposition within atherosclerotic plaques.

[0127] FIG. Ill shows non-limiting, representative images illustrating the effects of FAB-573 in a MASH and atherosclerosis mouse model. Male Ldlr- / - mice (n=13 / group) were fed a WD for 24 weeks. After the first 12 weeks, animals were orally administered with either vehicle (0.5% methyl cellulose) or FAB-573 (lOmg / kg body weight) once daily for the remaining 12 weeks. The images depict gross morphology of the thoracic cavity and isolated liver, as well as Oil Red O staining of the aortic sinus (scale bars, 100 pm).

[0128] FIG. 112 shows non-limiting, exemplary graphs illustrating the effects of FAB-596 in a MASH and atherosclerosis mouse model. Male Ldlr- / - mice (n=13 / group) were fed a WD for 24 weeks. After the first 12 weeks, animals were orally administered with either vehicle (0.5% methyl cellulose) or FAB-596 (lOmg / kg body weight) once daily for the remaining 12 weeks. Parameters measured included body weight, liver weight, liver to body weight, blood glucose, plasma ALT and AST activity, plasma triglycerides and total cholesterol levels, as well as Oil Red O positive area in the aortic sinus, indicative of lipid deposition within atherosclerotic plaques.

[0129] FIG. 113 shows non-limiting, representative images illustrating the effects of FAB-596 in a MASH and atherosclerosis mouse model. Male Ldlr- / - mice (n=13 / group) were fed a WD for 24 weeks. After the first 12 weeks, animals were orally administered with either vehicle (0.5% methyl cellulose) or FAB-596 (lOmg / kg body weight) once daily for the remaining 12 weeks. The images depict gross morphology of the thoracic cavity and isolated liver, as well as Oil Red O staining of the aortic sinus (scale bars, 100 pm).

[0130] FIG. 114 shows non-limiting, exemplary graphs illustrating the effects of FAB-599 in a MASH and atherosclerosis mouse model. Male Ldlr- / - mice (n=13 / group) were fed a WD for 24 weeks. After the first 12 weeks, animals were orally administered with either vehicle (0.5% methyl cellulose) or FAB-599 (lOmg / kg body weight) once daily for the remaining 12 weeks. Parameters measured included body weight, liver weight, liver to body weight, blood glucose, plasma ALT and AST activity, plasma triglycerides and total cholesterol levels, as well as Oil Red O positive area in the aortic sinus, indicative of lipid deposition within atherosclerotic plaques.

[0131] FIG. 115 shows non-limiting, representative images illustrating the effects of FAB-599 in a MASH and atherosclerosis mouse model. Male Ldlr- / - mice (n=13 / group) were fed a WD for 24 weeks. After the first 12 weeks, animals were orally administered with either vehicle (0.5% methyl cellulose) or FAB-599 (lOmg / kg body weight) once daily for the remaining 12 weeks. The images depict gross morphology of the thoracic cavity and isolated liver, as well as Oil Red O staining of the aortic sinus (scale bars, 100 pm).

[0132] FIG. 116 shows exemplary graphs indicating the systemic effects of compound 2 on LDH activity in mice. Male^grf " mice were orally administered either vehicle (0.6% methylcellulose and 0.5% Tween® 80 in water; n = 10) or compound 2 (20 mg / kg body weight; n = 10) daily for 10 days. LDH activity in gastrocnemius muscle LDH activity in kidney. LDH activity in heart. Data are presented as mean ± SEM. Normality was assessed using the Shapiro-Wilk test. Comparisons between two groups were performed using Student’s / -test when data met normality assumptions, or the Mann-Whitney test when normality was not assumed.

[0133] FIG. 117 shows a non-limiting, exemplary graph Comparison of intracellular and systemic distribution levels of compound 2 following treatment. Agxt primary hepatocytes were isolated and treated with compound 2 (50 pM, 24 h; n = 4) and male Agxf ' mice were orally administered compound 2 (20 mg / kg body weight, n = 10) daily for 10 days. Tissues were harvested and homogenized in methanol, and compound 2 concentrations were quantified by UPLC -Orbitrap HRMS / MS and normalized to protein concentration. Data are presented as mean ± SEM. Normality was assessed using the Shapiro-Wilk test, and group comparisons were performed using the Kruskal-Wallis test.DETAILED DESCRIPTION OF THE INVENTION

[0134] Aspects described herein provide for compositions and methods for treating an oxalate-related diseases and disorders.

[0135] Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the invention can be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the invention in any appropriate manner.

[0136] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0137] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.

[0138] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0139] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.

[0140] As used herein, the term “about” can refer to approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower). In embodiments, the term “about” can be denoted

[0141] As used herein, the term “substantially the same” or “substantially” can refer to variability typical for a particular method is taken into account.

[0142] The terms “sufficient” and “effective”, as used interchangeably herein, can refer to an amount (e.g., mass, volume, dosage, concentration, and / or time period) needed to achieve one or more desired result(s).

[0143] Before explaining at least one embodiment of the disclosure in detail, it is to be understood that the disclosure is not necessarily limited in its application to the details set forth in the following description or exemplified by the examples. The disclosure can be used for other embodiments or of being practiced or carried out in various ways. Other compositions, compounds, methods, features, and advantages of the disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. All such additional compositions, compounds, methods, features, and advantages can be included within this description, and be within the scope of the disclosure.

[0144] The term "alkyl" refers to the radical of saturated aliphatic groups, including straightchain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.

[0145] In some embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3-C30 for branched chains), 20 or fewer, 12 or fewer, or 7 or fewer. Likewise, in some embodiments cycloalkyls have from 3-10 carbon atoms in their ring structure, e.g., have 5, 6 or 7 carbons in the ring structure. The term "alkyl" (or "lower alkyl") as used throughout the specification, examples, and claims can include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a hosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.

[0146] Unless the number of carbons is otherwise specified, "lower alkyl" as used herein can refer to an alkyl group, as defined herein, but having from one to ten carbons, or from one to six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" havesimilar chain lengths. In some embodiments, alkyl groups are lower alkyls. In some embodiments, a substituent described herein as alkyl can be a lower alkyl.

[0147] It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl can include halogen, hydroxy, nitro, thiols, amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like. Cycloalkyls can be substituted in the same manner.

[0148] The term “heteroalkyl”, as used herein, refers to straight or branched chain, or cyclic carbon-containing radicals, or combinations thereof, containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quatemized. Heteroalkyls can be substituted as defined herein for alkyl groups.

[0149] The term "alkylthio" refers to an alkyl group, as defined herein, having a sulfur radical attached thereto. In some embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl. Representative alkylthio groups include methylthio, and ethylthio. The term “alkylthio” also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups. “Arylthio” refers to aryl or heteroaryl groups. Alkylthio groups can be substituted as defined herein for alkyl groups.

[0150] The terms "alkenyl" and "alkynyl", refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described herein, but that contain at least one double or triple bond respectively. For example,

[0151] The terms "alkoxyl" or "alkoxy" as used herein refers to an alkyl group, as defined herein, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, and tert-butoxy. An "ether," for example, can be two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O-alkenyl, and -O-alkynyl. Aroxy can be represented by -O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined herein. The alkoxy and aroxy groups can be substituted as described herein for alkyl.

[0152] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula:wherein R9, Rio, and Rio’ each independently represent a hydrogen, an alkyl, an alkenyl, -(CHa)™-Rs or R9 and Rio taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; Rs represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In some embodiments, only one of R9 or Rio can be a carbonyl, e.g., R9, Rio and the nitrogen together do not form an imide. In still other embodiments, the term “amine” does not encompass amides, e.g., wherein one of R9 and Rio represents a carbonyl. In additional embodiments, R9 and Rio (and optionally Rio ) each independently represent a hydrogen, an alkyl or cycloalkyl, an alkenyl or cycloalkenyl, or alkynyl. Thus, the term "alkylamine" as used herein can refer to an amine group, as defined herein, having a substituted (as described hereinfor alkyl) or unsubstituted alkyl attached thereto, i.e., at least one of R9 and Rio is an alkyl group.

[0153] As used herein, the term “imide” can refer to -C(O)NR’R”, wherein R’ and R” are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein. As used herein, the term “halogen” can refer to -F, -Cl, -Br or -I; the term "sulfhydryl" can refer to -SH; the term "hydroxyl" can refer to -OH; and the term "sulfonyl" can refer to -SO2-.

[0154] The term “substituted” as used herein, refers to permissible substituents of the compounds described herein. In the broadest sense, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, for example 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats. Representative substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic, substituted C3-C20 cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide groups. As used herein in reference to an “R” group, the name used to describe said “R” group can be the chemical name prior to the removal of a hydrogen. For example, wherein “R” is described as an “alkane” can refer to an “alkyl” group.

[0155] Heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is understood that “substitution” or “substituted” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0156] In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein. The permissible substituents can be one or more and the same or different for appropriate organic compounds. The heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms.

[0157] In various aspects, the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, each of which optionally is substituted with one or more suitable substituents. In some embodiments, the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, wherein each of the alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone can be further substituted with one or more suitable substituents.

[0158] Examples of substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, thioketone, ester, heterocyclyl, -CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroaralkoxy, azido, alkylthio, oxo, acylalkyl, carboxy esters, carboxamido, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl, arylamino, aralkylamino, alkylsulfonyl, carboxamidoalkylaryl, carboxamidoaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy, aminocarboxamidoalkyl, cyano, alkoxyalkyl, perhaloalkyl, arylalkyloxyalkyl, and the like. In some embodiments, the substituent is selected from cyano, halogen, hydroxyl, and nitro.

[0159] Aspects of the invention are drawn to a compound of Formula (I), Formula (II), Formula (III), or a pharmaceutically acceptable salt, prodrug, hydrate, or solvate thereof:Formula (I)wherein A-B is selected from -CH2-CH2-, -CH=CH- or -C=C-; C is selected from -CH2- or -CO-; D is selected from the group consisting of -CH2, -O-, or -NH-; E is selected from the group consisting of -CH2, -O-, or -NH-; F is selected from the group consisting of -O-, -NH-, -CO-NH-or -NFI-CO-; G = is selected from -CH2-, -CO- or -CO-CH2-; H-I is selected from -CH2-CH2- or -CH=CH-; m is 0 or 1; n is 0 or 1; o is 0, 1, 2, or 3; and p is 0, 1, 2, 3, or 4; or(Formula II)wherein A-B is selected from -CH2-CH2-, -CH=CH- or -C=C- C is selected from -CH2- or -CO-; D is selected from CH or N; E is selected from -CH2 or -CO-; F is selected from CH or N; G = is selected from -CH2-, -CO- or -CO-CH2-; H-I is selected from -CH2-CH2- or -CH=CH-.(Formula III)wherein A-B is selected from -CH2-CH2-, -CH=CH- or -C=C- C is selected from -CH2- or -CO-; D is selected from the group consisting of -CH2, -O-, or -NH-; E is selected from the group consisting of -CH2, -O-, or -NH-; H-I is selected from -CH2-CH2- or -CH=CH-; m is 0 or 1; n is 0 or 1; 0 is 0, 1, 2, or 3; and p is 0, 1, 2, 3, or 4. In some embodiments, either D = -O-, E = -O-, or both D = -O- and E = -O-.

[0160] In embodiments, the compound is:

[0161] As used herein, “FAB-599” can refer to “Compound 2,” and can be used interchangeably. Additionally, as used herein, “FAB-596” can refer to “Compound 26,” and can be used interchangeably.

[0162] As used herein, “FAB-583” can refer to “Compound 23,” and can be used interchangeably. Additionally, as used herein, “FAB-563” can refer to “Compound 20.”

[0163] Aspects of the invention are drawn to a compound of Formula (IV), Formula (V), Formula (VI), or a pharmaceutically acceptable salt, prodrug, hydrate, or solvate thereofFormula (IV)wherein A-B is selected from -CH2-CH2-, -CH=CH- or -C=C- C is selected from -CH2- or -CO-; D is selected from the group consisting of -CH2, -O-, or -NH-; E is selected from the group consisting of -CH2, -O-, or -NH-; F is selected from the group consisting of -O-, -NH-, -CO-NH-or -NH-CO-; G = is selected from -CH2-, -CO- or -CO-CH2-; H-I is selected from -CH2-CH2- or -CH=CH-; m is 0 or 1; n is 0 or 1; o is 0, 1, 2, or 3; and p is 0, 1, 2, 3, or 4; or(Formula V)wherein A-B is selected from -CH2-CH2-, -CH=CH- or -C=C-; C is selected from -CH2- or -CO-; D is selected from CH or N; E is selected from -CH2 or -CO-; F is selected from CH or N; G = is selected from -CH2-, -CO- or -CO-CH2-; H-I is selected from -CH2-CH2- or -CH=CH-.(Formula VI)wherein A-B is selected from -CH2-CH2-, -CH=CH- or -C=C-, C is selected from -CH2- or -CO-; D is selected from the group consisting of -CH2, -O-, or -NH-; E is selected from the group consisting of -CH2, -O-, or -NH-; H-I is selected from -CH2-CH2- or -CH=CH-; m is 0 or 1; n is 0 or 1; o is 0, 1, 2, or 3; and p is 0, 1, 2, 3, or 4. In some embodiments, C, D, E, or any combination thereof are -O-.

[0164] In embodiments, the compound is:

[0165] In embodiments, the pharmaceutically acceptable salt is an inorganic salt or an organic salt. Pharmaceutically acceptable salts can include, but are not limited to, amine salts, such as but not limited to N, N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, ammonium, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1 -para-chlorobenzyl-2-pyrrolidin- 1 '-ylmethylbenzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl) aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of mineral acids, such as but not limited to hydrochlorides, hydrobromides, phosphates, bicarbonates, bisulfates, and sulfates; and also including, but not limited to salts of sulfonates, such as but not limited to mesylates, tosylates, besylates, and camphrosulfonates; and also but not limited to, salts of organic acids, such as but not limited to acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates, formates, maleates, glutarates, and fumarates.

[0166] In some embodiments, the inorganic salt is selected from an ammonium salt, a sodium salt, a potassium salt, a lithium salt, a calcium salt, a magnesium salt, an aluminum salt, a zinc salt, a ferrous salt, a ferric salt, a hydrochloride salt, a hydrobromide salt, a bisulfate salt, a phosphate salt, or a bicarbonate salt.

[0167] In some embodiments, the organic salt a mesylate salt, a tosylate salt, a besylate salt, a camphrosulfonate salt, an acetate salt, a formate salt, a maleate salt, a succinate salt, a glutarate salt, a tartrate salt, a citrate salt, a malate salt, or a fumarate salt.

[0168] Compounds of those described herein, for example, can exist in the form of salts, for example acid addition salts or, in certain cases salts of organic and inorganic bases such as phenolate, carboxylate, sulphonate and phosphate salts. All such salts are within the scope of this disclosure.

[0169] The salts of the disclosure can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties. Selection, and Use, P. Heinrich Stahl (ed), Camille G. Wermuth (ed), ISBN:3-90639-026-8, Hardcover, 388 pages, August 2002. In embodiments, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; for example, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.

[0170] Aspects of the invention are drawn towards a pharmaceutical composition comprising a compound described herein or combination of compounds, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0171] In embodiments, the excipient comprises methyl cellulose. In some embodiment, the pharmaceutical composition can further comprise polysorbate 80.

[0172] In embodiments, the pharmaceutical composition can comprise an adjuvant, an additional active agent, or a combination thereof. In embodiments, the adjuvant can comprise an amino acid, a polyol, or a combination thereof. In embodiments, the amino acid is selected from the group consisting of L-Alanine, L-Arginine, L-Lysine, L-Histidine, L-Ornitine, L-Serine, L-Valine, L-Leucine, L-Proline, or Glycine. In embodiments, the polyol is selected from N-methyl-D-glucamine, tromethamine, choline, glucosamine, or diethanolamine.

[0173] In some embodiments, the pharmaceutical composition comprises an equimolar ratio to about 50 wt. % of either an amino acid or a polyol described herein with a compound described herein.

[0174] In some embodiments, the pharmaceutical composition comprises an equimolar ratio to about 50 % by wt. of either L- Alanine, L-Arginine, L-Lysine, L-Histidine, L-Ornitine, L-Serine, L-Valine, L-Leucine, L-Proline, or Glycine and a compound of Formula I, Formula II, or Formula III. In embodiments, the pharmaceutical composition comprises an equimolar ratio to about 50 % by wt. of either L- Alanine, L-Arginine, L-Lysine, L-Histidine, L-Ornitine, L-Serine, L-Valine, L-Leucine, L-Proline, or Glycine and FAB-599. In embodiments, the pharmaceutical compositioncomprises an equimolar ratio to about 50 % by wt. of either L-Alanine, L-Arginine, L-Lysine, L-Histidine, L-Ornitine, L-Serine, L-Valine, L-Leucine, L-Proline, or Glycine and FAB-596.

[0175] In some embodiments, the pharmaceutical composition comprises an equimolar ratio to about 50 % by wt. of either N-methyl-D-glucamine, tromethamine, choline, glucosamine, or diethanolamine and a compound of Formula I, Formula II, or Formula III. In some embodiments, the pharmaceutical composition comprises an equimolar ratio to about 50 % by wt. of either N-methyl-D-glucamine, tromethamine, choline, glucosamine, or diethanolamine and FAB-599. In some embodiments, the pharmaceutical composition comprises an equimolar ratio to about 50 % by wt. of either N-methyl-D-glucamine, tromethamine, choline, glucosamine, or diethanolamine and FAB-596.

[0176] In embodiments, the pharmaceutical composition can further comprise an additional active ingredient. In embodiments, the additional active ingredient can comprise a vitamin B6 derivative. In some embodiments, the vitamin B6 derivative is selected from the group consisting of pyridoxinium, pyridoxamine, piridoxal, or nicotinamide. The vitamin B6 derivatives can be a cofactor of AGXT. Without wishing to be bound by theory, vitamin B6 derivatives can be used in combination with the compounds described herein to treat Primary Hyperoxaluria. In some nonlimiting, exemplary embodiments, the vitamin B6 derivatives can act to reduce oxalate production in Hyperoxaluria Type 1. In embodiments, the pharmaceutical composition comprises a composition described herein and about 2 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg of a vitamin B6 derivative. In embodiments, the pharmaceutical composition comprises about 2 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg of a vitamin B6 derivative described herein.

[0177] In embodiments, the pharmaceutical composition comprises a compound described herein and about 2 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg of a vitamin B6 derivative. In embodiments, the pharmaceutical composition comprises about 2 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg of either pyridoxinium, pyridoxamine, piridoxal, or nicotinamide.

[0178] In embodiments, the pharmaceutical composition comprises a composition of Formula I and about 2 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg of a vitamin B6 derivative. In embodiments, the pharmaceutical composition comprises a composition of Formula I and about 2 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg of either pyridoxinium, pyridoxamine, piridoxal, or nicotinamide.

[0179] In embodiments, the pharmaceutical composition comprises FAB-599 and about 2 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg of a vitamin B6 derivative. In embodiments, the pharmaceutical composition comprises FAB-599 and about 2 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg of either pyridoxinium, pyridoxamine, piridoxal, or nicotinamide.

[0180] In embodiments, the pharmaceutical composition comprises FAB-596 and about 2 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg of a vitamin B6 derivative. In embodiments, the pharmaceutical composition comprises FAB-596 and about 2 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg of either pyridoxinium, pyridoxamine, piridoxal, or nicotinamide.

[0181] Pharmaceutically acceptable esters can include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids, and boronic acids.

[0182] Pharmaceutically acceptable enol ethers can include, but are not limited to, derivatives of formula C=C(OR) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, or heterocyclyl. Pharmaceutically acceptable enol esters can include, but are not limited to, derivatives of formula C=C(OC(O)R) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or cycloalkyl aralkyl heterocyclyl.

[0183] Pharmaceutically acceptable solvates and hydrates are complexes of a compound with one or more solvent or water molecules, 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules.

[0184] A reference to a compound of the disclosure and sub-groups thereof also includes ionic forms, salts, solvates, isomers, tautomers, esters, prodrugs, isotopes and protected forms thereof, such as, the salts or tautomers or isomers or solvates thereof; and more advantageously, the salts or tautomers or solvates thereof. As used herein, the term “isomer” can refer to molecules or polyamtoic ions with identical molecular formulas, but distinct arrangements of atoms in space. For example, constitutional isomers and stereoisomers of compounds described herein are also embodiments of the invention. For example, the enantiomers and diastereomers of compounds described herein can be aspects of the invention. For example, if (R, S) of a compound is described herein, (R, R), (S, R), and (S, S) can also be aspects of the invention. As used herein, the term “enantiomer” can refer to molecules which are nonsuperimposable mirror images of each other. As used herein, the term “diastereomer” can refer to a stereoisomer of a compound having two or more chiral centers that is not a mirror image of another stereoisomer of the same compound.

[0185] While the compounds described herein can be administered without formulation, they can also be formulated as pharmaceutical compositions. Aspects of the invention are drawn towards a pharmaceutical composition comprising a compound of any one of the compounds described herein, or a combination thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0186] The formulations or pharmaceutical composition can also be included, or packaged, with other non-toxic compounds, such as pharmaceutically acceptable carriers, excipients, binders and fdlers including, but not limited to, glucose, lactose, gum acacia, gelatin, mannitol, xanthan gum, locust bean gum, galactose, oligosaccharides and / or polysaccharides, starch paste, magnesium trisilicate, talc, com starch, starch fragments, keratin, colloidal silica, potato starch, urea, dextrans, dextrins, and the like. For example, the pharmaceutically acceptable carriers, excipients, binders, and fillers for use in the practice of the present invention are those which render the compounds of the invention amenable to intranasal delivery, oral delivery, parenteral delivery, intravitreal delivery, intraocular delivery, ocular delivery, subretinal delivery, intrathecal delivery, intravenous delivery, subcutaneous delivery, transcutaneous delivery, intracutaneous delivery, intracranial delivery, topical delivery and the like. Moreover, the packaging material can be biologically inertor lack bioactivity, such as plastic polymers or silicone, and can be processed internally by the subject without affecting the effectiveness of the composition / formulation packaged and / or delivered therewith.

[0187] In embodiments, compounds and compositions described herein can be administered via oral administration. In some embodiments, the disclosed compositions are formulated in a pharmaceutically acceptable oral dosage form. Oral dosage forms can comprise oral liquid dosage forms (such as tinctures, drops, emulsions, syrups, elixirs, suspensions, and solutions, and the like) and oral solid dosage forms. The pharmaceutical compositions can also be prepared as formulations suitable for parenteral administration, intramuscular, subcutaneous, intraperitoneal, or intravenous injection, comprising physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, liposomes, and sterile powders for reconstitution into sterile injectable solutions or dispersions.

[0188] “Parenteral administration” can refer to administration via injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, intramuscular administration. For example, compounds and compositions described herein can be administered via intraperitoneal injection (I. P.), intravenous injection (I. V.), and intramuscular injection (I. M.).

[0189] For oral preparations, the composition or pharmaceutical composition can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as com starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[0190] Oral solid dosage can comprise lozenges, troches, tablets, capsules, caplets, powders, pellets, multiparticulates, beads, spheres, and / or any combinations thereof. Oral solid dosage forms can be formulated as immediate release, controlled release, sustained release, extended release, or modified release formulations. Accordingly, in some embodiments, the disclosed oral solid dosage forms can be in the form of a tablet (including a suspension tablet, a fast-melt tablet, a bitedisintegration tablet, a rapid-disintegration tablet, an effervescent tablet, or a caplet), a pill, a powder (including a sterile packaged powder, a dispensable powder, or an effervescent powder),a capsule (including both soft or hard capsules, e.g., capsules made from animal-derived gelatin or plant-derived HPMC, or “sprinkle capsules”), solid dispersion, solid solution, bioerodible dosage form, controlled release formulations, pulsatile release dosage forms, multiparticulate dosage forms, pellets, granules, or an aerosol. In other embodiments, the pharmaceutical formulation can be in the form of a powder. In still other embodiments, the pharmaceutical formulation can be in the form of a tablet, including a fast-melt tablet. Additionally, pharmaceutical formulations can be administered as a single capsule or in multiple capsule dosage form. In some embodiments, the pharmaceutical formulation can be administered in two, three, four, or more capsules or tablets.

[0191] Oral solid dosage forms can contain pharmaceutically acceptable excipients such as fdlers, diluents, lubricants, surfactants, glidants, binders, dispersing agents, suspending agents, disintegrants, viscosity-increasing agents, film-forming agents, granulation aid, flavoring agents, sweetener, coating agents, solubilizing agents, and combinations thereof. Oral solid dosage forms also can comprise one or more pharmaceutically acceptable additives such as a compatible carrier, complexing agent, ionic dispersion modulator, disintegrating agent, surfactant, lubricant, colorant, moistening agent, plasticizer, stabilizer, penetration enhancer, wetting agent, anti-foaming agent, alone or in combination, as well as supplementary active compound(s).

[0192] Oral liquid dosage forms include tinctures, drops, emulsions, syrups, elixirs, suspensions, and solutions, and the like. These oral liquid dosage forms can be formulated with any pharmaceutically acceptable excipient known to those of skill in the art for the preparation of liquid dosage forms, and with solvents, diluents, carriers, excipients, and the like chosen as appropriate to the solubility and other properties of the active agents and other ingredients. Solvents can be, for example, water, glycerin, simple syrup, alcohol, medium chain triglycerides (MCT), and combinations thereof.

[0193] Liquid dosage forms for oral administration can be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions, which can contain an inactive diluent, such as water. Pharmaceutical formulations can be prepared as liquid suspensions or solutions using a sterile liquid, such as but not limited to, an oil, water, an alcohol, and combinations of these pharmaceutically suitable surfactants, suspending agents, emulsifying agents, can be added for oral or parenteral administration. Liquid formulations also can be prepared as single dose or multi-dose beverages. Suspensions can include oils. Such oils include peanut oil, sesame oil, cottonseed oil, corn oil, and olive oil. Suitable oils also include carrier oils such asMCT and long chain triglyceride (LCT) oils. Suspension preparation can also contain esters of fatty acids such as ethyl oleate, isopropyl myristate, fatty acid glycerides, and acetylated fatty acid glycerides. Suspension formulations can include alcohols, (such as ethanol, isopropyl alcohol, hexadecyl alcohol), glycerol, and propylene glycol. Ethers, such as poly(ethylene glycol), petroleum hydrocarbons such as mineral oil and petrolatum, and water can also be used in suspension formulations. Suspension can thus include an aqueous liquid or a non-aqueous liquid, an oil-in-water liquid emulsion, or a water-in-oil emulsion.

[0194] In some embodiments, formulations are provided comprising the disclosed compositions and at least one dispersing agent or suspending agent for oral administration to a subject. The formulation can be a powder and / or granules for suspension, and upon admixture with water, a substantially uniform suspension is obtained. The aqueous dispersion can comprise amorphous and non-amorphous particles consisting of multiple effective particle sizes such that a drug is absorbed in a controlled manner over time.

[0195] Supplementary active compounds include preservatives, antioxidants, antimicrobial agents including biocides and biostats such as antibacterial, antiviral and antifungal agents. Preservatives can be used to inhibit microbial growth or increase stability of the active ingredient thereby prolonging the shelf life of the formulation. Suitable preservatives are known in the art and include EDTA, EGTA, benzalkonium chloride or benzoic acid or benzoates, such as sodium benzoate. Antioxidants include vitamin A, vitamin C (ascorbic acid), vitamin E, tocopherols, other vitamins or provitamins, and compounds such as alpha lipoic acid.

[0196] Unit dosage forms for oral administration, such as syrups, elixirs, and suspensions, can be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contain a predetermined amount of the composition containing one or more compositions. Similarly, unit dosage forms for injection or intravenous administration can comprise the composition or pharmaceutical composition in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

[0197] Embodiments of the composition or pharmaceutical composition can be formulated into preparations for injection by dissolving, suspending, or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[0198] Embodiments of the composition or pharmaceutical composition can be utilized in aerosol formulation to be administered via inhalation. Embodiments of the composition or pharmaceutical composition can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

[0199] Embodiments of the composition or pharmaceutical composition can be formulated in an injectable composition in accordance with the disclosure. For example, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation can also be emulsified or the active ingredient (triamino-pyridine derivative and / or the labeled triamino-pyridine derivative) encapsulated in liposome vehicles in accordance with the disclosure.

[0200] In certain non-limiting, exemplary embodiments, any one of the compositions or pharmaceutical compositions described herein can be formulated with an appropriate vehicle. As used herein, the term “vehicle” can refer to an inert or inactive substance that serves as the carrier or medium in which the API is dissolved, suspended, or dispersed for administration to a patient in need thereof.

[0201] For example, a non-limiting vehicle formulation can comprise a polymeric component, a surfactant, water, or combinations thereof. In some non-limiting, exemplary embodiments the vehicle formulation described herein can comprise methyl cellulose, polyoxymethylene sorbitan monooleate, and molecular biology grade water. In some embodiments, the methyl cellulose can comprise a concentration of about 0.6 wt. %, the polyoxymethylene sorbitan monooleate can comprise a concentration of about 0.5 wt. %, and the molecular biology grade water can comprise a concentration of about 98.9 wt. %. In some embodiments the preparation of the vehicle mentioned herein can comprise; measuring about 240 mg of methyl cellulose into a 50 mb Falcon tube, adding 20 mL of water to the Falcon tube, vortexing the mixture until the methyl cellulose is dispersed, allowing the mixture to hydrate overnight in a water bath held at a temperature of about 40 °C, adding about 189 pL of polyoxymethylene sorbitan monooleate, then adjusting the final volume of the vehicle to about 40 mL.

[0202] In some non-limiting, exemplary embodiments the methyl cellulose can comprise a viscosity of about 400 cP and contain about 27.0% to about 32.0% methoxyl content. In some nonlimiting exemplary embodiments, the polymer component of the vehicle formulation describedherein can comprise a polymer with a viscosity of more than about 400 cP. In some embodiments, the polymeric component can comprise hydroxypropyl methylcellulose (HPMC),[002031 In some non-limiting, exemplary embodiments, any one of the compositions or pharmaceutical compositions described herein, can be formulated with the vehicle described herein by sonicating, until all visible clumps are dispersed, about 5mg to about 10 mg of any one of the compositions, or pharmaceutical compositions described herein, with about 70 % of the total volume of vehicle formulation, adjusting the pH to 7 by adding IM sodium hydroxide under stirring, then adjusting the final volume of the formulation with addition vehicle formulation. In some non-limiting, exemplary embodiments adjusting the pH to 7 by adding IM sodium hydroxide can produce a color change from orange to red. In some non-limiting, exemplary embodiments the solubility of the formulation described herein can comprise a solubility of about 2 mg / mL. In some non-limiting, exemplary embodiments, the solution is stable at room temperature for 24 hours.

[0204] In an embodiment, the composition or pharmaceutical composition can be formulated for delivery by a continuous delivery system. The term “continuous delivery system" is used interchangeably herein with "controlled delivery system” and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art.

[0205] Embodiments of the composition or pharmaceutical composition can be administered to a subject in one or more doses. Those of skill will readily appreciate that dose levels can vary as a function of the specific composition or pharmaceutical composition administered, the severity of the symptoms and the susceptibility of the subject to side effects. Dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

[0206] In an embodiment, multiple doses of the composition or pharmaceutical composition are administered. The frequency of administration of the composition or pharmaceutical composition can vary depending on any of a variety of factors, e.g., severity of the symptoms, and the like. For example, in an embodiment, the composition or pharmaceutical composition can be administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (ad), twice a day (qid), three times a day (tid), orfour times a day. As discussed above, in an embodiment, the composition or pharmaceutical composition is administered 1 to 4 times a day over a 1 to 10-day time period.

[0207] The duration of administration of the composition or pharmaceutical composition analogue, e.g., the period of time over which the composition or pharmaceutical composition is administered, can vary, depending on any of a variety of factors, including patient response. For example, the composition or pharmaceutical composition in combination or separately, can be administered over a period of time of about one day to one week, about one day to two weeks.

[0208] Embodiments of the composition or pharmaceutical composition can be administered to a subject using available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. Routes of administration can include, but are not limited to, enteral administration, parenteral administration, or inhalation.

[0209] Other compositions, compounds, methods, features, and advantages of the disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. It is intended that all such additional compositions, compounds, methods, features, and advantages be included within this description, and be within the scope of the disclosure.

[0210] Aspects of the invention are drawn towards methods of inhibiting the activity of glycolate oxidase (GO) and lactate dehydrogenase (LDHA) while activating degradation of glycolate oxidase (GO) and / or lactate dehydrogenase (LDHA) in a subject. In embodiments, the method comprises administering a therapeutically effective amount of a compound or pharmaceutical composition described herein.

[0211] In embodiments, the compounds described herein possess Small Molecule Mediated Protein Degradation (SMPD) activity. SMPD activity, as used herein, can refer to the ability of compounds to induce selective intracellular proteolysis. This process can be mediated through organelles such as the proteasome, endolysosome, or a combination thereof. Non-limiting mechanisms of SMPD activity can comprise proteolysis targeting chimera (PROTAC), molecular glue, ubiquitin independent degraders (UID), autophagy tethering compound (ATTEC), and autophagy targeting chimera (AUTOTAC). For example, a PROTAC molecule can refer to a molecule that degrades a target protein by hijacking the ubiquitin-proteasome system. For example, the molecule can be a heterobifunctional molecule comprising two ligands joined by a linker. For example, one of the two ligands can recruit and bind a protein of interest (POI), andone ligand can bind an E3 ubiquitin ligase. In some embodiments, the compounds can be primary alkylamines. (See, e.g., C. Kagiou, et al., Alkylamine-Tethered Molecules Recruit FBXO22 for Targeted Protein Degradation. Nat Commun 2024, 15 (1), 5409). For example, a molecular glue can refer to a molecule that degrades a target protein by hij acking the ubiquitin-proteasome system. For example, a molecular glue molecule can tightly bind together the E3 ubiquitin ligase and the POI. (See, e.g., Z. Kozicka, et al., Haven't got a glue: Protein surface variation for the design of molecular glue degraders, Cell Chemical Biology, 2021, 28 1), 1032-1047). For example, a UID molecule, can refer to a structure that generates a direct engagement of 26S proteosome and the POI without the involvement of ubiquitin. For example, without wishing to be bound by theory, an UID molecule can direct the protein of interest towards the 19S RP regulatory region in the proteasome. For example, a ATTEC molecule can refer to a molecule that degrades a target protein through the formation of an endolysosome. For example, the molecule can create a complex between a LC3 ligand and the POI. (See, e.g., Z. Li, et al., Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds, Nature, 2019, 575, 203-209). For example, a AUTOTAC molecule can refer to a molecule that degrades a target protein through the formation of an endolysosome. For example, an AUTOTAC can be a heterobifunctional molecule comprising two ligands joined by a linker. For example, one of the two ligands can recruit and bind aprotein of interest (POI), and one ligand can bind to the ZZ domain of SQSTM1. (See, e.g., C. H. Ji, et al., Targeted protein degradation via the autophagy-lysosome system: AUTOTAC (AUTOphagy-TArgeting Chimera), Autophagy, 2022, 18, 2259-2262).

[0212] Aspects of the invention are drawn towards methods of treating a subject afflicted with an oxalate production-related disease or disorder. In embodiments, the method comprises: administering to the subject a therapeutically effective amount of a compound described herein.

[0213] In embodiments, the disease or disorder can comprise a cardiometabolic disease or disorder, a cardiovascular disease or disorder, a metabolic disease or disorder, a liver disease or disorder, a renal disease or disorder, or a combination thereof.

[0214] As used herein, a metabolic disease can refer to a group of diseases and / or disorders in which errors of metabolism, imbalances in metabolism, or sub-optimal metabolism occur. The metabolic diseases described herein can comprise diseases that can be treated through the modulation of metabolism, although the disease itself can by a specific metabolic defect. Such metabolic diseases can involve, for example, glucose and fatty acid oxidation pathways.

[0215] As used herein, "metabolic disorder" or "metabolic disease" refers to a condition characterized by an alteration or disturbance in metabolic function. "Metabolic" and "metabolism" are terms known in the art and can comprise a range of biochemical processes that occur within a living organism. Metabolic and cardiovascular disease includes, but is not limited to, obesity, diabetes, atherosclerosis, metabolic syndrome, dyslipidemia, coronary heart disease, coronary artery disease, arteriosclerosis, atherothrombotic stroke, metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction associated steatohepatitis (MASH), compensated and noncompensated cirrhosis secondary to MASLD / MASH, hyperfattyacidemia or metabolic syndrome, diver fibrosis, obesity, heart disease, atherosclerosis, arthritis, cystinosis, phenylketonuria, proliferative retinopathy, Kearns-Sayre disease, or a combination thereof. In embodiments, the dyslipidemia can be hyperlipidemia. The hyperlipidemia can be hypercholesterolemia, hypertriglyceridemia, or both hypercholesterolemia and hypertriglyceridemia. The MASLD can be hepatic steatosis or steatohepatitis. The diabetes can be type 2 diabetes or type 2 diabetes with dyslipidemia.

[0216] In some exemplary embodiments, the metabolic disease or disorder can be diabetes, dyslipidemia, obesity, hyperoxaluria, primary hyperoxaluria (PH), PHI, PH2, PH3, systemic oxalosis, or any combination thereof.

[0217] In embodiments, methods are presented herein that are applicable to metabolic diseases related to glucose dysregulation and / or accumulation of lipids in the body, circulation or various organs, for example, the liver, and the pathological sequelae resulting therefrom, for example, metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction associated steatohepatitis (MASH), hyperglycemia, prediabetes, diabetes (type I and type II), obesity, insulin resistance, metabolic syndrome and diabetic dyslipidemia. Certain such metabolic diseases, disorders or conditions include, but are not limited to, hyperglycemia, prediabetes, diabetes (type I and type II), obesity, insulin resistance, metabolic syndrome and diabetic dyslipidemia. In embodiments, the metabolic disease, disorder or condition can be characterized by numerous physical symptoms. Any symptom known to one of skill in the art to be associated with the metabolic disease, disorder or condition can be prevented, treated, ameliorated or otherwise modulated with the compounds and methods described herein. In certain embodiments, the symptom can be any of, but not limited to, excessive urine production (polyuria), excessive thirst and increased fluid intake (polydipsia), blurred vision, unexplained weight loss and lethargy.

[0218] As used herein, cardiovascular diseases, disorders or conditions can comprise aortic stenosis, aneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular disease, coronary artery disease, carotid artery disease, coronary heart disease, dyslipidemia, heart failure, hypercholesterolemia, hyperlipidemia, hypertension, hypertriglyceridemia, myocardial infarction, peripheral vascular disease (e.g., peripheral artery disease, peripheral artery occlusive disease), thromboembolic diseases, retinal vascular occlusion, stroke, atherosclerotic cardiovascular disease (ASCVD), coronary artery disease (CAD), acute coronary syndrome (ACS), myocardial infarction (MI), ischemic stroke, peripheral artery disease (PAD), carotid atherosclerotic disease, heart failure (e.g., heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF)), myocardial ischemia-reperfusion injury, or vascular calcification and arterial stiffening.

[0219] In some exemplary embodiments, the cardiovascular disease or disorder is hypertension, atherosclerotic cardiovascular disease (ASCVD), coronary artery disease (CAD), acute coronary syndrome (ACS), myocardial infarction (MI), ischemic stroke, peripheral artery disease (PAD), carotid atherosclerotic disease, hear failure, myocardial ischemia-reperfusion injury, vascular calcification, arterial stiffening, an aneurysm, or any combination thereof. For example, the aneurysm can be a cerebral aneurysm, an abdominal aortic aneurysm, a thoracic aneurysm, or a peripheral aneurysm.

[0220] As used herein, the term “liver disease” and “hepatic disease” can be used interchangeably and can refer to damage to, a disorder involving, or a disease of the liver. Nonlimiting examples of liver disease include intrahepatic cholestasis (e.g., Alagille syndrome, biliary liver cirrhosis), fatty liver (e.g., alcoholic fatty liver, Reye's syndrome), hepatic vein thrombosis, hepatolenticular degeneration (i.e., Wilson's disease), hepatomegaly, liver abscess (e.g., amebic liver abscess), liver cirrhosis (e.g., alcoholic, biliary, and experimental liver cirrhosis), alcoholic liver diseases (e.g., fatty liver, hepatitis, cirrhosis), parasitic liver disease (e.g., hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice (e.g., hemolytic, hepatocellular, cholestatic jaundice), cholestasis, portal hypertension, liver enlargement, ascites, hepatitis (e.g., alcoholic hepatitis, animal hepatitis, chronic hepatitis (e.g., autoimmune, hepatitis B, hepatitis C, hepatitis D, drug induced chronic hepatitis), toxic hepatitis, viral human hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E), granulomatous hepatitis, secondary biliary cirrhosis, hepatic encephalopathy,varices, primary biliary cirrhosis, primary sclerosing cholangitis, hepatic steatosis or steatohepatitis, hepatocellular adenoma, hemangiomas, bile stones, liver failure (e.g., hepatic encephalopathy, acute liver failure), angiomyolipoma, calcified liver metastases, cystic liver metastases, fibrolamellar hepatocarcinoma, hepatic adenoma, hepatoma, hepatic cysts (e.g., Simple cysts, Polycystic liver disease, hepatobiliary cystadenoma, choledochal cyst), mesenchymal tumors (mesenchymal hamartoma, infantile hemangioendothelioma, hemangioma, peliosis hepatis, lipomas, inflammatory pseudotumor), epithelial tumors (e.g., bile duct hamartoma, bile duct adenoma), focal nodular hyperplasia, nodular regenerative hyperplasia, hepatoblastoma, hepatocellular carcinoma, cholangiocarcinoma, cystadenocarcinoma, tumors of blood vessels, angiosarcoma, Karposi's sarcoma, hemangioendothelioma, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous carcinoma, primary lymphoma, peliosis hepatis, erythrohepatic porphyria, hepatic porphyria (e.g., acute intermittent porphyria, porphyria cutanea tarda), and Zellweger syndrome. In embodiments, the liver disease can comprise fibrosis, a liver disease characterized in that the liver has excess cholesterol, triglycerides or other lipids that is illustrative of liver diseases such as MASLD, MASH or alcoholic related steatosis of the liver, liver cirrhosis or liver inflammation or hepatocellular carcinoma.

[0221] In some exemplary embodiments, the liver disease can be cirrhosis, hepatitis, metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction-associated steatohepatitis (MASH), compensated and decompensated cirrhosis secondary to MASLD / MASH, portal hypertension, ascites, hepatocellular carcinoma (HCC), alcohol-associated liver disease (ALD), specific aetiology steatoic liver disease, drug-induced liver injury, a monogenic steatotic liver disease, a cryptogenic steatotic liver disease, or a combination thereof. See e.g., Rinella et al. J Hepatol. 2023 Dec;79(6): 1542-1556 (pubmed. ncbi.nlm.nih.gov / 37364790 / ).

[0222] As used herein, the terms “renal disease” and “kidney disease” can be used interchangeably and can refer to a disease, disorder, or condition in which the function of a subject’s kidney is impaired. For example, the renal disease can comprise Abderhalden-Kaufmann-Lignac syndrome (Nephropathic Cystinosis), Nephrotoxicity, Acute Kidney Failure / Acute Kidney Injury, Acute Lobar Nephroma, Acute Phosphate Nephropathy, Acute Tubular Necrosis, Adenine Phosphoribosyltransferase Deficiency, Adenovirus Nephritis, AlagilleSyndrome, Alport Syndrome, Amyloidosis, ANCA Vasculitis Related to Endocarditis and Other Infections, Angiomyolipoma, Analgesic Nephropathy, Antiphospholipid Syndrome, Anti-TNF-a Therapy -related Glomerulonephritis, AP0L1 Mutations, Apparent Mineralocorticoid Excess Syndrome, Aristolochic Acid Nephropathy, Chinese Herbal Nephropathy, Balkan Endemic Nephropathy, Arteriovenous Malformations and Fistulas of the Urologic Tract, Autosomal Dominant Hypocalcemia, Bardet-Biedl Syndrome, Bartter Syndrome, P-Thalassemia Renal Disease, Bile Cast Nephropathy, Clq Nephropathy, C3 Glomerulopathy, C3 Glomerulopathy with Monoclonal Gammopathy, C4 Glomerulopathy, Calcineurin Inhibitor Nephrotoxicity, Callilepsis Laureola Poisoning, Cardiorenal syndrome, CFHR5 nephropathy, Charcot-Marie-Tooth Disease with Glomerulopathy, Cholesterol Emboli, chronic kidney disease (CKD), Churg-Strauss syndrome, Chyluria, Ciliopathy, Cold Diuresis, Collagenofibrotic Glomerulopathy, Collapsing Glomerulopathy, Collapsing Glomerulopathy Related to CMV, Combination Antiretroviral (cART) Related-Nephropathy, Congenital Anomalies of the Kidney and Urinary Tract (CAKUT), Congenital Nephrotic Syndrome, Congestive Renal Failure, Conorenal syndrome (Mainzer-Saldino Syndrome or Saldino-Mainzer Disease), Contrast Nephropathy, Cortical Necrosis, Cryocrystalglobulinemia, Cryoglobuinemia, Cystic Kidney Disease, Acquired, Cystinuria, Dasatinib-Induced Nephrotic-Range Proteinuria, Dense Deposit Disease (MPGN Type 2), Dent Disease (X-linked Recessive Nephrolithiasis), DHA Crystalline Nephropathy, Dialysis Disequilibrium Syndrome, Diabetes and Diabetic Kidney Disease, Diabetes Insipidus, Diffuse Mesangial Sclerosis, Diuresis, Duplicated Ureter, EAST syndrome, Ebola and the Kidney, Ectopic Kidney, Ectopic Ureter, Edema, Swelling, Erdheim-Chester Disease, Fabry's Disease, Familial Hypocalciuric Hypercalcemia, Fanconi Syndrome, Fraser syndrome, Fibronectin Glomerulopathy, Fibrillary Glomerulonephritis and Immunotactoid Glomerulopathy, Fraley syndrome, Fluid Overload, Hypervolemia, Focal Segmental Glomerulosclerosis, Focal Sclerosis, Focal Glomerulosclerosis, Galloway Mowat syndrome, Giant Cell (Temporal) Arteritis with Kidney Involvement, Gestational Hypertension, Gitelman Syndrome, Glomerular Diseases, Glomerular Tubular Reflux, Glycosuria, Goodpasture Syndrome, HANAC Syndrome, Heat Stress Nephropathy, Hematuria (Blood in Urine), Hemolytic Uremic Syndrome (HUS), Atypical Hemolytic Uremic Syndrome (aHUS), Hemophagocytic Syndrome, Hemorrhagic Cystitis, Hemorrhagic Fever with Renal Syndrome (HFRS, Hantavirus Renal Disease, Korean Hemorrhagic Fever, Epidemic Hemorrhagic Fever, NephropathisEpidemica), Hemosiderinuria, Hemosiderosis related to Paroxysmal Nocturnal Hemoglobinuria and Hemolytic Anemia, Hepatic Glomerulopathy, Hepatic Veno- Occlusive Disease, Sinusoidal Obstruction Syndrome, Hepatitis C-Associated Renal Disease, Hepatocyte Nuclear Factor 10-Associated Kidney Disease, Hepatorenal Syndrome, Herbal Supplements and Kidney Disease, High Altitude Renal Syndrome, High Blood Pressure and Kidney Disease, HIV- Associated Immune Complex Kidney Disease (HIVICK), HIV- Associated Nephropathy (HIV AN), HNF1B-related Autosomal Dominant Tubulointerstitial Kidney Disease, Horseshoe Kidney (Renal Fusion), Hunner's Ulcer, Hydroxychloroquine-induced Renal Phospholipidosis, Hyperaldosteronism, Hypercalcemia, Hyperkalemia, Hypermagnesemia, Hypernatremia, Hyperoxaluria, primary hyperoxaluria, Hyperphosphatemia, Hypocalcemia, Hypocomplementemic Urticarial Vasculitic Syndrome, Hypokalemia, Hypokalemia-induced renal dysfunction, Hypokalemic Periodic Paralysis, Hypomagnesemia, Hyponatremia, Hypophosphatemia, Hypertension, Hypertension, Immersion Diuresis, Immune-Checkpoint Therapy-Related Interstitial Nephritis, Interstitial Cystitis, Interstitial Nephritis, Interstitial Nephritis, Karyomegalic, JC Virus Nephropathy, Joubert Syndrome, Kidney Stones, Nephrolithiasis, Lecithin Cholesterol Acyltransferase Deficiency (LCAT Deficiency), Leptospirosis Renal Disease, Light Chain Deposition Disease, Monoclonal Immunoglobulin Deposition Disease, Light Chain Proximal Tubulopathy, Liddle Syndrome, Lightwood- Albright Syndrome, Lipoprotein Glomerulopathy, LMX1B Mutations Cause Hereditary FSGS, Loin Pain Hematuria, Lupus Kidney Disease, Lupus Nephritis, Lupus Nephritis with Antineutrophil Cytoplasmic Antibody Seropositivity, Lupus Podocytopathy, Lyme Disease- Associated Glomerulonephritis, Lysinuric Protein Intolerance, Lysozyme Nephropathy, Malarial Nephropathy, Malignancy- Associated Renal Disease, Malignant Hypertension, Malakoplakia, McKittrick- Wheelock Syndrome, Meatal Stenosis, Medullary Cystic Kidney Disease, Urolodulin-Associated Nephropathy, Juvenile Hyperuricemic Nephropathy Type 1, Medullary Sponge Kidney, Megaureter, Melamine Toxicity and the Kidney, MELAS Syndrome, Membranoproliferative Glomerulonephritis, Membranous Nephropathy, Membranous-like Glomerulopathy with Masked IgG Kappa Deposits, MesoAmerican Nephropathy, Metabolic Acidosis, Metabolic Alkalosis, Microscopic Polyangiitis, Milk-alkalai syndrome, Minimal Change Disease, Monoclonal Gammopathy of Renal Significance, Dysproteinemia, MUC1 Nephropathy, Multicystic dysplastic kidney, Multiple Myeloma, Myeloproliferative Neoplasmsand Glomerulopathy, Nail-patella Syndrome, NARP Syndrome, Nephrocalcinosis, Nephrogenic Systemic Fibrosis, Nephroptosis (Floating Kidney, Renal Ptosis), Nephrotic Syndrome, Neurogenic Bladder, Nodular Glomerulosclerosis, Non-Gonococcal Urethritis, Nutcracker syndrome, Oligomeganephronia, Orofaciodigital Syndrome, Orotic Aciduria, Orthostatic Hypotension, Orthostatic Proteinuria, Osmotic Diuresis, Osmotic Nephrosis, Ovarian Hyperstimulation Syndrome, Oxalate Nephropathy, Page Kidney, Papillary Necrosis, Papillorenal Syndrome (Renal-Coloboma Syndrome, Isolated Renal Hypoplasia), PARN Mutations and Kidney Disease, The Peritoneal-Renal Syndrome, POEMS Syndrome, Podocyte Infolding Glomerulopathy, Post-infectious Glomerulonephritis, Post-streptococcal Glomerulonephritis, Post-infectious Glomerulonephritis, Polyarteritis Nodosa, Polycystic Kidney Disease, Proliferative Glomerulonephritis with Monoclonal IgG Deposits (Nasr Disease), Proteinuria (Protein in Urine), Pseudohyperaldosteronism, Pseudohypobicarbonatemia, Pseudohypoparathyroidism, Pulmonary-Renal Syndrome, Pyelonephritis (Kidney Infection), Pyonephrosis, Pyridium and Kidney Failure, Radiation Nephropathy, Refeeding syndrome, Reflux Nephropathy, Rapidly Progressive Glomerulonephritis, Renal Abscess, Peripnephric Abscess, Renal Agenesis, Renal Arcuate Vein Microthrombi-Associated Acute Kidney Injury, Renal Artery Aneurysm, Renal Artery Dissection, Spontaneous, Renal Artery Stenosis, Renal Cell Cancer, Renal Cyst, Renal Hypouricemia with Exercise-induced Acute Renal Failure, Renal Infarction, Renal Osteodystrophy, Renal Tubular Acidosis, Renin Mutations and Autosomal Dominant Tubulointerstitial Kidney Disease, Renin Secreting Tumors (Juxtaglomerular Cell Tumor), Reset Osmostat, Retroperitoneal Fibrosis, Rhabdomyolysis, Rheumatoid Arthritis-Associated Renal Disease, Sarcoidosis Renal Disease, Salt Wasting, Renal and Cerebral, Schistosomiasis and Glomerular Disease, Schimke immuno-osseous dysplasia, Scleroderma Renal Crisis, Serpentine Fibula- Polycystic Kidney Syndrome, Exner Syndrome, Sickle Cell Nephropathy, Silica Exposure and Chronic Kidney Disease, Sri Lankan Farmers' Kidney Disease, Sjogren's Syndrome and Renal Disease, Kidney Disease Related to Stem Cell Transplantation, TAFRO Syndrome, Tea and Toast Hyponatremia, Tenofovir-Induced Nephrotoxicity, Thin Basement Membrane Disease, Benign Familial Hematuria, Thrombotic Microangiopathy Associated with Monoclonal Gammopathy, Trench Nephritis, Genitourinary, Tuberous Sclerosis, Tubular Dysgenesis, Immune Complex Tubulointerstitial Nephritis Due to Autoantibodies to the Proximal Tubule Brush Border, Tumor Lysis Syndrome, Uremia, Vasomotor Nephropathy,Vesicointestinal Fistula, Vesicoureteral Reflux, VGEF Inhibition and Renal Thrombotic Microangiopathy, Von Hippel-Lindau Disease, Waldenstrom's Macroglobulinemic Glomerulonephritis, Warfarin-Related Nephropathy, Wegener's Granulomatosis, Granulomatosis with Polyangiitis, Wunderlich syndrome, Zellweger Syndrome, or Cerebrohepatorenal Syndrome.

[0223] In some non-limiting, exemplary embodiments the renal disease can comprise nephrolithiasis, recurrent kidney stone disease, chronic kidney disease (CKD), or end-stage renal disease (ESRD).

[0224] In embodiments, the term hyperoxaluria can refer to a high concentration of oxalate in urine. In embodiments, the hyperoxaluria can comprise primary hyperoxalurias (PH-1, PH-2 and PH-3), secondary hyperoxaluria, idiopathic calcium oxalate urolithiasis. For example, the disease can comprise primary hyperoxaluria, for example PH-1.

[0225] The term hyperoxaluria refers to a high concentration of oxalate in urine. This situation can have a number of different causes. Hyperoxalurias can be classified as primary and secondary. Primary hyperoxalurias (PH) are a group of autosomal recessive genetic disorders involving enzyme failures that lead to an endogenous surplus production of oxalate. PH can comprise PHI, PH2, and PH3, with PHI being the most common and the most aggressive. [(1) Bhasin, B. Primary and Secondary Hyperoxaluria: Understanding the Enigma. World Journal of Nephrology 2015, 4 (2), 235], The same genetic error that gives rise to PH3 has also been linked to idiopathic oxalate lithiasis. [(1) Monico, C. G.; Rossetti, S.; Belostotsky, R.; Cogal, A. G.; Herges, R. M.; Seide, B. M.; Olson, J. B.; Bergstrahl, E. J.; Williams, H. J.; Haley, W. E.; et al. Primary Hyperoxaluria Type III Gene HOGA1 (Formerly DHDPSL) as a Possible Risk Factor for Idiopathic Calcium Oxalate Urolithiasis. CJASN 2011, 6 (9), 2289-2295.]

[0226] Secondary hyperoxalurias can be due to an excessive absorption of oxalate or its precursors in the bowel. This is linked to a diet rich in said precursors, or in the case of enteric hyperoxaluria, to an absorption disorder after bowel resection. [(1) Cochat, P.; Rumsby, G. Primary Hyperoxaluria. New England Journal of Medicine 2013, 369 (7), 649-658. (2) Lorenz, E. C.; Michet, C. J.; Milliner, D. S.; Lieske, J. C. Update on Oxalate Crystal Disease. Curr Rheumatol Rep 2013, 15 (7), 340. (3) Karaolanis, G.; Lionaki, S.; Moris, D.; Palla, V.-V; Vernadakis, S. Secondary Hyperoxaluria: A Risk Factor for Kidney Stone Formation and Renal Failure in Native Kidneys and Renal Grafts. Transplantation Reviews 2014, 28 (4), 182-187],

[0227] Primary hyperoxaluria type 1

[0228] Primary hyperoxaluria type 1 (PH-1) is a serious hereditary disease due to a deficiency of the AGT enzyme (encoded by the Agxtl gene) in hepatocytes [Zhang, X.; Roe, S. M.; Hou, Y.; Bartlam, M.; Rao, Z.; Pearl, L. H.; Danpure, C. J. J. Mol. Biol. 2003, 331, 643-652], This enzyme, AGT, is in charge of metabolizing glyoxylate in hepatic peroxisomes by transamination into glycine. In PH-1, where AGT activity is absent or the enzyme is erroneously located in the mitochondria, glyoxylate accumulation occurs as a result. Glyoxylate, then, comes to be metabolized by oxidation to oxalate, a process that is catalyzed by glycolate oxidase (GO) enzymes in peroxisomes and lactate dehydrogenase (LDH) in the cytoplasm. An excess production of oxalate, which can only be excreted in urine, produces renal capacity saturation and causes oxalate to precipitate in the form of insoluble calcium oxalate crystals. These crystals damage the renal tissue, reducing the excretory capacity of the kidney until ultimately leading to terminal kidney disease. As kidney damage progresses, oxalate accumulation becomes widespread and causes blood vessel, bone, joint, retinal, skin, bone marrow, heart and central nervous system disorders, ultimately leading to the patient’s death. PH-1 is a rare disease with an estimated incidence in Europe of 1:100000 births per year [Cochat, P.; Hulton, S. A.; Acquaviva, C.; Danpure, C. J.; Daudon, M.; Marchi, M. D.; Fargue, S.; Groothoff, J.; Harambat, J.; Hoppe, B.; et al. Nephrol. Dial. Transplant. 2012, 27, 1729-1736.], but it presents at an unusually high frequency in the Canary Islands [(1) Lorenzo, V.; Alvarez, A.; Torres, A.; Torregrosa, V.; Hernandez, D.; Salido, E. Kidney Int. 2006, 70, 1115-1119. (2) Santana, A.; Salido, E.; Torres, A.; Shapiro, L. J. PNAS 2003, 100, 7277-7282],

[0229] In embodiments, the disease comprises a GO-associated disease and / or lactate dehydrogenase (LDHA)-associated disease.

[0230] The term “subject” or “patient” can refer to any organism to which aspects of the invention can be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. For example, subjects to which compounds of the disclosure can be administered include animals, such as mammals. Non-limiting examples of mammals include primates, such as humans. For veterinary applications, a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals for example pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like. The term “livingsubject” can refer to a subject noted above or another organism that is alive. The term “living subject” can refer to the entire subject or organism and not just a part excised (e.g., a liver or other organ) from the living subject.

[0231] As used herein, "pharmaceutically acceptable derivatives" of a compound can include salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs thereof. Such derivatives can be readily prepared by those of skill in this art using known methods for such derivatization. The compounds produced can be administered to animals or humans without substantial toxic effects and either are pharmaceutically active or are prodrugs.

[0232] In embodiments, the method can comprise measuring the circulating oxalate levels of a subject. In embodiments, if the subjects circulating oxalate levels are elevated, therapeutic levels of the compounds described herein can be administered. In embodiments a subject’s oxalate levels can be measured by enzymatic assay, mass spectrometry, or a combination thereof. For example, the mass spectrometry can comprise LC-MS / MS, GC-MS and IC-MS. In embodiments, a clinician of ordinary skill in the art can determine if the subject’s oxalate levels are elevated.

[0233] In embodiments, circulating oxalate levels can be determined by an enzymatic assay known in the art. See, e.g., Enzymatic assay with the clinically validated enzymatic oxalate assay from Trinity Biotech (591-D) (Moya-Garzon et al. Eur. J. Med. Chem. 2022, PMID: 35500475; Donelan et al. PLoS One. 2023, PMID: 37167324; Pfau et al. Kidney Int. Rep. 2020. PMID: 33163722, Baum et al. Kidney Int. 2022, PMID: 36007597)

[0234] In embodiments, circulating oxalate levels can be determined by Ion Chromatography-Mass Spectrometry (IC-MS). See e.g., Das et al. Nat. Metab. 2024 PMID: 39333384).

[0235] In embodiments, elevated circulating oxalate levels can comprise less than about 1 uM, about 1 uM, about 1.5 uM, about 2 uM, about 2.5 uM, about 3 uM, about 4 uM, about 5 uM, about 6 uM, about 7 uM, about 8 uM, about 9 uM, about 10 uM, or greater than about 10 uM. See e.g., 163722.

[0236] As used herein, the term "administering" can refer to introducing a substance, such as the compounds described herein, or derivatives thereof, isomers thereof, or a combination thereof into a subject. Any route of administration can be utilized including, for example, intranasal, topical, oral, parenteral, intravitreal, intraocular, ocular, subretinal, intrathecal, intravenous, subcutaneous, transcutaneous, intracutaneous, intracranial and the like administration.

[0237] In embodiments, "administering" can also refer to providing a therapeutically effective amount of a formulation or pharmaceutical composition to a subject. The formulation or pharmaceutical compound can be administered alone, but can be administered with other compounds, excipients, fillers, binders, carriers or other vehicles selected based upon the chosen route of administration and standard pharmaceutical practice.

[0238] Administration can be by way of carriers or vehicles, such as injectable solutions, including sterile aqueous or non-aqueous solutions, or saline solutions; creams; lotions; capsules; tablets; granules; pellets; powders; suspensions, emulsions, or microemulsions; patches; micelles; liposomes; vesicles; implants, including microimplants; eye drops; other proteins and peptides; synthetic polymers; microspheres; nanoparticles; and the like.

[0239] In embodiments, the therapeutically effective amount of the composition inhibits GO activity, LDHA activity, or a combination thereof.

[0240] In embodiments, the therapeutically effective amount of the composition activates degradation of glycolate oxidase (GO), activates degradation of lactate dehydrogenase (LDHA), or activates degradation of both glycolate oxidase and lactate dehydrogenase.

[0241] In embodiments a therapeutically effective amount of a composition described herein can comprise less than about 0.1 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 120 mg / kg, about 135 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, about 300 mg / kg, about 325 mg / kg, about 350 mg / kg, about 375 mg / kg, about 400 mg / kg, about 425 mg / kg, about 450 mg / kg, about 475 mg / kg, about 500 mg / kg, about 525 mg / kg, about 550 mg / kg, about 575 mg / kg, about 600 mg / kg, about 625 mg / kg, about 650 mg / kg, about 675 mg / kg, about 700 mg / kg, about 725 mg / kg, about 750 mg / kg, about 775 mg / kg, about 800 mg / kg, about 825 mg / kg, about 850 mg / kg, about 875 mg / kg, about 900 mg / kg, about 1.0 g / kg, about 1.5 g / kg, about 2.0 g / kg, about 2.5 g / kg, about 5 g / kg, about 10 g / kg, about 25 g / kg, about 50 g / kg, or more than 50 g / kg of compound per body weight of a subject.

[0242] In some exemplary embodiments, the therapeutically effective amount can comprise about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, or about 20 mg / kg.

[0243] In embodiments a therapeutically effective amount of a composition described herein can comprise a concentration of less than about 1 nM, about 1 nM, about 5 nM, about 10 nM, about 20 nM, about 25 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 75 nM, about 80 nM, about 90 nM, about 100 nM, about 200 nM, about 250 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1000 nM, and greater than about 1000 nM. For example, the concentration can be about 500 nM. For example, the concentration can be about 700 nM.EXAMPLES

[0244] Examples are provided herein to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.EXAMPLE 1

[0245] Example 1: Synthesis and use of dual glycolate oxidase / 'lactate dehydrogenase inhibitors, activators of protein degradation

[0246] Non-limiting Description of the Invention

[0247] This disclosure comprises small molecule drugs with a pharmacological mechanism combining enzyme inhibition with activation of enzyme degradation pathways. The compounds are dual inhibitors of the enzymes glycolate oxidase (GO) and lactate dehydrogenase (LDHA) that simultaneously induce the degradation of at least one of these enzymes. Without wishing to be bound by theory, these compounds can induce degradation of both enzymes. GO and LDHA catalyze two consecutive metabolic steps in the hepatic biosynthesis of oxalate from glycolate and thus, these molecules decrease the production of oxalate in the liver. Therefore, these compounds can be drugs for the treatment of diseases related to overproduction of endogenous oxalate. Specially, the compounds are can be drugs for the treatment of primary hyperoxalurias (PHs) and metabolic dysfunction-associated steatotic liver disease (MASLD), along with its complications metabolic dysfunction associated steatohepatitis (MASH) and atherosclerosis (ACVD). In embodiments, the target population comprises patients of the three types of PHs (PHI, PH2 andPH3) and patients of MASLD with or without MASH and ACVD. Other groups comprise patients of oxalate urolithiasis of diverse etiologies.

[0248] The structure of the compounds contains a selectivity ligand (SL), a linker and a protein activation ligand (PAL). The SL that recognizes GO and LDHA (proteins of interest, Pols), is formed by a C5 substituted salicylate moiety. The PAL that activates the target degradation is a hydrophobic tag consisting of adamantane. The linker between SL and PAL is a hydrocarbon chain containing diverse heteroatoms (Figures 1 and 2). The length of the linkers can vary between molecules and can contain cyclic fragments.

[0249] Non-Limiting Surprising Aspects of the Disclosure

[0250] The molecules combine a new concept of GO and LDHA targeting including reversible enzyme inhibition together with activation of enzyme degradation (Small Molecule Mediated Protein Degradation, SMPD). Thus, once the inhibitor binds to the target enzyme, the protein is directed to the cellular protein degradation machinery. Thus, the compounds decrease the concentrations of the enzyme(s) they inhibit, an effect that is comparable to in vivo knockdown, with the advantage of avoiding any genetic manipulation.

[0251] -For the first time, we describe SMPD against lactic dehydrogenase A, PHs treated with SMPD drugs, and MASLD and MASH treated with SMPD drugs.

[0252] The described compounds exert their biological effect through a new dual mechanism combining enzyme inhibition and activation of protein degradation. Besides, the compounds are double targeted. A non-limiting summation of benefits of this strategy is:

[0253] Enzyme inhibition combined with activation of protein degradation presents the benefits of knockdown effect with the advantage of avoiding any genetic manipulation.

[0254] Double targeting GO and LDHA, ensures greater effectiveness in decreasing oxalate production with lower doses of drug. This translates into fewer secondary effects. Besides, it avoids the use of drug cocktails and thus, it minimizes the risk of pharmacological interactions.

[0255] Current treatment of PHI:

[0256] Despite of the growing interest of szRNAs therapeutics, their use presents serious drawbacks that question their utility when classical small molecule drugs are available. These limitations include their high cost, making them unaffordable for most of the patients. Moreover, these drugs have a single-target mechanism (either GO or LDHA), and are administered only byinjections. The unavailability of lumasiran and nedosiram, due to their high cost, is evident not only in developing countries but also in developed regions of Europe and America.

[0257] Compared to zRNA, the technologies described in this disclosure contain the following non-limiting advantages:

[0258] -Small inhibitors present better ADME properties.

[0259] -The compounds disclosed herein are effective after oral administration. Oral administration increases the quality of life for a patient receiving chronic treatment. It is also a safer route of administration.

[0260] -The compounds disclosed herein present a significantly lower cost of production, from inexpensive starting materials and in few synthetic steps. Further, the compounds require no strict conservation protocols. This can lower expenses and thereby reduce costs to patients and health authorities.

[0261] -The compounds disclosed herein are effective against two targets in the same metabolic route leading to oxalate formation. This double targeting approach possesses the benefits of multitarget therapies (lowering of doses) without the drawbacks of using drug cocktails (complicated pharmacokinetics and drug interactions). To date, no multi-target drug against PH has reached clinical trials.

[0262] While some drugs can reduce urinary oxalate in patients of PHI with good kidney function, they fail to do so in patients with impaired kidney function (e.g., chronic kidney disease or early end-stage renal disease).

[0263] Current treatment of PH2 and PH3:

[0264] There is no drug approved for the treatment of PH2 and PH3.

[0265] Current treatment of MASLD and MASH:

[0266] A drug against MASLD should address the intracellular accumulation of fat, the inflammation and fibrosis causing the progress of MASLD to MASH. These aspects are reduced after oral administration of MDMG-935P in a MASH mouse models (PCT / US24 / 17626). MDMG-935P is a double GO / LDHA inhibitor, prototype for the design of the new SMPD agents. Thus, targeting oxalate formation using dual GO / LDHA inhibitors, can provide a treatment approach for MASLD. This can be indicated by suppressed AGXT and enhanced LDHA activation found in livers from patients and mice with MASH.

[0267] The disclosure addresses issues such as severe diseases, the lack of effective / affordable treatments available and the chronic nature of the treatments. Besides, MASLD patients are a broad target population. MASLD affects over a third of the global population and MASH is found in up to 30% of patients with MASLD. In the United States alone, between 10 and 15 million people suffer from MASLD.

[0268] The progress of PHs leads to severe renal failure and end-stage renal disease (ESRD). During the progress of the disease, dialysis sessions are frequently required and finally, patients with PHs must undergo combined liver and kidney transplantations to survive. During the MASH stage of MASLD, the liver suffers a fibrotic process that culminates in liver failure and requires liver transplant. Kidney and liver transplantations require long-term immunosuppression and are associated with high morbidity and mortality. Moreover, dialysis and chirurgical procedures are resource consuming procedures and deeply affect the patient’s quality of life.

[0269] There is a lack of efficient drugs for MASLD and MASH. Resmetirom improves the readouts of markers for liver damage in only 10-15% of patients with MASH after placebo adjustment.

[0270] In the case of PHs, with the development of small-molecule oral drugs against PHs, the proposal contributes to substitute / complement chronic injectable treatments that impair lifequality of patients.

[0271] The high cost of the available siRNA treatments of PHs, will make the new small molecule-based treatment, the treatment of choice in developing and developed countries.EXAMPLE 2

[0272] Additional Non-Limiting Biological Results

[0273] Biological Evaluation Against Recombinant Enzymes hGO and hLDHA

[0274] The final compounds were evaluated against recombinant enzymes AGO and ALDH isozymes, ALDHA and ALDHB. For all the assays, we used fluorometric kinetic methods based on the fluorogenic reagent Amplex® Red (AGO) or on the fluorochrome NADH (ALDHA and ALDHB).

[0275] FAB-596 and FAB-599 are dual inhibitors of enzymes AGO and ALDHA. The two molecules are nanomolar inhibitors of ALDHA with IC50s of 200 nM (FAB-599) and 600 nM (FAB-596) and low micromolar inhibitors of AGO with IC50s of 1.1 pM and 3.1 pM, respectively.

[0276] Besides, inhibition kinetics were studied in competence with the enzymatic substrates, glycolic acid (AGO) and pyruvic acid (ALDHA and ALDHB). Affinity constants of the inhibitors (Ki) and mechanisms of inhibition were determined. Assays measured the enzymatic activity in the presence of four / five different inhibitor concentrations and ten different substrate concentrations (only seven substrate concentrations in the assay on ALDHB) (three replicates). FAB-599 presents nanomolar Ki values for both enzymes (390 nM and 40 nM for AGO and ALDHA, respectively) and a 42-fold higher affinity for ALDHA than for ALDHB [Ki(LDHB) = 1.7 pM], FAB-599 is a mixed-type noncompetitive inhibitor of AGO (a > 1) and a pure noncompetitive inhibitor of ALDHA and ALDHB (a = 1).EXAMPLE 3

[0277] Non-Limiting Synthetic Methods for FAB-599, FAB-596 and PLN-9

[0278] Scheme 1. Synthetic pathway for the preparation of compounds FAB-599, FAB-596 and PLN-9

[0279] 1. Methyl 5-(5-formylfuran-2-yl)-2-hydroxybenzoate (MDMG-409E) (step a, Scheme 1).

[0280] To a stirred solution of methyl 5-iodosalicylate (1) (1 eq) and 5-formyl-2-furanylboronic acid (2) (1.5 eq) in dimethylformamide (DMF) (2.5 mL / mmol), triethylamine (3 eq) and Pd(OAc)2 (0.05 eq) were added. The reaction mixture was stirred at room temperature (rt) overnight. After consumption of the starting material, determined by thin layer chromatography (TLC) (petrol etherethyl acetate) (70:30), the reaction mixture was concentrated in a high vacuum rotavapor. The solid residue was dissolved in ethyl acetate (AcOEt) and washed with water and brine. The organic layer was dried over MgSO₄, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (FCC) (gradient elution using petrol ether AcOEt) (80:20 — > 50:50) to yield MDMG-409E as a yellowish-orange solid (87% yield). 'HNMR (400 MHz, chloroform-d) 8 10.99 (s, 1H, OH), 9.61 (s, 1H), 8.31 (d, J= 2.3 Hz, 1H), 7.87 (dd, J= 8.7, 2.3 Hz, 1H), 7.31 (d, J= 3.7 Hz, 1H), 7.05 (d, J= 8.7 Hz, 1H), 6.74 (d, J= 3.7 Hz, 1H), 4.00 (s, 3H).13C NMR(101 MHz, chloroform-d) 6 177.0, 170.2, 162.7, 158.7, 151.9, 132.6, 127.2, 120.8, 118.6, 112.9, 106.8, 52.7. HRMS: m / z calcd. for [M+H] C13H11O5 247.0606; found, 247.0593 (deviation -1.1 ppm).

[0281] 2. General conditions for aldol condensation (step b, Scheme 1)

[0282] A solution of the corresponding acetophenone (1.5 equiv) in a suitable organic solvent (2.5 mL / mmol), was prepared and stirred in a round bottom flask. On this solution, aqueous sodium hydroxide (excess) was added dropwise. After the addition, the mixture was stirred during 30 min before the addition of MDMG-409E. Stirring at rt was maintained until the reaction was completed. The reaction was quenched by addition of HC1 or water and evaporation of the organic solvent under vacuum. The remaining aqueous layer was extracted with AcOEt (x3). The resulting organic phase was consecutively washed with HC1 (1 M), water and brine, dried over MgSO4, filtered and evaporated under vacuum. The final product was purified by FCC.

[0283] 2.1. Methyl (E)-2-hydroxy-5-{5-[3-(4-iodophenyl)-3-oxoprop-l-en-l-yl]furan-2-yl}benzoate (FAB-545) (step b, Scheme 1).

[0284] General protocol for aldol condensation. Acetophenone: / ?-iodoacetophenone (3a) (220 mg, 0.89 mmol, 2 equiv); organic solvent: tetrahydrofuran (THF):methanol (MeOH) (1:1) (2.5 mL / mmol); lONNaOH aqueous solution (0.45 mL, 4.5 mmol, 10 equiv); MDMG-409E (110 mg, 0.45 mmol, 1 equiv), added as a solution in THF: MeOH (1:1) (2.5 mL / mmol). Reaction time: 24 h. Quenching: addition of HC1 3N until pH 3. FCC purification: gradient elution using petrol ethenAcOEt mixtures (95:5 70:30). FAB-545: Reddish-orange solid (200 mg, 0.42 mmol, 94% yield).

[0285] 'H NMR (400 MHz, chloroform-d) 8 10.93 (s, 1H), 8.22 (d, J= 2.3 Hz, 1H), 7.87 (dq, J = 8.7, 2.2 Hz, 3H), 7.78 - 7.73 (m, 2H), 7.59 (d, J= 15.3 Hz, 1H), 7.39 (d, J= 15.3 Hz, 1H), 7.07 (d, J= 8.7 Hz, 1H), 6.82 (d, J= 3.6Hz, 1H), 6.70 (d, J= 3.6 Hz, 1H), 4.03 (s, 3H).13C NMR (101 MHz, chloroform-d) 8 189.27, 170.30, 162.06, 155.97, 150.93, 138.04, 137.81, 132.03, 131.09, 130.00, 126.25, 121.73, 119.59, 118.61, 118.07, 112.90, 107.58, 100.53, 52.78. HRMS (TOF, ES’ ): m z calcd. for C21H14O5I [M-H]', 472.9886; found, 472.9848 (deviation: -8.0 ppm).

[0286] 2.2. Methyl (£)-2-hydroxy-5-{5-[3-(4-bromophenyl)-3-oxoprop-l-en-l-yl]furan-2-yl}benzoate (FAB-535C).

[0287] General protocol for aldol condensation. Acetophenone: / ?-bromoacetophenone (3b) (400 mg, 1.83 mmol, 1.5 equiv); organic solvent: MeOH (2.5 mL / mmol); concentrated NaOH aqueous solution (1.2 g, 30 mmol, 30 equiv); MDMG-409E (300 mg, 1.22 mmol, 1 equiv). Reaction time: 4 h. Quenching: addition of water. FCC purification: gradient elution using petrol ether: AcOEt mixtures (10:0 — 8:2). FAB-535C: Solid (311 mg, 0.73 mmol, 60% yield).

[0288] 1HNMR(400 MHz, chloroform-d) 8 10.93 (s, 1H), 8.21 (d, J= 2.3 Hz, 1H), 7.93 - 7.89 (m, 2H), 7.87 (dd, J= 8.7, 2.3 Hz, 1H), 7.67 - 7.63 (m, 2H), 7.59 (d, J= 15.3 Hz, 1H), 7.40 (d, J= 15.3 Hz, 1H), 7.07 (d, J= 8.8 Hz, 1H), 6.82 (d, J= 3.6 Hz, 1H), 6.70 (d, J= 3.6 Hz, 1H), 4.03 (s, 3H). HRMS (TOF, ES+): m / z calcd. for C2iHi6O5Br [M+H]+, 427.0181; found: 427.0186 (deviation +1.2 ppm).

[0289] 3. Preparation of terminal alkenes for Heck reaction.

[0290] 3.1.7V-(Adamantan-l-ylmethyl)acrylamide (FAB-595).

[0291] A solution of (adamantan- 1 -ylmethyl)amine (200 mg, 1.21 mmol, 1 equiv) and di isopropyl ethyl amine (DIPEA) (312 mg, 2.42 mmol, 2 equiv) in anhydrous THF (8 mL), was prepared under argon. The solution was cooled to 0 °C and acryloyl chloride (120 mg, 1.33 mmol, 1.1 equiv) was added dropwise. The reaction was then warmed at rt and let to stir during 20 min. The reaction was then quenched by addition of a saturated solution of sodium bicarbonate (3 mL) followed by addition of water (3 mL). The organic phase was then separated, the THF was evaporated under vacuum and the residue was dissolved in AcOEt. The new organic phase was washed with HC1 (5%) and brine, dried on anhydrous MgSCh, fdtered and evaporated under vacuum. FCC purification (automatic): Gradient elution with mixtures petrol etherAcOEt (90:10^20:80). FAB-595: White solid.

[0292] 'H NMR (500 MHz, chloroform-d) 86.28 (d, J= 16.9 Hz, 1H), 6.12 (dd, J= 16.9, 10.3 Hz, 1H), 5.64 (d, J= 10.3 Hz, 1H), 3.04 (d, J= 6.4 Hz, 2H), 1.98 (s, 3H), 1.75-1.58 (m, 6H), 1.52 - 1.48 (m, 6H).13C NMR (126 MHz, chloroform-d) 6 165.89, 131.17, 126.43, 51.14, 40.37, 37.04, 34.00, 28.34.

[0293] 4. Preparation of terminal alkynes for Sonogashira coupling.

[0294] 4.1. A-(Adamantan-l-ylmethyl)hex-5-yn-l-amine (FAB-607).

[0295] Adamantan- l-ylmethyl)amine (268 mg, 270 pL, 1.62 mmol, 2 equiv) and potassium carbonate (112 mg, 0.81 mmol, 1 equiv) were introduced in a dry two-necked round-bottom flask and the mixture was purged with argon. Then, anhydrous acetonitrile (10 mL) was added, followed by 6-iodohexyne (168 mg, 106 pL, 0.81 mmol, 1 equiv). The reaction was then diluted with an additional volume of anhydrous acetonitrile (10 mL) and let to stir at 80 °C (reflux), under argon,for 18 h.2The reaction was then cooled down to rt and the solvents were evaporated under vacuum. The residue was suspended in DCM and the organic phase was washed with an aqueous solution of NaOH (5 M). The organic phase was dried over anhydrous MgSCh, filtered and evaporated under vacuum. FCC purification (automatic): Gradient elution with mixtures DCM: MeOH (99:1^90:10). FAB-607: Solid, 80 % yield (160 mg, 0.65 mmol).

[0296] 'H NMR (500 MHz, chloroform-d) 83.34 (bb, 1H), 2.70 (t, J= 7.5 Hz, 2H), 2.33 (s, 2H), 2.21 (td, J= 7.0, 2.4 Hz, 2H), 1.99-1.92 (m, 4H), 1.73 - 1.61 (m, 8H), 1.59-1.51 (m, 8H).13C NMR (126 MHz, chloroform-d) 8 84.29, 68.69, 62.17, 50.08, 40.84, 37.12, 33.32, 28.47, 27.98, 26.25, 18.38.

[0297] 4.2. Preparation of PLN-6.

[0298] 4.2.1. tert-Butyl-JV-(6-hydroxyhexyl)carbamate (PLN-5).

[0299] To a stirred solution of 6-aminohexan-l-ol (300 mg, 2.55 mmol, 1 equiv) in DCM (2.5 mL), a solution of BOC2O (834.8 mg, 3.8 mmol, 1.5 equiv) in dichloromethane (DCM) (2.5 mL) was slowly added at 0°C (water / ice bath). The addition was performed dropwise using an addition funnel. Once the addition was completed, the reaction mixture was stirred at rt overnight. The solvent was removed under vacuum and the residue was purified by FCC (gradient elution from hexane: AcOEt (2: 1) to AcOEt). PLN-5: Colourless oil, 94% yield. Product characterization agrees with reported data (P. Mader, R. Bartholomaus, S. Nicolussi, A. Baumann, M. Weis, A. Chicca, M. Rau, A. C. Simao, J. Gertsch, K.-H. Altmann, ChemMedChem 2021, 16, 145).

[0300] 4.2.2. te / 7-Butyl- / V-(6-hydroxyhexyl)carbamate (PLN-6).THF / DMF (dry),PLN-5 rt overnight PLN-6

[0301] A solution of PLN-5 (460.7 mg, 2.12 mmol, 1 equiv) was prepared under argon, in THF / DMF (1: 1) (dry, 11 mL) and cooled down to 0 °C. At this temperature, solid NaH (purity 60 %) (203.52 mg, 8.48 mmol, 2.4 equiv) was added. After stirring for 30 min at 0 °C (Ar), a solution of 6-iodohex-5-yne (5.96 mmol, 2.8 equiv) in dry THF (2 mL) was added dropwise. The mixture was stirred at rt overnight, then washed with brine and this aqueous phase was extracted with DCM(x3). The organic layers were combined, dried over anhydrous Na2SO4, fdtered and concentrated under reduced pressure. The crude was purified by FCC (silica gel) (gradient elution with mixtures hexane to hexane: AcOEt 1: 1). PLN-6: Yellow oil, 39% yield.

[0302] 'H NMR (400 MHz, chloroform-d) 54.48 (s, 1H), 3.42 (t, J= 6.3 Hz, 2H), 3.39 (t, J = 6.3 Hz, 2H), 3.10 (bb, 2H), 2.22 (td, J= 6.9, 2.5 Hz, 2H), 2.17 (s, 1H), 1.94 (t, J= 2.6 Hz, 1H), 1.72 - 1.45 (m, 10H), 1.44 (s, 9H), 1.36-1.30 (m, 2H).13C NMR (101 MHz, CDCh) 5 156.14, 84.55, 79.23, 70.95, 70.37, 68.49, 41.04, 30.20, 29.82, 28.93, 28.58, 26.80, 26.06, 25.42, 18.39. HRMS (TOF, ESI+): m / z calcd. for C17H31NNaO4[M+Na]+, 320.2196; found: 320.2203 (deviation -2.1 ppm).

[0303] 5. 5-{5-{(£)-3-{4-[(E)-3-(Adamantan-l-ylmethylamino)-3-oxoprop-l-enyl]phenyl}-3-oxoprop-l-enyl}furan-2-yl}-2-hydroxybenzoic acid (FAB-596) (Heck reaction, step c, Scheme 1).

[0304] All the solid reagents [FAB-535C (100 mg, 0.23 mmol, 1 equiv), FAB-595 (61 mg, 0.28 mmol, 1.2 equiv), Pd(OAc)2 (10 mg, 2 mol%), DABCO (10 mg, 4 mol%) andK2COs (31 mg, 0.23 mol, 1 equiv)] were mixed in a sealed vial. The vial was then purged with argon before the addition of anhydrous DMF. The reaction was then stirred at 120 °C for 12 h. After this time, the reaction was let to cool to rt and the solvent was partially removed under vacuum. The residue was filtered and the filtrate was diluted with AcOEt and washed with HC1 IN (x2) and brine. The organic phase was dried on anhydrous MgSO4, filtered and evaporated under vacuum. FCC purification: Gradient elution with mixtures petrol ether: DCM: MeOH (50:50:0— >-45:45:10 (1% AcOH)— >0:70:30). FAB-596: Pale orange solid.

[0305] rH NMR (500 MHz, DMSO-d6) 88.20 (s, 1H), 8.15 (d, J= 8.1 Hz, 2H), 8.03 (t, J= 5.9 Hz, 1H), 7.95 (d, J= 8.4 Hz, 1H), 7.75 (d, J= 8.0 Hz, 2H), 7.62 (d, J= 15 Hz, 1H), 7.59 (d, J = 15 Hz, 1H), 7.49 (d, J= 15.0 Hz, 1H), 7.21 (d, J= 3.3 Hz, 1H), 7.04 (d, J= 3.0 Hz, 1H), 6.94 (s, 1H), 6.91 (d, J= 15.0 Hz, 1H), 2.93 (d, J= 6.2 Hz, 2H), 1.94 (s, 3H), 1.91 (s, 1H), 1.67 (bd, J = 10 Hz, 3H), 1.60 (bd, J= 10 Hz, 3H), 1.48 (s, 6H).13C NMR (126 MHz, DMSO-cfc) 8 187.70,172.00, 171.06, 164.80, 156.40, 150.04, 139.30, 137.99, 137.31, 130.30, 128.95, 127.78, 126.30, 124.96, 120.67, 117.60, 117.17, 107.34, 50.40, 39.78, 36.52, 33.78, 27.70. HRMS (TOF, ESI+): m / z calcd. for C34H33NNaO6[M+Na]+, 574.2200; found: 574.2174 (deviation 4.6 ppm).

[0306] 6. General protocol for Sonogashira reaction (step d, Scheme 1)

[0307] The solid reagents [FAB-545 (1 equiv), bis(triphenylphosphine)palladium (II) dichloride (0.05 equiv) and copper (I) iodide (0.1 equiv)] were mixed in a well-dried, round-bottom flask and the mixture was purged under argon before the addition of anhydrous THF. Then a 1M solution of tetrabutylammonium fluoride (TBAF) in hexanes (1.6 equiv) and the corresponding terminal alkyne (1 equiv) were added to start the reaction. After overnight stirring at rt under argon, the crude was dissolved in AcOEt, filtered through Celite® and concentrated under reduced pressure. The final product was purified by FCC.

[0308] 6.1. Methyl 5-{5-{(£')-3-{4-[6-(adamantan-l-ylmethylamino)hex-l-ynyl]phenyl}-3-oxoprop-l-enyl}furan-2-yl}-2-hydroxybenzoate (FAB-599e).

[0309] General protocol for Sonogashira reaction. Reagents: FAB-545 (640 mg, 1.35 mmol, 1 equiv), bis(triphenylphosphine)palladium (II) dichloride (53 mg, 0.076 mmol, 0.05 equiv), copper (I) iodide (24 mg, 0.126 mmol, 0.1 equiv), 1M solution of tetrabutylammonium fluoride in hexanes (554 mg, 2.12 mL, 2.12 mmol, 1.6 equiv), FAB-607 (315 mg, 1.28 mmol, 1 equiv). FCC purification: Gradient elution with mixtures petrol ether: DCM: MeOH (50:50:2^50:50: 15). FAB-599e: Orange solid, 67% yield (497 mg, 0.86 mmol).

[0310] 'H NMR (400 MHz, chloroform-d) 8 8.20 (d, J= 2.2 Hz, 1H), 7.97 (d, J= 8.3 Hz, 2H), 7.87 (dd, J= 8.7, 2.2 Hz, 1H), 7.57 (d, J= 15.2 Hz, 1H), 7.55 (d, J= 8.3 Hz, 2H), 7.42 (d, J= 15.3 Hz, 1H), 7.07 (d, J= 8.7 Hz, 1H), 6.80 (d, J = 3.5 Hz, 1H), 6.69 (d, J = 3.6Hz, 1H), 4.02 (s, 3H), 3.06 (m, 2H), 2.62 (s, 2H), 2.49 (t, J= 6.9 Hz, 2H), 2.12 (m, 2H), 2.02 (s, 3H), 1.75-1.62 (m, 14H).

[0311] 6.2. Methyl 5-{5-{(£)-3-{4-{6-[6-(tert-butoxycarbonylamino)hexyloxy]hex-l-ynyl}phenyl}-3-oxoprop-l-enyl}furan-2-yl}-2-hydroxybenzoate (PLN-7).

[0312] General protocol for Sonogashira reaction. Reagents: FAB-545 (260 mg, 0.55 mmol, 1 equiv), bis(triphenylphosphine)palladium (II) dichloride (19.2 mg, 0.027 mmol, 0.05 equiv), copper (I) iodide (10.44 mg, 0.055 mmol, 0.1 equiv), 1M solution of tetrabutylammonium fluoride in hexanes (0.82 mL, 1.6 equiv), PLN-6 (181 mg, 0.61 mmol, 1.1 equiv). FCC purification: Gradient elution with mixtures hexane: AcOEt (4:1— >3:2). PLN-7: Orange hygroscopic solid, 52% yield (187 mg, 0.29 mmol).

[0313] 'H NMR (400 MHz, chloroform-d) 5 10.91 (s, 1H), 8.21 (d, J= 2.3 Hz, 1H), 7.96 (dd, J = 8.4, 1.9 Hz, 2H), 7.86 (dd, J= 8.7, 2.3 Hz, 1H), 7.57 (d, J= 15.3 Hz, 1H), 7.50 (dt, J= 8.4, 1.8 Hz, 2H), 7.44 (d, J= 15.3 Hz, 1H), 7.06 (d, J= 8.7 Hz, 1H), 6.80 (d, J= 3.6 Hz, 1H), 6.68 (d, J = 3.6 Hz, 1H), 4.50 (bb, 1H), 4.01 (s, 3H), 3.45 (t, J= 6.1 Hz, 2H), 3.40 (t, J= 6.6 Hz, 2H), 3.09 (t,.7= 7.1 Hz, 2H), 2.48 (t, J= 6.7 Hz, 2H), 1.79-1.65 (m, 4H), 1.61 - 1.53 (m, 2H), 1.51-1.41 (m, 11H), 1.39-1.30 (m, 4H).13C NMR (101 MHz, chloroform-d) 5 189.18 (C), 170.30 (C), 161.97 (C), 156.13 (C), 155.75 (C), 151.04 (C), 137.11 (C), 131.99 (CH), 131.83 (CH), 130.69 (CH), 128.76 (C), 128.43 (CH), 126.18 (CH), 121.78 (C), 119.21 (CH), 118.55 (CH), 118.48 (CH), 112.85 (C), 107.48 (CH), 94.01 (C), 80.63 (C), 79.18 (C), 70.99 (CH2), 70.41 (CH2), 52.74 (CH3), 40.73 (CH2), 30.18 (CH2), 29.80 (CH2), 29.14 (CH2), 28.55 (CH3), 26.77 (CH2), 26.04 (CH2), 25.53 (CH2), 19.55 (CH2). HRMS (ESI+): m / z calcd. for [M+H]+C38H46NO5: 644.3218; found: 644.3255 (deviation -5.8 ppm).

[0314] 7. 5-{5-{(E)-3-{4-[6-(Adamantan-1-ylmethylamino)hex-1-ynyl]phenyl}-3-oxoprop-l-enyl}furan-2-yl}-2-hydroxybenzoic acid (FAB-599).

[0315] FAB-599e (148 mg, 0.25 mmol, 1 equiv) was added on a solution of NaOH (40 mg, 1 mmol, 4 equiv) in MeOH:water (3: 1). The mixture was let to stir at 40 °C for 12 h. After this time, the organic solvent was removed under vacuum and a volume of DCM was added. The resulting organic phase was washed with aqueous HC1 (IN) (x2), dried over anhydrous MgSO₄, filtered and evaporated under vacuum. FCC purification (when necessary): Gradient elution with mixtures petrol ether: DCM: MeOH (44:44:2^40:40:20) + AcOH (1%). FAB-599: Orange solid, 90% yield (130 mg, 0.23 mmol)

[0316] 'H NMR (500 MHz, DMSO-d6) 88.17 (bb, 1H), 8.14 (s, 1H), 8.05 (d, J= 8.0 Hz, 2H), 7.75 (d, J= 8.7 Hz, 1H), 7.59 - 7.53 (m, 3H), 7.48 (d, J= 15.1 Hz, 1H), 7.17 (d, J= 3.0 Hz, 1H), 6.90 (d, J= 3.2 Hz, 1H), 6.75 (d, J= 8.5 Hz, 1H), 2.96 (bs, 2H), 2.64 (s, 2H), 2.55 (t, J= 6.6 Hz, 2H), 1.97 (s, 3H), 1.84 (bs, 2H), 1.70- 1.53 (m, 14H).13C NMR (126 MHz, DMSO-d6) 8 187.33, 170.71, 165.16, 157.69, 149.48, 136.88, 131.59, 130.40, 128.51, 128.41, 127.43, 126.38, 121.32, 120.17, 117.25, 117.10, 115.98, 106.43, 93.75, 80.71, 58.49, 48.07, 39.04, 36.01, 31.85, 27.33, 25.06, 24.30, 18.43. HRMS: m / z calcd. for [M+H] C37H40NO5 578.2901; found, 578.2909 (deviation -1.3 ppm).

[0317] 8. 5-{5-{(E)-3-{4-{6-[6-(tert-Butoxycarbonylamino)hexyloxy]hex-1-ynyl}phenyl}-3-oxoprop-l-enyl}furan-2-yl}-2-hydroxybenzoic acid (PLN-8).

[0318] Compound PLN-7 (141 mg, 0.22 mmol, 1 eq) was dissolved in pyridine (5 mL) and heated up to reflux (130 °C) overnight. After this time, the pyridine was removed using a high vacuum rotavapor. The residue was dissolved in AcOEt and the remaining trace of pyridine was extracted with aqueous HC1 5% (x2). The organic layer was dried over MgSCU, fdtered and concentrated under reduced pressure. The crude was purified by FCC (silica gel) (elution with a mixture hexane: DCM: MeOH 45:45:10). PLN-8: Red solid, 56% yield.

[0319] 'H NMR (400 MHz, methanol-d4) 88.36 (bs, 1H), 8.01 (d, J= 8.2 Hz, 2H), 7.89 (d, J = 8.3 Hz, 1H), 7.60 (d, J= 15.2 Hz, 1H), 7.54 - 7.46 (m, 3H), 7.01 - 6.96 (m, 2H), 6.85 (d, J= 3.5 Hz, 1H), 3.50 (t, J = 5.8 Hz, 2H), 3.46 (t, J= 6.5 Hz, 2H), 3.04 (t, J= 7.0 Hz, 3H), 2.48 (t, J= 6.7 Hz, 2H), 1.80-1.67 (m, 4H), 1.62-1.56 (m, 2H), 1.50-1.42 (m, 11H), 1.39-1.33 (m, 4H).13CNMR (101 MHz, methanol-d4) 8 190.75 (C), 163.61 (C), 158.56 (C), 158.15 (C), 151.99 (C), 138.24(C), 132.78 (CH), 132.03 (CH), 131.82 (CH), 130.24 (C), 129.48 (CH), 127.99 (CH), 122.21 (C), 121.19 (CH), 118.76 (CH), 118.51 (CH), 108.25 (CH), 94.80 (C), 81.40 (C), 79.79 (C), 71.87 (CH2), 71.32 (CH2), 41.32 (CH2), 30.95 (CH2), 30.70 (CH2), 29.97 (CH2), 28.80 (CH3), 27.69 (CH2), 26.99 (CH2), 26.54 (CH2), 19.95 (CH2). HRMS (ESI+): m / z calcd. for [M+H]+C37H44NO8 630.3061; found, 630.3089 (deviation -4.5 ppm).

[0320] 9. 5-{5-{(£)-3-{4-[6-(6-aminohexyloxy)hex-l-ynyl]phenyl}-3-oxoprop-l-enyl}furan-2-yl}-2-hydroxybenzoic acid (PLN-9).

[0321] To a 10 mL round bottom flask charged with a solution of PLN-8 (38.2 mg, 0.06 mmol) in DCM (7.6 mL), trifluoroacetic acid (2 mL) was added and the reaction was stirred for 4 h (rt). After complete reaction, the solvents were removed under reduce pressure. PLN-9: Purple solid, 64% yield.

[0322] NMR 'H NMR (400 MHz, methanol-d4) 8 8.32 (d, J= 2.3 Hz, 1H), 8.01 (d, J= 8.1 Hz, 2H), 7.91 (dd, J= 8.7, 2.2 Hz, 1H), 7.60 (d, J= 15.2 Hz, 1H), 7.53 (d, J= 15.2 Hz, 1H), 7.51 (d, J= 7.92 Hz, 2H), 7.03 - 6.97 (m, 2H), 6.85 (d, J= 3.6 Hz, 1H), 3.50 (t, J= 5.96 Hz, 2H), 3.46 (t, J = 6.48 Hz, 2H), 2.92 (t, J= 7.7 Hz, 3H), 2.49 (t, J= 6.7 Hz, 2H), 1.79 - 1.57 (m, 8H), 1.46 - 1.40 (m, 4H).13C NMR (101 MHz, MeOD) 8 190.71 (C), 163.64 (C), 157.91 (C), 152.08 (C), 138.27 (C), 132.77 (CH), 132.22 (CH), 132.02 (CH), 130.23 (C), 129.49 (CH), 127.78 (CH), 122.40 (C), 121.16 (CH), 118.92 (CH), 118.61 (CH), 108.41 (CH), 94.78 (C), 81.41 (C), 71.69 (CH2), 71.40 (CH2), 40.69 (CH2), 30.52 (CH2), 29.97 (CH2), 28.55 (CH2), 27.27 (CH2), 26.84 (CH2), 26.52 (CH2), 19.95 (CH2). HRMS (ESI+): m / z calcd. for [M+H]+C32H36NO6: 530.2537; found, 530.2549 (deviation -2.3 ppm).EXAMPLE 4

[0323] Targeting oxalate production by combining enzyme inhibition and proteolysis activation: A new therapeutic approach for primary hyperoxaluria type 1.

[0324] ABSTRACT

[0325] Primary hyperoxaluria type 1 (PHI) is a rare genetic disorder caused by hepatic overproduction of oxalate due to the deficiency of alanine-glyoxylate aminotransferase (AGXT). Therapeutic strategies targeting glycolate oxidase (GO) and lactate dehydrogenase A (LDHA), key enzymes in hepatic glyoxylate metabolism, have shown promise in reducing oxalate burden. However, recently approved siRNA approaches face limitations including high cost, unfavorable pharmacokinetics, interpatient variability, and limited global accessibility. Here, we report the development of compound 2, a dual GO / LDHA inhibitor (Ki = 390 nM and 40 nM, respectively) that also promotes hydrophobic tag-mediated autophagic degradation of LDHA. Its pharmacological efficacy was evaluated in Agxt- / - mice, both at the cellular level using primary hepatocytes and systemically via oral administration. Treatment resulted in a marked reduction in hepatic LDHA levels, urinary oxalate excretion, and renal calcium oxalate crystal deposition. These findings indicate compound 2 as a first-in-class, orally bioavailable small molecule with dual inhibitory and proteolytic activity, offering a new therapeutic candidate for PHI and other oxalate-related pathologies.

[0326] INTRODUCTION

[0327] Primary hyperoxaluria type 1 (PHI) is a rare autosomal recessive disorder characterized by the excessive hepatic production of oxalate, a metabolic end-product of glyoxylate (Figure 16A).1,2Oxalate accumulation leads to recurrent nephrolithiasis, nephrocalcinosis, and progressiverenal failure, often culminating in systemic oxalosis.2,3PHI is caused by mutations in the AGXT gene, which encodes alanine-glyoxylate aminotransferase (AGXT), a key hepatic enzyme that converts glyoxylate to glycine.4In the absence of AGXT activity, the hepatic enzyme glycolate oxidase (GO) converts glycolate to glyoxylate,5which is rapidly metabolized to oxalate by lactate dehydrogenase isozyme A (LDHA).6Beyond PHI,7dysregulated oxalate metabolism has been implicated in more prevalent conditions such as systemic inflammation,1metabolic dysfunction-associated steatotic liver disease (MASUD),8-10and cardiovascular disorders.11,12Thus, the pathological effects associated with elevated oxalate levels underscore the need for strategies to effectively reduce oxalate burden.

[0328] Therapeutic strategies for PHI have recently been transformed by RNA interference (RNAi)-based approaches. Liver-targeted small interfering RNAs (siRNAs) such as lumasiran13and nedosiran14— targeting GO and LDHA, respectively — have received regulatory approval (EMA and FDA, 2020; FDA, 2023) and are now in clinical use.15Additionally, small-molecule inhibitors such as stiripentol, a repurposed anticonvulsant with LDHA inhibitory activity have shown modest efficacy but are limited by hepatotoxicity.16,17Despite this array of different strategies, no multitargeted therapy for PHI has yet reached clinical trials.18,19Moreover, the existing therapies face several notable limitations, including high cost,20variability in patient response,21,22reduced efficacy in advanced disease stages,23and limited global accessibility.24,25Hence, there is a critical need for alternative therapeutic approaches that are cost-effective, pharmacologically efficient, and orally bioavailable.

[0329] Small molecules are attractive therapeutic alternatives due to their lower production cost,26oral bioavailability, and favorable pharmacokinetics.27Notably, dual inhibition of GO and LDHA addresses multiple upstream pathways of oxalate biosynthesis, which can offer superior therapeutic outcomes compared to monotherapy or siRNA-based combinations28Small molecule-induced protein degradation has emerged as a transformative strategy in drug development for metabolic diseases.29Unlike genetic silencing, small molecule-induced protein degradation enables selective post-translational cleavage of pathogenic proteins by hijacking the cell's proteolytic systems, such as the ubiquitin-proteasome system30or autophagy-lysosomal pathway.31Among the various strategies of small molecule-induced protein degradation, hydrophobic-tag protein degraders (HyT-PDs) are constructed by linking a selective ligand (SL) for the protein of interest (Pol) to a hydrophobic tag (HyT)32These bifunctional molecules induceproteolysis by mimicking misfolded proteins, effectively directing the Pol towards degradation pathways.33Without wishing to be bound by theory, this strategy can overcome the limitations of siRNA by using a single small molecule that selectively binds and degrades GO and LDHA.

[0330] Compound 1 (Figure 16B), a furyl salicylic acid (FSA), acts as a dual micromolar inhibitor of the human GO (AGO) and LDHA ( / / LDHA),34and could serve as a starting point for the development of SL. In the present study, we report the development of 5-[5-(3-oxo-3-phenylpropenyl)-2-furyl]salicylic acids (OPPFSAs), a new family of dual inhibitors with enhanced potency to the nanomolar range (Figure 16C). Moreover, we introduce compound 2, the first dual inhibitor that also promotes LDHA degradation via HyT-mediated proteolysis in the autophagy-lysosomal pathway (Figure 16D) a mechanism supported by extensive in silica characterization (Figure 16E).

[0331] Importantly, we indicate its in vivo efficacy through lowering urinary oxalate and elevating glycolate levels, resulting in a marked reduction of renal calcium oxalate crystal deposition in a murine PHI model (Figure 16F). These findings highlight the therapeutic effect of integrating dual enzymatic inhibition with targeted protein degradation as an approach for treating oxalate-related disorders.

[0332] RESULTS

[0333] Design strategy and synthesis of OPPFSAs and their derivatives

[0334] Compound 335(Figure 16B), which features a formyl group, serves as a precursor for the design of dual AGO / ALDHA inhibitors. Our docking studies suggest that this functionality plays a non-essential role in binding to the targeted proteins. Thus, in our previous work,34the chemical versatility of the formyl group was exploited to prepare / V-substituted aminomethylfuryl salicylic acids (AMFSAs, Figure 16B) via reductive amination.

[0335] Although AMFSAs act as dual inhibitors, their inhibitory activity remains at the micromolar range, suggesting that further structural optimization is needed to enhance their potency. To this end, we envisioned OPPFSAs as a vinylogous series of compound 3, incorporating a,0-unsaturated ketones and three aromatic rings, designated A-C (Figure 16C).

[0336] These new functionalities are designed to modulate the physicochemical properties of the molecules and to explore new interactions with biological targets, extending beyond the catalytic site. A family of OPPFSAs was developed by introducing various / z / -substituents on ring C, including terminal polar groups, halogens and alkyl chains (compounds 2 and 4-25, Table1). The polar functionalities at this position can influence the electronic character of ring C, enable polar interactions with the biological targets, and enhance the water solubility of the molecules. To this end, we incorporated electron withdrawing nitro and cyano groups (5 and 6) and the electron donating hydroxy group (7). Additionally, we included a benzylic alcohol (8), that can act as a hydrogen bond donor / acceptor, and aliphatic carboxylic acids (9 and 10) to facilitate hydrogen bonding or ionic interactions.

[0337] Table 1 Structure of oxophenylpropenyl furylsacylic acids (OPPFSAs) and their hydrogenated derivatives and total yields of synthesis.^Experimental total yields after purification.(b)In compound 29 the ketone group has been reduced to an alcohol, n = Number of steps in the synthetic route.

[0338] To increase molecular lipophilicity and promote hydrophobic interactions with the targets, halogens (11-12) and alkyl chains (2 and 13-26) were introduced as R substituents on ring C. Halogens also exert a mild electron-withdrawing effect, whereas alkyl chains act as weak electron donors. Linear alkyl chains of up to eight carbon in length (16, 18, 20-22) were employed to investigate the impact of extending the molecule beyond the catalytic site, following the design rationale of previous reported GO inhibitors.36

[0339] Further modifications on the alkyl chains were introduced to evaluate distinct chemical properties. To improve water solubility, and assess polar interactions with the targeted enzymes, isosteric substitutions at the benzylic methylene were carried out, incorporating oxygen (13), sulfur (14) and nitrogen (15). The isosteric replacement of a methyl group (16) with a trifluoromethyl group (17) aimed to probe electronic effects. Branching was explored using the A, A-dimethylamino group (15) and the isopropyl group (19). Unsaturations in compounds 23 and 24 were designed to reduce the rotational freedom of the side chain while increasing the electronic density beyond ring C, which can enable 7t-type interactions with the target enzymes. Finally, the / <? / 7-butyl carboxylates 24 and 25, which serve as precursors to carboxylic acids 9 and 10, respectively, were also included in the screening against recombinant enzymes. Importantly, compounds 2 and 26 were specifically designed as a HyT-PDs, incorporating adamantane HyT moieties linked via a six- or three-carbon spacer to the OPPF SA warhead, respectively. This feature was intended to promote target degradation through hydrophobic tagging. In OPPFSAs, the a,0-unsaturated ketone located between rings B and C contributes to a planar, highly conjugated structure. To explore structure-activity relationships, we synthesized a small set of hydrogenated derivatives of OPPFSAs (27-29, Table 1). The removal of this double bond increases the rotational freedom between rings B and C, disrupts the conjugation between the FSA core and ring C, andeliminates the coplanarity among the three aromatic rings. Without wishing to be bound by theory, these modifications can enhance the molecule’s adaptability to the binding sites of both targets.

[0340] Additionally, structurally related analogues that deviate from the canonical OPPFSA scaffold (pother structures") were designed by (i) replacing ring C with alkyl chains; (ii) substituting ring C with heteroaromatic rings such as pyrrole and furan; and (iii) fully hydrogenating ring B and the a,0-unsaturated ketone moiety (section SI and Figure 17).

[0341] A versatile synthetic route to OPPFSAs (4-25, Scheme 2 was developed, involving two to three steps (steps a-c) with total yields ranging between 32-82% (Table 1). The initial Suzuki-Miyaura coupling of methyl 5-iodosalicylate (30) and 5-formylfuran-2-boronic acid (31) yielded the ester-protected aldol substrate 32 (step a). Subsequent aldol condensation with selected acetophenones (33) afforded a, -unsaturated ketones type 34 (step b). In some cases, this step also induced simultaneous hydrolysis of the methyl ester, directly yielding the final acidic OPPFSA. When hydrolysis did not occur concurrently, it was achieved by refluxing in pyridine (step c).

[0342] The synthesis of compounds 2 and 26 required the preparation of the corresponding adamantane-functionalized terminal alkyne (35) or alkene (36), respectively. These intermediates were coupled with halogenated precursor 34-1 via a Sonogashira (step d) or Heck reaction (step e). While the coupling directly yielded the final acid 26, the synthesis of compound 2 required an additional ester hydrolysis step using aqueous sodium hydroxide (step f).

[0343] To obtain hydrogenated derivatives, selective hydrogenation of the double bond or non-selective hydrogenation of both the double bond and the ketone of intermediates type 34 was carried out using Raney Nickel® or palladium on charcoal catalysts, respectively (steps f and g, respectively). Following hydrolysis of the intermediate esters (step c), the final partial (27 and 28) or total (29) hydrogenated derivatives were obtained (Table 1).

[0344] Scheme 2. Synthesis of oxophenylpropenylfuryl salicylic acids (OPPFSAs) and hydrogenated derivatives.Conditions: (a) NEt3, Pd(OAc)2, DMF, rt (yield 87%). (b) (i) NaOH, THF: MeOH: H2O, rt; (ii) HC1 (yields 43-96%). (c) i) Py, reflux; ii) HC1 IN (yields 85-97%); (d) (Ph3P)2PdCl2, Cui, TBAF / hexane IM, THF (anhydrous), Ar, rt, 18 h (yield 67%). (e) Pd(OAc)2, DABCO, K2CO3, DMF (anhydrous), 120 °C, 12 h (91%). (f) (i) NaOH, MeOH: H2O (3:1), 40 °C, 12 h; (ii) HC1 IN (yield 90%). (g) H2, Raney Nickel®, THF, rt (yield 95%). (h) H2, Pd / C, THF, rt (yield 70%).

[0345] OPPFSAs exhibit enhanced potency compared to reference compound 1 as dual inhibitors of recombinant hGO and hLDHA[00346J Once a diverse array of final compounds was successfully synthesized, their inhibitory activity against the recombinant enzymes AGO and ALDHA was evaluated using fluorometric kinetic assays. An initial screening at 10 pM allowed us to assess inhibitory activity, compare with molecules from previous studies,34,35establish preliminary structure-activity relationships, and discard low potency molecules. The relative activity of AGO and ALDHA, as shown in Figure 18A-18B and section Table 4, ranged from 0% to 82% for AGO and 0% to 90% for ALDHA. Interestingly, 20 out of the 32 newly synthesized and tested compounds exhibited greater inhibitory effect against AGO and ALDHA than the reference compound 1 at 10 pM (yellow square, Figure 18A). Among them, 9 compounds inhibited both enzymes by 90% or more (red square, Figure 18A). Notably, approximately half of the new compounds achieved over 80% inhibition at 10 pM for at least one of the enzymes (Figure 18A-18B).

[0347] Among the different structural families (OPPFSAs, hydrogenated derivatives and other structures) OPPFSAs presenting alkyl chains (R = alkyl chain) were the most potent inhibitors. The presence of the phenyl ring C in the OPPFSA scaffold was found to be essential for potent inhibitory effect against both enzymes. Substitution of this ring with alkyl or heteroaryl moieties (S1-S5, other structures), resulted in a marked loss of inhibitory effect, particularly against AGO (Figures 18A-18B).

[0348] The nature of the para- substituent (R) on the phenyl ring C played a critical role. A direct correlation was observed between the hydrophobic character of R37and the inhibitory potency against both AGO and ALDHA (Figure 18A). Hydrophobic substituents, such as halogens (11 and 12) and alkyl chains (2 and 13-25) yielded potent dual inhibitors (Figure 18B). Linear alkyl chains up to eight carbon atoms (16-22) were well tolerated. Longer chains were not evaluated to avoid pharmacokinetic liabilities. Neither branching (e.g., Tr in 19) nor the presence of unsaturation (double or triple bonds in 9, 23 and 24) negatively impacted activity. Notably, butyl (20), 6-chlorohex-l-ynyl (23) and the bulky adamantyl-terminated chain (2) conferred particularly strong inhibition.

[0349] Isosteric replacement of methylene groups in R with heteroatoms such as oxygen, sulfur (13 and 14 vs. 18) or nitrogen (15 vs. 19) generally reduced dual inhibitory activity, with sulfur (14) being the best tolerated among these. The loss of activity affecting mainly ALDHA inhibition, observed when the methyl group (R) in compound 16 was replaced by either an electronwithdrawing trifluoromethyl group (17) or an electron-donating methoxy group (13) indicates that without wishing to be bound by theory, hydrophobicity, rather than electronic effects, modulate the inhibitory potency. In contrast, substituents with polar terminal groups were generally less effective, except for compound 9, which contains a carboxyvinyl group and showed good inhibition.

[0350] The presence of the planar a, P-unsaturated ketone moiety was found to be important for AGO inhibition, as hydrogenation of the alkene to alkane, or of both the alkene and ketone to an alkane-alcohol, generally resulted in reduced potency (“Hydrogenated derivatives”, Figure 18A), except for compound 28 (Figure 18B).

[0351] Following the initial screening, those compounds with inhibition percentages over 30% were selected for ICso determination (Table 2 and Figures 19-43 and Figures 82-105. For AGO, most ICso values ranged between 0.8 and 5 pM, whereas for ALDHA, many compounds exhibitednanomolar inhibitory activity. Notably, compounds 20 (R = butyl) and 23 (R = 6-chlorohex-l -inyl) showed IC50 values below 100 nM against ALDHA. Additionally, compound 23 demonstrated nanomolar potency against AGO. The incorporation of the adamantane HyT moiety in 2 preserved high affinity for both AGO and ALDHA, with excellent IC50 values of 1.05 pM and 160 nM, respectively.

[0352] Table 2. Summary of IC50 values obtained for representative compounds on recombinant glycolate oxidase and lactate dehydrogenase A. Comparison with the reference compound 1.Data are expressed as IC50 ± SD (pM). ND: IC50 not determined as the inhibition percentage was lower than 30% at 10 pM in the initial screening. ICsos are determined as a mean of four replicates with ten concentrations of inhibitor each (AGO concentration, 25 nM; ALDHA concentration, 0.05 units / mL; glycolate concentration, 180 pM; pyruvate concentration, 1 mM). Figure 19-43 and Figure 82-105 for semi logarithmic plots.

[0353] OPPFSAs reduce intracellular oxalate in PHI mouse primary hepatocytes, with compound 2 achieving levels below baseline

[0354] Encouraged by the results obtained in recombinant enzymes, we next evaluated the compounds in a more physiologically relevant model: primary hepatocytes isolated from Agxt" mice (Figure 44).8To induce oxalate overproduction, cells were stimulated with 5 mM glycolic acid (GA), and the compounds’ ability to reduce oxalate production was assessed.34In our previous study, compound 1 reduced extracellular oxalate to undetectable levels at a concentration of 10 pM suggesting strong activity. However, extracellular measurements alone can overestimate efficacy, as they do not capture intracellular metabolic dynamics. Under the same conditions, compound 1 did not significantly reduce intracellular oxalate levels compared to untreated, GA-stimulated hepatocytes. A substantial decrease to basal levels was only observed when the dose of compound 1 was increased to 50 pM (Figure 45). These results underscore the importance of directly quantifying intracellular oxalate as a more accurate and sensitive indicator of metabolic inhibition.

[0355] Based on the ICso results (Table 2), the top 17 compounds that outperformed compound 1 were selected for the in vitro cellular experiments. To rule out cytotoxic effects, we assessed cell viability in primary hepatocytes from Agxt1' mice using the CellTiter-Blue Assay. No evidence of cell death was observed for any of the compounds at 50 pM (Figure 46).

[0356] Next, Agxt ' primary hepatocytes were loaded with GA (5 mM) and either treated with vehicle (DMSO) or the selected inhibitors at 10 pM and 50 pM. To set a proper baseline, each set of cells had a control without GA stimulation. After 24 hours, intracellular oxalate levels were quantified to assess the extent of reduction achieved by each compound as fold change relative to GA loading (Figure 45). At 50 pM, all compounds reduced intracellular oxalate levels; however, statistically significant reductions were observed only for compounds 1, 2, 16, and 21. No significant differences were detected among these compounds. To draw more definitive conclusions, further evaluation was conducted at 10 pM. In contrast to compound 1, several OPPFSAs were able to significantly reduce intracellular oxalate levels at this lower concentration. Notably, significant reductions with respect to GA (*) and cells treated with compound 1 ('^) were observed with the alkyl-substituted derivatives 16 (R = -Me), 19 (R = -’Pr), 21 (R = -hexyl) and the adamantyl-terminated derivative 2. Among these, compound 2 exhibited the most pronounced effect, reducing intracellular oxalate to levels well below the physiological baseline observed in untreated control cells (Figure 45).

[0357] While many members of the OPPFSA family showed robust inhibition in vitro., their translation to cellular assays revealed substantial variability. One possible explanation lies in differences in solubility and membrane permeability — two key factors influencing cellular uptake and intracellular availability.

[0358] To explore this, we used Molecular Operative Environment (MOE) to calculate physicochemical parameters related to bioavailability: logP (octanol / water partition coefficient) and hlogD (distribution coefficient at pH 7.4). The logP values for the tested OPPFSAs ranged from 4.0 to 8.2, with compound 2 showing the highest lipophilicity. Corresponding hlogD values ranged between 3.8 to 8.1. Compounds 16 and 19 showed hlogD values of 4.7 and 5.7, respectively, while 20 and 23 exhibited higher values (6.21 and 6.25), suggesting that subtle differences in partitioning behavior can underline the observed variability in cellular performance.

[0359] Despite similar hlogD / logP ratios across the family, compound 2 displayed the lowest ratio (0.6), due to an hlogD value of 5.2, indicating a distinct and more favorable distribution behavior under physiological conditions (Figure 47).

[0360] These findings suggest that solubility and partitioning properties can contribute to the divergent cellular effects observed among OPPFSA analogs and should be considered when translating in vitro potency to complex biological models.

[0361] Compound 2 is a non-competitive inhibitor of hGO and hLDHA

[0362] The favorable phenotypic effect observed for compound 2 in PHI primary hepatocytes prompted further characterization of its biological profile. Thus, inhibition kinetics of compound 2 was carried out to determine the affinity constant of the inhibitor (K) and its mechanism of inhibition in competence with the enzymatic substrates, GA for AGO, and pyruvic acid (PA) for ALDHA (Table 3). For comparison, compounds 20 and 23, the most potent dual inhibitors based on ICso values (Table 2), were also analyzed. The enzymatic activity of recombinant hGO and ALDHA was measured in the presence of four / five different inhibitor concentrations and ten different substrate concentrations. As a result, K values ranged from 40 to 400 nM for hGO and from 26 to 90 nM for ALDHA (Table 3).

[0363] Table 3 K values obtained for selected compounds on recombinant human glycolate oxidase (AGO) and lactate dehydrogenase enzymes (ALDHA).Units: pM. Ki obtained in experiments using ten different substrate concentrations and four / five inhibitor concentrations each (n =3). Values of a = 1 indicate pure noncompetitive inhibition; values of a > 1 indicate noncompetitive mixed type inhibition (mixed competitive). “Competitive inhibitor.

[0364] These values represent an increase of 63 to 600-fold in the affinity for AGO and 68 to 235-fold in the affinity for ALDHA, with respect to I.34The three compounds, 2, 20 and 23, like I,34behave as non-competitive inhibitors of AGO (a > 1, mixed competitive).38In the case of ALDHA, compounds 2 and 20 exhibit a pure noncompetitive inhibition profile (a = 1) with respect to the substrate pyruvate, similar to compound I.34In contrast, compound 23 is a pyruvatecompetitive inhibitor of ALDHA with a Ki value of 26 nM (linear and nonlinear plots of all the studied compounds are presented in Figures 48-59). These results demonstrate that OPPFSAs exhibit a markedly improved inhibitory profile compared to their AMFSA and FSA predecessors; however, these findings do not fully explain why compound 2 has the most potent intracellular oxalate reduction among the compounds tested.

[0365] Compound 2 induces LDHA degradation via the autophagy-lysosomal pathway in PHI mouse hepatocytes

[0366] Compound 2 was designed to include an adamantane moiety aiming to promote target proteolysis as a HyT-PD. To evaluate whether this mechanism contributes to the compound's potent intracellular oxalate-lowering activity in PHI primary hepatocytes, we treated hepatocytes isolated from AgxtAmice with increasing concentrations of compound 2 (2, 10, 20, and 50 pM). While GO protein levels remained unchanged (Figure 60A), LDHA abundance was significantly reduced at 50 pM compared to vehicle-treated controls (Figure 60A). As expected, we observed no significant differences in the mRNA expression of Ldha or Haol (which encodes GO) at any concentration (Figure 60A). These results indicate that compound 2 induces LDHA degradation in Agxt'1' mouse primary hepatocytes.

[0367] Given the evidence that compound 2 promotes LDHA degradation, we next sought to elucidate the underlying proteolytic mechanism. Previous reports suggest that HyT-PDs can activate one or both of the major proteolytic systems, the ubiquitin-proteasome system and the autophagy-lysosomal pathway.32,33We thus investigated the involvement of these pathways in the degradation of LDHA upon binding to compound 2 (Figure 60B). To this end, we assessed LDHA protein abundance following treatment with 2 in the presence or absence of pathway-specific inhibitors. First, we co-treated cells with compound 2 and MG-132, a well-characterized proteasome inhibitor.39No significant increase in LDHA protein levels was observed in cells treated with 2 and MG-132, compared to cells treated with compound 2 alone, therefore ruling out proteasome involvement in this process (Figure 60C).

[0368] To determine whether compound 2 induces LDHA degradation via the autophagy-lysosomal pathway, we silenced autophagy-related gene 5 (Atg5) using siRNA (siAtg5). The encoded protein, ATG5, is essential for autophagosome formation and has previously been shown to be required for the action of compounds that engage this pathway.40Transfection with siAtg5 reduced ATG5 expression by approximately 70% (Figure 65). Under these conditions, the reduction in LDHA protein levels induced by compound 2 was abolished, and LDHA protein levels were significantly higher than in cells treated with the scrambled siRNA (siScr) (Figure 60C). These results indicate that compound 2 promotes LDHA degradation via an autophagydependent lysosomal pathway.

[0369] In silico analysis of compound 2 reveals enzyme-specific differences in binding dynamics and spatial disposition

[0370] To investigate the molecular basis for the differential HyT-PD activity of 2, and study its binding to hGO and ALDHA, we performed atomistic molecular dynamics (MD) simulation. Previous to MD simulation, docking poses of 2 were obtained on the crystal structures of ALDHA isozyme form (PDB ID: 1I10)41and hGO (PDB ID: 2RDT)36and are displayed in Figure 66-68.The docking pose of 2 on hGO served as a template to construct a homology model of hGO. The docking pose of 2 on TzLDHA (subunit B) was used on MOE to build a dimeric complex with subunit A of ALDHA containing cofactor 1,4-dihydronicotinamide adenine dinucleotide (NAI) and oxamic acid (OXM) inhibitor. The hGO homology model and the LDHA dimeric complex were later corrected and prepared using MOE QuickPrep module for further MD analysis (vide infra). The stability of protein-ligand complexes during MD simulations was assessed using Root MeanSquare Deviation (RMSD) analysis and validated by a Solvent Accessible Surface Area (SAS A) analysis in a timeframe of 100 ns (Figure 69-Figure 72).

[0371] Docked poses of 2 on hGO and hLDHA show the introduction of its salicylic acid warhead into the catalytic sites of the enzymes, interacting with key substrate-binding and catalytic residues (detailed binding interactions are presented in Figure 66-68). Given the size of 2, the furan ring and side chain are displayed towards the hydrophobic access channel of hGO and, in the case of ALDHA, towards the cofactor hydrophobic cleft. On AGO, the furan ring shows a it-stacking interaction with the key residue Trpl 10. On ALDHA the intermediate carbonylic group in 2 H-bonds the backbone chain of Ala29 at the entrance of the catalytic site pocket, as does the NAD cofactor through one of its oxygen-phosphate groups. In the case of AGO, the adamantane is displayed towards a hydrophobic cleft set by residues Ilel 15, Leul43, Vall39 and Tyrl34. This region has been identified as an allosteric binding site for new AGO inhibitors.42The adamantane orientation is also helped by a H-bond interaction established between the backbone chain of Alai 11 and the protonated methylamino moiety of 2. In the case of ALDHA, the adamantane methylamino moiety is displayed toward the end of the NAD+cofactor cleft, with the adamantane group on the hydrophobic region set by Ilel 15, Val52, Ilel 19, and Phel 18. In silico docking values of free energy of binding (AG) of 2 in AGO (PDB ID: 2RDT) and ALDHA are -13.15 and -11.93 Kcal / mol, respectively.

[0372] The MD simulation initiates with a homology model of AGO, using the docked pose of 2 on PDB ID 2RDT as template (Figure 69 - 73). RMDS cluster analysis on the 2-AGO ensemble after the simulation resulted in the most populated cluster-representative structure depicted in Figures 61A, 61C and 73 (blue). As shown, the salicylic moiety of ligand 2 remains inside the catalytic pocket establishing two H-bonds with catalytically important residue Arg263 through its carboxylic acid. Compared to frame 1 (t = 0, Figures 61A and 61C, pink), the ligand shifts towards the a3 helix, which is displaced outward. This displacement affects several catalytic site residues, including Trpl 10 and His260, which are repositioned away from the catalytic site. Additionally, a third H-bond is observed between the carbonyl group of 2 and the backbone of Lys211 (Figure 61A). This interaction seems to induce a distortion in the aE helix, which is essential for preserving the structural integrity of the catalytic site. The new ligand-protein conformation is partially driven by the adamantane moiety's propensity to occupy the hydrophobic cleft formed by residues Ilel 15, Leul43, Vall39, and Tyrl34. Furthermore, it establishes new interactions with loop 4 residues,including Phel86 and Glyl87, thereby promoting the closed loop conformation. However, this inward orientation of the adamantane moiety (Figure 61C, blue) is unfavorable for inducing protein degradation supporting the absence of hGO degradation activity observed in the presence of ligand 2

[0373] An RMDS cluster analysis was also carried out to the 2- / zLDHA dimeric ensemble MD simulation. The most populated cluster-representative structure is shown in Figures 61B, 61D and 78 (blue). Here, the pose of 2 on TzLDHA subunit B is noteworthy. Its salicylic moiety is inserted into the catalytic site establishing H-bonding interactions with important residues such as Argl05 and Arg 168 (Figure 5B), which are responsible for the pyruvate / lactate binding, and the backbone chain of Ala237.43The furan and unsaturated phenylketone are displayed toward the entrance of the catalytic site and along the NAD cofactor binding site, keeping a uniform distance with close residues such as Val30 and Arg98 among others. On the other hand, the adamantane moiety is set outside the substrate and cofactor binding site, protruding the protein surface and exposing both the amino and adamantane ring to the surrounding solvent. The outside disposition of the adamantane moiety is clearly observed when overlaying the initial 2-ALDHA dimeric ensemble (t = 0, pink) and the pose of 2 in the most populated cluster of ALDHA (blue) after the MD simulation (Figures 61B and 61D). The most notable differences between the two binding poses are the deeper insertion of 2 into the catalytic pocket after the simulation, caused by the outward movement of the a-lG / a-2G helix, and the external positioning of the adamantane ring. This outward orientation of the adamantane (Figure 61D), along with the stabilization of the dimeric complex by 2, likely accounts for the ligand-induced protein degradation observed in hepatocytes.32,33

[0374] Compound 2 presents molecular chameleonicity with solvent-triggered intramolecular bond formation.

[0375] To complete the in silico characterization of compound 2, we further analyzed its theoretical pharmacokinetic properties within the beyond Rule-of-5 (bRo5) chemical space. Protein degraders often display poor solubility and limited permeability due to their structural complexity. However, molecular chameleonicity — the ability of a compound to adapt its conformation to different environments — has been proposed as a compensatory mechanism that can overcome unfavorable ADME predictions.

[0376] This adaptive behavior refers to a molecule’s capacity to form intramolecular hydrogen bonds (IMHBs) in nonpolar media, effectively masking polar groups, while adopting more extended conformations in aqueous environments to improve solubility.44Chameleonicity directly correlates with dynamic molecular descriptors such as tridimensional polar surface area (3D-PSA), radius of gyration (Rgyr), and IMHB formation across solvent44,45Conformational clustering based on these parameters typically identifies three populations: (i) polar-open states in water, (ii) folded-nonpolar states in chloroform, and (iii) intermediate conformations. These clusters help relate chameleonic behavior to permeability indices like cChameCS and cChameP.

[0377] Conformational sampling of compound 2 revealed a strong, environment-dependent reorganization of polarity and shape. Conformers generated in water and chloroform occupied distinct regions across all three descriptors — 3D-PSA, Rgyr, and IMHBs.

[0378] In water, 3D-PSA values clustered at high levels, reflecting extended conformations that favor solvent hydrogen bonding. In contrast, chloroform exposure caused a ~10 A2decrease in PSA, indicating polarity masking via chameleonic folding (Figure 79A). Shielding polar surface area necessarily requires a geometrical contraction, and this is captured by the Rgyr distributions. Whereas the aqueous ensemble spans a wide range of sizes (4.8-10 A), the chloroform ensemble collapses into a narrow band centered at ~ 5 A (Figure 79B). This polarity-size trade-off is underpinned by a surge in IMHB formation: virtually all chloroform conformers form two or more internal hydrogen bonds, compared with a single IMHB in most aqueous structures. The data provide a direct mechanistic link between folding and polarity masking (Figure 79C).

[0379] Bringing these descriptors together highlights their synergistic interplay. Low-PSA, low-Rgyr conformers are exclusively associated with >2 IMHBs and are sampled only in chloroform, whereas high-PSA, high-Rgyr conformers with < 1 IMHB populate the aqueous ensemble. The scatter plot therefore visualizes the solvent-specific conformational partitioning that is the essence of IMHB-mediated chameleonicity (Figure 62A).

[0380] Clustering of the full dataset yielded three centroids (Figure 62B) that trace a step-wise opening pathway: a highly folded, double-IMHB ‘closed’ state, a partially unfolded ‘semi-closed’ state with one IMHB, and a fully extended ‘open’ state devoid of internal bonds. The progression between these states rationalizes the continuous distributions observed in Figures 62A and 79A-79C.

[0381] The separation between the aqueous and chloroform ensembles is captured quantitatively by cChameCS = 0.312 and cChameP = 0.093. Values in this range indicate moderate but meaningful chameleonicity — sufficient to reduce polarity in non-polar environments without compromising conformational diversity in water. Similar magnitudes have been associated with orally bioavailable bRo5 molecules in previous benchmark studies.44,45

[0382] Oral administration of compound 2 degrades hepatic LDHA, decreases urinary oxalate, and diminishes renal deposition of calcium-oxalate crystals in PHI mice

[0383] Once binding affinity, potency and mechanism of action for compound 2 had been successfully characterized, we sought to translate our findings in vivo. Agxt~'~ mice were orally administered once a day for ten consecutive days, with either a vehicle solution (0.6% methylcellulose and 0.5% Tween® 80 in water), or compound 2 at a dose of 20 mg / kg / day dissolved in the same formulation. To monitor changes in excreted levels of oxalic and glycolic acid, urine was collected starting five days prior to dosing to set a baseline and throughout the treatment regimen. On day ten, liver, kidney and plasma samples were harvested to further investigate the systemic effects of compound 2 (Figure 63A). We first focused on the liver, since it is the source of oxalate overproduction in PHI.46Consistent with the in vitro results in Agxf ' primary hepatocytes, we observed a significant reduction in hepatic LDHA expression in the treatment group as compared to vehicle (Figure 63B), with no changes in GO expression (Figure 7C). This reduction in LDHA abundance, together with the reported inhibition of this isozyme (Table 3) led to a 40% decrease in LDH hepatic activity (Figure 63D). Although compound 2 demonstrated potent pharmacological activity, we assessed its hepatotoxic effects was crucial. Notably, circulating levels of aspartate transaminase (AST), remained unchanged (Figure 63E), and a modest but statistically significant reduction in alanine aminotransferase (ALT) was observed in the treatment group compared to vehicle (Figure 63F). Finally, histological examination of liver sections using H& E staining revealed no morphological changes between the groups (Figure 63G). Moreover, there were no statistically significant differences in blood glucose, body weight, liver weight, or liver-to-body weight ratio between the groups (Figures 81A-81D) These findings indicate that treatment with compound 2 has no hepatotoxic effects.

[0384] After the behavior of compound 2 in the liver was fully characterized, we turned our attention to its effect on the renal system. The primary parameter used to monitor the progression of PHI is urinary oxalate concentration.46Mice subjected to treatment with 2 exhibited asignificant reduction in oxalate levels by day three when compared to the baseline, an effect that persisted up to day seven of treatment (Figure 64A). Concurrently, and consistent with GO inhibition, urinary glycolic acid levels progressively increased over the course of the treatment, reaching statistical significance by day seven (Figure 64B). This metabolic shift indicated that calcium-oxalate crystal deposition within the kidneys can also be altered. To test this, kidney sections from mice treated with either vehicle or compound 2 were stained with H& E and imaged under polarized light to visualize calcium-oxalate crystals as bright birefringent spots (Figure 64C). Quantification of the percentage area occupied by these crystals within the kidney sections showed a striking reduction in mice treated with compound 2 (Figure 64D). Altogether, these comprehensive studies highlight HyT-PD, and particularly compound 2, as an effective oxalate-lowering therapy for PHI.

[0385] DISCUSSION AND NON-LIMITING CONCLUSION

[0386] Studies on the FDA approved treatments for PHI, lumasiran and nedosiran, have raised concerns about inter-patient variability in therapeutic response.15,23,47,48Both therapies employ GalNAc-conjugated RNA interference strategies targeting either GO or LDHA, but are limited by their restrictive administration route and high commercialization costs.26To overcome these limitations, we aimed to develop an alternative, orally bioavailable, and cost-effective therapy that combines dual GO / LDHA targeting and a dual inhibition-degradation mechanism. In this study, we optimized the chemical structure of previous GO and LDHA dual inhibitors to (1) enhance their potency into the nanomolar range, (2) promote selective degradation of target enzymes, and (3) demonstrate potent in vivo efficacy. We report the synthesis and characterization of 33 new compounds, derived from oxophenylpropenyl furylsacylic acid. Based on their inhibitory profile in recombinant enzyme assays, 17 inhibitors were selected for intracellular oxalate quantification in AgxU primary hepatocytes. Among them compound 2 emerged as the most potent in lowering intracellular oxalate. Oral administration of compound 2 to Ag i" mice significantly decreased hepatic LDHA abundance and enzymatic activity, and consequently lowered urinary oxalate levels, and abolished renal calcium-oxalate crystal deposition without evidence of hepatotoxicity.

[0387] Traditionally, GO inhibitors have been designed based on the H-bond interactions with key amino acids in the active site and to engage the hydrophobic channel that provides access to it.36For example, in the case of CCPST inhibitor, its carboxylic acid interact with Arg263 and Tyr26 while the hydrophobic interactions are mediated through the / ?-chlorophenyl moiety.49Similarly, large LDHA inhibitors are known to occupy both the polar substrate site and the hydrophobic cleft associated with NAD+cofactor binding.50Due to this overlapping features, the general structure of active-site-directed GO and LDHA inhibitors can exhibit a high degree of similarity, particularly given that both enzymes act on the same a-hydroxy- or a-ketoacid substrates. This overlap has motivated simultaneous targeting of both enzymes using a single warhead, a strategy that was previously explored by our group.34However, no inhibitor has reached clinical use for PHI mainly due to lack of potency.

[0388] Building upon these insights we designed the OPPFSAs scaffold including the key structural and functional aspects of dual GO / LDHA inhibition. These compounds feature a planar P-hydroxyacid warhead derived from FSA core that mimics substrate binding at the catalytic site of both enzymes. Substituents at the furan C5 position fit in the hydrophobic channel of GO or the cofactor cleft in LDHA. Additionally, MD simulations of compound 2 bound to AGO and ALDHA support a critical role for the a, P-unsaturated ketone linker that bridges the FSA core and the side chain. This is supported by the observed loss of inhibitory potency happening after its partial (double bond) or total (double bond and carbonyl group) reduction. In addition, / rara-substitution on the ring C of OPPFSAs (R) significantly enhances inhibitory activity against both targets. The hydrophobic nature of R is particularly important, with alkyl chains between one and eight carbon lengths yielding potent dual inhibitors. Notably, OPPFSAs 2, 20 and 23 achieved nanomolar Ki values improving affinity up to 600 and 230-fold for AGO and ALDHA, respectively, compared to the lead 1.

[0389] Given that assays based on recombinant enzymes do not fully reflect the complexity of PHI pathophysiology, we evaluated the ability of the most promising compounds to reduce intracellular oxalate levels in Agxf ' primary hepatocytes. Surprisingly, compound 20 (bearing a butyl side chain) and compound 23 (bearing a chlorohexynyl chain), the two most potent inhibitors of recombinant AGO and ALDHA, were less effective than expected in this cellular setting. This discrepancy can be attributed to their structure containing acidic and highly hydrophobic ends. GO inhibitors are typically characterized by poor membrane translocation and poor water solubility that hinder their bioavailability and therapeutic utility.51In contrast, compounds 16 (R = Me) and 19 (R = Tr), featuring shorter and less hydrophobic alkyl chains, demonstrated greater efficacy in reducing intracellular oxalate levels compared to the positive control. Thus, the effectiveness of a GO inhibitor is believed to depend more on its ability to reach and bind GO inside the cell than onits intrinsic potency. Interestingly, although compound 2 shares the same polar head and hydrophobic side chain as other GO inhibitors, the presence of an amino group within the hydrocarbon side chain distinguishes it from compounds 20 and 23. This structural feature can contribute to its enhanced activity, as compound 2 effectively decreases intracellular oxalate level, even below baseline level, in the same GA-challenged Agxt' ' hepatocytes where 20 and 23 fail to do so. Although multiple factors can contribute to the enhanced cellular efficacy of compound 2, the two most likely properties could be: (1) favorable solubility and distribution properties in aqueous and hydrophobic media at physiological pH, and (2) a dual mechanism of action involving not only enzymatic inhibition but also targeted enzyme degradation.

[0390] To gain insight into the behavior of OPPFSAs in hydrophobic and aqueous environments, we performed in silico calculations of key physicochemical parameters. Theoretical values of logP and hlogD, indicates that without wishing to be bound by theoyr that compounds 2, 16 and 19 present a better solubility at physiological pH than compounds 20 and 23.

[0391] Notably, the presence of a distal amino group in the flexible side chain of compound 2 allows a balanced chameleonic behavior: it adopts extended, polar conformations in aqueous media while folding into compact, IMHB-stabilized states in lipophilic environments. This solvent-dependent reorganization enables the molecule to reconcile its unexpected water solubility with the low polarity required for passive membrane permeation, in agreement with its moderate cChameCS (0.312) and low cChameP (0.093) values. Collectively, these findings indicate that compound 2 possesses the conformational adaptability necessary to navigate the permeabilitysolubility trade-off typical of bRo5 chemical space, providing a mechanistic rationale for its favorable bioavailability.

[0392] To further explain why compound 2 still outperformed 16 and 19, despite displaying a similar behavior in aqueous environments, it is important to revisit the design rationale underlying its structure. Compound 2 was designed as an HyT-PD, featuring an adamantyl tail attached to the OPPFSA warhead through a flexible linker. In primary hepatocytes, compound 2 induces a dosedependent reduction in LDHA protein abundance. This shift in enzymatic activity is comparable to the effects of irreversible enzyme inhibitors32or siRNA-mediated knockdown,53where enzymatic function can only be restored through de novo protein synthesis. To further elucidate the mechanism, we explored how compound 2 mediates this reduction of LDHA in vitro. This effect could not be attributed to a downregulation at the transcriptional level, as no significantchanges in Ldha mRNA levels were observed across all tested concentrations. Notably, experiments in the presence of proteasome and autophagy inhibitors in Agxf1' mouse primary hepatocytes, demonstrated that compound 2 promotes the lysosome-dependent degradation of LDHA.

[0393] In addition, MD simulations of the 2-ALDHA complex offers a mechanistic rationale for the non-competitive behavior observed with respect to the substrate, while also supporting the role of the adamantane moiety in proteolysis activation. Initially, compound 2 binds at the cofactor site. However, during the simulation, conformational rearrangements lead to the outward displacement of the a-lG / a-2G helix. This structural shift allows the salicylic acid warhead of compound 2 to penetrate deeper into the catalytic site and interact with substrate-binding residues in a subsequent step. In this final conformation, the adamantane ring remains exposed above the protein surface, making it accessible for recognition by the cellular degradation machinery and thereby enabling proteolysis.

[0394] Following confirmation of compound 2’s oxalate-lowering efficacy in cellular models, we evaluated its therapeutic performance in vivo. To our knowledge, only one small-molecule LDHA degrader has been previously described, specifically in the context of pancreatic cancer. This PROTAC therapy recruits the E3 ligase van Hippel Lindau and induces LDHA proteolysis by the ubiquitin-proteasome system, although its efficacy has been observed only with intraperitoneal administration.54Oral administration is a key factor in improving treatment adherence and patient convenience during chronic therapies5,56Therefore, leveraging its chameleonic properties to enhance oral bioavailability,44compound 2 was administered to mice with PHI (Agxt'1') via oral gavage. Urinary oxalate excretion, a direct biomarker of hyperoxaluria, showed a significant reduction by the third day of treatment relative to baseline levels. Importantly, compound 2 retained its LDHA degrader activity in vivo, reducing hepatic LDHA protein levels by approximately 50%. This reduction in LDHA protein abundance corresponded to a similar decrease in LDH activity measured in liver tissues. Although no degradation of GO protein was observed, its inhibition was supported by a significant increase in urinary glycolate levels during treatment, consistent with GO blockade. The most compelling evidence of compound 2’s therapeutic efficacy was marked by more than 85% reduction in renal calcium-oxalate crystal deposition following treatment. This near-complete clearance of oxalate crystals from kidney tissue substantially lowers the risk of oxalate urolithiasis, representing a clinically meaningfuloutcome. Importantly, no signs of toxicity were observed in the in vivo study. These findings highlight compound 2 as a drug candidate for the treatment of PHI.

[0395] Despite the promising in vitro and in vivo results of compound 2, this molecule failed to induce degradation of GO, an enzyme whose knock-out is known to be harmless,57,58and plays a key role in managing hyperoxaluria.59We propose a mechanistic explanation for the lack of GO degradation, supported by MD simulations of the protein-ligand complex. One key advantage of MD simulations is their ability to capture conformational changes in the protein over time, providing insights into the spatial orientation of ligand moieties critical for degradation. To induce proteolysis, the adamantane HyT moiety must be exposed on the protein surface, serving as a recognizable motif for the degradation machinery. Interestingly, our simulations revealed that, unlike typical GO inhibitors, which stabilize an open conformation of loop 4 and keep the hydrophobic access channel exposed, compound 2 promotes closure of loop 4. This effect is driven by interactions between the adamantane group and hydrophobic residues within loop 4 and is further stabilized by a hydrogen bond between the ketone moiety of compound 2 and Lys211. This resulting distortion of the aE helix and the catalytic pocket buries the adamantyl group within the protein, preventing its surface exposure and thus precluding proteolysis. These findings indicate that redesigning the linker to be more rigid and extended, positioning the adamantyl group further from the catalytic site, can allow future derivatives to induce GO degradation.

[0396] In conclusion, compound 2 represents a first-in-class, orally bioavailable small-molecule that effectively reduces hepatic oxalate production and thereby nephrolithiasis through a dual mechanism: inhibition of GO and LDHA, and selective degradation of the latter. This dual-action profile results in significantly lowered urinary oxalate levels and kidney calcium-oxalate crystal burden in PHI mice, with no observed toxicity, offering a compelling alternative to current siRNAbased therapies targeting the same enzymes. As the first reported small-molecule dual GO / LDHA inhibitor capable of inducing LDHA proteolysis both in vitro and in vivo, compound 2 introduces anewHyT-PD-based chemical scaffold with therapeutic utility in PHI. Beyond PHI, this approach can have broader implications for treating other primary hyperoxalurias, and metabolic diseases involving oxalate overproduction, such as MASLD and cardiovascular diseases.

[0397] EXPERIMENTAL SECTION

[0398] CHEMICAL METHODS

[0399] General considerations

[0400] All solvents and chemicals were used as purchased without further purification. The progress of the reactions was controlled by: (a) thin layer chromatography (TLC) on aluminum plates (Merck AL Silicagel 60 F254) and visualized by UV lamp (254 nm) or by staining with iodine or solutions of (i) permanganate in distilled water (1% p / v), (ii) vanillin (0.5 g) in sulfuric acid: EtOH (1:3, 100 mL), (iii) ninhydrin (1 g) in EtOH (450 mL); (b) Liquid Chromatography-Mass Spectrometry (LC-MS) in an Agilent 1200 HPLC coupled with an Agilent 6110 single quadrupole, with electrospray ionization (ESI) in positive or negative mode. The HPLC column used was a Zorbax Eclipse XDB-C18 5 pM, 4.6 * 150 mm column or a Waters XBridge Column C18 3.5 pM, 2.1 x 100 mm. Purification by flash column chromatography (FCC) was performed on Silicagel Merck 60 (230-400 mesh ASTM). Automated FCC was carried out using a Biotage Isolera™One apparatus with UV-Vis detector. Purification by preparative layer chromatography (PLC) was made on Silicagel Merck 60 F254, 0,5 mm plates. The non-crystalline compounds were shown to be homogeneous by chromatographic methods and characterized by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). 'H-NMR and13C-NMR spectra have been recorded in a 2-channel 400 MHz Bruker Nanobay Avance III HD, a 2-channel 400 MHz Bruker Avance NEO, and a 2-channel 500 MHz Bruker Avance NEO spectrometers using DMSO-tfc, methanol-6 / 4, chloroform- / or acetone-rfc. Chemical shifts (5) are quoted in parts per million (ppm) and are referenced to residual H in the deuterated solvent as the internal standard. Coupling constants (J) are expressed in Hz. Splitting patterns are designated as follows: bb, broad band; bs, broad singlet; d, doublet; dd, double doublet; m, multiplet; pdt, pseudo double triplet; q, quadruplet; s, singlet; t, triplet. HRMS were recorded by time of flight (TOF) mass spectrometry with ESI in positive or negative mode, using a QTOF apparatus Bruker Compact. Purity of the compounds was assessed by HPLC using an Agilent 1200 instrument with diode-array detector equipped with a suitable column (General HPLC methods, below). Column temperature was set at 25 °C. Absorbance was measured at 214 and 254 nm. Purity of final compounds was higher than 95% by HPLC (except for compound 7, 15). An EnSpire® Multimode Plate Reader (PerkinElmer) was used to measure fluorescence. For fluorometric assays, OptiPlate black opaque 96-well microplates (PerkinElmer) were used. For all assays, the final volume in each well was 200 pL. Multi-channel pipettes were used for the addition of the reagents in the wells. Interferences in the kinetic fluorometric protocols were discarded as the slope in the linear interval was corrected by subtraction of the baseline reading registered before the addition of the substrate.

[0401] General HPI. C methods

[0402] General HPLC setup: Detection X = 214 and 254 nm; flow rate 0.8 mL / min; solvent A [water (0.1% HCOOH) / acetonitrile (0.1% HCOOH)]; solvent B [acetonitrile (0.1% HCOOH) 100%]. Injection volume 10 pL.

[0403] Column 1: Zorbax Eclipse XDB-C18 5pM, 4.6 x 150 mm.

[0404] Column 2: Waters XBridge Column-C182.5 pm, 4.6 x 75 mm.

[0405] Column 3: Waters XBridge Column-C82.5 pm, 4.6 x 75 mm.

[0406] HPLC method A. Solvent A (95 / 5). Isocratic A 2 min +gradient A— B 8 min +isocratic B 1 min (compound 8).

[0407] HPLC method B Solvent A (90 / 10). Isocratic A 2 min +gradient A— > B 8 min +isocratic B 1 min (compounds 1 and SI).

[0408] HPLC method C: Solvent A (70 / 30). Isocratic A 2 min +gradient A— B 8 min +isocratic B 1 min (compounds 7, 9, 10 and S3).

[0409] HPLC method D Solvent A (60 / 40). Isocratic A 2 min +gradient A— B 8 min +isocratic B 1 min (compound S5).

[0410] HPLC method E Solvent A (50 / 50). Isocratic A 2 min +gradient A— > B 8 min +isocratic B 1 min (compounds 6, 14-16, 25, 26, S2, S4 and S6).

[0411] HPLC method F: Solvent A (40 / 60). Isocratic A 2 min +gradient A— > B 8 min +isocratic B 1 min (compound 23).

[0412] HPLC method G Solvent A (30 / 70). Isocratic A 2 min +gradient A— B 8 min +isocratic B 1 min (compounds 12, 18-21, 24, 27 and 28).

[0413] HPLC method H. Solvent A (10 / 90). Isocratic A 2 min +gradient A— B 8 min +isocratic B 1 min (compound 22).

[0414] HPLC method I: Solvent A (60 / 40). Isocratic A 2 min +gradient A-^> B 17 min +isocratic B 1 min (compounds 4, 5, 11 and 13).

[0415] HPLC method J'. Solvent A (70 / 30). Isocratic A 2 min +gradient A— > B 17 min +isocratic B 1 min (compound 17).

[0416] HPLC method K Solvent A (80 / 20). Isocratic A 1 min +gradient A— B 5 min +isocratic B 1 min (compound 2).

[0417] General conditions for aldol condensation

[0418] Protocol A: To a stirred solution of compound type 32 (1.5 equiv) in solvent mixture containing mainly MeOH, a 10 N NaOH aqueous solution was added dropwise. After 10 min, a solution of 31 (1 equiv) was added. After 24 h stirring at rt, the reaction was quenched by addition of HC1 3N until pH 3 at rt. The organic solvents were evaporated under reduced pressure and 20 mb of AcOEt were added. The organic layer was washed with water and brine, dried over MgSO₄, filtered and concentrated under reduced pressure. The final products (salicylate esters or salicylic acids) were purified by FCC. Protocol B. To a stirred solution of compound type 32 (1.2 equiv) in EtOH (1 mL), 10 N NaOH aqueous solution (3 or 30 equiv) was added dropwise at 0°C and the resulting solution was stirred at the same temperature for 30 minutes. After this time, 334(50 mg, 0.215 mmol, 1 equiv) was added and the reaction was allowed to reach rt. Upon reaction completion (3 h or overnight), it was acidified with HC1 (1 N) to pH 1-2 at 0°C and the solvent was evaporated under reduced pressure. The residue was percolated through a short column of silica gel eluting with DCM / MeOH (80:20) and the product was purified by PLC.

[0419] General conditions for selective double bond hydrogenation in a, f -unsaturated ketones type 33

[0420] The corresponding a, -unsaturated ketone type 33 (1 equiv) was dissolved in THF at rt. Then, Raney Nickel ” activated catalyst (50% slurry in water) (1 mL) was added. For the reaction to take place, hydrogen (g) was bubbled through the solution for 1 h. Once completed, the reaction mixture was filtered through celite and concentrated under reduced pressure. FCC purification was performed by elution using mixtures of petrol ether and AcOEt.

[0421] General conditions for hydrolysis of methyl salicylates

[0422] The corresponding methyl salicylate (1 equiv) was dissolved in pyridine (10 mL / mmol) and heated up to reflux during 15 h. After this time, pyridine was removed using a high vacuum rotavapor. The residue was dissolved in AcOEt, and the remaining trace of pyridine was extracted with aqueous HC1 IN. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. FCC purification was performed using petrol ether: DCM: MeOH mixtures acidified with 1% AcOH.

[0423] 5-{5-{(£')-3-{4-[6-(adamantan-l-ylmethylamino)hex-l-ynyl]phenyl}-3-oxoprop-l-enyl}furan-2-yl}-2-hydroxybenzoic acid (2)

[0424] Compound 33-Ad (148 mg, 0.25 mmol, 1 equiv) was added on a solution of NaOH (40 mg, 1 mmol, 4 equiv) in MeOH:water (3:1). The mixture was let to stir at 40 °C for 12 h. After thistime, the organic solvent was removed under vacuum and a volume of DCM was added. The resulting organic phase was washed with aqueous HC1 (IN) (x2), dried over anhydrous MgSO4, filtered and evaporated under vacuum. FCC purification (when necessary): Gradient elution with mixtures petrol ether: DCM: MeOH (44:44:2— >40:40:20) + AcOH (1%). Orange solid, 90% yield (130 mg, 0.23 mmol). 'HNMR (500 MHz, DMSO-tL) 88.17 (bb, 1H), 8.14 (s, 1H), 8.05 (d, J = 8.0 Hz, 2H), 7.75 (d, J= 8.7 Hz, 1H), 7.59 - 7.53 (m, 3H), 7.48 (d, J= 15.1 Hz, 1H), 7.17 (d, J = 3.0 Hz, 1H), 6.90 (d, J= 3.2 Hz, 1H), 6.75 (d, J= 8.5 Hz, 1H), 2.96 (bs, 2H), 2.64 (s, 2H), 2.55 (t, J= 6.6 Hz, 2H), 1.97 (s, 3H), 1.84 (bs, 2H), 1.70 - 1.53 (m, 14H).13C NMR (126 MHz, DMSO-6) 8 187.33, 170.71, 165.16, 157.69, 149.48, 136.88, 131.59, 130.40, 128.51, 128.41, 127.43, 126.38, 121.32, 120.17, 117.25, 117.10, 115.98, 106.43, 93.75, 80.71, 58.49, 48.07, 39.04, 36.01, 31.85, 27.33, 25.06, 24.30, 18.43. HRMS: m / z calcd. for [M+H] C37H40NO5 578.2901; found, 578.2909 (deviation -1.3 ppm). HPLC (method K) (1 = 254 nm), 95.4%; (X = 214 nm), 97.2%; 'R = 7.16 min (column 3).

[0425] (E')-2-Hydroxy-5-[5-(3-phenyl-3-oxoprop-l-en-l-yl)furan-2-yl]benzoic acid (4)

[0426] General conditions for aldol condensation (protocol B) using acetophenone (30 pL, 0.26 mmol, 1.2 equiv) and 10 MNaOH aqueous solution (65 pL, 0.65 mmol, 3 equiv). Reaction time, 3 h. PLC purification: DCM / AcOH (99:1). Orange solid (48 mg, 0,14 mmol, 67% yield), mp, decomposes at approximately 180 °C. ’H NMR (400 MHz, methanol-cX) 88.35 (s, 1H), 8.05 (d, J = 7.3 Hz, 2H), 7.83 (d, J= 8.3 Hz, 1H), 7.64 - 7.50 (m, 5H), 6.96 (s, 2H), 6.81 (s, 1H).13C NMR (101 MHz, methanol-c / 4) 8 191.9, 163.7, 158.3, 151.9, 139.5, 134.0, 132.0, 131.4, 129.8 (2C), 129.5 (2C), 128.1, 122.0, 121.1, 118.7 (2C), 108.0. HRMS (TOF, ES’): m / z calcd for C20H13O5 (M - H+) 333.0763, found 333.0757 (deviation -1.8 ppm). HPLC (method I) (X = 254 nm), 96.4%; ( = 214 nm), 95.3%; ' = 12.75 min (column 1).

[0427] (£)-2-Hydroxy-5-{5-[3-(4-nitrophenyl)-3-oxoprop-l-en-l-yl]furan-2-yl} benzoic acid (5)

[0428] General conditions for aldol condensation (protocol B) using 4-nitroacetophenone (43 mg, 0.26 mmol, 1.2 equiv) and 10 M NaOH aqueous solution (65 pL, 0.65 mmol, 3 equiv). Reaction time, 3 h. PLC purification: DCM / AcOH (99:1). Reddish-brown solid (46 mg, 0,06 mmol, 56% yield), mp, decomposes at approximately 180 °C. 'H NMR (400 MHz, methanol -t ) 8 8.40-8.35 (m, 3H), 8.28 (d, J= 9 Hz, 2H), 7.78 (dd, J= 8.6, 2.2 Hz, 1H), 7.65 (d, J= 15.1 Hz, 1H), 7.57 (d, J= 15.1 Hz, 1H), 7.04 (d, J= 3.6 Hz, 1H), 6.89 (d, J= 8.6 Hz, 1H), 6.82 (d, J= 3.6Hz, 1H).13CNMR(101 MHZ, methanol-d4) 8189.9, 164.0, 159.5, 151.5, 144.5, 133.0, 130.6 (2C), 130.3, 128.4, 124.8 (2C), 123.9, 122.4, 121.2, 120.7, 118.1, 117.7, 107.9. HRMS (TOF, ES ): m / z calcd for C20H12NO7 (M - H+) 378.0614, found 378.0638. HPLC (method I) = 254 nm), 97.0%; (X = 214 nm), 97.9%; R = 13.01 min (column 1).

[0429] (£)-5-{5-[3-(4-Cyanophenyl)-3-oxoprop-l-en-l-yl]furan-2-yl}-2-hydroxybenzoic acid (6)

[0430] General conditions for hydrolysis of methyl salicylates, using 33-CN. FCC purification: Gradient elution using petrol ether: DCM: MeOH (49:49:2— >40:40:20) acidified with 1% AcOH. Reddish-orange solid (87% yield). 'H NMR (400 MHz, DMSO-t / 6) 8 8.25 (d, J= 8.6 Hz, 2 H), 8.25 (d, J= 2,3 Hz, 1H), 8.08 (dd, J= 8.1, 2.3 Hz, 1H), 8.07 - 8.03 (m, 2H), 7.62 (s, 2H), 7.26 (d, J= 3.6 Hz, 1H), 7.15 (d, J= 3.6 Hz, 1H), 7.08 (d, J= 8.7 Hz, 1H).13C NMR (101 MHz, DMSO-d&) 8 187.8, 171.3, 161.7, 155.8, 150.3, 141.1, 132.8, 131.6, 131.2, 128.9, 126.3, 121.2, 120.6, 118.3, 118.1, 117.5, 114.8, 114.2, 108.3. HRMS: m / z calcd. for [M-H] CisHisOe 327.0869; found, 327.0870 (deviation: 0.3 ppm). HPLC (method E) (X = 254 nm), 95.6%; (X = 214 nm), 97.9%; 'R = 6.23 min (column 2).

[0431] (E')-2-Hydroxy-5-{5-[3-(4-hydroxyphenyl)-3-oxoprop-l-en-l-yl]furan-2-yl}benzoic acid (7)

[0432] General conditions for aldol condensation (protocol A) using 31 (120 mg, 0.48 mmol, 1 equiv), / 2-hydroxyacetophenone (133 mg, 0.97 mmol, 2 equiv), NaOH 10 N (0.48 mL, 4.8 mmol, 10 equiv), THF (2 mL) and MeOH (5 mL). FCC purification: Gradient elution using petrol ether: DCM: MeOH (49:49:2 >45:45:10) acidified with 1% AcOH. Reddish-orange solid (75% yield). ’HNMR (500 MHz, methanol-d4) 88.31 (d, J= 2.2 Hz, 1H), 8.03 - 8.00 (m, 2H), 7.94 (dd, J= 8.7, 2.3 Hz, 1H), 7.59 (d, J= 15.3 Hz, 1H), 7.55 (d, J= 15.3 Hz, 1H), 7.02 (d, J= 8.7Hz, 1H), 6.95 (d, J= 3.6 Hz, 1H), 6.93 - 6.89 (m, 2H), 6.86 - 6.84 (m, 1H).13C NMR (126 MHz, methanol-c ) 8 190.3, 173.5, 163.9, 163.5, 157.3, 152.3, 132.6, 132.4, 132.2, 131.1, 127.6, 122.7, 120.2, 119.1, 119.0, 116.5, 108.3. HRMS: m / z calcd. for [M+H] C20H15O6 351.0869; found, 351.0881 (deviation +3.4 ppm). HPLC (method C) ( = 254 nm), 85.3%; (X = 214 nm), 84.6%; R = 9.13 min (column 2).

[0433]

[0434] (E)-2-Hydroxy-5-{5-{3-[4-(hydroxymethyl)phenyl]-3-oxoprop-l-en-l-yl}furan-2-yl}benzoic acid (8)

[0435] General conditions for hydrolysis of methyl salicylates, using 33-CH2OH (65 mg, 0,17 mmol). FCC purification: Gradient elution using petrol ether: DCM: MeOH (49:49:2 >45:45: 10) acidified with 1% AcOH. Reddish-orange solid (91% yield). 'H NMR (400 MHz, DMSO- e) 8 8.19 (d, J= 2.4 Hz, 1H), 8.07 (d, J= 8.3 Hz, 2H), 7.86 (dd, J= 8.6, 2.4 Hz, 1H), 7.57 (s, 2H), 7.51 (d, J= 8.3 Hz, 2H), 7.18 (d, J = 3.6 Hz, 1H), 6.97 (d, J= 3.6 Hz, 1H), 6.85 (d, J= 8.6 Hz, 1H), 5.37 (s, 1H), 4.61 (s, 2H).13CNMR(101 MHz, DMSO) 8 188.0, 171.1, 163.8, 156.7, 149.8, 147.9, 136.3, 130.0, 129.4, 128.2, 126.4, 126.3, 120.4, 118.5, 117.3, 117.0, 106.8, 62.5. HRMS: zw / zcalcd. for [M+H] C21H15O6363.0869; found, 363.0858 (deviation -3.0 ppm). HPLC (method A) (L = 254 nm), 100%; (k = 214 nm), 95.6%; 'R = 10.82 min (column 2).

[0436] 2-Hydroxy-5-{5-{(£)-3-[4-((£)-3-hydroxy-3-oxoprop-l-enyl)phenyl]-3-oxoprop-l-enyl}furan-2-yl} benzoic acid (9)

[0437] Compound 24 (20 mg, 0.04 mmol, 1 equiv) was dissolved in DCM (4 mL). Trifluoroacetic acid (1.49 g, 1 mL, 13 mmol, large excess), was added on the solution and stirring at rt was maintained for 2 h. After this time solvents were removed under vacuum. FCC purification: Gradient elution using petrol ether:dicloromethane:methanol (49:49:2^45:45: 10) acidified with 1% AcOH. Reddish-orange solid (93% yield). ’H NMR (400 MHz, DMSO-cfc) 8 8.25 (d, J = 2.3 Hz, 1H), 8.14 (d, J= 8.5 Hz, 2H), 8.10 (dd, J= 8.7, 2.3 Hz, 1H), 7.89 (d, J= 8.4 Hz, 2H), 7.68 (d, J= 16.0 Hz, 1H), 7.66 (d, J= 15.3 Hz, 1H), 7.59 (d, J= 15.3 Hz, 1H), 7.23 (d, J = 3.6 Hz, 1H), 7.14 (d, J= 3.6 Hz, 1H), 7.09 (d, J= 8.7 Hz, 1H), 6.70 (d, J= 16.1 Hz, 1H).13C NMR(101 MHz, DMSO-t / e) 8187.9, 171.4, 167.3, 161.4, 155.3, 150.5, 142.6, 138.6, 138.4, 131.6, 130.3, 128.8, 128.5, 126.1, 121.7, 120.9, 120.3, 118.1, 117.9, 113.9, 108.2. HRMS: m / z calcd. for C23H17O7 405.0974; found, 405.0976 (deviation +0.5 ppm). HPLC (method C) (X = 254 nm), 100%; (X = 214 nm), 100%; 'R = 9.77 min (column 2).

[0438] (£)-5-{5-{3-[4-(2-Carboxyethyl)phenyl]-3-oxoprop-l-en-l-yl}furan-2-yl}-2-hydroxybenzoic acid (10)

[0439] Compound 25 (1 equiv) was dissolved in DCM (4 mL). Trifluoroacetic acid (1.49 g, 1 mL, 13 mmol, large excess), was added on the solution and stirring at rt was maintained for 2 h. FCC purification: Gradient elution using petrol ether:dicloromethane:methanol (49:49:2^45:45: 10) acidified with 1% acetic acid. Reddish-orange solid (90% yield). 'H NMR (500 MHz, DMSO-tZe) 8 8.24 (d, J= 2.3 Hz, 1H), 8.09 (dd, J= 8.7, 2.3 Hz, 1H), 8.04 (d, J= 8.3 Hz, 2H), 7.63 (d, J= 15.0 Hz, 1H), 7.56 (d, J= 15.0 Hz, 1H), 7.44 (d, J= 8.3 Hz, 2H), 7.20 (d, J= 3.6 Hz, 1H), 7.13 (d, J= 3.6 Hz, 1H), 7.09 (d, J= 8.7 Hz, 1H), 2.93 (t, J= 7.5 Hz, 2H), 2.61 (t, J= 7.6 Hz, 2H).13C NMR (126 MHz, DMSO) 8 188.1, 173.6, 171.4, 161.3, 155.1, 150.5, 146.5, 135.7, 131.6, 129.9, 128.7, 128.5, 126.1, 120.9, 119.9, 118.1, 113.9, 108.1, 34.7, 30.3. HRMS: m / z calcd. for C23H17O7407.1053; found, 407.1090 (deviation +2.1 ppm). HPLC (method C) (X = 254 nm), 100%; (X = 214 nm), 100%; T = 9.54 min (column 2).

[0440] (2T)-2-Hydroxy-5-{5-[3-(4-bromophenyl)-3-oxoprop-l-en-l-yl]furan-2-yl}benzoic acid (11)

[0441] General conditions for aldol condensation (protocol B) using 4-bromoacetophenone (51 mg, 0.26 mmol, 1.2 equiv) and 10 M NaOH aqueous solution (650 pL, 6.5 mmol, 30 equiv). Reaction time: overnight. PLC purification: DCM / AcOH (99:1). Red-orange solid (33 mg, 37% yield), mp, decomposes at approximately 180 °C. 'H NMR (400 MHz, methanol-t / 4) 88.38 (d, J = 2.2 Hz, 1H), 8.00 - 7.96 (m, 2H), 7.79 (dd, J= 8.6, 2.2 Hz, 1H), 7.72 -7.68 (m, 2H), 7.60 (d, J = 15.2 Hz, 1H), 7.52 (d, J= 15.2 Hz, 1H), 6.98 (d, J = 3.6 Hz, 1H), 6.91 (d, J = 8.6 Hz, 1H), 6.80 (d, J= 3.6 Hz, 1H).13C NMR (101 MHz, methanol -t / 4) 8 190.5, 163.7, 158.9, 151.7, 138.5, 133.1 (2C), 132.3, 131.2 (2C), 130.7, 128.8, 128.3, 121.6, 121.6, 119.7, 118.3, 117.9, 107.9. HRMS (TOF, ES’): m / z calcd for C2oHi205Br (M - H+) 410.9868, found 410.9856. HPLC (method I) (X = 254 nm), 98.1%; (X = 214 nm), 98.2%;ZR= 14.63 min (column 1).

[0442] (£)-2-Hydroxy-5-{5-[3-(4-iodophenyI)-3-oxoprop-l-en-l-yl]furan-2-yl} benzoic acid (12)

[0443] General conditions for hydrolysis of methyl salicylates, using 33-1 (90 mg, 0.19 mmol, 1 equiv) FCC purification: Gradient elution using petrol ether: DCM: MeOH (49:49:2 >45:45:10) acidified with 1% AcOH. Reddish-orange solid (94% yield).1H NMR (400 MHz, methanol-d4) 8 8.39 (d, J= 2.3 Hz, 1H), 7.99 (pdt, J= 8.6, 2.5 Hz, 2H), 7.79 (dd, J= 8.59, 3.14 Hz, 1H), 7.70 (pdt, J= 8.6 Hz, 2.4 Hz, 2H), 7.60 (d, J = 15.2 Hz, 1H), 7.51 (d, J= 15.2 Hz, 1H), 6.98 (d, J= 3.6 Hz, 1H), 6.90 (d, J= 8.6 Hz, 1H), 6.80 (d, J= 3.6 Hz, 1H).13C NMR (101 MHz, methanol-d4) 8 190.5, 163.7, 158.9, 151.7, 138.5, 133.1, 132.3, 131.2, 130.7, 128.8, 128.2, 121.6, 121.6, 119.7, 118.3, 117.9, 107.9. HRMS: m / z calcd. for [M-H] C20H12O51458.9730; found, 458.9760 (deviation -8.0 ppm). HPLC (method G) (X = 254 nm), 95.9%; (X = 214 nm), 97.2%; 'R = 4.53 min (column 2).

[0444] (E)-2-Hydroxy-5-{5-[3-(4-methoxyphenyl)-3-oxoprop-l-en-l-yl]-2-furanyl}benzoic acid (13)

[0445] General conditions for aldol condensation (protocol B) using 4-methoxyacetophenone (43 mg, 0.26 mmol, 1.2 equiv) and 10 M NaOH aqueous solution (650 pL, 6.5 mmol, 30 equiv). Reaction time: overnight. PLC purification: DCM / AcOH (99:1). Red-orange solid (74 mg, 0.20 mmol, 94% yield), mp, decomposes at approximately 180 °C. 'HNMR (500 MHz, MeOD) 58.38 (s, 1H), 8.09 (d, J= 8.3 Hz, 2H), 7.78 (d, J= 8.5 Hz, 1H), 7.57 (m, 2H), 7.05 (d, J= 8.3 Hz, 2H), 6.92 (t, J= 5.6 Hz, 2H), 6.78 (d, J= 3.5 Hz, 1H), 3.89 (s, 3H).13C NMR (126 MHz, MeOD) 5 190.2, 165.3, 163.8, 158.5, 151.8, 132.2, 131.9 (2C), 131.3, 130.7, 128.2, 121.7, 120.7, 118.3, 118.2, 115.0 (2C), 107.7, 56.1. HRMS (TOF, ES’): mlz calcd for C21H13O6 (M - H+) 363.0869, found 363.0843. HPLC (method I) (X = 254 nm), 95.9%; (X = 214 nm), 96.4%; K = 11.50 min (column 1).

[0446] (E)-2-Hydroxy-5-{5-{3-(4-(methylthio)phenyl)-3-oxoprop-l-en-l-yl}furan-2-yl}benzoic acid (14)

[0447] General conditions for hydrolysis of methyl salicylates, using 33-SMe (41 mg, 0.1 mmol, 1 equiv). FCC purification: Gradient elution using petrol ether: DCM: MeOH (49:49:2^45:45: 10) acidified with 1% AcOH. Reddish-orange solid (85% yield). 'HNMR (500 MHz, methanol-t / i) 8 8.39 (d, J= 2.3 Hz, 1H), 8.01 (d, J= 8.5 Hz, 2H), 7.79 (dd, J= 8.6, 2.3 Hz, 1H), 7.56 (d, J= 3.7 Hz, 2H), 7.36 (d, J = 8.5 Hz, 2H), 6.95 (d, J = 3.6 Hz, 1H), 6.91 (d, J = 8.6 Hz, 1H), 6.79 (d, J = 3.6 Hz, 1H), 2.55 (s, 3H).13C NMR (126 MHz, methanol-d4) 8 190.6, 163.6, 158.6, 151.9, 147.8, 135.6, 131.7, 130.8, 130.0, 128.2, 126.1, 121.8, 121.1, 119.4, 118.3, 118.2, 107.8, 14.6. HRMS: m z calcd. for [M-H] C21H15O5S 379.0640; found, 379.0662 (deviation 5.8 ppm). HPLC (method E) ( = 254 nm), 98.4%; (X = 214 nm), 99.2%; 'R = 8.41 min (column 2).

[0448] ( )-5-{5-{3-[4-(7V, A-diinethylamino)phenyl]-3-oxoprop-l-en-l-yl}furan-2-yl}-2-hydroxybenzoic acid (15)

[0449] General conditions for aldol condensation (protocol A) using 31 (130 mg, 0.5 mmol, 1 equiv), / ?-(A, A-dimethylamino)acetophenone (129 mg, 0.79 mmol, 1.5 equiv), NaOH 10 N (0.53 mL, 5.3 mmol, 10 equiv), DCM (1 mL) and MeOH (5 mL). FCC purification: Gradient elution using petrol ether: DCM: MeOH (49:49:2— >-45:45:10) acidified with 1% AcOH. Reddish-orange solid (83% yield). 'H NMR (500 MHz, methanol-6 / 4) 8 8.39 (d, J = 2.2 Hz, 1H), 8.01 (d, J = 9.0 Hz, 2H), 7.79 (dd, J= 8.6, 2.2 Hz, 1H), 7.59 (d, J= 15.2 Hz, 1H), 7.51 (d, J= 15.2 Hz, 1H), 6.91 (d, J= 8.6 Hz, 1H), 6.89 (d, J= 3.5 Hz, 1H), 6.80 - 6.76 (m, 3H), 3.09 (s, 6H).13C NMR (126 MHz, methanol-6 / 4) 8 189.5, 163.5, 158.1, 155.4, 152.2, 132.0, 130.7, 130.2, 128.1, 126.8, 121.9,119.9, 118.9, 118.3, 112.1, 107.6, 40.1. HRMS: m z calcd. for [M+H] C22H20NO5378.1341; found, 378.1339 (deviation -0.5 ppm). HPLC (method E) (1 = 254 nm), 94.6%; (X = 214 nm), 93.2%; 'R = 6.92 min (column 2).

[0450] (7:)-2-Hydroxy-5- [5-[3-oxo-3-( / i-tolyl)prop-l -en-1 -yl|furan-2-yl!benzoic acid (16)

[0451] General conditions for hydrolysis of methyl salicylates, using 33-Me (110 mg, 0.3 mmol, 1 equiv) and pyridine (5 mL). FCC purification: Gradient elution using petrol ether: DCM: MeOH (49:49:2— >-45:45:10) acidified with 1% AcOH. Reddish-orange solid (86% yield). 'HNMR (500 MHz, DMSO- L) 8 8.17 (d, J= 2.4 Hz, 1H), 8.01 (d, J= 8.2 Hz, 2H), 7.85 (dd, J= 8.5, 2.2 Hz, 1H), 7.56 (s, 2H), 7.39 (d, J= 8.1 Hz, 2H), 7.17 (d, J= 3.6 Hz, 1H), 6.96 (d, J= 3.6 Hz, 1H), 6.84 (d, J= 8.6 Hz, 1H), 2.41 (s, 3H).13C NMR (126 MHz, DMSO-fifc) 8 187.9, 171.0, 159.1, 149.8, 143.3, 135.3, 130.0, 129.4, 129.4, 128.4, 126.3, 120.4, 117.3, 117.0, 106.8, 21.2. HRMS: m,'z calcd. for C21H15O5 347.0919; found, 347.0887 (deviation -9.2 ppm). HPLC (method E) = 254 nm), 98.2%; ( = 214 nm), 100%; < R = 8.10 min (column 2).

[0452] (E')-2-Hydroxy-5-{5-{3-[4-(trifluoromethyl)phenyl]-3-oxoprop-l-en-l-yl}furan-2-yl}-benzoic acid (17)

[0453] General conditions for aldol condensation (protocol B) using 4-trifluoromethylacetophenone (51 mg, 0.26 mmol, 1.2 equiv) and 10 M NaOH aqueous solution (65 pL, 0.65 mmol, 3 equiv). Reaction time: 3 h. PLC purification: DCM / petroleum ether / AcOH (90:9:1). Red solid (62 mg, 0.15 mmol, 71% yield), mp, decomposes at approximately 180 °C. 'H NMR (400 MHz, methanol-d4) 88.34 (s, 1H), 8.17 (d,,7 = 7.9 Hz, 2H), 7.80 (d,,7 = 8.1 Hz, 3H), 7.57 (d, J = 15.2 Hz, 1H), 7.46 (d, J= 15.2 Hz, 1H), 6.95 (s, 1H), 6.91 (d, J = 8.6 Hz, 1H), 6.78 (s, 1H).13C NMR (101 MHZ, methanol-6 / 4) 8 190.4, 174.9, 163.6, 158.6, 151.7, 142.6, 134.8 (q, J = 32 Hz), 132.7, 131.4, 130.4, 129.9 (2C), 128.1, 126.7 (q, J= 3.9 Hz, 2C), 125.2 (q, J = 272 Hz), 121.9, 121.8, 118.5, 118.0, 108.2. HRMS (TOF, ES ): m / z calcd for C21H12O5F3 (M - H+) 401.0637, found 401.0661. HPLC (method J) (X = 254 nm), 98.8%; ( = 214 nm), 97.8%; 'R = 14.29 min (column 1).

[0454] (E)-5-{5-[3-(4-Ethylphenyl)-3-oxoprop-l-en-l-yl]furan-2-yl}-2-hydroxybenzoic acid (18)

[0455] General conditions for aldol condensation (protocol A) using 31 (150 mg, 0.6 mmol, 1 equiv), / 2-ethyl acetophenone, (180 pL, 1.2 mmol, 2 equiv), NaOH 10N (0.3 mL, 3 mmol, 5 equiv), DCM (0.5 mL) and MeOH (10 mL). FCC purification: Gradient elution using petrolether: DCM: MeOH (49:49:2— >45:45:10) acidified with 1% AcOH. Reddish-orange solid (52% yield). ’HNMR (400 MHz, methanol-d4) 88.30 (d, J= 2.3 Hz, 1H), 7.99 (d, J= 8.3 Hz, 2H), 7.95 (dd, J= 8.7, 2.3 Hz, 1H), 7.57 (d, J= 3.8 Hz, 2H), 7.39 (d, J= 8.3 Hz, 2H), 7.03 (d, J= 8.7 Hz, 1H), 6.98 (d, J= 3.6 Hz, 1H), 6.86 (d, J= 3.6 Hz, 1H), 2.75 (q, J= 7.6 Hz, 2H), 1.28 (t, J= 7.6 Hz, 4H).13C NMR (101 MHz, methanol-d4) 8 191.5, 163.6, 157.5, 152.2, 151.5, 137.2, 132.6, 131.7, 129.8, 129.3, 127.6, 122.7, 120.6, 119.2, 119.1, 108.5, 29.9, 15.7. HRMS: m / z calcd. for C22H19O5 363.1232; found, 363.1242 (deviation +2.8 ppm). HPLC (method G) ( = 254 nm), 100%; (X = 214 nm),3.54 min (column 2).

[0456] (£)-2-Hydroxy-5-{5-[3-(4-isopropylphenyl)-3-oxoprop-l-en-l-yl]furan-2-yl] benzoic acid (19)

[0457] General conditions for hydrolysis of methyl salicylates, using 33-iPr (150 mg, 0.6 mmol, 1 equiv). FCC purification: Gradient elution using petrol ether: DCM: MeOH (49:49:2— >45:45:10) acidified with 1% AcOH. Reddish-orange solid (89% yield). 'HNMR (400 MHz, methanol-t / 4) 8 8.30 (d, J= 2.2 Hz, 1H), 8.00 (d, J= 8.4 Hz, 2H), 7.93 (dd, J= 8.7, 2.3 Hz, 1H), 7.56 (d, J= 3.8 Hz, 2H), 7.41 (d, J= 8.2 Hz, 2H), 7.02 (d, J= 8.7 Hz, 1H), 6.97 (d, J= 3.6 Hz, 1H), 6.85 (d, J = 3.6 Hz, 1H), 3.00 (p, J= 6.9 Hz, 1H), 1.30 (d, J= 6.9 Hz, 6H).13C NMR (101 MHz, methanol-d4) 8 191.5, 163.6, 157.5, 156.0, 152.2, 137.3, 132.5, 131.7, 129.8, 127.9, 127.6, 122.6, 120.7, 119.1, 119.0, 108.4, 35.5, 24.1. HRMS: m / z calcd. for C23H19O5 375.1232; found, 375.1219 (deviation -3.5 ppm). HPLC (method G) ( = 254 nm), 96.2%; (X = 214 nm), 97.9%; T = 4.28 min (column 2).

[0458] (£)-5-{5-[3-(4-ButylphenyI)-3-oxoprop-l-en-l-yl]furan-2-yl}-2-hydroxybenzoic acid (20)

[0459] General conditions for hydrolysis of methyl salicylates, using 33-Bu (83 mg, 0.20 mmol, 1 equiv) and pyridine (5 mL). FCC purification gradient elution using petrol ether: DCM: MeOH (49:49:2^45:45: 10) acidified with 1% AcOH. Reddish-orange solid (95% yield). 'H NMR (500 MHz, methanol-d4) 88.29 (d, J= 2.2 Hz, 1H), 7.96 (d, J= 8.3 Hz, 2H), 7.90 (dd, J= 8.7, 2.3 Hz, 1H), 7.54 (d, J= 9.5 Hz, 1H), 7.34 (d, J= 8.3 Hz, 2H), 6.99 (d, J= 8.7 Hz, 1H), 6.95 (d, J= 3.6 Hz, 1H), 6.82 (d, J = 3.6 Hz, 1H), 2.72 - 2.66 (m, 2H), 1.68 - 1.58 (m, 2H), 1.38 (h, J= 7.4 Hz, 3H), 0.95 (t, J= 7.4 Hz, 3H).13C NMR (126 MHz, methanol-d4) 8 191.4, 163.5, 152.1, 150.1, 137.1, 132.4, 131.7, 130.5, 129.9, 129.7, 129.7, 127.7, 120.7, 119.0, 119.0, 108.4, 36.7, 34.5, 23.4,Ill14.2. HRMS: nvz calcd. for C24H23O5 391.1545; found; 391.1557 (deviation +3.1 ppm). HPLC (method G) ( = 254 nm), 97.8%; (X = 214 nm), 98.5%; R. = 6.01 min (column 2).

[0460] (£')-5-{5-[3-(4-Hexylphenyl)-3-oxoprop-l-en-l-yl]furan-2-yl}-2-hydroxybenzoic acid (21)

[0461] General conditions for aldol condensation (protocol A) using 31 (150 mg, 0.6 mmol, 1 equiv), -hexylacetophenone (197 pL, 186 mg, 0.9 mmol, 1.5 equiv), NaOH 10 N (0.6 mL, 6 mmol, 10 equiv), DCM (1 mL) and MeOH (5 mL). FCC purification: Gradient elution using petrol ether: DCM: MeOH (49:49:2— >45:45:10) acidified with 1% AcOH. Reddish-orange solid (55% yield). 'H NMR (500 MHz, methanol-d4) 88.29 (d, J= 2.1 Hz, 1H), 7.98 (d, J= 8.1 Hz, 2H), 7.95 (dd, J= 8.7, 2.2 Hz, 1H), 7.60 - 7.52 (m, 2H), 7.35 (d, J= 8.1 Hz, 2H), 7.03 (d, J= 8.7 Hz, 1H), 6.97 (d, J= 3.6 Hz, 1H), 6.86 (d, J= 3.6 Hz, 1H), 2.70 (t, J = 7.7 Hz, 2H), 1.66 (p, J= 7.5 Hz, 2H), 1.38 - 1.30 (m, 6H), 0.93 - 0.88 (m, 3H).13C NMR (126 MHz, methanol-A) 8 191.5, 173.2, 163.6, 157.4, 152.2, 150.2, 137.1, 132.7, 131.7, 129.9, 129.7, 127.6, 122.7, 120.6, 119.1, 119.1, 114.6, 108.5, 37.0, 32.9, 32.3, 30.0, 23.7, 14.4. HRMS: m / z calcd. for [M+H] C26H27O5419.1839; found, 419.1853 (deviation 3.4 ppm). HPLC (method G) (X = 254 nm), 100%; (X = 214 nm), 100%; 'R = 9.83 min (column 2).

[0462] (£)-2-Hydroxy-5-{5-[3-(4-octylphenyl)-3-oxoprop-l-en-l-yl]furan-2-yl} benzoic acid (22)

[0463] General conditions for aldol condensation (protocol A) using 31 (179 mg, 0.73 mmol, 1 equiv), / 2-octyl acetophenone (200 mg, 0.81 mmol, 1.1 equiv), NaOH 10 N (0.7 mL, 7.3 mmol, 10 equiv), THF (1 mL) and MeOH (5 mL). FCC purification: Gradient elution using petrol ether: DCM: MeOH (49:49:2— >45:45:10) acidified with 1% AcOH. Reddish-orange solid (91% yield). ‘HNMR (500 MHz, chloroform-d) 8 10.71 (s, 1H), 8.31 (s, 1H), 7.99 (d, J= 7.4 Hz, 2H), 7.91 (d, J= 7.5 Hz, 1H), 7.62 (d, J= 15.0 Hz, 1H), 7.50 (d, J= 15.1 Hz, 1H), 7.32 (d, J= 7.6 Hz, 2H), 7.09 (d, J= 8.0 Hz, 1H), 6.75 (d, J= 49.6 Hz, 2H), 2.68 (t, J= 7.6 Hz, 2H), 1.64 (q, J= 7.0 Hz, 2H), 1.36 - 1.24 (m, 10H), 0.88 (t, J = 6.8 Hz, 3H).13C NMR (126 MHz, chloroform- / ) 8 190.1, 173.3, 162.5, 155.4, 151.2, 148.8, 136.0, 132.9, 130.6, 128.9, 127.0, 122.1, 119.1, 118.9, 118.7, 112.0, 107.6, 36.2, 32.0, 31.3, 29.6, 29.5, 29.4, 22.8, 14.3. HRMS: m / z calcd. for [M+H] C28H31O5 447.2152; found, 447.2166 (deviation +3.2 ppm). HPLC (method H) ( = 254 nm), 100%; (X = 214 nm), 100%; 'R =5.00 min (column 2).

[0464] (E)-5-{5-{3-[4-(6-Chlorohex-l-yn-l-yl)phenyl]-3-oxoprop-l-en-l-yl}furan-2-yl}-2-hydroxybenzoic acid (23)

[0465] General conditions for aldol condensation (protocol A) using the methylketone 34. FCC purification: Gradient elution using petrol ether: DCM: MeOH (49:49:2— >45:45:10) acidified with 1% AcOH. Reddish-orange solid (93% yield). *H NMR (500 MHz, methanol-t / i) 58.34 - 8.31 (m, 1H), 8.03 (d, J= 8.3 Hz, 2H), 7.95 (dd, J= 8.7, 1.8 Hz, 1H), 7.60 (t, J= 14.8 Hz, 2H), 7.53 (d, J = 8.2 Hz, 2H), 7.03 (d, J= 8.7 Hz, 1H), 7.01 (d, J= 3.6 Hz, 1H), 6.88 (d, J= 3.6 Hz, 1H), 3.66 (t, J= 6.5 Hz, 2H), 2.53 (t, J= 7.0Hz, 2H), 1.97 (dt, J= 14.6, 6.6 Hz, 2H), 1.79 (p, J= 7.1 Hz, 2H).13C NMR (126 MHz, methanol-d4) 6 190.8, 163.7, 157.8, 152.2, 138.3, 132.8, 132.5, 132.0, 130.1, 129.5, 127.7, 122.6, 121.1, 119.1, 118.7, 108.5, 94.2, 81.6, 45.3, 32.9, 27.0, 19.5. HRMS: m / z calcd. for C26H22CIO5 449.1143; found, 449.1150 (deviation +1.6 ppm). HPLC (method F) ( = 254 nm), 100%; (X = 214 nm), 100%; R =9.28 min (column 2).

[0466] 5-{5-{(£)-3-{4-[(E)-3-( rMJutoxy)-3-oxoprop-l-en-l-yl]phenyl}-3-oxoprop-l-en-l-yl}furan-2-yl}-2-hydroxybenzoic acid (24)

[0467] General conditions for aldol condensation (protocol A) using. 31 (81 mg, 0.33 mmol, 1 equiv), the methylketone 32-Acrylate-tBu (81 mg, 0.33 mmol, 1 equiv), NaOH 10 N (1 mL, 10 mmol, 30 equiv), DCM (1 mL) and MeOH (5 mL). FCC purification: Gradient elution using petrol ether: DCM: MeOH (49:49:2^45:45:10) acidified with 1% AcOH. Reddish-orange solid (73% yield). ’H NMR (400 MHz, methanol-d4) 88.26 (s, 1H), 8.05 (d, J= 7.8 Hz, 2H), 7.92 (d, J= 8.4 Hz, 1H), 7.72 (d,.7= 7.8 Hz, 2H), 7.64 - 7.54 (m, 2H), 7.49 (d,. / = 15.2 Hz, 1H), 7.01 (d,. / = 8.6 Hz, 1H), 6.98 (d, J=2.6Hz, 1H), 6.84 (d, J = 2.6Hz, 1H), 6.55 (d, J = 16.0 Hz, 1H), 1.54 (s, 9H).13CNMR(101 MHZ, methanol-d4) 8 190.8, 167.6, 157.6, 152.2, 143.6, 140.4, 140.2, 132.7, 132.1, 130.1, 129.4, 127.7, 123.5, 122.7, 121.2, 119.1, 118.8, 108.6, 82.1, 28.4. HPLC (method G) (X = 254 nm), 94.0%; (X = 214 nm), 97.7%; R =5.09 min (column 2).

[0468] (2 )-5-{5-{3-{4-[3-(terCButoxy)-3-oxopropyl]phenyl}-3-oxoprop-l-en-l-yl}furan-2-yl}-2-hydroxybenzoic acid (25)

[0469] General conditions for aldol condensation (protocol A) using 31 (147 mg, 0.6 mmol, 1 equiv), the methylketone 32-Propionate-tBu (176 mg, 0.7 mmol, 1.15 equiv), NaOH 10 N (3 mL, 30 mmol, 50 equiv), DCM (1 mL) and MeOH (5 mL). FCC purification: Gradient elution using petrol ether: DCM: MeOH (49:49:2^45:45:10) acidified with 1% AcOH. Reddish-orange solid (55% yield). 'H NMR (400 MHz, methanol-d4) 8 8.32 (d, J = 2.2 Hz, 1H), 8.01 (d, J = 8.3 Hz,2H), 7.95 (dd, J= 8.7, 2.2 Hz, 1H), 7.62- 7.53 (m, 2H), 7.41 (d, J= 8.3 Hz, 2H), 7.02 (d, J= 8.7 Hz, 1H), 6.99 (d, J= 3.6 Hz, 1H), 6.87 (d, J= 3.6 Hz, 1H), 2.99 (t, J= 7.5 Hz, 2H), 2.61 (t, J = 7.5 Hz, 2H), 1.41 (s, 9H).13CNMR (101 MHz, methanol-d4) 8 191.4, 173.8, 163.6, 157.7, 152.2, 148.0, 137.6, 132.5, 131.8, 130.0, 129.8, 127.7, 122.6, 120.8, 119.0, 108.4, 81.8, 37.4, 32.1, 28.3. HRMS: m / z calcd. for C23H17O7461.1679; found, 461.1621 (deviation -2.5 ppm). HPLC (method E) = 254 nm), 100%; ( = 214 nm), 98.7%; 'R =10.35 min (column 2).

[0470] 2-Hydroxy-5-{5-{3-oxo-3-[4-(trifluoromethyl)phenyl]propyl}furan-2-yl}benzoic acid (26)

[0471] General conditions for hydrolysis of methyl salicylates, using 35 (40 mg, 0.1 mmol, 1 equiv). FCC purification. Gradient elution using petrol ether: DCM: MeOH (49:49:2^45:45:10) acidified with 1% AcOH. Yellow solid (93% yield). 'H NMR (400 MHz, acetone-cX) 88.24 (d, J = 8.1 Hz, 2H), 8.15 (d, J = 2.2 Hz, 1H), 7.86 (d, J= 8.2 Hz, 2H), 7.74 (dd, J= 8.6, 2.1 Hz, 1H), 6.92 (d, J= 8.7 Hz, 1H), 6.60 (d, J= 3.2 Hz, 1H), 6.20 (d, J= 3.1 Hz, 1H), 3.53 (t, J= 7.3 Hz, 2H), 3.12(t, J = 7.3 Hz, 2H).13C NMR (101 MHz, acetone- de) 8 198.6, 173.2, 161.9, 155.1, 152.7, 141.0, 134.4 (q, J= 25.3 Hz), 131.2, 129.6, 126.5 (q, J= 3.8 Hz), 126.0, 123.8, 123.5, 118.4, 115.0, 108.4, 105.5, 37.9, 23.1. HRMS: m / z calcd. for [M+H] C21H14O5F3 403.0793; found, 403.0798 (deviation 1.2 ppm). HPLC (method E) ( = 254 nm), 100%; ( = 214 nm), 100%; T =10.19 min (column 2).

[0472] 5-{5-[3-(4-Butylphenyl)-3-oxopropyl]furan-2-yl}-2-hydroxybenzoic acid (27)

[0473] General conditions for hydrolysis of methyl salicylates, using 36 (57 mg, 0.14 mmol, 1 equiv). FCC purification: Gradient elution using petrol ether: DCM: MeOH (49:49:2 >45:45: 10) acidified with 1% AcOH. Clear oil (97% yield). 'H NMR (400 MHz, methanol-6 / 4) 8 8.15 (d, J = 2.4 Hz, 1H), 7.97 - 7.90 (m, 2H), 7.63 (dd, J= 8.6, 2.4 Hz, 1H), 7.31 (d, J= 8.2 Hz, 2H), 6.88 (d, J= 8.6 Hz, 1H), 6.47 (d, J= 3.2 Hz, 1H), 6.13 (d, J= 3.2 Hz, 1H), 3.40 (t, J= 7.3 Hz, 2H), 3.10 (t, J= 7.3 Hz, 2H), 2.68 (t, J= 7.7 Hz, 2H), 1.66 - 1.57 (m, 2H), 1.40 - 1.33 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H).13C NMR (101 MHz, methanol-d4) 8200.9, 162.0, 155.3, 153.5, 150.3, 135.9, 130.6, 129.8, 129.4, 126.5, 123.8, 118.1, 108.4, 105.2, 37.8, 36.6, 34.5, 23.9, 23.3, 14.2. HRMS: m / z calcd. for [M+Na] C24H24NaOs 415.1505; found, 415.1516 (deviation +2.6 ppm). HPLC (method G) (X = 254 nm), 98.0%; (X = 214 nm), 95.3%; 'R =5.95 min (column 2).

[0474] 5-{5-[3-(4-Butylphenyl)-3-hydroxypropyl]furan-2-yl}-2-hydroxybenzoic acid (28)

[0475] General conditions for hydrolysis of methyl salicylates, using 37 (37 mg, 0.09 mmol, 1 equiv). FCC purification: Gradient elution using petrol ether: DCM: MeOH (49:49:2 >45:45:10) acidified with 1% AcOH. Clear oil (96% yield). 'H NMR (400 MHz, methanol-6 / 4) 5 8.10 (d, J = 2.3 Hz, 1H), 7.72 (dd, J= 8.7, 2.3 Hz, 1H), 7.27 (d, J= 8.1 Hz, 2H), 7.15 (d, J= 8.0 Hz, 2H), 6.94 (d, J = 8.7 Hz, 1H), 6.50 (d, J= 3.2 Hz, 1H), 6.08 (d, J = 3.2Hz, 1H), 4.68 - 4.63 (m, 1H), 2.75 -2.68 (m, 2H), 2.61 - 2.56 (m, 2H), 2.16 - 2.03 (m, 2H), 1.57 (ddt, J= 9.0, 7.6, 3.5 Hz, 2H), 1.38 - 1.30 (m, 2H), 0.93 (t, J= 7.4 Hz, 3H).13C NMR (101 MHz, methanol-d4) 8 173.4, 162.1, 156.4, 152.8, 143.2, 143.2, 131.7, 129.4, 127.1, 126.0, 124.4, 118.6, 114.1, 108.2, 105.6, 74.2, 38.6, 36.3, 35.0, 25.5, 23.3, 14.3. HRMS: m / z calcd. for [M-H] C24H25O5 393.1706; found, 393.1707 (deviation +0.5 ppm). HPLC (method G) ( = 254 nm), 100%; ( = 214 nm), 96.4%; 'R =5.00 min (column 2).

[0476] Methyl 5-(5-formylfuran-2-yl)-2-hydroxybenzoate (31)

[0477] To a stirred solution of methyl 5-iodosalicylate (29) (200 mg, 0.719 mmol, 1 equiv) and 5-formyl-2-furanylboronic acid (30) (151 mg, 1.078 mmol, 1.5 equiv) in DMF (10 mL), triethylamine (300 pL, 2.157 mmol, 3 equiv) and Pd(OAc)2 (8 mg, 0.05 equiv) were added. The solvent was bubbled with argon. The reaction mixture was stirred at rt overnight, under Ar. After consumption of the starting material, determined by TLC (petrol etherAcOEt) (70:30), the reaction mixture was concentrated in a high vacuum rotavapor. The solid residue was dissolved in AcOEt and washed with water and brine. The organic layer was dried over MgSCh, filtered and concentrated under reduced pressure. The residue was purified by FCC (gradient elution using petrol ether: AcOEt) (80:20 — > 50:50) to yield 31 as a yellowish-orange solid (154 mg, 0.63 mmol, 87% yield). ’H NMR (400 MHz, chloroform- / ) 8 10.99 (s, 1H), 9.61 (s, 1H), 8.31 (d, J= 2.3 Hz, 1H), 7.87 (dd, J= 8.7, 2.3 Hz, 1H), 7.31 (d, J= 3.7 Hz, 1H), 7.05 (d, J= 8.7 Hz, 1H), 6.74 (d, J = 3.7 Hz, 1H), 4.00 (s, 3H).13C NMR (101 MHz, chloroform-d) 8 177.0, 170.2, 162.7, 158.7, 151.9, 132.6, 127.2, 120.8, 118.6, 112.9, 106.8, 52.7. HRMS: m / z calcd. for [M+H] C13H11O5 247.0606; found, 247.0593 (deviation -1.1 ppm).

[0478] tot- Butyl (E)-3-(4-acetylphenyl)acrylate (32-Acrylate-tBu)

[0479] Working in a sealed tube in an inert argon atmosphere, 4-bromoacetophenone (100 mg, 0.50 mmol, 1 equiv), palladium (II) acetate (9 mg, 0.04 mmol, 0.02 equiv), 1,4-diazabicylo[2,2,2]octane (DABCO) (9 mg, 0.08 mmol, 0.04 equiv) and potassium carbonate (69 mg, 0.50 mmol, 1 equiv) were dissolved in anhydrous N, A-dimethylformamide. After addition of / <? / / -butyl acrylate (1.5 equiv), the reaction mixture was heated to 120 °C during 15 h. Once cooled down, the reaction was fdtered through celite and concentrated under reduced pressure. FCC purification was performed by gradient elution using petrol etherAcOEt (100:0 —> 80:20). Yellowish oil (81% yield). 'H NMR (500 MHz, chloroform- / ) 8 7.95 (d, J= 8.4 Hz, 2H), 7.63 -7.55 (m, 3H), 6.45 (d, J = 16.0 Hz, 1H), 2.61 (s, 3H), 1.54 (s, 9H).13C NMR (126 MHz, chloroform-d) 8 197.5, 165.9, 142.1, 139.2, 137.9, 129.0, 128.1, 122.9, 81.1, 28.3, 26.8.

[0480] tert-Butyl 3-(4-acetylphenyl)propanoate (32-Propionate-tBu)

[0481] Compound 32-Acrylate-tBu (1 equiv) was dissolved in THF and 1 mL of Raney Nickel® activated catalyst (50% slurry in water) was then added. Hydrogen (g) was bubbled through the solution during Ih. Once completed, the reaction mixture was filtered through celite and concentrated under reduced pressure. FCC purification was performed by elution using mixtures of petrol ether and AcOEt. The final product was obtained as a yellowish oil (96% yield). *H NMR (400 MHz, chloroform-d) 87.88 (d, J= 8.2 Hz, 2H), 7.29 (d, J= 8.2 Hz, 2H), 2.96 (t, J = 7.7 Hz, 2H), 2.57 (s, 3H), 2.56 (t, J= 7.6 Hz, 2H), 1.40 (s, 9H) (spectral data agree with the ones reported previously).60

[0482] Methyl 5-{5-{(£)-3-{4-[6-(adamantan-l-ylmethylamino)hex-l-ynyl]phenyl}-3-oxoprop- l-enyl}furan-2-yl}-2-hydroxybenzoate (33-Ad)

[0483] The solid reagents, 33-1 (640 mg, 1.35 mmol, 1 equiv), bis(triphenylphosphine)palladium (II) dichloride (53 mg, 0.076 mmol, 0.05 equiv) and copper (I) iodide (24 mg, 0.126 mmol, 0.1 equiv) were mixed in a round-bottom flask and the mixture was purged under argon before the addition of anhydrous THF. Then a 1M solution of tetrabutylammonium fluoride in hexanes (554 mg, 2.12 mL, 2.12 mmol, 1.6 equiv) and 34 (315 mg, 1.28 mmol, 1 equiv) were added to start the reaction. After 18 h stirring at rt, the reaction was filtered through celite and concentrated under reduced pressure. FCC purification: Gradient elution with mixtures petrol ether: DCM: MeOH (50:50:2— >50:50:15). Orange solid, 67% yield (497 mg, 0.86 mmol). 'H NMR (500 MHz, chloroform-^ 8 10.92 (bb, IH), 8.20 (d, J= 2.3 Hz, 1 H), 7.97 (d, J= 8.5 Hz, 2H), 7.87 (dd, J= 8.9, 2.4 Hz, IH), 7.57 (d, J= 15.3 Hz, IH), 7.55 (d, J= 8.5 Hz, 2H), 7.42 (d, J =15.3 Hz, IH), 7.07 (d, J = 8.7 Hz, IH), 6.80 (d, J= 3.5 Hz, IH), 6.68 (d, J = 3.5 Hz, IH), 4.01 (s, 3H), 3.10 (m, 2H), 2.65 (m, 2H), 2.50 (t, J= 7.0 Hz, 2H), 2.14 (m, 2H), 2.02 (bs, 3H), 1.76-1.63 (m, 16H).13C NMR (126 MHz, chloroform-d) 8 189.2, 170.3, 162.0, 155.8, 151.0, 137.3, 132.0, 132.0, 130.8, 128.5, 128.3, 126.2, 121.8, 119.3, 118.6, 118.4, 112.9, 107.5, 92.6,81.4, 59.5, 52.8, 48.9, 40.1, 36.4, 32.9, 28.1, 25.9, 24.4, 19.2. HRMS: rn'z calcd. for [M+H] C38H42NO5 592.3057; found, 592.3082 (deviation -4.2 ppm).

[0484] Methyl (£)-5-{5-[3-(4-butylphenyl)-3-oxoprop-l-en-l-yl]furan-2-yl}-2-hydroxybenzoate (33-Bu)

[0485] General conditions for aldol condensation (protocol A) using 31 (90 mg, 0.36 mrnol),?-butylacetophenone (134 pL, 0.73 mmol), NaOH 10 N (1.8 mmol, 5 equiv), DCM (minimum amount for solids dissolution) and MeOH (5 mL). FCC purification: Gradient elution using petrol etherAcOEt (100:0 —> 80:20). Reddish-orange solid (87% yield). 'H NMR (400 MHz, chloroform-d) 5 10.92 (s, 1H), 8.22 (d, J= 2.3 Hz, 1H), 7.98 (d, J= 8.2 Hz, 2H), 7.87 (dd, J= 8.8, 2.3 Hz, 1H), 7.59 (d, J= 15.3 Hz, 1H), 7.48 (d, J= 15.3 Hz, 1H), 7.32 (d, J= 8.2 Hz, 2H), 7.07 (d, J = 8.7 Hz, 1H), 6.79 (d, J= 3.6Hz, 1H), 6.69 (d, J= 3.6 Hz, 1H), 4.03 (s, 3H), 2.70 (t, J= 7.7 Hz, 2H), 1.65 (m, 2H), 1.38 (m, 2H), 0.95 (t, J= 7.3 Hz, 3H).13C NMR (101 MHz, chloroform-d) 8 189.8, 170.4, 161.9, 155.6, 151.2, 148.6, 136.2, 132.0, 130.3, 128.8, 128.7, 126.2, 121.9, 119.0, 118.8, 118.6, 112.9, 107.4, 52.8, 35.9, 33.5, 22.5, 14.1. HRMS: m z calcd. for [M+H] C25H250S= 405.1702; found, 405.1689 (deviation -3.2 ppm).

[0486] Methyl (E)-2-hydroxy-5-{5-{3-oxo-3-[4-(trifluoromethyl)phenyl]prop-l-en-l-yl}furan-2-yl}benzoate (33-CF3)

[0487] General conditions for aldol condensation (protocol A) using 31 (114 mg, 0.46 mmol), / Mrifluoromethylacetophenorie (140 pL, 0.69 mmol), NaOH 10 N (460 pL, 4.6 mmol, 10 equiv), DCM (minimum amount for solids dissolution) and MeOH (5 mL). FCC purification: Gradient elution using petrol etherAcOEt (100:0 — > 80:20). Reddish-orange solid (93% yield). 'H NMR (400 MHz, chloroform-d) 8 10.93 (s, 1H), 8.22 (d, J= 2.3 Hz, 1H), 8.12 (d, J= 8.1 Hz, 2H), 7.87 (dd, J = 8.7, 2.3 Hz, 1H), 7.77 (d, J= 8.0 Hz, 2H), 7.60 (d, J= 15.3 Hz, 1H), 7.41 (d, J= 15.3 Hz, 1H), 7.07 (d, J= 8.7 Hz, 1H), 6.85 (d, J= 3.6 Hz, 1H), 6.71 (d, J= 3.6 Hz, 1H), 4.03 (s, 3H).13C NMR(101 MHz, chloroform-d) 6 189.1, 170.2, 162.0, 156.1, 150.7, 141.3 (q, J= 1.0Hz, C), 133.9 (q, J= 32.6 Hz, C), 131.9, 131.5, 128.7, 126.2, 125.7 (q, J= 3.7 Hz, CH), 123.7 (q, J = 272.7 Hz, CF3), 121.5, 119.9, 118.5, 118.0, 112.8, 107.5, 52.7. HRMS: m / z calcd. for [M+H] C22H16O5F3 417.0950; found, 417.0971 (deviation 5.0 ppm).

[0488] Methyl (£)-2-hydroxy-5-{5-{3-[4-(hydroxymethyl)phenyl]-3-oxoprop-l-en-l-yl}furan-2-yl}benzoate (33-CHiOH)

[0489] General conditions for aldol condensation (protocol A) using 31 (150 mg, 0.60 mmol), / ?-(hydroxy methyl) acetophenone (136 mg, 0.91 mmol), NaOH 10 N (0.3 mL, 3 mmol, 5 equiv), DCM (minimum amount for solids dissolution) and MeOH (10 mL). FCC purification: Gradient elution using petrol ether: AcOEt (80:20 — 20:80). Reddish-orange solid (83% yield).!H NMR (500 MHz, chloroform-606 10.92 (s, 1H), 8.22 (d, J= 2.3 Hz, 1H), 8.04 (d, J= 8.2 Hz, 2H), 7.87 (dd, J= 8.7, 2.3 Hz, 1H), 7.59 (d, J= 15.3 Hz, 1H), 7.51 (d, J= 8.1 Hz, 2H), 7.46 (d, J= 15.3 Hz, 1H), 7.07 (d, J= 8.7 Hz, 1H), 6.81 (d, J= 3.5 Hz, 1H), 6.69 (d, J= 3.5 Hz, 1H), 4.81 (s, 2H), 4.03 (s, 3H).13C NMR (126 MHz, chloroform-r / ) 8 189.8, 170.3, 162.0, 155.7, 151.0, 145.9, 137.7, 132.0, 130.7, 128.9, 126.9, 126.2, 121.8, 119.2, 118.7, 118.6, 112.9, 107.5, 64.9, 52.8. HRMS: m / z calcd. for [M+H] C22H19O6379.1182; found, 379.1194 (deviation 3.2 ppm).

[0490] Methyl (£)-5-{5-[3-(4-cyanophenyl)-3-oxoprop-l-en-l-yl]furan-2-yl}-2- hydroxybenzoate (33-CN)

[0491] General conditions for aldol condensation (protocol A) using 31 (95 mg, 0.38 mmol), / ?- cyanoacetophenone (85 mg, 0.58 mmol), NaOH 10 N (190 pL, 1.9 mmol, 5 equiv), DCM (minimum amount for solids dissolution) and MeOH (5 mL). FCC purification: Gradient elution using petrol etherAcOEt (100:0 — > 70:30). Reddish-orange solid (90% yield). 'HNMR (500 MHz, chloroform-60 8 10.94 (s, 1H), 8.23 (d, J= 2.4 Hz, 1H), 8.11 (d, J = 10.0 Hz, 2H), 7.88 (dd, J = 8.8, 2.3 Hz, 1H), 7.81 (d, J= 10.0 Hz, 2H), 7.62 (d, J= 15.2 Hz, 1H), 7.39 (d, J= 15.3 Hz, 1H), 7.08 (d, J= 8.8 Hz, 1H), 6.88 (d, J= 3.6 Hz, 1H), 6.73 (d, = 3.6 Hz, 1H), 4.03 (s, 3H).13CNMR (126 MHz, chloroform ) 8 188.7, 170.3, 162.2, 156.5, 150.7, 141.9, 132.6, 132.1, 132.0, 128.9, 126.4, 121.6, 120.5, 118.7, 118.3, 117.6, 115.9, 112.9, 107.8, 52.8. HRMS: m / z calcd. for [M+H] C22H14NO5393.0767; found, 393.0797 (deviation -7.6 ppm).

[0492] Methyl (£)-5-{5-[3-(4-ethylphenyl)-3-oxoprop-l-en-l-yl]furan-2-yl}-2- hydroxybenzoic acid (33-Et)

[0493] General conditions for aldol condensation (protocol A) using 31 (150 mg, 0.60 mmol), / >-ethylacetophenone (180 pL, 1.2 mmol, 2 equiv), NaOH 10 N (0.3 mL, 3 mmol, 5 equiv), DCM (minimum amount for solids dissolution) and MeOH (10 mL). FCC purification: Gradient elution using petrol etherAcOEt (100:0 — 70:30). Reddish-orange solid (43% yield). 'H NMR (400 MHz, chloroform-608 10.91 (s, 1H), 8.23 (d, J= 2.3 Hz, 1H), 7.99 (d, J= 8.2 Hz, 2H), 7.87 (dd, J= 8.7, 2.3 Hz, 1H), 7.59 (d, J= 15.3 Hz, 1H), 7.48 (d, J= 15.4 Hz, 1H), 7.34 (d, J= 8.3 Hz, 2H), 7.08 (d, J= 8.7 Hz, 1H), 6.79 (d, J= 3.6 Hz, 1H), 6.69 (d, J= 3.5 Hz, 1H), 4.03 (s, 3H), 2.74 (q, J =7.6 Hz, 2H), 1.29 (t, J= 7.6 Hz, 3H).13C NMR (101 MHz, chloroform-^ 8 189.8, 170.4, 162.0, 155.6, 151.2, 149.8, 136.2, 132.0, 130.4, 128.8, 128.3, 126.2, 121.9, 119.0, 118.8, 118.6, 112.9, 107.4, 77.4, 52.8, 29.2, 15.4. HRMS: m / z calcd. for [M+H] C23H2IO5377.1389; found, 377.1385 (deviation -1.1 ppm).

[0494] Methyl (£)-5-{5-[3-(4-hexylphenyl)-3-oxoprop-l-en-l-yl]furan-2-yl}-2- hydroxybenzoate (33-Hex) General conditions for aldol condensation (protocol A) using 31 (150 mg, 0.60 mmol), / ?-hexylacetophenone (197 pL, 0.9 mmol, 1.5 mmol equiv), NaOH 10N (0.6 mL, 6 mmol, 10 equiv), DCM (minimum amount for solids dissolution) and MeOH (5 mL). FCC purification: Gradient elution using petrol etherAcOEt) (100:0 — 70:30). Reddish-orange solid (36% yield).rH NMR (500 MHz, chloroform- ) 8 10.92 (s, 1H), 8.21 (d, J = 23 Hz, 1H), 7.98 (d, J = 8.2 Hz, 2H), 7.86 (dd, J= 8.7, 2.3 Hz, 1H), 7.58 (d, J= 15.3 Hz, 1H), 7.47 (d, J= 15.3 Hz, 1H), 7.32 (d, J = 8.0 Hz, 2H), 7.07 (d, J = 8.7 Hz, 1H), 6.78 (d, J = 3.5 Hz, 1H), 6.68 (d, J = 3.5 Hz, 1H), 4.02 (s, 3H), 2.69 (t, J= 7.7 Hz, 2H), 1.65 (m, 2H), 1.37 - 1.29 (m, 6H), 0.91 - 0.87 (m, 3H).13C NMR (126 MHz, chloroform-d) 8 189.7, 170.3, 161.9, 155.5, 151.1, 148.6, 136.1, 132.0, 130.3, 128.8, 128.7, 126.1, 121.9, 118.9, 118.8, 118.5, 112.8, 107.4, 52.7, 36.2, 31.8, 31.3, 29.1, 22.7, 14.2. HRMS: m / z calcd. for [M+H] C27H29O5433.2010; found 433.1987 (deviation +5.1 PPm).

[0495] Methyl (£)-2-hydroxy-5-{5-[3-(4-iodophenyl)-3-oxoprop-l-en-l-yl]furan-2-yl} benzoate (33-1)

[0496] General conditions for aldol condensation (protocol A) using 31 (110 mg, 0.45 mmol), / ?-iodoacetophenone (220 mg, 0.89 mmol), NaOH 10 N (0.45 mL, 4.5 mmol, 10 equiv), DCM (minimum amount for solids dissolution) and MeOH (5 mL). FCC purification: Gradient elution using petrol ether: AcOEt (95:5 — 70:30). Reddish-orange solid (200 mg, 0.42 mmol, 94% yield).! H NMR (400 MHz, chloroform- / ) 8 10.93 (s, 1H), 8.22 (d, J= 2.3 Hz, 1H), 7.89-7.84 (m, 3H), 7.75 (dt, J= 8.0 Hz, J = 4.0 Hz, 2H), 7.59 (d, J= 15.3 Hz, 1H), 7.39 (d, J= 15.3 Hz, 1H), 7.07 (d, J= 8.7 Hz, 1H), 6.82 (d, J= 3.6 Hz, 1H), 6.70 (d, J= 3.6 Hz, 1H), 4.03 (s, 3H).13C NMR (101 MHz, chloroform-d) 6 189.3, 170.3, 162.1, 156.0, 150.9, 138.0, 137.8, 132.0, 131.1, 130.0, 126.3, 121.7, 119.6, 118.6, 118.1, 112.9, 107.6, 100.5, 52.8. HRMS: m / z calcd. for [M-H] C21H14O5I 472.9886; found, 472.9848 (deviation: 6.5 ppm).

[0497] Methyl (E)-2-hydroxy-5-{5-[3-(4-isopropylphenyl)-3-oxoprop-l-en-l-yl]furan-2-yl}benzoate (33-iPr)

[0498] General conditions for aldol condensation (protocol A) using 31 (150 mg, 0.60 mmol), / ?-i sopropy I acetophenone (250 mg, 1.54 mmol, 2.6 equiv), NaOH 10N (0.5 mL, 5 mmol, 5 equiv), DCM (minimum amount for solids dissolution) and MeOH (10 mL). FCC purification: Gradient elution using petrol ether: AcOEt (100:0 — 70:30). Reddish-orange solid (91% yield).NMR (500 MHz, chloroform- ) 6 10.92 (s, 1H), 8.22 (d, J= 2.3 Hz, 1H), 8.00 (d, J= 8.2 Hz, 2H), 7.87 (dd, J= 8.7, 2.3 Hz, 1H), 7.59 (d, J= 15.3 Hz, 1H), 7.48 (d, J= 15.3 Hz, 1H), 7.37 (d, J= 8.3 Hz, 2H), 7.08 (d, J= 8.7 Hz, 1H), 6.80 (d, J= 3.6 Hz, 1H), 6.69 (d, J= 3.5 Hz, 1H), 4.03 (s, 3H), 3.00 (m, 1H), 1.31 (s, 3H), 1.29 (s, 3H).13C NMR (126 MHz, chloroform- ) 5 189.8, 170.4, 161.9, 155.6, 154.4, 151.2, 136.3, 132.0, 130.4, 128.9, 126.9, 126.2, 121.9, 119.0, 118.9, 118.6, 112.9, 107.4, 52.8, 34.4, 23.9. HRMS: m / z calcd. for [M+H] C24H23O5 391.1545; found, 391.1557 (deviation +3.1).

[0499] Methyl (£)-2-hydroxy-5-{5-[3-oxo-3-(p-tolyl)prop-l-en-l-yl]furan-2-yl} benzoate (33-Me)

[0500] General conditions for aldol condensation (protocol A) using 31 (150 mg, 0.60 mmol), £>-methylacetophenone (160 pL, 1.2 mmol), DCM (minimum amount for solids dissolution) and MeOH (10 mL). FCC purification: Gradient elution using petrol etherAcOEt (100:0 50:50). Reddish-orange solid (96% yield). 'H NMR (500 MHz, chloroform-d) 5 10.92 (s, 1H), 8.22 (d, J = 2.3 Hz, 1H), 7.96 (d, J= 8.2 Hz, 2H), 7.87 (dd, J= 8.7, 2.3 Hz, 1H), 7.58 (d, J = 15.4 Hz, 1H), 7.47 (d, J= 15.3 Hz, 1H), 7.32 (d, J= 7.8 Hz, 2H), 7.07 (d, J= 8.7 Hz, 1H), 6.79 (d, J= 3.5 Hz, 1H), 6.69 (d, J= 3.5 Hz, 1H), 4.03 (s, 3H), 2.45 (s, 3H).13C NMR (126 MHz, chloroform- ) 5 189.7, 170.4, 161.9, 155.6, 151.1, 143.6, 135.9, 132.0, 130.4, 129.5, 128.7, 126.1, 121.9, 118.9, 118.8, 118.5, 112.9, 107.4, 52.8, 21.8. HRMS: m z calcd. for [M+H] C22H19O5 363.1232; found, 363.1242 (deviation 2.8 ppm).

[0501] Methyl (E)-2-hydroxy-5-{5-{3-[4-(methylthio)phenyl]-3-oxoprop-l-en-l-yl}furan-2-yl}benzoate (33-SMe)

[0502] General conditions for aldol condensation (protocol A) using 31 (95 mg, 0.38 mmol), / ?- (methylthio)acetophenone (96 mg, 0.58 mmol), NaOH 10 N (0.19 mL, 1.9 mmol, 5 equiv), DCM (minimum amount for solids dissolution) and MeOH (5 mL). FCC purification: Gradient elution using petrol etherAcOEt (100:0 — > 80:20). Reddish-brown solid (89% yield). 'HNMR (500 MHz, chloroform-d) 5 10.92 (s, 1H), 8.20 (d, J= 2.3 Hz, 1H), 7.98 (d, J= 8.6 Hz, 2H), 7.86 (dd, J= 8.7, 2.3 Hz, 1H), 7.58 (d, J= 15.2 Hz, 1H), 7.45 (d, J= 15.3 Hz, 1H), 7.35 - 7.29 (m, 2H), 7.06 (d, J= 8.7 Hz, 1H), 6.79 (d, J= 3.6 Hz, 1H), 6.68 (d, J= 3.5 Hz, 1H), 4.02 (s, 3H), 2.54 (s, 3H).13C NMR (126 MHz, chloroforms / ) 8 188.8, 170.3, 161.9, 155.6, 151.1, 145.6, 134.7, 132.0, 130.4, 129.0, 126.1, 125.2, 121.8, 119.0, 118.5, 118.5, 112.8, 107.5, 52.8, 15.0. HRMS: m / z calcd. for [M+H] C22H17O5S 393.0767; found, 393.0797 (deviation -7.6 ppm).

[0503] 2V-(Adamantan-l-ylmethyl)hex-5-yn-l-amine (34)

[0504] Adamantan-l-ylmethyl)amine (268 mg, 270 pL, 1.62 mmol, 2 equiv) and potassium carbonate (112 mg, 0.81 mmol, 1 equiv) were introduced in a dry two-necked round-bottom flask and the mixture was purged with argon. Then, anhydrous acetonitrile (10 mb) was added, followed by 6-iodohexyne (168 mg, 106 pL, 0.81 mmol, 1 equiv). The reaction was then diluted with an additional volume of anhydrous acetonitrile (10 mb) and let to stir at 80 °C (reflux), under argon, for 18 h.61The reaction was then cooled down to rt and the solvents were evaporated under vacuum. The residue was suspended in DCM and the organic phase was washed with an aqueous solution of NaOH (5 M). The organic phase was dried over anhydrous MgSO4, filtered and evaporated under vacuum. FCC purification (automatic): Gradient elution with mixtures DCM MeOH (99:1^90:10). White solid, 80 % yield (160 mg, 0.65 mmol). ‘HNMR (500 MHz, chloroform-c / ) 83.34 (bb, 1H), 2.70 (t, J= 7.5 Hz, 2H), 2.32 (s, 2H), 2.21 (m, 2H), 1.98-1.93 (m, 4H), 1.73-1.60 (m, 8H), 1.59-1.51 (m, 8H).13C NMR (126 MHz, chloroform-d) 384.3, 68.7, 62.2, 50.1, 40.8, 37.1, 33.3, 28.5, 28.0, 26.3, 18.4.

[0505] Methyl 2-hydroxy-5-{5-{3-oxo-3-[4-(trifluoromethyl)phenyl]propyl}furan-2-yl} benzoate (35)

[0506] General conditions for selective double bond hydrogenation in a, P-unsaturated ketones using 33-CF3 (76 mg, 0.18 mmol, 1 equiv) in THF (5 mb). FCC purification (petrol ether: AcOEt) (100:0 90:10). Clear oil (95% yield). 'H NMR (400 MHz, chloroform-d) 8 10.75 (s, 1H), 8.08 (d, J= 8.1 Hz, 2H), 8.05 (d, J= 2.3 Hz, 1H), 7.73 (d, J= 8.2 Hz, 2H), 7.68 (dd, J= 8.7, 2.3 Hz, 1H), 6.98 (d, J= 8.8 Hz, 1H), 6.44 (d, J= 3.3 Hz, 1H), 6.13 (d, J= 3.3 Hz, 1H), 3.98 (s, 3H), 3.41 (t, J = 7.4 Hz, 2H), 3.17 (t, J = 7.4 Hz, 2H).13C NMR (101 MHz, chloroform-d) 8 197.8, 170.5, 160.8, 153.9, 151.9, 139.5, 134.6 (q, J= 32.7 Hz), 131.2, 128.5, 125.9 (q, J= 3.7 Hz), 124.7, 123.7 (q, J = 272.7 Hz), 123.1, 118.2, 112.6, 107.9, 104.9, 52.6, 37.6, 22.8.

[0507] Methyl 5-{5-[3-(4-butylphenyl)-3-oxopropyl]furan-2-yl}-2-hydroxybenzoate (36)

[0508] General conditions for selective double bond hydrogenation in a,[3-unsaturated ketones, using 33-Bu (147 mg, 0.36 mmol, 1 equiv). FCC purification (petroleum etherAcOEt) (100:070:30). Clear oil (94% yield). 'H NMR (500 MHz, chloroform-^ 8 10.76 (s, 1H), 8.07 (d, J= 2.3 Hz, 1H), 7.91 (d, J= 8.0 Hz, 2H), 7.69 (dd, J= 8.7, 2.3 Hz, 1H), 7.27 (d, J= 7.9 Hz, 2H), 6.98 (d, J= 8.7 Hz, 1H), 6.44 (d, J= 3.2 Hz, 1H), 6.12 (d, J= 3.2 Hz, 1H), 3.99 (s, 3H), 3.40 - 3.33 (m, 2H), 3.14 (t, J = 7.5 Hz, 2H), 2.66 (t, J = 7.7 Hz, 2H), 1.61 (p, J = 7.6 Hz, 2H), 1.35 (h, J = 7.4 Hz, 2H), 0.93 (t, J= 13 Hz, 3H).13C NMR (126 MHz, chloroform-d) 8 198.4, 170.6, 160.7, 154.5, 151.7, 149.1, 134.6, 131.2, 128.8, 128.3, 124.7, 123.2, 118.1, 112.5, 107.6, 104.8, 52.5, 37.1, 35.8, 33.4, 23.0, 22.5, 14.0.

[0509] Methyl 5-{5-[3-(4-butylphenyl)-3-hydroxypropyl]furan-2-yl}-2-hydroxybenzoate (37)

[0510] Compound 33-Bu (1 equiv) was dissolved in THE To the solution, palladium on activated charcoal 10% (0.05 equiv) was added. For the reaction to take place, H2 was bubbled in the solution for 3 h. Once completed, the reaction mixture was filtered through celite and concentrated under reduced pressure. FCC purification: Gradient elution using petrol etherAcOEt) (100:0 70:30). Clear oil (71% yield). ’H NMR (500 MHz, chloroform-c / ) 8 10.75 (s, 1H), 8.07 (d, J= 2.2 Hz, 1H), 7.70 (dd, J= 8.8, 2.3 Hz, 1H), 7.29 (d, J= 7.8 Hz, 2H), 7.18 (d, J= 7.8 Hz, 2H), 6.99 (d, J= 8.7 Hz, 1H), 6.44 (d, J= 3.2 Hz, 1H), 6.07 (d, J= 3.2 Hz, 1H), 4.74 (dd, J = 7.8, 5.5 Hz, 1H), 3.99 (s, 3H), 2.85 -2.71 (m, 2H), 2.61 (t, J = 7.8 Hz, 2H), 2.25 - 2.07 (m, 2H), 1.63 - 1.56 (m, 2H), 1.36 (h, J= 1A Hz, 2H), 0.93 (t, J= 13 Hz, 3H).13C NMR (126 MHz, chloroform-d) 8131.1, 128.6, 125.9, 124.5, 118.0, 107.2, 104.6, 73.7, 52.4, 37.1, 35.3, 33.7, 24.7, 22.4, 14.0.

[0511] BIOLOGICAL METHODS

[0512] Protein expression and purification

[0513] E. coli BL21 (DE3) cells were transformed with a recombinant pET-15b expression vector encoding the human GO or LDHA sequences (GenScript, Leiden, The Netherlands). For protein expression, 10 mL of LB medium supplemented with 0.1 mg mL1ampicillin were inoculated with transformed cells and incubated overnight at 37°C. A 5 mL aliquot of the overnight culture was then used to inoculate 500 mL of LB medium containing 0.1 mg mL1ampicillin. Cultures were grown at 37°C for 3 h until reaching an optical density (ODeoo) of approximately 0.8-1.0. The temperature was then lowered to 25°C, and protein expression was induced by adding 0.5 mM IPTG. After 5 h of induction, cells were harvested by centrifugation and the pellets were stored at -80°C.

[0514] Cell pellets were thawed the next day and resuspended in cold lysis buffer supplemented with PMSF, followed by sonication for cell disruption. For GO, the lysis buffer consisted of 20 mM NaH2PO4, 500 mM NaCl, 20 mM imidazole, and 50 pM FMN, pH 7.5. For LDHA, the buffer was identical except it contained 300 mM NaCl and no FMN. Lysates were clarified by centrifugation (30,000 x g;30 min, 4°C), and the supernatants were loaded onto immobilized metal affinity chromatography (IMAC) columns (Cytiva, Barcelona, Spain). Columns were washed with 20 mL of lysis buffer, and proteins were eluted using the same buffer containing 500 mM imidazole.

[0515] Eluted proteins were further purified by size-exclusion chromatography. Samples were loaded onto a Superdex 200 16 / 60 column (GE Healthcare, Madrid, Spain) pre-equilibrated with 20 mM NafLPCff 500 mM NaCl, pH 7.5 for GO, or 20 mM HEPES, 200 mM NaCl, pH 7.5 for LDHA. Fractions corresponding to the tetrameric species were pooled and concentrated. The purity of purified proteins was confirmed by SDS-PAGE.

[0516] Protein concentrations were determined spectrophotometrically using molar extinction coefficients calculated from the amino acid sequences of the human enzymes: E280 = 48000 M ' em1for GO and E280 = 44920 M ' em1for LDHA. Purified proteins were aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C.

[0517] Determination of GO inhibition percentage using a kinetic fluorometric assay

[0518] The inhibitory activity of test compounds against recombinant GO was assessed using a kinetic fluorometric assay in black 96-well OptiPlates (PerkinElmer). Each plate included four replicates of: (i) 100% activity controls (containing enzyme and substrate, but not inhibitor), (ii) 0% activity controls (containing enzyme but neither substrate nor inhibitor) and (iii) each tested compound at 10 pM. The final reaction volume per well was 200 pL consisting of: 50 mM phosphate buffer (pH 7), GO (25 nM), glycolate (180 pM), Amplex® Red (50 pM), Horseradish peroxidase (HRP, 2 units) and 1 % DMSO.

[0519] To each well, 20 pL of either phosphate buffer pH 7 with 10 % DMSO (for control wells) or a 100 pM solution of inhibitor in the same buffer was added. Then, 40 pL of GO solution (125 nM in phosphate buffer) was added, and the mixture was incubated at room temperature for 10 min. After incubation, 40 pL of an Amplex® Red / HRP mixture (composed by Amplex® Red 250 pM and 2 units of HRP) was added, followed by either 100 pL of MiliQ water for 0% activity control or 100 pL glycolate (solution 360 pM in water) which starts the reaction in the remainingwells. Fluorescence was immediately recorded every 60 seconds for the following 15 min at 27°C using a PerkinElmer Multimode Plate Reader Enspire (zcx 560 ± 10 nm and zem 590 ± 10 nm). The initial rate of reaction was determined by calculating the slope of fluorescence increase (RFU / min) over the first 4 minutes. The percentage of enzyme inhibition wasdetermined by comparing the slope for each compound to those of the 100% and 0 % activity controls.

[0520] Determination of GO inhibition using a kinetic fluorometric assay

[0521] The same protocol described above was used to determine dose-response inhibition curves. In this format, the inhibitor was added to each well using 1:3 serial dilution prepared in 10% DMSO / phosphate buffer. Atotal of 10 concentrations were tested, starting from 400 pM, 300 pM, 200 pM, or 100 pM depending on the compound. All other assay conditions, including reaction composition, timing, and fluorescence detection, remained unchanged.

[0522] Determination of GO Ki values using a kinetic fluorometric assay

[0523] Kinetic inhibition constants (Ki) were determined using the same fluorometric assay format described above, with measurements performed in black 96-well OptiPlates (PerkinElmer). For each Ki determination, three replicates of four to five different inhibitor concentrations were tested in the presence of ten different glycolate concentrations. The final reaction volume per well was 200 pL and consisted of: 50 mM phosphate buffer (pH 7, 15 pL), inhibitor at the corresponding concentration in DMSO (5 pL), GO (40 pL of 125 nM solution in phosphate buffer: final concentration of 25 nM), Amplex® Red / HRP mixture (40 pL containing Amplex® Red 250 pM and HRP 2 units; final Amplex® Red concentration: 50 pM) and glycolate (100 pL of a solution ranging from 20 mM to 1 pM; final concentration of 10 mM to 0.5 pM). In wells for KM determination DMSO alone was used instead of inhibitor solution.

[0524] The rection was initiated by the addition of glycolate and immediately monitored by fluorescence at zex 560 ± 10 nm and zem 590 ± 10 nm using a PerkinElmer Enspire Multimode Plate Reader. Fluorescence was recorded every 60 seconds over 15 min at 27°C. The initial velocity (vo) was calculated from the linear portion of the fluorescence-time curve, corresponding to a 10 min interval, and expressed as the slope (RFU / min). vo values were averaged across three replicates per condition.

[0525] Determination ofLDHA inhibition percentage using a kinetic fluorometric assay

[0526] The inhibitory activity of test compounds against recombinant LDHA was assessed using a kinetic fluorometric assay in black 96-well OptiPlates (PerkinElmer). Each plate includedfour replicates of: (i) 100% activity controls (containing enzyme and substrate, but not inhibitor), (ii) 0% activity controls (containing enzyme but neither substrate nor inhibitor) and (iii) each tested compound at 10 pM.

[0527] The final reaction volume per well was 200 pL consisting of: 50 mM phosphate buffer (pH 7), LDHA (0.015 U / ml), pyruvate (1 mM), NADH (150 pM) and 1 % DMSO. To each well, 40 pL of either phosphate buffer pH 7 with 10 % DMSO (for control wells) or a 50 pM solution of the test compound (in the same buffer) was added. A fresh solution of 600 pM NADH in phosphate buffer was prepared and mixed with LDHA at 0.3 U / mL in a 5: 1 ratio. Then, 60 pL of this NADH / LDHA mixture was added to each well, and background NADH degradation was monitored by measuring fluorescence every 60 seconds for 10 min at 27°C using a PerkinElmer Multimode Plate Reader Enspire (2ex340 ± 10 nm and / .em 460 ± 10 nm).

[0528] Subsequently, 100 pL of either phosphate buffer (for 0% activity control) or 100 pL pyruvate (2 mM) was added to initiate the enzymatic reaction. Fluorescence was then recorded again under the same conditions to monitor the NADH consumption associated with LDHA activity. Inhibition percentages were calculated by comparing the slopes of NADH consumption between treated and control wells, after correcting for background NADH degradation.

[0529] Determination of LDHA inhibition using a kinetic fluorometric assay

[0530] The same protocol described above was used to determine dose-response inhibition curves. In this format, the inhibitor was added to each well using 1:3 serial dilution prepared in 10% DMSO / phosphate buffer. Atotal of 10 concentrations were tested, starting from 400 pM, 300 pM, 200 pM, or 100 pM depending on the compound. All other assay conditions, including reaction composition, timing, and fluorescence detection, remained unchanged.

[0531] Determination of LDHA Ki values using a kinetic fluorometric assay

[0532] Kinetic inhibition constants ( ) were determined using the same fluorometric assay format described above, with measurements performed in black 96-well OptiPlates (PerkinElmer). For each Ki determination, three replicates of four to five different inhibitor concentrations were tested in the presence of ten different pyruvate concentrations. The final reaction volume per well was 200 pL and consisted of: 35 pL of 50 mM phosphate buffer (pH 7), 5 pL of the inhibitor at the corresponding concentration in DMSO, 60 pL of a freshly prepared NADH / LDHA mixture (500 pM NADH and 0.165 U / mL of LDHA; resulting in final concentrations of 150 pM for NADH and 0.015 U / ml for LDHA) and 100 pL of pyruvate prepared as a 3:2 serial dilution ranging from2 mM to 52 pM; (final concentrations of 1 mM to 26 nM). In wells for KM determination DMSO alone was used instead of inhibitor solution.

[0533] Fluorescence was registered as described above, and the initial velocity (vo) was calculated from the linear portion of the fluorescence-time curve, corresponding to a 10 min interval, and expressed as the slope (RFU / min). vo values were averaged across three replicates per condition.

[0534] Animal studies

[0535] All animal procedures were approved by the Institutional Animal Care & Use Committees of Louisiana State University Health Sciences Center-Shreveport (P-21-043, and P-24-025). All studies were performed in accordance with the institutional guidelines. Mice were randomly allocated to treatment groups followed by confirmation of equal body weights before treatment. AgxK' mice on the C57BL / 6J background were generated using CRISPR / Cas9, with guide-RNA targeting exon 1 of Agxt.8Agxf ' mice were housed under controlled temperature (22 ± 2 °C) and humidity conditions (40-60%) on a 12-h light-dark cycle and fed ad libitum with a standard chow diet (LabDiet, 5053, 13% of calories from fat). Twelve- week-old male Agxf1’ were orally gavaged with either an aqueous solution of 0.6% Methyl cellulose (Sigma-Aldrich) + 0.5% Tween® 80 (Sigma- Aldrich) or 2 at a 20 mg / kg / day dissolved in the same formulation. Urine was collected starting five days prior to dosing to set a baseline and throughout the treatment regimen. After 10 days, the mice were sacrificed, blood was collected, livers and kidneys were fixed in 4% formalin.

[0536] Hepatocyte Isolation

[0537] Primary hepatocytes were isolated from 8-10-week-old male Agxt / _mice. Following euthanasia, the left lateral lobe of the liver was carefully excised and rinsed with phosphate-buffered saline (PBS). Hepatocyte isolation was performed using the gentleMACS™ Perfuser system and associated kits from Miltenyi Biotec, following the described protocol.62Briefly, the gentleMACS Perfuser was assembled according to the manufacturer’s instructions, consisting of a lid, clamp, grid, and base. The liver lobe was secured onto the grid, and the lid was attached to the base to form a sealed chamber. A pre-warmed pre-digestion buffer was added to initiate the automated perfusion program. This program includes multiple steps: priming, initial perfusion, washing, equilibration, and enzymatic perfusion. An enzyme digestion solution containing collagenase and other matrix-degrading enzymes was perfused to facilitate hepatocytedissociation. Upon completion of enzymatic digestion, the digested liver lobe was transferred into a gentleMACS C Tube for further mechanical dissociation via gentle stirring. The resulting cell suspension was filtered through a MACS SmartStrainer (Miltenyi Biotec) to remove tissue debris and non-parenchymal cells. The filtrate containing hepatocytes was collected and subjected to low-speed centrifugation (50g for 3 min) to pellet the cells. Isolated primary hepatocytes were then resuspended in warm thawing and plating medium and plated at approximately 80% confluence into collagen-coated plates (0.01% w / v, Advanced Biomatrix NC0476635). Cells were allowed to adhere for approximately 3-4 h and non-adhered cells and debris were rinsed with warm PBS. Cells were maintained in hepatocyte maintenance medium (William’s E Medium (Gibco, A1217601) with maintenance supplements (Gibco, CM4000) at 37°C in a humidified atmosphere containing 5% CO2.

[0538] Cell culture treatments and measurements

[0539] Mouse primary hepatocytes were maintained in maintenance medium and utilized for experiments no longer than 48 h post-isolation. Hepatocytes were plated at approximately 80% confluence and allowed to adhere overnight. Next day, cells were challenged with glycolic acid at a concentration of 5 mM and treated with either vehicle (0.1% DMSO) or the desired compound at 2,10, 20, or 50 pM for additional 24 h. For proteasomal inhibition studies, overnight adhered primary hepatocytes were treated with 10 pM MG-132 (Sigma Aldrich, 474790) for 4 h, followed by treatment with either compound 2 or DMSO for 24 h. For silencing studies, primary hepatocytes were treated during seeding with either siAtg5 (Mouse Atg5 ON-TARGETplus siRNA, Dharmacon, CO, L-064838-00-0005) or scRNA (ON-TARGETplus non-targeting pool, Dharmacon, CO, D-001810-10-05) overnight using Lipofectamine RNAiMAX (Invitrogen, 13778150) transfection reagent following the manufacturer’s protocol. At the next day, the hepatocytes were treated with either compound 2 or DMSO for 24 h. For oxalate quantification following treatment, cells were washed twice with PBS, gently scraped into PBS, and collected by centrifugation. Cell pellets were stored at -80°C until further processing. Frozen cell pellets were thawed and lysed via sonication using a probe sonicator (Sonics Vibra-Cell, VCX130-50W). Oxalate levels in cell lysates were measured using the Oxalate Assay Kit (Abeam, ab 196990) according to the manufacturer’s instructions. Final oxalate concentrations were normalized to total protein content determined using a standard BCA protein assay (Thermo Fisher Scientific or equivalent).9

[0540] RNA isolation and qRT-PCR

[0541] Total RNA was extracted from approximately 50 mg of liver tissue. The tissue was lysed using TRIzol reagent (Invitrogen, 15596018) and homogenized with soft tissue ceramic beads (Cayman Chemical Company, 10011152) in a Precellys Evolution homogenizer (Bertin Technologies). After phase separation via chloroform extraction, RNA was purified using the RNeasy Mini Kit (QIAGEN, 74106) following the manufacturer’s protocol. For primary hepatocytes, lysis and RNA extraction were also carried out using the RNeasy Mini Kit per the supplier’s guidelines. Complementary DNA (cDNA) was synthesized using the SuperScript III First-Strand Synthesis System (Invitrogen, 18080-051) in accordance with the manufacturer’s instructions, utilizing a Mastercycler nexus gradient thermocycler (Eppendorf). Primers were obtained from Integrated DNA Technologies, and qRT-PCR was conducted with the SSoAdvanced Universal SYBR Green Supermix (Bio-Rad, 175271)onaCFX96TouchReal-Time PCR Detection System (Bio-Rad). Gene expression data were normalized to the Gapdh housekeeping gene and analyzed using theAACt method to determine fold changes relative to control groups.

[0542] Protein isolation and Western blot

[0543] For protein extraction from liver tissue, approximately 50 mg of sample was used. The tissue was homogenized in RIPA lysis and extraction buffer (G Biosciences, 786-489) containing 1% Halt protease inhibitor cocktail (Thermo Scientific, 78429), 1% phosphatase inhibitor cocktail A (Alfa Aesar, J65354. LQ), and ceramic homogenization beads (Cayman Chemical Company, 10011152), using the Precellys Evolution homogenizer (Bertin Technologies). For cell lysates, RIPA buffer with the same inhibitor cocktails (Bio-Rad, 1610737) was used. Protein concentration was determined using the Quick Start Bradford Dye Reagent (Bio-Rad, 5000205). Western blotting was performed using the following primary antibodies: rabbit anti-AGXT (Sigma, HPA035370, 1:1,000), rabbit anti-LDHA (Cell Signaling Technology, 2012, 1:1,000), rabbit anti-HAOl (Abeam, 194790, 1:1,000), mouse anti-P-actin (Cell Signaling Technology, 3700S, 1:1,000), mouse anti-GAPDH (Santa Cruz Biotechnology, sc365062, 1:5,000) and rabbit anti-ATG5(Cell Signaling Technology, 12994S, 1:1,000). Fluorescent secondary antibodies — donkey anti-rabbit (Li-Cor, 926-68073, 1:20,000) and donkey anti-mouse (Li-Cor, 926-32212, 1:20,000) — were used for detection with a Li-Cor Odyssey XF Imager. Band intensities were quantified using Image Studio Lite v.5.2 and normalized to GAPDH or P-actin levels.

[0544] Biochemical analyses of plasma and livers

[0545] Immediately after euthanizing the mice, plasma was obtained by centrifuging whole blood collected in lithium heparin-coated PST tubes (BD Microtainer, 365985). Levels of aspartate transaminase (AST) and alanine transaminase (ALT) in the plasma were determined using EnzyChrom assay kits specific for each enzyme (BioAssay Systems, EASTR-100 for AST and EALT-100 for ALT), following the protocols provided by the manufacturer. Lactate dehydrogenase (LDH) activity in liver samples was assessed using the LDH Assay Kit (Abeam, ab 102526) in accordance with the manufacturer’s guidelines.

[0546] Histology

[0547] Formalin-fixed tissues were sectioned on a M355S rotary microtome (Thermo Fisher Scientific) at 4-pm thickness and mounted on glass slides. Slides were stained for H& E (Thermo Fisher Scientific). Calcium-oxalate crystals were visualized using a Nikon Eclipse TS2R-FL microscope equipped with a polarized light filter.

[0548] Oxalic and glycolic acid analysis in urine using IC-MS

[0549] A volume of 80 pL of urine collected from mice before and during treatment, was diluted with 320 pL of ultrapure water and filtered through Centrifugal Filter Units (Merk), followed by injection for analysis by IC-MS. The IC mobile phase A (MPA; weak) was water and the mobile phase B (MPB; strong) was water containing 100 mM KOH. A Thermo Scientific Dionex ICS-6000+ system, including a Thermo lonPac ASH column (4-pm particle size, 250 x 2 mm), with the column compartment kept at 35°C, was used for separating metabolites. The autosampler tray was chilled to 4°C. The mobile phase flow rate was 360 pL min-1 and the gradient elution program was 0-5 min, 1% MPB; 5-25 min, 1-35% MPB; 25-39 min, 35-99% MPB; 39-49 min, 99% MPB; 49-50 min and99-l% MPB. The total run time was 55 min. To enhance sensitivity, methanol was delivered by an external pump and combined with the eluent via a low dead volume mixing tee. Data were acquired using a Thermo Orbitrap IQ-X Tribrid Mass Spectrometer under negative electrospray ionization.

[0550] COMPUTATIONAL METHODS

[0551] Docking protocol

[0552] Docking studies were carried out with Autodock 4.2.6 (AD4)63on the crystal structures of human lactate dehydrogenase M isozyme form (ALOHA, PDB ID: 1 II 0)41and human glycolate oxidase (AGO, PDB ID: 2RDT).36Ligand PDB structure was built and minimized on MOE.64Onceoptimized, ligand PDB was prepared for docking using the prepare_ligand4.py script included MGLTools 1.5.4.65Protein structures, on the other hand, were prepared for docking using MOE’s structure preparation module.64Water and ligand molecules were removed and charges and nonpolar hydrogen atoms were added at pH 7.0. The produced structures were saved as a pdb files and prepared for docking using the prepare_receptor4.py script from MGLTools. AD4 was used to automatically dock the ligands into the ALDHA and hGO. For ALDHA (PDB ID 1110) the docking grid was centered on the pyruvate-NADH binding site and set with the following grid parameters: 85 A x 90 A x 80 A with 0.375 A spacing. For AGO (PDB ID: 2RDT) the docking grid was centered on the FMN-CDST binding site and set with the following grid parameters: 80 A x 80 A x 80 A with 0.375 A spacing. In all calculations, AD4 parameter file was set to 100 GA runs, 2.500.000 energy evaluations and a population size of 150. The Lamarckian genetic algorithm local search (GALS) method was used for the docking calculations. All dockings were performed with a population size of 250 and a Solis and Wets local search of 300 rounds was applied with a probability of 0.06. A mutation rate of 0.02 and a crossover rate of 0.8 were used. The docking results from each of the 100 calculations were clustered based on root-mean square deviation (RMSD) (solutions differing by less than 2.0 A, except where stated) between the Cartesian coordinates of the atoms and were ranked on the basis of free energy of binding. UCSF Chimera 1.1566was used for molecules visualization and figures generation.66

[0553] Homology modeling and molecular dynamic protocol

[0554] A set of homology models of AGO were constructed using the 2-AGO (PDB ID 2RDT) docking pose obtained previously as a template. For this task, MOE Homology Model module was used with the default parameters. 10 models were created and classified according to their GB / VI energies, and the best performing model was selected. This model was next prepared using MOE QuickPrep module to correct the structure and to prepare the macromolecular complex for further MD analysis. For ALDHA, the obtained docking pose of ligand 2 on ALDHA (PDB ID 1110, subunit B) was used on MOE to build a dimeric complex with subunit A of ALDHA (PDB ID 1110), the latter containing cofactor 1,4-dihydronicotinamide adenine dinucleotide (NAI) and oxamic acid (OXM) inhibitor. The spatial conformation of the dimeric complex was based on the published crystal structure (1110), saved as a pdb file and prepared for further MD analysis using MOE QuickPrep module67Both 2-AGO and 2-ALDHA pdb files were next used for atomistic molecular dynamic (MD) simulation on NAMD 2.14. 2 ligand, FMN, NAI, and OXM wereparametrized using LigParGen server68-70and the MD simulations ran using OPLS-AA / M force field.71The initial ensembles were first submitted to a 30 picoseconds (ps) energy minimization at 300K on periodic boundary conditions (grid size: 84 x 73 x 42) containing the biomolecular complexes in a water box (0.15M NaCl concentration) to remove high-energy contacts, followed by successive 300 ps NVT and a 1 nanosecond (ns) NPT equilibration before the productive 100 ns MD simulation. During the MD simulation, water bonds were constrained using the SHAKE algorithm;72for every 2 fs, neighbors were searched in grid cells with 1 nm as the cutoff value for short-range neighbor list, electrostatic, and van der Waals; long- range electrostatics were treated with the particle mesh Ewald method73with a grid spacing of 1; constant temperature and pressure were maintained by coupling the system to an external bath at 300 K and 1 bar, using velocity rescaling74and Parrinello-Rahman,73respectively. A RESPA propagator with the integration time step of 1 fs was used.76TIP3P model was applied for water,77and UCSF Chimera 1.15 was used to perform the trajectory analyses and generate the final images. The trajectories data were subjected to an RMSD cluster analysis using the MD movie Tool in Chimera setting frame 0 as the starting one, and the default step size parameter of 34. The obtained most populated clusterrepresentative structures were saved as a pdb files and analyzed. Solvent accessible surface area (SASA), H-bonds and root-mean-square deviation (RMSD) calculations of the simulations were done using VMD78with in-house TLC scripts.

[0555] Chameleonicity evaluation.

[0556] Conformational ensembles were generated using the LowModeMD search engine implemented in MOE 2024 (6, Montreal, Canada)64with the MMFF94x force field and GB / VI implicit solvent model. Separate searches were performed in implicit water (s = 80) and implicit chloroform (s = 4.8). All conformers were energy -minimized to a root-mean-square (RMS) gradient < 0.005 kcal mol-1A"’.

[0557] Implicit Water (s = 80): Search method: LowModeMD (default); Rejection limit: 100; Iteration limit: 10,000; Energy window: 7 kcal mol-1; RMSD limit: 0.25 A; Conformation limit: 10,000. The search produced 526 unique conformers. The 200 lowest-energy conformers (AE < 10 kcal mol-1from the global minimum) were retained for further analysis.

[0558] Implicit Chloroform (e = 4.8): Search method: LowModeMD; Rejection limit: 500; Iteration limit: 10,000; Energy window: 12 kcal mol '; RMSD limit: 0.35 A; Conformation limit:10,000. The search produced 294 unique conformers. The 200 lowest-energy conformers (AE < 10 kcal moE1from the global minimum) were retained for further analysis.

[0559] 3D polar surface area (3D-PSA) was calculated with VEGA ZZ using the SRFCALC DOTS PSA 0.0 2 0 command. Intramolecular hydrogen bonds (IMHB) and radius of gyration (Rgyr) were calculated using custom Python scripts built on the RDKit cheminformatics library.79The resulting data from conformer ensembles were analyzed to compute environment-dependent distributions of 3D-PSA, Rgyr, and IMHB counts. Density plots and 2D / 3D scatter plots were constructed to compare conformational profiles across solvents. The data interpretation followed the established protocols from Rossi Sebastiano43and Garcia Jimenez.44

[0560] Statistical analyses

[0561] All statistical analyses were performed using GraphPad Prism v.10 software and Microsoft Excel 365. All data were expressed either as mean ± SD or mean ± SEM and repeated with at least three independent experiments. Biological replications were performed as indicated and averaged for each individual experiment. Each data point presented represents an independent experiment or an individual subject. Before statistical comparisons, data were tested for equal variance and normality using Shapiro-Wilk and Kolmogorov- Smirnov tests. If data passed, an unpaired z-test was used to compare two groups and a one-way ANOVA followed by Tukey’s post hoc test for comparisons among more than two groups. Otherwise, nonparametric tests (Mann-Whitney ( / -test or Kruskal-Wallis test followed by Dunn’s post hoc test) were used. P < 0.05 was considered statistically significant.

[0562] ABBREVIATIONS

[0563] 3D-PSA, tridimensional polar surface area; AcOEt, ethyl acetate; AD4, Autodock 4.2.6; AGXT, alanine-glyoxylate aminotransferase; ALT, alanine aminotransferase; AMFSA, aminomethylfuryl salicylic acid; AST, aspartate aminotransferase; Atg5, autophagy related gene 5; bRo5, beyond Rule of 5; Ca-Ox, calcium oxalate; EtOH, ethanol; FCC, flash column chromatography; FSA, furylsalicylic acid; GA, glycolic acid; GO, glycolate oxidase; AGO, human isoform of glycolate oxidase; LDHA, human isoform of lactate dehydrogenase A; HyT, hydrophobic-tag; HyT-PD, hydrophobic-tag protein degrader; IMHB, intramolecular hydrogen bond; LDHA, lactate dehydrogenase isozyme A; MASH, metabolic dysfunction-associated steatohepatitis; MASLD, metabolic dysfunction-associated steatotic liver disease; MeOH, methanol; MOE, molecular operative environment; NAI, 1,4-dihydronicotinamide adeninedinucleotide; OPPFSA, oxophenylpropenylfuryl salicylic acid; OXM, oxamic acid; PA, pyruvic acid; PHI, primary hyperoxaluria type 1; PLC, preparative layer chromatography; Pol, protein of interest; qRT-PCR, quantitative real-time polymerase chain reaction; Rgyr, radius of gyration;RMSD, root-mean square deviation; SA, salicylic acid; siRNA, small interfering RNA; siScr, control scrambled siRNA; SL, selectivity ligand.

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[0576] (12)Pfau, A.; Ermer, T.; Coca, S. G.; Tio, M. C.; Genser, B.; Reichel, M.; Finkelstein, F. O.; Marz, W.; Wanner, C.; Waikar, S. S.; Eckardt, K.-U.; Aronson, P. S.; Drechsler, C.; Knauf, F. High Oxalate Concentrations Correlate with Increased Risk for Sudden Cardiac Death inDialysis Patients. J. Am. Soc. Nephrol. 2021, 32 (9), 2375-2385. https: / / doi.org / 10.1681 / ASN.2020121793.

[0577] (13) Garrelfs, S. F.; Frishberg, Y.; Hulton, S. A.; Koren, M. J.; O’Riordan, W. D.; Cochat, P.; Deschenes, G.; Shasha-Lavsky, H.; Saland, J. M.; van’t Hoff, W. G.; Fuster, D. G.; Magen, D.; Moochhala, S. H.; Schalk, G.; Simkova, E.; Groothoff, J. W.; Sas, D. J.; Meliambro, K. A.; Lu, J.; Sweetser, M. T.; Garg, P. P.; Vaishnaw, A. K.; Gansner, J. M.; McGregor, T. L.; Lieske, J. C. Lumasiran, an RNAi Therapeutic for Primary Hyperoxaluria Type 1. N. Engl. J. Med.2021, 384 (13), 1216-1226. https: / / doi.org / 10.1056 / nejmoa2021712.

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[0586] (22) Saffe...

Claims

CLAIMSWhat is claimed is:

1. A compound of Formula (I):Formula (I)or a pharmaceutically acceptable salt, prodrug, hydrate, or solvate thereof, wherein A-B is selected from CH2-CH2-, -CH=CH- or -C=C~C is selected from -CH2- or -CO-;D is selected from the group consisting of -CH2, -O-, or -NH-;E is selected from the group consisting of -CH2, -O-, or -NH-;F is selected from the group consisting of -O-, -NH-, -CO-NH-, or -NH-CO-; G = is selected from -CH2-, -CO- or -CO-CH2-;H-I is selected from -CH2-CH2- or -CH=CH-;m is 0 or 1;n is 0 or 1;o is 0, 1, 2, or 3; andp is 0, 1, 2, 3, or 4.

2. A compound of Formula (II):(Formula II)or a pharmaceutically acceptable salt, prodrug, hydrate, or solvate thereof, whereinA-B is selected from -CH2-CH2-, -CH=CH- or -C=C- C is selected from -CH2- or -CO-;D is selected from CH or N;E is selected from -CH2 or -CO-;F is selected from CH or N; andG = is selected from -CH2-, -CO- or -CO-CH2-; andH-I is selected from -CH2-CH2- or -CH=CH-.

3. A compound of F ormula (III):(Formula III)or a pharmaceutically acceptable salt, prodrug, hydrate, or solvate thereof, wherein A-B is selected from -CH2-CH2-, -CH=CH- or -C=C-;C is selected from -CH2- or -CO- D is selected from the group consisting of -CH2, -O-, or -NH-;E is selected from the group consisting of -CH2, -O-, or -NH-;H-I is selected from -CH2-CH2- or -CH=CH- m is 0 or 1;n is 0 or 1;o is 0, 1, 2, or 3; andp is 0, 1, 2, 3, or 4; andwherein either D = -0-, E = -0-, or both D = -O- and E = -O-.

4. The compound of claim 1, wherein the compound is:YSL-ad3;YSL-ad1.

5. The compound of claim 2 wherein the compound is:

7. The compound of any one of claims 1-6, wherein the pharmaceutically acceptable salt is selected from an inorganic salt or an organic salt.

8. The compound of claim 7, wherein the inorganic salt is selected from an ammonium salt, a sodium salt, a potassium salt, a lithium salt, a calcium salt, a magnesium salt, an aluminum salt, a zinc salt, a ferrous salt, a ferric salt, a hydrochloride salt, a hydrobromide salt, a bisulfate salt, a phosphate salt, or a bicarbonate salt.

9. The compound of claim 7, wherein the organic salt is selected from a mesylate salt, a tosylate salt, a besylate salt, a camphrosulfonate salt, an acetate salt, a formate salt, a maleate salt, a succinate salt, a glutarate salt, a tartrate salt, a citrate salt, a malate salt, or a fumarate salt.

10. A pharmaceutical composition comprising a compound of any one of claims 1-9, or a combination thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.

11. The pharmaceutical composition of claim 10, wherein the excipient comprises methyl cellulose.

12. The pharmaceutical composition of claim 10 or claim 11, further comprising polysorbate 80.

13. The pharmaceutical composition of claim 10, wherein the pharmaceutical composition comprises an adjuvant, an additional active agent, or a combination thereof.

14. The pharmaceutical composition of claim 13, wherein the adjuvant comprises an amino acid, a polyol, or a combination thereof.

15. The pharmaceutical composition of claim 14, wherein the amino acid is selected from the group consisting of L-Alanine, L-Arginine, L-Lysine, L-Histidine, L-Omitine, L- Serine, L-Valine, L-Leucine, L-Proline, or Glycine.

16. The pharmaceutical composition of claim 14, wherein the polyol is selected from N-methyl-D-glucamine, tromethamine, choline, glucosamine, or diethanolamine.

17. The pharmaceutical composition of claim 10, further comprising a vitamin B6 derivative.

18. The pharmaceutical composition of claim 17, wherein the vitamin B6 derivative is selected from the group consisting of pyridoxinium, pyridoxamine, piridoxal, or nicotinamide.

19. A method of inhibiting the activity of glycolate oxidase (GO) and lactate dehydrogenase (LDHA) while activating degradation of glycolate oxidase (GO) and / or lactate dehydrogenase (LDHA) in a subject, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of claims 10-18.

20. The method of claim 19, wherein the therapeutically effective amount comprises less than about 0.1 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 120 mg / kg, about 135 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, and greater than about 200 mg / kg.

21. The method of claim 19, wherein the therapeutically effective amount comprises less than about 1 uM, about 1 uM, about 1.5 uM, about 2 uM, about 2.5 uM, about 3 uM, about 4 uM, about 5 uM, about 6 uM, about 7 uM, about 8 uM, about 9 uM, about 10 uM, about 12.5 uM, about 15 uM, about 20 uM, about 22.5 uM, about 25 uM, about 30 uM, about 35 uM, about 40 uM, about 45 uM, about 50 uM, about 60 uM, about 70 uM, or greater than about 70 uM.

22. A method of treating a subject afflicted with an oxalate production-related disease or disorder the method comprising:measuring the circulating oxalate levels of a subject; andadministering to the subject a therapeutically effective amount of the composition of any one of claims 10 to 18.

23. The method of claim 22, wherein the oxalate production-related disease or disorder comprises a cardiometabolic disease or disorder, a cardiovascular disease or disorder, a metabolic disease or disorder, a liver disease or disorder, a renal disease or disorder, or a combination thereof.

24. The method of claim 22, wherein the oxalate production-related disease or disorder comprises a GO-associated disease or disorder and / or an LDHA-associated disease or disorder.

25. The method of claim 23, wherein the cardiovascular disease or disorder comprises hypertension, atherosclerotic cardiovascular disease (ASCVD), coronary artery disease (CAD), acute coronary syndrome (ACS), myocardial infarction (MI), ischemic stroke, peripheral artery disease (PAD), carotid atherosclerotic disease, hear failure, myocardialischemia-reperfusion injury, vascular calcification, arterial stiffening, an aneurysm, or a combination thereof.

26. The method of claim 23, wherein the renal disease or disorder comprises nephrolithiasis, recurrent kidney stone disease, chronic kidney disease (CKD), end-stage renal disease (ESRD), or a combination thereof.

27. The method of claim 23, wherein the metabolic disease or disorder comprises diabetes, dyslipidemia, obesity, hyperoxaluria, primary hyperoxaluria (PH), PHI, PH2, PH3, systemic oxalosis, or a combination thereof.

28. The method of claim 23, wherein the liver disease or disorder comprises cirrhosis, hepatitis, metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction-associated steatohepatitis (MASH), compensated and decompensated cirrhosis secondary to MASLD / MASH, portal hypertension, ascites, hepatocellular carcinoma (HCC), alcohol-associated liver disease (ALD), specific aetiology steatoic liver disease, drug-induced liver injury, a monogenic steatotic liver disease, a cryptogenic steatotic liver disease, or a combination thereof.

29. The method of claim 22, wherein the therapeutically effective amount of the composition inhibits glycolate oxidase (GO) activity and lactate dehydrogenase (LDHA) activity.

30. The method of claim 22, wherein the therapeutically effective amount of the composition activates degradation of glycolate oxidase (GO) and / or lactate dehydrogenase (LDHA).

31. The method of claim 22, wherein measuring the circulating oxalate levels of a subject comprises an enzymatic assay, mass spectrometry, or a combination thereof.

32. The method of claim 22, wherein the subject’s circulating oxalate level is at least about 1 uM, about 1 uM, about 1.5 uM, about 2 uM, about 2.5 uM, about 3 uM, about 4 uM, about 5 uM, about 6 uM, about 7 uM, about 8 uM, about 9 uM, about 10 uM, or greater than about 10 uM.

33. The method of claim 22, wherein the therapeutically effective amount comprises less than about 0.1 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 120 mg / kg, about 135 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, and greater than about 200 mg / kg.

34. The method of claim 22, wherein the therapeutically effective amount comprises less than about 1 uM, about 1 uM, about 1.5 uM, about 2 uM, about 2.5 uM, about 3 uM, about 4 uM, about 5 uM, about 6 uM, about 7 uM, about 8 uM, about 9 uM, about 10 uM, about 12.5 uM, about 15 uM, about 20 uM, about 22.5 uM, about 25 uM, about 30 uM, about 35 uM, about 40 uM, about 45 uM, about 50 uM, about 60 uM, about 70 uM, or greater than about 70 uM.

35. The use of the compound of any one of claims 1-9 or the pharmaceutical composition of anyone of claims 10-18 for treating a subject afflicted with an oxalate production-related disease or disorder.

36. The use of the compound of any one of claims 1-9 or the pharmaceutical composition of anyone of claims 10-18 for inhibiting the activity of glycolate oxidase (GO)and lactate dehydrogenase (LDHA) while activating degradation of glycolate oxidase (GO) and / or lactate dehydrogenase (LDHA) in a subject.

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