Calcium oxalate crystal inhibitors for renal disorders

Abstract

The present invention relates to inositol derivatives comprising two or more cyclohexanol pentaester moieties connected by a common central linker and their use in the treatment or prevention of conditions associated with pathological crystallization. The present invention further relates to intermediates useful in the synthesis of the compounds of the present invention.

Description

Background Art

[0001] Kidney stones are mineral deposits in kidney tissue that form due to oversaturation of certain minerals in urine. The prevalence of kidney stones is high, up to 14.8%, and carries a huge risk of recurrence. Moreover, its incidence is increasing due to changes in lifestyle, diet, and increased prevalence of associated risk factors such as obesity, diabetes, and hypertension.

[0002] The underlying causes of kidney stone formation vary. However, a common component of most kidney stones is calcium oxalate (CaOx). CaOx crystallization and stone formation in the kidneys can physically block tubules, cause tissue inflammation and fibrosis, and impair organ function, creating a risk for developing chronic kidney disease and end-stage renal disease.

[0003] Another calcium precipitate of clinical importance in the kidney is calcium phosphate. Kidney stones composed primarily (50% or more) of calcium phosphate account for up to 10% of all stones and 15%-20% of calcium stones, 80% of which are composed of calcium oxalate. Calcium phosphate is also a minor component in up to 30% of calcium oxalate stones.

[0004] Different diseases such as hyperoxaluria, Dent's disease or Bartter's syndrome can lead to CaOx kidney stone formation. For example, in the case of primary hyperoxaluria, excessive oxalate is produced due to a deficiency or dysfunction of liver proteins involved in oxalate metabolism. The high oxalate load in the urine leads to CaOx crystallization in the renal tubules, kidney stone formation and consequently damage to the tissue. Patients suffer from renal failure and end-stage renal disease at a young age. Due to limited treatment options, 80% of patients require a combined liver-kidney transplant by the age of 30 years.

[0005] To date, treatment options for patients with kidney stones are merely palliative and far from disease modifying, thus placing patients at risk for stone recurrence and serious complications such as chronic kidney disease and end-stage renal disease. Existing stones can be surgically removed with concomitant medical management of pain associated with kidney stones. However, recurrence rates are high and preventive measures are limited. Recommended measures for patients with high urine calcium levels include high fluid intake, adaptive nutrition, and thiazide medications.

[0006] Although CaOx crystallization inhibitors such as citrate or phosphate have been suggested to prevent kidney stone formation, limited efficacy and adverse side effects limit their clinical use. Citrate has shown some efficacy as a preventive measure in patients with recurrent CaOx stones, but its mode of action remains unclear.

[0007] In summary, an agent that can reduce the propensity of CaOx crystals to nucleate, grow, and adhere to renal epithelial cells and ultimately reduce renal damage independent of the underlying cause would have important therapeutic value. Effective inhibitors of CaOx crystals in urine would be applicable to a variety of conditions associated with kidney stone formation and could reduce the risk of further serious complications.

[0008] Based on the above prior art, the object of the present invention is to provide a pharmaceutical compound for treating or preventing calcium oxalate and / or calcium phosphate crystals in conditions associated with pathological calcium oxalate crystals. This object is achieved by the subject matter of the independent claims of this specification. Summary of the invention

[0009] The first aspect of the present invention relates to a compound comprising two or more cyclohexanol pentaester moieties described by general formula (I) connected by a common central linker L,

[0010]

[0011] Where X is selected from OPO 3 2- OPSO 2 2- or OSO 3 - , L is a common central linker connecting n individual moieties represented by the general formula in square brackets and n is an integer from 2 to 10.

[0012] In certain embodiments, the compound is described by formula (II), (III) or (IV),

[0013]

[0014]

[0015] It will be understood that any compound of the present invention, when defined herein as a negatively charged compound, will be associated with a pharmaceutically acceptable counterion.

[0016] A second aspect of the invention relates to the use of the compounds described herein for the treatment of pathologies associated with the crystallization or formation of calcium and / or magnesium precipitates.

[0017] Likewise, the present invention relates to the use of the compounds described herein for treating pathologies associated with the crystallization or formation of oxalate precipitates.

[0018] Another aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of at least one schistosome compound of the present invention or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent or excipient.

[0019] Terms and Definitions

[0020] In the context of this specification, the term C 1 -C 4 Alkyl refers to a saturated straight or branched hydrocarbon having 1, 2, 3 or 4 carbon atoms, wherein in certain embodiments, one carbon-carbon bond may be unsaturated and one CH 2 Parts can be exchanged for oxygen (ether bridge) or nitrogen (NH or NR, R is methyl, ethyl or n-propyl or isopropyl; amino bridge). 1 -C 4 Non-limiting examples of alkyl groups are methyl, ethyl, propyl, prop-2-enyl, n-butyl, 2-methylpropyl, tert-butyl, but-3-enyl, prop-2-ynyl, and but-3-ynyl. 1 -C 4 Alkyl is a methyl, ethyl, propyl or butyl moiety.

[0021] In the context of this specification, C 1 -C 6 Alkyl denotes a saturated straight-chain or branched hydrocarbon having 1, 2, 3, 4, 5 or 6 carbon atoms, in which one carbon-carbon bond may be unsaturated and one CH 2 Parts can be exchanged for oxygen (ether bridge) or nitrogen (NH or NR, R is methyl, ethyl or n-propyl or isopropyl; amino bridge). 1 -C 6 Non-limiting examples of alkyl groups include the following: 1 -C 4 Examples of alkyl are given and additionally include 3-methylbut-2-enyl, 2-methylbut-3-enyl, 3-methylbut-3-enyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1,2-dimethylpropyl, pent-4-ynyl, 3-methyl-2-propyl, and 4-methyl-2-pentyl. In certain embodiments, C 5 The alkyl group is a pentyl or cyclopentyl moiety, and C 6 Alkyl is a hexyl or cyclohexyl moiety.

[0022] When used herein in a narrow sense, the term unsubstituted C n Alkyl refers to the part -C n H 2n -, or if used in the context of a terminal part, -C n H 2n+1 It may contain still fewer H atoms if ring structures or one or more (non-aromatic) double bonds are present.

[0023] The term unsubstituted C n Alkyl and substituted C n Alkyl includes straight chain alkyl groups containing or attached to a cyclic structure, such as a cyclopentane, cyclobutane, cyclopentane or cyclohexane moiety, which is unsubstituted or substituted according to the referenced annotation or context, with straight chain alkyl substituents. The sum of the total number of carbons and (where appropriate) N, O or other heteroatoms in a straight chain or cyclic structure is n.

[0024] When used in chemical formulae, the following abbreviations may be used: Me is a methyl group, CH 3 , Et is ethyl -CH 2 CH 3 , Prop is propyl-(CH 2 ) 2 CH 3 (n-propyl, n-pr) or -CH(CH 3 ) 2 (isopropyl, i-pr), but is butyl-C 4 H 9 、-(CH 2 ) 3 CH 3 、-CHCH 3 CH 2 CH 3 、-CH 2 CH(CH 3 ) 2 or -C(CH 3 ) 3 .

[0025] In the context of this specification, the term C 4 -C 7 Cycloalkyl refers to a saturated hydrocarbon ring having 4, 5, 6 or 7 carbon atoms, wherein in certain embodiments one carbon-carbon bond may be unsaturated and one CH 2 Parts can be exchanged for oxygen (ether bridge) or nitrogen (NH or NR, R is methyl, ethyl or propyl; amino bridge). 4 -C 7 Non-limiting examples of cycloalkyl moieties include cyclopropane (-C 4 H 7 ), cyclopentenyl (C 5 H 9 ) and cyclohexenyl (C 6 H 11 In certain embodiments, the cycloalkyl group is replaced by a C 1 To C 4 In certain embodiments, the cycloalkyl group is substituted with more than one C 1 To C 4The unsubstituted alkyl group is partially substituted.

[0026] The term substituted alkyl in its broadest sense refers to an alkyl as defined above in the broadest sense, which is covalently bonded to different carbon or hydrogen atoms, in particular to an atom selected from N, O, F, B, Si, P, S, Cl, Br and I, which atom itself may be bonded to one or more other atoms of the group, or to hydrogen, or to an unsaturated or saturated hydrocarbon (alkyl or aryl in the broadest sense), where applicable. In a narrower sense, substituted alkyl refers to an alkyl as defined above in the broadest sense, which is substituted on one or more carbon atoms by a group selected from the group consisting of: amine NH 2 , alkylamine NHR, imide NH, alkylimide NR, amino (carboxyalkyl) NHCOR or NRCOR, hydroxyl OH, oxyalkyl OR, oxy (carboxyalkyl) OCOR, carbonyl O and its ketal or acetal (OR) 2 、Nitrile CN、Isonitrile NC、Cyanate CNO、Isocyanate NCO、Thiocyanate CNS、Isothiocyanate NCS、Fluorine F、Chlorine Cl、Bromolecular Br、Iodine I、Phosphonate PO 3 H 2 ,PO 3 R 2 、Phosphate OPO 3 H 2 and OPO 3 R 2 , sulfhydryl SH, sulfanyl SR, sulfoxide SOR, sulfonyl SO 2 R, Sulfonamide SO 2 NHR, sulfuric acid SO 3 H and sulfate SO 3 R, where the R substituent used in this paragraph is different from any other use assigned to R in the body of the specification, is in its broadest sense unsubstituted or substituted C 1 To C 12 Alkyl, and in a more narrow sense, R is methyl, ethyl or propyl, unless otherwise indicated.

[0027] The term amino-substituted alkyl or hydroxy-substituted alkyl refers to a group substituted with one or more amine or hydroxyl NH 2 , NHR, NR 2 or OH modified alkyl according to the above definition, wherein the R substituent used in this paragraph is different from other uses specified for R in the main text of the specification and, in the broadest sense, is itself unsubstituted or substituted C 1 To C 12Alkyl, and in a more narrow sense, R is methyl, ethyl or propyl, unless otherwise specified. Alkyl groups with more than one carbon may contain more than one amine or hydroxyl group. Unless otherwise specified, the term "substituted alkyl" refers to an alkyl group that is substituted with at most one amine or hydroxyl group per C, except for bonds to the alkyl chain, terminal methyl groups or hydrogen.

[0028] The term carboxyl-substituted alkyl refers to an alkyl radical substituted with one or more carboxyl groups (COOH) or derivatives thereof (particularly carboxamides (CONH 2 , CONHR and CONR 2 , or carboxylate COOR, wherein R has the meaning given in the preceding paragraph and is different from the other meanings assigned to R in the body of the specification) modified by an alkyl group according to the above definition.

[0029] Non-limiting examples of amino-substituted alkyl groups include -CH 2 NH 2 、-CH 2 NHMe, -CH 2 NHEt, -CH 2 CH 2 NH 2 、-CH 2 CH 2 NHMe, -CH 2 CH 2 NHEt, -(CH 2 ) 3 NH 2 、-(CH 2 ) 3 NHMe, -(CH 2 ) 3 NHEt, -CH 2 CH(NH 2 )CH 3 、-CH 2 CH(NHMe)CH 3 、-CH 2 CH(NHEt)CH 3 、-(CH 2 ) 3 CH 2 NH 2 、-(CH 2 ) 3 CH 2 NHMe, -(CH 2 ) 3 CH 2 NHEt, -CH(CH 2 NH 2 )CH 2 CH 3 、-CH(CH 2NHMe)CH 2 CH 3 、-CH(CH 2 NHEt)CH 2 CH 3 、-CH 2 CH(CH 2 NH 2 )CH 3 、-CH 2 CH(CH 2 NHMe)CH 3 、-CH 2 CH(CH 2 NHEt)CH 3 、-CH(NH 2 )(CH 2 ) 2 NH 2 、-CH(NHMe)(CH 2 ) 2 NHMe、-CH(NHEt)(CH 2 ) 2 NHEt、-CH 2 CH(NH 2 )CH 2 NH 2 、-CH 2 CH(NHMe)CH 2 NHMe、-CH 2 CH(NHEt)CH 2 NHEt、-CH 2 CH(NH 2 )(CH 2 ) 2 NH 2 、-CH 2 CH(NHMe)(CH 2 ) 2 NHMe、-CH 2 CH(NHEt)(CH 2 ) 2 NHEt、-CH 2 CH(CH 2 NH 2 ) 2 、-CH 2 CH(CH 2 NHMe) 2 and -CH 2 CH(CH 2 NHEt) 2 (for terminal groups) and -CH 2 CHNH 2 -、-CH2 CHNHMe-、-CH 2 CHNHEt- (for an amino-substituted alkyl moiety bridging two other moieties).

[0030] Non-limiting examples of hydroxy-substituted alkyl groups include -CH 2 OH, -(CH 2 ) 2 OH, -(CH 2 ) 3 OH, -CH 2 CH(OH)CH 3 、-(CH 2 ) 4 OH, -CH(CH 2 OH)CH 2 CH 3 、-CH 2 CH(CH 2 OH)CH 3 、-CH(OH)(CH 2 ) 2 OH, -CH 2 CH(OH)CH 2 OH, -CH 2 CH(OH)(CH 2 ) 2 OH and -CH 2 CH(CH 2 OH) 2 (for this terminal part) and -CHOH-, -CH 2 CHOH-, -CH 2 CH(OH)CH 2 -、-(CH 2 ) 2 CHOHCH 2 -、-CH(CH 2 OH)CH 2 CH 2 -、-CH 2 CH(CH 2 OH)CH 2 -、-CH(OH)(CH 2 CHOH-, -CH 2 CH(OH)CH 2 OH, -CH 2 CH(OH)(CH 2 ) 2 OH and -CH2CHCH2OHCHOH- (for a hydroxy-substituted alkyl moiety bridging two other moieties).

[0031] The term halogen-substituted alkyl refers to an alkyl group according to the above definition modified by one or more halogen atoms selected from (independently) F, Cl, Br, I.

[0032] The term fluorine-substituted alkyl refers to an alkyl group according to the above definition modified by one or more fluoride groups F. Non-limiting examples of fluorine-substituted alkyl groups include -CH 2 F, -CHF 2 , -CF 3 、-(CH 2 ) 2 F, -(CHF) 2 H, -(CHF) 2 F, -C 2 F 5 、-(CH 2 ) 3 F, -(CHF) 3 H, -(CHF) 3 F, -C 3 F 7 、-(CH 2 ) 4 F, -(CHF) 4 H, -(CHF) 4 F and -C 4 F 9 .

[0033] Non-limiting examples of hydroxy and fluoro substituted alkyl groups include -CHFCH 2 OH, -CF 2 CH 2 OH, -(CHF) 2 CH 2 OH, -(CF 2 ) 2 CH 2 OH, -(CHF) 3 CH 2 OH, -(CF 2 ) 3 CH 2 OH, -(CH 2 ) 3 OH, -CF 2 CH(OH)CH 3 , -CF 2 CH(OH)CF 3 、-CF(CH 2 OH)CHFCH 3 and -CF(CH 2 OH)CHFCF 3 .

[0034] In the context of this specification, the term aryl refers to a cyclic aromatic C 5 -C 10 Hydrocarbon. Examples of aryl include, but are not limited to, phenyl and naphthyl, and any heteroaryl. Heteroaryl is an aryl group containing one or more nitrogen, oxygen and / or sulfur atoms. Examples of heteroaryl include, but are not limited to, pyrrole, thiophene, furan, imidazole, pyrazole, thiazole, oxazole, pyridine, pyrimidine, thiazine, quinoline, benzofuran and indole. In the context of this specification, aryl or heteroaryl may be additionally substituted with one or more alkyl groups.

[0035] As used herein, the term pharmaceutical composition refers to a compound of the present invention or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the present invention is provided in a form suitable for topical, parenteral or injection administration.

[0036] As used herein, the term pharmaceutically acceptable carrier includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, etc., and combinations thereof, as known to those skilled in the art (see, e.g., Remington: the Science and Practice of Pharmacy, ISBN 0857110624).

[0037] The first aspect of the present invention relates to a compound comprising two or more cyclohexanol pentaester moieties described by general formula (I) connected by a common central linker L,

[0038]

[0039] Where X is selected from OPO 3 2- OPSO 2 2- or OSO 3 - , L is a common central linker connecting n individual moieties represented by the general formula in square brackets and n is an integer from 2 to 10.

[0040] The straight lines in Formula I and elsewhere herein are intended to indicate that the stereochemistry of individual ring carbon atoms is indeterminate. The formula is intended to encompass any diastereomers.

[0041] In some embodiments, n has a value of 2 to 8. In some embodiments, n has a value of 2. In some embodiments, n has a value of 3. In some embodiments, n has a value of 4. In some embodiments, n has a value of 6. In some embodiments, n has a value of 8.

[0042] In certain specific embodiments, n is selected from 2, 3 and 4.

[0043] In certain embodiments, the central linker moiety L comprises or consists of a linear or branched poly(ethylene glycol) or polyglycerol. In certain embodiments, the central linker moiety L comprises or consists of a linear or branched alkyl group.

[0044] In certain embodiments, the molecular weight of the linking moiety L is <1000 g / mol, particularly <700 g / mol, more particularly <500 g / mol, or even <400 g / mol.

[0045] In certain embodiments, the compounds of the invention consist of a central linker L as defined above and two or more moieties described by general formula (I).

[0046] In certain embodiments, any one of the X's contained in any moiety (I) in the compounds according to the present invention is independently selected from OPO 3 2- OPSO 2 2- or OSO 3 - .

[0047] In certain embodiments, all X's contained in any particular moiety (I) in the compounds according to the present invention are identical and are selected from OPO 3 2- OPSO 2 2- or OSO 3 - .

[0048] In certain embodiments, all X in all moieties (I) included in the compounds according to the present invention are OPO. 3 2- .

[0049] In certain embodiments, all X in all moieties (I) included in the compounds according to the present invention are OPSO 2 2- .

[0050] In certain embodiments, all X in all moieties (I) included in the compounds according to the present invention are OSO 3- .

[0051] In certain embodiments, the linking moiety comprises or consists of a linear or branched poly(ethylene glycol) or polyglycerol having a molar mass of <1000 g / mol, particularly <700 g / mol, more particularly <500 g / mol, or even <400 g / mol.

[0052] In certain embodiments, the linking moiety comprises a central core A to which are attached polyglycerol or poly(ethylene glycol) chains separating the cyclohexanol ester moieties of formula (I).

[0053] In certain embodiments, the central core A (if present) can be selected from substituted or unsubstituted C 1 To C 4 Alkyl, substituted or unsubstituted C 4 To C 7 cycloalkyl, or a substituted or unsubstituted five-membered or six-membered aryl.

[0054] The linking moiety is typically a simple metabolically compatible linker designed to facilitate the assembly of two or more cyclohexanol pentaester moieties in a compound at a relatively low cost and ease of synthesis. In certain embodiments, the linker moiety may be a hydrocarbon substituted with a substituent other than hydrogen, such as one or more substituents selected from oxygen, nitrogen, fluorine, or other substituents known in the art of medicinal chemistry to modulate the absorption, distribution, metabolic process, excretion, or toxicity of the compound.

[0055] For the case where the linking moiety L is or comprises a substituted C 1 To C 4 Alkyl, substituted C 4 To C 7 The embodiments of cycloalkyl or substituted five-membered or six-membered aryl groups, wherein the substituents may be one or more oxygen-containing functional groups (non-limiting examples: -OH or =O), one or more nitrogen-containing functional groups (non-limiting examples: NH 2 、NHR N NR N 2 , R N Selected from unsubstituted C 1 To C 4 alkyl), one or more fluorine substituents, or any combination of oxygen, nitrogen or fluorine substituents.

[0056] In certain embodiments of the compounds described by general formula (I), the linking moiety L is a peptide linker. The moiety described by formula (I) can be covalently linked to the side chain of a proteinogenic amino acid, particularly a proteinogenic amino acid having a side chain that facilitates chemical attachment of a cyclohexanol ester moiety. Examples of such amino acids having favorable attachment functions are, but are not limited to, serine and threonine.

[0057] In certain embodiments, any one of the cyclohexanol pentaester moieties described by formula (I) is independently selected from (Ia) and (Ib),

[0058]

[0059] wherein each X (independently) has the above meaning, and wherein L is a linking moiety. In certain embodiments, the compound is described by the general formula (II),

[0060]

[0061] where each X is independent of any other X and can be an OPO 3 2- OPSO 2 2- 、OSO 3 - or CO 2 - , and wherein L is a common central linker (also referred to herein as a "linking moiety") as described in detail above.

[0062] In certain embodiments, the compound is described by formula (IIi), (IIIi) or (IIIi):

[0063]

[0064] where each X is independent of any other X and can be an OPO 3 2- OPSO 2 2- 、OSO 3 - or CO 2 - , and wherein L is a common central linker (also referred to herein as a "linking moiety") as described in detail above.

[0065] In certain embodiments, the compound is described by the general formula (II), (IIi), (IIIi) or (IIIi), wherein the linking moiety L is selected from the group consisting of -(O-CH 2 -CH 2 ) m-O- described poly(ethylene glycol), and m has a value between 1 and 12. In a specific embodiment, n is selected from 2, 3 or 4.

[0066] In certain embodiments, the linking moiety L is selected from the group consisting of -(O-CH 2 -CH(OH)-CH 2 ) m -O- described polyglycerol, and m has a value between 1 and 12. In a specific embodiment, m is selected from 2, 3 or 4.

[0067] In certain embodiments, all moieties described by formula (I) are (Ia) or (Ib).

[0068] In certain embodiments, the compound is described by formula (II), (IIi), (IIIi) or (IIIi), each X is OPO 3 2- And the linking moiety L is a linear poly(ethylene glycol) or polyglycerol.

[0069] In certain embodiments, the molecular weight of the compound described by Formula (II), (IIi), (IIIi) or (Iiiii) is from 1000 g / mol to 1500 g / mol.

[0070] In certain embodiments, the compound is described by formula (II), (IIi), (IIIi) or (IIIi), each X is OPO 3 2- , the linking portion L is a linear poly(ethylene glycol) or polyglycerol and the molecular weight of the compound is 1000 g / mol to 1500 g / mol.

[0071] In certain embodiments, the compound is described by formula (IIIv), (IIIv) or (IIIvi):

[0072]

[0073] wherein L is a common central linker (also referred to herein as a "linking moiety") as described in detail above. In certain embodiments, the compound is described by the general formula (IIvii), (IIviii) or (IIix):

[0074]

[0075] In certain embodiments, the compound is described by formula (IIa), (IIb), (IIc) or (IId):

[0076]

[0077] In certain embodiments, the compound is described by formula (IIe) or (IIf):

[0078]

[0079] In certain embodiments, compound (IIa) (OEG 2 -(IP5*) 2 ) and (IIb)(OEG 2 -(IP5) 2 ) has a molecular weight of 1204.04 g / mol. In one embodiment, the molecular weight of compounds (lIe) (PO1-1) and (IIe) is 1298.15 g / mol. In one embodiment, compound (IId) (OEG 8 -(IP5) 2 The molecular mass of ) and (IIf) is 1474.36 g / mol.

[0080] In certain embodiments, the compound is described by Formula (IIk), (IIm), or (IIn):

[0081]

[0082] In certain embodiments, the compounds of the invention are described by formula (IIg), (IIh) or (IIj):

[0083]

[0084] wherein k is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12.

[0085] In certain embodiments, the compounds of the present invention are described by the general formula (III),

[0086]

[0087] where each X is independent of any other X and can be an OPO 3 2- OPSO 2 2- or OSO 3 - , and wherein L is a common central linker (also referred to herein as a "linking moiety") as described in detail above.

[0088] In certain embodiments of the compounds described in (III), the linking moiety L is described by the general formula (IIIa):

[0089]

[0090] wherein o, p and q independently of one another have values ​​between 1 and 12 and the sum of o, p and q is <30.

[0091] In certain embodiments, A is selected from carbon atoms (C), hydroxy-, amino-, halogen- or carboxyl-substituted or unsubstituted C 4 To C 7 A is a cycloalkyl moiety, or a hydroxy-, amino-, halogen- or carboxyl-substituted or unsubstituted aryl group. In certain embodiments, A is a carbon atom.

[0092] In certain embodiments, R is selected from H and C 1 -C 3 unsubstituted or N-, O and / or halogen-substituted alkyl. In certain embodiments, is H or unsubstituted alkyl.

[0093] In certain specific embodiments of (III), L is described by (IIIa) and o, p and q independently have values ​​of 2 to 4.

[0094] In certain specific embodiments of (III), L is described by (IIIa) and the sum of o, p and q is ≤15.

[0095] In certain specific embodiments of (III), L is described by (IIIa), o, p and q independently have values ​​of 2 to 4 and the sum of o, p and q is ≤15.

[0096] In certain embodiments, R is selected from H and C 1 -C 3 Unsubstituted or N-, O- and / or halogen-substituted alkyl. In certain specific embodiments, R is H or unsubstituted alkyl.

[0097] In certain embodiments of compound (III), the linking moiety L is described by the general formula (IIIb),

[0098]

[0099] Therein, o, p and q independently of one another have values ​​between 1 and 12, and the sum of o, p and q is ≤30.

[0100] In certain embodiments, A is selected from carbon atoms (C), hydroxy-, amino-, halogen- or carboxyl-substituted or unsubstituted C 4 To C 7 A is a cycloalkyl moiety, or a hydroxy-, amino-, halogen- or carboxyl-substituted or unsubstituted aryl group. In certain embodiments, A is a carbon atom.

[0101] In certain embodiments, R is selected from H and C 1 -C 3unsubstituted or N-, O and / or halogen-substituted alkyl. In certain embodiments, is H or unsubstituted alkyl.

[0102] In certain specific embodiments of (III), L is described by (IIIb) and o, p and q independently have values ​​of 2 to 4.

[0103] In certain specific embodiments of (III), L is described by (IIIb) and the sum of o, p and q is ≤15.

[0104] In certain specific embodiments of (III), L is described by (IIIb), o, p and q independently have values ​​of 2 to 4 and the sum of o, p and q is ≤15.

[0105] In certain embodiments of (III), L is described by (IIIa) or (IIIb) and A is a carbon atom. In certain embodiments of (III), L is described by (IIIa) or (IIIb) and A is selected from substituted or unsubstituted C 1 To C 4 In certain embodiments of (III), L is described by (IIIa) or (IIIb) and A is selected from substituted or unsubstituted C 4 To C 7 In certain embodiments of (III), L is described by (IIIa) or (IIIb) and A is selected from substituted or unsubstituted five-membered or six-membered aryl.

[0106] In certain embodiments of compound (III), the linking moiety L is described by the general formula (IIIc),

[0107]

[0108] wherein A in formula (IIIb) is a carbon atom (C) and u, v and w independently of one another have values ​​of 1 to 12, in particular u, v and w independently of one another have values ​​of 2 to 4; the sum of u, v and w is ≤30, in particular the sum of u, v and w is ≤12.

[0109] In certain embodiments, R is selected from H and C 1 -C 3 Unsubstituted or N-, O- and / or halogen-substituted alkyl, in particular, wherein R is H or unsubstituted alkyl.

[0110] In certain embodiments, the molecular weight of the compound described by formula (III) is 1500 to 2500 g / mol.

[0111] In certain embodiments, the compound is described by formula (IIId) or (IIIe):

[0112]

[0113] In one embodiment, compound (IIId) ((OEG) 3 -(IP5) 3 ) has a molecular weight of 1922.21 g / mol. In one embodiment, compound (IIIe) ((OEG 4 ) 3 -(IP5) 3 ) has a molecular weight of 2318.69 g / mol.

[0114] In certain embodiments, the compound is described by the general formula (IV),

[0115]

[0116] where each X is independent of any other X and can be an OPO 3 2- OPSO 2 2- or OSO 3 - , and wherein L is a common central linker (also referred to herein as a "linking moiety") as described in detail above.

[0117] In certain embodiments of the compounds described by formula (IV), the linking moiety L is described by the formula,

[0118]

[0119] Therein, o, p, q and s independently of one another have values ​​from 1 to 12 and the sum of o, p, q and s is ≤40.

[0120] In certain embodiments, A is selected from carbon atoms (C), hydroxyl-, amino-, halogen- or carboxyl-substituted or unsubstituted C 4 To C 7 A is a cycloalkyl moiety, or a hydroxy-, amino-, halogen- or carboxyl-substituted or unsubstituted aryl group. In certain embodiments, A is a carbon atom.

[0121] In certain embodiments, o, p, q, and s independently have values ​​of 2 to 4. In certain specific embodiments, the sum of o, p, q, and s is ≤20.

[0122] In certain specific embodiments, o, p, q and s independently have values ​​of 2 to 4 and the sum of o, p, q and s is ≤20.

[0123] In certain embodiments of the compound described by (IV), L is (IVa) and A is selected from substituted or unsubstituted C1 To C 4 In certain embodiments, L is (IVa) and A is selected from substituted or unsubstituted C 4 To C 7 In certain embodiments, L is (IVa) and A is selected from substituted or unsubstituted five-membered or six-membered aryl.

[0124] In certain embodiments of the compounds described by formula (IV), the linking moiety L is described by the formula,

[0125]

[0126] wherein o, p, q and s independently of one another have values ​​1 to 12, in particular o, p, q and s independently of one another have values ​​2 to 4 and the sum of o, p, q and s is ≤40, in particular wherein the sum of o, p, q and s is ≤20.

[0127] In certain embodiments of the compounds described by formula (IV), the linking moiety L is described by the formula,

[0128]

[0129] wherein A of formula (IVb) is a carbon atom (C) and u, v, w and y independently of one another have values ​​of 1 to 12, in particular u, v, w and y independently of one another have values ​​of 2 to 4, and the sum of u, v, w and y is ≤40, in particular wherein the sum of u, v, w and y is ≤12.

[0130] In certain embodiments, the molecular weight of the compound described by formula (IV) is 2500 to 3000 g / mol.

[0131] In certain embodiments, the compound is described by formula (IVd) or (IVe),

[0132]

[0133] In certain embodiments, the compounds of the invention are provided as sodium salts. In certain embodiments, the compounds of the invention are provided as potassium salts. In certain embodiments, the compounds of the invention are provided as magnesium salts.

[0134] The present invention further encompasses intermediates useful in the synthesis of the compounds of the present invention, in particular compounds described by the general formula Va, Vb or Vc,

[0135]

[0136] wherein OPMB represents an O-(bis-p-methoxybenzyl) phosphate moiety, and wherein k is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12.

[0137] Another compound useful in the synthesis of the present invention is described by Formula VIa, VIb or VIc:

[0138]

[0139] wherein k is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12.

[0140] In one embodiment, compound (IVd) ((OEG) 4 -(IP5) 4 ) has a molecular weight of 2520.20 g / mol. In one embodiment, compound (IVe) ((OEG 4 ) 4 -(IP5) 4 ) has a molecular weight of 3048.84 g / mol.

[0141] Another aspect of the present invention relates to a compound according to the invention for use as a medicament.

[0142] A second aspect of the invention relates to the compounds of the invention for use in the treatment or prevention of conditions associated with pathological crystals, in particular pathological crystals of calcium salts and more in particular pathological crystals of calcium oxalate and / or calcium phosphate salts.

[0143] In the broadest sense, such use of the compounds according to the invention encompasses the use of any of the compounds described herein above according to the first aspect of the invention, but also encompasses a structurally slightly wider chemical space of compounds comprising, in particular consisting of, two or more cyclohexanol pentaester moieties described by general formula (I) linked by a common central linker L,

[0144]

[0145] Where X is selected from OPO 3 2- OPSO 2 2- 、OSO 3 - or CO 2 - , n is an integer from 2 to 10, wherein L is a linking portion comprising or consisting of a linear or branched alkyl group, the alkyl group being optionally substituted with oxygen, nitrogen, (fluorine), particularly wherein the molecular weight of the linking portion is <1000 g / mol, particularly <700 g / mol, more particularly <500 g / mol, or even <400 g / mol.

[0146] In certain embodiments, such compounds for treating or preventing conditions associated with pathological crystallization comprise 2, 3, 4, 6 or 8 moieties according to general formula (I). In certain specific embodiments, 2, 3 or 4 such moieties are contained in the molecule.

[0147] In certain embodiments, the compounds of the present invention for treating or preventing conditions associated with pathological crystallization are described by formula (VII),

[0148]

[0149] wherein k is an integer from 1 to 20, particularly from 1 to 10, more particularly an integer selected from 1, 2, 3, 4, 5 or 6.

[0150] The conditions associated with pathological calcium crystals caused by pathological calcium oxalate or calcium phosphate crystals in patients for which the compounds of the present invention are particularly useful include nephrocalcinosis, nephrolithiasis, calcinosis cutis, kidney stones, cardiovascular death (particularly in patients with chronic kidney disease), progression of chronic kidney disease and renal transplant failure. It has been shown that pathological crystals are associated with the morbidity of patients with chronic kidney disease, particularly where the condition is primary and secondary hyperoxaluria, Dent's disease, Bartter syndrome, distal renal tubular acidosis, neurogenic bladder, autosomal dominant polycystic kidney, calcium oxalate kidney stone formation, struvite and renal calcification. Therefore, the compounds of the present invention are generally used in patients with chronic kidney disease.

[0151] One embodiment of the present invention showing particularly high activity is to use a compound having a similar CaOx inhibitory effect ( Figure 2 ) of a novel bivalent IP5 compound (OEG 4 -(IP5) 2 , OEG 8 -(IP5) 2 , OEG 2 -(IP5) 2). The inventors have previously developed a series of small molecules based on inositol hexaphosphate (IP6) analogs (PCT / EP2012 / 004088, PCT / EP2016 / 080657), which can reduce the nucleation and growth of CaOx crystals in human urine and have improved pharmacokinetic (PK) properties. In addition, these compounds have been shown to prevent CaOx crystals from adhering to renal epithelial cells and show cytoprotective effects when treated with CaOx in vitro. Although these initial IP6 analogs failed to completely inhibit CaOx crystallization within the concentration range achievable in vivo, the inventors were surprised to find that the ability to inhibit CaOx crystallization was highly dependent on the combination of IP5 parts. Therefore, the inventors developed a new multivalent IP5 compound by connecting two inositol pentaphosphate (IP5) molecules using an oligo(ethylene glycol) linker (OEG), thereby increasing the overall negative charge of the molecule. Compared with the most effective compound previously developed, IP5-mono-PEG2 (OEG 2 -IP5), compared with the compound OEG of bis-IP5 linked by tri(ethylene glycol) 4 -(IP5) 2 The results showed that the efficacy of inhibiting CaOx crystallization to less than 50% of the control was improved by 2500 times ( Figure 1 ). This conclusion comes from the results of an in vitro assay measuring the crystallization tendency of CaOx in human urine.

[0152] The compounds may be formulated for intravenous, intraperitoneal, intramuscular, intraarterial, topical, intravesical or especially subcutaneous administration. Optionally, a pharmaceutically acceptable carrier and / or excipient may be present.

[0153] According to another aspect of the present invention, there is provided a method for treating or preventing any of the above-listed conditions associated with pathological calcium crystallization, comprising administering to a subject in need thereof any of the compounds detailed in the above formula.

[0154] It will be appreciated by those skilled in the art that any compound explicitly mentioned herein will be present in the form of a pharmaceutically acceptable salt of the compound. A pharmaceutically acceptable salt comprises an ionized compound and an oppositely charged counterion. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminum, benzathine, ethylenediamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine, and zinc.

[0155] Pharmaceutical compositions and administration

[0156] Another aspect of the present invention relates to a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.

[0157] In certain embodiments of the present invention, the compounds of the present invention are generally formulated into pharmaceutical dosage forms to provide an easily controlled dosage of the drug and to provide the patient with an elegant and easy to handle product.

[0158] The pharmaceutical composition can be formulated for oral administration, parenteral administration or rectal administration. In addition, the pharmaceutical composition of the present invention can be prepared in solid form (including but not limited to capsules, tablets, pills, granules, powders or suppositories) or liquid form (including but not limited to solutions, suspensions or emulsions). The dosage regimen of the compound of the present invention will vary depending on known factors, such as the pharmacodynamic properties of a particular agent and its mode and route of administration; the type, age, sex, health condition, medical condition and weight of the recipient; the nature and extent of symptoms; the type of concurrent treatment; the frequency of treatment; the route of administration, the patient's renal and liver function and the desired effect. In certain embodiments, the compound of the present invention can be administered in a single daily dose, or the total daily dose can be administered twice, three or four times a day.

[0159] In certain embodiments, for a subject of about 50-70kg, the pharmaceutical composition of the present invention or combination can be present in a unit dose of about 1-1000mg active ingredient. The therapeutically effective amount of a compound, pharmaceutical composition or combination thereof depends on the type, body weight, age and personal circumstances of the subject, the disorder or disease being treated or its severity. A physician, clinician or veterinarian with ordinary skills can easily determine the effective amount of each active ingredient necessary for preventing, treating or inhibiting a disorder or disease progression.

[0160] The pharmaceutical composition of the present invention can be subjected to conventional pharmaceutical operations (e.g., sterilization) and / or can contain conventional inert diluents, lubricants or buffers and adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc. They can be produced by standard processes, such as by conventional mixing, granulation, dissolution or lyophilization processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see, for example, L.Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th edition, 2013 (ISBN 8123922892).

[0161] Wherever alternatives of single separable features are arranged herein as "embodiments", it should be understood that such alternatives may be freely combined to form discrete embodiments of the invention disclosed herein.

[0162] The present invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be derived. These examples are intended to illustrate the present invention, but not to limit its scope. BRIEF DESCRIPTION OF THE DRAWINGS

[0163] Figure 1 IIc(OEG) at pH 6.2 and pH 5.5 4 -(IP5) 2 ) and compared with IP5-mono-PEG 2 (OEG 2 -IP5) inhibition of CaOx crystallization. CaOx crystallization was induced by adding 1 mM sodium oxalate to human urine and characterized by optical microscopy followed by automated image analysis to determine crystal number, hydrate form (COM - CaOx monohydrate, COD - CaOx dihydrate, nd - not defined) and crystal area. The surface areas of the different hydrate forms normalized to the total surface area of ​​the control were plotted. IIc / OEG 4 -(IP5) 2 Only at 300 nM did it lead to complete inhibition of COM crystals, followed by complete CaOx (COM and COD) inhibition at 4 μM. In contrast, for the previously dominant compound IP5-mono-PEG 2 , COM inhibition was only observed at 11 μM, while complete CaOx (COM and COD) inhibition was not observed in the tested range up to 100 μM.

[0164] Figure 2 The inhibition of CaOx crystallization by the compounds of the present invention at pH 6.2 is shown: IIb (OEG 2 -(IP5) 2 )、IIc(OEG 4 -(IP5) 2 ) and IId(OEG 8 -(IP5) 2 ).

[0165] Figure 3 The inhibition of CaOx crystallization by IP6 analogs previously described in PCT / EP2016 / 080657 at pH 6.2 is shown. CaOx crystallization is induced by adding 1 mM sodium oxalate to human urine and characterized by optical microscopy, followed by automatic image analysis to determine crystal number, hydrate form (COM-CaOx monohydrate, COD-CaOx dihydrate, nd-undefined) and crystal area. The surface areas of different hydrate forms normalized to the total surface area of ​​the control are plotted.

[0166] Figure 4 The synthesis of the compound of the invention described in Example 1 is shown.

[0167] Figure 5 The compound OEG of the present invention is shown 4 -(IP5)2 Image of suppressed CaOx crystal formation (scale bar: 50 μm).

[0168] Figure 6 The IP6 analog compound OEG previously described in PCT / EP2016 / 080657 is shown 2 - Image of IP5 inhibiting CaOx crystal formation (scale bar: 50 μm).

[0169] Figure 7 The compound (OEG 4 ) 3 -(IP5) 3 synthesis.

[0170] Figure 8 The changes in the crystallization pattern of CaOx induced by IP6 analogs are shown. The (OEG 4 ) 3 -(IP5) 3 Effect on bulk crystallization of CaOx in human urine spiked with 1 mM NaOx. COM - CaOx monohydrate, COD - CaOx dihydrate, nd - not defined; N = 3, normalized to the mean total area per field of view of each crystal type + SD of the control (no inhibitor).

[0171] Fig. 9 Display OEG 4 -(IP5) 2 Stabilizes early precursor particles and leads to delayed COD growth. (A) Characterization of OEG in Bis-Tris buffer (pH 6.2) by SEM at t=7h 4 -(IP5) 2 Dose-dependent inhibition of CaOx crystallization. Representative SEM images are shown (scale bar: 1 μm). (B) The samples were sampled at t=10 min with 500 nM OEG. 4 -(IP5) 2 Crystallization of CaOx with and without inhibitor and imaged by SEM (scale bar: 1 μm). Crystallinity was characterized by TEM with SAED (inlet).

[0172] Fig.10 Inhibition of CaOx adhesion to renal epithelial cells by IP6 analogs is shown. (A) OEG in vitro assessed by differential interference contrast microscopy 4 -(IP5) 2 、(B)OEG 2 -IP5 and (C) (OEG 2 ) 2- The anti-adhesion effect of IP4 on CaOx attachment to RPTEC / TERT1 cells was then quantified for crystal occupancy area. 150 μg / cm 2 Confluent cells were treated with CaOx premixed with compounds for 30 min, then washed, fixed and imaged. (D) Premixed with / without OEG adhered to confluent cell layer 4 -(IP5) 2 Example image of CaOx (Leica CTR6000, 63x oil objective). (E) To evaluate OEG 4 -(IP5) 2 To remove the bound CaOx from the cell layer, a modified version of the above assay was performed. RPTEC / TERT1 cells were incubated with CaOx for 30 min, washed with PBS to remove unbound CaOx, and assayed with OEG. 4 -(IP5) 2 The cells were further incubated for 2 h with or medium control (N=3, normalized to mean + SD of CaOx control, one-way ANOVA with Dunnett's multiple comparisons, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 compared with CaOx control).

[0173] Fig.11 OEG 4 -(IP5) 2 Prevented CaOx-induced cellular damage in renal epithelial cells. (A) Heat map and hierarchical clustering of relative gene expression levels determined by RNA sequencing (CaOx vs. CaOx-OEG 4 -(IP5) 2 Differentially expressed genes between 2 Ratio = 0.5, number of significant features 2000). (B) Gene set enrichment analysis of gene expression levels in CaOx and CaOx-OEG relative to the medium group 4 -(IP5) 2 The corresponding values ​​in the group. The top gene ontology terms and corresponding normalized enrichment scores of genes that were differentially up-regulated or down-regulated in the CaOx group relative to the medium control group are plotted (the top 10 up-regulated gene ontology terms with FDR≤0.05 and the down-regulated gene ontology terms with FDR≤0.05 are shown). The CaOx-OEG 4 -(IP5) 2Corresponding values ​​of differentially expressed genes in the groups (FDR≤0.05 is indicated in dark blue, FDR>0.05 is indicated in light blue). (C) Gene expression levels of genes involved in inflammation and immune response, cell signaling, and ECM production in different treatment groups. (D) Viability staining was used to assess the survival of RPTEC cells before treatment with OEG compared to cells treated with CaOx alone. 4 -(IP5) 2 Pre-incubation with CaOx resulted in a dose-dependent reduction in the number of dead cells (normalized to mean + SD of CaOx control, one-way ANOVA with Dunnett's multiple comparisons, ****p < 0.0001, **p < 0.01). (E) Example bright field images showing CaOx deposition (black dots) and cells that received no treatment, CaOx alone, or CaOx + 10 μM OEG. 4 -(IP5) 2 Red fluorescent images of cell death of treated cells (Leica CTR6000, 10x objective, scale bar: 100 μm). (N=3 for all experiments).

[0174] Table 1 shows the data of screening calcification inhibitors for their activity in inhibiting CaOx crystal growth. IP6 analogs previously described in PCT / EP2016 / 080657 and compounds of the present invention OEG are listed. 2 -(IP5) 2 , OEG 4 -(IP5) 2 , OEG 8 -(IP5) 2 and (OEG 4 ) 3 -(IP 5 ) 3 . DETAILED DESCRIPTION

[0175] Example 1: Synthesis of IIc(OEG 4 -(IP5) 2 )

[0176] Step 1:

[0177] Reagents: DMF, sodium hydride, p-methoxybenzyl chloride.

[0178] Under nitrogen atmosphere, inositol-1,3,5-orthoformate was dissolved in dry DMF, cooled to 0-5°C and sodium hydride (2.2 equivalents) was added in batches. After stirring at room temperature for 1 hour, p-methoxybenzyl chloride (2.3 equivalents) was added dropwise. The mixture was stirred at room temperature for 12 hours, then water was added and extracted with ethyl acetate. The solvent was removed in vacuo and the residue was crystallized from ether. The yield was 70%.

[0179] Step 2:

[0180] Reagents: MeOH; HCl 2N.

[0181] The product (1) of step 1 was dissolved in 10 volumes of methanol, 1 volume of HCl 2N was added and heated at 50°C for 3 hours. After cooling, the mixture was neutralized with 30% ammonium hydroxide and left overnight at room temperature. The solid formed was filtered, washed with water and dried. After crystallization with diethyl ether, compound 2 was obtained as a white solid. The yield was 80%.

[0182] Step 3:

[0183] Reagents: DMF, sodium hydride, p-methoxybenzyl chloride.

[0184] Under nitrogen atmosphere, the product (2) of step 2 was dissolved in anhydrous DMF, cooled to 0-5°C and sodium hydride (2 equivalents) was added in portions. After 1 hour at room temperature, the mixture was cooled at 0-5°C and p-methoxybenzyl chloride (2 equivalents) was added. After 12 hours at room temperature, water was added to the mixture and extracted with ethyl acetate. The residue was purified by column chromatography eluting with hexane / ethyl acetate 6 / 4 and then crystallized with ether. The yield was 35%.

[0185] Step 4:

[0186] Reagents: dichloromethane, tosyl chloride, triethylamine.

[0187] Dissolve tetraethylene glycol in anhydrous dichloromethane, add triethylamine (3 equivalents), then add p-toluenesulfonyl chloride (2.5 equivalents). After stirring at room temperature for 12 hours, remove the solvent in vacuo and purify the residue by column chromatography eluting with ethyl acetate / hexane 7 / 3. Yield 80%.

[0188] Step 5:

[0189] Reagents: DMF, sodium hydride, tetraethylene glycol ditosylate (step 4).

[0190] Under nitrogen atmosphere, the product (3) of step 3 was dissolved in dry DMF, cooled to 0-5°C, and sodium hydride (1.1 equivalents) was added in batches. After stirring at room temperature for 1 hour, the mixture was cooled to 0-5°C again, and a DMF (3 volumes) solution of compound 4 (0.5 equivalents) was added. After stirring at room temperature for 12 hours, water was added to the mixture and extracted with ethyl acetate. After removing the solvent of the mixture, it was purified by column chromatography using hexane / ethyl acetate elution. Yield 40%.

[0191] Step 6:

[0192] Reagents: dichloromethane, trifluoroacetic acid. Not purified.

[0193] The product of step 5 (5) was dissolved in dichloromethane (10 volumes) and trifluoroacetic acid (2 volumes) was added. After stirring at room temperature for 3 hours, the solvent was removed in vacuo and the residue was used in the next step without further purification. The yield was quantitative.

[0194] Step 7:

[0195] Reagents: DMF, 5-methyltetrazolyl, dibenzyloxy-diisopropylphosphoramide, then H 2 O 2 .

[0196] Under nitrogen atmosphere, the product of step 6 (6) was dissolved in anhydrous DMF (10 volumes), 5-methyltetrazolyl (9 equivalents) was added and cooled to -10°C, then dibenzyloxy-diisopropylphosphoramide (6 equivalents) was added dropwise. After stirring at room temperature for 12 hours, the mixture was cooled to 0-5°C, and 30% H 2 O 2 (9 equivalents). After stirring at room temperature for 1 hour, water was added and the mixture was extracted with ethyl acetate. After removal of the solvent, the residue was purified by column chromatography eluting with chloroform / methanol 96 / 4. Yield 40%.

[0197] Step 8:

[0198] Reagents: H2, Pd / C. Forms magnesium salt with magnesium ethoxide.

[0199] The product (7) of step 7 was dissolved in methanol (15 volumes), Pd / C 10% was added, and hydrogenated at room temperature and pressure for 24 hours. After filtering the catalyst, the solvent was removed under reduced pressure to obtain compound 8 as an acid. The yield was quantitative. A magnesium salt was formed by adding an aqueous solution of magnesium ethoxide to the aqueous solution of compound 8 and then distilling the solvent under vacuum.

[0200] Example 2: Synthesis (OEG 4 ) 4 -(IP5) 4

[0201] As synthetic OEG 4 -(IP5) 2 (Example 1; Figure 4 ) described in the preparation of compound 5 (PMB = p-methoxybenzyl; Figure 7 ).

[0202] Reagents for step A ( Figure 7 ): p-toluenesulfonyl chloride; dichloromethane, triethylamine. Column chromatography. The yield is 80%.

[0203] Reagents for step B ( Figure 7 ): trimethylolpropane, DMF, sodium hydride. Column chromatography. Yield: 45%.

[0204] Reagents for step C ( Figure 7 ): methanol, H 2 , Pd / C. The yield is 80%.

[0205] Reagents for step D ( Figure 7 ): p-toluenesulfonyl chloride; dichloromethane, triethylamine. Column chromatography. The yield is 70%.

[0206] Reagents of step E ( Figure 7 ): Figure 7 The compound 5 shown, DMF, sodium hydride, column chromatography, yield 20%.

[0207] Reagents of Step F ( Figure 7 ): dichloromethane, trifluoroacetic acid. Yield quantitative.

[0208] Reagents of step G ( Figure 7 ): DMF, dibenzyl-N,N-diisopropylphosphoramidite, 5-methyltetrazole. Column chromatography. The yield is 30%.

[0209] Reagents for Step H ( Figure 7 ): methanol, H2, Pd / C. The yield is 90%. Then the sodium salt is formed, quantitative.

[0210] Example 3: Inhibition of CaOx crystallization

[0211] To compare the efficacy of compounds in inhibiting calcium oxalate (CaOx) crystallization, a simple in vitro urine assay was used. Briefly, human urine (pH 6.2) was mixed with the compounds, and then 1 mM sodium oxalate was added to induce CaOx crystallization. After 7 hours of incubation at room temperature, crystallization was measured by optical microscopy, and automated image analysis was used to detect, classify, and quantify the types of CaOx crystals formed, namely CaOx monohydrate (COM) and CaOx dihydrate (COD).

[0212] The inventors found that all three novel bivalent IP5 structures showed similar in vitro CaOx inhibition power, and completely inhibited CaOx crystallization at 4 μmol / L ( Figure 1 The length of the linker had no significant effect on the inhibitory effect. 2 -IP5)(PCT / EP2012 / 004088&PCT / EP2016 / 080657) compared to OEG 4 -(IP5) 2 The efficacy of inhibiting CaOx crystallization to less than 50% of the control was increased by 2500 times ( Figure 1 ).

[0213] The inventors observed that, first, sub-inhibitory concentrations of the compound resulted in a shift in crystallization from COM to COD, and further increases in the compound concentration completely inhibited the growth of CaOx with unexpected efficiency. All tested divalent IP5 structures showed complete CaOx inhibition in in vitro assays at low micromolar concentrations, which is expected to be achieved in vivo ( Figure 2 , Table 1).

[0214] The inventors demonstrated that the efficacy of inhibiting CaOx crystallization depends on the number of phosphate groups on the molecule and that all OEGs at 4 μmol / L X -(IP5) 2 Complete inhibition of crystallization was observed for all compounds tested (Table 1). In addition, IP6 and other compounds tested at up to 30 μM or 100 μM, respectively, did not result in complete CaOx inhibition ( Figure 3 , Table 1). It was observed that the length of the OEG chain or the length of the linker had the least effect on the total CaOx crystallization, and mainly affected the COM-COD formation kinetics. In addition, lowering the pH to 5.5 had a greater effect than that of the OEG 4 -(IP5) 2 Significantly reduce OEG 2 -IP5 activity. 100 μM compound OEG 2 -IP5 did not result in <50% complete inhibition or COM inhibition. In addition, compound OEG 4 -(IP5) 2 COM inhibition was retained at 0.3 μM and 50% of full inhibition was retained at 2.4 μM ( Figure 1 , Table 1). Compound (OEG4)3-(IP5)3 exhibited Com inhibition at 0.04 μM and complete inhibition at 2.4 μM ( Figure 8 , Table 1).

[0215] Table 1

[0216]

[0217]

[0218] Example 4: OEG 4 -(IP5) 2 Suppression of CaOx crystallization by early precursor stabilization

[0219] Scanning electron microscopy (SEM) experiments of early crystallization at t = 10 minutes showed the presence of both COM-shaped particles and round nanoparticles, while the use of 500 nM OEG 4 -(IP5) 2 When , only nanoparticles ( Fig. 9B). At t = 1 h, nanoparticles were still present when 0.5-10 μM inhibitor was used, whereas micron-sized COM crystals dominated in the control sample, indicating that the OEG 4 -(IP5) 2 It can stabilize early nano-scale CaOx particles.

[0220] SEM experiments further confirmed the dose-dependent transition from predominantly COM to COD crystals, and OEG 4 -(IP5) 2 Inhibition of COD surface-specific growth ( Fig. 9 A) Together, these results suggest that OEG 4 -(IP5) 2 The crystallization process was altered at multiple stages, namely, crystal nucleation and growth kinetics, as well as crystal polymorphism and shape.

[0221] Example 5: with OEG 2 -IP5 compared to OEG 4 -(IP5) 2 More effectively blocked the interaction of CaOx and renal epithelial cells in vitro Adhesion

[0222] The anti-adhesion effect of selected IP6 analogs on CaOx crystals to renal proximal tubular epithelial cells (RPTEC) was studied in vitro by differential interference contrast microscopy. 4 -(IP5) 2 , OEG 2 -IP5 and (OEG 2 ) 2 Comparison of -IP4 showed that concentrations of 200 nM, 4 μM, and above 100 μM resulted in <25% CaOx adhesion ( Fig.10 AD). Citrate treatment up to 100 μM did not have any detectable protective effect on CaOx adhesion. 4 -(IP5) 2 Not only did it inhibit CaOx adhesion, but it also reversed the binding of pre-bound CaOx crystals at low micromolar concentrations ( Fig.10 E).

[0223] Example 6: OEG 4 -(IP5) 2 Cytoprotective effect against CaOx-induced cell damage

[0224] RNA sequencing of RPTEC in culture medium control, OEG 4 -(IP5) 2 Control and premixed with OEG 4 -(IP5) 2 The CaOx-treated cells showed similar gene expression profiles, whereas CaOx treatment alone caused dramatic changes ( Fig.11A). Gene set enrichment analysis of differentially expressed genes in CaOx relative to culture samples showed alterations in genes primarily involved in immune and inflammatory responses (which is consistent with the literature), as well as structural changes (e.g. microtubule organization) and protein modification and signaling roles (e.g. peptidylglutamate modifications, ER nuclear signaling pathways) ( Fig.11 B) The top 10 enriched gene sets of differentially expressed genes in CaOx relative to medium samples were compared with those in CaOx-OEG samples. 4 -(IP5) 2 The top 10 enriched gene sets of differentially expressed genes in samples and CaOx were compared, which showed similar changes, indicating that OEG 4 -(IP5) 2 Premixing with CaOx prevents CaOx-induced transcriptome changes ( Fig.11 B). Increased expression of TNFα and TGFβ signaling pathway genes, cell surface glycoproteins, CXC motif chemokine ligands, and complement cascade genes were detected in the CaOx group compared with all other treatment groups ( Fig.11 C) In addition, CaOx crystal treatment triggered plasma membrane damage, OEG, as demonstrated by ethylenediamine homodimer-1 staining. 4 -(IP5) 2 This damage was prevented in a dose-dependent manner ( Fig.11 D, E).

[0225] Materials and methods

[0226] Material

[0227] Compounds were custom synthesized by Chimete Sri (Tortona, Italy). Phytic acid dodecasodium salt was purchased from Biosynth AG (Thai, Switzerland). Pooled human urine was purchased from Lee Biosolutions (Maryland Heights, Missouri). Oxalate assay kit (MAK315), calcium colorimetric assay kit (MAK022), Bis-Tris, sodium oxalate, neutral buffered 10% formalin solution, and Mowiol 4-88 were purchased from Sigma-Aldrich (St. Louis, Missouri). Sodium chloride and calcium chloride dihydrate were obtained from Merck (Kenilworth, New Jersey). Calcium oxalate monohydrate was purchased from abcr (Karlsruhe, Germany). 8-well glass bottom slides (80827) were purchased from ibidi (Martinsried, Germany). Analytical grade trisodium citrate dihydrate, Nunc Lab-Tek II chamber slides, cell culture plates, live / dead viability / cytotoxicity kits for mammalian cells, and phosphate-buffered saline (PBS) were purchased from Thermo Fisher Scientific (Rochester, NY). RPTEC / TERT1 cells, ProxUp basal medium and supplements were obtained from Evercyte (Vienna, Austria). RNeasy kits were purchased from Quiagen (Hilden, Germany) and TrueSeq RNA kits were obtained from Illumina (San Diego, California).

[0228] CaOx screening assay and analysis

[0229] Human urine was stored at -20 °C in 50 mL aliquots. After thawing, the aliquots were centrifuged, filtered using a 0.45-μm syringe filter, and the pH was set to 6.2. According to the kit protocol, the oxalate concentration in human urine samples was determined using an oxalate assay kit, and the oxalate concentration of the samples used was found to be <100 μM. Sodium and compound dilutions were prepared in Bis Tris buffer (50 mM, 150 mM NaCl, pH 6.2). Urine was mixed with the compound in an Eppendorf tube, and sodium oxalate was then added. The final assay mixture with a total volume of 1 mL contained 90% urine, 5% sodium oxalate (1 mM) and 5% compound dilution. After sodium oxalate was added, the final assay mixture was vortexed and added to an imaging slide (8-well glass bottom chamber, ibidi) immediately. 400 μL was added to each well and two wells were prepared for each sample. After incubation for 7 hours at room temperature, crystallization was evaluated in bright field mode using a Leica CTR6000 microscope (Leica Microsystems, Wetzlar, Germany). For visualization, images of a 40x objective lens were used. For quantification, 2 wells / sample were used, each using 5 20x objective lens images. In the absence of sodium oxalate, no crystallization was observed.

[0230] Inhibition of seeded crystals was evaluated in a similar manner. Crystallization was induced with 1 mM sodium oxalate in urine as described above, followed by addition of compounds directly to each well after 1.5 h incubation at room temperature. Samples were imaged at t = 1.5 h prior to compound addition and then at a total incubation time of t = 7 h.

[0231] Quantification and classification of crystals were performed in Matlab (version R2016b or R2018b). In brief, crystals were segmented using edge detection and watershed algorithms, and then shape, intensity, and texture features were extracted for each crystal. The extracted features were used as input to a semi-supervised classification method to distinguish COM (class 0), COD (class 1), undetermined structures (class 2), and background noise (class 3). A cubic support vector machine (SVM) classifier was trained and subsequently used for test image classification.

[0232] All test images were processed using a batch processing application. The efficacy of the compounds in inhibiting CaOx crystallization was compared by analyzing the total area of ​​each field of view for each crystal type. In each experiment, the sum of the total area of ​​the three crystal categories (COM, COD, and unclassified) was normalized to the control (N=3). Due to the inherent variability of the crystallization process itself, normalization in each experiment reduced the impact of inter-experimental variability.

[0233] Scanning electron microscopy

[0234] In the presence of 1 mM NaOx, 2 mM CaCl 2 CaOx crystals for SEM were prepared in Bis Tris buffer (50 mM, 150 mM NaCl, pH 6.2) with the indicated concentrations of compounds. A 12 mm round glass coverslip was placed at the bottom of a 24-well plate. First, a final concentration of 20x CaCl was prepared in Bis Tris buffer. 2 The inhibitors and NaOx were prepared by first adding 800 μL Bis Tris buffer followed by 50 μL CaCl 2 (20x concentration) and 50 μL inhibitor (20x concentration) and vortex mix, prepare the assay mixture in an Eppendorf tube. Then add 100 μL NaOx (10x final concentration), vortex the assay mixture and add it immediately to the prepared 24-well plate. Prepare 400 μL per well and 2 wells per sample. The samples are incubated at room temperature for the specified time. Use a Leica CTR6000 microscope (Leica Microsystems) to take sample images in bright field mode, and then use double distilled water (ddH 2 The samples were washed once with 4% paraffin (0) and dried at room temperature. After drying, the samples were imaged by bright field microscopy again to confirm that the drying process had little effect on the crystal morphology. After drying, the glass cover slide was mounted on a SEM stub with silver paint and a 6nm platinum / palladium layer was coated using a CCU-010 metal sputtering coater (Safematic, Bad Ragaz, Switzerland). Magellan 400FEI SEM microscope (ThermoFisher Scientific, Rochester, NY) was used to image the samples using a TLD detector in secondary electron mode.

[0235] Cell experiments

[0236] According to the manufacturer's recommendations, the cells were maintained at 37°C and 5% CO 2 RPTEC / TERT1 human proximal tubule cells (Evercyte) were cultured in ProxUp basal medium (Evercyte, DMEM / Ham's F12, Hepes buffer and GlutaMAXTM) mixed with ProxUp supplement (Evercyte) at 4 °C. Cells were regularly tested for mycoplasma infection.

[0237] Cell viability was assessed using a Live / Dead Viability / Cytotoxicity Kit (ThermoFisher Scientific). Cells were cultured in 24-well plates and 48 h after seeding (after reaching confluence) were treated with 150 μg / cm2 of ProxUp basal medium premixed with the compound. 2 Calcium oxalate monohydrate (abcr) treatment. After 48 hours of treatment, cell viability was determined by staining with a live / dead cytotoxicity kit (ThermoFisher Scientific) according to the kit protocol. Images were obtained by epifluorescence microscopy using a Leica CTR6000 microscope (Leica Microsystems, 3 holes / sample, 3 images / hole), and the number of red fluorescent spots was quantified.

[0238] To quantify CaOx adhesion to RPTEC cells, cells were cultured on 8-well Nunc Lab Tek chamber slides (ThermoFisherScientific) and after 48 h of confluence, incubated at 37°C, 5% CO 2 The cells were treated with COM (abcr) premixed with compounds in ProxUp basal medium (pH set to 6.9) for 30 minutes. The cells were washed twice with phosphate buffered saline (PBS, pH 7.4), fixed with 10% neutral buffered formalin for 15 minutes, rinsed with PBS, and then fixed with ddH 2 O for final rinse. The chamber of the slide was removed and the cells were mounted with Mowiol. Images were acquired using a Leica CTR6000 microscope (Leica Microsystems) in DIC mode, 63x oil objective (3 wells / sample; 8 images / well), and the crystal occupancy area was quantified.

[0239] RNA sequencing

[0240] Cells were cultured as described in the cell viability assay. The four treatment groups consisted of medium control (ProxUp basal medium), 150 μg / cm 2 Calcium oxalate monohydrate (abcr), 150 μg / cm 2 Premixed with 10 μM OEG 4 -(IP5) 2 Calcium oxalate monohydrate (abcr) and only 10 μM OEG 4 -(IP5) 2composition. Total RNA was extracted using the RNeasy kit (Quiagen) according to the manufacturer's protocol. mRNA was purified and RNASeq libraries were prepared using the TruSeq RNA kit. Sequencing was performed on a Novasek 6000 (Illumina). Reads were aligned to the human reference genome GRCh38.p10 using the STAR tool, and transcripts were quantified using the Kallisto program. Ensembl version 91 was used for gene model definition. For heatmaps and hierarchical clustering of significantly different genes, the log2 fold change compared to the mean of all samples was calculated, and log2 fold changes >4 were set to 4. Data were analyzed on Webgestalt.org (v2019) 41 Gene set enrichment analysis was performed on . The input to GSEA is a gene list with an overall gene id and a ranking score (-log10*p-value*sign(log2 ratio)) as a measure of differential expression between two treatment groups. The differentially expressed genes were compared to the Gene Ontology (Biological Process Function Database) and the Human Genome-Protein Code was used as a reference set. To compare the expression levels of the selected genes, normalized gene counts were used.

[0241] Data analysis

[0242] Unless otherwise stated, GraphPad Prism (La Jolla, California) was used to prepare statistical analyses and graphs. Data are expressed as mean + SD. Conventional one-way ANOVA tests were used, followed by post hoc Dunnett's multiple comparisons to compare treatment groups with controls. Unless otherwise stated, image analysis was performed using Matlab versions R2018b and R2016b (MathWorks, Natick, Massachusetts).

Claims

1. A compound comprising two or three cyclohexanol pentaester moieties described by general formula (I) connected by a common central linker L, (I) Where each X is , L is a common central linker connecting n individual moieties represented by the formula in square brackets and n is 2 or 3, wherein the common central linker L has a molecular weight of <1000 g / mol, and wherein L consists of a linear or branched poly(ethylene glycol).

2. The compound according to claim 1, wherein the compound consists of two or three cyclohexanol pentaester moieties described by general formula (I) connected by a common central linker L.

3. The compound according to claim 1, wherein the molecular weight of L is <700 g / mol.

4. The compound according to claim 1, wherein the molecular weight of L is <500 g / mol.

5. The compound according to claim 1, wherein the molecular weight of L is <400 g / mol. The compound according to claim 1 , wherein n is 2.

7. The compound according to claim 1, wherein n is 3.

8. The compound of claim 1, wherein L is a linear poly(ethylene glycol).

9. The compound of claim 1, wherein L is branched poly(ethylene glycol).

10. The compound according to any one of claims 1 to 9, wherein any of the cyclohexanol pentaester moieties described by general formula (I) are independently selected from moieties described by general formula (Ia) or (Ib), (It) (Ib) wherein L is a common central linker and X has the meaning defined in claim 1 .

11. The compound according to any one of claims 1 to 9, wherein the compound is characterized by general formula (II): (II) Where L is , where m is a value from 1 to 12. The compound according to claim 11 , wherein the value of m is 2 to 8.

13. The compound according to claim 11, wherein the compound is characterized by the general formula (IIi), (IIIi), or (IIIi): (III) (III) (IIiii)。 14. The compound according to any one of claims 1 to 9, wherein the compound is characterized by formula (IIg), (IIh), or (IIj), (IIg) (IIh) (II) wherein k is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12.

15. The compound of claim 14, wherein the compound is characterized by formula (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (Ilk), (IIm), or (IIn), (IIa) (IIe) (If) (IIb) (IIc) (IId) (IIk) (IIm) (IIn)。 16. A compound according to any one of claims 1 to 9, wherein the compound is characterized by general formula (III), (III), wherein each X has the meaning indicated above, and The connecting part L is (IIIa) And among them - the values ​​of o, p and q are independently 1 to 12 and the sum of o, p and q is ≤ 30, -A is a carbon atom (C), and -R is selected from H and Unsubstituted or N-, O- and / or halogen-substituted alkyl.

17. The compound according to claim 16, wherein the values ​​of o, p and q are independently 2 to 4 and the sum of o, p and q is ≤12.

18. The compound of claim 16, wherein R is H or unsubstituted alkyl.

19. The compound according to claim 16, wherein the compound is characterized by formula (IIId) or (IIIe): (IIId) (3rd) 20. Use of a compound according to any one of claims 1 to 19 in the preparation of a medicament, wherein the condition associated with pathological crystals is nephrocalcinosis or nephrolithiasis caused by pathological calcium oxalate crystals in a patient.

21. The use according to claim 20, wherein the compound is formulated for intravenous, intraperitoneal, intramuscular, intraarterial, topical, intravesical or subcutaneous administration.

22. The use according to claim 20, wherein the compound is formulated for subcutaneous administration.

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