Cyclic oxaphosphorinane compounds and analogs thereof for the treatment of fibrotic diseases

Abstract

The present invention relates to the use of heterocyclic phosphine compounds or compositions comprising the same for the treatment of fibrotic diseases. In particular, the present invention relates to a compound of general formula (1) as claimed herein or a composition comprising the same for the treatment of fibrotic diseases.

Description

Technical Field

[0001] This invention relates to the use of heterocyclic phosphonic compounds or compositions containing them for the treatment of fibrotic diseases. Background Technology

[0002] Under normal circumstances, any organ or tissue in an organism responds to injury through a repair process aimed at restoring the functional integrity of the damaged tissue. The response involved in tissue repair is self-limiting and ceases when wound healing is complete. In pathological conditions, the response involved in tissue repair escapes regulatory mechanisms and evolves into an uncontrolled wound healing response, leading to fibrosis. This results in the long-term, gradual accumulation of excessive fibrous material, thereby altering and disrupting normal organ structure and function.

[0003] The deposition and accumulation of excessive extracellular matrix (ECM) components, such as collagen and fibronectin, leads to tissue hardening and scarring, causing abnormal organ remodeling and may ultimately result in organ failure in fibrotic diseases.

[0004] Injury to solid organs typically involves endothelial damage, platelet aggregation and activation, triggering an inflammatory response, with neutrophils, macrophages, eosinophils, and lymphocytes infiltrating the wound site. Infiltrating inflammatory cells and affected epithelial cells secrete various growth factors and cytokines to further amplify the inflammatory response. Molecules such as TGF-β, PDGF, and IL-13 activate macrophages and induce the recruitment, proliferation, and activation of fibroblasts at the wound site. Activated fibroblasts, or myofibroblasts, express smooth muscle actin and secrete collagen and other ECM components that stabilize the cytostromal matrix. This allows epithelial and endothelial cells to proliferate and migrate on a temporary matrix to regenerate damaged tissue. Once completed, the inflammatory process ceases, and fibroblasts undergo apoptosis, leading to the resolution of the wound response.

[0005] In fibrotic remodeling, persistent tissue damage or injury, or dysregulation of repair mechanisms, can lead to inappropriate trauma responses. Excessive deposition and cross-linking of collagen and ECM result in excessive ECM accumulation beyond normal requirements, which is associated with persistent myofibroblast activation, epithelial cell damage, and loss of normal tissue structure and function.

[0006] Abnormal wound healing (fibrosis) can involve any organ or tissue, such as the kidneys, lungs, intestines, skin, aorta, or liver, leading to a variety of fibrotic diseases. The causes of fibrotic diseases can vary depending on the organ or tissue involved and remain unknown in many cases. Liver fibrosis and eventually cirrhosis are caused by chronic liver damage resulting from exposure to various factors, including environmental and dietary factors or infectious agents. Continued excessive alcohol consumption or a high-fat / high-sugar diet can also lead to cirrhosis. Similarly, diabetes, hypertension, exposure to toxic substances, and various types of autoimmune diseases can damage the kidneys, leading to fibrotic remodeling and loss of kidney function. Many types of inflammatory bowel diseases, such as Crohn's disease or celiac disease, can cause fibrotic remodeling, resulting in narrowing and / or malabsorption.

[0007] Treatment for progressive fibrosis should aim to cure the underlying disease. For example, this might involve better blood pressure control to avoid hypertension, improved glycemic control in diabetes, or avoiding exposure to harmful allergens or toxins. However, some fibrotic diseases do not respond adequately to treatment of the underlying damage and may progress to organ failure regardless of the condition of the initial lesion that triggered the traumatic process. This is the case with chronic kidney disease, where progression to advanced kidney disease or end-stage renal failure may be observed even after treatment of immune damage, correction of hypertension, or improvement of diabetes (to name some of the underlying causes of kidney fibrosis).

[0008] Significant progress has been made in understanding the mechanisms leading to various fibrotic diseases. Many different cell types are involved in wound healing, but also in fibrosis and the self-sustaining nature of fibrotic diseases. Our understanding of the roles of different cellular products influencing fibrosis has also increased; many cellular products have pro-fibrotic effects, while others have protective anti-fibrotic effects. In the former case, CD4+Th2 cell responses producing IL-4, IL-5, IL-13, and IL-21 lead to increased fibrosis, while CD4+Th1 cells producing interferon-γ and IL-12 chemokines have protective anti-fibrotic effects. The transforming growth factor β (TGF-β) pathway has been shown to be involved in almost all types of fibrosis, although IL-4 has been shown to be more effective than TGF-β in inducing fibrotic responses. Other molecules or genes reported to be involved include procollagen I, III, and VI, arginase 1, lysyl oxidase, matrix metalloproteinase 2 (MMP-2), MMP-9, and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), as well as serooxygenase, procollagen III, secretory phosphoprotein 1, procollagen V, endoplasmic reticulum calcium-binding protein, and fibrinogen 1. The profibrotic role of angiotensin II has been extensively studied, particularly in cardiac fibrosis.

[0009] Despite the tremendous progress made in identifying the cells involved and the molecular mechanisms by which the necessary wound healing process transforms into abnormal and harmful self-sustaining fibrosis, there are currently very few specific treatment options available for fibrotic diseases.

[0010] Pirfenidone is a small molecule drug approved in Japan in 2008 and in Europe in 2011 for the treatment of idiopathic pulmonary fibrosis (IPF). It exhibits anti-fibrotic activity by downregulating TGF-β. Nintedanib is a three-angiokinase inhibitor that reduces or blocks VEGF, FGF, and PDGF-induced tyrosine kinase activity. Although both compounds can reduce fibrosis in IPF, their clinical benefits are limited (extending survival by 2 years) and they have numerous side effects.

[0011] To date, no other treatments have been approved for fibrosis-related diseases. However, 45% of deaths in developed countries are attributed to some type of chronic fibrotic disease. Therefore, there is an unmet medical need for specific treatments for fibrosis, a disease with extremely high social impact. Thus, one object of the present invention is to provide a novel method for treating fibrosis.

[0012] In this invention, the results disclosed herein demonstrate that certain glycomimetic compounds exhibit antifibrotic activity both in vitro and in vivo, and therefore these compounds can be used to design effective new methods for treating fibrotic diseases. Summary of the Invention

[0013] The present invention provides a family of heterocyclic phosphine compounds, specifically the compound 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl-2-oxo-2λ5-[1,2]oxaphosphacyclohexane, for the treatment of fibrotic diseases. Attached Figure Description

[0014] Figure 1 Staining of kidney sections from the control group and the renal failure rat group. (A) Red sirius staining as a marker of fibrosis. (B) Immunostaining with Mgat5. Magnification ×200.

[0015] (*p≤0.05;**p≤0.01)

[0016] Figure 2 Sirius red staining of kidney sections. C: Control + placebo. C+3.1: Control + compound 3.1. SNX: SNX + placebo. SNX+3.1: SNX + compound 3.1. Magnification ×200. (*p≤0.05)

[0017] Figure 3 Immunostaining of kidney sections containing collagen 1, 3, and 4. (A) Collagen 1. (B) Collagen 4. (C) Collagen 3. C: Control + placebo. C+3.1: Control + compound 3.1. SNX: SNX + placebo. SNX+3.1: SNX + compound 3.1. Magnification ×200. (*p≤0.05; **p≤0.01; ***p≤0.001; and ****p≤0.0001)

[0018] Figure 4 .L PHA staining as a glycan marker of MGAT5 activity in kidney sections. C: Control + placebo. C+3.1: Control + compound 3.1. SNX: SNX + placebo. SNX+3.1: SNX + compound 3.1. Magnification ×200. (*p≤0.05; **p≤0.01)

[0019] Figure 5 Staining of aortic ring sections cultured in control and calcified media. (A) Von Kossa staining as a calcification marker. (B) Sirius red staining. (C) Immunostaining with GnT-V. Magnification ×200.

[0020] Figure 6 Section pattern of the left lobe of mouse liver used for histological analysis. Sections a and c were stored at -80°C. Section b was used for immunostaining.

[0021] Figure 7 Weight tracking in NASH model mice over a 21-day period. Drug group: placebo, 10 ml / kg, orally, twice daily. Compound 3.1 group: 15 mg / kg, orally, twice daily. Telmisartan group: 10 mg / kg, orally, once daily. (*p≤0.05; **p≤0.01; ***p≤0.001; and ****p≤0.0001)

[0022] Figure 8 Sirius red staining of mouse liver tissue sections. Magnification ×200.

[0023] Figure 9 Sirius red was used to stain mouse liver tissue sections to quantify the area of ​​fibrosis. Area is expressed as a percentage and corresponds to the ratio: Sirius red-stained surface / cut surface. Magnification × 200. (p < 0.01) Invention Details

[0025] This invention relates to heterocyclic phosphine compounds of formula (1) as detailed below, and specifically to the use of compound 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl-2-oxo-2λ5-[1,2]oxaphosphacyclohexane (also known as compound 3.1) for the treatment of fibrotic diseases. These compounds have previously been described as anticancer agents, and specifically for reducing or preventing the occurrence of cancer metastasis, as disclosed in PCT patent applications WO2009 / 004096 and WO2014 / 128429.

[0026] The compound used according to the present invention has the following formula (1):

[0027]

[0028] Where Y represents an oxygen, sulfur, or selenium atom, with oxygen atom being preferred.

[0029] Z represents an O, S, Se, NH, or NR6 group, wherein R6 is an aryl or optionally substituted alkyl group, preferably with an oxygen atom.

[0030] R 1 Representing hydrogen atoms, optionally substituted alkyl or aryl groups,

[0031] R 2a Represents a hydrogen atom, halogen, azide (N3), carbonate or dithiocarbonate group, 1H-[1,2,3]triazolyl or the group -X-R2, wherein

[0032] X represents an oxygen, sulfur, selenium atom, NH or NR7 group, where R7 is an optionally substituted aryl or alkyl group; X preferably represents O or NH, and;

[0033] R2 represents an aryl group, an optionally substituted alkyl group, a hydrogen atom, a trichloroacetylimine ester group (-C(=NH)CCl3), an acyl group, a formyl group, a sulfonyl group, a sulfinyl group, a tert-butyldiphenylsilyl group, an allyl group, a glycosyl group, an ester group, an amide group, a thioamide group, or a sulfonamide group, or X-R2 represents a P(O)R2R6 group, wherein R2 and R6 independently represent an aryl group, an optionally substituted alkyl group, an OH group, an alkoxy group, or an aryloxy group.

[0034] R 3 and R 4 Each of these groups independently represents an aryl group, an optionally substituted alkyl group, a hydrogen atom, a trichloroacetylimine ester group, an acyl group, a formyl group, a sulfonyl group, a sulfinyl group, a tert-butyldiphenylsilyl group, an allyl group, a glycosyl group, an ester group, an amide group, a thioamide group, a sulfonamide group, or R. 3 and R 4 Together Formation - R 3 -R 4 - a divalent group, wherein -R 3 -R4 - Preferably, it represents isopropylidene, benzylidene, diphenylmethylene, cyclohexylmethylene, and their substituted analogs, such as 4-methoxybenzylidene, or straight-chain alkylene, such as ethylidene (to form a propane-1,2-diol group).

[0035] R 5 A hydrocarbon group representing a hydrogen atom or containing one or more heteroatoms, preferably selected from oxygen, sulfur, or nitrogen, more preferably oxygen.

[0036] In the description of the compounds of this invention, names are typically used according to their usual definition.

[0037] As used herein, "alkyl" means a straight or branched, saturated or unsaturated hydrocarbon group having 1 to 25 carbon atoms, particularly including acyclic groups having 1 to 8 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, butyl, n-hexyl; preferably cycloalkyl having 3 to 7 carbon atoms, and more preferably cycloalkylmethyl having 4 to 8 carbon atoms.

[0038] As used herein, “substituted alkyl” means an alkyl group as defined above, which is substituted via sp. 3 The carbon atom is bonded and substituted with one or more aryl groups and / or contains one or more heteroatoms (such as N, S, or O). Suitable examples include aralkyl groups, such as (-CPh3)-triphenylmethyl, benzyl (labeled Bn), or 4-methoxybenzyl; alkoxyalkyl groups, especially dialkoxymethyl groups, such as diethoxymethyl or dimethoxymethyl; CH2CO2R 11 Group, wherein R 11 Represents an alkyl or aryl group that may be substituted.

[0039] As used herein, "alkoxy" means an alkyl group, such as ethoxy, methoxy, or propoxy, that is bonded to the rest of the molecule via an oxygen atom.

[0040] As used in this article, "aryloxy group" refers to an aryl group that is bonded to the rest of the molecule via an oxygen atom, such as benzyloxy group.

[0041] As used herein, “acyl” means a group derived from a carboxylic acid by removing a hydroxyl group, preferably having the formula -C(O)R8, wherein R8 represents an aryl or optionally substituted alkyl group, such as acetyl, trifluoroacetyl, propionyl, oleyl, myristyl or benzoyl.

[0042] As used herein, “sulfonyl” means a group derived from sulfonic acid by removing a hydroxyl group, preferably having the formula -SO2R9, where R9 represents an optionally substituted alkyl or aryl group.

[0043] As used herein, "sulfinyl group" refers to a group derived from sulfinic acid by removing a hydroxyl group, preferably having the formula -SOR 10 , where R 10 Represents an alkyl or aryl group that may be substituted.

[0044] As used in this article, "dithiocarbonate group" refers to the formula -OC(S)SR 9c The group, wherein R 9c Represents an alkyl or aryl group that may be substituted.

[0045] As used in this article, "carbonate group" refers to the formula -OC(O)OR 9d The group, wherein R 9d Represents an alkyl or aryl group that may be substituted.

[0046] As used in this article, "ester group" refers to the formula -C(O)OR 10' The group, wherein R 10' Represents an alkyl or aryl group that may be substituted.

[0047] As used in this article, "amide group" refers to the formula -C(O)NR 9' R 9" The group, wherein R 9' Represents an optionally substituted alkyl or aryl group, and R 9" Indicates the substituted alkyl, aryl, or hydrogen atom.

[0048] As used in this article, "thioamide group" refers to the formula -C(S)NR 9a R 9b The group, wherein R 9a Represents an optionally substituted alkyl or aryl group, and R 9b Represents an alkyl, aryl, or hydrogen atom that may be substituted.

[0049] As used in this article, "sulfonamide group" refers to the formula -SO2NR. 11' R 11" The group, wherein R 11' Represents an optionally substituted alkyl or aryl group, and R 11" Represents an alkyl, aryl, or hydrogen atom that may be substituted.

[0050] As used herein, "aryl" means an aromatic monovalent carbocyclic group comprising only one ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl and triphenyl), which may optionally be substituted with one or more groups such as, but not limited to, alkyl (e.g., methyl), hydroxyalkyl, aminoalkyl, hydroxyl, thiol, amino, halogen (fluorine, bromine, iodine, chlorine), nitro, alkylthio, alkoxy (e.g., methoxy), aryloxy, monoalkylamino, dialkylamino, acyl, carboxyl, alkoxycarbonyl, aryloxycarbonyl, hydroxysulfonyl, alkoxysulfonyl, aryloxysulfonyl, alkylsulfonyl, alkylsulfinyl, cyano, trifluoromethyl, tetrazolyl, carbamoyl, alkylcarbamoyl, and dialkylcarbamoyl. Alternatively, two adjacent positions in the aromatic ring may be substituted with methylenedioxy or ethylenedioxy. As used herein, “aryl” also includes “heteroaryl” groups, which are aromatic rings in which one or more carbon atoms of one or more aromatic rings are replaced by a heteroatom, such as a nitrogen, oxygen, phosphorus, or sulfur atom. Heteroaryl groups can be structures containing one or more aromatic rings or structures in which only one or more aromatic rings are coupled to one or more non-aromatic rings. In structures with many rings, these rings can be fused, covalently bonded, or bonded to each other via divalent common groups (such as methylene, ethylene, or carbonyl). Suitable examples of heteroaryl groups include thiophene (2-thiophene, 3-thiophene), pyridinium (2-pyridinium, 3-pyridinium, 4-pyridinium), isoxazole, phthalimide, pyrazole, indole, and furan groups, as well as their benzofused analogs, phenylpyridinyl ketones, quinoline, phenothiazines, carbazole, and benzopyranones. As used herein, "glycosyl" includes all groups derived by removing a hydroxyl or hydrogen atom (preferably hydroxyl) from a natural or synthetic, protected or unprotected carbohydrate or sugar. Glycosyl groups may include monosaccharides or oligosaccharides, such as disaccharides. Glycosyl groups, such as glucosyl and mannose, can be derived from sugars such as, but not limited to, glucuronic acid, lactose, sucrose, maltose, allose, arbutin, glucose, mannose, idole, galactose, tarose, ribose, arabinose, xylose, lysose, fructose, threose, erythrose, [β]-DN-acetylgalactosamine, [β]-DN-acetylglucosamine, fucose, sialic acid, N-acetylneuraminic acid, N-acetylmucolonic acid, glucosamine, galactosamine, rhamnose, and their protected or substituted analogues, such as those substituted with acyl, alkyl, aryl, halogen, and amino groups, as well as their deoxygenated analogues.

[0051] As used herein, an oligosaccharide group refers to a glycosyl group derived from at least two covalently linked monosaccharides, preferably containing one to three sugar units. For a description of sugar structures, see “Essentials of Glycobiology”, Varki et al., eds., Chapter 2 (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1999). Preferred glycosyl groups are monosaccharide groups. In compounds of formula (1), when R… 2a When representing the -X-R2 group, R2 represents a glycosyl group, which is preferably linked via an X group representing O or NH, preferably O.

[0052] As used in this article, "sugar" refers to monosaccharides or oligosaccharides.

[0053] “Bn” represents benzyl, and “Ac” represents acetyl.

[0054] Some compounds of the present invention can exist in both solvated and non-solvated forms, for example, as hydrates. Generally, the solvated form is equivalent to the non-solvated form and is included within the framework of the present invention. Some compounds of the present invention can have a variety of different crystalline or amorphous forms. Generally, all physical forms are equivalent for the intended use according to the present invention and are included within the framework of the present invention.

[0055] The compounds of this invention have several asymmetric (optical) centers, and therefore may contain enantiomers or diastereomers. It should be understood that this invention includes all enantiomers and diastereomers of the compounds of formula (1), as well as mixtures thereof, especially those based on racemic compounds. Different isomers can be separated according to methods known to those skilled in the art, particularly methods based on silica gel chromatography or fractional crystallization.

[0056] The preferred compounds of formula (1) are those in which Y = Z = O, that is, 1,2-oxaphosphacyclohexane 2-oxide compounds.

[0057] In the compounds of this invention, the R1 substituent, when it does not represent a hydrogen atom, is always bonded to the phosphorus atom within the ring via a carbon atom.

[0058] Preferred R1 groups include H, alkyl groups such as 2-benzyloxyethyl, ethyl, n-butyl, 3-phenylpropyl, n-octyl, dialkoxymethyl groups such as diethoxymethyl or dimethoxymethyl, aryl groups such as phenyl, 4-methylphenyl, 4-nitrophenyl, 4-aminophenyl, 4-methoxyphenyl, 3,4-difluorophenyl, 2-thienyl, 4-fluorophenyl, 4-biphenyl, 3-methylphenyl, 3-methoxyphenyl, and 3,5-difluorophenyl, and the following groups:

[0059]

[0060] In one specific implementation plan, R 1 It is a phenyl group.

[0061] Preferred R2 groups include H, arylsulfonyl, methylsulfonyl, trichloroacetylimine ester, benzyl, glycosyl, and aryl, such as phenyl, 4-methylphenyl, 4-nitrophenyl, 4-aminophenyl, 3,4-difluorophenyl, 3,5-difluorophenyl, and 3,4-dinitrophenyl.

[0062] Preferred X-R2 groups include O-aryl, OH, NH2, NH-aryl, S-aryl, and dithiocarbonate groups, or NHCH2CO2R 11 , where R 11 As defined above; NHC(O)R 12 , where R 12 Represents an aryl or optionally substituted alkyl group; O-SO2R9, wherein R9 is as defined above; NH-Bn, O-glycosyl, OC(=NH)CCl3, phosphonic acid, hypophosphonic acid or phosphine oxide, urea, thiourea, carbamate and carbonate groups.

[0063] According to a specific implementation scheme, X-R2 is OH, and preferably R1 is phenyl.

[0064] Preferably, R 3 and R 4 Each group can independently represent a hydrogen atom, benzyl, benzoyl, or acetyl group, or they can be combined to form the formula -R. 3 -R 4 - The divalent group, preferably representing isopropylidene.

[0065] According to a specific implementation plan, R 3 and R 4 Represents benzyl and / or R 1 It is phenyl and / or X-R2 is OH.

[0066] According to another specific implementation plan, R 3 and R 4 Represents benzyl, and R is preferred. 1 It is phenyl and / or X-R2 is OH.

[0067] According to a preferred embodiment of the present invention, R 5 The compound of formula (1) has the following formula (2) or (3):

[0068]

[0069] Where R 1 R 2a R 3R 4 Y and Z are as defined above, R 14 R 15 and R 16 Each of these groups independently represents a hydrogen atom, aryl group, optionally substituted alkyl group, trichloroacetylimine ester group, acyl group, formyl group, sulfonyl group, sulfinyl group, tert-butyldiphenylsilyl group, allyl group, ester group, amide group, thioamide group, sulfonamide group, or R. 15 and R 16 Together Formation - R 15 -R 16 - a divalent group, wherein -R 15 -R 16 - Preferably, it represents isopropylidene, benzylidene, diphenylmethylene, cyclohexylmethylene, and their substituted analogs, such as 4-methoxybenzylidene, or linear alkylene, such as ethylidene.

[0070] According to a specific implementation plan, R 14 Representing benzyl, and preferably in at least one or more of the specific embodiments detailed above, including wherein R 3 and R 4 Represents benzyl and / or R 1 It is phenyl and / or X-R2 is OH.

[0071] R 5 When not representing hydrogen atoms, it is preferable to have 1 to 25 carbon atoms, more preferably 1 to 20 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 1 to 8 carbon atoms. 5 This can represent an optionally substituted alkyl group containing one or more heteroatoms, preferably selected from oxygen, sulfur, or nitrogen, more preferably oxygen. Preferred R 5 The groups include alkoxyalkyl groups, such as benzyloxymethyl (-CH2OBn), -CH2OH, 2,2-dimethyl-[1,3]-dioxacyclopentane-4-yl and 1,2-dihydroxy-ethyl CH(OH)CH2OH groups, which mean that in formulas (2) and (3), R 14 =H or Bn, and R 15 =R 16 =H or R 15 and R 16 Together they form isopropylidene.

[0072] According to a specific implementation plan, compounds that can be used to treat fibrosis are selected from the group consisting of:

[0073] 3-Hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl-2-oxo-2λ5-[1,2]oxaphosphacyclohexane,

[0074] 4-(2,2-dimethyl-[1,3]dioxacyclopentan-4-yl)-2,2-dimethyl-2-oxo-2-phenyl-tetrahydro- *5*-[1,3]dioxacyclopentenro[4,5-d][1,2]oxaphosphinine-3-aminobenzyl, more specifically (3aR,6S,7S,7aS)-7-(benzylamino)-4-((R)-2,2-dimethyl-1,3-dioxacyclopentan-4-yl)-2,2-dimethyl-6-phenyltetrahydro-[1,3]dioxacyclopentenro[4,5-d][1,2]oxaphosphinine 6-oxide (also referred to herein as compound 3.3),

[0075] N-((2S,3S,4S,5S,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-2-oxo-2-phenyl-1,2-oxaporhexane-3-yl)acetamide (also referred to herein as compound 2.2),

[0076] 4,5-Bis-benzyloxy-6-benzyloxymethyl-phenyl-2-oxo- -[1,2]oxaphosphacyclohexane-3-aminobenzyl, more specifically (2S,3S,4S,5S,6R)-3-(benzylamino)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-2-phenyl-1,2-oxaphosphacyclohexane (also referred to herein as compound 4.6),

[0077] (2S,3S,4S,5S,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-3-hydroxy-2-(4-phenoxyphenyl)-1,2-oxaphosphacyclohexane 2-oxide (also referred to herein as compound 3.0), and

[0078] Benzoic acid (3aR,6R,7R,7aS)-4-((R)-2,2-dimethyl-1,3-dioxacyclopentan-4-yl)-2,2-dimethyl-6-oxo-6-phenyltetrahydro-[1,3]dioxacyclopenteno[4,5-d][1,2]oxaphosphinin-7-yl ester (also referred to herein as compound 4.2).

[0079] In a more specific embodiment, the compound that can be used to treat fibrosis is 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl-2-oxo-2λ5-[1,2]oxaphosphacyclohexane.

[0080] The preparation of compound 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl-2-oxo-2λ5-[1,2]oxaphosphacyclohexane can be carried out, for example, as described in WO 2009 / 004096, WO 2014 / 128429 and WO 2018 / 054925.

[0081] The compound 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl-2-oxo-2λ5-[1,2]oxaphosphacyclohexane used according to the present invention preferably has the following formula (I):

[0082]

[0083] Therefore, the present invention relates to compounds of formula (1), preferably 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl-2-oxo-2λ5-[1,2]oxaphosphacyclohexane, and more preferably compounds of formula (I) (also known as PST3.1), in a method of treating fibrotic diseases.

[0084] On the other hand, the present invention relates to the compound of formula (1) as detailed above, which is used to treat fibrosis by inhibiting GnT-V activity.

[0085] In another respect, the present invention relates to compounds of formula (1) for treating fibrosis by inhibiting the production of collagen fibers (more specifically type 1, 3 and / or type 4 collagen) and / or by inhibiting mechanisms involving the cellular matrix and / or cell / cell interactions (including inhibiting fibroblast migration).

[0086] The present invention further provides the use of compounds of formula (1) as defined herein, specifically 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl-2-oxo-2λ5-[1,2]oxaphosphacyclohexane, and preferably compounds of formula (I) (also known as PST3.1), for the manufacture of medicaments or pharmaceutical compositions for the treatment of fibrotic diseases. In one particular aspect, fibrosis is treated by inhibiting GnT-V activity. In another particular aspect, fibrosis is treated by inhibiting the production of collagen fibers (more specifically type 1, 3, and / or type 4 collagen), and / or by inhibiting mechanisms involving the cellular matrix and / or cell / cell interactions (including inhibition of fibroblast migration).

[0087] The present invention further provides a method for treating fibrotic diseases in patients in need, the method comprising administering to a patient in need of such treatment an effective amount of a compound of formula (1) as defined herein, specifically 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl-2-oxo-2λ5-[1,2]oxaphosphacyclohexane, and preferably a compound of formula (I), also known as PST3.1. In one particular aspect, fibrosis is treated by inhibiting GnT-V activity. In another particular aspect, fibrosis is treated by inhibiting the production of collagen fibers (more specifically type 1, 3, and / or 4 collagen), and / or by inhibiting mechanisms involving the cellular matrix and / or cell / cell interactions (including inhibition of fibroblast migration).

[0088] According to the present invention, the term "fibrosis" specifically includes fibrosis of the lungs, kidneys, liver, heart, muscles, skin, soft tissues (e.g., mediastinum or retroperitoneum), bone marrow, intestines, aorta, and joints (e.g., knees, shoulders, or other joints). Specifically, the term "fibrotic disease" caused by fibrosis includes pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endocardial myocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis (a complication of pneumoconiosis in coal miners), renal systemic fibrosis, Crohn's disease, keloids, old myocardial infarction, scleroderma, systemic sclerosis, joint fibrosis, and certain forms of adhesive bursitis.

[0089] According to one specific implementation, the compound of formula (1) is used to treat liver, kidney or skin fibrosis, more specifically to treat kidney or skin fibrosis, including keloids or scleroderma.

[0090] According to one specific embodiment, the present invention relates to the use of compounds of formula (1) as defined herein for the manufacture of medicaments or pharmaceutical compositions for the treatment of liver, kidney or skin fibrosis.

[0091] More specifically, it is used to treat kidney or skin fibrosis, including keloids or scleroderma.

[0092] According to one specific embodiment, the present invention further relates to a method for treating liver, kidney or skin fibrosis in patients in need, more specifically for treating kidney or skin fibrosis, including keloids or scleroderma, by administering an effective amount of a compound of formula (1) as defined herein to patients in need of such treatment.

[0093] According to another specific implementation scheme, the compound of formula (1) is used to treat aortic fibrosis.

[0094] According to one specific embodiment, the present invention relates to the use of compounds of formula (1) as defined herein for the manufacture of medicaments or pharmaceutical compositions for the treatment of aortic fibrosis.

[0095] According to one specific embodiment, the present invention further relates to a method for treating aortic fibrosis in patients in need, the method being carried out by administering an effective amount of a compound of formula (1) as defined herein to patients in need of such treatment.

[0096] The compound of formula (1) may be provided in the pharmaceutical composition. The pharmaceutical composition may additionally contain pharmaceutically acceptable adjuvants and / or carriers.

[0097] As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonics, absorption delay agents, and similar carriers. Carriers may be suitable for parenteral administration, such as intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion. Alternatively, carriers may be suitable for non-parenteral administration, such as local, epidermal, or mucosal routes of administration. Carriers may be suitable for oral administration. Depending on the route of administration, the compounds of the present invention may be encapsulated in materials to protect them from acids and other natural conditions that may inactivate them. Pharmaceutical compositions of the present invention may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound without conferring any undesirable toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0098] Pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers for use in the pharmaceutical compositions of the present invention include water, buffered water, and saline. Other examples of carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). In many cases, isotonic agents, such as sugars, polyols (such as mannitol, sorbitol), or sodium chloride, will be required in the composition. Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations.

[0099] The pharmaceutical compositions of the present invention may contain additional active ingredients. Also within the scope of the present invention are kits containing a compound of formula (1) as defined herein and instructions for use (specifically, for the treatment of fibrotic diseases). The kit may additionally contain one or more other agents, such as other therapeutic or preventative agents as discussed above. The compounds of the present invention or compositions containing them may be administered for the treatment of fibrotic diseases.

[0100] In one implementation, the treatment of fibrotic disease is a therapeutic treatment. In a therapeutic application, a subject already suffering from the condition or disease described above is given an amount of compound sufficient to cure, alleviate, or partially suppress the condition or one or more symptoms thereof. Such therapeutic treatment may cause a reduction in the severity of disease symptoms, or an increase in the frequency or duration of symptom-free periods. An amount sufficient to achieve this is defined as a “therapeuticly effective amount.”

[0101] In one implementation, the treatment of fibrotic disease is preventative. In preventative application, a dose of the preparation is administered to a subject at risk of the condition or disease as described above, in an amount sufficient to prevent or reduce the subsequent effects of the condition or one or more of its symptoms. The amount sufficient to achieve this is defined as the "preventatively effective dose".

[0102] Treatment includes administering the compound or a pharmaceutical composition containing the compound to a patient declared to have a condition in order to cure, delay or slow the progression of the condition, thereby improving the condition of the patient or a healthy subject, specifically a subject at risk of developing fibrotic disease.

[0103] Subjects to be treated according to the present invention can be selected based on several criteria associated with fibrotic diseases, such as prior drug treatment, relevant pathology, genotype, exposure to risk factors, viral infection, and any other relevant biomarkers that can be assessed by imaging methods and immunological, biochemical, enzymatic, chemical, or nucleic acid detection methods.

[0104] The effective dose for each purpose will depend on the severity of the disease or injury, as well as the subject's weight and general condition.

[0105] The subjects to which the drug is administered can be humans or non-human animals. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Humans are typically the subjects to whom the drug is administered.

[0106] Compounds of formula (1) can be administered in an effective amount using a pharmaceutical composition as defined above. In the context of this invention, the term "effective amount" means the amount of compound sufficient to produce the desired therapeutic effect.

[0107] The frequency and / or dosage associated with administration may be adjusted by those skilled in the art based on the patient, their weight, pathology, form of administration, etc. If necessary, administration may be given once daily or even several times daily.

[0108] In a further embodiment, the present invention provides a method for treating fibrotic diseases, comprising administering to a subject in need of such treatment an effective amount of at least one compound of formula (1) or a pharmaceutical composition containing the compound.

[0109] The invention is further described with reference to the following non-limiting examples. Example

[0110] Example 1

[0111] Materials and Methods

[0112] Male Sprague-Dawley rats underwent 5 / 6 total nephrectomies (SNx) as a model of renal fibrosis, as SNx rats develop tubulointerstitial fibrosis. During this surgery, the right kidney was removed and two of the three branches of the left renal artery were ligated to induce necrosis of two-thirds of the kidney. The control kidney was the right kidney before nephrectomy. Tubulointerstitial renal fibrosis inevitably leads to renal function deterioration and failure, independent of primary kidney disease. Notably, in SNx rats, plasma creatinine concentration increased from 25.7 ± 1.3 μM to 120.0 ± 33.0 μM. Sirius red staining highlighted the fibrosis by collagen in paraffin-embedded kidney tissue sections. Five-micrometer-thick sections were cut and mounted on glass slides. The slides were dewaxed and stained with Sirius red to assess collagen localization. Positive collagen signals appeared as deep red. The sections were mounted in Entelan mounting medium and examined under a microscope. For each sample, 10 200x magnified fields of view images of the kidney sections were quantified using ImageJ software. The stained area was measured within the kidney section region. Immunohistochemical analysis of the kidney sections was also performed using paraffin sections. Primary antibody, anti-Mgat5, was incubated overnight at 4°C. Developing was performed using the Universal Vectastain ABC kit and ImmPACT AE according to the supplier Vector Laboratories' instructions. The sections were then mounted on the aqueous mounting medium VectaMount. TM The samples were examined in AQ staining under a microscope (Nikon Eclipse TE300). Quantification of histological staining was performed.

[0113] result

[0114] In a subtotal nephrectomy (SNx) model, the development of fibrosis and Mgat5 overexpression were confirmed in 12-week-old rats with renal failure. Figure 1Before nephrectomy, collagen fibers were uniformly distributed; 12 weeks post-surgery, a large amount of fibrosis was observed. The results showed a significant increase in fibrosis (15.4 ± 1.4%). Post-operative Mgat5 expression also significantly increased (14.5 ± 2.6%). These results indicate that increased renal fibrosis is associated with increased Mgat5 expression.

[0115] Example 2

[0116] Materials and Methods

[0117] Compound 3.1 was orally administered to control rats and SNx rats (as described above) at 20 mg / kg daily for 28 days. A control group was administered a mediator (without compound 3.1) in the same manner as compound 3.1. Sprague Dawley rats were used. Rats were randomly fed and placed in a 12-hour light, 12-hour dark cycle. Rats were divided into cages of 3. Some rats underwent 5 / 6 total nephrectomies (SNx) to develop chronic renal failure, which would lead to the development of renal fibrosis. Treatment 3.1a was administered to rats on the day of nephrectomy and continued throughout the regimen. It was delivered as a nano-suspension diluted in drinking water. Control rats received a single solvent (placebo). Histological and immunohistochemical methods were monitored as described in Example 1.

[0118] result

[0119] Plasma creatinine concentrations were measured at 4 weeks in control rats and SNx rats treated with 3.1 and placebo. Creatinine levels were significantly increased in nephrectomized rats (p<0.0001), validating the model. Serum creatinine levels in SNx-treated rats were lower than in SNx-treated rats. The development of fibrosis could be observed by staining tissue sections with Sirius red. Figure 2 At 4 weeks, a significant increase in fibrosis was observed in SNx rats (4.1 ± 0.2%) compared to control rats (7.7 ± 1.3%). A decreasing trend in fibrosis was observed in SNx rats (5.8 ± 1.2%) under treatment compared to control SNx rats. At the time of observation, treatment had no significant effect on fibrosis in either healthy animals or SNx rats. Other biomarkers that can measure the development of renal fibrosis include collagen (…). Figure 3 The results showed that the concentrations of collagen I, III, and IV were higher in SNx rats compared to control rats. Regarding collagen I, an effect was observed in SNx rats, with the percentage of collagen I increasing threefold after nephrectomy, from 0.7 ± 0.1% in the control group to 2.1 ± 0.2% in the SNx group (p = 0.0001). Figure 3A). Compared with untreated rats, collagen I expression was significantly reduced in treated rats with kidney injury (p = 0.014) (1.3 ± 0.3%). 3.1 Significantly reduced collagen I expression in renal fibrosis. Results obtained targeting collagen IV expression were similar to those obtained targeting collagen I ( Figure 3 B). IV collagen expression increased from 5.2 ± 0.8% in the control group to 9.4 ± 1.0% in the SNx group (p = 0.0002). 3.1 Significantly reduced IV collagen in renal fibrosis (9.4 ± 1.0% vs. 6.4 ± 0.3%; p = 0.015). An SNx effect was also observed for collagen III, i.e., the percentage of collagen III in nephrectomy rats was three times that of control rats (5.5 ± 1% vs. 1.7 ± 0.2%, p = 0.0007). Figure 3 C). Compared with untreated rats, SNx rats treated with 3.1 showed a trend toward reduced collagen III levels (4.0 ± 0.3%) (p = 0.27). In the control group, there was no therapeutic effect on collagen III.

[0120] Glycosylation resulting from MGAT5 activity will be specifically measured using L-PHA lectin staining. These results will potentially allow for the assessment of the effectiveness of 3.1 treatments along this pathway. Figure 4 PHA-L staining increased from 3.7±0.4% in the control group to 6.8±1.0% in the SNx group. Compared with untreated rats, PHA-L staining was significantly reduced in treated rats with kidney injury (6.8±1.0% vs. 5.1±0.5%).

[0121] Example 3

[0122] Materials and Methods

[0123] The model of vascular fibrosis was isolated aortic ring calcification. Aortic calcification is a typical complication of renal failure (chronic kidney disease) and also a model of vascular fibrosis closely related to cardiovascular disease. The thoracic aorta was harvested from the descending portion of the aortic crossing to the diaphragm. Adjacent connective tissue was gently removed, and the aorta was washed three times consecutively with PBS. The aorta was cut into rings approximately 2 mm thick and cultured in 24-well plates for 14 days. The basal medium was Dulbecco's modified Eagle's medium containing 4.5 g L⁻¹ glucose, 10 mM sodium pyruvate, and 50 mg / mL ascorbic acid, supplemented with 15% FCS and 3.8 mM NaH₂PO₄ / Na₂HPO₄ to induce calcification. Histological and immunohistochemical methods were monitored as described in Example 1.

[0124] result

[0125] Aortic rings cultured in calcified medium showed positive von Cussa staining distributed along the medial layer of the arterial explant, indicating medial calcification associated with fibrosis. Figure 5 (A and 5B). No calcium deposition was observed in the aortic rings in the control culture medium. Fibrosis was 5-fold higher in the in vitro model of calcified aortic rings compared to uncalcified aortic rings, and immunostaining with GnT-V showed a significant increase in the corresponding GnT-V protein (14.0 ± 2.7% vs. 3.5 ± 0.7%).

[0126] Example 4

[0127] Materials and Methods

[0128] NIH3T3 fibroblasts were seeded at 40,000 cells per square centimeter in 24-well plates in DMEM + 10% FBS.

[0129] Prior to trauma / treatment, cells were washed twice in serum-free DMEM and then cultured under appropriate conditions of DMEM + 2.5% FBS, TGFβ + / -, PST 3.1 + / -.

[0130] TGFβ 5 ng / ml; PST 3.1 1 μM, n = 6 wells per treatment.

[0131] At t0, the cell layer was scratched with a needle, and the reference was measured with a scale bar and observed under a microscope. Three photos were taken of each well to measure the width of the scratch using ImageJ software.

[0132] After culturing under the required conditions for 24 hours, the scratches were measured under a microscope by taking three photographs at the same scale using the same reference object.

[0133] The data is processed by calculating the percentage of closure.

[0134] result

[0135] The results are summarized in Table 1 below.

[0136] In the presence of TGFβ, wound healing was inhibited by % at 1 μM.

[0137] 24-hour - 1μM test compound

[0138]

[0139] Table 1

[0140] In this wound healing inhibition test, compound 3.1 appears to be the most active compound.

[0141] Example 5

[0142] The STAM model (developed and standardized by SMC Laboratories, Japan, https: / / www.smccro-lab.com / ) is a model that summarizes the same disease progression as non-alcoholic steatosis (NASH) and hepatocellular carcinoma (HCC) in humans: in 25% of affected patients (i.e., 20%–25% of the adult population), non-alcoholic fatty liver disease progresses to NASH, which increases the risk of developing cirrhosis, liver failure, and hepatocellular carcinoma. In NASH patients, liver fibrosis is the leading determinant of mortality (https: / / doi.org / 10.1053 / j.gastro.2019.11.311). In this model, two-day-old male C57BL / 6 mice were given a single dose of streptozotocin to reduce their insulin secretion capacity. When the mice were four weeks old, they were fed a high-fat diet. The background of this model was advanced type 2 diabetes that progressed to fatty liver, NASH, fibrosis, and thus hepatocellular carcinoma (HCC). Compared with other NASH-HCC mouse models, the disease progression time was relatively short, and 100% of the animals developed liver cancer at 20 weeks of age.

[0143] This model is able to reproduce many pathological features of human NASH:

[0144] Ballooning degeneration of cells is a characteristic pathological feature of human NASH; burn-out NASH is characterized by a decrease in lipid droplets as fibrosis progresses.

[0145] Fibrosis progresses around the central vein.

[0146] ALT (a marker of liver damage) was slightly elevated.

[0147] NASH markers, such as CK-18, have been added.

[0148] Increases in human HCC markers such as glutamine synthase, glypican-3, and AFP have been observed.

[0149] Materials and Methods

[0150] C57BL / 6JJmsSlc mice (females 14 days gestation) were obtained from Japan SLC, Inc. (Japan). Two days after birth, male mice were induced to develop NASH by a single subcutaneous injection of 200 μg streptozotocin (STZ, Sigma-Aldrich, USA) solution and then fed a high-fat diet (HFD, 57 kcal% fat, catalog number HFD32, CLEA Japan, Inc., Japan) after 4 weeks of age.

[0151] Sterile solid HFDs were provided free of charge and replaced every two days according to the manufacturer's instructions. Next, NASH model mice were randomly assigned to three groups of 10 six-week-old mice each, based on their body weight the day before treatment began. Randomization was performed using stratified random sampling by body weight using Excel software. NASH model mice were stratified by body weight to obtain SD (slowest possible) values, and the difference in mean body weight between groups was minimized.

[0152] Group 1: Mediator (10 NASH mice were orally administered placebo nanosuspension at a volume of 10 mL / kg twice daily during the period from 6 to 9 weeks of age).

[0153] Group 2: Compound 3.1 (PST3.1) (10 NASH mice were orally administered compound 3.1 nano-suspension at a dose of 15 mg / kg and a volume of 10 mL / kg twice daily (2*15 mg / kg / day) during the period from 6 to 9 weeks of age).

[0154] Group 3: Telmisartan (10 NASH mice were orally supplemented with a carrier containing telmisartan at a dose of 10 mg / kg and a volume of 10 mL / kg once daily during the period of 6 to 9 weeks of age). The carrier and compound 3.1 were provided by Phost'in Therapeutics SAS. Telmisartan ( It was purchased from Boehringer Ingelheim GmbH (Germany).

[0155] Telmisartan is a small-molecule antihypertensive drug used for its antifibrotic activity, but its clinical use is limited because it can cause systemic hypotension. https: / / doi.org / 10.1038 / s41551-018-0279- x ).

[0156] Table 2

[0157]

[0158] Mice were euthanized after 3 weeks of treatment, and whole livers were collected and washed with cold saline. Individual whole livers (parietal lobe and visceral lobe) were photographed. Liver weight was measured, and the liver-to-body weight ratio was calculated. The left lobe of the liver was dissected and removed, as shown below. Figure 6 As described above, and stored as follows:

[0159] a: Liver samples were stored at -80°C and embedded in compounds at the optimal cutting temperature (OCT, Sakura Finetek, Japan).

[0160] b: Liver samples were fixed in Bouin's solution (Sigma-Aldrich, Japan) for 24 hours. After fixation, the samples were embedded in paraffin for Sirius red staining.

[0161] c: Quick-freeze liver samples in liquid nitrogen and store them at -80°C.

[0162] Sections were cut from paraffin blocks of liver tissue using a rotary microtome (Leica Microsystems). Each section was coded with a number for blinded evaluation.

[0163] To visualize collagen deposition, liver sections fixed in Boone solution were stained with Sirius Red solution (FUJIFILMWako Pure Chemical Corporation). Briefly, the sections were dewaxed and hydrophilized with xylene, 100-70% alcohol series, and RO water, followed by treatment with 0.03% Sirius Red solution (catalog number: 194-16202) for 60 minutes. After washing with 0.5% acetic acid solution and RO water, the stained sections were dehydrated and clarified with 70-100% alcohol series and xylene, and then... new(Merck,Germany) is sealed and used for observation.

[0164] To quantify the fibrosis area, bright-field images of Sirius red stained sections around the central vein were captured at 200x magnification using a digital camera (DFC295; Leica, Germany), and the positive area in five fields per section was measured using ImageJ. Statistical analysis was performed using Prism Software 6 (GraphPad Software, USA). Bonferroni multiple comparison tests were used for statistical analysis. Results are expressed as mean ± SD. Comparisons were made between the following groups: 1) Group 1 (mediator) vs. Group 2 (PhOx430); 2) Group 1 (mediator) vs. Group 3 (telmisartan).

[0165] result

[0166] Three weeks after treatment, no deaths, weight loss, or clinical signs were observed in the second group of animals treated with compound 3.1 (see [link to treatment]). Figure 7 ).

[0167] Quantitative analysis of Sirius red-stained liver sections showed that oral administration of the 3.1 nm suspension of the compound significantly reduced the onset of fibrosis in this mouse model (see [link to study]). Figure 8 and 9 ).

[0168] Group 1, medium: positive area 0.84±0.31%

[0169] Group 2, compound 3.1: positive area 0.47±0.12%, p value <0.01.

[0170] Group 3, Telmisartan: positive area 0.50±0.24%, p value <0.01.

Claims

1. Use of the compound of formula (I) in the preparation of a medicament for the treatment of fibrotic diseases, wherein the compound has the following formula (I): (I) And the fibrotic disease mentioned therein is liver or kidney fibrosis.

2. The use according to claim 1, wherein the fibrotic disease is liver fibrosis.

3. The use according to claim 1, wherein the fibrotic disease is renal fibrosis.

4. Use of the compound of formula (I) as defined in claim 1 in the preparation of a kit for treating fibrotic diseases, wherein the fibrotic disease is liver or kidney fibrosis, and wherein the kit comprises the compound of formula (I) and instructions for use.

Images