WO2024214062A1

WO2024214062A1 - Mito-esculetin for the treatment of nafld and nash

Info

Publication number: WO2024214062A1
Application number: PCT/IB2024/053607
Authority: WO
Inventors: Srigiridhar Kotamraju; Gajalakshmi Singuru; Sriravali PULIPAKA; Altab SHAIKH; Balaji Sai ANDUGULAPATI; Rajamannar Thennati; Hridya CHEMPON
Original assignee: Council of Scientific and Industrial Research CSIR
Current assignee: Council of Scientific and Industrial Research CSIR
Priority date: 2023-04-12
Filing date: 2024-04-12
Publication date: 2024-10-17
Anticipated expiration: 2025-10-12

Abstract

The present invention relates to 6,7-dihydroxy coumarin phosphonium amphiphile compounds for use in the treatment of fatty liver diseases. Preferably, wherein the fatty liver 5 disease is selected from one or more conditions from the group of, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), fibrosis, Weber-Christian disease, Wolman's disease, acute fatty liver of pregnancy, and lip dystrophy, adipose tissue hypertrophy. The present invention also relates to compositions containing a compound of Formula I, II or III and a pharmaceutically acceptable excipient.

Description

MITO-ESCULETIN FOR THE TREATMENT OF NAFLD AND NASH

FIELD OF THE INVENTION

[0001] The present invention relates to a compound of formula I (shown below) for use in the treatment of faty liver diseases. Preferably, wherein the faty liver disease is selected from one or more conditions from the group of, non-alcoholic faty liver disease (NAFLD), non- alcoholic steatohepatitis (NASH), liver fibrosis, Weber-Christian disease, Wolman’s disease, acute faty liver of pregnancy, and lipodystrophy, adipose tissue hypertrophy. The present invention also relates to compositions containing a compound of Formula I and a pharmaceutically acceptable excipient.

Formula I

BACKGROUND OF THE INVENTION

[0002] Faty liver, i.e., steatosis, is a disease in which excessive amounts of lipids accumulate in the liver. Fatty liver may develop due to medicine or alcohol use, viral (e.g., Hepatitis C) or bacterial infections or obesity. Steatohepatitis is inflammation of the liver related to fat accumulation. Heavy alcohol use can lead to faty liver and inflammation and is usually referred to as alcoholic hepatitis. Steatohepatitis resembles alcoholic hepatitis, but can occur in people who seldom or never drink alcohol. In this instance, it is often called nonalcoholic steatohepatitis or NASH. Both alcoholic hepatitis and steatohepatitis can lead to scarring, e.g., cirrhosis, and hardening of the liver resulting in serious liver damage. Obesity is one of the risk factors for non-alcoholic fatty liver disease (NAFLD) and other chronic diseases such as atherosclerosis, type 2 diabetes, cancer. Obesity promotes faty liver disease by increasing lipid accumulation in the visceral fat tissue and excessive lipid build-up in the liver.

[0003] Non-alcoholic fatty liver disease (NAFLD) is a complex metabolic syndrome categorized by impaired liver metabolism due to excess fat accumulation leading to nonalcoholic steatohepatitis (NASH). Further, NAFLD is well-known hepatic manifestation of metabolic disease and is also associated with atherosclerosis and structural heart changes increasing the risk of cardiovascular disease (CVD). Patients with NAFLD may experience more CVD and that the mechanism may be related to insulin resistance, oxidative stress, inflammation, endothelial dysfunction and lipid metabolism.

[0004] The pathogenesis of NAFLD is strongly influenced by oxidative stress. Non-ETC sources of ROS, especially [3-oxidation, are the significant sources of mitochondrial ROS. Oxidative stress causes increased fatty acid oxidation (FAO) in response to FFA overload. A reduction in the activity of antioxidant enzymes such as catalase, glutathione peroxidase, and superoxide dismutase is a feature of NASH The failure to increase antioxidant levels in the mitochondria has been one of the limitations of antioxidant therapy in the treatment of mitochondrial diseases. To circumvent this, the concept of mitochondrial targeting of antioxidants is gaining much attention in the recent-past with reasonable success not only with respect to understanding the disease processes, but also with preclinical and clinical efficacies. In this, the antioxidant molecules were covalently coupled to an alkyl triphenylphosphonium cation (TPP⁺), and these compounds were taken up more readily by mitochondria owing to the greater mitochondrial membrane potential (140 to 170 mV; negative side) compared to the plasma membrane potential (30 to 60 mV). By this, the target antioxidant moiety accumulates selectively more in the mitochondrial compartment and facilitate the effective scavenging of mitochondria- derived ROS. With this concept, earlier, we synthesized octyl-TPP conjugated esculetin (Mito-Esc) and reported that Mito-Esc administration significantly ameliorates angiotensin (Ang)-II-induced atherosclerosis in Apoe'^/_ mice by enhancing AMPK- mediated nitric oxide levels (Kamewar et al., Sci Rep. 2016; 6: 1-18). However, a satisfactory treatment for fatty liver disease, such as NAFLD and NASH has not been presently available.

[0005] US9580452 describes the use of mito-esculetin for the treatment of atherosclerosis. PCT Application No. IB2020/061043 describes the use of mito-esculetin for the treatment of wounds, psoriasis and hair loss. Neither of these disclosures, however, suggests that mito-esculetin could be useful as for the treatment of fatty liver disease.

SUMMARY OF THE INVENTION

[0006] The present invention provides a method of treating a fatty liver disease comprising administering a therapeutically effective amount of a 6,7-dihydroxy coumarin phosphonium amphiphile compound selected from

Formula El wherein, Z is selected from a halide, mesylate, tosylate, citrate, tartrate, malate, acetate and trifluoroacetate;

R is hydrogen, deuterium, substituted or unsubstituted C1-5 alkyl, a substituted or unsubstituted C1-5 alkoxy, halide, C1-5 haloalkyl, substituted or unsubstituted Ce-ioaryl or C5-10 heteroaryl;

Ri is Ce-io aryl, C5-7 cycloalkyl or a C5-10 heteroaryl;

X is a C 1-30 saturated or unsaturated carbon chain, substituted or unsubstituted with C1-5 alkyl, C2-5 alkenyl or C2-5 alkynyl side chains; or X is

-(CH₂)_p-R2-(CH₂)n-, or

-(CH2)2-R2-(CH2)2-R2-(CH₂)m-; wherein p is an integer selected from 2 or 3, n is an integer selected from 3 to 6, and m is an integer selected from 2 to 4; and b is an integer selected from 0 to 2

R2 is O, NH or S.

[0007] In another aspect, the present invention provides a method of treating a fatty liver disease comprising administering a therapeutically effective amount of a 6,7-dihydroxy coumarin phosphonium amphiphile compound of Formula III

Formula III wherein, Z is selected from a halide, mesylate, tosylate, citrate, tartrate, malate, acetate and trifluoroacetate.

[0008] In another aspect, the present invention provides a 6,7-dihydroxy coumarin phosphonium amphiphile compound selected from:

wherein,

Z is selected from a halide, mesylate, tosylate, citrate, tartrate, malate, acetate and trifluoroacetate;

Ri is Ce-io aryl, C5-7 cycloalkyl or a C5-10 heteroaryl; X is a C 1-30 saturated or unsaturated carbon chain, substituted or unsubstituted with C1-5 alkyl, C2-5 alkenyl or C2-5 alkynyl side chains; or X is

-(CH₂)p-R2-(CH₂)n-, or

R2 is O, NH or S; for use in the treatment of fatty liver disease.

[0009] In one aspect, the present invention provides a 6,7-dihydroxy coumarin phosphonium amphiphile compound of Formula III

wherein, Z is selected from a halide, mesylate, tosylate, citrate, tartrate, malate, acetate and trifluoroacetate, for use in the treatment of fatty liver disease.

[0010] In another aspect, the present invention provides a composition comprising, a 6,7- dihydroxy coumarin phosphonium amphiphile compound selected from:

wherein,

Ri is Ce-io aryl, C5-7 cycloalkyl or a C5-10 heteroaryl; X is a C 1-30 saturated or unsaturated carbon chain, substituted or unsubstituted with C1-5 alkyl, C₂-5 alkenyl or C₂-5 alkynyl side chains; or X is

-(CH₂)_p-R₂-(CH₂)_n-, or

-(CH₂)₂-R₂-(CH₂)₂-R₂-(CH₂)_m-; wherein p is an integer selected from 2 or 3, n is an integer selected from 3 to 6, and m is an integer selected from 2 to 4; and b is an integer selected from 0 to 2

R₂ is O, NH or S; for use in the treatment of fatty liver disease.

[0011] In one aspect, the present invention provides a composition comprising, a 6,7- dihydroxy coumarin phosphonium amphiphile compound of Formula III

[0012] In yet another aspect, present invention provides a method of treating a fatty liver disease comprising administering a composition comprising a 6,7-dihydroxy coumarin phosphonium amphiphile compound selected from

wherein,

-(CH₂)p-R2-(CH₂)n-, or

R2 is O, NH or S.

[0013] In another aspect, the present invention provides a method of treating a fatty liver disease comprising administering a composition comprising a 6,7-dihydroxy coumarin phosphonium amphiphile compound of Formula III

wherein, Z is selected from a halide, mesylate, tosylate, citrate, tartrate, malate, acetate and trifluoroacetate.

[0014] In one aspect, the fatty liver disease is selected from one or more conditions from the group of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), Weber- Christian disease, Wolman’s disease, acute fatty liver of pregnancy, and lipodystrophy and adipose tissue hypertrophy.

[0015] The fatty liver disease is resulting from steatosis, obesity, diabetes, insulin resistance, hypertriglyceridemia, abetalipoproteinemia, glycogen storage diseases, metabolic syndrome, high fat diet, hepatitis and liver fibrosis.

[0016] In yet another aspect, the present invention provides method of reducing or reversing the progression of NAFLD into NASH comprising administering a 6,7-dihydroxy coumarin phosphonium amphiphile compound selected from

wherein,

-(CH₂)p-R2-(CH₂)n-, or

R2 is O, NH or S.

[0017] In another aspect, the present invention provides a method of reducing or reversing the progression of NAFLD into NASH comprising administering a compound of Formula III

[0018] In yet another aspect, the present invention provides method of reducing or reversing the progression of NAFLD into NASH comprising administering a composition comprising a 6,7-dihydroxy coumarin phosphonium amphiphile compound selected from

Formula E wherein,

R is hydrogen, deuterium, substituted or unsubstituted C1-5 alkyl, a substituted or unsubstituted C1-5 alkoxy, halide, C1-5 haloalkyl, substituted or unsubstituted Ce-ioaryl or C5-10 heteroaryl; Ri is Ce-io aryl, C5-7 cycloalkyl or a C5-10 heteroaryl;

X is a Ci-30 saturated or unsaturated carbon chain, substituted or unsubstituted with C1-5 alkyl,

C2-5 alkenyl or C2-5 alkynyl side chains; or X is

-(CH₂)p-R2-(CH₂)n-, or

R2 is O, NH or S.

[0019] In another aspect, the present invention provides a method of reducing or reversing the progression of NAFLD into NASH comprising administering a composition comprising a compound of Formula III

[0020] In another aspect, the present invention provides a 6,7-dihydroxy coumarin phosphonium amphiphile compound for use in reducing or reversing the progression of NAFLD into NASH, the compound is selected from

Formula IS wherein,

-(CH₂)_p-R₂-(CH₂)_n-, or

R₂ is O, NH or S.

[0021] In another aspect, the present invention provides a compound of Formula III for use in reducing or reversing the progression of NAFLD into NASH, the compound of Formula III is

BRIEF DESCRIPTION OF THE FIGURES

[0022] Figure 1. Mito-Esculetin administration causes reduction in high fat diet-induced gain in body weight and improves lipid profile in Apoe⁷ mice.

[0023] A-C), body weights, adipose tissue weight to body weight ratio, and liver weight to body weight ratio (n=7 per group). D-G), serum LDL, serum HDL, serum triglycerides, and serum TG/HDL ratio (n=6 per group). H, I) Serum AST and ALT activities (n=6 per group). GraphPad Prism 9 was used to perform the statistics. The 1-way or 2- way ANOVA method was used for multiple comparisons. Data represents Mean ± SD of the numbers shown in parenthesis (n). *, p< 0.05, **, p < 0.01, ***, p< 0.001 significantly different from the high-fat diet control group. #, p < 0.05, significantly different from the simvastatin treated group. $, p< 0.05 significantly different from the pioglitazone treated group. NS, not significant.

[0024] Figure 2. Mito-Esculetin administration improves HFD-induced insulin resistance.

[0025] A-D), fasting glucose, random glucose, oral glucose tolerance test, HbAlc levels (n=7 per group). E), plasma insulin levels (n=5 per group). Data represents Mean ± SD of the numbers shown in parenthesis (n). *, p< 0.05, **, p< 0.01, ***, p< 0.001 significantly different from the high-fat diet control group. #, p< 0.05 significantly different from the simvastatin treated group.

[0026] Figure 3. Mito-Esculetin administration reduces high fat diet induced serum pro- inflammatory cytokines levels in Apoe ^/_ mice.

[0027] A-F), TNF-a, IL-1J3, IL-6, IL-17, IL-23, and MCP-1 levels (n=4 per group) were measured using Genetix ELISA Assay kits as per the manufacturer’s instructions. G) The levels of

[0028] CD45.1, an inflammatory marker, were measured in mice peripheral blood samples by flow cytometry. Data represents Mean ± SD of six animals per group. *, p < 0.05, **, p < 0.01, significantly different from the high-fat diet control group; #, p < 0.05, significantly different from the simvastatin treated group. $, p < 0.05, $$, p < 0.01, significantly different from the pioglitazone treated group.

[0029] Figure 4. Mito-Esculetin administration inhibits HFD-induced adipose tissue hypertrophy, lipid accumulation, and adipocyte differentiation in Apoe ^/_ mice.

[0030] A), Adipose tissue weights (n=7 per group). C), quantification of adipocyte lipid content (n=3 per group), C-E), adipose tissue LDL cholesterol, HDL cholesterol, triglyceride levels (n=5 per group). F), non-Esterified free fatty acid levels in adipose tissue homogenates (n=5 per group) by NEFA assay kit (Genetix). G), adipose tissue homogenates were prepared and the indicated protein levels were measured by Western blot analysis (n=3 per group). Data represents Mean ± SD of the numbers shown in parenthesis (n). *, p< 0.05, **, p< 0.01, ***, p< O.OOlsignificantly different from the high-fat diet control group. #, p< 0.05 significantly different from the simvastatin treated group. $, p< 0.05, significantly different from the pioglitazone treated group. NS, not significant.

[0031] Figure 5. Mito-Esc treatment inhibits IBMX-induced differentiation of 3T3L1- adipocyte cells via the activation of AMPK.

[0032] A), 3T3-L1 cells were pretreated for Ih with either Mito-Esc (1.25 and 2.5 pM) or simvastatin (10 pM), or pioglitazone (10 pM) and then treated with IBMX for 4 days and then replaced the medium with insulin (IpL/lmL) containing low glucose DMEM + 10% FBS for another 4 days and intracellular triglyceride levels were measured in cell lysates. B), same as A, except that non-esterified free fatty acid levels were measured by NEFA assay kit. C), 3T3L-1 cells were treated with the differentiation cocktail (IBMX, lpL/lmL+ insulin IpL/lmL) for 2 to 8 days, and the indicated protein were measured by Western blot analysis. D), same as H except that cells were pretreated with Mito-Esc (1.25pM) before they were treated with differentiation cocktail for 2 to 8 days and indicated protein levels were measured by Western blot analysis E), Quantification of relative lipid content in cells after Oil red O staining. F), cells were transfected with siAMPK (40 nmol/L) for 16h and then pretreated with either Mito-Esculetin (1.25 and 2.5 pM) or simvastatin (10 pM) or pioglitazone (10 pM) for Ih and then treated with IBMX (IpL/lmL) for 8 days and SIRT-1 activity was measured in cell homogenates. ***, p<0.001 significantly different from late differentiated control. #, p< 0.05 significantly different from the simvastatin treated cells. $, p< 0.05 significantly different from the pioglitazone treated cells.

[0033] Figure 6. Mito-Esculetin treatment inhibits high-fat-diet-induced increase in lipid levels via the AMPK-SIRT-1-CD36 axis and attenuates fatty liver and hepatocellular steatosis in Apoe⁷ mice

[0034] A), Liver weights (n=6 per group). B), Quantification (%) of lipid content in the liver tissue by Oil red O staining (n=3). C), NAFLD Score (n=3 per group), d), indicated protein levels were measured in the liver tissue homogenates by immunoblot analysis (n=3 per group) . *, p < 0.05, **, p< 0.01, ***, p< 0.001 significantly different from the high-fat diet control group.

[0035] #, p< 0.05 significantly different from the simvastatin treated group. $, p< 0.05 significantly different from the pioglitazone treated group.

[0036] Figure 7. Mito-Es culetin inhibits palmitate-induced lipid accumulation by activating AMPK and SIRT-1 axis in HepG2 liver cells.

[0037] A), HepG2 cells were treated with BSA-palmitate (200 -3000 pM) for 24h and cell viability was measured by trypan blue dye exclusion assay. B), cells were pretreated with Mito-Esc 1.25 pM for Ih and then treated with BSA-palmitate for 8h and CD36 levels were measured by flowcytometry. C), cells were pretreated with Mito-Esc (1.25 pM) for Ih and then treated with BSA-palmitate for 8h and SIRT1 activity was measured in HepG2 cells homogenates. D), cells were transfected with siAMPK (40 nmol/L) for 16h and pretreated with Mito-Esculetin (1.25

[0038] pM), for Ih before they were treated with BSA-palmitate for 8h and SIRT-1 activity were measured in cells homogenates. E), cells were pretreated with Mito-Esc 1.25pM for Ih and then treated with BSA-palmitate for 8h intracellular triglyceride levels were measured. Graph

[0039] Pad Prism 9 was used to perform the statistics. The 1-way or 2-way ANOVA method was used for multiple comparisons wherever applicable. *, p <0.05 significantly different from BSA- palmitate treated cells.

[0040] Figure 8: Mito-Esc and metformin administration significantly improves cardiac fibrosis markers in aged Apoe-/- mice

[0041] Chronic administration of Mito-Esc, metformin and their combination showed a significant reduction of fibrotic makers expression like a-SMA, COL1A1 and COL3A1 compared to aged control heart tissues of Apoe'^/_ mice. *, statistically different at p<0.5 compared to the control group (n=3).

DETAILED DESCRIPTION OF THE INVENTION [0042] As used herein the following definitions apply unless clearly indicated otherwise. It should be understood that unless expressly stated to the contrary, the singular forms "a" "an" and "the" include plural reference unless the context clearly dictates otherwise.

[0043] Also, the use of "or" means "and/or" unless stated otherwise. Similarly, "comprise," "comprises," "comprising" "include," "includes," and "including" are interchangeable and not intended to be limiting.

[0044] By “alkyl,” in the present invention is meant a straight or branched hydrocarbon radical and includes C1-5, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, Sec-butyl, isobutyl, tert-butyl, n-pentyl, iso-pentyl, and the like.

[0045] “Alkenyl¹ means straight and branched hydrocarbon radicals and at least one double bond and includes C2-C5, for example, ethenyl, 3-butenl-yl, 2-ethenylbutyl, and the like.

[0046] “Alkynyl¹ means straight and branched hydrocarbon radicals and at least one triple bond and includes C2-C5, for example, ethynyl, 3-butyn 1-yl, propynyl, 2-butyn-l-yl, 3-pentyn- 1-yl, and the like.

[0047] By “aryl¹ is meant an aromatic carbocyclic group having a single ring (e.g., phenyl), multiple rings (e.g., biphenyl), or multiple condensed rings in which at least one is aromatic, (e.g., 1,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, or phenanthryl), which can be mono-, di-, or trisubstituted with, e.g., halogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl, lower acyloxy, aryl, heteroaryl, and hydroxy. A preferable aryl is Ce-io aryl, more preferably aryl is phenyl.

[0048] “Heteroatom”, means an atom of any element other than carbon or hydrogen.

[0049] Preferably, heteroatoms are nitrogen, oxygen, and sulfur.

[0050] “Cycloalkyl” means a monocyclic or polycyclic hydrocarbyl group having from 5 to 7 carbon atoms, for instance, cycloheptyl, cyclohexyl, and cyclopentyl.

[0051] By the term “halide¹ in the present invention is meant fluoride, bromide, chloride, and iodide.

[0052] By "heteroaryl¹ is meant one or more aromatic ring systems of 5-, 6-, or 7-membered rings with 5 to 10 carbon, containing at least one and up to four heteroatoms selected from nitrogen, oxygen, or sulfur. Such heteroaryl groups include, for example, (is)oxazolyl, oxadiazolyl, tetrazolyl, pyridyl, oxadiazolyl, oxathiadiazolyl, thiatriazolyl, pyrimidinyl, (iso) quinolinyl, napthyridinyl, phthalimidyl, benzimidazolyl, and benzoxazolyl.

[0053] As used herein, a "therapeutically effective amount" or "effective amount" refers to that amount of the compound sufficient to result in amelioration of symptoms, for example, treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions, typically providing a statistically significant improvement in the treated patient population.

[0054] When referencing an individual active ingredient, administered alone, a therapeutically effective close refers to that ingredient alone. When referring to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, including serially or simultaneously.

[0055] ‘ ‘Patient” as used herein refers to any human or nonhuman animal (e.g., primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles and the like). As used herein the term

[0056] ‘ ‘Treatment” refers to cure the disease and/or disorder as rapidly as possible and to prevent the progression to severe disease.

[0057] In one embodiment, the present disclosure provides a method of treating a fatty liver disease comprising administering a therapeutically effective amount of a 6,7-dihydroxy coumarin phosphonium amphiphile compound selected from

or;

or;

wherein,

Ri is Ce-io aryl, C5-7 cycloalkyl or a C5-10 heteroaryl;

-(CH₂)p-R2-(CH₂)n-, or

-(CH2)2-R2-(CH2)2-R2-(CH₂)m-; wherein p is an integer selected from 2 or 3, n is an integer selected from 3 to 6, and m is an integer selected from 2 to 4; and b is an integer selected from 0 to 2 R2 is O, NH or S.

[0058] In another embodiment, the present disclosure provides a method of treating a fatty liver disease comprising administering a therapeutically effective amount of a 6,7- dihydroxy coumarin phosphonium amphiphile compound of Formula II

wherein, Z is selected from a halide, mesylate, tosylate, citrate, tartrate, malate, acetate and trifluoroacetate;

-(CH₂)p-R2-(CH₂)n-, or

R2 is O, NH or S.

[0059] In another embodiment, the present disclosure provides a method of treating a fatty liver disease comprising administering a therapeutically effective amount of a 6,7- dihydroxy coumarin phosphonium amphiphile compound of Formula III

[0060] In another embodiment, the present disclosure provides a 6,7-dihydroxy coumarin phosphonium amphiphile compound selected from:

wherein,

R is hydrogen, deuterium, substituted or unsubstituted C1-5 alkyl, a substituted or unsubstituted Ci- 5 alkoxy, halide, C1-5 haloalkyl, substituted or unsubstituted Ce-ioaryl or C5-10 heteroaryl;

Ri is Ce-io aryl, C5-7 cycloalkyl or a C5-10 heteroaryl;

-(CH₂)p-R2-(CH₂)n-, or

R2 is O, NH or S; for use in the treatment of fatty liver disease.

[0061] In another embodiment, the present disclosure provides a 6,7-dihydroxy coumarin phosphonium amphiphile compound of Formula II

C2-5 alkenyl or C2-5 alkynyl side chains; or X is

-(CH₂)_p-R2-(CH₂)n-, or

R2 is O, NH or S; for use in the treatment of fatty liver disease.

[0062] In another embodiment, the present disclosure provides a 6,7-dihydroxy coumarin phosphonium amphiphile compound of Formula III

Formula ill wherein, Z is selected from a halide, mesylate, tosylate, citrate, tartrate, malate, acetate and trifluoroacetate, for use in the treatment of fatty liver disease.

[0063] In another embodiment, the present disclosure provides a composition comprising, a 6,7-dihydroxy coumarin phosphonium amphiphile compound selected from:

wherein,

Ri is Ce-io aryl, C5-7 cycloalkyl or a C5-10 heteroaryl;

-(CH₂)p-R2-(CH₂)n-, or

R2 is O, NH or S; for use in the treatment of fatty liver disease.

[0064] In one aspect, the fatty liver disease is selected from one or more conditions from the group of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, Weber-Christian disease, Wolman’s disease, acute fatty liver of pregnancy, and lipodystrophy and adipose tissue hypertrophy.

[0065] In another aspect, the fatty liver disease is resulting from steatosis, obesity, diabetes, insulin resistance, hypertriglyceridemia, abetalipoproteinemia, glycogen storage diseases, metabolic syndrome, high fat diet, hepatitis and liver fibrosis.

[0066] The inventors of the present invention, surprisingly found that the compounds, as disclosed in the preceding embodiments, for example compounds of Formula III, such as mitochondria- targeted esculetin (Mito-Esc) greatly improves HFD-induced NAFLD and the associated NASH etiology when tested in Apoc" mice. Further, the compounds, as disclosed in the preceding embodiments, for example compound of Formula III, such as Mito-Esc inhibited adipocyte differentiation as well as excess lipid uptake by modulating CD36, PPAR-y, and EBP-a and their target genes.

[0067] In another embodiment, the compounds, as disclosed in the preceding embodiments, for example compounds of Formula III, such as Mito-Esc administration reduced HFD- induced inflammatory processes in the adipose and liver tissues possibly by limiting lipid accumulation and lipotoxicity via the activation of AMPK-SIRT1 axis.

[0068] In another embodiment, the compounds, as disclosed in the preceding embodiments, for example compounds of Formula III, such as Mito-Esc increases the level of phospho- acetyl- Co A carboxylase (ACC).

[0069] In another embodiment, the compounds, as disclosed in the preceding embodiments, for example compounds of Formula III, such as Mito-Esc inhibits 3T3L1 preadipoctyte differentiation and adipogenesis.

[0070] In another embodiment, the compounds, as disclosed in the preceding embodiments, for example compounds of Formula III, such as Mito-Esc when administered in a therapeutically effective dose inhibits the CD36 receptor which facilitates the uptake of free fatty acids.

[0071] In another embodiment, the compounds, as disclosed in the preceding embodiments, for example compounds of Formula III, such as Mito-Esc when administered in a therapeutically effective dose results into significant reduction of LDLc, HDLc, and non- esterified free fatty acid (NEFA) levels.

[0072] In another embodiment, the compounds, as disclosed in the preceding embodiments, for example compounds of Formula III, such as Mito-Esc when administered in a therapeutically effective dose, decreases the transcript levels of adipogenic markers. More preferably, the adipogenic markers are PPAR-y, FABP4, EBP-[3, EBP-5, and SREBP1.

[0073] In another embodiment, the compounds, as disclosed in the preceding embodiments, for example compounds of Formula III, such as Mito-Esc when administered in a therapeutically effective dose inhibits resistin, leptin, and pro-inflammatory cytokine markers. Preferably, the pro-inflammatory cytokine markers are TNF-a, IL-1, IL-6, IL- 17, IL-23, and MCP-1.

[0074] In another embodiment, the present disclosure provides a composition comprising, a 6,7- dihydroxy coumarin phosphonium amphiphile compound of Formula II

C2-5 alkenyl or C2-5 alkynyl side chains; or X is

-(CH₂)p-R2-(CH₂)n-, or

R2 is O, NH or S; for use in the treatment of fatty liver disease. [0075] In another embodiment, the present disclosure provides a composition comprising, a

6,7- dihydroxy coumarin phosphonium amphiphile compound of Formula III

[0076] The present invention further provides a composition comprising a compound of Formula I, Formula II or Formula III according to the present invention. In some embodiments, the composition according to the invention is in a pharmaceutically acceptable form.

[0077] In certain embodiments, the pharmaceutical composition is formulated for oral or parenteral administration. In some embodiments, the pharmaceutical composition is administered as an oral dosage form. Preferably, the oral dosage form is in the form of tablet, capsule, dispersible tablets, sachets, sprinkles, liquids, solution, suspension, emulsion and the like. If the oral dosage form is a tablet, the tablet can be of any suitable shape such as round, spherical, or oval. The tablet may have a monolithic or a multi-layered structure. In some embodiments, the pharmaceutical composition of the present invention can be obtained by conventional approaches using conventional pharmaceutically acceptable excipients well known in the art. Examples of pharmaceutically acceptable excipients suitable for tablet preparation include, but are not limited to, diluents (e.g., calcium phosphate- dibasic, calcium carbonate, lactose, glucose, microcrystalline cellulose, cellulose powdered, silicified microcrystalline cellulose, calcium silicate, starch, starch pregelatinized, or polyols such as mannitol, sorbitol, xylitol, maltitol, and sucrose), binders (e.g., starch, pregelatinized starch, carboxymethyl cellulose, sodium cellulose, microcrystalline cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, crospovidone, or combinations thereof), disintegrants (e.g., crosslinked cellulose, cross-linked- polyvinylpyrrolidone (crosspovidone), sodium starch glycolate, polyvinylpyrrolidone (polyvidone, povidone), sodium carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose sodium), hydroxypropyl cellulose, hydroxypropyl methylcellulose, xanthan gum, alginic acid, or soy polysaccharides), wetting agents (e.g., polysorbate, sodium lauryl sulphate, or glyceryl stearate) or lubricants (e.g., sodium lauryl sulfate, talc, magnesium stearate, sodium stearyl fumarate, stearic acid, glyceryl behenate, hydrogenated vegetable oil, or zinc stearate). The tablets so prepared may be uncoated or coated for altering their disintegration, and subsequent enteral absorption of the active ingredient, or for improving their stability and/or appearance. In both cases, conventional coating agents and approaches well known in the art can be employed.

[0078] In certain embodiments, the parenteral administration can be formulated as a solution, suspension, emulsion, particle, powder, or lyophilized powder in association, or separately provided, with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and about 1-10% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. The vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by known or suitable techniques. In some embodiments, parenteral formulation may comprise a common excipient that includes, but not limited to, sterile water or saline, polyalkylene glycols, such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. Aqueous or oily suspensions for injection can be prepared by using an appropriate emulsifier or humidifier and a suspending agent, according to known methods. Parenteral route of administration includes, but is not limited to, subcutaneous route, intramuscular route, intravenous route, intrathecal route or intraperitoneal.

[0079] The formulations of the present invention can be prepared by a process known or otherwise described in the prior art, for example the process disclosed in Remington's Pharmaceutical Sciences.

[0080] In yet another embodiment, the present disclosure provides a method of treating a fatty liver disease comprising administering a composition comprising a 6,7-dihydroxy coumarin phosphonium amphiphile compound selected from

wherein,

-(CH₂)p-R2-(CH₂)n-, or

R2 is O, NH or S.

[0081] In yet another embodiment, the present disclosure provides a method of treating a fatty liver disease comprising administering a composition comprising a 6,7-dihydroxy coumarin phosphonium amphiphile compound of Formula II

-(CH₂)p-R2-(CH₂)n-, or -(CH₂)₂-R₂-(CH₂)₂-R₂-(CH₂)_m-; wherein p is an integer selected from 2 or 3, n is an integer selected from 3 to 6, and m is an integer selected from 2 to 4; and b is an integer selected from 0 to 2

R₂ is O, NH or S.

[0082] In yet another embodiment, the present disclosure provides a method of treating a fatty liver disease comprising administering a composition comprising a 6,7-dihydroxy coumarin phosphonium amphiphile compound of Formula III

[0083] In one aspect, the fatty liver disease is selected from one or more conditions from the group of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), Weber- Christian disease, Wolman’s disease, acute fatty liver of pregnancy, and lipodystrophy and adipose tissue hypertrophy.

[0084] In another aspect, the fatty liver disease is resulting from steatosis, obesity, diabetes, insulin resistance, hypertriglyceridemia, abetalipoproteinemia, glycogen storage diseases, metabolic syndrome, high fat diet, hepatitis and liver fibrosis.

[0085] In yet another embodiment, the present disclosure provides a method of reducing or reversing the progression of NAFLD into NASH comprising administering a 6,7- dihydroxy coumarin phosphonium amphiphile compound selected from

wherein,

-(CH₂)p-R2-(CH₂)n-, or

R2 is O, NH or S.

[0086] In another embodiment, the present disclosure provides a method of reducing or reversing the progression of NAFLD into NASH comprising administering a compound of Formula II

R₂ is O, NH or S.

[0087] In another embodiment, the present disclosure provides a method of reducing or reversing the progression of NAFLD into NASH comprising administering a compound of Formula III

[0088] In yet another aspect, the present invention provides method of reducing or reversing the progression of NAFLD into NASH comprising administering a composition comprising a 6,7-dihydroxy coumarin phosphonium amphiphile compound selected from

wherein,

Ri is Ce-io aryl, C5-7 cycloalkyl or a C5-10 heteroaryl;

-(CH₂)p-R2-(CH₂)n-, or

[0089] In another embodiment, the present disclosure provides a method of reducing or reversing the progression of NAFLD into NASH comprising administering a composition comprising a compound of Formula II

C2-5 alkenyl or C2-5 alkynyl side chains; or X is

-(CH₂)p-R2-(CH₂)n-, or

R2 is O, NH or S.

[0090] In yet another embodiment, the present disclosure provides a method of reducing or reversing the progression of NAFLD into NASH comprising administering a composition comprising a compound of Formula III

[0091] In another embodiment, the present disclosure provides a 6,7-dihydroxy coumarin phosphonium amphiphile compound for use in reducing or reversing the progression of NAFLD into NASH, the compound is selected from

wherein,

Ri is Ce-io aryl, C5-7 cycloalkyl or a C5-10 heteroaryl;

C2-5 alkenyl or C2-5 alkynyl side chains; or X is

-(CH₂)p-R2-(CH₂)n-, or

R2 is O, NH or S.

[0092] In another embodiment, the present disclosure provides a compound of Formula II for use in reducing or reversing the progression of NAFLD into NASH, the compound of Formula II is

wherein,

C2-5 alkenyl or C2-5 alkynyl side chains; or X is

-(CH₂)p-R2-(CH₂)n-, or

R2 is O, NH or S.

[0093] In another embodiment, the present disclosure provides a compound of Formula III for use in reducing or reversing the progression of NAFLD into NASH, the compound of Formula III is

[0094] In another embodiment, the present disclosure provides a method for the treatment and inhibition of fibrosis comprising administering a 6,7-dihydroxy coumarin phosphonium amphiphile compound selected from

wherein,

Ri is Ce-io aryl, C5-7 cycloalkyl or a C5-10 heteroaryl;

-(CH₂)p-R2-(CH₂)n-, or

R2 is O, NH or S.

[0095] In a preferable embodiment, the fibrosis is associated with fatty liver disease such as NAFLD.

[0096] In another embodiments, the fibrosis may be pulmonary fibrosis (e.g. idiopathic pulmonary fibrosis, diffuse interstitial pulmonary fibrosis, pleural fibrosis and fibrosis associated with asthma, fibrous dysplasia, cystic fibrosis), heart or cardiac fibrosis (e.g. endomyocardial fibrosis and fibrosis associated with cardiovascular disease), kidney fibrosis (e.g. associated with renal failure), dermal fibrosis (e.g., keloid), ocular fibrosis, mucosal fibrosis, fibrosis of the central nervous system, fibrosis in bone or bone marrow. More preferably, the fibrosis is cardiac fibrosis.

[0097] In another embodiment, the present disclosure provides a method of provides a method for the treatment and inhibition of fibrosis comprising administering a compound of Formula III

Formula IS wherein, Z is selected from a halide, mesylate, tosylate, citrate, tartrate, malate, acetate and trifluoroacetate.

[0098] In a preferable embodiment, the fibrosis is associated with fatty liver disease such as NAFLD. More preferably, the fibrosis is cardiac fibrosis.

[0099] In another embodiment, the present disclosure provides a method for the treatment and inhibition of fibrosis comprising administering a composition comprising a 6,7- dihydroxy coumarin phosphonium amphiphile compound selected from

or;

wherein,

Ri is Ce-io aryl, C5-7 cycloalkyl or a C5-10 heteroaryl;

-(CH₂)p-R2-(CH₂)n-, or

[00100] In a preferable embodiment, the fibrosis is associated with fatty liver disease such as NAFLD. More preferably, the fibrosis is cardiac fibrosis.

[00101] In another embodiment, the present disclosure provides a method for the treatment and inhibition of fibrosis comprising administering a composition comprising a compound of Formula III

[00102] In a preferable embodiment, the fibrosis is associated with fatty liver disease such as NAFLD. More preferably, the fibrosis is cardiac fibrosis.

[00103] In another embodiment the present disclosure provides a 6,7-dihydroxy coumarin phosphonium amphiphile compound for use in the treatment and inhibition of fibrosis, the compound is selected from

wherein,

Ri is Ce-io aryl, C5-7 cycloalkyl or a C5-10 heteroaryl;

-(CH₂)p-R2-(CH₂)n-, or

[00104] In a preferable embodiment, the fibrosis is associated with fatty liver disease such as NAFLD. More preferably, the fibrosis is cardiac fibrosis.

[00105] In another embodiment the present disclosure provides compound of Formula III for use in the treatment and inhibition of fibrosis, the compound of Formula III is

[00106] In a preferable embodiment, the fibrosis is associated with fatty liver disease such as NAFLD. More preferably, the fibrosis is cardiac fibrosis.

[00107] EXAMPLES

[00108] EXAMPLE 1

[00109] Synthesis of Mito-Esc. Mitochondria-targeted esculetin (The compounds of the invention can be prepared as described in U.S. Patent No.9, 580, 452).

[00110] EXAMPLE !

[00111] i) Animals experimentation

[00112] Experiments were conducted in 4 months old male Apo lipoprotein E knockout (Apoe'^/_) mice and protocols were approved by the IAEC at CSIR-IICT (IICT/IAEC/54/2018). Age-matched C57BL/6 mice served as a strain control. Mice were randomly divided into 8 groups (n=8 per group). Apoe'^/_mice were fed either standard chow diet (11.4% fat) or HFD (60% saturated fat) for 16 weeks. At the end of this period, plasma lipid and glucose profiles were monitored to confirm the dys-lipidemia and hyperglycemia. Apoe'^/_ mice groups were administered with 0.312, 0.625, and 1.25 mg/kg of Mito-Esc (made in 0.5% methylcellulose and tween-20) daily for 45 days by oral gavage. Similarly, simvastatin and pioglitazone were administered at 10 mg/kg.bd.wt respectively. Control mice groups received 0.5% methylcellulose and tween-20. Feed intake was monitored every day, and body weights were recorded weekly.

[00113] ii) Biochemical analysis

[00114] Mice were anaesthetized with ketamine and xylazine cocktail (90 mg/kg ketamine and 10 mg/kg xylazine) via intraperitoneal injection, and blood was collected via the orbital sinus puncture. The blood was centrifuged at 6000 rpm for 5 minutes, and the serum was collected. Serum HDL-cholesterol (cat# 89-6010977870), LDL-chole sterol (cat# 89-06010977900), and total triglycerides (cat# 89-06010977870) were measured using commercial kits (Coral Clinical Systems, India) according to the manufacturer’s protocol.

[00115] iii) AST and ALT enzyme activities:

[00116] Aspartate aminotransferase (MAK055-AST) and alanine aminotransferase (MAK052-ALT) enzyme activities were assayed in the plasma using commercially available kits (Sigma- Aldrich Chemicals, India) according to the manufacturer’s instructions.

[00117] iv) Blood glucose and HbAlc levels:

[00118] Fasting and random glucose levels were measured by the Accu-Check Performa blood glucose meter. Glycated hemoglobin (HbAlc) levels were measured in the whole blood using auto blood analyzer (Siemens, Germany).

[00119] v) OGTT measurement:

[00120] Oral glucose tolerance test (OGTT) was carried out at the end of the experimental protocol.

[00121] Mice were fasted overnight and given an oral glucose (2g/kg.bd.wt). A drop of blood was collected from each mouse at 0-, 30-, 60-, and 120-min intervals and measured the blood glucose levels.

[00122] vi) Cytokine levels measurement:

[00123] TNF-a (PG-711 OMO), IL- 1 [3 (PG-291 OMO), IL-6 (PG-9400MO), IL- 17 (PG-

1400MO), IL-23 (PG-9150MO), adipokines (Resistin (PG-3620MO), leptin (PG- 2560MO), adiponectin (PG- 6420MO) and insulin (PG-2600MO) levels were measured in serum and adipose tissue homogenates using respective ELISA kits (Genetix, India) according to the manufacturer’s instructions.

[00124] vii) Histologic analysis

[00125] Liver and adipose tissues were fixed with formalin and embedded in paraffin and made 3 pm sections and stained using hematoxylin and eosin stain. Collagen accumulation was detected by picrosirus red staining. Lipid accumulation was detected by Oil red O staining.

[00126] viii) Immunohistochemical staining

[00127] Cryosections of liver and adipose tissue sections (3 pm) were deparaffinized, and antigen retrieval was done in Tris-EDTA Buffer (10 mM Tris Base, 1 mM EDTA solution, 0.05% Tween-20, pH 9.0), blocked with normal serum containing 1% BSA, and incubated with primary antibody (1:50) overnight at 4 °C, followed by secondary fluorescent antibody, and incubated for 1 h at room temperature. Slides were washed with PBST solution. Images were captured using confocal microscope (Olympus).

[00128] ix) Real-time PCR

[00129] Total RNA was extracted with Tri reagent (Millipore) and cDNA prepared using akit (Thermo Fisher Scientific, Waltham, USA). qPCR was performed using SYBR Green PCR Master Mix (Thermo Fisher, Waltham, USA) with gene specific primers and the relative gene expression profiles were analyzed by the AACt method using 18S rRNA. GAPDH served as a normalization gene.

[00130] x) Western blot analysis

[00131] Protein extracts were prepared using RIPA-Lysis buffer and the proteins were resolved by 8- 10% SDS-PAGE. The proteins were transferred from the gels onto a nitrocellulose membrane, incubated with specific primary antibodies overnight at 4°C, followed by secondary antibodies for Ih at room temperature. A Luminal/Enhancer solution (Millipore Sigma) was used to detect protein signals using the Chemi Doc XRS⁺ (Bio-Rad).

[00132] xi) Non-Esterified free fatty acid (NEFA) measurement

[00133] NEFA were assayed in tissue homogenates using a commercial kit (Genetix, India) according to the manufacturer’s protocol. [00134] xii) Cell culture:

[00135] HepG2 cells were obtained from ATCC (Manassas, VA) and maintained (37°C, 5% CO2), cultured in DMEM supplemented with 10% FBS. Human Stellate cells (HUCLS) were procured from Lonza, and were grown in Stellate Growth Medium (Lonza).

[00136] xiii) Adipocyte differentiation:

[00137] Mouse-derived pre-adipocyte (3T3-L1, ATCC)) cells were grown in low- glucose DMEM with 10% FBS, L-glutamine (4 mmol/L), penicillin (100 units/mL), and streptomycin (100 pg/mL).

[00138] After two days, the medium was changed to adipocyte-differentiation medium (DIF-001, Reagent 1 and 2, Sigma), which contains high-glucose DMEM, 10% FBS, penicillin- streptomycin, 0.1 pM dexamethasone, and 0.5 mM 3-isobutyl-l- methylxanthine. Cells were incubated in this medium for 24h (for early differentiation), 48h (for mid-differentiation), and 72h (for late differentiation). Following 24h, 48h, and 72h, medium was aspirated and cells were incubated with fresh DMEM with 10% FBS, penicillin-streptomycin containing 10 pg/mL insulin (DIF-001, Reagent 3 Sigma) for another 24h. After this, medium was aspirated and fresh medium was added without the cocktails and were incubated further for 6 days (for early differentiation), 5 days (for mid differentiation), and 4 days (for late differentiation).

[00139] xiv) Cell viability by Trypan blue dye exclusion assay

[00140] At the end of the treatments, cells were mixed with 0.4% trypan blue solution (10 pL) in a 1: 1 ratio and the viability was measured based on the dye exclusion principle using a Countess automated cell counter (Invitrogen).

[00141] xv) Mitochondrial transmembrane potential

[00142] At the end of the treatments, cells were washed free of medium and incubated with 10 pM tetramethylrhodamine ethyl ester (TMRE) in serum-free medium for 30 min and then washed twice with DPBS and fluorescence was measured using Olympus fluorescence microscope equipped with Rhodamine filter.

[00143] xvi) Detection of mitochondrial superoxide

[00144] At the end of the treatments, cells were washed with PBS, added fresh serum free medium and incubated with 10 pM Mito-SOX red for 20 min. Fluorescence images were captured using an Olympus fluorescence microscope equipped with a Rhodamine fdter.

[00145] xvii) Oil red O staining:

[00146] Cells were stained with Oil Red O and evaluated the oil droplets as described previously (32). After capturing the images, isopropanol was added to the dishes and the lipid content was measured using a multimode reader at an absorbance of 490 nm. For in vivo samples, cryosections of 3 pm thickness were stained with Oil Red O as described previously (33). Cryosections were fixed briefly in formaldehyde, dehydrated with isopropanol, stained with Oil Red O solution for 10 minutes, washed with 60% isopropanol, counterstained with hematoxylin, washed with water, and mounted.

[00147] xviii) SIRT1 deacetylase activity

[00148] SIRT1 activity was measured using SIRT1 Activity Assay Kit (CS 1040, Millipore) following the manufacturer’s protocol.

[00149] xix) Transfection:

[00150] HepG2 and 3T3L-1 cells were transfected with AMPK and SIRT1 siRNAs (GeneX, India) using Xfect reagent (Takara Bio, USA) for 16h in antibiotic-free Opti- MEM (GIBCO, USA). Cells were supplemented with fresh DMEM or IBMX (for 3T3L-1 cells) containing DMEM with 10% FBS and were pre-treated with either Mito-Esc or simvastatin or pioglitazone for Ih before the addition of BSA-palmitate for 8h.

[00151] EXAMPLE 3

[00152] Mito-Esc administration dose-dependently improves high fat diet (HFD)- induced gain in body weight and lipid profile in Apoe ^/_ mice.

[00153] Apoe'^/_ mice were initially fed HFD for 16 weeks, and then administered with either Mito-Esc (0.312, 0.625, and 1.25 mg/kg.bd.wt), or simvastatin or pioglitazone (10 mg/kg.bd.wt) for 45 days. The mice were continued on HFD during the aforementioned treatment conditions. Since, HFD is known to cause dys-lipidemia and insulin resistance, simvastatin and pioglitazone were used as positive controls as they are widely used drugs to control dys-lipidemia and insulin resistance respectively. Mito-Esc dose-dependently reduced HFD-induced gain in body weight without affecting the feed intake (Fig. A). However, in comparison to Mito-Esc treated groups, simvastatin and pioglitazone treated groups failed to show these changes. The extent of body weight reduction was observed to be 34%, 57%, and 72% with 0.312, 0.625, and 1.25 mg Mito- Esc/kg.bd.wt respectively compared to HFD alone conditions. Accordingly, epididymal adipose tissue and liver weights in Mito-Esc administered groups were decreased, and were comparable to C57 and Apoe'^/_ mice fed with chow diet groups (Fig. IB and C). Mito-Esc administration significantly improved the lipid profile as well as TG/HDL ratio (Fig. 1D-G). Further, Mito- Esc along with simvastatin and pioglitazone dose-dependently reversed serum indicators of liver damage like aspartate amino transferase (AST) and alanine amino transferase (ALT) enzyme activities (Fig. 1H and I). Low doses of Mito-Esc seem to significantly reverse HFD-induced body weight gain and dyslipidemia.

[00154] EXAMPLE 4

[00155] Mito-Esc administration improves HFD-induced insulin resistance.

[00156] As NAFLD is closely linked to metabolic disorders such as obesity and insulin resistance, the blood glucose levels were measured, which has a direct bearing on insulin sensitivity. HFD- induced increase in fasting and random glucose levels were significantly reduced with all the concentrations of Mito-Esc as well as with pioglitazone (Fig. 2A and B). Although, simvastatin treatment group, compared to HFD alone fed group, showed reduced fasting and random glucose levels, it was statistically insignificant (Fig. 2A and B). Accordingly, the results on oral glucose tolerance test (OGTT) indicated that Mito-Esc administration caused a significant clearance of blood glucose by 2h (Fig. 2C). Interestingly, Mito-Esc administration showed better glucose clearance compared to pioglitazone group (Fig. 2C). Consistent with the above observations, Mito-Esc dose- dependently reduced HFD-induced HbAlc levels (Fig. 2D). Furthermore, the serum insulin levels in Mito-Esc administered groups were marginally, albeit, significantly reduced compared to high-fat diet alone group (Fig. 2E).

[00157] EXAMPLE 5

[00158] Mito-Esculetin administration attenuates high-fat diet induced inflammatory markers.

[00159] The efficacy of Mito-Esculetin was evaluated in reversing obesity-induced inflammatory signaling in Apoe'^/_ mouse serum samples. It was observed that feeding of HFD for 60 days significantly increased the levels of pro-inflammatory cytokines (TNF-a, IL-1, IL-6, IL-17, IL- 23, and MCP-1) compared to the chow diet fed control group (Fig. 3A-F). In contrast, the levels of the above mentioned pro-inflammatory cytokines were markedly decreased in Mito-Esc, simvastatin, and pioglitazone administrated mice groups compared to HFD fed group (Fig. 3 A- F). The levels of CD-45.1, a macrophage differentiation marker, in the peripheral blood was determined. The results indicate that Mito-Esc administration (1.25 mg/kg.bd.wt group) resulted in a significant decrease in CD-45.1 levels in comparison to all the other groups (Fig. 3G).

[00160] EXAMPLE 6

[00161] Mito-Esculetin administration ameliorates HFD-induced adipose tissue hypertrophy and dyslipidemia in Apoe ^/_ mice.

[00162] The weights of epididymal adipose tissue was measured and observed that Mito-Esc administration markedly reduced the HFD-induced epididymal adipose tissue fat mass compared to pioglitazone treated and high fat diet alone fed mice groups (Fig. 4A). H&E staining of adipose tissue sections showed increased size of adipocytes in the HFD group compared to chow diet fed group. Mito-Esc administration dose- dependently inhibited HFD- induced adipocyte hypertrophy. Pioglitazone administration failed to reverse HFD-induced adipocyte hypertrophy. Oil Red O staining showed a significant reduction in lipid accumulation in Mito-Esc administrated groups compared to pioglitazone administrated and HFD control group (Fig. 4B). Simvastatin and pioglitazone administrated groups showed lower adipocyte lipid content compared to HFD control group, the adipocyte lipid content in these groups was significantly higher in comparison to Mito-Esc administered group (Fig. 5C). Mito-Esc administration caused a significant reduction in adipocyte LDLc, HDLc, and non-esterified free fatty acids (NEFA) levels compared to pioglitazone treated and HFD alone fed groups (Fig. 4C-F). Following the above trends, compared to pioglitazone treated and HFD fed alone groups, Mito-Esc and simvastatin administrations showed decreased transcript levels of adipogenic markers like EBP-a, PPAR-y, FABP4, EBP-[3, EBP-5, and SREBP1. The adipogenic markers expression profiles of Mito-Esc administered groups were comparable to C57 mice group fed chow diet.

[00163] The adipose tissue of Mito-Esc administered mice groups showed increased AMPK activation as well as increased phospho-acetyl-CoA carboxylase (ACC) levels compared to HFD alone administered mice group. Mito-Esc administered mice groups showed increased SIRT1 protein and activity with a concomitant decrease in CD36 levels in the adipose tissue (Fig. 4G). CD36 is a receptor that facilitates the uptake of free fatty acids in adipose tissue and liver. In addition, the levels of F4/80, a macrophage marker, was significantly reduced in the adipose tissue of all the treatment groups compared to HFD control mice group (Fig. 4G). The levels of adiponectin, and pro-inflammatory cytokine markers in the adipose tissue were measured. In comparison to HFD control group, while the adiponectin levels were significantly increased in Mito-Esc, simvastatin, and pioglitazone administered mice groups, the levels of resistin, leptin, and pro-inflammatory cytokine markers (TNF-a, IL-1[3, IL-6, IL-17 and IL-23) were significantly inhibited. All these observations suggest that Mito-Esc administration hampers adipose tissue lipid accumulation by regulating the AMPK-SIRT1-CD36 axis, which in turn, may have a role in the inhibition of adipose tissue inflammation.

[00164] EXAMPLE 7

[00165] AMPK activation by Mito-Esc inhibits 3T3L1 preadipocytes differentiation and adipogenesis.

[00166] The effect of Mito-Esc, simvastatin, and pioglitazone on the 3T3-L1 cell viability was measured and found that none of these agents affect the cell viability (24- 72h) in the presence or absence of IB MX differentiation cocktail. The role of Mito-Esc (1.25 & 2.5 pM), simvastatin (10 pM), and pioglitazone (10 pM) in the regulation of early, post-mitotic or intermediate, and terminal differentiation of 3T3L1 preadipocytes was investigated. qPCR was performed to determine the stage-specific markers after the induction of pre-adipocyte differentiation by IBMX. Mito-Esc treatment dose- dependently, as well as simvastatin and pioglitazone, significantly inhibited the transcript levels of all the three stage-specific differentiation markers. IB MX -induced lipid accumulation was decreased with Mito-Esc and simvastatin treatments measured at the end of 8 days by Oil Red O and Nile red staining’s in the early, intermediate, and terminal stage differentiated adipocytes compared to pioglitazone treated cells (Fig. 5E). Accordingly, the levels of triglycerides, non-esterified free fatty acids, and CD36 were significantly decreased in Mito-Esc and simvastatin, treated conditions compared to late differentiated control cells and pioglitazone treated cells (Fig. 5A-B). Since Mito-Esc administration led to an increase in AMPK activation in the adipose tissue, the role of AMPK during Mito-Esc mediated inhibition of pre-adipocyte differentiation was tested. AMPK activation was significantly inhibited at all the stages of adipocyte differentiation compared to undifferentiated cells (Fig. 5C). The SIRT1 levels were markedly decreased during the differentiation process (Fig. 5C). Thereby indicating a crosstalk between AMPK activation and SIRT1 levels during the adipocyte differentiation process. In contrast, Mito-Esc treatment could greatly restore the AMPK activation as well as SIRT1 levels underthese conditions (Fig. 5D). Mito-Esc failed to regulate SIRT1 levels under AMPK depleted conditions (Fig. 5F). The functional relevance of AMPK activation and SIRT1 levels during Mito-Esc mediated inhibition of IB MX-induced pre- adipocyte differentiation was validated. Cells were pre-treated with either siAMPK or siSIRTl and measured the transcript levels of adipogenic markers like SREBP1C, EBP-a, PPAR-y, and CD36 during different stages of differentiation in the presence or absence of Mito-Esc. Mito-Esc treatment failed to regulate the levels of differentiation markers under AMPK or SIRT1 depleted conditions. All these observations suggest that Mito- Esc mediated inhibition adipocyte differentiation process is in part regulated by the activation of AMPK and SIRT1.

[00167] EXAMPLE 8

[00168] Mito-Esculetin administration improves high-fat diet-induced dys- regulation of lipid homeostasis in liver via the AMPK-SIRT-1-CD36 axis.

[00169] The effects of Mito-Esc, simvastatin, and pioglitazone administrations on HFD- induced fatty liver was assessed. Mito-Esc, simvastatin and pioglitazone treated mice groups showed a significant reduction in HFD-induced increase in liver weight (Fig . 6A) . H & E staining of liver sections in HFD control group revealed an aberrant morphology with an increase in the number and size of vacuoles, possibly indicating hepatic steatosis. In contrast, while simvastatin and pioglitazone treatment marginally improved the liver tissue integrity, Mito-Esc administration dose -dependently reversed the above pathological changes and demonstrated normal liver tissue integrity. Similar results were observed with Oil Red O staining wherein; Mito-Esc administration markedly reduced the extent of HFD- mediated increase in lipid accumulation (Fig. 6B). Mito-Esc administration dose- dependently inhibited the HFD-induced infiltration of macrophages into the liver tissue, as evidenced by the decreased levels of F4/80, a macrophage marker, by IHC. Compared to HFD control, although, simvastatin and pioglitazone treatments also reduced the levels of F4/80, the extent of reduction was significantly less compared to Mito-Esc (0.625 and 1.25 mg.kg.bd.wt) administration. Consistent with the above findings, Mito-Esc treatment dose dependently improved the HFD-induced alterations in lipid profile, non-esterified fatty acid levels, and AST and ALT activities in the liver tissue. Mito-Esc administration significantly increased the liver phospho-AMPK (Thr-172), phospho-ACC (Ser-80; inhibitory phosphorylation), SIRT1, and decreased the CD6 levels compared to HFD control group (Fig. 6D). Mito-Esc administration dose-dependently increased the levels of both PPAR-a and PPAR-y. Pioglitazone treatment also showed similar results. All these observations indicate that Mito-Esc elicit similar lipid lowering effects in adipose tissue and liver possibly by regulating the AMPK-SIRT1-CD36 axis during HFD conditions. The overall NAFLD score with different treatment conditions was calculated and presented the same in Fig. 6C.

[00170] The functional relevance of AMPK-SIRT1-CD36 axis in mediating Mito-Esc- mediated improvement in liver lipid homeostasis was investigated in HepG2 cells. Initially, cell viability was measured with BSA-palmitate (200-3000 pM) treated for 24h. Palmitate caused a dose- dependent cell death with an IC50 of -500 pM (Fig. 7A). Hence, 500 pM palmitate was used in all the subsequent experiments for 2-8h. BSA-palmitate time- dependently inhibited the AMPK activation as well as SIRT1 expression in HepG2 cells. On the contrary, Mito-Esc (1.25 pM) treatment time-dependently caused the activation of AMPK as well as increased the SIRT1 expression. Mito-Esc pre-treatment for Ih significantly rescued palmitate-induced inhibition of AMPK activation, ACC phosphorylation (Ser-80; inhibitory phosphorylation) and SIRT1 activity, and decreased the CD36 levels (Fig. 7B, and 7C). Conversely, Mito-Esc treatment failed to reverse the above alterations under AMPK or SIRT1 depleted conditions (Fig. 7D). Depletion of AMPK significantly inhibited the native ACC protein levels also. Next, cells were pre-treated with Mito-Esc for Ih before they were treated with BSA-palmitate for 8h and lipid levels were measured by Nile red staining. Mito-Esc treatment significantly inhibited the palmitate- induced intracellular accumulation of lipid and triglyceride levels (Fig. 7E). However, Mito-Esc treatment failed to regulate the intracellular lipid levels under AMPK or SIRT1 depleted conditions. In addition, Mito-Esc treatment greatly inhibited BSA- palmitate- induced mitochondrial superoxide production as measured by Mito-SOX staining. In tune with these results, Mito-Esc also preserved the palmitate-induced depolarization of mitochondrial membrane potential as measured by TMRE fluorescence. These results indicate that Mito-Esc treatment presumably reverses palmitate-mediated lipotoxicity via the regulation of AMPK-SIRT-1-CD36 axis in HepG2 cells.

[00171] EXAMPLE 9

[00172] Mito-Esc administration inhibits high fat diet-induced liver fibrosis in Apoe ^/_ mice.

[00173] Since, fibrosis is a significant feature of NAFLD progression to NASH, the effect of Mito-Esc, simvastatin, and pioglitazone on HFD-induced fibrosis markers was investigated. Picrosirus red staining revealed excessive accumulation of collagen in the high fat diet fed control group, which was rescued by Mito-Esc treatment in a dose-dependent manner. Simvastatin and pioglitazone treatments had marginal effect on HFD-induced collagen content. Moreover, the expression of a-smooth muscle action (SMA), a marker of fibrogenesis expressed by hepatic stellate cells, was remarkably reduced by Mito-Esc administration in comparison to HFD control group by immunofluorescence, qPCR, and Western blot analysis. Simvastatin and pioglitazone treatments also reduced a-SMA levels compared to HFD control group . Similarly, the transcript levels of the liver fibrosis encoding enzymes and structural proteins like COL1A and collagen-4; collagenases like MMP-1; tissue inhibitor of metalloproteinases like TIMP-1; and TGF-beta were significantly decreased in all the treatment groups compared to HFD control group. The effect of Mito- Esc on TGF-p-induced differentiation in human hepatic stellate cells (HUCLS) was tested. Hepatic stellate cells upon differentiation are activated and attain a fibroblast-like phenotype with collagen deposition. Mito-Esc (1.25-5 pM) treatment greatly inhibited TGF-p-induced HUCLS cells differentiation as visualized by phase contrast microscopy. Also, Mito-Esc treatment significantly reduced the TGF-p-induced transcript levels of hepatic fibrosis markers. All these results suggest that Mito-Esc treatment hampers hepatic stellate cell activation and thereby ameliorates HFD-mediated liver fibrosis.

[00174] EXAMPLE 10:

[00175] Mito-Esc, metformin and their combination showed a significant reduction of cardiac fibrotic makers expression like a-SMA, COL1A1 and COL3A1

[00176] The effect of Mito-Esc and metformin in the delay of age- associated cardiac fibrosis were assessed. The effect of Mito-Esc, metformin and their combination on fibrotic marker like a- SMA, COL1A1 and COL3A1 were evaluated. [00177] The experiments were conducted in 12 months old Apoe-/- mice and divided randomly into 4 groups (n=6 per group). Mice were fed a standard chow diet and administered with 50 mg/kg.bd.wt of metformin, 2.5 mg/kg.bd.wt of Mito-Esc, and their combination 2.5 mg/kg.bd.wt each of Mito-Esc and metformin (made in 0.5% methylcellulose and tween-20) daily for eight weeks by oral gavage. At the end of the experiment, heart tissues were fixed with formalin and embedded in paraffin and made 3 pm sections, stained with Masson’s trichrome stain to detect collagen accumulation as a marker of cardiac fibrosis. It was observed that chronic administration of Mito-Esc, metformin and their combination showed decreased collagen accumulation compared to the vehicle treated Apoe'^/_ aged control.

[00178] The Chronic administration of Mito-Esc, metformin and their combination showed a significant reduction of cardiac fibrotic makers expression like a-SMA, COL1A1 and COL3A1 compared to aged Apoe-/- control heart tissues. *, statistically different at p<0.5 compared to the control group (n=3).

Claims

1. A method of treating a fatty liver disease comprising administering a therapeutically effective amount of a 6,7-dihydroxy coumarin phosphonium amphiphile compound selected from

wherein,

R is hydrogen, deuterium, substituted or unsubstituted Cl -5 alkyl, a substituted or unsubstituted Cl-5 alkoxy, halide, Cl-5 haloalkyl, substituted or unsubstituted C6-10 aryl or C5-10 heteroaryl;

R1 is C6-10 aryl, C5-7 cycloalkyl or a C5-10 heteroaryl; X is a Cl-30 saturated or unsaturated carbon chain, substituted or unsubstituted with Cl-5 alkyl, C2-5 alkenyl or C2-5 alkynyl side chains; or X is

-(CH2)p-R2-(CH2)n-, or

-(CH2)2-R2-(CH2)2-R2-(CH2)m-; wherein p is an integer selected from 2 or 3, n is an integer selected from 3 to 6, and m is an integer selected from 2 to 4; and b is an integer selected from 0 to 2

R2 is O, NH or S.

2. A method of treating a fatty liver disease as claimed in claim 1, wherein the compound is Formula III:

3. A method of treating a fatty liver disease as claimed in claim 1 , wherein, the fatty liver disease resulting from Steatosis, obesity, diabetes, insulin resistance, hypertriglyceridemia, Abetalipoproteinemia, glycogen storage diseases, metabolic syndrome, high fat diet, hepatitis and liver fibrosis.

4. A method of treating a fatty liver disease as claimed in claim 1, wherein the fatty liver disease is selected from one or more conditions from the group of, Non-alcoholic fatty liver disease (NAFLD), Non-alcoholic Steatohepatitis (NASH), Weber-Christian disease, Wolman’s disease, acute fatty liver of pregnancy, and lipodystrophy, adipose tissue hypertrophy.

5. A method of treating a fatty liver disease as claimed in claim 1, wherein the compound of Formula I, II & III activates AMPK, SIRT1, and SIRT3.

6. A method of treating a fatty liver disease as claimed in claim 1 , wherein Formula I, II or III increases the level of phospho-acetyla-CoA carboxylase (ACC).

7. A method of treating a fatty liver disease as claimed in claim 1, wherein Formula I, II or III inhibits 3T3L1 preadipoctyte differentiation and adipogenesis.

8. A method of treating a fatty liver disease as claimed in claim 1, wherein, Formula I, II or III inhibits the CD36 receptor which facilitates the uptake of free fatty acids.

9. A method of treating fatty liver disease as claimed in claim 1, wherein Formula I, II or III results into significant reduction of LDLc, TG/HDLc ratio, and non-esterified free fatty acid (NEFA) levels.

10. A method of treating fatty liver disease as claimed in claim 1, wherein Formula I, II & III decreases the transcript levels of adipogenic markers.

11. A method of treating fatty liver disease as claimed in claim 10 wherein, the adipogenic markers are PPAR-y, FABP4, EBP-P, EBP-5, and SREBP 1.

12. A method of treating fatty liver disease as claimed claim 2, wherein Formula I, II & III inhibits resistin, leptin, and pro-inflammatory cytokine markers

13. A method of treating fatty liver disease as claimed claim 12, wherein, the pro-inflammatory cytokine markers are TNF-a, IL-1, IL-6, IL-17, IL-23, and MCP-1.

14. A method of reducing or reversing the progression of NAFLD into NASH comprising administering a therapeutically effective amount of a 6,7-dihydroxy coumarin phosphonium amphiphile compound selected from

(R Formula I or;

wherein,

R1 is C6-10 aryl, C5-7 cycloalkyl or a C5-10 heteroaryl;

X is a Cl-30 saturated or unsaturated carbon chain, substituted or unsubstituted with Cl-5 alkyl, C2-5 alkenyl or C2-5 alkynyl side chains; or X is

-(CH2)p-R2-(CH2)n-, or

R2 is O, NH or S.

15. A method of reducing or reversing the progression of NAFLD into NASH comprising administering a therapeutically effective amount of a 6,7-dihydroxy coumarin phosphonium amphiphile compound of formula III:

Formula III wherein, Z is selected from a halide, mesylate, tosylate, citrate, tartrate, malate, acetate and trifluoroacetate;

16. A composition comprising, a 6,7-dihydroxy coumarin phosphonium amphiphile use in treating fatty liver disease selected from:

Formula III wherein,

R is hydrogen, deuterium, substituted or unsubstituted Cl-5 alkyl, a substituted or unsubstituted Cl-5 alkoxy, halide, Cl-5 haloalkyl, substituted or unsubstituted C6-10 aryl or C5-10 heteroaryl;

R1 is C6-10 aryl, C5-7 cycloalkyl or a C5-10 heteroaryl;

-(CH2)p-R2-(CH2)n-, or

R2 is O, NH or S.

17. The compound as claimed in claim 16, wherein the compound is Formula III:

Formula III wherein,

Z is selected from a halide, mesylate, tosylate, citrate, tartrate, malate, acetate and trifluoroacetate.

18. A compound as claimed in claim 16, wherein, the fatty liver disease resulting from steatosis, obesity, diabetes, insulin resistance, hypertriglyceridemia, Abetalipoproteinemia, glycogen storage diseases, metabolic syndrome, high fat diet, hepatitis and liver fibrosis.

19. A compound as claimed in claim 16, wherein the fatty liver disease is selected from one or more conditions from the group of, Non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), Weber-Christian disease, Wolmans disease, acute fatty liver of pregnancy, and lipodystrophy, adipose tissue hypertrophy.

20. The compound as claimed in claim 16, wherein the compound wherein Formula I, II or III activates AMPK, SIRT1, and SIRT3.

21. The compound as claimed in claim 16, wherein Formula I, II or Ill increases the level of phospho-acetyl-CoA carboxylase (ACC).

22. The compound as claimed in claim 16, wherein Formula I, II or III inhibits 3T3L1 preadipocyte differentiation and adipogenesis.

23. The compound as claimed in claim 16, wherein, the compound wherein Formula I, II or III inhibits the CD36 receptor which facilitates the uptake of free fatty acids.

24. The compound as claimed in claim 16, wherein the compound Formula I, II or III, - esuits into significant reduction of LDLc, TG/HDLc, and non-esterified free fatty acid (NEFA) levels.

25. The compound as claimed in claim 16, wherein the compound wherein Formula I, II or III, decreases the transcript levels of adipogenic markers.

26. The compound as claimed in claim 25 wherein, adipogenic markers are PPAR-y, FABP4, EBP-p, EBP-5, and SREBP1.

27. The compound as claimed in claiml6, wherein Formula I, II or III inhibits resistin, leptin, and pro-inflammatory cytokine markers.

28. The compound as claimed in claim 27 wherein, the pro-inflammatory cytokine markers are TNF-a, IL-1, IL-6, IL- 17, IL-23, and MCP-1.

29. A composition comprising, a 6,7-dihydroxy coumarin phosphonium amphiphile use in reducing or reversing the progression of NAFLD into NASH selected from:

Formula III wherein,

R1 is C6-10 aryl, C5-7 cycloalkyl or a C5-10 heteroaryl;

-(CH2)p-R2-(CH2)n-, or

R2 is O, NH or S.

30. A compound of formula III for use in reducing or reversing the progression of NAFLD into NASH wherein the compound of Formula III: