Fatty acid compound

Abstract

Furan fatty acid synthesis compounds and furan fatty acid - amino acid conjugates are disclosed herein. Also disclosed herein are methods for treating non - alcoholic steatohepatitis (NASH) in a subject, comprising administering to the subject a therapeutically effective amount of any of the compounds disclosed herein. Also disclosed herein are methods for treating TG - induced pancreatitis and monogenic lipid disorders in a subject, comprising administering to the subject a therapeutically effective amount of any of the compounds disclosed herein.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 355,290, filed Jun. 24, 2022, which is incorporated herein by reference.

[0002] Confirmation of Government Support This invention was made with government support under grant numbers AT009806; DK112854 and GM125944 awarded by the National Institutes of Health. The government has certain rights in this invention.

Background Art

[0003] Background Obesity is known to be a multifactorial disease that affects more than 35% of the world's population and has reached epidemic proportions worldwide. The prevalence of overweight humans in Western countries poses a very high risk of metabolic diseases as well as high healthcare costs. Obesity-related conditions include heart disease, stroke, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis, type 2 diabetes (T2D), and certain types of cancer.

[0004] NAFLD is a chronic, increasingly common, and well-defined liver disorder in adults. NAFLD is ultimately associated with distinct pathophysiological changes that lead to hepatic steatosis, hypertriglyceridemia, and hyperglycemia, such as increased de novo lipogenesis (DNL) and production of very low density lipoprotein (VLDL), decreased hepatic fatty acid (FA) oxidation, and impaired insulin-mediated suppression of hepatic glucose production. All of these factors are known to be components of the metabolic syndrome (MetS) cluster.

[0005] Metabolic syndrome presents a cluster of metabolic deficits associated with insulin resistance, obesity, low-grade inflammation, and dyslipidemia. Atherosclerotic cardiovascular disease (ASCVD) is a major cause of mortality for patients with metabolic syndrome. Diabetic patients with dyslipidemia are significantly at higher risk of ASCVD than diabetic patients without lipoprotein abnormalities. A multi-drug approach (i.e., using specific treatments to normalize each dysregulated physiological risk factor) is generally employed to treat this subset of diabetic patients. Specifically, patients are primarily treated for their dyslipidemia with low-density lipoprotein cholesterol (LDL-C) lowering agents (e.g., statins, ezetimibe, and / or PCSK9 inhibitors) that are on top of the already prescribed antihyperglycemic medications. Clinical trials using diabetic patients (mainly type 2 diabetes) have shown that the reduction of LDL-C is positively correlated with a lower incidence of major adverse cardiac events. However, the benefits of statins are offset by a higher risk of muscle pain and can even lead to the occasional onset of diabetes in patients, resulting in lower maximum tolerated dosages or even discontinuation, and low compliance to treatment. More importantly, significant residual cardiovascular risk persists, particularly in patients with diabetes, even when patients show tolerance to statins for ASCVD treatment.

[0006] Non-alcoholic fatty liver disease (NAFLD) covers a range of liver conditions that affect people who drink little to no alcohol. In subjects with NAFLD, excessive amounts of fat are stored in liver cells. NAFLD is increasing in prevalence worldwide, particularly in Western countries, and is closely associated with obesity. In the United States, it is the most common form of chronic liver disease, affecting an estimated 80 million to 100 million people. NAFLD is particularly prevalent in the 40-60-year age group in subjects who have other co-existing diseases, including obesity and type 2 diabetes, and are at high risk of heart disease. Patients with alcoholic liver disease share a similar set of health problems resulting from fat accumulation caused by alcohol intake that leads to AFLD (alcoholic fatty liver disease).

[0007] NAFLD subjects may or may not exhibit physical symptoms of the disease. Symptoms include an enlarged liver, fatigue, and pain in the upper right abdomen. In the absence of physical symptoms, NAFLD can be diagnosed through biochemical tests, such as liver enzymes and ultrasound elastography. NAFLD can be treated with bariatric surgery, including Roux-en-Y gastric bypass, sleeve gastrectomy, or gastric banding, as these improve steatosis and steatohepatitis.

[0008] Non-alcoholic steatohepatitis (NASH) is a potentially serious form of the disease that includes liver inflammation that can lead to scarring and irreversible damage. In its most severe form, NASH can progress to cirrhosis and liver failure. NASH symptoms can include abdominal swelling, enlarged blood vessels just under the surface of the skin, enlarged breasts in men, an enlarged spleen, red palms, and yellowing of the skin and eyes (jaundice). NASH is also strongly associated with obesity, dyslipidemia, type 2 diabetes, and metabolic syndrome. Currently, there are no NASH-specific treatments approved by the US Food and Drug Administration.

[0009] Accumulating evidence suggests a beneficial effect of fish oil (FO) on weight loss. FO-derived diets provide essential omega-3 (Ω-3) FAs and have been shown to prevent hepatic steatosis, hypertriglyceridemia, and FA synthesis and reduce fat accumulation in human and animal models. FO and Ω-3, along with eicosapentaenoic acid and docosahexaenoic acid and their respective ethyl esters, which have been shown to reduce metabolic syndrome-related events, are an effective treatment for hypertriglyceridemia. Lovaza, an Ω-3 acid ethyl ester formulation TM is currently used to reduce hypertriglyceridemia in humans. Lovaza TM Treated patients have been reported to show high levels of specific metabolites identified as 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) in urine and plasma (Prentice, K. J. et al. CMPF, a Metabolite Formed Upon Prescription Omega-3-Acid Ethyl Ester Supplementation, Prevents and Reverses Steatosis. EBioMedicine 27, 200-213 (2018)).

[0010] In humans, CMPF is produced by the metabolism of furan fatty acids (FuFA), and is thus thought to be urofuranoic acid. FuFA are lipids synthesized by algae and bacteria and are found in plant- and fish-derived products. Nevertheless, it has been demonstrated that fish, rats, and humans are unable to synthesize FuFA de novo. FuFA have anti-inflammatory effects (Chang, F., Hsu, et al., Synthesis of anti-inflammatory furan fattyacids from biomass-derived 5-(chloromethyl)furfural. Sustain. Chem. Pharm. 1,14-18 (2015);Lee, E. S. et al. Potent analgesic and anti-inflammatoryactivities of 1-furan-2-yl-3-pyridin-2-yl-propenone with gastric ulcer sparingeffect. Biol Pharm Bull 29, 361-364 (2006);Tenikoff, D. et al, (Lyprinol(stabilised lipid extract of New Zealand green-lipped mussel): a potentialpreventative treatment modality for inflammatory bowel disease J.Gastroenterol. 361-365 (2005) doi:10.1007 / s00535-005-1551-x;Whitehouse, M. W.I. et al. Anti-inflammatory activity of a lipid fraction (Lyprinol) from the NZgreen-lipped mussel. Inflammooharmacology 5, 237-246 (1997));Wakimoto, T. etal.Furan fatty acid, as an anti-inflammatory component, is known to be derived from the green-lipped mussel Perna canaliculus Toshiyuki. Proc Natl Acad Sci U S A 108, 17533-17537 (2011) doi: 10.1073 / pnas.1110577108.) and to exhibit antioxidant effects (Xu, L. et al. Furan fatty acids - Beneficial or harmful to health? Prog. Lipid Res. 68, 119-137 (2017); Okada, Y. et al. Antioxidant Effect of Naturally Occurring Furan Fatty Acids on Oxidation of Linoleic Acid in Aqueous Dispersion Youji. J. Am. Oil Chem. Soc. 11, 858-862 (1990); Batna, A. & Spiteller, G. Effects of soybean lipoxygenase-1 on phosphatidylcholines containing furan fatty acids. Lipids 29, 397-403 (1994)).

[0011] Dysregulation of lipid metabolism has long been established as a cause of NAFLD and metabolic syndrome. More recently, alterations in amino acid metabolism have emerged as an important component of NASH progression that is shared among other metabolic disorders including cardiovascular disease (CVD). N-acyl amino acids (NAA) (e.g., N-oleoyl leucine or C18:1-Leu), which are composed of fatty acids and amino acids condensed by an amide bond, integrate the signaling of lipids and amino acids and have lower levels in plasma in NASH patients. Supplementation with C18:1-Leu via intraperitoneal administration in mice restored plasma levels and metabolic homeostasis, improved steatosis, and reduced inflammation and fibrosis in mice with established NASH (see PCT / US2021 / 46357). Along with the downregulation of pro-inflammatory / fibrotic pathways in the liver (ccl2, NF-kB inhibition), marked upregulation of the fatty acid β-oxidation (FAO) pathway via PPARα activation contributed to the improvement in NASH. Endogenous NAA have shown promising pharmacological benefits in metabolism and anti-inflammation, but their oral bioavailability is low because intestinal enzymes readily degrade the compounds, which hinders oral administration.

Prior Art Documents

Patent Documents

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Patent Document 1

Non-Patent Documents

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Non-Patent Document 1

Non-Patent Document 2

[0014] Summary

Chemical Formula

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[0015] A method for treating metabolic syndrome, lipid disorder, insulin resistance, inflammation, heart disease, cardiovascular disease (e.g., atherosclerotic cardiovascular disease (ASCVD), stroke, non-alcoholic fatty liver disease (NAFLD), steatosis (e.g., non-alcoholic steatohepatitis), type 2 diabetes (T2D), dyslipidemia, hypertriglyceridemia, immune states and inflammatory states that require a metabolic switch from oxidative phosphorylation to glycolysis in immune cells and inflammatory cells, skin-related lipid diseases, acne, hidradenitis suppurativa, osteoarthritis, Crohn's disease, psoriasis, rheumatoid arthritis, or obesity-related cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of any compound disclosed herein, is also disclosed herein. Other diseases include: metabolic abnormalities that contribute to the clinical findings of the syndrome, such as insulin resistance, glucose intolerance, and dyslipidemia, polycystic ovary syndrome (PCOS), a hormonal disorder that affects women and is characterized by enlarged ovaries containing small cysts. Wilson's disease, a rare hereditary disorder with mutations in copper transport that mainly affects the liver and brain and results in metabolic dysfunction and organ damage. Rheumatoid arthritis (RA), a chronic autoimmune disease characterized by joint inflammation in which macrophages, fibroblast-like synoviocytes, and T cells undergo a metabolic switch with increased dependence on glycolysis for energy production, which contributes to the inflammatory process. Psoriasis, a chronic inflammatory skin condition in which immune cells, including T cells and dendritic cells, undergo a metabolic shift towards glycolysis, which promotes pro-inflammatory cytokine production and disease pathogenesis. Inflammatory bowel diseases, including conditions such as Crohn's disease and ulcerative colitis, characterized by chronic inflammation of the gastrointestinal tract in which immune cells present in the inflamed intestine prefer glycolysis, which promotes inflammation and tissue damage. Systemic lupus erythematosus, a systemic autoimmune disease in which metabolic reprogramming of the immune system leads to increased dependence on glycolysis, which contributes to immune cell activation and tissue damage. Multiple sclerosis, a chronic inflammatory demyelinating disease of the central nervous system, characterized by metabolic reprogramming towards glycolysis in activated immune cells, particularly T cells and microglia, which supports neuroinflammation and neurodegeneration.A similar switch towards glycolysis that is prevented by the compounds disclosed by the inventors is mechanistically important and contributes to diseases including sepsis (macrophages and neutrophils), asthma (eosinophils and T cells), osteoarthritis (immune cells and chondrocytes), chronic obstructive pulmonary disease (COPD) (neutrophils and macrophages), cancer, including breast cancer (epithelial cells), leukemia (blood cells), and glioblastoma (brain cells), non-small cell lung cancer, depends on metabolic switches that support tumor growth, survival, and proliferation, heart failure and myocardial infarction in which cardiomyocytes show a metabolic change from fatty acid oxidation to glycolysis, Hashimoto's disease (rewiring of lymphocytes and macrophages towards glycolysis), chronic kidney disease (proximal tubular cells), Parkinson's disease (dopaminergic neurons in the brain) experience metabolic changes that include a shift towards glycolysis. This metabolic switch is thought to contribute to neuronal dysfunction and neurodegeneration. Amyotrophic lateral sclerosis (ALS) and its motor neurons shift towards glycolysis, mitochondrial dysfunction, scleroderma (metabolic changes in fibroblasts, immune cells, and endothelial cells), ischemic stroke (neurons and glial cells), osteosarcoma (malignant osteoblasts), glioblastoma (cancerous cells increased glycolysis), autoimmune hepatitis (lymphocytes and macrophages), colorectal cancer, primary biliary cholangitis, immune cells, e.g., lymphocytes show metabolic changes that include an increase in glycolysis.

[0016] A method for treating non-alcoholic steatohepatitis (NASH) in a subject, the method comprising administering to the subject a therapeutically effective amount of any compound disclosed herein, is further disclosed herein.

[0017] A method for treating TG-induced pancreatitis and monogenic lipid disorders in a subject, the method comprising administering to the subject a therapeutically effective amount of any compound disclosed herein, is further disclosed herein.

[0018] A method comprising co-administering to a subject any compound disclosed herein and fish oil is also disclosed herein.

[0019] Also disclosed herein are methods comprising co-administering to a subject any compound disclosed herein and omega-3.

[0020] Also disclosed herein are methods comprising co-administering to a subject any compound disclosed herein, fish oil, and omega-3.

[0021] The foregoing will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings.

Brief Description of the Drawings

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[0023] (Figure 1A) Chemical structures and nomenclature of the most common natural FuFAs. (Figure 1B) NEM-derivatization technique for detection and quantification by liquid chromatography-mass spectrometry (LC-MSMS), and Lovaza TM and various FuFAs detected in and present in fish oil. (Figure 1C) Percentages of detectable FuFAs and metabolites in fish oil, Lovaza TM and human plasma.

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DETAILED DESCRIPTION OF THE INVENTION

[0042] Detailed Description Technical Terms The following explanations of terms and methods are provided to better describe the compounds, compositions, and methods of the present invention and to guide those skilled in the art in the practice of this disclosure. It should also be understood that the technical terms used in this disclosure are for the sole purpose of describing specific embodiments and examples and are not intended to be limiting.

[0043] "Administration," as used herein, includes administration to a subject by another person or self - administration by the subject.

[0044] The term "alkyl" refers to a branched or unbranched saturated hydrocarbon group having 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. A "lower alkyl" group is a branched or unbranched saturated hydrocarbon having 1 to 6 carbon atoms. Preferred alkyl groups have 1 to 4 carbon atoms. The alkyl group can be a "substituted alkyl" in which one or more hydrogen atoms are substituted with substituents such as halogen, cycloalkyl, alkoxy, amino, hydroxyl, aryl, alkenyl or carboxyl. For example, lower alkyl or (C1-C6) alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl or hexyl; (C3-C6) cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; (C3-C6) cycloalkyl(C1-C6) alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl or 2-cyclohexylethyl; (C1-C6) alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy or hexyloxy; (C2-C6) alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl or 5-hexenyl; (C2-C6) alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl or 5-hexynyl; (C1-C6) alkanoyl can be acetyl, propanoyl or butanoyl;A halo(C1-C6)alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl or pentafluoroethyl; a hydroxy(C1-C6)alkyl can be hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl, 1-hydroxyhexyl or 6-hydroxyhexyl; a (C1-C6)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentyloxycarbonyl or hexyloxycarbonyl; a (C1-C6)alkylthio can be methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, pentylthio or hexylthio; a (C2-C6)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy or hexanoyloxy.;

[0045] An "analog" is a molecule that has a different chemical structure from the parent compound, e.g., a homolog (differing by an increase in chemical structure or mass, e.g., a difference in the length of an alkyl chain, or by the inclusion of one or more isotopes), a molecular fragment, a structure with one or more different functional groups, or a change in ionization. An analog is not necessarily synthesized from the parent compound. A derivative is a molecule derived from a basic structure.

[0046] "Animal" refers to a living, multi-cellular vertebrate organism that is a category including, for example, mammals and birds. The term "mammal" includes both humans and non-human mammals. Similarly, the term "subject" includes both humans and non-human subjects that are non-human mammals, birds, and the like. Exemplary non-human mammals include animal models (e.g., mice), non-human primates, companion animals (e.g., dogs and cats), livestock (e.g., pigs, sheep, cows), and non-domesticated animals such as big cats. The term "subject" applies regardless of the stage in the life cycle of the organism. Thus, the term "subject" applies to an organism in utero or in ovo, depending on the organism (i.e., whether the organism is a mammal, a bird such as a domesticated or wild fowl).

[0047] The term "co-administer" or "co-administering" refers to the administration of a compound disclosed herein and at least one other therapeutic agent or treatment within the same general period of time, and does not require administration at exactly the same moment in time (co-administration includes administration at exactly the same moment in time, however). Thus, co-administration can be on the same day or different days, or in the same week or different weeks. In some embodiments, the co-administration of two or more agents or treatments is concurrent. In other embodiments, the first agent / treatment is administered prior to the second agent / treatment. One of ordinary skill in the art understands that the formulations and / or routes of administration of the various agents or treatments used can vary. Appropriate dosages for co-administration can be readily determined by one of ordinary skill in the art. In some embodiments, when agents or treatments are co-administered, each agent or treatment is administered at a lower dosage than the dosage appropriate for their administration alone. Thus, co-administration is particularly desirable in embodiments where co-administration of agents or treatments reduces the required dosage of a potentially harmful (e.g., toxic) agent and / or reduces the frequency of administration of a potentially harmful (e.g., toxic) agent. "Co-administer" or "co-administering" encompasses the administration of two or more active agents to a subject such that both the active agents and / or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition containing two or more active agents therein.

[0048] Cyclic: Substantially indicates a hydrocarbon, a closed-ring compound, or a radical thereof. A cyclic compound or substituent can also contain one or more sites of unsaturation, but does not include aromatic compounds. An example of such a cyclic compound is cyclopentadienone.

[0049] The term "cycloalkyl" refers to a non-aromatic carbon-based ring composed of at least 3 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The term "heterocycloalkyl group" is a cycloalkyl group as defined above in which at least one of the carbon atoms of the ring is replaced by a heteroatom (e.g., but not limited to nitrogen, oxygen, sulfur or phosphorus).

[0050] The term "ester" refers to, for example, a carboxyl group-containing moiety in which hydrogen is replaced by, e.g., a C 1~6 alkyl group ("carboxyl C 1~6 alkyl" or "alkyl ester"), an aryl or aralkyl group ("aryl ester" or "aralkyl ester"), etc. CO2C 1~3 alkyl groups, such as methyl ester (CO2Me), ethyl ester (CO2Et) and propyl ester (CO2Pr), etc. are preferred, and CO2C 1~3 alkyl groups include their reverse esters (e.g., -OCOMe, -OCOEt and -OCOPr).

[0051] "Halo" or "halogen", as used herein, refers to fluoro, chloro, bromo and iodo.

[0052] The term "halogenated alkyl" or "haloalkyl group" refers to an alkyl group in which one or more hydrogen atoms present on those groups are replaced by a halogen (F, Cl, Br, I).

[0053] The term "heterocyclic" refers to a closed-ring compound or a radical thereof as a substituent bonded to another group, particularly another organic group, in which at least one atom in the ring structure is other than carbon and is typically oxygen, sulfur and / or nitrogen.

[0054] The term "N-acyl amino acid (NAA)" refers to a class of compounds in which an acyl group is bonded to an amino acid molecule. In these compounds, the acyl group is linked to the amino group (-NH2) of the amino acid to form an amide bond.

[0055] "Inhibit" refers to inhibiting the full onset of a disease or condition. "Inhibit" also refers to any quantitative or qualitative reduction in biological activity or binding compared to a control.

[0056] The term "subject" includes both human and non-human subjects, including birds and non-human mammals, such as non-human primates, companion animals (e.g., dogs and cats), domestic animals (e.g., pigs, sheep, cows), and non-domesticated animals (e.g., large cats). The term subject applies regardless of the stage in the life cycle of the organism. Thus, the term subject applies to organisms in utero or in ovo, depending on the organism (i.e., whether the organism is a mammal, a bird, e.g., a domesticated or wild fowl).

[0057] "Substituted" or "substitution" refers to the replacement of a hydrogen atom of a molecule or R group by one or more additional R groups. Unless otherwise defined, the terms "optionally substituted" or "optional substituent" as used herein, when referring to a group, refer to a group that may or may not be further substituted with one, two, three, four or more groups, preferably one, two or three groups, more preferably one or two groups. Substituents include, for example, C 1~6 alkyl, C 2~6 alkenyl, C 2~6 alkynyl, C 3~8 cycloalkyl, hydroxyl, oxo, C 1~6 alkoxy, aryloxy, C 1~6 alkoxyaryl, halo, C 1~6 alkylhalo (e.g., CF3 and CHF2), C 1~6Alkoxyhalo (e.g., OCF3 and OCHF2), carboxyl, ester, cyano, nitro, amino, substituted amino, disubstituted amino, acyl, ketone, amide, aminoacyl, substituted amide, disubstituted amide, thiol, alkylthio, thioxo, sulfate, sulfonate, sulfinyl, substituted sulfinyl, sulfonyl, substituted sulfonyl, sulfonamide, substituted sulfonamide, disubstituted sulfonamide, aryl, arC 1~6 Alkyl (arC 1-6 alkyl), heterocyclyl and heteroaryl may be selected, and each alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heterocyclyl and groups containing them may be further substituted as required. In the case of N - heterocycles, substituents as required include C 1~6 Alkyl, i.e., N - C 1~3 Alkyl, more preferably methyl, and in particular, N - methyl may also be included but is not limited thereto.

[0058] "Therapeutically effective amount" refers to the amount of a specified agent that is sufficient to achieve the desired effect in a subject being treated with that agent. Ideally, the therapeutically effective amount of an agent is an amount sufficient to inhibit or treat a disease or condition without causing a substantial cytotoxic effect on the subject. The therapeutically effective amount of an agent depends on the subject being treated, the severity of the affliction, and the method of administration of the therapeutic composition.

[0059] "Treatment" refers to therapeutic intervention that alleviates the signs or symptoms of a disease or pathological condition after the disease or pathological condition has begun to develop. As used herein, when referring to a disease or pathological condition, the term "alleviate" refers to any observable beneficial effect of treatment. Beneficial effects can be demonstrated, for example, by delay in the onset of clinical symptoms of a disease in a susceptible subject, reduction in the severity of some or all of the clinical symptoms of a disease, slower progression of a disease, improvement in the overall health or well-being of a subject, or by other parameters well known in the art that are specific to a particular disease. The phrase "treating a disease" refers, for example, to inhibiting the full development of a disease in a subject at risk of the disease. "Preventive" treatment is treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs thereof, for the purpose of reducing the risk of developing a pathology or condition or attenuating the severity of a pathology or condition.

[0060] A "pharmaceutical composition" is a composition that contains one or more non-toxic pharmaceutically acceptable additives, including carriers, diluents and / or adjuvants, and, optionally, one or more other biologically active components, together with an amount (e.g., unit dosage amount) of one or more of the disclosed compounds. Such pharmaceutical compositions can be prepared by standard pharmaceutical formulation techniques, such as those disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA (19th Edition).

[0061] The term "pharmaceutically acceptable salt or ester" refers to salts or esters prepared by conventional means that include, but are not limited to, salts of inorganic acids and organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid, etc. The "pharmaceutically acceptable salts" of the compounds disclosed herein include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and those formed from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts can be prepared by standard procedures, for example, by reacting the free acid with a suitable organic or inorganic base. Any chemical compound listed herein can alternatively be administered as its pharmaceutically acceptable salt. "Pharmaceutically acceptable salts" also include free acid, base, and zwitterionic forms. A description of suitable pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH (2002). When the compounds disclosed herein contain acidic functional groups such as carboxy groups, suitable pharmaceutically acceptable cationic counterparts for the carboxy groups are well known to those skilled in the art and include alkali, alkaline earth, ammonium, quaternary ammonium cations, etc. Such salts are known to those skilled in the art. For additional examples of "pharmacologically acceptable salts", see Berge et al., J. Pharm. Sci. 66:1 (1977).

[0062] "Pharmaceutically acceptable esters" include esters derived from the compounds described herein that have been modified to include a carboxyl group. An in vivo hydrolysable ester is an ester that is hydrolysed in the body of a human or animal to produce the parent acid or alcohol. Thus, representative esters include amino acid esters (e.g., L-glycil, L-leucyl or L-isoleucyl, or any of the other natural amino acids), esters in which the non-carbonyl portion of the carboxylic acid moiety of the ester group is a straight or branched chain alkyl (e.g., methyl, n-propyl, t-butyl or n-butyl), cycloalkyl, alkoxyalkyl (e.g., methoxymethyl), aralkyl (e.g., benzyl), aryloxyalkyl (e.g., phenoxymethyl), aryl (such as phenyl optionally substituted by, for example, halogen, C 1~4 alkyl or C 1~4 alkoxy), or amino); carboxylic acid esters selected from; sulfonic acid esters, such as alkylsulfonyl or aralkylsulfonyl (e.g., methanesulfonyl). "Pharmaceutically acceptable esters" also include inorganic esters, such as mono-, di- or tri-phosphate esters. In such esters, unless otherwise specified, any alkyl moiety present preferably contains from 1 to 18 carbon atoms, particularly from 1 to 6 carbon atoms, more particularly from 1 to 4 carbon atoms. Any cycloalkyl moiety present in such esters preferably contains from 3 to 6 carbon atoms. Any aryl moiety present in such esters preferably includes a phenyl group optionally substituted as shown in the definition of carbocycylyl above. Thus, pharmaceutically acceptable esters include C1-C 22Fatty acid esters, such as acetyl, t-butyl or long-chain straight or branched unsaturated or omega-6 monounsaturated fatty acids, such as palmoyl, stearoyl, etc. are included. Alternative aryl or heteroaryl esters include benzoyl, pyridylmethyloyl, etc., and any of them can be substituted as defined in the above carbocyclicryl. Additional pharmaceutically acceptable esters include aliphatic L-amino acid esters, such as leucyl, isoleucyl and especially valyl.

[0063] For therapeutic use, the salts of the compounds are salts in which the counterion is a pharmaceutically acceptable salt. However, salts of pharmaceutically unacceptable acids and bases can also be utilized, for example, in the preparation or purification of pharmaceutically acceptable compounds.

[0064] The pharmaceutically acceptable acid and base addition salts described above herein mean that they include therapeutically active non-toxic acid and base addition salt forms that the compound can form. Pharmaceutically acceptable acid addition salts can be readily obtained by treating the base form with an appropriate acid. Appropriate acids include, for example, inorganic acids such as hydrohalic acids such as hydrochloric acid or hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or organic acids such as acetic acid, propanoic acid, hydroxyacetic acid, lactic acid, pyruvic acid, oxalic acid (i.e., ethanedioic acid), malonic acid, succinic acid (i.e., butanedioic acid), maleic acid, fumaric acid, malic acid (i.e., hydroxybutanedioic acid), tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclamic acid, salicylic acid, p-aminosalicylic acid, pamoic acid and the like. Conversely, the salt form can be converted to the free base form by treatment with an appropriate base.

[0065] Compounds containing acidic protons can also be converted into their non-toxic metal or amine addition salt forms by treatment with suitable organic and inorganic bases. Suitable base salt forms include, for example, ammonium salts, alkali and alkaline earth metal salts such as lithium, sodium, potassium, magnesium, calcium salts, salts with organic bases such as benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as arginine, lysine, etc.

[0066] The term "addition salt" as used herein also includes solvates that can be formed by the compounds described herein when used above. Such solvates are, for example, hydrates, alcoholates, etc.

[0067] The term "quaternary amine" as used herein, when used previously, defines a quaternary ammonium salt that can be formed by the reaction of a compound between the basic nitrogen of the compound and a suitable quaternizing agent such as an optionally substituted alkyl halide, aryl halide or arylalkyl halide such as methyl iodide or benzyl iodide. Other reactants having good leaving groups such as alkyl trifluoromethanesulfonate, alkyl methanesulfonate and alkyl p-toluenesulfonate can also be used. Quaternary amines have a positively charged nitrogen. Pharmaceutically acceptable counterions include chloro, bromo, iodo, trifluoroacetate and acetate. The selected counterion can be introduced using an ion exchange resin.

[0068] Prodrugs of the disclosed compounds are also contemplated herein. A prodrug is an active or inactive compound that is chemically modified to the active compound via in vivo physiological actions such as hydrolysis, metabolism, conjugation, etc. after administration of the prodrug to a subject. The term "prodrug" as used throughout this specification refers to pharmacologically acceptable derivatives such as esters, amides and phosphates such that the in vivo biotransformation product resulting from the derivative is an active drug as defined in the compounds described herein. Prodrugs preferably have excellent water solubility, increased bioavailability and are readily metabolized or conjugated in vivo to the active inhibitor. Prodrugs of the compounds described herein can be prepared by modifying the functional groups present in the compounds in such a way that the modification is cleaved, either by conventional manipulation or in vivo, to the parent compound, and these modifications are used to form the active compound inside tissues and cells by conjugation to cellular organic molecules (e.g., coenzyme A, carnitine and glycerol for purposes of providing examples). The appropriateness and techniques involved in the preparation and use of prodrugs are well known to those skilled in the art. For a general discussion of prodrugs involving esters, see Svensson and Tunek, Drug Metabolism Reviews 165 (1988) and Bundgaard, Design of Prodrugs, Elsevier (1985).

[0069] The term "prodrug" is also intended to include any covalently attached carrier that releases the active parent drug of the present invention in vivo when the prodrug is administered to a subject. Prodrugs often have enhanced properties, such as solubility and bioavailability, compared to the active pharmaceutical agent, and thus the compounds disclosed herein can be delivered in prodrug form. Accordingly, prodrugs of the compounds disclosed herein, methods of delivering the prodrugs, and compositions containing such prodrugs are also contemplated. Prodrugs of the disclosed compounds are typically prepared by modifying one or more of the functional groups present in the compound in such a way that the modification is cleaved either by conventional manipulation or in vivo to yield the parent compound. Prodrugs can include compounds having phosphonate, hydroxy, thio, and / or amino groups functionalized with any group that is cleaved in vivo to yield the corresponding amino, hydroxy, thio, and / or phosphonate groups, respectively. Examples of prodrugs can include, without limitation, compounds having acylated amino groups and / or phosphonate ester or phosphonate amide groups.

[0070] Protected derivatives of the disclosed compounds are also contemplated. Various suitable protecting groups for use with the disclosed compounds are disclosed in Greene and Wuts, Protective Groups in Organic Synthesis; 3rd Ed.; John Wiley & Sons, New York, 1999.

[0071] Generally, protecting groups are removed under conditions that do not affect the rest of the molecule. These methods are well known in the art and include, for example, acid hydrolysis, hydrogenolysis, etc. One preferred method involves the cleavage of phosphonate esters under Lewis acidic conditions similar to the TMS-Br mediated ester cleavage to obtain free phosphonates, for example, the removal of phosphonate esters. A second preferred method involves the removal of protecting groups, for example, the removal of benzyl groups by hydrogenolysis using palladium on carbon in a suitable solvent system such as alcohols, acetic acid, etc. or mixtures thereof. t-Butoxy-based groups containing the t-butoxycarbonyl protecting group can be removed using inorganic or organic acids such as HCl or trifluoroacetic acid in a suitable solvent system such as water, dioxane, and / or methylene chloride. Another exemplary protecting group suitable for protecting amino and hydroxy functional groups is trityl. Other conventional protecting groups are known and the appropriate protecting group can be selected by one skilled in the art with reference to Greene and Wuts, Protective Groups in Organic Synthesis; 3rd Ed.; John Wiley & Sons, New York, 1999. When the amine is deprotected, the resulting salt can be easily neutralized to yield the free amine. Similarly, when the acid moiety, for example, the phosphonic acid moiety, is exposed, the compound can be isolated as an acid compound or as its salt.

[0072] Certain examples of the compounds disclosed herein may contain one or more asymmetric centers; thus, the compounds described may exist in different stereoisomeric forms. Accordingly, the compounds and compositions may be provided as individual pure enantiomers or as mixtures of stereoisomers (including racemic mixtures). In certain embodiments, the compounds disclosed herein may be synthesized or purified to be substantially enantiopure, for example, with an enantiomeric excess of 90%, 95%, 97%, or even higher than 99%, for example, in enantiopure form.

[0073] The compounds disclosed herein may have at least one asymmetric or geometric center (e.g., C=C, C=N cis-trans centers). All chiral, diasteromeric, racemic, meso, rotational, and geometric isomers of the structures are contemplated, unless otherwise specified. The compounds may be isolated as a single isomer or as a mixture of isomers. All tautomers of the compounds are also considered to be part of the present disclosure. The compounds disclosed herein may contain deuterium, tritium, 15 N, 13 C, and all other isotopes of the atoms present in the compounds, including but not limited to these.

[0074] Compound Non-natural synthetic fatty acids and their N-amino acid conjugates are disclosed herein. In certain embodiments, the compounds have improved physical / chemical properties compared to natural products. In certain embodiments, the compounds have an improved ADME profile. In certain embodiments, the compounds have improved biological activity and pharmacological effects.

[0075] In certain embodiments, certain moieties (e.g., halogen (e.g., fluoro) group(s), cycloaliphatic group(s), heteroatom(s), hydroxy group(s), oxo group(s), amide group(s), alkyl group(s)) are inserted into or along the fatty acid chain to inhibit beta-oxidation of the compound, allowing for a longer half-life. The increased permeability of these compounds and absorption via the portal vein also enable higher liver deposition, providing stronger organ-specific efficacy, as opposed to the lymphatic mechanism of natural fatty acids. In certain embodiments, the methyl groups at the 3,4-positions of the furan ring in natural products are replaced with moieties (e.g., hydrogen atom(s), fluorine atom(s), nitro, nitroso, halogenated alkyl group(s), alkyl group(s)) so that they do not provide a carboxylic acid functional group upon metabolism, thereby preventing the formation of CMPF-like metabolites that exhibit pancreatic toxicity. In certain embodiments, a heteroatom (e.g., O, N or S) is inserted into the carboxyalkyl chain to impede beta-oxidation of the compound.

[0076] In certain embodiments, a compound, or a pharmaceutically acceptable salt or ester thereof (including amino acid esters),

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

[0077] In certain embodiments, both R groups are not hydrogen.

[0078] In certain embodiments, both R groups are the same.

[0079] In certain embodiments, the R group is methyl, ethyl, propyl, isopropyl, fluorine or chlorine.

[0080] In certain embodiments, n is 1.

[0081] In certain embodiments, R’’’ is hydrogen or an isobutyl group, representing glycine and leucine respectively.

[0082] Other exemplary compounds include fatty acid-amino conjugates, such as

Chemical Structure

[0083] Additional exemplary compounds include

Chemical Structure

Chemical Structure

Chemical Structure

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0084] Exemplary synthetic procedures are shown below:

Chem.

[0085] Pharmaceutical compositions and methods of treatment In certain embodiments, the compounds disclosed herein can be used to treat metabolic syndrome. The compounds disclosed herein provide a novel treatment option for using overall metabolic reprogramming in a subject. For example, the compounds disclosed herein induce metabolic reprogramming from glycolysis to fatty acid oxidation.

[0086] In certain embodiments, the compounds disclosed herein can be used to treat dyslipidemia, insulin resistance, inflammation, heart disease, cardiovascular disease (e.g., atherosclerotic cardiovascular disease (ASCVD), stroke, non-alcoholic fatty liver disease (NAFLD), steatosis (e.g., non-alcoholic steatohepatitis), type 2 diabetes (T2D), dyslipidemia, hypertriglyceridemia, and certain types of obesity-related cancer).

[0087] In certain embodiments, the compounds disclosed herein can be used to treat non-alcoholic fatty liver disease (NAFLD) in a subject.

[0088] In certain embodiments, the compounds disclosed herein can be used to treat fatty liver disease in a subject.

[0089] In certain embodiments, the compounds disclosed herein can be used to treat non-alcoholic steatohepatitis (NASH) in a subject.

[0090] In certain embodiments, the compounds disclosed herein can be used to treat cardiovascular disease, particularly cardiovascular disease associated with other co-occurring underlying metabolic dysfunctions (e.g., diabetes, obesity, and metabolic syndrome).

[0091] In certain embodiments, the compounds disclosed herein can be used to treat those including ischemic stroke, heart failure, and myocardial infarction.

[0092] In certain embodiments, the compounds disclosed herein can be used to treat TG-induced pancreatitis and monogenic dyslipidemias, such as familial chylomicronemia.

[0093] In certain embodiments, the compounds disclosed herein can be used to treat immune disorders or immunometabolic disorders.

[0094] In certain embodiments, the compounds disclosed herein are used to treat primary biliary cholangitis. In certain embodiments, the compounds reduce atherosclerosis by simultaneously managing multiple metabolic risk factors for cardiovascular disease (i.e., elevated TG and cholesterol, insulin resistance, and inflammation).

[0095] In certain embodiments, the compounds disclosed herein activate fatty acid oxidation, reduce circulating TG and cholesterol, normalize fasting glucose levels, and / or reduce plasma pro-inflammatory biomarkers (e.g., TNFα).

[0096] In certain embodiments, the compounds disclosed herein for treating immune and inflammatory states characterized by a metabolic switch from oxidative phosphorylation to glycolysis.

[0097] In certain embodiments, the compounds disclosed herein are used to treat inflammatory and immune skin diseases, particularly acne, hidradenitis suppurativa, psoriasis, and osteoarthritis.

[0098] In certain embodiments, the compounds disclosed herein are used to treat inflammatory and autoimmune diseases including osteoarthritis, Crohn's disease, ulcerative colitis, rheumatoid arthritis, systemic lupus erythematosus, asthma, chronic obstructive pulmonary disease, and chronic kidney disease. In certain embodiments, the compounds disclosed herein are used to treat inflammatory states associated with underlying metabolic disorders including polycystic ovary syndrome (PCOS) and Wilson's disease. In certain embodiments, the compounds disclosed herein are used to treat cancers including leukemia, glioblastoma, breast cancer, pancreatic cancer, non-small cell lung cancer, lymphoma, liver cancer, lung cancer, colorectal cancer, melanoma, kidney cancer, osteosarcoma, and glioblastoma.

[0099] In certain embodiments, the compounds disclosed herein are used to treat neurological disorders including multiple sclerosis, Parkinson's disease, and amyotrophic lateral sclerosis.

[0100] In certain embodiments, the compounds disclosed herein may be an alternative treatment to current omega-3, which is an FDA-approved lipid-lowering treatment.

[0101] In certain embodiments, the above compounds inhibit acetyl-CoA carboxylase and exert a metabolic shift responsible for an anti-lipidosis effect.

[0102] In certain embodiments, the compounds disclosed herein exert an anti-inflammatory effect through acetyl-CoA carboxylase inhibition.

[0103] In certain embodiments, the compounds disclosed herein exert an anti-inflammatory effect through AMPK activation.

[0104] In certain compounds, the compounds disclosed herein exert an anti-inflammatory effect through ATP citrate lyase inhibition.

[0105] In certain embodiments, the above compounds regulate the function and level of FGF21.

[0106] In certain embodiments, the above compounds are used to reprogram metabolism in cancer to kill tumor cells.

[0107] In certain embodiments, the above compounds are anti-inflammatory and modulate immune cell responses, cell activation, and cytokine production.

[0108] In certain embodiments, a subject is in need of, or is recognized as being in need of, treatment for elevated TG, cholesterol, and / or insulin resistance.

[0109] In certain embodiments, the CoA conjugate can inhibit the enzyme lactate dehydrogenase (LDH). Glycolytic metabolism is important for supporting cancer cell growth, and LDH plays an important role in this pathway. Inhibition of LDH is an important target for developing drugs for treating cancer therapeutics.

[0110] The compounds disclosed herein can be co-administered with fish oil and / or omega-3 to enhance their effects.

[0111] In certain embodiments, the compound can be co-administered with an anti-obesity drug (dual glucose-dependent insulinotropic polypeptide (GIP) and GLP-1 receptor agonist [e.g., tirzepatide], an anti-diabetic drug (GLP-1 agonist [e.g., semaglutide], SGLT-2 inhibitor, metformin, thiazolidinedione, DPP-4 inhibitor, etc.); a cholesterol-lowering drug (e.g., statin, PCSK-9 inhibitor, ezetimibe); a fibrate; niacin; or an antihypertensive drug (e.g., ace inhibitor, angiotensin 2 receptor inhibitor, etc.).

[0112] In some embodiments, the methods disclosed herein include administering to a subject in need of treatment a pharmaceutical composition, e.g., a composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of one or more of the compounds disclosed herein. The compounds can be administered orally, parenterally (including subcutaneous injection (SC or depot-SC), intravenous (IV), intramuscular (IM or depot-IM), intrasternal injection or infusion techniques), sublingually, intranasally (inhalation), intrathecally, topically, ophthalmically or rectally. The pharmaceutical composition can be administered in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and / or vehicles. The compounds are preferably formulated into suitable pharmaceutical preparations, such as tablets, capsules or elixirs for oral administration, or sterile solutions or suspensions for parenteral administration. Typically, the compounds are formulated into pharmaceutical compositions using techniques and procedures well known in the art.

[0113] In some embodiments, one or more of the disclosed compounds (including compounds linked to a detectable label or a cargo moiety) are mixed or combined with a suitable pharmaceutically acceptable carrier to prepare a pharmaceutical composition. Pharmaceutically acceptable carriers or vehicles suitable for administration of the compounds provided herein include any such carrier known to be suitable for a particular mode of administration. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st Edition (2005) describes exemplary compositions and formulations suitable for pharmaceutical delivery of the compounds disclosed herein. Further, the compounds can be formulated as the sole pharmaceutically active ingredient in the composition or in combination with other active ingredients.

[0114] Upon mixing or adding a compound(s) to a pharmaceutically acceptable carrier, the resulting mixture can be a solution, suspension, emulsion, etc. Liposomal suspensions can also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. The form of the resulting mixture depends on several factors including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. If the above-mentioned compound exhibits insufficient solubility, methods for solubilization can be used. Such methods are known and include, but are not limited to, the use of co-solvents such as dimethyl sulfoxide (DMSO), surfactants such as Tween®, and dissolution in aqueous sodium bicarbonate. Derivatives of the compound, such as salts or prodrugs, can also be used in formulating an effective pharmaceutical composition. The disclosed compounds can also be prepared using carriers that protect them against rapid elimination from the body, such as time-release formulations or coatings. Such carriers include, but are not limited to, controlled release formulations such as microencapsulation delivery systems.

[0115] The disclosed compounds can be formulated as cyclodextrin inclusion complexes.

[0116] The disclosed compounds and / or compositions can be encapsulated in multiple or single-dose containers. The compounds and / or compositions can also be provided in a kit that includes component parts that can be assembled, for example, for use. For example, one or more of the disclosed compounds can be provided in lyophilized form, and a suitable diluent can be provided as a separate component for combination prior to use. In some examples, the kit can include a disclosed compound and a second therapeutic agent for co-administration. The compound and the second therapeutic agent can be provided as separate component parts. The kit can include multiple containers, each container holding one or more unit doses of the compound. The containers are preferably adapted for the desired mode of administration including, but not limited to, tablets, gel capsules, sustained release capsules, etc. for oral administration; depot products, pre-filled syringes, ampoules, vials, etc. for parenteral administration; and patches, medipads, creams, etc. for topical administration.

[0117] The active compound is contained in a pharmaceutically acceptable carrier in an amount sufficient to produce a therapeutically useful effect without the presence of undesirable side effects on the subject being treated. The therapeutically effective concentration can be determined experimentally by testing the compound in known in vitro and in vivo model systems for the disorder being treated. In some examples, the therapeutically effective amount of the compound is an amount that reduces or alleviates at least one symptom of the disorder for which the compound is administered. Typically, the composition is formulated for single dosage administration. The concentration of the active compound in the pharmaceutical composition depends on the rates of absorption, inactivation, and excretion of the active compound, the dosage schedule, and the amount administered, as well as other factors known to those of skill in the art.

[0118] In some examples, about 1 mg to 4000 mg of the disclosed compound, a mixture of such compounds, or a physiologically acceptable salt or ester thereof is formulated in unit dosage form with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavoring agent, etc. The amount of the active substance in the composition or preparation is an amount such that an appropriate dosage within the indicated range is obtained. The term "unit dosage form" refers to physically discrete units suitable as a single dosage for human subjects and other mammals, each unit containing a predetermined amount of the active material calculated to produce the desired therapeutic effect in association with the appropriate pharmaceutical excipients. In some examples, the composition is formulated in unit dosage form and each dosage contains one or more compounds of about 5 mg to about 4000 mg (e.g., about 1000 mg to about 4000 mg, about 100 mg to 1000 mg, about 10 mg to 100 mg or about 25 mg to 75 mg). In other examples, the unit dosage form contains about 0.1 mg, about 1 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 2000 mg, about 3000 mg, about 4000 mg or more of the disclosed compound(s).

[0119] The disclosed compounds or compositions can be administered as a single dose or divided into several smaller doses administered at intervals of time. The therapeutic compositions can be administered by single-dose delivery, by continuous delivery over an extended period of time, or by a repeated dosing protocol (e.g., by a multi-daily, daily, weekly, or monthly repeated dosing protocol). The exact dosage, timing, and duration of treatment are a function of the disease being treated and can be determined experimentally using known test protocols or by extrapolation from in vivo or in vitro test data. It is understood that the concentration and dosage values can also vary depending on the severity of the condition being alleviated. Further, for a particular subject, the dosing regimen can be adjusted over time according to the individual requirements and the professional judgment of the person administering or supervising the administration of the composition, and it is understood that the concentration ranges set forth herein are exemplary only.

[0120] When administered orally as a suspension, these compositions can be prepared according to techniques well known in the pharmaceutical formulation art and can contain microcrystalline cellulose for bulking, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and a sweetening / flavoring agent. As immediate release tablets, these compositions can contain microcrystalline cellulose, dibasic calcium phosphate, starch, magnesium stearate, and lactose and / or other excipients, binders, fillers, disintegrants, diluents, and lubricants. If oral administration is desired, the compound is typically provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition can also be formulated in combination with an antacid or other such ingredients.

[0121] Oral compositions generally contain an inert diluent or edible carrier and can be compressed into tablets or encapsulated in gelatin capsules. For oral therapeutic administration, the active compound(s) can be incorporated with excipients and used in the form of tablets, capsules or troches. Pharmaceutically compatible binders and adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients or compounds of similar nature: binders (e.g., but not limited to, tragacanth, acacia, corn starch or gelatin); excipients (e.g., microcrystalline cellulose, starch or lactose); disintegrants (e.g., but not limited to, alginic acid and corn starch); lubricants (e.g., but not limited to, magnesium stearate); glidants (e.g., but not limited to, colloidal silicon dioxide); sweetening agents (e.g., sucrose or saccharin); and flavoring and odor-correcting agents (e.g., peppermint, methyl salicylate or fruit flavors).

[0122] When the dosage unit form is a capsule, it can contain, in addition to the materials of the above type, a liquid carrier such as a fatty oil. Further, the dosage unit form can contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugars and other enteric agents. The compounds can also be administered as components of elixirs, suspensions, syrups, wafers, chewing gums, etc. Syrups can contain, in addition to the active compound, sucrose as a sweetening agent, as well as certain preservatives, dyes and colorants, and flavoring agents.

[0123] When administered orally, the compound can be administered in the usual dosage forms for oral administration. These dosage forms include the usual solid unit dosage forms of tablets and capsules, as well as liquid dosage forms such as solutions, suspensions, and elixirs. When solid dosage forms are used, they are preferably of the sustained release type such that the compound need only be administered once or twice a day. In some examples, the oral dosage form is administered to the subject once, twice, three times, four times, or more times a day. In additional examples, the compound can be orally administered to humans in a dosage range of 1 to 100 mg / kg body weight, either as a single dose or divided doses. One exemplary dosage range is 0.1 to 100 mg / kg body weight orally (e.g., 0.5 to 100 mg / kg body weight orally), either as a single dose or divided doses. For oral administration, the composition can be provided in the form of tablets containing from about 1 to 1000 milligrams of the active ingredient, particularly 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, or 1000 milligrams of the active ingredient. However, the specific dosage level and frequency of dosing for any particular patient can vary and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the various factors included in the subject being treated.

[0124] Injectable solutions or suspensions may also be formulated using suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting agents and suspending agents, such as sterile bland fixed oils containing synthetic monoglycerides, diglycerides or triglycerides, and fatty acids containing oleic acid, and phospholipids. Solutions or suspensions for parenteral, intradermal, subcutaneous or topical application may contain any of the following components: sterile diluents, such as water for injection, aqueous sodium chloride solution, fixed oils, naturally occurring vegetable oils, such as sesame oil, coconut oil, peanut oil, cottonseed oil, etc., or synthetic fatty vehicles, such as ethyl oleate, etc., polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol and methylparaben; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetate, citrate and phosphate; and agents for adjusting osmotic pressure, such as sodium chloride and dextrose. Parenteral preparations may be enclosed in ampoules, disposable syringes or multiple-dose vials made of glass, plastic or other suitable materials. Buffers, preservatives, antioxidants, etc. may be incorporated as required.

[0125] When administered intravenously, suitable carriers include physiological saline, phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents (e.g., glucose, polyethylene glycol, polypropylene glycol and mixtures thereof). Liposome suspensions containing tissue-targeted liposomes may also be suitable as pharmaceutically acceptable carriers.

[0126] The compound can be administered parenterally, for example, by IV, IM, depot-IM, SC or depot-SC. When administered parenterally, a therapeutically effective amount of from about 0.1 to about 500 mg / day (e.g., from about 1 mg / day to about 100 mg / day or from about 5 mg / day to about 50 mg / day) can be delivered. If a depot formulation is used once a month or once every two weeks for injection, the dosage can be a monthly dosage of from about 0.1 mg / day to about 100 mg / day, or from about 3 mg to about 3000 mg.

[0127] The compound can also be administered sublingually. When administered sublingually, the compound should be administered 1 to 4 times a day in the amounts described above for IM administration.

[0128] The compound can also be administered intranasally. When administered by this route, suitable dosage forms are nasal sprays or dry powders. The dosage of the compound for intranasal administration is the amount described above for IM administration. When administered by nasal aerosol or inhalation, these compositions can be prepared according to techniques well known in the pharmaceutical formulation art and can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents.

[0129] The compound can be administered intrathecally. When administered by this route, suitable dosage forms can be parenteral dosage forms. The dosage of the compound for intrathecal administration is the amount described above for IM administration.

[0130] The compound can be administered topically. When administered by this route, suitable dosage forms are creams, ointments or patches. When administered topically, exemplary dosages are from about 0.5 mg / day to about 200 mg / day. Since the amount that can be delivered by a patch is limited, two or more patches can be used.

[0131] The compound can be administered rectally by suppository. When administered by suppository, an exemplary therapeutically effective amount can range from about 0.5 mg to about 500 mg. When administered rectally in the form of a suppository, these compositions are prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquefies and / or dissolves in the rectal cavity to release the drug, such as synthetic glyceride esters of cocoa butter, polyethylene glycol.

[0132] As is well known to the administering physician or other clinician skilled in the treatment of retroviral infections, diseases and related disorders, the exact dosage and frequency of administration will depend on the particular compound being administered, the particular condition being treated, the severity of the condition being treated, the age, weight, general physical condition of the particular subject, and other drugs the individual may be taking, as will be apparent to those skilled in the art.

Example

[0133] Compound synthesis

Chemical formula

[0134]

Chemical formula

[0135]

Chemical Structure

[0136]

Chemical Structure

[0137] General experimental procedure for the synthesis of fatty acid - amino acid conjugates To a solution of free fatty acid (1.0 equiv) in DMF, hydroxybenzotriazole hydrate (HOBt, 1.5 equiv) and 1 - ethyl - 3 - (3’ - dimethylaminopropyl)carbodiimide HCl (EDC - HCl, 1.5 equiv) were added at rt. The mixture was stirred at rt for 1 h, then the alkyl ester HCl salt of amino acid (1.5 equiv) and potassium carbonate (3.0 equiv) were added. The mixture was stirred at rt for 16 h, then quenched with water. The mixture was then extracted with ethyl acetate. The organic layer was washed twice with water. The organic layer was then dried, evaporated, and purified by SiO2 column (0 - 30% ethyl acetate in hexane) to obtain the purified alkyl ester of fatty acid - amino acid conjugate. To a solution of the above - mentioned ester in THF / MeOH / H2O (4:1:1), lithium hydroxide (5.0 equiv) was added. The mixture was stirred at rt for 20 h, then quenched with 1N HCl solution. The mixture was extracted with ethyl acetate. The organic layer was then washed twice with water. The organic matter was dried, evaporated to obtain the corresponding fatty acid - amino acid conjugate. This product can be further purified by SiO2 column (0 - 10% MeOH in DCM) if necessary.

[0138] Specific examples of the synthesis of fatty acid - amino acid conjugates

Chemical formula

[0139] [Chemical formula] (2,2-Dimethyloctadecanoyl)leucine To a solution of 2,2-dimethyloctadecanoic acid (148 mg, 0.474 mmol) in DMF (5 mL) were added HOBt (96 mg, 0.71 mmol) and EDC-HCl (136 mg, 0.71 mmol). The mixture was stirred at rt for 1 h, then methyl L-leucine HCl (129 mg, 0.71 mmol) and K2CO3 (196 mg, 1.42 mmol) were added. The reaction was stirred at rt for 16 h and then quenched with water. The mixture was extracted with ethyl acetate and the organic layer was washed twice with water. The organic layer was dried over Na2SO4 and evaporated. The crude product was purified by SiO2 column (0 - 20% ethyl acetate in hexane) to give methyl (2,2-dimethyloctadecanoyl)leucinate (134 mg, 64%) as a white solid. To a solution of methyl (2,2-dimethyloctadecanoyl)leucinate (120 mg, 0.27 mmol) in THF (4 mL), MeOH (1 mL) and H2O (1 mL) was added lithium hydroxide (33 mg, 1.4 mmol). The mixture was stirred at rt for 20 h and then quenched with 1N HCl. The mixture was extracted with ethyl acetate. Then the organic layer was washed twice with water. The organics were dried and evaporated to give (2,2-dimethyloctadecanoyl)leucine (117 mg, 99%). 1H NMR (CDCl3): δ 0.88 (t, J = 7Hz, 3H), 0.96 (m, 6H), 1.18 (d, J = 4Hz, 6H), 1.25 (br, 28H), 1.49 (m, 2H), 1.6 (m, 1H), 1.72 (m, 2H), 4.58 (m, 1H), 5.97 (d, J = 8Hz, 1H); 13C NMR (CDCl3): δ 14.08, 21.87, 22.67, 22.83, 24.77, 24.98, 25.25, 25.28, 29.34, 29.54, 29.62, 29.65, 29.66, 29.69, 30.14, 31.91, 40.94, 41.30, 42.17, 50.96, 176.66, 178.73.

[0140]

Chem.

[0141] General procedure for the synthesis of fatty acid - coenzyme A conjugate To a solution of the fatty acid (1.0 equiv) in DCM, 1,1’-carbonyldiimidazole (CDI, 2.0 equiv) was added at rt. The reaction was stirred at rt until the free acid was completely consumed (ca. 2 h). The mixture was then washed four times with water. The organic layer was dried and evaporated. To the resulting oil in THF, coenzyme A or coenzyme A trilithium salt (0.15 equiv) in 0.1 M sodium bicarbonate solution (ca. 5 mL) was added. The mixture was stirred overnight at rt and then acidified to pH ca. 1 with 1 N HCl solution. The crude fatty acid-CoA conjugate was precipitated by the addition of ethyl acetate. The mixture was then filtered and the solid was washed with acetone and ethyl acetate to afford the corresponding purified fatty acid-CoA conjugate as a white solid.

[0142] Specific example of fatty acid-CoA conjugate synthesis

Chemical formula

[0143] Synthesis of C18:1-2M-Leu(2,2-dimethyloctadec-9-enoyl)leucine) A solution of 7-bromoheptanoic acid (2.08 g, 9.95 mmol) in THF (20 mL) was added with borane-THF solution (1 M, 21 mL, 21 mmol) at 0 °C. The mixture was stirred for 2 h while warming slowly to rt. The reaction mixture was quenched with saturated aqueous NaHCO3 and extracted with ethyl acetate. The organic layer was washed successively with saturated aqueous NaHCO3, saturated aqueous NaCl and H2O. The organic layer was then dried and evaporated to give 7-bromoheptan-1-ol as a colorless oil. To this crude product in dichloromethane (20 mL) were added 1-methylimidazole (2.37 mL, 29.8 mmol), tert-butyldimethylsilyl chloride (1.64 g, 10.9 mmol) and iodine (7.58 g, 29.8 mmol). Upon completion of the reaction, this was quenched with saturated aqueous Na2S2O3 and extracted three times with dichloromethane. The crude product was purified by silica gel column (0 - 5% ethyl acetate in hexane) to give purified ((7-bromoheptyl)oxy)(tert-butyl)dimethylsilane (1.78 g, 58%).

[0144] A solution of ethyl isobutyrate (2.78 mL, 20.7 mmol) in THF (10 mL) was added with lithium diisopropylamide THF solution (2 M, 17 mL, 34 mmol) at -78 °C. The mixture was stirred at -78 °C for 15 min and then at 0 °C for 15 min. The resulting enolate was slowly added at -78 °C to a solution of ((7-bromoheptyl)oxy)(tert-butyl)dimethylsilane (2.13 g, 6.89 mmol) in THF (8 mL). The mixture was warmed to rt over 3 h with stirring. The reaction was then quenched with saturated aqueous NH4Cl and extracted three times with ethyl acetate. The combined organic layers were dried and evaporated in vacuo. The crude product was purified by silica gel column (0 - 5% ethyl acetate in hexane) to give ethyl 9-((tert-butyldimethylsilyl)oxy)-2,2-dimethylnonanoate (1.84 g, 77%).

[0145] A solution of 9-((tert-butyldimethylsilyl)oxy)-2,2-dimethylnonanoate (1.84 g, 5.34 mmol) in THF (20 mL) was added dropwise with tetra-n-butylammonium fluoride THF solution (1 M, 8 mL, 8 mmol) at 0 °C. The desilylation reaction was stirred at rt until completion. The mixture was quenched with saturated aqueous NH4Cl and extracted three times with ethyl acetate. The crude product was purified by silica gel column (0 - 20% ethyl acetate in hexane) to give ethyl 9-hydroxy-2,2-dimethylnonanoate (1.07 g, 87%).

[0146] To a solution of ethyl 9-hydroxy-2,2-dimethylnonanoate (1.07 g, 4.65 mmol) in dichloromethane (20 mL) was added Dess-Martin periodinane (2.96 g, 6.98 mmol) at rt. Upon completion of the oxidation, saturated aqueous Na2S2O3 was added and the resulting mixture was extracted three times with diethyl ether. The combined organic layers were dried and evaporated. The crude product was purified by silica gel column (0 - 5% ethyl acetate in hexane) to give ethyl 2,2-dimethyl-9-oxononanoate (583 mg, 55%).

[0147] Nonyltriphenylphosphonium bromide (1.43 g, 3.05 mmol) was dissolved in DMSO (4 mL) and THF (4 mL). To this solution was slowly added KHMDS THF solution (1 M, 3 mL, 3 mmol) at 0 °C and the mixture was stirred for 30 minutes. The resulting orange ylide solution was slowly added to a solution of ethyl 2,2-dimethyl-9-oxononanoate (583 mg, 2.58 mmol) in THF (4 mL) at -78 °C. The mixture was slowly warmed to 0 °C with stirring. When the aldehyde was completely consumed based on TLC, the reaction mixture was quenched with ice-cold aqueous NH4Cl and extracted three times with ethyl acetate. The combined organic layers were dried and evaporated. The crude product was purified by silica gel column (0 - 5% ethyl acetate in hexane) to give the desired ethyl 2,2-dimethyloctadeca-9-enoate (475 mg, 55%).

[0148] To a solution of ethyl 2,2-dimethyloctadec-9-enoate (600 mg, 1.78 mmol) in THF (8 mL), MeOH (2 mL) and H2O (2 mL) was added lithium hydroxide (426 mg, 17.8 mmol). The mixture was stirred at 55 °C for 48 h. The reaction was then acidified to pH = 1 with 1 M aqueous HCl and extracted three times with ethyl acetate. The combined organic layers were dried and evaporated. The crude product was purified by silica gel column (0 - 20% ethyl acetate in hexane) to give the desired 2,2-dimethyloctadec-9-enoic acid or C18:1-2M (538 mg, 98%) as a colorless oil.

[0149] To a solution of C18:1-2M (82 mg, 0.26 mmol) in DMF (2 mL) were added HOBt (54 mg, 0.40 mmol) and EDC hydrochloride (76 mg, 0.40 mmol) at rt. The mixture was stirred for 1 h until C18:1-2M was consumed based on TLC. Then to this solution were added L-leucine methyl ester hydrochloride (72 mg, 0.40 mmol) and potassium carbonate (110 mg, 0.797 mmol) at rt. The reaction was stirred for 24 h and then quenched with aqueous HCl (pH = 1). The resulting mixture was extracted three times with ethyl acetate. The combined organic layers were dried and evaporated. The crude product was purified by silica gel column (0 - 20% ethyl acetate in hexane) to give ethyl (2,2-dimethyloctadec-9-enoyl)leucinate (85 mg, 75%).

[0150] To a solution of ethyl (2,2-dimethyloctadec-9-enoyl)leucinate (230 mg, 0.525 mmol) in THF (4 mL), H2O (1 mL) and MeOH (1 mL) was added LiOH (63 mg, 2.6 mmol) at rt. The reaction was stirred at rt for 2 h and then quenched with aqueous HCl (pH = 1). The mixture was extracted three times with ethyl acetate. The combined organic layers were dried and evaporated. The crude product was purified by silica gel column (0 - 10% MeOH in DCM) to give (2,2-dimethyloctadec-9-enoyl)leucine (210 mg, 94%).

[0151] Experimental procedure Detection of FuFA by NEM derivatization Detection, characterization, and quantification of FuFA in biological samples were performed during derivatization of the furan ring with 100 mM N-ethylmaleimide (NEM) (Sigma-Aldrich) for 30 minutes at 37 °C to form their respective Diels-Alder adducts. These adducts were specifically detected by monitoring the loss of NEM (m / z: 125) during the ionization process upon CID in a triple quadrupole mass spectrometer.

[0152] 11U3-2M chemical synthesis An example of the synthetic scheme for preparing 11U3-2M is shown in Figure 8.

[0153] Animals and experimental design Male C57BL / 6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Mice were acclimated for one week and then randomized to receive either a low-fat diet (LFD) or a high-fat diet (HFD) purchased from Research Diets (New Brunswick, NJ, USA). Obesity was induced by HFD (D12492, having 60% adjusted calories from fat) starting at 8 weeks of age for 16 weeks. Age-matched controls were maintained on LFD (D12450J matching the sucrose level of D12492). While maintaining the diet, mice on HFD were divided into four groups (n = 8 per group) and treated every other day for 7 weeks by oral gavage with 100 μl of 11D3 (50 mg / kg), 11U3 (50 mg / kg) or 11U3-2M (50 mg / kg) formulated in a 2:1 polyethylene glycol (Fisher Scientific, PEG400 #p167-1) and saline solution containing 1.5% octanoic acid (TCI, #O0027). The control group received 100 μl of PEG400 / saline / octanoic acid as vehicle (Veh). LFD mice were divided into two groups (n = 8 per group) and treated with veh or 11D3 (50 mg / kg) to detect any effect of native FuFA in LFD. Mice were grouped to start with the same body weight average before starting the treatment. During and prior to the gavage treatment, mice were fed ad libitum and given free access to water. Food intake, water consumption and mouse body weight were monitored weekly. At the 20-week (4-week treatment) time point, a glucose tolerance test (GTT) was performed on all cohorts. After a 2-week recovery (22 weeks), whole body composition was measured by EchoMRI TMIt was carried out using -100H. At the end of the 23-week study, the mice were euthanized with isoflurane and then laparotomy was performed. Blood was collected and the mice were perfused with 0.9% NaCl. Organs were harvested, snap-frozen in liquid nitrogen gas and stored at -80 °C for further analysis. The frozen tissue was disrupted into fine, easily recoverable powder using a Cellcrusher (Cell Crusher, Schull, Ireland) tissue grinder using dry ice. For immunohistochemistry, the same liver lobe was fixed for 48 hours in 10% formalin (Fisher Chemical, #SF98-4) for all mice, followed by a 75% ethanol bath and then paraffin embedding.

[0154] A second cohort of mice was planned to confirm the effect of 11U3-2M on mitochondrial and fatty acid (FA) β-oxidation using Oroboros analysis in both the HFD model and the LFD model. Male C57BL / 6J diet-induced obesity (C57BL / 6J DIO, #380050) and male C57BL / 6J diet-induced obesity control mice (C57BL / 6J DIO Control, #380056) were purchased from Jackson Laboratory (Bar Harbor, ME, USA) at 15 weeks of age. The mice were continuously fed either HFD or LFD during transportation and upon receipt. The mice were given food for an additional 4 weeks before starting the treatment. While continuing HFD (n = 8 per group) or LFD (n = 8 per group), the mice were treated with 11U3-2M only (50 mg / kg) or veh using the same formulation as the first cohort for 11 weeks. The mice were fasted for 5 hours and received a single intraperitoneal injection of 50 μl of D31-palmitic acid (Cambridge Isotope, 3 μM, #dlm-215-1) in PEG400 / saline (2:1) and 1.5% octanoic acid solution and were sacrificed 2 hours later to study FA metabolism. Blood and tissues were collected and stored as described above.

[0155] In vivo study GTT was performed on mice that had been fasted for 5 hours. The mice were intraperitoneally injected with 1.5 g / kg of glucose, which was filter-sterilized in 0.9% NaCl. Blood glucose levels were measured using a portable blood glucose meter (Accu-Chek Aviva, Roche Diagnostics, Indianapolis, IN, USA) at 0, 20, 40, 60, 90, and 120 minutes.

[0156] Plasma collection and measurement After collection, red blood cells and plasma were separated by centrifugation at 7500 g for 5 minutes at 4°C, aliquoted, and stored at -80°C. Insulin levels were measured by ELISA (Invitrogen, #EMINS) using 50 μl of plasma according to the manufacturer's instructions. Both aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured using 5 μl of plasma with ELISA (Abcam, #ab263882 and #ab282882, respectively). Metabolic hormones were determined using Luminex xMAP technology (Milliplex mouse metabolic hormone extended panel #MMHE-44K, Millipore, Burlington, MA, USA). The multiplex metabolic hormone plate was measured according to the manufacturer's instructions.

[0157] Pharmacokinetics study Male C57BL / 6J (12-week-old) mice were used to measure the absorption and metabolism of FuFA. In this procedure, 11D3 (50 mg / kg), 11U3 (50 mg / kg), 11U3-2M (50 mg / kg), and D31-palmitate (10 mg / kg) were administered simultaneously to a single animal (cassette dosing). As a result, the relative pharmacokinetics of multiple compounds can be rapidly evaluated using a small number of experimental animals. The compounds were injected intraperitoneally (n = 5) or by gavage (n = 5). At 1 hour, 2 hours, and 4 hours after injection, a small incision was made in the tail vein, and serial blood samples were collected (50 μl per collection) using a microhematocrit capillary tube (FisherBrand, #22-362566). At the 6-hour time point, the mice were anesthetized, and blood was collected from the large vein by bleeding from the terminal blood vessels. The blood samples were processed as described above to collect plasma. The pharmacokinetics (PK) of free FuFA and D31-palmitate and their esters found in complex lipids were evaluated using 10 μl of plasma. Briefly, 20 μl of water was added to 10 μl of plasma and divided into two glass tubes. The sample in one tube was measured directly using GC-MS upon derivatization, and the other sample was hydrolyzed before derivatization and quantification. Each sample was spiked with 500 nM heptadecanoic acid (Sigma, #H3500) and 11U3-CD3 standard. To obtain the free FA fraction, 200 μl of water was added and vortexed immediately, followed by the addition of 500 μl of ethyl acetate (Fisher, #E195-4). The mixture was vortexed and spun at 1500 g for 5 minutes at 4°C. The organic layer containing the free FA was collected and dried. For total FA quantification, basic hydrolysis was performed using 500 μl of 1 M KOH in MeOH and incubated at 60°C for 1 hour. The reaction was quenched by adding 300 μl of 1 M HCl. The mixture was extracted with ethyl acetate (1 mL) and spun at 1500 g for 5 minutes at 4°C. The organic layer containing the total FA was then collected and dried.For MS detection, pentafluorobenzyl bromide (PFB) derivatization was performed using 100 μl of 1% diisopropylethylamine and 100 μl of 2% PFB in acetonitrile (ACN, Fisher #A955), followed by incubation at room temperature (RT) for 30 minutes. The tubes were vortexed, centrifuged at 1500 g for 5 minutes at 4 °C, and dried under nitrogen gas. The samples were resuspended in 100 μl of ACN for GC analysis.

[0158] Immunohistochemistry Fixed liver tissues embedded in paraffin were cut into 4-μm sections. The liver sections were deparaffinized, rehydrated, and stained with hematoxylin-eosin (H&E) for histological evaluation of liver morphology. Quantification and scoring of hepatic steatosis, ballooning, and inflammation were performed blindly by a pathologist based on three parameters - steatosis, inflammation, and hepatocyte degeneration.

[0159] RNA extraction and quantitative real-time PCR Total RNA was extracted using 50 mg of frozen liver powder. The powder was homogenized in 1 mL of TRIzol solution (Invitrogen, #15-596-026) and 200 μl of chloroform, shaken vigorously for 15 seconds, and incubated at RT for 2 - 3 minutes. The lysate was centrifuged at 12,000 g for 15 minutes at 4°C. The upper aqueous phase was transferred into a new ice-cold tube, diluted with 500 μl of ice-cold isopropyl alcohol, and incubated at RT for 10 minutes. The tube was centrifuged at 12,000 g for 10 minutes at 4°C. The RNA pellet was washed twice using ice-cold 75% ethanol and centrifuged at 7,500 g for 5 minutes at 4°C. The RNA pellet was dissolved in RNase-free H2O, and the concentration was measured using a Nanodrop 2000 (Thermo Fisher Scientific). All RNA samples had an absorbance ratio of 260 nm / 280 nm between 1.95 and 2. RNA samples (1 μg) were subjected to reverse transcription using the iScript cDNA Synthesis Kit. The TaqMan Gene Expression Assay-On Demand System (Applied Biosystems, Foster City, CA, USA) was used for gene expression evaluation by quantitative PCR (qPCR) using the comparative Ct method and GAPDH as the housekeeping gene.

[0160] Bioinformatics and biostatistical analysis of RNA sequencing data Transcriptome sequencing was performed on mouse samples from each group in 4 replicates per condition. Quality control was first performed on the raw sequencing data by the tool FastQC. Next, low-quality reads and adapter sequences were trimmed by the tool Trimmomatic. After preprocessing, the remaining reads were aligned to the mouse reference genome mm10 by the STAR aligner. Gene counts per gene per sample were quantified by quantMode GeneCounts supported by the feature - STAR aligner. Gene expression normalization was performed by the R package DESeq2.

[0161] Differential expression analysis was performed based on gene counts by the DESeq2 package, considering gene expression profiles. Five pairwise comparisons were analyzed: LFD+Veh vs LFD+11D3, HFD+Veh vs HFD+11D3, HFD+Veh vs HFD+11U3, HFD+Veh vs HFD+11U3-2M, and LFD+Veh vs HFD+Veh. Differentially expressed genes (DEGs) were defined by an FDR = 5% and an absolute fold change equal to or greater than 1.5. For downstream analysis, Ingenuity Pathways Analysis (IPA) was used to perform pathway enrichment analysis on the DEGs. Significant pathways were defined by an FDR = 5%. The direction of regulation was measured by the Z-score, where a positive Z-score indicates pathway activation and a negative Z-score means inhibition, although some pathways show an unknown direction of regulation or a mix of regulation directions.

[0162] Fluorescent RNA in-situ hybridization Fluorescent RNA in-situ hybridization was performed on tissue sections using the LNA ISH Optimization Kits (Qiagen, #339459) according to the manufacturer's instructions. Briefly, the sectioned slides were deparaffinized and digested at 37°C for 15 minutes by the proteinase K approach. U6, scrambled RNA, and GM15441 (LCD0173184) probes were hybridized at final concentrations of 1 nM, 40 nM, and 40 nM, respectively, followed by stringent washing with serial dilutions of SSC (Sigma, #S6639-1L). After 30 minutes of blocking, the slides were incubated at RT for 60 minutes with anti-DIG-POD (Roche, #11207733910) diluted 1:400 according to the manufacturer's instructions. The TSA-plus Cyanine 3 (Akoya Bioscience, #NEL744001KT) substrate was added to the sections and incubated twice (5 minutes for each incubation) to amplify the fluorescent signal. NucBlue Live ReadyProbes Reagent was finally applied to the slides to facilitate cell nucleus staining. Images were acquired with a fluorescence microscope (Leica, DMi8) using the Ocular Advanced Scientific Camera Control software (Digital Optics Limited).

[0163] Western blot The liver sample (50 mg) was homogenized and vortexed in ice-cold RIPA buffer containing phosphatase (Fisher Scientific, #PIA32957) and protease (Fisher Scientific, #PIA32953) inhibitor cocktails. Protein concentration was determined using a BSA assay (Pierce). The protein lysate was mixed with LDS sample buffer (1:4) and heated at 95 °C for 2 min. The lysate was then loaded onto 10% Tris-Glycine eXtended (BioRad, Criterion #5671034) and electrophoresed. Proteins were electrophoretically transferred to a PVDF membrane (BioRad, #1620177), and the transfer was confirmed by staining the membrane with 0.1% Ponceau S solution (Sigma-Aldrich, #6226-79-5). The membrane was then blocked with 5% non-fat dry milk (LabScientific) or 5% BSA (MilliporeSigma) in TBS-T (Tris-buffered saline + 0.1% Tween® 20 [Thermo Fisher Scientific]) at RT for 90 min. After blocking, the membrane was incubated overnight at 4 °C with the following primary antibodies: anti-acetyl-CoA carboxylase (anti-ACC, Cell Signaling, #3662, 1 / 1000), anti-ATP citrate lyase (anti-ACLY, Cell Signaling, #4332, 1 / 1000), anti-β-actin (Cell Signaling, #4970, 1 / 1000). The blot was rinsed 3 times with TBS-T for 15 min each and incubated at RT for 90 min with the appropriate HRP-conjugated anti-rabbit (Cell Signaling, #7074, 1 / 10000). The membrane was washed 3 times in TBS-T for 15 min each and developed using Western Lightning Plus-ECL, Enhanced Chemiluminescence Substrate kit (Perkin Elmer LLC, #50-904-9323) or SuperSignal TMVisualized with the West Femto Maximum Sensitivity Substrate (Bio-Rad, #34094) and ChemiDoc imager (Bio-Rad). Quantitative concentration measurement analysis was performed using Image Lab software (Bio-Rad).

[0164] Thermal shift assay Proteins were extracted from liver powder (50 mg) using 1 mL of phosphate-buffered saline (PBS - Gibco, Paisley, UK) containing phosphatase (Fisher Scientific, #PIA32957) and protease (Fisher Scientific, #PIA32953) inhibitors. The suspension was vortexed and centrifuged at 16,000 g for 20 minutes at 4 °C, and the supernatant was collected. A 40 μl aliquot was transferred to a MicroAmp 8-tube strip (Applied Biosystems, #43 - 582 - 93) and heated at different temperatures (37; 45; 47; 49; 50.5; 52; 53.5; 55 °C) using a StepOne PCR system (Applied Biosystems). The tubes were centrifuged at 16,000 g for 20 minutes at 4 °C to remove denatured proteins. The supernatant was transferred to a new tube and analyzed by Western blot as described above.

[0165] Mouse primary hepatocyte isolation Primary mouse hepatocytes were isolated from 8-week-old male C57BL / 6J mice from Jackson Laboratories using a two-step collagenase perfusion. The mice were anesthetized using isoflurane (Piramal Critical Care, Andhra Pradesh, India). The fur in the abdominal region was cleaned with 70% alcohol and a midline incision was made to expose the viscera. An i.v. catheter (24 gauge × 3 / 4"; Terumo Medical Corporation, Elkton, MD) was cannulated into the portal vein. The catheter was secured and maintained by a surgical knot using Ethilon nylon monofilament 7-0 (Ethic, Johnson and Johnson, USA). The infusion tubing connected to the pump was inserted into the catheter and the liver was perfused with 50 ml of pre-warmed (40 °C) liver perfusion medium (Gibco, #17701038) supplemented with 2% penicillin-streptomycin (Sigma Chemical Company) at a rate of 5 ml / min. The vena cava was cut immediately after the start of perfusion to allow outflow. The liver was then perfused with 50 ml of pre-warmed (40 °C) liver digestion medium (Gibco, #17703034) at the same rate. During the entire perfusion process, occlusion of the vena cava was performed for a few seconds every 2 minutes to create backpressure and liver swelling to allow for good liver perfusion. At the end of perfusion, the liver was removed and placed in a 100 mm dish filled with 20 ml of cold plating medium composed of William's E medium (WEM) supplemented with primary hepatocyte plating supplement (Gibco, #CM3000) according to the manufacturer's instructions. The digested liver was torn with forceps to obtain a hepatocyte suspension. The hepatocytes were filtered through a 100 µm nylon cell strainer (Falcon, Durham, NC) into a 50 ml centrifuge tube to remove undigested debris. The filtrate was centrifuged at 50 × g for 3 minutes at 4 °C to pellet the hepatocytes. The hepatocytes were washed twice with plating medium and centrifuged to obtain a pellet.The hepatocytes were resuspended again in 20 ml of plating medium, mixed with 20 ml of 40% cold percoll (GE Healthcare, Uppsala, Sweden), and centrifuged at 150×g for 7 minutes at 4°C. The supernatant and dead cells were discarded, the lower phase was washed twice, and centrifuged at 50×g for 3 minutes at 4°C. The hepatocytes were resuspended again in 10 ml of plating medium and counted using a hemocytometer (Fisher Scientific). Cell viability (>90%) was evaluated using trypan blue staining (Gibco, Grand Island, NY). The hepatocytes were seeded at 2.2×10 5 cells / 6 well in collagen-coated plates (Gibco, Grand Island, NY). After a 3-hour attachment period in an incubator at 37°C with 5% CO2, the medium with unattached cells was removed and the cells were washed with PBS (Gibco, Paisley, UK). Fresh maintenance medium composed of WEM supplemented with primary hepatocyte maintenance supplement (Gibco, #CM4000) according to the manufacturer's instructions was added. Hepatocytes are easily distinguishable from other non-parenchymal cell types due to their large size. The hepatocytes were cultured in an incubator at 37°C with 5% CO2 for approximately 16 hours before the experimental procedure.

[0166] HPLC-MS analysis of cholesterol, cholesterol-esters, and triglycerides Cholesterol, total cholesterol - ester (TCE) and triglycerides (TG) were extracted from plasma samples. Briefly, 10 μl of plasma was used for extraction with 200 μl of ethyl acetate in the presence of internal standards for cholesterol (cholesterol - d7; Avanti Polar Lipids, Inc. #700041), 500 nM of TCE (16:0 cholesteryl - d7 ester; Avanti Polar Lipids, Inc. #700149), and 500 pM of TG (triheptadecanoin, Nu - Chek Prep., Inc.), vortexed, and spun at 1,500 g for 5 minutes at 4 °C. For cholesterol and TCE, 30 μl of the supernatant was mixed with 70 μl of ACN and run by LC - MS according to specific MRM transitions (369.3 / 147.3 and 376.3 / 147.3 for cholesterol or cholesteryl ester and d7 - cholesterol or d7 - cholesteryl ester, respectively) after in - source neutral loss of FA from cholesterol or TCE.

[0167] Plasma TG was analyzed by HPLC - HR - MS / MS using a C8 Luna column (2 × 150 mm, 5 μm, Phenomenex) with a mobile phase of acetonitrile / water (9:1, v / v) 0.1% ammonium acetate (solvent A) and isopropanol / acetonitrile 0.1% ammonium acetate (7:3, v / v) (solvent B) at a flow rate of 0.4 ml / min. The gradient program was 35% - 100% of solvent B (0.1 - 10 minutes), 100% of solvent B (10 - 13 minutes), followed by a 4 - minute re - equilibration to the initial conditions. A Q - Exactive hybrid quadrupole - orbitrap mass spectrometer (ThermoFisher) was used in positive mode with the following parameters: auxiliary gas heater temperature 250 °C, capillary temperature 300 °C, sheath gas flow rate 20, auxiliary gas flow rate 20, sweep gas flow rate 0, spray voltage 4 kV, S - lens RF level 60 (%). The continuous mass scan analysis was at a resolution of 17,500 in the range of 700 - 1500 m / z.

[0168] Detection and Analysis of FuFA-CoA by Mass Spectrometry Liver Liver powder (50 mg) was used and dissolved in plastic Eppendorf with 100 μl of milli-Q purified water and vortexed thoroughly. Then, 200 μl of ACN containing 400 nM of n-heptadecanoyl coenzyme A lithium salt as an internal standard (C17-CoA standard, NuCheck) was added, vortexed completely, and centrifuged at 10,000 g for 4 minutes at 4 °C. For protein precipitation, the supernatant was transferred to a new tube, and 20 μl was diluted with 180 μl of ACN / water (4:1 ratio) in a clean mass spectrometry vial for CoA analysis.

[0169] Primary hepatocytes As described above, cells were isolated and seeded in 6-well plates. The cells were treated with 11D3 (50 μM), 11U3 (50 μM) or 11U3-2M (50 μM) complexed with FA-free bovine serum albumin (Sigma, #A7511) for 1 hour. After 1 hour, the cells were washed 3 times with PBS and WEM medium was added to the wells. After fresh medium for 0 minutes, 15 minutes, 45 minutes and 90 minutes, the cells were washed, scraped, collected with PBS and centrifuged at 10,000 g for 4 minutes at 4 °C. PBS was discarded and the pellet was resuspended again in 150 μl of ice-cold methanol (MeOH, LC-MS grade, Alfa Aesar #UN1230) containing C17-CoA standard (400 nM), vortexed and centrifuged at 10,000 g for 4 minutes at 4 °C. The MeOH was transferred to a clean mass spectrometry vial for analysis.

[0170] FuFA-CoA in the liver and primary hepatocytes was started with 5% solvent B (ACN, 0.1% ammonium hydroxide) and 95% solvent A (water + 0.1% ammonium hydroxide) for 30 seconds, followed by a gradient increase from 5 to 65% of solvent B over 6 minutes, and then re-equilibration of the conditions using the system solvent. After chromatographic separation using a 150×2 mm Luna® 5μm C8 100 C8 Å LC column (00F-4040-B0) with a flow rate of 0.35 ml / min of 5% solvent B (ACN, 0.1% ammonium), API 5000 TM was detected using a mass spectrometer. Quantification of the analyte and internal standard peak areas was determined using the Quantitation Wizard in Sciex’s Analyst® software.

[0171] Untargeted / Targeted Metabolic Mass Spectrometry Untargeted Liver powder (50 mg) was resuspended again in 500 μL of H2O and extracted using ethyl acetate (1 mL). The phases were separated by centrifugation at 1500 g for 5 minutes at 4°C, the organic phase was collected and dried under nitrogen gas. For acetonitrile precipitation, 200 μL of ice-cold ACN was added to the homogenized liver tissue in water and vortexed thoroughly. The sample was centrifuged at 1500 g for 5 minutes at 4°C, all phases were removed and dried under nitrogen gas. The dried sample was reconstituted in MeOH and transferred to a clean mass spectrometry vial for analysis.

[0172] Mass spectrometry data were collected using a Q Exactive Orbitrap (ThermoFisher Scientific) in both positive and negative ion modes to obtain high-resolution accurate mass data. Two different chromatography systems were used: the organic phase from the bry and dier extracts was run on a 100×2 mm Luna® 5 μm C18(2) column (Phenomenex, 00D-4252-B0) using a solvent B (90:10 isopropyl alcohol, ACS reagent for HPLC (Honeywell #AH323): ACN, 0.1% formic acid, reagent grade (Sigma #F0507)) and solvent A (70:30 water, Optima TM LC / MS grade (Fisher #W6500): acetonitrile, 0.1% formic acid) gradient from 0 to 100% over 20 minutes, followed by 100% solvent B for 6 minutes, and then a return to 0% solvent B at a flow rate of 0.650 mL / min for 4 minutes. Chromatography for the bry and dier aqueous and acetonitrile precipitate extracts was performed using a solvent D (ACN, 0.1% formic acid) and solvent C (water, 0.1% formic acid) gradient from 10 to 100% over 18 minutes, followed by 100% solvent D for 6 minutes, and then a return to 10% solvent D at a flow rate of 0.650 mL / min for 6 minutes using the same column. Compound identification was carried out using Compound Discoverer 3.2 LCMS analysis software.

[0173] Targeted Ceramide and Carnitine Conjugate Evaluation Ceramides and carnitine conjugates were extracted using ethyl acetate in the presence of a ceramide standard (18:1 / 17:0) (N-heptadecanoyl-D-erythro-sphingosine, Avanti Polar Lipids #860517) and quantified using mass spectrometry.

[0174] Mass spectrometry data were collected using an API 5000 LC / MS / MS in positive ion mode. Chromatography for the organic phase of the blank and dialyzer sample extracts was performed using the same LC column with a 30% solvent B for 30 seconds, followed by a gradient of 30–100% solvent B in solvent A for 9 minutes, followed by 100% solvent B for 3 minutes, and then a return to 0% solvent B at a flow rate of 0.600 mL / min for 3 minutes. Quantification of the analyte and internal standard peak areas was determined using the Quantitation Wizard in Sciex’s Analyst® software.

[0175] Statistical analysis Results are presented as mean ± SEM. Statistical analysis was performed using GraphPad Prism, and the data were analyzed by one-way or two-way analysis of variance (ANOVA) with Holm-Sidak multiple comparison post hoc tests and Student’s t-tests. Normality of the data was measured using four normality tests (D’Agostino-Pearson, Anderson-Darling, Shapiro-Wilk, and Kolmogorov-Smirnov). All results were considered significant at p < 0.05. Statistical significance is shown as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0176] Results Detection and characterization of natural furan fatty acids FuFA can undergo oxidation, reduction, and metabolism, and thus multiple structures can be found in plants and fish, giving rise to various nomenclatures. For example, one of the natural FuFAs (11D3) is named as follows: “11” indicates the number of carbons on the carboxyalkyl chain, “D” indicates two methyl groups bonded at the β-position on the furan ring (one methyl is indicated by “M,” and the absence of a methyl is indicated by “U”), and “3” indicates the number of carbons on the alkyl chain (Figure 1A).

[0177] By using NEM-derivatization, the inventors were able to detect different FuFAs in the plasma of humans treated with FO, Lovaza TM drug application, and Lovaza TM (Figure 1B). Figure 1C shows higher concentrations of FuFAs with two methyl groups, FO, Lovaza TM drug application, and Lovaza TM and shows the percentage of FuFA molecules present in the plasma of humans treated with. 11D3 and 11U3 are two closely similar FuFAs except that 11U3 lacks two methyl groups on the furan ring, which slows its degradation and prevents its metabolism to CMPF.

[0178] The chemical synthesis of dimethyl 11U3 (11U3-2M) increases its stability and reduces its incorporation into fatty acids.

[0179] Structurally engineered 11U3-2M is chemically synthesized to present two methyl groups on the carbon adjacent to the carboxylic acid group. This modification prevents the oxidation and enzymatic metabolism that other native FuFAs (i.e., 11D3) undergo to yield CMPF as the end product (Figure 2A).

[0180] To confirm the increased stability and unique absorption pathway, a cassette pharmacokinetic (PK) study of 11U3-2M was conducted in mice compared to 11D3 and 11U3. All compounds were injected either by gavage (PO) or intraperitoneal injection (IP). D31-palmitic acid was used as the reference FA. Overall, 11D3, 11U3 and D31-palmitic acid were rapidly esterified. In contrast, 11U3-2M was present as the free acid and showed little to no incorporation into phospholipids or triglycerides. Furthermore, as expected, a longer half-life was observed, demonstrating that 11U3-2M had higher oral stability compared to native FuFAs (e.g., 11D3). Long-chain fatty acids are known to be distributed into chylomicrons upon re-esterification via the lymphatics, bypassing the initial hepatic transit via the portal vein. The resistance of 11U3-2M to esterification into TG and the absence of incorporation into chylomicrons indicate that 11U3-2M is absorbed via the hepatic portal vein, resulting in a liver-targeted therapeutic effect (Figure 2B).

[0181] 11U3-2M reverses steatosis and improves glucose clearance and insulin resistance. After the characterization and confirmation that 11U3-2M was more stable and had a longer effect after injection, we tested these three FuFAs (11D3, 11U3 and 11U3-2M) in a NAFLD mouse model (Figure 3A).

[0182] The effects of FuFA treatment on glucose metabolism, insulin resistance, and adiposity were analyzed using the HFD model in C57BL / 6J mice. As shown in Figure 3B, 11U3-2M significantly improved blood glucose clearance measured by GTT after 4 weeks of treatment compared to veh treatment. GTT showed 18% (p<0.01), 26% (p<0.05), and 22% (p<0.01) less glucose at 20, 40, and 60 minutes, respectively, in 11U3-2M vs. veh-treated mice. Fasting blood glucose showed a tendency to decrease in 11U3-2M-treated mice compared to veh, and the area under the curve from 0 to 120 minutes showed a 24% decrease in glucose with 11U3-2M treatment (p<0.05, Figure 3B). No significant differences were observed in 11D3 and 11U3 treatments compared to veh.

[0183] Consistent with glucose metabolism, 11U3-2M-treated mice showed lower plasma insulin levels measured by ELISA compared to veh (68%, p<0.05, Figure 3C). Mouse metabolic hormone multiplex analysis was used to find any hormones correlated with insulin level secretion. Two hormones known to have a role in insulin secretion, C-peptide 2 and gastric inhibitory polypeptide (GIP), were significantly lower in plasma from mice treated with 11U3-2M compared to veh (43%, p<0.01 and 36%, p<0.05, respectively. Figure 3C).

[0184] Mice fed with LFD showed hepatocytes with clear structure without inflammation, while mice in the HFD+veh group showed more severe steatosis with ballooning degeneration of hepatocytes (p<0.0001 for both), which was significantly reduced in mice treated with 11U3-2M (p<0.0001 for both). No difference was observed in 11D3- and 11U3-treated mice. Despite obvious inflammation being detected in the HFD+veh group compared with the LFD group, no difference was observed in the three FuFA-treated groups (Figure 3D). However, multiplex analysis of mouse metabolic hormones showed a significant reduction in plasma levels of AST and TNF-α in the 11U3-2M-treated group (Figure 3D).

[0185] These results indicate a strong effect of 11U3-2M in protecting against hepatic NAFLD with reduced glucose, insulin, and insulin-related hormones, accompanied by the accompanying preservation of liver structure and function.

[0186] 11U3-2M decreases plasma levels of cholesterol-ester and triglyceride and increases metabolic changes with a major effect on sphingolipids and acylcarnitine in the liver. Mass spectrometric analysis of plasma showed significant increases in triglyceride (TC, p<0.01), total cholesterol-ester (TCE, p<0.0001), and total cholesterol (p<0.01) in HFD+veh vs LFD+veh. Both TCE and TG were reduced by 11U3-2M treatment in HFD, with a reduction of TC in 11U3-2M (33%, p<0.01 and 16%, p<0.05, respectively) (Figure 4A), accompanied by the reduction of TC in 11U3-2M compared with the HFD+veh group (Figure 4A).

[0187] Untargeted metabolic analysis of the liver was performed to discover metabolites regulated by FuFA treatment. Notably, most compounds that showed a significant increase on HFD showed a corresponding decrease after 11U3-2M treatment and vice versa (Figure 4B), indicating that 11U3-2M treatment restored the liver to its basal state and recovered homeostasis. This analysis yielded sphingolipids during highly nonpolar organic extraction and acylcarnitine species during more polar ACN precipitation extraction (Figure 4B). Various sphingolipids were confirmed by the presence of fragments at 266.4 m / z for sphinganine, 264.4 m / z for ceramide (Cer) monohexose ceramide (monohexCer), sphingosine and sphingosine 1-phosphate (-1-P), and 184.0 m / z for sphingomyelin (SM). Using this approach, the identification of Cer(18:1 / 23:0) and Cer(18:1 / 25:0) was confirmed, although other possible identifications for these compounds are oxidized Cer(18:1 / 22:1) and Cer(18:1 / 24:1) species, respectively. Acylcarnitine species were confirmed by an 85.0 m / z fragment. Together, these results suggest changes in lipid metabolism in the liver upon administration of 11U3-2M treatment.

[0188] To confirm the changes in sphingolipids seen in the untargeted metabolomics data, the inventors quantified the levels of sphingolipid species identified in the Compound Discoverer analysis. Quantification of ceramide species (Figure 4C) confirmed a significant increase in common ceramides (e.g., Cer(18:1 / 16:0) and Cer(18:1 / 20:0)) in the HFD+veh group vs. LFD+veh and a significant reduction of these by 11U3-2M treatment. Total ceramide levels in each treatment reflected these results (Figure 4C). Interestingly, these effects were similar in SM(18:1 / 16:0), SM(18:1 / 20:0) and SM(18:1 / 24:0), which had not been observed previously (Figure 4C).

[0189] These results provide strong evidence that 11U3-2M alters hepatic metabolism and specifically affects FA metabolism.

[0190] 11U3-2M activates pathways related to fatty acid β-oxidation and PPARα. To understand the effects of FuFA, the inventors proceed to RNA-Sequencing (RNA-Seq) analysis of the liver. The FDR cut-off shows 1047 genes upregulated and 506 genes downregulated between LFD+veh vs HFD+veh, indicating a significant effect of diet on hepatic gene expression and physiology. Only a few genes were up- and downregulated by 11D3 (14 and 3) and 11U3 treatment (35 and 70) compared to HFD+veh. However, a greater number of genes were altered by 11U3-2M (296 and 312 respectively) compared to HFD+veh (Figure 5A).

[0191] By using Ingenuity Pathway Analysis (IPA) on these genes, the inventors observed that FA β-oxidation was the most activated pathway, followed by the mitochondrial L-carnitine shuttle pathway, triacylglycerol breakdown, and PPARα activation. These pathways show a clear increase in FA oxidation mediated by 11U3-2M (Figure 5A). Consistent with these data, the inventors also observed an increase (7-fold, p = 3.93e-22) in the pyruvate dehydrogenase kinase 4 (PDK4) gene between the 11U3-2M+veh group and the HFD+veh group. PDK4 is a contributing factor to the influential shift from glucose as the major energy fuel to FA after PPARα activation and is thus known to increase FA β-oxidation. RT-qPCR analysis confirmed the increase in PDK4 in the 11U3-2M-treated liver (9.3-fold, p<0.0001) (Figure 5C).

[0192] Furthermore, the most significantly upregulated gene was the long non-coding RNA (LncRNA) GM15441 (137-fold increase, p = 4.43e-65). This LncRNA has recently been reported to attenuate hepatic inflammasome activation in response to PPARα agonism and fasting. When GM15441 was confirmed by RT-qPCR, it was increased 320-fold in 11U3-2M compared to HFD+veh (p<0.0001). None of these genes were significantly upregulated by 11D3 and 11U3 treatment. These two highly significant genes modulated by 11U3-2M showed an effect towards increased PPARα-dependent FA β-oxidation (Figure 5C). To detect and localize this LncRNA in the liver, the inventors performed in-situ hybridization for GM15441 in the HFD-treated group. The signal for GM15441 was detected only in the nuclei of cells from 11U3-2M-treated livers (Figure 5D), which confirmed this large increase in GM15441 and its effect on FA β-oxidation mediated by PPARα.

[0193] Interestingly, one of the most significantly downregulated genes was LncRNA GM10804 (one-fifth, p = 9.46e-4). Knockdown of GM10804 has recently been implicated in the suppression of disorders of hepatic glucose and lipid metabolism in diabetes with NAFLD. 39 By RT-qPCR, it was confirmed that GM10804 was decreased to one-third (p<0.05) in 11U3-2M-treated HFD mice compared to HFD+veh (Figures 5B-D).

[0194] 11U3-2M increases the number and complex activity of mitochondria. To confirm the higher FA β-oxidation rate in mice treated with 11U3-2M and investigate mitochondrial function, the inventors used mice from a new cohort under the same diet as the first cohort and a 11U3-2M dosage. At the time of sacrifice, fresh liver sections were isolated and used directly for Oroboros analysis to measure oxygen consumption and mitochondrial complex activity (Figure 6A). The maximal activity of citrate synthase (CSA) indicates the mitochondrial content of the tissue. Measurement of CSA in the inventors' groups showed a significant increase in activity in both the LFD and HFD groups treated with 11U3-2M versus the corresponding veh-treated LFD and HFD groups (27% and 29%, respectively, Figure 6B). Oroboros analysis of mitochondrial flux using two different substrates (palmitoyl carnitine and pyruvate) showed a significantly higher flux in 11U3-2M-treated mice (Figure 6C). This increase could be explained by a larger number of mitochondria in 11U3-2M-treated mice. However, the flux normalized to CSA levels was still significantly increased (32.5% in LFD and 38.3% in HFD compared to the corresponding veh groups), indicating that 11U3-2M treatment increases flux levels independent of the number of mitochondria. This is due to the higher level of FA β-oxidation mediated by 11U3-2M, which increases the levels of NADH, the substrate of complex I in the electron transport system. When pyruvate was used as the substrate, no significant change was obtained (although a trend was observed), indicating that 11U3-2M-treated cells preferred FA β-oxidation over glycolysis. These observations were similar (23% for LFD+11U3-2M and 34% for HFD+11U3-2M in ratio to CSA compared to the veh-treated group), although slightly lower, after the addition of glutamate. The addition of succinate, which indicates maximal mitochondrial respiration, showed no significant difference between conditions (normalized by CSA), meaning that the activity of the succinate dehydrogenase complex was not modified by the inventors' treatment 11U3-2M (Figure 6C).

[0195] Formation of 11U3-2M-CoA adduct and its effect on fatty acid β-oxidation by direct inhibition of ACC and ACLY Significant effects on FA β-oxidation and the PPARα pathway were observed with 11U3-2M treatment. However, we investigated the effect of 11U3-2M on key enzymes via the formation of its CoA adduct in vivo. Quantification of the FuFA-CoA conjugate was performed on liver samples from FuFA-treated mice using mass spectrometry. The ratio of FuFA-CoA to the internal standard C17:0-CoA was calculated. The veh-treated group showed no presence of any FuFA-CoA conjugate. However, we also did not see any 11D3-CoA adduct or 11U3-CoA adduct in the treated groups (below the limit of detection). Nevertheless, 11U3-2M-CoA was significantly elevated (Figure 7A). We also investigated the formation of FuFA-CoA in primary mouse hepatocytes. Freshly isolated hepatocytes were incubated with FuFA for 1 hour and washed with PBS. Cells were then harvested at 0, 15, 45, or 90 minutes. The data showed the formation and metabolism of 11U3-2M-CoA over time, but no significant amounts of 11D3-CoA or 11U3-CoA were detected (Figure 7B).

[0196] To evaluate the effect of 11U3-2M-CoA on key enzymes in the FA synthesis pathway, the inventors proceeded to a thermal shift assay to elucidate the thermal stabilization of ACC and ACLY upon 11U3-2M-CoA binding. One random mouse from the HFD+veh group (#58) and the HFD+11U3-2M group (#69) was used to confirm general stability and determine the optimal temperature. ACC and ACLY showed higher stability at 52, 53.5, and 55 °C in the HFD+11U3-2M treatment compared to the veh treatment (Figure 7C). Next, the complete cohort was exposed to the critical temperatures determined in the first thermal shift assay (45 °C for total protein levels and 52, 53.5, and 55 °C to analyze stability). Mice from the HFD+11U3-2M group showed greatly increased ACC and ACLY stability at higher temperatures, indicating binding between 11U3-2M-CoA and the protein (Figure 7D). These data indicate that 11U3-2M also inhibits key enzymes that regulate FA biosynthesis and metabolism via its stable CoA conjugate.

Table 1

[0197] Fatty acids are enzymatically converted to NAA (N-acyl amino acids, also called fatty acyl amino acid conjugates FA-AA). Leucine and glycine conjugate levels are reduced and protective in metabolic diseases. As part of the present invention, FuFAs or dimethylated fatty acids with increased half-lives were combined with selected Gly and Leu amino acids to achieve additional protection.

[0198] As exemplified in the above table, a library of branched-chain fatty acid-amino acid (FA-AA) conjugates was synthesized. Significantly improved oral bioavailability was observed with dimethylated FA-AA conjugates. The absorption of selected linear vs. branched-chain FA-AA conjugates was compared in cassette PK studies as described in more detail below. A mixture of all FA-AA conjugates (with or without branches) was concomitantly orally administered to mice (n = 4 - 5, 30 mg / kg of each compound). All analogs with a dimethyl group introduced into the fatty acid moiety showed plasma and liver concentrations more than 20-fold higher upon oral administration than their linear counterparts. For example, 11U3-2M-Leu showed higher resistance to metabolism as its plasma concentration was maintained over a longer period than that of the linear counterpart 11U3-Leu of 11U3-2M-Leu (Figures 18A - 18D). The absorption improvement resulting from the branching of the fatty acid was universal across all FA-AA conjugates, independent of the amino acid selection. In contrast, all tested FA-AA conjugates administered intraperitoneally (n = 4 - 5, 30 mg / kg of each compound) reached significant levels in the circulation and bypassed first-pass metabolism. It should be noted that 11U3-2M-Leu still showed significantly higher C max , AUC 0~4 , and slower metabolism. Furthermore, FA-AA conjugates were found to achieve a dual action of slowly hydrolyzing and releasing secondary bioactive fatty acids (e.g., 11U3-2M) into the liver and plasma to protect against metabolic dysfunction.

[0199] Cassette PK experiment Two cassette PK experiments were conducted to compare the pharmacokinetics of dimethylacyl amino acids (acyl-2M-amino acids) with their native unbranched analogs and to evaluate the increased half-life, oral bioavailability, and liver concentration obtained by the present invention (introduction of dimethyl (2M) branching).

[0200] A group of mice (n = 4 per group per time point) was used. Compounds were delivered using a cassette pharmacokinetic design by oral gavage or intraperitoneal injection at a dose of 30 mg / kg. The groups are detailed in Table 1. It should be noted that the methyl ester of C18:0-2M-Gly was abbreviated as C18:0-2M-Gly-Me. Plasma and liver levels were demonstrated using HPLC-mass spectrometry-based determination and are shown in Figures 1 - 4 as relative ratios.

[0201] The following groups were used, and the time points were 0 hours, 1 hour, 2 hours, and 4 hours for intraperitoneal administration and 0 hours, 2 hours, and 4 hours for oral administration. [Table 2]

[0202] Plasma data indicate that the addition of the 2M group to acyl - amino acids makes the compounds orally bioavailable (Figures 26A - 26B). Without the introduced modification, these compounds reach only very low levels in plasma (Figures 26A - 26B).

[0203] Intraperitoneal administration shows that compounds containing the 2M group have a significantly increased half - life in plasma and reach significantly higher levels (Figures 25A - 25B).

[0204] Analysis of liver tissue reflects the effects observed in plasma, and the addition of the 2M group results in increased tissue levels and half - life (Figures 27A - 28B).

[0205] These effects are consistent among groups, and 11U3 - 2M - Gly and 11U3 - 2M - Leu reached the highest levels in plasma and liver under both intraperitoneal and oral administrations.

[0206] Benefits in NASH animals This study defined the protective properties of branched-chain FA-AA conjugates and the enhanced efficacy of 11U3-2M conjugated to leucine (Leu) when orally administered in an established NASH mouse model. C57BL6 mice were fed a NASH diet for 16 weeks and then, while maintaining the NASH diet, were orally administered vehicle (n = 10), C18:1-Leu (n = 10), C18:1-2M-Leu (n = 8), and 11U3-2M-Leu (n = 12) (10 mg / kg / day) for 6 weeks. The mice were sacrificed and organs were collected for further analysis. The inventors found a significant decrease in the liver and liver weight / body weight ratio in the 11U3-2Me-Leu treatment group compared to the vehicle control (Figure 19).

[0207] Furthermore, blood biomarkers related to liver injury, including aspartate transaminase (AST) and alanine transaminase (ALT), also significantly decreased in animals treated with 11U3-2M-Leu, along with cholesterol levels. Plasma AST and ALT also significantly decreased in animals treated with C18:1-2M-Leu (Figure 20).

[0208] Gene expression analysis of animal livers demonstrated that treatment with 11U3-2M-Leu, and to a lesser extent C18:1-2M-Leu, significantly induced the expression of important genes that regulate fatty acid oxidation via the PPARα signaling pathway (Figure 21) and suppressed the expression of genes that regulate lipogenesis (Figure 22), both of which contributed to the attenuation of hepatic steatosis, a feature of NASH.

[0209] Finally, it was found that inflammatory genes, including Ccl2 and Ccl5, which are responsible for monocyte recruitment and liver fibrosis, were significantly reduced in the liver (Figure 23). Together with the downregulation of various fibrosis and cell death genes (e.g., collagen and TGF-β), 11U3-2M-Leu and C18:1-2M-Leu treatments also demonstrated an antifibrotic effect beyond the reduction of hepatic steatosis (Figure 24). Notably, the suppression of hepatic and circulating Ccl2 is beneficial for both NASH and atherosclerosis by preventing monocyte recruitment in atherosclerotic plaques or the liver.

[0210] Considering the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are merely preferred examples of the invention and should not be construed as limiting the scope of the invention.

Claims

【Request Item 1】 【Chemistry 38】 【Chemistry 39】 【Chemistry 40】 【Chemistry 41】 A compound having the structure, or a pharmaceutically acceptable salt or ester thereof, Here, each R is independently hydrogen, C １ -C ６ alkyl, C １ -C ６ haloalkyl or halogen, provided that at least one R is C １ -C ６ alkyl, C １ -C ６ haloalkyl or halogen; X is O, S or NH; 'R is C １ -C ６ alkyl; Z is C １ -C ６ alkyl, C １ -C ６ haloalkyl or halogen; R” is hydrogen or alkyl; R’’’ is the side chain of a natural L-amino acid; n is 1 to 10; m is 0 to 10, a compound, or a pharmaceutically acceptable salt or ester thereof. 【Request Item 2】 【Chemistry 42】 【Chemistry 43】 A compound according to claim 1 having the structure, Here, each R is independently hydrogen, C １ ~C ６ Alkyl, C １ ~C ６ It is a haloalkyl or halogen, wherein at least one R is C １ ~C ６ Alkyl, C １ ~C ６ It is a haloalkyl or halogen; X is O, S or NH; R is C １ ~C ６ It is alkyl; Z is C １ ~C ６ Alkyl, C １ ~C ６ A compound that is a haloalkyl or halogen; R'' is hydrogen or alkyl; and n is 1 to 10.

3. The compound according to claim 1, wherein both R groups are not hydrogen.

4. The compound according to claim 1, wherein both R groups are the same.

5. The compound according to claim 1, wherein each R group is independently methyl, ethyl, propyl, isopropyl, fluorine, or chlorine.

6. The compound according to claim 5, wherein each R group is methyl.

7. The compound according to claim 6, wherein R''' is hydrogen or isobutyl. 【Request Item 8】 【Chemistry 44】 【Chemistry 45】 【Chemistry 46】 A compound having the structure.

9. A pharmaceutical composition comprising the compound described in claim 1 and at least one pharmaceutically acceptable excipient.

10. A composition for treating metabolic syndrome in a subject, comprising the compound described in Claim 1.

11. A composition comprising the compound described in Claim 1, wherein in the subject, dyslipidemia, insulin resistance, inflammation, heart disease, cardiovascular disease (e.g., atherosclerotic cardiovascular disease (ASCVD), stroke), type 2 diabetes (T2D), dyslipidemia, hypertriglyceridemia, immune and inflammatory states requiring metabolic switching from oxidative phosphorylation to glycolysis in immune and inflammatory cells, skin-related lipid disorders, acne, hidradenitis suppurativa, and osteoarthritis. Compositions for treating Crohn's disease, psoriasis, rheumatoid arthritis, obesity-related cancers, ulcerative colitis, rheumatoid arthritis, systemic lupus erythematosus, asthma, chronic obstructive pulmonary disease, chronic kidney disease, polycystic ovary syndrome (PCOS), Wilson's disease, leukemia, glioblastoma, breast cancer, pancreatic cancer, non-small cell lung cancer, lymphoma, liver cancer, lung cancer, colorectal cancer, melanoma, kidney cancer, osteosarcoma, glioblastoma, multiple sclerosis, Parkinson's disease, or amyotrophic lateral sclerosis.

12. A composition for treating non-alcoholic fatty liver disease (NAFLD) in a subject, comprising the compound described in Claim 1.

13. A composition for treating non-alcoholic steatohepatitis (NASH) in a subject, comprising the compound described in Claim 1.

14. A composition comprising the compound described in Claim 1 for treating TG-induced pancreatitis and monogenic dyslipidemia in a subject.

15. A combination for co-administration to a subject, comprising the compound described in claim 1 and at least one of fish oil and omega-3.

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