Compounds effective in treatment of hepatotoxicity and fatty liver diseases and uses thereof

Abstract

The present invention relates to compounds effective in the treatment of hepatotoxicity and fatty liver diseases and uses thereof.

Description

[0001] This application is a divisional application of a Chinese patent application with the application number 201680020530.X, the filing date of September 26, 2016, and the invention title of "Compounds Effective in Treating Hepatotoxicity and Fatty Liver Diseases and Their Uses".

[0002] Related Applications

[0003] This application claims the priority of U.S. Provisional Application No. 62 / 222,959, filed on September 24, 2015, U.S. Provisional Application No. 62 / 257,697, filed on November 19, 2015, and Patent Cooperation Treaty Application No. PCT / CN2016 / 078039, filed on March 31, 2016, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0004] The present invention relates to compounds effective in treating hepatotoxicity and fatty liver diseases and their uses. Background Art

[0005] Damage in an organ may be caused by a toxic agent. For example, therapeutic drugs, when administered in excessive amounts, often cause damage to organs, especially the liver or kidneys. Acetaminophen (also known as Panadol) is also known as paracetamol or N-acetyl-para-aminophenol (APAP), and is the most widely used drug on the market for relieving pain and reducing fever symptoms. There are many cases of drug poisoning or suicide due to improper use of APAP every year, and liver injury caused by APAP is a major cause of serious diseases and death. Alcohols or organic solvents such as carbon tetrachloride (CCl4) may also cause hepatotoxicity. Many clinical studies have confirmed that APAP-induced hepatotoxicity is preventable, and early diagnosis and timely administration of the antidote N-acetylcysteine (NAC) can prevent the occurrence of hepatotoxicity.

[0006] Early detection of overdose of paracetamol is necessary as the best prognosis can be achieved if the antidote is administered within 8 hours of poisoning. Early symptoms of drug poisoning include malaise, nausea and vomiting. However, some patients may not show signs of poisoning in the early stage (stage 1), even if the concentration of paracetamol in their blood reaches the toxic level and their abnormal liver function shows obvious abnormalities. Signs of hepatotoxicity, such as abdominal pain, persistent vomiting, jaundice, pain in the right upper abdomen, usually become obvious 24 - 48 hours (stage 2) after ingestion of a large amount of paracetamol. Serum transaminases usually begin to rise 16 hours after administration, accompanied by clinical symptoms. Stage 3 usually occurs 3 - 4 days after administration, and at this time, it is easy to predict the degree of liver injury and the prognosis. Signs of hepatotoxicity range from mild symptoms of elevated liver function indices (AST > 1,000 IU / L) to severe acute fulminant hepatitis accompanied by metabolic acidosis, jaundice, hyperglycemia, AST > 1,000 IU / L, abnormal coagulation and liver / brain lesions. In severe cases, stage 4 will cause oliguric renal failure or death.

[0007] Some patients with paracetamol poisoning only show mild liver injury but have severe nephrotoxicity, which is mainly caused by the direct metabolism of APAP in P-450s (cytochrome P450s, CYPs) in the renal tubules. However, acute renal failure may also be caused by hepatorenal syndrome caused by acute liver failure, and the fractional excretion of sodium (FeNa) can be used to distinguish primary renal injury (FeNa > 1) from hepatorenal syndrome (FeNa < 1). The formula for calculating FeNa is (urinary sodium concentration ÷ urinary creatinine concentration) ÷ (plasma sodium concentration ÷ plasma creatinine concentration) × 100.

[0008] The peak concentration of acetaminophen in the blood is reached 1-2 hours after oral administration, and the liver eliminates a large amount of acetaminophen. More than 90% of acetaminophen is conjugated with glutathione and sulfate to form non-toxic metabolites, and only less than 5% of acetaminophen is eliminated by different CYPs, including CYP2E1, CYP1A2, and CYP3A4, among which CYP2E1 and CYP1A2 are the main enzymes for metabolism. The metabolite N-acetyl-p-benzoquinoneimine (NAPQI) produced by these enzymes is a very active electrophile. Under normal conditions, NAPQI will immediately react with glutathione in the cell and form a non-toxic thiolate. Excessive acetaminophen causes the consumption rate of glutathione to be greater than its synthesis rate, and when the glutathione content in the cell is lower than 30% of the normal range, NAPQI will bind to macromolecules or nucleic acids containing cysteine and cause liver damage. The results of histochemical staining show that NAPQI will bind to the sulfhydryl group of cysteine and form a covalent bond in the centrilobular region before hepatocyte necrosis occurs.

[0009] Patients with liver disease, alcohol addiction, or who are taking drugs that may induce P450 activity, such as carbamazepine, ethanol, Isoniazid, Phenobarbital (possibly other barbiturates), Phenytoin, Sulfinpyrazone, Sulfonylureas, Rifampin, and Primidone, are a high-risk group for developing severe hepatotoxicity caused by APAP. Moreover, if the patient develops complications such as adult respiratory distress syndrome, cerebral edema, uncontrollable bleeding, infection, or multiple organ dysfunction syndrome (MODS), they may easily die. Taking ethanol as an example, ethanol is mainly eliminated by CYP2E1 in the liver, and its mechanism of APAP poisoning is divided into three stages: In the first stage, ethanol competes with APAP for the CYP2E1 receptor in the liver, and the concentration of NAPQI decreases in this stage. In the second stage, ethanol extends the half-life of CYP2E1 from 7 hours to 37 hours, which increases the content of CYP2E1 in the liver, and the concentration of NAPQ1 will slowly increase in this stage. In the third stage, during alcohol withdrawal, more CYP2E1 will appear in the liver to eliminate acetaminophen, so the toxic metabolites of acetaminophen increase significantly and cause liver damage. Recent studies have shown that diallyl sulfide can effectively prevent hepatotoxicity caused by acetaminophen in mice, further demonstrating that diallyl sulfide can inhibit the activity of CYP2E1. It is speculated that the protective mechanism of diallyl sulfide against acetaminophen-induced hepatotoxicity is achieved by inhibiting the production of the intermediate NAPQI from acetaminophen. Previous studies have shown that through inhibition, the consumption of reduced glutathione in hepatocytes, oxidative activation caused by NAPQI, mitochondrial dysfunction, and DNA damage can be reduced, thereby minimizing acetaminophen-induced liver injury. For example, Panax notoginseng, adenosine, and its derivatives adenosine monophosphate, adenosine diphosphate, and adenosine triphosphate can prevent acetaminophen-induced liver injury through this protective mechanism.

[0010] Fatty liver is considered another factor leading to liver injury. Under normal circumstances, fat accounts for 3% of the liver weight. Clinically, "fatty liver disease (FLD)" refers to fat in the liver exceeding 5% of the liver weight, or more than 10% of liver cells showing vacuolar fat changes in a liver tissue section. According to the cause of the disease, fatty liver can be divided into alcoholic fatty liver diseases (AFLD), non-alcoholic fatty liver diseases (NAFLD), or other fatty liver diseases caused by other factors such as drugs. The pathological features of fatty liver disease are the appearance of fat metamorphosis or steatosis, steatohepatitis or its analogues. Based on the percentage of hepatocytes with steatosis, fatty liver is classified as mild (<33%), moderate (33 - 66%), and severe (>66%). Previously, fatty liver was considered a benign and reversible condition and was thus less seriously regarded, but recent studies have found that it can lead to severe liver fibrosis and cirrhosis, and even liver cancer. With the increase in the obese population, the prevalence of FLD is also increasing.

[0011] In European and American countries, the main cause of liver diseases is chronic excessive alcohol consumption. Therefore, the vast majority of liver diseases are caused by alcohol damage. However, in the past 15 - 20 years, NAFLD has become the leading disease of liver dysfunction in European and American countries. Thaler described NAFLD in 1962. In 1980, Ludwig proposed "Non-alcoholic steatohepatitis (NASH)" from accompanying NAFLD, which he found in a group of obese female patients with diabetes and hyperlipidemia. Thereafter, in 1986, Schaffner again emphasized that NASH plays an important role in the mechanism of inducing fibrosis during the occurrence of NAFLD. It wasn't until 1998 that Day found that 15 - 50% of NASH patients had varying degrees of fibrosis, and thus clinicians began to pay attention to NAFLD. Today, in addition to AFLD, NASH is not only a stage in the natural progression of NAFLD clinically. Due to the existence of NASH, NAFLD is no longer considered a benign liver disease.

[0012] Regarding the mechanism of NAFLD, Day and James proposed the Two-hit hypothesis in the UK based on a large number of clinical studies and animal experiments. Fatty liver appears after the first hit, and steatohepatitis appears after the second hit. The first hit is caused by the excessive accumulation of fat in the liver due to obesity, hyperlipidemia, etc. The second hit is due to the action of oxidative stress and reactive oxygen species (ROS) in mitochondria, leading to lipid peroxidation on the liver cell membrane, the release of pro-inflammatory cytokines and free radicals, and fibrosis caused by the activation of stellate cells, resulting in hepatocyte necrosis. The mechanism of NASH involves the peroxidation of triglycerides, oxidative stress, ROS reaction, an increase in lipid peroxidation in hepatocytes, or an increase in cytokines and liver enzymes, leading to a series of autoimmune interactions.

[0013] The main causes of fatty liver are mainly related to the long-term excessive intake of animal fat, protein, and carbohydrates. Excessive calories are converted into fat and accumulate in the body, leading to obesity and fatty liver. The blood of patients with fatty liver may have normal GOT / GPT values. Therefore, the correct diagnosis of fatty liver must use abdominal ultrasound, and currently, abdominal ultrasound can provide an accuracy of over 97%.

[0014] Currently, there is no ideal drug that can provide specific therapeutic effects for FLD. The benchmark goal of its treatment is to improve potential risk factors or control the development of chronic diseases by using drugs. Generally, it is recommended to treat the symptoms according to the causes of fatty liver. For example, patients with fatty liver caused by overweight should lose weight moderately. Any patient with alcoholic fatty liver needs to abstain from alcohol and adopt a balanced diet to improve the condition. Chemicals or drugs that can damage the liver and cause fatty liver disease due to long-term exposure should be stopped immediately. Fatty liver caused by diseases such as hepatitis C, hyperlipidemia, etc. should be treated by treating the primary disease, such as treating hepatitis C or controlling blood lipids. However, if excessive triglycerides are caused by individual constitutional factors, it is difficult to improve fatty liver disease by losing weight.

[0015] However, existing drugs commonly used clinically to lower serum triglycerides and cholesterol usually come with side effects, such as hepatotoxicity, myopathy such as myalgia, myositis, rhabdomyolysis, etc. Regarding lipid-lowering drugs, muscle toxicity is the most significant side effect. In particular, Statins show the highest incidence of muscle toxicity and are accompanied by the production of fibrates. In addition, lipid-lowering drugs have a "fat driving" effect, which "drives" blood lipids to the liver, where fat has already accumulated. The influx of lipids is difficult to handle, resulting in an excessive accumulation of fat in the liver and making the condition of fatty liver worse. It can be seen that lipid-lowering drugs are not suitable for treating FLD. Summary of the Invention

[0016] On the one hand, the present invention provides a new compound, the structure of which is represented by formula (I) as follows

[0017]

[0018] Wherein,

[0019] L is a saturated or unsaturated aliphatic group;

[0020] R is selected from the group consisting of: hydrogen, a polyol group, and (G) p a sugar group, where G is a monosaccharide residue and p is an integer from 1 to 100, and at least one hydroxyl group in (G) p is substituted by a halogen atom; and

[0021] Q is an integer from 2 to 4, and each R is the same or different,

[0022] or a pharmaceutically acceptable salt thereof.

[0023] In certain specific embodiments, the compound of the present invention is represented by formula (II) as follows:

[0024] R1-O-X-(CH2) m -X-O-R2 Formula (II), wherein,

[0025] X is C=O;

[0026] R1 and R2 are the same or different and are selected from the group consisting of: hydrogen, a polyol group, and (G) p a sugar group, where G is a monosaccharide residue and p is an integer from 1 to 100, and at least one hydroxyl group in (G) p is substituted by a halogen atom, where when R1 is hydrogen, then R2 is not hydrogen; and m is an integer from 1 to 40.

[0027] On the other hand, the present invention provides a pharmaceutical composition comprising at least one compound as described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0028] On the other hand, the present invention provides a method of treatment by administering to an individual in need an effective amount of at least one compound as described herein or a pharmaceutically acceptable salt thereof.

[0029] In certain specific embodiments, the method of the present invention is provided for preventing or treating a disease or disorder characterized by increased cytochrome P450 activity or increased free radical content in an individual in need.

[0030] In certain specific embodiments, the method of the present invention is provided for preventing or treating organ damage in an individual in need.

[0031] In certain specific embodiments, the method of the present invention is provided for preventing or treating hepatotoxicity in an individual in need.

[0032] In certain specific embodiments, the method of the present invention is provided for preventing or treating fatty liver, protecting liver function or improving liver diseases caused by fatty liver or other related disorders.

[0033] On the other hand, the present invention provides the use of a compound as described herein or a pharmaceutically acceptable salt thereof for the preparation of a medicament. Specifically, the medicament can be used for preventing or treating (i) a disease or disorder characterized by increased cytochrome P450 activity or increased free radical content, (ii) organ damage, and / or (iii) hepatotoxicity, and / or (iv) preventing or treating fatty liver, protecting liver function or improving liver diseases caused by fatty liver or other related diseases.

[0034] Details of one or more specific embodiments of the invention are set forth in the description below. Other features or advantages of the invention will be apparent from the detailed description of several specific embodiments which follows and from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The foregoing summary of the invention and the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings presently preferred specific embodiments. It is to be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

[0036] In the drawings:

[0037] Figure 1 Shown is the percentage of prodrug residue or the formation of its related metabolites in blood (in vitro).

[0038] Figure 2 Shown are the plasma concentration-time curves of the prodrug and sucralose after oral administration of the prodrug in SD rats.

[0039] Figure 3 Shown is the plasma concentration-time curve of mannitol after oral administration of the prodrug in SD rats.

[0040] Figure 4 Shown are the H&E staining results of animal liver tissues. (A) Normal control group, (B) Control group for APAP-induced liver injury, (C) Positive control group treated with NAC, (D) Experimental group treated with mannitol (1.67 mg / kg), (E) Experimental group treated with sucralose (1.67 mg / kg), (F) Experimental group treated with mannitol (2.51 mg / kg) plus sucralose (2.51 mg / kg), (G) Experimental group treated with mannitol (3.34 mg / kg) plus sucralose (3.34 mg / kg), and (H) Experimental group treated with a combination of NAC and mannitol (3.34 mg / kg) and sucralose (3.34 mg / kg).

[0041] Figure 5 Shown are liver tissue sections taken from mice that were induced to develop fatty liver and then treated with different test compounds for four weeks.

[0042] Figure 6 Shown is a general way of the synthesis method of the compound of the present invention. Detailed Description of the Invention

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0044] As used herein, the articles "a" and "an" refer to one or more than one (i.e., at least one) of the grammatical object of the article. For example, "a component" refers to one component or more than one component.

[0045] I. Compounds

[0046] In one aspect, the present invention provides new compounds, the structures of which are represented by the following formula (I)

[0047]

[0048] Wherein

[0049] L is a saturated or unsaturated aliphatic group;

[0050] R is selected from the group consisting of: hydrogen, a polyol group, and (G)p a sugar group, wherein G is a monosaccharide residue and p is an integer from 1 to 100, and wherein (G) p at least one hydroxyl group therein is substituted by a halogen atom; and

[0051] Q is an integer from 2 to 4, and each R is the same or different,

[0052] or a pharmaceutically acceptable salt thereof.

[0053] As used herein, terms such as "aliphatic" or "aliphatic group" denote a hydrocarbon moiety that can be straight-chain (i.e., unbranched), branched, or cyclic (including fused, bridged, and spiro polycyclic), and can be fully saturated or can contain one or more unsaturated units, but is not aromatic. Generally, an aliphatic group contains 1 - 40 carbon atoms. In certain specific embodiments, the aliphatic group contains 1 - 20 carbon atoms, or 1 - 12 carbon atoms, 1 - 8 carbon atoms, or 1 - 4 carbon atoms. In certain specific embodiments, the aliphatic group contains 3 - 20 carbon atoms, or 3 - 12 carbon atoms, 3 - 8 carbon atoms, or 3 - 4 carbon atoms. Suitable aliphatic groups include, but are not limited to, straight-chain or branched alkyl, alkenyl, and alkynyl groups and their hybrids, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0054] In certain specific embodiments, the L group in formula (I) is selected from (a) a straight-chain alkyl group, (b) a branched alkyl group, (c) a straight-chain alkyl group substituted by a benzene ring, (d) a branched alkyl group substituted by a benzene ring, (e) a phenyl group, wherein the benzene ring contains a straight-chain aliphatic group, and (f) a phenyl group, wherein the benzene ring contains an aliphatic branch.

[0055] As used herein, the term "polyol group" denotes an alcohol that contains multiple hydroxyl groups (two or more hydroxyl groups) per molecule. Specifically, the polyol group can be straight-chain or cyclic, substituted or unsubstituted, or a mixture thereof, provided that the resulting complex is water-soluble and pharmaceutically acceptable.

[0056] In certain specific embodiments, the polyol group is a C3 - 24 polyol, particularly a C3 - 20 polyol, more particularly a C3 - 12 polyol or C3 - 12 polyol, containing two or more hydroxyl groups.

[0057] In more specific specific embodiments, the polyol group is -CH-(CHOH) nCH2OH represents, where n is 1 - 22, 1 - 18, 1 - 10, or 1 - 6. In a specific instance, n is 4.

[0058] Preferred polyols are sugar alcohols. Examples of polyols include, but are not limited to, 3 - carbon polyols (e.g., glycerol, erythritol, and threitol); 5 - carbon polyols (e.g., arabitol, xylitol, and ribitol); 6 - carbon polyols (e.g., mannitol, sorbitol, galactitol, fucitol, iditol, and inositol); 12 - carbon polyols (e.g., oleitol, isomaltitol, maltitol, and lactitol); 18 - carbon polyols (e.g., maltotriitol); and 24 - carbon polyols (maltotetritol).

[0059] In formula (I), G represents a monosaccharide residue. The monosaccharides used herein are preferably 6 - carbon monosaccharides having the chemical formula C6H 12 O6 (i.e., hexoses). The hexose can be of D - configuration, L - configuration, or a combination thereof. Hexoses are generally classified according to functional groups. For example, aldoses have an aldehyde at position 1, such as allose, altrose, glucose, mannose, gulose, idose, galactose, and talose; while ketoses have a ketone at position 2, such as psicose, fructose, sorbose, and tagatose. Hexoses also contain 6 hydroxyl groups, and the aldehyde or ketone functional group in the hexose can react with adjacent hydroxyl functional groups to form intramolecular hemiacetals or hemiketals, respectively. If the resulting cyclic sugar is a 5 - membered ring, it is a furanose. If the resulting cyclic sugar is a 6 - membered ring, it is a pyranose. The ring spontaneously opens and closes, allowing rotation of the bond between the carbonyl group and the adjacent carbon atom, resulting in two different configurations (α and β). Hexoses can be of S - configuration or R - configuration.

[0060] According to the present invention, at least one hydroxyl group in one or more monosaccharide residues in formula (I) is substituted with a halogen atom. Examples of the halogen atom include chlorine, bromine, and iodine. Specifically, the halogen atom is chlorine.

[0061] As used herein, terms such as "S" or "R" name optical isomers by their configuration without reference to the naming of the reference molecule, which is called the R / S system. According to the Cahn Ingold Prelog priority rules, based on the atomic number, each chiral center is labeled R or S according to a system in which its ligands are each assigned a priority. This system labels each chiral center in a molecule (and also has an extension to chiral molecules that do not involve chiral centers). If a compound has two chiral centers, it can be labeled, for example, as an (S,S) isomer or an (S,R) isomer.

[0062] As used herein, the term "pharmaceutically acceptable salts" includes acid addition salts. "Pharmaceutically acceptable acid addition salts" refer to those salts that retain the biological effectiveness and the properties of the free base, and the free base forms salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, pyruvic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, trifluoroacetic acid and the like.

[0063] In certain specific embodiments, in formula (I), q is 2, 3 or 4, and at least one of the R groups is different from the other in R.

[0064] In certain specific embodiments, in formula (I), q is 2.

[0065] In these specific embodiments, the compounds of the present invention can be represented by formula (II) as follows:

[0066] R1-O-X-(CH2) m -X-O-R2 Formula (II),

[0067] wherein

[0068] X is C=O;

[0069] R1 and R2 are the same or different and are selected from the group consisting of hydrogen, polyol groups and sugar groups of (G) p where G is a monosaccharide residue and p is an integer from 1 to 100, wherein at least one hydroxyl group in (G) p is substituted by a halogen atom, wherein when R1 is hydrogen, then R2 is not hydrogen; and m is an integer from 1 to 40,

[0070] or a pharmaceutically acceptable salt thereof.

[0071] In certain specific embodiments, in formula (II), R1 is a polyol group and R2 is a sugar group of (G) p In this case, the compound of formula (II) is considered a conjugate that connects the polyol moiety and the sugar moiety through an ester bond by a linker. Specifically, the linker is represented by -O-X-(CH2) m -X-O- (Formula (L)), wherein X is C=O, m is 1-40, 1-20, 1-12, 1-8 or 1-4, more specifically, m is 3- twenty, 3-12, 3-8 or 3-4. In a specific instance, m is 4.

[0072] In some specific embodiments, p is 2. The sugar group is represented by -G1-O-G2, where G1 and G2 are the same or different and are selected from the group consisting of aldohexoses and ketohexoses, and at least one hydroxyl group in G1 or at least one hydroxyl group in G2 is substituted by a halogen atom.

[0073] In some specific embodiments, G1 is glucose, where one hydroxyl group is substituted by chlorine; and G2 is fructose, where two hydroxyl groups are substituted by chlorine.

[0074] In certain specific embodiments, the sugar group is represented by formula (Ia),

[0075]

[0076] Certain examples of the compounds of the present invention are as follows:

[0077] ((2R,3R,4R,5R,6R)-6-(((2R,5R)-2,5-bis(chloromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)oxy)-3-chloro-4,5-dihydroxytetrahydro-2H-pyran-2-yl)methyl((2R,3R,4R)-2,3,4,5,6-pentahydroxyhexyl)adipate of formula 1

[0078]

[0079] and

[0080] C6-mannitol of formula 2

[0081]

[0082] On the other hand, the present invention provides an intermediate of formula C as follows:

[0083]

[0084] where Ph is phenyl and Bn is benzyl.

[0085] The compounds of formula (I) can be chemically synthesized, for example, by the method shown in the general scheme such as Figure 6 as follows.

[0086] Specifically, the present invention provides a linker that can provide one or more -COOH groups for esterification with alcohols. In step 1, the linker providing the first -COOH group (protected if any others) reacts with R having the first free hydroxyl group (protected if any others) to carry out the first esterification, producing a compound of formula (I), where q is 1. In step 2, the linker providing the second -COOH group (protected if any others) reacts with R having the second free hydroxyl group (protected if any others) to carry out the second esterification, producing a compound of formula (I), where q is 2. In step 3, the linker providing the third -COOH group (protected if any others) reacts with R having the third free hydroxyl group (protected if any others) to carry out the third esterification, producing a compound of formula (I), where q is 3. In step 4, the linker providing the fourth -COOH group (protected if any others) reacts with R having the fourth free hydroxyl group (protected if any others) to carry out the fourth esterification, producing a compound of formula (I), where q is 4.

[0087] In some specific embodiments, the linker for carrying out the esterification is represented by formula (La),

[0088] P1 - O - X - (CH2) m -X - O - P2 Formula (La)

[0089] where X and m are as defined above, and P1 and P2 are the same or different, and are protecting groups or H.

[0090] In some specific embodiments, the linker for carrying out the esterification is represented by formula (Lb)

[0091]

[0092] As used herein, a "protecting group" is a chemical group attached to a functional moiety (e.g., the oxygen in a hydroxyl group or the nitrogen in an amine group, replacing hydrogen) to protect the functional group from reacting in an undesired manner. Protecting groups include, for example, tert - butyl, cycloalkyl (e.g., cyclohexyl), aryl (e.g., 2,4 - dinitrophenyl), aralkyl (e.g., benzyl, 2,6 - dichlorobenzyl, 3 - bromobenzyl, 2 - nitrobenzyl, 4 - dimethylaminocarbonylbenzyl, and triphenylmethyl), tetrahydropyranyl, acyl, alkoxycarbonyl (e.g., tert - butoxycarbonyl), aralkyloxycarbonyl (e.g., benzyloxycarbonyl, 2 - bromobenzyloxycarbonyl), dialkylthiophosphoryl (e.g., dimethylthiophosphoryl), and diarylthiophosphoryl (e.g., diphenylthiophosphoryl). Preferred protecting groups include acyl and its analogs.

[0093] In a specific instance, in Example 1, Scheme 1 was provided, showing the specific synthesis method of the compounds of the present invention.

[0094] II. Uses of the Compounds of the Present Invention

[0095] The compounds of the present invention can be used as drugs for therapeutic methods. Generally, the compounds of formula (I) are prodrugs and can be converted into metabolites after administration, providing the desired therapeutic effects as described herein. In one instance, the compound of formula (I) is Compound F, which can be converted into mannitol, sucralose, and C6-mannitol after administration, all of which can act as P450 inhibitors and provide, for example, an anti-hepatotoxic effect. See the following examples.

[0096] The present invention provides a therapeutic method by administering to an individual in need an effective amount of at least one compound as described herein or a pharmaceutically acceptable salt thereof.

[0097] The compounds of the present invention have been found to be effective as, for example, P450 inhibitors.

[0098] In some specific embodiments, the methods of the present invention are provided for preventing or treating diseases or disorders characterized by increased cytochrome P450 activity in an individual in need.

[0099] Examples of these diseases or disorders are listed in Table A.

[0100] Table A

[0101]

[0102]

[0103] In some specific embodiments, the methods of the present invention are provided for preventing or treating diseases or disorders characterized by increased free radical content in an individual in need.

[0104] In some specific embodiments, the methods of the present invention are provided for preventing or treating organ damage in an individual in need.

[0105] In a specific embodiment, the organ damage is in the liver or kidney.

[0106] In a specific embodiment, the organ damage or hepatotoxicity is caused by a therapeutic drug, CCl4, or lipid accumulation.

[0107] In a specific embodiment, the therapeutic drug is acetaminophen.

[0108] In some specific embodiments, the methods of the present invention are provided for preventing or treating hepatotoxicity in an individual in need.

[0109] In some specific embodiments, the method of the present invention is provided to prevent or treat fatty liver, protect liver function or improve liver diseases caused by fatty liver or other related diseases.

[0110] As used herein, the term "liver fat content" refers to the content of fat accumulated in the liver of an individual and includes lipids in a broad sense, such as triglyceride (TG) and cholesterol. As used herein, the term "reducing liver fat content" generally refers to reducing the abnormal liver fat content in an individual, that is, reducing the abnormal liver fat content, and more specifically, reducing the abnormal liver fat content to a normal level. For example, under normal circumstances, fat accounts for 3% of the liver weight. If the fat in the liver exceeds 5% of the liver weight, it is judged as abnormal fat accumulation (the above liver fat content is an exemplary relative percentage and may vary due to race and other factors). In a specific aspect, the term "reducing liver fat content" used herein may represent the content of abnormal liver fat in an individual, for example, reducing from 5% or more of the liver weight to 3% of the liver weight. The liver fat content can be evaluated by standard analytical methods, including but not limited to ultrasonic analysis, magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), computed tomography (CT), and liver biopsy.

[0111] As used herein, the term "liver function" refers to one or more physiological functions performed by the liver. Liver function can be analyzed by many conventional determination methods, such as alanine aminotransferase (ALT) analysis or aspartate transaminase (AST) analysis. According to the present invention, the compounds described herein can be used to maintain liver function, including improving liver function and preventing liver damage.

[0112] As used herein, the term "liver disease" refers to injury to liver cells or injury caused by certain factors that potentially leads to liver dysfunction. According to the present invention, in some specific embodiments, the compounds proposed herein can be used to improve liver diseases caused by fatty liver. More specifically, the "liver injury" as used herein refers to a liver having histological or biochemical dysfunction as compared to a normal liver. In a particular specific embodiment, "liver injury" refers to liver injury caused by alcohol or non-alcohol factors such as high-fat diet or obesity, or therapeutic drugs or organic solvents. In a particular specific embodiment, "liver injury" can be liver tissue injury having one or more characteristics selected from steatosis, lobular inflammation, hepatocyte balloning, and vesicular lipid droplets produced by hepatocytes. In a particular specific embodiment, "liver injury" can be a biochemical dysfunction of the liver, which can be determined by the activity of alanine transaminase (ALT) or aspartate transaminase (AST). Higher activity of ALT or AST indicates severe dysfunction of the biochemical function of the liver.

[0113] As used herein, the term "liver antioxidant activity" refers to the activity or ability against oxidative stress. Improving the liver antioxidant activity of an individual by the compounds according to the present invention means including, but not limited to, reducing oxidative stress or enhancing the enzyme activity or content of members of the antioxidant system. Members of the antioxidant system can be glutathione peroxidase (GPx), glutathione (GSH), glutathione reductase (GRd), and / or superoxide dismutase (SOD).

[0114] According to the present invention, the compounds described herein include common excipients and bioflavonoids, which can be used to reduce liver fat content and improve related conditions. The term "related conditions" as used herein includes conditions caused by abnormal accumulation of liver fat, including, but not limited to, fatty liver disease, acute and chronic alcoholic fatty liver disease, acute and chronic non-alcoholic fatty liver disease, acute and chronic alcoholic hepatitis, acute and chronic non-alcoholic steatohepatitis, non-alcoholic cirrhosis, and alcoholic cirrhosis (ICD-9-CM diagnostic codes: 571.8, 571.0, 571.1, 571.2, 571.3, 571.4, 571.5, 571.9).

[0115] As used herein, the term "prevention" refers to preventive measures against a disease or the symptoms or conditions of a disease. Preventive measures include, but are not limited to, administering or dispensing one or more active agents to an individual who has not been diagnosed with the disease or the symptoms or conditions of the disease but is likely to be susceptible or prone to contracting the disease. The purpose of preventive measures is to avoid, prevent, or delay the occurrence of a disease or the symptoms or manifestations of a disease.

[0116] As used herein, the term "treatment" refers to therapeutic measures against a disease or the symptoms or conditions of a disease. Therapeutic measures include, but are not limited to, administering or dispensing one or more active agents to an individual who has a disease or the symptoms or manifestations of a disease or the exacerbation of a disease. The purpose of therapeutic measures is to treat, cure, alleviate, relieve, alter, remedy, improve, ameliorate, or affect the disease, the symptoms or manifestations of the disease, the disability caused by the disease, or the exacerbation of the disease.

[0117] As used herein, a "CYP2E1 inhibitor" is any compound, substance, or material that can inhibit CYP2E1 activity. Many assays can be used to analyze CYP2E1 activity, such as the analysis of human or rat liver microsomes.

[0118] As used herein, an individual in need of treatment according to the present invention includes humans and non-human mammals. Non-human mammals include, but are not limited to, companion animals such as cats, dogs, and the like, and farm animals such as cows, horses, sheep, goats, pigs, and the like.

[0119] The term "effective amount" or a similar term refers to the amount of an active agent sufficient to achieve a desired therapeutic, preventive, and / or biological effect in an individual, such as reducing drug-induced side effects, or inhibiting, improving, alleviating, reducing, or preventing one or more symptoms or manifestations or the progression of a disease. The actual effective amount may vary depending on various factors such as the route and frequency of administration, the weight and species of the individual receiving the drug, and the purpose of administration. Those skilled in the art can determine the dose in each case based on the disclosure herein, the methods, and their own experience.

[0120] As used herein, the term "standard dose" refers to the effective dose of a therapeutic agent recommended by an authoritative source in the medical community, including the Food and Drug Administration, and commonly used in routine practice. As used herein, the term "reduced dose" refers to a dose that is lower than the standard dose but still maintains substantially the same therapeutic effect as the same therapeutic agent. Specifically, according to the present invention, the reduced dose of a therapeutic drug is about 90% or less, 80% or less, 70% or less, 60% or less, 50% or less of the standard therapeutic dose of the therapeutic drug.

[0121] In some specific embodiments, the effective amount of the active ingredient used herein can be formulated with a pharmaceutically acceptable carrier into a pharmaceutical composition in a suitable form for delivery and absorption.

[0122] As used herein, "pharmaceutically acceptable" means that the carrier is compatible with the active ingredient in the composition and preferably can stabilize the active ingredient and is safe for the individual receiving the treatment. The carrier can be a diluent, carrier, excipient or matrix for the active ingredient. The composition can additionally contain lubricants, wetting agents, emulsifiers and suspending agents, preservatives, sweeteners, and flavoring agents. The compositions of the present invention can provide a rapid, sustained or delayed release effect of the active ingredient after administration to a patient.

[0123] According to the present invention, the composition can be in the form of tablets, pills, powders, lozenges, packages, troches, elixirs, suspensions, lotions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and packaged powders.

[0124] The compositions of the present invention can be delivered by any physiologically acceptable route, for example, orally, parenterally (e.g., intramuscularly, intravenously, subcutaneously and intraperitoneally), transdermal delivery, suppository and intranasal routes. For parenteral administration, it is preferably used in the form of a sterile aqueous solution, which can contain other substances, such as salts or glucose sufficient to make the solution isotonic with blood. Standard pharmacological techniques well known to those skilled in the art can be used to prepare appropriate parenteral compositions under sterile conditions and no additional creative effort is required.

[0125] In certain specific embodiments, the compound of formula (I) or its pharmaceutically acceptable salts of the present invention can be used for preventing or treating damage in organs, for example, in the liver or kidney, which can be caused by an overdose of a therapeutic drug (e.g., acetaminophen) or exposure to alcohols, chemical agents, biomolecules or any substance that can cause toxic effects in these organs.

[0126] Specifically, the damage in the liver can include damage, impairment or loss of hepatocytes or tissues, resulting in abnormal liver function or abnormal liver protein content. In some specific embodiments, the liver damage as described herein is acute liver damage, which refers to liver damage with a relatively rapid onset, for example, a period of less than 12 weeks, specifically less than 6 weeks from the onset of symptoms. In some specific embodiments, patients with acute liver damage have not had chronic liver disease.

[0127] Specifically, the injury in the kidney can include injury, damage or loss of renal cells or tissues, resulting in abnormal renal function. Such renal injury can be determined, for example, by a decrease in glomerular filtration rate, a decrease in urine output, an increase in serum creatinine, an increase in serum cystatin C, etc. In some specific embodiments, the renal injury described herein is acute renal injury, which can represent a sudden or rapid decline in the kidney's filtration function, for example, within 14 days, preferably within 7 days, more preferably within 72 hours, and even more preferably within 48 hours.

[0128] In a specific embodiment, the compound of formula (I) of the present invention or a pharmaceutically acceptable salt thereof can prevent or treat an undesirable condition caused by NAPQI (N-acetyl-p-benzoquinoneimine).

[0129] Accordingly, the present invention provides the use of the compound of formula (I) of the present invention or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating an undesirable condition caused by NAPQI (N-acetyl-p-benzoquinoneimine) in an individual. The present invention also provides a method for preventing or treating an undesirable condition caused by NAPQI (N-acetyl-p-benzoquinoneimine) in an individual in need thereof, comprising administering to the individual a compound of formula (I) of the present invention or a pharmaceutically acceptable salt thereof in an amount effective to prevent or treat the undesirable condition.

[0130] III. Combinatorial use of the compounds of the present invention with other active agents

[0131] The compounds of the present invention and / or their metabolites can be administered in combination with one or more additional active agents, which are specifically active agents acting as P450 inhibitors and / or providing anti-hepatotoxic activity and / or having anti-steatosis activity, thereby providing, for example, a synergistic effect.

[0132] Some active agents as P450 inhibitors (named "the first active agent") are described in PCT / CN2013 / 087049 (USSN 14 / 441,317, the content of which is incorporated herein by reference in its entirety). Specific examples of such P450 inhibitors include, but are not limited to, polyethylene glycol sorbitan monolaurate (Tween 20), microcrystalline cellulose, dicalcium phosphate dihydrate, Brij 35, saccharin, mannitol, Cremophor RH40, sucralose, crospovidone, sodium starch glycolate, Eudragit S100, sodium croscarmellose, Pluronic F68, menthol, low-substituted hydroxypropyl cellulose, pregelatinized starch, Dextrates NF hydrate, citric acid, Cremophor EL, Aerosil 200, Myrj 52, sorbic acid, lemon oil, hydroxypropyl cellulose, sorbitol, acesulfame potassium, hydroxypropyl methylcellulose, lactose monohydrate, maltodextrin, Brij 58, Brij 76, Tween 80, Tween 40, PEG 400, PEG 4000, PEG 8000, Span60, sodium benzoate, hydroxyethyl methylcellulose, methylcellulose, Span 80, sodium cyclohexylsulfamate, glyceryl behenate, red oxide, glycerol monostearate, copovidone K28, starch acetate, magnesium stearate, sodium lauryl sulfate, povidone K30, PEG2000, and N-acetylcysteamine (NAC) and any combination thereof.

[0133] In certain specific embodiments, one or more of the first active agents used in combination with the compound of formula (I) of the present invention are selected from the group consisting of dicalcium phosphate dehydrate, menthol, mannitol, sucralose, N-acetylcysteamine (NAC) and any combination thereof.

[0134] Some active agents having anti - fatty liver activity (named "second active agents") are described in PCT / CN2016 / 078039, the content of which PCT application is incorporated herein by reference in its entirety. Specific examples of active agents having anti - fatty liver activity include, but are not limited to, (ii) second active agents selected from the group consisting of: sodium dodecyl sulfate, menthol, sucralose, mannitol, sorbitol, saccharin, glycerol, sodium benzoate, erythrosine, pre - gelatinized starch, sodium cyclohexanesulfamate, sorbic acid, lemon oil, citric acid, butylated hydroxyanisole, wolfberry fruit, isovitexin, eriodictyol, ergosterol, β - myrcene, hypercholesterolemia, (+) - catechin, galangin, morin, sciadopitysin, apigenin - 7 - glucoside, (+) - taxifolin, trans - cinnamic acid, diosmin, mongolicin, xylitol, luteolin, swertiamarin, puerarin, phloridzin, sinensetin, (-) - epigallocatechin, kaempferol, ursolic acid, silymarin, (+) - limonene, hesperidin, (-) - epicatechin - 3 - gallate, silybin, formononetin, ethyl myristate, eicosapentaenoic acid (EPA), wogonin, povidone K - 30, protocatechuic acid, umbelliferone, hesperetin, nordihydroguaiaretic acid, neohesperidin, naringin, (-) - epicatechin, glycyrrhizin, baicalin, quercitrin, baicalein, and any combination thereof.

[0135] In certain specific embodiments, one or more second active agents used in combination with the compound of formula (I) of the present invention are selected from the group consisting of: sodium dodecyl sulfate, menthol, sucralose, mannitol, sorbitol, saccharin, glycerol, sodium benzoate, erythrosine, pre - gelatinized starch, sodium cyclohexanesulfamate, sorbic acid, lemon oil, citric acid, butylated hydroxyanisole, wolfberry fruit, isovitexin, eriodictyol, ergosterol, β - myrcene, hypercholesterolemia, (+) - catechin, galangin, morin, sciadopitysin, apigenin - 7 - glucoside, (+) - taxifolin, trans - cinnamic acid, diosmin, mongolicin, xylitol, luteolin, swertiamarin, and any combination thereof.

[0136] In certain specific embodiments, one or more second active agents used in combination with the compound of formula (I) of the present invention are selected from the group consisting of: puerarin, phloridzin, sinensetin, (-)-epigallocatechin, kaempferol, ursolic acid, silymarin, (+)-limonene, hesperidin, (-)-epicatechin-3-gallate, silybin, formononetin, ethyl myristate, eicosapentaenoic acid (EPA), wogonin, polyvinylpyrrolidone K-30, protocatechuic acid, umbelliferone, hesperetin, nordihydroguaiaretic acid, neohesperidin, naringin, (-)-epicatechin, glycyrrhizin, baicalin, quercitrin, baicalein, and any combination thereof.

[0137] In certain specific embodiments, one or more second active agents used in combination with the compound of formula (I) of the present invention are selected from the group consisting of: eriodictyol, mannitol, menthol, sucralose, saccharin, and any combination thereof.

[0138] In certain specific embodiments, one or more second active agents used in combination with the compound of formula (I) of the present invention are selected from the group consisting of: (1) a combination of saccharin and mannitol, (2) a combination of menthol and mannitol, (3) a combination of sucralose and mannitol, (4) a combination of eriodictyol and mannitol, (5) a combination of eriodictyol and sucralose, (6) a combination of menthol, mannitol, and eriodictyol, and (7) a combination of sucralose, mannitol, and eriodictyol.

[0139] Specifically, the compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more additional agents can be administered simultaneously or sequentially.

[0140] In the present invention, there is also provided that the compound of formula (I) of the present invention or a pharmaceutically acceptable salt thereof can prevent or treat an undesirable condition caused by NAPQI (N-acetyl-p-benzoquinone imine).

[0141] As a specific embodiment, the present invention provides a combination of the compound of formula (I) and / or its metabolite with N-acetylcysteine (NAC). The present invention also provides a method for administering N-acetylcysteine (NAC) to an individual in need, comprising administering to the individual a combination of NAC and the compound of formula (I) and / or its metabolite. In one specific embodiment, the combination or method of the present invention is effective in preventing or treating a disease or condition for which NAC is effective. In some specific embodiments, the diseases or conditions treated or prevented by NAC are selected from the group consisting of: myoclonic epilepsy, acute respiratory distress syndrome, heavy metal poisoning, influenza virus infection, heart disease, Sjogren's syndrome, chronic bronchitis, epilepsy (Unverricht-Lundborg type), and HIV infection.

[0142] The present invention is further illustrated by the following examples, which are provided for illustrative purposes only and not for limitation.

[0143] Example

[0144] Example 1: Synthesis of the compound of Formula 1 (Compound F) of the present invention

[0145] Synthesis of ((2R,3R,4R,5R,6R)-6-(((2R,5R)-2,5-bis(chloromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)oxy)-3-chloro-4,5-dihydroxytetrahydro-2H-pyran-2-yl)methyl ((2R,3R,4R)-2,3,4,5,6-pentahydroxyhexyl) adipate (Formula 1) (Compound F)

[0146] The synthetic strategy for the synthesis of Formula 1 (Compound F) is shown in Scheme 1.

[0147] Scheme 1

[0148]

[0149] HMDS = hexamethyldisilazane; TMSOTf = trimethylsilyl trifluoromethanesulfonate; TBAF = tetrabutylammonium fluoride

[0150] THF = tetrahydrofuran

[0151] TMS = trimethylsilyl; DCC = dicyclohexylcarbodiimide; DMAP = 4-dimethylaminopyridine; DCM = dichloromethane

[0152] DMF = N,N'-dimethylformamide; DIBAL = diisobutylaluminum; Bn = benzyl ether

[0153] General method Unless otherwise specified, all chemicals were obtained from commercial sources and used directly. The chromatographic purity of the products was evaluated under the following conditions:

[0154] Mobile phase composition A: methanol:H2O = 5 / 95 (v / v), containing 0.05% NH4OH

[0155] B: methanol:H2O = 95 / 5 (v / v), containing 0.05% NH4OH

[0156] Chromatographic system:

[0157]

[0158] MS analysis was performed under the following conditions:

[0159] Mass spectrometer settings:

[0160]

[0161] Using Bruker standard pulse programs, in MeOH-d4 (δ H 3.30, δ C 49.0) or CDCl3 (δ H 7.24, δ C 77.0), Bruker AMX-500 NMR spectra were obtained; in the HMQC and HMBC experiments, Δ = 1 s and J were 140, 8 Hz respectively, the correlation maps consisted of 512×1K data points for each spectrum, and each spectrum consisted of 16 to 64 transients.

[0162] 1.1 Mannitol (Compound (i)) to Compound (B)

[0163] 1.1.1 Mannitol (Compound (i)) to Compound (i)-1

[0164]

[0165] At room temperature and one atmosphere, benzaldehyde (30 mL, 0.345 mmol) was added to a solution of D-mannitol (25 g, 0.137 mol) in DMF (250 mL). At 0 °C, concentrated sulfuric acid (10 mL) was added dropwise to the mixture. After gradually warming to room temperature, the mixture was stirred for 3 days. Then the mixture was poured into ice water (250 mL) and n-hexane (200 mL) with vigorous stirring. After warming the mixture to room temperature, the precipitate was filtered and washed with n-hexane. The precipitate was suspended in chloroform and heated under reflux with vigorous stirring for 15 minutes. When the mixture reached room temperature, the undissolved precipitate was collected and recrystallized from ethanol to give the desired product as a white solid (9.86 g, 20%). Rf = 0.45 (EA / Hex = 1 / 1).

[0166] 1.1.2 Compound (i)-1 to Compound (i)-2

[0167]

[0168] At room temperature and one atmosphere, benzyl bromide (7.96 mL, 66.96 mmol) was added to a solution of 1,3,4,6-dibenzylidene (10 g, 27.9 mmol) in DMF (100 mL). The mixture was cooled to 0 °C, and then 60% NaH (2.68 g, 66.96 mmol) was added within a few minutes. After gradually warming to room temperature, the mixture was stirred overnight. Then the reaction was quenched with water (dropwise), with NaHCO 3(水溶液)Extract with dichloromethane. Dry the organic layer over MgSO4 and concentrate in vacuo. Purify the residue by silica gel column chromatography to give the desired product (10.39 g, 69%). R f = 0.2 (EA / Hex = 1 / 6).

[0169] 1.1.3 Compound (i)-2 to Compound (B)

[0170]

[0171] Cool a solution of 2,5-dibenzyl-1,3,4,6-dibenzyliene (1.5 g, 2.78 mmol) in toluene (12.5 mL) to -18 °C (ice-salt bath). Add dropwise 1.2 M DIBAL (18.5 mL, 22.3 mmol) and warm to room temperature. After 1.5 h, cool the reaction to 0 °C and then quench with MeOH and 15% KOH (水溶液) Quench. Extract the mixture with DCM, dry the organic layer over MgSO4 and concentrate in vacuo. Purify the residue by silica gel column chromatography to give the desired product (709 mg, 47%), R f = 0.1 (EA / HEX = 1 / 5).

[0172] 1.2 Sucralose (Compound (ii)) to Compound (D)

[0173] 1.2.1 Compound (ii) to Compound (ii)-1

[0174]

[0175] To a solution of sucralose (1 g, 2.5 mmol) in DCM (10 mL) add HMDS (2.6 mL, 12.57 mmol) and TMSOTf (45 μL, 0.25 mmol). Stir the reaction overnight at room temperature. Concentrate the reaction in vacuo and pass through cotton, washing with hexane. Concentrate the filtrate again in vacuo to give the product, quantitatively. (1.9 g, quantitative). R f = 0.9 (EA / HEX = 1 / 8).

[0176] 1.2.2 Compound (ii)-1 to Compound (D)

[0177]

[0178] To a solution of penta-TMS sucralose (5 g, 6.6 mmol) in pyridine (150 mL) was added 0.1 M pyridine-TcCl solution (6.6 mL) and stirred in an open flask for 3 days. The reaction solution was concentrated in vacuo and purified by silica gel column chromatography to obtain the desired product (1.4 g, 30%). f =0.5(EA / HEX=1 / 8).

[0179] 1.3 Synthesis of 6-oxo-6-((2R,3R,4R)-2,3,4-tris(benzyloxy)-4-(2-phenyl-1,3-dioxolane-4-yl)butoxy)hexanoic acid (Compound (C))

[0180]

[0181] At 0 ℃, in a flame-dried RB flask, compound A (165 mg, 1 equivalent) was dissolved in DCM (5 mL), and pyridine (0.2 mL) and DMAP (50 mg) were then added thereto. The reaction mixture was then stirred for 10 minutes and compound B (59 mg, 1.5 equivalents) was added. The reaction mixture was then stirred at room temperature for 5 hours. TLC confirmed that the reaction was complete. The reaction mixture was evaporated to dryness on a rotary evaporator under reduced pressure. The crude compound was further purified by column chromatography to obtain the desired compound as a colorless oil (136 mg, 67%).

[0182] 1. Synthesis of 4-((2R,3S,4R,5R,6R)-6-(((2R,5R)-2,5-bis(chloromethyl)-3,4-bis((trimethylsilyl)oxy)tetrahydrofuran-2-yl)oxy)-3-chloro-4,5-bis((trimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)methyl((2R,3R,4R)-2,3,4-tris(benzyloxy)-4-(2-phenyl-1,3-dioxolan-4-yl)butyl) adipate

[0183]

[0184] To a cold solution of compound C (100 mg, 1.0 equiv) in DCM was added DCC (35 mg, 1.15 equiv) and the mixture was stirred for 10 min. Then to this was added DMAP (5 mg, 0.25 equiv, catalytic) to compound D (112 mg, 1.2 equiv). The reaction mixture was warmed to room temperature and stirred for 4 h. TLC confirmed the completion of the reaction. The reaction mixture was evaporated to dryness on a rotary evaporator under reduced pressure. The crude compound was then purified by column chromatography using neutral silica gel and a hexane solution of 5 to 15% ethyl acetate with 1% triethylamine as the eluent to give the desired compound E as a colorless oil (84 mg, 42%).

[0185] Synthesis of 1.5((2R,3R,4R,5R,6R)-6-(((2R,5R)-2,5-bis(chloromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)oxy)-3-chloro-4,5-dihydroxytetrahydro-2H-pyran-2-yl)methyl ((2R,3R,4R)-2,3,4,5,6-pentahydroxyhexyl) adipate (Compound F)

[0186]

[0187] In a flame-dried single-necked R.B. flask, compound E (500 mg, 1 equiv) was dissolved in anhydrous MeOH (20 mL), and then the solution was degassed by nitrogen (nitrogen syringe deep inside the solution, nitrogen purge for 15 min). Then 10% Pd-C (200 mg, 33% w / w) was carefully added to the reaction mixture. Finally, the reaction mixture was stirred under a hydrogen balloon pressure for 6 h. TLC confirmed the completion of the reaction. The reaction mixture was then filtered through a bed of celite and the celite bed was washed with anhydrous methanol. The filtrate was evaporated to dryness on a rotary evaporator under reduced pressure. The final compound was then kept under high vacuum to give the desired final compound F as a colorless semi-solid or white solid (190 mg, 73%). The structure of compound F was identified by high-resolution mass spectrometry and 13 CNMR.

[0188] Example 2: Compound F as a prodrug generates metabolite 2.1 when incubated with blood (in vitro). Materials and methods

[0189] Fresh human whole blood was used for the study of drug hydrolysis. The drug (10 mg, compound F) was dissolved in 1 mL of solution (20% methanol). In a 50 mL flask, 20 mL aliquots of fresh whole blood containing 1.0 mg of the drug were incubated at 37 °C in a shaking water bath for drug hydrolysis (n = 3). At time 0, the drug was added, and after incubation at different times, blood samples were collected at 0.25, 0.5, 0.33, 0.75, 1, 2, 4, 6, 12, and 24 hours. The blood samples were quenched with 1 mL of acetonitrile to stop the enzymatic hydrolysis of the drug, and thus the samples were obtained. The prodrug and its related metabolites in the blood, such as C6-mannitol, mannitol, and sucralose, were determined by an API QTrap 5500 triple quadrupole mass spectrometer equipped with an ion-spray (ESI) source. The ESI interface was used in the negative ion mode.

[0190] 2.2 Results

[0191] The prodrug was monitored at the transition of m / z 688.9 → 180.9. Sucralose was monitored at the transition of m / z 395 → 359; mannitol was monitored at the transition of m / z 452.3 → 273.3; C6-mannitol was monitored at the transition of m / z 309 → 101.1. All compounds were identified by high-resolution mass spectrometry and 13 13C NMR. The structure of C6-mannitol (Formula (2)) is as follows:

[0192]

[0193] The percentage of the remaining prodrug after incubation of the prodrug in blood, and the percentage increases of sucralose, mannitol, and C6-mannitol were plotted against time to represent the hydrolysis of the prodrug in blood ( Figure 1 ). The results showed that with compound F as the prodrug, it was converted to its metabolites, including sucralose, mannitol, and C6-mannitol, after incubation with blood in vitro.

[0194] Example 3: Pharmacokinetic Study in SD (Sprague Dawley) - Rats (In Vivo)

[0195] 3.1 Materials and Methods

[0196] The prodrug was orally administered to SD - rats at a dose of 3.67 mg / kg body weight. Blood samples were collected into heparinized microcentrifuge tubes at intervals of 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours. Plasma samples were obtained immediately after centrifuging the blood samples at 8,000 rpm for 10 minutes. Then the plasma samples were stored at -80 °C for later use. The plasma samples were analyzed for the prodrug and its related metabolites, such as mannitol and sucralose, by an APIQTrap5500 triple quadrupole mass spectrometer equipped with an ion spray (ESI) source. The ESI interface was used in the negative ion mode.

[0197] 3.2 Results The prodrug was monitored at the transition of m / z 688.9→180.9. Sucralose was monitored at the transition of m / z 395→359; mannitol was monitored at the transition of m / z 452.3→273.3; C6 - mannitol was monitored at the transition of m / z 309→101.1.

[0198] Figure 2 and Figure 3 respectively show the plasma concentration - time curves of the prodrug and its related metabolites, such as sucralose and mannitol, in SD rats orally administered a single dose of 3.67 mg / kg of the prodrug. The results show that compound F, as a prodrug, is converted into its metabolites, including sucralose, mannitol, and C6 - mannitol, in the animal body after administration.

[0199] Example 4: CYP2E1 Inhibitory Activity Analysis

[0200] 4.1 Materials and Methods Microsomes were prepared from human liver for in vitro screening of CYP450 isozyme inhibitors. Based on the reaction of microsomal CYP450 isozymes prepared from livers of different sources with their specific substrate chlorzoxazone (CZX), effective human liver CYP450 isozyme inhibitors were tested, and the principle of CYP450 isozyme inhibitors was tested. After adding the test sample, the amount of the CYP450 isozyme metabolite standard 6 - OH - CZX (6 - hydroxy - chlorzoxazone) was specifically used to calculate the inhibition rate of the CYP450 isozyme (CYP2E1) of the test sample, using the amount of 6 - OH - CZX in the control group as the baseline.

[0201] All samples were tested in triplicate. To determine the percentage inhibition, each test compound was dissolved at three different concentrations of 1, 2, and 4 μg / mL. The amount of CYP2E1 activity in the presence of the test compound was compared to that of the control cultures. A 500-μL reaction mixture containing 0.5 mg of microsomal protein was incubated with 320 μM CZX in 50 mM phosphate buffer containing 5 mM MgCl2 and 1 mM NADPH at pH 7.4 for 30 minutes at 37 °C. The reaction was terminated with ice-cold acetonitrile, and then 4-hydroxytoluenesulfonamide was added as an internal standard. Before liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis, the organic phase was evaporated to dryness and reconstituted in the mobile phase (methanol:water = 1:1). A API 3000 triple quadrupole mass spectrometer equipped with an ion spray (ESI) source was used to determine 6-OH-CZX in human liver microsomes. The ESI interface was used in the positive ion mode. 6-OH-CZX was monitored at the transition of m / z 284.5→185.9.

[0202] Results analysis: Using the control group as the baseline, the detected signal values obtained from LC / MS / MS were converted to the amount (pmol) of the CYP450 isozyme metabolite standard 6-hydroxy-chlorzoxazone, i.e., the inhibition rate of the CYP450 isozyme in the control group was 0%. In the presence of the test compound, the amount of CYP450 isozyme activity was compared to that of the control cultures.

[0203] 4.2 Results Diethyldithiocarbamic acid (DDTC) is a known CYP2E1 inhibitor. At a concentration of 100 μM, treatment with DDTC resulted in a 90.9% inhibition rate of CYP2E1 in human liver microsomes (measured using CZX as the substrate for CYP2E1). Based on the observed inhibitory activity of DDTC, we tested a new compound (prodrug) and its related metabolites for CYP2E1 inhibition at concentrations of 4, 2, and 1 μg / mL. The results are summarized in Table 1.

[0204] Table 1. Inhibition rates of CYP2E1 inhibitors screened in vitro from human liver microsomes

[0205]

[0206] The CYP 2E1 inhibition rates of the test compounds detected in human liver microsomes are shown in Table 1. From this result, it is known that the test compounds, including the prodrug (Compound F) and its metabolites, namely mannitol, sucralose, and C6-mannitol with a protecting group (Formula C), have been confirmed to be effective as P450 2E1 inhibitors, and the intermediate metabolite of the 4 μg / mL prodrug (i.e., C6-mannitol with a protecting group, Formula C) showed the best inhibitory effect (70.3 ± 2.8%).

[0207] Example 5: Analysis of liver injury induced by acetaminophen (APAP) and CCl4

[0208] 5.1 Materials and methods

[0209] 5.1.1 Reagents All organic solvents were of HPLC grade and purchased from Tedia Company (Fairfield, Ohio, USA). APAP was purchased from Sigma Company (St. Louis, Missouri, USA), and the galactose injectable solution was manufactured by Southern Photochemical Co. by dissolving 400 g of galactose (Sigma Company) in 1 L of a buffer solution containing isotonic salts for injection.

[0210] 5.1.2 Animals Male SD (Sprague-Dawley) rats weighing 175 - 280 g were purchased from an experimental animal institution in Taiwan, China. This study was conducted in accordance with the guidelines for animal research of the health research institutions in Taiwan, China, and all rats were placed in an air / humidity-controlled environment under a 12-hour day / 12-hour night cycle and with unrestricted access to water and food. During the study, the body weights of the rats were continuously monitored with normal water supply.

[0211] 5.1.3 Treatments

[0212] 5.1.3.1 Liver injury induced by APAP Based on the liver injury induced by APAP, animal experiments (rats) were conducted using mannitol and sucralose.

[0213] In the normal control group (Group 1), the animals were not fed APAP. In the control group for APAP-induced liver injury (Group 2), the animals were fed a single dose of APAP at a dosage of 2,000 mg per kilogram of body weight to induce hepatotoxicity. In the positive control group treated with NAC (Group 3), the animals were fed a single dose of APAP at a dosage of 2,000 mg per kilogram of body weight to induce hepatotoxicity. Four hours later, a 24-hour treatment period via gavage was initiated, including first administering 140 mg of NAC (per kilogram of body weight), and subsequently administering 70 mg of NAC (per kilogram of body weight) five times every 4 hours. In the experimental group (Group 4), the animals were fed a single dose of APAP at a dosage of 2,000 mg per kilogram of body weight to induce hepatotoxicity. Four hours later, a 24-hour treatment period via gavage was initiated, including administering the components of the present invention six times every 4 hours. Each subgroup is as follows:

[0214] (a) (Group 4.1): Mannitol was administered at a dose of less than or equal to 100 mg per person every 4 hours for 24 hours,

[0215] (b) (Group 4.2): Mannitol was administered at a dose twice that of Group 4.1 every 4 hours for 24 hours. (c) (Group 4.3): Sucralose was administered at a dose of less than or equal to 100 mg per person every 4 hours for 24 hours,

[0216] (d) (Group 4.4): Sucralose was administered at a dose twice that of Group 4.3 every 4 hours for 24 hours. (e) (Group 4.5): A combination of mannitol at a dose 0.5 times that of Group 4.1 and sucralose at a dose 0.5 times that of Group 4.3 was administered every 4 hours for 24 hours,

[0217] (f) (Group 4.6): A combination of mannitol at the dose of Group 4.1 and sucralose at the dose of Group 4.3 was administered every 4 hours for 24 hours,

[0218] (g) (Group 4.7): A combination of mannitol at a dose 1.5 times that of Group 4.1 and sucralose at a dose 1.5 times that of Group 4.3 was administered every 4 hours for 24 hours,

[0219] (h) (Group 4.8): A combination of mannitol at a dose twice that of Group 4.1 and sucralose at a dose twice that of Group 4.3 was administered every 4 hours for 24 hours, and (i) (Group 4.9): First, 140 mg of NAC per kilogram of body weight was administered, and subsequently, 70 mg of NAC plus a combination of mannitol at a dose twice that of Group 4.1 and sucralose at a dose twice that of Group 4.3 was administered every 4 hours for five times.

[0220] After a 24-hour treatment period, blood was collected from the tail artery of the rats for AST / SLT determination. Subsequently, the rats were subjected to the GSP test. Finally, the rats were sacrificed and histological analysis was performed.

[0221] 5.1.3.2 CCl4-induced liver injury Based on CCl4-induced liver injury, animal experiments (mice) were conducted using mannitol and sucralose selected from the active ingredients described herein.

[0222] In the normal control group, animals were given physiological saline by intraperitoneal injection. In the control group of CCl4-induced liver injury, animals were given 10 ml / kg CCl4 (40% in corn oil) by intraperitoneal injection to induce hepatotoxicity. In the experimental group, animals were given 10 ml / kg CCl4 (40% in corn oil) by intraperitoneal injection to induce hepatotoxicity. Four hours later, different components of the present invention were given by gavage. Before or 24 hours after administration of the components of the present invention, blood was collected from the mice for AST / ALT analysis. Finally, on the second day, the animals were sacrificed, blood was collected for AST / ALT analysis, and histological analysis was performed.

[0223] On the other hand, mice in other experimental groups were fed the components of the present invention for 12 weeks, and the mice were subjected to the GSP test.

[0224] 5.1.4 Blood sample processing After completion, the rats were sacrificed under ether anesthesia, blood was collected from the tail artery of the rats, and placed in a test tube containing EDTA. The plasma was centrifuged at 13,000 rpm for 15 minutes at 4 °C, the separated plasma was aliquoted and transferred to a centrifuge tube, and stored at -80 °C.

[0225] 5.1.5 Biochemical analysis Liver injury was quantified by measuring plasma AST and ALT activities. AST and ALT are common indicators of hepatotoxicity and were measured using a Synchron LXi 725 system (Beckman Instruments, Inc., USA).

[0226] 5.1.6 Optical microscopy After sacrificing the rats, histological analysis was performed. Liver samples were fixed in a phosphate buffer solution containing 10% formaldehyde, dehydrated, embedded in paraffin, sections with a thickness of 5 μm were prepared, then stained with hematoxylin and eosin, and periodic acid Schiff stain (PAS) was performed. The stained sections were observed under an optical microscope.

[0227] 5.1.7 Quantitative Tests of Liver Function After the study was completed, GSP tests were performed on all rats. A galactose solution at a concentration of 0.4 g / ml body weight (0.5 g / kg) was injected intravenously into the rats within 30 seconds. Blood samples were collected from the tail artery of the rats at 5, 10, 15, 30, 45, and 60 minutes after injection. Colorimetric galactose dehydrogenase was used to quantify the concentration of galactose, and the test concentration range was 50 to 1,000 μg / ml. The within-day variation for each concentration was calculated using the standard deviation and coefficient of variation (CV), and the maximum allowable coefficient of variation was 10% CV. The between-day variation was examined by comparing the slopes and intercepts of the calibration curves. GSP was the blood galactose concentration obtained at 60 seconds after stopping the 30-second injection.

[0228] 5.1.8 Statistical Analysis All data were expressed as mean ± standard deviation (SD), and ANOVA was used to calculate the results to determine significance. Calculations were performed using the Statistical Package for the Social Sciences (version 13, SPSS Inc.). Subsequently, post hoc tests were used to examine the least significant difference for multiple comparisons to confirm the significant differences between groups. When p < 0.05, the mean differences between groups were significant.

[0229] 5.2 Results

[0230] 5.2.1 Mannitol, Sucralose, and Other Ingredients Can Effectively Treat APAP-Induced Liver Injury The results are shown in Table 2.

[0231] Table 2

[0232]

[0233]

[0234]

[0235] *p < 0.05, **p < 0.??, ***p < 0.005: Comparison of each experimental group with the APAP control group

[0236] The results showed that liver injury occurred in the APAP hepatotoxicity group. In contrast, this liver injury and survival rate could be improved in a dose-dependent manner by using mannitol and / or sucralose. In particular, the combination of mannitol and sucralose achieved a synergistic effect; the results were similar to those of the normal control group and even better than those of the positive control group treated with NAC as the standard treatment. In addition, other ingredients including Aerosil 200, sodium starch glycolate, crospovidone, microcrystalline cellulose, and povidone K-30 were also found to be effective in treating liver injury and were also better than the positive control group treated with NAC as the standard treatment.

[0237] The improved results were also reflected in the corresponding liver tissues.

[0238] Figure 4 The results of histological analysis are shown. Liver tissue sections from rats in the APAP hepatotoxicity group showed that hepatocytes around the central vein were damaged, with visible vacuolization and a decrease in the number of cell nuclei. Some hepatocytes even showed signs of necrosis, and the liver injury was more severe compared to hepatocytes from rats in the normal control group ( Figure 4 B). Conversely, the liver structure of the control group rats was normal, with intact hepatocytes arranged in order and no vacuolization ( Figure 4 A). In the liver sections of the experimental groups treated with mannitol and / or sucralose, the hepatocytes were relatively intact, with visible nuclei and less vacuolization ( Figure 4 D, E, F, G, H). In particular, the combination of mannitol and sucralose achieved the best protective effect ( Figure 4 G); the results were even better than those of the positive control group treated with the standard NAC treatment ( Figure 4 C).

[0239] 5.2.2 Mannitol can effectively treat CCl4-induced liver injury

[0240] The results are shown in Table 3.

[0241] Table 3

[0242]

[0243] Statistical analysis: Anova and LSD tests.

[0244] ***p < 0.005, **p < 0.01, *p < 0.05, comparison between the experimental group and the CCl4 control group.

[0245] The results showed that liver injury occurred in the CCl4 control group. Conversely, this liver injury could be improved by using mannitol.

[0246] Example 6: Determination of fatty liver

[0247] 6.1 Materials and methods

[0248] 6.1.1 Cell lines and cell culture media

[0249] The activities of various components (including mannitol, sucralose, and other components) described herein for reducing fat content were analyzed by using the human hepatoma cell line Hep G2.

[0250] Prepare DMEM medium numbered A - F listed in Table 4 for subsequent experiments using Dulbecco's Modified Eagle's Medium (DMEM).

[0251] Table 4: Preparation of DMEM medium numbered A - F

[0252]

[0253]

[0254] Store DMEM medium numbered A - F at 2 - 8°C and warm it in a 37°C water bath before the experiment.

[0255] 6.1.2 Cell counting and viability test Dead cells absorb 0.4% trypan blue and become colored; while live cells exclude certain dyes due to their intact cell membranes and appear transparent. Mix 100 μl of cell suspension evenly with an equal volume of 0.4% trypan blue to form a mixture. Add some of the mixture (about 20 μl) to the grooves on a hemocytometer, then cover with a coverslip and observe under an optical microscope. Live cells are not stained and dead cells appear blue.

[0256] 6.1.3 Induce fatty liver cells from cells of the HepG2 cell line Incubate the HepG2 cell line (15×10 6 cells) in DMEM medium numbered B, incubate in an incubator at 37°C with 5% CO2 for 24 hours, then culture in DMEM medium numbered C (serum - free medium) for 24 hours, and finally culture in DMEM medium numbered D (containing oleate / albumin complex) for another 48 hours to induce the HepG2 cell line to form fatty liver cells.

[0257] 6.1.4 Treatment of each group of fatty liver cells Divide the HepG2 cell line into six groups, including: (1) blank group: no treatment; (2) DMSO group: cells from the blank group are treated with dimethyl sulfoxide (DMSO); (3) control group: fatty liver cells are induced with oleic acid; (4) vector group: fatty liver cells induced with oleic acid are further treated with DMSO; (5) positive control group: fatty liver cells are treated with silymarin; and (6) test group: fatty liver cells are treated with various compounds of the present invention.

[0258] 6.1.5 Determination of triglyceride (TG) in cells After incubation for 72 hours, the treated cells from each group were washed twice continuously in PBS, and then incubated with 0.5 ml of trypsin / EDTA for 3 minutes. After that, the cells were scraped with 2 ml of PBS and then transferred to a centrifuge tube for sonication. A 20 μl volume of cell extract was taken to measure the protein content. A commercially available reagent kit (Randox) was used for TG determination. The TG content obtained above was divided by the protein content, and the resulting ratio represents the relative content of TG in the cells.

[0259] 6.1.6 Selection of experimental animals B6 mice recommended in the specification "Methods for Evaluating the Hepatoprotective and Health Care Effects of Health Foods" published by the health management agency in Taiwan, China, were used for animal experiments. More than four mice were used in each group for the pretest, and more than twelve mice were used in each group for the confirmation test. Male mice weighing 18 - 23 g, bred in an animal house at 23 ± 2 °C with a relative humidity of 55 ± 15% under a normal light / dark cycle (lights on from 7:00 am to 7:00 pm / off from 7:00 pm to 7:00 am), were purchased from BioLASCO Co., Ltd. (Taipei, Taiwan, China) and placed in the experimental animal center of a medical research institution in Taiwan, China. Animal experiments were conducted according to the animal experiment guidelines of the health research institution in Taiwan, China. The mice were fed a normal diet at 3 - 5 g / day, with unlimited access to water for 1 - 2 weeks, and their health status was studied. The body weight of the mice was recorded once a week.

[0260] 6.1.7 Grouping of animals The tested animals were randomly divided into a blank group, a high-fat diet control group (High Fat Diet, HFD), a positive control group (Positive Control, PS), and a test group. The animals in the blank group were fed a normal diet. The animals in the HFD group were fed a high-fat diet. The animals in the PS group were fed a high-fat diet and additionally fed silymarin (5 mg / kg / day) by gavage. The animals in the test group were fed a high-fat diet and additionally fed the test compound by gavage.

[0261] 6.1.8 Test method

[0262] The animals in the blank group were fed a normal diet ad libitum for 12 weeks, while the animals in the HFD group, PS group, and test group were fed a high-fat diet ad libitum for 12 weeks. After 8 weeks of feeding, deionized water was added to the animals in the blank group and HFD group once a day by gavage; the animals in the PS group were fed silymarin once a day by gavage; and the animals in the test group were fed the test compound once a day by gavage for a period of 4 or 8 weeks.

[0263] Before the test and at the eighth, twelfth, and sixteenth weeks after the test, blood was collected from the cheek or heart. At the end of the experiment, all the mice were weighed, then sacrificed, and blood was collected from the cheek or heart simultaneously. The blood samples of the mice were left to clot at room temperature for 1 hour, and then centrifuged at 15,700×g at 4°C for 5 minutes in a refrigerated centrifuge to separate the serum. Subsequently, biochemical indicators of liver function, including aspartate transaminase (AST), alanine transaminase (ALT), triglyceride (TG), total cholesterol (TCHO / TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C), were detected using an automatic blood biochemical analyzer.

[0264] In addition, abdominal fat and liver samples were removed from the sacrificed mice's abdomen and weighed to compare the weights of the fat and liver, and to obtain the ratio of liver weight to body weight. Two tissue blocks with a volume of approximately 1 cm 3 were cut from the largest right hepatic lobe, fixed in 10% neutral formaldehyde solution, then embedded in paraffin and sectioned. The cut sections were stained with H&E for histopathological observation. In addition, the remaining liver was frozen for storage and the contents of triglyceride and total cholesterol in the liver were detected. Additionally, the liver function of each group of animals was analyzed by the galactose single-point method, which is recognized and recommended by the US Food and Drug Administration (FDA) and the health management agency in Taiwan, China, for clinical use to quantify the remaining liver function. At the end of the experiment, each animal was given 0.5 g of galactose ( 0.4 g / mL) / kg of animal by intravenous injection. One hour after administration, approximately 0.5 ml of whole blood was taken with a filter paper to analyze the liver function of the mice. The higher the GSP value, the worse the remaining liver function. (FDA: "Industrial Guideline: Pharmacokinetics in Impaired Patients, Study Design, Data Analysis, and Effects on Dosage and Labeling, 2003".)

[0265] 6.1.9 Histopathological tissue sectioning:

[0266] At the end of the experiment, all the mice were sacrificed. One tissue block with a volume of approximately 1 cm 3 was cut from the largest right hepatic lobe, fixed in 10% neutral formaldehyde, and then dehydrated and cleared in ethanol (30, 50, 70, 95, 99.5%) and xylene at various concentrations. Then, the xylene was replaced with a hot paraffin solution. Finally, the tissue was embedded in a paraffin solution. The finished paraffin sample was cut into 5-μm-thick paraffin sections using a microtome. The sections were attached to clean glass slides, dried at 37°C, and then stained using the H&E staining method.

[0267] 6.1.10 Hematoxylin and eosin staining (H&E)

[0268] The liver tissue sections were dewaxed in xylene for 30 minutes, and then successively rehydrated in 99.5%, 95%, 70%, 50% and 30% aqueous ethanol for 30 minutes each. After soaking in distilled water for 10 minutes, the sections could be stained. First, the sections were immersed in hematoxylin for 30 seconds to stain the cell nuclei, then washed with distilled water for several minutes, and then stained with eosin for 2 - 5 minutes and washed with distilled water again for several minutes. After staining, the sections were dehydrated twice in 50%, 70%, 95% and 100% aqueous ethanol for 30 seconds each time, cleared twice in xylene, and finally sealed and stored with a mounting medium.

[0269] 6.1.11 Histopathological Observation

[0270] When there is persistent liver injury, to observe the changes in injury, fat accumulation, necrosis or fibrosis in hepatocytes, the liver tissue is stained with H&E to evaluate the degree of liver fat accumulation. All histopathological sections were excised from the same location on the largest right lobe of the liver to eliminate bias in subjective observation, and then subjected to pathological staining. For the semi - quantitative analysis evaluation in pathology, it must be confirmed by a physician or veterinary pathologist, who performs a double - blind analysis to score (NAS score) and compares all the sections without knowing the experimental design. Finally, the difference analysis of each group is performed by statistical methods.

[0271] 6.1.12 Analysis of Liver Antioxidant Capacity Approximately 0.1 g of liver tissue was taken from the sacrificed animals and homogenized by centrifugation in a biological homogenizer for 10 minutes. Nine times the weight (w / w) of buffer (pH 7.4, 50 mmol / L Tris - HCl, 180 mmol / L KCl) was added to the homogenized tissue, and then it was thoroughly mixed with a shaker for standby. The obtained liver tissue homogenate solution sample was used to analyze various members in the liver antioxidant system, including glutathione peroxidase (GPx), glutathione (GSH), glutathione reductase (Grd) and superoxide dismutase (SOD). The methods for related analysis can be found in known literature, for example, the draft of "Methods for Evaluating the Hepatoprotective and Health - Care Effects of Health Foods" published by the health management agency in Taiwan, China.

[0272] 6.1.13 Statistical Analysis All data were expressed as mean ± standard deviation (SD). One - way analysis of variance (ANOVA) was calculated using the Statistical Package for the Social Sciences, version 13, SPSS Inc., to determine the statistically significant differences in the experimental results. Then, multiple comparisons were performed using the least significant difference method in the post - hoc test to confirm the significant differences between groups. When p < 0.05, the mean difference between groups was judged to be significant.

[0273] 6.2 Results

[0274] 6.2.1 Cell Experiment In the cell experiment, the results of measuring the decrease in TG content in HepG2 cells in the positive control group (silymarin) are shown in Table 5.

[0275] Table 5: Effect of silymarin on reducing TG content in HepG2 adipocytes of the positive control group

[0276]

[0277] Table 6 shows the results of measuring the reduction in TG content in HepG2 adipocytes using a constant concentration of the test compound. As shown, under constant concentration conditions, the test compound exhibited varying degrees of TG-reducing effects in steatotic hepatocytes derived from induced HepG2 cells relative to the control group. The TG reduction rate (%) was calculated using the following equation: [1 - (TG content in the test group - TG content in the blank group) / (TG content in the oleic acid-induced group - TG content in the blank group)] × 100%.

[0278] Table 6: TG content in fatty liver cells reduced by test compounds

[0279]

[0280]

[0281]

[0282] Table 6-1: Some test compounds from Table 6 that can reduce TG content in fatty liver cells

[0283]

[0284]

[0285] Table 6-2: Some test compounds (flavonoids) from Table 6 that reduce TG content in fatty liver cells

[0286]

[0287]

[0288] Table 6-3: Some test compounds (excipients) from Table 6 that reduce TG content in fatty liver cells

[0289] Test substance (1.0 μM) TG reduction rate (%) Sodium dodecyl sulfate 38.73±4.65 Sucralose 26.68±2.93 Mannitol 22.35±5.74 Sorbitol 20.06±2.57 Saccharin 17.53±6.96 Glycerol 16.23±4.25 Sodium benzoate 14.35±4.86 Red oxide 13.59±2.08 Butylated hydroxyanisole 6.21±3.8 Sodium cyclohexanesulfamate 4.77±4.49 Menthol 66.24±1.87 Citric acid 2.55±4.43 Lemon oil 0.56±1.07 Pregelatinized starch 7.18±13.41 Sorbic acid 2.03±1.96

[0290] 6.2.2 Animal experiments

[0291] In animal experiments, all animals except those in the blank group fed with normal feed were treated to induce fatty liver. After eight weeks, in addition to the original feed, each group of animals was given different treatments for four or eight weeks. Animals in the blank group and the HFD group were fed with deionized water; animals in the PS group were fed with silymarin; and animals in the test groups were fed with different test compounds, including puerarin, phloridzin, eriodictyol, sucralose, mannitol, saccharin, hesperetin, menthol, and their combinations.

[0292] 6.2.2.1 Effects on animal body weight, liver weight, and body fat weight and safety evaluation of test compounds

[0293] The results from the animal experiments showed that the liver weight, body fat weight, and weight gain of each group of animals were as listed in Tables 7-1 and 7-2.

[0294] Table 7-1: Analysis results of liver weight and body fat weight caused by test compounds

[0295]

[0296]

[0297]

[0298] Table 7-2: Analysis results of weight gain caused by test compounds

[0299]

[0300]

[0301] The results showed that the abdominal fat weight of animals induced with fatty liver increased. Among the test compounds administered individually, mannitol, menthol, and sucralose could significantly reduce the abdominal fat weight of animals.

[0302] In addition, no abnormal conditions were observed in the animals of the test groups after the test compounds were given. No animals died during the test. After the test, autopsy examinations were performed on the sacrificed animals, and no diseases or clinical symptoms caused by the test compounds were observed. Therefore, the test compounds are safe.

[0303] 6.2.2.2 Test compounds effectively reduce lipids in the liver

[0304] Figure 5 Shown are mice induced to exhibit fatty liver, where hepatocytes near the porta hepatis region (including bile ducts, portal veins, and hepatic arteries) are covered with many large vesicular fat droplets, and hepatocyte swelling is observed, indicating that an animal model of fatty liver has been successfully established by the induction method.

[0305] The results of animal experiments showed that after 4 weeks or 8 weeks of administration, various test compounds exhibited the effect of reducing lipids in the livers of animals. The results are shown in Tables 8-1 and 8-2.

[0306] Table 8-1: Test compounds can reduce hepatic lipids in animals (4-week dosing period)

[0307]

[0308]

[0309] Table 8-2: Test compounds can reduce hepatic lipids in animals (8-week dosing period)

[0310]

[0311] The results showed that triglyceride (TG) and total cholesterol (TC) levels increased in the livers of mice induced with fatty liver. Among the test compounds administered individually, hesperidin, puerarin, shikonol, phloridzin, mannitol, menthol, and sucralose significantly reduced triglyceride (TG) in the liver. In particular, after 4 weeks of treatment with eriodictyol, an excellent effect was achieved with a reduction of approximately 67% (p < 0.005) in the triglyceride (TG) content in the liver. In addition, hesperetin, eriodictyol, phloridzin, mannitol, menthol, sucralose, and saccharin significantly reduced total cholesterol (TC) in the liver. Specifically, after 4 weeks of treatment with saccharin, an excellent effect was achieved with a reduction of approximately 56% (p < 0.005) in the total cholesterol (TC) content in the liver.

[0312] When combinations of two test compounds were administered, the combinations of saccharin and mannitol, menthol and mannitol, sucralose and mannitol, eriodictyol and mannitol, or eriodictyol and sucralose significantly reduced triglyceride (TG) in the liver. In particular, after 4 weeks of treatment with the combination of menthol and mannitol, an excellent effect was achieved with a reduction of approximately 77% (p < 0.005) in the triglyceride (TG) content in the liver; and after 8 weeks of treatment with the combination of eriodictyol and sucralose, an excellent effect was achieved with a reduction of approximately 78% (p < 0.005) in the triglyceride (TG) content in the liver. In addition, the combinations of sucralose and mannitol, eriodictyol and mannitol, or eriodictyol and sucralose significantly reduced the total cholesterol (TC) content in the liver, and after 8 weeks of treatment with the combination of eriodictyol and sucralose, an excellent effect was achieved with a reduction of approximately 77% (p < 0.005) in the total cholesterol (TC) content in the liver.

[0313] When a combination of three test compounds is administered, the combination of menthol, mannitol, and eriocitrin or the combination of sucralose, mannitol, and eriocitrin can significantly reduce triglycerides (TG) in the liver. In particular, after 8 weeks of treatment with the combination of sucralose, mannitol, and eriocitrin, an excellent effect of reducing the triglyceride (TG) content in the liver by about 79% (p < 0.005) can be achieved. In addition, the combination of sucralose, mannitol, and eriocitrin can significantly reduce total cholesterol (TC) in the liver.

[0314] 6.2.2.3 Test compounds effectively reduce liver injury

[0315] 6.2.2.3.1 Effects of reducing liver fat and liver injury in liver tissue The results of animal experiments showed that multiple test compounds exhibited the efficacy of reducing liver fat and liver tissue injury during the 4-week test period. Figure 5 Liver tissue injury of animals with fatty liver is shown. Liver tissue injury includes many large vesicular fat droplets covering hepatocytes and hepatocyte swelling near the porta hepatis area (including bile ducts, portal veins, and hepatic arteries). In contrast, after treatment with silymarin, menthol, eriocitrin, or mannitol for 4 weeks, the large vesicular fat droplets in hepatocytes in liver tissue sections were significantly reduced. A part of small fragmented fat droplets was still observed in the mice treated with silymarin, but the liver tissue type of the mice treated with menthol, eriocitrin, or mannitol was close to that of the animals in the blank group, which represents mild fatty liver disease. In addition, the results of NAS scores are shown in Table 9.

[0316] Table 9: Test compounds can reduce the liver injury condition of animals

[0317]

[0318]

[0319] NAS (Non-alcoholic Fatty Liver Activity Score) represents the activity score of non-alcoholic fatty liver disease [Hepatology, June 2005; Vol. 41, No. 6: pp. 1313-21], and comprehensively evaluates the degree of steatosis, lobular inflammation, and hepatocyte ballooning. The scoring table is shown in Table 10. A high score indicates severe liver injury.

[0320]

[0321]

[0322] The results showed that liver tissue damage occurred in mice induced with fatty liver (increased NAS score). Among the test compounds administered individually, eriodictyol and mannitol could significantly reduce liver injury. Notably, when a combination of two compounds was administered, the combination of menthol and mannitol achieved excellent results. Almost no liver injury occurred. Its NAS score was the same as that of the blank group.

[0323] 6.2.2.3.2 Effect of reducing liver dysfunction The results of animal experiments showed that during the 4-week or 8-week dosing period, various test compounds exhibited the effect of alleviating liver dysfunction in animals. The results are shown in Tables 11-1 and 11-2.

[0324] Table 11-1: Test compounds can reduce liver dysfunction in animals (dosing period is 4 weeks)

[0325]

[0326]

[0327]

[0328] Table 11-2: Test compounds can reduce liver dysfunction in animals (dosing period is 8 weeks)

[0329]

[0330] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are most commonly used as enzyme indicators to reflect liver biochemical dysfunction. Under normal circumstances, these enzymes are present in hepatocytes. However, when hepatocytes are damaged, they will leak out. An increase in the ALT and AST values in serum usually indicates liver inflammation and liver dysfunction.

[0331] The results showed that animals induced with fatty liver (increased ALT and AST values) suffered from liver dysfunction. Among the test compounds administered individually, hesperetin, puerarin, eriodictyol, phloridzin, mannitol, menthol, sucralose, and saccharin could all significantly reduce the ALT and AST values. In particular, after treatment with mannitol for 4 weeks, excellent results were achieved with a reduction of approximately 64% (p < 0.005) in the ALT value and approximately 60% (p < 0.005) in the AST value.

[0332] When a combination of two test compounds is administered, the combination of menthol and mannitol, and the combination of eriocitrin and sucralose can significantly reduce the ALT value. In addition, the combination of menthol and mannitol, the combination of sucralose and mannitol, or the combination of saccharin and mannitol can significantly reduce the AST value. In particular, after 4 weeks of treatment with the combination of menthol and mannitol, excellent effects of reducing the ALT value by about 76% (p < 0.005) and the AST value by about 62% (p < 0.005) can be achieved.

[0333] When a combination of three test compounds is administered, the combination of sucralose, mannitol, and eriocitrin can significantly reduce the ALT value (p < 0.005).

[0334] 6.2.2.4 Test compounds can improve the antioxidant activity of the liver

[0335] The results of animal experiments showed that during the 4-week test period, various test compounds showed the effect of improving the antioxidant activity of the animal liver. The results are shown in Tables 12-1 and 12-2.

[0336] Table 12-1: Test compounds can improve the antioxidant activity of the animal liver (Gpx and GSH)

[0337]

[0338]

[0339] Table 12-2: Test compounds can improve the antioxidant activity of the animal liver (Grd and SOD)

[0340]

[0341] Gpx, GSH, Grd, and SOD are common members of the liver antioxidant system, and these members can reduce the oxidative stress in the liver and prevent liver damage caused by oxidative stress. An increase in the values of Gpx, GSH, Grd, and SOD indicates that the liver maintains better antioxidant activity.

[0342] The results showed that the antioxidant activity of fatty liver-induced mice was reduced. Among the test compounds administered individually, hesperetin, puerarin, eriocitrin, phloridzin, mannitol, and sucralose could all significantly improve the antioxidant activity. In particular, after 4 weeks of treatment with mannitol, excellent effects of a significant increase in the contents of Gpx, GSH, Grd, and SOD (p < 0.005) were achieved.

[0343] In summary, test compounds (including mannitol and sucralose, etc.) can reduce the fat content in the liver, reduce liver damage, and improve the antioxidant activity of the liver. These compounds have been proven to be safe through animal experiments and have been found to have the potential to be developed into health foods or drugs for reducing liver fat and improving related diseases, such as fatty liver disease, acute and chronic alcoholic fatty liver disease, acute and chronic non-alcoholic fatty liver diseases (NAFLD), acute and chronic alcoholic hepatitis, acute and chronic non-alcoholic steatohepatitis, non-alcoholic cirrhosis and alcoholic cirrhosis (ICD-9-CM diagnostic codes: 571.8, 571.0, 571.1, 571.2, 571.3, 571.4, 571.5, 571.9).

Claims

1. A compound represented by formula (II), R1-O-X-(CH2) m -X-O-R2 of formula (II), wherein X is C=O; R1 and R2 are the same or different and are selected from the group consisting of: hydrogen, a polyol group, and (G) p a saccharide group of, the polyol group being -CH2(CHOH) n CH2OH, where n is an integer from 1 to 18, where G is a monosaccharide residue and p is an integer from 1 to 100, where at least one hydroxyl group in (G) p is substituted by a halogen atom, where when R1 is hydrogen, then R2 is not hydrogen; when R1 and R2 are polyol groups, then n is not simultaneously 1; and m is an integer from 3 to 40, or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein n in the polyol group is an integer from 2 to 18.

3. The compound according to claim 1, wherein two or more of the hydroxyl groups in (G) p are substituted by halogen atoms.

4. The compound according to claim 1, wherein the monosaccharide residue is a hexose.

5. The compound according to claim 4, wherein the hexose is selected from the group consisting of aldohexoses and ketohexoses.

6. The compound according to claim 1, wherein the sugar group R1 or R2 is represented by -G1-O-G2, where G1 and G2 are the same or different and are selected from the group consisting of aldohexoses and ketohexoses, and at least one hydroxyl group in G1 or at least one hydroxyl group in G2 is substituted by a halogen atom.

7. The compound according to claim 6, wherein the halogen atom is selected from the group consisting of chlorine, bromine, and iodine.

8. The compound according to claim 7, wherein the halogen atom is chlorine.

9. The compound according to claim 6, wherein G1 is glucose with one hydroxyl group substituted by chlorine; and G2 is fructose with two hydroxyl groups substituted by chlorine.

10. The compound according to claim 6, wherein R1 or R2 is represented by formula (Ia) 11. The compound according to claim 2, wherein m is an integer from 3 to 12.

12. A compound represented by formula (II), R1-O-X-(CH2) m -X-O-R2 of formula (II), wherein X is C=O; R1 and R2 are the same or different and are selected from the group consisting of: hydrogen and (G) p a sugar group of, the polyol group being -CH2(CHOH) n CH2OH, where n is an integer from 1 to 18, where G is a monosaccharide residue and p is an integer from 1 to 100, where at least one hydroxyl group in (G) p is substituted by a halogen atom, where when R1 is hydrogen, then R2 is not hydrogen; when R1 and R2 are polyol groups, then n is not simultaneously 1; and m is an integer from 3 to 40, or a pharmaceutically acceptable salt thereof.

13. The compound according to claim 12, wherein two or more of the hydroxyl groups in (G) p are substituted by halogen atoms.

14. The compound according to claim 12, wherein the monosaccharide residue is a hexose.

15. The compound according to claim 14, wherein the hexose is selected from the group consisting of aldohexoses and ketohexoses.

16. The compound according to claim 12, wherein the sugar group R1 or R2 is represented by -G1-O-G2, where G1 and G2 are the same or different and are selected from the group consisting of aldohexoses and ketohexoses, and at least one hydroxyl group in G1 or at least one hydroxyl group in G2 is substituted by a halogen atom.

17. The compound according to claim 16, wherein the halogen atom is selected from the group consisting of chlorine, bromine, and iodine.

18. The compound according to claim 17, wherein the halogen atom is chlorine.

19. The compound according to claim 16, wherein G1 is glucose with one hydroxyl group substituted by chlorine; and G2 is fructose with two hydroxyl groups substituted by chlorine.

20. The compound according to claim 16, wherein R1 or R2 is represented by formula (Ia) 21. The compound according to claim 12, wherein m is an integer from 3 to 12.

22. A compound represented by formula (II), R1-O-X-(CH2) m -X-O-R2 of formula (II), wherein X is C=O; R1 and R2 are the same or different and are selected from the group consisting of: hydrogen, a polyol group, and (G) p a sugar group, the polyol group being a 6-carbon polyol, a 12-carbon polyol, or an 18-carbon polyol, where G is a monosaccharide residue and p is an integer of 1 or 2, wherein at least one hydroxyl group in (G) p is substituted by a halogen atom, where when R1 is hydrogen, then R2 is not hydrogen; when R1 and R2 are polyol groups, then n is not simultaneously 1; and m is an integer from 3 to 40, or a pharmaceutically acceptable salt thereof.

23. The compound according to claim 22, wherein the polyol group is a 6-carbon polyol.

24. The compound according to claim 22, wherein two or more of the hydroxyl groups in (G) p are substituted by halogen atoms.

25. The compound according to claim 22, wherein the monosaccharide residue is a hexose.

26. The compound according to claim 25, wherein the hexose is selected from the group consisting of aldohexoses and ketohexoses.

27. The compound according to claim 22, wherein the glycosyl group R1 or R2 is represented by -G1-O-G2, wherein G1 and G2 are the same or different and are selected from the group consisting of aldohexoses and ketohexoses, and at least one hydroxyl group in G1 or at least one hydroxyl group in G2 is substituted by a halogen atom.

28. The compound according to claim 27, wherein the halogen atom is selected from the group consisting of chlorine, bromine, and iodine.

29. The compound according to claim 28, wherein the halogen atom is chlorine.

30. The compound according to claim 27, wherein G1 is glucose with one hydroxyl group substituted by chlorine; and G2 is fructose with two hydroxyl groups substituted by chlorine.

31. The compound according to claim 27, wherein R1 or R2 is represented by formula (Ia) 32. The compound according to claim 23, wherein m is an integer from 3 to 12.

33. The compound according to claim 23, wherein m is 4.

34. The compound according to claim 23, wherein m is 4 and p is 2.

35. The compound according to claim 22, which is selected from the group consisting of: ((2R,3R,4R,5R,6R)-6-(((2R,5R)-2,5-bis(chloromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)oxy)-3-chloro-4,5-dihydroxytetrahydro-2H-pyran-2-yl)methyl ((2R,3R,4R)-2,3,4,5,6-pentahydroxyhexyl) adipate of formula 1 36. The compound according to claim 22, which is C6-mannitol of formula 2 37. A pharmaceutical composition comprising the compound according to any one of claims 1 to 36 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

38. The pharmaceutical composition according to claim 37, wherein the pharmaceutical composition further comprises one or more additional reagents selected from the group consisting of: i) The first active agent is selected from the group consisting of: polyethylene glycol sorbitan monolaurate, microcrystalline cellulose, dicalcium phosphate dihydrate, Brij 35, saccharin, mannitol, Cremophor RH40, sucralose, crospovidone, sodium starch glycolate, Eudragit S100, sodium carboxymethylcellulose cross-linked, Pluronic F68, menthol, low-substituted hydroxypropyl cellulose, pregelatinized starch, Dextrates NF hydrate, citric acid, Cremophor EL, Aerosil 200, Myrj52, sorbic acid, lemon oil, hydroxypropyl cellulose, sorbitol, acesulfame potassium, hydroxypropyl methylcellulose, lactose monohydrate, maltodextrin, Brij 58, Brij 76, Tween 80, Tween 40, PEG 400, PEG 4000, PEG 8000, Span 60, sodium benzoate, hydroxyethyl methylcellulose, methylcellulose, Span 80, sodium cyclohexanesulfamate, glyceryl behenate, red oxide, glyceryl monostearate, copovidone K28, starch acetate, magnesium stearate, sodium lauryl sulfate, povidone K30, PEG2000, and N-acetylcysteine and any combination thereof; ii) The second active agent is selected from the group consisting of: sodium dodecyl sulfate, menthol, sucralose, mannitol, sorbitol, saccharin, glycerol, sodium benzoate, red oxide, pregelatinized starch, sodium cyclohexanesulfamate, sorbic acid, lemon oil, citric acid, butylated hydroxyanisole, wolfberry, isovitexin, eriodictyol, ergosterol, β-myrcene, hypercholesterolemia, (+)-catechin, galangin, morin, sciadopitysin, apigenin-7-glucoside, (+)-taxifolin, trans-cinnamic acid, evening primrose inclusion, mongolicain, xylitol, luteolin, swertiamarin, puerarin, phloridzin, sinensetin, (-)-epigallocatechin, kaempferol, ursolic acid, silymarin, (+)-limonene, hesperidin, (-)-epicatechin-3-gallate, silybin, formononetin, ethyl myristate, eicosapentaenoic acid, wogonin, povidone K-30, protocatechuic acid, umbelliferone, hesperetin, nordihydroguaiaretic acid, neohesperidin, naringin, (-)-epicatechin, glycyrrhizin, baicalin, quercitrin, baicalein, and any combination thereof; and Any combination of i) and ii).

39. The pharmaceutical composition according to claim 38, wherein the one or more additional agents are selected from the group consisting of: dehydrated dicalcium phosphate, menthol, mannitol, sucralose, N-acetylcysteine and any combination thereof.

40. The pharmaceutical composition according to claim 38, wherein the one or more additional reagents are selected from the group consisting of: 1) a combination of saccharin and mannitol, 2) a combination of menthol and mannitol, 3) a combination of sucralose and mannitol, 4) a combination of eriodictyol and mannitol, 5) a combination of eriodictyol and sucralose, 6) a combination of menthol, mannitol and eriodictyol, and 7) a combination of sucralose, mannitol and eriodictyol.

41. Use of a compound according to any one of claims 1 to 36 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for preventing or treating a disease or disorder characterized by increased cytochrome P450 activity or increased free radical content.

42. Use of a compound according to any one of claims 1 to 36 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for preventing or treating organ damage.

43. Use of a compound according to any one of claims 1 to 36 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for preventing or treating hepatotoxicity.

44. Use of a compound according to any one of claims 1 to 36 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for preventing or treating fatty liver, protecting liver function or improving liver diseases caused by fatty liver or other related diseases.

45. The use according to any one of claims 41 to 44, wherein the compound or its pharmaceutically acceptable salt is administered in combination with one or more additional reagents selected from the group consisting of: i) a first active agent selected from the group consisting of: polyethylene glycol sorbitan monolaurate, microcrystalline cellulose, dicalcium phosphate dihydrate, Brij 35, saccharin, mannitol, Cremophor RH40, sucralose, crospovidone, sodium starch glycolate, Eudragit S100, cross-linked carboxymethylcellulose sodium, Pluronic F68, menthol, low-substituted hydroxypropyl cellulose, pregelatinized starch, Dextrates NF hydrate, citric acid, Cremophor EL, Aerosil 200, Myrj52, sorbic acid, lemon oil, hydroxypropyl cellulose, sorbitol, acesulfame potassium, hydroxypropyl methylcellulose, lactose monohydrate, maltodextrin, Brij 58, Brij 76, Tween 80, Tween 40, PEG 400, PEG 4000, PEG 8000, Span 60, sodium benzoate, hydroxyethyl methylcellulose, methylcellulose, Span 80, sodium cyclohexanesulfamate, glyceryl behenate, red oxide, glycerol monostearate, copovidone K28, starch acetate, magnesium stearate, sodium lauryl sulfate, povidone K30, PEG2000, and N-acetylcysteamine and any combination thereof; ii) A second active agent selected from the group consisting of: sodium lauryl sulfate, menthol, sucralose, mannitol, sorbitol, saccharin, glycerol, sodium benzoate, erythrosine, pregelatinized starch, sodium cyclohexanesulfamate, sorbic acid, lemon oil, citric acid, butylated hydroxyanisole, wolfberry fruit, isovitexin, eriodictyol, ergosterol, β-myrcene, hypercholesterolemia, (+)-catechin, galangin, morin, sciadopitysin, apigenin-7-glucoside, (+)-taxifolin, trans-cinnamic acid, evening primrose extract, buddleoside, xylitol, luteolin, swertiamarin, puerarin, phloridzin, sinensetin, (-)-epigallocatechin, kaempferol, ursolic acid, silymarin, (+)-limonene, hesperidin, (-)-epicatechin-3-gallate, silybin, formononetin, ethyl myristate, eicosapentaenoic acid, wogonin, povidone K-30, protocatechuic acid, umbelliferone, hesperetin, nordihydroguaiaretic acid, neohesperidin, naringin, (-)-epicatechin, glycyrrhizin, baicalin, quercitrin, baicalein, and any combination thereof; and any combination of (i) and (ii).

46. The use according to claim 45, wherein the one or more additional reagents are selected from the group consisting of: dicalcium phosphate dehydrate, menthol, mannitol, sucralose, N-acetylcysteamine, and any combination thereof.

47. The use according to claim 45, wherein the one or more additional reagents are selected from the group consisting of: 1) a combination of saccharin and mannitol, 2) a combination of menthol and mannitol, 3) a combination of sucralose and mannitol, 4) a combination of eriodictyol and mannitol, 5) a combination of eriodictyol and sucralose, 6) a combination of menthol, mannitol, and eriodictyol, and 7) a combination of sucralose, mannitol, and eriodictyol.

48. The use according to claim 45, wherein the compound or a pharmaceutically acceptable salt thereof and the one or more additional reagents are administered simultaneously or sequentially.

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