Compositions and methods for decreasing body weight and treating liver disease

Abstract

The present invention relates to methods of decreasing body weight, maintaining body weight, decreasing liver weight, and decreasing levels of triglycerides, total cholesterol, and low-density lipoprotein cholesterol in the blood. The present invention also relates to methods for treating liver disease, e.g., fatty liver disease, in human subjects by administering an activator of sirtuin 6 (SIRT6). Exemplary fatty liver diseases or disorders include, but are not limited to, Metabolic Dysfunction-Associated Steatohepatitis, and hepatic steatosis.

Description

COMPOSITIONS AND METHODS FOR DECREASING BODY WEIGHT ANDTREATING LIVER DISEASESTATEMENT OF PRIORITY

[0001] This application claims the benefit, under 35 U.S.C. §119(e), of U.S. Provisional Application No. 63 / 662,058, filed June 20, 2024, the entire contents of which is incorporated by reference herein in its entirety.FIELD OF THE INVENTION

[0002] The present invention relates to methods of decreasing body weight, maintaining body weight, decreasing liver weight, decreasing levels of triglycerides, total cholesterol, and low- density lipoprotein cholesterol, and treating liver disease in human subjects by administering an activator of SIRT6.BACKGROUND OF THE INVENTION

[0003] Increased body and liver weights, and raised triglycerides, total cholesterol, low-density lipoprotein cholesterol levels, hypertension, high fasting blood glucose, obesity, and insulin resistance (sometimes collectively referred to as metabolic syndrome) are some of the largest issues that affect the modern healthcare system. Additionally, fatty liver disease, such as Metabolic Dysfunction-Associated Steatohepatitis (MASH), and hepatic deficiency can lead to dysregulation of hepatic metabolic homeostasis and increased hepatic lipogenesis, inflammation, fibrogenesis, and steatosis. The group of proteins called sirtuins have been linked to such weight gain and hepatic lipid and metabolic homeostasis. They are shown to play a role in human hepatic functioning. Noticeably, sirtuin 6 (SIRT6) protein levels are decreased in the liver of MASH patients as well as other metabolic disorders.

[0004] Obesity is worldwide health problem that is reaching epidemic proportions. The United States has the highest rates of obesity in the developed world. It was reported in 2010 that 35.7% of US adults are obese. Overweight and obesity are the fifth leading risk for global deaths. A new class of antidiabetic drugs called glucagon-like peptide 1 receptor agonists (GLP-1 RA) has had some success in inducing body weight loss.

[0005] There is a need in the art for effective therapeutics for promoting decreases in body and liver weights, lowering triglyceride, total cholesterol, and low-density lipoprotein cholesterol levels, and treating liver disease.SUMMARY OF THE INVENTION

[0006] Sirtuin 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD+) dependent histone deacetylase / deacylase that is known to have various roles in body weight maintenance, hepatic lipogenesis, and hepatic metabolic homeostasis functions such as regulation of blood and / or hepatic triglycerides, cholesterol, and low-density lipoprotein cholesterol homeostasis, induction of hepatic fatty acid oxidation (FAO) genes, reduction of oxidative stress, reduction of hepatic inflammation, reduction of hepatic fibrosis, and reduction of hepatic steatosis. The importance of SIRT6 is further shown in mice studies using liver-specific SIRT6 knockout (LKO) mice, where the LKO mice are more susceptible to Western diet-induced fatty liver disease compared to WT mice (Dong, 2023). SIRT6 LKO mice have also been shown to exhibit elevated cholesterol levels in the blood and liver, and on the other hand, hepatic SIRT6 overexpression has improved hypercholesterolemia in these mice (Dong, 2023).

[0007] The present invention is based on the determination that the administration of SIRT6 activators leads to broad systemic homeostasis by normalizing body weight, liver weight, levels of triglycerides, total cholesterol, and LDL cholesterol in the blood, and provides a significant therapeutic effect for fatty liver disease and metabolic syndrome. Additionally, the present invention is based on the determination that SIRT6 activators, alone or in combination with weight loss inducing agents such as GLP-1 RAs, and / or estrogen may provide a significant reduction in weight loss for patients in need thereof.

[0008] Thus, one aspect of the invention relates to a method of decreasing body weight in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby decreasing body weight in the subject. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for decreasing body weight in the subject. In some embodiments, the method further comprises administering to the subject an estrogen for further decreasing body weight in the subject.

[0009] Another aspect of the invention relates to a method of maintaining body weight in a subject in need thereof (e.g., after weight loss due to administration of weight loss drugs, diet, exercise, or a combination thereof), comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby maintaining body weight in the subject. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for maintaining body weight in the subject.

[0010] Another aspect of the invention relates to a method of decreasing triglycerides in the blood in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby decreasing triglycerides in the blood in the subject. In some embodiments, the method further comprises administering to the subj ect a therapeutically effective amount of an additional therapeutic agent for decreasing triglycerides in the blood in the subject.

[0011] Another aspect of the invention relates to a method of decreasing total cholesterol in the blood in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby decreasing total cholesterol in the blood in the subject. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for decreasing total cholesterol in the blood in the subject.

[0012] Another aspect of the invention relates to a method of decreasing low-density lipoprotein cholesterol in the blood in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby decreasing low-density lipoprotein cholesterol in the blood in the subject. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for decreasing low-density lipoprotein cholesterol in the blood in the subject.

[0013] Another aspect of the invention relates to a method of decreasing liver weight in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby decreasing liver weight in the subject. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for decreasing liver weight in the subject.

[0014] Another aspect of the invention relates to a method of treating a liver disease, e.g., a fatty liver disease, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby treating the liver disease in the subject. In someembodiments, the method further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for treating the liver disease in the subject.

[0015] Another aspect of the invention relates to a method of suppressing hepatic lipogenesis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby suppressing hepatic lipogenesis in the subject. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for suppressing hepatic lipogenesis in the subject.

[0016] Another aspect of the invention relates to a method of increasing hepatic fatty acid oxidation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby increasing hepatic fatty acid oxidation in the subject. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for increasing hepatic fatty acid oxidation in the subject.

[0017] Another aspect of the invention relates to a method of reducing oxidative stress in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby reducing oxidative stress in the subject. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for reducing oxidative stress in the subject.

[0018] Another aspect of the invention relates to a method of decreasing hepatic inflammation in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby decreasing hepatic inflammation in the subject. In some embodiments, the method further comprises administering to the subj ect a therapeutically effective amount of an additional therapeutic agent for further decreasing hepatic inflammation in the subject.

[0019] Another aspect of the invention relates to a method of decreasing hepatic steatosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby decreasing hepatic steatosis in the subject. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for decreasing hepatic steatosis in the subject.

[0020] Another aspect of the invention relates to a method of decreasing hepatic fibrosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amountof a SIRT6 activator, thereby decreasing hepatic fibrosis in the subject. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for decreasing hepatic fibrosis in the subject.

[0021] Another aspect of the invention relates to a method of preserving muscle mass in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby preserving muscle mass in the subject. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for preserving muscle mass in the subject.

[0022] These and other aspects of the invention are set forth in more detail in the description of the invention below.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 is a table showing a study design (Model Number: [518510-customized] high fat diet-induced obesity) testing the results of administering SP-624 to high fat diet-induced obese male and female mice compared to high fat diet-induced obese mice administered orlistat and mice fed a normal diet. Male and female C57BL / 6 mice at 6 weeks of age were used in this study. Animals were fed either normal diet (Group 1) or high fat diet (HFD: 20% protein, 60% fat, and 20% carbohydrate; Groups 2-5) for 4 weeks before treatment. Vehicle and test articles were administered by oral gavage (PO) once daily (QD) for 4 weeks (from Day 28 to Day 55) after 4 weeks of HFD feeding (total 8 weeks, from Day 0 to Day 56). The reference compound Orlistat was administered by oral gavage (PO) once daily (QD) for 4 weeks (from Day 28 to Day 55) after 4 weeks of HFD feeding. Vehicle for Orlistate: WFI; vehicle for test articles: 0.5% MC.

[0024] FIG. 2 is a graph showing the results of administering the respective vehicle or test article to male mice fed a high fat diet on body weight over time in comparison to male mice fed a normal diet.

[0025] FIG. 3 is a graph showing the results of administering the respective vehicle or test article to female mice fed a high fat diet on body weight over time in comparison to female mice fed a normal diet.

[0026] FIG. 4 is a graph showing the measured fasting blood glucose levels (mg / dL) of male C57BL / 6 mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to male mice fed a normal diet.

[0027] FIG. 5 is a graph showing the measured total cholesterol levels in the blood (mg / dL) of male C57BL / 6 mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to male mice fed a normal diet.

[0028] FIG. 6 is a graph showing the measured low-density lipoprotein cholesterol levels in the blood (mg / dL) of male C57BL / 6 mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to male mice fed a normal diet.

[0029] FIG. 7 is a graph showing the measured triglyceride levels in the blood (mg / dL) of male C57BL / 6 mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to male mice fed a normal diet.

[0030] FIG. 8 is a graph showing the measured fasting blood glucose levels (mg / dL) of female C57BL / 6 mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to female mice fed a normal diet.

[0031] FIG. 9 is a graph showing the measured total cholesterol levels in the blood (mg / dL) of female C57BL / 6 mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to female mice fed a normal diet.

[0032] FIG. 10 is a graph showing the measured low-density lipoprotein cholesterol levels in the blood (mg / dL) of female C57BL / 6 mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to female mice fed a normal diet.

[0033] FIG. 11 is a graph showing the measured triglyceride levels in the blood (mg / dL) of female C57BL / 6 mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to female mice fed a normal diet.

[0034] FIG. 12 is a diagram showing a dissected mouse subject indicating the scapular fat pad, the inguinal fat pad, the epididymal fat pad, and the epididymus of the mouse.

[0035] FIG. 13 is a graph showing the measured epididymal fat weight (g) of male C57BL / 6 mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to male mice fed a normal diet.

[0036] FIG. 14 is a graph showing the measured inguinal fat weight (g) of male C57BL / 6 mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to male mice fed a normal diet.

[0037] FIG. 15 is a graph showing the measured liver weight (g) of male C57BL / 6 mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or Orlistat compared to male mice fed a normal diet.

[0038] FIG. 16 is a graph showing the calculated epidi dymal fat-to-body weight ratio (%) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to male C57BL / 6 mice fed a normal diet.

[0039] FIG. 17 is a graph showing the calculated inguinal fat-to-body weight ratio (%) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to male mice C57BL / 6 fed a normal diet.

[0040] FIG. 18 is a graph showing the calculated liver-to-body weight ratio (%) of male C57BL / 6 mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to male mice fed a normal diet.

[0041] FIG. 19 is a graph showing the measured peri ovarian fat weight (g) of female C57BL / 6 mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to female mice fed a normal diet.

[0042] FIG. 20 is a graph showing the measured inguinal fat weight (g) of female C57BL / 6 mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to female mice fed a normal diet.

[0043] FIG. 21 is a graph showing the measured liver weight (g) of female C57BL / 6 mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to female mice fed a normal diet.

[0044] FIG. 22 is a graph showing the calculated periovarian fat-to-body weight ratio (%) of female C57BL / 6 mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to female mice fed a normal diet.

[0045] FIG. 23 is a graph showing the calculated inguinal fat-to-body weight ratio (%) of female C57BL / 6 mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to female mice fed a normal diet.

[0046] FIG. 24 is a graph showing the calculated liver-to-body weight ratio (%) of female mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage, or orlistat compared to female mice fed a normal diet.

[0047] FIG. 25 is a table showing the study design for testing the administration of SP-624 and / or Semaglutide to high fat diet-induced obese male mice compared to male mice fed a normal diet. Male C57BL / 6J mice and controls are placed on diet at 6 weeks of age in the Jackson Laboratory. Mice at 15 weeks of age will be purchased from the Jackson Laboratory and fed 3 weeks with High-fat diet (HFD) in PDS. Male C57BL / 6J DIO mice (Strain #:380050, Jackson Laboratory) and C57BL / 6J DIO Control mice (Strain #:380056, Jackson Laboratory) at 18 weeks of age are used in the study. Test article SP-624 will be given by oral gavage (PO) once daily (QD) starting after 12 weeks of HFD feeding for consecutive 4 weeks (Days 0-27)(Groups 3-4). The first day of dosing is designated as Day 0. GLP-1R agonist Semaglutide is administered subcutaneously (SC) once every 3 days starting on Day 0 for a total of ten (10) injections (Days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27)(Groups 5-6). Animals in combo groups (Groups 7-10) receive both SP-624 and Semaglutide. Animals in Group 2 receive vehicle of SP-624 given PO, QD starting after 12 weeks of HFD feeding for consecutive 4 weeks (Days 0-27) as well as the vehicle Semaglutide administered SC once every 3 days starting at Day 0 for a total of ten (10) injections (Days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27). Group 11 : animals receive high dose of SP-624 given PO, QD starting after 12 weeks of HFD feeding for consecutive 8 weeks (Days 0-55) as well as high dose Semaglutide given SC Q3D starting at Day 0 for a total of ten (10) injections (Days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27). Group 12: animals receive vehicle of SP-624 given PO, QD starting after 12 weeks of HFD feeding for consecutive 8 weeks (Days 0-55) as well as high dose Semaglutide given SC Q3D starting at Day 0 for a total of ten (10) injections (Days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27). Vehicle 1 for SP-624: 0.5% MC; vehicle 2 for Semaglutide 20 mM citrate buffer (pH 7.0).

[0048] FIGS. 26A-26B are graphs showing the measured total cholesterol levels in the blood (mg / dL) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 12 weeks, 16 weeks (FIG. 26A), and 20 weeks (FIG. 26B).

[0049] FIGS. 27A-27B are graphs showing the measured triglycerides level in the blood (mg / dL) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 12 weeks, 16 weeks (FIG. 27A), and 20 weeks (FIG. 27B).

[0050] FIGS. 28A-28B are graphs showing the measured alanine transaminase levels in the blood (U / L) of male mice fed a high fat diet after being administered their respective vehicle, SP-624dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 12 weeks, 16 weeks (FIG. 28A), and 20 weeks (FIG. 28B).

[0051] FIGS. 29A-29B are graphs showing the measured lactate dehydrogenase levels in the blood (IU / L) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 12 weeks, 16 weeks (FIG. 29A), and 20 weeks (FIG. 29B).

[0052] FIGS. 30A-30B are graphs showing the measured creatinine levels in the blood (mg / dL) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 12 weeks, 16 weeks (FIG. 30A), and 20 weeks (FIG. 30B).

[0053] FIGS. 31 A- 3 IB are graphs showing the measured fasting blood glucose levels in the blood (mg / dL) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 12 weeks, 16 weeks (FIG. 31 A), and 20 weeks (FIG. 3 IB).

[0054] FIGS. 32A-32B are graphs showing the measured insulin levels in the blood (ng / mL) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 12 weeks, 16 weeks (FIG. 32A), and 20 weeks (FIG. 32B).

[0055] FIGS. 33A-33B are graphs showing the measured leptin levels in the blood (ng / mL) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 12 weeks, 16 weeks (FIG. 33A), and 20 weeks (FIG. 33B).

[0056] FIGS. 34A-34B are graphs showing the measured high density lipoprotein levels in the blood (mg / dL) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 16 weeks (FIG. 34A) and 20 weeks (FIG. 34B).

[0057] FIGS. 35A-35B are graphs showing the measured low density lipoprotein levels in the blood (mg / dL) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 16 weeks (FIG. 35A) and 20 weeks (FIG. 35B).

[0058] FIGS. 36A-36B are graphs showing the measured aspartate transaminase levels in the blood (U / L) of male mice fed a high fat diet after being administered their respective vehicle, SP- 624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 16 weeks (FIG. 36A) and 20 weeks (FIG. 36B).

[0059] FIGS. 37A-37B are graphs showing the measured blood urea nitrogen levels in the blood (mg / dL) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 16 weeks (FIG. 37A) and 20 weeks (FIG. 37B).

[0060] FIGS. 38A-38B are graphs showing the measured serum IL-6 levels in the blood (pg / mL) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 16 weeks (FIG. 38 A) and 20 weeks (FIG. 38B).

[0061] FIGS. 39A-39B are graphs showing the measured serum TNF-a levels in the blood (pg / mL) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 16 weeks (FIG. 39A) and 20 weeks (FIG. 39B).

[0062] FIGS. 40A-40D are graphs showing the measured epididymal fat weight (g) (FIGS. 40A and 40C) or epididymal fat-to-body weight ratio (%) (FIGS. 40B and 40D) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 16 weeks (FIGS. 40A-40B) and 20 weeks (FIGS. 40C-40D). 1 : Sham (normal diet); 2: Vehicle 1, 10 mL / kg QDx28, PO + Vehicle 2, 5 mL / kg Q3Dxl0, SC (HFD); 3: SP-264, 3 mg / kg QDx28, PO (HFD); 4: SP-264, 10 mg / kg QDx28, PO (HFD); 5: Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 6: Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD); 7: SP-264, 3 mg / kg QDx28, PO + Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 8: SP-264, 3 mg / kg QDx28, PO + Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD); 9: SP-264, 10 mg / kg QDx28, PO + Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 10: SP-264, 10 mg / kg QDx28, PO + Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD) (FIGS. 40A and 40B).

[0063] FIGS. 41 A-41D are graphs showing the measured inguinal fat weight (g) (FIGS. 41 A and 41C) or inguinal fat-to-body weight ratio (%) (FIGS. 4 IB and 4 ID) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 16 weeks (FIGS. 41A-41B) and 20 weeks (FIGS.41C-41D). 1 : Sham (normal diet); 2: Vehicle 1, 10 mL / kg QDx28, PO + Vehicle 2, 5 mL / kg Q3Dxl0, SC (HFD); 3: SP-264, 3 mg / kg QDx28, PO (HFD); 4: SP-264, 10 mg / kg QDx28, PO (HFD); 5: Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 6: Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD); 7: SP-264, 3 mg / kg QDx28, PO + Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 8: SP-264, 3 mg / kg QDx28, PO + Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD); 9: SP-264, 10 mg / kg QDx28, PO + Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 10: SP-264, 10 mg / kg QDx28, PO + Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD) (FIGS. 41A and 41B).

[0064] FIGS. 42A-42D are graphs showing the measured liver weight (g) (FIGS. 42 A and 42C) or liver-to-body weight ratio (%) (FIGS. 42B and 42D) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 16 weeks (FIGS. 42A-42B) and 20 weeks (FIGS. 42C-42D). 1 : Sham (normal diet); 2: Vehicle 1, 10 mL / kg QDx28, PO + Vehicle 2, 5 mL / kg Q3Dxl0, SC (HFD); 3: SP-264, 3 mg / kg QDx28, PO (HFD); 4: SP-264, 10 mg / kg QDx28, PO (HFD); 5: Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 6: Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD); 7: SP-264, 3 mg / kg QDx28, PO + Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 8: SP-264, 3 mg / kg QDx28, PO + Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD); 9: SP-264, 10 mg / kg QDx28, PO + Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 10: SP-264, 10 mg / kg QDx28, PO + Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD) (FIGS. 42A and 42B).

[0065] FIGS. 43A-43D are graphs showing the measured gastrocnemius weight (g) (FIGS. 43A and 43 C) or gastrocnemius-to-body weight ratio (%) (FIGS. 43B and 43D) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 16 weeks (FIGS. 43A-43B) and 20 weeks (FIGS. 43C-43D). 1 : Sham (normal diet); 2: Vehicle 1, 10 mL / kg QDx28, PO + Vehicle 2, 5 mL / kg Q3Dxl0, SC (HFD); 3: SP-264, 3 mg / kg QDx28, PO (HFD); 4: SP-264, 10 mg / kg QDx28, PO (HFD); 5: Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 6: Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD); 7: SP-264, 3 mg / kg QDx28, PO + Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 8: SP-264, 3 mg / kg QDx28, PO + Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD); 9: SP-264, 10 mg / kg QDx28, PO + Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 10: SP-264, 10 mg / kg QDx28, PO + Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD) (FIGS. 43A and 43B)

[0066] FIGS. 44A-44D are graphs showing the measured soleus weight (g) (FIGS. 44A and 44C) or soleus-to-body weight ratio (%) (FIGS. 44B and 44D) of male mice fed a high fat diet afterbeing administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 16 weeks (FIGS. 44A-44B) and 20 weeks (FIGS. 44C-44D). 1 : Sham (normal diet); 2: Vehicle 1, 10 mL / kg QDx28, PO + Vehicle 2, 5 mL / kg Q3Dxl0, SC (HFD); 3: SP-264, 3 mg / kg QDx28, PO (HFD); 4: SP-264, 10 mg / kg QDx28, PO (HFD); 5: Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 6: Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD); 7: SP-264, 3 mg / kg QDx28, PO + Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 8: SP-264, 3 mg / kg QDx28, PO + Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD); 9: SP-264, 10 mg / kg QDx28, PO + Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 10: SP-264, 10 mg / kg QDx28, PO + Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD) (FIGS. 44A and 44B).

[0067] FIGS. 45A-45D are graphs showing the measured tibialis anterior weight (g) (FIGS. 45A and 45C) or tibialis anterior-to-body weight ratio (%) (FIGS. 45B and 45D) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 16 weeks (FIGS. 45A-45B) and 20 weeks (FIGS. 45C-45D). 1 : Sham (normal diet); 2: Vehicle 1, 10 mL / kg QDx28, PO + Vehicle 2, 5 mL / kg Q3Dxl0, SC (HFD); 3: SP-264, 3 mg / kg QDx28, PO (HFD); 4: SP-264, 10 mg / kg QDx28, PO (HFD); 5: Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 6: Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD); 7: SP-264, 3 mg / kg QDx28, PO + Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 8: SP-264, 3 mg / kg QDx28, PO + Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD); 9: SP-264, 10 mg / kg QDx28, PO + Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 10: SP-264, 10 mg / kg QDx28, PO + Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD) (FIGS. 45A and 45B).

[0068] FIGS. 46A-46D are graphs showing the measured quadriceps weight (g) (FIGS. 46A and 46C) or quadriceps-to-body weight ratio (%) (FIGS. 46B and 46D) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for 16 weeks (FIGS. 46A-46B) and 20 weeks (FIGS. 46C-46D). 1 : Sham (normal diet); 2: Vehicle 1, 10 mL / kg QDx28, PO + Vehicle 2, 5 mL / kg Q3Dxl0, SC (HFD); 3: SP-264, 3 mg / kg QDx28, PO (HFD); 4: SP-264, 10 mg / kg QDx28, PO (HFD); 5: Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 6: Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD); 7: SP-264, 3 mg / kg QDx28, PO + Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 8: SP-264, 3 mg / kg QDx28, PO + Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD); 9: SP-264, 10 mg / kg QDx28, PO + Semaglutide, 10 nmol / kg Q3Dxl0, SC (HFD); 10: SP-264, 10 mg / kg QDx28, PO + Semaglutide, 30 nmol / kg Q3Dxl0, SC (HFD) (FIGS. 46A and 46B).

[0069] FIGS. 47A-47B are graphs showing the body weight (g) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage compared to male mice fed a normal diet for up to 28 days (FIG. 47A) and 56 days (FIG. 47B).

[0070] FIG. 48 is a graph showing the food intake (g) of male mice fed a high fat diet after being administered their respective vehicle, SP-624 dosage and / or Semaglutide dosage for up to 55 days.

[0071] FIG. 49 is a table and graph showing the oil red O (%) stain in the liver of treatment groups 1-12, as represented in FIG. 25. Data are presented as Mean ± SEM. Group 1 : Sham (Normal diet); Group 2: Vehicle 1 / Vehicle 2 (10 mL / kg, QDx28, PO / 5 mL / kg, Q3Dxl0, SC, HFD; Group 3: SP- 624 low (3 mg / kg, QDx28, PO, HFD); Group 4: SP-624 high (10 mg / kg, QDx28, PO, HFD); Group 5: Semaglutide low (10 nmol / kg, Q3Dxl0, SC, HFD); Group 6: Semaglutide high (30 nmol / kg, Q3Dxl0, SC, HFD); Group 7: SP-624 low / Semaglutide low (3 mg / kg, QDx28, PO / 10 nmol / kg Q3Dxl0, SC, HFD); Group 8: SP-624 low / Semaglutide high (3 mg / kg, QDx28, PO / 30 nmol / kg Q3Dxl0, SC, HFD); Group 9: SP-624 high / Semaglutide low (10 mg / kg, QDx28, PO / 10 nmol / kg Q3Dxl0, SC, HFD); Group 10: SP-624 high / Semaglutide high (10 mg / kg, QDx28, PO / 30 nmol / kg Q3Dxl0, SC, HFD). Group 11 : SP-624 high / Semaglutide high (10 mg / kg, QDx56, PO / 30 nmol / kg Q3Dxl0, SC, HFD). Group 12: Vehicle 1 / Semaglutide high (NA, QDx56, PO / 30 nmol / kg Q3Dxl0, SC, HFD). For Groups 1-10, * / ?<0.05; ** <0.01; Sham (Group 1) vs. Vehicle 1 / Vehicle 2 (Group 2); unpaired Student’s / -test;# / ?<0.05;##><0.01 : Treated (Groups 3-10) vs. Vehicle 1 / Vehicle 2 (Group 2); one-way ANOVA followed by Dunnett’s test. For Groups 11-12, * / ?<0.05; ** <0.01: Group 11 vs. Group 12; unpaired Student’s / -test.DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0072] The present invention is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant invention. Hence, the following specification is intended to illustrate some particular embodimentsof the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

[0073] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0074] 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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0075] All publications, patent applications, patents, nucleotide sequences, amino acid sequences and other references mentioned herein are incorporated by reference in their entirety.Definitions

[0076] As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0077] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0078] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0079] Furthermore, the term “about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0080] As used herein, the transitional phrase “consisting essentially of’ is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic andnovel character! stic(s) of the claimed invention. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0081] By the term “treat,” “treating,” or “treatment of’ (or grammatically equivalent terms) is meant to reduce or to at least partially improve or ameliorate the severity of the subject’s condition and / or to alleviate, mitigate or decrease in at least one clinical symptom and / or to delay the progression of the condition.

[0082] As used herein, the term “prevent,” “prevents,” or “prevention” (and grammatical equivalents thereof) means to delay or inhibit the onset of a disease. The terms are not meant to require complete abolition of disease, and encompass any type of prophylactic treatment to reduce the incidence of the condition or delays the onset of the condition.

[0083] A “treatment effective” or “therapeutically effective” amount as used herein is an amount that is sufficient to provide some improvement or benefit to the subject. Alternatively stated, a “treatment effective” amount is an amount that will provide some alleviation, mitigation, decrease or stabilization in at least one clinical symptom in the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0084] A “prevention effective” amount as used herein is an amount that is sufficient to prevent and / or delay the onset of a disease, disorder and / or clinical symptoms in a subject and / or to reduce and / or delay the severity of the onset of a disease, disorder and / or clinical symptoms in a subject relative to what would occur in the absence of the methods of the invention. Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject.

[0085] “Pharmaceutically acceptable,” as used herein, means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with the compositions of this invention, without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The material would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art (see, c. ., Remington's Pharmaceutical Science,' 21sted. 2005). Exemplary pharmaceutically acceptable carriers for the compositions of this invention include, but are not limited to, sterile pyrogen-free water and sterile pyrogen-free physiological saline solution.Methods of Use

[0086] A first aspect of the invention relates to a method of decreasing body weight in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby decreasing body weight in the subject. In some embodiments, the subject in need thereof is characterized by decreased levels and / or activity of SIRT6 (e.g., the subject in need thereof has 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% decreased levels and / or activity of SIRT6 relative to a control subject that is not overweight).

[0087] In some embodiments, the method may result in a decrease in body weight of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more after the subject is administered a therapeutically effective amount of a SIRT6 activator. In some embodiments, the subject who is administered a SIRT6 activator is one that has been diagnosed and / or suspected of having obesity or being overweight.

[0088] GLP-1 RAs promote the reduction of body weight by mimicking the functions of endogenous glucagon-like peptide 1, acting via modulating hormonal regulation processes. GLP- 1 RAs are shown to stimulate insulin and inhibit glucagon secretion, induce satiety by delaying the rate of gastric emptying and decreasing gastrointestinal (GI) motility, and have a role in the modification of gastric volume in expectation of or response to a meal. Further, studies indicate that there is a more prominent effect of GLP-1 RAs regarding weight reduction in female patients. One possible reason for this result is the higher presence of estrogen in females. In support of this, animal studies have shown that estrogen signaling plays a critical role in GLP-1 RA’s modification of the food-reward aspect of food seeking (Rentzeperi et al., 2022). Without being bound by theory, it is thought that this finding demonstrates that sex hormones are at least partially responsible for the differential responses of GLP-1 RAs between males and females (Rentzeperi et al., 2022). To further increase body weight reduction, administering an estrogen conjugated to a SIRT6 activator or a GLP-1 RA to a patient may more effectively target estrogen delivery to cells expressing GLP-1 receptors. This targeted delivery of estrogen controls food intake by greater modification of the food-reward behavior of the patient (Rentzeperi et al., 2022).

[0089] In some embodiments, the method of decreasing body weight further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agentfor decreasing body weight. In some embodiments, the additional therapeutic includes, but is not limited to, orlistat, phentermine, phentermine-topiramate, naltrexone-bupropion, setmelanotide, a GLP-1 RA (e.g., tirzepatide, liraglutide, semaglutide, exenatide, albiglutide, lixisenatide, dulaglutide) or any combination thereof. In some embodiments, the additional therapeutic is a GLP-1 RA.

[0090] In some embodiments, the method of decreasing body weight further comprises administering to a subject an effective amount of an estrogen for decreasing body weight. In some embodiments, the estrogen is conjugated to the SIRT6 activator or the additional therapeutic agent. In some embodiments, body weight may decrease by about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more after the subject is administered a therapeutically effective amount of the estrogen conjugated to the SIRT6 activator or the additional therapeutic agent.

[0091] In some embodiments, the method of decreasing body weight is administered to a female subject for a more prominent body weight loss result compared to male subjects. In some embodiments, the female subject who is administered the estrogen conjugated to the SIRT6 activator or the additional therapeutic agent is one that has been diagnosed and / or suspected of having obesity or being overweight.

[0092] Another aspect of the invention relates to a method of maintaining body weight in a subject in need thereof (e.g., after weight loss due to administration of weight loss drugs, gastric bypass surgery, diet, exercise, or a combination thereof), comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby maintaining body weight in the subject. In some embodiments, the subject in need thereof is characterized by decreased levels and / or activity of SIRT6 (e.g., the subject in need thereof has 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% decreased levels and / or activity of SIRT6 relative to a control subject that is not in need of body weight maintenance).

[0093] In some embodiments, the method comprises maintaining body weight in the subject after withdrawal of a weight loss treatment e.g., GLP-1 RA treatment). In some embodiments, the method further comprises maintaining the benefits of the weight loss treatment (e.g., GLP-1 RA treatment in the subject, for example, improved glycemic control, enhanced insulin secretion, reduced glucagon release, delayed gastric emptying, decreased postprandial glucose levels, appetite suppression, weight loss, lowered blood pressure, improved lipid profiles, reducedcardiovascular risk, renal protection, decreased hepatic fat accumulation, and potential neuroprotective and anti-inflammatory effects.

[0094] In some embodiments, the method may result in maintenance of body weight within about 0.1%, 0.5%, 1%, 2%, 5%, or 10% of the goal weight or initial weight of the subject. In some embodiments, the subject who is administered a SIRT6 activator is one that is currently or was previously diagnosed and / or suspected of having obesity or being overweight.

[0095] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for maintaining body weight (e.g., one or more of the agents listed above for weight loss), diet, exercise, or a combination thereof.

[0096] A second aspect of the invention relates to a method of decreasing triglycerides in the blood in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby decreasing triglycerides in the blood in the subject. In some embodiments, the subject in need thereof is characterized by decreased levels and / or activity of SIRT6 (e.g., the subject in need thereof has 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% decreased levels and / or activity of SIRT6 relative to a control subject that does not have elevated triglyceride levels). In some embodiments, the method may result in a decrease in triglyceride levels in the blood of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more after the subject is administered a therapeutically effective amount of a SIRT6 activator. In some embodiments, the subject who is administered a SIRT6 activator is one that has been diagnosed and / or suspected of having elevated triglyceride levels in the blood or liver or has been diagnosed and / or suspected of having a condition characterized by the subject having elevated triglyceride levels.

[0097] In some embodiments, the method of decreasing triglycerides in the blood further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for decreasing triglycerides in the blood in the subject. In some embodiments, the additional therapeutic agent includes, but is not limited to, niacin, a fibrate or a fibric acid derivative, a statin, icosapent, omega-3 fatty acid or omega-3 fatty acid ethyl ester (e.g., docosahexaenoic acid, eicosapentaenoic acid), or any combination thereof.

[0098] A third aspect of the invention relates to a method of decreasing total cholesterol in the blood in a subject in need thereof, comprising administering to the subject a therapeuticallyeffective amount of a SIRT6 activator, thereby decreasing total cholesterol in the blood in the subject. In some embodiments, the subject in need thereof is characterized by decreased levels and / or activity of SIRT6 (e.g., the subject in need thereof has 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% decreased levels and / or activity of SIRT6 relative to a control subject that does not have elevated total cholesterol levels). In some embodiments, the method may result in a decrease in total cholesterol levels in the blood of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more after the subject is administered a therapeutically effective amount of a SIRT6 activator. In some embodiments, the subject who is administered a SIRT6 activator is one that has been diagnosed and / or suspected of having elevated total cholesterol levels in the blood or has been diagnosed and / or suspected of having a condition characterized by the subject having elevated total cholesterol levels.

[0099] In some embodiments, the method of decreasing total cholesterol in the blood further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for decreasing total cholesterol in the blood in the subject. In some embodiments, the additional therapeutic agent includes, but is not limited to, niacin, a fibrate or a fibric acid derivative, a statin, a resin or a bile acid sequestrant, a cholesterol absorption inhibitor, omega-3 fatty acid or omega-3 fatty acid ethyl ester (e.g., docosahexaenoic acid, eicosapentaenoic acid), marine-derived omega-3 polyunsaturated fatty acid (PUFA), a PCSK9 inhibitor, an adenosine triphosphate-citrate lyase (ACLY) inhibitor, or any combination thereof.

[0100] A fourth aspect of the invention relates to a method of decreasing LDL cholesterol in the blood in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby decreasing LDL cholesterol in the blood in the subject. In some embodiments, the subject in need thereof is characterized by decreased levels and / or activity of SIRT6 (e.g., the subject in need thereof has 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% decreased levels and / or activity of SIRT6 relative to a control subject that does not have elevated LDL cholesterol levels). In some embodiments, the method may result in a decrease in LDL cholesterol levels in the blood of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more after the subject is administered a therapeutically effective amount of a SIRT6 activator. In some embodiments, the subject who is administered a SIRT6 activator is one that has been diagnosed and / or suspected of having elevatedLDL cholesterol levels in the blood or has been diagnosed and / or suspected of having a condition characterized by the subject having elevated LDL cholesterol levels.

[0101] In some embodiments, the method of decreasing LDL cholesterol in the blood further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for decreasing LDL cholesterol in the blood in the subject. In some embodiments, the additional therapeutic agent includes, but is not limited to, niacin, a fibrate or a fibric acid derivative, a statin, a resin or a bile acid sequestrant, omega-3 fatty acid or omega-3 fatty acid ethyl ester (e.g., docosahexaenoic acid, eicosapentaenoic acid), a PCSK9 inhibitor, or any combination thereof.

[0102] Without being bound by theory, it is thought that the PCSK9 gene transcription can be suppressed by the SIRT6 and a FOXO3 transcription factor regulating the gene together, causing a reduction of LDL cholesterol in blood circulation.

[0103] A fifth aspect of the invention relates to a method of decreasing liver weight in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby decreasing liver weight in the subject. In some embodiments, the subject in need thereof is characterized by decreased levels and / or activity of SIRT6 (e.g., the subject in need thereof has 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% decreased levels and / or activity of SIRT6 relative to a control subject that does not have increased liver weight). In some embodiments, the method may result in a decrease in liver weight of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more after the subject is administered a therapeutically effective amount of a SIRT6 activator. In some embodiments, the subject who is administered a SIRT6 activator is one that has been diagnosed and / or suspected of having increased liver weight or has been diagnosed and / or suspected of having a condition characterized by the subject having an increased liver weight.

[0104] A sixth aspect of the invention relates to a method of treating a liver disease, e.g., a fatty liver disease, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby treating liver disease in the subject. In some embodiments, the fatty liver disease is related to a dysfunction in hepatic metabolic homeostasis. In some embodiments, the fatty liver disease is steatohepatitis, e.g., Metabolic Dysfunction- Associated Steatohepatitis (MASH). In some embodiments, the subject in need thereof is characterized by decreased levels and / or activity of SIRT6 (e.g., the subject in need thereof has1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% decreased levels and / or activity of SIRT6 relative to a control subject that does not have liver disease). In some embodiments, the subject who is administered a SIRT6 activator is one that has been diagnosed and / or suspected of having a liver disease, e.g., a fatty liver disease.

[0105] In some embodiments, the method for treating a liver disease further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for treating the liver disease in the subject. In some embodiments, the additional therapeutic agent includes, but is not limited to, an insulin sensitizer, a lipid-lowering drug (e.g., statins), an angiotensin receptor blocker, pentoxifylline, a steroid (corticosteroid), a thyroid hormone receptor (THR)-beta agonist (resmetirom), or any combination thereof.

[0106] A seventh aspect of the invention relates to a method of suppressing hepatic lipogenesis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby suppressing hepatic lipogenesis in the subject. In some embodiments, the subject in need thereof is characterized by decreased levels and / or activity of SIRT6 (e.g., the subject in need thereof has 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% decreased levels and / or activity of SIRT6 relative to a control subject that does not have increased hepatic lipogenesis). In some embodiments, the method may result in a decrease in hepatic lipogenesis of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more after the subject is administered a therapeutically effective amount of a SIRT6 activator. In some embodiments, the subject who is administered a SIRT6 activator is one that has been diagnosed and / or suspected of having increased or dysregulated hepatic lipogenesis or has been diagnosed and / or suspected of having a condition characterized by the subject having increased or dysregulated hepatic lipogenesis.

[0107] Without being bound by theory, it is thought that the SIRT6 activator suppresses hepatic lipogenesis by regulating hepatic triglycerides, cholesterol, and low-density lipoprotein cholesterol. The SIRT6 activator is thought to suppress several major transcription factors promoting lipogenesis through deacetylation, including liver X receptor (LXR), carbohydrate response element binding protein (ChREBP), sterol regulatory element binding protein 1 (SREBP1), and X-box binding protein 1 (XBP1). Further, without being bound by theory, it is thought that the SIRT6 activator also regulates SREBP1 transcriptional activity through an interaction of core clock components. Acting with transcription factor FOXO3, the SIRT6activator is thought to decrease hepatic cholesterol biosynthesis by deacetylating the SREBP2 gene, thus suppressing SREBP2 gene transcription.

[0108] An eighth aspect of the invention relates to a method of increasing hepatic fatty acid oxidation (FAO) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby increasing FAO in the subject. In some embodiments, the subject in need thereof is characterized by decreased levels and / or activity of SIRT6 (e.g., the subject in need thereof has 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% decreased levels and / or activity of SIRT6 relative to a control subject that does not have decreased FAO). In some embodiments, the method may result in an increase in hepatic fatty acid oxidation of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more after the subject is administered a therapeutically effective amount of a SIRT6 activator. In some embodiments, the subject who is administered a SIRT6 activator is one that has been diagnosed and / or suspected of having decreased hepatic fatty acid oxidation or has been diagnosed and / or suspected of having a condition characterized by the subject having decreased hepatic fatty acid oxidation.

[0109] Without being bound by theory, it is thought that the SIRT6 activator increases hepatic fatty acid oxidation by suppressing cell death inducing DNA Fragmentation Factor alpha (DFFA)- like effector C (CIDEC) gene expression. By repressing CIDEC, the SIRT6 activator promotes fasting or ketogenic diet-induced ketogenesis. The SIRT6 activator further increases hepatic fatty acid oxidation by directly inducing FAO gene expression through deacetylating nuclear receptor coactivator 2 (NCOA2) and enhancing NCOA2’s coactivation activity. The SIRT6 activator also directly induces FAO genes through regulating peroxisome proliferator activated receptor alpha (PPARa) Furthermore, without being bound by theory, it is thought that the SIRT6 activator also indirectly promotes FAO gene expression by downregulating microRNA-122. Additionally, the SIRT6 activator is thought to work by deacetylating acyl-CoA synthetase long-chain family member 5 (ACSL5), where a deacetylated ACSL5 is more active for promoting fatty acid oxidation by converting long-chain fatty acids to long-chain fatty acyl-CoAs.

[0110] A ninth aspect of the invention relates to a method of reducing oxidative stress in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby reducing oxidative stress in the subject. In some embodiments, the subject in need thereof is characterized by decreased levels and / or activity of SIRT6 (e g., thesubject in need thereof has 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% decreased levels and / or activity of SIRT6 relative to a control subject that does not have increased oxidative stress). In some embodiments, the method nay result in a decrease in oxidative stress of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more after the subject is administered a therapeutically effective amount of a SIRT6 activator. In some embodiments, the subject who is administered a SIRT6 activator is one that has been diagnosed and / or suspected of having elevated oxidative stress or has been diagnosed and / or suspected of having a condition characterized by the subject having elevated oxidative stress.

[0111] Without being bound to theory, it is thought that the SIRT6 activator reduces oxidative stress by inducing PPARG coactivator 1 alpha and endonuclease G (ENDOG). Further, it is thought that the SIRT6 activator can be effective reducing oxidative stress stemming from a high- fat diet.

[0112] A tenth aspect of the invention relates to a method of decreasing hepatic inflammation in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby decreasing hepatic inflammation in the blood in the subject. In some embodiments, the subject in need thereof is characterized by decreased levels and / or activity of SIRT6 (e.g., the subject in need thereof has 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% decreased levels and / or activity of SIRT6 relative to a control subject that does not have increased hepatic inflammation). In some embodiments, the method may result in a decrease in hepatic inflammation of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more after the subject is administered a therapeutically effective amount of a SIRT6 activator. In some embodiments, the subject who is administered a SIRT6 activator is one that has been diagnosed and / or suspected of having increased hepatic inflammation or has been diagnosed and / or suspected of having a condition characterized by the subject having increased hepatic inflammation.

[0113] Without being bound to theory, it is thought that the SIRT6 activator decreases hepatic inflammation by modulating immune cells, specifically macrophages. SIRT6’s impact on hepatic inflammation pathways has been demonstrated in studies testing myeloid-specific SIRT6 knockout mice under a high-fat diet, where the mice showed worse systemic insulin resistance and glucose intolerance, heavier liver weights, higher hepatic triglyceride and cholesterol levels, higher hepatic macrophage infiltration, and higher non-alcoholic fatty liver disease activity scores in comparisonto wildtype mice (Dong, 2023). The SIRT6 activator is thought to repress multiple inflammatory pathways, including c-JUN, nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-kB), and signal transducer and activator of transcription 3 (STAT3). Furthermore, by inhibiting these inflammatory pathways, the SIRT6 activator suppresses Ml macrophage polarization, which decreases hepatic inflammation.

[0114] An eleventh aspect of the invention relates to a method of decreasing hepatic fibrosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby decreasing hepatic fibrosis in the subject. In some embodiments, the subject in need thereof is characterized by decreased levels and / or activity of SIRT6 (e.g., the subject in need thereof has 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% decreased levels and / or activity of SIRT6 relative to a control subject that does not have increased hepatic fibrosis). In some embodiments, the method may result in a decrease in hepatic fibrosis of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more after the subject is administered a therapeutically effective amount of a SIRT6 activator. In some embodiments, the subject who is administered a SIRT6 activator is one that has been diagnosed and / or suspected of having increased hepatic fibrosis or has been diagnosed and / or suspected of having a condition characterized by the subject having increased hepatic fibrosis.

[0115] Without being bound to theory, it is thought that the SIRT6 activator decreases hepatic fibrosis by regulating hepatic stellate cell activation pathways. It is thought that the SIRT6 activator inhibits the transforming growth factor beta (TGFp)-SMAD signaling by suppressing SMAD2 and SMAD3, dampening their transcriptional activities. Additionally, without being bound by theory, the SIRT6 activator is also thought to suppress the coactivator activities of YAP and TAZ / WWTR1 by deacetylation. Studies have demonstrated SIRT6’s impact on the YAP and 'f AZ pathways through comparing SIRT6 hepatic stellate cell-specific knockout mice to wildtype mice, in which the former showed significantly increased levels of YAP and TAZ acetylation (Dong, 2023). Further, the SIRT6 activator is thought to decrease the YAP / TAZ-TEA domain transcription factor 1 activation complexes, thereby altering their protein complex formation.

[0116] A twelfth aspect of the invention relates to a method of decreasing hepatic steatosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby decreasing hepatic steatosis in the blood in the subject. In some embodiments, the subject in need thereof is characterized by decreased levels and / or activity ofSIRT6 (e.g., the subject in need thereof has 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% decreased levels and / or activity of SIRT6 relative to a control subject that does not have hepatic steatosis). In some embodiments, the method may result in a decrease in hepatic steatosis of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more after the subject is administered a therapeutically effective amount of a SIRT6 activator. In some embodiments, the subject who is administered a SIRT6 activator is one that has been diagnosed and / or suspected of having increased hepatic steatosis or has been diagnosed and / or suspected of having a condition characterized by the subject having increased hepatic steatosis.

[0117] Without being bound by theory, it is thought that the SIRT6 activator decreases hepatic steatosis by regulating hepatic lipogenesis pathways. One way SIRT6 is believed to decrease hepatic steatosis is by deacetylating the X-box binding protein 1 (XBP1), resulting in XBP1 undergoing ubiquitin-proteasome-mediated degradation. The result from deacetylating XBP1 stabilizes SIRT6 and protects against hepatic steatosis.

[0118] A thirteenth aspect of the invention relates to a method of preserving muscle mass in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby preserving muscle mass in the subject. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an additional therapeutic agent for preserving muscle mass in the subject. In some embodiments, the subject in need thereof is characterized by decreased levels and / or activity of SIRT6 (e.g., the subject in need thereof has 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% decreased levels and / or activity of SIRT6 relative to a control subject). In some embodiments, the method may result in a preservation of muscle mass of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more after the subject is administered a therapeutically effective amount of a SIRT6 activator relative to a subject that has not been administered a SIRT6 activator. In some embodiments, the subject who is administered a SIRT6 activator is one that has been diagnosed and / or suspected of having loss of muscle mass or has been diagnosed and / or suspected of having a condition characterized by loss of muscle mass.

[0119] In some embodiments, the methods of the invention may further include administering NAD+ or an NAD+ precursor. The NAD+ or NAD+ precursor may be administered at the same time as the SIRT6 activator or at different time, e.g., on a different administration schedule. TheNAD+ or NAD+ precursor may be administered in the same composition as the SIRT6 activator or in a separate composition.

[0120] The methods of the invention may be carried out with any NATH precursor known in the art or later developed. The term “NAD+ precursor” is intended to mean compounds that are known to increase the level of NATH in a subject after administration. Example NATH precursors useful to the present invention include, but are not limited to, nicotinamide riboside, nicotinic acid riboside, nicotinic acid, nicotinamide, nicotinamide mononucleotide, niacin, and tryptophan. NATH precursors may be converted into NATH by any pathway, reaction, or synthesis method known to those in the art. Example synthesis pathways of NATH from NATH precursors include, but are not limited to, the Kynurenine pathway, the Preiss-Handler pathway, the NATH salvage pathway, and / or the NRH salvage pathway. The methods of the invention may be carried out with NATH by itself or in addition to a NATH precursor.Compounds

[0121] The methods of the invention may be carried out with any SIRT6 activator known in the art or later developed. Examples of SIRT6 activators include, without limitation, quercetin, isoquercetin, kaempferol, luteolin, cyanidin, fisetin, delphinidin, icariin, N-acetylethanolamines, oleic acid, linoleic acid, fucoidan, MDL-800, MDL-811, UBCS038 (You et al., Angew. Chem. Int. Ed. 56:1007 (2017)), UBCS039, UBCS040, UBCS058, UBCS060, UBCS068, myristic acid, OEA, CL5D, 10b, 5-C1-PZA, BHJH-TM3, 15f, 17a (catechin gallate), 19b (OSSJ28167), 20b, 21b, 22a (A127-(CONHPr)-B178), 23, and forvisirvat (SP-624). Exemplary compounds are described in more detail in Fiorentino et al., J. Med. Chem. 64:9732 (2021) and Akter et al., Int. J. Mol. Sci. 22:4180 (2021), each incorporated by reference herein in its entirety.

[0122] The term “SIRT6 activator” also includes SIRT6 itself, either in the form of the protein or a functional fragment or variant thereof, or in the form of a nucleic acid encoding SIRT6 or a functional fragment or variant thereof. The nucleic acid may be in a vector, e.g., a viral vector such as an adeno-associated virus vector (e.g., as disclosed in WO 2025 / 032584 or US 2023 / 0018934, incorporated herein by reference). SIRT6 variants may include those disclosed in US 2024 / 0277868, incorporate herein by reference).

[0123] A further example of a SIRT6 activator is a compound of Formula 1 or a pharmaceutically acceptable salt thereof:(D wherein:R1is a C1-C6 alkyl group optionally substituted with the same or different one to two substituents selected from a substituent group X, a C3-C6 cycloalkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, or a 4-7 membered saturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X,R2is a C1-C6 alkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C3-C6 cycloalkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, or a 4-7 membered saturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X,A is a 5-membered aromatic heterocyclic ring, a 6-membered aromatic heterocyclic ring, an 8-10 membered condensed aromatic heterocyclic ring, a 5-7 membered unsaturated heterocyclic ring, a 4-7 membered saturated heterocyclic ring, a benzene ring, -CH=, or a cyano group, wherein when A is a cyano group, R3and R3do not exist, R3and R3are each independently a hydrogen atom, a halogen atom, a cyano group, a hydroxy group, an oxo group, a C1-C6 alkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X,a C1-C6 alkoxy group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C2-C6 alkenyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C2-C6 alkynyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C3-C6 cycloalkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, an amino group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C1-C6 alkoxycarbonyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a carbamoyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a phenyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a 5-membered aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, a 6-membered aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, a 5-7 membered unsaturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, a 4-7 membered saturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, an 8-10 membered condensed aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, orR3and R3may form a 5-7 membered unsaturated heterocyclic ring, a 4-7 membered saturated heterocyclic ring, or a C3-C6 cycloalkyl ring as a ring that binds to each other and condenses with A, and the ring is optionally substituted with the same or different one to two substituents selected from the substituent group X,substituent group X is a halogen atom, a cyano group, a hydroxy group, an oxo group, a C1-C6 alkyl group, a hydroxy C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a C1-C6 haloalkyl group, a C3-C6 cycloalkyl group, a C3-C6 halocycloalkyl group, a phenyl group optionally substituted with the same or different one to two substituents selected from a substituent group Y, a 5-membered aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 6-membered aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 4-7 membered saturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a C1-C6 alkoxy group, a C1-C6 haloalkoxy group, a C3-C6 cycloalkoxy group, a C3-C6 halocycloalkoxy group, a phenoxy group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 5-membered aromatic heterocyclic oxy group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 6-membered aromatic heterocyclic oxy group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 4-7 membered saturated heterocyclic oxy group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a C1-C6 alkoxy carbonyl group, a C3-C6 cycloalkoxy carbonyl group, a carboxy group, a C1-C6 alkylcarbonyl group, a C3-C6 cycloalkylcarbonyl group, a phenylcarbonyl group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a carbamoyl group, a mono (C 1-C6 alkyl) aminocarbonyl group, a di (C 1-C6 alkyl) aminocarbonyl group, a mono (C1-C6 alkyl) aminosulfonyl group, a di (C1-C6 alkyl) aminosulfonyl group, an amino group, a mono (C1-C6 alkyl) amino group, a di (C1-C6 alkyl) amino group, a C1-C6 alkoxy carbonylamino group, a mono (C1-C6 alkyl) aminocarbonylamino group, a di (C1-C6 alkyl) aminocarbonylamino group, a C1-C6 alkylcarbonylamino group,a phenylcarbonylamino group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 5-membered aromatic heterocyclic carbonylamino group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 6-membered aromatic heterocyclic carbonylamino group optionally substituted with the same or different one to two substituents selected from the substituent group Y, or a C1-C6 alkylsulfonylamino group, substituent group Y is a C1-C6 alkyl group, a C1-C6 alkoxy group, a halogen atom, or a hydroxy group.

[0124] In some embodiments, the compound of Formula 1 is any compound selected from the following group:(2S,5’R)-7-chloro-6-(5-ethyl-l,3,4-oxadiazol-2-yl)-3’,4-dimethoxy-5’-methyl-spiro [benzofuran- 2,4’ -cyclohex-2-ene]- 1 ’ ,3 -di one;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(5-tetrahydropyran-4-yl-l,3,4-oxadiazol-2-yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5 ’R)-7-chl oro-3 ’ ,4-dimethoxy-5 ’ -methyl-6-[5-( 1 -methyl-4-piperidyl)- 1 ,3 ,4-oxadiazol-2-yl] spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-6-[5-(4-fluoro-l-methyl-4-piperidyl)-l,3,4-oxadiazol-2-yl]-3’,4-dimethoxy- 5’-methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chl oro-3 ’,4-dimethoxy-6-[5-[(lS)-l -methoxyethyl]- 1,3, 4-oxadi azol -2 -yl]-5 ’ -methylspiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-4-ethoxy-3’-methoxy-5’-methyl-6-(5-methyl-l,3,4-oxadiazol-2-yl) spiro[benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-4-(difluoromethoxy)-3’-methoxy-5’-methyl-6-(5-methyl-l,3,4-oxadiazol-2-yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chl oro-3 ’,4-dimethoxy-6-[3-(l -methoxy ethyl)-l, 2, 4-oxadiazol-5-yl]-5’-methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chloro-6-[3-(l -hydroxy- l-methyl-ethyl)-l, 2, 4-oxadi azol-5-yl]-3’, 4-dimethoxy-5’- methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(lH-pyrazol-5-yl) spiro [benzofuran-2,4’- cyclohex-2-ene]-l ’,3-dione;(2 S, 5 ’ R)-7-chl oro-3 ’ ,4-di m ethoxy-6- [ 1 -(2-methoxy ethyl) py razol -3 -y 1 ] - 5 ’ -m ethy 1 - spi ro[b enzofuran-2, 4 ’ -cy cl ohex-2-ene] - 1 ’ , 3 -di one;(2S,5’R)-7-chloro-6-(l,8-dioxa-2-azaspiro [4.5] dec-2-en-3-yl)-3’,4-dimethoxy-5’-methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l ’, 3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(8-methyl-l-oxa-2,8-diazaspiro [4.5] dec-2-en-3- yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l ’, 3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-6-(2-methoxypyrimidin-5-yl)-5’-methyl-spiro [benzofuran- 2,4’ -cy clohex-2-ene]- 1 ’ ,3 -di one;(2S,5’R)-7-chloro-3’,4-dimethoxy-6-(6-methoxy-3-pyridyl)-5’-methyl-spiro [benzofuran-2,4’- cyclohex-2-ene]-l ’, 3-dione; or(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(3-pyridyl) spiro [benzofuran-2, 4’ -cyclohex-2 - ene]-l ’, 3-dione.

[0125] In some embodiments, the compound of Formula 1 is a compound of Formula 1’ or a pharmacologically acceptable salt thereof:(D wherein:R1is a C1-C6 alkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C3-C6 cycloalkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, or a 4-7 membered saturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group XR2is a C1 -C6 alkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X,a C3-C6 cycloalkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, or a 4-7 membered saturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X.A is a 5-membered aromatic heterocyclic ring, a 6-membered aromatic heterocyclic ring, an 8-10 membered condensed aromatic heterocyclic ring, a 5-7 membered unsaturated heterocyclic ring, a 4-7 membered saturated heterocyclic ring, a benzene ring, or a single bond, wherein when it is a single bond, one or the other of R3and R3is not present,R3and R3are each independently a hydrogen atom, a halogen atom, a cyano group, a hydroxy group, a C1-C6 alkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C1-C6 alkoxy group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C2-C6 alkenyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C2-C6 alkynyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C3-C6 cycloalkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, an amino group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C1-C6 alkoxycarbonyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a carbamoyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a phenyl group optionally substituted with the same or different one to two substituents selected from the substituent group X,a 5-membered aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, a 6-membered aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, a 5-7 membered unsaturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, a 4-7 membered saturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, an 8-10 membered condensed aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, orR3and R3may form a 5-7 membered unsaturated heterocyclic ring, a 4-7 membered saturated heterocyclic ring, or a C3-C6 cycloalkyl ring as a ring that binds to each other and condenses with A, and the ring is optionally substituted with the same or different one to two substituents selected from the substituent group X, substituent group X is a halogen atom, a cyano group, a hydroxy group, an oxo group, a C1-C6 alkyl group, a hydroxy C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a C1-C6 haloalkyl group, a C3-C6 cycloalkyl group, a C3-C6 halocycloalkyl group, a phenyl group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 5-membered aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 6-membered aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 4-7 membered saturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a C1-C6 alkoxy group, a C1-C6 haloalkoxy group, a C3-C6 cycloalkoxy group, a C3-C6 halocycloalkoxy group, a phenoxy group optionally substituted with the same or different one to two substituents selected from the substituent group Y,a 5-membered aromatic heterocyclic oxy group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 6-membered aromatic heterocyclic oxy group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 4-7 membered saturated heterocyclic oxy group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a C1-C6 alkoxycarbonyl group, a C3-C6 cycloalkoxycarbonyl group, a carboxy group, a C1-C6 alkylcarbonyl group, a C3-C6 cycloalkylcarbonyl group, a phenylcarbonyl group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a carbamoyl group, a mono (C 1 -C6 alkyl) aminocarbonyl group, a di (C 1 -C6 alkyl) aminocarbonyl group, a mono (C1-C6 alkyl) aminosulfonyl group, a di (C1-C6 alkyl) aminosulfonyl group, an amino group, a mono (C1-C6 alkyl) amino group, a di (C1-C6 alkyl) amino group, a C1-C6 alkoxy carbonylamino group, a mono (C1-C6 alkyl) aminocarbonylamino group, a di (C1-C6 alkyl) aminocarbonylamino group, a C1-C6 alkyl carbonylamino group, a phenylcarbonylamino group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 5-membered aromatic heterocyclic carbonylamino group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 6-membered aromatic heterocyclic carbonylamino group optionally substituted with the same or different one to two substituents selected from the substituent group Y, or a C1-C6 alkylsulfonylamino group, and substituent group Y is a C1-C6 alkyl group, a C1-C6 alkoxy group, a halogen atom, or a hydroxy group.

[0126] In some embodiments of the compound of Formula 1 or Formula 1’, R1is a C1-C6 alkyl group, R2is a C1-C6 alkyl group, A is a 5-membered aromatic heterocyclic ring, and R3and R3are each independently a hydrogen or a C1-C6 alkyl group.

[0127] In some embodiments of the compound of Formula 1 or Formula 1’, R1is a methyl group, an ethyl group, or a hydroxy ethyl group.

[0128] In some embodiments of the compound of Formula 1 or Formula 1’, R2is a methyl group.

[0129] In some embodiments of the compound of Formula 1 or Formula 1 ’, A is a 5-membered aromatic heterocyclic ring, R3is a methyl group, an ethyl group, a hydroxy C1-C3 alkyl group, or a methoxy C1-C3 alkyl group, and R3is a hydrogen atom.

[0130] In some embodiments, the compound of Formula 1 is a compound of a Formula 1” or a pharmacologically acceptable salt thereof:d ”) wherein R1is a methyl group or an ethyl group;R2is a methyl group;

[0131] A is any ring selected from the following group:indicates a binding group; andR3is a methyl group or an ethyl group.

[0132] In some embodiments, the compound of Formula 1’ is any compound selected from the following group:(2S,5’R)-7-chloro-6-(2-hydroxyethoxy)-3’,4-dimethoxy-5’-methyl-spiro [benzofuran-2,4’- cyclohex-2-ene]- 1 ’ ,3 -di one;(2S,5’R)-7-chloro-3’,4-dimethoxy-6-(2-methoxyethoxy)-5’-methyl-spiro [benzofuran-2,4’- cyclohex-2-ene]-l’,3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(l-methylpyrazol-3-yl) spiro [benzofuran-2,4’- cyclohex-2-ene]-l ’, 3-dione;(2S,5’R)-7-chloro-6-(l-ethylpyrazol-3-yl)-3’,4-dimethoxy-5’-methyl-spiro [benzofuran-2,4’- cyclohex-2-ene]- 1 ’ ,3 -di one;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(5-methyl-l,3,4-oxadiazol-2-yl) spiro[b enzofuran-2, 4 ’ -cy cl ohex-2-ene] - 1’ , 3 -di one;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(3-methyl-l,2,4-oxadiazol-5-yl) spiro[benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(5-methyl)-l,2,4-oxadiazol-3-yl) spiro[benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-6-[5-(l-hydroxy-l-methyl-ethyl)-l,3,4-oxadiazol-2-yl]-3’,4-dimethoxy-5’- methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chloro-6-[5-[(lS)-l -hydroxy ethyl]-l, 3, 4-oxadiazol-2-yl]-3’,4-dimethoxy-5’ -methylspiro [benzofuran-2,4’-cyclohex-2-ene]-T, 3-dione;(2S, 5 ’R)-7-chloro-6-[5-[(lR)-l -hydroxy ethyl]- 1,3, 4-oxadiazol-2-yl]-3’,4-dimethoxy-5 ’-methyl- spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-4-ethoxy-6-[5-(l-hydroxy-l-methyl-ethyl)-l,3,4-oxadiazol-2-yl]-3’-methoxy- 5 ’-methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chloro-4-ethoxy-6-[5-[(lS)-l -hydroxy ethyl]- 1,3, 4-oxadiazol-2-yl]-3’-methoxy-5’- methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chloro-6-[3-(l -hydroxy ethyl)- 1, 2, 4-oxadiazol-5-yl]-3’,4-dimethoxy-5’ -methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-4-(2-hydroxyethoxy)-3’-methoxy-5’-methyl-6-(5-methyl-l,3,4-oxadiazol-2- yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chloro-4-(2 -hydroxy ethoxy)-3’-methoxy-5’-methyl-6-(3-methyl- 1,2, 4-oxadiazol-5- yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chloro-4-(2 -hydroxy ethoxy)-3’-methoxy-5’-methyl-6-(5-m ethyl- 1,2, 4-oxadiazol-3- yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chloro-6-(5-ethyl-l, 3, 4-oxadiazol-2-yl)-3’,4-dimethoxy-5 ’-methyl-spiro [benzofuran- 2,4’ -cyclohex-2-ene]- 1 ’ ,3 -dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(5-tetrahydropyran-4-yl-l,3,4-oxadiazol-2-yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5 ’R)-7-chl oro-3 ’ ,4-dimethoxy-5 ’ -methyl-6-[5-( 1 -methyl-4-piperidyl)- 1 ,3 ,4-oxadiazol-2-yl] spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-6-[5-(4-fluoro-l-methyl-4-piperidyl)-l,3,4-oxadiazol-2-yl]-3’,4-dimethoxy- 5’- methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chl oro-3 ’,4-dimethoxy-6-[5-[(lS)-l -methoxy ethyl]- 1,3, 4-oxadiazol -2 -yl]-5’ -methylspiro [benzofuran-2,4’-cyclohex-2-ene]-r, 3-dione;(2S, 5 ’R)-7-chloro-4-ethoxy-3’-methoxy-5’-methyl-6-(5-methyl-l, 3, 4-oxadiazol -2-yl) spiro[benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-4-(difluoromethoxy)-3’-methoxy-5’-methyl-6-(5-methyl-l,3,4-oxadiazol-2-yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chl oro-3 ’,4-dimethoxy-6-[3-(l -methoxy ethyl)-l, 2, 4-oxadiazol-5-yl]-5 ’-methyl-spiro [b enzofuran-2, 4 ’ -cy cl ohex-2-ene] - 1’ , 3 -di one;(2S,5’R)-7-chloro-6-[3-(l-hydroxy-l-methyl-ethyl)-l,2,4-oxadiazol-5-yl]-3’,4-dimethoxy-5’- methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l ’, 3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(lH-pyrazol-5-yl) spiro [benzofuran-2,4’- cyclohex-2-ene]- 1 ’ ,3 -di one;(2S,5’R)-7-chloro-3’,4-dimethoxy-6-[l-(2-methoxyethyl) pyrazol-3-yl]-5’ -methyl-spiro[benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione; (2S,5’R)-7-chloro-6-(l,8-dioxa-2-azaspiro [4.5] dec-2-en-3-yl)-3’,4-dimethoxy-5’ -methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-r, 3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(8-methyl-l-oxa-2,8-diazaspiro [4.5] dec-2-en-3- yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chl oro-3’, 4-dimethoxy-6-(2-methoxypyrimidin-5-yl)-5’ -methyl-spiro [benzofuran-2,4’ -cyclohex-2-ene]- 1 ’ ,3 -dione;(2S, 5 ’R)-7-chl oro-3 ’,4-dimethoxy-6-(6-methoxy-3-pyridyl)-5 ’-methyl-spiro [benzofuran-2,4’- cyclohex-2-ene]-l ’,3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(3-pyridyl) spiro [benzofuran-2,4’-cyclohex-2- ene]-l’,3-dione; or(2S, 5 ’R)-7-chl oro-3’, 4, 6-trimethoxy-5’ -methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’,3- dione.

[0133] In one embodiment, the compound is (2S,5’R)-7-chloro-6-(l-ethylpyrazol-3-yl)-3’,4- dimethoxy-5’ -methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’,3-dione or a pharmacologically acceptable salt thereof.

[0134] In one embodiment, the compound is (2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(5- methyl-l,3,4-oxadiazol-2-yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione or a pharmacologically acceptable salt thereof.

[0135] In one embodiment, the compound is (2S,5’R)-7-chloro-6-(5-ethyl-l,3,4-oxadiazol-2-yl)- 3’,4-dimethoxy-5’-methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione or a pharmacologically acceptable salt thereof.

[0136] In one embodiment, the compound is (2S,5’R)-7-chloro-6-[3-(l-hydroxy-l-methyl-ethyl)- l,2,4-oxadiazol-5-yl]-3’,4-dimethoxy-5’-methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’,3- dione or a pharmacologically acceptable salt thereof.

[0137] In one embodiment, the compound is (2S,5’R)-7-chloro-4-ethoxy-3’-methoxy-5’-methyl- 6-(5-methyl-l,3,4-oxadiazol-2-yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione or a pharmacologically acceptable salt thereof.

[0138] In certain embodiments, the 5-membered aromatic heterocyclic ring for A is the same as described above, but more preferably, it represents the following 5-membered ring. It should be noted that in this case, R3is not present.wherein * indicates a binding group.

[0139] In the present specification, the “5-membered aromatic heterocyclic ring” is a monocyclic 5-membered aromatic heterocyclic ring containing one to four atoms selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom. For example, rings such as those shown below are included.

[0140] In the present specification, the “6-membered aromatic heterocyclic ring” is a monocyclic 6-membered aromatic heterocyclic ring containing one to four atoms selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom. For example, rings such as those shown below are included.

[0141] In the present specification, the “8-10 membered condensed aromatic heterocyclic ring” is an 8-10 membered condensed aromatic heterocyclic ring containing one to four atoms selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom. For example, rings such as those shown below are included.

[0142] In the present specification, the “5-7 membered unsaturated heterocyclic ring” is a ring in which a monocyclic 5-7 membered saturated heterocyclic ring is partially oxidized or a ring in which an aromatic heterocyclic ring is partially reduced containing one to four atoms selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom. For example, rings such as those shown below are included.

[0143] In the present specification, the “4-7 membered saturated heterocyclic ring” is a monocyclic 4-7 membered saturated heterocyclic ring containing one to four atoms selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom. For example, rings such as those shown below are included.

[0144] The “halogen atom” in the present specification is a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, and is preferably a fluorine atom or a chlorine atom.

[0145] The “C1-C6 alkyl group” in the present specification is a linear or branched alkyl group having one to six carbon atoms. Examples thereof include a methyl group, an ethyl group, a 1- propyl group, an isopropyl group, a 1 -butyl group, a 2-butyl group, a 2 -m ethyl- 1 -propyl group, a 2-methyl-2-propyl group, a 1 -pentyl group, a 2-pentyl group, a 3 -pentyl group, a 2-methyl-2-butyl group, a 3-methyl-2-butyl group, a 1 -hexyl group, a 2-hexyl group, a 3-hexyl group, a 2-methyl-1 -pentyl group, a 3-methyl-l -pentyl group, a 2-ethyl-l -butyl group, a 2,2-dimethyl-l -butyl group, and a 2,3 -dimethyl- 1 -butyl group, and it is preferably a methyl group or an ethyl group.

[0146] The “C2-C6 alkenyl group” in the present specification is a linear or branched alkenyl group having two to six carbon atoms, and it may have one or two or more carbon-carbon double bonds. For example, it is a vinyl group, a 2-propenyl (allyl) group, a 2-butenyl group, a 2-pentenyl group, a 3-methyl-2-butenyl group, a 2-hexenyl group, or a 3-methyl-2-pentenyl group, and preferably, it is a vinyl group or an allyl group.

[0147] The “C2-C6 alkynyl group” in the present specification is a linear or branched alkynyl group having two to six carbon atoms, and it may have one or two or more carbon-carbon triple bonds. For example, it is an ethynyl group, a 1-propynyl group, a 2-propynyl group, a 1-butynyl group, a 2-butynyl group, a 1 -pentynyl group, a 2-pentynyl group, or 1 -hexynyl group, and it is preferably an ethynyl group or a 1-propynyl group.

[0148] The “C1-C6 alkoxy group” in the present specification is a group in which an oxygen atom is bonded to a C1-C6 alkyl group. Examples thereof include a methoxy group, an ethoxy group, a 1 -propoxy group, a 2-propoxy group, a 1 -butoxy group, a 2-butoxy group, a 2 -m ethyl- 1 -propoxy group, a 2-methyl-2-propoxy group, a 1 -pentyloxy group, a 2-pentyloxy group, a 3 -pentyloxy group, a 2-methyl-2-butoxy group, a 3 -methyl-2 -butoxy group, a 1-hexyloxy group, a 2-hexyloxy group, a 3 -hexyloxy group, a 2-methyl-l -pentyloxy group, and a 3-methyl-l -pentyloxy group. Preferably, it is a methoxy group, an ethoxy group, a 1 -propoxy group, or a 2-propoxy group.

[0149] The “C3-C6 cycloalkyl group” in the present specification is a cyclic alkyl group having three to six carbon atoms, and it is preferably a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, or a cyclohexyl group.

[0150] The “hydroxy C1-C6 alkyl group” in the present specification is a group in which a hydroxyl group is bonded to a C1-C6 alkyl group. For example, it is a hydroxymethyl group or a hydroxy ethyl group.

[0151] The “C1-C6 alkoxy C1-C6 alkyl group” in the present specification is a group in which a C1-C6 alkoxy is bonded to a C1-C6 alkyl group. Examples thereof include a methoxymethyl group, a methoxyethyl group, an ethoxymethyl group, and an ethoxyethyl group.

[0152] The “C1-C6 haloalkyl group” in the present specification is a group in which a halogen atom is bonded to a C1-C6 alkyl group. Examples thereof include a fluoromethyl group, a difluoromethyl group, a dichloromethyl group, a dibromomethyl group, a trifluoromethyl group, atri chloromethyl group, a 2-fluoroethyl group, a 2-bromoethyl group, a 2-chloroethyl group, a 2- iodoethyl group, a 2,2-difluoroethyl group, a 2,2,2-trifluoroethyl group, a trichloroethyl group, a pentafluoroethyl group, a 3 -fluoropropyl group, a 3 -chloropropyl group, and a 4-fluorobutyl group. It is preferably a trifluoromethyl group.

[0153] The “C3-C6 halocycloalkyl group” in the present specification is a group in which a halogen atom is bonded to a C3-C6 cycloalkyl group, and examples thereof include a fluorocyclopropyl group, a fluorocyclobutyl group, a fluorocyclopentyl group, and a fluorocyclohexyl group.

[0154] The “C1-C6 haloalkoxy group” in the present specification is a group in which a halogen atom is bonded to a C1-C6 alkoxy group, and examples thereof include a fluoromethoxy group, a difluoromethoxy group, a dichloromethoxy group, a dibromomethoxy group, a trifluoromethoxy group, a tri chloromethoxy group, a 2-fluoroethoxy group, a 2-bromoethoxy group, a 2- chloroethoxy group, a 2-iodoethoxy group, a 2,2-difluoroethoxy group, a 2,2,2-trifluoroethoxy group, a tri chloroethoxy group, a pentafluoroethoxy group, a 3 -fluoropropoxy group, a 3- chloropropoxy group, and a 4-fluorobutoxy group. It is preferably a trifluoromethoxy group.

[0155] The “C3-C6 cycloalkoxy group” in the present specification is a group in which a C3-C6 cycloalkyl group is bonded to an oxygen atom, and it is preferably a cyclopropyloxy group, a cyclobutyloxy group, a cyclopentyloxy group, or a cyclohexyloxy group.

[0156] The “C3-C6 halocycloalkoxy group” in the present specification is a group in which a C3- C6 halocycloalkyl group is bonded to an oxygen atom, and examples thereof include a fluorocyclopropoxy group, a fluorocyclobutoxy group, a fluorocyclopentyloxy group, and a fluorocyclohexyloxy group.

[0157] The “5-membered aromatic heterocyclic oxy group” in the present specification is a group in which a 5-membered aromatic heterocyclic ring is bonded to an oxygen atom.

[0158] The “6-membered aromatic heterocyclic oxy group” in the present specification is a group in which a 6-membered aromatic heterocyclic ring is bonded to an oxygen atom.

[0159] The “4-7 membered saturated heterocyclic oxy group” in the present specification is a group in which a 4-7 membered saturated heterocyclic ring is bonded to an oxygen atom.

[0160] The “C1-C6 alkoxycarbonyl group” in the present specification is a group in which a Cl- C6 alkoxy group is bonded to a carbonyl group, and examples thereof include a methoxy carbonyl group, an ethoxy carbonyl group, and a propoxycarbonyl group.

[0161] The “C3-C6 cycloalkoxy carbonyl group” in the present specification is a group in which a C3-C6 cycloalkoxy group is bonded to a carbonyl group, and it is preferably a cyclopropyloxycarbonyl group, a cyclobutyloxycarbonyl group, a cyclopentyloxycarbonyl group, or a cyclohexyloxycarbonyl group.

[0162] The “C1-C6 alkyl carbonyl group” in the present specification is a group in which a Cl- C6 alkyl group is bonded to a carbonyl group, and examples thereof include a methyl carbonyl group, an ethyl carbonyl group, or a propyl carbonyl group.

[0163] The “mono (C1-C6 alkyl) aminocarbonyl group” in the present specification is a group in which one C1-C6 alkyl group is bonded to the amino group of an aminocarbonyl group, and it is preferably a methylaminocarbonyl group, an ethylaminocarbonyl group, or a propylaminocarbonyl group.

[0164] The “di (C1-C6 alkyl) aminocarbonyl group” in the present specification is a group in which two C1-C6 alkyl groups are bonded to the amino group of an aminocarbonyl group, and it is preferably a dimethylaminocarbonyl group, a diethylaminocarbonyl group, or a dipropylaminocarbonyl group.

[0165] The “mono (C1-C6 alkyl) aminosulfonyl group” in the present specification is a group in which one C1-C6 alkyl group is bonded to the amino group of an aminosulfonyl group, and it is preferably a methylaminosulfonyl group, an ethylaminosulfonyl group, or a propylaminosulfonyl group

[0166] The “di (C1-C6 alkyl) aminosulfonyl group” in the present specification is a group in which two C1-C6 alkyl groups are bonded to the amino group of the aminosulfonyl group, and it is preferably a dimethylaminosulfonyl group, a diethylaminosulfonyl group, or a dipropylaminosulfonyl group.

[0167] The “mono (C1-C6 alkyl) amino group” in the present specification is a group in which one C1-C6 alkyl group is bonded to an amino group, and it is preferably a methylamino group, an ethylamino group, or a propylamino group.

[0168] The “di (C1-C6 alkyl) amino group” in the present specification is a group in which two C1-C6 alkyl groups are bonded to an amino group, and it is preferably a dimethylamino group, a diethylamino group, or a dipropyl amino group.

[0169] The “C1-C6 alkoxy carbonylamino group” in the present specification is a group in which a C1-C6 alkoxy carbonyl group is bonded to an amino group, and for example, it is amethoxycarbonylamino group, an ethoxycarbonylamino group, or a propoxycarbonylamino group.

[0170] The “mono (C1-C6 alkyl) aminocarbonylamino group” in the present specification is a group in which a mono (C1-C6 alkyl) aminocarbonyl group is bonded to an amino group, and it is preferably a methylaminocarbonylamino group, an ethylaminocarbonylamino group, or a propylaminocarbonylamino group.

[0171] The “di (C1-C6 alkyl) aminocarbonylamino group” in the present specification is a group in which a di (C1-C6 alkyl) aminocarbonyl group is bonded to an amino group, and it is preferably a dimethylaminocarbonylamino group, a diethylaminocarbonylamino group, or a dipropylaminocarbonylamino group.

[0172] The “5-membered aromatic heterocyclic carbonylamino group” in the present specification is a group in which a 5-membered aromatic heterocyclic carbonyl group is bonded to an amino group.

[0173] The “6-membered aromatic heterocyclic carbonylamino group” in the present specification is a group in which a 6-membered aromatic heterocyclic carbonyl group is bonded to an amino group.

[0174] The “C1-C6 alkyl sulfonylamino group” in the present specification is a group in which a C1-C6 alkyl group is bonded to the sulfonyl group of a sulfonylamino group, and it is preferably a methyl sulfonylamino group, an ethylsulfonylamino group, or a propylsulfonylamino group.

[0175] In some embodiments, the methods of the invention may be carried out by administering SIRT6 protein or a polynucleotide encoding SIRT6 protein. The SRT6 protein may be a wild-type protein or a modified protein. Examples of modified SIRT6 proteins include, without limitation, the modified proteins disclosed in WO 2022 / 241228 and WO 2024 / 021228, incorporated by reference herein in their entirety. In some embodiments, the polynucleotide encoding SIRT6 protein may be administered in a non-viral (e.g., a lipid nanoparticle) or viral (e.g., adeno- associated virus) vector. Methods of delivering SIRT6 by a vector may be carried out, for example, by the methods disclosed in US 2023 / 0018934, incorporated by reference herein in its entirety. In some embodiments, the SIRT6 protein or polynucleotide encoding SIRT6 protein may be administered together with a SIRT6 activator (e.g., any of the SIRT6 activators disclosed herein) in order to activate the newly administered SIRT6, thereby increasing SIRT6 activity additively or synergistically.Pharmaceutical compositions

[0176] The term “pharmaceutically acceptable salt” indicates a salt of the compounds that can be used as a pharmaceutical. When the compounds have an acidic group or a basic group it can be converted to a basic salt or an acidic salt by reacting with a base or an acid to form a salt thereof.

[0177] The pharmaceutically acceptable “basic salt” of the compounds preferably includes an alkali metal salt such as a sodium salt, a potassium salt, and a lithium salt; an alkaline earth metal salt such as a magnesium salt and a calcium salt; organic base salts such as an N-methyl morpholine salt, a triethylamine salt, a tributylamine salt, a diisopropylethylamine salt, a dicyclohexylamine salt, an N-methylpiperidine salt, a pyridine salt, a 4-pyrrolidinopyridine salt, and a picoline salt; and an amino acid salt such as glycine salt, a lysine salt, an arginine salt, an ornithine salt, a glutamate, and an aspartate, and it is preferably an alkali metal salt.

[0178] The pharmaceutically acceptable “acidic salt” of the compounds preferably includes an inorganic acid salt such as a hydrohalide such as a hydrofluoride, a hydrochloride, a hydrobromide, and a hydroiodide, a nitrate, a perchlorate, a sulfate, and a phosphate; an organic salt such as a lower alkanesulfonate such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate, an aryl sulfonate such as a benzenesulfonates, and a p-toluene sulfonate, an acetate, a malate, a fumarate, a succinate, a citrate, an ascorbate, a tartrate, an oxalate, a maleate, and the like; and an amino acid salt such as glycine salt, a lysine salt, an arginine salt, an ornithine salt, a glutamate, and an aspartate, and it is most preferably a hydrohalide (in particular, a hydrochloride).

[0179] The compounds of the present invention or the pharmaceutically acceptable salt thereof may absorb moisture, adhere to adsorbed water, or become a hydrate by leaving in the air or recrystallization. The present invention also encompasses compounds of such various hydrates, solvates, and crystalline polymorphs.

[0180] The compounds of the present invention, their pharmaceutically acceptable salts or solvates thereof, depending on the type and combination of substituents, may have various isomers such as geometric isomers such as a cis isomer and a trans isomer, tautomers, or optical isomers such as a d isomer and an 1 isomer, while the compounds include those all isomers, stereoisomers, and mixtures of these isomers and stereoisomers in any ratio unless otherwise specified. Mixtures of these isomers may be resolved by known resolution means.

[0181] The compounds of the present invention also include labels, that is, a compound in which one or more atoms of the compounds are substituted with an isotope (for example,2H,3H,13C,14C,3’S, and the like).

[0182] In addition, the present invention also encompasses a prodrug of the described compounds. The prodrug is a compound having a group which can be converted to an amino group, a hydroxyl group, a carboxyl group, or the like of the compound by hydrolysis or under physiological conditions, and as a group forming such a prodrug, it is a group described in Prog. Med., Vol. 5, pp. 2157 to 2161 (1985) or the like. As the prodrug, more specifically, when an amino group is present in the compound, a compound in which the amino group is acylated, alkylated, or phosphorylated (for example, it is a compound in which the amino group is eicosanoylated, alanylated, pentylaminocarbonylated, (5-methyl-2-oxo-l,3-dioxolen-4-yl) methoxy carbonylated, tetrahydrofuranylated, pyrrolidinyl methylated, pivaloyloxymethylatied, or tert-butyl ated, or the like) and the like are included, and when a hydroxyl group is present in the compound, a compound in which the hydroxyl group is acylated, alkylated, phosphorylated, or borated (for example, it is a compound in which the hydroxyl group is acetylated, palmitoylated, propanoylated, pivaloylated, succinylated, fumarylated, alanylated, or dimethylaminomethyl carbonylated, or the like) and the like are included. In addition, when a carboxy group is present in the compound, a compound in which the carboxy group is esterified or amidated (for example, it is a compound in which the carboxy group is ethyl esterified, phenyl esterified, carboxymethyl esterified, dimethylaminomethyl esterified, pivaloyloxymethyl esterified, ethoxycarbonyloxyethyl esterified, amidated, or methylamidated, or the like.), and the like are included.

[0183] The SIRT6 activator compounds of the present invention may be produced by synthetic methods known in the art and as described in WO 2017 / 170623 and WO 2019 / 065928, incorporated by reference herein in their entirety.Administration methods

[0184] Administration of the compounds of the present invention may be carried out by any form of oral administration by a tablet, a pill, a capsule, a granule, a powder, a solution, or the like, or by any form of parenteral administration by an injection for intra-articular, intravenous, intramuscular, intraocular, or the like, a suppository, an eye drop, an eye ointment, a transdermalsolution, an ointment, a transdermal patch, a transmucosal solution, a transmucosal patch, an inhalant, or the like.

[0185] In some embodiments, a solid composition for oral administration, a tablet, a powder, a granule, and the like are used. Such a solid composition is composed of one or more active ingredients and at least one inert excipient such as lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, polyvinyl pyrrolidone, magnesium metasilicate aluminate, and / or the like. The solid composition may contain, according to a conventional method, one or more of an inert additive such as a lubricant such as magnesium stearate, a disintegrant such as sodium carboxymethyl starch, a stabilizer, and a solubilizer. The tablet or pill may be coated with a sugar coating or a film of a substance soluble in the stomach or intestine, if necessary.

[0186] In some embodiments, a liquid composition for oral administration, a pharmaceutically acceptable emulsion, solution, suspension, syrup, elixir, or the like is used. To such a liquid composition, it is possible to add a generally used inert diluent such as purified water or ethanol. The liquid composition may contain, in addition to an inert diluent, one or more of a solubilizer, an adjuvant such as a wetting agent, a sweetening agent, a flavoring agent, a fragrance, and a preservative.

[0187] In some embodiments, an injection for parenteral administration, a sterile aqueous or nonaqueous solution, a suspension or an emulsion, and the like are used. The aqueous solvent includes, for example, distilled water for injection, physiological saline, and the like. The nonaqueous solvents include, for example, propylene glycol, polyethylene glycol, and vegetable oil such as olive oil, alcohols such as ethanol, Polysorbate 80, and the like. Such an injection composition may further contain a one or more of a tonicity agent, a preservative, a wetting agent, an emulsion, a dispersing agent, a stabilizer, or a solubilizer. These injection compositions can be sterilized by, for example, fdtration through a bacteria retention filter, application of a bactericide, or irradiation. In addition, these injection compositions may be used by producing a sterile solid composition and dissolved or suspended in sterile water or a sterile solvent for injection prior to use.

[0188] In some embodiments, an external preparation, an ointment, a plaster, a cream, a jelly, a cataplasm, a spray, a lotion, an eye drop, an eye ointment, and the like are used. These external preparations include generally used ointment bases, lotion bases, aqueous or non-aqueoussolutions, suspensions, emulsions, and the like. For example, as an ointment or lotion base, polyethylene glycol, propylene glycol, white petrolatum, bleached beeswax, polyoxyethylene hydrogenated castor oil, glycerin monostearate, stearyl alcohol, cetyl alcohol, lauromacrogol, sorbitan sesquioleate, and the like are used.

[0189] A transmucosal agent such as an inhalant and a transnasal agent are used in solid, liquid, or semisolid form, and it may be produced according to a conventionally known method. For example, a known excipient, and furthermore, one or more of a pH adjuster, a preservative, a surfactant, a lubricant, a stabilizer, a thickener, and the like may be added as appropriate. With these transmucosal agents, devices appropriate for inhalation or insufflation may be used as the method of administration. For example, the compound may be administered alone or as a powder of a formulated mixture, or as a solution or suspension in combination with a pharmaceutically acceptable carrier, using known devices and nebulizers, such as metered dose inhalation devices. A dry powder inhaler or the like may be for single or multiple administration, and a dry powder or powder containing capsule may be also used. Alternatively, an appropriate ejector may be used. For example, it may be in the form of a pressurized aerosol spray or the like using a suitable gas such as chlorofluoroalkane, hydrofluoroalkane, or carbon dioxide.

[0190] In the case of normal oral administration, the appropriate daily dose is about 0.001 to 100 mg / kg, preferably 0.1 to 30 mg / kg, and more preferably 0.1 to 10 mg / kg of body weight. In some embodiments, the appropriate daily dose is about 1 to 500 mg, e.g., about 5 to 200 mg, e.g., about 10-100 mg, e.g., about 15-30 mg. This is administered in one dose or separated into two or more doses. When administered intravenously, the appropriate daily dose is about 0.0001 to 10 mg / kg of body weight, which is administered once or separated into several times a day. In addition, as a transmucosal agent, about 0.001 to 100 mg / kg of body weight is administered once or separated into several times a day. The dose is appropriately determined depending on the individual case in consideration of symptoms, age, sex, and the like.

[0191] In some embodiments, the additional therapeutic agent is administered in the same composition as the SIRT6 activator. In some embodiments, the additional therapeutic agent is administered in a separate composition as the SIRT6 activator.

[0192] In the methods of the present invention, the compounds may be administered in combination with additional various therapeutic agents or preventive agents for diseases that are considered to exhibit the efficacy thereof. The combination may be administered simultaneously,separately, concurrently, and continuously or at desired time intervals. The co-administered agents may be blended or formulated separately. The therapeutic agent may be, for example, one that is known to decrease body weight, decrease liver weight, decrease triglyceride, total cholesterol, and / or LDL cholesterol level(s), or treat the targeted, or another, fatty liver disease. In an embodiment, the therapeutic agent is one that treats Metabolic Dysfunction-Associated Steatohepatitis, hepatic steatosis, hepatic fibrosis, hepatic inflammation, and / or a dysfunction of hepatic lipogenesis.

[0193] The methods of the present invention find use in both veterinary and medical applications. Suitable subjects include avians, reptiles, amphibians, fish, and mammals. The term “mammal” as used herein includes, but is not limited to, humans, primates, non-human primates (e.g., monkeys and baboons), cattle, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g., rats, mice, hamsters, and the like), etc. Human subjects include neonates, infants, juveniles, and adults. Optionally, the subject is “in need of’ the methods of the present invention, e.g., because the subject has or is believed at risk for one or more of the disorders or conditions described herein or that would benefit from the delivery of a compound as described herein. As a further option, the subject can be a laboratory animal and / or an animal model of disease. Preferably, the subject is a human.EXAMPLES

[0194] The present invention will be explained below in further detail with reference to an example, though the present invention is in no way limited by this Example.

[0195] Example 1: Effects of SP-624 on Diet-Induced Obesity.

[0196] A study was designed to determine the results of administering a SIRT6 activator (SP-624) to male and female C57BL / 6 mice in an obesity model (FIG. 1). The duration of the study covered eight total weeks, from Day 0 to Day 56. The mice were six weeks of age. The mice were divided into five groups, with Group 1 being fed a normal diet and Groups 2-5 being fed a high fat diet consisting of 20% protein, 60% fat, and 20% carbohydrate (Research Diets DI 2492) to induce obesity. Each Group were fed for four weeks prior to the administration of any treatment or control, from Day 0 to Day 27. Group 1 was not administered any treatment. Group 2 was administered a vehicle. Group 3 was administered SP-624 at a concentration of 0.1 mg / mL, using 0.5% methylcellulose (MC) as the vehicle for administration. Group 4 was administered SP-624 at aconcentration of 1.0 mg / mL, using 0.5% MC as the vehicle for administration. Group 5 was administered reference compound orlistat at a concentration of 10.0 mg / mL, using water for injection (WFI) as the vehicle for administration. Starting at Day 28 and continuing until Day 55 of the study, each vehicle, test article, or reference compound was administered by oral gavage once daily for four weeks, after four weeks of feeding the mice of Groups 2-5 a high fat diet.

[0197] FIG. 2 and FIG. 3 depict the body weight of the male and female mice over the course of the study. Both male and female mice in Groups 3 and 4, who were administered SP-624 at different concentrations, observed a decrease in body weight after the administration of SP-624 compared to mice in Group 2, that were only administered a vehicle.

[0198] The male mice of Groups 3 and 4, that received SP-624, on average had lower levels of fasting blood glucose than the male mice of Group 2 (FIG. 4). Similarly, the male mice of Groups 3 and 4 had lower total cholesterol levels in the blood on average than the male mice of Group 2 (FIG. 5). Notably, the male mice of Group 4, that received a higher concentration of SP-624, had lower levels of total cholesterol in the blood than the male mice of Group 3. The male mice in Group 4 on average had lower levels of LDL cholesterol in the blood than the male mice in Groups 2 and 5, who received the vehicle and orlistat, respectively (FIG. 6). Similarly, the male mice of Group 4 on average had lower levels of triglycerides in the blood than the male mice in either of Groups 2 or 5 (FIG. 7).

[0199] The female mice of Groups 3 and 4 on average had slightly lower levels of fasting blood glucose compared to the female mice of Group 1, who were fed the normal diet, and the female mice of Group 2, who were fed the high fat diet and received only the vehicle during the study (FIG. 8). The female mice of Groups 3 and 4 on average had lower levels of total cholesterol measured in the blood than the female mice of Group 2 (FIG. 9). The female mice of Group 4 had slightly lower total cholesterol in the blood than the female mice of Group 3. The female mice of Groups 3 and 4 on average had lower blood levels of LDL cholesterol than the female mice of Group 2, and the female mice of Group 5, who received orlistat (FIG. 10). The female mice of Groups 3 and 4 on average had lower levels of triglycerides in the blood in comparison to the female mice in Group 2 (FIG. 11).

[0200] FIG. 12 depicts a dissection of one of the mice, indicating different areas of body fat reduced by the administration of SP-624 to the subject.

[0201] FTG. 13 shows that on average, the male mice administered SP-624 had a lower epididymal fat weight compared to the male mice administered only vehicle. Similarly, FIG. 14 shows that the male mice administered SP-624 on average had lower inguinal fat weight in comparison to the male mice only administered vehicle. FIG. 15 portrays that the male mice administered SP-624 on average had lower liver weights in comparison to the liver weights of the male mice who only received vehicle. Additionally, FIG. 15 shows that the Group 3 male mice who received the lower dosage of SP-624 on average had roughly the same liver weight as the male mice who were fed the normal diet. The male mice of Group 4, who received the higher dosage of SP-624, had a lower liver weight on average in comparison to the Group 1 male mice who were fed the normal diet. The male mice administered SP-624 had lower epididymal fat-to-body weight ratios than the male mice who were administered only a vehicle (FIG. 16). Similarly, FIG. 17 shows that the male mice administered SP-624 had lower inguinal fat-to-body weight ratios compared to the male mice who received just a vehicle. The male mice administered SP-624 on average had lower liver-to- body weight ratios than the male mice who received only a vehicle or the male mice who received the reference compound orlistat (FIG. 18).Female mice who were administered SP-624 on average had a lower periovarian fat weight than the female mice who received only a vehicle (FIG. 19). FIG. 20 shows that the female mice who received SP-624 also had lower inguinal fat weight on average in comparison to the female mice administered just a vehicle. FIG. 21 shows that the female mice administered SP-624 had a lower liver weight on average than the female mice administered orlistat and had a slightly lower liver weight on average than the female mice who were only administered vehicle. The female mice receiving SP-624 had lower periovarian fat-to-body weight ratios on average than the female mice administered just a vehicle (FIG. 22). Similarly, FIG. 23 shows that the female mice receiving SP-624 had lower inguinal fat-to-body weight ratios on average in comparison to the inguinal fat- to-body weight ratios of the female mice only receiving a vehicle. FIG. 24 indicates that while the female mice administered SP-624 had higher average liver-to-body weight ratios compared to the female mice receiving just a vehicle, the SP-624-administered female mice had a lower liver-to- body weight ratio average than the female mice administered orlistat.

[0202] Example 2: Effects of SP-624 and / or Semaglutide on Diet-Induced Obesity.

[0203] A study was designed to determine the results of administering a SIRT6 activator (SP-624) and / or Semaglutide to male C57BL / 6 mice in an obesity model (FIG. 25). The duration of thestudy covered eight total weeks, from Day 0 to Day 56. Starting at six weeks of age the mice were given a high fat diet consisting of 20% protein, 60% fat, and 20% carbohydrate (Research Diets D12492) for 12 weeks in order to induce obesity. The mice were 18 weeks of age at the time of the study. The mice were divided into 12 groups, with Group 1 being fed a normal diet and Groups 2-12 being fed a high fat diet. Group 2 was administered vehicle 1 of SP-624 (0.5% MC) by oral gavage once daily for 4 weeks, and vehicle 2 of Semaglutide (20 mM citrate buffer (pH 7.0)) by subcutaneous injection once every three days for up to 27 days. Groups 3 and 4 were administered a 3 mg / kg low dose (Group 3) or a 10 mg / kg high dose (Group 4) of SP-624 by oral gavage once daily for 4 weeks. Groups 5 and 6 were administered a 10 nmol / kg low dose (Group 5) or a 30 nmol / kg high dose (Group 6) of Semaglutide subcutaneously once every three days for up to 27 days. Groups 7-10 received both SP-624 and Semaglutide at low / low dosages (Group 7), low / high dosages (Group 8), high / low dosages (Group 9), and high / high dosages (Group 10), respectively. Group 11 was administered a high dose of SP-624 by oral gavage once daily for 8 weeks and a high dose of Semaglutide subcutaneously once every three days for up to 27 days. Group 12 was administered vehicle 1 of SP-624 by oral gavage once daily for 8 weeks and a high dose of Semaglutide subcutaneously once every three days for up to 27 days.

[0204] Blood Biochemistry Index

[0205] Male mice fed a high fat diet for 16 weeks significantly increased total cholesterol levels (FIG. 26A), triglyceride levels (FIG. 27A), and alanine transaminase levels (FIG. 28A) on weeks 12 and 16, indicating a successful induction of diet-induced obesity compared to the normal diet fed mice. SP-624 at 3 and 10 mg / kg showed, in a dose-dependent manner, significant reduction in total cholesterol levels on week 16, when compared to the high fat diet fed mice (FIG. 26A). Semaglutide at 10 and 30 nmol / kg also significantly reduced total cholesterol levels (FIG. 26A) and alanine transaminase levels (FIG. 28A) on week 16. Moreover, SP-624 at 3 and 10 mg / kg in combination with Semaglutide at 10 and 30 nmol / kg significantly reduced, in a dose-dependent manner, total cholesterol levels (FIGS. 26A-26B) and alanine transaminase levels (FIGS. 28A- 28B) on week 16. Further, SP-624 at 3 and 10 mg / kg in combination with Semaglutide at 30 nmol / kg also significantly lowered triglyceride levels on week 16 (FIGS. 27A-27B).

[0206] Lactate dehydrogenase levels were significantly elevated in the high fat diet fed mice on week 16 (FIG. 29A). Only SP-624 at 10 mg / kg in combination with Semaglutide at 10 nmol / kg significantly lowered lactate dehydrogenase levels on week 16 (FIGS. 29A-29B).

[0207] Creatinine levels between the vehicle control and treated groups were similar (FIG. 30A). Only SP-624 at 3 mg / kg in combination with Semaglutide at 30 nmol / kg significantly reduced creatinine levels on week 16 (FIG. 30A).

[0208] On week 20, total cholesterol levels were increased in Semaglutide withdrawal vehicle group (Group 12) (FIG. 26B). Semaglutide- withdrawn mice continuously treated with SP-624 at 10 mg / kg for additional 4 weeks significantly maintained total cholesterol reduction from week 16 to week 20, when compared to Semaglutide withdrawal vehicle group (Group 12) (FIG. 26B).

[0209] Male mice fed a high fat diet for 16 weeks moderately to significantly increased the fasting blood glucose (FIG. 31A), insulin (FIG. 32A), and leptin (FIG. 33A) levels on weeks 12 and 16, indicating a development of insulin and leptin resistance compared to the normal diet fed mice. Semaglutide at 10 and 30 nmol / kg alone or in combination with SP-624 at 3 and 10 mg / kg significantly reduced fasting blood glucose (FIG. 31A), insulin (FIG. 32A), and leptin (FIG. 33A) levels on week 16, when compared to the high fat diet fed mice. SP-624 alone at 3 and 10 mg / kg did not affect fasting blood glucose (FIG. 31A), insulin (FIG. 32A), and leptin (FIG. 33A) levels.

[0210] Fasting blood glucose (FIG. 31B), insulin (FIG. 32B), and leptin (FIG. 33B) levels were increased in Semaglutide withdrawal vehicle group (Group 12) on week 20, and Semaglutide- withdrawn mice continuously treated with SP-624 at 10 mg / kg for additional 4 weeks significantly reduced insulin (FIG. 32B) and leptin (FIG. 33B) levels on week 20, when compared to Semaglutide withdrawal vehicle group (Group 12).

[0211] Compared to the normal diet fed mice, male mice fed a high fat diet for 16 weeks had significantly increased levels of high-density lipoprotein (FIG. 34A) and low-density lipoprotein (FIG. 35A) on week 16. SP-624 at 3 and 10 mg / kg alone, Semaglutide at 10 and 30 nmol / kg alone or in combination with SP-624 at 3 and 10 mg / kg moderately to significantly lowered high-density lipoprotein (FIG. 34A) and low-density lipoprotein (FIG. 35A) levels on week 16, when compared to the high fat diet fed.

[0212] Blood urea nitrogen levels were significantly decreased in high fat diet fed mice on week 16 (FIG. 37A). SP-624 at 3 and 10 mg / kg in combination with Semaglutide at 10 and 30 nmol / kg significantly lowered blood urea nitrogen levels on week 16 (FIG. 37A).

[0213] Cytokine Levels

[0214] Serum TNF-a levels were significantly increased in the high fat diet fed mice on week 16 compared to the normal diet fed mice (FIG. 39A). SP-624 at 3 and 10 mg / kg in combination withSemaglutide at 10 and 30 nmol / kg moderately to significantly lowered TNF-a levels on week 16 (FIG. 39A).

[0215] Serum IL-6 (FIG. 38A) and aspartate transaminase (FIG. 36A) levels between the vehicle control and treated groups was similar. Only SP-624 at 10 mg / kg in combination with Semaglutide at 10 nmol / kg significantly reduced aspartate transaminase levels on week 16 (FIG. 36A).

[0216] On week 20, Semaglutide-withdrawn mice continuously treated with SP-624 at 10 mg / kg for additional 4 weeks significantly reduced high-density lipoprotein (FIG. 34B), low-density lipoprotein (FIG. 35B), aspartate transaminase (FIG. 36B), and TNF-ct levels (FIG. 39B), when compared to Semaglutide withdrawal vehicle group (Group 12).

[0217] Tissue Weight

[0218] Male mice fed a high fat diet for 16 weeks had significant increase in epididymal fat weight (FIG. 40A), inguinal fat mass (FIG. 41A), and liver weight (FIG. 42A) as well as epididymal fat- to-body weight ratio (FIG. 40B) and inguinal fat-to-body weight ratio (FIG. 41B), when compared to the normal diet fed mice. Semaglutide at 10 and 30 nmol / kg alone or in combination with SP- 624 at 3 and 10 mg / kg caused moderate to significant reductions in epididymal fat weight (FIG. 40A), inguinal fat mass (FIG. 41A), and inguinal fat-to-body weight ratio (FIG. 41B) on week 16. Moreover, SP-624 at 1 and 10 mg / kg alone, Semaglutide at 10 and 30 nmol / kg alone, or in combination with SP-624 at 1 and 10 mg / kg induced significant reductions in liver weight (FIG. 42A) and liver-to-body weight ratio (FIG. 42B), when compared to the high fat diet fed mice.

[0219] On week 20, Semaglutide-withdrawn mice continuously treated with SP-624 at 10 mg / kg for additional 4 weeks significantly maintained inguinal fat weight (FIG. 41C) and inguinal fat- to-body weight ratio (FIG. 41D) reduction.

[0220] At week 16 or week 20, liver was stained for lipid content by oil red O staining (FIG. 49). Histopathological examination of the livers showed that the oil red O-stained area (%) of the high fat diet fed mice was observed to be significantly different from the normal diet fed mice. All treatments tended to decrease the oil red O-stained red tissue (FIG. 49). Also, the oil red O-stained percent (%) value of all the treated groups (Groups 3-10) was significantly lower than that of vehicle group (FIG. 49). Moreover, Semaglutide-withdrawn mice continuously treated with SP- 624 at 10 mg / kg for additional 4 weeks significantly decreased the oil red O-staining on week 20, when compared to Semaglutide withdrawal vehicle group (Group 12) (FIG. 49).

[0221] Muscle Weight

[0222] Compared to the normal diet fed mice, the high fat diet fed mice had significantly reduced relative weight of lower limb muscles (FIGS. 43A, 44A, 45A, 46A), while Semaglutide at 10 and 30 nmol / kg alone or in combination with SP-624 at 1 and 10 mg / kg significantly reversed the effect of high fat diet on soleus muscle-to-body weight ratio (FIG. 44B), tibialis anterior muscle- to-body weight ratio (FIG. 45B), and quadriceps muscle-to-body weight ratio (FIG. 46B). Moreover, Semaglutide at 30 nmol / kg alone or in combination with SP-624 at 1 and lOmg / kg significantly reversed the effect of high fat diet on gastrocnemius muscle-to-body weight ratio (FIG. 43B). When compared to Semaglutide withdrawal vehicle group (Group 12), Semaglutide- withdrawn mice continuously treated with SP-624 at 10 mg / kg for additional 4 weeks significantly increased soleus muscle-to-body weight ratio on week 20 (FIGS. 44C, 44D).

[0223] Group 2 mice fed a high fat diet for 16 weeks caused significant increase in body weight, when compared to the normal diet fed mice of Group 1 (FIG. 47A). SP-624 at 3 and 10 mg / kg alone (Groups 3 and 4) showed moderate decrease in body weight, when compared to Group 2. Moreover, Semaglutide at 10 and 30 nmol / kg alone (Groups 5 and 6) or in combination with SP- 624 at 3 and 10 mg / kg (Groups 7-12) significantly decreased the body weight from Day 7 to Day 28 (FIG. 47A), when compared to Group 2. Mice regained their body weight after Semaglutide withdrawal (Groups 11 and 12) (FIG. 47B). Semaglutide-withdrawn mice continuously treated with SP-624 at 10 mg / kg for additional 4 weeks showed mild decrease in body weight during the study period (Group 11), when compared to Semaglutide withdrawal vehicle group (Group 12) (FIG. 47B)

[0224] Food intake was measured once weekly from week 13 to week 20 for Groups 11 and 12. SP-624 at 10 mg / kg in combination with Semaglutide at 30 nmol / kg caused mild increase in food intake (FIG. 48).

[0225] The foregoing examples are illustrative of the present invention and are not to be construed as limiting thereof. Although the invention has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the invention as described and defined in the following claims.

Claims

What is claimed is:

1. A method of decreasing body weight in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a sirtuin 6 (SIRT6) activator, thereby decreasing body weight in the subject.

2. A method of maintaining body weight in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a sirtuin 6 (SIRT6) activator, thereby maintaining body weight in the subject.

3. A method of maintaining body weight in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a sirtuin 6 (SIRT6) activator after withdrawal of a weight loss treatment (e.g., glucagon-like peptide 1 receptor agonist (GLP- 1 RA) treatment), thereby maintaining body weight in the subject.

4. A method of decreasing triglycerides in the blood in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a sirtuin 6 (SIRT6) activator, thereby decreasing triglycerides in the blood in the subject.

5. A method of decreasing total cholesterol in the blood in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a sirtuin 6 (SIRT6) activator, thereby decreasing total cholesterol in the blood in the subject.

6. A method of decreasing low-density lipoprotein (LDL) cholesterol in the blood in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a sirtuin 6 (SIRT6) activator, thereby decreasing LDL cholesterol in the blood in the subject.

7. A method of decreasing liver weight in a subject in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a sirtuin 6 (SIRT6) activator, thereby decreasing liver weight in the subject.

8. A method of treating a fatty liver disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a sirtuin 6 (SIRT6) activator, thereby treating the fatty liver disease in the subject.

9. The method of claim 8, wherein the fatty liver disease is related to a dysfunction in hepatic metabolic homeostasis.

10. The method of claim 9, wherein the fatty liver disease is Metabolic Dysfunction- Associated Steatohepatitis (MASH).

11. A method of suppressing hepatic lipogenesis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby suppressing hepatic lipogenesis in the subject.

12. A method of increasing hepatic fatty acid oxidation (FAO) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby increasing hepatic fatty acid oxidation in the subject.

13. A method of reducing oxidative stress in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby reducing oxidative stress.

14. A method of decreasing hepatic inflammation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby decreasing hepatic fibrosis.

15. A method of decreasing hepatic steatosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby decreasing hepatic steatosis.

16. A method of decreasing hepatic fibrosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby decreasing hepatic fibrosis.

17. A method of preserving muscle mass in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SIRT6 activator, thereby preserving muscle mass.

18. The method of any one of claims 1-17, further comprising administering a therapeutically effective amount of an additional therapeutic agent.

19. The method of claim 18, wherein the additional therapeutic agent is an agent that decreases body weight.

20. The method of claim 19, wherein the additional therapeutic agent is, orlistat, phentermine, phentermine-topiramate, naltrexone-bupropion, setmelanotide, a glucagon-like peptide-1 receptor agonist (e.g., tirzepatide, liraglutide, semaglutide, exenatide, albiglutide, lixisenatide, dulaglutide), or any combination thereof.

21. The method of claim 19, wherein the agent is a glucagon-like peptide 1 receptor agonist (GLP-1 RA).

22. The method of any one of claims 1-21, further comprising administering an effective amount of an estrogen.

23. The method of claim 22, wherein the subject is a female.

24. The method of claim 22 or 23, wherein the estrogen is conjugated to the SIRT6 activator or the additional therapeutic agent.

25. The method of claim 22, comprising administering the therapeutically effective amounts of the SIRT6 activator and the estrogen in combination with a therapeutically effective amount of a GLP-1 RA.

26. The method of claim 18, wherein the additional therapeutic agent is an agent that decreases triglycerides in the blood.

27. The method of claim 26, wherein the additional therapeutic agent is niacin, a fibrate or a fibric acid derivative, a statin, icosapent, omega-3 fatty acid or omega-3 fatty acid ethyl ester (e.g. docosahexaenoic acid, eicosapentaenoic acid), or any combination thereof.

28. The method of claim 18, wherein the additional therapeutic agent is an agent that decreases total cholesterol in the blood.

29. The method of claim 28, wherein the additional therapeutic agent is niacin, a fibrate or a fibric acid derivative, a statin, a resin or a bile acid sequestrant, a cholesterol absorption inhibitor, omega-3 fatty acid or omega-3 fatty acid ethyl ester (e.g., docosahexaenoic acid, eicosapentaenoic acid), marine-derived omega-3 polyunsaturated fatty acid (PUFA), a PCSK9 inhibitor, an adenosine triphosphate-citrate lyase (ACLY) inhibitor, or any combination thereof.

30. The method of claim 18, wherein the additional therapeutic agent is an agent that decreases low-density lipoprotein cholesterol in the blood.

31. The method of claim 30, wherein the additional therapeutic agent is niacin, a fibrate or a fibric acid derivative, a statin, a resin or a bile acid sequestrant, omega-3 fatty acid or omega-3 fatty acid ethyl ester (e.g., docosahexaenoic acid, eicosapentaenoic acid), a PCSK9 inhibitor, or any combination thereof.

32. the method of claim 18, wherein the additional therapeutic agent is an agent that treats fatty liver disease.

33. The method of claim 32, wherein the additional therapeutic agent is an insulin sensitizer, a lipid-lowering drug (e g., statins), an angiotensin receptor blocker, pentoxifylline, a steroid (corticosteroid), a thyroid hormone receptor (THR)-beta agonist (resmetirom), or any combination thereof.

34. The method of any one of claims 1-33, further comprising administering to the subject a therapeutically effective amount of nicotinamide adenine dinucleotide (NAD+) and / or an NAD+ precursor.

35. The method of claim 34, wherein the SIRT6 activator and the NAD+ and / or NAD+ precursor are administered in the same composition.

36. The method of claim 34, wherein the SIRT6 activator and the NAD+ and / or NAD+ precursor are administered in separate compositions.

37. The method of any one of claims 34-36, wherein the NAD+ precursor is nicotinamide riboside, nicotinic acid riboside, nicotinic acid, nicotinamide, nicotinamide mononucleotide, tryptophan, or any combination thereof.

38. The method of any one of claims 1-37, wherein the SIRT6 activator is quercetin, isoquercetin, kaempferol, luteolin, cyanidin, fisetin, delphinidin, icariin, N-acetylethanolamines, oleic acid, linoleic acid, fucoidan, MDL-800 (CAS No. 2275619-53-7), MDL-811 (CAS No. 2275619-98-0), UBCS038 (CAS No. 358721-70-7), UBCS039 (CAS No. 358721-70-7), UBCS040 (l-(4,5-Dihydropyrrolo[l,2-a']quinoxalin-4-yl)naphthalen-2-ol), UBCS058 (4- (Pyridin-3-yl)pyrrolo[l,2-rz]quinoxaline), UBCS060 (4-(Pyridin-2-yl)-4,5-dihydropyrrolo[l,2- tz]quinoxaline), UBCS068 (l-(5-((3-(Trifluoromethyl)phenyl)sulfonyl)-4,5-dihydropyrrolo[l,2- rz]quinoxalin-4-yl)naphthalen-2-ol), myristic acid, oleoylethanolamide, CL5D (CAS No. 2488745-53-3), 10b (2-(l-benzofuran-2-yl)- / V-(diphenylmethyl) quinoline-4-carboxamide), or forvisirvat (SP-624).

39. The method of any one of claims 1-37, wherein the SIRT6 activator is a compound of Formula 1 or a pharmaceutically acceptable salt thereof(D wherein:R1is a C1-C6 alkyl group optionally substituted with the same or different one to two substituents selected from a substituent group X, a C3-C6 cycloalkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, or a 4-7 membered saturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X;R2is a C1-C6 alkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C3-C6 cycloalkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, or a 4-7 membered saturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X;A is a 5-membered aromatic heterocyclic ring, a 6-membered aromatic heterocyclic ring, an 8-10 membered condensed aromatic heterocyclic ring, a 5-7 membered unsaturated heterocyclic ring, a 4-7 membered saturated heterocyclic ring, a benzene ring, -CH=, or a cyano group, wherein when A is a cyano group, R3and R3do not exist;R3and R3are each independently a hydrogen atom, a halogen atom, a cyano group, a hydroxy group, an oxo group, a C1-C6 alkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C1-C6 alkoxy group optionally substituted with the same or different one to two substituents selected from the substituent group X,a C2-C6 alkenyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C2-C6 alkynyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C3-C6 cycloalkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, an amino group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C1-C6 alkoxy carbonyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a carbamoyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a phenyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a 5-membered aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, a 6-membered aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, a 5-7 membered unsaturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, a 4-7 membered saturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, an 8-10 membered condensed aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, orR3and R3may form a 5-7 membered unsaturated heterocyclic ring, a 4-7 membered saturated heterocyclic ring, or a C3-C6 cycloalkyl ring as a ring that binds to each other and condenses with A, and the ring is optionally substituted with the same or different one to two substituents selected from the substituent group X; substituent group X is a halogen atom, a cyano group, a hydroxy group, an oxo group, a C1-C6 alkyl group, a hydroxy C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a C1-C6 haloalkyl group, a C3-C6 cycloalkyl group, a C3-C6 halocycloalkyl group,a phenyl group optionally substituted with the same or different one to two substituents selected from a substituent group Y, a 5-membered aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 6-membered aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 4-7 membered saturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a C1-C6 alkoxy group, a C1-C6 haloalkoxy group, a C3-C6 cycloalkoxy group, a C3-C6 halocycloalkoxy group, a phenoxy group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 5-membered aromatic heterocyclic oxy group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 6-membered aromatic heterocyclic oxy group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 4-7 membered saturated heterocyclic oxy group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a C1-C6 alkoxycarbonyl group, a C3-C6 cycloalkoxycarbonyl group, a carboxy group, a C1-C6 alkylcarbonyl group, a C3-C6 cycloalkylcarbonyl group, a phenyl carbonyl group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a carbamoyl group, a mono (C1-C6 alkyl) aminocarbonyl group, a di (C1-C6 alkyl) aminocarbonyl group, a mono (C1-C6 alkyl) aminosulfonyl group, a di (C1-C6 alkyl) aminosulfonyl group, an amino group, a mono (C1-C6 alkyl) amino group, a di (C1-C6 alkyl) amino group, a C1-C6 alkoxycarbonylamino group, a mono (C1-C6 alkyl) aminocarbonylamino group, a di (C1-C6 alkyl) aminocarbonylamino group, a C1-C6 alkylcarbonylamino group, a phenylcarbonylamino group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 5-membered aromatic heterocyclic carbonylamino group optionally substituted with the same or different one to two substituents selected from the substituent group Y,a 6-membered aromatic heterocyclic carbonylamino group optionally substituted with the same or different one to two substituents selected from the substituent group Y, or a C1-C6 alkylsulfonylamino group; and substituent group Y is a C1-C6 alkyl group, a C1-C6 alkoxy group, a halogen atom, or a hydroxy group.

40. The method of claim 39, wherein the 5-membered aromatic heterocyclic ring or the 5- membered aromatic heterocyclic group in A, R3, or R3is any one selected from the group:

41. The method of claim 39 or 40, wherein the 6-membered aromatic heterocyclic ring or the 6-membered aromatic heterocyclic group in A, R3, or R3is any one selected from the group:

42. The method of any one of claims 39-41, wherein the 8-10 membered condensed aromatic heterocyclic ring or the 8-10 membered condensed aromatic heterocyclic group in A, R3, or R3is any one selected from the group:

43. The method of any one of claims 39-42, wherein the 5-7 membered unsaturated heterocyclic ring or 5-7 membered unsaturated heterocyclic group in A, R3, or R3is any one selected from the group:

44. The method of any one of claims 39-43, wherein the 4-7 membered saturated heterocyclic ring or the 4-7 membered saturated heterocyclic group in A, R1, R2, or R3is any one selected from the group:

45. The method any one of claims 39-44, wherein the compound of Formula 1 is any compound selected from the following group:(2S,5’R)-7-chloro-6-(5-ethyl-l,3,4-oxadiazol-2-yl)-3’,4-dimethoxy-5’-methyl-spiro [benzofuran- 2,4’ -cyclohex-2-ene]- 1 ’ ,3 -dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(5-tetrahydropyran-4-yl-l,3,4-oxadiazol-2-yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5 ’R)-7-chl oro-3 ’ ,4-dimethoxy-5 ’ -methyl-6-[5-( 1 -methyl-4-piperidyl)- 1 ,3 ,4-oxadiazol-2-yl] spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-6-[5-(4-fluoro-l-methyl-4-piperidyl)-l,3,4-oxadiazol-2-yl]-3’,4-dimethoxy- 5’-methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chl oro-3 ’,4-dimethoxy-6-[5-[(lS)-l -methoxy ethyl]- 1,3, 4-oxadi azol -2 -yl]-5 ’ -methylspiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-4-ethoxy-3’-methoxy-5’-methyl-6-(5-methyl-l,3,4-oxadiazol-2-yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-4-(difluoromethoxy)-3’-methoxy-5’-methyl-6-(5-methyl-l,3,4-oxadiazol-2-yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chl oro-3 ’,4-dimethoxy-6-[3-(l -methoxy ethyl)-l, 2, 4-oxadiazol-5-yl]-5’-methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-6-[3-(l-hydroxy-l-methyl-ethyl)-l,2,4-oxadiazol-5-yl]-3’,4-dimethoxy-5’- methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(lH-pyrazol-5-yl) spiro [benzofuran-2,4’- cyclohex-2-ene]-l’,3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-6-[l-(2-methoxyethyl) pyrazol-3-yl]-5’-methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-6-(l,8-dioxa-2-azaspiro [4.5] dec-2-en-3-yl)-3’,4-dimethoxy-5’-methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(8-methyl-l-oxa-2,8-diazaspiro [4.5] dec-2-en-3- yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-6-(2-methoxypyrimidin-5-yl)-5’-methyl-spiro [benzofuran-2,4’ -cyclohex-2-ene]- 1 ’ ,3 -dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-6-(6-methoxy-3-pyridyl)-5’-methyl-spiro [benzofuran-2,4’- cyclohex-2-ene]-l ’, 3-dione; or(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(3-pyridyl) spiro [benzofuran-2,4’-cyclohex-2- ene]-l’,3-dione.

46. The method of any one of claims 39-45, wherein the compound of Formula 1 is a compound of Formula T or a pharmaceutically acceptable salt thereof:wherein:R1is a C1-C6 alkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C3-C6 cycloalkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, or a 4-7 membered saturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X;R2is a C1-C6 alkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X,a C3-C6 cycloalkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, or a 4-7 membered saturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X;A is a 5-membered aromatic heterocyclic ring, a 6-membered aromatic heterocyclic ring, an 8-10 membered condensed aromatic heterocyclic ring, a 5-7 membered unsaturated heterocyclic ring, a 4-7 membered saturated heterocyclic ring, a benzene ring, or a single bond, wherein when it is a single bond, one or the other of R3and R3is not present;R3and R3are each independently a hydrogen atom, a halogen atom, a cyano group, a hydroxy group, a C1-C6 alkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C1-C6 alkoxy group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C2-C6 alkenyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C2-C6 alkynyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C3-C6 cycloalkyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, an amino group optionally substituted with the same or different one to two substituents selected from the substituent group X, a C1-C6 alkoxy carbonyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a carbamoyl group optionally substituted with the same or different one to two substituents selected from the substituent group X, a phenyl group optionally substituted with the same or different one to two substituents selected from the substituent group X,a 5-membered aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, a 6-membered aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, a 5-7 membered unsaturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, a 4-7 membered saturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, an 8-10 membered condensed aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group X, orR3and R3may form a 5-7 membered unsaturated heterocyclic ring, a 4-7 membered saturated heterocyclic ring, or a C3-C6 cycloalkyl ring as a ring that binds to each other and condenses with A, and the ring is optionally substituted with the same or different one to two substituents selected from the substituent group X; substituent group X is a halogen atom, a cyano group, a hydroxy group, an oxo group, a C1-C6 alkyl group, a hydroxy C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a C1-C6 haloalkyl group, a C3-C6 cycloalkyl group, a C3-C6 halocycloalkyl group, a phenyl group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 5-membered aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 6-membered aromatic heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 4-7 membered saturated heterocyclic group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a C1-C6 alkoxy group, a C1-C6 haloalkoxy group, a C3-C6 cycloalkoxy group, a C3-C6 halocycloalkoxy group, a phenoxy group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 5-membered aromatic heterocyclic oxy group optionally substituted with the same or different one to two substituents selected from the substituent group Y,a 6-membered aromatic heterocyclic oxy group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 4-7 membered saturated heterocyclic oxy group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a C1-C6 alkoxycarbonyl group, a C3-C6 cycloalkoxycarbonyl group, a carboxy group, a C1-C6 alkylcarbonyl group, a C3-C6 cycloalkylcarbonyl group, a phenylcarbonyl group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a carbamoyl group, a mono (C1-C6 alkyl) aminocarbonyl group, a di (C1-C6 alkyl) aminocarbonyl group, a mono (C1-C6 alkyl) aminosulfonyl group, a di (C1-C6 alkyl) aminosulfonyl group, an amino group, a mono (C1-C6 alkyl) amino group, a di (C1-C6 alkyl) amino group, a C1-C6 alkoxycarbonylamino group, a mono (C1-C6 alkyl) aminocarbonylamino group, a di (C1-C6 alkyl) aminocarbonylamino group, a C1-C6 alkylcarbonylamino group, a phenylcarbonylamino group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 5-membered aromatic heterocyclic carbonylamino group optionally substituted with the same or different one to two substituents selected from the substituent group Y, a 6-membered aromatic heterocyclic carbonylamino group optionally substituted with the same or different one to two substituents selected from the substituent group Y, or a C1-C6 alkylsulfonylamino group; and substituent group Y is a C1-C6 alkyl group, a C1-C6 alkoxy group, a halogen atom, or a hydroxy group.

47. The method of claim 46, wherein R1is a methyl group, an ethyl group, or a hydroxyethyl group.

48. The method of claim 46 or 47, wherein R2is a methyl group.

49. The method of any one of claims 46-48, wherein the 5-membered aromatic heterocyclic ring or the 5-membered aromatic heterocyclic group in A, R3, or R3is any one selected from the group:

50. The method of any one of claims 46-49, wherein the 6-membered aromatic heterocyclic ring or the 6-membered aromatic heterocyclic group in A, R3, or R3is any one selected from the group:

51. The method of any one of claims 46-50, wherein the 5-7 membered unsaturated heterocyclic ring or the 5-7 membered unsaturated heterocyclic group in A, R3, or R3is any one selected from the group:

52. The method of any one of claims 46-51, wherein the 4-7 membered saturated heterocyclic ring or the 4-7 membered saturated heterocyclic group in A, R1, R2, or R3is any one selected from the group:

53. The method of any one of claims 46-52, wherein A is a 5-membered aromatic heterocyclic ring; R3is a methyl group, an ethyl group, a hydroxy C1-C3 alkyl group, or a methoxy C1-C3 alkyl group; and R3is a hydrogen atom.

54. The method of any one of claims 46-53, wherein A is any ring selected from the following group, and in the case of two binding groups, R3is not present:wherein * indicates a binding group.

55. The method of any one of claims 46-48, wherein the compound of Formula 1 is a compound of a Formula 1" or a pharmaceutically acceptable salt thereof:whereinR1is a methyl group or an ethyl group;R2is a methyl group;A is any ring selected from the following group:wherein * indicates a binding group; and R3is a methyl group or an ethyl group.

56. The method of any one of claims 46-53, wherein the compound of Formula 1' is any compound selected from the following group: (2S,5’R)-7-chloro-6-(2-hydroxyethoxy)-3’,4-dimethoxy-5’-methyl-spiro [benzofuran-2,4’ - cyclohex-2-ene] - 1’ ,3 -di one;(2S,5’R)-7-chloro-3’,4-dimethoxy-6-(2-methoxyethoxy)-5’-methyl-spiro [benzofuran-2,4’ - cyclohex-2-ene]- 1 ’ ,3 -di one;(2S,5 ’R)-7-chl oro-3 ’ ,4-dimethoxy-5 ’ -methyl-6-( 1 -methylpyrazol-3 -yl) spiro [benzofuran-2,4’ - cyclohex-2-ene]- 1 ’ ,3 -di one;(2S,5’R)-7-chloro-6-(l-ethylpyrazol-3-yl)-3’,4-dimethoxy-5’-methyl-spiro [benzofuran-2,4’ - cyclohex-2-ene]-l’,3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(5-methyl-l,3,4-oxadiazol-2-yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(3-methyl-l,2,4-oxadiazol-5-yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(5-methyl)-l,2,4-oxadiazol-3-yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-6-[5-(l-hydroxy-l-methyl-ethyl)-l,3,4-oxadiazol-2-yl]-3’,4-dimethoxy-5’- methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l ’,3-dione;(2S, 5 ’R)-7-chloro-6-[5-[(lS)-l -hydroxy ethyl]-l, 3, 4-oxadiazol-2-yl]-3’,4-dimethoxy-5’ -methylspiro [benzofuran-2,4’ -cy cl ohex-2-ene]-l ’, 3-dione;(2S, 5 ’R)-7-chloro-6-[5-[(lR)-l -hydroxy ethyl]- 1,3, 4-oxadiazol-2-yl]-3’,4-dimethoxy-5 ’-methylspiro [benzofuran-2,4’ -cy cl ohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-4-ethoxy-6-[5-(l-hydroxy-l-methyl-ethyl)-l,3,4-oxadiazol-2-yl]-3’-methoxy- 5’-methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-4-ethoxy-6-[5-[(l S)-l -hydroxy ethyl]- 1, 3, 4-oxadiazol-2-yl]-3’-methoxy-5’- methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l ’,3-dione;(2S,5’R)-7-chloro-6-[3-(l-hydroxyethyl)-l,2,4-oxadiazol-5-yl]-3’,4-dimethoxy-5’-methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chloro-4-(2 -hydroxy ethoxy)-3’-methoxy-5’-methyl-6-(5-m ethyl- 1,3, 4-oxadiazol-2- yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l ’,3-dione;(2S, 5 ’R)-7-chloro-4-(2 -hydroxy ethoxy)-3’-methoxy-5’-methyl-6-(3-methyl- 1,2, 4-oxadiazol-5- yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chloro-4-(2 -hydroxy ethoxy)-3’-methoxy-5’-methyl-6-(5-m ethyl- 1,2, 4-oxadiazol-3- yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-6-(5-ethyl-l,3,4-oxadiazol-2-yl)-3’,4-dimethoxy-5’-methyl-spiro [benzofuran- 2,4’ -cyclohex-2-ene]- 1 ’ ,3 -di one;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(5-tetrahydropyran-4-yl-l,3,4-oxadiazol-2-yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-[5-(l-methyl-4-piperidyl)-l,3,4-oxadiazol-2-yl] spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-6-[5-(4-fluoro-l-methyl-4-piperidyl)-l,3,4-oxadiazol-2-yl]-3’,4-dimethoxy- 5’- methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chl oro-3 ’,4-dimethoxy-6-[5-[(lS)-l -methoxy ethyl]- 1, 3, 4-oxadiazol -2 -yl]-5’ -methylspiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-4-ethoxy-3’-methoxy-5’-methyl-6-(5-methyl-l,3,4-oxadiazol-2-yl) spiro[b enzofuran-2, 4 ’ -cy cl ohex-2-ene] - 1’ , 3 -di one;(2S,5’R)-7-chloro-4-(difluoromethoxy)-3’-methoxy-5’-methyl-6-(5-methyl-l,3,4-oxadiazol-2-yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S, 5 ’R)-7-chl oro-3 ’,4-dimethoxy-6-[3-(l -methoxy ethyl)-!, 2, 4-oxadiazol-5-yl]-5 ’-methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-6-[3-(l-hydroxy-l-methyl-ethyl)-l,2,4-oxadiazol-5-yl]-3’,4-dimethoxy-5’- methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l ’, 3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(lH-pyrazol-5-yl) spiro [benzofuran-2,4’- cyclohex-2-ene]- 1 ’ ,3 -di one;(2 S, 5 ’ R)-7-chl oro-3 ’ ,4-di m ethoxy-6-[ 1 -(2-methoxy ethyl ) pyrazol -3 -y 1 ] -5 ’ -methyl -spi ro [b enzofuran-2, 4 ’ -cy cl ohex-2-ene] - 1’ , 3 -di one;(2S,5’R)-7-chloro-6-(l,8-dioxa-2-azaspiro [4.5] dec-2-en-3-yl)-3’,4-dimethoxy-5’-methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(8-methyl-l-oxa-2,8-diazaspiro [4.5] dec-2-en-3- yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l ’,3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-6-(2-methoxypyrimidin-5-yl)-5’-methyl-spiro [benzofuran- 2,4’ -cy clohex-2-ene]- 1 ’ ,3 -di one;(2S,5’R)-7-chloro-3’,4-dimethoxy-6-(6-methoxy-3-pyridyl)-5’-methyl-spiro [benzofuran-2,4’- cyclohex-2-ene]-l’,3-dione;(2S,5’R)-7-chloro-3’,4-dimethoxy-5’-methyl-6-(3-pyridyl) spiro [benzofuran-2,4’-cyclohex-2- ene]-l’,3-dione; or(2S,5’R)-7-chloro-3’,4,6-trimethoxy-5’-methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’,3- dione.

57. The method of claim 56, wherein the compound is (2S,5’R)-7-chloro-6-(l-ethylpyrazol- 3-yl)-3’,4-dimethoxy-5’-methyl-spiro [benzofuran-2,4’-cyclohex-2-ene]-l’, 3-dione or a pharmacologically acceptable salt thereof.

58. The method of claim 56, wherein the compound is (2S,5’R)-7-chloro-3’,4-dimethoxy-5’- methyl-6-(5-methyl-l,3,4-oxadiazol-2-yl) spiro [benzofuran-2,4’-cyclohex-2-ene]-l ’,3-dione or a pharmacologically acceptable salt thereof.

59. The method of claim 56, wherein the compound is (2S,5’R)-7-chloro-6-(5-ethyl-l,3,4- oxadiazol-2-yl)-3 ’ ,4-dimethoxy-5 ’ -methyl-spiro [benzofuran-2,4’ -cyclohex-2-ene]- 1 ’ ,3 -dione or a pharmacologically acceptable salt thereof.

60. The method of claim 56, wherein the compound is (2S,5’R)-7-chloro-6-[3-(l-hydroxy-l- methyl-ethyl)-l, 2, 4-oxadiazol-5-yl]-3’,4-dimethoxy-5’ -methyl-spiro [benzofuran-2,4’-cyclohex- 2-ene]-l ’,3-dione or a pharmacologically acceptable salt thereof.61 . The method of claim 56, wherein the compound is (2S,5’R)-7-chloro-4-ethoxy-3’- methoxy-5’-methyl-6-(5-methyl-l,3,4-oxadiazol-2-yl) spiro [benzofuran-2,4’-cyclohex-2-ene]- 1 ’,3-dione or a pharmacologically acceptable salt thereof.

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