RBP4 antagonists for the treatment and prevention of nonalcoholic fatty liver disease and gout

文档序号：862300 发布日期：2021-03-16 浏览：23次中文

阅读说明：本技术 用于治疗和预防非酒精性脂肪肝疾病和痛风的rbp4拮抗剂 (RBP4 antagonists for the treatment and prevention of nonalcoholic fatty liver disease and gout ) 是由康斯坦丁·彼得鲁欣博格拉卡·拉奇安德拉斯·瓦拉迪于 2019-08-01 设计创作，主要内容包括：本发明提供了一种方法用于在患有非酒精性脂肪肝疾病(NAFLD)的受试者中治疗该病的方法,所述方法包括向所述受试者施用包含一定量对治疗所述受试者有效的化合物的药物组合物,从而治疗受试者,所述化合物是非类视黄醇的视黄醇结合蛋白4(RBP4)拮抗剂。本发明提供了一种用于在患有痛风的受试者中治疗该病的方法,所述方法包括向所述受试者施用包含一定量对治疗所述受试者有效的化合物的药物组合物,从而治疗受试者,所述化合物是视黄醇结合蛋白4(RBP4)拮抗剂。(The present invention provides a method for treating non-alcoholic fatty liver disease (NAFLD) in a subject suffering therefrom, comprising administering to the subject a pharmaceutical composition comprising an amount of a compound effective to treat the subject, the compound being a non-retinoid retinol binding protein 4(RBP4) antagonist, thereby treating the subject. The present invention provides a method for treating gout in a subject having the disease, the method comprising administering to the subject a pharmaceutical composition comprising an amount of a compound effective to treat the subject, the compound being a retinol binding protein 4(RBP4) antagonist, thereby treating the subject.)

1. A method for treating non-alcoholic fatty liver disease (NAFLD) in a subject having the disease, the method comprising administering to the subject a pharmaceutical composition comprising an amount of a compound effective to treat the subject, the compound being a non-retinoid retinol binding protein 4(RBP4) antagonist, thereby treating the subject.

2. A method for treating gout in a subject having the disease, the method comprising administering to the subject a pharmaceutical composition comprising an amount of a compound effective to treat the subject, the compound being a retinol binding protein 4(RBP4) antagonist, thereby treating the subject.

3. A method for treating nonalcoholic fatty liver disease (NAFLD) or gout in a subject having the disease, the method comprising administering to the subject a pharmaceutical composition comprising an amount of a compound effective to treat the subject, wherein the compound has the structure

Wherein L is a linking group having the structure:

and Z is a group having the structure:

wherein:

R₁、R₂、R₃、R₄and R₅Each independently of the others being H, halogen, CF₃Or C₁-C₄Alkyl, aryl or heteroaryl;

R₆h, OH or halogen or absent;

ψ is absent or present and, when present, is a key;

b is a substituted or unsubstituted heterobicyclic, pyridazine, pyrazole, pyrazine, thiadiazole or triazole,

wherein the heterobicyclic ring is not a chloro-substituted indole; and is

The pyrazole, when substituted, is not substituted with trifluoromethyl;

b' is substituted or unsubstituted phenyl, pyridine, pyrimidine, benzyl, pyrrolidine, sulfolane, oxetane, CO₂H or (C)₁-C₄Alkyl) -CO₂H，

Wherein the substituted phenyl is not substituted with trifluoromethyl or 3- (carboxylic acid methyl ester), the substituted pyridine is not substituted with trifluoromethyl, and the substituted pyrrolidine is not substituted with hydroxamic acid, and

the substituted or unsubstituted pyrrolidine is bonded to the carbonyl group by a carbon-carbon bond;

a is absent or present and when present is

B₁Is a substituted or unsubstituted monocyclic, bicyclic, heteromonocyclic, heterobicyclic, benzyl, CO2H or (C1-C4 alkyl) -CO2H,

wherein when B₁Is CO₂When H, then A is present and is

R₇Is an alkyl group;

x is N or CR₈Wherein R is₈H, OH or halogen;

B₂has the following structure:

wherein:

α and β are each a bond, present or absent;

X₁is N, NH or NR₉₉，

Wherein R is₉₉Is alkyl, alkenyl or alkynyl;

X₂is C or N;

X₃is CH or N;

R₉、R₁₀and R₁₁Each independently is H, halogen, alkyl, alkenyl, alkynyl, alkyl-OH, alkyl-NH₂alkyl-OAc alkyl-O (CO) -alkyl, alkyl-O-alkyl, haloalkyl, cycloalkyl, O-alkyl, NH-alkyl, C (O) OH, C (O) -NH₂、C(O)-N(CH₃)₂、C(O)-NHCH₃、NHC(O)-N(CH₃)₂CN or CF₃，

Wherein:

X₁、X₂and X₃Each is N, α is present and β is absent; or

X₁Is NH, X₂Is C, X₃Is CH, α is absent and β is present; or

X1 is N, X2 is N, X3 is CH, α is present and β is absent; or

X₁Is NH or NR₉₉、X₂Is C, X₃Is N, alpha is absent and beta is present, wherein when X is present₁Is NH,X₂Is C, X₃Is N, alpha is absent and beta is present, then R₉、R₁₀And R₁₁Is not H, but is not H,

or, B₂Has the following structure:

wherein:

R₁₂、R₁₃and R₁₄Each independently is H, halogen, alkyl, alkenyl, alkynyl, alkyl-OH, alkyl-NH₂alkyl-Oac, alkyl-O (CO) -alkyl, alkyl-O-alkyl, haloalkyl, cycloalkyl, O-alkyl, NH-alkyl, C (O) OH, C (O) -NH₂、C(O)-N(CH₃)₂、C(O)-NHCH₃、NHC(O)-N(CH₃)₂CN or CF₃，

Or a pharmaceutically acceptable salt thereof.

4. The method of claim 3, wherein R₁、R₂、R₃、R₄And R₅Each independently of the others being H, halogen, CF₃Or C₁-C₄An alkyl group.

5. The method of any one of claims 1-4, wherein the subject has a NAFLD disease selected from the group consisting of: hepatic steatosis (fatty liver), nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma.

6. The method of any one of claims 1-5, wherein the method further comprises the step of determining or having determined the level of RBP4 in adipose tissue of the subject, and administering the pharmaceutical composition if the level of RBP4 in adipose tissue is elevated.

7. The method of any one of claims 1-6, wherein the method further comprises the step of determining or having determined the level of RBP4 in the serum of the subject, and administering the pharmaceutical composition if the level of RBP4 in the serum is elevated.

8. The method of any one of claims 1-7, wherein the amount of the compound is effective to reduce the level of RBP4 in adipose tissue of the subject or to reduce the level of RBP4 in serum of the subject.

9. The method of any one of claims 1-8, wherein the amount of the compound is effective to reduce uric acid levels in serum of the subject.

10. The method of any one of claims 1-9, wherein the amount of the compound is effective to normalize the concentration of triglycerides in the liver of the subject, or to normalize the concentration of free fatty acids in the serum of the subject, to normalize the concentration of free fatty acids in the liver of the subject.

11. The method of any one of claims 1-10, wherein the amount of the compound is effective to prevent fatty acid transport through RBP4, or prevent fatty acid transport to the liver through RBP4, or inhibit binding between RBP4 and a fatty acid.

12. The method of any one of claims 10-11, wherein the fatty acid is from adipose tissue.

13. The method of any one of claims 1-12, wherein the subject has elevated serum RBP4 levels.

14. The method of claim 13, wherein said serum RBP 4level is elevated more than 3 micrograms/ml.

15. The method according to any one of claims 1-14, wherein the compound is not a ligand of a nuclear receptor RAR.

16. The method of any one of claims 2 and 4-15, wherein the RBP4 antagonist is a non-retinoid antagonist.

17. The method of any one of claims 2 and 4-5, wherein the RBP4 antagonist is not fenritide.

18. The method of any one of claims 3-17, wherein

L isAnd Z is

19. The method of any one of claims 3-17, wherein

L isAnd Z is

20. The method of any one of claims 3-17, wherein

L is

Z is

R₁、R₂、R₃、R₄Or R₅Two or more of which are not H,

and

when R is₁Is CF₃、R₂Is H, R₃Is F, R₄Is H and R₅Is H, or R₁Is H, R₂Is CF₃、R₃Is H, R₄Is CF₃And R is₅Is H, or R₁Is Cl, R₂Is H, R₃Is H, R₄Is F and R₅Is H, or R₁Is CF₃、R₂Is H, R₃Is F, R₄Is H and R₅Is H, or R₁Is CF₃、R₂Is F, R₃Is H, R₄Is H and R₅Is H, or R₁Is Cl, R₂Is F, R₃Is H, R₄Is H and R₅When is H, then B is not

21. The method of any one of claims 3-17, wherein

L isAnd Z is

22. The method of any one of claims 3-17, wherein

L is

Z isAnd is

R₆Is absent or presentPresent and when present is H, OH or halogen

And when ψ exists, then R₆Is absent, and when ψ is absent, then R is present₆Is present.

23. The method of any one of claims 3-17, wherein

L isAnd is

Z is

24. The method of any one of claims 3-17, wherein

L isAnd is

Z is

25. The method of any one of claims 3-17, wherein

L is

Z is

R6 is H, and A is

26. The method of any one of claims 3-17, wherein

L isAnd Z is

27. The method of any one of claims 3-26, wherein the compound is

Or a pharmaceutically acceptable salt thereof.

28. The method of any one of claims 3-26, wherein the compound is

Or a pharmaceutically acceptable salt thereof.

29. The method of any one of claims 3-26, wherein the compound is

Or a pharmaceutically acceptable salt thereof.

30. The method of any one of claims 3-26, wherein the compound is

Or a pharmaceutically acceptable salt thereof.

31. The method of any one of claims 3-26, wherein the compound is

Or a pharmaceutically acceptable salt thereof.

32. The method of any one of claims 3-226, wherein the compound is

Or a pharmaceutically acceptable salt thereof.

33. The method of any one of claims 3-26, wherein the compound is

Or a pharmaceutically acceptable salt thereof.

34. The method of any one of claims 1-26, wherein the compound is

Or a pharmaceutically acceptable salt thereof.

35. The method of any one of claims 1-34, wherein the amount of the compound is 5-1000mg, 5-800mg, 5-200mg, 45-1000mg, 45-800mg, 10-50mg, 96mg, 24mg, or 10mg per day.

36. The method of any one of claims 1-35, wherein the method further comprises administering an amount of a second agent that is (R) - (+) - (5, 6-dichloro-2, 3,9,9 a-tetrahydro 3-oxo-9 a-propyl-1H-fluoren-7-yl) oxy ] acetic acid (DPOFA), a non-steroidal anti-inflammatory drug (NSAID) (such as indomethacin, colchicine, rasidone), a corticosteroid, betamethasone, prednisone, dexamethasone, cortisone, hydrocortisone, methylprednisolone, prednisolone, a biologic anti-IL-1 alpha/beta drug, conatinumab, linaglip, anakinra, allopurinol, benzbromarone, a form of uricase, goleplerenase, topiroxostat (FYX-051), ulodesine (BCX4208), KUX-1151, RLBN1001, RDEA3170, alfofenphen ester (MBX-102), levotofisopam (levotofisopam), UR-1102, PF-06743649, BCX4208, SHR4640, Lumiracoxib (Lumiracoxib), Tranilast (Tranilast), topirostat, LC350189, Bucillamine (Bucilamine), AC-201, Huzhen capsule, MPC-004, FYU-981, sodium bicarbonate, SEL-212, SEL-037, Apremilast (Apremilast), TMX-67, SSS11, D-0120, febuxostat or probenecid, or an ester or salt effective to treat a subject, thereby treating the subject.

37. The method of any one of claims 1 and 3-36, wherein the subject has gout.

38. The method of claim 37, wherein the amount of the second agent and/or the amount of the compound is effective to reduce uric acid levels in the blood of the subject or reduce uric acid reabsorption in the kidney of the subject.

39. The method according to claim 37 or 38, wherein the gout is chronic gout or acute gout.

Technical Field

Throughout this application, certain publications are referenced in parentheses. Full citations for these publications may be found at the end of the specification. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

The invention was made with government support awarded by the national institutes of health under accession numbers NS074476 and EY 027027027. The government has certain rights in this invention.

Background

Nonalcoholic fatty liver disease (NAFLD)

Non-alcoholic fatty liver disease (NAFLD) encompasses a range of conditions associated with lipid deposition in liver hepatocytes. Hepatic steatosis refers to the accumulation of lipids in the liver. NAFLD is characterized by hepatic steatosis caused by other causes than excessive alcohol consumption. Clinically, hepatic steatosis is defined as hepatic triglyceride content exceeding 5% of the total weight of the liver. While simple hepatic steatosis is the least extreme in the NAFLD range, it can progress to more severe conditions in the NAFLD range, such as mild hepatic steatosis and nonalcoholic steatohepatitis (NASH). NASH is an extreme form of NAFLD, characterized by accumulation of lipids in the liver, with accompanying inflammation and hepatocellular injury or fibrosis. NASH often leads to severe liver complications, such as cirrhosis and hepatocellular carcinoma.

NAFLD is the most common form of chronic liver disease in the united states, with an estimated impact of 7 thousand 5 million to 1 million people. There is currently no approved drug therapy for any form of NAFLD. The development of drug therapies for NAFLD is of paramount importance.

Gouty arthritis

Gouty arthritis (gout) is the most common form of inflammatory arthritis, affecting over 800 million people in the united states (Lawrence, r.c. et al 2008). Uric acid is a metabolite resulting from purine metabolism that is present in many food and human tissues (Terkeltaub, R.A.2001; Burns, C. et al 2013). Gout is caused by excessive uric acid levels in the blood that result in the deposition of mono-sodiuurate crystals in the tissue. These crystals form when uric acid concentrations in tissues and in the circulation exceed the solubility limit, resulting in gout attacks. Risk factors for gout include being overweight or obese, suffering from hypertension, drinking, use of diuretics, high meat and seafood in the diet, excessive fructose intake, and renal dysfunction (Choi, h.k. et al 2004 a; Choi, h.k.2004b; Krishnan, e.2012).

Acute attacks occur when urate crystals in the joints cause acute inflammation. Episodes are characterized by pain, redness, swelling, and heat that last from days to weeks. The pain may be mild or severe. Most of the initial attacks occur in the lower extremities. The typical manifestations of the metatarsophalangeal joint of the metatarsophalangeal toe (podagra) are those of 50% of gout patients. Chronic gout is characterized by chronic arthritis with joint soreness and pain. Persons with gout may also deposit tophus or lumps of urate crystals in soft tissue. The clinically inactive (critical) segment between gout attacks occurs after the acute attack has subsided. People with gout continue to suffer from hyperuricemia, which results in the continued deposition of urate crystals in the tissue and damage. As the disease progresses, the critical segment becomes shorter.

Uric acid is synthesized from its precursor xanthine by an enzyme called Xanthine Oxidase (XO). Thus, XO inhibitors (e.g., allopurinol and febuxostat) dominate the market (Stamp, L.K. et al 2015; Love, B.L. et al 2010). However, the most common cause of elevated circulating uric acid levels is insufficient uric acid secretion in the kidneys. Slightly effective probenecid and the recently approved lesinurad are therapeutic approaches to increase kidney uric acid secretion.

The incidence and prevalence of gout is rising. This is due to factors such as an increase in the aging population and lifestyle factors, many of which are characterized by excessive intake of fructose and alcohol in the diet promoting hyperuricemia, lack of physical exercise and abdominal fat accumulation, which are taken by many of the aging people and are characterized by hyperuricemic dietary hyperuricemia and prophylactic aspirin (Burns, c. et al 2013; Choi, h.k. et al 2004 a).

There remains a significant unmet clinical need in the treatment of gout. Of 800 million patients with gout, over 300 million are receiving urate-lowering therapy (primarily XO inhibitors). Nevertheless, 100 million patients continue to experience 3 or more episodes per year, indicating a need for better urate-lowering therapy.

Disclosure of Invention

The present invention provides a method for treating non-alcoholic fatty liver disease (NAFLD) in a subject suffering therefrom, comprising administering to the subject a pharmaceutical composition comprising an amount of a compound effective to treat the subject, the compound being a non-retinoid retinol binding protein 4(RBP4) antagonist, thereby treating the subject.

The present invention provides a method for treating gout in a subject having the disease, the method comprising administering to the subject a pharmaceutical composition comprising an amount of a compound effective to treat the subject, the compound being a retinol binding protein 4(RBP4) antagonist, thereby treating the subject.

The present invention provides a method for treating nonalcoholic fatty liver disease (NAFLD) or gout in a subject having the disease, the method comprising administering to the subject a pharmaceutical composition comprising an amount of a compound effective to treat the subject, thereby treating the subject, wherein the compound has the structure

Wherein L is a linking group having the structure:

and Z is a group having the structure:

wherein:

R₁、R₂、R₃、R₄and R₅Each independently of the others being H, halogen, CF₃Or C₁-C₄An alkyl group;

R₆h, OH or halogen or absent;

ψ is absent or present and, when present, is a key;

b is a substituted or unsubstituted heterobicyclic, pyridazine, pyrazole, pyrazine, thiadiazole or triazole,

wherein the heterobicyclic ring is not a chloro-substituted indole; and is

The pyrazole, when substituted, is not substituted with trifluoromethyl;

b' is substituted or unsubstituted phenyl, pyridine, pyrimidine, benzyl, pyrrolidine, sulfolane, oxetane, CO₂H or (C)₁-C₄Alkyl) -CO₂H，

the substituted or unsubstituted pyrrolidine is bonded to the carbonyl group by a carbon-carbon bond;

a is absent or present and when present is

B₁Is a substituted or unsubstituted monocyclic, bicyclic, heteromonocyclic, heterobicyclic, benzyl, CO₂H or (C)₁-C₄Alkyl) -CO₂H，

Wherein when B₁Is CO₂When H, then A is present and is

R₇Is an alkyl group;

x is N or CR₈Wherein R is₈H, OH or halogen;

B₂has the following structure:

wherein:

α and β are each a bond, present or absent;

X₁is N, NH or NR₉₉，

Wherein R is₉₉Is alkyl, alkenyl or alkynyl;

X₂is C or N;

X₃is CH or N;

Wherein:

X₁、X₂and X₃Each is N, alphaIs present and β is absent; or

X₁Is NH, X₂Is C, X₃Is CH, α is absent and β is present; or

X₁Is N, X₂Is N, X₃Is CH, α is present and β is absent; or

X₁Is NH or NR₉₉、X₂Is C, X₃Is N, alpha is absent and beta is present, wherein when X is present₁Is NH, X₂Is C, X₃Is N, alpha is absent and beta is present, then R₉、R₁₀And R₁₁Is not H, but is not H,

or B₂Has the following structure:

wherein:

R₁₂、R₁₃and R₁₄Each independently is H, halogen, alkyl, alkenyl, alkynyl, alkyl-OH, alkyl-NH₂alkyl-OAc, alkyl-O (CO) -alkyl, alkyl-O-alkyl, haloalkyl, cycloalkyl, O-alkyl, NH-alkyl, C (O) OH, C (O) -NH₂、C(O)-N(CH₃)₂、C(O)-NHCH₃、NHC(O)-N(CH₃)₂CN or CF₃，

Or a pharmaceutically acceptable salt thereof.

Drawings

FIG. 1: a single oral administration (PO) or intravenous administration (IV) of one dose of compound 1 induced a strong reduction in circulating levels of RBP4 in wild type mice. The upper panel shows data for intravenous administration of 2mg compound 1/kg body weight of mice. The lower panel shows data for oral administration of 5mg compound 1/kg body weight of mice.

RBP 4levels were determined in plasma samples collected at baseline and 10 time points after compound 1 administration. Three groups of mice were used for oral and intravenous administration experiments (mouse group 1, mouse group 2 and mouse group 3). Each mouse group consisted of 5 animals. Plasma RBP 4levels were determined using RBP4 (mouse/rat) dual ELISA kit (AdipoGen, switzerland) according to the manufacturer's instructions.

FIG. 2: an adi-hRBP4 transgenic mouse model was generated as described by Lee 2016. The human hRBP4 transgene was introduced at the ROSA26 locus. The human transgene comprises a loxP-flanked termination cassette that prevents expression. These mice were bred with adiponectin-Cre expressing mice, thus removing the termination cassette only in adipocytes. Thus, in this model, human RBP4 was specifically expressed in adipose tissue.

FIG. 3: experimental design for assessing the efficacy of compound 1 in the adi-hRBP4 genetic model of hepatic steatosis.

FIG. 4: in transgenic mouse strains, compound 1 induced a decrease in serum levels of human and mouse RBP 4. The left bar in each pair of bars is shaded in light gray and represents baseline data. The right bar of each pair of bars is shaded in dark grey and represents the serum levels of human and mouse RBP4 at the end of the study.

FIG. 5: body weight change dynamics of three groups of transgenic animals: both groups had High Fat Diets (HFD) accumulated more weight than the control group. Weight gain in the HFD group was significant compared to the standard diet group (. SP < 0.05;. SP < 0.01; two-factor RM anova, combined with Holm-Sidak post hoc comparison test)

FIG. 6: significance between HFD and HFD + compounds. Treatment with compound 1 significantly reduced HFD-induced weight gain at several time points. Significant body weight differences were detected between the HFD only group and the treated group (HFD + compound 1) (. P < 0.05;. P < 0.01; two-factor RM anova, combined with Holm-Sidak post hoc comparative test).

FIG. 7: as shown by the comparison between the treatment group (HFD + compound 1) and the HFD only group, treatment with compound 1 significantly reduced body weight at the end of the 29 day treatment period. Significant differences were observed between the HFD group and the treatment group (HFD + compound 1) (P ═ 0.0153; one-way RM analysis of variance, combined with Holm-Sidak post hoc comparison test).

FIG. 8: there was no difference in food intake between the treatment (HFD + compound 1) and HFD only groups (two-factor RM anova, combined with Holm-Sidak post hoc comparative test).

FIG. 9: compound 1 significantly reduced liver free fatty acid concentration. At the end of the 29 day study, there was a significant difference between the HFD only group and the treatment group (HFD + compound 1) (P ═ 0.011; one-way anova, combined with Holm-Sidak post hoc comparative test). The data displayed was determined at the end of the 29 day study.

FIG. 10: compound 1 significantly reduced hepatic triglycerides. There was a significant difference between the HFD only group and the treatment group (HFD + compound 1) (P ═ 0.01; one-way anova, combined with Holm-Sidak post hoc comparison test). The data displayed was determined at the end of the 29 day study.

FIG. 11: compound 1 significantly reduced lipid deposition in the liver of treated animals. A. Liver histology scoring.

FIG. 11B: compound 1 significantly reduced lipid deposition in the liver of treated animals. B. The average score of the normal food group was 0.

FIG. 11C: compound 1 significantly reduced lipid deposition in the liver of treated animals. C. The average score of the HFD only group was 2.9.

FIG. 11D: compound 1 significantly reduced lipid deposition in the liver of treated animals. D. The average score for the treatment group (HFD + compound 1) was 1.6. Significant differences were observed between only the HFD group and the treatment group (HFD + compound 1) (P < 0.001; one-way anova, combined with Holm-Sidak post hoc comparison test). The data displayed was determined at the end of the 29 day study.

FIG. 12: compound 1 significantly reduced serum uric acid. There was a significant difference between the HFD only group and the treatment group (HFD + compound 1) (P ═ 0.03; one-way anova, combined with Holm-Sidak post hoc comparison test). However, there was no statistically significant difference between the regular food group and the HFD only group. The data displayed was determined at the end of the 29 day study.

FIG. 13: no steatosis was observed in the kidneys of the animals under HFD. The figure is a representative image of only the HFD set. In any of the three experimental groups, no fat droplets were observed in the renal tubules. The data displayed was determined at the end of the 29 day study.

FIG. 14: representative isotherms of a1120 (positive control) and fatty acids bound to human RBP 4. 10nM of³H-retinol was used as the radioligand. Fatty acids (e.g., palmitic acid, oleic acid, and linoleic acid) can replace the radioactive retinol from RBP4, suggesting that they can bind to the retinol binding pocket of RBP 4. This data suggests that RBP4 expressed in adipose tissue may be involved in the transport of fatty acids from adipose tissue to the liver. Compound 1 and other RBP4 antagonists can inhibit this fatty acid transport because they compete for binding to the same retinol binding pocket in RBP 4.

FIG. 15: in vivo PK data for analog compounds 1 and 2 following IV and PO administration in rodents^a。

^aThe administration group consisted of three non-drug-administered male CD-1 mice or adult male Sprague-Dawley rats. Data are shown as mean ± SD.^bTotal body clearance.^cMaximum observed concentration of compound in plasma.^dTime to maximum observed concentration of compound in plasma after oral administration.^eThe terminal apparent half-life of the compound was eliminated from plasma.^fDispensing amount in steady state. The area under the plasma concentration-time curve of the compound from 0 to the last time point in plasma is quantifiable.^hBioavailability; f ═ (AUCINFpo × dose IV) ÷ (aucnfiv × dose po) Intravenous (IV) formulation ═ 3% dimethacrylate/45% polyethylene glycol 300/12% ethanol/40% sterile water; the dosage of IV is 2 mL/kg; oral (PO) formulation 2% Tween 80 in 0.9% saline; PO was administered at 5 mL/kg. Dosing regimen of compound 3 in mice and rats: 2.0mg/kg IV, 5mg/kg PO. Dosing regimen of compound 1 in mice: 2.0mg/kg IV, 5mg/kgAnd PO. Dosing regimen of compound 1 in rats: 1.0mg/kg IV, 2.0mg/kg IV, 5mg/kg PO.

FIG. 16A PK/PD characteristics of Compound 1 in mice. Plasma RBP 4levels in CD-1 mice following a single oral administration of 5mg/kg Compound 1.

FIG. 16B: PK/PD characteristics of Compound 1 in mice. Plasma RBP 4levels in CD-1 mice following a single 2mg/kg intravenous administration of compound 1.

Intravenous dose (D) of 2mg/kg of Compound 1. Data are shown as mean ± SD. For each time point of blood collection, three mice were used in the study.

FIG. 16C: PK/PD characteristics of Compound 1 in mice. Plasma compound levels following a single oral administration of compound 1 at a 5mg/kg dose. Data are shown as mean ± SD. For each time point of blood collection, three mice were used in the study.

FIG. 16D: PK/PD characteristics of Compound 1 in mice. Plasma compound levels following a single oral administration of compound 1 at a dose of 2 mg/kg. Data are shown as mean ± SD. For each time point of blood collection, three mice were used in the study.

FIG. 17A Effect of oral administration of Compound 1 on circulating serum RBP 4levels in adi-hRBP4 mice. Serum levels of mouse RBP4 were measured at baseline (black circles) and at the end of 29 days of compound treatment (red squares) using a species-specific rodent or human enzyme-linked immunosorbent assay. At the end of the study, a statistically significant 90% reduction in RBP4 was seen in both human and mouse mice compared to baseline (two-way analysis of variance, combined with Holm-Sidak post hoc test,. P < 0.0001). A significant decrease in human and mouse RBP4 concentrations was detected in compound 1-treated adi-hRBP4 mice compared to vehicle-treated knockout controls (two-way anova, combined with Holm-Sidak post hoc test, P < 0.0001). Error bar display SD; the graph bar shows the average. Each data point on the graph represents serum RBP4 concentration from a single animal. The number of male adi-hRBP4 mice per treatment group was: normal diet 8, HFD 7 and HFD 8 with compound 1.

FIG. 17B: effect of oral administration of compound 1 on circulating levels of serum RBP4 in adi-hRBP4 mice. Serum levels of human RBP4 were measured at baseline (black circles) and at the end of 29 days of compound treatment (red squares) using a species-specific rodent or human enzyme-linked immunosorbent assay. At the end of the study, a statistically significant 90% reduction in RBP4 was seen in both human and mouse mice compared to baseline (two-way analysis of variance, combined with Holm-Sidak post hoc test,. P < 0.0001). A significant decrease in human and mouse RBP4 concentrations was detected in compound 1-treated adi-hRBP4 mice compared to vehicle-treated knockout controls (two-way anova, combined with Holm-Sidak post hoc test, P < 0.0001). Error bar display SD; the graph bar shows the average. Each data point on the graph represents serum RBP4 concentration from a single animal. The number of male adi-hRBP4 mice per treatment group was: normal diet 8, HFD 7 and HFD 8 with compound 1.

FIG. 18A Compound 1 partially prevents high fat diet-induced obesity in adi-hRBP4 mice. Body weight gain in male adi-hRBP4 mice fed normal diet (n-8), HFD (n-7) and HFD with 58 (n-8). Compound-treated HFD mice had significantly reduced weight gain at four time points from day 19 compared to the untreated HFD group (two-factor Repeated Measures (RM) analysis of variance, combined with Holm-Sidak post hoc test,. P < 0.05;. P < 0.01). The weight gain of food-fed mice was lower than the weight gain of the HFD group at all time points studied (two-factor RM anova, combined with Holm-Sidak post hoc test;. P < 0.0001). Values represent the average percent of body weight change from baseline. Error bars show SD.

FIG. 18B: compound 1 partially prevented high fat diet-induced obesity in adi-hRBP4 mice. Daily food consumption was normalized to body weight in male adi-hRBP4 mice fed normal food, HFD and HFD with compound 1. There was no difference in food consumption between the untreated HFD group and the compound-treated HFD mice (two-factor RM anova, combined with Holm-Sidak post hoc comparative test). Values represent average normalized daily food consumption. Error bars show SD.

Figure 19A effect of oral administration of compound 1 at a dose of 20mg/kg on liver free fatty acid and triglyceride levels in obese adihRBP4 mice. Liver ffa (a) levels in male adi-hRBP4 mice fed normal diet (n-8), HFD (n-7) and HFD with compound 58 (n-8). Hepatic FFA (P ═ 0.0107, one-way anova, combined with Holm-Sidak post hoc test) and triglyceride (P ═ 0.0104, one-way anova, combined with Holm-Sidak post hoc test) levels were significantly reduced in compound 1-treated HFD mice compared to untreated HFD group. The graph bar shows the average value; error bar display SD; p < 0.05; p < 0.0001. Each data point on the graph represents the FFA or TG concentration from a single animal.

FIG. 19B: effect of oral administration of compound 1 at a dose of 20mg/kg on liver free fatty acid and triglyceride levels in obese adihRBP4 mice. Liver tg (b) levels in male adi-hRBP4 mice fed normal diet (n-8), HFD (n-7) and HFD with compound 58 (n-8). Hepatic FFA (P ═ 0.0107, one-way anova, combined with Holm-Sidak post hoc test) and triglyceride (P ═ 0.0104, one-way anova, combined with Holm-Sidak post hoc test) levels were significantly reduced in compound 1-treated HFD mice compared to untreated HFD group. The graph bar shows the average value; error bar display SD; p < 0.05; p < 0.0001. Each data point on the graph represents the FFA or TG concentration from a single animal.

FIG. 20A Effect of Compound 1 on liver lipid deposition in adi-hRBP4 mice. Representative liver frozen sections stained with oil red O showed fatty liver status in food-fed, HFD and compound 1-treated HFD adi-hRBP4 mice. The compound was administered orally at a dose of 20 mg/kg.

FIG. 20B: effect of Compound 1 on liver lipid deposition in adi-hRBP4 mice. Histological scoring of oil red O stained liver cryosections of food-fed, HFD and compound 1 treated HFD adi-hRBP4 mice. Hepatic steatosis was classified into grade 0 (0% of hepatocytes with vesicular steatosis), grade 1 (< 33% of hepatocytes with vesicular steatosis), grade 2 (33-66% of hepatocytes with vesicular steatosis), and grade 3 (> 66% of hepatocytes with vesicular steatosis). Data were analyzed using one-way analysis of variance (combined with Holm-Sidak post hoc tests). The graph bar shows the average value; error bar display SD; p < 0.001. Each data point on the graph represents the steatosis histology score from a single animal. The number of male adi-hRBP4 mice per treatment group was: normal diet 8, HFD 7 and HFD 8 with compound 1.

FIG. 21: binding of fatty acids to RBP 4. (A) Isotherms of palmitic, oleic, linoleic and docosahexaenoic acids bound to human RBP 4. 10nM of 3H-retinol was used as radioligand. (B) Superposition of minimal binding conformations of retinol (black, 5NU7), antagonist compound 3 (purple, 3FMZ) and palmitic, oleic, linoleic and docosahexaenoic acids (orange). Phe36 was dark green in 3FMZ (next to the fatty acid carboxylic acid group) and light green in the 5NU7 model (next to the 1 alcohol group). The contact residue is marked and depicted in bar format (MOE, Chemical Computing Group, Inc., Montreal, Canada).

Detailed Description

Wherein L is a linking group having the structure:

and Z is a group having the structure:

wherein:

R₁、R₂、R₃、R₄and R₅Each independently of the others being H, halogen, CF₃Or C₁-C₄An alkyl group;

R₆h, OH or halogen or absent;

ψ is absent or present and, when present, is a key;

b is a substituted or unsubstituted heterobicyclic, pyridazine, pyrazole, pyrazine, thiadiazole or triazole,

wherein the heterobicyclic ring is not a chloro-substituted indole; and is

The pyrazole, when substituted, is not substituted with trifluoromethyl;

b' is substituted or unsubstituted phenyl, pyridine, pyrimidine, benzyl, pyrrolidine, sulfolane, oxetane, CO₂H or (C)₁-C₄Alkyl) -CO₂H，

the substituted or unsubstituted pyrrolidine is bonded to the carbonyl group by a carbon-carbon bond;

a is absent or present and when present is

B₁Is a substituted or unsubstituted monocyclic, bicyclic, heteromonocyclic, heterobicyclic, benzyl, CO2H or (C1-C4 alkyl) -CO2H,

wherein when B₁Is CO₂When H, then A is present and is

R₇Is an alkyl group;

x is N or CR₈Wherein R is₈H, OH or halogen;

B₂has the following structure:

wherein:

α and β are each a bond, present or absent;

X₁is N, NH or NR₉₉，

Wherein R is₉₉Is alkyl, alkenyl or alkynyl;

X₂is C or N;

X₃is CH or N;

Wherein:

X₁、X₂and X₃Each is N, α is present and β is absent; or

X₁Is NH, X₂Is C, X₃Is CH, α is absent and β is present; or

X1 is N, X2 is N, X3 is CH, α is present and β is absent; or

X₁Is NH or NR₉₉、X₂Is C, X₃Is N, alpha is absent and beta is present, wherein when X is present₁Is NH, X₂Is C, X₃Is N, alpha is absent and beta is present, then R₉、R₁₀And R₁₁Is not H, but is not H,

or B₂Has the following structure:

wherein:

Or a pharmaceutically acceptable salt thereof.

In one embodiment, the subject has a NAFLD disease selected from the group consisting of: hepatic steatosis (fatty liver), nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma.

In some embodiments, the method further comprises the step of determining or having determined the level of RBP4 in adipose tissue of the subject, and administering the pharmaceutical composition if the level of RBP4 in adipose tissue is elevated.

In one embodiment, the method further comprises the step of determining or having determined the level of RBP4 in the serum of the subject, and administering the pharmaceutical composition if the level of RBP4 in the serum is elevated.

In one embodiment, the amount of the compound is effective to reduce RBP 4levels in adipose tissue of the subject. In another embodiment, the amount of the compound is effective to reduce RBP 4levels in the serum of the subject. In another embodiment, the amount of the compound is effective to reduce uric acid levels in the serum of the subject. In another embodiment, the amount of the compound is effective to normalize the concentration of triglycerides in the liver of the subject.

In one embodiment, the amount of the compound is effective to normalize the concentration of free fatty acids in the serum of the subject. In another embodiment, the amount of the compound is effective to normalize the concentration of free fatty acids in the liver of the subject. In some embodiments, the amount of the compound is effective to prevent the transport of fatty acids through RBP 4. In additional embodiments, the amount of the compound is effective to prevent the transport of fatty acids to the liver via RBP 4. In one embodiment, the amount of the compound is effective to inhibit binding between RBP4 and a fatty acid.

In one embodiment, the fatty acid is from adipose tissue.

In one embodiment, the subject does not have elevated serum RBP4 levels. In another embodiment, the subject has elevated serum RBP4 levels. In some embodiments, the serum RBP 4level is elevated above 3 micrograms/ml.

In some embodiments, the NAFLD is hepatic steatosis selected from simple hepatic steatosis and mild hepatic steatosis.

In one embodiment, the compound is not a ligand for the nuclear receptor RAR. In another embodiment, the RBP4 antagonist is a non-retinoid antagonist. In another embodiment, the RBP4 antagonist is not fenritinide.

In some embodiments, L isAnd Z is

In a further embodiment, L isAnd Z is

In a further embodiment, L is

Z is

R₁、R₂、R₃、R₄Or R₅Two or more of which are not H,

and is

When R is₁Is CF₃、R₂Is H, R₃Is F, R₄Is H and R₅Is H, or R₁Is H, R₂Is CF₃、R₃Is H, R₄Is CF₃And R is₅Is H, or R₁Is Cl, R₂Is H, R₃Is H, R₄Is F and R₅Is H, or R₁Is CF₃、R₂Is H, R₃Is F, R₄Is H and R₅Is H, or R₁Is CF₃、R₂Is F, R₃Is H, R₄Is H and R₅Is H, or R₁Is Cl, R₂Is F, R₃Is H, R₄Is H and R₅When is H, then B is not

In some embodiments, L isAnd Z is

Is that

Z isAnd is

R₆Is absent or present, and when present is H, OH or

Halogen element

And when ψ exists, then R₆Is absent, and when ψ is absent, then R is present₆Is present.

In some embodiments, L isAnd is

Z is

In some embodiments, L isAnd is

Z is

In some embodiments, L is

Z is

R6 is H, and A is

In some embodiments, L isAnd Z is

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

The methods of the present invention include pharmaceutical compositions wherein the compound has the structure:

wherein:

R₁、R₂、R₃、R₄and R₅Each independently of the others being H, halogen, CF₃Or C₁-C₄An alkyl group;

x is N or CR₆，

Wherein R is₆Is H, OH or halogen;

a is absent or present and when present is

B has the following structure:

wherein:

α and β are each a bond, present or absent;

X₁is N, NH or NR₁₀，

Wherein R is₁₀Is alkyl, alkenyl or alkynyl;

X₂is C or N;

X₃is CH or N;

R₇、R₈and R₉Each independently is H, halogen, alkyl, alkenyl, alkynyl, alkyl-OH, alkyl-NH₂alkyl-OAc alkyl-O (CO) -alkyl, alkyl-O-alkyl, haloalkyl, cycloalkyl, O-alkyl, NH-alkyl, C (O) OH, C (O) -NH₂、C(O)-N(CH₃)₂、C(O)-NHCH₃、NHC(O)-N(CH₃)₂CN or CF₃，

Wherein:

X₁、X₂and X₃Each is N, α is present and β is absent; or

X₁Is NH, X₂Is C, X₃Is CH, α is absent and β is present; or

X1 is N, X2 is N, X3 is CH, α is present and β is absent; or

X₁Is NH or NR₁₀、X₂Is C, X₃Is N, alpha is absent and beta is present, wherein when X is present₁Is NH, X₂Is C, X₃Is N, alpha is absent and beta is present, then R₇、R₈And R₉Is not H, but is not H,

alternatively, B has the following structure:

wherein:

R₁₁、R₁₂and R₁₃Each independently is H, halogen, alkyl, alkenyl, alkynyl, alkyl-OH, alkyl-NH₂alkyl-OAc, alkyl-O (CO) -alkyl, alkyl-O-alkyl, haloalkyl, cycloalkyl, O-alkyl, NH-alkyl, C (O) OH, C (O) -NH₂、C(O)-N(CH₃)₂、C(O)-NHCH₃、NHC(O)-N(CH₃)₂CN or CF₃，

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the compounds of the methods have the following structure:

in some embodiments, the compounds of the methods have the following structure:

wherein:

R₁、R₂、R₃、R₄and R₅Each independently of the others being H, halogen, CF₃Or C₁-C₄An alkyl group; and is

B has the following structure:

wherein:

α and β are each a bond, present or absent;

X₁is N, NH or NR₁₀，

Wherein R is₁₀Is alkyl, alkenyl or alkynyl;

X₂is C or N;

X₃is CH or N;

R₇、R₈and R₉Each independently is H, halogen, alkyl, alkenyl, alkynyl, alkyl-OH, alkyl-NH₂alkyl-OAc, alkyl-O-alkyl, haloalkyl, cycloalkyl, O-alkyl, NH-alkyl, C (O) OH, C (O) -NH₂、C(O)-N(CH₃)₂、C(O)-NHCH₃、NHC(O)-N(CH₃)₂CN or CF₃Wherein:

X₁、X₂and X₃Each is N, α is present and β is absent; or

X₁Is NH, X₂Is C, X₃Is CH, α is absent and β is present; or

X1 is N, X2 is N, X3 is CH, α is present and β is absent; or

alternatively, B has the following structure:

wherein:

R₁₁、R₁₂and R₁₃Each independently of the others being H, halogen, alkyl-OH, alkyl-NH₂alkyl-OAc, alkyl-O-alkyl, haloalkyl, cycloalkyl, O-alkyl, NH-alkyl, C (O) OH, C (O) -NH₂、C(O)-N(CH₃)₂、C(O)-NHCH₃、NHC(O)-N(CH₃)₂CN or CF₃，

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the compounds of the methods have the following structure:

wherein:

R₁、R₂、R₃、R₄and R₅Each independently of the others being H, halogen, CF₃Or C₁-C₄An alkyl group;

y is an alkyl group;

a is absent or present and when present isAnd is

B has the following structure:

wherein:

α and β are each a bond, present or absent;

X₁is N, NH or NR₁₀，

Wherein R is₁₀Is alkyl, alkenyl or alkynyl;

X₂is C or N;

X₃is CH or N;

Wherein:

X₁、X₂and X₃Each is N, α is present and β is absent; or

X₁Is NH, X₂Is C, X₃Is CH, α is absent and β is present; or

X1 is N, X2 is N, X3 is CH, α is present and β is absent; or

alternatively, B has the following structure:

wherein:

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the compounds of the methods have the following structure:

in some embodiments, the compounds of the methods have the following structure:

wherein:

α and β are each a bond, present or absent;

X₁is N, NH or NR₁₀，

Wherein R is₁₀Is alkyl, alkenyl or alkynyl;

X₂is C or N;

X₃is CH or N;

Wherein:

X₁、X₂and X₃Each is N, α is present and β is absent; or

X₁Is NH, X₂Is C, X₃Is CH, α is absent and β is present; or

X1 is N, X2 is N, X3 is CH, α is present and β is absent; or

X₁Is NH or NR₁₀、X₂Is C, X₃Is N, alpha is absent and beta is present, wherein when X is present₁Is NH, X₂Is C, X₃Is N, alpha is absent and beta is present, then R₇、R₈And R₉Is not H.

In some embodiments, B or B₂Has the following structure:

wherein:

R₁₁、R₁₂and R₁₃Each independently is H, halogen, alkyl, alkenyl, alkynyl,alkyl-OH, alkyl-NH₂alkyl-OAc, alkyl-O (CO) -alkyl, alkyl-O-alkyl, haloalkyl, cycloalkyl, O-alkyl, NH-alkyl, C (O) OH, C (O) -NH₂、C(O)-N(CH₃)₂、C(O)-NHCH₃、NHC(O)-N(CH₃)₂CN or CF₃。

In some embodiments, B or B₂Has the following structure:

R₇、R₈and R₉Each independently of the others being H, halogen, alkyl-OH, alkyl-NH₂alkyl-OAc, alkyl-O-alkyl, haloalkyl, cycloalkyl, O-alkyl, NH-alkyl, C (O) OH, C (O) -NH₂、C(O)-N(CH₃)₂、C(O)-NHCH₃、NHC(O)-N(CH₃)₂CN or CF₃。

In some embodiments, B or B₂Has the following structure:

R₇、R₈and R₉Each independently is H,Halogen, alkyl-OH, alkyl-NH₂alkyl-OAc, alkyl-O-alkyl, haloalkyl, cycloalkyl, O-alkyl, NH-alkyl, C (O) OH, C (O) -NH₂、C(O)-N(CH₃)₂、C(O)-NHCH₃、NHC(O)-N(CH₃)₂CN or CF₃。

In some embodiments, B or B₂Has the following structure:

In some embodiments, R₇、R₈And R₉Each independently is H, Cl, Br, F, OCH₃、OCH₂CH₃、CF₃、CN、CH₃、CH₃CH₃C (O) OH or C (O) -NH₂。

In some embodiments, R₇、R₈And R₉Each independently is H, CH₂CH₂OH、CH₂CH₂OCH₃、CH₂CH₂OAc、CH₂CH₂Cl、CH₂CH₂F or CH₂CH₂Br。

In some embodiments, R₇、R₈And R₉Each independently is H, halogen or alkyl.

In some embodiments, R₇、R₈And R₉Both of which are H, and R₇、R₈And R₉The remaining one of which is not H.

In some embodiments, R₇、R₈And R₉One of them is H, and R₇、R₈And R₉The remaining two of (a) are not H.

In some embodiments, B or B₂Has the following structure:

in some embodiments, R₇、R₈And R₉Each independently is H, CH₃、Br、Cl、F、CH₂CH₂OH、CH₂CH₂OCH₃、CH₂CH₂OAc、CH₂CH₂Cl、CH₂CH₂F or CH₂CH₂Br。

In some embodiments, B or B₂Has the following structure:

in some embodiments, R₇And R₉Each independently is H, CH₃、Br、Cl、F、CH₂CH₂OH、CH₂CH₂OCH₃、CH₂CH₂OAc、CH₂CH₂Cl、CH₂CH₂F or CH₂CH₂Br。

In some embodiments, B or B₂Has the following structure:

in some embodiments, R₇、R₈And R₉Each independently is H, CH₃、Br、Cl、F、CH₂CH₂OH、CH₂CH₂OCH₃、CH₂CH₂OAc、CH₂CH₂Cl、CH₂CH₂F or CH₂CH₂Br。

In some embodiments, B or B₂Has the following structure:

in some embodiments, R₇And R₉Each independently is H, CH₃、Br、Cl、F、CH₂CH₂OH、CH₂CH₂OCH₃、CH₂CH₂OAc、CH₂CH₂Cl、CH₂CH₂F or CH₂CH₂Br。

In some embodiments, B in the compound has the following structure:

in some embodiments, R₇、R₈And R₉Each independently is H, CH₃、Br、Cl、F、CH₂CH₂OH、CH₂CH₂OCH₃、CH₂CH₂OAc、CH₂CH₂Cl、CH₂CH₂F or CH₂CH₂Br。

In some embodiments, B or B₂Has the following structure:

in some embodiments, R₇And R₉Each independently is H, CH₃、Br、Cl、F、CH₂CH₂OH、CH₂CH₂OCH₃、CH₂CH₂OAc、CH₂CH₂Cl、CH₂CH₂F or CH₂CH₂Br。

In some embodiments, B or B₂Has the following structure:

in some embodiments, R in the compound₇、R₈And R₉Each independently is H, CH₃、Br、Cl、F、CH₂CH₂OH、CH₂CH₂OCH₃、CH₂CH₂OAc、CH₂CH₂Cl、CH₂CH₂F or CH₂CH₂Br; and R is₁₀Is an alkyl group.

In some embodiments, B or B₂Has the following structure:

in some embodiments, R₇And R₉Each independently is H, CH₃、Br、Cl、F、CH₂CH₂OH、CH₂CH₂OCH₃、CH₂CH₂OAc、CH₂CH₂Cl、CH₂CH₂F or CH₂CH₂Br; and R is₁₀Is an alkyl group.

In some embodiments, B or B₂Has the following structure:

In some embodiments, R₁₁、R₁₂And R₁₃Each independently is H, Cl, Br, F, OCH₃、OCH₂CH₃、CF₃、CN、CH₃、CH₃CH₃C (O) OH or C (O) -NH₂。

In some embodiments, R₁₁、R₁₂And R₁₃Each independently is H, CH₂CH₂OH、CH₂CH₂OCH₃、CH₂CH₂OAc、CH₂CH₂Cl、CH₂CH₂F or CH₂CH₂Br。

In some embodiments, R₁₁、R₁₂And R₁₃Each independently is H, halogen or alkyl.

In some embodiments, R₁₁、R₁₂And R₁₃Both of which are H, and R₁₁、R₁₂And R₁₃The remaining one of which is not H.

In some embodiments, R₁₁、R₁₂And R₁₃One of them is H, and R₁₁、R₁₂And R₁₃The remaining two of (a) are not H.

In some embodiments, B or B₂Has the following structure:

in some embodiments, R₁₁、R₁₂And R₁₃Each independently is H, CH₃、Br、Cl、F、CH₂CH₂OH、CH₂CH₂OCH₃、CH₂CH₂OAc、CH₂CH₂Cl、CH₂CH₂F or CH₂CH₂Br。

In some embodiments, B or B₂Has the following structure:

in some embodiments, R₁₁And R₁₃Each independently is H, CH₃、Br、Cl、F、CH₂CH₂OH、CH₂CH₂OCH₃、CH₂CH₂OAc、CH₂CH₂Cl、CH₂CH₂F or CH₂CH₂Br。

In some embodiments, X is N. In some embodiments, X of the compound is CH.

In some embodiments, R₁、R₂、R₃、R₄And R₅Each H, t-Bu, Cl, F or CF₃。

In some embodiments, R₁、R₂、R₃And R₄Are respectively H; and is

R₅Is CF₃Or t-Bu.

In some embodiments of the present invention, the substrate is,

R₁、R₃and R₄Are respectively H;

R₂is halogen;

R₅is CF₃Or t-Bu.

In some embodiments of the present invention, the substrate is,

R₁、R₂、R₃and R₄Are respectively H;

R₅is CF₃Or t-Bu.

In some embodiments of the present invention, the substrate is,

R₁、R₂、R₃and R₄Are respectively H;

R₅is CF₃。

In some embodiments, R₁、R₂、R₃、R₄And R₅Is not H.

In some casesIn embodiments, R₁、R₂、R₃、R₄And R₅Are not H.

In some embodiments, R₁、R₂、R₃、R₄And R₅Two or more of which are not H.

In some embodiments, R₁、R₂、R₃、R₄And R₅Three of which are not H.

In some embodiments, R₁、R₂、R₃、R₄And R₅Three or more of which are not H.

In some embodiments, the compounds of the methods have the following structure:

a pharmaceutically acceptable salt thereof.

In some embodiments, B or B₂Is not provided with

The present invention provides compounds having the structure:

wherein:

R₁、R₂、R₃、R₄and R₅Each independently is H, halogenAlkyl, haloalkyl, O-haloalkyl, aryl or heteroaryl;

x is N or CR₆，

Wherein R is₆Is H, OH or halogen;

a is absent or present and when present is

B has the following structure:

wherein:

α and β are each a bond, present or absent;

X₁is N, NH or NR₁₀，

Wherein R is₁₀Is alkyl, alkenyl or alkynyl;

X₂is C or N;

X₃is CH or N;

Wherein:

X₁、X₂and X₃Each is N, α is present and β is absent; or

X₁Is NH, X₂Is C, X₃Is CH, α is absent and β is present; or

X1 is N, X2 is N, X3 is CH, α is present and β is absent; or

X₁Is NH or NR₁₀、X₂Is C, X₃Is N, αIs absent and beta is present, wherein when X is present₁Is NH, X₂Is C, X₃Is N, alpha is absent and beta is present, then R₇、R₈And R₉Is not H, but is not H,

alternatively, B has the following structure:

wherein

X₄And X₅Each independently is N or CH; and is

R₁₁、R₁₂And R₁₃Each independently is H, halogen, alkyl, alkenyl, alkynyl, alkyl-OH, alkyl-NH₂alkyl-OAc, alkyl-O (CO) -alkyl, alkyl-O-alkyl, haloalkyl, cycloalkyl, O-alkyl, NH-alkyl, C (O) OH, C (O) -NH₂、C(O)-N(CH₃)₂、C(O)-NHCH₃、NHC(O)-N(CH₃)₂CN or CF₃，

Or a pharmaceutically acceptable salt thereof.

54. A compound having the structure:

wherein:

R₁、R₂、R₃、R₄and R₅Each independently of the others being H, halogen, CF₃Or C₁-C₄Alkyl, aryl or heteroaryl; and is

B has the following structure:

wherein:

α and β are each a bond, present or absent;

X₁is N, NH or NR₁₀，

Wherein R is₁₀Is alkyl, alkenyl or alkynyl;

X₂is C or N;

X₃is CH or N;

Wherein:

X₁、X₂and X₃Each is N, α is present and β is absent; or

X₁Is NH, X₂Is C, X₃Is CH, α is absent and β is present; or

X1 is N, X2 is N, X3 is CH, α is present and β is absent; or

alternatively, B has the following structure:

wherein:

Or a pharmaceutically acceptable salt thereof.

A compound having the structure:

wherein:

R₁、R₂、R₃、R₄and R₅Each independently of the others being H, halogen, CF₃Or C₁-C₄Alkyl, aryl or heteroaryl;

y is an alkyl group;

a is absent or present and when present isAnd is

B has the following structure:

wherein:

α and β are each a bond, present or absent;

X₁is N, NH or NR₁₀，

Wherein R is₁₀Is alkyl, alkenyl or alkynyl;

X₂is C or N;

X₃is CH or N;

Wherein:

X₁、X₂and X₃Each is N, α is present and β is absent; or

X₁Is NH, X₂Is C, X₃Is CH, α is absent and β is present; or

X1 is N, X2 is N, X3 is CH, α is present and β is absent; or

alternatively, B has the following structure:

wherein

R₁₁、R₁₂And R₁₃Each independently of the others being H, halogen, alkyl-OH, alkyl-NH₂alkyl-OAc, alkyl-O-alkyl, haloalkyl, cycloalkyl, O-alkyl, NH-alkyl, C (O) OH, C (O) -NH₂、C(O)-N(CH₃)₂、C(O)-NHCH₃、NHC(O)-N(CH₃)₂CN or CF₃，

Or a pharmaceutically acceptable salt thereof.

In some embodiments of the above compound, wherein R is₁、R₂、R₃、R₄And R₅Is not H.

In some embodiments of the above compound, wherein R is₁、R₂、R₃、R₄And R₅Are not H.

In some embodiments of the above compound, wherein R is₁、R₂、R₃、R₄And R₅Are respectively H, methyl and BPhenyl, t-Bu, i-Pr, OCF₃、CF₃、OCF₂CF₃、CF₂CF₃Cl, Br or F.

In some embodiments of the above compound, wherein R is₁、R₂、R₃And R₄Are all H; and R is₅is-H, OCF₃、CF₂CF₃Methyl, ethyl, i-Pr or phenyl.

In some embodiments, the compound has the following structure:

a pharmaceutically acceptable salt thereof.

The compounds and classes of compounds disclosed in PCT international publication nos. WO 2014/152013, WO 2015/168286, WO 2014/151959, WO 2014/152018, and WO 2014/151936 may be used in the methods of the present invention, and thus, the compounds and classes of compounds disclosed in each of PCT international publication nos. WO 2014/152013, WO 2015/168286, WO 2014/151959, WO 2014/152018, and WO 2014/151936 are hereby incorporated by reference into the methods of the present invention.

In embodiments, the compound is an RBP4 antagonist.

In one embodiment, the amount of the compound is 5-1000mg, 5-800mg, 5-200mg, 45-1000mg, 45-800mg, 10-50mg, 96mg, 24mg, or 10mg per day.

In some embodiments, the method further comprises administering an amount of a second agent that is (R) - (+) - (5, 6-dichloro-2, 3,9,9 a-tetrahydro-3-oxo-9 a-propyl-1H-fluoren-7-yl) oxy ] acetic acid (DPOFA), a non-steroidal anti-inflammatory drug (NSAID) (such as indomethacin, colchicine, rasidone), a corticosteroid (such as betamethasone, prednisone, dexamethasone, cortisone, hydrocortisone, methylprednisolone, prednisolone), a biologic anti-IL-1 alpha/a drug (such as nanoconzumab, rituximab, anakinra), allopurinol, benzbromarone, peroxose (peglocyme), and other forms of uricase, topiroxot (FYX-051), ulodesine (BCX4208), KUX-1151, RLBN1001, RDEA3170, allofen ester (MBX-102), levo-tofisopam (levotofisopam), UR-1102, PF-06743649, BCX4208, SHR4640, Lumiracoxib (Lumiracoxib), Tranilast (Tranilast), topirostat, LC350189, Bucillamine (Bucilamine), AC-201, Huzhen capsules (including Polygonum cuspidatum (Polygonum cuspidatum) and Ligustrum lucidum (Ligustrum lucidum)), MPC-004, FYU-981, sodium bicarbonate, SEL-212, SEL-037, Apremilast (TMApreX-67, SSS11, D-0120, febuxostat or probucotan, or a sultam or a salt effective to treat a subject, thereby treating the subject.

In one embodiment, the second agent is DPOFA.

In some embodiments, the subject has gout.

In one embodiment, the amount of the second agent or the amount of the compound is effective to reduce uric acid levels in the blood of the subject. In another embodiment, the amount of the second agent or the amount of the compound is effective to reduce uric acid reabsorption in the kidney of the subject.

In one embodiment, the amount of the second agent is effective to increase uric acid clearance in the subject.

In some embodiments, the amount of the second agent is effective to increase uric acid levels in urine of the subject.

In one embodiment, the amount of the second agent is effective to increase renal clearance of uric acid in the subject. In additional embodiments, the amount of the second agent or the amount of the compound is effective to reduce one or more symptoms associated with gout in the subject.

In some embodiments, the one or more symptoms associated with gout is joint pain, arthritis, joint redness, decreased range of motion of the joint. In additional embodiments, the amount of the second agent or the amount of the compound is effective to prevent gout in the subject.

In one embodiment, the preventing comprises increasing uric acid levels in urine of the subject. In another embodiment, the preventing comprises reducing uric acid levels in blood of the subject. In another embodiment, the preventing comprises increasing uric acid clearance in the subject. In another embodiment, the preventing comprises reducing uric acid reabsorption in the kidney of the subject.

In one embodiment, the preventing comprises increasing renal clearance of uric acid in the subject. In another embodiment, the preventing comprises alleviating one or more symptoms associated with gout in the subject.

In some embodiments, the one or more symptoms associated with gout is joint pain, arthritis, joint redness, decreased range of motion of the joint.

In one embodiment, the gout is chronic gout. In various embodiments, the gout is acute gout.

In some embodiments, the amount of the second agent or the amount of the compound prevents recurrence of chronic gout.

In one embodiment of the invention, the subject is a mammal.

In some embodiments, the subject is female, and administration of the compound reduces uric acid levels by 2.4-6.0 mg/dL. In another embodiment, the subject is male and administration of the compound reduces uric acid levels by 3.4-7.0 mg/dL.

In another embodiment, administration of the compound reduces uric acid levels in the subject to less than 7 mg/dL.

The present invention also provides a pharmaceutical composition for treating a subject having non-alcoholic fatty liver disease (NAFLD) disease or gout comprising an amount of a retinol binding protein 4(RBP4) antagonist or a compound as defined above.

In some embodiments, the pharmaceutical composition further comprises an amount of a second agent for treating a subject having non-alcoholic fatty liver disease (NAFLD) disease or gout.

In some embodiments, the RBP4 antagonist or compound and second agent are prepared for simultaneous, concurrent, or combined administration.

The present invention further provides a pharmaceutical composition comprising a retinol binding protein 4(RBP4) antagonist or a compound as defined above for use in combination therapy together with a pharmaceutical composition comprising a second medicament for the treatment of non-alcoholic fatty liver disease (NAFLD) disease or gout.

The present invention further provides a pharmaceutical composition comprising an amount of a retinol binding protein 4(RBP4) antagonist or a compound as defined above, for use in treating a subject having non-alcoholic fatty liver disease (NAFLD) disease or gout, as adjunctive therapy to a second agent or in combination with a second medicament.

The present invention also provides the use of a retinol binding protein 4(RBP4) antagonist or a compound as defined above in the manufacture of a medicament for the treatment of a subject suffering from non-alcoholic fatty liver disease (NAFLD) disease or gout.

As used herein, "combination" refers to a combination of agents for use in therapy by simultaneous or contemporaneous administration. Simultaneous administration refers to the administration of a mixture of a first compound and a second compound, whether an actual mixture, suspension, emulsion, or other physical combination. In this case, the combination may be a mixture or separate containers of the first compound and the second compound that are combined just prior to administration. Contemporaneous administration refers to the administration of the first compound and the second compound separately, either simultaneously or at sufficiently close times so that additive and or preferred synergistic activity is observed relative to the activity of the first compound or the second compound alone.

As used herein, "concomitantly administering" or "concomitantly" administration refers to the administration of two agents administered within a sufficiently close time interval to allow the separate therapeutic effects of each agent to overlap.

As used herein, "additional" or "additional therapy" refers to a combination of agents for therapy, wherein a subject receiving the therapy begins a first treatment regimen of one or more agents before beginning a second treatment regimen of one or more different agents other than the first treatment regimen, whereby not all agents used in the therapy are simultaneously activated. For example, compound 1 therapy is added to a subject who has received DPOFA therapy.

As used herein, a "non-retinoid RBP4 antagonist" is an RBP4 antagonist that is not a retinoid. Retinoids are natural or synthetic analogs of retinol, which consist of four isoprenoid units joined in a head-to-tail fashion. Retinoids are described in the IUPAC-IUB Joint Commission on Biochemical nomenclature (1982).

Unless otherwise indicated, when the structures of the compounds of the present invention include asymmetric carbon atoms, it is understood that the compounds exist as racemates, racemic mixtures and as separate single enantiomers. All such isomeric forms of these compounds are expressly included in the present invention. Unless otherwise indicated, each stereoisomeric carbon may be in the R or S configuration. It is therefore to be understood that isomers resulting from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of the invention unless otherwise indicated. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis (as described in "enertiomers, racemes and solutions" by j.jacques, a.collet and s.wilen, published by John Wiley & Sons, NY, 1981). For example, the separation can be carried out by preparative chromatography on a chiral column.

The present invention is also intended to include all isotopes of atoms occurring on the compounds disclosed herein. Isotopes include those atoms of the same atomic number but different mass numbers. By way of general example, and not limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

It should be noted that any notation of carbon in the structures throughout this application, when used without additional notation, is intended to denote all isotopes of carbon, for example¹²C、¹³C or¹⁴C. In addition, any of¹³C or¹⁴The compound of C may specifically have the structure of any of the compounds disclosed herein.

It should be noted that any notation of hydrogen in structures throughout this application, when used without additional notation, is intended to denote all isotopes of hydrogen, such as¹H、²H or³H. In addition, any of²H or³The compound of H may specifically have the structure of any of the compounds disclosed herein.

Isotopically labeled compounds can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically labeled reagents in place of the non-labeled reagents employed.

The terms "substituted", "substituted" and "substituent" refer to a functional group as described above in which one or more bonds to a hydrogen-containing atom therein is replaced with a bond to a non-hydrogen or non-carbon atom, provided that normal valency is maintained, and that the substitution results in a stable compound. Substituted groups also include groups in which one or more bonds to one or more carbon or one or more hydrogen atoms are replaced with one or more bonds (including double or triple bonds) to a heteroatom. Examples of the substituent include the above functional groups and halogens (i.e., F, Cl, Br, and I); alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl and trifluoromethyl; a hydroxyl group; alkoxy groups such as methoxy, ethoxy, n-propoxy and isopropoxy; aryloxy groups such as phenoxy; arylalkoxy groups, such as benzyloxy (phenylmethoxy) and p-trifluoromethylbenzyloxy (4-trifluoromethylphenylmethoxy); a heteroaryloxy group; sulfonyl groups such as trifluoromethanesulfonyl, methylsulfonyl and p-toluenesulfonyl; nitro, nitrosyl; a mercapto group; thioalkyl groups such as methylsulfanyl, ethylsulfanyl and propylsulfanyl; a cyano group; amino groups such as amino, methylamino, dimethylamino, ethylamino, and diethylamino; and a carboxyl group. When multiple substituent moieties are disclosed or claimed, the substituted compounds may be independently substituted, singly or multiply, with one or more of the disclosed or claimed substituent moieties. Independently substituted means that the (two or more) substituents may be the same or different.

In the compounds used in the method of the present invention, the substituents may be substituted or unsubstituted, unless otherwise specifically defined.

In the compounds used in the methods of the present invention, the alkyl, heteroalkyl, monocyclic, bicyclic, aryl, heteroaryl, and heterocyclic groups may be further substituted by substituting one or more hydrogen atoms with an alternative non-hydrogen group. These include, but are not limited to, halogen, hydroxy, mercapto, amino, carboxy, cyano, and carbamoyl.

It will be appreciated that substituents and substitution patterns on the compounds used in the methods of the invention may be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials. If the substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure is obtained.

In selecting compounds for use in the methods of the present invention, one of ordinary skill in the art will recognize that the various substituents, i.e., R, will be selected in accordance with well-known principles of chemical structure connectivity₁、R₂And the like.

As used herein, "alkyl" is intended to include both branched and straight chain saturated aliphatic hydrocarbon groups having the indicated number of carbon atoms. Thus, for example, "C₁-C_nC in alkyl₁-C_nIs defined to include groups having a linear or branched arrangement of 1, 2, 1, n-1, or n carbons, and specifically includes methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, isopropyl, isobutyl, sec-butyl, and the like. One embodiment may be C₁-C₁₂Alkyl radical, C₂-C₁₂Alkyl radical, C₃-C₁₂Alkyl radical, C₄-C₁₂Alkyl groups, and the like. One embodiment may be C₁-C₈Alkyl radical, C₂-C₈Alkyl radical, C₃-C₈Alkyl radical, C₄-C₈Alkyl groups, and the like. "alkoxy" means an alkyl group as described above attached through an oxygen bridge.

The term "alkenyl" refers to straight or branched chain nonaromatic hydrocarbon radicals containing at least 1 carbon-carbon double bond and up to the maximum number of nonaromatic carbon-carbon double bonds that may be present. Thus, C₂-C_nAlkenyl is defined to include groups having 1, 2, n. For example, "C₂-C₆Alkenyl "means alkenyl having 2,3, 4, 5 or 6 carbon atoms and, respectively, at least one carbon-carbon double bond and, for example, at C₆In the case of alkenyl groups, up to 3 carbon-carbon double bonds. Alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl. As described above for alkyl, the linear, branched, or cyclic portion of the alkenyl group can contain a double bond and can be substituted if a substituted alkenyl group is indicated. One embodiment may be C₂-C₁₂Alkenyl or C₂-C₈An alkenyl group.

The term "alkynyl" refers to a straight or branched chain nonaromatic hydrocarbon radical containing at least 1 carbon-carbon triple bond and up to the maximum number of nonaromatic carbon-carbon triple bonds that may be present. Thus, C₂-C_nAlkynyl is defined to include groups having 1, 2, n. For example, "C₂-C₆Alkynyl "refers to alkynyl groups having 2 or 3 carbon atoms and 1 carbon-carbon triple bond, or alkynyl groups having 4 or 5 carbon atoms and up to 2 carbon-carbon triple bonds, or alkynyl groups having 6 carbon atoms and up to 3 carbon-carbon triple bonds. Alkynyl includes ethynyl, propynyl and butynyl. As described above for alkyl, the straight or branched chain portion of the alkynyl group can contain triple bonds and can be substituted if a substituted alkynyl group is indicated. One embodiment may be C₂-C_nAlkynyl. One embodiment may be C₂-C₁₂Alkynyl or C₃-C₈Alkynyl.

Alkyl groups may be unsubstituted or substituted with one or more substituents including, but not limited to, halogen, alkoxy, alkylthio, trifluoromethyl, difluoromethyl, methoxy, and hydroxy.

As used herein, "C" is₁-C₄Alkyl "includes branched and straight chain C₁-C₄An alkyl group.

As used herein, "heteroalkyl" includes both branched and straight-chain saturated aliphatic hydrocarbon groups having at least 1 heteroatom in the chain or branch.

As used herein, "cycloalkyl" includes a ring of an alkane having a total number of carbon atoms of three to eight or any number within this range (i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl).

As used herein, "heterocycloalkyl" is intended to mean a 5 to 10 membered non-aromatic ring containing 1 to 4 heteroatoms selected from O, N and S, and includes bicyclic groups. Thus, "heterocyclyl" includes, but is not limited to, the following: imidazolyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, dihydropiperidinyl, tetrahydrothienyl, and the like. If the heterocyclic ring contains a nitrogen, it is understood that its corresponding N-oxide is also encompassed by this definition.

As used herein, "aryl" is intended to mean any stable monocyclic, bicyclic, or polycyclic carbocyclic ring of up to 10 atoms in each ring, wherein at least one ring is aromatic and may be unsubstituted or substituted. Examples of such aryl elements include, but are not limited to: phenyl, p-tolyl (4-methylphenyl), naphthyl, tetrahydronaphthyl, indanyl, phenanthryl, anthryl or acenaphthenyl. Where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that the attachment is through an aromatic ring.

The term "alkylaryl" refers to an alkyl group as described above wherein one or more bonds to a hydrogen contained therein is replaced with a bond to an aryl group as described above. It is understood that an "alkylaryl" group is attached to the core molecule through a bond from an alkyl group, and that the aryl group serves as a substituent on the alkyl group. Examples of arylalkyl moieties include, but are not limited to, benzyl (phenylmethyl), p-trifluoromethylbenzyl (4-trifluoromethylphenylmethyl), 1-phenylethyl, 2-phenylethyl, 3-phenylpropyl, 2-phenylpropyl, and the like.

The term "heteroaryl" as used herein denotes a stable monocyclic, bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein at least one ring is aromatic and contains 1 to 4 heteroatoms selected from O, N and S. Bicyclic aromatic heteroaryl groups include, but are not limited to, phenyl, pyridine, pyrimidine, or pyridazine rings that (a) are fused to a 6-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom; (b) fused with a 5 or 6 membered aromatic (unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused with a 5-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom and one oxygen or one sulfur atom; or (d) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one heteroatom selected from O, N or S. Heteroaryl groups within this definition include, but are not limited to: benzimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothienyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, indolinyl, indolyl, indolizinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl, 1, 4-dioxanyl, hexahydroazepinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, Dihydrobenzothienyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dithianyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzyl, tetrahydrofuranyl, tetrahydrothienyl, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, benzoxazolyl, isoxazolyl, isothiazolyl, furyl, thienyl, benzothienyl, benzofuranyl, quinolyl, isoquinolyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, and the like, Pyridyl, pyrimidyl, pyrrolyl, tetrahydroquinolyl. In the case where the heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that the attachment is via an aromatic ring or via a heteroatom-containing ring, respectively. If the heteroaryl group contains a nitrogen atom, it is understood that its corresponding N-oxide is also encompassed by this definition.

As used herein, "monocyclic" includes any stable polycyclic carbocyclic ring of up to 10 atoms, and may be unsubstituted or substituted. Examples of such non-aromatic monocyclic elements include, but are not limited to: cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. Examples of such aromatic monocyclic elements include, but are not limited to: a phenyl group. As used herein, "heteromonocyclic" includes any monocyclic ring containing at least one heteroatom.

As used herein, "bicyclic" includes any stable polycyclic carbocyclic ring of up to 10 atoms fused to a polycyclic carbocyclic ring of up to 10 atoms, wherein each ring is independently unsubstituted or substituted. Examples of such non-aromatic bicyclic elements include, but are not limited to: decahydronaphthalene. Examples of such aromatic bicyclic elements include, but are not limited to: naphthalene. As used herein, "heterobicyclic" includes any bicyclic ring containing at least one heteroatom.

The term "phenyl" is intended to mean an aromatic six-membered ring containing six carbons and any substituted derivatives thereof.

The term "benzyl" is intended to mean a methylene group directly attached to a benzene ring. Benzyl is methyl in which the hydrogen is replaced by phenyl and any substituted derivatives thereof.

The term "pyridine" is intended to mean heteroaryl groups having a six-membered ring containing 5 carbon atoms and 1 nitrogen atom, and any substituted derivatives thereof.

The term "pyrazole" is intended to denote heteroaryl groups having a five-membered ring comprising three carbon atoms and two nitrogen atoms, wherein the nitrogen atoms are adjacent to each other, and any substituted derivatives thereof.

The term "indole" is intended to mean a heteroaryl group having a five-membered ring fused to a benzene ring, wherein the five-membered ring contains 1 nitrogen atom directly attached to the benzene ring.

The term "oxetane" is intended to mean a non-aromatic four-membered ring comprising three carbon atoms and one oxygen atom and any substituted derivatives thereof.

The compounds used in the process of the invention can be prepared by techniques well known in organic synthesis and familiar to those of ordinary skill in the art. However, these may not be the only means of synthesizing or obtaining the desired compound.

The compounds of the invention can be prepared by techniques described in the following documents: vogel's Textbook of Practical Organic Chemistry, A.I. Vogel, A.R. Tatchell, B.S. Furnis, A.J. Hannaford, P.W.G.Smith, (Prentice Hall) fifth edition (1996), March's Advanced Organic Chemistry: Reactions, mechanics, and Structure, Michael B.Smith, Jerry March, (Wiley-Interscience) fifth edition (2007), and references therein, which are incorporated herein by reference. However, these may not be the only means of synthesizing or obtaining the desired compound.

The compounds of the invention may be prepared by the techniques described herein or by the techniques described in PCT International publication Nos. WO/2014/152013, WO/2015/168286, WO/2014/151959, WO/2014/152018 and WO/2014/151936, the contents of each of which are incorporated herein by reference.

The various R groups attached to the aromatic ring of the compounds disclosed herein can be added to the ring by standard procedures such as those shown in the following documents: advanced Organic Chemistry Part B: Reaction and Synthesis, Francis Carey and Richard Sundberg, fifth edition (Springer) (2007), the contents of which are incorporated herein by reference.

Another aspect of the invention includes the compounds of the invention as pharmaceutical compositions.

As used herein, the term "pharmaceutically active agent" refers to any substance or compound that is suitable for administration to a subject and provides a biological activity or other direct effect in the treatment, cure, mitigation, diagnosis or prevention of a disease, or affects the structure or any function of the subject. Pharmaceutically active agents include, but are not limited to, the substances and compounds described in the following references: physicians' Desk Reference (PDR Network, LLC; version 64; 11/15 2009) And "applied Drug Products with Therapeutic Equisition events" (U.S. department Of Health And Human Services, version 30, 2010), which are incorporated herein by Reference. Pharmaceutically active agents having a pendant carboxylic acid group can be modified according to the present invention using methods readily available and known to those of ordinary skill in the art of standard esterification reactions and chemical synthesis. In the case of pharmaceutically active agents having no carboxylic acid groups, one of ordinary skill would be able to design and incorporate carboxylic acid groups into pharmaceutically active agents, where esterification may be subsequently performed, so long as the modification does not interfere with the biological activity or effect of the pharmaceutically active agent.

The compounds of the invention may be in the form of salts. As used herein, "salts" are salts of the compounds of the present invention that are modified by making acid or base salts of the compounds. In the case of compounds for the treatment of diseases, the salts are pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues (e.g., amines); alkali metal salts or organic salts of acidic residues, such as phenols. The salts may be made with organic or inorganic acids. Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates and the like. The phenate is an alkaline earth metal salt, sodium salt, potassium salt or lithium salt. In this regard, the term "pharmaceutically acceptable salts" refers to the relatively non-toxic inorganic and organic acid or base addition salts of the compounds of the present invention. These salts may be prepared in situ during the final isolation and purification of the compounds of the invention, or by reacting the purified compounds of the invention in their free base or free acid form, respectively, with a suitable organic or inorganic acid or base, and isolating the salt thus formed. Representative Salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthenate, mesylate, gluconate, lactate, and laurylsulfate Salts and the like (see, e.g., Berge et al (1977) "Pharmaceutical Salts", J.pharm.Sci.66: 1-19).

Salts or pharmaceutically acceptable salts are contemplated for all compounds disclosed herein. In some embodiments, a pharmaceutically acceptable salt or salt of any of the above compounds of the invention.

As used herein, "treating" or "treatment" refers to preventing, slowing, stopping or reversing the progression of a disease or infection. Treatment may also mean amelioration of one or more symptoms of the disease or infection. One embodiment of "treating gout" is to delay or prevent the onset, progression, or lessen the severity of gout.

As used herein, "normalize," with respect to normalizing a concentration in a subject with a disease, refers to increasing or decreasing the concentration such that the concentration is closer to the concentration in a subject without the disease.

As used herein, "increased," such as an increased concentration of RBP4 in a subject with a disease, refers to an increased concentration of RBP4 as compared to the concentration in a subject without the disease.

The compounds of the present invention may be administered in various forms, including those detailed herein. The treatment with the compounds may be part of a combination therapy or an adjuvant therapy, i.e. the treatment of a subject in need of such a drug in combination with one or more compounds of the invention, or the administration of another drug for such a disease. The combination therapy may be a sequential therapy in which the patient is treated first with one drug and then with the other drug, or the two drugs are administered simultaneously. Depending on the dosage form employed, they may be administered independently by the same or two or more different routes of administration.

As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering a compound of the invention to an animal or human. The vehicle may be liquid or solid and is selected according to the intended mode of administration in question. Liposomes are also pharmaceutically acceptable carriers.

The dosage of the compound administered in treatment will vary depending upon a variety of factors, such as the pharmacodynamic properties of the particular chemotherapeutic agent and its mode and route of administration; age, sex, metabolic rate, absorption efficiency, health status and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent therapy administered; the frequency of treatment; and the desired therapeutic effect.

Dosage units of the compounds for use in the methods of the invention may comprise a single compound or a mixture thereof with other agents. The compounds can be administered in oral dosage forms of tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered intravenously (bolus or infusion), intraperitoneally, subcutaneously, or intramuscularly, or introduced directly into or onto the site of infection, for example by injection, topical application, or other methods, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.

The compounds used in the methods of the present invention may be administered in admixture with a suitable pharmaceutical diluent, extender, excipient or carrier (collectively referred to herein as pharmaceutically acceptable carriers), suitably selected with regard to the intended form of administration and in accordance with conventional pharmaceutical practice. The units will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration. The compounds may be administered alone or in admixture with a pharmaceutically acceptable carrier. The carrier may be a solid or a liquid, and the type of carrier is typically selected based on the type of administration used. The active agents may be co-administered in the form of tablets or capsules, liposomes, in agglomerated powder or liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin, and agar. The capsule or tablet is easy to formulate and make into capsule or tablet easy to swallow or chew; other solid forms include granules and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow inducing agents and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents (including esters), emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent formulations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally comprise flavoring and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

Techniques and compositions for preparing dosage forms useful in the present invention are described in the following references: 7Modern pharmaceuticals, chapters 9 and 10 (Banker & Rhodes, Editors, 1979); pharmaceutical Dosage Forms, Tablets (Lieberman et al, 1981); ansel, Introduction to Pharmaceutical Dosage Forms second edition (1976); remington's Pharmaceutical Sciences, 17 th edition (Mack Publishing Company, Easton, Pa., 1985); advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, eds., 1992); advances in Pharmaceutical Sciences Vol.7 (David Ganderton, Trevor Jones, James McGinity, eds., 1995); aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36(James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Pharmaceutical Applications: Drugs and the Pharmaceutical Sciences, Vol 61(Alain Rolland, Ed., 1993); Drug Delivery to Pharmaceutical Particulate track (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J.G.Hardy, S.S.Vision, Cll.Wilson, Ecal pharmaceuticals and Pharmaceutical Dosage Forms (published by T.40, supra) are incorporated by reference.

Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow inducing agents and melting agents. For example, for oral administration in the form of a tablet or capsule dosage unit, the active pharmaceutical ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Suitable binders include starch, gelatin, natural sugars (such as glucose or beta-lactose), corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like.

The compounds used in the methods of the invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. The compounds may be administered as a component of a tissue-targeting emulsion.

The compounds used in the methods of the invention may also be coupled to soluble polymers as targetable drug carriers or as prodrugs. Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxyethylaspartame-resorcinol, or polyethylene oxide-polylysine substituted with palmitoyl residues. In addition, the compounds may be coupled to a class of biodegradable polymers useful for achieving controlled release of a drug, such as polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross-linked or amphiphilic block copolymers of hydrogels.

Gelatin capsules may contain the active ingredient compound and powdered carriers such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be made as immediate release products or sustained release products to provide continuous release of the drug over a period of hours. Compressed tablets may be sugar-coated or film-coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated to selectively disintegrate in the gastrointestinal tract.

For oral administration in liquid form, the oral pharmaceutical ingredients are combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents (including esters), emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent formulations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, thickeners, and melting agents.

Liquid dosage forms for oral administration may contain coloring and flavoring agents to increase patient acceptance. Generally, water, suitable oils, saline, aqueous dextrose (glucose) and related sugar solutions and glycols (e.g., propylene glycol or polyethylene glycol) are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water-soluble salt of the active ingredient, a suitable stabilizer, and if desired, a buffer substance. Antioxidants, such as sodium bisulfite, sodium sulfite, or ascorbic acid, alone or in combination, are suitable stabilizers. Citric acid and its salts and sodium edetate are also used. In addition, parenteral solutions may contain preservatives such as benzalkonium chloride, methyl or propyl parabens and chlorobutanol. Suitable pharmaceutical carriers are described in standard reference books in the field: remington's Pharmaceutical Sciences, Mack Publishing Company.

The compounds used in the methods of the invention may also be administered in intranasal form by use of suitable intranasal vehicles or by transdermal routes using those forms of transdermal skin patches well known to those of ordinary skill in the art. For administration in the form of a transdermal delivery system, administration is generally continuous, rather than intermittent, throughout the dosage regimen.

Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

Each embodiment disclosed herein is to be considered as applying to each other disclosed embodiment. Accordingly, all combinations of the various elements described herein are within the scope of the invention.

The present invention will be better understood by reference to the following experimental details, but those skilled in the art will readily appreciate that the specific experiments described in detail are only illustrative of the invention as more fully described in the claims that follow.

Details of the experiment

Example 1:

as shown in figure 1, a single oral and intravenous administration of compound 1 induced a significant reduction in circulating serum RBP 4levels in wild-type mice.

The primary objective of this study was to determine the targeting of compound 1 in mice and to determine whether compound 1 is active in mice. To perform this assay, the effect of oral and intravenous administration of compound 1 on the dynamics of plasma RBP 4levels in male CD-1 mice was investigated. Aliquots of the collected plasma samples were used to analyze plasma RBP4 concentrations using standard ELISA protocols (Adipogen RBP4 (mouse/rat) dual ELISA kit) as described by the ELISA kit manufacturer. Plasma RBP4 concentrations were determined at 10 time points after IV or PO compound administration. Blood samples can be taken up 3 times at most from a single animal due to the small blood volume in the mice. Samples from different animals were analyzed for RBP4 concentration at different time points. Pre-dose plasma samples were collected from all animals enrolled in the study and the mean pre-dose RBP4 concentration was used as baseline. The experimental design of this study is shown in table 1.

Table 1: experimental design and mouse grouping

^aBlood was collected from 3 untreated mice as baseline controls.

^bThe intravenous route is via the tailA vein.

For IV administration, the compounds were prepared as solutions in 3% DMA/45% PEG 300/12% ethanol/40% sterile water. For PO application, the compound was prepared as 2% in 0.9% saline80. In summary, oral (5mg/kg) and intravenous (2mg/kg) administration of compound 1 resulted in a significant reduction in plasma RBP4 concentration in male CD-1 mice. The maximum mean plasma RBP4 reduction was 84.9% after 12 hours Post Oral (PO) administration and 81.4% after 8 hours post Intravenous (IV) administration. The results of the study provide evidence for the in vivo activity of the test compounds in mice.

Example 2:

emerging evidence suggests that in addition to its retinol transport function, RBP4 may play a pathogenic role in several common diseases, such as insulin resistance, type 2 diabetes (T2D), metabolic syndrome, and low grade vascular inflammation. There is a large body of evidence supporting the association of elevated circulating levels of RBP4 with the development of NAFLD.

Lee 2016 recently reported a transgenic mouse model ("adi-hRBP 4 mouse") in which human RBP4 can be specifically expressed in adipocytes. The transgenic mice had slightly elevated circulating levels of RBP4 due to increased production of human RBP4 in adipose tissue of the mice. The transgenic mice gained weight and developed hepatic steatosis. Indeed, when fed regular food, these mice develop NAFLD at 3-4 months of age; and when fed a High Fat Diet (HFD), the metabolic phenotype worsened more rapidly than littermate controls. Thus, the adi-hRBP4 mouse is a transgenic model of hepatic steatosis.

Compound 1, an advanced RBP4 antagonist, was evaluated in adi-hRBP4 mice. In this experiment, male adi-hRBP4 mice were divided into three age-matched treatment groups. The first group was fed with normal food (hereinafter referred to as "normal food group"), the second group was fed with High Fat Diet (HFD) (hereinafter referred to as "HFD-only group"), and the third group was fed with HFD and compound 1 (hereinafter referred to as "treatment group"). Compound 1 was formulated into food to provide a daily oral dose to the treatment group of 20mg compound 1/kg mouse body weight. The treatment time was four (4) weeks. The following variables were measured: body weight, food consumption, liver lipids, blood chemistry, urinalysis, liver histology, kidney histology, retina-histology, and ERG.

The experimental design is summarized in fig. 2, and the experimental data for this experiment is shown in fig. 3-14.

Both human and mouse RBP4 were present in the mouse circulatory system (serum) as shown in figure 4. Human RBP4 is expressed and secreted in adipose tissue, while mouse RBP4 is produced primarily in the liver. However, circulating concentrations of human RBP4 produced in adipose tissue in untreated animals were approximately 5% of mouse RBP4 produced primarily in the liver. As shown in figure 4, administration of compound 1 significantly reduced circulating levels of human and mouse serum RBP 4levels in HFD-fed adi-hRBP4 mice.

As shown in fig. 6 and 7, compound 1 was also found to significantly reduce HFD-induced weight gain, although there was no difference in food intake between the HFD-only group and the treatment group (fig. 8). Compound 1 was also found to significantly reduce the concentration of hepatic triglycerides and hepatic free fatty acids compared to the HFD group only (figures 9 and 10).

Fig. 14 shows that fatty acids can act as ligands for RBP4, suggesting that RBP4 may be involved in fatty acid transport from adipose tissue to liver. Compound 1 and other RBP4 antagonists can inhibit this trafficking because they compete for binding to the same ligand binding pocket in RBP 4.

Thus, reducing the levels of human and mouse RBP4 was associated with a significant efficacy of compound 1 in reducing weight gain and normalizing the concentrations of triglycerides and free fatty acids in the liver.

This data indicates that compound 1 and similar compounds can be used to treat a range of diseases in human NAFLD. Compound 1 and similar compounds are particularly useful for treating NAFLD-range diseases in patients with elevated serum RBP4 levels.

Example 3: treatment of gout

Hepatic steatosis in NAFLD induces up-regulation of xanthine oxidase in the liver, leading to increased uric acid production. In people with NAFLD, elevated circulating uric acid concentrations can lead to gout symptoms. NAFLD is closely associated with the development of hyperuricemia.

Administration of compound 1 to the transgenic mice described in example 2 significantly reduced circulating levels of uric acid. Thus, compound 1 and similar compounds are useful for treating gout and gouty arthritis in patients with NAFLD. Compound 1 and similar compounds are also useful for treating gout and gouty arthritis in patients without NAFLD.

Example 4: combination therapy for gout

Since compound 1 inhibits the production of uric acid in the liver, compound 1 and similar compounds can be used in combination with drugs that induce the excretion of uric acid in the kidney. One example of such a drug is DPOFA, exemplified in PCT International publication No. WO/2017/083652, which is incorporated herein by reference. In view of the disclosure herein, these two compounds have complementary mechanisms of action. Specifically, DPOFA increased uric acid excretion through the urine, while compound 1 inhibited NAFLD-induced Xanthine Oxidase (XO) upregulation in the liver. XO is an enzyme responsible for uric acid synthesis in the liver, which is the main site of uric acid production. Thus, in combination, compound 1 decreased uric acid production, while DPOFA increased uric acid excretion from the body, thereby treating the subject.

Example 5: efficacy in mammalian models

The effectiveness of the compounds listed herein was tested in adi-hRBP4 mice. The adi-hRBP4 mouse model showed a modest increase in human RBP4 expression in mouse adipose tissue and was considered a model of hepatic steatosis. The compound was administered orally at 20mg/kg for 4 weeks. Serum levels of human and mouse RBP4 were reduced in treated animals. RBP4 and Free Fatty Acid (FFA) levels were reduced in the liver in treated mice. (1) The levels of free fatty acids in serum, (2) triglycerides in liver, (3) serum uric acid are also reduced.

Example 6: administering to a subject

Administering to a subject having NAFLD an amount of compound 1. The amount of the compound is effective to treat the subject.

An amount of compound 1 is administered to a patient suffering from NAFLD and elevated circulating levels of serum RBP 4. The amount of the compound is effective to treat the subject.

Administering to a subject having hepatic steatosis an amount of compound 1. The amount of the compound is effective to treat the subject.

A subject having nonalcoholic steatohepatitis (NASH) is administered an amount of compound 1. The amount of the compound is effective to treat the subject.

Administering to a subject having cirrhosis an amount of compound 1. The amount of the compound is effective to treat the subject.

Administering to a subject having hepatocellular carcinoma an amount of compound 1. The amount of the compound is effective to treat the subject.

Administering to a subject with gout an amount of compound 1. The amount of the compound is effective to treat the subject.

The structures of compounds 2-4 are shown below.

Administering to a subject having NAFLD an amount of compound 2. The amount of the compound is effective to treat the subject.

Administering to a subject having hepatic steatosis an amount of compound 2. The amount of the compound is effective to treat the subject.

A subject with nonalcoholic steatohepatitis (NASH) is administered an amount of compound 2. The amount of the compound is effective to treat the subject.

Administering to a subject having cirrhosis an amount of compound 2. The amount of the compound is effective to treat the subject.

Administering to a subject having hepatocellular carcinoma an amount of compound 2. The amount of the compound is effective to treat the subject.

Administering to a subject with ventilation an amount of compound 2. The amount of the compound is effective to treat the subject.

Administering to a subject having NAFLD an amount of compound 3. The amount of the compound is effective to treat the subject.

Administering to a subject having hepatic steatosis an amount of compound 3. The amount of the compound is effective to treat the subject.

A subject having nonalcoholic steatohepatitis (NASH) is administered an amount of compound 3. The amount of the compound is effective to treat the subject.

Administering to a subject having cirrhosis an amount of compound 3. The amount of the compound is effective to treat the subject.

Administering to a subject having hepatocellular carcinoma an amount of compound 3. The amount of the compound is effective to treat the subject.

Administering to a subject with ventilation an amount of compound 3. The amount of the compound is effective to treat the subject.

Administering to a subject having NAFLD an amount of compound 4. The amount of the compound is effective to treat the subject.

Administering to a subject having hepatic steatosis an amount of compound 4. The amount of the compound is effective to treat the subject.

A subject with nonalcoholic steatohepatitis (NASH) is administered an amount of compound 4. The amount of the compound is effective to treat the subject.

Administering to a subject having cirrhosis an amount of compound 4. The amount of the compound is effective to treat the subject.

Administering to a subject having hepatocellular carcinoma an amount of compound 4. The amount of the compound is effective to treat the subject.

Administering to a subject with gout an amount of compound 4. The amount of the compound is effective to treat the subject.

Example 7: DPOFA in combination with Compound 1

Administering to a subject with gout an amount of compound DPOFA in combination with compound 1. The amounts of DPOFA and compound 1 are effective to treat the subject with gout. The combination is more effective in treating gout than each compound administered alone.

Example 8: DPOFA in combination with Compound 1

Administering to a subject with gout and NAFLD an amount of compound DPOFA in combination with compound 1. The amounts of DPOFA and compound 1 are effective to treat the subject with gout and NAFLD. The combination is more effective in treating gout and NAFLD than each compound administered alone.

Example 9: DPOFA in combination with Compound 1 for preventing gout in a subject

Administering to a subject having a history of, or at risk of, a gout attack an amount of compound DPOFA in combination with compound 1. The amounts of DPOFA and compound 1 are effective to prevent a gout attack in the subject. The combination is more effective in preventing gout than each compound administered alone.

Example 10: in vivo activity: PK profile of compound 1 and compound 3 in rodents.

Both compound 1 and compound 3 had good PK profiles in naive male CD-1 mice and adult Sprague-Dawley male rats (figure 15). The compounds show moderate to low clearance in both species and good half-life (t)_1/2). Full exposure (AUC) was also obtained_last) And good oral bioavailability (% F). The good oral bioavailability, moderate half-life and proper exposure and good ADME profile obtained in mice, fully confirm the establishment of PK/PD and final proof of concept using compound 1 and compound 3 in mouse animal models.

Example 11: in vivo activity: PK/PD correlation of compound 1 in mice.

Prior to testing compound 1 in a transgenic model of hepatic steatosis in mice, acute dosing studies were performed with compound 1 in mice to measure the effect of compound on circulating plasma RBP 4levels and establish PK/PD correlations (figure 16). A maximum 85% reduction in plasma RBP4 was observed following a single oral administration (5mg/kg) of compound 1 (fig. 16A), while a 81% reduction in plasma RBP4 was observed following 2mg/kg intravenous administration (fig. 16B). The reduction in serum RBP4 in vivo after oral and intravenous administration of compound 1 (fig. 16A, B) showed a good correlation with the concentration of compound in plasma (fig. 16C, D). The long-term exposure and moderate to low clearance of compound 1 obtained after a single oral administration correlated well with the extent of reduction in RBP4 (85% reduction at the 12 hour time point) and the duration of the reduction in RBP4 (71% reduction at the 24 hour time point).

Furthermore, the magnitude of the RBP4 reduction correlates closely with the predicted free drug concentration of compound 1 in plasma, which exceeds the concentration required to disrupt the RBP4-TTR interaction as measured in an in vitro HTRF assay. These data confirm the in vivo targeting of compound 1 and reveal a good PK/PD relationship between compound exposure and mouse biological responses, and in addition confirm the properties of compound 1 in transgenic models of mouse hepatic steatosis.

Example 12: compound 1 reduced circulating levels of fat-derived RBP4 in a transgenic model of hepatic steatosis in mice.

As previously reported by Blaner and co-workers (Lee, s. -a. et al 2016), a genetic model of mouse liver steatosis can be generated by targeting a human RBP4(hRBP) cDNA construct with a loxP-neor-stop cassette to the mouse Rosa26 locus. To specifically express hRBP4 in mouse adipocytes, knock-in mice were bred with adiponectin-Cre mice. 22-specific expression of human RBP4 in mouse adipocytes did not result in a significant increase in circulating levels of RBP4, and there was no change in retinoid levels in plasma, liver and adipose tissue, while resulting in significant increases in obesity, impaired glucose tolerance and hepatic Triglyceride (TG) levels (Lee, s.

To examine the effect of compound 1 on the metabolic parameters of male adi-hRBP4 mice, the compound was administered as a formulation to HFD food at a dose of 20mg/kg per day for 29 days. When animals were changed from standard food to a high fat diet (60% of the calories from fat), administration of the compound began at 20 weeks of age. Chronic oral administration of compound 1 caused a 90% reduction in circulating levels of mouse and human (adipose tissue secreted) serum RBP4 (fig. 17). In untreated adi-hRBP4 mice, circulating levels of adipose-derived human RBP4 (2-3 μ g/mL) accounted for a small fraction of 3-5% of the mouse-specific serum RBP4 that was produced primarily in the liver (FIG. 17). Notably, this modest increase in circulating levels of RBP4 conferred by adipocyte secretion is sufficient to trigger induction of a strong metabolic phenotype.

Example 13: compound 1 reduced the weight gain of obese adi-hRBP4 mice.

The weight gain of the adihRBP4 mice on the high fat diet was significantly higher than that of the transgenic animals kept on the standard diet over the 29 day study period (fig. 18A). There was a significant statistical difference in the percent weight gain between the diet-fed mice and the HFD adi-hRBP mice 5 days after the initiation of high fat feeding (fig. 18A). By administering compound 1, the body weight gain of HFD animals was significantly reduced. There was a significant statistical difference in body weight gain between compound 1-treated and untreated HFD mice after 19 days of high fat diet feeding (fig. 18A). At the end of treatment on day 29 (2.2. + -. 1.7g), 53% less than untreated HFD animals (4.7. + -. 1.6 g). The reduction in body weight gain in compound 1 treated adi-RBP4 mice was not associated with a reduction in food intake, as compound 1 did not alter HFD food consumption (fig. 10B).

Example 14: compound 1 reduced liver lipid levels in obese adi-hRBP4 mice.

According to previous reports (Lee, s. -a. et al 2016), liver Free Fatty Acid (FFA) and Triglyceride (TG) levels were significantly higher in adi-hRBP4 mice maintained on a high fat diet than in transgenic animals kept on standard chow (fig. 19; P <0.0001 for FFA and TG). Administration of compound 1 reduced FFA levels by 30% (fig. 11A; P ═ 0.0107) and TG levels by 29% (fig. 11B; P ═ 0.0104).

Consistent with the dynamics of liver TG accumulation, histological examination of oil red O-stained frozen liver sections (fig. 20A) and liver steatosis grading (fig. 20A) demonstrated that mice maintaining HFD were significantly more steatosis in adi-hRBP4 mice compared to food-fed transgenic mice that showed no evidence of liver steatosis.

A significant improvement in hepatic steatosis was observed in adi-hRBP4 obese mice treated with compound 1, which showed fewer and fewer lipid droplets compared to untreated adihRBP4 mice maintained on HFD (fig. 18B). Liver steatosis staging (fig. 12B) showed that the degree of steatosis was significantly reduced by 43% (P <0.001) in adihRBP4 mice fed HFD treated with compound 1, further confirming the ability of compound 1 to alleviate liver steatosis in adi-hRBP4 mice.

Example 15: binding of fatty acid ligand to RBP 4.

It is not clear how a modest increase in RBP4 expression in adipose tissue promotes the development of hepatic steatosis. It is speculated that this ability of RBP4 may be related to the binding and trafficking of endogenous non-retinoid ligands, and in this regard, the nature and quantity are unknown. Consistent with the ability of RBP4 to interact with non-retinoid ligands, previous crystallographic studies of heterologously expressed RBP4 demonstrated that the accidentally discovered fatty acid ligands (oleic and linoleic acids) from the expression host bind in the ligand-binding pocket of RBP4 (Huang, h.

Recent X-ray crystallography and mass spectrometry findings reported by Monaco and colleagues confirmed that RBP4 was able to bind fatty acids (Perduca, m. et al 2018). The ability of retinol binding proteins to bind hydrophobic non-retinoid ligands may be general, as demonstrated by the recently described interaction of cellular retinol binding protein 1 with certain cannabinoids (Silvaroli, j.a. et al 2019).

The affinity of palmitic, oleic, linoleic and docosahexaenoic acids for RBP4 was tested using the SPA assay, which can measure the substitution of radioactive retinol in purified human RBP4 (fig. 21A). These competitive binding experiments confirmed that the fatty acids tested functioned as weak RBP4 ligands. Our docking model is also consistent with the ability of RBP4 to bind fatty acid ligands. Figure 13B shows the overlap of compound 3 and the fatty acids palmitic, oleic, linoleic and docosahexaenoic acids docked in our 3FMZ model, as well as all-trans retinol docked in our 5NU7(holo-RBP4) model. The figure shows a good overlap between all-trans retinol and compound 3, as both present similar geometric constitutions and project their polar fragments to the opening of the binding cavity, where they undergo H-bond interactions with Gln98 and Arg121, respectively. Similar to all-trans retinol and compound 3, the fatty acid also extends its hydrophobic tail through the narrow β -barrel and into the β -ionone pocket, and its polar carboxylic acid binds to residues closer to the binding cavity opening, namely Leu36 and Phe 36.

Discussion of the related Art

In one aspect, the invention relates to small molecules for use in the treatment of NAFLD disease. Disclosed herein are specific uses of small molecules as non-retinoid RBP4 antagonists for the treatment of non-alcoholic fatty liver disease (NAFLD) diseases. The compounds listed herein have been shown to bind RBP4 in vitro and/or antagonize RBP4-TTR interactions in vitro at biologically significant concentrations. Additional compounds described herein are analogs of the compounds listed herein that similarly bind RBP4 in vitro at biologically significant concentrations and antagonize RBP4-TTR interactions in vitro.

fenritidine [ N- (4-hydroxy-phenyl) retinoamide, 4HRP ] is a previously developed retinoid drug for the treatment of cancer. Like other retinoid drugs, fenritinide is non-specific and therefore capable of binding to multiple drug targets. Its anticancer activity is attributed to the ability to produce reactive oxygen species in malignant tumor cells and to induce apoptosis. Some of the other activities of this compound are mediated by its action as a ligand for the nuclear receptor RAR. The ability to act as RBP4 ligand is well characterized over the range of different activities of fenritinide. For example, fenritinide was found to bind to RBP4, displace all-trans retinol from RBP4 (Berni 1992), disrupt RBP4-TTR interactions (Berni 1992, Schafer 1993), reduce serum RBP4 and retinol (Adams 1995), and inhibit intraocular all-trans retinol uptake and slow the visual cycle (Radu 2005). However, it is known from the literature that the beneficial effects of fenritidine in models of diabetes and obesity are not mediated by its activity as an RBP4 antagonist. Koh 2012 reported that fenritinide can improve insulin sensitivity and reduce liver lipid levels in obese mice. This observation is consistent with earlier reports indicating that long-term administration of fenritidine can prevent obesity, insulin resistance and liver steatosis caused by high fat diet (Preitner 2009). However, it is convincingly shown in Preitner that its activity as an RBP4 antagonist does not mediate the beneficial effect of fenritide on reducing HFD-induced obesity, given that fenritide is equally effective in reducing fat loss in RBP4 knockout animals. Motani 2009 also demonstrated that the ability of fenritinide to increase insulin sensitivity in HFD-fed mice was independent of its activity as an RBP4 antagonist. In general, although Koh (and earlier work by Preitner and Motani) showed beneficial effects of fenritide in the relevant HFD-induced obesity model, earlier work by Preitner and Motani convincingly attributed these beneficial effects to fenritide's activity unrelated to the involvement of RBP 4. For these reasons, one of ordinary skill in the art would not reasonably expect administration of an RBP4 antagonist to treat NAFLD disease in view of Koh 2012 and Preitner 2009.

fenritinide is also toxic and teratogenic. Thus, the safety profile of fenritinide may be incompatible with long-term administration to subjects with non-life threatening conditions.

As briefly discussed above, Koh 2012 discloses the use of fenritinide ("FEN") in genetically obese (ob/ob) mice. In Koh, ob/ob mice are divided into two groups, and both groups are fed a High Fat Diet (HFD), but FEN is administered to only one group. Koh 2012 reported that FEN decreased weight gain, insulin resistance and fatty liver in ob/ob mice fed a high fat diet. Koh also reported that FEN reduced RBP4 and TTR levels in the circulation (page 371). Furthermore, Koh reported a decrease in RBP 4levels in adipose tissue of FEN-treated obese mice (page 374, right panel). Koh states that "lower levels of circulating lipids may be transported to tissues including liver after fenritinide treatment" (page 374, right panel). In contrast to Koh 2012, Lee 2016 showed that in mice not administered a high fat diet, free fatty acids redistributed from adipose tissue to the liver, not to all tissues implied by Koh.

Koh 2012 further showed that plasma adiponectin levels differed between mice treated with vehicle and FEN, which could be associated with decreased Triglyceride (TG) synthesis and increased Free Fatty Acid (FFA) oxidation in FEN treated mice. However, due to the increase in the level of the adiponectin-stimulated transcription factor PPAR α, Koh later indicated that FEN may affect fatty acid oxidase in obese mice universally (pages 374 to 375). Koh further concluded that lower liver fat accumulation results from increased fatty acid oxidation, rather than decreased adipogenesis (page 375). In contrast, Lee 2016 found that increased expression of TNF and leptin resulted in RBP4 inducing inflammation in adipose tissue, resulting in increased lipolysis. Lee 2016 further states that free fatty acids produced by lipolysis are absorbed by the liver, which results in elevated TG levels and hepatic steatosis.

Thus, not only was the positive attribute of fenritide in the HFD-induced obesity model shown to be independent of its activity as an RBP4 antagonist, but also Koh 2012 suggested its proposed error mechanism for treatment with fenritide.

Furthermore, Koh 2012 does not disclose the use of any RBP4 antagonist for the treatment of non-alcoholic fatty liver disease (NAFLD), and the mice used in Koh's study are not specific models of NAFLD. In contrast, the data in this application are from a transgenic model of hepatic steatosis, a NAFLD disease.

Thus, Koh 2012 does not provide any reasonable hint that RBP4 antagonists could successfully treat NAFLD.

While Lee 2016 discloses sufficient evidence to correlate elevated circulating levels of RBP4 with the occurrence of NAFLD (page 1534), Lee 2016 also points out that some studies report evidence for a lack of correlation between RBP4 and NAFLD (page 1543). Thus, the complete role of elevated circulating levels of RBP4 in the development of NAFLD is not clear.

Lee 2016 further states that the development of obesity leads to an increase in the expression of RBP4 by adipocytes (page 1534). However, Lee explicitly states: "whether RBP4 actually stimulates hepatic steatosis in a liver-autonomous manner remains to be determined". Similar to Koh 2012, Lee 2016 found no increase in the rate of neonatal adipogenesis in the liver of adi-hRBP4 mice (p 1540). However, unlike Koh, Lee did not find any statistically significant genotype-dependent differences in Free Fatty Acid (FFA) oxidation. In contrast, Lee states that its data support the conclusion that increased uptake of circulating fatty acids (FFA) by the liver "substantially" resulted in the fatty liver phenotype observed in adi-hRBP4 mice.

However, Lee 2016 did not show what effect an RBP4 antagonist had on adi-hRBP4 mice or what effect an RBP4 antagonist had on any subject. For example, none of the experiments disclosed in Lee 2016 administered any RBP4 antagonist to a subject. Indeed, Lee 2016 does not suggest that RBP4 antagonists could be used to treat NAFLD.

Applicants herein have shown, among other things, that RBP4 antagonists can normalize the concentrations of triglycerides and free fatty acids in the liver, thereby treating NAFLD disease.

Currently, there is no FDA approved drug therapy for any form of NAFLD. The present invention identifies non-retinoid RBP4 antagonists useful for treating NAFLD and other conditions characterized by excessive accumulation of RBP4 in adipocytes. Without wishing to be bound by any scientific theory, because the accumulation of RBP4 in adipocytes appears to be the direct cause of fatty liver in NAFLD, the compounds described herein are disease modulators because they directly address the cause of NAFLD. The present invention provides novel therapeutic methods that will treat patients with NAFLD as well as patients with conditions characterized by excess RBP4 in adipocytes.

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