阅读说明：本技术 一种新型FFA1和PPARα/γ/δ四重激动剂、其制备方法及其作为药物的用途 (Novel FFA1 and PPAR alpha/gamma/delta quadruple agonist, preparation method thereof and application thereof as medicament ) 是由 李政 张陆勇 周宗涛 于 2020-12-28 设计创作，主要内容包括：本发明涉及一种式(I)所示的新型FFA1和PPAR-α/γ/δ四重激动剂、其制备方法及含有该化合物的药物组合物,作为制备治疗、预防或缓解一种或多种疾病或功能障碍的药物,相比现有技术,其具有更优的代谢稳定性、更强的体内药理效应,具有更广阔的应用前景。(The invention relates to a novel FFA1 and PPAR-alpha/gamma/delta quadruple agonist shown in formula (I), a preparation method thereof and a pharmaceutical composition containing the compound, which are used for preparing a medicament for treating, preventing or relieving one or more diseases or dysfunctions.)

1. A compound (I) or pharmaceutically acceptable salts, prodrugs, esters and solvates thereof:

。

2. a pharmaceutical composition comprising a compound (I) as claimed in claim 1 or a pharmaceutically acceptable salt, prodrug, ester and solvate thereof, together with a pharmaceutically acceptable adjuvant, carrier or diluent.

3. The use of compound (I) as claimed in claim 1 or pharmaceutically acceptable salts, prodrugs, esters and solvates thereof as FFA1 and PPAR- α/γ/δ quadruple agonists.

4. Use of a compound (I) as claimed in claim 1 or a pharmaceutically acceptable salt, prodrug, ester and solvate thereof for the manufacture of a medicament for the treatment, prevention or alleviation of one or more diseases or disorders selected from fatty liver, cholestatic liver disease, mitochondrial disease, liver graft-versus-host disease, virally induced chronic liver disease, alcoholic liver disease, pharmaceutical liver injury, diabetes, diabetic complications, pre-diabetes, hyperlipidemia, obesity, metabolic syndrome, gout, atherosclerosis, organ fibrosis, inflammation and cancer.

5. The use of claim 4, wherein the one or more diseases or disorders comprise non-alcoholic fatty liver disease, primary biliary cholangitis, primary sclerosing cholangitis, alcoholic fatty liver.

Technical Field

The invention relates to a novel FFA1 and PPAR alpha/gamma/delta quadruple agonist, a preparation method and application thereof, belonging to the technical field of medicines. The structures of the compounds involved in the present invention are novel and unique in this field.

Background

The metabolic syndrome is a common disease characterized by insulin resistance and visceral fat accumulation, and is accompanied by low-density lipoprotein increase and high-density lipoprotein cholesterol reduction, the common diseases comprise obesity, diabetes, hyperlipidemia, atherosclerosis, fatty liver and the like, and the diabetes patients are also frequently complicated with diseases such as hyperlipidemia, cardiovascular diseases, diabetic nephropathy, diabetic neuropathy and the like. Metabolic syndrome can be treated by diet regulation and exercise, and when these fail to relieve symptoms, medication is required. In the aspect of drug treatment of metabolic syndrome, the prior clinically used hypoglycemic drugs or lipid-lowering drugs have single effect and have ideal effect of improving various pathological indexes of metabolic syndrome when the hypoglycemic drugs or lipid-lowering drugs have different effects. Therefore, research on drugs for improving metabolic syndrome is being conducted in various fields, in order to bring safer and more effective new drugs to patients with metabolic syndrome. Among them, free fatty acid receptor 1(FFA1) agonists and peroxisome proliferator-activated receptor (PPAR) multiple agonists have become hot research points in this field in recent years.

Free fatty acid receptors 1(FFA1) are G protein-coupled receptors, also known as G protein-coupled receptors 40 (GPR 40). It is coupled to the Gq family of G protein α -subunits to perform important functions in various physiological processes. FFA1 is expressed primarily in pancreatic beta cells and increases phospholipase c (plc) activity via the Gq protein to promote insulin secretion. Recent studies have shown that FFA1 is also expressed in the liver, improving hepatic insulin sensitivity. In High Fat Diet (HFD) -induced diabetic mice, activation of FFA1 can lower blood glucose, lower plasma insulin and improve glucose intolerance and insulin resistance. The efficacy and safety of FFA1 agonists have also been demonstrated in type 2 diabetic patients. In addition, it was also found that activation of FFA1 contributes to improvement of high fat diet-induced hepatic steatosis in C57BL/6 mice. Recent studies have shown that FFA1 can be a potential target for anti-organ fibrosis, and the FFA1 agonist PBI-4050 is now in phase II clinical studies of organ fibrosis.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor transcription factor superfamily which regulate the expression of target genes, and the PPARs can be divided into three types of alpha, beta (or delta) and gamma according to the difference of subtype structures, wherein the PPARs are mainly distributed in liver and brown fat and are closely related to the regulation of blood fat level and insulin resistance, namely inflammatory response; PPAR gamma is mainly expressed in adipose tissue and immune system, has close relation with adipocyte differentiation, body immunity and insulin resistance, and is a target of action of Thiazolidinediones (TZDs) serving as insulin sensitizers; PPAR δ is mainly distributed in fat, skeletal muscle, heart and liver, and mainly regulates glycolipid metabolism, improves inflammatory response, and the like. Research has now confirmed that: the PPAR multiple agonist can activate and regulate the expression of related genes, plays an important role in adipogenesis and glycolipid metabolism, and can regulate and control various diseases including obesity, diabetes, hyperlipidemia and the like [ Azadeh Matin and the like, J.Med. chem.2009, 52, 6835-K6850; shen et al, J.Nutr.2006, 899-905 ]. The PPAR alpha/delta dual agonist GFT505 is also in non-alcoholic fatty liver phase III clinical research and shows excellent pharmacological activity (Bertrand Cariou et al, Expert Opin investig. drugs. 2014, 23, 1441-1448). The PPAR alpha/gamma/delta pan-agonist Lanifibranor achieves a primary endpoint and multiple critical secondary endpoints in phase 2b clinical trials, has good overall tolerance, and is currently the only on-going therapy that simultaneously meets the dual histological endpoints established by FDA and EMA of the european union for accelerated NASH drug approval in the united states. The FDA awarded lanifibrane "breakthrough therapy title" for NASH treatment on day 13/10/2020, which was the first NASH treatment drug obtained since 2015. However, the doses used in clinical trials were 800mg and 1200 mg/day, with larger doses.

Due to the improvement effect of FFA1 on hepatic steatosis and fibrosis, the FFA1 can generate a synergistic effect with PPAR-alpha/gamma/delta, so that the FFA 1/PPAR-alpha/gamma/delta quadruple agonist is expected to target complex multiple pathogenesis of fatty liver and improve the treatment effect. Recent studies have shown that the therapeutic effect of FFA 1/PPAR-alpha/gamma/delta quadruple agonist RLA8 on fatty liver model mice is superior to that of the drug obeticholic acid in the research (Meng Hua Li et al, j. pharmacol. exp. ther.2019, 369, 67-77). The inventors have conducted extensive studies on this compound and found that the long chain carboxylic acid of RLA8 is susceptible to β oxidation, making it a significant pharmacokinetic defect. Aiming at the defect of the drug property of RLA8, the inventor purposefully introduces different substituents at the beta position of carboxylic acid to reduce the beta oxidation of long-chain carboxylic acid, and through a large amount of structure-activity relationship researches, the FFA 1/PPAR-alpha/gamma/delta quadruple agonist with better in-vivo curative effect and more stable metabolism is preferably selected.

Disclosure of Invention

The invention aims to provide an FFA 1/PPAR-alpha/gamma/delta quadruple agonist with better curative effect and more stable metabolism, and provides a new potential medicament for preventing or/and treating fatty liver, cholestatic liver disease, liver graft-versus-host disease, virus-induced chronic liver disease, alcoholic liver disease, drug-induced liver injury, diabetes, diabetic complication, prediabetes, hyperlipidaemia, obesity, metabolic syndrome, atherosclerosis, organ fibrosis, inflammation, cancer and other diseases. The inventors have fully themselves made extensive studies, practices and experience to prefer FFA1 and PPAR-alpha/gamma/delta quadruple agonists according to the invention which have unexpected metabolic stability and pharmacological characteristics.

Summary of the invention:

in one aspect, the present invention provides the following compounds (I) or pharmaceutically acceptable salts, prodrugs, esters, and solvates thereof, wherein said salts include pharmaceutically acceptable sodium salts, potassium salts, organic base salts, and the like; prodrugs include pharmaceutically acceptable carboxylic acid esters, amides, and the like.

The invention relates to the use of compounds or pharmaceutically acceptable salts, prodrugs, esters and solvates thereof as FFA1 and PPAR α/γ/δ quadruple agonists.

Another aspect of the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of said compounds or pharmaceutically acceptable salts, prodrugs, esters and solvates thereof, together with a suitable carrier, diluent or excipient.

The invention also relates to the use of the compounds, or pharmaceutically acceptable salts, prodrugs, esters and solvates thereof, for the manufacture of a medicament for the treatment, prevention or alleviation of one or more diseases or disorders selected from fatty liver, cholestatic liver disease, mitochondrial disease, liver graft-versus-host disease, viral-induced chronic liver disease, alcoholic liver disease, pharmaceutical liver injury, diabetes, diabetic complications, prediabetes, hyperlipidemia, obesity, metabolic syndrome, gout, atherosclerosis, organ fibrosis, inflammation and cancer.

The compounds of the present invention can be synthesized by the following steps:

the compound represented by the formula (II) and the compound represented by the formula (III) undergo a condensation reaction under basic conditions to give a compound I (ZLY 18).

As the base, inorganic bases and organic bases are included, and as the inorganic bases, there can be mentioned, for example, alkali metal carbonates such as sodium carbonate, potassium carbonate, cesium carbonate and the like; alkali metal bicarbonates such as potassium bicarbonate and the like; alkali metal hydroxides such as lithium hydroxide, sodium hydroxide, potassium hydroxide, etc.; as the organic base, there may be mentioned, for example, triethylamine, pyridine, lutidine, n-butyllithium, potassium tert-butoxide, sodium methoxide, sodium ethoxide and the like.

Drawings

FIG. 1: ZLY18 induced hyperlipidemia mice by Triton WR-1339Influence of blood lipid index (n is 7, P is less than 0.0001, is relative to the model control group,^####p.ltoreq.0.0001 is the result of a statistical test relative to the positive control (RLA 8).

FIG. 2: the effect of ZLY18 on high fat + high sugar + carbon tetrachloride induced fatty liver model blood lipid and liver lipid. Statistical test results relative to the model control group,^#is a statistical test result relative to the positive control (RLA 8).

FIG. 3: results of liver section HE staining (A), F4/80 immunohistochemistry (B) and NAS activity score (C).

FIG. 4: liver section masson staining (A), alpha-SMA immunohistochemistry (B) and positive area analysis (C).

Detailed Description

The present invention will be further described with reference to the following examples. It should be noted that the following examples are only for illustration and are not intended to limit the present invention. Variations of those skilled in the art in light of the teachings of this invention are intended to be within the scope of the claims appended hereto.

Example 1

(E) -2- (2-fluoro-4- ((3-methoxy-5- (4-methoxystyryl) phenoxy) methyl) phenoxy) acetic acid

1a (0.5g, 1.9mmol) and 1b (0.49g, 1.9mmol) were dissolved in acetonitrile (20mL), potassium carbonate (0.79g, 5.7mmol) was added, the reaction was carried out at 60 ℃ for 4h, after completion of the TLC check, suction filtration was carried out, the residue was purified by column chromatography (petroleum ether/ethyl acetate, 1: 1, v/v) after drying of the mother liquor to give 0.71g of a white solid in 85.2% yield.

¹H NMR(400MHz，DMSO-d₆)δ7.53(d，J＝8.1Hz，2H)，7.36-7.09(m，3H)，7.10-6.91(m，4H)，6.84(s，1H)，6.75(s，1H)，6.47(s，1H)，5.02(s，2H)，4.36(s，2H)，3.77(s，6H).¹³C NMR(75MHz，DMSO-d₆)δ170.78， 161.12，161.09，160.09，159.52，139.93，130.01，130.01，129.43，129.35，129.12，128.36，126.54，120.05，114.90， 114.68，110.45，105.39，105.38，102.53，100.73，68.99，68.00，55.69，55.63.ESI-MS m/z：437.1[M-H]^-.Anal. calcd.For C₂₅H₂₃FO₆：C，68.49；H，5.29；Found：C，68.57；H，5.21.

Example 2

FFA1 and PPAR agonistic activity, metabolic stability, in vivo lipid lowering, anti-fatty liver and anti-fibrotic activity of the compounds of the present invention can be determined by using the assay system described below.

The following description of the biological test example illustrates the present invention.

The experimental procedures for the specific conditions in the test examples of the present invention are generally carried out under conventional conditions or under conditions recommended by commercial manufacturers. Reagents with no specific source are indicated, and are commonly purchased in the market.

Test example 1 the agonistic activity of the compounds of the invention on hFFA1-CHO stably transfected cells and PPAR

The present invention uses the following method to determine the agonistic activity of the compound of the present invention FFA 1:

hFFA1-CHO stably transfected cells at 3X 10⁴The density of each well was inoculated into a 96-well plate and placed at 37 ℃ in 5% CO₂The cell culture box is used for overnight culture; discarding the culture medium, adding 100ul HBSS into each well, cleaning, adding 100ul Fluo-4 dye solution containing Probenecid, and incubating at 37 deg.C for 90 min; after the incubation is finished, sucking out Fluo-4 dye solution, adding 100 mu l of HBSS buffer solution, and washing off the dye; adding 100 μ l HBSS containing Probenecid into each well, and incubating at 37 deg.C for 10 min; drugs were added at different concentrations to each well of a 96-well plate and read using flipr (molecular devices) according to the parameter set-up table. And analyzing the experimental result. Agonistic activity ═ (compound well fluorescence value-blank well fluorescence value)/(linoleic acid well fluorescence value-blank well fluorescence value) × 100%, the results are shown in table 1.

The present invention measures the agonistic activity of the PPAR of the present compound using the following method:

transfection: HEK293 cells at 5X 10 before transfection⁴The density of each well was inoculated into a 96-well plate and placed at 37 ℃ in 5% CO₂One day (for PPAR γ and PPAR δ transfections); HepG2 cells at 6X 10⁴The density of each well was inoculated into a 96-well plate and placed at 37 ℃ in 5% CO₂One day (for PPAR α transfection); transfection was performed with FuGENE HD transfection reagent (purchased from Roche) separately: 25ng/well pBIND-PPAR α or PPAR δ or PPAR γ, 25ng/well pG5Luc and 0.15 μ l/well FuGENE HD.

Agonist activity assay: after 24h transfection, the test compound was added to the transfected cell well plate, incubated for 18h, lysed by adding 20. mu.l of cell lysate and 30. mu.l of luciferase assay reagent II (purchased from Promega), mixed well, assayed for fluorescence, delayed for 2 seconds, and read for 10 seconds. Transfection efficiency was corrected using the internal reference Renilla luciferase activity. All transfection experiments were repeated at least three times independently, at least 2 replicates per experimental group. Relative fluorescence intensity ═ firefly fluorescence intensity/nephrotic fluorescence intensity. PPAR agonistic activity (%) [ (X-Min)/(Max-Min) ] × 100%, where X represents the relative fluorescence intensity of the compound group, Min represents the relative fluorescence intensity of the blank control group, and Max represents the relative fluorescence intensity of the positive control compound group at a concentration of 10 μ M. The agonist activities of the example compounds PPAR α, PPAR δ and PPAR γ are shown in table 1.

Table 1: FFA1 and PPAR agonist activity

And (4) conclusion: the compounds of the invention have relatively balanced agonist activity against FFA1, PPAR α, PPAR γ and PPAR δ, with in vitro activity comparable to that of RLA 8.

Test example 2 in vitro hepatic microparticle metabolic stability of the Compound of the present invention

Rat liver microsomes (0.25mg/ml) and test compound (500ng/ml) were mixed well in 0.1M PBS buffer (pH 7.4), preincubated at 37 ℃ for 5 minutes, and then NADPH was added to catalyze the metabolic reaction according to the instructions of the metabolic stability kit. After co-incubation at 37 ℃ for various times (0, 15, 30 and 60 min), cold methanol with internal standard was added and vortexed for 3 min, the reaction was stopped, and the free drug concentration in the supernatant was measured by LC/MS and the half-life and clearance calculated, the results are shown in table 1.

Table 2: the compounds of the invention are stable to in vitro liver microparticle metabolism.

And (4) conclusion: compared with RLA8, the compound ZLY18 has better metabolic stability and 2.6-fold prolonged metabolic half life.

Test example 3 evaluation of lipid-lowering Activity

ICR mice of 8 weeks of age were randomly divided into a normal group, a vehicle control group (blank vehicle: 0.5% sodium carboxymethylcellulose solution), and a test compound group (80 mg/kg). The group is administered by intragastric administration for 5 days, and injected with Triton WR-1339600 mg/kg to the abdominal cavity on day 4 to prepare model for hyperlipidemia, and administered for 2 hr on day 5, and then blood is taken, and the content of TG (triglyceride) and TC (total cholesterol) in serum is determined by kit method, and the result is shown in figure 1. Results of experiments on hyperlipidemia induced by Triton WR-1339 show that: the blood fat reducing effect of ZLY18 in vivo is far stronger than that of RLA8, and the blood fat level of ZLY18 treatment group mice is unexpectedly found to reach a normal state.

Test example 4 in vivo anti-fatty liver and hepatic fibrosis activities of the compounds of the present invention can be determined by using an assay system as described below:

8-week-old C57BL/6 mice, male, randomly divided into 4 groups of 7 mice each, blank control group (blank vehicle: 0.5% sodium carboxymethylcellulose solution), model group, positive control RLA8 group (100mg/kg), test compound ZLY18 group (100mg/kg), western diet (Teklad diets, TD.120528) feed supplemented with 23.1g/L fructose and 18.9g/L glucose in drinking water, and intraperitoneally injected with CCl₄Olive oil solution (0.2 μ L/g) twice weekly for 12 weeks. After 8 weeks from the start of the experiment, the test compound and the vehicle were administered once a day by gavage for 4 weeks simultaneously with continuous molding. After the last administration, blood plasma was collected from mice after fasting for 12hMeasuring blood lipid level of mouse with full-automatic biochemical analyzer, and measuring TG (triglyceride), TC (total cholesterol) and free fatty acid (NEFA) content in liver according to kit instruction method, and the result is shown in figure 2; the liver tissues were taken and examined for improvement of fatty liver and hepatic fibrosis by HE staining, Masson staining, F4/80 and alpha-SMA, and the staining results are shown in FIG. 3 and FIG. 4.

The experimental results show that: after long-term administration of non-alcoholic fatty liver model mice, ZLY18 has stronger effects of improving blood lipid and liver lipid deposition than RLA8 (figure 2); the results of HE staining of liver tissues (fig. 3) show that ZLY18 significantly improved the degree of hepatic steatosis, inflammatory infiltration and ballooning lesions, while the NAS score of the liver was significantly reduced compared to RLA 8. F4/80 staining shows that ZLY18 has stronger inflammation improving effect; the results of the liver tissue masson staining and alpha-SMA staining (FIG. 4) show that the degree of liver fibrosis of the ZLY 18-administered group was more significantly improved than that of RLA 8. In conclusion, the effect of ZLY18 on improving nonalcoholic fatty liver and fibrosis is obviously better than that of RLA8, and the medicine has wider medicinal development prospect.