CN112759515B

CN112759515B - Novel FFA1 and PPAR alpha/gamma/delta quadruple agonist, preparation method thereof and application thereof as medicine

Info

Publication number: CN112759515B
Application number: CN202011595384.8A
Authority: CN
Inventors: 李政; 张陆勇; 周宗涛
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2023-06-13
Anticipated expiration: 2040-12-28
Also published as: CN112759515A

Abstract

The invention relates to a novel FFA1 and PPAR-alpha/gamma/delta quadruple agonist shown in a formula (I), a preparation method thereof and a pharmaceutical composition containing the compound, which are used for preparing medicines for treating, preventing or relieving one or more diseases or dysfunctions.

Description

Novel FFA1 and PPAR alpha/gamma/delta quadruple agonist, preparation method thereof and application thereof as medicine

Technical Field

The invention relates to a novel FFA1 and PPAR alpha/gamma/delta quadruple agonist, a preparation method and application thereof, and belongs to the technical field of medicines. The structures of the compounds referred to 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 elevation and high density lipoprotein cholesterol reduction, and common diseases are obesity, diabetes, hyperlipidemia, atherosclerosis, fatty liver and the like, wherein 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 dietary regulation and exercise, and when these fail to relieve symptoms, medication is required. In the aspect of the drug treatment of the metabolic syndrome, the blood sugar-reducing drugs or lipid-lowering drugs used clinically at present have single effect and have different ideal effects of improving various pathological indexes of the metabolic syndrome. Therefore, research on drugs for improving metabolic syndrome is ongoing in various fields in an effort to bring safer and more effective new drugs for patients with metabolic syndrome. Among them, free fatty acid receptor 1 (FFA 1) agonists and peroxisome proliferator-activated receptor (PPAR) multiple agonists are the focus of research in this field in recent years.

The free fatty acid receptor (Free fatty acid receptors, ffa 1) is a G protein-coupled receptor, also known as G protein-coupled receptor 40 (G protein coupled receptor, gpr 40). Which is coupled to the alpha-subunit of the Gq family of G proteins to perform important functions in a variety of physiological processes. FFA1 is expressed primarily in pancreatic beta cells and can increase phospholipase C (PLC) activity via Gq protein to promote insulin secretion. Recent studies have shown that FFA1 is also expressed in the liver, improving liver insulin sensitivity. In High Fat Diet (HFD) induced diabetic mice, FFA1 activation can lower blood glucose, lower plasma insulin and improve glucose intolerance and insulin resistance. Efficacy and safety of FFA1 agonists were also demonstrated in type 2 diabetics. In addition, the study also found that activation of FFA1 helped improve liver steatosis in high fat diet induced C57BL/6 mice. Recent studies have shown that FFA1 can be a potential target against organ fibrosis, and FFA1 agonist PBI-4050 is now in clinical studies of organ fibrosis phase II.

Peroxisome proliferator-activated receptors (peroxisome proliferator-activated receptor, PPARs) are members of the nuclear receptor transcription factor superfamily that regulate target gene expression, and PPARs can be classified into three types, α, β (or δ) and γ, depending on subtype structures, wherein pparα is mainly distributed in the liver and brown fat, and is closely related to regulation of blood lipid levels, insulin resistance, i.e., inflammatory response; PPARgamma is mainly expressed in adipose tissues and immune systems, has close relation with adipocyte differentiation, organism immunity and insulin resistance, and is a target of the action of an insulin sensitizer Thiazolidinedione (TZDs); pparδ is mainly distributed in fat, skeletal muscle, heart and liver, and mainly regulates glycolipid metabolism, improves inflammatory response, and the like. The research has now confirmed that: PPAR multiple agonists are capable of activating and regulating the expression of related genes, playing an important role in adipogenesis, glycolipid metabolism, and regulating a variety of diseases including obesity, diabetes, hyperlipidemia, etc. (Azadeh matrix, etc., j.med.chem.2009, 52, 6835-6850; shen et al, j.nutr.2006, 899-905]. The PPARα/δ dual agonist GFT505 is also in clinical studies of non-alcoholic fatty liver III, exhibiting superior pharmacological activity (Bertrand Cariou et al, expert Opin. Invest. Drugs.2014, 23, 1441-1448). The PPAR alpha/gamma/delta pan agonist laniferror reached a primary endpoint and a number of critical secondary endpoints in phase 2b clinical trials, and overall tolerability was good, being the only current treatment under investigation that satisfied both the U.S. FDA and eu EMA as dual histological endpoints for accelerated NASH drug approval. The FDA granted the "breakthrough therapy designation" to lanibror for NASH at 10 months 13 in 2020, which was the first NASH treatment drug to obtain this designation since 2015. However, the doses used in the clinical trials were 800mg and 1200 mg/day, with larger doses.

In view of the improvement effect of FFA1 on liver 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 the 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 in-flight drug obeticholic acid [ Meng Hua Li et al, J.Pharmacol.exp. Ther.2019, 369, 67-77]. The inventor has conducted a great deal of research on the compound, and found that the long-chain carboxylic acid of RLA8 is easy to undergo beta oxidation, so that the compound has obvious pharmacokinetic defects. Aiming at the RLA8, the inventor purposefully introduces different substituents at the beta position of carboxylic acid to reduce beta oxidation of long-chain carboxylic acid, and through a great amount of structure-activity relationship researches, FFA 1/PPAR-alpha/gamma/delta quadruple agonists with better in-vivo curative effect and more stable metabolism are preferred.

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, chronic liver disease caused by viruses, alcoholic liver disease, drug-induced liver injury, diabetes, diabetic complications, prediabetes, hyperlipidemia, obesity, metabolic syndrome, atherosclerosis, organ fibrosis, inflammation, cancer and other diseases. The inventors have, through extensive research, practice and experience in and of themselves, preferably found that FFA1 and PPAR-alpha/gamma/delta quadruple agonists according to the invention 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 the 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 present invention relates to the use of compounds or pharmaceutically acceptable salts, prodrugs, esters, and solvates thereof as FFA1 and PPAR alpha/gamma/delta quadruple agonists.

Another aspect of the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of the compound, or a pharmaceutically acceptable salt, prodrug, ester, and solvate thereof, in combination with a suitable carrier, diluent, or excipient.

The invention also relates to the use of said 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 dysfunctions selected from the group consisting of fatty liver, cholestatic liver disease, mitochondrial disease, liver graft versus host disease, viral induced chronic liver disease, alcoholic liver disease, drug induced liver injury, diabetes, diabetic complications, pre-diabetes, hyperlipidemia, obesity, metabolic syndrome, gout, atherosclerosis, organ fibrosis, inflammation and cancer.

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

and (3) carrying out condensation reaction on the compound shown in the formula (II) and the compound shown in the formula (III) under alkaline conditions to obtain a compound I (ZLY 18).

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

Drawings

Fig. 1: influence of ZLY18 on hyperlipidemia induced mouse blood lipid index by Triton WR-1339

n=7), p.ltoreq.0.0001 is the statistical test result with respect to the model control group, ^#### p.ltoreq.0.0001 is the result of the statistical test with respect to the positive control (RLA 8).

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

Fig. 3: liver sections were HE stained (A), F4/80 immunohistochemistry (B) and NAS activity scoring (C) results.

Fig. 4: liver sections were stained with masson (a), a-SMA immunohistochemistry (B) and positive area analysis (C) results.

Detailed Description

The invention is further illustrated below with reference to examples. It should be noted that the following examples are given by way of illustration only and are not intended to limit the present invention. Variations that occur to those skilled in the art in light of the teachings of the present invention are intended to be within the scope of the claims of the present application.

Example 1

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

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

¹ H NMR(400MHz，DMSO-d ₆ )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). ¹³ C NMR(75MHz，DMSO-d ₆ )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 C ₂₅ H ₂₃ FO ₆ ：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 invention can be measured by using the assay system described below.

The following biological test examples illustrate the invention.

The experimental methods for the specific conditions in the test cases of the present invention are generally conventional or according to the conditions recommended by the commercial manufacturers. The reagents of specific origin are not noted and are commonly used reagents purchased in the market.

Test example 1 agonistic Activity of the inventive Compounds against hFFA1-CHO stable cells and PPARs

The present invention uses the following method to determine the agonistic activity of the compound FFA1 of the present invention:

hFFA1-CHO stable cells at 3X 10 ⁴ Density of wells/density of wells was seeded in 96-well plates at 37℃in 5% CO ₂ Overnight culture in a cell incubator; the medium was discarded, 100ul of HBSS was added to each well and washed, and then 100ul of Fluo-4 dye solution containing Probenecid was added thereto and incubated at 37℃for 90min; after the incubation, the Fluo-4 dye solution was aspirated, 100. Mu.l of HBSS buffer was added, and the dye was washed away; 100 μl HBSS containing Probenecid was added to each well and incubated at 37deg.C for 10min; different concentrations of drug were added to each well of the 96-well plate and read FLIPR (Molecular Devices) according to the parameter set-up table. And analyzing the experimental result. Agonist activity = (compound pore fluorescence value-blank Kong Yingguang value)/(linoleic acid pore fluorescence value-blank Kong Yingguang value) ×100% and the results are shown in table 1.

The present invention uses the following methods to determine the agonistic activity of the compound PPAR of the invention:

transfection: HEK293 cells were grown at 5X 10 prior to transfection ⁴ Density of wells/density of wells was seeded in 96-well plates at 37℃in 5% CO ₂ One day (for pparγ and pparδ transfection) in cell culture; hepG2 cells at 6X 10 ⁴ Density of wells/density of wells was seeded in 96-well plates at 37℃in 5% CO ₂ One day (for pparα transfection); transfection was performed with FuGENE HD transfection reagent (available from Roche), respectively: 25ng/well pBIND-PPARα or PPARδ or PPARγ,25ng/well pG5Luc and 0.15 μl/well FuGENE HD.

Agonist activity assay: after 24h of transfection, the test compound was added to the post-transfection cell well plate, incubated for 18h, lysed by addition of 20. Mu.l of cell lysate and 30. Mu.l of luciferase assay reagent II (from Promega), mixed well, fluorescence measured, 2 seconds delay, read for 10 seconds. Transfection efficiency was corrected using the reference Renilla luciferase activity. All transfection experiments were independently repeated at least three times, at least 2 duplicate wells per experimental group. Relative fluorescence intensity = firefly fluorescence intensity/renilla fluorescence intensity. PPAR agonist activity (%) = [ (X-Min)/(Max-Min) ] ×100%, where X represents compound group relative fluorescence intensity, min represents blank group relative fluorescence intensity, max represents positive control compound group relative fluorescence intensity at a concentration of 10 μm. The pparα, pparδ and pparγ agonistic activities of the example compounds are shown in table 1.

Table 1: FFA1 and PPAR agonistic Activity

Conclusion: the compounds of the invention have relatively balanced agonistic activity on FFA1, pparα, pparγ and pparδ, and in vitro activity is comparable to RLA 8.

Test example 2 in vitro liver microparticle metabolic stability of the inventive Compounds

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

Table 2: in vitro liver microparticle metabolic stability of the compounds of the present invention.

Conclusion: compared with RLA8, the compound ZLY18 has better metabolic stability and prolonged metabolic half-life by 2.6 times.

Test example 3 evaluation of lipid-lowering Activity

ICR mice at 8 weeks of age were randomized into the normal group, vehicle control group (blank vehicle: 0.5% sodium carboxymethyl cellulose solution) and test compound group (80 mg/kg). Each group was subjected to intragastric administration for 80mg/kg for 5 consecutive days and to intraperitoneal injection of Triton WR-1339 600mg/kg on day 4 to prepare a hyperlipidemia model, blood was collected 2 hours after the administration on day 5, and the TG (triglyceride) and TC (total cholesterol) contents in serum were measured by the kit method, and the results are shown in FIG. 1. The experimental result of Triton WR-1339 induced hyperlipidemia shows that: the blood lipid reducing effect of ZLY18 in vivo is far stronger than that of RLA8, and it is unexpectedly found that the blood lipid level of ZLY 18-treated mice reaches a normal state.

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

c57BL/6 mice of 8 weeks old, males were randomly divided into 4 groups of 7, blank control group (blank vehicle: 0.5% sodium carboxymethyl cellulose solution), model group, positive control RLA8 group (100 mg/kg), test compound ZLY18 group (100 mg/kg), and drinking water given western diet (Teklad diet, TD.120528) supplemented with 23.1g/L fructose and 18.9g/L glucose, while intraperitoneally injecting CCl ₄ Olive oil solution (0.2 μl/g) twice weekly for 12 weeks. After 8 weeks from the start of the experiment, blank vehicle and test compound were administered by intragastric administration once daily, respectively, and the administration was continued for 4 weeks while molding. After the last administration, the mice are taken blood and plasma is taken after 12h of fasting, the blood fat level of the mice is measured by a full-automatic biochemical analyzer, and the contents of TG (triglyceride), TC (total cholesterol) and free fatty acid (NEFA) in the liver are measured according to the method of a kit instruction, and the result is shown in the figure 2; liver tissue is respectively dyed by HE, pinus massoniana and F4/80 and alpha-SMA to observe fatty liver and liver fibrosis improvement, and the dyeing results are shown in figure 3 and figure 4.

The experimental results show that: after the non-alcoholic fatty liver model mice are administrated for a long time, ZLY18 has stronger effect of improving blood lipid and liver lipid deposition than RLA8 (figure 2); the results of HE staining of liver tissue (figure 3) showed that ZLY18 was more significantly improved in liver steatosis, inflammatory infiltrate and balloon-like lesions than RLA8, while the liver NAS score was significantly reduced. F4/80 staining shows that ZLY18 has stronger effect of improving inflammation; the results of the masson staining and the alpha-SMA staining (fig. 4) of liver tissue showed a more significant improvement in liver fibrosis compared to RLA8, ZLY 18-dosed group. In conclusion, the effect of ZLY18 in improving non-alcoholic fatty liver and fibrosis is obviously better than that of RLA8, and the method has wider medicinal development prospect.

Claims

1. A compound (I) or a pharmaceutically acceptable salt thereof:

。

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

3. The use of compound (I) as claimed in claim 1 or a pharmaceutically acceptable salt thereof as FFA1 and PPAR-alpha/gamma/delta quadruple agonists.

4. Use of compound (I) or a pharmaceutically acceptable salt thereof as claimed in claim 1 for the manufacture of a medicament for the treatment, prevention or alleviation of one or more diseases or disorders selected from the group consisting of fatty liver, cholestatic liver disease, mitochondrial disease, liver graft versus host disease, virally induced chronic liver disease, alcoholic liver disease, drug induced 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 comprises non-alcoholic fatty liver disease, primary cholangitis, primary sclerosing cholangitis, alcoholic fatty liver disease.