WO2019120257A1 - 作为ppar激动剂的吡咯烷衍生物的无定形及其制备方法 - Google Patents

作为ppar激动剂的吡咯烷衍生物的无定形及其制备方法 Download PDF

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WO2019120257A1
WO2019120257A1 PCT/CN2018/122423 CN2018122423W WO2019120257A1 WO 2019120257 A1 WO2019120257 A1 WO 2019120257A1 CN 2018122423 W CN2018122423 W CN 2018122423W WO 2019120257 A1 WO2019120257 A1 WO 2019120257A1
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compound
amorphous
formula
stirring
solvent
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PCT/CN2018/122423
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English (en)
French (fr)
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袁之渿
江志赶
贺海鹰
张晓�
许继文
周卫祥
黎健
陈曙辉
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广东众生睿创生物科技有限公司
南京明德新药研发股份有限公司
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Priority to CN201880070995.5A priority Critical patent/CN111356676B/zh
Priority to RU2020117795A priority patent/RU2749056C1/ru
Priority to AU2018391211A priority patent/AU2018391211B2/en
Priority to EP18893065.5A priority patent/EP3696166B1/en
Priority to KR1020207015135A priority patent/KR102397011B1/ko
Priority to JP2020529232A priority patent/JP6969001B2/ja
Priority to US16/759,672 priority patent/US11396493B2/en
Priority to CA3080091A priority patent/CA3080091C/en
Publication of WO2019120257A1 publication Critical patent/WO2019120257A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/08—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon radicals, substituted by hetero atoms, attached to ring carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/402—1-aryl substituted, e.g. piretanide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/06—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with radicals, containing only hydrogen and carbon atoms, attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • the present invention relates to an amorphous form of a pyrrolidine derivative as a PPAR agonist and a process for the preparation thereof.
  • Nonalcoholic fatty liver disease is the most common liver disease in developed countries. It means that excess fat accumulates in the liver in the form of triglycerides (steatosis >5% hepatocyte tissue). ). In addition to excess fat, patients with NAFLD are associated with hepatocyte damage and inflammation (steatohepatitis), the latter being NASH (Nonalcoholic steatohepatitis). There was no correlation between simple steatosis in NAFLD and short-term morbidity or mortality, but once advanced to NASH, the risk of cirrhosis, liver failure, and hepatocellular carcinoma (HCC) was significantly increased. Liver cirrhosis due to NASH is a cause of increasing liver transplantation.
  • NASH nonalcoholic steatohepatitis
  • NASH nonalcoholic fatty liver disease
  • NAFLD nonalcoholic fatty liver disease
  • Most patients with NAFLD are male, elderly, hypertensive and diabetic. 60-76% of people with diabetes have NAFLD and 22% have NASH.
  • Pediatric patients with NAFLD are also growing year by year, and 38-53% of obese children have NAFLD. In China, the incidence of nonalcoholic fatty liver disease has increased to the first.
  • Peroxisome proliferator-activated receptor is a member of the nuclear hormone receptor superfamily, a ligand-activated transcription factor that regulates gene expression, mainly in three subtypes: PPAP Alpha is mainly in brown fat Expression in tissues, liver, heart and skeletal muscle plays a major role in the metabolism of bile acids, lipids and sugars; PPAP Delta has no specific expression and may have anti-inflammatory effects; Gamma has a certain effect on insulin resistance.
  • the receptor is associated with a variety of disease states, including dyslipidemia, hyperlipidemia, hypercholesterolemia, atherosclerosis, atherogenesis, hypertriglyceridemia, heart failure, myocardial infarction, blood vessels Disease, cardiovascular disease, hypertension, obesity, inflammation, arthritis, cancer, Alzheimer's disease, skin disease, respiratory disease, eye condition, IBD (stress bowel disease), ulcerative colitis and gram Ron disease.
  • PPAR agonists are one of the most effective potential drugs for the treatment of fatty liver from the various mechanisms of PPAR beneficial to liver function.
  • the following compounds are PPAR agonist compounds which have been reported in the literature.
  • a first object of the present invention is to provide an amorphous form of the compound of the formula (I), which has considerable stability and further has a certain pharmaceutical prospect, from the viewpoint of solving the deficiencies of the prior art.
  • the development of the compounds shown in I) as a clinical drug provides a viable choice of drug substance.
  • the amorphous form of the compound of formula (I) is characterized by the absence of sharp diffraction peaks in the amorphous X-ray powder diffraction pattern (XRPD).
  • Amorphous belongs to the thermodynamic high energy state and is a thermodynamic metastable structure.
  • the basic particles constituting the compound appear as disordered arrangement in three dimensions, and the X-ray powder diffraction spectrum is judged to be amorphous.
  • the amorphous X-ray powder diffraction pattern of the compound of the above formula (I) has a broad and gentle diffraction peak at a 2 ⁇ angle of 10 to 25°.
  • amorphous X-ray powder diffraction pattern of the compound of the formula (I) is shown in Fig. 1.
  • the amorphous form described above has a differential scanning calorimetry (DSC) having a starting point for two endothermic peaks at 69.28 ⁇ 3 ° C and 239.33 ⁇ 3 ° C.
  • DSC differential scanning calorimetry
  • the above amorphous form has a DSC spectrum as shown in FIG.
  • the amorphous form described above has a thermogravimetric analysis curve (TGA) having a weight loss of 0.9958% at 120.00 ⁇ 3 °C.
  • TGA thermogravimetric analysis curve
  • the amorphous form described above has a TGA pattern as shown in FIG.
  • a second object of the present invention is to provide a process for the preparation of an amorphous compound of the formula (I) which comprises adding a compound of the formula (I) to a solvent, heating or stirring or recrystallizing; From: methanol, ethanol, tetrahydrofuran, ethyl acetate and n-heptane, the stirring temperature of the heating and stirring is 25 ° C to 45 ° C, and the heating and stirring (beating) time is 2 hours to 48 hours, in the preparation method
  • the mass/volume ratio of the compound to the solvent was 1:3.5 to 6 g/mL.
  • the method has stable process, mild reaction conditions and easy availability of raw materials, and can be used for large-scale industrial production of the amorphous form of the compound of the formula (I).
  • the inventors have surprisingly found that, unlike conventional defects in the form of amorphous forms, such as poor stability and poor pharmaceutical properties, the amorphous compound of the formula (I) mentioned in the present invention has high stability and specific expression.
  • the amorphous form of the compound of the formula (I) has high stability under high temperature, high humidity and the like, and based on the existing stability data, it can be judged that the amorphous form of the compound of the formula (I) has certain medicinal prospects;
  • the amorphous form of the compound of the formula (I) referred to in the present invention has an obvious inhibitory effect on the cytokine of the PPAR-associated pathway, and is induced by CCl 4 in the C57BL/6 mouse acute liver injury test and the MCD diet-induced db/
  • the db mouse NASH model found that the compound of formula (I) had a significant effect on the improvement of liver damage, NAS Score and liver fibrosis.
  • the amorphous form of the compound of the formula (I) of the present invention has good stability and has certain medicinal prospects; therefore, if the compound represented by the formula (I) is proved by the detection means in the drug substance and/or Part or all of the formulation product is present in an amorphous form and should be considered as amorphous using the compound of formula (I) provided by the present invention.
  • the detecting means may further comprise, in addition to the aforementioned X-ray powder diffraction, differential scanning calorimetry (DSC), infrared spectroscopy (IR), Raman spectroscopy (Raman), solid nuclear magnetic resonance ( The method of SSNMR) and everything else can support the amorphous detection method using the compound of the formula (I) of the present invention, and can remove effects such as pharmaceutical excipients, etc., by methods commonly used by those skilled in the art, such as Subtraction map method, etc.
  • intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, combinations thereof with other chemical synthesis methods, and those skilled in the art.
  • Well-known equivalents, preferred embodiments include, but are not limited to, embodiments of the invention.
  • the solvent used in the present invention is commercially available.
  • the present invention employs the following abbreviations: DCM stands for dichloromethane; DMF stands for N,N-dimethylformamide; DMSO stands for dimethyl sulfoxide; EtOH stands for ethanol; MeOH stands for methanol; TFA stands for trifluoroacetic acid; TsOH stands for P-toluenesulfonic acid; mp represents melting point; EtSO 3 H represents ethanesulfonic acid; MeSO 3 H stands for methanesulfonic acid; ATP stands for adenosine triphosphate; HEPES stands for 4-hydroxyethylpiperazineethanesulfonic acid; EGTA stands for ethylene glycol double (2 -Aminoethyl ether)tetraacetic acid; MgCl 2 represents magnesium dichloride; MnCl 2 represents manganese dichloride; DTT represents dithiothreitol.
  • Test method Approximately 10-20 mg samples were used for XRPD detection.
  • Test method The sample ( ⁇ 1 mg) was placed in a DSC aluminum pan for testing under the condition of 50 mL/min N 2 , heating the sample from 25 ° C to 350 ° C, and the heating rate was 10 ° C / min.
  • Test method Take the sample (2 ⁇ 5mg) and put it into the TGA platinum pot for testing.
  • the method is: heating the sample from room temperature to -350 °C at a heating rate of 10 °C/min under the condition of 25 mL/min N 2 The rate is 10 ° C / min.
  • Figure 1 is an XRPD spectrum of amorphous Cu-K alpha radiation of a compound of formula (I).
  • Figure 2 is an amorphous DSC spectrum of the compound of formula (I).
  • Figure 3 is an amorphous TGA spectrum of the compound of formula (I).
  • Acetonitrile (30 L) was added to a 50 L autoclave at 25 ° C, stirring was started, then Compound A (2.00 kg, 13.32 mol, 1.0 eq), ethyl bromoisobutyrate (7.79 kg, 39.95 mol, 3.0 eq) was added. And potassium carbonate (5.52 kg, 39.95 mol, 3.0 eq).
  • the reaction solution was stirred at 80 ° C for 16 hours. The reaction temperature was lowered to 25 ° C, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was dissolved in ethyl acetate (5L). The filter cake was washed with ethyl acetate (5 L x 2) and ethyl acetate was combined.
  • Hydrogen chloride gas (3.67 kg, 100.54 mol, 5.3 eq) was introduced into ethanol (12 L) under a dry ice ethanol bath (-60 ° C), and the temperature of the system was controlled below 0 ° C.
  • Ethanol (13 L) and a freshly prepared hydrogen chloride ethanol solution were added to a 50 L reactor, and the mixture was stirred, and the temperature was naturally raised to 25 °C.
  • Compound B (5.01 kg, 18.97 mol, 1.0 eq) was then added. After the starting material was completely dissolved, p-methylthioacetophenone (2.83 kg, 17.07 mol, 0.9 eq) was added in portions. The mixture was stirred at 25 ° C for 16 hours.
  • N,N-dimethylformamide (15 L) to a 50 L reactor, start stirring, add trimethylsulfoxonium iodide (3.78 kg, 16.01 mol 1.2 eq), then cool to 0 ° C, add unseen in batches. Potassium butoxide (1.79 kg, 16.01 mol, 1.2 eq). After stirring at 0 °C for 30 minutes, a solution of compound C (5.5 kg, 13.34 mol, 1.0 eq) in N,N-dimethylformamide (15L) was slowly added. The mixture was stirred at 0 ° C for 2 hours.
  • reaction solution was slowly poured into ice water (0-5 ° C, 30 L), and then extracted with petroleum ether / ethyl acetate (1:1, 10L ⁇ 3). The combined organic phases were washed with EtOAc EtOAc EtOAc.
  • Ethanol (35.0 L) was added to a dry 50 liter reactor, stirring was started, then compound D (5.45 kg, 12.79 mol, 1.0 eq) and glacial acetic acid (2.30 kg, 38.37 mol, 3.0 eq) were added. After heating the reaction mixture to 80 ° C, zinc powder (2.45 kg, 38.37 mol, 3.0 eq) was added in portions. The resulting suspension was stirred at 80 ° C for an additional 16 hours. The reaction mixture was filtered, and then filtered and evaporated. The concentrate was dissolved in ethyl acetate (10 L) and taken to a 50L sep. funnel.
  • reaction solution was poured into a 50 L separatory funnel, washed with 5% aqueous sodium carbonate (10 L ⁇ 2) and saturated aqueous sodium chloride (10L ⁇ 2), dried over anhydrous sodium sulfate , 3.92 kg of compound G was obtained.
  • the concentrated liquid was pumped into a 50 L separatory funnel, and the stirring was started, and then ethyl acetate (20 L) was added thereto, and a 10% aqueous potassium hydrogensulfate solution (10 L ⁇ 2) and a saturated aqueous sodium chloride solution (10 L ⁇ 2) were used. It was washed with anhydrous sodium sulfate (1.5 kg) and evaporated. It was concentrated to about 8 L of solvent remaining, and a large amount of solid was precipitated, and concentration was stopped to fall to 25 °C. The concentrated suspension was filtered, and the filter cake was washed with ethyl acetate (2L.times.3), dried, and dried under reduced pressure in vacuo to give 2.44 kg of Compound H, yield: 89.85%.
  • Chiral separation conditions chiral column: Chiralpak AD-3 100 x 4.6 mm I.D., 3 um; mobile phase: 40% methanol (0.05% DEA) - CO2; flow rate: 4 mL/min; column temperature: 40 °C.
  • Compound I corresponds to a retention time of 1.604 minutes.
  • Chiral separation conditions chiral column: Chiralpak AD-3 100 x 4.6 mm I.D., 3 um; mobile phase: 40% methanol (0.05% DEA)-CO2; flow rate: 4 mL/min; column temperature: 40 °C.
  • Compound J corresponds to a retention time of 1.576 minutes.
  • lithium hydroxide (165.60 g, 3.94 mol, 3.0 eq) was added and stirring was continued at 25 °C for 20 minutes.
  • the reaction mixture was separated, EtOAc m m m m m m m
  • the concentrate was dissolved in ethyl acetate (1.5 L) then n-heptane (5.6L) was slowly added with stirring. Stirring was continued for 30 minutes after the addition and filtration.
  • the filter cake was added to n-heptane / ethyl acetate (4:1, 3.5 L x 3), and then stirred for 30 minutes with rapid stirring and filtered.
  • the obtained filter cake was dissolved in tert-butyl methyl ether (5 L), washed successively with 5% aqueous potassium hydrogensulfate solution (1.5 L ⁇ 2), deionized water (1 L ⁇ 2), dried over anhydrous sodium sulfate (300 g), filtered, reduced Concentrated by pressure. The resulting solid is dried in a vacuum oven (40-45 ° C) to give the compound of formula (I).
  • Chiral separation conditions chiral column: Chiralpak AD-3 100 x 4.6 mm I.D., 3 um; mobile phase: 40% of methanol (0.05% DEA) in CO2; flow rate: 2.8 mL/min; column temperature: 40 °C.
  • the crude light yellow solid formula (I) compound (310.5g) was added to a 3L reaction bottle, then n-heptane (1500mL) was added, the addition was completed, the reaction was stirred at 25 ° C for 2h, filtered, filter cake It was washed with n-heptane (500 mL), then filtered to give a crude material.
  • the crude product was dried in a vacuum oven, and the morphology was determined by XRPD.
  • the XRPD spectrum of Cu-K ⁇ radiation of the obtained final product is shown in Fig. 1; the DSC spectrum is shown in Fig. 2; and the TGA spectrum is shown in Fig. 3.
  • the compound of the formula (I) (40.0 mg) was weighed into a 4.0 mL glass vial, and 150 ⁇ L of ethyl acetate was added to make it a turbid liquid. After stirring at 40 ° C for 2 days on a magnetic stirrer, the sample was centrifuged, and the supernatant was taken out in a fume hood to evaporate until the solvent evaporated. The resulting solid was then dried in a vacuum oven at 40 ° C overnight. The resulting final product was the same amorphous form as in Example 1.
  • the compound of the formula (I) (39.9 mg) was weighed into a 4.0 mL glass vial, and 150 ⁇ L of tetrahydrofuran was added to make it a turbid liquid. After stirring at 40 ° C for 2 days on a magnetic stirrer, the sample was centrifuged, and the supernatant was taken out in a fume hood to evaporate until the solvent evaporated. The resulting solid was then dried in a vacuum oven at 40 ° C overnight. The resulting final product was the same amorphous form as in Example 1.
  • Example 5 Solid stability test of amorphous compound of formula (I) under high temperature and high humidity conditions
  • the above experimental data shows that the amorphous form of the compound of the formula (I) provided by the present invention has no significant change in content and impurities under high temperature and high humidity conditions, and has high high temperature and high humidity stability.
  • Example 6 Solid physical stability test of amorphous compound of formula (I) under high humidity conditions
  • the above experimental data shows that the amorphous form of the compound of the formula (I) provided by the present invention has no change in morphology and properties under high-humidity conditions, and has high high-humidity stability.
  • Example 7 Stability test of amorphous form of compound of formula (I) in different solvents
  • ETC Enzyme Fragmentation Complementation
  • the NHR protein test is based on the detection of a standard length NHR protein in an activated state, and a protein containing a steroid receptor coactivator (SRCP) region with one or more standard LXXLL sequences of nuclear fusion proteins - Protein interactions.
  • SRCP steroid receptor coactivator
  • the NHR is labeled on the ProLinkTM component of the EFC test system, while the SRCP region and the enzyme receptor component (EA) are fused and expressed in the nucleus.
  • EA enzyme receptor component
  • NHR is transferred to the nucleus and the SRCP region is obtained, where a complementary effect is produced, producing an equivalent of activated-galactose (-Gal) with the generation of a chemical light signal.
  • Benefits associated with this approach include reduced compound incubation time, direct testing of NHR targets, use of standard length human NHR sequences, and selection of new classes of compounds based on disrupted protein-protein interactions.
  • the NHR NT assay detected the transfer of an NHR between the cytoplasm and the nuclear compartment.
  • the receptor is labeled on the ProLinkTM component of the EFC test system, while the EA and nuclear sequence are fused, thereby limiting the expression of EA on the nucleus.
  • the transfer of NHR to the nucleus results in a complementary effect with EA, producing an equivalent of activated-galactose (-Gal) with the generation of a chemical light signal.
  • PathHunter NHR cell lines were developed from frozen stocks according to standard procedures.
  • cells need to be incubated with the compound to elicit a response.
  • the compound was formulated into a stock solution from a buffer solution and diluted 5 times.
  • cells are pre-incubated with the antagonist and then challenged with an agonist at the EC80 concentration.
  • the compound was formulated into a stock solution from a buffer solution and diluted 5 times.
  • the experimental signal is generated from a single addition of a 12.5 uL or 15 uL (50% v/v) PathHunter test reagent mixture which is then incubated for one hour at room temperature.
  • the chemiluminescent signal generated for the microplate will be detected by a PerkinElmer Envision instrument.
  • % activity 100% ⁇ (measured compound RLU mean - medium background RLU mean) / (ligand maximum control mean - media background RLU mean)
  • % reverse agonistic activity 100% ⁇ ((media background RLU mean - measured compound RLU mean) / (media background RLU mean - ligand maximum control RLU mean))
  • the compound of the formula (I) provided by the present invention has a high degree of chemical properties and physics, unlike the conventional knowledge of the amorphous form of the drug in the art. Morphological stability, and the amorphous form of the compound of formula (I) has obvious inhibitory effects on cytokines of PPAR-related pathways, and has significant effects on liver damage, NAS Score and liver fibrosis; it is known that the compound of formula (I) has amorphous Good medicinal prospects.

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Abstract

本发明涉及一种作为PPAR激动剂的吡咯烷衍生物的无定形及其制备方法。

Description

作为PPAR激动剂的吡咯烷衍生物的无定形及其制备方法
相关申请的引用
本申请主张如下优先权：
CN201711394677.8，申请日2017-12-21。
技术领域
本发明涉及一种作为PPAR激动剂的吡咯烷衍生物的无定形及其制备方法。
背景技术
非酒精性脂肪肝病(Nonalcoholic fatty liver disease，NAFLD)是发达国家或地区最常见的肝脏疾病，是指过多的脂肪以甘油三酯的形式堆积在肝脏中(脂肪变性>5％的肝细胞组织)。除了有过多脂肪外，NAFLD的患者伴有肝细胞损伤和炎症(脂肪性肝炎)，后者即NASH(Nonalcoholic steatohepatitis)。NAFLD中单纯的脂肪变性和短期的发病率或死亡率的增加没有相关性，但一旦进展到NASH则显著提高肝硬化、肝衰竭和肝细胞癌(HCC)的风险。由于NASH引起的肝硬化是肝移植日益增加的一个原因。在NASH患者中肝病所致的发病率和死亡率都大大增加，并且和心血管疾病发病率和死亡率增加密切相关。对无症状的中年男性患者的诊断显示：46％的患者为非酒精性脂肪肝病(NAFLD)，12.2％为NASH。NAFLD患者多为男性、老年人、高血压患者和糖尿病人。60-76％的糖尿病人患有NAFLD，22％患有NASH。NAFLD的小儿患者也在逐年增长，肥胖儿童中有38-53％患有NAFLD。在中国，非酒精性脂肪肝发病已经增至第一。
目前尚无FDA批准的药物治疗该疾病，中国临床常用多烯磷脂酰胆碱、水飞蓟素、熊去氧胆酸、甘草酸等护肝药物。
过氧化物酶体增殖物激活受体(PPAR)是细胞核激素受体超家族的成员，其是调节基因表达的配体-激活的转录因子，主要有3个亚型:PPAP Alpha主要在棕色脂肪组织、肝脏、心脏和骨骼肌中表达，在胆酸、脂类及糖的代谢中发挥主要作用；PPAP Delta表达特异性不明显，可能具有抗炎作用；Gamma对胰岛素抵抗有一定作用。该受体与多种疾病状态有关，包括血脂异常症、高脂血症、高胆固醇血症、动脉粥样硬化、动脉粥样化形成、高甘油三酯血症、心力衰竭、心肌梗死、血管疾病、心血管疾病、高血压、肥胖症、炎症、关节炎、癌症、阿尔茨海默病、皮肤病、呼吸疾病、眼部病症、IBD(应激性肠病)、溃疡性结肠炎及克罗恩病。从PPAR多种对肝脏功能有益的条件机理来看，PPAR激动剂是治疗脂肪肝最有效的潜在药物之一。
如下化合物为已有文献报道的PPAR激动剂化合物。
Figure PCTCN2018122423-appb-000001
发明内容
本发明的第一个目的在于从解决现有技术的不足出发，提供了式(I)所示化合物的无定形，该无定形具有相当的稳定性，进而具有一定的药用前景，为式(I)所示化合物开发成临床用药提供了一种可行的原料药选择。
Figure PCTCN2018122423-appb-000002
本发明的上述目的通过如下技术方案予以实现：
式(I)所示化合物的无定形，其特征在于所述无定形的X-射线粉末衍射图谱(XRPD)中没有尖锐的衍射峰。
本领域的技术人员公知，无定形(Amorphous)属于热力学高能态，为热力学亚稳态结构，构成其化合物的基本微粒在三维空间呈现为无序排列，X-射线粉末衍射谱图是判断无定形形态最直观的方式之一；具体的，当化合物以无定形的形态存在时，其X-射线粉末衍射图谱通常表现为没有尖锐的衍射峰，即XRPD谱图中体现为没有衍射峰，或者有一个或者数个宽且平缓的衍射峰(业内常形象的称之为“馒头峰”)；本领域的技术人员可以理解，所述的无定形XRPD谱图中宽且平缓的衍射峰为相对于晶型XRPD谱图中窄且尖锐的衍射峰而言，一般来说，无定形XRPD谱图中宽且平缓的衍射峰2θ角跨度可达5°甚至更大。
具体的，前述式(I)所示化合物的无定形的X-射线粉末衍射图谱在2θ角为10～25°之间存在一个宽且平缓的衍射峰。
本发明的一个具体的方案中，所述式(I)所示化合物的无定形其X-射线粉末衍射图谱如图1所示。
本发明的一些方案中，上述无定形，其差示扫描量热曲线(DSC)在69.28±3℃和239.33±3℃有两 个吸热峰的起始点。
本发明的一个具体的方案中，上述无定形，其DSC图谱如图2所示。
本发明的一些方案中，上述无定形，其热重分析曲线(TGA)在120.00±3℃处失重达0.9958％。
本发明的一个具体的方案中，上述无定形，其TGA图谱如图3所示。
本发明的第二个目的在于提供了一种式(I)化合物无定形的制备方法，所述制备方法包括将式(I)化合物加入到溶剂中加热搅拌或重结晶制得；所述溶剂选自：甲醇、乙醇、四氢呋喃、乙酸乙酯和正庚烷，所述加热搅拌的搅拌温度为25℃～45℃，所述加热搅拌(打浆)的时间为2小时～48小时，所述制备方法中化合物与溶剂的质量/体积比为1:3.5～6g/mL。
该方法工艺稳定，反应条件温和，原料易得，可用于大规模工业化生产式(I)化合物无定形。
技术效果
发明人惊讶的发现，不同于常规以无定形形态存在之化合物稳定性不佳、药用性能不好等缺陷，本发明提及的式(I)化合物无定形具有较高的稳定性，具体表现为所述式(I)化合物无定形在高温、高湿等条件下具有较高的稳定性，基于已有的稳定性数据可以判断：式(I)化合物无定形具有一定的药用前景；
进一步的，本发明提及的式(I)化合物无定形对与PPAR相关通路的细胞因子的抑制作用明显，并通过CCl 4诱导的C57BL/6小鼠急性肝损伤实验和MCD饮食诱导的db/db小鼠NASH模型发现，式(I)化合物对肝损伤、NAS Score和肝纤维化的改善作用显著。
综上可知，本发明式(I)所示化合物的无定形具有较好的稳定性，具有一定的药用前景；因此，如果通过检测手段证明式(I)所示化合物在原料药和/或制剂产品中部分或全部以无定形存在，则应被视为使用了本发明提供的式(I)所示化合物的无定形。所述检测手段除了前述提及的X-射线粉末衍射外，还可以进一步包括差示扫描量热法(DSC)，红外光谱法(IR)，拉曼光谱法(Raman)，固体核磁共振法(SSNMR)的方法及其他一切可以佐证使用了本发明所述式(I)所示化合物的无定形的检测方法，并可以采用本领域技术人员常用方法去除诸如药物辅料等所带来的影响，诸如差减图谱法等。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在含有下列含义。一个特定的短语或术语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文出现商品名 时，旨在指代其对应的商品或其活性成分。
本发明的中间体化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本发明的化学变化及其所需的试剂和物料。为了获得本发明的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。
下面会通过实施例具体描述本发明，这些实施例并不意味着对本发明的任何限制。
本发明所使用的所有溶剂是市售的，无需进一步纯化即可使用。
本发明所使用的溶剂可经市售获得。本发明采用下述缩略词：DCM代表二氯甲烷；DMF代表N，N-二甲基甲酰胺；DMSO代表二甲亚砜；EtOH代表乙醇；MeOH代表甲醇；TFA代表三氟乙酸；TsOH代表对甲苯磺酸；mp代表熔点；EtSO 3H代表乙磺酸；MeSO 3H代表甲磺酸；ATP代表三磷酸腺苷；HEPES代表4-羟乙基哌嗪乙磺酸；EGTA代表乙二醇双(2-氨基乙基醚)四乙酸；MgCl 2代表二氯化镁；MnCl 2代表二氯化锰；DTT代表二硫苏糖醇。
1.1粉末X-射线衍射(X-ray powder diffractometer,XRPD)
仪器型号：布鲁克D8 advance(Bruker D8 Advance)X-射线衍射仪
测试方法：大约10～20mg样品用于XRPD检测。
详细的XRPD参数如下：
光管：Cu,kα,
Figure PCTCN2018122423-appb-000003
光管电压：40kV，光管电流：40mA
发散狭缝：0.60mm
探测器狭缝：10.50mm
防散射狭缝：7.10mm
扫描范围：4-40deg
步径：0.02deg
步长：0.12秒
样品盘转速：15rpm
1.2差热分析(Differential Scanning Calorimeter,DSC)
仪器型号：TA Q2000差示扫描量热仪
测试方法：取样品(～1mg)置于DSC铝锅内进行测试，方法为：在50mL/min N 2条件下，加热样品从25℃-350℃，升温速率为10℃/min。
1.3热重分析(Thermal Gravimetric Analyzer,TGA)
仪器型号：TA Q5000IR热重分析仪
测试方法：取样品(2～5mg)置于TGA铂金锅内进行测试，方法为：在25mL/min N 2条件下，以10℃/min的升温速率，加热样品从室温到-350℃，升温速率为10℃/min。
附图说明
图1为式(I)化合物无定形的Cu-Kα辐射的XRPD谱图。
图2为式(I)化合物无定形的DSC谱图。
图3为式(I)化合物无定形的TGA谱图。
具体实施方式
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
实施例1：式(I)化合物的制备
Figure PCTCN2018122423-appb-000004
第一步：化合物B的制备
在25℃下向50L的反应釜中加入乙腈(30L),启动搅拌，然后加入化合物A(2.00kg，13.32mol,1.0eq)，溴代异丁酸乙酯(7.79kg，39.95mol,3.0eq)和碳酸钾(5.52kg,39.95mol,3.0eq)。反应液在 80℃下搅拌16小时。将反应温度降至25℃后过滤，滤液减压浓缩。所得残余物溶解于乙酸乙酯(5L)。滤饼用乙酸乙酯(5L×2)洗涤，合并乙酸乙酯溶液。合并的有机相用氢氧化钠水溶液(1mol/L,5L/次)洗涤，直至通过薄层层析法(石油醚：乙酸乙酯＝5:1)检测至有机相中没有原料点A。有机相通过饱和食盐水(5L×2)洗涤，无水硫酸钠干燥，过滤，减压浓缩。得到1.51kg化合物B,产率：42.9％。
1H NMR(400MHz,CHLOROFORM-d)δppm 9.85(s,1H),7.50(s,2H),4.31-4.23(m,2H),2.25(s,6H),1.45(s,6H),1.33(t,J＝7.2Hz,1H).
第二步：化合物C的制备
在干冰乙醇浴下(-60℃)，向乙醇(12L)中通入氯化氢气体(3.67kg，100.54mol,5.3eq)，并将体系温度控制在0℃以下。向50L的反应釜中加入乙醇(13L)和新制备的氯化氢乙醇溶液，开启搅拌，自然升温至25℃。然后加入化合物B(5.01kg,18.97mol,1.0eq)。待原料完全溶解后，分批加入对甲硫基苯乙酮(2.83kg,17.07mol,0.9eq)。混合物在25℃下搅拌16小时。将反应体系抽滤，滤饼溶于乙酸乙酯(30L),用水(10L×2)，氢氧化钠水溶液(1N,8L×2)和饱和食盐水(8L×2)洗涤，无水硫酸钠(1.5kg)干燥，过滤，减压浓缩，得到6.10kg化合物C，产率：76.8％。
MS m/z(ESI):413.1[M+1].
1H NMR(400MHz,CHLOROFORM-d)δppm 7.95(d,J＝8.28Hz,2H),7.71(d,J＝15.56Hz,1H),7.42(d,J＝15.56Hz,1H),7.31-7.28(m,4H),4.30(q,J＝7.28Hz,2H),2.54(s,3H),2.25(s,6H),1.50(s,6H),1.36(t,J＝7.15Hz,3H).
第三步：化合物D的制备
在50L反应釜中加入N,N-二甲基甲酰胺(15L),开启搅拌，加入三甲基碘化亚砜(3.78kg,16.01mol 1.2eq)，然后降温至0℃，分批加入叔丁醇钾(1.79kg,16.01mol,1.2eq)。在0℃下搅拌30分钟后，缓慢加入化合物C(5.5kg,13.34mol,1.0eq)的N,N-二甲基甲酰胺(15L)溶液。混合物在0℃下搅拌2小时。将反应液缓慢倒入冰水(0-5℃，30L)中，然后用石油醚/乙酸乙酯(1:1,10L×3)萃取。合并的有机相用水(10L×2)和饱和食盐水(10L×2)洗涤，无水硫酸钠(2kg)干燥，过滤，减压浓缩，得到5.48kg化合物D,产率：96.3％。
MS m/z(ESI):427.2[M+1].
1H NMR(400MHz,CHLOROFORM-d)δppm 7.91(d,J＝8.5Hz,2H),7.27(d,J＝8.5Hz,2H),6.81-6.70(m,1H),6.75(s,1H),4.29(q,J＝7.0Hz,2H),2.83-2.74(m,1H),2.56(m,1H),2.51(s,3H),2.18(s,6H),1.84(m,1H),1.46(s,6H),1.35(t,J＝7.2Hz,3H)。
第四步：化合物E的制备
向干燥的50升反应釜中加入乙醇(35.0L)，开启搅拌，然后加入化合物D(5.45kg，12.79mol，1.0eq)和冰醋酸(2.30kg，38.37mol，3.0eq)。将反应混合物加热到80℃后，分批加入锌粉(2.45kg，38.37mol，3.0eq)。得到的悬浊液在80℃下继续搅拌16小时。将反应液过滤，滤饼用乙酸乙酯(3L×2)洗涤，合并有机相减压浓缩。将浓缩液溶于乙酸乙酯(10L),抽入50L分液漏斗中。向50L分液漏斗中抽入乙酸乙酯(15L)和水(10L),搅拌5分钟后静置分液。有机相依次用10％碳酸钠水溶液(10L×2)和饱和食盐水(10L×1)洗涤，无水硫酸钠干燥，过滤，减压浓缩，得到5.15kg化合物E,产率：92.9％。
MS m/z(ESI):429.2[M+1].
1H NMR(400MHz,CHLOROFORM-d)δppm 7.74(d,J＝8.5Hz,2H),7.17(d,J＝8.5Hz,2H),6.71(s,2H),4.21(q,J＝7.1Hz,2H),2.82(t,J＝7.3Hz,2H),2.51(t,J＝7.7Hz,2H),2.45(s,3H),2.09(s,6H),1.94(quin,J＝7.5Hz,2H),1.39(s,6H),1.28(t,J＝7.0Hz,3H)
第五步：化合物F的制备
向50L反应釜中加入无水二氯甲烷(20L)，启动搅拌,然后加入化合物E(5.21kg,12.02mol,1.0eq)和2,6-二甲基吡啶(4.50kg,42.07mol,3.5eq)，降温至0℃。向反应液中滴加三甲硅基三氟甲磺酸酯(8.01kg,36.06mol,3.0eq)，在0℃下继续搅拌30分钟左右。取反应液通过薄层层析法(石油醚：乙酸乙酯＝5:1)检测。向50L分液漏斗中抽入冷水(5-10℃，10L),随后在搅拌下抽入反应液，搅拌5分钟后分液。有机相用饱和食盐水(10L)洗涤，减压浓缩。将浓缩液加到甲醇/水(2:1，30L)的混合溶液中，搅拌20分钟左右，析出黄色固体。将混合物过滤，减压抽干得到黄色固体粗品。
在50L反应釜中加入无水甲苯(35L)，启动搅拌，将减压抽干的粗品加入反应釜。降温至0℃,将二氯二氰基苯醌(2.99kg,13.22mol,1.1eq)分批加入反应釜中，在0℃下继续搅拌1小时。取反应液通过薄层层析法(石油醚：乙酸乙酯＝5:1)检测。在120L的桶中加入水(50L)和亚硫酸钠(3.00kg),搅拌澄清后，将反应液缓慢倒入该亚硫酸钠溶液中，并加入乙酸乙酯(15L)。快速搅拌10分钟，析出大量黄色固体，抽滤，滤饼用石油醚/乙酸乙酯(3:1,10L×2)洗涤。合并滤液并分出有机相。有机相用5％亚硫酸钠溶液(10L×2)和饱和食盐水(10L×2)洗涤，无水硫酸钠(2.00kg)干燥。过滤，减压浓缩(40-50℃)。得到4.29kg粗产品。随后将粗产品加入12L无水乙醇中，25℃下搅拌0.5小时，随后过滤。收集滤饼并减压干燥得到3.50kg化合物F，产率:69.9％。
MS m/z(ESI):427.2[M+1].
1H NMR(400MHz,CHLOROFORM-d)δppm 7.85(d,J＝8.5Hz,2H),7.28-7.27(m,2H),7.22-7.14(m,1H),7.02(s,1H),6.83(s,2H),4.30(s,2H),3.53(d,J＝6.8Hz,2H),2.55(s,3H),2.21(s,6H),1.49(s,6H),1.38(s,3H).
第六步：化合物G的制备
向干燥的50升反应釜中加入2-甲基四氢呋喃(30L),开启搅拌，加入化合物F(3.0kg，6.50mol，1.0eq)和三氟乙酸(37.05g,0.33mol,0.05eq)，然后缓慢加入N-甲氧基甲基-N-(三甲基硅甲基)苄胺(1.85kg,7.80mol,1.2eq)，控制内温在30℃以下。滴加完毕在25℃下继续搅拌12小时。将反应液抽入50L分液漏斗中，依次用5％碳酸钠水溶液(10L×2)和饱和氯化钠水溶液(10L×2)洗涤，无水硫酸钠(2kg)干燥，过滤，减压浓缩，得到3.92kg化合物G。
MS m/z(ESI):560.0[M+1].
1H NMR(400MHz,CHLOROFORM-d)δppm 7.71(d,J＝8.5Hz,1H),7.31-7.20(m,9H),6.71(s,1H),4.30-4.23(m,2H),3.76-3.36(m,6H),3.07-2.96(m,2H),2.68(t,J＝7.3Hz,2H),2.51(s,3H),2.10-2.03(m,6H),1.36-1.22(m,9H).
第七步：化合物H的制备
将化合物G(3.92kg,5.04mol,1.0eq)用无水乙醇(20L)溶解，加到50升反应釜中，开启搅拌。将氢氧化钠(604.8g,15.12mol,3.0eq)溶于水(6L)中，然后缓慢加到反应液中。加料完毕，在25℃下继续搅拌16小时。将反应液减压浓缩，除去大部分溶剂(乙醇)。将浓缩后的液体抽入50L分液漏斗中,开启搅拌，然后抽入乙酸乙酯(20L),用10％的硫酸氢钾水溶液(10L×2)和饱和氯化钠水溶液(10L×2)洗涤，无水硫酸钠(1.5kg)干燥，减压浓缩。大约浓缩至剩余8L左右溶剂，有大量固体析出，停止浓缩，降至25℃。将浓缩的悬浮液过滤,滤饼用乙酸乙酯(2L×3)洗涤，抽干，通过真空干燥箱减压干燥，得到2.44kg化合物H,产率：89.85％。
MS m/z(ESI):532.1[M+1].
1H NMR(400MHz,CHLOROFORM-d)δppm 7.66(dd,J＝4.0,8.3Hz,2H),7.41-7.33(m,2H),7.23-7.08(m,5H),6.79(d,J＝2.0Hz,2H),4.12(q,J＝7.0Hz,2H),3.93-3.60(m,4H),3.48-3.32(m,1H),2.97-2.84(m,1H),2.67(d,J＝7.8Hz,2H),2.57-2.49(m,3H),2.25-2.13(m,6H),1.46(s,6H),1.36(t,J＝7.2Hz,3H).
第八步：化合物I的制备
向干燥的50升反应釜中加入乙腈(24L)和异丙醇(6L),开启搅拌，然后加化合物H(3.04kg,5.65mol,1.0eq)。在80℃下缓慢加入(S)-(-)-(1-萘基)乙胺(724.79g)。加料完毕，在80℃下继续搅拌1小时。停止加热，自然冷却，在30℃下继续搅拌16小时。停止搅拌，抽滤，滤饼用异丙醇(2L×2)洗涤，抽干后，将固体转移至旋蒸中减压干燥，得到1.63kg化合物I。
手性拆分条件：手性柱：Chiralpak AD-3 100×4.6mm I.D.,3um；流动相：40％甲醇(0.05％DEA)- CO2；流速：4mL/min；柱温：40℃。
化合物I对应的保留时间：1.604分钟。
第九步：化合物J的制备
向干燥的50L反应釜中加入无水乙醇(30L)和无水甲醇(4.5L)，开启搅拌，加入化合物I(2.93kg，4.17mol,1.0eq)。升温至80℃搅拌1小时，停止加热，自然冷却，在30℃下继续搅拌16小时。将悬浮液抽滤，滤饼用乙醇(2L×2)洗涤后减压干燥。向残余物中加入甲醇(1.5L)和乙酸乙酯(15L),搅拌后抽入50L分液器中。有机相用10％的硫酸氢钾水溶液(10L×5)，饱和氯化钠水溶液(5L×2)洗涤，无水硫酸钠(1kg)干燥，过滤，减压浓缩，得到0.97kg化合物J。
手性拆分条件：手性柱：Chiralpak AD-3 100×4.6mm I.D.,3um；流动相：40％甲醇(0.05％DEA)-CO2；流速：4mL/min；柱温：40℃。
化合物J对应的保留时间：1.576分钟。
第十步：式(I)化合物的制备
向干燥的50L反应釜中加入无水二氯甲烷(7L),开启搅拌，加入化合物J(700g,1.31mol,1.0eq)和三乙胺(1.33kg,13.1mol,10.0eq)。在0℃下向反应液中滴加氯甲酸苯酯(2.24kg,13.1mol,10.0eq)。加料完毕，在0℃下继续搅拌1小时。向反应液中加入碳酸钾(543.37g,3.94mol,3.0eq)的纯水(3L)溶液,升温至40℃继续搅拌20分钟。然后加入氢氧化锂(165.60g,3.94mol,3.0eq),在25℃下继续搅拌20分钟。将反应混合物分液，有机相用饱和氯化钠水溶液(2L)洗涤，无水硫酸钠(500g)干燥，过滤，减压浓缩。浓缩液用乙酸乙酯(1.5L)溶解，然后在快速搅拌下缓慢加入正庚烷(5.6L)。加完继续搅拌30分钟，过滤。将滤饼加到正庚烷/乙酸乙酯(4:1,3.5L×3)中，快速搅拌打浆30分钟，过滤。得到的滤饼用叔丁基甲醚(5L)溶解，依次用5％硫酸氢钾水溶液(1.5L×2),去离子水(1L×2)洗涤，无水硫酸钠(300g)干燥，过滤，减压浓缩。得到的固体通过真空烘箱干燥(40-45℃)，得到式(I)化合物。
MS m/z(ESI):584.1[M+23].
1H NMR(400MHz,MeOD-d 4)δppm 7.65(d,J＝6.8Hz,2H),7.43-7.37(m,2H),7.29-7.22(m,3H),7.16(t,J＝7.2Hz,2H),6.87(s,2H),4.13-3.92(m,2H),3.89-3.80(m,1H),3.69(dd,J＝5.4,10.7Hz,1H),3.58-3.49(m,1H),2.81(d,J＝14.1Hz,2H),2.71-2.63(m,1H),2.55(s,3H),2.23(s,6H),1.43(s,6H).
手性拆分条件：手性柱：Chiralpak AD-3 100×4.6mm I.D.,3um；流动相：40％of methanol(0.05％DEA)in CO2；流速：2.8mL/min；柱温：40℃。
式(I)化合物对应的保留时间：2.018分钟。
实施例2.式(I)化合物无定形的制备
控制温度在25℃，将粗品淡黄色固体式(I)化合物(310.5g)加入到3L反应瓶中，然后加入正庚烷(1500mL)，加料完毕，反应在25℃搅拌2h，过滤，滤饼用正庚烷(500mL)洗涤，然后过滤得到粗品，粗品经真空干燥箱干燥，XRPD检测其形态，所得终产物的形态为无定形。
所得终产物的Cu-Kα辐射的XRPD谱图如图1所示；DSC谱图为图2所示；TGA谱图如图3所示。
实施例3.式(I)化合物无定形的制备
称取式(I)化合物(40.0mg)加到4.0mL玻璃瓶中，加入150μL的乙酸乙酯，使其成浑浊液。在磁力搅拌器上40℃搅拌2天后，将样品离心，取上清液置于通风橱中挥发至溶剂挥发干。然后将得到固体置于40℃真空干燥箱中干燥过夜。所得终产物为与实施例1相同的无定形。
实施例4.式(I)化合物无定形的制备
称取式(I)化合物(39.9mg)加到4.0mL玻璃瓶中，加入150μL的四氢呋喃，使其成浑浊液。在磁力搅拌器上40℃搅拌2天后，将样品离心，取上清液置于通风橱中挥发至溶剂挥发干。然后将得到固体置于40℃真空干燥箱中干燥过夜。所得终产物为与实施例1相同的无定形。
实施例5：式(I)化合物无定形在高温、高湿条件下的固体稳定性试验
平行称取2份式(I)化合物无定形样品，每份大约100mg，置于玻璃样品瓶的底部，摊成薄薄一层。样品用铝箔纸封瓶口，并在铝箔纸上扎些小孔，保证样品能与环境空气充分接触，置于40℃/75％湿度条件恒温恒湿箱。在上述条件下放置的样品于第10天，30天，60天，90天取样检测，检测结果与0天的初始检测结果进行比较，HPLC分析方法如表1所示，试验结果见下表2所示：
表1.HPLC分析方法
Figure PCTCN2018122423-appb-000005
Figure PCTCN2018122423-appb-000006
表2.式(I)化合物无定形的固体稳定性试验
时间点(天) 外观 纯度(％) 总杂质(％)
0 灰白色粉末 97.37 2.63
10 灰白色粉末 97.49 2.51
30 灰白色粉末 97.31 2.69
60 灰白色粉末 97.59 2.41
90 灰白色粉末 97.28 2.72
以上实验数据表明：本发明所提供的式(I)化合物无定形在高温、高湿条件下未发现含量及杂质情况的明显变化，具有较高的高温、高湿稳定性。
实施例6：式(I)化合物无定形在高湿条件下固体物理稳定性试验
平行称取2份式(I)化合物无定形，每份大约100mg，放置于玻璃样品瓶的底部，摊成薄薄一层，铝箔纸封瓶口，并在铝箔纸上扎些小孔，保证样品能与环境空气充分接触。把制备的样品分别放置于25℃/92.5％的相对条件下，考察样品第10天的物理稳定性。同时，单独称取一份大约100mg式(I)化合物无定形，放置于玻璃样品瓶的底部，用螺纹瓶盖密封后，保存于-20℃条件下，作为对照品使用。在第10天，取出所有样品，恢复至室温，观察样品外观变化，并用XRPD检测样品形态。通过对加速样品与对照样品的比较，判断式(Ⅰ)化合物无定形的固体物理稳定性。下表3为式(I)化合物无定形固体物理稳定性实验结果。
表3.式(I)化合物无定形在高湿条件下固体物理稳定性试验
Figure PCTCN2018122423-appb-000007
以上实验数据表明：本发明所提供的式(I)化合物无定形在高湿条件下未发现形态及性状的变化，具有较高的高湿稳定性。
实施例7：式(I)化合物无定形在不同溶剂中的稳定性试验
取约20mg的式(I)化合物无定形多份，分别加入0.3-0.4mL的下表中的单一或混合溶剂，40℃条件下搅拌。搅拌2天后，若样品为溶液状态或接近溶液状态，则过滤后自然挥发除去溶剂；若样品仍为混悬液，则离心样品，收集沉淀，上清液置于通风橱中挥发至溶剂挥发干。然后将得到沉淀和挥干溶剂后的固体置于40℃真空干燥箱中干燥过夜。收集所有样品中的固体，XRPD检测其状态。结果见表4。
表4.式(I)化合物无定形在不用溶剂中的稳定性实验
序号 溶剂 外观 结果
1 甲醇 挥发除去溶剂后析出固体 无定形
2 乙醇 混悬液(2天)/挥发除去溶剂后析出固体 均为无定形
3 乙腈 混悬液(2天)/挥发除去溶剂后析出固体 均为无定形
4 丙酮 混悬液(2天)/挥发除去溶剂后析出固体 均为无定形
5 乙酸乙酯 混悬液(2天)/挥发除去溶剂后析出固体 均为无定形
6 四氢呋喃 混悬液(2天)/挥发除去溶剂后析出固体 均为无定形
7 1,4-二氧六环 混悬液(2天)/挥发除去溶剂后析出固体 均为无定形
8 甲醇：水＝3:1 挥发除去溶剂后析出固体 无定形
9 乙醇：水＝3:1 混悬液(2天)/挥发除去溶剂后析出固体 均为无定形
10 乙腈：水＝3:1 混悬液(2天)/挥发除去溶剂后析出固体 均为无定形
11 丙酮：水＝3:1 混悬液(2天)/挥发除去溶剂后析出固体 均为无定形
以上实验数据表明：本发明所提供的式(I)化合物无定形在常规溶剂中未发生形态变化，具有较高的稳定性。
生物活性测试
实验例8：体外评价
PPAR激动活性体外测试原理
细胞核激素受体(NHR)测试
PathHunter的NHR蛋白相互作用和核转移测试是用来检测在均一的、非成像的实验中的细胞核核激素受体的活化能力。该技术被称为酶碎片互补(ETC)，由DiscoverX开发。
NHR蛋白测试是基于检测：在活化状态下标准长度的NHR蛋白，和一个含有类固醇受体共激活肽(SRCP)区域，并带有一个或多个标准的LXXLL作用序列的细胞核融合蛋白的蛋白-蛋白相互作用。
NHR被标记在EFC测试体系的ProLinkTM组分上，而SRCP区域和酶受体组分(EA)融合并在细胞核中表达。当与配体结合时，NHR会转移到细胞核并且得到SRCP区域，在这里就产生了互补作用，生成了一当量的活化-牛乳糖(-Gal)，并伴有化学光信号的生成。和这种途径所相关的效益，包括降低化合物孵育时间，对NHR靶点的直接测试，使用标准长度的人类NHR序列，以及基于破坏蛋白-蛋白相互作用去选择一些全新种类的化合物。
NHR NT实验检测了一个NHR在细胞质和细胞核隔室之间的转移。受体被标记在EFC测试体系的ProLinkTM组分上，同时EA和细胞核序列融合，从而限制了EA对细胞核的表达。NHR向细胞核的转移导致了和EA的互补作用，生成了一当量的活化-牛乳糖(-Gal)，并伴有化学光信号的生成。
细胞处理：
1.PathHunter NHR细胞株按照标准操作从冷冻库存中展开。
2.将细胞接种在20uL/孔的384孔白色细胞板，并于测试前在37℃孵育适当的时间。培养基中含有滤除血清的活性炭葡聚糖，以降低荷尔蒙表达的等级。
激动剂实验模式：
1.对于激动活性测定，细胞需要和化合物一起孵育以诱发响应。
2.由缓冲溶液将化合物配成库溶液，5倍稀释。
3.将5uL已5倍稀释的化合物溶液加入到细胞中，并在37℃(或室温)孵育3-16小时。保证最终的媒介浓度为1％。
抑制剂实验模式：
1.对于抑制活性测定，需将细胞预先与拮抗剂孵育，然后用激动剂在EC80浓度处挑战。
2.由缓冲溶液将化合物配成库溶液，5倍稀释。
3.将5uL已5倍稀释的化合物溶液加入到细胞中，并在37℃(或室温)孵育60分钟。保证最终的媒介浓度为1％。
4.将5uL用缓冲溶液6倍稀释的EC80激动剂加入到细胞中，并在37℃(或室温)孵育3-16小时。
信号检测：
1.实验信号由单次加入的12.5uL或15uL(50％v/v)PathHunter测试试剂混合物生成，该试剂之后需在室温孵育一小时。
2.对微孔板的生成的化学发光信号将由PerkinElmer Envision仪器检测。
数据分析：
1.化合物活性由CIBS数据分析软件分析得到(ChemInnovation,CA)。
2.对于激动剂模式的实验，百分比活性由以下公式计算得到：
％活性＝100％×(所测化合物RLU均值-介质背景RLU均值)/(配体最大控制均值-介质背景RLU均值)
3.对于拮抗剂模式的实验，百分比活性由以下公式计算得到：
％抑制＝100％×(1-(所测化合物RLU均值-介质背景RLU均值)/(EC80对照化合物的RLU均值-介质背景RLU均值))
4.需要注意的是，配体的响应会造成受体活性的下降(具有持续活性靶点的反向激动剂)。这些反向激动剂的活性由以下公式计算得到：
％反向激动活性＝100％×((介质背景RLU均值-所测化合物RLU均值)/(介质背景RLU均值-配体最大控制RLU均值))
实验结果见表5：
表5.本发明化合物体外筛选试验结果
Figure PCTCN2018122423-appb-000008
注1：以已知的PPARα激动剂GW7647、PPARδ激动剂L-165,041和PPARγ激动剂Troglitazone的体外平台最高激发响应值为100％，其他化合物的最高响应值与其做对比，得到相应的最高激发响应值。一般认为最高激发响应值大于80％为完全激动剂，大于50％小于80％为部分激动剂，而小于50％则激动效应不完全。
注2：A≤100nM；100nM<B≤150nM；150nM<C≤200nM；200nM<D≤250nM；E>250nM。
注3：100％≥I≥80％；80％>II≥50％；III<50％。
以上实验数据表明：式(I)化合物对PPAR Alpha和Delta受体的激活作用显著，并选择性地对PPAR Gamma受体具有激活作用。
综合以上对于式(I)化合物无定形稳定性及活性的评价实验可知：不同于本领域对于药物无定形的通常 认识，本发明提供的式(I)化合物无定形具有较高的化学性质和物理形态的稳定性，并且式(I)化合物无定形对与PPAR相关通路的细胞因子的抑制作用明显，对肝损伤、NAS Score和肝纤维化的改善作用显著；可知式(I)化合物无定形具有较好的药用前景。

Claims (8)

  1. 式(I)所示化合物的无定形，
    Figure PCTCN2018122423-appb-100001
    ，其特征在于所述无定形的X-射线粉末衍射图谱中没有尖锐的衍射峰。
  2. 根据权利要求1所述的式(I)所示化合物的无定形，其特征在于所述无定形的X-射线粉末衍射图谱在2θ角为10～25°之间存在一个宽且平缓的衍射峰。
  3. 根据权利要求1所述的式(I)所示化合物的无定形，其特征在于所述无定形的X-射线粉末衍射图谱如图1所示。
  4. 根据权利要求1所述的无定形，其差示扫描量热曲线在69.28±3℃和239.33±3℃有两个吸热峰的起始点。
  5. 根据权利要求4所述的无定形，其DSC图谱如图2所示。
  6. 根据权利要求1所述的无定形，其热重分析曲线在120.00±3℃处失重达0.9958％。
  7. 根据权利要求6所述的无定形，其TGA图谱如图3所示。
  8. 式(I)化合物无定形的制备方法，包括将式(I)化合物加入到溶剂中加热搅拌或重结晶制得，所述溶剂选自：甲醇、乙醇、四氢呋喃、乙酸乙酯和正庚烷，所述加热搅拌的搅拌温度为25℃～45℃，所述打浆的时间为2小时～48小时，所述制备方法中化合物与溶剂的质量/体积比为1:3.5～6g/mL。
PCT/CN2018/122423 2017-12-21 2018-12-20 作为ppar激动剂的吡咯烷衍生物的无定形及其制备方法 WO2019120257A1 (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10526320B2 (en) * 2016-07-12 2020-01-07 Guangdong Raynovent Biotech Co., Ltd. Pyrrolidine derivatives as PPAR agonists

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8642660B2 (en) * 2007-12-21 2014-02-04 The University Of Rochester Method for altering the lifespan of eukaryotic organisms
WO2018010656A1 (zh) * 2016-07-12 2018-01-18 南京明德新药研发股份有限公司 作为ppar激动剂的吡咯烷衍生物

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3637974B2 (ja) 1993-03-25 2005-04-13 エーザイ株式会社 ピロリジン誘導体
WO2002017912A1 (en) 2000-08-31 2002-03-07 Abbott Laboratories Endothelin antagonists
EP1702916A1 (en) 2005-03-18 2006-09-20 Santhera Pharmaceuticals (Schweiz) GmbH DPP-IV inhibitors
WO2007053819A2 (en) 2005-10-31 2007-05-10 Bristol-Myers Squibb Company Pyrrolidinyl beta-amino amide-based inhibitors of dipeptidyl peptidase iv and methods
EA015106B1 (ru) * 2006-04-24 2011-06-30 Эли Лилли Энд Компани Ингибиторы 11-бета-гидроксистероид дегидрогеназы 1
FR2908766B1 (fr) 2006-11-20 2009-01-09 Sanofi Aventis Sa Derives de pyrrole,leur preparation et leur utilisation en therapeutique.

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8642660B2 (en) * 2007-12-21 2014-02-04 The University Of Rochester Method for altering the lifespan of eukaryotic organisms
WO2018010656A1 (zh) * 2016-07-12 2018-01-18 南京明德新药研发股份有限公司 作为ppar激动剂的吡咯烷衍生物

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10526320B2 (en) * 2016-07-12 2020-01-07 Guangdong Raynovent Biotech Co., Ltd. Pyrrolidine derivatives as PPAR agonists

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