CN111825695B

CN111825695B - Oxazolidinone compound, preparation method, application and pharmaceutical composition thereof

Info

Publication number: CN111825695B
Application number: CN201910300665.7A
Authority: CN
Inventors: 沈建华; 许叶春; 黄福宝; 胡杭晨
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2019-04-15
Filing date: 2019-04-15
Publication date: 2022-04-12
Anticipated expiration: 2039-04-15
Also published as: CN111825695A

Abstract

The invention relates to the use as Lp-PLA₂Oxazolidinone compounds of covalent inhibitor and pharmaceutical compositions thereof, wherein the oxazolidinone compounds have a structure shown as general formula I, R₁，R₂And R₃Is defined as shown in the specification and the claims. The compound of the general formula I, the stereoisomer or the pharmaceutically acceptable salt thereof can be used as Lp-PLA₂Covalent inhibitors, prevention and/or treatment and/or amelioration of Lp-PLA₂Diseases associated with enzymatic activity. Meanwhile, the compound of the general formula I, the stereoisomer or the pharmaceutically acceptable salt thereof can be used as Lp-PLA₂Specific molecular probes.

Description

Oxazolidinone compound, preparation method, application and pharmaceutical composition thereof

Technical Field

The invention relates to the field of medicinal chemistry, in particular to oxazolidinone compounds with novel structures, a preparation method thereof, a medicinal composition taking the compounds as active ingredients, and an application of the compounds and the medicinal composition in preparation of Lp-PLA (low-cholesterol-lactic acid) for treatment₂The application of enzyme activity related disease medicine. Meanwhile, the invention relates to the field of molecular probe recognition, in particular to high-selectivity Lp-PLA₂Fluorescent probes and their use in Lp-PLA₂And (3) application in detection.

Background

Lipoprotein-associated phospholipase A₂(Lp-PLA₂) Also known as plasma type platelet activating factor acetylhydrolase (plasma PAF-AH), is capable of specifically hydrolyzing the ester bond at the sn-2 position of Platelet Activating Factor (PAF) and its analogs in vivo. Lp-PLA₂Belonging to the phospholipase A group₂The GIIA subfamily of superfamily G7 (GVIII) is secreted mainly by inflammatory cells, such as macrophages, lymphocytes and monocytes. The enzyme contains 441 amino acids, and the relative molecular mass is 45 kD. Except Lp-PLA₂In addition, the GVIII family also comprises another subfamily, the GVIB subfamily. It is also known as platelet activating factor acetylhydrolase II (PAF-AHII) and is a cytoplasmic enzyme with a relative molecular mass of 40kD and contains a myristoylation site at the N-terminus. With Lp-PLA₂In contrast, it has 41% amino acid homology and highly similar substrate specificity (chem. Rev 2011,111, 6130-.

80% of Lp-PLA in human plasma ₂20% Lp-PLA bound to Low Density Lipoprotein (LDL)₂Binding to High Density Lipoprotein (HDL). Lp-PLA₂Has strong affinity for oxidation-damaged phospholipids (ox-PL), and can be rapidly hydrolyzed to generate pro-inflammatory stimulators, such as oxidized phosphatidylcholine (ox-PC) to lysophosphatidylcholine (lyso-PC) and oxidized non-esterified fatty acids (ox-NEFA), to promote the production of non-inflammatory drugsInflammation/immune responses in a variety of cells including endothelial cells, smooth muscle cells, monocytes/macrophages, T cells, and neutrophils (The enzymes.2015; Volume 38, ISSN: 1874-.

Oxidized lipids in Lp-PLA₂The process of hydrolysis into inflammatory substances in the body may cause various diseases. These include atherosclerosis, coronary heart disease, angina pectoris, stroke, myocardial infarction, ischemia-reperfusion injury, multiple sclerosis, psoriasis, sepsis, and tumor. Thus, Lp-PLA₂Inhibitors may be universally useful for interfering with The development or progression of these diseases (Curr Pharm Des 2014; 20: 6256-.

The pathological mechanisms of atherosclerosis mainly include abnormal blood lipid level, oxidative stress, vascular endothelial cell inflammation and dysfunction, and the like. Inflammation and oxidative stress play an important role in the development and progression of atherosclerosis, from the formation of plaque initially to its instability, to subsequent rupture of the plaque. Therefore, inhibition of inflammatory factors of atherosclerosis is a new approach to treat this disease. Research shows that Lp-PLA is proved in experiments carried out on a diabetic high-cholesterol pig model₂Inhibitors can affect the status of atherosclerotic plaque volume, composition and gene expression in diabetic/hypercholesterolemic pigs and effectively inhibit the continued growth of atherosclerotic plaques (J Am Heart Assoc 2015; 4: e 001477).

Several epidemiological studies have also revealed Lp-PLA in plasma₂A positive correlation between levels and cardiovascular disease risk. A nested case-control Study named West of Scotland Coronary survival Study (WOSCOPS) recruited 580 people who had suffered from myocardial infarction, ischemic reperfusion, or Coronary heart disease-related death and 1168 controls, demonstrated for the first time Lp-PLA₂Level correlation with Coronary Heart Disease (CHD) events, indicating Lp-PLA₂Is an independent risk factor for coronary heart disease prediction, has small correlation with fibrinogen except the expression of LDL, and has high sensitivity C-reactive protein (hs-CRP)There is little correlation between, and the levels of, white blood cell count and other risk factors, and the levels are not affected by smoking. Lp-PLA₂The incidence of coronary events increased by 22% for each standard deviation increase in levels. Similar to this, the Monitoring of Trends and Determinants in Cardiovascular Disease (MONICA) -Augsburg cohort study and the Rotterdams study, both reveal to varying degrees that Lp-PLA₂The level correlates strongly with the occurrence of a cardiovascular event. Furthermore, some pertain to Lp-PLA₂The study of ischemic stroke risk shows that the increased Lp-PLA₂Levels can cause up to twice the risk of disease (Current Opinion in Pharmacology 2006,6: 154-. Thus, Lp-PLA₂The inhibitor can be used for treating cardiovascular and cerebrovascular related diseases, such as atherosclerosis, coronary heart disease, angina pectoris, apoplexy, myocardial infarction, and ischemia reperfusion injury.

Lp-PLA₂Hydrolysis of ox-PC to form lyso-PC, etc., stimulates downstream pathways associated with lyso-PC, a pro-inflammatory factor, and induces the release of multiple cytotoxic inflammatory cytokines. When these cytotoxic substances act on brain microvascular endothelial cells, the permeability of the endothelial cells is increased, and the expression of tight junction protein among endothelial cells is affected, thereby disrupting the Blood Brain Barrier (BBB). And Lp-PLA₂The inhibitor can inhibit the production of proinflammatory substances, reduce inflammatory response, and improve permeability of blood brain barrier (Journal of Alzheimer's Disease 2013,35: 179-198; Diabetes)&Vascular Disease Research2017,14(3): 200-. Thus, Lp-PLA₂The inhibitors can be used for treating diseases related to the increase of blood brain barrier permeability, such as but not limited to multiple sclerosis, Alzheimer's disease and the like. Plasma Lp-PLA in an Experimental Allergic Encephalomyelitis (EAE) rat model₂The activity of (a), oxidized low density lipoprotein and the concentration of lyso-PC increase with the progression of the disease. Lp-PLA developed by Kurarian Schker₂Inhibitor 5- ((9-methoxy-4-oxo-6, 7-dihydropyrimidine [6,1-a ]]The benzisothiazolin-2-yl) oxy) -2- (3- (trifluoromethyl) phenoxy) benzonitrile is capable of alleviating the development of disease in this EAE model (WO 20)14/114248). In addition, Lp-PLA developed by Kurarian Schker₂The results of the second phase clinical trials of the inhibitor rilapladib against Alzheimer's disease show (Alzheimer's)&Dementia:Translational Research&Clinical Interventions 1 2015,131-140)，Lp-PLA₂The inhibitor is safe and effective after oral administration, and can significantly improve executive function/working memory (EF/WM) (effective amount: 0.45; P ═ 0.026) of patients with Alzheimer's disease.

It was shown that lyso-PC was able to activate vascular endothelial growth factor receptor 2(VEGFR2) (Proc Natl Acad Sci 2016,113,7213.). Activation of VEGFR2 initiates multiple signaling pathways including MAPK, AKT, PKC, which are involved in the biological effects of endothelial cell survival, proliferation, migration, increased vascular permeability, vascular growth, vasodilation, etc. (Cardiovasular Research 2001,49: 568-581). Abnormalities in these biological effects can lead to the formation of a variety of diseases, for example, endothelial cell proliferation and migration can promote the formation of atherosclerotic plaques, leading to coronary heart disease, myocardial infarction, stroke, angina pectoris, and the like; increased vascular permeability can lead to central nervous system disorders based pathologically on blood brain barrier disruption; vascular growth can lead to tumors, etc. Lp-PLA₂And lyso-PC plays an important role in regulating or controlling the production of the biological effects and diseases. It has been shown that Lp-PLA₂High expression in cancer patients such as gastric cancer, pancreatic cancer and breast cancer (nat. Rev. cancer 2012,12(11): 782. 792.). Thus, Lp-PLA₂The inhibitors can be used for treating diseases related to VEGFR2 activation, such as but not limited to tumors, atherosclerosis, central nervous system diseases, and the like.

50% of the total weight in the tumor is indeed macrophages. These tumor-associated macrophages (TAMs) have previously been considered to be important anti-tumor effector cells that can directly kill tumor cells or eliminate tumors by presenting tumor-associated antigens to induce an immune response in the body. However, recent studies show that tumor-associated macrophages can act on tumor-associated stroma or endothelial cells by secreting special cytokines, growth factors and the like, and participate in promoting tumor development and stimulating tumor cellsGrowth and metastasis, induction of tumor neovascularization and lymphangiogenesis/immunosuppression, etc. (Cancer Res 2018,78 (19); 5492-503). Lp-PLA secreted by TAMs₂Have been shown to promote the migration of nasopharyngeal carcinoma cells (Oncotarget 2016, 23; 7(34): 55473-55490). In addition, highly expressed Lp-PLA₂Levels can be observed in a variety of tumor cells, such as breast, ovarian, colon, kidney, liver and lung cancer cells, especially in invasive and metastatic cancer samples (J Path: Clin Res 2017,3: 123. sup. 138; Genome Biology 2016,17: 108). Thus, Lp-PLA₂The inhibitors can be used for treating the formation or development of tumors in which TAMs are involved.

Lp-PLA₂Has the effects of promoting inflammation and promoting atherosclerosis, and can be used for detecting Lp-PLA in blood₂The inflammation degree and stability of the atheromatous plaque can be effectively known. The dynamic monitoring of inflammatory mediators in the change process can track the severity of atherosclerosis and warn myocardial infarction and cerebral thrombosis, and is a supplementary means for traditional risk assessment. Lipoprotein-associated phospholipase A organized by the Special Committee for cardiovascular and cerebrovascular diseases of the China Association of the elderly school and the China Association of physicians' examination Committee for cardiovascular and cerebrovascular diseases₂Clinical application expert advice states: the american heart society foundation (ACCF)/American Heart Association (AHA)2010 asymptomatic adult cardiovascular risk assessment guidelines recommend that Lp-PLA be considered for asymptomatic adults at intermediate risk₂Testing to further assess the risk of cardiovascular events (recommendation level IIb). The 2013 ACCF/AHA cardiovascular risk assessment guidelines suggest that asymptomatic first-degree prevention patients, after risk assessment, still cannot be sure whether patients in need of treatment can be considered for new marker assessment. The 2011 AHA/American stroke Association primary guidelines for stroke prevention suggest that inflammatory markers such as hs-CRP or Lp-PLA be detected₂A high risk patient in a stroke (recommendation level ib, evidence level B) can be identified. The european cardiology society 2012 clinical practice guideline for cardiovascular disease prevention suggests: Lp-PLA can be detected by patients with high recurrence risk of acute atherosclerotic thrombotic events₂To further assess the risk of relapse: (Recommendation level iib, evidence level B). Based on the above-mentioned research evidence and recommendations of international guidelines, it is recommended that the following population detect Lp-PLA₂Levels to predict risk of cardiovascular events: (1) screening of asymptomatic high risk population: especially people at high risk such as atherosclerotic cardiovascular disease, can detect Lp-PLA based on traditional risk factor evaluation₂To further assess the risk of future cardiovascular disease. (2) Lp-PLA, a statin-treated patient with better control of cholesterol levels₂Levels may increase the value of risk prediction of cardiovascular events. (3) Patients with acute thrombotic events, including ACS and atherosclerotic ischemic stroke patients, Lp-PLA₂The kit is helpful for long-term risk assessment, and the prediction value can be improved by combined detection with hs-CRP. (China society of Others and society of Engineers, China Committee of physicians and medical experts for testing, China Association of physicians' Committee of cardiovascular and cerebrovascular diseases, expert recommendation for clinical application of lipoprotein-related phospholipase A2 [ J]Journal of cardiovascular disease, 2015,43 (10)).

In addition to its role in predicting cardiovascular events, Lp-PLA₂It has also been reported that it can be used to predict the occurrence of certain tumors or the prognosis of Tumor therapy, such as the prediction of the occurrence of esophageal Cancer (Tumor biol.2016,37: 6349-. Considering Lp-PLA₂Potential wide application in clinical prediction and development of highly selective Lp-PLA₂The molecular probe is helpful for more accurately and sensitively monitoring Lp-PLA in vivo₂The change of the level, thereby exerting clinical guidance significance.

Fragment-based drug molecule design (FBDD), which has been widely used in industry and academia in recent years, has become an effective method for identifying small molecules that bind to a variety of therapeutic targets. FBDD is a novel drug discovery method which starts with fragment molecules (MW <300Da) and develops drug design based on compound-target protein structural information. The fragment molecule has the advantages of wide chemical structure coverage rate, high molecule combination efficiency and the like. FBDD facilitates drug discovery of target proteins for new chemical frameworks or inhibitors that are difficult to obtain with traditional screening approaches. The main technical route is to screen small fragments with low molecular weight and low affinity by a high-sensitivity physical or chemical method to obtain the binding information of the fragments and the atomic level of a target protein. And then extending or connecting the fragments based on the structure information of the drug target to finally obtain a new molecular entity with high affinity with the drug target and strong drug-like property. The technology is helpful to obtain lead compounds with better physicochemical property, stronger framework novelty or new binding sites. In the development process of the last two decades, marketed drugs using FBDD technology are emerging, such as Verofinib developed by Plexikon for treating melanoma and Venetosalax developed by Erberwein for treating leukemia, and many drugs under clinical study also show good therapeutic effects (Nature Reviews Drug Discovery 2016,15: 605-. However, the application of FBDD strategy in the development of covalent inhibitors is rarely reported, and most of the current highly selective covalent inhibitors are covalently bound to lysine or serine near the active center of the target protein and directly act on the active center.

Presently disclosed about Lp-PLA₂The inhibitors were mostly developed by the Kurarin Schker company. In the first patent published, Kurarin Schker developed a class of Lp-PLA₂The potent inhibitors of (WO99/24420, WO01/60805, WO02/30911, WO03/016287, WO03/042179, WO03/042206, WO08/048867, etc.) are characterized by containing a pyrimidinone or pyridone group in the structure, and represent compounds darapladib and rilapladib. Kulansu Schk subsequently discloses a class of Lp-PLA₂Inhibitors (US2012/0142717, WO2012/075917, WO2012/037782, WO2013/013503, WO2013/014185, WO2014/114248, WO2014/114249, WO2014/114694, WO2016/011931, WO2016/012916 and WO2016/012917) are still characterized by pyrimidinone groups, but are distinguished from previous inhibitors in that such structures are linear structures and also have relatively small molecular weights. Recently, Abide Therapeutics, USA, has disclosed a class of Lp-PLA with novel structure₂Inhibitors of (9) (WO2017/059135), Lp-PLA of such carbamates₂The inhibitor may be covalently linked to Lp-PLA₂Binding, resulting in an inhibitory effect. Lp-PLA reported so far₂Less covalent inhibitor and poor selectivity, especially for PLA2 VIIB.

Disclosure of Invention

The invention discloses an orally effective oxazolidinone Lp-PLA with good selectivity₂A covalent inhibitor. Meanwhile, based on the compounds, a class of oxazolidinone Lp-PLA with strong specificity is obtained₂A fluorescent probe molecule. Firstly, an oxazolidinone compound with a novel structure is selected as an electrophilic warhead. The warhead is derived from bacterial fermentation and is reported to be Lp-PLA₂But the warhead is linked to Lp-PLA₂The mechanism of action is unclear and the isolated bacterial fermentation containing the warhead is less selective to PLA2VIIB, only about 9 times (Journal of Antibiotics 2000,53(7): 664-669). After the warhead is synthesized, the warhead and Lp-PLA are analyzed₂The crystal structure of the interaction, the first demonstration of the warhead, is through the formation of covalent bonds between the carbamate moiety of the parent nucleus and the serine of the protein catalytic center. Then starting from the electrophilic fragment, the bullet is grown by adopting an FBDD strategy, and a series of compounds with better activity and selectivity on PLA2VIIB reaching more than 1000 times are obtained. Finally, a compound with better in vitro activity and selectivity is subjected to more systematic selectivity evaluation and in vivo evaluation, a compound which is effective in oral administration and has better selectivity on other proteins is obtained, and a fluorescent probe synthesized based on the compound can sensitively detect Lp-PLA₂And horizontal, thereby completing the present invention.

In one aspect, the present invention provides a class of compounds represented by the following general formula I, stereoisomers thereof, or pharmaceutically acceptable salts thereof:

in the above general formula I, R₁Selected from hydrogen, C₁-C₆Alkyl or-O- (CH)₂)_m-R₄Wherein, the C is₁-C₆Alkyl is unsubstituted or optionally substituted with a group selected from: halogen, cyano, nitro, C₁-C₄Alkoxy radical, C₆-C₁₀An aryloxy group, a carboxyl group, or a hydroxyl group; r₄Is selected from C₁-C₆Alkyl radical, C₆-C₁₀Aryl or C₆-C₁₀Heteroaryl radical, R₄C in (1)₁-C₆Alkyl radical, C₆-C₁₀Aryl or C₆-C₁₀Heteroaryl is unsubstituted or optionally substituted by a group selected from: halogen, cyano, nitro, C₁-C₄Alkoxy, carboxyl, or hydroxyl; m is an integer of 0 to 2;

R₂、R₃each independently selected from hydrogen and C₁-C₆Alkyl, or- (CH)₂)_n-NR₅R₆Wherein, the C is₁-C₆Alkyl is unsubstituted or optionally substituted with a group selected from: halogen, cyano, nitro, C₁-C₄Alkoxy, carboxyl, or hydroxyl; r₅、R₆Each independently selected from hydrogen and C₁-C₅Acyloxy, or a fluorophore; n is an integer of 1 to 5;

in the compound represented by the general formula I, the absolute configuration of a chiral center in an oxazole ring parent nucleus is optionally R or S configuration; the relative configuration of the exocyclic double bond in the oxazole ring parent nucleus is optionally in the Z or E configuration.

Preferably, R₁Selected from hydrogen, C₁-C₄Alkyl or-O- (CH)₂)_m-R₄Wherein, the C is₁-C₄Alkyl is unsubstituted or optionally substituted with a group selected from: halogen or C₆-C₁₀An aromatic oxy group; r₄Is selected from C₁-C₆Alkyl or C₆-C₁₀Aryl radical, R₄C in (1)₁-C₆Alkyl or C₆-C₁₀Aryl is unsubstituted or optionally substituted with a group selected from: halogen or cyano; m is 0 or 1; more preferably, R₁Selected from the group consisting of 1,1, 1-trifluoroethoxy, phenoxymethyl which is unsubstituted or substituted by cyano, benzyloxy which is unsubstituted or substituted by cyano; optimally, R₁Is 1,1, 1-trifluoroethoxy;

preferably, R₂、R₃Each independently selected from C₁-C₄Alkyl, or- (CH)₂)_n-NR₅R₆Wherein, the C is₁-C₄Alkyl is unsubstituted or optionally substituted with a group selected from: cyano radicals, C₁-C₄Alkoxy, carboxyl, or hydroxyl; r₅、R₆Each independently selected from hydrogen, tert-butyloxycarbonyl, Diaminorhodamine (DARs) or BODIPY fluorophores; n is 4 or 5; more preferably, R₂、R₃Each independently selected from ethyl or- (CH)₂)_n-NR₅R₆；R₅、R₆One is hydrogen and the other is selected from lisiane rhodanese B sulfonyl or 3-boropyrrolyl; n is 5;

wherein the lissamine rhodanine B sulfonyl structure is:

the structure of the 3-fluoroboropyrrolyl propionyl group is as follows:

in a particular embodiment, R₂、R₃Each independently selected from ethyl, 5-amino-n-pentyl, 5-tert-butoxycarbonylamino-n-pentyl, and,

In particular embodiments, the absolute configuration of the chiral center in the mother nucleus of the oxazole ring is optionally in the R or S configuration; preferably in the S configuration; relative configuration E of the exocyclic double bond in the oxazole ring parent nucleus.

In a more specific embodiment, the compound of formula I is selected from any one of the compounds represented by the following structural formulae.

In a particular embodiment, when R₁、R₂、R₃When containing a basic functional group, such as, but not limited to, an amino group, the pharmaceutically acceptable salts are hydrochloride, hydrobromide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, oxalate, succinate, malate, tosylate, tartrate, fumarate, glutamate, glucuronate, lactate, glutarate, arginate, maleate; when R is₁、R₂、R₃When the functional group contains an acidic functional group, for example, but not limited to, a carboxyl group and a sulfonic acid group, the pharmaceutically acceptable salts include sodium salt, potassium salt, calcium salt, aluminum salt, ammonium salt, methylamine salt, ethylamine salt, ethanolamine salt and the like.

In a particular embodiment, when R₁、R₂、R₃When the compound contains a chiral center, a cyclic substituent group or an unsaturated substituent group, the stereoisomer is enantiomer, diastereomer, racemate, cis-trans isomer and the like.

In another aspect, the present invention provides a process for the preparation of a compound of formula I as described above, which can be synthesized by the following reaction scheme:

wherein R is₁、R₂And R₃The definition of (a) is the same as the above definition;

(1) performing addition elimination reaction on the compound Ia and the compound Ib in the presence of an aprotic solvent, evaporating the reaction solvent to dryness, and then adding a protic solvent and a reducing agent to perform reductive amination reaction to obtain a compound Ic;

(2) and carrying out condensation reaction on the compound Ic and the compound Id to obtain the compound I.

Preferably, the aprotic solvent of step (1) is selected from one or more of dichloromethane, dichloroethane, tetrahydrofuran, or acetonitrile, etc.;

preferably, step (1) is carried out in the presence of an inert water scavenger selected from the group consisting of activated molecular sieves, anhydrous magnesium sulfate or other inert water scavengers;

preferably, the addition elimination, reductive amination and condensation reaction of step (1) are carried out at a temperature of 0 ℃ to room temperature for 3 to 24 hours;

preferably, the protic solvent of step (1) is selected from one or more of methanol or ethanol, etc.;

preferably, the reducing agent in step (1) is selected from one or more of sodium borohydride, sodium acetate borohydride, sodium cyanoborohydride and the like.

Preferably, step (2) is carried out in the presence of a solvent, a condensing agent, a base; the solvent is selected from one or more of Dichloromethane (DCM), N-Dimethylformamide (DMF), acetonitrile, or dimethyl sulfoxide (DMSO). The condensing agent is selected from carbodiimide type condensing agents, phosphorus positive ion type condensing agents and urea positive ion type condensing agents; the alkali is one or more of organic alkali or inorganic alkali.

Specifically, the preparation method may include dissolving the compound Ia and the compound Ib in a dry aprotic solvent such as dichloromethane, dichloroethane, tetrahydrofuran, or acetonitrile, reacting the compound Ia and the compound Ib at 0 ℃ to room temperature for 4 to 24 hours with a molecular sieve, anhydrous magnesium sulfate, or other inert water scavengers activated under stirring, directly evaporating the reaction solvent to dryness, and obtaining the intermediate Ic compound from the residue with a protic solvent such as methanol or ethanol, and a reducing agent such as sodium borohydride, sodium acetate, or sodium cyanoborohydride as a reaction reagent. Subsequently, intermediate Id (prepared by the method described in j. org. chem.2006,71, 8579-.

On the other hand, when R₂And R₃One of which is ethyl and the other is- (CH)₂)_n-NR₅R₆When this is the case, the compounds of formula If can be synthesized by the following reaction scheme:

wherein R is₁、R₂、R₃、R₅And R₆And n is as defined above.

Specifically, the method comprises the following two methods:

the method comprises the following steps: dissolving compound Ie in dry dichloromethane, dichloroethane, tetrahydrofuran, acetonitrile, DMF or DMSO as reaction solvent, sequentially adding organic base or inorganic base under stirring, and adding compound R₅-Cl and/or compound R₆-Cl at 0 ℃ to room temperature for 4-24 hours to obtain the compound of formula If;

or the second method: the compound R₅-OH and/or compound R₆-OH is dissolved in DCM, DMF, acetonitrile or DMSO, and under stirring a condensing agent such as but not limited to N, N ' -Dicyclohexylcarbodiimide (DCC), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) or 2- (7-azobenzotriazol) -N, N, N ', N ' -tetramethylurea Hexafluorophosphate (HATU) is first added followed by the sequential addition of an organic or inorganic base, compound Ie, and the reaction is carried out at 0 ℃ to room temperature for 4-24 hours to obtain the compound of formula If.

The target product obtained in the above steps can be respectively purified by appropriate methods such as column chromatography, recrystallization and the like to obtain pure products.

In the above reaction step, the organic base includes, but is not limited to, pyridine, triethylamine, N-diisopropylamine, tetrabutylammonium bromide, N-butyllithium, potassium tert-butoxide, preferably triethylamine, N-diisopropylamine, and pyridine; the inorganic base includes, but is not limited to, sodium hydride, sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, potassium bicarbonate, preferably lithium hydroxide, potassium carbonate, cesium carbonate.

In yet another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula I as described above, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; and pharmaceutically acceptable adjuvants. Wherein the pharmaceutically acceptable excipients include, but are not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (e.g. human serum albumin), buffer substances (e.g. phosphates), glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, beeswax, lanolin, polyethylene glycol water, polyvinylpyrrolidone, cellulosic substances, colloidal silica, salts (e.g. protamine sulfate, zinc salts, sodium carboxymethylcellulose, or magnesium trisilicate) or electrolytes (e.g. sodium chloride).

In particular embodiments, the pharmaceutical composition may further comprise one or more of a hypolipidemic agent, a cholesterol-lowering agent, an anti-atherosclerotic agent, an anti-multiple sclerosis agent, an antineoplastic agent, a hypoglycemic agent, an insulin sensitizer, an anti-anginal agent, an anti-inflammatory agent, a hypotensive agent, or a lipoprotein a-lowering agent. For example, the pharmaceutical composition further comprises an anti-multiple sclerosis drug, such as one or more of interferon-beta, glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, natalizumab, alemtuzumab, rituximab, azathioprine, cyclophosphamide, methotrexate, or cyclosporine a. For example, the pharmaceutical composition may also include conventional antineoplastic agents, such as alkylating agents, antimetabolites, antitumor antibiotics, antitumor botanicals, or hormone-and molecule-targeted antineoplastic agents, such as interfering cell signaling agents, ubiquitination-proteasome inhibitors, or addivo and curidan antibody drugs. For example, the pharmaceutical composition may also include statins, such as atorvastatin, simvastatin, pravastatin, lovastatin, and the like.

In particular embodiments, the pharmaceutical compositions may be in a variety of forms, such as tablets, capsules, powders, syrups, solutions, suspensions, aerosols, and the like, and may be presented in a suitable solid or liquid carrier or diluent; it can also be stored in a suitable injection or drip disinfectant device; may also contain flavoring agent, etc.

In specific embodiments, the pharmaceutical composition comprises a safe and effective amount (e.g., 0.1 to 99.9 parts by weight, preferably 1 to 90 parts by weight) of a compound of formula I or a pharmaceutically acceptable salt thereof, based on 100 parts by weight of the total weight of the pharmaceutical composition; and the balance of pharmaceutically acceptable auxiliary materials. Alternatively, the pharmaceutical composition comprises 0.1-99.9% by total weight, preferably 1-90% by total weight, based on 100% by total weight of the pharmaceutical composition, of a compound of formula I or a pharmaceutically acceptable salt thereof; and the balance of pharmaceutically acceptable auxiliary materials.

In a specific embodiment, the ratio of the compound of formula I to the pharmaceutically acceptable adjuvant is preferably, the compound of formula I as an active ingredient accounts for more than 60% of the total weight, the rest accounts for 0-40% of the total weight, the amount of the rest is preferably 1-20%, and most preferably 1-10%.

In another aspect, the present invention provides the use of a compound of formula I, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for the preparation of a medicament for inhibiting Lp-PLA₂Use in active medicine.

In still another aspect, the present invention provides a use of the compound of formula I, its stereoisomer or its pharmaceutically acceptable salt, or the pharmaceutical composition in the preparation of a medicament for preventing and/or treating and/or improving atherosclerosis and/or multiple sclerosis and/or tumors.

In a specific embodiment, the and Lp-PLA₂The enzyme activity-related disease is selected from atherosclerosis, angina pectoris, rheumatoid arthritis, apoplexy, myocardial infarction, psoriasis, brain inflammation diseases, ischemia reperfusion injury, septicemia, neurodegenerative disease, multiple sclerosisSexual sclerosis and Alzheimer's disease. In particular, the said copolymers with Lp-PLA₂The disease associated with enzyme activity is atherosclerosis, multiple sclerosis, or tumor.

In a further aspect, the present invention provides a method of using a pharmaceutical composition comprising a compound of formula I, a stereoisomer or a pharmaceutically acceptable salt thereof, as described above, for the treatment of Lp-PLA₂A method for treating a disease associated with enzymatic activity comprising administering to a subject in need thereof a safe and effective amount of a compound of formula I; the subject in need thereof includes cells cultured in vitro, human or non-human mammals, preferably, humans, dogs.

In the present invention, the "safe and effective dose" means: diseases, disorders, side effects, etc. are cured, ameliorated, effectively prevented or have a significantly reduced incidence without serious side effects in subjects treated with the dose as compared to subjects not treated with the dose. In addition, effective dosages to enhance normal physiological function are also included.

The compounds of the general formula I according to the invention can also be used in combination with the following drugs in the treatment of the above-mentioned diseases: a blood fat reducing drug, an anti-atherosclerosis drug, an anti-multiple sclerosis drug, an anti-tumor drug, a blood sugar reducing drug, an anti-angina drug, an anti-inflammatory drug, a blood pressure lowering drug or a lipoprotein a lowering drug. For example in combination with statins which inhibit cholesterol synthesis, the antioxidant probucol, insulin sensitizers, calcium channel antagonists or non-steroidal anti-inflammatory drugs.

The compounds of formula I of the present invention may be used in combination with cholesterol lowering agents, such as statins. Statins are HM-CoA reductase inhibitors, specifically, such as atorvastatin, simvastatin, pravastatin, lovastatin, and the like. The two drugs may be administered simultaneously or separately as recommended by the physician. Up to 30% of high cholesterol patients are not effective for statin therapy. The compounds of formula I of the present invention may be suitable for use in this part of the patient.

Since cardiovascular events are the leading cause of death in diabetic patients, the compounds of formula I of the present invention may be used in combination with hypoglycemic agents or insulin sensitizers to reduce the incidence of cardiovascular events in diabetic patients.

Due to the research and discovery of Lp-PLA in recent years₂Plays an important role in The formation and migration of tumors and The formation of new blood vessels (The enzymes.2015; Volume 38, ISSN:1874-6047), so The compound of The general formula I can be combined with anti-tumor drugs to relieve The symptoms of tumor patients. For example, the compounds of formula I of the present invention may be used in combination with conventional antineoplastic agents, such as alkylating agents, antimetabolites, antitumor antibiotics, antitumor botanicals or hormones, molecularly targeted antineoplastic agents, such as interfering cell signaling agents or ubiquitination-proteasome inhibitors, and European Divos and Cruda antibody drugs.

In addition, the compound of the general formula I can be used together with a medicament for resisting multiple sclerosis to prevent the occurrence of the multiple sclerosis in advance or delay the progress of the disease. For example, the compounds of formula I of the present invention may be used in combination with interferon-beta, glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, natalizumab, alemtuzumab, rituximab, azathioprine, cyclophosphamide, methotrexate, or cyclosporin A.

Drawings

FIG. 1 shows the administration of compound 4 of the present invention orally to Lp-PLA in SD rats₂The change in activity affects the results.

FIG. 2 shows Lp-PLA derived from human-derived recombinase using fluorescent probes (Compounds 9 and 10) of the present invention₂The result of fluorescent labeling of (2).

Figure 3 shows the results of the selectivity of compound 4 of the invention for proteomes in lysates of HEK293t cells.

FIG. 4 shows Lp-PLA in the present invention₂Selective results of specific fluorescent probes (compound 9 and compound 10) on HEK293t cell lysate proteome.

Term(s) for

In the present invention, the term "C" unless otherwise specified₁-C₆Alkyl "refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms, including without limitation methyl, ethyl, propyl, isopropyl, butyl, and the like(ii) a The term "C₁-C₄Alkyl "has a similar meaning.

In the present invention, the term "C" unless otherwise specified₁-C₄Alkoxy "means a straight or branched chain alkoxy group having 1 to 4 carbon atoms, including, but not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, and the like;

in the present invention, the term "C" unless otherwise specified₆-C₁₀Aryl "refers to an aromatic hydrocarbon group containing 6 to 10 atoms and conforming to the Huckel rule, including, without limitation, phenyl or naphthyl, and the like. The term "C₆-C₁₀By "aryloxy" is meant an aromatic hydrocarbonoxy group containing 6 to 10 atoms and conforming to the Huckel rule, including without limitation phenoxy or naphthoxy, and the like. The term "C₆-C₁₀The term "heteroarylene" denotes an aromatic ring group containing 6 to 10 atoms, conforming to the Huckel rule, and containing 1 to 4 heteroatoms selected from N, O, S, including, without limitation, pyridyl, thienyl, furyl, pyrrolyl, oxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, thiazolyl, quinolinyl, pyrimidinyl, purinyl, carbazolyl, pyrazolyl, isothiazolyl, imidazolyl, isoxazolyl. The term "fused ring aryl" contains 2-4 rings and conforms to the Huckel rule aromatic system and includes, without limitation, benzopyranyl, benzopyranonyl, benzofuranyl, benzothienyl, indolinyl, indolyl, azaindolyl, azaindolinyl, chromanyl, chromalinyl, pyrazolopyrimidinyl, azaquinazolinyl, azaquinazolinonyl, pyridofuranyl, pyridothienyl, thienopyrimidinyl, quinazolinyl, quinazolinonyl, pyrimidonyl, pyridazinyl, triazinyl, benzoxazinyl, benzoxazinonyl, benzothiazinyl, benzothiazinonyl, benzoxazinonyl, benzothiazinonyl, benzothiazolyl, benzimidazolyl, benzotriazolyl, naphthyridinyl, and the like.

In the present invention, unless otherwise specified, the term "halogen" means fluorine, chlorine, bromine, iodine, preferably fluorine and chlorine; "Hydrogen" means-H; "hydroxy group"Refers to-OH; "cyano" means-CN; "nitro" means-NO₂(ii) a "trifluoromethyl" means-CF₃(ii) a "methoxy" means OCH₃(ii) a "carboxyl" means-COOH.

In the present invention, the term "aprotic solvent" means an aprotic solvent comprising an aprotic polar solvent and an aprotic non-polar solvent, and includes, without limitation, dichloromethane, dichloroethane, benzene, diethyl ether, carbon tetrachloride, tetrahydrofuran, acetonitrile, pyridine, dimethyl sulfoxide, N-dimethylformamide, acetone and the like, unless otherwise specified.

As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. "independent" or "independently" means that the described objects are independent of each other and do not affect each other.

In the present invention, the "substitution" is a mono-or poly-substitution. In specific embodiments, the substitution is mono-, di-, tri-, or tetra-substituted. In particular embodiments, the polysubstituted refers to comprising a plurality of identical or different groups, such as two, three, four.

Detailed Description

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments.

Examples

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

In the following preparations, the structure of the compound was determined by Nuclear Magnetic Resonance (NMR) or Mass Spectrometry (MS). NMR hydrogen spectrum and carbon spectrum are measured by BrukeraVANCE-400/500 type nuclear magnetic instrument, and the measuring solvent is deuterated dimethyl sulfoxide (DMSO-d6) and deuterated chloroform (CDCl)₃) Internal standard Tetramethylsilane (TMS) and chemical shift of 10^-6(ppm) is given as a unit.

Mass spectral data were determined by an Agilent model 6120 mass spectrometer and an Agilent G6520Q-TOF.

The silica gel plate for Thin Layer Chromatography (TLC) is HSGF254 silica gel plate of cigarette platform yellow sea, and the silica gel plate for TLC is 0.1-0.2 mm.

The column chromatography generally uses 200-300 mesh silica gel of Taiwan yellow sea as a carrier.

Known starting materials for the present invention may be synthesized by or according to methods known in the art, or may be purchased from companies such as carbofuran, hydrania, alatin, piper, tetan, alfa aesar, chinese medicine, etc.

Hydrogen or nitrogen atmosphere means that the reaction flask is connected with a hydrogen or nitrogen balloon of about 1L solvent.

In the examples, unless otherwise specified, the reaction temperature is room temperature, and the temperature range is 20-25 ℃; developing with an ultraviolet lamp; the term "concentration" means that the solvent in the solution of the preparation compound is distilled off by a rotary evaporator.

The progress of the reaction in the examples was checked by Thin Layer Chromatography (TLC) using a developing system of: a: dichloromethane and methanol system, B: petroleum ether and ethyl acetate system, C; acetone and petroleum ether system, the volume ratio of the solvent is adjusted according to the polarity of the compound.

The system of column chromatography eluent used for purifying the compound comprises: a: dichloromethane and methanol system, B: petroleum ether and ethyl acetate system, C; the volume ratio of the acetone to the petroleum ether system is adjusted according to the different polarities of the compounds, and a small amount of triethylamine and an acidic or basic reagent can be added for adjustment. Wherein the boiling range of the petroleum ether is 60-90 ℃.

Preparation of intermediates

Intermediate 1- (S) -tetrahydro-1H, 3H-pyrrolo [1,2-c ] oxazole-1, 3-dione

L-proline (500mg, 4.35mmol) is added into a double-neck flask, the ultra-dry tetrahydrofuran is dissolved, and after nitrogen protection, the mixture is cooled in an ice bath under stirring, and then the ultra-dry tetrahydrofuran solution of the triphosgene (516mg, 1.74mmol) is added dropwise. After the addition was complete, the reaction was carried out in ice bath for 5 minutes at room temperature for 10 minutes and in an oil bath at 40 ℃ until the solution was completely clear (about 60 to 90 minutes). The reaction solvent was directly evaporated to dryness to obtain intermediate 1a, which was used directly as the next step without purification.

The intermediate 1a is dissolved in ultra-dry tetrahydrofuran (8mL), and cooled in an ice bath after being protected by nitrogen. A solution of triethylamine (605. mu.L, 4.35mmol) in extra dry tetrahydrofuran (2mL) was added dropwise with stirring, and after completion of the addition, reaction was carried out in an ice bath for 30 minutes to obtain intermediate 1, which was quickly used as a starting material for the next step.

Intermediate 2- (triphenyl-lambda)⁵-Phosphine ylide) acetic acid 2- (trimethylsilyl) ethyl ester

Bromoacetyl bromide (729. mu.L, 8.37mmol) was added to a two-necked flask and dissolved in ultra-dry dichloromethane, after nitrogen protection, the temperature was lowered in an ice bath. A solution of 2- (trimethylsilyl) ethanol (1.2mL, 8.37mmol) and triethylamine (1.16mL, 8.37mmol) in dichloromethane was added dropwise with stirring, and the ice bath was removed after the addition. After reaction at room temperature overnight, poured into water, dichloromethane extracted three times, the organic phases were combined, washed once with 2M hydrochloric acid, twice with saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated under reduced pressure to give intermediate 2a as a pale yellow oil, which was used directly as the next starting material.

Dissolving the intermediate 2a in ethyl acetate, adding triphenylphosphine (4.39g, 16.74mmol) under stirring, reacting at room temperature for 24 hours to obtain a large amount of white solid, directly filtering, washing the filter cake (easy to absorb moisture) with ethyl acetate, vacuum drying or quickly transferring the filter cake to a eggplant-shaped bottle, and evaporating by rotation of an oil pump to obtain an intermediate 2b (white solid, 3.39g) as a white solid with a yield of 80%. MS (ESI, M/z) 421[ M-Br^-]⁺。

Intermediate 2b was dissolved in toluene (8mL), and a toluene solution of triethylamine (2mL) was added dropwise with stirring at room temperature. After the dropwise addition, the reaction solution was reacted at room temperature for 30 minutes to obtain a yellow reaction solution, which was directly used for the next reaction.

Intermediate 3- (3-oxotetrahydro-1H, 3H-pyrrolo [1,2-c ] oxazole-1-ethylene) acetic acid 2- (trimethylsilyl) ethyl ester

The tetrahydrofuran (10mL, 4.35mmol) solution of the intermediate 1 and the toluene (10mL, 6.69mmol) solution of the intermediate 2 obtained above are directly mixed, and then the mixture is reacted at the temperature of 150 ℃ by microwave for 10 minutes or heated to the temperature of 150 ℃ by oil bath for 3 hours, silica gel is directly added for stirring sample, and the intermediate 3 (colorless oil, 76mg) is obtained by column chromatography, and the yield is 6.2%.¹H NMR (400MHz, chloroform-d) δ 5.62(d, J ═ 2.0Hz,1H),4.90(ddd, J ═ 8.9,6.7,1.7Hz,1H), 4.24-4.16 (M,2H), 3.72-3.63 (M,1H),3.28(ddd, J ═ 11.3,8.7,4.7Hz,1H),2.62(dtd, J ═ 13.1,6.8,3.4Hz,1H), 2.16-1.99 (M,2H), 1.64-1.53 (M,1H), 1.05-0.97 (M,2H),0.04(s,9H). MS (ESI, M/z):284[ M + H ]: M + H)]⁺。

Intermediate 4- (S, E) -2- (3-oxotetrahydro-1H, 3H-pyrrolo [1,2-c ] oxazole-1-ethylene) acetic acid

Intermediate 3(100mg, 0.35mmol) was dissolved in tetrahydrofuran, and 1M tetrabutylammonium fluoride (1.4mL, 1.40mmol) in tetrahydrofuran (5% water) was added with stirring, reacted overnight at room temperature, evaporated directly to dryness and sample stirred, and column chromatographed to give intermediate 4 (pale yellow solid, 50mg) in 79% yield. MS (ESI, M/z):182[ M-H]^-。

Intermediate 5-4 '- (trifluoromethyl) - [1,1' -biphenyl ] -4-carbaldehyde

P-bromotrifluorotoluene (500mg, 2.23mmol), 4-formylphenylboronic acid (335mg, 2.23mmol), sodium carbonate (473mg, 4.46mmol), palladium tetratriphenylphosphine (258mg, 0.223mmol) were dissolved in toluene: methanol: water (v: v: v) is 2: 1: 1, under the protection of nitrogen, stirring at 80 ℃ overnight. Subsequently, the reaction solution was poured into a saturated aqueous ammonium chloride solution, extracted three times with ethyl acetate, the organic phases were combined, washed three times with a saturated saline solution, dried over anhydrous magnesium sulfate, filtered to dryness, and subjected to column chromatography to obtain intermediate 5 (white solid, 463mg) with a yield of 83%.¹H NMR (400MHz, chloroform-d) delta 10.08(s,1H),7.99(d, J ═ 8.3Hz,2H),7.76(d, J ═ 8.2Hz,2H),7.74(s,4H), MS (ESI, M/z):251[ M + H ]: 251[ M + H]⁺。

Intermediate 6-2 ' -benzyloxy-4 ' -trifluoromethyl- [1,1' -biphenyl ] -4-carbaldehyde

2-bromo-5-trifluoromethylphenol (500mg, 2.08mmol) was dissolved in N, N-Dimethylformamide (DMF), and potassium carbonate (374mg, 2.71mmol), benzyl bromide (249. mu.L, 2.08mmol) and potassium iodide (17mg, 0.104mmol) were added successively with stirring, and reacted at 120 ℃ for three hours. The reaction solution was poured into a saturated aqueous ammonium chloride solution, extracted with ethyl acetate three times, the organic phases were combined, washed with saturated brine three times, dried over anhydrous magnesium sulfate, filtered, evaporated to dryness, and subjected to column chromatography to give intermediate 6a (colorless oil, 521mg) in a yield of 76%.¹H NMR (400MHz, chloroform-d) δ 7.71(d, J ═ 8.1Hz,1H),7.52(d, J ═ 7.3Hz,2H),7.45(t, J ═ 7.3Hz,2H),7.38(t, J ═ 7.2Hz,1H),7.19(s,1H),7.15(d, J ═ 8.2Hz,1H),5.21(s, 2H).

Intermediate 6a (521mg, 1.58mmol), 4-formylphenylboronic acid (237mg, 1.58mmol), sodium carbonate (335mg, 3.16mmol), tetrakistriphenylphosphine palladium (183mg, 0.158mmol) were dissolved in toluene: methanol: the water content is 2: 1: 1, under the protection of nitrogen, stirring at 80 ℃ overnight. Subsequently, the reaction solution was poured into a saturated aqueous ammonium chloride solution, extracted with ethyl acetate three times, the organic phases were combined, washed with saturated brine three times, dried over anhydrous magnesium sulfate, filtered to dryness, and subjected to column chromatography to give intermediate 6 (white solid, 444mg) in 79% yield.¹H NMR (400MHz, chloroform-d) δ 10.06(s,1H),7.93(d, J ═ 8.3Hz,2H),7.73(d, J ═ 8.2Hz,2H),7.47(d, J ═ 7.9Hz,1H),7.33(ddd, J ═ 12.3,7.4,4.3Hz,7H),5.14(s,2H). MS (ESI, M/z):357[ M + H: (M, H): 357 ═ 12.3,7.4,4.3Hz,7H)]⁺。

Intermediate 7-2 ' - (2,2, 2-trifluoroethyl) -4' -trifluoromethyl- [1,1' -biphenyl ] -4-carbaldehyde

Intermediate 7a (a colorless liquid, 583mg) was prepared in 87% yield based on the synthetic method for intermediate 6a starting from 2-bromo-5-trifluoromethylphenol (500mg, 2.08mmol) and 1,1, 1-trifluoro-2-iodoethane (205 μ L, 2.08 mmol).¹H NMR (400MHz, chloroform-d) δ 7.72(d, J ═ 8.2Hz,1H), 7.25-7.20 (m,1H),7.13(s,1H),4.46(q, J ═ 7.9Hz,2H).

Intermediate 7 (white solid, 510mg) was prepared in 81% yield based on the synthetic method for intermediate 6 using intermediate 7a (583mg, 1.81mmol) as a starting material.¹H NMR (400MHz, chloroform-d) delta 10.08(s,1H), 8.01-7.93 (M,2H),7.69(d, J ═ 8.2Hz,2H),7.53(d, J ═ 7.9Hz,1H), 7.49-7.41 (M,1H),7.20(s,1H),4.38(q, J ═ 7.9Hz,2H). MS (ESI, M/z):349[ M + H ]: 349[ M + H]⁺。

Intermediate 8-2- (4 '-formyl-4- (trifluoromethyl) - [1,1' -biphenyl ] -2-oxy) methylbenzonitrile

Intermediate 8a (colorless liquid, 613mg) was prepared in 83% yield based on the synthetic method for intermediate 6a starting from 2-bromo-5-trifluoromethylphenol (500mg, 2.08mmol) and 2-bromomethylbenzonitrile (406mg, 2.08 mmol).¹H NMR (400MHz, chloroform-d) δ 7.85(d, J ═ 7.8Hz,1H), 7.77-7.66 (m,3H),7.49(t, J ═ 7.6Hz,1H),7.22(s,1H),7.18(d, J ═ 8.2Hz,1H),5.36(s, 2H).

Intermediate 8 (white solid, 520mg) was prepared in 79% yield based on the synthetic method for intermediate 6 using intermediate 8a (613mg, 1.73mmol) as the starting material.¹H NMR (400MHz, chloroform-d) delta 10.06(s,1H),7.93(d, J ═ 8.2Hz,2H), 7.78-7.66 (M,3H),7.57(t, J ═ 7.7Hz,1H),7.49(d, J ═ 7.9Hz,1H), 7.47-7.38 (M,3H),7.34(s,1H),5.29(s,2H). MS (ESI, M/z):382[ M + H ], (M,3H) ]]⁺。

Intermediate 9-2- (4 '-formyl-4- (trifluoromethyl) - [1,1' -biphenyl ] -2-) methoxy benzonitrile

2-bromo-5-trifluoromethylbenzyl alcohol (500mg, 1.97mmol) was dissolved in ultra-dry dichloromethane and cooled in an ice bath. At 0 ℃, dropwise adding an ultra-dry dichloromethane solution of phosphorus tribromide (185 mu L, 1.97mmol), reacting at room temperature for three hours after dropwise adding, slowly adding the reaction solution into a saturated aqueous solution of sodium bicarbonate, extracting dichloromethane for three times, combining organic phases, washing the organic phases for three times by using saturated saline solution, drying the organic phases by using anhydrous magnesium sulfate, filtering and evaporating to dryness, and performing column chromatography to obtain an intermediate 9a (colorless oil, 510mg) with the yield of 82%.¹H NMR (400MHz, chloroform-d) δ 7.75-7.9 (m,2H),7.43(dd, J ═ 8.2,1.9Hz,1H),4.62(s, 2H).

Intermediate 9b (a colorless oil, 448mg) was prepared in 78% yield based on the synthetic method for intermediate 6a starting from intermediate 9a (510mg, 1.62mmol) and 2-hydroxybenzonitrile (193mg, 1.62 mmol).¹H NMR(400MHz, chloroform-d) δ 7.95(s,1H),7.74(d, J ═ 8.3Hz,1H),7.64(dd, J ═ 7.6,1.3Hz,1H), 7.60-7.54 (m,1H),7.49(d, J ═ 8.2Hz,1H),7.09(t, J ═ 7.6Hz,1H),7.03(d, J ═ 8.5Hz,1H),5.26(s, 2H).

Intermediate 9 (white solid, 394mg) was prepared in 82% yield based on the synthetic procedure for intermediate 6 starting from intermediate 9b (448mg, 1.26 mmol).¹H NMR (400MHz, chloroform-d) δ 10.07(s,1H), 8.00-7.93 (M,3H),7.74(d, J ═ 8.0Hz,1H), 7.63-7.56 (M,3H),7.48(d, J ═ 7.9Hz,2H), 7.08-7.01 (M,1H),6.81(d, J ═ 8.5Hz,1H),5.02(s,2H). MS (ESI, M/z):382[ M + H: (M,1H) ]]⁺。

Intermediate 10-N¹,N¹-diethyl-N²- ((4'- (trifluoromethyl) - [1,1' -biphenyl)]-4-yl) methyl) ethane-1, 2-ethylenediamine

Will N¹,N¹After dissolving diethyl-1, 2-ethylenediamine (281 μ L, 2.00mmol) and intermediate 5(500mg, 2.00mmol) in ultra-dry dichloromethane, adding two activated molecular sieves with stirring, reacting overnight at room temperature, directly evaporating the solvent to dryness, redissolving the residue with anhydrous methanol, adding sodium borohydride (76mg, 2.00mmol) with stirring, reacting for three hours at room temperature, directly adding silica gel, stirring, and performing column chromatography to obtain intermediate 10 (609 mg as a colorless oil) with a yield of 87%.¹H NMR (400MHz, chloroform-d) δ 7.68(s,4H),7.56(d, J ═ 8.1Hz,2H),7.43(d, J ═ 8.1Hz,2H),3.87(s,2H),2.72(t, J ═ 6.1Hz,2H),2.61(t, J ═ 6.1Hz,2H),2.54(q, J ═ 7.1Hz,4H),2.30(s,1H),1.02(t, J ═ 7.1Hz,6H), MS (ESI, M/z):351[ M + H:]⁺。

intermediate 11-N¹- ((2' - (benzyloxy) -4' (trifluoromethyl) - [1,1' -biphenyl)]-4-yl) methyl) -N²,N²-diethyl-1, 2-ethanediamine

Starting with intermediate 6 (500mg, 1.40)mmol), intermediate 11 (colorless oil, 531mg) was synthesized with yield 83% according to the synthesis method of intermediate 10.¹H NMR (400MHz, chloroform-d) δ 7.53(d, J ═ 7.9Hz,2H),7.44(d, J ═ 7.8Hz,1H),7.37(d, J ═ 7.9Hz,2H), 7.35-7.23 (M,7H),5.11(s,2H),3.85(s,2H),2.72(t, J ═ 6.1Hz,2H),2.59(t, J ═ 6.1Hz,2H),2.51(q, J ═ 7.1Hz,4H),1.00(t, J ═ 7.1Hz,6H), MS (ESI, M/z):457[ M + H: (M, J:, 7.7.1 Hz,6H)]⁺。

Intermediate 12-N¹,N¹-diethyl-N²- ((2'- (2,2, 2-trifluoroethoxy) - [1,1' -biphenyl)]-4-yl) methyl) ethane-1, 2-ethylenediamine

Intermediate 12 (colorless oil, 470mg) was synthesized in 73% yield according to the synthesis method for intermediate 10 using intermediate 7 as a starting material (500mg, 1.44 mmol).¹H NMR (400MHz, chloroform-d) δ 7.49(d, J ═ 8.2Hz,3H), 7.44-7.38 (M,3H),7.19(s,1H),4.30(q, J ═ 8.0Hz,2H),3.87(s,2H),2.73(t, J ═ 6.1Hz,2H),2.61(t, J ═ 6.1Hz,2H),2.53(q, J ═ 7.1Hz,4H),1.02(t, J ═ 7.1Hz,6H), MS (ESI, M/z):449[ M + H, 6H): 449[ M + H:]⁺。

intermediate 13-2- ((4'- ((((2- (diethylamino) ethyl) amino) methyl) -4- (trifluoromethyl) - [1,1' -biphenyl ] -2-oxy) methyl) benzonitrile

Intermediate 13 (colorless oil, 486mg) was synthesized in a yield of 77% by the method for synthesizing intermediate 10 using intermediate 8 as a starting material (500mg, 1.31 mmol).¹H NMR (400MHz, chloroform-d) δ 7.68(d, J ═ 7.6Hz,1H), 7.59-7.54 (M,1H),7.52(d, J ═ 8.1Hz,2H), 7.49-7.45 (M,2H),7.39(p, J ═ 8.9,8.4Hz,4H),7.29(s,1H),5.27(s,2H),3.86(s,2H),2.75(t, J ═ 6.1Hz,2H),2.63(t, J ═ 6.1Hz,2H),2.55(q, J ═ 7.1Hz,4H),1.03(t, J ═ 7.1Hz,6H), MS (ESI 482, M/z) ([ M + H): M, J ═ 7.1H), and M]⁺。

Intermediate 14, 2- ((4'- ((((2- (diethylamino) ethyl) amino) methyl) -4- (trifluoromethyl) - [1,1' -biphenyl ] -2-yl) methoxy) benzonitrile

Intermediate 13 (colorless oil, 499mg) was synthesized in a yield of 79% according to the synthesis method of intermediate 10 using intermediate 9 as a starting material (500mg, 1.31 mmol).¹H NMR (400MHz, chloroform-d) δ 7.94(s,1H),7.67(d, J ═ 8.0Hz,1H),7.57(d, J ═ 7.6Hz,1H), 7.52-. 41(M,4H),7.36(d, J ═ 8.0Hz,2H), 7.05-6.98 (M,1H),6.81(d, J ═ 8.5Hz,1H),5.08(s,2H),3.95(s,2H), 3.13-2.97 (M,8H),1.31(t, J ═ 7.3Hz,6H), MS (ESI, M/z):482[ M + H, 1H ], 1H ], 7.7.57 (d), 7.7.0, 1H), and 7.]⁺。

Intermediate 15- (5- ((2-aminoethyl) (ethyl) amino) pentyl) carbamic acid tert-butyl ester

N-Cbz ethanolamine (2g, 10.25mmol) and p-dimethylaminopyridine (DMAP, 250mg, 2.05mmol) were dissolved in dichloromethane and p-toluenesulfonyl chloride (TsCl, 2.15g, 11.28mmol) and triethylamine (Et) were added dropwise in this order under ice bath₃N, 1.56mL, 11.28mmol) in dichloromethane. After the dropwise addition, the reaction solution was slowly warmed to room temperature, stirred for 5 hours, poured into saturated aqueous ammonium chloride solution, extracted with dichloromethane three times, the organic phases were combined, washed with saturated brine three times, dried over anhydrous magnesium sulfate, filtered and evaporated to dryness, and subjected to column chromatography to obtain intermediate 15a (white solid, 3.26g) with a yield of 91%.¹H NMR (400MHz, chloroform-d) δ 7.76(d, J ═ 8.2Hz,2H), 7.40-7.27 (M,7H),5.21(s,1H),5.05(s,2H),4.08(t, J ═ 5.0Hz,2H),3.43(q, J ═ 5.4Hz,2H),2.42(s,3H). MS (ESI, M/z):350[ M + H: (M, H): 350: (M, J ═ 5.4Hz,2H)]⁺。

Intermediate 15a (3.26g, 9.34mmol), N-Boc cadaverine (1.45g, 7.18mmol) and potassium carbonate (2.6g, 18.68mmol) were dissolved in acetonitrile and heated to 50 ℃ in an oil bath overnight. After filtration, silica gel was added directly to stir the sample and column chromatography afforded intermediate 15b (colorless liquid, 1.39g) in 51% yield. MS (ES)I,m/z):380[M+H]⁺。

Intermediate 15b (1.39g, 3.67mmol) and N, N-diisopropylethylamine (DIPEA, 1.28mL, 7.34mmol) were dissolved in N, N-Dimethylformamide (DMF), and iodoethane (441. mu.L, 5.51mmol) was added with stirring and reacted at room temperature overnight. The reaction solution was poured into an aqueous ammonium chloride solution, extracted three times with ethyl acetate, the organic phases were combined, washed three times with saturated brine, dried over anhydrous magnesium sulfate, filtered and evaporated to dryness, and subjected to column chromatography to give intermediate 15c (a colorless liquid, 1.02g) in a yield of 68%. MS (ESI, M/z) 408[ M + H ]]⁺。

Intermediate 15C (1.02g, 2.50mmol) was dissolved in methanol, and after nitrogen substitution, 100mg of Pd/C was added, followed by hydrogen substitution. The reaction was allowed to react overnight at room temperature, filtered directly to dryness to give intermediate 15 as a colourless liquid, 634mg), 93% yield. Without purification, was used directly as next step. MS (ESI, M/z):274[ M + H]⁺。

Intermediate 16- (tert-butyl 5- (ethyl (2- (((2' - ((trifluoromethoxy) methyl) -4' - (trifluoromethyl) - [1,1' -biphenyl ] -4-yl) methyl) amino) ethyl) amino) pentyl) carbamate

Intermediate 13 (colorless oil, 741mg) was synthesized in 85% yield according to the synthesis method of intermediate 10, starting from intermediate 7(500mg, 1.44mmol) and intermediate 15(393mg, 1.44 mmol). MS (ESI, M/z):606[ M + H]⁺。

Intermediate 17-N¹- (2- (diethylamino) ethyl) -N¹- ((4'- (trifluoromethyl) - [1,1' -biphenyl)]-4-yl) methyl) propane-1, 3-diamine

Intermediate 10(500mg, 1.43mmol), N-Boc-3-aminopropylbromide (339mg, 1.43mmol), and potassium carbonate (257mg, 1.86mmol) were dissolved in N, N-Dimethylformamide (DMF) and stirred at room temperature overnight. Pouring the reaction solutionAdding into ammonium chloride aqueous solution, extracting with ethyl acetate for three times, combining organic phases, washing the organic phases with saturated saline solution for three times, drying with anhydrous magnesium sulfate, filtering, evaporating to dryness, and performing column chromatography to obtain an intermediate 17a (colorless liquid, 625mg) with the yield of 86%. MS (ESI, M/z):508[ M + H]⁺。

Intermediate 17a (625mg, 1.23mmol) was dissolved in a mixed solution of dichloromethane and trifluoroacetic acid (in DCM: TFA ═ 5:1 by volume) and reacted at room temperature overnight. The reaction solution was poured into an aqueous ammonium chloride solution, extracted three times with dichloromethane, the organic phases were combined, washed three times with saturated brine, dried over anhydrous magnesium sulfate, filtered and evaporated to dryness, and subjected to column chromatography to give intermediate 17 (a colorless liquid, 370mg) in 74% yield. MS (ESI, M/z) 408[ M + H ]]⁺。

Example 1- (S, E) -N, N-dimethyl-2- (3-oxotetrahydro-1H, 3H-pyrrolo [1,2-c ] oxazol-1-ylidene) acetamide (Compound 1)

Intermediate 4(20mg, 0.11mmol) was dissolved in N, N-Dimethylformamide (DMF), 2- (7-benzotriazole oxide) -N, N' -tetramethyluronium hexafluorophosphate (HATU, 62.7mmol, 0.17mmol), N-diisopropylethylamine (DIPEA, 39 μ L, 0.22mmol), dimethylamine hydrochloride (10mg, 0.12mmol) were added sequentially with stirring, reacted overnight at room temperature, the reaction solution was poured into an aqueous ammonium chloride solution, the aqueous phase was extracted 3 times with ethyl acetate, the organic phases were combined, washed 3 times with saturated saline, dried over anhydrous magnesium sulfate, filtered to dryness, and column chromatography was performed to give the title compound (light yellow solid, 20mg), yield 85%. MS (ESI, M/z) 211[ M + H]⁺。

Example 2- (S, E) -N- (2- (diethylamino) ethyl) -2- (3-oxotetrahydro-1H, 3H-pyrrolo [1,2-c ] oxazol-1-ylidene) -N- ((4'- (trifluoromethyl) - [1,1' -biphenyl ] -4-yl) methyl) acetamide (Compound 2)

Compound 2 (white solid, 48mg) was prepared with a yield of 84% according to the preparation method of compound 1, starting from intermediate 4(20mg, 0.11mmol) and intermediate 10(38mg, 0.11 mmol).¹H NMR (400MHz, chloroform-d) δ 7.67(t, J ═ 7.1Hz,4H),7.58(d, J ═ 8.2Hz,2H),7.28(d, J ═ 8.2Hz,2H),6.00(d, J ═ 1.8Hz,1H), 5.04-4.92 (m,1H),4.70(s,2H), 3.88-3.59 (m,3H), 3.36-3.13 (m,7H),2.60(dq, J ═ 12.1,5.7Hz,1H),2.11(dq, J ═ 14.0,7.1Hz,2H), 1.73-1.57 (m,1H),1.33(t, J ═ 7.2Hz,6H).¹³C NMR(126MHz,Chloroform-d)δ169.36,166.80,156.37,143.73,139.72,135.33,129.63(q,J＝32.6Hz),128.05(2C),127.36(2C),127.33(2C),125.83,125.80,124.21(d,J＝272.0Hz),93.49,64.52,53.29,52.73,48.27(2C),45.94,44.23,30.66,26.14,9.28(2C).HRMS(ESI):m/z[M+H]⁺C₂₈H₃₃F₃N₃O₃Computing 516.2469; measured, 516.2472.

Example 3- (S, E) -N- ((2' - (benzyloxy)) -4' - (trifluoromethyl) - [1,1' -biphenyl ] -4-yl) methyl) -N- (2- (diethylamino) ethyl) -2- (3-oxotetrahydro-1H, 3H-pyrrolo [1,2-c ] oxazol-1-ethylidene) acetamide (Compound 3)

Compound 3 (white solid, 55mg) was prepared in 80% yield according to the preparation method of compound 1 starting from intermediate 4(20mg, 0.11mmol) and intermediate 11(50mg, 0.11 mmol).¹H NMR (400MHz, chloroform-d) δ 7.59(d, J ═ 8.1Hz,2H),7.44(d, J ═ 7.9Hz,1H),7.34(t, J ═ 6.9Hz,6H),7.28(s,1H),7.25(d, J ═ 8.2Hz,2H),6.06(s,1H),5.13(s,2H),5.01(t, J ═ 7.9Hz,1H),4.72(s,2H),3.81(dt, J ═ 14.4,5.4Hz,1H), 3.76-3.66 (m,2H), 3.37-3.13 (m,7H), 2.65-2.60 (m,1H), 2.17-2.11 (m,2H), 1.70-1.64 (m,1H), t, 1.6H (t, 6H).¹³C NMR (126MHz, chloroform-d) δ 169.18,166.69,156.36,155.67,137.33,136.12,134.59,133.80,131.20,130.95(d, J ═ 32.6Hz),130.37(2C),128.62(2C),128.09,127.07(2C),126.48(2C),123.92(d, J ═ 272.4Hz),118.10(d, J ═ 3.5Hz),1009.84(d, J ═ 3.5Hz),93.54,70.75,64.50,53.01,48.26(2C),45.96,44.31,30.70,29.71,26.17,9.46(2C).HRMS(ESI):m/z[M+H]⁺C₃₅H₃₉F₃N₃O₄computing 622.2887; measured, 622.29.

Example 4- (S, E) -N- (2- (diethylamino) ethyl) -2- (3-oxotetrahydro-1H, 3H-pyrrolo [1,2-c ] oxazol-1-ylidene) -N- ((2' - (2,2, 2-trifluoroethoxy) -4' - (trifluoromethyl) - [1,1' -biphenyl ] -4-yl) methyl) acetamide (Compound 4)

Compound 4 (white solid, 56mg) was prepared in 82% yield according to the preparation method of compound 1 starting from intermediate 4(20mg, 0.11mmol) and intermediate 11(49mg, 0.11 mmol).¹H NMR (400MHz, chloroform-d) δ 7.51(d, J ═ 8.2Hz,2H),7.46(d, J ═ 8.0Hz,1H),7.39(d, J ═ 8.0Hz,1H),7.25(d, J ═ 8.1Hz,2H),7.18(s,1H),6.01(d, J ═ 1.7Hz,1H), 5.03-4.95 (m,1H),4.70(s,2H),4.35(q, J ═ 8.0Hz,2H), 3.87-3.60 (m,3H), 3.32-3.15 (m,7H), 2.66-2.57 (m,1H), 2.17-2.04 (m,2H), 1.71-1.56 (m,1H),1.32(t, 7H), 2.6H (t, 6H).¹³C NMR (126MHz, chloroform-d) δ 169.48,166.89,156.42,154.26,136.16,135.17,134.36,131.73,131.02(q, J ═ 32.7Hz),130.13(2C),126.68(2C),123.6(q, J ═ 272.2Hz),123.10(q, J ═ 278.5Hz),119.90(d, J ═ 3.5Hz),110.49(d, J ═ 3.5Hz),93.46,66.44(q, J ═ 35.9Hz),64.53,53.48,52.89,48.47(2C),45.90,44.38,30.63,26.08,9.19(2C), hrms (esi): M/z [ M + H: [ M + H ], (M, J ═ 3.5Hz),110.49(d, 3.6, 66.19 (2C)]⁺C₃₀H₃₄F₆N₃O₄Computing 614.2448; measured, 614.2448.

Example 5- (S, E) -N- ((2' - (2-cyanobenzyloxy) -4' - (trifluoromethyl) - [1,1' -biphenyl ] -4-yl) methyl) -N- (2- (diethylamino) ethyl) -2- (3-oxotetrahydro-1H, 3H-pyrrolo [1,2-c ] oxazol-1-ethylidene) acetamide (Compound 5)

Intermediate 4(20mg, 0.11 mmo)l) and intermediate 13(53mg, 0.11mmol) as starting materials, compound 5 (white solid, 52mg) was prepared in 73% yield according to the preparation method of compound 1.¹H NMR (400MHz, chloroform-d) δ 7.70(d, J ═ 7.5Hz,1H), 7.66-7.60 (m,1H),7.57(d, J ═ 8.1Hz,2H), 7.50-7.42 (m,4H),7.38(d, J ═ 8.1Hz,1H),7.32(s,1H),7.25(d, J ═ 8.1Hz,2H),6.03(d, J ═ 1.8Hz,1H),5.27(s,2H), 5.05-4.96 (m,1H),4.73(s,2H), 3.86-3.68 (m,3H), 3.40-3.14 (m,7H),2.62(dq, J ═ 12.2,6.3, 1H), 2.20-2.20 (m,1H), 2.35H, 1H), 1H, 71H, 1H, 7H, and 7.35H.¹³C NMR (126MHz, chloroform-d) δ 169.08,166.58,156.36,155.14,139.32,137.02,134.79,134.31,133.29,133.06,131.40,131.14(d, J ═ 32.7Hz),130.33(2C),128.89,128.62,126.46(2C),123.80(d, J ═ 273.4Hz),118.90(d, J ═ 3.5Hz),117.00,111.16,110.30(d, J ═ 3.5Hz),93.58,68.80,64.50,52.93,48.11(2C),45.96,44.19,30.69,29.70,26.18,9.36(2C). MS (ESI, M/z):647[ M + H,3, 5Hz ], 3, 36(2C). MS (ESI, M/z):647[ M + H]⁺。

Example 6- (S, E) -N- ((2' - (2-cyanophenoxy) methyl) -4' - (trifluoromethyl) - [1,1' -biphenyl ] -4-yl) methyl) -N- (2- (diethylamino) ethyl) -2- (3-oxotetrahydro-1H, 3H-pyrrolo [1,2-c ] oxazol-1-ethylidene) acetamide (Compound 6)

Compound 6 (white solid, 54mg) was prepared with a yield of 75% according to the preparation method of compound 1, starting from intermediate 4(20mg, 0.11mmol) and intermediate 14(53mg, 0.11 mmol). MS (ESI, M/z):647[ M + H]⁺。

Example 7 tert-butyl (S, E) -N- (5- (ethyl (2- (2- (3-oxotetrahydro-1H, 3H-pyrrolo [1,2-c ] oxazol-1-ylidene) -N- ((2' - (2,2, 2-trifluoroethoxy) -4' - (trifluoromethyl) - [1,1' -biphenyl ] -4-yl) methyl) acetylamino) ethyl) amino) pentyl) carbamate (Compound 7)

Intermediate 4(20mg, 0.11mmol) andintermediate 16(59mg, 0.11mmol) was used as a starting material, and compound 7 (white solid, 55mg) was prepared in 65% yield according to the preparation method of compound 1. MS (ESI, M/z):771[ M + H]⁺。

Example 8- (S, E) -N- (2- ((5-Aminopentyl) (ethyl) amino) ethyl) -2- (3-oxotetrahydro-1H, 3H-pyrrolo [1,2-c ] oxazol-1-ethylene-N- ((2' - (2,2, 2-trifluoroethoxy) -4' - (trifluoromethyl) - [1,1' -biphenyl ] -4-yl) methyl) acetamide (Compound 8)

Compound 7(55mg, 0.072mmol) was dissolved in Dichloromethane (DCM): trifluoroacetic acid (TFA) ═ 5:1, and stirred at room temperature overnight. The reaction solution was poured into a saturated aqueous sodium bicarbonate solution, extracted with dichloromethane three times, the organic phases were combined, washed with a saturated aqueous sodium chloride solution three times, and subjected to column chromatography to give compound 8 (colorless oil, 37mg) in 77% yield. MS (ESI, M/z) 671[ M + H]⁺。

Example 9- (S, E) -3- (5, 5-difluoro-7, 9-dimethyl-5H-4. lambda⁴,5λ⁴-dipyrrole [1,2-c:2',1' -f][1,3,2]Diazoleborane-3-alkenyl-N- (5- (ethyl (2- (2- (3-oxotetrahydro-1H, 3H-pyrrole [1, 2-c))]And oxazole-1-ethylene) -N- ((2' - (2,2, 2-trifluoroethoxy) -4' - (trifluoromethyl) - [1,1' -biphenyl]-4-yl) methyl) acetamido) ethyl) amino) pentyl) propionamide (compound 9)

Starting from compound 8(20mg, 0.030mmol) and 3-Bodipy-propionic acid (9mg, 0.030mmol), compound 9 (dark red solid, 24mg) was prepared in 85% yield in light-shielding conditions according to the preparation method for compound 1. HRMS (ESI) M/z [ M + H]⁺C₄₇H₅₄[¹¹B]F₈N₆O₅Computing 945.4116; measured, 945.4132.

Example 10- (S, E) -2- (6- (diethylamino) -3H-xanthen-9-yl) -5- (N- (5- (ethyl (2- (2- (3-oxotetrahydro-1H, 3H-pyrrolo [1,2-c ] oxazol-1-ethylene) -N- ((2' - (2,2, 2-trifluoroethoxy) -4' - (trifluoromethyl) - [1,1' -biphenyl ] -4-yl) methyl) acetylamino) ethyl) amino) pentyl) sulfamoyl) benzenesulfonic acid (Compound 10)

Dissolving the compound 8(20mg, 0.030mmol) in N, N-Dimethylformamide (DMF), sequentially adding N, N-diisopropylethylamine (DIPEA, 11 μ L, 0.060mmol) and lissamine rhodamine B sulfonyl chloride (18mg, 0.030mmol), stirring overnight at room temperature in the dark, directly evaporating to dryness, and performing column chromatography to obtain the compound 10 (bright red solid, 29mg) with a yield of 81%. HRMS (ESI) M/z [ M + H]⁺C₆₀H₆₉F₆N₆O₁₀S₂Computing 1211.4415; measured, 1211.4409.

Example 11- (S, E) -N- (3- ((2- (diethylamino) ethyl) ((4'- (trifluoromethyl) - [1,1' -biphenyl ] -4-yl) methyl) amino) propyl) -2- (3-oxotetrahydro-1H, 3H-pyrrolo [1,2-c ] oxazol-1-ethylidene) acetamide (Compound 11)

Compound 11 (white solid, 49mg) was prepared in 78% yield according to the preparation method of compound 1, starting from intermediate 4(20mg, 0.11mmol) and intermediate 17(45mg, 0.11 mmol). MS (ESI, M/z) 572[ M + H]⁺。

Pharmacological examples

Experimental example 1: in vitro inhibitory Activity test

Preparation of reagents

1) Reaction buffer

50mM Tris-HCl，1mM EGTA，pH 7.2

2) Thioester analogs of phosphatidylcholine (2-thio-PAF, Cayman, Lot 60945)

Dissolving with anhydrous ethanol, preparing 25mg/ml mother liquor, packaging, and storing at-80 deg.C. Diluted 900 times with the reaction buffer and used (concentration: about 50. mu.M)

3)DTNB(5,5’-dithio-bis-(2-nitrobenzoic acid),Sigma,Lot D8130)

Is prepared when in use. The solution was made up to 1.1mg/ml with triple distilled water and the appropriate amount of 0.1M NaOH was added until DTNB was just dissolved.

4) Compounds were dissolved in DMSO to appropriate concentrations.

Principle of experiment

Determination of Lp-PLA by using 2-thio-PAF as substrate₂And (4) activity. The sulfydryl generated after 2-thio-PAF hydrolysis can react with DTNB to generate a yellow substance, and the light density value is detected at 412nm, so that Lp-PLA is reflected₂And (4) activity.

1.1 Lp-PLA with human-derived recombinase as enzyme source₂And assay of PLA2VIIB enzyme inhibitory Activity (in vitro)

1.1.1 Experimental methods

1) The corresponding reagents were added according to the reaction system of table 1 below and mixed well with shaking.

Table 1: reaction system

	Blank hole	Sample well	Control well
				Reaction buffer	10μL
Human recombinant enzyme (enzyme source)		10μL	10μL
				DMSO	5μL	5μL
Sample (DMSO dissolution)		5μL

2) Add 10. mu.L DTNB per well.

3) 175. mu.L of reaction buffer 2-thio-PAF was added to each well and shaken on a microplate reader for 15s to mix the well liquids. The slope was determined at 412nM for 10min and once per minute. The inhibition rate was calculated according to the following formula:

the inhibition rate was 1- (slope sample well-slope blank)/(slope control well-slope blank well) × 100%.

1.1.2 the results of the experiment are shown in Table 2 below.

Table 2: Lp-PLA with partial compounds as enzyme sources for human-derived recombinase₂Inhibiting activity of

Compound numbering	IC₅₀(nM)
		1	311000
2	25
		3	14
4	13
		5	15
6	24
		9	14
10	25
		11	2500

Wherein compounds 2, 3,4, 5,6, 9 and 10 are each more than 200-fold selective for the PLA2VIIB enzyme, in particular, compound 2 is more than 1000-fold selective and compound 4 is more than 3000-fold selective.

Experimental example 2: inhibition of Lp-PLA in SD rats₂Activity assay

2.1 Experimental methods

3 SD rats per group, orally administered 25mg/kg and 50mg/kg, compound dissolved in carboxymethyl celluloseMixed solution of sodium ascorbate (sodium CMC) and DMSO (ratio of 20: 1). Collecting blood in orbit 0h before administration, 1h, 2h, 3h, 5h, 7h, and 24h after administration, anticoagulating with heparin, centrifuging at 3500rpm at 4 deg.C for 10min, collecting plasma, and measuring Lp-PLA in plasma₂And (4) activity.

2.2 results of the experiment

According to the test result of in vitro activity, compound 4 is selected to carry out Lp-PLA in SD rats₂The inhibitory activity was tested and the results are shown in FIG. 1.

The results show that after single administration, the compound 4 can remarkably inhibit Lp-PLA in SD rat plasma₂And there is a certain dose-effect relationship. The high selectivity and oral effectiveness of compound 4 indicates that Lp-PLA is designed based on the compound₂The feasibility of specific probe molecules of (1).

Experimental example 3: Lp-PLA₂Fluorescence labeling experiment of recombinant protein (in vitro)

Preparation of reagents

1) SDS-PAGE Loading buffer

Adding 5mL of 1.5M Tris-HCl (pH6.8), 2g of SDS, 10mL of glycerol, 30mg of bromophenol blue, 1mL of beta-mercaptoethanol in sequence, dissolving with ultrapure water to a constant volume of 20mL, and subpackaging with 1 mL/tube for preservation at-20 ℃.

2)10 Xelectrophoresis buffer

376g of glycine, 60.6g of Tris-Base and 20g of SDS are sequentially added, the mixture is dissolved by ultrapure water to be constant volume to 2L, the mixture is stored at normal temperature, and deionized water is added for dilution to 10 times when the mixture is used.

3) The fluorescent probe sample of the present invention is dissolved in DMSO to an appropriate concentration

4) Lp-PLA (human recombinant enzyme source)₂Diluted to the appropriate concentration with buffer (100mM Tris7.2, 1mM EDTA).

Principle of experiment

Lp-PLA (human recombinant enzyme source)₂After being incubated with a specific fluorescent probe, the probe is mixed with Lp-PLA₂Covalent reaction, fluorescence labeling the protein specificity band, SDS-PAGE, and fluorescence detecting the gel to obtain Lp-PLA₂The fluorescence signal of (1).

3.1 Experimental methods

1) Lp-PLA (human recombinant enzyme source)₂Incubate with fluorescent probes for 40min at 37 ℃.

2) SDS-PAGE, 80V constant pressure, 120 min.

3) Performing fluorescence scanning to obtain Lp-PLA with fluorescence signals₂Protein bands.

3.2 results of the experiment

Compounds 9 and 10 were selected for this experiment based on the results of the in vitro activity and selectivity assays, as shown in figure 2. The results show that all three compounds can sensitively detect Lp-PLA₂Protein and has obvious dose-effect relationship.

Experimental example 4: selective assay of Compound 4 on proteome in HEK293t cell lysates (in vitro)

Preparation of reagents

1)5 xSDS-PAGE loading buffer

2)10 Xelectrophoresis buffer

376g of glycine, 60.6g of Tris-Base and 20g of SDS are sequentially added, dissolved by ultrapure water to be constant volume to 2L, stored at normal temperature and diluted to 10 times by adding deionized water when in use.

3) The test probe sample and the compound of the invention are dissolved in DMSO to the appropriate concentration

5) HEK293t cell lysates were diluted to appropriate concentrations with lysis buffer.

Principle of experiment

The fluorescent probe FP-TAMAR can be combined with most serine hydrolases and fluorescently labeled. Therefore, we obtained the selectivity of the compound of the present invention by comparing the difference of the labeled protein species under the FP-TAMAR effect between the HEK293t cell lysate that had been treated with the compound of the present invention and that had not been treated. If the fluorescence signals of a protein in the two groups are similar, it is indicated that the protein does not bind to the covalent inhibitor, whereas if the fluorescence signal of the covalent inhibitor-treated group is significantly less than that of the non-treated group, it is indicated that the covalent inhibitor has a binding effect on the protein.

4.1 Experimental methods

1) Adding a proper amount of Lp-PLA of human recombinant enzyme source into HEK293t cell lysate₂Or the same amount of Lp-PLA of the human recombinant enzyme source₂And (4) a buffer solution.

2) Appropriate amount of compound 4 or equivalent DMSO was added to HEK293t cell lysate and incubated at 37 ℃ for 30 minutes.

3) The universal fluorescent probe FP-TAMAR was added to all groups to a final concentration of 10. mu.M and incubated for 30 min at 37 ℃.

4) SDS-PAGE, 80V constant pressure, 120 min.

5) And performing fluorescence scanning to obtain a protein band with a fluorescence signal.

4.2 results of the experiment

Based on the results of the in vitro activity and selectivity tests, compound 4 was selected for this experiment and the results are shown in figure 3. The result shows that the compound 4 has better selectivity on other proteins in HEK293t cell lysate and can be specifically contacted with Lp-PLA₂And (4) combining.

Experimental example 5: Lp-PLA₂Selective assay (in vitro) of specific fluorescent probes (Compound 9 and Compound 10) on the proteome of HEK293t cell lysates

Preparation of reagents

1) SDS-PAGE Loading buffer

2) Electrophoresis buffer solution

3) Lp-PLA in the present invention₂Fluorescent probes (Compound 9 and Compound 10) were dissolved in DMSO to appropriate concentrations

Principle of experiment

HEK293t cell lysate and Lp-PLA₂After the specific fluorescent probe is incubated, the probe is mixed with Lp-PLA₂Or other unknown protein covalent reaction, the protein specificity is marked with fluorescence, and the fluorescence signal of the marked protein is obtained by SDS-PAGE and fluorescence detection of the gel.

5.1 Experimental methods

2) Adding an appropriate amount of Lp-PLA in the invention into HEK293t cell lysate₂Fluorescent probes (compound 9 and compound 10) or equal amounts of DMSO and incubated at 37 ℃ for 30 minutes.

3) SDS-PAGE, 80V constant pressure, 120 min.

4) And performing fluorescence scanning to obtain a protein band with a fluorescence signal.

5.2 results of the experiment

Compounds 9 and 10 were selected for this experiment based on the results of the in vitro activity and selectivity assays, and the results are shown in figure 4. The result shows that the compound 9 has better selectivity on other proteins in HEK293t cell lysate and can be specifically contacted with Lp-PLA₂Binding Lp-PLA₂Characterized from a number of proteins.

Claims

1. A compound represented by the general formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

wherein R is₁Selected from hydrogen, C₁-C₆Alkyl or-O- (CH)₂)_m-R₄Wherein, the C is₁-C₆Alkyl is unsubstituted or optionally substituted with a group selected from: halogen, cyano, nitro, C₁-C₄Alkoxy radical, C₆-C₁₀An aryloxy group, a carboxyl group, or a hydroxyl group; r₄Is selected from C₁-C₆Alkyl radical, C₆-C₁₀Aryl or C₆-C₁₀Heteroaryl radical, R₄C in (1)₁-C₆Alkyl radical, C₆-C₁₀Aryl or C₆-C₁₀Heteroaryl is unsubstituted or optionally substituted by a group selected from: halogen, cyano, nitro, C₁-C₄Alkoxy, carboxyl, or hydroxyl; m is an integer of 0 to 2;

R₂、R₃each independently selected from hydrogen and C₁-C₆Alkyl, or- (CH)₂)_n-NR₅R₆Wherein, the C is₁-C₆Alkyl is unsubstituted or optionally substituted with a group selected from: halogen, cyano, nitro, C₁-C₄Alkoxy, carboxyl, or hydroxyl; r₅、R₆Each independently selected from hydrogen and C₁-C₅Acyloxy group,

n is an integer of 1 to 5;

the absolute configuration of the chiral center is optionally in the R or S configuration; the relative configuration of the exocyclic double bond in the parent nucleus of the oxazole ring is either the Z or E configuration.

2. A class of compounds represented by formula I, stereoisomers thereof or pharmaceutically acceptable salts thereof, according to claim 1, wherein:

R₁selected from hydrogen, C₁-C₄Alkyl or-O- (CH)₂)_m-R₄Wherein, the C is₁-C₄Alkyl is unsubstituted or optionally substituted with a group selected from: halogen or C₆-C₁₀An aromatic oxy group; r₄Is selected from C₁-C₆Alkyl or C₆-C₁₀Aryl radical, R₄C in (1)₁-C₆Alkyl or C₆-C₁₀Aryl is unsubstituted or optionally substituted with a group selected from: halogen or cyano; m is 0 or 1;

R₂、R₃each independently selected from C₁-C₄Alkyl, or- (CH)₂)_n-NR₅R₆Wherein, the C is₁-C₄Alkyl is unsubstituted or optionally substituted with a group selected from: cyano radicals, C₁-C₄Alkoxy, carboxyl, or hydroxyl; r₅、R₆Each independently selected from hydrogen, t-butyloxycarbonyl, methyl tert-butyloxycarbonyl, ethyl tert-butyloxycarbonyl, and ethyl-butyloxycarbonyl,

n is 4 or 5.

3. A class of compounds represented by formula I, stereoisomers thereof or pharmaceutically acceptable salts thereof, according to claim 1, wherein:

R₂、R₃each independently selected from ethyl, 5-amino-n-pentyl, 5-tert-butoxycarbonylamino-n-pentyl, and,

4. A class of compounds represented by formula I, stereoisomers thereof or pharmaceutically acceptable salts thereof, according to claim 1, wherein:

R₁selected from the group consisting of 1,1, 1-trifluoroethoxy, phenoxymethyl which is unsubstituted or substituted by cyano, benzyloxy which is unsubstituted or substituted by cyano;

R₂、R₃each independently selected from ethyl or- (CH)₂)_n-NR₅R₆；R₅、R₆One is hydrogen and the other is selected from lisiane rhodanese B sulfonyl or 3-boropyrrolyl; n is 5;

wherein the lissamine rhodanine B sulfonyl structure is:

the structure of the 3-fluoroboropyrrolyl propionyl group is as follows:

5. the class of compounds represented by formula I, stereoisomers thereof, or pharmaceutically acceptable salts thereof, according to claim 4, wherein:

R₁is 1,1, 1-trifluoroethoxy.

6. A class of compounds represented by formula I, stereoisomers thereof or pharmaceutically acceptable salts thereof, according to claim 1, wherein: the compound of the general formula I is selected from any one of the compounds represented by the following structural formula:

7. the method for preparing the compound represented by the general formula I according to any one of claims 1 to 6, which comprises the following steps:

(2) carrying out condensation reaction on the compound Ic and the compound Id to obtain a compound I;

wherein R is₁、R₂And R₃Are as defined in the respective claims.

8. The process for preparing a compound of formula I according to claim 1 or 2, wherein R is₂And R₃One of which is ethyl and the other is- (CH)₂)_n-NR₅R₆When compounds of formula If are synthesized by the following reaction scheme:

wherein R is₁、R₅And R₆And n is as defined in the respective claims; the method is selected from one of the following two methods:

or the second method: the compound R₅-OH and/or compound R₆Dissolving OH in DCM, DMF, acetonitrile or DMSO, adding condensing agent under stirring, sequentially adding organic base or inorganic base and compound Ie, and reacting at 0 deg.C to room temperature for 4-24 hrTo obtain the compound of the general formula If.

9. A pharmaceutical composition characterized by: comprising a compound of general formula I according to any one of claims 1 to 6, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; and pharmaceutically acceptable adjuvants.

10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition further comprises one or more of a hypolipidemic agent, a cholesterol-lowering agent, an anti-atherosclerotic agent, an anti-multiple sclerosis agent, an antineoplastic agent, a hypoglycemic agent, an insulin sensitizer, an anti-anginal agent, an anti-inflammatory agent, a hypotensive agent, or a lipoprotein a-lowering agent.

11. Use of a compound of general formula I according to any one of claims 1 to 6, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 9 or 10 for the preparation of a medicament for inhibiting Lp-PLA₂Use in active medicine.

12. Use of a compound of general formula I according to any one of claims 1 to 6, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 9 or 10, for the preparation of a medicament for the prevention and/or treatment and/or amelioration of atherosclerosis and/or multiple sclerosis and/or tumors.