CN107459511B - Anti-enterovirus 71(EV71) 4-iminooxazolidine-2-ketone compound and preparation method and application thereof - Google Patents

Abstract

4-iminooxazolidine-2-one enterovirus 71(EV71)3C protease inhibitor has a structural general formula shown as compound (M), variables in the structure are defined in the specification, and the compounds effectively inhibit or block the replication of enterovirus 71. The invention relates to discovery and application of a compound containing a structure shown as a formula (M), various optical isomers, pharmaceutically active metabolites, pharmaceutically acceptable salts, solvates and prodrugs thereof in preparation of antiviral drugs for treating hand-foot-and-mouth virus infection diseases. The invention also relates to an intermediate and a synthetic method for preparing the compound with the structure of the formula (M).

Description

Anti-enterovirus 71(EV71) 4-iminooxazolidine-2-ketone compound and preparation method and application thereof

Technical Field

The present invention relates to compounds of formula (M) and pharmaceutical compositions for the treatment of enterovirus 71(EV71) infection, and methods of synthesis, formulation and compounds for use in such synthesis. In particular, the invention provides 4-iminooxazolidine-2-ones, pharmaceutical compositions containing such compounds and methods of treating EV71 infection with such compounds.

Background

Hand-Foot-Mouth disease (HFMD) is a common global infectious disease caused by enteroviruses, and the prevalence of the disease is reported in most regions and countries of the world. The hand-foot-and-mouth disease is mainly transmitted through a feces-mouth path and a respiratory tract, and is strong in infectivity and easy to cause epidemic or outbreak. The disease is mainly developed by infants under 6 years old, most patients have slight symptoms, and the disease is mainly characterized by fever and rash or herpes at the parts of hands, feet, oral cavity and the like. A small number of patients can be complicated with aseptic meningitis, encephalitis, acute flaccid paralysis, respiratory tract infection, myocarditis and the like, and individual severe children are quick in disease progression and easy to die. The clinical manifestations are fever and rashes in the hands, feet, mouth and other parts. More seriously, the virus can also invade the respiratory system, the central nervous system and the like of a patient to cause symptoms such as encephalitis, pulmonary edema, flaccid paralysis, myocarditis and the like, the disease progresses quickly, and death is easy to occur. Southeast Asia and China are high-incidence areas of hand-foot-and-mouth disease all the time, particularly in recent years, with the common influence of various factors such as personnel mobility and virus variation, the disease has a large outbreak in provinces such as Shandong, Henan and the like in China, thousands of cases of infant death are caused, and huge economic and mental damage is brought to infant families.

There are more than 20 enteroviruses causing hand-foot-and-mouth disease, enterovirus 71 and coxsackievirus A group 16, 4, 5, 9 and 10 types, and coxsackievirus B group 2 and 5 types are all the more common viruses of hand-foot-and-mouth disease, wherein the most common are enterovirus 71(EV71) and coxsackievirus A16 (Cox A16). Virology and epidemiological studies have confirmed that human Enterovirus type 71 (Enterovirus 71, EV71) is the main causative agent of human hand-foot-and-mouth disease which has developed in recent years, and is also responsible for a variety of nervous system-related diseases such as aseptic meningitis (aseptic meningitis), brainstem encephalitis (brain encephalitis) and polio-like paralysis (poliomyelitis syndrome). In recent years, EV71 has caused multiple outbreaks, and researches show that the genotype of EV71 is changed continuously in the circulation, and gene mutation at certain sites causes the change of the pathogenicity of EV71, so that the prevention and treatment of EV71 face considerable pressure. At present, the specific mechanism of the EV71 virus for forming virus persistence and causing hand-foot-and-mouth disease is not clarified, a specific and effective therapeutic drug is still lack clinically, and only a traditional Chinese medicine or other antiviral drugs can be adopted for treatment, and researches show that a great number of participants have no favorable effect on the treatment and death cases continuously occur clinically. Therefore, there is a need to develop anti-EV 71 virus formulations for hand-foot-and-mouth disease with high efficacy specificity.

EV71 was first isolated from a stool specimen of an infant with CNS disease in 1969 by Schmit et al, and the virus particles are of a typical regular icosahedral structure and belong to the genus Enterovirus of the family picornaviridae. The gene consists of about 7400 nucleotides, belongs to a single-stranded positive-strand RNA virus, only contains an open-reading frame (ORF), encodes polyprotein (polyprotein) consisting of 2194 amino acids, and is respectively 746bp 5 'non-coding region (UTR) and 83bp 3' non-coding region on two sides of the genome. The EV71 genome encodes a polyprotein (polyprotein) of about 2193 amino acids. In infected cells, the polyprotein is hydrolyzed to three precursor proteins, P1, P2, and P3. Through further shearing of protease of cells and viruses, the P1 precursor protein can be further matured into four virus structural proteins of VP1, VP2, VP3 and VP4, and is responsible for assembly and stabilization of virus particles; the P2 precursor protein is further matured into non-structural proteins (nsp) 2A (specific protease), 2B and 2C; the P3 precursor protein was used to form the nonstructural proteins 3A, 3B (VPg, 5' terminal binding protein), 3C (specific protease) and 3D (RNA-dependent RNA polymerase, RdRp).

Of the seven non-structural proteins, the 3C protease is thought to function mainly as a specific protease, belonging to the cysteine protease, and the active center of the 3C protease is a catalytic triad consisting of Cys147, His40, and Glu71, which is responsible for specifically cleaving each protein encoded by the virus from multimers to form proteins with independent functions, so that once the function of the protein is lost, further transcription and replication of the virus will not proceed normally. Therefore, the effect of inhibiting 3C protease by the specifically recognized selective small molecules is an effective means for treating EV71 virus infection, and the 3C protease becomes an important drug target for treating hand-foot-and-mouth disease.

The invention designs a series of 4-imino oxazolidine-2-ketone 3C protease inhibitors by combining the combination sites of active regions based on the crystal structure characteristics of EV 713C protease.

The first advantage of the invention is that the 4-imino oxazolidine-2-ketone inhibitor provided by the invention can effectively inhibit EV 713C protease.

The second advantage of the invention is that the provided 4-imino oxazolidine-2-ketone inhibitor can effectively inhibit SARS virus and MERS-CoV main protein Nsp5 protease.

The third advantage of the invention is that the invention can be used for preparing the medicine for curing diseases induced by cysteine protease, such as poliomyelitis, common cold, etc.

The compounds I-VI are found to have good activity on EV71 virus 3C protease and NSP5 protease of SARS virus and MERS-CoV through biological activity experiments, show very good inhibitory activity on EV71 virus in cell culture, and have good pharmacokinetic properties in a living body.

The object of the present invention is to find a particularly effective class of small molecule compounds against enterovirus 71 and to provide intermediates for the synthesis of said protease inhibitor compounds and methods for the synthesis of these.

The invention content is as follows:

the invention aims at overcoming the defects of the prior art of EV71 virus 3C protease inhibitor, provides a virus 3C protease inhibitor containing EV71 of a formula (M), and also aims at providing synthesis of the intermediate of the protease inhibitor compound and a synthesis method for the synthesis.

The invention relates to application of 4-imino oxazolidine-2-ketone compounds and/or pharmaceutically acceptable salts and/or hydrates in preparation of medicines for treating enterovirus 71(EV71) infection diseases. These compounds are useful for inhibiting the 3C protease of the EV71 virus, or for the prevention/treatment of one or more symptoms of infection by the EV71 virus, as a pharmaceutically acceptable salt and/or hydrate thereof, or as a component of a pharmaceutical composition, whether or not co-administered with other antiviral agents, anti-infective agents, immunomodulators or antibiotics for the treatment of hand-foot-and-mouth disease.

More specifically, the invention relates to an application of a compound of formula (M) and/or a pharmaceutically acceptable salt and/or a hydrate in preparing a medicament for treating enterovirus 71(EV71) infectious diseases:

wherein

R₁Represents-aryl, -aralkyl, -heteroaralkyl;

R₂represents-H, -F;

R₃represents C1-6 alkyl, -aryl, -heteroaryl.

The pharmaceutical compositions encompassed within the scope of the present invention comprise an anti-EV 71 virus effective amount of a compound of formula (M) or a therapeutically acceptable salt thereof, in admixture with a pharmaceutically acceptable pharmaceutical carrier or adjuvant.

An important aspect of the present invention is directed to a method of treating an enterovirus 71(EV71) infectious disease in a mammal by administering to the mammal an amount of a compound of formula M, or a therapeutically acceptable salt or ester thereof, or a composition thereof, effective against EV71 virus.

Another important aspect of the present invention relates to the search for effective agents for the treatment of hand-foot-and-mouth disease by exposing the virus to a composition of formula (M), or a therapeutically acceptable salt or ester thereof, which inhibits the EV71 virus, such as the above-mentioned compositions.

Other aspects relate to pharmaceutical compositions that may additionally include other anti-EV 71 agents, and may also include inhibitors of other targets of the EV71 virus, such as 3D protease inhibitors and VP1 protein inhibitors.

Detailed description of the preferred embodiments

Defining:

as used herein, the following definitions apply unless otherwise noted:

in referring to each example, (R) or (S) is used to indicate the absolute configuration of the asymmetric center, which is an indication for the entire compound and not for the individual substituents.

When identified herein as "P1, P2, P3", it is intended to refer to the position of the residue of an amino acid beginning at the C-terminus and extending towards the N-terminus of the peptide analog, i.e., P1 represents the first position from the C-terminus and P2 is the second position from the C-terminus (see Berger A. & Schechter I., Transactions of the Royal Society London series B257, 249-264 (1970)).

The term "C1-6 alkyl" as used herein, whether used alone or in combination with another substituent, refers to a non-cyclic, straight or branched alkyl substituent containing from 1 to 6 carbon atoms and includes, for example, methyl, ethyl, propyl, butyl, hexyl, 1-methylethyl, 1, 1-dimethylethyl, 1-methylpropyl and 2-methylpropyl.

The term "aryl" as used herein means an aromatic monocyclic ring system containing 6 carbon atoms, or an aromatic bicyclic ring system containing 10 atoms, such as phenyl and naphthyl-ring systems.

The term "heteroaryl" as used herein, alone or in combination with another substituent, means a monovalent substituent derived from a five, six or seven membered unsaturated heterocyclic ring having 1, 2 or 3 heteroatoms selected from N, O, S, attached through a ring carbon or nitrogen atom, with the removal of hydrogen. Examples of suitable heterocycles are: thiophene, furan, pyrrole, imidazole, pyrazole, thiazole, oxazole, isoxazole, 1, 2, 3-triazole, 1, 2-thiadiazole, pyridine, pyrazine, pyrimidine, 1, 2, 4-triazine, benzoxazole, benzothiazole, quinoline.

The term "pharmaceutically acceptable salt" as used herein refers to salts of the compounds of formula (M) which are suitable for use in normal medical treatment in contact with the tissues of humans and animals without toxicity, irritation, allergic response, or the like. Generally water-soluble or oil-soluble, or readily dispersible, and are effective in their use. The term includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

The term "pharmaceutically acceptable acid addition salts" refers to salts formed with inorganic acids such as sulfuric acid, nitric acid, phosphoric acid, hydrochloric acid, hydrobromic acid, sulfamic acid and the like, and organic acids such as acetic acid, trifluoroacetic acid, trichloroacetic acid, cinnamic acid, citric acid, maleic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzoic acid, benzenesulfonic acid, glycolic acid, malic acid, lactic acid, malonic acid, oxalic acid, nicotinic acid, succinic acid, salicylic acid, stearic acid, tartaric acid, sulfanilic acid, trimethylbenzenesulfonic acid, p-methylbenzenesulfonic acid, mandelic acid, pectinic acid, picric acid, propionic acid and the like, which retain the biological activity and the properties of the free base, and which are not biologically or otherwise desired.

The term "pharmaceutically acceptable base addition salts" refers to salts formed with inorganic bases such as ammonia or ammonium or metal cations such as hydroxides or carbonates of sodium, magnesium, copper, zinc, calcium, potassium, aluminum and the like, particularly preferably ammonium, potassium, sodium, calcium, magnesium salts, which retain the properties of the biologically active and free acid and are not biologically or otherwise undesirable. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts formed from primary, secondary and tertiary amines, quaternary ammonium compounds, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, tripropylamine, isopropylamine, tributylamine, ethanolamine, diethanolamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, piperazine, piperidine, purine, tetramethylammonium compounds, tetraethylammonium compounds, pyridine, N, N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, N, N-dibenzylphenethylamine and the like. Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, caffeine.

The 4-iminooxazolidine-2-ones of the invention may be present in free form or in salt form. Pharmaceutically acceptable salts of many compound types and methods for their preparation are known to those skilled in the art. Pharmaceutically acceptable salts include conventional non-toxic salts, including the quaternary ammonium salts of such compounds formed from bases and inorganic or organic acids.

The compounds of the present invention may form hydrates or solvates. The person skilled in the art knows the hydrates formed when the compounds are lyophilized together with water or the solvates formed when concentrated in solution with a suitable organic solvent.

The invention encompasses pharmaceutical compositions comprising a therapeutic amount of a compound of the invention, and one or more pharmaceutically acceptable carriers and/or excipients. Carriers include, for example, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier or excipient may also include time delay materials known in the art, such as glyceryl monostearate or glyceryl distearate, and may also include waxes, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate and the like. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. The composition can be in the form of a liquid, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder. The composition can be formulated into suppository with conventional binder and carrier such as triglyceride. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose and magnesium carbonate, and the like. Depending on the desired formulation, the formulation may be designed to mix, granulate and compress or dissolve the ingredients. In another approach, the composition may be formulated as nanoparticles.

The pharmaceutical compositions of the present invention may be administered in a wide variety of pharmaceutical forms. The pharmaceutical carrier used may be solid or liquid.

If a solid carrier is used, the preparation may be in the form of a tablet, powder or pellet placed in a hard gelatin capsule or in the form of a lozenge or troche. The amount of solid carrier varies widely, but is preferably from about 25mg to about 1.0 g. Typical solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. The solid carrier may comprise one or more substances which may act simultaneously as flavouring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrants; it may also be an encapsulating material. In powders, the carrier is a finely divided solid which is in admixture with the finely divided active ingredient. In tablets the active ingredient is mixed with the carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. Powders and tablets preferably contain up to 99% active ingredient.

If a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable solution or suspension in an ampoule or vial or in a nonaqueous liquid suspension. Typical liquid carriers include syrup, peanut oil, olive oil, water, and the like. Liquid carriers are used to prepare solutions, suspensions, emulsions, syrups, tinctures and sealed compositions. The active ingredient may be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or a pharmaceutically acceptable oil or fat. The liquid carrier may contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, pigments, viscosity regulators, and tonicity agents. Suitable examples of liquid carriers for oral and parenteral administration include water (containing in part additives as described above, e.g., cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). The carrier for parenteral administration may also be an oil or fat such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are sterile liquid compositions for parenteral administration. The liquid carrier for the pressurized composition may be a halogenated hydrocarbon or other pharmaceutically acceptable propellant. Sterile solutions or suspension solutions liquid pharmaceutical compositions may be used, for example, for intravenous, intramuscular, intraperitoneal or subcutaneous injection. The suspension may be formulated according to the known art using suitable dispersing or wetting agents, such as tween 80, and suspending agents. The injection can be performed by single push or gradual injection for 30 min. The compounds may also be administered orally in the form of liquid or solid compositions. The term parenteral as used herein includes subcutaneous, intradermal, intramuscular, intravenous, intraarticular, intrasynovial, intrasternal, intrathecal and intralesional injection or infusion techniques.

To obtain a stable water-soluble dosage form, the compound or a pharmaceutically acceptable salt thereof may be dissolved in an aqueous solution of an organic or inorganic acid, 0.3M succinic or citric acid solution. Alternatively, the acidic derivative may be dissolved in a suitable alkaline solution. If a soluble form is not obtained, the compound may be dissolved in a suitable co-solvent or combination thereof. Examples of such suitable co-solvents include, but are not limited to, ethanol at a concentration ranging from 0 to 60% by volume of the total volume, propylene glycol, polyethylene glycol 300, polysorbate 80, glycerol, polyoxyethylene fatty acid esters, fatty alcohols or glycerol hydroxy fatty acid esters, and the like.

The pharmaceutical compositions of the present invention may be administered orally, parenterally or via an implanted reservoir, oral administration or administration by injection being preferred. Various delivery systems are known and may be used for the administration of compounds or other various formulations including tablets, capsules, injectable solutions, capsules in liposomes, microparticles, microcapsules, and the like. Methods of introduction include, but are not limited to, cutaneous, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular and (generally preferred) oral routes. The compounds may be administered by any convenient or other suitable route, for example by infusion or bolus injection, by absorption through epithelial or mucosal lines (e.g., oral mucosa, rectal and intestinal mucosa, etc.) or by drug-loaded stents and may be administered together with other biologically active agents. Can be administered systemically or locally. For use in the treatment or prevention of a nasal, bronchial or pulmonary disorder, the preferred route of administration is oral, nasal or bronchial aerosol or nebulizer.

Other suitable excipients or carriers that may be used with the formulations and compositions mentioned above may be found in standard pharmacological texts, for example in "Remington's Pharmaceutical Sciences", 19 th edition. For the prevention and treatment of hand-foot-and-mouth disease caused by the EV71 virus, the 3C protease inhibitor compounds described herein are useful in monotherapy in a dosage range of between about 0.01 to about 100mg/kg body weight per day, preferably between 0.5 to 75mg/kg body weight per day. Typically, the pharmaceutical compositions of the present invention will be administered from about 1 to 5 times per day, or in addition to a continuous infusion. Such drugs are useful as chronic or acute treatments. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form may vary depending upon the host treated and the particular mode of administration. A representative formulation will contain from about 5% to about 95% active ingredient (weight/weight). Preferably, such formulations contain from about 20% to about 80% of the active compound.

This will be understood by those familiar with the art that higher or lower doses than those mentioned above may be required. The particular dosage and mode of treatment for a particular patient will depend upon a variety of factors including the activity of the particular compound employed, the age, weight, sex, general health, diet, time of administration, metabolic rate, drug combination of the patient, and the severity and course of the infection, the patient's predisposition to infection, and the judgment of the treating physician. In general, treatment is initiated at small doses substantially below the optimal dose of the compound. The dose is then increased by a small increment until the optimum effect is reached in this case. In general, it is desirable to administer the compounds at concentrations that are generally sufficient to produce effective antiviral results, but do not cause any harmful or adverse side effects.

When the compositions of the present invention comprise a compound of formula (M) in combination with one or more additional therapeutic or prophylactic agents, the compound and additional agent(s) should be present in an amount to provide a dosage level of between about 10 and 100%, more preferably about 10 to 80%, typically in a single treatment regimen.

The resulting compositions are administered in vivo to a mammal, such as a human, when these compounds or pharmaceutically acceptable salts thereof are formulated with a pharmaceutically acceptable carrier, to treat or prevent EV71 viral infection.

In preferred embodiments, these methods are useful for reducing EV71 replication capacity in mammals. If the pharmaceutical composition includes only a compound of the invention as an active ingredient, such methods may additionally comprise administering to the mammal an agent selected from the group consisting of an immunomodulator, an antiviral agent, another EV71 virus 3C protease inhibitor, or another target in the EV71 life cycle, such as a 3D protease inhibitor and a VP1 protein inhibitor. Such additional formulations may be administered to the mammal prior to, concurrently with, or subsequent to the administration of the compositions of the present invention.

Process flow

The compounds of formula (M) of the present invention can be efficiently prepared by the process of the present invention, including the following general synthetic methods. R in these synthetic methods₁，R₂，R₃As defined above.

The process flow I:

the intermediate 1-4 is synthesized by the route of the scheme I, in the scheme, glutamic acid 1-1 is used as a raw material, carboxyl is firstly subjected to methyl esterification protection, then amino is subjected to functional group protection to obtain an intermediate 1-2, and then the compound 1-2 is subjected to deprotonation by LiHMDS (or other strong basic reagents) to introduce cyanoethyl groups to obtain an intermediate 1-3. Then reducing the cyano group of the intermediate 1-3, further carrying out intramolecular amidation reaction, and carrying out intramolecular cyclization to obtain the key intermediate 1-4.

Preparation of the Compound N-Boc-glutamic acid dimethyl ester (1-2) of step I-1

Acetyl chloride (5.0mL) was slowly added dropwise to methanol (100.0mL) at 0 deg.C, stirred for 5 minutes, then glutamic acid (10.0g, 67.9mmol) was added, stirring was continued and heated to reflux, and the reflux temperature was maintained for reaction for 2 hours. The reaction was stopped, and the solvent was distilled off under reduced pressure. The resulting oil was dissolved in THF, TEA (28.54mL, 203.7mmol) was added dropwise at 0 ℃ and stirred at 0 ℃ for 5 minutes, followed by addition of di-tert-butyl dicarbonate (17.78g, 81.5mmol) dissolved in THF (30.0mL) and stirring to room temperature for reaction for 2.5 hours. After completion of the reaction, the solvent was removed under reduced pressure, the residue was dissolved in water (200.0mL), a citric acid solution was added to acidify the mixture to PH 4, DCM (2 × 100.0mL) was added for extraction, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, which was purified by flash chromatography (PE: EA ═ 5: 1) to obtain the objective compound (1-2) (17.8g, yield 95.2%).

Step I-preparation of 2-t-Butoxycarbonylamino-4-cyanoethyl-glutaric acid dimethyl ester (1-3) as compound

Lithium bis (trimethylsilyl) amide (78.5mL of 1.0M in THF, 78.5mmol) was added to a-78 deg.C solution of dimethyl N-Boc-glutamate (1-2) (10.0g, 36.4mmol) in anhydrous THF (200.0mL) and the resulting solution was stirred at this temperature for 30 min. Bromopropionitrile (3.4mL) was then slowly added dropwise and the reaction mixture was stirred at-78 ℃ for an additional 2 hours. After the reaction was complete, the reaction was quenched by the addition of glacial acetic acid (5.0mL) and stirred to room temperature. The solvent was removed under reduced pressure, and the residue was dissolved with water (100.0mL), extracted with DCM (100.0mL × 3), the organic phases were combined, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, and then concentrated to obtain a crude product, which was purified by flash chromatography (PE: EA ═ 2: 1) to obtain the objective compound (1-3) (7.1g, yield 59.5%).

Step I-3 preparation of Compound 2-tert-Butoxycarbonylamino-3- (2-carbonyl-3-piperidinoalkyl) -propionic acid methyl ester (1-4)

To a solution of dimethyl 2-tert-butoxycarbonylamino-4-cyanoethyl-glutarate (1-4) (5.0g, 15.9mmol) in methanol (80.0mL) was added cobalt chloride hydrate (4.0g, 15.9mmol), followed by addition of sodium borohydride (6.0g, 159.5mmol) in portions to the resulting pink solution at 0 deg.C and stirring at room temperature for 18 hours. The reaction was monitored by TLC, and after completion of the reaction, the reaction was quenched by addition of saturated aqueous ammonium chloride (30.0mL) and stirred for 10 min. The solid impurities were removed by suction filtration, the volatile solvent was removed under reduced pressure, the residual liquid was extracted with DCM (100.0 mL. times.3), and the organic phase was washed with water (2X 50.0 mL). The combined organic phases were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered to remove the solvent and the crude product was purified by flash chromatography (EA) to give the key intermediate (1-4) (2.9g, 60.7% yield).

Scheme II

Structural units 2-4 are synthesized by scheme IITo obtain, wherein R is₁，R₂The groups represented have already been described above. Taking a compound 2-1 as a raw material, firstly carrying out carboxyl methyl esterification protection to obtain a compound 2-2, and then carrying out condensation with different organic acids 2-3 to obtain an intermediate 2-4.

Scheme III

The compound of formula (M) in the invention is prepared by removing amino protecting group from compound 1-4 and removing carboxyl protecting group from compound 2-4, condensing, and further derivatizing to obtain compound 3-1.

Procedure IV

The scheme is to carry out structural derivatization of a compound 3-1 to obtain R₁，R₂Compounds of formula (M) are different groups.

Step IV-1 Compound 2- [2-R₂-phenyl-amino-1-carbonyl-3-R₁]Preparation of propylamino-3- (2-carbonyl-3-piperidinoalkane) -propanol (4-1)

To 2- [2-R₂-phenyl-amino-1-carbonyl-3-R₁]To a methanol solution (10.0mL) of propylamino-3- (2-carbonyl-3-piperidinoalkane) -propionic acid methyl ester (1.0equiv.) was added sodium borohydride (10.0equiv.) in portions, and the mixture was stirred at room temperature for 2 hours. The reaction was quenched by the addition of saturated aqueous ammonium chloride (5.0mL) and methanol was removed under reduced pressure. Extraction was performed with DCM (3 × 50.0ml), the combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated to give the crude product which was purified by flash chromatography (DCM: MeOH ═ 50: 1) to give the title compound (4-1) (87.8% yield).

Step IV-2 Compound 2- [2-R₂-phenyl-amino-1-carbonyl-3-R₁]Preparation of propylamino-3- (2-carbonyl-3-piperidinoalkane) -propionaldehyde (4-2)

To 2- [2-R₂-phenyl-amino-1-carbonyl-3-R₁]Absence of propylamino-3- (2-carbonyl-3-piperidinoalkane) -propanol (1.0equiv.)Aqueous DCM (10.0mL) solution was added DMP (1.0equiv.) and stirred for 2 hours. The reaction was quenched by the addition of saturated sodium bicarbonate (2.0mL) and sodium thiosulfate (2.0equiv.) was added and stirred until the organic phase was clear. Extraction with DCM (3 × 50.0mL), drying of the combined organic phases over anhydrous sodium sulphate and concentration gave a crude product which was purified by flash chromatography (DCM: MeOH ═ 30: 1) to give the title compound 4-2 (81.3% yield).

Step IV-3 Compound 1-cyano-2- [2-R₂-phenyl-amino-1-carbonyl-3-R₁]Preparation of (E) -propylamino-3- (2-carbonyl-3-piperidinoalkane) -propanol (4-3)

To 2- [2-R₂-phenyl-amino-1-carbonyl-3-R₁]To a solution of propylamino-3- (2-carbonyl-3-piperidinoalkane) -propionaldehyde (1equiv.) in DCM (1mL) was added a saturated aqueous solution (0.1mL) (40%) of sodium hydrogen sulfite (5equiv.) and stirred for 30 minutes. Then, an aqueous solution (0.5ml) of potassium cyanide (52.1mg, 0.8mmol) was added dropwise at 0 ℃ and reacted at room temperature for 24 hours. Extraction with DCM (3 × 10mL), drying of the combined organic phases over anhydrous sodium sulphate and concentration gave the crude product which was purified by flash chromatography (DCM: MeOH ═ 40: 1) to give the desired compound 4-3 (52.3% yield).

Step IV-4 α preparation of monosubstituted Carbamate cyanamide (4-4)

4-3(500mg, 1.06mmol) was dissolved in 50.0mL of anhydrous dichloromethane, and N, N' -carbonyldiimidazole (390mg, 1.17mmol) in dichloromethane was slowly added dropwise over 1 hour under nitrogen protection and ice bath. After the addition, the reaction was carried out at room temperature for 30 minutes under nitrogen protection. After completion of the reaction, triethylamine (150mg, 1.27mmol) was added. After the addition, the reaction was carried out at room temperature for 2 hours under nitrogen protection. After completion of the reaction, the reaction mixture was washed with water (50 mL. times.2), a saturated ammonium chloride solution (50 mL. times.2), a saturated sodium bicarbonate solution (50 mL. times.2) and a saturated saline solution (50 mL. times.2) in this order. The organic phase was collected. Anhydrous Na for organic phase₂SO₄Drying, filtration and collection of the filtrate, evaporation of the solvent under reduced pressure and column chromatography of the residue (dichloromethane: methanol 60: 1v/v) gave the product α -monosubstituted carbamate cyanide (4-4) as a white solid (48% yield).

Step IV preparation of 54-Iminooxazolidin-2-one (4-5)

4-4(400mg, 0.72mmol) was dissolved in 50.0mL of anhydrous dichloromethane and triethylamine (145mg, 1.44mmol) was added. After the addition, the reaction was carried out at room temperature for 24 hours. After completion of the reaction, the reaction mixture was washed with water (50 mL. times.2). The organic phase was collected. Anhydrous Na for organic phase₂SO₄Drying, filtration and collection of the filtrate, evaporation of the solvent under reduced pressure and column chromatography of the residue (dichloromethane: methanol 45: 1v/v) gave the product 4-5 as a white solid (25% yield).

Examples of the invention

The present invention is illustrated in more detail by the following non-limiting examples, but the present invention is not limited to the following examples. The temperatures are provided in degrees celsius in the examples below. Unless otherwise stated, solution percentages represent weight to volume and solution ratios represent volume to volume relationships. The structure of the compounds of the examples was determined by one or more of the following methods: nuclear magnetic resonance spectrometer, high resolution mass spectrometry, thin layer chromatography. If a given structural formula representing a compound does not conform to its chemical name, the structural formula is taken as the standard.

Nuclear magnetic resonance spectrum (¹H NMR and¹³c NMR) were measured on a Bruker400 spectrometer at 400MHz field strength. Chemical shifts are expressed in parts per million (ppm, δ) shifts relative to the tetramethylsilane standard internal standard.¹The multiplicity of peaks in H-NMR is shown below: s is singlet; d is doublet; t is a triplet; m is multiplet. Coupling constants are expressed in hertz. The solvent peak is referenced to the internal deuterated reagent. The commercial reagents used are obtained separately from their respective suppliers and are described elsewhere herein if the conditions to be treated are present. Tetrahydrofuran (THF) is obtained by distilling with sodium-benzophenone system before use; dichloromethane (DCM) was distilled from calcium hydride before use.

The following abbreviations are used herein: me: a methyl group; MeOH: methanol; boc: tert-butoxycarbonyl; TEA: triethylamine; EtOAc: ethyl acetate; DMP: dess-martin reagent; PE: petroleum ether; et (Et)₂O: diethyl ether; TFA: trifluoroacetic acid; EDC: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride); HOBt: 1-hydroxybenzotriazole hydrate. Further, "L" represents a natural oneAmino acids present. FBS: fetal bovine serum; PBS solution: a phosphate buffer; PBST: adding Tween-20 into the phosphate buffer; ESMS: carrying out electrospray mass spectrometry; MS: mass spectrometry analysis; HPLC: high performance liquid chromatography.

Examples of embodiments of the invention are as follows:

EXAMPLE 1 preparation of N-Boc-L- (+) -glutamic acid dimethyl ester (1-2)

Acetyl chloride (5mL) was slowly added dropwise to methanol (100mL) at 0 deg.C, stirred for 5 minutes, then glutamic acid (10g, 67.9mmol) was added, stirring was continued and heated to reflux, and the reflux temperature was maintained for reaction for 2 hours. The reaction was stopped, the solvent was removed under reduced pressure and recrystallized from ether. The resulting oil was dissolved in THF (150mL), TEA (28.5mL, 203.7mmol) was added dropwise at 0 ℃ and stirred at 0 ℃ for 5 minutes, followed by addition of di-tert-butyl dicarbonate (17.8g, 81.5mmol) dissolved in THF (30mL) and stirring to room temperature for 2.5 hours. After the reaction was completed, the solvent was evaporated under reduced pressure, water (200mL) was added to the residue, and extracted from the aqueous phase with DCM (2 × 200mL), the combined organic phases were dried over anhydrous sodium sulfate and then concentrated, and the resulting crude product was purified by flash chromatography (PE: EA ═ 5: 1) to give N-Boc-L- (+) -dimethyl glutamate (17.7g, yield 95.2%) as a colorless oily liquid, TLC: r_f＝0.5 (PE∶EA＝5∶1)；¹H-NMR(400MHz，CDCl₃)：δ5.36(m，1H)，4.32(m，1H)，3.75(s，3H)，3.68(s，3H)，2.43(m，2H)，2.19(m，1H)，1.96(m，1H)，1.440(s，9H)；¹³C-NMR (100MHz，CDCl₃)：δ173.0，172.6，155.3，79.7，52.7，52.2，51.6，30.0，28.1(3)，27.5。

EXAMPLE 22 preparation of tert-Butoxycarbonylamino-4-cyanoethyl-glutaric acid dimethyl ester (1-3)

At-78 deg.C, the bisLithium (trimethylsilyl) amide (78.5mL of a 1.0M solution in THF, 78.5mmol) was added slowly dropwise to a solution of N-Boc-L- (+) -glutamic acid dimethyl ester (1-2) (10g, 36.4mmol) in anhydrous THF (200mL) and the resulting solution was stirred at this temperature for 30 min. Then, while maintaining the temperature, bromopropionitrile (3.4mL) was slowly added dropwise and the reaction mixture was stirred at-78 ℃ for an additional 2 hours. After the reaction was complete, glacial acetic acid (5mL) was added to quench the reaction and stir to room temperature. The solvent was removed under reduced pressure, water (200mL) was then added, the aqueous phase was extracted with DCM (2 × 200mL), the combined organic phases were dried over anhydrous sodium sulphate and concentrated to give the crude product which was purified by flash chromatography (PE: EA ═ 2: 1) to give dimethyl 2-tert-butoxycarbonylamino-4-cyanoethyl-glutarate (7.1g, 59.5% yield) as a pale yellow oily liquid, TLC: r_f＝0.4(PE∶EA＝2∶1)；¹H-NMR(400MHz，CDCl₃)：δ 5.07(d，J＝8.8Hz，1H)，4.36(dd，J＝12.4Hz，1H)，3.75(s，3H)，3.72(s，3H)，2.63(m， 1H)，2.39(m，2H)，1.96-2.06(m，4H)，1.45(s，9H)；¹³C-NMR(100MHz，CDCl₃)：δ174.38，172.34，155.37，118.67，80.33，52.56，52.17，51.56，40.78，34.46，28.26， 27.32，15.15。

EXAMPLE 32 preparation of tert-Butoxycarbonylamino-3- (2-carbonyl-3-piperidinoalkane) -propionic acid methyl ester (1-4)

To a solution of dimethyl 2-tert-butoxycarbonylamino-4-cyanoethyl-glutarate (1-4) (5g, 15.9mmol) in methanol (80mL) was added cobalt chloride hydrate (4g, 14.6mmol), followed by slow addition of sodium borohydride (6g, 157.9mmol) in portions to the resulting pink mixture at 0 deg.C and stirring at room temperature for 18 hours. The reaction was quenched by the addition of saturated aqueous ammonium chloride (30mL) and stirred for 10 min. Filtering to remove solid impurities, and evaporating to remove the volatile solvent under reduced pressure. The aqueous phase was extracted with DCM (3X 100mL), the combined organic phases were dried over anhydrous sodium sulfate and concentrated to give the crude product which was purified by flash column (EA) to give 2-tert-butoxycarbonylamino-3- (2-carbonyl-3-piperidinane)) Methyl propionate (2.9g, yield 60.7%) as white foamy solid, TLC: r_f＝0.4(EA)；¹H-NMR(400MHz，CDCl₃)：δ6.007(s，1H)， 5.60(d，J＝8.4Hz，1H)，4.32(m，1H)，3.73(s，3H)，3.2(t，J＝3.2Hz，2H)，2.32(m，1H)， 1.60-1.90(m，4H)，1.44(s，9H)；¹³C-NMR(100MHz，CDCl₃)：δ174.54，173.21， 155.92，79.74，52.26，51.75，42.29，38.03，34.25，28.29，26.57，21.57。

Example 4 preparation of Cinnamomylcarbonylphenylalanine methyl ester (2-4)

To a solution of methyl phenylalanine (10g, 55.8mmol) in DCM (100mL) at 0 deg.C was added cinnamic acid (9.9g, 67.0mmol), EDC (16.1g, 83.8mmol), HOBt (11.3 g, 83.8mmol) in that order, then TEA (35.2mL, 251.4mmol) was added dropwise and stirred to room temperature for 2 hours. The solvent was evaporated under reduced pressure, water (200mL) was added, DCM (2 × 200mL) was used for extraction from the aqueous phase, the combined organic phases were dried over anhydrous sodium sulfate and concentrated to give the crude product which was purified by flash chromatography (PE: EA ═ 2: 1) to give cinnamoylcarbonylamino-phenylalanine methyl ester (13.8g, 80.2% yield) as a white solid, TLC: r_f＝0.5(PE∶EA＝2∶1)；¹HNMR(400MHz，CDCl₃)：δ7.64-7.60(d，1H， J＝16Hz)，7.47-7.11(m，10H)，6.42-6.38(d，1H，J＝16Hz)，5.06-5.02(m，1H)，3.74(s， 3H)，3.26-3.14(m，2H)；¹³C-NMR(100MHz，CDCl₃)：δ172.2，165.4，141.8，135.9， 134.6，129.8，129.3(2)，128.8(2)，128.6(2)，127.9(2)，127.2，120.0，53.4，52.4，37.9。

EXAMPLE 5 preparation of Cinnamomoylcarbonylphenylalanine (2-5)

To cinnamoylcarbonylamino-phenylalanine methyl ester (2-4) (10g, 32.3mmol) in methanol and water (100 mL: 200 m)L) to the solution was added lithium hydroxide (2.0g, 48.5mmol), and the mixture was stirred at room temperature for 1.5 hours. The solvent was evaporated under reduced pressure and 1N hydrochloric acid was added to adjust the pH to 3. Extraction with ethyl acetate (3 × 100mL), combining the organic phases, drying over anhydrous sodium sulfate, and concentration gave cinnamoylcarbonylamino-phenylalanine (8.6g, 90.6% yield) as a white solid, TLC: r_f＝0.1(PE∶EA＝2∶1)；¹H-NMR(400MHz， CDCl₃)：δ7.65-7.61(d，1H，J＝16Hz)，7.35-7.18(m，10H)，6.39-6.35(d，1H，J＝16Hz)， 5.02-5.00(m，1H)，3.30-3.20(m，2H)；¹³C-NMR(100MHz，CDCl₃)：δ166.4，142.6，135.7，134.4，130.1，129.4(2)，128.9(2)，128.6(2)，128.8(2)，128.0，127.3，119.4，53.7，53.7，37.2。

EXAMPLE 62 preparation of methyl- [2- (cinnamoylcarbonyl) -amino-1-carbonyl-3-phenyl ] propylamino-3- (2-carbonyl-3-piperidinoalkane) -propionate (3-1)

To 2-tert-butoxycarbonylamino-3- (2-carbonyl-3-piperidinoalkyl) -propionic acid methyl ester (5g, 16.6mmol) in dry DCM (80mL) was added TFA (13mL) dropwise at 0 deg.C, and the mixture was stirred in ice bath for 1.5 h. The solvent was evaporated under reduced pressure and the pH was adjusted to neutral with triethylamine. Then dissolved in DCM (100mL), cinnamyl carbonylamino-phenylalanine (5.8g, 19.9mmol), EDC (4.7g, 24.9mmol), HOBt (3.4g, 24.9mmol) were added sequentially at 0 deg.C, then TEA (10.5mL, 74.7mmol) was added dropwise, and stirred to room temperature for 2 hours. Washed with water (3 × 50mL), the organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude product which was purified by flash chromatography (DCM: MeOH ═ 20: 1) to give 2- [2- (cinnamoylcarbonyl) -amino-1-carbonyl-3-phenyl ] carbonyl]Propylamino-3- (2-carbonyl-3-piperidine) -propionic acid methyl ester (6.9g, 87.8%) as a white foamy solid, TLC: r_f＝0.5(DCM∶MeOH＝20∶1)；¹H-NMR(400MHz，CDCl₃)：δ7.49(d， J＝16Hz，1H)，7.21-7.35(10H)，6.46(d，J＝16Hz，1H)，4.87(q，J＝3.2Hz，J＝4Hz，1H)， 3.73(s，3H)，3.42(m，1H)，3.09-3.25(m，4H)，2.41(m，1H)，2.23(m，1H)，1.30-1.80 (6H)；¹³C-NMR(100MHz，CDCl₃)：δ172.05，165.26，141.89，135.83，134.63， 129.89，128.85，128.63，127.92，127.19，119.96，53.31，52.42，37.91。

EXAMPLE 71 preparation of cyano-2- [2- (cinnamoylcarbonyl) -amino-1-carbonyl-3-phenyl ] propylamino-3- (2-carbonyl-3-piperidinoalkane) -propanol (3-2)

To 2- [2- (cinnamoylcarbonyl) -amino-1-carbonyl-3-phenyl]A solution of propylamino-3- (2-carbonyl-3-piperidinoalkane) -propionic acid methyl ester (3-1) (1g, 2.2mmol) in methanol (10mL) was added dropwise to sodium borohydride (0.8g, 21.6mmol) and stirred at room temperature for 2 hours. The reaction was quenched by addition of saturated aqueous ammonium chloride (5mL), the volatile solvent was evaporated under reduced pressure, extracted with DCM (3 × 50mL), the combined organic phases were dried over anhydrous sodium sulfate and concentrated to give a crude product which was purified by flash chromatography (DCM: MeOH ═ 20: 1) to give 2- [2- (cinnamoylcarbonyl) -amino-1-carbonyl-3-phenyl ] carbonyl]Propylamino-3- (2-carbonyl-3-piperidinoalkane) -propanol (0.8g, 80.9% yield) was a white foamy solid, TLC: r_f＝0.4(DCM∶MeOH＝20∶1)；¹H-NMR(400MHz，CDCl₃)：δ7.49(d，J＝16Hz，1H)，7.21-7.35(10H)，6.46(d，J＝16Hz，1H)，4.87 (q，J＝3.2Hz，J＝4Hz，1H)，3.71(m，2H)，3.42(m，1H)，3.09-3.25(m，4H)，2.41(m， 1H)，2.23(m，1H)，1.30-1.80(6H)；¹³C-NMR(100MHz，CDCl₃)：δ179.89，171.21， 166.12，141.64，136.76，134.62，129.87，129.37，128.82，128.50，127.94，126.86， 120.28，66.2，54.89，54.81，41.08，39.11，38.96，29.42，28.10，26.72。

EXAMPLE 82 preparation of- [2- (cinnamoylcarbonyl) -amino-1-carbonyl-3-phenyl ] propylamino-3- (2-carbonyl-3-piperidinoalkane) -propionaldehyde (3-3)

To 2- [2- (cinnamoylcarbonyl) -amino-1-carbonyl-3-phenyl]To a solution of propylamino-3- (2-carbonyl-3-piperidinoalkane) -propanol (3-2) (1g, 2.3mmol) in anhydrous DCM (10mL) was added DMP (1.5g, 3.4mmol), and the mixture was stirred for 2 hours. Adding saturated hydrogen carbonateThe reaction was quenched with sodium (2.0mL) and sodium thiosulfate (1.1 g, 6.9mmol) was added and stirred until the organic phase was clear. DCM (3 × 50mL) was added for extraction, the combined organic phases were dried over anhydrous sodium sulfate and concentrated to give the crude product which was purified by flash chromatography (DCM: MeOH ═ 30: 1) to give 2- [2- (cinnamoylcarbonyl) -amino-1-carbonyl-3-phenyl ] carbonyl]Propylamino-3- (2-carbonyl-3-piperidinoalkane) -propionaldehyde (0.83g, 80.7% yield) was a white foamy solid, TLC: r_f＝0.5(DCM∶MeOH ＝20∶1)；¹H-NMR(400MHz，CDCl₃)：δ9.40(s，1H)，8.41(d，J＝6.4Hz)，7.58(d，J＝16Hz，1H)，7.23(10H)，6.49(d，J＝16Hz，1H)，5.08(m，1H)，4.32(m，4H)，1.47-2.27 (7H)；¹³C-NMR(100MHz，CDCl₃)：δ200.20，175.09，172.29，165.80，141.51，136.46，134.72，129.79，129.49，128.81，128.53，127.91，126.97，120.38，57.09，54.38，42.19， 38.82，37.18，30.74，27.06，21.1。

EXAMPLE 91 preparation of cyano-2- [2- (cinnamoylcarbonyl) -amino-1-carbonyl-3-phenyl ] propylamino-3- (2-carbonyl-3-piperidinoalkane) -propanol (3-4)

To 2- [2- (cinnamoylcarbonyl) -amino-1-carbonyl-3-phenyl]To a solution of propylamino-3- (2-carbonyl-3-piperidinoalkyl) -propionaldehyde (3-3) (100mg, 0.2mmol) in DCM (1mL) was added a saturated aqueous solution (0.1mL) (40%) of sodium bisulfite (84.2mg, 0.8mmol), and the mixture was stirred for 30 minutes. Then, an aqueous solution (0.5mL) of potassium cyanide (52.3mg, 0.8mmol) was added dropwise at 0 ℃ and the reaction was carried out at room temperature for 24 hours. Extraction with DCM (3 × 10mL), drying of the combined organic phases over anhydrous sodium sulphate and concentration, purification of the crude product by flash chromatography (DCM: MeOH ═ 40: 1) gave 1-cyano-2- [2- (cinnamoylcarbonyl) -amino-1-carbonyl-3-phenyl-carbonyl]Propylamino-3- (2-carbonyl-3-piperidinoalkane) -propanol (50.6mg, yield 50.2%) as a white foamy solid, TLC: r_f＝0.5(DCM∶MeOH＝10∶1)；¹H-NMR(400MHz， CDCl₃)：δ8.35(d，J＝7.6Hz，1H)，7.54(d，J＝16Hz，1H)，7.23(10H)，6.97(d，J＝7.6Hz， 1H)，6.46(d，J＝16Hz，1H)，4.96(q，J＝6.8Hz；14.0Hz)，4.53(1H)，4.22(1H)，3.19 (4H)，1.43-2.24(8H).¹³C-NMR(100MHz，CDCl₃)：δ175.42，172.86，166.08，141.67， 136.36，134.62，129.83，129.48，128.80，128.58，127.92，126.99，120.23，118.72， 64.30，54.57，51.30，42.25，38.47，37.66，31.50，26.99，21.15。

EXAMPLE 101 preparation of cyano-2- [2- (3-methyl-oxazolecarbonyl) -amino-1-carbonyl-3-phenyl ] propylamino-3- (2-carbonyl-3-piperidinoalkyl) -propanol (3-5)

To 2- [2- (3-methyl-oxazolecarbonyl) -amino-1-carbonyl-3-phenyl]To a solution of propylamino-3- (2-carbonyl-3-piperidinoalkyl) -propionaldehyde (100mg, 0.2mmol) in DCM (1mL) was added a saturated aqueous solution (0.1mL) (40%) of sodium bisulfite (84.2mg, 0.8mmol), and the mixture was stirred for 30 minutes. Then, an aqueous solution (0.5mL) of potassium cyanide (52.3mg, 0.8mmol) was added dropwise at 0 ℃ and the reaction was carried out at room temperature for 24 hours. Extraction with DCM (3 × 10mL), drying of the combined organic phases over anhydrous sodium sulphate and concentration gave the crude product which was purified by flash chromatography (DCM: MeOH ═ 40: 1) to give 1-cyano-2- [2- (3-methyl-oxazolecarbonyl) -amino-1-carbonyl-3-phenyl-carbonyl]Propylamino-3- (2-carbonyl-3-piperidinoalkane) -propanol (50.2mg, yield 50%) was a white foamy solid,¹H NMR(400MHz，CDCl₃)δ：8.38(d，J＝7.4Hz，1H)，7.61(d， J＝8.1Hz，1H)，7.23(d，J＝7.2Hz，2H)，6.96(t，J＝8.2Hz，2H)，6.35(s，1H)，4.93 (q，J＝14.1，7.0Hz，1H)，4.57(d，J＝4.3Hz，1H)，4.24(m，1H)，3.36-3.17(m，3H)， 3.12(dd，J＝14.0，7.5Hz，1H)，3.29(m，2H)，3.22(dd，J＝13.7，7.8Hz，1H)，3.12 (dd，J＝14.0，7.5Hz，1H)，2.46(s，3H)，2.36-2.14(m，2H)，2.10-1.97(m，1H)， 1.77(m，3H)，1.51(dd，J＝21.8，10.7Hz，1H)；¹³C NMR(100MHz，CDCl₃)δ： 175.56，172.00，171.32，161.96(d，J_C-F＝245.0Hz)，159.10，158.18，131.86(d，J_C-F＝3.1Hz)，131.01(d，J_C-F＝8.0Hz)，118.62，115.45(d，J_C-F＝21.3Hz)，101.38， 64.19，54.51，51.25，42.31，37.72，37.64，31.43，26.95，21.19，12.30.HRMS(ESMS)： C₂₃H₂₆FN₅NaO₅(M+Na)⁺，calcd.494.1810，found494.1812。

example 11 α preparation of carbamate cyanides (3-6)

3-5(500mg, 1.06mmol) was dissolved in 50.0mL of anhydrous dichloromethane, and N, N' -carbonyldiimidazole (390mg, 1.17mmol) in dichloromethane was slowly added dropwise over 1 hour under nitrogen protection and ice bath. After the addition, the reaction was carried out at room temperature for 30 minutes under nitrogen protection. After completion of the reaction, triethylamine (150mg, 1.27mmol) was added. After the addition, the reaction was carried out at room temperature for 2 hours under nitrogen protection. After completion of the reaction, the reaction mixture was washed with water (50 mL. times.2), a saturated ammonium chloride solution (50 mL. times.2), a saturated sodium bicarbonate solution (50 mL. times.2) and a saturated saline solution (50 mL. times.2) in this order. The organic phase was collected. Anhydrous Na for organic phase₂SO₄Drying, filtration and collection of the filtrate, evaporation of the solvent under reduced pressure and column chromatography of the residue (dichloromethane: methanol 60: 1v/v) gave product 3-6(283mg, 0.51mmol, 48%) as a white solid.¹H NMR(400MHz，CDCl₃)δ7.25-7.15(m，2H)， 6.94(td，J＝8.7，3.1Hz，2H)，6.34-6.27(s，1H)，5.47(q，J＝4.5Hz，1H)，5.02(dd，J ＝14.6，7.4Hz，1H)，4.38(dd，J＝8.1，4.0Hz，1H)，3.31-3.13(m，3H)，3.08(dt，J＝ 13.9，7.0Hz，1H)，2.77(s，3H)，2.43(s，3H)，2.40-2.30(m，1H)，2.30-2.20(m，1H)， 2.06-1.93(m，1H)，1.90-1.77(m，1H)，1.76-1.59(m，2H)，1.43(m，1H)；¹³C NMR(101MHz，CDCl₃)δ174.48，171.53，171.20，161.91(d，J_C-F＝245.0Hz)， 158.91，158.24，154.45，132.07(d，J_C-F＝3.1Hz)，130.91(d，J_C-F＝8.0Hz)，115.98， 115.39(d，J_C-F＝21.3Hz)，101.31，64.03，54.39，48.15，42.18，37.96，37.35，31.40， 29.66，27.68，21.30，12.24.HRMS(ESMS)：C₂₅H₃₀FN₆O₆(M+H)⁺，calcd.514.2096， found 514.2095。

EXAMPLE 124 preparation of iminooxazolidine-2-ones (III)

Compound 3-6(400mg, 0.72mmol) was dissolved in 50.0mL of anhydrous dichloromethane, and triethylamine (145mg, 1.44mmol) was added. After the addition, the reaction was carried out at room temperature for 24 hours. After completion of the reaction, the reaction mixture was washed with water (50 mL. times.2). The organic phase was collected. Anhydrous Na for organic phase₂SO₄Drying, filtration and collection of the filtrate, evaporation of the solvent under reduced pressure and column chromatography of the residue (dichloromethane: methanol ═ 45: 1v/v) gave the products 3-7(100mg, 0.18mmol, 25%) as white solids.¹H NMR(400MHz，CDCl₃)δ7.17-7.10(m，2H)，6.93-6.86(m， 2H)，6.29(s，1H)，5.30(s，1H)，5.02-4.86(m，1H)，4.66(d，J＝0.5Hz，1H)，3.42- 3.23(m，4H)，3.11(dd，J＝13.8，4.8Hz，1H)，2.99(dd，J＝13.7，9.1Hz，1H)，2.42(m， 4H)，2.23(dd，J＝14.9，9.7Hz，1H)，2.09(dd，J＝3.0，1.4Hz，1H)，1.83(d，J＝1.9 Hz，1H)，1.76-1.61(m，2H)，1.60-1.44(m，3H)，0.82(t，J＝7.3Hz，3H)；¹³C NMR(101MHz，CDCl₃)δ174.82，171.66，171.08，168.85，161.83(d，J_C-F＝244.9Hz)， 158.70，158.29，156.06，132.07(d，J_C-F＝2.8Hz)，130.75(d，J_C-F＝7.9Hz)，115.31 (d，J_C-F＝21.3Hz)，101.60，79.83，54.67，47.25，42.26，41.75，37.81，37.72，32.43， 26.48，21.20，20.46，12.24，11.07.HRMS(ESMS)：C₂₇H₃₃FN₆NaO₆(M+Na)⁺， calcd.579.2338，found579.2341。

EXAMPLE 134 preparation of iminooxazolidine-2-ones (I)

The synthesis step is carried out by using a compound III.¹H NMR(400MHz，CDCl₃)δ7.25-7.15(m，2H)，6.94(td， J＝8.7，3.1Hz，2H)，6.34-6.27(s，1H)，5.47(q，J＝4.5Hz，1H)，5.02(dd，J＝14.6， 7.4Hz，1H)，4.38(dd，J＝8.1，4.0Hz，1H)，3.31-3.13(m，3H)，3.08(dt，J＝13.9，7.0 Hz，1H)，2.77(s，3H)，2.43(s，3H)，2.40-2.30(m，1H)，2.30-2.20(m，1H)，2.06- 1.93(m，1H)，1.90-1.77(m，1H)，1.76-1.59(m，2H)，1.43(m，1H)；¹³C NMR(101 MHz，CDCl₃)δ174.48，171.53，171.20，161.91(d，J_C-F＝245.0Hz)，158.91，158.24， 154.45，132.07(d，J_C-F＝3.1Hz)，130.91(d，J_C-F＝8.0Hz)，115.98，115.39(d，J_C-F＝21.3Hz)，101.31，64.03，54.39，48.15，42.18，37.96，37.35，31.40，29.66，27.68， 21.30，12.24.HRMS(ESMS)：C₂₅H₃₀FN₆O₆(M+H)⁺，calcd.514.2096，found 514.2095。

Example 144 preparation of iminooxazolidine-2-ones (II)

The synthesis step is carried out by using a compound III.¹H NMR(400MHz，CDCl₃)δ8.11(d，J＝8.0Hz，1H)，7.62 (d，J＝8.2Hz，1H)，7.13(dd，J＝8.0，5.6Hz，2H)，6.87(t，J＝8.5Hz，2H)，6.23(s， 1H)，5.36(t，J＝5.5Hz，1H)，4.93(dd，J＝14.3，7.3Hz，1H)，4.36-4.22(m，1H)， 3.24-3.06(m，5H)，3.00(dd，J＝13.8，7.6Hz，1H)，2.35(s，3H)，2.30-2.23(m，1H)， 2.18(d，J＝4.0Hz，1H)，2.00-1.87(m，1H)，1.75(dd，J＝8.8，4.9Hz，1H)，1.68- 1.52(m，2H)，1.37(dd，J＝22.1，10.0Hz，1H)，1.06(t，J＝7.2Hz，3H)；¹³C NMR (101MHz，CDCl₃)δ174.55，171.53，171.19，161.91(d，J_C-F＝244.9Hz)，158.92， 158.24，153.67，132.06(d，J_C-F＝2.9Hz)，130.92(d，J_C-F＝8.0Hz)，115.98，115.40 (d，J_C-F＝21.2Hz)，101.32，63.87，54.42，48.22，42.17，37.94，37.35，36.24，31.89， 29.32，22.66，14.86，12.22.HRMS(ESMS)：C₂₆H₃₁FN₆NaO₆(M+Na)⁺，calcd.565.2181，found 565.2184。

EXAMPLE 154 preparation of iminooxazolidine-2-ones (IV)

The synthesis step is carried out by using a compound III.¹H NMR(400MHz，CDCl₃)δ8.10(d，J＝7.9Hz，1H)， 7.61(d，J＝8.4Hz，1H)，7.13(dd，J＝13.3，5.4Hz，2H)，6.87(t，J＝8.5Hz，2H)，6.24 (s，1H)，5.30(t，J＝5.8Hz，1H)，4.96(dd，J＝14.4，7.3Hz，1H)，4.29(dd，J＝7.8，4.1 Hz，1H)，3.18(m，2H)，3.05(dtd，J＝27.8，13.9，7.1Hz，4H)，2.35(s，3H)，2.32-2.23 (m，1H)，2.23-2.11(m，1H)，2.01-1.86(m，1H)，1.82-1.69(m，1H)，1.61(dd，J＝ 26.5，16.8Hz，2H)，1.49-1.30(m，3H)，0.83(dd，J＝9.0，5.8Hz，3H)；¹³C NMR (101MHz，CDCl₃)δ174.47，171.49，171.17，161.91(d，J_C-F＝245.0Hz)，158.88， 158.25，153.80，132.06(d，J_C-F＝3.0Hz)，130.91(d，J_C-F＝7.7Hz)，115.96，115.39 (d，J_C-F＝21.2Hz)，101.32，63.91，54.35，48.16，43.08，42.17，38.03，37.35，31.34， 29.66，21.30，12.23，11.16.HRMS(ESMS)：C₂₇H₃₃FN₆NaO₆(M+Na)⁺，calcd.579.2338，found 579.2340。

EXAMPLE 164 preparation of iminooxazolidine-2-ones (V)

The synthesis step is carried out by using a compound III.¹H NMR(400MHz，CDCl₃)δ8.17(d，J＝7.8Hz，1H)， 7.67(d，J＝8.2Hz，1H)，7.27-7.12(m，2H)，6.95(t，J＝8.4Hz，2H)，6.30(s，1H)， 5.36-5.30(m，1H)，5.09-4.92(m，1H)，4.36(d，J＝3.4Hz，1H)，3.26(s，2H)，3.22 -3.13(m，3H)，3.07(dd，J＝13.6，7.5Hz，1H)，2.42(s，3H)，2.35(d，J＝10.4Hz，1H)， 2.25(d，J＝4.5Hz，1H)，2.00(m，1H)，1.82(m，1H)，1.68(d，J＝9.3Hz，2H)，1.48(dt， J＝14.8，7.2Hz，2H)，1.35(dd，J＝14.9，7.1Hz，3H)，0.91(t，J＝7.2Hz，3H)；¹³C NMR(101MHz，CDCl₃)δ174.52，171.49，171.16，161.93(d，J_C-F＝245.1Hz)， 158.90，158.25，153.78，132.05(d，J_C-F＝2.9Hz)，130.92(d，J_C-F＝7.8Hz)，115.95， 115.40(d，J_C-F＝21.3Hz)，101.32，63.90，54.39，48.23，42.18，41.11，38.01，37.37， 31.48，30.13，29.33，26.54，21.29，19.83，13.64，12.22.HRMS(ESMS)：C₂₈H₃₆FN₆O₆(M+H)⁺，calcd.571.2675，found 571.2674。

Example 174 preparation of iminooxazolidine-2-ones (VI)

The synthesis step is carried out by using a compound III.¹H NMR(400MHz，CDCl₃)δ8.25(d，J＝5.9Hz，1H)， 7.69(d，J＝7.9Hz，1H)，7.40(d，J＝7.1Hz，2H)，7.34-7.24(m，2H)，7.24-7.12(m， 2H)，7.08(t，J＝7.2Hz，1H)，6.92(t，J＝8.2Hz，2H)，6.20(s，1H)，5.39(d，J＝4.4Hz， 1H)，4.94(dd，J＝14.0，6.9Hz，1H)，4.48(d，J＝1.7Hz，1H)，3.19(dd，J＝18.4，10.1 Hz，3H)，3.08(dd，J＝13.7，7.8Hz，1H)，2.70(m，1H)，2.36(s，3H)，2.27(m，1H)， 2.07-1.93(m，1H)，1.85-1.75(m，1H)，1.69(dd，J＝21.7，10.5Hz，2H)，1.46(dd，J ＝18.8，7.8Hz，1H)；¹³C NMR(101MHz，CDCl₃)δ174.76，171.72，171.25，161.92 (d，J_C-F＝245.1Hz)，159.11，158.12，151.10，137.16，131.88(d，J_C-F＝2.9Hz)， 130.84(d，J_C-F＝7.9Hz)，129.09，124.09，118.95，115.77，115.47(d，J_C-F＝21.3Hz)， 101.35，64.04，54.74，48.30，42.23，37.70，37.39，31.66，29.69，21.13，12.21.HRMS (ESMS)：C₃₀H₃₁FN₆NaO₆(M+Na)⁺，calcd.613.2181，found613.2182。

Example 18 in vitro enzyme Activity screening of EV 713C protease inhibitors

The enzyme activity of inhibitors against 3C protease was determined using Fluorescence Resonance Energy Transfer (FRET) technique, designing the substrate according to the 3C protease recognition site: Dabcyl-RTATVQGPSLDFE-Edans, final concentrations of inhibitor were: 1mM, 500. mu.M, 250. mu.M, 125. mu.M, 62.5. mu.M, 31.25. mu.M, 15.625. mu.M, 7.8125. mu.M, 3.9. mu.M, 1.95. mu.M, 976nM, 488nM, 244nM, 122nM, 61nM, 30.5nM, 15.3nM, 3.8nM, 1.9nM, 0.95nM, with negative controls. The enzyme activity was measured using a 96-well plate, and a 100. mu.l reaction system included: 20mM MES pH6.5, 10ug/ml BSA, 10. mu.M EV 713C protein, 150. mu.M fluorogenic substrate and different concentrations of inhibitor, reacting at 37 ℃, detecting fluorescence intensity by a microplate reader, and processing the obtained data by using software GraphPad Prism 5 to obtain IC of the inhibitor₅₀. The observations indicate that IC for compounds I-VI₅₀Is 0.5-2 μ M.

Example 19 in vitro enzyme Activity screening of SARS and MERS Main protease Nsp5 inhibitors

The enzyme activity of inhibitors against SARS Nsp5 and MERS protease was determined using Fluorescence Resonance Energy Transfer (FRET) technique, designing substrates based on Nsp5 protease recognition sites: MCA-AVLQSGFRL-L-Dnp, inhibitor final concentrations 1mM, 500. mu.M, 250. mu.M, 125. mu.M, 62.5. mu.M, 31.25. mu.M, 15.625. mu.M, 7.8125. mu.M, 3.9. mu.M, 1.95. mu.M, 976nM, 488nM, 244nM, 122nM, 61nM, 30.5nM, 15.3nM, 3.8nM, 1.9nM, 0.95nM, respectively, with negative controls. The enzyme activity was measured using a 96-well plate, and a 100. mu.l reaction system included: 50mM Tris-HCl pH7.3, 1mM EDTA, 0.5. mu.M SARS nsp5 protein, 16. mu.M fluorogenic substrate and various concentrations of inhibitor, reacted at 37 ℃ byDetecting fluorescence intensity by a microplate reader, processing obtained data by using software GraphPad Prism 5 to obtain IC of the inhibitor₅₀. The observation result shows that the compounds I-VI have good inhibitory activity on the protease of SARS and MERS, and the IC thereof₅₀Is 2-10 μ M.

Example 20 virus replication inhibition capacity Cell-based immunological detection (CID) assay was screened:

in CID experiments RD (human embryo rhabdomyospora) cells were trypsinized and then diluted to 3X 10 with DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FBS and 1% PS⁴One/ml, 100. mu.l/well RD cells were added to a 96-well plate at 37 ℃ with 5% CO₂Overnight incubation followed by the next day of addition of 50. mu.l/well diluted inhibitor to final concentrations: 25 μ M, 10 μ M, 5 μ M, 2.5 μ M, 1.5 μ M, 1.3 μ M, 1.1 μ M, 0.9 μ M, 0.7 μ M, 0.5 μ M, 0.166 μ M, 0.05 μ M. After 2h 50. mu.l/well of EV71 virus (titer 100 TCID) was added₅₀)，37℃，5％CO₂Culturing, washing twice with PBS after 30h, fixing the cells with 50. mu.l/well of anhydrous methanol for 10min, then washing twice with PBS, adding 100. mu.l/well PBS + 0.5% Tween20+ 10% FBS for 1h at 37 ℃, adding 100. mu.l/well (1: 500) diluted primary antibody for 3h at 37 ℃, washing the plate with 0.5% PBST for 3 times, adding 100. mu.l/well (1: 2500) diluted secondary antibody for i-mouse immunoglobulin B, acting for 1h at 37 ℃, washing the plate with 0.5% PBST for 3 times, adding 100. mu.l/well of OPD substrate for 5min at room temperature, and developing with 50. mu.l/well of 1M H₂SO₄The reaction was stopped and the fluorescence was read from each well on an ELISA meter (490 nM). The inhibitor was repeated 3 times in 4 wells per concentration in the above experiment. EC was calculated using GraphPad Prism 5₅₀The value is obtained. Compounds I to VI all show good activity against EV71 with EC₅₀The values were all less than 1. mu.M.

Claims (3)

1. A4-iminooxazolidine-2-one enterovirus 71(EV71)3C protease inhibitor having the structure shown below:

2. the use of the inhibitor of claim 1 for the manufacture of a medicament for the treatment of an enterovirus infectious disease caused by enterovirus 71(EV 71).

3. Use of an inhibitor according to claim 1 for the manufacture of a medicament for the treatment of a disease caused by a coronavirus infection, wherein said coronavirus comprises: severe acute respiratory syndrome virus (SARS), middle east respiratory syndrome coronavirus (MERS-CoV).