CN115385983A

CN115385983A - Azabicyclo compound, preparation method, pharmaceutical composition and application thereof

Info

Publication number: CN115385983A
Application number: CN202211142137.1A
Authority: CN
Inventors: 朱孝云; 蒋维平
Original assignee: Jiaxing'an Dikang Biotechnology Co ltd
Current assignee: Jiaxing'an Dikang Biotechnology Co ltd
Priority date: 2022-01-11
Filing date: 2022-01-11
Publication date: 2022-11-25
Also published as: CN115583984A; CN114426568A; CN114426568B

Abstract

The invention provides an azabicyclo compound, a preparation method, a pharmaceutical composition and application thereof, and a method for treating coronavirus infection by using the composition. The compound provided by the invention has higher inhibition activity for inhibiting 3CL protease, and has the advantages of high metabolic stability, high oral absorption, better bioavailability, high lung distribution, quick toxin expelling, good SARS-CoV-2 resistant activity, high selectivity, high therapeutic index and the like.

Description

Azabicyclo compound, preparation method, pharmaceutical composition and application thereof

The application is a divisional application with the application date of 2022, 01 month and 11 day, the application number of 202210026867.9, and the name of the invention is '2-oxo-3-pyrrolidinylpropionitrile compound and its pharmaceutical composition and use'.

Technical Field

The present invention provides a substituted 2-oxo-3-pyrrolidinylpropionitrile compound, a prodrug, an oxide, a salt, a metal complex or a stereochemical isomer, a preparation method thereof, a pharmaceutical composition containing the same, and a method for preventing or treating coronavirus or Ebola virus infection by using the composition.

Technical Field

Coronaviruses (Coronavirus) belong to the genus Coronaviridae (Coronaviridae) and the genus coronaviruses (Coronavirus) in the phylogenetic classification, and mature coronaviruses are coronal or coronal under an electron microscope, and are named as coronaviruses. Coronaviruses belong to the mononegavirales, which can be classified into four genera, alpha, beta, gamma and delta, based on serological characteristics, and are important pathogens for many species such as avian species, bat family, feline species and human species. SARS-CoV, MERS-CoV and COVID-19 are members of the same beta coronavirus family, in which the persistence of COVID-19 variants, such as the Deltay and the Onckron variants (Omicron), results in a diminished or even ineffective protective capacity of the new crown vaccine. The new coronavirus is a brand-new coronavirus, and a new coronavirus particle comprises five components: the virus particle comprises an RNA gene chain and four proteins, wherein the outermost layer of the particle is Spike glycoprotein (S), a virus Envelope which is formed by small Envelope glycoprotein (E) and Membrane glycoprotein (M) is arranged below the Spike, and the core which is hidden in the virus Envelope is a spiral folding structure formed by the RNA gene chain and Nucleocapsid Protein (N). There is an urgent need in the world for safe and effective anti-coronavirus small molecule drugs to end the global pandemic of new coronavirus.

The 3C-like protease (3 CLpro), also called main protease (Mpro), is entirely composed of 306 amino acids, and can further cleave new corona polyprotein, thereby generating helicase, RNA-dependent RNA polymerase and other related replication elements, which play an important role in virus proliferation and assembly. The native 3CLpro monomer consists of three domains, and the two monomers interact to form a pocket structure containing the substrate binding site. The active center is located in the gap between domains I and II, and the catalytic sites are Cys at position 145 and His at position 41. Paxlovid acts on the target of 3C-like protease (3 CLpro), and inhibits the replication of the virus by inhibiting the 3CLpro of the virus, inhibiting the RNA replication and the generation of related non-structural proteins.

The action target of the pff-07321332 in the pfizer is 3C-like protease (3 CLpro), and inhibits the replication of the virus by inhibiting the 3CLpro virus, inhibiting the replication of RNA and the generation of related non-structural proteins. PF-07321332 has been approved by FDA emergency drug marketing for the treatment of new coronavirus infection, and PF-07321332 combined with low-dose Ritonavir (Ritonavir) in clinical application helps to slow down the metabolism or decomposition of PF-07321332, so that the effective concentration in vivo can be maintained for a long time to improve the antiviral activity. However, when PF-07321332 is used for treating new coronary pneumonia, other prescription drugs, such as antilipemic statins, anticoagulants and antidepressants, increase the toxic and side effects of these drugs, and even may cause death and other serious adverse reactions.

In view of the above, there is an urgent need in the art for safer, more effective, and more convenient anti-neocoronal drugs. The novel anti-new crown medicine is not only a hotspot of international anti-new crown virus research, but also an important tool for preventing and treating infectious diseases in China.

Disclosure of Invention

The invention provides a substituted 2-oxo-3-pyrrolidinylpropionitrile compound shown in a formula (I), a formula (II) and/or a formula (III), a stereoisomer, a hydrate, a solvate, a polymorphic substance, an active metabolite, a pharmaceutically acceptable salt thereof or a prodrug thereof:

wherein:

R ¹ deuterium and H;

R ² is a CD ₃ 、CH ₃ ；

R ³ Is a CD ₃ 、CH ₃ 、CHF ₂ H, deuterium, halogen, hydroxy, amino, cyano, haloalkyl, cycloalkyl.

And/or the presence of a gas in the gas,

wherein:

R ¹ deuterium, H;

R ² is a CD ₃ 、CH ₃ ；

And/or the presence of a gas in the gas,

wherein:

n is 0 to 3;

R ¹ deuterium, H;

R ² is a CD ₃ 、CH ₃ ；

The substituted 2-oxo-3-pyrrolidinylpropionitrile compound shown in the formula (I), the formula (II) and/or the formula (III) preferably has a structure shown in any one of the following structural formulas:

the compounds of the invention are generally used in the form of the free acid or free base. Alternatively, the compounds of the present invention may be used in the form of acid or base salts. The acid addition salts of the free amino compounds of the present invention may be prepared by methods well known in the art and may be prepared from organic and inorganic acids. Suitable organic acids include maleic acid, fumaric acid, benzoic acid, ascorbic acid, succinic acid, methanesulfonic acid, acetic acid, trifluoroacetic acid, oxalic acid, propionic acid, tartaric acid, salicylic acid, citric acid, gluconic acid, lactic acid, mandelic acid, phenylacetic acid, aspartic acid, stearic acid, palmitic acid, glycolic acid, glutamic acid, and benzenesulfonic acid. Suitable inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and nitric acid. Base salts include those formed with the carboxylate anion and include those formed with organic and inorganic cations such as those selected from the group consisting of alkali metal ions, alkaline earth metal ions (e.g., lithium, sodium, potassium, magnesium, barium, calcium), and ammonium ions, as well as substituted derivatives thereof (e.g., dibenzylamine, benzylamine, 2-hydroxyethylamine, and the like). Thus, the term "pharmaceutically acceptable salts" of the general formula (I), formula (II) and/or formula (III) shall include and all acceptable salt forms.

In addition, prodrugs are also included within the scope of the present invention. A prodrug is any covalently bound carrier that releases a compound of formula (I), formula (II) and/or formula (III) in vivo when the prodrug is administered to a patient. Prodrugs are generally prepared by modifying functional groups in a manner that enables the modification to be cleaved, either by conventional exchange or in vivo, to yield the parent compound. Prodrugs include, for example, compounds of the present invention wherein a hydroxy, amino, or sulfhydryl group is bonded to any group that, when administered to a patient, is cleaved to yield the hydroxy, amino, or sulfhydryl group.

Thus, representative examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups of compounds of formula (I), formula (II) and/or formula (III). Further, in the case of carboxylic acid (-COOH), esters such as methyl ester, ethyl ester, and the like may be included. In the case of hydroxyl groups, mixed anhydrides such as methoxy, ethoxy, propoxy, tert-butoxy, and the like may be included.

For stereoisomers, the compounds of formula (I), formula (II) and/or formula (III) may have chiral centers and may exist as racemates, racemic mixtures, as well as individual enantiomers or diastereomers. All isomeric forms are included within the present invention, including mixtures thereof. Furthermore, certain crystalline forms of the compounds of formula (I), formula (II) and/or formula (III) may exist in the form of polymorphs, which are also included in the present invention. Furthermore, some of the compounds of formula (I), formula (II) and/or formula (III) may also form solvates with water or other organic solvents, such as hydrates and tert-butanol solvates. Such solvates are likewise included within the scope of the present invention.

It will be understood by those skilled in the art that any compound may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. In the substituted 2-oxo-3-pyrrolidinylpropionitrile compound related to the invention, the deuteration means that the atom on the relevant site of the compound contains a deuterium atom with a natural ratio exceeding the natural abundance ratio of deuterium. Thus, any 2-oxo-3-pyrrolidinylpropionitrile compound containing a deuterium atom at the relevant site in a ratio higher than the natural abundance of deuterium is within the scope of the present invention. For example, it is understood that the corresponding 2-oxo-3-pyrrolidinylpropionitrile compounds having the corresponding deuteration rate or deuterium content obtained by introducing deuterium atoms using commercially available deuteration reagents by the same or similar chemical synthesis means as shown in the examples of the present invention are within the scope of the present invention. The chemical synthesis and deuteration reagents are not limited to those exemplified in the examples, but are to be understood as all synthetic methods or routes that can be used in the art to obtain the compounds of the present invention, and all deuteration reagents that can incorporate deuterium atoms into target molecules by the aforementioned synthetic methods or routes.

According to the specific embodiments of the present invention disclosed hereinafter, the same or similar principles and methods can be used by those skilled in the art to prepare each specific compound involved in the 2-oxo-3-pyrrolidinylpropionitrile group of the general formula (I), formula (II) and/or formula (III) of the present invention.

The invention further provides an application of the 2-oxo-3-pyrrolidinylpropionitrile compound shown in the formula (I), the formula (II) and/or the formula (III), a stereoisomer, a hydrate, a solvate, a polymorph, an active metabolite, a pharmaceutically acceptable salt or a prodrug thereof in preparation of anti-coronavirus medicines.

The invention further provides an application of the 2-oxo-3-pyrrolidinylpropionitrile compound shown in the formula (I), the formula (II) and/or the formula (III), a stereoisomer, a hydrate, a solvate, a polymorph, an active metabolite, a pharmaceutically acceptable salt or a prodrug thereof in preparation of medicines for preventing, treating and/or relieving diseases related to coronavirus infection. Wherein, the disease related to coronavirus infection specifically refers to symptoms and/or diseases caused by coronavirus. The symptoms include, but are not limited to, cold-like symptoms accompanied by fever, chill, headache, myalgia, loss or deterioration of taste, general malaise, and the like, or gastrointestinal symptoms such as respiratory inflammation, abdominal pain, vomiting, diarrhea, and the like, such as pharyngalgia, rhinorrhea, nasal obstruction, cough, sputum, and the like. In particular, it can prevent or treat pneumonia caused by coronavirus, especially pneumonia caused by new coronavirus (COVID-19), and symptoms including inflammatory reaction, pulmonary fibrosis, and pulmonary exudative reaction characterized by deep airway and pulmonary alveoli injury, and further complicated symptoms such as acute encephalopathy, kidney injury, liver injury, heart failure, myocarditis, etc.

The present invention further provides a method for preventing, treating and/or ameliorating diseases associated with coronavirus infection, the method comprising administering to a subject in need thereof a therapeutically effective amount of a 2-oxo-3-pyrrolidinylpropionitrile compound represented by formula (I), formula (II) and/or formula (III), a stereoisomer, a hydrate, a solvate, a polymorph, an active metabolite, a pharmaceutically acceptable salt thereof or a prodrug thereof. Wherein the diseases related to coronavirus infection are specifically described above.

The invention further provides a pharmaceutical composition, which comprises a therapeutically effective amount of 2-oxo-3-pyrrolidinylpropionitrile compounds shown in formula (I), formula (II) and/or formula (III), stereoisomers, hydrates, solvates, polymorphs, active metabolites, pharmaceutically acceptable salts or prodrugs thereof, and a pharmaceutically acceptable carrier.

The invention further provides a pharmaceutical composition, which not only comprises a therapeutically effective amount of the 2-oxo-3-pyrrolidinylpropionitrile compound shown in the formula (I), the formula (II) and/or the formula (III), a stereoisomer, a hydrate, a solvate, a polymorph, an active metabolite, a pharmaceutically acceptable salt or a prodrug thereof, and a pharmaceutically acceptable carrier, but also comprises other pharmaceutically active ingredients. The additional pharmaceutical active ingredient is selected from the group consisting of antibody drugs, RNA drugs, viral protease inhibitors, lysosome-tropic drugs, M2 proton channel inhibitors, RNA polymerase inhibitors (e.g., ridciclovir, molnupiravir), neuraminidase inhibitors, reverse transcriptase inhibitors, drugs that inhibit viral entry into cells, interferons (e.g., type I, II, III), nucleoside drugs, neocoronaviral neutralizing antibodies, pharmacokinetic potentiators, reduced coenzyme II (NADPH) inhibitors, and cytochrome P450 A4 enzyme (CYP 3 A4) inhibitors (e.g., ritonavir, nelfinavir, darunavir).

The compounds of the present invention may be used in combination with other pharmaceutical agents in order to enhance the efficacy of the compounds or to reduce the dosage of the compounds. The other pharmaceutical agent is selected from the group consisting of antibody drugs, RNA drugs, viral protease inhibitors, lysosomotropic drugs, M2 proton channel inhibitors, RNA polymerase inhibitors (e.g., ridivir, molnupiravir), neuraminidase inhibitors, reverse transcriptase inhibitors, drugs that inhibit viral entry into cells, interferons (e.g., types I, II, III), nucleoside drugs, neutralizing antibodies to neocoronaviruses, pharmacokinetic potentiators, reduced coenzyme II (NADPH) inhibitors, and cytochrome P450 3A4 enzyme (CYP 3 A4) inhibitors (e.g., ritonavir, nelfinavir, darunavir).

The above-mentioned other pharmaceutical active ingredient or other pharmaceutical preparation includes an antiviral agent selected from efavirenz, elbasvir, nevirapine, rilpivirine, paritaprevir, simeprevir, daclatasvir, gradoprevir, pibrentavir, adefovir, amprenavir, amprigen, apliviroc, anti-caprine antagonist, balavir, ledipasvir, glecaprevir, sofosbuvir, bicegravir, dasabuvir, lamivudine, atazanavir, ombitasvir, lamivudine, navir, basivirin, favipiravir, ST-193, oseltamivir, zanamivir, pervirir, danoviriir, daunoprevir, ritionir, remdesivir, cobicistat, elvitegravir, emtricitabine, tenofovir, tenofovir disoproxil, nitazoxanide, norvir, plerixafor, PRO140, raltegravir, pyramidine, saquinavir, telbivudine, tenofovir alamidine hemifumarate, abacavir, dolutegravir, stavudine, cabotegravir, cytarabine, ecoliever, epidiadocatachin gate, etravirine, fostemsevavir, gemcitabine, griffithsin, imunovir, indinavir, maraviroc, methisazone, MK-2048, nelfmavir, nevirapine, TNX-355, valacyvir, VIR-576, and zalcitabine. Viral protease inhibitors (e.g., nafamostat, camostat, gabexate, epsilon-aminocaproic acid, aprotinin), fusion protease inhibitors (e.g., BMY-27709, CL 61917, CL 62554), M2 proton channel inhibitors (e.g., amantadine, rimantadine), polymerase inhibitors, endonucleases inhibitors (e.g., L-735822, flutamide), neuraminidase inhibitors (e.g., zanamivir, oseltamivir, peramivir, ABT-675), reverse transcriptase inhibitors (e.g., abacavir, adefovir, delavirdine, didanosine, avanz, emtricitabine, lamivudine, nevirapine, stavudine, tenoviir, tenovivir disoproxil, zaitabassine, protease inhibitors (e.g., amantidazole, kinavirenz, narivir, aprotinin), arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir, daurnavir, docosanol, edoxudine, viral entry inhibitors (e.g., enfuvirtide, maraviroc), entecavir, immunovir, idoxuridine, imiquimod, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibatine, inosine, integrin enzyme inhibitors (e.g., raltegravir), interferons (type I, II, III), lopinavir, loviride, moroxydine, nexavir, nucleoside drugs (such as aciclovir), truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zodovudine, penciclovir, pleconaril, podophylotoxin, ribivirin, tipranavir, triflouridine, trizivir, tromitadine.

The above-mentioned other pharmaceutically active ingredients or other pharmaceutical preparations include immunomodulators, PD-1 or PDL-1 monoclonal antibody drugs, such as nivolumab, atezolizumab, pembrolizumab, durvalumab, BMS-936559, or avelumab, immunomodulators further include TSR-022, SGN-40, CP-870893, RO7009789, MBG453, LAG525, TSR-033, MK-1454, ASAY 404, amidobenzimidazoles, hu5F9-G4, ADU-S100.

The compounds or compositions of the present invention are used in combination with a traditional Chinese medicine or a Chinese patent medicine in order to enhance the efficacy of the compound, improve the symptoms of a patient, reduce the dosage of the compound or reduce the side effects of the medicine. For example, it can be used in combination with HUOXIANGZHENGQI Capsule (pill, water, oral liquid), JINHUAQINGGAN granule, LIANHUAQINGWEN Capsule (granule), SHUFENGJIEDU Capsule (granule), DIFFUSHUANQI granule, HUASHIBAIDU granule, HUANFUQIBADU granule, QINGFEIDU decoction (decoction), XINGUANYAN pneumonia No. 1 (decoction), XINGUANYAN pneumonia No. 2 (decoction), XINGUANYAN pneumonia No. 3 (decoction), YANXIAOPING injection, XUEBIJING injection, HONGDUNING injection, TANQINGREQING injection, XINGNING injection, SHENFU injection, SHENMAI injection, and SHENMAI injection.

Glucocorticoids, including inhaled corticosteroids that alleviate the severe side effects of oral steroids, non-limiting examples of corticosteroids that may be used in combination with the compounds or compositions of the invention are dexamethasone, dexamethasone sodium phosphate, fluorometholone acetate, loteprednol etabonate, hydrocortisone, prednisolone, fludrocortisone, triamcinolone acetonide, betamethasone, beclomethasone dipropionate, methylprednisolone, fluocinolone, fluocinonide, flunisolide, flucortolide-2-butyl ester, flumethasone pivalate, budesonide, halobetasol propionate, mometasone furoate, fluticasone propionate, ciclesonide, or pharmaceutically acceptable salts thereof.

The compounds and compositions of the present invention are also intended to provide general care for patients with coronavirus infections, including parenteral fluids (including glucose saline and ringer's lactate) and nutrition, antibiotics (including metronidazole and cephalosporin antibiotics, such as ceftriaxone and cefuroxime, macrolide antibiotics, such as azithromycin, aminoglycoside antibiotics, such as etimicin, prazolamide, quinolone antibiotics, such as levofloxacin, moxifloxacin, oxazolidinones, such as linezolid and tedizolid), and/or antifungal prophylaxis, fever and pain killers, anti-emetics (such as metoclopramide) and/or antidiarrheals, probiotics, vitamin and mineral supplements (including vitamin K and octyl sulfate), non-steroidal anti-inflammatory drugs (such as ibuprofen, paracetamol, celecoxib), antitussives (ambroxol), quinine drugs (quinine, chloroquine, hydroxychloroquine).

Pure forms or suitable pharmaceutical compositions of the compounds of the present invention or pharmaceutically acceptable salts thereof may be administered by any acceptable mode of administration of agents that serve similar utilities. The pharmaceutical compositions of the present invention may be prepared by combining a compound of the present invention with a suitable pharmaceutically acceptable carrier, diluent or excipient, and may be formulated into solid, semi-solid, liquid or gaseous form preparations such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of administration of the pharmaceutical composition include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal administration. As used herein, the term parenteral includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. The pharmaceutical compositions of the present invention are formulated to allow the cleansing component contained therein to be bioavailable upon administration of the composition to a patient. The compound of the invention may be administered to a subject or patient in the form of one or more dosage units in a composition, wherein, for example, a tablet may be a single dosage unit and a container containing the compound in aerosol form may contain a plurality of dosage units. The actual methods of preparing the dosage forms are known, or will be known, to those skilled in the art. The compositions to be administered will contain, in any event, a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, in order to treat the disease or condition of interest in accordance with the teachings of the present invention.

The pharmaceutical compositions of the present invention may be in solid or liquid form. In one aspect, the carrier is a microparticle, such that the composition is in the form of, for example, a tablet or powder. The carrier can be a liquid and the composition can be, for example, an oral syrup, an injectable liquid, or an aerosol suitable for administration, for example, by inhalation. When intended for oral administration, the pharmaceutical composition is preferably selected from solid or liquid forms, wherein semi-solid, semi-liquid, suspension and gel forms are included in the forms herein considered to be solid or liquid. For oral solid compositions, the pharmaceutical compositions may be formulated into powders, granules, compressed tablets, pills, capsules, chewable tablets, powder tablets, and the like. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of a binder such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, xanthan gum, or gelatin; excipients, such as starch, lactose or dextrin; disintegrating agents such as alginic acid, sodium alginate, primogel, corn starch, and the like; lubricants, such as magnesium stearate or hydrogenated vegetable oil (Sterotex); glidants, such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; a flavoring agent, such as peppermint, methyl salicylate, or orange flavoring; and a colorant.

In the preparation of compositions for oral administration, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid compositions (e.g., suspensions, syrups, free-forms, emulsions and solutions) or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrants and the like in the case of solid compositions. For parenteral compositions, the carrier will typically comprise sterile water, at least in large part, but other ingredients, such as solubilizers, emulsifiers or additional adjuvants may also be added. Injectable solutions may be prepared in which the carrier comprises a physiological saline solution, a dextrose solution, or a mixture of both. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations, such as powders for reconstitution.

When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier, for example polyethylene glycol or an oil. The pharmaceutical composition may be in liquid form, such as a tincture, syrup, solution, emulsion or suspension. Such liquids may be administered orally, or delivered by injection, as two examples. When intended for oral administration, the compositions preferably contain one or more of sweeteners, preservatives, dyes/colorants and flavor enhancers in addition to the compounds of the present invention. In compositions intended for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents and isotonicity agents may be included.

The liquid pharmaceutical compositions of the present invention, whether in solution, suspension or other similar form, may include one or more adjuvants selected from sterile diluents, such as water for injection, physiological saline solutions, preferably physiological saline, ringer's solution, isotonic sodium chloride, fixed oils (e.g., synthetic mono-or diglycerides which may be used as a solvent or suspending medium), polyethylene glycols, glycerol, propylene glycol and the like; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium oxide or dextrose. Parenteral formulations can be packaged in ampules, disposable syringes or multi-dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

Liquid pharmaceutical compositions of the invention intended for parenteral or oral administration should contain an amount of a compound of the invention such that a suitable dosage is obtained. The pharmaceutical compositions of the invention may be intended for topical administration, in which case the carrier preferably comprises a solution, emulsion, ointment or gel base. For example, the base may comprise one or more of paraffin oil, lanolin, polyethylene glycol, beeswax, mineral oil, diluents (e.g., water and alcohol), and emulsifiers and stabilizers. Thickeners may be present in the pharmaceutical compositions for topical administration. If transdermal administration is intended, the composition may comprise a transdermal patch or an iontophoretic device.

The pharmaceutical compositions of the present invention may be administered rectally, in the form of suppositories, for example, which melt in the rectum and release the drug. Compositions for rectal administration may contain an oily base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycols.

The pharmaceutical compositions of the present invention may include a variety of substances that alter the physical form of the solid or liquid dosage unit. For example, the composition may include a material that forms a coating shell around the active ingredient. The material forming the coating shell is generally inert and may be selected from, for example, sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

Pharmaceutical compositions of the invention in solid or liquid form may include an agent that binds to a compound of the invention and thereby aids in the delivery of the compound. Suitable agents with this capability include monoclonal or polyclonal antibodies, proteins or liposomes.

The pharmaceutical compositions of the present invention may be comprised of dosage units that can be administered in the form of an aerosol. The term aerosol is used to denote a variety of systems ranging from the jelly-like variety to systems consisting of pressurized packs. Delivery may be by liquefied or compressed gas, or by a suitable pump system that dispenses the active ingredient. Aerosols of the compounds of the invention may be delivered as single, two, or three phase systems to deliver the active ingredient. The delivery of the aerosol comprises the necessary containers, actuators, valves, sub-containers, etc., which together may form a kit. Preferred aerosols are determined by one of skill in the art without undue experimentation.

The pharmaceutical compositions of the present invention may be prepared by methods well known in the pharmaceutical arts. For example, pharmaceutical compositions to be administered by injection may be prepared by combining a compound of the present invention with sterile distilled water to form a solution. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the compounds of the present invention, thereby facilitating dissolution or uniform suspension of the compounds in an aqueous delivery system.

The compounds of the invention, or pharmaceutically acceptable salts thereof, are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the particular compound employed; metabolic stability and length of action of the compound; the age, weight, general health, sex, and diet of the patient; mode and time of administration; the rate of excretion; a drug combination; the severity of the particular disorder or condition; and individuals undergoing therapy.

The compounds of the present invention or pharmaceutically acceptable derivatives thereof can also be administered simultaneously with, before, or after the administration of one or more other therapeutic agents. Such combination therapy includes the administration of a single pharmaceutical dosage formulation containing a compound of the present invention and one or more other active agents, as well as the administration of separate pharmaceutical dosage formulations of a compound of the present invention with each active agent itself. For example, a compound of the invention and another active agent can be administered to a patient in a single oral composition (e.g., a tablet or capsule), or each agent can be administered as a separate oral formulation. In the case of separate administration formulations, the compound of the invention and the one or more additional active agents may be administered substantially at the same time (i.e., simultaneously) or at separately staggered times (i.e., sequentially); combination therapy is understood to include all such regimens.

The pharmaceutical combination of the present invention can be administered in a dose adjusted depending on the disease state, the administration route, the age or body weight of the patient. Orally administered, considering a daily antiviral effective dose of 0.01mpk to 500mpk, more preferably 0.1mpk to 50mpk. More suitably, the required dose is administered in one, two, three, four or more sub-doses at appropriate intervals throughout the day. The sub-doses may be presented in unit dosage form, e.g. containing from 1 to 1000mg, more preferably from 5 to 500mg, of active ingredient per unit dosage form. In pediatric applications, or when used by patients having substantially different body weights.

The present invention relates to substituted 2-oxo-3-pyrrolidinylpropionitrile compounds and their prodrugs, deuterated compounds, pharmaceutical compositions containing them, and methods of using the compositions to inhibit the proliferation of syncytial virus.

The present invention provides substituted 2-oxo-3-pyrrolidinylpropionitrile compounds, prodrugs, oxides, salts, metal complexes or stereochemically isomeric forms thereof, pharmaceutical compositions containing the same, and methods of using the compositions for treating new coronavirus infections. The compound provided by the invention has higher inhibition activity for inhibiting SARS-CoV-2 3CL protease, has the advantages of high metabolic stability, high oral absorption, better bioavailability, high lung distribution, quick toxin expelling, good SARS-CoV-2 resistant activity, high selectivity, high therapeutic index and the like, is clinically orally taken, and does not need the combination potential with a drug substitute synergist CYP3A4 enzyme inhibitor.

Unless otherwise specified, reagents and starting materials for use in the invention are commercially available.

Unless otherwise specified, the compounds of the invention are prepared by hand or

The software names, and the commercial compounds used the supplier catalog names.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

Detailed Description

Certain preferred embodiment aspects of the invention are shown by way of illustration in the following non-limiting examples. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions. The starting materials may be obtained from commercial sources, or prepared by methods known in the art, or prepared according to the methods described herein.

Example 1.

Compound CN-01: (1S, 3aR, 6aS) -N- { (1S) -1-cyano-2- [ (3S) -2-oxo-3-pyrrolidinyl ] ethyl } -2- [ (2S) -3, 3-dimethyl-2- (2, 2-trifluoroacetylamino) butanoyl ] octahydrocyclopenta [ C ] pyrrole-1-carboxamide

The synthetic route is as follows:

preparation of intermediate 3

16g (58.1 mmol) of N-tert-butoxycarbonyl-L-glutamic acid dimethyl ester were dissolved in 250ml of anhydrous THF, cooled to-78 ℃ and N was added ₂ And (3) protecting, dropwise adding 133.7ml (133.7 mmol) of 1M lithium bis (trimethylsilyl) amide (LiHDMS) solution, stirring and reacting for 2 hours at the temperature of minus 78 ℃, then dropwise adding 10.5g (87.2 mmol) of 2-bromoacetonitrile, continuously reacting for 3 hours, and detecting by TLC that the reaction is complete. Then, 25ml of methanol and 20ml of acetic acid were added in this order, and the reaction mixture was warmed to room temperature to quench the reaction. The solvent was evaporated under reduced pressure, flash column chromatography on silica gel, gradient elution with ethyl acetate/petroleum ether, and concentration under reduced pressure to give 14.7g of a yellow oil as intermediate 3.

Preparation of intermediate 4

10g of intermediate 3 (31.8 mmol) are dissolved in 160ml MeOH and 4.5g (19.1 mmol) CoCl are added ₂ .6H ₂ And O, cooling to 0 ℃. 7.2g (191 mmol) of NaBH are added portionwise ₄ After the addition, the reaction mixture was warmed to room temperature, stirred for 10hr, TLC checked for completion of the reaction, and 80ml of saturated ammonium chloride solution was added to quench the reaction. Filtering, evaporating solvent under reduced pressure, adding water and ethyl acetate, separating organic layer, concentrating under reduced pressure, performing rapid silica gel column chromatography, gradient eluting with ethyl acetate/petroleum ether, and concentrating under reduced pressure to obtain 4.1g white solid as intermediate 4.

Preparation of intermediate 5

8g (28 mmol) of intermediate 4 and 96ml of 7M ammonia-methanol solution were added to the reaction vessel, and the reaction was stirred at 25 ℃ for 50hr and checked by TLC. Concentration under reduced pressure gave 6.9g of a yellow solid as intermediate 5.

Preparation of intermediate 6

Dissolving 10g (31.3 mmol) of intermediate 5 in 100ml of isopropanol, cooling to 0 ℃, adding 5.5M HCl-isopropanol 30ml, stirring at room temperature for 5hr, then heating to 50 ℃, stirring for reaction for 3hr, detecting by TLC that the reaction is complete, and concentrating the reaction solution under reduced pressure to obtain 5.5g of white solid as intermediate 6.

¹ H-NMR(MeOH-d ₄ )δppm：1.89-1.91(1H，m)，1.97-2.22(2H,m)，2.44-2.46 (1H，m)，2.68–2.91(1H，m),3.40(2H，dd),4.07(1H，dd)。LC-MS:ESI 172.2[M+H] ⁺ 。

Preparation of intermediate 10

To a reaction flask were added 10g (48.6 mmol) of methyl (1S, 3aR,6 aS) -octahydrocyclopenta [ C ] pyrrole-1-carboxylate hydrochloride, 12.4g (53.4 mmol) of N-tert-butoxycarbonyl-3-methyl-L-valine, 20ml of DMF and 180ml of MeCN, followed by addition of 20.4g (53.4 mmol) of HATU, cooling to 0 ℃ with stirring, dropwise addition of 25.4ml (145.8 mmol) of DIPEA, warming of the reaction solution to room temperature, followed by stirring for reaction for 15hr, monitoring completion of the reaction by TLC, and concentration of the reaction solution under reduced pressure. Extracting with water and ethyl acetate, washing the organic layer with water and 1M hydrochloric acid once respectively, separating the organic layer, concentrating under reduced pressure, performing rapid silica gel column chromatography, performing gradient elution with ethyl acetate/petroleum ether, and concentrating under reduced pressure to obtain 15.6g colorless oily substance as intermediate 10.

LC-MS:ESI 383.3[M+H] ⁺ 。

Preparation of intermediate 11

After 15g (39.3 mmol) of intermediate 10 was dissolved in 40ml of THF, 2.8g (117.7 mmol) of LiOH and 10ml of water were added, the reaction was stirred at room temperature for 2.5hr, TLC was used to monitor completion of the reaction, the organic solvent was distilled off under reduced pressure, and 1M hydrochloric acid was added to adjust the pH to about 2. Ethyl acetate is added for extraction, the organic layer is washed by saturated sodium chloride solution, the organic layer is separated out, and 11.9g of white solid which is the intermediate 11 is obtained by decompression and concentration.

Preparation of intermediate 12

10g (27.1 mmol) of intermediate 11 was dissolved in 30ml DCM, added with 35ml of 4M HCl-1, 4-dioxane solution, stirred at room temperature for 14hr, TLC monitored the completion of the reaction, and filtered to obtain 11.7g of white solid as intermediate 12.

Preparation of intermediate 13

Dissolving 17g (56 mmol) of the intermediate 12 in 40ml of methanol, adding 30ml (215 mmol) of triethylamine, then adding 12.8g (90.2 mmol) of ethyl trifluoroacetate, heating the reaction liquid to 50 ℃, stirring for reaction for 15hr, monitoring the reaction by TLC, concentrating under reduced pressure, evaporating to remove the organic solvent, adding water, and adjusting the pH to 3-4 by hydrochloric acid. Extraction with ethyl acetate, washing of the organic layer with saturated sodium chloride solution, separation of the organic layer, concentration under reduced pressure to obtain 12.8g of white solid as intermediate 13, with a content of 79.8% (qNMR).

LC-MS:ESI 365.1[M+H] ⁺ 。

Preparation of intermediate 14

12.8g (27.9 mmol) of intermediate 13 (content: 79.8%), 6.5g (31.7 mmol) of intermediate 6 and 100ml of 2-butanone are charged into a reaction flask, then 0.8g (7.2 mmol) of hydroxypyridone is added, stirring is carried out to reduce the temperature to 0 ℃, 15ml (85 mmol) of DIPEA is added dropwise, then 6.4g (33.5 mmol) of EDCI is added, the temperature of the reaction solution is increased to room temperature, then stirring is carried out for reaction for 15hr, TLC is carried out to monitor the reaction completion, 50ml of ethyl acetate/50 ml of MTBE mixed solvent is added, 40ml of water and 40ml of saturated sodium chloride solution are added for extraction, the organic layer is washed twice with saturated sodium chloride solution, 1M hydrochloric acid is washed twice, the organic layer is separated, and reduced pressure concentration is carried out to obtain 12.4g of white solid as the intermediate 14.

LC-MS:ESI 518.4[M+H] ⁺ 。

Preparation of Compound CN-01

9.5g (18.3 mmol) of the intermediate 14 was dissolved in 100ml of DCM, followed by addition of 10.8g (45.2 mmol) of methyl N- (triethylaminothioacyl) carbamate (Burgess reagent), stirring at room temperature for reaction for 2hr, monitoring completion of the reaction by TLC, dropwise addition of 50ml of a saturated sodium bicarbonate solution and 25ml of a saturated sodium chloride solution for extraction, separation of an organic layer, washing twice with a saturated sodium chloride solution, washing once with 1M hydrochloric acid, separation of an organic layer, drying over anhydrous magnesium sulfate, filtration, concentration under reduced pressure, flash column chromatography on silica gel, gradient elution with petroleum ether/ethyl acetate/methanol, and concentration under reduced pressure to obtain 6.6g of a white solid, i.e., compound CN-01.

¹ H-NMR(DMSO-d ₆ )δppm：0.88-0.90(1H,m)，0.96(9H，s)，1.14-1.18(2H， m)，1.34-1.40(2H,m)，1.52-1.54(1H,m)，1.63–1.74(2H,m)，2.13–2.16(1H,m)， 2.25-2.30(2H,m)，2.37-2.42(1H,m)，2.77–2.89(1H,m)，3.03-3.10(1H,m)，3.12 –3.17(1H,m)，3.21–3.24(1H,m)，3.56–3.70(2H,m)，3.91(1H,dd)，4.15(1H,s)， 4.44(1H,d)，4.95-5.01(1H,m)，7.70(1H,br)，9.05(1H,d)，9.47(1H,d)。LC-MS: ESI 500.3[M+H] ⁺ 。

Example 2.

Compound CN-02: (1S, 3aR, 6aS) -N- { (1S) -1-cyano-2- [ (3S) -2-oxo-3-pyrrolidinyl-5, 5-d ₂ ]Ethyl } -2- [ (2S) -3, 3-dimethyl-2- (2, 2-trifluoroacetylamino) butanoyl group]Octahydrocyclopenta [ C]Pyrrole-1-carboxamides

The synthetic route is as follows:

preparation of intermediate 15

10g of intermediate 3 (31.8 mmol) are dissolved in 150ml of MeOD, and 4.5g (19.1 mmol) of CoCl are added ₂ .6H ₂ And O, cooling to 0 ℃.8g (191 mmol) of NaBD were added portionwise ₄ After the addition, the reaction mixture was warmed to room temperature, stirred for 10hr, TLC checked for completion of the reaction, and 80ml of saturated ammonium chloride solution was added to quench the reaction. Filtering, evaporating under reduced pressure to remove solvent, adding water and ethyl acetate, separating organic layer, concentrating under reduced pressure, and concentratingPerforming silica gel column chromatography, performing gradient elution with ethyl acetate/petroleum ether, and concentrating under reduced pressure to obtain 4.5g of white solid as intermediate 15.

Preparation of intermediate 16

16.1g (56 mmol) of intermediate 15 and 192ml of 7M ammonia-methanol solution were added to the reaction vessel, and the reaction was stirred at 25 ℃ for 50hr and checked by TLC. Concentration under reduced pressure gave 14.2g of a yellow solid as intermediate 16.

Preparation of intermediate 7

Dissolving 10g (31.3 mmol) of intermediate 16 in 100ml of isopropanol, cooling to 0 ℃, adding 5.5M HCl-isopropanol 30ml, stirring at room temperature for 6hr, then heating to 50 ℃, stirring for reaction for 3hr, detecting by TLC that the reaction is complete, and concentrating the reaction solution under reduced pressure to obtain 6.1g of white solid which is intermediate 7.

¹ H-NMR(MeOH-d ₄ )δppm：1.88-1.91(1H，m)，1.96-2.22(2H,m)，2.43-2.45 (1H，m)，2.67–2.90(1H，m),4.06(1H，dd)。LC-MS:ESI 174.2[M+H] ⁺ . Deuterium substitution rate: 99.2% (LC-MS).

Preparation of intermediate 18

The preparation of intermediate 18 was as described for intermediate 14 except that intermediate 6 was replaced with intermediate 7.

LC-MS:ESI 520.4[M+H] ⁺ 。

Preparation of Compound CN-02

The preparation of compound CN-02 was as described for compound CN-01, except that intermediate 14 was replaced with intermediate 18.

¹ H-NMR(DMSO-d ₆ )δppm：0.87-0.91(1H,m)，0.94(9H，s)，1.16-1.19(2H， m)，1.35-1.41(2H,m)，1.53-1.55(1H,m)，1.64–1.76(2H,m)，2.15–2.18(1H,m)， 2.23-2.29(2H,m)，2.38-2.89(2H,m)，3.23–3.26(1H,m)，3.55–3.72(2H,m)，3.90 (1H,dd)，4.18(1H,s)，4.46(1H,d)，4.94-5.03(1H,m)，7.71(1H,br)，9.04(1H,d)， 9.48(1H,d)。LC-MS:ESI 502.3[M+H] ⁺ . Deuterium substitution rate: 99.0% (LC-MS).

Example 3.

Compound CN-08: (S) -N- { (1S) -1-cyano-2- [ (3S) -2-oxo-3-pyrrolidinyl ] ethyl } -2- [ (2S) -3, 3-dimethyl-2- (2, 2-trifluoroacetylamino) butanoyl ] -5-azaspiro [2.4] heptane-6-carboxamide

The synthetic route is as follows:

preparation of intermediate 20

To a reaction flask were added 9.3g (48.6 mmol) of (S) -5-azaspiro [2.4] heptane-6-carboxylic acid methyl ester hydrochloride, 12.4g (53.4 mmol) of N-tert-butoxycarbonyl-3-methyl-L-valine, 20ml of DMF and 180ml of MeCN, followed by addition of 20.4g (53.4 mmol) of HATU, cooling to 0 ℃ with stirring, dropwise addition of 25.4ml (145.8 mmol) of DIPEA, warming to room temperature with stirring, reaction with stirring for 15hr, completion of TLC monitoring, and concentration of the reaction solution under reduced pressure. Extracting with water and ethyl acetate, washing the organic layer with water and 1M hydrochloric acid once respectively, separating the organic layer, concentrating under reduced pressure, performing rapid silica gel column chromatography, performing gradient elution with ethyl acetate/petroleum ether, and concentrating under reduced pressure to obtain 13.0g colorless oily substance as intermediate 20.

LC-MS:ESI 369.3[M+H] ⁺ 。

Preparation of intermediate 21

14.5g (39.3 mmol) of intermediate 20 was dissolved in 40ml of THF, 2.8g (117.7 mmol) of LiOH and 10ml of water were added, the reaction was stirred at room temperature for 2.5hr, the completion of the reaction was monitored by TLC, the organic solvent was distilled off under reduced pressure, and 1M hydrochloric acid was added to adjust the pH to about 2. Ethyl acetate is added for extraction, the organic layer is washed by saturated sodium chloride solution, the organic layer is separated out, and 10.1g of white solid is obtained by decompression and concentration as an intermediate 21.

Preparation of intermediate 22

9.6g (27.1 mmol) of intermediate 21 was dissolved in 30ml DCM, then 35ml of 4M HCl-1, 4-dioxane solution was added, the reaction was stirred at room temperature for 14hr, TLC monitored for completion of the reaction, and filtration was carried out to give 10.9g of white solid as intermediate 22.

Preparation of intermediate 23

Dissolving 16.2g (56 mmol) of intermediate 22 in 40ml of methanol, adding 30ml (215 mmol) of triethylamine, then adding 12.8g (90.2 mmol) of ethyl trifluoroacetate, heating the reaction solution to 50 ℃, stirring for reaction for 15 hours, monitoring the reaction by TLC, concentrating under reduced pressure, evaporating to remove the organic solvent, adding water, and adjusting the pH to 3-4 by hydrochloric acid. Ethyl acetate extraction, washing the organic layer with saturated sodium chloride solution, separating the organic layer, and concentrating under reduced pressure to obtain 13.2g of white solid as intermediate 23 with a content of 87.9% (qNMR).

LC-MS:ESI 351.1[M+H] ⁺ 。

Preparation of intermediate 24

11.6g (27.9 mmol) of intermediate 23 (content: 87.9%), 6.5g (31.7 mmol) of intermediate 6 and 100ml of 2-butanone are added to a reaction flask, then 0.8g (7.2 mmol) of hydroxypyridone is added, stirring is carried out to reduce the temperature to 0 ℃, 15ml (85 mmol) of DIPEA is added dropwise, then 6.4g (33.5 mmol) of EDCI is added, the reaction solution is heated to room temperature, then stirring is carried out for reaction for 15hr, TLC is used for monitoring the completion of the reaction, 50ml of ethyl acetate/50 ml of MTBE mixed solvent is added, 40ml of water and 40ml of saturated sodium chloride solution are added for extraction, the organic layer is washed twice with saturated sodium chloride solution, 1M hydrochloric acid is washed twice, the organic layer is separated, and reduced pressure concentration is carried out to obtain 11.6g of white solid as intermediate 24.

LC-MS:ESI 504.4[M+H] ⁺ 。

Preparation of Compound CN-08

9.2g (18.3 mmol) of intermediate 24 was dissolved in 100ml of DCM, followed by addition of 10.8g (45.2 mmol) of methyl N- (trisethylamine sulfonyl) carbamate (Burgess reagent), reaction was stirred at room temperature for 2hr, completion of the reaction was monitored by TLC, extraction was performed by dropwise addition of 50ml of a saturated sodium bicarbonate solution and 25ml of a saturated sodium chloride solution, separation of an organic layer, washing twice with a saturated sodium chloride solution, washing once with 1M hydrochloric acid, separation of an organic layer, drying over anhydrous magnesium sulfate, filtration, concentration under reduced pressure, flash column chromatography on silica gel, gradient elution with petroleum ether/ethyl acetate/methanol, concentration under reduced pressure to obtain 5.7g of a white solid as compound CN-08.

¹ H-NMR(DMSO-d ₆ )δppm：0.57-0.88(4H，m)，0.97(9H，s)，1.22-1.63(1H， m)，1.64–1.75(2H,m)，2.04–2.13(1H,m)，2.14-2.91(3H,m)，3.03-3.17(2H,m)， 3.53-4.10(2H,m)，4.29-4.48(2H,m)，4.66-4.87(1H,m)，7.68(1H,br)，9.01(1H,d)， 9.39(1H,d)。LC-MS:ESI 486.3[M+H] ⁺ 。

Example 4.

Chemical combinationThe compound CN-09: (S) -N- { (1S) -1-cyano-2- [ (3S) -2-oxo-3-pyrrolidinyl-5, 5-d ₂ ]Ethyl } -2- [ (2S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) butanoyl group]-5-azaspiro [2.4]Heptane-6-carboxamide

The synthetic route is as follows:

preparation of intermediate 25

Intermediate 25 was prepared as described for intermediate 24 except intermediate 6 was replaced with intermediate 7.

LC-MS:ESI 506.4[M+H] ⁺ 。

Preparation of Compound CN-09

The preparation of compound CN-09 was as described for compound CN-08, except that intermediate 24 was replaced with intermediate 25.

¹ H-NMR(DMSO-d ₆ )δppm：0.58-0.90(4H，m)，0.99(9H，s)，1.21-1.62(1H， m)，1.64–1.75(2H,m)，2.03–2.14(1H,m)，2.15-2.93(3H,m)，3.51-4.11(2H,m)， 4.30-4.49(2H,m)，4.68-4.89(1H,m)，7.69(1H,br)，9.05(1H,d)，9.36(1H,d)。LC-MS: ESI 488.3[M+H] ⁺ . Deuterium substitution rate: 99.1% (LC-MS).

Example 5.

The compound CN-10: (1R, 2S, 5S) -N- { (1S) -1-cyano-2- [ (3S) -2-oxo-3-pyrrolidinyl-5, 5-d ₂ ]Ethyl } -3- [ (2S) -3, 3-dimethyl-2- (2, 2-trifluoroacetylamino) butanoyl group]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide

The synthetic route is as follows:

preparation of intermediate 27

To a reaction flask were added 10g (48.6 mmol) of (1R, 2S, 5S) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxylic acid methyl ester hydrochloride, 12.4g (53.4 mmol) of N-tert-butoxycarbonyl-3-methyl-L-valine, 20ml of DMF and 180ml of MeCN, followed by addition of 20.4g (53.4 mmol) of HATU, cooling to 0 ℃ with stirring, dropwise addition of 25.4ml (145.8 mmol) of DIPEA, warming of the reaction solution to room temperature, followed by stirring for reaction for 15hr, monitoring by TLC for completion of the reaction, and concentration of the reaction solution under reduced pressure. Extracting with water and ethyl acetate, washing the organic layer with water and 1M hydrochloric acid once respectively, separating the organic layer, concentrating under reduced pressure, performing rapid silica gel column chromatography, performing gradient elution with ethyl acetate/petroleum ether, and concentrating under reduced pressure to obtain 16.3g colorless oily substance as intermediate 27.

LC-MS:ESI 383.3[M+H] ⁺ 。

Preparation of intermediate 28

After 15g (39.3 mmol) of intermediate 27 was dissolved in 40ml of THF, 2.8g (117.7 mmol) of LiOH and 9ml of water were added, the reaction was stirred at room temperature for 2.5hr, TLC was used to monitor completion of the reaction, the organic solvent was distilled off under reduced pressure, and 1M hydrochloric acid was added to adjust the pH to about 2. Adding ethyl acetate for extraction, washing an organic layer by using a saturated sodium chloride solution, separating the organic layer, and concentrating under reduced pressure to obtain 12g of white solid serving as an intermediate 28.

Preparation of intermediate 29

15g (40.6 mmol) of intermediate 28 were dissolved in 40ml DCM and then 51ml of 4M HCl-1, 4-dioxane solution was added, the reaction was stirred at room temperature for 14hr, TLC monitored for completion of the reaction and filtered to give 11.4g of white solid as intermediate 29.

Preparation of intermediate 30

17.1g (56 mmol) of the intermediate 29 is dissolved in 40ml of methanol, 30ml (215 mmol) of triethylamine is added, then 12.8g (90.2 mmol) of ethyl trifluoroacetate is added, the temperature of the reaction solution is raised to 50 ℃, the reaction is stirred for 15 hours, TLC monitors the complete reaction, the organic solvent is evaporated by concentration under reduced pressure, water is added, and the pH is adjusted to 3-4 by hydrochloric acid. Ethyl acetate extraction, washing the organic layer with saturated sodium chloride solution, separating the organic layer, and concentrating under reduced pressure to obtain 13.7g of white solid as intermediate 30 with a content of 83.4% (qNMR).

LC-MS:ESI 365.1[M+H] ⁺ 。

Preparation of intermediate 31

12.2g (27.9 mmol) of intermediate 30 (content: 83.4%), 6.6g (31.7 mmol) of intermediate 7 and 110ml of 2-butanone are added into a reaction flask, then 0.78g (7.1 mmol) of hydroxypyridone is added, stirring is carried out to reduce the temperature to 0 ℃, 14.6ml (84 mmol) of DIPEA is added dropwise, then 6.4g (33.5 mmol) of EDCI is added, the temperature of the reaction solution is increased to room temperature, then stirring is carried out for reaction for 15hr, TLC is used for monitoring the reaction completion, 50ml of ethyl acetate/50 ml of MTBE mixed solvent is added, 40ml of water and 40ml of saturated sodium chloride solution are added for water extraction, the organic layer is washed twice with saturated sodium chloride solution, 1M hydrochloric acid is washed twice, the organic layer is separated, and 13.6g of white solid is obtained by decompression concentration and is intermediate 31.

LC-MS:ESI 520.4[M+H] ⁺ 。

Preparation of Compound CN-10

9.5g (18.2 mmol) of the intermediate 31 was dissolved in 90ml of DCM, followed by addition of 10.8g (45.2 mmol) of methyl N- (triethylaminothioacyl) carbamate (Burgess reagent), stirring at room temperature for reaction for 2hr, monitoring completion of the reaction by TLC, dropwise addition of 40ml of a saturated sodium bicarbonate solution and 20ml of a saturated sodium chloride solution for extraction, separation of an organic layer, washing twice with a saturated sodium chloride solution, washing once with 1M hydrochloric acid, separation of an organic layer, drying over anhydrous magnesium sulfate, filtration, concentration under reduced pressure, flash column chromatography on silica gel, gradient elution with petroleum ether/ethyl acetate/methanol, and concentration under reduced pressure to obtain 7.5g of a white solid, i.e., the compound CN-10.

¹ H-NMR(DMSO-d ₆ )δppm：0.84(3H,s)，0.97(9H，s)，1.02(3H,s)，1.31(1H, d)，1.58(1H,dd),1.64–1.75(2H,m)，2.04–2.13(1H,m)，2.14–2.15(1H,m)，2.39- 2.41(1H,m)，3.70(1H,d)，3.93(1H,dd)，4.16(1H,s)，4.43(1H,d)，4.96-5.00(1H, m)，7.69(1H,br)，9.04(1H,d)，9.45(1H,d)。LC-MS:ESI 502.4[M+H] ⁺ . Deuterium substitution rate: 99.1% (LC-MS).

Compound CN-10 was able to form a solvate with methyl tert-butyl ether (MTBE).

Reference is made to the above examples to prepare control compounds: (1R, 2S, 5S) -N- { (1S) -1-cyano-2- [ (3S) -2-oxo-3-pyrrolidinyl ] ethyl } -3- [ (2S) -3, 3-dimethyl-2- (2, 2-trifluoroacetylamino) butanoyl ] -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide (PF-07321332).

PF-07321332

LC-MS:ESI 500.4[M+H] ⁺ 。

Referring to the synthetic schemes of the above examples, the compounds shown in table 1 below were prepared.

Table 1 other compound structures and mass spectral data

Effect example 1 evaluation of SARS-CoV-2 3CL protease inhibitory Activity

Examples compounds were tested for their inhibitory activity against the novel coronavirus 3CL protease (SARS-CoV-2 3 CLpro).

< technical method > the enzyme level inhibitory activity of an inhibitor against 3CL protease was determined using Fluorescence Resonance Energy Transfer (FRET) technique.

< step > on a 96-well plate, 27.5. Mu.L of enzyme buffer (20 mM Tris-HCl,100mM NaCl,1mM EDTA, pH 7.3) was added to each well, along with 2.5. Mu.L of different concentrations of compound and 5. Mu.L of EV71 Cpro (final concentration 3. Mu.M). Co-incubation was performed at 37 ℃ for 20min. Then 15. Mu.L of fluorogenic substrate diluted in buffer (final concentration 20. Mu.M) was added. Fluorescence parameters were measured using a Gen5 fluorescence gradiometer with excitation and emission wavelengths of 340nm and 490nm respectively, and data were read after 10min at 37 ℃. Negative control, no compound added, the rest of the procedure was the same.

<Results>The obtained data were processed using the software GraphPad prism7.0 to calculate IC ₅₀ The value is obtained. The results of the experiment are shown in table 2.

TABLE 2 inhibitory Activity of the Compounds of the examples on SARS-CoV-2 3CL protease

From the above results, all the tested compounds showed higher inhibitory activity against SARS-CoV-2 3CL protease than that of the SARS-CoV-2 3CL protease, and particularly, the inhibitory activity against the 3CL protease of the compounds CN-02, CN-09 and CN-10 was about 2 times that of PF-07321332. Therefore, the compounds of the examples can be prepared as preventive/therapeutic drugs for symptoms/diseases induced by infection with a novel coronavirus, and have superior therapeutic effects.

Effect example 2 evaluation of SARS-CoV-2 Virus CPE Activity

The compounds of the examples were tested for their ability to inhibit the replication of SARS-CoV-2 virus.

<Step (ii) of>In BSL-3 laboratory, veroE6 cell line was inoculated (10) ⁴ Individual cells/100. Mu.L/well) in 96-well plates, then maintained at 37 ℃ 5% CO ₂ Cultured in a cell culture box. After 24 hours, the supernatants of the Vero E6 cell lines were replaced with infection medium (with 2% fbs) containing different concentrations of the compounds of the examples. Using SARS-CoV-2[ nCoV-2019 ]]The virus infected VeroE6 cells at a multiplicity of infection (MOI) of 0.01. Cells inoculated with virus were added to test compound wells and maintained in DMEM with 2% heat-inactivated FBS. The inoculated culture was subjected to 5% CO at 37 ℃% ₂ Incubate for 3 days. 3-fold serial dilutions of the example compounds were prepared in triplicate starting at 1uM and added to VeroE6 cell culture medium on the outside of the culture substrate approximately 30 minutes prior to infection. The experiment was performed in a blank control well (normal cells), a virus control well (no drug was added before virus infection), and a positive drug control well (PF-07321332 was added before infection).

< results > results of anti-neocoronaviruses activity of samples:

the inhibition rate (%)EC was then determined using curve fitting software GraphPad prism7.0 ₅₀ The value is obtained. Concentration EC causing 50% inhibitory effect ₅₀ As shown. Toxic effects of drugs on VeroE6 cells, CC ₅₀ The selection index for the inhibition of the sample against the novel coronavirus is indicated as SI ₅₀ And (4) showing. The CPE test results are shown in table 3.

TABLE 3 results of the test for inhibiting SARS-CoV-2 virus replication with the compounds of the examples

From the above results, it is clear that all the tested compounds showed higher anti-new crown virus effect and higher selectivity index (therapeutic index), especially the anti-SARS-CoV-2 effect of compounds CN-09 and CN-10 is about 2.5 times of PF-07321332, and the therapeutic index of compounds CN-09 and CN-10 is 6 times or more of PF-07321332. Thus, the compounds of the examples can be prepared as prophylactic/therapeutic agents for symptoms/diseases induced by SARS-CoV-2 infection, and have a higher therapeutic index and lower toxicity.

Effect example 3 study of pharmacokinetics of pulmonary tissue and plasma of SD rat

Plasma distribution of drug: SD rats were dosed orally with the example compound at 200mpk, with the administration of the lysin: 2% (v/v) Tween 80+0.5% (w/v) MC, and the plasma concentrations of the corresponding compounds in SD rats at 0.1, 0.25, 0.5, 0.75, 1, 2, 4, and 8 hours after administration were determined by tandem mass spectrometry to calculate the plasma PK parameters of the drug.

Drug lung tissue distribution: SD rats were orally administered the example compound at a dose of 200mpk, with the administration of a lysin: 2% (v/v) Tween 80 in 0.5% (w/v) MC solution, 0.25, 0.5, 1, 2, 4, 8 hours after administration, taking SD rat lung tissue, adding homogenized lysozyme (0.02M acetate buffer) hand-held tissue homogenizer for homogenization. Tandem mass spectrometry was used to determine the concentration of the compound in lung tissue and calculate the PK parameters for drug lung tissue.

The lung and plasma pharmacokinetic parameters are shown in table 4.

Table 4 test results of lung/plasma ratio of compound drug of examples

From the above results, it can be seen that all the tested compounds have excellent oral absorption pharmacokinetic parameters and excellent drug distribution in target organ lung tissue, especially the pulmonary drug distribution amounts of compounds CN-09 and CN-10 are 5.2 times and 3.1 times of PF-07321332, respectively. The lung/plasma ratio of the drugs for compounds CN-09 and CN-10 was about 2 times that of PF-07321332. Therefore, the compound of the embodiment can be used for preparing an oral medicament for preventing/treating symptoms/diseases induced by SARS-CoV-2 infection, has higher exposure and higher lung targeting property, is clinically orally administered, and has the combined potential without a drug-induced synergist CYP3A4 enzyme inhibitor.

It will be appreciated by those skilled in the art that the present disclosure is not limited to the foregoing illustrative embodiments, but may be embodied in other specific forms without departing from the essential attributes thereof. It is therefore intended that the embodiments described herein are to be considered in all respects as illustrative and not restrictive, the embodiments referenced by the appended claims being other than the foregoing embodiments, the references being made to the appended claims rather than to the foregoing examples, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

All patents, patent applications, and literature references cited in this specification are hereby incorporated by reference in their entirety. In the event of inconsistencies, the present disclosure, including definitions, will be convincing.

Claims

1. A compound represented by the formula:

2. a pharmaceutical composition comprising an effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

3. The pharmaceutical composition according to claim 2, further comprising an additional pharmaceutically active ingredient selected from the group consisting of ritonavir in a therapeutically effective amount.

4. Use of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a condition or disease caused by a virus having a 3C-like protease.

5. Use of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a condition or disease caused by the β -coronavirus.

6. Use of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a condition or disease caused by SARS-CoV-2 infection.

7. Use of a pharmaceutical composition according to any one of claims 2 to 3 for the preparation of a medicament for the treatment of a condition or disease caused by a virus having a 3C-like protease.

8. Use of a pharmaceutical composition according to any one of claims 2 to 3 for the preparation of a medicament for the treatment of a condition or disease caused by the β -coronavirus.

9. Use of a pharmaceutical composition according to any one of claims 2-3 for the preparation of a medicament for the treatment of symptoms or diseases caused by SARS-CoV-2 infection.