CN108929329A - 2- azacyclo- -5- trifluoromethyl -8- nitro benzo (thio) pyrans -4- ketone compounds - Google Patents

Abstract

The invention discloses 2-azacyclic-5-trifluoromethyl-8-nitrobenzo(thio)pyran-4-ketone compounds, their preparation method and their application in the treatment and/or prevention of Mycobacterium tuberculosis Application of drugs caused by infectious diseases. Specifically, the present invention relates to compounds represented by formula (I) and their isomers, pharmaceutically acceptable salts thereof, and pharmaceutical compositions containing the compounds of the present invention, wherein X, Y, R ₁ , R ₂ , and n are as described in the description mentioned. The present invention aims to prepare a new compound with anti-tuberculosis mycobacterium activity, which can be used as a potential new drug for the treatment or prevention of infectious diseases caused by bacteria, especially tuberculosis (TB) disease caused by mycobacteria sexual therapy and can be used to overcome problems associated with drug resistance in Mycobacterium tuberculosis.

Description

2-Azacyclo-5-trifluoromethyl-8-nitrobenzo(thio)pyran-4-ones compound

technical field

The invention belongs to the technical field of medicine. In particular, it relates to 2-azacyclo-5-trifluoromethyl-8-nitrobenzo(thio)pyran-4-one compounds represented by the general formula (I), and its preparation method uses the compound as Pharmaceutical compositions of active ingredients, and their use in the treatment and/or prophylaxis of infectious diseases caused by Mycobacterium tuberculosis.

Background technique

Tuberculosis (TB) is a chronic fatal disease caused by Mycobacterium tuberculosis. It is a major infectious disease that endangers human health and causes human death. Like AIDS, tuberculosis has become one of the leading causes of death in the world. According to the World Health Organization (WHO) estimate (Global tuberculosis report 2016), the number of new tuberculosis cases in the world in 2015 was approximately 10.4 million, of which 5.9 million were males (56%), 3.5 million were females (34%), and 100 million for children (accounting for 10%). 1.2 million new TB cases were among people living with HIV (11%), and an estimated 1.4 million people and 400,000 HIV-infected people died from TB.

Chemotherapy is the mainstay of tuberculosis treatment. The use of streptomycin in 1944 ushered in a new era of anti-tuberculosis drug treatment. With the successive appearance of isoniazid, rifampicin and pyrazinamide, the course of treatment for tuberculosis was shortened to 6 months, and entered the "short-course chemotherapy" era. era". Nevertheless, long-term drug combination therapy causes adverse reactions in patients, and it is difficult to adhere to regular drug use. In addition, most of the drugs used were born in the 1950s and 1960s. Long-term, widespread and irregular use made the development of drug-resistant bacteria increasingly serious, and the emergence of multi-drug Drug-resistant TB (MDR-TB), extensively drug-resistant TB (XDR-TB) and totally drug-resistant TB (TDR-TB). In the face of drug-resistant tuberculosis, it is necessary to use expensive and highly toxic second-line or even third-line anti-tuberculosis drugs. Therefore, it is extremely urgent to develop anti-tuberculosis drugs with new skeletons and novel mechanisms of action to treat and control tuberculosis, especially drug-resistant tuberculosis. New anti-tuberculosis drugs need to have the characteristics of high efficacy, low toxicity, and the ability to shorten treatment time.

Contents of the invention

The technical problem to be solved in the present invention is to provide a 2-azacyclic-5-trifluoromethyl-8-nitrobenzo(thio)pyran-4 with novel structure and strong anti-mycobacterium tuberculosis activity. - Ketones. The present invention finds that 2-azacyclic-5-trifluoromethyl-8-nitrobenzo(thio)pyran-4-one compounds have strong anti-tuberculosis Mycobacterium effect and can be used for The therapeutic or preventive treatment of infectious diseases, especially tuberculosis (TB) disease caused by mycobacteria, can also be used to overcome problems related to drug resistance. The present invention has been accomplished based on the above findings.

Summary of the invention

To this end, the first aspect of the present invention provides compounds represented by general formula (I) and their isomers, or pharmaceutically acceptable salts thereof,

in,

X is O or S;

Y is C or N;

R is H, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, F, _Cl , Br, CN, OH, (=O), (=S);

n is 0, 1 or 2;

R ₂ is F, substituted or unsubstituted C ₃ -C ₆ cycloalkyl, substituted or unsubstituted C ₃ -C ₆ heterocyclyl, substituted or unsubstituted C ₆ -C ₁₀ aryl, substituted or unsubstituted The C ₂ -C ₉ heteroaryl group, or together with the carbon atoms connected together represent a substituted or unsubstituted cycloalkyl group containing 3-8 carbon atoms or a group containing 1-3 heteroatoms selected from oxygen and sulfur Substituted or unsubstituted 4-6 membered heterocyclic group;

The C ₃ -C ₆ -membered heterocyclic group and C ₂ -C ₉ heteroaryl group contain at least one heteroatom selected from N, O, and S;

_The substituted or unsubstituted substituents in R can be optionally selected from the following groups: F, Cl, Br, hydroxyl, amino, nitro, cyano, trifluoromethyl, trifluoromethoxy, C ₁ - C ₃ alkyl, halogenated C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy or C ₁ -C ₃ alkylamino.

In a preferred example, the structural formula of the compound is as shown in (II):

Wherein, the definitions of Y, R ₁ , R ₂ and n are the same as those described in the first aspect of the present invention.

In another preferred example, the structural formula of the compound is as shown in (III):

Wherein, the definitions of Y, R ₁ , R ₂ and n are the same as those described in the first aspect of the present invention.

In some aspects, the compound of formula (I) is selected from the compound of formula (II-a):

in,

R ₁ is H, C ₁ -C ₃ alkyl, F, Cl or (=O);

n is 0 or 1;

_R2 is F, substituted or unsubstituted cyclohexyl, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted pyridyl, substituted or unsubstituted quinolinyl, substituted or unsubstituted The pyrrolyl group, substituted or unsubstituted furyl group, substituted or unsubstituted thienyl group, substituted or unsubstituted thiazolyl group, or together with the carbon atoms connected together represent a substituted or unsubstituted cyclopentyl group or a 1-2 A substituted or unsubstituted 5-membered heterocyclic group selected from oxygen and sulfur heteroatoms;

_The substituted or unsubstituted substituents in R can be optionally selected from the following groups: F, Cl, Br, hydroxyl, amino, nitro, cyano, trifluoromethyl, trifluoromethoxy, C ₁ - C ₃ alkyl, halogenated C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy or C ₁ -C ₃ alkylamino.

In some aspects, the compound of formula (I) is selected from the compound of formula (II-b):

in,

R ₁ is H, C ₁ -C ₃ alkyl, F, Cl or (=O);

n is 0 or 1;

_R is substituted or unsubstituted cyclohexyl, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted pyridyl, substituted or unsubstituted quinolinyl, substituted or unsubstituted pyrrole substituted or unsubstituted furyl, substituted or unsubstituted thienyl, substituted or unsubstituted thiazolyl;

_The substituted or unsubstituted substituents in R can be optionally selected from the following groups: F, Cl, Br, hydroxyl, amino, nitro, cyano, trifluoromethyl, trifluoromethoxy, C ₁ - C ₃ alkyl, halogenated C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy or C ₁ -C ₃ alkylamino;

In some aspects, the compound of formula (I) is selected from the compound of formula (III-a):

in,

R ₁ is H, C ₁ -C ₃ alkyl, F, Cl or (=O);

n is 0 or 1;

_R2 is F, substituted or unsubstituted cyclohexyl, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted pyridyl, substituted or unsubstituted quinolinyl, substituted or unsubstituted The pyrrolyl group, substituted or unsubstituted furyl group, substituted or unsubstituted thienyl group, substituted or unsubstituted thiazolyl group, or together with the carbon atoms connected together represent a substituted or unsubstituted cyclopentyl group or a 1-2 A substituted or unsubstituted 5-membered heterocyclic group selected from oxygen and sulfur heteroatoms;

_The substituted or unsubstituted substituents in R can be optionally selected from the following groups: F, Cl, Br, hydroxyl, amino, nitro, cyano, trifluoromethyl, trifluoromethoxy, C ₁ - C ₃ alkyl, halogenated C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy or C ₁ -C ₃ alkylamino;

In some aspects, the compound of formula (I) is selected from the compound of formula (III-b):

in,

R ₁ is H, C ₁ -C ₃ alkyl, F, Cl or (=O);

n is 0 or 1;

_R is substituted or unsubstituted cyclohexyl, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted pyridyl, substituted or unsubstituted quinolinyl, substituted or unsubstituted pyrrole substituted or unsubstituted furyl, substituted or unsubstituted thienyl, substituted or unsubstituted thiazolyl;

_The substituted or unsubstituted substituents in R can be optionally selected from the following groups: F, Cl, Br, hydroxyl, amino, nitro, cyano, trifluoromethyl, trifluoromethoxy, C ₁ - C ₃ alkyl, halogenated C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy or C ₁ -C ₃ alkylamino;

In formula (II) and (III) scheme,

n is preferably 0, 1 or 2;

R ₁ is preferably H, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, F, Cl, Br, CN, OH, (=O) or (=S);

When Y is C,

_R2 is preferably F,

When Y is N,

_R2 is preferably

Rx is F, Cl, Br, hydroxyl, amino, nitro, cyano, trifluoromethyl, trifluoromethoxy, C ₁ -C ₃ alkyl, halogenated C ₁ -C ₃ alkyl, C ₁ - C ₃ alkoxy or C ₁ -C ₃ alkylamino;

The pharmaceutically acceptable salt described in the present invention is a salt formed by the compound of the present invention and an acid selected from the following: hydrochloric acid, p-toluenesulfonic acid, tartaric acid, maleic acid, lactic acid, methanesulfonic acid, sulfuric acid, phosphoric acid, lemon acid, acetic acid or trifluoroacetic acid. Preference is given to hydrochloric acid, p-toluenesulfonic acid or trifluoroacetic acid.

The compound according to any one of the first aspect of the present invention is the target compound of the present invention (represented by structural formula or described by systematic name) prepared in the examples, its isomers, and pharmaceutically acceptable salts thereof.

According to any one compound of the first aspect of the present invention, it is a compound selected from the following:

The second aspect of the present invention provides a method for preparing any one of the compounds described in the first aspect of the present invention, which comprises the following steps:

Compound A is in a suitable solvent (such as dichloromethane, tetrahydrofuran, acetonitrile, preferably dichloromethane), and amine compound B, under the action of a condensation reagent (such as CDI, DCC, EDCI\HOBT, HATU, preferably DCC) , under the protection of air or inert gas (Ar or N ₂ ), put it at -10°C-50°C for 1-24 hours, preferably at room temperature for 8-15 hours, to obtain the compound shown in formula C;

The compound shown in formula C is in a suitable solvent (such as DMF, DMSO, preferably DMF), under basic conditions (such as sodium carbonate, potassium carbonate or cesium carbonate, preferably potassium carbonate), under an inert gas (Ar or N ₂ ) Under protection, react at 20-140°C for 0.5-12 hours, preferably at 110°C for 1-5 hours, to obtain the compound of formula (II).

Compound A in the present invention can be easily prepared by referring to known methods in existing publications, for example (J. Med. Chem. 2007, 50, 3369-3379).

Compound D and carbon disulfide in a suitable solvent (such as toluene, acetone, tetrahydrofuran, DMF, DMSO, preferably DMSO), under basic conditions (such as NaH, sodium hydroxide, sodium methoxide, sodium ethoxide, potassium tert-butoxide, tert-butyl Sodium alkoxide, preferably sodium hydroxide), under the protection of air or inert gas (Ar or N ₂ ), react at -10-30°C for 10-60 minutes, preferably at 15-25°C for 15-30 minutes, then Add MeI at 15-25°C and react for 30-60 minutes to obtain compound E;

Compound E and amine compound B, in a suitable solvent (such as tert-butanol, isopropanol, ethylene glycol, ethylene glycol dimethyl ether, DMF, DMSO, preferably isopropanol), in air or inert gas ( Under the protection of Ar or N ₂ ), react at 80-160°C for 1-48 hours, preferably at 120-140°C for 20-25 hours, to obtain the compound of formula (III).

Dissolve or suspend the compound of formula (III) in a suitable solvent (such as dichloromethane, tetrahydrofuran, methanol, ethanol, isopropanol, preferably ethanol, isopropanol) in air or inert Under the protection of gas (Ar or N ₂ ), add the corresponding acid solution (such as hydrochloric acid, sulfuric acid, ethanol hydrochloride, preferably ethanol hydrochloride), stir at room temperature or under heating for 1-48 hours, preferably at room temperature for 2 hours, and filter , washed with the corresponding solvent, and dried to obtain the corresponding salt of the compound of formula (III).

The third aspect of the present invention provides a pharmaceutical composition, which comprises a therapeutically effective amount of any one of the compounds described in the first aspect of the present invention and pharmaceutically acceptable salts thereof, and optionally one or more pharmaceutically acceptable Accepted excipients.

The fourth aspect of the present invention provides the compound described in any one of the first aspect of the present invention and the pharmaceutically acceptable salt thereof, or the pharmaceutical composition described in any one of the third aspect of the present invention in the preparation of treatment and/or prevention of tuberculosis Application in drugs for infectious diseases caused by bacilli.

The foregoing merely outlines certain aspects of the invention, but is not limited thereto. These and other aspects will be described more specifically and fully below.

Detailed description of the invention

Various aspects and features of the present invention are further described below.

All the documents cited in the present invention are incorporated herein by reference in their entirety, and if the meaning expressed in these documents is inconsistent with the present invention, the expression of the present invention shall prevail. In addition, various terms and phrases used in the present invention have common meanings known to those skilled in the art. Even so, the present invention still hopes to make a more detailed description and explanation of these terms and phrases here. The terms and phrases mentioned are as follows: If there is any inconsistency with the known meaning, the meaning expressed in the present invention shall prevail. The following are definitions of various terms used in the present invention, and these definitions apply to the terms used throughout the specification of this application, unless otherwise stated in specific cases.

In general, the term "substituted or unsubstituted" means that one or more hydrogen atoms in a given structure are replaced by a particular substituent. Unless otherwise indicated, an optional substituent may be substituted at each substitutable position of the group. When more than one position of a given structure is substituted at all times by one or more substituents selected from a particular group, then the substituents may be substituted at each position identically or differently.

C _i -C _j represents a moiety having an integer "i" (inclusive) to an integer "j" (inclusive) of carbon atoms. Thus, for example, C ₁ -C ₃ alkyl refers to an alkyl group having 1 to 3 carbon atoms inclusive. For example, C ₂ -C ₉ heteroaryl refers to a heteroaryl group with 2 to 9 (including 2 and 9) carbon atoms, including tetrazolyl, triazolyl, thienyl, pyridyl, pyrimidyl, quinoline base.

As used herein, the term "alkyl" refers to an alkyl group having the specified number of carbon atoms, which is straight or branched, and which may include subgroups thereof, for example referring to "C ₁ - When C ₃ alkyl", it may also include sub-range groups represented by C ₁ -C ₂ alkyl, and specific groups such as methyl, ethyl, n-propyl, isopropyl.

As used herein, the terms "alkoxy" and "alkylamino" are conventional expressions referring to an alkyl group attached to the rest of the molecule through an oxygen atom or an amine group, respectively, where the alkyl group is Invention described.

As used herein, the term "haloalkyl" means that the hydrogens on the alkyl group are replaced by one or more halogen atoms, examples of which include, but are not limited to, monofluoromethyl, monofluoromethoxy, etc. .

As used herein, the term "cycloalkyl" refers to a cyclic alkyl group having the indicated number of ring carbon atoms, and which may include subgroups thereof, for example when referring to "C ₃ -C ₆ cycloalkyl" , which may also include sub-range groups represented by C ₃ -C ₅ cycloalkyl, C ₄ -C ₆ cycloalkyl, etc., and specific groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl .

As used herein, the term "C ₃ -C ₆ heterocycloalkyl", unless otherwise stated or limited, refers to a saturated or partially unsaturated monocyclic, bicyclic or tricyclic ring containing 3-6 ring carbon atoms Ring systems wherein at least one ring atom is selected from nitrogen, sulfur and oxygen atoms. Unless otherwise stated, a heterocyclyl group can be carbonyl or nitrogenyl, and a _-CH2- group can optionally be replaced by a carbonyl group. Ring sulfur atoms can optionally be oxidized to S-oxides. Heterocyclic groups include, but are not limited to, oxetanyl, azetidinyl, tetrahydrofuranyl, tetrahydropyrrolyl, tetrahydrothiazolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholine , Homopiperazinyl, etc.

As used herein, the term "C ₆ -C ₁₀ aryl" denotes monocyclic and bicyclic carbocyclic ring systems containing 6 ring atoms or 6-10 ring atoms, wherein at least one ring is aromatic, wherein Each ring contains rings of 3-6 atoms, and the ring system has one or more points of attachment to the rest of the molecule. Aromatic groups may include phenyl and naphthyl.

As used herein, the term "C2 _{- C9} heteroaryl" refers herein to an aromatic group having 1 to 3 heteroatoms as ring atoms, the remaining ring atoms being carbon, including oxygen, sulfur and nitrogen . Examples of heteroaryl include, but are not limited to, pyridyl, pyridazinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, pyrimidinyl, imidazolyl, furyl, thienyl, pyrazinyl Wait.

As used herein, the term "ring" means a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl. The so-called ring includes fused rings. The number of atoms in a ring is usually defined as the number of ring members, eg "C ₃ -C ₆ ring" means 3-6 atoms arranged around.

As used herein, the term "heteroatom" refers to the form of O, S, N, including any oxidation state of N, S; the form of primary, secondary, tertiary amine and quaternary ammonium salts; or on the nitrogen atom in a heterocyclic ring. The hydrogen-substituted form.

As used herein, the terms "halogen", "halo" and the like mean fluorine (F), chlorine (Cl) or bromine (Br).

"Room temperature" in the present invention refers to a temperature from 10°C to 40°C. In some embodiments, "room temperature" refers to a temperature ranging from 20°C to 30°C; in other embodiments, room temperature refers to 25°C.

As used herein, the term "effective amount" refers to the amount of drug that can achieve the desired treatment of the disease or condition of the present invention in a subject.

As described herein, the term "pharmaceutically acceptable", for example, when describing a "pharmaceutically acceptable salt", means that the salt is not only physiologically acceptable to the subject, but also refers to a pharmaceutically useful synthetic compound. substance.

As used herein, the term "pharmaceutical composition", which may also refer to a "composition", is used to achieve the treatment of the diseases or conditions of the present invention in a subject, especially a mammal.

"Treatment" of a disease includes:

(1) prevention of the disease, that is, the absence of clinical symptoms of the disease in mammals exposed to or susceptible to the disease but not experiencing or showing symptoms of the disease,

(2) inhibit the disease, that is, arrest or reduce the progression of the disease or its clinical symptoms,

(3) Alleviating the disease, ie, causing reversion of the disease or its clinical symptoms.

A "therapeutically effective amount" refers to an amount of a compound which, when administered to a mammal for the purpose of treating a disease, is sufficient to effect treatment of that disease. A therapeutically effective amount will vary depending on the compound, the disease to be treated and its severity, and the age, weight, sex and other factors of the mammal. A therapeutically effective amount can also refer to any amount of the compound sufficient to achieve the desired beneficial effect, including preventing disease, inhibiting disease or alleviating disease as described in (1)-(3) above. For example, the amount of compound may be between 0.1-250 mg/kg, or preferably, 0.5-100 mg/kg, or more preferably, 1-50 mg/kg, or even more preferably, 2-20 mg/kg. Preferably, said amount of compound is administered to the mammal twice daily. More preferably, said amount of compound is administered to the mammal once a day.

As used herein, the term "disease and/or condition" refers to a physical state of the subject that is associated with the disease and/or condition of the present invention. For example, the diseases and/or conditions mentioned in the present invention refer to Mycobacterium tuberculosis infectious diseases.

As used herein, the term "subject" may refer to a patient or other animals, particularly mammals, such as humans, dogs, Monkeys, cows, horses, etc.

Another aspect of the present invention also relates to a pharmaceutical composition containing the compound of the present invention as an active ingredient. The pharmaceutical composition can be prepared according to methods known in the art. Any dosage form suitable for human or animal use can be prepared by combining the compounds of the present invention with one or more pharmaceutically acceptable solid or liquid excipients and/or adjuvants.

The compound in the present invention or the pharmaceutical composition containing it can be administered in the form of unit dosage, and the route of administration can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eye, Lungs and respiratory tract, skin, vagina, rectum, etc.

The dosage form for administration may be a liquid dosage form, a solid dosage form or a semi-solid dosage form. Liquid dosage forms can be solutions (including true solutions and colloid solutions), emulsions (including o/w type, w/o type and double emulsion), suspensions, injections (including aqueous injections, powder injections and infusion solutions), eye drops Agents, nasal drops, lotions and liniments, etc.; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules ( Including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, dripping pills, suppositories, films, patches, gas (powder) aerosols, sprays, etc.; semi-solid dosage forms can be ointments, Gels, pastes, etc.

The compound of the present invention can be made into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

To prepare the compound of the present invention into tablets, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, solubilizers. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, iso Propanol, etc.; binders can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, arabic mucilage, gelatin slurry, sodium carboxymethylcellulose, methylcellulose, hypromellose Base cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked poly Vinylpyrrolidone, croscarmellose sodium, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecylsulfonate, etc.; lubricants and co-solvents It can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol and the like.

Tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

In order to make the administration unit into a capsule, the compound of the present invention as an active ingredient can be mixed with a diluent and a solubilizer, and the mixture can be directly placed in a hard capsule or a soft capsule. The active ingredient compound of the present invention can also be made into granules or pellets with diluents, binders, and disintegrants, and then placed in hard capsules or soft capsules. Various diluents, binders, wetting agents, disintegrants, and solubilizers used in the preparation of tablets of the compound of the present invention can also be used to prepare capsules of the compound of the present invention.

In order to make the compound of the present invention into injection, water, ethanol, isopropanol, propylene glycol or their mixtures can be used as solvent and appropriate amount of solubilizer, cosolvent, pH regulator and osmotic pressure regulator commonly used in this field can be added. The solubilizer or co-solvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be Sodium chloride, mannitol, glucose, phosphate, acetate, etc. For preparation of freeze-dried powder injection, mannitol, glucose, etc. can also be added as proppants.

In addition, coloring agents, preservatives, fragrances, flavoring agents or other additives can also be added to the pharmaceutical preparations, if necessary.

In order to achieve the purpose of medication and enhance the therapeutic effect, the medicine or pharmaceutical composition of the present invention can be administered by any known administration method.

The compound or composition of the present invention can be taken alone, or used in combination with other therapeutic drugs or symptomatic drugs. When the compound of the present invention has a synergistic effect with other therapeutic drugs, its dose should be adjusted according to the actual situation.

Beneficial technical effect

The inventor of the present application synthesized a series of compounds through extensive research, and carried out the minimum inhibitory concentration MIC (Minimuminhibitory concentration) measurement with the M.tuberculosis H ₃₇ Rv bacterial strain by MABA (Microplatealamar blue assay) method, showed better The activity against Mycobacterium tuberculosis, of which 14 compounds obtained MIC<0.5 μg/mL, MIC of 13 compounds reached 10 ^-8 g/mL, comparable to the first-line anti-tuberculosis drug isoniazid, and low toxicity to Vero cells ( IC ₅₀ greater than 64μg/mL) showed good safety, and showed higher safety than benzothiazinone compound PBTZ169 (EMBO Mol.Med.2014,6,372-383) in HepG2 cytotoxicity test . Some compounds in the present invention have better metabolic properties than PBTZ169 in liver microsome and hepatocyte metabolic stability tests. The in vivo pharmacokinetics results in mice showed that compound 11 had better in vivo pharmacokinetic properties than PBTZ169. The present invention provides a class of new compounds with novel structure, strong anti-tuberculosis activity in vivo, and good pharmacokinetic properties. The parent nucleus structure is a benzopyrone or benzothiopyrone structure, which can be used for infections caused by bacteria Disease, especially tuberculosis caused by Mycobacterium tuberculosis, can also be used to overcome problems related to drug resistance.

Detailed ways

The present invention can be described in detail by the following examples, but it does not imply any adverse limitation on the present invention. The present invention has been described in detail herein, and its specific embodiments are also disclosed. For those skilled in the art, it is necessary to make various changes and improvements to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. Obvious.

For all of the following examples, standard manipulations and purification methods known to those skilled in the art can be used. All temperatures are in °C (degrees Celsius) unless otherwise indicated. The structures of the compounds were determined by nuclear magnetic resonance spectroscopy (NMR).

Preparation of the Example section

The structure of the compound was confirmed by hydrogen nuclear magnetic resonance spectroscopy ( ¹ H NMR). Proton and Carbon NMR shifts (δ) are given in parts per million (ppm). Coupling constants (J) are in Hertz (Hz). The nuclear magnetic resonance spectrum was measured with a Mercury-400 nuclear magnetic resonance instrument, deuterated chloroform (CDCl ₃ ) was used as a solvent, and tetramethylsilane (TMS) was used as an internal standard.

The electronic balance adopts the Japanese Yanaco LY-300 electronic balance.

Column chromatography generally uses 200-300 mesh silica gel as the carrier.

Anhydrous solvents were all worked up by standard methods. Other reagents were commercially available analytically pure.

The following abbreviations are used in the present invention:

CDI is carbonyldiimidazole.

DCC is dicyclohexylcarbodiimide.

DMF is N,N-dimethylformamide.

DMSO is dimethylsulfoxide.

EDCI is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

HOBT is 1-hydroxybenzotriazole.

HATU is 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

Example

Example 1

(S)-2-(2-Methyl-1,4-dioxa-8-azaspiro[4.5]decane-8-yl)-6-trifluoromethyl-8-nitro-benzo Pyran-4-one

route:

Experimental steps:

The first step (S)-1-(2-chloro-3-nitro-5-(trifluoromethyl)phenyl)-3-(2-methyl-1,4-dioxa-8-nitrogen Preparation of heterospiro[4.5]decane-8-yl)propane-1,3-dione B-1

In a 25mL reaction bottle, the compound 3-(2-chloro-3-nitro-5-(trifluoromethyl)phenyl)-3-oxopropionic acid A (311mg, 1.0mmol), DCC (201mg, 1.0mmol) was dissolved in dry dichloromethane (5mL), under Ar protection, (S)-2-methyl-1,4-dioxa-8-azaspiro[4.5]decane (157mg, 1.0 mmol), stirred at room temperature for 12 hours. The insoluble solids were filtered off, concentrated, and separated by silica gel (200-300 mesh) column chromatography, using ethyl acetate-petroleum ether (V:V=5-15:100) mixture as the eluent. Intermediate B-1 was obtained, 250 mg of light yellow solid, yield 55.5%.

¹ H NMR (400MHz, CDCl ₃ )δ: 15.64(brs,1H),8.01(s,1H),8.00(s,1H),5.72(s,1H),4.30-4.13(m,1H),4.12- 4.08(m,1H),3.75-3.47(m,5H),1.79-1.75(m,4H),1.30(m,3H).

The second step (S)-2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]decane-8-yl)-6-trifluoromethyl-8-nitro - Preparation of benzopyran-4-one II-1 (compound 1)

In a 10mL reaction bottle, compound (S)-1-(2-chloro-3-nitro-5-(trifluoromethyl)phenyl)-3-(2-methyl-1,4-dioxo Hetero-8-azaspiro[4.5]decane-8-yl)propane-1,3-dione B-1 (90 mg, 0.2 mmol), potassium carbonate (28 mg, 0.2 mmol) were dissolved in DMF (2 mL) , protected by Ar gas, the temperature was raised to 110° C. for 1 hour. Evaporate the solvent to dryness, add 25 mL of dichloromethane, wash with water and brine successively, dry over anhydrous sodium sulfate, filter, concentrate, silica gel (200-300 mesh) column chromatography, methanol-dichloromethane (V:V=1:100 ) mixture is the eluent. Compound II-1 (compound 1) was obtained, 58 mg of light yellow solid, yield 69.9%.

¹ H NMR (400MHz, CDCl ₃ )δ:8.74(s,1H),8.51(s,1H),5.66(s,1H),4.32-4.27(m,1H),4.14-4.10(m,1H), 3.77-3.71(m,4H),3.53-3.49(m,1H),1.88-1.85(m,4H),1.31(d,J=6.0Hz,3H).

Example 2

2-(4-(Cyclohexylmethyl)piperazin-1-yl)-6-trifluoromethyl-8-nitro-benzopyran-4-one

route:

Experimental steps:

The first step 1-(2-chloro-3-nitro-5-trifluoromethylphenyl)-3-(4-(cyclohexylmethyl)piperazin-1-yl)propane-1,3-di Preparation of Ketone B-2

Using 1-(cyclohexylmethyl)piperazine (182mg, 1.0mmol) as the raw material, the procedure similar to the first step in Example 1 was used to obtain intermediate B-2, 272mg of light yellow solid, with a yield of 57.1%.

¹ H NMR (400MHz, CDCl ₃ ) δ: 15.62 (brs, 1H), 8.01 (s, 1H), 8.00 (s, 1H), 5.67 (s, 1H), 3.73-3.49 (m, 4H), 2.45 ( m,4H),2.17(m,2H),1.79-1.71(m,5H),1.26-1.20(m,4H),0.90-0.87(m,2H).

The second step of 2-(4-(cyclohexylmethyl)piperazin-1-yl)-6-trifluoromethyl-8-nitro-benzopyran-4-one II-2 (compound 2) preparation

With 1-(2-chloro-3-nitro-5-trifluoromethylphenyl)-3-(4-(cyclohexylmethyl)piperazin-1-yl)propane-1,3-dione ( 95mg, 0.2mmol) as raw materials, using the similar operation steps in the second step in Example 1, to obtain compound II-2 (compound 2), light yellow solid 46mg, yield 52.3%.

¹ H NMR (400MHz, CDCl ₃ ) δ: 8.74(d, J=2.0Hz, 1H), 8.50(d, J=2.0Hz, 1H), 5.59(s, 1H), 3.65(m, 4H), 2.55 (m,4H),2.20(m,2H),1.80-1.62(m,5H),1.28-1.19(m,4H),0.91-0.86(m,2H).

Example 3

2-(4-(Cyclohexylmethyl)piperazin-2-on-1-yl)-6-trifluoromethyl-8-nitro-benzopyran-4-one

Using 1-(cyclohexylmethyl)piperazin-2-one (39mg, 0.2mmol) as raw material, the similar operation steps in Example 2 were used to obtain compound II-3 (compound 3), 42mg of light yellow solid, yield 46.4%.

¹ H NMR (400MHz, CDCl ₃ )δ: 8.76(d, J=2.0Hz, 1H), 8.55(d, J=2.0Hz, 1H), 5.54(s, 1H), 4.14(s, 2H), 3.98 -3.96(m,2H),3.58-3.56(m,2H),3.34(d,J＝7.2Hz,2H),1.77-1.66(m,5H),1.29-1.19(m,4H),1.02-0.99 (m,2H).

Example 4

2-(2-Methyl-1,4-dioxa-8-azaspiro[4.5]decane-8-yl)-6-(trifluoromethyl)-8-nitro-benzothio Pyran-4-one

route

Experimental steps:

Preparation of the first step 2-methylthio-8-nitro-6-(trifluoromethyl)-4H-thiochromen-4-one D

In a 50mL reaction flask, NaOH (800mg, 20mmol) was dissolved in DMSO (5mL), carbon disulfide (2.34g, 30mmol) was added at 20°C, and 2-chloro-3-nitro-5-trifluoromethylbenzene Ethanone (2.67 g, 10 mmol) was added therein in portions, and after 15 minutes, methyl iodide (10 mmol) was added at a temperature of 20° C. and reacted for 30 minutes. Add 100mL of ethyl acetate, wash with 100mL of water and brine successively, dry over anhydrous sodium sulfate, filter, concentrate, and separate by silica gel (200-300 mesh) column chromatography, ethyl acetate-petroleum ether (V:V=7:100) The mixed solution is the eluent. Intermediate D was obtained, 1.83 g of light yellow solid, yield 60.0%.

¹ H NMR (400MHz, CDCl ₃ ) δ: 9.12(d, J=2.0Hz, 1H), 8.84(d, J=2.0Hz, 1H), 6.91(s, 1H), 2.70(s, 3H).

The second step (2-methyl-1,4-dioxa-8-azaspiro[4.5]decane-8-yl)-6-(trifluoromethyl)-8-nitro-benzothio Preparation of pyran-4-one III-1 (compound 4)

In a 25mL reaction flask, 2-methylthio-8-nitro-6-(trifluoromethyl)-4H-thiochromen-4-one (64mg, 0.2mmol), (S)-2- Methyl-1,4-dioxa-8-azaspiro[4.5]decane (157mg, 1.0mmol) was dissolved in isopropanol (5mL) and reacted at 140°C for 24 hours under the protection of Ar to obtain compound III -1 (compound 4), 57 mg of light yellow solid, yield 66.3%.

¹ H NMR (300MHz, CDCl ₃ )δ:9.13(s,1H),8.77(s,1H),6.45(s,1H),4.28(m,1H),4.12(m,1H),3.82(m, 4H), 3.51(m, 1H), 1.88(m, 4H), 1.32(d, J=6Hz, 3H).

Example 5

2-(1,4-Dioxa-8-azaspiro[4.5]decane-8-yl)-6-(trifluoromethyl)-8-nitro-benzothiopyran-4- ketone

Using 1,4-dioxa-8-azaspiro[4.5]decane (143mg, 1.0mmol) as a raw material, the second step in Example 4 was used to obtain compound III-2 (compound 5), Light yellow solid 60mg, yield 72.2%.

¹ H NMR (400MHz, CDCl ₃ )δ:9.13(s,1H),8.76(s,1H),6.41(s,1H),4.03(s,4H),3.81(m,4H),1.88(m, 4H).

Example 6

2-(1,4-Dithia-8-azaspiro[4.5]decane-8-yl)-6-(trifluoromethyl)-8-nitro-benzothiopyran-4- ketone

Using 1,4-dithia-8-azaspiro[4.5]decane (175mg, 1.0mmol) as a raw material, the second step in Example 4 was similarly operated to obtain compound III-3 (compound 6), Light yellow solid 70mg, yield 77.8%.

¹ H NMR (400MHz, CDCl ₃ )δ: 9.12(d, J=1.6Hz, 1H), 8.78(d, J=1.6Hz, 1H), 6.54(s, 1H), 3.83(m, 4H), 3.39 (s,4H),2.28(m,4H).

Example 7

2-(1-Oxa-4-thia-8-azaspiro[4.5]decane-8-yl)-6-(trifluoromethyl)-8-nitro-benzothiopyran- 4-keto

Using 1-oxa-4-thia-8-azaspiro[4.5]decane (159mg, 1.0mmol) as raw material, the second step in Example 4 was used to obtain compound III-4 (compound 7 ), light yellow solid 52mg, yield 60.5%.

¹ H NMR (400MHz, CDCl ₃ )δ:9.13(s,1H),8.80(s,1H),6.78(s,1H),4.23(m,2H),3.86(m,4H),3.16(m, 2H),2.17(m,4H).

Example 8

2-(4-(cyclohexylmethyl)-3-oxo-piperazin-1-yl)-6-(trifluoromethyl)-8-nitro-benzothiopyran-4-one

Using 1-(cyclohexylmethyl)-piperazin-2-one (196mg, 1.0mmol) as a raw material, the second step in Example 4 was followed to obtain Compound III-5 (Compound 8), a pale yellow solid 61 mg, yield 64.8%.

¹ H NMR (400MHz, CDCl ₃ )δ:9.16(s,1H),8.80(s,1H),6.19(s,1H),4.24(m,2H),3.90(m,2H),3.59(m, 2H),3.36(m,2H),1.73-1.66(m,6H),1.26-1.21(m,3H),1.02-1.00(m,2H).

Example 9

2-(4,4-Difluoropiperidin-1-yl)-6-(trifluoromethyl)-8-nitro-benzothiopyran-4-one

Using 4,4-difluoropiperidine (121mg, 1.0mmol) as a raw material, the second step in Example 4 was followed to obtain Compound III-6 (Compound 9), 63mg of a light yellow solid, with a yield of 79.7%.

¹ H NMR (400MHz, CDCl ₃ ) δ: 9.13(s,1H), 8.80(s,1H), 6.41(s,1H), 3.83(m,4H), 2.20(m,4H).

Example 10

2-(4-(thiazol-2-yl)piperazin-1-yl)-6-(trifluoromethyl)-8-nitro-benzothiopyran-4-one

Using (piperazin-1-yl)thiazole (169mg, 1.0mmol) as a raw material, the second step in Example 4 was used to obtain compound III-7 (compound 10), 43mg of light yellow solid, yield 48.9% .

¹ H NMR (400MHz, CDCl ₃ ) δ: 9.14(s, 1H), 8.80(s, 1H), 7.28(d, J=3.6Hz, 1H), 6.70(d, J=3.6Hz, 1H), 6.31 (s,1H),3.84(m,8H).

Example 11

2-(4-(Cyclohexylmethyl)piperazin-1-yl)-6-(trifluoromethyl)-8-nitro-benzothiopyran-4-one

Using 1-(cyclohexylmethyl)piperazine (182mg, 1.0mmol) as a raw material, the second step in Example 4 was used to obtain Compound III-8 (Compound 11), 66mg of orange solid, yield 72.5% .

¹ H NMR (400MHz, CDCl ₃ )δ:9.12(s,1H),8.75(s,1H),6.25(s,1H),3.66(m,4H),2.56(m,4H),2.20(m, 2H),1.80-1.67(m,5H),1.51(m,1H),1.26-1.16(m,3H),0.94-0.88(m,2H).

Example 12

2-(4-(cyclohexylmethyl)-3-methyl-piperazin-1-yl)-6-(trifluoromethyl)-8-nitro-benzothiopyran-4-one

Using 1-(cyclohexylmethyl)-2-methylpiperazine (196mg, 1.0mmol) as a raw material, the second step in Example 4 was followed to obtain Compound III-9 (Compound 12), a pale yellow solid 67 mg, yield 71.3%.

¹ H NMR (400MHz, CDCl ₃ ) δ: 9.12(s,1H), 8.74(s,1H), 6.24(s,1H), 3.80-3.77(m,2H), 3.47(m,1H), 3.19( m,1H),2.95(m,1H),2.58(m,1H),2.50(m,1H),2.36(m,1H),2.01(m,1H),1.87(m,1H),1.71(m ,4H),1.46(m,1H),1.25-1.11(m,6H),0.91-0.88(m,2H).

Example 13

2-(4-(Benzyl)piperazin-1-yl)-6-(trifluoromethyl)-8-nitro-benzothiopyran-4-one

Using N-benzylpiperazine (176 mg, 1.0 mmol) as the raw material, the second step in Example 4 was followed to obtain compound III-10 (compound 13), 55 mg of light yellow solid, and the yield was 61.1%.

¹ H NMR (400MHz, CDCl ₃ )δ:9.12(s,1H),8.75(s,1H),7.35(m,5H),6.24(s,1H),3.68(m,4H),3.60(s, 2H),2.63(m,4H).

Example 14

2-(4-Benzyl-3-methyl-piperazin-1-yl)-6-(trifluoromethyl)-8-nitro-benzothiopyran-4-one

Using 1-benzyl-2-methylpiperazine (190mg, 1.0mmol) as a raw material, the second step in Example 4 was used to obtain compound III-11 (compound 21), a light yellow solid 47mg, yield 50.7%.

¹ H NMR (400MHz, CDCl ₃ )δ: 9.12(s, 1H), 8.75(s, 1H), 7.35-7.26(m, 5H), 6.23(s, 1H), 4.06(d, J=13.2Hz, 1H), 3.84(t, J=15.2Hz, 2H), 3.40(t, J=9.6Hz, 1H), 3.27-3.20(m, 2H), 2.85(d, J=12.4Hz, 1H), 2.71( brs,1H),2.32(t,J=9.2Hz,1H),1.26(d,J=6.0Hz,3H).

Example 15

2-(4-(Cyclohexylmethyl)piperazin-1-yl)-6-(trifluoromethyl)-8-nitro-benzothiopyran-4-one hydrochloride

2-(4-(Cyclohexylmethyl)piperazin-1-yl)-6-(trifluoromethyl)-8-nitro-benzothiopyran-4-one (46mg, 0.1mmol) Place in 5mL of ethanol, heat until dissolved, add 0.1mL of 1N hydrochloric acid ethanol solution dropwise under stirring, stir for 2 hours after the dropwise addition, cool and filter, wash with a small amount of cold ethanol, and dry to obtain compound III-12 (compound 22), yellow Solid 47mg, yield 95.6%.

¹ H NMR (400MHz,DMSO-d ₆ )δ:10.48(brs,1H),8.87(s,1H),8.86(s,1H),6.44(s,1H),4.31-4.28(m,2H), 3.77-3.70(m,2H),3.61-3.58(m,2H),3.19-3.17(m,2H),2.99(brs,2H),1.83-1.80(m,3H),1.70-1.67(m,2H ),1.64-1.60(m,1H),1.26-1.12(m,3H),0.98-0.95(m,2H).

Example 16

2-(4-(Benzyl)piperazin-1-yl)-6-(trifluoromethyl)-8-nitro-benzothiopyran-4-one hydrochloride

2-(4-(Benzyl)piperazin-1-yl)-6-(trifluoromethyl)-8-nitro-benzothiopyran-4-one (45 mg, 0.1 mmol) was placed in In 5mL of ethanol, heat until dissolved, add dropwise 0.1mL of 1N hydrochloric acid ethanol solution under stirring, stir for 2 hours after the dropwise addition, cool and filter, wash with a small amount of cold ethanol, and dry to obtain compound III-13 (compound 26), yellow solid 46mg , yield 94.7%.

¹ H NMR (400MHz, DMSO-d ₆ ) δ: 11.3 (brs, 1H), 8.88 (s, 1H), 8.85 (s, 1H), 7.58 (brs, 2H), 7.47 (brs, 3H), 6.42 ( s,1H),4.37-4.33(m,4H),3.66-3.60(m,2H),3.41-3.39(m,2H),3.23-3.21(m,2H).

Biological activity test

1. In vitro anti-tuberculosis activity test

Determination method: Microplate Alamar Blue Assay (MABA) method was used to determine the anti-tuberculosis activity in vitro.

Experimental principle: Adding Alamar Blue to the medium can be used as a redox indicator, and the color changes from blue to red, reflecting the consumption of oxygen molecules by the microorganisms studied. The color change of Alamar Blue can be measured photometrically with an emission wavelength of 590nm.

Experimental method: sterile 96-well plate (Falcon3072; Becton Dickinson, Lincoln Park, NJ), the test compound was dissolved in DMSO to make an initial solution with a concentration of 5 mg/mL, 199 μL 7H9 medium was added to the highest concentration well, and 1 μL compound initial solution After mixing evenly, dilute to the remaining wells successively by 2 times, and the final compound concentrations are: 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.2, 0.1, 0.05, 0.025 μg/mL. Select Mycobacterium tuberculosis H ₃₇ R _v (or clinically isolated drug-resistant strains) cultured for 2 to 3 weeks to make a bacterial suspension, inoculate into 7H9 medium containing 0.05% Tween 80, 10% ADC, 37 Cultivate statically at ℃ for 1-2 weeks, grow to McFarland 1 (equivalent to 10 ⁷ CFU/mL), dilute 1:20, add 100 μL to each well, and the final concentration of the bacterial solution is 10 ⁶ CFU/mL. Two growth control wells without antibiotics were set on each plate, and the 96-well plate was incubated at 37°C. After 7 days, add 20 μL of a mixture of 10×Alamar Blue and 5% Tween80 to growth control wells, and incubate at 37°C for 24 hours. If the color changes from blue to pink, add the above-mentioned amount of Alamar Blue and The Tween 80 mixture was incubated at 37°C for 24 hours to record the color of each well, and the 590nm fluorescence value was measured with a microplate reader to calculate the MIC ₉₀ .

Table 1. In vitro anti-tuberculosis _{H37Rv activity} of some compounds of the present invention

Table 2, the anti-drug-resistant Mycobacterium tuberculosis activity of some compounds of the present invention in vitro

^a Resistance to isoniazid, rifampicin, streptomycin, ethambutol, rifapentine, rifabutin, and benzamidosalicylic acid.

^b Resistance to isoniazid, ethambutol, rifampicin, rifapentine, rifabutin, amikacin, and capreomycin.

From the data in Table 1 and Table 2, it can be seen that the compounds of the present invention have strong in vitro activity against tuberculosis-sensitive bacteria and drug-resistant bacteria.

2. Cytotoxicity test

Determination method: MTT method

Experimental principle: cell activity converts the oxidized 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium through mitochondrial dehydrogenase (such as succinate dehydrogenase) Bromide (trade name: thiazolium blue)/MTT[3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide] reduced to insoluble blue formazan compound, dissolved in DMSO The amount of transformation was positively correlated with the number of viable cells, as determined by post-chromogenesis.

Experimental method: 1. Preparation of cell suspension. Digest the Vero/HepG2 cells that have been cultured to the logarithmic growth phase with 0.25% trypsin for 2 to 3 minutes, discard the digestion solution, add an appropriate amount of culture solution, mix well, take 20 μL and count it under a microscope with a hemocytometer, and prepare a suitable Concentration of cell suspension, set aside. At the same time, prepare 5g/L MTT solution with PBS (phosphate buffered solution), filter and sterilize, and set aside. 2. Drug preparation and cytotoxicity testing. The test drug was dissolved in DMSO, diluted 50 times with the culture medium to make the highest concentration tested, and then serially diluted 1:3 with the culture medium on a 96-well plate, with 6 concentrations for each compound, the highest The concentration was 64 μg/mL, and 6 parallel wells were set up for each concentration, 50 μL/well. The prepared cell suspension was inoculated into a 96-well plate, 50 μL/well, and the cell concentration was 4×10 ⁵ cells/mL. At the same time, a cell control well without drugs and a medium blank control well were set up. After culturing for 48 hours, 10 μL/well of MTT was added to continue culturing for 4 hours. Take out the culture plate, carefully discard the medium in the wells, add 100 μL of DMSO to each well, shake until the formazan particles are completely dissolved, and measure the optical density (OD ₅₇₀ ) at a wavelength of 570 nm with an enzyme-linked immunosorbent detector. 3. Data processing. Cell inhibition percentage (%)=[(cell control OD ₅₇₀ value-medication group OD ₅₇₀ value)/(cell control OD ₅₇₀ value-blank OD ₅₇₀ value)]×100%. Dose-response curve fitting was carried out with Origin7.0 software, and the concentration (IC ₅₀ ) of various compounds at 50% inhibition rate of cells was calculated.

Table 3, Vero cytotoxicity of some compounds of the present invention

Table 4, HepG2 cytotoxicity of some compounds of the present invention

From the data in Table 3 and Table 4, it can be known that the compound of the present invention has low cytotoxicity and shows higher safety than the positive drug PBTZ169.

3. Metabolic stability test of liver microsomes

experimental method:

Target compounds were selected for metabolic stability study of liver microsomes (mixed human source (Bioreclamation)), and the specific method was as follows: the synthesized target compounds were respectively prepared into 1 μM test solution. The microsomal protein concentration was 1 mg/mL. Reactions were initiated by the addition of NADPH (1 mM) and samples were incubated in a shaking incubator at 37°C for up to 60 minutes. Reactions were stopped at 0, 5, 15 and 30 minutes by the addition of ice-cold acetonitrile/methanol (50:50) with internal standard. Aliquots of the reaction mixture were withdrawn at 0, 5, 15, 30, and 60 minutes, and ice-cold acetonitrile/methanol (50:50, v/v) containing the internal standard was added. Samples were centrifuged at 4°C for 15 minutes (4,000 rpm) and the supernatant analyzed by LC-MS/MS. Chromatographic conditions: chromatographic column: Kinetex C18 100A (30mm×3.0mm, 2.6μm); column temperature: room temperature, mobile phase: acetonitrile-water (containing 0.1% formic acid) gradient; flow rate: 0.9mL/min. Metabolic stability of compounds was assessed by measuring the residual amount of compounds with and without the NADPH cofactor.

Table 5, human liver microsome metabolic stability data of some compounds of the present invention

From the data in Table 5, it can be seen that the compounds of the present invention have higher metabolic stability of liver microsomes.

4. Hepatocyte metabolic stability test

experimental method:

Mixed CD-1 mice (Bioreclamation IVT), mixed SD rats (Bioreclamation IVT), mixed male dogs (Bioreclamation IVT), mixed male cynomolgus monkeys (RILD) and mixed humans (Bioreclamation IVT) were used hepatocytes were measured. Compounds (compound 11 and PBTZ169) were tested at a concentration of 1 μΜ with a final hepatocyte concentration of 1 million cells/mL. Reactions were initiated by adding pre-warmed hepatocyte solution (2 million cells/mL) to compound solution (2 μM). Incubate the reaction mixture at 37 °C for 120 min in a _CO incubator at 100 rpm. At predetermined time points (0, 15, 30, 60, 90 and 120 min), 30 μL of the reaction mixture was withdrawn and the reaction was terminated by adding 200 μL of ice-cold ACN/MeOH (50:50) with internal standard. The samples were mixed well, centrifuged at 4°C for 15 minutes (4,000 rpm), and the supernatant analyzed by LC-MS/MS. Chromatographic conditions: chromatographic column: Kinetex C18100A (30mm×3.0mm, 2.6μm); column temperature: room temperature, mobile phase: acetonitrile-water (containing 0.1% formic acid) gradient; flow rate: 0.9mL/min. Metabolic stability of compounds in hepatocytes was assessed by determining the amount of compound remaining.

Table 6, Hepatocyte Metabolic Stability of Compound 11 and PBTZ169

It can be seen from the data in Table 6 that the compound 11 of the present invention has a longer half-life in five species of hepatocytes than the positive control drug PBTZ169, and its metabolic stability is significantly better than that of PBTZ169.

5. In vivo pharmacokinetic test of compound 11, compound 22 and PBTZ169 in mice

experimental method:

Each group used three Balb/c mice (male) weighing 22-23 g to conduct pharmacokinetic studies of compound 11, compound 22 and PBTZ169. Compound 11 was prepared as 2.5 mg/mL suspension with 0.5% carboxymethyl cellulose and 0.5% Tween 80, compound 22 was prepared as 2.5 mg/mL suspension with 0.5% carboxymethyl cellulose, and PBTZ169 was prepared as 0.5% carboxymethyl cellulose Methylcellulose and 4 molar equivalents of 1N hydrochloric acid were prepared as a 2.5 mg/mL suspension, and all compounds were administered at a dose of 25 mg/kg. Plasma samples were collected at 5, 15, 30 minutes, and 1, 2, 4, 7, 24 hours after oral administration. Collected plasma samples were stored at -80°C until analysis. Plasma samples were extracted with acetonitrile containing terfenadine internal standard at a ratio of extractant to plasma of 20:1. Analyte quantification was performed by LC/TSQQuantum Access mass spectrometer (AB Sciex 5500). Chromatographic conditions: Chromatographic column: Kinetex C18 100A (30mm×3.0mm, 2.6μm); column temperature: room temperature, mobile phase: acetonitrile/water (80:20, v/v) (containing 0.1% formic acid); flow rate: 0.8mL /min. Compound detection on the mass spectrometer was performed in electrospray positive ionization mode. MRM method detected m/z 456.17/359.80 (compound 11), 456.17/359.80 (compound 22), m/z 457.16/344.20 (PBTZ169). Pharmacokinetic parameters were calculated using WinNonlin software (6.3Pharsight Corporation, Mountain View, USA).

Table 7 Plasma pharmacokinetic parameters after oral administration of compound 11, compound 22 and PBTZ169 in mice

It can be seen from Table 7 that Compound 11 and Compound 22 of the present invention have a longer half-life in mice than PBTZ169, and their pharmacokinetic properties are better than PBTZ169, which indicates that the number of administrations can be reduced in clinical use, thereby improving the efficacy of medication for patients. compliance.

6. Stability investigation of compound 11, compound 22 and PBTZ169

The stability of compound 11, compound 22 and PBTZ169 placed under light, high temperature and high humidity conditions for 10 days was investigated by HPLC, and the results are shown in Table 8.

Table 8 Compound 11, Compound 22 and PBTZ169 Stability Investigation Results

The purity of the compound was detected by Waters e2695-PDA HPLC system. Chromatographic conditions: chromatographic column: KromasilC18 (250mm×4.6mm, 5μm); column temperature: 30°C, mobile phase: acetonitrile/water (84:16, v/v) isogradient; flow rate: 1.0mL/min. It can be seen from Table 8 that the stability of compound 11 and compound 22 of the present invention under light conditions is significantly better than that of PBTZ169. Compared with PBTZ169, compound 11 and compound 22 have better physicochemical properties.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention, those skilled in the art can make the above-mentioned The embodiments are subject to changes, modifications, substitutions and variations.

Claims (13)

1. A compound of formula (I) and isomers, or pharmaceutically acceptable salts thereof:

wherein,

x is O or S;

y is C or N;

R₁is H, C₁-C₃Alkyl radical, C₁-C₃Alkoxy, F, Cl, Br, CNOH, (═ O), or (═ S);

n is 0,1 or 2;

R₂is F, substituted or unsubstituted C₃-C₆Cycloalkyl, substituted or unsubstituted C₃-C₆Heterocyclic radical, substituted or unsubstituted C₆-C₁₀Aryl, substituted or unsubstituted C₂-C₉Heteroaryl, or together with the carbon atoms to which they are both attached, represents a substituted or unsubstituted cycloalkyl group containing 3 to 8 carbon atoms or a substituted or unsubstituted 4-to 6-membered heterocyclic group containing 1 to 3 heteroatoms selected from oxygen, sulfur;

said C₃-C₆Heterocyclic group, C₂-C₉Heteroaryl contains at least one heteroatom selected from N, O, S;

the R is₂Wherein the substituted or unsubstituted substituents may optionally be selected from the group consisting of: F. cl, Br, hydroxyl, amino, nitro, cyano, trifluoromethyl, trifluoromethoxy and C₁-C₃Alkyl, halo C₁-C₃Alkyl radical, C₁-C₃Alkoxy or C₁-C₃An alkylamino group.

2. The compound according to claim 1, which is represented by the general formula (II);

wherein,

y is C or N;

R₁is H, C₁-C₃Alkyl radical, C₁-C₃Alkoxy, F, Cl, Br, CN, OH, (═ O), or (═ S);

n is 0,1 or 2;

R₂is F, substituted or unsubstituted C₃-C₆Cycloalkyl, substituted or unsubstituted C₃-C₆Heterocyclic radical, substituted or unsubstituted C₆-C₁₀Aryl, substituted or unsubstituted C₂-C₉Heteroaryl, or together with the carbon atoms to which they are both attached, represents a substituted or unsubstituted cycloalkyl group containing 3 to 8 carbon atoms or a substituted or unsubstituted 4-to 6-membered heterocyclic group containing 1 to 3 heteroatoms selected from oxygen, sulfur;

said C₃-C₆Heterocyclic group, C₂-C₉Heteroaryl contains at least one heteroatom selected from N, O, S;

the R is₂Wherein the substituted or unsubstituted substituents may optionally be selected from the group consisting of: F. cl, Br, hydroxyl, amino, nitro, cyano, trifluoromethyl, trifluoromethoxy and C₁-C₃Alkyl, halo C₁-C₃Alkyl radical, C₁-C₃Alkoxy or C₁-C₃An alkylamino group.

3. The compound according to claim 1, which is represented by the general formula (III);

wherein,

y is C or N;

R₁is H, C₁-C₃Alkyl radical, C₁-C₃Alkoxy, F, Cl, Br, CN, OH, (═ O), or (═ S);

n is 0,1 or 2;

R₂is F, substituted or unsubstituted C₃-C₆Cycloalkyl, substituted or unsubstituted C₃-C₆Heterocyclic radical, substituted or unsubstituted C₆-C₁₀Aryl, substituted or unsubstituted C₂-C₉Heteroaryl, or together with the carbon atoms to which they are both attached, represents a substituted or unsubstituted cycloalkyl group containing 3 to 8 carbon atoms or a substituted or unsubstituted 4-to 6-membered heterocyclic group containing 1 to 3 heteroatoms selected from oxygen, sulfur;

said C₃-C₆Heterocyclic group, C₂-C₉Heteroaryl contains at least one heteroatom selected from N, O, S;

the R is₂Wherein the substituted or unsubstituted substituents may optionally be selected from the group consisting of: F. cl, Br, hydroxyl, amino, nitro, cyano, trifluoromethyl, trifluoromethoxy and C₁-C₃Alkyl, halo C₁-C₃Alkyl radical, C₁-C₃Alkoxy or C₁-C₃An alkylamino group.

4. The compound according to claim 1 or 2, which is represented by the general formula (II-a):

wherein,

R₁is H, C₁-C₃Alkyl, F, Cl or (═ O);

n is 0 or 1;

R₂is F, a substituted or unsubstituted cyclohexyl group, a substituted or unsubstituted phenyl group, a substituted or unsubstituted naphthyl group, a substituted or unsubstituted pyridyl group, a substituted or unsubstituted quinolyl group, a substituted or unsubstituted pyrrolyl group, a substituted or unsubstituted furyl group, a substituted or unsubstituted thienyl group, a substituted or unsubstituted thiazolyl group, or together with the carbon atom to which they are commonly attached represents a substituted or unsubstituted cyclopentyl group or a substituted or unsubstituted 5-membered heterocyclic group containing 1 to 2 heteroatoms selected from oxygen, sulfur;

the R is₂Wherein the substituted or unsubstituted substituents may optionally be selected from the group consisting of: F. cl, Br, hydroxyl, amino, nitro, cyano, trifluoromethyl, trifluoromethoxy and C₁-C₃Alkyl, halo C₁-C₃Alkyl radical, C₁-C₃Alkoxy or C₁-C₃An alkylamino group.

5. The compound according to claim 1 or 2, which is represented by the general formula (II-b):

wherein,

R₁is H, C₁-C₃Alkyl, F, Cl or (═ O);

n is 0 or 1;

R₂is substituted or unsubstituted cyclohexyl, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted pyridyl, substituted or unsubstituted quinolyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted furyl, substituted or unsubstituted thienyl, or substituted or unsubstituted thiazolyl;

the R is₂Wherein the substituted or unsubstituted substituents may optionally be selected from the group consisting of: F. cl, Br, hydroxyl, amino, nitro, cyano, trifluoromethyl, trifluoromethoxy and C₁-C₃Alkyl, halo C₁-C₃Alkyl radical, C₁-C₃Alkoxy or C₁-C₃An alkylamino group.

6. The compound according to claim 1 or 3, which is represented by the general formula (III-a):

wherein,

R₁is H, C₁-C₃Alkyl, F, Cl or (═ O);

n is 0 or 1;

R₂is F, substituted or unsubstituted cyclohexyl, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted pyridyl, substituted or unsubstituted quinolyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted furyl, substituted or unsubstituted thienyl, substituted or unsubstituted thiazolyl, or one to the carbon atoms to which they are both attachedThe substituent represents a substituted or unsubstituted cyclopentyl group or a substituted or unsubstituted 5-membered heterocyclic group containing 1 to 2 heteroatoms selected from oxygen and sulfur;

the R is₂Wherein the substituted or unsubstituted substituents may optionally be selected from the group consisting of: F. cl, Br, hydroxyl, amino, nitro, cyano, trifluoromethyl, trifluoromethoxy and C₁-C₃Alkyl, halo C₁-C₃Alkyl radical, C₁-C₃Alkoxy or C₁-C₃An alkylamino group.

7. The compound according to claim 1 or 3, which is represented by the general formula (III-b):

wherein,

R₁is H, C₁-C₃Alkyl, F, Cl or (═ O);

n is 0 or 1;

R₂is substituted or unsubstituted cyclohexyl, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted pyridyl, substituted or unsubstituted quinolyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted furyl, substituted or unsubstituted thienyl, substituted or unsubstituted thiazolyl;

the R is₂Wherein the substituted or unsubstituted substituents may optionally be selected from the group consisting of: F. cl, Br, hydroxyl, amino, nitro, cyano, trifluoromethyl, trifluoromethoxy and C₁-C₃Alkyl, halo C₁-C₃Alkyl radical, C₁-C₃Alkoxy or C₁-C₃An alkylamino group.

8. The compound according to claim 2 and isomers thereof, or pharmaceutically acceptable salts thereof,

wherein,

R₁is H, C₁-C₃Alkyl radical, C₁-C₃Alkoxy, F, Cl, Br, CN, OH, (═ O), or (═ S);

n is 0,1 or 2;

when the Y is C, the compound has the structure of,

R₂is F,

When the Y is an amino group represented by the formula (I),

R₂is composed of

Rx is F, Cl, Br, hydroxyl, amino, nitro, cyano, trifluoromethyl, trifluoromethoxy or C₁-C₃Alkyl, halo C₁-C₃Alkyl radical, C₁-C₃Alkoxy or C₁-C₃An alkylamino group.

9. A compound according to claim 3 and isomers thereof, or pharmaceutically acceptable salts thereof,

wherein,

R₁is H, C₁-C₃Alkyl radical, C₁-C₃Alkoxy, F, Cl, Br, CN, OH, (═ O), or (═ S);

n is 0,1 or 2;

when the Y is C, the compound has the structure of,

R₂is F,

When the Y is an amino group represented by the formula (I),

R₂is composed of

Rx is F, Cl, Br, hydroxyl, amino, nitro, cyano, trifluoromethyl, trifluoromethoxy or C₁-C₃Alkyl, halo C₁-C₃Alkyl radical, C₁-C₃Alkoxy or C₁-C₃An alkylamino group.

10. A compound according to any one of claims 1 to 9, and isomers thereof, or pharmaceutically acceptable salts thereof, selected from the following compounds:

11. a process for the preparation of a compound according to any one of claims 1 to 10, comprising the steps of:

(1)

condensing the compound A and a nitrogen heterocyclic side chain B to obtain a compound shown in a formula C, and then closing a ring under an alkaline condition to obtain a compound shown in a formula (II);

or (2)

The compound D reacts with carbon disulfide under alkaline conditions, then is methylated to obtain a compound E, and then is subjected to substitution reaction with a nitrogen heterocyclic side chain B to obtain a compound shown in a formula (III), the compound shown in the formula (III) can be treated by corresponding acid in a known proper solvent and is converted into pharmaceutically acceptable salt, wherein the acceptable salt is selected from hydrochloride, p-toluenesulfonate, tartrate, maleate, lactate, methanesulfonate, sulfate, phosphate, citrate, acetate or trifluoroacetate,

wherein, Y, R₁、R₂N is as defined in any one of claims 1 to 9.

12. A pharmaceutical composition comprising a therapeutically and/or prophylactically effective amount of a compound of any one of claims 1 to 10, and isomers thereof, or pharmaceutically acceptable salts thereof, and optionally one or more pharmaceutically acceptable carriers, excipients, diluents, adjuvants and vehicles.

13. Use of a compound according to any one of claims 1 to 10, an isomer thereof or a pharmaceutically acceptable salt thereof, or a composition according to claim 12 for the manufacture of a medicament for the treatment and/or prevention of infectious diseases caused by mycobacterium tuberculosis.