CN113527331A

CN113527331A - Nitroimidazole derivative and preparation method and application thereof

Info

Publication number: CN113527331A
Application number: CN202010307399.3A
Authority: CN
Inventors: 赵传生; 向军; 刘广才; 董吉; 宋海峰
Original assignee: Nanjing Changao Pharmaceutical Science and Technology Co Ltd
Current assignee: Nanjing Changao Pharmaceutical Science and Technology Co Ltd
Priority date: 2020-04-17
Filing date: 2020-04-17
Publication date: 2021-10-22
Anticipated expiration: 2040-04-17
Also published as: WO2021208850A1; CN113527331B

Abstract

The invention discloses a nitroimidazole derivative, a preparation method and application thereof. The compounds have the following general formula (I):

Description

Nitroimidazole derivative and preparation method and application thereof

Technical Field

The invention belongs to the fields of pharmacology, medicinal chemistry and pharmacology, and particularly relates to a novel nitroimidazole compound, a preparation method thereof and application of the compound in treating diseases related to infection caused by mycobacterium tuberculosis.

Background

Tuberculosis is caused by infection of Mycobacterium tuberculosis (Mycobacterium tuberculosis) and is one of the oldest diseases of human beings, and as of today, tuberculosis still seriously harms human health. According to WHO statistics, about 1/3 people in the world are infected with mycobacterium tuberculosis, which is the infectious disease (higher than HIV) causing the most deaths.

According to a '2019 global tuberculosis report' issued by the World Health Organization (WHO), about 1000 thousands of new tuberculosis patients are discovered globally in 2018, and the number of the patients is relatively stable in recent years. The number of HIV-negative tuberculosis deaths was estimated to be about 120 million globally in 2018, and in addition, the number of HIV-positive tuberculosis deaths was about 25.1 million. Meanwhile, about 48.4 million people are estimated to be newly-discovered rifampicin-resistant tuberculosis (RR-TB) globally in 2018, and 78% of the people are multi-drug-resistant tuberculosis (MDR-TB). Of the MDR-TB patients, 6.2% were estimated to be broadly drug resistant tuberculosis (XDR-TB). There are three countries that account for half of the worldwide cases of MDR/RR-TB: india (27%), china (14%) and russia (9%).

At present, a first-line treatment for sensitive tuberculosis adopts a combined treatment strategy of rifampicin, isoniazid, ethambutol and pyrazinamide, although the treatment success rate can reach more than 85%, the treatment period is as long as 6 months, and the treatment side effect is large, for example, the combined treatment of rifampicin and isoniazid may cause serious hepatotoxicity, ethambutol may cause optic nerve damage and the like. Some people fail to receive regular treatment, and some people develop drug-resistant tuberculosis (rifampin-resistant or multidrug-resistant) due to incomplete or improper treatment. For drug-resistant tuberculosis, the treatment period is longer, the treatment side effect is larger, and the treatment success rate is only about 55 percent. Drug-resistant tuberculosis, especially multi-drug resistant tuberculosis and widely drug resistant tuberculosis, is a leading cause of death in tuberculosis patients, especially patients with immunodeficiency people, such as AIDS and tuberculosis double-infection patients.

WO9701562 discloses a plurality of nitroimidazole compounds, which represent a compound PA-824(pretomanid), have a brand new action mechanism and can be used for treating tuberculosis. In 8 months 2019, the FDA announced the approval of pretomanids developed by the non-profit organization global tuberculosis drug development consortium (TB Alliance) to be marketed for the treatment of specific highly resistant Tuberculosis (TB) patients in combination with bedaquiline and linezolid. However, PA-824 has low water solubility and bioavailability, requires a complex tablet formulation for oral administration, and further improves its antitubercular activity [ bioorg.med.chem.lett,2008,18(7), 2256-.

OPC-67683 from tsukamur Pharmaceutical co Ltd (Otsuka Pharmaceutical co., Ltd) [ j.med.chem.,2006,49(26),7854-7860 ], the mechanism of action of which is similar to that of PA-824, is used for the treatment of tuberculosis. The compound was marketed approved by the european union committee in 5 months 2014 for the treatment of adult patients with multi-drug resistant pulmonary tuberculosis (MDR-TB). Although highly active, the compound has the same problems as PA-824, the compound has low solubility in water and poor plasma stability, and limits its pharmacokinetic properties, and it needs to be taken 2 times a day. Meanwhile, PA-824 and OPC-67683 have strong hERG potassium current inhibition activity, and have serious cardiotoxicity problem due to the side effect of prolonging QT-QTc interval clinically. Therefore, the target medicine has further optimization and perfection space. We have conducted systematic studies on the target drug and achieved good results (patent publication No. CN105732659A), but when the representative compound was subjected to toxicological experiments, PK-PD studies showed that the representative compound had a "capping" phenomenon, which was difficult to further study, and the drug effect in mice did not exceed PA-824.

PA-824 and OPC-67683 structural formulas

In view of the above, there is still a great need in the art to develop new antitubercular drugs. This new drug should have the following characteristics: effective against drug-resistant bacteria, particularly multidrug-resistant bacteria; can be used in combination with the first-line antituberculosis drugs currently used; has ideal metabolic property, can be orally taken and is taken once a day; the safety is superior to the existing medicines.

Disclosure of Invention

The invention aims to provide a novel antituberculosis drug, which is effective on drug-resistant bacteria, in particular multi-drug resistant bacteria; has ideal metabolic property, can be orally taken and is taken once a day; the safety is superior to the existing medicines.

In a first aspect of the present invention, there is provided an anti-tuberculosis compound, which is a compound represented by formula (I) or an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

in formula (I): m, n represent an integer between 0 and 4;

x is oxygen or NH;

R¹selected from hydrogen, alkyl, cycloalkyl, or cycloalkylalkyl, said alkyl, cycloalkyl, or cycloalkylalkyl being unsubstituted or optionally substituted with one to three groups independently selected from halogen, alkyl;

R²selected from hydrogen, alkyl, cycloalkyl, alkoxy, alkylthio, cycloalkoxy, halogen, cyano, or nitro, said alkyl, cycloalkyl, alkoxy, alkylthio, or cycloalkoxy being unsubstituted or optionally substituted with one to three groups independently selected from halogen, alkyl, alkoxy.

In another mode, R¹Is hydrogen or C_1-4Alkyl radical, said C_1-4Alkyl is unsubstituted or optionally substituted with one to three halogens.

In another mode, R²Is C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkylthio, halogen, or cyano, said C_1-4Alkyl radical, C_1-4Alkoxy, or C_1-4Alkylthio is unsubstituted or optionally substituted with one to three halogen.

In another form, the pharmaceutically acceptable salt comprises: a salt of a compound represented by the general formula (I) with an acid; wherein the acid comprises: inorganic acids, organic acids or acidic amino acids; the inorganic acid includes: hydrochloric, hydrobromic, hydrofluoric, sulfuric, nitric or phosphoric acids; the organic acid includes: formic acid, acetic acid, propionic acid, oxalic acid, trifluoroacetic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, p-toluenesulfonic acid, ethanesulfonic acid, or benzenesulfonic acid; the acidic amino acids include: aspartic acid or glutamic acid.

In another preferred embodiment, the compound is a compound of formula 1, a compound of formula 2, a compound of formula 3, a compound of formula 4, a compound of formula 5, a compound of formula 6, a compound of formula 7, a compound of formula 8, a compound of formula 9, a compound of formula 10, a compound of formula 11, a compound of formula 12, a compound of formula 13, a compound of formula 14, a compound of formula 15, a compound of formula 16, a compound of formula 17, a compound of formula 18, a compound of formula 19, a compound of formula 20, a compound of formula 21, a compound of formula 22, a compound of formula 23, a compound of formula 24, a compound of formula 25, a compound of formula 26, a compound of formula 27, a compound of formula 28, a compound of formula 29, a compound of formula 30, a compound of formula 31, a compound of formula 32, or a compound of formula 33:

in a second aspect of the present invention, there is provided a method for preparing an anti-tubercular compound provided by the present invention as described above, the method comprising the steps of:

(1) reducing the compound with the structure shown as the formula I-5 to obtain the compound with the structure shown as the formula I-6;

(2) chlorinating the compound with the structure shown as the formula I-6 to obtain a compound with the structure shown as the formula I-7; and

(3) mixing a compound with a structure shown as a formula I-7 and a compound with a structure shown as a formula I-8, and reacting to obtain a compound with a structure shown as a formula I;

m，n，R¹and R²The definition of (1) is as before; x is oxygen.

The present invention also provides a method for preparing the anti-tubercular compound provided by the present invention as described above, the method comprising the steps of: mixing a compound with a structure shown as a formula I-5 and a compound with a structure shown as a formula II-1, and reacting in the presence of a reducing agent to obtain a compound with a structure shown as a formula I;

m，n，R¹and R²The definition of (1) is as before; x is NH.

In another embodiment, the compounds of formula I-5 are obtained by the following steps:

(a) carrying out coupling reaction on a compound with a structure shown as a formula I-3 and tri-n-butylvinyl tin to obtain a compound with a structure shown as a formula I-4; and

(b) oxidizing and cutting off double bonds of the compound shown in the structural formula I-4 to obtain a compound shown in the structural formula I-5;

in a third aspect of the present invention there is provided the use of an anti-tubercular compound as hereinbefore described with reference to the present invention in the manufacture of a medicament for the treatment of a disease associated with an infection caused by mycobacterium tuberculosis.

In another preferred embodiment, the antituberculous compound provided by the invention is used for preparing a medicament for treating infectious diseases caused by multidrug-resistant mycobacterium tuberculosis.

In a fourth aspect of the present invention, there is provided a pharmaceutical composition for treating a disease associated with infection by mycobacterium tuberculosis, comprising a therapeutically effective amount of an anti-tubercular compound provided by the present invention as described above and a pharmaceutically acceptable excipient or carrier.

Accordingly, the present invention provides a novel antituberculous drug, which is effective against drug-resistant bacteria, particularly multidrug-resistant bacteria; can be used in combination with the first-line antituberculosis drugs currently used; has ideal metabolic property, can be orally taken and is taken once a day; the safety is superior to the existing medicines.

Detailed Description

The inventor synthesizes and screens a large number of compounds through extensive research, and finds that the compound shown in the formula (I) has unexpected advantages in the aspects of pharmacokinetics and pharmacodynamics, has strong inhibitory activity on tubercle bacillus, and is effective on drug-resistant bacteria, particularly multi-drug-resistant bacteria. The present invention has been completed based on this finding.

The invention provides a compound shown as a formula (I), or an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

m, n, X, R in the formula (I)¹And R²As defined above.

Some representative compounds of the invention and their structural formulas are set forth in the following table:

unless otherwise indicated, the following terms used in the specification and claims have the following meanings:

"alkyl" refers to a saturated aliphatic hydrocarbon group, including straight and branched chain groups of 1 to 6 carbon atoms. Lower alkyl groups having 1 to 4 carbon atoms are preferred, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl.

"cycloalkyl" means a 3-to 7-membered all-carbon monocyclic aliphatic hydrocarbon group or a ring in which one carbon atom is replaced by a heteroatom such as oxygen, sulfur, etc., wherein one or more rings may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system. For example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexane, cyclohexadiene and the like.

"alkoxy" refers to an alkyl group bonded to the rest of the molecule through an ether oxygen atom. Representative of alkoxy groups are alkoxy groups having 1 to 4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy and tert-butoxy. As used herein, "alkoxy" includes unsubstituted and substituted alkoxy groups, especially alkoxy groups substituted with one or more halogens. Preferred alkoxy groups are selected from OCH₃，OCF₃，CHF₂O，CF₃CH₂O, iPrO, nPro, iBuO, cPro, nBuO or tBuO.

"alkylthio" refers to an alkyl group bonded to the rest of the molecule through a sulfur atom. Representative alkylthio groups are alkylthio groups having 1 to 4 carbon atoms, such as methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, sec-butylthio and tert-butylthio. As used herein, "alkylthio" includes unsubstituted and substituted alkylthio, especially alkylthio substituted with one or more halogen. Preferred alkylthio groups are selected from SCH3, SCF3, CHF2S, CF3CH2S, iPrS, nPrS, iBuS, cPrS, nBuS or tBuS.

"halogen" means fluorine, chlorine, bromine or iodine.

"chemical bond" refers to the general term for a strong interaction between two or more adjacent atoms (or ions) within a pure molecule or crystal.

"optionally substituted" or "substituted" means that the reference group may be substituted with one or more additional groups independently and independently selected from alkyl, alkoxy, and halo.

"an integer between 0 and 4" means 0, 1,2, 3, 4; "an integer between 1 and 4" means 1,2, 3, 4.

The compounds of the present invention contain at least 1 asymmetric center and thus occur as racemates, racemic mixtures, single enantiomers, diastereomeric compounds and individual diastereomers. Asymmetric centers that may be present depend on the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers and all possible optical isomers and diastereomeric mixtures and pure or partially pure compounds are included within the scope of the invention. The present invention is meant to include all such isomeric forms of these compounds.

The "pharmaceutically acceptable salt" used herein is not particularly limited as long as it is a pharmaceutically acceptable salt, and includes inorganic salts and organic salts. Specifically, the salts of the compounds of the present invention with acids are exemplified, and acids suitable for salt formation include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, phosphoric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, trifluoroacetic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc., and acidic amino acids such as aspartic acid, glutamic acid, etc.

The invention also provides a preparation method of the novel antituberculous compound or the pharmaceutically acceptable inorganic or organic salt thereof.

The following specifically describes the preparation method of the compound having the structure of the general formula (I) of the present invention, but these specific methods do not limit the present invention at all.

The compound having the structure of the general formula (I) of the present invention can be produced by the following method, however, the conditions of the method, such as reactants, solvent, base, amount of the compound used, reaction temperature, time required for the reaction, etc., are not limited to the following explanation. The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art to which the present invention pertains.

The process for preparing the antituberculous antibacterial compound of the present invention may include:

scheme 1: procedure when X ═ O

Firstly, reducing a compound with a structure shown as a formula I-5 to obtain a compound with a structure shown as a formula I-6;

secondly, chlorinating the compound with the structure shown as the formula I-6 to obtain a compound with the structure shown as the formula I-7;

and thirdly, mixing the compound with the structure shown as the formula I-7 and the compound with the structure shown as the formula I-8, and reacting to obtain the compound with the structure shown as the formula I.

The reduction reaction in the first step can be carried out in a suitable solvent by selecting a suitable reducing agent; such solvents include, but are not limited to, alcohols such as methanol, ethanol, ethers such as tetrahydrofuran, and the like; reducing agents used include, but are not limited to, sodium borohydride, potassium borohydride, lithium borohydride, and lithium aluminum hydride.

The chlorination reaction of the second step can be carried out in a suitable solvent by using a suitable chlorinating agent; such solvents include, but are not limited to, dichloromethane, chloroform, toluene; the chlorinating agents used include, but are not limited to, thionyl chloride, phosphorus oxychloride, phosphorus trichloride, phosphorus pentachloride.

In one embodiment of the invention, the compound with the structure shown in formula I-8 in the third step is mixed with a suitable solvent and then placed at a low temperature (such as-20-0 ℃), then strong base is added for reaction for a period of time (such as 0.5-2h), and then the compound with the structure shown in formula I-7 is added for continuous reaction; the suitable solvent is selected from Dimethylformamide (DMF), dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), tetrahydrofuran; the strong base includes, but is not limited to, sodium hydride, potassium hydride, sodium tert-butoxide, potassium tert-butoxide.

In one embodiment of the invention, after the reaction in the third step is finished, a compound with a structure shown as a formula I is obtained through conventional treatment and separation; such conventional processing means include, but are not limited to, extraction, washing, drying, concentration, chromatography, and the like.

In one embodiment of the present invention, a compound having the structure of formula I-5 can be obtained from a compound having the structure of formula I-3, and the process can include the steps of:

the method comprises the following steps of firstly, carrying out coupling reaction on a compound with a structure shown as a formula I-3 and tri-n-butylvinyltin to obtain a compound with a structure shown as a formula I-4; and

and secondly, oxidizing and cutting off double bonds of the compound shown in the structural formula I-4 to obtain the compound shown in the structural formula I-5.

In one embodiment of the present invention, the catalyst for the coupling reaction of the first step includes, but is not limited to, Pd (PPh)₃)₄、Pd(dppf)₂Cl₂、Pd₂(dba)₃(ii) a The reaction solvent for the coupling reaction may include, but is not limited to, Dimethylformamide (DMF), dimethylacetamide (DMAc), N-methylpyrrolidone (NMP); the coupling reaction may be carried out at a temperature of between 50 and 120 ℃.

In one embodiment of the present invention, the oxidizing agents that can be used for the oxidative cleavage in the second step include, but are not limited to, sodium periodate, potassium periodate; catalysts that may be used include, but are not limited to, potassium osmate, ruthenium trichloride; suitable solvents for the oxidative cleavage include, but are not limited to, a mixed solvent of dioxane and water, and suitable temperatures may be in the range of 0 to 50 ℃.

In one embodiment of the present invention, the process 1 may include the following steps:

the method comprises the following steps of firstly, carrying out coupling reaction on a compound with a structure shown as a formula I-3 and tri-n-butylvinyltin to obtain a compound with a structure shown as a formula I-4;

secondly, oxidizing and cutting off double bonds of the compound shown as the structural formula I-4 to obtain a compound shown as the structural formula I-5;

thirdly, reducing the compound with the structure shown as the formula I-5 to obtain the compound with the structure shown as the formula I-6;

fourthly, chlorinating the compound with the structure shown as the formula I-6 to obtain a compound with the structure shown as the formula I-7;

and fifthly, mixing the compound with the structure shown as the formula I-7 and the compound with the structure shown as the formula I-8, and reacting to obtain the compound with the structure shown as the formula I.

The compound with the structure shown in the formula I-3 in the first step can be obtained by taking 5-bromo-2-chloropyrimidine as an initial raw material through two ways:

one approach is to dissolve 5-bromo-2-chloropyrimidine and a primary amine compound in a solvent (such as but not limited to N-butanol, N-propanol, isobutanol), and perform a substitution reaction at a certain temperature (such as 80-120 ℃) by using a suitable base (such as but not limited to N, N-Diisopropylethylamine (DIPEA), triethylamine) as an acid-binding agent to obtain a compound with a structure shown as formula I-1; the compound with the structure shown as the formula I-1 is mixed with a proper solvent and then placed at a low temperature (such as-20-0 ℃), then strong base is added for reaction for a period of time (such as 0.5-2h), and then iodo matter or bromo matter is added for continuous reaction to obtain the compound with the structure shown as the formula I-3; the suitable solvent is selected from Dimethylformamide (DMF), dimethylacetamide (DMAc), N-methylpyrrolidone (NMP); the strong base includes, but is not limited to, sodium hydride, potassium hydride, LDA, NaHMDS, LiHMDS.

The other way is to dissolve 5-bromo-2-chloropyrimidine and a primary amine compound in a solvent (such as, but not limited to, ethanol, isopropanol, n-butanol and isobutanol) to carry out substitution reaction at a certain temperature (such as 80-100 ℃) to obtain a compound with a structure shown as a formula I-2; the compound with the structure shown as the formula I-2 and halogenated aryl are subjected to coupling reaction under the conditions that copper (such as but not limited to cuprous iodide and cuprous bromide) is used as a catalyst and amino acid (such as but not limited to L-proline, dimethylethylenediamine and N, N' -dimethyl-1, 2-cyclohexanediamine) is used as a ligand, so that the compound with the structure shown as the formula I-3 can be obtained.

In one embodiment of the present invention, the process 1 can be performed according to the following reaction formula and its related description:

R¹、R²m, n and the same as defined in formula (I) herein, wherein R¹Not equal to hydrogen.

(1) Dissolving 5-bromo-2-chloropyrimidine and primary amine compounds serving as starting materials in a solvent (such as n-butanol), taking a proper base (such as DIPEA) as an acid-binding agent, and carrying out substitution reaction at a certain temperature (such as 80-120 ℃) to obtain an intermediate I-1;

(2) reacting the intermediate I-1 in a suitable solvent (such as DMF or DMAc) at low temperature (-20-0 ℃) for a certain time (such as 0.5-2h) after adding strong base (such as sodium hydride), then adding iodo or bromo, and separating after the reaction is finished to obtain an intermediate I-3;

(3) the starting materials 5-bromo-2-chloropyrimidine and primary amine compound are dissolved in a solvent (such as ethanol) and subjected to substitution reaction at a certain temperature (such as 80-100 ℃) to obtain an intermediate I-2. The intermediate I-2 and halogenated aryl are subjected to coupling reaction under the conditions that copper (such as cuprous iodide) is used as a catalyst and amino acid (such as L-proline) is used as a ligand to obtain an intermediate I-3;

(4) intermediate I-3 and tri-n-butylvinyltin in the presence of a palladium catalyst (e.g., Pd (PPh)₃)₄) In a suitable solvent (e.g.: DMF) and at a suitable temperature (e.g.: carrying out coupling reaction at 50-120 ℃, and obtaining an intermediate I-4 after the reaction is finished and conventional column chromatography separation;

(5) the double bond of the intermediate I-4 is oxidized and cut off by taking sodium periodate as an oxidant and potassium osmate as a catalyst in a proper solvent (for example, a mixed solvent of dioxane and water) and at a proper temperature (for example, 0-50 ℃) to obtain an aldehyde intermediate I-5;

(6) reduction of intermediate I-5 in a suitable solvent (e.g., methanol) with a suitable reducing agent (e.g., sodium borohydride) affords alcohol intermediate I-6;

(7) the intermediate I-6 is chlorinated in a suitable solvent (e.g., dichloromethane) with a suitable chlorinating agent (e.g., thionyl chloride) to provide a chlorinated intermediate I-7;

(8) after the intermediate I-8 is reacted in a suitable solvent (e.g., DMF or DMAc) at a low temperature (-20-0 ℃) with a strong base (e.g., sodium hydride) for a certain period of time (e.g., 0.5-2 hours), the chlorinated intermediate I-7 is added and the reaction is continued. After the reaction is finished, the target compound (I) is obtained through conventional post-treatment separation.

And (2) a flow scheme: procedure when X is NH

The compound with the structure shown in the formula I-5 and the compound with the structure shown in the formula II-1 are mixed and then react in the presence of a reducing agent to obtain the compound with the structure shown in the formula I.

In one embodiment of the invention, the compound with the structure shown in the formula I-5 and the compound with the structure shown in the formula II-1 react for 2-20 hours in the presence of organic base to obtain intermediate imine, and then a reducing agent is added to react for 1-22 hours to obtain the compound with the structure shown in the formula I. Such organic bases include, but are not limited to, triethylamine, N-Diisopropylethylamine (DIPEA); the reaction solvent to obtain the intermediate imine includes, but is not limited to, dichloromethane, dichloroethane; the reducing agent includes, but is not limited to, sodium triacetoxyborohydride, sodium borohydride, sodium cyanoborohydride.

The compound with the structure shown in the formula I-5 can be obtained by the method.

In one embodiment of the present invention, the process scheme 2 can be performed according to the following reaction formula and its related description:

The intermediate I-5 and II-1 are reacted in a solvent (such as dichloromethane) in the presence of an organic base (such as triethylamine) for a period of time (such as 2-20h) to produce an intermediate imine, and then a reducing agent (such as sodium triacetoxyborohydride) is added for a suitable period of time (such as 1-22h) to obtain the target compound (I) with reduced imine.

In one embodiment of the present invention, there is also provided scheme 3, which proceeds as follows and is described in relation thereto:

(1) the intermediate I-1 is used as a raw material, and the intermediate III-1 is synthesized according to the method of the document WO 2017/176817.

(2) Reduction of intermediate III-1 in a suitable solvent (e.g., methanol) with a suitable reducing agent (e.g., sodium borohydride) affords alcohol intermediate III-2;

(3) the intermediate III-2 is chlorinated in a suitable solvent (e.g. dichloromethane) with a suitable chlorinating agent (e.g. thionyl chloride) to give the chlorinated intermediate III-3;

(4) after the intermediate I-8 is reacted in a suitable solvent (e.g., DMF or DMAc) at a low temperature (-20-0 ℃) with a strong base (e.g., sodium hydride) for a certain period of time (e.g., 0.5-2 hours), the chloro intermediate III-3 is added and the reaction is continued. After the reaction is finished, the target compound 33 is obtained by conventional post-treatment and separation.

The general structural formula related in the preparation method provided by the invention is shown in the following table:

wherein, m, n, R¹And R²As defined above.

The invention also provides application of the novel antituberculous compound, or the optical isomer thereof, or the pharmaceutically acceptable salt thereof in treating diseases related to infection caused by tubercle bacillus.

The compound of the general formula (I) has strong anti-mycobacterium tuberculosis effect, and particularly has excellent effect on multi-drug resistant and widely drug resistant mycobacterium tuberculosis.

The compound of the general formula (I) has better in vitro and in vivo activity, no inhibition on hERG potassium current and better drug pharmacokinetic property. The compound has important significance for improving the activity of the mycobacterium tuberculosis, improving the drug effect, reducing side effects and saving the cost.

In the present invention, the "active ingredient" means a compound represented by the general formula (I) and a pharmaceutically acceptable inorganic or organic salt of the compound of the general formula (I). The compounds of the present invention may contain one or more asymmetric centers and thus occur as racemates, racemic mixtures, single enantiomers, diastereomeric compounds and individual diastereomers. Asymmetric centers that may be present depend on the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers and all possible optical isomers and diastereomeric mixtures and pure or partially pure compounds are included within the scope of the invention. The present invention is meant to include all such isomeric forms of these compounds.

In addition, some of the compounds of the present invention may be prepared by reacting a polar protic solvent, such as methanol, ethanol, isopropanol, and a pharmaceutically acceptable acid, to form a pharmaceutically acceptable salt, if desired. The pharmaceutically acceptable inorganic or organic acid may be: hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, aspartic acid, or glutamic acid, and the like.

The term "tubercle bacillus caused", as used herein, refers to caused by drug-sensitive tubercle bacillus for clinical tuberculosis, drug-resistant tubercle bacillus for clinical certain drug, drug-resistant tubercle bacillus for clinical multiple drugs, and widely drug-resistant tubercle bacillus.

The terms "disease of infection by tubercle bacillus" or "tubercle bacillus infectious disease" are used interchangeably and as used herein refer to tuberculosis of the lung, lymphoid tuberculosis, intestinal tuberculosis, bone tuberculosis, tuberculous pleuritis, tuberculous meningitis and the like.

The compound of the present invention and pharmaceutically acceptable inorganic or organic salts thereof, and pharmaceutical compositions containing the compound as a main active ingredient can be used for treating diseases associated with tubercle bacillus, since the compound of the present invention has excellent anti-tubercle bacillus activity. According to the prior art, the compounds of the invention are useful for the treatment of tuberculosis and other infectious diseases.

The invention also provides a pharmaceutical composition for treating diseases related to infection caused by tubercle bacillus, which comprises the nitroimidazole compounds with effective dose and pharmaceutically acceptable excipient or carrier.

The pharmaceutical composition of the present invention comprises a safe and effective amount of the nitroimidazole compounds of the present invention and pharmaceutically acceptable excipients or carriers. Wherein "safe, effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-1000mg of a compound of the invention per dose, preferably 5-500mg of a compound of the invention per dose, more preferably 10-200mg of a compound of the invention per dose.

The compound and the pharmaceutically acceptable salt thereof can be prepared into various preparations, wherein the preparation comprises the compound or the pharmaceutically acceptable salt thereof in a safe and effective amount range and a pharmaceutically acceptable excipient or carrier. Wherein "safe, effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. The safe and effective amount of the compound is determined according to the age, condition, course of treatment and other specific conditions of a treated subject.

"pharmaceutically acceptable excipient or carrier" refers to: aOne or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of being blended with the compounds of the present invention and with each other without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable excipients or carrier moieties are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate etc.), gelatin, talc, solid lubricants (e.g. stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g. soybean oil, sesame oil, peanut oil, olive oil etc.), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol etc.), emulsifiers (e.g. propylene glycol, glycerol, mannitol, sorbitol etc.)

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

When the compounds of the present invention are administered, they may be administered orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), topically.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

When using pharmaceutical compositions, a safe and effective amount of a compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, wherein the administration is a pharmaceutically acceptable and effective dose, and the daily dose for a human of 60kg body weight is usually 1-1000mg, preferably 10-500 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The features mentioned above with reference to the invention, or the features mentioned with reference to the embodiments, can be combined arbitrarily. All features disclosed in this specification may be combined in any combination, provided that there is no conflict between such features and the combination, and all possible combinations are to be considered within the scope of the present specification. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

The main advantages of the invention include:

1. the compound has a specific effect on mycobacterium tuberculosis and has excellent effects on multi-drug resistant and widely drug resistant mycobacterium tuberculosis.

2. The compound of the invention has better drug pharmacokinetic property. The compound has important significance for improving the activity of the mycobacterium tuberculosis, improving the drug effect, reducing side effects and saving the cost.

3. The compound of the invention has no inhibition on the hERG potassium current, which shows that the compound has better safety on the cardiovascular system.

4. The compound has excellent bactericidal effect in mice and can obviously reduce the colony number of infected mice lungs.

The various specific aspects, features and advantages of the compounds, methods and pharmaceutical compositions described above are set forth in detail in the following description, which makes the present invention clear. It should be understood herein that the detailed description and examples, while indicating specific embodiments, are given by way of illustration only. After reading the description of the invention, one skilled in the art can make various changes or modifications to the invention, and such equivalents fall within the scope of the invention as defined in the application.

The present invention is more specifically explained in the following examples. It should be understood, however, that these examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention in any way. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Parts and percentages are parts and percentages by weight unless otherwise indicated.

All examples of the invention¹H-NMR was recorded using a Varian Mercury 400M or 600M nuclear magnetic resonance apparatus, and chemical shifts are expressed in delta (ppm); the Ms was determined using Shimadzu LC-Ms-2020 Mass spectrometer. The silica gel used for separation is not illustrated to be 200-300 meshes, and the proportions of the eluents are volume ratios.

Example 1: (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (Compound 1)

(1) 4-Trifluoromethoxyaniline (26.7g, 0.15mol), 5-bromo-2-chloropyrimidine (19.34g, 0.1mmol), DIPEA (25.85g, 0.2mol) were dissolved in n-butanol (180mL) and stirred overnight at 120 ℃. TLC (petroleum ether: ethyl acetate: 10:1), after the reaction was completed, the reaction solution was spin-dried, a small amount of dichloromethane was added to dissolve the concentrate, petroleum ether was added dropwise, the solid was precipitated, and a pale yellow solid 1-1 was obtained by filtration (13.85g, yield: 42%).

¹H-NMR(400MHz,CDCl₃)δ8.44(s,2H),7.66-7.56(m,2H),7.37(s,1H),7.24-7.15(m,2H).ESI-Ms:233.9[M+1]+.

(2) 1-1(6.68g, 20mmol) was dissolved in dry DMF (70mL), NaH (1.2g, 30mmol) was added thereto under argon protection under ice bath, stirred for 30min under ice bath, then methyl iodide (5.68g, 40mmol) was added thereto, slowly warmed to room temperature, and stirred overnight for reaction. And monitoring by LC-Ms, and finishing the reaction. Ethyl acetate, water, liquid separation, ethyl acetate extraction, combined organic phases, brine washing, drying, concentration, column chromatography (petroleum ether: ethyl acetate: 20:1) to obtain light yellow solid 1-3(5.46g, yield: 78.5%).

¹H-NMR(400MHz,CDCl₃)δ8.33(s,2H),7.36-7.30(m,2H),7.26-7.23(m,2H),3.50(s,3H).ESI-Ms:348.0[M+1]⁺.

(3) 1-3(5.22g, 15mmol) was dissolved in dry DMF (60mL) and tributylvinyltin (7.13g, 22.5mmol), Pd (PPh) were added₃)₄(0.87g, 0.75mmol) was then reacted overnight at 120 ℃ under argon. The reaction was complete as monitored by LC-MS. KF, water and diatomite are added into the system for suction filtration, ethyl acetate extraction is carried out, the combined organic phase is washed by saturated saline solution, dried, concentrated and subjected to column chromatography (petroleum ether: ethyl acetate: 20:1) to obtain white solid 1-4(2.82g, yield: 63.8%).

¹H-NMR(400MHz,CDCl₃)δ8.39(s,2H),7.36-7.30(m,2H),7.26-7.22(m,2H),6.50(dd,J＝17.2Hz,0.4Hz,1H),5.61(dd,J＝17.2Hz,0.4Hz,1H),5.17(dd,J＝11Hz,0.4Hz,1H),3.53(s,3H).ESI-Ms:296.1[M+1]⁺.

(4) Dissolving 1-4(2.36g, 8.0mmol) in dioxane/water (30mL, 1: 1), adding sodium periodate (6.82g, 32mmol), and adding K₂OsO₄.2H₂O (29mg, 0.08mmol) was reacted at room temperature. The reaction was complete as monitored by LC-MS. Adding water into the system, extracting with ethyl acetate, mixing the organic phases, washing with dilute hydrochloric acid, saturated sodium bicarbonate water solution and saturated salt solution in sequence, drying, concentrating to obtain 1-5(2.80g) white solid, and directly feeding the crude product to the next step without purification. ESI-Ms:298.1[ M +1]]⁺.

(5) 1-5(2.80g) was dissolved in methanol (30mL) and NaBH was added thereto under ice-bath₄(0.45g, 12mmol) and then slowly warmed to the latter room temperature for 2 hours. The reaction was complete as monitored by LC-MS. The system was quenched with water, concentrated, and column chromatographed (petroleum ether: ethyl acetate: 3: 1) to give 1-6(1.48g, yield: 62.1%) as a white solid.

¹H-NMR(400MHz,CDCl₃)δ8.25(s,2H),7.35-7.29(m,2H),7.25-7.22(m,2H),4.48(s,2H),3.49(s,3H).ESI-Ms:300.1[M+1]⁺.

(6) 1-6(598mg, 2.0mmol) was dissolved in methylene chloride (10mL), and 5mL of thionyl chloride (5mL) was added thereto and reacted at room temperature. Monitoring by LC-MS, after the reaction is completed, concentrating, adding dichloromethane, and rotary evaporating SOCl₂Twice, to obtain white solid 1-7(623mg), and directly feeding into the next step. ESI-Ms:318.1[ M +1]]⁺.

(7) Intermediate I-8(400mg,2.14mmol) was added to dry DMF (5mL), NaH (340mg,8.5mmol) was added thereto under ice bath and argon protection, reaction was carried out for half an hour under ice bath, a solution of intermediate 1-7(623mg) in DMF (5mL) was added, and reaction was carried out for 1h after the ice bath was warmed to room temperature. The reaction was monitored by LC-MS to be complete, and ethyl acetate and water were added to the system, followed by liquid separation, extraction, combination of organic phases, washing with saturated brine, drying, concentration, and column chromatography (dichloromethane: methanol: 20:1) to obtain compound 1(191mg, yield: 20.5%) as a pale yellow solid.

¹H NMR(400MHz,CDCl₃)δ8.28(s,2H),7.38(s,1H),7.34–7.29(m,2H),7.24-7.21(m,2H),4.63-4.59(m,1H),4.56(d,J＝11.5Hz,1H),4.44(d,J＝11.4Hz,1H),4.34(d,J＝12.3Hz,1H),4.19(dd,J＝13.3,4.6Hz,1H),4.12-4.07(m,2H),3.51(s,3H).ESI-Ms:467.1[M+1]⁺.

Example 2: (S) -N-Ethyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (Compound 2)

Compound 2 was prepared in the same manner as in example 1.

¹H-NMR(400MHz,CDCl₃)δ8.20(s,2H),7.33(s,1H),7.19(s,4H),4.55(d,J＝11.8Hz,1H),4.49(d,J＝11.6Hz,1H),4.38(d,J＝11.3Hz,1H),4.29(d,J＝12.2Hz,1H),4.15(dd,J＝13.6,4.8Hz,1H),4.08-4.03(t,J＝8.5Hz,2H),3.95(q,J＝6.9Hz,2H),1.15(t,J＝6.9Hz,3H).ESI-Ms:481.1[M+1]⁺.

Example 3: (S) -N-propyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (Compound 3)

Compound 3 was prepared in the same manner as in example 1.

¹H-NMR(400MHz,CDCl₃)δ8.19(s,2H),7.33(s,1H),7.21-7.17(m,4H),4.60–4.51(m,1H),4.48(d,J＝11.4Hz,1H),4.37(d,J＝11.4Hz,1H),4.30(d,J＝12.3Hz,1H),4.15(dd,J＝13.4,4.6Hz,1H),4.08-4.01(m,2H),3.87–3.80(m,2H),1.61-1.56(m,2H),0.85(t,J＝7.4Hz,3H).ESI-Ms:494.2[M+1]⁺.

Example 4: (S) -N-isopropyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (Compound 4)

Compound 4 was prepared in the same manner as in example 1.

¹H NMR(400MHz,CDCl₃)δ8.17(s,2H),7.32(s,1H),7.22(d,J＝8.7Hz,2H),7.07(d,J＝8.7Hz,2H),5.12-5.05(m,1H),4.53(d,J＝12.1Hz,1H),4.46(d,J＝11.5Hz,1H),4.36(d,J＝11.5Hz,1H),4.29(d,J＝12.0Hz,1H),4.14(dd,J＝13.5,4.4Hz,1H),4.04(d,J＝10.9Hz,2H),1.08(d,J＝6.7Hz,6H).ESI-Ms:494.2[M+1]⁺.

Example 5: (S) -N-trifluoroethyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (Compound 5)

Compound 5 was prepared in the same manner as in example 1.

¹H NMR(600MHz,CDCl₃)δ8.25(s,2H),7.33(s,1H),7.23(d,J＝6.1Hz,4H),4.66-4.60(m,2H),4.57(d,J＝11.6Hz,1H),4.53(d,J＝10.9Hz,1H),4.42(d,J＝10.8Hz,1H),4.31(d,J＝11.6Hz,1H),4.17(d,J＝10.3Hz,1H),4.09(s,2H).ESI-Ms:535.1[M+1]⁺.

Example 6: (S) -N-Cyclopropylmethyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (Compound 6)

Compound 6 was prepared in the same manner as in example 1.

¹H NMR(600MHz,CDCl₃)δ8.19(s,2H),7.33(s,1H),7.24(d,J＝8.8Hz,2H),7.19(d,J＝8.7Hz,2H),4.57–4.52(m,1H),4.48(d,J＝11.5Hz,1H),4.37(d,J＝11.4Hz,1H),4.30(d,J＝12.2Hz,1H),4.15(dd,J＝13.5,4.6Hz,1H),4.05(d,J＝12.4Hz,2H),3.77(d,J＝6.9Hz,2H),1.08–1.03(m,1H),0.39-0.34(m,2H),0.10-0.06(m,2H).ESI-Ms:507.2[M+1]⁺.

Example 7: (S) -N-Cyclopentylmethyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (Compound 7)

Compound 7 was prepared by the same method as in example 1.

¹H-NMR(400MHz,CDCl₃)δ8.20(s,2H),7.35(s,1H),7.24(d,J＝8.8Hz,2H),7.19(d,J＝8.7Hz,2H),4.56–4.52(m,1H),4.45(d,J＝11.8Hz,1H),4.32(d,J＝11.6Hz,1H),4.29(d,J＝12.0Hz,1H),4.13(dd,J＝13.4,4.4Hz,1H),4.06(d,J＝12.2Hz,2H),3.75(d,J＝6.9Hz,2H),1.88-1.72(m,2H),1.52-1.40(m,7H).ESI-Ms:535.2[M+1]⁺.

Example 8: (S) -N- ((4, 4-difluoromethylcyclohexyl) methyl) -5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (Compound 8)

Compound 8 was prepared in the same manner as in example 1.

¹H-NMR(400MHz,CDCl₃)δ8.18(s,2H),7.20(s,1H),7.22(d,J＝8.7Hz,,2H),718(d,J＝8.8Hz,2H),4.58-4.49(m,1H),4.48(d,J＝12.0Hz,1H),4.34(d,J＝12.2Hz,1H),4.0(d,J＝12.3Hz,1H),4.18-4.14(m,1H),4.08(d,J＝12.0Hz,2H),3.65(d,J＝7.0Hz,2H),1.68-1.34(m,8H),1.42-1.37(m,1H).ESI-Ms:585.2[M+1]⁺.

Example 9: (S) -N-cyclopropyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (Compound 9)

(1) Cyclopropylamine (11.1g, 0.195mol), 5-bromo-2-chloropyrimidine (12.5g, 65mmol) were dissolved in ethanol (130mL) and stirred at 80 ℃ for 3 h. After cooling, a solid was precipitated, and the reaction mixture was filtered to obtain 9-2(5.67g, yield: 68%) as a white solid, which was then directly subjected to the next step without further purification. ESI-Ms:213.9[ M +1] +.

(2) Intermediate 9-2(4.27g,0.02mol), CuI (762mg,0.004mol), Cs₂CO₃(16.3g,0.05mol), L-proline (920mg,0.008mol), p-trifluoromethoxyiodobenzene (12.6g,0.045mol) were dissolved in DMSO (40ml), and reacted overnight at 120 ℃ under argon shield. And (5) monitoring by LC-MS, and finishing the reaction. Adding ethyl acetate and water into the system, separating, extracting, mixing organic phases, washing with saturated saline water, concentrating, and performing column chromatography (petroleum ether: ethyl acetate)Ethyl acid ester ═ 4: 1) this gave 9-3 as a pale yellow solid (1.5g, 20% yield).

¹H-NMR(400MHz,CDCl₃)δ8.32(s,2H),7.34-7.29(m,2H),7.24-7.20(m,2H),3.10-3.05(m,1H),1.06-0.93(m,2H),0.60-0.51(m,2H).ESI-Ms:374.0[M+1]⁺.

(3) Compound 9 was subsequently prepared in the same manner as in example 1, starting from intermediate 9-3.

¹H-NMR(400MHz,CDCl₃)δ8.32(s,2H),7.38(s,1H),7.24–7.19(m,4H),4.62-4.58(m,1H),4.56(d,J＝11.7Hz,1H),4.46(d,J＝11.6Hz,1H),4.35(d,J＝12.3Hz,1H),4.21(dd,J＝13.4,4.5Hz,1H),4.15–4.08(m,2H),3.17–3.10(m,1H),0.96-0.91(m,2H),0.55–0.47(m,2H).ESI-Ms:492.1[M+1]⁺.

Example 10: (S) -N-cyclopentyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (Compound 10)

Compound 10 was prepared by the same method as in example 9.

Compound 10:¹H-NMR(400MHz,CDCl₃)δ8.23(s,2H),7.35(s,1H),7.23(m,-7.18(m,4H),4.60-4.56(m,1H),4.55(d,J＝11.8Hz,1H),4.42(d,J＝12.0Hz,1H),4.30(d,J＝12.1Hz,1H),4.19(dd,J＝13.1,4.7Hz,1H),4.14-4.06(m,2H),3.09-3.04(m,1H),1.82-1.52(m,8H).ESI-Ms:521.2[M+1]⁺.

example 11: (S) -N- (3-chloro-4- (trifluoromethoxy) phenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine (Compound 11)

Compound 11 was prepared in the same manner as in example 1.

¹H NMR(400MHz,CDCl₃)δ8.30(s,2H),7.44(d,J＝2.5Hz,1H),7.39(s,1H),7.32(dd,J＝8.9,1.2Hz,1H),7.25-7.22(m,1H),4.65–4.59(m,1H),4.57(d,J＝11.5Hz,1H),4.46(d,J＝11.5Hz,1H),4.35(d,J＝12.3Hz,1H),4.20(dd,J＝13.3,4.6Hz,1H),4.14–4.08(m,2H),3.51(s,3H).ESI-Ms:501.1[M+1]⁺.

Example 12: (S) -N- (3-bromo-4- (trifluoromethoxy) phenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine (Compound 12)

Compound 12 was prepared in the same manner as in example 1.

¹H NMR(400MHz,CDCl₃)δ8.30(s,2H),7.59(d,J＝2.1Hz,1H),7.39(s,1H),7.34–7.26(m,2H),4.64–4.59(m,1H),4.57(d,J＝11.4Hz,1H),4.46(d,J＝11.4Hz,1H),4.35(d,J＝12.2Hz,1H),4.21(dd,J＝13.3,4.6Hz,1H),4.15–4.07(m,2H),3.51(s,3H).ESI-Ms:544.0[M+1]⁺.

Example 13: (S) -4- (methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine) benzonitrile (Compound 13)

Compound 13 was prepared in the same manner as in example 1.

¹H-NMR(400MHz,CDCl₃)δ8.28(s,2H),7.46(d,J＝8.8Hz,2H),7.40(d,J＝8.8Hz,2H),7.38(s,1H),4.64-4.58(m,1H),4.57(d,J＝11.6Hz,1H),4.45(d,J＝11.6Hz,1H),4.34(d,J＝12.3Hz,1H),4.19(dd,J＝13.3,4.6Hz,1H),4.12-4.08(m,2H),3.52(s,3H).ESI-Ms:408.1[M+1]⁺.

Example 14: (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (3, 4, 5-trifluorophenyl) pyrimidin-2-amine (Compound 14)

Compound 14 was prepared in the same manner as in example 1.

¹H-NMR(400MHz,CDCl₃)δ8.28(s,2H),7.38(s,1H),7.23-7.17(m,2H),4.60-4.57(m,1H),4.56(d,J＝11.4Hz,1H),4.43(d,J＝11.4Hz,1H),4.34(d,J＝12.3Hz,1H),4.19(dd,J＝13.3,4.6Hz,1H),4.12-4.06(m,2H),3.50(s,3H).ESI-Ms:437.1[M+1]⁺.

Example 15: (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) benzyl) pyrimidin-2-amine (Compound 15)

Compound 15 was prepared in the same manner as in example 1.

¹H NMR(600MHz,CDCl₃)δ8.22(s,2H),7.34(s,1H),7.19(J＝8.2Hz,2H),7.08(d,J＝8.2Hz,2H),4.82(s,2H),4.58–4.53(m,1H),4.49(d,J＝11.4Hz,1H),4.38(d,J＝11.4Hz,1H),4.31(d,J＝12.2Hz,1H),4.16(dd,J＝13.5,4.6Hz,1H),4.12-4.07(m,2H),3.07(s,3H).ESI-Ms:481.1[M+1]⁺.

EXAMPLE 16 (S) -N-cyclopropyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) benzyl) pyrimidin-2-amine (Compound 16)

Compound 16 was prepared in the same manner as in example 1.

¹H NMR(600MHz,CDCl₃)δ8.27(s,2H),7.34(s,1H),7.16(d,J＝8.6Hz,2H),7.05(d,J＝8.2Hz,2H),4.83(s,2H),4.59–4.54(m,1H),4.51(d,J＝11.4Hz,1H),4.39(d,J＝11.4Hz,1H),4.30(d,J＝12.2Hz,1H),4.15(dd,J＝13.5,4.7Hz,1H),4.10–4.04(m,2H),2.73–2.67(m,1H),0.82-0.79(m,2H),0.63–0.58(m,2H).ESI-Ms:507.2[M+1]⁺.

Example 17 (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenethyl) pyrimidin-2-amine (Compound 17)

Compound 17 was prepared in the same manner as in example 1.

¹H NMR(600MHz,CDCl₃)δ8.20(s,2H),7.34(s,1H),7.18(J＝8.6Hz,2H),7.06(d,J＝8.6Hz,2H),4.57–4.53(m,1H),4.48(d,J＝11.4Hz,1H),4.36(d,J＝11.4Hz,1H),4.30(d,J＝12.2Hz,1H),4.15(dd,J＝13.5,4.6Hz,1H),4.03-4.07(m,2H),3.52(t,J＝7.2Hz,2H),3.05(s,3H),2.82(t,J＝7.2Hz,2H).ESI-Ms:495.2[M+1]⁺.

EXAMPLE 18 (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (3- (trifluoromethoxy) phenyl) pyrimidin-2-amine (Compound 18)

Compound 18 was prepared in the same manner as in example 1.

¹H NMR(400MHz,CDCl₃)δ8.30(s,2H),7.42–7.36(m,2H),7.25(d,J＝8.0Hz,1H),7.19(s,1H),7.07(d,J＝8.2Hz,1H),4.63–4.59(m,1H),4.57(d,J＝11.5Hz,1H),4.45(d,J＝11.4Hz,1H),4.35(d,J＝12.1Hz,1H),4.20(dd,J＝13.5,4.6Hz,1H),4.14–4.07(m,2H),3.53(d,J＝0.4Hz,3H).ESI-Ms:467.2[M+1]⁺.

Example 19 (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (2- (trifluoromethoxy) phenyl) pyrimidin-2-amine (Compound 19)

Compound 19 was prepared in the same manner as in example 1.

¹H NMR(400MHz,CDCl₃)δ8.27(s,2H),7.38(s,1H),7.34(s,4H),4.60–4.52(m,2H),4.44(d,J＝11.6Hz,1H),4.33(d,J＝12.1Hz,1H),4.18(dd,J＝13.4,4.5Hz,1H),4.13–4.05(m,2H),3.44(s,3H).ESI-Ms:467.2[M+1]⁺.

EXAMPLE 20 ((S) -N-Ethyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (3- (trifluoromethoxy) phenyl) pyrimidin-2-amine (Compound 20)

Compound 20 was prepared in the same manner as in example 1.

¹H NMR(400MHz,CDCl₃)δ8.29(s,2H),7.42–7.36(m,2H),7.26(d,J＝8.0Hz,1H),7.19(s,1H),7.06(d,J＝8.2Hz,1H),4.64–4.59(m,1H),4.57(d,J＝11.5Hz,1H),4.45(d,J＝11.4Hz,1H),4.36(d,J＝12.1Hz,1H),4.21(dd,J＝13.5,4.6Hz,1H),4.14–4.07(m,2H),3.93(q,J＝6.9Hz,2H),1.13(t,J＝6.9Hz,3H).ESI-Ms:481.1[M+1]⁺.

EXAMPLE 21 (S) -N- (4-methoxyphenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -pyrimidin-2-amine (Compound 21)

Compound 21 was prepared in the same manner as in example 1.

¹H-NMR(400MHz,CDCl₃)δ8.24(s,2H),7.33(s,1H),7.23(d,J＝8.4Hz,2H),7.17(d,J＝8.4Hz,2H),4.54-4.43(m,2H),4.42(d,J＝12.1Hz,1H),4.32(d,J＝11.2Hz,1H),4.17(dd,J＝13.5Hz,4.6Hz,1H),4.10-4.07(m,2H),3.92(s,3H),3.50(s,3H).ESI-Ms:413.1[M+1]⁺.

EXAMPLE 22 (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethyl) phenyl) pyrimidin-2-amine (Compound 22)

Compound 22 was prepared in the same manner as in example 1.

¹H-NMR(400MHz,CDCl₃)δ8.29(s,2H),7.43(d,J＝8.6Hz,2H),7.37(s,1H),7.35(d,J＝8.8Hz,2H),4.64-4.55(m,2H),4.56(d,J＝11.4Hz,1H),4.45(d,J＝11.4Hz,1H),4.36(d,J＝12.2Hz,1H),4.20(dd,J＝13.3Hz,4.6Hz,1H),4.13-4.09(m,2H),3.53(s,3H).ESI-Ms:451.1[M+1]⁺.

Example 23 (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethylthio) phenyl) pyrimidin-2-amine (Compound 23)

Compound 23 was prepared in the same manner as in example 1.

¹H-NMR(400MHz,CDCl₃)δ8.26(s,2H),7.42(d,J＝8.6Hz,2H),7.38(s,1H),7.29(d,J＝8.8Hz,2H),4.62-4.57(m,1H),4.56(d,J＝11.6Hz,1H),4.44(d,J＝11.6Hz,1H),4.32(d,J＝12.3Hz,1H),4.17(dd,J＝13.3,4.6Hz,1H),4.13-4.08(m,2H),3.51(s,3H).ESI-Ms:483.1[M+1]⁺.

EXAMPLE 24 (S) -N- (4- (difluoromethoxy) phenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -pyrimidin-2-amine (compound 24)

Compound 24 was prepared in the same manner as in example 1.

¹H NMR(600MHz,CDCl₃)δ8.26(s,2H),7.36-7.33(m,2H),7.28(J＝8.6Hz,2H),7.17(d,J＝8.6Hz,2H),4.57–4.52(m,1H),4.47(d,J＝11.4Hz,1H),4.35(d,J＝11.4Hz,1H),4.30(d,J＝12.2Hz,1H),4.17(dd,J＝13.5,4.6Hz,1H),4.12-4.07(m,2H),3.50(s,3H).ESI-Ms:449.1[M+1]⁺.

EXAMPLE 25 (S) -N- (3-fluoro-4- (trifluoromethoxy) phenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine (Compound 25)

Compound 25 was prepared in the same manner as in example 1.

¹H NMR(400MHz,CDCl₃)δ8.30(s,2H),7.39(s,1H),7.29(t,J＝8.7Hz,1H),7.21(dd,J＝11.2,2.4Hz,1H),7.13–7.08(m,1H),4.64–4.59(m,1H),4.57(d,J＝11.4Hz,1H),4.46(d,J＝11.4Hz,1H),4.35(d,J＝12.1Hz,1H),4.21(dd,J＝13.4,4.6Hz,1H),4.14-4.09(m,2H),3.51(s,3H).ESI-Ms:485.1[M+1]⁺.

EXAMPLE 26 (S) -N- (2-chloro-4- (trifluoromethoxy) phenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine (Compound 26)

Compound 26 was prepared in the same manner as in example 1.

¹H NMR(400MHz,CDCl₃)δ8.28(s,2H),7.38(s,1H),7.36(d,J＝1.8Hz,1H),7.32(d,J＝8.7Hz,1H),7.21–7.16(m,1H),4.63–4.57(m,1H),4.55(d,J＝11.5Hz,1H),4.44(d,J＝11.5Hz,1H),4.35(d,J＝12.2Hz,1H),4.21(dd,J＝13.4,4.5Hz,1H),4.14–4.07(m,2H),3.42(s,3H).ESI-Ms:501.1[M+1]⁺.

EXAMPLE 27 (S) -N- (2-methyl-4- (trifluoromethoxy) phenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine (Compound 27)

Compound 27 was prepared in the same manner as in example 1.

¹H NMR(400MHz,CDCl₃)δ8.29(s,2H),7.40(s,1H),7.19-7.14(m,2H),7.11(d,J＝8.9Hz,1H),4.66–4.59(m,1H),4.56(d,J＝11.4Hz,1H),4.44(d,J＝11.4Hz,1H),4.36(d,J＝12.0Hz,1H),4.21(dd,J＝13.3,4.4Hz,1H),4.15–4.08(m,2H),3.41(s,3H),2.14(s,3H).ESI-Ms:481.1[M+1]⁺.

EXAMPLE 28 (S) -N- (4-fluoro-3- (trifluoromethoxy) phenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine (Compound 28)

Compound 28 was prepared in the same manner as in example 1.

¹H NMR(600MHz,CDCl₃)δ8.23(s,2H),7.35(s,1H),7.22(d,J＝5.5Hz,1H),7.17-7.13(m,2H),4.56(d,J＝11.9Hz,1H),4.50(d,J＝11.3Hz,1H),4.41(d,J＝11.4Hz,1H),4.32(d,J＝12.0Hz,1H),4.19(dd,J＝13.0,4.6Hz,1H),4.10(s,2H),3.44(s,3H).ESI-Ms:485.1[M+1]⁺.

Example 29 (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxo) methyl) -N-phenylpyrimidin-2-amine (Compound 29)

Compound 29 was prepared by the same method as in example 1.

¹H NMR(400MHz,CDCl₃)δ8.26(s,2H),7.35(s,1H),7.33–7.26(m,4H),7.18-7.14(m,1H),4.62-4.57(m,1H),4.56(d,J＝11.5Hz,1H),4.44(d,J＝11.5Hz,1H),4.34(d,J＝12.3Hz,1H),4.19(dd,J＝13.4,4.6Hz,1H),4.14-4.07(m,2H),3.43(s,3H).ESI-Ms:383.1[M+1]⁺.

Example 30 (S) -N- (4-cyclopropylphenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine (Compound 30)

Compound 30 was prepared by the same method as in example 1.

¹H NMR(400MHz,CDCl₃)δ8.26(s,2H),7.36(s,1H),7.32(d,J＝8.6Hz,2H),7.28(d,J＝8.7Hz,2H),4.64-4.59(m,1H),4.55(d,J＝11.5Hz,1H),4.43(d,J＝11.5Hz,1H),4.34(d,J＝12.3Hz,1H),4.19(dd,J＝13.3,4.5Hz,1H),4.13-4.07(m,2H),3.46(s,3H),2.01–1.95(m,1H),1.59-1.52(m,2H),1.16-1.08(m,2H).ESI-Ms:423.2[M+1]⁺.

Example 31 (S) -N- ((2- (methyl (4- (trifluoromethoxy) phenyl) amino) pyrimidin-5-yl) methyl) -2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-amine (Compound 31)

Intermediate 1-5(148mg,0.50mmol), triethylamine (66mg,0.65mmol) were dissolved in dichloromethane (10mL), then added to starting material II-1(84mg,0.50mmol), reacted overnight at room temperature, NaBH (OAc) added₃(424mg,2.0mmol) and the reaction was continued at room temperature overnight. Sodium bicarbonate solution was added, the layers were separated, the aqueous layer was extracted with dichloromethane, the dichloromethane layers were combined, washed with saturated sodium chloride solution, dried, concentrated, and column-chromatographed (dichloromethane:methanol 50:1) gave compound 31(116mg, yield 52.7%) as a pale yellow powder.

¹H-NMR(400MHz,CDCl₃)δ8.21(s,2H),7.46(s,1H),7.35(d,J＝8.6Hz,2H),7.26(d,J＝8.6Hz,2H),4.51(dd,J＝11.4,2.6Hz,1H),4.43(dd,J＝11.4,5.6Hz,1H),4.23(dd,J＝12.2,4.4Hz,1H),3.98(dd,J＝12.2,4.4Hz,1H),3.81(q,J＝13.4Hz,2H),3.50–3.40(m,1H),3.20(s,3H).ESI-Ms:466.1[M+1]⁺.

Example 32(S) -N- ((2- (methyl (4- (trifluoromethoxy) benzyl) amino) pyrimidin-5-yl) methyl) -2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-amine (Compound 32)

Compound 32 was prepared in the same manner as in example 31.

¹H NMR(400MHz,CDCl₃)δ8.33(s,2H),7.45(s,1H),7.30(d,J＝8.2Hz,2H),7.20(d,J＝8.2Hz,2H),4.94(s,2H),4.50(dd,J＝11.6,2.6Hz,1H),4.43(dd,J＝11.8,5.5Hz,1H),4.24(dd,J＝12.2,4.3Hz,1H),3.99(dd,J＝12.2,4.4Hz,1H),3.81(q,J＝13.4Hz,2H),3.50–3.41(m,1H),3.19(s,3H).ESI-Ms:480.2[M+1]⁺.

EXAMPLE 33 (S) -5- (((2-Nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (Compound 33)

Starting from 1-1, intermediate IIII-1 was synthesized according to the method of document WO2017/176817, and then compound 33 was prepared by the same method as in example 1.

¹H NMR(600MHz,CDCl₃)δ8.32(s,2H),7.57(d,J＝9.0Hz,2H),7.35(s,1H),7.13(d,J＝8.6Hz,2H),4.62–4.58(m,1H),4.56(d,J＝11.4Hz,1H),4.44(d,J＝11.4Hz,1H),4.32(d,J＝12.2Hz,1H),4.18(dd,J＝14.0,4.9Hz,1H),4.12–4.06(m,2H).ESI-Ms:453.1[M+1]⁺.

Pharmacological examples

Example 34: in vitro efficacy test of partial compounds on mycobacterium tuberculosis H37Rv strain

Transferring the tested strain H37Rv into liquid culture medium, culturing at 37 deg.C for 2 weeks, sucking a little of culture solution, placing in 4mL liquid culture medium, adding 10-20 particles of sterile glass beads with diameter of 2-3mm, shaking for 20-30S, standing for l0-20 min, sucking supernatant of bacterial suspension, adjusting turbidity to 1 McLee unit with liquid culture medium, which is equivalent to 1 × 10⁷CFU/mL is ready for use. Each drug was dissolved in an appropriate amount of DMSO to 1mg/mL and filtered through a 0.22 μm filter. Then diluted with liquid medium to the desired experimental concentration. The final concentrations of the test drugs were set as follows: 0.0039. mu.g/mL, 0.0078. mu.g/mL, 0.0156. mu.g/mL, 0.03125. mu.g/mL, 0.0625. mu.g/mL, 0.125. mu.g/mL, 0.25. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL, 2. mu.g/mL, 4. mu.g/mL, for a total of 11 concentration gradients. Adding 100 μ L of the above medicinal solution into 96-well microporous plate, adding 100 μ L of 1mg/mL bacterial solution to reach set final concentration, and culturing at 37 deg.C. Three groups of parallel controls were set for the same drug dilution, with the control group containing no drug and the inoculum size set at 100%, 10% and 1%, respectively. After 14 days of incubation at 37 ℃, each colony was observed and the lowest concentration of the colony-free drug group was used as the MIC value of the test compound against the strain. The Minimal Inhibitory Concentration (MIC) of each compound against M.tuberculosis was observed and compared with the MIC results of the control drug PA-824. The results are shown in Table 1.

TABLE 1 in vitro Activity of Mycobacterium tuberculosis type H37 Rv-Minimum Inhibitory Concentration (MIC)

As can be seen from Table 1, the in vitro MIC of the compound of the present invention shows significantly better in vitro activity than the control drug PA-824. For example, compound 1, compound 13, compound 18, compound 22, compound 23, compound 28, compound 31, and compound 33 had an in vitro activity (MIC) of 0.03125 μ g/mL, which was 4 times as potent as the control drug PA-824 in vitro.

Example 35: in vitro efficacy experiment of part of compounds on drug-resistant mycobacterium tuberculosis strains

Clinical isolates of tested strains (1146-14: streptomycin resistance; 4061-15: isoniazid resistance; 3997-7: rifampicin resistance; B2, MDR-TB; B6, B29 and B53, XDR-TB) mycobacterium tuberculosis are obtained from the clinical separation in the pulmonaceae hospital of Shanghai city. The method comprises the following steps: a. collecting sputum samples of inpatients of tuberculosis in pulmonary hospitals in Shanghai city, treating with alkali, inoculating to improved Roche medium, and culturing for 2 weeks; b. the absolute concentration method is used for measuring drug sensitivity: fresh culture was scraped from the culture medium slant, suspended by grinding with physiological saline to 1 McLeod cell (1mg/mL), diluted to 10-2mg/mL, and 0.1mL was inoculated onto a drug-sensitive medium and observed after four weeks. Reference material: laboratory test protocol for tuberculosis diagnosis, authored by the national institute of advanced technology for tuberculosis diagnosis, published by the national institute of advanced education and culture, 2006, 1 month) into liquid medium, culturing at 37 deg.C for 2 weeks, sucking a little amount of culture broth, placing into 4mL of liquid medium, adding 10-20 particles of sterile glass beads with diameter of 2-3mm, shaking for 20-30S, standing for l0-20 min, sucking supernatant of bacterial suspension, adjusting turbidity to 1 McLee unit with liquid medium, which is equivalent to 1 × 10⁷CFU/mL is ready for use. Each drug was dissolved in an appropriate amount of DMSO to 1mg/mL and filtered through a 0.22 μm filter. Then diluted to the required experimental concentration by liquid culture. The final concentrations of the test drugs were set as follows: 0.0039. mu.g/mL, 0.0078. mu.g/mL, 0.0156. mu.g/mL, 0.03125. mu.g/mL, 0.0625. mu.g/mL, 0.125. mu.g/mL, 0.25. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL, 2. mu.g/mL, 4. mu.g/mL, and in 11 concentration gradient tests, 100. mu.L of each of the above-mentioned drug solutions was added to a 96-well microplate, and 100. mu.L of a 1 mg/mL-concentration bacterial solution was added to bring the drug concentration to the set final concentration, and the culture was carried out at 37 ℃. Three groups of parallel controls were set for the same drug dilution, with the control group containing no drug and the inoculum size set at 100%, 10% and 1%, respectively. Observe the drug pair tuberculosisThe Minimum Inhibitory Concentration (MIC) of Mycobacterium was also compared to the MIC results for PA-824. The results are shown in Table 2.

TABLE 2 in vitro Activity against drug-resistant Mycobacterium tuberculosis-Minimal Inhibitory Concentration (MIC)

S is streptomycin, H is isoniazid, R is rifampicin B2, MDR-TB, B53, B29, B6 is XDR-TB.

As can be seen from Table 2, the compounds of the present invention and the control compounds PA-824 all showed excellent in vitro antibacterial activity against streptomycin-resistant strains, isoniazid-resistant strains, rifampin-resistant strains, multi-resistant strains (B2) and extensively resistant strains (B53, B29 and B6), and also showed that the in vitro activity of the compounds of the present invention against various drug-resistant strains was superior to that of the control PA-824. This shows that the compounds of the present invention, like PA-824, can be used for the treatment of diseases caused by drug-resistant tubercle bacillus, especially multi-drug resistant and extensively drug resistant tubercle bacillus.

Meanwhile, the compound of the invention has the same action mechanism with PA-824, is a brand new action mechanism and has no cross drug resistance with the existing drugs. Therefore, in addition to the compounds listed in Table 2, other compounds described in the present invention have inhibitory activity against H37Rv strain, and also against various drug-resistant strains.

Example 36: in vivo pharmacokinetic experiments with partial Compounds

Test compounds were formulated as a homogeneous suspension at a final concentration of 2mg/mL using 0.5% CMC-Na in water for oral administration. Orally administered by intragastric administration, the single administration dosage is 10mg/kg, and the administration volume is 10mL/kg., and 0.15mL of blood sample is taken after 15min, 30min, 1h, 2h, 4h, 6h, 10h, 12h and 24h of administration through retrobulbar venous plexus of mice.

Preparing a test article with a final concentration of 0.5mg/mL for intravenous administration, wherein the test article preparation solvent is 5% DMSO + 20% EA + 50% PEG400+ 25% Saline (normal Saline) water solution, and the single administration dose is 2 mg/kg.: blood samples were collected 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 24h after administration.

Selecting CD-1 mice, male, the week age at the beginning of administration is 6-8 weeks, the body weight at the beginning of administration is 20-30g, and the number of marking pen is numbered. Animal weights were measured prior to dosing, and healthy animals of similar weights were selected for inclusion in the experiment without random grouping, with all animals drinking water freely during the experiment.

The plasma is collected and placed in a marked centrifugal tube, the plasma is rapidly separated at 3500 rpm/min, 10 min and 4 ℃, and then the plasma is stored below-40 ℃ to be tested. The drug concentration in plasma was measured by LC-MS/MS method and the pharmacokinetic parameters were calculated.

TABLE 3 results of pharmacokinetic experiments in CD-1 mice

As can be seen from the data in Table 3, some of the compounds of the present invention have better pharmacokinetic properties after a single oral administration in mice. Table 3 is representative of only a portion of the compounds, and the remaining compounds may also have superior pharmacokinetic properties.

These results show that the compound of the present invention has excellent drug properties and is likely to be developed into an effective tuberculosis treatment drug. In addition, since the in vitro activity of the partial compound of the present invention is significantly higher than that of the control drug, it is reasonable to believe that the partial compound of the present invention exhibits excellent in vivo efficacy.

Example 37 mouse model of acute infection partial Compounds tested for in vivo efficacy

BALB/c mice, females, weighing approximately 20 grams, were infected with Mycobacterium tuberculosis H37Rv (ATCC strain 27274) by aerosol route using an inhalation exposure system at a dose of approximately 5000 CFU. 5 untreated mice were euthanized on the day of treatment to determine the infection dose. The drugs to be tested were formulated as suspensions using 0.5% w/v carboxymethylcellulose (CMC). Stored at 4 ℃ before use. Control mice were treated with 0.5% CMC only.

Mice were grouped and weighed, 5 mice per group, and gavage was started five days per week, once per day for four weeks. After a 3 day washout period following the last dose, the experimental mice were euthanized, the two lungs were aseptically removed and ground and homogenized in 3mL Hank's Balanced Salt Solution (HBSS). The HBSS solution was ten-fold diluted and cultured on Middlebrook 7H11 agar plates for three weeks, and colony forming units were counted. Results are expressed as mean LogCFU values for each group of mice.

TABLE 4 in vivo efficacy test in H37Rv acutely infected BALB/c female mice

a 5 non-dosed mice were euthanized at day 24 post-infection due to a fulminant infection.

For H37Rv acutely infected BALB/c female mice, none of the mice in three groups, PA-824, Compound 1 and Compound 2, died after the dosing was completed. As can be seen from Table 4, the CFU value of each dose group at the end of the administration of compound 2 was lower than that of the control PA-824, and was significantly better than that of the control drug, especially in the 30mg/kg dose group, compound 2 had significant bactericidal effect on tubercle bacillus, with a CFU decrease of 0.73 log (compared with 0 day), and a CFU value increase of PA-824 (compared with 0 day). Compound 1 decreased CFU by 0.43 log units over PA-824 in the 100mg/kg dose group.

The data show that the compound of the invention has more excellent in-vivo bactericidal activity compared with PA-824, and particularly the advantage of the compound 2 is more obvious at medium dosage, namely, the compound with lower dosage can play a better treatment effect and simultaneously reduce side effects.

Example 38 inhibition of hERG Potassium ion channel by Compounds

HEK-293 cells stably expressing hERG (CreacellTM, France) were used to record hERG potassium channel currents using whole-cell patch-clamp technology at room temperature. The whole-cell patch clamp voltage stimulation protocol for recording whole-cell hERG potassium current was as follows: after forming a whole cell seal, the cell membrane voltage was clamped at-80 mV. The clamping voltage is depolarized from-80 mV to-50 mV for 0.5s (measured as leakage current), then stepped to 30mV for 2.5s, and then rapidly returned to-50 mV for 4s to excite the tail current of the hERG channel. Data were collected repeatedly every 10s and the effect of the drug on the hERG tail current was observed. Leakage current was measured at-50 mV stimulus of 0.5 s. Experimental data were collected from EPC-10 amplifiers (HEKA) and stored in PatchMaster (HEKA) software.

And (3) data analysis: the current after each drug concentration was first normalized to the blank current

Then calculating the inhibition rate corresponding to each drug concentration

The mean and standard error were calculated for each concentration and the half inhibitory concentration for each compound was calculated using the following equation:

the dose-dependent effect was fitted non-linearly using the above equation, where C represents drug concentration, IC50 is the half inhibitory concentration, and h represents the hill coefficient. Curve fitting and calculation of IC50 were done using IGOR software.

Sample preparation: 1. preparing a weighed test sample into a stock solution with a corresponding concentration by using DMSO (dimethyl sulfoxide); 2. sequentially diluting the stock solution of the test sample into 0.5mM, 2mM and 8mM of diluent by using DMSO; 3. diluting the sample diluent with extracellular fluid to obtain liquid of 0.5 μ M, 2 μ M, 8 μ M and 32 μ M, and subjecting to ultrasonic treatment for 20 min; 4. the solubility of the sample to be tested was visually and microscopically examined and then tested.

Table 5 inhibition of hERG by part of the compounds:

compound (I)	IC₅₀(μM)
		Compound 2	>32
PA-824	5.8

Table 5 shows that the compound of the invention has no inhibition on the hERG potassium current, which indicates that the compound of the invention has good safety on the cardiovascular system and the safety is better than that of a control medicament (PA-824).

The foregoing is merely a preferred embodiment of the invention and is not intended to limit the scope of the invention, which is defined by the claims appended hereto, and any other technical entity or method that is encompassed by the claims as broadly defined herein, or equivalent variations thereof, is contemplated as being encompassed by the claims.

Claims

1. An anti-tuberculosis compound, which is a compound represented by formula (I) or an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

in formula (I): m, n represent an integer between 0 and 4;

x is oxygen or NH;

R¹selected from hydrogen, alkyl, cycloalkyl, or cycloalkylalkyl, said alkyl, cycloalkyl, or cycloalkylalkyl being unsubstituted or optionally substituted with one to three substituents independently selected from halogen, alkylSubstituted by groups;

2. The anti-tuberculosis compound of claim 1, wherein R¹Is hydrogen or C_1-4Alkyl radical, said C_1-4Alkyl is unsubstituted or optionally substituted with one to three halogens.

3. The anti-tuberculosis compound of claim 1, wherein R²Is C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkylthio, halogen, or cyano, said C_1-4Alkyl radical, C_1-4Alkoxy, or C_1-4Alkylthio is unsubstituted or optionally substituted with one to three halogen.

4. The anti-tuberculosis compound of claim 1, wherein the pharmaceutically acceptable salt comprises: a salt of a compound represented by the general formula (I) with an acid; wherein the acid comprises: inorganic acids, organic acids or acidic amino acids; the inorganic acid includes: hydrochloric, hydrobromic, hydrofluoric, sulfuric, nitric or phosphoric acids; the organic acid includes: formic acid, acetic acid, propionic acid, oxalic acid, trifluoroacetic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, p-toluenesulfonic acid, ethanesulfonic acid, or benzenesulfonic acid; the acidic amino acids include: aspartic acid or glutamic acid.

5. The anti-tuberculosis compound according to claim 1, wherein the compound is a compound of formula 1, a compound of formula 2, a compound of formula 3, a compound of formula 4, a compound of formula 5, a compound of formula 6, a compound of formula 7, a compound of formula 8, a compound of formula 9, a compound of formula 10, a compound of formula 11, a compound of formula 12, a compound of formula 13, a compound of formula 14, a compound of formula 15, a compound of formula 16, a compound of formula 17, a compound of formula 18, a compound of formula 19, a compound of formula 20, a compound of formula 21, a compound of formula 22, a compound of formula 23, a compound of formula 24, a compound of formula 25, a compound of formula 26, a compound of formula 27, a compound of formula 28, a compound of formula 29, a compound of formula 30, a compound of formula 31, a compound of formula 32, a compound of formula 33:

6. a method for preparing the anti-tubercular compound according to claim 1, comprising the steps of:

(2) chlorinating the compound with the structure shown as the formula I-6 to obtain a compound with the structure shown as the formula I-7;

m，n，R¹and R²The definition of (1) is as before; x is oxygen.

7. A method for preparing the anti-tubercular compound according to claim 1, comprising the steps of: mixing a compound with a structure shown as a formula I-5 and a compound with a structure shown as a formula II-1, and reacting in the presence of a reducing agent to obtain a compound with a structure shown as a formula I;

m，n，R¹and R²The definition of (1) is as before; x is NH.

8. The process according to claim 6 or 7, wherein the compound of formula I-5 is obtained by the following steps:

(a) carrying out coupling reaction on a compound with a structure shown as a formula I-3 and tri-n-butylvinyl tin to obtain a compound with a structure shown as a formula I-4;

9. use of an anti-tubercular compound according to any one of claims 1 to 5 for the manufacture of a medicament for the treatment of a disease associated with an infection caused by mycobacterium tuberculosis; preferably preparing the medicament for treating infectious diseases caused by multi-drug resistant mycobacterium tuberculosis.

10. A pharmaceutical composition for the treatment of diseases associated with infection by mycobacterium tuberculosis comprising a therapeutically effective amount of an anti-tubercular compound according to any one of claims 1 to 5 and a pharmaceutically acceptable excipient or carrier.