A phenoxazine compound and use thereof

Abstract

The application provides a phenoxazine compound and application thereof. The phenoxazine compound is shown as formula (I). The phenoxazine compound of the application has higher in-vitro safety and excellent in-vitro broad-spectrum activity on mycobacteria.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular, the present application relates to a phenoxazine compound and application thereof. BACKGROUND

[0002] Mycobacterium tuberculosis (M.tuberculosis, MTB) and Nontuberculous mycobacteria (NTM) belong to the same genus of Mycobacterium. The clinical symptoms and chest images of MTB and NTM infections are very similar. In the absence of strain identification, NTM infection is often misdiagnosed as TB in clinic, and treated as TB. However, due to the fact that traditional anti-TB drugs have little or no activity against most NTM, when the clinical effect is poor or even the disease worsens, it is mistakenly believed to be drug-resistant TB and continues to be treated. Unfortunately, so far there is no specific and efficient anti-NTM (especially drug-resistant NTM) drug available in clinic. In recent years, NTM infection has become a thorny clinical problem, and the incidence of NTM even exceeds that of TB in many countries. NTM infection has become another major challenge to global public health security after TB. Therefore, it is urgent to develop new anti-MTB and NTM drugs with novel mechanisms of action to achieve effective treatment and control of MTB and NTM.

[0003] 2-amino-phenoxazin-3-one (questiomycin, as shown in the following structure) is widely present in nature, but its anti-mycobacterial activity has not been reported. Through the screening of compounds, the present application found that phenoxazine has anti-MTB activity. Further, through the structural modification of the benzene ring part, a series of phenoxazine compounds were designed and synthesized, and some of the compounds showed better anti-MTB activity than questiomycin. At the same time, some of the compounds also showed better anti-NTM activity than questiomycin. At the same time, the compounds showed higher in vitro safety.

[0004] SUMMARY

[0005] One object of the present application is to provide a phenoxazine compound, a stereoisomer, a deuterated compound or a pharmaceutically acceptable salt thereof;

[0006] Another object of the present application is to provide a pharmaceutical composition;

[0007] Still another object of the present application is to provide the pharmaceutical use of the phenoxazine compound, the stereoisomer, the deuterated compound or the pharmaceutically acceptable salt thereof.

[0008] To achieve the above object, in one aspect, the present application provides a phenoxazine compound, a stereoisomer, a deuterated compound or a pharmaceutically acceptable salt thereof, wherein the phenoxazine compound has a structure as shown in formula (I):

[0009]

[0010] wherein,

[0011] R1 is H, F, Cl, Br, I, hydroxyl, substituted or unsubstituted C 1-4 linear or branched alkyl, substituted or unsubstituted C 1-4 linear or branched alkoxy, COOH, COOR3, CONHR3, or 3- to 12-membered saturated or unsaturated heterocyclyl; when substituted, the alkyl or alkoxy alkyl is substituted with 1, 2, or 3 substituents selected from F, Cl, Br, I, hydroxyl, C 1-4 linear or branched alkyl or 3- to 12-membered saturated or unsaturated heterocyclyl; optionally, the heterocyclyl is substituted with 1, 2, or 3 substituents selected from F, Cl, Br, or I; the heterocyclyl contains 1, 2, or 3 heteroatoms selected from N, O, or S;

[0012] R2 is H, F, Cl, Br, I, or substituted or unsubstituted C 1-4 linear or branched alkyl; when substituted, the alkyl is substituted with 1, 2, or 3 substituents selected from F, Cl, Br, I, or C 1-4 alkyl;

[0013] R3 is substituted or unsubstituted C 1-4 linear or branched alkyl, or 3- to 8-membered heterocyclyl, wherein, when substituted, the alkyl is substituted with 1, 2, or 3 substituents selected from F, Cl, Br, I, hydroxyl, carboxyl, or C 1-4 linear or branched alkyl; the heterocyclyl contains 1, 2, or 3 heteroatoms selected from N, O, or S.

[0014] According to some embodiments of the present application, wherein,

[0015] R1 is H, F, Cl, Br, I, hydroxyl, substituted or unsubstituted C 1-4 linear or branched alkyl, substituted or unsubstituted C 1-4 linear or branched alkoxy, COOH, or COOR3, CONHR3, or 3- to 10-membered saturated or unsaturated heterocyclyl; when substituted, the alkyl or alkoxy alkyl is substituted with 1, 2, or 3 substituents selected from F, Cl, Br, I, C 1-4straight or branched alkyl; when substituted, said alkyl is substituted with 1, 2, or 3 substituents selected from the group consisting of F, Cl, Br, or I;

[0016] R2is H, F, Cl, Br, I, or substituted or unsubstituted C 1-4 straight or branched alkyl; when substituted, said alkyl is substituted with 1, 2, or 3 substituents selected from the group consisting of F, Cl, Br, or I;

[0017] R3is substituted or unsubstituted C 1-4 straight or branched alkyl, or a 3- to 8-membered heterocycle, wherein, when substituted, said alkyl is substituted with 1, 2, or 3 substituents selected from the group consisting of F, Cl, Br, I, or carboxyl.

[0018] According to some embodiments of the application, wherein,

[0019] R1is H, F, Cl, Br, I, hydroxyl, substituted or unsubstituted C 1-4 straight or branched alkyl, substituted or unsubstituted C 1-4 straight or branched alkoxy, COOH or COOR3, CONHR3, or a 3- to 8-membered saturated or unsaturated heterocycle; when substituted, said alkyl or alkoxy alkyl is substituted with 1, 2, or 3 substituents selected from the group consisting of F, Cl, Br, I, C 1-4 straight or branched alkyl or a 3- to 10-membered saturated or unsaturated heterocycle; optionally, said heterocycle is substituted with 1, 2, or 3 substituents selected from the group consisting of F, Cl, Br, or I; said heterocycle contains 1 or 2 heteroatoms selected from the group consisting of N, O, or S;

[0020] R2is H, F, Cl, Br, I, or substituted or unsubstituted C 1-4 straight or branched alkyl; when substituted, said alkyl is substituted with 1, 2, or 3 substituents selected from the group consisting of F, Cl, Br, or I;

[0021] R3is substituted or unsubstituted C 1-4 straight or branched alkyl, or a 3- to 8-membered heterocycle, wherein, when substituted, said alkyl is substituted with 1, 2, or 3 substituents selected from the group consisting of F, Cl, Br, I, or carboxyl.

[0022] According to some embodiments of the application, wherein,

[0023] when R1is C 1-4When R1is C1-C6alkyl, the 3- to 10-membered saturated or unsaturated heterocyclyl is selected from 3- to 6-membered mono-heterocyclyl, 5-, 6-, 7-, 8-, 9-, or 10-membered heterospirocyclyl, 7-, 8-, 9-, or 10-membered annulated heterocyclyl.

[0024] According to some embodiments of the present application, wherein,

[0025] When R1is C1-C6alkyl, the 3- to 10-membered saturated or unsaturated heterocyclyl is selected from 3- to 6-membered mono-heterocyclyl, 5-, 6-, 7-, 8-, 9-, or 10-membered heterospirocyclyl, 7-, 8-, 9-, or 10-membered annulated heterocyclyl. 1-4 When R1is C1-C6alkyl, the 3- to 10-membered saturated or unsaturated heterocyclyl is selected from 3- to 6-membered mono-heterocyclyl, 5-, 6-, 7-, 8-, 9-, or 10-membered heterospirocyclyl, 7-, 8-, 9-, or 10-membered annulated heterocyclyl.

[0026] According to some embodiments of the present application, wherein, when R1is C1-C6alkyl, the 3- to 10-membered saturated or unsaturated heterocyclyl is selected from 3- to 6-membered mono-heterocyclyl, 5-, 6-, 7-, 8-, 9-, or 10-membered heterospirocyclyl, 7-, 8-, 9-, or 10-membered annulated heterocyclyl. 1-4 When R1is C1-C6alkyl, the 3- to 10-membered saturated or unsaturated heterocyclyl is selected from 3- to 6-membered mono-heterocyclyl, 5-, 6-, 7-, 8-, 9-, or 10-membered heterospirocyclyl, 7-, 8-, 9-, or 10-membered annulated heterocyclyl.

[0027] The mono-heterocyclylalkyl is selected from:

[0028] The mono-heterocyclylalkyl is selected from:

[0029] The annulated heterocyclyl is selected from:

[0030] The annulated heterocyclyl is selected from:

[0031] According to some embodiments of the present application, wherein, the mono-heterocyclyl is selected from one of the following structures:

[0032]

[0033] wherein X1and X2are each independently selected from N, O, or S.

[0034] According to some embodiments of the present application, wherein, X1is N; and X2is N, O, or S.

[0035] According to some embodiments of the present application, wherein, the mono-heterocyclyl is selected from one of the following structures:

[0036]

[0037] According to some embodiments of the present application, wherein, the annulated heterocyclyl is selected from one of the following structures:

[0038]

[0039] According to some embodiments of the application, wherein said fused heterocyclic ring is selected from one of the following structures:

[0040]

[0041] According to some embodiments of the application, wherein,

[0042] R1is H, F, Cl, Br, I, hydroxyl, substituted or unsubstituted C 1-4 straight or branched chain alkyl, substituted or unsubstituted C 1-4 straight or branched chain alkoxy, COOH or COOR3, CONHR3, or 4- to 6- membered saturated or unsaturated heterocyclic ring;

[0043] R2is H, F, Cl, Br, I, or substituted or unsubstituted alkyl, methyl, ethyl, n- propyl, iso-propyl, n-butyl, iso-butyl, t-butyl or sec-butyl;

[0044] R3is substituted or unsubstituted C 1-4 straight or branched chain alkyl, or 3- to 6-membered heterocyclic ring; said heterocyclic ring containing 1 or 2 N heteroatoms.

[0045] According to some embodiments of the application, wherein,

[0046] R1is H, F, Cl, Br, I, substituted or unsubstituted C 1-2 straight chain alkyl, CONHR3, or 4- to 6- membered saturated or unsaturated heterocyclic ring; when substituted, said alkyl or alkoxy alkyl is substituted with 1, 2, or 3 substituents selected from F, Cl, Br, or I; said heterocyclic ring containing 1 or 2 heteroatoms selected from N, O or S;

[0047] R2is H, F, Cl, Br, I, or substituted or unsubstituted alkyl, methyl, ethyl, n- propyl; when substituted, said alkyl is substituted with 1, 2, or 3 substituents selected from F, Cl, Br, or I;

[0048] R3is substituted or unsubstituted C1, C2, or C3straight chain alkyl; when substituted, said alkyl is substituted with 1, or 2 substituents selected from F, Cl, Br, I or carboxyl.

[0049] According to some embodiments of the application, wherein, when R1is a 4- to 6- membered saturated or unsaturated heterocyclic ring, said heterocyclic ring is selected from one of the following heterocyclic rings:

[0050] pyrrolyl, thienyl, furanyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, isoxazolyl, oxazolyl, tetrahydrofuranyl, piperidinyl, tetrahydropiperidinyl, morpholinyl, piperazinyl, thiomorpholinyl, pyridinyl, pyridazinyl, pyrimidinyl, or pyrazinyl.

[0051] According to some embodiments of the present application, when R1is a 4- to 6- membered saturated or unsaturated heterocyclyl, the 4- to 6- membered saturated or unsaturated heterocyclyl structure is as follows:

[0052]

[0053] wherein X3and X4are each independently selected from N, O, or S.

[0054] According to some embodiments of the present application, X3is N; and X4is N, O, or S.

[0055] According to some embodiments of the present application, the 4- to 6- membered saturated or unsaturated heterocyclyl structure is as follows:

[0056]

[0057] According to some embodiments of the present application, the phenoxazine compound is selected from one of the following structures:

[0058]

[0059] R1is F, Cl, Br, I, or a 4- to 6- membered saturated heterocyclyl; the heterocyclyl contains 1 or 2 heteroatoms selected from N, O, or S;

[0060] R2is H, F, Cl, Br, or I.

[0061] According to some embodiments of the present application, the phenoxazine compound is selected from one of the following structures:

[0062]

[0063] In another aspect, the present application also provides a method for preparing the phenoxazine compound, the method comprising preparing the phenoxazine compound of formula (I) using compound (III) and compound (IV) as raw materials:

[0064]

[0065] According to some embodiments of the present application, the compound of formula (III) and the compound of formula (IV) are reacted at 10-40 °C for 24-48 h to obtain the phenoxazine compound of formula (I).

[0066] According to some embodiments of the present application, the compound of formula (III) and the compound of formula (IV) are reacted in an oxygen atmosphere.

[0067] According to some embodiments of the present application, the method comprises dissolving the compound of formula (III) and the compound of formula (IV) in organic solvents respectively, and then slowly adding the solution containing the compound of formula (III) into the solution containing the compound of formula (IV).

[0068] According to some embodiments of the present application, the organic solvent is a mixed solution of an alkyl alcohol with 1-4 carbon atoms and water, and the volume ratio of the two is 1:3 to 3:1.

[0069] According to some embodiments of the present application, the organic solvent is a mixed solution of methanol and water.

[0070] According to some embodiments of the present application, the volume ratio of the alkyl alcohol and water is 1:2 to 2:1; preferably 1:1.

[0071] In another aspect, the present application also provides a pharmaceutical composition, wherein the pharmaceutical composition contains the phenoxazine compound, the stereoisomer, the deuterated compound or the pharmaceutically acceptable salt thereof according to any one of the present application, and a pharmaceutically acceptable carrier.

[0072] According to some embodiments of the present application, the weight percentage of the phenoxazine compound, the stereoisomer, the deuterated compound or the pharmaceutically acceptable salt thereof in the composition is 0.1%-99.9%.

[0073] The pharmaceutical composition of the present application can be prepared into any pharmaceutically acceptable dosage form. Preferably, the pharmaceutical preparation is a tablet, a sugar-coated tablet, a film-coated tablet, an enteric-coated tablet, a sustained-release tablet, a capsule, a hard capsule, a soft capsule, a sustained-release capsule, a powder.

[0074] The pharmaceutical composition of the present application, as a preparation form, contains the effective amount of the phenoxazine compound, the stereoisomer, the deuterated compound or the pharmaceutically acceptable salt thereof according to the present application in each dose, which is 0.1-1000 mg.

[0075] The "each dose" refers to each preparation unit, such as each tablet, each capsule, and also refers to each dose taken, such as 100 mg each time.

[0076] The pharmaceutical composition of the present application, when prepared into a solid pharmaceutical preparation in the form of a powder, tablet, dispersible powder, capsule, or sachet, can use a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier that can be used is preferably one or more substances selected from the group consisting of diluents, flavoring agents, solubilizing agents, lubricants, suspending agents, binders, bulking agents, and the like, or can be an encapsulating material.

[0077] Suitable pharmaceutically acceptable carriers include magnesium carbonate, magnesium stearate, talc, sucrose, lactose, pectin, dextrin, starch, gelatin, methylcellulose, sodium carboxymethylcellulose, cocoa butter, and the like.

[0078] Because of their ease of administration, tablets, powders, sachets, and capsules represent the most advantageous oral dosage unit forms. In order to facilitate the administration of a precise dosage, the pharmaceutical preparations described above are advantageously formulated in the form of dosage units.

[0079] The dosage unit form of the preparation refers to a physically discrete unit suitable for administration as a single dose, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect. Such dosage unit forms can be packaged in a variety of ways, such as in tablets, capsules, or in powders packed in vials or ampoules.

[0080] While the quantity of active ingredient contained in the dosage unit form can vary, it is generally adjusted to produce a dosage of between 1 and 800 mg, depending on the potency of the active ingredient selected.

[0081] In another aspect, the present application also provides use of the phenoxazine compound, stereoisomer, deuterated product, or pharmaceutically acceptable salt thereof according to any one of the present application, or the pharmaceutical composition according to the present application in the preparation of a medicament for treating mycobacterial infection.

[0082] According to some embodiments of the present application, the mycobacterium is selected from Mycobacterium tuberculosis or non-tuberculous mycobacteria.

[0083] According to some embodiments of the present application, the non-tuberculous mycobacteria is selected from Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, or Mycobacterium kansasii.

[0084] In the treatment of mycobacterial infection, a dose of 6 to 14 mg / kg of body weight is administered in the first stage. However, the dose administered can vary depending on the patient's needs, the severity of the infection to be treated, the selected compound, and the like.

[0085] The preferred dose suitable for a given situation can be determined by a person skilled in the art in a routine manner. Generally, the amount at which treatment is initiated is lower than the optimal dose of the active ingredient, and the dose administered is then gradually increased until the optimal therapeutic effect is achieved. For the sake of convenience, the total daily dose can be divided into several parts, administered in fractions at several times.

[0086] In the absence of a specific definition in the present invention, the following terms are explained as follows (the following explanations are only general examples and not a limitation of the present invention):

[0087] "Substituted" means that a hydrogen atom on a carbon atom or a heteroatom is replaced by one or more defined substituents, the upper limit of the number of substituents being equal to the sum of the number of hydrogens that can be replaced by the substituents, and in general, the number of substituents is any integer between 1 and the upper limit, and when the number of halogen substituents is greater than 1, the substituents can be the same or different.

[0088] "Deuterated" means that a hydrogen atom on a group containing hydrogen atoms is replaced by at least one deuterium, the upper limit of the number of deuteriums being equal to the sum of the number of hydrogens that can be replaced by the deuteriums, and in general, the number of deuteriums is any integer between 1 and the upper limit, preferably 1 to 20 deuterium atoms are replaced, more preferably 1 to 10 deuterium atoms are replaced, still more preferably 1 to 5 deuterium atoms are replaced, further more preferably 1 to 3 deuterium atoms are replaced.

[0089] "Alkyl" means a monovalent linear or branched saturated aliphatic hydrocarbon group, and in general, an alkyl group of 1 to 20 carbon atoms, preferably an alkyl group of 1 to 10 carbon atoms, more preferably an alkyl group of 1 to 5 carbon atoms, further more preferably an alkyl group of 1 to 3 carbon atoms, further more preferably an alkyl group of 1 to 2 carbon atoms. For example, it includes methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, neopentyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and various branched isomers thereof.

[0090] "Heterocycle", "heterocyclyl" means a substituted or unsubstituted, saturated or unsaturated, aromatic or non-aromatic ring containing heteroatoms, typically containing 1 to 4 heteroatoms selected from N, O or S, including monocyclic heterocycles, bicyclic bridged heterocycles, bicyclic fused heterocycles and bicyclic spiro heterocycles, typically 3 to 14 membered heterocycles, preferably 4-12 membered heterocycles, more preferably 4-10 membered heterocycles, further preferably 5-7 membered heterocycles. Heterocyclyl groups can be attached at a heteroatom or a carbon atom, such as epoxyl, aziridinyl, oxetanyl, azetidinyl, 1,3-dioxolanyl, 1,4-dioxolanyl, 1,3-dioxanyl, azepanyl, pyridyl, furanyl, thienyl, pyranyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyridazinyl, imidazolyl, piperidinyl, morpholinyl, thiomorpholinyl, 1,3-dithianyl, dihydrofuranyl, dihydropyranyl, dihydrothiopyranyl, tetrahydrofuranyl, tetrahydropyrrolyl, tetrahydroimidazolyl, oxazolyl, dihydrooxazolyl, tetrahydrooxazolyl, tetrahydrothiazolyl, tetrahydropyranyl, benzimidazolyl, benzopyridyl, pyrrolopyridyl, benzodihydrofuranyl, azabicyclo[3.2.1]octanyl, azabicyclo[5.2.0]nonanyl, oxatricyclo[5.3.1.1]dodecanyl, azadamantyl and oxaspiro[3.3]heptanyl, and the like.

[0091] "Alkoxy" means -O-alkyl, which is -O-C 1-8 alkyl, preferably -O-C 1-6 alkyl, more preferably -O-C 1-4 alkyl, further preferably -O-C 1-2 alkyl, unless otherwise specified. For example, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, sec-butoxy, t-butoxy, n-pentoxy, n-hexoxy, cyclopropoxy and cyclobutoxy, and the like.

[0092] In summary, the present application provides a phenoxazine compound and its application. The compound of the present application has the following advantages:

[0093] The phenoxazine compound of the present application shows higher in vitro safety and excellent in vitro broad-spectrum activity against mycobacteria. DETAILED DESCRIPTION

[0094] The following detailed description of the implementation process and the beneficial effects of the present application are intended to help the reader better understand the essence and characteristics of the present application, and are not intended to limit the scope of the present application.

[0095] Example 1, 2-amino-6-fluoro-3H-phenoxazin-3-one

[0096]

[0097] 2-amino-6-fluorophenol (500 mg, 3.93 mmol) was dissolved in a mixture of 15 ml methanol and 15 ml water, then o-aminophenol (429 mg, 3.93 mmol) was dissolved in a mixture of 15 ml methanol and 15 ml water and added slowly to the above solution and stirred for 24 hours at room temperature under oxygen atmosphere. Work-up: concentrated, preparative column separation (water (0.225% formic acid) - acetonitrile) to give a red solid. (Yield: 10.27%).

[0098] 1 H NMR: (400 MHz, DMSO-d6) δ 7.52 (br d, J = 8.0 Hz, 1H), 7.45-7.30 (m, 2H), 7.08-6.84 (s, 2H), 6.52-6.29 (m, 2H). MS-ESI (m / z): 231.2 (M+H) + .

[0099] Example 2, 2-amino-7-fluoro-3H-phenoxazin-3-one

[0100]

[0101] Prepared as in example 1, 2-amino-5-fluorophenol reacted with o-aminophenol to give a yellow solid.

[0102] 1H NMR (400 MHz, DMSO-d6) δ 6.36 (d, J = 10.88 Hz, 2H) 6.65-6.97 (m, 2H) 7.28 (td, J = 8.76, 2.75 Hz, 1H) 7.49 (dd, J = 9.19, 2.69 Hz, 1H) 7.75 (dd, J = 8.88, 6.13 Hz, 1H). MS-ESI (m / z): 231.2 (M+H) + .

[0103] Example 3, 2-amino-8-fluoro-3H-phenoxazin-3-one

[0104]

[0105] Prepared as in example 1, 2-amino-4-fluorophenol reacted with o-aminophenol to give a yellow solid.

[0106] 1H NMR (400 MHz, DMSO-d6) δ 6.36 (d, J = 7.00 Hz, 2H) 6.79 - 7.17 (m, 2H) 7.33 (td, J = 8.66, 3.06 Hz, 1H) 7.50 - 7.60 (m, 2H). MS-ESI (m / z): 231.2 (M+H) + .

[0107] Example 4, 2-amino-9-fluoro-3H-phenoxazin-3-one

[0108]

[0109] Preparation method is the same as example 1, 2-amino-3-fluorophenol reacts with o-aminophenol to produce yellow solid.

[0110] 1 H NMR (400 MHz, DMSO-d6) δ 6.39 (d, J = 5.00 Hz, 2H) 6.80 - 7.09 (m, 2H) 7.27 (ddd, J = 9.94, 8.38, 1.19 Hz, 1H) 7.32 - 7.37 (m, 1H) 7.40 - 7.48 (m, 1H). ESI (m / z): 231.1 (M+H) + .

[0111] Example 5, 2-amino-6-hydroxy-3H-phenoxazin-3-one

[0112]

[0113] 2-amino phenol (5.80 g, 53.19 mmol) was dissolved in a mixture of methanol (70 mL) and water (70 mL), then 2-amino-6-bromo-phenol (10 g, 53.19 mmol) was added and stirring was continued for 72 hours at 25 °C under oxygen atmosphere (15 psi). Work-up: the reaction was concentrated, then ethyl acetate (100 mL) was added, washed with water, the organic phase was concentrated to produce red solid compound 2 (5 g, 32.30%).

[0114] Compound 2 was protected by Boc and dissolved in dioxane solution (8 mL), then Pd(dppf)Cl2(42 mg, 0.06 mmol), bis(pinacolato)diboron (321 mg, 1.27 mmol) and potassium acetate (339 mg, 3.45 mmol) were added to the system. Stirring was continued for 2 hours at 80 °C under nitrogen protection. Work-up: the reaction was concentrated, the concentrated solution was dissolved in ethyl acetate and washed with water, the organic phase was concentrated and column chromatography (SiO2, PE:EtOAc = 10:1 to 3:1) to produce yellow solid intermediate 4 (120 mg, yield 23.80%).

[0115] Compound 4 was dissolved in dioxane solution (1 mL), then 1 mL water and oxone (131 mg, 0.21 mmol) were added under ice bath condition. Stirring at 25 °C for 4 h. Work-up: sodium thiosulfate (30 mg) was added to the reaction solution and stirred for 30 min, then the reaction solution was concentrated, the concentrated solution was dissolved in ethyl acetate and washed with water, the organic phase was prepared to give yellow solid 5 (30 mg, 47.11 %), compound 5 was deprotected to give red solid product (12 mg, 86.32 %).

[0116] 1 H NMR: (400 MHz, DMSO-d6) δ 8.23-8.08 (m, 1H), 7.23-7.13 (m, 2H), 6.98 (dd, J = 2.4, 6.8 Hz, 1H), 6.73 (s, 2H), 6.41-6.24 (m, 2H). MS-ESI (m / z): 229.1 (M+H) + .

[0117] Example 6, 2-amino-6-methyl-3H-phenoxazin-3-one

[0118]

[0119] Preparation method is the same as example 1, 2-amino-6-methylphenol reacts with o-aminophenol to produce yellow solid.

[0120] 1 H NMR: (400 MHz, DMSO-d6) δ 7.67 (br d, J = 7.8 Hz, 1H), 7.57-7.48 (m, 1H), 7.44 (br t, J = 7.6 Hz, 1H), 7.40-7.32 (m, 1H), 6.86 (s, 2H), 6.31 (s, 1H), 2.08 (s, 3H). MS-ESI (m / z): 227.2 (M+H) + .

[0121] Example 7, methyl-2-amino-3-oxo-3H-phenoxazine-8-carboxylate

[0122]

[0123] Preparation method is the same as example 1, methyl-3-amino-4-hydroxybenzoate reacts with o-aminophenol to produce yellow solid.

[0124] 1H NMR (400 MHz, DMSO-d6) δ 7.77 (dd, J = 7.94, 1.19 Hz, 1H), 7.44 - 7.53 (m, 2H), 7.37 - 7.43 (m, 1H), 6.36 - 6.69 (m, 1H), 6.31 (s, 1H), 2.23 (s, 3H). MS-ESI (m / z): 227.1 (M+H) + .

[0125] Example 8, 2-Amino-l-methyl-3H-phenoxazin-3-one

[0126]

[0127] Prepared according to the procedure of Example 1 by reacting 2-amino-3- methylphenol with o-aminophenol to give a yellow solid.

[0128] 1 H NMR (400 MHz, DMSO-d6) δ 7.77 (dd, J = 7.94, 1.19 Hz, 1H), 7.44 - 7.53 (m, 2H), 7.37 - 7.43 (m, 1H), 6.36 - 6.69 (m, 1H), 6.31 (s, 1H), 2.23 (s, 3H). MS-ESI (m / z): 227.1 (M+H) + .

[0129] Example 9, 2-Amino-l,9-difluoro-3H-phenoxazin-3-one

[0130]

[0131] Prepared according to the procedure of Example 1 by reacting 2-amino-3- methylphenol with o-aminophenol to give a yellow solid.

[0132] 1 H NMR (400 MHz, DMSO-d6) δ 7.77 (dd, J = 7.94, 1.19 Hz, 1H), 7.44 - 7.53 (m, 2H), 7.37 - 7.43 (m, 1H), 6.36 - 6.69 (m, 1H), 6.31 (s, 1H), 2.23 (s, 3H). MS-ESI (m / z): 227.1 (M+H) + .

[0133] Example 10, 2-Amino-6-morpholinomethyl-3H-phenoxazin-3-one

[0134]

[0135] Compound 1A (2.00 g, 11.9 mmol) and morpholine (1.56 g, 17.9 mmol) were dissolved in EtOH (20 mL), then sodium borohydride (5.07 g, 23.9 mmol) was added portionwise, and the reaction was allowed to proceed at 20 °C for 2 h. Work-up: the reaction was quenched with H2O (20 mL) and extracted with ethyl acetate (20 mL x 3), and the organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and subjected to column chromatography (SiO2, Petroleum ether / Ethyl acetate = 1 / 0 to 2 / 1) to give white intermediate 2A (2.10 g, 8.60 mmol, 71.8% yield, 97.5% purity).

[0136] Compound 2A (1.1 g, 4.62 mmol) was dissolved in a mixture of EtOH (9 mL) and H2O (3 mL), then NH4Cl (1.23 g, 23.1 mmol) was added portionwise, and Fe (1.29 g, 23.0 mmol) was added portionwise under nitrogen protection, and the reaction was allowed to proceed at 80 °C for 2 h. Work-up: cooled to room temperature, filtered, and the filtrate was concentrated and subjected to column chromatography (SiO2, Petroleum ether / Ethyl acetate = 1 / 0 to 1 / 3) to give white solid 3A (400 mg, 1.92 mmol, 41.6% yield).

[0137] Referring to Example 1, compound 2A was reacted with o-aminophenol to give the target product

[0138] 1 H NMR (400 MHz, DMSO-d6) δ 7.80 (t, J = 4.0 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.47 (t, J = 7.8 Hz, 1H), 6.54 (s, 1H), 6.37 (s, 1H), 4.59 (s, 2H), 3.98 (d, J = 8.8 Hz, 2H), 3.65-3.60 (m, 2H), 3.44 (d, J = 11.2 Hz, 2H), 3.27 (t, J = 6.8 Hz, 2H). MS-ESI (m / z): 312.1 (M+H) + .

[0139] Example 11, 2-amino-6-piperazinylmethyl-3H-phenoxazin-3-one

[0140]

[0141] Referring to Example 10, compound 1A was reacted with N-Boc piperazine, reduced by Fe / ammonium chloride, reacted with o-aminophenol, and de-Boc by TFA to give the target product.

[0142] 1 H NMR (400 MHz, DMSO-d6) δ 8.63 (br s, 2H), 7.70 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 7.2 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 6.90 (br s, 1H), 6.44 (s, 1H), 6.37 (s, 1H), 4.08 (s, 2H), 3.17 (s, 4H), 2.89 (s, 4H). MS-ESI (m / z): 311.1 (M+H) + .

[0143] Example 12, 2-Amino-6-morpholino-3H-phenoxazin-3-one

[0144]

[0145]

[0146] Pd(OAc)2(483 mg, 2.15 mmol), BINAP (2.01 g, 3.23 mmol) and Cs2CO3(14.0 g, 43.1 mmol) were dissolved in THF (50 mL) under N2protection at room temperature, then compound T-l (5.00 g, 21.5 mmol) and morpholine (5.63 g, 64.6 mmol) were added, and the mixture was reacted at 70 °C for 12 hours. Post-treatment: cooled to room temperature, diluted with water, extracted with ethyl acetate (30 mL x 3). The organic layer was concentrated and column chromatography (SiO2, Petroleum ether / Ethyl acetate = 1 / 0 to 9 / 1) to give T-2 (5.00 g, 17.4 mmol, 40.4% yield, 83% purity) as a light yellow solid.

[0147] The above solid (5.00 g, 21.0 mmol) was dissolved in dichloromethane (50 mL), and BBr3(21.0 g, 83.9 mmol) was added dropwise under ice bath, and the mixture was reacted under ice bath for 1 hour. Post-treatment: the reaction was quenched by adding ice water (30 mL) into the solution, and the pH was adjusted to 8 by dissolving saturated sodium bicarbonate solution, extracted with ethyl acetate (30 mL x 3), the organic layer was concentrated and column chromatography (SiO2, Petroleum ether / Ethyl acetate = 1 / 10 to 9 / 1) to give T-3 (3.46 g, 15.4 mmol, 73.5% yield) as a light yellow solid.

[0148] Referring to the method of Example 10, a brown solid was prepared by reduction and o-aminophenol reaction.

[0149] 1H NMR (400 MHz, DMSO-d6) δ 7.36 - 7.28 (m, 2H), 7.12 - 7.05 (m, 1H), 6.79 (br s, 2H), 6.42 (s, 1H), 6.35 (s, 1H), 3.82 (t, J = 4.0 Hz, 4H), 3.10 (t, J = 4.4 Hz, 4H). MS-ESI (m / z): 298.1 (M+H) + .

[0150] Example 13, 2-amino-6-thiomorpholinyl-3H-phenoxazin-3-one

[0151]

[0152] The target product was prepared by reacting with thiomorpholine according to the reaction of Example 12.

[0153] 1 H NMR (400 MHz, DMSO-d6) δ 7.36 - 7.28 (m, 2H), 7.12 - 7.05 (m, 1H), 6.79 (br s, 2H), 6.42 (s, 1H), 6.35 (s, 1H), 3.82 (t, J = 4.0 Hz, 4H), 3.10 (t, J = 4.4 Hz, 4H). MS-ESI (m / z): 298.1 (M+H) + .

[0154] Example 14, 2-amino-6-piperazinyl-3H-phenoxazin-3-one

[0155]

[0156] The target product was prepared by reacting with N-Boc piperazine according to the reaction of Example 12, and removing Boc by TFA.

[0157] 1 H NMR (400 MHz, DMSO-d6) δ 7.36 - 7.28 (m, 2H), 7.12 - 7.05 (m, 1H), 6.79 (br s, 2H), 6.42 (s, 1H), 6.35 (s, 1H), 3.82 (t, J = 4.0 Hz, 4H), 3.10 (t, J = 4.4 Hz, 4H). MS-ESI (m / z): 298.1 (M+H) + .

[0158] Example 15, 2-amino-6-((5-fluoro-lH-indol-3-yl)methyl)-3H-phenoxazin-3-one

[0159]

[0160] Referring to Example 10, compound 1A was reacted with indole, reduced by Fe / ammonium chloride, and reacted with o-aminophenol to produce the target product.

[0161] 1 H NMR (400 MHz, DMSO-d6) δ 10.99 (s, 1H), 7.53 (d, J = 8.00 Hz, 1H), 7.43-7.37 (m, 2H), 7.35-7.29 (m, 3H), 6.89-6.88 (m, 1H), 6.82-6.77 (m, 2H), 6.51 (s, 1H), 6.36 (s, 1H), 4.25 (s, 2H). MS-ESI (m / z): 360.1 (M+H) + .

[0162] Example 16, (2-amino-3-oxo-3H-phenoxazin-8-yl) glycine

[0163]

[0164] Compound 7 (270 mg, 1.0 mmol) was dissolved in 2 mL of a mixture of methanol and 2 mL of water, then lithium hydroxide solid (48 mg, 2.0 mmol) was added, and stirred at room temperature for 2 hours. Work-up: concentrated, and dried with anhydrous toluene three times, and directly subjected to the next reaction. The above solid was dissolved in DMF (2 mL) solution, 2 mL of pyridine and 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 380 mg, 1.0 mmol) were added, stirred at room temperature for 0.5 hours, then methyl glycinate (89 mg, 1.0 mmol) was added, and reacted at room temperature for 2 hours. Work-up: the reaction solution was concentrated, and separated by a preparative column (water (0.225% formic acid)-acetonitrile) to produce a red solid. The above solid was hydrolyzed with lithium hydroxide in methanol / water solution, and separated by a preparative column to produce the target product.

[0165] 1 H NMR (400 MHz, DMSO-d6) δ 1 H NMR (500 MHz, DMSO-d6) δ 13.00 (br s, 1H), 8.10 (s, 1H), 7.87-7.66 (m, 3H), 7.12-7.10 (m, 1H), 6.86 (br s, 1H), 6.21 (br s, 2H), 4.00 (s, 2H). MS-ESI (m / z): 314.1 (M+H) + .

[0166] Biological Example 1

[0167] In vitro anti-mycobacterial activity test

[0168] The anti-Mycobacterium tuberculosis activity of the compounds of the present application is expressed by determining the minimum inhibitory concentration (MIC, μg / mL) against Mycobacterium tuberculosis standard strain H37Rv ATCC 27294. In this test, phenoxazone was used as a control drug.

[0169] The minimum inhibitory concentration was determined as follows: a sterile 48-well plate (Tuberculosis Rapid Susceptibility Test Special Micro-culture Plate) was used, and each well was added with a drug diluted with 2 times concentration of culture medium (modified Middlebrook 7H9 liquid culture medium) according to the requirements of the drug susceptibility test design. Each compound was prepared into an initial solution of an appropriate concentration, and diluted with the culture medium (2x) to twice the concentration of each compound to be used, and 10 gradients of each compound were added to each well of the 48-well plate at 100 μL, and the final concentration of the test drug was 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015 μg / mL, respectively. The standard strain H 37 Rv ATCC 27294 was inoculated with 100 μL per well, and the bacterial amount per well was 4 x 10 -3 mg. Two growth positive control wells without an antibacterial drug and two growth negative control wells in which the culture medium was replaced with distilled water were provided for each plate, and the 48-well plate was covered and sealed around the periphery with transparent tape, and incubated in a humid box at 37°C. After 3 days, the positive growth control wells and the negative growth control wells were observed, and when a clear difference was observed between the two, the number and morphology of bacterial growth in each test well were observed, and inhibition or resistance was determined and the results were recorded, and the observation and recording were performed again after 7 days for confirmation. The minimum concentration of the drug contained in the sterile growth control well was the minimum inhibitory concentration (MIC). The results of the determination are shown in Table 1. The compounds all showed an anti-Mycobacterium tuberculosis standard strain H 37 Rv ATCC 27294 in vitro activity. Among them, the activity of Examples 1, 8-9, 12-13, and 16 is superior to that of questiomycin.

[0170] Table 1 In vitro activity of the compounds of the examples against Mycobacterium tuberculosis

[0171]

[0172]

[0173] Biological Example 2

[0174] In vitro anti-NTM bacterial activity test

[0175] Mycobacterium avium (ATCC 25291), M. intracellulare (ATCC 13950), M. abscessus (ATCC 19977) and M. kansasii (ATCC 12478) were cultured to log phase in 7H9 broth or CAMHB broth, and the bacterial suspension was diluted to 5 x 10 5 CFU / mL in reserve.

[0176] The 96-well plates were diluted by doubling dilution. After 7 days of incubation at 37°C (3 days for M. abscessus), 20 μL of Alamar Blue and 12.5 μL of 20% Tween-80 were added to each well, and the plates were incubated at 37°C for another 24 hours. The color of each well was recorded, with blue indicating no growth of the strain and red indicating growth of the strain. The drug concentration at which the color changed from blue to red was defined as the minimum inhibitory concentration (MIC). The test was repeated three times. The cytotoxicity test used two types of cells, Vero and HepG2, and the MTT test method (European Journal of Medicinal Chemistry 218 (2021) 113398) was used. The results are shown in Table 2.

[0177] Table 2 In vitro activity of the example compounds against NTM mycobacteria and cytotoxicity

[0178]

[0179]

[0180] The results in Table 2 show that questiomycin has no activity against the four strains of NTM, while the example compounds in Table 2 exhibit activity against at least two strains of NTM. For example, Examples 1-4, 8 and 16 have significant activity against M. avium, M. intracellulare and M. kansasii infection; Example 6 exhibits good activity against M. avium; and Example 16 also exhibits activity against M. abscessus. At the same time, Examples 1-16 have better in vitro safety than questiomycin.

Claims

1. A phenoxazine compound or a pharmaceutically acceptable salt thereof, wherein, the phenoxazine compound is selected from one of the following structures: 。 2. A process for preparing the phenoxazine compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein, the method comprises preparing the phenoxazine compound of formula (I) using compound (III) and compound (IV) as raw materials: ； wherein R1 and R2 are the same as the corresponding positions of the compound of claim 1.

3. A pharmaceutical composition, wherein, the pharmaceutical composition comprises the phenoxazine compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

4. Use of the phenoxazine compound of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 3 in the preparation of a medicament for treating mycobacterial infection.

5. Use according to claim 4, wherein, the mycobacterium is selected from Mycobacterium tuberculosis or non-tuberculous mycobacteria.

6. Use according to claim 5, wherein, the non-tuberculous mycobacteria is Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare or Mycobacterium kansasii.

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