An n-amide substituted phenoxazine compound, preparation and application thereof

Abstract

The application provides a kind of N amide-substituted phenoxazine compound and its preparation and application.The compound of the application shows excellent anti-MTB activity and excellent anti-NTM activity.In addition, compared with existing products, the compound of the application shows higher in vitro safety.The phenoxazine compound has the structure shown in formula (I).

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular, the present application relates to a kind of N amide-substituted phenoxazine compound and its preparation and application. BACKGROUND

[0002] Mycobacterium tuberculosis and non-tuberculous mycobacteria belong to the genus Mycobacterium. Among them, TB caused by MTB is still one of the top ten causes of death in the world. NTM refers to mycobacteria other than MTB and M. leprae (M. leprae), which is a conditional pathogen and widely distributed in nature. Common NTM pathogenic bacteria in clinical practice include M. abscessus, M. avium, M. intracellulare and M. kansasii, etc. NTM is one of the important pathogens of pulmonary and extrapulmonary infections in AIDS patients, and is also the main reason for the increase in hospitalization and mortality of such patients. In recent years, NTM infection has become a thorny clinical problem, and the incidence of NTM even exceeds TB in many countries. Unfortunately, so far there is no specific and efficient anti-NTM (especially drug-resistant NTM) drug available in clinical practice. Therefore, NTM infection has become another major challenge to global public health security after TB. At the same time, the clinical symptoms and chest images of MTB and NTM infection are very similar, and the sputum specimens also show acid-fast staining positive. Without strain identification, NTM infection is often misdiagnosed as TB in clinical practice, and treated as TB. However, since traditional anti-TB drugs have little or no activity against most NTM, when the clinical effect is poor or even the condition worsens, it is misdiagnosed as drug-resistant TB and continues to be treated. Therefore, it is urgent to develop new anti-MTB and NTM drugs with new 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 and also has a wide range of drug properties, but its anti-mycobacterial activity has not been reported. The present discovery found that questiomycin has anti-MTB activity through screening of compounds, and further designed and synthesized a series of 2-amido-modified questiomycin compounds through structural modification, and some compounds showed better anti-MTB activity than questiomycin. Further, some compounds also showed anti-NTM activity, while questiomycin had no activity against NTM.

[0004] At the same time, the compounds show higher in vitro safety.

[0005] SUMMARY

[0006] An object of the present application is to provide a kind of N amide-substituted phenoxazine compound;

[0007] Another object of the present application is to provide the preparation method of the N amide-substituted phenoxazine compound described in the application;

[0008] Still another object of the present application is to provide a kind of pharmaceutical composition;

[0009] Still another object of the present application is to provide the use of the N amide-substituted phenoxazine compound described in the application.

[0010] To achieve the above object, in one aspect, the present application provides a kind of N amide-substituted phenoxazine compound, its stereoisomer, deuterium or its pharmaceutically acceptable salt, wherein the phenoxazine compound structure is as shown in formula (I):

[0011]

[0012] Wherein,

[0013] R1 is selected from 4-10 membered aryl, C 1-6 Linear or branched alkyl, or-(CH2) n R3;Optionally, the aryl and alkyl are substituted by 1, 2 or 3 substituents selected from F, Cl, Br, I or C 1-6 Linear or branched alkyl; optionally, 1 or 2 CH2 in the alkyl carbon chain is replaced by O or S;

[0014] R2 is selected from H, F, Cl, Br, I, C 1-6 Linear or branched alkyl or C 1-6 Alkoxy; optionally, the alkyl or alkoxy is substituted by substituents selected from F, Cl, Br, I, C 1-6 Linear or branched alkyl;

[0015] R3 is selected from C 3-8 Cycloalkyl, C 1-6 Alkoxy, OH, NH2, nitro or carboxyl; optionally, the cycloalkyl or alkoxy is substituted by substituents selected from F, Cl, Br, I, C 1-6 Linear or branched alkyl;

[0016] n is 0, 1, 2, 3, 4 or 5.

[0017] According to some specific embodiments of the present application, wherein,

[0018] R1 is selected from 6- or 8-membered aryl, C 1-6 Linear or branched alkyl, or-(CH2) nR3; optionally, said aryl and alkyl are substituted with 1, 2 or 3 substituents selected from F, Cl, Br, I or C 1-4 straight or branched alkyl, or -(CH2)1-3-; optionally, 1 or 2 CH2in the alkyl carbon chain is replaced by O or S;

[0019] R2is selected from H, F, Cl, Br, I, methyl, ethyl, n-propyl, i-propyl, methoxy, ethoxy or propoxy; optionally, the alkyl or alkoxy is substituted with 1, 2 or 3 substituents selected from F, Cl, Br, I or C 1-4 straight or branched alkyl, or C 1-4 alkoxy; optionally, said alkyl or alkoxy is substituted with 1, 2 or 3 substituents selected from F, Cl, Br, I or C 1-4 straight or branched alkyl, or C

[0020] R3is selected from C 3-8 cycloalkyl, C 1-5 alkoxy, OH, NH2, nitro or carboxyl; optionally, said cycloalkyl or alkoxy is substituted with 1, 2 or 3 substituents selected from F, Cl, Br, I or C 1-4 straight or branched alkyl, or C

[0021] According to some embodiments of the application, wherein said aryl is selected from one of the following structures:

[0022]

[0023] According to some embodiments of the application, wherein,

[0024] R1is selected from phenyl, C 1-6 straight or branched alkyl, or -(CH2) n R3; optionally, said phenyl and alkyl are substituted with 1, 2 or 3 substituents selected from F, Cl, Br, I or C 1-4 straight alkyl; optionally, 1 or 2 CH2in the alkyl carbon chain is replaced by O or S;

[0025] R2is selected from H, F, Cl, Br, I, methyl, ethyl, n-propyl, i-propyl, methoxy, ethoxy or propoxy; optionally, the alkyl or alkoxy is substituted with 1, 2 or 3 substituents selected from F, Cl, Br, I or C 1-4 straight alkyl;

[0026] R3is selected from C 3-6 cycloalkyl, C 1-4 alkoxy, OH, or NH2; optionally, said cycloalkyl or alkoxy is substituted with 1, 2 or 3 substituents selected from F, Cl, Br, I or C 1-4 straight alkyl.

[0027] According to some embodiments of the application, wherein,

[0028] R1is selected from C 1-4linear alkyl; optionally, said alkyl is substituted with 1, 2, or 3 substituents selected from the group consisting of F, Cl, Br, or I;

[0029] R2is selected from the group consisting of H, F, Cl, Br, I, C 1-4 linear or branched alkyl or C 1-4 alkoxy; optionally, said alkyl or alkoxy is substituted with a substituent selected from the group consisting of F, Cl, Br, I, C 1-4 linear alkyl.

[0030] According to some embodiments of the present application, wherein R2is selected from the group consisting of H, C 1-4 linear or branched alkyl or C 1-4 alkoxy.

[0031] According to some embodiments of the present application, wherein,

[0032] R1is selected from the group consisting of methyl, R2is selected from the group consisting of F, Cl, Br, I, C 1-4 linear alkyl; or

[0033] R1is selected from the group consisting of C 2-4 linear alkyl, R2is selected from the group consisting of H or C 1-4 alkoxy.

[0034] According to some embodiments of the present application, wherein R1is selected from the group consisting of linear or branched alkyl, and when a CH2in the alkyl carbon chain is replaced by O or S, the C atom to which the carbonyl of the mother nucleus is attached is replaced by O or S.

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

[0036]

[0037] In another aspect, the present application also provides a preparation method of the N-amide substituted phenoxazine compound, stereoisomer, deuterated compound or pharmaceutically acceptable salt thereof according to the present application, wherein the method comprises preparing the N-amide substituted phenoxazine compound shown in formula (I) from a compound shown in formula (II), and R1C(O)Cl or (R1C(O))2O:

[0038]

[0039] According to some embodiments of the present application, wherein the compound shown in formula (II) and R1C(O)Cl or (R1C(O))2O are reacted at 10-50°C to prepare the N-amide substituted phenoxazine compound shown in formula (I).

[0040] According to some embodiments of the present application, the compound of formula (II) and R1C(O)Cl or (R1C(O))2O are reacted to prepare the N-amide substituted phenoxazine compound of formula (I) at room temperature to 50°C.

[0041] According to some embodiments of the present application, the compound of formula (II) and R1C(O)Cl or (R1C(O))2O are reacted for 3-12 hours to prepare the N-amide substituted phenoxazine compound of formula (I).

[0042] According to some embodiments of the present application, the compound of formula (II) and R1C(O)Cl or (R1C(O))2O are reacted in pyridine to prepare the N-amide substituted phenoxazine compound of formula (I).

[0043] According to some embodiments of the present application, after the reaction of the compound of formula (II) and R1C(O)Cl or (R1C(O))2O, the N-amide substituted phenoxazine compound of formula (I) is further isolated by column chromatography.

[0044] According to some embodiments of the present application, the column chromatography uses SiO2as the packing material.

[0045] According to some embodiments of the present application, the column chromatography uses PE / EA = 1:2-2:1 as the mobile phase.

[0046] According to some embodiments of the present application, the column chromatography uses PE / EA = 1 / 1 as the mobile phase.

[0047] According to some embodiments of the present application, the method further comprises using a compound of formula (III) and a compound of formula (IV) as raw materials to prepare the compound of formula (II):

[0048]

[0049] According to some embodiments of the present application, the compound of formula (III) and the compound of formula (IV) are reacted to prepare the compound of formula (II) at 10-40°C.

[0050] According to some embodiments of the present application, the compound of formula (III) and the compound of formula (IV) are reacted to prepare the compound of formula (II) at room temperature.

[0051] According to some embodiments of the present application, the compound of formula (III) and the compound of formula (IV) are reacted for 24-144 hours to prepare the compound of formula (II).

[0052] According to some embodiments of the present application, the compound of formula (III) and the compound of formula (IV) are reacted to prepare the compound of formula (II) in a mixed solvent of an alkyl alcohol having 1-4 carbon atoms and water.

[0053] According to some embodiments of the present application, the alkyl alcohol having 1-4 carbon atoms is selected from methanol, ethanol or propanol.

[0054] According to some embodiments of the present application, the volume ratio of the alkyl alcohol having 1-4 carbon atoms and water is 1:3 to 3:1.

[0055] According to some embodiments of the present application, the volume ratio of the alkyl alcohol having 1-4 carbon atoms and water is 1:2 to 2:1; preferably 1:1.

[0056] According to some embodiments of the present application, the compound of formula (III) and the compound of formula (IV) are reacted to prepare the compound of formula (II) in an oxygen atmosphere.

[0057] According to some embodiments of the present application, the step of preparing the compound of formula (II) from the compound of formula (III) and the compound of formula (IV) comprises:

[0058] The compound of formula (IV) is dissolved in methanol / water (volume ratio 1 / 1), and then the compound of formula (III) is dissolved in methanol / water (volume ratio 1 / 1) and slowly added to the above solution, and the reaction system is reacted at room temperature for 24 hours in an oxygen atmosphere to obtain the compound of formula (II).

[0059] In another aspect, the present application also provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the N-amide substituted 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.

[0060] 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%.

[0061] 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.

[0062] According to some embodiments of the present application, the pharmaceutical composition is a tablet, a powder, a sachet or a capsule.

[0063] When the pharmaceutical composition of the present application is in solid form, the pharmaceutically acceptable carrier is selected from one or more of diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, bulking agents, etc.

[0064] When the pharmaceutical composition of the present application is in encapsulated form, the pharmaceutically acceptable carrier is selected from one or more of magnesium carbonate, magnesium stearate, talc, sucrose, lactose, pectin, dextrin, starch, gelatin, methylcellulose, sodium carboxymethylcellulose, cocoa butter.

[0065] It is especially advantageous to formulate the aforementioned pharmaceutical preparations in dosage unit form for ease of administration and uniformity of dosage. Dosage unit forms can be in the form of tablets, capsules, or powders packaged in vials or sachets.

[0066] The pharmaceutical composition of the present application, as a preparation form, contains an effective amount of the compound of the present application in each dose is 0.1-1000 mg.

[0067] Although the amount of active ingredient contained in dosage unit forms can vary, the range is generally between 1 and 800 mg, adjusted according to the potency of the active ingredient selected.

[0068] The term "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.

[0069] In another aspect, the present application also provides the use of the N-amide substituted phenoxazine compound, stereoisomer, deuterated compound or pharmaceutically acceptable salt thereof according to the present application, or the pharmaceutical composition according to the present application in the preparation of a medicament for treating mycobacterial infection.

[0070] According to some specific embodiments of the present application, the mycobacterium is selected from Mycobacterium tuberculosis or non-tuberculous mycobacteria.

[0071] According to some specific embodiments of the present application, the non-tuberculous mycobacteria is selected from Mycobacterium avium or Mycobacterium kansasii.

[0072] According to some specific embodiments of the present application, when the active compound of formula (I) of the present application is used as a medicament for treating Mycobacterium tuberculosis infection, it is preferred to administer in an amount of 6-14 mg / kg body weight in the first phase.

[0073] In certain situations, the person skilled in the art can determine the preferred dosage for a certain situation in a routine manner. Generally, the amount at which the treatment is initiated is lower than the optimal dosage of the active ingredient, and the dosage is then gradually increased until the optimal therapeutic effect is achieved. For the sake of convenience, the total daily dosage can be divided into several portions, which are administered in fractions at different times.

[0074] It is understood that the embodiments of the present application can be combined with each other without contradiction.

[0075] In the absence of a specific definition in the present application, the following terms are explained as follows (the following explanations are only general examples and are not a limitation of the present application):

[0076] "Substituted" means that the hydrogen atoms on a carbon atom or a heteroatom are 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 generally, 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.

[0077] "Optional" is an alternative expression, which means that the following event can occur or not occur, for example, "optionally, the aryl and alkyl are substituted by 1, 2 or 3 substituents selected from F, Cl, Br, I or C 1-6 "Substituted" means that the hydrogen atoms on a carbon atom or a heteroatom are 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 generally, 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.

[0078] "Deuterated" means that the hydrogen atoms on a group containing hydrogen atoms are 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 generally, the number of deuteriums is any integer between 1 and the upper limit, preferably 1-20 deuterium atoms are replaced, more preferably 1-10 deuterium atoms are replaced, still more preferably 1-5 deuterium atoms are replaced, further preferably 1-3 deuterium atoms are replaced.

[0079] "Alkyl" means a monovalent linear or branched saturated aliphatic hydrocarbon group, and generally, it is 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 preferably an alkyl group of 1 to 3 carbon atoms, further 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.

[0080] "Alkoxy" means -O-alkyl, and when not specifically limited, it is -O-C 1-8 alkyl, preferably -O-C 1-6alkyl, more preferably -O-C 1-4 alkyl, more preferably -O-C 1-2 alkyl. For example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, t-butoxy, n-pentoxy, n-hexoxy, cyclopropoxy, and cyclobutoxy, and the like.

[0081] "Aryl" means a substituted or unsubstituted aromatic hydrocarbon group having a single ring or fused rings wherein the ring atoms are, without limitation, 4 to 10, 4 to 8, or 6 to 8 carbon atoms. The aryl ring can be fused to a saturated or unsaturated carbocyclic ring wherein the ring that is attached to the parent structure is an aryl ring, non-limiting examples include benzene, naphthalene, indane, tetralin, indene, xanthene, biphenyl, fluorene, and the like.

[0082] In summary, the present application provides a kind of N amide substituted phenoxazine compound and its preparation and application.

[0083] The N amide substituted phenoxazine compound of the present application has the following advantages:

[0084] The compound of the present application shows better anti-MTB activity than questiomycin, and better anti-NTM activity than questiomycin. At the same time, the compound of the present application shows higher in vitro safety than questiomycin. DETAILED DESCRIPTION

[0085] The following specific examples will illustrate the implementation process and beneficial effects of the present application in detail, which are intended to help the reader better understand the essence and characteristics of the present application, and not as a limitation on the scope of the present application.

[0086] Example 1, N-(3-oxo-3H-phenoxazin-2-yl)acetamide

[0087]

[0088] 2-Aminophenol (25 g, 229.09 mmol) was dissolved in a mixture of 100 mL of methanol and 100 mL of water, and stirred at room temperature for 24 hours under an oxygen atmosphere. Post-processing: the reaction solution was concentrated, then 50 mL of ethyl acetate was added, washed with water, and the organic phase was concentrated to obtain 2-amino-3H-phenoxazin-3-one (1.6 g, yield: 3.29%).

[0089] 2-Amino-3H-phenoxazin-3-one (100 mg, 471.25 μmol) was dissolved in 5 mL of acetic acid, then acetic anhydride (96 mg, 0.94 mmol) was added, and the reaction solution was reacted at 50°C for 12 hours. Post-processing, the solid in the reaction system was filtered, and the filter cake was dried to obtain the product (92 mg, yield: 76.79%).

[0090] 1 H NMR: (400 MHz, DMSO-d6) δ 9.73 (s, 1H), 8.29 (s, 1H), 7.87 (dd, J = 1.2, 8.0 Hz, 1H), 7.67-7.61 (m, 1H), 7.60-7.55 (m, 1H), 7.52-7.45 (m, 1H), 6.50 (s, 1H), 2.24 (s, 3H). MS-ESI (m / z): 255.2 (M+H) + .

[0091] Example 2, N-(3-oxo-3H-phenoxazin-2-yl)benzamide

[0092]

[0093] Prepared according to the procedure of Example 1 by reacting compound 2-amino-3H- phenoxazin-3-one with benzoyl chloride to give a yellow solid.

[0094] 1 H NMR: (400 MHz, DMSO-d6) δ 9.73 (s, 1H), 8.29 (s, 1H), 7.87 (dd, J = 1.2, 8.0 Hz, 1H), 7.67-7.61 (m, 1H), 7.60-7.55 (m, 1H), 7.52-7.45 (m, 1H), 6.50 (s, 1H), 2.24 (s, 3H). MS-ESI (m / z): 255.2 (M+H) + .

[0095] Example 3, 2-cyclopropyl-N-(3-oxo-3H-phenoxazin-2-yl)acetamide

[0096]

[0097] Prepared according to the procedure of Example 1 by reacting compound 2-amino-3H- phenoxazin-3-one with 2-cyclopropylacetyl chloride to give a yellow solid.

[0098] 1H NMR (400 MHz, DMSO-d6) δ 0.91 (t, J = 7.38 Hz, 3H) 1.60 (sxt, J = 7.33 Hz, 2H) 2.55 (t, J = 7.32 Hz, 2H) 6.50 (s, IH) 7.45 - 7.51 (m, IH) 7.54 - 7.60 (m, IH) 7.61 - 7.68 (m, IH) 7.87 (dd, J = 7.94, 1.31 Hz, IH) 8.30 (s, IH) 9.62 (s, IH). MS-ESI (m / z): 282.1 (M+H) + .

[0099] Example 4, N-(3-oxo-3H-phenoxazin-2-yl) n-butyramide

[0100]

[0101] Prepared according to the procedure of Example 1 by reacting the compound 2-amino-3H- phenoxazin-3-one with n-butyryl chloride to give a yellow solid.

[0102] 1 H NMR (400 MHz, DMSO-d6) δ 0.91 (t, J = 7.38 Hz, 3H) 1.60 (sxt, J = 7.33 Hz, 2H) 2.55 (t, J = 7.32 Hz, 2H) 6.50 (s, IH) 7.45 - 7.51 (m, IH) 7.54 - 7.60 (m, IH) 7.61 - 7.68 (m, IH) 7.87 (dd, J = 7.94, 1.31 Hz, IH) 8.30 (s, IH) 9.62 (s, IH). MS-ESI (m / z): 282.1 (M+H) + .

[0103] Example 5, 3,3-dimethyl-N-(3-oxo-3H-phenoxazin-2-yl) butyramide

[0104]

[0105] Prepared according to the procedure of Example 1 by reacting the compound 2-amino-3H- phenoxazin-3-one with 3,3-dimethyl n-butyryl chloride to give a yellow solid.

[0106] 1H NMR (400 MHz, DMSO-d6) δ 1.01 (s, 9 H) 2.48 (br s, 2 H) 6.50 (br s, 1 H) 7.45 - 7.52 (m, 1 H) 7.55 - 7.60 (m, 1 H) 7.62 - 7.68 (m, 1 H) 7.87 (dd, J=7.91, 1.38 Hz, 1 H) 8.31 (s, 1 H) 9.51 (s, 1 H). MS-ESI (m / z): 311.1 (M+H) + .

[0107] Example 6, Ethyl-(3-oxo-3H-phenoxazin-2-yl)carbamate

[0108]

[0109] Prepared according to the procedure of Example 1, reacting compound 2-amino-3H- phenoxazin-3-one with ethyl chloroformate to give a yellow solid.

[0110] 1 1H NMR (400 MHz, DMSO-d6) δ 1.27 (t, J=7.07 Hz, 3 H) 4.22 (q, J=7.09 Hz, 2 H) 6.50 (s, 1 H) 7.46 - 7.52 (m, 1 H) 7.56 - 7.60 (m, 1 H) 7.62 - 7.68 (m, 1 H) 7.86 - 7.91 (m, 2 H) 8.75 (br s, 1 H). MS-ESI (m / z): 285.2 (M+H) + .

[0111] Example 7, 2-Amino-N-(3-oxo-3H-phenoxazin-2-yl)acetamide

[0112]

[0113] Prepared according to the procedure of Example 1, reacting compound 2-amino-3H- phenoxazin-3-one with N-Boc-glycine using condensing agent HATU, followed by deprotection to give a black solid.

[0114] 1 1H NMR (400 MHz, DMSO-d6) δ 4.02 (s, 2 H) 6.55 (s, 1 H) 7.47 - 7.54 (m, 1 H) 7.57 - 7.62 (m, 1 H) 7.64 - 7.71 (m, 1 H) 7.90 (dd, J=7.94, 1.19 Hz, 1 H) 8.11 (br s, 3 H) 8.30 (s, 1 H) 10.39 (br s, 1 H). MS-ESI (m / z): 270.1 (M+H) + .

[0115] Example 8, N-(8-methyl 3-oxo-3H-phenoxazin-2-yl)acetamide

[0116]

[0117] Referring to the reaction of Example 1, o-aminophenol was reacted with 2-amino-4-methylphenol to give 2-amino-8-methyl-3H-phenoxazin-3-one, which was then reacted with acetic anhydride to give a red solid.

[0118] 1 H NMR: (400 MHz, DMSO-d6) δ 8.52 (s, 1H), 7.33-7.21 (m, 2H), 7.13 (s, 1H), 6.88-6.70 (m, 1H), 6.43 (s, 1H), 2.24 (s, 3H), 2.20 (3, 3H). MS-ESI (m / z): 269.2 (M+H) + .

[0119] Example 9, N-(9-methoxy 3-oxo-3H-phenoxazin-2-yl) n-propionamide

[0120]

[0121] Referring to the reactions of Examples 1 and 2, o-aminophenol was reacted with 2-amino-3-methoxyphenol to give 2-amino-9-methoxy-3H-phenoxazin-3-one, which was then reacted with propionyl chloride to give a red solid.

[0122] 1 H NMR: (400 MHz, DMSO-d6) δ 8.53 (s, 1H), 7.26-7.11 (m, 2H), 6.80-6.68 (m, 1H), 6.62-6.50 (m, 1H), 6.39 (s, 1H), 4.08 (s, 3H), 2.48 (q, J = 7.0 Hz, 2H), 1.20 (t, J = 7.0 Hz, 3H). MS-ESI (m / z): 299.2 (M+H) + .

[0123] Example 10, N-(7-methyl 3-oxo-3H-phenoxazin-2-yl) n-hexanamide

[0124]

[0125] Referring to the reaction of Example 1, o-aminophenol was reacted with 2-amino-5-methylphenol to give 2-amino-7-methyl-3H-phenoxazin-3-one, which was then reacted with n-hexanoyl chloride to give a red solid.

[0126] 1H NMR: (400 MHz, DMSO-d6) δ 8.53 (s, 1H), 8.12-7.83 (m, 2H), 7.61-7.52 (m, 1H), 7.21-7.18 (m, 1H), 6.51 (s, 1H), 2.43-2.15 (m, 2H), 2.21 (s, 3H), 1.50-1.33 (m, 6H), 1.20 (t, J = 7.0 Hz, 3H). MS-ESI (m / z): 325.2 (M+H) + .

[0127] Example 11, N-(6-fluoro-3-oxo-3H-phenoxazin-2-yl)acetamide

[0128]

[0129] Referring to the reaction of Example 1, o-aminophenol was reacted with 2-amino-6-fluorophenol to produce 2-amino-6-fluoro-3H-phenoxazin-3-one, which was then reacted with acetic anhydride to produce a red solid.

[0130] 1 H NMR: (400 MHz, CDCl3) δ 8.54 (s, 1H), 8.42 (s, 1H), 7.67 (t, J = 4.6 Hz, 1H), 7.34 (t, J = 6.0 Hz, 2H), 6.54 (s, 1H), 2.29 (s, 3H). MS-ESI (m / z): 273.1 (M+H) + .

[0131] Example 12, N-(8-fluoro-3-oxo-3H-phenoxazin-2-yl)acetamide

[0132]

[0133] Referring to the reaction of Example 1, o-aminophenol was reacted with 2-amino-6-fluorophenol to produce 2-amino-6-fluoro-3H-phenoxazin-3-one, which was then reacted with acetic anhydride to produce a red solid.

[0134] 1 H NMR: (400 MHz, DMSO-d6) δ 9.79 (s, 1H), 8.27 (s, 1H), 7.76-7.73 (m, 1H), 7.65-7.61 (m, 1H), 7.56-7.52 (m, 1H), 6.49 (s, 1H), 2.24 (s, 3H). MS-ESI (m / z): 273.2 (M+H) + .

[0135] Biological Example 1

[0136] In vitro anti-mycobacterial activity test

[0137] The anti-Mycobacterium tuberculosis activity of the compounds of the present application is expressed by determining the minimum inhibitory concentration (MIC, μg / mL) against M. tuberculosis standard strain H 37 Rv ATCC 27294. In this test, phenoxazone was used as a control drug. The minimum inhibitory concentration was determined as follows: a sterile 48-well plate (Mycobacteria Rapid Susceptibility Test Special Microculture 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 used, 10 gradients for each compound, 100 μL was added to each well of the 48-well plate, 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, 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, inhibition or drug resistance was determined, and the results were recorded. After 7 days, the results were observed and recorded again for confirmation. The minimum concentration of the drug contained in the sterile growth control well was the minimum inhibitory concentration (MIC). The determination results are shown in Table 1. The in vitro activity of the compounds of the examples against the standard strain H 37 Rv ATCC 27294 was superior to or equivalent to questiomycin; among them, the activity of Examples 1, 8-9, 11-12 was superior to questiomycin.

[0138] Table 1 In vitro activity of the compounds of the examples against Mycobacterium tuberculosis

[0139] Compound MIC (μg / mL) Compound MIC (μg / mL) Example 1 0.186 Example 9 0.312 Example 3 0.461 Example 10 0.652 Example 4 0.658 Example 11 0.402 Example 6 0.444 Example 12 0.368 Example 8 0.289 questiomycin 0.412

[0140] Biological Example 2

[0141] In vitro anti-NTM bacterial activity test

[0142] Mycobacterium avium (ATCC 25291) and Mycobacterium kansasii (ATCC 12478) were cultured to the logarithmic growth phase, 7H9 liquid culture medium or CAMHB culture, and the bacterial suspension was diluted to 5 x 10 5CFU / mL. The 96-well plates were prepared by diluting each drug-containing well by a factor of two. 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 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 of the determination are shown in Table 2.

[0143] Table 2 In vitro activity of the compounds of the examples on NTM mycobacteria and cytotoxicity

[0144]

[0145] The results in Table 2 show that the compounds of Examples 1-12 all have obvious activity against both M. avium and M. kansasii infections, while questiomycin does not. At the same time, the compounds of the examples of the present application all show better in vitro safety than questiomycin.

Claims

1. The use of an N-amide-substituted phenoloxazine compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating mycobacterial infections, wherein the mycobacteria are selected from Mycobacterium avium, and the phenoloxazine compound has the following structure: 。

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