N-hydrocarbyl-substituted phenoxazine compounds, their preparation and use

Abstract

The application provides an N-hydrocarbyl-substituted phenoxazine compound and preparation and application thereof. The compound of the application exhibits excellent anti-MTB activity and excellent anti-NTM activity. Meanwhile, compared with existing products, the compound of the application exhibits higher in-vitro safety. The phenoxazine compound is shown as 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 alkyl-substituted phenoxazine compound and its preparation and application. BACKGROUND

[0002] Phenoxazine compounds are widely distributed in nature, it has anti-tumor, antibacterial and other biological activities, its unique three-ring structure also becomes the object of focus of drug research and development.This paper through to the compound library carries out anti-tubercle bacillus (Mycobacterium tuberculosis, M.tuberculosis, MTB) activity screening, first discovers that phenoxazine compound questiomycin (structure is as shown below) has anti-MTB activity (Table 2), however, further research finds that its in vitro cytotoxicity is greater (Table 2).Based on this, further through structural modification, design and synthesis a series of 2-amino modified phenoxazine compounds, show comparable anti-MTB activity with questiomycin.At the same time, the compounds also show activity to abscess bacillus, M.avium, M.intracellulare and M.kansasii, and questiomycin has no activity to the above-mentioned mycobacterium.More importantly, all compounds show good in vitro safety.It is known that MTB and non-tuberculous mycobacteria (Nontuberculous mycobacteria, NTM) belong to the same genus of mycobacterium.Among them, TB caused by MTB is still one of the top ten causes of death in the world, NTM is a conditional pathogen, but in recent years, NTM infection has increasingly become a thorny clinical problem, and the incidence of NTM even exceeds TB in many countries.Moreover, there is still no specific and efficient anti-NTM (especially drug-resistant NTM) drug available in clinic.Therefore, it is very meaningful to develop new anti-MTB and NTM drugs with new mechanism of action to achieve effective treatment and control of MTB and NTM.

[0003] SUMMARY

[0004] One object of the present application is to provide a kind of N alkyl-substituted phenoxazine compound;

[0005] Another object of the present application is to provide the preparation method of the N alkyl-substituted phenoxazine compound;

[0006] Still another object of the present application is to provide a kind of pharmaceutical composition;

[0007] Still another object of the present application is to provide the use of the N alkyl-substituted phenoxazine compound.

[0008] To achieve the above objectives, in one respect, the present invention provides an N-alkyl-substituted phenoloxazine compound, its stereoisomers, deuterated derivatives, or pharmaceutically acceptable salts thereof, wherein the structure of the phenoloxazine compound is shown in formula (I):

[0009]

[0010] in,

[0011] R1 is selected from aryl groups ranging from 4 to 10 francs, C 1-6 Straight-chain or branched alkyl groups, or -(CH2) n R3; optionally, the aryl group, -(CH2) n The alkylene or alkyl group of R3 is surrounded by one, two, or three atoms selected from F, Cl, Br, I, or C. 1-6 The alkyl group is substituted with a straight-chain or branched alkyl group; optionally, the alkyl group or -(CH2) is substituted with a substituent. n One or two CH2 atoms in the carbon chain of R3 are replaced by O or S;

[0012] R2 is selected from H, F, Cl, Br, I, C 1-6 Straight-chain or branched alkyl or C 1-6 Alkoxy group; optionally, the alkyl or alkoxy group is selected from F, Cl, Br, I, C. 1-6 Substituents of straight-chain or branched alkyl groups;

[0013] R3 is selected from C 3-8 cycloalkyl, C 1-6 Alkyl, 4- to 10-membered aryl, OH, NH2, nitro, or carboxyl; optionally, the cycloalkyl, alkoxy, or aryl group is selected from F, Cl, Br, I, OH, C. 1-6 Substituents of straight-chain or branched alkyl groups;

[0014] n can be 1, 2, 3, 4 or 5.

[0015] According to some specific embodiments of the present invention, the aryl group is selected from one of the following structures:

[0016]

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

[0018] R1 is selected from hexavalent or octyl aryl, C 1-6 Straight-chain or branched alkyl groups, or -(CH2) n R3; optionally, the aryl group, -(CH2) n The alkylene or alkyl group of R3 is surrounded by one, two, or three atoms selected from F, Cl, Br, I, or C.1-4 optionally substituted by one or more substituents selected from F, Cl, Br, I, OH, C n one or two CH2in the carbon chain of R3is replaced by O or S;

[0019] R2is selected from H, F, Cl, Br, I, C 1-4 optionally substituted by one or more substituents selected from F, Cl, Br, I, OH, C 1-4 optionally substituted by one or more substituents selected from F, Cl, Br, I, OH, C 1-4 optionally substituted by one or more substituents selected from F, Cl, Br, I, OH, C

[0020] R3is selected from C 3-8 optionally substituted by one or more substituents selected from F, Cl, Br, I, OH, C 1-6 optionally substituted by one or more substituents selected from F, Cl, Br, I, OH, C 1-4 optionally substituted by one or more substituents selected from F, Cl, Br, I, OH, C

[0021] According to some embodiments of the application, wherein,

[0022] R1is selected from C 1-6 optionally substituted by one or more substituents selected from F, Cl, Br, I, OH, C n optionally substituted by one or more substituents selected from F, Cl, Br, I, OH, C n optionally substituted by one or more substituents selected from F, Cl, Br, I, OH, C 1-4 optionally substituted by one or more substituents selected from F, Cl, Br, I, OH, C n one or two CH2in the carbon chain of R3is replaced by O or S;

[0023] R2is selected from H, F, Cl, Br, I, C 1-4 optionally substituted by one or more substituents selected from F, Cl, Br, I, OH, C

[0024] R3is selected from C 3-6 optionally substituted by one or more substituents selected from F, Cl, Br, I, OH, C 1-5 optionally substituted by one or more substituents selected from F, Cl, Br, I, OH, C 1-4 optionally substituted by one or more substituents selected from F, Cl, Br, I, OH, C

[0025] According to some embodiments of the application, wherein,

[0026] R1is selected from C 2-4 optionally substituted by one or more substituents selected from F, Cl, Br, I, OH, C nR3; optionally, said alkyl or -(CH2) n R3is alkylene substituted with 1, 2 or 3 substituents selected from the group consisting of F, Cl, Br, I; optionally, said alkyl or -(CH2) n 1 or 2 CH2in the carbon chain of R3is replaced by O or S;

[0027] R2is selected from the group consisting of H, F, Cl, Br, I, C 1-4 straight chain 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;

[0028] R3is selected from the group consisting of C 3-6 cycloalkyl, C 1-5 alkoxy or OH; optionally, said cycloalkyl, alkoxy is substituted with a substituent selected from the group consisting of F, Cl, Br, I, OH.

[0029] According to some embodiments of the present application, wherein,

[0030] R1is selected from the group consisting of C 2-4 straight chain alkyl, or -(CH2) n R3; optionally, said alkyl or -(CH2) n R3is alkylene substituted with 1, 2 or 3 substituents selected from the group consisting of F, Cl, Br, I;

[0031] R2is selected from the group consisting of H or C 1-4 straight chain alkyl; optionally, said alkyl or alkoxy is substituted with a substituent selected from the group consisting of F, Cl, Br, I;

[0032] R3is selected from the group consisting of C 3-6 cycloalkyl or C 1-5 alkoxy; optionally, said cycloalkyl, alkoxy is substituted with a substituent selected from the group consisting of F, Cl, Br, I, OH.

[0033] According to some embodiments of the present application, wherein, the phenoxazine compound is selected from one of the following structures:

[0034]

[0035] In another aspect, the present application also provides a method for preparing the N-alkyl substituted phenoxazine compound, stereoisomer, deuterated compound or pharmaceutically acceptable salt thereof, wherein the method comprises reacting a compound of formula (II) to obtain the N-alkyl substituted phenoxazine compound of formula (I):

[0036]

[0037] According to some specific embodiments of the present application, the method comprises preparing the N-alkyl-substituted phenoxazine compound of formula (I) by reacting the compound of formula (II) with R1C(O)Cl or (R1C(O))2O, and then reducing with BH3.THF;

[0038] or

[0039] The compound of formula (I) is prepared by directly alkylating the compound of formula (II) with R1I.

[0040] According to some specific embodiments of the present application, the compound of formula (II) and R1C(O)Cl or (R1C(O))2O are reacted at 10-50°C to prepare the N-alkyl-substituted phenoxazine compound of formula (I).

[0041] According to some specific embodiments of the present application, the compound of formula (II) and R1C(O)Cl or (R1C(O))2O are reacted at room temperature to 50°C to prepare the N-alkyl-substituted phenoxazine compound of formula (I).

[0042] According to some specific 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-alkyl-substituted phenoxazine compound of formula (I).

[0043] According to some specific 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-alkyl-substituted phenoxazine compound of formula (I).

[0044] According to some specific embodiments of the present application, the alkylating reaction of the compound of formula (II) with R1I is carried out at 15-40°C.

[0045] According to some specific embodiments of the present application, the alkylating reaction of the compound of formula (II) with R1I is carried out at 20-30°C.

[0046] According to some specific embodiments of the present application, the alkylating reaction of the compound of formula (II) with R1I is carried out at 25°C.

[0047] According to some specific embodiments of the present application, the alkylating reaction of the compound of formula (II) with R1I is carried out for 10-40 hours.

[0048] According to some specific embodiments of the present application, the alkylating reaction of the compound of formula (II) with R1I is carried out for 20-30 hours.

[0049] According to some embodiments of the present application, the reaction time for the reaction of the compound of formula (II) with R1I is 24 hours.

[0050] According to some embodiments of the present application, after the reaction of the compound of formula (II) with R1C(O)Cl or (R1C(O))2O is completed, the method further comprises a step of separating the N-hydrocarbyl-substituted phenoxazine compound of formula (I) by column chromatography.

[0051] According to some embodiments of the present application, the column chromatography uses SiO2as the packing material.

[0052] According to some embodiments of the present application, the method further comprises a step of preparing the compound of formula (II) using a compound of formula (III) and a compound of formula (IV) as raw materials:

[0053]

[0054] According to some embodiments of the present application, the compound of formula (II) is prepared by reacting the compound of formula (III) and the compound of formula (IV) at a temperature of 10-40°C.

[0055] According to some embodiments of the present application, the compound of formula (II) is prepared by reacting the compound of formula (III) and the compound of formula (IV) at room temperature.

[0056] According to some embodiments of the present application, the compound of formula (II) is prepared by reacting the compound of formula (III) and the compound of formula (IV) for 24-144 hours.

[0057] According to some embodiments of the present application, the compound of formula (II) is prepared by reacting the compound of formula (III) and the compound of formula (IV) for 24-48 hours.

[0058] According to some embodiments of the present application, the compound of formula (II) is prepared by reacting the compound of formula (III) and the compound of formula (IV) using a mixed solvent of an alkyl alcohol having 1-4 carbon atoms and water as the reaction solvent.

[0059] According to some embodiments of the present application, the alkyl alcohol having 1-4 carbon atoms is selected from the group consisting of methanol, ethanol, and propanol.

[0060] According to some embodiments of the present application, the volume ratio of the alkyl alcohol having 1-4 carbon atoms to water is 1:3 to 3:1.

[0061] 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.

[0062] 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 to prepare the compound of formula (II).

[0063] 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: dissolving the compound of formula (III) and the compound of formula (IV) in a reaction solvent, respectively, and then adding the reaction solvent dissolving the compound of formula (III) dropwise into the reaction solvent dissolving the compound of formula (IV) to react.

[0064] 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:

[0065] The compound of formula (II) can be obtained by dissolving the compound of formula (IV) in methanol / water (volume ratio 1 / 1), then dissolving the compound of formula (III) in methanol / water (volume ratio 1 / 1) and slowly adding it into the above solution, and reacting the system in an oxygen atmosphere at room temperature for 24 hours.

[0066] In another aspect, the present application also provides a pharmaceutical composition, wherein the pharmaceutical composition contains the N-hydrocarbyl-substituted phenoxazine compound, the stereoisomer, the deuterated compound or the pharmaceutically acceptable salt thereof according to the present application, and a pharmaceutically acceptable carrier.

[0067] 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%.

[0068] 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.

[0069] According to some embodiments of the present application, the pharmaceutical composition is a tablet, a powder, a sachet or a capsule.

[0070] When the pharmaceutical composition of the present application is in a solid form, the pharmaceutically acceptable carrier is selected from one or more of diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, bulking agents, etc.

[0071] When the pharmaceutical composition of the present application is in an 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.

[0072] It is especially advantageous to formulate the aforementioned pharmaceutical preparations in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used in the specification herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such dosage unit forms are tablets (including scored or coated tablets), capsules, and the like.

[0073] 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.

[0074] Although the amount of active ingredient that can be included in the dosage unit form varies, the drug dosage unit form generally contains between 1-800 mg, adjusted to the potency of the active ingredient selected.

[0075] 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.

[0076] In another aspect, the present application also provides the use of the N-alkyl 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.

[0077] According to some embodiments of the present application, the mycobacterium is selected from Mycobacterium tuberculosis or non-tuberculous mycobacteria.

[0078] According to some embodiments of the present application, the non-tuberculous mycobacteria is selected from Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare or Mycobacterium kansasii.

[0079] According to some 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 stage.

[0080] In some cases the optimal dose to be administered to a subject can be determined by routine methods. Typically, the amount effective at the beginning of the treatment is lower than the optimal dose of the active ingredient, and then the dose is gradually increased until the optimal therapeutic effect is achieved. For the convenience, the total daily dose can be divided into several parts, and administered in several fractions.

[0081] It is understood that the embodiments of the present application can be combined with each other without contradiction.

[0082] In the present application, the following terms are explained as follows (the following explanations are only general examples and not limitations of the present application) unless otherwise defined in the present application:

[0083] "Substituted" means that the hydrogen atoms on carbon atoms or heteroatoms are replaced by one or more defined substituents, the upper limit of the number of substituents is 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 same or different substituents can be replaced.

[0084] "Optional" is an optional expression, which means that the following event can occur or not occur, for example, "optionally, the aryl and alkyl groups 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 carbon atoms or heteroatoms are replaced by one or more defined substituents, the upper limit of the number of substituents is 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 same or different substituents can be replaced.

[0085] "Deuterated" means that the hydrogen atoms on the hydrogen-containing group are replaced by at least one deuterium, and the upper limit of the number of deuteriums is equal to the sum of the number of hydrogens that can be replaced by the substituents, 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, more preferably 1-5 deuterium atoms are replaced, further preferably 1-3 deuterium atoms are replaced.

[0086] "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, and 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.

[0087] "Alkoxy" means -O-alkyl, and unless otherwise specified, it 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. For example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, t-butoxy, n-pentoxy, n-hexoxy, cyclopropoxy and cyclobutoxy, etc.

[0088] "Aryl" means a substituted or unsubstituted aromatic hydrocarbon group having a single ring or multiple rings in which rings are fused together, wherein the aryl ring can be substituted with one or more of the following: alkyl, alkenyl, alkynyl, halide, haloalkyl, haloalkenyl, haloalkynyl, aldehyde, ketone, carboxylate, ester, carboxamide, cyano, hydroxyl, oxo, amine, nitro, thiol, sulfonyl, sulfonamide, sulfide, or any combination thereof, and wherein the number of carbon atoms in the aryl group, including any substituents, is from 4 to 10, 4 to 8, or 6 to 8. The aryl group can be fused to a saturated or unsaturated carbocyclic ring, wherein the ring attached to the parent structure is an aryl ring, non-limiting examples include phenyl, naphthyl, indanyl, indenyl, indenonyl, and the like.

[0089] In summary, the present application provides a kind of N hydrocarbon group substituted phenoxazine compound and its preparation and application.The compound of the present application has the following advantages:

[0090] The compound of the present application shows better anti-MTB activity than questiomycin, and better anti-NTM activity than questiomycin.At the same time, relative to questiomycin, the compound of the present application shows higher in vitro safety. DETAILED DESCRIPTION

[0091] 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.

[0092] Example 1, 2-methylamino-3H-phenoxazin-3-one

[0093]

[0094] 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 oxygen atmosphere. Post-processing: concentrate the reaction solution, then add 50 mL of ethyl acetate, wash with water, concentrate the organic phase, and prepare to obtain 2-amino-3H-phenoxazin-3-one (1.6 g, yield: 3.29%).

[0095] The above compound (300 mg, 1.41 mmol) and methyl iodide (401.32 mg, 176.02 μL, 2.83 mmol) were dissolved in DMF (7 mL), then anhydrous sodium carbonate (599.35 mg, 235.96 μL, 5.65 mmol) was added, and stirred at 80°C for 4 hours under nitrogen protection. Post-processing, concentration, and separation by preparative column (Water (0.225% FA)-ACN) to obtain the target product (yield: 15.79%).

[0096] 1H NMR (400 MHz, DMSO-d6) δ 7.71 (dd, J = 7.6, 1.6 Hz, 1H), 7.56 - 7.37 (m, 3H), 7.24 (d, J = 5.6 Hz, 1H), 6.39 (s, 1H), 6.11 (s, 1H), 2.85 (d, J = 5.2 Hz, 3H). ESI (m / z): 227.2 (M+H) + .

[0097] Example 2, 2-ethylamino-3H-phenoxazin-3-one

[0098]

[0099] 2-amino-3H-phenoxazin-3-one (100 mg, 471.25 μmol) was dissolved in 5 mL acetic acid, then acetic anhydride (96 mg, 0.94 mmol) was added, and the reaction was allowed to proceed at 50 °C for 12 hours. After work-up, the solid in the reaction system was filtered, and the filter cake was dried and dissolved in 5 mL tetrahydrofuran solution, then BH3.THF (1.4 mL, 1M in THF) was added, and the reaction was allowed to proceed at 80 °C for 2 hours. After work-up, 10 mL methanol was added and stirred for 0.5 hours, and the reaction was concentrated to give a yellow product (yield: 9.41%).

[0100] 1 1H NMR (400 MHz, DMSO-d6) δ 7.70 (d, J = 8.0 Hz, 1H), 7.54 - 7.37 (m, 3H), 7.04 (t, J = 5.2 Hz, 1H), 6.41 (s, 1H), 6.26 (s, 1H), 3.29 - 3.23 (m, 2H), 1.22 (t, J = 7.2 Hz, 3H). MS-ESI (m / z): 241.1 (M+H) + .

[0101] Example 3, 2-(n-butylamino)-3H-phenoxazin-3-one

[0102]

[0103] The preparation method was the same as Example 2, and the compound 2-n-butyl-N-(3-oxo-3H-phenoxazin-2-yl)acetamide was reacted with BH3.THF to give a yellow solid.

[0104] 1H NMR (400 MHz, DMSO-d6) δ 0.92 (t, J = 7.38 Hz, 3H) 1.36 (sxt, J = 7.43 Hz, 2H) 1.59 (quin, J = 7.32 Hz, 2H) 3.23 (q, J = 6.75 Hz, 2H) 6.17 (s, 1H) 6.39 (s, 1H) 7.09 (br t, J = 6.00 Hz, 1H) 7.37 - 7.56 (m, 3H) 7.70 (dd, J = 7.82, 1.44 Hz, 1H). 13 C NMR (151 MHz, DMSO) δ 179.87, 149.30, 148.01, 145.90, 141.88, 133.82, 128.63, 127.79, 125.32, 115.92, 103.36, 95.07, 41.69, 29.65, 19.76, 13.70, MS-ESI (m / z): 269.3 (M+H) + .

[0105] Example 4, 2-((cyclopropylmethyl)amino)-3H-phenoxazin-3-one

[0106]

[0107] Preparation method same as Example 2, compound 2-cyclopropyl-N-(3-oxo-3H- phenoxazin-2-yl)acetamide reacted with BH3.THF to produce red solid.

[0108] 1 H NMR (400 MHz, METHANOL-d4) δ 0.31 - 0.38 (m, 2H) 0.58 - 0.66 (m, 2H) 1.15 - 1.26 (m, 1H) 3.17 (d, J = 6.88 Hz, 2H) 6.22 (s, 1H) 6.39 (s, 1H) 7.36 - 7.43 (m, 1H) 7.44 - 7.49 (m, 2H) 7.69 (d, J = 7.88 Hz, 1H). 13 C NMR (151 MHz, DMSO) δ 179.85, 149.31, 148.03, 145.85, 141.90, 133.81, 128.72, 127.86, 125.35, 115.94, 103.32, 95.37, 46.26, 9.84, 3.48, MS-ESI (m / z): 267.2 (M+H) + .

[0109] Example 5, 2-benzylamino-3H-phenoxazin-3-one

[0110]

[0111] Preparation method same as example 2, compound N-(3-oxo-3H-phenoxazin-2-yl)benzamide reacted with BH3.THF to produce red solid.

[0112] 1 H NMR: (400 MHz, DMSO-d6) δ 7.77 (t, J = 6.4 Hz, 1H), 7.66 (dd, J = 1.2, 8.0 Hz, 1H), 7.51 - 7.42 (m, 2H), 7.41 - 7.31 (m, 5H), 7.28 - 7.21 (m, 1H), 6.40 (s, 1H), 6.06 (s, 1H), 4.49 (d, J = 6.4 Hz, 2H). MS-ESI (m / z): 303.2 (M+H) + .

[0113] Example 6, 2-(4-fluorophenyl)amino-3H-phenoxazin-3-one

[0114]

[0115] 2-amino-3H-phenoxazin-3-one (500 mg, 2.36 mmol, 1 eq) and 1-fluoro-4- iodobenzene (679.99 mg, 353.24 μL, 3.06 mmol) were dissolved in DMF (10 mL), then K3PO4 (1.50 g, 585.19 μL, 7.07 mmol) and CuI (67.31 mg, 11.98 μL, 353.42 μmol) were added, under nitrogen protection, 80 °C for 16 hours. After treatment, preparation liquid phase (Water (0.1% NH3H2O)-ACN) to produce the above red product (5%).

[0116] 1H NMR (400 MHz, DMSO-d6) δ 8.89 (s, 1H), 7.71 (d, J = 7.3 Hz, 1H), 7.56 - 7.48 (m, 2H), 7.48 - 7.37 (m, 3H), 7.25 (t, J = 8.8 Hz, 2H), 6.65 (s, 1H), 6.50 (s, 1H). MS-ESI (m / z): 307.1 (M+H) + .

[0117] Example 7, 2-(2-hydroxyethyl)amino-3H-phenoxazin-3-one

[0118]

[0119] Preparation method same as example 1, compound 2-amino-3H-phenoxazin-3-one reacted with iodoethanol to produce red solid.

[0120] 1 H NMR: (400 MHz, DMSO-d6) δ 7.73 (d, J = 8.0 Hz, 1H), 7.57-7.36 (m, 3H), 7.02 (t, J = 5.0 Hz, 1H), 6.39 (s, 1H), 6.27 (s, 1H), 3.55-3.47 (m, 2H), 3.30-3.26 (m, 2H). MS-ESI (m / z): 257.1 (M+H) + .

[0121] Example 8, 2-(2-Hydroxyethoxyethyl)amino-3H-phenoxazin-3-one

[0122]

[0123] Prepared by the method of Example 1, reacting compound 2-amino-3H-phenoxazin-3-one with 2-(2-iodoethoxy)ethanol to give a red solid.

[0124] 1 H NMR: (400 MHz, DMSO-d6) δ 7.72 (d, J = 8.0 Hz, 1H), 7.57-7.33 (m, 3H), 7.03 (t, J = 5.2 Hz, 1H), 6.41 (s, 1H), 6.30 (s, 1H), 3.55-3.27 (m, 6H), 3.31-3.26 (m, 2H). MS-ESI (m / z): 301.1 (M+H) + .

[0125] Example 9, 2-Ethylamino-6-fluoro-3H-phenoxazin-3-one

[0126]

[0127] Prepared by the method of Examples 1 and 2, reacting o-aminophenol with 2-amino-6-fluorophenol to give 2-amino-6-fluoro-3H-phenoxazin-3-one, which was then reacted with acetic anhydride and BH3.THF to give a red solid.

[0128] 1 H NMR: (400 MHz, DMSO-d6) δ 7.53 (d, J = 8.0 Hz, 1H), 7.44-7.32 (m, 2H), 7.10-6.91 (m, 2H), 3.30-3.25 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H). MS-ESI (m / z): 259.2 (M+H) + .

[0129] Example 10, 2-Ethylamino-8-methyl-3H-phenoxazin-3-one

[0130]

[0131] Referring to the reactions of Examples 1 and 2, o-aminophenol was reacted with 2-amino-4-methylphenol to produce 2-amino-8-methyl-3H-phenoxazin-3-one, which was then reacted with acetic anhydride and BH3.THF to produce a red solid.

[0132] 1 H NMR: (400 MHz, DMSO-d6) δ 7.35 (d, J = 7.6 Hz, 1H), 7.11 (s, 1H), 6.89 (d, J = 7.6 Hz, 1H), 6.43 (s, 1H), 6.39 (s, 1H), 3.17 (q, J = 7.2 Hz, 2H), 2.10 (s, 3H), 1.21 (t, J = 7.2 Hz, 3H). MS-ESI (m / z): 255.2 (M+H) + .

[0133] Example 11, 2-n-Propylamino-9-methoxy-3H-phenoxazin-3-one

[0134]

[0135] Referring to the reactions of Examples 1 and 2, o-aminophenol was reacted with 2-amino-4-methylphenol to produce 2-amino-8-methyl-3H-phenoxazin-3-one, which was then reacted with acetic anhydride and BH3.THF to produce a red solid.

[0136] 1 H NMR: (400 MHz, DMSO-d6) δ 7.35 (d, J = 7.6 Hz, 1H), 7.11 (s, 1H), 6.89 (d, J = 7.6 Hz, 1H), 6.43 (s, 1H), 6.39 (s, 1H), 3.17 (q, J = 7.2 Hz, 2H), 2.10 (s, 3H), 1.21 (t, J = 7.2 Hz, 3H). MS-ESI (m / z): 255.2 (M+H) + .

[0137] Example 12, 2-n-Hexylamino-7-methyl-3H-phenoxazin-3-one

[0138]

[0139] Referring to the reactions of Examples 1 and 2, o-aminophenol was reacted with 2-amino-4-methylphenol to produce 2-amino-8-methyl-3H-phenoxazin-3-one, which was then reacted with acetic anhydride and BH3.THF to produce a red solid.

[0140] 1 H NMR: (400 MHz, DMSO-d6) δ 7.28-6.83 (m, 3H), 6.71 (s, 1H), 6.49 (s, 1H), 6.39 (s, 1H), 3.19-3.15 (m, 2H), 1.52-1.48 (m, 2H), 1.37-1.32 (m, 4H), 1.18 (t, J = 7.0 Hz, 3H). MS-ESI (m / z): 311.2 (M+H) + .

[0141] Biological Example 1

[0142] In vitro anti-mycobacterial activity test

[0143] The anti-Mycobacterium tuberculosis activity of the compounds of the present application was expressed by determining the minimum inhibitory concentration (MIC, μg / mL) against Mycobacterium tuberculosis standard strain H 37 Rv ATCC 27294. In this test, questiomycin was used as the control drug. The minimum inhibitory concentration was determined as follows: aseptic 48-well plates (tuberculosis rapid drug sensitivity special micro-culture plates) were prepared according to the requirements of the drug sensitivity test design, and each well was added with the drug diluted with 2-fold concentration medium (modified Middlebrook 7H9 liquid culture medium). Each compound was prepared into an initial solution of an appropriate concentration, diluted with the medium (2x) to a two-fold concentration of each compound used, and 10 gradients of each compound were added to each well of the 48-well plate, 100 μL per well, 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 4x10 -3 mg. Two growth positive control wells without the antibacterial drug and two growth negative control wells with distilled water instead of the medium were set for each plate, the 48-well plate was covered and sealed around the periphery with transparent tape, and then 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 observation and recording were repeated for confirmation. The minimum concentration of the drug contained in the aseptic 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 equivalent to or better than that of questiomycin. Among them, the activity of compounds 2, 3-4, 8, 10, 12 was better than that of questiomycin.

[0144] Table 1 in vitro activity of example compounds against M. tuberculosis

[0145] Compound MIC (μg / mL) Compound MIC (μg / mL) Example 1 0.525 Example 9 0.422 Example 2 0.400 Example 10 0.310 Example 3 0.370 Example 11 0.421 Example 4 0.376 Example 12 0.385 Example 8 0.389 questiomycin 0.412

[0146] Biological Example 2

[0147] In vitro anti-NTM activity test

[0148] M. avium (ATCC 25291), M. intracellulare (ATCC 13950), M. abscessus (ATCC 19977) and M. kansasii (ATCC 12478) were cultured to the logarithmic growth phase in 7H9 liquid medium or CAMHB medium, and the bacterial suspension was diluted to 5x10 5 CFU / mL for standby. A 96-well plate was prepared by sequentially diluting each drug-containing well by a factor of two. After incubation at 37°C for 7 days (3 days for M. abscessus), 20 μL of Alamar Blue and 12.5 μL of 20% Tween-80 were added to each well, and incubation was continued 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 of the well that changed from blue to red was defined as the minimum drug 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.

[0149] Table 2 in vitro activity of example compounds against NTM mycobacteria and cytotoxicity

[0150]

[0151] The results in Table 2 show that the compound of Example 2 has obvious activity against M. abscessus, M. avium, M. intracellulare and M. kansasii infection; the compounds of Examples 1, 3-12 also have obvious activity against M. avium, M. intracellulare and M. kansasii infection, while questiomycin has no activity against NTM. At the same time, all the compounds of the present application also show better in vitro safety than questiomycin.

Claims

1. An N-hydrocarbyl-substituted phenoxazine compound of the formula: ###000001### wherein, The N-alkyl-substituted phenoxazine compound is selected from one of the following structures: 。 2. A pharmaceutical composition, wherein, The pharmaceutical composition contains the N-alkyl-substituted phenoxazine compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

3. Use of the N-alkyl-substituted phenoxazine compound of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2 in the manufacture of a medicament for the treatment of Mycobacterium avium infection.

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