WO2018233670A1 - Benzoisoxazole spiropyrimidinetrione compound, preparation method therefor and use thereof - Google Patents

Abstract

Disclosed in the present invention are a benzoisoxazole spiropyrimidinetrione compound, a preparation method therefor and use thereof, said compound having a structure as represented by formula (I). In said formula, the definition of each substituent is as described in the description and claims. The benzoisoxazole spiropyrimidinetrione compound of the present invention has higher antibacterial activities and better metabolic properties in vitro and in vivo.

Description

Benzoisoxazole spiropyrimidine compound, preparation method and use thereof Technical field

The present invention relates to a benzopyrazole group-containing spiropyrimidine compound, a pharmaceutical composition thereof, a process for the preparation thereof and use thereof in an anti-infective drug.

Background technique

With the widespread use and even abuse of antibacterial drugs, the multi-drug resistance of pathogenic bacteria has become a serious problem threatening human health. A series of drug-resistant pathogens represented by multidrug-resistant Staphylococcus aureus MRSA, Bringing great challenges to clinical treatment. In recent years, although some new antibacterial drugs such as linezolid and daptomycin have been marketed, it can effectively control the infection of Gram-positive bacteria such as MRSA. However, with more than 10 years of clinical application, bacteria have begun to slow down these drugs. Slowly producing resistant strains. Therefore, a new type of antibacterial drug with novel mechanism of research and development, unique structure and effective against drug-resistant bacteria is an urgent scientific research task and an unmet major clinical requirement.

Benzoisoxazole spirotriones are a new class of bacterial type II topoisomerase inhibitors. Although they interact with quinolones for bacterial type II topoisomerases, their specific regions of action and The mechanism of action is completely different. The novel chemical structure and new mechanism of action of benzoisoxazole spiropyrimidines have good antibacterial activity against sensitive and resistant Gram-positive bacteria, and there is no cross-resistance with clinical antibacterial drugs. Medicinal.

AstraZeneca and other companies reported a series of spirotriazol compounds, and a preliminary study on its structure-activity relationship found that the methyl-substituted spiropyrimidine compounds on isoxazole have good antibacterial activity. AstraZeneca continued to conduct derivatization studies on such compounds, and found AZD0914 with better activity, while eliminating the genotoxicity and bone marrow toxicity of such compounds. Currently, AZD0914 is undergoing Phase II clinical studies.

However, these spirotrione compounds, such as AZD0914, still have the problem that the antibacterial activity is not strong and the metabolic properties are not ideal, resulting in a large amount of clinical drug overdose, which limits the possibility of treating the systemic infection drug to some extent.

Therefore, there is still a need in the art to develop a spiropyrimidine compound for use in human or animal systemic infection with better activity and superior metabolic properties to overcome the above problems.

Summary of the invention

The object of the present invention is to provide a benzoisoxazole spiropyrimidine compound having better activity and superior metabolic properties, the enantiomers, diastereomers, racemates and mixtures thereof. And its pharmaceutically acceptable salts.

In a first aspect of the invention, there is provided a compound of the formula (I), or an enantiomer, diastereomer, racemate thereof and mixtures thereof, or a pharmaceutically acceptable thereof salt,

Wherein R ₁ is hydrogen, halogen or cyano;

R ₂ and R _{3 are} independently a hydrogen atom or a C ₁ -C ₃ alkyl group; or R ₂ and R ₃ together with a carbon atom to be bonded form a 3-7 membered aliphatic ring or a 3-7 membered oxygen-containing heterocyclic ring;

R ₄ is a hydrogen atom or a C ₁ -C ₃ alkyl group;

Each * indicates the racemization, S type or R type independently;

Provided that when R ₁ is a halogen, R ₂ and R ₃ together with the attached carbon atom form a 3-7 membered aliphatic ring or a 3-7 membered oxygen-containing heterocyclic ring.

The general formula (I) of the present invention means that the compound contains at least three chiral centers in the presence of enantiomers and diastereomers. For enantiomers, two enantiomers can be obtained using a general chiral resolution method or an asymmetric synthesis method. For the diastereomers, separation can be carried out by fractional recrystallization or chromatography. The compound represented by the formula (I) of the present invention includes any one of the above isomers or a mixture thereof.

In another preferred embodiment, R ₂ and R ₃ together with the attached carbon atom form a 3-7 membered aliphatic ring or a 3-7 membered oxygen-containing heterocyclic ring.

In another preferred embodiment, R ₂ and R _{3 are} simultaneously hydrogen or hydrogen at the same time.

In another preferred embodiment, R ₁ is hydrogen, fluorine, chlorine or cyano.

In another preferred embodiment, R ₁ is hydrogen, fluorine or chlorine; and R ₂ and R ₃ together with the attached carbon atoms form a 3-7 membered aliphatic ring or a 3-7 membered oxygen-containing heterocyclic ring.

In another preferred embodiment, R ₁ is hydrogen, fluorine or chlorine; and R ₄ is hydrogen.

In another preferred embodiment, R ₂ and R ₃ together with the attached carbon atom form a 3-7 membered aliphatic ring or a 3-7 membered oxygen-containing heterocyclic ring; and R ₄ is hydrogen.

In another preferred embodiment, R ₁ is a cyano group;

R ₂ and R _{3 are} independently a hydrogen atom or a C ₁ -C ₃ alkyl group; or R ₂ and R ₃ together with the attached carbon atom form a 3-7 membered aliphatic ring.

In another preferred embodiment, the compound is:

According to a second aspect of the present invention, there is provided a pharmaceutical composition comprising the compound of the first aspect or an enantiomer thereof, a diastereomer, a racemate, and a mixture thereof, Or a pharmaceutically acceptable salt thereof;

A pharmaceutically acceptable carrier or excipient.

The present invention provides a novel benzisoxazole spiropyrimidine compound which can be used alone or mixed with a pharmaceutically acceptable adjuvant (for example, an excipient, a diluent, etc.) to prepare a tablet for oral administration. , capsules, granules or syrups, etc. The pharmaceutical composition can be obtained by a conventional method of pharmacy.

A third aspect of the invention provides a process for the preparation of the compound of the first aspect, the method comprising the steps of:

(i) intermediate Ia by intramolecular nucleophilic substitution reaction to form intermediate Ib;

(ii) intermediate Ib is nucleophilic substituted to form intermediate Ic;

(iii) intermediate Ic is catalytically hydrolyzed to form intermediate Id;

(iv) intermediate Id by nucleophilic substitution reaction to form intermediate Ie;

(v) reacting intermediate Ie with barbituric acid to form a compound of formula I,

In the formulas, *, R ₁ , R ₂ , R ₃ and R ₄ are as defined above.

In another preferred embodiment, intermediate Ia is reacted with cesium carbonate by intramolecular nucleophilic substitution to form intermediate Ib.

In another preferred embodiment, the intermediate Ib is reacted with N,N'-carbonyldiimidazole by nucleophilic substitution to form the intermediate Ic.

In another preferred embodiment, the intermediate Id is reacted with 2R,6R-dimethylmorpholine by nucleophilic substitution to form the intermediate Ie.

In another preferred embodiment, the intermediate Ic is hydrolyzed by hydrochloric acid to form an intermediate Id.

A fourth aspect of the invention provides the process for the preparation of the compound of the first aspect, wherein when R ₁ is a cyano group, the method comprises the steps of:

(i') intermediate I-12 is hydrolyzed to form intermediate I-13;

(ii') intermediate I-13 is subjected to intramolecular nucleophilic substitution to give intermediate I-14;

(iii') intermediate I-14 is subjected to nucleophilic substitution to give intermediate I-15;

(iv') intermediate I-15 is subjected to nucleophilic substitution to give intermediate I-16;

(v') intermediate I-16 is reacted with barbituric acid to form the compound of claim 1 represented by formula C,

In the formulas, *, R ₂ , R ₃ and R ₄ are as defined above.

In another preferred embodiment, the intermediate I-12 is hydrolyzed by hydrochloric acid to form an intermediate I-13.

In another preferred embodiment, intermediate I-13 is reacted with sodium bicarbonate by intramolecular nucleophilic substitution to form intermediate I-14.

In another preferred embodiment, intermediate I-14 is reacted with 2R,6R-dimethylmorpholine by nucleophilic substitution to give intermediate I-15.

In another preferred embodiment, intermediate I-15 is reacted with N,N'-carbonyldiimidazole by nucleophilic substitution to give intermediate I-16.

A fifth aspect of the invention provides the compound of the first aspect, or an enantiomer, diastereomer, racemate or mixture thereof, or a pharmaceutically acceptable salt thereof, or a second The use of the pharmaceutical composition of the aspect for the preparation of a medicament for the treatment of a bacterial infectious disease.

In another preferred embodiment, the infectious disease is an infectious disease caused by Gram-positive bacteria.

In another preferred embodiment, the infectious disease is an infectious disease caused by a multidrug resistant bacteria.

In another preferred embodiment, the multidrug resistant bacteria are selected from the group consisting of MRSA, MSSA, MRSE, MSSE, PRSP, and Spy.

According to a sixth aspect of the present invention, there is provided an in vitro bacteriostatic method for administering a compound of the above aspect or an enantiomer thereof, a diastereomer, a racemate and a mixture thereof to a subject or the environment. Or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the second aspect.

According to a seventh aspect of the invention, there is provided a compound of the formula I, the structure of which is represented by the formula Ia, Ib, Ic, Id or Ie:

In the formulas, *, R ₁ , R ₂ , R ₃ and R ₄ are as defined above.

The compound of the invention has better in vitro and in vivo antibacterial activity, excellent in vivo metabolic properties, exposure and peak concentration are far superior to the positive control drug AZD0914, and the compound of the invention has better medicine than the positive control drug AZD0914. Sex, is expected to become a better antibacterial drug.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to form a new or preferred technical solution. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equal or similar purpose. Due to space limitations, we will not repeat them here.

Detailed ways

The inventors of the present application have extensively and intensively studied to carry out various structural modifications to AZD0914, especially to introduce a spiro ring structure on an oxazolidinone ring, and synthesize a series of novel spiropyrimidine compounds, which are found in vitro. In vivo antibacterial activity, drug metabolism properties are much better than AZD0914, it is suitable as a new antibacterial drug for human or animal antibacterial treatment of infection. On the basis of this, the present invention has been completed.

the term

In the present invention, the term "C ₁ -C ₃ alkyl" means a straight or branched alkyl group having 1 to 3 carbon atoms, and includes, without limitation, methyl, ethyl, propyl, isopropyl. .

In the present invention, the term "3-7 membered aliphatic ring" means a cyclic alkyl group having 3 to 7 carbon atoms in the ring, and includes, without limitation, a cyclopropane ring, a cyclobutane ring, a cyclopentane ring. , a cyclohexane ring, a cycloheptane ring, and the like.

In the present invention, the term "3-7 membered oxygen-containing heterocyclic ring" means a cycloalkyl ring having 3 to 7 ring atoms and containing 1, 2 or 3 O atoms, and includes, without limitation, a propylene oxide ring. , butylene oxide ring, epoxy heptane ring and the like.

The "pharmaceutically acceptable salt" includes pharmaceutically acceptable base addition salts. These include, but are not limited to, salts of inorganic bases such as sodium, potassium, calcium and magnesium salts. These include, but are not limited to, salts of organic bases such as ammonium salts, triethylamine salts, lysine salts, arginine salts and the like. These salts can be prepared by methods known in the art.

Preparation

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

Route 1

In a preferred embodiment, Compound 1, Compound 2, Compound 3 and Compound 4 are prepared in Scheme 1.

R ₂ , R ₃ and R ₄ are as defined above.

a, intermediate A [J. Med. Chem, 2015, 58 (15): 6264-6282.] and N-chlorosuccinimide in a polar aprotic solvent, from room temperature to 50 ° C conditions The lower reaction is carried out for 20-60 minutes (minutes) to obtain intermediate I-1. The polar aprotic solvent may be: 1,4-dioxane, toluene, tetrahydrofuran, N,N-dimethylformamide, The optimum reaction temperature was 40 ° C and the optimum reaction time was 30 min.

b, intermediate I-1 in the presence of different primary amines in a polar aprotic solvent, 0 ° C to room temperature reaction for 20-60min, nucleophilic substitution to form the corresponding intermediate I-2, The primary amine is a variety of suitable primary amines, and the polar aprotic solvent may be: 1,4-dioxane, toluene, tetrahydrofuran, N,N-dimethylformamide, optimum reaction temperature The optimum reaction time was 30 min at 0 °C.

c. Intermediate I-2 is reacted in a polar aprotic solvent in a polar aprotic solvent at room temperature to 70 ° C for 2-5 h (hours) in the presence of cesium carbonate to form the corresponding intermediate I-3. The aprotic solvent may be: 1,4-dioxane, toluene, tetrahydrofuran, N,N-dimethylformamide, the optimum reaction temperature is 60 ° C, and the optimum reaction time is 3 h.

d, intermediate I-3 and N,N'-carbonyldiimidazole are catalyzed by 4-dimethylaminopyridine in a polar aprotic solvent at 60-90 ° C for 3-8 h to form a corresponding Intermediate I-4, the polar aprotic solvent may be: 1,4-dioxane, toluene, tetrahydrofuran, N,N-dimethylformamide, the optimum reaction temperature is 80 ° C, the best The reaction time was 5 h.

e, intermediate I-4 and hydrochloric acid, reacted in a solvent at room temperature to 50 ° C for 2-5 h to form the corresponding intermediate I-5, the solvent may be 1,4-dioxane, tetrahydrofuran, N, N-dimethylformamide, the optimum reaction temperature is 30 ° C, and the optimum reaction time is 3 h.

f, intermediate I-5 and 2R, 6R-dimethylmorpholine, in the presence of an organic base, in a polar aprotic solvent, reacted at 70-100 ° C for 12-20h to form the corresponding intermediate I -6, the organic base may be triethylamine or N,N-diisopropylethylamine, and the polar aprotic solvent may be acetonitrile, 1,4-dioxane, tetrahydrofuran, N, N. - dimethylformamide, the optimum reaction temperature is 80 ° C, and the optimum reaction time is 15 h.

g, intermediate I-6 and barbituric acid, in a mixed solvent of acetic acid and water, reacted with 100-120 ° C for 4-7h, the corresponding compound B is formed, the optimal reaction temperature is 100 ° C, the best reaction The time is 5h.

Route 2

In a preferred embodiment, Compound 5, Compound 6 and Compound 7 are prepared in Scheme 2.

a, 2,6-difluorobenzonitrile and lithium diisopropylamide, N,N-dimethylformamide, reacted in a polar aprotic solvent at -80 ~ -65 ° C for 3-8min, resulting Intermediate I-7, the polar aprotic solvent may be tetrahydrofuran, dichloromethane, the optimum reaction temperature is -80 ° C, and the optimum reaction time is 5 min.

b. Intermediate I-7 and ethylene glycol are reacted in a toluene solvent at 100-130 ° C for 3-8 h under catalytic conditions of p-toluenesulfonic acid to form intermediate I-8. The optimum reaction temperature is 120 ° C. The optimal reaction time is 4h.

c. Intermediate I-8, lithium diisopropylamide and N,N-dimethylformamide are reacted in a polar aprotic solvent at -80 to -65 ° C for 20-60 min to form intermediate I. -9, the polar aprotic solvent may be tetrahydrofuran, dichloromethane, the optimum reaction temperature is -80 ° C, and the optimum reaction time is 30 min.

d, intermediate I-9 and hydroxylamine hydrochloride, in an organic base, in a polar solvent, at 0 ° C to room temperature for 1-3h to form intermediate I-10, the organic base can be pyridine, triethyl Amine or N,N-diisopropylethylamine, the polar solvent may be methanol, dichloromethane, 1,4-dioxane, tetrahydrofuran, N,N-dimethylformamide, preferably The reaction temperature was 0 ° C and the optimum reaction time was 2 h.

e, intermediate I-10 and N-chlorosuccinimide, reacted in a polar aprotic solvent at room temperature to 50 ° C for 20-60 min to obtain intermediate I-11, the polarity The aprotic solvent may be: 1,4-dioxane, toluene, tetrahydrofuran, N,N-dimethylformamide, the optimum reaction temperature is 40 ° C, and the optimum reaction time is 30 min.

f, intermediate I-11 and various qualified primary amines, in a polar aprotic solvent, reacted at 0 ° C to room temperature for 10-60 min, nucleophilic substitution to form intermediate I-12, the polarity is not The protic solvent may be: 1,4-dioxane, toluene, tetrahydrofuran, N,N-dimethylformamide, the optimum reaction temperature is 0 ° C, and the optimum reaction time is 30 min.

g, intermediate I-12 and hydrochloric acid, reacted in a solvent at room temperature to 50 ° C for 2-5h to form intermediate I-13, the solvent may be: 1,4-dioxane, tetrahydrofuran, N, N-dimethylformamide, the optimum reaction temperature is 40 ° C, and the optimum reaction time is 3 h.

h, intermediate I-13 and aqueous sodium hydrogencarbonate, reacting in a solvent at 0 ° C to 40 ° C for 1-4 h to form intermediate I-14, the solvent may be: 1,4-dioxane, Tetrahydrofuran, N,N-dimethylformamide, the optimum reaction temperature is 25 ° C, and the optimum reaction time is 2 h.

i, intermediate I-14 and 2R, 6R-dimethylmorpholine, in the presence of an organic base, in a polar aprotic solvent, reacted at 70-100 ° C for 12-20h to form intermediate I-15, The organic base may be triethylamine or N,N-diisopropylethylamine, and the polar aprotic solvent may be acetonitrile, 1,4-dioxane, tetrahydrofuran, N,N-dimethyl The optimum reaction temperature is 80 ° C and the optimum reaction time is 12 h.

j, intermediate I-15 and N, N'-carbonyldiimidazole, catalyzed by 4-dimethylaminopyridine, reacted in a polar aprotic solvent at 60-90 ° C for 3-8 h, corresponding to the corresponding The intermediate I-16, the polar aprotic solvent may be: 1,4-dioxane, toluene, tetrahydrofuran, N, N-dimethylformamide, the optimum reaction temperature is 80 ° C, the most The reaction time is 5h.

k, intermediate I-16 and barbituric acid, in a mixed solvent of acetic acid and water, reacted at 100-120 ° C for 4-7h to form compound C, the optimal reaction temperature is 110 ° C, the optimal reaction time is 5h .

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising an active ingredient in a safe and effective amount, together with a pharmaceutically acceptable carrier.

As used herein, "active ingredient" refers to a compound of formula I as described herein.

The "active ingredient" and pharmaceutical composition of the present invention are useful for the preparation of a medicament for treating an infectious disease. In another preferred embodiment, a medicament for the preparation of an infectious disease caused by a multidrug-resistant bacterium is prepared and/or treated.

By "safe and effective amount" is meant that the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects. In general, the pharmaceutical compositions contain from 1 to 2000 mg of active ingredient per dose, more preferably from 10 to 200 mg of active ingredient per dose. Preferably, the "one dose" is a tablet.

"Pharmaceutically acceptable carrier" means: one or more compatible solid or liquid fillers or gel materials which are suitable for human use and which must be of sufficient purity and of sufficiently low toxicity. By "compatibility" it is meant herein that the components of the composition are capable of intermingling with the active ingredients of the present invention and with respect to each other without significantly reducing the efficacy of the active ingredients. Examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid). , magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifier

Wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The administration form of the active ingredient or the pharmaceutical composition of the present invention is not particularly limited, and representative administration methods include

(but not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous).

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs.

The compounds of the invention may be administered alone or in combination with other therapeutic agents such as antibacterials.

When a pharmaceutical composition is used, a safe and effective amount of a compound of the invention is administered to a mammal (e.g., a human) in need of treatment wherein the dosage is a pharmaceutically effective effective dosage, for a 60 kg body weight, The dose to be administered is usually from 1 to 2000 mg, preferably from 20 to 500 mg. Of course, specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples, which do not specify the specific conditions, are usually manufactured according to conventional conditions (such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)). The conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight and parts by weight.

Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described may be employed in the methods of the invention. The preferred embodiments and materials described herein are for illustrative purposes only.

In all of the following examples, ¹ H-NMR was recorded on a Varian Mercury 400 nuclear magnetic resonance apparatus, ¹³ C-NMR was recorded on a BRUKER 500 MHz nuclear magnetic resonance apparatus (low temperature), and the chemical shift was expressed in δ (ppm); For the 200-300 mesh, the ratio of the eluents is a volume ratio.

Example 1: (2R, 4S, 4aS)-11-fluoro-2,4-dimethyl-8-(5-oxo-4-oxo-6-azaspiro[2.4]heptane-6-yl -1,2,4,4a-tetrahydro-2'H,6H-spiro[isoxazole [4,5-g][1,4]oxazine [4,3-a]quinoline-5, 5'-pyrimidine]-2',4',6'(1'H,3'H)-trione (Compound 1)

(a) 1-(((5-(1,3-dioxolan-2-yl)-6,7-difluorobenzo[d]isoxazol-3-yl)amino)methyl)- 1-cyclopropanol (I-3-1)

Intermediate A (1 g, 4.048 mmol) was added to 20 ml of N,N-dimethylformamide, and the mixture was stirred and dissolved. N-chlorosuccinimide (650 mg, 4.668 mmol) was added at room temperature. The reaction was carried out at 40 ° C for 30 min, and the reaction was monitored by TLC (thin-layer chromatography). The reaction mixture was taken to 0 ° C, and 1-aminomethyl-1-cyclopropanol (1.06 g, 12.144 mmol) was slowly added and returned to room temperature for 1 h. The reaction was monitored by TLC. EtOAc (EtOAc: EtOAc (EtOAc: EtOAc) dried over anhydrous sodium sulfate, and concentrated under reduced pressure and column chromatography [petroleum ether: ethyl acetate = 1: 1] to give a white solid 810 mg, yield ^{65%, 1 H NMR (400MHz} , CDCl 3) δ7.53 (dd, J = 5.4, 1.5 Hz, 1H), 6.13 (s, 1H), 4.79 (t, J = 5.6 Hz, 1H), 4.22 - 4.09 (m, 4H), 3.58 (d, J = 5.5 Hz, 2H), 2.65 (s, 1H), 0.95 (t, J = 6.2 Hz, 2H), 0.75 (t, J = 6.2 Hz, 2H).

(b) 6-(5-((1,3-dioxolan-2-yl)-6,7-difluorobenzo[d]isoxazol-3-yl)-4-oxo-6- Azaspiro[2.4]heptane-5-one (I-4-1)

Intermediate 1-3-1 (344 mg, 1.036 mmol) was added to 15 ml of N,N-dimethylformamide, and dissolved by stirring, and carbonyldiimidazole (671 mg, 4.414 mmol) and 4-dimethylamino group were sequentially added, respectively. The pyridine (63 mg, 0.52 mmol) was heated to 115 ° C for 8 h. The reaction was taken from EtOAc EtOAc. Column chromatography [petroleum ether: ethyl acetate = 2:1] afforded 337 mg of white solid (yield: 96%, ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.55 (dd, J = 5.9, 1.9 Hz, 1H) , 6.16 (s, 1H), 4.31 (s, 2H), 4.22 - 4.09 (m, 4H), 1.44 - 1.39 (m, 2H), 0.98 - 0.92 (m, 2H).

(c) 6,7-Difluoro-3-(5-oxo-4-oxo-6-azaspiro[2.4]heptan-6-yl)benzo[d]isoxazole-5-carbaldehyde ( I-5-1)

The intermediate I-4-1 (330 mg, 0.976 mmol) was dissolved in 15 ml of 1,4-dioxane, and 6M hydrochloric acid (5 ml) was added, and the mixture was stirred at room temperature for 2 h. The organic layer was combined, washed with water, brine, dried over anhydrous sodium sulfate sulfatesssssssssssssssssss , ¹ H NMR (400MHz, CDCl ₃ ) δ 10.30 (s, 1H), 9.00 (dd, J = 5.9, 1.9 Hz, 1H), 4.32 (s, 2H), 1.44 (m, 2H), 1.00 - 0.95 (m, 2H).

(d) 6-((2R,6R)-2,6-dimethylmorpholine)-7-fluoro-3-(5-oxo-4-oxo-6-azaspiro[2.4]heptane- 6-yl)benzo[d]isoxazole-5-formaldehyde (I-6-1)

Intermediate 1-5-1 (200 mg, 0..67 mmol) was dissolved in 10 ml of acetonitrile and stirred to dissolve. 2R,6R-dimethylmorpholine (0.31 ml, 2.54 mmol) and N, N were sequentially added. -diisopropylethylamine (0.42 ml, 2.54 mmol), the reaction mixture was refluxed for 10 h at 90 ° C, the reaction was completed by TLC, cooled to room temperature, extracted with water and ethyl acetate. washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure and column chromatography [petroleum ether: ethyl acetate = 5: 1] to give a pale yellow solid 80mg, yield ^{31%, 1 H NMR (400MHz} , CDCl3) δ10.39 (s, 1H), 8.83 (s, 1H), 4.30 (s, 2H), 4.29 - 4.22 (m, 2H), 3.45 (d, J = 11.7 Hz, 2H), 3.06 (dd, J = 11.7, 5.4 Hz, 2H), 1.45 - 1.40 (m, 2H), 1.34 (d, J = 6.5 Hz, 6H), 0.98 - 0.93 (m, 2H).

(e) (2R, 4S, 4aS)-11-fluoro-2,4-dimethyl-8-(5-oxo-4-oxo-6-azaspiro[2.4]heptane-6-yl) -1,2,4,4a-tetrahydro-2'H,6H-spiro[isoxazole [4,5-g][1,4]oxazine [4,3-a]quinoline-5,5 '-Pyrimidine]-2',4',6'(1'H,3'H)-Trione (Compound 1)

Intermediate I-6-1 (80 mg, 0.206 mmol) was added to a mixed solvent of 4 ml of acetic acid and 1 ml of water, stirred to dissolve, and barbituric acid (32 mg, 0.246 mmol) was added thereto, and the mixture was heated to 100 ° C for 3 h. The reaction was completed by TLC, and the mixture was cooled to room temperature. EtOAc (EtOAc) White solid 78 mg, yield 76%, mp 266-267 ° C; ¹ H NMR (400 MHz, DMSO) δ 11.82 (s, 1H), 11.46 (s, 1H), 7.76 (s, 1H), 4.29 - 4.21. , 2H), 4.11 (d, J = 12.9 Hz, 1H), 3.94 (d, J = 8.8 Hz, 1H), 3.78 (br s, 1H), 3.72 - 3.62 (m, 2H), 3.17 - 3.06 (m , 1H), 2.92 (d, J = 14.1 Hz, 1H), 1.24-1.19 (m, 2H), 1.15 (d, J = 6.3 Hz, 3H), 1.00 - 0.95 (m, 2H), 0.90 (d, J = 6.3 Hz, 3H). ¹³ C NMR (126 MHz, DMSO) δ 171.40, 168.15, 154.20 (d, J = 12.8 Hz), 153.79, 153.17, 149.98, 135.30, 133.76 (d, J = 238.8 Hz), 122.85 , 118.83, 106.52, 72.58, 72.14, 64.88, 62.33, 56.73 (d, J = 9.3 Hz), 53.38, 50.71, 39.02, 18.64, 18.61, 10.32, 10.26; MS (ESI) m/z: [(M+1) ) ⁺ , 500.1].

Example 2: (2R, 4S, 4aS)-11-fluoro-2,4-dimethyl-8-(6-oxo-2,5-dioxo-7-azaspiro[3.4]octane- 7-yl)-1,2,4,4a-tetrahydro-2'H,6H-spiro[isoxazole[4,5-g][1,4]oxazine[4,3-a]quinoline -5,5'-pyrimidine]-2',4',6'(1'H,3'H)-trione (Compound 2)

(a) 3-(((5-(1,3-dioxolan-2-yl)-6,7-difluorobenzo[d]isoxazol-3-yl)amino)methyl)oxy Heterocyclic butanol (I-3-2)

According to the synthesis method of the intermediate I-3-1, intermediate A (1.2 g, 4.857 mmol), N-chlorosuccinimide (778 mg, 5.828 mmol), 3-(aminomethyl) oxacyclohexane Butanol (1.25g, 12.142mmol) and cesium carbonate (4.748g, 14.571mmol) were used as raw materials to synthesize 1-(((5-(1,3-dioxolan-2-yl)-6,7-di) Fluorobenzo[d]isoxazol-3-yl)amino)methyl)oxybutanol 949 mg, white solid, yield 59.5%, ¹ H NMR (400 MHz, DMSO) δ 8.08 (d, J = 5.7 Hz, 1H), 7.41 (t, J = 5.7 Hz, 1H), 6.09 (s, 1H), 6.01 (s, 1H), 4.48 (dd, J = 15.1, 6.6 Hz, 4H), 4.12 - 3.99 ( m, 4H), 3.55 (d, J = 5.8 Hz, 2H).

(b) 7-(5-((1,3-Diethoxylan-2-yl)-6,7-difluorobenzo[d]isoxazol-3-yl)-2,5-dioxo -7-azaspiro[3.4]octane-6-one (I-4-2)

Intermediate I-3-2 (940 mg, 2.86 mmol), N,N'-carbonyldiimidazole (1.857 g, 11.45 mmol) and 4-dimethylaminopyridine according to the procedure of Intermediate 1-4-1. (175 mg, 1.43 mmol) as a starting material for the synthesis of 7-(5-((1,3-dioxolan-2-yl)-6,7-difluorobenzo[d]isoxazole-3-yl) -2,5-Dioxa-7-azaspiro[3.4]octane-6-one 872 mg, white solid, yield 86%, ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.44 (dd, J = 5.9 , 1.8 Hz, 1H), 6.12 (s, 1H), 5.10 (d, J = 8.8 Hz, 2H), 4.82 (d, J = 8.7 Hz, 2H), 4.49 (s, 2H), 4.20 - 4.04 (m , 4H).

(c) 6,7-Difluoro-3-(6-oxo-2,5-dioxo-7-azaspiro[3.4]octane-7-yl)benzo[d]isoxazole-5 - Formaldehyde (I-5-2)

Synthesis of 6,7-difluoro-3-(6-oxo) from Intermediate I-4-2 (885 mg, 2.498 mmol) and 6M hydrochloric acid (30 mL). -2,5-Dioxa-7-azaspiro[3.4]octane-7-yl)benzo[d]isoxazole-5-formaldehyde 580 mg, white solid, yield 75%, ¹ H NMR (400 MHz , CDCl ₃ ) δ 10.31 (s, 1H), 8.91 (dd, J = 5.8, 1.8 Hz, 1H), 5.14 (d, J = 9.0 Hz, 2H), 4.85 (d, J = 8.7 Hz, 2H) , 4.53 (s, 2H).

(d) 6-((2R,6R)-2,6-Dimethylmorpholine)-7-fluoro-3-(6-oxo-2,5-dioxo-7-azaspiro[3.4] Octane-7-yl)benzo[d]isoxazole-5-carbaldehyde (I-6-2)

Intermediate I-5-2 (150 mg, 0.484 mmol), 2R,6R-dimethylmorpholine (139 mg, 1.209 mmol) and N,N-diisopropyl Synthesis of 6-((2R,6R)-2,6-dimethylmorpholine)-7-fluoro-3-(6-oxo-2,5-di) by using ethylamine (187 mg, 1.451 mmol) as a starting material Oxy-7-azaspiro[3.4]octane-7-yl)benzo[d]isoxazole-5-carboxaldehyde 172 mg, white solid, yield 87%, ¹ H NMR (400 MHz, CDCl ₃ ) δ ₁₀ . 37 (s, 1H), 8.71 (s, 1H), 5.14 (d, J = 8.3 Hz, 2H), 4.85 (d, J = 8.2 Hz, 2H), 4.50 (s, 2H), 4.30 - 4.22 (m) , 2H), 3.45 (d, J = 12.0 Hz, 2H), 3.06 (dd, J = 11.7, 5.6 Hz, 2H), 1.34 (d, J = 6.5 Hz, 6H).

(e) (2R, 4S, 4aS)-11-fluoro-2,4-dimethyl-8-(6-oxo-2,5-dioxo-7-azaspiro[3.4]octane-7 -yl)-1,2,4,4a-tetrahydro-2'H,6H-spiro[isoxazole [4,5-g][1,4]oxazine [4,3-a]quinoline- 5,5'-pyrimidine]-2',4',6'(1'H,3'H)-trione (compound 2)

(2R, 4S, 4aS)-11-fluoro-2 was synthesized according to the synthesis method of compound 1, using intermediate I-6-2 (93 mg, 0.229 mmol) and barbituric acid (33 mg, 0.252 mmol) as raw materials. 4-Dimethyl-8-(6-oxo-2,5-dioxo-7-azaspiro[3.4]octane-7-yl)-1,2,4,4a-tetrahydro-2' H,6H-spiro[isoxazole [4,5-g][1,4]oxazine [4,3-a]quinoline-5,5'-pyrimidine]-2',4',6' ( 1'H,3'H)-trione 90 mg, white solid, yield 76%, ¹ H NMR (400 MHz, DMSO) δ 11.63 (s, 2H), 7.73 (s, 1H), 4.86 - 4.76 (m) , 4H), 4.43–4.36 (m, 2H), 4.10 (d, J = 12.5 Hz, 1H), 3.93 (d, J = 8.9 Hz, 1H), 3.83–3.74 (m, 1H), 3.72–3.60 ( m, 2H), 3.18 - 3.05 (m, 1H), 2.90 (d, J = 13.7 Hz, 1H), 1.14 (d, J = 6.2 Hz, 3H), 0.89 (d, J = 6.4 Hz, 3H); ¹³ C NMR (126 MHz, DMSO) δ 171.46, 168.17, 154.15 (d, J = 13.0 Hz), 152.88, 152.86, 150.07, 135.28, 133.74 (d, J = 238.9 Hz), 122.83, 118.85, 106.49, 81.72, 81.52, 80.54, 72.57, 72.12, 64.90, 56.68, 53.38, 52.55, 39.02, 18.62, 18.62; MS (EI) m/z: [M ⁺ , 515].

Example 3: (2R, 4S, 4aS)-11-fluoro-2,4-dimethyl-8-(6-oxo-5-oxo-7-azaspiro[3.4]octane-7-yl -1,2,4,4a-tetrahydro-2'H,6H-spiro[isoxazole [4,5-g][1,4]oxazine [4,3-a]quinoline-5, 5'-pyrimidine]-2',4',6'(1'H,3'H)-trione (compound 3)

(a) 1-(((5-(1,3-dioxolan-2-yl)-6,7-difluorobenzo[d]isoxazol-3-yl)amino)methyl)) Butanol (I-3-3)

According to the synthesis method of Intermediate 1-3-1, Intermediate A (750 mg, 3.036 mmol), N-chlorosuccinimide (527 mg, 3.947 mmol), 1-(aminomethyl)cyclobutanol ( 675 mg, 6.679 mmol) and cesium carbonate (3.957 g, 12.144 mmol) were used as starting materials to synthesize 1-(((5-(1,3-dioxolan-2-yl)-6,7-difluorobenzo[ d] isoxazol-3-yl)amino)methyl)cyclobutanol 710 mg, white solid, yield 72%, ¹ H NMR (400 MHz, DMSO) δ 8.15 (d, J = 5.9 Hz, 1H), 7.23 (t, J = 5.4 Hz, 1H), 6.08 (s, 1H), 5.27 (s, 1H), 4.11 - 3.99 (m, 4H), 3.34 (d, J = 5.6 Hz, 2H), 2.12 - 1.92 (m, 4H), 1.72–1.45 (m, 2H).

(b) 7-(5-((1,3-dioxolan-2-yl)-6,7-difluorobenzo[d]isoxazol-3-yl)-5-oxo-7- Azaspiro[3.4]octane-6-one (I-4-3)

Intermediate I-3-3 (533 mg, 1.63 mmol), N,N'-carbonyldiimidazole (1.059 g, 6.53 mmol) and 4-dimethylaminopyridine according to the procedure of Intermediate 1-4-1. (100 mg, 0.82 mmol) was used as a starting material to synthesize 7-(5-((1,3-dioxolan-2-yl)-6,7-difluorobenzo[d]isoxazol-3-yl) -5-oxo-7-azaspiro[3.4]octane-6-one 468 mg, white solid, yield 82%, ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.51 (dd, J = 5.9, 1.8 Hz , 1H), 6.15 (s, 1H), 4.26 (s, 2H), 4.22 - 4.07 (m, 4H), 2.71 (ddd, J = 19.3, 10.1, 2.9 Hz, 2H), 2.38 (ddd, J = 11.4) , 8.5, 3.7 Hz, 2H), 2.09 - 1.97 (m, 1H), 1.85 - 1.71 (m, 1H).

(c) 6,7-Difluoro-3-(6-oxo-5-oxo-7-azaspiro[3.4]octane-7-yl)benzo[d]isoxazole-5-carbaldehyde ( I-5-3)

The synthesis of 6,7-difluoro-3-(6-oxo) was carried out according to the synthesis of the intermediate I-5-1 using the intermediate I-4-3 (468 mg, 1.328 mmol) and 6M hydrochloric acid (15 ml). -5-oxo-7-azaspiro[3.4]octane-7-yl)benzo[d]isoxazole-5-carbaldehyde 380 mg, white solid, yield 93%, ¹ H NMR (400 MHz, CDCl ₃ ) δ 10.29 (s, 1H), 8.95 (dd, J = 5.8, 1.7 Hz, 1H), 4.27 (s, 2H), 2.73 (ddd, J = 19.5, 10.1, 2.9 Hz, 2H), 2.44 - 2.35 (m, 2H), 2.10 - 2.00 (m, 1H), 1.87 - 1.74 (m, 1H).

(d) 6-((2R,6R)-2,6-dimethylmorpholine)-7-fluoro-3-(6-oxo-5-oxo-7-azaspiro[3.4]octane- 7-yl)benzo[d]isoxazole-5-carbaldehyde (I-6-3)

According to the synthesis of the intermediate I-6-1, intermediate I-5-3 (100 mg, 0.324 mmol), 2R, 6R-dimethylmorpholine (75 mg, 0.649 mmol) and N,N-diisopropyl Synthesis of 6-((2R,6R)-2,6-dimethylmorpholine)-7-fluoro-3-(6-oxo-5-oxo-7) with ethylamine (126 mg, 0.973 mmol) as starting material - azaspiro[3.4]octane-7-yl)benzo[d]isoxazole-5-carbaldehyde 103 mg, white solid, yield 79%, ¹ H NMR (400 MHz, CDCl ₃ ) δ 10.38 (s , 1H), 8.79 (s, 1H), 4.31 - 4.20 (m, 4H), 3.44 (d, J = 12.1 Hz, 2H), 3.05 (dd, J = 11.9, 5.6 Hz, 2H), 2.72 (ddd, J=19.8, 10.0, 3.2 Hz, 2H), 2.43–2.34 (m, 2H), 2.10–1.98 (m, 1H), 1.85–1.73 (m, 1H), 1.34 (d, J=6.4 Hz, 6H) .

(e) (2R, 4S, 4aS)-11-fluoro-2,4-dimethyl-8-(6-oxo-5-oxo-7-azaspiro[3.4]octane-7-yl) -1,2,4,4a-tetrahydro-2'H,6H-spiro[isoxazole [4,5-g][1,4]oxazine [4,3-a]quinoline-5,5 '-pyrimidine]-2',4',6'(1'H,3'H)-trione (compound 3)

(2R, 4S, 4aS)-11-fluoro-2 was synthesized according to the synthesis method of compound 1, using intermediate I-6-3 (92 mg, 0.228 mmol) and barbituric acid (32 mg, 0.251 mmol) as raw materials. 4-Dimethyl-8-(6-oxo-5-oxo-7-azaspiro[3.4]octane-7-yl)-1,2,4,4a-tetrahydro-2'H,6H - snail [isoxazole [4,5-g][1,4]oxazine [4,3-a]quinoline-5,5'-pyrimidine]-2',4',6'(1'H , 3'H) - 99 mg of trione, as a white solid, yield ^{84%, 1 H NMR (400MHz} , DMSO) δ11.73 (s, 1H), 11.47 (s, 1H), 7.70 (s, 1H), 4.23 (s, 2H), 4.10 (d, J = 12.5 Hz, 1H), 3.93 (d, J = 8.8 Hz, 1H), 3.84 - 3.74 (m, 1H), 3.71 - 3.62 (m, 2H), 3.17 - 3.05 (m, 1H), 2.91 (d, J = 13.9 Hz, 1H), 2.50 - 2.32 (m, 4H), 1.89 - 1.63 (m, 2H), 1.15 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H). ¹³ C NMR (126 MHz, DMSO) δ 171.41, 168.15, 154.12 (d, J = 12.9 Hz), 153.34, 153.24, 150.00, 135.26, 133.76 (d, J = 238.8 Hz) , 122.77, 118.81, 106.65, 81.39, 72.57, 72.13, 64.87, 56.73 (d, J = 9.4 Hz), 55.57, 53.37, 39.01, 34.95, 34.79, 18.64, 18.61, 11.96; MS (EI) m/z: [ M ⁺ , 513].

Example 4: (2R,4S,4aS)-11-fluoro-2,4-dimethyl-8-(2-oxo-1-oxo-3-azaspiro[4.4]decane-3-yl -1,2,4,4a-tetrahydro-2'H,6H-spiro[isoxazole [4,5-g][1,4]oxazine [4,3-a]quinoline-5, 5'-pyrimidine]-2',4',6'(1'H,3'H)-trione (compound 4)

(a) 1-(((5-(1,3-dioxolan-2-yl)-6,7-difluorobenzo[d]isoxazol-3-yl)amino)methyl)) Pentanol (I-3-4)

According to the synthesis method of Intermediate 1-3-1, Intermediate A (600 mg, 2.430 mmol), N-chlorosuccinimide (422 mg, 3.160 mmol), 1-(aminomethyl)cyclopentanol ( 615 mg, 5.346 mmol) and cesium carbonate (3.167 g, 9.720 mmol) were used as starting materials to synthesize 1-(((5-(1,3-dioxolan-2-yl)-6,7-difluorobenzo[ d] isoxazol-3-yl)amino)methyl)cyclopentanol 582 mg, white solid, yield 70%, ¹ H NMR (400 MHz, DMSO) δ 8.13 (d, J = 5.0 Hz, 1H), 7.19 (t, J = 5.6 Hz, 1H), 6.08 (s, 1H), 4.51 (s, 1H), 4.14 - 3.99 (m, 4H), 3.31 (d, J = 5.8 Hz, 2H), 1.78 - 1.50 (m, 8H).

(b) 3-(5-((1,3-dioxolan-2-yl)-6,7-difluorobenzo[d]isoxazol-3-yl)-1-oxo-3- Azaspiro[4.4]decane-2-one (I-4-4)

Intermediate 1-3-4 (526 mg, 1.546 mmol), N,N'-carbonyldiimidazole (1.002 g, 6.182 mmol) and 4-dimethylaminopyridine according to the procedure of Intermediate 1-4-1. (95mg, 0.773mmol) was used as raw material to synthesize 3-(5-((1,3-dioxolan-2-yl)-6,7-difluorobenzo[d]isoxazole-3-yl) 1-oxo-3-azaspiro[4.4]nonan-2-one 450 mg, white solid, yield 79%, ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.54 (dd, J = 5.9, 1.7 Hz , 1H), 6.16 (s, 1H), 4.23 - 4.07 (m, 6H), 2.32 - 2.21 (m, 2H), 2.03 - 1.80 (m, 6H).

(c) 6,7-Difluoro-3-(2-oxo-1-oxo-3-azaspiro[4.4]decane-3-yl)benzo[d]isoxazole-5-carbaldehyde ( I-5-4)

Synthesis of 6,7-difluoro-3-(2-oxo) from the intermediate I-4-4 (158 mg, 0.431 mmol) and 6M hydrochloric acid (5 ml). -1-Oxo-3-azaspiro[4.4]decane-3-yl)benzo[d]isoxazole-5-carboxaldehyde 135 mg, white solid, yield 97%, ¹ H NMR (400 MHz, CDCl ₃ ) δ 10.30 (s, 1H), 8.98 (dd, J = 5.9, 1.8 Hz, 1H), 4.17 (s, 2H), 2.33 - 2.22 (m, 2H), 2.08 - 1.81 (m, 6H).

(d) 6-((2R,6R)-2,6-dimethylmorpholine)-7-fluoro-3-(2-oxo-1-oxo-3-azaspiro[4.4]decane- 3-yl)benzo[d]isoxazole-5-formaldehyde (I-6-4)

Intermediate 1--5-4 (100 mg, 0.310 mmol), 2R,6R-dimethylmorpholine (72 mg, 0.620 mmol) and N,N-diisopropyl. Synthesis of 6-((2R,6R)-2,6-dimethylmorpholine)-7-fluoro-3-(2-oxo-1-oxo-3) by using ethylamine (120 mg, 0.931 mmol) as a starting material - azaspiro[4.4]decane-3-yl)benzo[d]isoxazole-5-formaldehyde 114 mg, pale white solid, yield 88%, ¹ H NMR (400 MHz, CDCl ₃ ) δ 10.38 ( s, 1H), 8.81 (s, 1H), 4.31 - 4.21 (m, 2H), 4.15 (s, 2H), 3.44 (d, J = 12.1 Hz, 2H), 3.05 (dd, J = 11.8, 5.5 Hz , 2H), 2.28 (m, 2H), 2.06 - 1.80 (m, 6H), 1.34 (d, J = 6.4 Hz, 6H).

(e) (2R, 4S, 4aS)-11-fluoro-2,4-dimethyl-8-(2-oxo-1-oxo-3-azaspiro[4.4]decane-3-yl) -1,2,4,4a-tetrahydro-2'H,6H-spiro[isoxazole [4,5-g][1,4]oxazine [4,3-a]quinoline-5,5 '-pyrimidine]-2',4',6'(1'H,3'H)-trione (Compound 4)

(2R, 4S, 4aS)-11-fluoro-2 was synthesized according to the synthesis method of compound 1, using intermediate I-6-4 (99 mg, 0.237 mmol) and barbituric acid (34 mg, 0.261 mmol) as raw materials. 4-Dimethyl-8-(2-oxo-1-oxo-3-azaspiro[4.4]decane-3-yl)-1,2,4,4a-tetrahydro-2'H,6H - snail [isoxazole [4,5-g][1,4]oxazine [4,3-a]quinoline-5,5'-pyrimidine]-2',4',6'(1'H , 3'H)-trione 94 mg, white solid, yield 75%, ¹ H NMR (400 MHz, DMSO) δ 11.82 (s, 1H), 11.46 (s, 1H), 7.73 (s, 1H), 4.14 –4.08(m,3H),3.93(d,J=8.8Hz,1H),3.83–3.74(m,1H), 3.70–3.62(m,2H),3.15–3.07(m,1H),2.91(d , J = 13.7 Hz, 1H), 2.12 - 2.04 (m, 2H), 1.96 - 1.87 (m, 2H), 1.80 - 1.71 (m, 4H), 1.15 (d, J = 6.2 Hz, 3H), 0.89 ( d, J = 6.4 Hz, 3H); ¹³ C NMR (151 MHz, DMSO) δ 170.96, 167.69, 153.68 (d, J = 12.9 Hz), 153.66, 152.90, 149.54, 134.78, 133.29 (d, J = 238.8 Hz), 122.23, 118.53, 106.19, 90.29, 72.12, 71.68, 64.42, 56.27 (d, J = 8.9 Hz), 53.33, 52.92, 38.55, 37.72, 37.55, 23.11, 23.08, 18.19, 18.17; MS (EI) m/z: [M ⁺ , 527].

Example 5: (2R, 4S, 4aS)-2,4-dimethyl-2',4',6'-trioxo-8-(5-oxo-4-oxa-6-aza Spirulina [2.4]heptane-6-yl)-1,1',2,3',4,4a,4',6'-octahydro-2'H,6H-spiro[isoxazole [4,5 -g][1,4]oxazine [4,3-a]quinoline-5,5'-pyrimidine]-11-carbonitrile (Compound 5)

(a) 2,6-difluoro-3-cyanobenzaldehyde (I-7)

2M diisopropylamide lithium tetrahydrofuran solution (14 ml, 28.04 mmol) was added to 20 ml of dry tetrahydrofuran under argon atmosphere, cooled to -80 ° C, and 2,6-difluorobenzonitrile was slowly added dropwise. (3.00 g, 21.57 mmol) in tetrahydrofuran solution. After 5 min, N,N-dimethylformamide (5.00 ml, 64.71 mmol) was slowly added dropwise. After 5 min, the reaction was monitored by TLC and quenched with saturated ammonium chloride. ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 1.72g of oil, yield 48%, ¹ H NMR (400MHz, CDCl ₃ ) δ 10.32 (d, J = 0.6 Hz, 1H), 8.20 (ddd, J = 8.9, 7.8, 6.2 Hz, 1H), 7.27 –7.22 (m, 1H).

(b) 3-(1,3-dioxolan-2-yl)-2,6-difluorobenzonitrile (I-8)

Intermediate I-7 (2.00 g, 11.97 mmol) was added to 15 ml of toluene at room temperature, stirred and dissolved, and ethylene glycol (2.00 ml, 35.92 mmol) and p-toluenesulfonic acid monohydrate (227 mg, 1.197 mmol) were added. , equipped with a water separator, raised to 110 ° C for 3 h, TLC monitoring reaction was completed, cooled to room temperature, extracted with water and ethyl acetate, combined organic layer, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate Analysis [petroleum ether: ethyl acetate = 4:1] afforded 1.92 g of colorless oil, yield 76%, ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.85 - 7.79 (m, 1H), 7.12 - 7.06 (m, 1H), 6.04 (s, 1H), 4.18 - 4.07 (m, 4H).

(c) 3-(1,3-dioxolan-2-yl)-2,6-difluoro-5-formylbenzonitrile (I-9)

2M diisopropylamide lithium tetrahydrofuran solution (7.7 ml, 15.41 mmol) was added to 15 ml of dry tetrahydrofuran under argon atmosphere, cooled to -80 ° C, and slowly added dropwise intermediate I-8 (2.71) g, 12.84 mmol) in tetrahydrofuran solution. After 30 min, N,N-dimethylformamide (2.97 ml, 38.52 mmol) was slowly added dropwise. After 30 min, the reaction was completely monitored by TLC, and saturated ammonium chloride was added to quench the reaction. The mixture was combined with EtOAc. EtOAc. 1.95g, yield 64%, ¹ H NMR (400MHz, CDCl ₃ ) δ 10.31 (s, 1H), 8.36 (t, J = 7.7 Hz, 1H), 6.07 (s, 1H), 4.21 - 4.06 (m) , 4H).

(d) 3-(1,3-dioxolan-2-yl)-2,6-difluoro-5-((indolyl)methyl)benzonitrile (I-10)

Intermediate I-9 (700 mg, 2.929 mmol) was added to 20 ml of methanol under ice-cooling, and stirred to dissolve. Hydrochloric acid hydrochloride (224 mg, 3.222 mmol) and pyridine (0.31 ml, 3.808 mmol) were added to the mixture. After stirring at room temperature for 1 h, the reaction was completed with EtOAc EtOAc (EtOAc)EtOAc. , 720 mg of white solid, yield 96%, ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.28 (s, 1H), 8.23 (t, J = 7.8 Hz, 1H), 7.79 (s, 1H), 6.02 ( s, 1H), 4.17 - 4.05 (m, 4H).

(e) 3-cyano-5-(1,3-dioxolan-2-yl)-2,4-difluoro-N'-hydroxy-N-((1-hydroxycyclopropyl)methyl Benzoquinone (I-12-5)

Intermediate I-10 (400 mg, 1.574 mmol) was added to 20 ml of N,N-dimethylformamide at room temperature, stirred to dissolve, and N-chlorosuccinimide (252 mg, 1.889 mmol) was added. The reaction was carried out to 40 ° C for 30 min, and the reaction was completely monitored by TLC. The reaction mixture was dropped to 0 ° C, and 1-aminomethyl-1-cyclopropanol (343 mg, 3.935 mmol) was slowly added dropwise. After 10 min, the reaction was completely monitored by TLC. After adding water and ethyl acetate, the organic layer was combined, washed with water, brine, dried over anhydrous sodium sulfate 28%, ¹ H NMR (400 MHz, DMSO) δ 10.05 (s, 1H), 7.85 (t, J = 8.0 Hz, 1H), 6.07 - 6.02 (m, 2H), 5.27 (s, 1H), 4.10– 3.97 (m, 4H), 2.90 (d, J = 6.4 Hz, 2H), 0.48 (dd, J = 6.7, 4.8 Hz, 2H), 0.37 (dd, J = 6.5, 4.6 Hz, 2H).

(f) 6-Fluoro-5-formyl-3-(((1-hydroxycyclopropyl)methyl)amino)benzo[d]isoxazole-7-carbonitrile (I-14-5)

Intermediate I-12-5 (150 mg, 0.442 mmol) was added to 10 ml of 1,4-dioxane, 6M hydrochloric acid (4 ml) was added, and the mixture was stirred at 35 ° C for 2 h, and the reaction was completely monitored by TLC. The reaction mixture was cooled to 0 ° C, and the mixture was stirred and evaporated to dryness. Column chromatography under reduced pressure [petroleum ether: ethyl acetate = 1 : 3] afforded a yellow powder (yield: 68 mg, yield: 68%, ¹ H NMR (400 MHz, DMSO) δ 10.17 (s, 1H), 8.97 (d, J = 6.6 Hz, 1H), 7.79 (t, J = 5.4 Hz, 1H), 5.49 (s, 1H), 3.36 (d, J = 5.9 Hz, 2H), 0.63 (d, J = 7.3 Hz, 4H) .

(g) 6-((2R,6R)-2,6-dimethylmorpholine)-5-formyl-3-(((1-hydroxycyclopropyl)methyl)amino)benzo[d] Isoxazole-7-carbonitrile (I-15-5)

Intermediate I-14-5 (83 mg, 0.323 mmol) was added to 20 ml of acetonitrile at room temperature, and 2R,6R-dimethylmorpholine (0.065 ml, 0.484 mmol) and N,N-diisopropyl The amine (0.110 ml, 0.646 mmol) was stirred at 40 ° C for 2 h, and the reaction was completed by TLC. The mixture was taken to room temperature, and the mixture was directly subjected to column chromatography [ petroleum ether: ethyl acetate = 2:3] to obtain 50 mg of yellow solid. Yield 42%, ¹ H NMR (400MHz, CDCl ₃ ) δ 10.18 (s, 1H), 8.22 (s, 1H), 5.23 (t, J = 5.3 Hz, 1H), 4.37 - 4.28 (m, 2H) , 3.76 (dd, J = 12.3, 3.0 Hz, 2H), 3.56 (d, J = 5.4 Hz, 2H), 3.27 (dd, J = 12.3, 5.7 Hz, 2H), 1.34 (d, J = 6.5 Hz, 6H), 0.95 (dd, J = 6.5, 5.7 Hz, 2H), 0.76 (dd, J = 6.8, 5.5 Hz, 2H).

(h) 6-((2R,6R)-2,6-dimethylmorpholine)-5-formyl-3-(5-oxo-4-oxa-6-azaspiro[2.4]g Alkan-6-yl)benzo[d]isoxazole-7-carbonitrile (I-16-5)

Intermediate I-15-5 (50 mg, 0.135 mmol), N,N-carbonyldiimidazole (44 mg, 0.270 mmol), 4-dimethylaminopyridine (17 mg, 0.135 mmol) as a starting material, 20 mg of a yellow solid, yield 38%, ¹ H NMR (400 MHz, CDCl ₃ ) δ 10.06 (s, 1H), 9.17 (s, 1H), 4.40 - 4.32 (m, 2H) , 4.30 (d, J = 2.3 Hz, 2H), 3.80 (dd, J = 12.7, 2.9 Hz, 2H), 3.23 (dd, J = 12.7, 6.0 Hz, 2H), 1.43 (t, J = 7.4 Hz, 2H), 1.32 (d, J = 6.4 Hz, 6H), 0.99 - 0.94 (m, 2H).

(i) (2R, 4S, 4aS)-2,4-dimethyl-2',4',6'-trioxo-8-(5-oxo-4-oxa-6-aza snail [2.4]heptane-6-yl)-1,1',2,3',4,4a,4',6'-octahydro-2'H,6H-spiro[isoxazole [4,5- g][1,4]oxazine [4,3-a]quinoline-5,5'-pyrimidine]-11-carbonitrile (Compound 5)

Starting from intermediate I-16-5 (20 mg, 0.050 mmol) and barbituric acid (8 mg, 0.060 mmol), according to the synthesis of compound 1, 15 mg of pale yellow solid, yield 59%, ¹ H NMR (400MHz, DMSO) δ11.75 (s, 2H), 8.08 (s, 1H), 4.56 (d, J = 13.4 Hz, 1H), 4.23 (s, 2H), 4.05 (d, J = 8.9 Hz, 1H) ), 3.87–3.80 (m, 1H), 3.75 (d, J = 14.6 Hz, 1H), 3.70–3.63 (m, 1H), 3.26 (dd, J = 14.6, 10.3 Hz, 1H), 2.86 (d, J = 14.3 Hz, 1H), 1.20 (t, J = 7.1 Hz, 2H), 1.18 (d, J = 6.2 Hz, 3H), 0.97 (t, J = 7.1 Hz, 2H), 0.92 (d, J = 6.3 Hz, 3H); ¹³ C NMR (126MHz, DMSO) δ 171.22, 168.39, 166.83, 153.87, 153.32, 151.45, 150.24, 127.89, 121.77, 115.55, 104.11, 76.30, 72.83, 72.72, 66.01, 62.47, 56.45, 52.58, 50.54, 38.31, 18.52, 18.16, 10.30, 10.24; MS (EI) m/z: [M ⁺ , 506].

Example 6, (2R, 4S, 4aS)-2,4-dimethyl-8-((S)-4-methyl-2-oxooxazol-3-yl)-2',4', 6'-Trioxo-1,1',2,3',4,4a,4',6'-octahydro-2'H,6H-spiro[isoxazole [4,5-g][1 , 4]oxazine [4,3-a]quinoline-5,5'-pyrimidine]-11-carbonitrile (compound 6)

(a) (S)-3-cyano-5-(1,3-dioxolan-2-yl)-2,4-difluoro-N'-hydroxy-N-(1-hydroxypropane-2 -based) benzamidine (I-12-6)

Starting from intermediate I-10 (300 mg, 1.181 mmol), N-chlorosuccinimide (189 mg, 1.417 mmol) and (S)-2-amino-1-propanol (0.200 ml, 2.598 mmol) According to the synthesis method of the intermediate I-12-5, 299 mg (yield: 77%) of pale brown oil, ¹ H NMR (400 MHz, CD ₃ OD) δ 10.03 (s, 1H), 7.86 (t, J) = 7.9 Hz, 1H), 6.05 (s, 1H), 5.79 (d, J = 10.5 Hz, 1H), 4.67 (t, J = 5.4 Hz, 1H), 4.09 - 3.99 (m, 5H), 3.23 (t , J = 5.4 Hz, 2H), 0.99 (d, J = 6.5 Hz, 3H).

(b) (S)-6-Fluoro-5-formyl-3-((1-hydroxypropan-2-yl)amino)benzo[d]isoxazole-7-carbonitrile (I-14-6 )

The intermediate I-12-6 (200 mg, 0.612 mmol), 6M hydrochloric acid (5 ml) and a saturated aqueous solution of sodium hydrogencarbonate were used as a starting material to obtain a white solid 70 mg (yield: 44). %, ¹ H NMR (400MHz, CDCl ₃ ) δ 10.35 (s, 1H), 8.40 (d, J = 6.2 Hz, 1H), 5.00 (d, J = 6.4 Hz, 1H), 3.97 (dd, J = 21.6, 7.8 Hz, 2H), 3.76 - 3.70 (m, 1H), 1.92 (t, J = 4.6 Hz, 1H), 1.40 (d, J = 6.6 Hz, 3H).

(c) 6-((2R,6R)-2,6-dimethylmorpholine)-5-formyl-3-(((S)-1-hydroxypropan-2-yl)amino)benzo[ d] Isoxazole-7-carbonitrile (I-15-6)

Intermediate I-14-6 (70 mg, 0.266 mmol), 2R, 6R-dimethylmorpholine (0.037 ml, 0.277 mmol), N,N-diisopropylethylamine (0.057 ml, 0.333 mmol) As a raw material, according to the synthesis method of Intermediate I-15-5, 67 mg of a yellow solid was obtained, yield 70%, ¹ H NMR (400 MHz, CDCl ₃ ) δ 10.18 (s, 1H), 8.18 (s, 1H) , 4.90 (d, J = 7.2 Hz, 1H), 4.35 - 4.29 (m, 2H), 4.00 - 3.91 (m, 2H), 3.75 (dd, J = 12.9, 3.3 Hz, 2H), 3.72 - 3.68 (m , 1H), 3.27 (dd, J = 12.4, 5.8 Hz, 2H), 1.37 (d, J = 6.6 Hz, 3H), 1.34 (d, J = 6.5 Hz, 6H).

(d) 6-((2R,6R)-2,6-Dimethylmorpholine)-5-formyl-3-((S)-4-methyl-2-oxooxazol-3-yl Benzo[d]isoxazole-7-carbonitrile (I-16-6)

Synthesis of Intermediate I-4-1 with Intermediate I-15-6 (67 mg, 0.187 mmol), carbonyldiimidazole (61 mg, 0.374 mmol) and 4-dimethylaminopyridine (23 mg, 0.187 mmol). Method, 50 mg of yellow solid was obtained, yield 70%, ¹ H NMR (400 MHz, CDCl ₃ ) δ 10.06 (s, 1H), 9.03 (s, 1H), 4.79 - 4.73 (m, 2H), 4.39 - 4.33 (m, 2H), 4.28 - 4.23 (m, 1H), 3.79 (dd, J = 12.6, 3.1 Hz, 2H), 3.22 (dd, J = 12.7, 5.9 Hz, 2H), 1.61 (d, J = 5.9) Hz, 3H), 1.31 (d, J = 6.5 Hz, 6H).

(e) (2R, 4S, 4aS)-2,4-dimethyl-8-((S)-4-methyl-2-oxooxazol-3-yl)-2',4',6 '-Trioxo-1,1',2,3',4,4a,4',6'-octahydro-2'H,6H-spiro[isoxazole [4,5-g][1, 4] Oxazine [4,3-a]quinoline-5,5'-pyrimidine]-11-carbonitrile (Compound 6)

Using Intermediate I-16-6 (50 mg, 0.130 mmol) and barbituric acid (18 mg, 0.143 mmol) as a starting material, according to the synthesis of compound 1 to give a pale yellow solid, 35 mg, yield 54%, ¹ H NMR (500MHz, DMSO) δ 11.88 (s, 1H), 11.57 (s, 1H), 7.93 (s, 1H), 4.72 - 4.61 (m, 2H), 4.56 (dd, J = 14.3, 1.3 Hz, 1H) , 4.19 (dd, J = 8.0, 5.3 Hz, 1H), 4.05 (d, J = 8.9 Hz, 1H), 3.87 - 3.79 (m, 1H), 3.73 (d, J = 14.6 Hz, 1H), 3.65 ( Dq, J = 12.8, 6.3 Hz, 1H), 3.27 (dd, J = 14.6, 10.4 Hz, 1H), 2.89 (d, J = 14.5 Hz, 1H), 1.42 (d, J = 6.0 Hz, 3H), 1.18 (d, J = 6.2 Hz, 3H), 0.92 (d, J = 6.3 Hz, 3H); ¹³ C NMR (126 MHz, DMSO) δ 170.97, 168.19, 166.56, 154.63, 152.77, 151.48, 149.88, 127.72, 121.66, 115.51, 104.94, 76.44, 72.86, 72.69, 70.80, 65.94, 56.49, 52.93, 52.65, 38.29, 18.54, 18.15, 17.74; MS (ESI) m/z: [(M-1) ^- , 493.2].

Example 7, (2R, 4S, 4aS)-2,4-dimethyl-8-((S)-5-methyl-2-oxothiazol-3-yl)-2',4',6 '-Trioxo-1,1',2,3',4,4a,4',6'-octahydro-2'H,6H-spiro[isoxazole [4,5-g][1, 4] Oxazine [4,3-a]quinoline-5,5'-pyrimidine]-11-carbonitrile (Compound 7)

(a) (R)-3-cyano-5-(1,3-dioxolan-2-yl)-2,4-difluoro-N'-hydroxy-N-(2-hydroxypropyl) Benzoquinone (I-12-7)

Intermediate I-10 (800 mg, 3.149 mmol), N-chlorosuccinimide (463 mg, 3.464 mmol) and (R)-(-)-1-amino-2-propanol (0.545 ml, 6.928) Methyl) 456 mg, yield 44%, ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.89 (t, J = 7.7 Hz, 1H). ), 6.05 (s, 1H), 5.77 (s, 1H), 4.18 - 4.05 (m, 4H), 3.82 - 3.75 (m, 1H), 2.97 (d, J = 13.1 Hz, 1H), 2.87 - 2.80 ( m, 1H), 1.11 (d, J = 6.3 Hz, 3H).

(b) (R)-6-fluoro-5-formyl-3-((2-hydroxypropyl)amino)benzo[d]isoxazole-7-carbonitrile (I-14-7)

The intermediate I-12-7 (443 mg, 1.354 mmol), 6M hydrochloric acid (8 ml) and a saturated aqueous solution of sodium hydrogencarbonate were used as a starting material. %, ¹ H NMR (400 MHz, DMSO) δ 10.17 (s, 1H), 8.90 (d, J = 6.7 Hz, 1H), 7.71 (t, J = 5.7 Hz, 1H), 4.86 (d, J = 4.8) Hz, 1H), 3.97 - 3.87 (m, 1H), 3.17 (td, J = 6.0, 2.2 Hz, 2H), 1.13 (d, J = 6.2 Hz, 3H).

(c) 6-((2R,6R)-2,6-dimethylmorpholine)-5-formyl-3-(((R)-2-hydroxypropyl)amino)benzo[d]iso Oxazole-7-carbonitrile (I-15-7)

Intermediates I-14-7 (150 mg, 0.570 mmol), 2R, 6R-dimethylmorpholine (0.090 ml, 0.684 mmol) and N,N-diisopropylethylamine (0.200 ml, 1.140 mmol) As a raw material, 162 mg (yield: 79%) of pale yellow solid (yield: 79%, ¹ H NMR (400 MHz, CDCl ₃ ) δ 10.18 (s, 1H), 8.22 (s, 1H). ), 5.28–5.24 (m, 1H), 4.36–4.28 (m, 2H), 4.24–4.16 (m, 1H), 3.75 (dd, J = 12.3, 3.0 Hz, 2H), 3.60–3.54 (m, 1H) ), 3.32–3.22 (m, 3H), 1.33 (d, J = 6.4 Hz, 9H).

(d) 6-((2R,6R)-2,6-Dimethylmorpholine)-5-formyl-3-((R)-5-methyl-2-oxooxazol-3-yl Benzo[d]isoxazole-7-carbonitrile (I-16-7)

The synthesis of intermediate I-4-1 was carried out using intermediate I-15-7 (120 mg, 0.335 mmol), carbonyldiimidazole (109 mg, 0.670 mmol) and 4-dimethylaminopyridine (41 mg, 0.335 mmol). Method, 82 mg of a yellow solid was obtained in a yield of 64%, ¹ H NMR (400 MHz, CDCl ₃ ) δ10.05 (s, 1H), 9.11 (s, 1H), 5.03 (dd, J = 14.0, 7.5 Hz, 1H) ), 4.39 - 4.29 (m, 3H), 3.84 (dd, J = 8.5, 5.8 Hz, 1H), 3.80 (dd, J = 12.3, 2.6 Hz, 2H), 3.23 (dd, J = 12.7, 5.9 Hz, 2H), 1.64 (d, J = 6.3 Hz, 3H), 1.32 (d, J = 6.4 Hz, 6H).

(e) (2R, 4S, 4aS)-2,4-dimethyl-8-((R)-5-methyl-2-oxooxazol-3-yl)-2',4',6 '-Trioxo-1,1',2,3',4,4a,4',6'-octahydro-2'H,6H-spiro[isoxazole [4,5-g][1, 4] Oxazine [4,3-a]quinoline-5,5'-pyrimidine]-11-carbonitrile (Compound 7)

Using Intermediate I-16-7 (82 mg, 0.213 mmol) and barbituric acid (30 mg, 0.235 mmol) as a starting material, according to the synthesis of Compound 1, a white solid, 34 mg, yield 32%, ¹ H NMR ( 400 MHz, DMSO) δ 11.77 (s, 1H), 11.64 (s, 1H), 8.08 (s, 1H), 5.02 - 4.92 (m, 1H), 4.60 - 4.54 (m, 1H), 4.22 (dd, J = 9.5, 8.3 Hz, 1H), 4.04 (d, J = 8.9 Hz, 1H), 3.88 - 3.81 (m, 1H), 3.77 (d, J = 14.7 Hz, 1H), 3.74 - 3.62 (m, 2H) , 3.27 (dd, J = 14.7, 10.3 Hz, 1H), 2.85 (dd, J = 14.5, 1.2 Hz, 1H), 1.46 (d, J = 6.2 Hz, 3H), 1.19 (d, J = 6.3 Hz, 3H), 0.93 (d, J = 6.4 Hz, 3H); ¹³ C NMR (151 MHz, DMSO) δ 170.98, 168.12, 166.72, 154.18, 153.44, 151.29, 149.90, 128.07, 121.48, 115.52, 104.19, 76.22, 73.07, 72.78 , 72.64, 65.95, 56.39, 52.60, 51.24, 38.22, 20.32, 18.48, 18.10; MS (ESI) m/z: [(M-1) ^- , 493.2].

Example 8 Determination of in vitro antibacterial activity

Experimental strain

In vitro antibacterial activity experiments were performed with 3 strains of methicillin-resistant Staphylococcus aureus MRSA, 3 strains of methicillin-sensitive Staphylococcus aureus MSSA, 3 strains of methicillin-resistant Staphylococcus epidermidis MRSE, and 3 strains of methoxy Xilin sensitive Staphylococcus epidermidis MSSE, 3 strains of penicillin-resistant Streptococcus pneumoniae PRSP, 3 strains of Streptococcus pyogenes.

The above strains were clinical isolates collected in Sichuan and Beijing in December 2015. The collection unit was identified by VITEK-60 automatic microbiological identification instrument and then re-identified by conventional methods. Each strain of bacteria was divided into single colonies by agar plates before the experiment, and the freshly cultured cells at 37 ° C overnight were appropriately diluted for the experiment.

Quality control strain: Staphylococcus aureus ATCC25923. Purchased from the Clinical Testing Center of the Ministry of Health of the People's Republic of China

2. Medium and culture conditions

Medium: MH (Mueller-Hinton, hydrolyzed casein) broth medium (OXOID);

Culture conditions: incubation at 35-37 ° C for 16-18 h.

3. Test method

Adopted the Clinical and Laboratory Standards Institute (CLSI) Antimicrobial Susceptibility Testing (Twenty-Third Informational Supplement) M02-A11, M07-A9 and M11-A8 , 2013] The recommended micro-broth dilution method is used to determine the minimum inhibitory concentration (MIC) of each test sample against the tested strain.

After diluting each test sample to different concentrations in MH broth medium, 100 μl of each test solution of different concentrations was separately aspirated into a sterilized 96-well polystyrene plate. The first to twelfth wells were filled with a drug solution, and the final concentrations of the drugs in each well were 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, and 0.03 mg/L, respectively. There is no additional medicine and bacteria as a blank control. Adding bacteria without dosing as a bacterial growth control.

The test bacterial liquid was adjusted to a bacterial suspension equivalent to 0.5 McMulster standard with physiological saline, diluted with MH broth 1:100, and added to the chemical solution to make the final concentration of the bacterial liquid about 10 ⁴ CFU/ml; Then, 100 μl of the bacterial solution was separately added to the above wells (the total volume in each well was 200 μl), sealed, and placed in a 35-37 ° C incubator for 18-20 hours to judge the result. The OD ₆₀₀ value was measured by a microplate reader to minimize the minimum drug concentration of the bacteria in the wells to be the minimum inhibitory concentration (MIC). When testing Streptococcus pneumoniae, it is necessary to add a final concentration of 5% sterile defibrinated sheep blood to a broth of Colombian broth, 5% CO ₂ , and culture in a 35-37 ° C incubator for 18-20 h.

The results of in vitro antibacterial activity are shown in Table 1. It can be seen from Table 1 that the compound of the present invention has good in vitro antibacterial activity, and the in vitro antibacterial activity of Compound 1 on MRSA, MSSA, MRSE, MSSE, PRSP and Spy is much better than that of the control drug AZD0914, which is about 8 times that of AZD0914. The in vitro antibacterial activity of Compounds 2, 3 and 4 was comparable to that of the control drug.

Table 1 Experimental results of in vitro antibacterial activity of some compounds of the present invention

^a Methicillin resistant S.aureus. ^b Methicillin sensitive S.aureus. ^c Methicillin resistant S.epidermidis. ^d Methicillin sensitive S.epidermidis. ^e Penicillin resistant S.pneumoniae. ^f S.pyogenes.

Example 9 Determination of Compound Solubility

Blank matrix

Preparation of artificial gastric juice (without pepsin): Take 20 mg of sodium chloride, add 70 μL of concentrated hydrochloric acid and sufficient water to make up to 10 mL. The pH of the artificial gastric juice is about 1.2.

Preparation of artificial intestinal juice (without trypsin): Take 68mg of potassium dihydrogen phosphate dissolved in 2500μL of water, add 770μL of 0.2N sodium hydroxide solution and 5mL of water, adjust the pH to 6.8 with 0.2N sodium hydroxide or 0.2N hydrochloric acid. ±0.1, add water to a volume of 10mL.

Deionized water.

2. Sample preparation

About 3 mg of the compound was weighed and added to 500 μL of a blank substrate, and shaken at 37 ° C for 24 hours. After the incubation, the sample was centrifuged for 30 min, and the supernatant was transferred to a new EP tube and centrifuged for 30 min.

3. Chromatographic conditions:

Analytical column: Acquity BEH C18 (1.5 μm; 2.1 x 50 mm, Waters)

Mobile phase: 0.1% FA + 5 mM NH4AC in Water: 0.1% FA in ACN

4. Standard curve production

Standard curve samples (12.5, 25, 50, 100, 200, 400, 2000, 10000 nM) were prepared with acetonitrile/water (1/1, v/v) and the peak area of the analyte was quantified by Waters MassLynx (version V4.1) software. The concentration of the analyte is calculated using an external standard method. Taking the peak area (y) of the object to be tested as the ordinate, the concentration (x) of the object to be tested is the abscissa, and the linear regression is performed by the weighted (1/x2) least squares method to obtain the standard curve equation y=ax+b. a is the slope and b is the intercept.

5. Sample analysis

Take 50 μL of the supernatant from each sample of the matrix, add 450 μL of acetonitrile/water (1/1, v/v), mix by vortexing, and then add 20 μL to 180 μL of acetonitrile/water (1/1, v/v). After vortex mixing, LC-MS/MS analysis was carried out, and the obtained peak area was brought into the standard curve equation to obtain the saturation solubility of each matrix. Each sample was measured four times for each matrix, and the results were averaged to obtain different matrix saturation solubility of the final compound.

The experimental results are shown in Table 2. Solubility is an important property in evaluating the drug-forming properties of drug molecules. It affects the absorption, distribution, drug exposure, oral bioavailability of drug molecules in animals and humans. At present, most drug molecules cannot be successfully marketed, and poor solubility is a key factor. It can be seen from Table 2 that the solubility of the compound of the present invention in different matrices is greatly improved relative to the positive control drug AZD0914, the biopharmaceutical properties are greatly improved, and the drug-forming property is superior.

Table 2 Solubility data of compounds in different matrices

Example 10 Pharmacokinetic test in mice

Healthy female CD-1 mice were randomly divided into two groups, 3 in each group, and the test compounds were orally administered. The specific arrangement is shown in Table 3 below.

Table 3 dosage regimen

Each animal took 0.030 mL of blood through the eye, and EDTAK2 was anticoagulated. The time of collection was: PO group: 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h after administration of the test substance. Blood samples were collected and placed on ice, and plasma was separated by centrifugation within 30 minutes (centrifugation conditions: 5000 rpm, 10 minutes, room temperature). Store at –80 °C before analysis. The experimental results are shown in Table 4.

Table 4 oral pharmacokinetic data of mice

The excellent metabolic properties are the key indicators of the drug-forming properties of the compounds. The pharmacokinetic experiments prove that the compounds of the present invention have ideal metabolic characteristics, and the exposure and peak concentrations are far superior to the positive control drug AZD0914.

Example 11

Pharmacokinetic test in rats

Nine SD rats were randomly divided into three groups of three, each of which is shown in Table 5.

Table 5 dosing schedule

Each animal took 0.100 mL of blood through the eye, and EDTAK _{2 was} anticoagulated. The time of collection was: (1) PO group: 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h after administration of the test article; 2) Group IV: 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h after administration of the test substance. Blood samples were collected and placed on ice, and plasma was centrifuged within 30 minutes (centrifugation conditions: 5000 rpm, 10 minutes, 4 ° C). Store at –80 °C before analysis. The experimental results are shown in Table 6.

Table 6 Main pharmacokinetic parameters of SD rats after oral administration (PO) or intravenous (IV) compound 1

Displays the 10mg / kg and 100mg / kg Comparative results obtained are shown in the case of oral administration dosage and dose both C _max plasma showed significant positive correlation between the administration of the compound. 1 100mg / kg was administered 10mg of Cmax 15 times of /kg, indicating that the absorption of Compound 1 at the dose of 100 mg / kg was not significantly affected, oral bioavailability also increased gradually, from 22.6% to 45.8%.

Based on the pharmacokinetic parameters of various aspects of Compound 1, it can be seen that Compound 1 has excellent metabolic properties in rats and is worthy of further research and development.

Example 12

In vivo antibacterial activity of the compounds of the invention against MRSA

Test strain

According to the results of the in vitro test, clinical isolates of methicillin-resistant Staphylococcus aureus MRSA15-3 (see Table 7 below) were selected as spare test strains for in vivo protection experiments.

Table 7 In vitro antibacterial activity of the compound of the present invention and a control drug against Staphylococcus aureus MRSA15-3 - MIC (mg / L)

2. Test animals

Healthy Kunming mice around 4 weeks old, weighing 18-22g, male and female, SPF

Estimated number of mice used: 200

3. In vivo protection test method

3.1 bacterial liquid equipment

One day before the infection of the test bacteria, 2-3 single colonies were picked and inoculated into 2 ml of MH broth, and cultured at 37 ° C for 6 h. 0.1 ml of the bacterial solution was transferred to 10 ml of MH broth and cultured at 37 ° C for 18 h. The bacterial liquid is the original bacterial liquid. The bacterial solution was diluted with 5% dry yeast solution for use (prepared freshly on the day).

3.2 Minimum lethal bacteria (MLD) test

Take healthy Kunming mice, weighing 18-22 grams, randomly divided into groups, 5-10 mice in each group, male and female, absorb the above different dilutions of bacterial solution, intraperitoneal injection into mice, re-injection every 20g mouse 0.5ml, observed for 7-14 days after infection, and recorded the number of mouse deaths, the minimum amount of bacteria causing 100% death of mice as the minimum lethal bacteria amount (MLD), using the amount of bacteria as the amount of infectious bacteria in the in vivo protection test .

3.3 drug solution and route of administration:

Liquid preparation: test with medicinal 0.5% sodium carboxymethyl cellulose and dilute to the desired concentration of solution

Route of administration: intragastric administration

Dosing capacity: 0.5ml / 20g BW (body weight, body weight)

3.4 group design

It is proposed to set the dose range of the test sample to 20-2.5mg/kg, accurately weigh the drug, convert it to the effective drug weight according to the potency, and use the 0.5% sodium carboxymethyl cellulose to prepare the solution of the desired concentration. The moment between groups is 1:0.5.

Group design: AZD0914 (20, 10, 5, 2.5g·kg ^-1 ) group, compound 1 (20, 10, 5, 2.5g·kg ^-1 ) group, infection control group, blank control group, ABT toxicity control .

3.5 In vivo protection experimental methods and results statistics

The mice were stopped from feeding water for 18 hours before the test, and were randomly divided into groups according to their body weight. Each group of 8 mice, male and female, were injected intraperitoneally with the infection test bacterial solution. Each 20g mouse was injected with 0.5ml, 0.5h after infection, 4h. The designed dose was intragastrically administered, and 0.5 ml was perfused into the stomach every 20 g of mice; the number of deaths of the mice was observed and recorded, and the number of deaths was observed for 7-14 days. According to the number of deaths of mice, Sun Ruiyuan and other editors DAS1.0 software were used to calculate half of the Bliss method. Effective dose ED ₅₀ and 95% confidence limit.

The control drug AZD0914 group received oral cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) 50 mg/kg two hours before infection, and 50 mg/kg ABT once orally after 12 hours. Other compounds did not require oral ABT.

Infected control group: only the infected bacteria were not administered, and the same volume of physiological saline was administered after the infection of the bacteria; the blank control group: no infection of the bacteria, intraperitoneal injection of an equal volume of physiological saline, and an equal volume of physiological saline.

4. Test results

The results of in vivo antibacterial activity of the systemic systemic infection model are shown in Table 8.

Table 8 Protective effect of compounds of the invention against S. aureus infection in mice in vivo MRSA15-3 (ED ₅₀₎

The positive control drug AZD0914 needs to be combined with the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT), and the ED ₅₀ for S. aureus MRSA15-3 infected mice is 11.51 mg/kg, while Compound 1 does not need to be combined. ABT, ED ₅₀ for oral administration to S. aureus MRSA15-3 infected mice was 3.87 mg/kg, which was significantly better than AZD0914.

In summary, the compound of the present invention has better drug-forming properties than the positive control drug AZD0914, and is expected to be a better antibacterial drug.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the present invention.

Claims (10)

1. a compound of the formula (I), or an enantiomer, diastereomer, racemate or mixture thereof, or a pharmaceutically acceptable salt thereof,

   

   Wherein R ₁ is hydrogen, halogen or cyano;

   R ₂ and R _{3 are} independently a hydrogen atom or a C ₁ -C ₃ alkyl group; or R ₂ and R ₃ together with a carbon atom to be bonded form a 3-7 membered aliphatic ring or a 3-7 membered oxygen-containing heterocyclic ring;

   R ₄ is a hydrogen atom or a C ₁ -C ₃ alkyl group;

   Each * indicates the racemization, S type or R type independently;

   Provided that when R ₁ is a halogen, R ₂ and R ₃ together with the attached carbon atom form a 3-7 membered aliphatic ring or a 3-7 membered oxygen-containing heterocyclic ring.
2. The compound of claim 1 wherein R ₁ is fluoro, chloro or cyano.
3. The compound according to claim 1, wherein R ₁ is fluorine; and R ₂ and R ₃ together with the carbon atom to be bonded form a 3-7 membered aliphatic ring or a 3-7 membered oxygen-containing heterocyclic ring.
4. The compound according to claim 1, wherein R ₁ is a cyano group; R ₂ and R _{3 are} independently a hydrogen atom or a C ₁ - C ₃ alkyl group; or R ₂ and R ₃ are formed together with a carbon atom to which it is bonded. 3-7 yuan fat ring.
5. The compound of claim 1 wherein said compound is:

   

   
6. A pharmaceutical composition comprising the compound of claim 1 or an enantiomer thereof, a diastereomer, a racemate, and a mixture thereof, or a pharmaceutical thereof Acceptable salt;

   A pharmaceutically acceptable carrier or excipient.
7. The method of preparing a compound according to claim 1, wherein the method comprises the steps of:

   

   (i) intermediate Ia by intramolecular nucleophilic substitution reaction to form intermediate Ib;

   (ii) intermediate Ib is nucleophilic substituted to form intermediate Ic;

   (iii) intermediate Ic is hydrolyzed to form intermediate Id;

   (iv) intermediate Id by nucleophilic substitution reaction to form intermediate Ie;

   (v) reacting intermediate Ie with barbituric acid to form a compound of formula I,

   In the formulas, *, R ₁ , R ₂ , R ₃ and R ₄ are as defined in claim 1.
8. A method of producing a compound according to claim 1, wherein when R ₁ is a cyano group, the method comprises the steps of:

   

   (i') intermediate I-12 is hydrolyzed to form intermediate I-13;

   (ii') intermediate I-13 is subjected to intramolecular nucleophilic substitution to give intermediate I-14;

   (iii') intermediate I-14 is subjected to nucleophilic substitution to give intermediate I-15;

   (iv') intermediate I-15 is subjected to nucleophilic substitution to give intermediate I-16;

   (v') intermediate I-16 is reacted with barbituric acid to form the compound of claim 1 represented by formula C,

   In the formulas, *, R ₂ , R ₃ and R ₄ are as defined in claim 1.
9. The compound of claim 1 or an enantiomer thereof, a diastereomer, a racemate, and mixtures thereof, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 7. Use for the preparation of a medicament for the treatment of a bacterial infectious disease.
10. A compound intermediate of formula I, characterized in that the structure of the intermediate of the compound of formula I is as shown in formula Ia, Ib, Ic, Id or Ie:

    

    In the formulas, *, R ₁ , R ₂ , R ₃ and R ₄ are as defined in claim 1.