CN109111466B - Benzisoxazole spiropyrimidine triones compound, preparation method and application thereof - Google Patents

Benzisoxazole spiropyrimidine triones compound, preparation method and application thereof Download PDF

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CN109111466B
CN109111466B CN201711116329.4A CN201711116329A CN109111466B CN 109111466 B CN109111466 B CN 109111466B CN 201711116329 A CN201711116329 A CN 201711116329A CN 109111466 B CN109111466 B CN 109111466B
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nucleophilic substitution
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CN109111466A (en
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杨玉社
张银勇
石程辉
陈乾
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Shanghai Institute of Materia Medica of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
    • C07D261/20—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/10—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings
    • C07D317/14—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D317/30—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
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    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
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    • C07—ORGANIC CHEMISTRY
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    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
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    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
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Abstract

The invention discloses a benzisoxazole spiropyrimidine trione compound and a preparation method and application thereof, and the structure is shown as formula I. Wherein the substituents are as defined in the description and claims. The benzisoxazole spiropyrimidine trione compound has higher in-vitro and in-vivo antibacterial activity and better metabolic property.

Description

Benzisoxazole spiropyrimidine triones compound, preparation method and application thereof
Technical Field
The invention relates to a spiropyrimidine trione compound containing a benzisoxazole group, a pharmaceutical composition thereof, a preparation method thereof and application thereof in anti-infective drugs.
Background
With the wide use of antibacterial drugs, even abuse, the multi-drug resistance of pathogenic bacteria becomes a serious problem threatening human health, and a series of drug-resistant pathogens represented by multi-drug resistant staphylococcus aureus MRSA bring huge challenges to clinical treatment. In recent years, although some novel antibacterial drugs such as linezolid, daptomycin and the like are marketed and can effectively control gram-positive bacteria such as MRSA and the like, with the clinical application of more than 10 years, the bacteria gradually generate drug-resistant strains to the drugs. Therefore, the research and development of novel antibacterial drugs with novel action mechanisms, unique structures and effective drug-resistant bacteria are urgent scientific research tasks and are also unmet important clinical requirements.
The benzisoxazole spiropyrimidine triones are novel bacterial II type topoisomerase inhibitors, and although the benzisoxazole spiropyrimidine triones and quinolone drugs act on the bacterial II type topoisomerase, the specific action regions and action mechanisms of the benzisoxazole spiropyrimidine triones are completely different. The novel chemical structure and the novel action mechanism of the benzisoxazole spiropyrimidine trione compound enable the benzisoxazole spiropyrimidine trione compound to have good antibacterial activity on sensitive and drug-resistant gram-positive bacteria and have no cross drug resistance with the clinical existing antibacterial drugs.
A series of spiropyrimidine triones compounds are reported by companies such as Aslicon and the like, and preliminary research on structure-activity relationship of the spiropyrimidine triones shows that the methyl-substituted spiropyrimidine triones on isoxazole have good antibacterial activity. The Aslicon continues to perform derivatization research on the compounds, and further discovers AZD0914 with better activity, and simultaneously eliminates the genotoxicity and bone marrow toxicity of the compounds, and currently, AZD0914 is undergoing phase II clinical research.
However, the spiropyrimidinetrione compounds such as AZD0914 still have the problems of weak antibacterial activity and undesirable metabolic properties, which result in large clinical drug overdose, and the possibility of using the spiropyrimidinetrione compounds as drugs for treating systemic infection is limited to a certain extent.
Therefore, there is still a need in the art to develop spiropyrimidinetriones with better activity and better metabolic properties for systemic infection of humans or animals to overcome the above problems.
Disclosure of Invention
The invention aims to provide a benzisoxazole spiropyrimidine trione compound with better activity and metabolic property, an enantiomer, a diastereoisomer, a racemate, a mixture of the enantiomer, the diastereoisomer, the racemate and a pharmaceutically acceptable salt of the benzisoxazole spiropyrimidine trione compound.
In a first aspect of the invention, there is provided a compound of formula (I), or an enantiomer, diastereomer, racemate, or mixture thereof, or a pharmaceutically acceptable salt thereof,
Figure BDA0001466313540000021
in the formula, R 1 Is hydrogen, halogen or cyano;
R 2 、R 3 independently of a hydrogen atom or C 1 -C 3 An alkyl group; or R 2 、R 3 Together with the carbon atom to which they are attached form a 3-7 membered alicyclic or 3-7 membered oxygen containing heterocyclic ring;
R 4 is a hydrogen atom or C 1 -C 3 An alkyl group;
each independently represents racemic, S-or R-form;
with the proviso that when R 1 When it is halogen, R 2 、R 3 Together with the carbon atom to which they are attached form a 3-7 membered alicyclic or 3-7 membered oxygen containing heterocyclic ring.
The compound represented by the general formula (I) of the invention contains at least 3 chiral centers, and enantiomers and diastereomers exist. For enantiomers, two enantiomers can be obtained by a general chiral resolution method or an asymmetric synthesis method. Diastereoisomers can be separated by fractional recrystallization or chromatographic separation. The compound represented by the general formula (I) of the present invention includes any one of the above isomers or a mixture thereof.
In another preferred embodiment, R 2 、R 3 Together with the carbon atom to which they are attached form a 3-7 membered alicyclic or 3-7 membered oxygen containing heterocyclic ring.
In another preferred embodiment, R 2 、R 3 With or without 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; r 2 、R 3 Together with the carbon atom to which they are attached form a 3-7 membered alicyclic or 3-7 membered oxygen containing heterocyclic ring.
In another preferred embodiment, R 1 Is hydrogen, fluorine or chlorine; r is 4 Is hydrogen.
In another preferred embodiment, R 2 、R 3 Together with the carbon atom to which they are attached form a 3-7 membered alicyclic or 3-7 membered oxygen containing heterocyclic ring; r 4 Is hydrogen.
In another preferred embodiment, R 1 Is cyano;
R 2 、R 3 independently of a hydrogen atom or C 1 -C 3 An alkyl group; or R 2 、R 3 Together with the carbon atom to which they are attached form a 3-7 membered alicyclic ring.
In another preferred embodiment, the compound is:
Figure BDA0001466313540000031
in a second aspect of the invention, there is provided a pharmaceutical composition comprising a compound of the first aspect or enantiomers, diastereomers, racemates and mixtures thereof, or a pharmaceutically acceptable salt thereof; and
a pharmaceutically acceptable carrier or excipient.
The invention provides a novel benzisoxazole spiropyrimidine trione compound which can be used alone or mixed with medicinal auxiliary materials (such as excipient, diluent and the like) to be prepared into tablets, capsules, granules, syrups and the like for oral administration. The pharmaceutical composition can be prepared according to a conventional method in pharmacy.
In a third aspect of the present invention, there is provided a process for the preparation of a compound according to the first aspect, the process comprising the steps of:
Figure BDA0001466313540000032
(i) the intermediate Ia is subjected to intramolecular nucleophilic substitution reaction to generate an intermediate Ib;
(ii) carrying out nucleophilic substitution reaction on the intermediate Ib to generate an intermediate Ic;
(iii) carrying out catalytic hydrolysis reaction on the intermediate Ic to generate an intermediate Id;
(iv) carrying out nucleophilic substitution reaction on the intermediate Id to generate an intermediate Ie;
(v) the intermediate Ie reacts with barbituric acid to generate a compound shown as a general formula I,
in the formulae 1 、R 2 、R 3 And R 4 As defined above.
In another preferred embodiment, the intermediate Ia and cesium carbonate undergo an intramolecular nucleophilic substitution reaction to form the intermediate Ib.
In another preferred embodiment, intermediate Ib is subjected to nucleophilic substitution reaction with N, N' -carbonyldiimidazole to form intermediate Ic.
In another preferred embodiment, intermediate Id and 2R, 6R-dimethyl morpholine undergo nucleophilic substitution reaction to produce intermediate Ie.
In another preferred embodiment, intermediate Ic is hydrolyzed with hydrochloric acid to form intermediate Id
In a fourth aspect of the invention, there is provided a process for the preparation of a compound according to the first aspect, R 1 When cyano, the method comprises the following steps:
Figure BDA0001466313540000041
(I') subjecting the intermediate I-12 to hydrolysis reaction to produce an intermediate I-13;
(ii') subjecting the intermediate I-13 to intramolecular nucleophilic substitution reaction to produce an intermediate I-14;
(iii') subjecting intermediate I-14 to nucleophilic substitution to produce intermediate I-15;
(iv') subjecting the intermediate I-15 to nucleophilic substitution to produce an intermediate I-16;
(v') reaction of intermediate I-16 with barbituric acid to produce the compound of claim 1 of formula C,
in the formulae 2 、R 3 And R 4 As defined above.
In another preferred embodiment, intermediate I-12 is hydrolyzed under the catalysis of hydrochloric acid to form intermediate I-13
In another preferred embodiment, intermediate I-13 is subjected to intramolecular nucleophilic substitution reaction with sodium bicarbonate to form intermediate I-14.
In another preferred embodiment, intermediate I-14 is subjected to nucleophilic substitution reaction with 2R, 6R-dimethylmorpholine to produce intermediate I-15.
In another preferred embodiment, intermediate I-15 is subjected to nucleophilic substitution reaction with N, N' -carbonyldiimidazole to produce intermediate I-16.
In a fifth aspect of the present invention, there is provided a use of the compound of the first aspect, or enantiomers, diastereomers, racemates thereof, and mixtures thereof, or pharmaceutically acceptable salts thereof, or the pharmaceutical composition of the second aspect, for preparing a medicament for treating bacterial infectious diseases.
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 multidrug-resistant bacteria.
In another preferred embodiment, the multi-drug resistant bacteria is selected from the group consisting of: MRSA, MSSA, MRSE, MSSE, PRSP, and Spy.
In a sixth aspect of the invention, there is provided an in vitro method of bacteriostasis by administering to a subject or environment a compound of the first aspect or enantiomers, diastereomers, racemates and mixtures thereof, or pharmaceutically acceptable salts thereof, or a pharmaceutical composition of the second aspect.
In a seventh aspect of the present invention, there is provided a compound intermediate of formula I, having a structure represented by formula Ia, Ib, Ic, Id or Ie:
Figure BDA0001466313540000051
in the formulae 1 、R 2 、R 3 And R 4 As defined above.
The compound of the invention has better in vitro and in vivo antibacterial activity, excellent in vivo metabolic property, far better exposure and peak concentration than that of a positive control medicament AZD0914, better medicament forming property than that of the positive control medicament AZD0914, and is expected to become a better antibacterial medicament.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Not to be reiterated herein, but to the extent of space.
Detailed Description
The inventor of the application researches extensively and deeply, carries out various structural modifications on AZD0914, particularly introduces a spiro structure on an oxazolidone ring, synthesizes a series of spiropyrimidinetrione compounds with novel structures, finds that the in vitro and in vivo antibacterial activity and the drug metabolism property of the spiropyrimidinetrione compounds are greatly superior to those of AZD0914, and is very suitable for being used as a novel antibacterial drug for the antibacterial treatment of infection of human beings or animals. On the basis of this, the present invention has been completed.
Term(s) for
In the present invention, the term "C 1 -C 3 Alkyl "refers to a straight or branched chain having 1 to 3 carbon atomsAlkyl groups, including 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 on 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 3-7 ring atoms and containing 1,2 or 3O atoms, including without limitation a propylene oxide ring, a butylene oxide ring, a heptane oxide ring and the like.
The "pharmaceutically acceptable salts" include pharmaceutically acceptable base addition salts. Including but not limited to salts with inorganic bases such as sodium, potassium, calcium, and magnesium salts, and the like. Including but not limited to salts with organic bases such as ammonium, triethylamine, lysine, arginine, and the like. These salts can be prepared by methods known in the art.
Preparation method
The compound represented by the present invention (I) can be produced by the following method, however, the conditions of the method, such as reactants, solvent, base, amount of the compound used, reaction temperature, time required for the reaction, etc., are not limited to the following explanation. The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art to which the present invention pertains.
Route 1
In a preferred embodiment, compound 1, compound 2, compound 3 and compound 4 are prepared as in scheme 1.
Figure BDA0001466313540000061
R 2 、R 3 And R 4 As defined above.
a. Intermediate A [ J.Med.chem,2015,58(15):6264-6282 ] is reacted with N-chlorosuccinimide in a polar aprotic solvent, which may be: 1, 4-dioxane, toluene, tetrahydrofuran and N, N-dimethylformamide, wherein the optimal reaction temperature is 40 ℃, and the optimal reaction time is 30 min.
b. The intermediate I-1 reacts for 20-60min at 0 ℃ to room temperature in polar aprotic solvents in the presence of different primary amines respectively to generate corresponding intermediate I-2 through nucleophilic substitution, wherein the primary amines are various primary amines meeting requirements, and the polar aprotic solvents can be: 1, 4-dioxane, toluene, tetrahydrofuran and N, N-dimethylformamide, wherein the optimal reaction temperature is 0 ℃, and the optimal reaction time is 30 min.
c. Intermediate I-2 is reacted in the presence of cesium carbonate in a polar aprotic solvent, which may be: 1, 4-dioxane, toluene, tetrahydrofuran and N, N-dimethylformamide, wherein the optimal reaction temperature is 60 ℃, and the optimal reaction time is 3 hours.
d. The intermediate I-3 reacts with N, N' -carbonyldiimidazole at 60-90 ℃ for 3-8h in a polar aprotic solvent under the catalysis of 4-dimethylaminopyridine to generate the corresponding intermediate I-4, wherein the polar aprotic solvent can be: 1, 4-dioxane, toluene, tetrahydrofuran and N, N-dimethylformamide, wherein the optimal reaction temperature is 80 ℃, and the optimal reaction time is 5 hours.
e. And (3) reacting the intermediate I-4 with hydrochloric acid in a solvent at room temperature to 50 ℃ for 2-5h to generate the corresponding intermediate I-5, wherein the solvent can be 1, 4-dioxane, tetrahydrofuran or N, N-dimethylformamide, the optimal reaction temperature is 30 ℃, and the optimal reaction time is 3 h.
f. The intermediate I-5 and 2R, 6R-dimethyl morpholine react for 12-20h at 70-100 ℃ in a polar aprotic solvent in the presence of an organic base to generate the corresponding intermediate I-6, wherein the organic base can be triethylamine or N, N-diisopropylethylamine, the polar aprotic solvent can be acetonitrile, 1, 4-dioxane, tetrahydrofuran and N, N-dimethylformamide, the optimal reaction temperature is 80 ℃, and the optimal reaction time is 15 h.
g. The intermediate I-6 and barbituric acid react with 100-120 ℃ in a mixed solvent of acetic acid and water for 4-7h to generate a corresponding compound B, wherein the optimal reaction temperature is 100 ℃ and the optimal reaction time is 5 h.
Route 2
In a preferred embodiment, compound 5, compound 6 and compound 7 are prepared as in scheme 2.
Figure BDA0001466313540000071
a. 2, 6-difluorobenzonitrile, lithium diisopropylamide and N, N-dimethylformamide are reacted in a polar aprotic solvent at a temperature of between 80 ℃ below zero and 65 ℃ below zero for 3 to 8min to generate an intermediate I-7, wherein the polar aprotic solvent can be tetrahydrofuran and dichloromethane, the optimal reaction temperature is 80 ℃ below zero, and the optimal reaction time is 5 min.
b. The intermediate I-7 and ethylene glycol react for 3-8h at the temperature of 130 ℃ in a toluene solvent under the catalysis of p-toluenesulfonic acid to generate the intermediate I-8, the optimal reaction temperature is 120 ℃, and the optimal reaction time is 4 h.
c. And (3) reacting the intermediate I-8 with lithium diisopropylamide and N, N-dimethylformamide in a polar aprotic solvent at the temperature of between 80 ℃ below zero and 65 ℃ below zero for 20 to 60min to generate an intermediate I-9, wherein the polar aprotic solvent can be tetrahydrofuran and dichloromethane, the optimal reaction temperature is 80 ℃ below zero, and the optimal reaction time is 30 min.
d. The intermediate I-9 and hydroxylamine hydrochloride react for 1-3h at 0 ℃ to room temperature in a polar solvent under the condition of organic base to generate an intermediate I-10, wherein the organic base can be pyridine, triethylamine or N, N-diisopropylethylamine, the polar solvent can be methanol, dichloromethane, 1, 4-dioxane, tetrahydrofuran or N, N-dimethylformamide, the optimal reaction temperature is 0 ℃, and the optimal reaction time is 2 h.
e. Reacting the intermediate I-10 with N-chlorosuccinimide in a polar aprotic solvent at room temperature to 50 ℃ for 20-60min to obtain an intermediate I-11, wherein the polar aprotic solvent can be: 1, 4-dioxane, toluene, tetrahydrofuran and N, N-dimethylformamide, wherein the optimal reaction temperature is 40 ℃, and the optimal reaction time is 30 min.
f. The intermediate I-11 reacts with various primary amines meeting the conditions in a polar aprotic solvent at 0 ℃ to room temperature for 10-60min to generate the intermediate I-12 through nucleophilic substitution, wherein the polar aprotic solvent can be: 1, 4-dioxane, toluene, tetrahydrofuran and N, N-dimethylformamide, wherein the optimal reaction temperature is 0 ℃, and the optimal reaction time is 30 min.
g. Reacting the intermediate I-12 with hydrochloric acid in a solvent at room temperature to 50 ℃ for 2-5h to generate an intermediate I-13, wherein the solvent can be: 1, 4-dioxane, tetrahydrofuran and N, N-dimethylformamide, wherein the optimal reaction temperature is 40 ℃, and the optimal reaction time is 3 hours.
h. Reacting the intermediate I-13 with an aqueous solution of sodium bicarbonate at 0 to 40 ℃ for 1 to 4 hours in a solvent to form an intermediate I-14, wherein the solvent can be: 1, 4-dioxane, tetrahydrofuran and N, N-dimethylformamide, wherein the optimal reaction temperature is 25 ℃, and the optimal reaction time is 2 hours.
i. The intermediate I-14 and 2R, 6R-dimethyl morpholine react for 12-20h at 70-100 ℃ in a polar aprotic solvent in the presence of an organic base to generate the intermediate I-15, wherein the organic base can be triethylamine or N, N-diisopropylethylamine, the polar aprotic solvent can be acetonitrile, 1, 4-dioxane, tetrahydrofuran and N, N-dimethylformamide, the optimal reaction temperature is 80 ℃, and the optimal reaction time is 12 h.
j. The intermediate I-15 reacts with N, N' -carbonyldiimidazole at 60-90 ℃ for 3-8h in a polar aprotic solvent under the catalysis of 4-dimethylaminopyridine to generate the corresponding intermediate I-16, wherein the polar aprotic solvent can be: 1, 4-dioxane, toluene, tetrahydrofuran and N, N-dimethylformamide, wherein the optimal reaction temperature is 80 ℃, and the optimal reaction time is 5 hours.
k. The intermediate I-16 and barbituric acid react for 4-7h at the temperature of 120 ℃ in a mixed solvent of acetic acid and water to generate a compound C, the optimal reaction temperature is 110 ℃, and the optimal reaction time is 5 h.
Pharmaceutical composition
The invention also provides a pharmaceutical composition comprising a safe and effective amount of the active ingredient, and a pharmaceutically acceptable carrier.
The active ingredient refers to the compound of the formula I.
The active ingredient and the pharmaceutical composition are used for preparing the drugs for treating infectious diseases. In another preferred embodiment, the compound is used for preparing a medicament for preventing and/or treating infectious diseases caused by multi-drug resistant bacteria.
"safe and effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000mg of active ingredient per dose, more preferably, 10-200mg of active ingredient per dose. Preferably, said "dose" is a tablet.
"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of being combined with the active ingredients of the present invention and with each other without significantly diminishing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), and the like
Figure BDA0001466313540000091
) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.
The mode of administration of the active ingredient or pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and the like.
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 tinctures.
The compounds of the present invention may be administered alone or in combination with other therapeutic agents, such as antibacterial agents.
When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, and for a human body with a weight of 60kg, the daily administration dose is usually 1 to 2000mg, preferably 20 to 500 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for which specific conditions are not indicated in the following examples are generally carried out according to conventional conditions (e.g.as described in Sambrook et al, molecular cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
In all of the examples that follow, 1 H-NMR was recorded using a Varian Mercury 400 NMR spectrometer, 13 C-NMR was recorded using a BRUKER 500MHz NMR spectrometer (low temperature) and chemical shifts are expressed in delta (ppm); the silica gel for separation is 200-300 mesh, and the ratio of the eluent is volume ratio.
Example 1: (2R,4S,4aS) -11-fluoro-2, 4-dimethyl-8- (5-oxo-4-oxo-6-azaspiro [2.4] heptan-6-yl) -1,2,4,4 a-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)
Figure BDA0001466313540000101
Adding the intermediate A (1g, 4.048mmol) into 20ml of N, N-dimethylformamide, stirring to dissolve, adding N-chlorosuccinimide (650mg, 4.868mmol) at room temperature, raising the temperature to 40 ℃ for reaction for 30min, monitoring the reaction completion by TLC (thin layer chromatography), bringing the reaction solution to 0 ℃, slowly adding 1-aminomethyl-1-cyclopropanol (1.06g, 12.144mmol), returning to room temperature for reaction for 1h, monitoring the reaction completion by TLC, adding cesium carbonate (4.567g, 12.144mmol), raising the temperature to 60 ℃ for reaction for 2h, monitoring the reaction completion by TLC, lowering the temperature to room temperature, adding ethyl acetate for extraction, combining organic layers, washing with water, washing with saturated water, drying with anhydrous sodium sulfate, and concentrating under reduced pressure column chromatography [ petroleum ether: ethyl acetate 1:1]To obtain 810mg of white solid with the yield of 65 percent, 1 H NMR(400MHz,CDCl 3 )δ7.53(dd,J＝5.4,1.5Hz,1H),6.13(s,1H),4.79(t,J＝5.6Hz,1H),4.22–4.09(m,4H),3.58(d,J＝5.5Hz,2H),2.65(s,1H),0.95(t,J＝6.2Hz,2H),0.75(t,J＝6.2Hz,2H)。
(b)6- (5- ((1, 3-dioxolan-2-yl) -6, 7-difluorobenzo [ d ] isoxazol-3-yl) -4-oxo-6-azaspiro [2.4] heptan-5-one (I-4-1)
Figure BDA0001466313540000102
Adding intermediate I-3-1(344mg, 1.036mmol) into 15ml of N, N-dimethylformamide, stirring to dissolve, sequentially adding carbonyldiimidazole (671mg, 4.414mmol) and 4-dimethylaminopyridine (63mg, 0.52mmol), heating to 115 deg.C, reacting for 8h, monitoring by TLC, cooling to room temperature,water was added, ethyl acetate was added, and extraction was performed, organic layers were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure for column chromatography [ petroleum ether: ethyl acetate 2:1]To obtain 337mg of white solid with the yield of 96 percent, 1 H NMR(400MHz,CDCl 3 )δ8.55(dd,J＝5.9,1.9Hz,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)
Figure BDA0001466313540000103
Intermediate I-4-1(330mg, 0.976mmol) was dissolved in 15ml1, 4-dioxane, 6M hydrochloric acid (5ml) was added, stirred at room temperature for 2h, TLC monitored for reaction completion, water was added and ethyl acetate was added for extraction, organic layers were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure for column chromatography [ petroleum ether: dichloromethane: ethyl acetate 5:5:1]285mg of white solid is obtained, the yield is 99 percent, 1 H NMR(400MHz,CDCl 3 )δ10.30(s,1H),9.00(dd,J＝5.9,1.9Hz,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] heptan-6-yl) benzo [ d ] isoxazole-5-carbaldehyde (I-6-1)
Figure BDA0001466313540000111
Dissolving the intermediate I-5-1(200mg, 0..67mmol) in 10ml of acetonitrile, stirring to dissolve the intermediate, respectively adding 2R, 6R-dimethylmorpholine (0.31ml, 2.54mmol) and N, N-diisopropylethylamine (0.42ml, 2.54mmol) in sequence, refluxing the reaction solution at 90 ℃ for 10h, monitoring the reaction completion by TLC, cooling to room temperature, adding water and ethyl acetate for extraction, combining organic layers, washing with water, washing with saturated salt water, drying with anhydrous sodium sulfate, and concentrating under reduced pressure for column chromatography [ petroleum ether: ethyl acetate 5:1]Obtaining light yellow solid 80mg with 31 percent of yield, 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＝11.7Hz,2H),3.06(dd,J＝11.7,5.4Hz,2H),1.45–1.40(m,2H),1.34(d,J＝6.5Hz,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] heptan-6-yl) -1,2,4,4 a-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)
Figure BDA0001466313540000112
Adding the intermediate I-6-1(80mg, 0.206mmol) into a mixed solvent of 4ml acetic acid and 1ml water, stirring to dissolve the intermediate, adding barbituric acid (32mg, 0.246mmol), raising the temperature to 100 ℃ for reaction for 3h, monitoring the reaction completion by TLC, cooling to room temperature, adding water and ethyl acetate for extraction, combining organic layers, washing with water, washing with saturated salt water, drying with anhydrous sodium sulfate, concentrating under reduced pressure and performing reverse phase column preparation [ methanol: 50:50 of water]To obtain 78mg of white solid with the yield of 76 percent, and the m.p.266-267 ℃; 1 H NMR(400MHz,DMSO)δ11.82(s,1H),11.46(s,1H),7.76(s,1H),4.29–4.21(m,2H),4.11(d,J＝12.9Hz,1H),3.94(d,J＝8.8Hz,1H),3.78(br s,1H),3.72–3.62(m,2H),3.17–3.06(m,1H),2.92(d,J＝14.1Hz,1H),1.24-1.19(m,2H),1.15(d,J＝6.3Hz,3H),1.00–0.95(m,2H),0.90(d,J＝6.3Hz,3H). 13 C NMR(126MHz,DMSO)δ171.40,168.15,154.20(d,J＝12.8Hz),153.79,153.17,149.98,135.30,133.76(d,J＝238.8Hz),122.85,118.83,106.52,72.58,72.14,64.88,62.33,56.73(d,J＝9.3Hz),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] octan-7-yl) -1,2,4,4 a-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) oxetane (I-3-2)
Figure BDA0001466313540000121
According to the method for synthesizing the intermediate I-3-1, 1- (((5- (1, 3-dioxolan-2-yl) -6, 7-difluorobenzo [ d ] is synthesized from the intermediate A (1.2g, 4.857mmol), N-chlorosuccinimide (778mg, 5.828mmol), 3- (aminomethyl) oxetane (1.25g, 12.142mmol) and cesium carbonate (4.748g, 14.571mmol)]Isoxazol-3-yl) amino) methyl) oxetane 949mg, white solid, yield 59.5%, 1 H NMR(400MHz,DMSO)δ8.08(d,J＝5.7Hz,1H),7.41(t,J＝5.7Hz,1H),6.09(s,1H),6.01(s,1H),4.48(dd,J＝15.1,6.6Hz,4H),4.12–3.99(m,4H),3.55(d,J＝5.8Hz,2H)。
(b)7- (5- ((1, 3-dioxolan-2-yl) -6, 7-difluorobenzo [ d ] isoxazol-3-yl) -2, 5-dioxo-7-azaspiro [3.4] octan-6-one (I-4-2)
Figure BDA0001466313540000122
According to the synthesis method of the intermediate I-4-1, the intermediate I-3-2(940mg, 2.86mmol), N' -carbonyldiimidazole (1.857g, 11.45mmol) and 4-dimethylaminopyridine (175mg, 1.43mmol) are used as raw materials to synthesize 7- (5- ((1, 3-dioxolan-2-yl) -6, 7-difluorobenzo [ d ] b]Isoxazol-3-yl) -2, 5-dioxo-7-azaspiro [3.4]872mg of octane-6-one, white solid, yield 86%, 1 H NMR(400MHz,CDCl 3 )δ8.44(dd,J＝5.9,1.8Hz,1H),6.12(s,1H),5.10(d,J＝8.8Hz,2H),4.82(d,J＝8.7Hz,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] octan-7-yl) benzo [ d ] isoxazole-5-carbaldehyde (I-5-2)
Figure BDA0001466313540000131
According to the synthesis method of the intermediate I-5-1, the intermediate I-4-2(885mg, 2.498mmol) and 6M hydrochloric acid (30ml) are used as raw materials to synthesize the 6, 7-difluoro-3- (6-oxo-2, 5-dioxo-7-azaspiro [3.4]]Octane-7-yl) benzo [ d]580mg of isoxazole-5-carbaldehyde which is a white solid with a yield of 75 percent, 1 H NMR(400MHz,CDCl 3 )δ10.31(s,1H),8.91(dd,J＝5.8,1.8Hz,1H),5.14(d,J＝9.0Hz,2H),4.85(d,J＝8.7Hz,2H),4.53(s,2H)。
(d)6- ((2R,6R) -2, 6-dimethylmorpholine) -7-fluoro-3- (6-oxo-2, 5-dioxo-7-azaspiro [3.4] octan-7-yl) benzo [ d ] isoxazole-5-carbaldehyde (I-6-2)
Figure BDA0001466313540000132
Synthesis of 6- ((2R,6R) -2, 6-dimethylmorpholine) -7-fluoro-3- (6-oxo-2, 5-dioxo-7-azaspiro [3.4] from intermediate I-5-2(150mg, 0.484mmol), 2R, 6R-dimethylmorpholine (139mg, 1.209mmol) and N, N-diisopropylethylamine (187mg, 1.451mmol) according to the Synthesis of intermediate I-6-1]Octane-7-yl) benzo [ d]172mg of isoxazole-5-carbaldehyde in the yield of 87% as a white solid, 1 H NMR(400MHz,CDCl 3 )δ10.37(s,1H),8.71(s,1H),5.14(d,J＝8.3Hz,2H),4.85(d,J＝8.2Hz,2H),4.50(s,2H),4.30–4.22(m,2H),3.45(d,J＝12.0Hz,2H),3.06(dd,J＝11.7,5.6Hz,2H),1.34(d,J＝6.5Hz,6H)。
(e) (2R,4S,4aS) -11-fluoro-2, 4-dimethyl-8- (6-oxo-2, 5-dioxo-7-azaspiro [3.4] octan-7-yl) -1,2,4,4 a-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)
Figure BDA0001466313540000133
According to the synthesis method of the compound 1, (2R,4S,4aS) -11-fluoro-2, 4-dimethyl-8- (6-oxo-2, 5-dioxo-7-azaspiro [3.4] is synthesized by taking the intermediate I-6-2(93mg, 0.229mmol) and barbituric acid (33mg, 0.252mmol) aS raw materials]Octane-7-yl) -1,2,4,4 a-tetrahydro-2' H, 6H-spiro [ isoxazole [4,5-g][1,4]Oxazines [4,3-a ]]Quinoline-5, 5' -pyrimidines]90mg of 2',4',6' (1' H,3' H) -trione, white solid, yield 76%, 1 H NMR(400MHz,DMSO)δ11.63(s,2H),7.73(s,1H),4.86–4.76(m,4H),4.43–4.36(m,2H),4.10(d,J＝12.5Hz,1H),3.93(d,J＝8.9Hz,1H),3.83–3.74(m,1H),3.72–3.60(m,2H),3.18–3.05(m,1H),2.90(d,J＝13.7Hz,1H),1.14(d,J＝6.2Hz,3H),0.89(d,J＝6.4Hz,3H)； 13 C NMR(126MHz,DMSO)δ171.46,168.17,154.15(d,J＝13.0Hz),152.88,152.86,150.07,135.28,133.74(d,J＝238.9Hz),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] octan-7-yl) -1,2,4,4 a-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) cyclobutanol (I-3-3)
Figure BDA0001466313540000141
1- (((5- (1, 3-dioxolan-2-yl) -6, 7-difluorobenzo [ d ] was synthesized according to the method for synthesizing intermediate I-3-1, starting from intermediate A (750mg, 3.036mmol), N-chlorosuccinimide (527mg, 3.947mmol), 1- (aminomethyl) cyclobutanol (675mg, 6.679mmol) and cesium carbonate (3.957g, 12.144mmol)]Isoxazol-3-yl) amino) methyl) cyclobutanol 710mg, white solid, yield 72%, 1 H NMR(400MHz,DMSO)δ8.15(d,J＝5.9Hz,1H),7.23(t,J＝5.4Hz,1H),6.08(s,1H),5.27(s,1H),4.11–3.99(m,4H),3.34(d,J＝5.6Hz,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] octan-6-one (I-4-3)
Figure BDA0001466313540000142
According to the synthesis of intermediate I-4-1, intermediate I-3-3(533mg, 1.63mmol), N' -carbonyldiimidazole (1.059g, 6.53mmol) and 4-dimethylaminopyridine (10.059 g, 6.53mmol) were used0mg, 0.82mmol) as raw material to synthesize 7- (5- ((1, 3-dioxolan-2-yl) -6, 7-difluorobenzo [ d ]]Isoxazol-3-yl) -5-oxo-7-azaspiro [3.4]468mg of octane-6-ketone, white solid, 82% of yield, 1 H NMR(400MHz,CDCl 3 )δ8.51(dd,J＝5.9,1.8Hz,1H),6.15(s,1H),4.26(s,2H),4.22–4.07(m,4H),2.71(ddd,J＝19.3,10.1,2.9Hz,2H),2.38(ddd,J＝11.4,8.5,3.7Hz,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] octan-7-yl) benzo [ d ] isoxazole-5-carbaldehyde (I-5-3)
Figure BDA0001466313540000151
According to the synthesis method of the intermediate I-5-1, the intermediate I-4-3(468mg, 1.328mmol) and 6M hydrochloric acid (15ml) are used as raw materials to synthesize the 6, 7-difluoro-3- (6-oxo-5-oxo-7-azaspiro [3.4]]Octane-7-yl) benzo [ d]380mg of isoxazole-5-carbaldehyde which is a white solid with a yield of 93 percent, 1 H NMR(400MHz,CDCl 3 )δ10.29(s,1H),8.95(dd,J＝5.8,1.7Hz,1H),4.27(s,2H),2.73(ddd,J＝19.5,10.1,2.9Hz,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] octan-7-yl) benzo [ d ] isoxazole-5-carbaldehyde (I-6-3)
Figure BDA0001466313540000152
According to the synthesis method of the intermediate I-6-1, the intermediate I-5-3(100mg, 0.324mmol), 2R, 6R-dimethylmorpholine (75mg, 0.649mmol) and N, N-diisopropylethylamine (126mg, 0.973mmol) are used as raw materials to synthesize 6- ((2R,6R) -2, 6-dimethylmorpholine) -7-fluoro-3- (6-oxo-5-oxo-7-azaspiro [3.4]]Octane-7-yl) benzo [ d]103mg of isoxazole-5-carbaldehyde which is a white solid with a yield of 79%, 1 H NMR(400MHz,CDCl 3 )δ10.38(s,1H),8.79(s,1H),4.31–4.20(m,4H),3.44(d,J＝12.1Hz,2H),3.05(dd,J＝11.9,5.6Hz,2H),2.72(ddd,J＝19.8,10.0,3.2Hz,2H),2.43–2.34(m,2H),2.10–1.98(m,1H),1.85–1.73(m,1H),1.34(d,J＝6.4Hz,6H)。
(e) (2R,4S,4aS) -11-fluoro-2, 4-dimethyl-8- (6-oxo-5-oxo-7-azaspiro [3.4] octan-7-yl) -1,2,4,4 a-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)
Figure BDA0001466313540000153
According to the synthesis method of the compound 1, (2R,4S,4aS) -11-fluoro-2, 4-dimethyl-8- (6-oxo-5-oxo-7-azaspiro [3.4] is synthesized by taking the intermediate I-6-3(92mg, 0.228mmol) and barbituric acid (32mg, 0.251mmol) aS raw materials]Octane-7-yl) -1,2,4,4 a-tetrahydro-2' H, 6H-spiro [ isoxazole [4,5-g][1,4]Oxazines [4,3-a ]]Quinoline-5, 5' -pyrimidines]99mg of 2',4',6' (1' H,3' H) -trione, 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＝12.5Hz,1H),3.93(d,J＝8.8Hz,1H),3.84–3.74(m,1H),3.71–3.62(m,2H),3.17–3.05(m,1H),2.91(d,J＝13.9Hz,1H),2.50–2.32(m,4H),1.89–1.63(m,2H),1.15(d,J＝6.3Hz,3H),0.89(d,J＝6.3Hz,3H). 13 C NMR(126MHz,DMSO)δ171.41,168.15,154.12(d,J＝12.9Hz),153.34,153.24,150.00,135.26,133.76(d,J＝238.8Hz),122.77,118.81,106.65,81.39,72.57,72.13,64.87,56.73(d,J＝9.4Hz),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] nonan-3-yl) -1,2,4,4 a-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) cyclopentanol (I-3-4)
Figure BDA0001466313540000161
Synthesis method according to intermediate I-3-11- (((5- (1, 3-dioxolan-2-yl) -6, 7-difluorobenzo [ d ] was synthesized from intermediate A (600mg, 2.430mmol), N-chlorosuccinimide (422mg, 3.160mmol), 1- (aminomethyl) cyclopentanol (615mg, 5.346mmol) and cesium carbonate (3.167g, 9.720mmol)]Isoxazol-3-yl) amino) methyl) cyclopentanol 582mg, white solid, yield 70%, 1 H NMR(400MHz,DMSO)δ8.13(d,J＝5.0Hz,1H),7.19(t,J＝5.6Hz,1H),6.08(s,1H),4.51(s,1H),4.14–3.99(m,4H),3.31(d,J＝5.8Hz,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] nonan-2-one (I-4-4)
Figure BDA0001466313540000162
3- (5- ((1, 3-dioxolan-2-yl) -6, 7-difluorobenzo [ d ] was synthesized according to the method for synthesizing intermediate I-4-1, starting from intermediate I-3-4(526mg, 1.546mmol), N' -carbonyldiimidazole (1.002g, 6.182mmol) and 4-dimethylaminopyridine (95mg, 0.773mmol)]Isoxazol-3-yl) -1-oxo-3-azaspiro [4.4]450mg of nonane-2-one, white solid, yield 79%, 1 H NMR(400MHz,CDCl 3 )δ8.54(dd,J＝5.9,1.7Hz,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] nonan-3-yl) benzo [ d ] isoxazole-5-carbaldehyde (I-5-4)
Figure BDA0001466313540000171
According to the method for synthesizing the intermediate I-5-1, the intermediate I-4-4(158mg, 0.431mmol) and 6M hydrochloric acid (5ml) are used as raw materials to synthesize the 6, 7-difluoro-3- (2-oxo-1-oxo-3-azaspiro [ 4.4%]Nonan-3-yl) benzo [ d]135mg of isoxazole-5-carbaldehyde is a white solid with the yield of 97 percent, 1 H NMR(400MHz,CDCl 3 )δ10.30(s,1H),8.98(dd,J＝5.9,1.8Hz,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] nonan-3-yl) benzo [ d ] isoxazole-5-carbaldehyde (I-6-4)
Figure BDA0001466313540000172
Synthesis of 6- ((2R,6R) -2, 6-dimethylmorpholine) -7-fluoro-3- (2-oxo-1-oxo-3-azaspiro [4.4] from intermediate I-5-4(100mg, 0.310mmol), 2R, 6R-dimethylmorpholine (72mg, 0.620mmol) and N, N-diisopropylethylamine (120mg, 0.931mmol) according to the Synthesis of intermediate I-6-1]Nonan-3-yl) benzo [ d]114mg of isoxazole-5-carbaldehyde which is a pale white solid with a yield of 88%, 1 H NMR(400MHz,CDCl 3 )δ10.38(s,1H),8.81(s,1H),4.31–4.21(m,2H),4.15(s,2H),3.44(d,J＝12.1Hz,2H),3.05(dd,J＝11.8,5.5Hz,2H),2.28(m,2H),2.06–1.80(m,6H),1.34(d,J＝6.4Hz,6H)。
(e) (2R,4S,4aS) -11-fluoro-2, 4-dimethyl-8- (2-oxo-1-oxo-3-azaspiro [4.4] nonan-3-yl) -1,2,4,4 a-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)
Figure BDA0001466313540000181
According to the synthesis method of the compound 1, (2R,4S,4aS) -11-fluoro-2, 4-dimethyl-8- (2-oxo-1-oxo-3-azaspiro [4.4] azaspiro [ 4.261 mmol) is synthesized by taking the intermediate I-6-4(99mg, 0.237mmol) and barbituric acid (34mg, 0.261mmol) aS raw materials]Nonan-3-yl) -1,2,4,4 a-tetrahydro-2' H, 6H-spiro [ isoxazole [4,5-g][1,4]Oxazines [4,3-a ]]Quinoline-5, 5' -pyrimidines]94mg of 2',4',6' (1' H,3' H) -trione, white solid, yield 75%, 1 H NMR(400MHz,DMSO)δ11.82(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＝13.7Hz,1H),2.12–2.04(m,2H),1.96–1.87(m,2H),1.80–1.71(m,4H),1.15(d,J＝6.2Hz,3H),0.89(d,J＝6.4Hz,3H)； 13 C NMR(151MHz,DMSO)δ170.96,167.69,153.68(d,J＝12.9Hz),153.06,152.90,149.54,134.78,133.29(d,J＝238.8Hz),122.23,118.53,106.19,90.29,72.12,71.68,64.42,56.27(d,J＝8.9Hz),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-azaspiro [2.4] heptan-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)
Figure BDA0001466313540000182
Adding 2M lithium diisopropylamide tetrahydrofuran solution (14ml, 28.04mmol) into 20ml dry tetrahydrofuran at room temperature, cooling to-80 ℃ under the protection of argon, slowly adding 2, 6-difluorobenzonitrile (3.00g, 21.57mmol) tetrahydrofuran solution dropwise, after 5min, slowly adding N, N-dimethylformamide (5.00ml, 64.71mmol), after 5min, monitoring the reaction by TLC, adding saturated ammonium chloride to quench the reaction, heating to room temperature, adding water, adding ethyl acetate to extract, combining organic layers, washing with water, washing with saturated salt water, drying with sodium sulfate, concentrating column chromatography under reduced pressure [ petroleum ether: ethyl acetate 2:1]To obtain 1.72g of colorless oily substance with the yield of 48 percent, 1 H NMR(400MHz,CDCl 3 )δ10.32(d,J＝0.6Hz,1H),8.20(ddd,J＝8.9,7.8,6.2Hz,1H),7.27–7.22(m,1H)。
(b)3- (1, 3-Dioxolan-2-yl) -2, 6-difluorobenzonitrile (I-8)
Figure BDA0001466313540000183
Adding the intermediate I-7(2.00g, 11.97mmol) into 15ml of toluene at room temperature, stirring for dissolving, adding ethylene glycol (2.00ml, 35.92mmol) and p-toluenesulfonic acid monohydrate (227mg, 1.197mmol), loading a water separator, raising the temperature to 110 ℃ for reaction for 3 hours, monitoring the reaction completion by TLC, reducing the temperature to room temperature, adding water and extracting with ethyl acetate, combining organic layers, washing with water, saturating, and collecting the organic layersWashed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure for column chromatography [ petroleum ether: ethyl acetate 4:1]To obtain 1.92g of colorless oily substance with the yield of 76%, 1 H NMR(400MHz,CDCl 3 )δ7.85–7.79(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)
Figure BDA0001466313540000191
Adding a 2M tetrahydrofuran solution (7.7ml, 15.41mmol) of lithium diisopropylamide into 15ml of dry tetrahydrofuran at room temperature, cooling to-80 ℃ under the protection of argon, slowly dropwise adding a tetrahydrofuran solution of an intermediate I-8(2.71g, 12.84mmol), slowly dropwise adding N, N-dimethylformamide (2.97ml, 38.52mmol) after 30min, monitoring the reaction completion by TLC after 30min, adding saturated ammonium chloride to quench the reaction, heating to room temperature, adding water, adding ethyl acetate for extraction, combining organic layers, washing with water, washing with saturated common salt water, drying with sodium sulfate, and concentrating column chromatography under reduced pressure [ petroleum ether: ethyl acetate 5:1]To obtain 1.95g of colorless oily substance with a yield of 64%, 1 H NMR(400MHz,CDCl 3 )δ10.31(s,1H),8.36(t,J＝7.7Hz,1H),6.07(s,1H),4.21–4.06(m,4H)。
(d)3- (1, 3-Dioxolan-2-yl) -2, 6-difluoro-5- ((hydroxyimino) methyl) benzonitrile (I-10)
Figure BDA0001466313540000192
Under ice bath conditions, intermediate I-9(700mg, 2.929mmol) was added to 20ml of methanol and stirred to dissolve it, hydroxylamine hydrochloride (224mg, 3.222mmol) and pyridine (0.31ml, 3.808mmol) were added in this order, the mixture was warmed to room temperature and stirred for 1 hour, TLC monitored for reaction completion, water was added and ethyl acetate was extracted, organic layers were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure for column chromatography [ petroleum ether: ethyl acetate 2:1]720mg of white solid is obtained, the yield is 96 percent, 1 H NMR(400MHz,CDCl 3 )δ8.28(s,1H),8.23(t,J＝7.8Hz,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) benzamidine (I-12-5)
Figure BDA0001466313540000193
Adding the intermediate I-10(400mg, 1.574mmol) into 20ml of N, N-dimethylformamide at room temperature, stirring to dissolve the intermediate I-10, adding N-chlorosuccinimide (252mg, 1.889mmol), raising the temperature to 40 ℃ for reaction for 30min, monitoring the reaction completion by TLC, reducing the reaction liquid to 0 ℃, slowly and dropwise adding 1-aminomethyl-1-cyclopropanol (343mg, 3.935mmol), after 10min, monitoring the reaction completion by TLC, adding water and ethyl acetate for extraction, combining organic layers, washing with water, washing with saturated salt water, drying with anhydrous sodium sulfate, concentrating the column chromatography under reduced pressure [ petroleum ether: ethyl acetate 1:3]Obtaining light yellow powder 150mg with yield of 28%, 1 H NMR(400MHz,DMSO)δ10.05(s,1H),7.85(t,J＝8.0Hz,1H),6.07–6.02(m,2H),5.27(s,1H),4.10–3.97(m,4H),2.90(d,J＝6.4Hz,2H),0.48(dd,J＝6.7,4.8Hz,2H),0.37(dd,J＝6.5,4.6Hz,2H)。
(f) 6-fluoro-5-formyl-3- (((1-hydroxycyclopropyl) methyl) amino) benzo [ d ] isoxazole-7-carbonitrile (I-14-5)
Figure BDA0001466313540000201
At room temperature, adding the intermediate I-12-5(150mg, 0.442mmol) into 10ml of 1, 4-dioxane, adding 6M hydrochloric acid (4ml), raising the temperature to 35 ℃, stirring for 2h, monitoring the reaction completion by TLC, reducing the reaction liquid to 0 ℃, adding saturated sodium bicarbonate aqueous solution to adjust the pH value to 9, stirring for 30min, monitoring the reaction completion by TLC, adding water, extracting with ethyl acetate, combining organic layers, washing with water, washing with saturated salt water, drying with anhydrous sodium sulfate, and concentrating under reduced pressure for column chromatography [ petroleum ether: ethyl acetate 1:3]83mg of yellow powder is obtained, the yield is 68 percent, 1 H NMR(400MHz,DMSO)δ10.17(s,1H),8.97(d,J＝6.6Hz,1H),7.79(t,J＝5.4Hz,1H),5.49(s,1H),3.36(d,J＝5.9Hz,2H),0.63(d,J＝7.3Hz,4H)。
(g)6- ((2R,6R) -2, 6-dimethylmorpholine) -5-formyl-3- (((1-hydroxycyclopropyl) methyl) amino) benzo [ d ] isoxazole-7-carbonitrile (I-15-5)
Figure BDA0001466313540000202
Intermediate I-14-5(83mg, 0.323mmol) was added to 20ml acetonitrile at room temperature, 2R, 6R-dimethylmorpholine (0.065ml, 0.484mmol) and N, N-diisopropylethylamine (0.110ml, 0.646mmol) were added, the mixture was raised to 40 ℃ and stirred for 2h, TLC monitored for reaction completion, cooled to room temperature, and column chromatography was performed directly with a pad column [ petroleum ether: ethyl acetate 2:3]Obtaining yellow solid 50mg with 42% yield, 1 H NMR(400MHz,CDCl 3 )δ10.18(s,1H),8.22(s,1H),5.23(t,J＝5.3Hz,1H),4.37–4.28(m,2H),3.76(dd,J＝12.3,3.0Hz,2H),3.56(d,J＝5.4Hz,2H),3.27(dd,J＝12.3,5.7Hz,2H),1.34(d,J＝6.5Hz,6H),0.95(dd,J＝6.5,5.7Hz,2H),0.76(dd,J＝6.8,5.5Hz,2H)。
(h)6- ((2R,6R) -2, 6-dimethylmorpholine) -5-formyl-3- (5-oxo-4-oxa-6-azaspiro [2.4] heptan-6-yl) benzo [ d ] isoxazole-7-carbonitrile (I-16-5)
Figure BDA0001466313540000211
According to the synthesis method of the intermediate I-4-1, the intermediate I-15-5(50mg, 0.135mmol), N-carbonyldiimidazole (44mg, 0.270mmol) and 4-dimethylaminopyridine (17mg, 0.135mmol) are used as raw materials to prepare 20mg of yellow solid with the yield of 38%, 1 H NMR(400MHz,CDCl 3 )δ10.06(s,1H),9.17(s,1H),4.40–4.32(m,2H),4.30(d,J＝2.3Hz,2H),3.80(dd,J＝12.7,2.9Hz,2H),3.23(dd,J＝12.7,6.0Hz,2H),1.43(t,J＝7.4Hz,2H),1.32(d,J＝6.4Hz,6H),0.99–0.94(m,2H)。
(i) (2R,4S,4aS) -2, 4-dimethyl-2 ',4',6' -trioxo-8- (5-oxo-4-oxa-6-azaspiro [2.4] heptan-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)
Figure BDA0001466313540000212
Taking intermediate I-16-5(20mg, 0.050mmol) and barbituric acid (8mg, 0.060mmol) as raw materials, preparing 15mg of light yellow solid according to the synthesis method of the compound 1, with the yield of 59%, 1 H NMR(400MHz,DMSO)δ11.75(s,2H),8.08(s,1H),4.56(d,J＝13.4Hz,1H),4.23(s,2H),4.05(d,J＝8.9Hz,1H),3.87–3.80(m,1H),3.75(d,J＝14.6Hz,1H),3.70–3.63(m,1H),3.26(dd,J＝14.6,10.3Hz,1H),2.86(d,J＝14.3Hz,1H),1.20(t,J＝7.1Hz,2H),1.18(d,J＝6.2Hz,3H),0.97(t,J＝7.1Hz,2H),0.92(d,J＝6.3Hz,3H)； 13 C NMR(126MHz,DMSO)δ171.22,168.39,166.83,153.87,153.32,151.45,150.24,127.89,121.77,115.55,104.11,76.30,72.83,72.72,66.01,62.47,56.45,52.58,50.54,38.31,18.52,18.16,10.30,10.24；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-hydroxypropan-2-yl) benzamidine (I-12-6)
Figure BDA0001466313540000221
299mg of a brown-yellow oily substance was obtained in a 77% yield from intermediate I-10(300mg, 1.181mmol), N-chlorosuccinimide (189mg, 1.417mmol) and (S) -2-amino-1-propanol (0.200ml, 2.598mmol) as starting materials by the method for synthesizing intermediate I-12-5, 1 H NMR(400MHz,CD 3 OD)δ10.03(s,1H),7.86(t,J＝7.9Hz,1H),6.05(s,1H),5.79(d,J＝10.5Hz,1H),4.67(t,J＝5.4Hz,1H),4.09–3.99(m,5H),3.23(t,J＝5.4Hz,2H),0.99(d,J＝6.5Hz,3H)。
(b) (S) -6-fluoro-5-formyl-3- ((1-hydroxypropan-2-yl) amino) benzo [ d ] isoxazole-7-carbonitrile (I-14-6)
Figure BDA0001466313540000222
Taking intermediate I-12-6(200mg, 0.612mmol), 6M hydrochloric acid (5ml) and saturated aqueous solution of sodium bicarbonate as raw materials, according to the synthesis method of intermediate I-14-5, 70mg of white solid is prepared, the yield is 44%, 1 H NMR(400MHz,CDCl 3 )δ10.35(s,1H),8.40(d,J＝6.2Hz,1H),5.00(d,J＝6.4Hz,1H),3.97(dd,J＝21.6,7.8Hz,2H),3.76–3.70(m,1H),1.92(t,J＝4.6Hz,1H),1.40(d,J＝6.6Hz,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)
Figure BDA0001466313540000223
Using intermediate I-14-6(70mg, 0.266mmol), 2R, 6R-dimethylmorpholine (0.037ml, 0.277mmol) and N, N-diisopropylethylamine (0.057ml, 0.333mmol) as raw materials, preparing 67mg of yellow solid with 70% yield according to the synthesis method of intermediate I-15-5, 1 H NMR(400MHz,CDCl 3 )δ10.18(s,1H),8.18(s,1H),4.90(d,J＝7.2Hz,1H),4.35–4.29(m,2H),4.00–3.91(m,2H),3.75(dd,J＝12.9,3.3Hz,2H),3.72–3.68(m,1H),3.27(dd,J＝12.4,5.8Hz,2H),1.37(d,J＝6.6Hz,3H),1.34(d,J＝6.5Hz,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)
Figure BDA0001466313540000231
50mg of yellow solid was obtained in a yield of 70% by the synthetic method of intermediate I-4-1 using intermediate I-15-6(67mg, 0.187mmol), carbonyldiimidazole (61mg, 0.374mmol) and 4-dimethylaminopyridine (23mg, 0.187mmol) as raw materials, 1 H NMR(400MHz,CDCl 3 )δ10.06(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＝12.6,3.1Hz,2H),3.22(dd,J＝12.7,5.9Hz,2H),1.61(d,J＝5.9Hz,3H),1.31(d,J＝6.5Hz,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)
Figure BDA0001466313540000232
Taking intermediate I-16-6(50mg, 0.130mmol) and barbituric acid (18mg, 0.143mmol) as raw materials, preparing 35mg of light yellow solid according to the synthesis method of the compound 1, with the yield of 54 percent, 1 H NMR(500MHz,DMSO)δ11.88(s,1H),11.57(s,1H),7.93(s,1H),4.72–4.61(m,2H),4.56(dd,J＝14.3,1.3Hz,1H),4.19(dd,J＝8.0,5.3Hz,1H),4.05(d,J＝8.9Hz,1H),3.87–3.79(m,1H),3.73(d,J＝14.6Hz,1H),3.65(dq,J＝12.8,6.3Hz,1H),3.27(dd,J＝14.6,10.4Hz,1H),2.89(d,J＝14.5Hz,1H),1.42(d,J＝6.0Hz,3H),1.18(d,J＝6.2Hz,3H),0.92(d,J＝6.3Hz,3H)； 13 C NMR(126MHz,DMSO)δ170.97,168.19,166.56,154.63,152.77,151.48,149.88,127.72,121.66,115.51,104.94,76.44,72.86,72.69,70.80,65.94,56.49,52.93,52.65,38.29,18.54,18.15,17.74；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) benzamidine (I-12-7)
Figure BDA0001466313540000241
Using intermediate I-10(800mg, 3.149mmol), N-chloroSuccinimide (463mg, 3.464mmol) and (R) - (-) -1-amino-2-propanol (0.545ml, 6.928mmol) as raw materials, and according to the synthesis method of intermediate I-12-5, pale yellow powder 456mg was obtained with a yield of 44%, 1 H NMR(400MHz,CDCl 3 )δ7.89(t,J＝7.7Hz,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＝13.1Hz,1H),2.87–2.80(m,1H),1.11(d,J＝6.3Hz,3H)。
(b) (R) -6-fluoro-5-formyl-3- ((2-hydroxypropyl) amino) benzo [ d ] isoxazole-7-carbonitrile (I-14-7)
Figure BDA0001466313540000242
Taking intermediate I-12-7(443mg, 1.354mmol), 6M hydrochloric acid (8ml) and saturated sodium bicarbonate aqueous solution as raw materials, obtaining 150mg of light yellow powder according to the synthesis method of intermediate I-14-5 with the yield of 42%, 1 H NMR(400MHz,DMSO)δ10.17(s,1H),8.90(d,J＝6.7Hz,1H),7.71(t,J＝5.7Hz,1H),4.86(d,J＝4.8Hz,1H),3.97–3.87(m,1H),3.17(td,J＝6.0,2.2Hz,2H),1.13(d,J＝6.2Hz,3H)。
(c)6- ((2R,6R) -2, 6-dimethylmorpholine) -5-formyl-3- (((R) -2-hydroxypropyl) amino) benzo [ d ] isoxazole-7-carbonitrile (I-15-7)
Figure BDA0001466313540000243
Using intermediate I-14-7(150mg, 0.570mmol), 2R, 6R-dimethylmorpholine (0.090ml, 0.684mmol) and N, N-diisopropylethylamine (0.200ml, 1.140mmol) as raw materials, according to the synthesis method of intermediate I-15-5, obtaining light yellow solid 162mg with 79% yield, 1 H NMR(400MHz,CDCl 3 )δ10.18(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＝12.3,3.0Hz,2H),3.60–3.54(m,1H),3.32–3.22(m,3H),1.33(d,J＝6.4Hz,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)
Figure BDA0001466313540000251
82mg of yellow solid was obtained with a yield of 64% by the synthesis method of intermediate I-4-1 using intermediate I-15-7(120mg, 0.335mmol), carbonyldiimidazole (109mg, 0.670mmol) and 4-dimethylaminopyridine (41mg, 0.335mmol) as starting materials, 1 H NMR(400MHz,CDCl 3 )δ10.05(s,1H),9.11(s,1H),5.03(dd,J＝14.0,7.5Hz,1H),4.39–4.29(m,3H),3.84(dd,J＝8.5,5.8Hz,1H),3.80(dd,J＝12.3,2.6Hz,2H),3.23(dd,J＝12.7,5.9Hz,2H),1.64(d,J＝6.3Hz,3H),1.32(d,J＝6.4Hz,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)
Figure BDA0001466313540000252
Using intermediate I-16-7(82mg, 0.213mmol) and barbituric acid (30mg, 0.235mmol) as raw materials, according to the synthesis method of the compound 1, 34mg of white solid is prepared with the yield of 32%, 1 H NMR(400MHz,DMSO)δ11.77(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＝9.5,8.3Hz,1H),4.04(d,J＝8.9Hz,1H),3.88–3.81(m,1H),3.77(d,J＝14.7Hz,1H),3.74–3.62(m,2H),3.27(dd,J＝14.7,10.3Hz,1H),2.85(dd,J＝14.5,1.2Hz,1H),1.46(d,J＝6.2Hz,3H),1.19(d,J＝6.3Hz,3H),0.93(d,J＝6.4Hz,3H)； 13 C NMR(151MHz,DMSO)δ170.98,168.12,166.72,154.18,153.44,151.29,149.90,128.07,121.48,115.52,104.19,76.22,73.07,72.78,72.64,65.95,56.39,52.60,51.24,38.22,20.32,18.48,18.10；MS(ESI)m/z:[(M-1) - ,493.2]。
example 8
In vitro antimicrobial Activity assay
1. Experimental strains
The in vitro antibacterial activity experiment selects 3 methicillin-resistant staphylococcus aureus MRSA, 3 methicillin-sensitive staphylococcus aureus MSSA, 3 methicillin-resistant staphylococcus epidermidis MRSE, 3 methicillin-sensitive staphylococcus epidermidis MSSE, 3 penicillin-resistant streptococcus pneumoniae PRSP and 3 pyogenes which are clinically separated.
The above strains are clinical isolated pathogenic bacteria collected in Sichuan and Beijing areas 12 months in 2015. The collected cells were identified by VITEK-60 automatic microorganism identification instrument and then re-identified by conventional method. Each strain of bacteria was subjected to single colony streaking on an agar plate before the experiment, and the freshly cultured cells at 37 ℃ overnight were appropriately diluted for the experiment.
Quality control of the strain: staphylococcus aureus ATCC 25923. Purchased from the clinical testing center of Ministry of health of the people's republic of China.
2. Culture medium and culture conditions
Culture medium: MH (Mueller-Hinton, hydrolyzed casein) broth (OXOID);
the culture conditions are as follows: incubating at 35-37 deg.C for 16-18 h.
3. Test method
Minimum Inhibitory Concentration (MIC) values for each test sample were determined using the American society for Clinical and Laboratory Standards Institute (CLSI) Antimicrobial Susceptibility Testing protocol [ Performance Standards for Antimicrobial Susceptibility Testing; minute-Third information Supplement ] M02-A11, M07-A9 and M11-A8, 2013 ] recommended broth dilutions.
After each test sample was diluted to different concentrations in MH broth, 100 μ l of each test sample solution was pipetted into a sterilized 96-well polystyrene plate. Adding the medicinal liquid into the 1 st to 12 th holes, wherein the final concentration of the medicinal liquid in each hole is 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06 and 0.03 mg/L. No drug and bacteria were added as a blank control. Bacteria were added and no drug was added as a control for bacterial growth.
Adjusting test bacterial liquid to bacterial suspension corresponding to 0.5 McLeod's ratio standard with normal saline, diluting with MH broth 1: 100, adding into the medicinal liquidFinal concentration of bacterial liquid is about 10 4 CFU/ml; then respectively sucking 100 mul of bacterial liquid, adding the bacterial liquid into the holes (the total volume in each hole is 200 mul), sealing, and then placing in an incubator at 35-37 ℃ for culturing for 18-20h, and judging the result. OD determination with microplate reader 600 The value is given as the Minimum Inhibitory Concentration (MIC) at which the lowest drug Concentration that completely inhibits bacterial growth in the well is taken as the minimum Inhibitory Concentration. For testing Streptococcus pneumoniae, 5% sterile defibrinated sheep blood and 5% CO were added to Columbia broth to a final concentration 2 Culturing at 35-37 deg.C for 18-20 hr.
The in vitro antibacterial activity results are shown in table 1. As can be seen from Table 1, the compounds of the present invention have very good in vitro antibacterial activity, the in vitro antibacterial activity of compound 1 to 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, while the in vitro antibacterial activity of compounds 2,3 and 4 is equivalent to that of the control drug.
TABLE 1 in vitro antibacterial Activity test results for some of the compounds of the invention
Figure BDA0001466313540000261
Figure BDA0001466313540000271
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 solubility of Compounds
1. Blank matrix
Preparation of artificial gastric juice (without pepsin): 20mg of sodium chloride was taken and 70. mu.L of concentrated hydrochloric acid and enough water were added to make 10 mL. The pH of the artificial gastric fluid is about 1.2.
Preparation of artificial intestinal juice (without pancreatin): 68mg of potassium dihydrogen phosphate is dissolved in 2500. mu.L of water, 770. mu.L of 0.2N sodium hydroxide solution and 5mL of water are added, the pH is adjusted to 6.8 +/-0.1 by 0.2N sodium hydroxide or 0.2N hydrochloric acid, and the volume is adjusted to 10mL by adding water.
Deionized water.
2. Sample preparation
Approximately 3mg of the compound was weighed, and 500. mu.L of the blank matrix was added thereto, respectively, and shaken at 37 ℃ for 24 hours. After incubation, the samples were centrifuged for 30min, and the supernatant was transferred to a new EP tube and centrifuged for another 30 min.
3. Chromatographic conditions are as follows:
and (3) analyzing the column: acquisty BEH C18(1.5 μm; 2.1X 50mm, Waters)
Mobile phase: 0.1% FA +5mM NH4AC in Water 0.1% FA in ACN
4. Standard Curve preparation
Standard curve samples (12.5,25,50,100,200,400,2000,10000nM) were prepared in acetonitrile/water (1/1, V/V) and the analyte peak areas were quantified by Waters MassLynx (version V4.1) software. And calculating the concentration of the substance to be detected by using an external standard method. Taking the peak area (y) of the object to be measured as the ordinate and the concentration (x) of the object to be measured as the abscissa, linear regression is performed by using a weighted (1/x2) least square method to obtain a standard curve equation y as ax + b, wherein a is the slope and b is the intercept.
5. Sample analysis
Respectively taking 50 mu L of supernatant of each matrix sample, adding 450 mu L of acetonitrile/water (1/1, v/v), mixing uniformly by vortex, taking 20 mu L of supernatant, adding 180 mu L of acetonitrile/water (1/1, v/v), mixing uniformly by vortex, performing LC-MS/MS analysis, and substituting the obtained peak area into a standard curve equation to obtain the saturated solubility of each matrix. Each sample was measured four times for each matrix and the results averaged to obtain different matrix saturation solubilities of the final compound.
The results are shown in Table 2.
Solubility is an important property in evaluating the potency of a drug molecule, and it affects the absorption, distribution, drug exposure, oral bioavailability, etc. of the drug molecule in animals and humans. The vast majority of drug molecules are not successfully marketed at the end and poor solubility is a key factor. As can be seen from Table 2, the solubility of the compound of the present invention in different matrixes is greatly improved compared with that of the positive control drug AZD0914, the biological pharmaceutical property is greatly improved, and the pharmaceutical property is better.
Table 2 solubility data for compounds under different matrices
Figure BDA0001466313540000281
Example 10
In vivo pharmacokinetic testing in mice
Healthy female CD-1 mice, randomly divided into two groups of 3, were orally administered with the test compounds, as specified in Table 3 below.
TABLE 3 dosing regimen
Figure BDA0001466313540000282
Each animal was anticoagulated with EDTAK2 by drawing 0.030mL of blood through the orbit at each time point: PO group: 15min, 30min, 1h, 2h, 4h, 6h, 8h and 24h after the test substance is administered. Blood samples were collected on ice and plasma was centrifuged within 30 minutes (centrifugation conditions: 5000 rpm, 10 minutes, room temperature). The samples were stored at-80 ℃ before analysis. The results are shown in Table 4.
TABLE 4 pharmacokinetic data for oral drugs in mice
Figure BDA0001466313540000283
The excellent metabolic property is a key index of the druggability of the compound, and a pharmacogenetic experiment proves that the compound has ideal metabolic characteristics, and the exposure and peak concentration are far better than those of a positive control medicament AZD 0914.
Example 11
In vivo pharmacokinetic testing in rats
SD rats 9, randomly divided into three groups of 3 rats each, and the specific arrangement is shown in Table 5.
TABLE 5 dosing regimen
Figure BDA0001466313540000291
Each animal draws 0.100mL of blood, EDTAK, through the orbit each time 2 Anticoagulation, the collection time points were: (1) PO group: 15min, 30min, 1h, 2h, 4h, 6h, 8h and 24h after the administration of the test substance; (2) group IV: the administration time of the test substance is 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h and 24 h. Blood samples were collected on ice and plasma was centrifuged within 30 minutes (centrifugation conditions: 5000 rpm, 10 minutes, 4 ℃). The samples were stored at-80 ℃ before analysis. The results are shown in Table 6.
TABLE 6 Main pharmacokinetic parameters after oral administration (PO) or intravenous Injection (IV) of Compound 1 in SD rats
Figure BDA0001466313540000292
A comparison of the results obtained from the oral doses of 10mg/kg and 100mg/kg shows the Cmax in plasma max The Cmax of 100mg/kg administered compound 1 was 15 times higher than that of 10mg/kg administered, indicating that the absorption of compound 1 at the dose of 100mg/kg was not significantly affected and that the oral bioavailability gradually increased from 22.6% to 45.8%.
By combining all aspects of pharmacokinetic parameters of the compound 1, the compound 1 has excellent metabolic property in rats, and is worthy of further research and development.
Example 12
In vivo antibacterial Activity of Compounds of the invention against MRSA
1. Test strains
According to the in vitro test results, clinically isolated methicillin-resistant staphylococcus aureus MRSA15-3 (see table 7 below) is selected as a standby test strain for in vivo protection experiments.
TABLE 7 inventive Compounds and control drug vs. Staphylococcus aureus MRSA15-3
In vitro antibacterial Activity of-MIC (mg/L)
Figure BDA0001466313540000301
2. Test animal
Healthy Kunming mouse of about 4 weeks old, with body weight of 18-22g, half female and half male, SPF grade
Expected number of mice used: 200 are
3. In vivo protection test method
3.1 preparation of bacterial liquid
The test bacteria are inoculated in 2ml MH broth one day before infection, 2-3 single colonies are selected and inoculated in the MH broth, the culture is carried out for 6h at 37 ℃, 0.1ml of the bacteria liquid is taken and transferred into 10ml of the MH broth, the culture is carried out for 18h at 37 ℃, and the bacteria liquid is the original bacteria liquid. The resulting suspension was diluted in 5% dry yeast solution in multiple proportions (freshly prepared on the day).
3.2 Minimum Lethal Dose (MLD) assay
Healthy Kunming white mice are taken, the weight of the white mice is 18-22g, the white mice are randomly grouped, 5-10 mice are taken in each group, the half of the mice and the half of the mice are female and male, bacterial liquids with different dilution concentrations are absorbed, the bacterial liquids are respectively injected into the bodies of the mice in an abdominal cavity, 0.5ml of the bacterial liquids are injected into each 20g of the mice, the observation is carried out for 7-14 days after infection, the death number of the mice is recorded, the minimum bacterial quantity causing 100% death of the mice is used as the minimum lethal bacterial quantity (MLD), and the bacterial quantity is used as the infectious bacterial quantity of an in vivo protection test.
3.3 liquid medicine preparation and administration route:
preparing liquid medicine: the test is prepared by 0.5 percent of medicinal sodium carboxymethylcellulose and diluted to a solution with required concentration for standby
The administration route is as follows: administration by intragastric administration
Dose capacity: 0.5ml/20g BW (body weight)
3.4 group design
Setting the test sample dosage range to be 20-2.5mg/kg, accurately weighing the drugs, converting the drug dosage into the effective drug weight according to the potency, preparing the drugs into a solution with the required concentration by using 0.5% sodium carboxymethylcellulose, and setting the torque between the dosage groups to be 1: 0.5.
group design: a. theZD0914(20、10、5、2.5g·kg -1 ) Group, Compound 1(20, 10, 5, 2.5 g.kg) -1 ) Group, infection control group, blank control group, ABT toxicity control.
3.5 in vivo protection test methods and statistics of results
Stopping feeding and supplying water for the mice 18 hours before the test, randomly grouping the mice according to the weight, carrying out intraperitoneal injection on 8 mice in each group, respectively carrying out intraperitoneal injection on infection test bacteria liquid, carrying out intragastric injection on 0.5ml of the mice per 20g, carrying out intragastric administration on 0.5h and 4h after infection according to the designed dose, and carrying out intragastric administration on 0.5ml of the mice per 20 g; observing and recording the death number of the mouse, continuously observing for 7-14 days, and calculating half effective dose ED according to the death number of the mouse by using DAS1.0 software compiled by Sunruie et al and a Bliss method 50 And a 95% confidence limit.
The control AZD0914 group orally administered the cytochrome P450 inhibitor 1-Aminobenzotriazole (ABT)50mg/kg two hours prior to infection and another 50mg/kg ABT 12 hours later, other compounds did not require oral ABT.
Infection control group: only infected bacteria are not administrated, and after the infected bacteria, the normal saline with the same volume as the stomach is perfused; blank control group: no bacteria infection, and injecting equal volume of normal saline into the abdominal cavity, and infusing equal volume of normal saline into the stomach.
4. Test results
The results of the in vivo antibacterial activity test in the systemic infection model are shown in Table 8.
TABLE 8 Compounds of the invention in mice infected with Staphylococcus aureus MRSA15-3
In vivo protection (ED) 50 )
Figure BDA0001466313540000311
The positive control drug AZD0914 needs to be combined with the cytochrome P450 inhibitor 1-Aminobenzotriazole (ABT) to treat ED of mice infected with Staphylococcus aureus MRSA15-3 50 The dose was 11.51mg/kg, whereas Compound 1 was administered orally without ABT for ED in mice infected with Staphylococcus aureus MRSA15-3 50 3.87mg/kg, which is obviously better than AZD 0914.
In conclusion, the compound has better drug forming property than the positive control drug AZD0914, and is expected to become a better antibacterial drug.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (25)

1. A compound shown in a general formula (I), or enantiomer, diastereoisomer, racemate and mixture thereof, or pharmaceutically acceptable salt thereof,
Figure FDA0003686906580000011
in the formula, R 1 Is halogen or cyano;
R 4 is a hydrogen atom or C 1 -C 3 An alkyl group;
each independently represents racemic, S-or R-form;
with the proviso that when R 1 When it is halogen, R 2 、R 3 Together with the carbon atom to which they are attached form a 3-7 membered alicyclic or 3-7 membered oxygen containing heterocyclic ring;
R 1 is cyano, R 2 、R 3 Together with the carbon atom to which they are attached form a 3-7 membered alicyclic ring.
2. The compound of claim 1, wherein R is 1 Is fluorine, chlorine or cyano.
3. The compound of claim 1, wherein R is 1 Is fluorine or chlorine; r 2 、R 3 Together with the carbon atom to which they are attached form a 3-7 membered alicyclic or 3-7 membered oxygen containing heterocyclic ring.
4. The compound of claim 1, wherein R is 1 Is fluorine or chlorine; r 4 Is hydrogen.
5. The compound of claim 1, wherein R is 2 、R 3 Together with the carbon atom to which they are attached form a 3-7 membered alicyclic or 3-7 membered oxygen containing heterocyclic ring; r 4 Is hydrogen.
6. The compound of claim 1, wherein R is 1 Is fluorine; r 2 、R 3 Together with the carbon atom to which they are attached form a 3-7 membered alicyclic or 3-7 membered oxygen containing heterocyclic ring.
7. The compound of claim 1, wherein R is 1 Is cyano, R 2 、R 3 Together with the carbon atom to which they are attached form a 3-membered alicyclic ring.
8. The compound of claim 1, wherein said compound is:
Figure FDA0003686906580000021
9. a pharmaceutical composition comprising a compound of claim 1 or enantiomers, diastereomers, racemates and mixtures thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier or excipient.
10. A process for the preparation of a compound according to claim 1, comprising the steps of:
Figure FDA0003686906580000022
(i) the intermediate Ia is subjected to intramolecular nucleophilic substitution reaction to generate an intermediate Ib;
(ii) carrying out nucleophilic substitution reaction on the intermediate Ib to generate an intermediate Ic;
(iii) the intermediate Ic is hydrolyzed to generate an intermediate Id;
(iv) carrying out nucleophilic substitution reaction on the intermediate Id to generate an intermediate Ie;
(v) the intermediate Ie reacts with barbituric acid to generate a compound shown as a general formula I,
in the formulae 1 、R 2 、R 3 And R 4 Is as defined in claim 1.
11. A process according to claim 10, wherein intermediate Ia is subjected to intramolecular nucleophilic substitution with cesium carbonate to form intermediate Ib.
12. The process of claim 10, wherein intermediate Ib is reacted with N, N' -carbonyldiimidazole by nucleophilic substitution to form intermediate Ic.
13. The process of claim 10, wherein intermediate Id is reacted with 2R, 6R-dimethylmorpholine via nucleophilic substitution to form intermediate Ie.
14. The process of claim 10 wherein intermediate Ic is hydrolyzed catalyzed by hydrochloric acid to form intermediate Id.
15. A process for the preparation of a compound according to claim 1, wherein R is 1 When cyano, the method comprises the following steps:
Figure FDA0003686906580000031
(I') subjecting the intermediate I-12 to hydrolysis reaction to produce an intermediate I-13;
(ii') subjecting the intermediate I-13 to intramolecular nucleophilic substitution reaction to produce an intermediate I-14;
(iii') subjecting intermediate I-14 to nucleophilic substitution to produce intermediate I-15;
(iv') subjecting the intermediate I-15 to nucleophilic substitution to produce an intermediate I-16;
(v') reaction of intermediate I-16 with barbituric acid to produce the compound of claim 1 of formula C,
in the formulae 2 、R 3 And R 4 Is as defined in claim 1.
16. The process of claim 15, wherein intermediate I-12 is hydrolyzed catalyzed by hydrochloric acid to form intermediate I-13.
17. The process of claim 15, wherein intermediate I-13 is reacted with sodium bicarbonate via intramolecular nucleophilic substitution to form intermediate I-14.
18. The process of claim 15, wherein intermediate I-14 is nucleophilic-substituted with 2R, 6R-dimethylmorpholine to form intermediate I-15.
19. The method of claim 15, wherein intermediate I-15 is subjected to nucleophilic substitution with N, N' -carbonyldiimidazole to form intermediate I-16.
20. Use of the compounds according to claim 1 or enantiomers, diastereomers, racemates and mixtures thereof, or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions according to claim 9, for the preparation of a medicament for the treatment of bacterial infectious diseases.
21. The use according to claim 20, wherein the infectious disease is an infectious disease caused by gram-positive bacteria.
22. The use of claim 20, wherein the infectious disease is an infectious disease caused by a multidrug-resistant bacterium.
23. The use of claim 22, wherein the multi-drug resistant bacteria is selected from the group consisting of: MRSA, MSSA, MRSE, MSSE, PRSP, and Spy.
24. A method of in vitro non-diagnostic, non-therapeutic bacteriostatic administration comprising administering to a subject or environment a compound of claim 1 or enantiomers, diastereomers, racemates thereof and mixtures thereof or pharmaceutically acceptable salts thereof or a pharmaceutical composition of claim 9.
25. An intermediate of a compound of formula I, having a structure according to formula Ia, Ib, Ic, Id or Ie:
Figure FDA0003686906580000041
in the formulae 1 、R 2 、R 3 And R 4 Is as defined in claim 1.
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