CN111362967A

CN111362967A - Benzoxadiazatetetradecene derivatives and use thereof

Info

Publication number: CN111362967A
Application number: CN202010347120.4A
Authority: CN
Inventors: 范文华; 唐春雷; 范为正; 吴露婷; 张晴; 张永杰
Original assignee: Nanjing Leizheng Pharmaceutical Technology Co ltd; Jiangnan University
Current assignee: Nanjing Leizheng Pharmaceutical Technology Co ltd; Jiangnan University
Priority date: 2020-04-28
Filing date: 2020-04-28
Publication date: 2020-07-03
Anticipated expiration: 2040-04-28
Also published as: CN111362967B

Abstract

The invention discloses a benzoxadiazacyclotetradecene derivative and application thereof, belonging to the field of medicines. The benzoxadiazacyclotetradecene derivative with the structure shown in the general formula (I) has excellent anaplastic lymphoma enzyme (ALK) inhibition activity and excellent pharmacodynamic performance, and can remarkably prolong the large metabolic half-life of a medicament; can be safely and effectively used for treating anaplastic lymphoma kinase positive (ALK +) metastatic (advanced) non-small cell lung cancer (NSCLC) and the like, therebyProvides a new means for treating cancer, metabolic and immune diseases, cardiovascular diseases, neurological diseases and the like.

Description

Benzoxadiazatetetradecene derivatives and use thereof

Technical Field

The invention relates to a benzoxadiazacyclotetradecene derivative and application thereof, belonging to the field of medicines.

Background

It is well known that lung cancer is one of the most threatening malignancies to humans, with morbidity and mortality ranking first in men and second in women. Among lung cancers, the most common one belongs to non-small cell lung cancer (NSCLC), accounting for about 85% of lung cancers; wherein the positive incidence rate of Anaplastic Lymphoma Kinase (ALK) accounts for 3 to 5 percent of that of non-small cell lung cancer (NSCLC).

ALK is a member of the receptor tyrosine kinase superfamily and the amino acid sequence level is most closely related to members such as Ros-1, leukocyte tyrosine kinase, insulin receptor and cMet (liver growth factor receptor), as are all members of this gene family, which have extracellular ligand binding regions, transmembrane sequences and intracellular kinase catalytic/signaling regions. The signaling ligand properties of anaplastic lymphoma enzyme (ALK) have not been elucidated, and different mechanisms have been proposed in the literature (Stoica G. E. et a., ]. Bio l. chem.,2001,276, 16772-. Anaplastic lymphoma enzyme (ALK) is abundantly expressed primarily in the developing nervous system, and although its expression is retained in certain regions of the brain, spinal cord and eye, its relative content in adult animals tends to decrease. Anaplastic Lymphoma Kinase (ALK) plays an important role in oncology, and point mutations of the full-length ALK enzyme that cause activation of this enzyme and increase expression of the full-length enzyme have all been shown to result in neuroblastoma. Furthermore, fusion of ALK and other proteins due to gene translocation has also been shown to result in activated kinase regions associated with cancer. Many such ALK translocations leading to gene fusions are seen in lymphomas, the most common being the nuclear phosphoprotein NPM-ALK fusions seen in anaplastic large cell lymphomas. ALK and EML4 fusion produces a chimeric protein (EML4-ALK) that is thought to be associated with 3 to 5% of non-small cell lung adenocarcinoma (NSCLC).

In 2011, the ALK/ROS1/c-MET inhibitor crizotinib was approved for the treatment of locally advanced or metastatic NSCLC patients that were ALK-positive as detected by FDA-approved tests. Crizotinib also showed efficacy in treating NSCLC with ROS1 translocation, as clinically observed for other tyrosine kinase inhibitors, mutations in ALK and ROS1 that have conferred resistance to ALK inhibitors.

Therefore, ALK and ROS1 are attractive molecular targets for cancer therapeutic intervention. There remains a need to identify compounds with novel activity profiles for both wild-type and mutant forms of ALK and ROS 1.

Currently, the target drugs for ALK mutation are first-generation ALK targeting drug Crizotinib (Crizotinib), and second-generation ALK targeting drugs include Ceritinib (Ceritinib), aletinib (Alectinib), bugatinib (brigatinib) and the newly-marketed third-generation ALK targeting drug loratinib (loretinanib).

Loratinib (lorelatinb, PF-06463922) is a novel anti-drug resistant ALK inhibitor developed by fevered for the investigative treatment of anaplastic lymphoma kinase positive (ALK +) metastatic (advanced) non-small cell lung cancer (NSCLC). Moreover, the medicine has high blood brain barrier permeability, and is approved to exert better efficacy on the non-small cell lung cancer with central nervous system metastasis.

However, the drug has poor metabolic stability, short half-life period, large dosage, and easy generation of drug resistance and toxic and side effects.

Disclosure of Invention

In order to solve the problems, the inventor develops a novel benzoxadiazacyclotetradecene derivative used as an ALK inhibitor, and the compound has the advantages of lower toxic and side effects, effectively increased blood brain barrier passing rate, longer half life and better safety. The inhibitor is expected to have good curative effect, is expected to overcome the problems of drug resistance and toxic and side effects, and has good development prospect. The novel benzoxadiazacyclotetradecene derivatives and salts of the present invention are effective in inhibiting anaplastic lymphoma enzyme (ALK) and are useful for treating or ameliorating abnormal cell proliferative disorders.

The invention provides a novel benzoxadiazacyclotetradecene derivative serving as an anaplastic lymphoma enzyme (ALK) inhibitor, and the structural compound has better pharmacodynamic performance and higher metabolic stability.

The invention provides a benzoxadiazepitetradecene derivative with a structure shown as a general formula (I) or a pharmaceutically acceptable salt thereof:

wherein when R is₁When it is hydrogen, R₂、R₃Each independently selected from C₃-C₆Cycloalkyl, deuterated methyl; wherein cycloalkyl may be optionally mono-to pentasubstituted with the same or different substituents selected from halogen, trifluoromethyl, cyano, nitro, hydroxy or C₁-C₄An alkyl group;

when R is₁Is selected from C₁-C₄Alkyl radical, C₃-C₆Cycloalkyl, -CH₂OR₄、-COOR₅、-COR₆or-CH₂OP(O)OR₇OR₈When R is₂、R₃Each independently selected from C₁-C₄Alkyl, deuterated methyl or C₃-C₆A cycloalkyl group; wherein saidThe alkyl and cycloalkyl groups may be optionally mono-to pentasubstituted with the same or different substituents selected from halogen, trifluoromethyl, cyano, nitro, hydroxy or C₁-C₄An alkyl group; wherein R is₄，R₅，R₆Are respectively and independently selected from hydrogen and C₁-C₄Alkyl or C₃-C₆A cycloalkyl group; r₇，R₈Each independently selected from hydrogen and C₁-C₄Alkyl or C₃-C₆A cycloalkyl group; wherein said alkyl, cycloalkyl may be optionally mono-to pentasubstituted with the following same or different substituents selected from: halogen, trifluoromethyl, cyano, nitro, hydroxy or C₁-C₄An alkyl group.

The invention provides a benzoxadiazepitetradecene derivative with a structure shown as a general formula (I) or a pharmaceutically acceptable salt thereof, wherein when R is₁When it is hydrogen, R₂、R₃At least one of each is deuterated methyl and the other is selected from C₁-C₄Alkyl radical, C₃-C₆Cycloalkyl, deuterated methyl. Wherein, C₃-C₆Cycloalkyl is preferably cyclopropyl; c₁-C₄The alkyl group is preferably a methyl group.

The invention provides a benzoxadiazepitetradecene derivative with a structure shown as a general formula (I) or a pharmaceutically acceptable salt thereof, R₄Preferably hydrogen, C₁-C₄An alkyl group. Further preferred are methyl and hydrogen.

The invention provides a benzoxadiazepitetradecene derivative with a structure shown as a general formula (I) or a pharmaceutically acceptable salt thereof, R₅Preferably selected from hydrogen, methyl, ethyl or isopropyl;

the invention provides a benzoxadiazepitetradecene derivative with a structure shown as a general formula (I) or a pharmaceutically acceptable salt thereof, R₆Hydrogen is preferred.

The invention provides a benzoxadiazepitetradecene derivative with a structure shown as a general formula (I) or a pharmaceutically acceptable salt thereof, R₇，R₈Independently of one another, preferably hydrogen and methylEthyl, isopropyl, tert-butyl; further preferred are ethyl and tert-butyl.

The invention provides a benzoxadiazepitetradecene derivative with a structure shown as a general formula (I) or a pharmaceutically acceptable salt thereof, which is selected from the following groups:

the invention relates to a benzoxadiazacyclotetradecene derivative with a structure shown in a general formula (I) or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is an inorganic salt or an organic salt, and the inorganic salt comprises hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate and acid phosphate; the organic salt is selected from acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, benzenesulfonate, salicylate.

The invention also aims to provide a pharmaceutical composition, which contains the compound with the general formula (I) or the pharmaceutically acceptable salt thereof, and pharmaceutically acceptable carriers and pharmaceutical excipients.

The drug carrier comprises: microcapsules, microspheres, nanoparticles, liposomes.

The pharmaceutic adjuvant comprises: comprising solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, and release retardants.

The compounds of the present invention or pharmaceutically acceptable salts thereof may be formulated as solid formulations for oral administration, including, but not limited to, capsules, tablets, pills, powders, granules, and the like.

The single dose of the compound of the present invention or a pharmaceutically acceptable salt thereof may be 0.01 to 1000mg, for example, 0.05 to 800mg, 0.1 to 500mg, 0.01 to 300mg, 0.01 to 200mg, 0.05 to 150mg, 0.05 to 50mg, etc.

In another aspect, the present invention provides the use of a compound of formula (I) as described above, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use as an ALK inhibitor.

In another aspect, the present invention provides the use of a compound of formula (I) as described above, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer.

The cancer mentioned in the invention can be selected from non-small cell lung cancer (NSCLC), squamous cell carcinoma, hormone refractory prostate cancer, papillary renal cell carcinoma, colorectal adenocarcinoma, neuroblastoma, Anaplastic Large Cell Lymphoma (ALCL), gastric cancer, metastatic brain cancer and the like.

The invention has the beneficial effects that:

the benzoxadiazacyclotetradecene derivative with the structure shown in the general formula (I) has a convenient synthesis method, good anaplastic lymphoma enzyme (ALK) inhibition activity and excellent pharmacodynamic performance, and can remarkably prolong the large metabolic half-life of a medicament.

The novel benzoxadiazacyclotetradecene derivative has the biological function of inhibiting ALK, is used for treating anaplastic lymphoma kinase positive (ALK +) metastatic (advanced) non-small cell lung cancer (NSCLC), and provides a new means for searching and treating cancers, metabolic and immune diseases, cardiovascular diseases, neurological diseases and the like.

Detailed Description

The technical solution of the present invention will be described in detail with reference to the following examples.

In the present invention, "C₁-C₄Alkyl "refers to a saturated straight or branched chain monovalent hydrocarbon group of 1 to 4 carbon atoms, respectively. Examples include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, and 2-methyl-2-propyl.

In the present invention, "C₃-C₆Cycloalkyl "refers to cycloalkyl groups of 3 to 6 carbon atoms each.

In the present invention "administering" or "administering" an individual compound means providing a compound of the invention to an individual in need of treatment.

< Compound or pharmaceutically acceptable salt thereof >

The invention provides a novel benzoxadiazacyclotetradecene derivative serving as an anaplastic lymphoma enzyme (ALK) inhibitor or a pharmaceutically acceptable salt thereof, wherein the structural formula of the derivative is shown as a general formula (I):

the present invention also provides a process for the preparation of a compound of formula (I) by the reaction of:

reaction scheme 1:

wherein R is₄Selected from hydrogen, C₁-C₄Alkyl or C₃-C₆Cycloalkyl when R₄When hydrogen is obtained by reacting a compound of formula (II) with formaldehyde under tetrabutylammonium fluoride conditions, when R is₄Is C₁-C₄Alkyl radical, C₃-C₆Cycloalkyl radicals can be formed by reaction with R₄OCH₂Br is obtained under alkaline conditions;

alternatively, reaction scheme 2:

wherein R is₅Selected from hydrogen, C₁-C₄Alkyl or C₃-C₆Cycloalkyl radicals obtainable by reacting a compound of the formula (II) with R₅OCOCl is obtained under alkaline conditions;

alternatively, reaction scheme 3:

wherein R is₆Selected from hydrogen, C₁-C₄Alkyl or C₃-C₆Cycloalkyl when R₆When hydrogen, by reaction of a compound of formula (II) with formic acid, when R is₆Is C₁-C₄Alkyl or C₃-C₆Cycloalkyl radicals obtainable by reacting a compound of the formula (II) with R₆COCl is obtained under alkaline condition;

alternatively, reaction scheme 4:

wherein R is₇,R₈Independently selected from hydrogen, C₁-C₄Alkyl or C₃-C₆A cycloalkyl group;

suitable bases include tertiary amine bases such as Diisopropylethylamine (DIEA) and triethylamine, inorganic bases, and the like.

< pharmaceutical composition >

The invention also provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, as defined above, and a pharmaceutically acceptable carrier, excipient or diluent.

The compounds of the present invention or pharmaceutically acceptable salts thereof may be formulated as solid formulations for oral administration, including, but not limited to, capsules, tablets, pills, powders, granules, and the like. In these solid dosage forms, the compounds of general formula (I) according to the invention as active ingredient are mixed with at least one customary inert excipient (or carrier), for example with sodium citrate or dicalcium phosphate. Or mixing with the following components: (1) fillers or solubilizers, for example, starch, lactose, sucrose, glucose, mannitol, silicic acid, and the like; (2) binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, gum arabic and the like; (3) humectants, such as glycerol and the like; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, and the like; (5) a slow solvent such as paraffin and the like; (6) absorption accelerators such as quaternary ammonium compounds and the like; (7) wetting agents such as cetyl alcohol and glyceryl monostearate and the like; (8) adsorbents, for example, kaolin, and the like; (9) lubricants, for example, talc, calcium stearate, solid polyethylene glycols, sodium lauryl sulfate, and the like, or mixtures thereof. Capsules, tablets, pills, etc. may also contain buffering agents.

The solid dosage forms, e.g., tablets, dragees, capsules, pills, and granules, can be coated or microencapsulated with coating and shell materials such as enteric coatings and other crystalline forms of materials well known in the art. They may contain opacifying agents and the release of the active ingredient in such compositions may be delayed in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active ingredient may also be in microencapsulated form with one or more of the above excipients.

The compounds of the present invention or pharmaceutically acceptable salts thereof may be formulated in liquid dosage forms for oral administration, including, but not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, tinctures, and the like. In addition to the compounds of formula (I) or pharmaceutically acceptable salts thereof as active ingredients, the liquid dosage forms may contain inert diluents conventionally employed in the art, such as water and other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn oil, olive oil, castor oil, sesame oil and the like or mixtures of such materials and the like. In addition to these inert diluents, the liquid dosage forms of the present invention may also include conventional adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, perfuming agents and the like.

Such suspending agents include, for example, ethoxylated stearyl alcohol, polyoxyethylene sorbitol, and sorbitan, microcrystalline cellulose, agar, and the like, or mixtures of these materials.

The compounds of the present invention and pharmaceutically acceptable salts thereof may be formulated for parenteral injection in dosage forms including, but not limited to, physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions and dispersions. Suitable carriers, diluents, solvents, excipients include water, ethanol, polyols and suitable mixtures thereof.

The compounds of the present invention or pharmaceutically acceptable salts thereof may be formulated into dosage forms for topical administration, including, for example, ointments, powders, suppositories, drops, sprays, inhalants and the like. The compounds of the general formula (I) according to the invention or their pharmaceutically acceptable salts as active ingredients are mixed under sterile conditions with physiologically acceptable carriers and optionally preservatives, buffers and, if desired, propellants.

The pharmaceutical composition comprises the compound of the general formula (I) or pharmaceutically acceptable salt thereof as an active ingredient, and pharmaceutically acceptable carriers, excipients and diluents. In preparing the pharmaceutical compositions, the compounds of formula (I) or pharmaceutically acceptable salts thereof of the present invention are typically mixed with a pharmaceutically acceptable carrier, excipient or diluent. Wherein the content of the compound of the general formula (I) or the pharmaceutically acceptable salt thereof may be 0.01-1000mg, for example, 0.05-800mg, 0.1-500mg, 0.01-300mg, 0.01-200mg, 0.05-150mg, 0.05-50mg, etc.

< use >

The invention also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer in a mammal.

A compound of general formula (I), or a pharmaceutically acceptable salt thereof, for use in a method of treating a metastatic (advanced) non-small cell lung cancer (NSCLC) disease in a mammal, including a human, by inhibiting anaplastic lymphoma enzyme (ALK) activity, which method comprises administering to said mammal, including a human, a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, as defined above.

A "therapeutically effective amount" is an amount of a compound of the invention effective to produce a biological or medical response (e.g., decrease or inhibit enzymatic protein activity, or ameliorate symptoms, alleviate a condition, slow or delay disease progression, or prevent disease) in an individual.

Cancers to which the present invention refers include lung cancer, bone cancer, membrane adenocarcinoma, skin cancer, cancer of the head or neck, skin melanoma or endobulbar melanoma, uterine cancer, ovarian cancer, rectal cancer, carcinoma of the biliary tree, stomach cancer, colon cancer, breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, hodgkin's disease, carcinoma of the esophagus, carcinoma of the small intestine, carcinoma of the endocrine system, carcinoma of the thyroid gland, carcinoma of the parathyroid gland, carcinoma of the adrenal gland, carcinoma of soft tissue, carcinoma of the urethra, carcinoma of the penis, carcinoma of the prostate, chronic or acute leukemia, lymphocytic lymphomas, cancer of the shoulder, carcinoma of the kidney or ureter, renal cell carcinoma, menamandoma, carcinoma of the Central Nervous System (CNS), primary central nervous system lymphoma, spinal axis carcinoma, brain stem glioma, pituitary adenoma, and the like.

The compounds of the present invention or pharmaceutically acceptable salts thereof may be administered to mammals including humans, either orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), topically (powders, ointments, drops) or intratumorally.

The compound or the pharmaceutically acceptable salt thereof can be independently administered or combined with other pharmaceutically acceptable therapeutic agents to be combined with other antitumor drugs. Such combination therapy may be achieved by the simultaneous, sequential or separate use of the individual components of the therapy. Such therapeutic agents include, but are not limited to: antineoplastic acting on DNA chemical structure, such as cisplatin, antineoplastic acting on nucleotide synthesis, such as methotrexate, 5-fluorouracil and the like, antineoplastic acting on nucleic acid transcription, such as adriamycin, epirubicin, aclacinomycin and the like, antineoplastic acting on tubulin synthesis, such as taxol, vinorelbine and the like, aromatase inhibitors, such as aminoglutethimide, letrozole, rennin and the like, cell signaling pathway inhibitors, such as ALK inhibitors crizotinib, ceritinib, exetinib, loratinib and the like. Anti-tumor monoclonal antibodies, immunosuppressants PD-1, PD-L1 and the like, and the components to be combined can be administered simultaneously or sequentially, in a single preparation form or in different preparation forms. Such combinations include not only combinations of one or other active agents of the compounds of the present invention, but also combinations of two or more other active agents of the compounds of the present invention.

The invention will be further illustrated with reference to the following specific examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, which are not specified under specific conditions, are generally performed under conventional conditions.

The following intermediate substances are involved in the specific embodiment and can be synthesized by the following process route:

intermediate 9: preparation of tert-butyl ((4-bromo-5-cyano-1-methyl-1H-pyrazol-3-yl) methyl) (methyl) carbamate

The route is as follows:

the specific process comprises the following steps:

step a: preparation of methyl 2, 4-dicarbonyl valerate (intermediate 1):

dimethyl oxalate (23.6g,0.2mol) and acetone (29.2g,0.2mol) in an ice salt bath are slowly dropped into 25% sodium methoxide methanol (200mL,0.2mol), the mixture is stirred for 8 hours at 0 ℃, the reaction solution is poured into 200mL of water, ethyl acetate is used for extraction, the pH of an aqueous phase is adjusted to be 2-3 by concentrated hydrochloric acid, the aqueous phase is extracted by ethyl acetate, an organic phase is washed by water, saturated salt is washed by water, and the product is concentrated after drying to obtain 26g of a product, wherein the yield is as follows: 90 percent.

Step b: preparation of methyl 3-methyl-1H-pyrazole-5-carboxylate (intermediate 2):

dissolving methyl 2, 4-dicarbonyl valerate (intermediate 1) (12g,0.076mol) in 100mL dichloromethane, slowly adding hydrazine hydrate (85%, 5.3g,0.091mol) dropwise under ice salt bath, stirring at 0 ℃ for 3 hours, pouring the reaction solution into 200mL water, extracting with dichloromethane, washing the organic phase with water, drying, and concentrating to obtain 8.8g of a product, wherein the yield is as follows: 83 percent.

Step c: preparation of methyl 1, 3-dimethyl-1H-pyrazole-5-carboxylate (intermediate 3):

dissolving 3-methyl-1H-pyrazole-5-carboxylic acid methyl ester (intermediate 2) (5g,0.036mol) in 50mL tetrahydrofuran, adding methyl iodide (6.1g,0.043mol), reacting at 50 ℃ for 3 hours, pouring the reaction solution into 100mL water, extracting with ethyl acetate, washing the organic phase with water, drying, and concentrating to obtain 4.5g of a product, wherein the yield is as follows: 82 percent.

Step d: preparation of methyl 4-bromo-1, 3-dimethyl-1H-pyrazole-5-carboxylate (intermediate 4):

methyl 1, 3-dimethyl-1H-pyrazole-5-carboxylate (intermediate 3) (1g,6.49mmol) was dissolved in 5mL DMF and NBS (1.73g,9.73mmol) was added portionwise and stirred at room temperature for 4 hours after the addition. And (3) dropwise adding the reaction solution into 50mL of ice water, separating out a white solid, filtering, washing a filter cake with water, and drying to obtain 1.1g of a product, wherein the yield is as follows: 73 percent.

Step e: preparation of 4-bromo-1, 3-dimethyl-1H-pyrazole-5-carboxamide (intermediate 5)

Adding 4-bromo-1, 3-dimethyl-1H-pyrazole-5-carboxylic acid methyl ester (intermediate 4) (1g,0.0043mol) into 10mL of ammonia water, stirring overnight at room temperature, precipitating a solid, performing suction filtration, and drying to obtain 0.65g of a product, wherein the yield is as follows: 69.5 percent.

Step f: preparation of 4-bromo-1, 3-dimethyl-1H-pyrazole-5-carbonitrile (intermediate 6)

Suspending 4-bromo-1, 3-dimethyl-1H-pyrazole-5-carboxamide (intermediate 5) (400mg,1.83mmol) in 5mL acetonitrile, adding phosphorus oxychloride (844mg,3.0eq), heating to 80 ℃ for reaction for 3 hours, cooling the reaction solution to room temperature, slowly pouring into ice water, precipitating a solid, filtering, washing with water, and drying to obtain a product 200mg, wherein the yield is 54.5%.

Step g: preparation of 4-bromo-3- (bromomethyl) -1-methyl-1H-pyrazole-5-carbonitrile (intermediate 7)

4-bromo-1, 3-dimethyl-1H-pyrazole-5-carbonitrile (intermediate 6) (500mg,2.5mmol), NBS (489mg,2.75mmol), AIBN (82mg,0.5mmol) was dissolved in 10mL of carbon tetrachloride, protected with nitrogen and refluxed overnight. After the reaction solution was cooled to room temperature, it was filtered, and the filtrate was spin-dried to obtain 600mg of the product with a yield of 86%.

Step h: preparation of 4-bromo-1-methyl-3- ((methylamino) methyl) -1H-pyrazole-5-carbonitrile (intermediate 8)

Dissolving 4-bromo-3- (bromomethyl) -1-methyl-1H-pyrazole-5-carbonitrile (intermediate 7) (1g,3.6mmol) in 10mL of methylamine alcohol solution, stirring overnight at room temperature, rotary evaporating to dryness, adjusting pH to 2-3 with dilute hydrochloric acid, washing with ethyl acetate to remove impurities, adjusting pH of the aqueous phase to 9-10 with 1M aqueous NaOH, extracting with ethyl acetate, combining the organic phases, washing with water, drying, and concentrating to give 600mg of product, yield: 73 percent.

Step i: preparation of tert-butyl ((4-bromo-5-cyano-1-methyl-1H-pyrazol-3-yl) methyl) (methyl) carbamate (intermediate 9)

4-bromo-1-methyl-3- ((methylamino) methyl) -1H-pyrazole-5-carbonitrile (intermediate 8) (500mg,2.18mmol) was dissolved in 10mL tetrahydrofuran, triethylamine (330mg,3.27mmol) and di-tert-butyl dicarbonate (713mg,3.27mmol) were added, stirring was carried out at room temperature for 3 hours, the reaction was diluted with ethyl acetate, washed with aqueous citric acid, washed with water, dried, and concentrated to give 680mg of product, yield: 95 percent.¹H NMR(400MHz,CDCl3)δ4.46(br s,2H),4.01(s,3H),2.83(brs,3H),1.47(s,9H).

Intermediate 14: preparation of methyl (R) -2- (1- ((2-amino-5-bromopyridin-3-yl) oxy) ethyl) -4-fluorobenzoate

The route is as follows:

step j: preparation of (S) -1- (5-fluoro-2-iodophenyl) ethan-1-ol (intermediate 10)

A solution of (-) -DIPCI (5.7g,17.8mmol) in tetrahydrofuran (50mL) was cooled to-20 to-30 deg.C, then a solution of 1- (5-fluoro-2-iodophenyl) ethanone (3.13g,11.9mmol) in tetrahydrofuran (50mL) was added dropwise and the reaction was heated to room temperature. After 2h, cool to-30 deg.C, add another portion of (-) -DIPCl (3.8g,11.9mmol), incubate for 30 min, then warm to room temperature, concentrate to dryness after 1h, and dissolve the residue with methyl tert-butyl ether (100 mL). A solution of diethanolamine (3.1g,29.6mmol) in ethanol/tetrahydrofuran (7.5mL/15mL) was slowly added dropwise to the reaction solution under ice-cooling, heated under reflux for 2 hours, then cooled to room temperature, filtered, and the filtrate was concentrated. The residue was taken up in 7:3 heptane/ethyl acetate (100mL) to precipitate a solid which was filtered. This procedure was repeated until no more solids were observed after the liquid was concentrated. Purifying by column chromatography to obtain oily substance. The colorless oil was further purified by recrystallization from heptane to give 1.8g of white crystals, yield: 57 percent.

Step k: preparation of (S) -1- (5-fluoro-2-iodophenyl) methanesulfonic acid ethyl ester (intermediate 11)

A solution of intermediate 10(2.2g,8.3mmol) in methyl tert-butyl ether (35mL) was cooled to 0 deg.C, triethylamine (2.3mL,16.6mmol) and methanesulfonyl chloride (0.96mL,12.4mmol) were added dropwise in that order, and the reaction was heated to room temperature and stirred for 3 hours. Filter and wash the solid with ethyl acetate. The filtrate was concentrated to give 3.5g of a pale yellow oil, yield: 80 percent of

Step l: preparation of (R) -3- (1- (5- (5-fluoro-2-iodophenyl) ethoxy) pyridin-2-amine (intermediate 12)

Cesium carbonate (6.5g,20.1mmol) was added to a mixed solvent of 2-amino-3-hydroxypyridine (1.33g,12.1mmol) in methyltetrahydrofuran (60mL) and acetone (30mL), stirred at room temperature for 30 minutes, then heated to 40 ℃, and a solution of intermediate 11(3.44g,80mmol) in methyltetrahydrofuran (30mL) was added dropwise. The reaction was carried out at 80 ℃ for 24 hours. The reaction solution was filtered, the filtrate was concentrated, and column chromatography purification was performed to obtain 1.4g of a white solid, yield: 38 percent of

Step m: preparation of methyl (R) -2- (1- (((2-aminopyridin-3-yl) oxy) ethyl) -4-fluorobenzoate (intermediate 13)

Intermediate 12(2g,5.7mmol) was dissolved in 20mL of methanol and triethylamine (1.54mL,11.3mmol) was added to the solution, followed by Pd (dppf) C1₂(0.41g,0.57 mmol). The reaction was carried out at 100 ℃ for 16 hours under an atmosphere of carbon monoxide at a pressure of 100 psi. The reaction mixture was filtered, the filtrate was concentrated, and column chromatography purification gave 1.3g of a brown oil, yield: 79 percent.

Step n: preparation of methyl (R) -2- (1- ((2-amino-5-bromopyridin-3-yl) oxy) ethyl) -4-fluorobenzoate (intermediate 14)

Intermediate 13(1.3g,4.5mmol) was dissolved in 20mL acetonitrile and NBS (0.79g,4.5mmol) in acetonitrile (20mL solution) was added slowly at 0 ℃ and the mixture was stirred for 2h after addition was complete. Concentrate to dryness, dissolve the residue with ethyl acetate and wash with 2M aqueous sodium hydroxide and 10% aqueous sodium thiosulfate. Drying, concentration, column chromatography gave 1.2g, yield: 73 percent.

^lHNMR(400MHz,DMSO-d₆)δ:7.97-7.93(m,1H),7.61-7.51(m,1H),7.54(s,1H),7.29-7.24(m,1H),6.89(s,1H),6.25-6.08(m,3H),3.91(s,3H),1.59(d,J＝8.0Hz,3H).

Intermediate 18: preparation of (10R) -7-amino-12-fluoro-2, 10, 16-trimethyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathizatetradecyl-3-carbonitrile

The route is as follows:

step o: preparation of methyl (R) -2- (1- ((5-amino-2- (3- ((((tert-butoxycarbonyl) (methyl) amino) methyl ] - -5-cyano-1-methyl-1H-pyrazol-4-) pyridin-3-yl) oxy) ethyl-4-fluorobenzoate (intermediate 15)

Intermediate 14(0.9g,2.4 mmols), intermediate 9(1.0g,3.0 mmols), pinacol diboron ester (0.9g,3.6 mmols), cesium fluoride (1.9g,12.6 mmols) were dissolved in a 9:1 methanol/water (12mL) solution, heated to 60 deg.C, and Pd (dppf) Cl was added under nitrogen protection₂A solution of (175mg,0.24mmol) in toluene (1.5mL) was stirred at reflux for 3 h. Adding another part of Pd (dppf) Cl₂A solution of (87mg,0.12mmol) in toluene (1.5mL) was stirred at 60 ℃ overnight. After cooling to room temperature, ethyl acetate was added, filtered, and the filtrate was washed with water and brine in sequence, dried, and concentrated. Purification by column chromatography gave 570mg of a yellow oil, yield: and 43 percent.

Step p preparation of (R) -2- (1- ((5-amino-2- (3- ((((tert-butoxycarbonyl) (methyl) amino) methyl ] -5-cyano-1-methyl-1H-pyrazol-4-yl) pyridin-3-yl) oxy) ethyl) -4-fluorobenzoic acid (intermediate 16)

Intermediate 15(2.7g,5mmol) was dissolved in methanol (20mL), a solution of sodium hydroxide (1.0g,25mmol) in water (2mL) was added, and the mixture was stirred at 40 ℃ for 3 hours. The reaction was diluted with water, concentrated to remove methanol, and the aqueous phase was washed with ethyl acetate. The aqueous phase was neutralized with dilute hydrochloric acid, extracted with ethyl acetate, dried, and concentrated to give 2g of a pale yellow solid, yield: 76 percent.

Step q: preparation of (R) -2- (1- ((5-amino-2- (5-cyano-1-methyl-3- ((methylamino) methyl) -1H-pyrazol-4-yl) pyridin-3-yl) oxy) ethyl) -4-fluorobenzoic acid (intermediate 17)

Intermediate 16(960mg,1.82mmol) was dissolved in 4M hydrogen chloride-dioxane solution (15mL) and the reaction was stirred at room temperature for 2.5 h. Concentration gave 700mg of an off-white solid, yield: 83.1 percent.

Step r: preparation of (10R) -7-amino-12-fluoro-2, 10, 16-trimethyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathizatetradecane-3-carbonitrile (intermediate 18)

Intermediate 17(100mg,0.217mmol) was dissolved in DMF (10mL), DIPEA (42mg,0.325mmol) was added, the temperature was reduced to 0 ℃ and HATU (123.5mg,0.325mmol) was slowly added and reacted at 0 ℃ for 2 hours. Adding the reaction solution into water, extracting with ethyl acetate, washing the organic phase with water, drying, concentrating, and performing column chromatography to obtain 40mg, wherein the yield is as follows: 45 percent.

^lHNMR(400MHz,DMSO-d₆)δ:7.61-7.58(m,2H),7.48-7.45(m,1H),7.20-7.16(m,1H),6.81(s,1H),6.19(s,2H),5.61(q,J＝8.0Hz,1H),4.44(d,J＝16.0Hz,1H),4.20(d,J＝12.0Hz,1H),4.04(s,3H),2.99(s,3H),1.68(d,J＝8.0Hz,3H).

Intermediate 25: preparation of tert-butyl ((4-bromo-5-cyano-1-deuterated methyl-1H-pyrazol-3-yl) methyl) (cyclopropyl) carbamate

The route is as follows:

the synthesis method refers to the synthesis of intermediate 9, wherein iodomethane is replaced by deuterated iodomethane, methylamine is replaced by cyclopropylamine, ESI-MS M/z:358.2/360.2[ M + H ]]⁺。

Intermediate 27: preparation of tert-butyl ((4-bromo-5-cyano-1-deuterated methyl-1H-pyrazol-3-yl) methyl) (deuterated methyl) carbamate

The route is as follows:

synthetic methods reference intermediate 25In which cyclopropylamine is replaced with deuterated methylamine, ESI-MS M/z:335.2/337.2[ M + H]⁺。

Intermediate 34: preparation of tert-butyl ((4-bromo-5-cyano-1-cyclopropyl-1H-pyrazol-3-yl) methyl) (deuterated methyl) carbamate

The route is as follows:

the synthesis method refers to the synthesis of an intermediate 9, wherein methyl iodide is replaced by bromocyclopropane, methylamine is replaced by deuterated methylamine, ESI-MS M/z is 358.2/360.2[ M + H ]]⁺。

Intermediate 36: preparation of tert-butyl ((4-bromo-5-cyano-1-cyclopropyl-1H-pyrazol-3-yl) methyl) (methyl) carbamate

Synthetic methods reference the synthesis of intermediate 25, where cyclopropylamine was replaced with methylamine, ESI-MS M/z:332.2/334.2[ M + H]⁺。

Intermediate 38: preparation of tert-butyl ((4-bromo-5-cyano-1-methyl-1H-pyrazol-3-yl) methyl) (deuterated methyl) carbamate:

the route is as follows:

the synthesis method refers to the synthesis of an intermediate 9, wherein methylamine is replaced by deuterated methylamine, ESI-MS M/z is 332.2/334.2[ M + H ]]⁺。

Intermediate 40: preparation of tert-butyl ((4-bromo-5-cyano-1-methyl-1H-pyrazol-3-yl) methyl) (cyclopropyl) carbamate

The route is as follows:

synthetic methods reference the synthesis of intermediate 9, where methylamine was replaced with cyclopropylamine, ESI-MS M/z 355.2/357.2[ M + H]⁺。

Example 1: (10R) -7-hydroxymethyl-amino-12-fluoro-2, 10, 16-trimethyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathizatetradecane-3-carbonitrile (Compound 1)

Intermediate 18(100mg,0.245mmol) was suspended in water (1mL), dichloromethane (1mL), methanol (1mL), and aqueous formaldehyde (37%, 0.4mL) and tetrabutylammonium fluoride (1M,0.08mL) were added. The reaction mixture was extracted with dichloromethane overnight at room temperature, the organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and column purified to give 80mg of a solid in 74.7% yield.

ESI-MS m/z:437.2[M+H]⁺。

^lHNMR(400MHz,DMSO-d₆)δ:9.29(s,1H),7.61-7.58(m,2H),7.48-7.45(m,1H),7.20-7.16(m,1H),6.81(s,1H),6.39(d,J＝4.0Hz,2H),5.61(q,J＝8.0Hz,1H),4.44(d,J＝16.0Hz,1H),4.20(d,J＝12.0Hz,1H),4.04(s,3H),2.99(s,3H),1.68(d,J＝8.0Hz,3H).

Example 2: (10R) -7-carbamic acid ethyl ester-12-fluoro-2, 10, 16-trimethyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathizatetradecane-3-carbonitrile (Compound 2)

Intermediate 18(100mg,0.245mmol) was dissolved in THF, DIPEA (38mg,0.294mmol) was added, ethyl chloroformate (26.5mg,0.245mmol) was slowly added at 0 ℃, then stirred at room temperature overnight, the reaction was washed with water, extracted with ethyl acetate, the organic phase was dried and spun dry, and the column was purified to 35mg, yield: 29.7 percent.

ESI-MS m/z:479.2[M+H]⁺。

^lHNMR(400MHz,DMSO-d₆)δ:9.51(s,1H),7.98(s,1H),7.83-7.80(m,1H),7.49-7.47(m,1H),7.21-7.16(m,2H),5.66(q,J＝8.0Hz,1H),4.48(d,J＝16.0Hz,1H),4.25(d,J＝12.0Hz,1H),4.18(q,J＝8.0Hz,2H),4.08(s,3H),3.00(s,3H),1.68(d,J＝8.0Hz,3H),1.28(t,J＝8.0Hz,3H).

Example 3: (10R) -7-Aminocarboxaldehyde-12-fluoro-2, 10, 16-trimethyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathizatetradecane-3-carbonitrile (Compound 3)

Intermediate 18(100mg,0.245mmol) was slowly added to formic acid (113mg,2.45mmol) under ice-bath conditions, acetic anhydride (37mg,0.367mmol) was slowly added, and after the addition was completed, the mixture was stirred at room temperature overnight. Diluting the reaction solution with ethyl acetate, washing with water, drying, concentrating, and performing column chromatography to obtain a solid 42mg, wherein the yield is as follows: 39.2 percent.

ESI-MS m/z:435.2[M+H]⁺。

^lHNMR(400MHz,DMSO-d₆)δ:10.42(d,J＝12.0Hz,1H),9.23(s,1H),7.89(s,1H),7.81(d,J＝8.0Hz,1H),7.50-7.46(m,1H),7.21-7.17(m,2H),5.73(q,J＝8.0Hz,1H),4.48(d,J＝16.0Hz,1H),4.23(d,J＝12.0Hz,1H),4.08(s,3H),3.01(s,3H),1.72(d,J＝8.0Hz,3H).

Example 4: (10R) -7-amino-12-fluoro-2-deuterated methyl-10-methyl-16-cyclopropyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathizazepine-3-carbonitrile (Compound 4)

Synthetic methods reference the synthesis of intermediate 18, with intermediate 9 replaced intermediate 25.

ESI-MS m/z:436.2[M+H]⁺

lHNMR(400MHz,DMSO-d₆)δ:7.61-7.58(m,2H),7.48-7.45(m,1H),7.20-7.16(m,1H),6.81(s,1H),6.19(s,2H),5.61(q,J＝8.0Hz,1H),4.44(d,J＝16.0Hz,1H),4.20(d,J＝12.0Hz,1H),2.30-2.22(m,1H),1.68(d,J＝8.0Hz,3H),0.83-0.56(m,4H).

Example 5: (10R) -7-amino-12-fluoro-2, 16-dideuteromethyl-10-methyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathizazepine-3-carbonitrile (Compound 5)

Synthetic methods reference the synthesis of intermediate 18, wherein intermediate 9 is replaced with intermediate 27.

ESI-MS m/z:413.2[M+H]⁺

lHNMR(400MHz,DMSO-d₆)δ:7.61-7.58(m,2H),7.48-7.45(m,1H),7.20-7.16(m,1H),6.81(s,1H),6.19(s,2H),5.61(q,J＝8.0Hz,1H),4.44(d,J＝16.0Hz,1H),4.20(d,J＝12.0Hz,1H),1.68(d,J＝8.0Hz,3H).

Example 6: (10R) -7-amino-12-fluoro-2-cyclopropyl-10-methyl-16-deuterated methyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathizazepine-3-carbonitrile (Compound 6)

Synthetic methods reference the synthesis of intermediate 18, wherein intermediate 9 is replaced with intermediate 34.

ESI-MS m/z:436.2[M+H]⁺

lHNMR(400MHz,DMSO-d₆)δ:7.61-7.58(m,2H),7.48-7.45(m,1H),7.20-7.16(m,1H),6.81(s,1H),6.19(s,2H),5.61(q,J＝8.0Hz,1H),4.44(d,J＝16.0Hz,1H),4.20(d,J＝12.0Hz,1H),2.32-2.25(m,1H),1.68(d,J＝8.0Hz,3H),0.81-0.58(m,4H).

Example 7: (10R) -7-hydroxymethyl-amino-12-fluoro-2, 10-dimethyl-16-deuterated methyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathizazepine-3-carbonitrile (Compound 7)

Synthetic methods reference the synthesis of example 1, wherein intermediate 9 is replaced with intermediate 38.

ESI-MS m/z:440.2[M+H]⁺

^lHNMR(400MHz,DMSO-d₆)δ:9.29(s,1H),7.61-7.58(m,2H),7.48-7.45(m,1H),7.20-7.16(m,1H),6.81(s,1H),6.39(d,J＝4.0Hz,2H),5.61(q,J＝8.0Hz,1H),4.44(d,J＝16.0Hz,1H),4.20(d,J＝12.0Hz,1H),4.04(s,3H),1.68(d,J＝8.0Hz,3H).

Example 8: (10R) -7-hydroxymethyl-amino-12-fluoro-2, 16-dideuteromethyl-10-methyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathizazepine-3-carbonitrile (Compound 8)

Synthetic methods reference the synthesis of example 1, wherein intermediate 9 is replaced with intermediate 27.

ESI-MS m/z:443.2[M+H]⁺

^lHNMR(400MHz,DMSO-d₆)δ:9.29(s,1H),7.61-7.58(m,2H),7.48-7.45(m,1H),7.20-7.16(m,1H),6.81(s,1H),6.39(d,J＝4.0Hz,2H),5.61(q,J＝8.0Hz,1H),4.44(d,J＝16.0Hz,1H),4.20(d,J＝12.0Hz,1H),1.68(d,J＝8.0Hz,3H).

Example 9: (10R) -7-hydroxymethyl-amino-12-fluoro-2-deuterated methyl-10, 16-dimethyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathizazepine-3-carbonitrile (Compound 9)

Synthetic methods reference the synthesis of example 1, wherein intermediate 9 is replaced with intermediate 36.

ESI-MS m/z:440.2[M+H]⁺

^lHNMR(400MHz,DMSO-d₆)δ:9.29(s,1H),7.61-7.58(m,2H),7.48-7.45(m,1H),7.20-7.16(m,1H),6.81(s,1H),6.39(d,J＝4.0Hz,2H),5.65(q,J＝8.0Hz,1H),4.44(d,J＝16.0Hz,1H),4.20(d,J＝12.0Hz,1H),2.99(s,3H),1.68(d,J＝8.0Hz,3H).

Example 10: (10R) -7-Aminomethylphosphonic acid di-tert-butyl-12-fluoro-2, 10, 16-trimethyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathizatetradecane-3-carbonitrile (Compound 10)

Dissolving the intermediate 18(100mg,0.246mmol) in 1mL DMF, adding NaH (10mg,0.246mmol) under ice bath, reacting at 0 ℃ for 0.5h after the addition is finished, then adding DMF (0.5mL) of di-tert-butyl chloromethyl phosphate (67mg,0.246mmol) into the reaction solution, reacting at room temperature for 2h, pouring the reaction solution into water, extracting DCM, washing the organic phase with water, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, and purifying the column to obtain 20mg of a solid, wherein the yield is: 13 percent.

ESI-MS m/z:629.4[M+H]⁺

^lHNMR(400MHz,DMSO-d₆)δ:9.28(s,1H),7.61-7.58(m,2H),7.48-7.45(m,1H),7.20-7.16(m,1H),6.81(s,1H),6.36(d,J＝4.0Hz,2H),5.63(q,J＝8.0Hz,1H),4.44(d,J＝16.0Hz,1H),4.20(d,J＝12.0Hz,1H),4.04(s,3H),2.99(s,3H),1.68(d,J＝8.0Hz,3H)，1.32(s,18H)

Example 11: (10R) -7-Aminophosphoric acid dihydromethyl ester-12-fluoro-2, 10, 16-trimethyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathidiazacyclo-3-carbonitrile (Compound 11)

Compound 13(100mg,0.159mmol) was dissolved in 1mL of dilute hydrochloric acid (1M) and 1mL of tetrahydrofuran, stirred at room temperature for 1 hour, extracted with dichloromethane, dried and then spin-dried to give a solid 25mg, yield: 30.5 percent.

ESI-MS m/z:517.1[M+H]⁺

^lHNMR(400MHz,DMSO-d₆)δ:9.28(s,1H),7.61-7.58(m,2H),7.48-7.45(m,1H),7.20-7.16(m,1H),6.81(s,1H),6.34(d,J＝4.0Hz,2H),5.61(q,J＝8.0Hz,1H),4.44(d,J＝16.0Hz,1H),4.20(d,J＝12.0Hz,1H),4.04(s,3H),2.99(s,3H),1.68(d,J＝8.0Hz,3H).

Example 12: (10R) -7-Aminophosphoric acid diethyl ester-12-fluoro-2, 10, 16-trimethyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathidiazacyclo-3-carbonitrile (Compound 12)

The synthesis was as in example 10, replacing di-tert-butyl chloromethyl phosphate with diethyl chloromethyl phosphate.

ESI-MS m/z:573.2[M+H]⁺

^lHNMR(400MHz,DMSO-d₆)δ:9.28(s,1H),7.61-7.58(m,2H),7.48-7.45(m,1H),7.20-7.16(m,1H),6.81(s,1H),6.36(d,J＝4.0Hz,2H),5.61(q,J＝8.0Hz,1H),4.44(d,J＝16.0Hz,1H),4.20(d,J＝12.0Hz,1H),4.04(s,3H),4.03(q,J＝8.0Hz,4H),2.99(s,3H),1.68(d,J＝8.0Hz,3H),1.41(t,J＝8.0Hz,6H).

Example 13: (10R) -7-amino acid dimethyl ester-12-fluoro-2, 10, 16-trimethyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathizatetradecane-3-carbonitrile (Compound 13)

The synthesis was as in example 10, but di-tert-butyl chloromethyl phosphate was replaced by dimethyl chloromethyl phosphate.

ESI-MS m/z:545.2[M+H]⁺

lHNMR(400MHz,DMSO-d₆)δ:9.28(s,1H),7.61-7.58(m,2H),7.48-7.45(m,1H),7.20-7.16(m,1H),6.81(s,1H),6.36(d,J＝4.0Hz,2H),5.63(q,J＝8.0Hz,1H),4.44(d,J＝16.0Hz,1H),4.20(d,J＝12.0Hz,1H),4.04(s,3H),3.78(s,6H),2.99(s,3H),1.68(d,J＝8.0Hz,3H).

Example 14: (10R) -7-Methoxymethylmethylamino-12-fluoro-2, 10, 16-trimethyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathizatetradecane-3-carbonitrile (Compound 14)

Dissolving the intermediate 18(100mg,0.246mmol) in 1mL DMF, adding NaH (10mg,0.246mmol) under ice bath, reacting at 0 ℃ for 0.5h after the addition is finished, then adding bromomethyl methyl ether (30.7mg,0.246mmol), reacting at room temperature for 2h, pouring the reaction liquid into water, extracting with DCM, washing the organic phase with water, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, and purifying with a column to obtain a solid 32mg, yield: 28.9 percent.

ESI-MS m/z:451.2[M+H]⁺

^lHNMR(400MHz,DMSO-d₆)δ:9.29(s,1H),7.61-7.58(m,2H),7.48-7.45(m,1H),7.20-7.16(m,1H),6.81(s,1H),6.39(d,J＝4.0Hz,2H),5.61(q,J＝8.0Hz,1H),4.44(d,J＝16.0Hz,1H),4.20(d,J＝12.0Hz,1H),4.04(s,3H),3.32(s,3H),2.99(s,3H),1.68(d,J＝8.0Hz,3H).

Example 15: (10R) -7-Aminomethylphosphonic acid di-tert-butyl-12-fluoro-2, 10-dimethyl-16-deuterated methyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathidiazacyclo-3-carbonitrile (Compound 15)

The synthesis was as in example 10, with intermediate 9 replacing intermediate 38.

ESI-MS m/z:632.3[M+H]⁺

^lHNMR(400MHz,DMSO-d₆)δ:9.28(s,1H),7.61-7.58(m,2H),7.48-7.45(m,1H),7.20-7.16(m,1H),6.81(s,1H),6.36(d,J＝4.0Hz,2H),5.61(q,J＝8.0Hz,1H),4.44(d,J＝16.0Hz,1H),4.20(d,J＝12.0Hz,1H),4.04(s,3H),1.68(d,J＝8.0Hz,3H)，1.32(s,18H).

Example 16: (10R) -7-Aminomethylphosphonic acid di-tert-butyl-12-fluoro-2-deuterated methyl-10, 16-dimethyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathizatetradecyl-3-carbonitrile (Compound 16)

The synthesis was as in example 10, with intermediate 9 replacing intermediate 36.

ESI-MS m/z:632.3[M+H]⁺

^lHNMR(400MHz,DMSO-d₆)δ:9.28(s,1H),7.61-7.58(m,2H),7.48-7.45(m,1H),7.20-7.16(m,1H),6.81(s,1H),6.36(d,J＝4.0Hz,2H),5.62(q,J＝8.0Hz,1H),4.44(d,J＝16.0Hz,1H),4.20(d,J＝12.0Hz,1H),2.99(s,3H),1.68(d,J＝8.0Hz,3H)，1.32(s,18H).

Example 17: (10R) -7-Aminomethylphosphonic acid di-tert-butyl-12-fluoro-2, 10-dimethyl-16-cyclopropyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathizazepine-3-carbonitrile (Compound 17)

The synthesis was as in example 10, with intermediate 9 replacing intermediate 40.

ESI-MS m/z:655.3[M+H]⁺

^lHNMR(400MHz,DMSO-d₆)δ:9.28(s,1H),7.61-7.58(m,2H),7.48-7.45(m,1H),7.20-7.16(m,1H),6.81(s,1H),6.36(d,J＝4.0Hz,2H),5.61(q,J＝8.0Hz,1H),4.44(d,J＝16.0Hz,1H),4.20(d,J＝12.0Hz,1H),4.04(s,3H),2.30-2.22(m,1H),1.68(d,J＝8.0Hz,3H),1.32(s,18H),0.83-0.56(m,4H).

Example 18: (10R) -7-hydroxymethyl-amino-12-fluoro-2, 10-dimethyl-16-cyclopropyl-5-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methine bridge) -pyrazolo [4,3-H ] [2,5,11] benzoxathizazepine-3-carbonitrile (Compound 18)

Synthetic methods reference the synthesis of example 1, wherein intermediate 9 is replaced with intermediate 40.

ESI-MS m/z:463.3[M+H]⁺。

^lHNMR(400MHz,DMSO-d₆)δ:9.29(s,1H),7.61-7.58(m,2H),7.48-7.45(m,1H),7.20-7.16(m,1H),6.81(s,1H),6.39(d,J＝4.0Hz,2H),5.61(q,J＝8.0Hz,1H),4.44(d,J＝16.0Hz,1H),4.20(d,J＝12.0Hz,1H),4.03(s,3H),2.30-2.22(m,1H),1.67(d,J＝8.0Hz,3H),0.83-0.56(m,4H).

Example 19 investigation of ALK inhibitory Effect of novel benzoxadiazacyclotetradecene derivatives

Enzyme analysis of wild type ALK and L1196M mutant ALK

Inhibition of the ALK enzyme by wild-type ALK and L1196M mutant ALK was measured by microfluidic mobility shift analysis. Reactions were performed in 96-well plates in 50 μ L volumes containing preactivated human recombinant wild-type (1.3nM) or L1196M (0.5nM) ALK kinase domain (amino acids 1093 to 1411), 1.5 μ M phosphorus receptor, 5' FAM-KKSRGDYMTMQIG-CONH₂(CPCScientific, Sunnyva1e, Calif.), test compound (11-dose three-fold serial dilution, final 2% maple dimethyl) or pure dimethylsulfoxide, 1mM DTT, 0.002% Tween-20 with 5mM magnesium chloride/25 mM Hepes (pH 7.1), preincubated for 20 minutes, initiated by addition of ATP (60 μ M final concentration, km grade). The reaction was incubated at room temperature for 1 hour, stopped by the addition of 0.1M EDTA (pH8), and the extent of reaction (5% to 20% conversion without inhibitor) was determined by separating the fluorescently labeled peptide substrate from the phosphorylated product by LabChip EZ Reader II (Ca1iperLife Sciences, Hopkinton, Mass.) electrophoresis. Kinetic and crystallographic studies confirmed that the inhibitor is ATP competitive. Ki values were calculated by fitting the conversion to an equation for competitive inhibition using non-linear regression (GraphPad Prism, GraphPad software, San Diego, Calif.), and wild-type ATP K was experimentally determined_mATP K of 58. mu.M and L1196M kinase_mThe value was 55 μ M. Self-preparing ATThe pase (baculovirus expressed), preactivated for 1 hour at room temperature in the presence of 2mM ATP, 10mM magnesium chloride and 4mM DTT/20mM Hepes (pH 7.5) using autophosphorylation of 16 μ M unactivated enzyme, confirmed complete phosphorylation of the ALK kinase domain (4 phosphates per protein molecule) by Q-TOF mass spectrometry.

Cellular Phospho-ALK (Tyr1604) ELISA for EML 4-ALK:

cell lines:

NIH-3T3EML4-ALK wt v1 and NIH-3T3EML4-ALK v1 Ll196M cells are human stable cell lines. The cells were stored in DMEM (Invitrogen, Car1sbad, CA) medium supplemented with 1% L-glutamine, 1% penicillin and streptomycin, 1. mu.g/mL puromycin, and 10% newborn bovine serum (NCS) in a T-75 flask in a 5% carbon dioxide incubator at 37 ℃.

And (3) analysis:

the cells were washed with PBS, suspended in DMEM medium supplemented with 0.5% NCS and 1% penicillin and streptomycin, seeded into 96-well plates at a density of 20,000 cells/well/100 μ L, and incubated at 37 ℃ in a 5% carbon dioxide incubator. After 20 hours of incubation, 100 μ L of test medium DMEM containing the indicated PF-compound concentration or control group (dimethyl sulfoxide) was added to the plate and incubated for 1 hour with an incubator. The medium was removed, and the lysate containing phosphatase inhibitor and phenylmethanesulfonyl fluoride (PMSF) was added to the wells and shaken at 4 ℃ for 30 minutes to obtain a protein lysate. Phosphorylation of ALK was then assessed using a pathscanphos-ALK (Tyr1604) chemiluminescent sandwich ELISA kit (Cell Signal Technology inc., cat #7020), see below:

Pho-ALK (Tyr1604) rabbit antibody was coated on 96-well micro-plates. 50 μ L of cell lysate was added to the antibody-coated plate and incubated at room temperature for 2 hours. Unbound material was removed by bulk washing with 0.1% Tween20/PBS and ALK mouse mAb was added to detect captured phospho-ALK (Tyr1604) and phospho-ALK fusion proteins. The binding check antibody was then recognized with anti-mouse IgG (HRP enzyme-bound antibody). Finally, a chemiluminescent agent was added and incubated for 10 minutes to allow signal development. The assay plate was read in luminescence mode using an Envision plate reader. Using four parameter analysisMethod for calculating IC by fitting concentration response curve₅₀The value is obtained.

Ki and IC obtained by ALK enzyme analysis and cell Phospho-ALK (Tyr1604) ELISA analysis for WT EML4-ALK and L1196M EML4-ALK₅₀The data are shown in Table 1.

TABLE 1 results of cytostatic Properties of different compounds

Through the analysis of enzyme activity data of the wild ALK and the L1196M mutant ALK, compounds 4, 5 and 6 show better effects than loratinib on the level of kinase and the level of cells, and compounds 1,2, 3, 7, 8, 9 and 18 show the same performance as loratinib on the level of kinase and the level of cells; although compounds 10, 11, 12, 13, 15,16,17 were slightly weaker than loratinib at the kinase level as phosphate prodrugs, among them, compounds 10, 11, 12, 13, 14 were all precursors of compound 1, compound 15 was a precursor of compound 7, compound 16 was a precursor of compound 9, and compound 17 was a precursor of compound 18, and their effects at the cellular level were comparable to loratinib.

Example 20 exploration of the metabolic stability of novel benzoxadiazacyclotetradecene derivatives

Evaluation of compound stability was performed using human liver microsomes. The liver microsomal enzyme stability of the example compounds was compared to Lorlatinib (PF-06463922).

Measurement System: the metabolic stability of the compounds of the present invention was tested using 1mM NADPH for liver microparticles mixed by men and women. The samples were analyzed using a mass spectrometer. HRMS was used to determine peak area response ratios (peak area corresponding to test compound or control divided by peak area of the analytical internal standard) without running a standard curve. In order to detect all possible metabolites, HRMS scans were performed at the appropriate m/z range.

The measurement conditions were as follows: the assay was performed with one incubation (N ═ 1) v. Test compounds were incubated at 37 ℃ in buffer containing 0.5 mg/ml liver microsomal protein. Reactions were initiated by addition of cofactors and samples taken at 0, 2,4, 8, 16, 24, 36, 48 hours, positive controls (5 μ M testosterone) were incubated in parallel and samples taken at 0, 2,4, 8, 16, 24, 36, 48 hours.

And (3) measuring quality control: the control compound testosterone was performed in parallel to confirm the enzymatic activity of the (liver) microsomes. After the final time point, the addition of NADPH to the reaction mixture was confirmed using fluorimetry. The T1/2 of the control met acceptable internal standards.

The analysis method comprises the following steps:

liquid chromatography column: thermo BDS Hypersil C1830X2.0 mM, 3 μm, with guard column M.P., buffer 25mM formic acid buffer, pH 3.5;

aqueous phase (a): 90% water, 10% buffer;

organic phase (B): 90% acetonitrile, 10% buffer;

flow rate: 300 microliter/min

Automatic sample injector: injection volume 10 microliter

See table 2 for gradient program.

TABLE 2 gradient program

By using human liver microsomes, examples 4, 5, 6 showed a metabolic half-life of greater than 36 hours, 10, 12, 15,16,17 showed a metabolic half-life of 24-36 hours, significantly greater than 20 hours for Lorlatinib (PF-06463922), and examples 1,2, 3, 7, 8, 9, 11, 13, 14, 18 showed similar metabolic half-lives for Lorlatinib (PF-06463922), as described in the present invention. The results show that the relatively long metabolic half-life makes them potentially useful for lowering therapeutic doses and extending the time interval between administrations.

Enzymatic Activity, IC from Compounds of examples 1-17₅₀Values and metabolic half-life data show that for compounds of the general formula (I), the linker and substituent groups have important effects on the pharmacodynamic properties and metabolic stability of the compounds. While the invention has been illustrated by the foregoing specific embodiments, it is not to be construed as being limited thereby; but that the present invention encompass the generic aspects previously disclosed. Various modifications and embodiments can be made without departing from the spirit and scope of the invention.

Claims

1. A benzoxadiazepitetradecene derivative or its pharmaceutically acceptable salt has a structure shown in general formula (I),

when R is₁Is selected from C₁-C₄Alkyl radical, C₃-C₆Cycloalkyl, -CH₂OR₄、-COOR₅、-COR₆or-CH₂OP(O)OR₇OR₈When R is₂、R₃Each independently selected from C₁-C₄Alkyl, deuterated methyl or C₃-C₆A cycloalkyl group; wherein said alkyl, cycloalkyl may optionally be mono-to pentasubstituted with the same or different substituents selected from halogen, trifluoromethyl, cyano, nitro, hydroxy or C₁-C₄An alkyl group; wherein R is₄，R₅，R₆Are respectively and independently selected from hydrogen and C₁-C₄Alkyl or C₃-C₆A cycloalkyl group; r₇，R₈Each independently selected from hydrogen and C₁-C₄Alkyl or C₃-C₆A cycloalkyl group; wherein said alkyl, cycloalkyl may be optionally mono-to pentasubstituted with the following same or different substituents selected from: halogen, trifluoromethyl, cyano, nitro, hydroxy or C₁-C₄An alkyl group.

2. A benzoxadiazepitetradecene derivative or its pharmaceutically acceptable salt has a structure shown in general formula (I),

R₁is hydrogen; r₂、R₃At least one of each is deuterated methyl and the other is selected from C₁-C₄Alkyl radical, C₃-C₆Cycloalkyl, deuterated methyl.

3. The benzoxadiazacyclotetradecene derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein R is₄Selected from methyl and hydrogen.

4. The benzoxadiazacyclotetradecene derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein R is₅Selected from hydrogen, methyl, ethyl, isopropyl.

5. The benzoxadiazepidodecene derivative or a pharmaceutically acceptable salt thereof, according to claim 1, R₇，R₈Each independently selected from hydrogen, methyl, ethyl, isopropyl, tert-butyl.

6. The benzoxadiazepitetradecene derivative according to claim 1, or a pharmaceutically acceptable salt thereof, which is selected from the following compounds:

7. the benzoxadiazepitetradecene derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein said pharmaceutically acceptable salt is selected from an inorganic salt or an organic salt; wherein the inorganic salt is selected from hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate, and acid phosphate; the organic salt is selected from acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, benzenesulfonate, salicylate.

8. A pharmaceutical composition comprising a benzoxadiazacyclotetradecene derivative according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and a pharmaceutically acceptable adjuvant.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable carrier comprises: microcapsules, microspheres, nanoparticles, liposomes.

10. Use of a benzoxadiazacyclotetradecene derivative according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of cancer.