CN112390797A

CN112390797A - Novel spirocyclic K-Ras G12C inhibitor

Info

Publication number: CN112390797A
Application number: CN201910756965.6A
Authority: CN
Inventors: 樊后兴; 谢雨礼
Original assignee: Wigen Biomedicine Technology Shanghai Co Ltd
Current assignee: Wigen Biomedicine Technology Shanghai Co Ltd
Priority date: 2019-08-15
Filing date: 2019-08-15
Publication date: 2021-02-23
Also published as: WO2021027911A1; CN113767103A; CN113767103B

Abstract

The invention relates to a novel spiro compound, a preparation method and application thereof. Specifically, the invention relates to a compound shown in a formula (1) and a preparation method thereof, and application of the compound shown in the formula (1) and each optical isomer, each crystal form and pharmaceutically acceptable salt thereof as an irreversible inhibitor of a G12C mutant K-Ras protein in preparation of medicines for resisting Ras related diseases such as tumors and the like.

Description

Novel spirocyclic K-Ras G12C inhibitor

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a novel K-Ras G12C inhibitor, a preparation method thereof and a use method of the compound.

Background

Ras protein family is an important signal transduction transmission molecule in cells, and plays an important role in growth and development. Analysis and studies of a large number of in vitro tumor cells, animal models, and human tumor samples indicate that overactivation of Ras family proteins is an early event in human tumor development and an important cause of the development and progression of a variety of cancers. Targeting and inhibition of Ras protein activity is therefore an important tool for the treatment of related tumors.

Ras proteins exist in two forms, which bind to GDP in an unactivated resting state; and when cells receive signals such as growth factor stimulation, Ras proteins bind to GTP and are activated. Activated Ras proteins recruit a variety of signal-transfer proteins, promoting phosphorylation of downstream signaling molecules such as ERK, S6, thereby activating the Ras signaling pathway, regulating cell growth, survival, migration and differentiation. The Ras protein itself GTPase enzyme activity can be the hydrolysis of GTP back to GDP. And the interaction of GTP enzyme activator proteins (GAPs) and Ras in cells greatly promotes the activity of Ras GTPase, so that the over-activation of Ras protein is prevented.

Mutations in the K-Ras, H-Ras and N-Ras proteins in the Ras protein family are one of the common genetic mutations in many tumors, and are the major factors that lead to the overactivation of Ras proteins in tumors. Compared with wild Ras protein, these mutations result in unregulated Ras protein activity, stable binding of GTP, and sustained activation, thereby promoting growth, migration, and differentiation of tumor cells. Among these, K-Ras protein mutations are the most common, accounting for 85% of all Ras mutations, while N-Ras (12%) and H-Ras (3%) are relatively rare. K-Ras mutations are very common in a variety of cancers: including pancreatic (95%), colorectal (45%), and lung (25%), among others, while relatively rare (< 2%) in breast, ovarian, and brain cancers. The K-Ras mutation site is mainly concentrated at position G12, with mutations at G12C being most common. For example, in non-small cell lung cancer (NSCLC), K-Ras G12C accounts for 50% of all K-Ras mutations, followed by G12V and G12D. Genomics research shows that K-Ras mutation in non-small cell lung cancer does not coexist with EGFR, ALK, ROS1, RET and BRAF mutation, but coexists with STK11, KEAP1, TP53 and other mutations, and suggests that the K-Ras mutation and the STK11, KEAP1, TP53 mutation and other synergistic effects are possibly involved in malignant transformation, proliferation and invasion of cells. In addition to tumors, abnormal activation of Ras protein is also involved in non-neoplastic diseases including diabetes, neurodegenerative diseases, and the like, and thus it can be seen that small molecule compounds targeting Ras protein can benefit a large number of cancer patients carrying specific genetic variations and non-cancer patients with overactivation of the Ras pathway.

However, since the discovery of Ras mutations in tumors for forty years, although we have made a more thorough understanding of the pathogenesis of the Ras pathway, there has been no effective therapeutic approach to target Ras proteins for a large number of patients carrying mutations in Ras proteins and overactivation of the Ras pathway. Therefore, the development of a high-activity small-molecule inhibitor aiming at the Ras protein, in particular to the K-Ras G12C protein with high mutation frequency has important clinical significance.

Disclosure of Invention

The invention aims to provide a compound with a structural general formula shown in formula (1), or optical isomers, crystal forms, pharmaceutically acceptable salts, hydrates or solvates thereof:

in formula (1):

l is a bond or NH;

a is a bivalent 4-12 membered saturated or partially saturated monocyclic, bicyclic, bridged or spirocyclic ring containing 1-2N atoms, optionally substituted with one or more R²Substituted when by more than one R²When substituted, R²Which may be the same or different, R²Is H, CN, C1-C3 alkyl, halogen substituted C1-C3 alkyl or cyano substituted C1-C3 alkyl;

R¹is aryl or heteroaryl, which may be substituted with 1 to 3 of the following groups: halogen, hydroxy, amino, C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C1-C3 alkoxy, halogen-substituted C1-C3 alkyl or halogen-substituted C1-C3 alkoxy, and when substituted with a plurality of substituents, the substituents may be the same or different;

represents a single or double bond between V and Z, when Z is CO and V is

When the V and the Z are connected by a single bond; or, when V is N and Z is

When, V and Z are connected by double bond;

R_a、R_b、R_cand R_dAre all H, R_eAnd R_fAnd the carbon atoms to which they are attached form a C3-C6 cycloalkyl or C4-C6 heterocycle; or, R_c、R_d、R_eAnd R_fAre all H, R_aAnd R_bAnd the carbon atoms to which they are attached form a C3-C6 cycloalkyl or C4-C6 heterocycle; or, R_a、R_b、R_eAnd R_fWhen both are H, R_cAnd R_dAnd the carbon atoms to which they are attached form a C3-C6 cycloalkyl or C4-C6 heterocycle; and

e is an electrophilic moiety capable of forming a covalent bond with the cysteine residue at position 12 of the K-Ras mutant protein,

wherein the content of the first and second substances,

y is a bond or C1-C6 alkylene;

R³is aminoalkyl, cycloalkyl, alkyl-substituted amido, heterocyclyl, aryl or heteroaryl, each of which may be substituted with 1 to 3 of the following groups: halogen, O, CN, OH, alkylhydroxy, dialkylamino, C1-C6 alkyl, C3-C6 cycloalkyl, halogen substituted C1-C3 alkyl or halogen substituted C1-C3 alkoxy, which when substituted with a plurality of substituents, may be the same or different.

In another preferred embodiment, the compound of formula (1) has a structure represented by formula (1A) or formula (1B):

wherein:

l is a bond or NH;

y is a bond or C1-C6 alkylene;

R³is aminoalkyl, cycloalkyl, alkyl-substituted amido, heterocyclyl, aryl or heteroaryl, each of which may be substituted with 1 to 3 of the following groups: halogen, O, CN, OH, alkylhydroxy, dialkylamino, C1-C6 alkyl, C3-C6 cycloalkyl, halogen substituted C1-C3 alkyl or halogen substituted C1-C3 alkoxy, which when substituted with a plurality of substituents, may be the same or different;

e is an electrophilic moiety capable of forming a covalent bond with the cysteine residue at position 12 of the K-Ras mutant protein.

In another preferred embodiment, wherein in said formula (1), formula (1A) and formula (1B), E is a group containing an electrophilic carbon-carbon double bond or carbon-carbon triple bond.

In another preferred embodiment, wherein in the general formula (1), the general formula (1A) and the general formula (1B), E is:

wherein R is⁴Is H, F, CF₃OMe or-CH₂OMe，R⁵Is H, Me, Et, CN, -CONH₂、-CH₂F、-CHF₂、CF₃、-CH₂OH、CH₂OMe、

In another preferred embodiment, wherein in said general formula (1), general formula (1A) and general formula (1B), -A-L-E is:

wherein n is 1 or 2, L is a bond or NH, R²Is H, CN, C1-C3 alkyl, halogen substituted C1-C3 alkyl or cyano substituted C1-C3 alkyl.

In another preferred embodiment, wherein in said general formula (1), general formula (1A) and general formula (1B), Y is a bond, -CH₂-, -CH (Me) -or-CH₂CH₂-。

In another preferred embodiment, wherein in said general formula (1), general formula (1A) and general formula (1B), R¹Comprises the following steps:

wherein R is⁶And R⁷Independently H, halogen, hydroxyl, amino, C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C1-C3 alkoxy, halogen substituted C1-C3 alkyl or halogen substituted C1-C3 alkoxy.

In another preferred embodiment, wherein in said general formula (1), general formula (1A) and general formula (1B), R³Comprises the following steps:

wherein n is 1, 2 or 3, R⁸And R⁹Independently H, halogen, hydroxyl, amino, C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C1-C3 alkoxy, halogen substituted C1-C3 alkyl or halogen substituted C1-C3 alkoxy, R¹⁰Is C1-C3 alkyl, C3-C6 cycloalkyl, C3-C6 cycloalkylalkyl, C1-C3 alkoxyalkyl, halogen substituted C1-C3 alkyl, halogen substituted C3-C6 cycloalkyl or

In various embodiments, the compound has one of the structures listed in table 1 below:

table 1 list of representative compounds of the invention:

another object of the present invention is to provide a pharmaceutical composition comprising a pharmaceutically acceptable excipient or carrier and, as an active ingredient, a compound of the general formula (1) of the present invention, or each of its optical isomers, a pharmaceutically acceptable inorganic or organic salt.

Still another object of the present invention is to provide the use of the above-mentioned compound of the present invention, or each optical isomer, pharmaceutically acceptable inorganic or organic salt thereof, for the preparation of a medicament for treating RAS-related diseases.

It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.

Synthesis of Compounds

The following specifically describes the production process of the compound of the general formula (1) of the present invention, but these specific processes do not set any limit to the present invention.

The compounds of formula (1) described above may be synthesized using standard synthetic techniques or known techniques in combination with the methods described herein. In addition, the solvents, temperatures and other reaction conditions mentioned herein may vary. Starting materials for the synthesis of the compounds may be obtained synthetically or from commercial sources, such as, but not limited to, Aldrich Chemical co. (Milwaukee, Wis.) or Sigma Chemical co. (st. The compounds described herein and other related compounds having various substituents can be synthesized using well-known techniques and starting materials, including those found in M argon ch, ADVANCED ORGANIC CHEMISTRY 4^thEd., (Wiley 1992); c argon ey and Sundberg, ADVANCED ORGANIC CHEMISTRY 4^thEd, Vols.A and B (Plenum 2000, 2001), Green and Wuts, PROTECTIVE GROUPS IN ORGANIC synthieSIS 3^rdThe recipe in ed., (Wiley 1999)The method is carried out. The general method of compound preparation may be varied by the use of appropriate reagents and conditions for introducing different groups into the formulae provided herein.

In one aspect, the compounds described herein are according to methods well known in the art. However, the conditions of the method, such as reactants, solvent, base, amount of the compound used, reaction temperature, time required for the reaction, and the like, 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. In one aspect, the present invention also provides a method for preparing the compound represented by the general formula (1), which is prepared by the following general reaction scheme 1 or 2:

general reaction scheme 1

Embodiments of the compounds of formula (1) may be prepared according to general reaction scheme 1 (method A), wherein R_a、R_b、R_c、R_d、R_e、R_f、R¹、R²、R³A, E and Y are as defined above. As shown in general reaction scheme 1, starting material S (synthesized by the methods described in preparations 1 to 9 of this patent) and fragment A produce A1 under basic conditions, A1 removes the protecting group (e.g., Boc) to give A2, A2 and R1-X are coupled to give A3, A3 compound is reacted in the presence of an oxidizing agent to give A4, A4 is reacted under appropriate conditions and R³The reaction of the-Y-OH fragment to form A5, removal of the protecting group (e.g., Cbz) from A5 to form A6, and reaction of A6 with an acid chloride or anhydride compound to form A7.

General reaction scheme 2

Embodiments of the compounds of formula (1) may be prepared according to general reaction scheme 2 (method B), wherein R_a、R_b、R_c、R_d、R_e、R_f、R¹、R²、R³A, E and Y are as hereinbefore defined and X represents chlorine, bromine, iodine or OTf. As shown in general scheme 2, intermediate A1 is formed into B1 in the presence of an oxidant, B1 is hydrolyzed under basic conditions to B2, B2 is hydrolyzed under appropriate conditions and R³Reaction of the-Y-X fragment to give B3, deprotection of B3 (e.g., Boc) to give B4, coupling of B4 and R1-X to give B5, deprotection of B5 (e.g., Cbz) to give B6, reaction of B6 with an acid chloride or anhydride compound to give B7.

Further forms of the compounds

"pharmaceutically acceptable" as used herein refers to a substance, such as a carrier or diluent, which does not diminish the biological activity or properties of the compound and which is relatively non-toxic, e.g., by being administered to an individual without causing unwanted biological effects or interacting in a deleterious manner with any of the components it contains.

The term "pharmaceutically acceptable salt" refers to a form of a compound that does not cause significant irritation to the organism to which it is administered and does not abrogate the biological activity and properties of the compound. In certain particular aspects, pharmaceutically acceptable salts are obtained by reacting a compound of formula (1) with an acid, such as an inorganic acid, e.g., hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, phosphoric acid, nitric acid, phosphoric acid, etc., an organic acid, e.g., formic acid, acetic acid, propionic acid, oxalic acid, trifluoroacetic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc., and an acidic amino acid, e.g., aspartic acid, glutamic acid, etc.

References to pharmaceutically acceptable salts are understood to include solvent addition forms or crystalline forms, especially solvates or polymorphs. Solvates contain either stoichiometric or non-stoichiometric amounts of solvent and are selectively formed during crystallization with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is ethanol. Solvates of the compounds of formula (1) are conveniently prepared or formed as described herein. Illustratively, the hydrate of the compound of formula (1) is conveniently prepared by recrystallization from a mixed solvent of water/organic solvent, using an organic solvent including, but not limited to, dioxane, tetrahydrofuran, ethanol or methanol. In addition, the compounds mentioned herein can exist in unsolvated as well as solvated forms. In general, the solvated forms are considered equivalent to unsolvated forms for purposes of the compounds and methods provided herein.

In other embodiments, the compound of formula (1) is prepared in different forms, including, but not limited to, amorphous, pulverized, and nano-sized forms. In addition, the compound of formula (1) includes crystalline forms, and may also be polymorphic forms. Polymorphs include different lattice arrangements of the same elemental composition of a compound. Polymorphs typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal forms, optical and electrical properties, stability and solubility. Different factors such as recrystallization solvent, crystallization rate and storage temperature may cause a single crystal form to dominate.

In another aspect, the compounds of formula (1) have one or more stereogenic centers and thus occur as racemates, racemic mixtures, single enantiomers, diastereomeric compounds and single diastereomers. Asymmetric centers that may be present depend on the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers and all possible optical isomers and diastereomeric mixtures and pure or partially pure compounds are included within the scope of the invention. The present invention is meant to include all such isomeric forms of these compounds.

Term(s) for

Unless otherwise defined, terms used in this application, including the specification and claims, are defined as follows. It must be noted that, in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Conventional methods of mass spectrometry, nuclear magnetism, HPLC, protein chemistry, biochemistry, recombinant DNA technology and pharmacology are used, if not otherwise stated. In this application, "or" and "means" and/or "are used unless otherwise stated.

"alkyl" refers to a saturated aliphatic hydrocarbon group, including straight and branched chain groups of 1 to 6 carbon atoms. Lower alkyl groups having 1 to 4 carbon atoms are preferred, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl. As used herein, "alkyl" includes unsubstituted and substituted alkyl groups, especially alkyl groups substituted with one or more halogens. Preferred alkyl groups are selected from CH₃，CH₃CH₂，CF₃，CHF₂，CF₃CH₂，ⁱPr，ⁿPr，ⁱBu，^cPr，ⁿBu or^tBu。

"cycloalkyl" refers to a 3-to 6-membered all-carbon monocyclic aliphatic hydrocarbon group in which one or more rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron system. For example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexane, cyclohexadiene and the like.

"alkoxy" refers to an alkyl group bonded to the rest of the molecule through an ether oxygen atom. Representative of alkoxy groups are alkoxy groups having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy and tert-butoxy. As used herein, "alkoxy" includes unsubstituted and substituted alkoxy groups, especially alkoxy groups substituted with one or more halogens. Preferred alkoxy groups are selected from OCH₃，OCF₃，CHF₂O，CF₃CH₂O，ⁱPrO，ⁿPrO，ⁱBuO，^cPrO，ⁿBuO or^tBuO。

"aryl" refers to a group having at least one aromatic ring structure, i.e., a carbocyclic aryl group having a conjugated pi-electron system, such as a benzene ring and a naphthalene ring.

"heteroaryl" refers to an aromatic group containing one or more heteroatoms (O, S or N), the heteroaryl being monocyclic or polycyclic, e.g., a monocyclic heteroaryl ring fused to one or more carbocyclic aromatic groups or other monocyclic heterocyclic groups. Examples of heteroaryl groups include, but are not limited to, pyridyl, pyridazinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, indolyl, benzimidazolyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, benzopyridyl, and pyrrolopyrimidinyl.

"halogen" means fluorine, chlorine, bromine or iodine.

The term "bond" or "single bond" refers to a chemical bond between two atoms or between two moieties when the atoms connected by the bond are considered part of a larger structure. In one aspect, when a group described herein is a bond, the absence of a reference group allows for the formation of a bond between the remaining defined groups.

The term "membered ring" includes any cyclic structure. The term "element" is intended to mean the number of backbone atoms constituting a ring. Thus, for example, cyclohexyl, pyridyl, pyranyl, thiopyranyl are six-membered rings and cyclopentyl, pyrrolyl, furanyl and thienyl are five-membered rings.

The term "fragment" refers to a specific part or functional group of a molecule. Chemical moieties are generally considered to be chemical entities contained in or attached to a molecule.

Specific pharmaceutical and medical terms

The term "acceptable", as used herein, means that a prescribed component or active ingredient does not unduly adversely affect the health of the general therapeutic target.

The terms "treat," "treatment process," or "therapy" as used herein include alleviating, inhibiting, or ameliorating a symptom or condition of a disease; inhibiting the generation of complications; ameliorating or preventing underlying metabolic syndrome; inhibiting the development of a disease or condition, such as controlling the development of a disease or condition; alleviating the disease or symptoms; regression of the disease or symptoms; alleviating a complication caused by the disease or symptom, or preventing or treating a symptom caused by the disease or symptom.

As used herein, a compound or pharmaceutical composition, when administered, can ameliorate a disease, symptom, or condition, particularly severity, delay onset, slow progression, or reduce duration of a condition. Whether fixed or temporary, continuous or intermittent, may be attributed to or associated with administration.

"active ingredient" means a compound represented by the general formula (1), and a pharmaceutically acceptable inorganic or organic salt of the compound of the general formula (1). The compounds of the present invention may contain one or more asymmetric centers and thus occur as racemates, racemic mixtures, single enantiomers, diastereomeric compounds and individual diastereomers. Asymmetric centers that may be present depend on the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers and all possible optical isomers and diastereomeric mixtures and pure or partially pure compounds are included within the scope of the invention. The present invention is meant to include all such isomeric forms of these compounds.

The terms "compound", "composition", "medicament" or "drug" are used interchangeably herein and refer to a compound or composition that, when administered to an individual (human or animal), is capable of inducing a desired pharmaceutical and/or physiological response through local and/or systemic action.

The term "administered" as used herein refers to the direct administration of the compound or composition, or the administration of a prodrug (produg), derivative (derivative), or analog (analog) of the active compound, which results in an equivalent amount of the active compound in the individual to whom it is administered.

Although numerical ranges and parameters setting forth the broad scope of the invention are approximate, the values set forth in the specific examples are presented as precisely as possible. Any numerical value, however, inherently contains certain standard deviations found in their respective testing measurements. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within the acceptable standard error of the mean, as considered by those skilled in the art. Except in the experimental examples, or where otherwise expressly indicated, it is to be understood that all ranges, amounts, values and percentages herein used (e.g., to describe amounts of materials, length of time, temperature, operating conditions, quantitative ratios, and the like) are to be modified by the word "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, these numerical parameters are to be understood as meaning both the number of significant digits indicated and the number resulting from applying ordinary rounding techniques.

Unless defined otherwise herein, the scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Furthermore, as used herein, the singular tense of a noun, unless otherwise conflicting with context, encompasses the plural form of that noun; the use of plural nouns also covers the singular form of such nouns.

Therapeutic uses

The invention provides methods of treating diseases, including but not limited to conditions (e.g., cancer) involving G12C K-Ras, G12C H-Ras and/or G12C N-Ras mutations, using the compounds or pharmaceutical compositions of the invention.

In some embodiments, there is provided a method for the treatment of cancer, the method comprising administering to an individual in need thereof an effective amount of a pharmaceutical composition of any of the foregoing compounds of protective structure (1). In some embodiments, the cancer is mediated by K-Ras, H-Ras and/or G12C N-Ras mutations. In other embodiments, the cancer is lung cancer, pancreatic cancer, colon cancer, MYH-related polyposis, or colorectal cancer.

Route of administration

The compound and the pharmaceutically acceptable salt thereof can be prepared into various preparations, wherein the preparation comprises the compound or the pharmaceutically acceptable salt thereof in a safe and effective amount range and a pharmaceutically acceptable excipient or carrier. Wherein "safe, effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. The safe and effective amount of the compound is determined according to the age, condition, course of treatment and other specific conditions of a treated subject.

"pharmaceutically acceptable excipient or 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 blended with the compounds of the present invention and with each other without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable excipients or 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, glycerol, mannitol, sorbitol etc.), emulsifiers (e.g. propylene glycol, glycerol, mannitol, sorbitol etc.)

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

When the compounds of the present invention are administered, they may be administered orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), topically.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release 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 compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or 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, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds. 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 1000mg, preferably 10 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 features mentioned above with reference to the invention, or the features mentioned with reference to the embodiments, can be combined arbitrarily. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

The various specific aspects, features and advantages of the compounds, methods and pharmaceutical compositions described above are set forth in detail in the following description, which makes the present invention clear. It should be understood herein that the detailed description and examples, while indicating specific embodiments, are given by way of illustration only. After reading the description of the invention, one skilled in the art can make various changes or modifications to the invention, and such equivalents fall within the scope of the invention as defined in the application.

In all of the embodiments described herein, the first,¹H-NMR was recorded using a Vian Mercury 400 NMR spectrometer with chemical shifts expressed in delta (ppm); the silica gel used for separation is not illustrated to be 200-300 meshes, and the proportions of the eluents are volume ratios.

The invention employs the following abbreviations: ar represents argon; aq represents an aqueous solution; (Boc)₂O represents di-tert-butyl dicarbonate; CDCl₃Represents deuterated chloroform; CD (compact disc)₃OD represents deuterated methanol; CH (CH)₃CN represents acetonitrile; CH (CH)₃NO₂Represents nitromethane; (COCl)₂Represents oxalyl chloride; cs₂CO₃Represents cesium carbonate; CuI represents cuprous iodide; DBU represents 1, 8-diazacyclo [5,4, 0%]Undecene-7; DCM represents dichloromethane; DIPEA stands for diisopropylethylamine; dioxane represents 1, 4-Dioxane; DMF represents dimethylformamide; DMSO represents divaricate knotweed herb; EA represents ethyl acetate; EtOH stands for ethanol; EtONa stands for sodium ethoxide; h represents hour; NaOH represents sodium hydroxide; NaOMe represents sodium methoxide; LC-MS stands for liquid-mass spectrometry; m-CPBA represents m-chloroperoxybenzoic acid; MeOH represents methanol; min represents min; MS represents mass spectrum; na (Na)₂CO₃Represents sodium carbonate; NaH represents sodium hydride; NMR stands for nuclear magnetic resonance; pd₂(dba)₃Represents tris (dibenzylideneacetone) dipalladium; PE represents petroleum ether; SOCl₂Represents thionyl chloride;^t-BuOK for potassium tert-butoxide;^t-BuONa represents sodium tert-butoxide; TFA (CF)₃COOH) represents trifluoroacetic acid; tf₂O represents trifluoromethanesulfonic anhydride; THF represents tetrahydrofuran; xantphos stands for 4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene.

The specific implementation mode is as follows:

preparation example Synthesis of tert-butyl ' - (methylthio) -4' - (((trifluoromethyl) sulfonyl) oxy) -5',8' -dihydro-7 ' H-spiro [ cyclopropane-1, 6' -pyrido [3,4-d ] pyrimidine ] -7' -carboxylate (S-1)

(E) -methyl 3- (1- ((tert-butoxycarbonyl) amino) cyclopropyl) acrylate

Adding 2- (diethoxyphosphoryl) methyl acetate (21.0g,0.1mol) into a 500mL three-necked flask, drying THF (200mL), cooling to-20 ℃ under the protection of Ar gas, then dropwise adding LiHMDS (130mL,1M in THF,0.13mol) at-20 ℃ to-10 ℃, after dropwise adding, stirring the mixed solution at-10 ℃ for 30min, then dropwise adding a THF (30mL) solution of (1-aldehyde cyclopropyl) carbamic acid tert-butyl ester (16.7g, 90mmol) at-10 ℃, and stirring the mixed solution for 5h at-10 ℃ to r.t. After the completion of the reaction, the reaction was monitored by LC-MS, and then quenched by adding a saturated ammonium chloride solution (100mL),EA (100mL) extraction, organic phase washed with saturated sodium chloride solution (100mL), concentrated to dryness, and residue column chromatography (EA/PE 0/5 to 1/5) purified to give light brown oil (13.46g, yield: 62%), ESI-MS M/z:242.0[ M + H ]/[]⁺。

(E) -methyl 3- (1- ((tert-butoxycarbonyl) (2-methoxy-2-oxoethyl) amino) cyclopropyl) acrylate

Adding (E) -methyl 3- (1- ((tert-butoxycarbonyl) amino) cyclopropyl) acrylate (13.4g, 55.5mmol) and DMF (135mL) into a 500mL single-neck flask, cooling to 0 ℃ under the protection of Ar gas, adding NaH (2.89g, 60% in minute, 72.2mmol) in batches, stirring at 0-5 ℃ for 30min after the addition is finished, adding ethyl bromoacetate (10.2g,66.6mmol), and stirring the mixed solution at room temperature for reaction for 20 h. After the reaction was monitored by LC-MS, the system was quenched with saturated ammonium chloride solution (100mL), extracted with EA (100 mL. times.2), the combined organic phases were washed with saturated sodium chloride solution (100mL), concentrated to dryness, and the residue was purified by column chromatography (EA/PE: 0/5 to 1/5) to give a light brown oil (8.87g, 51% yield), ESI-MS M/z:336.0[ M + Na ]: 336.0]⁺。

3- (1- ((tert-Butoxycarbonyl) (2-methoxy-2-oxoethyl) amino) cyclopropyl) propionic acid methyl ester

A500 mL single-neck flask was charged with methyl (E) -3- (1- ((tert-butoxycarbonyl) (2-methoxy-2-oxoethyl) amino) cyclopropyl) acrylate (8.87g,28.34mmol), MeOH (170mL), 10% Pd/C (800mg, wet% ═ 50%), H₂After three times of replacement, hydrogenation is carried out for 20h at room temperature under normal pressure. After the reaction was completed as monitored by LC-MS, the system was filtered through celite, the filtrate was concentrated to dryness to give a colorless oil (9.1g, yield 100%), ESI-MS M/z:338.0[ M + Na ]]⁺。

4- (tert-butyl) 7-ethyl-6-oxo-4-azaspiro [2.5] octane-4, 7-dicarboxylate

Dissolving methyl 3- (1- ((tert-butoxycarbonyl) (2-methoxy-2-oxoethyl) amino) cyclopropyl) propionate (9.1g,28.34mmol) in absolute methanol (180mL), adding fresh sodium methoxide (2.30g,42.51mmol) under the protection of Ar, heating to reflux for 5H, monitoring the reaction by LC-MS, adjusting the pH of the mixed solution to 5-6, concentrating, adding ethyl acetate (100mL) into the residue, and adding H₂O (100mL), the aqueous phase was extracted with EA (50mL), and the combined organic phases were extracted with saturated sodium chlorideThe solution (50mL) was washed, concentrated and purified by column chromatography (PE/EA: 10/1 to 2/1) to give a nearly colorless oil (3.3g, 41% yield), ESI-MS M/z:284.0[ M + H ], (ESI-MS M/z)]⁺。

2' - (methylthio) -4' -oxo-3 ',4',5',8' -tetrahydro-7 ' H-spiro [ cyclopropane-1, 6' -pyrido [3,4-d ] ] pyrimidine ] -7' -carboxylic acid tert-butyl ester

Into a 250mL single-necked flask was added 4- (tert-butyl) 7-ethyl 6-oxo-4-azaspiro [2.5]]Octane-4, 7-dicarboxylate (3.3g, 11.65mmol) and MeOH (66mL), sodium methoxide (3.14g, 58.2mmol) and S-methylisothiourea sulfate (3.5g, 17.5mmol) were added, respectively, under Ar protection, and the mixture was reacted at room temperature for 20 h. And monitoring by LC-MS and TLC (PE/EA is 2/1), adjusting the pH of the mixed solution to 5-6 by using 2N HCl under ice bath after the reaction is finished, and concentrating under reduced pressure. Adding H to the residue₂O (60mL) and EA (15mL), slurrying and stirring for half an hour, filtering, and drying to obtain a white-like solid (3.43g, yield 91%), ESI-MS M/z:324.1[ M + H ]]⁺。

2' - (methylthio) -4' - (((trifluoromethyl) sulfonyl) oxy) -5',8' -dihydro-7 ' H-spiro [ cyclopropane-1, 6' -pyrido [3,4-d ] pyrimidine ] -7' -carboxylic acid tert-butyl ester

To a 250mL three-necked flask was added 2'- (methylthio) -4' -oxo-3 ',4',5',8' -tetrahydro-7 'H-spiro [ cyclopropane-1, 6' -pyrido [3,4-d ]]]Pyrimidines]-7' -Carboxylic acid tert-butyl ester (3.43g, 10.6mmol), DIPEA (4.1g, 31.8mmol) and DCM (70mL) were cooled to 0-5 ℃ in an ice bath under Ar protection, and Tf was added dropwise₂A solution of O (3.89g, 13.78mmol) in DCM (20mL) was added dropwise, and the mixture was stirred at room temperature for 20 h. TLC monitoring (PE/EA: 2/1) after completion of the reaction, H was added₂Quenching with O (100mL), dropwise adding 1N HCl to adjust the pH value to 5-6, separating liquid, washing an organic phase with a saturated sodium chloride solution (50mL), concentrating, and purifying a residue by column chromatography (PE/EA is 10/0 to 5/1) to obtain an off-white solid (3.5g, yield is 72.6%), ESI-MS M/z:456.1[ M + H ], (ESI-MS M/z: 456.1)]⁺。

Preparation example Synthesis of tert-butyl 22 ' - (methylthio) -4' - (((trifluoromethyl) sulfonyl) oxy) -5',8' -dihydro-7 ' H-spiro [ cyclobutane-1, 6' -pyrido [3,4-d ] pyrimidine ] -7' -carboxylate (S-2)

Using (1-aldehyde cyclobutyl) carbamic acid tert-butyl ester as a raw material, and adopting a synthesis method of an intermediate S-1 to obtain a target intermediate S-2, ESI-MS M/z:470.1[ M + H ]]⁺。

Preparation example Synthesis of tert-butyl 32 ' - (methylthio) -4' - (((trifluoromethyl) sulfonyl) oxy) -5',8' -dihydro-7 ' H-spiro [ oxetane-3, 6' -pyrido [3,4-d ] pyrimidine ] -7' -carboxylate (S-3)

Using (3-formyl oxetan-3-yl) tert-butyl carbamate as a raw material, and adopting a synthesis method of an intermediate S-1 to obtain a target intermediate S-3, ESI-MS M/z:472.1[ M + H ]]⁺。

Preparation example Synthesis of tert-butyl 42 ' - (methylthio) -4' - (((trifluoromethyl) sulfonyl) oxy) -5',6' -dihydro-7 ' H-spiro [ cyclopropane-1, 8' -pyrido [3,4-d ] pyrimidine ] -7' -carboxylate (S-4)

1-Aminocyclopropane-1-carboxylic acid ethyl ester

Dissolving 1-aminocyclobutane-1-carboxylic acid (10.1g,0.1mol) in ethanol (100mL), slowly adding thionyl chloride (23.8g, 0.2mol) dropwise under ice bath, reacting at room temperature for 20H after dropwise addition, monitoring the reaction by LC-MS, concentrating the mixed solution, adding DCM (100mL) and aqueous sodium bicarbonate solution (100mL) into the residue, stirring, separating the solution, extracting the aqueous phase with (100mL) 2 again, combining the organic phases, washing with saturated sodium chloride solution (100mL 2), drying with anhydrous sodium sulfate, filtering, concentrating to obtain pale yellow oily substance (9.08g, yield 70%), ESI-MS M/z:130.1[ M + H ], [ M-M/z%]⁺。

1- ((4-ethoxy-4-oxobutyl) amino) cyclopropane-1-carboxylic acid ethyl ester

Dissolving 1-aminocyclopropane-1-carboxylic acid ethyl ester (9.08g,70.4mmol) in acetonitrile (200mL), adding DIPEA (18.2g,141mmol), ethyl 4-bromobutyrate (27.5g,141mmol), and under the protection of ArHeating to 80 deg.C, reacting for 20H, monitoring by LC-MS, concentrating, adding ethyl acetate (200mL), and H₂O (100mL), stirred, separated, the aqueous phase extracted with ethyl acetate (100mL), the combined organic phases washed with saturated sodium chloride (100mL), concentrated, and the residue purified by column chromatography (DCM/MeOH 20/0 to 20/1) to afford a pale yellow oil (8.70g, 51% yield), ESI-MS M/z:244.2[ M + H20/0 to 20/1)]⁺。

1- ((tert-Butoxycarbonyl) (4-ethoxy-4-oxobutyl) amino) cyclopropane-1-carboxylic acid ethyl ester

Ethyl 1- ((4-ethoxy-4-oxobutyl) amino) cyclopropane-1-carboxylate (8.70g,35.8mmol) was washed with THF (100mL) and H₂O (50mL) was dissolved and Boc was added₂O (15.7g,72mmol) and Na₂CO₃(11.4g, 0.107mol), the mixture was heated to 60 ℃ and stirred for 20H, and after completion of the reaction was monitored by LC-MS, EA (100mL) and H were added to the reaction mixture₂O (100mL), stirred, separated, the organic phase concentrated and the residue purified by column chromatography (PE/EA: 5/0 to 5/1) to give a colorless oil (7.61g, 62% yield), ESI-MS M/z:344.2[ M + H/z]⁺。

4- (tert-butyl) 7-ethyl-8-oxo-4-azaspiro [2.5] octane-4, 7-dicarboxylate

Dissolving ethyl 1- ((tert-butoxycarbonyl) (4-ethoxy-4-oxobutyl) amino) cyclopropane-1-carboxylate (7.61g, 22.2mmol) in absolute ethyl alcohol (140mL), adding fresh sodium ethoxide (2.26g,33.3mmol) under the protection of Ar, then heating to reflux for 5H, monitoring the reaction completion by LC-MS, adjusting the pH of the mixture to 5-6, concentrating, adding ethyl acetate (100mL) and H into the residue₂O (100mL), extraction of the aqueous phase with EA (50mL), combined organic phases washed with saturated sodium chloride solution (50mL), concentrated, and purified by column chromatography (PE/EA: 10/1 to 2/1) to give a nearly colorless oil (2.31g, 35% yield), ESI-MS M/z:298.0[ M + H/z: (M + H): 298.0]⁺。

2' - (methylthio) -4' -carbonyl-3 ',4',5',6' -tetrahydro-7 ' H-spiro [ cyclopropane-1, 8' -pyrido [3,4-d ] pyrimidine ] -7' -carboxylic acid tert-butyl ester

Into a 250mL single-necked flask was added 4- (tert-butyl) 7-ethyl-8-oxo-4-azaspiro [ 2.5%]Octane-4, 7-dicarboxylate (4.76g,16.0mmol) and EtOH (100mL) were added with sodium ethoxide (sodium ethoxide) (sodium ethox5.44g, 80mmol) and S-methylisothiouronium sulfate (4.8g,24mmol) at room temperature for 20 h. And monitoring by LC-MS and TLC (PE/EA is 2/1), adjusting the pH of the mixed solution to 5-6 by using 2N HCl under ice bath after the reaction is finished, and concentrating under reduced pressure. Adding H to the residue₂O (60mL) and EA (30mL), slurrying, stirring for half an hour, filtering, and oven drying to obtain white solid (4.24g, yield 82%), ESI-MS M/z:324.1[ M + H ]]⁺。

2' - (methylthio) -4' - (((trifluoromethyl) sulfonyl) oxy) -5',6' -dihydro-7 ' H-spiro [ cyclopropane-1, 8' -pyrido [3,4-d ] pyrimidine ] -7' -carboxylic acid tert-butyl ester

To a 250mL three-necked flask was added 2'- (methylthio) -4' -carbonyl-3 ',4',5',6' -tetrahydro-7 'H-spiro [ cyclobutane-1, 8' -pyrido [3,4-d ]]Pyrimidines]-7' -Carboxylic acid tert-butyl ester (4.24g, 13.11mmol), DIPEA (5.08g, 39.40mmol) and DCM (100mL) were cooled to 0-5 ℃ in an ice bath under protection of Ar, and Tf was added dropwise₂O (4.45g, 15.75mmol) in DCM (20mL) was added dropwise, and the mixture was stirred at RT for 20 h. TLC monitoring (PE/EA: 2/1) after completion of the reaction, H was added₂Quenching with O (100mL), dropwise adding 1N HCl to adjust the pH value to 5-6, separating liquid, washing an organic phase with a saturated sodium chloride solution (50mL), concentrating, and purifying a residue by column chromatography (PE/EA is 10/0 to 10/1) to obtain an off-white solid (4.35g, yield 73%), ESI-MS M/z:456.1[ M + H ], (ESI-MS M/z: 456.1)]⁺。

Preparation example Synthesis of tert-butyl 52 ' - (methylthio) -4' - (((trifluoromethyl) sulfonyl) oxy) -5',6' -dihydro-7 ' H-spiro [ cyclobutane-1, 8' -pyrido [3,4-d ] pyrimidine ] -7' -carboxylate (S-5)

1-aminocyclobutyl-1-formic acid is taken as a raw material, a target intermediate S-5 is obtained by adopting a synthesis method of an intermediate S-4, ESI-MS M/z is 470.1[ M + H ]]⁺。

Preparation example Synthesis of tert-butyl 62 ' - (methylthio) -4' - (((trifluoromethyl) sulfonyl) oxy) -5',6' -dihydro-7 ' H-spiro [ oxetane-1, 8' -pyrido [3,4-d ] pyrimidine ] -7' -carboxylate (S-6)

3-aminooxetane-3-formic acid is taken as a raw material, a target intermediate S-6 is obtained by adopting a fragment S-4 synthesis method, ESI-MS M/z is 472.1[ M + H ]]⁺。

Preparation example Synthesis of 72 '- (methylthio) -4' - ((((trifluoromethyl) sulfonyl) oxy) -6 'H-spiro [ oxetane-3, 5' -pyrido [3,4-d ] pyrimidine ] -7'(8' H) -carboxylic acid tert-butyl ester (S-7)

2- (Oxetayclobut-3-ylidene) acetic acid ethyl ester

Dissolving oxetan-3-one (10.8g,150mm0l) in dichloromethane (150mL), adding ethoxyformylmethylenetriphenylphosphonium (52.2g,150mmol) under ice bath, reacting at room temperature overnight, monitoring the reaction by LC-MS, concentrating to obtain white solid, pulping the solid with PE/EA (10/1,100mL), concentrating the filtrate, and performing column chromatography (PE/EA is 20/1 to 5/1) to obtain near colorless gum (15g, yield 70%), ESI-MS M/z:143.0[ M + H ])]⁺。

2- (3- (nitromethyl) oxetan-3-yl) acetic acid ethyl ester

Dissolving ethyl 2- (oxetan-3-ylidene) acetate (14.2g,100mL) in nitromethane (100mL), adding DBU (1.52g,10mmol), reacting overnight at room temperature, monitoring completion of the reaction by LC-MS, concentrating, dissolving the residue in ethyl acetate, washing with dilute acid, concentrating, and performing column chromatography (PE/EA: 20/1 to 5/1) to obtain a nearly colorless gum (10g, yield 50%), ESI-MS M/z:204.0[ M + H ])]⁺。

2- (3- (aminomethyl) oxetan-3-yl) acetic acid ethyl ester

Ethyl 2- (3- (nitromethyl) oxetan-3-yl) acetate (6.1g,30mmol) was dissolved in ethanol (100mL), trifluoroacetic acid (6.8g,60mmol) was added, 10% Pd/C (1.36g, wet% ═ 20%) was added, and H was bubbled through₂Reacting at 50 ℃ overnight, monitoring by LC-Ms, filtering after the reaction is finished, concentrating to obtain a primary product, and directly putting the primary product into the next reaction, wherein ESI-MS m/z is 174.1[M+H]⁺。

N- (tert-Butoxycarbonyl) -N- ((3- (2-ethoxy-2-carbonylethyl) oxetan-3-yl) methyl) glycine ethyl ester

Dissolving ethyl 2- (3- (aminomethyl) oxetan-3-yl) acetate (theoretical amount, 30mmol) in acetonitrile (150mL), adding ethyl bromoacetate (5.0g,30mmol), potassium carbonate (20g,150mmol), heating to 50 deg.C for reaction overnight, monitoring by LC-MS, filtering after reaction completion, concentrating, dissolving with dichloromethane (150mL), adding DIPEA (7.74g,60mmol), Boc₂O (7.8g,36mmol), reacted overnight at RT, monitored by LC-MS, after completion of the reaction, the reaction was washed with water, concentrated and chromatographed (PE/EA: 20/1 to 5/1) to give a nearly colourless gum (5.4g, 50% yield) ESI-MS M/z:360.1[ M + H/z ]]⁺。

6- (tert-butyl) 9-ethyl 8-carbonyl-2-oxa-6-azaspiro [3.5] nonane-6, 9-dicarboxylate

Dissolving ethyl N- (tert-butoxycarbonyl) -N- ((3- (2-ethoxy-2-carbonylethyl) oxetan-3-yl) methyl) glycinate (7.2g,20mmol) in absolute ethanol (60mL), adding fresh sodium ethoxide (2.7g,40mmol), heating to reflux for 5H, monitoring by LC-MS, adjusting pH to neutral, concentrating, adding ethyl acetate (100mL) to dissolve, washing with water, washing with saturated sodium chloride solution, concentrating, and performing column chromatography (PE/EA: 10/1 to 2/1) to obtain nearly colorless gum (2.5g, yield 40%), ESI-MS M/z:314.1[ M + H ])]⁺。

2'- (methylthio) -4' -carbonyl-4 ',8' -dihydro-3 'H-spiro [ oxetane-3, 5' -pyrido [3,4-d ] pyrimidine ] -7'(6' H) -carboxylic acid tert-butyl ester

Into a 250mL single-neck flask was added 6- (tert-butyl) 9-ethyl 8-carbonyl-2-oxa-6-azaspiro [ 3.5%]Nonane-6, 9-dicarboxylate (5g, 15.96mmol) and EtOH (100mL), respectively, sodium ethoxide (5.43g, 79.8mmol) and S-methylisothiourea sulfate (4.8g, 23.9mmol) were added under Ar protection and allowed to react at room temperature overnight. LC-MS monitoring and TLC spot plates (PE/EA. RTM. 2/1), cooled in an ice bath, adjusted to pH 5 with 2N HCl, concentrated, and H added to the crude product₂O (30mL) and EA (30mL), slurrying, stirring for half an hour, filtering, and oven drying to obtain white solid (4.3g, yield 79%), ESI-MS M/z:340.1[ M + H ]]⁺。

2'- (methylthio) -4' - (((trifluoromethyl) sulfonyl) oxy) -6 'H-spiro [ oxetane-3, 5' -pyrido [3,4-d ] pyrimidine ] -7'(8' H) -carboxylic acid tert-butyl ester

To a 250mL single-neck bottle was added 2'- (methylthio) -4' -carbonyl-4 ',8' -dihydro-3 'H-spiro [ oxetane-3, 5' -pyrido [3,4-d ]]Pyrimidines]-7'(6' H) -Carboxylic acid tert-butyl ester (4.0g, 11.80mmol), DIPEA (4.57g, 35.40mmol) and DCM (50mL) cooled to 0-5 ℃ in an ice bath under protection of Ar, Tf was added dropwise₂O (5.0g, 17.7mmol) in DCM (10mL) was added dropwise and reacted at room temperature, TLC plate (PE/EA: 2/1) was added and after the starting material had reacted substantially completely, the reaction was concentrated and purified by column chromatography (PE/EA: 1/0 to 10/1) to give a yellow solid (3.3g, 60% yield) ESI-MS M/z:472.0[ M + H/z: [ M + H ] ]]⁺。

Preparation example Synthesis of 82 '- (methylthio) -4' - (((trifluoromethyl) sulfonyl) oxy) -6 'H-spiro [ cyclobutane-1, 5' -pyrido [3,4-d ] pyrimidine ] -7'(8' H) -carboxylic acid tert-butyl ester (S-8)

Uses cyclobutanone as raw material, adopts the synthetic method of fragment S-7 to obtain target intermediate S-8, ESI-MS M/z is 470.1[ M + H ]]⁺。

Preparation example Synthesis of 92 '- (methylthio) -4' - (((trifluoromethyl) sulfonyl) oxy) -6 'H-spiro [ cyclopropane-1, 5' -pyrido [3,4-d ] pyrimidine ] -7'(8' H) -carboxylic acid tert-butyl ester (S-9)

2-cyclopropyl methylene ethyl acetate is used as a raw material, a target intermediate S-9 is obtained by adopting a fragment S-7 synthesis method, ESI-MS M/z is 456.1[ M + H ]]⁺。

Example Synthesis of- ((S) -1-acryloyl-4- (2' - (((S) -1-methylpyrrolidin-2-yl) methoxy) -7' - (naphthalen-1-yl) -7',8' -dihydro-5 ' H-spiro [ cyclopropane-1, 6' -pyrido [3,4-d ] pyrimidin ] -4' -yl) piperazin-2-yl) acetonitrile (Compound 1)

Compound 1 was prepared according to method a as described below:

(S) -4' - (4- ((benzyloxy) carbonyl) -3- (cyanomethyl) piperazin-1-yl) -2' - (methylthio) -5',8' -dihydro-7 ' H-spiro [ cyclopropane-1, 6' -pyrido [3,4-d ] pyrimidine ] -7' -carboxylic acid tert-butyl ester (1-1)

A500 mL single vial was charged with S-1(9.1g, 20.0mmol), DIPEA (5.16g, 40mmol), benzyl (S) -2- (cyanomethyl) piperazine-1-carboxylate (5.22g, 20.0mmol) and DMF (91mL), heated to 100 deg.C under Ar protection for 1 hour, monitored by TLC (PE/EA: 10/1), the starting material was allowed to react completely, the reaction mixture was cooled to room temperature, water (100mL) was added, EA (100 mL: 2) was extracted, the organic phases were combined, washed with saturated sodium chloride solution, the organic phase was concentrated, purified by column chromatography (PE/EA: 1/0 to 2/1) to give 1-1(10.6 g) of a white solid with a yield of 94%), ESI-MS M/z:565.3[ M + H ], [ ESI-MS M/z ] (565.3 [ (. M + H ])]⁺。

(S) -4' - (4- ((benzyloxy) carbonyl) -3- (cyanomethyl) piperazin-1-yl) -2' - (methylsulfonyl) -5',8' -dihydro-7 ' H-spiro [ cyclopropane-1, 6' -pyrido [3,4-d ] pyrimidine ] -7' -carboxylic acid tert-butyl ester (1-2)

Add 1-1(10.6g, 18.8mmol) and DCM (210mL) to a 500mL single neck flask, cool to 0-5 deg.C in an ice bath under Ar protection, add m-CPBA (11.4g, 65.8mmol), and react for 2h in an ice bath. TLC (PE/EA: 1/5), the starting material was reacted completely, saturated sodium bicarbonate solution (60mL) was added to the reaction mixture, stirred, separated, the aqueous phase was extracted with DCM (100 mL. times.2), the organic phases were combined, washed with saturated sodium chloride solution, the organic phase was concentrated and purified by column chromatography (PE: EA: 1: 0to 1:1) to give 1-2(9.1g, 81%) white solid (ESI-MS M/z:597.2[ M + H ])]⁺。

4' - ((S) -4- ((benzyloxy) carbonyl) -3- (cyanomethyl) piperazin-1-yl) -2' - ((((S) -1-methylpyrrolidin-2-yl) methoxytert-butyl) -5',8' -dihydro 7' H-spiro [ cyclopropane-1, 6' -pyrido [3,4-d ] pyrimidine ] -7' -carboxylic acid tert-butyl ester (1-3)

Into a 500mL single-neck flask were added (S) - (1-methylpyrrolidin-2-yl) methanol (2.108g,18.30mmol) and toluene (180mL) under Ar gas protectionCooling the bath to 0-5 ℃, adding potassium tert-butoxide (2.57g,22.88mmol), stirring at 0-5 ℃ for reaction for 15min, adding 1-2(9.1g, 15.25mmol), and stirring the mixed solution at 0-10 ℃ for reaction for 2 h. LC-MS and TLC monitoring (PE/EA: 1/1), after the reaction of the raw materials is completed, adding saturated ammonium chloride (100mL) into the system for quenching, stirring, separating, extracting the aqueous phase with EA (90 mL: 2), combining the organic phases, washing with saturated sodium chloride solution (90 mL: 2), concentrating, purifying the residue by column chromatography (EA/PE: 0/1 to 1/2) to obtain white-like solid 1-3(7.23g, yield 75%), ESI-MS M/z:632.3[ M + H ]/(ESI + M0/1 to 1/2)]⁺。

Benzyl (S) -2- (cyanomethyl) -4- (2'- (((S) -1-methylpyrrolidin-2-yl) methoxy) -7',8 '-dihydro-5' H-spiro [ cyclopropane-1, 6 '-pyrido [3,4-d ] pyrimidin ] -4' -yl) piperazine-1-carboxylate (1-4)

A250 mL one-neck flask was charged with 1-3(7.23g, 11.44mmol), DCM (140mL) and TFA (25.2g, 221mmol), and the mixture was stirred at RT for 2 h. After LC-MS monitoring reaction completion, the system was concentrated, DCM (200mL) and sodium bicarbonate solution (100mL) were added to the residue, stirring was carried out at room temperature for 30min, liquid separation was carried out, the aqueous phase was extracted with DCM (100mL), the combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated to dryness to obtain 1-4(5.66g, yield 93%) as a yellowish brown oil, ESI-MS M/z:532.3[ M + H ]/(M + H)]⁺。

(S) -benzyl 2- (cyanomethyl) -4- (2' - (((S) -1-methylpyrrolidin-2-yl) methoxy) -7' - (naphthalen-1-yl) -7',8' -dihydro-5 ' H-spiro [ cyclopropane-1, 6' -pyrido [3,4-d ] pyrimidine ] -4' -yl) piperazine-1-carboxylate (1-5)

A100 mL single-necked flask was charged with 1-4(266mg,0.5mmol), 1-bromonaphthalene (166mg,0.75mmol),^t-BuONa (144mg, 1.5mmol) and Dioxane (20mL) were replaced with Ar for protection, Sphos-G3-Pd (87mg,0.1mmol) was added, and the mixture was heated to reflux with Ar for reaction for 20 h. After the completion of the reaction was monitored by LC-MS, the system was quenched with water, extracted with EA (20 mL. multidot.2), the organic phase was washed with saturated sodium chloride, concentrated and purified by column chromatography (DCM/MeOH/NH)₄OH 40/1/0.02) to give 1-5(72mg, 22% yield) as a light brown oil, ESI-MS M/z 658.4[ M + H ]]⁺。

2- ((S) -4- (2' - (((S) -1-methylpyrrolidin-2-yl) methoxy) -7' - (naphthalen-1-yl) -7',8' -dihydro-5 ' H-spiro [ cyclopropane-1, 6' -pyrido [3,4-d ] pyrimidin ] -4' -yl) piperazin-2-yl) acetonitrile (1-6)

In a 50mL single-neck flask were added 1-5(72mg,0.11mmol), MeOH (10mL) and 10% Pd/C (20mg, wet% ═ 50%), H₂After three times of replacement, the mixture was stirred at room temperature under normal pressure for 20 hours. After LC-MS monitoring reaction, the system is filtered, the filtrate is concentrated to obtain 1-6(58mg, yield 100%) yellow foamy solid, ESI-MS M/z is 524.3[ M + H ]]⁺。

2- ((S) -1-acryloyl-4- (2' - (((S) -1-methylpyrrolidin-2-yl) methoxy) -7' - (naphthalen-1-yl) -7',8' -dihydro-5 ' H-spiro [ cyclopropane-1, 6' -pyrido [3,4-d ] pyrimidin ] -4' -yl) piperazin-2-yl) acetonitrile (compound 1)

A50 mL single-neck flask is added with 1-6(58mg,0.11mmol), DCM (5mL), DIPEA (42mg,0.33mmol), cooled to 0 ℃ in an ice bath under the protection of Ar, and then a DCM solution of acryloyl chloride (9mg,0.10mmol) is added dropwise, and the system is stirred for 1h at 0-5 ℃. After completion of the reaction monitored by LC-MS, water was added for quenching, the aqueous phase was separated, the aqueous phase was extracted with DCM (10mL), the combined organic phases were washed with saturated sodium chloride solution (10mL), and the residue after concentration was purified by pre-TLC to give Compound 1 as an off-white solid (13mg, yield 20%).

¹H NMR(400MHz,CDCl₃)δ:7.97(m,1H),7.72-7.21(m,6H),6.63-6.52(m,1H),6.24(dd,J＝16.9,1.9Hz,1H),5.72(ddd,J＝10.5,3.2,1.9Hz,1H),4.66(d,J＝11.9Hz,1H),4.32(m,3H),3.64-3.51(m,3H),3.48-3.22(m,4H),2.74(s,3H),2.53-2.32(m,7H),1.86-1.65(m,4H),0.65-0.46(m,4H)；ESI-MS m/z:578.3[M+H]⁺。

Example 2-example 93 Synthesis of Compound 2-Compound 93

Compound 2-compound 93 can be obtained by using intermediates S-1 to S-9 as starting materials and employing synthesis method a, similarly to the synthesis method of compound 1.

Example Synthesis of- ((S) -1- (2-Fluoroacryloyl) -4- (7'- (8-Methylnaphthalen-1-yl) -1' - (((S) -1-Methylpyrrolidin-2-yl) methyl) -2 '-oxo-1', 5',7',8 '-tetrahydro-2' H-spiro [ cyclopropane-1, 6 '-pyrido [3,4-d ] pyrimidin ] -4' -yl) piperazin-2-yl) acetonitrile (Compound 94)

Compound 94 was prepared according to procedure B as described below:

(S) -4' - (4- ((benzyloxy) carbonyl) -3- (cyanomethyl) piperazin-1-yl) -2' -oxo-1 ',2',5',8' -tetrahydro-7 ' H-spiro [ cyclopropane-1, 6' -pyrido [3,4-d ] pyrimidine ] -7' -carboxylic acid tert-butyl ester (94-1)

Adding 1-2(5.27g,8.83mmol) and 1, 4-dioxane (100mL) into a 250mL single-neck flask, adding sodium hydroxide solution (44.1mL,2N), reacting at room temperature overnight, monitoring by TLC (PE/EA ═ 1/1) that the raw materials are completely reacted, adjusting the pH to 7 with 2N hydrochloric acid, precipitating a solid, filtering to obtain a white solid 94-1, extracting the filtrate with EA (50mL), washing with saturated sodium chloride solution, drying with anhydrous sodium sulfate, filtering, adding EA (5mL) into the concentrated crude product, pulping, stirring, filtering to obtain a white solid 94-1, combining to obtain the product 94-1(3.82g, yield 81%), ESI-MS M/z:535.2[ M + H/z ]: 535.2]⁺。

4'- ((S) -4- ((benzyloxy) carbonyl) -3- (cyanomethyl) piperazin-1-yl) -1' - ((((S) -1-methylpyrrolidin-2-yl) methyl) -2 '-oxo-1', 2',5',8 '-tetrahydro-7' -spiro [ cyclopropane-1, 6 '-pyrido [3,4-d ] pyrimidine ] -7' -carboxylic acid tert-butyl ester (94-2)

94-1(1.26g,2.36mmol), Cs and water were added to a 100mL closed-loop reactor₂CO₃(1.53g,4.70mmol), (S) -2- (bromomethyl) -1-methylpyrrolidine (632mg,3.55mmol), CuI (89mg,0.47mmol) and DMSO (24mL), sealed and warmed to 100 ℃ and the reaction stirred for 20 h. Cooling to room temperature, adding saturated ammonium chloride solution (20mL), quenching, stirring at room temperature for 30min, adding EA (20mL 2) for extraction, combining organic phases, washing with saturated sodium chloride solution (20mL 2), concentrating, and purifying residue by column chromatography (DCM/MeOH 30/0 to 20/1) to obtain yellow-brown solid 94-2(610mg, yield 41%), ESI-MS M/z 632.3[ M + H ] M + H]⁺。

Benzyl (S) -2- (cyanomethyl) -4- (1' - (((S) -1-methylpyrrolidin-2-yl) methyl) -2' -oxo-1 ',5',7',8' -tetrahydro-2 ' H-spiro [ cyclopropane-1, 6' -pyrido [3,4-d ] pyrimidine ] -4' -yl) piperazine-1-carboxylate (94-3)

A250 mL single neck flask was charged with 94-2(610mg,0.966mmol), DCM (10mL) and TFA (2.20g,19.31mmol), and the mixture was stirred at RT for 4 h. LC-MS monitoringAfter completion, the system was concentrated, the residue was added with DCM (20mL) and sodium bicarbonate solution (20mL), stirred at room temperature for 30min, the aqueous phase was separated, extracted with DCM (20mL), the combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated to give 94-3 as a yellowish-brown oil (493mg, 96% yield), ESI-MS M/z:532.3[ M + H ], [ ESI-MS M/z ]]⁺。

Benzyl (S) -2- (cyanomethyl) -4- (7'- (8-methylnaphthalen-1-yl) -1' - (((S) -1-methylpyrrolidin-2-yl) methyl) -2 '-oxo-1', 5',7',8 '-tetrahydro-2' H-spiro [ cyclopropane-1, 6 '-pyrido [3,4-d ] pyrimidine ] -4' -yl) piperazine-1-carboxylate (94-4)

A250 mL single-neck flask was charged with 94-3(272mg,0.512mmol), 1-bromo-8-methylnaphthalene (170mg,0.768mmol), sodium tert-butoxide (98mg,1.024mmol), Xantphos (58mg,0.1mmol) and Dioxane (10mL), and after Ar displacement protection, Pd was added₂(dba)₃(46mg,0.05mmol), heating to reflux and stirring under the protection of mixed solution Ar for reaction for 20 h. After completion of the reaction, LC-MS monitored. The system was quenched with water. EA (20mL 2) extraction. The organic phase was washed with saturated sodium chloride. After concentration, column chromatography purification (MeOH/DCM. RTM. 0/1 to 1/40to 1/20) gave 94-4(62mg, 18% yield) as a light brown oil, ESI-MS M/z:672.4[ M + H ])]⁺。

2- ((S) -4- (7'- (8-methylnaphthalen-1-yl) -1' - (((S) -1-methylpyrrolidin-2-yl) methyl) -2 '-oxo-1', 5',7',8 '-tetrahydro-2' H-spiro [ cyclopropane-1, 6 '-pyrido [3,4-d ] pyrimidin ] -4' -yl) piperazin-2-yl) acetonitrile (94-5)

A100 mL single-neck flask was charged with 94-4(62mg,0.092mmol), MeOH (10mL), and 10% Pd/C (20mg, wet% ═ 50%), H₂After three times of replacement, the mixture was stirred at room temperature under normal pressure for 20 hours. After LC-MS monitoring reaction, the system was filtered and the filtrate was concentrated to give 94-5(55mg, yield 100%) as a yellow foamy solid, ESI-MSm/z:538.3[ M + H ]]⁺。

2- ((S) -1- (2-Fluoroacryloyl) -4- (7'- (8-methylnaphthalen-1-yl) -1' - ((((S) -1-methylpyrrolidin-2-yl) methyl) -2 '-oxo-1', 5',7',8 '-tetrahydro-2' H-spiro [ cyclopropane-1, 6 '-pyrido [3,4-d ] pyrimidin ] -4' -yl) piperazin-2-yl) acetonitrile (Compound 94)

A50 mL single vial was charged with 2-fluoro-acrylic acid (25mg, 0.277mmol), DCM (5mL) and DMF (2mg) with Ar displacement protection and then cooled to 0 deg.C, followed by dropwise addition of oxalyl chloride (30mg, 0.236mmol) in DCM (2mL) and stirring of the mixture at room temperature for 2 h. The mixture was then cooled to 0 ℃ in an ice bath and a solution of 94-5(47mg, 0.088mmol) in DCM (2mL) and DIPEA (57mg, 0.44mmol) in DCM (2mL) was added dropwise. The mixture was stirred for 1h in ice bath. After LC-MS monitoring completion of the reaction, the reaction was quenched with water (10mL), separated, the aqueous phase extracted with DCM (10mL), the combined organic phases washed with saturated sodium chloride solution (10mL), dried over anhydrous sodium sulfate, and the crude pre-TLC purified to give off-white solid 94(22mg, 41% yield).

¹H NMR(400MHz,CDCl₃)δ:7.64(d,J＝19.2Hz,2H),7.31(m,4H),5.40(d,J＝47.5Hz,1H),5.23(d,J＝16.7Hz,1H),4.49(m,1H),4.33-3.97(m,1H),3.74-3.51(m,4H),3.52-3.21(m,5H)3.18-2.75(m,6H),2.65(s,3H),2.56-2.35(m,4H),1.94-1.82(m,4H),0.62-0.35(m,4H)；ESI-MS m/z:610.3[M+H]⁺.

Example 95-example 165 Synthesis of Compound 95-Compound 165

Compound 95-compound 165 can be obtained by using intermediates S-1 to S-9 as starting materials and employing synthesis method B in a similar manner to the synthesis method of compound 94.

EXAMPLE 166 assay of the amount of pERK and ERK proteins in H358 cells with Compounds

H358 cells were seeded in a 24-well plate, after one day of growth, a test compound (1. mu.M concentration) was added, after 24 hours of compound action, after cell lysis, cell lysates were transferred to a 96-well ELISA plate, levels of pERK and ERK in the lysates were determined using an ELISA kit (abcam 176660), the ratio of pERK to ERK was calculated and compared to DMSO group, the percentage of inhibition of pERK activity by the compound was calculated, and the results are shown in Table 2 below.

TABLE 2 inhibitory Activity of Compounds of the invention against H358 intracellular pERK levels

+ represents an inhibition rate of less than or equal to 50%

+ indicates an inhibition of 50% to 90%

And +++ indicates an inhibition of greater than 90%.

EXAMPLE 167 antiproliferative Activity of Compounds on H358 cells

2500H 358 cells were plated in ultra low adsorption 96-well plates (corning,7007) and after one day of growth, a gradient dilution compound (up to 30M, 5-fold dilution, five total doses) was added, and after three days of compound addition, Cell Titer Glow (Promega, G9681) was added to evaluate pellet growth and calculate IC₅₀The values, results are given in table 3 below.

TABLE 3 antiproliferative activity of the compounds of the invention on H358 cells

+ represents a compound of IC₅₀Greater than 30 μ M

+ represents the IC of the compound₅₀Is 1 to 30 mu M

+ + + + + denotes IC of the Compound₅₀Less than 1. mu.M.

Claims

1. A compound with a structure shown as a general formula (1) or each optical isomer, each crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof:

in formula (1):

l is a bond or NH;

represents a single or double bond between V and Z, when Z is CO and V is

When the V and the Z are connected by a single bond; or, when V is N and Z is

When, V and Z are connected by double bond;

wherein the content of the first and second substances,

y is a bond or C1-C6 alkylene;

2. The compound of claim 1, having the structure of formula (1A) or formula (1B):

wherein:

l is a bond or NH;

y is a bond or C1-C6 alkylene;

R³is aminoalkyl, cycloalkyl, alkyl-substituted amido, heterocyclyl, aryl or heteroaryl, said heterocyclyl, aryl or heteroarylHeteroaryl groups may each be substituted with 1 to 3 of the following groups: halogen, O, CN, OH, alkylhydroxy, dialkylamino, C1-C6 alkyl, C3-C6 cycloalkyl, halogen substituted C1-C3 alkyl or halogen substituted C1-C3 alkoxy, which when substituted with a plurality of substituents, may be the same or different;

3. The compound according to claim 1 or 2, wherein in the general formula (1), the general formula (1A) and the general formula (1B), E is a group having an electrophilic carbon-carbon double bond or carbon-carbon triple bond.

4. The compound of claim 3, wherein in the general formula (1), the general formula (1A) and the general formula (1B), E is:

5. The compound according to claim 1 or 2, wherein in the general formula (1), the general formula (1A) and the general formula (1B), -a-L-E is:

6. The compound according to claim 1 or 2, wherein in the general formula (1), the general formula (1A) and the general formula (1B), Y is a bond, -CH₂-, -CH (Me) -or-CH₂CH₂-。

7. The compound according to claim 1 or 2, wherein in the general formula (1), the general formula (1A) and the general formula (1B), R is¹Comprises the following steps:

8. The compound according to claim 1 or 2, wherein in the general formula (1), the general formula (1A) and the general formula (1B), R is³Comprises the following steps:

9. The compound of claims 1-8, or a pharmaceutically acceptable salt thereof, wherein the compound has one of the following structures:

10. a pharmaceutical composition for the treatment, regulation and/or prevention of diseases associated with the K-Ras G12C mutant protein, which comprises a pharmaceutically acceptable excipient or carrier, and as an active ingredient a compound according to any one of claims 1 to 9, or each optical isomer, pharmaceutically acceptable salt, hydrate or solvate thereof.

11. The pharmaceutical composition of claim 10, wherein said composition is in an oral dosage form.

12. The pharmaceutical composition of claim 10, wherein the composition is in an injectable dosage form.

13. Use of a compound according to any one of claims 1 to 9, or each optical isomer, each crystalline form, a pharmaceutically acceptable salt, hydrate or solvate thereof, for the treatment of a disorder mediated by the K-Ras G12C mutation in a subject in need thereof.

14. The method of claim 13 wherein the condition is cancer, the cancer being hematological cancer and solid tumor.