CN117777197A - Phosphorus-containing compound, and preparation and application thereof - Google Patents

Abstract

The present invention relates to a class of phosphorus-containing compounds and their preparation and application. Specifically, the compound of the present invention has a structure represented by formula (I), in which the definitions of each group and substituent are as described in the specification. The invention also discloses the preparation method of the compound and its use in regulating CDK2 kinase activity or treating CDK2-related diseases.

Description

A type of phosphorus-containing compound and its preparation and application

Technical Field

The present invention relates to the field of medical technology, and in particular to a class of phosphorus-containing compounds used as CDK2 kinase inhibitors, and pharmaceutically acceptable salts thereof, to pharmaceutical compositions comprising such compounds and salts, and to uses thereof, and to applications thereof in regulating CDK2 kinase activity or treating CDK2-related diseases.

Background Art

Cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases, important cellular enzymes that perform essential functions in regulating cell division and proliferation.

Heterodimerization of CDKs and regulatory subunits (Cyclins) fully activates and regulates key cellular processes including cell cycle progression and cell division (Morgan, d.o., Annu Rev Cell Dev Biol, 1997, 13: 261-91). Uncontrolled proliferation is a characteristic of cancer cells. Abnormal regulation of CDK activity is associated with abnormal regulation of the cell cycle and can be detected in almost all forms of human cancer (Sherr, c.j., Science, 1996, 274 (5293): 1672-7).

CDK2 plays a vital role in G1/S transition and S phase progression. CDK2 and cyclin E (CCNE) complex phosphorylates retinoblastoma pocket protein family members (p107, p130, pRb), releases E2F transcription factor, and promotes the transition from G1 phase to S phase (Henley, S.A. and F.A. Dick, Cell Div, 2012, 7 (1): 10). CDK2/cyclin A is then activated to phosphorylate endogenous substrates, thereby allowing DNA synthesis, replication and centrosome duplication (Ekholm, S.V. and S.I. Reed, Curr Opin Cell Biol, 2000, 12 (6): 67684). It is reported that the CDK2 pathway mainly affects tumorigenesis through the amplification and/or overexpression of CCNE1 and the mutational inactivation of CDK2 endogenous inhibitors (such as p27).

CCNE1 copy number increase and overexpression have been confirmed in ovarian, gastric, endometrial, breast and other tumors and are associated with poor prognosis in these tumors (Keyomarsi, K., et al., N Engl J Med., 2002, 347(20):1566-75; Nakayama, N., et al., Cancer, 2010, 116(11):2621-34; Au-Yeung, G., et al., Clin Cancer Res, 2017, 23(7):1862-1874; Rosen, D.G., et al., Cancer, 2006, 106(9):1925-32). It has been reported that CCNE1 amplification and/or overexpression can also lead to trastuzumab resistance in HER2+ breast cancer and CDK4/6 inhibitor resistance in estrogen receptor-positive breast cancer. Inhibitors targeting CDK2 have been shown to induce apoptosis and inhibit tumor proliferation.

However, due to the high similarity of the ATP-binding domains of CDKs family kinases, there is currently a lack of selective inhibitors for CDK2. Most small molecules can often inhibit multiple CDK kinase subtypes and even other non-CDK family kinases at the same time. Such off-target effects bring about potent cell killing or inhibition, but also bring about significant side effects. Therefore, the development of new selective CDK2 kinase inhibitors has broad application prospects.

Summary of the invention

The object of the present invention is to provide a compound represented by formula (I), a preparation method thereof and use thereof in CDK2 kinase inhibitor drugs.

In the first aspect of the present invention, a compound is provided, wherein the compound is a compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof,

in:

Ring A is selected from the following group: _C5-7 cycloalkyl, 5-7 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, _C5-6 aryl, 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the cycloalkyl, heterocycloalkyl, aryl, heteroaryl are optionally substituted by 0, 1, 2 or 3 _R1 , and Ring A and Ring B form a cyclic structure;

Ring B is selected from the group consisting of C _5-6 aryl, 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the aryl and heteroaryl are optionally substituted by 0, 1, 2 or 3 R ₂ ;

R ₁ is selected from the following group: hydrogen, deuterium, halogen, hydroxyl, cyano, =O, C _1-4 alkyl, C _{1-4 haloalkyl, 1-4} membered heteroalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, 3-5 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, C _3-5 cycloalkyl, C _2-4 alkenyl, C _2-4 alkynyl;

_R2 is selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl, cyano, _C1-4 alkyl, C1-4 haloalkyl, _1-4 membered heteroalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S;

R ₃ is NR ₆ R ₇ ;

^Ra is selected from the group consisting of hydrogen, deuterium, halogen, hydroxy, cyano, _C1-4 alkyl, _C1-4 fluoroalkyl, = _CH2 ;

_X1 is selected from the group consisting of _CH2 , O; or _X1 is absent, such that Four-membered cyclobutyl

X ₂ is selected from the group consisting of O, -CH ₂ -, -CH ₂ O-;

R ₄ and R ₅ are independently selected from the following group: C _1-6 alkyl, C _1-6 haloalkyl, 1-6 membered heteroalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, C _3-5 cycloalkyl, 3-5 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the C _1-6 alkyl, C _1-6 haloalkyl, heteroalkyl, C _3-5 cycloalkyl, 3-5 membered heterocycloalkyl are optionally substituted with one or more substituents selected from the following group: deuterium, hydroxyl, halogen, cyano, =O, C _1-4 alkyl, C _1-4 haloalkyl, C _1-4 alkoxy, C _1-4 haloalkoxy,

Alternatively, _R4 and _R5 together with the phosphorus atom to which they are attached form a 4-7 membered heterocycloalkyl group, wherein the heterocycloalkyl group is optionally substituted by 0-3 substituents selected from the group consisting of deuterium, hydroxyl, halogen, cyano, =O, _C1-4 alkyl, _C1-4 haloalkyl, _C1-4 alkoxy, _C1-4 haloalkoxy;

R ₆ and R ₇ are independently selected from the following group: hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, -L-(C _3-6 cycloalkyl), -L-(3-6 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S), wherein the C _1-6 alkyl, C _3-6 cycloalkyl, 3-6 membered heterocycloalkyl are optionally substituted by one or more substituents selected from the following group: deuterium, hydroxyl, halogen, cyano, C _1-4 alkyl, C _1-4 alkoxy, halogenated C _1-4 alkyl, halogenated C _1-4 alkoxy,

Alternatively, _R6 and _R7 connected to the same N atom are connected to form a 3-7 membered heterocyclic group, wherein the 3-7 membered heterocyclic group is optionally substituted by one or more substituents selected from the group consisting of deuterium, hydroxyl, halogen, cyano, =O, _C1-4 alkyl, _C1-4 alkoxy, halogenated _C1-4 alkyl, halogenated _C1-4 alkoxy;

L is a bond or a C _1-6 alkylene group.

In another preferred embodiment, Represents the junction site.

In another preferred embodiment, the compound has a structure selected from the following group:

in,

Ring A is selected from the following group: _C5-7 cycloalkyl, 5-7 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, _C5-6 aryl, 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the cycloalkyl, heterocycloalkyl, aryl, heteroaryl are optionally substituted by 0, 1, 2 or 3 _R1 , and Ring A and Ring B form a cyclic structure;

Ring B is selected from the group consisting of C _5-6 aryl, 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the aryl and heteroaryl are optionally substituted by 0, 1, 2 or 3 R ₂ ;

R ₁ is selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl, cyano, =O, C _1-4 alkyl, C _{1-4 haloalkyl, 1-4} membered heteroalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, 3-5 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, C _3-5 cycloalkyl, C _2-4 alkenyl, C _2-4 alkynyl;

_R2 is selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl, cyano, _C1-4 alkyl, C1-4 haloalkyl, _1-4 membered heteroalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S;

R ₃ is NR ₆ R ₇ ;

R ₄ and R ₅ are independently selected from the following group: C _1-6 alkyl, C _1-6 haloalkyl, 1-6 membered heteroalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, C _3-5 cycloalkyl, 3-5 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the C _1-6 alkyl, C _1-6 haloalkyl, heteroalkyl, C _3-5 cycloalkyl, 3-5 membered heterocycloalkyl are optionally substituted with 1-6 substituents selected from the following group: deuterium, hydroxyl, halogen, cyano, =O, C _1-4 alkyl, C _1-4 haloalkyl, C _1-4 alkoxy, C _1-4 haloalkoxy,

Alternatively, _R4 and _R5 together with the phosphorus atom to which they are attached form a 4-7 membered heterocycloalkyl group, wherein the heterocycloalkyl group is optionally substituted by 0-3 substituents selected from the group consisting of deuterium, hydroxyl, halogen, cyano, =O, _C1-4 alkyl, _C1-4 haloalkyl, _C1-4 alkoxy, _C1-4 haloalkoxy;

R ₆ and R ₇ are independently selected from the following group: hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, -L-(C _3-6 cycloalkyl), -L-(3-6 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O and S), wherein the C _1-6 alkyl, C _3-6 cycloalkyl and 3-6 membered heterocycloalkyl are optionally substituted by 1 to 6 substituents selected from the following group: deuterium, hydroxyl, halogen, cyano, C _1-4 alkyl, C _1-4 alkoxy, halogenated C _1-4 alkyl and halogenated C _1-4 alkoxy,

Alternatively, _R6 and _R7 connected to the same N atom are connected to form a 3-7 membered heterocyclic group, wherein the 3-7 membered heterocyclic group is optionally substituted by 1-6 substituents selected from the group consisting of deuterium, hydroxyl, halogen, cyano, =O, _C1-4 alkyl, _C1-4 alkoxy, halogenated _C1-4 alkyl, halogenated _C1-4 alkoxy;

L is a bond or a C _1-6 alkylene group.

In another preferred embodiment, when R ₄ and R ₅ are different substituents, the compound has a structure selected from the following group:

wherein ring A, ring B, R ₃ , R ₄ and R ₅ are as defined in claim 2.

In another preferred embodiment, the A ring and the B ring are selected from the following group:

in:

X ₃ and Y are each independently selected from the following group: C, N;

G, H, Q, T, W, E, and Z are each independently selected from the group consisting of C, CH, N, NH, O, and S;

Ring A and Ring B are optionally substituted by R ₁ or R ₂ ;

R ₁ and R ₂ are as described in claim 1.

In another preferred embodiment, Represents the junction site.

In another preferred embodiment, the A ring and the B ring have a structure selected from the following group:

In another preferred embodiment, the A ring and the B ring are preferably

In another preferred embodiment, the A ring and the B ring are preferably

In another preferred embodiment, the A ring and the B ring are preferably

In another preferred embodiment, _R3 is selected from the following group:

In another preferred embodiment, the compound is selected from the following group:

In another preferred embodiment, the compound is the compound prepared in the examples.

In another preferred embodiment, the compound is compound C1-C40.

In another preferred embodiment, the compound is compound C3, C10, C12, C22, C24, C28, C33.

The second aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds described in the first aspect of the present invention and a pharmaceutically acceptable carrier.

The third aspect of the present invention provides a use of the compound described in the first aspect of the present invention for preparing a drug for regulating CDK2 kinase activity or treating CDK2-related diseases.

In another preferred embodiment, the CDK2-related disease is cancer or tumor.

In another preferred embodiment, the cancer or tumor is selected from the following group: breast cancer, ovarian cancer, bladder cancer, uterine cancer, lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, thyroid cancer, esophageal cancer, kidney cancer, liver cancer, head and neck cancer, glioblastoma, mantle cell lymphoma (MCL), chronic myeloid leukemia (CML) and acute myeloid leukemia (AML).

In another preferred embodiment, the lung cancer is non-small cell lung cancer.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

DETAILED DESCRIPTION

After long-term and in-depth research, the inventor unexpectedly prepared a phosphorus-containing compound with excellent CDK kinase inhibitory activity and a preparation method thereof. Specifically, through structural optimization, the inventor obtained a class of compounds with excellent CDK2 selective inhibitory activity. On this basis, the inventor completed the present invention.

the term

In the present invention, unless otherwise specified, the terms used have the general meanings well known to those skilled in the art.

In the present invention, the term "halogen" refers to F, Cl, Br or I.

In the present invention, the term "C _1-4 alkyl" refers to a straight or branched alkyl group including 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, or the like. The term "C _1-6 alkyl" has a similar meaning.

In the present invention, the term " _C2-6 alkenyl" refers to a straight or branched alkenyl group having 2 to 6 carbon atoms and containing one double bond, including but not limited to ethenyl, propenyl, butenyl, isobutenyl, pentenyl and hexenyl, etc. The term " _C2-4 alkenyl" has a similar meaning.

In the present invention, the term "C _2-6 alkynyl" refers to a straight or branched alkynyl group having 2 to 6 carbon atoms and containing one triple bond, including but not limited to ethynyl, propynyl, butynyl, isobutynyl, pentynyl and hexynyl, etc. The term "C _2-4 alkynyl" has a similar meaning.

In the present invention, the term "C _3-8 cycloalkyl" refers to a cyclic alkyl group having 3-8 carbon atoms in the ring, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. Similar terms "C _5-7 cycloalkyl", "C _3-6 cycloalkyl", "C _3-5 cycloalkyl" have similar meanings.

In the present invention, the term "C _1-4 alkoxy" refers to a straight or branched alkoxy group having 1 to 4 carbon atoms, including but not limited to methoxy, ethoxy, propoxy, isopropoxy, butoxy and the like.

In the present invention, the term "1-6 membered heteroalkyl" refers to a 1-6 membered straight or branched alkyl group containing a heteroatom selected from N, O or S, including but _not limited to methoxymethyl, _ethoxymethyl , propoxymethyl, isopropoxymethyl, butoxymethyl, _-CH2OH , _-CH2NH2 , _{-CH2NHCH3 , -CH2N} (CH3 _{) 2 ,} -CH2CH2OCH3 _{, -CH2CH2NHCH3 , -CH2CH2N} ( _CH3 ) _{2 ,} etc. The term "1-4 membered heteroalkyl" has _a similar meaning.

In the present invention, the term "heterocyclyl" or "heterocycloalkyl" refers to a 3-7 membered heterocyclic group containing 1, 2 or 3 heteroatoms selected from N, O and S, including (but not limited to) the following groups:

In the present invention, the term "aromatic ring" or "aryl group" has the same meaning, preferably "C _6-10 aryl group". The term "C _6-10 aryl group" refers to an aromatic ring group having 6 to 10 carbon atoms without heteroatoms in the ring, such as phenyl, naphthyl, etc. The term "C _5-6 aryl group" has a similar meaning.

In the present invention, the term "aromatic heterocycle" or "heteroaryl" has the same meaning and refers to a heteroaromatic group containing one to multiple heteroatoms. Non-limiting examples include: furanyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring. The heteroaryl group can be optionally substituted or unsubstituted.

In the present invention, the term "halo" means substituted with halogen.

In the present invention, the term "substituted" refers to one or more hydrogen atoms on a specific group being replaced by a specific substituent. The specific substituent is the substituent described above, or the substituent appearing in each embodiment. Unless otherwise specified, a substituted group may have a substituent selected from a specific group at any substitutable site of the group, and the substituent may be the same or different at each position. It will be understood by those skilled in the art that the combination of substituents contemplated by the present invention is those that are stable or chemically feasible. The substituents include, for example (but not limited to): halogen, hydroxyl, C1-C4 alkyl, C3-C7 cycloalkyl, 3- to 7-membered heterocyclic group, aryl, heteroaryl, C2-C10 acyl, C2-C10 ester group, amino, C1-C4 alkoxy, etc.

In the present invention, the term 1-6 means 1, 2, 3, 4, 5 or 6. Other similar terms have similar meanings.

In the present invention, the term "plurality" refers to 2, 3, 4, 5 or 6 each independently.

It should be understood that when a group is present in multiple different positions of a compound at the same time, its definition at each position is independent of each other and may be the same or different. That is, the term "selected from the following group:" and the term "each independently selected from the following group:" have the same meaning.

Compound

The present invention provides a compound, which is a compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof,

wherein Ring A, Ring B, R ₃ , R ₄ , R ₅ , ^Ra , X ₁ and X ₂ are as defined above.

In another preferred embodiment, in the compound, any one of ring A, ring B, R ₃ , R ₄ , R ₅ , ^Ra , X ₁ , and X ₂ is independently a corresponding specific group in the specific compound.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention and an acid or base that is suitable for use as a drug. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is a salt formed by a compound of the present invention and an acid. Suitable acids for forming salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid.

Another preferred salt is a salt of the compound of the present invention and a base, such as an alkali metal salt (e.g., sodium salt or potassium salt), an alkaline earth metal salt (e.g., magnesium salt or calcium salt), an ammonium salt (e.g., lower alkanolammonium salt and other pharmaceutically acceptable amine salts), for example, methylamine salt, ethylamine salt, propylamine salt, dimethylamine salt, trimethylamine salt, diethylamine salt, triethylamine salt, tert-butylamine salt, ethylenediamine salt, hydroxyethylamine salt, dihydroxyethylamine salt, trihydroxyethylamine salt, and amine salts formed from morpholine, piperazine, and lysine, respectively.

The term "solvate" refers to a complex in which the compound of the present invention is coordinated with solvent molecules to form a specific ratio.

In addition, the compounds of the present invention also include prodrugs of the compounds shown in formula (I). The term "prodrug" includes a compound that may be biologically active or inactive, and when taken by an appropriate method, it undergoes metabolism or chemical reactions in the human body and is converted into a class of compounds of formula (I), or a salt or solution composed of a compound of formula (I). The prodrug includes (but is not limited to) carboxylate, carbonate, phosphate, nitrate, sulfate, sulfone, sulfoxide, amino compound, carbamate, azo compound, phosphoramide, glucoside, ether, acetal and the like forms of the compound.

It should be understood that the specific preparation method of the compound of formula (I) of the present invention does not constitute any limitation to the present invention. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such combination can be easily performed by a person skilled in the art to which the present invention belongs.

Typically, the compounds of the present invention can be prepared by the process described in the examples, wherein the raw materials and reagents used can be purchased from commercial sources unless otherwise specified.

Pharmaceutical compositions and methods of administration

The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of one or more of the compounds and a pharmaceutically acceptable carrier.

Since the compounds of the present invention have excellent anti-tumor activity, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate tumor-related diseases.

The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, and more preferably, contains 10-1000 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

"Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The pharmaceutical composition is in the form of injection, capsule, tablet, pill, powder or granule.

There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administrations include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

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, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl 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 the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into 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 compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

Besides such inert diluents, the composition may also include 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 such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may include 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 non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration of the compounds of the 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 that may be required.

The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds (such as anti-tumor drugs).

The treatment method of the present invention can be used alone or in combination with other treatment methods or therapeutic drugs.

When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 50 to 1000 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

Compared with the prior art, the present invention has the following main advantages:

1) The compound has excellent CDK2 selective inhibitory activity;

2) The compound has excellent pharmacokinetic properties, safety and drugability.

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

Example Synthesis Route

Synthesis of intermediates

Synthesis of intermediate int1:

1. Synthesis of int1-2

Dissolve int1-1 (10 g, 64.0 mmol) in 150 mL of methanol, add NaBH ₄ (2.44 g, 64.0 mmol) in batches under nitrogen protection and ice-water bath cooling, then heat the reaction solution to room temperature and stir for 2 hours. After the reaction is completed, slowly add 1 M dilute hydrochloric acid solution under ice-water bath cooling to quench the reaction, concentrate under reduced pressure to evaporate the solvent methanol, then dilute with water, extract with dichloromethane, separate the organic phase, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 8.27 g of crude product. Ms[M+Na] ⁺ 181.1

2. Synthesis of int1-3

Dissolve int1-2 (8.27 g, 52.33 mmol) in 100 mL of ethanol, then add imidazole (10.67 g, 157.0 mmol), slowly dropwise add TBDPSCl (15.83 g, 57.58 mmol) under nitrogen protection in an ice-water bath, and after the addition is complete, warm the reaction solution to room temperature and stir for 16 hours. After the reaction is completed, evaporate the solvent ethanol under reduced pressure, then dilute the residue with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and column chromatograph to obtain 19.86 g of the product. Ms[M+H]+397.2. 1HNMR(400MHz, CDCl3)δ7.73-7.58(m,4H),7.43-7.33(m,6H),4.31-4.20(m,1H),4.18-4.03(m,2H),2.71-2.55(m,1H),2.19-2.08(m,1H) ),2.01(d,J＝5.6Hz,2H),1.86-1.70(m,2H),1.64-1.53(m,1H),1.25(t,J＝7.1Hz,3H),1.05(s,9H).

3. Synthesis of int1-4

Under nitrogen protection, n-butyl lithium (88 mL, 2.5 M in hexane) was added to 500 mL of anhydrous tetrahydrofuran, the system was cooled to -65 ° C, and then acetonitrile (9.2 g, 224.8 mmol) was slowly added dropwise. After the addition was complete, the temperature was maintained and stirred for 1 hour. Then, an anhydrous tetrahydrofuran solution (100 mL) of int1-3 (44.7 g, 112.4 mmol) was slowly added dropwise, and the temperature was maintained and stirred for 2 hours. After the reaction was completed, water was added dropwise at -65 ° C to quench the reaction, and then stirred and warmed to room temperature. The reaction solution was extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 49.0 g of crude product. Ms [M + H] + 392.2

4. Synthesis of int1-5

Dissolve tert-butylhydrazine hydrochloride (9.35 g, 75.0 mmol) and triethylamine (10.43 mL, 75.0 mmol) in 150 mL of ethanol, stir for 0.5 hours, then add ethanol solution (50 mL) of int1-4 (19.6 g, 50.0 mmol), and then heat the reaction system to 75 ° C and stir for 16 hours. After the reaction, the reaction solution was cooled to room temperature, concentrated, diluted with water, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified to obtain 19.1 g. Ms[M+H]+462.3 1HNMR(400MHz,CDCl3)δ7.70-7.66(m,4H),7.40-7.31(m,6H),5.46(s,1H),5.19(s,1H),4.46-4.27(m,1H),3.55-3.27(m,2H),2.90-2.8 1(m,1H),2.29-2.04(m,1H),1.93-1.64(m,4H),1.57(s,9H),1.06(s,9H).

5. Synthesis of int1-6

Dissolve int1-5 (19.12 g, 41.0 mmol) in 160 mL of acetonitrile, add benzyl chloroformate (11.7 mL, 82.0 mmol) dropwise under ice-water cooling, and after the addition is complete, warm to room temperature and stir for 2 h, then add NaHCO3 (11.02 g, 130.0 mmol) and stir at room temperature for 16 h. After the reaction is completed, filter the reaction solution, wash the filter cake with ethyl acetate several times, concentrate the filtrate under reduced pressure, dilute with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify to obtain 21.2 g. Ms[M+H]+596.4

6. Synthesis of int1-7

Int1-6 (21.0 g, 35.0 mmol) was dissolved in 150 mL of anhydrous tetrahydrofuran under stirring at room temperature, and glacial acetic acid (4.2 mL, 70.0 mmol) and tetrabutylammonium fluoride (35.0 mL, 1 M in THF) were added in sequence, and heated to 40 ° C under nitrogen atmosphere and stirred for 23 hours. After the reaction was completed, the reaction solution was cooled to room temperature, concentrated under reduced pressure, and the residue was diluted with water, extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified to obtain 4.41 g. Ms[M+H]+358.2. 1HNMR(400MHz, DMSO-d6)δ9.06(s,1H),7.59-7.22(m,5H),5.92(s,1H),5.12(s,2H),4.57(d,J＝4.4Hz,1H),4.22-4.09(m,1H),2.99-2.78 (m,1H),2.23-2.14(m,1H),1.90-1.79(m,1H),1.77-1.64(m,3H),1.47(s,9H).

7. Synthesis of int1-8

Dissolve int1-7 (4.41 g, 12.35 mmol) in 80 mL of dichloromethane, add p-nitrophenyl chloroformate (3.73 g, 18.53 mmol), pyridine (2.93 g, 37.05 mmol), DMAP (149 mg, 1.24 mmol) in sequence, and stir at room temperature for 16 h. After the reaction, the reaction solution was washed with saturated brine for several times, dried over anhydrous sodium sulfate, concentrated and purified to obtain 4.34 g of the product. Ms[M+H]+523.2

8. Synthesis of int1-9

Dissolve int1-8 (4.34 g, 8.31 mmol) in 80 mL of tetrahydrofuran, add isopropylamine (1.07 mL, 12.47 mmol) and diisopropylethylamine (4.33 mL, 24.93 mmol) in turn, and stir at room temperature overnight. After the reaction is completed, dilute the reaction system with water, extract with ethyl acetate, combine the organic phases, wash with saturated brine, dry with anhydrous sodium sulfate, concentrate, and purify to obtain 1.17 g of the product. Ms[M+H]+443.3

9. Synthesis of int1, int1A, int1B

Int1-9 (1.07 g, 2.42 mmol) was dissolved in a mixed solvent of 10 mL methanol and 10 mL ethyl acetate, and then Pd/C (214 mg, 20 mol%) was added and stirred at room temperature for 5 h under a hydrogen atmosphere. After the reaction was completed, the reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure and purified to obtain 640 mg of int1. The peak time of int1A (300 mg) was 28.878 min using chiral preparative chromatography, and the peak time of int1B (291 mg) was 33.753 min. The chromatographic column used was Phenomenex 3μm Cellulose-4, mobile phase is n-hexane:isopropanol=90:10, column temperature is 30℃.

Synthesis of intermediate int2:

1. Synthesis of int2-2

In a 250mL single-necked flask, first add dry tetrahydrofuran (80mL) dissolved with int2-1 (5g, 23.04mmol, 1.0eq), then add triethylamine (4.7g, 46.08mmol, 2.0eq) and 2-(trimethylsilyl)ethoxymethyl chloride (5.77g, 34.56mmol, 1.5eq) under ice bath conditions, then remove the ice bath and stir at room temperature for 16h. Then add water under ice bath, and then extract with ethyl acetate (3*30mL), combine the organic phases, wash the organic phases with saturated sodium chloride aqueous solution (3*30mL), dry over anhydrous sodium sulfate, and vacuum rotary evaporation to obtain a crude product. The crude product is separated and purified by silica gel column chromatography to obtain the product int2-2 (4.6g). MS[M+H] ⁺ :349.2.

2. Synthesis of int2-3

The reaction was carried out in parallel in two 25 mL reaction tubes. Int2-2 (4 g, 11.5 mmol, 1.5 eq), Pd(OAc) ₂ (176 mg, 0.76 mmol, 0.1 eq), XantPhos (880 mg, 1.52 mmol, 0.2 eq), and KOAc (80 mg, 0.76 mmol, 0.1 eq) were added in sequence. The tubes were sealed and N ₂ was replaced. Then tetrahydrofuran (15 mL*2) and diisopropylethylamine (1.2 mL*2) were added in sequence through a syringe. The reaction solution was placed in a 70°C oil bath and stirred for 20 min. Another reactant, dimethylphosphine oxide (600 mg, 7.64 mmol, 1.0 eq), was added and stirred at 70°C for 10 min. Then the temperature was raised to 100°C and stirred overnight. The next day, the solid in the reaction solution was filtered out with a triangular funnel, and the solid was washed with ethyl acetate 2-3 times. The reaction solution was vacuum dried, and water (30 mL) and ethyl acetate (3*20 mL) were added, extracted 3 times, and the organic phases were combined. The organic phase was washed with a saturated sodium chloride aqueous solution (3*30 mL), dried over anhydrous sodium sulfate, and vacuum evaporated to obtain a crude product. Silica gel column chromatography was used for separation and purification to obtain the product int2-3 (2 g). Ms[M+H]+: 347.3.

3. Synthesis of int2-4

In a 100mL single-necked flask, int2-3 (2g, 5.76mmol, 1.0eq) was added, and then dichloromethane (3mL) was added to stir and dissolve, and trifluoroacetic acid (3mL) was added at room temperature to react for 2 hours. After the reaction was completed, the mixture was concentrated, and dichloromethane was added to evaporate the remaining trifluoroacetic acid, and the reaction was repeated 3 times. The product int2-4 (1.2g) was obtained by vacuum drying using an oil pump.

4. Synthesis of int2-5

In a 25 mL reaction tube, int2-4 (750 mg, 3.456 mmol) was added, and then aminoacetaldehyde dimethyl acetal (12 mL) was added, and stirred at 110°C overnight. The reaction solution was concentrated to obtain the crude product int2-5 (1 g), which was directly used in the next step. Ms[M+H] ⁺ :276.1.

5. Synthesis of int2-6

In a 25 mL reaction tube, add int2-5 (1 g, 3.636 mmol, 1 eq) in acetone solution (15 mL), then add TsOH·H ₂ O (940 mg, 5.454 mmol, 1.5 eq) in water solution (1.5 mL), and react overnight at 70°C. Concentrate, separate and purify by silica gel column chromatography to obtain product int2-6 (1 g). Ms[M+H] ⁺ :230.1.

6. Synthesis of int2-7

In a 50 mL single-mouth bottle, int2-6 (600 mg) was added, and then 15 mL of trifluoroacetic acid was added, and stirred at 80° C. for 48 h. After the reaction was completed, the reaction solution was concentrated and separated and purified by silica gel column chromatography to obtain the product int2-7 (350 mg). Ms[M+H] ⁺ :212.1.

7. Synthesis of int2

Int2-7 (300 mg, 1.0 eq) was added to a 25 mL reaction tube, followed by 10 mL of phosphorus oxychloride and N,N-dimethylformamide (10 mg, 0.1 eq), and the mixture was reacted at 110°C for 40 minutes. The reaction solution was concentrated and purified by Prep-TLC to obtain the product int2 (87 mg). Ms[M+H] ⁺ : 230.6.

Synthesis of intermediate int3:

Synthesis of int3-2

Add compound int3-1 (21 g, 1.00 eq), potassium carbonate (33.6 g, 2 eq) and N,N-dimethylformamide (300 mL) to a 1000 mL single-mouth bottle. Slowly add benzyl bromide (22.7 g, 1.1 eq) at room temperature, and heat to 80 °C for 1 h. Monitor the reaction by TLC. Lower the temperature to room temperature, filter, and wash the filter residue with ethyl acetate. Dilute the filtrate with ethyl acetate, wash three times with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, and concentrate. Purify with a silica gel column and concentrate to obtain 29 g of the product. Ms[M+H] ⁺ 264.

Synthesis of int3-3

Add compound int3-2 (16 g, 1.00 eq), CuI (1.16 g, 0.1 eq), ethyl acetate (30.7 g, 5 eq) and N,N-dimethylformamide (240 mL) to a 500 mL single-mouth bottle. Add trimethylsilyl acetylene (17.9 g, 3 eq) and Pd(PPh ₃ ) ₂ Cl ₂ (2.14 g, 0.05 eq) at room temperature, and react at 70°C for 1 h under nitrogen atmosphere. Monitor the reaction by TLC. Lower the temperature to room temperature. Add water to the reaction solution, extract three times with ethyl acetate, dry the organic phase with anhydrous sodium sulfate, filter and concentrate. Purify on a silica gel column and concentrate to obtain 14 g of product. Ms[M+H] ⁺ 282.

Synthesis of int3-4

Add compound int3-3 (10 g, 1.00 eq) and acetone (150 mL) to a 500 mL single-mouth bottle, add AgNO ₃ (1.82 g, 0.3 eq), cool to 0°C, add NBS (8.87 g, 1.4 eq) in batches, and react at room temperature in a dark room for 1 h. Monitor the reaction by TLC. Filter, wash with dichloromethane, and concentrate the organic phase. Purify on a silica gel column and concentrate to obtain 4.3 g of product. Ms[M+H] ⁺ 288.

Synthesis of int3-5

Add 2,4,6-trimethylbenzenesulfonylhydroxylamine (3.68g, 1.2eq) and dichloromethane (50mL) to a 100mL single-mouth bottle, add int3-4 (4.1g, 1eq) in dichloromethane (10mL) dropwise at 0℃, and react at room temperature for 1h. Monitor the disappearance of the raw material by thin layer chromatography. Add petroleum ether to the reaction solution, filter, and dry the filter cake to obtain 7g of product. Ms[M+H] ⁺ 303.

Synthesis of int3-6

Add int3-5 (4 g, 1 eq) and acetic acid (72 mL) to a 250 mL single-mouth bottle. Heat to 80 °C and react for 16 h. Liquid quality indicates that the reaction is complete. Cool the reaction solution and concentrate. Dilute with ethyl acetate, wash with saturated sodium bicarbonate aqueous solution, extract with ethyl acetate three times, dry the organic phase with anhydrous sodium sulfate, filter, and concentrate. Purify with silica gel column and concentrate to obtain 1.6 g of product. Ms[M+H] ⁺ 303.

Synthesis of int3-7

In a 50mL single-mouth bottle, int3-6 (600mg, 1eq), KOAc (39mg, 0.2eq), diisopropylethylamine (774mg, 3eq), Pd(OAc) ₂ (89mg, 0.2eq), Xantphos (463mg, 0.4eq) and tetrahydrofuran (13mL) were added. The temperature was raised to 70°C in a nitrogen atmosphere and stirred for 20min. Dimethylphosphine oxide (330mg, 2eq) was dissolved in tetrahydrofuran (2mL) and slowly added dropwise to the reaction solution at 70°C and stirred for 10min. The reaction solution was heated to 100°C and stirred for 16h. The reaction was monitored by thin layer chromatography and liquid chromatography-mass spectrometry. The reaction solution was cooled to room temperature, filtered, and washed with dichloromethane. Water was added to the organic phase, the layers were separated, and the aqueous phase was extracted three times with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. Purification was performed on a silica gel column and 300mg of the product was obtained after concentration. Ms[M+H] ⁺ 301.

Synthesis of int3-8

Add int3-7 (600 mg, 1 eq) and methanol (20 mL) to a 100 mL single-mouth bottle. Add 10% Pd/C (1 g) at room temperature to replace the hydrogen. Stir the reaction solution at room temperature for 16 h in a hydrogen atmosphere. TLC and HPLC showed that the reaction was complete. Filter, wash with methanol, and concentrate the organic phase to obtain 350 mg of product. Ms[M+H] ⁺ 211.

Synthesis of int3

Add int3-8 (350 mg, 1 eq), ethyl acetate (895 mg, 6 eq) and dichloromethane (15 mL) to a 50 mL single-mouth bottle. Cool to 0 ° C, add N-phenylbis(trifluoromethanesulfonyl)imide (790 mg, 1.5 eq) in batches, and react at room temperature for 30 min. Thin layer chromatography shows that the reaction is complete. Filter, wash with dichloromethane, add saturated sodium bicarbonate aqueous solution to the organic phase, separate the layers, and extract the aqueous phase with ethyl acetate three times. Combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate. Purify on a silica gel column and concentrate to obtain 370 mg of the product. Ms[M+H] ⁺ 343.

Synthesis of intermediate int4:

1. Synthesis of int4-2

Dissolve compound int4-1 (10 g, 76.8 mmol) in 80 mL of N,N-dimethylformamide, then add imidazole (15.7 g, 230.4 mmol), cool and stir under ice bath conditions. Slowly add TBDPSCl (23.2 g, 84.5 mmol), then warm the reaction solution to room temperature and stir overnight. TLC control shows that the raw material reacts completely. Add water to the system, extract with ethyl acetate, dry and concentrate the organic phase, and purify by column chromatography to obtain 24.12 g of compound int4-2. Ms[M+H] ⁺ 369.1

2. Synthesis of int4-3

Under nitrogen protection, n-BuLi (52 mL, 2.5 mol/L) and THF (70 mL) were added to the flask, and the mixture was stirred in a dry ice bath to cool down. Acetonitrile (5.3 g, 128.4 mmol) was added dropwise while controlling the internal temperature not to exceed -55°C. After the addition was complete, the mixture was stirred for 1 hour. Then a solution of compound int4-2 (23.62 g, 64.2 mmol) in THF (47 mL) was added dropwise. After the addition was complete, the mixture was stirred at -78°C for 2 hours.

TLC control showed that the raw material was completely reacted. Water was added to the system, extracted with ethyl acetate, and the organic phase was dried and concentrated to obtain 28.66 g of compound int4-3, which was used directly in the next step.

3. Synthesis of compound int4-4

Add tert-butylhydrazine hydrochloride (14.2 g, 114 mmol) and 143 mL of ethanol to the flask, then add triethylamine (11.52 g, 114 mmol) and stir at room temperature for 30 minutes. Add a solution of compound int4-23 (28.66 g, 76 mmol) in ethanol (143 mL) and heat to 75°C to react overnight.

The reaction of the raw material was complete according to TLC control. The system was concentrated until no liquid was distilled out, water was added for stirring, ethyl acetate was extracted, dried, concentrated, and purified by column chromatography to obtain 28.77 g of compound int4-4.

4. Synthesis of compound int4-5

Under nitrogen protection, compound int4-24 (24.27 g, 54.3 mmol) was dissolved in 170 mL of acetonitrile and cooled with stirring in an ice bath. Cbz-cl (18.5 g, 108.6 mmol) was slowly added dropwise, and stirred at room temperature for 1 to 2 hours after the addition was complete. Sodium bicarbonate (14.6 g, 174 mmol) was added, and the temperature was slowly raised to room temperature and stirred overnight.

The reaction of the raw material was complete according to TLC control. The system was filtered, the filtrate was concentrated until no liquid was distilled out, water was added and stirred, ethyl acetate was extracted, dried and concentrated, and purified by column chromatography to obtain 24.88 g of compound int4-5.

5. Synthesis of compound int4-6

Dissolve compound int4-5 (8.28 g, 14.2 mmol) in 82.8 mL of methanol, and cool under ice bath with stirring. Slowly add concentrated hydrochloric acid (1.7 mL) dropwise, and stir at room temperature overnight after the addition is complete.

TLC control showed that the raw material was completely reacted. Sodium bicarbonate solution was added to the system to adjust the pH to alkaline, and the excess methanol was removed under reduced pressure. Ethyl acetate was extracted, dried, concentrated, and purified by column chromatography to obtain 3.57 g of compound int4-6. Ms[M+H] ⁺ 344.2

6. Synthesis of compound int4-7

Dissolve compound int4-6 (3 g, 8.74 mmol) and p-nitrophenyl chloroformate (2.64 g, 13.11 mmol) in 60 mL of dichloromethane, slowly add pyridine (2.07 g, 26.22 mmol) and DMAP (0.11 g, 0.874 mmol), and react at room temperature for 5 hours.

TLC control, the raw material reaction is complete. Add water to the system and wash the liquid. Add saturated sodium chloride solution to the organic phase and wash the liquid 5 times. The organic phase is concentrated and purified by column chromatography to obtain 4.16g of compound int4-7. Ms[M+H] ⁺ 509.3

7. Synthesis of compound int4-8

Dissolve compound int4-7 (4.16 g, 8.2 mmol) and isopropylamine (726 mg, 12.3 mmol) in 83 mL of tetrahydrofuran, add DI ethyl acetate (3.2 g, 24.6 mmol), and react at room temperature for 5 hours.

TLC control, the raw material reaction is complete. Add water to the system, extract the liquid with ethyl acetate. Add saturated sodium chloride solution to the organic phase and wash the liquid. The organic phase is concentrated and purified by column chromatography to obtain 2.67g of compound int4-8. Ms[M+H] ⁺ 429.3

8. Synthesis of compound int4

Compound int4-8 (2.67 g, 6.2 mmol) was dissolved in 26 mL of methanol/26 mL of ethyl acetate, palladium carbon (0.534 g) was added, and the mixture was stirred overnight under hydrogen atmosphere.

TLC detection showed that the raw material was completely reacted. The filtrate was filtered, concentrated under reduced pressure, and purified by column chromatography to obtain 1.85 g of compound int4.

Synthesis of intermediate int5:

1. Synthesis of int5-2

In a 100mL single-necked flask, first add 25mL of water, add int5-1 (4.6g, 1.0eq) while stirring, and slowly add bromine (1.64mL, 1.0eq) after the system is evenly mixed. After the addition is complete, stir and react at room temperature for 2h. Cool to 0°C, slowly add saturated sodium bicarbonate aqueous solution, adjust the pH value to greater than 7, then add ethyl acetate (100X3mL) for extraction, separate the liquids, dry over anhydrous sodium sulfate, filter and concentrate, and then separate and purify by column chromatography to obtain int5-2 (5.6g).

2. Synthesis of int5-3

In a 250mL single-necked flask, add dry tetrahydrofuran (70mL) dissolved with int5-2 (5.6g, 1.0eq), then add diisopropylethylamine (7.7g, 2.5eq) and SEMCl (8.01g, 2.0eq) under ice bath conditions, and naturally warm to room temperature and stir for 16h. After the reaction is completed, extract with ethyl acetate (3*30mL), combine the organic phases, wash the organic phases with saturated brine (30mL), dry over anhydrous sodium sulfate, concentrate in vacuo, and separate and purify by silica gel column chromatography to obtain the product int5-3 (8.6g).

3. Synthesis of int5-4

In a 250mL three-necked flask, int5-3 (8.6g, 1.2eq), Pd(OAc) ₂ (442mg, 0.1eq), XantPhos (2.28g, 0.2eq), KOAc (193mg, 0.1eq) were added in sequence to replace N ₂ , and tetrahydrofuran (40mL*2) and diisopropylethylamine (4.5g, 1.8eq) were added in sequence through a syringe. The reaction solution was placed in a 70℃ oil bath and stirred for 20min, then dimethylphosphine oxide (1.54g, 1.0eq) was added, and the stirring was continued at 70℃ for 10min, and the temperature was raised to 100℃ and stirred overnight. After the reaction is completed, the solid in the reaction solution is filtered out with a triangular funnel, and the solid is washed with ethyl acetate 2-3 times. The reaction solution is vacuum-dried and then water (30 mL) and ethyl acetate (3*20 mL) are added and extracted 3 times. The organic phases are combined, washed with NaCl(aq) (3*30 mL), dried over Na ₂ SO ₄ , concentrated in vacuo, and separated and purified by silica gel column chromatography to obtain the product int5-4 (2 g).

4. Synthesis of int5-5

In a 100mL single-necked flask, int5-4 (2g, 1.0eq) was added, and then dichloromethane (3mL) was added to stir and dissolve, and TFA (3mL) was added at room temperature to react for 2 hours. After the reaction was completed, the reaction solution was removed by vacuum rotary evaporator to remove dichloromethane and trifluoroacetic acid, and dichloromethane was added to rotary evaporate the remaining TFA, and the reaction was repeated 3 times, and the product int5-5 (1.2g) was obtained by vacuum drying with an oil pump.

5. Synthesis of int5-6

In a 25 mL microwave tube, int5-5 (1.2 g) was added, and then aminoacetaldehyde dimethyl acetal (12 mL) was added, and the reaction was stirred overnight at 110° C. After the reaction was completed, the reaction solution was rotary evaporated using an oil pump to remove aminoacetaldehyde dimethyl acetal, and the crude product int5-6 (1.4 g) was obtained, which can be directly used in the next step without purification.

6. Synthesis of int5-7

In a 25mL microwave tube, an acetone solution (15mL) containing int5-7 (1.4g, 1eq) was added, and then an aqueous solution (1.5mL) containing TsOH·H2O (1.25g, 1.5eq) was added, and the mixture was reacted at 70°C and stirred overnight. On the second day, the reaction solution was rotary evaporated using an oil pump to remove acetone and water, and then silica gel was directly added to mix the sample, and the crude product was separated and purified by silica gel column chromatography to obtain the product int5-7 (1.0g).

7. Synthesis of int5-8

In a 50 mL single-mouth bottle, int5-7 (1.0 g) was added, and then 15 mL of trifluoroacetic acid was added, and stirred at 80°C for 48 h. After the reaction was completed, the reaction solution was concentrated, and a saturated sodium bicarbonate aqueous solution was added to adjust the pH to > 7, and then the aqueous phase was spin-dried and methanol was added for washing, filtered, and the filtrate was collected and concentrated, and silica gel was added to mix the sample, and the crude product was separated and purified by silica gel column chromatography to obtain the product int5-8 (810 mg).

8. Synthesis of int5

Int5-8 (810 mg, 1.0 eq) was added to a 25 mL microwave tube, and then 10 mL of phosphorus oxychloride and N,N-dimethylformamide (22 mg, 0.1 eq) were added. After reacting at 110°C for 40 minutes, the reaction solution was concentrated and the crude product was separated and purified by silica gel column chromatography to obtain the product int5 (630 mg).

Synthesis of compounds:

The structural formula marked with an "&" symbol indicates that the compound is a cis racemate, the structural formula marked with an "or" symbol indicates that the compound is a single-configuration compound but the absolute configuration is unknown, the structural formula marked with an "abs" symbol indicates that the absolute configuration of the compound is the structure shown, and the structural formula marked with a "rac" symbol indicates that the compound is a racemate.

Examples 1 and 2

Compounds of the present invention

The synthetic route and experimental process are as follows:

1. Synthesis of C1-1

In a 25mL single-mouth bottle, int1 (117mg, 1.0eq) was added, followed by 2mL N,N-dimethylformamide, and potassium tert-butoxide (85mg, 2.0eq) was added at 0°C. After stirring for 10 minutes, a solution of int2 (87mg, 1.0eq) in N,N-dimethylformamide (1.0mL) was added dropwise, and the mixture was reacted at room temperature for 30 minutes. Water was added to the reaction solution, and ethyl acetate was added for extraction twice. The ethyl acetate phase was dried and concentrated, and purified by Prep-TLC to obtain C1-1 (25mg). Ms[M+H] ⁺ :502.5.

2. Synthesis of C1-2

Add 25 mg of C1-1 to a 25 mL single-mouth bottle, then add 3 ml of formic acid, and stir at 80 ° C for 30 minutes. After concentrating the reaction solution, add saturated sodium bicarbonate aqueous solution to the reaction system to adjust the pH value to greater than 7, then add ethyl acetate to extract 3 times, dry and concentrate the ethyl acetate phase, and purify by Prep-TLC to obtain 15 mg of C1-2. The analysis method is: chromatographic column: phenomenex Lux○R5um Cellulose-1 (250X30min), mobile phase: n-hexane: ethanol = 7:3; running time: 13min; flow rate: 30mL/min. Prepared by chiral

C1 (3.2 mg), peak time 11.59 min. Ms [M+H] ⁺ : 446.4, ¹ H NMR (400MHz, DMSO-d6) δ8.30-8.00(m,1H),7.67-7.30(s,1H),6.98(d,J＝8.0Hz,1H),6.71-6.29(m,1H),6.42(s,1H),5.10-4.94(m,1H),3.60-3.55(m,1H) ),3.15-3.04(m,1H),2.10-2.00(m,1H),1.98-1.89(m,2H),1.86(s,3H),1.83(s,3H),1.80-1.69(m,2H),1.69-1.56(m,1H),1.08-1.03(m,3H),1. 03-0.97(m,3H);

C2 (4.4 mg), peak time 9.40 min. Ms [M+H] ⁺ : 446.4, ¹ H NMR (400MHz, DMSO-d6) δ8.34-8.00(m,1H),7.67-7.32(m,1H),6.98(d,J＝8.0Hz,1H),6.73-6.30(m,1H),6.43(s,1H),5.10-4.93(m,1H),3.61-3.54(m,1H) ),3.15-3.04(m,1H),2.11-1.99(m,1H),1.94-1.88(m,2H),1.86(s,3H),1.83(s,3H),1.79-1.70(m,2H),1.70-1.58(m,1H),1.09-1.03(m,3H),1. 03-0.96(m,3H).

The above synthesis method was used to synthesize the compounds in the following table:

Examples 27 and 28

Compounds of the present invention

The synthetic route and experimental process are as follows:

Synthesis of C27-1

In a 50mL single-mouth bottle, int3 (100mg, 1eq), int1 (108mg, 1.2eq), Cs ₂ CO ₃ (285mg, 3eq), Xantphos (34mg, 0.2eq), Pd(OAc) ₂ (7mg, 0.1eq) and 1,4-dioxane (15mL) were added. The temperature was raised to 100°C in a nitrogen atmosphere and the reaction was continued for 16h. The liquid chromatography indicated that the reaction was complete. The reaction solution was cooled, filtered, washed with ACN three times, and the organic phase was concentrated. 80mg of the product was obtained by TLC purification. Ms[M+H] ⁺ 501.

Synthesis of C27-2

C27-1 (80 mg, 1 eq) and formic acid (6 mL) were added to a 25 mL single-mouth bottle. The temperature was raised to 80 °C and the reaction was continued for 16 h. The liquid chromatography indicated that the reaction was complete. The reaction solution was cooled and concentrated. The solution was dissolved in ethyl acetate, the pH was adjusted to neutral with saturated sodium bicarbonate aqueous solution, and the solution was extracted with ethyl acetate three times. The organic phase was dried over anhydrous sodium sulfate and concentrated. The product was purified by TLC to obtain 40 mg. Ms[M+H] ⁺ 445.

Synthesis of C27 and C28

C27-2 (40 mg) was separated and prepared by chiral high performance liquid chromatography to give compounds C27 and C28, respectively.

The separation conditions are as follows:

Instrument: Agilent 1260 InfinityⅡ

Column: Phenomenex 5μm Cellulose-4 (250X 30mm)

Mobile phase: n-hexane: ethanol = 30:70

Flow rate: 15mL/min, detection wavelength: 254nm

Column temperature: 25:, solvent: isopropanol

Injection concentration: 10 mg/mL, injection volume: 400 μL

Running time: 20 minutes

C28 peak time: 14.05min, 13mg, ¹ HNMR (400MHz, Chloroform-d) δ8.10 (s, 1H), 8.00 (d, J = 6.8Hz, 1H), 7.69-7.62 (m, 1H), 7.04 (d,J＝7.6Hz,1H),6.77(t,J＝7.2Hz,1H),5.98(s,1H),5.18(s,1H),4.89(s,1H),3.78(s,1H) ,3.20(p,J＝8.4Hz,1H),2.59-2.44(m,1H),2.14(s,1H),2.00-1.66(m,10H),1.12(d,J＝6.8Hz,6H). Ms[M+H] ⁺ 445.

C27 peak time: 15.52min, 6mg, ¹ HNMR (400MHz, Chloroform-d) δ8.05-7.92 (m, 2H), 7.64 (s, 1H), 7.06 (d, J = 7.6Hz, 1H), 6.77 (t,J＝7.2Hz,1H),5.98(s,1H),5.19(s,1H),4.84(s,1H),3.79(s,1H),3.20(p,J＝8.4Hz,1H) ,2.58-2.48(m,1H),2.14(s,1H),2.00-1.70(m,10H),1.12(t,J＝6.8Hz,6H).Ms[M+H] ⁺ 445.

The above synthesis method was used to synthesize the compounds in the following table:

Examples 33 and 34

Compounds of the present invention

The synthetic route and experimental process are as follows:

1. Synthesis of C33-1

In a 25mL single-mouth bottle, int1 (119mg, 1.0eq) was added, followed by 2mL of N,N-dimethylformamide, and potassium tert-butoxide (96mg, 2.0eq) was added at 0 methyl, and after stirring for 10 minutes, a solution of int5 (100mg, 1.0eq) in N,N-dimethylformamide (1.0mL) was added dropwise, and the mixture was reacted at room temperature for 30 minutes. Water was added to the reaction solution, and ethyl acetate was added to extract twice, the ethyl acetate phase was dried and concentrated, and Prep-TLC was used to purify C33-1 (84mg).

2. Synthesis of C33-2

C33-1 (79 mg) was added to a 25 mL single-mouth bottle, followed by 3 ml formic acid, and the mixture was stirred at 80°C for 30 minutes. After the reaction solution was concentrated, a saturated sodium bicarbonate aqueous solution was added to the reaction system to adjust the pH value to greater than 7, and then ethyl acetate was added for extraction 3 times. The ethyl acetate phase was dried and concentrated to prepare and purify 42 mg of the product. Ms[M+H] ⁺ 460.2.

3. Synthesis of C33 and C34

The following chiral preparation method was used to chirally separate C33-2:

Instrument: Agilent 1260 InfinityⅡ

Chromatographic column: phenomenex Lux ^○R 5um Cellulose-1 (250X30min)

Solvent: Isopropyl alcohol

Mobile phase: n-hexane: ethanol = 7:3

Run time: 13 minutes

Flow rate: 30mL/min

Wavelength: 254nm

Column temperature: 25°C

Injection volume: 750 μL

Concentration: 8mg/mL

C33 peak time: 11.12min, 15.2mg, Ms[M+H] ⁺ 460.2

C34 peak time: 9.10min, 15.3mg, Ms[M+H] ⁺ 460.2

The above synthesis method was used to synthesize the compounds in the following table:

Examples 35 and 36

Compounds of the present invention

The synthetic route and experimental process are as follows:

1. Synthesis of C35-2

C35-1 (2.26 g, 10.3 mmol) was added to a 100 ml single-necked flask, and 45 ml of acetonitrile was added for dissolution and stirring, and NCS (1.65 g, 12.4 mmol) was added, and the temperature was raised to 70°C and stirred for 14 h. After the reaction was completed, the product was concentrated under reduced pressure and purified to obtain 2.59 g of the product, Ms[M+H] ⁺ 253.

2. Synthesis of C35-3

C35-2 (2.59 g, 10.2 mmol) was added to a 100 ml single-necked flask, followed by 45 ml of THF/MeOH/H ₂ O (v: 1:1:1) and NaOH (2.05 g, 51 mmol), and stirred at room temperature for 1 hour. After the reaction was completed, the mixture was concentrated under reduced pressure, extracted with water and ethyl acetate, and purified to obtain 2.35 g of the product, Ms[M+H] ⁺ 225.

3. Synthesis of C35-4

C35-3 (2.35 g, 10.4 mmol) was added to a 100 ml single-necked flask, and 40 ml of N,N-dimethylformamide was dissolved, and then HATU (5.95 g, 15.6 mmol), aminoacetaldehyde dimethyl acetal (1.32 g, 12.5 mmol), and diisopropylethylamine (4.04 g, 31.3 mmol) were added, and then stirred at room temperature for 1 hour. After the reaction was completed, water was added to quench the reaction, and the organic phase was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 2.99 g of crude product, Ms[M+H] ⁺ 312.

4. Synthesis of C35-5

C35-4 (2.99 g, 9.6 mmol), p-TSA (2.47 g, 14.4 mmol) were added to a 100 ml single-necked flask, and acetone/water (v: 10:1) was added, the N ₂ was replaced, the temperature was raised to 70°C, and stirred for 12 h. After the reaction was completed, the mixture was concentrated under reduced pressure, and ethyl acetate/dichloromethane was added to slurry to obtain 1.6 g of crude product, Ms[M+H] ⁺ 266.

5. Synthesis of C35-6

C35-5 (1.6 g, 6.0 mmol) was added to a 100 ml single-necked flask, and 20 ml of trifluoroacetic acid was added, and the mixture was stirred for 3 h with 80 ethanol. After the reaction was completed, the mixture was concentrated under reduced pressure and purified to obtain 724 mg of the product, Ms[M+H] ⁺ 248.

6. Synthesis of C35-7

C35-6 (716 mg, 2.9 mmol) was added to a 100 ml single-necked flask, 20 ml POCl ₃ and 1 ml N,N-dimethylformamide, and the mixture was heated to 80 °C and stirred for 2 h. After the reaction was completed, the mixture was concentrated under reduced pressure and purified to obtain 560 mg of the product, Ms[M+H] ⁺ 266.

7. Synthesis of C35-8

In a 50ml single-necked flask, int1 (418mg, 1.36mmol) was added, 10ml DMSO was added, stirred evenly, cooled to 0, NaH (108mg, 2.72mmol) was added, stirred evenly, C35-7 (360mg, 1.36mmol) was added, warmed to room temperature, and stirred for 1h. After the reaction was completed, the temperature was lowered, ethyl acetate was added to dilute the organic phase, the reaction was quenched with water, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified to obtain 176mg of the product, Ms[M+H] ⁺ 538.

8. Synthesis of C35-9

C35-8 (165 mg, 0.31 mmol), Pd(OAc) ₂ (27 mg, 0.12 mmol), Xantphos (142 mg, 0.25 mmol), 5 ml Dioxane, and diisopropylethylamine (118 mg, 0.92 mmol) were added to a 10 ml single-necked flask. After uniform stirring, the mixture was moved to 100 °C and stirred for 0.5 h. A solution of dimethylphosphine oxide (48 mg, 0.61 mmol) in Dioxane was added dropwise and stirred for 2 h. After the reaction was completed, the mixture was concentrated under reduced pressure and purified to obtain 146 mg of the product, Ms[M+H] ⁺ 536.

9. Synthesis of C35-10

C35-9 (146 mg, 0.27 mmol) was added to a 50 ml single-necked flask, and 10 ml of HCO ₂ H solution was added, and the mixture was moved to 80°C and stirred overnight. After the reaction was completed, the mixture was concentrated under reduced pressure and purified to obtain 111 mg of the product, Ms[M+H] ⁺ 480.

10. Synthesis of C35 and C36

Compound C35-10 was separated and prepared by chiral high performance liquid chromatography to obtain the following compounds:

C35: Peak time: 13.01 min, 32.6 mg, Ms[M+H] ⁺ 480, ¹ H NMR (400 MHz, DMSO-d ₆ )δ8.34-8.00(m,1H),7.67-7.32(m,1H),6.98(d,J＝8.0Hz,1H),6.73-6.30(m,1H),5.10-4.93(m,1H),3.61-3.54(m,1H),3.15-3.04(m,1H),2.11-1 .99(m,1H),1.94-1.88(m,1H),1.86(s,3H),1.83(s,3H),1.79-1.70(m,2H),1.70-1.58(m,1H),1.09-1.03(m,3H),1.03-0.96(m,3H).

C36: Peak time: 20.58 min, 29 mg, Ms [M+H] ⁺ 480, ¹ H NMR (400 MHz, DMSO-d ₆ )δ8.30-8.00(m,1H),7.67-7.30(s,1H),6.98(d,J＝8.0Hz,1H),6.71-6.29(m,1H),5.10-4.94(m,1H),3.60-3.55(m,1H),3.15-3.04(m,1H),2.10-2 .00(m,1H),1.98-1.89(m,1H),1.86(s,3H),1.83(s,3H),1.80-1.69(m,2H),1.69-1.56(m,1H),1.08-1.03(m,3H),1.03-0.97(m,3H).

Examples 37 and 38

Compounds of the present invention

The synthetic route and experimental process are as follows:

1. Synthesis of compound C37-2

Compound C37-1 (357 mg, 1.16 mmol) was dissolved in DMSO (15 mL), potassium tert-butoxide (289 mg, 2.58 mmol) was added under ice bath conditions, and a mixture of int1 (300 mg, 1.29 mmol) dissolved in DMSO (15 mL) was added dropwise to the reaction system under stirring. Then the mixture was heated to room temperature and reacted for 1.5 hours. Liquid chromatography showed that the raw materials reacted completely. Under ice bath conditions, water was added to the system to quench the reaction, and the liquid was separated by ethyl acetate extraction; the organic phase was then washed with saturated sodium chloride solution, and the organic phase was concentrated in vacuo without liquid distillation. The product was purified by column chromatography to obtain 300 mg. Ms[M+H] ⁺ 504.2

2. Synthesis of compound C37-3

Under nitrogen protection, compound C37-2 (150 mg, 0.30 mmol) was dissolved in 1,4-dioxane (11 mL), and then palladium acetate (27 mg, 0.12 mmol), Xantphos (139 mg, 0.24 mmol), and diisopropylethylamine (116 mg, 0.90 mmol) were added. After addition, the temperature was raised to 100 ° C and the reaction was continued for 0.5 hours. Then 11 mL of 1,4-dioxane solution of dimethylphosphine (46.8 mg, 0.6 mmol) was added, and the reaction was continued at 100 ° C for 2 hours. Liquid chromatography showed that the raw material reacted completely. The reaction system was vacuum concentrated until no liquid flowed out, and 80 mg of the product was prepared and purified. Ms[M+H] ⁺ 502

3. Synthesis of C37-4

Dissolve compound C37-3 (80 mg, 0.16 mmol) in formic acid (10 mL). React at 80°C for 1 to 4 hours. The liquid mass shows a product ion peak, and the system is vacuum concentrated until no liquid is distilled out. Add water to the system, adjust the pH to weak alkalinity with sodium bicarbonate solution under ice bath, and extract with ethyl acetate. After the organic phase is concentrated, 90 mg of the product is prepared.

4. Synthesis of C37 and C38

C37-4 was isolated using chiral preparative chromatography:

C38: 8.5mg, Ms[M+H] ⁺ : 446.2

C37: 9.4mg, Ms[M+H] ⁺ : 446.2

Examples 39 and 40

Compounds of the present invention

The synthetic route and experimental process are as follows:

1. Synthesis of C39-2

C39-1 (5.00 g, 26.4 mmol) was added to a 100 ml single-necked flask, and 40 ml of N,N-dimethylformamide was dissolved, and then HATU (15.08 g, 39.5 mmol), aminoacetaldehyde dimethyl acetal (3.31 g, 31.6 mmol), and diisopropylethylamine (10.18 g, 40.3 mmol) were added, and then stirred at room temperature for 1 hour. After the reaction was completed, water was added to quench the reaction, and the organic phase was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 5.58 g of crude product, Ms[M+H] ⁺ 277.

2. Synthesis of C39-3

C39-2 (4.00 g, 14.5 mmol), p-TSA (3.74 g, 21.7 mmol) were added to a 100 ml single-necked flask, and acetone/water (v: 10:1) was added. The atmosphere was replaced with _N2 , and the temperature was raised to 70°C, and stirred for 12 h. After the reaction was completed, the mixture was concentrated under reduced pressure and purified to obtain 4.18 g of crude product, Ms[M+H] ⁺ 230.

3. Synthesis of C39-4

C39-3 (4.18 g, 18.2 mmol) was added to a 100 ml single-necked flask, and 20 ml of trifluoroacetic acid was added, and the mixture was stirred for 3 h with 80 ethanol. After the reaction was completed, the mixture was concentrated under reduced pressure and purified to obtain 765 mg of the product, Ms[M+H] ⁺ 213.

4. Synthesis of C39-5

C39-4 (700 mg, 3.3 mmol) was added to a 100 ml single-necked flask, 8 ml POCl ₃ and 30 ml MeCN were added, the temperature was raised to 80°C and stirred for 2 h. After the reaction was completed, the mixture was concentrated under reduced pressure and purified to obtain 450 mg of the product, Ms[M+H] ⁺ 231.

5. Synthesis of C39-6

In a 50ml single-necked flask, int1 (401mg, 1.3mmol) was added, 10ml DMSO was added, stirred evenly, cooled to 0, ^t BuOK (438mg, 3.9mmol) was added, stirred evenly, C39-5 (300mg, 1.3mmol) was added, the temperature was raised to room temperature, and stirred for 1h. After the reaction was completed, the temperature was lowered, ethyl acetate was added to dilute the organic phase, the reaction was quenched with water, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified to obtain 217mg of the product, Ms[M+H] ⁺ 502.

6. Synthesis of C39-7

C39-6 (200 mg, 0.40 mmol), Pd(OAc) ₂ (36 mg, 0.16 mmol), Xantphos (184 mg, 0.32 mmol), 10 ml Dioxane, diisopropylethylamine (154 mg, 1.19 mmol) were added to a 10 ml single-necked flask, stirred evenly, then stirred at 100 °C for 0.5 h, and a solution of dimethylphosphine (62 mg, 0.80 mmol) in Dioxane was added dropwise, and stirred for 2 h. After the reaction was completed, the mixture was concentrated under reduced pressure and purified to obtain 159 mg of the product, Ms[M+H] ⁺ 501.

7. Synthesis of C39-8

C39-7 (159 mg, 0.32 mmol) was added to a 50 ml single-necked flask, and 10 ml of formic acid solution was added, and the mixture was stirred overnight at 80 °C. After the reaction was completed, the mixture was concentrated under reduced pressure and purified to obtain 100 mg of the product, Ms[M+H] ⁺ 445.

8. Synthesis of C39 and C40

Compound C39-8 was separated and prepared by chiral high performance liquid chromatography to obtain compounds

Instrument: Agilent 1260Ⅱ

Column: Phenomenex 5μm Cellulose-4 (250X 21.2mm)

Mobile phase: n-hexane: ethanol (0.1% diethylamine) = 30:70

Flow rate: 15 mL/min, detection wavelength: 254 nm

Column temperature: 25°C, solvent: isopropanol: 50% acetonitrile water = 2:1

Injection concentration: 7 mg/mL, injection volume: 500 μL

Run time: 25 minutes

C39: Peak time: 13.41 min, 20 mg, Ms [M + H] ⁺ 445, ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.85-9.62 (m, 1H), 7.90-7.84 (m, 1H), 7.75-7.66 (m, 1H), 755-7.43 (m, 1H), 7.18-7.09 (m, 1H), 6.95 (m, J = 8.0 Hz, 1H), 6.58-6.40 (m, 1H), 5.02-4.97 (m, 1H), 3.59-3.56 (m, 1H), 3.08-3.04(m,1H),2.67-2.58(m,1H),2.08-1.97(m,1H),1.93-1.88(1,H),1.77-1.72(m,2H),1.68(s,3H),1.64(s,3H),1.24-1.22(m,2H),1.04 -1.03(m,3H),1.02-1.01(m,3H).

C40: Peak time: 22.36 min, 20 mg, Ms [M + H] ⁺ 445, ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.85-9.61 (m, 1H), 7.91-7.84 (m, 1H), 7.75-7.66 (m, 1H), 7.54-7.43 (m, 1H), 7.18-7.09 (m, 1H), 6.95 (d, J = 8.0 Hz, 1H), 6.58-6.40 (m, 1H), 5.02-4.97 (m, 1H), 3.59-3.56 (m, 1H), 3.08-3.04(m,1H),2.67-2.59(m,1H),2.06-2.00(m,1H),1.93-1.88(m,1H),1.77-1.71(m,2H),1.68(s,3H),1.64(s,3H),1.24-1.22(m,2H),1.0 4-1.03(m,3H),1.02-1.01(m,3H).

Cell proliferation inhibition assay

1. Experimental Materials

Cell lines supplier OVCAR3 Beina Biotechnology TOV21G Beina Biotechnology

Comparative compound Palbociclib

2. Experimental plan

1. Cell plating

a) Preparation of cell suspension

b) removing the culture medium from the culture flask;

c) Rinse the cells once with PBS;

d) digest with trypsin and collect by centrifugation;

e) Resuspend in culture medium, count and adjust to appropriate concentration.

f) Add the cell suspension to each well of a 96-well plate at a volume of 80uL, and place one plate for each cell type. Culture overnight in a 37°C, 5% CO ₂ incubator.

2. Compound treatment

Compound dilution

a) Prepare gradient dilution solutions of the test compounds: Palbociclib and the example compounds were stored at 10 mM. Then 2.5 uL of the stock solution was dissolved in 497.5 uL of DMSO-free culture medium, and then 3-fold continuous gradient dilutions were performed with 0.1% DMSO culture medium, for a total of 9 concentrations. The compound concentrations after dilution were as follows:

10000nM, 3333.33nM, 1111.11nM, 370.34nM, 123.45nM, 41.15nM, 13.72nM, 4.57nM, 1.52nM

b) After thorough mixing, 20uL of the compound solution was added to the cell culture plate containing 80uL of cells, with 4 replicate wells for each concentration.

c) Transfer the cells to an incubator and incubate for 5 days.

3. MTT assay

a) Take out the cell culture plate and add 10μL of 5mg/ml MTT into the hood.

b) Place the cell culture plate back into the incubator and continue incubating for 3 hours

c) Take out the cell culture plate and remove the culture medium, add 100uL of isopropanol (0.4mM HCl, 0.4% NP40), shake at room temperature for 30 minutes, and read the plate at 570nm.

4. Data Analysis

The survival rate (%Cell Survival) was calculated using the following formula:

%Cell Survival＝100%×(OD_Sample-OD_LCave)/(OD_HC-OD_

LCave)

OD_HC: 0.1‰ DMSO control group cell reading

OD_Sample: Cell reading after adding compound

OD_LC: Blank culture medium reading

Analyzed by Prizm:Dose-response-Inhibition-Log(inhibitor) vs response(three parameters for the best fit).

The results of the cell proliferation inhibition test are shown in Table 1.

Table 1

A means IC50≤300nM; B means 300nM＜IC50≤1000nM; C means 1000nM＜IC50≤3000nM; D means 3000nM＜IC50.

The experimental results show that: for the CyclinE amplified cell line OVCAR3, the example compounds showed a good inhibitory effect, while for the non-CyclinE amplified cell line TOV21G, the example compounds had no obvious inhibitory effect, showing good selectivity of the compounds.

Enzyme activity test

Prepare the compound:

1. Prepare the test compound and palbociclib into 0.5 nM DMSO solution;

2. Use Echo550 to transfer 20 nL of the stock solution to a 384-well plate. Use DMSO as a blank control.

Experimental steps:

1. Prepare 1.3X enzyme solution containing enzyme, substrate, and coenzyme factor according to the table below;

2. Add 15uL of 1.3x enzyme solution to each well and incubate at room temperature for 30 minutes;

3. Add 5uL of 4x ATP solution to start the reaction. The final volume of each well is 20uL;

4. After incubation at room temperature for 150 min, add 75uL of stop buffer solution to terminate the experiment;

5. Analyze samples using EZ reader.

Data Analysis:

Calculate the % inhibition using the following equation

The positive control (PC) was treated with DMSO

The negative control (NC) was without enzyme.

% inhibition rate = 100-100*((CR _PC -CR _sample )/(CR _PC -CR _NC ))

Table 2

A means IC50≤10nM; B means 10nM＜IC50≤100nM; C means 100nM＜IC50≤500nM; D means 500nM＜IC50.

The data show that the compounds of the present invention have good selectivity and activity against CDK2 kinase.

The present invention provides a series of compounds with excellent CDK2 kinase activity and selectivity, which have good inhibitory effects on cell overproliferation caused by abnormal expression or amplification of CyclineE, especially compounds C3, C10, C12, C22, C24, C28, C33, etc., and therefore have broad application prospects.

In addition, compared with compound C27, compound C28 with (1R,3S) conformation has higher selectivity.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims (10)

1. A compound, characterized in that the compound is a compound of formula (I), or a pharmaceutically acceptable salt or stereoisomer thereof, in: Ring A is selected from the following group: _C5-7 cycloalkyl, 5-7 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, _C5-6 aryl, 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the cycloalkyl, heterocycloalkyl, aryl, heteroaryl are optionally substituted by 0, 1, 2 or 3 _R1 , and Ring A and Ring B form a cyclic structure; Ring B is selected from the group consisting of C _5-6 aryl, 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the aryl and heteroaryl are optionally substituted by 0, 1, 2 or 3 R ₂ ; R ₁ is selected from the following group: hydrogen, deuterium, halogen, hydroxyl, cyano, =O, C _1-4 alkyl, C _{1-4 haloalkyl, 1-4} membered heteroalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, 3-5 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, C _3-5 cycloalkyl, C _2-4 alkenyl, C _2-4 alkynyl; _R2 is selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl, cyano, _C1-4 alkyl, C1-4 haloalkyl, _1-4 membered heteroalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S; R ₃ is NR ₆ R ₇ ; ^Ra is selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl, cyano, _C1-4 alkyl, _C1-4 fluoroalkyl, = _CH2 ; _X1 is selected from the group consisting of _CH2 , O; or _X1 is absent, such that Four-membered cyclobutyl X ₂ is selected from the group consisting of O, -CH ₂ -, -CH ₂ O-; R ₄ and R ₅ are independently selected from the following group: C _1-6 alkyl, C _1-6 haloalkyl, 1-6 membered heteroalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, C _3-5 cycloalkyl, 3-5 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the C _1-6 alkyl, C _1-6 haloalkyl, heteroalkyl, C _3-5 cycloalkyl, 3-5 membered heterocycloalkyl are optionally substituted with one or more substituents selected from the following group: deuterium, hydroxyl, halogen, cyano, =O, C _1-4 alkyl, C _1-4 haloalkyl, C _1-4 alkoxy, C _1-4 haloalkoxy, Alternatively, _R4 and _R5 together with the phosphorus atom to which they are attached form a 4-7 membered heterocycloalkyl group, wherein the heterocycloalkyl group is optionally substituted by 0-3 substituents selected from the group consisting of deuterium, hydroxyl, halogen, cyano, =O, _C1-4 alkyl, _C1-4 haloalkyl, _C1-4 alkoxy, _C1-4 haloalkoxy; R ₆ and R ₇ are independently selected from the following group: hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, -L-(C _3-6 cycloalkyl), -L-(3-6 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S), wherein the C _1-6 alkyl, C _3-6 cycloalkyl, 3-6 membered heterocycloalkyl are optionally substituted by one or more substituents selected from the following group: deuterium, hydroxyl, halogen, cyano, C _1-4 alkyl, C _1-4 alkoxy, halogenated C _1-4 alkyl, halogenated C _1-4 alkoxy, Alternatively, _R6 and _R7 connected to the same N atom are connected to form a 3-7 membered heterocyclic group, wherein the 3-7 membered heterocyclic group is optionally substituted by one or more substituents selected from the group consisting of deuterium, hydroxyl, halogen, cyano, =O, _C1-4 alkyl, _C1-4 alkoxy, halogenated _C1-4 alkyl, halogenated _C1-4 alkoxy; L is a bond or a C _1-6 alkylene group. 2. The compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound has a structure selected from the group consisting of: in, Ring A is selected from the following group: _C5-7 cycloalkyl, 5-7 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, _C5-6 aryl, 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the cycloalkyl, heterocycloalkyl, aryl, heteroaryl are optionally substituted by 0, 1, 2 or 3 _R1 , and Ring A and Ring B form a cyclic structure; Ring B is selected from the group consisting of C _5-6 aryl, 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the aryl and heteroaryl are optionally substituted by 0, 1, 2 or 3 R ₂ ; R ₁ is selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl, cyano, =O, C _1-4 alkyl, C _{1-4 haloalkyl, 1-4} membered heteroalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, 3-5 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, C _3-5 cycloalkyl, C _2-4 alkenyl, C _2-4 alkynyl; _R2 is selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl, cyano, _C1-4 alkyl, C1-4 haloalkyl, _1-4 membered heteroalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S; R ₃ is NR ₆ R ₇ ; R ₄ and R ₅ are independently selected from the following groups: C _1-6 alkyl, C _1-6 haloalkyl, 1-6 membered heteroalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, C _3-5 cycloalkyl, 3-5 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, wherein the C _1-6 alkyl, C _1-6 haloalkyl, heteroalkyl, C _3-5 cycloalkyl, 3-5 membered heterocycloalkyl are optionally substituted with 1-6 substituents selected from the following groups: deuterium, hydroxyl, halogen, cyano, =O, C _1-4 alkyl, C _1-4 haloalkyl, C _1-4 alkoxy, C _1-4 haloalkoxy, Alternatively, _R4 and _R5 together with the phosphorus atom to which they are attached form a 4-7 membered heterocycloalkyl group, wherein the heterocycloalkyl group is optionally substituted by 0-3 substituents selected from the group consisting of deuterium, hydroxyl, halogen, cyano, =O, _C1-4 alkyl, _C1-4 haloalkyl, _C1-4 alkoxy, _C1-4 haloalkoxy; R ₆ and R ₇ are independently selected from the following group: hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, -L-(C _3-6 cycloalkyl), -L-(3-6 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O and S), wherein the C _1-6 alkyl, C _3-6 cycloalkyl and 3-6 membered heterocycloalkyl are optionally substituted by 1 to 6 substituents selected from the following group: deuterium, hydroxyl, halogen, cyano, C _1-4 alkyl, C _1-4 alkoxy, halogenated C _1-4 alkyl and halogenated C _1-4 alkoxy, Alternatively, _R6 and _R7 connected to the same N atom are connected to form a 3-7 membered heterocyclic group, wherein the 3-7 membered heterocyclic group is optionally substituted by 1-6 substituents selected from the group consisting of deuterium, hydroxyl, halogen, cyano, =O, _C1-4 alkyl, _C1-4 alkoxy, halogenated _C1-4 alkyl, halogenated _C1-4 alkoxy; L is a bond or a C _1-6 alkylene group. 3. The compound according to claim 2, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein when R ₄ and R ₅ are different substituents, the compound has a structure selected from the following group: wherein ring A, ring B, R ₃ , R ₄ and R ₅ are as defined in claim 2. 4. The compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the ring A and the ring B are selected from the following group: in: X ₃ and Y are each independently selected from the following group: C, N; G, H, Q, T, W, E, and Z are each independently selected from the group consisting of C, CH, N, NH, O, and S; Ring A and Ring B are optionally substituted by R ₁ or R ₂ ; R ₁ and R ₂ are as described in claim 1. 5. The compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the A ring and the B ring have a structure selected from the group consisting of: 6. The compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein _R3 is selected from the group consisting of: 7. The compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound is selected from the group consisting of: 8. A pharmaceutical composition, characterized in that it comprises a therapeutically effective amount of one or more compounds according to claim 1 and a pharmaceutically acceptable carrier. 9. Use of the compound according to claim 1, characterized in that it is used for preparing a drug for regulating CDK2 kinase activity or treating CDK2-related diseases. 10. The use according to claim 9, characterized in that the disease is a cancer or tumor selected from the group consisting of breast cancer, ovarian cancer, bladder cancer, uterine cancer, lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, thyroid cancer, esophageal cancer, kidney cancer, liver cancer, head and neck cancer, glioblastoma, mantle cell lymphoma (MCL), chronic myeloid leukemia (CML) and acute myeloid leukemia (AML).