CN117285590A - Compound, preparation method thereof, composition comprising compound and application of compound - Google Patents

Abstract

The application provides a compound, a preparation method thereof, a composition comprising the compound and application thereof. The compound has the formula IStructures are shown, M-L-E formula I wherein: m represents KRAS ^G12C Ligand of protein, L represents a connecting chain, E represents ligand of E3 ubiquitin ligase. The compound provided by the application shows good anti-tumor activity and excellent KRASG12C protein degradation, can be used for preventing and treating various cancers, and has a huge application prospect in the field of medicines.

Description

A compound, a preparation method thereof, a composition comprising the same and its application

Technical Field

The present application relates to the field of medicine, and specifically to a compound targeting KRASG12C protein degradation, its pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph, a method for preparing the compound, a pharmaceutical composition comprising the compound, and the application of the compound.

Background Art

The full name of the KRAS gene is Kirsten ratsarcoma viral oncogene homolog. The protein encoded by this gene is a small GTPase, which belongs to the RAS superprotein family. There are three genes in the Ras gene family related to human tumors - HRAS, KRAS and NRAS, which are located on chromosomes 11, 12 and 1 respectively. Among them, KRAS mutation is the most common, accounting for about 80% of RAS mutations. Among the KRAS gene mutations, the mutations located at codon 12 account for a large proportion, including G12V, G12C, G12D, etc. In lung adenocarcinoma, G12 mutations account for 85% of KRAS gene mutations, of which G12C accounts for as high as 46%. When the 12th codon in the KRAS gene mutates from glycine to cysteine, it will affect the binding of GAP to KRAS, thereby inhibiting the hydrolysis of GTP bound to KRAS. As GTP continues to accumulate, KRAS binds to GTP more easily, thereby activating more KRAS proteins, ultimately abnormally activating multiple downstream signaling pathways, stimulating cells to continue to grow and proliferate, and causing the occurrence and development of tumors.

AMG510 and MRTX849 have been launched for KRASG12C mutations and have achieved good results in clinical applications, breaking the bottleneck of KRAS protein being undruggable. However, continuous administration of inhibitors will inevitably lead to acquired drug resistance.

In recent years, PROTAC has attracted widespread attention. As a new type of targeted protein degradation (TPD), PROTAC is a type of dual-target chimeric molecule consisting of three parts: a ligand that recruits E3 ubiquitin ligase, a ligand that targets the target protein (POI), and a linker between the two. PROTAC forms a stable ternary complex with the target protein and E3 ubiquitin ligase, leading to polyubiquitination of POI, which is subsequently degraded by the 26S proteasome, thereby regulating the level of proteins overexpressed in cells due to various abnormal signals, thereby achieving the effect of inhibiting tumor growth. Compared with traditional "occupancy-driven" small molecule drugs and macromolecule drugs, PROTAC has significant advantages, mainly including the following aspects: (1) PROTAC molecules can target targets that are traditionally considered "undruggable", expanding the space of drug targets; (2) PROTAC molecules can be recycled during the degradation of target proteins, with longer-lasting pharmacological activity, low doses, and reduced drug toxicity; (3) PROTAC molecules can effectively catalyze the complete degradation of multiple mutant target proteins, have the characteristics of high activity and high selectivity, and can overcome drug resistance caused by target protein mutations. Based on the above advantages, PROTAC has become one of the important development directions of drug research and development. Therefore, PROTAC drugs can effectively solve the problem of drug resistance of KRAS inhibitors. KRAS-PROTAC drug development will create a new direction for KRAS drug development, and the problem of KRAS undruggability and drug resistance of inhibitors in clinical applications is expected to be solved.

Summary of the invention

The present application provides a compound and a preparation method and application thereof. The compound not only has excellent KRASG12C protein degradation effect and anti-tumor activity, but also has little toxic side effects on the human body, and can be used to prepare anti-tumor drugs.

The first aspect of the present application provides a compound having a structure shown in Formula I, a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph thereof,

M-L-E

Formula I

Wherein: M represents the ligand of KRAS ^G12C protein, L represents the linker chain, and E represents the ligand of E3 ubiquitin ligase.

In any embodiment of the first aspect, the above-mentioned M is a structure shown in Formula II:

wherein ^X1 and ^X2 are each independently halogen, and the number of ^X1 is 1 or 2, and the number of ^X2 is 1, 2, 3 or 4, preferably the number of ^X1 and ^X2 is both 1;

^R1 and ^R2 are each independently a substituted or unsubstituted C1-C6 alkyl group, the number of ^R2 is 1, 2 or 3, preferably the number of ^R2 is 1; preferably ^R1 and ^R2 are each independently a substituted or unsubstituted C1-C3 alkyl group, preferably ^R1 is n-propyl or isopropyl, preferably ^R2 is methyl or ethyl;

Preferably, M is

In any embodiment of the first aspect, the above L is The terminal is connected to M. Connect the end to E.

Wherein, L ¹ is C1-C20 alkylene, C1-C20 haloalkylene, (CH ₂ CH ₂ O) m-(C1-C6 alkylene), -(C1-C6 alkylene)-(3-8 membered heterocycloalkylene)-(C1-C6 alkylene), (C1-C6 alkylene)-(5-10 membered heteroarylene)-(C1-C6 alkylene), and m is any integer between 1 and 20;

n is 0 or 1.

In any embodiment of the first aspect, n is 0, and ^L1 is (C1-C6 alkylene)-(3-8 membered heterocycloalkylene)-(C1-C6 alkylene).

In any embodiment of the first aspect, the above n is 1, ^L1 is C1-C20 alkylene, C1-C20 halogenated _{alkylene, (CH2CH2O )} m-(C1-C6 alkylene), (C1-C6 alkylene)-(3-8 membered heterocycloalkylene)-(C1-C6 alkylene), (C1-C6 alkylene)-(5-10 membered heteroarylene)-(C1-C6 alkylene), and m is any integer between 1 and 20.

In any embodiment of the first aspect, L ¹ is C1-C10 alkylene, C1-C10 haloalkylene, (CH ₂ CH ₂ O) m-(C1-C3 alkylene), (C1-C3 alkylene)-(4-6 membered heterocycloalkylene)-(C1-C6 alkylene), (C1-C3 alkylene)-(5-6 membered heteroarylene)-(C1-C6 alkylene), m is any integer between 1 and 6, and n is 0 or 1;

Preferably, the 4-6 membered heterocycloalkylene in the (C1-C3 alkylene)-(4-6 membered heterocycloalkylene)-(C1-C6 alkylene) is selected from aziridinyl, aziridinyl and aziridinyl; preferably, the 4-6 membered heterocycloalkylene in the (C1-C3 alkylene)-(4-6 membered heterocycloalkylene)-(C1-C6 alkylene) is selected from piperidinyl, morpholinyl and piperazinyl; preferably, the 4-6 membered heterocycloalkylene in the (C1-C3 alkylene)-(4-6 membered heterocycloalkylene)-(C1-C6 alkylene) is selected from

Preferably, the 5-6 membered heteroarylene group in the (C1-C3 alkylene)-(5-6 membered heteroarylene)-(C1-C6 alkylene) is selected from pyrrolylene, pyrazolylene, imidazolylene, triazolylene; preferably, the 5-6 membered heteroarylene group in the (C1-C3 alkylene)-(5-6 membered heteroarylene)-(C1-C6 alkylene) is selected from triazolylene, 1,2,4-triazolylene, 1,2,5-triazolylene, 1,3,4-triazolylene; preferably, the 5-6 membered heteroarylene group in the (C1-C3 alkylene)-(5-6 membered heteroarylene)-(C1-C6 alkylene) is selected from

In any embodiment of the first aspect, the above L is any one of the following structures:

Wherein, n1 represents any integer between 1-20, preferably any integer between 1-6; m represents any integer between 1-20, preferably any integer between 1 and 6; n2 represents any integer between 1-6, The terminal is connected to M. Connect the E end.

In any embodiment of the first aspect, the above E is any one of the following structures:

In any embodiment of the first aspect, the above E is L is any of the following structures:

In any embodiment of the first aspect, the above E is L is

In any embodiment of the first aspect, the above E is L is

In any embodiment of the first aspect, the structural formula of the compound represented by the above formula I is any one of the structures YN1-YN21:

The second aspect of the present application provides a method for preparing any one of the compounds provided in the first aspect, which comprises any one or more of the following synthetic routes.

Synthesis route A: AMG510 is used as a raw material and reacted with tert-butyl bromate to prepare intermediate _A1 ; the intermediate _A1 is hydrolyzed under trifluoroacetic acid to form intermediate _A2 ; the intermediate _A2 is condensed with VHL ligand VH032 amide to form target compound _A3 .

Wherein: n1 represents any integer between 1 and 20, preferably any integer between 1 and 6;

Synthesis route B: AMG510 is used as a raw material and reacted with tert-butyl bromide to prepare intermediate _B1 ; intermediate _B1 is hydrolyzed under trifluoroacetic acid to form intermediate _B2 ; intermediate _B2 is condensed with E3 ligase ligand amide to generate target compound _B3 or _B4 .

Wherein: m represents any integer between 1 and 20, preferably any integer between 1 and 6;

Synthesis route C: AMG510 is used as a raw material, and reacted with tert-butyl bromoester to prepare intermediate _C1 ; the intermediate _C1 is hydrolyzed under trifluoroacetic acid to form intermediate _C2 ; the intermediate _C2 is reacted with tert-butyl bromopropionate to prepare intermediate _C3 , the intermediate _C3 is hydrolyzed under trifluoroacetic acid to form intermediate _C4 , and the intermediate _C4 is condensed with VHL ligand VH032 amide to generate target compound _C5 ;

Wherein: n3 represents any integer between 1 and 6, preferably represents any integer between 1 and 3, and X is C or N;

Synthesis route D: AMG510 is used as a raw material and reacted with 3-bromoprop-1-yne to prepare intermediate D ₁ ; hydrazoic acid and VHL ligand VH032 amide are condensed to form intermediate D ₂ ; intermediate D ₁ and intermediate D ₂ are reacted by click chemistry to generate target compound D ₃ ,

Where: n2 represents any integer between 1 and 6;

Synthesis route E: 1-bromo-3-chloropropane reacts with VHL ligand VH101 to form intermediate E ₁ , and intermediate E ₁ reacts with intermediate C ₂ in synthesis route C to obtain target compound E ₂ ,

Wherein: n3 represents any integer between 1 and 6, and X represents C or N.

The third aspect of the present application provides a use of any one of the compounds shown in Formula I provided in the first aspect, its pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph in the preparation of a degradation agent for targeted degradation of KRASG12C.

In any embodiment of the third aspect, the above-mentioned targeted degradation agent for degradation of KRASG12C is a degradation agent for treating or preventing tumor diseases, and the tumor diseases include one or more of gastric cancer, oral cancer, esophageal cancer, thyroid cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, colorectal cancer, kidney cancer, osteosarcoma, medulloblastoma, rhabdomyosarcoma, and glioblastoma.

The fourth aspect of the present application provides the use of any one of the compounds as shown in Formula I provided in the first aspect, its pharmaceutically acceptable salts, prodrugs, stable isotope derivatives, isomers, solvates or polymorphs and other drugs in the preparation of drugs for treating or preventing tumor diseases, wherein the other drugs include any one or more of infigratinib, crizotinib, MK-2206, selumetinib, Dactolisib, Naporafenib, SHP099, AZD457, linsitinib, ibrutinib, cyclophosphamide, doxorubicin, cytarabine, azacitidine, decitabine, carfilzomib, thalidomide, lenalidomide, pomadomine, gefitinib, osimertinib, erlotinib, tepotinib, ositinib, afatinib, flutamide and nilutamide.

In any embodiment of the fourth aspect, the tumor disease includes one or more of gastric cancer, oral cancer, esophageal cancer, thyroid cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, colorectal cancer, kidney cancer, osteosarcoma, medulloblastoma, rhabdomyosarcoma, and glioblastoma.

The fifth aspect of the present application provides a pharmaceutical composition comprising a compound as shown in Formula I provided by any embodiment of the first aspect, a pharmaceutically acceptable salt, a prodrug, a stable isotope derivative, an isomer, a solvate or a polymorph thereof, the pharmaceutical composition also comprising a pharmaceutically acceptable carrier or excipient thereof, the carrier comprising any one or more of an ion exchanger, alumina, aluminum stearate, lecithin, serum protein, a buffer substance, glycerol, sorbic acid, potassium sorbate, a partial glyceride mixture of saturated vegetable fatty acids, water, salts, electrolytes, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, a cellulosic substance, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, beeswax and lanolin.

In some embodiments, the pharmaceutical composition can be administered in a therapeutically effective amount by a suitable route, such as oral, sublingual, rectal, parenteral, injection (intradermal, subcutaneous, intramuscular, intravenous, arterial injection), pulmonary, nasal, tongue, cheek, skin, mucosa, conjunctiva, topical administration or in the form of an implant.

Suitable for oral administration are known administration forms which deliver the active ingredient rapidly and/or in a modified manner, such as tablets (uncoated or coated, such as tablets with enteric or mol coatings), capsules, dragees, granules, pellets, powders, emulsions, suspensions and aerosols.

With parenteral administration it is possible to avoid the absorption step (intravenous, intraarterial, intracardiac, intraspinal or intralumbar administration) or to include the absorption (intramuscular, subcutaneous, intradermal, transdermal or intraperitoneal administration). Suitable administration forms for parenteral administration are, in particular, preparations in the form of solutions, suspensions, emulsions, lyophilizates and sterile powders for injection and infusion.

Suitable for other routes of administration are medicines for inhalation (especially powder inhalation, spray), nasal drops/solutions, sprays; tablets or capsules for lingual, sublingual or buccal administration, suppositories, preparations for the ears and eyes, vaginal capsules, aqueous suspensions (lotions, shaken mixtures), lipophilic suspensions, ointments, creams, emulsions, pastes, dusting powders or implants, such as stents.

The active ingredient can be converted into the administration form by methods known per se. It can be achieved with inert, non-toxic, suitable pharmaceutical excipients. It particularly includes carriers (e.g. microcrystalline cellulose), solvents (e.g. liquid polyethylene glycol), emulsifiers (e.g. sodium lauryl sulfate), dispersants (e.g. polyvinyl pyrrolidone), synthetic and natural biopolymers (e.g. proteins), stabilizers (e.g. antioxidants and ascorbic acid), colorants (e.g. inorganic pigments such as iron oxide) or flavoring agents and/or taste masking agents. In suitable cases, the active ingredient can be present in one or more of the above-mentioned carriers in the form of microencapsulation.

Definition and Description

Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered ambiguous or unclear without special definition, but should be understood according to the ordinary meaning.

A dash ("-") not between two letters or symbols indicates a substituent's attachment point, and the order of attachment is arbitrary. However, when the substituent's attachment point is obvious to those skilled in the art, for example, a halogen substituent, the "-" may be omitted.

The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salt" refers to salts of compounds of the present application, prepared from compounds with specific substituents discovered in the present application and relatively non-toxic acids or bases. When the compounds of the present application contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the compounds contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, trifluoroacetic acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid, etc.; also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the present application contain basic and acidic functional groups, and can be converted into any base or acid addition salt.

The pharmaceutically acceptable salts of the present application can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, the preparation method of such salts is: in water or an organic solvent or a mixture of the two, these compounds in free acid or base form are reacted with a stoichiometric amount of an appropriate base or acid to prepare.

The compound isomers of the present application may exist in any form of tautomers, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may exist in substituents such as alkyl. All of these isomers and their mixtures are included within the scope of the present application.

The present application also includes all suitable stable isotope derivatives of the compounds of the present invention. In this case, the stable isotope derivatives of the compounds of the present application should be understood as such compounds, wherein at least one atom in the compounds of the present application is replaced by another atom with the same atomic number but atomic mass being different from the atomic mass common in nature or mainly existing. The isotope examples that can be introduced into the compounds of the present application are isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine and iodine, such as ² H (deuterium), ³ H (tritium), ¹¹ C, ¹³ C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ³³ S, ³⁴ S, ³⁵ S, ³⁶ S, ¹⁸ F, ³⁶ Cl, ⁸² Br, ¹²³ I, 124 I, ¹²⁹ I ^{and 131 I.} Certain stable isotope derivatives of the compounds of the present application (such as those in which one or more radioactive isotopes are introduced in particular) can be used, for example, to study the mechanism of action or distribution of the active substance in vivo; because they can be relatively easily prepared and detected, compounds labeled with ³ H- or ¹⁴ C-isotopes are particularly suitable for this. In addition, the introduction of isotopes (such as deuterium) can produce certain therapeutic advantages due to the higher metabolic stability of the compound, such as an increase in the half-life in vivo or a reduction in the effective dose required; the modifications of the compounds of the present application may therefore optionally also represent preferred embodiments of the present invention. Isotope labels of the compounds of the present application can be prepared by methods known to those skilled in the art, such as by the specifications given in the following methods and embodiments, using the corresponding isotopic modifications of the respective reagents and/or starting compounds.

The compounds of the present application may contain non-natural proportions of atomic isotopes on one or more atoms constituting the compound. For example, compounds may be labeled with radioactive isotopes, such as tritium (3H), iodine-125 (125I) or C-14 (14C). For another example, deuterated drugs may be formed by replacing hydrogen with heavy hydrogen. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have the advantages of reducing toxic side effects, increasing drug stability, enhancing therapeutic effects, and extending the biological half-life of drugs. All isotopic composition changes of the compounds of the present invention, whether radioactive or not, are included in the scope of the present application. "Optional" or "optionally" refers to the possibility that the event or situation described subsequently may but does not necessarily occur, and the description includes the situation in which the event or situation occurs and the situation in which the event or situation does not occur.

In the present application, "polymorph" refers to a crystalline form of a compound (or its salt, hydrate or solvate) in a specific crystal packing arrangement. All polymorphs have the same elemental composition. Different crystal forms usually have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardnesses, crystal shapes, optical and electrical properties, stability and solubility. Recrystallization solvents, crystallization rates, storage temperatures and other factors may lead to one crystal form being dominant.

In the present application, "solvate" refers to a mixture produced by dissolving a compound in a solvent.

In the present application, "prodrug" refers to a compound obtained by chemically modifying a drug, which is inactive or has low activity in vitro and releases active drugs through enzymatic or non-enzymatic conversion in vivo to exert its efficacy.

The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted compound is stable.

Unless otherwise specified, the number of atoms in a ring is generally defined as the ring member number, for example, "3-6 membered ring" refers to a "ring" having 3-6 atoms arranged around it.

Unless otherwise specified, the term "C1-C6 alkylene" is used to represent a straight or branched divalent saturated hydrocarbon group consisting of 1 to 6 carbon atoms. The C1-C6 alkylene includes C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C4, C6, C5, C4, C3, C2C1 alkylene, etc. C1-C6 alkyl is also understood in the above manner; except that the alkyl is monovalent (such as CH ₃ ) and the alkylene is divalent (such as -CH ₂ -).

The term "cycloalkyl" itself or in combination with other terms represents a cyclic form of an alkyl, alkenyl or alkynyl group or a mixture thereof. In addition, the cycloalkyl group may contain fused rings, but does not include fused aryl and heteroaryl groups, unless otherwise specified as unsubstituted, otherwise the cycloalkyl group may be substituted. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cyclohexynyl, cyclohexynyl, cyclohexadienyl, cyclopentadienyl, cyclopentenyl, cycloheptyl, norbornyl, etc. If the size of the ring is not specified, the cycloalkyl group described herein contains 3-10 ring members, 3-8 ring members, or 3-6 ring members. Cycloalkylene is also understood in the above manner, except that the cycloalkyl group is a monovalent ring and the cycloalkylene group is a divalent ring.

The term "heterocycle" or "heterocycloalkyl" or "heterocyclyl" by itself or in combination with other terms represents a cycloalkyl group containing at least one ring carbon atom and at least one ring heteroatom selected from O, N, P, Si and S, preferably selected from N, O and S, wherein the ring is non-aromatic but may contain unsaturation. The nitrogen and sulfur atoms in the heterocyclic group may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. In various embodiments, the ring heteroatoms are selected from N, O and S. If not otherwise specified, the heterocyclic group described herein contains 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 4-5, 4-6, 4-7, 4-8, 5-10, 5-8 ring members, and at least one ring member is a heteroatom selected from N, O and S; usually no more than 3 of these heteroatoms are contained in the heterocyclic group, and usually no more than 2 of these heteroatoms are contained in a single ring of the heterocyclic group. The heterocyclic group may be fused to other carbocyclic, heterocyclic or aromatic rings. The heterocyclic group may be attached to the rest of the molecule at a ring carbon or a ring heteroatom and may be substituted as described for alkyl groups. In addition, the heterocyclic ring may contain fused rings, but does not include fused systems containing a heteroaryl group as part of the fused ring system. Examples of heterocyclic groups include, but are not limited to, 1-(1,2,5,6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, 1,2,3,4-tetrahydropyridinyl, dihydroindole (indoline), tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl, dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1,2-oxazinyl or dioxepanyl, etc. Heterocycloalkylene is also understood in the same manner as above, except that the heterocyclyl group is a monovalent ring and the heterocycloalkylene group is a divalent ring.

Unless otherwise indicated, the term "aryl" refers to an aromatic hydrocarbon group, which can be a single ring or multiple rings (e.g., 1-3 rings) with rings fused together. An aryl group may contain fused rings, wherein one or more rings are optionally cycloalkyl, but do not include heterocyclic or heteroaromatic rings; a fused system containing at least one heteroaromatic ring is referred to as a heteroaryl group, and a phenyl ring fused to a heterocyclic ring is referred to herein as a heterocyclic group. Aryl groups include fused ring systems in which a phenyl ring is fused to a cycloalkyl ring. Examples of aryl groups include, but are not limited to, phenyl, 1-naphthyl, tetralin, and the like. Arylene groups are also understood in the above manner, except that aryl groups are monovalent rings and arylene groups are divalent rings.

The term "heteroaryl" as used herein refers to a group containing a monocyclic or two or three fused rings, wherein at least one ring is an aromatic ring containing 1-4 heteroatoms selected from N, O and S as ring members (i.e., it contains at least one heteroaromatic ring), wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. The heteroaryl group can be connected to the rest of the molecule through a ring carbon or a ring heteroatom, and if the group is a bicyclic or tricyclic ring, it can be connected through any ring of the heteroaryl group. The heteroaryl group can contain fused rings, wherein one or more rings are optionally cycloalkyl or heterocycloalkyl or aryl, provided that at least one ring is a heteroaromatic ring. Non-limiting examples of heteroaryl are 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, triazole, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl. Substituents for each of the above aryl and heteroaryl ring systems are selected from the following acceptable substituents. Heteroarylene is also understood in the above manner, except that heteroaryl is a monovalent ring and heteroarylene is a divalent ring. Non-limiting examples of heteroarylene are pyrrolylene, pyrazolylene, imidazolylene, triazoleylene, pyridazinylene, pyrazinylene, oxazolylene, phenyl-4-oxazolylene, isoxazolylene, thiazolylene, furanylene, thienylene, pyridinylene, 3-pyridylene, and pyrimidinylene.

Aryl and/or heteroaryl groups typically contain up to 4 substituents (0-4) per ring, and sometimes contain 0-3 or 0-2 substituents.

As used herein, "therapeutically effective amount" refers to an amount capable of producing the desired pharmacological and/or physiological effect. The effect may be preventive, capable of completely or partially preventing a disease or its symptoms; and/or may be therapeutic, capable of partially or completely curing a disease and/or side effects associated with the disease. The therapeutically effective amount of the compound of the present application generally includes any amount sufficient to inhibit AR activity that can be detected by any experiment described herein, by other AR activity assays known to those skilled in the art, or by detecting inhibition or relief of cancer symptoms.

The compounds were named according to conventional nomenclature in the art or using software, and commercially available compounds were named according to the supplier's catalog name.

Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

The compounds provided in the present application exhibit good anti-tumor activity and excellent KRASG12C protein degradation effect, can be used to prevent and treat various cancers, and have great application prospects in the medical field.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments of the present application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the drawings without creative work.

FIG1 shows the Western-Blot test results of compound YN4 and compound YN14 on KRAS protein in non-small cell lung cancer cell line NCI-H358 and pancreatic cancer cell line Miacapa-2.

FIG. 2 shows the experimental results of compound YN14 inducing tumor cell apoptosis.

FIG3 shows the experimental results of compound YN14 blocking tumor cell cycle.

FIG4 shows the experimental results of compound YN14 inhibiting tumor cell migration.

FIG5 shows the anti-tumor proliferation test results of compound YN14 in nude mice xenografted with human pancreatic cancer Miacapa-2 cells.

FIG6 shows the immunohistochemistry (A) and Western-Blot test results (B) of compound YN14 in nude mouse xenograft tumors of human pancreatic cancer Miacapa-2 cells.

DETAILED DESCRIPTION

The following examples are provided to provide those skilled in the art with a complete disclosure of how to perform and evaluate the methods and compounds claimed herein. This example merely illustrates the present invention and does not limit the scope of the present invention.

The preparation method of the compound of formula I of the present invention is described in more detail below, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention can also be prepared more conveniently by combining the various synthetic methods described in the optional specification or known in the art, and such a combination can be more easily performed by a technician in the field to which the present invention belongs. The following examples are preferred illustrative preferred embodiments of the present invention and do not constitute any limitation to the present invention.

The raw materials can be obtained from commercial sources, or prepared by methods known in the art, or prepared according to the methods described herein. The structure of the compound is determined by nuclear magnetic resonance (1H-NMR) and/or mass spectrometry (MS). NMR determination is AVANCENEO 600MHz nuclear magnetic resonance instrument, the determination solvent is deuterated dimethyl sulfoxide (DMSO-d6), and TMS is internal standard. MS is determined using Agilent 1100LC/MSD Trap SL version Mass Spectrometer (ESI) liquid chromatography-mass spectrometry. Column chromatography uses 200-300 mesh silica gel from Qingdao Ocean Chemical Plant.

For all the following examples, standard operations and purification methods known to those skilled in the art can be used. Unless otherwise stated, all temperatures are expressed in ° C (Celsius). The structure of the compound is confirmed by nuclear magnetic resonance (NMR) or mass spectrometry (MS). 1HNMR was measured by a JNM-ECA-400 nuclear magnetic resonance instrument of Japan Electronics, and the measuring solvent was deuterated dimethyl sulfoxide (DMSO-d6), and TMS was an internal standard. The mass spectrum was measured by an API3000 (ESI) mass spectrometer. All reaction solvents were not subjected to standardization pretreatment unless otherwise noted. In the following examples, unless otherwise specified, % refers to mass percentage. Silica gel for column chromatography was produced by Qingdao Ocean Chemical Plant (200-300 mesh); silica gel plates for thin layer chromatography were thin layer chromatography silica gel prefabricated plates produced by Yantai Institute of Chemical Industry. The compounds of the present invention can be prepared by referring to conventional methods in the art and using suitable reagents, raw materials and purification methods known to those skilled in the art.

Example 1: Synthesis of (2R,4S)-1-((2R)-2-(2-(4-(((R)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)acetylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(-4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN1).

In the above reaction route, the reagents and conditions used are as follows: i) acetone, potassium carbonate, 55°C; ii) trifluoroacetic acid, dichloromethane, room temperature; iii) 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature

i: Synthesis of tert-butyl 2-(2-(4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)acetate

4-((S)-4-acryloyl-2-methylpiperazine-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (AMG510) (400.0 mg, 0.71 mmol), tert-butyl 2-bromoacetate (164.9 mg, 0.85 mmol), potassium carbonate (117.3 mg, 0.85 mmol) were dissolved in acetone (40 mL), and 2 drops of water were added, and the mixture was stirred and refluxed at 55°C for 12 h. After the reaction was completed, 20 mL of water was added, and the mixture was extracted 3 times with 30 mL of ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and dried by spin drying, and purified by column chromatography (dichloromethane: methanol = 20: 1) to obtain a yellow oil (287.12 mg, 60%). LC/MS (ESI) m/z: [M+H]+675.30.

ii: Synthesis of 2-(2-(4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)acetic acid

Tert-butyl 2-(2-(4-((S)-4-acryloyl-2-methylpiperazine-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)acetate (200 mg, 0.30 mmol) was dissolved in dichloromethane (20 mL), trifluoroacetic acid (240 mg, 2 mmol) was added, and stirred at room temperature overnight. After the reaction was completed, it was dried by spin drying and purified by column chromatography (dichloromethane: methanol = 20: 1) to obtain a yellow oil (148.32 mg, 80%). LC/MS (ESI) m/z: [M+H] + 619.24.

iii: Synthesis of (2S,4R)-1-((2S)-2-(2-(2-(4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(-4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide.

2-(2-(4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)acetic acid (148 mg, 0.24 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N- (4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (124.7 mg, 0.29 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (136.88 mg, 0.36 mmol), N,N-diisopropylethylamine (46.53 mg, 0.36 mmol) were dissolved in N,N-dimethylformamide (8 mL) and stirred at room temperature for 10 h. After the reaction was completed, 20 mL of water was added, and the mixture was extracted three times with 30 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and dried by spin drying. The mixture was purified by column chromatography (dichloromethane: methanol = 20: 1) to obtain a yellow solid (91.46 mg, 37%). 1H NMR(600MHz, DMSO-d6)δ8.98(d,J＝4.5Hz,1H),8.60–8.49(m,1H),8.34(dd,J＝37.8,11.8Hz,2H),7.41(ddd,J＝30.7,21.9,12.3Hz,6H),7.31–7.13(m,2H),6.9 8–6.84(m,3H),6.21(t,J＝13.7Hz,1H),5.76(d,J＝9.4Hz,1H),5.15(d,J＝9.4H z,1H),4.86(d,J＝38.6Hz,1H),4.71–4.17(m,11H),3.60(dd,J＝54.3,11.3Hz,4H),3.11(d,J＝12.9Hz,1H),2.72(d,J＝32.8Hz,1H),2.44(d,J＝3.7Hz,3H), 1.93–1.79(m,4H),1.41–1.19(m,7H),1.10–0.91(m,7H),0.88–0.67(m,12H).13CNM R(151MHz,DMSO)δ172.12,169.45,169.32,165.51,162.88,154.18,153.11,151.97,151.44,150.74,149.33,148.32,146.21,144.06,140.71,133.53 ,131.60,131.22,130.26,129.37,129.22,128.62,128.49,128.25,127.47,123.69,1 12.66,109.26,107.31,70.30,68.41,61.97,59.05,57.14,56.82,55.83,55.25,51.87,42.15,38.65,38.52,36.01,31.71,27.27,26.35,23.10 ,22.32,22.02,21.22,18.66,16.41,16.00,15.08,14.54.LC/MS(ESI)m/z:[M+H]+1031.41.

Example 2: Synthesis of (2R,4S)-1-((2R)-2-(3-(2-(4-(((R)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyridin[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)propionamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(-4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN2)

Synthesis method: Refer to Example 1, and separate by column chromatography (dichloromethane-methanol, volume ratio 20:1) to obtain 95.48 mg of yellow solid with a yield of 38%. NMR(600MHz, DMSO-d6)δ9.03(s,1H),8.63(t,J＝6.1Hz,1H),8.44(t,J＝4.6Hz,1H),8.39–8.26(m,1H),8.21–7.94(m,1H),7.47(dt,J＝18.9,12.0Hz,5H),7.2 8–7.20(m,1H),7.06(d,J＝7.6Hz,1H),6.97–6.86(m,2H),6.26(t,J＝14.0Hz,1H),5.81(dd,J＝14.8,6.3Hz,1H),5.31–5. 19(m,1H),4.97(s,1H),4.54–4.40(m,4H),4.34(d,J＝17.4Hz,1H),4.27(dt,J＝12.2,5.7Hz,2H),4.18(d,J＝14.5Hz,2H),3.77–3.65(m,4H),2.73(s,2 H),2.50(s,4H),2.10(t,J＝11.1Hz,1H),2.01–1.88(m,4H),1.45–1.26(m,5H),1.12(q,J＝6.8Hz,3H),1.04–0.85(m,13H).13C NMR(151MHz,DMSO-d6)δ172.40,170.44,169.96,169.91,169.61,165.52,163.00,159.37,151.94,150.00,148.86,148.21,145.65,139.98,139.95,13 1.65,131.23,130.13,129.13,128.48,128.25,127.90,123.7 7,69.36,69.31,59.20,56.89,56.84,42.13,38.42,35.84,35.44,34.96,34.17,30.27,30.23,29.48,26.75,26.71,22.50,22.43,22.26,21.87 ,17.71,17.41,16.40,15.99,15.24.LC/MS(ESI)m/z:[M+H]+1045.44.

Example 3: Synthesis of (2R,4S)-1-((2R)-2-(4-(4-(((R)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyridine[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)butyrylamide)-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN3).

Synthesis method: Refer to Example 1, and separate by column chromatography (dichloromethane-methanol, volume ratio 20:1) to obtain 101.39 mg of yellow solid with a yield of 41%. NMR (600MHz, DMSO-d6) δ9.04(s,1H),8.63(t,J＝5.8Hz,1H),8.43(d,J＝4.9Hz,1H),8.40–8.34(m,1H),7.97–7.91(m,1H),7.53–7.41(m,6H),7.24(dd,J＝20.1 ,4.9Hz,1H),7.01(d,J＝8.5Hz,1H),6.96–6.87(m,2H),6.30–6.22(m,1H),5.82(dd,J＝10.4,2.4Hz,1H),5.19(dd,J＝8.1,3.3Hz,1H),5.00(d,J＝57.2Hz, 1H),4.59 (d,J＝9.5Hz,1H),4.51–4.46(m,2H),4.41(s,2H),4.37(d,J＝14.9Hz,1H),4.28(dd,J＝16.0,5.4Hz,1H),4.00(dt,J＝16.7,7.3Hz,2H),3.75–3.67(m,3H), 2.81–2.69(m,1H),2.26–2.07(m,4H),2.00–1.89(m,4H),1.83–1.68(m,2H),1.44–1.37(m,3H),1.30(d,J＝6.6Hz,2H),1.12(d,J＝6.7Hz,3H),1.03– 0.84(m,15H).13C NMR (151MHz, DMSO) δ172.40,171.87,171.70,170.27,165.51,163.95,162.99,161.27,157.88,154.29,153.31,151.94,151.51,150.08,148.84,148.2 1,144.26,144.21,139.99,132.84,131.64,131.26,130.14,129.13,128.50,128.43,127.91, 123.74,109.44,108.27,106.04,97.62,69.33,68.69,59.17,56.84,56.20,49.35,42.12,38.46,35.70,31.27,30.22,29.52,26.81,26.71,25. 22,22.43,22.19,22.03,17.54,16.41,15.99,15.35,14.46,12.16.LC/MS(ESI)m/z:[M+H]+1059.46.

Example 4: Synthesis of (2R,4S)-1-((2R)-2-(5-(2-(4-(((R)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)pentanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(-4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN4).

Synthesis method: Refer to Example 1, and separate by column chromatography (dichloromethane-methanol, volume ratio 20:1) to obtain 97.46 mg of yellow solid, with a yield of 39%. 1H NMR (600MHz, DMSO-d6) δ9.04 (s, 1H), 8.63 (d, J = 6.3 Hz, 1H), 8.47 (d, J = 4.9 Hz, 1H), 8.36 (dd, J = 20.3, 8.9 Hz, 1H), 7.92 (t, J = 10.7 Hz, 1H), 7.47 (td, J = 22.5, 19.9, 8.0 Hz, 6H),7.32(d,J＝17.6Hz,1H),7.01(d,J＝8.6Hz,1H),6.91(dd,J＝12.0,6.5Hz,2H),6.26(t,J＝13.7Hz,1H),5.82(d,J＝10.9Hz,1H),5.18(s,1H),4.98(s,1 H),4.60(d,J＝9 .4Hz,1H),4.49(t,J＝8.2Hz,3H),4.40(d,J＝17.5Hz,3H),4.28(dd,J＝15.5,5.3Hz,1H),4.00(t,J＝6.4Hz,2H),3.75–3.67(m,3H),2.85–2.73(m,1H),2.29 (dt,J＝14.4,6 .9Hz,1H),2.11(dt,J＝20.1,9.1Hz,2H),2.02–1.91(m,4H),1.58–1.49(m,3H),1.41(d,J＝6.9Hz,4H),1.35–1.26(m,3H),1.15(d,J＝6.8Hz,3H),1.05–0 .89(m,15H).13C NMR (151MHz, DMSO) δ172.42,172.29,172.23,170.12,165.53,162.93,159.35,157.93,154.17,153.27,151.94,151.61,149.95,148.19,144.32,144.2 0,139.99,132.65,132.58,131.66,130.13,129.30,129.12,128.47,128.38,128.25,127.91,11 1.98,108.22,69.36,68.77,59.18,56.84,56.72,53.89,42.13,38.44,35.73,34.78,34.68,30.10,28.41,28.34,26.83,22.25,22.10,22.00,2 1.78,18.98,17.67,17.58,17.18,16.40,16.00,15.27,14.36,12.77.LC/MS(ESI)m/z:[M+H]+1073.48.

Example 5: Synthesis of (2R,4S)-1-((2R)-2-(6-(2-(4-(((R)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyridine[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)hexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(-4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN5).

Synthesis method: Refer to Example 1, and separate by column chromatography (dichloromethane-methanol, volume ratio 20:1) to obtain 95.47 mg of yellow solid with a yield of 36%. NMR (600MHz, DMSO-d6) δ8.98(s,1H),8.57(d,J＝7.3Hz,1H),8.41–8.30(m,2H),7.86(d,J＝9.4Hz,1H),7.45–7.34(m,6H),7.19(d,J＝14.3Hz,1H),6.95(d,J＝ 8.4Hz,1H),6.85(t,J＝10.2Hz,2H),6.20(t,J＝13.2Hz,1H),5.76(d,J＝10.5Hz,1H),5.13(s,1H),4.89(d,J＝19.6Hz,1H),4.54(d,J＝9.2Hz,1H ),4.43(d,J＝11.1Hz,3H),4.36–4.18(m,4H),3.96–3.87(m,2H),3.65(q,J＝10.5Hz,4H),2.88(s,2H),2.73(s,3H),2.44(s,3H),2.19(dt,J＝15.3,7. 9Hz,1H),2.05(q,J＝9.8Hz,3H),1.90(d,J＝26.3Hz,5H),1.55–1.41(m,4H),1.34(s,4H),1.26–1.12(m,4H),1.07(d,J＝6.6Hz,4H),0.92(s,15H).13C NMR (151MHz, DMSO) δ172.43,172.39,170.17,165.50,164.06,162.97,162.79,157.89,154.25,154.20,151.94,151.56,150.05,148.97,148.20,144.3 5,144.23,139.99,132.62,132.56,131.65,131.26,130.13,129.12,128.43,128.26,127.90,123.77,10 9.56,108.38,105.95,69.35,69.08,59.17,56.84,56.73,49.62,42.12,38.44,36.25,35.70,35.20,31.24,30.23,29.66,28.39,26.83,25.52, 25.41,25.29,22.41,22.30,22.19,21.98,17.55,17.41,16.41,15.99,15.43.LC/MS(ESI)m/z:[M+H]+1087.50.

Example 6: Synthesis of (2R,4S)-1-((2R)-2-(9-(2-(4-(((R)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)nonanamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(-4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN6).

The synthesis method was as described in Example 1. After separation by column chromatography (dichloromethane-methanol, volume ratio 20:1), 86.45 mg of yellow solid was obtained with a yield of 35%. NMR (600MHz, DMSO-d6) δ8.97(s,1H),8.56(t,J＝6.2Hz,1H),8.41–8.27(m,2H),7.83(d,J＝10.4Hz,1H),7.46–7.35(m,6H),7.21–7.14(m,1H),6.95(d,J＝8. 5Hz,1H),6.85(t,J＝9.5Hz,2H),6.20(t,J＝14.2Hz,1H),5.76(d,J＝10.5Hz,1H),5.13(s,1H),4.90(d,J＝28.3Hz,1H),4.53(d,J＝9.3Hz,1 H),4.47–4.18(m,8H),3.91(dt,J＝16.0,9.2Hz,2H),3.65(q,J＝22.8,16.6Hz,6H),2.44(s,4H),2.24(dt,J＝14.9,7.7Hz,1H),2.05(dt,J＝33.4,8.0Hz, 3H),1.95–1.82(m,5H),1.47(s,4H),1.42–1.27(m,6H),1.24(d,J＝11.4Hz,2H),1.14(s,10H),1.06(d,J＝6.6Hz,4H),1.01–0.78(m,17H).13C NMR (151MHz, DMSO) δ172.52,172.43,170.20,165.51,164.01,163.71,162.95,157.95,154.24,154.15,151.92,150.06,148.86,148.31,139.99,132.6 2,132.54,131.65,131.25,130.13,129.12,128.44,128.26,127.91,123.75,109.33,109.07,108.18,108.05, 105.92,69.35,69.13,68.89,68.66,67.59,59.17,58.52,56.82,56.74,42.13,38.43,35.69,35.31,30.29,30.23,29.12,29.02,28.94,28.65, 26.91,26.84,25.87,25.52,22.42,22.30,22.22,21.98,17.48,17.41,16.41,15.98.LC/MS(ESI)m/z:[M+H]+1129.54.

Example 7: Synthesis of (2R,4S)-1-((2R)-2-(2-(2-(4-(((R)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)acetylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN7).

Synthesis method: Refer to Example 1, and separate by column chromatography (dichloromethane-methanol, volume ratio 20:1) to obtain 89.47 mg of yellow solid with a yield of 37%. 1H NMR (600MHz, DMSO-d6) δ8.98 (s, 1H), 8.62-8.56 (m, 1H), 8.38 (d, J = 4.8 Hz, 1H), 8.28 (td, J = 23.1, 19.2, 8.8 Hz, 1H), 7.47-7.35 (m, 6H), 7.30 (d, J = 9.4 Hz, 1H), 7.18 (dd, J = 20.4, 5.0Hz,1H),7.00(d,J＝8.4Hz,1H),6.91–6.80(m,2H),6.22(d,J＝15.8Hz,1H),5.77(d,J＝11.3Hz,1H),5.16(d,J＝8.6Hz,1H),4.93(d,J＝8.8Hz,1H),4.53 (t,J＝8.9Hz,1H),4.47– 4.33(m,4H),4.28(ddd,J＝15.6,10.0,5.2Hz,2H),4.20–4.01(m,3H),3.81(dd,J＝15.0,5.4Hz,1H),3.75–3.61(m,6H),3.58(d,J＝10.1Hz,1H),2.74–2. 68(m,1H),2.43(s,3H), 2.08(dt,J＝13.3,5.4Hz,1H),1.90(dd,J＝15.3,5.7Hz,4H),1.36(td,J＝9.3,7.1,3.4Hz,4H),1.26–1.22(m,3H),1.07(d,J＝6.7Hz,3H),0.94(d,J＝6.6Hz ,2H),0.90(s,11H).13C NMR (151MHz, DMSO) δ172.19,169.95,168.77,165.71,164.09,157.97,154.24,153.29,151.92,151.54,150.08,149.01,148.24,146.04,144.31,139.9 1,132.88,131.61,131.25,130.20,129.19,128.54,128.43,127.95,124.06,112.18,109.85 ,108.73,106.50,103.98,70.42,69.58,69.33,59.23,57.45,56.12,51.61,49.69,44.96,42.15,38.43,36.26,31.16,30.24,29.48,29.16,26. 62,25.29,22.42,22.22,21.93,17.45,16.37,16.03,15.31,14.56.LC/MS(ESI)m/z:[M+H]+1075.46.

Example 8: Synthesis of (2R,4S)-1-((2R)-2-(2-(2-(4-(((R)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)acetylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN8).

Synthesis method: Refer to Example 1, and separate by column chromatography (dichloromethane-methanol, volume ratio 20:1) to obtain 88.97 mg of yellow solid, with a yield of 39%. 1H NMR (600MHz, DMSO-d6) δ8.96 (s, 1H), 8.58 (t, J = 6.1 Hz, 1H), 8.37 (d, J = 4.8 Hz, 1H), 8.31 (p, J = 8.7, 7.6 Hz, 1H), 7.40 (d, J = 21.3 Hz, 7H), 7.22-7.16 (m, 1H), 6.97 (d, J = 8.5 Hz, 1H), 6.86(q,J＝7.2,6.7Hz,2H),6.20(dd,J＝16.4,11.8Hz,1H),5.76(dd,J＝10.3,2.4Hz,1H),5.16(s,1H),4.90(s,1H),4.56(d,J＝9.6Hz,1H),4.47–4.33(m, 5H),4.28(tt,J＝15.8,5 .1Hz,3H),4.18–3.99(m,4H),3.92(d,J＝4.5Hz,2H),3.67(dd,J＝11.2,4.4Hz,3H),3.60(dd,J＝10.6,6.4Hz,3H),3.49(t,J＝4.7Hz,3H),3.42(d,J＝6.8Hz,3H ),2.44(s,1H),2.42 (d,J＝4.0Hz,3H),2.06(dd,J＝12.9,8.1Hz,1H),1.94–1.86(m,5H),1.34(p,J＝6.1,5.2Hz,5H),1.28–1.22(m,7H),1.07(d,J＝6.7Hz,4H),0.91(s,13H),0 .85(t,J＝6.7Hz,3H).13C NMR(151MHz,DMSO-d6)δ172.65,169.62,169.08,165.53,163.88,162.96,161.14,159.38,157.81,154.23,151.89,151.60,150.06,148.83,148.22,14 4.28,144.16,139.92,132.62,132.56,132.00,131.25,130.20,129.36,129.16,128.59,128.42,128. 25,127.96,123.81,106.01,70.90,70.24,70.02,69.35,69.22,69.17,69.07,59.63,57.06,56.18,42.16,38.42,36.17,30.25,29.48,26.72,2 6.61,22.42,22.31,22.23,21.98,17.51,17.41,16.41,16.36,15.98,15.25.LC/MS(ESI)m/z:[M+H]+1119.49.

Example 9: (2R,4S)-1-((2R)-2-(2-(2-2-(2-(4-(((R)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)propionylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN9).

Synthesis method: Refer to Example 1, and separate by column chromatography (dichloromethane-methanol, volume ratio 20:1) to obtain 92.36 mg of yellow solid, 38%. 1H NMR (600MHz, DMSO-d6) δ8.98 (s, 1H), 8.56 (t, J = 6.1 Hz, 1H), 8.38 (d, J = 4.9 Hz, 1H), 8.34-8.27 (m, 1H), 7.90 (d, J = 9.4 Hz, 1H), 7.46-7.35 (m, 6H), 7.19 (dd, J = 11.5, 4.9 Hz, 1H), 6 .99(d,J＝8.6Hz,1H),6.88(tt,J＝10.1,4.8Hz,2H),6.20(t,J＝14.0Hz,1H),5.76(dd,J＝10.4,2.4Hz,1H),5.13(d,J＝3.5Hz,1H),4.90(s,1H),4.55(d,J＝ 9.3Hz,1H),4.45–4.40( m,3H),4.35(s,1H),4.30(t,J＝13.1Hz,2H),4.22(dd,J＝15.8,5.5Hz,1H),4.09(d,J＝5.8Hz,1H),4.04(dd,J＝6.5,3.4Hz,2H),3.69–3.60(m,3H),3.58–3. 49(m,6H),2.44(s,3H) ,2.31(dt,J＝14.6,6.1Hz,1H),2.07–2.00(m,1H),1.92–1.87(m,4H),1.36–1.32(m,4H),1.25(d,J＝14.0Hz,5H),1.07(d,J＝6.7Hz,3H),0.91(s,12H) ,0.85(t,J＝6.8Hz,2H).13C NMR (151MHz, DMSO) δ172.40,171.54,170.35,170.04,168.96,168.49,165.79,163.19,159.79,151.94,151.55,150.04,149.18,148.20,146.76,146.2 7,144.53,139.99,132.89,131.64,131.27,130.61,130.13,129.12,128.41,128.36,127.91,124 .09,112.03,109.99,108.74,106.23,80.31,70.28,69.86,69.34,68.95,67.41,59.19,56.85,56.74,42.12,38.42,36.39,35.82,31.96,30.58 ,29.48,26.77,22.97,22.42,22.17,22.10,17.80,17.52,16.82,15.54.LC/MS(ESI)m/z:[M+H]+1118.49.

Example 10: Synthesis of 2-(2-(4-(((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)ethoxy)-N-(2-(6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)acetamide (YN10).

In the above reaction route, the reagents and conditions used are as follows: i) acetone, potassium carbonate, 55°C; ii) trifluoroacetic acid, dichloromethane, room temperature; iii) 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature.

i: Synthesis of tert-butyl 2-(2-(4-(((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)ethoxy)acetate.

4-((S)-4-acryloyl-2-methylpiperazine-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (AMG510) (400.0 mg, 0.71 mmol), tert-butyl 2-(2-bromoethoxy)acetate (202.32 mg, 0.85 mmol), potassium carbonate (117.3 mg, 0.85 mmol) were dissolved in acetone (40 mL), and 2 drops of water were added, and the mixture was stirred and refluxed at 55°C for 12 h. After the reaction was completed, 20 mL of water was added, and the mixture was extracted 3 times with 30 mL of ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and dried by spin drying, and purified by column chromatography (dichloromethane: methanol = 20: 1) to obtain a yellow oil (285.47 mg, 56%). LC/MS (ESI) m/z: [M+H]+719.33.

ii: Synthesis of 2-(2-(4-(((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)ethoxy)acetic acid.

Tert-butyl 2-(2-(4-(((S)-4-acryloyl-2-methylpiperazine-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)ethoxy)acetate (215.42 mg, 0.30 mmol) was dissolved in dichloromethane (20 mL), trifluoroacetic acid (240 mg, 2 mmol) was added, and stirred at room temperature overnight. After the reaction was completed, it was dried by spin drying and purified by column chromatography (dichloromethane: methanol = 20: 1) to obtain a yellow oil (216.81 mg, 80%). LC/MS (ESI) m/z: [M+H] + 663.27.

iii: Synthesis of 2-(2-(4-(((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)ethoxy)-N-(2-(6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)acetamide.

2-(2-(4-(((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)ethoxy)acetic acid (216.81 mg, 0.24 mmol), 3-(4-amino-1-oxoisoindol-2-yl)piperidine-2,6-dione (124.7 mg, 0.29 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (136.88 mg, 0.36 mmol), and N,N-diisopropylethylamine (46.53 mg, 0.36 mmol) were dissolved in N,N-dimethylformamide (8 mL) and stirred at room temperature for 10 h. After the reaction was completed, 20 mL of water was added, and the mixture was extracted three times with 30 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and dried by spin drying. The mixture was purified by column chromatography (dichloromethane: methanol = 20: 1) to obtain a yellow solid (80.19 mg, 37%). 1H NMR (600 MHz, DMSO-d6) δ 11.07–10.95 (m, 1H), 9.65 (dd, J = 18.3, 7.1 Hz, 1H), 8.37 (dd, J = 12.6, 4.8 Hz, 1H), 8.28 (td, J = 18.7, 17.1, 8.7 Hz, 1H), 7.68 (dt, J = 19.1, 5.3 Hz, 1H ),7.58(d,J＝7.1Hz,1H),7.49(dt,J＝34.0,7.6Hz,2H),7.18(dd,J＝28.9,5.0Hz,1H),7.02(d,J＝8.5Hz,1H),6.90(t,J＝8.7Hz,1H),6.80(ddt,J＝19.3,13.2 ,7.8Hz,1H),6. 20(t,J＝14.5Hz,1H),5.76(t,J＝5.7Hz,1H),5.19–5.09(m,1H),4.96–4.81(m,1H),4.41–4.10(m,6H),4.03–3.81(m,3H),3.79–3.57(m,4H),2.92(d p,J＝19.3,6.1Hz,1H) ,2.76–2.65(m,1H),2.58(dd,J＝34.4,17.1Hz,1H),2.37–2.26(m,1H),2.03–1.92(m,1H),1.92–1.81(m,3H),1.35–1.19(m,5H),1.09–1.00(m,3H), 0.99–0.80(m,4H).13C NMR (151MHz, DMSO) δ173.32,171.48,168.69,168.63,168.24,165.48,163.99,163.76,163.05,162.86,159.43,157.75,154.26,153.22,151.56,150.1 2,148.83,145.82,145.58,144.53,143.97,135.35,133.27,1 33.22,132.68,131.25,129.08,128.22,126.95,123.71,120.34,108.65,106.16,70.72,69.64,55.38,51.99,46.83,31.67,30.26,30.20,26.8 1,23.03,22.46,22.23,21.91,17.39.LC/MS(ESI)m/z:[M+H]+904.35.

Example 11: Synthesis of 2-(2-(2-)(2-(4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)ethoxy)-ethoxy-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)acetamide (YN11).

Synthesis method: Refer to Example 10, and separate by column chromatography (dichloromethane-methanol, volume ratio 20:1) to obtain 86.58 mg of yellow solid with a yield of 38%. NMR (600MHz, DMSO-d6) δ11.02(s,1H),9.71(d,J＝9.2Hz,1H),8.41–8.29(m,2H),7.71(t,J＝10.0Hz,1H),7.55(d,J＝7.6Hz,1H),7.50–7.41(m,2H),7.22–7.1 5(m,1H),6.98(d,J＝8.4Hz,1H),6.87(dt,J＝23.4,11.0Hz,2H),6.20(t,J＝14.3Hz,1H),5.75(d,J＝9.2Hz,2H),5.18–5.11(m, 1H),4.89(s,1H),4.35(dt,J＝42.7,15.3Hz,5H),4.08(d,J＝18.0Hz,6H),3.53(d,J＝61.2Hz,10H),2.92(t,J＝14.8Hz,1H),2.71(t,J＝11.8Hz,1H),2.60 (d,J＝17.2Hz,1H),2.39–2.31(m,1H),1.96(dd,J＝57.2,11.1Hz,5H),1.43–1.19(m,9H),1.11–1.03(m,4H),0.98–0.83(m,5H).13C NMR (151MHz, DMSO) δ173.34,171.52,168.86,168.24,165.51,162.93,161.36,159.57,158.18,154.20,150.08,148.84,145.81,144.00,136.36,133.3 5,133.24,132.62,131.26,129.12,128.09,126.68,123.76,120.2 0,112.18,109.75,108.66,106.00,71.53,70.35,69.26,69.22,56.09,52.75,49.34,46.94,45.32,44.39,42.36,32.42,30.79,29.48,23.04,2 2.34,22.24,21.98,17.51,15.95,15.20.LC/MS(ESI)m/z:[M+H]+948.38.

Example 12: Synthesis of (2S,4R)-1-((2S)-2-(4-(2-(4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)methyl)piperidin-1-yl)butanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN14).

In the above reaction route, the reagents and conditions used are as follows: i) acetone, potassium carbonate, 55°C; ii) trifluoroacetic acid, dichloromethane, room temperature; iii) potassium carbonate, N,N-dimethylformamide, room temperature; iv) trifluoroacetic acid, dichloromethane, room temperature; v) 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature.

i: Synthesis of tert-butyl 4-((2-(4-(((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)methyl)piperidine-1-carboxylate.

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (AMG510) (800.0 mg, 1.42 mmol), tert-butyl 4-(bromomethyl)piperidine-1-carboxylate (471.02 mg, 1.70 mmol), and potassium carbonate (234.6 mg, 1.70 mmol) were dissolved in acetone (40 mL), and 2 drops of water were added. The mixture was stirred and refluxed at 55 °C for 12 h. After the reaction was completed, 20 mL of water was added, and the mixture was extracted three times with 30 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and dried by spin drying. The mixture was purified by column chromatography (dichloromethane: methanol = 20: 1) to obtain a yellow oil (516.24 mg, 48%). LC/MS (ESI) m/z: [M+H] + 758.38.

ii: Synthesis of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-(piperidin-4-ylmethoxy)phenyl)-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one.

4-((2-(4-(((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)methyl)piperidine-1-carboxylic acid tert-butyl ester (516.24 mg, 0.68 mmol) was dissolved in dichloromethane (20 mL), trifluoroacetic acid (480 mg, 4 mmol) was added, and stirred at room temperature overnight. After the reaction was completed, the mixture was dried by spin drying and purified by column chromatography (dichloromethane: methanol = 20: 1) to obtain a yellow oil (357.42 mg, 80%). LC/MS (ESI) m/z: [M+H]+ 658.32.

iii: Synthesis of tert-butyl 4-(4-(2-(4-(((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)methyl)piperidin-1-yl)butanoate.

4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-(piperidin-4-ylmethoxy)phenyl)-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (357.42 mg, 0.54 mmol), tert-butyl 4-bromobutyrate (146.52 mg, 0.66 mmol), potassium carbonate (91.08 mg, 0.66 mmol) were dissolved in N,N-dimethylformamide (10 mL) and stirred at room temperature for 10 h. After the reaction was completed, 20 mL of water was added, and the mixture was extracted three times with 30 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and dried by spin drying. The mixture was purified by column chromatography (dichloromethane: methanol = 20: 1) to obtain a yellow oil (211.52 mg, 49%). LC/MS (ESI) m/z: [M+H]+800.42.

iv: Synthesis of 4-(4-((2-(4-(S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)methyl)piperidin-1-yl)butanoic acid.

Tert-butyl 4-(4-(2-(4-(((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)methyl)piperidin-1-yl)butanoate (211.52 mg, 0.28 mmol) was dissolved in dichloromethane (20 mL), trifluoroacetic acid (480 mg, 4 mmol) was added, and stirred at room temperature overnight. After the reaction was completed, it was dried by spin drying and purified by column chromatography (dichloromethane: methanol = 20: 1) to obtain a yellow oil (166.44 mg, 80%). LC/MS (ESI) m/z: [M+H] + 744.36.

v: Synthesis of (2S,4R)-1-((2S)-2-(4-(2-(4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)methyl)piperidin-1-yl)butanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide.

4-(4-((2-(4-(S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)methyl)piperidin-1-yl)butanoic acid (166.44 mg, 0.22 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

(111.08 mg, 0.26 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (98.86 mg, 0.26 mmol), N,N-diisopropylethylamine (32.16 mg, 0.26 mmol) were dissolved in N,N-dimethylformamide (10 mL) and stirred at room temperature for 10 h. After the reaction was completed, 20 mL of water was added, and 30 mL of ethyl acetate was used for extraction three times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and dried by spin drying. The yellow solid (94.06 mg, 37%) was obtained by column chromatography purification (dichloromethane: methanol = 20: 1). 1H NMR(600MHz, DMSO-d6)δ8.99(s,1H),8.61(t,J＝6.3Hz,1H),8.41–8.31(m,2H),7.99(d,J＝9.1Hz,1H),7.46–7.36(m,6H),7.19(dd,J＝13.6,5.0Hz,1H),6.98( d,J＝8.5Hz,1H),6.88(dd,J＝12.7,5.9Hz,2H),6.21(dd,J＝16.8,10.3Hz,1H),5.77(d,J＝10.4Hz,1H),5.21(s,1H),4.92(d,J＝19.7Hz,1H),4.54(d,J＝9.1 Hz,1H),4.4 6–4.20(m,8H),4.15(d,J＝13.1Hz,1H),4.04(t,J＝11.0Hz,1H),3.89–3.62(m,8H),3.18(d,J＝40.9Hz,4H),2.70(tt,J＝15.3,7.3Hz,3H),2.45(s,4H),2 .33–2.14 (m,3H),2.06(dd,J＝12.8,7.8Hz,1H),1.96–1.68(m,9H),1.55(d,J＝34.8Hz,3H),1.42–1.20(m,10H),1.12–1.04(m,5H),0.94(s,13H),0.84(t,J＝8.5 Hz,2H).13C NMR (151MHz, DMSO) δ172.45,172.08,171.72,170.07,165.54,164.00,163.78,163.00,161.21,159.36,157.65,154.26,153.33,151.93,151.53,150.1 0,148.83,148.20,145.83,145.55,144.11,143.98,139.99,132.61,131.67,131.27,130.20,130.12,129.34,129.17,1 29.11,128.43,128.26,127.91,123.76,111.92,109.17,108.98,108.50,69.36,59.21,57.01,56.85,51.66,49.55,42.13,38.45,35.70,32.49 ,30.27,26.86,26.46,23.03,22.30,22.15,21.87,21.02,17.55,17.38,16.40,16.04,15.28.LC/MS(ESI)m/z:[M+H]+1156.55.

Example 13: Synthesis of (2S,4R)-1-((2S)-2-(4-(2-(4-(((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)ethyl)piperazin-1-ylbutanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(-4-(4-(methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN12)

Synthesis method: Refer to Example 12, and separate by column chromatography (dichloromethane-methanol, volume ratio 20:1) to obtain 97.58 mg of yellow solid, with a yield of 38%. 1H NMR (600MHz, DMSO-d6) δ8.99 (s, 1H), 8.57 (s, 1H), 8.34 (s, 1H), 7.87 (s, 1H), 7.46-7.37 (m, 5H), 7.20 (s, 1H), 6.99 (d, J = 9.2 Hz, 1H), 6.88 (s, 2H), 6.22 (d, J = 14.6 Hz, 1H), 5.77 (s, 1H), 5.14 (s, 1H), 4.90 (s, 1H), 4.45-4.41 (m, 3H), 4.29 (d, J = 1 3.8Hz,1H),4.22(d,J＝15.3Hz,1H),4.17(s,2H),4.04(s,3H),3.67(s,4H),2.72(s,1H),2.28–2.23(m,3H),2.03(s,2H),1.90(d,J＝15.3Hz,4H),1.6 4(s,2H),1.34(s,6H),1.25(d,J＝12.9Hz,8H),1.08(s,4H),0.93(s,15H),0.86(s,2H).13C NMR (151MHz, DMSO) δ172.94,172.44,170.41,165.52,164.02,163.72,163.02,160.98,159.35,157.79,154.19,153.22,151.94,151.59,150.49,148.8 5,148.23,146.27,145.44,144.28,144.15,139.69,133.02,131.63,131.23,130.22,129.15,128.46,128.42,127.93 ,123.76,109.19,108.99,108.33,106.03,69.58,59.01,57.71,56.08,55.38,52.88,51.67,49.39,46.12,45.33,44.33,42.26,37.41,35.16,3 2.87,31.61,30.30,29.48,26.85,23.06,22.29,22.14,21.88,17.47,16.40,15.99,15.27.LC/MS(ESI)m/z:[M+H]+1171.56.

Example 14: Synthesis of (2S, 4R)-1-((2S)-2-(3-(4-(2-(4-(((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)ethyl)piperazine-1-ylpropionamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(-4-(4-(methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN13).

Synthesis method: Refer to Example 12, and separate by column chromatography (dichloromethane-methanol, volume ratio 20:1) to obtain 92.37 mg of yellow solid, 39%. 1H NMR(600MHz, DMSO-d6)δ8.98(s,1H),8.57(t,J＝6.1Hz,1H),8.35(dt,J＝37.3,7.4Hz,2H),7.86(d,J＝9.2Hz,1H),7.48–7.36(m,6H),7.19(dd,J＝11.7,5.1Hz,1 H),6.98(d,J＝8.6Hz,1H),6.86(q,J＝18.4,13.5Hz,2H),6.21(t,J＝14.8Hz,1H),5.76(d,J＝10.5Hz,1H),5.14(s,1 H),4.90(s,1H),4.54(d,J＝9.3Hz,1H),4.43(h,J＝10.6,9.7Hz,3H),4.36(s,1H),4.32–4.19(m,3H),4.04(q,J＝17.2,15.3Hz,3H),2.44(s,4H),2.13(d t,J＝14.3,7.2Hz,2H),2.03(d,J＝8.9Hz,2H),1.90(d,J＝15.8Hz,4H),1.68–1.59(m,2H),1.34(d,J＝6.3Hz,4H),1.25(d,J ＝14.5Hz,11H),1.07(d,J＝6.3Hz,4H),0.94(d,J＝9.5Hz,12H).13CNMR(151MHz,DMSO)δ176.47,175.17,173.17,172.88,171.28,170.61,166.94,166.48,15 4.37,151.94,150.75,150.73,149.02,148.87,139.99,133.38,131.65,131.11,131.00,130.12,129.73,129.12,1 28.59,128.47,128.42,127.91,125.12,124.20,123.70,110.25,106.35,70.33,68.46,59.83,57.30,56.41,53.17,42.96,38.43,36.36,33.84 ,31.92,30.30,29.49,29.16,29.06,26.84,23.47,22.44,22.15,21.86,17.17,16.41,14.84.LC/MS(ESI)m/z:[M+H]+1171.56.

Example 15: Synthesis of (2S,4R)-1-((2S)-2-(2-(4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)methyl)-1H-1,2,3-triazol-1-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(4-(methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN15).

1) Synthesis of 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-(but-3-yn-1-yloxy)-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one.

4-((S)-4-acryloyl-2-methylpiperazine-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (AMG510) (400.0 mg, 0.71 mmol), 3-bromoprop-1-yne (100.25 mg, 0.85 mmol), potassium carbonate (117.3 mg, 0.85 mmol) were dissolved in acetone (40 mL), and 2 drops of water were added, and the mixture was stirred and refluxed at 55°C for 12 h. After the reaction was completed, 20 mL of water was added, and the mixture was extracted 3 times with 30 mL of ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and dried by spin drying, and purified by column chromatography (dichloromethane: methanol = 20: 1) to obtain a yellow oil (195.39 mg, 46%). LC/MS (ESI) m/z: [M+H]+613.27.

2) Synthesis of (2S,4R)-1-((S)-2-(2-azidoacetylamino)-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide.

(2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (215.31 mg, 0.5 mmol), azidoacetic acid (60.61 mg, 0.6 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (228.14 mg, 0.6 mmol), and N,N-diisopropylethylamine (77.55 mg, 0.6 mmol) were dissolved in N,N-dimethylformamide (8 mL) and stirred at room temperature for 10 h. After the reaction was completed, 20 mL of water was added, and the mixture was extracted three times with 30 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and dried by spin drying. The mixture was purified by column chromatography (dichloromethane: methanol = 20: 1) to obtain a yellow solid (146.21 mg, 57%). LC/MS (ESI) m/z: [M+H] + 514.22.

3) Synthesis of (2S,4R)-1-((2S)-2-(2-(4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)methyl)-1H-1,2,3-triazol-1-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(4-(methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN15)

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-(but-3-yn-1-yloxy)-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (195.39 mg, 0.33 mmol), (2S,4R)-1-((S)-2-(2-azidoacetamido)-1-yl)-2-(2-(but-3-yn-1-yloxy)-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (195.39 mg, 0.33 mmol), 1H 2 O 2 0.75 mg, 1.3 mmol)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (200.15 mg, 0.39 mmol), copper sulfate pentahydrate (97.50 mg, 0.39 mmol) and sodium ascorbate (77.26 mg, 0.39 mmol) were dissolved in tetrahydrofuran (10 ml), and 2 ml of water was added. The mixture was reacted at room temperature for 10 h. After the reaction was completed, 20 mL of water was added, and the mixture was extracted 3 times with 30 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and dried by spin drying. The mixture was purified by column chromatography (dichloromethane: methanol = 20: 1) to obtain a yellow solid (245.75 mg, 67%). NMR (600MHz, DMSO-d6) δ8.99(s,1H),8.62(t,J＝6.1Hz,1H),8.57(d,J＝8.6Hz,1H),8.37(d,J＝4.7Hz,1H),8.29(dd,J＝20.5,8.9Hz,1H),7.94(d,J＝22.9Hz,1H), 7.49–7.38(m,6H), 7.19(dd,J＝36.7,6.8Hz,3H),6.88(dq,J＝35.2,10.3,9.5Hz,2H),6.21(dd,J＝17.0,9.8Hz,1H),5.78–5.73(m,1H),5.23(t,J＝10.5Hz,2H),5.19–5.09(m ,3H),4.90(s,1H),4.54(d d,J＝9.5,4.2Hz,1H),4.49–4.43(m,3H),4.36–4.19(m,5H),4.14(dq,J＝10.8,6.2,5.8Hz,1H),4.02(d,J＝14.6Hz,1H),3.72–3.63(m,3H),3.58(d,J＝ 10.5Hz,1H),2.05(t,J＝10.3H z,1H),1.91(dq,J＝8.1,4.4Hz,3H),1.75(d,J＝4.4Hz,2H),1.35(d,J＝6.8Hz,4H),1.25(d,J＝14.7Hz,3H),1.07(t,J＝4.7Hz,4H),0.94(d,J＝22.8Hz,14H),0 .81(d,J＝6.8Hz,1H).13C NMR (151MHz, DMSO) δ172.36,169.49,165.65,165.52,164.15,162.85,161.09,159.48,157.63,154.24,153.31,150.05,149.33,148.22,146.02,144.1 7,143.93,142.15,139.98,132.75,131.66,131.24,130.15,129.13,128.43,128.27,127.89,126 .53,123.82,110.03,108.66,106.12,69.36,62.69,59.26,57.32,57.02,51.78,49.57,46.08,45.20,44.29,42.14,38.55,36.06,30.20,29.48 ,26.81,26.73,22.33,22.24,22.09,17.39,16.42,15.98,15.24,14.50.LC/MS(ESI)m/z:[M+H]+1112.47.

Example 16: Synthesis of (2S,4R)-1-((2S)-2-(3-(4-(2-(4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)methyl)-1H-1-1,2,3-triazolyl)propionamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(4-(methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN16).

Synthesis method: With reference to Example 15, the product was separated by column chromatography (dichloromethane-methanol, volume ratio 20:1) to give a yellow solid (267.13 mg, 63%). 1H NMR (600 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.58 (t, J = 6.1 Hz, 1H), 8.38 (d, J = 4.9 Hz, 1H), 8.33–8.26 (m, 1H), 8.14 (dd, J = 9.3, 5.2 Hz, 1H), 7.90 (d, J = 7.2 Hz, 1H), 7.51–7.33 (m, 6 H),7.21–7.15(m,2H),6.89(dt,J＝14.2,7.5Hz,2H),6.21(dd,J＝16.9,9.7Hz,1H),5.76(dd,J＝10.2,2.4Hz,1H),5.17(d,J＝3.5Hz,1H),5.13–5.07(m,2H) ,4.90(s,1H),4. 58–4.48(m,3H),4.45–4.29(m,5H),4.22(dd,J＝15.9,5.5Hz,1H),3.69–3.62(m,3H),2.93(dq,J＝16.6,8.5,8.1Hz,1H),2.78–2.67(m,2H),2.44(s,3 H),2.06(dd,J=13.1 ,7.9Hz,1H),1.93–1.89(m,2H),1.78(s,2H),1.36–1.33(m,3H),1.23(d,J＝4.3Hz,1H),1.07(d,J＝6.7Hz,3H),0.92(d,J＝6.7Hz,2H),0.85(s,10H),0.8 2–0.80(m,1H).13C NMR (151MHz, DMSO) δ172.39,169.84,169.35,165.50,163.76,162.85,161.84,160.58,159.58,159.51,159.17,157.38,157.03,154.20,152.86,151.9 3,151.15,150.05,148.81,148.20,145.78,144.03,143.62,142.28,139.98,132.56,131.65,131.25,130 .13,129.12,128.43,128.27,127.90,124.95,123.78,123.49,109.57,108.67,106.07,69.37,63.47,59.19,56.95,56.89,49.33,46.45,45.77 ,44.96,42.12,38.44,35.70,35.58,30.21,26.69,22.35,22.09,17.40,16.41.LC/MS(ESI)m/z:[M+H]+1126.58.

Example 17: Synthesis of (2S,4R)-1-((2S)-2-(4-(2-(4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)methyl)-1H-1-1,2,3-triazol-1-yl)butanamido)-3-dimethylbutyryl)-4-hydroxy-N-(4-(4-(-4-(4-(methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN17).

Synthesis method: Refer to Example 15, and separate by column chromatography (dichloromethane-methanol, volume ratio 20:1) to obtain a yellow solid (243.63 mg, 59%) NMR(600MHz, DMSO-d6)δ8.99(s,1H),8.58(dt,J＝9.4,4.7Hz,1H),8.38(d,J＝4.8Hz,1H),8.29(dd,J＝18.3,7.2Hz,1H),8.02–7.97(m,2H),7.48–7.37(m,6H), 7.23–7.15(m,2H),6.89(dt,J＝17.5,10.5Hz,2H),6.24–6.19(m,1H),5.77(dd,J＝10.5,2.3Hz,1H),5.17–5.12(m,3H),4.90(s,1H),4.56(d,J＝9.3Hz,1 H),4.46–4.42(m ,2H),4.38–4.30(m,5H),4.23(dd,J＝15.8,5.5Hz,1H),3.69–3.62(m,3H),2.45(s,3H),2.29(dt,J＝15.0,7.5Hz,1H),2.19(td,J＝11.0,10.4,4.7Hz,1 H),2.08–1. 96(m,4H),1.94–1.88(m,3H),1.76(s,2H),1.34(t,J＝5.5Hz,4H),1.24(d,J＝6.4Hz,2H),1.07(t,J＝6.2Hz,4H),0.93(d,J＝11.3Hz,14H),0.79(d,J＝6. 8Hz,1H).13C NMR (151MHz, DMSO) δ172.43,171.51,170.08,165.51,163.78,162.86,159.55,157.40,157.36,154.20,153.21,151.93,151.43,150.04,148.79,148.2 0,145.76,144.14,142.44,139.99,132.60,131.66,131.24,130.13,129.32,129.12,128.43,128.26,127.90 ,124.79,124.70,123.77,106.09,69.37,62.79,59.19,56.94,56.87,49.49,42.13,38.42,35.73,32.07,30.20,29.49,26.89,26.84,26.70,24 .96,22.56,22.34,22.27,22.24,22.09,17.40,17.34,16.41,15.99,15.24,14.41.LC/MS(ESI)m/z:[M+H]+1141.27.

Example 18: Synthesis of (2S,4R)-1-((2S)-2-(5-(4-(2-(4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)methyl)-1H-1-1,2,3-triazol-1-yl)pentanamido)-3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(-4-(4-(methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN18).

Synthesis method: With reference to Example 15, the product was separated by column chromatography (dichloromethane-methanol, volume ratio 20:1) to give a yellow solid (253.26 mg, 67%). 1H NMR (600 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.59 (t, J = 6.1 Hz, 1H), 8.38 (d, J = 4.9 Hz, 1H), 8.30 (dt, J = 17.7, 7.3 Hz, 1H), 7.99-7.90 (m, 2H), 7.50-7.37 (m, 6H), 7.22-7.17 (m, 2H), 7.14 ( d,J＝4.9Hz,1H),6.89(dt,J＝21.3,10.5Hz,2H),6.26–6.18(m,1H),5.79–5.74(m,1H),5.18–5.10(m,3H),4.94–4.87(m,1H),4.55(d,J＝9.3Hz,1H),4 .45(ddd,J＝10.1,6.7,3.2H z,3H),4.38–4.31(m,5H),4.23(dd,J＝15.9,5.3Hz,1H),3.71–3.63(m,3H),2.45(s,3H),2.31(dt,J＝14.8,7.6Hz,1H),2.22–2.13(m,1H),2.05(ddd,J＝ 10.5,7.6,2.6Hz,1H),1. 96–1.88(m,3H),1.77(q,J＝6.5,5.8Hz,4H),1.43(ddt,J＝29.0,14.0,6.8Hz,3H),1.35(t,J＝5.7Hz,3H),1.23(s,2H),1.10–1.04(m,4H),0.93(s,12H) ,0.79(d,J＝6.7Hz,1H).13C NMR (151MHz, DMSO) δ172.45,172.13,170.16,165.53,163.79,162.86,161.06,159.43,157.38,154.21,151.92,150.05,148.77,148.36,145.79,144.0 3,142.42,142.38,139.99,132.55,131.66,131.25,130.13,129.32,129.12,128.54,128.46,128.40,128.25,1 27.90,124.75,124.67,123.78,123.55,110.15,109.77,106.06,69.37,62.83,62.70,59.19,56.87,56.83,49.52,42.13,40.51,38.43,35.67, 34.49,30.20,29.79,22.72,22.33,22.24,22.09,17.40,17.34,16.40,15.99,15.25.LC/MS(ESI)m/z:[M+H]+1155.36.

Example 19: Synthesis of (2S,4R)-1-((2S)-2-(6-(4-(2-(4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)methyl)-1H-1-1,2,3-triazolyl)hexanamido)-3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-(4-(methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN19).

Synthesis method: Refer to Example 15, and separate by column chromatography (dichloromethane-methanol, volume ratio 20:1) to obtain a yellow solid (243.63 mg, 62%). 1H NMR (600 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.58 (t, J = 6.1 Hz, 1H), 8.38 (d, J = 4.9 Hz, 1H), 8.30 (dt, J = 19.3, 7.9 Hz, 1H), 7.94 (d, J = 12.8 Hz, 1H), 7.87 (d, J = 9.3 Hz, 1H), 7.49–7.37 (m,6H),7.19(d,J＝7.1Hz,2H),7.14(d,J＝4.9Hz,1H),6.93–6.81(m,2H),6.21(t,J＝14.2Hz,1H),5.77(dd,J＝10.4,2.3Hz,1H),5.16–5.09(m,3H),4.90( s,1H),4.55(d,J＝ 9.4Hz,1H),4.46–4.41(m,3H),4.38–4.26(m,6H),4.23(dd,J＝15.8,5.3Hz,1H),3.73–3.63(m,4H),2.45(s,4H),2.25(dt,J＝14.7,7.6Hz,1H),2.14–2. 02(m,2H),1.95–1. 88(m,3H),1.76(q,J＝8.2Hz,4H),1.50(ddt,J＝21.5,13.7,6.8Hz,3H),1.35(t,J＝6.1Hz,3H),1.23(s,2H),1.17(q,J＝7.7Hz,2H),0.93(s,13H),0.78(d ,J=6.7Hz,1H).13C NMR (151MHz, DMSO) δ172.44,172.38,170.19,165.51,163.79,162.85,154.20,151.92,151.47,150.05,148.79,148.20,145.76,142.40,139.99,139.9 7,132.55,131.66,131.25,130.13,129.53,129.12,128.52,128.43,128.26,127.91,124.67,124.61,123.77,123 .57,112.10,110.17,109.46,108.67,108.18,106.08,69.36,62.83,62.69,59.18,56.85,56.78,49.70,42.13,38.43,35.67,35.08,30.20,29. 92,26.84,25.92,25.23,22.77,22.34,22.25,22.08,17.36,16.41,15.98,15.25,14.41.LC/MS(ESI)m/z:[M+H]+1169.34.

Example 20: Synthesis of (2S,4S)-1-((2S)-2-(4-(2-(4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)methyl)piperidin-1-yl)butanamido)-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-(-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YN20).

Synthesis method: Refer to Example 12, and separate by column chromatography (dichloromethane-methanol, volume ratio 20:1) to obtain a yellow solid (226.57 mg, 39%) 1H NMR (600 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.68-8.65 (m, 1H), 8.40-8.35 (m, 2H), 7.92 (d, J = 8.6 Hz, 1H), 7.46-7.38 (m, 7H), 7.19 (dd, J = 18.8, 5.0 Hz, 1H), 6.96 (d, J = 8.3 Hz, 1H), 6.87 (tt ,J＝8.6,4.1Hz,2H),6.22(dd,J＝16.9,10.6Hz,1H),5.77(d,J＝11.2Hz,2H),5.48(s,1H),4.92(s,1H),4.47(d,J＝8.4Hz,2H),4.42–4.36(m,2H),4.30(d ,J＝10.2Hz,2H),4.26–4.19 (m,2H),3.94(dd,J＝10.2,5.7Hz,1H),3.86–3.78(m,3H),3.71–3.66(m,1H),3.56–3.42(m,5H),2.94(s,2H),2.76–2.62(m,2H),2.38–2.31(m,2H),2. 26(dt,J＝14.6,7.4Hz,2H),2 .20–2.13(m,2H),1.94(s,2H),1.88(s,2H),1.76(dt,J＝12.3,6.0Hz,2H),1.50(q,J＝12.9Hz,3H),1.34(q,J＝5.7,5.1Hz,5H),1.23(s,3H),1.08(t,J ＝7.2Hz,5H),0.96(s,15H).13C NMR (151MHz, DMSO) δ172.94,172.45,170.41,165.52,164.02,163.71,163.01,161.16,159.42,159.35,157.79,154.24,154.18,151.94,150.08,150.0 3,148.86,148.23,145.84,145.43,144.29,144.15,139.70,132.61,131.63,131.23,130.22,129.15,128.43,128.26,1 27.93,123.76,123.69,123.44,111.59,109.19,109.00,108.30,106.02,69.58,59.00,57.67,56.08,55.38,52.52,42.66,37.41,35.16,32.88 ,30.30,30.25,29.48,26.85,22.48,22.28,22.14,21.88,17.47,17.39,16.40,15.99,15.27.LC/MS(ESI)m/z:[M+H]+1156.55.

Example 21: Synthesis of (2S,4R)-N-(2-(3-(4-(2-(4-(((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyridine[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)methyl)piperidin-1-yl)propoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-(((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidinecarboxamide (YN21).

1) Synthesis of (2S,4R)-N-(2-(3-chloropropoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutyryl)-4-hydroxypyrrolidine-2-carboxamide.

(2S, 4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (200 mg, 0.38 mmol), 1-bromo-3-chloropropane (71.73 mg, 0.46 mmol), potassium carbonate (63.48 mg, 0.46 mmol) were dissolved in acetone (40 mL), and 2 drops of water were added, and the mixture was stirred and refluxed at 55°C for 12 h. After the reaction was completed, 20 mL of water was added, and the mixture was extracted three times with 30 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and dried by spin drying. The mixture was purified by column chromatography (dichloromethane: methanol = 20: 1) to obtain a yellow oil (131.74 mg, 57%). LC/MS (ESI) m/z: [M+H]+ 609.22.

2), Synthesis of (2S,4R)-N-(2-(3-(4-(2-(4-(((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyridin[2,3-d]pyrimidin-7-yl)-3-fluorophenoxy)methyl)piperidin-1-yl)propoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-(((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidinecarboxamide (YN21).

4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-(piperidin-4-ylmethoxy)phenyl)-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (200 mg, 0.30 mmol), (2S,4R)-N-(2-(3-chloropropoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (182.40 mg, 0.30 mmol), and potassium carbonate (41.40 mg, 0.30 mmol) were dissolved in N,N-dimethylformamide (8 mL) and stirred at room temperature for 10 h. After the reaction was completed, 20 mL of water was added, and the mixture was extracted three times with 30 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and dried by spin drying. The mixture was purified by column chromatography (dichloromethane: methanol = 20: 1) to obtain a yellow solid (173.37 mg, 47%). NMR(600MHz, DMSO-d6)δ8.98(s,1H),8.53(t,J＝6.2Hz,1H),8.38(d,J＝5.1Hz,2H),7.43(dd,J＝11.7,7.6Hz,2H),7.31–7.27(m,1H),7.19(dd,J＝23.6,5.0Hz,2H) ,7.03–6.95(m,4H),6.89–6.83(m,2H),6.21(dd,J＝16.7,10.7Hz,1H),5.76(dd,J＝10.1,2.4Hz,1H),5.21(s,1H),4.92(d,J＝22.7Hz,1H),4.61(d,J＝9.2Hz , 1H),4.54(t,J＝8.2Hz,1H),4.39–4.21(m,7H),4.14–4.02(m,4H),3.84(dt,J＝20.2,8.3Hz,3H),3.71–3.59(m,4H),2.89(s,1H),2.73(d,J＝9.8Hz,3H ),2.46(s,4H),2.14–2.08(m,2H),2.03–1.85(m,9H),1.36(dt,J＝16.3,10.2Hz,8H),1.28–1.21(m,6H),1.16(d,J＝8.4Hz,2H),1.07(d,J＝6.6Hz,5H) ,0.96(s,16H).13C NMR (151MHz, DMSO) δ172.29,169.40,168.62,168.49,165.50,164.00,163.76,163.02,162.79,157.79,156.21,154.18,153.50,151.92,151.55,150.0 8,148.84,148.35,145.94,145.44,132.61,131.77,131.38,131.23,128.42,128.28,127.48,123.75,121.74,112.15,79.79 ,78.24,69.38,68.98,66.50,60.60,59.69,57.17,57.02,56.30,54.91,38.38,37.70,37.03,36.24,31.23,30.30,30.07,26.68,22.47,22.27, 22.15,21.89,21.22,17.91,17.48,17.39,16.45,15.98,15.27,14.55,13.48,13.42,13.22,13.15.LC/MS(ESI)m/z:[M+H]+1230.57.

Performance evaluation:

1. Evaluation of the anti-proliferation activity of the compounds in human non-small cell lung cancer cells NCI-H358 and human pancreatic cancer cells Miapaca-2

In this experiment, the cell models selected were the KRASG12C-overexpressing NCI-H358 non-small cell lung cancer cell line and the Miapaca-2 human pancreatic cancer cell line. At the same time, the KRASG12C-independent HepG2 liver cancer cell line was selected, in which the KRASG12C expression level was low. At the same time, the normal human embryonic kidney cell 293T cell line was selected, and the inhibitory activity of the 21 compounds in the embodiment on the above cell lines was determined. The evaluation method and results are described below.

1. Experimental materials and instruments

1.1 Experimental Materials and Instruments

2. Experimental steps

(1) Solution preparation

① Dissolve each of the above compounds in DMSO to prepare a 10 mM stock solution. Compounds used within three months should be stored in a desiccator at room temperature, and others can be stored long-term at -20°C.

② The above compound stock solutions and the positive reference compound AMG510 were diluted with DMSO, with a starting concentration of 10 μM, and 3-fold gradient dilutions were performed in NCI-H358, Miapaca-2, HepG2, and 293T cell experiments, with 10 concentration points. Oscillate on an oscillator for 5 minutes.

The chemical formula of the positive reference compound AMG510 is as follows:

(2) Cell dosing

① NCI-H358, Miacapa-2, HepG2, and 293T cells in the logarithmic growth phase were seeded in a 96-well plate, with 3000-4000 cells per well, and the culture plate was pre-cultured in an incubator for 24 hours (under the conditions of 37° C. and 5% CO 2 ).

② Replace the culture medium in the well plate and add 50 μL of compounds of corresponding concentrations and positive controls to the culture plate.

③ Incubate the culture plate in an incubator for 144 hours, add 10 μl of CCK-8 solution to each well, and then incubate the culture plate in an incubator for 1-4 hours.

④Measure the absorbance at 450nm using an enzyme-labeled instrument.

(3) Data processing

① Calculate %Inhibition:

%Inhibition=100-(Signalcmpd-SignalAve_PC)/(SignalAve_VC-SignalAve_PC)×100.

②Calculate the IC50 and Plot effect dose curve of the compound:

IC50 values were calculated by fitting the logarithm of % Inhibition and compound concentration to a nonlinear regression (dose response - variable slope) using GraphPad 6.0.

Y＝Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope))

X: log of inhibitor concentration; Y: %Inhibition.

3. Experimental results

After calculating %Inhibition, the dose-effect curve was drawn with the logarithm of the test compound concentration as the abscissa and the average cell survival rate as the ordinate, and the IC50 value was fitted. The results showed:

As shown in Table 1, each compound exhibited excellent anti-proliferative activity against KRASG12C-overexpressing NCI-H358 non-small cell lung cancer cells and Miapaca-2 human pancreatic cancer cells, among which YN14 and YN21 had comparable activities; they exhibited relatively weak anti-proliferative activity against HepG2 cells expressing wild-type KRASWT and normal 293T cells, and their IC50 values were all greater than 10 μM, indicating that the compounds of the present application specifically target KRASG12C cells and have high selectivity.

Table 1. Antiproliferative activity (IC50 values) of KRASG12C degraders and AMG510 on NCI-H358, Miapaca-2, HepG2, and 293T cells

2. Western-Blot test to determine the degradation effect of compounds on KRASG12C protein In this experiment, the cell models selected KRASG12C overexpressing NCI-H358 non-small cell lung cancer cell line and Miapaca-2 human pancreatic cancer cell line, and the compounds YN4 and YN14 with excellent anti-tumor proliferation activity in Table 1 were selected to determine their degradation effect on KRASG12C protein. The evaluation method and results are described below.

1. Experimental Materials and Instruments

1.1 Experimental consumables for biological activity evaluation

Semi-dry transfer apparatus, cell counting chamber, micropipette, gel imaging system and chemiluminescence imaging system were purchased from Bio-Rad, USA.

2. Experimental Procedure

2.1 Preparation of working solution:

The above compounds and the positive reference compound AMG510 were diluted with DMSO, with a starting concentration of 2430 nM, and in NCI-H358 and Miapaca-2 cell experiments, a 3-fold gradient dilution with 11 concentration points was performed. The mixture was shaken on an oscillator for 5 minutes.

2.2 Cell Dosing

2.2.1 NCI-H358 and Miapaca-2 cells in the logarithmic growth phase were seeded in 6-well plates with 1 million cells per well. The culture plates were pre-cultured in an incubator for 24 hours (at 37° C., 5% CO ₂ ).

2.2.1 Replace the culture medium in the well plate and add 100 μL of the corresponding concentration of compound and positive drug to the culture plate.

2.3 Preparation of total cell protein samples:

Wash the cells 2-3 times with PBS. Aspirate the residual liquid thoroughly for the last time. Add an appropriate volume of total cell protein extraction reagent and collect the cells into a 1.5 ml centrifuge tube with a cell scraper. Centrifuge at 12000g for 5 minutes, collect the supernatant and determine the protein concentration.

2.4 SDS-PAGE electrophoresis, membrane transfer, blocking, primary antibody and secondary antibody incubation were performed in sequence, and finally the compound was luminescently developed, and the grayscale value was quantitatively analyzed using imageJ, and the % degradation and maximum degradation rate (Dmax) were calculated. GraphPad 6.0 was used to draw the dose-effect curve with the logarithm of the test compound concentration as the horizontal axis and the average % degradation value as the vertical axis, and the half effective degradation concentration DC50 value was fitted.

3. Test results

As shown in Figure 1 and Table 2, it can be observed from the Western-Blot test that YN4 and YN14 both have significant KRASG12C degradation effects in NCI-H358 and Miapaca-2 cell lines, and their DC50s are both at the nanomolar level, and Dmax is greater than 90%. Among them, the DC50 of YN14 in NCI-H358 and Miapaca-2 cells is 67nM and 28nM, respectively, and Dmax>95%, indicating that the compound YN14 of this example is a highly effective KRASG12C degrader.

Table 2. Effects of KRAS degraders and AMG510 on KRAS protein degradation in NCI-H358 and Miapaca-2 cells

Note: —— indicates that no obvious degradation results were detected.

3. Determination of the induction effect of YN14 on tumor cell apoptosis

In this example, the cell models selected were NCI-H358 non-small cell lung cancer cell line overexpressing KRASG12C and Miapaca-2 human pancreatic cancer cell line, and compound YN14 with excellent KRASG12C protein degradation effect in Table 2 was selected to measure its induction effect on tumor cell apoptosis. The evaluation method and results are described below.

1. Experimental Materials and Instruments

1.1 Experimental consumables for biological activity evaluation

2. Experimental Procedure

2.1 Take cells in the logarithmic growth phase, digest them with 0.25% trypsin and beat them into single cells, and suspend the cells in DMEM culture medium containing 10% fetal bovine serum for later use.

2.2 Dilute the cell suspension in multiples and seed it in a 6-well plate, with 3-5×10 ⁴ cells per well. Gently rotate the plate to disperse the cells evenly. Culture the plate overnight in a cell culture incubator at 37°C, 5% CO ₂ and saturated humidity.

2.3 Preparation of working solution: The above compounds were diluted with DMSO and shaken on a shaker for 5 minutes.

2.4 Replace the culture medium in the culture plate with complete culture medium containing different concentrations of the test substance and treat for 24-48 hours.

2.5 The adherent cells were collected by digestion with EDTA-free trypsin (3000 rpm, centrifugation for 5 min), and the cells were washed twice with PBS (3000 rpm centrifugation for 5 min), and 10 ⁵ cells were collected.

2.6 Add 500 μL of Binding Buffer to suspend the cells, then add 5 μL of Annexin V-PE and 5 μL of PI and mix well.

2.7 After 5-15 minutes of reaction at room temperature in the dark, flow cytometry observation and detection were performed. Excitation wavelength Ex = 488 nm; emission wavelength Em = 578 nm. Normal cells treated with apoptosis induction were used as controls for fluorescence compensation adjustment to remove spectral overlap and set the position of the cross gate.

3. Test results

As shown in Figure 2 and Table 3, the experimental results showed that YN14 significantly induced apoptosis of NCI-H358 and Miapaca-2 cells at concentrations of 100 nM and 300 nM, and had a dose-dependent effect.

Table 3 Effect of KRAS degrader YN14 on the percentage of apoptotic cells in NCI-H358 and Miapaca-2

IV. Determination of the inhibitory effect of YN14 on tumor cell cycle

In this experiment, the cell models selected were NCI-H358 non-small cell lung cancer cell line overexpressing KRASG12C and Miapaca-2 human pancreatic cancer cell line, and the compound YN14 with excellent KRASG12C protein degradation in Table 1 was selected to measure its blocking effect on tumor cell cycle. The evaluation method and results are described below.

1. Experimental Materials and Instruments

1.1 Experimental consumables for biological activity evaluation

2. Experimental Procedure

2.1 Preparation of cell samples

2.1.1 Take cells in the logarithmic growth phase, digest them with 0.25% trypsin and beat them into single cells, and suspend the cells in DMEM culture medium containing 10% fetal bovine serum for later use.

2.1.2 Dilute the cell suspension in multiples and seed it in a 6-well plate, with 3-5×10 ⁴ cells per well. Gently rotate the plate to disperse the cells evenly. Culture the plate overnight in a cell culture incubator at 37°C, 5% CO ₂ and saturated humidity.

2.1.3 Preparation of working solution: The above compounds and positive reference compound AMG510 were diluted with DMSO and shaken on a shaker for 5 minutes.

2.1.4 Replace the culture medium in the culture plate with complete culture medium containing different concentrations of the test substance and treat for 24-48 hours.

2.1.5 Digest the cells with trypsin (without EDTA), collect all the adherent cells, and gently blow away the cells to collect about 10 ⁶ cells.

2.1.6 After washing twice with PBS, resuspend the cells in 250 μL pre-cooled PBS.

2.2 Cell fixation

Slowly add 750 μL of pre-cooled anhydrous ethanol, mix gently by pipetting, fix at -20°C overnight, and seal with a sealing film.

2.3PI staining

2.3.1 Take the fixed cells at -20℃ and centrifuge at 3000r/min for 3-5min to precipitate the cells. Carefully remove the supernatant.

2.3.2 After washing twice with PBS, add 200 μL pre-cooled PBS to resuspend the cells.

2.3.3 Add 0.2 mL RNase A (1 mg/mL, dissolved in PBS) and incubate in a 37°C water bath for 30 min.

2.3.4 Add 5 μL of propidium iodide staining solution to each tube of cell sample, then add 0.3 mL of PBS, mix slowly and thoroughly, and incubate at 4°C in the dark for 30 minutes.

2.4 Flow cytometry and analysis

The cytometer detected red fluorescence at an excitation wavelength of 488 nm and simultaneously detected light scattering. FlowJoX10.0 software was used to analyze the cell DNA content.

3. Test results

As shown in Figure 3, the experimental results showed that the G2/M phase content increased significantly under the action of 100nM and 300nM of YN14, indicating that YN14 blocked the NCI-H358 and Miapaca-2 cell cycle in the G2/M phase in a dose-dependent manner.

V. Determination of the inhibitory activity of YN14 on tumor cell metastasis

In this experiment, the cell model selected was the KRASG12C overexpressing NCI-H358 non-small cell lung cancer cell line, and the compound YN14 with excellent KRASG12C protein degradation in Table 1 was selected, and its inhibitory effect on tumor cell metastasis was determined by scratch test. The evaluation method and results are described below.

1. Experimental Materials and Instruments

1.1 Experimental consumables for biological activity evaluation

2. Experimental Procedure

2.1 All sterilizable instruments must be sterilized, and rulers and marker pens must be exposed to ultraviolet light for 30 minutes before operation.

2.2 Use a marker pen to draw even horizontal lines on the back of the 6-well plate, using a ruler to measure, approximately every 0.5 to 1 cm, across the holes. Make at least 5 lines across each hole.

2.3 Take cells in the logarithmic growth phase, digest them with 0.25% trypsin and blow them into single cells, and suspend the cells in DMEM culture medium containing 10% fetal bovine serum for later use.

2.4 Add about 5-10×10 ⁵ cells to a 6-well plate. After overnight, the cells will cover the entire 6-well plate. The next day, use a 200 μL pipette tip to vertically measure the ruler and scratch horizontally.

2.5 Preparation of working solution: The above compounds and positive reference compound AMG510 were diluted with DMSO and shaken on a shaker for 5 minutes.

2.6 Wash the cells three times with PBS to remove the scratched cells and replace the culture medium with the target conditioned medium.

2.7 Randomly select several points to take pictures under a microscope and record the initial position of the cells. Place the cells in a 37°C, 5% CO2 incubator for culture.

2.8 Samples were taken every 12 hours, and photos were taken again under a microscope at the location of the initial photo to record the location of cell migration. Image processing software was used to measure the distance of cell migration.

3. Test results

As shown in Figure 4, the cell scratch assay showed that YN14 inhibited the migration ability of NCI-H358 cells in a dose-dependent manner at nanomolar concentrations, and had a dose-dependent effect.

VI. Determination of the inhibitory and degradation activity of YN14 on the growth of nude mouse xenograft tumors In this experiment, the human pancreatic cancer Miacapa-2 cell line was selected as the cell model, and YN14, a compound with excellent KRASG12C protein degradation ability in Table 1, was selected to determine its inhibitory effect on the growth of tumor cell nude mouse xenograft tumors. The evaluation method and results are described below.

1. Experimental Materials and Instruments

1.1 Experimental consumables for biological activity evaluation

2. Experimental Procedure

The cells in logarithmic growth phase were digested with trypsin, counted and resuspended in PBS, and 5×10 ⁶ Miapaca-2 cells were inoculated into the mammary pads of female BALB/c nude mice aged 4 to 6 weeks (generally selected vascular-rich sites such as the axilla or groin), and each nude mouse was injected with 0.1 mL of cell suspension. According to different experimental requirements, 8 mice per group can be set. The dosing regimen for animal experiments was: solvent control, YN14 (15 mg/kg), YN14 (30 mg/kg), intraperitoneal administration, once a day.

Starting from the inoculation time, the size of the tumor growth was observed and measured every 2 days, and the maximum diameter (L) and minimum diameter (D) of the tumor were measured. The calculation formula of the tumor volume was V = L × D2 × 1/2. The nude mice were weighed every 2 days. 24 hours after the last administration, the tumor inhibition rate TGI (%) was calculated according to the tumor volume size = (Vc-Vt)/(Vc-V0)*100, where Vc, Vt and V0 were the initial administration tumor volume, the tumor volume of the administration group and the tumor volume of the control group, respectively.

The mice were killed, and the subcutaneously transplanted tumors were dissected and weighed. Part of the tumor tissue was fixed with paraformaldehyde solution for 48 hours, embedded in paraffin, and sliced. Then, HE (hematoxylin-eosin staining) and immunohistochemical staining were performed for analysis.

3. Test results

As shown in Figures 5 and 6 and Table 4, the experimental results show that compound YN14 significantly inhibits the growth of Miacapa-2 transplanted tumors and has a dose-dependent effect. When the dose of YN14 was 30 mg/kg, the tumor inhibition rate exceeded 100%. There was no weight loss and other toxic side effects in nude mice during the experiment, indicating that YN14 has good tolerance. HE staining, immunohistochemistry and western-blot were performed on the tumors of each group. The results showed (Figure 6) that YN14 significantly inhibited KRAS, tumor vascular marker CD31, reduced the content of p-ERK and p-AKT in tumors, and increased the content of cleaved-caspase 3, a cell apoptosis marker.

Table 4. Inhibitory effect of the degradation agent YN14 on human pancreatic cancer Miacapa-2 xenograft tumors in nude mice

It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the main purpose of the present application, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims (11)

1. A compound having a structure as shown in Formula I, or a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph thereof, M-L-E Formula I Wherein: M represents the ligand of KRAS ^G12C protein, L represents the linker chain, and E represents the ligand of E3 ubiquitin ligase. 2. The compound according to claim 1, its pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph, characterized in that M is a structure shown in Formula II: wherein ^X1 and ^X2 are each independently halogen, and the number of ^X1 is 1 or 2, and the number of ^X2 is 1, 2, 3 or 4, preferably the number of ^X1 and ^X2 is both 1; ^R1 and ^R2 are each independently a substituted or unsubstituted C1-C6 alkyl group, the number of ^R2 is 1, 2 or 3, preferably the number of ^R2 is 1; preferably ^R1 and ^R2 are each independently a substituted or unsubstituted C1-C3 alkyl group, preferably ^R1 is n-propyl or isopropyl, preferably ^R2 is methyl or ethyl; Preferably, M is 3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph thereof, characterized in that: L is Wherein, L ¹ is C1-C20 alkylene, C1-C20 haloalkylene, (CH ₂ CH ₂ O) m-(C1-C6 alkylene), (C1-C6 alkylene)-(3-8 membered heterocycloalkylene)-(C1-C6 alkylene), (C1-C6 alkylene)-(5-10 membered heteroarylene)-(C1-C6 alkylene), and m is any integer between 1 and 20; n is 0 or 1, Preferably, n is 0, and L ¹ is (C1-C6 alkylene)-(3-8 membered heterocycloalkylene)-(C1-C6 alkylene); Preferably, n is 1, L ¹ is C1-C20 alkylene, C1-C20 haloalkylene, (CH ₂ CH ₂ O) m-(C1-C6 alkylene), (C1-C6 alkylene)-(3-8 membered heterocycloalkylene)-(C1-C6 alkylene), (C1-C6 alkylene)-(5-10 membered heteroarylene)-(C1-C6 alkylene), and m is any integer between 1 and 20; Preferably, L ¹ is C1-C10 alkylene, C1-C10 haloalkylene, (CH ₂ CH ₂ O) m-(C1-C3 alkylene), (C1-C3 alkylene)-(4-6 membered heterocycloalkylene)-(C1-C6 alkylene), (C1-C3 alkylene)-(5-6 membered heteroarylene)-(C1-C6 alkylene), m is any integer between 1 and 6, and n is 0 or 1; Preferably, the 4-6 membered heterocycloalkylene in the (C1-C3 alkylene)-(4-6 membered heterocycloalkylene)-(C1-C6 alkylene) is selected from aziridinyl, aziridinyl and aziridinyl; preferably, the 4-6 membered heterocycloalkylene in the (C1-C3 alkylene)-(4-6 membered heterocycloalkylene)-(C1-C6 alkylene) is selected from piperidinyl, morpholinyl and piperazinyl; preferably, the 4-6 membered heterocycloalkylene in the (C1-C3 alkylene)-(4-6 membered heterocycloalkylene)-(C1-C6 alkylene) is selected from Preferably, the 5-6 membered heteroarylene group in the (C1-C3 alkylene)-(5-6 membered heteroarylene)-(C1-C6 alkylene) is selected from pyrrolylene, pyrazolylene, imidazolylene, triazolylene; preferably, the 5-6 membered heteroarylene group in the (C1-C3 alkylene)-(5-6 membered heteroarylene)-(C1-C6 alkylene) is selected from triazolylene, 1,2,4-triazolylene, 1,2,5-triazolylene, 1,3,4-triazolylene; preferably, the 5-6 membered heteroarylene group in the (C1-C3 alkylene)-(5-6 membered heteroarylene)-(C1-C6 alkylene) is selected from Preferably, L is any one of the following structures: Wherein, n1 represents any integer between 1-20, preferably any integer between 1-6; m represents any integer between 1-20, preferably any integer between 1 and 6; n2 represents any integer between 1-6, The terminal is connected to M. Connect the E end. 4. The compound, pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph thereof according to any one of claims 1 to 3, characterized in that E is any one of the following structures: 5. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph thereof, characterized in that the structural formula of the compound represented by formula I is any one of the structures YN1-YN21: 6. A method for preparing the compound according to any one of claims 1 to 5, comprising any one or more of the following synthetic routes: Synthesis route A: AMG510 is used as a raw material and reacted with tert-butyl bromate to prepare intermediate _A1 ; the intermediate _A1 is hydrolyzed under trifluoroacetic acid to form intermediate _A2 ; the intermediate _A2 is condensed with VHL ligand VH032 amide to form target compound _A3 . Wherein: n1 represents any integer between 1 and 20, preferably any integer between 1 and 6; Synthesis route B: AMG510 is used as a raw material and reacted with tert-butyl bromide to prepare intermediate _B1 ; the intermediate _B1 is hydrolyzed under trifluoroacetic acid to form intermediate _B2 ; the intermediate _B2 is condensed with E3 ligase ligand amide to generate target compound _B3 or _B4 . Wherein: m represents any integer between 1 and 20, preferably any integer between 1 and 6; Synthesis route C: AMG510 is used as a raw material, and reacted with tert-butyl bromoester to prepare intermediate _C1 ; the intermediate _C1 is hydrolyzed under trifluoroacetic acid to form intermediate _C2 ; the intermediate _C2 is reacted with tert-butyl bromopropionate to prepare intermediate _C3 , the intermediate _C3 is hydrolyzed under trifluoroacetic acid to form intermediate _C4 , and the intermediate _C4 is condensed with VHL ligand VH032 amide to generate target compound _C5 ; Wherein: n3 represents any integer between 1 and 6, preferably represents any integer between 1 and 3, and X is C or N; Synthesis route D: AMG510 is used as a raw material and reacted with 3-bromoprop-1-yne to prepare intermediate D ₁ ; hydrazoic acid and VHL ligand VH032 amide are condensed to form intermediate D ₂ ; intermediate D ₁ and intermediate D ₂ are reacted by click chemistry to generate target compound D ₃ , Where: n2 represents any integer between 1 and 6; Synthesis route E: 1-bromo-3-chloropropane reacts with VHL ligand VH101 to form intermediate E ₁ , and intermediate E ₁ reacts with intermediate C ₂ in synthesis route C to obtain target compound E ₂ , Wherein: n3 represents any integer between 1 and 6, and X represents C or N. 7. Use of a compound as shown in formula I according to any one of claims 1 to 5, a pharmaceutically acceptable salt, a prodrug, a stable isotope derivative, an isomer, a solvate or a polymorph thereof in the preparation of a degradation agent for targeted degradation of KRASG12C. 8. The use according to claim 7, characterized in that the targeted degradation agent for KRASG12C is a degradation agent for treating or preventing tumor diseases, and the tumor diseases include one or more of gastric cancer, oral cancer, esophageal cancer, thyroid cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, colorectal cancer, kidney cancer, osteosarcoma, medulloblastoma, rhabdomyosarcoma, and glioblastoma. 9. Use of the compound of formula I as described in any one of claims 1 to 5, its pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph and other drugs in the preparation of a drug for treating or preventing tumor diseases, wherein the other drugs include any one or more of infigratinib, crizotinib, MK-2206, selumetinib, Dactolisib, Naporafenib, SHP099, AZD457, linsitinib, ibrutinib, cyclophosphamide, doxorubicin, cytarabine, azacitidine, decitabine, carfilzomib, thalidomide, lenalidomide, pomadomine, gefitinib, osimertinib, erlotinib, tepotinib, ositinib, afatinib, flutamide and nilutamide. 10. The use according to claim 9 is characterized in that the tumor disease includes one or more of gastric cancer, oral cancer, esophageal cancer, thyroid cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, colorectal cancer, kidney cancer, osteosarcoma, medulloblastoma, rhabdomyosarcoma, and glioblastoma. 11. A pharmaceutical composition comprising a compound as shown in any one of claims 1 to 5, a pharmaceutically acceptable salt, a prodrug, a stable isotope derivative, an isomer, a solvate or a polymorph thereof, the pharmaceutical composition also comprising a pharmaceutically acceptable carrier or excipient thereof, the carrier comprising any one or more of an ion exchanger, aluminum oxide, aluminum stearate, lecithin, serum protein, a buffer substance, glycerol, sorbic acid, potassium sorbate, a partial glyceride mixture of saturated vegetable fatty acids, water, salt, electrolyte, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, colloidal silicon oxide, magnesium trisilicate, polyvinyl pyrrolidone, a cellulosic substance, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, beeswax and lanolin.