Method for synthesizing cyclic α,β-unsaturated compound, and coupling thereof with sulfydryl

Abstract

Disclosed in the present invention are a method for synthesizing a cyclic α,β-unsaturated compound, and the coupling thereof with a sulfhydryl. Specifically, the present invention relates to a method for synthesizing a 5- or 6-membered α,β-unsaturated cyclic compound by reacting a compound containing an aldehyde group with a compound containing an active methylene at the α-position as starting materials.

Description

Synthetic methods of a class of cyclic α,β-unsaturated compounds and their coupling with thiol groups

[0001] This application claims priority to Chinese patent application 2024118016012, filed on December 9, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention provides a cyclic α,β-unsaturated compound and its synthesis method, belonging to the field of pharmaceutical technology involving thiol coupling, including but not limited to the preparation and development of covalent drugs and antibody-drug conjugates. Background Technology

[0003] The typical mode of drug binding to target proteins is through non-covalent interactions. Non-covalent interactions, including electrostatic interactions, van der Waals interactions, hydrogen bonds, and hydrophobic interactions, can enhance the affinity and specificity of ligands with receptors, enzymes, or ion channels. However, these non-covalent bindings are reversible and can be competitively discontinued by endogenous substrates, affecting drug efficacy.

[0004] Covalent drugs are small molecule compounds that can covalently bind to specific target proteins, thereby regulating their biological functions. These compounds often contain functional groups such as acrylamide, β-lactam, sulfonyl fluoride, and ethylene oxide, which can chemically react with specific amino acid residues in the target protein, such as cysteine, serine, lysine, and glutamate residues, to form covalent bonds, thereby regulating the biological functions of the corresponding protein. Compared with non-covalent compounds, covalent compounds have advantages such as longer duration of action. Several covalent drug molecules are currently on the market, such as afatinib and ibrutinib. These compounds form stable covalent bonds by coupling the acrylamide functional group to the thiol group of the cysteine ​​residue in the target protein, thereby inhibiting the activity of the target protein. However, the reactivity and selectivity of existing covalent functional groups are still not ideal, and they also have disadvantages such as poor water solubility, which is not conducive to drug formulation.

[0005] Conjugation technology has wide applications in tumor therapy, disease diagnosis, and efficient screening. Antibody-drug conjugates (ADCs) consist of an antibody, a linker, and an effector molecule linked together. After entering tumor tissue via the bloodstream, ADCs bind to receptors on the surface of tumor cells. Subsequently, under receptor-mediated endocytosis, effector molecules that specifically release tumor cell growth inhibition or apoptosis occur. The conjugation of the linker to the antibody is a crucial step in ADC drug preparation, fundamentally altering the pharmacokinetics and therapeutic index of the ADC. Conjugation of functional groups such as maleimide to cysteine ​​residues containing thiol groups in antibodies is currently the most common conjugation technique in ADC drug preparation. However, existing techniques suffer from drawbacks such as low reaction yields and easy drug polymerization. Summary of the Invention

[0006] This invention provides a method for synthesizing compounds containing novel cyclic α,β-unsaturated functional groups. Surprisingly, compounds containing these cyclic α,β-unsaturated functional groups can undergo rapid covalent reactions with thiol groups, thereby enabling the preparation and development of various drugs targeting or involving thiol coupling, including but not limited to covalent drugs and antibody-drug conjugates.

[0007] Synthetic methods for cyclic α,β-unsaturated compounds include preparation methods based on the following general formulas (1) and (2):

[0008] in,

[0009] Z can be NR4, O, or S independently;

[0010] m can be 0, 1, 2, 3, or 4;

[0011] R1 and R2 are each independently selected from halogens, H, deuterium, substituted or unsubstituted (C1-C6) alkyl, (C1-C6) alkoxy, 6-10 aryl or 5-10 heteroaryl, or R1 and R2 together with the commonly linked atoms form (C3-C6) cycloalkyl, 3-10 saturated or partially unsaturated heterocycloalkyl, 7-11 heterospirocycloyl, 5-11 heterobridged cycloyl;

[0012] R3 and R4 are each independently selected from H, (C1-C6)alkyl, (C3-C12)cycloalkyl, (C1-C6)alkoxy, -OR k -OH, -NR k R l -CN, -C(O)R k -C(O)OR k -S(O)2R k -S(O)R k-CH2OC(O)R k -CH2OC(O)OR k -C(S)R k -C(S)OR k -C(O)SR k -C(O)NR k R l -C(S)NR k R l -C(O)ONR k R l -(C1-C6)alkyl-OR k -(C1-C6)alkyl NR k R l -S(O)2NR k R l -P(O)R k R l -(C1-C6)alkyl-(C3-C12)cycloalkyl, -(C1-C6)alkyl-(C3-C12)heterocyclic alkyl, 3- to 12-membered saturated or partially unsaturated heterocyclic alkyl, 7- to 12-membered heterospirocyclic alkyl or 5- to 12-membered heterobridged cycloalkyl, 6- to 10-membered aryl, 5- to 15-membered heteroaryl, wherein the (C1-C6)alkyl, (C3-C12)cycloalkyl, (C1-C6)alkoxy, -(C1-C6)alkyl-(C3-C12)cycloalkyl, -(C1-C6)alkyl-(C3-C12)heterocyclic alkyl, 3- to 12-membered saturated or partially unsaturated heterocyclic alkyl, 7- to 12-membered heterospirocyclic alkyl or 5- to 12-membered heterobridged cycloalkyl, 6- to 10-membered aryl, 5- to 15-membered heteroaryl are optionally further surrounded by 1, 2, 3, 4 or 5 Rs. m Substitution; two hydrogen atoms on the same carbon atom can be replaced by oxygen to form an oxo group or by... Replaced;

[0013] Alternatively, in general formula (1), R2 and R3 together with the atoms between them form 4-10 saturated or partially unsaturated heterocyclic alkyl groups, 7-11 heterospirocyclic groups, and 5-11 heterobridged cyclic groups;

[0014] Alternatively, in general formula (2), one R1 and R3 together with the atoms between them form a 4- to 10-membered saturated or partially unsaturated heterocyclic alkyl group, a 7- to 11-membered heterospirocyclic group, or a 5- to 11-membered heterobridged cyclic group;

[0015] R k and R lEach of the following is independently H, -C(O)R, -C(O)OR, (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C12)cycloalkyl, 3- to 12-membered saturated or partially unsaturated heterocyclic alkyl, 7- to 12-membered heterospirocyclic alkyl or 5- to 12-membered heterobridged cycloalkyl, 6- to 10-membered aryl, 5- to 12-membered heteroaryl, wherein the (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C12)cycloalkyl, 3- to 12-membered saturated or partially unsaturated heterocyclic alkyl, 7- to 12-membered heterospirocyclic alkyl or 5- to 12-membered heterobridged cycloalkyl, 6- to 10-membered aryl, 5- to 12-membered heteroaryl is optionally further surrounded by 1, 2, 3, 4 or 5 Rs. m Substitution; two hydrogen atoms on the same carbon atom can be replaced by oxygen to form an oxo group or by... Replaced;

[0016] Or R k and R l Together with the commonly linked atoms, they form 3- to 10-membered saturated or partially unsaturated heterocyclic alkyl groups, 7- to 11-membered heterospirocyclic groups, and 5- to 11-membered heterobridged cyclic groups, wherein the heterocyclic alkyl groups, heterospirocyclic groups, and heterobridged cyclic groups may optionally be further bound by 1, 2, 3, 4, or 5 R atoms. n replace;

[0017] R a and R b Each is independently selected from hydrogen, deuterium, alkyl, deuterated alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, cycloalkyl, or heterocycloalkyl; or R a and R b Together with the commonly linked atoms, they form 3- to 10-membered cycloalkyl or heterocycloalkyl groups, which may optionally be further surrounded by 1, 2, 3, 4, or 5 R groups. n replace;

[0018] R mSelected from deuterium, H, halogen, OH, SH, NH2, -CN, -NC, -NCS, -N3, NO2, -CONH2, (C1-C6)alkyl, (C1-C6)alkyl-(C3-C6)cycloalkyl, (C1-C6)alkyl-(C3-C6)heterocyclicalkyl, (C1-C6)haloalkyl, (C2-C6)alkynyl, (C2-C6)alkenyl, (C3-C6)cycloalkyl, -N=S(O)(R)2, -S(O)=(NR)R, -SCN, -S(O)2CN, -SNC, -S(O)2NC, -S(O)2N(R)2, -SF5, -P(O)(R)2, -P(S)(R)2, -P(O )(OR)R, -P(O)(OR)2, -B(OR)2, -B(R)2, -BH(OR), -SeR, -SeCN, -NCSe, -Si(R)3, -Si(OR)3, -SiR(OR)2, -Si(R)2(OR), -N(R)2, -N(R)OR, -C(O)N R(OR), -C(O)NRCN, -C(O)R, -C(O)OR, -C(O)CH2R, -S(O)2R, -S(O)R, -CH2OC(O)R, -CH2OC(O)OR, -C(S)R, -C(S)OR, -C(O)SR, -C(O)N(R)2, -(CH2) 1～3 OR, -(CH2) 1～3 N(R)2、-(CH2) 1～3 S(O)R、-(CH2) 1～3 S(O)2R、-(CH2) 1～3 NS(O)(R)2, -OC(O)R, (C1-C6)alkyl-C(=O)-, (C1-C6)alkoxy, (C1-C6)alkylthio, -NHC(O)OR, -NHC(O)N(R)2, -NHC(O)R, -NRS(O)2R, -NRCN, 3-12 saturated or partially unsaturated heterocyclic alkyl, 7-12 heterospirocyclic alkyl or 5-12 heterobridged cycloalkyl, 6-10 aryl, 5-12 heteroaryl, wherein the (C1-C6)alkyl, (C1-C6)alkyl-(C3-C6)cycloalkyl, (C1-C 6) Alkyl-(C3-C6)heterocyclic alkyl, (C2-C6)alkynyl, (C2-C6)alkenyl, (C3-C6)cycloalkyl, -OC(O)-(C1-C6)alkyl, -OC(O)-(C3-C6)cycloalkyl, (C1-C6)alkyl-C(=O)-, (C1-C6)alkoxy, (C1-C6)alkylthio or (C1-C6)alkylamine, 3- to 12-membered saturated or partially unsaturated heterocyclic alkyl, 7- to 12-membered heterospirocyclic alkyl or 5- to 12-membered heterobridged cycloalkyl, 6- to 10-membered aryl, 5- to 12-membered heteroaryl, optionally surrounded by 1, 2, 3, 4 or 5 Rs. nreplace;

[0019] Each R n Each of the following is independently selected from H, halogen, (C1-C6)alkyl, (C1-C6)alkoxy, cyclopropyl, OH, NH2, CN, MeNH-, Me2N-, CH3, CH2F, CHF2 or CF3;

[0020] Each R is independently H, a substituted or unsubstituted (C1-C6) alkyl, a substituted or unsubstituted (C3-C6) cycloalkyl, a substituted or unsubstituted phenyl, a substituted or unsubstituted 3- to 7-membered saturated or partially unsaturated heterocyclic alkyl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; or each of the two independently selected Rs can form a 3- to 10-membered saturated or partially unsaturated heterocyclic alkyl, a 7- to 11-membered heterospirocyclic, or a 5- to 11-membered heterobridged cycloalkyl;

[0021] When Z is NH, the compound of general formula (1) can be prepared by the following method to obtain the compound of general formula (3):

[0022] or

[0023] or

[0024] in, Represents substituted or unsubstituted amino, hydroxyl, aliphatic or aromatic residues. PG is a protecting group that can be deprotected under certain conditions to reveal the naked α-NH.

[0025] FG is a structural unit that is an aldehyde group, a protecting group of an aldehyde group, or a unit that generates an aldehyde group through a reaction;

[0026] When Z is NH, the compound of general formula (2) can be prepared by the following method to obtain the compound of general formula (4):

[0027] or

[0028] or

[0029] When Z is O, the compound of general formula (1) can be prepared by the following method to obtain the compound of general formula (5):

[0030] or

[0031] or

[0032] or

[0033] R5 is a substituted or unsubstituted (C1-C6) alkyl, a substituted or unsubstituted (C3-C6) cycloalkyl, a substituted or unsubstituted phenyl, a substituted or unsubstituted 3-7 member saturated or partially unsaturated heterocycloalkyl, or a substituted or unsubstituted 5-10 member heteroaryl, and LP is a leaving group, such as Br, OMs, etc.

[0034] When Z is O, the compound of general formula (2) can be prepared by the following method to obtain the compound of general formula (6):

[0035] In another preferred embodiment, the synthesis method includes the following steps:

[0036] Compound of formula (int_9)

[0037] Reaction with compounds of formula (int_10)

[0038] Compounds with the formula (int_1)

[0039] Or the expression (int_11)

[0040] Oxidation forms a compound of formula (int_1).

[0041] R1, R2, and R3 are as defined above;

[0042] Compounds of formula (int_11-B)

[0043] Reaction with compounds of formula (int_10)

[0044] Compounds with the formula (int_3)

[0045] Or the expression (int_12)

[0046] Oxidation forms a compound of formula (int_3).

[0047] Among them, R1, R2, R3 and PG are as defined above;

[0048] Formula (int_13) after oxidation

[0049] Compounds with formula (int_5)

[0050] Where R1, R3 and m are as defined above;

[0051] Formula (int_14) is oxidized

[0052] Compounds with formula (int_6)

[0053] R1, R3, m, and PG are as defined above.

[0054] In another preferred embodiment, in the synthesis method described therein, R1 and R2 are phenyl, methyl, trifluoromethyl or ethyl; or R1 and R2 together with the commonly linked atoms form (C3-C6) cycloalkyl, 3-6 membered heterocyclic alkyl, preferably cyclopropyl or cyclobutyl;

[0055] Alternatively, R2 and R3 together with the atoms between them can form 4-10 saturated or partially unsaturated heterocyclic alkyl groups, 7-11 heterospirocyclic groups, or 5-11 heterobridged cyclic groups.

[0056] Alternatively, in general formula (2), an R1 and an R3 together with the atoms between them form a 4- to 10-membered saturated or partially unsaturated heterocyclic alkyl group, a 7- to 11-membered heterospirocyclic group, or a 5- to 11-membered heterobridged cyclic group.

[0057] In another preferred embodiment, in the synthesis method described therein, Z is NH or O;

[0058] R3 is a phenyl group, a 5- to 7-membered monocyclic heteroaryl group containing 1, 2, 3, or 4 heteroatoms independently selected from N, S, Se, and O, or an 8- to 10-membered bicyclic heteroaryl group containing 1, 2, 3, 4, 5, or 6 heteroatoms independently selected from N, S, Se, and O, wherein the phenyl or heteroaryl group is optionally further surrounded by 1, 2, 3, 4, or 5 R3 atoms. m replace;

[0059] In another preferred embodiment, in the synthesis method, R3 is composed of 1, 2, 3, 4, or 5 R... m The substituted phenyl group, or a 5- to 6-membered monocyclic heteroaryl group containing one, two, three, or four heteroatoms independently selected from N, S, Se, and O, preferably surrounded by one, two, three, four, or five R atoms. m Substituted phenyl, pyrrolyl, thiophenyl, furanyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, 1,2,4-triazolyl, 1,2,3-triazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, 1,2,5-thiadiazolyl, 1,2,5-oxadiazolyl.

[0060] In another preferred embodiment, in the synthesis method, R3 is composed of 1, 2, 3, 4, or 5 R... m The substituted 8- to 10-membered bicyclic heteroaryl group containing one, two, three, four, five, or six heteroatoms independently selected from N, S, and O is preferred.

[0061] In another preferred embodiment, in the synthesis method described therein, R m For H, F, Cl, Br, OH, NO2, NH2, CN, -NHCH3, -N(CH3)2, -NHC(O)OC(CH3)3, -S(O)2CH3, -CH2OCH3, -S(O)2CH3, -N=S(O)(CH3)2, -S(O)=(NH)CH3, -C(O)OH, -C(O)OC H3, -C(O)OC(CH3)3, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -C(CH3)3, -OCH3, -OCH2CH3, -CF3, -CHF2, -CH2F, -CH2CF3, -OCF3, -OCHF2, -OCH2F, -CH=CH2, -CH2CH=CH2, -CH=CH2CH3, -CH=CH2CH2OCH3, -CH=CH2CH2N(CH3)2, -C≡CH, -C≡CHCH3, -CH2C≡CH, -C≡CHCH2OCH3, -C≡CHCH2N(CH3)2, cyclopropyl, cyclobutyl, oxacyclobutane, azacyclobutane, -SCN, -S(O)2CN, -SNC, -S(O)2NC, -S(O)2N(CH3)2, -SF5, -P(O)(CH3)2, -P(S)(CH3)2, -P(O)(OCH3)CH3, -P(O)(OCH3)2, -B(OH)2, -SeR, -NHS(O)2CH3, -NHCN.

[0062] In another preferred embodiment, in the synthesis method described therein, Z is NH or O;

[0063] R3 is -C(O)R k or -C(O)OR k ;

[0064] R kThe (C1-C3) alkyl, (C3-C6) cycloalkyl, or 3- to 6-membered saturated or partially unsaturated heterocyclic alkyl containing 1 to 2 heteroatoms independently selected from N, S, and O, wherein the (C1-C3) alkyl, (C3-C6) cycloalkyl, or 3- to 6-membered saturated or partially unsaturated heterocyclic alkyl containing 1 to 2 heteroatoms independently selected from N, S, and O is optionally further surrounded by 1, 2, 3, 4, or 5 R atoms. m Substitution; two hydrogen atoms on the same carbon atom can be replaced by oxygen to form an oxo group or by... Replaced; R a and R b Each can be H or F independently;

[0065] In another preferred embodiment, in the synthesis method described therein, Z is NH or O;

[0066] R3 is -C(O)R k or -C(O)OR k ;

[0067] R k The phenyl group is a 5- to 6-membered monocyclic heteroaryl group containing one, two, three, or four heteroatoms independently selected from N, S, and O; or an 8- to 10-membered bicyclic heteroaryl group containing one, two, three, or four heteroatoms independently selected from N, S, and O, wherein the phenyl or heteroaryl group is optionally further surrounded by one, two, three, four, or five R atoms. m replace;

[0068] In another preferred embodiment, in the synthesis method described therein, Z is NH or O;

[0069] R3 is -C(O)NR k R l ;

[0070] R k and R l Each of the following is independently H, (C1-C3)alkyl, (C1-C3)haloalkyl, (C3-C6)cycloalkyl, 3-6 member saturated or partially unsaturated heterocyclic alkyl, 7-12 member heterospirocyclic alkyl or 5-12 member heterobridged cycloalkyl, phenyl, 5-12 member heteroaryl containing 1-4 heteroatoms independently selected from N, S and O; the two hydrogens on the same carbon may be replaced by oxygen to form an oxo group or by... Replaced; R a and R b Each can be H or F independently.

[0071] In another preferred embodiment, in the synthesis method described therein, Z is NH or O;

[0072] R3 is H, (C1-C3)alkyl, or (C3-C6)cycloalkyl, wherein the (C1-C3)alkyl or (C3-C6)cycloalkyl is further surrounded by 1, 2, 3, 4, or 5 R3 groups. m Substitution; two hydrogen atoms on the same carbon atom can be replaced by oxygen to form an oxo group or by... Replaced; R a and R b Each can be H or F independently;

[0073] In another preferred embodiment, in the synthesis method described therein, Z is NH or O;

[0074] R3 is a 3- to 12-membered saturated or partially unsaturated heterocyclic alkyl group, a 7- to 12-membered heterospirocyclic alkyl group, or a 5- to 12-membered heterobridged cycloalkyl group containing 1 to 2 heteroatoms independently selected from N, S, and O, wherein the 3- to 12-membered saturated or partially unsaturated heterocyclic alkyl group, a 7- to 12-membered heterospirocyclic alkyl group, or a 5- to 12-membered heterobridged cycloalkyl group containing 1 to 2 heteroatoms independently selected from N, S, and O is optionally further surrounded by 1, 2, 3, 4, or 5 R3 atoms. m Substitution; two hydrogen atoms on the same carbon atom can be replaced by oxygen to form an oxo group or by... Replaced; R a and R b Each can be H or F independently;

[0075] In another preferred embodiment, in the synthesis method described therein, R3 is...

[0076] In another preferred embodiment, in the synthesis method, wherein Represents any substituted (C1-C8) alkyl; any substituted (C3-C12) cycloalkyl; any substituted amino; any substituted hydroxyl; any substituted heterocyclic alkyl; any substituted partially unsaturated heterocyclic alkyl; any substituted phenyl; radioactive or non-radioactive nuclides, biotin, reporter enzymes, nucleotides, oligonucleotides, fluorophores, amino acids, peptides, and any substituted 5- to 10-membered heteroaromatic systems.

[0077] In another preferred embodiment, in the synthesis method, wherein Represents any substituted (C1-C8) alkyl; any substituted (C3-C6) cycloalkyl; any substituted amino; any substituted hydroxyl; any substituted heterocyclic alkyl; any substituted partially unsaturated heterocyclic alkyl; any substituted phenyl; any substituted 5- to 6-membered heteroaromatic systems.

[0078] In another preferred embodiment, in the synthesis method, wherein Represents a linker, a drug, or a linker-drug conjugate.

[0079] This invention also provides a class of compounds as shown in formula (1):

[0080] in Z, R1, R2 and R3 are as defined in any of the preceding claims.

[0081] This invention also provides a class of compounds as shown in formula (2):

[0082] in Z, R1, R3 and m are as defined in any of the preceding claims.

[0083] In another preferred embodiment, the Knoevenagel condensation reaction between the aldehyde-containing intermediate and the α-position active methylene intermediate in the synthesis method is characterized by the following: the reaction is carried out in an organic solvent, using inorganic substances such as organic bases, inorganic bases, amine salts, Lewis acid-amine complexes, potassium fluoride, and aluminum phosphate as catalysts.

[0084] In another preferred embodiment, in the synthesis method described therein, the organic solvent is preferably one or more selected from dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, 1,1,1-trichloroethane, acetonitrile, acetone, tetrahydrofuran, 2-methyltetrahydrofuran, diethyl ether, methyl tert-butyl ether, 1,4-dioxane, acetone, ethyl acetate, petroleum ether, n-hexane, cyclohexane, isopentane, n-pentane, cyclopentane, toluene, dichloroethylene, pyridine, nitromethane, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, methanol, ethanol, isopropanol, tert-butanol, n-butanol, cyclohexanol, 1,2-propanediol, and ethylene glycol, more preferably one or more selected from dichloromethane, chloroform, 1,2-dichloroethane, acetonitrile, acetone, tetrahydrofuran, 2-methyltetrahydrofuran, ethyl acetate, and pyridine.

[0085] In another preferred embodiment, in the synthesis method, the organic base is preferably one or more of pyrrolidine, piperidine, aziridine, diethylamine, dimethylamine, ethylamine, methylamine, ethylenediamine, triethylamine, triethylenediamine, 1,8-diazabicyclo[5,4,0]undecene-7, 1,5-diazabicyclo[4,3,0]non-5-ene, 4-dimethylaminopyridine, pyridine, quinoline, N-methylmorpholine, morpholine, and tetramethylethylenediamine; more preferably, the organic base is one or more of pyrrolidine, piperidine, morpholine, aziridine, diethylamine, and dimethylamine.

[0086] In another preferred embodiment, in the synthesis method described therein, the complex of the Lewis acid and the amine is preferably one or more of titanium tetrachloride / pyridine, titanium tetrachloride / triethylamine, and trimethylchlorosilane / pyrrolidine.

[0087] In another preferred embodiment, the synthesis method described herein may further include water removal during the reaction, preferably by using a water separator, adding a molecular sieve or other water removal reagent.

[0088] In another preferred embodiment, the dehydrating agent in the synthesis method is preferably anhydrous sodium sulfate or anhydrous magnesium sulfate.

[0089] In another preferred embodiment, the reaction in the synthesis method is carried out in a temperature range of about -15°C to 100°C.

[0090] In another preferred embodiment, the reaction temperature in the synthesis method is from -15°C to 100°C, preferably from 20°C to 40°C, and more preferably from 20°C to 30°C.

[0091] In another preferred embodiment, the reaction temperature in the synthesis method is 25°C.

[0092] In another preferred embodiment, in the synthesis method described therein, the molar equivalent ratio of the α-position active methylene intermediate compound and the aldehyde intermediate is preferably in the range of 5:1 to 1:5.

[0093] In another preferred embodiment, in the synthesis method described therein, the molar equivalent ratio of the intermediate compound containing the α-position active methylene group to the intermediate containing the aldehyde group is preferably 1:1 to 1:5, more preferably 1:1 to 1:2, more preferably 1:1.2 to 1:2, and even more preferably 1:1.5 to 1:2.

[0094] In another preferred embodiment, in the synthetic method described therein, the Knoevenagel condensation reaction conditions for the aldehyde-containing intermediate and the α-position active methylene intermediate are as follows:

[0095] Dissolve 1 molar equivalent of an aldehyde-containing intermediate and more than 1 molar equivalent of pyrrolidine in dichloromethane, then add less than 1 molar equivalent of an α-active methylene intermediate, and continue stirring at 25°C until the reaction is complete.

[0096] Alternatively, under ice-water bath cooling, dissolve 1 molar equivalent of the aldehyde-containing intermediate and more than 1 molar equivalent of pyrrolidine in dichloromethane, then add trimethylchlorosilane less than the molar equivalent of pyrrolidine, heat to about 25°C and stir for a period of time, then add less than 1 molar equivalent of the intermediate containing α-active methylene, and continue stirring at about 25°C until the reaction is complete.

[0097] Compared with existing technologies, the novel cyclic α,β-unsaturated functional groups or compounds involved in this invention can undergo faster covalent reactions with thiol groups, thus enabling efficient coupling with thiol-containing proteins. This significantly increases the binding affinity of these compounds to target proteins and enhances their biological activity. Therefore, the method of this invention is particularly suitable for target proteins with low thiol reactivity that are difficult to target with existing technologies, providing a new development strategy for the preparation of covalent drugs. Furthermore, using the method of this invention, effector molecules can be coupled to antibodies through a linker containing such cyclic α,β-unsaturated functional groups to prepare various novel ADC drugs. Compared with existing coupling techniques, the linker obtained by the method of this invention has the advantages of high coupling efficiency and less drug polymerization.

[0098] It should be understood that the foregoing general description of the invention and the following detailed description are exemplary and illustrative, and are intended to provide further explanation of the claimed invention.

[0099] the term

[0100] Unless otherwise specified, the terms used in this application, including the specification and claims, are defined as follows. It must be noted that in the specification and appended claims, unless otherwise clearly indicated, the singular form "a" includes the plural meaning. Unless otherwise specified, substituents (such as alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, etc.) in this application are optionally substituted, i.e., they can be substituted or unsubstituted. Unless otherwise specified, conventional methods such as mass spectrometry, nuclear magnetic resonance, HPLC, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology are used. In this application, unless otherwise specified, "or" or "and" refers to "and / or".

[0101] Unless otherwise specified, "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain and branched groups with 1 to 6 carbon atoms. Lower alkyl groups containing 1 to 4 carbon atoms are preferred, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, and tert-butyl. Lower alkyl groups containing 1 to 3 carbon atoms are more preferred, such as methyl, ethyl, propyl, and 2-propyl. As used herein, "alkyl" includes unsubstituted and substituted alkyl groups, especially alkyl groups substituted with one or more halogens. Preferred alkyl groups are selected from CH3, CH3CH2, CF3, CHF2, CF3CH2, CF3(CH3)CH, etc. i Pr、 n Pr、 i Bu、 n Bu or t Bu.

[0102] Unless otherwise specified, "alkylene" means a divalent alkyl group as defined above. Examples of alkylene include, but are not limited to, methylene and ethylene.

[0103] Unless otherwise specified, "alkenyl" refers to an unsaturated aliphatic hydrocarbon group containing a carbon-carbon double bond, including straight-chain or branched groups with 1 to 14 carbon atoms. Lower alkenyl groups containing 1 to 4 carbon atoms are preferred, such as vinyl, 1-propenyl, 1-butenyl, or 2-methylpropenyl. Lower alkenyl groups containing 1 to 2 carbon atoms are more preferred.

[0104] Unless otherwise specified, “alkenyl” refers to a divalent alkenyl group as defined above.

[0105] Unless otherwise specified, "alkynyl" refers to an unsaturated aliphatic hydrocarbon group containing a carbon-carbon triple bond, including straight-chain and branched groups with 1 to 14 carbon atoms. Lower alkynyl groups containing 1 to 4 carbon atoms are preferred, such as ethynyl, 1-propynyl, or 1-butynyl. Lower alkynyl groups containing 1 to 2 carbon atoms are more preferred.

[0106] Unless otherwise specified, “ethynyl” means a divalent ethynyl group as defined above.

[0107] Unless otherwise specified, "cycloalkyl" refers to a non-aromatic hydrocarbon ring system (monocyclic, bicyclic, or polycyclic), preferably containing 3-14 ring carbon atoms (C14-C24). 3-14 A non-aromatic hydrocarbon ring system (cycloalkyl). In some embodiments, the cycloalkyl group has 3-10 ring carbon atoms (C60-C70). 3-10 (Cycloalkyl). In some embodiments, the cycloalkyl group has 3-8 cyclic carbon atoms (C60-C85). 3-8 (Cycloalkyl). In some embodiments, the cycloalkyl group has 3-7 cyclic carbon atoms (C60-C75). 3-7 (Cycloalkyl). In some embodiments, the cycloalkyl group has 3-6 cyclic carbon atoms (C66-C66). 3-6(Cycloalkyl). In some embodiments, the cycloalkyl group has 4-6 cyclic carbon atoms (C66-C66). 4-6 (Cycloalkyl). In some embodiments, the cycloalkyl group has 5-6 cyclic carbon atoms (C66-C66). 5-6 (Cycloalkyl). In some embodiments, the cycloalkyl group has 5-10 cyclic carbon atoms (C10-C20). 5-10 Cycloalkyl groups. A partially unsaturated cycloalkyl group may be referred to as a "cycloalkenyl" if the carbide ring contains at least one double bond, or as a "cycloynyl" if the carbide ring contains at least one triple bond. Cycloalkyl groups may include monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) groups and spirocyclic groups. In some embodiments, the cycloalkyl group is monocyclic. In some embodiments, the cycloalkyl group is bicyclic. In some embodiments, the cycloalkyl group is either monocyclic or bicyclic. In some embodiments, the cycloalkyl group is tricyclic. The cycloforming carbon atom of the cycloalkyl group may optionally be oxidized to form an oxo or thio group. Cycloalkyl groups also include cycloalkylene groups. In some embodiments, the cycloalkyl group contains 0, 1, or 2 double bonds. In some embodiments, the cycloalkyl group contains 1 or 2 double bonds (partially unsaturated cycloalkyl). In some embodiments, the cycloalkyl group may be fused with aryl, heteroaryl, cycloalkyl, and heterocyclic alkyl groups. In some embodiments, the cycloalkyl group may be fused with aryl, cycloalkyl, and heterocyclic alkyl groups. In some embodiments, the cycloalkyl group may be fused with aryl and heterocyclic alkyl groups. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cyclohepttrienyl, norcamphenyl, norpinel, norcarel, bicyclo[1.1.1]pentyl, bicyclo[2.1.1]hexyl, and so on.

[0108] Unless otherwise specified, “cycloalkylene” means a divalent cycloalkyl group as defined above.

[0109] Unless otherwise specified, "alkoxy" refers to an alkyl group bonded to the remainder of the molecule via an ether oxygen atom. Representative alkoxy groups are those having 1-6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, and tert-butoxy. As used herein, "alkoxy" includes unsubstituted and substituted alkoxy groups, particularly those substituted with one or more halogens. Preferred alkoxy groups are selected from OCH3, OCF3, CHF2O, CF3CH2O, etc. i- PrO, n- PrO, i- BuO、 n- BuO or t- BuO.

[0110] Unless otherwise specified, "alkathioyl" refers to an alkyl group bonded to the remainder of the molecule via a sulfur atom. Representative alkathioyl groups are those having 1-6 carbon atoms, such as methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, sec-butylthio, and tert-butylthio. Preferred alkathioyl groups are selected from -SCH3, -SCF3, CHF2S-, CF3CH2S-, etc. i- PrS-、 n- PrS-、 i- BuS-、 n- BuS- or t- BuS-.

[0111] Unless otherwise specified, "alkylamine" refers to an alkyl group bonded to the remainder of the molecule via a nitrogen atom. Representative alkylamine groups are those having 1-6 carbon atoms, such as methylamino, dimethylamino, methylethylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, sec-butylamino, and tert-butylamino. Preferred alkylamine groups are selected from -NHCH3, -N(CH3)2, -NHCF3, CHF2NH-, CF3CH2NH-, etc. i- PrNH-、 n- PrNH-、 i- BuNH-、 n- BuNH- or t- BuNH-.

[0112] Unless otherwise specified, "aryl" refers to a hydrocarbon aromatic group, which can be monocyclic or polycyclic, such as a monocyclic aryl ring fused with one or more carbocyclic aromatic groups. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and phenanthrene.

[0113] Unless otherwise specified, "aryloxy group" refers to an aryl group bonded to the rest of the molecule via an ether oxygen atom. Examples of aryloxy groups include, but are not limited to, phenoxy and naphthoxy groups.

[0114] Unless otherwise specified, "arylene" refers to a divalent aryl group as defined above. Examples of arylene groups include, but are not limited to, 1,4-phenylene, 1,3-phenylene, 1,2-phenylene, naphthylene, and phenanthrene.

[0115] Unless otherwise specified, "heteroaryl" refers to a substituted or unsubstituted aromatic group containing one or more heteroatoms, wherein the heteroatoms are independently selected from O, N, P, Se, or S, and the number of heteroatoms is preferably 1, 2, 3, or 4. It is more preferably a 5-14-membered aromatic group containing 1-4 heteroatoms selected from oxygen, sulfur, and nitrogen, more preferably a 5-9-membered aromatic group containing 1-2 heteroatoms optionally selected from oxygen, sulfur, or nitrogen, and even more preferably a 5-6-membered aromatic group containing 1-3 heteroatoms optionally selected from oxygen, sulfur, or nitrogen. The heteroaryl group can be monocyclic or polycyclic. Monocyclic heteroaryl groups are preferably 5-6-membered aromatic groups containing 1-3 heteroatoms optionally selected from oxygen, nitrogen, or sulfur. More preferably, they are 5-6-membered aromatic groups containing 1-2 heteroatoms optionally selected from oxygen, nitrogen, or sulfur. Even more preferably, they are 5-6-membered aromatic groups containing 1 heteroatom optionally selected from oxygen, nitrogen, or sulfur. In some embodiments, the monocyclic heteroaryl ring is fused with one or more carbocyclic aromatic groups or other monocyclic heterocyclic alkyl groups. In some embodiments, the nitrogen on the heteroaryl ring can be oxidized to an N-oxide, such as... Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, quinolinyl, isoquinolinyl, quinazolinyl, furanyl, thiopheneyl, isoxazolyl, thiazolyl, oxazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, isothiazolyl, pyrroloyl, indolyl, benzimidazolyl, benzofuranyl, benzothiazolyl, benzothiaphenyl, benzooxazolyl, benzopyridyl, pyrrolopyrimidinyl, 1H-pyrrolo[3,2-b]pyridyl, 1H-pyrrolo[2,3-c]pyridyl, 1H-pyrrolo[3,2-c]pyridyl, 1H-pyrrolo[2,3-b]pyridyl.

[0116] Unless otherwise specified, “hybrid aryl” refers to a divalent heteroaryl group as defined above.

[0117] Unless otherwise specified, "heterocyclic alkyl" refers to a non-aromatic ring or ring system that may optionally contain one or more alkenyl groups as part of a ring structure, having at least one heteroatom ring member independently selected from boron, phosphorus, nitrogen, sulfur, oxygen, selenium, and silicon, preferably containing 1-4 heteroatoms selected from oxygen, sulfur, or nitrogen, and more preferably containing 1-2 heteroatoms selected from oxygen, sulfur, or nitrogen. In some embodiments, the heterocyclic alkyl is a 5-14 membered non-aromatic ring containing a cyclic carbon atom and 1-4 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, or sulfur (5-14 membered heterocyclic alkyl). In some embodiments, the heterocyclic alkyl is a 3-9 membered non-aromatic ring containing a cyclic carbon atom and 1-4 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, or sulfur (3-9 membered heterocyclic alkyl). In some embodiments, the heterocyclic alkyl group is a 5-8 membered non-aromatic ring containing a cyclic carbon atom and 1-4 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, or sulfur (5-8 membered heterocyclic alkyl group). In some embodiments, the heterocyclic alkyl group is a 5-6 membered non-aromatic ring containing a cyclic carbon atom and 1-4 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, or sulfur (5-6 membered heterocyclic alkyl group). In some embodiments, the 5-6 membered heterocyclic alkyl group contains 1-3 cyclic heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclic alkyl group contains 1-2 cyclic heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclic alkyl group contains 1 cyclic heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the heterocyclic alkyl group is a 10-13 membered non-aromatic ring containing a cyclic carbon atom and 1-4 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, or sulfur (10-13 membered heterocyclic alkyl group). In some embodiments, the 10-13 membered heterocyclic alkyl group contains 1-3 cyclic heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the 10-13 membered heterocyclic alkyl group contains 1-2 cyclic heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the 10-13 membered heterocyclic alkyl group contains 1 cyclic heteroatom independently selected from nitrogen, oxygen, and sulfur. If the heterocyclic alkyl group contains at least one double bond, then the partially unsaturated heterocyclic alkyl group may be referred to as a "heterocyclic alkenyl group," or if the heterocyclic alkyl group contains at least one triple bond, then the partially unsaturated heterocyclic alkyl group may be referred to as a "heterocyclic ynyl group." Heterocyclic alkyl groups may include monocyclic, bicyclic, spirocyclic, or polycyclic (e.g., having two fused or bridging rings) ring systems. In some embodiments, the heterocyclic alkyl group is a monocyclic group having 1, 2, or 3 heteroatoms independently selected from nitrogen, sulfur, and oxygen. The cyclic carbon atoms and heteroatoms of the heterocyclic alkyl group may optionally be oxidized to form oxo or thio groups or other oxidized bonds (e.g., C(O), S(O), C(S) or S(O)2, N-oxides, etc.), or the nitrogen atom may be quaternized. The heterocyclic alkyl group may be linked via cyclic carbon atoms or cyclic heteroatoms. In some embodiments, the heterocyclic alkyl group contains 0 to 3 double bonds.In some embodiments, the heterocyclic alkyl group contains 0 to 2 double bonds. The definition of heterocyclic alkyl also includes portions of an aromatic ring (also called partially unsaturated heterocycles) having one or more aromatic rings fused to (i.e., sharing bonds with) the heterocyclic alkyl ring, such as benzo[a] derivatives of piperidine, morpholine, aziridine, or tetrahydrothiophene, and pyrido[a] derivatives of piperidine, morpholine, aziridine, or tetrahydrothiophene. Heterocyclic alkyl groups containing fused aromatic rings can be linked via any cyclizing atom, including the cyclizing atom of the fused aromatic ring. Examples of heterocyclic alkyl groups include, but are not limited to, azirrobutyl, azirroheptyl, dihydrobenzofuranyl, dihydrofuranyl, dihydropyranyl, N-morpholinyl, 3-oxa-9-azaspiro[5.5]undecyl, 1-oxa-8-azaspiro[4.5]decyl, piperidinyl, piperazinyl, oxoperazinyl, pyranyl, pyrrolidinyl, quininyl, tetrahydrofuranyl, tetrahydropyranyl, 1,2,3,4-tetrahydroquinolinyl, scopolamine, 4,5,6,7-tetrahydrothiazo[5,4-c]pyridinyl, and 4,5,6,7-tetrahydro-1H-imidazolium. [4,5-c]pyridine, N-methylpiperidinyl, tetrahydroimidazolyl, pyrazolyl, butyrolactam, valproic acid, imidazolinone, hydantoin, dioxolane, phthalimide, pyrimidin-2,4(1H,3H)-diketoyl, 1,4-dioxane, morpholinyl, thiomorpholinyl, thiomorpholin-S-oxide, thiomorpholin-S,S-oxide, piperazine, pyranyl, pyridinone, 3-pyrrololinyl, thiaranyl, pyranone, tetrahydrothiophene, 2-azaspiro[3.3]heptyl, indololinyl.

[0118] Unless otherwise specified, "heterocyclic alkylene" refers to a divalent heterocyclic alkylene group as defined above, and specific examples of heterocyclic alkylene groups include, but are not limited to, those that ... specifically defined above.

[0119] Unless otherwise specified, "heterocyclic spirocyclic alkyl" / "heterocyclic alkyl" / "heterocyclic group" refers to a polycyclic cyclic hydrocarbon group formed by two or more saturated or partially unsaturated monocyclic rings sharing a single carbon atom (called a spiro atom), wherein one or more (e.g., 1, 2, or 3) ring atoms are selected from nitrogen, oxygen, or S(O). pThe heteroatom (where p is 0, 1, or 2) is a carbon atom, and the remaining ring atoms are carbon. When the heteroatom is a nitrogen atom, the nitrogen atom can be substituted or unsubstituted (i.e., N or NR, where R is hydrogen or other substituents defined herein). Each monocycle may contain one or more double bonds, but no ring has a fully conjugated π-electron system. Spirocycloheterocyclic groups are classified as monospirocycloheterocyclic, bispirocycloheterocyclic, or polyspirocycloheterocyclic groups based on the number of shared spiro atoms between rings. The term "(5-15-membered) heterocyclic spirocycloalkyl" refers to a heterocyclic spirocycloalkyl having 5 to 15 ring atoms, wherein the monocycles sharing the spiro atom are 3 to 8-membered monocycles, and at least one monocycle is a heterocyclic alkyl ring. Preferably, it is a (6-18-membered) heterocyclic spirocycloalkyl having 6 to 18 ring atoms, wherein 1 to 3 ring atoms are heteroatoms, more preferably a (7-15-membered) heterocyclic spirocycloalkyl having 7 to 15 ring atoms, wherein 1 to 3 ring atoms are heteroatoms. The most preferred are 9-membered (4-membered monocyclic (heterocyclic) alkyl groups / 6-membered monocyclic (heterocyclic) alkyl groups, 5-membered monocyclic (heterocyclic) alkyl groups / 5-membered monocyclic (heterocyclic) alkyl groups, 10-membered (5-membered monocyclic (heterocyclic) alkyl groups / 6-membered monocyclic (heterocyclic) alkyl groups, or 11-membered (6-membered monocyclic (heterocyclic) alkyl groups / 6-membered monocyclic (heterocyclic) alkyl groups. Specific examples of heterocyclic spirocyclic alkyl groups include, but are not limited to, those listed below.

[0120] Unless otherwise specified, "subheterospirocyclic" refers to a divalent heterospirocyclic group as defined above. Specific examples of subheterospirocyclic groups include, but are not limited to, those described above.

[0121] Unless otherwise specified, "heterobridged cycloalkyl" or "heterobridged cycloyl group" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two non-directly connected atoms. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered. Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged heterocyclic groups, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic.

[0122] Unless otherwise specified, "hybridized cycloalkylene" refers to a divalent heterobridged cycloalkylene group as defined above, and specific examples of hybridized cycloalkylene groups include, but are not limited to, those that are not limited to, heterobridged cycloalkylene groups.

[0123] Unless otherwise specified, "oxo" refers to =O; for example, a group formed by substituting carbon with an oxo group is a "carbonyl". The group formed by the substitution of sulfur with one oxo group is called "thionyl". The group formed by the substitution of sulfur with two oxo groups is called a sulfonyl group. ".

[0124] Unless otherwise specified, "halogen" (or halogenated group) means fluorine, chlorine, bromine or iodine. The term "halogenated" (or "halogen substituted") appearing before the group name indicates that the group is partially or completely halogenated, that is, substituted by F, Cl, Br or I in any combination, preferably substituted by F or Cl.

[0125] Unless otherwise specified, "acyl" means -C(=O)-R, where R is selected from optionally substituted alkyl, alkenyl, ynyl, cycloalkyl, cycloalkenyl, cycloynyl, aryl, heteroaryl or heterocycloalkyl.

[0126] Unless otherwise specified, the word “comprising”, or variations thereof such as “including” or “containing”, may be understood to mean including the stated element or integer, or a group of elements or integers, but does not exclude any other element or integer, or a group of elements or integers.

[0127] The substituent "-O-CH2-O-" indicates that the two oxygen atoms in the substituent are connected to two adjacent carbon atoms of a heterocyclic alkyl, aryl, or heteroaryl group. For example:

[0128] When the number of a linking group is 0, such as -(CH2)0-, it indicates that the linking group is a single bond.

[0129] When one of the variables is selected as a chemical bond, it means that the two groups connected are directly linked. For example, when L in XLY represents a chemical bond, it means that the structure is actually XY.

[0130] The term "membered ring" includes any ring structure. The term "membered" refers to the number of skeleton atoms that make up the ring. For example, cyclohexyl, pyridyl, pyranyl, and thioranyl are six-membered rings, while cyclopentyl, pyrroleyl, furanyl, and thiophenyl are five-membered rings.

[0131] The term "fragment" refers to a specific part or functional group of a molecule. Chemical fragments are generally considered to be chemical entities contained in or attached to a molecule.

[0132] The term "isomer" refers to any tautomer, stereoisomer, transisomer, isotopic isomer, enantiomer, or diastereomer of any compound of the present invention. The compounds of the present invention may have one or more chiral centers or double bonds, and thus exist in stereoisomeric form, such as double-bonded isomers (i.e., E / Z geometric isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). Therefore, the compounds of the present invention encompass all corresponding stereoisomers, i.e., stereoisomerically pure (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) forms, as well as mixtures of enantiomers and stereoisomers, such as racemates. The enantiomers and stereoisomers of the compounds of this invention can be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral gas chromatography, chiral high-performance liquid chromatography, and by crystallizing the compounds as chiral salt complexes or by crystallizing the compounds in chiral solvents. The enantiomers and stereoisomers can also be obtained by well-known asymmetric synthetic methods using stereoisomerically pure or enantiomerically pure intermediates, reagents, and catalysts.

[0133] The term "isotope isomers" refers to different molecules that are identical in structure except for their isotopes.

[0134] The term "restricted transisomer" refers to a conformational stereoisomer that arises when rotation around a single bond within a molecule is prevented or significantly slowed by spatial interactions with other parts of the molecule, and when the substituents at the ends of the single bond are asymmetrical; that is, a restricted transisomer does not require a stereocenter. When the rotational barrier around the single bond is sufficiently high and the interconversion between conformations is sufficiently slow, the separation of individual isomers is permissible (LaPlante et al., J.Med.Chem. 2011, 54, 20, 7005), preferably by chiral resolution.

[0135] Unless otherwise specified, use wedge-shaped solid line keys. and wedge-shaped dashed key The absolute configuration of the center of a solid is represented by a straight solid line key. and straight dashed key The relative configuration of the center of a solid is indicated by a wavy line. Indicates wedge-shaped solid line key or wedge-shaped dashed key Or use wavy lines Indicates a straight solid line key Or straight dashed key

[0136] Unless otherwise stated, use Indicates a single bond or a double bond.

[0137] Unless otherwise stated, use The E-type or Z-type, or a mixture of both, represent carbon-carbon double bonds, such as... express Or a mixture of the two.

[0138] Unless otherwise stated, use The E-type or Z-type, or a mixture of both, represent carbon-carbon double bonds, such as... express Or a mixture of the two.

[0139] Unless otherwise stated, the structures of amidines in this invention all include their tautomers, such as:

[0140] Unless otherwise specified, the term "substituted" means that one or more hydrogen atoms on a specified atom or group are substituted by one or more substituents other than hydrogen atoms, without exceeding the normal valence of the specified atom. For example, one or more hydrogen atoms of alkyl, alkylene, alkenyl, alkynyl, hydroxyl, or amino groups may be substituted by one or more substituents. The substituents mentioned include, but are not limited to, alkyl, alkenyl, alkynyl, acyl, amino, amide, amidyl, aryl, azide, carbamoyl, carboxyl, carboxylic acid ester, cyano, guanidinyl, halogen, haloalkyl, heteroalkyl, heteroaryl, heterocyclic, hydroxyl, hydrazyl, imino, oxo, nitro, alkylsulfinyl, sulfonic acid, alkylsulfonyl, thiocyanate, thiol, thion, or combinations thereof. The definition of "substituted" does not include similar indeterminate structures obtained by defining substituents having further substituents attached to infinity (e.g., a substituted aryl group having a substituted alkyl group itself substituted by a substituted aryl group, which is further substituted by a substituted heteroalkyl group, etc.). Unless otherwise specified, the maximum number of successive substitutions in the compounds described herein is three. For example, the successive substitution of a substituted aryl group by two other substituted aryl groups is limited to ((substituted aryl) substituted aryl) substituted aryl. Similarly, the above definition does not include disallowed substitution patterns (e.g., a methyl group substituted with five fluorine atoms or a heteroaryl group having two adjacent oxygen ring atoms). Such disallowed substitution patterns are well known to those skilled in the art. Whenever used to modify a chemical group, “substituted” may describe other chemical groups as defined herein. For example, the term “substituted aryl” includes, but is not limited to, “alkylaryl.” Unless otherwise specified, if a group is described as optionally substituted, any substituted elements of that group are themselves unsubstituted.

[0141] "Optional" or "optionally" means that the event or condition described below may, but is not required, occur, and the description includes both the scenario in which the event or condition occurs and the scenario in which the event or condition does not occur.

[0142] The terms “optionally substituted X” / “arbitrarily substituted X” (e.g., “optionally substituted alkyl” / “arbitrarily substituted alkyl”) are intended to be equivalent to “X, wherein X is optionally substituted” (e.g., “alkyl, wherein the alkyl is optionally substituted”). It is not intended to mean that the characteristic “X” (e.g., alkyl) itself is optional. As described in this specification, certain target compounds may contain one or more “optionally substituted” moieties. Generally, the term “substituted”, whether or not preceded by the term “optionally”, means that one or more hydrogens of the specified moieties are replaced by suitable substituents, such as any of the substituents or groups described in this specification. Unless otherwise specified, the “optionally substituted” group may have suitable substituents at each substituted position of said group, and the substituents at each position may be the same or different when more than one position in any given structure is substituted by more than one substituent selected from the specified group. For example, in the term "optionally substituted (C1-C6)alkyl-(5-9-membered)heteroaryl", the alkyl moiety, the heteroaryl moiety, or both may be optionally substituted. The combinations of substituents in this disclosure are preferably combinations that form stable or chemically viable compounds. As used in this specification, the term "stable" means that a compound remains substantially unchanged when subjected to conditions that allow it to be generated, detected, and, in some embodiments, recovered, purified, and used for one or more of the purposes disclosed herein.

[0143] The suitable monovalent substituent on the substituted carbon atom of the "optionally substituted" group can independently be deuterium; halogen; -(CH2). 0-4 R o ;-(CH2) 0-4 OR o ;-O(CH2) 0-4 R o -O-(CH2) 0-4 C(O)OR o ;-(CH2) 0-4 CH(OR o )2;-(CH2) 0-4 SR o ;R o Substituted -(CH2) 0-4 Ph;R o Substituted -(CH2) 0-4 O(CH2) 0-1 Ph;R o Substituted -CH=CHPh; R o Substituted -(CH2) 0-4 O(CH2) 0-1-(5-9) heteroaryl; 4-8 saturated or unsaturated heterocyclic alkyl (e.g., pyridyl); 3-8 saturated or unsaturated cycloalkyl (e.g., cyclopropyl, cyclobutyl, or cyclopentyl); -NO2; -CN; -N3; ​​-(CH2) 0-4 N(R o )2;-(CH2) 0-4 N(R o )C(O)R o ;-N(R o )C(S)R o ;-(CH2) 0-4 N(R o )C(O)NR o 2; -N(R) o )C(S)NR o 2;-(CH2) 0-4 N(R o )C(O)OR o ;-N(R o )N(R o )C(O)R o ;-N(R o )N(R o )C(O)NR o 2; -N(R) o )N(R o )C(O)OR o ;-(CH2) 0-4 C(O)R o ;-C(S)R o ;-(CH2) 0-4 C(O)OR o ;-(CH2) 0-4 -C(O)-N(R o )2;-(CH2) 0-4 -C(O)-N(R o )-S(O)2-R o ;-C(NCN)NR o 2;-(CH2) 0-4 C(O)SR o ;-(CH2) 0-4 C(O)OSiR o 3; -(CH2) 0-4 OC(O)R o ;-OC(O)(CH2) 0-4 SR o ;-SC(S)SR o ;-(CH2) 0-4 SC(O)R o ;-(CH2) 0-4 C(O)NRo 2; -C(S)NR o 2;-C(S)SR o ;-(CH2) 0-4 OC(O)NR o 2; -C(O)N(OR) o )R o ;-C(O)C(O)R o ;-C(O)CH2C(O)R o ;-C(NOR) o )R o ;-(CH2) 0-4 SSR o ;-(CH2) 0-4 S(O)2R o ;-(CH2) 0-4 S(O)2OR o ;-(CH2) 0-4 OS(O)2R o ;-S(O)2NR o 2;-(CH2) 0-4 S(O)R o ;-N(R o )S(O)2NR o 2; -N(R) o )S(O)2R o ;-N(OR) o )R o ;-C(NOR) o )NR o 2;-C(NH)NR o 2; -P(O)2R o ;-P(O)R o 2; -P(O)(OR o )2;-OP(O)R o 2; -OP(O)(OR o )2;-OP(O)(OR o )R o ;-SiR o 3; -(C 1-4 )alkylene-ON(R o )2; or -(C 1-4 )alkylene-C(O)ON(R o )2, where each R o It can be substituted and independently of hydrogen, -C as defined below. 1-6 Aliphatic groups, -CH2Ph, -O(CH2) 0-1Ph, -CH2-(5-6-membered) heteroaryl, or a 3-6-membered saturated, partially unsaturated, or aryl ring having 0-4 independently selected heteroatoms chosen from nitrogen, oxygen, or sulfur, or, despite the above definitions, two independently existing R... o Together with the atoms connected thereto, they form 3-12 saturated or partially unsaturated cycloalkyl or heterocycloalkyl groups having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur; 3-12 aryl or heteroaryl groups having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, and may be substituted as defined below.

[0144] R o (or two independently existing R) o Suitable monovalent substituents on the ring formed together with the atoms it is attached to can be halogens, -(CH2), or other similar substances independently. 0-2 R o1 -(halogenated R) o1 -(CH2) 0-2 OH, -(CH2) 0-2 OR o1 -(CH2) 0-2 CH(OR o1 )2、-O(halogenated R o1 -CN, -N3, -(CH2) 0-2 C(O)R o1 -(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR o1 -(CH2) 0-2 SR o1 -(CH2) 0-2 SH, -(CH2) 0-2 NH2、-(CH2) 0-2 NHR o1 -(CH2) 0-2 NR o1 2, -NO2, -SiR o1 3. -OSiR o1 3. -C(O)SR o1 -(C 1-4 )alkylene-C(O)OR o1 or -SSR o1 , where each R o1 Unsubstituted, or substituted by one or more halogens when preceded by "halogenated", and each R o1 Selected independently from C 1-4 Aliphatic groups, -CH2Ph, -O(CH2) 0-1Ph, a 3-6 membered saturated or partially unsaturated cycloalkyl or heterocycloalkyl group having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, and a 5-6 membered aryl or heteroaryl group having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. R o Suitable divalent substituents on saturated carbon atoms include =O and =S.

[0145] Suitable divalent substituents on the saturated carbon atom of the "optionally substituted" group include: =O, =S, =NNR o2 2、=NNHC(O)R o2 =NNHC(O)OR o2 =NNHS(O)2R o2 =NR o2 =NOR o2 -O(C(R) o2 2)) 2-3 O- or -S(C(R) o2 2)) 2-3 S-, where R o2 Each time it appears, it is independently selected from hydrogen; the C that can be substituted can be defined as follows: 1-6 Aliphatic group; or 5-6 membered saturated or partially unsaturated cycloalkyl or heterocycloalkyl groups having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or 5-6 membered aryl or heteroaryl groups having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents attached to the adjacent substituted carbon of the "optionally substituted" group include: -O(CR o2 2) 2-3 O-, where R o2 Each time it appears, it is independently selected from hydrogen; the C that can be substituted can be defined as follows: 1-6 Aliphatic group; or 3-6 saturated or partially unsaturated cycloalkyl or heterocycloalkyl groups having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or 5-6 aryl or heteroaryl groups having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0146] R o2 Suitable substituents on aliphatic groups include halogens, -R o3 -(halogenated R) o3 -OH, -OR o3 -O (halogenated R) o3 -CN, -C(O)OH, -C(O)OR o3 -NH2, -NHR o3 -NR o3 2 or -NO2, where each R o3 Unsubstituted, or substituted by one or more halogens when preceded by "halogenated", and each R o3Selected independently from C 1-4 Aliphatic groups, -CH2Ph, -O(CH2) 0-1 Ph, a 3-6 saturated or partially unsaturated cycloalkyl or heterocycloalkyl group having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or a 5-6 aryl or heteroaryl group having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0147] Suitable substituents on the substituted nitrogen of the "optionally substituted" group include -R o4 -NR o4 2. -C(O)R o4 -C(O)OR o4 -C(O)C(O)R o4 -C(O)CH2C(O)R o4 -S(O)2R o4 -S(O)2NR o4 2. -C(S)NR o4 2. -C(NH)NR o4 2 or -N(R) o4 )S(O)2R o4 ; where each R o4 It is hydrogen on its own; the C that can be substituted can be defined as follows. 1-6 Aliphatic group; unsubstituted -OPh; 3-6 membered saturated or partially unsaturated cycloalkyl or heterocycloalkyl groups having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; 5-6 membered aryl or heteroaryl groups having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Or, despite the above definitions, two independently existing R groups. o4 Together with the atoms attached thereto, they form 3-12 saturated or partially unsaturated cycloalkyl or heterocycloalkyl groups having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur; or 3-12 aryl or heteroaryl groups having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0148] R o4 Suitable substituents on the aliphatic group are independently halogens, -R o5 -(halogenated R) o5 -OH, -OR o5 -O (halogenated R) o5 -CN, -C(O)OH, -C(O)OR o5 -NH2, -NHR o5 -NR o5 2 or -NO2, where each R o5 Unsubstituted, or substituted by one or more halogens when preceded by "halogenated", and each R o5 C is independent 1-4Aliphatic groups, -CH2Ph, -O(CH2) 0-1 Ph, a 3-6 membered saturated or partially unsaturated cycloalkyl or heterocycloalkyl group having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, and a 5-6 membered aryl or heteroaryl group having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. R o4 Suitable divalent substituents on saturated carbon atoms include =O and =S.

[0149] Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of that group can be linked to other groups by chemical bonds. When the chemical bond connection is non-directional and the connectable site contains H atoms, the number of H atoms at that site will decrease accordingly with the number of chemical bonds being connected, resulting in a group with a corresponding valence. For example, "pyridyl" indicates... "Oxazolyl" indicates

[0150] As used herein, the term "aliphatic or aromatic residue" refers to an aliphatic substituent that can be arbitrarily substituted, such as an alkyl residue. However, this alkyl residue can be further substituted with halogen, amino, nitro, carbonyl, aliphatic, and / or aromatic substituents. For example, an aliphatic residue can be a nucleic acid, peptide, protein, enzyme, isoenzyme, antibody, nucleotide, oligonucleotide, monosaccharide, polysaccharide, polymer, fluorophore, or arbitrarily substituted benzene, as long as the direct connection between the molecule and the core structure is aliphatic. An aromatic residue refers to a substituent that is part of an aromatic system and is directly connected to the core structure, such as an arbitrarily substituted phenyl or pyridyl group.

[0151] As used in this article, the term "substituted hydroxyl group" refers to a hydroxyl group where the hydrogen atom is further replaced by any aliphatic or aromatic residue, meaning the core structure is connected to this aliphatic or aromatic residue via an oxygen atom (e.g., ). ).

[0152] As used in this article, the term "substituted amino" refers to an amino group in which the hydrogen atom is further replaced by any aliphatic or aromatic residue, meaning that the core structure is connected to this aliphatic or aromatic residue via a nitrogen atom (e.g., ). ).

[0153] As used herein, the term "peptide" refers to an organic compound containing two or more amino acids covalently linked by peptide bonds (amide bonds). Peptides are named according to the number of amino acids they make up; for example, a dipeptide contains two amino acid residues, a tripeptide contains three amino acid residues, and so on. Peptides containing ten or fewer amino acids are called oligopeptides, while peptides containing more than ten amino acid residues are called polypeptides. These amino acids may form at least one ring or a branched or unbranched chain, or a mixture thereof. Proteins and antibodies are peptides and therefore, although covered by this terminology, they may be named separately due to their importance.

[0154] As used herein, the term "amino acid" refers to an organic compound having a -CH(NH3)-COOH group. In one embodiment, the term "amino acid" refers to naturally occurring amino acids: arginine, lysine, aspartic acid, glutamic acid, glutamine, asparagine, histidine, serine, threonine, tyrosine, cysteine, methionine, tryptophan, alanine, isoleucine, leicine, phenylalanine, valine, proline, and glycine. However, the term also broadly includes non-naturally occurring amino acids.

[0155] The amino acids and peptides according to the invention can also be modified with functional groups. Non-limiting examples are glycosyl groups, such as N-acetylgalactosamine (GalNAc), or protecting groups, such as fluorenylmethoxycarbonyl (Fmoc)-modified or esterified groups.

[0156] The term "protein" refers to a peptide, a long chain containing one or more amino acid residues. Proteins perform a wide range of functions both in vivo and in vitro, including catalyzing metabolic reactions, DNA replication, responding to stimuli, and transporting molecules and catalyzing reactions. Proteins are folded into specific three-dimensional structures. Residues in proteins are frequently chemically modified, such as through post-translational modifications, thereby altering the protein's physical and chemical properties, folding, stability, activity, and ultimately its function. Sometimes proteins attach non-peptide groups, called prosthetic groups or cofactors. Many proteins, including enzymes and coenzymes, can also work synergistically to perform a specific function, and they often combine to form stable protein complexes. All of these forms are included in the term "protein."

[0157] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule, preferably composed of four polypeptide chains: two heavy (H) chains and two light (L) chains, which are typically linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (hereinafter referred to as VH) and a heavy chain constant region. The heavy chain constant region may include, for example, three domains: CH1, CH2, and CH3. Each light chain consists of one light chain variable region (hereinafter referred to as VL) and one light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL typically consists of three CDRs and up to four FRs, which are arranged, for example, in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0158] The term "antibody-drug conjugate" or the abbreviation ADC is well known to those skilled in the art and, as used herein, refers to the linking of an antibody or its antigen-binding fragment to a drug substance (e.g., chemotherapeutic agents, toxins, immunotherapeutic agents, imaging probes, etc.). As used herein, a "linker" is any chemical part of a drug substance covalently linked to an antibody or its antigen-binding fragment. As used herein, the term "linker-drug conjugate" refers to a molecule or chemical group that includes or comprises a linker as defined above and a drug substance. In this respect, the term "linker-drug conjugate" generally refers to a portion of an antibody-drug conjugate that is not an antibody or its antigen-binding fragment. Typically, in a linker-drug conjugate, the linker is covalently linked to the drug substance.

[0159] The term "DAR" or "drug antibody ratio" refers to the ratio of small molecule compounds or novel protein degraders to antibodies in a conjugate, i.e., the average number of small molecule compounds or novel protein degraders linked to each antibody. In some embodiments, the DAR of a conjugate is a value from 1 to 10. In some embodiments, the DAR of a conjugate is a value of 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the DAR of the coupling is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.

[0160] The terms "conjugating" and "linking" are used interchangeably. "Conjugating" and "linking" refer to the covalent or non-covalent attachment of a molecule with therapeutic effects, such as a drug, a novel protein degrader, or a drug component, to an antibody.

[0161] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features. Detailed Implementation

[0162] This invention provides a method for synthesizing a class of compounds containing novel cyclic α,β-unsaturated functional groups. This invention provides a method for coupling a cyclic α,β-unsaturated compound with a thiol-containing compound, including peptides, proteins, and antibodies. In the embodiments, the cyclic α,β-unsaturated compound is a compound capable of Michael addition with a thiol group. The covalent drug and antibody conjugates produced in the embodiments have not been reported in previous literature. The bioactivity of representative drugs was determined in the embodiments.

[0163] Schemes 1 and 2 illustrate the general strategy for synthesis according to the present invention using five-membered cyclic α,β-unsaturated compounds as examples. R1, R2, R3, and R5 are as defined in any of the preceding claims.

[0164] Option 1

[0165] Option 2

[0166] Scheme 3 describes the general strategy for synthesis according to the present invention using a six-membered cyclic α,β-unsaturated compound as an example. R1, R3, m, and PG are as defined in any of the preceding claims.

[0167] Option 3

[0168] The method described in this paper allows for use in R1, R2, R3, A large number of different organic compounds merge at the location.

[0169] Those skilled in the art will understand that embodiments of the present invention can be combined with each other, provided that combinations that may violate any laws of nature are excluded.

[0170] Synthesis of cyclic α,β-unsaturated compounds of formula (3)

[0171] Intermediates (int_1) and (int_2) undergo a Knoevenagel condensation reaction to generate an unstable intermediate. The α-NH group of this intermediate then undergoes an intramolecular ring-closure reaction with CN to yield the target compound. Alternatively, intermediates (int_3) and (int_2) undergo a Knoevenagel condensation reaction to generate a stable intermediate (int_4). Under certain conditions, intermediate (int_4) removes the protecting group PG from the N group, and the exposed α-NH group directly undergoes a ring-closure reaction with CN to yield the target compound.

[0172] Synthesis of cyclic α,β-unsaturated compounds of formula (4)

[0173] Intermediate (int_6) and intermediate (int_2) undergo a Knoevenagel condensation reaction to generate a stable intermediate (int_7). Under certain conditions, intermediate (int_7) removes the protecting group PG on N, and the exposed NH directly undergoes a cyclization reaction with CN to obtain the target compound.

[0174] Synthesis of cyclic α,β-unsaturated compounds of formula (5)

[0175] Intermediate (int_1) and intermediate (int_8) undergo a Knoevenagel condensation reaction to first generate an unstable reaction intermediate. The α-position NH of the reaction intermediate then undergoes an intramolecular cyclization reaction with CN to obtain the target compound.

[0176] Preferably, the Knoevenagel condensation reaction between the aldehyde-containing intermediate and the α-position active methylene intermediate described herein has the following characteristics:

[0177] The reaction is carried out in an organic solvent, preferably dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, 1,1,1-trichloroethane, acetonitrile, acetone, tetrahydrofuran, 2-methyltetrahydrofuran, diethyl ether, methyl tert-butyl ether, 1,4-dioxane, acetone, ethyl acetate, petroleum ether, n-hexane, cyclohexane, isopentane, n-pentane, cyclopentane, toluene, dichloroethylene, pyridine, nitromethane, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, methanol, ethanol, isopropanol, tert-butanol, n-butanol, cyclohexanol, 1,2-propanediol, ethylene glycol, and more preferably dichloromethane, chloroform, 1,2-dichloroethane, acetonitrile, acetone, tetrahydrofuran, 2-methyltetrahydrofuran, ethyl acetate, or pyridine.

[0178] The reaction commonly uses inorganic catalysts such as organic bases, inorganic bases, amine salts, Lewis acid-amine complexes, potassium fluoride, and aluminum phosphate. The organic bases are preferably pyrrolidine, piperidine, aziridine, diethylamine, dimethylamine, ethylamine, methylamine, ethylenediamine, triethylamine, triethylenediamine, 1,8-diazabicyclo[5,4,0]undecene-7, 1,5-diazabicyclo[4,3,0]non-5-ene, 4-dimethylaminopyridine, pyridine, quinoline, N-methylmorpholine, morpholine, and tetramethylethylenediamine. The Lewis acid-amine complexes are preferably titanium tetrachloride / pyridine, titanium tetrachloride / triethylamine, and trimethylchlorosilane / pyrrolidine. More preferably, the organic bases are pyrrolidine, piperidine, morpholine, aziridine, diethylamine, and dimethylamine.

[0179] The reaction produces water. Removing water can shift the reaction equilibrium toward the product. Therefore, water can be removed by using a water separator, adding molecular sieves or other water-removing reagents to increase the yield.

[0180] The reaction can be carried out at room temperature (i.e., about 25°C). However, it can also be carried out in a temperature range of -15°C to 100°C. The reaction time depends on the reaction volume and the amount of reactants. The ratio of the aldehyde-containing intermediate to the α-active methylene intermediate is preferably in the range of 5:1 to 1:5.

[0181] Example 1: Synthesis of Compound 1

[0182] Step 1: Synthesis of compound int_1-2:

[0183] Compound int_1-1 (80 mg), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (331 mg, 0.87 mmol), and cyanoacetic acid (68 mg, 0.80 mmol) were dissolved in N,N-dimethylformamide (30 mL), and diisopropylethylamine (281 mg, 2.18 mmol) was added. The reaction mixture was stirred at 25 °C for 16 hours. Water (300 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The filtrate was then purified by slurrying with ethyl acetate (20 mL) to obtain a white solid compound int_1-2.

[0184] LCMS m / z(ESI): 161 [M+H] + .

[0185] Step 2: Synthesis of compound int_1-4:

[0186] 2-Amino-2-methyl-1-propanol (2.5 g, 28.05 mmol) was dissolved in acetonitrile (50 mL), and potassium carbonate (7.75 g, 56.09 mmol) was added. The reaction solution was cooled to 0 °C, and methyl chloroformate (2.65 g, 28.05 mmol) was added. The mixture was heated to 25 °C and stirred for 5 hours. The reaction solution was concentrated to dryness under reduced pressure, quenched with water (50 mL), and extracted with ethyl acetate (50 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The compound int_1-4 was then purified by silica gel chromatography.

[0187] LC-MS m / z (ESI): 148.1 [M+H] + .

[0188] Step 3: Synthesis of compounds int_1-5:

[0189] Compound int_1-4 (0.8 g) was dissolved in dichloromethane (50 mL). The reaction solution was cooled to 0 °C, and Dys-Martin oxidant (3.46 g, 8.15 mmol) was added. The mixture was heated to 25 °C and stirred for 2 hours. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution until the pH reached 8. The solution was then extracted with dichloromethane (50 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain a colorless oily compound int_1-5. The crude product was used directly in the next reaction.

[0190] LC-MS m / z (ESI): 146.1 [M+H] + .

[0191] Step 4: Synthesis of Compound 1:

[0192] Compound int_1-5 (1.2 g) was dissolved in dichloromethane (20 mL). The reaction solution was cooled to 0 °C, and pyrrolidine (2 mL) and trimethylchlorosilane (1 mL) were added. The mixture was heated to 25 °C and stirred for 0.5 hours. Then, compound int_1-2 (92 mg, 0.57 mmol) was added, and the mixture was stirred at 25 °C for another hour. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution (30 mL), and then extracted with dichloromethane (30 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. Compound 1 was then purified by silica gel chromatography.

[0193] LC-MS m / z(ESI): 288 [M+H] + .

[0194] Example 2: Synthesis of Compound 2

[0195] Step 1: Synthesis of compound int_2-2:

[0196] Compound int_2-1 (2 g, 13.33 mmol) was dissolved in dichloromethane (60 mL), and 3-aminopyridine (1.4 g, 14.89 mmol) was added. The mixture was stirred at 25 °C for 48 hours under nitrogen protection. The reaction solution was quenched in ice water, and the aqueous phase was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated sodium chloride aqueous solution (50 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to finally obtain compound int_2-2.

[0197] LC-MS m / z (ESI): 164.9 [M+H] + .

[0198] Step 2: Synthesis of compound int_2-4:

[0199] Compound int_2-2 (600 mg, 3.65 mmol) was dissolved in dichloromethane (20 mL). Under nitrogen protection, pyrrolidine (5.2 g, 73 mmol) was added, followed by diethyl malonate (580 mg, 3.65 mmol). The mixture was stirred at room temperature for 12 hours. The reaction solution was concentrated and purified by C18 reverse-phase column chromatography to obtain compound int_2-4.

[0200] LC-MS m / z (ESI): 261.1 [M+H] + .

[0201] Step 3: Synthesis of compound int_2-5:

[0202] Compound int_2-4 (430 mg, 1.65 mmol) was dissolved in methanol (5 mL), water (5 mL) was added, and then lithium hydroxide (346 mg, 8.25 mmol) was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated, the pH was adjusted to 3-4 with dilute hydrochloric acid, and purified by C18 reverse-phase column chromatography to obtain compound int_2-5.

[0203] LC-MS m / z (ESI): 233.1 [M+H] + .

[0204] Step 4: Synthesis of Compound 2:

[0205] Compound int_203-4 (62 mg, 0.267 mmol) was dissolved in dichloromethane (20 mL) under nitrogen protection. Aniline (25 mg, 0.267 mmol) was added, followed by triethylamine (81 mg, 0.801 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (203 mg, 0.534 mmol). The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated and purified sequentially by C18 reversed-phase column chromatography and silica gel chromatography to obtain compound 2.

[0206] LC-MS m / z(ESI): 308 [M+H] + .

[0207] Example 3: Synthesis of Compound 3

[0208] Step 1: Synthesis of compound int_3-2:

[0209] Tert-butyl (2,2-dimethyl-3-oxopropyl)carbamate (65 mg, 0.323 mmol), 4A molecular sieve (0.2 g), tetrahydropyrrole (173 mg, 2.43 mmol), and compound int_1-2 (26 mg, 0.162 mmol) were mixed in dichloromethane (3 mL) and reacted at room temperature for 20 hours under argon protection. The mixture was filtered, the filtrate was concentrated, and the residue was purified by silica gel chromatography to obtain compound int_3-2.

[0210] LC-MS m / z(ESI): 344 [M+H] + .

[0211] Step 2: Synthesis of Compound 3:

[0212] Compound int_3-2 (60 mg, 0.17 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (159 mg) was added. The mixture was stirred at room temperature for 2 hours under argon protection. N,N-diisopropylethylamine (0.4 mL) was added to the mixture, and the mixture was concentrated. The residue was purified by silica gel chromatography to obtain compound 3.

[0213] LC-MS m / z(ESI): 244 [M+H] + .

[0214] Example 4: Synthesis of Compound 4

[0215] Step 1: Synthesis of compound int_4-2:

[0216] 3-Fluoroaniline (1 g, 8.999 mmol) and triethylamine (2.7 g, 26.998 mmol) were dissolved in dichloromethane (15 mL), and then compound int_2-1 (2 g, 13.499 mmol) was added. The mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated and purified by silica gel chromatography to obtain compound int_4-2.

[0217] LC-MS m / z (ESI): 182.1 [M+H] + .

[0218] Step 2: Synthesis of Compound 4:

[0219] Compound int_4-2 (500 mg, 2.759 mmol), 2-cyanoacetic acid (470 mg, 5.519 mmol), and pyrrolidine (3.9 g, 55.18 mmol) were dissolved in dichloromethane (10 mL) and stirred at room temperature for 24 hours under argon protection. The reaction intermediate and intramolecular cyclization product 4 were simultaneously detected by LC-MS. The reaction solution was concentrated and purified by C18 reversed-phase column chromatography to obtain compound 4 (the reaction intermediate was converted to compound 4 during purification).

[0220] LC-MS m / z (ESI): 249.1 [M+H] + .

[0221] 1 H NMR (400MHz, DMSO-d6) δ10.28(s,1H),8.29(s,1H),7.68–7.63(m,2H),7.45–7.40(m,2H),7.30–7.26(m,1H),1.35(s,6H).

[0222] Example 5: Synthesis of Compound 5

[0223] Step 1: Synthesis of compound int_5-2:

[0224] 2-Amino-2-methylpropanol (5.46 g, 61.3 mmol) was dissolved in dichloromethane (150 mL), and a solution of benzoyl isothiocyanate (10.0 g, 61.3 mmol) in dichloromethane (20 mL) was added dropwise at room temperature. After the addition was complete, the mixture was stirred at room temperature for 20 hours. The mixture was concentrated to dryness, and the residue was added to ethyl acetate (20 mL) and petroleum ether (100 mL), stirred at room temperature for 1 hour, filtered, and dried to give a pale yellow solid compound int_5-2.

[0225] LC-MS m / z(ESI): 253.0 [M+H] + .

[0226] Step 2: Synthesis of compound int_5-3:

[0227] Compound int_5-2 (13.3 g, 52.7 mmol) was dissolved in a tetrahydrofuran / methanol / water mixture (60 mL / 60 mL / 20 mL), and then lithium hydroxide monohydrate (4.42 g, 105.4 mmol) was added. The mixture was stirred at room temperature for 1 hour. The pH of the mixture was adjusted to 7–8 with 2N hydrochloric acid, and then concentrated to about one-quarter of its original volume before being directly added to the next reaction step.

[0228] LC-MS m / z (ESI): 149.0 [M+H] + .

[0229] Step 3: Synthesis of compound int_5-5:

[0230] In the previous step, ethanol (50 mL) and 3-bromo-1,1,1-trifluoroprop-2-one (8.05 g, 42.16 mmol) were added to the crude solution of compound int_5-3 (52.7 mmol). The mixture was heated to 80 °C and stirred for 2 hours. After cooling, the reaction solution was concentrated. The residue was added to ethyl acetate (100 mL) and saturated sodium bicarbonate aqueous solution (100 mL), stirred, and separated. The organic phase was washed with saturated sodium chloride solution (50 mL), concentrated, and the residue was purified by silica gel chromatography to obtain compound int_5-5.

[0231] LC-MS m / z(ESI): 241.0 [M+H] + .

[0232] Step 4: Synthesis of compounds int_5-6:

[0233] Compound int_5-5 (4.2 g, 17.5 mmol) was dissolved in dimethyl sulfoxide (84 mL), and 2-iodobenzoic acid (7.3 g, 26.2 mmol) was added. The mixture was stirred at room temperature for 20 hours. The reaction solution was filtered, and the filter cake was washed with ethyl acetate. The filtrate was cooled in an ice bath, and saturated sodium thiosulfate aqueous solution (100 mL) and saturated sodium bicarbonate aqueous solution (100 mL) were added. The mixture was stirred for 20 minutes and then extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed successively with saturated sodium bicarbonate solution (100 mL) and saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to give a pale yellow solid compound int_5-6.

[0234] LC-MS m / z(ESI): 239.0 [M+H] + .

[0235] Step 5: Synthesis of compound int_5-8:

[0236] (S)-1-phenylbut-1-amine (210 mg, 1.407 mmol) was dissolved in N,N-dimethylformamide (5 mL), followed by the addition of cyanoacetic acid (133 mg, 1.548 mmol), N,N-diisopropylethylamine (545 mg, 4.221 mmol), and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (800 mg, 2.111 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated and purified by C18 reversed-phase column chromatography to obtain compound int_5-8.

[0237] LC-MS m / z (ESI): 217.1 [M+H] + .

[0238] Step 6: Synthesis of Compound 5:

[0239] Compound int_5-8 (50 mg, 0.231 mmol), compound int_5-6 (83 mg, 0.347 mmol), and pyrrolidine (200 mg, 2.812 mmol) were dissolved in dichloromethane (5 mL) and stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure to remove the solution, and then purified by reverse-phase Flash chromatography using a C18 reverse-phase column to obtain compound 5.

[0240] LC-MS m / z (ESI): 437.2 [M+H] + .

[0241] 1 H NMR(400MHz, DMSO-d6)δ9.03(s,1H),8.91(d,J=8.3Hz,1H),7.85(q,J=1.1Hz,1H),7.77–7.68(m,1H),7.41–7.27 (m,3H),7.27–7.13(m,1H),4.93(m,1H),1.79(m,2H),1.68(d,J=10.4Hz,6H),1.29(m,2H),0.88(t,J=7.3Hz,3H).

[0242] Example 6: Synthesis of Compound 6

[0243] Compound int_6-1 (50 mg, 0.231 mmol), compound int_4-2 (63 mg, 0.347 mmol), and pyrrolidine (200 mg, 2.812 mmol) were dissolved in dichloromethane (5 mL) and stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure to remove the solution, and then purified by C18 reversed-phase column chromatography to obtain compound 6.

[0244] LC-MS m / z (ESI): 380.3 [M+H] + .

[0245] 1 H NMR (400MHz, DMSO-d6) δ10.40(s,1H),7.72(s,1H),7.56(td,J=8.2,6.7Hz,1H),7.30(h,J=5.3Hz,4H),7.24–7.14 (m,2H),7.11(d,J=7.9Hz,1H),4.96(q,J=7.6Hz,1H),1.77–1.57(m,2H),1.36–1.12(m,8H),0.85(t,J=7.3Hz,3H).

[0246] Example 7 Synthesis of Compound 7

[0247] Step 1: Synthesis of compound int_7-2:

[0248] Compound int_7-1 (489 mg, 2.27 mmol) was dissolved in dichloromethane (10 mL), and Dys-Martin oxidant (1.01 g, 2.38 mmol) was added. The mixture was stirred overnight at room temperature. The reaction was quenched with saturated sodium bicarbonate solution to pH 8, and then extracted with dichloromethane (20 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound int_7-2.

[0249] Step 2: Synthesis of compound int_7-3:

[0250] Compound int_7-2 (385 mg, 1.81 mmol) was dissolved in dichloromethane (4 mL) and trifluoroacetic acid (1 mL) and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to remove the solvent, yielding a yellow liquid compound int_7-3. The crude product was used directly in the next reaction step.

[0251] Step 3: Synthesis of compound int_7-6:

[0252] Compound int_7-4 (27 mg, 196 μmol) was dissolved in dichloromethane (5 mL), and triethylamine (59 mg, 584 μmol) was added. The reaction solution was cooled to 0 °C under argon protection, and then methyl malonate chloride (28 mg, 205 μmol) dissolved in dichloromethane (0.5 mL) was added. The mixture was stirred for 1 hour under ice bath cooling. The reaction solution was concentrated to dryness under reduced pressure, and the residue was purified by C18 reversed-phase column chromatography to obtain compound int_7-6.

[0253] LC-MS m / z(ESI): 237 [M+H] + .

[0254] Step 4: Synthesis of Compound 7:

[0255] Compound int_7-6 (29 mg, 0.124 mmol), compound int_7-3 (205 mg, crude trifluoroacetate), pyrrolidine (214 mg), and trimethylchlorosilane (108 mg) were dissolved in dichloromethane (5 mL) and stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure to remove the solution, and compound 7 was purified by C18 reversed-phase column chromatography.

[0256] LC-MS m / z(ESI): 300 [M+H] + .

[0257] Example 8: Synthesis of Compound 8

[0258] Step 1: Synthesis of compound int_8-2:

[0259] Compound int_8-1 (389 mg, 1.81 mmol) was dissolved in dichloromethane (5 mL), and Dys-Martin oxidant (1.16 g, 2.74 mmol) was added. The mixture was stirred overnight at room temperature. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution to pH 8. The mixture was extracted with dichloromethane (20 mL × 3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound int_8-2.

[0260] Step 2: Synthesis of compound int_8-3:

[0261] Compound int_8-2 (72 mg, 0.338 mmol), compound int_5-8 (65 mg, 0.301 mmol), pyrrolidine (242 mg), and 4A molecular sieve (200 mg) were mixed in dichloromethane (2.5 mL) and stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure to remove the solution, and then purified by C18 reversed-phase column chromatography to obtain compound int_8-3.

[0262] LC-MS m / z(ESI): 412 [M+H] + .

[0263] Step 3: Synthesis of Compound 8:

[0264] Compound int_8-3 (80 mg, 0.194 mmol) was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (1 mL) and stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure to remove the solution, and then purified by C18 reversed-phase column chromatography to obtain compound 8.

[0265] LC-MS m / z(ESI): 312 [M+H] + .

[0266] Example 9: Synthesis of Compound 9

[0267] Step 1: Synthesis of compound int_9-2:

[0268] Diethyl malonate (485 mg, 3.031 mmol), tert-butyl (2,2-dimethyl-3-oxopropyl)carbamate (400 mg, 1.987 mmol), and tetrahydropyrrole (2.83 g, 39.7 mmol) were mixed in dichloromethane (10 mL), and the mixture was stirred at 30 °C for 20 hours under argon protection. A saturated sodium bicarbonate aqueous solution (20 mL) was added to the mixture, and the mixture was separated. The aqueous phase was extracted again with dichloromethane (20 mL × 3). The combined organic phases were washed with a saturated sodium chloride aqueous solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to obtain a colorless oily compound, int_9-2.

[0269] LC-MS m / z (ESI): 344.2 [M+H] + .

[0270] Step 2: Synthesis of compound int_9-3:

[0271] Compound int_9-2 (120 mg, 0.349 mmol) and sodium tert-butoxide (3 mg) were mixed in tetrahydrofuran (2 mL), and the mixture was stirred at room temperature for 3 hours under argon protection. The resulting mixture of compound int_9-3 was used directly in the next reaction step.

[0272] Step 3: Synthesis of compound int_9-4:

[0273] Methanol / water (1 mL / 0.5 mL) and lithium hydroxide monohydrate (100 mg, 2.38 mmol) were added to the reaction solution of compound int_9-3, and the mixture was stirred at room temperature for 5 hours. The pH of the reaction mixture was adjusted to 5-6 with 2N hydrochloric acid, and the mixture was freeze-dried to obtain crude compound int_9-4, which was used directly in the next reaction.

[0274] Step 4: Synthesis of compound int_9-5:

[0275] Compound int_9-4 (crude, 0.349 mmol), 3-aminopyridine (22 mg, 0.232 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (176 mg, 0.349 mmol), and triethylamine (117 mg, 1.16 mmol) were mixed in N,N-dimethylformamide (5 mL). The mixture was stirred at room temperature for 5 hours under argon protection. The mixture was purified by C18 reverse-phase column chromatography to obtain compound int_9-5.

[0276] LC-MS m / z (ESI): 346 [M+H] + .

[0277] Step 5: Synthesis of Compound 9:

[0278] Compound int_9-5 (30 mg, 0.087 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) was added. The mixture was stirred at room temperature for 2 hours under argon protection. N,N-diisopropylethylamine (0.4 mL) was added to the reaction mixture, and the mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 9.

[0279] LC-MS m / z(ESI): 246 [M+H] + .

[0280] Example 10: Synthesis of Compound 10

[0281] Step 1: Synthesis of compound int_10-2:

[0282] Compound int_10-1 (172 mg) was dissolved in dichloromethane (20 mL). The reaction solution was cooled to 0 °C, and pyrrolidine (2 mL) and trimethylchlorosilane (1 mL) were added. The mixture was heated to 25 °C and stirred for 0.5 hours. Then, compound int_1-2 (92 mg, 0.57 mmol) was added, and the mixture was stirred at 25 °C for another hour. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution (30 mL), and then extracted with dichloromethane (30 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. Compound int_10-2 was then purified by silica gel chromatography.

[0283] LC-MS m / z(ESI): 344 [M+H] + .

[0284] Step 2: Synthesis of Compound 10:

[0285] Compound int_10-5 (50 mg, 0.146 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) was added. The mixture was stirred at room temperature for 2 hours under argon protection. N,N-diisopropylethylamine (0.4 mL) was added to the reaction mixture, and the mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 10.

[0286] LC-MS m / z(ESI): 244 [M+H] + .

[0287] Example 11 Synthesis of Compound 11

[0288] Step 1: Synthesis of compound int_11-2:

[0289] Compounds int_11-1 (2.87 g, 13.33 mmol) and int_1-2 (2.13 g, 13.33 mmol) were dissolved in methanol (30 mL), and sodium hydroxide aqueous solution (10 M, 2 mL) was added. The mixture was stirred at room temperature for 1 hour. Water (80 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (100 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. Compound int_11-2 was then purified by silica gel chromatography.

[0290] LC-MS m / z(ESI): 376 [M+H] + .

[0291] Step 2: Synthesis of Compound 11:

[0292] Compound int_11-2 (100 mg, 0.266 mmol) was dissolved in dichloromethane (2 mL), and then a methanol solution of hydrogen chloride (3 M, 5 mL) was added. The mixture was heated to 40 °C and stirred for 16 hours. The reaction solution was concentrated to dryness under reduced pressure, and then purified by silica gel chromatography to obtain compound 11.

[0293] LC-MS m / z(ESI): 312 [M+H] + .

[0294] Example 12 Synthesis of Compound 12

[0295] Step 1: Synthesis of compound int_12-2:

[0296] Compound int_12-1 (69 mg, 0.323 mmol), 4A molecular sieve (0.2 g), tetrahydropyrrole (173 mg, 2.43 mmol), and compound int_5-8 (35 mg, 0.162 mmol) were mixed in dichloromethane (3 mL), and the mixture was stirred at room temperature for 20 hours under argon protection. The mixture was filtered, the filtrate was concentrated, and the residue was purified by silica gel chromatography to obtain compound int_12-2.

[0297] LC-MS m / z (ESI): 414 [M+H] + .

[0298] Step 2: Synthesis of Compound 12:

[0299] Compound int_12-2 (70 mg, 0.17 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (159 mg) was added. The mixture was stirred at room temperature for 2 hours under argon protection. N,N-diisopropylethylamine (0.4 mL) was added to the mixture, and the mixture was concentrated. The residue was purified by silica gel chromatography to obtain compound 12.

[0300] LC-MS m / z(ESI): 314 [M+H] + .

[0301] Example 13 Synthesis of Compound 13

[0302] Step 1: Synthesis of compound int_13-2:

[0303] Compounds int_13-1 (2.52 g, 13.33 mmol) and int_1-2 (2.13 g, 13.33 mmol) were dissolved in methanol (30 mL), and sodium hydroxide aqueous solution (10 M, 2 mL) was added. The mixture was stirred at room temperature for 1 hour. Water (80 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (100 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. Compound int_13-2 was then purified by silica gel chromatography.

[0304] LC-MS m / z(ESI): 350 [M+H] + .

[0305] Step 2: Synthesis of Compound 13:

[0306] Compound int_13-2 (93 mg, 0.266 mmol) was dissolved in dichloromethane (2 mL), and then a methanol solution of hydrogen chloride (3 M, 5 mL) was added. The mixture was heated to 40 °C and stirred for 16 hours. The reaction solution was concentrated to dryness under reduced pressure, and then purified by silica gel chromatography to obtain compound 13.

[0307] LC-MS m / z(ESI): 258 [M+H] + .

[0308] Example 14 Synthesis of Compound 14

[0309] Compound int_14-1 (100 mg, 0.418 mmol) was dissolved in tetrahydrofuran (3 mL), and sodium hydride (60%, 67 mg) was added. The mixture was stirred at 25 °C for 0.5 h under nitrogen protection. Then, compound int_2-1 (126 mg, 0.836 mmol) was added, and the mixture was heated to 50 °C and stirred for 16 h. After cooling, the reaction solution was quenched in ice water (10 mL). The aqueous phase was extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated sodium chloride aqueous solution (5 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. Compound 14 was obtained by silica gel chromatography.

[0310] LC-MS m / z(ESI): 292 [M+H] + .

[0311] Example 15 Synthesis of Compound 15

[0312] Step 1: Synthesis of compound int_15-3:

[0313] Compound int_15-2 (100 mg, 0.365 mmol) was dissolved in tetrahydrofuran (3 mL), and sodium hydride (60% (60 mg)) was added. The mixture was stirred at 25 °C for 0.5 h under nitrogen protection. Then, compound int_15-1 (108 mg, 0.547 mmol) was added, and the mixture was heated to 50 °C and stirred for 16 h. After cooling, the reaction solution was quenched in ice water (10 mL). The aqueous phase was extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated sodium chloride aqueous solution (5 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. Compound int_15-3 was obtained by silica gel chromatography.

[0314] LC-MS m / z(ESI): 390 [M+H] + .

[0315] Step 2: Synthesis of Compound 15:

[0316] Compound int_15-3 (100 mg, 0.256 mmol) was dissolved in dichloromethane (2 mL), and then a methanol solution of hydrogen chloride (3 M, 5 mL) was added. The mixture was heated to 40 °C and stirred for 16 hours. The reaction solution was concentrated to dryness under reduced pressure, and then purified by silica gel chromatography to obtain compound 15.

[0317] LC-MS m / z(ESI): 326 [M+H] + .

[0318] Example 16: Screening of Synthetic Conditions for Cyclic α,β-Unsaturated Compounds

[0319] The following investigation, through the synthesis of compound 16, examined the Knoevenagel condensation reaction conditions of the aldehyde-containing intermediate (e.g., int_4-2) and the α-active methylene-containing intermediate (e.g., int_1-2) involved in this invention. The reaction procedures are as follows:

[0320] At a specified temperature, an aldehyde-containing intermediate and a base are mixed in an organic solvent. A Lewis acid (e.g., trimethylchlorosilane) may be added or not. After stirring, an intermediate containing an α-active methylene group is added, and stirring continues until the reaction is complete. The proportions of the starting material, reaction intermediate, and target product in the reaction solution are determined by HPLC (excluding the proportion of the aldehyde-containing intermediate in the reaction solution).

[0321] At 25 °C, compound int_4-2 (85 mg, 0.468 mmol) and pyrrolidine (222 mg, 3.12 mmol) were dissolved in a solvent (5 mL), and then compound int_1-2 (50 mg, 0.312 mmol) was added. The mixture was stirred for 16 hours. The product ratios under different solvent conditions are shown in Table 1.

[0322] Table 1. Screening of reaction solvents

[0323] At 25°C, compound int_4-2 (85 mg, 0.468 mmol) and a base (3.12 mmol) were dissolved in a solvent (5 mL), and then compound int_1-2 (50 mg, 0.312 mmol) was added. The mixture was stirred for 16 hours. The product ratios under different conditions are shown in Table 2.

[0324] Table 2 Screening of Alkali and Solvents

[0325] Compound int_4-2 (85 mg, 0.468 mmol) and pyrrolidine (3.12 mmol) were dissolved in a solvent (5 mL), and then compound int_1-2 (50 mg, 0.312 mmol) was added. The mixture was stirred for 16 hours. The product ratios under different conditions are shown in Table 3.

[0326] Table 3 Reaction Temperature and Solvent Screening

[0327] At 25°C, compound int_4-2 and pyrrolidine (3.12 mmol) were dissolved in dichloromethane (5 mL), and then compound int_1-2 (50 mg, 0.312 mmol) was added. The mixture was stirred for 16 hours. The product ratios under different conditions are shown in Table 4.

[0328] Table 4. Screening of equivalent numbers for intermediates containing aldehyde groups.

[0329] At 25°C, compound int_4-2 (85 mg, 0.468 mmol) and pyrrolidine (222 mg, 3.12 mmol) were dissolved in dichloromethane (5 mL), followed by the addition of a dehydrating agent (200 mg) and compound int_1-2 (50 mg, 0.312 mmol), and the mixture was stirred for 16 hours. The product ratios under different conditions are shown in Table 5.

[0330] Table 5 Screening of Dehydrating Agents

[0331] At 25 °C, compound int_4-2 (85 mg, 0.468 mmol) and pyrrolidine (222 mg, 3.12 mmol) were dissolved in dichloromethane (5 mL), followed by the addition of compound int_1-2 (50 mg, 0.312 mmol). The reaction was then monitored after stirring for different times. The product ratios under different conditions are shown in Table 6.

[0332] Table 6 Reaction Time Screening

[0333] Using the above synthetic method, the target compounds in Table 7 can be obtained through Knoevenagel condensation reactions involving different aldehyde-containing intermediates and intermediates containing α-active methylene groups.

[0334] Table 7

[0335] Example 17: Synthesis of compound L-1

[0336] Step 1: Synthesis of compound int_L1-2

[0337] Compounds int_L1-1 (375 mg, 2 mmol) and int_4-3 (340 mg, 4 mmol) were dissolved in dichloromethane (10 mL), and tetrahydropyrrole (2.85 g, 40 mmol) was added. The reaction mixture was reacted at room temperature for 24 hours under argon protection. The reaction solution was concentrated and purified by C18 reversed-phase column chromatography to obtain compound int_L1-2.

[0338] LC-MS m / z (ESI): 255.1 [M+H] + .

[0339] Step 2: Synthesis of compound int_L1-4

[0340] Compounds int_L1-2 (76 mg, 0.3 mmol) and int_L1-3 (267 mg, 0.33 mmol) were dissolved in N,N-dimethylformamide (3 mL). Under ice bath conditions, N,N-diisopropylethylamine (78 mg, 0.6 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (137 mg, 0.36 mmol) were added sequentially. The reaction mixture was slowly heated to room temperature for 5 hours under argon protection. The reaction solution was purified by C18 reverse-phase column chromatography to obtain compound int_L1-4.

[0341] LC-MS m / z (ESI): 1045.4 [M+H] + .

[0342] Step 3: Synthesis of compound L-1

[0343] Compound int_L1-4 (105 mg, 0.1 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.2 mL) was added under ice bath conditions. The mixture was then slowly heated to room temperature for 2 hours. The reaction solution was concentrated and purified by C18 reversed-phase column chromatography to obtain compound L-1.

[0344] LC-MS m / z (ESI): 945.4 [M+H] + .

[0345] Example 18: Synthesis of compound L-2

[0346] Step 1: Synthesis of compound int-L2-1

[0347] Compounds int_L1-3 (840 mg, 1 mmol) and int_4-3 (94 mg, 1.1 mmol) were dissolved in N,N-dimethylformamide (10 mL). Under ice bath conditions, N,N-diisopropylethylamine (388 mg, 3 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (456 mg, 1.2 mmol) were added sequentially. The mixture was slowly heated to room temperature and stirred for 16 hours under argon protection. Water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by C18 reversed-phase column chromatography to obtain compound int_L2-1.

[0348] LC-MS m / z (ESI): 908.3 [M+H] + .

[0349] Step 2: Synthesis of compound L2

[0350] Compounds int_L2-1 (91 mg, 0.1 mmol) and int_4-2 (36.2 mg, 0.2 mmol) were dissolved in dichloromethane (2 mL), and tetrahydropyrrole (71 mg, 1 mmol) was added. The reaction was carried out at room temperature for 12 hours under argon protection. The reaction solution was concentrated and purified by C18 reversed-phase column chromatography to obtain compound L-2.

[0351] LC-MS m / z (ESI): 1071.4 [M+H] + .

[0352] Example 19: Synthesis of compound L-3

[0353] Step 1: Synthesis of compound int_L3-2

[0354] Compounds int_4-3 (468 mg, 5.5 mmol) and int_L3-1 (866 mg, 5 mmol) were dissolved in N,N-dimethylformamide (50 mL). Under ice bath conditions, N,N-diisopropylethylamine (1.94 g, 15 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (2.28 g, 6 mmol) were added sequentially. The mixture was slowly heated to room temperature and stirred for 4 hours under argon protection. Water (200 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to obtain compound int_L3-2.

[0355] LC-MS m / z (ESI): 241.2 [M+H] + .

[0356] Step 2: Synthesis of compound int_L3-3

[0357] Compounds int_L3-2 (721 mg, 3 mmol) and int_2-2 (985 mg, 6 mmol) were dissolved in dichloromethane (30 mL), and tetrahydropyrrole (2.13 g, 30 mmol) was added. The reaction was carried out at room temperature for 12 hours under argon protection. The reaction solution was concentrated and purified by C18 reversed-phase column chromatography to obtain compound int_L3-3.

[0358] LC-MS m / z (ESI): 387.2 [M+H] + .

[0359] Step 3: Synthesis of compound int_L3-4

[0360] Compound int_L3-3 (386 mg, 1 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (2 mL) was added under ice bath conditions. The mixture was then slowly brought to room temperature and reacted for 2 hours. The reaction solution was concentrated to obtain a crude product, which was used directly in the next reaction step.

[0361] LC-MS m / z (ESI): 331.2 [M+H] + .

[0362] Step 4: Synthesis of compound L-3

[0363] The crude product int_L3-4 (109 mg, 0.33 mmol) and compound int_L1-3 (252 mg, 0.3 mmol) were dissolved in N,N-dimethylformamide (3 mL). Under ice bath conditions, N,N-diisopropylethylamine (116 mg, 0.9 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (137 mg, 0.36 mmol) were added sequentially. The reaction mixture was slowly heated to room temperature for 12 hours under argon protection. The reaction solution was purified by C18 reverse-phase column chromatography to obtain compound L-3.

[0364] LC-MS m / z (ESI): 1053.5 [M+H] + .

[0365] Example 20: Synthesis of compound L-4

[0366] Step 1: Synthesis of compound int_L4-2

[0367] Compounds int_4-3 (468 mg, 5.5 mmol) and int_L4-1 (876 mg, 5 mmol) were dissolved in N,N-dimethylformamide (50 mL). Under ice bath conditions, N,N-diisopropylethylamine (1.94 g, 15 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (2.28 g, 6 mmol) were added sequentially. The mixture was slowly heated to room temperature and stirred for 12 hours under argon protection. Water (200 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to obtain compound int_L4-2.

[0368] LC-MS m / z (ESI): 243.1 [M+H] + .

[0369] Step 2: Synthesis of compound int_L4-3

[0370] Compounds int_L4-2 (606 mg, 2.5 mmol) and int_2-2 (821 mg, 5 mmol) were dissolved in dichloromethane (25 mL), and tetrahydropyrrole (1.78 g, 25 mmol) was added. The reaction mixture was reacted at room temperature for 12 hours under argon protection. The reaction solution was concentrated and purified by C18 reversed-phase column chromatography to obtain compound int_L4-3.

[0371] LC-MS m / z (ESI): 389.2 [M+H] + .

[0372] Step 3: Synthesis of compound int_L4-4

[0373] Compound int_L4-3 (311 mg, 0.8 mmol) was dissolved in dichloromethane (8 mL), and trifluoroacetic acid (1.6 mL) was added under ice bath conditions. The mixture was then slowly heated to room temperature for 2 hours. The reaction solution was concentrated to obtain a crude product, which was used directly in the next step of the reaction.

[0374] LC-MS m / z (ESI): 333.2 [M+H] + .

[0375] Step 4: Synthesis of compound L-4

[0376] The crude product int_L4-4 (37 mg, 0.11 mmol) and compound int_L1-3 (84 mg, 0.1 mmol) were dissolved in N,N-dimethylformamide (1 mL). Under ice bath conditions, N,N-diisopropylethylamine (39 mg, 0.3 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (46 mg, 0.12 mmol) were added sequentially. The reaction mixture was slowly heated to room temperature for 5 hours under argon protection. The reaction solution was purified by C18 reverse-phase column chromatography to obtain compound L-4.

[0377] LC-MS m / z (ESI): 1055.5 [M+H] + .

[0378] Example 21: Synthesis of compound L-5

[0379] Step 1: Synthesis of compound int_L5-2

[0380] Compounds int_L1-3 (840 mg, 1 mmol) and int_L5-1 (373 mg, 1.1 mmol) were dissolved in N,N-dimethylformamide (10 mL). Under ice bath conditions, N,N-diisopropylethylamine (388 mg, 3 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (456 mg, 1.2 mmol) were added sequentially. The reaction mixture was slowly heated to room temperature for 12 hours under argon protection. The reaction solution was purified by C18 reverse-phase column chromatography to obtain compound int_L5-2.

[0381] LC-MS m / z (ESI): 1162.5 [M+H] + .

[0382] Step 2: Synthesis of compound int_L5-3

[0383] Compound int_L5-2 (639 mg, 0.55 mmol) was dissolved in N,N-dimethylformamide (5.5 mL), and tetrahydropyrrole (47 mg, 0.66 mmol) was added sequentially under ice bath conditions. The reaction was carried out under argon protection and slowly heated to room temperature for 3 hours. The reaction solution was purified by C18 reverse-phase column chromatography to obtain compound int_L5-3.

[0384] LC-MS m / z (ESI): 940.4 [M+H] + .

[0385] Step 3: Synthesis of compound int_L5-4

[0386] Compounds int_L5-3 (226 mg, 0.24 mmol) and int_4-3 (23 mg, 0.264 mmol) were dissolved in N,N-dimethylformamide (2.5 mL). Under ice bath conditions, N,N-diisopropylethylamine (93 mg, 0.72 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (110 mg, 0.288 mmol) were added sequentially. The mixture was slowly heated to room temperature and stirred for 12 hours under argon protection. The reaction solution was purified by C18 reverse-phase column chromatography to obtain compound int_L5-4.

[0387] LC-MS m / z (ESI): 1007.4 [M+H] + .

[0388] Step 4: Synthesis of compound L-5

[0389] Compounds int_L5-4 (101 mg, 0.1 mmol) and int_L5-6 (29 mg, 0.2 mmol) were dissolved in dichloromethane (1 mL), and tetrahydropyrrole (71 mg, 1 mmol) was added. The reaction was carried out at room temperature for 12 hours under argon protection. The reaction solution was concentrated and purified by C18 reverse-phase column chromatography to obtain compound L-5.

[0390] LC-MS m / z (ESI): 1152.5 [M+H] + .

[0391] Example 22: Synthesis of compound L-6

[0392] Step 1: Synthesis of compound int_L6-1

[0393] Compound int_L1-1 (1.87 g, 10 mmol) was dissolved in dichloromethane (100 mL), followed by tetrahydropyrrole (14.2 g, 200 mmol) and diethyl malonate (1.61 g, 10 mmol) in sequence. The reaction was carried out at room temperature for 12 hours under argon protection. The reaction solution was concentrated and purified by silica gel chromatography to obtain compound int_L6-1.

[0394] LC-MS m / z (ESI): 284.1 [M+H] + .

[0395] Step 2: Synthesis of compound int_L6-2

[0396] Compound int_L6-1 (1.42 g, 5 mmol) was dissolved in methanol (15 mL), water (15 mL) was added, and then lithium hydroxide (600 mg, 25 mmol) was added. The reaction was carried out at room temperature for 2 hours. The pH was adjusted to 4-5 with dilute hydrochloric acid, the reaction solution was concentrated, and the compound int_L6-2 was obtained by separation and purification using a C18 reversed-phase column.

[0397] LC-MS m / z (ESI): 256.1 [M+H] + .

[0398] Step 3: Synthesis of compound int_L6-3

[0399] Compounds int_L6-2 (84 mg, 0.33 mmol) and int_L5-3 (282 mg, 0.3 mmol) were dissolved in N,N-dimethylformamide (3 mL). Under ice bath conditions, N,N-diisopropylethylamine (116 mg, 0.9 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (137 mg, 0.36 mmol) were added sequentially. The reaction was carried out under argon protection and slowly increased to room temperature for 12 hours. Compound int_L6-3 was obtained by purification using a C18 reversed-phase column.

[0400] LC-MS m / z (ESI): 1163.5 [M+H] + .

[0401] Step 4: Synthesis of compound L-6

[0402] Compound int_L6-3 (118 mg, 0.1 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.2 mL) was added under ice bath conditions. The mixture was then slowly heated to room temperature for 2 hours. The reaction solution was concentrated and purified by C18 reversed-phase column chromatography to obtain compound L-6.

[0403] LC-MS m / z (ESI): 1077.4 [M+H] + .

[0404] Example 23: Synthesis of compound L-7

[0405] Compounds int_2-5 (51 mg, 0.22 mmol) and int_L5-3 (188 mg, 0.2 mmol) were dissolved in N,N-dimethylformamide (2 mL). Under ice bath conditions, N,N-diisopropylethylamine (78 mg, 0.6 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (91 mg, 0.24 mmol) were added sequentially. The reaction was carried out under argon protection and slowly increased to room temperature for 12 hours. Compound L-7 was obtained by purification using a C18 reversed-phase column.

[0406] LC-MS m / z (ESI): 1154.5 [M+H] + .

[0407] Example 24: Synthesis of compound L-8

[0408] Step 1: Synthesis of compound int_L8-1

[0409] Compounds int_9-4 (148 mg, 0.55 mmol) and int_L5-3 (470 mg, 0.5 mmol) were dissolved in N,N-dimethylformamide (5 mL). Under ice bath conditions, N,N-diisopropylethylamine (194 mg, 1.5 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (228 mg, 0.6 mmol) were added sequentially. The reaction was carried out under argon protection and slowly increased to room temperature for 12 hours. Compound int_L8-1 was obtained by purification using a C18 reversed-phase column.

[0410] LC-MS m / z (ESI): 1191.5 [M+H] + .

[0411] Step 2: Synthesis of compound L-8

[0412] Compound int_L8-1 (119 mg, 0.1 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.2 mL) was added under ice bath conditions. The mixture was then slowly heated to room temperature for 2 hours. The reaction solution was concentrated and purified by C18 reversed-phase column chromatography to obtain compound L-8.

[0413] LC-MS m / z (ESI): 1091.5 [M+H] + .

[0414] Example 25: Synthesis of compound L-9

[0415] Step 1: Synthesis of compound int_L9-1

[0416] Compound int_7-3 (1.13 g, 10 mmol) was dissolved in dichloromethane (100 mL), followed by tetrahydropyrrole (14.2 g, 200 mmol) and diethyl malonate (1.61 g, 10 mmol) in sequence. The reaction mixture was then reacted at room temperature for 12 hours under argon protection. The reaction solution was concentrated and purified by C18 reversed-phase column chromatography to obtain compound int_L9-1.

[0417] LC-MS m / z (ESI): 210.1 [M+H] + .

[0418] Step 2: Synthesis of compound int_L9-2

[0419] Compound int_L8-1 (879 mg, 4.2 mmol) was dissolved in methanol (10 mL), water (10 mL) was added, and then lithium hydroxide (503 mg, 21 mmol) was added. The reaction was carried out at room temperature for 2 hours. The pH was adjusted to 3-4 with dilute hydrochloric acid, the reaction solution was concentrated, and the compound int_L9-2 was obtained by separation and purification using a C18 reverse-phase column.

[0420] LC-MS m / z (ESI): 182.1 [M+H] + .

[0421] Step 3: Synthesis of compound L-9

[0422] Compounds int_L9-2 (40 mg, 0.22 mmol) and int_L5-3 (188 mg, 0.2 mmol) were dissolved in N,N-dimethylformamide (2 mL). Under ice bath conditions, N,N-diisopropylethylamine (78 mg, 0.6 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (91 mg, 0.24 mmol) were added sequentially. The reaction was carried out under argon protection and slowly increased to room temperature for 12 hours. Compound L-9 was obtained by purification using a C18 reversed-phase column chromatography.

[0423] LC-MS m / z (ESI): 1103.5 [M+H] + .

[0424] Example 26 Michael addition activity test of the compound of the present invention with mercaptoethanol

[0425] The compound was co-incubated with excess mercaptoethanol in pH 7.0 buffer at 37°C for 12 h using HPLC (column: Agilent Zorbax SB-Aq C18 (4.6 x 250 mm, particle size: 5 μm); column temperature: 25°C; injection volume: 5 μL; mobile phase: methanol:water = 10:90, v / v; flow rate: 0.8 mL / min; detection wavelength: 215 nm). The formation of its covalent addition product was detected to simulate its interaction with the target protein and determine its Michael addition reactivity.

[0426] Example 27 Michael addition activity test of the compound of the present invention with glutathione

[0427] The compound was co-incubated with excess glutathione ethanol in pH 7.0 buffer at 37°C for 12 h using HPLC (column: Agilent Zorbax SB-Aq C18 (4.6 x 250 mm, particle size: 5 μm); column temperature: 25°C; injection volume: 5 μL; mobile phase: methanol:water = 10:90, v / v; flow rate: 0.8 mL / min; detection wavelength: 215 nm). The formation of the covalent addition product was detected to simulate its interaction with the target protein and determine its Michael addition reactivity.

[0428] Example 28: Preparation of antibody-drug conjugates (ADCs) by adding the compounds of the present invention to antibody thiol groups.

[0429] At 37°C, 63 μL of an aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (CAS: 51805-45-9) (5 mg / mL) was added to 1 mL of PBS buffered solution of the antibody Trastuzumab (pH 7.0, 50 mM, containing 1 mM EDTA, 10.8 mg / mL), and incubated at 37°C for 1 h. The antibody solution was then transferred to an ultrafiltration centrifuge tube and diluted to 15 mL with citrate buffered saline (pH 5.0, 10 mM, containing 1 mM EDTA). The solution was ultrafiltered and centrifuged (12 min, 4500 rpm) six times. After ultrafiltration concentration, 1.74 mL of sample was obtained. The sample concentration was determined using a UV-Vis spectrophotometer, and the sample recovery rate was found to be 91%, with a concentration of 5.90 mg / mL. Take 1.71 mL of the above sample (containing 10.1 mg of antibody), adjust the pH to 6.5 with 1 M sodium citrate buffer solution, add 70.8 μL of dehydroascorbic acid (CAS: 490-83-5) aqueous solution (1 mg / mL) to the antibody solution, and react at 4 °C for 2 h. Transfer to an ultrafiltration centrifuge tube, dilute to 15 mL with citrate buffer solution (pH 5.0, 10 mM, containing 1 mM EDTA), and ultrafilter centrifuge (12 min, 4500 rpm) 6 times. Transfer the solution to a centrifuge tube to obtain 1.73 mL of sample. Determine the sample concentration using a UV-Vis spectrophotometer, and the sample recovery rate is determined to be 91%, with a concentration of 5.37 mg / mL.

[0430] Add 1.70 mL of the above antibody solution (containing 9.10 mg of antibody) to a test tube, adjust the pH to 6.0 with 1 M sodium citrate buffer solution, add DMSO solution (5 mg / mL, 3.5 equiv) of the cyclic α,β-unsaturated compound of this invention to the antibody solution, mix well, and react at room temperature for 90 min. After the reaction, filter through a 0.22 μM filter and transfer to an ultrafiltration centrifuge tube. Dilute to 15 mL with histidine buffer (pH 6.0, 25 mM, containing 6% sucrose), and ultrafilter and centrifuge (12 min, 4500 rpm) six times to obtain the antibody-conjugated compound buffer, which is then stored frozen at 4 °C. Calculate the average value (DAR value) using UV chromatography. Analyze the polymer content by size exclusion chromatography (SEC).

[0431] Example 29 Preparation of ADC-L-3

[0432] At 37°C, a prepared aqueous solution of tris(2-carbonylethyl) phosphate hydrochloride (10 mM, 84.5 μL, 845 nmol) was added to a PBS-buffered aqueous solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 2.5 mL, 10 mg / mL, 169 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0433] Add 127 μL of a pre-prepared dimethyl sulfoxide (DMSO) solution (20 mM) of compound L-3 dropwise to the above reaction solution. Place the solution in a water bath shaker and shake at 25 °C for 3 hours, then stop the reaction. Purify the reaction solution by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain 4.9 mL of PBS buffer solution containing the product ADC-L-3 (3.5 mg / mL). Store frozen at 4 °C.

[0434] RP-HPLC calculated average DAR value: n = 7.7.

[0435] Table 8

[0436] Biological Example 1: Thiol Addition Reaction of the Compounds of the Invention at the Protein Level

[0437] The experiments on the protein level of the cyclic α,β-unsaturated compounds of this invention were first conducted on compound 6. The purified PI3Kα protein was diluted in binding buffer (20 mM Tris (pH 7.5), 150 mM NaCl). Compound 6 was added to the buffer at a protein:compound molar ratio of 1:10. After incubation at room temperature for 2 hours, mass spectrometry was performed. The mass spectrometry results showed that after incubation with the compound, the mass spectrum peak position shifted from the original molecular weight of the purified PI3Kα protein, proving that PI3Kα covalently binds to compound 6.

[0438] The purified PI3Kα protein was diluted in binding buffer. The compound was added to the buffer at a protein:compound molar ratio of 1:10. After incubation at room temperature for 2 hours, protein electrophoresis was performed. The protein bands were stained with Coomassie Brilliant Blue, excised, and digested with trypsin in the gel before mass spectrometry identification. Mass spectrometry results showed that compound 6 bound to the thiol group of the PI3Kα peptide.

[0439] Biological Example 2: In vitro experiment to inhibit PI3Kα kinase activity by the compound of the present invention.

[0440] The ADP-Glo ​​Kinase Assay kit was used in the experiment, following the manufacturer's instructions. The following buffer solutions were prepared: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, and 2 mM DTT. The test compound sample was dissolved in DMSO and diluted 3-fold at a predetermined initial concentration, e.g., 10 μM, and added to the screening system. A DMSO control and a control without kinase were also included. The optimal concentrations of PI3Kα enzyme, substrate (PIP2), and ATP were prepared using buffer. The enzyme reaction system included: buffer, 25 μM ATP, kinase substrate (PIP2, 50 μg / mL), and PI3Kα kinase (0.15 μg / mL). The reaction was allowed to proceed at room temperature for 1 hour. The reaction was terminated by adding a stop reagent (ADP-Glo ​​reagent, 5 μL), and the ADP content in the system was detected using a detection reagent (Kinase Detection Reagent, 10 μL). Signal data were collected using an Envision instrument. Calculate the inhibition rate using the following formula: % Inhibition rate = (DMSO control signal value - Sample signal value) / (DMSO control signal value - Untreated kinase control signal value). Use Y = Bottom + (Top - Bottom) / (1 + (IC) 50 The formula / X)^HillSlope) is fitted to a curve to obtain IC. 50 Values. The results are shown in Table 9 below.

[0441] Table 9. Inhibitory activity of the compounds of this invention against PI3K kinase (IC50) 50 (nM)

[0442] Biological Example 3: In vitro inhibition of HCC1954 cell proliferation by the compound of the present invention.

[0443] HCC1954 (PI3KαH1047R mutant) cells were seeded in 384-well plates (Fisher 142762) with 2000 cells per well. On the second day, serially diluted compounds were added. 144 hours after compound addition, CellTiter-Lumi (Beyotime C0068XL) was added to measure ATP levels in the cells, assess cell growth, and calculate the IC50 of the compound in inhibiting cell growth. 50 The results are shown in Table 10 below.

[0444] Table 10 Inhibitory activity of the compounds of the present invention against HCC1954 cells (IC50) 50 (μM)

[0445] Biological Example 4: In vitro inhibition of MCF-7 cell proliferation by the compound of the present invention.

[0446] MCF-7 (PI3KαE545K mutant) cells were seeded in 384-well plates (Fisher 142762) with 2000 cells per well. On the second day, serially diluted compounds were added. 144 hours after compound addition, CellTiter-Lumi (Beyotime C0068XL) was added to measure ATP levels in the cells, assess cell growth, and calculate the IC50 of the compound in inhibiting cell growth. 50 The results are shown in Table 11 below.

[0447] Table 11 Inhibitory activity of the compounds of the present invention against MCF-7 cells (IC50) 50 (μM)

[0448] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

Methods for preparing cyclic α,β-unsaturated compounds include synthetic methods of the following general formulas (1) and (2): in, Z can be NR4, O, or S independently; m can be 0, 1, 2, 3, or 4; R1 and R2 are each independently selected from halogens, H, deuterium, substituted or unsubstituted (C1-C6) alkyl, (C1-C6) alkoxy, 6-10 aryl or 5-10 heteroaryl, or R1 and R2 together with the commonly linked atoms form (C3-C6) cycloalkyl, 3-10 saturated or partially unsaturated heterocycloalkyl, 7-11 heterospirocycloyl, 5-11 heterobridged cycloyl; R3 and R4 are each independently selected from H, (C1-C6)alkyl, (C3-C12)cycloalkyl, (C1-C6)alkoxy, -OR k -OH, -NR k R l -CN, -C(O)R k -C(O)OR k -S(O)2R k -S(O)R k -CH2OC(O)R k -CH2OC(O)OR k -C(S)R k -C(S)OR k -C(O)SR k -C(O)NR k R l -C(S)NR k R l -C(O)ONR k R l -(C1-C6)alkyl-OR k -(C1-C6)alkyl NR k R l -S(O)2NR k R l -P(O)R k R l -(C1-C6)alkyl-(C3-C12)cycloalkyl, -(C1-C6)alkyl-(C3-C12)heterocyclic alkyl, 3- to 12-membered saturated or partially unsaturated heterocyclic alkyl, 7- to 12-membered heterospirocyclic alkyl or 5- to 12-membered heterobridged cycloalkyl, 6- to 10-membered aryl, 5- to 15-membered heteroaryl, wherein the (C1-C6)alkyl, (C3-C12)cycloalkyl, (C1-C6)alkoxy, -(C1-C6)alkyl-(C3-C12)cycloalkyl, -(C1-C6)alkyl-(C3-C12)heterocyclic alkyl, 3- to 12-membered saturated or partially unsaturated heterocyclic alkyl, 7- to 12-membered heterospirocyclic alkyl or 5- to 12-membered heterobridged cycloalkyl, 6- to 10-membered aryl, 5- to 15-membered heteroaryl are optionally further surrounded by 1, 2, 3, 4 or 5 Rs. m Substitution; two hydrogen atoms on the same carbon atom can be replaced by oxygen to form an oxo group or by... Replaced; Alternatively, in general formula (1), R2 and R3 together with the atoms between them form 4-10 saturated or partially unsaturated heterocyclic alkyl groups, 7-11 heterospirocyclic groups, and 5-11 heterobridged cyclic groups; Alternatively, in general formula (2), one R1 and R3 together with the atoms between them form a 4- to 10-membered saturated or partially unsaturated heterocyclic alkyl group, a 7- to 11-membered heterospirocyclic group, or a 5- to 11-membered heterobridged cyclic group; R k and R l Each of the following is independently H, -C(O)R, -C(O)OR, (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C12)cycloalkyl, 3- to 12-membered saturated or partially unsaturated heterocyclic alkyl, 7- to 12-membered heterospirocyclic alkyl or 5- to 12-membered heterobridged cycloalkyl, 6- to 10-membered aryl, 5- to 12-membered heteroaryl, wherein the (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C12)cycloalkyl, 3- to 12-membered saturated or partially unsaturated heterocyclic alkyl, 7- to 12-membered heterospirocyclic alkyl or 5- to 12-membered heterobridged cycloalkyl, 6- to 10-membered aryl, 5- to 12-membered heteroaryl is optionally further surrounded by 1, 2, 3, 4 or 5 Rs. m Substitution; two hydrogen atoms on the same carbon atom can be replaced by oxygen to form an oxo group or by... Replaced; Or R k and R l Together with the commonly linked atoms, they form 3- to 10-membered saturated or partially unsaturated heterocyclic alkyl groups, 7- to 11-membered heterospirocyclic groups, and 5- to 11-membered heterobridged cyclic groups, wherein the heterocyclic alkyl groups, heterospirocyclic groups, and heterobridged cyclic groups may optionally be further bound by 1, 2, 3, 4, or 5 R atoms. n replace; R a and R b Each is independently selected from hydrogen, deuterium, alkyl, deuterated alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, cycloalkyl, or heterocycloalkyl; or R a and R b Together with the commonly linked atoms, they form 3- to 10-membered cycloalkyl or heterocycloalkyl groups, which may optionally be further surrounded by 1, 2, 3, 4, or 5 R groups. n replace; R m Selected from deuterium, H, halogen, OH, SH, NH2, -CN, -NC, -NCS, -N3, NO2, -CONH2, (C1-C6)alkyl, (C1-C6)alkyl-(C3-C6)cycloalkyl, (C1-C6)alkyl-(C3-C6)heterocyclicalkyl, (C1-C6)haloalkyl, (C2-C6)alkynyl, (C2-C6)alkenyl, (C3-C6)cycloalkyl, -N=S(O)(R)2, -S(O)=(NR)R, -SCN, -S(O)2CN, -SNC, -S(O)2NC, -S(O)2N(R)2, -SF5, -P(O)(R)2, -P(S)(R)2, -P(O )(OR)R, -P(O)(OR)2, -B(OR)2, -B(R)2, -BH(OR), -SeR, -SeCN, -NCSe, -Si(R)3, -Si(OR)3, -SiR(OR)2, -Si(R)2(OR), -N(R)2, -N(R)OR, -C(O)N R(OR), -C(O)NRCN, -C(O)R, -C(O)OR, -C(O)CH2R, -S(O)2R, -S(O)R, -CH2OC(O)R, -CH2OC(O)OR, -C(S)R, -C(S)OR, -C(O)SR, -C(O)N(R)2, -(CH2) 1～3 OR, -(CH2) 1～3 N(R)2、-(CH2) 1～3 S(O)R、-(CH2) 1～3 S(O)2R、-(CH2) 1～3 NS(O)(R)2, -OC(O)R, (C1-C6)alkyl-C(=O)-, (C1-C6)alkoxy, (C1-C6)alkylthio, -NHC(O)OR, -NHC(O)N(R)2, -NHC(O)R, -NRS(O)2R, -NRCN, 3-12 saturated or partially unsaturated heterocyclic alkyl, 7-12 heterospirocyclic alkyl or 5-12 heterobridged cycloalkyl, 6-10 aryl, 5-12 heteroaryl, wherein the (C1-C6)alkyl, (C1-C6)alkyl-(C3-C6)cycloalkyl, (C1-C 6) Alkyl-(C3-C6)heterocyclic alkyl, (C2-C6)alkynyl, (C2-C6)alkenyl, (C3-C6)cycloalkyl, -OC(O)-(C1-C6)alkyl, -OC(O)-(C3-C6)cycloalkyl, (C1-C6)alkyl-C(=O)-, (C1-C6)alkoxy, (C1-C6)alkylthio or (C1-C6)alkylamine, 3- to 12-membered saturated or partially unsaturated heterocyclic alkyl, 7- to 12-membered heterospirocyclic alkyl or 5- to 12-membered heterobridged cycloalkyl, 6- to 10-membered aryl, 5- to 12-membered heteroaryl, optionally surrounded by 1, 2, 3, 4 or 5 Rs. n replace; Each R n Each of the following is independently selected from H, halogen, (C1-C6)alkyl, (C1-C6)alkoxy, cyclopropyl, OH, NH2, CN, MeNH-, Me2N-, CH3, CH2F, CHF2 or CF3; Each R is independently H, a substituted or unsubstituted (C1-C6) alkyl, a substituted or unsubstituted (C3-C6) cycloalkyl, a substituted or unsubstituted phenyl, a substituted or unsubstituted 3- to 7-membered saturated or partially unsaturated heterocyclic alkyl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; or each of the two independently selected Rs can form a 3- to 10-membered saturated or partially unsaturated heterocyclic alkyl, a 7- to 11-membered heterospirocyclic, or a 5- to 11-membered heterobridged cycloalkyl; When Z is NH, the compound of general formula (1) is prepared from an aldehyde-containing intermediate and an α-position active methylene intermediate by the following method to obtain the compound of general formula (3): or, or, in, Represents substituted or unsubstituted amino, hydroxyl, aliphatic or aromatic residues. PG is a protecting group, which can be deprotected under certain conditions to reveal the naked α-NH. FG is a structural unit that is an aldehyde group, a protecting group of an aldehyde group, or a unit that generates an aldehyde group through a reaction; When Z is NH, the compound of general formula (2) is prepared from an aldehyde-containing intermediate and an α-position active methylene intermediate by the following method to obtain the compound of general formula (4): or, or, When Z is O, the compound of general formula (1) is prepared from an aldehyde-containing intermediate and an α-position active methylene intermediate by the following method to obtain the compound of general formula (5): or, or, or, R5 is a substituted or unsubstituted (C1-C6) alkyl, a substituted or unsubstituted (C3-C6) cycloalkyl, a substituted or unsubstituted phenyl, a substituted or unsubstituted 3-7 member saturated or partially unsaturated heterocycloalkyl, or a substituted or unsubstituted 5-10 member heteroaryl, and LP is a leaving group, such as Br, OMs, etc. When Z is O, the compound of general formula (2) is prepared from an aldehyde-containing intermediate and an α-position active methylene intermediate by the following method to obtain the compound of general formula (6): The method according to claim 1 includes the following steps: Compounds of formula (int_9): Reaction with compounds of formula (int_10): Compounds with the formula (int_1): Alternatively, the expression (int_11): Oxidation forms a compound of formula (int_1). in, R1, R2, and R3 are as defined above; Compounds of formula (int_11-B): Reaction with compounds of formula (int_10): Compounds with the formula (int_3): Alternatively, the expression (int_12): Oxidation forms a compound of formula (int_3), wherein R1, R2, R3, and PG are as previously defined; oxidation of formula (int_13) results in: Compounds with the formula (int_5): Where R1, R3 and m are as defined above; Formula (int_14) after oxidation: Compounds with the formula (int_6): R1, R3, m, and PG are as defined above. According to the method of claim 1 or 2, R1 and R2 are phenyl, methyl, trifluoromethyl or ethyl; or R1 and R2 together with the commonly linked atoms form (C3-C6) cycloalkyl, 3- to 6-membered heterocycloalkyl, preferably cyclopropyl or cyclobutyl; Alternatively, R2 and R3 together with the atoms between them can form 4-10 saturated or partially unsaturated heterocyclic alkyl groups, 7-11 heterospirocyclic groups, or 5-11 heterobridged cyclic groups. Alternatively, in general formula (2), an R1 and an R3 together with the atoms between them form a 4- to 10-membered saturated or partially unsaturated heterocyclic alkyl group, a 7- to 11-membered heterospirocyclic group, or a 5- to 11-membered heterobridged cyclic group. In the method according to any one of claims 1 to 3, Z is NH or O; R3 is a phenyl group, a 5- to 7-membered monocyclic heteroaryl group containing 1, 2, 3, or 4 heteroatoms independently selected from N, S, Se, and O, or an 8- to 10-membered bicyclic heteroaryl group containing 1, 2, 3, 4, 5, or 6 heteroatoms independently selected from N, S, Se, and O, wherein the phenyl or heteroaryl group is optionally further surrounded by 1, 2, 3, 4, or 5 R3 atoms. m replace. According to the method of claim 4, R3 is 1, 2, 3, 4 or 5 Rs. m The substituted phenyl group, or a 5- to 6-membered monocyclic heteroaryl group containing one, two, three, or four heteroatoms independently selected from N, S, Se, and O, preferably surrounded by one, two, three, four, or five R atoms. m Substituted phenyl, pyrrolyl, thiophenyl, furanyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, 1,2,4-triazolyl, 1,2,3-triazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, 1,2,5-thiadiazolyl, 1,2,5-oxadiazolyl. According to the method of claim 4, R3 is 1, 2, 3, 4 or 5 Rs. m The substituted 8- to 10-membered bicyclic heteroaryl group containing one, two, three, four, five, or six heteroatoms independently selected from N, S, and O is preferred. The method according to any one of claims 1 to 6, R m For H, F, Cl, Br, OH, NO2, NH2, CN, -NHCH3, -N(CH3)2, -NHC(O)OC(CH3)3, -S(O)2CH3, -CH2OCH3, -S(O)2CH3, -N=S(O)(CH3)2, -S(O)=(NH)CH3, -C(O)OH, -C(O)OC H3, -C(O)OC(CH3)3, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -C(CH3)3, -OCH3, -OCH2CH3, -CF3, -CHF2, -CH2F, -CH2CF3, -OCF3, -OCHF2, -OCH2F, -CH=CH2, -CH2CH=CH2, -CH=CH2CH3, -CH=CH2CH2OCH3, -CH=CH2CH2N(CH3)2, -C≡CH, -C≡CHCH3, -CH2C≡CH, -C≡CHCH2OCH3, -C≡CHCH2N(CH3)2, cyclopropyl, cyclobutyl, oxacyclobutane, azacyclobutane, -SCN, -S(O)2CN, -SNC, -S(O)2NC, -S(O)2N(CH3)2, -SF5, -P(O)(CH3)2, -P(S)(CH3)2, -P(O)(OCH3)CH3, -P(O)(OCH3)2, -B(OH)2, -SeR, -NHS(O)2CH3, -NHCN. In the method according to any one of claims 1 to 3, Z is NH or O; R3 is -C(O)R k or -C(O)OR k ; R k The (C1-C3) alkyl, (C3-C6) cycloalkyl, or 3- to 6-membered saturated or partially unsaturated heterocyclic alkyl containing 1 to 2 heteroatoms independently selected from N, S, and O, wherein the (C1-C3) alkyl, (C3-C6) cycloalkyl, or 3- to 6-membered saturated or partially unsaturated heterocyclic alkyl containing 1 to 2 heteroatoms independently selected from N, S, and O is optionally further surrounded by 1, 2, 3, 4, or 5 R atoms. m Substitution; two hydrogen atoms on the same carbon atom can be replaced by oxygen to form an oxo group or by... Replaced; R a and R b Each can be H or F independently. In the method according to any one of claims 1 to 3, Z is NH or O; R3 is -C(O)R k or -C(O)OR k ; R k The phenyl group is a 5- to 6-membered monocyclic heteroaryl group containing one, two, three, or four heteroatoms independently selected from N, S, and O; or an 8- to 10-membered bicyclic heteroaryl group containing one, two, three, or four heteroatoms independently selected from N, S, and O, wherein the phenyl or heteroaryl group is optionally further surrounded by one, two, three, four, or five R atoms. m replace. In the method according to any one of claims 1 to 3, Z is NH or O; R3 is -C(O)NR k R l ; R k and R l Each of the following is independently H, (C1-C3)alkyl, (C1-C3)haloalkyl, (C3-C6)cycloalkyl, 3-6 member saturated or partially unsaturated heterocyclic alkyl, 7-12 member heterospirocyclic alkyl or 5-12 member heterobridged cycloalkyl, phenyl, 5-12 member heteroaryl containing 1-4 heteroatoms independently selected from N, S and O; the two hydrogens on the same carbon may be replaced by oxygen to form an oxo group or by... Replaced; R a and R b Each can be H or F independently. In the method according to any one of claims 1 to 3, Z is NH or O; R3 is H, (C1-C3)alkyl, or (C3-C6)cycloalkyl, wherein the (C1-C3)alkyl or (C3-C6)cycloalkyl is further surrounded by 1, 2, 3, 4, or 5 R3 groups. m Substitution; two hydrogen atoms on the same carbon atom can be replaced by oxygen to form an oxo group or by... Replaced; R a and R b Each can be H or F independently. In the method according to any one of claims 1 to 3, Z is NH or O; R3 is a 3- to 12-membered saturated or partially unsaturated heterocyclic alkyl group, a 7- to 12-membered heterospirocyclic alkyl group, or a 5- to 12-membered heterobridged cycloalkyl group containing 1 to 2 heteroatoms independently selected from N, S, and O, wherein the 3- to 12-membered saturated or partially unsaturated heterocyclic alkyl group, a 7- to 12-membered heterospirocyclic alkyl group, or a 5- to 12-membered heterobridged cycloalkyl group containing 1 to 2 heteroatoms independently selected from N, S, and O is optionally further surrounded by 1, 2, 3, 4, or 5 R3 atoms. m Substitution; two hydrogen atoms on the same carbon atom can be replaced by oxygen to form an oxo group or by... Replaced; R a and R b Each can be H or F independently. The method according to any one of claims 1 to 12, wherein R3 is The method according to any one of claims 1 to 13, wherein Represents any substituted (C1-C8) alkyl; any substituted (C3-C12) cycloalkyl; any substituted amino; any substituted hydroxyl; any substituted heterocyclic alkyl; any substituted partially unsaturated heterocyclic alkyl; any substituted phenyl; radioactive or non-radioactive nuclides, biotin, reporter enzymes, nucleotides, oligonucleotides, fluorophores, amino acids, peptides, and any substituted 5- to 10-membered heteroaromatic systems. The method according to any one of claims 1 to 13, wherein Represents any substituted (C1-C8) alkyl; any substituted (C3-C6) cycloalkyl; any substituted amino; any substituted hydroxyl; any substituted heterocyclic alkyl; any substituted partially unsaturated heterocyclic alkyl; any substituted phenyl; any substituted 5- to 6-membered heteroaromatic systems. The method according to any one of claims 1 to 13, wherein Represents a linker, a drug, or a linker-drug conjugate. The synthesis method according to claim 1 or 2 is characterized in that, The Knoevenagel condensation reaction between the aldehyde-containing intermediate and the α-position active methylene intermediate is carried out in an organic solvent, using one or more inorganic substances such as organic bases, inorganic bases, amine salts, Lewis acid-amine complexes, potassium fluoride, and aluminum phosphate as catalysts. According to the synthesis method of claim 17, the organic solvent is preferably one or more of dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, 1,1,1-trichloroethane, acetonitrile, acetone, tetrahydrofuran, 2-methyltetrahydrofuran, diethyl ether, methyl tert-butyl ether, 1,4-dioxane, acetone, ethyl acetate, petroleum ether, n-hexane, cyclohexane, isopentane, n-pentane, cyclopentane, toluene, dichloroethylene, pyridine, nitromethane, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, methanol, ethanol, isopropanol, tert-butanol, n-butanol, cyclohexanol, 1,2-propanediol, and ethylene glycol, more preferably one or more of dichloromethane, chloroform, 1,2-dichloroethane, acetonitrile, acetone, tetrahydrofuran, 2-methyltetrahydrofuran, ethyl acetate, and pyridine. According to the synthesis method of claim 17 or 18, the organic base is preferably one or more of pyrrolidine, piperidine, aziridine, diethylamine, dimethylamine, ethylamine, methylamine, ethylenediamine, triethylamine, triethylenediamine, 1,8-diazabicyclo[5,4,0]undecene-7, 1,5-diazabicyclo[4,3,0]non-5-ene, 4-dimethylaminopyridine, pyridine, quinoline, N-methylmorpholine, morpholine, and tetramethylethylenediamine; more preferably, the organic base is one or more of pyrrolidine, piperidine, morpholine, aziridine, diethylamine, and dimethylamine. According to the synthesis method of claim 17 or 18, the complex of the Lewis acid and the amine is preferably one or more of titanium tetrachloride / pyridine, titanium tetrachloride / triethylamine, and trimethylchlorosilane / pyrrolidine. The synthesis method according to any one of claims 17 to 20 may further include water removal during the reaction, preferably by water separator removal, addition of molecular sieve or other water removal reagents. The dehydrating agent according to claim 21 is preferably anhydrous sodium sulfate or anhydrous magnesium sulfate. The synthesis method according to any one of claims 17 to 22, wherein the reaction temperature is from -15°C to 100°C, preferably from 20°C to 40°C, and more preferably from 20°C to 30°C. According to the synthesis method of claim 23, the reaction temperature is 25°C. According to any one of claims 17 to 24, the molar equivalent ratio of the α-position active methylene intermediate compound and the aldehyde intermediate is preferably in the range of 5:1 to 1:

5. According to the synthesis method of claim 25, the molar equivalent ratio of the intermediate compound containing the α-position active methylene group to the intermediate containing the aldehyde group is preferably 1:1 to 1:5, more preferably 1:1 to 1:2, more preferably 1:1.2 to 1:2, and even more preferably 1:1.5 to 1:

2. The synthesis method according to any one of claims 1-2 and 17-26 is characterized in that, The Knoevenagel condensation reaction conditions for the intermediate containing an aldehyde group and the intermediate containing an α-position active methylene group are as follows: Dissolve 1 molar equivalent of an aldehyde-containing intermediate and more than 1 molar equivalent of pyrrolidine in dichloromethane, then add less than 1 molar equivalent of an α-active methylene intermediate, and continue stirring at 25°C until the reaction is complete. Alternatively, under ice-water bath cooling, dissolve 1 molar equivalent of the aldehyde-containing intermediate and more than 1 molar equivalent of pyrrolidine in dichloromethane, then add trimethylchlorosilane less than the molar equivalent of pyrrolidine, heat to about 25°C and stir for a period of time, then add less than 1 molar equivalent of the intermediate containing α-active methylene, and continue stirring at about 25°C until the reaction is complete.

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