Novel ecteinin derivative, preparation method and application thereof, antibody drug conjugate and application thereof

Abstract

The invention relates to a novel ascitein derivative, a preparation method and application thereof, an antibody drug conjugate and application thereof. Some of the novel ascitein derivatives provided by the invention have relatively strong in-vitro cytotoxicity (1-10nM), and some of the novel ascitein derivatives have relatively strong in-vitro cytotoxicity (lt; 1nM). Meanwhile, antibody drug conjugates based on the novel ascitein derivative have relatively strong in-vitro cytotoxicity (1-10nM), and some antibody drug conjugates have relatively strong in-vitro cytotoxicity (lt; 1nM).

Description

Technical Field

[0001] The invention relates to a novel ecteinascidin derivative, a preparation method and use thereof, an antibody-drug conjugate and application thereof. Background Art

[0002] Ecteinascidin is a family of natural products isolated from ascidian, which includes many biologically active compounds, which usually contain two to three tetrahydroisoquinoline subunits.

[0003] Trabectedin (Yondelis, ET-743) is one of them. It has significant biological activity against a variety of tumor cells. It was first isolated from the Caribbean sea squirt (Ecteinascidia turbinata) by Kenneth L. Rinehart in 1990 and can now be prepared by artificial synthesis (Nat. Prod. Rep., 2015, 32, 328-347). Trabectedin has a unique mechanism of action. It binds to the minor groove of DNA, interfering with cell division, gene transcription process and DNA repair mechanism. On October 23, 2015, it was approved by the U.S. Food and Drug Administration (FDA) for the treatment of specific soft tissue sarcomas. Clinical data showed that in more than 500 patients, the median PFS was 4.2 months (n = 345; 95% confidence interval (CI): 3.0-4.8 months), and the risk of disease progression or death was reduced by 45% (HR = 0.55; 95% CI: 0.44-0.70; p < 0.001). The median OS was 13.7 months. The most serious risks associated are neutropenic sepsis (serious infection caused by leukopenia), rhabdomyolysis (serious muscle problems), cardiomyopathy (myocardial problems, including heart failure), hepatotoxicity (liver problems, including liver failure), allergic reactions, as well as extravasation (leakage of trabectedin from the vein during infusion), tissue necrosis (tissue cell damage or death) and embryotoxicity. Among the 378 patients who received at least one dose of trabectedin in the randomized trial, the most common (≥20%) adverse reactions were nausea (75%), fatigue (69%), vomiting (46%), constipation (37%), decreased appetite (37%), diarrhea (35%), peripheral edema (28%), dyspnea (25%), and headache (25%). The most common (≥5%) grade 3-4 laboratory abnormalities were neutropenia (43%), increased alanine aminotransferase (ALT) (31%), thrombocytopenia (21%), anemia (19%), increased aspartate aminotransferase (AST) (17%), and increased creatine kinase (6.4%).

[0004] In summary, although ecteinascidin derivatives have been used clinically as chemotherapeutic drugs, there is still a high clinical demand for the continued development of new ecteinascidin derivatives and corresponding antibody-drug conjugates, and for further improving effectiveness and safety. Summary of the invention

[0005] The present invention provides novel ecteinascidin derivatives. The ecteinascidin compounds have good anti-tumor activity, and antibody-coupled drugs based thereon have the potential to be used for the treatment of tumor diseases.

[0006] The ecteinascidin derivative is characterized in that its structure is a compound represented by the general formula (I):

[0007]

[0008] Wherein: the R1 is selected from hydroxyl or cyano;

[0009] The R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, mercapto, halogen, deuterated alkyl, halogenated alkyl, halogenated alkoxy, hydroxy-substituted alkyl, amino-substituted alkyl, alkylamino-substituted alkyl, alkyl, alkoxy, alkylthio, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, alkyl-substituted amino, cycloalkyl-substituted alkyl, heterocyclic group, aryl, heteroaryl, sulfonic acid group, The R2 and R3 together with the carbon atoms to which they are connected form a saturated carbocyclic ring, a saturated heterocyclic ring, a cycloalkyl group, a heterocycloalkyl group or an acyclic ring; the x is selected from an integer of 1-10;

[0010] The R a and R b Each is independently selected from hydrogen, deuterium, hydroxyl, hydroxyl-substituted alkyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkyl-substituted sulfonyl, aryl-substituted sulfonyl, heteroaryl-substituted sulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a saturated heterocyclic ring, a heterocycloalkyl ring or an acyclic ring;

[0011] R4 is selected from hydrogen, alkyl, deuterated alkyl, halogenated alkyl, amino-substituted alkyl, alkyl substituted with alkylamino or The s is selected from an integer of 1-10;

[0012] Said R5 and R6 are both hydrogen or alkyl, said R5, R6 together with the carbon atoms to which they are connected form a saturated carbocyclic ring, a saturated heterocyclic ring, a cycloalkyl group, a heterocycloalkyl group or an acyclic ring; or R5 and R6 are different and are independently selected from hydrogen, alkyl, cycloalkyl or alkyl substituted with cycloalkyl;

[0013] The X a , X c , X1, X2, X3, and X4 are independently selected from hydrogen, halogen, C 1-4 Alkyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCF3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from -C(=O)-, -S(=O)2- or -CHCF3; R c is selected from a 3- to 10-membered saturated or partially unsaturated heterocyclic group, a C 3-10 Saturated or partially unsaturated carbocyclic group or n is selected from an integer of 0-10, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0014] The R d and R e are the same or different and are independently selected from hydrogen, deuterium, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, cycloalkyl-substituted alkyl, heterocyclyl, aryl or heteroaryl; said R d , R e Together with the carbon atoms to which they are attached, they form a saturated carbocyclic ring, a saturated heterocyclic ring, a cycloalkyl group, a heterocycloalkyl group, or an acyclic ring;

[0015] When one of R2 or R3 is hydrogen, the other is not C 1-4 or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

[0016] According to one embodiment of the present invention, in the general formula (I):

[0017] The R1 is selected from hydroxyl or cyano;

[0018] The R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, mercapto, halogen, deuterated alkyl, halogenated alkyl, halogenated alkoxy, hydroxyalkyl, amino-substituted alkyl, alkylamino-substituted alkyl, alkyl, alkoxy, alkylthio, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, alkyl-substituted amino, cycloalkylalkyl, heterocyclic, aryl, heteroaryl, sulfonic acid, The R2 and R3 together with the carbon atoms to which they are connected constitute a cycloalkyl group, a heterocycloalkyl group or a non-cyclic group;

[0019] The x is selected from an integer of 1-10;

[0020] The R a and R b Each is independently selected from hydrogen, deuterium, hydroxyl, hydroxyalkyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a heterocycloalkyl group or is not ring-forming;

[0021] R4 is selected from hydrogen, alkyl, deuterated alkyl, halogenated alkyl, amino-substituted alkyl, alkyl substituted with alkylamino or The s is selected from an integer of 1-10;

[0022] Said R5 and R6 are both hydrogen or alkyl, said R5, R6 together with the carbon atoms to which they are connected constitute a cycloalkyl, a heterocycloalkyl or acyclic; or R5 and R6 are different and are independently selected from hydrogen, alkyl, cycloalkyl or cycloalkylalkyl;

[0023] Said X1 and X4 are hydrogen;

[0024] The X a , X c , X2, X3 are independently selected from hydrogen, halogen, C 1-4 Alkyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCF3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from C(=O), S(=O)2 or -CHCF3; R c is selected from a 3- to 10-membered saturated or partially unsaturated heterocyclic group, a C 3-10 Saturated or partially unsaturated carbocyclic group or n is selected from an integer of 0-10, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0025] The R d and R e are the same or different and are independently selected from hydrogen, deuterium, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 alkynyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, aryl or heteroaryl; R d , R e Together with the carbon atoms to which it is attached, it forms a cycloalkyl group, a heterocycloalkyl group or is not ring-forming;

[0026] When one of R2 or R3 is hydrogen, the other is not C 1-4 When both R2 and R3 are hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 Alkyl

[0027] According to one embodiment of the present invention, in the general formula (I):

[0028] The R1 is selected from hydroxyl or cyano;

[0029] The R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, mercapto, halogen, deuterated alkyl, halogenated alkyl, halogenated alkoxy, hydroxy-substituted alkyl, amino-substituted alkyl, alkylamino-substituted alkyl, alkyl, alkoxy, alkylthio, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, alkyl-substituted amino, cycloalkyl-substituted alkyl, heterocyclic group, aryl, heteroaryl, The R2 and R3 together with the carbon atoms to which they are connected form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring; the x is an integer selected from 1-10;

[0030] The R a and R b Each is independently selected from hydrogen, deuterium, hydroxyl, hydroxyl-substituted alkyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkyl-substituted sulfonyl, aryl-substituted sulfonyl, heteroaryl-substituted sulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a saturated heterocyclic ring or an acyclic ring;

[0031] R4 is selected from hydrogen, alkyl, deuterated alkyl, halogenated alkyl, amino-substituted alkyl, alkyl substituted with alkylamino or The s is selected from an integer of 1-10;

[0032] Said R5 and R6 are both hydrogen or alkyl, said R5, R6 together with the carbon atoms to which they are connected form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring; or R5 and R6 are different and are independently selected from hydrogen, alkyl, cycloalkyl or alkyl substituted with cycloalkyl;

[0033] The X a , X c , X1, X2, X3, and X4 are independently selected from hydrogen, halogen, C1-4 Alkyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCF3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from -C(=O)-, -S(=O)2- or -CHCF3; R c is selected from a 3- to 10-membered saturated or partially unsaturated heterocyclic group, a C 3-10 Saturated or partially unsaturated carbocyclic group or n is selected from an integer of 0-10, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0034] The R d and R e are the same or different and are independently selected from hydrogen, deuterium, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, cycloalkyl-substituted alkyl, heterocyclyl, aryl or heteroaryl; said R d , R e Together with the carbon atoms to which they are attached, they form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring;

[0035] When one of R2 or R3 is hydrogen, the other is not C 1-4 or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

[0036] According to one embodiment of the present invention, in the general formula (I):

[0037] The R1 is selected from hydroxyl or cyano;

[0038] The R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, thiol, fluorine, chlorine, deuterated C 1-6 Alkyl, halogenated C 1-6 Alkyl, halogenated C 1-6 Alkoxy, C 1-6 Hydroxyl substituted alkyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, C2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 Cycloalkyl, C 1-6 Alkyl-substituted amino, cycloalkyl-substituted alkyl, heterocyclic group, aryl, heteroaryl, The R2 and R3 together with the carbon atoms to which they are connected form a 3- to 10-membered saturated carbocyclic ring, a 3- to 10-membered saturated heterocyclic ring or an acyclic ring; the x is an integer selected from 1-6;

[0039] The R a and R b Each is independently selected from hydrogen, hydroxy, hydroxy-substituted alkyl, alkyl, cycloalkyl, aryl, alkyl-substituted sulfonyl, aryl-substituted sulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a saturated heterocyclic ring or is not a ring;

[0040] R4 is selected from hydrogen, C 1-6 Alkyl, deuterated C 1-6 Alkyl, halogenated C 1-6 Alkyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl or The s is selected from an integer of 1-6;

[0041] R5 and R6 are both hydrogen or C 1-6 alkyl; said R5, R6 together with the carbon atoms to which they are connected constitute a 3-10 membered saturated carbocyclic ring, a 3-10 membered saturated heterocyclic ring or an acyclic ring; or R5 and R6 are different and are independently selected from hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl or cycloalkyl-substituted alkyl;

[0042] The X a , X c , X1, X2, X3, X4 are independently selected from hydrogen, fluorine, chlorine, methyl, ethyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCF3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from -C(=O)-; R c is selected from a 3- to 6-membered saturated heterocyclic group, a C 3-6 Saturated carbocyclic group or n is selected from an integer of 0-6, Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0043] The R d and R eare the same or different and are independently selected from hydrogen, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, cycloalkyl-substituted alkyl or aryl; R d , R e Together with the carbon atoms to which they are attached, they form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring;

[0044] When one of R2 or R3 is hydrogen, the other is not C 1-4 or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

[0045] According to one embodiment of the present invention, in the general formula (I):

[0046] The R1 is selected from hydroxyl or cyano;

[0047] The R2 and R3 are the same or different and are independently selected from hydrogen, hydroxyl, mercapto, fluorine, chlorine, trideuterated methyl, trifluoromethyl, trifluoromethoxy, C 1-6 Hydroxyl substituted alkyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 Cycloalkyl, C 1-6 Alkyl-substituted amino, cycloalkyl-substituted alkyl, heterocyclic group, aryl, heteroaryl, The R2 and R3 together with the carbon atoms to which they are connected form a 3- to 6-membered saturated carbocyclic ring, a 3- to 6-membered saturated heterocyclic ring or an acyclic ring; the x is an integer selected from 1-6;

[0048] The R a and R b Each is independently selected from hydrogen, hydroxy, hydroxy-substituted alkyl, alkyl, cycloalkyl, aryl, alkyl-substituted sulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a saturated heterocyclic ring or is not a ring;

[0049] R4 is selected from hydrogen, C 1-6Alkyl, trideuterated methyl, trifluoromethyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl or The s is selected from an integer of 1-6;

[0050] R5 and R6 are both hydrogen or C 1-6 alkyl; said R5, R6 together with the carbon atoms to which they are connected constitute a 3-6 membered saturated carbocyclic ring, a 3-6 membered saturated heterocyclic ring or an acyclic ring; or R5 and R6 are different and are independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl or cycloalkyl-substituted alkyl;

[0051] The X a , X c , X1, X2, X3, X4 are independently selected from hydrogen, fluorine, chlorine, methyl, ethyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from -C(=O)-; R c is selected from a 3- to 6-membered saturated heterocyclic group, a C 3-6 Saturated carbocyclic group or n is selected from an integer of 0-6, Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0052] The R d and R e are the same or different and are independently selected from hydrogen, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, cycloalkyl-substituted alkyl or aryl; R d , R e Together with the carbon atoms to which they are attached, they form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring;

[0053] When one of R2 or R3 is hydrogen, the other is not C 1-4 or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

[0054] According to one embodiment of the present invention, in the general formula (I):

[0055] The R1 is selected from hydroxyl or cyano;

[0056] The R2 and R3 are the same or different and are independently selected from hydrogen, hydroxy, fluorine, trifluoromethyl, trifluoromethoxy, C 1-6 Hydroxyl substituted alkyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, C 1-6 Alkyl-substituted amino, The R2 and R3 together with the carbon atoms to which they are connected form a 3- to 6-membered saturated carbocyclic ring, a 3- to 6-membered saturated heterocyclic ring or an acyclic ring; the x is an integer selected from 1-2;

[0057] The R a and R b Each is independently selected from hydrogen, hydroxy substituted alkyl, alkyl, alkyl substituted sulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a saturated heterocyclic ring or is not a ring;

[0058] R4 is selected from hydrogen, C 1-4 Alkyl, amino substituted C 1-4 Alkyl, C 1-4 Alkylamino substituted C 1-4 Alkyl or The s is selected from an integer of 1-2;

[0059] R5 and R6 are both hydrogen or C 1-6 alkyl; said R5, R6 together with the carbon atoms to which they are connected constitute a 3-6 membered saturated carbocyclic ring, a 3-6 membered saturated heterocyclic ring or an acyclic ring; or R5 and R6 are different and are independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl or cycloalkyl-substituted alkyl;

[0060] The X a , X c , X1, X2, X3, X4 are independently selected from hydrogen, fluorine, methyl or wherein A is selected from oxygen, -NCH3 or -NH; Y is selected from -C(=O)-; R c Selected from C 3-6 Saturated carbocyclic group or n is selected from an integer of 0-1, Z is selected from hydrogen or hydroxyl;

[0061] The R d and R eare the same or different and are independently selected from hydrogen, trifluoromethyl, alkyl, cycloalkyl or cycloalkyl substituted alkyl; R d , R e Together with the carbon atoms to which they are attached, they form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring;

[0062] When one of R2 or R3 is hydrogen, the other is not C 1-4 or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

[0063] According to one embodiment of the present invention, in the general formula (I):

[0064] The R1 is selected from hydroxyl or cyano;

[0065] The R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, mercapto, halogen, deuterated alkyl, halogenated alkyl, halogenated alkoxy, hydroxy-substituted alkyl, amino-substituted alkyl, alkylamino-substituted alkyl, alkyl, alkoxy, alkylthio, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, alkyl-substituted amino, cycloalkyl-substituted alkyl, heterocyclic group, aryl, heteroaryl, The R2 and R3 together with the carbon atoms to which they are connected form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring;

[0066] The x is selected from an integer of 1-10;

[0067] The R a and R b Each is independently selected from hydrogen, deuterium, hydroxyl, hydroxyl-substituted alkyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkyl-substituted sulfonyl, aryl-substituted sulfonyl, heteroaryl-substituted sulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a saturated heterocyclic ring or an acyclic ring;

[0068] R4 is selected from hydrogen, alkyl, deuterated alkyl, halogenated alkyl, amino-substituted alkyl, alkyl substituted with alkylamino or The s is selected from an integer of 1-10;

[0069] Said R5 and R6 are both hydrogen or alkyl, said R5, R6 together with the carbon atoms to which they are connected form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring; or R5 and R6 are different and are independently selected from hydrogen, alkyl, cycloalkyl or alkyl substituted with cycloalkyl;

[0070] Said X1 and X4 are hydrogen;

[0071] The X a , X c , X2, X3 are independently selected from hydrogen, halogen, C 1-4 Alkyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCF3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from -C(=O)-, -S(=O)2- or -CHCF3; R c is selected from 3 to 10 membered saturated or partially unsaturated heterocyclic groups, C 3-10 Saturated or partially unsaturated carbocyclic group or n is selected from an integer of 0-10, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0072] The R d and R e are the same or different and are independently selected from hydrogen, deuterium, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 alkynyl, cycloalkyl, cycloalkyl-substituted alkyl, heterocyclyl, aryl or heteroaryl; R d , R e Together with the carbon atoms to which they are attached, they form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring;

[0073] When one of R2 or R3 is hydrogen, the other is not C 1-4 When both R2 and R3 are hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

[0074] According to one embodiment of the present invention, in the general formula (I):

[0075] The R1 is selected from hydroxyl or cyano;

[0076] The R2 and R3 are the same or different and are independently selected from hydrogen, hydroxy, fluorine, trifluoromethyl, trifluoromethoxy, methyl, methoxy, cyclopropyl substituted methyl, The R a and R b Each is independently selected from hydrogen or methyl; R2, R3 together with the carbon atom to which they are connected form a 3-membered saturated carbocyclic ring, a 4-membered oxygen-containing saturated heterocyclic ring or a 4-membered sulfur-containing saturated heterocyclic ring; x is selected from an integer of 1-2;

[0077] Said R4 is selected from hydrogen or methyl;

[0078] R5 and R6 are independently selected from hydrogen or methyl. When both R5 and R6 are methyl, the carbon atom connecting the two forms a 3-membered saturated carbocyclic ring.

[0079] Said X1 and X4 are independently selected from hydrogen or methoxy;

[0080] The X a , X2, X3 are independently selected from hydrogen, fluorine, methyl or wherein A is selected from oxygen, -NCH3 or -NH; Y is selected from -C(=O)-; R c Selected from C 3-6 Saturated carbocyclic group or n is selected from an integer of 0-1, Z is selected from hydrogen or hydroxyl;

[0081] The R d and R e are the same or different and are independently selected from hydrogen, trifluoromethyl, cyclopropyl or cyclopropyl-substituted methyl; R d , R e Together with the carbon atom to which it is attached, it forms a 3- to 5-membered saturated carbocyclic ring;

[0082] When one of R2 or R3 is hydrogen, the other is not methyl; or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3, where R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

[0083] According to one embodiment of the present invention, the compound in the general formula (I) is selected from at least one of the following compounds:

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090] According to one embodiment of the present invention, the compound in the general formula (Ⅰ) is selected from at least one of the compounds of the structural formulas P1-P32, P36-P38, P43-P91, P93, P95-P97, P101-P108, P110, P112-P114, P118, P119, P136, P140-P142, P144, P146-P148, P152, P153, P155-P158, P160, P163-P175, P186-P188, P241, P253, P260-P262, P264, and P265.

[0091] According to one embodiment of the present invention, the compound in the general formula (I) is selected from at least one of the compounds of the structural formulas T1-T19.

[0092] According to one embodiment of the present invention, in the P1-P32, P36-P38, P58-P91, P93, P95-P97, P101-P108, P110, P112-P114, P118, P119, P136, P140-P142, P144, P146-P148, P152, P166, P167, P170, P172, P174, P186-P188, P241, P253, P261-P262, P264, and T1-T19 compounds, the -CN at the R1 position is replaced by -OH.

[0093] According to one embodiment of the present invention, the derivative comprises a single chiral component or is a mixture of diastereomers.

[0094] According to one embodiment of the present invention, the pharmaceutically acceptable salts, tautomers and stereoisomers of the compound of the general formula (I) are also included.

[0095] In the compounds of the aforementioned general formula (I), alkyl groups substituted with hydroxyl groups are also referred to as hydroxyalkyl groups; alkyl groups substituted with cycloalkyl groups are also referred to as cycloalkylalkyl groups; sulfonyl groups substituted with alkyl groups are also referred to as alkylsulfonyl groups; sulfonyl groups substituted with aryl groups are also referred to as arylsulfonyl groups; and sulfonyl groups substituted with heteroaryl groups are also referred to as heteroarylsulfonyl groups.

[0096] According to one embodiment of the present invention, in the general formula (I):

[0097] The R1 is selected from hydroxyl or cyano;

[0098] The R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, thiol, fluorine, chlorine, deuterated C 1-6 Alkyl, halogenated C 1-6 Alkyl, halogenated C 1-6 Alkoxy, C 1-6 Hydroxyalkyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 Cycloalkyl, C 1-6 Alkyl-substituted amino, cycloalkylalkyl, heterocyclic, aryl, heteroaryl, sulfonic acid, The R2, R3 together with the carbon atom to which they are connected constitute C 3-10 Cycloalkyl, 3- to 10-membered heterocycloalkyl or acyclic;

[0099] The R a and R b Each is independently selected from hydrogen, hydroxyl, hydroxyalkyl, alkyl, cycloalkyl, aryl, alkylsulfonyl, arylsulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a heterocycloalkyl group or is not ring-forming;

[0100] The x is selected from an integer of 1-6;

[0101] R4 is selected from hydrogen, C 1-6 Alkyl, deuterated C 1-6 Alkyl, halogenated C 1-6 Alkyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl or The s is selected from an integer of 1-6;

[0102] R5 and R6 are both hydrogen or C 1-6 Alkyl; said R5, R6 together with the carbon atom to which they are connected constitute C 3-10 cycloalkyl, 3 to 10 membered heterocycloalkyl or acyclic; or R5 and R6 are different and independently selected from hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl or cycloalkylalkyl;

[0103] The Xa , X c , X1, X2, X3, X4 are independently selected from hydrogen, fluorine, chlorine, methyl, ethyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCF3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from C(=O); R c is selected from a 3- to 6-membered saturated heterocyclic group, a C 3-6 Saturated carbocyclic group or n is selected from an integer of 0-6, Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0104] The R d and R e are the same or different and are independently selected from hydrogen, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, cycloalkylalkyl or aryl; R d , R e Together with the carbon atoms to which it is attached, it forms a cycloalkyl group, a heterocycloalkyl group or is not ring-forming;

[0105] When one of R2 or R3 is hydrogen, the other is not C 1-4 or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

[0106] According to one embodiment of the present invention, in the general formula (I):

[0107] The R1 is selected from hydroxyl or cyano;

[0108] The R2 and R3 are the same or different and are independently selected from hydrogen, hydroxyl, mercapto, fluorine, chlorine, trideuterated methyl, trifluoromethyl, trifluoromethoxy, C 1-6 Hydroxyalkyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 Cycloalkyl, C1-6 Alkyl-substituted amino, cycloalkylalkyl, heterocyclic, aryl, heteroaryl, sulfonic acid, The R2, R3 together with the carbon atom to which they are connected constitute C 3-6 Cycloalkyl, 3- to 6-membered heterocycloalkyl or acyclic;

[0109] The R a and R b Each is independently selected from hydrogen, hydroxyl, hydroxyalkyl, alkyl, cycloalkyl, aryl, alkylsulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a heterocycloalkyl group or is not ring-forming;

[0110] The x is selected from an integer of 1-6;

[0111] R4 is selected from hydrogen, C 1-6 Alkyl, trideuterated methyl, trifluoromethyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl or The s is selected from an integer of 1-6;

[0112] R5 and R6 are both hydrogen or C 1-6 Alkyl; said R5, R6 together with the carbon atom to which they are connected constitute C 3-6 cycloalkyl, 3 to 6 membered heterocycloalkyl or acyclic; or R5 and R6 are different and independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl or cycloalkylalkyl;

[0113] The X a , X c , X1, X2, X3, X4 are independently selected from hydrogen, fluorine, chlorine, methyl, ethyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from C(=O); R c is selected from a 3- to 6-membered saturated heterocyclic group, a C 3-6 Saturated carbocyclic group or n is selected from an integer of 0-6, Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0114] The R d and R e are the same or different and are independently selected from hydrogen, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6Alkynyl, cycloalkyl, cycloalkylalkyl or aryl; R d , R e Together with the carbon atoms to which it is attached, it forms a cycloalkyl group, a heterocycloalkyl group or is not ring-forming;

[0115] When one of R2 or R3 is hydrogen, the other is not C 1-4 or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

[0116] According to one embodiment of the present invention, in the general formula (I):

[0117] The R1 is selected from hydroxyl or cyano;

[0118] The R2 and R3 are the same or different and are independently selected from hydrogen, hydroxy, fluorine, trifluoromethyl, trifluoromethoxy, C 1-6 Hydroxyalkyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, C 1-6 Alkyl-substituted amino, sulfonic acid, The R2, R3 together with the carbon atom to which they are connected constitute C 3-6 Cycloalkyl, 3- to 6-membered heterocycloalkyl or acyclic;

[0119] The R a and R b Each is independently selected from hydrogen, hydroxyalkyl, alkyl, alkylsulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a heterocycloalkyl group or is not ring-forming;

[0120] The x is selected from an integer of 1-2;

[0121] R4 is selected from hydrogen, C 1-4 Alkyl, amino substituted C 1-4 Alkyl, C 1-4 Alkylamino substituted C 1-4 Alkyl or The s is selected from an integer of 1-2;

[0122] R5 and R6 are both hydrogen or C1-6 Alkyl; said R5, R6 together with the carbon atom to which they are connected constitute C 3-6 cycloalkyl, 3 to 6 membered heterocycloalkyl or acyclic; or R5 and R6 are different and independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl or cycloalkylalkyl;

[0123] The X a , X c , X1, X2, X3, X4 are independently selected from hydrogen, fluorine, methyl or wherein A is selected from oxygen, -NCH3 or -NH; Y is selected from C(=O); R c Selected from C 3-6 Saturated carbocyclic group or n is selected from an integer of 0-1, Z is selected from hydrogen or hydroxyl;

[0124] The R d and R e are the same or different and are independently selected from hydrogen, trifluoromethyl, alkyl, cycloalkyl or cycloalkylalkyl; R d , R e Together with the carbon atoms to which it is attached, it forms a cycloalkyl group, a heterocycloalkyl group or is not ring-forming;

[0125] When one of R2 or R3 is hydrogen, the other is not C 1-4 or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

[0126] According to one embodiment of the present invention, in the general formula (I):

[0127] The R1 is selected from hydroxyl or cyano;

[0128] The R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, mercapto, halogen, deuterated alkyl, halogenated alkyl, halogenated alkoxy, hydroxyalkyl, amino-substituted alkyl, alkylamino-substituted alkyl, alkyl, alkoxy, alkylthio, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, alkyl-substituted amino, cycloalkylalkyl, heterocyclic, aryl, heteroaryl, sulfonic acid, The R2 and R3 together with the carbon atoms to which they are connected constitute a cycloalkyl group, a heterocycloalkyl group or a non-cyclic group;

[0129] The x is selected from an integer of 1-10;

[0130] The R a and R b Each is independently selected from hydrogen, deuterium, hydroxyl, hydroxyalkyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a heterocycloalkyl group or is not ring-forming;

[0131] R4 is selected from hydrogen, alkyl, deuterated alkyl, halogenated alkyl, amino-substituted alkyl, alkyl substituted with alkylamino or The s is selected from an integer of 1-10;

[0132] Said R5 and R6 are both hydrogen or alkyl, said R5, R6 together with the carbon atoms to which they are connected constitute a cycloalkyl, a heterocycloalkyl or acyclic; or R5 and R6 are different and are independently selected from hydrogen, alkyl, cycloalkyl or cycloalkylalkyl;

[0133] Said X1 and X4 are hydrogen;

[0134] The X a , X c , X2, X3 are independently selected from hydrogen, halogen, C 1-4 Alkyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCF3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from C(=O), S(=O)2 or -CHCF3; R c is selected from 3 to 10 membered saturated or partially unsaturated heterocyclic groups, C 3-10 Saturated or partially unsaturated carbocyclic group or n is selected from an integer of 0-10, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0135] The R d and R e are the same or different and are independently selected from hydrogen, deuterium, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 alkynyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, aryl or heteroaryl; R d , R e Together with the carbon atoms to which it is attached, it forms a cycloalkyl group, a heterocycloalkyl group or is not ring-forming;

[0136] When one of R2 or R3 is hydrogen, the other is not C 1-4When both R2 and R3 are hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

[0137] According to one embodiment of the present invention, in the general formula (I): wherein: the R1 is selected from hydroxyl or cyano;

[0138] The R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, mercapto, halogen, deuterated alkyl, halogenated alkyl, halogenated alkoxy, hydroxy-substituted alkyl, amino-substituted alkyl, alkylamino-substituted alkyl, alkyl, alkoxy, alkylthio, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, alkyl-substituted amino, cycloalkyl-substituted alkyl, heterocyclic group, aryl, heteroaryl, sulfonic acid group, The R2 and R3 together with the carbon atoms to which they are connected constitute a cycloalkyl group, a heterocycloalkyl group or a non-cyclic group;

[0139] The R a and R b Each is independently selected from hydrogen, deuterium, hydroxyl, hydroxyl-substituted alkyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkyl-substituted sulfonyl, aryl-substituted sulfonyl, heteroaryl-substituted sulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a heterocycloalkyl group or is not ring-forming;

[0140] The x is selected from an integer of 1-10;

[0141] R4 is selected from hydrogen, alkyl, deuterated alkyl, halogenated alkyl, amino-substituted alkyl, alkyl substituted with alkylamino or The s is selected from an integer of 1-10;

[0142] R5 and R6 are both hydrogen or alkyl; R5, R6 together with the carbon atoms to which they are connected constitute a cycloalkyl group, a heterocycloalkyl group or do not form a ring; or R5 and R6 are different and are independently selected from hydrogen, alkyl, cycloalkyl or alkyl substituted with cycloalkyl;

[0143] The X a , X c , X1, X2, X3, and X4 are independently selected from hydrogen, halogen, C 1-4 Alkyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCF3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from -C(=O)-, -S(=O)2- or -CHCF3; R c is selected from 3 to 10 membered saturated or partially unsaturated heterocyclic groups, C 3-10 Saturated or partially unsaturated carbocyclic group or n is selected from an integer of 0-10, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0144] The R d and R e are the same or different and are independently selected from hydrogen, deuterium, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, cycloalkyl-substituted alkyl, heterocyclyl, aryl or heteroaryl; said R d , R e Together with the carbon atoms to which it is attached, it forms a cycloalkyl group, a heterocycloalkyl group or is not ring-forming;

[0145] When one of R2 or R3 is hydrogen, the other is not C 1-4 or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

[0146] According to one embodiment of the present invention, in the general formula (I):

[0147] The R1 is selected from hydroxyl or cyano;

[0148] The R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, thiol, fluorine, chlorine, deuterated C 1-6 Alkyl, halogenated C 1-6 Alkyl, halogenated C 1-6 Alkoxy, C 1-6 Hydroxyl substituted alkyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, C 2-6 Alkenyl, C 2-6Alkynyl, C 3-8 Cycloalkyl, C 1-6 Alkyl-substituted amino, cycloalkyl-substituted alkyl, heterocyclic group, aryl group, heteroaryl group, sulfonic acid group, The R2, R3 together with the carbon atom to which they are connected constitute C 3-10 Cycloalkyl, 3- to 10-membered heterocycloalkyl or acyclic;

[0149] The R a and R b Each is independently selected from hydrogen, hydroxy, hydroxy-substituted alkyl, alkyl, cycloalkyl, aryl, alkyl-substituted sulfonyl, aryl-substituted sulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a heterocycloalkyl group or is not ring-forming;

[0150] The x is selected from an integer of 1-6;

[0151] R4 is selected from hydrogen, C 1-6 Alkyl, deuterated C 1-6 Alkyl, halogenated C 1-6 Alkyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl or The s is selected from an integer of 1-6;

[0152] R5 and R6 are both hydrogen or C 1-6 Alkyl; said R5, R6 together with the carbon atom to which they are connected constitute C 3-10 cycloalkyl, 3 to 10 membered heterocycloalkyl or acyclic; or R5 and R6 are different and independently selected from hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl or cycloalkyl-substituted alkyl;

[0153] The X a , X c , X1, X2, X3, X4 are independently selected from hydrogen, fluorine, chlorine, methyl, ethyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCF3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from -C(=O)-; R c is selected from a 3- to 6-membered saturated heterocyclic group, a C 3-6 Saturated carbocyclic group or n is selected from an integer of 0-6, Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0154] The R dand R e are the same or different and are independently selected from hydrogen, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, cycloalkyl-substituted alkyl or aryl; R d , R e Together with the carbon atoms to which it is attached, it forms a cycloalkyl group, a heterocycloalkyl group or is not ring-forming;

[0155] When one of R2 or R3 is hydrogen, the other is not C 1-4 or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

[0156] According to one embodiment of the present invention, in the general formula (I):

[0157] The R1 is selected from hydroxyl or cyano;

[0158] The R2 and R3 are the same or different and are independently selected from hydrogen, hydroxyl, mercapto, fluorine, chlorine, trideuterated methyl, trifluoromethyl, trifluoromethoxy, C 1-6 Hydroxyl substituted alkyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 Cycloalkyl, C 1-6 Alkyl-substituted amino, cycloalkyl-substituted alkyl, heterocyclic group, aryl group, heteroaryl group, sulfonic acid group, The R2, R3 together with the carbon atom to which they are connected constitute C 3-6 Cycloalkyl, 3- to 6-membered heterocycloalkyl or acyclic;

[0159] The R a and R b Each is independently selected from hydrogen, hydroxy, hydroxy-substituted alkyl, alkyl, cycloalkyl, aryl, alkyl-substituted sulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a heterocycloalkyl group or is not ring-forming;

[0160] The x is selected from an integer of 1-6;

[0161] R4 is selected from hydrogen, C 1-6 Alkyl, trideuterated methyl, trifluoromethyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl or The s is selected from an integer of 1-6;

[0162] R5 and R6 are both hydrogen or C 1-6 Alkyl; said R5, R6 together with the carbon atom to which they are connected constitute C 3-6 cycloalkyl, 3 to 6 membered heterocycloalkyl or acyclic; or R5 and R6 are different and independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl or cycloalkyl-substituted alkyl;

[0163] The X a , X c , X1, X2, X3, X4 are independently selected from hydrogen, fluorine, chlorine, methyl, ethyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from -C(=O)-; R c is selected from a 3- to 6-membered saturated heterocyclic group, a C 3-6 Saturated carbocyclic group or n is selected from an integer of 0-6, Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0164] The R d and R e are the same or different and are independently selected from hydrogen, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, cycloalkyl-substituted alkyl or aryl; R d , R e Together with the carbon atoms to which it is attached, it forms a cycloalkyl group, a heterocycloalkyl group or is not ring-forming;

[0165] When one of R2 or R3 is hydrogen, the other is not C 1-4 or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

[0166] According to one embodiment of the present invention, in the general formula (I):

[0167] The R1 is selected from hydroxyl or cyano;

[0168] The R2 and R3 are the same or different and are independently selected from hydrogen, hydroxy, fluorine, trifluoromethyl, trifluoromethoxy, C 1-6 Hydroxyl substituted alkyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, C 1-6 Alkyl-substituted amino, sulfonic acid, The R2, R3 together with the carbon atom to which they are connected constitute C 3-6 Cycloalkyl, 3- to 6-membered heterocycloalkyl or acyclic;

[0169] The R a and R b Each is independently selected from hydrogen, hydroxy substituted alkyl, alkyl, alkyl substituted sulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a heterocycloalkyl group or is not ring-forming;

[0170] The x is selected from an integer of 1-2;

[0171] R4 is selected from hydrogen, C 1-4 Alkyl, amino substituted C 1-4 Alkyl, C 1-4 Alkylamino substituted C 1-4 Alkyl or The s is selected from an integer of 1-2;

[0172] R5 and R6 are both hydrogen or C 1-6 Alkyl; said R5, R6 together with the carbon atom to which they are connected constitute C 3-6 cycloalkyl, 3 to 6 membered heterocycloalkyl or acyclic; or R5 and R6 are different and independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl or cycloalkyl-substituted alkyl;

[0173] The X a , X c , X1, X2, X3, X4 are independently selected from hydrogen, fluorine, methyl or wherein A is selected from oxygen, -NCH3 or -NH; Y is selected from -C(=O)-; R c Selected from C3-6 Saturated carbocyclic group or n is selected from an integer of 0-1, Z is selected from hydrogen or hydroxyl;

[0174] The R d and R e are the same or different and are independently selected from hydrogen, trifluoromethyl, alkyl, cycloalkyl or cycloalkyl substituted alkyl; R d , R e Together with the carbon atoms to which it is attached, it forms a cycloalkyl group, a heterocycloalkyl group or is not ring-forming;

[0175] When one of R2 or R3 is hydrogen, the other is not C 1-4 or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

[0176] According to one embodiment of the present invention, in the general formula (I):

[0177] The R1 is selected from hydroxyl or cyano;

[0178] The R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, mercapto, halogen, deuterated alkyl, halogenated alkyl, halogenated alkoxy, hydroxy-substituted alkyl, amino-substituted alkyl, alkylamino-substituted alkyl, alkyl, alkoxy, alkylthio, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, alkyl-substituted amino, cycloalkyl-substituted alkyl, heterocyclic group, aryl, heteroaryl, sulfonic acid group, The R2 and R3 together with the carbon atoms to which they are connected constitute a cycloalkyl group, a heterocycloalkyl group or a non-cyclic group;

[0179] The x is selected from an integer of 1-10;

[0180] The R a and R b Each is independently selected from hydrogen, deuterium, hydroxyl, hydroxyl-substituted alkyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkyl-substituted sulfonyl, aryl-substituted sulfonyl, heteroaryl-substituted sulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a heterocycloalkyl group or is not ring-forming;

[0181] R4 is selected from hydrogen, alkyl, deuterated alkyl, halogenated alkyl, amino-substituted alkyl, alkyl substituted with alkylamino or The s is selected from an integer of 1-10;

[0182] Said R5 and R6 are both hydrogen or alkyl, said R5, R6 together with the carbon atoms to which they are connected constitute a cycloalkyl, a heterocycloalkyl or acyclic; or R5 and R6 are different and are independently selected from hydrogen, alkyl, cycloalkyl or alkyl substituted with cycloalkyl;

[0183] Said X1 and X4 are hydrogen;

[0184] The X a , X c , X2, X3 are independently selected from hydrogen, halogen, C 1-4 Alkyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCF3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from -C(=O)-, -S(=O)2- or -CHCF3; R c is selected from 3 to 10 membered saturated or partially unsaturated heterocyclic groups, C 3-10 Saturated or partially unsaturated carbocyclic group or n is selected from an integer of 0-10, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0185] The R d and R e are the same or different and are independently selected from hydrogen, deuterium, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 alkynyl, cycloalkyl, cycloalkyl-substituted alkyl, heterocyclyl, aryl or heteroaryl; R d , R e Together with the carbon atoms to which it is attached, it forms a cycloalkyl group, a heterocycloalkyl group or is not ring-forming;

[0186] When one of R2 or R3 is hydrogen, the other is not C 1-4 When both R2 and R3 are hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

[0187] The use of the aforementioned ecteinascidin derivatives in the preparation of anticancer drugs.

[0188] An antibody-drug conjugate, characterized in that it comprises the ecteinascidin derivative as claimed in claim 1 and an antibody.

[0189] Use of the aforementioned antibody-drug conjugates in the preparation of anticancer drugs.

[0190] A compound of general formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0191] M-L1-L2-D

[0192] General formula (III)

[0193] Wherein: the M is selected from

[0194]

[0195] The L1 is selected from

[0196] One or more combinations of; n1 = an integer of 1-20, n2 = an integer of 0-20, n3 = an integer of 1-20, n4 = an integer of 0-20, n5 = an integer of 0-20, n6 = an integer of 0-20, n7 = an integer of 0-20;

[0197] The L2 is

[0198]

[0199] n8=an integer from 0 to 20, n9=an integer from 0 to 20, n 10 = integer from 0 to 20, n 11 = integer from 0 to 20;

[0200] D is selected from the product group formed by the aforementioned ecteinascidin derivative losing one or more atoms or groups;

[0201] Where D is covalently bonded to L2 through the nitrogen or oxygen in D;

[0202] According to one embodiment of the present invention, in the general formula (III),

[0203] Wherein: the M is selected from

[0204] The L1 is selected from One or more combinations of; n1 = an integer of 1-20, n2 = an integer of 0-20, n3 = an integer of 1-20 or n4 = an integer of 0-20;

[0205] The L2 is

[0206] n8=an integer from 0 to 20, n 10 = integer from 0 to 20 or n 11 = integer from 0 to 20;

[0207] D is selected from the product group formed by the aforementioned ecteinascidin derivative losing one or more atoms or groups;

[0208] D is covalently bound to L2 through the nitrogen or oxygen in any of X1, X2, X3, X4, R2, R3 or R4 in D;

[0209] According to one embodiment of the present invention, in the general formula (III),

[0210] Wherein: the M is selected from

[0211] The L1 is selected from One or more combinations of;

[0212] n1=an integer of 1-18, n2=an integer of 0-18, n3=an integer of 1-18, n4=an integer of 0-18;

[0213] The L2 is n8=an integer from 0 to 18 or n 11 = an integer from 0 to 18;

[0214] D is selected from the product group formed by the product of the aforementioned ecteinascidin derivative losing one or more atoms or groups;

[0215] D is covalently bonded to L2 through the nitrogen or oxygen in any of X1, X2, X3, X4, R2, R3 or R4 in D.

[0216] According to one embodiment of the present invention, in the general formula (III),

[0217] The M is selected from

[0218] The L1 is selected from One or more combinations of; n1 = 1-20, n2 = 1-20, n3 = 1-20, n4 = 1-20;

[0219] The L2 is n8=1-20、n 10 =1-20;

[0220] D is selected from the product group formed by the aforementioned ecteinascidin derivative losing one or more atoms or groups;

[0221] D is covalently bonded to L2 through the nitrogen or oxygen in any of X1, X2, X3, X4, R2, R3 or R4 in D.

[0222] According to one embodiment of the present invention, in the compound of the general formula (III), M is selected from

[0223] The L1 is selected from One or more combinations of; n1=1-20, n2=1-20, n3=1-20, n4=1-20, n5=1-20, n6=1-20 or n7=1-20;

[0224] The L2 is

[0225] n8=1-20、n9=1-20 or n 10 =1-20;

[0226] D is selected from the aforementioned ecteinascidin derivatives, or pharmaceutically acceptable salts, tautomers, and product groups formed by the loss of one or more atoms or groups of the stereoisomers thereof;

[0227] Where D is covalently bonded to L2 through the nitrogen or oxygen in D;

[0228] According to one embodiment of the present invention, in the general formula (III),

[0229] Wherein: the M is selected from

[0230] The L1 is selected from One or more combinations of; n1=1-20, n2=1-20, n3=1-20 or n4=1-20;

[0231] The L2 is n8=1-20 or n 10 =1-20;

[0232] D is selected from the aforementioned ecteinascidin derivatives, or pharmaceutically acceptable salts, tautomers, and product groups formed by the loss of one or more atoms or groups of the stereoisomers thereof;

[0233] D is covalently bonded to L2 through the nitrogen or oxygen in any of X1, X2, X3, X4, R2, R3 or R4 in D.

[0234] According to one embodiment of the present invention, in the general formula (III),

[0235] Wherein: the M is selected from

[0236] The L1 is selected from One or more combinations of;

[0237] n1=1-20, n2=1-20, n3=1-20, n4=1-20;

[0238] The L2 is n8=1-20;

[0239] D is selected from the aforementioned ecteinascidin derivatives, or pharmaceutically acceptable salts, tautomers, and product groups formed by the loss of one or more atoms or groups of the stereoisomers thereof;

[0240] D is covalently bonded to L2 through the nitrogen or oxygen in any of X1, X2, X3, X4, R2, R3 or R4 in D.

[0241] Among them, n1, n2, n3, n4, n5, n6, n7, n8, n9, n 10 is an integer.

[0242] According to one embodiment of the present invention, the compound of general formula (III) is selected from at least one of the compounds in the following table:

[0243]

[0244]

[0245]

[0246]

[0247]

[0248] An antibody-drug conjugate, characterized in that it comprises a structure as shown in general formula (IV):

[0249]

[0250] wherein Ab is an antibody, an antibody fragment or a protein, wherein the antibody is selected from a mouse antibody, a rabbit antibody, a phage display antibody, a yeast display antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody and a multispecific antibody; M-L1-L2-D is selected from a compound of the aforementioned general formula (III), or a pharmaceutically acceptable salt thereof, a tautomer, or a stereoisomer thereof; n 12 Select any value between 0-20.

[0251] The carbon-carbon double bond of M in the M-L1-L2-D is connected to the thiol group in the antibody Ab by a Michael addition reaction or a nucleophilic substitution reaction between the thiol sulfur atom in the antibody Ab and the bromomethylene of M to form a CS bond.

[0252] According to one embodiment of the present invention, the n 12 Select any value between 1-20.

[0253] According to one embodiment of the present invention, in the general formula (IV), the Ab is a monoclonal antibody.

[0254] According to one embodiment of the present invention, in the general formula (IV), the Ab is selected from abciximab, alemtuzumab, anetuzumab, atezolizumab, avelumab, basiliximab, bevacizumab, blinatumomab, brentuximab, catumaxomab, cetuximab, cetuximab, cotuximab, daclizumab, daratumumab, deninutuzumab, denosumab, depatuxizumab, denutumab, durvalumab, elotuzumab, enroku, gebatumumab, gemtuzumab, ibritumomab tiuxetan, indalatuzumab, indoximab, inotuzumab, ipilimumab, labetuzumab, latuzumab, latuximab, rifatuzumab, lovotuzumab, miratumumab, metuximab, Antibody, natalizumab, nesituzumab, nimotuzumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, omalizumab, palivizumab, panitumumab, pertrastuzumab, pembrolizumab, pertuzumab, pinatuzumab, polotuzumab, ramucirumab, lovatuzumab, sacetuzumab, setuximab, setuximab, sofetozumab, vadatuximab, worsotuzumab, trastuzumab, anti-CD4 antibody, anti-CD5 antibody, anti-CD13 antibody and anti-CD30 antibody, antigen-binding fragments or immunologically active portions thereof; M-L1-L2-D is selected from the aforementioned compound of formula (III), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof; n 12 Select any value between 0-12.

[0255] The English name of Abciximab is: Abciximab; the English name of Alemtuzumab is: Alemtuzumab; the English name of Anetumab is: Anetumab; the English name of Atezolizumab is: Atezolizumab; the English name of Avelumab is: Avelumab; the English name of Basiliximab is: Basiliximab; the English name of Bevacizumab is: Bevacizumab; the English name of Blinatomab is: Brentuximab; the English name of Catumaxomab is: Catumaxomab; the English name of Cetuximab is: Cetuximab; the English name of Cituzumab is: Cir mtuzumab; Coltuximab is called Coltuximab; Daclizumab is called Daclizumab; Daratumumab is called Daratumumab; Denintuzumab is called Denintuzumab; Denosumab is called Depatuxizumab; Dinutuximab is called Dinutuximab; Durvalumab is called Elotuzumab; Enfortumab is called Enfortumab; Glembatu is called Glembatu mumab; Gemtuzumab is called Gemtuzumab; Ibritumomab is called Ibritumomab; Indatuximab is called Indutuximab; Indusatumab is called Indusatumab; Inotuzumab is called Ipilimumab; Labetuzumab is called Ladiratuzumab; Laprituximab is called Laprituximab; Lifastuzumab is called Lifastuzumab; Lorvotozumab is called Lorvotozumab uzumab; Milatuzumab is called Milatuzumab; Mirvetuximab is called Mirvetuximab; Naratuximab is called Naratuximab; Necitumumab is called Necitumumab; Nimotuzumab is called Nivolumab; Obinutuzumab is called Obinutuzumab; Ofatumumab is called Ofatumumab; Olaratumab is called Olaratumab; Omalizumab is called Omalizumab; Palivizumab is called Palivizumab;The English name of Panitumumab is Panitumumab; the English name of Patratuzumab is Patritumab; the English name of Pembrolizumab is Pembrolizumab; the English name of Pertuzumab is Pertuzumab; the English name of Pinatuzumab is Pinatuzumab; the English name of Polotuzumab is Polatuzumab; the English name of Ramucirumab is Ramucirumab; the English name of Lovatuzumab is Rovalpituzumab; the English name of Sacituzumab is Sacituzumab; the English name of Siltuximab is Siltuximab); the English name of Sitratuzumab is Sirtratumab; the English name of Sofituzumab is Sofituzumab; the English name of Vadastuximab is Vadastuximab; the English name of Vorsetuzumab is Vorsetuzumab; the English name of Trastuzumab is Trastuzumab. ;

[0256] According to one embodiment of the present invention, the antibody-drug conjugate in the general formula (IV) is selected from at least one of the compounds of the following structural formulas:

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267] n 12 Select any value between 0-8.

[0268] According to one embodiment of the present invention, the n 12 Select any value between 1-8

[0269] Another aspect of the present invention is to provide the ecteinascidin derivatives described in the present invention, or their pharmaceutically acceptable salts, tautomers, and stereoisomers for use in treating cancer, preferably non-small cell lung cancer, small cell lung cancer, sarcoma, gastric cancer, ovarian cancer, colorectal cancer, breast cancer, pancreatic cancer, prostate cancer, liver cancer, kidney cancer and blood tumors.

[0270] Another aspect of the present invention is to provide the antibody-drug conjugate based on the ectodermin derivative of the present invention for treating cancer, preferably for treating non-small cell lung cancer, small cell lung cancer, sarcoma, gastric cancer, ovarian cancer, colorectal cancer, breast cancer, pancreatic cancer, prostate cancer, liver cancer, kidney cancer and blood tumors.

[0271] The main advantages of the present invention are:

[0272] 1. Some of the novel ecteinascidin derivatives provided by the present invention have relatively strong in vitro cytotoxicity (1-10 nM), and some have relatively stronger in vitro cytotoxicity (<1 nM).

[0273] 2. The antibody-drug conjugates based on the novel ecteinascidin derivatives provided by the present invention have strong in vitro cytotoxicity (1-10 nM), and some antibody-drug conjugates have stronger in vitro cytotoxicity (<1 nM). DETAILED DESCRIPTION

[0274] The following are examples of the synthesis and analysis of the compounds (including ecteinascidin derivatives, linkers and linker-drug conjugates) and antibody drug conjugates described in this patent. It should be noted that these examples do not limit the scope of protection of the present invention.

[0275] Unless otherwise stated, all anhydrous reagents were purchased directly from the suppliers and stored under nitrogen. All other reagents and solvents were purchased of high purity and used without further purification.

[0276] Synthesis and Characterization of Ecteinascidin Derivatives

[0277] Example 1: Synthesis of Compound P1

[0278]

[0279] Step 1: Synthesis of 2-(4-fluoro-3-nitrophenyl)ethylenediamine

[0280] Add borane dimethyl sulfide (2.5M, 0.69mL, 2.5eq) to a THF solution of 2-(4-fluoro-3-nitrobenzene)acetonitrile (100mg, 0.55mmol, 1.0eq) and reflux for 1 hour. Quench with MeOH and concentrate. Purify by thin layer chromatography to obtain the target product (70mg, yield: 97.8%) as a colorless oil.

[0281] LC-MS (ESI) m / z: 185.3 [M+H] +

[0282] Step 2: Synthesis of 5-(2-aminoethyl)-2-fluoroaniline

[0283] Under N2 protection, 20 mg of 5% wet Pd / C was added to a solution of 2-(4-fluoro-3-nitrophenyl)ethylenediamine (70 mg, 454 mmol, 1.0 eq) in MeOH (20 mL). The suspension was replaced with H2 several times under vacuum and stirred at room temperature overnight under H2 protection. The suspension was filtered through diatomaceous earth and the filter cake was washed with methanol. The combined filtrate was concentrated to dryness and used directly in the next step.

[0284] Step 3: Synthesis of P1

[0285] A mixture of intermediate Int-A (10 mg, 0.016 mmol, 1.0 eq), 5-(2-aminoethyl)-2-fluoroaniline (24.7 mg, 0.16 mmol, 10 eq) and NaOAc (6.56 mg, 0.08 mmol, 10 eq) was dissolved in anhydrous ethanol and stirred at 25°C overnight. After the reaction was completed, the solution was filtered and the residue was purified by preparative HPLC to obtain the target product (1.5 mg, yield: 12.3%) as a white solid. LC-MS (ESI) m / z: 758.1 [M+H] +

[0286] 1 H NMR (400MHz, DMSO-d6) δ8.78 (s, 1H), 6.46 (s, 1H), 6.40 (d, J = 13.1Hz, 1H), 6.36 ( d,J=9.4Hz,1H),6.25(s,1H),6.16(s,1H),5.06(d,J=12.1Hz,3H),4.49–4.42(m ,2H),4.20(d,J=4.5Hz,1H),4.08(s,1H),4.00(s,1H),3.64(s,4H),3.22(s,1H) ,3.08(s,1H),2.79(s,2H),2.35(s,1H),2.30(s,3H),2.23(s,3H),2.05(s,3H).

[0287] Example 2: Synthesis of Compound P5

[0288]

[0289] Step 1: Synthesis of 2-(4-(dimethylamino)-3-nitrophenyl)acetonitrile

[0290] 2-(4-Fluoro-3-nitrophenyl)acetonitrile (100 mg, 0.55 mmol, 1.0 eq), dimethylamine (40%, w / w, 25 mg, 0.55 mmol, 1 eq) and K2CO3 (115.05 mg, 0.832 mmol, 1.5 eq) were added to a DMSO (1.5 mL) sealed tube solution. The mixture was stirred at 120 ° C overnight under argon protection. After quenching with water, it was extracted with ethyl acetate and the organic layer was washed twice with saturated brine. The organic layer was dried over sodium sulfate, filtered and concentrated to give a crude product (125 mg, yield: 100%) as a brown solid, which was used directly in the next step without purification.

[0291] LC-MS (ESI) m / z: 206.4 [M+H] +

[0292] Step 2: Synthesis of 4-(2-aminoethyl)-N,N-dimethyl-2-nitroaniline

[0293] The crude product 2-(4-(dimethylamino)-3-nitrophenyl)acetonitrile (125 mg, 0.61 mmol, 1.0 eq) was dissolved in THF (3 mL), and then borane dimethyl sulfide (2.5 M, 0.76 mL, 2.5 eq) was added and refluxed for 1 hour. After quenching with MeOH, the mixture was concentrated. The residue was purified by thin layer chromatography to obtain the target compound (90 mg, yield: 70.0%) as a colorless oil.

[0294] LC-MS (ESI) m / z: 210.5 [M+H] +

[0295] Step 3: Synthesis of 4-(2-aminoethyl)-N,N-dimethylbenzene-1,2-diamine

[0296] Under N2 protection, 20 mg of 5% wet Pd / C was added to a solution of 4-(2-aminoethyl)-N,N-dimethyl-2-nitroaniline (90 mg, 430 mmol, 1.0 eq) in MeOH (10 mL). The suspension was degassed under vacuum and replaced with H2 several times. Stirred at room temperature overnight under H2 protection. The suspension was filtered through diatomaceous earth and the filter cake was washed with methanol. The combined filtrate was concentrated and used directly in the next step.

[0297] LC-MS (ESI) m / z: 180.2 [M+H] +

[0298] Step 4: Synthesis of P5

[0299] A mixture of intermediate Int-A (10 mg, 0.016 mmol, 1.0 eq), 4-(2-aminoethyl)-N,N-dimethylbenzene-1,2-diamine (28.83 mg, 0.16 mmol, 10 eq) and NaOAc (6.56 mg, 0.08 mmol, 10 eq) in anhydrous ethanol was stirred overnight at 25°C. After the reaction was completed, the reaction solution was filtered and the filtrate was purified by preparative high performance liquid chromatography to obtain the target product (5.47 mg, yield:

[0300] 43.4%) as a white solid.

[0301] LC-MS (ESI) m / z: 783.0 [M+H] +

[0302] 1 H NMR (500MHz, DMSO-d6) δ8.68 (s, 1H), 6.40 (d, J = 11.3Hz, 2H), 6.17 (d, J = 8.0Hz, 2H),6.12(s,1H),5.00(d,J=11.5Hz,1H),4.61(s,2H),4.45(d,J=2.9Hz,2H),4. 21–4.13(m,1H),4.08(d,J=2.3Hz,1H),3.94(dd,J=11.6,2.5Hz,1H),3.62(s,3H ),3.24(d,J=4.7Hz,1H),2.97(td,J=11.5,3.7Hz,1H),2.83–2.61(m,3H),2.43–

[0303] 2.34(m,2H),2.33(s,6H),2.28(s,3H),2.22(d,J=3.3Hz,1H),2.19(s,4H),2.12(d,J=14.6Hz,1H),2.03(s,3H),1.96(s,4H).

[0304] Example 3: Synthesis of Compounds P9 and P10

[0305]

[0306] Step 1: Synthesis of compound 2-amino-1-(3-aminophenyl)ethylene glycol

[0307] Under N2 protection, 5% wet Pd / C (20 mg) was added to a solution of 2-amino-1-(3-nitrophenyl)ethane 1-ol (200 mg, 0.0188 mmol, 1.0 eq.) in methanol (50 mL). The suspension was degassed under vacuum and replaced with H2 several times. Stir at room temperature for 16 hours under H2 protection. The suspension was filtered through diatomaceous earth and the filter cake was washed with methanol. The combined filtrate was concentrated to give a crude product (190 mg, crude product) as an oil.

[0308] LC-MS (ESI) m / z: 153.2 [M+H] + .

[0309] 1 H NMR(400MHz,DMSO-d6)δ7.97(s,2H),7.01-6.98(m,1H),6.59-6.57(m,1H),6.47–6.50(m ,2H),6.92(s,1H),5.12(s,2H),4.66-4.62(m,1H),2.90-2.94(m,1H),2.77-2.72(m,1H).

[0310] Step 2: Synthesis of P9 and P10

[0311] The intermediate Int-A (10 mg, 0.016 mmol, 1.0 eq.), 3-(2-aminoethyl)aniline (12 mg, 0.081 mmol, 5.0 eq.) and NaOAc (19 mg, 0.16 mmol, 10 eq.) were dissolved in anhydrous ethanol (2 mL), stirred at room temperature overnight, the solvent was removed under reduced pressure, and purified by preparative HPLC to obtain P9 (1.67 mg, yield: 13.91%) and P10 (2.43 mg, yield: 20%) as white solids. LC-MS (ESI) m / z: 756.2 [M+H] +

[0312] 1 H NMR (400MHz, DMSO-d6) δ8.76 (s, 1H), 7.62-7.56 (d, J = 24Hz 2H),6.63(s,1H),6.51(d,J=8Hz,2H),6.46(s,1H),6.25(s,2H),6.15(s,1H),5.05-4.98(m,7 H),4.45(s,5H),4.19(s,4H),3.96(s,3H),2.79(s,4H),2.28-2.23(m,6H),2.04-1.98(m,6H). 1H NMR (400MHz, DMSO-d6) δ8.85(s,1H),6.63(d,J=8Hz,1H),6.51(d,J=12Hz,2H),6.35-6.32(m,2H),5.14-5.15(m,3H),4.74-4.72( m,1H),4.49(s,2H),4.26-4.19(m,8H),4.15-4.01(m,3H),3.17(s,5H),2.85-2.81(m,4H),2.35-2.30(m,10H),2.10-2.04(m,8H).

[0313] Example 4: Synthesis of Compounds P12 and P13

[0314]

[0315] Step 1: Synthesis of compound 2-hydroxy-2-(4-methoxy-3-nitrophenyl)acetonitrile

[0316] 4-Methoxy-3-nitrobenzaldehyde (4.0g, 22mmol, 1eq.) and trimethylsilyl cyanide (8.7g, 88mmol, 4eq.) were added to a reaction flask, dissolved with dichloromethane (50mL), and LiClO4·3H2O (3.53g, 22mmol, 1eq.) was added dropwise. Stirred overnight at room temperature, detected by TLC (EA: n-heptane = 1 / 3), the reaction was completed. The reaction solution was filtered, the filtrate was washed twice with 10% dilute hydrochloric acid, the organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product (3.9g, yield: 85%) as a yellow solid, which was directly used in the next step.

[0317] Step 2: Synthesis of compound 2-amino-1-(4-methoxy-3-nitrophenyl)ethane-1-ol

[0318] The compound 2-hydroxy-2-(4-methoxy-3-nitrophenyl)acetonitrile (3.9 g, 18.7 mmol, 1 eq.) was dissolved in anhydrous THF (80 mL), 1 M borane dimethyl sulfide (46.7 mL, 46.75 mmol, 2.5 eq.) was slowly added, the temperature was raised to 60 ° C and stirring was continued. LC-MS showed that the reaction of the raw material was completed, the temperature was lowered to 0 ° C, methanol was slowly added to quench, the solution gradually turned purple, concentrated, and purified by silica gel flash column chromatography (DCM / MeOH=30 / 1 to 10 / 1) to obtain the target compound (3.8 g, yield: 96%) as a light yellow solid.

[0319] LC-MS (ESI) m / z: 195.2 [M-OH] +

[0320] 1 H NMR(400MHz,DMSO-d6)δ7.79(d,J=2.2Hz,1H),7.59(dd,J=8.7,2.2Hz,1H),7.3 1(d,J=8.7Hz,1H),4.48(dd,J=7.3,4.5Hz,1H),3.90(s,3H),2.72–2.56(m,2H).

[0321] Step 3: Synthesis of compound 2-amino-1-(3-amino-4-methoxyphenyl)ethane-1-ol

[0322] To a 100 mL methanol solution of the compound 2-amino-1-(4-methoxy-3-nitrophenyl)ethane-1-ol (3.8 g, 17.9 mmol) was added 5% wet Pd / C (200 mg). The suspension was degassed under vacuum and replaced with H2 several times. Stir at room temperature for 2 hours under H2 protection. LC-MS showed that the raw material was completely reacted, and the palladium carbon was removed by filtration and concentrated to obtain the target compound (1.76 g, yield: 54%) as a brown solid.

[0323] Step 4: Synthesis of P12 and P13

[0324] The intermediate Int-A (200 mg, 0.321 mmol, 1 eq.), the compound 2-amino-1-(3-amino-4-methoxyphenyl)ethane-1-ol (586 mg, 3.2 mmol, 10 eq.), and glacial acetic acid (192 mg, 3.2 mmol, 10 eq.) were added to a reaction bottle, and 20 mL of anhydrous ethanol was added and stirred at 60°C for 1 hour. The reaction was completed by LC-MS detection. The anhydrous ethanol was concentrated, and a saturated sodium carbonate solution was added to adjust the pH to 7-8, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel flash column chromatography (MeOH / DCM=1 / 50) to obtain P12 (96 mg, yield: 76%) and P13 (100 mg, yield: 79%) as brown solids.

[0325] P12

[0326] LC-MS (ESI) m / z: 786.1 [M+H] +

[0327] 1H NMR(400MHz,Chloroform-d)δ6.59(s,1H),6.51(s,1H),6.34(s,1H),6.01(d,J=25.3Hz, 2H),5.78(s,1H),4.99(d,J=11.6Hz,1H),4.57(s,1H),4.36–4.26(m,2H),4.23–4.14(m,2 H),4.11(d,J=11.7Hz,1H),3.81(s,3H),3.59(s,3H),3.51(d,J=4.8Hz,1H),3.42(s,1H) ,3.34(s,1H),2.94(d,J=5.3Hz,2H),2.33(s,4H),2.29(s,3H),2.21(s,3H),2.04(s,3H).

[0328] P13

[0329] LC-MS (ESI) m / z: 786.1 [M+H] +

[0330] 1 H NMR(400MHz,Chloroform-d)δ7.89(d,J=2.3Hz,3H),7.54(dd,J=8.7,2.4Hz,3H),7.05(d,J=8.6Hz,3H),6.6 4(d,J=6.0Hz,1H),6.42(s,1H),6.03(ddd,J=21.2,10.1,4.2Hz,4H),5.49(d,J=17.3Hz,3H),5.35(d,J=10. 5Hz,4H),4.70(d,J=5.0Hz,6H),4.46(s,6H),4.36–4.26(m,2H),4.20–4.12(m,1H),3.79(s,2H),3.61(s,2H ),3.50(d,J=4.8Hz,1H),2.92(dd,J=14.3,5.2Hz,3H),2.35(s,3H),2.27(s,3H),2.20(s,3H),2.03(s,3H).

[0331] Example 5: Synthesis of Compound P14

[0332]

[0333] P13 (5.0 mg, 0.0063 mmol, 1 eq.) was dissolved in 1.5 mL of anhydrous acetonitrile, cooled to 0°C, and chlorosulfonic acid (9.0 mg, 0.075 mmol, 12 eq.) was added. The reaction was completed by LC-MS detection. Saturated ammonium chloride solution was slowly added to the reaction solution for quenching. The resulting solution was purified by preparative HPLC to obtain the target product (0.2 mg, yield: 3.6%) as a brown solid.

[0334] LC-MS (ESI) m / z: 866.1 [M+H] +

[0335] Example 6: Synthesis of Compound P19

[0336]

[0337] Step 1: Synthesis of 2-(4-methoxy-3-nitrophenyl)-2-methylpropionitrile

[0338] To a solution of 2-(4-methoxyphenyl)-2-methylpropionitrile (100 mg, 0.57 mmol, 1.0 eq) in dichloromethane (1.5 mL) was added a mixture of HNO3 (65%, w / w, 82.9 mg, 0.86 mmol, 1.5 eq) and H2SO4 (88.8 mg, 0.86 mmol, 1.5 eq). The mixture was stirred at room temperature overnight. After the reaction was completed, the pH was adjusted to 7 with a saturated aqueous solution of NaHCO3. The organic layer was extracted with dichloromethane, separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by preparative thin layer chromatography (EA: heptane = 1:2) to obtain the target product (75 mg, yield: 60.1%) as a white solid.

[0339] LC-MS (ESI) m / z: 220.8 [M+H] +

[0340] Step 2: Synthesis of 2-(4-methoxy-3-nitrophenyl)-2-methylpropylamine

[0341] Borane dimethyl sulfide (2.5M, 0.43mL, 2.5eq) was added to a solution of 2-(4-methoxy-3-nitrophenyl)-2-methylpropionitrile (75mg, 0.34mmol, 1.0eq) in anhydrous tetrahydrofuran (3mL) and stirred under reflux for 1 hour. The mixture was quenched with MeOH and concentrated. The residue was purified by preparative thin layer chromatography to give the title compound (50mg, yield: 65.1%) as a colorless oil.

[0342] LC-MS (ESI) m / z: 225.2 [M+H] +

[0343] Step 3: Synthesis of 5-(1-amino-2-methylpropane-2-yl)-2-methoxyaniline

[0344] Under N2 protection, 20 mg of 5% wet Pd / C was added to a solution of 2-(4-methoxy-3-nitrophenyl)-2-methylpropane-1-amine (50 mg, 223 mmol, 1.0 eq) in MeOH (10 mL). The suspension was degassed under vacuum and replaced with H2 several times. Stirred at room temperature overnight under H2 protection. The suspension was filtered through diatomaceous earth and the filter cake was washed with methanol. The combined filtrate was concentrated and used directly in the next step.

[0345] LC-MS (ESI) m / z: 194.9 [M+H] +

[0346] Step 4: Synthesis of P19

[0347] A mixture of intermediate Int-A (10 mg, 0.016 mmol, 1.0 eq) and 5-(1-amino-2-methylpropane-2-yl)-2-methoxyaniline (20 mg, 0.08 mmol, 5 eq) was dissolved in anhydrous ethanol (1 mL) and stirred overnight at 25° C. After the reaction was completed, the reaction solution was filtered and the filtrate was purified by preparative HPLC to obtain P19 (4.67 mg, yield: 36.3%) as a white solid.

[0348] LC-MS (ESI) m / z: 798.0 [M+H] +

[0349] 1 H NMR(500MHz,DMSO-d6)δ8.71(s,1H),6.43(d,J=1.5Hz,2H),6.21–6.09(m,3H),5.02(d,J=11 .5Hz,1H),4.59(s,2H),4.47(d,J=2.9Hz,2H),4.22–4.16(m,1H),4.09(s,1H),3.99(dd,J=1 1.6,2.5Hz,1H),3.63(s,3H),3.46(s,4H),3.26(d,J=4.8Hz,1H),2.91–2.69(m,3H),2.30(s ,4H),2.20(s,3H),2.12(d,J=14.9Hz,1H),2.04(s,3H),1.97(s,3H),0.99(d,J=13.0Hz,6H).

[0350] Example 7: Synthesis of Compound P30

[0351]

[0352] Step 1: Synthesis of 2-(dimethylamino)-2-(4-methoxy-3-nitrophenyl)acetonitrile

[0353] To a solution of 4-methoxy-3-nitrobenzaldehyde (1 g, 5.52 mmol, 1.0 eq) in dichloromethane (10 mL) was added LiClO4 (1.17 g, 11.04 mmol, 2 eq) and stirred for 1 minute. Then N, N, 1,1,1-pentamethylsilylamine (1.94 g, 16.56 mmol, 3 eq) was added and stirred for 5 minutes. TMSCN (657 mg, 6.62 mmol, 1.2 eq) was added and stirred for 5 minutes. LC-MS showed that the reaction was complete. Dichloromethane (50 mL) was added to the reaction solution and washed twice with saturated NaHCO3 aqueous solution. The organic layer was dried over sodium sulfate, filtered and concentrated. The resulting orange oil was purified by silica gel flash column chromatography to obtain the target compound (840 mg, yield: 65%).

[0354] LC-MS (ESI) m / z: 236 [M+H] +

[0355] Step 2: Synthesis of 3-butyl (2-(3-amino-4-methoxyphenyl)-2-(dimethylamino)ethyl)carbamate

[0356] To a solution of 2-(dimethylamino)-2-(4-methoxy-3-nitrophenyl)acetonitrile (100 mg, 0.425 mmol, 1.0 eq) in THF (3 mL) was added Boc2O (139 mg, 0.638 mmol, 1.5 eq) and 20 mg Raney Ni. Stir overnight under H2 balloon protection at room temperature. LC-MS showed that the reaction was complete. Filter and concentrate the filtrate to remove the solvent. The residue was used directly in the next step.

[0357] LC-MS (ESI) m / z: 310 [M+H] +

[0358] Step 3: Synthesis of 1-(3-amino-4-methoxyphenyl)-N1,N1-dimethylethane-1,2-diamine TFA salt

[0359] The above 3-butyl (2- (3-amino-4-methoxyphenyl) -2- (dimethylamino) ethyl) carbamate was dissolved in DCM / TFA (3 / 1, v / v, 6 mL) and stirred at room temperature for 1 hour. The solvent was removed by concentration to obtain 256 mg of crude product, which was used directly in the next step.

[0360] LC-MS (ESI) m / z: 310 [M+H] +

[0361] Step 4: Synthesis of P30

[0362] The intermediate Int-A (4 mg, 0.0064 mmol, 1.0 eq), 1-(3-amino-4-methoxyphenyl)-N1, N1-dimethylethylenediamine-1,2-diamine TFA salt (16 mg, 0.08 mmol, 10 eq) and NaOAc (6.4 mg, 0.08 mmol, 10 eq) were dissolved in anhydrous EtOH (1 mL) and stirred overnight at 25 ° C. After the reaction was completed, the crude product was purified by preparative HPLC to obtain P30 (1.37 mg, yield: 26%) as a white solid.

[0363] LC-MS (ESI) m / z: 813 [M+H] +

[0364] Example 8: Synthesis of Compound P36

[0365]

[0366] Step 1: Synthesis of 1-(4-methoxy-3-nitrophenyl)cyclopropanenitrile

[0367] To a solution of 1-(4-methoxyphenyl)-1'-cyclopropanenitrile (100 mg, 0.577 mmol, 1.0 eq) in dichloromethane (1 mL) was slowly added a mixture of 65% HNO3 (454.56 mg, 0.865 mmol) and conc. H2SO4 (84.83 mg, 0.865 mmol) at 0°C and stirred overnight. LC-MS showed that the reaction was complete. The pH was adjusted to 7 with saturated NaHCO3 aqueous solution and the aqueous phase was extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by preparative thin layer chromatography to obtain the target compound (110 mg, yield: 87%) as a yellow solid.

[0368] LC-MS (ESI) m / z: 219 [M+H] +

[0369] Step 2: Synthesis of (1-(4-methoxy-3-nitrophenyl)cyclopropyl)methylamine

[0370] To a solution of 1-(4-methoxy-3-nitrophenyl)cyclopropanenitrile (120 mg, 0.549 mmol, 1.0 eq) in THF (3 mL) was added 2.5 MB H3﹒Me2S (0.1 mL, 2.5 eq). The reaction mixture was refluxed for 1 hour. It was quenched with MeOH and concentrated. The residue was purified by preparative thin layer chromatography to obtain the target product (30 mg, yield: 24.5%).

[0371] LC-MS (ESI) m / z: 223 [M+H] +

[0372] Step 3: Synthesis of 5-(1-(aminomethyl)cyclopropyl)-2-methoxyaniline

[0373] Under nitrogen protection, 10 mg of 5% wet Pd / C was added to a solution of (1-(4-methoxy-3-nitrophenyl)cyclopropyl)methylamine (30 mg, 0.135 mmol, 1.0 eq) in MeOH (2 mL). After evacuation and replacement with hydrogen three times, the mixture was stirred at room temperature overnight under hydrogen protection, Pd / C was filtered off, and the filtrate was concentrated to obtain 37 mg of crude product as brown oil, which was used directly in the next step.

[0374] LC-MS (ESI) m / z: 193 [M+H] +

[0375] Step 4: Synthesis of P36

[0376] The intermediate Int-A (4 mg, 0.0064 mmol, 1.0 eq), 5-(1-(aminomethyl)cyclopropyl)-2-methoxyaniline (12 mg, 0.064 mmol, 10 eq) and HOAc (3.84 mg, 0.064 mmol, 10 eq) were dissolved in anhydrous EtOH (1 mL) and stirred at 25 ° C overnight. After the reaction was completed, the crude product was purified by preparative HPLC to obtain P36 (3.95 mg, yield: 61%) as a white solid. LC-MS (ESI) m / z: 796 [M+H] +

[0377] Example 9: Synthesis of Compound P52

[0378]

[0379] To a suspension of P19 (15 mg, 0.0188 mmol) in CH3CN / H2O (3 / 2, v / v, 2 mL) was added AgNO3 (80 mg, 0.47 mmol). The mixture was stirred at room temperature overnight. LC-MS showed that the reaction was complete, and a saturated NaHCO3 solution was added to quench the reaction and filter. The product was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by high performance preparative liquid chromatography to obtain the title compound (3.87 mg, yield: 26%) as a white solid.

[0380] LC-MS (ESI) m / z: 789.6 [M+H] +

[0381] 1H NMR(400MHz,Chloroform-d)δ6.63(s,1H),6.50(s,1H),6.35(s,1H),6.04(d,J=1.3Hz,1H),5.96(d,J=1.2Hz,1H),5.7 6(s,1H),5.13(d,J=11.1Hz,1H),4.82(s,1H),4.49(d,J=3.3Hz,1H),4.18(d,J=5.2Hz,1H),4.03(d,J=10.1Hz,1H),3. 83(d,J=3.5Hz,3H),3.68(s,1H),3.62–3.56(m,5H),3.24(s,1H),3.05(d,J=11.6Hz,1H),2.89(d,J=11.3Hz,2H),2.44 (d,J=11.6Hz,2H),2.36(s,4H),2.32(s,3H),2.24(d,J=7.6Hz,1H),2.21(s,3H),2.05(s,3H),1.11(d,J=11.7Hz,6H).

[0382] Example 10: Synthesis of Compound P58

[0383]

[0384] HATU (99 mg, 0.26 mmol, 10 eq) was added to a DMF (2 mL) solution of P74-6 (see Example 12) (20 mg, 0.026 mmol, 1.0 eq) and 2-cyclopropyl-2-hydroxyacetic acid (30 mg, 0.26 mmol, 10 eq). The reaction mixture was stirred for 5-10 minutes, and DIPEA (33.6 mg, 0.26 mmol, 10 eq) was added. Stir at room temperature for 3 hours. Compound P58 (9.25 mg, yield: 41.1%) was obtained by purification by preparative HPLC as a white solid.

[0385] LC-MS (ESI) m / z: 868.2 [M+H] +

[0386] 1H NMR (400MHz, DMSO-d6) δ8.99(s,1H),8.70(s,1H),7.89(d,J=2.7Hz,1H),6.43(d,J=5.9Hz,2H),6.15(s,2H),6.07(d ,J=4.8Hz,1H),5.04(d,J=11.4Hz,1H),4.49(d,J=2.8Hz,2H),4.20(s,1H),4.11(s,1H),4.06(d,J=11.5Hz,1H),3.72 (s,1H),3.63(s,3H),3.58(s,3H),3.08–2.94(m,3H),2.86–2.65(m,3H),2.41–2.32(m,1H),2.29(s,3H),2.21(s,3H) ,2.19–2.12(m,1H),2.05(s,3H),1.98(s,3H),1.08(t,J=7.1Hz,1H),0.40(dd,J=8.9,4.6Hz,2H),0.32–0.25(m,2H).

[0387] Example 11: Synthesis of Compound P65

[0388]

[0389] HATU (49.5 mg, 0.13 mmol, 10 eq) was added to a DMF (2 mL) solution of P74-6 (see Example 12) (10 mg, 0.013 mmol, 1.0 eq) and 1-hydroxycyclopropane-1-carboxylic acid (13.3 mg, 0.13 mmol, 10 eq). The reaction mixture was stirred for 5-10 minutes, and then DIPEA (16.8 mg, 0.13 mmol, 10 eq) was added and stirred at room temperature for 1 hour. The mixture was purified by preparative HPLC to obtain compound P65 (3.14 mg, yield: 30.7%) as a white solid.

[0390] LC-MS (ESI) m / z: 845.0 [M+H] +

[0391] 1H NMR (400MHz, DMSO-d6) δ9.24(s,1H),8.73(s,1H),7.89(s,1H),6.83(s,1H),6.46(s,2H),6.17(s,2H),5.07( d,J=11.5Hz,1H),4.51(d,J=2.9Hz,2H),4.22(d,J=4.8Hz,1H),4.16–4.05(m,2H),3.64(d,J=14.7Hz,6H),3. 30(s,1H),3.19(d,J=5.2Hz,1H),3.08–2.96(m,2H),2.89–2.66(m,2H),2.37(d,J=16.3Hz,1H),2.32(s,3H), 2.23(s,3H),2.19(d,J=14.5Hz,1H),2.07(s,3H),2.00(s,3H),1.13(q,J=3.7Hz,2H),0.97(q,J=3.8Hz,2H).

[0392] Example 12: Synthesis of Compound P74

[0393]

[0394] Step 1: Synthesis of P74-2

[0395] P74-1 (10 g, 0.0729 mol, 1.0 eq) was dissolved in 100 mL of dichloromethane, 50 mL of saturated sodium bicarbonate was added, the temperature was lowered to 0-10 ° C, Cbz-Cl (22.3 g, 0.13 mol, 1.8 eq) was added dropwise, and after the addition was complete, the temperature was raised to room temperature and stirred overnight. The layers were separated, the aqueous phase was extracted twice with 100 mL of dichloromethane, the organic phases were combined, and dried over anhydrous sodium sulfate. Concentration, ethyl acetate was added to the residue until it was completely dissolved, n-heptane was added dropwise for crystallization, and the solid was filtered out. The filtrate can be concentrated, and the residue is crystallized again with ethyl acetate / n-heptane. After the combined solids were dried, compound P74-2 (16.2 g, yield 81.8%) was obtained.

[0396] LC-MS (ESI) m / z: 272.0 [M+H] +

[0397] 1 H NMR (300MHz, Chloroform-d) δ7.35(s,5H),7.05(d,J=7.8Hz,2H),6.77(d,J=7.8Hz,2H),5.10(s,2H),4.73(s,1H),3.42(d,J=6.7Hz,2H),2.74(s,2H).

[0398] Step 2: Synthesis of P74-3

[0399] P74-2 (14 g, 0.0516 mol, 1.0 eq.) was dissolved in 140 mL of acetic acid, and a mixed solution of 140 mL of acetic acid and 65% nitric acid (3.57 mL, 0.0516 mol, 1.0 eq.) was added dropwise at room temperature. The mixture was reacted for 1 hour, and ethyl acetate and saturated sodium bicarbonate were added to wash the layers. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel flash column chromatography (eluted with EA / n-heptane (1:4, v / v)) to obtain the target product P74-3 (15.4 g, yield: 94%).

[0400] LC-MS (ESI) m / z: 317.0 [M+H] +

[0401] 1 H NMR(300MHz,DMSO-d6)δ10.49(s,1H),7.93(s,1H),7.48–7.31(m,6H),7.10(d,J=8.5Hz,1H),5.3 2(d,J=5.7Hz,1H),5.13(d,J=15.2Hz,2H),4.83(s,1H),3.45(q,J=6.8Hz,2H),2.99–2.66(m,2H).

[0402] Steps 3 and 4: Synthesis of P74-5

[0403] P74-3 (12 g, 0.0379 mol, 1.0 eq.) and potassium carbonate (15.7 g, 0.113 mol, 3.0 eq.) were added to a reaction flask, 240 mL of acetonitrile was added and stirred to dissolve, iodomethane (8.08 g, 0.0569 mol, 1.5 eq.) was added, and refluxed for 2 hours. Cool down, filter, and concentrate the filtrate. 200 mL of dichloromethane and 100 mL of water were added to extract the layers, the aqueous phase was extracted with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain P74-4. P74-4 was dissolved in 300 mL of methanol, 1.5 g of 5% wet Pd / C was added, the suspension was degassed under vacuum conditions, and replaced with H2 several times. Stir at room temperature overnight under H2 protection. The suspension was filtered through celite, the filtrate was concentrated, dissolved with ethyl acetate, 1.0 eq. 2N HCl / EtOH solution was added, the solid precipitated, and filtered to obtain the target product P74-5 (7.08 g, two-step yield: 93%).

[0404] P74-4:

[0405] 1H NMR(300MHz,Chloroform-d)δ7.80–7.61(m,1H),7.37(d,J=9.8Hz,6H),7.02(d,J=8.6Hz,1H),5 .10(s,2H),4.97–4.77(m,1H),3.95(d,J=5.5Hz,3H),3.45(q,J=6.9Hz,2H),2.97–2.68(m,2H).

[0406] P74-5:

[0407] 1 H NMR (300MHz, D2O) δ6.98–6.88(m,1H),6.88–6.73(m,2H),3.79(d,J=3.4Hz,3H),3.15(t,J=7.2Hz,2H),2.92–2.75(m,2H).

[0408] Step 5: Synthesis of P74-6

[0409] Dissolve the intermediate Int-A (20 mg, 0.0322 mmol, 1.0 eq.), P74-5 (65.2 mg, 0.322 mmol, 10.0 eq.) and sodium acetate (29 mg, 0.354 mmol, 11 eq.) in 1.5 mL of anhydrous ethanol and stir overnight at room temperature. TLC indicates that the reaction of the raw material is complete. Filter, wash the inorganic salt with ethyl acetate, concentrate the filtrate, and purify the crude product by silica gel flash column chromatography (eluted with EA / n-heptane (1:1, v / v)) to obtain the target product P74-6 (19 mg, yield: 76.7%).

[0410] 1 H NMR(300MHz,Chloroform-d)δ6.60(s,1H),6.37(s,1H),6.27(s,1H),6.06(s,1H),5.99(s,1H),5.31(s, 1H),5.02(d,J=11.4Hz,1H),4.33(s,1H),4.29(d,J=4.5Hz,1H),4.19(s,1H),4.12(d,J=12.3Hz,1H),3. 82(d,J=8.3Hz,3H),3.67–3.57(m,3H),3.52(d,J=5.1Hz,1H),3.43(s,1H),3.13(s,1H),2.96(s,2H),2. 83(s,2H),2.60(s,3H),2.33(s,3H),2.32(s,1H),2.28(d,J=2.7Hz,3H),2.21(s,3H),2.11–2.02(m,4H).

[0411] Step 6: Synthesis of P74

[0412] HATU (49.5 mg, 0.13 mmol, 10 eq.) was added to a DMF (1 mL) solution of P74-6 (10 mg, 0.013 mmol, 1.0 eq.) and (1s, 3s)-3-hydroxycyclobutane-1-carboxylic acid (15 mg, 0.13 mmol, 10 eq.). The reaction mixture was stirred for 5-10 minutes, and DIPEA (16.8 mg, 0.13 mmol, 10 eq.) was added. Stirring was continued at room temperature for 1 hour. Compound P74 (6.59 mg, yield 58.4%) was obtained by purification by preparative HPLC as a white solid.

[0413] LC-MS (ESI) m / z: 868.2 [M+H] +

[0414] 1 H NMR (400MHz, DMSO-d6) δ6.50 (s, 1H), 6.36 (s, 1H), 6.19 (d, J = 7.9Hz, 2H), 5.13 (s, 1H), 4 .85(dt,J=13.2,6.9Hz,1H),4.53(s,1H),4.21(d,J=13.6Hz,3H),3.95(q,J=8.3Hz,2H), 3.66(s,4H),3.56(s,4H),2.87–2.67(m,4H),2.39(d,J=8.0Hz,3H),2.32(s,4H),2.29( s,1H),2.25(s,4H),2.20–2.08(m,3H),2.05(s,4H),1.99(s,4H),1.96(d,J=9.9Hz,3H).

[0415] Example 13: Synthesis of Compound P75

[0416]

[0417] HATU (49.5 mg, 0.13 mmol, 10 eq.) was added to a DMF (1 mL) solution of P74-6 (10 mg, 0.013 mmol, 1.0 eq.) and (1r, 3r)-3-hydroxycyclobutane-1-carboxylic acid (15 mg, 0.13 mmol, 10 eq.). The reaction mixture was stirred for 5-10 minutes, and DIPEA (16.8 mg, 0.13 mmol, 10 eq.) was added. Stir at room temperature for 1 hour. Purification by preparative HPLC gave compound P75 (4.25 mg, yield: 37.7%) as a white solid.

[0418] LC-MS (ESI) m / z: 868.1 [M+H] +

[0419] 1 H NMR(400MHz,DMSO-d6)δ8.88(s,1H),8.77(d,J=14.2Hz,2H),7.64(s,1H),6.47(s,1H),6.39(s,1H) ,6.17(d,J=5.4Hz,3H),5.05(dt,J=13.4,7.0Hz,3H),4.51(s,2H),4.25(dd,J=15.3,8.2Hz,3H),4. 12(d,J=18.3Hz,2H),3.65(s,4H),3.54(s,4H),3.23–2.67(m,9H),2.41–2.32(m,4H),2.31(s,4H), 2.28(d,J=3.0Hz,1H),2.23(s,4H),2.14–2.07(m,2H),2.06(s,3H),2.03–2.00(m,2H),1.99(s,3H).

[0420] Example 14: Synthesis of Compound P85

[0421]

[0422] Oxalyl chloride (0.25 mL, 0.48 mmol) and 0.1 eq of catalytic DMF were added to a solution of 2-(tert-butyldimethylsilyl)oxyacetic acid (200 mg, 0.96 mmol) in dichloromethane (3 mL) at 0°C. The reaction was monitored with BnNH2. After the reaction was completed, the obtained 2-((tert-butyldimethylsilyl)oxy)acetyl chloride solution was directly used in the next step.

[0423] To a DMF solution of P1 (4 mg, 0.0052 mmol) was added Et3N (2.6 mg, 0.026 mmol) and heated to 60°C. The above 2-((tert-butyldimethylsilyl)oxy)acetyl chloride solution (0.1 mL) was added to the reaction mixture. 2-((tert-butyldimethylsilyl)oxy)acetyl chloride solution (0.1 mL) was then added until P1 was completely reacted. Under this reaction condition, TBS was also deprotected to directly obtain the target product P85. P85 (1.64 mg, yield: 38%) was purified by preparative HPLC to obtain a white solid.

[0424] LC-MS (ESI) m / z: 816 [M+H] +

[0425] 1H NMR(400MHz,DMSO-d6)δ8.80(s,1H),6.48(s,1H),6.26(s,1H),6.17(s,1H), 5.88(t,J=5.8Hz,1H),4.49(d,J=2.8Hz,1H),3.99(d,J=5.7Hz,2H),3.65(s, 3H),3.24(d,J=4.6Hz,1H),2.81(d,J=7.3Hz,1H),2.76–2.65(m,1H),2.38–2 .33(m,1H),2.30(s,3H),2.24(s,3H),2.09(s,1H),2.06(s,3H),2.01(s,3H).

[0426] Example 15: Synthesis of Compound P90

[0427]

[0428] Step 1: Synthesis of P90-1

[0429] HATU (29.64 mg, 0.078 mmol, 3.0 eq.) and DIPEA (16.77 mg, 0.13 mmol, 5 eq.) were added to a 2 mL dichloromethane solution of P74-6 (20 mg, 0.026 mmol, 1.0 eq.) and glycolic acid (8 mg, 0.105 mmol, 4.0 eq.). After the addition, the mixture was stirred at room temperature overnight. Purification by preparative HPLC gave compound P90-1 (3.3 mg, yield: 15.3%) as a white solid.

[0430] LC-MS (ESI) m / z: 828.0 [M+H] +

[0431] 1H NMR (400MHz, DMSO-d6) δ9.03(s,1H),8.75(s,1H),7.90(s,1H),6.47(s,2H),6.18(s,2H),6.13(t,J=5 .5Hz,1H),5.07(d,J=11.6Hz,1H),4.52(d,J=2.8Hz,2H),4.22(d,J=4.8Hz,1H),4.18–4.05(m,2H),3. 96(d,J=5.5Hz,2H),3.66(s,3H),3.61(s,3H),3.30(s,1H),3.01(s,1H),2.78(s,2H),2.70(t,J=1.9H z,1H),2.39–2.34(m,1H),2.32(s,3H),2.24(s,3H),2.18(d,J=14.6Hz,1H),2.07(s,4H),2.00(s,3H).

[0432] Step 2: Synthesis of P90

[0433] To a solution of P90-1 (3.77 mg, 0.0045 mmol, 1.0 eq.) in acetonitrile (0.5 mL) at room temperature, 200 equivalents of formaldehyde (37 wt%) and 10 equivalents of NaCNBH3 were added. After stirring for 1 hour, 40 equivalents of HOAc were added. The reaction mixture was stirred at room temperature for 2 hours. Purification by preparative HPLC gave compound P90 (0.23 mg, yield: 6%) as a white solid.

[0434] LC-MS (ESI) m / z: 842.1 [M+H] +

[0435] 1H NMR(400MHz,DMSO-d6)δ9.03(s,1H),8.76(s,1H),7.84(s,1H),6.35(s,1H),6.25(s,1H),6.22(s,1H),6. 13(s,1H),4.94(d,J=11.4Hz,1H),4.50(d,J=2.7Hz,1H),4.19(d,J=13.4Hz,2H),3.94(s,2H),3.78(d,J=1 1.1Hz,1H),3.66(s,3H),3.52(s,4H),3.25(d,J=4.7Hz,1H),3.10–2.98(m,2H),2.86(dd,J=17.6,9.2Hz,1 H),2.57(d,J=18.6Hz,3H),2.42(s,1H),2.35(s,3H),2.14(s,3H),2.11(s,3H),2.03(s,3H),1.98(s,3H).

[0436] Example 16: Synthesis of Compound P96

[0437]

[0438] Steps 1 and 2: Synthesis of P96

[0439] To a solution of P19 (19 mg, 0.024 mmol) in DMF (1 mL) was added 2-((tert-butyldimethylsilyl)oxy)acetic acid (45.6 mg, 0.24 mmol), HATU (91.3 mg, 0.24 mmol), and DIEA (31 mg, 0.24 mmol). LC-MS showed that the reaction was complete, and water was added to quench, extracted with ethyl acetate, and concentrated. Anhydrous THF (1 mL) was added, and then 3HF·3Et3N (0.9 mL) was added and reacted at room temperature for 1 hour. LC-MS showed that the reaction was complete. Saturated aqueous NaHCO3 solution was added to the reaction solution, extracted with dichloromethane, the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by preparative high-performance liquid chromatography gave the target product P96 (5.86 mg, yield 28.7%) as a white solid. LC-MS (ESI) m / z: 856.7 [M+H] +

[0440] 1H NMR(400MHz,Chloroform-d)δ8.51(s,1H),8.15(s,1H),6.52(s,1H),6.37(s,1H),5.97(d,J=1.3Hz,1H),5.90(d,J=1.2Hz,1H),5. 78(s,1H),5.23(s,1H),5.07–4.94(m,1H),4.92(d,J=11.4Hz,1H),4.50(s,1H),4.24(s,1H),4.21(dd,J=4.8,1.5Hz,1H),4.11(s,2 H),4.08(d,J=2.8Hz,1H),4.05–4.00(m,1H),3.73(s,3H),3.52(s,3H),3.46–3.42(m,1H),3.34(s,1H),2.94(d,J=11.8Hz,1H),2.8 7(d,J=5.3Hz,2H),2.38(s,1H),2.26(s,3H),2.22(s,3H),2.14(s,3H),2.02(d,J=14.0Hz,2H),1.96(s,3H),1.06(d,J=1.6Hz,6H).

[0441] Example 17: Synthesis of Compound P166

[0442]

[0443] Step 1: Synthesis of 2-methoxy-3-nitrobenzaldehyde

[0444] Dissolve 2-hydroxy-3-nitrobenzaldehyde (5 g, 30 mmol), Cs2CO3 (29 g, 89 mmol), and MeI (5.1 g, 35.9 mmol) in DMF (40 mL). Stir at 80°C for 1.5 hours. Filter the mixture, wash with water, and extract with MTBE. Dry the organic layer over anhydrous sodium sulfate and concentrate to obtain the target compound (4 g, yield 73.8%) as a yellow solid.

[0445] Step 2: Synthesis of (2-methoxy-3-nitrophenyl)methanol

[0446] 2-Methoxy-3-nitrobenzaldehyde (370 mg, 2.04 mmol) was dissolved in EtOH (5 mL) solution, and NaBH4 (85 mg, 2.24 mmol) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, it was quenched with 1N HCl and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 350 mg of a crude product as a solid, which was directly used in the next step.

[0447] 1H NMR (400MHz, DMSO-d6) δ7.81(m,2H),7.37(t,1H),5.45(t,J=5.7Hz,1H),4.63(d,J=5.7Hz,2H),3.85(s,3H).

[0448] Step 3: Synthesis of 1-(bromomethyl)-2-methoxy-3-nitrobenzene

[0449] Pyridine (37 mg) was added to a solution of (2-methoxy-3-nitrophenyl)methanol (120 mg, 0.655 mmol) in dichloromethane (3 mL) and cooled to 0°C. PBr3 (266 mg, 0.982 mmol) was gradually added. After the reaction was completed, water was poured into the solution and extracted with dichloromethane. The organic phase was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by preparative thin layer chromatography to obtain the target compound (65 mg, yield 40%).

[0450] 1 H NMR (400MHz, DMSO-d6) δ7.95(dd,J=8.2,1.7Hz,1H),7.86(dd,J=7.7,1.7Hz,1H),7.38(t,J=7.9Hz,1H),4.78(s,2H),3.93(s,3H).

[0451] Step 4: Synthesis of 2-(2-methoxy-3-nitrophenyl)acetonitrile

[0452] To a solution of TMSCN (31.42 mg, 0.29 mmol) in THF (2 mL) was added TBAF (1 M THF solution, 0.29 ml) and stirred for 1 hour. Then, a solution of 1-(bromoethyl)-2-methoxy-3-nitrobenzene (65 mg, 0.264 mmol) in acetonitrile was added. After the reaction was completed, the concentrated residue was purified by preparative thin layer chromatography to obtain the target compound (25 mg, yield 48.9%).

[0453] LC-MS (ESI) m / z: 166.3 [M-CN] +

[0454] Step 5: Synthesis of 2-(3-amino-2-methoxyphenyl)acetonitrile

[0455] Under N2 protection, 5% wet Pd / C was added to a solution of 2-(2-methoxy-3-nitrophenyl)acetonitrile (25 mg, 0.56 mmol, 1.0 equivalent) in 3 mL THF. The suspension was degassed under vacuum and replaced with H2 several times. Stirred overnight at room temperature under H2 protection. The suspension was filtered through diatomaceous earth and the filter cake was washed with methanol. The combined filtrate was concentrated and used directly in the next step.

[0456] LC-MS (ESI) m / z: 163.2 [M+H] +

[0457] Step 6: Synthesis of 3-(2-aminoethyl)-2-methoxyaniline

[0458] Under Ar protection, the crude product 2-(3-amino-2-methoxyphenyl)acetonitrile was dissolved in (1 mL) anhydrous tetrahydrofuran. Borane dimethyl sulfide (2M, 0.5 mL) was added dropwise to the solution at 0°C, and then heated under reflux for 1 hour. LC-MS showed that the reaction was complete and cooled to 0°C. It was quenched with 1 mL of MeOH and concentrated to remove the solvent. The residue was purified by preparative thin layer chromatography to obtain 10 mg of the target product.

[0459] LC-MS (ESI) m / z: 167.3 [M+H] +

[0460] Step 7: Synthesis of P166

[0461] The intermediate Int-A (5 mg, 0.008 mmol), 3-(2-aminoethyl)-2-methoxyaniline (14.6 mg, 0.08 mmol) and AcOH (6.4 mg, 0.08 mmol) were dissolved in anhydrous ethanol (1 mL) and stirred at 25 ° C overnight. After the reaction was completed, the solvent was concentrated to obtain a solid crude product. The crude product was purified by preparative high performance liquid chromatography to obtain the target compound (0.62 mg, yield 10%) as a white solid.

[0462] LC-MS (ESI) m / z: 770.4 [M+H] +

[0463] Example 18: Synthesis of Compound P170

[0464]

[0465] Step 1: Synthesis of P170-1

[0466] At 0°C, oxalyl chloride (2M in dichloromethane, 0.08 mL, 0.15 mmol) was added to a solution of 2-((tert-butyldimethylsilyl)oxy)acetic acid (57 mg, 0.3 mmol) in dichloromethane (5 mL), and 2 drops of catalytic DMF were added. After stirring at 0°C for 1 hour, the reaction solution was added to a solution of P166 (24 mg, 0.03 mmol) and triethylamine (30.3 mg, 0.3 mmol) in anhydrous DMF (1 mL). Stirring was continued at room temperature for 2 hours. LC-MS showed that the reaction was complete, and water was added to quench the reaction, and the reaction was extracted with ethyl acetate. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel flash column chromatography to obtain the target product (15 mg, yield: 53.2%) as a yellow liquid.

[0467] LC-MS(ESI)m / z:[M+H] + 942

[0468] Step 2: Synthesis of P170

[0469] Triethylamine trifluoroacetate (1 mL) was added to a solution of P170-1 (14 mg, 0.014 mmol, 1 eq) in anhydrous THF (1 mL) and stirred at room temperature for one hour. LC-MS showed that the reaction was complete, neutralized with saturated aqueous NaHCO3, extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by HPLC to obtain the target product (2.35 mg, yield: 19.1%) as a white solid. LC-MS (ESI) m / z: [M+H] + 828

[0470] 1 H NMR (400MHz, DMSO-d6) δ9.06(s,1H),8.77(s,1H),7.88(d,J=8.8Hz,1H),6.73(d,J=8.8Hz,1H),6.46(s,1H),6.25(s,1H) ,6.17(s,1H),6.12(m,1H),5.06(d,J=11.5Hz,1H),4.45(d,J=2.8Hz,3H),4.20(d,J=4.5Hz,2H),4.08(s,1H),4.03(d,J=

[0471] 11.3Hz,2H),3.97(d,J=5.4Hz,3H),3.64(s,3H),3.61(s,3H),3.23(m,2H),3.08(m,1H),2.80(m,2H ),2.75–2.56(m,3H),2.41(m,2H),2.28(s,3H),2.24(s,3H),2.08(s,1H),2.05(s,3H),1.98(s,3H).

[0472] Example 19: Synthesis of Compound P172

[0473]

[0474] Step 1: Synthesis of 2-(4-methyl-3-nitrophenyl)acetonitrile

[0475] To a solution of TMSCN (1.3 g, 13 mmol) in THF (15 mL) was added TBAF (1 M THF solution, 12 mL) and stirred for 1 hour. Then a solution of 4-(chloromethyl)-1-methyl-2-nitrobenzene (1 g, 5.4 mmol) in CH3CN (9 mL) was added. After the reaction was completed, the mixture was concentrated and the crude product was purified by silica gel flash column chromatography to obtain the target compound (688 mg, yield 72.3%).

[0476] LC-MS (ESI) m / z: 177 [M+H] +

[0477] 1 H NMR (400MHz, Chloroform-d) δ7.98(d,J=2.0Hz,1H),7.55(dd,J=7.9,2.0Hz,1H),7.43(d,J=7.9Hz,1H),3.86(s,2H),2.65(s,3H).

[0478] Step 2: Synthesis of 2-(3-amino-4-methylphenyl)acetonitrile

[0479] Under N2 protection, 5% wet Pd / C (40 mg) was added to a solution of 2-(4-methyl-3-nitrobenzene)acetonitrile (680 mg) in THF (10 mL). The suspension was degassed under vacuum and replaced with H2 several times. Stirred at room temperature overnight under H2 protection. The suspension was filtered through diatomaceous earth and the filter cake was washed with methanol. The combined filtrate was concentrated and used directly in the next step.

[0480] LC-MS (ESI) m / z: 147 [M+H] +

[0481] Step 3: Synthesis of 5-(2-aminoethyl)-2-methylaniline

[0482] To a MeOH solution of 2-(3-amino-4-methylphenyl)acetonitrile (38 mg) was added Raney Ni (5 mg) and 0.1-0.5 eq. NH3.H2O. Stir at room temperature for 2 hours. After the reaction was completed, the solid was filtered off and concentrated. The target product (27 mg, yield: 69%) was obtained by preparative thin layer chromatography.

[0483] LC-MS (ESI) m / z: 151 [M+H] +

[0484] 1 H NMR (400MHz, Chloroform-d) δ7.01(d,J=7.4Hz,1H),6.58(d,J=8.2Hz,2H),2.98(t,J=6.8Hz,2H),2.69(t,J=6.8Hz,2H),2.18(s,3H).

[0485] Step 4: Synthesis of P172

[0486] The intermediate Int-A (4 mg, 0.0065 mmol), 5-(2-aminoethyl)-2-methylaniline (9.8 mg, 0.065 mmol) and HOAc (3.9 mg, 0.065 mmol) were dissolved in anhydrous ethanol (1 mL) and stirred at 25 ° C overnight. After the reaction was completed, the solvent was removed under reduced pressure to obtain a solid crude product. The crude product was purified by preparative HPLC to obtain the target product P172 (3.32 mg, yield: 68%) as a white solid. LC-MS (ESI) m / z: 754 [M+H] +

[0487] 1H NMR(400MHz,DMSO-d6)δ6.45(s,1H),6.41(s,1H),6.24–6.15(m,3H),5.03(d,J=11.6Hz,1H),4.71 (s,2H),4.47(d,J=2.8Hz,1H),4.20(d,J=4.7Hz,1H),4.08(s,1H),4.01(d,J=12.2Hz,1H),3.65(s ,4H),3.24(d,J=4.8Hz,2H),3.08–3.00(m,1H),2.79(d,J=7.5Hz,2H),2.60(s,1H),2.47–2.33(m, 1H),2.31(s,4H),2.27(dt,J=4.8,2.1Hz,1H),2.23(s,3H),2.05(s,3H),2.01(s,4H),1.86(s,3H).

[0488] Example 20: Synthesis of Compound P241

[0489]

[0490] Step 1: Synthesis of 1-(4-methoxybenzyl)cyclopropane-1-amine

[0491] At 0°C, Ti(O-ipr)4(15.46g, 54.39mmol, 2eq) and EtMgBr(7.2g, 54.39mmol, 2eq) were added to a solution of 2-(4-methoxyphenyl)acetonitrile(4g, 27.19mmol, 1eq) in THF(50mL) and MTBE(50mL) in turn and kept for 1 hour. BF3˙OEt2(7.6g, 54.39mmol, 2eq) was added to the reaction solution and stirred for 1 hour. After the reaction was completed, it was quenched with 2M NaOH and filtered. Extracted with ethyl acetate(100mL), the organic phase was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel flash column chromatography to obtain the target product (2.7g, yield: 56.5%) as a white solid.

[0492] 1 H NMR (400MHz, DMSO-d6) δ7.05–6.96(m,2H),6.73–6.65(m,2H),3.63(s,3H),2.50(s,2H),0.51–0.34(m,4H).

[0493] Step 2: Synthesis of benzyl (1-(4-methoxybenzyl) cyclopropyl) carbamate

[0494] Cbz-Cl (4.6 mg, 27.41 mmol, 1.8 eq) was slowly dripped into dichloromethane (30 mL) and saturated NaHCO3 aqueous solution of 1-(4-methoxybenzyl)cyclopropane-1-amine (2.7 g, 15.23 mmol, 1 eq). Stir overnight at room temperature. Extract with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel flash column chromatography to obtain the target product (3 g, yield: 63.8%) as a white solid.

[0495] LC-MS (ESI) m / z: 312 [M+H] +

[0496] Step 3: Synthesis of benzyl (1-(4-methoxy-3-nitrobenzyl) cyclopropyl) carbamate

[0497] At 0°C, 65% HNO3 (467 mg, 4.81 mmol, 1.5 eq) was added to a solution of benzyl (1-(4-methoxybenzyl) cyclopropyl) carbamate (1 g, 3.21 mmol, 1 eq) in TFA (10 mL) and stirred for 2 hours. After the reaction was completed, saturated NaHCO3 aqueous solution was added to quench the reaction, the solvent was removed under reduced pressure, and the pH was adjusted to 7-8. The mixture was extracted and separated with dichloromethane, dried with anhydrous sodium sulfate and filtered, and purified by silica gel flash column chromatography to obtain the target compound (250 mg, yield: 21%) as a yellow solid.

[0498] LC-MS (ESI) m / z: 357 [M+H] +

[0499] Step 4: Synthesis of 5-((1-aminocyclopropyl)methyl)-2-methoxyaniline

[0500] Under N2 protection, HOAc (57.3 mg, 0.954 mmol, 2 eq) and 50 mg of 10% wet Pd / C were added to a solution of (1-(4-methoxy-3-nitrobenzyl)cyclopropyl)carbamate (170 mg, 0.477 mmol) in methanol (3 mL). The suspension was degassed under vacuum and replaced with H2 several times. Stir at room temperature overnight under H2 protection. The suspension was filtered through diatomaceous earth and the filter cake was washed with methanol. The combined filtrate was concentrated and used directly in the next step.

[0501] LC-MS (ESI) m / z: 193 [M+H] +

[0502] Step 5: Synthesis of P241

[0503] The intermediate Int-A (10 mg, 0.016 mmol, 1 eq) and 5-((1-aminocyclopropyl)methyl)-2-methoxyaniline (5.19 mg, 0.16 mmol, 10 eq) were dissolved in anhydrous ethanol (2 mL) and stirred at 60 ° C for 1 hour. After the reaction was completed, the solvent was removed under reduced pressure to obtain a solid. The crude product was purified by preparative HPLC to obtain the target compound P241 (1.87 mg, yield: 11%) as a white solid. LC-MS (ESI) m / z: 796 [M+H] +

[0504] Example 21: Synthesis of Compounds P261, P262

[0505]

[0506] Step 1: Synthesis of 3-cyclopropyl-2-(4-methoxyphenyl)propionitrile

[0507] 2-(4-methoxyphenyl)acetonitrile (1.0 g, 6.8 mmol) was dissolved in DMF (10 mL) solution, NaH (60% w / w, 300 mg, 7.48 mmol) was added at 0°C and stirred for 1 hour. (Bromoethyl)cyclopropane (918 mg, 6.8 mmol) was added at 0°C. After the reaction was completed, it was poured into saturated NH4Cl aqueous solution and extracted with MTBE. The organic phase was dried over sodium sulfate, filtered and concentrated. The crude product was purified by silica gel flash column chromatography to obtain the target compound (1.2 g, yield: 87.7%) as a solid.

[0508] LC-MS (ESI) m / z: 202 [M+H] +

[0509] Step 2: Synthesis of 3-cyclopropyl-2-(4-methoxy-3-nitrophenyl)propionitrile

[0510] To a solution of 3-cyclopropyl-2-(4-methoxyphenyl)propionitrile (1.2 g, 5.96 mmol) in dichloromethane (20 mL), add 20 mL of a mixture of conc. HNO3 (65%, 2.6 g, 26.8 mmol) and conc. H2SO4 (2.77 g, 26.8 mmol). After the reaction is complete, adjust the pH to 7-8 with a saturated aqueous solution of NaHCO3. Extract with dichloromethane, separate the organic phase, dry with anhydrous sodium sulfate, filter, concentrate, and purify with silica gel flash column chromatography to obtain the target product (400 mg, yield 27%).

[0511] LC-MS (ESI) m / z: 247 [M+H] +

[0512] Step 3: Synthesis of 2-(3-amino-4-methoxyphenyl)-3-cyclopropylpropionitrile

[0513] Under N2 protection, 5% wet Pd / C was added to a solution of 3-cyclopropyl-2-(4-methoxy-3-nitrophenyl)propionitrile (110 mg, 0.45 mmol) in THF (3 mL). The suspension was degassed under vacuum and replaced with H2 several times. Stirred at room temperature overnight under H2 protection. The suspension was filtered through diatomaceous earth and the filter cake was washed with methanol. The combined filtrate was concentrated and used directly in the next step.

[0514] LC-MS (ESI) m / z: 217 [M+H] +

[0515] Step 4: Synthesis of 5-(1-amino-3-cyclopropylpropane-2-yl)-2-methoxyaniline

[0516] Under Ar protection, at 0°C, borane dimethyl sulfide (2M, 0.5 ml) was added dropwise to a solution of the crude product 2-(3-amino-4-methoxyphenyl)-3-cyclopropylpropionitrile in anhydrous tetrahydrofuran. The reaction solution was heated to reflux for 1 hour. After LC-MS showed that the reaction was complete, it was cooled to 0°C. It was quenched with 1 mL of MeOH and concentrated to remove the solvent. The residue was purified by preparative thin layer chromatography to obtain the target product (10 mg, yield of two steps: 10%).

[0517] Step 5: Synthesis of P261, P262

[0518] The intermediate Int-A (5 mg, 0.008 mmol), 5-(1-amino-3-cyclopropylpropane-2-yl)-2-methoxyaniline (10 mg, 0.04 mmol), HOAc (4 mg, 0.064 mmol) were dissolved in anhydrous ethanol (1 mL) and stirred at 25 ° C overnight. After the reaction was completed, the solvent was removed under reduced pressure to obtain a solid. The crude product was purified by preparative high performance liquid chromatography to obtain P261: (0.58 mg, yield: 17%), P262: (1 mg, yield: 30%).

[0519] LC-MS (ESI) m / z: 824 [M+H] +

[0520] LC-MS (ESI) m / z: 824 [M+H] +

[0521] Example 22: Synthesis of Compound T1

[0522]

[0523] Step 1: Synthesis of 2-(3-methoxy-5-nitrophenyl)acetonitrile

[0524] To a solution of TMSCN (97 mg, 0.97 mmol) in THF (5 mL) was added TBAF (1 M THF solution, 0.9 mL) and stirred for 1 hour. Then, a solution of 1-(bromoethyl)-3-methoxy-5-nitrobenzene (200 mg, 0.81 mmol) in acetonitrile was added. After the reaction was completed, the crude product was concentrated and purified by preparative thin layer chromatography to obtain the target compound (50 mg, yield: 31.7%).

[0525] LC-MS (ESI) m / z: 193 [M+H] +

[0526] Step 2: Synthesis of 2-(3-amino-5-methoxyphenyl)acetonitrile

[0527] Under N2 protection, 10 mg of 5% wet Pd / C was added to 2-(3-methoxy-5-nitrophenyl)acetonitrile (50 mg) in THF (3 mL). The suspension was degassed under vacuum and replaced with H2 several times. Stirred at room temperature overnight under H2 protection. The suspension was filtered through diatomaceous earth and the filter cake was washed with methanol. The combined filtrate was concentrated and used directly in the next step.

[0528] LC-MS (ESI) m / z: 163 [M+H] +

[0529] Step 3: Synthesis of 3-(2-aminoethyl)-5-methoxyaniline

[0530] Under Ar protection, borane dimethyl sulfide (2M, 0.6mL) was added dropwise to a solution of the crude product 2-(3-amino-5-methoxyphenyl)acetonitrile (40mg, 0.25mmol) in anhydrous tetrahydrofuran at 0°C. The above reaction solution was heated to reflux for 1 hour. After LC-MS showed that the reaction was complete, it was cooled to 0°C. It was quenched with 1mL of MeOH and concentrated to remove the solvent. The residue was purified by preparative thin layer chromatography to obtain the target compound (7mg, two-step yield: 16%).

[0531] LC-MS (ESI) m / z: 167 [M+H] +

[0532] Step 4: Synthesis of T1

[0533] The intermediate Int-A (5 mg, 0.008 mmol), 3-(2-aminoethyl)-5-methoxyaniline (5 mg, 0.032 mmol) and HOAc (4 mg, 0.064 mmol) were dissolved in anhydrous ethanol (1 mL) and stirred at 25°C overnight. After the reaction was completed, the solvent was removed under reduced pressure to obtain a solid crude product. The crude product was purified by preparative HPLC to obtain the target compound T1: (3.35 mg, yield: 54%) as a white solid.

[0534] LC-MS (ESI) m / z: 770 [M+H] +

[0535] Example 23: Synthesis of Compound T2

[0536]

[0537] Step 1: Synthesis of 2-amino-2-(3-hydroxy-4-methoxyphenyl)acetonitrile

[0538] To a solution of 3-hydroxy-4-methoxybenzaldehyde (10 g, 65.72 mmol, 1 eq) in methanol (50 mL) was added NH3 / MeOH (30% w / w, 50 mL) and the resulting solution was stirred at room temperature for 1 hour. Cyanotrimethylsilane (20 mL) was added and stirred overnight. After the reaction was completed, it was quenched with ice water and the solvent was removed under reduced pressure. The residue was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel flash column chromatography to obtain the target compound (880 mg, yield: 7.5%) as a white solid.

[0539] LC-MS (ESI) m / z: 179 [M+H] +

[0540] 1 H NMR (400MHz, DMSO-d6) δ9.12(brs,1H),6.98–6.91(m,2H),6.88(m,1H),4.88(s,1H),3.78(s,3H).

[0541] Step 2: Synthesis of tert-butyl (cyano (3-hydroxy-4-methoxyphenyl) methyl) carbamate

[0542] To a solution of 2-amino-2-(3-hydroxy-4-methoxyphenyl)acetonitrile (620 mg, 3.48 mmol, 1.0 eq) in THF (10 mL) was added Boc2O (836.2 mg, 3.83 mmol, 1.1 eq) and Et3N (702.9 mg, 6.96 mmol, 2.0 eq). Stir at room temperature overnight. After the reaction was completed, it was concentrated and extracted with ethyl acetate (50 mL) and water (50 mL) respectively. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and purified by silica gel flash column chromatography to obtain the target compound (500 mg, yield: 38%) as a white solid.

[0543] Step 3: Synthesis of tert-butyl (2-amino-1-(3-hydroxy-4-methoxyphenyl)ethyl) carbamate

[0544] Tert-butyl (cyano (3-hydroxy-4-methoxyphenyl) methyl) carbamate (50 mg, 0.179 mmol, 1.0 eq) was dissolved in anhydrous THF (2 mL). LiAlH4 (2 M, 1.1 eq) was added and stirred at room temperature for 2 hours. After the reaction was completed, it was quenched with Na2SO4·10H2O, filtered, and the filtrate was concentrated and used directly in the next step.

[0545] LC-MS (ESI) m / z: 283.4 [M+H] +

[0546] Step 4: Synthesis of T2-5

[0547] The intermediate Int-A (10 mg, 0.016 mmol, 1.0 eq), tert-butyl (2-amino-1-(3-hydroxy-4-methoxyphenyl)ethyl) carbamate (44.9 mg, 0.16 mmol, 10 eq) and HOAc (9.54 mg, 0.16 mmol, 10 eq) were dissolved in anhydrous EtOH (2 mL) and stirred at room temperature for 1 hour. After the reaction was completed, the solvent was removed under reduced pressure to obtain a solid crude product. The crude product was purified by preparative HPLC to obtain the target product (11 mg, yield 77%) as a white solid.

[0548] LC-MS (ESI) m / z: 886.3 [M+H] +

[0549] Step 5: Synthesize T2

[0550] To a solution of T2-5 (11 mg) in dichloromethane (2 mL) was added HCl / 1,4-dioxane (4 M, 2 mL). The mixture was stirred at room temperature overnight. After the reaction was completed, the mixture was purified by preparative HPLC to obtain the target product T2 (2.37 mg, yield: 26.9%) as a white solid.

[0551] LC-MS (ESI) m / z: 786.9 [M+H] +

[0552] Example 24: Synthesis of Compound T3

[0553]

[0554] Step 1: Synthesis of 2-(4-fluoro-3-nitrophenyl)-2-methylpropionitrile

[0555] At 0°C, add fuming nitric acid (2 mL) dropwise into a round-bottom flask containing 2-(4-fluorophenyl)-2-methylpropionitrile (500 mg, 3.0 mmol). Continue stirring the mixture at 0°C for 30 minutes. Pour the reaction mixture into ice and then extract with ethyl acetate. The combined organic layer is washed with saturated brine, then dried over MgSO4, filtered, concentrated, and the crude product is purified by silica gel flash column chromatography (eluted with DCM / MeOH 100 / 0 to 96 / 4) to obtain the target product (100 mg, yield: 16%).

[0556] Step 2: Synthesis of 2-(3-amino-4-fluorophenyl)-2-methylpropionitrile

[0557] Under N2 protection, 2-(4-fluoro-3-nitrophenyl)-2-methylpropionitrile (100 mg, 0.56 mmol) was dissolved in THF (3 mL) and 20 mg of 5% wet Pd / C was added. The suspension was degassed under vacuum and replaced with H2 several times. Stir at room temperature overnight under H2 protection. The suspension was filtered through diatomaceous earth and the filter cake was washed with methanol. The combined filtrate was concentrated and used directly in the next step.

[0558] LC-MS (ESI) m / z: 179.2 [M+H] +

[0559] Step 3: Synthesis of 5-(1-amino-2-methylpropane-2-yl)-2-fluoroaniline

[0560] Under Ar protection, borane dimethyl sulfide (2M, 0.5 ml) was added dropwise to a solution of the crude product 2-(3-amino-4-fluorophenyl)-2-methylpropionitrile in anhydrous tetrahydrofuran (3 mL) at 0°C. After the above solution was heated to reflux for 1 hour, LC-MS showed that the reaction was complete and cooled to 0°C. It was quenched with MeOH (1 mL) and concentrated to remove the solvent. The residue was purified by preparative thin layer chromatography to obtain the target product (10 mg, two-step yield: 11%).

[0561] LC-MS (ESI) m / z: 165.9 [M+H] +

[0562] 1 H NMR (400MHz, Chloroform-d) δ6.95(m,1H),6.78(m,1H),6.68(m,1H),3.73(s,2H),2.77(s,2H),1.28(s,6H).

[0563] Step 4: Synthesize T3

[0564] The intermediate Int-A (5 mg, 0.008 mmol), 5-(1-amino-2-methylpropane-2-acyl)-2-fluoroaniline (14.6 mg, 0.08 mmol) and NaOAc (6.4 mg, 0.08 mmol) were dissolved in anhydrous ethanol (1 mL) and stirred at 25 ° C overnight. After the reaction was completed, the solvent was removed under reduced pressure to obtain a solid crude product. The crude product was purified by preparative high performance liquid chromatography to obtain the target product (1.69 mg, yield: 26.7%) as a white solid.

[0565] LC-MS (ESI) m / z: 786.3 [M+H] +

[0566] 1 H NMR(400MHz, DMSO-d6)δ8.80(s,1H),6.62(d,J=9.7Hz,1H),6.47(s,1H),6.36(d,J=13.1Hz ,1H),6.26(s,1H),6.16(s,1H),5.05(s,1H),5.03(s,2H),4.47(m,2H),4.20(d,J=4.5Hz,1H ),4.08(s,1H),3.99(d,J=11.2Hz,2H),3.64(s,4H),3.24–3.20(m,1H),3.05(m,1H),2.81( m,2H),2.35(m,1H),2.32(s,3H),2.30–2.25(m,2H),2.23(s,3H),2.06(s,3H),2.01(s,3H).

[0567] Example 25: Synthesis of Compound T4

[0568]

[0569] HATU (25.1 mg, 0.066 mmol) was added to a DMF (2 mL) solution of P1 (5 mg, 0.0066 mmol) and 1-hydroxycyclopropane-1-carboxylic acid (6.74 mg, 0.066 mmol). The reaction mixture was stirred for 5-10 minutes, and DIPEA (8.58 mg, 0.066 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. Compound T4 (1.72 mg, yield: 30.9%) was purified by preparative HPLC as a white solid. LC-MS (ESI) m / z: 842 [M+H] +

[0570] Example 26: Synthesis of Compound T5

[0571]

[0572] HATU (19.8 mg, 0.053 mmol) was added to a DMF (2 mL) solution of P1 (4 mg, 0.0053 mmol) and (1s, 3s)-3-hydroxycyclobutane-1-carboxylic acid (6 mg, 0.053 mmol). The reaction mixture was stirred for 5-10 minutes, and then DIPEA (6.8 mg, 0.053 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour and then purified by preparative HPLC to obtain the target compound T5 (1.69 mg, yield: 36.9%) as a white solid.

[0573] (ESI)m / z:856.4[M+H] +

[0574] Example 27: Synthesis of Compound T6

[0575]

[0576] Step 1: Synthesis of 2-methoxy-3-nitrobenzaldehyde

[0577] Cs2CO3 (29.24 g, 90 mmol, 3 eq) and CH3I (5.1 g, 36 mmol, 1.2 eq) were added to a solution of 2-hydroxy-3-nitrobenzaldehyde (5 g, 30 mmol, 1.0 eq) in anhydrous DMF (40 mL) and stirred at 80 °C for 1.5 hours. After the reaction was completed, the solid was filtered out. After dilution with water, the resulting solution was extracted with MTBE. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the target product (4.02 g, yield: 73.6%) as a yellow solid.

[0578] LC-MS (ESI) m / z: 182.2 [M+H] +

[0579] Step 2: Synthesis of (2-methoxy-3-nitrophenyl)methanol

[0580] Dissolve 2-methoxy-3-nitrobenzaldehyde (3.5 g, 19.3 mmol, 1.0 eq) in ethanol (30 mL) and add NaBH4 (804 mg, 21.3 mmol, 1.1 eq). Stir at room temperature for 2.5 hours. After the reaction is complete, adjust the pH to 3 with 1N HCl. Extract with dichloromethane, separate the organic layer, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the desired product (2.89 g, crude) as a yellow solid.

[0581] LC-MS (ESI) m / z: 184.2 [M+H] +

[0582] Step 3: Synthesis of 1-(bromomethyl)-2-methoxy-3-nitrobenzene

[0583] Dissolve (2-methoxy-3-nitrobenzene)methanol (2.89 g, 15.8 mmol, 1.0 eq) in anhydrous dichloromethane (40 mL), and then add pyridine (891 mg, 11.4 mmol, 0.72 eq). Slowly drop PBr3 (6.4 g, 23.7 mmol, 1.5 eq) into the above reaction mixture. Stir at room temperature for 3 hours. After the reaction is completed, add water and extract with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, concentrate, and purify by silica gel flash column chromatography to obtain the target product (1.8 g, yield: 46.3%) as a yellow solid.

[0584] LC-MS (ESI) m / z: 245.8 [M+H] +

[0585] Step 4: Synthesis of 2-(2-methoxy-3-nitrophenyl)acetonitrile

[0586] Dissolve TBAF (8.1 mmol, 1.1 eq) and trimethylsilyl cyanide (872 mg, 8.8 mmol, 1.2 eq) in THF (20 mL) and stir at room temperature for 1 hour. Add 1-(bromomethyl)-2-methoxy-3-nitrobenzene (1.8 g, 7.32 mmol, 1.0 eq) in acetonitrile (20 mL). Continue stirring at room temperature for 1 hour. After the reaction mixture is concentrated, purify it with silica gel flash column chromatography to obtain the target product (1.2 g, yield: 85.7%) as a yellow solid.

[0587] LC-MS (ESI) m / z: 193 [M+H] +

[0588] Step 5: Synthesis of 2-(2-methoxy-3-nitrophenyl)ethyl-1-amine

[0589] 2-(2-methoxy-3-nitrophenyl)acetonitrile (1.1 g, 5.7 mmol, 1.0 eq) was dissolved in anhydrous THF (40 mL) solution, and 2.5 M borane dimethyl sulfide (14.3 mL, 2.5 eq) was added. The reaction mixture was refluxed for 1 hour. The above reaction mixture was quenched with MeOH and concentrated. Purification by silica gel flash column chromatography gave the target product (650 mg, yield: 55%).

[0590] LC-MS (ESI) m / z: 196 [M+H] +

[0591] 1 H NMR(400MHz,Chloroform-d)δ7.69(dd,J=8.1,1.6Hz,1H),7.45(dd,J=7.6,1.7Hz,1H), 7.16(t,J=7.9Hz,1H),3.90(s,3H),3.73–3.65(m,2H),2.87(m,2H),1.74–1.67(m,2H).

[0592] Step 6: Synthesis of 3-(2-aminoethyl)-2-methoxyaniline acetate

[0593] Under N2 protection, 5% wet Pd / C and HOAc (398 mg, 7 mmol, 2.0 eq) were added to a THF solution of 2-(2-methoxy-3-nitrophenyl)ethyl-1-amine (650 mg, 3.57 mmol, 1 eq). The suspension was degassed under vacuum and replaced with H2 several times. Stirred at room temperature overnight under H2 protection. The suspension was filtered through diatomaceous earth and the filter cake was washed with methanol. The combined filtrate was concentrated to obtain (780 mg, crude product), which was used directly in the next step.

[0594] LC-MS (ESI) m / z: 167 [M+H] +

[0595] 1 H NMR (400MHz, DMSO-d6) δ6.76(m,1H),6.58(m,1H),6.39(m,1H),4.86(brs,2H),3.64(s,3H),2.84(m,2H),2.71(m,2H).

[0596] Step 7: Synthesis of benzyl (3-(2-((benzyloxy)carbonyl)amino)ethyl)-2-methoxyphenyl)carbamate

[0597] To a mixture of 3-(2-aminoethyl)-2-methoxyaniline acetate (780 mg, 3.45 mmol, 1.0 eq) in dichloromethane (20 mL) and saturated sodium bicarbonate (50 mL) aqueous solution at 0-10°C, CbzCl (1.76 g, 10.4 mmol, 3.0 eq) was added. The resulting mixture was stirred at room temperature overnight. LC-MS showed that the reaction was complete, and the aqueous phase was separated and extracted twice with dichloromethane (20 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by silica gel flash column chromatography gave the target product (550 mg, yield: 76.5%) as a yellow solid.

[0598] LC-MS (ESI) m / z: 435 [M+H] +

[0599] 1H NMR(400MHz,Chloroform-d)δ8.00(s,1H),7.44–7.27(m,10H),7.20(s,1H),7.06(t,J=7 .9Hz,1H),6.85(m,1H),5.22(s,2H),5.08(s,2H),3.71(s,3H),3.45(m,2H),2.84(m,2H).

[0600] Step 8: Synthesis of benzyl (3-(2-((benzyloxy)carbonyl)amino)ethyl)-2-hydroxyphenyl)carbamate

[0601] At -78 ° C, BCl3 (1M dichloromethane solution, 12.7 mL, 12.7 mmol, 10 eq) was added to a solution of benzyl (3-(2-((benzyloxy)carbonyl)amino)ethyl)-2-methoxyphenyl)carbamate (550 mg, 1.27 mmol, 1.0 eq) in dichloromethane (40 mL) and stirred at the same temperature for 2 hours. LC-MS showed that the reaction was complete. The cooled solution was quenched with saturated aqueous NaHCO3 and extracted with dichloromethane (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel flash column chromatography to obtain the target product (400 mg, yield: 74.9%) as a yellow solid.

[0602] LC-MS (ESI) m / z: 421 [M+H] +

[0603] 1H NMR(400MHz,Chloroform-d)δ8.47(brs,1H),7.51(brs,1H),7.44–7.28(m,10H) ,7.19(m,1H),6.80(m,2H),5.22(s,2H),5.14(s,2H),3.35(m,2H),2.89(m,2H).

[0604] Step 9: Synthesis of benzyl(3-(2-((benzyloxy)carbonyl)amino)ethyl)-2-(methoxymethoxy)phenyl)carbamate.

[0605] To a solution of benzyl (3-(2-((benzyloxy)carbonyl)amino)ethyl)-2-hydroxyphenyl)carbamate (150 mg, 0.36 mmol, 1.0 eq) in anhydrous acetonitrile (20 mL) at 0°C, cesium carbonate (235 mg, 0.72 mmol, 2.0 eq) and MOMBr (67.5 mg, 0.54 mmol, 1.5 eq) were added. The mixture was stirred at room temperature for 4 hours. LC-MS monitored the completion of the reaction. After adding water, the mixture was extracted with ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel flash column chromatography to obtain the target product (80 mg, yield 47.8%) as a yellow liquid.

[0606] LC-MS (ESI) m / z: 465 [M+H] +

[0607] Step 10: Synthesis of 3-(2-aminoethyl)-2-(methoxymethoxy)aniline

[0608] To a solution of benzyl (3-(2-((benzyloxy)carbonyl)amino)ethyl)-2-(methoxymethoxy)phenyl)carbamate (80 mg, 0.17 mmol, 1.0 eq) in MeOH (5 mL) under N2 protection, 5% wet Pd / C (20 mg) and HOAc (20.4 mg, 0.34 mmol, 2.0 eq) were added. The suspension was degassed under vacuum and replaced with H2 several times. Stir at room temperature under H2 protection for 0.5 h. The suspension was filtered through celite and the filter cake was washed with methanol. The combined filtrate was concentrated and used directly in the next step.

[0609] LC-MS (ESI) m / z: 197 [M+H] +

[0610] Step 11: Synthesis of T6-12

[0611] The intermediate Int-A (9 mg, 0.015 mmol, 1.0 eq), 3-(2-aminoethyl)-2-(methoxymethoxy)aniline (14.1 mg, 0.072 mmol, 5 eq) and HOAc (9.54 mg, 0.16 mmol, 10 eq) were dissolved in anhydrous EtOH (2 mL) and stirred at room temperature for 2.5 hours. After the reaction was completed, it was concentrated to obtain a solid. The crude product can be used directly in the next step without further purification.

[0612] LC-MS (ESI) m / z: 800 [M+H] +

[0613] Step 12: Synthesize T6

[0614] T6-12 (10 mg) was dissolved in a solution of dichloromethane (2 mL), and TFA (1 mL) was added. The mixture was stirred at room temperature overnight. After the reaction was completed, it was neutralized with a saturated aqueous solution of NaHCO3. Dichloromethane was added for extraction, and the organic layer was separated and concentrated. Purification by preparative HPLC gave T6 (3.86 mg, yield: 40.8%) as a brown solid.

[0615] LC-MS (ESI) m / z: 756 [M+H] +

[0616] Example 28: Synthesis of Compound T7

[0617]

[0618] Step 1: Synthesis of T7-1

[0619] 2-(tert-Butyldimethylsilyl)oxy)acetic acid (38 mg, 0.2 mmol, 10 eq) was dissolved in anhydrous dichloromethane (2 mL), and oxalyl chloride (2M dichloromethane solution, 0.05 mL, 0.1 mmol, 5 eq) and 2 drops of catalytic DMF were added at 0 ° C. After stirring at 0 ° C for 1 hour, the reaction mixture was added to an anhydrous DMF solution of T6-12 (16 mg, 0.02 mmol, 1.0 eq) and triethylamine (20 mg, 0.2 mmol, 10 eq) in another flask. Stir at room temperature for 2 hours. LC-MS showed that the reaction was complete, quenched with water, and extracted with ethyl acetate. The organic layers were combined, dried, filtered, and purified by silica gel flash column chromatography to obtain the target product (12 mg, yield: 60%) as a yellow liquid.

[0620] LC-MS (ESI) m / z: 972 [M+H] +

[0621] Step 2: Synthesize T7

[0622] T7-1 (9 mg, 0.0093 mmol, 1.0 eq) was dissolved in anhydrous tetrahydrofuran (1 mL), triethylamine trihydrofluoride (0.5 mL) was added and maintained for 1 hour. LC-MS showed that the reaction was complete and neutralized with saturated NaHCO3 aqueous solution. The mixture was extracted with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by preparative HPLC to obtain the target product T7 (2.29 mg, yield: 30%) as a white solid.

[0623] LC-MS (ESI) m / z: 814 [M+H] +

[0624] Example 29: Synthesis of T8

[0625]

[0626] Step 1: Synthesis of 2-amino-4-(2-aminoethyl)phenol

[0627] Under N2 protection, a mixture of T8-1 (50 mg, 0.16 mmol) and 10% wet Pd / C (30 mg) was added to a MeOH (3 mL) solution. The suspension was degassed under vacuum and replaced with H2 several times. Stir at room temperature for 4-5 hours under H2 protection, and the reaction was complete when detected by TLC. The suspension was filtered through diatomaceous earth and the filter cake was washed with methanol. The combined filtrate was concentrated and used directly in the next step.

[0628] LC-MS(ESI)m / z:[M+H] + 153.2

[0629] Step 2: Synthesis of T8

[0630] The intermediate Int-A (20 mg, 0.032 mmol), T8-2 (24 mg, 0.16 mmol) and HOAc (20 mg, 0.32 mmol) were added to anhydrous EtOH (1 mL) solution and stirred at room temperature overnight. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography to obtain the target product (10 mg, yield: 41.7%) as a white solid.

[0631] LC-MS(ESI)m / z:[M+H] + 756.4

[0632] 1H NMR(400MHz,DMSO-d6)δ8.78(s,1H),8.70(s,1H),6.44(s,1H),6.28(s,1H),6.21(d,J=8H z,2H),6.16(s,1H),5.06(d,J=12Hz,3H),4.44(s,2H),4.19(s,1H),4.04(s,1H),3.98-3.9 5(m,1H),3.63(s,3H),3.19-3.08(m,5H),2.79(s,2H),2.58(s,2H),2.56(s,1H),2.43(s,1 H),2.29(s,3H),2.21(s,3H),2.03(s,3H),1.99(s,3H),1.95(s,1H),1.69(d,J=16Hz,1H).

[0633] Example 30: Synthesis of Compound T9

[0634]

[0635] T8 (6 mg, 0.008 mmol), glycolic acid (1.8 mg, 0.024 mmol), DIEA (9.2 mg, 0.072 mmol) and HATU (4.5 mg, 0.012 mmol) were dissolved in anhydrous DMF (0.4 mL) and stirred at room temperature overnight. LC-MS showed the target product. The target product (3.0 mg, yield: 46.9%) was purified by HPLC as a white solid.

[0636] LC-MS(ESI)m / z:[M+H] + 814.22

[0637] Example 31: Synthesis of Compound T10

[0638]

[0639] Step 1: Synthesis of 4-(dimethoxymethyl)-1-methoxy-2-nitrobenzene

[0640] To a solution of 4-methoxy-3-nitrobenzaldehyde (2g, 11mmol) and trimethoxymethane (3.76g, 25.4mmol) in anhydrous methanol, p-toluenesulfonic acid monohydrate (21mg, 0.11mmol) was added, and the reaction solution was stirred at room temperature for 2 hours. The reaction was completed by thin layer chromatography. Saturated NaHCO3 aqueous solution was added for neutralization, and ethyl acetate was used for extraction. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel flash column chromatography to obtain the target compound (2.2g, yield 87.7%) as a colorless oil.

[0641] LC-MS (ESI) m / z: 197 [M-CH3O] +

[0642] 1 H NMR (400MHz, DMSO-d6) δ7.85(d,J=2.2Hz,1H),7.69–7.63(m,1H),7.40(d,J=8.7Hz,1H),5.44(s,1H),3.95(s,3H),3.27(s,6H).

[0643] Step 2: Synthesis of 2-methoxy-2-(4-methoxy-3-nitrophenyl)acetonitrile

[0644] SnCl2 (66.8 mg, 0.352 mmol) was added to a solution of 4-(dimethoxymethyl)-1-methoxy-2-nitrobenzene (1 g, 4.4 mmol) and TMSCN (437 mg, 4.4 mmol) in THF (10 mL). The reaction was completed by LC-MS monitoring. Saturated NaHCO3 aqueous solution was added to quench, extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel flash column chromatography to obtain the target product (690 mg, yield 70.5%).

[0645] LC-MS (ESI) m / z: 223 [M+H] +

[0646] Step 3: Synthesis of 5-(2-amino-1-methoxyethyl)-2-methoxyaniline

[0647] To a THF solution of 2-methoxy-2-(4-methoxy-3-nitrophenyl)acetonitrile (50 mg, 0.22 mmol), add Raney Ni and 0.1-0.5 eq NH3﹒H2O. Stir at room temperature for 2 hours. After the reaction is complete, filter and concentrate. Purify by preparative thin layer chromatography to obtain the target product (34 mg, yield: 77.3%).

[0648] LC-MS (ESI) m / z: 197 [M+H] +

[0649] Step 4: Synthesis of T10

[0650] The intermediate Int-A (4 mg, 0.0065 mmol), 5-(2-amino-1-methoxyethyl)-2-methoxyaniline (12.56 mg, 0.065 mmol), HOAc (3.9 mg, 0.065 mmol) and NaOAc (5.25 mg, 0.064 mmol) were dissolved in anhydrous ethanol (1 mL) and stirred at 25°C overnight. After the reaction was completed, the solvent was removed under reduced pressure to obtain a solid crude product. The crude product was purified by preparative HPLC to obtain the target product T10 (1.53 mg, yield: 29.7%) as a white solid.

[0651] LC-MS (ESI) m / z: 800 [M+H] +

[0652] Example 32: Synthesis of Compound T11

[0653]

[0654] Step 1: Synthesis of T11

[0655] P36 (20 mg, 0.025 mmol), 2-hydroxyacetic acid (3.8 mg, 0.05 mmol), EDCI (23 mg, 0.125 mmol), DMAP (1.5 mg, 0.0125 mmol) and HOAt (16 mg, 0.125 mmol) were dissolved in DMF (0.2 mL) and stirred at room temperature for 2 hours. LC-MS showed the target product, which was purified by preparative HPLC to obtain the target product (8.3 mg, yield: 38%).

[0656] LC-MS (ESI) m / z: 854.3 [M+H] +

[0657] 1 H NMR(400MHz,DMSO-d6)δ9.01(s,1H),8.74(s,1H),7.55(s,1H),6.43(s,2H),6.19-6.17(m,2H ),6.11(s,1H),5.09-5.05(m,1H),4.52(s,2H),4.23(s,1H),4.14-4.07(m,2H),3.93(s,2H), 3.65(s,3H),3.59(s,3H),3.32-3.30(m,3H),2.81-2.77(m,2H),2.32(s,3H),2.23-2.21(m,4 H),2.19-1.14(m,5H),2.00(s,3H),1.92-1.90(m,1H),0.85-0.81(m,2H),0.70-0.53(m,2H).

[0658] Example 33: Synthesis of Compound T12

[0659]

[0660] Step 1: Synthesis of 3-(bromomethyl)-5-nitrophenol

[0661] At 0°C, PPh3 (7.2 g, 27.5 mmol) and CBr4 (9.2 g, 27.5 mmol) were added to a solution of 3-(hydroxymethyl)-5-nitrophenol (3.1 g, 18.33 mmol) in THF (50 mL) and stirred at room temperature for 1 hour. LC-MS showed that the reaction was complete. After quenching with water, the mixture was extracted twice with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by silica gel flash column chromatography gave the target product (3.7 g, yield: 87.6%) as a yellow solid.

[0662] LC-MS (ESI) m / z: 231.7 [M+H] +

[0663] Step 2: Synthesis of 2-(3-hydroxy-5-nitrophenyl)acetonitrile

[0664] 3-(Bromomethyl)-5-nitrophenol (2.7 g, 11.64 mmol) was dissolved in anhydrous acetonitrile (20 mL), and TBAF (18.63 mL, 18.63 mmol) and TMSCN (2.1 g, 20 mmol) in anhydrous THF were added at room temperature and reacted for 4 hours. LC-MS showed that the reaction was complete. The solvent was removed by concentration, diluted with ethyl acetate, washed with water several times, the organic phase was dried over sodium sulfate, filtered, concentrated, and the residue was purified by silica gel flash column chromatography to obtain the target product (1.74 g, yield: 84.2%) as a yellow solid.

[0665] LC-MS (ESI) m / z: 176.8 [MH] -

[0666] Step 3: Synthesis of 3-(2-aminoethyl)-5-nitrophenol

[0667] Add BH3·THF (1M, 7 mL, 7 mmol) to a solution of 2-(3-hydroxy-5-nitrophenyl)acetonitrile (500 mg, 2.8 mmol) in anhydrous THF and reflux for 1 hour. LC-MS showed that the reaction was complete. Quench with methanol at 0°C and reflux for half an hour. Concentrate the reaction solution and use it directly in the next step.

[0668] LC-MS (ESI) m / z: 183.3 [M+H] +

[0669] Step 4: Synthesis of benzyl (3-hydroxy-5-nitrophenylethyl ester) carbamate

[0670] To a solution of the crude product 3-(2-aminoethyl)-5-nitrophenol (511 mg, 2.8 mmol) in anhydrous dichloromethane, Cbz-Cl (860 mg, 5.04 mmol) and NaHCO3 aqueous solution (283 mg, 3.36 mmol, 1.2 eq.) were added. Stir at room temperature for 1.5 hours. LC-MS showed that the reaction was complete. The organic phase was separated by washing the solution with water, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the target compound (840 mg, crude product) as a yellow liquid. The crude product was used directly in the next step without further purification.

[0671] LC-MS (ESI) m / z: 317.1 [M+H] +

[0672] Step 5: Synthesis of benzyl (3-(methoxymethoxy)-5-nitrophenylethyl ester) carbamate

[0673] Cs2CO3 (1.7 g, 5.27 mmol) and MOMBr (495 mg, 3.95 mmol) were added to a solution of benzyl (3-hydroxy-5-nitrophenylethyl) carbamate (840 mg, 2.63 mmol) in anhydrous acetonitrile (20 mL) and stirred at room temperature for 1 hour. LC-MS showed that the reaction was complete, and water was added to quench the reaction. The reaction solution was extracted with dichloromethane, and the organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel flash column chromatography to obtain the target product (160 mg, yield: 15.8%) as a yellow liquid.

[0674] LC-MS (ESI) m / z: 361.0 [M+H] +

[0675] 1 H NMR(400MHz,Chloroform-d)δ7.76(t,J=2.2Hz,1H),7.71(s,1H),7.39–7.29(m, 5H),7.18(s,1H),5.21(s,2H),5.10(s,2H),3.48(s,5H),2.90(t,J=7.0Hz,2H).

[0676] Step 6: Synthesis of 3-(2-aminoethyl)-5-(methoxymethoxy)aniline

[0677] Under N2 protection, 5% wet Pd / C (20 mg) and HOAc (53 mg, 0.88 mmol) were added to a solution of benzyl (3-(methoxymethoxy)-5-nitrophenylethyl) carbamate (160 mg, 0.44 mmol) in THF (10 mL). The suspension was degassed under vacuum and replaced with H2 several times. Stirred at room temperature overnight under H2 protection, and the reaction was complete when detected by TLC. The suspension was filtered through diatomaceous earth and the filter cake was washed with methanol. The combined filtrate was concentrated and used directly in the next step. LC-MS (ESI) m / z: 196.9 [M+H] +

[0678] Steps 7 and 8: Synthesis of T12

[0679] The intermediate Int-A (10 mg, 0.016 mmol) and 3-(2-aminoethyl)-5-(methoxymethoxy)aniline (10.6 mg, 0.041 mmol) were dissolved in EtOH (10 mL) and stirred at 50 ° C for 3 hours. LC-MS showed that the reaction was complete. After concentration, the solvent was removed and TFA / DCM = 1:1 (v / v) (1 mL) was added. After the reaction was completed, it was concentrated and purified by preparative high performance liquid chromatography to obtain the target product (0.46 mg, yield: 3.78%) as a white solid.

[0680] LC-MS (ESI) m / z: 756.4 [M+H] +

[0681] Example 34: Synthesis of Compound T13

[0682]

[0683] Step 1: Synthesis of T13-1

[0684] To a solution of crude product T12-8 (20 mg, 0.025 mmol) in DMF (2 mL), 2-((tert-butyldimethylsilyl)oxy)acetic acid (47.6 mg, 0.25 mmol), HATU (95 mg, 0.25 mmol) and DIEA (32.3 mg, 0.25 mmol) were added. LC-MS showed that the reaction was complete, and the target product (4.2 mg, yield: 13.4%) was obtained as a white solid after purification by preparative HPLC.

[0685] LC-MS (ESI) m / z: 972.6 [M+H] +

[0686] Step 2: Synthesis of T13

[0687] To a solution of T13-1 (3 mg, 0.003 mmol) in THF (0.7 mL) was added 3HF·3Et3N (0.7 mL), stirred at room temperature for 1 hour, and LC-MS showed that the reaction was complete. Saturated aqueous NaHCO3 solution was added to the reaction solution, then extracted with dichloromethane, the organic layer was separated, dried, filtered, concentrated, and dissolved in DCM / TFA = 3 / 1. After the reaction was completed, the pH was adjusted to 7-8 with saturated aqueous NaHCO3 solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by preparative high performance liquid chromatography to obtain the target product (0.97 mg, yield: 38.6%) as a white solid.

[0688] LC-MS (ESI) m / z: 814.4 [M+H] +

[0689] 1 H NMR(400MHz,Chloroform-d)δ8.00(s,1H),6.86(s,1H),6.65(s,1H),6.57(s,1H),6.06(d,J=2 4.5Hz,2H),5.85(s,1H),4.86(s,1H),4.50(s,2H),4.34–4.24(m,2H),4.14(s,1H),4.06(s,2H) ,3.80(s,3H),3.52(s,1H),3.42(d,J=8.1Hz,1H),2.97–2.88(m,2H),2.83–2.68(m,2H),2.56(d ,J=16.6Hz,2H),2.33(s,3H),2.30(s,3H),2.25(s,1H),2.22(s,4H),2.06(s,3H),2.04(s,1H).

[0690] Example 35: Synthesis of Compound T14

[0691]

[0692] Step 1: Synthesis of benzyl (4-hydroxy-3-methoxy-5-nitrophenylethyl) carbamate

[0693] At 0°C, 65% nitric acid (0.6 mL) was slowly added to a solution of benzyl (4-hydroxy-3-methoxyphenethyl) carbamate (300 mg, 0.99 mmol) in dichloromethane (1.5 mL) and stirred for 20 minutes at the same temperature. LC-MS showed that the reaction was complete, and the reaction solution was poured into ice water to quench. Extracted with dichloromethane, the organic layers were combined, dried, filtered, concentrated, and purified by silica gel flash column chromatography to obtain the target compound (80 mg, yield: 23%) as a yellow solid.

[0694] LC-MS(ESI)m / z:[M+H] + 347.0

[0695] 1 H NMR (400MHz, DMSO-d6) δ10.18 (s, 1H), 7.39–7.24 (m, 7H), 7.15 (d, J = 2.0Hz, 1H), 5.00 (s, 2H), 3.84 (s, 3H), 3.26 (q, J = 6.7Hz, 2H), 2.70 (t, J = 7.0Hz, 2H).

[0696] Step 2: Synthesis of benzyl (3,4-dimethoxy-5-nitrophenylethyl ester) carbamate

[0697] A solution of benzyl (4-hydroxy-3-methoxy-5-nitrophenylethyl) carbamate (80 mg, 0.2 mmol), Cs2CO3 (197.45 mg, 0.606 mmol) and MeI (34.4 mg, 0.242 mmol) in acetonitrile (5 mL) was added to a sealed tube and stirred at 80°C for 1 hour. LC-MS showed that the reaction was complete, the reaction mixture was filtered and the filtrate was concentrated, and the target compound (55 mg, yield: 60%) was purified by column chromatography as a yellow solid.

[0698] LC-MS(ESI)m / z:[M+H] + 361.1

[0699] Step 3: Synthesis of 5-(2-aminoethyl)-2,3-dimethoxyaniline

[0700] Under N2 protection, HOAc (18.31 mg, 0.305 mmol) and 10% wet Pd / C (20 mg, 20% w / w) were added to a solution of benzyl (3,4-dimethoxy-5-nitrophenylethyl) carbamate (55 mg, 0.152 mmol) in TFE (10 mL). The suspension was degassed under vacuum and replaced with H2 several times. Stirred at room temperature overnight under H2 protection, and the reaction was complete by TLC detection. The suspension was filtered through diatomaceous earth and the filter cake was washed with methanol. The combined filtrate was concentrated and used directly in the next step. LC-MS (ESI) m / z: [M+H] + 197.4

[0701] Step 4: Synthesis of T14

[0702] The intermediate Int-A (10 mg, 0.016 mmol) and 5-(2-aminoethyl)-2,3-dimethoxyaniline (18.9 mg, 0.096 mmol) were dissolved in 2,2,2-trifluoroethanol (2 mL). Stirred at 50°C overnight. LC-MS showed that the reaction was complete. Purification by preparative HPLC gave the target product (6.72 mg, yield: 52.9%) as a white solid.

[0703] LC-MS(ESI)m / z:[M+H] + 800.5

[0704] 1 H NMR(400MHz,Chloroform-d)δ6.48(s,1H),6.02(s,1H),5.98(d,J=1.4Hz,1H),5.92(d,J=1.4Hz,1H),5.68(s,1 H),4.62–4.44(m,2H),4.24–4.18(m,1H),4.18–4.15(m,1H),4.12–4.05(m,1H),4.04(d,J=5.2Hz,1H),3.72(s, 3H),3.55(d,J=1.1Hz,3H),3.49–3.43(m,1H),3.29(d,J=0.9Hz,3H),3.06(d,J=15.0Hz,1H),2.91–2.75(m,2H) ,2.59–2.49(m,1H),2.49–2.41(m,2H),2.24(s,3H),2.22(s,3H),2.19–2.14(m,1H),2.13(s,3H),1.98(s,3H).

[0705] Example 36: Synthesis of Compound T15

[0706]

[0707] Steps 1 and 2: Synthesis of T15

[0708] To a solution of T14 (10 mg, 0.0125 mmol) in DMF (1 mL) were added 2-(tert-butyldimethylsilyl)oxy)acetic acid (23.9 mg, 0.125 mmol), HATU (47 mg, 0.125 mmol) and DIEA (16.1 mg, 0.125 mmol). LC-MS showed that the reaction was complete, and the mixture was diluted with ethyl acetate and washed with water. The organic phase was separated, dried, concentrated and dissolved in anhydrous THF (1 mL). Then, 3HF·3Et3N (0.9 mL) was added and stirred at room temperature for 1 hour. LC-MS showed that the reaction was complete, and saturated NaHCO3 aqueous solution and dichloromethane were added to the mixture, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated and purified by preparative HPLC to obtain the target product (1.6 mg, yield:

[0709] 14.9%) as a white solid.

[0710] LC-MS(ESI)m / z:[M+H] + 858.5

[0711] Example 37: Synthesis of Compound T16

[0712]

[0713] Step 1: Synthesis of benzyl (3-amino-4-methoxyphenethyl) carbamate

[0714] Dissolve benzyl (4-methoxy-3-nitrophenylethyl) carbamate (500 mg, 1.51 mmol) in EtOH / H2O=4 / 1 (10 mL) solution, add iron powder (339 mg, 6.05 mg) and NH4Cl (320 mg, 6.05 mmol) and reflux for 3 hours. LC-MS shows that the reaction is complete and filtered. The filtrate is washed with water and extracted with ethyl acetate. The organic phases are combined, dried over anhydrous sodium sulfate, filtered, concentrated and used directly in the next step. LC-MS (ESI) m / z: [M+H] + 301.2

[0715] Step 2: Synthesis of benzyl (4-methoxy-3-(methylamino)phenethyl) carbamate

[0716] The crude product benzyl (3-amino-4-hydroxyphenylethyl) carbamate (200 mg, 0.665 mmol) was dissolved in methanol (5 mL), and then paraformaldehyde (79.8 mg, 2.66 mmol) was added and stirred at room temperature for 1 hour. NaBH3CN (209 mg, 3.32 mmol) was added at 0°C and stirred for 2 hours. LC-MS showed that the reaction was complete, and the reaction solution was diluted with ethyl acetate and water. The organic layer was separated and concentrated, and purified by silica gel flash column chromatography to obtain the target product (50 mg, yield: 22.7%).

[0717] LC-MS(ESI)m / z:[M+H] + 315.1

[0718] 1 H NMR(400MHz,Chloroform-d)δ7.38(s,5H),6.71(d,J=8.0Hz,1H),6.50(d,J=8.0Hz,1H),6.43 (s,1H),5.13(s,2H),3.86(s,3H),3.49(d,J=6.9Hz,2H),2.87(s,3H),2.77(t,J=7.0Hz,2H).

[0719] Steps 3 and 4: Synthesis of 5-(2-aminoethyl)-2,3-dimethoxyaniline

[0720] Under N2 protection, HOAc (17.16 mg, 0.286 mmol) and 10% wet Pd / C (20 mg, 20% w / w) were added to a solution of benzyl (4-methoxy-3-(methylamino)phenethyl)carbamate (45 mg, 0.143 mmol) in trifluoroethanol (10 mL). The suspension was degassed under vacuum and replaced with H2 several times. Stirred at room temperature overnight under H2 protection, and the reaction was complete by TLC. Filtered, intermediate Int-A (20 mg, 0.0322 mmol) was added to the filtrate, stirred at room temperature for 16 hours, and LC-MS showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain the target compound (11 mg, yield: 43.6%) as a white solid.

[0721] LC-MS(ESI)m / z:[M+H] + 784.3

[0722] 1H NMR(400MHz,Chloroform-d)δ6.63(s,1H),6.36(s,1H),6.14(s,1H),6.08(s,1H),6.01(s,1H),5.76(s, 1H),5.05(d,J=11.5Hz,1H),4.59(s,1H),4.36(s,1H),4.31(d,J=4.8Hz,1H),4.22(s,1H),4.13(d,J=6.4 Hz,1H),3.83(s,3H),3.61(s,3H),3.55(d,J=4.9Hz,1H),3.45(s,1H),3.18(s,1H),2.98(d,J=9.0Hz,2H ),2.78(s,3H),2.67(s,1H),2.51(d,J=16.4Hz,1H),2.36(s,5H),2.30(s,3H),2.23(s,4H),2.08(s,3H).

[0723] Example 38: Synthesis of Compound T17

[0724]

[0725] Step 1: Synthesis of T17-1

[0726] T16 (11 mg, 0.014 mmol), Mukaiyama reagent (7.12 mg, 0.0278 mmol) and 2-((tert-butyldimethylsilyl)oxy)acetic acid (3.99 mg, 0.0209 mmol) were dissolved in anhydrous dichloromethane (3 mL) and Et3N (7.0 mg, 0.0693 mmol) was added. Stir at room temperature for 5 hours. LC-MS showed that the reaction was complete, concentrated, and purified by silica gel flash column chromatography to obtain the target product (6 mg, yield: 44.7%) as a white solid.

[0727] LC-MS(ESI)m / z:[M+H] + 956.7

[0728] Step 2: Synthesis of T17

[0729] To a solution of T17-1 (6 mg, 0.00628 mmol) in anhydrous THF was added 3HF·3Et3N (1 mL) and stirred at room temperature for 30 minutes. LC-MS showed that the reaction was complete. The mixture was quenched with saturated aqueous NaHCO3 and extracted with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by silica gel flash column chromatography gave the desired product (1.55 mg, yield: 29.3%) as a white solid.

[0730] LC-MS(ESI)m / z:[M+H] + 842.4

[0731] Example 39: Synthesis of Compound T18

[0732]

[0733] Step 1: Synthesis of 2-(2-methoxyphenyl)-2-methylpropionitrile

[0734] 2-Fluoroanisole (5 g, 39.66 mmol), isobutyronitrile (10.9 g, 158.67 mmol) and KHMDS (1 M THF solution, 59.49 mL) were dissolved in dry toluene (50 mL) and stirred at 60 ° C for 16 hours. After the reaction was completed, it was quenched with saturated NH4Cl aqueous solution, extracted with ethyl acetate, and the organic layer was washed with an appropriate amount of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel flash column chromatography to obtain the target product (6.5 g, yield: 93.5%) as a colorless liquid.

[0735] LC-MS (ESI) m / z: 176.3 [M+H] +

[0736] Step 2: Synthesis of 2-(2-methoxy-3-nitrophenyl)-2-methylpropionitrile

[0737] 2-(2-methoxyphenyl)-2-methylpropionitrile (2 g, 11.4 mmol) and KNO3 (1.5 g, 14.85 mmol) were dissolved in dichloromethane (10 mL). Trifluoroacetic anhydride (10 mL) was added dropwise and stirred at room temperature for 1 hour. After the reaction was completed, it was diluted with 20 mL of water and 20 mL of dichloromethane. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The target product (555 mg, yield: 22%) was purified by silica gel flash column chromatography as a yellow solid.

[0738] LC-MS (ESI) m / z: 221.5 [M+H] +

[0739] 1 H NMR (400MHz, DMSO-d6) δ7.95 (dd, J=8.2Hz, 1.6Hz, 1H), 7.73 (dd, J=8.0, 1.6Hz, 1H), 7.35 (t, J=8.0Hz, 1H), 1.74 (s, 6H).

[0740] Step 3: Synthesis of 3-(1-amino-2-methylpropan-2-yl)-2-methoxyaniline

[0741] 2-(2-methoxy-3-nitrophenyl)-2-methylpropionitrile (10 mg) was dissolved in EtOH (1 mL) and 1 drop of NH3﹒H2O was added. A small amount of Raney Ni was added and stirred for 16 hours under H2 protection. After the reaction was completed, the filtrate was filtered and concentrated and used directly in the next step.

[0742] LC-MS (ESI) m / z: 195.1 [M+H] +

[0743] Step 4: Synthesize T18

[0744] Int-A (10 mg, 0.008 mmol), 3-(1-amino-2-methylpropan-2-yl)-2-methoxyaniline (8.8 mg, 0.045 mmol) and AcOH (2.7 mg, 0.045 mmol) were dissolved in anhydrous EtOH (1 mL) and stirred overnight at 25 °C. After the reaction was completed, the solvent was removed by distillation under reduced pressure. The crude product was purified by preparative HPLC to obtain the target product (2.39 mg, yield: 18.6%) as a white solid. LC-MS (ESI) m / z: 798.7 [M+H] +

[0745] 1 H NMR(400MHz,Chloroform-d)δ6.66(d,J=8.5Hz,1H),6.61(s,1H),6.44(d,J=8.5Hz,1H),6.15(d,J=1.4Hz,1H),6.0 0(d,J=1.4Hz,1H),5.79(s,1H),5.00(d,J=11.5Hz,1H),4.49(s,1H),4.25(d,J=3.9Hz,2H),4.16(d,J=2.7Hz,1H), 4.05(dd,J=11.5,2.0Hz,1H),3.78(s,3H),3.69(s,3H),3.46–3.37(m,3H),3.15(d,J=11.8Hz,1H),3.05–2.88(m,2 H),2.53(d,J=14.8Hz,1H),2.34(s,4H),2.28(s,3H),2.23(d,J=7.9Hz,1H),2.21(s,3H),2.05(s,3H),1.25(s,6H).

[0746] Synthesis and characterization of intermediates

[0747] Example 40: Synthesis of Compound L1

[0748]

[0749] At 0°C, (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxo-5,8,11,14-tetraazahexadecanoic acid (500 mg, crude product) and N,N-diisopropylethylamine (609 mg, 4.72 mmol) were added to a DMF (4.00 mL) solution of 2,5-dioxopyrrolidin-1-yl-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15,18-hexaoxobenzofuran-21-oate (626 mg, 1.18 mmol). After the addition was complete, the reaction solution was stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete. The mixture was concentrated to give a crude product, which was purified by preparative high performance liquid chromatography (0-60% acetonitrile in water) to give (S)-10-benzyl-38-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18-pentaoxo-3,21,24,27,30,33,36-heptaoxo-5,8,11,14,17-pentaazaoctadecanoic acid (90 mg, yield: 9%) as a yellow oil.

[0750] LC-MS (ESI) m / z: 861.4 [M+Na] + .

[0751] 1 H NMR (400MHz, DMSO-d6): δ8.51-8.57(m,1H),8.26-8.29(m,1H),8.12-8.15(m,1H),8.08-8.10(m,1H),7.96-7.99(m,1H),7.15-7.27(m,6H),7. 01(s,2H),4.61(d,J=6.8Hz,2H),4.47-4.52(m,1H),3.98(s,2H),3.49- 3.73(m,32H),3.03-3.08(m,1H),2.77-2.83(m,1H),2.37-2.41(m,2H).

[0752] Example 41: Synthesis of Compound L2

[0753]

[0754] Steps 1 and 2: Synthesis of L2-3

[0755] Under nitrogen protection at room temperature, L2-1 (4.2 g, 5.7 mmol, 1.0 eq) was dissolved in 15 mL of anhydrous DMF, and DBU (608 mg, 3.99 mmol, 0.7 eq) was added to react at room temperature for 0.5 hours, and TLC was used for detection (DCM / MeOH=20 / 1). If there was any unreacted raw material, 0.2 eq DBU was added, and the reaction was completed by TLC detection to generate L2-2, and 0.7 eq-0.9 eq 4-methylbenzenesulfonic acid pyridine was added. Then 1.0 eq HOBt, 1.0 eq (S)-42-(((benzyloxy)carbonyl)amino)-39-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38-azatetraconane-43-oleic acid, and 0.9 eq EDCI were added in sequence, and the reaction was carried out at room temperature for 1-2 hours. The reaction was completed by LCMS detection. 500 mL of water / 500 mL of DCM were added to the reaction solution, the layers were separated by extraction, the organic phase was concentrated, DMF was added to dissolve, and the product was purified by reverse phase column chromatography (CH3CN / H2O=10%-100%) to obtain product L2-3 (4 g, yield 53%, two steps).

[0756] LC-MS (ESI) m / z: 681.6 [M / 2 + Na] +

[0757] Step 3: Synthesis of L2-4

[0758] Compound L2-3 (4 g, 3 mmol, 1 eq) was dissolved in DMF, a small amount of palladium carbon was added, and the mixture was replaced with a hydrogen balloon. The mixture was reacted at 70°C for 3 hours. When the reaction was completed by LCMS, the palladium carbon was removed by filtration, and the filtrate was used directly in the next step.

[0759] LC-MS (ESI) m / z: 1094.5 [M+H] +

[0760] Step 4: Synthesis of L2

[0761] To the DMF solution of compound L2-4, add 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (0.74 g, 2.4 mmol, 0.8 eq) and DIEA (1.17 g, 9 mmol, 3 eq), stir at room temperature for 1 hour, and purify by reverse phase column chromatography (CH3CN / H2O=10%-50%) to obtain the product L2 (1.56 g, yield 40%, two steps).

[0762] LC-MS (ESI) m / z: 666.2 [M / 2 + Na] +

[0763] Example 42: Synthesis of Compound L3

[0764]

[0765] Step 1: Synthesis of benzyl 1-((2-aminoacetylamino)methoxy)cyclopropane-1-carboxylate

[0766] Keeping at 0-5°C, add t-BuOK (300 mg, 2.7 mmol) to a THF (15 mL) solution of L3-1 (500 mg, 1.4 mmol) and 1-hydroxycyclopropane-1-carboxylic acid benzyl ester (500 mg, 2.6 mmol). Then raise the temperature of the reaction solution to room temperature and stir for 10 minutes. Add ice water to the reaction solution, extract with ethyl acetate 3 times, combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate and use directly in the next step.

[0767] LC-MS (ESI) m / z: 279 [M+H] +

[0768] Step 2: Synthesis of (S)-1-((11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxy-2-oxy-4,7,10,13,16-pentaazaheptadecan-17-yl)oxy)cyclopropane-1-carboxylic acid benzyl ester

[0769] A mixture of L3-2 (920 mg, crude), (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine (701 mg, 1.4 mmol), EDCI (382 mg, 2.0 mmol) and HOAT (270 mg, 2.0 mmol) was dissolved in DMF (15 mL) and stirred at room temperature overnight. LC-MS showed the target product, which was used directly in the next step without purification.

[0770] LC-MS (ESI) m / z: 762 [M+H] +

[0771] Step 3: Synthesis of benzyl (S)-1-((13-amino-7-benzyl-3,6,9,12-tetraoxy-2,5,8,11-tetraazatridecyl)oxy)cyclopropane-1-carboxylate

[0772] Triethylamine (0.3 mL) was added to the reaction solution of L3-3 and stirred overnight. LC-MS showed the target product, and the reaction solution was purified by preparative HPLC to obtain the target product (123 mg, three-step yield: 12%) as a white solid.

[0773] LC-MS (ESI) m / z: 540 [M+H] +

[0774] Step 4: Synthesis of L3-5

[0775] A solution of L3-4 (100 mg, 1.85 mmol), (S)-42-(((benzyloxy)carbonyl)amino)-39-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38-azatetraconane-43-oleic acid (180 mg, 2.25 mmol), EDCI (95 mg, 5.55 mmol) and HOBt (65 mg, 5.55 mmol) in DMF (0.6 mL) was stirred at room temperature overnight. LC-MS showed the target product. The target product was purified by preparative HPLC to obtain the target product (60 mg, yield: 40.2%) as a white solid.

[0776] LC-MS (ESI) m / z: 1344.5 [M+H] +

[0777] Step 5: Synthesis of L3-6

[0778] Under N2 protection, L3-5 (60 mg, 0.045 mmol) and 10% wet Pd / C (10 mg) were added to a DMF (2 mL) solution. The suspension was degassed under vacuum and replaced with H2 several times. Stir at room temperature under H2 protection for 4-5 hours. The reaction was complete when detected by TLC and used directly in the next step.

[0779] Step 6: Synthesis of L3

[0780] 6-(Maleimido)hexanoic acid succinimidyl ester (20 mg, 0.065 mmol) and DIEA (0.02 mL, 0.11 mmol) were added to the DMF solution of L3-6 and stirred at room temperature overnight. The target compound (18 mg, two-step yield: 30%) was obtained by purification by preparative HPLC.

[0781] LC-MS (ESI) m / z: 1313.3 [M+H] +

[0782] Example 43: Synthesis of Compound L4

[0783]

[0784] Step 1: Synthesis of (S)-benzyl 2-(2-(2-aminoacetamido)acetamido)-3-phenylpropionamido)acetate

[0785] At room temperature, diethylamine (508 mg, 6.95 mmol) was added to a solution of (S)-benzyl 11-benzyl-1-(9H-fluorene-9-yl)-3,6,9,12-tetraoxo-2-oxo-4,7,10,13-tetraazapentadecan-15-acid (3.00 g, 4.63 mmol) in DMF (20.0 mL). The reaction solution was stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete. Filtered, the filtrate was concentrated in vacuo to obtain (S)-benzyl 2-(2-(2-aminoacetamido)acetamido)-3-phenylpropionamido)acetate (2.50 g, crude product) as a yellow oil, which was used directly in the next step without purification.

[0786] LC-MS (ESI) m / z: 426.9 [M+H] +

[0787] Step 2: Synthesis of (5S, 14S)-1-benzyl 17-tert-butyl 5-benzyl-14-((benzyloxy)carbonyl)amino)-4,7,10,13-tetraoxo-3,6,9,12-tetraazaheptadecan-1,17-dioate

[0788] To a solution of (S)benzyl 2-(2-(2-aminoacetamido)acetamido)-3-phenylpropionamido)acetate (2.50 g, crude) and HATU (4.46 g, 11.7 mmol) in DMF (30.0 mL) at 0°C were added (S)-2-((benzyloxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (2.96 g, 8.78 mmol) and DIEA (3.03 g, 23.5 mmol). After stirring at room temperature for 2 hours, the mixture was concentrated and the crude product was purified by silica gel flash column chromatography (MeOH / DCM 0-10%) to give (5S,14S)-1-benzyl 17-tert-butyl 5-benzyl-14-((benzyloxy)carbonyl)amino)-4,7,10,13-tetraoxo-3,6,9,12-tetraazaheptadecan-1,17-dioic acid ester (1.20 g, yield: 27%) as a yellow solid.

[0789] LC-MS (ESI) m / z: 745.7 [M+H] +

[0790] Step 3: Synthesis of (7S, 16S)-7-benzyl-16-((benzyloxy)carbonyl)amino)-3,6,9,12,15-pentaoxo-1-phenyl-2-oxo-5,8,11,14-tetraazanonadecan-19-oic acid

[0791] At room temperature, TFA (5.00 mL) was added to a solution of (5S, 14S)-1-benzyl 17-tert-butyl-5-benzyl-14-((benzyloxy)carbonyl)amino)-4,7,10,13-tetraoxo-3,6,9,12-tetraazaheptadecane-1,17-dioic acid ester (1.20 g, 1.61 mmol) in dichloromethane (5.00 mL). The reaction mixture was stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete. The reaction mixture was concentrated to give (7S, 16S)-7-benzyl-16-((benzyloxy)carbonyl)amino)-3,6,9,12,15-pentaoxo-1-phenyl-2-oxo-5,8,11,14-tetraazanonadecan-19-acid (1.20 g, crude product) as a yellow oil, which was used directly in the next step without purification.

[0792] LC-MS (ESI) m / z: 690.4 [M+H] +

[0793] Step 4: Synthesis of (42S,51S)-benzyl 51-benzyl-42-((benzyloxy)carbonyl)amino)-39,43,46,49,52-pentaoxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38,44,47,50,53-pentaazapentaenoate

[0794] Aminododecanol monomethyl ether (778 mg, 1.39 mmol) and DIEA (898 mg, 6.96 mmol) were added to a solution of (7S,16S)-7-benzyl 16-(((benzyloxy)carbonyl)amino)-3,6,9,12,15-pentaoxo-1-phenyl-2-oxo-5,8,11,14-tetraazanonadecan-19-oic acid (1.20 g, crude) and HATU (1.32 g, 3.47 mmol) in DMF (20 mL) at 0°C. The reaction solution was stirred at room temperature for 2 hours and concentrated to give a crude product, which was purified by reverse phase column chromatography (CH3CN / H2O 0-65%) to give (42S, 51S)-benzyl 51-benzyl-42-((benzyloxy)carbonyl)amino)-39,43,46,49,52-pentaoxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38,44,47,50,53-pentaazapentacarbonate ester (645 mg, two-step yield: 30%) as a white solid.

[0795] LC-MS (ESI) m / z: 616.3 [M+H] +

[0796] Step 5: Synthesis of (42S,51S)-benzyl 51-benzyl-42-((benzyloxy)carbonyl)amino)-39,43,46,49,52-pentaoxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38,44,47,50,53-pentaazapentaenoate

[0797] Under nitrogen protection, 10% wet Pd / C was added to a solution of benzyl ((S)-42-((benzyloxy)carbonyl)amino)-39-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecyl-38-azatricarbonylcyclobutane 43-acyl)glycylglycyl-L-phenylalanine glycine (645 mg, 0.52 mmol) in MeOH (10.0 mL), vacuumed and replaced with hydrogen three times, and the mixture was stirred at room temperature for 12 hours. LC-MS showed that the reaction was complete. Pd / C was removed by filtration and the filtrate was concentrated to give (42S,51S)-42-amino-51-benzyl-39,43,46,49,52-pentaoxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38,44,47,50,53-pentaazapentacarbonyl-55-oic acid (320 mg, crude product) as a yellow oil which was used in the next step without purification.

[0798] LC-MS (ESI) m / z: 1006.9 [M+H] +

[0799] Step 6: Synthesis of (42S, 51S)-51-benzyl-42-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-39,43,46,49,52-pentaoxo-25,8,11,14,17,20,23,26,29,35-dodeca-38,44,47,50,53-pentaazapentaenoic acid

[0800] To a solution of (42S,51S)-42-amino-51-benzyl-39,43,46,49,52-pentaoxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38,44,47,50,53-pentaazapentaenoic acid (320 mg, crude) and succinimidyl 6-(maleimido)hexanoate (147 mg, 0.48 mmol) in DMF (4.00 mL) was added DIEA (164 mg, 1.27 mmol) at 0°C. After the reaction solution was stirred at room temperature for 2 hours, the mixture was concentrated to give a crude product, which was purified by preparative high performance liquid chromatography to give (42S, 51S)-51-benzyl-42-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-39,43,46,49,52-pentaoxo-25,8,11,14,17,20,23,26,26,29,32,32,35-dodec-38,44,47,50,50-53-pentaazapentaenoic acid (100 mg, two-step yield: 16%) as a yellow oil.

[0801] LC-MS (ESI) m / z: 1201.6 [M+H] +

[0802] 1 H NMR (400MHz, DMSO-d6): δ8.36-8.38(m,1H),8.10-8.13(m,2H),8.01-8.03(m,2H),7.85-7 .87(m,1H),7.19-7.33(m,5H),7.01(s,2H),4.52-4.58(m,1H),4.19-4.23(m,1H),3.59-3 .79(m,44H),3.36-3.46(m,9H),3.25(s,3H),3.01-3.08(m,1H),2.75-2.81(m,1H),2.10- 2.12(m,5H),1.86-1.89(m,2H),1.70-1.75(m,1H),1.47-1.51(m,5H),1.19-1.22(m,2H).

[0803] Example 44: Synthesis of Compound L5

[0804]

[0805] Step 1: Synthesis of L5-2

[0806] L2-4 (2.0 g, 1.83 mmol), 2,5-dioxopyrrolidin-1-yl 6-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)hexanoate (846 mg, 1.9 mmol) and DIEA (0.63 mL, 3.7 mmol) were dissolved in DMF (10 mL) and stirred at room temperature overnight. LC-MS showed the target product, which was used directly in the next reaction without purification.

[0807] LC-MS (ESI) m / z: 1429.7 [M+H] +

[0808] Step 2: Synthesis of L5-3

[0809] Triethylamine (3 mL) was added to the above solution, and the reaction solution was stirred at room temperature overnight. LC-MS showed the target product, which was purified by preparative HPLC (2.0 g, yield: 87%) as an oil.

[0810] LC-MS (ESI) m / z: 1428.4 [M+H] +

[0811] Step 3: Synthesis of L5

[0812] L5-3 (1.5 g, 1.3 mmol), 2,5-dioxopyrrolidin-1-yl (S)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dihydro-2,5-dihydro-1H-pyrrol-1-yl) propanoate (104 mg, 1.3 mmol) and DIEA (0.45 mL, 2.6 mmol) were dissolved in DMF (2 mL) and stirred at room temperature overnight. LC-MS showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain the target compound (1.07 mg, yield: 58.6%) as a white solid.

[0813] LC-MS (ESI) m / z: 1473.3 [M+H] +

[0814] Example 45: Synthesis of Compound L6

[0815]

[0816] Step 1: Synthesis of L6-2

[0817] At room temperature, L6-1 (600 mg, 0.73 mmol), NHS (84 mg, 0.73 mmol) and EDCI (140 mg, 0.73 mmol) were dissolved in DMF (2 mL) solution and stirred for 3 hours. LC-MS showed that the reaction was complete, and valyl-alanine (135 mg, 0.72 mmol) and DIEA (0.249 mL, 1.45 mmol) were added to the solution and continued to stir overnight. LC-MS showed that the reaction was complete, and the reaction solution was purified by preparative high performance liquid chromatography to obtain the target product (394 mg, yield: 54%) as a white solid.

[0818] LC-MS (ESI) m / z: 993 [M+H] +

[0819] Step 2: Synthesis of L6-3

[0820] Under N2 protection, L6-3 (300 mg, 0.29 mmol) and 5% wet Pd / C (30 mg) were added to a DMF (2 mL) solution. The suspension was degassed under vacuum and replaced with H2 several times. Stirred at 45 °C overnight under H2 protection. The reaction was complete when LC-MS detected. The reaction solution was filtered and directly proceeded to the next step.

[0821] LC-MS (ESI) m / z: 859.4 [M+H] +

[0822] Step 3: Synthesis of L6-4

[0823] L6-3 (258 mg, 0.3 mmol), 2,5-dioxopyrrolidin-1-yl-6-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)hexanoate (135 mg, 0.3 mmol) and DIEA (0.105 mL, 0.6 mmol) were added to a solution of DMF (2 mL) and stirred at room temperature for 3 hours. LC-MS showed that the reaction was complete, and the reaction solution was directly used in the next step.

[0824] LC-MS (ESI) m / z: 598.0 [M+H] +

[0825] Step 4: Synthesis of L6-5

[0826] Triethylamine (0.5 mL) was added to the reaction solution of L6-4, and stirring was continued overnight. LC-MS showed that the reaction was completed. The reaction solution was purified by preparative HPLC to obtain the target product (112 mg, 3-step yield: 33%) as an oil.

[0827] LC-MS (ESI) m / z: 972 [M+H] +

[0828] Step 5: Synthesis of L6

[0829] (S)-3-((tert-butyloxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid (16 mg, 0.056 mmol), EDCI (10.6 mg, 0.056 mmol) and NHS (6.5 mg, 0.056 mmol) were added to DMF (0.5 mL) and stirred at room temperature for 2 hours, then L6-5 (50 mg, 0.051 mmol) and DIEA (0.02 mL, 0.12 mmol) were added to the reaction solution and stirred at room temperature for 3 hours. LC-MS showed that the reaction was complete, and the target product (45 mg, yield: 71%) was purified by preparative HPLC as an oil.

[0830] LC-MS (ESI) m / z: 1238.6 [M+H] +

[0831] Example 46: Synthesis of Compound L7

[0832]

[0833] At room temperature, L6-3 (60 mg, 0.09 mmol), 6-(maleimido)hexanoic acid succinimidyl ester (23 mg, 0.07 mmol) and DIEA (0.034 mL, 0.2 mmol) were added to a DMF (2 mL) solution and stirred overnight. LC-MS showed that the reaction was complete, and the reaction solution was purified by preparative HPLC to obtain the target compound (24 mg, yield: 33%) as a white solid.

[0834] LC-MS(ESI)m / z:[M / 2+H] + 902.8

[0835] Synthesis and characterization of linker-drug conjugates

[0836] Example 47: Synthesis of Compound LP1

[0837]

[0838] P13 (30 mg, 0.038 mmol, 1 eq.), L1 (96.06 mg, 0.114 mmol, 3 eq.), HOAt (25.86 mg, 0.19 mmol, 5 eq.) and DMAP (2.32 mg, 0.019 mmol, 0.5 eq.) were dissolved in 1.5 mL of anhydrous DMF, EDCI (19.66 mg, 0.19 mmol, 5 eq.) was added, stirred at room temperature for 2 hours, and LC-MS showed that the reaction was complete. The target product (34.58 mg, yield: 56%) was purified by preparative HPLC as a white solid.

[0839] LC-MS (ESI) m / z: 803.4 [M / 2+H] +

[0840] 1 H NMR (400MHz, DMSO-d6) δ8.76(s,1H),8.71(s,1H),8.63(t,J=6.7Hz,1H),8.28(t,J=5.7Hz,1 H),8.14(t,J=5.7Hz,1H),8.09(d,J=7.9Hz,1H),8.02–7.94(m,2H),7.28–7.14(m,6H),7.00 (s,2H),6.42(d,J=10.3Hz,2H),6.13(s,2H),5.03(d,J=11.6Hz,1H),4.77(d,J=7.8Hz,1H), 4.67(d,J=6.8Hz,2H),4.46(s,3H),4.19(d,J=4.7Hz,1H),4.15(d,J=7.4Hz,1H),4.11(s,1H ),4.06(d,J=11.3Hz,1H),4.02(s,2H),3.78–3.67(m,5H),3.64(s,3H),3.60(d,J=6.7Hz,3H ),3.57(s,4H),3.55(d,J=4.9Hz,2H),3.51(d,J=5.0Hz,3H),3.48(d,J=1.7Hz,10H),3.46(s ,10H),3.45(d,J=2.4Hz,5H),3.28(s,2H),3.04(d,J=9.6Hz,2H),2.78(dd,J=25.0,14.5Hz, 2H),2.67(s,1H),2.38(t,J=6.5Hz,2H),2.29(s,3H),2.22(s,3H),2.04(s,3H),1.97(s,3H).

[0841] Example 48: Synthesis of Compound LP62

[0842]

[0843] Step 1: Synthesis of LP62

[0844] P1 (8 mg, 0.011 mmol), L2 (141.6 mg, 0.11 mmol), DMAP (0.68 mg, 0.0055 mmol) and HOAt (15 mg, 0.11 mmol) were added to 1 mL of anhydrous DMF solution, and EDCI (11.4 mg, 0.11 mmol) was added in batches. After the addition, the reaction solution was stirred at room temperature overnight. The target product (1.4 mg, yield: 29%) was purified by preparative HPLC as a brown oil.

[0845] LC-MS (ESI) m / z: 1014.3 [M / 2+H] +

[0846] Example 49: Synthesis of Compound LP63

[0847]

[0848] L3 (18 mg, 0.014 mmol), P74-6 (8.2 mg, 0.014 mmol), DMAP (0.2 mg, 0.007 mmol) and HOAt (9 mg, 0.07 mmol) were dissolved in anhydrous DMF (200 μL) solution, and EDCI (13 mg, 0.07 mmol) was added. The reaction solution was stirred at room temperature overnight. LC-MS showed the target product. The target product (10 mg, yield: 45.5%) was purified by preparative HPLC as a white solid.

[0849] LC-MS(ESI)m / z:[M / 2+H] + 1033.51

[0850] 1H NMR(400MHz,DMSO-d6)δ9.14(s,2H),8.75-8.70(m,1H),8.47(s,1H),8.32(s,1H),8.13(s,2H),8.09(s,1H),8.09(s,1H),7.98(s, 1H),7.85(s,1H),7.59(s,1H),7.26-7.17(m,6H),6.98(s,2H),6.44(s,2H),6.11(s,2H),5.05(d,J=12Hz,1H),4.51(s,2H),4.49( s,3H),4.21(s,2H),4.11-4.04(m,2H),3.71-3.64(m,12H),3.57-3.50(m,54H),3.43-3.36(m,8H),3.23(s,3H),3.20-3.15(m,3H) ,3.04-2.99(m,2H),2.37(s,3H),2.33(s,3H),2.21-2.05(m,9H),1.96(s,3H),1.95-1.86(m,2H),1.49(s,4H),1.21-1.14(m,6H).

[0851] Example 50: Synthesis of LP64

[0852]

[0853] P166 (25 mg, 0.032 mmol), L2 (125.41 mg, 0.097 mmol), DMAP (7 mg, 0.006 mmol) and HOAt (43.55 mg, 0.32 mmol) were dissolved in a DMF (1 mL) solution, and EDCI (61.34 mg, 0.32 mmol) was added in batches. After the addition, the reaction solution was stirred at room temperature overnight. LC-MS showed the target product, and the reaction mixture was purified by preparative HPLC to obtain the target product LP64 (18.5 mg, yield: 27.9%) as a white solid.

[0854] LC-MS(ESI)m / z:[M / 2+H] + 1020

[0855] 1H NMR(400MHz,DMSO-d6)δ8.87(s,1H),8.75(s,1H),8.68(t,J=6.7Hz,1H),8.32(m,2H),8 .12(m,2H),8.07–7.96(m,2H),7.85(t,J=5.7Hz,1H),7.77(d,J=8.8Hz,1H),7.29–7.12( m,5H),6.99(s,2H),6.73(d,J=8.9Hz,1H),6.66(brs,1H),6.47(s,1H),6.26(s,1H),6. 18(s,1H),5.33(t,J=4.9Hz,1H),5.07(d,J=11.5Hz,1H),4.68(d,J=6.6Hz,2H),4.54–4. 41(m,3H),4.24–4.13(m,2H),4.13–3.97(m,4H),3.71(m,6H),3.64(s,3H),3.60(s,3H) ,3.51(d,J=1.4Hz,48H),3.45–3.38(m,8H),3.24(s,3H),3.17(m,3H),3.14–2.99(m,3H) ,2.80(m,3H),2.42–2.32(m,2H),2.28(s,3H),2.24(s,3H),2.10(t,J=7.8Hz,5H),2.05( s,3H),2.02(d,J=7.4Hz,2H),1.99(s,3H),1.88(m,1H),1.72(m,2H),1.53–1.38(m,6H).

[0856] Example 51: Synthesis of Compound LP65

[0857]

[0858] P13 (13 mg, 0.016 mmol, 1.0 eq), L2 (63.89 mg, 0.049 mmol, 3.0 eq), DMAP (0.97 mg, 0.008 mmol, 0.5 eq) and HOAt (21.77 mg, 0.16 mmol, 10 eq) were dissolved in DMF (1 mL) solution, and EDCI (16.56 mg, 0.16 mmol, 10 eq) was added. After the addition, the reaction solution was stirred at room temperature overnight. The reaction solution was purified by preparative HPLC to obtain the target product (1.3 mg, yield: 3.7%) as a white solid.

[0859] LC-MS (ESI) m / z: 1028 [M / 2+H] +

[0860] 1 H NMR(400MHz,DMSO-d6)δ8.78(s,1H),8.74(s,1H),8.16(m,3H),8.02(m,2H),7.88(s,1H),7.26(m,5H),7.20(s,1H),7.01 (s,2H),6.47(s,1H),6.40(s,1H),6.16(s,2H),5.20(m,1H),5.07(m,1H),4.69(m,2H),4.52(m,3H),4.21(m,3H),4.13(s, 1H),3.73(s,3H),3.65(s,3H),3.59(s,2H),3.52(m,44H),3.44(m,2H),3.26(m,2H),3.19(m,4H),2.91(m,3H),2.81(m,5 H),2.75(m,2H),2.69(m,1H),2.35(m,2H),2.31(m,3H),2.24(s,3H),2.12(t,3H),2.07(s,2H),1.99(s,3H),1.48(m,6H).

[0861] Example 52: Synthesis of Compound LP66

[0862]

[0863] L2 (18 mg, 0.014 mmol), P19 (11 mg, 0.014 mmol), DMAP (0.8 mg, 0.007 mmol) and HOAt (9 mg, 0.07 mmol) were dissolved in anhydrous DMF (400 μL) solution, EDCI (13 mg, 0.07 mmol) was added, and the reaction solution was stirred at room temperature overnight. LC-MS showed the target product. The target product (12 mg, yield: 41.5%) was purified by preparative HPLC to obtain a white solid.

[0864] LC-MS (ESI) m / z: 1034.14 [M / 2+H] +

[0865] 1H NMR(400MHz,DMSO-d6)δ8.83(s,2H),8.69(s,1H),8.37(s,1H),8.16(s,2H),8.05(s,2H),8.02(s,1H),7.88(s,1H),7.25-7.22(m,4 H),7.19-7.17(m,1H),7.00(s,2H),6.50(s,1H),6.25(s,1H),6.19-6.17(m,1H),5.06(s,1H),4.68(s,1H),4.58(s,1H),4.50(s,1H) ,4.26(s,1H),4.20(s,2H),4.10-4.08(m,2H),4.04(s,2H),3.64(s,5H),3.56(s,4H),3.50(s,45H),3.43-3.33(m,10H),2.24(s,3H ),3.19-2.77(m,7H),2.32(s,3H),2.23(s,3H),2.11-2.06(m,7H),1.98(s,3H),1.48-1.45(m,2H),1.45(s,4H),1.24-1.08(m,10H).

[0866] Example 53: Synthesis of LP67

[0867]

[0868] A mixture of P36 (18 mg, 0.022 mmol), L2 (29 mg, 0.014 mmol), DMAP (0.8 mg, 0.007 mmol) and HOAt (9 mg, 0.07 mmol) was added to anhydrous DMF (0.6 mL) solution, and EDCI (13 mg, 0.07 mmol) was added to the reaction solution in batches. The reaction solution was stirred at room temperature overnight. LC-MS showed the target product, which was purified by preparative HPLC to obtain the target product (14 mg, yield: 30%) as a white solid.

[0869] LC-MS(ESI)m / z:[M / 2+H] + 1033.3

[0870] 1H NMR(400MHz, DMSO-d6)δ8.86(s,2H),8.67-8.70(m,1H),8.34-8.40(m,1H),8.12-8.15(m,1H),8.00-8.05(m,2H),7.85-7.87(m,1H),7.27-7. 19(m,5H),7.01(s,2H),6.51(s,1H),6.29(s,1H),6.23(s,1H),6.19(s ,1H),5.20-5.22(m,1H),4.68(d,J=8Hz,1H),4.59(s,1H),4.50-4.55(m ,1H),4.19-4.27(m,4H),4.04(s,2H),3.77-3.52(m,61H),3.45-3.36( m,9H),3.26(s,3H),3.22-3.19(m,2H),3.06-3.03(m,1H),2.84-2.69( m,4H),2.35(s,3H),2.24(s,3H),2.14-2.10(m,4H),2.04-2.00(m,6H) ,1.95-1.86(m,2H),1.52-1.46(m,4H),1.26-1.19(m,4H),1.03(s,2H).

[0871] Example 54: Synthesis of Compound LP68

[0872]

[0873] L4 (3 mg, 0.0025 mmol), T2 (2 mg, 0.0025 mmol), DMAP (0.14 mg, 0.0012 mmol) and HOAt (4.5 mg, 0.035 mmol) were dissolved in anhydrous DMF (0.40 mL) solution, EDCI (6.5 mg, 0.035 mmol) was added, and the reaction solution was stirred at room temperature overnight. The target product (0.27 mg, yield: 5.4%) was purified by preparative HPLC as a white solid.

[0874] LC-MS (ESI) m / z: 984.67 [M / 2+H] +

[0875] Example 55: Synthesis of Compound LP69

[0876]

[0877] Step 1: Synthesis of LP69

[0878] A mixture of T8 (3 mg, 0.004 mmol), L2 (5 mg, 0.004 mmol), EDCI (7 mg, 0.04 mmol), HOAt (5 mg, 0.04 mmol) and DMAP (0.3 mg, 0.002 mmol) was dissolved in anhydrous DMF (0.4 mL) at room temperature and stirred for 2 hours. LC-MS showed the target product, which was purified by preparative HPLC to obtain the target product (1.1 mg, yield: 13.7%) as a white solid.

[0879] LC-MS (ESI) m / z: 1012.8 [M / 2+H] +

[0880] Example 56: Synthesis of Compound LP70

[0881]

[0882] Step 1: Synthesis of LP70

[0883] EDCI (2.4 mg, 0.0125 mmol) was added to a solution of P241 (2 mg, 0.0025 mmol), L2 (5 mg, 0.0038 mmol), HOAt (1.7 mg, 0.0125 mmol) and DMAP (0.15 mg, 0.0012 mmol) in anhydrous DMF (0.4 mL). The mixture was stirred at room temperature overnight. LC-MS showed that the reaction was complete. The target product (0.3 mg, yield: 5.7%) was obtained as a white solid after purification by preparative HPLC.

[0884] LC-MS (ESI) m / z: 1032.8 [M / 2+H] +

[0885] Example 57: Synthesis of Compound LP71

[0886]

[0887] Step 1: Synthesis of LP71-1

[0888] A solution of L5 (28 mg, 0.019 mmol), P19 (14 mg, 0.017 mmol), EDCI (16 mg, 0.085 mmol), DMAP (1 mg, 0.009 mmol) and HOAt (11 mg, 0.085 mmol) in DMF (0.4 mL) was stirred at room temperature for 2 hours. LC-MS showed the target product, which was purified by preparative HPLC to obtain the target compound (16 mg, yield: 41%) as a white solid.

[0889] LC-MS (ESI) m / z: 1127.4 [M / 2+H] +

[0890] Step 2: Synthesis of LP71

[0891] LP71-1 (14 mg, 0.006 mmol) was dissolved in 10% TFA / DCM (4 mL) and stirred at 0°C for 3 hours. TLC showed that the reaction was complete, and DMF (2 mL) was added. The mixture was concentrated to remove TFA and dichloromethane, and the residue was purified by preparative HPLC to obtain the target product (6.3 mg, yield: 46%) as a white solid.

[0892] LC-MS (ESI) m / z: 1077.4 [M / 2+H] +

[0893] Example 58: Synthesis of Compound LP72

[0894]

[0895] EDCI (23 mg, 0.06 mmol) was added to a solution of T14 (10 mg, 0.012 mmol), L2 (38.6 mg, 0.03 mmol), HOAt (16.3 mg, 0.06 mmol), and DMAP (0.73 mg, 0.006 mmol) in anhydrous DMF (2 mL). The reaction solution was stirred at 40°C for 1.5 hours and purified by preparative HPLC to obtain the target compound (1.50 mg, yield: 11.5%) as a white solid.

[0896] LC-MS(ESI)m / z:[M / 2+H] + 1035.4

[0897] Example 59: Synthesis of Compound LP73

[0898]

[0899] Step 1: Synthesis of LP73-1

[0900] L6 (16 mg, 0.013 mmol), P166 (10 mg, 0.015 mmol), EDCI (12 mg, 0.065 mmol), DMAP (0.8 mg, 0.007 mmol) and HOAt (9 mg, 0.056 mmol) were added to DMF (0.8 mL) and stirred at room temperature for 2 hours. LC-MS showed the target product, which was purified by preparative HPLC to obtain the target product (3 mg, yield: 11.6%) as a white solid.

[0901] LC-MS (ESI) m / z: 995.5 [1 / 2M+H] +

[0902] Step 2: Synthesis of LP73

[0903] A solution of LP73-1 (3 mg, 0.006 mmol) in 10% TFA / DCM (1 mL) was stirred at 0°C for 3 hours. TLC showed that the reaction was complete, and DMF (2 mL) was added. The mixture was concentrated to remove TFA and dichloromethane, and the resulting residue was purified by preparative HPLC to give the target product (2.6 mg, yield: 83%) as a white solid.

[0904] LC-MS (ESI) m / z: 946.2 [M / 2+H] +

[0905] Example 60: Synthesis of Compound LP74

[0906]

[0907] Step 1: Synthesis of LP74-1

[0908] L6 (16 mg, 0.013 mmol), P19 (10 mg, 0.013 mmol), EDCI (12 mg, 0.065 mmol), DMAP (0.8 mg, 0.007 mmol) and HOAt (9 mg, 0.056 mmol) were dissolved in DMF (0.8 mL) and stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete, and the target product (6 mg, yield: 23%) was purified by preparative HPLC as a white solid.

[0909] LC-MS (ESI) m / z: 993.6 [M / 2+H] +

[0910] Step 2: Synthesis of LP74

[0911] A solution of LP74-1 (6 mg, 0.0019 mmol) in 10% TFA / DCM (1 mL) was stirred at 0°C for 3 hours. TLC showed that the reaction was complete, and DMF (2 mL) was added. The mixture was concentrated to remove TFA and dichloromethane, and the resulting residue was purified by preparative HPLC to obtain the target product (1.4 mg, yield: 22%) as a white solid.

[0912] LC-MS (ESI) m / z: 960.2 [M / 2+H] +

[0913] Example 61: Synthesis of Compound LP75

[0914]

[0915] At room temperature, a solution of L7 (10 mg, 0.01 mmol), P166 (8 mg, 0.01 mmol), EDCI (16 mg, 0.085 mmol), DMAP (1 mg, 0.009 mmol) and HOAt (11 mg, 0.08 mmol) in DMF (0.4 mL) was stirred for 2 hours. LC-MS showed that the reaction was complete, and the reaction solution was purified by preparative HPLC to obtain the target product (1.7 mg, yield: 8.5%) as a white solid.

[0916] LC-MS(ESI)m / z:[M / 2+H] + 1052.5

[0917] Example 62: Synthesis of Compound LP76

[0918]

[0919] At room temperature, a solution of L7 (10 mg, 0.01 mmol), P19 (8 mg, 0.01 mmol), EDCI (16 mg, 0.085 mmol), DMAP (1 mg, 0.009 mmol) and HOAt (11 mg, 0.08 mmol) in DMF (0.4 mL) was stirred for 2 hours. LC-MS showed that the reaction was complete, and the reaction solution was purified by preparative HPLC to obtain the target product (3.4 mg, yield: 20%) as a white solid.

[0920] LC-MS(ESI)m / z:[M / 2+H] + 916.9

[0921] Example 63: Synthesis of Compound LP77

[0922]

[0923] L4 (2 mg, 0.0017 mmol), P19 (1.5 mg, 0.0017 mmol), EDCI (1.6 mg, 0.008 mmol), DMAP (0.1 mg, 0.009 mmol) and HOAt (1.1 mg, 0.008 mmol) were added to anhydrous DMF (0.4 mL), and the reaction solution was stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete, and the target product (0.57 mg, yield: 17%) was purified by preparative HPLC as a white solid. LC-MS (ESI) m / z: 1981.6 [M+H] +

[0924] Example 64: Synthesis of Compound LP78

[0925]

[0926] Step: Synthesis of LP78

[0927] L4 (33 mg, 0.03 mmol), P166 (15 mg, 0.03 mmol), EDCI (30 mg, 0.15 mmol), DMAP (1.8 mg, 0.015 mmol) and HOAt (21 mg, 0.15 mmol) were added to DMF (0.4 mL) and stirred at room temperature for 2 hours. LC-MS showed the target product, which was purified by preparative HPLC to obtain the target product (14.7 mg, yield: 38%) as a white solid.

[0928] LC-MS (ESI) m / z: 1953.6 [M+H] +

[0929] 1 H NMR(400MHz,DMSO-d6)δ9.5(s,1H),8.95(s,1H),8.41–8.38(m,1H),8.06-8.02(m,2H),7.95-7.90(m,2H),7.79-7.76(m,1H),7.71(s,1H),7 .16-7.14(m,4H),7.11-7.08(s,1H),6.92(s,2H),6.7(s,1H),6.45(s ,1H),6.19(s,1H),5.13(s,1H),4.49-4.44(m,3H),4.19-4.08(m,4H), 3.86(d,J=1.0Hz,2H),3.68-3.53(m,13H),3.46-3.42(m,46H),3.31- 3.27(m,3H),3.17(s,3H),3.12-3.08(m,3H),3.30-2.95(m,2H),2.80- 2.59(m,6H),2.22-2.21(m,6H),2.04-1.98(m,7H),1.91(s,3H),1.84- 1.76(m,1H),1.68-1.61(m,1H),1.43-1.35(m,4H),1.17-1.08(m,3H).

[0930] Preparation and characterization of antibody-drug conjugates

[0931] The following are examples of methods for preparing and analyzing antibody drug conjugates. It should be noted that the following examples are only partial embodiments and are not limited to the embodiments described herein. For example, the antibody is not limited to the antibodies currently shown, but may also be any other antibody.

[0932] General conjugation process for the preparation of novel ecteinascidin derivative-based antibody drug conjugates

[0933] Place the monoclonal antibody (1.0 eq.) in a 0.5 mL centrifuge tube, add 25 mM Na2B4O7, 25 mM NaCl, 1 mM DTPA buffer, dilute the antibody concentration to 5-10 mg / mL, shake and mix, and add 2 mg / mL TCEP (5 eq. to 8 eq.) aqueous solution to the mixture for antibody reduction. After shaking and mixing, place the reaction on a heating and cooling constant temperature mixer and reduce it at 20-25°C for 2-3 hours. Add an equal volume of 500 mM Tris, pH 8.8 or other suitable buffer, and then add a toxin linker solution (5 eq. to 8 eq.) dissolved in 10 mg / mL DMSO, add DMSO according to 10-20% of the total volume of the final reaction solution, shake and mix, and place it on a heating and cooling constant temperature mixer for reaction, and couple at 25°C for 16-24 hours.

[0934] Use 300 mg / mL dextran-coated activated carbon (manufacturer: Sigma) to remove the residual toxin linkers in the reaction solution that are not coupled to the antibody. Add 10% volume of activated carbon solution to the reaction solution, mix and shake, and shake at 4°C for 2 hours. Take the supernatant to detect the residual toxin linker content. If the free toxin linker content is greater than 1%, the above process should be repeated several times until the test result meets the standard (usually 3 treatments can meet the requirements). After the treatment is completed, the mixture is centrifuged, the supernatant is collected with a syringe and a hydrophilic membrane filter, and the activated carbon is filtered out. The buffer of the desired antibody-drug conjugate is replaced with a suitable storage buffer by 4 ultrafiltrations and stored at -80°C.

[0935] General Characterization Methods for Antibody Drug Conjugates

[0936] (a) DAR value of ADC analyzed by HIC-HPLC

[0937] High performance liquid chromatography: e2695 high performance liquid chromatography system or 1260 Agilent liquid chromatograph.

[0938] Column: MabPac TM HIC-Butyl 5μm 4.6×100mm (manufacturer: Thermo).

[0939] Mobile phase: Mobile phase A (MPA): 1.5M (NH4)2SO4+50mM potassium phosphate (pH7.0); Mobile phase B (MPB): 50mM sodium phosphate (pH 7.0) / isopropanol (75:25V / V); Elution was performed according to the following elution procedure (5%-95%), wherein, from 0 to 2 min, the volume of mobile phase A was 100%-95%, and the volume of mobile phase B was 0%-5%; from 2 to 22 min, the volume of mobile phase A was 95%-5%, and the volume of mobile phase B was 5%-95%; from 22 to 24 min, the volume of mobile phase A was 5%-0%, and the volume of mobile phase B was 95%-100%; from 24 to 26 min, the volume of mobile phase A was 0%-100%, and the volume of mobile phase B was 100%-0%; from 26 to 30 min, the volume of mobile phase A was 100%-100%, and the volume of mobile phase B was 0%-0%.

[0940] Detection conditions: Set the mobile phase flow rate to 1 ml / min, the detection wavelength to 280 nm, and the column temperature to 30 °C.

[0941] Experimental steps: Take 50 μg of the coupled sample (the volume depends on the sample concentration), inject it into the high performance liquid chromatograph, elute using the above elution procedure, and record the chromatogram.

[0942] DAR value calculation formula: DAR = Σ (relative peak area × number of loaded drugs) / 100

[0943] (b) Free Linker Payload (mol / mol%) value analyzed by C18-HPLC

[0944] High performance liquid chromatography: e2695 high performance liquid chromatography system.

[0945] Chromatographic column: C18 3.5 μm 4.6×150 mm (manufacturer: Waters).

[0946] Mobile phase: Mobile phase A (MPA): 0.1% TFA-H2O; Mobile phase B (MPB): 0.1% TFA-ACN; Elution was performed according to the following elution procedure (5%-95%), wherein the volume of mobile phase A was 90%-20% from 0 to 30 min, and the volume of mobile phase B was 10%-80%; the volume of mobile phase A was 20%-90% from 30 to 31 min, and the volume of mobile phase B was 80%-10%; the volume of mobile phase A was 90%-90% from 31 to 35 min, and the volume of mobile phase B was 10%-10%

[0947] Detection conditions: Set the mobile phase flow rate to 0.5 ml / min, the detection wavelength to 254 nm, and the column temperature to 30 °C.

[0948] Preparation example of reagent I: Measure 30 mL of anhydrous methanol and 50 mL of acetonitrile, weigh 10 g of sodium chloride, mix in a container, stir at room temperature for more than 1 hour, and then let stand for 1 hour. Take the supernatant, filter with a 0.22 μm organic membrane, store at room temperature, and the validity period is 3 months.

[0949] Example of preparing reagent II: measure 100mL of reagent I, 15mL of DMSO, and 85mL of ADC sample storage buffer respectively, mix them evenly in a container, and store at room temperature. The validity period is 2 months.

[0950] Experimental steps:

[0951] Sample solution preparation:

[0952] 1) Free toxin reference: Take the toxin corresponding to the test sample as the reference and prepare it with the above reagent II to a final concentration of 1.0 mg / ml.

[0953] 2) Take 10 μl of the above-mentioned 1.0 mg / ml toxin reference substance and add it to 90 μl of Reagent II to prepare a concentration of 100 μg / ml; then dilute the samples in sequence with Reagent II according to the following table to prepare samples of the required concentration.

[0954] Sample No. Concentration of sample to be taken Sample volume to be taken Volume of reagent II to be taken Prepare the sample to the desired concentration 1 100μg / ml 40μl 360μl 10μg / ml 2 10μg / ml 180μl 180μl 5.0μg / ml 3 5.0μg / ml 120μl 180μl 2.0μg / ml 4 2.0μg / ml 180μl 180μl 1.0μg / ml 5 1.0μg / ml 180μl 180μl 0.5μg / ml 6 0.5μg / ml 120μl 180μl 0.2μg / ml 7 0.2μg / ml 180μl 180μl 0.1μg / ml

[0955] Note: Inject the samples prepared above to the required concentration into HPLC in ascending order.

[0956] Sample test: Take 85 μg of the coupled sample, mix the sample with 3 μl DMSO for 5 minutes, then take 60 μl of reagent I and add it to the sample system, mix for 5-10 minutes, centrifuge at 2000 rpm for 2 minutes, take 20 μL of the supernatant, inject it into the HPLC, elute using the above elution procedure, and record the chromatogram.

[0957] According to the calculated standard binary linear regression curve equation, the content of free toxin in ADC was calculated.

[0958] Free Drug (mol / mol%) = residual small molecule molar concentration / antibody molar concentration × 100.

[0959] (c) Determination of free linker-payload content by LC-MS / MS

[0960] Internal reference compounds: 50 ng / ml tolbutamide, labetalol, etofesamide and buspirone

[0961] Extraction solvent: ethyl acetate

[0962] Serial dilution of the standard: dilute the standard to 20000, 16000, 10000, 4000, 1000, 200, 40, 20 ng / ml with 50% methanol in water.

[0963] Sample pre-treatment: Take 20 μL of sample, then add 20 μL of blank buffer and 240 μL of extractant. After vortex mixing, let stand until stratification, then take 160 μL of supernatant and put it into a 96-well plate to dry, add 200 μL of 50% acetonitrile water, mix well, and finally enter LC-MS / MS analysis.

[0964] Pre-treatment of standard products: Take 2μL of working solution and add it to 38μL of blank buffer, then add 240μL of extractant. Vortex and let stand until stratification, take 160μL of supernatant and put it into a 96-well plate to dry, add 200μL of 50% acetonitrile water and mix well.

[0965] Mass spectrometry conditions:

[0966]

[0967]

[0968] Data processing: A standard curve was made based on the concentration of the standard and the corresponding mass spectrometry response value. The residual amount of free linker-payload was calculated through the linear fitting curve (formula) and the mass spectrometry response value of the detected sample.

[0969] (d) SEC-HPLC analysis of ADC aggregates

[0970] High performance liquid chromatography: e2695 high performance liquid chromatography system or 1260 Agilent liquid chromatograph.

[0971] Chromatographic column: Waters Xbridge BEH200 SEC (7.8×300mm, 3.5μm)

[0972] Mobile phase: 50 mM PB + 200 mM Arg (pH 6.80) + 10% IPA, eluted according to the following elution program, 0-30 min mobile phase A volume 100%-100%;

[0973] Detection conditions: Set the mobile phase flow rate to 0.5 ml / min, the detection wavelength to 280 nm, and the column temperature to 26 °C.

[0974] Experimental steps: Take 20 μg of the coupled sample (the volume depends on the sample concentration), inject it into the high performance liquid chromatograph, elute using the above elution procedure, and record the chromatogram.

[0975] Calculation formula: Monomer purity (%) = A monomer / A total × 100%; polymer purity (%) = A polymer / A total × 100%

[0976] Example 65: Preparation of Antibody Drug Conjugate ADC-1

[0977]

[0978] According to the general coupling process for preparing antibody drug conjugates, TCEP (10mM, 1.8eq.) aqueous solution was added to 50mM PBS pH 7.4 buffer of trastuzumab (1.0mg, 38.9mg / mL, 1.0eq.) and reduced at 22°C for 2 hours; then LP1 (10mg / mL, 4.0eq.) DMSO solution was added and coupled at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment for 3 times, the buffer of the desired antibody drug conjugate was ultrafiltered 8 times to obtain ADC-1 (C ADC (mg / mL): 10.56, V (mL): 0.08, yield 84.5%).

[0979] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0980] HIC-DAR: 2.30, SEC purity: 98.85%, Free Linker Payload (mol / mol%): notdetected.

[0981] Example 66: Preparation of Antibody Drug Conjugate ADC-85

[0982]

[0983] According to the general coupling process for preparing antibody drug conjugates, TCEP (10mM, 2eq.) aqueous solution was added to trastuzumab (1.0mg, 21.07mg / mL, 1.0eq.) in 50mM PBS pH 7.0 buffer, and reduced at 22°C for 2 hours; then LP62 (10mg / mL, 5.0eq.) DMSO solution was added and coupled at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment for 3 times, the buffer of the desired antibody drug conjugate was ultrafiltered 5 times to obtain ADC-85 (C ADC (mg / mL): 6.68, V (mL): 0.1, yield 66.8%).

[0984] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0985] HIC-DAR: 1.20, SEC purity: 100%, Free Linker Payload (mol / mol%): notdetected.

[0986] Example 67: Preparation of Antibody Drug Conjugate ADC-86

[0987]

[0988] According to the general coupling process for preparing antibody drug conjugates, TCEP (1mM, 15eq.) aqueous solution was added to trastuzumab (1.5mg, 10mg / mL, 1.0eq.) in 20mM PBS pH 7.4 buffer, and reduced at 37°C for 1.5 hours; placed on ice for 5 minutes, and then LP63 (1mM, 15.0eq.) DMSO solution was added, and the coupling reaction was carried out at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment three times, the buffer of the desired antibody drug conjugate was ultrafiltered five times to obtain ADC-86 (C ADC (mg / mL): 8, V (mL): 0.08, yield 40%).

[0989] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0990] HIC-DAR: 6.5, SEC purity: 65%, Free Linker Payload (mol / mol%): not detected.

[0991] Example 68: Preparation of Antibody Drug Conjugate ADC-87

[0992]

[0993] According to the general coupling process for preparing antibody drug conjugates, TCEP (10mM, 8eq.) aqueous solution was added to trastuzumab (1.0mg, 21.07mg / mL, 1.0eq.) in 50mM PBS pH 7.0 buffer, and reduced at 22°C for 2 hours; then LP65 (10mg / mL, 12.0eq.) DMSO solution was added and coupled at 22°C for 3 hours. After purification by dextran-coated activated carbon treatment for 3 times, the buffer of the desired antibody drug conjugate was ultrafiltered 5 times to obtain ADC-87 (C ADC (mg / mL): 5.53, V (mL): 0.15, yield 63.6%).

[0994] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0995] HIC-DAR: 8.0, SEC purity: 89%, Free Linker Payload (mol / mol%): not detected.

[0996] Example 69: Preparation of Antibody Drug Conjugate ADC-88

[0997]

[0998] According to the general coupling process for preparing antibody drug conjugates, TCEP (10mM, 2eq.) aqueous solution was added to trastuzumab (1.0mg, 21.07mg / mL, 1.0eq.) in 50mM PBS pH 7.0 buffer, and reduced at 22°C for 2 hours; then LP66 (10mg / mL, 8.0eq.) DMSO solution was added and coupled at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment for 3 times, the buffer of the desired antibody drug conjugate was ultrafiltered 5 times to obtain ADC-88 (C ADC (mg / mL): 6.09, V (mL): 0.11, yield 66.9%).

[0999] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[1000] HIC-DAR: 4.0, SEC purity: 99.08%, Free Linker Payload (mol / mol%): notdetected.

[1001] Example 70: Preparation of Antibody Drug Conjugate ADC-89

[1002]

[1003] According to the general coupling process for preparing antibody drug conjugates, TCEP (10mM, 2eq.) aqueous solution was added to trastuzumab (1.0mg, 21.07mg / mL, 1.0eq.) in 50mM PBS pH 7.0 buffer, and reduced at 22°C for 2 hours; then LP67 (10mg / mL, 8.0eq.) DMSO solution was added and coupled at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment for 3 times, the buffer of the desired antibody drug conjugate was ultrafiltered 5 times to obtain ADC-89 (C ADC (mg / mL): 4.73, V (mL): 0.12, yield 56.8%).

[1004] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[1005] HIC-DAR: 3.8, SEC purity: 92.87%, Free Linker Payload (mol / mol%): notdetected.

[1006] Example 71: Preparation of Antibody Drug Conjugate ADC-90

[1007]

[1008] According to the general coupling process for preparing antibody drug conjugates, TCEP (10mM, 2eq.) aqueous solution was added to trastuzumab (1.0mg, 21.07mg / mL, 1.0eq.) in 50mM PBS pH 7.0 buffer, and reduced at 22°C for 2 hours; then LP68 (10mg / mL, 9.0eq.) DMSO solution was added and coupled at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment for 3 times, the buffer of the desired antibody drug conjugate was ultrafiltered 5 times to obtain ADC-90 (C ADC (mg / mL): 2.81, V (mL): 0.12, yield 33.7%).

[1009] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[1010] HIC-DAR: 2.08, SEC purity: 93.64%, Free Linker Payload (mol / mol%): notdetected.

[1011] Example 72: Preparation of Antibody Drug Conjugate ADC-91

[1012]

[1013] According to the general coupling process for preparing antibody drug conjugates, TCEP (10mM, 2eq.) aqueous solution was added to trastuzumab (1.0mg, 21.07mg / mL, 1.0eq.) in 50mM PBS pH 7.0 buffer, and reduced at 22°C for 2 hours; then LP64 (10mg / mL, 5.0eq.) DMSO solution was added and coupled at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment for 3 times, the buffer of the desired antibody drug conjugate was ultrafiltered 8 times to obtain ADC-91 (C ADC (mg / mL):8.18, V (mL):0.13, yield 70.9%).

[1014] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[1015] HIC-DAR: 7.93, SEC purity: 95.63%, Free Linker Payload (mol / mol%): notdetected.

[1016] Example 73: Preparation of Antibody Drug Conjugate ADC-92

[1017]

[1018] According to the general coupling process for preparing antibody drug conjugates, TCEP (10mM, 2eq.) aqueous solution was added to trastuzumab (1.0mg, 21.07mg / mL, 1.0eq.) in 50mM PBS pH 7.0 buffer, and reduced at 22°C for 2 hours; then LP70 (10mg / mL, 5.0eq.) DMSO solution was added, and the coupling reaction was carried out at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment for 3 times, the buffer of the desired antibody drug conjugate was ultrafiltered 8 times to obtain ADC-92 (C ADC (mg / mL): 5.82, V (mL): 0.1, yield: 58.2%).

[1019] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[1020] HIC-DAR: 4.6, SEC purity: 98.92%, Free Linker Payload (mol / mol%): notdetected.

[1021] Example 74: Preparation of Antibody Drug Conjugate ADC-93

[1022]

[1023] According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mM, 2 eq.) aqueous solution was added to 50 mM PBS pH 7.0 buffer solution of trastuzumab (2.0 mg, 21.07 mg / mL, 1.0 eq.), and reduced at 22°C for 2 hours; then LP71 (10 mg / mL, 5.0 eq.) DMSO solution was added, and the coupling reaction was carried out at 22°C for 1 hour.

[1024] Then, 500 mM Tris-HCl, pH 8.8 solution, which is 80% of the total volume of ADC, was added to adjust the pH to 8.0, and the mixture was incubated at 22°C for 4 hours. After purification by dextran-coated activated carbon treatment for 3 times, the buffer of the desired antibody-drug conjugate was ultrafiltered 8 times to obtain ADC-93 (C ADC(mg / mL):8.76, V (mL):0.1, yield: 43.8%).

[1025] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[1026] HIC-DAR: 1.6, SEC purity: 94.6%, Free Linker Payload (mol / mol%): notdetected.

[1027] Example 75: Preparation of Antibody Drug Conjugate ADC-94

[1028]

[1029] According to the general coupling process for preparing antibody drug conjugates, TCEP (10mM, 2eq.) aqueous solution was added to trastuzumab (2mg, 21.07mg / mL, 1.0eq.) in 50mM PBS pH 7.0 buffer, and reduced at 22°C for 2 hours; then LP73 (10mg / mL, 5.0eq.) DMSO solution was added, and the coupling reaction was carried out at 22°C for 1 hour. 500mM Tris-Hcl, pH8.8 solution with 80% of the total volume of ADC was added to adjust the pH to 8.0, incubated at 22°C for 4 hours, and purified by dextran-coated activated carbon treatment for 3 times, and the buffer of the desired antibody drug conjugate was ultrafiltered 8 times to obtain ADC-94 (C ADC (mg / mL):8.99, V (mL):0.1, yield: 45%).

[1030] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[1031] HIC-DAR: 1.6, SEC purity: 97.67%, Free Linker Payload (mol / mol%): notdetected.

[1032] Example 76: Preparation of Antibody Drug Conjugate ADC-95

[1033]

[1034] According to the general coupling process for preparing antibody drug conjugates, TCEP (10mM, 2eq.) aqueous solution was added to trastuzumab (1.5mg, 21.07mg / mL, 1.0eq.) in 50mM PBS pH 7.0 buffer, and reduced at 22°C for 2 hours; then LP75 (10mg / mL, 5.0eq.) DMSO solution was added and coupled at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment for 3 times, the buffer of the desired antibody drug conjugate was ultrafiltered 8 times to obtain ADC-95 (C ADC (mg / mL): 6.18, V (mL): 0.11, yield: 45.3%).

[1035] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[1036] HIC-DAR: 2.9, SEC purity: 99.61%, Free Linker Payload (mol / mol%): notdetected.

[1037] Example 77: Preparation of Antibody Drug Conjugate ADC-96

[1038]

[1039] According to the general coupling process for preparing antibody drug conjugates, TCEP (10mM, 2eq.) aqueous solution was added to trastuzumab (1.5mg, 21.07mg / mL, 1.0eq.) in 50mM PBS pH 7.0 buffer, and reduced at 22°C for 2 hours; then LP76 (10mg / mL, 5.0eq.) DMSO solution was added and coupled at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment for 3 times, the buffer of the desired antibody drug conjugate was ultrafiltered 8 times to obtain ADC-96 (C ADC (mg / mL): 6.82V(mL): 0.11, yield: 50%).

[1040] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[1041] HIC-DAR: 4.3, SEC purity: 98.38%, Free Linker Payload (mol / mol%): notdetected.

[1042] Example 78: Preparation of Antibody Drug Conjugate ADC-97

[1043]

[1044] According to the general coupling process for preparing antibody drug conjugates, TCEP (10mM, 2eq.) aqueous solution was added to trastuzumab (0.8mg, 21.07mg / mL, 1.0eq.) in 50mM PBS pH 7.0 buffer, and reduced at 22°C for 2 hours; then LP77 (10mg / mL, 5.0eq.) DMSO solution was added and coupled at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment for 3 times, the buffer of the desired antibody drug conjugate was ultrafiltered 8 times to obtain ADC-97 (C ADC (mg / mL): 3.46, V (mL): 0.1, yield: 43.3%).

[1045] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[1046] HIC-DAR: 3.4, SEC purity: 98.9%, Free Linker Payload (mol / mol%): notdetected.

[1047] Example 79: Preparation of Antibody Drug Conjugate ADC-98

[1048]

[1049] According to the general coupling process for preparing antibody drug conjugates, TCEP (10mM, 2eq.) aqueous solution was added to trastuzumab (1.0mg, 21.07mg / mL, 1.0eq.) in 50mM PBS pH 7.0 buffer, and reduced at 22°C for 2 hours; then LP78 (10mg / mL, 7.0eq.) DMSO solution was added and coupled at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment three times, the buffer of the desired antibody drug conjugate was ultrafiltered three times to obtain ADC-98 (C ADC (mg / mL): 6.21, V (mL): 0.1, yield: 62.1%).

[1050] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[1051] HIC-DAR: 1.7, SEC purity: 97.85%, Free Linker Payload (mol / mol%): notdetected.

[1052] Biological evaluation

[1053] 1) Experiment on tumor cell proliferation inhibition of novel ecteinascidin derivatives

[1054] MDA-MB-231, OV90, and NCI-N87 cells (1500 cells per well, 80 μL volume) at a certain cell concentration were added to a 96-well plate. The cells were cultured in a constant temperature incubator (37°C, 5% CO2) overnight. First, a solution of 2 mM ectinacidin derivatives was prepared with DMSO solution, and after a 1:4 gradient dilution, 1 μL of the dilution prepared with DMSO solution was added to each well and added to 99 μL of PBS solution containing 10% FBS per well, and mixed thoroughly. Take the diluted solution (2 μL of the diluted solution per well, 18 μL of PBS containing 10% FBS), incubate with the cells (37°C, 5% CO2) for five days, add CellTitre-Glo reagent (40 μL per well), and use i3X microplate reader to measure the fluorescence value.

[1055] In vitro cytotoxicity of the ecteinascidin derivatives of the present invention (++++: <1 nM; +++: 1-10 nM; ++: 10-200 nM; +: >200 nM)

[1056]

[1057]

[1058]

[1059]

[1060]

[1061]

[1062]

[1063] All ecteinascidin derivatives showed certain antitumor activity, among which the in vitro cell killing activity of P1, P9, P10, P12, P13, P19, P30, P36, P52, P58, P65, P74, P75, P85, P90, P96, P166, P170, P172, T1, T3, T4, T10, T11 and T17 was stronger than or equivalent to that of trabectedin and rubectin.

[1064] 2) Tumor cell proliferation inhibition experiment based on antibody-drug conjugates of novel ecteinascidin derivatives

[1065] 1500 cells were seeded in a 96-well plate (excluding the edge wells filled with PBS), and the cells were cultured in 80uL of appropriate culture medium and placed in a constant temperature cell culture incubator at 37°C and 5% CO2. After the cells were cultured overnight in a constant temperature incubator (37°C, 5% CO2), 20uL of diluted antibody-drug conjugate solution based on the new ectenes derivative was added to each well to make the final concentration of the conjugate range from 3.8pM to 250nM. After the cells were incubated with the conjugate for 5 days, the 96-well plate was placed at room temperature for equilibration for 30 minutes. 40uL of (Promega, G7572) reagent, after 10 minutes of reaction in the dark, the fluorescence value was measured using SpectraMax i3X microplate reader, and the curve of luminescence value versus conjugate concentration (nM) was fitted using Graphpad Prism software.

[1066] In vitro cytotoxicity of antibody-drug conjugates (++++: <1 nM; +++: 1-10 nM; ++: 10-200 nM; +: >200 nM)

[1067]

[1068] All ADCs exhibited certain anti-tumor activity. For example, in NCI-N87 cells, the in vitro cell killing activities of ADC-1, ADC-86, ADC-88, ADC-89, ADC-91, ADC-92, ADC-93, ADC-95 and ADC-97 were 1-10 nM; while the in vitro cell killing activities of ADC-87, ADC-90 and ADC-94 were less than 1 nM.

[1069] The main advantages of the present invention are:

[1070] 1. Some of the novel ecteinascidin derivatives provided by the present invention have relatively strong in vitro cytotoxicity (1-10 nM), and some have relatively stronger in vitro cytotoxicity (<1 nM).

[1071] 2. The antibody-drug conjugates based on the novel ecteinascidin derivatives provided by the present invention have strong in vitro cytotoxicity (1-10 nM), and some antibody-drug conjugates have stronger in vitro cytotoxicity (<1 nM).

Claims

1. An ecteinascidin derivative, characterized in that Its structure is shown in the general formula (Ⅰ): Wherein: the R1 is selected from hydroxyl or cyano; The R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, mercapto, halogen, deuterated alkyl, halogenated alkyl, halogenated alkoxy, hydroxy-substituted alkyl, amino-substituted alkyl, alkylamino-substituted alkyl, alkyl, alkoxy, alkylthio, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, alkyl-substituted amino, cycloalkyl-substituted alkyl, heterocyclic group, aryl, heteroaryl, sulfonic acid group, The R2 and R3 together with the carbon atoms to which they are connected form a saturated carbocyclic ring, a saturated heterocyclic ring, a cycloalkyl group, a heterocycloalkyl group or an acyclic ring; the x is selected from an integer of 1-10; The R a and R b Each is independently selected from hydrogen, deuterium, hydroxyl, hydroxyl-substituted alkyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkyl-substituted sulfonyl, aryl-substituted sulfonyl, heteroaryl-substituted sulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a saturated heterocyclic ring, a heterocycloalkyl ring or an acyclic ring; R4 is selected from hydrogen, alkyl, deuterated alkyl, halogenated alkyl, amino-substituted alkyl, alkyl substituted with alkylamino or The s is selected from an integer of 1-10; Said R5 and R6 are both hydrogen or alkyl, said R5, R6 together with the carbon atoms to which they are connected form a saturated carbocyclic ring, a saturated heterocyclic ring, a cycloalkyl group, a heterocycloalkyl group or an acyclic ring; or R5 and R6 are different and are independently selected from hydrogen, alkyl, cycloalkyl or alkyl substituted with cycloalkyl; The X a , X c , X1, X2, X3, and X4 are independently selected from hydrogen, halogen, C 1-4 Alkyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCF3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from -C(=O)-, -S(=O)2- or -CHCF3; R c is selected from 3 to 10 membered saturated or partially unsaturated heterocyclic groups, C 3-10 Saturated or partially unsaturated carbocyclic group or n is selected from an integer of 0-10, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3; The R d and R e are the same or different and are independently selected from hydrogen, deuterium, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, cycloalkyl-substituted alkyl, heterocyclyl, aryl or heteroaryl; said R d , R e Together with the carbon atoms to which they are attached, they form a saturated carbocyclic ring, a saturated heterocyclic ring, a cycloalkyl group, a heterocycloalkyl group, or an acyclic ring; When one of R2 or R3 is hydrogen, the other is not C 1-4 or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

2. The ecteinascidin derivative according to claim 1, characterized in that In the general formula (I): The R1 is selected from hydroxyl or cyano; The R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, mercapto, halogen, deuterated alkyl, halogenated alkyl, halogenated alkoxy, hydroxyalkyl, amino-substituted alkyl, alkylamino-substituted alkyl, alkyl, alkoxy, alkylthio, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, alkyl-substituted amino, cycloalkylalkyl, heterocyclic, aryl, heteroaryl, sulfonic acid, The R2 and R3 together with the carbon atoms to which they are connected constitute a cycloalkyl group, a heterocycloalkyl group or a non-cyclic group; The x is selected from an integer of 1-10; The R a and R b Each is independently selected from hydrogen, deuterium, hydroxyl, hydroxyalkyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a heterocycloalkyl group or is not ring-forming; R4 is selected from hydrogen, alkyl, deuterated alkyl, halogenated alkyl, amino-substituted alkyl, alkyl substituted with alkylamino or The s is selected from an integer of 1-10; Said R5 and R6 are both hydrogen or alkyl, said R5, R6 together with the carbon atoms to which they are connected constitute a cycloalkyl, a heterocycloalkyl or acyclic; or R5 and R6 are different and are independently selected from hydrogen, alkyl, cycloalkyl or cycloalkylalkyl; Said X1 and X4 are hydrogen; The X a , X c , X2, X3 are independently selected from hydrogen, halogen, C 1-4 Alkyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCF3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from C(=O), S(=O)2 or -CHCF3; R c is selected from 3 to 10 membered saturated or partially unsaturated heterocyclic groups, C 3-10 Saturated or partially unsaturated carbocyclic group or n is selected from an integer of 0-10, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3; The R d and R e are the same or different and are independently selected from hydrogen, deuterium, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 alkynyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, aryl or heteroaryl; R d , R e Together with the carbon atoms to which it is attached, it forms a cycloalkyl group, a heterocycloalkyl group or is not ring-forming; When one of R2 or R3 is hydrogen, the other is not C 1-4 When both R2 and R3 are hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

3. The ecteinascidin derivative according to claim 1, characterized in that In the general formula (I): The R1 is selected from hydroxyl or cyano; The R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, mercapto, halogen, deuterated alkyl, halogenated alkyl, halogenated alkoxy, hydroxy-substituted alkyl, amino-substituted alkyl, alkylamino-substituted alkyl, alkyl, alkoxy, alkylthio, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, alkyl-substituted amino, cycloalkyl-substituted alkyl, heterocyclic group, aryl, heteroaryl, The R2 and R3 together with the carbon atoms to which they are connected form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring; the x is an integer selected from 1-10; The R a and R b Each is independently selected from hydrogen, deuterium, hydroxyl, hydroxyl-substituted alkyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkyl-substituted sulfonyl, aryl-substituted sulfonyl, heteroaryl-substituted sulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a saturated heterocyclic ring or an acyclic ring; R4 is selected from hydrogen, alkyl, deuterated alkyl, halogenated alkyl, amino-substituted alkyl, alkyl substituted with alkylamino or The s is selected from an integer of 1-10; Said R5 and R6 are both hydrogen or alkyl, said R5, R6 together with the carbon atoms to which they are connected form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring; or R5 and R6 are different and are independently selected from hydrogen, alkyl, cycloalkyl or alkyl substituted with cycloalkyl; The X a , X c , X1, X2, X3, and X4 are independently selected from hydrogen, halogen, C 1-4 Alkyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCF3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from -C(=O)-, -S(=O)2- or -CHCF3; R c is selected from 3 to 10 membered saturated or partially unsaturated heterocyclic groups, C 3-10 Saturated or partially unsaturated carbocyclic group or n is selected from an integer of 0-10, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3; The R d and R e are the same or different and are independently selected from hydrogen, deuterium, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, cycloalkyl-substituted alkyl, heterocyclyl, aryl or heteroaryl; said R d , R e Together with the carbon atoms to which they are attached, they form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring; When one of R2 or R3 is hydrogen, the other is not C 1-4 or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

4. The ecteinascidin derivative according to claim 1 or 3, characterized in that In the general formula (I): The R1 is selected from hydroxyl or cyano; The R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, thiol, fluorine, chlorine, deuterated C 1-6 Alkyl, halogenated C 1-6 Alkyl, halogenated C 1-6 Alkoxy, C 1-6 Hydroxyl substituted alkyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 Cycloalkyl, C 1-6 Alkyl-substituted amino, cycloalkyl-substituted alkyl, heterocyclic group, aryl, heteroaryl, The R2 and R3 together with the carbon atoms to which they are connected form a 3- to 10-membered saturated carbocyclic ring, a 3- to 10-membered saturated heterocyclic ring or an acyclic ring; the x is an integer selected from 1-6; The R a and R b Each is independently selected from hydrogen, hydroxy, hydroxy-substituted alkyl, alkyl, cycloalkyl, aryl, alkyl-substituted sulfonyl, aryl-substituted sulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a saturated heterocyclic ring or is not a ring; R4 is selected from hydrogen, C 1-6 Alkyl, deuterated C 1-6 Alkyl, halogenated C 1-6 Alkyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl or The s is selected from an integer of 1-6; R5 and R6 are both hydrogen or C 1-6 alkyl; said R5, R6 together with the carbon atoms to which they are connected constitute a 3-10 membered saturated carbocyclic ring, a 3-10 membered saturated heterocyclic ring or an acyclic ring; or R5 and R6 are different and are independently selected from hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl or cycloalkyl-substituted alkyl; The X a , X c , X1, X2, X3, X4 are independently selected from hydrogen, fluorine, chlorine, methyl, ethyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCF3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from -C(=O)-; R c is selected from a 3- to 6-membered saturated heterocyclic group, a C 3-6 Saturated carbocyclic group or n is selected from an integer of 0-6, Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3; The R d and R e are the same or different and are independently selected from hydrogen, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, cycloalkyl-substituted alkyl or aryl; R d , R e Together with the carbon atoms to which they are attached, they form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring; When one of R2 or R3 is hydrogen, the other is not C 1-4 or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

5. The ecteinascidin derivative according to claim 1 or 3, characterized in that In the general formula (I): The R1 is selected from hydroxyl or cyano; The R2 and R3 are the same or different and are independently selected from hydrogen, hydroxyl, mercapto, fluorine, chlorine, trideuterated methyl, trifluoromethyl, trifluoromethoxy, C 1-6 Hydroxyl substituted alkyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 Cycloalkyl, C 1-6 Alkyl-substituted amino, cycloalkyl-substituted alkyl, heterocyclic group, aryl, heteroaryl, The R2 and R3 together with the carbon atoms to which they are connected form a 3- to 6-membered saturated carbocyclic ring, a 3- to 6-membered saturated heterocyclic ring or an acyclic ring; the x is an integer selected from 1-6; The R a and R b Each is independently selected from hydrogen, hydroxy, hydroxy-substituted alkyl, alkyl, cycloalkyl, aryl, alkyl-substituted sulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a saturated heterocyclic ring or is not a ring; R4 is selected from hydrogen, C 1-6 Alkyl, trideuterated methyl, trifluoromethyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl or The s is selected from an integer of 1-6; R5 and R6 are both hydrogen or C 1-6 alkyl; said R5, R6 together with the carbon atoms to which they are connected constitute a 3-6 membered saturated carbocyclic ring, a 3-6 membered saturated heterocyclic ring or an acyclic ring; or R5 and R6 are different and are independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl or cycloalkyl-substituted alkyl; The X a , X c , X1, X2, X3, X4 are independently selected from hydrogen, fluorine, chlorine, methyl, ethyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from -C(=O)-; R c is selected from a 3- to 6-membered saturated heterocyclic group, a C 3-6 Saturated carbocyclic group or n is selected from an integer of 0-6, Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3; The R d and R e are the same or different and are independently selected from hydrogen, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, cycloalkyl-substituted alkyl or aryl; R d , R e Together with the carbon atoms to which they are attached, they form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring; When one of R2 or R3 is hydrogen, the other is not C 1-4 or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

6. The ecteinascidin derivative according to claim 1 or 3, characterized in that In the general formula (I): The R1 is selected from hydroxyl or cyano; The R2 and R3 are the same or different and are independently selected from hydrogen, hydroxy, fluorine, trifluoromethyl, trifluoromethoxy, C 1-6 Hydroxyl substituted alkyl, amino substituted C 1-6 Alkyl, C 1-6 Alkylamino substituted C 1-6 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, C 1-6 Alkyl-substituted amino, The R2 and R3 together with the carbon atoms to which they are connected form a 3- to 6-membered saturated carbocyclic ring, a 3- to 6-membered saturated heterocyclic ring or an acyclic ring; the x is an integer selected from 1-2; The R a and R b Each is independently selected from hydrogen, hydroxy substituted alkyl, alkyl, alkyl substituted sulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a saturated heterocyclic ring or is not a ring; R4 is selected from hydrogen, C 1-4 Alkyl, amino substituted C 1-4 Alkyl, C 1-4 Alkylamino substituted C 1-4 Alkyl or The s is selected from an integer of 1-2; R5 and R6 are both hydrogen or C 1-6 alkyl; said R5, R6 together with the carbon atoms to which they are connected constitute a 3-6 membered saturated carbocyclic ring, a 3-6 membered saturated heterocyclic ring or an acyclic ring; or R5 and R6 are different and are independently selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl or cycloalkyl-substituted alkyl; The X a , X c , X1, X2, X3, X4 are independently selected from hydrogen, fluorine, methyl or wherein A is selected from oxygen, -NCH3 or -NH; Y is selected from -C(=O)-; R c Selected from C 3-6 Saturated carbocyclic group or n is selected from an integer of 0-1, Z is selected from hydrogen or hydroxyl; The R d and R e are the same or different and are independently selected from hydrogen, trifluoromethyl, alkyl, cycloalkyl or cycloalkyl substituted alkyl; R d , R e Together with the carbon atoms to which they are attached, they form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring; When one of R2 or R3 is hydrogen, the other is not C 1-4 or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

7. The ecteinascidin derivative according to claim 1 or 3, characterized in that In the general formula (I): The R1 is selected from hydroxyl or cyano; The R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, mercapto, halogen, deuterated alkyl, halogenated alkyl, halogenated alkoxy, hydroxy-substituted alkyl, amino-substituted alkyl, alkylamino-substituted alkyl, alkyl, alkoxy, alkylthio, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, alkyl-substituted amino, cycloalkyl-substituted alkyl, heterocyclic group, aryl, heteroaryl, The R2 and R3 together with the carbon atoms to which they are connected form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring; The x is selected from an integer of 1-10; The R a and R b Each is independently selected from hydrogen, deuterium, hydroxyl, hydroxyl-substituted alkyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkyl-substituted sulfonyl, aryl-substituted sulfonyl, heteroaryl-substituted sulfonyl; said R a , R b Together with the nitrogen atom to which it is attached, it forms a saturated heterocyclic ring or an acyclic ring; R4 is selected from hydrogen, alkyl, deuterated alkyl, halogenated alkyl, amino-substituted alkyl, alkyl substituted with alkylamino or The s is selected from an integer of 1-10; Said R5 and R6 are both hydrogen or alkyl, said R5, R6 together with the carbon atoms to which they are connected form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring; or R5 and R6 are different and are independently selected from hydrogen, alkyl, cycloalkyl or alkyl substituted with cycloalkyl; Said X1 and X4 are hydrogen; The X a , X c , X2, X3 are independently selected from hydrogen, halogen, C 1-4 Alkyl or wherein A is selected from oxygen, sulfur, -NH, -NCD3, -NCF3, -NCH3, -NCH2CH3 or -NCH2CH2CH3; Y is selected from -C(=O)-, -S(=O)2- or -CHCF3; R c is selected from 3 to 10 membered saturated or partially unsaturated heterocyclic groups, C 3-10 Saturated or partially unsaturated carbocyclic group or n is selected from an integer of 0-10, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3; The R d and R e are the same or different and are independently selected from hydrogen, deuterium, trifluoromethyl, alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, cycloalkyl-substituted alkyl, heterocyclyl, aryl or heteroaryl; R d , R e Together with the carbon atoms to which they are attached, they form a saturated carbocyclic ring, a saturated heterocyclic ring or an acyclic ring; When one of R2 or R3 is hydrogen, the other is not C 1-4 When both R2 and R3 are hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

8. The ecteinascidin derivative according to claim 1 or 3, characterized in that In the general formula (I): The R1 is selected from hydroxyl or cyano; The R2 and R3 are the same or different and are independently selected from hydrogen, hydroxy, fluorine, trifluoromethyl, trifluoromethoxy, methyl, methoxy, cyclopropyl substituted methyl, The R a and R b Each is independently selected from hydrogen or methyl; R2, R3 together with the carbon atom to which they are connected form a 3-membered saturated carbocyclic ring, a 4-membered oxygen-containing saturated heterocyclic ring or a 4-membered sulfur-containing saturated heterocyclic ring; x is selected from an integer of 1-2; Said R4 is selected from hydrogen or methyl; R5 and R6 are independently selected from hydrogen or methyl. When both R5 and R6 are methyl, the carbon atoms connected to the two form a 3-membered saturated carbocyclic ring. Said X1 and X4 are independently selected from hydrogen or methoxy; The X a , X2, X3 are independently selected from hydrogen, fluorine, methyl or wherein A is selected from oxygen, -NCH3 or -NH; Y is selected from -C(=O)-; R c Selected from C 3-6 Saturated carbocyclic group or n is selected from an integer of 0-1, Z is selected from hydrogen or hydroxyl; The R d and R e are the same or different and are independently selected from hydrogen, trifluoromethyl, cyclopropyl or cyclopropyl-substituted methyl; R d , R e Together with the carbon atom to which it is attached, it forms a 3- to 5-membered saturated carbocyclic ring; When one of R2 or R3 is hydrogen, the other is not methyl; or when R2, R3, X1, and X4 are all hydrogen, X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3, where R aa , R ab and R ac are each independently selected from hydrogen or C 1-4 of alkyl.

9. The ecteinascidin derivative according to claim 1, characterized in that The compound in the general formula (I) is selected from at least one of the following compounds:

10. The ecteinascidin derivative according to claim 9, characterized in that In the compounds P1-P32, P36-P38, P58-P91, P93, P95-P97, P101-P108, P110, P112-P114, P118, P119, P136, P140-P142, P144, P146-P148, P152, P166, P167, P170, P172, P174, P186-P188, P241, P253, P261-P262, P264, and T1-T19, the -CN at the R1 position is replaced by -OH.

11. The ecteinascidin derivative according to claim 9, characterized in that The derivatives include a single chiral component or a mixture of diastereoisomers.

12. The ecteinascidin derivative according to any one of claims 1 to 3, characterized in that The compounds of the general formula (I) also include pharmaceutically acceptable salts, tautomers and stereoisomers thereof.

13. Use of the ecteinascidin derivative according to any one of claims 1 to 12 in the preparation of anticancer drugs.

14. An antibody-drug conjugate, characterized in that: The invention comprises the ecteinascidin derivative and antibody according to any one of claims 1 to 12.

15. Use of the antibody-drug conjugate according to claim 14 in the preparation of anticancer drugs.

16. A compound of general formula (III), or a pharmaceutically acceptable salt thereof, or a tautomer or stereoisomer thereof, characterized in that: MA1L2-D General formula (III) Wherein: the M is selected from The L1 is selected from One or more combinations of; n1 = an integer of 1-20, n2 = an integer of 0-20, n3 = an integer of 1-20, n4 = an integer of 0-20, n5 = an integer of 0-20, n6 = an integer of 0-20, n7 = an integer of 0-20; The L2 is n8=an integer from 0 to 20, n9=an integer from 0 to 20, n 10 = integer from 0 to 20, n 11 = integer from 0 to 20; D is selected from the product group formed by the ecteinascidin derivative according to any one of claims 1 to 12 losing one or more atoms or groups; Wherein D is covalently bonded to L2 via the nitrogen or oxygen in D.

17. The compound according to claim 16, characterized in that: In the general formula (III), Wherein: the M is selected from The L1 is selected from One or more combinations of; n1 = an integer of 1-20, n2 = an integer of 0-20, n3 = an integer of 1-20 or n4 = an integer of 0-20; The L2 is n8=an integer from 0 to 20, n 10 = integer from 0 to 20 or n 11 = integer from 0 to 20; D is selected from the product group formed by the ecteinascidin derivative according to any one of claims 1 to 12 losing one or more atoms or groups; D is covalently bonded to L2 through the nitrogen or oxygen in any of X1, X2, X3, X4, R2, R3 or R4 in D.

18. The compound according to claim 16, characterized in that: In the general formula (III), Wherein: the M is selected from The L1 is selected from One or more combinations of; n1=an integer of 1-18, n2=an integer of 0-18, n3=an integer of 1-18, n4=an integer of 0-18; The L2 is n8=an integer from 0 to 18 or n 11 = an integer from 0 to 18; D is selected from the product group formed by the product of the ecteinascidin derivative according to any one of claims 1 to 12 losing one or more atoms or groups; D is covalently bonded to L2 through the nitrogen or oxygen in any of X1, X2, X3, X4, R2, R3 or R4 in D.

19. The compound according to claim 16, characterized in that: In the general formula (III), The M is selected from The L1 is selected from One or more combinations of; n1 = an integer of 1-20, n2 = an integer of 1-20, n3 = an integer of 1-20, n4 = an integer of 1-20; The L2 is n8=integer from 1 to 20, n 10 = integer from 1 to 20; D is selected from the product group formed by the ecteinascidin derivative according to any one of claims 1 to 12 losing one or more atoms or groups; D is covalently bonded to L2 through the nitrogen or oxygen in any of X1, X2, X3, X4, R2, R3 or R4 in D.

20. The compound according to claim 16, characterized in that: The compound of general formula (III) is selected from at least one of the compounds in the following table:

21. An antibody-drug conjugate, characterized in that: Including the structure shown in general formula (IV): wherein Ab is an antibody, an antibody fragment or a protein, wherein the antibody is selected from a mouse antibody, a rabbit antibody, a phage display antibody, a yeast display antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody and a multispecific antibody; M-L1-L2-D is selected from any compound according to any one of claims 16 to 20, or a pharmaceutically acceptable salt thereof, a tautomer, a stereoisomer thereof; n 12 Select any value between 0-20.

22. The antibody-drug conjugate according to claim 21, characterized in that: The 12 Select any value between 1-20.

23. The antibody-drug conjugate according to claim 21, characterized in that: In the general formula (IV), Ab is selected from abciximab, alemtuzumab, anetuzumab, atezolizumab, avelumab, basiliximab, bevacizumab, blinatumomab, brentuximab, catumaxomab, cetuximab, cetuximab, cotuximab, daclizumab, daratumumab, deninutuzumab, denosumab, depatuxizumab, denutumab, durvalumab, elotuzumab, enroku, gebatumumab, gemtuzumab, ibritumomab tiuxetan, indalatuzumab, indoximab, inotuzumab, ipilimumab, labetuzumab, latuzumab, latuximab, rifatuzumab, lovotuzumab, miratumumab, metuximab, natalizumab, natuximab, natuximab, Tocilizumab, nimotuzumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, omalizumab, palivizumab, panitumumab, pertrastuzumab, pembrolizumab, pertuzumab, pinatuzumab, polotuzumab, ramucirumab, lovatuzumab, tasitumab, setuximab, setuximab, sofetozumab, vadatuximab, worthertuzumab, trastuzumab, anti-CD4 antibody, anti-CD5 antibody, anti-CD13 antibody and anti-CD30 antibody, antigen-binding fragments or immunologically active portions thereof; M-L1-L2-D is selected from the compound of any one of claims 16 to 20, or a pharmaceutically acceptable salt thereof, a tautomer, a stereoisomer thereof; n 12 Select any value between 0-12.

24. The antibody-drug conjugate according to claim 21, characterized in that: In the general formula (IV), Ab is selected from pertuzumab, saccharotuzumab, mituximab, cetuximab, trastuzumab or cetuzumab; M-L1-L2-D is selected from the compound according to any one of claims 16 to 20, or a pharmaceutically acceptable salt thereof, a tautomer or a stereoisomer thereof; n 12 Select any value between 0-8.

25. The antibody-drug conjugate according to claim 21, characterized in that: The antibody-drug conjugate in the general formula (IV) is selected from at least one of the compounds of the following structural formulas: n 12 Select any value between 0-8.

26. The antibody-drug conjugate according to claim 25, characterized in that: The 12 Select any value between 1-8.

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