Novel ecteinascidin derivative, preparation method therefor and use thereof, and antibody-drug conjugate and use thereof

Abstract

The present invention relates to a novel ecteinascidin derivative, a preparation method therefor and a use thereof, and an antibody-drug conjugate and a use thereof. Provided are novel ecteinascidin derivatives, wherein some of the novel ecteinascidin derivatives have relatively strong in-vitro cytotoxicity (1-10 nM), and the remainder have stronger in-vitro cytotoxicity (less than 1 nM). Additionally, provided are antibody-drug conjugates based on the novel ecteinascidin derivatives, wherein some of the novel antibody-drug conjugates have relatively strong in-vitro cytotoxicity (1-10 nM), and the remainder have stronger in-vitro cytotoxicity (less than 1 nM).

Description

Novel ecteinascidin derivatives, preparation methods and uses thereof, antibody-drug conjugates and applications thereof Technical Field

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

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

[0003] Trabectedin (Yondelis, ET-743) is one of these drugs, exhibiting 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 synthesized (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, and DNA repair mechanisms. 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 were neutropenic sepsis (severe 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 chemotherapy 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 their effectiveness and safety.

[0005] Summary of the Invention

[0006] The present invention provides novel ecteinascidin derivatives. The ecteinascidin compounds have good anti-tumor activity, and antibody-drug conjugates based thereon have the potential to be used for the treatment of tumor diseases.

[0007] The ecteinascidin derivative is characterized in that its structure is a compound represented by the general formula (I):

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

[0009] Said R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, mercapto, halogen, deuterated alkyl, halogenated alkyl, halogenated alkoxy, hydroxyl 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 are each independently selected from hydrogen, deuterium, hydroxy, hydroxy-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] Said R4 is selected from hydrogen, alkyl, deuterated alkyl, halogenated alkyl, amino-substituted alkyl, alkylamino-substituted alkyl or Said s is selected from an integer of 1-10;

[0012] R5 and R6 are both hydrogen or alkyl, and R5, R6 together with the carbon atoms to which they are attached 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, an alkyl group, a cycloalkyl group, or an alkyl group substituted with a cycloalkyl group;

[0013] The X a 、X c , X1, X2, X3, 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 to 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, and X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa 、R ab and R ac 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] Said R1 is selected from hydroxyl or cyano;

[0018] Said 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 2-6Alkynyl, 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] Said R4 is selected from hydrogen, alkyl, deuterated alkyl, halogenated alkyl, amino-substituted alkyl, alkylamino-substituted alkyl or Said s is selected from an integer of 1-10;

[0022] R5 and R6 are both hydrogen or alkyl, and R5, R6 together with the carbon atoms to which they are attached form a cycloalkyl, 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 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 to 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 、Re Together with the carbon atoms to which it is attached, it forms a cycloalkyl group, a heterocycloalkyl group, or is not a ring;

[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 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] Said R1 is selected from hydroxyl or cyano;

[0029] Said R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, mercapto, halogen, deuterated alkyl, halogenated alkyl, halogenated alkoxy, hydroxyl 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, 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 are each independently selected from hydrogen, deuterium, hydroxy, hydroxy-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 uncyclic ring;

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

[0032] R5 and R6 are both hydrogen or alkyl, and R5, R6 together with the carbon atoms to which they are attached 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 cycloalkyl-substituted alkyl;

[0033] The X a 、Xc , X1, X2, X3, 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 to 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, and X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa 、R ab and R ac 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] Said R1 is selected from hydroxyl or cyano;

[0038] Said 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, C1-6 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, R2 and R3 together with the carbon atoms to which they are attached form a 3- to 10-membered saturated carbocyclic ring, a 3- to 10-membered saturated heterocyclic ring, or an acyclic ring; x is an integer selected from 1 to 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] Said 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 Said 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 atom 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 3 to 6 membered saturated heterocyclic group, C 3-6 Saturated carbocyclic group or n is selected from an integer of 0 to 6, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0043] 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;

[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, and X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa 、R ab and R ac 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] Said R1 is selected from hydroxyl or cyano;

[0047] Said R2 and R3 are the same or different and are independently selected from hydrogen, hydroxyl, mercapto, fluorine, chlorine, trideuteromethyl, 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, R2 and R3 together with the carbon atoms to which they are attached form a 3- to 6-membered saturated carbocyclic ring, a 3- to 6-membered saturated heterocyclic ring, or an acyclic ring; x is selected from an integer of 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] Said 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 Said 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 atom 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 3 to 6 membered saturated heterocyclic group, C 3-6 Saturated carbocyclic group or n is selected from an integer of 0 to 6, and 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, and X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa 、R ab and R ac 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] Said R1 is selected from hydroxyl or cyano;

[0056] Said 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 groups, R2 and R3 together with the carbon atoms to which they are attached form a 3- to 6-membered saturated carbocyclic ring, a 3- to 6-membered saturated heterocyclic ring, or an acyclic ring; x is an integer selected from 1 to 2;

[0057] The R a and R b are 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] Said 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 Said 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 atom 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 an integer selected from 0 to 1, and Z is selected from hydrogen or hydroxyl;

[0061] 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;

[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, and X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa 、R ab and R ac 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] Said R1 is selected from hydroxyl or cyano;

[0065] Said R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, mercapto, halogen, deuterated alkyl, halogenated alkyl, halogenated alkoxy, hydroxyl 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, 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 are each independently selected from hydrogen, deuterium, hydroxy, hydroxy-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 uncyclic ring;

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

[0069] R5 and R6 are both hydrogen or alkyl, and R5, R6 together with the carbon atoms to which they are attached 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 cycloalkyl-substituted alkyl;

[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 to 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 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] Said R1 is selected from hydroxyl or cyano;

[0076] Said 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 attached 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, together with the carbon atom to which they are connected, they form 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 an integer selected from 0 to 1, and 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 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] According to one embodiment of the present invention, the compound in the general formula (I) is selected from at least one compound 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.

[0085] 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.

[0086] 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.

[0087] According to one embodiment of the present invention, the derivative comprises a single chiral component or is a mixture of diastereomers.

[0088] According to one embodiment of the present invention, the present invention also includes pharmaceutically acceptable salts, tautomers and stereoisomers of the compound of general formula (I).

[0089] In the compound of the aforementioned general formula (I), a hydroxyl-substituted alkyl group is also referred to as a hydroxyalkyl group; a cycloalkyl-substituted alkyl group is also referred to as a cycloalkylalkyl group; an alkyl-substituted sulfonyl group is also referred to as an alkylsulfonyl group; an aryl-substituted sulfonyl group is also referred to as an arylsulfonyl group; and a heteroaryl-substituted sulfonyl group is also referred to as a heteroarylsulfonyl group.

[0090] According to one embodiment of the present invention, in the general formula (I):

[0091] Said R1 is selected from hydroxyl or cyano;

[0092] Said 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, C1-6 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;

[0093] The R a and R b Each is independently selected from hydrogen, hydroxy, 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;

[0094] The x is selected from an integer of 1-6;

[0095] Said 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 Said s is selected from an integer of 1-6;

[0096] R5 and R6 are both hydrogen or C 1-6 Alkyl; the 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;

[0097] 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 cis selected from 3 to 6 membered saturated heterocyclic group, C 3-6 Saturated carbocyclic group or n is selected from an integer of 0 to 6, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0098] 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 a ring;

[0099] 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, and X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa 、R ab and R ac Each independently selected from hydrogen or C 1-4 of alkyl.

[0100] According to one embodiment of the present invention, in the general formula (I):

[0101] Said R1 is selected from hydroxyl or cyano;

[0102] Said R2 and R3 are the same or different and are independently selected from hydrogen, hydroxyl, mercapto, fluorine, chlorine, trideuteromethyl, 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, 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 3-6 Cycloalkyl, 3- to 6-membered heterocycloalkyl, or acyclic;

[0103] The R aand 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;

[0104] The x is selected from an integer of 1-6;

[0105] Said 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 Said s is selected from an integer of 1-6;

[0106] R5 and R6 are both hydrogen or C 1-6 Alkyl; the 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;

[0107] 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 3 to 6 membered saturated heterocyclic group, C 3-6 Saturated carbocyclic group or n is selected from an integer of 0 to 6, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0108] 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 a ring;

[0109] When one of R2 or R3 is hydrogen, the other is not C 1-4or when R2, R3, X1, and X4 are all hydrogen, and X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa 、R ab and R ac Each independently selected from hydrogen or C 1-4 of alkyl.

[0110] According to one embodiment of the present invention, in the general formula (I):

[0111] Said R1 is selected from hydroxyl or cyano;

[0112] Said 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;

[0113] 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;

[0114] The x is selected from an integer of 1-2;

[0115] Said 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 Said s is selected from an integer of 1-2;

[0116] R5 and R6 are both hydrogen or C 1-6 Alkyl; the 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;

[0117] 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 an integer selected from 0 to 1, and Z is selected from hydrogen or hydroxyl;

[0118] 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 a ring;

[0119] 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, and X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa 、R ab and R ac Each independently selected from hydrogen or C 1-4 of alkyl.

[0120] According to one embodiment of the present invention, in the general formula (I):

[0121] Said R1 is selected from hydroxyl or cyano;

[0122] Said 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;

[0123] The x is selected from an integer of 1-10;

[0124] The R a and R bEach 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;

[0125] Said R4 is selected from hydrogen, alkyl, deuterated alkyl, halogenated alkyl, amino-substituted alkyl, alkylamino-substituted alkyl or Said s is selected from an integer of 1-10;

[0126] R5 and R6 are both hydrogen or alkyl, and R5, R6 together with the carbon atoms to which they are attached form a cycloalkyl, heterocycloalkyl or acyclic; or R5 and R6 are different and are independently selected from hydrogen, alkyl, cycloalkyl or cycloalkylalkyl;

[0127] Said X1 and X4 are hydrogen;

[0128] 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 to 10, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0129] 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 a ring;

[0130] 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 Raa 、R ab and R ac Each independently selected from hydrogen or C 1-4 of alkyl.

[0131] According to one embodiment of the present invention, in the general formula (I): wherein: the R1 is selected from hydroxyl or cyano;

[0132] Said R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, mercapto, halogen, deuterated alkyl, halogenated alkyl, halogenated alkoxy, hydroxyl 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;

[0133] The R a and R b are each independently selected from hydrogen, deuterium, hydroxy, hydroxy-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;

[0134] The x is selected from an integer of 1-10;

[0135] Said R4 is selected from hydrogen, alkyl, deuterated alkyl, halogenated alkyl, amino-substituted alkyl, alkylamino-substituted alkyl or Said s is selected from an integer of 1-10;

[0136] R5 and R6 are both hydrogen or alkyl; R5, R6 together with the carbon atoms to which they are attached constitute a cycloalkyl, heterocycloalkyl or acyclic; or R5 and R6 are different and are independently selected from hydrogen, alkyl, cycloalkyl or cycloalkyl-substituted alkyl;

[0137] The X a 、X c , X1, X2, X3, 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 cis 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 to 10, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0138] 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 a ring;

[0139] 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, and X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa 、R ab and R ac Each independently selected from hydrogen or C 1-4 of alkyl.

[0140] According to one embodiment of the present invention, in the general formula (I):

[0141] Said R1 is selected from hydroxyl or cyano;

[0142] Said 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, 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;

[0143] 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;

[0144] The x is selected from an integer of 1-6;

[0145] Said 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 Said s is selected from an integer of 1-6;

[0146] R5 and R6 are both hydrogen or C 1-6 Alkyl; the 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;

[0147] 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 3 to 6 membered saturated heterocyclic group, C 3-6 Saturated carbocyclic group or n is selected from an integer of 0 to 6, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0148] 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, 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 a ring;

[0149] 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, and X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa 、R ab and R ac Each independently selected from hydrogen or C 1-4 of alkyl.

[0150] According to one embodiment of the present invention, in the general formula (I):

[0151] Said R1 is selected from hydroxyl or cyano;

[0152] Said R2 and R3 are the same or different and are independently selected from hydrogen, hydroxyl, mercapto, fluorine, chlorine, trideuteromethyl, 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, 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;

[0153] 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;

[0154] The x is selected from an integer of 1-6;

[0155] Said R4 is selected from hydrogen, C 1-6 Alkyl, trideuterated methyl, trifluoromethyl, amino substituted C1-6 Alkyl, C 1-6 Alkylamino-substituted C 1-6 Alkyl or Said s is selected from an integer of 1-6;

[0156] R5 and R6 are both hydrogen or C 1-6 Alkyl; the 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;

[0157] 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 3 to 6 membered saturated heterocyclic group, C 3-6 Saturated carbocyclic group or n is selected from an integer of 0 to 6, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0158] 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 a ring;

[0159] 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, and X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa 、R ab and R ac Each independently selected from hydrogen or C 1-4 of alkyl.

[0160] According to one embodiment of the present invention, in the general formula (I):

[0161] Said R1 is selected from hydroxyl or cyano;

[0162] Said 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;

[0163] The R a and R b are 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;

[0164] The x is selected from an integer of 1-2;

[0165] Said 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 Said s is selected from an integer of 1-2;

[0166] R5 and R6 are both hydrogen or C 1-6 Alkyl; the 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;

[0167] 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 an integer selected from 0 to 1, and Z is selected from hydrogen or hydroxyl;

[0168] 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 a ring;

[0169] 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, and X2 or X3 is not -NHCH3, -NHCH2CH3 or -NHCH2CH2CH3; wherein R aa 、R ab and R ac Each independently selected from hydrogen or C 1-4 of alkyl.

[0170] According to one embodiment of the present invention, in the general formula (I):

[0171] Said R1 is selected from hydroxyl or cyano;

[0172] Said R2 and R3 are the same or different and are independently selected from hydrogen, deuterium, hydroxyl, mercapto, halogen, deuterated alkyl, halogenated alkyl, halogenated alkoxy, hydroxyl 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;

[0173] The x is selected from an integer of 1-10;

[0174] The R a and R b are each independently selected from hydrogen, deuterium, hydroxy, hydroxy-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;

[0175] Said R4 is selected from hydrogen, alkyl, deuterated alkyl, halogenated alkyl, amino-substituted alkyl, alkylamino-substituted alkyl or Said s is selected from an integer of 1-10;

[0176] R5 and R6 are both hydrogen or alkyl, and R5, R6 together with the carbon atoms to which they are attached form a cycloalkyl, heterocycloalkyl or acyclic; or R5 and R6 are different and are independently selected from hydrogen, alkyl, cycloalkyl or cycloalkyl-substituted alkyl;

[0177] Said X1 and X4 are hydrogen;

[0178] 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 to 10, and Z is selected from hydrogen, hydroxyl, thiol, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -CD3 or -CF3;

[0179] 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 a ring;

[0180] 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 Each independently selected from hydrogen or C 1-4 of alkyl.

[0181] The use of the aforementioned ecteinascidin derivatives in the preparation of anticancer drugs.

[0182] An antibody-drug conjugate, characterized by comprising the ecteinascidin derivative according to claim 1 and an antibody.

[0183] Application of the aforementioned antibody-drug conjugates in the preparation of anticancer drugs.

[0184] A compound of general formula (III), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0185] M-L1-L2-D

[0186] General formula (III)

[0187] Wherein: the M is selected from

[0188] 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;

[0189] 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 = an integer from 0 to 20;

[0190] D is selected from the product group formed by the aforementioned ecteinascidin derivative losing one or more atoms or groups;

[0191] Where D is covalently bound to L2 through the nitrogen or oxygen in D;

[0192] According to one embodiment of the present invention, in the general formula (III),

[0193] Wherein: said M is selected from

[0194] 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;

[0195] The L2 is n8=an integer from 0 to 20, n 10 = integer from 0 to 20 or n 11 = an integer from 0 to 20;

[0196] D is selected from the product group formed by the aforementioned ecteinascidin derivative losing one or more atoms or groups;

[0197] D is covalently bound to L2 through the nitrogen or oxygen in any of X1, X2, X3, X4, R2, R3 or R4 in D;

[0198] According to one embodiment of the present invention, in the general formula (III),

[0199] Wherein: said M is selected from

[0200] 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;

[0201] The L2 is n8=an integer from 0 to 18 or n 11 = an integer from 0 to 18;

[0202] D is selected from the product group formed by the product of the aforementioned ecteinascidin derivative losing one or more atoms or groups;

[0203] D is covalently bonded to L2 through the nitrogen or oxygen in any of X1, X2, X3, X4, R2, R3 or R4 in D.

[0204] According to one embodiment of the present invention, in the general formula (III),

[0205] The M is selected from

[0206] The L1 is selected from One or more combinations of; n1 = 1-20, n2 = 1-20, n3 = 1-20, n4 = 1-20;

[0207] The L2 is n8=1-20、n 10 =1-20;

[0208] D is selected from the product group formed by the aforementioned ecteinascidin derivative losing one or more atoms or groups;

[0209] D is covalently bonded to L2 through the nitrogen or oxygen in any of X1, X2, X3, X4, R2, R3 or R4 in D.

[0210] According to one embodiment of the present invention, in the compound of the general formula (III), M is selected from

[0211] 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;

[0212] The L2 is n8=1-20、n9=1-20 or n 10 =1-20;

[0213] D is selected from the aforementioned ecteinascidin derivatives, or pharmaceutically acceptable salts, tautomers, and product groups formed by losing one or more atoms or groups from their stereoisomers;

[0214] Where D is covalently bound to L2 through the nitrogen or oxygen in D;

[0215] According to one embodiment of the present invention, in the general formula (III),

[0216] Wherein: said M is selected from

[0217] The L1 is selected from One or more combinations of; n1 = 1-20, n2 = 1-20, n3 = 1-20 or n4 = 1-20;

[0218] The L2 is n8=1-20 or n 10 =1-20;

[0219] D is selected from the aforementioned ecteinascidin derivatives, or pharmaceutically acceptable salts, tautomers, and product groups formed by losing one or more atoms or groups from their stereoisomers;

[0220] D is covalently bonded to L2 through the nitrogen or oxygen in any of X1, X2, X3, X4, R2, R3 or R4 in D.

[0221] According to one embodiment of the present invention, in the general formula (III),

[0222] Wherein: said 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;

[0224] The L2 is n8=1-20;

[0225] D is selected from the aforementioned ecteinascidin derivatives, or pharmaceutically acceptable salts, tautomers, and product groups formed by losing one or more atoms or groups from their stereoisomers;

[0226] D is covalently bonded to L2 through the nitrogen or oxygen in any of X1, X2, X3, X4, R2, R3 or R4 in D.

[0227] Among them, n1, n2, n3, n4, n5, n6, n7, n8, n9, n 10 is an integer.

[0228] According to one embodiment of the present invention, the compound of formula (III) is selected from at least one of the compounds in the following table:

[0229] An antibody-drug conjugate, characterized in that it comprises a structure as shown in general formula (IV):

[0230] 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 the 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.

[0231] 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.

[0232] According to one embodiment of the present invention, the n 12 Select any value between 1-20.

[0233] According to one embodiment of the present invention, in the general formula (IV), the Ab is a monoclonal antibody.

[0234] 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, coxutuximab, daclizumab, daratumumab, deninutuzumab, denosumab, depatuxizumab, dinutuximab, durvalumab, elotuzumab, enroku, gebatuzumab, gemtuzumab, ibritumomab tiuxetan, indalatuzumab, indoximab, inutuzumab, ipilimumab, labetuzumab, latuzumab, latuximab, rifatuzumab, lovutuzumab, miratumumab, mituximab, miratumumab ... Antibody, natalizumab, nesituzumab, nimotuzumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, omalizumab, palivizumab, panitumumab, pertrastuzumab, pembrolizumab, pertuzumab, pinatuzumab, polotuzumab, ramucirumab, lovatuzumab, tasitumumab, 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 compound of the aforementioned general formula (III), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof; n 12 Select any value between 0-12.

[0235] The English name of Abciximab is: Abciximab; the English name of Alemtuzumab is: Alemtuzumab; the English name of Anetuzumab 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 Cituximab 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 Durvalumab; Elotuzumab is called Elotuzumab; Enfortumab is called Enfortumab; Glembatu is called Glembatu mumab; Gemtuzumab is called Gemtuzumab; Ibritumomab is called Ibritumomab; Indatuximab is called Indusatumab; Inotuzumab is called Inotuzumab; Ipilimumab is called Ipilimumab; Labetuzumab is called Ladiratuzumab; Laprituximab is called Laprituximab; Lifastuzumab is called Lifastuzumab; Lorvotuzumab is called Lorvotuzumab uzumab; Milatuzumab; Mirvetuximab; Naratuximab; Necitumumab; Nimotuzumab; Nivolumab; Obinutuzumab; Ofatumumab; Olaratumab; Omalizumab; Palivizumab;Panitumumab is called Panitumumab; Patritumab is called Patritumab; Pembrolizumab is called Pembrolizumab; Pertuzumab is called Pertuzumab; Pinatuzumab is called Pinatuzumab; Polotuzumab is called Polatuzumab; Ramucirumab is called Ramucirumab; Rovalpituzumab is called Rovalpituzumab; Sacituzumab is called Sacituzumab; Siltuximab is called Siltuximab; Sirtratumab is called Sofituzumab; Vadastuximab is called Vadastuximab; Vorsetuzumab is called Vorsetuzumab; Trastuzumab is called Trastuzumab.

[0236] According to one embodiment of the present invention, the antibody-drug conjugate in the general formula (IV) is selected from at least one compound of the following structural formula:

[0237] n 12 Select any value between 0-8.

[0238] According to one embodiment of the present invention, the n 12 Select any value between 1-8

[0239] Another aspect of the present invention is to provide the ecteinascidin derivatives of the present invention, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof 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.

[0240] Another aspect of the present invention is to provide an antibody-drug conjugate based on an ecteinascidin derivative according to the present invention for use in 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.

[0241] The main advantages of the present invention are:

[0242] 1. Some of the novel ecteinascidin derivatives provided by the present invention have relatively strong in vitro cytotoxicity (1-10 nM), while others have even stronger in vitro cytotoxicity (<1 nM).

[0243] 2. The antibody-drug conjugates based on novel ecteinascidin derivatives provided by the present invention have strong in vitro cytotoxicity (1-10 nM), and some antibody-drug conjugates have even stronger in vitro cytotoxicity (<1 nM). DETAILED DESCRIPTION

[0244] 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.

[0245] 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.

[0246] Synthesis and Characterization of Eteinascidin Derivatives

[0247] Example 1: Synthesis of Compound P1

[0248] Step 1: Synthesis of 2-(4-fluoro-3-nitrophenyl)ethylenediamine

[0249] To a THF solution of 2-(4-fluoro-3-nitrobenzene)acetonitrile (100 mg, 0.55 mmol, 1.0 eq) was added borane dimethyl sulfide (2.5 M, 0.69 mL, 2.5 eq) and the mixture was refluxed for 1 hour. The mixture was quenched with MeOH and concentrated. Purification by thin-layer chromatography afforded the desired product (70 mg, 97.8% yield) as a colorless oil.

[0250] LC-MS (ESI) m / z: 185.3 [M+H] +

[0251] Step 2: Synthesis of 5-(2-aminoethyl)-2-fluoroaniline

[0252] To a solution of 2-(4-fluoro-3-nitrophenyl)ethylenediamine (70 mg, 454 mmol, 1.0 eq) in MeOH (20 mL) was added 20 mg of 5% wet Pd / C. The suspension was repeatedly replaced with H2 under vacuum and stirred overnight at room temperature under H2. The suspension was filtered through Celite, and the filter cake was washed with methanol. The combined filtrates were concentrated to dryness and used directly in the next step.

[0253] Step 3: Synthesis of P1

[0254] 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 completion of the reaction, the solution was filtered, and the residue was purified by preparative HPLC to obtain the desired product (1.5 mg, 12.3% yield) as a white solid. LC-MS (ESI) m / z: 758.1 [M+H] +

[0255] 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).

[0256] Example 2: Synthesis of Compound P5

[0257] Step 1: Synthesis of 2-(4-(dimethylamino)-3-nitrophenyl)acetonitrile

[0258] 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 KCO (115.05 mg, 0.832 mmol, 1.5 eq) were added to a DMSO (1.5 mL) sealed tube. The mixture was stirred at 120°C overnight under argon. After quenching with water, the mixture 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 the crude product (125 mg, yield: 100%) as a brown solid, which was used directly in the next step without purification.

[0259] LC-MS (ESI) m / z: 206.4 [M+H] +

[0260] Step 2: Synthesis of 4-(2-aminoethyl)-N,N-dimethyl-2-nitroaniline

[0261] The crude product, 2-(4-(dimethylamino)-3-nitrophenyl)acetonitrile (125 mg, 0.61 mmol, 1.0 eq), was dissolved in THF (3 mL). Borane dimethyl sulfide (2.5 M, 0.76 mL, 2.5 eq) was added and stirred at reflux for 1 hour. The mixture was quenched with MeOH and concentrated. The residue was purified by thin-layer chromatography to afford the title compound (90 mg, 70.0% yield) as a colorless oil.

[0262] LC-MS (ESI) m / z: 210.5 [M+H] +

[0263] Step 3: Synthesis of 4-(2-aminoethyl)-N,N-dimethylbenzene-1,2-diamine

[0264] To a solution of 4-(2-aminoethyl)-N,N-dimethyl-2-nitroaniline (90 mg, 430 mmol, 1.0 eq) in MeOH (10 mL) was added 20 mg of 5% wet Pd / C under N₂ protection. The suspension was degassed under vacuum and replaced with H₂ several times. The mixture was stirred at room temperature under H₂ protection overnight. The suspension was filtered through celite, and the filter cake was washed with methanol. The combined filtrates were concentrated and used directly in the next step.

[0265] LC-MS (ESI) m / z: 180.2 [M+H] +

[0266] Step 4: Synthesis of P5

[0267] 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 completion of the reaction, the reaction solution was filtered, and the filtrate was purified by preparative HPLC to obtain the desired product (5.47 mg, 43.4% yield) as a white solid.

[0268] LC-MS (ESI) m / z: 783.0 [M+H] +

[0269] 1H 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),2.43–2.34(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).

[0270] Example 3: Synthesis of Compounds P9 and P10

[0271] Step 1: Synthesis of compound 2-amino-1-(3-aminophenyl)ethylene glycol

[0272] To a solution of 2-amino-1-(3-nitrophenyl)ethan-1-ol (200 mg, 0.0188 mmol, 1.0 eq.) in methanol (50 mL) was added 5% wet Pd / C (20 mg) under N₂ protection. The suspension was degassed under vacuum and replaced with H₂ several times. The mixture was stirred at room temperature under H₂ protection for 16 hours. The suspension was filtered through celite, and the filter cake was washed with methanol. The combined filtrates were concentrated to give the crude product (190 mg, crude) as an oil.

[0273] LC-MS (ESI) m / z: 153.2 [M+H] + .

[0274] 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).

[0275] Step 2: Synthesis of P9 and P10

[0276] 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) and stirred at room temperature overnight. The solvent was removed under reduced pressure and purified by preparative HPLC to afford P9 (1.67 mg, 13.91% yield) and P10 (2.43 mg, 20% yield) as white solids. LC-MS (ESI) m / z: 756.2 [M+H] +

[0277] 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).

[0278] 1 H 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).

[0279] Example 4: Synthesis of Compounds P12 and P13

[0280] Step 1: Synthesis of compound 2-hydroxy-2-(4-methoxy-3-nitrophenyl)acetonitrile

[0281] 4-Methoxy-3-nitrobenzaldehyde (4.0 g, 22 mmol, 1 eq.) and trimethylsilyl cyanide (8.7 g, 88 mmol, 4 eq.) were added to a reaction flask and dissolved in dichloromethane (50 mL). LiClO₄·3H₂O (3.53 g, 22 mmol, 1 eq.) was added dropwise and stirred at room temperature overnight. The reaction was complete by TLC (EA:n-heptane = 1 / 3). The reaction solution was filtered, and the filtrate was washed twice with 10% dilute hydrochloric acid. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product (3.9 g, 85% yield) as a yellow solid, which was used directly in the next step.

[0282] Step 2: Synthesis of compound 2-amino-1-(4-methoxy-3-nitrophenyl)ethane-1-ol

[0283] 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), and 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 raw material reaction was complete. The temperature was lowered to 0°C and methanol was slowly added to quench the solution. The solution gradually turned purple and was concentrated. The solution was purified by silica gel flash column chromatography (DCM / MeOH = 30 / 1 to 10 / 1) to obtain the title compound (3.8 g, yield: 96%) as a light yellow solid.

[0284] LC-MS (ESI) m / z: 195.2 [M-OH] +

[0285] 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).

[0286] Step 3: Synthesis of compound 2-amino-1-(3-amino-4-methoxyphenyl)ethane-1-ol

[0287] To a solution of 2-amino-1-(4-methoxy-3-nitrophenyl)ethane-1-ol (3.8 g, 17.9 mmol) in 100 mL of methanol was added 5% wet Pd / C (200 mg). The suspension was degassed under vacuum and replaced with H₂ several times. Stirred at room temperature under H₂ for 2 hours. LC-MS indicated complete reaction of the starting material. The mixture was filtered to remove Pd-C and concentrated to afford the title compound (1.76 g, 54% yield) as a brown solid.

[0288] Step 4: Synthesis of P12 and P13

[0289] Intermediate Int-A (200 mg, 0.321 mmol, 1 eq.), 2-amino-1-(3-amino-4-methoxyphenyl)ethan-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 flask. 20 mL of anhydrous ethanol was added and stirred at 60°C for 1 hour. LC-MS confirmed the reaction was complete. The anhydrous ethanol was concentrated, and the pH was adjusted to 7-8 by adding saturated sodium carbonate solution. The organic phase was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column chromatography on silica gel (MeOH / DCM = 1 / 50) to afford P12 (96 mg, 76% yield) and P13 (100 mg, 79% yield) as brown solids.

[0290] P12

[0291] LC-MS (ESI) m / z: 786.1 [M+H] +

[0292] 1 H 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).

[0293] P13

[0294] LC-MS (ESI) m / z: 786.1 [M+H] +

[0295] 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).

[0296] Example 5: Synthesis of Compound P14

[0297] P13 (5.0 mg, 0.0063 mmol, 1 eq.) was dissolved in 1.5 mL of anhydrous acetonitrile and cooled to 0°C. 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 to quench the reaction. The resulting solution was purified by preparative HPLC to obtain the desired product (0.2 mg, yield: 3.6%) as a brown solid.

[0298] LC-MS (ESI) m / z: 866.1 [M+H] +

[0299] Example 6: Synthesis of Compound P19

[0300] Step 1: Synthesis of 2-(4-methoxy-3-nitrophenyl)-2-methylpropionitrile

[0301] 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 completion, the pH was adjusted to 7 with saturated aqueous NaHCO₃. Extraction was performed with dichloromethane, and the organic layer was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by preparative thin-layer chromatography (EA:heptane = 1:2) to afford the desired product (75 mg, 60.1% yield) as a white solid.

[0302] LC-MS (ESI) m / z: 220.8 [M+H] +

[0303] Step 2: Synthesis of 2-(4-methoxy-3-nitrophenyl)-2-methylpropylamine

[0304] To a solution of 2-(4-methoxy-3-nitrophenyl)-2-methylpropionitrile (75 mg, 0.34 mmol, 1.0 eq) in anhydrous tetrahydrofuran (3 mL) was added borane dimethyl sulfide (2.5 M, 0.43 mL, 2.5 eq). The mixture was stirred at reflux for 1 hour. The mixture was quenched with MeOH and concentrated. The residue was purified by preparative thin-layer chromatography to afford the title compound (50 mg, 65.1% yield) as a colorless oil.

[0305] LC-MS (ESI) m / z: 225.2 [M+H] +

[0306] Step 3: Synthesis of 5-(1-amino-2-methylpropane-2-yl)-2-methoxyaniline

[0307] To a solution of 2-(4-methoxy-3-nitrophenyl)-2-methylpropane-1-amine (50 mg, 223 mmol, 1.0 eq) in MeOH (10 mL) under N₂ protection was added 20 mg of 5% wet Pd / C. The suspension was degassed under vacuum and replaced with H₂ several times. Stirred at room temperature under H₂ protection overnight. The suspension was filtered through celite, and the filter cake was washed with methanol. The combined filtrates were concentrated and used directly in the next step.

[0308] LC-MS (ESI) m / z: 194.9 [M+H] +

[0309] Step 4: Synthesis of P19

[0310] 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 at 25°C overnight. After completion of the reaction, the reaction solution was filtered and the filtrate was purified by preparative HPLC to afford P19 (4.67 mg, 36.3% yield) as a white solid.

[0311] LC-MS (ESI) m / z: 798.0 [M+H] +

[0312] 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).

[0313] Example 7: Synthesis of Compound P30

[0314] Step 1: Synthesis of 2-(dimethylamino)-2-(4-methoxy-3-nitrophenyl)acetonitrile

[0315] 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. N,N,1,1,1-pentamethylsilylamine (1.94 g, 16.56 mmol, 3 eq) was then added and stirred for 5 minutes. TMSCN (657 mg, 6.62 mmol, 1.2 eq) was then added and stirred for 5 minutes. LC-MS indicated the reaction was complete. Dichloromethane (50 mL) was added to the reaction mixture and the mixture was washed twice with saturated aqueous NaHCO₃. The organic layer was dried over sodium sulfate, filtered, and concentrated. The resulting orange oil was purified by silica gel flash column chromatography to yield the title compound (840 mg, 65% yield).

[0316] LC-MS (ESI) m / z: 236 [M+H] +

[0317] Step 2: Synthesis of 3-butyl (2-(3-amino-4-methoxyphenyl)-2-(dimethylamino)ethyl)carbamate

[0318] 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 BocO (139 mg, 0.638 mmol, 1.5 eq) and 20 mg of Raney Ni. The mixture was stirred overnight at room temperature under a H balloon. LC-MS indicated the reaction was complete. The mixture was filtered and the filtrate was concentrated to remove the solvent. The residue was used directly in the next step.

[0319] LC-MS (ESI) m / z: 310 [M+H] +

[0320] Step 3: Synthesis of 1-(3-amino-4-methoxyphenyl)-N1,N1-dimethylethane-1,2-diamine TFA salt

[0321] 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 concentrated to obtain 256 mg of crude product, which was used directly in the next step.

[0322] LC-MS (ESI) m / z: 310 [M+H] +

[0323] Step 4: Synthesis of P30

[0324] 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 at 25°C overnight. After completion of the reaction, the crude product was purified by preparative HPLC to afford P30 (1.37 mg, 26% yield) as a white solid.

[0325] LC-MS (ESI) m / z: 813 [M+H] +

[0326] Example 8: Synthesis of Compound P36

[0327] Step 1: Synthesis of 1-(4-methoxy-3-nitrophenyl)cyclopropanecarbonitrile

[0328] To a solution of 1-(4-methoxyphenyl)-1'-cyclopropanecarbonitrile (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 concentrated H₂SO₄ (84.83 mg, 0.865 mmol) at 0°C and stirred overnight. LC-MS indicated the reaction was complete. The pH was adjusted to 7 with saturated aqueous NaHCO₃, and the aqueous phase was extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative thin-layer chromatography to afford the title compound (110 mg, 87% yield) as a yellow solid.

[0329] LC-MS (ESI) m / z: 219 [M+H] +

[0330] Step 2: Synthesis of (1-(4-methoxy-3-nitrophenyl)cyclopropyl)methanamine

[0331] To a solution of 1-(4-methoxy-3-nitrophenyl)cyclopropanecarbonitrile (120 mg, 0.549 mmol, 1.0 eq) in THF (3 mL) was added 2.5 M B H₃﹒ Me₂S (0.1 mL, 2.5 eq). The reaction mixture was refluxed for 1 hour. The mixture was quenched with MeOH and concentrated. The residue was purified by preparative thin-layer chromatography to afford the desired product (30 mg, 24.5% yield).

[0332] LC-MS (ESI) m / z: 223 [M+H] +

[0333] Step 3: Synthesis of 5-(1-(aminomethyl)cyclopropyl)-2-methoxyaniline

[0334] To a solution of (1-(4-methoxy-3-nitrophenyl)cyclopropyl)methanamine (30 mg, 0.135 mmol, 1.0 eq) in MeOH (2 mL) was added 10 mg of 5% wet Pd / C under nitrogen. The mixture was evacuated and replaced with hydrogen three times. The mixture was then stirred at room temperature overnight under hydrogen. The Pd / C was removed by filtration, and the filtrate was concentrated to afford 37 mg of the crude product as a brown oil, which was used directly in the next step.

[0335] LC-MS (ESI) m / z: 193 [M+H] +

[0336] Step 4: Synthesis of P36

[0337] 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 overnight at 25°C. After completion of the reaction, the crude product was purified by preparative HPLC to afford P36 (3.95 mg, 61% yield) as a white solid. LC-MS (ESI) m / z: 796 [M+H] +

[0338] Example 9: Synthesis of Compound P52

[0339] 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 indicated that the reaction was complete, and 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 HPLC to afford the title compound (3.87 mg, yield: 26%) as a white solid.

[0340] LC-MS (ESI) m / z: 789.6 [M+H] +

[0341] 1 H 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).

[0342] Example 10: Synthesis of Compound P58

[0343] To a 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) in DMF (2 mL) was added HATU (99 mg, 0.26 mmol, 10 eq). The reaction mixture was stirred for 5-10 minutes, followed by the addition of DIPEA (33.6 mg, 0.26 mmol, 10 eq). The mixture was stirred at room temperature for 3 hours. Purification by preparative HPLC afforded compound P58 (9.25 mg, 41.1% yield) as a white solid.

[0344] LC-MS (ESI) m / z: 868.2 [M+H] +

[0345] 1 H 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).

[0346] Example 11: Synthesis of Compound P65

[0347] To a 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) in DMF (2 mL) was added HATU (49.5 mg, 0.13 mmol, 1.0 eq). The reaction mixture was stirred for 5-10 minutes, followed by the addition of DIPEA (16.8 mg, 0.13 mmol, 10 eq) and stirring at room temperature for 1 hour. Purification by preparative HPLC afforded compound P65 (3.14 mg, 30.7% yield) as a white solid.

[0348] LC-MS (ESI) m / z: 845.0 [M+H] +

[0349] 1 H 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).

[0350] Example 12: Synthesis of Compound P74

[0351] Step 1: Synthesis of P74-2

[0352] 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, and the temperature was cooled to 0-10°C. Cbz-Cl (22.3 g, 0.13 mol, 1.8 eq) was added dropwise. After the addition was complete, the mixture was warmed to room temperature and stirred overnight. The layers were separated, and the aqueous phase was extracted twice with 100 mL of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate. The residue was concentrated, and ethyl acetate was added until completely dissolved. n-heptane was added dropwise to crystallize the residue, and the solid was filtered. The filtrate was concentrated, and the residue was crystallized again with ethyl acetate / n-heptane. The combined solid was dried to obtain compound P74-2 (16.2 g, 81.8% yield).

[0353] LC-MS (ESI) m / z: 272.0 [M+H] +

[0354] 1 H NMR (300MHz, Chloroform-d) δ7.35 (s, 5H), 7.05 (d, J = 7.8 Hz, 2H), 6.77 (d, J = 7.8 Hz, 2H), 5.10 (s, 2H), 4.73 (s, 1H), 3.42 (d, J = 6.7Hz, 2H), 2.74 (s, 2H).

[0355] Step 2: Synthesis of P74-3

[0356] P74-2 (14 g, 0.0516 mol, 1.0 eq.) was dissolved in 140 mL of acetic acid. A mixture 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 allowed to react for 1 hour. Ethyl acetate and saturated sodium bicarbonate were added for washing. The layers were separated, extracted with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel flash column chromatography (eluted with EA / n-heptane (1:4, v / v)) to obtain the desired product, P74-3 (15.4 g, 94% yield).

[0357] LC-MS (ESI) m / z: 317.0 [M+H] +

[0358] 1H 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).

[0359] Steps 3 and 4: Synthesis of P74-5

[0360] 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. Methyl iodide (8.08 g, 0.0569 mol, 1.5 eq.) was added and refluxed for 2 hours. The mixture was cooled, filtered, and the filtrate was concentrated. The mixture was separated by adding 200 mL of dichloromethane and 100 mL of water. The aqueous phase was extracted with dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to yield P74-4. P74-4 was dissolved in 300 mL of methanol, and 1.5 g of 5% wet Pd / C was added. The suspension was degassed under vacuum and replaced with H2 several times. Stirred at room temperature under H2 protection overnight. The suspension was filtered through celite, the filtrate was concentrated, dissolved with ethyl acetate, and 1.0 eq. 2N HCl / EtOH solution was added to precipitate a solid which was filtered to give the target product P74-5 (7.08 g, two-step yield: 93%).

[0361] P74-4:

[0362] 1 H 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).

[0363] P74-5:

[0364] 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).

[0365] Step 5: Synthesis of P74-6

[0366] 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.) were dissolved in 1.5 mL of anhydrous ethanol and stirred at room temperature overnight. TLC confirmed the completion of the reaction. The mixture was filtered, the inorganic salts were washed with ethyl acetate, and the filtrate was concentrated. The crude product was purified by silica gel flash column chromatography (eluting with EA / n-heptane (1:1, v / v)) to obtain the desired product P74-6 (19 mg, 76.7% yield).

[0367] 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).

[0368] Step 6: Synthesis of P74

[0369] To a 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.) in DMF (1 mL) was added HATU (49.5 mg, 0.13 mmol, 10 eq.). The reaction was stirred for 5-10 minutes, followed by the addition of DIPEA (16.8 mg, 0.13 mmol, 10 eq.). Stirring was continued at room temperature for 1 hour. Compound P74 (6.59 mg, 58.4% yield) was purified by preparative HPLC to afford a white solid.

[0370] LC-MS (ESI) m / z: 868.2 [M+H] +

[0371] 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).

[0372] Example 13: Synthesis of Compound P75

[0373] To a 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.) in DMF (1 mL) was added HATU (49.5 mg, 0.13 mmol, 10 eq.). The reaction mixture was stirred for 5-10 minutes, followed by the addition of DIPEA (16.8 mg, 0.13 mmol, 10 eq.). The mixture was stirred at room temperature for 1 hour. Purification by preparative HPLC afforded compound P75 (4.25 mg, 37.7% yield) as a white solid.

[0374] LC-MS (ESI) m / z: 868.1 [M+H] +

[0375] 1H 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).

[0376] Example 14: Synthesis of Compound P85

[0377] To a solution of 2-(tert-butyldimethylsilyl)oxyacetic acid (200 mg, 0.96 mmol) in dichloromethane (3 mL) was added oxalyl chloride (0.25 mL, 0.48 mmol) and a catalytic amount of DMF (0.1 eq) at 0°C. The reaction was monitored with BnNH2. Upon completion, the resulting 2-((tert-butyldimethylsilyl)oxy)acetyl chloride solution was used directly in the next step.

[0378] 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-mentioned 2-((tert-butyldimethylsilyl)oxy)acetyl chloride solution (0.1 mL) was added to the reaction mixture. Further 2-((tert-butyldimethylsilyl)oxy)acetyl chloride solution (0.1 mL) was added until P1 was completely reacted. Under these reaction conditions, TBS was also simultaneously deprotected, directly yielding the target product P85. P85 was purified by preparative HPLC to afford P85 (1.64 mg, 38% yield) as a white solid.

[0379] LC-MS (ESI) m / z: 816 [M+H] +

[0380] 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).

[0381] Example 15: Synthesis of Compound P90

[0382] Step 1: Synthesis of P90-1

[0383] To a solution of P74-6 (20 mg, 0.026 mmol, 1.0 eq.) and glycolic acid (8 mg, 0.105 mmol, 4.0 eq.) in 2 mL of dichloromethane were added HATU (29.64 mg, 0.078 mmol, 3.0 eq.) and DIPEA (16.77 mg, 0.13 mmol, 5 eq.). After the addition was complete, the mixture was stirred at room temperature overnight. Purification by preparative HPLC afforded compound P90-1 (3.3 mg, 15.3% yield) as a white solid.

[0384] LC-MS (ESI) m / z: 828.0 [M+H] +

[0385] 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).

[0386] Step 2: Synthesis of P90

[0387] To a solution of P90-1 (3.77 mg, 0.0045 mmol, 1.0 eq.) in acetonitrile (0.5 mL) at room temperature were added 200 equivalents of formaldehyde (37 wt%) and 10 equivalents of NaCNBH3. The mixture was stirred for 1 hour, and 40 equivalents of HOAc were added. The reaction mixture was stirred at room temperature for 2 hours. Purification by preparative HPLC afforded compound P90 (0.23 mg, 6% yield) as a white solid.

[0388] LC-MS (ESI) m / z: 842.1 [M+H] +

[0389] 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).

[0390] Example 16: Synthesis of Compound P96

[0391] Steps 1 and 2: Synthesis of P96

[0392] To a solution of P19 (19 mg, 0.024 mmol) in DMF (1 mL) were 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 indicated the reaction was complete, and the product was quenched with water, extracted with ethyl acetate, and concentrated. Anhydrous THF (1 mL) was added, followed by 3HF·3Et3N (0.9 mL), and allowed to react at room temperature for 1 hour. LC-MS indicated the reaction was complete. Saturated aqueous NaHCO3 was added to the reaction solution, and the product was extracted with dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by preparative HPLC afforded the desired product, P96 (5.86 mg, 28.7% yield), as a white solid. LC-MS (ESI) m / z: 856.7 [M+H] +

[0393] 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,2H),4.08(d,J=2.8Hz,1H),4.05–4.00(m,1 H),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),2.87(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).

[0394] Example 17: Synthesis of Compound P166

[0395] Step 1: Synthesis of 2-methoxy-3-nitrobenzaldehyde

[0396] 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. The organic layer is dried over anhydrous sodium sulfate and concentrated to yield the title compound (4 g, 73.8% yield) as a yellow solid.

[0397] Step 2: Synthesis of (2-methoxy-3-nitrophenyl)methanol

[0398] Dissolve 2-methoxy-3-nitrobenzaldehyde (370 mg, 2.04 mmol) in EtOH (5 mL) and add NaBH4 (85 mg, 2.24 mmol). Stir at room temperature for 1 hour. After completion, quench with 1N HCl and extract with dichloromethane. The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated to give 350 mg of the crude product as a solid, which is used directly in the next reaction.

[0399] 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).

[0400] Step 3: Synthesis of 1-(bromomethyl)-2-methoxy-3-nitrobenzene

[0401] To a solution of (2-methoxy-3-nitrophenyl)methanol (120 mg, 0.655 mmol) in dichloromethane (3 mL) was added pyridine (37 mg) and cooled to 0°C. PBr3 (266 mg, 0.982 mmol) was gradually added. After the reaction was complete, 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 yield the title compound (65 mg, 40% yield).

[0402] 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).

[0403] Step 4: Synthesis of 2-(2-methoxy-3-nitrophenyl)acetonitrile

[0404] To a solution of TMSCN (31.42 mg, 0.29 mmol) in THF (2 mL) was added TBAF (1 M in THF, 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 completion of the reaction, the concentrated residue was purified by preparative thin-layer chromatography to yield the title compound (25 mg, 48.9% yield).

[0405] LC-MS (ESI) m / z: 166.3 [M-CN] +

[0406] Step 5: Synthesis of 2-(3-amino-2-methoxyphenyl)acetonitrile

[0407] Under N2 protection, a solution of 2-(2-methoxy-3-nitrophenyl)acetonitrile (25 mg, 0.56 mmol, 1.0 equivalent) in 3 mL of THF was added with 5% wet Pd / C. 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 celite and the filter cake was washed with methanol. The combined filtrate was concentrated and used directly in the next step.

[0408] LC-MS (ESI) m / z: 163.2 [M+H] +

[0409] Step 6: Synthesis of 3-(2-aminoethyl)-2-methoxyaniline

[0410] Under Ar protection, the crude 2-(3-amino-2-methoxyphenyl)acetonitrile was dissolved in 1 mL of anhydrous tetrahydrofuran. Borane dimethyl sulfide (2M, 0.5 mL) was added dropwise to the solution at 0°C, followed by heating under reflux for 1 hour. LC-MS indicated the reaction was complete, and the mixture was cooled to 0°C. The mixture was quenched with 1 mL of MeOH and the solvent was concentrated. The residue was purified by preparative thin-layer chromatography to yield 10 mg of the desired product.

[0411] LC-MS (ESI) m / z: 167.3 [M+H] +

[0412] Step 7: Synthesis of P166

[0413] 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 completion of the reaction, the solvent was concentrated to yield a crude solid. The crude product was purified by preparative HPLC to afford the title compound (0.62 mg, 10% yield) as a white solid.

[0414] LC-MS (ESI) m / z: 770.4 [M+H] +

[0415] Example 18: Synthesis of Compound P170

[0416] Step 1: Synthesis of P170-1

[0417] 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) at 0°C, followed by the addition of two catalytic drops of DMF. After stirring at 0°C for 1 hour, the reaction mixture 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 indicated the reaction was complete, and the mixture was quenched with water and extracted with ethyl acetate. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel flash column chromatography to afford the desired product (15 mg, 53.2% yield) as a yellow liquid.

[0418] LC-MS (ESI) m / z: [M+H] + 942

[0419] Step 2: Synthesis of P170

[0420] 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 indicated the reaction was complete. The mixture was neutralized with saturated aqueous NaHCO₃ and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by HPLC to yield the desired product (2.35 mg, 19.1% yield) as a white solid. LC-MS (ESI) m / z: [M+H] + 828

[0421] 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=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).

[0422] Example 19: Synthesis of Compound P172

[0423] Step 1: Synthesis of 2-(4-methyl-3-nitrophenyl)acetonitrile

[0424] To a solution of TMSCN (1.3 g, 13 mmol) in THF (15 mL) was added TBAF (1 M in THF, 12 mL) and stirred for 1 hour. Then, a solution of 4-(chloromethyl)-1-methyl-2-nitrobenzene (1 g, 5.4 mmol) in CHCN (9 mL) was added. After completion of the reaction, the mixture was concentrated and the crude product was purified by silica gel flash column chromatography to obtain the title compound (688 mg, 72.3% yield).

[0425] LC-MS (ESI) m / z: 177 [M+H] +

[0426] 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).

[0427] Step 2: Synthesis of 2-(3-amino-4-methylphenyl)acetonitrile

[0428] Under N₂ protection, to a solution of 2-(4-methyl-3-nitrobenzene)acetonitrile (680 mg) in THF (10 mL) was added 5% wet Pd / C (40 mg). The suspension was degassed under vacuum and replaced with H₂ several times. Stirred at room temperature under H₂ protection overnight. 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.

[0429] LC-MS (ESI) m / z: 147 [M+H] +

[0430] Step 3: Synthesis of 5-(2-aminoethyl)-2-methylaniline

[0431] To a solution of 2-(3-amino-4-methylphenyl)acetonitrile (38 mg) in MeOH, add Raney Ni (5 mg) and 0.1-0.5 eq. NH₃﹒H₂O. Stir at room temperature for 2 hours. After completion of the reaction, filter out the solid and concentrate. Purify by preparative thin-layer chromatography to obtain the desired product (27 mg, 69% yield).

[0432] LC-MS (ESI) m / z: 151 [M+H] +

[0433] 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).

[0434] Step 4: Synthesis of P172

[0435] 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 completion of the reaction, the solvent was removed under reduced pressure to yield a crude solid. The crude product was purified by preparative HPLC to yield the desired product, P172 (3.32 mg, 68% yield), as a white solid. LC-MS (ESI) m / z: 754 [M+H] +

[0436] 1 H 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).

[0437] Example 20: Synthesis of Compound P241

[0438] Step 1: Synthesis of 1-(4-methoxybenzyl)cyclopropan-1-amine

[0439] To a solution of 2-(4-methoxyphenyl)acetonitrile (4 g, 27.19 mmol, 1 eq) in THF (50 mL) and MTBE (50 mL) was added Ti(O-ipr)4 (15.46 g, 54.39 mmol, 2 eq) and EtMgBr (7.2 g, 54.39 mmol, 2 eq) at 0°C and maintained for 1 hour. BF3˙OEt2 (7.6 g, 54.39 mmol, 2 eq) was then added to the reaction mixture and stirred for 1 hour. After completion, the reaction was quenched with 2M NaOH and filtered. The organic phase was extracted with ethyl acetate (100 mL), separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel flash column chromatography to obtain the desired product (2.7 g, 56.5% yield) as a white solid.

[0440] 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).

[0441] Step 2: Synthesis of benzyl (1-(4-methoxybenzyl) cyclopropyl) carbamate

[0442] Cbz-Cl (4.6 mg, 27.41 mmol, 1.8 eq) was slowly added dropwise to a solution of 1-(4-methoxybenzyl)cyclopropan-1-amine (2.7 g, 15.23 mmol, 1 eq) in dichloromethane (30 mL) and saturated aqueous NaHCO₃. The mixture was stirred overnight at room temperature. The mixture was extracted 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 desired product (3 g, 63.8% yield) as a white solid.

[0443] LC-MS (ESI) m / z: 312 [M+H] +

[0444] Step 3: Synthesis of benzyl (1-(4-methoxy-3-nitrobenzyl) cyclopropyl) carbamate

[0445] To a solution of benzyl (1-(4-methoxybenzyl)cyclopropyl)carbamate (1 g, 3.21 mmol, 1 eq) in TFA (10 mL) at 0°C was added 65% HNO₃ (467 mg, 4.81 mmol, 1.5 eq) and stirred for 2 hours. After completion, the reaction was quenched by the addition of saturated aqueous NaHCO₃. The solvent was removed under reduced pressure, and the pH was adjusted to 7-8. The residue was extracted with dichloromethane, separated, dried over anhydrous sodium sulfate, filtered, and purified by silica gel flash column chromatography to obtain the title compound (250 mg, 21% yield) as a yellow solid.

[0446] LC-MS (ESI) m / z: 357 [M+H] +

[0447] Step 4: Synthesis of 5-((1-aminocyclopropyl)methyl)-2-methoxyaniline

[0448] To a solution of (1-(4-methoxy-3-nitrobenzyl)cyclopropyl)carbamate (170 mg, 0.477 mmol) in methanol (3 mL) was added HOAc (57.3 mg, 0.954 mmol, 2 eq) and 50 mg of 10% wet Pd / C under N₂ protection. The suspension was degassed under vacuum and replaced with H₂ several times. Stirred at room temperature under H₂ protection overnight. The suspension was filtered through celite, and the filter cake was washed with methanol. The combined filtrates were concentrated and used directly in the next step.

[0449] LC-MS (ESI) m / z: 193 [M+H] +

[0450] Step 5: Synthesis of P241

[0451] 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 completion of the reaction, the solvent was removed under reduced pressure to yield a solid. The crude product was purified by preparative HPLC to afford the target compound P241 (1.87 mg, 11% yield) as a white solid.

[0452] LC-MS (ESI) m / z: 796 [M+H] +

[0453] Example 21: Synthesis of Compounds P261 and P262

[0454] Step 1: Synthesis of 3-cyclopropyl-2-(4-methoxyphenyl)propionitrile

[0455] Dissolve 2-(4-methoxyphenyl)acetonitrile (1.0 g, 6.8 mmol) in DMF (10 mL). Add NaH (60% w / w, 300 mg, 7.48 mmol) at 0°C and stir for 1 hour. Add (bromoethyl)cyclopropane (918 mg, 6.8 mmol) at 0°C. After completion of the reaction, pour into saturated aqueous NH4Cl and extract with MTBE. The organic phase is dried over sodium sulfate, filtered, and concentrated. The crude product is purified by silica gel flash column chromatography to obtain the title compound (1.2 g, 87.7% yield) as a solid.

[0456] LC-MS (ESI) m / z: 202 [M+H] +

[0457] Step 2: Synthesis of 3-cyclopropyl-2-(4-methoxy-3-nitrophenyl)propionitrile

[0458] To a solution of 3-cyclopropyl-2-(4-methoxyphenyl)propionitrile (1.2 g, 5.96 mmol) in dichloromethane (20 mL) was added 20 mL of a mixture of concentrated HNO₃ (65%, 2.6 g, 26.8 mmol) and concentrated H₂SO₄ (2.77 g, 26.8 mmol). After completion of the reaction, the pH was adjusted to 7-8 with saturated aqueous NaHCO₃. Extraction with dichloromethane was performed, and the organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel flash column chromatography to obtain the desired product (400 mg, 27% yield).

[0459] LC-MS (ESI) m / z: 247 [M+H] +

[0460] Step 3: Synthesis of 2-(3-amino-4-methoxyphenyl)-3-cyclopropylpropionitrile

[0461] To a solution of 3-cyclopropyl-2-(4-methoxy-3-nitrophenyl)propionitrile (110 mg, 0.45 mmol) in THF (3 mL) was added 5% wet Pd / C under N₂ protection. 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 celite, and the filter cake was washed with methanol. The combined filtrates were concentrated and used directly in the next step.

[0462] LC-MS (ESI) m / z: 217 [M+H] +

[0463] Step 4: Synthesis of 5-(1-amino-3-cyclopropylpropane-2-yl)-2-methoxyaniline

[0464] Under Ar protection, borane dimethyl sulfide (2M, 0.5 ml) was added dropwise to a solution of the crude 2-(3-amino-4-methoxyphenyl)-3-cyclopropylpropionitrile in anhydrous tetrahydrofuran at 0°C. The reaction mixture was heated under reflux for 1 hour. LC-MS indicated the reaction was complete, and the mixture was cooled to 0°C. The mixture was quenched with 1 mL of MeOH and the solvent was removed by concentration. The residue was purified by preparative thin-layer chromatography to yield the desired product (10 mg, 10% yield over two steps).

[0465] Step 5: Synthesis of P261 and P262

[0466] Intermediate Int-A (5 mg, 0.008 mmol), 5-(1-amino-3-cyclopropylpropane-2-yl)-2-methoxyaniline (10 mg, 0.04 mmol), and HOAc (4 mg, 0.064 mmol) were dissolved in anhydrous ethanol (1 mL) and stirred overnight at 25°C. After completion of the reaction, the solvent was removed under reduced pressure to yield a solid. The crude product was purified by preparative HPLC to afford P261 (0.58 mg, 17% yield) and P262 (1 mg, 30% yield).

[0467] LC-MS (ESI) m / z: 824 [M+H] +

[0468] LC-MS (ESI) m / z: 824 [M+H] +

[0469] Example 22: Synthesis of Compound T1

[0470] Step 1: Synthesis of 2-(3-methoxy-5-nitrophenyl)acetonitrile

[0471] To a solution of TMSCN (97 mg, 0.97 mmol) in THF (5 mL) was added TBAF (1 M in THF, 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 completion of the reaction, the crude product was concentrated and purified by preparative thin-layer chromatography to yield the title compound (50 mg, 31.7% yield).

[0472] LC-MS (ESI) m / z: 193 [M+H] +

[0473] Step 2: Synthesis of 2-(3-amino-5-methoxyphenyl)acetonitrile

[0474] Under N₂ protection, to a solution of 2-(3-methoxy-5-nitrophenyl)acetonitrile (50 mg) in THF (3 mL) was added 10 mg of 5% wet Pd / C. 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 celite, and the filter cake was washed with methanol. The combined filtrate was concentrated and used directly in the next step.

[0475] LC-MS (ESI) m / z: 163 [M+H] +

[0476] Step 3: Synthesis of 3-(2-aminoethyl)-5-methoxyaniline

[0477] To a solution of the crude 2-(3-amino-5-methoxyphenyl)acetonitrile (40 mg, 0.25 mmol) in anhydrous tetrahydrofuran (40 mg, 0.25 mmol) was added dropwise borane dimethyl sulfide (2 M, 0.6 mL) at 0°C under Ar protection. The reaction mixture was heated at reflux for 1 hour. LC-MS indicated the reaction was complete, and the mixture was cooled to 0°C. The mixture was quenched with 1 mL of MeOH and the solvent was removed by concentration. The residue was purified by preparative thin-layer chromatography to yield the title compound (7 mg, 16% yield over two steps).

[0478] LC-MS (ESI) m / z: 167 [M+H] +

[0479] Step 4: Synthesis of T1

[0480] 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 completion of the reaction, the solvent was removed under reduced pressure to yield a crude solid. This crude product was purified by preparative HPLC to afford the target compound T1 (3.35 mg, 54% yield) as a white solid.

[0481] LC-MS (ESI) m / z: 770 [M+H] +

[0482] Example 23: Synthesis of Compound T2

[0483] Step 1: Synthesis of 2-amino-2-(3-hydroxy-4-methoxyphenyl)acetonitrile

[0484] 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 completion of the reaction, the reaction 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 title compound (880 mg, yield: 7.5%) as a white solid.

[0485] LC-MS (ESI) m / z: 179 [M+H] +

[0486] 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).

[0487] Step 2: Synthesis of tert-butyl (cyano (3-hydroxy-4-methoxyphenyl) methyl) carbamate

[0488] 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). The mixture was stirred at room temperature overnight. After completion of the reaction, the mixture was concentrated and extracted with ethyl acetate (50 mL) and water (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel flash column chromatography to obtain the title compound (500 mg, 38% yield) as a white solid.

[0489] Step 3: Synthesis of tert-butyl (2-amino-1-(3-hydroxy-4-methoxyphenyl)ethyl)carbamate

[0490] Dissolve tert-butyl (cyano(3-hydroxy-4-methoxyphenyl)methyl)carbamate (50 mg, 0.179 mmol, 1.0 eq) in anhydrous THF (2 mL). Add LiAlH₄ (2 M, 1.1 eq) and stir at room temperature for 2 hours. After completion, quench with Na₂SO₄·10H₂O, filter, and concentrate the filtrate for use in the next step.

[0491] LC-MS (ESI) m / z: 283.4 [M+H] +

[0492] Step 4: Synthesis of T2-5

[0493] 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 completion of the reaction, the solvent was removed under reduced pressure to obtain a crude solid. The crude product was purified by preparative HPLC to yield the desired product (11 mg, 77% yield) as a white solid.

[0494] LC-MS (ESI) m / z: 886.3 [M+H] +

[0495] Step 5: Synthesis of T2

[0496] 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 completion of the reaction, the product was purified by preparative HPLC to afford the desired product, T2 (2.37 mg, 26.9% yield), as a white solid.

[0497] LC-MS (ESI) m / z: 786.9 [M+H] +

[0498] Example 24: Synthesis of Compound T3

[0499] Step 1: Synthesis of 2-(4-fluoro-3-nitrophenyl)-2-methylpropionitrile

[0500] Fuming nitric acid (2 mL) was added dropwise to a round-bottom flask containing 2-(4-fluorophenyl)-2-methylpropionitrile (500 mg, 3.0 mmol) at 0°C. The mixture was stirred at 0°C for 30 minutes. The reaction mixture was poured into ice and extracted with ethyl acetate. The combined organic layers were washed with saturated brine, dried over MgSO4, filtered, and concentrated. The crude product was purified by silica gel flash column chromatography (eluting with DCM / MeOH 100 / 0 to 96 / 4) to obtain the desired product (100 mg, yield: 16%).

[0501] Step 2: Synthesis of 2-(3-amino-4-fluorophenyl)-2-methylpropionitrile

[0502] 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. Stirred at room temperature under H₂ protection overnight. 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.

[0503] LC-MS (ESI) m / z: 179.2 [M+H] +

[0504] Step 3: Synthesis of 5-(1-amino-2-methylpropane-2-yl)-2-fluoroaniline

[0505] Under Ar protection, borane dimethyl sulfide (2M, 0.5 ml) was added dropwise to a solution of the crude 2-(3-amino-4-fluorophenyl)-2-methylpropionitrile in anhydrous tetrahydrofuran (3 mL) at 0°C. After heating the solution under reflux for 1 hour, LC-MS indicated the reaction was complete. The solution was then cooled to 0°C. The reaction was quenched with MeOH (1 mL) and the solvent was removed by concentration. The residue was purified by preparative thin-layer chromatography to yield the desired product (10 mg, 11% yield over two steps).

[0506] LC-MS (ESI) m / z: 165.9 [M+H] +

[0507] 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).

[0508] Step 4: Synthesize T3

[0509] Intermediate Int-A (5 mg, 0.008 mmol), 5-(1-amino-2-methylpropane-2-yl)-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 completion of the reaction, the solvent was removed under reduced pressure to yield a crude solid. The crude product was purified by preparative HPLC to yield the desired product (1.69 mg, 26.7% yield) as a white solid.

[0510] LC-MS (ESI) m / z: 786.3 [M+H] +

[0511] 1H 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).

[0512] Example 25: Synthesis of Compound T4

[0513] To a solution of P1 (5 mg, 0.0066 mmol) and 1-hydroxycyclopropane-1-carboxylic acid (6.74 mg, 0.066 mmol) in DMF (2 mL) was added HATU (25.1 mg, 0.066 mmol). The reaction mixture was stirred for 5-10 minutes, and then 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, 30.9% yield) was purified by preparative HPLC to obtain a white solid. LC-MS (ESI) m / z: 842 [M+H] +

[0514] Example 26: Synthesis of Compound T5

[0515] To a solution of P1 (4 mg, 0.0053 mmol) and (1s,3s)-3-hydroxycyclobutane-1-carboxylic acid (6 mg, 0.053 mmol) in DMF (2 mL) was added HATU (19.8 mg, 0.053 mmol). The reaction mixture was stirred for 5-10 minutes, followed by the addition of DIPEA (6.8 mg, 0.053 mmol). The reaction mixture was stirred at room temperature for 1 hour and then purified by preparative HPLC to afford the target compound T5 (1.69 mg, 36.9% yield) as a white solid.

[0516] (ESI)m / z:856.4[M+H] +

[0517] Example 27: Synthesis of Compound T6

[0518] Step 1: Synthesis of 2-methoxy-3-nitrobenzaldehyde

[0519] To a solution of 2-hydroxy-3-nitrobenzaldehyde (5 g, 30 mmol, 1.0 eq) in anhydrous DMF (40 mL) were added CsCO (29.24 g, 90 mmol, 3 eq) and CHI (5.1 g, 36 mmol, 1.2 eq) and stirred at 80°C for 1.5 hours. After completion of the reaction, the solid was filtered. 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 yield the desired product (4.02 g, 73.6% yield) as a yellow solid.

[0520] LC-MS (ESI) m / z: 182.2 [M+H] +

[0521] Step 2: Synthesis of (2-methoxy-3-nitrophenyl)methanol

[0522] 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 completion, 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.

[0523] LC-MS (ESI) m / z: 184.2 [M+H] +

[0524] Step 3: Synthesis of 1-(bromomethyl)-2-methoxy-3-nitrobenzene

[0525] Dissolve (2-methoxy-3-nitrobenzene)methanol (2.89 g, 15.8 mmol, 1.0 eq) in anhydrous dichloromethane (40 mL) and add pyridine (891 mg, 11.4 mmol, 0.72 eq). Slowly add PBr3 (6.4 g, 23.7 mmol, 1.5 eq) dropwise to the reaction mixture. Stir at room temperature for 3 hours. After completion of the reaction, add water and extract with dichloromethane. The combined organic phases are dried over anhydrous sodium sulfate, concentrated, and purified by silica gel flash column chromatography to obtain the desired product (1.8 g, 46.3% yield) as a yellow solid.

[0526] LC-MS (ESI) m / z: 245.8 [M+H] +

[0527] Step 4: Synthesis of 2-(2-methoxy-3-nitrophenyl)acetonitrile

[0528] 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 a solution of 1-(bromomethyl)-2-methoxy-3-nitrobenzene (1.8 g, 7.32 mmol, 1.0 eq) in acetonitrile (20 mL). Stirring is continued at room temperature for 1 hour. The reaction mixture is concentrated and purified by silica gel flash column chromatography to obtain the desired product (1.2 g, 85.7% yield) as a yellow solid.

[0529] LC-MS (ESI) m / z: 193 [M+H] +

[0530] Step 5: Synthesis of 2-(2-methoxy-3-nitrophenyl)ethyl-1-amine

[0531] Dissolve 2-(2-methoxy-3-nitrophenyl)acetonitrile (1.1 g, 5.7 mmol, 1.0 eq) in anhydrous THF (40 mL) and add 2.5 M borane dimethyl sulfide (14.3 mL, 2.5 eq). Reflux the reaction mixture for 1 hour. Quench the reaction mixture with MeOH and concentrate. Purify the product by flash column chromatography on silica gel to obtain the desired product (650 mg, 55% yield).

[0532] LC-MS (ESI) m / z: 196 [M+H] +

[0533] 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).

[0534] Step 6: Synthesis of 3-(2-aminoethyl)-2-methoxyaniline acetate

[0535] To a THF solution of 2-(2-methoxy-3-nitrophenyl)ethan-1-amine (650 mg, 3.57 mmol, 1 eq) was added 5% wet Pd / C and HOAc (398 mg, 7 mmol, 2.0 eq) under N₂ protection. 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 celite, and the filter cake was washed with methanol. The combined filtrates were concentrated to yield 780 mg of crude product, which was used directly in the next step.

[0536] LC-MS (ESI) m / z: 167 [M+H] +

[0537] 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).

[0538] Step 7: Synthesis of benzyl (3-(2-((benzyloxy)carbonyl)amino)ethyl)-2-methoxyphenyl)carbamate

[0539] To a mixture of 3-(2-aminoethyl)-2-methoxyaniline acetate (780 mg, 3.45 mmol, 1.0 eq) in dichloromethane (20 mL) and saturated aqueous sodium bicarbonate (50 mL) at 0-10°C was added CbzCl (1.76 g, 10.4 mmol, 3.0 eq). The resulting mixture was stirred at room temperature overnight. LC-MS indicated the reaction was complete. The aqueous phase was separated and extracted twice with dichloromethane (20 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by silica gel flash column chromatography afforded the desired product (550 mg, 76.5% yield) as a yellow solid.

[0540] LC-MS (ESI) m / z: 435 [M+H] +

[0541] 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).

[0542] Step 8: Synthesis of benzyl (3-(2-((benzyloxy)carbonyl)amino)ethyl)-2-hydroxyphenyl)carbamate

[0543] 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) was added BCl (1 M solution in dichloromethane, 12.7 mL, 12.7 mmol, 10 eq) at -78°C and stirred at the same temperature for 2 hours. LC-MS indicated the reaction was complete. The cooled solution was quenched with saturated aqueous NaHCO 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 desired product (400 mg, yield: 74.9%) as a yellow solid.

[0544] LC-MS (ESI) m / z: 421 [M+H] +

[0545] 1 H 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).

[0546] Step 9: Synthesis of benzyl(3-(2-((benzyloxy)carbonyl)amino)ethyl)-2-(methoxymethoxy)phenyl)carbamate.

[0547] 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 were added cesium carbonate (235 mg, 0.72 mmol, 2.0 eq) and MOMBr (67.5 mg, 0.54 mmol, 1.5 eq). The mixture was stirred at room temperature for 4 hours. LC-MS monitored the reaction completion. After addition of water, the mixture was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel flash column chromatography to afford the desired product (80 mg, 47.8% yield) as a yellow liquid.

[0548] LC-MS (ESI) m / z: 465 [M+H] +

[0549] Step 10: Synthesis of 3-(2-aminoethyl)-2-(methoxymethoxy)aniline

[0550] 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) was added 5% wet Pd / C (20 mg) and HOAc (20.4 mg, 0.34 mmol, 2.0 eq) under N₂ protection. The suspension was degassed under vacuum and replaced with H₂ several times. The mixture was stirred 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 filtrates were concentrated and used directly in the next step.

[0551] LC-MS (ESI) m / z: 197 [M+H] +

[0552] Step 11: Synthesis of T6-12

[0553] 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 complete, the mixture was concentrated to obtain a solid. The crude product was used directly in the next reaction without further purification.

[0554] LC-MS (ESI) m / z: 800 [M+H] +

[0555] Step 12: Synthesize T6

[0556] Dissolve T6-12 (10 mg) in dichloromethane (2 mL) and add TFA (1 mL). Stir at room temperature overnight. After completion, neutralize with saturated aqueous NaHCO₃. Extract with dichloromethane, separate the organic layer, and concentrate. Purify by preparative HPLC to obtain T6 (3.86 mg, 40.8% yield) as a brown solid.

[0557] LC-MS (ESI) m / z: 756 [M+H] +

[0558] Example 28: Synthesis of Compound T7

[0559] Step 1: Synthesis of T7-1

[0560] 2-(tert-Butyldimethylsilyloxy)acetic acid (38 mg, 0.2 mmol, 10 eq) was dissolved in anhydrous dichloromethane (2 mL). Oxalyl chloride (2 M in dichloromethane, 0.05 mL, 0.1 mmol, 5 eq) and two drops of catalytic DMF were added at 0°C. After stirring at 0°C for 1 hour, the reaction mixture was added to a solution of T6-12 (16 mg, 0.02 mmol, 1.0 eq) and triethylamine (20 mg, 0.2 mmol, 10 eq) in anhydrous DMF in a separate flask. The mixture was stirred at room temperature for 2 hours. LC-MS indicated the reaction was complete, and the product was quenched with water and extracted with ethyl acetate. The combined organic layers were dried, filtered, and purified by silica gel flash column chromatography to yield the desired product (12 mg, 60% yield) as a yellow liquid.

[0561] LC-MS (ESI) m / z: 972 [M+H] +

[0562] Step 2: Synthesize T7

[0563] T7-1 (9 mg, 0.0093 mmol, 1.0 eq) was dissolved in anhydrous tetrahydrofuran (1 mL), and triethylamine trihydrofluoride (0.5 mL) was added and maintained for 1 hour. LC-MS indicated the reaction was complete, and the mixture was neutralized with saturated aqueous NaHCO₃. Extraction was performed with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by preparative HPLC to yield the desired product, T7 (2.29 mg, 30% yield), as a white solid.

[0564] LC-MS (ESI) m / z: 814 [M+H] +

[0565] Example 29: Synthesis of T8

[0566] Step 1: Synthesis of 2-amino-4-(2-aminoethyl)phenol

[0567] 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 under H2 protection for 4-5 hours. TLC confirmed the reaction was complete. 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.

[0568] LC-MS (ESI) m / z: [M+H] + 153.2

[0569] Step 2: Synthesis of T8

[0570] 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) and stirred at room temperature overnight. After completion of the reaction, the reaction mixture was purified by preparative HPLC to afford the desired product (10 mg, 41.7% yield) as a white solid.

[0571] LC-MS (ESI) m / z: [M+H] + 756.4

[0572] 1 H 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).

[0573] Example 30: Synthesis of Compound T9

[0574] 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 overnight at room temperature. LC-MS indicated the desired product. Purification by HPLC afforded the desired product (3.0 mg, 46.9% yield) as a white solid.

[0575] LC-MS (ESI) m / z: [M+H] + 814.22

[0576] Example 31: Synthesis of Compound T10

[0577] Step 1: Synthesis of 4-(dimethoxymethyl)-1-methoxy-2-nitrobenzene

[0578] To a solution of 4-methoxy-3-nitrobenzaldehyde (2 g, 11 mmol) and trimethoxymethane (3.76 g, 25.4 mmol) in anhydrous methanol, p-toluenesulfonic acid monohydrate (21 mg, 0.11 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. Completion of the reaction was monitored by thin-layer chromatography. Neutralization was performed by adding saturated aqueous NaHCO₃ solution, followed by extraction with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel flash column chromatography to yield the title compound (2.2 g, 87.7% yield) as a colorless oil.

[0579] LC-MS (ESI) m / z: 197 [M-CH3O] +

[0580] 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).

[0581] Step 2: Synthesis of 2-methoxy-2-(4-methoxy-3-nitrophenyl)acetonitrile

[0582] 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) was added SnCl₂ (66.8 mg, 0.352 mmol). The reaction was monitored for completion by LC-MS. The mixture was quenched with saturated aqueous NaHCO₃ and extracted with ethyl acetate. 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 desired product (690 mg, 70.5% yield).

[0583] LC-MS (ESI) m / z: 223 [M+H] +

[0584] Step 3: Synthesis of 5-(2-amino-1-methoxyethyl)-2-methoxyaniline

[0585] To a THF solution of 2-methoxy-2-(4-methoxy-3-nitrophenyl)acetonitrile (50 mg, 0.22 mmol) was added Raney Ni and 0.1-0.5 eq of NH₃﹒H₂O. The mixture was stirred at room temperature for 2 hours. After completion of the reaction, the mixture was filtered and concentrated. Purification by preparative thin-layer chromatography afforded the desired product (34 mg, 77.3% yield).

[0586] LC-MS (ESI) m / z: 197 [M+H] +

[0587] Step 4: Synthesis of T10

[0588] 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 overnight at 25°C. After completion of the reaction, the solvent was removed under reduced pressure to yield a crude solid. The crude product was purified by preparative HPLC to yield the desired product, T10 (1.53 mg, 29.7% yield), as a white solid.

[0589] LC-MS (ESI) m / z: 800 [M+H] +

[0590] Example 32: Synthesis of Compound T11

[0591] Step 1: Synthesis of T11

[0592] 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 indicated the desired product, which was purified by preparative HPLC to yield the desired product (8.3 mg, 38% yield).

[0593] LC-MS (ESI) m / z: 854.3 [M+H] +

[0594] 1H 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).

[0595] Example 33: Synthesis of Compound T12

[0596] Step 1: Synthesis of 3-(bromomethyl)-5-nitrophenol

[0597] To a solution of 3-(hydroxymethyl)-5-nitrophenol (3.1 g, 18.33 mmol) in THF (50 mL) at 0°C was added PPh3 (7.2 g, 27.5 mmol) and CBr4 (9.2 g, 27.5 mmol). The mixture was stirred at room temperature for 1 hour. LC-MS indicated 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 afforded the desired product (3.7 g, 87.6% yield) as a yellow solid.

[0598] LC-MS (ESI) m / z: 231.7 [M+H] +

[0599] Step 2: Synthesis of 2-(3-hydroxy-5-nitrophenyl)acetonitrile

[0600] Dissolve 3-(Bromomethyl)-5-nitrophenol (2.7 g, 11.64 mmol) in anhydrous acetonitrile (20 mL). Add TBAF (18.63 mL, 18.63 mmol) and TMSCN (2.1 g, 20 mmol) in anhydrous THF at room temperature and allow to react for 4 hours. LC-MS indicates the reaction is complete. Concentrate to remove the solvent, dilute with ethyl acetate, and wash several times with water. The organic phase is dried over sodium sulfate, filtered, and concentrated. The residue is purified by silica gel flash column chromatography to obtain the desired product (1.74 g, 84.2% yield) as a yellow solid.

[0601] LC-MS (ESI) m / z: 176.8 [MH] -

[0602] Step 3: Synthesis of 3-(2-aminoethyl)-5-nitrophenol

[0603] To a solution of 2-(3-hydroxy-5-nitrophenyl)acetonitrile (500 mg, 2.8 mmol) in anhydrous THF was added BH3·THF (1 M, 7 mL, 7 mmol) and refluxed for 1 hour. LC-MS indicated the reaction was complete. The mixture was quenched with methanol at 0°C and refluxed for half an hour. The reaction mixture was concentrated and used directly in the next step.

[0604] LC-MS (ESI) m / z: 183.3 [M+H] +

[0605] Step 4: Synthesis of benzyl (3-hydroxy-5-nitrophenylethyl ester) carbamate

[0606] To a solution of the crude product 3-(2-aminoethyl)-5-nitrophenol (511 mg, 2.8 mmol) in anhydrous dichloromethane were added Cbz-Cl (860 mg, 5.04 mmol) and a NaHCO3 aqueous solution (283 mg, 3.36 mmol, 1.2 eq.). The mixture was stirred at room temperature for 1.5 hours. LC-MS showed that the reaction was complete. The organic phase was washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the title compound (840 mg, crude product) as a yellow liquid. The crude product was used directly in the next step without further purification.

[0607] LC-MS (ESI) m / z: 317.1 [M+H] +

[0608] Step 5: Synthesis of benzyl (3-(methoxymethoxy)-5-nitrophenylethyl ester) carbamate

[0609] To a solution of benzyl (3-hydroxy-5-nitrophenylethyl) carbamate (840 mg, 2.63 mmol) in anhydrous acetonitrile (20 mL) was added CsCO (1.7 g, 5.27 mmol) and MOMBr (495 mg, 3.95 mmol) and stirred at room temperature for 1 hour. LC-MS indicated the reaction was complete. The mixture was quenched with water and extracted with dichloromethane. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel flash column chromatography to afford the desired product (160 mg, 15.8% yield) as a yellow liquid.

[0610] LC-MS (ESI) m / z: 361.0 [M+H] +

[0611] 1H 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).

[0612] Step 6: Synthesis of 3-(2-aminoethyl)-5-(methoxymethoxy)aniline

[0613] Under N2 protection, to a solution of benzyl (3-(methoxymethoxy)-5-nitrophenylethyl)carbamate (160 mg, 0.44 mmol) in THF (10 mL) was added 5% wet Pd / C (20 mg) and HOAc (53 mg, 0.88 mmol). The suspension was degassed under vacuum and replaced with H2 several times. Stirred at room temperature under H2 protection overnight. TLC confirmed the reaction was complete. 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. LC-MS (ESI) m / z: 196.9 [M+H] +

[0614] Steps 7 and 8: Synthesis of T12

[0615] 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 indicated the reaction was complete. After concentration, the solvent was removed, and TFA / DCM (1:1 v / v) (1 mL) was added. After completion of the reaction, the mixture was concentrated and purified by preparative HPLC to yield the desired product (0.46 mg, 3.78% yield) as a white solid.

[0616] LC-MS (ESI) m / z: 756.4 [M+H] +

[0617] Example 34: Synthesis of Compound T13

[0618] Step 1: Synthesis of T13-1

[0619] To a solution of the crude product T12-8 (20 mg, 0.025 mmol) in DMF (2 mL) were added 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). LC-MS indicated the reaction was complete. Purification by preparative HPLC afforded the desired product (4.2 mg, 13.4% yield) as a white solid.

[0620] LC-MS (ESI) m / z: 972.6 [M+H] +

[0621] Step 2: Synthesis of T13

[0622] To a solution of T13-1 (3 mg, 0.003 mmol) in THF (0.7 mL) was added 3HF·3Et3N (0.7 mL) and stirred at room temperature for 1 hour. LC-MS indicated the reaction was complete. Saturated aqueous NaHCO3 was added to the reaction solution, followed by extraction with dichloromethane. The organic layer was separated, dried, filtered, concentrated, and dissolved in DCM / TFA (3 / 1). After completion of the reaction, the pH was adjusted to 7-8 with saturated aqueous NaHCO3. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative HPLC to afford the desired product (0.97 mg, 38.6% yield) as a white solid.

[0623] LC-MS (ESI) m / z: 814.4 [M+H] +

[0624] 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).

[0625] Example 35: Synthesis of Compound T14

[0626] Step 1: Synthesis of benzyl (4-hydroxy-3-methoxy-5-nitrophenylethyl) carbamate

[0627] To a solution of benzyl (4-hydroxy-3-methoxyphenethyl) carbamate (300 mg, 0.99 mmol) in dichloromethane (1.5 mL) was slowly added 65% nitric acid (0.6 mL) at 0°C and stirred at the same temperature for 20 minutes. LC-MS indicated the reaction was complete, and the reaction solution was quenched by pouring into ice water. The mixture was extracted with dichloromethane, and the combined organic layers were dried, filtered, concentrated, and purified by silica gel flash column chromatography to afford the title compound (80 mg, 23% yield) as a yellow solid.

[0628] LC-MS (ESI) m / z: [M+H] + 347.0

[0629] 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).

[0630] Step 2: Synthesis of benzyl (3,4-dimethoxy-5-nitrophenylethyl ester) carbamate

[0631] A solution of benzyl (4-hydroxy-3-methoxy-5-nitrophenethyl) carbamate (80 mg, 0.2 mmol), CsCO (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 indicated the reaction was complete. The reaction mixture was filtered, and the filtrate was concentrated and purified by column chromatography to afford the title compound (55 mg, 60% yield) as a yellow solid.

[0632] LC-MS (ESI) m / z: [M+H] + 361.1

[0633] Step 3: Synthesis of 5-(2-aminoethyl)-2,3-dimethoxyaniline

[0634] To a solution of benzyl (3,4-dimethoxy-5-nitrophenylethyl) carbamate (55 mg, 0.152 mmol) in TFE (10 mL) was added HOAc (18.31 mg, 0.305 mmol) and 10% wet Pd / C (20 mg, 20% w / w) under N2 protection. The suspension was degassed under vacuum and replaced with H2 several times. Stirred overnight at room temperature under H2 protection. The reaction was complete by TLC. 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. LC-MS (ESI) m / z: [M+H] + 197.4

[0635] Step 4: Synthesis of T14

[0636] 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 overnight at 50°C. LC-MS indicated the reaction was complete. Purification by preparative HPLC afforded the desired product (6.72 mg, 52.9% yield) as a white solid.

[0637] LC-MS (ESI) m / z: [M+H] + 800.5

[0638] 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).

[0639] Example 36: Synthesis of Compound T15

[0640] Steps 1 and 2: Synthesis of T15

[0641] To a solution of T14 (10 mg, 0.0125 mmol) in DMF (1 mL) were added 2-(tert-butyldimethylsilyloxy)acetic acid (23.9 mg, 0.125 mmol), HATU (47 mg, 0.125 mmol), and DIEA (16.1 mg, 0.125 mmol). LC-MS indicated 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). 3HF·3Et3N (0.9 mL) was then added and stirred at room temperature for 1 hour. LC-MS indicated the reaction was complete, and saturated aqueous NaHCO3 and dichloromethane were added to the mixture. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by preparative HPLC to yield the desired product (1.6 mg, 14.9% yield) as a white solid.

[0642] LC-MS (ESI) m / z: [M+H] + 858.5

[0643] Example 37: Synthesis of Compound T16

[0644] Step 1: Synthesis of benzyl (3-amino-4-methoxyphenethyl) carbamate

[0645] Benzyl (4-methoxy-3-nitrophenylethyl) carbamate (500 mg, 1.51 mmol) was dissolved in EtOH / H₂O = 4 / 1 (10 mL). Iron powder (339 mg, 6.05 mg) and NH₄Cl (320 mg, 6.05 mmol) were added and refluxed for 3 hours. LC-MS indicated the reaction was complete and the mixture was filtered. The filtrate was washed with water and extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and used directly in the next step. LC-MS (ESI) m / z: [M+H] + 301.2

[0646] Step 2: Synthesis of benzyl (4-methoxy-3-(methylamino)phenethyl) carbamate

[0647] Crude benzyl (3-amino-4-hydroxyphenethyl) carbamate (200 mg, 0.665 mmol) was dissolved in methanol (5 mL), and paraformaldehyde (79.8 mg, 2.66 mmol) was added. The mixture was stirred at room temperature for 1 hour. NaBH3CN (209 mg, 3.32 mmol) was added at 0°C, and stirring was continued for 2 hours. LC-MS indicated the reaction was complete, and the reaction solution was diluted with ethyl acetate and water. The organic layer was separated and concentrated, and then purified by silica gel flash column chromatography to obtain the desired product (50 mg, 22.7% yield).

[0648] LC-MS (ESI) m / z: [M+H] + 315.1

[0649] 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).

[0650] Steps 3 and 4: Synthesis of 5-(2-aminoethyl)-2,3-dimethoxyaniline

[0651] Under N₂ protection, to a solution of benzyl (4-methoxy-3-(methylamino)phenethyl)carbamate (45 mg, 0.143 mmol) in trifluoroethanol (10 mL) was added HOAc (17.16 mg, 0.286 mmol) and 10% wet Pd / C (20 mg, 20% w / w). The suspension was degassed under vacuum and replaced with H₂ several times. Stirring was carried out at room temperature under H₂ protection overnight. TLC confirmed the reaction was complete. The mixture was filtered, and Intermediate Int-A (20 mg, 0.0322 mmol) was added to the filtrate. The mixture was stirred at room temperature for 16 hours. LC-MS indicated the reaction was complete. The reaction solution was purified by preparative HPLC to obtain the title compound (11 mg, 43.6% yield) as a white solid.

[0652] LC-MS (ESI) m / z: [M+H] + 784.3

[0653] 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).

[0654] Example 38: Synthesis of Compound T17

[0655] Step 1: Synthesis of T17-1

[0656] 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). Et3N (7.0 mg, 0.0693 mmol) was added. Stirring was continued at room temperature for 5 hours. LC-MS indicated the reaction was complete. The product was concentrated and purified by silica gel flash column chromatography to afford the desired product (6 mg, 44.7% yield) as a white solid.

[0657] LC-MS (ESI) m / z: [M+H] + 956.7

[0658] Step 2: Synthesis of T17

[0659] 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 indicated the reaction was complete. The mixture was quenched with saturated aqueous NaHCO3 and extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by silica gel flash column chromatography afforded the desired product (1.55 mg, 29.3% yield) as a white solid.

[0660] LC-MS (ESI) m / z: [M+H] +842.4

[0661] Example 39: Synthesis of Compound T18

[0662] Step 1: Synthesis of 2-(2-methoxyphenyl)-2-methylpropionitrile

[0663] 2-Fluoroanisole (5 g, 39.66 mmol), isobutyronitrile (10.9 g, 158.67 mmol), and KHMDS (1 M solution in THF, 59.49 mL) were dissolved in dry toluene (50 mL) and stirred at 60°C for 16 hours. After completion, the reaction was quenched with saturated aqueous NH4Cl and extracted with ethyl acetate. The organic layer was washed with an appropriate amount of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The desired product (6.5 g, 93.5% yield) was purified by silica gel flash column chromatography to obtain the desired product as a colorless liquid.

[0664] LC-MS (ESI) m / z: 176.3 [M+H] +

[0665] Step 2: Synthesis of 2-(2-methoxy-3-nitrophenyl)-2-methylpropionitrile

[0666] 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 complete, 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 desired product (555 mg, yield: 22%) was purified by silica gel flash column chromatography as a yellow solid.

[0667] LC-MS (ESI) m / z: 221.5 [M+H] +

[0668] 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).

[0669] Step 3: Synthesis of 3-(1-amino-2-methylprop-2-yl)-2-methoxyaniline

[0670] Dissolve 2-(2-methoxy-3-nitrophenyl)-2-methylpropionitrile (10 mg) in EtOH (1 mL) and add 1 drop of NH₃﹒H₂O. Add a small amount of Raney Ni and stir under H₂ protection for 16 hours. After the reaction is complete, filter and concentrate the resulting filtrate, which is used directly in the next step.

[0671] LC-MS (ESI) m / z: 195.1 [M+H] +

[0672] Step 4: Synthesize T18

[0673] 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 complete, the solvent was removed by distillation under reduced pressure. The crude product was purified by preparative HPLC to obtain the desired product (2.39 mg, 18.6% yield) as a white solid. LC-MS (ESI) m / z: 798.7 [M+H] +

[0674] 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).

[0675] Synthesis and characterization of intermediates

[0676] Example 40: Synthesis of Compound L1

[0677] (S)-16-Amino-10-benzyl-6,9,12,15-tetraoxo-3-oxo-5,8,11,14-tetraazahexadecanoic acid (500 mg, crude) and N,N-diisopropylethylamine (609 mg, 4.72 mmol) were added to a 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) in DMF (4.00 mL) at 0°C. After complete addition, the reaction mixture was stirred at room temperature for 2 hours. LC-MS indicated 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.

[0678] LC-MS (ESI) m / z: 861.4 [M+Na] + .

[0679] 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).

[0680] Example 41: Synthesis of Compound L2

[0681] Steps 1 and 2: Synthesis of L2-3

[0682] Under nitrogen at room temperature, L2-1 (4.2 g, 5.7 mmol, 1.0 eq) was dissolved in 15 mL of anhydrous DMF. DBU (608 mg, 3.99 mmol, 0.7 eq) was added and allowed to react at room temperature for 0.5 h. TLC was used to monitor the reaction (DCM / MeOH = 20 / 1). If any starting material remained unreacted, 0.2 eq of DBU was added. The reaction was complete by TLC to yield L2-2. 0.7-0.9 eq of 4-methylbenzenesulfonic acid pyridinium chloride was then added. Subsequently, 1.0 eq of HOBt, 1.0 eq of (S)-4,2-(((benzyloxy)carbonyl)amino)-3,9-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38-azatetraconane-4,3-oleic acid, and 0.9 eq of EDCI were added. The reaction was allowed to react at room temperature for 1-2 h. The reaction was complete by LCMS. 500 mL of water / 500 mL of DCM was added to the reaction solution, the layers were separated by extraction, the organic phase was concentrated, DMF was added to dissolve the clear phase, 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).

[0683] LC-MS (ESI) m / z: 681.6 [M / 2+Na] +

[0684] Step 3: Synthesis of L2-4

[0685] 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 hydrogen balloon. The mixture was reacted at 70°C for 3 hours. When the reaction was complete by LCMS, the palladium carbon was removed by filtration, and the filtrate was used directly in the next step.

[0686] LC-MS (ESI) m / z: 1094.5 [M+H] +

[0687] Step 4: Synthesis of L2

[0688] To the DMF solution of compound L2-4, 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) were added, stirred at room temperature for 1 hour, and purified by reverse phase column chromatography (CH3CN / H2O=10%-50%) to give product L2 (1.56 g, 40% yield, two steps).

[0689] LC-MS (ESI) m / z: 666.2 [M / 2+Na] +

[0690] Example 42: Synthesis of Compound L3

[0691] Step 1: Synthesis of benzyl 1-((2-aminoacetamido)methoxy)cyclopropane-1-carboxylate

[0692] Maintaining the temperature at 0-5°C, add t-BuOK (300 mg, 2.7 mmol) to a solution of L3-1 (500 mg, 1.4 mmol) and benzyl 1-hydroxycyclopropane-1-carboxylate (500 mg, 2.6 mmol) in THF (15 mL). The reaction mixture was then heated to room temperature and stirred for 10 minutes. Ice water was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and used directly in the next step.

[0693] LC-MS (ESI) m / z: 279 [M+H] +

[0694] 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

[0695] 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.

[0696] LC-MS (ESI) m / z: 762 [M+H] +

[0697] 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

[0698] 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.

[0699] LC-MS (ESI) m / z: 540 [M+H] +

[0700] Step 4: Synthesis of L3-5

[0701] 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, and purification by preparative HPLC gave the target product (60 mg, yield: 40.2%) as a white solid.

[0702] LC-MS (ESI) m / z: 1344.5 [M+H] +

[0703] Step 5: Synthesis of L3-6

[0704] 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 by TLC and used directly in the next step.

[0705] Step 6: Synthesis of L3

[0706] Succinimidyl 6-(maleimido)hexanoate (20 mg, 0.065 mmol) and DIEA (0.02 mL, 0.11 mmol) were added to a DMF solution of L3-6 and stirred at room temperature overnight. Purification by preparative HPLC afforded the title compound (18 mg, 30% yield over two steps).

[0707] LC-MS (ESI) m / z: 1313.3 [M+H] +

[0708] Example 43: Synthesis of Compound L4

[0709] Step 1: Synthesis of (S)-benzyl 2-(2-(2-aminoacetamido)acetamido)-3-phenylpropionamido)acetate

[0710] To a solution of (S)-benzyl 11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12-tetraoxo-2-oxo-4,7,10,13-tetraazapentadecan-15-oate (3.00 g, 4.63 mmol) in DMF (20.0 mL) was added diethylamine (508 mg, 6.95 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete. The mixture was filtered and the filtrate was concentrated in vacuo to give (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.

[0711] LC-MS (ESI) m / z: 426.9 [M+H] +

[0712] 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

[0713] 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 flash column chromatography on silica gel (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-dioate (1.20 g, yield: 27%) as a yellow solid.

[0714] LC-MS (ESI) m / z: 745.7 [M+H] +

[0715] 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

[0716] 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-dioate (1.20 g, 1.61 mmol) in dichloromethane (5.00 mL) was added TFA (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-oic acid (1.20 g, crude) as a yellow oil, which was used directly in the next step without purification.

[0717] LC-MS (ESI) m / z: 690.4 [M+H] +

[0718] 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

[0719] 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 mixture was stirred at room temperature for 2 hours and then 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.

[0720] LC-MS (ESI) m / z: 616.3 [M+H] +

[0721] 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

[0722] Under nitrogen, to a solution of benzyl((S)-42-((benzyloxy)carbonyl)amino)-39-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38-azatricarbonyl-43-yl)glycylglycyl-L-phenylalanine glycine (645 mg, 0.52 mmol) in MeOH (10.0 mL) was added 10% wet Pd / C. The mixture was evacuated and replaced with hydrogen three times, and stirred at room temperature for 12 hours. LC-MS showed 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) as a yellow oil which was used in the next step without purification.

[0723] LC-MS (ESI) m / z: 1006.9 [M+H] +

[0724] 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

[0725] 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)hexanamido)-39,43,46,49,52-pentaoxo-25,8,11,14,17,20,23,26,26,29,32,32,35-dodeca-38,44,47,50,50-53-pentaazapentaenoic acid (100 mg, two-step yield: 16%) as a yellow oil.

[0726] LC-MS (ESI) m / z: 1201.6 [M+H] +

[0727] 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).

[0728] Example 44: Synthesis of Compound L5

[0729] Step 1: Synthesis of L5-2

[0730] 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.

[0731] LC-MS (ESI) m / z: 1429.7 [M+H] +

[0732] Step 2: Synthesis of L5-3

[0733] Triethylamine (3 mL) was added to the above solution, and the reaction solution was stirred at room temperature overnight. LC-MS showed the desired product, which was purified by preparative HPLC (2.0 g, yield: 87%) as an oil.

[0734] LC-MS (ESI) m / z: 1428.4 [M+H] +

[0735] Step 3: Synthesis of L5

[0736] 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 give the target compound (1.07 mg, yield: 58.6%) as a white solid.

[0737] LC-MS (ESI) m / z: 1473.3 [M+H] +

[0738] Example 45: Synthesis of Compound L6

[0739] Step 1: Synthesis of L6-2

[0740] 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) and stirred for 3 hours at room temperature. LC-MS indicated the reaction was complete. Valyl-alanine (135 mg, 0.72 mmol) and DIEA (0.249 mL, 1.45 mmol) were added to the solution and stirred overnight. LC-MS indicated the reaction was complete. The reaction solution was purified by preparative HPLC to yield the desired product (394 mg, 54% yield) as a white solid.

[0741] LC-MS (ESI) m / z: 993 [M+H] +

[0742] Step 2: Synthesis of L6-3

[0743] 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 overnight at 45°C under H2 protection. The reaction was complete as determined by LC-MS. The reaction mixture was filtered and directly proceeded to the next step.

[0744] LC-MS (ESI) m / z: 859.4 [M+H] +

[0745] Step 3: Synthesis of L6-4

[0746] 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 indicated the reaction was complete, and the reaction solution was used directly in the next step.

[0747] LC-MS (ESI) m / z: 598.0 [M+H] +

[0748] Step 4: Synthesis of L6-5

[0749] 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 complete. The reaction solution was purified by preparative HPLC to obtain the target product (112 mg, 3-step yield: 33%) as an oil.

[0750] LC-MS (ESI) m / z: 972 [M+H] +

[0751] Step 5: Synthesis of L6

[0752] (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. L6-5 (50 mg, 0.051 mmol) and DIEA (0.02 mL, 0.12 mmol) were then added to the reaction mixture, which was stirred at room temperature for 3 hours. LC-MS indicated the reaction was complete, and the desired product (45 mg, 71% yield) was purified by preparative HPLC to obtain an oil.

[0753] LC-MS (ESI) m / z: 1238.6 [M+H] +

[0754] Example 46: Synthesis of Compound L7

[0755] L6-3 (60 mg, 0.09 mmol), succinimidyl 6-(maleimido)hexanoate (23 mg, 0.07 mmol), and DIEA (0.034 mL, 0.2 mmol) were added to a DMF (2 mL) solution at room temperature and stirred overnight. LC-MS indicated the reaction was complete, and the reaction solution was purified by preparative HPLC to yield the title compound (24 mg, 33% yield) as a white solid.

[0756] LC-MS (ESI) m / z: [M / 2+H] + 902.8

[0757] Synthesis and characterization of linker-drug conjugates

[0758] Example 47: Synthesis of Compound LP1

[0759] 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 and stirred at room temperature for 2 hours. LC-MS indicated the reaction was complete. The desired product (34.58 mg, 56% yield) was obtained by preparative HPLC purification as a white solid.

[0760] LC-MS (ESI) m / z: 803.4 [M / 2+H] +

[0761] 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).

[0762] Example 48: Synthesis of Compound LP62

[0763] Step 1: Synthesis of LP62

[0764] To 1 mL of anhydrous DMF, add 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). EDCI (11.4 mg, 0.11 mmol) was then added portionwise. After complete addition, the reaction mixture was stirred at room temperature overnight. Purification by preparative HPLC afforded the desired product (1.4 mg, 29% yield) as a brown oil.

[0765] LC-MS (ESI) m / z: 1014.3 [M / 2+H] +

[0766] Example 49: Synthesis of Compound LP63

[0767] 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), and EDCI (13 mg, 0.07 mmol) was added. The reaction mixture was stirred at room temperature overnight. LC-MS indicated the desired product. Purification by preparative HPLC afforded the desired product (10 mg, 45.5% yield) as a white solid.

[0768] LC-MS (ESI) m / z: [M / 2+H] + 1033.51

[0769] 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).

[0770] Example 50: Synthesis of LP64

[0771] 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 DMF (1 mL). EDCI (61.34 mg, 0.32 mmol) was added portionwise. After complete addition, the reaction mixture was stirred at room temperature overnight. LC-MS indicated the desired product. The reaction mixture was purified by preparative HPLC to yield the desired product, LP64 (18.5 mg, 27.9% yield), as a white solid.

[0772] LC-MS (ESI) m / z: [M / 2+H] + 1020

[0773] 1H NMR (400MHz, DMSO-d6) δ8.87(s,1H),8.75(s,1H),8.68(t,J=6.7Hz,1H),8.32(m,2 H),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).

[0774] Example 51: Synthesis of Compound LP65

[0775] 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). EDCI (16.56 mg, 0.16 mmol, 10 eq) was added. After addition, the reaction mixture was stirred at room temperature overnight. The reaction mixture was purified by preparative HPLC to obtain the desired product (1.3 mg, 3.7% yield) as a white solid.

[0776] LC-MS (ESI) m / z: 1028 [M / 2+H] +

[0777] 1H 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).

[0778] Example 52: Synthesis of Compound LP66

[0779] 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). EDCI (13 mg, 0.07 mmol) was added, and the reaction mixture was stirred at room temperature overnight. LC-MS indicated the desired product. Purification by preparative HPLC afforded the desired product (12 mg, 41.5% yield) as a white solid.

[0780] LC-MS (ESI) m / z: 1034.14 [M / 2+H] +

[0781] 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).

[0782] Example 53: Synthesis of LP67

[0783] 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). EDCI (13 mg, 0.07 mmol) was added portionwise to the reaction mixture. The reaction mixture was stirred at room temperature overnight. LC-MS indicated the presence of the desired product, which was then purified by preparative HPLC to afford the desired product (14 mg, 30% yield) as a white solid.

[0784] LC-MS (ESI) m / z: [M / 2+H] + 1033.3

[0785] 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).

[0786] Example 54: Synthesis of Compound LP68

[0787] 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). EDCI (6.5 mg, 0.035 mmol) was added, and the reaction mixture was stirred at room temperature overnight. Purification by preparative HPLC afforded the desired product (0.27 mg, 5.4% yield) as a white solid.

[0788] LC-MS (ESI) m / z: 984.67 [M / 2+H] +

[0789] Example 55: Synthesis of Compound LP69

[0790] Step 1: Synthesis of LP69

[0791] 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 indicated the desired product, which was purified by preparative HPLC to yield the desired product (1.1 mg, 13.7% yield) as a white solid. LC-MS (ESI) m / z: 1012.8 [M / 2+H] +

[0792] Example 56: Synthesis of Compound LP70

[0793] Step 1: Synthesis of LP70

[0794] 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) was added EDCI (2.4 mg, 0.0125 mmol). The mixture was stirred at room temperature overnight. LC-MS indicated the reaction was complete. Purification by preparative HPLC afforded the desired product (0.3 mg, 5.7% yield) as a white solid.

[0795] LC-MS (ESI) m / z: 1032.8 [M / 2+H] +

[0796] Example 57: Synthesis of Compound LP71

[0797] Step 1: Synthesis of LP71-1

[0798] 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 indicated the desired product, which was purified by preparative HPLC to afford the desired compound (16 mg, 41% yield) as a white solid.

[0799] LC-MS (ESI) m / z: 1127.4 [M / 2+H] +

[0800] Step 2: Synthesis of LP71

[0801] LP71-1 (14 mg, 0.006 mmol) was dissolved in 10% TFA / DCM (4 mL) and stirred at 0°C for 3 hours. TLC indicated 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 yield the desired product (6.3 mg, 46% yield) as a white solid.

[0802] LC-MS (ESI) m / z: 1077.4 [M / 2+H] +

[0803] Example 58: Synthesis of Compound LP72

[0804] 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) was added EDCI (23 mg, 0.06 mmol). The reaction mixture was stirred at 40°C for 1.5 hours and purified by preparative HPLC to afford the title compound (1.50 mg, 11.5% yield) as a white solid.

[0805] LC-MS (ESI) m / z: [M / 2+H] + 1035.4

[0806] Example 59: Synthesis of Compound LP73

[0807] Step 1: Synthesis of LP73-1

[0808] 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 indicated the desired product, which was purified by preparative HPLC to yield the desired product (3 mg, 11.6% yield) as a white solid.

[0809] LC-MS (ESI) m / z: 995.5 [1 / 2M+H] +

[0810] Step 2: Synthesis of LP73

[0811] 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 indicated 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 afford the desired product (2.6 mg, 83% yield) as a white solid.

[0812] LC-MS (ESI) m / z: 946.2 [M / 2+H] +

[0813] Example 60: Synthesis of Compound LP74

[0814] Step 1: Synthesis of LP74-1

[0815] 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 indicated the reaction was complete, and the desired product was purified by preparative HPLC (6 mg, 23% yield) as a white solid.

[0816] LC-MS (ESI) m / z: 993.6 [M / 2+H] +

[0817] Step 2: Synthesis of LP74

[0818] 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 indicated 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 afford the desired product (1.4 mg, 22% yield) as a white solid.

[0819] LC-MS (ESI) m / z: 960.2 [M / 2+H] +

[0820] Example 61: Synthesis of Compound LP75

[0821] 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 at room temperature for 2 hours. LC-MS indicated the reaction was complete, and the reaction solution was purified by preparative HPLC to afford the desired product (1.7 mg, 8.5% yield) as a white solid.

[0822] LC-MS (ESI) m / z: [M / 2+H] + 1052.5

[0823] Example 62: Synthesis of Compound LP76

[0824] 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 at room temperature for 2 hours. LC-MS indicated the reaction was complete, and the reaction solution was purified by preparative HPLC to afford the desired product (3.4 mg, 20% yield) as a white solid.

[0825] LC-MS (ESI) m / z: [M / 2+H] + 916.9

[0826] Example 63: Synthesis of Compound LP77

[0827] 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 mixture was stirred at room temperature for 2 hours. LC-MS indicated the reaction was complete, and the desired product (0.57 mg, 17% yield) was purified by preparative HPLC to obtain a white solid. LC-MS (ESI) m / z: 1981.6 [M+H] +

[0828] Example 64: Synthesis of Compound LP78

[0829] Step: Synthesis of LP78

[0830] 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 indicated the desired product, which was purified by preparative HPLC to yield the desired product (14.7 mg, 38% yield) as a white solid.

[0831] LC-MS (ESI) m / z: 1953.6 [M+H] +

[0832] 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).

[0833] Preparation and characterization of antibody-drug conjugates

[0834] The following are examples of methods for preparing and analyzing antibody-drug conjugates. It should be noted that the following examples are merely partial examples and are not limited to the examples described herein. For example, the antibodies described are not limited to the antibodies currently presented but may be any other antibodies.

[0835] General conjugation process for the preparation of antibody-drug conjugates based on novel ecteinascidin derivatives

[0836] Place the monoclonal antibody (1.0 eq.) in a 0.5 mL centrifuge tube and add 25 mM Na2B4O7, 25 mM NaCl, and 1 mM DTPA buffer to a dilution concentration of 5-10 mg / mL. After vortexing, add 2 mg / mL TCEP (5 to 8 eq.) in water to reduce the antibody. After vortexing, place the reaction mixture on a thermomixer with heating and cooling at 20-25°C for 2-3 hours. Add an equal volume of 500 mM Tris, pH 8.8, or other suitable buffer, followed by a 10 mg / mL DMSO-dissolved toxin linker solution (5 to 8 eq.). Supplement the reaction mixture with DMSO to 10-20% of the final volume. After vortexing, place the mixture on a thermomixer with heating and cooling at 25°C for 16-24 hours.

[0837] Use 300 mg / mL dextran-coated activated carbon (manufacturer: Sigma) to remove 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 results meet the standard (usually 3 times of treatment 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.

[0838] General Characterization Methods for Antibody Drug Conjugates

[0839] (a) DAR value of ADC analyzed by HIC-HPLC

[0840] High performance liquid chromatography: e2695 high performance liquid chromatography system or 1260 Agilent liquid chromatograph.

[0841] Column: MabPac TM HIC-Butyl 5 μm 4.6×100 mm (manufacturer: Thermo).

[0842] Mobile phase: Mobile phase A (MPA): 1.5M (NH4)2SO4 + 50mM potassium phosphate (pH 7.0); Mobile phase B (MPB): 50mM sodium phosphate (pH 7.0) / isopropanol (75:25V / V); Elution was performed according to the following elution program (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%.

[0843] 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.

[0844] 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.

[0845] DAR value calculation formula: DAR = Σ (relative peak area × number of loaded drugs) / 100

[0846] (b) C18-HPLC analysis of Free Linker Payload (mol / mol%)

[0847] High performance liquid chromatography: e2695 high performance liquid chromatography system.

[0848] Chromatographic column: C18 3.5 μm 4.6×150 mm (manufacturer: Waters).

[0849] 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 program (5%-95%): 0-30 min: 90%-20% mobile phase A, 10%-80% mobile phase B; 30-31 min: 20%-90% mobile phase A, 80%-10% mobile phase B; 31-35 min: 90%-90% mobile phase A, 10%-10% mobile phase B

[0850] 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.

[0851] Reagent I preparation example: 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 at least 1 hour, then let it stand for 1 hour. Remove the supernatant, filter it through a 0.22 μm organic membrane, and store at room temperature. It is valid for 3 months.

[0852] Reagent II preparation example: Measure 100 mL of Reagent I, 15 mL of DMSO, and 85 mL of ADC Sample Storage Buffer, mix well in a container, and store at room temperature. The shelf life is 2 months.

[0853] Experimental steps:

[0854] Sample solution preparation:

[0855] 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.

[0856] 2) Take 10 μl of the 1.0 mg / ml toxin reference sample and add it to 90 μl of Reagent II to prepare a concentration of 100 μg / ml. Then, dilute the sample with Reagent II according to the table below to prepare the sample of the desired concentration. Note: Inject the samples prepared above to the required concentration into HPLC in ascending order.

[0857] 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 high performance liquid chromatograph, elute using the above elution procedure, and record the chromatogram.

[0858] According to the calculated standard binary linear regression curve equation, the content of free toxin in ADC was calculated.

[0859] Free Drug (mol / mol%) = residual small molecule molar concentration / antibody molar concentration × 100.

[0860] (c) Determination of free linker-payload content by LC-MS / MS

[0861] Internal reference compounds: 50 ng / ml tolbutamide, labetalol, etofesamide, and buspirone

[0862] Extraction solvent: ethyl acetate

[0863] Serial dilution of the standard: dilute the standard to 20,000, 16,000, 10,000, 4,000, 1,000, 200, 40, and 20 ng / ml with 50% methanol in water.

[0864] Sample pre-treatment: Take 20 μL of sample, then add 20 μL of blank buffer and 240 μL of extraction agent. After vortex mixing, let stand until layers separate. Then, take 160 μL of supernatant, place it in a 96-well plate, air dry, add 200 μL of 50% acetonitrile water, mix thoroughly, and finally perform LC-MS / MS analysis.

[0865] Standard pre-treatment: Add 2 μL of working solution to 38 μL of blank buffer, then add 240 μL of extraction agent. Vortex to mix thoroughly, let stand until layers separate. Transfer 160 μL of supernatant to a 96-well plate, air dry, and add 200 μL of 50% acetonitrile water. Mix thoroughly.

[0866] 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 by linear fitting the curve (formula) and the mass spectrometry response value of the test sample.

[0867] (d) SEC-HPLC analysis of ADC aggregates

[0868] High performance liquid chromatography: e2695 high performance liquid chromatography system or 1260 Agilent liquid chromatograph.

[0869] Chromatographic column: Waters Xbridge BEH200 SEC (7.8×300mm, 3.5μm)

[0870] Mobile phase: 50 mM PB + 200 mM Arg (pH 6.80) + 10% IPA, elution was performed according to the following elution program, 0-30 min mobile phase A volume was 100%-100%;

[0871] 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.

[0872] 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.

[0873] Calculation formula: Monomer purity (%) = A monomer / A total × 100%; polymer purity (%) = A polymer / A total × 100%

[0874] Example 65: Preparation of Antibody Drug Conjugate ADC-1

[0875] n 12 =2.3

[0876] According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mM, 1.8 eq.) was added to trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.4 buffer and reduced at 22°C for 2 hours; then LP1 (10 mg / mL, 4.0 eq.) in DMSO 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 eight times to obtain ADC-1 (C ADC (mg / mL):10.56, V (mL):0.08, yield 84.5%).

[0877] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0878] HIC-DAR: 2.30, SEC purity: 98.85%, Free Linker Payload (mol / mol%): not detected.

[0879] Example 66: Preparation of Antibody Drug Conjugate ADC-85

[0880] n 12 =1.2

[0881] According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mM, 2 eq.) aqueous solution was added to trastuzumab (1.0 mg, 21.07 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer and reduced at 22°C for 2 hours; then LP62 (10 mg / mL, 5.0 eq.) in DMSO 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-85 (C ADC (mg / mL): 6.68, V (mL): 0.1, yield 66.8%).

[0882] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0883] HIC-DAR: 1.20, SEC purity: 100%, Free Linker Payload (mol / mol%): not detected.

[0884] Example 67: Preparation of Antibody Drug Conjugate ADC-86

[0885] n 12 =6.5

[0886] 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%).

[0887] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0888] HIC-DAR: 6.5, SEC purity: 65%, Free Linker Payload (mol / mol%): not detected.

[0889] Example 68: Preparation of Antibody Drug Conjugate ADC-87

[0890] n 12 =8.0

[0891] According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mM, 8 eq.) aqueous solution was added to trastuzumab (1.0 mg, 21.07 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer and reduced at 22°C for 2 hours; then LP65 (10 mg / mL, 12.0 eq.) in DMSO was added and the coupling reaction was carried out at 22°C for 3 hours. 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-87 (C ADC (mg / mL): 5.53, V (mL): 0.15, yield 63.6%).

[0892] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0893] HIC-DAR: 8.0, SEC purity: 89%, Free Linker Payload (mol / mol%): not detected.

[0894] Example 69: Preparation of Antibody Drug Conjugate ADC-88

[0895] n 12 =4.0

[0896] According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mM, 2 eq.) aqueous solution was added to trastuzumab (1.0 mg, 21.07 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer and reduced at 22°C for 2 hours; then LP66 (10 mg / mL, 8.0 eq.) in DMSO 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-88 (C ADC (mg / mL):6.09, V (mL):0.11, yield 66.9%).

[0897] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0898] HIC-DAR: 4.0, SEC purity: 99.08%, Free Linker Payload (mol / mol%): not detected.

[0899] Example 70: Preparation of Antibody Drug Conjugate ADC-89

[0900] n 12 =3.8

[0901] According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mM, 2 eq.) aqueous solution was added to trastuzumab (1.0 mg, 21.07 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer and reduced at 22°C for 2 hours; then LP67 (10 mg / mL, 8.0 eq.) in DMSO 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-89 (C ADC (mg / mL):4.73, V (mL):0.12, yield 56.8%).

[0902] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0903] HIC-DAR: 3.8, SEC purity: 92.87%, Free Linker Payload (mol / mol%): not detected.

[0904] Example 71: Preparation of Antibody Drug Conjugate ADC-90

[0905] n 12 =2.1

[0906] According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mM, 2 eq.) aqueous solution was added to trastuzumab (1.0 mg, 21.07 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer and reduced at 22°C for 2 hours; then LP68 (10 mg / mL, 9.0 eq.) in DMSO 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-90 (C ADC (mg / mL): 2.81, V (mL): 0.12, yield 33.7%).

[0907] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0908] HIC-DAR: 2.08, SEC purity: 93.64%, Free Linker Payload (mol / mol%): not detected.

[0909] Example 72: Preparation of Antibody Drug Conjugate ADC-91

[0910] n 12 =7.9

[0911] According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mM, 2 eq.) aqueous solution was added to trastuzumab (1.0 mg, 21.07 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer and reduced at 22°C for 2 hours; then LP64 (10 mg / mL, 5.0 eq.) in DMSO 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 eight times to obtain ADC-91 (C ADC(mg / mL):8.18, V (mL):0.13, yield 70.9%).

[0912] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0913] HIC-DAR: 7.93, SEC purity: 95.63%, Free Linker Payload (mol / mol%): not detected.

[0914] Example 73: Preparation of Antibody Drug Conjugate ADC-92

[0915] n 12 =4.6

[0916] According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mM, 2 eq.) aqueous solution was added to trastuzumab (1.0 mg, 21.07 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer and reduced at 22°C for 2 hours; then LP70 (10 mg / mL, 5.0 eq.) in DMSO 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 eight times to obtain ADC-92 (C ADC (mg / mL): 5.82, V (mL): 0.1, yield: 58.2%).

[0917] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0918] HIC-DAR: 4.6, SEC purity: 98.92%, Free Linker Payload (mol / mol%): not detected.

[0919] Example 74: Preparation of Antibody Drug Conjugate ADC-93

[0920] n 12 =1.6

[0921] According to the general coupling procedure for preparing antibody drug conjugates, TCEP (10 mM, 2 eq.) aqueous solution was added to trastuzumab (2.0 mg, 21.07 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer, and the mixture was reduced at 22°C for 2 hours. Then, LP71 (10 mg / mL, 5.0 eq.) in DMSO was added, and the coupling reaction was carried out at 22°C for 1 hour.

[0922] Then, 500 mM Tris-HCl, pH 8.8 solution (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 three times, the buffer of the desired antibody drug conjugate was ultrafiltered eight times to obtain ADC-93 (C ADC (mg / mL):8.76, V (mL):0.1, yield: 43.8%).

[0923] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0924] HIC-DAR: 1.6, SEC purity: 94.6%, Free Linker Payload (mol / mol%): not detected.

[0925] Example 75: Preparation of Antibody Drug Conjugate ADC-94

[0926] n 12 =1.6

[0927] According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mM, 2 eq.) was added to trastuzumab (2 mg, 21.07 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer and reduced at 22°C for 2 hours; then LP73 (10 mg / mL, 5.0 eq.) in DMSO was added and the coupling reaction was carried out at 22°C for 1 hour. 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 three times. The buffer of the desired antibody drug conjugate was ultrafiltered eight times to obtain ADC-94 (C ADC (mg / mL):8.99, V (mL):0.1, yield: 45%).

[0928] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0929] HIC-DAR: 1.6, SEC purity: 97.67%, Free Linker Payload (mol / mol%): not detected.

[0930] Example 76: Preparation of Antibody Drug Conjugate ADC-95

[0931] n 12 =2.9

[0932] According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mM, 2 eq.) aqueous solution was added to trastuzumab (1.5 mg, 21.07 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer and reduced at 22°C for 2 hours; then LP75 (10 mg / mL, 5.0 eq.) in DMSO 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 eight times to obtain ADC-95 (C ADC (mg / mL): 6.18, V (mL): 0.11, yield: 45.3%).

[0933] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0934] HIC-DAR: 2.9, SEC purity: 99.61%, Free Linker Payload (mol / mol%): not detected.

[0935] Example 77: Preparation of Antibody Drug Conjugate ADC-96

[0936] n 12 =4.3

[0937] According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mM, 2 eq.) aqueous solution was added to trastuzumab (1.5 mg, 21.07 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer and reduced at 22°C for 2 hours; then LP76 (10 mg / mL, 5.0 eq.) in DMSO 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 eight times to obtain ADC-96 (C ADC (mg / mL): 6.82V (mL): 0.11, yield: 50%).

[0938] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0939] HIC-DAR: 4.3, SEC purity: 98.38%, Free Linker Payload (mol / mol%): not detected.

[0940] Example 78: Preparation of Antibody Drug Conjugate ADC-97

[0941] n 12 =3.4.

[0942] According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mM, 2 eq.) aqueous solution was added to trastuzumab (0.8 mg, 21.07 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer and reduced at 22°C for 2 hours; then LP77 (10 mg / mL, 5.0 eq.) in DMSO 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 eight times to obtain ADC-97 (C ADC (mg / mL): 3.46, V (mL): 0.1, yield: 43.3%).

[0943] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0944] HIC-DAR: 3.4, SEC purity: 98.9%, Free Linker Payload (mol / mol%): not detected.

[0945] Example 79: Preparation of Antibody Drug Conjugate ADC-98

[0946] n 12 =1.7

[0947] According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mM, 2 eq.) aqueous solution was added to trastuzumab (1.0 mg, 21.07 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer and reduced at 22°C for 2 hours; then LP78 (10 mg / mL, 7.0 eq.) in DMSO 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 three times to obtain ADC-98 (C ADC (mg / mL): 6.21, V (mL): 0.1, yield: 62.1%).

[0948] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0949] HIC-DAR: 1.7, SEC purity: 97.85%, Free Linker Payload (mol / mol%): not detected.

[0950] Biological evaluation

[0951] 1) Tumor cell proliferation inhibition experiment of new ecteinascidin derivatives

[0952] MDA-MB-231, OV90, and NCI-N87 cells (1500 cells per well, 80 μL volume) were plated into 96-well plates at various cell concentrations. The cells were cultured overnight in a constant temperature incubator (37°C, 5% CO2). A solution of an ecteinascidin derivative was prepared in DMSO, with a maximum concentration of 2 mM. After a 1:4 serial dilution, 1 μL of this DMSO-based solution was added to 99 μL of 10% FBS-containing PBS per well and mixed thoroughly. The diluted solution (2 μL per well, 18 μL of 10% FBS-containing PBS) was incubated with the cells (37°C, 5% CO2) for five days. CellTitre-Glo reagent (40 μL per well) was then added, and fluorescence was measured using an i3X microplate reader.

[0953] In vitro cytotoxicity of the ecteinascidin derivatives of the present invention (++++: <1 nM; +++: 1-10 nM; ++: 10-200 nM; +: >200 nM)

[0954] All ecteinascidin derivatives exhibited certain antitumor activity, among which 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 had in vitro cytotoxicity that was stronger than or equivalent to that of trabectedin and rubectin.

[0955] 2) Tumor cell proliferation inhibition experiment based on antibody-drug conjugates based on novel ecteinascidin derivatives

[0956] 1500 cells were seeded in a 96-well plate (excluding the edge wells filled with PBS), 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 novel ectenes derivative was added to each well to give a final conjugate concentration range of 3.8pM-250nM. After incubation of the cells with the conjugate for 5 days, the 96-well plate was placed at room temperature for 30 minutes. 40uL of CellTiter- (Promega, G7572) reagent was reacted in the dark for 10 minutes, and fluorescence was measured using a SpectraMax i3X microplate reader. Graphpad Prism software was used to fit the curve of luminescence value versus conjugate concentration (nM).

[0957] In vitro cytotoxicity of antibody-drug conjugates (++++: <1 nM; +++: 1-10 nM; ++: 10-200 nM; +: >200 nM)

[0958] 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.

[0959] The main advantages of the present invention are:

[0960] 1. Some of the novel ecteinascidin derivatives provided by the present invention have relatively strong in vitro cytotoxicity (1-10 nM), while others have even stronger in vitro cytotoxicity (<1 nM).

[0961] 2. The antibody-drug conjugates based on novel ecteinascidin derivatives provided by the present invention have strong in vitro cytotoxicity (1-10 nM), and some antibody-drug conjugates have even 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 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 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 its connected The nitrogen atom forms a saturated heterocyclic ring or no 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, R2, R3 together with the carbon atoms to which they are connected constitute a 3-6-membered saturated carbon atom. cyclic, 3- to 6-membered saturated heterocyclic or acyclic; said x is selected from an integer of 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 Selected from 3 to 10 membered saturated or partially unsaturated heterocyclic group, 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: M-L1-L2-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; Where D is covalently bonded to L2 through 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 = integer of 1-18, n2 = integer of 0-18, n3 = integer of 1-18, n4 = 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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