Cyclic dinucleotide compound, conjugate and application thereof

Abstract

The invention discloses a cyclic dinucleotide compound as well as a conjugate and application thereof, and particularly discloses an antibody-immunostimulatory conjugate as shown in a formula II or pharmaceutically acceptable salt thereof. The antibody-immunostimulation conjugate provided by the invention has good tumor specificity and has an obvious inhibition effect on tumors. # imgabs0 #

Description

[0001] This application claims the priority of Chinese Patent Application No. 2023118185393 with the filing date of December 27, 2023. This application incorporates the entire text of the above-mentioned Chinese patent application by reference. Technical Field

[0002] The present invention relates to a cyclic dinucleotide compound, its conjugate, a pharmaceutical composition and uses thereof. Background Art

[0003] Stimulator of interferon genes (STING), also known as T1MEM173, MITA, MPYS, and ERIS, is an important signaling molecule in innate immune signaling. The protein encoded by this gene contains a five-transmembrane structure and plays an important regulatory role in the immune response related to viral or bacterial infections. As a pattern recognition receptor, STING can detect and recognize exogenous nucleic acids in the cytoplasm and activate the signal transduction pathway related to the type I interferon response. In addition, studies have shown that STING participates in the regulation of apoptosis signals by interacting with major histocompatibility complex class II (MHCII). Studies on human tumors with spontaneous T cell infiltration have shown that CD8+ T cell infiltration is closely related to the transcriptional signature of type I interferon (Harlin et al, Cancer Res, 2009; 69(7):OF1). Mechanistic studies in mouse models have shown that in experimental animals with defective type I interferon signaling, the process of T cell activation against tumor-associated antigens is abnormal (Diamond et al, J. Exp. Med., 2011; 208(10):1989; Fuerte et al, J. Exp. Med., 2011; 208(10):2005). Further studies on the process of innate immune system recognition of tumors in vivo and related signaling pathways such as IFN expression mediated by antigen-presenting cells (APCs) during this process have revealed that the STING signaling pathway can be activated by DNA in the cytoplasm. These exogenous nucleic acids can be recognized by cyclic-GMP-AMP synthase (cGAS), which then catalyzes the generation of cyclic nucleic acids such as cyclic GMP-AMP (cGAMP). These cyclic nucleic acids can act as endogenous ligands to activate the STING signal (Sun et al, Science, 2013; 339(15):786). Activated STING can then induce the autophosphorylation of TBK1 kinase and the phosphorylation of interferon regulatory factor 3 (IRF-3). Phosphorylated IRF3 can further activate the gene transcription process of type I interferon, regulate the synthesis and secretion of type I interferon, and thus trigger an immune response. In summary, existing studies have shown that the STING signaling pathway plays an extremely important role in the process of tumor recognition by the innate immune system. The activation of this signaling pathway on antigen-presenting cells is directly related to the process of T cell activation against tumor-associated antigens. Based on its role in tumor immune recognition, it is expected that activating the STING signal by drugs or other pharmacological methods can enhance IFN expression and have a positive effect on tumor treatment. Therefore, the development of STING signal agonists for the treatment of tumor diseases has become a research hotspot.

[0004] In addition, studies have shown that stimulating the activation of the STING signaling pathway also contributes to the antiviral response. The loss of functional response at the cellular or organismal level demonstrates that viral load cannot be controlled in the absence of STING. Activation of the STING signaling pathway triggers an immune response leading to antivascular and proinflammatory cytokines against the virus and mobilizes the innate and adaptive immune systems. Therefore, small molecule compounds with agonistic effects on the STING signaling pathway have the potential to treat chronic viral infections, such as being used to treat HBV.

[0005] Currently, cyclic dinucleotide (CDN) analog STING agonists are mostly administered via intratumoral injection, and this administration method has several drawbacks, such as issues like tumor accessibility by intratumoral injection, tumor exposure, and nonspecificity. Currently, there are no marketed ADC drugs of CDN analogs. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defects of the existing cyclic dinucleotide analog STING agonists, such as single administration method, poor tumor accessibility, and poor specificity of action with tumors, and to provide a cyclic dinucleotide compound, its antibody conjugate, its pharmaceutical composition, and applications thereof. The antibody conjugate of the cyclic dinucleotide compound of the present invention has good tumor specificity and has a significant inhibitory effect on tumors.

[0007] The present invention solves the above technical problems through the following technical solutions.

[0008] The present invention provides an antibody-immunostimulatory conjugate of formula II or a pharmaceutically acceptable salt thereof,

[0009]

[0010] wherein Ab is an antibody;

[0011] t is 1 to 8;

[0012] L is a linker having the following combination: -(L1)a-(Z)b-M-; L1 is connected to D, and M is connected to Ab;

[0013] a is 0, 1, 2, 3, 4, 5, or 6; b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0014] D is the group formed by the shown compound losing one hydrogen atom; the *-labeled P stereoconfigurations are independently R, S, or R / S;

[0015] B1 and B2 are each independently and at least one of B1 and B2 is

[0016] R1 and R2 are independently -OH, -N3, F, -OCH3 or -SCF3;

[0017] R3 and R 3’ are independently H, -C(O)R4, -C(O)OR4, -OR4 or -R4;

[0018] R4 is C 1-10 alkyl, C 6-10 aryl, 5-10 membered heteroaryl, C 3-10 cycloalkyl, 3-10 membered heterocycloalkyl, C 6-10 arylC 1-10 alkyl, 5-10 membered heteroarylC 1-10 alkyl, C 3-10 cycloalkylC 1-10 alkyl, 3-10 membered heterocycloalkylC 1-10 alkyl; said R4 is unsubstituted or optionally substituted at any position by 1 to 3 substituents selected from hydroxy, mercapto, dithiol, amino, C 1-6 alkylamino, C 1-6 alkylaminoC 1-10 alkyl;

[0019] R5 is H, -(L2) d -(Z) e -(maleimidyl) or -(L2) d -(Z) e -H;

[0020] d is 0, 1, 2, 3, 4, 5 or 6; e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0021] Each L1 and each L2 are independently The c2 side of L1 is connected to D; the c2 side of L2 is connected to the mercapto group in D;

[0022] X is

[0023] p is independently 1, 2 or 3;

[0024] Q is a linking bond, -C(O)O-, -C(O)N(R6)- or -C 1-3 alkylene-O-;

[0025] Q1 is phenyl or pyridyl;

[0026] R6 is H or C 1-6 alkyl; said C 1-6The alkyl group is unsubstituted or optionally substituted at any position by 1 C 1-6 -alkylamino or C 1-6 -alkylsulfonyl;

[0027] R7 and R 7’ are each independently H or C 1-6 -alkyl;

[0028] R8 is phenyl or a 5- to 10-membered heteroaryl group; said R8 is unsubstituted or optionally substituted at any position by 1 to 3 substituents selected from hydroxy, amino, carboxy, cyano, nitro, mercapto, C 1-6 -alkyl, C 3-8 -cycloalkyl, halogen, halo-C 1-6 -alkyl, C 1-6 -alkylamino, C 1-6 -alkoxy, halo-C 1-6 -alkoxy, C 1-6 -alkylthio, amido, hydroxyamino, formyl, acetyl, methoxycarbonyl, amino-C 1-6 -alkyl, hydroxy-C 1-6 -alkyl and C 1-6 -alkylsulfonyl;

[0029] Each Z is independently -(A) v -, -PEG-, -C(O)-(CH2) x -, -NR9-(CH2) y -, -O-(CH2) y -, -S-(CH2) y -, -(CH2) x -C 6-14 -arylene-(CH2) y -, -(CH2) x -5- to 6-membered heteroarylene-(CH2) y -, -(CH2) x -C 3-6 -cycloalkylene-(CH2) y -, -(CH2) x -3- to 6-membered heterocycloalkylene-(CH2) y -, -NR9-(CH2) x -C(O)-, -O-(CH2) x -C(O)-, -S-(CH2) x -C(O)-, -(CH2) x CH(-L3-R9)-C(O)-, -(CH2) x CH(-L3-R 9a )-C(O)-, -(CH2) x -S-S-(CH2)y -, C 1-6 Alkylene or C 2-6 Alkenylene;

[0030] L3 is a linking bond, -NH-, or

[0031] R9 is independently hydrogen, C 1-6 Alkyl, -PO(OH)2, -PO(OCH3)2, -C(O)-(CH2CH2O) n -CH3, -(CH2CH2O) n -CH3,

[0032] R 9a is independently

[0033] -PEG- is -(CH2CH2O) n -(CH2) u - or -(CH2CH2O) n -(CH2) u -C(O)-;

[0034] A is independently an amino acid residue;

[0035] x and y are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; v is independently 1, 2, 3, 4, or 5; n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50; u is independently 0, 1, 2, 3, 4, or 5;

[0036] M is a linker that is linked to Ab.

[0037] In some embodiments, in the antibody-immunostimulatory conjugate or a pharmaceutically acceptable salt thereof shown in Formula II, the definitions of some groups are as described below, and the definitions of the remaining groups are as described in any other embodiment (hereinafter referred to as "in some embodiments"):

[0038] Ab is an antibody;

[0039] t is 1 to 8;

[0040] L is a linker having the following combination: -(L1) a -(Z) b-M-; L1 is connected to D, and M is connected to Ab;

[0041] a is 0, 1, 2, 3, 4, 5 or 6; b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0042] D is a group formed by the shown compound losing a hydrogen atom; the *-labeled P-configuration is independently R, S or R / S;

[0043] B1 and B2 are independently And at least one of B1 and B2 is

[0044] R1 and R2 are independently -OH, -N3, F, -OCH3 or -SCF3;

[0045] R3 and R 3’ are independently H, -C(O)R4, -C(O)OR4, -OR4 or -R4;

[0046] R4 is C 1-10 alkyl, C 6-10 aryl, 5-10 membered heteroaryl, C 3-10 cycloalkyl, 3-10 membered heterocycloalkyl, C 6-10 arylC 1-10 alkyl, 5-10 membered heteroarylC 1-10 alkyl, C 3-10 cycloalkylC 1-10 alkyl, 3-10 membered heterocycloalkylC 1-10 alkyl; the said R4 is unsubstituted or optionally substituted at any position by 1 to 3 substituents selected from hydroxyl, mercapto, dithiol, amino, C 1-6 alkylamino, C 1-6 alkylaminoC 1-10 alkyl;

[0047] R5 is H, -(L2) d -(Z) e -(maleimide group) or -(L2) d -(Z) e -H;

[0048] d is 0, 1, 2, 3, 4, 5 or 6; e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0049] Each L1 and each L2 are independently connected to D at the c2 side;

[0050] X is

[0051] p is independently 1, 2 or 3;

[0052] Q is a linking group, -C(O)O-, -C(O)N(R6)- or -C 1-3 alkylene-O-;

[0053] Q1 is phenyl or pyridyl;

[0054] R6 is H or C 1-6 alkyl; the C 1-6 alkyl is unsubstituted, or optionally substituted at any position by 1 C 1-6 alkylamino or C 1-6 alkylsulfonyl;

[0055] R7 and R 7’ are each independently H or C 1-6 alkyl;

[0056] R8 is phenyl or a 5- to 10-membered heteroaryl; the R8 is unsubstituted, or optionally substituted at any position by 1 to 3 substituents selected from hydroxy, amino, carboxy, cyano, nitro, mercapto, C 1-6 alkyl, C 3-8 cycloalkyl, halogen, halo-C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, halo-C 1-6 alkoxy, C 1-6 alkylthio, amido, hydroxyamino, formyl, acetyl, methoxycarbonyl, amino-C 1-6 alkyl, hydroxy-C 1-6 alkyl and C 1-6 alkylsulfonyl;

[0057] Each Z is independently -(A) v -, -PEG-, -C(O)-(CH2) x -, -NR9-(CH2) y -, -O-(CH2) y -, -S-(CH2) y -, -(CH2) x -C 6-14 arylene-(CH2) y -, -(CH2) x -5- to 6-membered heteroarylene-(CH2) y -, -(CH2) x -C 3-6 cycloalkylene-(CH2) y -, -(CH2) x -3- to 6-membered heterocycloalkylene-(CH2)y -, -NR9-(CH2) x -C(O)-, -O-(CH2) x -C(O)-, -S-(CH2) x -C(O)-, -(CH2) x CH(NHR9)-C(O)-, -(CH2) x -S-S-(CH2) y -, C 1-6 alkylene or C 2-6 alkenylene;

[0058] R9 is independently hydrogen, C 1-6 alkyl, -PO(OH)2, -PO(OCH3)2, -C(O)-(CH2CH2O) n -CH3 or -(CH2CH2O) n -CH3; -PEG- is -(CH2CH2O) n -(CH2) u - or -(CH2CH2O) n -(CH2) u -C(O)-;

[0059] A is independently an amino acid residue;

[0060] x and y are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; v is independently 1, 2, 3, 4 or 5; n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50; u is independently 0, 1, 2, 3, 4 or 5;

[0061] M is a linker that links to Ab.

[0062] In some embodiments, the antibody may comprise one or more antigen-binding domains that can bind to an antigen.

[0063] In some embodiments, the antibody may comprise one or two antigen-binding domains that can bind to an antigen.

[0064] In some embodiments, the antibody may comprise one antigen-binding domain that can bind to an antigen.

[0065] In some embodiments, the antibody may comprise only one antigen-binding domain that can bind to an antigen.

[0066] In some embodiments, the antibody may comprise an Fc segment.

[0067] In some embodiments, the antibody may comprise only an Fc segment.

[0068] In some embodiments, the antibody may comprise only one antigen-binding domain capable of binding to an antigen and an Fc segment.

[0069] In some embodiments, the antibody may be a monoclonal antibody.

[0070] In some embodiments, the antibody is an anti-HER2 antibody, an anti-EGFR antibody or an anti-5T4 antibody.

[0071] In some embodiments, the anti-HER2 monoclonal antibodies include, but are not limited to: Trastuzumab, Trastuzumab biosimilar, Pertuzumab, Pertuzumab biosimilar, Margetuximab, HT-19, etc.

[0072] In some embodiments, the antibody is Trastuzumab or Pertuzumab.

[0073] In some embodiments, the anti-EGFR antibodies include, but are not limited to: Cetuximab.

[0074] In some embodiments, the anti-EGFR antibody is Cetuximab or Nimotuzumab.

[0075] In some embodiments, the anti-5T4 antibodies include, but are not limited to: huA1.

[0076] In some embodiments, the antibody is Trastuzumab, Pertuzumab, Cetuximab or huA1.

[0077] In some embodiments, the antibody is Trastuzumab, Pertuzumab, Cetuximab, huA1, Cetuximab, Nimotuzumab; or variants thereof.

[0078] In the antibody-immunostimulatory conjugate or a pharmaceutically acceptable salt thereof as shown in Formula II, t is an integer or a non-integer. When it is a non-integer, it means that the antibody-immunostimulatory conjugate as shown in Formula II is a mixture of antibody-immunostimulatory conjugates with different conjugation ratios; when it is an integer, it may mean that the antibody-immunostimulatory conjugate as shown in Formula II is a single antibody-immunostimulatory conjugate with a fixed conjugation ratio, or it may also mean that the antibody-immunostimulatory conjugate as shown in Formula II is a mixture of antibody-immunostimulatory conjugates with different conjugation ratios.

[0079] In some embodiments, t can be any value from 2 to 8.

[0080] In some embodiments, t can be any value from 1 to 3.

[0081] In some embodiments, t can be any value from 3 to 5.

[0082] In some embodiments, t can be any value from 6 to 8.

[0083] In some embodiments, a is 0, 1, 2, 3 or 4; a is preferably 0, 1 or 2.

[0084] In some embodiments, b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; b is preferably 1, 2, 3, 4, 5, 6, 7 or 8; b is more preferably 1, 2, 3, 4, 5 or 6.

[0085] In some embodiments, d is 0, 1, 2 or 3; d is preferably 1 or 2; d is more preferably 1.

[0086] In some embodiments, e is 0, 1, 2, 3, 4, 5 or 6; e is preferably 0, 1, 2, 3 or 4; e is more preferably 0, 1, 2 or 3.

[0087] In some embodiments, R6 is H or C 1-6 alkyl; the C 1-6 alkyl is unsubstituted or optionally substituted at any position by 1 dimethylamino or methylsulfonyl group.

[0088] In some embodiments, R6 is H, -CH3, -CH2CH2N(CH3)2 or -CH2CH2S(O)2CH3.

[0089] In some embodiments, each L1 and each L2 are each independently The c2 side of L1 is connected to D; the c2 side of L2 is connected to the sulfhydryl group in D.

[0090] In some embodiments, each L1 and each L2 are independently The c2 side of L1 is connected to D; the c2 side of L2 is connected to the thiol group in D.

[0091] In some embodiments, -(L1) a - is The c2 side is connected to D.

[0092] In some embodiments, -(L1) a - is The c2 side is connected to the thiol group in D.

[0093] In some embodiments, -(L1) a - is The c2 side is connected to the amino group in D.

[0094] In some embodiments, -(L1) a - is The c2 side is connected to the amino group in D.

[0095] In some embodiments, -(L1) a - is absent, The c2 side is connected to the thiol group in D.

[0096] In some embodiments, Z is independently -(A) v -, -PEG-, -C(O)-(CH2) x -, -NR9-(CH2) y -, -O-(CH2) y -, -S-(CH2) y -, -(CH2) x -C 6-14 Arylene-(CH2) y -, -(CH2) x -5- to 6-membered heteroarylene-(CH2) y -, -(CH2) x -C 3-6 Cycloalkylene-(CH2) y -, -(CH2) x -3- to 6-membered heterocycloalkylene-(CH2) y -, -NR9-(CH2) x -C(O)-, -O-(CH2) x -C(O)-, -S-(CH2) x -C(O)-, -(CH2)x CH(NHR9)-C(O)-, -(CH2) x -S-S-(CH2) y -, C 1-6 alkylene or C 2-6 alkenylene; x and y are independently any integer from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; v is independently any integer from 1, 2, 3, 4 or 5; said -(CH2) x -5- to 6-membered heteroarylene-(CH2) y - in the "5- to 6-membered heteroarylene", the heteroatoms are selected from one or more of N, O and S, and the number of heteroatoms is 1, 2 or 3; said -(CH2) x -3- to 6-membered heterocycloalkylene-(CH2) y - in the "3- to 6-membered heterocycloalkylene", the heteroatoms are selected from one or more of N, O and S, and the number of heteroatoms is 1, 2 or 3.

[0097] In some embodiments, in A, the amino acid is a natural amino acid or a non-natural amino acid.

[0098] In some embodiments, in A, the amino acid residue is modified by 1 to 3 R 10 ; R 10 is independently C 1-6 alkyl, C 1-6 alkyl acyl, C 1-6 alkoxy acyl, -C(O)-(CH2CH2O) n -CH3 or -(CH2CH2O) n -CH3; said C 1-6 alkyl, C 1-6 alkyl acyl or C 1-6 alkoxy acyl may further be substituted by 1 R8 at any position.

[0099] In some embodiments, each A is independently each R A is independently an amino acid side chain or an amino acid side chain modified by 1 to 3 R 10 ; or R A and the adjacent nitrogen atom form a five-membered heterocyclic group;

[0100] said R 10 is independently C 1-6 alkyl, C 1-6 alkyl acyl, C 1-6 alkoxy acyl, -C(O)-(CH2CH2O) n -CH3 or -(CH2CH2O) n-CH3; The C 1-6 alkyl, C 1-6 alkyl acyl or C 1-6 alkoxycarbonyl may be further substituted by one R8 at any position.

[0101] In some embodiments, the may be or may also be

[0102] In some embodiments, in A, each R A is independently hydrogen, methyl, ethyl, n-propyl, isopropyl, isobutyl, or R A and the adjacent nitrogen atom form a five-membered heterocyclic group.

[0103] In some embodiments, -(A) v - is each R A is independently hydrogen, methyl, ethyl, n-propyl, isopropyl, isobutyl, or R A and the adjacent nitrogen atom form a five-membered heterocyclic group; v is 1, 2, 3 or 4, and v is preferably 1 or 2.

[0104] In some embodiments, -(A) v - is

[0105] In some embodiments, -(A) v - is

[0106] In some embodiments, -(Z) b - is any of the following combinations:

[0107] 1)-(CH2) x -(c1),

[0108] 2)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -(c1),

[0109] 3)-C(O)-(CH2) x -(c1),

[0110] 4)-C(O)-(CH2) x -C 3-6 subcycloalkyl-(CH2)y -(c1),

[0111] 5)-C(O)-(CH2) x -phenylene-(CH2) y -(c1),

[0112] 6)-C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -(c1),

[0113] 7)-C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -phenylene-(CH2) y -(c1),

[0114] 8)-(A) v -(c1),

[0115] 9)-(A) v -C(O)-(CH2) x -(c1),

[0116] 10)-(A) v -C(O)-(CH2) x -phenylene-(CH2) y -(c1),

[0117] 11)-(A) v -C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -(c1) or

[0118] 12)-(A) v -C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -phenylene-(CH2) y -(c1); wherein, the c1 side is connected to M.

[0119] In some embodiments, -(Z) b -is any one of the following combinations:

[0120] 13)-C(O)-(CH2) x -(A) v -C(O)-(CH2) x-(c1);

[0121] 14)-(A) v -C(O)-(CH2CH2O) n -(CH2) u -(c1);

[0122] 15)-C(O)-(CH2) x -NH-(c1);

[0123] 16)-C(O)-CH(-L3-R9)-NHC(O)-(CH2) x -phenylene-(CH2) y -;

[0124] 17)-C(O)-CH(-L3-R9)-NHC(O)-(CH2) x -, wherein the c1 side is connected to M.

[0125] In some embodiments, in M, the linker is a group formed by click reaction, click-like reaction, thiol ligation, amine ligation, oxime ligation or hydrazone ligation.

[0126] In some embodiments, in M, the linker is a group formed by thiol ligation on the side chain of an antibody cysteine residue or amine ligation on the side chain of a lysine residue.

[0127] In some embodiments, in M, the linker is a group formed by thiol ligation, preferably M is more preferably

[0128] R 11 is hydrogen or C 1-4 alkyl; the linker is connected through the c side and the rest of L.

[0129] In some embodiments, R 11 is hydrogen, methyl or ethyl.

[0130] In some embodiments, in M, the linker is a group formed by oxime ligation, preferably the linker is connected through the c side and the rest of L.

[0131] In some embodiments, in M, the linker is a group formed by hydrazone linkage, preferably The linker is connected to the rest of L through the c side.

[0132] In some embodiments, in M, the linker is a group formed by amino linkage, and the group formed by amino linkage is preferably a group formed by the amino group of the lysine side chain; more preferably

[0133] In some embodiments, the linker is a group formed by click chemistry connection or click-like chemistry connection, preferably: The linker is connected to the rest of L through the c side.

[0134] In some embodiments, in M, the linker is The linker is connected to the rest of L through the c side.

[0135] In some embodiments, L is

[0136]

[0137]

[0138]

[0139] In some embodiments, L is

[0140]

[0141]

[0142] In some embodiments, D is The group formed by the compound losing a hydrogen atom;

[0143] In some embodiments, d is 1.

[0144] In some embodiments, R5 is H, -L2-(Z) e -(maleimidyl) or -L2-(Z) e -H.

[0145] In some embodiments, R5 is -L2-(Z) e -H; -L2- is Connected to the sulfhydryl group in D through the c2 side; e is 1; -(Z) e-H is C 1-6 alkyl group.

[0146] In some embodiments, R5 is -L2-(Z) e -H; -L2- is connected to the sulfhydryl group in D on the c2 side; e is 3, -(Z) e -H is -C(O)NH-(CH2) y -O-(CH2) y -H.

[0147] In some embodiments, R5 is -L2-(Z) e -H; -L2- is connected to the sulfhydryl group in D on the c2 side; e is 0 or 1, -(Z) e -H is H or methyl.

[0148] In some embodiments, R5 is Q is a linking bond, -C(O)O- or -C 1-3 alkylene-O-; R7 and R 7’ are each independently H or methyl.

[0149] In some embodiments, R5 is Q1 is phenyl; R7 and R 7’ are each independently H or methyl.

[0150] In some embodiments, R8 is phenyl or a 5-6 membered heteroaryl group; the 5-6 membered heteroaryl group is preferably pyridyl, pyrimidinyl, pyrazinyl, pyrrolyl, thienyl, furyl, pyrazolyl, thiazolyl or oxazolyl; the R8 is unsubstituted or optionally substituted by 1 to 3 substituents selected from hydroxyl, amino, cyano, carboxyl, nitro, mercapto, C 1-6 alkyl, halogen, halo C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, halo C 1-6 alkoxy, C 1-6 alkylthio, amido, aldehyde, acetyl, methyl ester, amino C 1-6 alkyl, hydroxy C 1-6 alkyl and methylsulfonyl, and the substituents are substituted at any position.

[0151] In some embodiments, R5 is -L2-(Z) e -(maleimide group); -L2- is connected to the sulfhydryl group in D on the c2 side; -(Z) e - is -(A) v -C(O)-(CH2) x-(c3); The c3 side is linked to the maleimide group.

[0152] In some embodiments, R5 is The c2 side is linked to the mercapto group in D.

[0153] In some embodiments, R5 is The c2 side is linked to the mercapto group in D.

[0154] In some embodiments, B1 and B2 are each independently And at least one of B1 and B2 is

[0155] In some embodiments, D is A group formed by the loss of a hydrogen atom from an amino group or a mercapto group in the indicated compound.

[0156] In some embodiments, D is A group formed by the loss of a hydrogen atom from the amino group in the indicated compound; R3 or R 3’ Are each independently

[0157] In some embodiments, D is A group formed by the loss of a hydrogen atom from the mercapto group in the indicated compound.

[0158] In some embodiments, D is A group formed by the loss of a hydrogen atom from the mercapto group in the indicated compound.

[0159] In some embodiments, R1 is -OCH3; R2 is F.

[0160] In some embodiments, D is any one of the following structures or a pharmaceutically acceptable salt thereof:

[0161] Wherein, the definitions of R1, R2 and R5 are as described above.

[0162] In some embodiments, D is any one of the following structures:

[0163]

[0164]

[0165] Or a pharmaceutically acceptable salt thereof.

[0166] In some embodiments, D is any one of the following structures:

[0167] or a pharmaceutically acceptable salt thereof.

[0168] In some embodiments, Ab is an anti-HER2 antibody, an anti-EGFR antibody, or an anti-5T4 antibody;

[0169] L is a linker having the following combination: -(L1) a -(Z) b -M-; L1 is connected to D, and M is connected to Ab;

[0170] -(L1) a - is

[0171] -(Z) b - is -(CH2) x -(c1) or -C(O)-(CH2) x -phenylene-(CH2) y -;

[0172] x and y are independently 0, 1, 2, 3, 4, 5, or 6;

[0173] M is a linker that connects to Ab;

[0174] D is the group formed by the loss of a hydrogen atom from the mercapto group in the indicated compound;

[0175] R5 is H.

[0176] In some embodiments, the antibody-immunostimulatory conjugate as shown in Formula II or a pharmaceutically acceptable salt thereof, and the antibody-immunostimulatory conjugate is any one of the antibody-immunostimulatory conjugates in Table A:

[0177] Table A

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203] Among them, antibody 1 is Trastuzumab; antibody 2 is Pertuzumab; antibody 3 is Trastuzumab-LALA (L234A / L235A); antibody 4 is Trastuzumab-AAG (L234A / L235A / P329G); antibody 5 is engineered Trastuzumab with a cysteine inserted between heavy chain positions 239 and 240; antibody 7 is Cetuximab; antibody 8 is huA1(V H v2.0+V Lv2.4); Antibody 9 is Trastuzumab (HC-s239.5, L234A / L235A / P329G); Antibody 11 is Enfortumab (Ha22-2); Antibody 12 is an antibody prepared by using the method disclosed in Example 25 of CN115209921A with Cetuximab; Antibody 13 is Nimotuzumab.

[0204] In some embodiments, Antibody 3 is an antibody purchased from Shanghai Bayi Biotechnology Co., Ltd. with the catalog number B801901.

[0205] In some embodiments, Antibody 4 is an antibody purchased from Shanghai Bayi Biotechnology Co., Ltd. with the catalog number MHDDD001.

[0206] In some embodiments, Antibody 5 is an antibody purchased from Shanghai Bayi Biotechnology Co., Ltd. with the catalog number LPDDFD001.

[0207] In some embodiments, Antibody 8 is a huA1 (V H v2.0 + V L v2.4) antibody prepared by expression and purification according to the sequence information table (SEQ ID NO:54 and SEQ ID NO:70) disclosed in US8044178B2.

[0208] In some embodiments, Antibody 9 is an antibody purchased from Shanghai Bayi Biotechnology Co., Ltd. with the catalog number CZ7DUD001.

[0209] The present invention provides a cyclic dinucleotide compound represented by Formula I or a pharmaceutically acceptable salt thereof,

[0210] D-LX

[0211] (I)

[0212] wherein, LX is a linker precursor having the following combination: -(L1) a -(Z) b -M'; L1 is connected to D;

[0213] a is 0, 1, 2, 3, 4, 5 or 6; b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0214] D is the group formed by removing one hydrogen atom from the shown compound;

[0215] B1 and B2 are each independently And at least one of B1 and B2 is

[0216] R1 and R2 are independently -OH, -N3, F, -OCH3 or -SCF3;

[0217] R3 and R 3’ are independently H, -C(O)R4, -C(O)OR4, -OR4 or -R4;

[0218] R4 is C 1-10 alkyl, C 6-10 aryl, 5-10 membered heteroaryl, C 3-10 cycloalkyl, 3-10 membered heterocycloalkyl, C 6-10 arylC 1-10 alkyl, 5-10 membered heteroarylC 1-10 alkyl, C 3-10 cycloalkylC 1-10 alkyl, 3-10 membered heterocycloalkylC 1-10 alkyl; said R4 is unsubstituted, or optionally substituted at any position by 1 to 3 substituents selected from hydroxy, mercapto, dithiol, amino, C 1-6 alkylamino, C 1-6 alkylaminoC 1-10 alkyl;

[0219] R5 is H, -(L2) d -(Z) e -(maleimidyl) or -(L2) d -(Z) e -H;

[0220] d is 0, 1, 2, 3, 4, 5 or 6; e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0221] Each L1 and each L2 are independently The c2 side of L1 is connected to D; the c2 side of L2 is connected to the mercapto group in D;

[0222] X is

[0223] p is independently 1, 2 or 3;

[0224] Q is a linking bond, -C(O)O-, -C(O)N(R6)- or -C 1-3 alkylene-O-;

[0225] Q1 is phenyl or pyridyl;

[0226] R6 is H or C 1-6 alkyl; said C 1-6 alkyl is unsubstituted, or optionally substituted by 1 C 1-6Alkylamino or C 1-6 alkylsulfonyl is substituted at any position;

[0227] R7 and R 7’ are each independently H or C 1-6 alkyl;

[0228] R8 is phenyl or a 5- to 10-membered heteroaryl; said R8 is unsubstituted or optionally substituted by 1 to 3 substituents selected from hydroxyl, amino, carboxyl, cyano, nitro, mercapto, C 1-6 alkyl, C 3-8 cycloalkyl, halogen, halo C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, halo C 1-6 alkoxy, C 1-6 alkylthio, amido, hydroxyamino, aldehyde, acetyl, methyl ester, amino C 1-6 alkyl, hydroxy C 1-6 alkyl and C 1-6 alkylsulfonyl is substituted at any position;

[0229] Each Z is independently -(A) v -, -PEG-, -C(O)-(CH2) x -, -NR9-(CH2) y -, -O-(CH2) y -, -S-(CH2) y -, -(CH2) x -C 6-14 arylene-(CH2) y -, -(CH2) x -5- to 6-membered heteroarylene-(CH2) y -, -(CH2) x -C 3-6 cycloalkylene-(CH2) y -, -(CH2) x -3- to 6-membered heterocycloalkylene-(CH2) y -, -NR9-(CH2) x -C(O)-, -O-(CH2) x -C(O)-, -S-(CH2) x -C(O)-, -(CH2) x CH(-L3-R9)-C(O)-, -(CH2) x CH(-L3-R 9a )-C(O)-, -(CH2) x -S-S-(CH2) y -, C 1-6Alkylene or C 2-6 Alkenylene;

[0230] L3 is a linking bond, -NH-, or

[0231] R9 is independently hydrogen, C 1-6 Alkyl, -PO(OH)2, -PO(OCH3)2, -C(O)-(CH2CH2O) n -CH3, -(CH2CH2O) n -CH3,

[0232] R 9a is independently

[0233] -PEG- is -(CH2CH2O) n -(CH2) u - or -(CH2CH2O) n -(CH2) u -C(O)-;

[0234] A is independently an amino acid residue;

[0235] x and y are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; v is independently 1, 2, 3, 4, or 5; n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50; u is independently 0, 1, 2, 3, 4, or 5;

[0236] M’ is a linker precursor.

[0237] In some embodiments, LX is a linker precursor having the following combination: -(L1) a -(Z) b -M’; L1 is connected to D;

[0238] a is 0, 1, 2, 3, 4, 5, or 6; b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0239] D is the group formed by the shown compound losing a hydrogen atom;

[0240] B1 and B2 are each independently And at least one of B1 and B2 is

[0241] R1 and R2 are independently -OH, -N3, F, -OCH3 or -SCF3;

[0242] R3 and R 3’ are independently H, -C(O)R4, -C(O)OR4, -OR4 or -R4;

[0243] R4 is C 1-10 alkyl, C 6-10 aryl, 5-10 membered heteroaryl, C 3-10 cycloalkyl, 3-10 membered heterocycloalkyl, C 6-10 arylC 1-10 alkyl, 5-10 membered heteroarylC 1-10 alkyl, C 3-10 cycloalkylC 1-10 alkyl, 3-10 membered heterocycloalkylC 1-10 alkyl; said R4 is unsubstituted or optionally substituted at any position by 1 to 3 substituents selected from hydroxy, mercapto, dithiol, amino, C 1-6 alkylamino, C 1-6 alkylaminoC 1-10 alkyl;

[0244] R5 is H, -(L2) d -(Z) e -(maleimidyl) or -(L2) d -(Z) e -H;

[0245] d is 0, 1, 2, 3, 4, 5 or 6; e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0246] Each L1 and each L2 are each independently The c2 side of L1 is connected to D; the c2 side of L2 is connected to the mercapto group in D;

[0247] X is

[0248] p is independently 1, 2 or 3;

[0249] Q is a linking bond, -C(O)O-, -C(O)N(R6)- or -C 1-3 alkylene-O-;

[0250] Q1 is phenyl or pyridyl;

[0251] R6 is H or C 1-6 alkyl; said C1-6 The alkyl group is unsubstituted or optionally substituted at any position by 1 C 1-6 alkylamino or C 1-6 alkylsulfonyl;

[0252] R7 and R 7’ are each independently H or C 1-6 alkyl;

[0253] R8 is phenyl or a 5- to 10-membered heteroaryl group; said R8 is unsubstituted or optionally substituted at any position by 1 to 3 substituents selected from hydroxy, amino, carboxy, cyano, nitro, mercapto, C 1-6 alkyl, C 3-8 cycloalkyl, halogen, halo C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, halo C 1-6 alkoxy, C 1-6 alkylthio, amido, hydroxyamino, formyl, acetyl, methyl ester, amino C 1-6 alkyl, hydroxy C 1-6 alkyl and C 1-6 alkylsulfonyl at any position;

[0254] Each Z is independently -(A) v -, -PEG-, -C(O)-(CH2) x -, -NR9-(CH2) y -, -O-(CH2) y -, -S-(CH2) y -, -(CH2) x -C 6-14 arylene-(CH2) y -, -(CH2) x -5- to 6-membered heteroarylene-(CH2) y -, -(CH2) x -C 3-6 cycloalkylene-(CH2) y -, -(CH2) x -3- to 6-membered heterocycloalkylene-(CH2) y -, -NR9-(CH2) x -C(O)-, -O-(CH2) x -C(O)-, -S-(CH2) x -C(O)-, -(CH2) x CH(NHR9)-C(O)-, -(CH2) x -S-S-(CH2) y -, C 1-6 alkylene or C2-6 Vinylene;

[0255] R9 is independently hydrogen, C 1-6 alkyl, -PO(OH)2, -PO(OCH3)2, -C(O)-(CH2CH2O) n -CH3 or -(CH2CH2O) n -CH3;

[0256] -PEG- is -(CH2CH2O) n -(CH2) u - or -(CH2CH2O) n -(CH2) u -C(O)-;

[0257] A is independently an amino acid residue;

[0258] x and y are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; v is independently 1, 2, 3, 4 or 5; n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50; u is independently 0, 1, 2, 3, 4 or 5;

[0259] M' is a linker precursor.

[0260] In some embodiments, in the cyclic dinucleotide compound of formula I or a pharmaceutically acceptable salt thereof, the definitions of some groups are as described below, and the definitions of the remaining groups are as described in any other embodiment (hereinafter referred to as "in some embodiments"): a is 0, 1, 2, 3 or 4; a is preferably 0, 1 or 2.

[0261] In some embodiments, b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; b is preferably 1, 2, 3, 4, 5, 6, 7 or 8; b is more preferably 1, 2, 3, 4, 5 or 6.

[0262] In some embodiments, d is 0, 1, 2 or 3; d is preferably 1 or 2; d is more preferably 1.

[0263] In some embodiments, e is 0, 1, 2, 3, 4, 5 or 6; e is preferably 0, 1, 2, 3 or 4; e is more preferably 0, 1, 2 or 3.

[0264] In some embodiments, R6 is H or C 1-6alkyl; said C 1-6 The alkyl is unsubstituted or optionally substituted at any position with one dimethylamino or methylsulfonyl group.

[0265] In some embodiments, R6 is H, -CH3, -CH2CH2N(CH3)2 or -CH2CH2S(O)2CH3.

[0266] In some embodiments, each L1 and each L2 are each independently The c2 side of L1 is connected to D; the c2 side of L2 is connected to the thiol group in D.

[0267] In some embodiments, each L1 and each L2 are each independently The c2 side of L1 is connected to D; the c2 side of L2 is connected to the thiol group in D.

[0268] In some embodiments, -(L1) a - is The c2 side is connected to D.

[0269] In some embodiments, -(L1) a - is The c2 side is connected to the thiol group in D.

[0270] In some embodiments, -(L1) a - is The c2 side is connected to the amino group in D.

[0271] In some embodiments, -(L1) a - is The c2 side is connected to the amino group in D.

[0272] In some embodiments, -(L1) a - is absent, The c2 side is connected to the thiol group in D.

[0273] In some embodiments, Z is independently -(A) v -, -PEG-, -C(O)-(CH2) x -, -NR9-(CH2) y -, -O-(CH2) y -, -S-(CH2) y -, -(CH2) x -C 6-14 arylene-(CH2)y -, -(CH2) x - a 5- to 6-membered heteroaryl-(CH2) y -, -(CH2) x -C 3-6 cycloalkylene-(CH2) y -, -(CH2) x - a 3- to 6-membered heterocycloalkylene-(CH2) y -, -NR9-(CH2) x -C(O)-, -O-(CH2) x -C(O)-, -S-(CH2) x -C(O)-, -(CH2) x CH(NHR9)-C(O)-, -(CH2) x -S-S-(CH2) y -, C 1-6 alkylene or C 2-6 alkenylene; x and y are independently any integer from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; v is independently any integer from 1, 2, 3, 4 or 5; said -(CH2) x - a 5- to 6-membered heteroaryl-(CH2) y - in the "5- to 6-membered heteroaryl", said heteroatoms are selected from one or more of N, O and S, and the number of said heteroatoms is 1, 2 or 3; said -(CH2) x - a 3- to 6-membered heterocycloalkylene-(CH2) y - in the "3- to 6-membered heterocycloalkylene", said heteroatoms are selected from one or more of N, O and S, and the number of said heteroatoms is 1, 2 or 3.

[0274] In some embodiments, in A, said amino acid is a natural amino acid or a non-natural amino acid.

[0275] In some embodiments, in A, said amino acid residue is substituted with 1 to 3 R 10 groups; R 10 is independently C 1-6 alkyl, C 1-6 alkylacyl, C 1-6 alkoxycarbonyl, -C(O)-(CH2CH2O) n -CH3 or -(CH2CH2O) n -CH3; said C 1-6 alkyl, C 1-6 alkylacyl or C 1-6 alkoxycarbonyl may further be substituted with 1 R8 at any position.

[0276] In some embodiments, each A is independently Each R A is independently an amino acid side chain or an amino acid side chain modified with 1 to 3 Rs 10 ; or R A forms a five-membered heterocyclic group with the adjacent nitrogen atom;

[0277] The aforementioned R 10 is independently C 1-6 alkyl, C 1-6 alkyl acyl, C 1-6 alkoxy acyl, -C(O)-(CH2CH2O) n -CH3 or -(CH2CH2O) n -CH3; the C 1-6 alkyl, C 1-6 alkyl acyl or C 1-6 alkoxy acyl may further be substituted with one R8 at any position.

[0278] In some embodiments, the may be and may also be

[0279] In some embodiments, in A, each R A is independently hydrogen, methyl, ethyl, n-propyl, isopropyl, isobutyl, or R A forms a five-membered heterocyclic group with the adjacent nitrogen atom.

[0280] In some embodiments, -(A) v - is each R A is independently hydrogen, methyl, ethyl, n-propyl, isopropyl, isobutyl, or R A forms a five-membered heterocyclic group with the adjacent nitrogen atom; v is any integer from 1 to 4, and v is preferably 1 or 2.

[0281] In some embodiments, -(A) v - is

[0282] In some embodiments, -(A) v - is

[0283] In some embodiments, -(Z) b - is any one of the following combinations:

[0284] 1)-(CH2) x -(c1),

[0285] 2)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -(c1),

[0286] 3)-C(O)-(CH2) x -(c1),

[0287] 4)-C(O)-(CH2) x -C 3-6 Sub-cycloalkyl-(CH2) y -(c1),

[0288] 5)-C(O)-(CH2) x -Sub-phenyl-(CH2) y -(c1),

[0289] 6)-C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -(c1),

[0290] 7)-C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -Sub-phenyl-(CH2) y -(c1),

[0291] 8)-(A) v -(c1),

[0292] 9)-(A) v -C(O)-(CH2) x -(c1),

[0293] 10)-(A) v -C(O)-(CH2) x -Sub-phenyl-(CH2) y -(c1),

[0294] 11)-(A) v -C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -(c1) or

[0295] 12)-(A) v -C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -Phenylene-(CH2) y -(c1); wherein the c1 side is connected to M'.

[0296] In some embodiments, -(Z) b -is any of the following combinations:

[0297] 13)-C(O)-(CH2) x -(A) v -C(O)-(CH2) x -(c1);

[0298] 14)-(A) v -C(O)-(CH2CH2O) n -(CH2) u -(c1);

[0299] 15)-C(O)-(CH2) x -NH-(c1);

[0300] 16)-C(O)-CH(-L3-R9)-NHC(O)-(CH2) x -Phenylene-(CH2) y -;

[0301] 17)-C(O)-CH(-L3-R9)-NHC(O)-(CH2) x -, wherein the c1 side is connected to M'.

[0302] In some embodiments, M' is a linker precursor for reacting with the side chain of an antibody amino acid residue, preferably a linker precursor for reacting with the amino group, sulfhydryl group, azide group, aldehyde group, acyl group or phenolic hydroxyl group of the side chain of an antibody amino acid residue.

[0303] In some embodiments, M' is a linker precursor for reacting with the side chain of an antibody amino acid residue, preferably a linker precursor for reacting with the amino group or sulfhydryl group of the side chain of an antibody amino acid residue.

[0304] In some embodiments, M' is ethynyl, vinyl, hydroxylamine,

[0305] R 11 is hydrogen or C 1-4 alkyl;

[0306] Each R 12and R 12’ are each independently a halogen (such as bromine or iodine), nitro or -SO3 - ;

[0307] R 13 and R 13’ are each independently hydrogen, a halogen (such as bromine), phenylthio or pyridylthio.

[0308] In some embodiments, R 11 is hydrogen, methyl or ethyl.

[0309] In some embodiments, M' is

[0310] In some embodiments, M' is In some embodiments, LX is

[0311]

[0312]

[0313] In some embodiments, LX is

[0314]

[0315] In some embodiments, D is the group formed by the shown compound losing a hydrogen atom;

[0316] In some embodiments, d is 1.

[0317] In some embodiments, R5 is H, -L2-(Z) e -(maleimidyl) or -L2-(Z) e -H.

[0318] In some embodiments, R5 is -L2-(Z) e -H; -L2- is connected to the sulfhydryl group in D at the c2 side; e is 1; -(Z) e -H is C 1-6 alkyl.

[0319] In some embodiments, R5 is -L2-(Z) e -H; -L2- is The c2 side is connected to the mercapto group in D; e is 3, -(Z) e -H is -C(O)NH-(CH2) y -O-(CH2) y -H.

[0320] In some embodiments, R5 is -L2-(Z) e -H; -L2- is The c2 side is connected to the mercapto group in D; e is 0 or 1, -(Z) e -H is H or methyl.

[0321] In some embodiments, R5 is Q is a linking bond, -C(O)O- or -C 1-3 alkylene-O-; R7 and R 7’ are each independently H or methyl.

[0322] In some embodiments, R5 is Q1 is phenyl; R7 and R 7’ are each independently H or methyl.

[0323] In some embodiments, R8 is phenyl or a 5- to 6-membered heteroaryl group; the 5- to 6-membered heteroaryl group is preferably pyridyl, pyrimidinyl, pyrazinyl, pyrrolyl, thienyl, furyl, pyrazolyl, thiazolyl or oxazolyl; the R8 is unsubstituted or optionally substituted with 1 to 3 substituents selected from hydroxyl, amino, cyano, nitro, mercapto, C 1-6 alkyl, halogen, halo-C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, halo-C 1-6 alkoxy, C 1-6 alkylthio, amido, aldehyde, acetyl, methyl ester, amino-C 1-6 alkyl, hydroxy-C 1-6 alkyl and methylsulfonyl, and the substituents are substituted at any position.

[0324] In some embodiments, R5 is -L2-(Z) e -(maleimidyl); -L2- is the c2 side is connected to the mercapto group in D; -(Z) e - is -(A) v -C(O)-(CH2) x -(c3); the c3 side is connected to the maleimidyl group.

[0325] In some embodiments, R5 is The thiol group on the c2 side is connected to D.

[0326] In some embodiments, R5 is The thiol group on the c2 side is connected to D.

[0327] In some embodiments, B1 and B2 are each independently And at least one of B1 and B2 is

[0328] In some embodiments, D is A group formed by the loss of one hydrogen atom from the amino group or the thiol group in the indicated compound.

[0329] In some embodiments, D is A group formed by the loss of one hydrogen atom from the amino group in the indicated compound; R3 or R 3’ Are each independently

[0330] In some embodiments, D is A group formed by the loss of one hydrogen atom from the thiol group in the indicated compound.

[0331] In some embodiments, D is A group formed by the loss of one hydrogen atom from the thiol group in the indicated compound.

[0332] In some embodiments, D is any of the following structures or a pharmaceutically acceptable salt thereof:

[0333] Wherein, the definitions of R1, R2 and R5 are as described above.

[0334] In some embodiments, D is any of the following structures:

[0335]

[0336]

[0337] Or a pharmaceutically acceptable salt thereof.

[0338] In some embodiments, D is any of the following structures:

[0339] Or a pharmaceutically acceptable salt thereof.

[0340] In some embodiments, the cyclic dinucleotide derivative represented by Formula I or a pharmaceutically acceptable salt thereof, the cyclic dinucleotide derivative is selected from:

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362] or a pharmaceutically acceptable salt thereof.

[0363] The present invention also provides a compound represented by Formula D'-1 or D'-2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof;

[0364]

[0365] B1 and B2 are each independently and at least one of B1 and B2 is

[0366]

[0367] R1 and R2 are independently -OH, -N3, F, -OCH3 or -SCF3;

[0368] R5 is -(L2) d -(Z) e -H;

[0369] d is 0, 1, 2, 3, 4, 5 or 6; e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0370] Each L2 is independently connected to D at the c2 side;

[0371] X is

[0372] p is independently 1, 2 or 3;

[0373] Q is a linking bond, -C(O)O-, -C(O)N(R6)- or -C 1-3 alkylene-O-;

[0374] Q1 is phenyl or pyridyl;

[0375] R6 is H or C 1-6 alkyl; the C 1-6 alkyl is unsubstituted or optionally substituted at any position by 1 C 1-6 alkylamino or C 1-6 alkylsulfonyl;

[0376] R7 and R 7’ are each independently H or C 1-6 alkyl;

[0377] R8 is phenyl or a 5- to 10-membered heteroaryl; the R8 is unsubstituted or optionally substituted by 1 to 3 selected from hydroxy, amino, carboxy, cyano, nitro, mercapto, C 1-6 alkyl, C 3-8 cycloalkyl, halogen, halo C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, halo C 1-6 alkoxy, C 1-6Alkylthio, amido, hydroxyamino, aldehyde, acetyl, methyl ester, amino C 1-6 Alkyl, hydroxy C 1-6 Alkyl and C 1-6 Substituents of alkylsulfonyl are substituted at any position;

[0378] Each Z is independently -C(O)-(CH2) x -, -NH-(CH2) y -, -O-(CH2) y -, C 1-6 Alkylene or C 2-6 Alkenylene;

[0379] Each x and each y are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0380] In some embodiments, in the cyclic dinucleotide derivative represented by formula D'-1 or D'-2 or a pharmaceutically acceptable salt thereof, the definitions of some groups are as described below, and the definitions of the remaining groups are as described in any other embodiment (hereinafter referred to as "in some embodiments"): R1 is -OCH3; R2 is F.

[0381] In some embodiments, d is 1.

[0382] In some embodiments, e is 0, 1, 2, 3 or 4; e is preferably 0, 1, 2 or 3.

[0383] In some embodiments, R5 is -L2-(Z) e -H.

[0384] In some embodiments, R5 is -L2-(Z) e -H; -L2- is Connected to the thiol group in D on the c2 side; e is 1; -(Z) e -H is C 1-6 Alkyl.

[0385] In some embodiments, R5 is -L2-(Z) e -H; -L2- is Connected to the thiol group in D on the c2 side; e is 3, -(Z) e -H is -C(O)NH-(CH2) y -O-(CH2) y -H.

[0386] In some embodiments, R5 is -L2-(Z) e -H; -L2- is Connected to the thiol group in D on the c2 side; e is 0 or 1, -(Z)e -H is H or methyl.

[0387] In some embodiments, R5 is Q is a linking bond, -C(O)O- or -C 1-3 alkylene-O-; R7 and R 7’ are each independently H or methyl.

[0388] In some embodiments, R5 is Q1 is phenyl; R7 and R 7’ are each independently H or methyl.

[0389] In some embodiments, R8 is phenyl or a 5- or 6-membered heteroaryl; the 5- or 6-membered heteroaryl is preferably pyridyl, pyrimidinyl, pyrazinyl, pyrrolyl, thienyl, furyl, pyrazolyl, thiazolyl or oxazolyl; the R8 is unsubstituted or optionally substituted with 1 to 3 substituents selected from hydroxy, amino, cyano, nitro, mercapto, C 1-6 alkyl, halogen, halo-C 1-6 alkyl, C 1-6 alkylamino, C 1-6 alkoxy, halo-C 1-6 alkoxy, C 1-6 alkylthio, amido, aldehyde, acetyl, methyl ester, amino-C 1-6 alkyl, hydroxy-C 1-6 alkyl and methylsulfonyl, and the substituents are substituted at any position.

[0390] In some embodiments, R5 is The c2 side is linked to the mercapto group in D.

[0391] In some embodiments, R5 is The c2 side is linked to the mercapto group in D.

[0392] In some embodiments, D'-1 or D'-2 is any one of the following structures:

[0393]

[0394] or a pharmaceutically acceptable salt thereof.

[0395] The present invention provides a pharmaceutical composition comprising substance K and a pharmaceutically acceptable excipient;

[0396] The substance K is substance K-1, substance K-2 or substance K-3;

[0397] The substance K-1 is the antibody-immunostimulatory conjugate shown in formula II above or a pharmaceutically acceptable salt thereof;

[0398] The aforementioned substance K-2 is the cyclic dinucleotide compound shown in Formula I above or a pharmaceutically acceptable salt thereof;

[0399] The aforementioned substance K-3 is the compound shown in Formula D'-1 or D'-2 above, its stereoisomer or a pharmaceutically acceptable salt thereof.

[0400] In some embodiments, the dosage of the aforementioned substance K can be a therapeutically effective amount.

[0401] In some embodiments, the antibody-immunostimulatory conjugate shown in Formula II above or a pharmaceutically acceptable salt thereof, the cyclic dinucleotide compound shown in Formula I above or a pharmaceutically acceptable salt thereof, or the compound shown in Formula D'-1 or D'-2 above, its stereoisomer or a pharmaceutically acceptable salt thereof can be a therapeutically effective amount.

[0402] In the aforementioned pharmaceutical composition, the pharmaceutically acceptable excipients can include pharmaceutically acceptable carriers, diluents, and / or excipients.

[0403] The aforementioned pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, topical administration (such as intratumoral injection), etc.

[0404] The present invention also provides the use of a substance K or the aforementioned pharmaceutical composition in the preparation of a drug for regulating T cells and other immune cells, wherein the substance K is substance K-1, substance K-2, or substance K-3;

[0405] The aforementioned substance K-1 is the antibody-immunostimulatory conjugate shown in Formula II above or a pharmaceutically acceptable salt thereof;

[0406] The aforementioned substance K-2 is the cyclic dinucleotide compound shown in Formula I above or a pharmaceutically acceptable salt thereof;

[0407] The aforementioned substance K-3 is the compound shown in Formula D'-1 or D'-2 above, its stereoisomer or a pharmaceutically acceptable salt thereof.

[0408] In some embodiments, the dosage of the aforementioned substance K can be a therapeutically effective amount.

[0409] The present invention provides the use of a substance K or the aforementioned pharmaceutical composition in the preparation of a drug for treating and / or alleviating tumors, wherein the substance K is substance K-1, substance K-2, or substance K-3;

[0410] The aforementioned substance K-1 is the antibody-immunostimulatory conjugate shown in Formula II above or a pharmaceutically acceptable salt thereof;

[0411] The substance K-2 described above is the cyclic dinucleotide compound shown in Formula I above or a pharmaceutically acceptable salt thereof;

[0412] The substance K-3 described above is the compound shown in Formula D'-1 or D'-2 above, its stereoisomer or a pharmaceutically acceptable salt thereof.

[0413] In some embodiments, the dosage of the substance K can be a therapeutically effective amount.

[0414] The present invention provides the use of a substance K or the above-mentioned pharmaceutical composition in the preparation of a drug for treating, alleviating and / or preventing STING-mediated related diseases, wherein the substance K is substance K-1, substance K-2 or substance K-3;

[0415] The substance K-1 described above is the antibody-immunostimulatory conjugate shown in Formula II above or a pharmaceutically acceptable salt thereof;

[0416] The substance K-2 described above is the cyclic dinucleotide compound shown in Formula I above or a pharmaceutically acceptable salt thereof;

[0417] The substance K-3 described above is the compound shown in Formula D'-1 or D'-2 above, its stereoisomer or a pharmaceutically acceptable salt thereof.

[0418] In some embodiments, the STING-mediated related diseases refer to tumors or viral infectious diseases.

[0419] In some embodiments, the dosage of the substance K can be a therapeutically effective amount.

[0420] The tumor described above can be a malignant tumor, including metastatic and non-metastatic cancers, also including familial hereditary and sporadic cancers, and can also include solid tumors and non-solid tumors.

[0421] In the present invention, the terms "tumor" and "cancer" have the same meaning.

[0422] In the present invention, unless otherwise specified, the term "selectively substituted by one or more groups at any position" means that any one or more hydrogen atoms of one or more atoms specified on the group are replaced by the specified group, provided that the normal valence of the specified atoms is not exceeded, and the substitution at any position is a reasonable substitution common in the art.

[0423] In the present invention, when the bonding of the substituent shows an intersection with the bonding of two atoms in the connecting ring, then such a substituent can be bonded to any bondable ring atom on the ring.

[0424] In the present invention, any combination of variables is allowed only if such a combination results in a stable compound.

[0425] In the present invention, when any variable appears more than once in the composition or structure of a compound, its definition in each case is independent. For example, when R is substituted by one or more groups, each substituent is an independent substituent and may be the same or different.

[0426] Unless otherwise specified, the following terms used in the specification and claims of the present invention have the following meanings:

[0427] The term "antibody" refers to any form of an antibody that exhibits the desired biological activity (e.g., inhibiting the binding of a ligand to its receptor or inhibiting ligand-induced receptor signal transduction). Thus, "antibody" is administered in its broadest sense and expressly includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (including bispecific antibodies). A naturally occurring "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region and a heavy chain constant region. The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region and a light chain constant region. The light chain constant region contains a CL domain. The variable regions of the heavy and light chains contain binding domains (antigen-binding domains) that interact with an antigen, and the antigen-binding domain can be provided by one or more variable regions on the antibody. Specifically, the antigen-binding domain comprises the variable domain of the antibody light chain (VL) and the variable domain of the antibody heavy chain (VH). In some cases, the antigen that the antibody can bind is a tumor-associated antigen. In some cases, the antigen that the antibody can bind is a tumor-specific antigen. The antibody can be a monoclonal antibody, a human antibody, a humanized antibody, or a chimeric antibody. These antibodies can have any class (IgG, IgE, IgM, IgD, IgA, and IgY) or subtype (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The present invention includes not only intact antibodies but also fragments of antibodies having immunological activity (including Fab, F(ab')2, scFv, or Fv fragments) or fusion proteins formed by antibodies and other sequences. Thus, the "antibody" as used in the present invention also includes fragments, derivatives, and analogs of the antibody.

[0428] In the present invention, the "antibody" may further comprise an engineered antibody. The engineered antibody may 1) comprise one or more non-naturally encoded amino acids incorporated into the heavy chain, light chain, or both the heavy chain and the light chain, and the one or more non-naturally encoded amino acids include, but are not limited to, one or more of the following non-natural amino acids: p-acetylphenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-acetyl-L-phenylalanine, p-acyl-L-phenylalanine, p-propargyloxy-L-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, p-azidomethyl-phenylalanine, etc.; 2) insert cysteine residues at different positions of the heavy or light chain of the antibody, or replace specific amino acid residues of the heavy or light chain of the antibody with cysteine residues, thereby forming unpaired cysteines for conjugation.

[0429] As used herein, the "antibody" or its antigen-binding fragment may comprise an Fc region, which may be further modified. In some cases, one or more mutations in the Fc region result in improvements in drugs containing such modified Fc regions, such as, for example, a decrease in effector function, an alteration in the regulation of the drug metabolic half-life, and an alteration in drug stability. In some cases, the modified Fc region may comprise one or more mutations that reduce or eliminate the interaction between the antibody and the immune system. Key interactions may include the interaction between the antibody Fc and Fcγ receptors, as well as the interaction with C1q of the complement system. In the case of using IgG1 as the isotype of the antibody of the present invention, the effector function can be adjusted by substituting a part of the amino acid residues of the constant region. Variants of IgG1 that reduce or attenuate effector function include, but are not limited to: IgG1 LALA (IgG1-L234A, L235A), IgG1 LAGA (IgG1-L235A, G237A), IgG1 AAG (IgG1-L234A, L235A, P329G), etc. The above L234A, L235A indicate that the leucines at positions 234 and 235 determined by the EU index (Proc. Natl. Acad. Sci. U.S.A., Vol. 63, No. 1 (May 15, 1969), p78-85) are replaced by alanines, G237A indicates that the glycine at position 237 determined by the EU index is replaced by alanine, and P329G indicates that the proline at position 329 determined by the EU index is replaced by alanine. In some cases, modification of the Fc segment glycosylation can alter the drug efficacy and pharmacokinetic properties (Journal of Pharmaceutical Sciences. 2015, 104(6), 1866-1884). For example, natural antibodies produced by mammalian cells usually contain branched, biantennary oligosaccharides, which are usually attached to Asn297 of the CH2 domain of the Fc segment through an N-linkage. The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the stem of the biantennary oligosaccharide structure. Modification of the oligosaccharides in the antibody can produce antibody variants with some improved properties. For example, afucylation can enhance antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADCP); reducing terminal sialylation or increasing terminal acetylglycosamination can enhance the ADCC effect; treating CHO cells with mannosidase inhibitors can increase mannosylation and afucylation, thereby enhancing the ADCC effect and slightly reducing the CDC effect, etc.In addition, glycosylation modification can also be carried out by mutation, such as the N297Q mutation, where the asparagine at position 297 is mutated to glutamine.

[0430] The term "mAb" also referred to as "monoclonal antibody" means a polypeptide (including antibodies, bispecific antibodies, etc.) having substantially the same amino acid sequence or derived from the same genetic origin. Monoclonal antibodies are highly specific and can target a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations that typically include multiple different antibodies against multiple different determinants (epitopes), each monoclonal antibody targets only a single determinant on the antigen. In some specific embodiments, the antigen that the mAb can bind is a tumor-associated antigen; in some specific embodiments, the antigen that the mAb can bind is a tumor-specific antigen; in some specific embodiments, the mAb binds to optional antigens including but not limited to: HER2, 5T4 (TPBG), EGFR, or Nectin-4.

[0431] The terms "fragment", "derivative", and "analogue" refer to polypeptides that substantially maintain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogues of the present invention can be a) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or b) polypeptides having a substituent group in one or more amino acid residues, or c) polypeptides formed by fusing the mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or d) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with a 6His tag).

[0432] In the present invention, the biosimilar of the mAb means that although there are minor differences in its inactive ingredients clinically, the biosimilar of the mAb is highly similar to the mAb and there are no clinically significant differences in terms of safety and / or efficacy, etc.

[0433] The term "HER2" (also known as ERBB2, NEU, NGL, TKR1, CD340, p185, MLN19, HER-2 / neu) refers to a transmembrane tyrosine kinase receptor of the epidermal growth factor (EGF) receptor family. HER2 contains an extracellular binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain. HER2 does not have its own ligand-binding domain and thus cannot bind growth factors. However, HER2 binds tightly to other ligand-binding EGF receptor family members (such as HER1 or HER3) to form heterodimers, stabilizing ligand binding and enhancing the activation of kinase-mediated downstream signaling pathways. In humans, there are species HER2 isoforms: A, B, C, D, and E. "HER2" as described in the present invention includes all HER2 isoforms.

[0434] The term "anti-HER2 antibody" refers to an antibody that targets HER2 as the target protein, and the anti-HER2 antibody can be derived from any species, such as humans, rats, mice, and rabbits. The anti-HER2 antibody is preferably a monoclonal anti-HER2 antibody, and more preferably a humanized anti-HER2 antibody. The anti-HER2 antibodies include but are not limited to: pertuzumab, trastuzumab, trastuzumab biosimilar (such as inetetamab), pertuzumab biosimilar, margetuximab, HT-19, etc.;

[0435] Among them, Trastuzumab (also known as Herceptin or Herclon) is a humanized monoclonal antibody that can bind to the juxtamembrane region of the extracellular domain of the HER2 receptor (Hudis CA, N Engl J Med. 2007; 357(1): 39-51). The amino acid sequences of the variable regions of the heavy and light chains of Trastuzumab are disclosed in US Patent 5,821,337. Trastuzumab interacts with the tricyclic region formed by human HER2 residues 557-561, 570-573, and 593-603 (Cho et al., Nature 421: 756-760, 2003). Trastuzumab can interfere with HER2 signaling by preventing HER2 receptor dimerization, promoting HER2 receptor endocytosis, and inhibiting the shedding of the extracellular domain. Additionally, another important mechanism of action of anti-HER2 antibodies is to mediate antibody-dependent cell cytotoxicity (ADCC). In ADCC, the anti-HER2 antibody binds to tumor cells and then recruits immune cells, such as macrophages, through interaction with Fcγ receptors (FcγR). Trastuzumab has a conserved human IgG Fc region and is capable of recruiting immune effector cells responsible for antibody-dependent cell cytotoxicity (Hudis CA, N Engl J Med. 2007; 357(1): 39-51). Trastuzumab was approved by the US FDA in September 1998 for the treatment of patients with metastatic breast cancer whose tumors overexpress HER2 and who are receiving one or more chemotherapy regimens. In some embodiments, to achieve site-specific conjugation, engineered Trastuzumab with a cysteine inserted between positions 239 and 240 of the heavy chain can be prepared by the method disclosed in Molecular Pharmaceutics. 2017, 14, 1501-1516; in some embodiments, variants of IgG1 of Trastuzumab are obtained by replacing a portion of the amino acid residues in the constant region to reduce or attenuate effector functions, including but not limited to: IgG1 LALA (IgG1-L234A, L235A), IgG1 LAGA (IgG1-L235A, G237A), IgG1 AAG (IgG1-L234A, L235A, P329G), etc. The above L234A, L235A indicate that the leucine at positions 234 and 235 determined by the EU index (Proc. Natl. Acad. Sci. U.S.A., Vol. 63, No. 1 (May 15, 1969), p78-85) is replaced by alanine, G237A indicates that the glycine at position 237 determined by the EU index is replaced by alanine, and P329G indicates that the proline at position 329 determined by the EU index is replaced by alanine.

[0436] Pertuzumab (also known as Perjeta, Omnitarg) is a humanized monoclonal antibody that binds to the extracellular domain of the HER2 receptor and inhibits the dimerization of HER2 and other HER receptors. The amino acid sequences of the heavy and light chain variable regions of pertuzumab are disclosed in US Patent 7,560,111. Pertuzumab mainly interacts with residues in the 245-333 region of human HER2, especially residues His245, Val286, Ser288, Leu295, His296 or Lys311 (Franklin et al., Cancer Cell 5:317-328, 2004). Studies have shown that pertuzumab is more effective than trastuzumab in disrupting the formation of HER1-HER2 and HER3-HER2 complexes in breast and prostate cancer cell lines (Agus et al., J Clin Oncol. 2005; 23(11):2534-43. Epub Feb 7, 2005). Pertuzumab was approved by the US FDA in June 2012 and is used in combination with trastuzumab and docetaxel to treat HER2-positive metastatic breast cancer patients who have not received anti-HER2 therapy or chemotherapy.

[0437] Margetuximab (also known as MGAH22, Margenza) is an Fc-engineered monoclonal antibody that targets the HER2 protein and binds to the extracellular region of HER2. It differs from the variable region sequence of trastuzumab by only a few amino acids, and the Fc has been mutated at five sites: F243L / R292P / Y300L / L235V / P396L, which improves the affinity for CD16A and enhances the ADCC activity. The modified Fc region of Margetuximab increases the binding to the activating Fc receptor FCGR3A (CD16A) and reduces the binding to the inhibitory Fc receptor FCGR2B (CD32B), resulting in stronger ADCC effects and NK cell activation (Nordstrom J. et al., Breast Cancer Research, 2011; 13:R123). Margetuximab was approved by the US FDA in December 2020 for the treatment of adult metastatic HER2-positive breast cancer (MBC) patients who have received 2 or more anti-HER2 targeted therapies, and at least 1 anti-HER2 targeted therapy was used to treat metastatic breast cancer.

[0438] In the present invention, the anti-HER2 antibody only needs to specifically bind to HER2 (for example, an anti-HER2 antibody that has the activity of internalizing in HER2-expressing cells through HER2 binding), and is not limited to the antibodies listed above.

[0439] The isotypes of the "anti-HER2 antibody" in the present invention include IgG1, IgG2, IgG3, IgG4, etc., preferably IgG1, IgG2 or IgG4.

[0440] The term "HER2 low expression" generally refers to a HER2 expression level of IHC 1+ in clinical tests, or IHC 2+ / FISH negative (i.e., IHC 2+ and at the same time FISH test is negative). The terms "HER2 high expression" and "HER2 positive" can be used interchangeably and generally refer to a HER2 expression level of IHC 2+ / FISH positive (i.e., IHC 2+ and at the same time FISH test is positive), or IHC 3+ in clinical tests. When the IHC staining intensity is reported as a range, the term "HER2 low expression" in this article, in addition to including IHC 1+ or IHC 2+ / FISH negative, also includes the ranges of IHC 0 to 1+ and IHC 1+ to 2+. The terms "HER2 high expression" and "HER2 positive" each, in addition to including IHC 2+ / FISH positive or IHC 3+, also include the range of IHC 2+ to 3+. In the present invention, FISH negative means that the FISH test result shows no amplification of the HER2 gene, and FISH positive means that the FISH test result shows amplification of the HER2 gene.

[0441] The term "EGFR", which is the epidermal growth factor receptor, belongs to the ErbB family. Homodimerization or heterodimerization of EGFR and other ErbB family members activates the cytoplasmic tyrosine kinase domain to initiate intracellular signal transduction. Overexpression or activating mutations of EGFR are associated with the development of various types of cancers, such as pancreatic cancer, breast cancer, glioblastoma multiforme, head and neck cancer, and non-small cell lung cancer. "Anti-EGFR monoclonal antibodies" include but are not limited to: cetuximab, panitumumab, nimotuzumab, necitumumab, depatuxizumab, amivantamab, etc.;

[0442] Among them, Cetuximab (also known as Erbitux or Iressa) is a human-mouse chimeric antibody. In addition to competitively binding to the extracellular binding epitope of the natural ligand EGF to block the conduction of the signaling pathway, Cetuximab can also induce receptor internalization, downregulation and degradation. In addition, Cetuximab can also block the cell cycle and cause antibody-dependent cytotoxicity. Cetuximab was first approved by the US FDA in 2004 for the treatment of metastatic colorectal cancer patients with positive EGFR who are ineffective after treatment with the chemotherapy drug irinotecan; it was also approved by the US FDA in 2006 for the combined radiotherapy of locally or regionally advanced head and neck squamous cell carcinoma.

[0443] Panitumumab (also known as Vectibix) is a fully humanized IgG2 antibody. Like Cetuximab, Panitumumab can rapidly bind to EGFR and has a low dissociation constant, so it can effectively competitively prevent the binding of EGF and TGFα ligands. Panitumumab shows good anti-tumor activity in xenograft models of various tumor cells, including pancreatic cancer, breast cancer, renal cancer, prostate cancer and head and neck cancer, etc. (Yang X.D. et al., Critical Reviews in Oncology / hematology. 2001; 38(1): 17-23). Panitumumab was first approved by the US FDA in 2006 for the treatment of metastatic colorectal cancer patients with positive EGFR who have been treated with irinotecan, oxaliplatin and 5-fluorouracil.

[0444] Nimotuzumab (also known as Tai Xinsheng) is a fully humanized IgG1 antibody. The heavy chain variable region of Nimotuzumab is quite different from that of Cetuximab. After Nimotuzumab binds to EGFR, it does not completely assume an inhibited conformation like Cetuximab. The extracellular domains I and III of EGFR may still bind to each other to form an activated equilibrium conformation. Its affinity for binding to the EGFR epitope is also much weaker than that of Cetuximab. Clinically, a larger dose is required to achieve the efficacy of Cetuximab, but at the same time, the probability of side effects such as rash is relatively low (Talavera A. et al., Cancer Research. 2009; 69(14): 5851-9). In 2014, the FDA approved Nimotuzumab for the treatment of head and neck cancer and glioma.

[0445] Necitumumab (also known as Portrazza) is a fully humanized IgG1 antibody. The cavity between the heavy and light chains of Necitumumab is larger than that of cetuximab. Some cetuximab-resistant mutations that are highly prevalent in metastatic colorectal cancer, such as S492R, are exactly located in this binding region. Therefore, Necitumumab can overcome the antibody-drug resistance phenomenon caused by these EGFR extracellular point mutations. In 2015, the FDA approved Necitumumab for the treatment of non-small cell lung cancer.

[0446] In the present invention, the anti-EGFR antibody only needs to specifically bind to EGFR (for example, an anti-EGFR antibody that has the activity of internalizing in EGFR-expressing cells through EGFR binding), and is not limited to the antibodies listed above.

[0447] The isotypes of the "anti-EGFR antibody" in the present invention include IgG1, IgG2, IgG3, IgG4, etc., and preferably IgG1, IgG2 or IgG4.

[0448] The term "5T4", also known as 5T4 carcinoembryonic antigen or trophoblast glycoprotein TPBG, is a 72 kDa glycoprotein defined by a monoclonal antibody produced against a glycoprotein isolated by wheat germ agglutinin from human placental syncytiotrophoblast microvilli membranes. It has limited expression in normal tissues, but is overexpressed in various types of cancer cells. "Anti-5T4 monoclonal antibody" includes but is not limited to: H8 and its humanized monoclonal antibody disclosed in WO2006 / 031653A1, A1, A2, A3 and their humanized monoclonal antibodies (such as huA1) disclosed in US8044178B2, the engineered A1 monoclonal antibody disclosed in WO2013068874, naptumomab, etc.

[0449] The term "Nectin-4", also known as 191P4D12 protein, is a cell adhesion molecule and a surface molecule belonging to the nectin protein family, which plays a key role in various biological processes of epithelial cells, endothelial cells, immune cells and nerve cells during development and adulthood. Nectin-4 is a tumor-associated antigen on tumors with mainly poor prognosis. Anti-Nectin-4 monoclonal antibodies include but are not limited to: enfortumab (Ha22-2), etc.

[0450] The term "linker" refers to a degradable or non-degradable linking fragment used to link a small molecule drug D and an antibody. An antibody molecule can be linked to multiple linkers carrying small molecule drugs D. Usually, each linker can link one or more small molecule drugs. In the present invention, it is preferred that each linker links one small molecule drug D. Usually, each antibody can be linked to multiple linkers. In the present invention, it is preferred that each antibody is linked to 1 to 8 linkers.

[0451] In the present invention, the non-degradable linker refers to the linker having enzyme stability and / or chemical stability in vivo and in vitro, and the release of the small molecule drug D may not depend on the differential properties of plasma, tumor tissue and intracellular enzyme levels. The release of the small molecule drug D can be achieved by antigen-mediated phagocytosis to endocytose the antibody-immunostimulatory conjugate and then degrade the antibody to the amino acid level, thereby releasing a derivative of the small molecule drug D, which is composed of the small molecule drug D, the linker and the amino acid residue or the residue covalently linked by the small molecule drug D and the linker. The antibody-immunostimulatory conjugate constructed with such non-degradable linkers has better stability. The non-degradable linkers include alkylene chains and their polymers (such as: alkylene amide polymers, alkylene glycol polymers or combinations including alkylene glycol and alkylene amide polymer fragments) or ethylene glycol fragments and polyethylene glycol fragments, and their combinations.

[0452] In the present invention, the degradable linker can be degraded in vivo or in vitro, and it includes a linker that can be degraded by specific enzymes in vivo or in vitro or a linker that is chemically unstable itself. The degradable linker can be degraded intracellularly to release the small molecule drug D. For example, it can be reduced in the cytoplasm, degraded under lysosomal acidic conditions or degraded by specific proteases or other enzymes in the cell. The degradable linker includes one or more enzymatically degradable linkers, chemically unstable linkers or other degradable linkers, and other parts can be linkers that are not degraded by enzymes or are chemically stable. The chemically unstable linkers include oxime, hydrazone and / or disulfide groups (such as: ). The linker specifically degraded by enzymes is based on 1) a linker formed by amino acid residues or peptides. The peptide bond can have good serum stability because the lysosomal proteolytic enzymes are much less active in the blood than in some tumor tissues. Therefore, the linker can be selectively degraded in some tumor tissues or cells to release the small molecule drug D. The lysosomal enzymes can be selected from cathepsin B, cathepsin S, plasmin, elastase, β-glucuronidase or β-galactosidase, etc. The linker formed based on peptides (-(A) v-) It can be a tetrapeptide (including but not limited to: -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Gly-Gly-Phe-Gly-, -Leu-Ser-Gly-lys-, -Ala-Ala-Pro-Val-, -Glu-Val-Ala-Gly-, -Glu-Val-Cit-Gly-, -Leu-Ala-Glu-Gly-), a tripeptide (including but not limited to: -Val-Leu-Lys-, -Ala-Pro-Val-, -Ala-Ala-Asn-, -Glu-Val-Cit-, -Leu-Ala-Glu-, -Glu-Val-Ala-, -Val-Cit-Gly-, -Val-Ala-Gly-, -Leu-Ala-Glu-), a dipeptide (including but not limited to: -Val-Cit-, -Cit-Val-, -Val-Ala-, -Ala-Val-, -Ala-Cit-, -Cit-Ala-, -Asn-Cit-, -Cit-Asn-, -Cit-Cit-, -Val-Glu-, -Glu-Val-, -Ser-Cit-, -Cit-Ser-, -Lys-Cit-, -Cit-Lys-, -Asp-Cit-, -Cit-Asp-, -Ala-Val-, -Val-Ala-, -Phe-Lys-, -Lys-Phe-, -Val-Lys-, -Lys-Val-, -Ala-Lys-, -Lys-Ala-, -Phe-Cit-, -Cit-Phe-, -Leu-Cit-, -Cit-Leu-, -Ile-Cit-, -Cit-Ile-, -Phe-Arg-, -Arg-Phe-, -Cit-Trp-, -Trp-Cit-, -Pro-Val-) or an amino acid monomer (including but not limited to: -Lys-, -Gly-, -Cit-). The linker formed based on peptides is preferably a dipeptide linker, a tripeptide linker or a tetrapeptide linker, more preferably a dipeptide linker. The other cleavable linkers can include ester linkages formed by the reaction of PEG carboxylic acid or activated PEG carboxylic acid with the hydroxyl group on the small molecule drug D, and such ester linkages can be hydrolyzed under physiological conditions to release the small molecule drug D. Hydrolyzable cleavable linkages include but are not limited to carbonate linkages, imine linkages generated by the reaction of amines and aldehydes, phosphate ester linkages obtained by the reaction of hydroxyl groups and phosphate groups, acetal linkages obtained by the reaction of hydroxyl groups and aldehydes, orthoester linkages obtained by the reaction of formates and hydroxyl groups, etc. The cleavable linker can also include non-cleavable fragments, such as polyethylene glycol (PEG) and its related polymers. 2) Linkers formed by pyrophosphate esters or phosphate esters.Lysosomal acid pyrophosphatase and acid phosphatase are enzymes that hydrolyze pyrophosphate esters and terminal monophosphate esters to their parent alcohols, respectively, in lysosomes. Targeting these enzymes can effectively release the small molecule drug D with an alkyl alcohol at the end.

[0453] In the present invention, the "linker" may further include a spacer group. The linker head can be directly connected to the linker in the linker body, or connected to the linker in the linker body through a spacer group. The spacer group can be polyethylene glycol and its related polymers, alkylene groups containing 1 to 10 carbon atoms, cyclohexyl groups, phenyl groups, 1,3-dioxane groups, amide groups, ester groups, oxo groups, substituted amino groups, triazole groups or a combination of any one or more of the above spacer groups.

[0454] In the present invention, the "linker" may further include a self-immolative group, which spatially separates the small molecule drug D and the enzyme degradation site.

[0455] In the present invention, the "linker" further includes a linker head, which can be connected to an antibody. It can be formed by reacting a linker head precursor with a thiol group (e.g., cysteine), an amino group (e.g., lysine), a carbonyl group (e.g., p-acetylphenylalanine), an aldehyde group, an azide group (e.g., p-azidomethylphenylalanine), a phenol group (e.g., tyrosine), etc. in the antibody.

[0456] In the present invention, the "linker head precursor" can react with an antibody and form a linker head connected to the antibody.

[0457] In the present invention, the linker head precursor can react with a thiol group (e.g., cysteine), an amino group (e.g., lysine), a carbonyl group (e.g., p-acetylphenylalanine), an aldehyde group, an azide group (e.g., p-azidomethylphenylalanine), a phenolic hydroxyl group (e.g., tyrosine), etc. in the antibody; the linker head precursor can also be connected to a specific group of the antibody through an enzyme-catalyzed reaction.

[0458] The linker head precursors that can react with the thiol group on the side chain of an antibody amino acid residue include, but are not limited to: ethynyl, vinyl, hydroxylamine, R 12 、R 13 、R 13’ and R 11 are as defined above.

[0459] The reaction between the linker head precursor and the thiol group (e.g., cysteine) in the antibody is specific, for example, as shown in Formulas 1 to 6 and 12:

[0460]

[0461]

[0462] When the linker is maleimide or succinimide, it can be further hydrolyzed to obtain hydrolysis products, as shown in, for example, Reaction Scheme 1' or Reaction Scheme 2':

[0463]

[0464] The linker precursors that can react with the azide group on the side chain of the antibody amino acid residue include, but are not limited to: ethynyl, The click chemical reaction between the linker precursor and the azide group (e.g., p-azidomethylphenylalanine) in the antibody is specific, for example, as shown in Formula 7 or 13:

[0465]

[0466]

[0467] The linker precursors that can react with the carbonyl or aldehyde group on the side chain of the antibody amino acid residue include, but are not limited to: hydroxylamine or hydrazine group; the reaction between the linker precursor and the acyl group (e.g., p-acetylphenylalanine) or aldehyde group in the antibody is specific, for example, as shown in Formulas 8 - 9:

[0468]

[0469] The linker precursors that can react with the phenol group on the side chain of the antibody amino acid residue include, but are not limited to: The click-like chemical reaction between the linker precursor and the phenol group (e.g., tyrosine) in the antibody is specific, for example, as shown in Formula 10:

[0470]

[0471] The linker precursor that directly reacts with the amino group on the side chain of the antibody amino acid residue through a reaction to form a linker for connecting the small molecule drug D and the antibody is an active ester group, including, but not limited to: R 12 and R 12’ are as defined above; the reaction between the linker precursor and the amino group (e.g., lysine) in the antibody is specific, for example, as shown in Formula 11:

[0472]

[0473] The term "linker precursor" can directly react with the antibody and form a degradable or non-degradable linker for connecting the small molecule drug D and the antibody.

[0474] The term "and / or" includes any and all combinations of one or more of the associated listed items.

[0475] The term "C A-B " refers to a range from a starting point to an ending point, where A and B and each point within their ranges are integers representing the number of carbon atoms. For example, C 1-4 represents 1, 2, 3, or 4 carbon atoms; C 1-6 represents 1, 2, 3, 4, 5, or 6 carbon atoms; C 3-8 represents 3, 4, 5, 6, 7, or 8 carbon atoms; C A-B can be used in combination with any group containing carbon atoms to define the number of carbon atoms. For example, C 1-6 alkyl, C 3-8 cycloalkyl, C 6-10 aryl, C 1-4 alkoxy, C 3-8 cycloalkyl C 1-4 alkyl, etc.

[0476] The term "alkyl" refers to a saturated straight-chain or branched-chain hydrocarbon group containing 1 - 20 carbon atoms, preferably 1 - 10 carbon atoms, more preferably 1 - 8, 1 - 6, 1 - 4, or 1 - 3 carbon atoms. Representative examples of alkyl include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, n-pentyl, n-hexyl, n-heptyl, octyl, nonyl, decyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 4,4-dimethylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, 2,2,4-trimethylpentyl, undecyl, dodecyl, and their various isomers, etc.

[0477] The term "alkenyl" refers to a straight-chain or branched-chain non-aromatic hydrocarbon group containing at least 1 carbon-carbon double bond. One to three carbon-carbon double bonds can be present, preferably 1 carbon-carbon double bond. The term "C 2-4 alkenyl" refers to an alkenyl having 2 - 4 carbon atoms, and the term "C 2-6 alkenyl" refers to an alkenyl having 2 - 6 carbon atoms, including vinyl, propenyl, butenyl, 2-methylbutenyl.

[0478] The term "alkynyl" refers to a straight-chain or branched-chain hydrocarbon group containing at least 1 carbon-carbon triple bond. One to three carbon-carbon triple bonds may be present, preferably 1 carbon-carbon triple bond. The term "C 2-6 alkynyl" refers to an alkynyl group having 2 to 6 carbon atoms, including ethynyl, propynyl, butynyl, and 3-methylbutynyl.

[0479] The term "alkylene" refers to a saturated straight-chain or branched-chain non-bridging divalent alkyl group containing 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Examples include, but are not limited to, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH(CH3)-, -CH2C(CH3)2CH2-, -CH2C(CH3)2CH2CH2-, =CH2, =CHCH3, =C(CH3)2.

[0480] The term "alkenylene" refers to a straight-chain or branched-chain non-aromatic divalent hydrocarbon group containing at least 1 carbon-carbon double bond. One to three carbon-carbon double bonds may be present, preferably 1 carbon-carbon double bond. The alkenylene preferably contains 2 to 10 carbon atoms, more preferably 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

[0481] The term "alkynylene" refers to a straight-chain or branched-chain non-aromatic divalent hydrocarbon group containing at least 1 carbon-carbon triple bond. One to three carbon-carbon triple bonds may be present, preferably 1 carbon-carbon triple bond. The alkynylene preferably contains 2 to 10 carbon atoms, more preferably 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

[0482] The term "cycloalkyl" refers to a saturated or partially unsaturated (containing 1 or 2 double bonds) monocyclic or polycyclic group containing 3 to 20 carbon atoms. "Monocyclic cycloalkyl" is preferably a 3- to 10-membered monocyclic cycloalkyl, more preferably a 3- to 8-membered monocyclic cycloalkyl, such as: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, cyclohexenyl. "Polycyclic cycloalkyl" includes "bridged cycloalkyl", "fused cycloalkyl", and "spirocycloalkyl". Representative examples of "bridged cycloalkyl" include, but are not limited to: borneol, bicyclo[2.2.1]heptenyl, bicyclo[3.1.1]heptanyl, bicyclo[2.2.1]heptanyl, bicyclo[2.2.2]octanyl, bicyclo[3.2.2]nonanyl, bicyclo[3.3.1]nonanyl, bicyclo[4.2.1]nonanyl, and adamantyl, etc. "Fused cycloalkyl" contains a cycloalkyl ring fused to a phenyl, cycloalkyl, or heteroaryl group. Fused cycloalkyl includes, but is not limited to: benzocyclobutenyl, 2,3-dihydroindenyl, decahydronaphthyl, etc. The monocyclic cycloalkyl or polycyclic cycloalkyl can be linked to the parent molecule through any carbon atom on the ring.

[0483] The term "cycloalkanediyl" refers to a divalent cycloalkyl group. Thus, "cycloalkanediyl" encompasses the definitions of the above cycloalkyl groups. "Cycloalkanediyl" is preferably C 3-10 cycloalkanediyl, more preferably C 3-8 cycloalkanediyl or C 3-6 cycloalkanediyl.

[0484] The term "heterocycloalkyl" refers to a saturated or partially unsaturated (including 1 or 2 double bonds) non-aromatic cyclic group composed of carbon atoms and heteroatoms selected from nitrogen, oxygen, sulfur, boron, etc. and / or sulfur-containing heteroatom groups. This cyclic group can be a monocyclic or polycyclic group. Among them, the sulfur-containing heteroatom groups are selected from, but not limited to, S(O), S(O)2, and S(O)(NH). In the present invention, the number of heteroatoms and / or heteroatom groups in the heterocycloalkyl is preferably 1, 2, 3, or 4, and the boron, nitrogen, or carbon atoms in the heterocycloalkyl can be optionally oxidized. The nitrogen atom can be further optionally substituted by other groups to form a tertiary amine or quaternary ammonium salt. "Monocyclic heterocycloalkyl" is preferably a 3- to 10-membered monocyclic heterocycloalkyl, more preferably a 3- to 8-membered monocyclic heterocycloalkyl. For example: pyrrolidinyl, dihydropyrrolidinyl, dihydroimidazolyl, dihydropyrazolyl, tetrahydrofuryl, tetrahydropyrazinyl, dihydrofuryl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, tetrahydropyridyl, tetrahydropyrimidinyl, piperidinyl, aziridinyl, morpholinyl, thiomorpholinyl, thiomorpholin-S-oxide-4-yl, piperidinyl, piperazinyl, 1,4-dioxanyl, homopiperazinyl, 1-imino-1-oxidotetrahydro-2H-thiopyranyl, 1,1-dioxidotetrahydrothienyl, 1-imino-1-oxidotetrahydrothienyl, 1,1-dioxido-3,4-dihydro-2H-thiopyranyl, 1-imino-1-oxidotetrahydro-2H-thiopyranyl, 1,1-dioxido-2,3-dihydrothienyl, 1-imino-1-oxidotetrahydro-2H-thienyl, etc. "Polycyclic heterocycloalkyl" includes "fused heterocycloalkyl", "spiroheterocyclic group", and "bridged heterocycloalkyl". "Fused heterocycloalkyl" contains a monocyclic heterocycloalkyl ring fused to a phenyl group, cycloalkyl group, heterocycloalkyl group, or heteroaryl group. Fused heterocycloalkyl includes, but is not limited to: 2,3-dihydrobenzofuranyl, 1,3-dihydroisobenzofuranyl, dihydroindolyl, 2,3-dihydrobenz[b]thienyl, dihydrobenzopyranyl, 1,2,3,4-tetrahydroquinolinyl, 1,2,3,4-tetrahydroisoquinolinyl, 1,2,3,4-tetrahydronaphthyridinyl, 5,6,7,8-tetrahydronaphthyridinyl, 1-hydroxy-1,3-dihydrobenz[c][1,2]oxaborolyl, etc. The monocyclic heterocycloalkyl and polycyclic heterocycloalkyl can be linked to the parent molecule through any ring atom on the ring. The above ring atoms specifically refer to the carbon atoms and / or nitrogen atoms that make up the ring skeleton.

[0485] The term "heterocycloalkylene" refers to divalent heterocycloalkyl. Thus, "heterocycloalkylene" encompasses the definitions of the above-mentioned heterocycloalkyl. "Heterocycloalkylene" is preferably a 3- to 10-membered heterocycloalkylene, more preferably a 3- to 8-membered heterocycloalkylene or a 3- to 6-membered heterocycloalkylene.

[0486] The term "cycloalkylalkyl" means that cycloalkyl is linked to the parent nucleus structure through an alkyl group. Thus, "cycloalkylalkyl" encompasses the definitions of the above-mentioned alkyl and cycloalkyl.

[0487] The term "heterocycloalkylalkyl" means that heterocycloalkyl is linked to the parent nucleus structure through an alkyl group. Thus, "heterocycloalkylalkyl" encompasses the definitions of the above-mentioned alkyl and heterocycloalkyl.

[0488] The term "alkoxy" refers to an alkyloxy group having the specified number of carbon atoms linked through an oxygen bridge. Thus, "alkoxy" encompasses the definition of the above-mentioned alkyl.

[0489] The term "alkylthio" refers to an alkylthio group having the specified number of carbon atoms linked through a thiol bridge. Thus, "alkylthio" encompasses the definition of the above-mentioned alkyl.

[0490] The term "hydroxyalkyl" means that any one hydrogen atom on the alkyl group is replaced by a hydroxyl group, including but not limited to: -CH2OH, -CH2CH2OH, -CH2CH2C(CH3)2OH.

[0491] The term "aryl" refers to any stable 6- to 20-membered monocyclic or polycyclic aromatic group, such as: phenyl, naphthyl, tetrahydronaphthyl, 2,3-dihydroindenyl or biphenyl, etc.

[0492] The term "arylene" refers to divalent heteroaryl. Thus, "arylene" encompasses the definition of the above-mentioned aryl. "Arylene" is preferably C 6-10 arylene, more preferably phenylene.

[0493] The term "heteroaryl" refers to an aromatic ring group formed by replacing at least one carbon atom on the ring with a heteroatom selected from nitrogen, oxygen, or sulfur, which can be a 5- to 7-membered monocyclic structure or a 7- to 20-membered fused ring structure, preferably a 5- to 6-membered heteroaryl. In the present invention, the number of heteroatoms is preferably 1, 2, or 3, including: pyridyl, pyridone, pyrimidinyl, pyrimidine-2,4(1H,3H)-dione, pyrimidinone, piperazinyl, pyridazinone, furyl, thienyl, thiazolyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-thiadiazole, 1,2,4-triazolyl, 1,2,3-triazolyl, tetrazolyl, indazolyl, isoindazolyl, indolyl, isoindolyl, benzofuryl, benzothienyl, benzo[d][1,3]dioxolyl, benzothiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, isoquinolinone, quinazolinyl, 4-hydroxythieno[3,2-c]pyridyl, 4,5-dihydro-4-oxofuro[3,2]pyridyl, 4-hydroxy-5-azaindolyl, furo[2,3-c]pyridin-7(6H)-one, thieno[2,3-c]pyridin-7(6H)-one, etc.

[0494] The term "heteroarylene" refers to a divalent heteroaryl. Thus, "heteroarylene" includes the definition of the above-mentioned heteroaryl. "Heteroarylene" is preferably a 5- to 10-membered heteroarylene, more preferably a 5- to 6-membered heteroarylene.

[0495] The term "arylalkyl" means that an aryl is connected to the parent nucleus structure through an alkyl. Thus, "arylalkyl" includes the definitions of the above-mentioned alkyl and aryl.

[0496] The term "heteroarylalkyl" means that a heteroalkyl is connected to the parent nucleus structure through an alkyl. Thus, "heteroarylalkyl" includes the definitions of the above-mentioned alkyl and heteroaryl.

[0497] The term "halogen" means fluorine, chlorine, bromine, or iodine.

[0498] The term "haloalkyl" means an alkyl that is arbitrarily substituted by a halogen. Thus, "haloalkyl" includes the definitions of the above-mentioned halogen and alkyl.

[0499] The term "haloalkoxy" means an alkoxy that is arbitrarily substituted by a halogen. Thus, "haloalkoxy" includes the definitions of the above-mentioned halogen and alkoxy.

[0500] The term "amino" means -NH2, and the term "alkylamino" means that at least one hydrogen atom on the amino group is substituted by an alkyl, including but not limited to: -NHCH3, -N(CH3)2, -NHCH2CH3, -N(CH2CH3)2. Thus, "alkylamino" includes the definitions of the above-mentioned alkyl and amino.

[0501] The term "aminoalkyl" means that any one hydrogen atom on the alkyl group is replaced by an amino group, including but not limited to: -CH2NH2, -CH2CH2NH2, -CH2CH2C(CH3)2NH2.

[0502] The term "acyl" means -C(O)-C 1-6 alkyl group; preferably formyl, acetyl, propionyl or isopropionyl.

[0503] The term "nitro" means -NO2.

[0504] The term "cyano" means -CN.

[0505] The term "oxo group" means =O.

[0506] The term "mercapto group" means -SH.

[0507] The term "amide group" means -C(O)NH2.

[0508] The term "hydroxyamino" means -NHOH.

[0509] The term "carboxyl group" means -C(O)OH.

[0510] The term "aldehyde group" means -C(O)H.

[0511] The term "methyl ester group" means -C(O)OCH3.

[0512] The term "maleimide group" means

[0513] In the present invention, the abbreviations of amino acids are conventional abbreviations (refer to the Nomenclature Principles of Organic Chemistry of the Chinese Chemical Society 2017), for example: alanine (Ala), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), glutamic acid (Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), valine (Val). In the present invention, the symbolic representation of a peptide (taking -Gly-(-NH-CH2-CO-) as an example) means that when the short horizontal line representing the peptide bond is on the right side of Gly, it represents the removal of the OH group from the -COOH group of the amino acid, and when the short horizontal line is on the left side of Gly, it represents the removal of a H atom from the -NH2- of the amino acid.

[0514] In the present invention, generally it is considered that 31 in 31P NMR31 The chemical shift of P is related to the shape of the electron cloud outside the nucleus. When the spherical symmetry of the electron cloud is better, the chemical shift moves towards the high field; when the ligands of phosphorus are the same or the electronic properties of the ligands are similar, 31 the chemical shift of P moves towards the high field. Especially when the ligand is a group with high electronegativity, the change is particularly obvious. Aromatic hydrocarbon substitution makes the 31 chemical shift of P shift towards the high field compared to aliphatic hydrocarbon substitution. This is because the conjugation of the groups adjacent to the phosphorus atom will also affect 31 the chemical shift of P. The enhancement of the electron-donating conjugation makes 31 the chemical shift of P shift towards the high field (Hu WX, et al., Chin Chem Lett 1992, 3(4)). In the present invention, when phosphorus is substituted, the chemical shift also follows the above principle. For example, by comparing the chemical shifts of the phosphorus at position 1 and the phosphorus at position 2 of compound 1M and compound 1-3, it can be judged that the chemical shift of the phosphorus at position 2 is biased towards the high field. When the mercapto hydrogen connected to phosphorus in compound 1-3 is substituted with a benzyl group, through 31 the change in the chemical shift of P, it can be speculated that the benzyl substitution site in compound A is on the mercapto group at position 2.

[0515]

[0516] 31 P NMR (162 MHz, DMSO-d6 + D2O): δ55.847.

[0517]

[0518] 31 P NMR (DMSO-d6 + D2O): δ55.759, 51.401.

[0519]

[0520] 31 P NMR (DMSO-d6 + D2O): δ53.113, 27.573.

[0521] "Room temperature" as used in the present invention means 15 - 30 °C.

[0522] In the "antibody-immunostimulatory conjugate", "compound" and "pharmaceutically acceptable salt" of the present invention, the substituents R1, R2, R3, R4, R 4’ , R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 12’ , R 13 , R 13’ , R A and L1～2 If there are stereoisomers, they may exist in the form of a single stereoisomer or a mixture thereof.

[0523] The "pharmaceutically acceptable salts" of the present invention are discussed in Berge, et al., "Pharmaceutically acceptable salts", J. Pharm. Sci., 66, 1-19 (1977) and are obvious to pharmaceutical chemists. The salts are substantially non-toxic and can provide the desired pharmacokinetic properties, palatability, absorption, distribution, metabolism or excretion, etc. The compounds of the present invention may have acidic groups, basic groups or amphoteric groups, and typical pharmaceutically acceptable salts include salts prepared by reacting the compounds of the present invention with acids.

[0524] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain the preferred examples of the present invention.

[0525] The reagents and raw materials used in the present invention are all commercially available. Detailed Description of the Invention

[0526] The structures of all compounds of the present invention can be identified by nuclear magnetic resonance ( 1 H NMR) and / or mass spectrometry (MS).

[0527] 1 The H NMR chemical shift (δ) is recorded in PPM (10 -6 ). NMR is performed using a Bruker AVANCE-400 spectrometer. Suitable solvents are chloroform-d (CDCl3), methanol-d (CD3OD), dimethyl sulfoxide-d6 (DMSO-d6), and tetramethylsilane as an internal standard (TMS).

[0528] Liquid chromatography-mass spectrometry (LCMS) was determined by an Agilent 1200 HPLC / 6120 mass spectrometer using a chromatographic column: Xtimate C18, 3.0×50 mm, 3 μm, column temperature 40°C; or determined by a Thermo UltiMate 3000 HPLC / MSQ PLUS mass spectrometer using a chromatographic column XBridge C18, 3.0×50 mm, 3.5 μm, column temperature 30°C. Agilent gradient elution condition 1: 95 - 5% solvent A1 and 5 - 95% solvent B1 (0 - 2.0 minutes), then 95% solvent B1 and 5% solvent A1 (held for 1.1 minutes), the percentages being the volume percentages of a certain solvent in the total solvent volume. Solvent A1: aqueous solution of 0.01% trifluoroacetic acid (TFA); Solvent B1: acetonitrile solution of 0.01% trifluoroacetic acid; the percentages being the volume percentages of the solute in the solution. Thermo gradient elution condition 2: 95 - 5% solvent A2 and 5 - 95% solvent B2 (0 - 2 minutes), then 95% solvent B2 and 5% solvent A2 (held for 1.8 minutes), the percentages being the volume percentages of a certain solvent in the total solvent volume. Solvent A2: aqueous solution of 10 mM ammonium bicarbonate; Solvent B2: acetonitrile.

[0529] All compounds of the present invention can be separated by preparative high performance liquid chromatography or flash column chromatography.

[0530] Preparative high performance liquid chromatography (prep-HPLC) used an Agela FLEXA-HP preparative liquid chromatography with a chromatographic column: Welch Xtimate C18, 10um, 21.2mm x 250mm. Separation conditions: mobile phase A: acetonitrile, mobile phase B: aqueous solution of 10 mmol / L ammonium bicarbonate; detection wavelength: 214 nm & 254 nm; flow rate: 15.0 mL / minute; gradient elution conditions:

[0531] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0.00 0.0 100.0 20.00 15.0 85.0 21.00 85.0 15.0 24.00 85.0 15.0 25.00 0.0 100.0

[0532] Flash column chromatography (flash system / CheetahTM) used an Agela Technologies MP200, and the normal phase separation column used was Flash columm Silica-CS (25g, 40g, 80g, 120g or 330g), Tianjin Bonna-Agilent, and the elution system was ethyl acetate / petroleum ether, or dichloromethane / methanol; the reverse phase separation column was a C18 reverse phase column (Spherical C18, 40 - 75um, Model: SW-040), and the elution system was acetonitrile / 0.1% aqueous ammonium bicarbonate solution.

[0533] All compounds of the present invention can be analyzed by high performance liquid chromatography. The high performance liquid chromatography (HPLC) uses Waters e2695 and 2498 UV / VIS Detector;

[0534] Condition 1: The chromatographic column is: Waters Xselect CHS C18 (4.6 * 150 mm) 5 μm, mobile phase A: methanol, mobile phase B: 10 mM potassium dihydrogen phosphate buffer (pH adjusted to 8.0 with ammonia water); flow rate: 1.0 mL / min; column temperature: 35 °C; detection wavelength: 214 nm & 254 nm; gradient elution conditions:

[0535] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0.00 5.0 95.0 5.00 5.0 95.0 10.00 35.0 65.0 14.00 60.0 40.0 15.00 5.0 95.0 30.00 5.0 95.0

[0536] Condition 2: The chromatographic column is: Waters Xselect CHS C18 (4.6 * 250 mm) 5 μm, mobile phase A: methanol, mobile phase B: 10 mM potassium dihydrogen phosphate buffer (pH adjusted to 8.0 with ammonia water); flow rate: 1.0 mL / min; column temperature: 35 °C; detection wavelength: 214 nm & 254 nm; gradient elution conditions:

[0537] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0.00 10.0 90.0 5.00 25.0 75.0 17.50 35.0 65.0 17.60 10.0 90.0 23.00 10.0 90.0

[0538] The ultra-high performance liquid chromatography (UPLC) uses Waters H-Class UPLC. Conditions: The chromatographic column is: Waters ACQUITY UPLC BEH Shield RP18 2.1 mm * 100 mm, 1.7 μm, mobile phase A: acetonitrile, mobile phase B: 5 mM potassium dihydrogen phosphate buffer (pH adjusted to 2.5 with phosphoric acid); flow rate: 0.4 ml / min; column temperature: 40 °C; detection wavelength: 214 nm & 262 nm; gradient elution conditions:

[0539] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0.00 10.0 90.0 5.00 40.0 60.0 7.00 90.0 10.0 13.00 90.0 10.0 13.10 10.0 90.0 15.00 10.0 90.0

[0540] The meanings of the abbreviations used in the examples of the present invention are as follows:

[0541] (Boc)2O: Di-tert-butyl dicarbonate; BINAP: 1,1'-Binaphthalene-2,2'-bis(diphenylphosphine); DBAD: Di-tert-butyl azodicarboxylate; DCA: Dichloroacetic acid; DDTT: N,N-Dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formamidine; DPCP: Diphenyl chlorophosphate; DMF: N,N-Dimethylformamide; DMSO: Dimethyl sulfoxide; DIPEA: N,N-Diisopropylethylamine; EDCI: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; EEDQ: 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; HATU: O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; PyBOP: 1H-Benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate; TBSCl: tert-Butyldimethylchlorosilane.

[0542] Synthesis of intermediates:

[0543] Synthesis of Linker-1

[0544]

[0545] Under ice bath conditions, cesium iodide (127 mg, 0.49 mmol) and boron trifluoride diethyl etherate (45% BF3, 69 mg, 0.49 mmol) were successively added to a dry dichloromethane (10 mL) solution of Mc-Val-Ala-PAB-OH (200 mg, 0.41 mmol). The reaction mixture was stirred at room temperature overnight. The reaction was quenched by adding water at 0 °C. The aqueous phase was extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (petroleum ether / ethyl acetate = 1 / 1) to obtain Linker-1 (50 mg) as an off-white solid. m / z: [M+H] + 597.0.

[0546] Synthesis of Linker-2

[0547]

[0548] Under ice bath conditions, cesium iodide (140 mg, 0.54 mmol) and boron trifluoride diethyl etherate (45% BF3, 77 mg, 0.54 mmol) were successively added to a dry dichloromethane (10 mL) solution of Mc-Val-Cit-PAB-OH (260 mg, 0.45 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (dichloromethane / ethyl acetate = 1 / 1) to obtain Linker-2 (120 mg) as an off-white solid. m / z: [M+H] +682.5。

[0549] Synthesis of Linker-3

[0550]

[0551] To a solution of compound Mc-Val-Cit-PAB-OH (150 mg, 0.26 mmol) in DMF (4 mL), bis(p-nitrophenyl) carbonate (158 mg, 0.52 mmol) and DIPEA (101 mg, 0.78 mmol) were added successively. The reaction mixture was stirred overnight at room temperature. After filtration, the reaction mixture was concentrated under reduced pressure. The residue was purified by Flash column chromatography (C18, eluent: 0 - 60% acetonitrile in 10 mM aqueous ammonium bicarbonate) to obtain Linker-3 (125 mg) as a pale yellow solid. m / z: [M+H] + 738.1。

[0552] Synthesis of Linker-4

[0553]

[0554] To a solution of Mc-Val-Ala-PAB-OH (120 mg, 0.25 mmol) in DMF (3 mL), bis(p-nitrophenyl) carbonate (152 mg, 0.50 mmol) and DIPEA (97 mg, 0.75 mmol) were added successively. The reaction mixture was stirred overnight at room temperature. After filtration, the reaction mixture was concentrated under reduced pressure. The residue was purified by Flash column chromatography (C18, eluent: 0 - 85% acetonitrile in 10 mM aqueous ammonium bicarbonate) to obtain Linker-4 (115 mg) as a pale yellow solid. m / z: [M+H] + 652.1。

[0555] Synthesis of Linker-5

[0556]

[0557] Step 1: Under ice bath conditions, methanesulfonyl chloride (65 mg, 0.57 mmol) and pyridine (75 mg, 0.95 mmol) were added dropwise successively to a solution of 1-(6-hydroxyhexyl)-1H-pyrrole-2,5-dione (75 mg, 0.38 mmol) in dichloromethane (5 mL). The reaction mixture was slowly warmed to room temperature and stirred for an additional 2 hours. The reaction was quenched with ice water, and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with saturated brine and concentrated under reduced pressure to obtain 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl methanesulfonate (100 mg). m / z: [M+H] + 276.0。

[0558] Step 2: Sodium iodide (250 mg, 1.65 mmol) was added to a solution of the product obtained in Step 1 (90 mg, 0.33 mmol) in acetone (10 mL). The reaction mixture was stirred at 50 °C for 12 h. The residue was dissolved in ethyl acetate, washed successively with water and saturated brine, the organic layer was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give Linker-5 (105 mg). m / z: [M+H] + 308.0

[0559] Synthesis of Linker-6

[0560]

[0561] Step 1: Under ice-bath conditions, HATU (3.85 g, 10.1 mmol) and DIPEA (2.18 g, 16.9 mmol) were successively added to a solution of 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylic acid (2 g, 8.43 mmol) in N,N-dimethylformamide (15 mL). After the reaction mixture was stirred at room temperature for 5 min, (4-aminophenyl)methanol (1.14 g, 9.27 mmol) was added, and the reaction mixture was further stirred at room temperature for 3 h. The reaction was quenched by adding ice water, the aqueous phase was extracted with ethyl acetate, the combined organic phases were concentrated under reduced pressure to remove most of the organic solvents, petroleum ether was added to the residue, and the mixture was sonicated and then filtered. The filter cake was dried to give 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-N-(4-(hydroxymethyl)phenyl)cyclohexane-1-carboxamide (2.8 g). m / z: [M+H] + 343.0

[0562] Step 2: Cesium iodide (610 mg, 2.34 mmol) and boron trifluoride diethyl etherate (310 mg, 2.19 mmol) were added to a solution of the product obtained in Step 1 (500 mg, 1.46 mmol) in acetonitrile (50 mL). The reaction mixture was slowly warmed to room temperature and stirred for another 12 h, and then concentrated directly under reduced pressure to give Linker-6 (530 mg). m / z: [M+H] + 453.1

[0563] Synthesis of Linker-8

[0564]

[0565]

[0566] Step 1: To a solution of Fmoc-Val-Cit-OH (480 mg, 0.97 mmol), DIPEA (238 mg, 1.84 mmol), and HATU (420 mg, 1.1 mmol) in DMF (5 mL) was added 4-(hydroxymethyl)phenylglycine ester hydrochloride (200 mg, 0.92 mmol). The reaction mixture was stirred at room temperature for 2 days and purified directly by flash column chromatography (C18, eluent: 0 - 60% acetonitrile in 10 mM aqueous ammonium bicarbonate) to give L1 (200 mg) as a white solid. m / z: [M+H] + 660.2.

[0567] Step 2: To a solution of L1 (200 mg, 0.3 mmol) in DMF (2 mL) was added diethylamine (350 mg, 4.85 mmol). The reaction mixture was stirred at room temperature for 2 hours and purified directly by flash column chromatography (C18, eluent: 100% 0.1% aqueous trifluoroacetic acid) to give L2 (160 mg) as an off-white solid. m / z: [M+H] + 438.2.

[0568] Step 3: To a solution of L2 (160 mg, 0.29 mmol) in DMF (2 mL) were successively added DIPEA (45 mg, 0.35 mmol) and N-succinimidyl 6-(maleimidyl)hexanoate (98 mg, 0.32 mmol). The reaction mixture was stirred at room temperature for 3 hours and purified directly by flash column chromatography (C18, eluent: 0 - 60% acetonitrile in 0.1% aqueous trifluoroacetic acid) to give L3 (20 mg) as an off-white solid. m / z: [M+H] + 631.2.

[0569] Step 4: Using the synthetic method of Linker-1, Linker-8 was obtained by reacting with L3. m / z: [M+H] + 741.1.

[0570] Synthesis of Linker-9

[0571]

[0572] Step 1: Under ice-bath conditions, hydrobromic acid acetic acid solution (33%, 50 mL) was added dropwise to a solution of methyl 1,2,3,4-tetra-O-acetyl-β-D-glucuronate (16 g, 42.5 mmol) in dichloromethane (160 mL). The reaction mixture was stirred at room temperature for 12 hours. Then the reaction mixture was concentrated under reduced pressure, and the residue was diluted with dichloromethane. The organic phase was washed successively with saturated aqueous sodium bicarbonate and saturated brine, and the organic phase was separated and concentrated under reduced pressure to give L4 (15.9 g) as a white solid. m / z: [M+Na]+ 421.1。

[0573] Step 2: Silver oxide (4.16 g, 18.0 mmol) was added to a solution of L4 (5.23 g, 13.2 mmol) and 4-hydroxy-3-nitrobenzaldehyde (2 g, 12.0 mmol) in acetonitrile (25 mL). The reaction system was stirred overnight in the dark at room temperature. Then the reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (dichloromethane / methanol = 20 / 1) to obtain L5 (5 g) as a white solid. m / z: [M+Na] + 506.0。

[0574] Step 3: L5 (2.3 g, 4.76 mmol) was dissolved in a mixed solution of methanol (10 mL) and dichloromethane (30 mL), and then 10% palladium on carbon (400 mg) was added. The reaction system was purged with hydrogen three times and then stirred under a hydrogen atmosphere for 5 hours. Then the reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain L6 (1.5 g) as a white solid. m / z: [M+H] + 456.2。

[0575] Step 4: HATU (120 mg, 0.31 mmol), DIPEA (50 mg, 0.39 mmol) and L6 (95 mg, 0.21 mmol) were added to a solution of Boc-glycine (46 mg, 0.26 mmol) in DMF (5 mL). The reaction system was stirred at room temperature for 2 hours. The reaction solution was directly purified by Flash column chromatography (C18, eluent: 0% - 75% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain L7 (160 mg) as a white solid. m / z: [M+H] + 613.2。

[0576] Step 5: Trifluoroacetic acid (1 mL) was added dropwise to a solution of L7 (150 mg, 0.24 mmol) in dichloromethane (3 mL) under ice bath conditions. The reaction solution was stirred at room temperature for 1.5 hours. The reaction solution was directly purified by Flash column chromatography (C18, eluent: 0% - 50% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain L8 (110 mg) as a white solid. m / z: [M+H] + 513.2。

[0577] Step 6: To a solution of L8 (80 mg, 0.16 mmol) in DMF (5 mL) was added N-succinimidyl 6-(maleimidyl)hexanoate (54 mg, 0.18 mmol), and the reaction system was stirred at room temperature for 2 h. The reaction solution was directly purified by Flash column chromatography (C18, eluent: 0% - 70% acetonitrile in 0.1% aqueous trifluoroacetic acid) to obtain L9 (110 mg) as a white solid. m / z: [M+H] + 706.2。

[0578] Step 7: Under ice bath conditions, cesium iodide (58 mg, 0.22 mmol) and boron trifluoride diethyl etherate (36 mg, 0.25 mmol) were added to a solution of L9 (100 mg, 0.14 mmol) in acetonitrile (5 mL). After the reaction solution was stirred at room temperature for 3 h, it was directly concentrated under reduced pressure to obtain Linker-9 (110 mg). m / z: [M+H] + 816.2。

[0579] Synthesis of Linker-10

[0580]

[0581] Step 1: A solution of Boc-Val-Ala-PAB-OH (2 g, 5.08 mmol), bis(p-nitrophenyl) carbonate (3.1 g, 10.2 mmol) and DIPEA (1.97 g, 15.2 mmol) in DMF (10 mL) was stirred at room temperature for 3 h. The reaction solution was directly purified by Flash column chromatography (C18, eluent: 0% - 80% acetonitrile in 10 mM aqueous ammonium bicarbonate) to obtain L10 (1.6 g). m / z: [M+Na] + 581.2。

[0582] Step 2: A solution of L10 (1 g, 1.79 mmol), 2-((methylamino)methyl)benzoic acid (300 mg, 1.79 mmol) and DIPEA (470 mg, 3.64 mmol) in DMF (5 mL) was stirred at room temperature for 3 h. The reaction solution was directly purified by Flash column chromatography (C18, eluent: 0% - 80% acetonitrile in 0.1% aqueous trifluoroacetic acid) to obtain L11 (1.01 g). m / z: [M+Na] + 607.2。

[0583] Step 3: Under ice bath conditions, a solution of the Ghosez reagent (449 mg, 3.36 mmol) in dichloromethane (2 mL) was added to a suspension of L11 (1 g, 1.71 mmol) in dichloromethane (20 mL) and tetrahydrofuran (2 mL). The reaction mixture was stirred at 0 °C for 1 hour. It was concentrated under reduced pressure to obtain Linker-10 (1.2 g). m / z: [M+H] + 603.4。

[0584] Synthesis of Linker-11

[0585]

[0586] Step 1: Under ice bath conditions, HATU (110 mg, 0.29 mmol) and DIPEA (62 mg, 0.48 mmol) were successively added to a solution of 6-maleimidocaproic acid (50 mg, 0.24 mmol) in DMF (3 mL). After the reaction mixture was stirred for 10 minutes, L6 (87 mg, 0.19 mmol) was added. The reaction system was stirred at 0 °C for 1 hour and directly purified by Flash column chromatography (C18, eluent: 0 - 60% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to obtain L12 (110 mg). m / z: [M+H] + 648.5。

[0587] Step 2: Using the synthesis method of linker-1, Linker-11 was obtained by reacting with L12. m / z: [M+H] + 759.0。

[0588] Synthesis of Linker-12

[0589]

[0590] Step 1: EDCI (187 mg, 0.98 mmol) and HOBT (132 mg, 0.98 mmol) were successively added to a solution of amino-pentaethylene glycol-tert-butyl propionate (266 mg, 0.65 mmol) in DMF (5 mL). After the reaction mixture was stirred at room temperature for 5 minutes, 2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetic acid (150 mg, 0.65 mmol) was added. The reaction mixture was continuously stirred at room temperature for 1 hour and directly purified by Flash column chromatography (C18, eluent: 0 - 40% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to obtain L13 (260 mg) as a light yellow solid. m / z: [M+H] + 623.2。

[0591] Step 2: Under ice-bath cooling, trifluoroacetic acid (2 mL) was added dropwise to a solution of L13 (130 mg, 0.21 mmol) in dichloromethane (5 mL). After the addition was complete, the reaction mixture was stirred at room temperature for 2 hours. It was concentrated under reduced pressure, and the residue was purified by Flash column chromatography (C18, eluent: 0 - 45% acetonitrile in 0.1% aqueous trifluoroacetic acid) to obtain Linker-12 (66 mg) as a pale yellow oil. m / z: [M+H] + 567.2。

[0592] Synthesis of Linker-14

[0593]

[0594] Using the synthesis method of Linker-6, Linker-14 was synthesized using L14 (synthesized by the synthesis method of L8, replacing Boc-glycine in step 4 with Boc-L-alanine to obtain L14) and Fmoc-L-valine. m / z: [M+Na] + 980.0。

[0595] Synthesis of Linker-18

[0596]

[0597] Step 1: Under ice-bath conditions, chlorosulfonic acid neopentyl ester (4.3 g, 23.1 mmol) was slowly added dropwise to a solution of 4-(hydroxymethyl)-2-nitrophenol (3 g, 17.7 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (3.5 g, 23.1 mmol) in dichloromethane (40 mL). The reaction mixture was stirred at room temperature for 6 hours. The reaction was quenched by adding saturated aqueous ammonium chloride solution, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 - 2 / 1) to obtain L16 (960 mg) as a yellow oil. m / z: [M+H2O] + 337.2。

[0598] Step 2: Under ice-bath conditions, zinc powder (6.14 g, 93.9 mmol) was slowly added to a mixed solution of L16 (3 g, 9.39 mmol) and ammonium chloride (5.02 g, 93.9 mmol) in methanol (15 mL), tetrahydrofuran (30 mL) and water (30 mL). The reaction system was stirred at room temperature for 3 hours. It was concentrated under reduced pressure, and the residue was extracted with ethyl acetate. The combined organic phases were concentrated under reduced pressure, and the residue was purified by Flash column chromatography (C18, eluent: 0% - 60% acetonitrile in 0.1% aqueous trifluoroacetic acid) to obtain L17 (1.5 g) as a yellow solid. m / z: [M+H]+ 290.1。

[0599] Step 3: To a solution of L17 (500 mg, 1.73 mmol) in dichloromethane (15 mL) were added TBSCl (313 mg, 2.08 mmol) and imidazole (153 mg, 2.25 mmol). The reaction mixture was stirred at room temperature for 2 h, concentrated under reduced pressure, and the residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 4 / 1) to give L18 (650 mg) as a yellow solid. m / z: [M+H] + 404.2。

[0600] Step 4: To a solution of L18 (620 mg, 1.54 mmol) and Fmoc-glycine (504 mg, 1.69 mmol) in DMF (5 mL) were successively added HATU (761 mg, 2 mmol) and DIPEA (398 mg, 3.08 mmol). The reaction mixture was stirred at room temperature for 16 h, quenched by adding water, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were concentrated under reduced pressure. The residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 4 / 1) to give L19 (880 mg) as a pale yellow oil. m / z: [M+H] + 683.2。

[0601] Step 5: Under ice-bath conditions, to a solution of L19 (850 mg, 1.24 mmol) in tetrahydrofuran (10 mL) was added triethylamine trihydrofluoride (1 g, 6.2 mmol). The reaction mixture was stirred at room temperature for 5 h, and the pH was adjusted to 7 with saturated aqueous sodium bicarbonate at 0 °C. The mixture was extracted with ethyl acetate, and the combined organic phases were concentrated under reduced pressure. The residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 1 / 3) to give L20 (550 mg) as a colorless oil. m / z: [M+H] + 569.2。

[0602] Step 6: Under ice-bath conditions, to a solution of L20 (200 mg, 0.35 mmol) in acetonitrile (20 mL) were added cesium iodide (164 mg, 0.63 mmol) and boron trifluoride diethyl etherate (1396 mg, 0.98 mmol). The reaction mixture was stirred at room temperature for 48 h and then concentrated directly under reduced pressure. The residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 1 / 1) to give Linker-18 (140 mg) as a white solid. m / z: [M+H] + 679.0。

[0603] Synthesis of Linker-20

[0604]

[0605] Step 1: Add lead tetraacetate (17.5 g, 39.5 mmol) to a mixed solution of Fmoc-Gly-Gly-OH (10 g, 28.2 mmol) in tetrahydrofuran (75 mL) and toluene (25 mL). The reaction mixture was stirred at 85 °C for 2 hours, cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 9 / 1 - 2 / 3) to obtain L21 (9.5 g) as a white solid. m / z: [M+Na] + 391.2.

[0606] Step 2&3: Add trimethylchlorosilane (148 mg, 1.36 mmol) to a solution of L21 (500 mg, 1.36 mmol) in dichloromethane (10 mL). The reaction mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure to obtain L22. Dissolve L22 in acetonitrile (10 mL), add 4-mercaptobutyric acid (160 mg, 1.36 mmol), and the reaction mixture was stirred at room temperature overnight and then directly purified by Flash column chromatography (C18, eluent: 0 - 85% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to obtain L23 (400 mg) as a white solid. m / z: [M+H] + 429.2.

[0607] Step 4: Under ice bath conditions, add triethylamine (100 mg, 1 mmol) and isobutyl chloroformate (140 mg, 1 mmol) to a solution of L23 (390 mg, 0.91 mmol) in dichloromethane (5 mL) in sequence. The reaction mixture was stirred at 0 °C for 1 hour, filtered through a short silica gel column, and the filtrate was concentrated under reduced pressure to obtain Linker-20 (380 mg).

[0608] Synthesis of Linker-21

[0609]

[0610] Step 1: Under ice bath conditions, add EDCI (17.3 g, 90.3 mmol), HOBT (16.3 g, 120 mmol), DIPEA (15.56 g, 120 mmol) and 2-methoxyethylamine (5.43 g, 72.2 mmol) to a solution of 5-formyl-2-hydroxybenzoic acid (10 g, 60.2 mmol) in dichloromethane (100 mL) in sequence. The reaction mixture was stirred at room temperature for 12 hours and directly purified by Flash column chromatography (C18, eluent: 0 - 65% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to obtain L24 (7.9 g). m / z: [M+H] + 224.2.

[0611] Step 2: To a solution of L24 (1.5 g, 6.72 mmol) in acetonitrile (80 mL) was added L4 (3.09 g, 8.06 mmol) and silver oxide (4.67 g, 20.2 mmol). The reaction system was stirred at room temperature for 12 h and then filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (dichloromethane / methanol = 20 / 1) to give L25 (2.7 g) as a brown foamy solid. m / z: [M+H] + 540.2

[0612] Step 3: Under ice-bath conditions, sodium borohydride (310 mg, 8.08 mmol) was added to a solution of L25 (2.18 g, 4.05 mmol) in ethanol (10 mL) and tetrahydrofuran (20 mL). The reaction mixture was stirred at 0 °C for 2 h, quenched by adding water, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, separated, and concentrated under reduced pressure. The residue was purified by Flash column chromatography (dichloromethane / methanol = 20 / 1) to give L26 (2.7 g) as a brown foamy solid. m / z: [M+H] + 542.2

[0613] Step 4: Using the synthesis method of Linker-1, Linker-21 was obtained by reacting with L26. m / z: [M+H] + 652.2

[0614] Synthesis of Linker-22

[0615]

[0616] Step 1: Under ice-bath conditions, EDCI (58 mg, 0.3 mmol), HOBT (43 mg, 0.32 mmol), and Val-Ala-PAB-OH (59 mg, 0.2 mmol) were successively added to a solution of 4-(3-p-toluenesulfonyl-2-(tosylmethyl)propanoyl)benzoic acid (100 mg, 0.2 mmol) in DMF (30 mL). The reaction mixture was stirred at room temperature for 1 h and directly purified by Flash column chromatography (C18, eluent: 0 - 75% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to give L27 (100 mg).

[0617] Step 2: Using the synthesis method of Linker-1, Linker-22 was obtained by reacting with L27. m / z: [M+H] + 886.0

[0618] Synthesis of Linker-23

[0619]

[0620]

[0621] Step 1: Palladium on carbon (70 mg, 10% wt) was added to a mixed solution of methanol (15 mL) and dichloromethane (5 mL) of Cbz-Gly-Gly-Phe-Gly-OtBu (350 mg, 0.66 mmol). The reaction system was purged with hydrogen three times and then stirred under a hydrogen atmosphere for 2 hours. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain L28 (250 mg) as a yellow oil. m / z: [M+H] + 393.2。

[0622] Steps 2&3: Using the synthesis method of Linker-12, L30 was synthesized from L28 and Linker-12. m / z: [M+H] + 884.4。

[0623] Step 4: Under ice bath conditions, N,N'-diisopropylcarbodiimide (56.8 mg, 0.45 mmol) was added to a solution of L30 (130 mg, 0.15 mmol) and N-hydroxysuccinimide (51.8 mg, 0.45 mmol) in DMF (2 mL). The reaction solution was stirred at room temperature overnight and directly purified by flash column chromatography (C18, eluent: 0% - 50% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) to obtain Linker-23 (60 mg) as a yellow solid. m / z: [M+H] + 982.4。

[0624] Synthesis of Linker-24

[0625]

[0626] Step 1: Under ice bath conditions, 33% hydrobromic acid acetic acid solution (15 mL) was added dropwise to a solution of β-D-galactose pentaacetate (6.20 g, 15.9 mmol) in dichloromethane (60 mL). The reaction system was stirred at room temperature for 2 hours. Then the reaction mixture was poured into ice water (300 mL), extracted with ice-cold dichloromethane, the organic phases were combined, washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain L31 (6.53 g) as a white solid. m / z: [M+Na] + 433.0。

[0627] Step 2: Silver oxide (13.1 g, 56.4 mmol) was added to a solution of L31 (6.53 g, 15.9 mmol) and 4-hydroxy-3-nitrobenzaldehyde (2.92 g, 17.5 mmol) in acetonitrile (100 mL). The reaction system was stirred overnight in the dark at room temperature. Then the reaction mixture was filtered through diatomaceous earth and concentrated under reduced pressure. The residue was purified by Flash column chromatography (dichloromethane / methanol = 20 / 1) to obtain L32 (7.78 g) as a white solid. m / z: [M+Na] + 520.2。

[0628] Step 3: L32 (2 g, 4.02 mmol) was dissolved in ethyl acetate (100 mL) solution, then 10% palladium on carbon (0.50 g) was added, and a drop of triethylamine was added dropwise. The reaction system was stirred overnight under a hydrogen balloon. Then the reaction mixture was filtered through diatomaceous earth and concentrated under reduced pressure to obtain L33 (1.88 g) as a white solid. m / z: [M+H] + 470.2。

[0629] Step 4: Under ice bath conditions, EDCI (0.61 g, 3.18 mmol) and HOBT (0.43 g, 3.18 mmol) were added to a solution of L33 (1 g, 2.13 mmol) in DMF (10 mL). Then a solution of 4-maleimidophenylacetic acid (0.49 g, 2.12 mmol) in DMF (2 mL) was slowly added dropwise to the above mixture. The reaction system was stirred overnight at room temperature. The reaction solution was directly purified by Flash column chromatography (C18, eluent: 0% - 52% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) to obtain L34 (442 mg) as a yellow solid. m / z: [M+Na] + 705.2。

[0630] Step 5: Under ice bath conditions, boron trifluoride diethyl etherate (82.3 mg, 0.58 mmol) was added dropwise to a solution of L34 (200 mg, 0.29 mmol) and cesium iodide (151 mg, 0.58 mmol) in acetonitrile (5 mL). The reaction system was stirred at room temperature for 2 hours. Then the reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by prep-TLC (petroleum ether / ethyl acetate = 1 / 1) to obtain Linker-24 (144 mg) as a yellow solid. m / z: [M+Na] + 815.1。

[0631] Synthesis of Linker-26

[0632]

[0633] Step 1: Under ice bath conditions, imidazole (140 mg, 2.11 mmol) and TBSCl (530 mg, 3.52 mmol) were added to a solution of L6 (800 mg, 1.76 mmol) in dichloromethane (10 mL). The reaction mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure. The residue was purified by Flash column chromatography (C18, eluent: 0% - 85% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain L35 (920 mg). m / z: [M+H] + 570.2.

[0634] Step 2: Under ice bath conditions, HATU (210 mg, 0.56 mmol) and DIPEA (110 mg, 0.86 mmol) were added to a solution of 6-((tert-butoxycarbonyl)amino)hexanoic acid (100 mg, 0.43 mmol) in DMF (3 mL). After the reaction mixture was stirred at 0 °C for 5 minutes, L35 (240 mg, 0.43 mmol) was added to the above system. The reaction system was continuously stirred at room temperature for 12 hours and then directly purified by Flash column chromatography (C18, eluent: 0 - 85% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain L36 (300 mg). m / z: [M+H] + 783.3.

[0635] Step 3: A mixed solution of L36 (165 mg, 0.21 mmol) in dichloromethane (2 mL) and trifluoroacetic acid (1.5 mmol) was stirred at room temperature for 1.5 hours and concentrated under reduced pressure. The residue was purified by Flash column chromatography (C18, eluent: 0 - 65% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain L37 (115 mg). m / z: [M+H] + 569.2.

[0636] Steps 4 & 5: Using the synthetic method of Linker-22, 4-(3-p-toluenesulfonyl-2-(tosylmethyl)propanoyl)benzoic acid was reacted with L37 to obtain Linker-26. m / z: [M+H] + 1161.2.

[0637] Synthesis of Linker-27

[0638]

[0639] Step 1: Sodium hydride (60%, 8.61 g, 215 mmol) was added to a solution of N,N'-diacetyldiaminomethane (20 g, 154 mmol) in anhydrous tetrahydrofuran (1 L). The reaction mixture was stirred at room temperature for 10 minutes, and 3-chloro-2-(chloromethyl)propene (20.2 g, 161 mmol) was added dropwise to the above reaction mixture. The reaction mixture was stirred under reflux for 48 hours, cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (methanol / dichloromethane = 50 / 1) to obtain L39 (12 g) as a yellow oil. m / z: [M+H] + 183.3.

[0640] Step 2: Sodium hydroxide (4.39 g, 110 mmol) was added to a suspension of L39 (4 g, 22.0 mmol) in water (20 mL). The reaction system was stirred at 110 °C overnight. After cooling the reaction system in an ice bath, solid sodium hydroxide (1.4 g) was added, and the aqueous phase was extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product of L40 (4.3 g), which was directly used in the next step.

[0641] Step 3: Under ice bath conditions, HOBT (11.8 g, 87.6 mmol), EDCI (16.8 g, 87.6 mmol), diethylphosphonoacetic acid (14 g, 71.4 mmol), and DIPEA (14.2 g, 110 mmol) were successively added to a solution of L40 (2.15 g, 21.9 mmol) in dichloromethane (40 mL). After the reaction mixture was slowly warmed to room temperature and stirred overnight, it was diluted with dichloromethane (20 mL). The organic phase was washed with water, separated, and concentrated under reduced pressure. The residue was purified by Flash column chromatography (C18, eluent: 0% - 60% acetonitrile in 0.05% aqueous trifluoroacetic acid) to obtain L41 (3.5 g) as a colorless oil. m / z: [M+H] + 455.2.

[0642] Step 4: At -70 °C, ozone was introduced into a mixed solution of L41 (1 g, 2.2 mmol) in dichloromethane (20 mL) and methanol (5 mL), and the mixture was stirred for 10 minutes. Nitrogen was introduced into the reaction system to displace the excess ozone, and dimethyl sulfide (409 mg, 6.6 mmol) was added. After the reaction mixture was stirred and warmed to room temperature, it was concentrated under reduced pressure to obtain L42 (1 g) as a yellow oil. m / z: [M+H] + 457.2.

[0643] Step 5: To a solution of L42 (500 mg, 1.1 mmol) in methanol (5 mL) were successively added 3-(aminooxy)propanoic acid (220 mg, 2.09 mmol) and sodium acetate (451 mg, 5.5 mmol). The reaction mixture was stirred at room temperature overnight, quenched with water, and directly purified by Flash column chromatography (C18, eluent: 0% - 35% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) to obtain L43 (560 mg) as a pale yellow oil. m / z: [M+H] + 544.2。

[0644] Step 6: Under ice bath conditions, sodium hydride (60%, 36 mg, 0.9 mmol) was added to a solution of L43 (97 mg, 0.18 mmol) in anhydrous tetrahydrofuran (5 mL). The reaction mixture was stirred at 0 °C for 30 minutes, and a freshly prepared formaldehyde tetrahydrofuran solution (1.38 mL, 0.29 M) was added dropwise to the above reaction mixture. The resulting reaction mixture was stirred at room temperature for 10 minutes, quenched with water, and directly purified by Flash column chromatography (C18, eluent: 0.05% trifluoroacetic acid aqueous solution, 1 minute) to obtain Linker-27 (133 mg) as a yellow oil. m / z: [M+H] + 296.2。

[0645] Synthesis of Linker-32

[0646]

[0647] Step 1: Under ice bath conditions, TBSCl (390 mg, 2.56 mmol) and imidazole (190 mg, 2.77 mmol) were added to a solution of L33 (1 g, 2.13 mmol) in dichloromethane (10 mL). The reaction mixture was stirred at room temperature for 2 hours, quenched with water, the aqueous phase was extracted with dichloromethane, the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (ethyl acetate / petroleum ether = 1 / 3) to obtain L45 (900 mg) as an off-white solid. m / z: [M+H] + 584.2。

[0648] Step 2: Under ice bath conditions, Linker-27 (124 mg, 0.21 mmol), DIPEA (54 mg, 0.42 mmol), and HATU (120 mg, 0.32 mmol) were successively added to a solution of L45 (120 mg, 0.21 mmol) in DMF (3 mL). The reaction mixture was stirred at room temperature for 2 days and directly purified by Flash column chromatography (C18, eluent: 0% - 75% acetonitrile in 10 mM ammonium bicarbonate aqueous solution) to obtain L46 (10 mg) as an off-white solid. m / z: [M+Na]+ 883.4。

[0649] Step 3: Under ice bath conditions, triethylamine trihydrofluoride (10 mg, 0.06 mmol) was added dropwise to a solution of L46 (10 mg, 0.012 mmol) in tetrahydrofuran (1 mL). The reaction mixture was stirred overnight at room temperature, quenched with water, the aqueous phase was extracted with ethyl acetate, the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain L47 (9 mg) as an off-white solid. m / z: [M+Na] + 769.2。

[0650] Step 4: Under ice bath conditions, thionyl chloride (3 mg, 0.024 mmol) was added dropwise to a solution of L47 (9 mg, 0.012 mmol) in dichloromethane (2 mL). The reaction mixture was stirred at room temperature for 2 hours and directly concentrated under reduced pressure to obtain Linker-32 (9 mg) as a yellow solid. m / z: [M+Na] + 787.2。

[0651] Synthesis of Linker-33

[0652]

[0653] Step 1: Acetyl bromo-α-D-glucose (1.85 g, 4.5 mmol) and silver oxide (3.13 g, 13.5 mmol) were added to a solution of tert-butyl 5-formylsalicylate (1 g, 4.50 mmol) in acetonitrile (40 mL). The reaction system was stirred at room temperature for 12 hours, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by Flash column chromatography (methanol / dichloromethane = 0%-5%) to obtain L48 (2.2 g) as a light yellow solid. m / z: [M+Na] + 575.2。

[0654] Step 2: A mixed solution of L48 (2.2 g, 3.98 mmol) in dichloromethane (10 mL) and trifluoroacetic acid (2.5 mL) was stirred at room temperature for 1 hour. The reaction mixture was diluted with dichloromethane, and the pH was adjusted to 6.0 - 6.5 by slowly adding saturated aqueous sodium bicarbonate. The aqueous phase was extracted with ethyl acetate, the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain L49 (1.5 g) as a light yellow solid. m / z: [M+Na] + 519.2。

[0655] Step 3: Under ice bath conditions, sodium borohydride (110 mg, 3.02 mmol) was added portionwise to a mixed solution of L49 (1.5 g, 3.02 mmol) in tetrahydrofuran (5 mL) and ethanol (1 mL). The reaction system was stirred at room temperature for 3 hours. Acetone was added to quench the reaction, and the mixture was filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by Flash column chromatography (C18, 0%-50% acetonitrile in 0.1% aqueous trifluoroacetic acid) to obtain L50 (1.3 g) as a white solid. m / z: [M+Na] + 521.2。

[0656] Step 4: Under ice bath conditions, thionyl chloride (240 mg, 2 mmol) was added to a solution of L50 (500 mg, 1 mmol) in dichloromethane (2 mL). The reaction solution was stirred at room temperature overnight. It was concentrated under reduced pressure, dichloromethane (5 mL) was added to the residue, and it was concentrated under reduced pressure again. The operation was repeated twice to obtain L51 (500 mg) as a white solid. m / z: [M+Na] + 539.0。

[0657] Step 5: To a solution of L51 (260 mg, 0.5 mmol) in methanol (5 mL) was added (69.1 mg, 0.5 mmol). The reaction system was stirred at room temperature for 0.5 hour. After the reaction solution was concentrated under reduced pressure, it was directly purified by Flash column chromatography (C18, 0%-60% acetonitrile in 0.1% aqueous trifluoroacetic acid) to obtain Linker-33 (80 mg) as a white solid. m / z: [M+Na] + 371.0。

[0658] Synthesis of Linker-34

[0659]

[0660] Synthesis of L52: Using the synthesis method of Linker-27, 3-(aminooxy)propanoic acid in Step 5 was replaced with tert-butyl (2-(aminooxy)ethyl)carbamate to react to obtain L52. m / z: [M+Na] + 389.2。

[0661] Under ice bath conditions, trifluoroacetic acid (1 mL) was added to a solution of L52 (1 g, 2.73 mmol) in dichloromethane (9 mL). The reaction solution was stirred at room temperature for 1 hour and then directly concentrated under reduced pressure. The residue was purified by Flash column chromatography (C18, 0%-60% acetonitrile in 10 mM aqueous ammonium bicarbonate) to obtain Linker-34 (400 mg) as a colorless oil. m / z: [M+H] + 267.2。

[0662] Synthesis of Linker-35

[0663]

[0664]

[0665] Step 1: Under ice bath conditions, to a solution of L53 (prepared by the synthetic method of L33, reacted with 1,2,3,4,6-penta-O-acetyl-5-thio-D-glucopyranose to obtain L53) (400 mg, 0.82 mmol) in dichloromethane (40 mL), tert-butyldimethylsilyl chloride (TBSCl) (160 mg, 1.07 mmol) and imidazole (150 mg, 2.13 mmol) were successively added. After the reaction mixture was stirred at room temperature for 2 hours, the reaction was quenched with methanol, concentrated under reduced pressure at low temperature, and the residue was purified by flash column chromatography (C18, 0%-95% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain L54 (470 mg) as a white solid. m / z: [M+H] + 600.2。

[0666] Step 2: Under ice bath conditions, to a solution of L54 (300 mg, 0.5 mmol) in DMF (2 mL), 2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetyl chloride (153 mg, 0.6 mmol) and pyridine (79 mg, 1 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours and directly purified by flash column chromatography (C18, 0%-85% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain L55 (400 mg) as a white solid. m / z: [M+H] + 813.2。

[0667] Step 3: Under ice bath conditions, hydrofluoric acid triethylamine (0.25 mL, 1.55 mmol) was added dropwise to a solution of L55 (390 mg, 0.31 mmol) in tetrahydrofuran (5 mL). The reaction mixture was stirred at room temperature for 2.5 hours and directly purified by flash column chromatography (C18, 0%-49% acetonitrile in 0.05% aqueous trifluoroacetic acid solution) to obtain L56 (210 mg) as a yellow solid. m / z: [M+H] + 699.2。

[0668] Step 4: Under ice bath conditions, thionyl chloride (26 mg, 0.22 mmol) was added dropwise to a solution of L56 (50 mg, 0.07 mmol) in dichloromethane (10 mL). The reaction mixture was stirred at room temperature for 3 hours, and after concentration under reduced pressure, Linker-35 (50 mg) was obtained as a yellow solid. m / z: [M+Na] + 739.2。

[0669] Synthesis of Linker-38

[0670]

[0671]

[0672] Step 1: Under ice bath conditions, imidazole (90 mg, 1.32 mmol) and TBSCl (330 mg, 2.2 mmol) were successively added to a solution of L6 (500 mg, 1.1 mmol) in dichloromethane (10 mL). The reaction mixture was stirred at room temperature for 1 hour, and the solvent was removed by concentration under reduced pressure. The residue was purified by flash column chromatography (C18, 90% acetonitrile in 10 mM aqueous ammonium bicarbonate) to obtain L57 (600 mg) as a white solid. m / z: [M+H] + 570.2。

[0673] Step 2: Under ice bath conditions, HATU (280 mg, 0.74 mmol), DIPEA (160 mg, 1.24 mmol) and L57 (390 mg, 0.68 mmol) were successively added to a solution of L57 (200 mg, 0.62 mmol) in DMF (8 mL). The reaction mixture was stirred at room temperature for 2 hours and then directly purified by flash column chromatography (C18, 95% acetonitrile in 10 mM aqueous ammonium bicarbonate) to obtain L58 (500 mg) as a white solid. m / z: [M+Na] + 897.3。

[0674] Step 3: Palladium on carbon (10%, 355 mg) was added to a solution of L58 (350 mg, 0.4 mmol) in ethyl acetate (5 mL). After the reaction system was purged with hydrogen, it was stirred at room temperature under a hydrogen atmosphere for 2 hours. The reaction system was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain L59 (300 mg) as a white solid. m / z: [M+H] + 785.0。

[0675] Step 4: Under ice bath conditions, EDCI (110 mg, 0.58 mmol), HOBT (78 mg, 0.58 mmol), DIPEA (75 mg, 0.58 mmol) and 2,5,8,11,14,17 - hexaoxanonadecane - 19 - amine (86 mg, 0.29 mmol) were successively added to a solution of L59 (230 mg, 0.29 mmol) in DMF (10 mL). The reaction mixture was stirred at room temperature for 2 hours and then directly purified by flash column chromatography (C18, 95% acetonitrile in 10 mM aqueous ammonium bicarbonate) to obtain L60 (300 mg) as a colorless oil. m / z: [M+H] + 1062.5。

[0676] Steps 5 & 6: Using the synthetic method of Linker-6, Linker-38 was synthesized from L60 and 2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetic acid. m / z: [M+H] + 1171.1

[0677] Synthesis of Linker-39

[0678]

[0679] Step 1: Potassium carbonate (0.36 g, 2.58 mmol) was added to a solution of compound L45 (500 mg, 0.86 mmol) in methanol (15 mL). The reaction system was stirred at room temperature for 1 hour, the pH was adjusted to 6 - 7 with acetic acid, and after concentration under reduced pressure at low temperature, it was directly purified by flash column chromatography (C18, eluent: 10% - 90% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain L63 (330 mg) as a white solid. m / z: [M+H] + 416.3

[0680] Step 2: Under ice bath conditions, 2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetic acid, HOBT (100 mg, 0.77 mmol), and EDCI (150 mg, 0.77 mmol) were added to a solution of L63 (180 mg, 0.43 mmol) in DMF (5 mL). The reaction solution was stirred at room temperature for 2 hours and then directly purified by flash column chromatography (C18, eluent: 0% - 85% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain L64 (270 mg) as a white solid. m / z: [M+Na] + 651.2

[0681] Step 3: Under ice bath conditions, triethylamine hydrofluoride (150 mg, 0.96 mmol) was added to a solution of L64 (200 mg, 0.32 mmol) in tetrahydrofuran (5 mL). The reaction solution was stirred at room temperature for 2 hours and then directly purified by flash column chromatography (C18, eluent: 0% - 75% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain L65 (160 mg) as a white solid. m / z: [M+H] + 515.3

[0682] Step 4: Thionyl chloride (21 mg, 0.17 mmol) and one drop of DIPEA were added to a solution of L65 (30 mg, 0.058 mmol) in dichloromethane (3 mL). The reaction solution was stirred at room temperature for 12 hours, and after concentration under reduced pressure, Linker-39 (33 mg) was obtained as a pale yellow oil. m / z: [M+H]+ 579.0。

[0683] Synthesis of Linker-40

[0684]

[0685] Step 1: To a solution of L35 (1.33 g, 2.34 mmol) and (S)-2-((tert-butoxycarbonyl)amino)penta-4-ynoic acid (500 mg, 2.34 mmol) in DMF (6 mL) were successively added HATU (1.16 g, 3.04 mmol) and DIPEA (756 mg, 5.85 mmol). The reaction mixture was stirred at room temperature for 5 h, quenched by adding water, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1) to give L66 (1.4 g) as a white solid. m / z: [M+Na] + 787.2。

[0686] Step 2: Under nitrogen protection, sodium ascorbate (166 mg, 0.84 mmol), copper(I) iodide (80 mg, 0.42 mmol) and DIPEA (54 mg, 0.42 mmol) were added to a solution of L66 (320 mg, 0.42 mmol) and 19-azido-2,5,8,11,14,17-hexaoxanonadecane (148 mg, 0.46 mmol) in DMF (2 mL). The reaction mixture was stirred at 60 °C for 2 h, cooled to room temperature, quenched by adding water, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined and concentrated under reduced pressure. The residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 1 / 1) to give L67 (400 mg) as a white solid. m / z: [M+H] + 1086.5。

[0687] Step 3: Trifluoroacetic acid (2 mL) was added to a solution of L67 (347 mg, 0.32 mmol) in dichloromethane (4 mL). The reaction mixture was stirred at room temperature for 2 h and then concentrated directly under reduced pressure. The residue was purified by prep-HPLC (mobile phase A: 0.1% aqueous trifluoroacetic acid, mobile phase B: acetonitrile; gradient elution of mobile phase B from 10% to 60%, elution time: 25 min) to give L68 (280 mg) as a pale yellow solid. m / z: [M+H] + 872.4。

[0688] Step 4: Under ice bath conditions, HATU (105 mg, 0.28 mmol) and DIPEA (60 mg, 0.46 mmol) were added to a solution of L68 (200 mg, 0.23 mmol) and 4-maleimidophenylacetic acid (53 mg, 0.23 mmol) in DMF (2 mL). After the reaction mixture was stirred at 0 °C for 2 h, it was directly purified by prep-HPLC (mobile phase A: 0.1% aqueous trifluoroacetic acid solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 10% to 60%, elution time: 20 min) to obtain L69 (300 mg) as a yellow solid. m / z: [1 / 2M+H] + 543.2。

[0689] Step 5: Under ice bath conditions, thionyl chloride (26 mg, 0.22 mmol) was added to a solution of L69 (60 mg, 0.06 mmol) in dichloromethane (2 mL). The reaction mixture was slowly warmed to room temperature and stirred for an additional 1 h. It was concentrated under reduced pressure to obtain Linker-40 (60 mg) as a yellow solid. m / z: [M+H] + 1103.4。

[0690] Synthesis of Linker-41 to Linker-44

[0691]

[0692] Step 1: To a solution of CTC resin (2 g, 2.2 mmol) in dichloromethane (20 mL) were added Fmoc-sarcosine (1.03 g, 3.30 mmol) and N,N-diisopropylethylamine (2.13 g, 16.5 mmol). After the reaction mixture was shaken on a shaker for 2 h, it was washed 4 times with DMF and DCM, and 2 times with methanol (10 min each time). The resin was washed 2 times with a 20% diethylamine / DMF mixed solution (once every 15 min), 4 times with DMF and DCM, and then DMF (30 mL) was added. Then, Fmoc-sarcosine (1.34 g, 4.32 mmol), HATU (1.67 g, 4.4 mmol) and DIPEA (1.4 g, 10.8 mmol) were added in sequence. The reaction mixture was shaken on a shaker for 2 h and washed 4 times with DMF and DCM. The resin was washed 2 times with a 20% diethylamine / DMF mixed solution (once every 15 min), 4 times with DMF and DCM, and then dried under reduced pressure. The above steps were repeated until the CTC resin-poly(sarcosine) with the target i value was obtained.

[0693] Step 2: Add bromoacetic acid (2.45 g, 17.6 mmol) and N,N'-diisopropylcarbodiimide (3.6 g, 28.6 mmol) to the CTC resin - poly(sarcosine) DMF (30 mL) solution obtained in Step 1. The reaction solution is shaken on a shaker for 1 hour and washed 4 times with DMF to obtain CTC resin - poly(sarcosine) - 2 - bromoacetamide.

[0694] Step 3: Add 2 - azidoethylamine (280 mg, 3.3 mmol) and N,N'-diisopropylcarbodiimide (430 mg, 3.3 mmol) to the CTC resin - poly(sarcosine) - 2 - bromoacetamide DMF (20 mL) solution. After the reaction solution is shaken for 2 hours, it is washed 4 times with DMF and DCM, and then the resin is dissociated with a 1% TFA / DMF mixed solution (2 times, 5 minutes each time). The solution is concentrated under reduced pressure. The residue is directly purified by prep - HPLC (mobile phase A: 0.1% aqueous trifluoroacetic acid solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 10% to 35%, elution time: 20 minutes) to obtain Linker41 - Linker44, which are white solids.

[0695] Linker - 41, i = 6, m / z: [M + H] + 571.4.

[0696] Linker - 42, i = 12, m / z: [M + H] + 997.6.

[0697] Linker - 43, i = 18, m / z: [1 / 2M + H] + 712.4.

[0698] Linker - 44, i = 24, m / z: [1 / 2M + H] + 925.5.

[0699] Synthesis of Linker - 47

[0700]

[0701] Step 1: Add HATU (129 mg, 0.34 mmol) and DIPEA (67 mg, 0.52 mmol) to a solution of L45 (150 mg, 0.26 mmol) and Boc-glycine (46 mg, 0.26 mmol) in DMF (3 mL). Stir the reaction mixture at room temperature for 3 hours, and directly perform prep-HPLC (mobile phase A: 0.1% aqueous trifluoroacetic acid solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 10% to 95%, elution time: 20 minutes). Add 0.1% aqueous trifluoroacetic acid solution (20 mL) to the collected eluate, stir at room temperature, and after monitoring the reaction to completion by LCMS, directly lyophilize the reaction mixture to obtain L70 (95 mg) as a white solid. m / z: [M+Na] + 649.2.

[0702] Step 2: Under ice bath conditions, add thionyl chloride (36 mg, 0.3 mmol) to a solution of L70 (95 mg, 0.15 mmol) in dichloromethane (2 mL). Slowly warm the reaction mixture to room temperature and continue stirring for 1 hour. Concentrate under reduced pressure to obtain Linker-47 (100 mg) as a white solid. m / z: [M+Na] + 667.2.

[0703] Synthesize the Linkers shown in Table 1 using the above synthesis method:

[0704] Table 1

[0705]

[0706]

[0707]

[0708] Synthesis of tert-butyl (chloromethyl)ethane-1,2-diyl bis(methylcarbamate)

[0709]

[0710] Under ice bath conditions, add pyridine (1.14 g, 14.4 mmol) and chloroformyl chloride (1.48 g, 11.5 mmol) to a solution of tert-butyl methyl(2-(methylamino)ethyl)carbamate (1.8 g, 9.56 mmol) in dichloromethane (20 mL). Slowly warm the reaction system to room temperature and continue stirring for 12 hours. Quench the reaction with water, extract the aqueous phase with dichloromethane, separate the organic phase and concentrate under reduced pressure. Purify the residue by Flash column chromatography (petroleum ether / ethyl acetate = 1 / 3) to obtain tert-butyl (chloromethyl)ethane-1,2-diyl bis(methylcarbamate) (2.3 g).

[0711] Synthesis of 4-((tert-Butoxycarbonyl)(methyl)amino)butane(isobutylcarbon)ic Anhydride

[0712]

[0713] Under ice-bath conditions, isobutyl chloroformate (230 mg, 1.70 mmol) was slowly added to a solution of 4-(tert-butylcarbonyl(methyl)amino)butyric acid (300 mg, 1.38 mmol) and triethylamine (170 mg, 1.70 mmol) in dichloromethane (7 mL). The reaction mixture was stirred at room temperature for 2 hours, concentrated under reduced pressure. The residue was dissolved in dichloromethane and filtered through a short silica gel column chromatography. The filtrate was concentrated under reduced pressure to obtain 4-((tert-butoxycarbonyl)(methyl)amino)butane(isobutylcarbon)ic anhydride (110 mg) as a colorless oil. m / z: [M+Na] + 340.2。

[0714] (S)-2-(((Chloromethoxy)carbonyl)(2-(dimethylamino)ethyl)amino)methyl)pyrrolidine-1-carboxylic Acid tert-Butyl Ester Synthesis

[0715]

[0716] Step 1: Under ice-bath conditions, N,N-dimethylethylenediamine (7 g, 79.4 mmol) and sodium triacetoxyborohydride (21 g, 99.3 mmol) were successively added to a solution of (S)-2-acetylpyrrolidine-1-carboxylic acid tert-butyl ester (15.8 g, 79.4 mmol) in dichloromethane (20 mL). The reaction system was slowly warmed to room temperature and stirred for an additional 3 hours. The reaction was quenched by adding water. The aqueous phase was extracted with dichloromethane. The combined organic phases were concentrated under reduced pressure. The residue was purified by Flash column chromatography (dichloromethane / methanol = 1 / 8) to obtain (S)-2-(((2-(dimethylamino)ethyl)amino)methyl)pyrrolidine-1-carboxylic acid tert-butyl ester (900 mg). m / z: [M+H] + 272.2。

[0717] Step 2: Under ice-bath conditions, pyridine (180 mg, 2.22 mmol) and chloroformyl chloride (190 mg, 1.44 mmol) were successively added to a solution of the product obtained in Step 1 (300 mg, 1.11 mmol) in dichloromethane (15 mL). The reaction mixture was slowly warmed to room temperature and stirred for 12 hours. The reaction was quenched by adding water (15 mL). The aqueous phase was extracted with dichloromethane. The combined organic phases were concentrated under reduced pressure. The residue was purified by Flash column chromatography (dichloromethane / methanol = 1 / 5) to obtain (S)-2-(((chloromethoxy)carbonyl)(2-(dimethylamino)ethyl)amino)methyl)pyrrolidine-1-carboxylic acid tert-butyl ester (110 mg). m / z: [M+H] + 364.2。

[0718] Synthesis of 4-((4-((tert-Butoxycarbonyl)amino)benzyl)thio)butane(isobutylcarbon)ic anhydride

[0719]

[0720] Step 1: Under ice bath conditions, boron trifluoride diethyl etherate (2.03 g, 14.3 mmol) was slowly added dropwise to a solution of tert-butyl (4-(hydroxymethyl)phenyl)carbamate (2 g, 8.96 mmol) and cesium carbonate (3.72 g, 14.3 mmol) in acetonitrile (40 mL). After the addition was complete, the reaction system was stirred at room temperature for 2 hours. A solution of 4-mercaptobutyric acid (1.08 g, 8.96 mmol) and DIPEA (3.47 g, 26.9 mmol) in acetonitrile (25 mL) was slowly added to the above reaction system. The reaction solution was continuously stirred at room temperature for 3 hours, concentrated under reduced pressure, and the residue was purified by Flash column chromatography (C18, eluent: 0 - 60% acetonitrile in 0.1% aqueous trifluoroacetic acid) to obtain 4-((4-((tert-butoxycarbonyl)amino)benzyl)oxy)butyric acid (860 mg). m / z: [M+Na] + 348.2。

[0721] Step 2: Under ice bath conditions, isobutyl chloroformate (134 mg, 0.98 mmol) was slowly added to a solution of the product obtained in Step 1 (350 mg, 1.08 mmol) and triethylamine (142 mg, 1.4 mmol) in dichloromethane (10 mL). The reaction solution was stirred at 0 °C for 1 hour and then directly concentrated under reduced pressure. Dichloromethane (3 mL) was added to the residue, and it was filtered through a short silica gel column chromatography. The filtrate was concentrated under reduced pressure to obtain 4-((4-((tert-butoxycarbonyl)amino)benzyl)thio)butane(isobutylcarbon)ic anhydride (500 mg) as a pale yellow oil. m / z: [M+Na] + 448.2。

[0722] Synthesis of 4-((4-((tert-Butoxycarbonyl)amino)benzyl)dithio)butyric acid

[0723]

[0724] Step 1: A mixed solution of benzyl p - nitro - bromide (7.56 g, 35 mmol) in toluene (60 mL) and tetrahydrofuran (30 mL) was slowly added dropwise to a solution of thioacetic acid (5.3 g, 70 mmol) and triethylamine (8.85 g, 87.5 mmol) in toluene (75 mL). After the addition was complete, the reaction mixture was stirred overnight at room temperature. It was concentrated under reduced pressure. The residue was redissolved in a small amount of ethyl acetate, and 10 volumes of n - heptane were added. A solid precipitated, which was filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 3 / 1) to obtain S-(4 - nitrophenyl) thioacetate (7.7 g) as a yellow solid. m / z: [M + H] + 212.0。

[0725] Step 2: S-(4 - nitrophenyl) thioacetate (2 g, 9.5 mmol), iron powder (2.6 g, 47.4 mmol) and ammonium chloride (2.53 g, 47.4 mmol) were added to ethanol (50 mL) and water (15 mL). The reaction mixture was stirred at 90 °C for 1.5 hours, filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (C18, eluent: 0 - 30% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to obtain S-(4 - aminophenyl) thioacetate (1.3 g) as a yellow solid. m / z: [M + H] + 182.0。

[0726] Step 3: A solution of S-(4 - ((tert - butoxycarbonyl)amino)phenyl) thioacetate (1.2 g, 6.62 mmol), di - tert - butyl dicarbonate (2.89 g, 13.2 mmol) and triethylamine (1.34 g, 13.2 mmol) in dichloromethane (20 mL) was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and the residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 3 / 1) to obtain S-(4 - ((tert - butoxycarbonyl)amino)phenyl) thioacetate (840 mg) as a white solid. m / z: [M + Na] + 304.2。

[0727] Step 4: Under ice - bath conditions, lithium hydroxide monohydrate (94 mg, 3.9 mmol) was added to a solution of S-(4 - ((tert - butoxycarbonyl)amino)phenyl) thioacetate (550 mg, 1.95 mmol) and 4 - mercaptobutyric acid (9 mg, 0.072 mmol) in ethanol (1 mL). The reaction mixture was stirred at 0 °C for 50 minutes and then directly concentrated under reduced pressure. The residue was purified by Flash column chromatography (C18, eluent: 0 - 70% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to obtain tert - butyl (4-(mercaptomethyl)phenyl)carbamate (430 mg) as a white solid. m / z: [M + Na] + 262.2。

[0728] Step 5: Slowly add a solution of tert-butyl (4-(mercaptomethyl)phenyl)carbamate (420 mg, 1.75 mmol) in acetonitrile (20 mL) to a solution of 4-(pyridin-2-yldithio)butyric acid (1 g, 4.38 mmol) in acetonitrile (10 mL) over 30 minutes. After the addition is complete, stir the reaction mixture at room temperature for 2 hours and then purify it directly by flash column chromatography (C18, eluent: 0 - 80% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to obtain 4-((4-((tert-butoxycarbonyl)amino)benzyl)dithio)butyric acid (480 mg) as a white solid. m / z: [M+Na] + 380.0。

[0729] Synthesis of 5-((4-(chloromethyl)phenoxy)methyl)-1-methyl-2-nitro-1H-imidazole

[0730]

[0731] Step 1: Stir a solution of (1-methyl-2-nitro-1H-imidazol-5-yl)methanol (400 mg, 2.55 mmol), 4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)phenol (930 mg, 4.47 mmol), triphenylphosphine (1.34 g, 5.1 mmol) and DBAD (1.17 g, 5.1 mmol) in toluene (10 mL) at 50 °C for 5 hours. Concentrate the reaction mixture under reduced pressure and purify the residue by flash column chromatography (ethyl acetate / petroleum ether = 1 / 3) to obtain 1-methyl-2-nitro-5-((4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)phenoxy)methyl)-1H-imidazole (600 mg, 60%). m / z: [M+Na] + 370.2。

[0732] Step 2: Add trifluoroacetic acid (1.5 mL) to a mixed solution of the product obtained in Step 1 (600 mg, 1.51 mmol) in tetrahydrofuran (2 mL) and methanol (2 mL). Stir the reaction mixture at room temperature overnight and then concentrate it directly under reduced pressure. Purify the residue by flash column chromatography (C18, eluent: 0 - 50% acetonitrile in 10 mM ammonium bicarbonate aqueous solution) to obtain (4-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)phenyl)methanol (260 mg). m / z: [M+H] + 264.2。

[0733] Step 3: Under ice bath conditions, thionyl chloride (90 mg, 0.076 mmol) was added to a mixed solution of the product obtained in Step 2 (20 mg, 0.076 mmol) in dichloromethane (5 mL). After the reaction solution was stirred at 0 °C for 1 hour, it was directly concentrated under reduced pressure to obtain 5-((4-(chloromethyl)phenoxy)methyl)-1-methyl-2-nitro-1H-imidazole (20 mg). m / z: [M+H] + 282.2.

[0734] (Synthesis of (1-methyl-2-nitro-1H-imidazol-5-yl)methyl (4-nitrophenyl) carbonate)

[0735]

[0736] (1-Methyl-2-nitro-1H-imidazol-5-yl)methanol (225 mg, 1.43 mmol), DIPEA (554 mg, 4.29 mmol) and a solution of bis(p-nitrophenyl) carbonate (870 mg, 2.86 mmol) in DMF (2 mL) were stirred at room temperature for 2 hours. The reaction solution was directly purified by Flash column chromatography (C18, 0%-65% acetonitrile in 0.05% aqueous trifluoroacetic acid) to obtain (1-methyl-2-nitro-1H-imidazol-5-yl)methyl (4-nitrophenyl) carbonate (350 mg) as a white solid. m / z: [M+H] + 323.0.

[0737] (Synthesis of 5-(chloromethyl)-1,4-dimethyl-2-nitro-1H-imidazole)

[0738]

[0739] Step 1: Under nitrogen protection, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (414 mg, 0.51 mmol) and cesium carbonate (1.10 g, 3.38 mmol) were added to a solution of compound 1.1 (400 mg, 1.69 mmol) in 1,4-dioxane (5 mL). After addition, the reaction system was purged with nitrogen three times, trimethylcyclotriboroxane (0.97 mL, 3.38 mmol) was added, and the reaction system was purged with nitrogen three times again. The reaction mixture was sealed and stirred at 100 °C for 3 hours. Then the reaction mixture was cooled to room temperature, filtered through diatomaceous earth, the filtrate was concentrated under reduced pressure, and the residue was purified by Flash column chromatography (C18, eluent: 0%-23% acetonitrile in 0.05% aqueous trifluoroacetic acid) to obtain (1,4-dimethyl-2-nitro-1H-imidazol-5-yl)methanol (80.7 mg) as a yellow solid. m / z: [M+H] + 172.2.

[0740] Step 2: Under ice bath conditions, DIPEA (51.7 mg, 0.40 mmol) was added to a solution of (1,4-dimethyl-2-nitro-1H-imidazol-5-yl)methanol (27.4 mg, 0.16 mmol) in tetrahydrofuran (2 mL), and then methanesulfonyl chloride (40.3 mg, 0.35 mmol) was added dropwise. The reaction system was stirred at room temperature for 1 hour. Then the reaction mixture was cooled to 0 °C, diluted with ethyl acetate (20 mL), washed with hydrochloric acid (1 M, 20 mL) and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at low temperature to obtain 5-(chloromethyl)-1,4-dimethyl-2-nitro-1H-imidazole (28.0 mg) as a yellow oil. m / z: [M+H] + 190.0。

[0741] (E)-4-((4-(Chloromethyl)phenyl)diazenyl)-2-hydroxybenzoic acid synthesis

[0742]

[0743] Step 1: Sodium nitrite (2.1 g, 30.5 mmol) and hydrogen chloride (6 g, 165 mmol) were added to an aqueous solution of (4-aminophenyl)methanol (100 mL). The reaction solution was stirred at 0 °C for 20 minutes, and the above reaction solution was added to an aqueous solution of 2-hydroxybenzoic acid (3.08 g, 22.3 mmol), sodium carbonate (3.23 g, 30.5 mmol) and sodium hydroxide (1.06 g, 26.4 mmol) in water (100 mL). The reaction system was continuously stirred at 0 °C for 1 hour, and the pH was adjusted to 3 - 4 with hydrochloric acid (1 M). The aqueous phase was extracted with ethyl acetate, and the combined organic phases were concentrated under reduced pressure. The residue was purified by Flash column chromatography (C18, eluent: 0% - 85% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to obtain (E)-2-hydroxy-4-((4-(hydroxymethyl)phenyl)diazenyl)benzoic acid (500 mg) as a yellow solid. m / z: [M+H] + 273.2。

[0744] Step 2: Under ice bath conditions, thionyl chloride (39 mg, 0.33 mmol) was added to a solution of (E)-2-hydroxy-4-((4-(hydroxymethyl)phenyl)diazenyl)benzoic acid (35 mg, 0.13 mmol) in dichloromethane (5 mL). The reaction solution was slowly warmed to room temperature and continued to stir for 1 hour, and concentrated under reduced pressure to obtain (E)-4-((4-(chloromethyl)phenyl)diazenyl)-2-hydroxybenzoic acid (36 mg) as a yellow solid. m / z: [M+H] + 291.0。

[0745] Synthesis of N-(5-(Chloromethyl)-2-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)phenyl)-2,5,8,11,14,17,20,23-oxahexacosane-26-amine

[0746]

[0747] Step 1: To a solution of 4-hydroxy-3-nitrobenzyl alcohol (2.0 g, 11.8 mmol) in dichloromethane (100 mL) were added 3,4-dihydro-2H-pyran (1.02 g, 12.2 mmol) and pyridinium p-toluenesulfonate (0.3 g, 1.18 mmol). The reaction system was stirred at room temperature for 16 h. Then it was washed with saturated aqueous sodium bicarbonate solution and water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 4 / 1) to obtain 2-nitro-4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)phenol (2.89 g) as a yellow oil. m / z: [M+Na] + 276.1.

[0748] Step 2: Under nitrogen protection, to a solution of the product from Step 1 (2.89 g, 11.4 mmol) in methanol (150 mL) was added palladium on carbon (10%, 364 mg). Then it was purged with hydrogen three times, and the reaction system was stirred at room temperature under a hydrogen atmosphere for 2 h. Then the reaction mixture was filtered through diatomaceous earth and concentrated under reduced pressure to obtain 2-amino-4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)phenol (2.54 g) as a grayish-black oil. m / z: [M+Na] + 246.2.

[0749] Step 3: Under ice bath conditions, to a solution of 3-methyl-2-nitroimidazole-4-methanol (1.0 g, 6.36 mmol), the product from Step 2 (1.70 g, 7.63 mmol), and triphenylphosphine (2.50 g, 9.54 mmol) in tetrahydrofuran (10 mL) was added di-tert-butyl azodicarboxylate (2.20 g, 9.54 mmol). The reaction system was stirred at room temperature for 2 h. The reaction solution was directly purified by Flash column chromatography (C18, eluent: 0% - 25% acetonitrile in 0.1% aqueous trifluoroacetic acid) to obtain 2-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)-5-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)aniline (0.51 g) as a yellow solid. m / z: [M+Na] + 385.2.

[0750] Step 4: Under ice bath conditions, trifluoroacetic acid (2 mL) was added dropwise to a solution of the product from Step 3 (510 mg, 1.41 mmol) in dichloromethane (6 mL). The reaction system was stirred at room temperature for 1 hour. After the reaction solution was concentrated under reduced pressure, it was purified by Flash column chromatography (C18, eluent: 0% - 42% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain (3-amino-4-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)phenyl)methanol (391 mg) as a yellow solid. m / z: [M+H] + 279.2。

[0751] Step 5: Under ice bath conditions, tert-butyldimethylsilyl trifluoromethanesulfonate (320 mg, 1.21 mmol) and imidazole (89.4 mg, 1.31 mmol) were added to a solution of the product from Step 4 (280 mg, 1.01 mmol) in dichloromethane (10 mL). The reaction system was stirred at room temperature for 10 minutes. Then, water was added for washing, the aqueous phase was extracted with dichloromethane, the combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 2 / 1) to obtain 5-(((tert-butyldimethylsilyl)oxy)methyl)-2-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)aniline (233 mg) as a yellow solid. m / z: [M+Na] + 415.2。

[0752] Steps 6 & 7: Under ice bath conditions, HATU (79.1 mg, 0.21 mmol) and DIPEA (41.4 mg, 0.32 mmol) were added to a solution of 4,7,10,13,16,19,22,25-octaoxahexacosanoic acid (65.8 mg, 0.16 mmol) in DMF (3 mL). The reaction system was stirred at 0 °C for 5 minutes. The product from Step 5 (60 mg, 0.15 mmol) was added to the above reaction system, and the resulting mixture was slowly warmed to room temperature and stirred for an additional 16 hours. The reaction solution was directly purified by Flash column chromatography (C18, eluent: 0% - 64% acetonitrile in 0.05% aqueous trifluoroacetic acid solution). The collected solution was stirred at room temperature for 4.5 hours and then lyophilized to obtain N-(5-(hydroxymethyl)-2-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)phenyl)-2,5,8,11,14,17,20,23-oxahexacosane-26-amine (88.7 mg) as a yellow oil. m / z: [M+H] + 673.4。

[0753] Step 8: Under ice bath conditions, thionyl chloride (7.6 mg, 0.064 mmol) was added dropwise to a solution of the product from Step 7 (21.7 mg, 0.032 mmol) in dichloromethane (3 mL), and the reaction system was stirred at 0 °C for 1 hour. The reaction solution was concentrated under reduced pressure at low temperature to obtain N-(5-(chloromethyl)-2-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)phenyl)-2,5,8,11,14,17,20,23-oxahexacosane-26-amine (22.3 mg) as a yellow oil. m / z: [M+H] + 691.2

[0754] (2R,3R,4R,5R,6R)-5-Acetamido-2-(acetoxymethyl)-6-azidotetrahydro-2H-pyran-3,4-diyl diacetate synthesis

[0755]

[0756] Under nitrogen protection, iron(III) chloride (0.08 g, 0.51 mmol) and trimethylsilyl azide (0.44 g, 3.85 mmol) were added to a solution of β-D-galactosamine pentaacetate (1.0 g, 2.57 mmol) in dichloromethane (10 mL). After addition, the reaction system was purged with nitrogen three times and then stirred at room temperature for 72 hours. The reaction mixture was washed successively with saturated aqueous sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (C18, eluent: 0% - 39% acetonitrile in 0.02% aqueous trifluoroacetic acid) to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-azidotetrahydro-2H-pyran-3,4-diyl diacetate (0.52 g) as a white solid. m / z: [M+H] + 373.2

[0757] (2R,3R,4S,5R,6R)-2-(Acetoxymethyl)-6-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(azidomethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate synthesis and (2R,3S,4S,5R,6R)-2-(azidomethyl)-6-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triol synthesis

[0758]

[0759] Step 1: Under nitrogen protection, triphenylphosphine (15.3 g, 58.4 mmol) was added to a solution of trehalose (10 g, 29.2 mmol) in DMF (100 mL), and the reaction system was stirred at room temperature for 15 minutes. Under ice bath conditions, N-bromosuccinimide (6.76 g, 38 mmol) was added to the above solution, and the reaction system was stirred at 80 °C for 8 hours. Then the reaction mixture was cooled to room temperature, quenched with methanol, and the solvent was removed by concentration under reduced pressure. It was dissolved in water (100 mL), and the aqueous phase was back-extracted with dichloromethane. The aqueous phase was concentrated under reduced pressure to obtain (2S,3S,4S,5R,6R)-2-(bromomethyl)-6-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triol (11.8 g) as a yellow transparent solid. m / z: [M+Na] + 427.0。

[0760] Step 2: Acetic anhydride (37 mL, 395 mmol) was added dropwise to a solution of (2S,3S,4S,5R,6R)-2-(bromomethyl)-6-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triol (11.8 g, 29.2 mmol) in pyridine (110 mL), and the reaction system was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (100 mL), washed successively with saturated aqueous sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(bromomethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (17.4 g) as a yellow solid. m / z: [M+Na] + 721.2。

[0761] Step 3: Sodium azide (0.55 g, 8.45 mmol) was added to a solution of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(bromomethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (1.18 g, 1.69 mmol) in DMF (11.8 mL). The reaction system was stirred overnight at 55 °C. Then the reaction mixture was cooled to room temperature, diluted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Flash column chromatography (C18, eluent: 0% - 57% acetonitrile in 0.05% aqueous trifluoroacetic acid) to obtain (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(azidomethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (0.73 g) as a white solid. m / z: [M+Na] + 684.2。

[0762] Step 4: Under nitrogen protection, sodium methoxide (35 mg, 0.65 mmol) was added to a solution of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(azidomethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (430 mg, 0.65 mmol) in methanol (13 mL). The reaction system was stirred at room temperature for 20 hours. Then ion exchange resin AMBERLITE IR-120(H) was added to adjust the pH to neutral, the resin was filtered off, and the filtrate was concentrated under reduced pressure to obtain (2R,3S,4S,5R,6R)-2-(azidomethyl)-6-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triol (256 mg) as a pink solid. m / z: [M+Na] + 390.2。

[0763] Synthesis of 2-azido-N-((3R,4R,5R,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide

[0764]

[0765] Step 1: Under nitrogen protection, hexamethyldisilazane (5.2 mL, 24.9 mmol) was added portionwise to a solution of D-(+)-galactosamine hydrochloride (2.15 g, 9.97 mmol) in acetonitrile (20 mL). The reaction system was stirred at room temperature for 3 hours. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain (3R,4R,5S,6R)-2,4,5-tris((trimethylsilyl)oxy)-6-(((trimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3-amine (4.36 g) as a yellow oil. m / z: [M - TMS + H] + 396.2

[0766] Step 2: Under ice bath conditions, N-hydroxysuccinimide bromoacetate (3.32 g, 14.1 mmol) was added to a solution of (3R,4R,5S,6R)-2,4,5-tris((trimethylsilyl)oxy)-6-(((trimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3-amine (4.36 g, 9.32 mmol) in dichloromethane (20 mL). The reaction system was stirred at room temperature for 2 hours and then directly concentrated under reduced pressure. The residue was triturated with petroleum ether / ethyl acetate = 10 / 1 (22 mL), the solid was filtered off, and the filtrate was concentrated under reduced pressure to obtain 2-bromo-N-((3R,4R,5S,6R)-2,4,5-tris((trimethylsilyl)oxy)-6-(((trimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3-yl)acetamide (5 g) as a yellow semi-oily semi-solid. m / z: [M + H] + 588.2, 590.2

[0767] Step 3: Sodium azide (0.68 g, 10.5 mmol) was added to a solution of 2-bromo-N-((3R,4R,5S,6R)-2,4,5-tris((trimethylsilyl)oxy)-6-(((trimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3-yl)acetamide (5 g, 8.49 mmol) in DMF (15 mL). The reaction system was stirred at 20 °C overnight. Then the reaction mixture was diluted with ethyl acetate (20 mL), washed with a large amount of water, the aqueous phase was extracted with ethyl acetate again, and the combined organic phases were concentrated under reduced pressure to obtain 2-azido-N-((3R,4R,5S,6R)-2,4,5-tris((trimethylsilyl)oxy)-6-(((trimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3-yl)acetamide (3.9 g) as a yellow oil.

[0768] Step 4: Dowex 50wx8 cation exchange resin (1 g) was added to a methanol (15 mL) solution of 2-azido-N-((3R,4R,5S,6R)-2,4,5-tris((trimethylsilyl)oxy)-6-(((trimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3-yl)acetamide (3.9 g, 7.1 mmol), and the reaction system was stirred at room temperature for 2 hours. It was filtered through diatomaceous earth, the filter cake was rinsed with a small amount of methanol, the filtrates were combined and concentrated under reduced pressure, and the residue was slurried with ethanol (20 mL), and the solid was collected and dried to obtain 2-azido-N-((3R,4R,5R,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (1 g) as a white solid. m / z: [M+Na] + 285.2.

[0769] Synthesis of compounds:

[0770] Example 1: Synthesis of Compounds 1-1, 1-2, 1-3 and 1-4

[0771]

[0772] Step 1: Compound 1A (1.2 g, 2.49 mmol) and 1B (2.4 g, 2.74 mmol) were co-evaporated with acetonitrile (10 mL) twice respectively, and then dissolved in acetonitrile (5 mL) respectively for use; under nitrogen protection and in an ice bath, the acetonitrile solution of Compound 1B was slowly added to the acetonitrile solution of Compound 1A containing 4A molecular sieve. After the obtained mixture was stirred for 1 hour, DDTT (0.61 g, 2.99 mmol) was added to the above reaction system, and after stirring for another half an hour, the molecular sieve was filtered off, and the filtrate was concentrated under reduced pressure to obtain Compound 1C (1.3 g, crude product). m / z: [M+H] + 1289.2.

[0773] Step 2: Under nitrogen protection and in an ice-water bath, a dichloromethane solution of DCA (0.6 M, 9 ml, 5.39 mmol) was added to a dichloromethane (10.0 mL) solution of Compound 1C (1.0 g, 0.77 mmol). After the reaction system was stirred at room temperature for 0.5 hour, triethylsilane (0.5 ml) and pyridine (1.0 mL) were added, and it was concentrated under reduced pressure. The residue was purified by Flash column chromatography (0.1% NH4HCO3 / CH3CN = 0 - 50%) to obtain Compound 1D (510 mg, pyridine salt, crude product) as a white solid. m / z: [M+H] + 987.0.

[0774] Step 3: Compound 1D (500 mg, 0.51 mmol) and pyridine (2 mL) were co-azeotropically concentrated three times, then redissolved in pyridine (2 mL), and slowly added dropwise to a pyridine solution of DPCP (685 mg, 2.55 mmol) at -40 °C. Then, the mixture was stirred at -20 °C for 1 hour, and the reaction solution containing compound 1E was directly used for the next step reaction.

[0775] Step 4: Water (459 mg, 25.5 mmol) and 3H-1,2-benzodithiol-3-one (102 mg, 0.61 mmol) were directly added to the reaction solution of compound 1E prepared in Step 3. After stirring at room temperature for 20 minutes, the reaction solution was directly purified by Flash column chromatography (0.1% NH4HCO3 / CH3CN = 0 - 40%) to obtain compound 1F (200 mg) as a white solid. m / z: [M+H] + 1000.1.

[0776] Step 5: To a solution of compound 1F (180 mg, 0.18 mmol) in acetonitrile (2 mL), tert-butylamine (2 mL) was added. The resulting mixture was stirred at room temperature for 0.5 hour, concentrated under reduced pressure, and the residue was purified by Flash column chromatography (C18, eluent: 0 - 35% acetonitrile in 10 mM aqueous ammonium bicarbonate solution). First, compound 1G-1 (130 mg) was collected, and then compound 1G-2 (35 mg) was collected, both as white solids. m / z: [M+H] + 948.0.

[0777] Step 6: To a solution of compound 1G-1 (120 mg) in methanol (3 mL), ammonia water (3 mL) was added. The mixture was stirred in a sealed tube at 50 °C for 5 hours. After the reaction solution was cooled to room temperature, the reaction was quenched with acetic acid, and directly freeze-dried to obtain compound 1-A.

[0778] Compound 1-A was purified by prep-HPLC to obtain the following compounds, all of which are ammonium salts

[0779] 1-1: White solid, 10.6 mg, m / z: [M+H] + 740.0, HPLC-RT (Condition 2): 10.814 minutes, 1 1H NMR (400 MHz, D2O): δ 7.70 - 8.35 (m, 3H), 6.38 (d, J = 13.6 Hz, 1H), 6.02 - 6.12 (m, 2H), 5.27 - 5.54 (m, 2H), 4.35 - 4.49 (m, 4H), 3.95 - 4.11 (m, 3H), 3.48 (s, 3H); 31P NMR (162 MHz, D2O): δ 54.72, 54.22, 53.46; 19 F NMR (376 MHz, D2O): δ -201.09;

[0780] 1-2: White solid, 8.2 mg, m / z: [M+H] + 740.0, HPLC-RT (Condition 2): 11.380 min, 1 H NMR (400 MHz, D2O): δ 8.08 - 8.35 (m, 3H), 6.34 - 6.36 (m, 1H), 6.07 (d, J = 7.6 Hz, 1H), 5.73 - 5.85 (m, 1H), 4.88 - 5.00 (m, 2H), 4.47 (s, 3H), 4.02 - 4.17 (m, 4H), 3.50 (s, 3H); 31 P NMR (162 MHz, D2O): δ 54.94, 52.83, 51.54; 19 F NMR (376 MHz, D2O): δ -202.86;

[0781] 1-3: White solid, 8.2 mg, m / z: [M+H] + 740.0, HPLC-RT (Condition 2): 10.370 min, 1 H NMR (400 MHz, D2O): δ 8.36 (s, 2H), 8.12 (s, 1H),.61 (s, 1H), 6.11 (s, 1H), 5.79 (s, 1H), 5.39 (d, J = 51.6 Hz, 1H), 4.93 - 5.02 (m, H), 4.36 - 4.48 (m, 3H), 3.84 - 4.03 (m, 4H), 3.51 (s, 3H); 31 P NMR (162 MHz, DMSO-d6 + D2O): δ 55.759, 51.401; 19 F NMR (376 MHz, D2O): δ -202.92.

[0782] Using the synthesis method of compound 1-A, reacting compound 1G-2 to obtain compound 1-B, and purifying by prep-HPLC to obtain compound 1-4: ammonium salt, white solid, 5.2 mg, m / z: [M+H] + 740.0, HPLC-RT (Condition 1): 11.650 min, 11H NMR (400 MHz, D2O): δ 8.51 (s, 1H), 8.28 (s, 1H), 8.18 (s, 1H), 6.64 (s, 1H), 6.45 (d, J = 16.0 Hz, 1H), 5.68 - 5.82 (m, 1H), 5.26 - 5.43 (m, 2H), 4.07 - 4.57 (m, 7H), 3.41 (s, 3H); 31 31P NMR (162 MHz, D2O): δ 54.64, 52.83; 19 19F NMR (376 MHz, D2O): δ -201.55.

[0783] Single crystal X-ray diffraction experiment of compounds 1 - 3

[0784] 1. Single crystal cultivation: Compounds 1 - 3 (8 mg) were dissolved in a mixed solution of water, acetonitrile and tetrahydrofuran (1 / 8 / 1, 0.5 mL), and slowly crystallized by evaporation at room temperature. Single crystals were collected for single crystal diffraction testing.

[0785] 2. The test parameters are shown in Table 2 below:

[0786] Table 2

[0787]

[0788]

[0789] 3. Test results: The stereostructures of compounds 1 - 3 are as follows, and the absolute configurations of all chiral centers in their molecules are R configurations.

[0790]

[0791] Example 2: Synthesis of compound 1M and compound A

[0792]

[0793] Step 1: To a solution of compound 1H (2.5 g, 7.9 mmol) in DMF (12 mL) were successively added TBSCl (1.44 g, 10 mmol) and imidazole (1.62 g, 23.8 mmol). The reaction mixture was stirred at room temperature for 4 hours, quenched with water, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1) to obtain compound 1I (2.9 g, yield: 86%) as a white solid. m / z: [M + H] + 429.2.

[0794] Step 2: To a solution of Compound 1I (2.9 g, 6.8 mmol) in anhydrous acetonitrile (25 mL) was added PSI (3.92 g, 8.8 mmol) and DBU (1.34 g, 8.8 mmol). The reaction mixture was stirred at room temperature for half an hour and then directly concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1) to give Compound 1J (3 g, yield: 65%) as a white solid. m / z: [M+H] + 675.2。

[0795] Step 3: Under nitrogen protection, a solution of Compound IJ (3 g, 4.4 mmol), 1K (1.7 g, 4.4 g) and DBU (2 g, 13.2 mmol) in anhydrous acetonitrile (30 mL) was stirred at room temperature for 1 hour and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1 - 1 / 4) to give Compound 1L (2.7 g, yield: 69%) as a white solid.

[0796] Step 4: A solution of Compound 1L (2.7 g, 3.0 mmol) in hydrochloric acid - 1,4 - dioxane (40 mL, 2 M) was stirred at room temperature for 16 hours and then directly concentrated under reduced pressure. The residue was purified by Flash column chromatography (C18, eluent: 0 - 35% acetonitrile in 10 mM aqueous ammonium bicarbonate) to first collect Compound 1M (1.1 g, yield: 55%) as a pale yellow solid. m / z: [M+H] + 662.1。 31 P NMR (162 MHz, DMSO - d6 + D2O): δ55.847。

[0797]

[0798] To a solution of Compound 1 - 3 (300 mg, 0.41 mmol) and benzyl chloride (52 mg, 0.41 mmol) in DMF (1 mL) was added sodium iodide (3.6 mg, 0.024 mmol). The reaction mixture was stirred at room temperature for 24 hours and the reaction solution was directly purified by prep - HPLC (eluent: 10% - 55% acetonitrile in 0.1% aqueous ammonium bicarbonate) to give Compound A (50 mg, yield: 15%) as a white solid. m / z: [M+H] + 830.0; 31 P NMR (162 MHz, DMSO - d6 + D2O): δ53.113, 27.573。

[0799] Example 3: Synthesis of Compound 2

[0800]

[0801] Under ice bath conditions, 5-((4-(chloromethyl)phenoxy)methyl)-1-methyl-2-nitro-1H-imidazole (20 mg, 0.07 mmol) and sodium iodide (31 mg, 0.21 mmol) were successively added to a mixed solution of anhydrous acetonitrile (1 mL) and dimethylacetamide (1 mL) of compound 1-3 (62 mg, 0.084 mmol). After the reaction mixture was stirred at room temperature for 2 hours, it was directly purified by flash column chromatography (C18, eluent: 0-60% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain compound 2 (40 mg, yield: 58%) as a white solid. m / z: [1 / 2M+H] + 493.2。

[0802] Example 4: Synthesis of Compound 3 and Compound 4

[0803]

[0804] Step 1: Linker-21 (180 mg, 0.27 mmol) was added to a solution of compound 1-3 (200 mg, 0.27 mmol) in DMF (5 mL). The reaction mixture was stirred at room temperature for 2 hours and directly purified by flash column chromatography (C18, eluent: 0-70% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain 3 (135 mg, yield: 40%) as a white solid. m / z: [1 / 2M+H] + 632.2。

[0805] Step 2: Hydrochloric acid-1,4-dioxane solution (2 mL, 4 M) was added to a solution of compound 3 (80 mg, 0.063 mmol) in water (2 mL). The reaction mixture was stirred at 35 °C for 12 hours and directly purified by flash column chromatography (C18, eluent: 0-50% acetonitrile in 0.1% aqueous trifluoroacetic acid solution) to obtain compound 4 (50 mg, yield: 70%) as a white solid. m / z: [1 / 2M+H] + 562.2。

[0806] Example 5: Synthesis of Compound 5

[0807]

[0808] Using the synthesis method of compound 3, compound 5 was obtained by reacting compound 1-3 with iodomethyl pivalate. m / z: [M+H] + 853.9。

[0809] Example 6: Synthesis of Compound 6

[0810]

[0811] Synthesis method of compound 2: React compound 1-3 with 2-(chloromethyl)-5-nitrothiophene to obtain compound 6. m / z: [M+H] + 880.9

[0812] Example 7: Synthesis of compound 7

[0813]

[0814] Synthesis method of compound 2: React compound 1-3 with (E)-4-((4-(chloromethyl)phenyl)diazenyl)-2-hydroxybenzoic acid to obtain compound 7. m / z: [M+H] + 994.0

[0815] Example 8: Synthesis of I-1

[0816]

[0817] Dropwise add a solution of Linker-1 (5.9 mg, 0.01 mmol) in anhydrous DMF (0.3 mL) to a solution of compound 1-1 (10 mg, 0.01 mmol) in anhydrous DMF (2 mL). Stir the reaction mixture at 50 °C for 3 hours, and then directly purify the reaction solution by prep-HPLC (mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 15% to 50%, elution time: 20 minutes) to obtain I-1 (3.6 mg) as a white solid. UPLC RT = 6.003 minutes; m / z: [M+H] + 1208.2

[0818] Example 9: Synthesis of I-2

[0819]

[0820] Dropwise add a solution of Linker-1 (5.9 mg, 0.01 mmol) in anhydrous DMF (0.3 mL) to a solution of compound 1-3 (10 mg, 0.01 mmol) in anhydrous DMF (2 mL). Stir the reaction mixture at room temperature for 3 hours, and then directly purify the reaction solution by prep-HPLC (mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 15% to 65%, elution time: 25 minutes) to obtain I-2 (3.6 mg) as a white solid. UPLC RT = 6.609 minutes; m / z: [M+H] + 1208.1

[0821] Example 10: Synthesis of I-3

[0822]

[0823] A solution of Linker-2 (13.7 mg, 0.01 mmol) in anhydrous DMF (0.3 mL) was added dropwise to a solution of Compound 1-3 (15 mg, 0.02 mmol) in anhydrous DMF (2 mL). The reaction mixture was stirred at room temperature for 3 hours, and then the reaction solution was directly purified by prep-HPLC (mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 15% to 65%, elution time: 25 minutes) to obtain I-3 (8.9 mg) as a white solid. UPLC RT = 6.329 minutes; m / z: [M+H] + 1294.3. 31 P NMR (162 MHz, DMSO-d6 + D2O): δ 54.318, 27.733.

[0824] Example 11: Synthesis of I-4

[0825]

[0826] Linker-1 (17 mg, 0.029 mmol) was added to a solution of Compound 1-1 (10 mg, 0.013 mmol) in anhydrous DMF (2 mL). The reaction solution was stirred at 50 °C for 5 hours and directly purified by prep-HPLC (mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 15% to 85%, elution time: 25 minutes) to obtain I-4 (6.3 mg) as a white solid. UPLC RT = 6.893 minutes; m / z: [M+H] + 1677.8.

[0827] Example 12: Synthesis of I-5

[0828]

[0829] Linker-2 (340 mg, 0.50 mmol) was added to a solution of Compound 1-3 (150 mg, 0.20 mmol) in anhydrous DMF (3.5 mL). The reaction solution was stirred at room temperature for 2 hours and directly purified by Flash column chromatography (C18, eluent: 0 - 65% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain I-5 (140 mg) as a white solid. UPLC RT = 6.577 minutes; m / z: [1 / 2M+H] + 924.8.

[0830] Example 13: Synthesis of I-6

[0831]

[0832] Using the synthesis method of I-3, reacting compound 1-1 with Linker-2 to obtain I-6 as a white solid. HPLC RT = 13.448 minutes; m / z: [1 / 2M+H] + 647.8

[0833] Example 14: Synthesis of I-7

[0834]

[0835] Step 1: To a solution of compound 1-3 (140 mg, 0.19 mmol) in DMF (3 mL), sequentially add tert-butyl (chloromethyl)ethane-1,2-diyl bis(methylcarbamate) (69 mg, 0.25 mmol) and sodium iodide (290 mg, 1.9 mmol). The reaction system was stirred at room temperature for 3 hours and then directly purified by flash column chromatography (C18, eluent: 0 - 55% acetonitrile in 10 mM aqueous ammonium bicarbonate) to obtain compound 4A (110 mg, yield: 59%). m / z: [M+H] + 983.9

[0836] Step 2: Under ice bath conditions, trifluoroacetic acid (1 mL) was added dropwise to a solution of compound 4A (80 mg, 0.081 mmol) in dichloromethane (2 mL). The reaction solution was stirred at room temperature for 2 hours, concentrated under reduced pressure, and the residue was purified by flash column chromatography (C18, eluent: 0 - 50% acetonitrile in 10 mM aqueous ammonium bicarbonate) to obtain compound 4B (70 mg, yield 96%). m / z: [M+H] + 883.9

[0837] Step 3: To a solution of compound 4B (40 mg, 0.045 mmol) in DMF (2 mL), sequentially add Linker-3 (43 mg, 58 mmol) and DIPEA (15 mg, 0.11 mmol). The reaction solution was stirred at room temperature for 2 hours, concentrated under reduced pressure, and the residue was purified by flash column chromatography (C18, eluent: 0 - 50% acetonitrile in 10 mM aqueous ammonium bicarbonate) to obtain compound I-7 (55 mg, yield 82%) as a white solid. HPLC RT = 14.510 minutes; m / z: [1 / 2M+H] + 741.8

[0838] Example 15: Synthesis of I-8

[0839]

[0840] To a solution of Compound 1-3 (25 mg, 0.034 mmol) in anhydrous DMF (2 mL) was added Linker-5 (10 mg, 0.034 mmol). The reaction mixture was stirred at room temperature for 12 hours and purified directly by prep-HPLC (mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution with mobile phase B from 10% to 45%, elution time: 20 minutes) to obtain I-8 (3.2 mg) as a white solid. HPLC RT = 14.350 minutes; m / z: [M+H] + 919.1。

[0841] Example 16: Synthesis of I-9

[0842]

[0843] To a solution of Compound 1-3 (20 mg, 0.027 mmol) in anhydrous DMF (3 mL) was added Linker-6 (18 mg, 0.041 mmol). The reaction mixture was stirred at room temperature for 2 hours and purified directly by prep-HPLC (mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution with mobile phase B from 5% to 65%, elution time: 20 minutes) to obtain I-9 (3.2 mg) as a white solid. HPLC RT = 14.622 minutes; m / z: [M+H] + 1064.0。

[0844] Example 17: Synthesis of I-10

[0845]

[0846]

[0847] Step 1: To a solution of Compound 1-3 (80 mg, 0.11 mmol) in DMF (3 mL) was added tert-butyl (2-(2-iodoethoxy)ethyl)carbamate (35 mg, 0.11 mmol). The reaction mixture was stirred at room temperature for 12 hours and purified directly by Flash column chromatography (C18, eluent: 0 - 70% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain Compound 5A (32 mg, yield: 52%). m / z: [M+H] + 927.0。

[0848] Steps 2 & 3: Using the synthetic method of steps 2 and 3 of III-8, reacting with compound 5A to obtain compound I-10 (prep-HPLC separation: mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution mobile phase B from 3% to 45%, elution time: 20 minutes) as a white solid. HPLC RT = 14.034 minutes; m / z: [M+H] + 1424.9。

[0849] Example 18: Synthesis of I-11

[0850]

[0851] Using the synthetic method of compound I-8, reacting with compound 1-3 and 1-(2-iodoethyl)-1H-pyrrole-2,5-dione to obtain compound I-11 (prep-HPLC separation: mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution mobile phase B from 5% to 45%, elution time: 20 minutes) as a white solid. HPLC RT = 12.959 minutes; m / z: [M+H] + 863.1。

[0852] Example 19: Synthesis of I-12

[0853]

[0854] Using the synthetic method of compound I-10, reacting with compound 1-3 and tert-butyl (17-iodo-3,6,9,12,15-pentaoxaheptadecyl)carbamate to obtain compound I-12 (prep-HPLC: mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution mobile phase B from 5% to 45%, elution time: 20 minutes) as a white solid. HPLC RT = 13.701 minutes; m / z: [M+H] + 1196.2。

[0855] Example 20: Synthesis of I-13

[0856]

[0857] Steps 1 & 2: Using the synthetic method of 5B, reacting with 1-3 and tert-butyl (2-iodoethyl)carbamate to obtain compound 6B as a pale yellow solid. m / z: [1 / 2M+H] + 392.0。

[0858] Step 3: Under ice bath conditions, to a solution of compound 6B (25 mg, 0.032 mmol) in DMF (2 mL) were successively added Mc-Val-Cit-OH (18 mg, 0.038 mmol), HATU (20 mg, 0.038 mmol) and DIPEA (60 mg, 0.038 mmol). After the reaction mixture was stirred at room temperature for 1 hour, it was directly purified by prep-HPLC (mobile phase A: 0.1% aqueous trifluoroacetic acid solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 45%, elution time: 20 minutes) to obtain compound I-13 (5.2 mg, yield: 13%) as an off-white solid. UPLC RT = 5.299 minutes; m / z: [1 / 2M + H] + 616.8。

[0859] Example 21: Synthesis of I-14

[0860]

[0861] Using the synthetic method of compound I-9, reacting compound 1-3 with Linker-7 gave compound I-14 (prep-HPLC: mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 50%, elution time: 20 minutes) as a white solid. HPLC RT = 13.968 minutes; m / z: [1 / 2M + H] + 650.7。

[0862] Example 22: Synthesis of I-15

[0863]

[0864] Steps 1 & 2: Using the synthetic method of compound 4B, reacting compound 1-3 with tert-butyl (2-(((chloromethoxy)carbonyl)oxy)ethyl)(methyl)carbamate gave compound 7B as a pale yellow solid. m / z: [M + H] + 871.0。

[0865] Step 3: To a solution of Compound 7B (10 mg, 0.011 mmol) in DMF (2 mL), Linker-3 (8.1 mg, 0.11 mmol), DIPEA (2.8 mg, 0.022 mmol) and HOBT (2.2 mg, 0.017 mmol) were added successively. The reaction mixture was stirred at room temperature for 2 h, concentrated under reduced pressure, and the residue was purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution with mobile phase B from 5% to 55%, elution time: 20 min) to obtain Compound I-15 (5 mg, yield: 28%) as an off-white solid. HPLC RT = 14.228 min; m / z: [1 / 2M+H] + 735.2。

[0866] Example 23: Synthesis of I-16

[0867]

[0868] Step 1: Compound 1-3 (50 mg, 0.068 mmol) was dissolved in dry pyridine (2 mL), concentrated under reduced pressure, and the above operation was repeated twice. Under ice bath conditions and nitrogen protection, pyridine (2 mL) and trimethylchlorosilane (44 mg, 0.41 mmol) were added successively to 1-3. After addition, the reaction mixture was stirred at room temperature for 0.5 h. Then, a solution of 4-((tert-butoxycarbonyl)(methyl)amino)butane (isobutylcarbon)ic anhydride (110 mg) in dry pyridine (0.5 mL) was added, and stirring was continued for 3 h. The reaction mixture was directly purified by Flash column chromatography (C18, eluent: 0-60% acetonitrile in 0.1% aqueous trifluoroacetic acid) to obtain Compound 8A (30 mg, yield: 47%) as a white solid. m / z: [M+H] + 939.3。

[0869] Step 2: Using the synthesis method of 7B, reacting with 8A to obtain 8B as a white solid. m / z: [M+H] + 839.2。

[0870] Step 3: Using the synthesis method of Step 3 of I-15, reacting with Compound 8B to obtain Compound I-16 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution with mobile phase B from 15% to 70%, elution time: 20 min) as a white solid. HPLC RT = 13.067 min; m / z: [1 / 2M+H] + 719.3。

[0871] Example 24: Synthesis of I-17

[0872]

[0873] Steps 1 & 2: Using the synthesis method of compound 4B, react compounds 1 - 3 with tert-butyl (S)-2-((((chloromethoxy)carbonyl)(2-(dimethylamino)ethyl)amino)methyl)pyrrolidine-1-carboxylate to obtain compound 9B as a pale yellow solid. m / z: [M+H] + 967.2.

[0874] Step 3: Using the synthesis method of step 3 of compound I-15, react 9B to obtain compound I-17 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 2% to 65%, elution time: 20 minutes) as a white solid. HPLC RT = 13.891 minutes; m / z: [1 / 2M+H] + 783.0.

[0875] Example 25: Synthesis of I-18

[0876]

[0877] Using the synthesis method of III-3, react compounds 1 - 3 with Linker-8 to obtain compound I-18 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 10% to 55%, elution time: 20 minutes) as a white solid. HPLC RT = 13.777 minutes; m / z: [M+H] + 1352.2.

[0878] Example 26: Synthesis of I-19

[0879]

[0880] Steps 1 & 2: Using the synthesis method of compound 8B, react compounds 1 - 3 with 4-((4-((tert-butoxycarbonyl)amino)benzyl)thio)butane (isobutyl carbon)ic anhydride to obtain compound 10B as a yellow solid. m / z: [M+H] + 947.0.

[0881] Step 3: Using the synthesis method of step 3 of I-15, react compound 10B to obtain compound I-19 (prep-HPLC separation: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 70%, elution time: 20 minutes) as a white solid. HPLC RT = 13.762 minutes; m / z: [1 / 2M+H] + 773.2.

[0882] Example 27: Synthesis of I-20

[0883]

[0884] A mixed solution of Mc-Val-Cit-OH (7.9 mg, 17 μM), Compound 10B (8 mg, 8.4 μM) and EEDQ (4.15 mg, 17 μM) in dichloromethane (0.4 mL) and methanol (0.2 mL) was stirred at room temperature for 2 hours. The reaction solution was directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 70%, elution time: 20 minutes) to obtain I-20 (3.73 mg, yield: 30%) as a white solid. HPLC RT = 13.114 minutes; m / z: [1 / 2M+H] + 698.6

[0885] Example 28: Synthesis of I-21

[0886]

[0887] Using the synthesis method of I-3, reacting Compound 1-3 with Linker-9 gave I-21 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 65%, elution time: 20 minutes) as a white solid. HPLC RT = 14.432 minutes; m / z: [M+H] + 1427.4

[0888] Example 29: Synthesis of I-22

[0889]

[0890]

[0891] Steps 1&2: Using the synthesis method of Compound 8B, reacting Compound 1-3 with Linker-10 gave Compound 11B as a pale yellow solid. m / z: [1 / 2M+H] + 603.6

[0892] Step 3: To a mixed solution of compound 11B (5.6 mg, 0.018 mmol) and N-succinimidyl 6-(maleimidyl)hexanoate (6 mg, 0.02 mmol) in tetrahydrofuran (0.5 mL) and DMF (0.5 mL) was added DIPEA (3.1 mg, 0.024 mmol). The reaction mixture was stirred at room temperature for 1 hour and then directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 70%, elution time: 20 minutes) to obtain compound I-22 (1.35 mg, yield: 8%) as a white solid. HPLC RT = 13.496 minutes; m / z: [1 / 2M+H] + 700.2。

[0893] Example 30: Synthesis of I-23

[0894]

[0895]

[0896] Step 1: Under ice bath conditions, isobutyl chloroformate (134 mg, 0.98 mmol) was added to a solution of 4-((4-((tert-butoxycarbonyl)amino)benzyl)disulfanyl)butanoic acid (350 mg, 0.98 mmol) and triethylamine (141 mg, 1.4 mmol) in dichloromethane (10 mL). The reaction mixture was stirred at 0 °C for 0.5 hour and then concentrated under reduced pressure. The residue was diluted with a small amount of dichloromethane and passed through a short silica gel column. The obtained filtrate was reserved. Compound 1-3 (100 mg, 0.14 mmol) was dissolved in dry pyridine (2 mL) and concentrated under reduced pressure. The above steps were repeated twice, and it was redissolved in dry pyridine (3 mL). Under nitrogen protection and ice bath conditions, trimethylchlorosilane (91 mg, 0.84 mmol) was added to the pyridine solution of compound 1-3. After addition, the reaction mixture was stirred at room temperature for 0.5 hour, then the above dichloromethane filtrate was added, and the reaction mixture was continuously stirred at room temperature for 3 hours and concentrated under reduced pressure. The residue was purified by Flash column chromatography (C18, eluent: 0 - 40% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain compound 12A (125 mg, yield: 86%) as a white solid. m / z: [1 / 2M+H] + 540.0。

[0897] Step 2: Under ice bath conditions, trifluoroacetic acid (3 mL) was added to 12A (120 mg, 0.11 mmol) in dichloromethane (10 mL). The reaction solution was stirred at 0 °C for 2 hours, concentrated under reduced pressure, and the residue was purified by Flash column chromatography (C18, eluent: 0 - 30% acetonitrile in 0.1% aqueous trifluoroacetic acid) to obtain compound 12B (55.6 mg, yield: 51%) as a white solid. m / z: [M+H] + 979.0。

[0898] Step 3: Using the synthesis method of I-20, I-23 was obtained by reacting with 12B (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 70%, elution time: 20 minutes) as a white solid. HPLC RT = 13.798 minutes; m / z: [1 / 2M+H] + 671.8。

[0899] Example 31: Synthesis of I-24

[0900]

[0901]

[0902] Step 1: Under ice bath conditions, Linker-11 (52 mg, 0.068 mmol) was added to a solution of compound 1-3 (50 mg, 0.068 mmol) in DMF (3 mL). The reaction solution was stirred at room temperature for 1.5 hours and then directly purified by Flash column chromatography (C18, eluent: 0 - 75% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain compound 13A (38 mg, yield: 41%) as a pale yellow solid. m / z: [1 / 2M+H] + 685.8。

[0903] Step 2: A mixed solution of compound 13A (20 mg, 0.015 mmol) in water (0.5 mL) and hydrochloric acid - 1,4-dioxane (0.5 mL, 4 M) was stirred at 38 °C for 12 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 55%, elution time: 20 minutes) to obtain compound I-24 (5.76 mg, yield: 31%) as a white solid. HPLC RT = 11.793 minutes; m / z: [1 / 2M+H] + 615.8。

[0904] Example 32: Synthesis of I-25

[0905]

[0906] Using the synthesis method of compound I-20, reacting compound 10B with Mc-Val-Ala-OH to obtain compound I-25 (Flash column chromatography: C18, eluent is 0 - 60% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) as a white solid. HPLC RT = 13.447 minutes; m / z: [M+H] + 1310.0.

[0907] Example 33: Synthesis of I-26

[0908]

[0909] Using the synthesis method of compound I-17, replacing Linker-3 in step 3 with Linker-4 to react to obtain compound I-26 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution mobile phase B from 5% to 60%, elution time: 20 minutes) as a white solid. HPLC RT = 14.249 minutes; m / z: [M+H] + 1478.9.

[0910] Example 34: Synthesis of I-27

[0911]

[0912] Using the synthesis method of I-3, reacting compound 1-3 with Linker-13 to obtain I-27 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution mobile phase B from 3% to 50%, elution time: 20 minutes) as a white solid. HPLC RT = 13.656 minutes; m / z: [1 / 2M+H] + 825.3.

[0913] Example 35: Synthesis of I-28

[0914]

[0915]

[0916] Step 1: Using the synthesis method of I-3, reacting compound 1-3 with Linker-14 to obtain 14A as a white solid. m / z: [M+H] + 1569.0.

[0917] Step 2: Diethylamine (16 mg, 0.22 mmol) was added to a solution of Compound 14A (70 mg, 0.045 mmol) in DMF (2 mL). The reaction mixture was stirred at room temperature for 1 hour and then purified directly by flash column chromatography (C18, eluent: 0 - 65% acetonitrile in 10 mM aqueous ammonium bicarbonate) to give Compound 14B (55 mg, yield: 92%). m / z: [M+H] + 1347.0。

[0918] Step 3: Compound 14B (40 mg, 0.03 mmol) was stirred in a solution of water and hydrochloric acid - 1,4 - dioxane (2 mL, 4 M) at 20 °C for 12 hours. The reaction mixture was purified directly by flash column chromatography (C18, eluent: 0 - 70% acetonitrile in 10 mM aqueous ammonium bicarbonate) to give Compound 14C (35 mg, yield: 98%). m / z: [M+H] + 1207.2。

[0919] Step 4: N - succinimidyl 6 - (maleimidyl)hexanoate (14 mg, 0.045 mmol) and 3 drops of DIPEA were added to a solution of Compound 14C (18 mg, 0.015 mmol) in DMF (2 mL). The reaction system was stirred at room temperature for 1.5 hours. The reaction mixture was purified directly by prep - HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution, mobile phase B from 2% to 50%, elution time: 20 minutes) to give Compound I - 28 (3.1 mg, yield: 14%) as a white solid. HPLC RT = 13.946 minutes; m / z: [M+H] + 1400.4。

[0920] Example 36: Synthesis of I - 29

[0921]

[0922] Using the synthetic method of Compound I - 17, Linker - 3 in Step 3 was replaced with Linker - 16 to obtain Compound I - 29 (prep - HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution, mobile phase B from 3% to 65%, elution time: 20 minutes) as a white solid. UPLC RT = 6.378 minutes; m / z: [1 / 2M+H] + 917.0。

[0923] Example 37: Synthesis of I - 30

[0924]

[0925] Synthesis method using compound I-20: React compound 10B with Linker-17 to obtain compound I-30 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 70%, elution time: 20 minutes), which is a white solid. UPLC RT = 13.414 minutes; m / z: [1 / 2M+H] + 833.4。

[0926] Example 38: Synthesis of I-31

[0927]

[0928]

[0929] Step 1: Using the synthesis method of I-3, react compound 1-3 with Linker-18 to obtain 15A as a white solid. m / z: [1 / 2M+H] + 645.2。

[0930] Step 2: Add diethylamine (14.2 mg, 0.20 mmol) to a DMF (2 mL) solution of compound 15A (50 mg, 0.039 mmol). After the reaction mixture is stirred at room temperature for 1 hour, it is directly purified by flash column chromatography (C18, eluent: 0 - 60% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain compound 15B (55 mg, yield: 92%). m / z: [1 / 2M+H] + 534.6。

[0931] Step 3: Add N-(6-Maleimidocaproyl)succinimide (13 mg, 0.042 mmol) and 3 drops of DIPEA to a DMF (2 mL) solution of compound 15B (18 mg, 0.015 mmol). The reaction system is stirred at room temperature for 1 hour. The reaction mixture is directly purified by flash column chromatography (C18, eluent: 0 - 60% acetonitrile in 0.1% aqueous trifluoroacetic acid solution) to obtain 15C (40 mg, yield: 97%) as a white solid. m / z: [1 / 2M+H] + 631.2。

[0932] Step 4: Sodium iodide dihydrate (12 mg, 0.06 mmol) was added to a solution of compound 15C (15 mg, 0.012 mmol) in acetonitrile (3 mL). The reaction mixture was stirred at 40 °C for 20 h. After cooling the reaction mixture to room temperature, it was directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 2% to 60%, elution time: 20 min) to obtain compound I-31 (1.2 mg, yield: 9%) as a white solid. HPLC RT = 12.657 min; m / z: [M+H] + 1191.1。

[0933] Example 39: Synthesis of I-32

[0934]

[0935]

[0936] Step 1: Using the synthesis method of 8A, compound 1-3 and Linker-20 were reacted to obtain 16A as a white solid.

[0937] Step 2: Diethylamine (25 mg, 0.34 mmol) was added to a solution of 16A (65 mg, 0.057 mmol) in DMF (2 mL). The reaction mixture was stirred at room temperature for 2 h and directly purified by flash column chromatography (C18, eluent: 0 - 40% acetonitrile in 0.1% aqueous trifluoroacetic acid) to obtain 16B (30 mg, yield: 57%) as a white solid. m / z: [M+H] + 928.2。

[0938] Step 3: Under ice bath conditions, a solution of Linker-19 (3 mg, 0.004 mmol), DIPEA (0.5 mL) and HATU (1.6 mg, 0.004 mmol) in DMF (0.5 mL) was stirred for 5 min. Compound 16B (3.3 mg, 0.004 mmol) was added to the above reaction mixture. The reaction mixture was stirred at room temperature overnight and then directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 70%, elution time: 20 min) to obtain compound I-32 (1.8 mg, yield: 27%) as an off-white solid. HPLC RT = 13.021 min; m / z: [1 / 2M+H] + 869.3。

[0939] Example 40: Synthesis of I-33

[0940]

[0941] Synthesis method of I-32: Replace Linker-19 in Step 3 with Mc-Gly-Gly-Phe-OH and react to obtain I-33 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 80%, elution time: 20 minutes), which is a white solid. UPLC RT = 10.141 minutes; m / z: [1 / 2M+H] + 691.8

[0942] Example 41: Synthesis of I-34

[0943]

[0944] Add Linker-2 (7.3 mg, 0.011 mmol) to a solution of Compound 4 (10 mg, 0.009 mmol) in DMF (2 mL). After stirring the reaction solution at room temperature for 1 hour, directly use prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 65%, elution time: 20 minutes) to obtain Compound I-34 (2 mg, yield: 13%), which is a white solid. HPLC RT = 13.860 minutes; m / z: [1 / 2M+H] + 838.8

[0945] Example 42: Synthesis of I-35

[0946]

[0947] Add Linker-2 (16 mg, 0.023 mmol) to a solution of Compound 5 (20 mg, 0.023 mmol) in DMF (2 mL). After stirring the reaction solution at room temperature for 1 hour, directly use prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 50%, elution time: 20 minutes) to obtain Compound I-35 (12.3 mg, yield: 35%), which is a white solid. HPLC RT = 16.147 minutes; m / z: [1 / 2M+H] + 704.8

[0948] Example 43: Synthesis of I-36

[0949]

[0950] To a solution of Compound 2 (18 mg, 0.018 mmol) in DMF (1 mL) was added Linker-2 (16 mg, 0.023 mmol). After the reaction mixture was stirred at room temperature for 1 hour, it was directly subjected to prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution with mobile phase B from 10% to 30%, elution time: 20 minutes) to obtain Compound I-36 (7.8 mg, yield: 27%) as a white solid. HPLC RT = 15.783 minutes; m / z: [1 / 2M + H] + 770.4。

[0951] Example 44: Synthesis of I-37

[0952]

[0953] Using the synthesis method of I-3, reacting Compound 1-3 with Linker-22 gave I-37 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution with mobile phase B from 3% to 75%, elution time: 20 minutes) as a white solid. HPLC RT = 15.308 minutes; m / z: [M + H] + 1497.4。

[0954] Example 45: Synthesis of I-38

[0955]

[0956] Using the synthesis method of I-3, reacting Compound 1-3 with Linker-24 gave I-38 (Flash column chromatography: C18, eluent was 0% - 48% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) as a white solid. UPLC RT = 7.599 minutes; m / z: [M + H] + 1404.4。 31 P NMR (162 MHz, DMSO-d6 + D2O): δ 52.848, 27.645。

[0957] Example 46: Synthesis of I-39

[0958]

[0959] To a solution of compound I-38 (25 mg, 0.018 mmol) in water (1.5 mL) was added hydrochloric acid / 1,4-dioxane (4 M, 1.5 mL), and the reaction system was stirred at room temperature for 20 hours. Then it was directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution with mobile phase B from 5% to 70%, elution time: 20 minutes) to obtain compound I-39 (0.77 mg, yield: 3%) as a white solid. UPLC RT = 4.985 minutes; m / z: [M+H] + 1236.1。

[0960] Example 47: Synthesis of I-40

[0961]

[0962] Under ice bath conditions, to a solution of Linker-23 (12 mg, 0.012 mmol) and compound 9B (11.6 mg, 0.012 mmol) in DMF (0.5 mL) was added DIPEA (2.3 mg, 0.02 mmol). After the reaction solution was stirred at room temperature for 1 hour, it was directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution with mobile phase B from 5% to 50%, elution time: 20 minutes) to obtain compound I-40 (4.3 mg, yield: 19%) as a white solid. UPLC RT = 5.459 minutes; m / z: [1 / 2M+H] + 917.5。

[0963] Example 48: Synthesis of I-41

[0964]

[0965] Using the synthesis method of I-3, compound 1-3 and Linker-25 were reacted to obtain I-41 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution with mobile phase B from 5% to 75%, elution time: 20 minutes) as a white solid. UPLC RT = 7.650 minutes; m / z: [M+H] + 1583.3。

[0966] Example 49: Synthesis of I-42

[0967]

[0968] Synthesis method of I-3. React compound 1-3 with Linker-26 to obtain I-42 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 85%, elution time: 20 minutes), which is a white solid. UPLC RT = 8.348 minutes; m / z: [1 / 2M+H] + 886.8.

[0969] Example 50: Synthesis of I-43

[0970]

[0971] Add sodium iodide (7.64 mg, 0.051 mmol) and 4-(chloromethyl)-1-methyl-2-nitro-1H-imidazole (3 mg, 0.017 mmol) to a solution of compound I-3 (22 mg, 0.017 mmol) in DMF (2 mL). After stirring the reaction mixture at room temperature for 2.5 hours, purify it directly by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 60%, elution time: 20 minutes) to obtain compound I-43 (2.5 mg, yield: 10%), which is a white solid. UPLC RT = 6.180 minutes; m / z: [1 / 2M+H] + 717.2.

[0972] Example 51: Synthesis of I-44

[0973]

[0974] Using the synthesis method of I-43, react compound I-3 with 5-(chloromethyl)-1-methyl-4-nitro-1H-imidazole to obtain I-44 (Flash column chromatography: eluent is 0 - 55% acetonitrile in 0.1% aqueous trifluoroacetic acid solution), which is a white solid. UPLC RT = 6.039 minutes; m / z: [1 / 2M+H] + 717.2.

[0975] Example 52: Synthesis of I-45

[0976]

[0977] Using the synthesis method of I-36, react compound 2 with Linker-26 to obtain I-45 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 75%, elution time: 20 minutes), which is a white solid. UPLC RT = 7.765 minutes; m / z: [1 / 2M+CH3CN+H]+ 1050.4。

[0978] Example 53: Synthesis of I-46

[0979]

[0980] Step 1: A solution of Compound 1-3 (90 mg, 0.12 mmol) and Linker-29 (94 mg, 0.13 mmol) in DMF (5 mL) was stirred at room temperature for 1 hour and then purified directly by flash column chromatography (C18, eluent: 0 - 45% acetonitrile in 10 mM aqueous ammonium bicarbonate) to obtain 17A (40 mg, yield: 20%) as an off-white solid. m / z: [1 / 2M + H] + 661.5。

[0981] Step 2: Diethylamine (22 mg, 0.3 mmol) was added to a solution of 17A (40 mg, 0.03 mmol) in DMF (5 mL). The reaction mixture was stirred at room temperature overnight and then purified directly by flash column chromatography (C18, eluent: 0 - 45% acetonitrile in 0.05% aqueous trifluoroacetic acid) to obtain 17B (25 mg, yield: 52%) as a white solid. m / z: [1 / 2M + H] + 551.2。

[0982] Step 3: Under ice-bath conditions, a solution of 17B (8 mg, 0.007 mmol), Linker-27 (4.1 mg, 0.008 mmol), DIPEA (3 mg, 0.022 mmol), and HATU (3 mg, 0.008 mmol) in DMF (3 mL) was stirred for 2 hours. The reaction mixture was purified directly by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 45%, elution time: 20 minutes) to obtain Compound I-46 (1.03 mg, yield: 11%) as a white solid. UPLC RT = 5.702 minutes; m / z: [M + H] + 1378.5。

[0983] Example 54: Synthesis of I-47

[0984]

[0985] Synthesis of 18B: Using the synthesis method of 17B, Compound 1-3 was reacted with Linker-30 to obtain 18B as a white solid. m / z: [1 / 2M + H] + 582.5。

[0986] Synthesis method of I-46: React compound 18B with Linker-31 to obtain I-47 (prep-HPLC: mobile phase A: 0.1% aqueous trifluoroacetic acid solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 45%, elution time: 20 minutes), which is a white solid. UPLC RT = 6.211 minutes; m / z: [1 / 2M+H] + 713.8

[0987] Example 55: Synthesis of I-48

[0988]

[0989] Synthesis method of I-46: React compound 18B with Linker-27 to obtain I-48 (prep-HPLC: mobile phase A: 0.1% aqueous trifluoroacetic acid solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 45%, elution time: 20 minutes), which is a white solid. UPLC RT = 6.021 minutes; m / z: [1 / 2M+H] + 720.7

[0990] Example 56: Synthesis of I-49

[0991]

[0992] Synthesis method of I-46: React compound 18B with Linker-28 to obtain I-49 (prep-HPLC: mobile phase A: 0.1% aqueous trifluoroacetic acid solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 45%, elution time: 20 minutes), which is a white solid. UPLC RT = 6.420 minutes; m / z: [1 / 2M+H] + 764.8

[0993] Example 57: Synthesis of I-50

[0994]

[0995] Add sodium iodide (4 mg, 0.028 mmol) to a DMF (2 mL) solution of compound 1-3 (10 mg, 0.014 mmol) and Linker-32 (9 mg, 0.013 mmol). The reaction system is stirred at room temperature for 2 days and then directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 75%, elution time: 20 minutes) to obtain compound I-50 (3 mg, yield: ) as a white solid. UPLC RT = 7.140 minutes; m / z: [1 / 2M+H] + 734.831 1H NMR (162 MHz, DMSO-d6 + D2O): δ 52.902, 27.667.

[0996] Example 58: Synthesis of I-51

[0997]

[0998] Step 1: A solution of Compound 1-3 (74 mg, 0.1 mmol), Linker-33 (35 mg, 0.1 mmol), and sodium iodide (18 mg, 0.12 mmol) in DMF (1.2 mL) was stirred at room temperature for 5 hours and then purified directly by flash column chromatography (C18, 0 - 65% acetonitrile in 0.1% aqueous trifluoroacetic acid) to obtain 19A (45 mg, yield: 43%) as a pale yellow solid. m / z: [1 / 2M + H] + 526.5.

[0999] Step 2: Under ice bath conditions, a solution of 19A (45 mg, 0.043 mmol), Linker-34 (10 mg, 0.038 mmol), DIPEA (14.7 mg, 0.11 mmol), and HATU (21.7 mg, 0.057 mmol) in DMF (1.2 mL) was stirred for 2 hours. The reaction solution was purified directly by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution with mobile phase B from 15% to 70%, elution time: 20 minutes) to obtain Compound I-51 (22 mg, yield: 44%) as a white solid. UPLC RT = 4.785 minutes; m / z: [1 / 2M + H] + 650.7.

[1000] Example 59: Synthesis of I-52

[1001]

[1002] Using the synthesis method of I-50, Compound 1-3 was reacted with Linker-35 to obtain I-52 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution with mobile phase B from 5% to 75%, elution time: 20 minutes) as a white solid. HPLC RT = 14.703 minutes; m / z: [M + H] + 1420.9.

[1003] Example 60: Synthesis of I-53

[1004]

[1005] Steps 1 & 2: Using the synthetic method of Compound 4B, react Compound 1-3 with Linker-37 to obtain Compound 20B as a white solid. m / z: [M+H] + 1265.5.

[1006] Step 3: Add (1-Methyl-2-nitro-1H-imidazol-5-yl)methyl (4-nitrophenyl) carbonate (6 mg, 0.018 mmol) and DIPEA (4 mg, 0.032 mmol) to a solution of Compound 20B (20 mg, 0.016 mmol) in DMF (1 mL). After stirring the reaction mixture at room temperature for 3 hours, purify it directly by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 10% to 85%, elution time: 20 minutes) to obtain Compound I-53 (5 mg, yield: 22%) as a white solid. UPLC RT = 7.240 minutes; m / z: [M+H] + 1448.5.

[1007] Example 61: Synthesis of I-54

[1008]

[1009] Using the synthetic method of I-3, react Compound 1-3 with Linker-38 to obtain I-54 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 70%, elution time: 20 minutes) as a white solid. UPLC RT = 7.233 minutes; m / z: [M+H] + 1782.5.

[1010] Example 62: Synthesis of I-55

[1011]

[1012] Under ice bath conditions, a solution of N-(5-(chloromethyl)-2-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)phenyl)-2,5,8,11,14,17,20,23-oxahexacosane-26-amine (22.3 mg, 0.023 mmol) in acetonitrile (1 mL) was added dropwise to a solution of I-3 (22 mg, 0.017 mmol) and sodium iodide (10.3 mg, 0.069 mmol) in DMF (2 mL). The reaction system was stirred at room temperature for 2 days. The reaction solution was directly purified by prep-HPLC (mobile phase A: 0.1% aqueous trifluoroacetic acid solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 55%, elution time: 20 minutes) to obtain I-55 (6.25 mg, yield: 12%) as a white solid. UPLC RT = 6.864 minutes; m / z: [1 / 2M+H] + 974.8

[1013] Example 63: Synthesis of I-56

[1014]

[1015] Using the synthesis method of I-46, Linker-27 in step 3 was replaced with Linker-31 to react to obtain I-56 (C18, eluent: 0 - 40% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) as a white solid. UPLC RT = 5.722 minutes; m / z: [1 / 2M+H] + 682.8

[1016] Example 64: Synthesis of I-57

[1017]

[1018] Using the synthesis method of I-43, I-56 and 5-(chloromethyl)-1-methyl-2-nitro-1H-imidazole were reacted to obtain I-57 (prep-HPLC: mobile phase A: 0.1% aqueous trifluoroacetic acid solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 85%, elution time: 20 minutes) as a white solid. UPLC RT = 6.160 minutes; m / z: [1 / 2M+H] + 752.8

[1019] Example 65: Synthesis of I-58

[1020]

[1021] Synthesis method using I-43: React I-3 with 4-bromo-5-(chloromethyl)-1-methyl-2-nitro-1H-imidazole to obtain I-58 (prep-HPLC: mobile phase A: 0.1% aqueous trifluoroacetic acid solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 50%, elution time: 20 minutes), which is a white solid. UPLC RT = 6.730 minutes; m / z: [1 / 2M+H] + 756.2

[1022] Example 66: Synthesis of I-59

[1023]

[1024] Synthesis method using I-3: React compound 6 with Linker-2 to obtain I-59 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 70%, elution time: 20 minutes), which is a white solid. UPLC RT = 7.007 minutes; m / z: [M+H] + 1435.3

[1025] Example 67: Synthesis of I-60

[1026]

[1027] Synthesis method using I-43: React I-3 with 5-(chloromethyl)-1-methyl-2-nitro-1H-imidazole to obtain I-60 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 85%, elution time: 20 minutes), which is a white solid. UPLC RT = 6.539 minutes; m / z: [M+H] + 1433.2

[1028] Example 68: Synthesis of I-61

[1029]

[1030] Synthesis method using I-43: React I-3 with 5-(chloromethyl)-1,4-dimethyl-2-nitro-1H-imidazole to obtain I-61 (prep-HPLC: mobile phase A: 0.1% aqueous trifluoroacetic acid solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 55%, elution time: 20 minutes), which is a white solid. UPLC RT = 6.448 minutes; m / z: [1 / 2M+H] + 724.2

[1031] Example 69: Synthesis of I-62

[1032]

[1033] Using the synthesis method of I-43, I-50 was reacted with 4-bromo-5-(chloromethyl)-1-methyl-2-nitro-1H-imidazole to obtain I-62 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 65%, elution time: 20 minutes) as a white solid. UPLC RT = 7.154 minutes; m / z: [M+H] + 1686.1

[1034] Example 70: Synthesis of I-63

[1035]

[1036] Using the synthesis method of I-43, I-50 was reacted with 5-(chloromethyl)-1-methyl-2-nitro-1H-imidazole to obtain I-63 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 65%, elution time: 20 minutes) as a white solid. UPLC RT = 6.919 minutes; m / z: [M+H] + 1607.5

[1037] Example 71: Synthesis of I-64

[1038]

[1039] Using the synthesis method of I-3, compound 7 was reacted with Linker-2 to obtain I-64 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 55%, elution time: 20 minutes) as a white solid. UPLC RT = 7.388 minutes; m / z: [M+H] + 1548.6

[1040] Example 72: Synthesis of I-65

[1041]

[1042] Using the synthesis method of I-3, compound 7 was reacted with Linker-39 to obtain I-65 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 80%, elution time: 20 minutes) as a white solid. UPLC RT = 9.125 minutes; m / z: [M+H] + 1490.5

[1043] Example 73: Synthesis of I-66

[1044]

[1045] Sodium iodide (4 mg, 0.03 mmol) was added to a solution of Linker-40 (60 mg, 0.05 mmol) and Compound 1-3 (40 mg, 0.05 mmol) in DMF (1.5 mL). After the reaction mixture was stirred at 30 °C for 2 days, it was directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 10% to 65%, elution time: 20 minutes) to obtain I-66 (14.7 mg, yield: 98%) as a white solid. HPLC RT = 14.541 minutes; m / z: [1 / 2M+H] + 903.8。

[1046] Example 74: Synthesis of I-67

[1047]

[1048]

[1049] Step 1: Sodium iodide (20 mg, 0.13 mmol) was added to a solution of Linker-45 (102 mg, 0.13 mmol) and Compound 1-3 (80 mg, 0.11 mmol) in DMF (1.5 mL). After the reaction mixture was stirred at 30 °C for 1 day, it was directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 10% to 60%, elution time: 20 minutes) to obtain 21A (65 mg, yield: 34%) as a white solid. m / z: [1 / 2M+H] + 743.8。

[1050] Step 2: Under nitrogen protection, cuprous iodide (3.8 mg, 0.02 mmol) and DIPEA (4 mg, 0.03 mmol) were added to a DMF (1 mL) solution of 21A (15 mg, 0.01 mmol) and 37-azido-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxatriacontane (12 mg, 0.02 mmol). The reaction mixture was stirred at 60 °C for 2 hours, cooled to room temperature, quenched with water, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were concentrated under reduced pressure. The residue was purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution with mobile phase B from 10% to 60%, elution time: 20 minutes) to obtain compound I-67 (3.4 mg, yield: 95%) as a white solid. HPLC RT = 17.565 minutes; m / z: [1 / 2M + H] + 1035.8。

[1051] Example 74: Synthesis of I-68

[1052]

[1053] Using the synthetic method of I-67, reacting 21A with 73-azido-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetraoxaheptatriacontane gave I-68 (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution with mobile phase B from 10% to 60%, elution time: 20 minutes) as a white solid. HPLC RT = 14.705 minutes; m / z: [1 / 2M + H] + 1300.8。

[1054] Example 75: Synthesis of I-69

[1055]

[1056] Under nitrogen protection, cuprous iodide (4 mg, 0.02 mmol) and N,N'-diisopropylcarbodiimide (4 mg, 0.03 mmol) were added to a solution of compound 21A (15 mg, 0.01 mmol) and Linker-41 (11 mg, 0.02 mmol) in DMF (1 mL). The reaction system was stirred at 60 °C for 2 hours, cooled to room temperature, and the reaction solution was directly purified by prep-HPLC (mobile phase A: 0.1% aqueous trifluoroacetic acid solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 10% to 65%, elution time: 20 minutes) to obtain compound I-69 (4.17 mg, yield: 20%) as a white solid. UPLC RT = 5.785 minutes; m / z: [1 / 2M+H] + 1028.6。

[1057] Example 76: Synthesis of I-70

[1058]

[1059] Using the synthesis method of I-69, I-70 was obtained by reacting 21A with Linker-42 (mobile phase A: 0.1% aqueous trifluoroacetic acid solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 10% to 65%, elution time: 20 minutes) as a white solid. HPLC RT = 13.507 minutes; m / z: [1 / 2M+H] + 1241.8。

[1060] Example 77: Synthesis of I-71

[1061]

[1062] Using the synthesis method of I-69, compound I-71 was obtained by reacting 21A with Linker-43 (mobile phase A: 0.1% aqueous trifluoroacetic acid solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 10% to 65%, elution time: 20 minutes) as a white solid. UPLC RT = 5.478 minutes; m / z: [1 / 2M+H] + 1454.2。

[1063] Example 78: Synthesis of I-72

[1064]

[1065] Synthesis method using I-69: React 21A with Linker-44 to obtain compound I-72 (mobile phase A: 0.1% aqueous trifluoroacetic acid solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 10% to 65%, elution time: 20 minutes), which is a white solid. HPLC RT = 13.407 minutes; m / z: [1 / 2M+H] + 1668.1

[1066] Example 79: Synthesis of I-73

[1067]

[1068] Synthesis method using I-7: React compound 1-3 with Linker-47 to obtain compound I-73 (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 10% to 45%, elution time: 20 minutes), which is a white solid. HPLC RT = 15.289 minutes; m / z: [M+H] + 1549.9

[1069] Example 80: Synthesis of I-74

[1070]

[1071] Synthesis of compound 22A: Using the synthesis method of steps 1 & 2 of compound I-7, react compound 1-3 with Linker-48 to obtain 22A as a white solid. m / z: [M+H] + 1607.4

[1072] Under ice bath conditions, add PyBOP (8 mg, 14 μM) and DIPEA (3 mg, 19 μM) to a DMF (1 mL) solution of compound 22A (15 mg, 9.3 μM) and compound 6-1 (5 mg, 14 μM). The reaction solution is slowly warmed to room temperature and stirred for 2 hours. The reaction solution is directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 10% to 50%, elution time: 20 minutes) to obtain compound I-74 (4.6 mg, yield: 26%) as a white solid. HPLC RT = 15.276 minutes; m / z: [M+H] + 1908.7

[1073] Example 81: Synthesis of I-75

[1074]

[1075] Synthesis method of I-74: React compound 1-3 with Linker-49 to obtain compound I-75 (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 10% to 55%, elution time: 20 minutes), which is a white solid. HPLC RT = 14.320 minutes; m / z: [1 / 2M+H] + 1292.8

[1076] Example 82: Synthesis of I-76

[1077]

[1078] Synthesis method of I-67: React 21A with 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl azide to obtain compound I-76 (mobile phase A: 1 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 65%, elution time: 20 minutes), which is a white solid. HPLC RT = 15.305 minutes; m / z: [1 / 2M+H] + 929.8

[1079] Example 83: Synthesis of I-77

[1080]

[1081] Synthesis method of I-67: React 21A with 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl azide to obtain compound I-77 (mobile phase A: 1 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 65%, elution time: 20 minutes), which is a white solid. HPLC RT = 15.360 minutes; m / z: [1 / 2M+H] + 929.8

[1082] Example 84: Synthesis of I-78

[1083]

[1084] Synthesis method of I-67: React 21A with 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl azide to obtain compound I-78 (mobile phase A: 1 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 65%, elution time: 20 minutes), which is a white solid. HPLC RT = 15.334 minutes; m / z: [1 / 2M+H] + 929.3

[1085] Example 85: Synthesis of I-79

[1086]

[1087] Synthetic method using I-67: React 21A with (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-azidotetrahydro-2H-pyran-3,4-diyl diacetate to obtain compound I-79 (mobile phase A: 1 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 65%, elution time: 20 minutes) as a white solid. UPLC RT = 7.300 minutes; m / z: [1 / 2M+H] + 929.4

[1088] Example 86: Synthesis of I-80

[1089]

[1090] Synthetic method using I-67: React 21A with 2,3,6,2',3',4',6'-hepta-O-acetyl-β-lactose azide to obtain compound I-80 (mobile phase A: 1 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 65%, elution time: 20 minutes) as a white solid. UPLC RT = 7.781 minutes; m / z: [1 / 2M+H] + 1073.7

[1091] Example 87: Synthesis of I-81

[1092]

[1093] Synthetic method using I-67: React 21A with (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(azidomethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate to obtain compound I-80 (mobile phase A: 1 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 50%, elution time: 20 minutes) as a white solid. UPLC RT = 7.818 minutes; m / z: [1 / 2M+H] + 1073.8

[1094] Example 88: Synthesis of I-82

[1095]

[1096] Synthetic method using I-67: React 21A with 2-azido-N-((3R,4R,5R,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide to obtain compound I-82 (mobile phase A: 0.05% aqueous trifluoroacetic acid solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 50%, elution time: 20 minutes) as a white solid. HPLC RT = 14.408 minutes; m / z: [1 / 2M+H] + 874.3。

[1097] Example 89: Synthesis of I-83

[1098]

[1099] Synthetic method using I-67: React 21A with 1-azido-1-deoxy-β-D-galactose to obtain compound I-83 (mobile phase A: 1 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 65%, elution time: 20 minutes) as a white solid. UPLC RT = 6.499 minutes; m / z: [1 / 2M+H] + 845.6。

[1100] Example 90: Synthesis of I-84

[1101]

[1102] Synthetic method using I-67: React 21A with (2R,3S,4S,5R,6R)-2-(azidomethyl)-6-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triol to obtain compound I-84 (mobile phase A: 0.05% aqueous trifluoroacetic acid solution, mobile phase B: acetonitrile; gradient elution, mobile phase B from 5% to 50%, elution time: 20 minutes) as a white solid. HPLC RT = 13.832 minutes; m / z: [1 / 2M+H] + 926.8。

[1103] Example 91: Antibody Conjugation Reaction

[1104] I. General method A for antibody-drug conjugation reaction (as shown in Formula 1):

[1105] Method 1: The antibody was ultrafiltered and replaced with phosphate buffer (50 mM PBS, 2 mM EDTA, pH 6.5), and the protein concentration was adjusted to 5 - 10 mg / mL. An aqueous solution of TCEP (6 - 12 equivalents relative to one molecule of antibody) was added, and the reaction was carried out at room temperature for 60 - 120 minutes. After the reduction reaction was completed, the antibody concentration was diluted to approximately 5 mg / mL with the above phosphate buffer, and then a compound of formula I (Linker-payload) 10 - 20 times the molar amount of the antibody was dissolved in an organic solvent (DMA or DMSO) with a total volume of 5% - 20%, added to the reaction system, and stirred at room temperature for 30 - 120 min. After the conjugation reaction was completed, the solution was changed to MES buffer (25 mM, pH 6.5) using an ultrafiltration centrifugal tube with a molecular weight cut-off of 30 KDa to obtain the antibody-immunostimulatory conjugate.

[1106] Method 2: The antibody was ultrafiltered and replaced with phosphate buffer (50 mM sodium dihydrogen phosphate - disodium hydrogen phosphate + 50 mM sodium chloride + 2 mM EDTA, pH 7.4 ± 0.2), and the protein concentration was adjusted to 10 ± 2 mg / ml. An aqueous solution of TCEP (2.2 equivalents relative to one molecule of antibody) was added. Stirring was carried out at 25 ± 2 °C for more than 2 hours. The protein concentration was adjusted to 5.0 ± 0.5 mg / mL with phosphate buffer, and under stirring conditions, an appropriate amount of DMA was added dropwise to the reaction system. After mixing evenly, a DMA solution of the compound of formula I (Linker-payload) (5.5 equivalents relative to one molecule of antibody) was added dropwise. Stirring was carried out at 25 ± 2 °C for more than 1 hour. Ultrafiltration replacement was carried out with a replacement buffer (50 mM sodium dihydrogen phosphate - disodium hydrogen phosphate + 6% trehalose, pH 6.5 + 0.1) to obtain the antibody-immunostimulatory conjugate.

[1107] Method 3: The antibody concentration was diluted to 5 mg / mL with phosphate buffer (25 mM PBS, 1 mM EDTA, pH 7.4). After shaking and mixing evenly, an aqueous solution of 2 mg / mL TCEP (8 equivalents relative to one molecule of antibody) was added for antibody reduction. After shaking and mixing evenly, the reaction was carried out at 37 °C for 2 hours on a refrigerated constant temperature mixer. Ultrafiltration was carried out 4 times with phosphate buffer to recover the ultrafiltered antibody. A dimethyl sulfoxide solution of the compound of formula I (Linker-payload) (4 equivalents relative to one molecule of antibody) was added, and the dimethyl sulfoxide solution was supplemented according to 10% of the total volume of the reaction solution. After shaking and mixing evenly, the reaction was carried out at 20 °C for 2 hours on a refrigerated constant temperature mixer, and then a dimethyl sulfoxide solution of Linker-payload (4 equivalents relative to one molecule of antibody) was added and the reaction was carried out overnight at 20 °C. The sample storage buffer was replaced with an ultrafiltration tube, and ultrafiltration was carried out 10 times with 30 mM His-HAc, pH 5.5 to obtain the antibody-immunostimulatory conjugate.

[1108] Method 4: Adjust antibody 5 or 8 to a concentration of 3 mg / mL with phosphate buffer (50 mM PBS, 2 mM EDTA, pH 6.5). Add an aqueous solution of TCEP at 20 equivalents (TCEP / antibody), mix well, and place it in a thermostatic mixer for reaction at room temperature for 17 hours. Centrifuge at 4300 rpm for 10 minutes using an ultrafiltration centrifugal tube (Millipore UFC803096), and repeat ultrafiltration three times. Add DHAA oxidant at 30 equivalents (DHAA / antibody) to the sample collected in the previous step in a centrifuge tube, mix well, and place it in a thermostatic mixer for stirring reaction at room temperature for 2 hours, followed by ultrafiltration three times. Cool the sample to about 4 °C, add the compound shown in Formula I (Linker-payload) (10 equivalents relative to one molecule of antibody), mix well, and place it in a thermostatic mixer for reaction at 4 °C for 1 hour. Ultrafilter three times to obtain the antibody-immunostimulatory conjugate.

[1109] II. General method B for antibody-drug conjugation reaction (as shown in Formula 1'):

[1110] Replace the antibody-immunostimulatory conjugate obtained by General method A into phosphate buffer (50 mM PBS, 2 mM EDTA, PH 8.0) using an ultrafiltration tube (MWCO 30KD, 4 mL, manufacturer: Millipore), and react overnight at 37 °C. Ultrafilter 10 times with 30 mM His-HAc without dimethyl sulfoxide, pH 5.5, to obtain the antibody-immunostimulatory conjugate.

[1111] III. General method C for antibody-drug conjugation reaction (as shown in Formula 5 or 12):

[1112] Dilute the antibody to a concentration of 5 mg / mL with phosphate buffer (25 mM PBS, 1 mM EDTA, pH 7.4), mix well by shaking, add an aqueous solution of 2 mg / mL TCEP (8 equivalents relative to one molecule of antibody) for antibody reduction, mix well by shaking, and place it on a refrigerated thermostatic mixer for reaction at 37 °C for 2 hours. Ultrafilter 4 times with 50 mM BBS, pH 8.0 buffer, recover the ultrafiltered antibody, add a dimethyl sulfoxide solution of the compound shown in Formula I (Linker-payload) (6 - 8 equivalents relative to one molecule of antibody), supplement the dimethyl sulfoxide solution according to 10% of the total volume of the reaction solution, mix well by shaking, and place it on a refrigerated thermostatic mixer for reaction at 20 °C for 1 hour. Replace the sample storage buffer with an ultrafiltration tube, and ultrafilter 10 times with 30 mM His-HAc, pH 5.5 to obtain the antibody-immunostimulatory conjugate.

[1113] IV. General method D for antibody-drug conjugation reaction (as shown in Formula 13):

[1114] React the phosphate buffer solution of antibody 12 (25 mM PBS, 1 mM EDTA, pH 7.4) with a dimethyl sulfoxide solution of the compound shown in Formula I (Linker - payload) (10 mM, 6 - 8 equivalents relative to one molecule of antibody) overnight at 37 °C. Replace the sample storage buffer with an ultrafiltration tube and ultrafilter 5 times with 30 mM His - HAc, pH 5.5 to obtain the antibody - immunostimulatory conjugate.

[1115] In the above general method, the antibody molecules used were anti - HER2 antibody 1: Trastuzumab, Roche; anti - HER2 antibody 2: Pertuzumab, Roche; anti - HER2 antibody 3: Trastuzumab - LALA (L234A / L235A), B801901, Shanghai Bioy Ying Biotechnology Co., Ltd.; anti - HER2 antibody 4: Trastuzumab - AAG (L234A / L235A / P329G), MHDDD001, Shanghai Bioy Ying Biotechnology Co., Ltd.; anti - HER2 antibody 5: Trastuzumab(HC - s239.5) (an engineered Trastuzumab with a cysteine inserted between heavy chain positions 239 and 240, which can be prepared by the method disclosed in Molecular Pharmaceutics. 2017, 14, 1501 - 1516), LPDDFD001, Shanghai Bioy Ying Biotechnology Co., Ltd.; antibody 6: Anti - HEL human IgG1 - Kappa Isotype control, B117901, Shanghai Bioy Ying Biotechnology Co., Ltd.; anti - EGFR antibody 7: Cetuximab, Merck; anti - 5T4 antibody 8: huA1(V H v2.0+V Lv2.4) (It can be expressed, purified and prepared through the sequence information tables (SEQ ID NO: 54 and SEQ ID NO: 70) disclosed in US8044178B2); Anti-HER2 antibody 9: Trastuzumab (HC-s239.5, L234A / L235A / P329G), CZ7DUD001, Shanghai Bioyiming Biotechnology Co., Ltd.; Antibody 10: Anti-HEL human IgG1 (L234A / L235A / P329G)-Kappa Isotype control, B422203, Shanghai Bioyiming Biotechnology Co., Ltd.; Antibody 11: Enfortumab (Ha22-2) (It can be expressed, purified and prepared through the sequence information table disclosed in US10894090B2); Anti-EGFR antibody 12: Prepared from Cetuximab by using the method disclosed in Example 25 of CN115209921A; Anti-EGFR antibody 13: Nimotuzumab, Biotech Pharmaceuticals Co., Ltd.

[1116] V: Determination of the average number of drug linkages (DAR) per molecule of antibody in the antibody-drug conjugate

[1117] For the average number of drug linkages per molecule of antibody in the antibody-drug conjugate, it can be determined by high performance liquid chromatography (HPLC) analysis using the following method.

[1118] HPLC instrument: Waters / Waters e2695; Mobile phase A: 1.5M (NH4)2SO4 + 50mM potassium phosphate (pH 7.0); Mobile phase B: 50mM sodium phosphate (pH 7.0) / isopropanol (75:25 V / V); Analytical column: Thermo MabPac TM HIC-Butyl 5μm 4.6×100mm, PN.088558; Injection volume: 5μL; Flow rate: 1mL / min; Column temperature: 30°C; Detector: PDA detector; Detection wavelength: 280nm. Elution gradient 1: Mobile phase B from 20% to 80%, elution time 20 minutes, mobile phase B from 80% to 100%, elution time 2 minutes, mobile phase B held at 100% for 6 minutes; Elution gradient 1: Mobile phase B from 10% to 65%, elution time 20 minutes, mobile phase B from 65% to 100%, elution time 2 minutes, mobile phase B held at 100% for 6 minutes.

[1119] Hydrophobic interaction chromatography can be used to determine the drug-antibody conjugate ratio (DAR) in antibody-drug conjugates. The uncoupled antibody drug has the weakest hydrophobicity and is eluted first; the antibody conjugated with 8 drugs has the strongest hydrophobicity and is eluted last. The percentage of peak area represents the relative distribution of ADCs conjugated with a specific number of drugs. The weighted average DAR is calculated as SUM (peak area of each component * corresponding DAR value) / total peak area through the percentage of peak area and the number of conjugated drugs.

[1120] VI: Analysis of the molecular size heterogeneity (SEC) of antibody-immunostimulatory conjugates

[1121] Method A: Chromatographic column: Waters XBridge 7.8 * 300 mm, 3.5 μm; Mobile phase A: 100 mM PB + 200 mM L-arginine hydrochloride, pH 6.8; Mobile phase B: isopropanol; Injection volume: 10 μl; Column temperature: 25 °C; Flow rate: 0.8 ml / min; Isocratic elution. Method B: HPLC instrument: Waters Acquity Arc; Mobile phase: 100 mM PB + 200 mM Arg·HCl + 5% IPA, pH 6.8; Analytical column: TOSOH TSKgel G3000 SWxl, 7.8 * 300 mm, 5 μM, PN0008541; Injection volume: 10 μL; Flow rate: 0.5 mL / min; Column temperature: 30 °C; Detector: PDA detector; Detection wavelength: 280 nm; Gradient: isocratic elution.

[1122] Antibody-immunostimulatory conjugates were prepared using the general method A as shown in Table 3 below:

[1123] Table 3

[1124]

[1125]

[1126]

[1127] Antibody-immunostimulatory conjugates were prepared using the general method B as shown in Table 4 below:

[1128] Table 4

[1129] Number Linker-payload number Antibody DAR (HIC) SEC% II-54 I-38 Antibody 1 7.82 98.27％ II-55 I-38 Antibody 5 2.00 95.25％ II-56 I-38 Antibody 9 2.32 99.48％

[1130] Antibody-immunostimulatory conjugates were prepared using the general method C as shown in Table 5 below:

[1131] Table 5

[1132] Number Linker-payload number Antibody DAR (HIC) SEC% II-7 I-57 Antibody 1 3.87 98.96％ II-57 I-41 Antibody 1 3.82 90.90％ II-58 I-46 Antibody 1 3.95 98.47％ II-59 I-47 Antibody 1 4.11 96.76％ II-60 I-48 Antibody 1 3.66 97.17％ II-61 I-50 Antibody 1 3.80 98.24％ II-62 I-50 Antibody 10 4.08 95.58％ II-63 I-50 Antibody 7 4.23 95.66％ II-64 I-49 Antibody 1 3.94 97.96％ II-65 I-51 Antibody 1 3.80 98.61％ II-66 I-37 Antibody 1 3.83 91.66％

[1133] The antibody-immunostimulatory conjugate was prepared using General Method D as shown in Table 6 below:

[1134] Table 6

[1135] Number Linker-payload number Antibody DAR (HIC) SEC% II-84 I-73 Antibody 12 1.51 97.36％

[1136] Example 92: Synthesis of C-7

[1137]

[1138] Using Preparation Method 2 in General Method A, C-7 was prepared using Antibody 1 and Compound 17 (WO2019129880A1), DAR: 2.5, SEC: 99.23%.

[1139] Biological Example:

[1140] Experimental Example 1: Test for the activation level of type I interferon

[1141] THP-1 dual cells (purchased from Invivogen) were seeded into a 96-well plate at 100,000 cells / well, and phorbol 12-myristate 13-acetate (PMA) with a final concentration of 30 ng / ml was added and induced for 24 hours. After 24 hours, the supernatant was discarded, and the cells were rinsed twice with fresh medium. Then, the test compound diluted in a 3-fold concentration gradient prepared with PB buffer (50 mM HEPES, 100 mM KCl, 3 mM MgCl2, 0.1 mM DTT, 85 mM sucrose, 1 mM ATP, 0.1 mM GTP, 0.2% bovine serum albumin, and 5 μg / ml digitonin) was added to the cells, with the highest final concentration of the compound being 10 μM and the lowest final concentration being 0.0015 μM. After the cells were incubated in the incubator for 30 minutes, the supernatant was discarded, and the cells were rinsed twice again with fresh medium and fresh medium was added, and then the cells were returned to the incubator for continued culture for 24 hours. After 24 hours, 10 μL of the supernatant from each well was taken into a new 96-well plate, and 50 μL / well of QUANTI-Luc reagent (purchased from Invivogen) was added, and the Luciferase reading was immediately measured using a TECAN M1000. The activation level of type I interferon is positively correlated with the detected luciferin intensity. A dose-response curve was plotted using Graphpad Prism software, and the EC of the test compound was analyzed 50 .

[1142] Experimental Example 2: Detection of cytokines in the hPBMC system

[1143] After the cryopreserved human peripheral blood mononuclear cells (Allcells) were rapidly thawed in a 37°C water bath, they were added to 9 mL of RPMI 1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 1 mM sodium pyruvate (all from Gibco). Centrifuge at 400×g for 5 minutes at room temperature, discard the supernatant, resuspend with the medium, and adjust the cell density to 4×10 6 / mL. Add them to a 96-well flat-bottom plate (Corning), 50 μL per well. Add 50 μL of the medium to the culture system.

[1144] Prepare a dilution of the test sample at 3-fold working concentration with the medium and add it to the cell suspension, 50 μL per well. The total volume of the system is 150 μL, and the initial concentration of the test sample is 0.67 μM, with 3-fold serial dilution. Add 50 μL of the medium to the blank control and incubate in a 37°C, 5% CO2 incubator for 20 hours. Centrifuge at 500×g for 5 minutes at room temperature and collect the supernatant. The concentration of TNFα was detected and analyzed using HTRF (Cisbio) and Infinite M1000 PRO (TECAN).

[1145] The test results showed that the TNFα secretion values of II-1, II-7, II-10-II-18, II-22, II-27, II-28, II-30, II-32, II-34, II-35, II-37, II-39, II-40, II-41, II-45, II-46, II-50, II-51, II-52, II-54, II-55, II-58-II-61, II-67-II-82 were all less than 500 pg / mL at a concentration of 500 nM.

[1146] Experimental Example 3: Detection of cytokines in the co-culture system of hPBMC and BT474

[1147] After the cryopreserved human peripheral blood mononuclear cells (Allcells) were rapidly thawed in a 37°C water bath, they were added to 9 mL of RPMI 1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 1 mM sodium pyruvate (all from Gibco). Centrifuge at 400×g for 5 minutes at room temperature, discard the supernatant, resuspend with the medium, and adjust the cell density to 4×10 6 / mL. Add it to a 96-well flat-bottom plate (Corning), 50 μL per well. Collect human breast cancer epithelial cells BT474 (Nanjing Kebai Biotechnology Co., Ltd.) in normal growth state, resuspend them in RPMI 1640 complete medium, and adjust the density to 4×10 5 / mL. Add it to a 96-well plate, 50 μL per well, and mix well with hPBMC.

[1148] Prepare a dilution of the test sample at 3 times the working concentration with the medium, add it to the cell suspension, 50 μL per well. The total volume is 150 μL. The initial concentration of Compounds 1-3 is 0.8 μM, and it is diluted in a 5-fold gradient; the initial concentration of other test samples is 0.1 μM, and it is diluted in a 5-fold gradient. Add 50 μL of medium to the blank control, and incubate in an incubator at 37 °C and 5% CO2 for 20 hours. Centrifuge at 500 rpm at room temperature for 5 minutes, and collect the supernatant. The concentration of TNF-α is detected and analyzed using HTRF (Cisbio) and Infinite M1000 PRO (TECAN).

[1149] The test results show that: at the same concentration, II-5 has an obvious inducing effect on TNF-α in the BT474 co-culture system, and the inducing effect of II-5 on TNF-α is significantly weakened in the single hPBMC system. The inducing effects of II-2 and II-5 on TNF-α in the BT474 co-culture system are significantly better than those of Compounds 1-3, Trastuzumab, and the combination of Trastuzumab and 1-3.

[1150] The TNFα secretion values of II-1, II-3, II-4, II-5, II-7, II-8, II-19, II-20, II-23-II-31, II-33, II-34, II-36, II-39-II-43, II-49, II-53, II-57-II-60, II-64, II-66, II-67, II-73, II-78, II-81, II-82 are between 1500 and 9000 pg / mL at a concentration of 100 nM.

[1151] Experimental Example 4: Detection of cytokines in the hPBMC and MDA-MB-468 co-culture system

[1152] Cryopreserved human peripheral blood mononuclear cells (Saili Biotech) were quickly thawed in a 37°C water bath and then added to 9 mL of RPMI 1640 medium containing 10% fetal bovine serum, 1× penicillin-streptomycin-glutamine, 1 mM sodium pyruvate, 1× MEM non-essential amino acids, and 10 mM HEPES (all from Gibco). Centrifuge at 400×g for 5 minutes at room temperature, discard the supernatant, resuspend with the medium, and adjust the cell density to 4×106 / mL. Add it to a 96-well flat-bottom plate (Corning), 50 μL per well. Collect human breast cancer cells MDA-MB-468 (Nanjing Kebai Biotech Co., Ltd.) in normal growth state, resuspend them in complete RPMI 1640 medium, and adjust the density to 4×10 5 / mL. Add it to the 96-well plate, 50 μL per well, and mix well with hPBMC.

[1153] Prepare a dilution of the test sample at 3 times the working concentration with the medium and add 50 μL to the cell suspension. The total volume is 150 μL, and the initial concentration of the compound is 500 nM, diluted in a 5-fold gradient. Add 50 μL of the medium to the blank control. Incubate in a 37°C, 5% CO2 incubator for 20 hours. Centrifuge at 500 rpm for 5 minutes at room temperature and collect the supernatant. The concentration of TNF-α was detected and analyzed using HTRF (VKEY-BIO) and Infinite M1000 PRO (TECAN).

[1154] Experimental Example 5: Cell Proliferation Assay

[1155] On the first day, SK-BR-3 human breast cancer cells in logarithmic growth phase (Nanjing Kebai Biotechnology Co., Ltd.) were digested with an appropriate amount of trypsin, and then prepared into a single-cell suspension with RPMI 1640 (Gibco) medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. After counting the cells, the density was adjusted, and they were seeded in a 96-well plate at 100 μL per well, with 2500 cells per well. Then, the cell culture plate was placed in an incubator at 37 °C and 5% CO2 overnight. On the second day, the ADC was serially diluted 5-fold with the above RPMI 1640 medium and added to the cell culture plate at 50 μL / well, with the highest concentration of ADC being 500 nM and the lowest concentration being 0.00128 nM. The control group was the medium. After brief centrifugation, the cell culture plate was placed in a carbon dioxide incubator and incubated for 120 hours. On the fifth day, the cell culture plate was taken out, and 50 μL of Cell Titer-Glo (CellTiter-glo, Promega) detection reagent was added to each well. After incubating in the dark for ten minutes, the luminescence was detected using a Microplate reader (Tecan, Infinite M1000 Pro). Graphpad software was used to analyze the data, and a three-parameter equation was used to fit the curve and calculate the IC 50 value.

[1156] Experimental Example 6: Plasma Stability Experiment

[1157] The test samples were separately added to human and mouse plasma at a concentration of 0.05 mg / mL. On days 0, 2, 4, 7, and 9, 50 μL of the test sample solution was taken and placed in a new plate. Prepare 1 μg / mL human Her2 / ErbB2 protein (acro, Cat#HE2-H5225) and 5 μg / mL anti-small molecule antibody (4222-03) in Coating Buffer. Add 30 μL to each well of a high-binding 384-well plate (High Bind Microplate, SpectraPlate, Cat#: 6007500) and incubate overnight at 4°C. Wash three times with the washing solution (90 μL per well), add 60 μL of blocking buffer (PBS buffer solution containing 5% BSA) to each well, and incubate at 37°C for 1 hour. Then add 30 μL of the sample or standard and incubate at room temperature for 2 hours. Wash three times with the washing solution (PBS buffer solution containing 0.05% Tween-20, 90 μL per well), add 30 μL of the detection antibody (Anti-Human IgG(Fab specific)-peroxidase: Sigma, Cat#A0293), and incubate at room temperature for 1 hour. Wash three times with the washing solution (90 μL per well), add 30 μL of TMB solution (A+B) (Solarbio, Cat#: PR1210-2*50 mL), and incubate in the dark at room temperature for 5 minutes. Add 30 μL of the stop solution (2 M dilute sulfuric acid) and read the OD value at a wavelength of 450 nm. The results showed that the antibody-immunostimulatory conjugate shown in Formula II had good plasma stability, especially for II-4, II-38, II-42, II-43, II-46, II-49, II-57, II-58, II-61, II-66, whose active half-lives in human and rat plasma were both greater than 4 days.

[1158] Experimental Example 7: PK Concentration Detection

[1159] 384-well plates were coated with human Her2 / ErbB2 protein (acro, Cat#HE2-H5225) or 5 μg / mL anti-small molecule antibody (4222-03), and kept in a refrigerator at 4°C overnight. The next day, the plates were washed three times with a washing solution (PBS buffer solution containing 0.05% Tween-20), and then blocked with a blocking buffer (PBS buffer solution containing 5% BSA), incubated at 37°C for 1-2 hours, and then washed three times with a washing solution. The diluted standard and the sample to be tested were added to the wells, incubated at room temperature for 2 hours, and then washed three times with a washing solution. Then, the detection antibody (Anti-Human IgG (Fab specific)-peroxidase: Sigma, Cat#A0293) diluted with PBS buffer was added and incubated at room temperature for 1 hour. Finally, the color development solution was added, and the reaction was terminated with a stop solution (2M dilute sulfuric acid) after incubation at room temperature for 10 minutes, and the OD value was read at a wavelength of 450nm.

[1160] Experimental Example 8: In vivo efficacy study of mouse colon cancer CT26-hHER2 subcutaneous transplant tumor model

[1161] Cell culture: Mouse colon cancer CT26-hHER2 cells were maintained as monolayers in RPMI-1640 medium containing 10% fetal bovine serum in a constant temperature incubator at 37°C with 5% CO2. After treatment with trypsin-EDTA, the cells were subcultured twice a week. Cells in the exponential growth phase were harvested and counted for inoculation.

[1162] Experimental animals: BALB / c mice, 6-8 weeks, 16-18 g, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[1163] Six experimental groups were set for 1-3, II-1, II-2, II-3, and II-4, as shown in Table 7:

[1164] Table 7

[1165]

[1166] Two experimental groups were set for Trastuzumab, as shown in Table 8 below:

[1167] Table 8

[1168] Group Number of mice Test compound Dose Route of administration Dosing schedule 7 8 Vehicle -- i.t. QW×1 8 8 Trastuzumab 25mg / kg i.p. QW×1

[1169] Six experimental groups were set for Trastuzumab, II-22, II-25, II-30, and II-37, as shown in Table 9 below:

[1170] Table 9

[1171] Group Number of mice Test compound Dose Route of administration Dosing schedule 9 5 Vehicle -- i.p. QW×2 10 3 Trastuzumab 1mg / kg i.p. QW×2 11 3 II-22 1mg / kg i.p. QW×2 12 3 II-25 1mg / kg i.p. QW×2 13 3 II-30 1mg / kg i.p. QW×2 14 3 II-37 1mg / kg i.p. QW×2

[1172] Set up 12 experimental groups for Trastuzumab + I-3, II-4, II-6, II-38, II-43, II-42, II-44, II-49, II-53, II-57, II-58, II-59 as shown in Table 10 below:

[1173] Table 10

[1174] Group Number of mice Test compound Dose Route of administration Dosing schedule 15 5 Vehicle -- i.p. QW×2 16 5 Trastuzumab+I-3 1mg / kg+0.06mg / kg i.p. QW×2 17 5 II-43 1mg / kg i.p. QW×2 18 5 II-6 1mg / kg i.p. QW×2 19 5 II-44 1mg / kg i.p. QW×2 20 5 II-42 1mg / kg i.p. QW×2 21 5 II-57 1mg / kg i.p. QW×2 22 5 II-58 1mg / kg i.p. QW×2 23 5 II-53 1mg / kg i.p. QW×2 24 5 II-38 1mg / kg i.p. QW×2 25 5 II-49 1mg / kg i.p. QW×2 26 5 II-4 1mg / kg i.p. QW×2

[1175] Set up 6 experimental groups for II-45, II-51, II-54, II-61, II-62 as shown in Table 11 below:

[1176] Table 11

[1177] Group Number of mice Test compound Dose Route of administration Dosing schedule 27 5 Vehicle -- i.p. QW×2 28 5 II-61 1mg / kg i.p. QW×2 29 5 II-62 1mg / kg i.p. QW×2 30 5 II-45 1mg / kg i.p. QW×2 31 5 II-54 1mg / kg i.p. QW×2 32 5 II-51 1mg / kg i.p. QW×2

[1178] Set up 6 experimental groups for II-46, II-55, II-64, II-65, I-65 as shown in Table 12 below:

[1179] Table 12

[1180] Group Number of mice Test compound Dose Route of administration Dosing schedule 33 5 Vehicle -- i.p. QW×2 34 5 II-46 1mg / kg i.p. Q2W×2 35 5 II-55 1mg / kg i.p. Q2W×2 36 5 II-64 1mg / kg i.p. Q2W×2 37 5 II-65 1mg / kg i.p. Q2W×2 38 5 I-65 0.15 mg / kg i.p. QW×2

[1181] Note: i.t.: intratumoral injection, i.v.: intravenous injection, i.p.: intraperitoneal injection

[1182] Experimental method: Inoculate CT26-hHER2 cell line (3.0×10 6 cells / mouse) subcutaneously into the right back of experimental mice, with an inoculation volume of 0.1 mL per mouse. Regularly observe the growth of tumors. When the tumors grow to about 100 mm 3 or so, randomly group the mice according to tumor size and mouse body weight, and administer drugs according to the dosing plan. During the whole experiment, measure the body weight and tumor size of mice twice a week.

[1183] Tumor size calculation formula: Tumor volume (mm 3 ) = 0.5 × (tumor long diameter × tumor short diameter 2 ).

[1184] The experimental results are shown in Tables 13 - 18:

[1185] Table 13

[1186]

[1187] Table 14

[1188]

[1189] Table 15

[1190]

[1191]

[1192] Table 16

[1193]

[1194] Table 17

[1195]

[1196] Table 18

[1197]

[1198]

[1199] The results showed that: compared with Compounds 1-3 and Trastuzumab, the compounds of the present invention not only achieved systemic administration, but also showed better pharmacodynamic effects in the subcutaneous xenograft tumor model of mouse colon cancer CT26-hHER2.

[1200] Experimental Example 9: In Vivo Pharmacodynamic Experiment of Subcutaneous Xenograft Tumor Model of Mouse Colon Cancer CT26

[1201] Cell culture: Mouse colon cancer CT26 cells were maintained in a monolayer in RPMI-1640 medium containing 10% fetal bovine serum in a constant temperature incubator at 37 °C with 5% CO2. After treatment with trypsin-EDTA, the cells were subcultured twice a week. Cells in the exponential growth phase were harvested and counted for inoculation.

[1202] Experimental animals: BALB / c mice, 6-8 weeks old, 16-18 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[1203] Twelve experimental groups were set up for II-42, II-58, I-3, I-38, I-43, I-44, I-53, I-58, I-59, I-61, I-64 as shown in Table 19 below:

[1204] Table 19

[1205] Group Number of mice Test compound Dose Route of administration Dosing schedule 1 4 Vehicle -- i.p. QW×2 2 4 II-42 1 mg / kg i.p. QW×2 3 4 II-58 1 mg / kg i.p. QW×2 4 4 I-3 0.15 mg / kg i.p. QW×2 5 4 I-43 0.15 mg / kg i.p. QW×2 6 4 I-44 0.15 mg / kg i.p. QW×2 7 4 I-58 0.15 mg / kg i.p. QW×2 8 4 I-61 0.15 mg / kg i.p. QW×2 9 4 I-64 0.15 mg / kg i.p. QW×2 10 4 I-53 0.15 mg / kg i.p. QW×2 11 4 I-38 0.15 mg / kg i.p. QW×2 12 4 I-59 0.15 mg / kg i.p. QW×2

[1206] Note, i.p.: intraperitoneal injection

[1207] Experimental method: The CT26 cell line (3.0×10 6 cells / mouse) was inoculated subcutaneously into the right back of experimental mice, and the inoculation volume for each mouse was 0.1 mL. The growth of tumors was observed regularly. When the tumors grew to about 100 mm 3When the tumor size and mouse body weight were around [specific value], the mice were randomly grouped and administered according to the dosing plan. During the entire experiment, the body weight and tumor size of the mice were measured twice a week.

[1208] Tumor size calculation formula: Tumor volume (mm 3 ) = 0.5 × (tumor major axis × tumor minor axis 2 ).

[1209] The experimental results are shown in Table 20:

[1210] Table 20

[1211]

[1212]

[1213] Experimental Example 10: In Vivo Pharmacodynamic Experiment of Mouse Melanoma B16F10-hHER2 Subcutaneous Transplantation Tumor Model

[1214] Cell culture: Mouse melanoma B16F10-hHER2 cells were maintained in a monolayer in DMEM medium containing 10% fetal bovine serum and 1 μg / mL puromycin in a constant temperature incubator at 37°C with 5% CO2. The tumor cells were passaged twice a week. Cells in the exponential growth phase were harvested and counted for inoculation.

[1215] Experimental animals: C57 mice, 6 - 9 weeks old, 18 - 22 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[1216] Four experimental groups were set up for II-5, C-7, and Trastuzumab as shown in Table 21 below:

[1217] Table 21

[1218] Group Number of mice Test compound Dose Dose (small molecule content) Route of administration Dosing schedule 1 6 Vehicle -- -- i.t. 2 doses 2 6 Trastuzumab 10 mg / kg -- i.p. 2 doses 3 6 II-5 0.25 mg / kg 0.01 mg / kg i.p. 2 doses 4 6 C-7 0.25 mg / kg 0.01 mg / kg i.p. 2 doses

[1219] Note: i.t.: Intratumoral injection, i.p.: Intraperitoneal injection

[1220] Experimental method: The B16F10-hHER2 cell line (1.0×10 5 cells / mouse) was inoculated subcutaneously on the right back of the experimental mice, and the inoculation volume for each mouse was 0.1 mL. The growth of the tumor was observed regularly. When the tumor grew to about 100 mm 3 or so, the mice were randomly grouped according to the tumor size and mouse body weight, and administered on the 14th and 21st days according to the dosing plan. During the entire experiment, the body weight and tumor size of the mice were measured three times a week.

[1221] Tumor size calculation formula: Tumor volume (mm 3 ) = 0.5 × (tumor major axis × tumor minor axis2 )。

[1222] The experimental results are shown in Table 22:

[1223] Table 22

[1224]

[1225]

[1226] The results show that: in the subcutaneous xenograft tumor model of murine melanoma B16F10-hHER2, the efficacy of II-5 is significantly better than that of C-7 and Trastuzumab.

[1227] Experimental Example 11: In Vivo Efficacy Experiment of Subcutaneous Xenograft Tumor Model of Human Lung Cancer Cells NCI-H1373

[1228] Cell culture: Human lung cancer NCI-H1373 cells were maintained in a monolayer in a constant temperature incubator containing 10% fetal bovine serum and RPMI-1640 medium at 37°C with 5% CO2. The tumor cells were passaged twice a week. Cells in the exponential growth phase were harvested and counted for inoculation.

[1229] Experimental animals: Balb / c nude mice, 6 - 8 weeks old, 20 - 25 g, purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.

[1230] Two experimental groups were set up for II-63 as shown in Table 23 below:

[1231] Table 23

[1232] Group Number of mice Test compound Dose Route of administration Dosing schedule 1 3 Vehicle -- i.v. QW×2 2 3 II-63 1 mg / kg i.v. QW×2

[1233] Note: i.v.: intravenous injection

[1234] Experimental method: The NCI-H1373 cell line (5×10 6 cells / mouse) was inoculated subcutaneously on the right back of the experimental mice, and the inoculation volume for each mouse was 0.1 mL. The growth of the tumor was observed regularly. When the tumor grew to about 100 mm 3 or so, the mice were randomly grouped according to the tumor size and body weight, and administered drugs according to the dosing plan. During the whole experimental process, the body weight and tumor size of the mice were measured twice a week.

[1235] Tumor size calculation formula: Tumor volume (mm 3 ) = 0.5×(tumor long diameter × tumor short diameter 2 ).

[1236] The experimental results are shown in Table 24:

[1237] Table 24

[1238]

[1239] Experimental Example 12: In Vivo Efficacy Experiment of Subcutaneous Xenograft Tumor Model of Human Pancreatic Cancer Cells HPAC

[1240] Cell culture: Human pancreatic cancer HPAC cells were maintained in a monolayer in a constant temperature incubator containing 10% fetal bovine serum and RPMI-1640 medium at 37°C with 5% CO2. The tumor cells were passaged twice a week. Cells in the exponential growth phase were harvested and counted for inoculation.

[1241] Experimental animals: Balb / c nude mice, 6 - 8 weeks old, 20 - 25 g, purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.

[1242] Two experimental groups were set up for II-63 as shown in Table 25 below:

[1243] Table 25

[1244] Group Number of mice Test compound Dose Route of administration Dosing schedule 1 3 Vehicle -- i.v. QW×2 2 3 II-63 1 mg / kg i.v. QW×2

[1245] Note: i.v.: Intravenous injection

[1246] Experimental method: The HPAC cell line (5×10 6 cells / mouse) was inoculated subcutaneously on the right back of the experimental mice, and the inoculation volume for each mouse was 0.1 mL. The growth of the tumor was observed regularly. When the tumor grew to about 100 mm 3 or so, the mice were randomly grouped according to the tumor size and body weight, and administered drugs according to the dosing plan. During the whole experiment, the body weight and tumor size of the mice were measured twice a week.

[1247] Tumor size calculation formula: Tumor volume (mm 3 ) = 0.5×(tumor long diameter × tumor short diameter 2 ).

[1248] The experimental results are shown in Table 26:

[1249] Table 26

[1250]

[1251] Experimental Example 13: In Vivo Efficacy Experiment of Subcutaneous Xenograft Tumor Model of Human Breast Cancer MDA-MB-468

[1252] Cell culture: Human breast cancer MDA-MB-468 cells were maintained in a monolayer in a constant temperature incubator containing 10% fetal bovine serum and a culture medium containing 90% DMEM at 37°C with 5% CO2. The tumor cells were passaged twice a week. Cells in the exponential growth phase were harvested and counted for inoculation.

[1253] Experimental animals: BALB / c-nude mice, 6 - 8 weeks old, 18 - 22 g, purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[1254] Four experimental groups were set up for II - 48 and II - 63 as shown in Table 27 below:

[1255] Table 27

[1256] Group Number of mice Test compound Dose Route of administration Dosing schedule 1 5 Vehicle -- i.v. QW×3 2 5 Antibody 8 10 mg / kg i.v. QW×3 3 5 II-48 1 mg / kg i.v. QW×3 4 5 II-63 1 mg / kg i.v. QW×3

[1257] Note: i.v.: intravenous injection

[1258] Experimental method: The MDA - MB - 468 cell line (2.5×10 6 cells / mouse) was inoculated subcutaneously into the right back of experimental mice, with an inoculation volume of 0.2 mL per mouse. The growth of tumors was observed regularly. When the tumors grew to about 250 mm 3 or so, the mice were randomly grouped according to the tumor size and body weight, and administered drugs according to the dosing plan. During the whole experimental process, the body weight and tumor size of the mice were measured twice a week.

[1259] Tumor size calculation formula: Tumor volume (mm 3 ) = 0.5×(long diameter of tumor × short diameter of tumor 2 ).

[1260] The experimental results are shown in Table 28:

[1261] Table 28

[1262]

[1263]

[1264] Experimental Example 14: In - vivo pharmacodynamic experiment of subcutaneous xenograft tumor model of colon cancer B - hNECTIN4 MC38

[1265] Cell culture: Colon cancer B - hNECTIN4 MC38 cells (Beijing Bioocytogen Co., Ltd.) were maintained in a monolayer in a constant temperature incubator at 37°C with 5% CO2 in RPMI - 1640 medium containing 10% fetal bovine serum. The tumor cells were passaged twice a week. Cells in the exponential growth phase were harvested and counted for inoculation.

[1266] Experimental animals: C57BL / 6 mice, 6 - 8 weeks old, 17 - 21 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[1267] Three experimental groups were set up for II - 83 as shown in Table 29 below:

[1268] Table 29

[1269] Group Number of mice Test compound Dose Route of administration Dosing schedule 1 5 Vehicle -- i.v. QW×2 2 5 Antibody 11 1 mg / kg i.v. QW×2 3 5 II-83 1 mg / kg i.v. QW×2

[1270] Note: i.v.: intravenous injection

[1271] Experimental method: Inoculate the B-hNECTIN4 MC38 cell line (5×10 5 cells / mouse) subcutaneously into the right dorsal side of experimental mice, with the inoculation volume of 0.1 mL per mouse. Regularly observe the growth of tumors. When the tumors grow to about 100 mm 3 or so, randomly group the mice according to the tumor size and body weight, and administer drugs according to the dosing plan. During the whole experimental process, measure the body weight and tumor size of mice twice a week.

[1272] Tumor size calculation formula: Tumor volume (mm 3 ) = 0.5×(long diameter of tumor × short diameter of tumor 2 ).

[1273] The experimental results are shown in Table 30:

[1274] Table 30

[1275]

[1276] Experimental Example 15: In Vivo Efficacy Experiment of Subcutaneous Xenograft Tumor Model of Mouse Colon Cancer CT26-hEGFR

[1277] Cell culture: Mouse colon cancer CT26-hEGFR cells are maintained in a monolayer in a constant temperature incubator containing 10% fetal bovine serum and RPMI-1640 medium at 37°C with 5% CO2. The tumor cells are passaged twice a week. Collect the cells in the exponential growth phase and count them for inoculation.

[1278] Experimental animals: Balb / c mice, 6 - 8 weeks old, 18 - 22 g, purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.

[1279] Set 4 experimental groups for II-47, II-85, II-86 as shown in Table 31 below:

[1280] Table 31

[1281] Group Number of mice Test compound Dose Route of administration Dosing schedule 1 5 Vehicle -- s.c. QW×2 2 5 II-47 1 mg / kg s.c. QW×2 3 5 II-85 1 mg / kg s.c. QW×2 4 5 II-86 1 mg / kg s.c. QW×2

[1282] Note: s.c.: subcutaneous injection

[1283] Experimental method: Inoculate the CT26-hEGFR cell line (5×10 6 cells / mouse) subcutaneously into the right dorsal side of experimental mice, with the inoculation volume of 0.1 mL per mouse. Regularly observe the growth of tumors. When the tumors grow to about 100 mm 3Around [time], they were randomly grouped according to tumor size and mouse body weight, and administered drugs according to the dosing plan. During the entire experiment, the body weight and tumor size of the mice were measured twice a week.

[1284] Tumor size calculation formula: Tumor volume (mm 3 ) = 0.5 × (tumor major axis × tumor minor axis 2 ).

[1285] The experimental results are shown in Table 32:

[1286] Table 32

[1287]

Claims

1. An antibody-immunostimulatory conjugate as shown in formula II or a pharmaceutically acceptable salt thereof, in, Ab is antibody; t is 1 to 8; L is a linker having the following combination: -(L1) a -(Z) b -M-; L1 is connected to D, M is connected to Ab; a is 0, 1, 2, 3, 4, 5 or 6; b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; D is The group formed by the compound shown losing a hydrogen atom; the stereo configurations marked with * are independently R, S or R / S; B1 and B2 are independently And, at least one of B1 and B2 is R1 and R2 are independently -OH, -N3, F, -OCH3 or -SCF3; R3 and R 3’ are independently H, -C(O)R4, -C(O)OR4, -OR4 or -R4; R4 is C 1-10 Alkyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 3-10 Cycloalkyl, 3-10 membered heterocycloalkyl, C 6-10 Aryl C 1-10 Alkyl, 5-10 membered heteroaryl C 1-10 Alkyl, C 3-10 Cycloalkyl C 1-10 Alkyl, 3-10 membered heterocycloalkyl C 1-10 Alkyl; said R4 is unsubstituted, or is selectively substituted by 1 to 3 groups selected from hydroxyl, thiol, dithiol, amino, C 1-6 Alkylamino, C 1-6 Alkylamino C 1-10 The substituents of the alkyl group are substituted at any position; R5 is H, -(L2) d -(Z) e -(maleimido) or -(L2) d -(Z) e -H; d is 0, 1, 2, 3, 4, 5 or 6; e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Each L1 and each L2 are independently The c2 side of L1 is connected to D; the c2 side of L2 is connected to the thiol group of D; X is p is independently 1, 2 or 3; Q is a linking bond, -C(O)O-, -C(O)N(R6)- or -C 1-3 Alkylene-O-; Q1 is phenyl or pyridyl; R6 is H or C 1-6 Alkyl; the C 1-6 The alkyl group is unsubstituted or optionally substituted with 1 C 1-6 Alkylamino or C 1-6 Alkylsulfonyl is substituted at any position; R7 and R 7’ are independently H or C 1-6 alkyl; R8 is phenyl or 5-10 membered heteroaryl; said R8 is unsubstituted or selectively substituted with 1-3 groups selected from hydroxyl, amino, carboxyl, cyano, nitro, thiol, C 1-6 Alkyl, C 3-8 Cycloalkyl, halogen, halogenated C 1-6 Alkyl, C 1-6 Alkylamino, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, C 1-6 Alkylthiol, amide, hydroxyamino, aldehyde, acetyl, methyl ester, amino C 1-6 Alkyl, hydroxyl C 1-6 Alkyl and C 1-6 The substituent of the alkylsulfonyl group is substituted at any position; Each Z is independently -(A) v -、-PEG-、-C(O)-(CH2) x -、-NR9-(CH2) y -、-O-(CH2) y -、-S-(CH2) y -、-(CH2) x -C 6-14 Arylene-(CH2) y -、-(CH2) x -5-6 membered heteroarylene-(CH2) y -、-(CH2) x -C 3-6 Cycloalkylene-(CH2) y -、-(CH2) x -3-6 membered heterocycloalkylene-(CH2) y -、-NR9-(CH2) x -C(O)-, -O-(CH2) x -C(O)-, -S-(CH2) x -C(O)-, -(CH2) x CH(-L3-R9)-C(O)-, -(CH2) x CH(-L3-R 9a )-C(O)-、-(CH2) x -SS-(CH2) y -、C 1-6 Alkylene or C 2-6 alkenylene; L3 is a connecting bond, -NH- or R9 is independently hydrogen, C 1-6 Alkyl, -PO(OH)2, -PO(OCH3)2, -C(O)-(CH2CH2O) n -CH3, -(CH2CH2O) n -CH3, R 9a Independently -PEG- is -(CH2CH2O) n -(CH2) u -or-(CH2CH2O) n -(CH2) u -C(O)-; A is independently an amino acid residue; x and y are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; v is independently 1, 2, 3, 4, or 5; n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50; u is independently 0, 1, 2, 3, 4, or 5; M is a connector connected to Ab.

2. The antibody-immunostimulatory conjugate of formula II or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The antibody-immunostimulatory conjugate as shown in formula II satisfies one or more of the following conditions: (1) Ab is anti-HER2 antibody, anti-EGFR antibody or anti-5T4 antibody; (2) t is any value from 2 to 8; (3) Each L1 and each L2 are independently The c2 side of L1 is connected to D; the c2 side of L2 is connected to the thiol group of D; (4) Z is independently -(A) v -、-PEG-、-C(O)-(CH2) x -、-NR9-(CH2) y -、-O-(CH2) y -、-S-(CH2) y -、-(CH2) x -C 6-14 Arylene-(CH2) y -、-(CH2) x -5-6 membered heteroarylene-(CH2) y -、-(CH2) x -C 3-6 Cycloalkylene-(CH2) y -、-(CH2) x -3-6 membered heterocycloalkylene-(CH2) y -、-NR9-(CH2) x -C(O)-, -O-(CH2) x -C(O)-, -S-(CH2) x -C(O)-, -(CH2) x CH(NHR9)-C(O)-、-(CH2) x -SS-(CH2) y -、C 1-6 Alkylene or C 2-6 alkenylene; x and y are independently any integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; v is independently any integer of 1, 2, 3, 4 or 5; the -(CH2) x -5-6 membered heteroarylene-(CH2) y -, in the "5- to 6-membered heteroarylene" group, the heteroatom is selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3; the -(CH2) x -3-6 membered heterocycloalkylene-(CH2) y -In the "3- to 6-membered heterocycloalkylene" in, the heteroatom is selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3; (5) In M, the linker is a group formed by click chemistry reaction connection, click-like chemistry reaction connection, sulfhydryl connection, amino connection, oxime connection or hydrazone connection; (6)D is The group formed by the loss of a hydrogen atom from the compound shown; for for 3. The antibody-immunostimulatory conjugate of formula II or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: The antibody-immunostimulatory conjugate as shown in formula II satisfies one or more of the following conditions: (1) Ab is trastuzumab, pertuzumab, cetuximab, huA1, cetuximab, nimotuzumab or a variant thereof; (2) t is any value from 6 to 8; (3)-(L1) a - does not exist, The c2 side is connected to D; (4)-(Z) b - is any of the following combinations: -(CH2) x -(c1), -(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -(c1), -C(O)-(CH2) x -(c1), -C(O)-(CH2) x -C 3-6 Cycloalkylene-(CH2) y -(c1), -C(O)-(CH2) x -phenylene-(CH2) y -(c1), -C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -(c1), -C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -phenylene-(CH2) y -(c1), -(A) v -(c1), -(A) v -C(O)-(CH2) x -(c1), -(A) v -C(O)-(CH2) x -phenylene-(CH2) y -(c1), -(A) v -C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -(c1), -(A) v -C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -phenylene-(CH2) y -(c1), -C(O)-(CH2) x -(A) v -C(O)-(CH2) x -(c1), -(A) v -C(O)-(CH2CH2O) n -(CH2) u -(c1), -C(O)-(CH2) x -NH-(c1), -C(O)-CH(-L3-R9)-NHC(O)-(CH2) x -phenylene-(CH2) y -or-C(O)-CH(-L3-R9)-NHC(O)-(CH2) x -, where the c1 side is connected to M; (5) In M, the connector is The connector is connected to the rest of L via the c side; (6) R5 is H, -L2-(Z) e -(maleimido) or -L2-(Z) e -H.

4. The antibody-immunostimulatory conjugate of formula II or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The antibody-immunostimulatory conjugate as shown in formula II satisfies one or more of the following conditions: (1)-(L1) a -for The c2 side is connected to D; (2) Each A is independently Each R A Independently amino acid side chains or 1 to 3 R 10 Modified amino acid side chain; or R A Together with the adjacent nitrogen atom, it forms a five-membered heterocyclic group; The R 10 Independently for C 1-6 Alkyl, C 1-6 Alkyl acyl, C 1-6 Alkoxyacyl, -C(O)-(CH2CH2O) n -CH3 or -(CH2CH2O) n -CH3; said C 1-6 Alkyl, C 1-6 Alkyl acyl or C 1-6 Alkoxyacyl is optionally substituted by 1 R8 at any position; (3) R6 is H or C 1-6 Alkyl; the C 1-6 The alkyl group is unsubstituted or selectively substituted with one dimethylamino or methylsulfonyl group at any position; (4) B1 and B2 are independently And, at least one of B1 and B2 is 5. The antibody-immunostimulatory conjugate of formula II or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: -(A) v -for Each R A are independently hydrogen, methyl, ethyl, n-propyl, isopropyl, isobutyl, or R A and the adjacent nitrogen atom form a five-membered heterocyclic group; v is 1, 2, 3 or 4; preferably, -(A) v -for 6. The antibody-immunostimulatory conjugate of formula II or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R5 is -L2-(Z) e -H;-L2- The c2 side is connected to the thiol group in D; e is 1; -(Z) e -H is C 1-6 alkyl; Or, R5 is -L2-(Z) e -H;-L2- The c2 side is connected to the thiol in D; e is 3, -(Z) e -H is -C(O)NH-(CH2) y -O-(CH2) y -H; Or, R5 is Q is a connecting bond, -C(O)O- or -C 1-3 Alkylene-O-; R7 and R 7’ are each independently H or methyl; Or, R5 is Q1 is phenyl; R7 and R 7’ are each independently H or methyl; Or, R5 is -L2-(Z) e -(maleimide); -L2- is The c2 side is connected to the thiol group in D; -(Z) e -for-(A) v -C(O)-(CH2) x -(c3); the c3 side is connected to the maleimide group; and / or, R8 is phenyl or 5-6 membered heteroaryl; the 5-6 membered heteroaryl is preferably pyridyl, pyrimidinyl, pyrazinyl, pyrrolyl, thienyl, furyl, pyrazolyl, thiazolyl or oxazolyl; R8 is unsubstituted or selectively substituted with 1 to 3 groups selected from hydroxyl, amino, cyano, carboxyl, nitro, thiol, C 1-6 Alkyl, halogen, halogenated C 1-6 Alkyl, C 1-6 Alkylamino, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, C 1-6 Alkylthiol, amide, aldehyde, acetyl, methyl ester, amino C 1-6 Alkyl, hydroxyl C 1-6 The substituents of the alkyl group and the methylsulfonyl group may be substituted at any position.

7. The antibody-immunostimulatory conjugate of formula II or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: D is The group formed by the loss of a hydrogen atom from an amino or thiol group in the compound shown; Or, D is The group formed by the amino group in the compound shown loses a hydrogen atom; R3 or R 3’ Separately Or, D is The group formed by the loss of a hydrogen atom from the thiol group in the compound shown; Or, D is The group formed by the loss of a hydrogen atom from the thiol group in the compound shown.

8. The antibody-immunostimulatory conjugate of formula II or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Or, D is any of the following structures: or a pharmaceutically acceptable salt thereof.

9. The antibody-immunostimulatory conjugate of formula II or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The antibody-immunostimulatory conjugate shown in formula II is any antibody-immunostimulatory conjugate in Table A.

10. A cyclic dinucleotide compound as shown in formula I or a pharmaceutically acceptable salt thereof, D—LX (I) in, LX is a linker precursor having the following combination: - (L1) a -(Z) b -M'; L1 is connected to D; M' is a connector precursor; L1, Z, D, a and b are as defined in any one of claims 1-9.

11. The cyclic dinucleotide compound of formula I or a pharmaceutically acceptable salt thereof according to claim 10, characterized in that: M' is a linker precursor for reacting with the amino or thiol side chains of the antibody amino acid residues; preferably ethynyl, vinyl, hydroxylamine, R 11 is hydrogen or C 1-4 alkyl; Each R 12 and R 12’ are independently halogen, nitro or -SO3 - ; R 13 and R 13’ are each independently hydrogen, halogen, phenylthio or pyridylthio; More preferably 12. The cyclic dinucleotide compound of formula I or a pharmaceutically acceptable salt thereof according to claim 10, characterized in that: LX 13. The cyclic dinucleotide compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 10 to 12, characterized in that: The cyclic dinucleotide compound as shown in Formula I is or a pharmaceutically acceptable salt thereof.

14. A compound of formula D'-1 or D'-2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; B1 and B2 are independently And, at least one of B1 and B2 is for for R1 and R2 are independently -OH, -N3, F, -OCH3 or -SCF3; R5 is -(L2) d -(Z) e -H; d is 0, 1, 2, 3, 4, 5 or 6; e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Each L2 is independently The c2 side is connected to D; X is p is independently 1, 2 or 3; Q is a linking bond, -C(O)O-, -C(O)N(R6)- or -C 1-3 Alkylene-O-; Q1 is phenyl or pyridyl; R6 is H or C 1-6 Alkyl; the C 1-6 The alkyl group is unsubstituted or optionally substituted with 1 C 1-6 Alkylamino or C 1-6 Alkylsulfonyl is substituted at any position; R7 and R 7’ are independently H or C 1-6 alkyl; R8 is phenyl or 5-10 membered heteroaryl; said R8 is unsubstituted or selectively substituted with 1 to 3 groups selected from hydroxyl, amino, carboxyl, cyano, nitro, thiol, C 1-6 Alkyl, C 3-8 Cycloalkyl, halogen, halogenated C 1-6 Alkyl, C 1-6 Alkylamino, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, C 1-6 Alkylthiol, amide, hydroxyamino, aldehyde, acetyl, methyl ester, amino C 1-6 Alkyl, hydroxyl C 1-6 Alkyl and C 1-6 The substituent of the alkylsulfonyl group is substituted at any position; Each Z is independently -C(O)-(CH2) x -、-NH-(CH2) y -、-O-(CH2) y -、C 1-6 Alkylene or C 2-6 alkenylene; Each x and each y is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

15. The compound of formula D'-1 or D'-2, its stereoisomer or pharmaceutically acceptable salt thereof according to claim 14, characterized in that: R5 is -L2-(Z) e -H;-L2- The c2 side is connected to the thiol group in D; e is 1; -(Z) e -H is C 1-6 alkyl; Or, R5 is -L2-(Z) e -H;-L2- The c2 side is connected to the thiol in D; e is 3, -(Z) e -H is -C(O)NH-(CH2) y -O-(CH2) y -H; Or, R5 is Q is a connecting bond, -C(O)O- or -C 1-3 Alkylene-O-; R7 and R 7’ are each independently H or methyl; Or, R5 is Q1 is phenyl; R7 and R 7’ are each independently H or methyl; and / or, R8 is phenyl or 5-6 membered heteroaryl; the 5-6 membered heteroaryl is preferably pyridyl, pyrimidinyl, pyrazinyl, pyrrolyl, thienyl, furanyl, pyrazolyl, thiazolyl or oxazolyl; R8 is unsubstituted or selectively substituted with 1 to 3 groups selected from hydroxyl, amino, cyano, nitro, mercapto, C 1-6 Alkyl, halogen, halogenated C 1-6 Alkyl, C 1-6 Alkylamino, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, C 1-6 Alkylthiol, amide, aldehyde, acetyl, methyl ester, amino C 1-6 Alkyl, hydroxyl C 1-6 The substituents of the alkyl group and the methylsulfonyl group may be substituted at any position.

16. The compound of formula D'-1 or D'-2, its stereoisomer or pharmaceutically acceptable salt thereof according to claim 15, characterized in that: R5 is The c2 side is connected to the thiol group in D.

17. The compound of formula D'-1 or D'-2, its stereoisomer or pharmaceutically acceptable salt thereof according to claim 14, characterized in that: The formula D'-1 or D'-2 is any of the following structures: or a pharmaceutically acceptable salt thereof.

18. A pharmaceutical composition comprising a substance K and a pharmaceutically acceptable excipient; the substance K is an antibody-immunostimulatory conjugate as shown in formula II as described in any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, or a cyclic dinucleotide compound as shown in formula I as described in any one of claims 10 to 13 or a pharmaceutically acceptable salt thereof, or a compound as shown in formula D'-1 or D'-2 as described in any one of claims 14 to 17, a stereoisomer or a pharmaceutically acceptable salt thereof.

19. Use of a substance K or a pharmaceutical composition as claimed in claim 17 in the preparation of a drug for treating and / or alleviating tumors, wherein the substance K is an antibody-immunostimulatory conjugate as shown in formula II as described in any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, or a cyclic dinucleotide compound as shown in formula I as described in any one of claims 10 to 13 or a pharmaceutically acceptable salt thereof, or a compound as shown in formula D'-1 or D'-2 as described in any one of claims 14 to 17, a stereoisomer or a pharmaceutically acceptable salt thereof.

20. Use of a substance K or a pharmaceutical composition as claimed in claim 17 in the preparation of a drug for treating and / or alleviating tumors, wherein the substance K is an antibody-immunostimulatory conjugate as shown in formula II as described in any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, or a cyclic dinucleotide compound as shown in formula I as described in any one of claims 10 to 13 or a pharmaceutically acceptable salt thereof, or a compound as shown in formula D'-1 or D'-2 as described in any one of claims 14 to 17, a stereoisomer or a pharmaceutically acceptable salt thereof, and the substance K or the pharmaceutical composition is used in combination with one or more other types of therapeutic agents.

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