Cyclic dinucleotide compound, conjugate thereof, and use thereof

Abstract

Disclosed in the present invention are a cyclic dinucleotide compound, a conjugate thereof, and use thereof, and particularly disclosed is an antibody-immunostimulant conjugate represented by formula II or a pharmaceutically acceptable salt thereof. The antibody-immunostimulant conjugate provided by the present invention has good tumor specificity and exhibits a significant inhibition effect on tumors.

Description

Cyclic dinucleotide compound, conjugate and application thereof

[0001] This application claims priority to Chinese Patent Application No. 2023118185393, filed December 27, 2023. This application incorporates the entirety of the aforementioned Chinese Patent Application. Technical Field

[0002] The present invention relates to a cyclic dinucleotide compound, a conjugate thereof, a pharmaceutical composition and applications. 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 five transmembrane structures and plays an important regulatory role in the immune response process associated with viral or bacterial infection. As a pattern recognition receptor, STING can detect and recognize exogenous nucleic acids in the cytoplasm and activate signal transduction pathways related to type I interferon response. In addition, studies have shown that STING participates in the regulation of cell apoptosis signals by interacting with type II major histocompatibility complex (MHCII). Studies on human tumors with spontaneous T cell infiltration have shown that CD8+ T cell infiltration is closely related to the transcriptional characteristics of type I interferon (Harlin et al, Cancer Res, 2009; 69(7):OF1). Mechanistic studies conducted in mouse models have shown that in experimental animals with type I interferon signaling defects, 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 innate immune system's tumor recognition process in vivo and on related signaling pathways such as tumor-induced IFN expression mediated by antigen-presenting cells (APCs) 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) and catalyzed to produce cyclic nucleic acid substances, such as cyclic GMP-AMP (cGAMP), which can act as endogenous ligands to activate STING signaling (Sun et al, Science, 2013; 339(15): 786). Activated STING subsequently induces autophosphorylation of TBK1 kinase and phosphorylation of interferon regulatory factor 3 (IRF-3). Phosphorylated IRF3 further activates the transcription of type I interferon genes, regulating their synthesis and secretion, and ultimately inducing an immune response. In summary, existing studies have demonstrated that the STING signaling pathway plays a crucial role in tumor recognition by the innate immune system. Activation of this signaling pathway on antigen-presenting cells is directly linked to T cell activation against tumor-associated antigens. Given its role in tumor immune recognition, it is anticipated that activating STING signaling through drugs or other pharmacological approaches could enhance IFN expression and potentially have a positive effect on tumor treatment. Therefore, the development of STING signaling agonists for the treatment of tumors has become a research hotspot.

[0004] Additionally, studies have shown that stimulating the activation of the STING signaling pathway also contributes to antiviral responses. Loss of a 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 the production of anti-vascular and pro-inflammatory cytokines to combat the virus and mobilizing both the innate and adaptive immune systems. Therefore, small molecule compounds that agonize the STING signaling pathway have the potential to treat chronic viral infections, such as HBV.

[0005] Currently, cyclic dinucleotide (CDN) analogs of STING agonists are mostly administered via intratumoral injection. This approach has several drawbacks, including limited tumor accessibility, tumor exposure, and nonspecificity. Currently, no CDN analogs are available for ADC therapy. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of existing cyclic dinucleotide analog STING agonists, such as their single administration route, poor tumor accessibility, and low tumor specificity. The present invention provides a cyclic dinucleotide compound, an antibody conjugate thereof, a pharmaceutical composition thereof, and uses thereof. The cyclic dinucleotide compound-antibody conjugate of the present invention exhibits good tumor specificity and 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 as shown in Formula II or a pharmaceutically acceptable salt thereof,

[0009] Wherein, Ab is antibody;

[0010] t is 1 to 8;

[0011] L is a linker having the following combination: -(L1) a -(Z) b -M-; L1 is connected to D, M is connected to Ab;

[0012] a is 0, 1, 2, 3, 4, 5 or 6; b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0013] D is The group formed by the loss of a hydrogen atom from the compound shown; * indicates that the stereo configuration of P is independently R, S or R / S;

[0014] B1 and B2 are independently Moreover, at least one of B1 and B2 is

[0015] R1 and R2 are independently -OH, -N3, F, -OCH3 or -SCF3;

[0016] R3 and R 3’ are independently H, -C(O)R4, -C(O)OR4, -OR4 or -R4;

[0017] 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;

[0018] R5 is H, -(L2) d -(Z) e -(maleimido) or -(L2) d -(Z) e -H;

[0019] d is 0, 1, 2, 3, 4, 5 or 6; e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0020] 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 sulfhydryl group of D;

[0021] X is

[0022] p is independently 1, 2 or 3;

[0023] Q is a connecting bond, -C(O)O-, -C(O)N(R6)- or -C 1-3 Alkylene-O-;

[0024] Q1 is phenyl or pyridyl;

[0025] R6 is H or C 1-6 Alkyl; the C 1-6The alkyl group is unsubstituted or optionally substituted with 1 C 1-6 Alkylamino or C 1-6 Alkylsulfonyl is substituted at any position;

[0026] R7 and R 7’ are independently H or C 1-6 alkyl;

[0027] R8 is phenyl or 5-10 membered heteroaryl; said R8 is unsubstituted or selectively substituted by 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;

[0028] 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 -3-6 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;

[0029] L3 is a connecting bond, -NH- or

[0030] R9 is independently hydrogen, C 1-6 Alkyl, -PO(OH)2, -PO(OCH3)2, -C(O)-(CH2CH2O) n -CH3, -(CH2CH2O) n -CH3,

[0031] R 9a Independently

[0032] -PEG- is -(CH2CH2O) n -(CH2) u -or-(CH2CH2O) n -(CH2) u -C(O)-;

[0033] A is independently an amino acid residue;

[0034] 47, 48, 49, or 50; u is independently 0, 1, 2, 3, 4, or 5;

[0035] M is the connector connected to Ab.

[0036] In some embodiments, in the antibody-immunostimulatory conjugate of Formula II or a pharmaceutically acceptable salt thereof, some of the groups are defined as follows, and the remaining groups are defined as described in any other embodiment (hereinafter referred to as "in some embodiments"):

[0037] Ab is antibody;

[0038] t is 1 to 8;

[0039] L is a linker having the following combination: -(L1) a -(Z) b-M-; L1 is connected to D, M is connected to Ab;

[0040] a is 0, 1, 2, 3, 4, 5 or 6; b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0041] D is The group formed by the loss of a hydrogen atom from the compound shown; * indicates that the stereo configuration of P is independently R, S or R / S;

[0042] B1 and B2 are independently Moreover, at least one of B1 and B2 is

[0043] R1 and R2 are independently -OH, -N3, F, -OCH3 or -SCF3;

[0044] R3 and R 3’ are independently H, -C(O)R4, -C(O)OR4, -OR4 or -R4;

[0045] 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;

[0046] R5 is H, -(L2) d -(Z) e -(maleimido) or -(L2) d -(Z) e -H;

[0047] d is 0, 1, 2, 3, 4, 5 or 6; e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0048] Each L1 and each L2 are independently The c2 side is connected to D;

[0049] X is

[0050] p is independently 1, 2 or 3;

[0051] Q is a connecting bond, -C(O)O-, -C(O)N(R6)- or -C 1-3 Alkylene-O-;

[0052] Q1 is phenyl or pyridyl;

[0053] 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;

[0054] R7 and R 7’ are independently H or C 1-6 alkyl;

[0055] R8 is phenyl or 5-10 membered heteroaryl; said R8 is unsubstituted or selectively substituted by 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;

[0056] 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-3-6 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;

[0057] 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)-;

[0058] A is independently an amino acid residue;

[0059] 47, 48, 49, or 50; u is independently 0, 1, 2, 3, 4, or 5;

[0060] M is the connector connected to Ab.

[0061] In some embodiments, the antibody may comprise one or more antigen binding domains that can bind to an antigen.

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

[0063] In some embodiments, the antibody may comprise an antigen binding domain that can bind to an antigen.

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

[0065] In some embodiments, the antibody may comprise an Fc region.

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

[0067] In some embodiments, the antibody may comprise only an antigen-binding domain capable of binding to an antigen and an Fc region.

[0068] In some embodiments, the antibody may be a monoclonal antibody.

[0069] In some embodiments, the antibody is an anti-HER2 antibody, an anti-EGFR antibody, or an anti-5T4 antibody.

[0070] In some embodiments, the anti-HER2 monoclonal antibody includes but is not limited to Trastuzumab, Trastuzumab biosimilar, Pertuzumab, Pertuzumab biosimilar, Margetuximab, HT-19, etc.

[0071] In some embodiments, the antibody is trastuzumab or pertuzumab.

[0072] In some embodiments, the anti-EGFR antibodies include but are not limited to Cetuximab.

[0073] In some embodiments, the anti-EGFR antibody is Cetuximab or Nimotuzumab.

[0074] In some embodiments, the anti-5T4 antibodies include but are not limited to huA1.

[0075] In some embodiments, the antibody is trastuzumab, pertuzumab, cetuximab, or huA1.

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

[0077] In the antibody-immunostimulatory conjugate of Formula II or a pharmaceutically acceptable salt thereof, t is an integer or a non-integer. When it is a non-integer, it means that the antibody-immunostimulatory conjugate of Formula II is a mixture of antibody-immunostimulatory conjugates with different coupling ratios. When it is an integer, it may mean that the antibody-immunostimulatory conjugate of Formula II is a single antibody-immunostimulatory conjugate with a fixed coupling ratio, or it may mean that the antibody-immunostimulatory conjugate of Formula II is a mixture of antibody-immunostimulatory conjugates with different coupling ratios.

[0078] In some embodiments, the t can be any value between 2 and 8.

[0079] In some embodiments, the t can be any value from 1 to 3.

[0080] In some embodiments, the t can be any value between 3 and 5.

[0081] In some embodiments, the t can be any value between 6 and 8.

[0082] In some embodiments, a is 0, 1, 2, 3, or 4; preferably, a is 0, 1, or 2.

[0083] In some embodiments, b is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably b is 1, 2, 3, 4, 5, 6, 7, or 8; more preferably b is 1, 2, 3, 4, 5, or 6.

[0084] In some embodiments, d is 0, 1, 2, or 3; preferably d is 1 or 2; more preferably d is 1.

[0085] In some embodiments, e is 0, 1, 2, 3, 4, 5, or 6; preferably, e is 0, 1, 2, 3, or 4; more preferably, e is 0, 1, 2, or 3.

[0086] In some embodiments, R6 is H or C 1-6 Alkyl; the C 1-6 The alkyl group is unsubstituted or optionally substituted with one dimethylamino group or methylsulfonyl group at any position.

[0087] In some embodiments, R6 is H, -CH3, -CH2CH2N(CH3)2, or -CH2CH2S(O)2CH3.

[0088] In some embodiments, each L1 and each L2 are independently The c2 side in L1 is connected to D; the c2 side in L2 is connected to the thiol group in D.

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

[0090] In some embodiments, -(L1) a -for The c2 side is connected to D.

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

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

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

[0094] In some embodiments, -(L1) a - does not exist, The c2 side is connected to the sulfhydryl group in D.

[0095] 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 -3-6 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- to 6-membered heteroarylene-(CH2) y In the "5- to 6-membered heteroarylene" 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; the -(CH2) x -3-6 membered heterocycloalkylene-(CH2) y In the "3- to 6-membered heterocycloalkylene" in -, the heteroatoms are selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3.

[0096] In some embodiments, in A, the amino acid is a natural amino acid or an unnatural amino acid.

[0097] In some embodiments, in A, the amino acid residue is replaced by 1 to 3 R 10 Modification; R 10 Independently C 1-6 Alkyl, C 1-6 Alkyl acyl, C 1-6 Alkoxyacyl, -C(O)-(CH2CH2O) n -CH3 or -(CH2CH2O) n -CH3; the C 1-6 Alkyl, C 1-6 Alkyl acyl or C 1-6 The alkoxyacyl group may be further substituted by one R8 at any position.

[0098] In some embodiments, 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 and the adjacent nitrogen atom to form a five-membered heterocyclic group;

[0099] The R 10 Independently C 1-6 Alkyl, C 1-6 Alkyl acyl, C 1-6 Alkoxyacyl, -C(O)-(CH2CH2O) n-CH3 or -(CH2CH2O) n -CH3; the C 1-6 Alkyl, C 1-6 Alkyl acyl or C 1-6 The alkoxyacyl group may be further substituted by one R8 at any position.

[0100] In some embodiments, the Can It can also be

[0101] In some embodiments, in A, each R A are independently hydrogen, methyl, ethyl, n-propyl, isopropyl, isobutyl, or R A Together with the adjacent nitrogen atom, it forms a five-membered heterocyclic group.

[0102] In some embodiments, -(A) v -for Each R A are independently hydrogen, methyl, ethyl, n-propyl, isopropyl, isobutyl, or R A Together with the adjacent nitrogen atom, it forms a five-membered heterocyclic group; v is 1, 2, 3 or 4, and v is preferably 1 or 2.

[0103] In some embodiments, -(A) v -for

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

[0105] In some embodiments, -(Z) b -Any combination of the following:

[0106] 1)-(CH2) x -(c1),

[0107] 2)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -(c1),

[0108] 3)-C(O)-(CH2) x -(c1),

[0109] 4)-C(O)-(CH2) x -C 3-6 Cycloalkylene-(CH2) y -(c1),

[0110] 5)-C(O)-(CH2) x -phenylene-(CH2) y -(c1),

[0111] 6)-C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -(c1),

[0112] 7)-C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -phenylene-(CH2) y -(c1),

[0113] 8)-(A) v -(c1),

[0114] 9)-(A) v -C(O)-(CH2) x -(c1),

[0115] 10)-(A) v -C(O)-(CH2) x -phenylene-(CH2) y -(c1),

[0116] 11)-(A) v -C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -(c1) or

[0117] 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.

[0118] In some embodiments, -(Z) b -Any combination of the following:

[0119] 13)-C(O)-(CH2)x -(A) v -C(O)-(CH2) x -(c1);

[0120] 14)-(A) v -C(O)-(CH2CH2O) n -(CH2) u -(c1);

[0121] 15)-C(O)-(CH2) x -NH-(c1);

[0122] 16)-C(O)-CH(-L3-R9)-NHC(O)-(CH2) x -phenylene-(CH2) y -;

[0123] 17)-C(O)-CH(-L3-R9)-NHC(O)-(CH2) x -, where the c1 side is connected to M.

[0124] 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.

[0125] In some embodiments, in M, the linker is a group formed by linking through a sulfhydryl group on the side chain of a cysteine ​​residue or an amino group on the side chain of a lysine residue of the antibody.

[0126] In some embodiments, in M, the linker is a group formed by a thiol connection, preferably M is more preferably

[0127] R 11 is hydrogen or C 1-4 Alkyl; the linker is connected to the rest of L via the C side.

[0128] In some embodiments, R 11 is hydrogen, methyl or ethyl.

[0129] In some embodiments, in M, the linker is a group formed by oxime connection, preferably The connector is connected to the rest of L via the c-side.

[0130] In some embodiments, in M, the linker is a group formed by hydrazone connection, preferably The connector is connected to the rest of L via the c-side.

[0131] In some embodiments, in M, the linker is a group formed by connecting via an amino group, and the group formed by connecting via an amino group is preferably a group formed by connecting via a lysine side chain amino group; more preferably

[0132] In some embodiments, the linker is a group formed by click chemistry reaction connection or click chemistry-like reaction connection, preferably: The connector is connected to the rest of L via the c-side.

[0133] In some embodiments, in M, the linker is The connector is connected to the rest of L via the c-side.

[0134] In some embodiments, L is

[0135] In some embodiments, L is

[0136] In some embodiments, D is The group formed by the loss of a hydrogen atom from the compound shown; for and / or for and / or

[0137] In some embodiments, d is 1.

[0138] In some embodiments, R5 is H, -L2-(Z) e -(maleimido) or -L2-(Z) e -H.

[0139] In some embodiments, 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.

[0140] In some embodiments, R5 is -L2-(Z) e -H;-L2- The c2 side is connected to the thiol group in D; e is 3, -(Z) e -H is -C(O)NH-(CH2) y -O-(CH2) y -H.

[0141] In some embodiments, R5 is -L2-(Z) e -H;-L2- The c2 side is connected to the thiol group in D; e is 0 or 1, -(Z) e -H is H or methyl.

[0142] In some embodiments, 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.

[0143] In some embodiments, R5 is Q1 is phenyl; R7 and R 7’ are each independently H or methyl.

[0144] In some embodiments, 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; the R8 is unsubstituted or selectively substituted by 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.

[0145] In some embodiments, 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.

[0146] In some embodiments, R5 is The c2 side is connected to the thiol group in D.

[0147] In some embodiments, R5 is The c2 side is connected to the thiol group in D.

[0148] In some embodiments, B1 and B2 are each independently Moreover, at least one of B1 and B2 is

[0149] In some embodiments, D is The group formed by losing a hydrogen atom from an amino or thiol group in the compound shown.

[0150] In some embodiments, D is The group formed by the amino group in the compound shown loses a hydrogen atom; R3 or R 3’ Independently

[0151] In some embodiments, D is The group formed by losing a hydrogen atom from the thiol group in the compound shown.

[0152] In some embodiments, D is The group formed by losing a hydrogen atom from the thiol group in the compound shown.

[0153] In some embodiments, R1 is -OCH3; R2 is F.

[0154] In some embodiments, D is any of the following structures or a pharmaceutically acceptable salt thereof:

[0155] Wherein, R1, R2 and R5 are as defined above.

[0156] In some embodiments, D is any of the following structures:

[0157] or a pharmaceutically acceptable salt thereof.

[0158] In some embodiments, D is any of the following structures: or a pharmaceutically acceptable salt thereof.

[0159] In some embodiments, Ab is an anti-HER2 antibody, an anti-EGFR antibody, or an anti-5T4 antibody;

[0160] L is a linker having the following combination: -(L1) a -(Z) b -M-; L1 is connected to D, M is connected to Ab;

[0161] -(L1) a -for

[0162] -(Z) b - is -(CH2) x -(c1) or -C(O)-(CH2) x -phenylene-(CH2) y -;

[0163] x and y are independently 0, 1, 2, 3, 4, 5 or 6;

[0164] M is the connector connected to Ab;

[0165] D is The group formed by the loss of a hydrogen atom from the thiol group in the compound shown;

[0166] R5 is H.

[0167] In some embodiments, the antibody-immunostimulatory conjugate of Formula II or a pharmaceutically acceptable salt thereof is any one of the antibody-immunostimulatory conjugates in Table A:

[0168] Table A

[0169] 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 an engineered Trastuzumab with a cysteine ​​inserted between positions 239 and 240 of the heavy chain; antibody 7 is Cetuximab; antibody 8 is huA1 (V H v2.0+V L v2.4); Antibody 9 is Trastuzumab (HC-s239.5, L234A / L235A / P329G); Antibody 11 is Enfortumab (Ha22-2); Antibody 12 is an antibody prepared using Cetuximab using the method disclosed in Example 25 of CN115209921A; Antibody 13 is Nimotuzumab.

[0170] In some embodiments, Antibody 3 is purchased from Shanghai Bio-Technology Co., Ltd. with catalog number B801901.

[0171] In some embodiments, antibody 4 is purchased from Shanghai Bio-Technology Co., Ltd. with catalog number MHDDD001.

[0172] In some embodiments, antibody 5 is purchased from Shanghai Bio-Technology Co., Ltd. with the product number LPDDFD001.

[0173] [Corrected 28.02.2025 according to Rule 91] In some embodiments, antibody 8 is huA1 (V) expressed and purified according to the sequence information table disclosed in US8044178B2 (SEQ ID NO: 54 and SEQ ID NO: 70). H v2.0+V L v2.4) Antibodies.

[0174] In some embodiments, antibody 9 is purchased from Shanghai Bio-Technology Co., Ltd. with the product number CZ7DUD001.

[0175] The present invention provides a cyclic dinucleotide compound as shown in Formula I or a pharmaceutically acceptable salt thereof,

[0176] D-LX

[0177] (I)

[0178] Wherein, LX is a linker precursor having the following combination: -(L1) a -(Z) b -M'; L1 is connected to D;

[0179] a is 0, 1, 2, 3, 4, 5 or 6; b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0180] D is The group formed by the loss of a hydrogen atom from the compound shown;

[0181] B1 and B2 are independently Moreover, at least one of B1 and B2 is

[0182] R1 and R2 are independently -OH, -N3, F, -OCH3 or -SCF3;

[0183] R3 and R 3’ are independently H, -C(O)R4, -C(O)OR4, -OR4 or -R4;

[0184] 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;

[0185] R5 is H, -(L2) d -(Z) e -(maleimido) or -(L2) d -(Z) e -H;

[0186] d is 0, 1, 2, 3, 4, 5 or 6; e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0187] 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 sulfhydryl group of D;

[0188] X is

[0189] p is independently 1, 2 or 3;

[0190] Q is a connecting bond, -C(O)O-, -C(O)N(R6)- or -C 1-3 Alkylene-O-;

[0191] Q1 is phenyl or pyridyl;

[0192] 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;

[0193] R7 and R 7’ are independently H or C 1-6 alkyl;

[0194] R8 is phenyl or 5-10 membered heteroaryl; said R8 is unsubstituted or selectively substituted by 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;

[0195] 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 -3-6 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;

[0196] L3 is a connecting bond, -NH- or

[0197] R9 is independently hydrogen, C 1-6 Alkyl, -PO(OH)2, -PO(OCH3)2, -C(O)-(CH2CH2O) n -CH3, -(CH2CH2O) n -CH3,

[0198] R 9a Independently

[0199] -PEG- is -(CH2CH2O) n -(CH2) u -or-(CH2CH2O) n -(CH2) u -C(O)-;

[0200] A is independently an amino acid residue;

[0201] 47, 48, 49, or 50; u is independently 0, 1, 2, 3, 4, or 5;

[0202] M' is a linker precursor.

[0203] In some embodiments, LX is a linker precursor having the following combination: - (L1) a -(Z) b -M'; L1 is connected to D;

[0204] a is 0, 1, 2, 3, 4, 5 or 6; b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0205] D is The group formed by the loss of a hydrogen atom from the compound shown;

[0206] B1 and B2 are independently Moreover, at least one of B1 and B2 is

[0207] R1 and R2 are independently -OH, -N3, F, -OCH3 or -SCF3;

[0208] R3 and R 3’ are independently H, -C(O)R4, -C(O)OR4, -OR4 or -R4;

[0209] 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;

[0210] R5 is H, -(L2) d -(Z) e -(maleimido) or -(L2) d -(Z) e -H;

[0211] d is 0, 1, 2, 3, 4, 5 or 6; e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0212] 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 sulfhydryl group of D;

[0213] X is

[0214] p is independently 1, 2 or 3;

[0215] Q is a connecting bond, -C(O)O-, -C(O)N(R6)- or -C 1-3 Alkylene-O-;

[0216] Q1 is phenyl or pyridyl;

[0217] 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;

[0218] R7 and R 7’ are independently H or C 1-6 alkyl;

[0219] R8 is phenyl or 5-10 membered heteroaryl; said R8 is unsubstituted or selectively substituted by 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;

[0220] 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 -3-6 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;

[0221] R9 is independently hydrogen, C 1-6 Alkyl, -PO(OH)2, -PO(OCH3)2, -C(O)-(CH2CH2O) n -CH3 or -(CH2CH2O) n -CH3;

[0222] -PEG- is -(CH2CH2O) n -(CH2) u -or-(CH2CH2O) n -(CH2) u -C(O)-;

[0223] A is independently an amino acid residue;

[0224] 47, 48, 49, or 50; u is independently 0, 1, 2, 3, 4, or 5;

[0225] M' is a linker precursor.

[0226] In some embodiments, in the cyclic dinucleotide compound of Formula I or a pharmaceutically acceptable salt thereof, some groups are defined as follows, and the remaining groups are defined as described in any other scheme (hereinafter referred to as "in some embodiments"): a is 0, 1, 2, 3 or 4; a is preferably 0, 1 or 2.

[0227] In some embodiments, b is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably b is 1, 2, 3, 4, 5, 6, 7, or 8; more preferably b is 1, 2, 3, 4, 5, or 6.

[0228] In some embodiments, d is 0, 1, 2, or 3; preferably d is 1 or 2; more preferably d is 1.

[0229] In some embodiments, e is 0, 1, 2, 3, 4, 5, or 6; preferably, e is 0, 1, 2, 3, or 4; more preferably, e is 0, 1, 2, or 3.

[0230] In some embodiments, R6 is H or C 1-6 Alkyl; the C 1-6 The alkyl group is unsubstituted or optionally substituted with one dimethylamino group or methylsulfonyl group at any position.

[0231] In some embodiments, R6 is H, -CH3, -CH2CH2N(CH3)2, or -CH2CH2S(O)2CH3.

[0232] In some embodiments, each L1 and each L2 are independently The c2 side in L1 is connected to D; the c2 side in L2 is connected to the thiol group in D.

[0233] In some embodiments, each L1 and each L2 are independently The c2 side in L1 is connected to D; the c2 side in L2 is connected to the thiol group in D.

[0234] In some embodiments, -(L1) a -for The c2 side is connected to D.

[0235] In some embodiments, -(L1) a -for The c2 side is connected to the sulfhydryl group in D.

[0236] In some embodiments, -(L1) a -for The c2 side is connected to the amino group in D.

[0237] In some embodiments, -(L1) a -for The c2 side is connected to the amino group in D.

[0238] In some embodiments, -(L1) a - does not exist, The c2 side is connected to the sulfhydryl group in D.

[0239] 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 -3-6 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- to 6-membered heteroarylene-(CH2) y In the "5- to 6-membered heteroarylene" 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; the -(CH2) x -3-6 membered heterocycloalkylene-(CH2) y In the "3- to 6-membered heterocycloalkylene" in -, the heteroatoms are selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3.

[0240] In some embodiments, in A, the amino acid is a natural amino acid or an unnatural amino acid.

[0241] In some embodiments, in A, the amino acid residue is replaced by 1 to 3 R 10 Modification; R 10 Independently C 1-6 Alkyl, C 1-6 Alkyl acyl, C 1-6 Alkoxyacyl, -C(O)-(CH2CH2O) n -CH3 or -(CH2CH2O) n -CH3; the C 1-6 Alkyl, C 1-6 Alkyl acyl or C 1- The 6-alkoxyacyl group may be further substituted by one R8 at any position.

[0242] In some embodiments, each A is independently Each R A Each independently represents an amino acid side chain or is surrounded by 1 to 3 R 10 Modified amino acid side chain; or R A and the adjacent nitrogen atom to form a five-membered heterocyclic group;

[0243] The R 10 Independently C 1-6 Alkyl, C 1-6 Alkyl acyl, C 1-6 Alkoxyacyl, -C(O)-(CH2CH2O) n -CH3 or -(CH2CH2O) n -CH3; the C 1-6 Alkyl, C 1-6 Alkyl acyl or C1-6 The alkoxyacyl group may be further substituted by one R8 at any position.

[0244] In some embodiments, the Can It can also be

[0245] In some embodiments, in A, each R A are independently hydrogen, methyl, ethyl, n-propyl, isopropyl, isobutyl, or R A Together with the adjacent nitrogen atom, it forms a five-membered heterocyclic group.

[0246] In some embodiments, -(A) v -for Each R A are independently hydrogen, methyl, ethyl, n-propyl, isopropyl, isobutyl, or R A Together with the adjacent nitrogen atom, it forms a five-membered heterocyclic group; v is any integer from 1 to 4, and v is preferably 1 or 2.

[0247] In some embodiments, -(A) v -for

[0248] In some embodiments, -(A) v -for

[0249] In some embodiments, -(Z) b -Any combination of the following:

[0250] 1)-(CH2) x -(c1),

[0251] 2)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -(c1),

[0252] 3)-C(O)-(CH2) x -(c1),

[0253] 4)-C(O)-(CH2) x -C 3-6 Cycloalkylene-(CH2) y -(c1),

[0254] 5)-C(O)-(CH2) x -phenylene-(CH2) y -(c1),

[0255] 6)-C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -(c1),

[0256] 7)-C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -phenylene-(CH2) y -(c1),

[0257] 8)-(A) v -(c1),

[0258] 9)-(A) v -C(O)-(CH2) x -(c1),

[0259] 10)-(A) v -C(O)-(CH2) x -phenylene-(CH2) y -(c1),

[0260] 11)-(A) v -C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -(c1) or

[0261] 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'.

[0262] In some embodiments, -(Z) b -Any combination of the following:

[0263] 13)-C(O)-(CH2) x -(A) v -C(O)-(CH2) x -(c1);

[0264] 14)-(A) v -C(O)-(CH2CH2O) n -(CH2) u -(c1);

[0265] 15)-C(O)-(CH2) x -NH-(c1);

[0266] 16)-C(O)-CH(-L3-R9)-NHC(O)-(CH2) x -phenylene-(CH2) y -;

[0267] 17)-C(O)-CH(-L3-R9)-NHC(O)-(CH2) x -, where the c1 side is connected to M'.

[0268] 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, sulfhydryl, azide, aldehyde, acyl or phenolic hydroxyl groups of the side chain of an antibody amino acid residue.

[0269] 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 thiol group of the side chain of an antibody amino acid residue.

[0270] In some embodiments, M' is ethynyl, vinyl, hydroxylamino,

[0271] R 11 is hydrogen or C 1-4 alkyl;

[0272] Each R 12 and R 12’ are independently halogen (such as bromine or iodine), nitro or -SO3 - ;

[0273] R 13 and R 13’ are each independently hydrogen, halogen (eg, bromine), phenylthio or pyridylthio.

[0274] In some embodiments, R 11 is hydrogen, methyl or ethyl.

[0275] In some embodiments, M' is

[0276] In some embodiments, M' is

[0277] In some embodiments, LX is

[0278] In some embodiments, LX is

[0279] In some embodiments, D is The group formed by the loss of a hydrogen atom from the compound shown; for and / or for and / or

[0280] In some embodiments, d is 1.

[0281] In some embodiments, R5 is H, -L2-(Z) e -(maleimido) or -L2-(Z) e -H.

[0282] In some embodiments, 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.

[0283] In some embodiments, R5 is -L2-(Z) e -H;-L2- The c2 side is connected to the thiol group in D; e is 3, -(Z) e -H is -C(O)NH-(CH2) y -O-(CH2) y -H.

[0284] In some embodiments, R5 is -L2-(Z) e -H;-L2- The c2 side is connected to the thiol group in D; e is 0 or 1, -(Z) e -H is H or methyl.

[0285] In some embodiments, R5 is Q is a connecting bond, -C(O)O- or -C 1-3 Alkylene-O-; R7 and R7’ are each independently H or methyl.

[0286] In some embodiments, R5 is Q1 is phenyl; R7 and R 7’ are each independently H or methyl.

[0287] In some embodiments, 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; the R8 is unsubstituted or selectively substituted with 1 to 3 groups selected from hydroxyl, amino, cyano, 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.

[0288] In some embodiments, 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.

[0289] In some embodiments, R5 is The c2 side is connected to the thiol group in D.

[0290] In some embodiments, R5 is The c2 side is connected to the thiol group in D.

[0291] In some embodiments, B1 and B2 are each independently Moreover, at least one of B1 and B2 is

[0292] In some embodiments, D is The group formed by losing a hydrogen atom from an amino or thiol group in the compound shown.

[0293] In some embodiments, D is The group formed by the amino group in the compound shown loses a hydrogen atom; R3 or R 3’ Independently

[0294] In some embodiments, D is The group formed by losing a hydrogen atom from the thiol group in the compound shown.

[0295] In some embodiments, D is The group formed by losing a hydrogen atom from the thiol group in the compound shown.

[0296] In some embodiments, D is any of the following structures or a pharmaceutically acceptable salt thereof:

[0297] Wherein, R1, R2 and R5 are as defined above.

[0298] In some embodiments, D is any of the following structures:

[0299] or a pharmaceutically acceptable salt thereof.

[0300] In some embodiments, D is any of the following structures: or a pharmaceutically acceptable salt thereof.

[0301] In some embodiments, the cyclic dinucleotide derivative as shown in Formula I or a pharmaceutically acceptable salt thereof is selected from:

[0302] or a pharmaceutically acceptable salt thereof.

[0303] The present invention also provides a compound as shown in formula D'-1 or D'-2, its stereoisomers or pharmaceutically acceptable salts thereof;

[0304] B1 and B2 are independently Moreover, at least one of B1 and B2 is

[0305] for and / or for and / or

[0306] R1 and R2 are independently -OH, -N3, F, -OCH3 or -SCF3;

[0307] R5 is -(L2) d -(Z) e -H;

[0308] d is 0, 1, 2, 3, 4, 5 or 6; e is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0309] Each L2 is independently The c2 side is connected to D;

[0310] X is

[0311] p is independently 1, 2 or 3;

[0312] Q is a connecting bond, -C(O)O-, -C(O)N(R6)- or -C 1-3 Alkylene-O-;

[0313] Q1 is phenyl or pyridyl;

[0314] 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;

[0315] R7 and R 7’ are independently H or C 1-6 alkyl;

[0316] R8 is phenyl or 5-10 membered heteroaryl; said R8 is unsubstituted or selectively substituted by 1 to 3 groups selected from hydroxyl, amino, carboxyl, cyano, nitro, thiol, C 1-6 Alkyl, C3-8 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;

[0317] Each Z is independently -C(O)-(CH2) x -、-NH-(CH2) y -、-O-(CH2) y -、C 1-6 Alkylene or C 2-6 alkenylene;

[0318] Each x and each y is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0319] In some embodiments, in the cyclic dinucleotide derivatives shown in formula D'-1 or D'-2 or pharmaceutically acceptable salts thereof, some groups are defined as follows, and the remaining groups are defined as described in any other scheme (hereinafter referred to as "in some embodiments"): R1 is -OCH3; R2 is F.

[0320] In some embodiments, d is 1.

[0321] In some embodiments, e is 0, 1, 2, 3, or 4; preferably, e is 0, 1, 2, or 3.

[0322] In some embodiments, R5 is -L2-(Z) e -H.

[0323] In some embodiments, 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.

[0324] In some embodiments, R5 is -L2-(Z) e -H;-L2- The c2 side is connected to the thiol group in D; e is 3, -(Z) e -H is -C(O)NH-(CH2) y -O-(CH2) y-H.

[0325] In some embodiments, R5 is -L2-(Z) e -H;-L2- The c2 side is connected to the thiol group in D; e is 0 or 1, -(Z) e -H is H or methyl.

[0326] In some embodiments, 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.

[0327] In some embodiments, R5 is Q1 is phenyl; R7 and R 7’ are each independently H or methyl.

[0328] In some embodiments, 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; the R8 is unsubstituted or selectively substituted with 1 to 3 groups selected from hydroxyl, amino, cyano, 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.

[0329] In some embodiments, R5 is The c2 side is connected to the thiol group in D.

[0330] In some embodiments, R5 is The c2 side is connected to the thiol group in D.

[0331] In some embodiments, D'-1 or D'-2 is any of the following structures:

[0332] or a pharmaceutically acceptable salt thereof.

[0333] The present invention provides a pharmaceutical composition comprising a substance K and a pharmaceutically acceptable excipient;

[0334] The substance K is substance K-1, substance K-2 or substance K-3;

[0335] The substance K-1 is the above-mentioned antibody-immunostimulatory conjugate represented by Formula II or a pharmaceutically acceptable salt thereof;

[0336] The substance K-2 is the cyclic dinucleotide compound represented by Formula I or a pharmaceutically acceptable salt thereof;

[0337] The substance K-3 is the compound represented by the above formula D'-1 or D'-2, or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0338] In some embodiments, the amount of substance K can be a therapeutically effective amount.

[0339] In some embodiments, the antibody-immunostimulatory conjugate of Formula II or a pharmaceutically acceptable salt thereof, the cyclic dinucleotide compound of Formula I or a pharmaceutically acceptable salt thereof, or the compound of Formula D'-1 or D'-2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof may be a therapeutically effective amount.

[0340] In the pharmaceutical composition, the pharmaceutically acceptable excipients may include pharmaceutically acceptable carriers, diluents and / or excipients.

[0341] The pharmaceutical composition can be administered through conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, local administration (eg, intratumoral injection), etc.

[0342] The present invention also provides a use of a substance K or the above-mentioned 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;

[0343] The substance K-1 is the above-mentioned antibody-immunostimulatory conjugate represented by Formula II or a pharmaceutically acceptable salt thereof;

[0344] The substance K-2 is the cyclic dinucleotide compound represented by Formula I or a pharmaceutically acceptable salt thereof;

[0345] The substance K-3 is the compound represented by the above formula D'-1 or D'-2, or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0346] In some embodiments, the amount of substance K can be a therapeutically effective amount.

[0347] The present invention provides a use of a substance K or the above-mentioned 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;

[0348] The substance K-1 is the above-mentioned antibody-immunostimulatory conjugate represented by Formula II or a pharmaceutically acceptable salt thereof;

[0349] The substance K-2 is the cyclic dinucleotide compound represented by Formula I or a pharmaceutically acceptable salt thereof;

[0350] The substance K-3 is the compound represented by the above formula D'-1 or D'-2, or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0351] In some embodiments, the amount of substance K can be a therapeutically effective amount.

[0352] The present invention provides a use of a substance K or the pharmaceutical composition described above 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;

[0353] The substance K-1 is the above-mentioned antibody-immunostimulatory conjugate represented by Formula II or a pharmaceutically acceptable salt thereof;

[0354] The substance K-2 is the cyclic dinucleotide compound represented by Formula I or a pharmaceutically acceptable salt thereof;

[0355] The substance K-3 is the compound represented by the above formula D'-1 or D'-2, or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0356] In some embodiments, the STING-mediated disease is a tumor or a viral infectious disease.

[0357] In some embodiments, the amount of substance K can be a therapeutically effective amount.

[0358] The tumor may be a malignant tumor, including metastatic and non-metastatic cancers, hereditary and sporadic cancers, and may also include solid tumors and non-solid tumors.

[0359] In the present invention, the "tumor" and "cancer" have the same meaning.

[0360] 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 atom is not exceeded, and the substitution at any position is a reasonable substitution commonly used in the art.

[0361]

[0046] In the present invention, when a bond to a substituent is shown to intersect a bond connecting two atoms in a ring, then such substituent may be bonded to any bondable ring atom on the ring.

[0362] Any combination of variables in this invention is permissible only if such combination results in a stable compound.

[0363] In the present invention, when any variable occurs more than once in the composition or structure of a compound, its definition on each occurrence is independent. For example, when R is substituted with one or more groups, each substituent is an independent substituent and may be the same or different.

[0364] Unless otherwise specified, the following terms appearing in the present specification and claims have the following meanings:

[0365] The term "antibody" refers to any form of an antibody that exhibits a desired biological activity (e.g., inhibiting the binding of a ligand to its receptor or by inhibiting ligand-induced receptor signal transduction). Therefore, "antibody" is used in its broadest sense and explicitly includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (including bispecific antibodies). Naturally occurring "antibodies" are glycoproteins 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 comprises a CL domain. The variable regions of the heavy and light chains contain a binding domain (antigen binding domain) that interacts with an antigen. The antigen binding domain can be provided by one or more variable regions on the antibody. Specifically, the antigen binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH). In some cases, the antigen to which the antibody can bind is a tumor-associated antigen. In some cases, the antigen to which 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 not only includes complete antibodies, but also includes fragments of antibodies with immunological activity (including Fab, F(ab')2, scFv or Fv fragments) or fusion proteins formed by antibodies and other sequences. Therefore, the "antibody" described in the present invention also includes fragments, derivatives and analogs of the antibody.

[0366] In the present invention, the "antibody" may further include an engineered antibody. The engineered antibody may 1) include one or more non-naturally encoded amino acids incorporated into the heavy chain, light chain, or both the heavy chain and the light chain, wherein 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 into different positions of the antibody heavy chain or light chain, or replace specific amino acid residues of the antibody heavy chain or light chain with cysteine ​​residues, thereby forming unpaired cysteines for coupling.

[0367] The "antibodies" or antigen-binding fragments thereof described herein 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 the drug comprising such a modified Fc region, such as reduced effector function, altered regulation of drug metabolic half-life, and improved drug stability. In some cases, the modified Fc region may comprise one or more mutations that reduce or eliminate interactions between the antibody and the immune system. Key interactions may include interactions between the antibody Fc and Fcγ receptors and with C1q of the complement system. When IgG1 is used as the isotype of the antibodies of the present invention, effector function can be modulated by substituting amino acid residues in a portion of the constant region. IgG1 variants that reduce or attenuate effector function include, but are not limited to, IgG1 LALA (IgG1-L234A, L235A), IgG1 LAGA (IgG1-L235A, G237A), and IgG1 AAG (IgG1-L234A, L235A, P329G). The above L234A and L235A indicate that the leucine at positions 234 and 235 as determined by the EU index (Proc. Natl. Acad. Sci. USA, Vol. 63, No. 1 (May 15, 1969), p78-85) is substituted by alanine, G237A indicates that the glycine at position 237 as determined by the EU index is substituted by alanine, and P329G indicates that the proline at position 329 as determined by the EU index is substituted by alanine. In some cases, the efficacy and pharmacokinetic properties of drugs can be altered by glycosylation modification of the Fc segment (Journal of Pharmaceutical Sciences. 2015, 104(6), 1866-1884). For example, natural antibodies produced by mammalian cells generally contain branched, biantennary oligosaccharides, which are generally attached to Asn297 of the CH2 domain of the Fc segment via an N-linked bond. Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the stem of biantennary oligosaccharide structures. Modification of oligosaccharides in antibodies can produce antibody variants with improved properties. For example, afucosylation can enhance antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADCP); reducing terminal sialylation or increasing terminal acetylglucosamine can enhance ADCC; treating CHO cells with mannosidase inhibitors can increase mannosylation and afucosylation, thereby enhancing ADCC and slightly reducing CDC.In addition, glycosylation modification can also be performed through mutation, such as the N297Q mutation, in which asparagine at position 297 is mutated to glutamine.

[0368] The term "monoclonal antibody", also known as "monoclonal antibody", refers to polypeptides (including antibodies, bispecific antibodies, etc.) having substantially the same amino acid sequence or derived from the same genetic source. Monoclonal antibodies are highly specific and can be directed against a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations that typically include a variety of different antibodies directed against multiple different determinants (epitopes), each monoclonal antibody is directed against only a single determinant on the antigen. In some specific embodiments, the antigen to which the monoclonal antibody can bind is a tumor-associated antigen; in some specific embodiments, the antigen to which the monoclonal antibody can bind is a tumor-specific antigen; in some specific embodiments, the monoclonal antibody binds to, including but not limited to, the following optional antigens: HER2, 5T4 (TPBG), EGFR, or Nectin-4.

[0369] The terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. A polypeptide fragment, derivative, or analog of the present invention may be a) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code; b) a polypeptide having a substituent group in one or more amino acid residues; c) a polypeptide formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or d) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence, a sequence used to purify the polypeptide, a proprotein sequence, or a fusion protein with a 6His tag).

[0370] In the present invention, the biosimilar of the monoclonal antibody refers to a monoclonal antibody that is highly similar to the monoclonal antibody, although there are slight differences in its inactive ingredients in clinical practice, and has no clinically significant differences in safety and / or efficacy.

[0371] 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 comprises an extracellular binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain. HER2 does not have its own ligand binding domain and therefore cannot bind growth factors. However, HER2 tightly binds to other ligand-binding EGF receptor family members (e.g., HER1 or HER3) to form heterodimers, stabilizing ligand binding and enhancing activation of kinase-mediated downstream signaling pathways. In humans, there are species HER2 isoforms: A, B, C, D, and E. "HER2" as used herein includes all HER2 isoforms.

[0372] The term "anti-HER2 antibody" refers to an antibody that uses HER2 as a 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 the anti-HER2 antibody is more preferably a humanized anti-HER2 antibody. The anti-HER2 antibodies include but are not limited to: pertuzumab, trastuzumab, trastuzumab biosimilars (e.g., inetetamab), pertuzumab biosimilars, margetuximab, HT-19, etc.

[0373] Among them, trastuzumab (also known as Herceptin or Herclon) is a humanized monoclonal antibody that can bind to the membrane-proximal region of the extracellular structure of the HER2 receptor (Hudis CA, N Engl J Med. 2007; 357(1): 39-51). The amino acid sequences of the heavy and light chain variable regions of trastuzumab are disclosed in U.S. Patent 5,821,337. Trastuzumab interacts with the three-ring 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. In addition, another important mechanism of anti-HER2 antibodies is to mediate antibody-dependent cellular cytotoxicity (ADCC). In ADCC, anti-HER2 antibodies bind to tumor cells and then recruit immune cells, such as macrophages, through interactions with Fcγ receptors (FcγRs). Trastuzumab has a conserved human IgG Fc region and is able to recruit immune effector cells responsible for antibody-dependent cellular 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 whose tumors overexpress HER2 and who have received one or more chemotherapy regimens for metastatic breast cancer. In some embodiments, in order to achieve site-specific conjugation, an engineered Trastuzumab in which a cysteine ​​is 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, IgG1 variants of Trastuzumab are obtained by reducing or weakening the effector function by replacing a portion of the amino acid residues in the constant region, including but not limited to: IgG1 LALA (IgG1-L234A, L235A), IgG1 LAGA (IgG1-L235A, G237A), IgG1 AAG (IgG1-L234A, L235A, P329G), etc. The above-mentioned L234A and L235A indicate that the leucine at positions 234 and 235 determined by the EU index (Proc. Natl. Acad. Sci. USA, Vol. 63, No. 1 (May 15, 1969), p78-85) are 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.

[0374] 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 U.S. Patent 7,560,111. Pertuzumab primarily interacts with residues within the 245-333 region of human HER2, particularly 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 for use in combination with trastuzumab and docetaxel in the treatment of patients with HER2-positive metastatic breast cancer who have not received prior anti-HER2 therapy or chemotherapy.

[0375] Margetuximab (also known as MGAH22 and Margenza) is an Fc-engineered monoclonal antibody targeting the HER2 protein, binding to the extracellular domain of HER2. Its variable region sequence differs only by a few amino acids from that of trastuzumab. The Fc region of margetuximab undergoes five mutations: F243L / R292P / Y300L / L235V / P396L, which increase its affinity for CD16A and enhance ADCC activity. This modification enhances the binding of the Fc region of margetuximab to the activating Fc receptor FCGR3A (CD16A) and reduces its binding to the inhibitory Fc receptor FCGR2B (CD32B), resulting in enhanced ADCC and NK cell activation (Nordstrom J. et al., Breast Cancer Research, 2011;13:R123). Margetuximab was approved by the U.S. FDA in December 2020 for the treatment of adult patients with metastatic HER2-positive breast cancer (MBC) who have received two or more anti-HER2 targeted therapies, at least one of which was used to treat metastatic breast cancer.

[0376] In the present invention, the anti-HER2 antibody is not limited to the antibodies listed above as long as it specifically binds to HER2 (for example, an anti-HER2 antibody that has the activity of internalizing in HER2-expressing cells through binding to HER2).

[0377] The isotype of the "anti-HER2 antibody" in the present invention includes IgG1, IgG2, IgG3, IgG4, etc., preferably IgG1, IgG2 or IgG4.

[0378] The term "HER2 low expression" generally refers to a HER2 expression level of IHC 1+, or IHC 2+ / FISH negative (i.e., IHC 2+ and FISH negative) in clinical testing. The terms "HER2 high expression" and "HER2 positive" are used interchangeably and generally refer to a HER2 expression level of IHC 2+ / FISH positive (i.e., IHC 2+ and FISH positive) or IHC 3+ in clinical testing. When IHC staining intensity is reported as a range, the term "HER2 low expression" herein includes, in addition to IHC 1+ or IHC 2+ / FISH negative, IHC 0 to 1+ and IHC 1+ to 2+ ranges. The terms "HER2 high expression" and "HER2 positive" each include, in addition to IHC 2+ / FISH positive or IHC 3+, IHC 2+ to 3+ ranges. In the present invention, FISH-negative means that the FISH test results show that the HER2 gene is not amplified, and FISH-positive means that the FISH test results show that the HER2 gene is amplified.

[0379] The term "EGFR" refers to epidermal growth factor receptor, which 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 cancer, 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.;

[0380] Among them, cetuximab (also known as Erbitux) is a human-mouse chimeric antibody. In addition to competitively binding to the extracellular binding epitope of the natural ligand EGF to block signaling, cetuximab can also induce receptor internalization, downregulation, and degradation. Furthermore, cetuximab can arrest the cell cycle and induce antibody-dependent cytotoxicity. Cetuximab was first approved by the US FDA in 2004 for the treatment of patients with EGFR-positive metastatic colorectal cancer who have failed treatment with the chemotherapy drug irinotecan. In 2006, it was approved by the US FDA for combination therapy with radiation therapy for the treatment of locally or regionally advanced head and neck squamous cell carcinoma.

[0381] Panitumumab (also known as Vectibix) is a fully humanized IgG2 antibody. Like cetuximab, panitumumab rapidly binds to EGFR with a low dissociation constant, effectively competitively blocking the binding of EGF and TGFα ligands. Panitumumab has demonstrated strong anti-tumor activity in various tumor cell xenograft models, including pancreatic cancer, breast cancer, renal cancer, prostate cancer, and head and neck cancer (Yang X 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 patients with EGFR-positive metastatic colorectal cancer who have been previously treated with irinotecan, oxaliplatin, and 5-fluorouracil.

[0382] Nimotuzumab (also known as Taixinsheng) is a fully humanized IgG1 antibody. The heavy chain variable region of Nimotuzumab differs significantly from that of cetuximab. After binding to EGFR, Nimotuzumab does not completely inhibit the EGFR in the same way as cetuximab. The extracellular domains I and III of EGFR may still bind to each other to form an equilibrium conformation in an activated state. Its affinity for the EGFR epitope is also much weaker than that of cetuximab. Clinically, a higher dose is required to achieve the same efficacy as cetuximab, but the probability of side effects such as rash is also lower (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.

[0383] 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, such as S492R, that are common in metastatic colorectal cancer are located in this binding region. Therefore, necitumumab can overcome the antibody drug resistance caused by these extracellular point mutations in EGFR. Necitumumab was approved by the FDA in 2015 for the treatment of non-small cell lung cancer.

[0384] In the present invention, the anti-EGFR antibody is not limited to the antibodies listed above as long as it specifically binds to EGFR (for example, an anti-EGFR antibody having an activity of internalization in EGFR-expressing cells through binding to EGFR).

[0385] The isotype of the "anti-EGFR antibody" in the present invention includes IgG1, IgG2, IgG3, IgG4, etc., preferably IgG1, IgG2 or IgG4.

[0386] The term "5T4," also known as 5T4 carcinoembryonic antigen or trophoblast glycoprotein TPBG, is a 72 kDa glycoprotein defined by a monoclonal antibody raised against a glycoprotein isolated from wheat germ agglutinin of the microvilli of the human placental syncytiotrophoblast. It has limited expression in normal tissues but is overexpressed by various types of cancer cells. "Anti-5T4 monoclonal antibodies" include, but are not limited to, H8 and its humanized monoclonal antibodies disclosed in WO2006 / 031653A1, A1, A2, A3 and their humanized monoclonal antibodies (e.g., huA1) disclosed in US8044178B2, the engineered A1 monoclonal antibody disclosed in WO2013068874, naptumomab, and the like.

[0387] Nectin-4, also known as 191P4D12 protein, is a cell adhesion molecule belonging to the nectin family of surface molecules. It plays a key role in various biological processes in epithelial, endothelial, immune, and neural cells during development and adulthood. Nectin-4 is a tumor-associated antigen primarily found in tumors with poor prognosis. Anti-nectin-4 monoclonal antibodies include, but are not limited to, enfortumab (Ha22-2).

[0388] The term "linker" refers to a degradable or non-degradable linker fragment used to connect a small molecule drug D to an antibody. A single antibody molecule can be linked to multiple linkers carrying small molecule drugs D. Typically, each linker can be linked to one or more small molecule drugs. In the present invention, preferably, each linker is linked to one small molecule drug D. Typically, each antibody can be linked to multiple linkers. In the present invention, preferably, each antibody is linked to 1 to 8 linkers.

[0389] In the present invention, the non-degradable linker means that the linker has 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 differentiated properties of the enzyme levels in plasma, tumor tissue and cells. The release of the small molecule drug D can be achieved by antigen-mediated phagocytosis of the antibody-immunostimulatory conjugate, which then degrades the antibody to the amino acid level, thereby releasing a derivative of the small molecule drug D. The derivative of the small molecule drug D is composed of the small molecule drug D, the linker and the amino acid residue or the residue to which the small molecule drug D and the linker are covalently linked. The antibody-immunostimulatory conjugate constructed by such non-degradable linkers has better stability. Non-degradable linkers include alkylene chains and polymers thereof (for example: 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 combinations thereof.

[0390] 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 a specific enzyme in vivo or in vitro or a linker that is chemically unstable itself. The degradable linker can be degraded in the cell to release the small molecule drug D, for example, it can be reduced in the cytoplasm, degraded under the acidic conditions of the lysosome or degraded by a specific protease or other enzyme in the cell. The degradable linker includes one or more enzymatically degradable linkers, chemically unstable linkers or other degradable linkers, and the other part can be a linker that is not degraded by enzymes or is chemically stable. The chemically unstable linker includes oxime, hydrazone and / or disulfide groups (for example: ). The linker that is specifically degraded by the enzyme is a linker based on 1) amino acid residues or peptides. The peptide bond can have good serum stability because the activity of lysosomal proteolytic enzymes in the blood is much lower than that in certain tumor tissues. Therefore, the linker can be selectively degraded in certain tumor tissues or cells to release small molecule drugs D. The lysosomal enzyme can be selected from cathepsin B, cathepsin S, plasmin, elastase, β-glucuronidase or β-galactosidase, etc. Peptide-based linker (-(A) v-) 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-), dipeptides (including but not limited to: -Val-Cit-, -Cit-Val-, -Val-Ala-, -Ala-Val-, -Ala-Cit-, -Cit-Ala-, -Asn-), n-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 amino acid monomers (including but not limited to: -Lys-, -Gly-, -Cit-), the peptide-based linker is preferably a dipeptide linker, a tripeptide linker or a tetrapeptide linker, more preferably a dipeptide linker. The other degradable linkers may include an ester link formed by the reaction of PEG carboxylic acid or activated PEG carboxylic acid with a hydroxyl group on the small molecule drug D, wherein such an ester link can be hydrolyzed under physiological conditions to release the small molecule drug D. Hydrolytically degradable links include but are not limited to carbonate bonds, imine links generated by the reaction of amines and aldehydes, phosphate ester links obtained by the reaction of hydroxyl groups and phosphate groups, acetal links obtained by the reaction of hydroxyl groups and aldehydes, orthoester links obtained by the reaction of formate and hydroxyl groups, etc. The cleavable linker may also include a non-cleavable fragment, such as polyethylene glycol (PEG) and related polymers. 2) Linkers formed by pyrophosphate or phosphate.Lysosomal acid pyrophosphatase and acid phosphatase are enzymes that hydrolyze pyrophosphates and terminal monophosphates, respectively, into their parent alcohols within the lysosome. Targeting these enzymes can effectively release small molecule drugs D that terminate in alkyl alcohols.

[0391] In the present invention, the "linker" may further include an extension group (Spacer), and the connector may be directly connected to the linker in the linker, or connected to the linker in the linker through an extension group (Spacer). The extension group may be polyethylene glycol and related polymers, an alkylene group containing 1 to 10 carbon atoms, a cyclohexyl group, a phenyl group, a 1,3-dioxane group, an amide group, an ester group, an oxo group, a substituted amino group, a triazole group or and any one or more combinations of the above-mentioned extending groups.

[0392] 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.

[0393] In the present invention, the "linker" also includes a linker, which can be connected to the antibody, and can be formed by the reaction of a linker precursor and a thiol group (e.g., cysteine), an amino group (e.g., lysine), a carbonyl group (e.g., p-acetylphenylalanine), an aldehyde group, an azido group (e.g., p-azidomethylphenylalanine), a phenol group (e.g., tyrosine), etc. in the antibody.

[0394] In the present invention, the "linker precursor" can react with an antibody to form a linker connected to the antibody.

[0395] In the present invention, the linker precursor can react with sulfhydryl groups (e.g., cysteine), amino groups (e.g., lysine), carbonyl groups (e.g., p-acetylphenylalanine), aldehyde groups, azido groups (e.g., p-azidomethylphenylalanine), phenolic hydroxyl groups (e.g., tyrosine), etc. in the antibody; the linker precursor can also be linked to specific antibody groups through an enzyme-catalyzed reaction.

[0396] The linker precursors that can react with the side chain sulfhydryl groups of the antibody amino acid residues include but are not limited to: acetylene, vinyl, hydroxylamine, R 12 、R 13 、R 13’ and R 11 The definition of is as mentioned above.

[0397] The reaction between the linker precursor and the sulfhydryl group (e.g., cysteine) in the antibody is specific, as shown in Formulas 1 to 6 and 12:

[0398] When the linker is maleimide or succinimide, it can be further hydrolyzed to obtain a hydrolysis product, such as shown in Reaction Formula 1' or Reaction Formula 2':

[0399] The linker precursors that can react with the azide groups on the side chains of the antibody amino acid residues include but are not limited to: acetylene, The click chemistry reaction between the linker precursor and the azide group (e.g., p-azidomethylphenylalanine) in the antibody is specific, as shown in Formula 7 or 13:

[0400] The linker precursors that can react with the side chain carbonyl or aldehyde groups of the antibody amino acid residues include but are not limited to: hydroxylamine or hydrazine groups; the reaction of the linker precursor with the acyl group (e.g., p-acetylphenylalanine) or aldehyde group in the antibody is specific, for example, as shown in Formulas 8 to 9:

[0401] The linker precursors that can react with the phenol groups on the side chains of antibody amino acid residues include but are not limited to: The click-chemistry-like reaction between the linker precursor and the phenol group (e.g., tyrosine) in the antibody is specific, as shown in Formula 10:

[0402] The linker precursor that directly reacts the linker with the side chain amino group of the antibody amino acid residue is an active ester group, including but not limited to: R 12 and R 12’ The definition is as described 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:

[0403] The term "linker precursor" can directly react with an antibody and form a degradable or non-degradable linker for linking a small molecule drug D and an antibody.

[0404] The term "and / or" includes any and all combinations of one or more of the associated listed items.

[0405] The term "C A-B " refers to a range including the starting point and the end point, wherein A and B and each point in the range are integers representing the number of carbon atoms, for example, C 1-4 Indicates the number of carbon atoms is 1, 2, 3 or 4; C 1-6 Indicates the number of carbon atoms is 1, 2, 3, 4, 5 or 6; C 3-8 Indicates the number of carbon atoms is 3, 4, 5, 6, 7 or 8; CA-B It can be used in conjunction with any group containing carbon atoms to limit the number of carbon atoms, such as 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.

[0406] The term "alkyl" refers to a saturated straight or branched chain hydrocarbon group containing 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 8, 1 to 6, 1 to 4 or 1 to 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 various isomers thereof.

[0407] The term "alkenyl" refers to a straight or branched non-aromatic hydrocarbon group containing at least one carbon-carbon double bond. There may be 1 to 3 carbon-carbon double bonds, preferably one carbon-carbon double bond. 2-4 "Alkenyl" refers to an alkenyl group having 2 to 4 carbon atoms. 2-6 The term "alkenyl" refers to an alkenyl group having 2 to 6 carbon atoms, including ethenyl, propenyl, butenyl, and 2-methylbutenyl.

[0408] The term "alkynyl" refers to a straight or branched hydrocarbon group containing at least one carbon-carbon triple bond. There may be 1 to 3 carbon-carbon triple bonds, preferably one carbon-carbon triple bond. 2-6 The term "alkynyl" refers to an alkynyl group having 2 to 6 carbon atoms, including ethynyl, propynyl, butynyl and 3-methylbutynyl.

[0409] The term "alkylene" refers to a saturated straight or branched non-bridging divalent alkyl group containing 1-20 carbon atoms, preferably 1-10 carbon atoms, more preferably 1-8, 1-6 or 1-4 carbon atoms, examples of which include but are not limited to -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH(CH3)-, -CH2C(CH3)2CH2-, -CH2C(CH3)2CH2CH2-, =CH2, =CHCH3, =C(CH3)2.

[0410] The term "alkenylene" refers to a straight or branched non-aromatic divalent hydrocarbon radical containing at least one carbon-carbon double bond, wherein 1 to 3 carbon-carbon double bonds may be present, and preferably 1 carbon-carbon double bond is present. The alkenylene preferably contains 2 to 10 carbon atoms, more preferably 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

[0411] The term "alkynylene" refers to a straight or branched non-aromatic divalent hydrocarbon radical containing at least one carbon-carbon triple bond. There may be 1 to 3 carbon-carbon triple bonds, preferably 1. The alkynylene radical preferably contains 2 to 10 carbon atoms, more preferably 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

[0412] The term "cycloalkyl" refers to a saturated or partially unsaturated (containing one or two double bonds) monocyclic or polycyclic group containing 3 to 20 carbon atoms. "Monocyclic cycloalkyl" is preferably a 3-10 membered monocyclic alkyl, more preferably a 3-8 membered monocyclic alkyl, for example: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, cyclohexenyl. "Polycyclic cycloalkyl" includes "bridged ring group", "fused cycloalkyl" and "spirocyclic alkyl". Representative examples of "bridged ring group" include, but are not limited to: bornyl, bicyclo[2.2.1]heptenyl, bicyclo[3.1.1]heptenyl, bicyclo[2.2.1]heptenyl, bicyclo[2.2.2]octyl, bicyclo[3.2.2]nonanyl, bicyclo[3.3.1]nonanyl, bicyclo[4.2.1]nonanyl and adamantyl. "Fused cycloalkyl" includes a cycloalkyl ring fused to a phenyl, cycloalkyl, or heteroaryl group, including but not limited to benzocyclobutenyl, indanyl, decahydronaphthyl, etc. Monocyclic or polycyclic cycloalkyl groups can be attached to the parent molecule through any carbon atom in the ring.

[0413] The term "cycloalkylene" refers to a divalent cycloalkyl group. Thus, "cycloalkylene" includes the above definition of cycloalkyl. "Cycloalkylene" is preferably C 3-10 Cycloalkylene, more preferably C 3-8 Cycloalkylene or C 3-6 Cycloalkylene.

[0414] The term "heterocycloalkyl" refers to a saturated or partially unsaturated (containing 1 or 2 double bonds) non-aromatic cyclic group consisting of carbon atoms and heteroatoms selected from nitrogen, oxygen, sulfur, boron, and / or sulfur-containing heteroatom groups. This cyclic group may be a monocyclic or polycyclic group, wherein the sulfur-containing heteroatom group is 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 may be optionally oxidized. The nitrogen atom may optionally be further substituted with other groups to form a tertiary amine or a quaternary ammonium salt. "Monocyclic heterocycloalkyl" is preferably a 3-10 membered monocyclic heterocycloalkyl, more preferably a 3-8 membered monocyclic heterocycloalkyl. For example: pyrrolidinyl, dihydropyrrolidinyl, dihydroimidazolyl, dihydropyrazolyl, tetrahydrofuranyl, tetrahydropyrazinyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, piperidinyl, aziridinyl, morpholinyl, thiomorpholinyl, thiomorpholine-S-oxide-4-yl, piperidinyl, piperazinyl, 1,4-dioxane, homopiperazine The term "polycyclic heterocycloalkyl" includes "fused heterocycloalkyl", "spiroheterocyclyl" and "bridged heterocycloalkyl". "Fused heterocycloalkyl" includes a monocyclic heterocycloalkyl ring fused to a phenyl, cycloalkyl, heterocycloalkyl, or heteroaryl group. Examples of fused heterocycloalkyl include, but are not limited to, 2,3-dihydrobenzofuranyl, 1,3-dihydroisobenzofuranyl, dihydroindole, 2,3-dihydrobenzo[b]thienyl, dihydrobenzopyranyl, 1,2,3,4-tetrahydroquinolyl, 1,2,3,4-tetrahydroisoquinolyl, 1,2,3,4-tetrahydronaphthyridinyl, 5,6,7,8-tetrahydronaphthyridinyl, and 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborolanyl. Monocyclic and polycyclic heterocycloalkyl groups may be attached to the parent molecule via any ring atom. The ring atoms specifically refer to the carbon and / or nitrogen atoms that constitute the ring backbone.

[0415] The term "heterocycloalkylene" refers to a divalent heterocycloalkyl group. Thus, "heterocycloalkylene" includes the above-mentioned definition of heterocycloalkyl. "Cycloalkylene" is preferably a 3-10 membered heterocycloalkylene, more preferably a 3-8 membered heterocycloalkylene or a 3-6 membered heterocycloalkylene.

[0416] The term "cycloalkylalkyl" refers to a cycloalkyl group connected to a parent core structure via an alkyl group. Thus, "cycloalkylalkyl" encompasses the above definitions of alkyl and cycloalkyl.

[0417] The term "heterocycloalkylalkyl" refers to a heterocycloalkyl group connected to a parent core structure via an alkyl group. Thus, "heterocycloalkylalkyl" encompasses the above definitions of alkyl and heterocycloalkyl.

[0418] The term "alkoxy" refers to an alkyloxy group having the stated number of carbon atoms attached through an oxygen bridge. Thus, "alkoxy" encompasses the above-mentioned definition of alkyl.

[0419] The term "alkanethiol" refers to an alkylthiol group having the stated number of carbon atoms attached via a thiol bridge. Thus, "alkanethiol" encompasses the above definition of alkyl.

[0420] The term "hydroxyalkyl" refers to an alkyl group in which any hydrogen atom is replaced by a hydroxy group, including but not limited to: -CH2OH, -CH2CH2OH, -CH2CH2C(CH3)2OH.

[0421] The term "aryl" refers to any stable 6-20 membered monocyclic or polycyclic aromatic group, for example, phenyl, naphthyl, tetrahydronaphthyl, 2,3-dihydroindanyl or biphenyl.

[0422] The term "arylene" refers to a divalent heteroaryl group. Thus, "arylene" includes the above-mentioned definition of aryl. "Arylene" is preferably C 6-10 The arylene group is more preferably a phenylene group.

[0423] 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 5-7 membered monocyclic structure or a 7-20 membered fused ring structure, preferably a 5-6 membered heteroaryl group. In the present invention, the number of heteroatoms is preferably 1, 2 or 3, including: pyridyl, pyridonyl, pyrimidinyl, pyrimidine-2,4 (1H, 3H) -dione, pyrimidonyl, piperazinyl, pyridazinonyl, furanyl, 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, benzofuranyl, benzothiophenyl, benzo[d][1,3]dioxolanyl, benzothiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, isoquinolinonyl, quinazolinyl, 4-hydroxythieno[3,2-c]pyridinyl, 4,5-dihydro-4-oxofurano[3,2]pyridinyl, 4-hydroxy-5-azaindolyl, furano[2,3-c]pyridin-7(6H)-onyl, thien[2,3-c]pyridin-7(6H)-onyl, etc.

[0424] The term "heteroarylene" refers to a divalent heteroaryl group. Thus, "heteroarylene" includes the above-mentioned definition of heteroaryl. "Heteroarylene" is preferably a 5-10 membered heteroarylene, more preferably a 5-6 membered heteroarylene.

[0425] The term "arylalkyl" refers to an aryl group connected to a parent core structure via an alkyl group. Thus, "arylalkyl" includes the above definitions of alkyl and aryl.

[0426] The term "heteroarylalkyl" refers to a heterocyclic alkyl group connected to a parent core structure via an alkyl group. Thus, "heteroarylalkyl" includes the above definitions of alkyl and heteroaryl.

[0427] The term "halogen" denotes fluorine, chlorine, bromine or iodine.

[0428] The term "haloalkyl" refers to an alkyl group optionally substituted by a halogen. Thus, "haloalkyl" encompasses the above definitions of halogen and alkyl.

[0429] The term "haloalkoxy" refers to an alkoxy group optionally substituted by a halogen. Thus, "haloalkoxy" encompasses the above definitions of halogen and alkoxy.

[0430] The term "amino" refers to -NH2, and the term "alkylamino" refers to an amino group in which at least one hydrogen atom is replaced by an alkyl group, including but not limited to: -NHCH3, -N(CH3)2, -NHCH2CH3, -N(CH2CH3)2. Thus, "alkylamino" includes the above definitions of alkyl and amino.

[0431] The term "aminoalkyl" refers to an alkyl group in which any hydrogen atom is replaced by an amino group, including but not limited to: -CH2NH2, -CH2CH2NH2, -CH2CH2C(CH3)2NH2.

[0432] The term "acyl" refers to -C(O)-C 1-6 Alkyl; preferably formyl, acetyl, propionyl or isopropionyl.

[0433] The term "nitro" refers to -NO2.

[0434] The term "cyano" refers to -CN.

[0435] The term "oxo" refers to =0.

[0436] The term "mercapto" refers to -SH.

[0437] The term "amido" refers to -C(O)NH2.

[0438] The term "hydroxyamino" refers to -NHOH.

[0439] The term "carboxy" refers to -C(O)OH.

[0440] The term "aldehyde" refers to -C(O)H.

[0441] The term "carbomethoxy" refers to -C(O)OCH3.

[0442] The term "maleimido" refers to

[0443] In the present invention, the abbreviations of amino acids are conventional abbreviations (refer to the Principles of Organic Chemistry Nomenclature 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 symbol of the peptide is represented (taking -Gly-(-NH-CH2-CO-) as an example) as follows: when the dash representing the peptide bond is to the right of Gly, it represents the removal of the OH group from the -COOH group of the amino acid; when the dash is to the left of Gly, it represents the removal of an H atom from the -NH2- of the amino acid.

[0444] In the present invention, it is generally considered that 31 P NMR 31 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 toward 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 upfield, especially when the ligand is a group with high electronegativity. 31 The chemical shift of P is shifted to the upfield because the conjugation of the adjacent groups of the phosphorus atom also affects 31 The chemical shift of P, the enhancement of the conjugation of the electrons 31 The chemical shift of P is shifted to the upfield (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 phosphorus at position 1 and phosphorus at position 2 of compound 1M and compound 1-3, it can be judged that the chemical shift of phosphorus at position 2 is biased to the upfield. When the thiol hydrogen connected to phosphorus in compound 1-3 is replaced by benzyl, the chemical shift of phosphorus at position 2 is biased to the upfield. 31 Based on the change in chemical shift of P, it can be inferred that the benzyl substitution site in compound A is at the 2-thiol group.

[0445] 31 P NMR (162MHz, DMSO-d6+D2O): δ55.847.

[0446] 31 P NMR (DMSO-d6+D2O): δ55.759, 51.401.

[0447] 31 P NMR(DMSO-d6+D2O): δ53.113,27.573.

[0448] The "room temperature" mentioned in the present invention refers to 15-30°C.

[0449] 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 L 1～2 If stereoisomers exist, they may exist as single stereoisomers or as mixtures thereof.

[0450] 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 readily apparent to pharmaceutical chemists. Such salts are substantially non-toxic and provide desired pharmacokinetic properties, palatability, absorption, distribution, metabolism or excretion. The compounds of the present invention may have acidic groups, basic groups or amphoteric groups. Typical pharmaceutically acceptable salts include salts prepared by reacting the compounds of the present invention with acids.

[0451] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0452] The reagents and raw materials used in the present invention are commercially available. DETAILED DESCRIPTION

[0453] The structures of all compounds of the present invention can be determined by nuclear magnetic resonance ( 1 H NMR) and / or mass spectrometry (MS).

[0454] 1 H NMR chemical shifts (δ) are reported in PPM (10 -6 NMR spectra were performed using a Bruker AVANCE-400 spectrometer. Suitable solvents were deuterated chloroform (CDCl3), deuterated methanol (CD3OD), deuterated dimethyl sulfoxide (DMSO-d6), and tetramethylsilane as an internal standard (TMS).

[0455] Liquid chromatography-mass spectrometry (LCMS) was performed on an Agilent 1200 HPLC / 6120 mass spectrometer using an Xtimate C18 column, 3.0 × 50 mm, 3 μm, at 40°C; or on a Thermo Fisher Scientific UltiMate 3000 HPLC / MSQ PLUS mass spectrometer using an XBridge C18 column, 3.0 × 50 mm, 3.5 μm, at 30°C. Agilent gradient elution conditions were: 95-5% solvent A1 and 5-95% solvent B1 (0-2.0 min), followed by 95% solvent B1 and 5% solvent A1 (hold for 1.1 min). Percentages are the volume percentage of a particular solvent relative to the total solvent volume. Solvent A1: 0.01% trifluoroacetic acid (TFA) in water; Solvent B1: 0.01% trifluoroacetic acid in acetonitrile. Percentages are the volume percentage of the solute relative to the total solvent volume. Thermo gradient elution condition 2: 95-5% solvent A2 and 5-95% solvent B2 (0-2 minutes), followed by 95% solvent B2 and 5% solvent A2 (hold for 1.8 minutes). The percentage represents the volume percentage of a particular solvent relative to the total solvent volume. Solvent A2: 10 mM ammonium bicarbonate in water; Solvent B2: acetonitrile.

[0456] All the compounds of the present invention can be separated by preparative high performance liquid chromatography or flash column chromatography.

[0457] Preparative high-performance liquid chromatography (prep-HPLC) was performed using an Agela FLEXA-HP preparative liquid chromatography column: Welch Xtimate C18, 10 μm, 21.2 mm x 250 mm. Separation conditions: Mobile phase A: acetonitrile, mobile phase B: 10 mmol / L ammonium bicarbonate aqueous solution; detection wavelength: 214 nm & 254 nm; flow rate: 15.0 mL / min; gradient elution conditions:

[0458] Flash column chromatography (flash column chromatography) (flash system / Cheetah™) was performed using an Agela Technologies MP200. The normal phase separation column used was a Flash column Silica-CS (25 g, 40 g, 80 g, 120 g, or 330 g) from Tianjin Bona Agele, 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, 40-75 μm, Model: SW-040), the elution system is acetonitrile / 0.1% ammonium bicarbonate aqueous solution.

[0459] All compounds of the present invention can be analyzed by high performance liquid chromatography (HPLC) using Waters e2695, 2498 UV / VIS Detector;

[0460] Condition 1: Chromatographic column: Waters Xselect CHS C18 (4.6*150mm) 5μm, mobile phase A: methanol, mobile phase B: 10mM potassium dihydrogen phosphate buffer (pH adjusted to 8.0 with ammonia water); flow rate: 1.0mL / min; column temperature: 35℃; detection wavelength: 214nm & 254nm; gradient elution conditions:

[0461] Condition 2: Chromatographic column: Waters Xselect CHS C18 (4.6*250mm) 5μm, mobile phase A: methanol, mobile phase B: 10mM potassium dihydrogen phosphate buffer (pH adjusted to 8.0 with ammonia water); flow rate: 1.0mL / min; column temperature: 35℃; detection wavelength: 214nm & 254nm; gradient elution conditions:

[0462] Ultra-high performance liquid chromatography (UPLC) was performed using a Waters H-Class UPLC. The following conditions were used: chromatographic column: 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 wavelengths: 214 nm & 262 nm; gradient elution conditions:

[0463] The abbreviations used in the embodiments of the present invention have the following meanings:

[0464] (Boc)2O: di-tert-butyl dicarbonate; BINAP: 1,1'-binaphthyl-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'-tetramethylurea; PyBOP: 1H-benzotriazol-1-oxytris(1-pyrrolidinyl)phosphine hexafluorophosphate; TBSCl: tert-butyldimethylchlorosilane.

[0465] Synthesis of intermediates:

[0466] Synthesis of Linker-1

[0467] To a solution of Mc-Val-Ala-PAB-OH (200 mg, 0.41 mmol) in dry dichloromethane (10 mL) was added cesium iodide (127 mg, 0.49 mmol) and boron trifluoride etherate (45% BF3, 69 mg, 0.49 mmol) in an ice bath. The reaction was stirred at room temperature overnight. Water was added to quench the reaction at 0°C. The aqueous phase was extracted with dichloromethane, and 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 afford Linker-1 (50 mg) as an off-white solid. m / z: [M+H] + 597.0.

[0468] Synthesis of Linker-2

[0469] To a solution of Mc-Val-Cit-PAB-OH (260 mg, 0.45 mmol) in dry dichloromethane (10 mL) was added cesium iodide (140 mg, 0.54 mmol) and boron trifluoride etherate (45% BF3, 77 mg, 0.54 mmol) in an ice bath. 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 afford Linker-2 (120 mg) as an off-white solid. m / z: [M+H] + 682.5.

[0470] Synthesis of Linker-3

[0471] To a solution of Mc-Val-Cit-PAB-OH (150 mg, 0.26 mmol) in DMF (4 mL) were added di(p-nitrobenzene) carbonate (158 mg, 0.52 mmol) and DIPEA (101 mg, 0.78 mmol), respectively. 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 (C18, eluent: 0-60% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford Linker-3 (125 mg) as a pale yellow solid. m / z: [M+H] + 738.1.

[0472] Synthesis of Linker-4

[0473] To a solution of Mc-Val-Ala-PAB-OH (120 mg, 0.25 mmol) in DMF (3 mL) were added di(p-nitrobenzene) carbonate (152 mg, 0.50 mmol) and DIPEA (97 mg, 0.75 mmol) in sequence. 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 (C18, eluent: 0-85% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford Linker-4 (115 mg) as a pale yellow solid. m / z: [M+H] + 652.1.

[0474] Synthesis of Linker-5

[0475] Step 1: To a solution of 1-(6-hydroxyhexyl)-1H-pyrrole-2,5-dione (75 mg, 0.38 mmol) in dichloromethane (5 mL) was added dropwise methylsulfonyl chloride (65 mg, 0.57 mmol) and pyridine (75 mg, 0.95 mmol) in an ice bath. The reaction mixture was slowly warmed to room temperature and stirred for 2 hours. Ice water was added to quench the reaction. The aqueous phase was extracted with dichloromethane. The combined organic phases were washed with saturated brine and concentrated under reduced pressure to yield 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl methanesulfonate (100 mg). m / z: [M+H] + 276.0.

[0476] 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 hours. The residue was dissolved in ethyl acetate, washed with water and saturated brine, respectively. The organic phase was separated and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain Linker-5 (105 mg). m / z: [M+H] + 308.0.

[0477] Synthesis of Linker-6

[0478] Step 1: 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) was added HATU (3.85 g, 10.1 mmol) and DIPEA (2.18 g, 16.9 mmol) in an ice bath. The reaction mixture was stirred at room temperature for 5 minutes, followed by the addition of (4-aminophenyl)methanol (1.14 g, 9.27 mmol). The reaction mixture was stirred at room temperature for 3 hours and quenched with ice water. The aqueous phase was extracted with ethyl acetate, and the combined organic phases were concentrated under reduced pressure to remove most of the organic solvent. Petroleum ether was added to the residue, and the mixture was sonicated and filtered. The filter cake was dried to obtain 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.

[0479] Step 2: To a solution of the product obtained in Step 1 (500 mg, 1.46 mmol) in acetonitrile (50 mL) was added cesium iodide (610 mg, 2.34 mmol) and boron trifluoride etherate (310 mg, 2.19 mmol). The reaction solution was slowly warmed to room temperature and stirred for 12 hours. The reaction solution was then directly concentrated under reduced pressure to yield Linker-6 (530 mg). m / z: [M+H] + 453.1.

[0480] Synthesis of Linker-8

[0481] 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 was stirred at room temperature for 2 days and then purified directly by flash column chromatography (C18, eluent: 0-60% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford L1 (200 mg) as a white solid. m / z: [M+H] + 660.2.

[0482] 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 was stirred at room temperature for 2 hours and then purified directly by Flash column chromatography (C18, eluent: 100% 0.1% trifluoroacetic acid in water) to afford L2 (160 mg) as an off-white solid. m / z: [M+H] + 438.2.

[0483] Step 3: To a solution of L2 (160 mg, 0.29 mmol) in DMF (2 mL) were added DIPEA (45 mg, 0.35 mmol) and 6-(maleimido)hexanoic acid succinimidyl ester (98 mg, 0.32 mmol) in sequence. The reaction mixture was stirred at room temperature for 3 hours and then directly purified by Flash column chromatography (C18, eluent: 0-60% acetonitrile in 0.1% trifluoroacetic acid in water) to afford L3 (20 mg) as an off-white solid. m / z: [M+H] + 631.2.

[0484] Step 4: Using the synthesis method of Linker-1, react with L3 to obtain Linker-8. m / z: [M+H] + 741.1.

[0485] Synthesis of Linker-9

[0486] Step 1: To a solution of 1,2,3,4-tetra-O-acetyl-β-D-glucuronic acid methyl ester (16 g, 42.5 mmol) in dichloromethane (160 mL) was added dropwise a solution of hydrobromic acid and acetic acid (33%, 50 mL) under ice-cooling conditions. The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was then concentrated under reduced pressure, and the residue was diluted with dichloromethane. The organic phase was washed with saturated aqueous sodium bicarbonate and saturated brine. The organic phase was separated and concentrated under reduced pressure to yield L4 (15.9 g) as a white solid. m / z: [M+Na] + 421.1.

[0487] Step 2: 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) was added silver oxide (4.16 g, 18.0 mmol). The reaction system was stirred overnight at room temperature in the dark. The reaction mixture was then filtered through celite, the filtrate was concentrated under reduced pressure, and 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.

[0488] Step 3: Dissolve L5 (2.3 g, 4.76 mmol) in a mixture of methanol (10 mL) and dichloromethane (30 mL), then add 10% palladium on carbon (400 mg). The reaction system is purged with hydrogen three times and stirred under a hydrogen atmosphere for 5 hours. The reaction mixture is then filtered through celite, and the filtrate is concentrated under reduced pressure to afford L6 (1.5 g) as a white solid. m / z: [M+H] + 456.2.

[0489] Step 4: To a solution of Boc-glycine (46 mg, 0.26 mmol) in DMF (5 mL) were added HATU (120 mg, 0.31 mmol), DIPEA (50 mg, 0.39 mmol), and L6 (95 mg, 0.21 mmol), respectively. The reaction system was stirred at room temperature for 2 hours. The reaction solution was directly purified by flash column chromatography (C18, eluent: 0% to 75% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford L7 (160 mg) as a white solid. m / z: [M+H] + 613.2.

[0490] 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-cooling conditions. The reaction mixture was stirred at room temperature for 1.5 hours. The reaction mixture was directly purified by flash column chromatography (C18, eluent: 0% to 50% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford L8 (110 mg) as a white solid. m / z: [M+H] + 513.2.

[0491] Step 6: To a solution of L8 (80 mg, 0.16 mmol) in DMF (5 mL) was added succinimidyl 6-(maleimido)hexanoate (54 mg, 0.18 mmol), and the reaction system was stirred at room temperature for 2 hours. The reaction solution was directly purified by flash column chromatography (C18, eluent: 0% to 70% acetonitrile in 0.1% trifluoroacetic acid in water) to afford L9 (110 mg) as a white solid. m / z: [M+H] + 706.2.

[0492] Step 7: To a solution of L9 (100 mg, 0.14 mmol) in acetonitrile (5 mL) was added cesium iodide (58 mg, 0.22 mmol) and boron trifluoride etherate (36 mg, 0.25 mmol) under ice-bath conditions. The reaction mixture was stirred at room temperature for 3 hours and then concentrated under reduced pressure to obtain Linker-9 (110 mg). m / z: [M+H] + 816.2.

[0493] Synthesis of Linker-10

[0494] Step 1: A solution of Boc-Val-Ala-PAB-OH (2 g, 5.08 mmol), di(p-nitrobenzene) 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 hours. The reaction solution was directly purified by flash column chromatography (C18, eluent: 0% to 80% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford L10 (1.6 g). m / z: [M+Na] + 581.2.

[0495] 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 hours. The reaction solution was directly purified by flash column chromatography (C18, eluent: 0% to 80% acetonitrile in 0.1% trifluoroacetic acid in water) to afford L11 (1.01 g). m / z: [M+Na] + 607.2.

[0496] Step 3: To a suspension of L11 (1 g, 1.71 mmol) in dichloromethane (20 mL) and tetrahydrofuran (2 mL) was added a solution of Ghosez reagent (449 mg, 3.36 mmol) in dichloromethane (2 mL) under ice-cooling conditions. The reaction mixture was stirred at 0°C for 1 hour. The mixture was concentrated under reduced pressure to afford Linker-10 (1.2 g). m / z: [M+H] + 603.4.

[0497] Synthesis of Linker-11

[0498] Step 1: To a solution of 6-maleimidocaproic acid (50 mg, 0.24 mmol) in DMF (3 mL) was added HATU (110 mg, 0.29 mmol) and DIPEA (62 mg, 0.48 mmol) in an ice bath. The reaction mixture was stirred for 10 minutes, and then 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 give L12 (110 mg). m / z: [M+H] + 648.5.

[0499] Step 2: Using the synthesis method of linker-1, react with L12 to obtain linker-11. m / z: [M+H] + 759.0.

[0500] Synthesis of Linker-12

[0501] Step 1: To a solution of amino-pentaethylene glycol-tert-butyl propionate (266 mg, 0.65 mmol) in DMF (5 mL) were added EDCI (187 mg, 0.98 mmol) and HOBT (132 mg, 0.98 mmol) in sequence. The reaction was stirred at room temperature for 5 minutes, followed by the addition of 2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetic acid (150 mg, 0.65 mmol). The reaction was stirred at room temperature for 1 hour and then purified directly by flash column chromatography (C18, eluent: 0-40% acetonitrile in 0.1% trifluoroacetic acid in water) to afford L13 (260 mg) as a light yellow solid. m / z: [M+H] + 623.2.

[0502] Step 2: Under ice-cooling, trifluoroacetic acid (2 mL) was added dropwise to a solution of L13 (130 mg, 0.21 mmol) in dichloromethane (5 mL). After addition, the reaction mixture was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure, and the residue was purified by flash column chromatography (C18, eluent: 0-45% acetonitrile in 0.1% trifluoroacetic acid in water) to afford Linker-12 (66 mg) as a light yellow oil. m / z: [M+H] + 567.2.

[0503] Synthesis of Linker-14

[0504] Using the synthesis method of Linker-6, Linker-14 was synthesized from L14 (L14 was obtained by replacing Boc-glycine in step 4 with Boc-L-alanine using the synthesis method of L8) and Fmoc-L-valine. m / z: [M+Na] + 980.0.

[0505] Synthesis of Linker-18

[0506] Step 1: Under ice bath conditions, neopentyl chlorosulfonate (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 solution was stirred at room temperature for 6 hours. The reaction was quenched by adding saturated aqueous ammonium chloride solution, the aqueous phase was extracted with ethyl acetate, the organic phases were combined and washed with saturated brine, and the organic phase was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 to 2 / 1) to obtain L16 (960 mg) as a yellow oil. m / z: [M+H2O] + 337.2.

[0507] Step 2: 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) was slowly added zinc powder (6.14 g, 93.9 mmol) under ice-bath conditions. The reaction system was stirred at room temperature for 3 hours. The mixture was concentrated under reduced pressure, and the residue was extracted with ethyl acetate. The organic phases were combined and concentrated under reduced pressure, and the residue was purified by flash column chromatography (C18, eluent: 0% to 60% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to obtain L17 (1.5 g) as a yellow solid. m / z: [M+H] + 290.1.

[0508] Step 3: To a solution of L17 (500 mg, 1.73 mmol) in dichloromethane (15 mL) was added TBSCl (313 mg, 2.08 mmol) and imidazole (153 mg, 2.25 mmol). The reaction mixture was stirred at room temperature for 2 hours, concentrated under reduced pressure, and the residue was purified by flash column chromatography (petroleum ether / ethyl acetate = 4 / 1) to afford L18 (650 mg) as a yellow solid. m / z: [M+H] + 404.2.

[0509] 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 added HATU (761 mg, 2 mmol) and DIPEA (398 mg, 3.08 mmol) in sequence. The reaction solution was stirred at room temperature for 16 hours, quenched with 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 = 4 / 1) to give L19 (880 mg) as a light yellow oil. m / z: [M+H] + 683.2.

[0510] Step 5: To a solution of L19 (850 mg, 1.24 mmol) in tetrahydrofuran (10 mL) was added triethylamine trihydrofluoride (1 g, 6.2 mmol) under ice-bath conditions. The reaction mixture was stirred at room temperature for 5 hours and then adjusted to pH 7 with saturated aqueous sodium bicarbonate at 0°C. The mixture was extracted with ethyl acetate, and the organic phases were combined and concentrated under reduced pressure. The residue was purified by flash column chromatography (petroleum ether / ethyl acetate = 1 / 3) to afford L20 (550 mg) as a colorless oil. m / z: [M+H] + 569.2.

[0511] Step 6: To a solution of L20 (200 mg, 0.35 mmol) in acetonitrile (20 mL) was added cesium iodide (164 mg, 0.63 mmol) and boron trifluoride etherate (1396 mg, 0.98 mmol) in an ice bath. The reaction mixture was stirred at room temperature for 48 hours and then concentrated under reduced pressure. The residue was purified by flash column chromatography (petroleum ether / ethyl acetate = 1 / 1) to afford Linker-18 (140 mg) as a white solid. m / z: [M+H] + 679.0.

[0512] Synthesis of Linker-20

[0513] Step 1: To a solution of Fmoc-Gly-Gly-OH (10 g, 28.2 mmol) in tetrahydrofuran (75 mL) and toluene (25 mL) was added lead tetraacetate (17.5 g, 39.5 mmol). 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 to 2 / 3) to afford L21 (9.5 g) as a white solid. m / z: [M+Na] + 391.2.

[0514] Steps 2 & 3: To a solution of L21 (500 mg, 1.36 mmol) in dichloromethane (10 mL) was added trimethylsilyl chloride (148 mg, 1.36 mmol). The reaction was stirred at room temperature for 1 hour and then concentrated under reduced pressure to afford L22. L22 was dissolved in acetonitrile (10 mL) and 4-mercaptobutyric acid (160 mg, 1.36 mmol) was added. The reaction was stirred at room temperature overnight and then purified directly by flash column chromatography (C18, eluent: 0-85% acetonitrile in 0.1% trifluoroacetic acid in water) to afford L23 (400 mg) as a white solid. m / z: [M+H] + 429.2.

[0515] Step 4: To a solution of L23 (390 mg, 0.91 mmol) in dichloromethane (5 mL) was added triethylamine (100 mg, 1 mmol) and isobutyl chloroformate (140 mg, 1 mmol) in an ice bath. 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).

[0516] Synthesis of Linker-21

[0517] Step 1: To a solution of 5-formyl-2-hydroxybenzoic acid (10 g, 60.2 mmol) in dichloromethane (100 mL) was added 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) in an ice bath. The reaction mixture was stirred at room temperature for 12 hours and then purified directly by flash column chromatography (C18, eluent: 0-65% acetonitrile in 0.1% trifluoroacetic acid in water) to afford L24 (7.9 g). m / z: [M+H] + 224.2.

[0518] 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 hours, then filtered through celite, and 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.

[0519] Step 3: To a solution of L25 (2.18 g, 4.05 mmol) in ethanol (10 mL) and tetrahydrofuran (20 mL) was added sodium borohydride (310 mg, 8.08 mmol) under ice-bath conditions. The reaction mixture was stirred at 0°C for 2 hours, quenched with 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 afford L26 (2.7 g) as a brown foamy solid. m / z: [M+H] + 542.2.

[0520] Step 4: Using the synthesis method of Linker-1, react with L26 to obtain Linker-21. m / z: [M+H] + 652.2.

[0521] Synthesis of Linker-22

[0522] Step 1: To a solution of 4-(3-tosyl-2-(tosylmethyl)propionyl)benzoic acid (100 mg, 0.2 mmol) in DMF (30 mL) was added EDCI (58 mg, 0.3 mmol), HOBT (43 mg, 0.32 mmol), and Val-Ala-PAB-OH (59 mg, 0.2 mmol) in an ice bath. The reaction mixture was stirred at room temperature for 1 hour and then purified directly by flash column chromatography (C18, eluent: 0-75% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford L27 (100 mg).

[0523] Step 2: Using the synthetic method of Linker-1, react with L27 to obtain Linker-22. m / z: [M+H] + 886.0.

[0524] Synthesis of Linker-23

[0525] Step 1: To a solution of Cbz-Gly-Gly-Phe-Gly-OtBu (350 mg, 0.66 mmol) in methanol (15 mL) and dichloromethane (5 mL) was added palladium on carbon (70 mg, 10% wt). The reaction system was purged with hydrogen three times and stirred under a hydrogen atmosphere for 2 hours. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to afford L28 (250 mg) as a yellow oil. m / z: [M+H] + 393.2.

[0526] Steps 2 & 3: L30 was synthesized from L28 and Linker-12 using the synthesis method of Linker-12. m / z: [M+H] + 884.4.

[0527] Step 4: To a solution of L30 (130 mg, 0.15 mmol) and N-hydroxysuccinimide (51.8 mg, 0.45 mmol) in DMF (2 mL) was added N,N'-diisopropylcarbodiimide (56.8 mg, 0.45 mmol) under ice-cooling conditions. The reaction mixture was stirred at room temperature overnight and then directly purified by flash column chromatography (C18, eluent: 0% to 50% acetonitrile in 0.05% trifluoroacetic acid in water) to afford Linker-23 (60 mg) as a yellow solid. m / z: [M+H] + 982.4.

[0528] Synthesis of Linker-24

[0529] Step 1: To a solution of β-D-galactose pentaacetate (6.20 g, 15.9 mmol) in dichloromethane (60 mL) was added dropwise 33% hydrobromic acid in acetic acid (15 mL) under ice-cooling conditions. The reaction system was stirred at room temperature for 2 hours. The reaction mixture was then poured into ice water (300 mL) and extracted with icy dichloromethane. The combined organic phases were washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to yield L31 (6.53 g) as a white solid. m / z: [M+Na] + 433.0.

[0530] Step 2: 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) was added silver oxide (13.1 g, 56.4 mmol). The reaction system was stirred at room temperature in the dark overnight. The reaction mixture was then filtered through celite and concentrated under reduced pressure. The residue was purified by flash column chromatography (dichloromethane / methanol = 20 / 1) to afford L32 (7.78 g) as a white solid. m / z: [M+Na]+ 520.2.

[0531] Step 3: Dissolve L32 (2 g, 4.02 mmol) in ethyl acetate (100 mL), then add 10% palladium on carbon (0.50 g) and one drop of triethylamine. Stir the reaction mixture under a hydrogen balloon overnight. Filter the reaction mixture through celite and concentrate under reduced pressure to afford L33 (1.88 g) as a white solid. m / z: [M+H] + 470.2.

[0532] Step 4: To a solution of L33 (1 g, 2.13 mmol) in DMF (10 mL) was added EDCI (0.61 g, 3.18 mmol) and HOBT (0.43 g, 3.18 mmol) under ice-cooling conditions. A solution of 4-maleimidophenylacetic acid (0.49 g, 2.12 mmol) in DMF (2 mL) was then slowly added dropwise to the mixture. The reaction was stirred overnight at room temperature. The reaction solution was directly purified by flash column chromatography (C18, eluent: 0% to 52% acetonitrile in 0.05% trifluoroacetic acid in water) to afford L34 (442 mg) as a yellow solid. m / z: [M+Na] + 705.2.

[0533] Step 5: To a solution of L34 (200 mg, 0.29 mmol) and cesium iodide (151 mg, 0.58 mmol) in acetonitrile (5 mL) was added dropwise boron trifluoride etherate (82.3 mg, 0.58 mmol) under ice-bath conditions. The reaction system was stirred at room temperature for 2 hours. The reaction mixture was then filtered and concentrated under reduced pressure. The residue was purified by prep-TLC (petroleum ether / ethyl acetate = 1 / 1) to afford Linker-24 (144 mg) as a yellow solid. m / z: [M+Na] + 815.1.

[0534] Synthesis of Linker-26

[0535] Step 1: To a solution of L6 (800 mg, 1.76 mmol) in dichloromethane (10 mL) was added imidazole (140 mg, 2.11 mmol) and TBSCl (530 mg, 3.52 mmol) under ice-cooling conditions. 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% to 85% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford L35 (920 mg). m / z: [M+H] + 570.2.

[0536] Step 2: To a solution of 6-(tert-butyloxycarbonyl)amino)hexanoic acid (100 mg, 0.43 mmol) in DMF (3 mL) was added HATU (210 mg, 0.56 mmol) and DIPEA (110 mg, 0.86 mmol) under ice-cooling conditions. The reaction mixture was stirred at 0°C for 5 minutes, and then L35 (240 mg, 0.43 mmol) was added. The reaction mixture was stirred at room temperature for 12 hours and then purified directly by flash column chromatography (C18, eluent: 0-85% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford L36 (300 mg). m / z: [M+H] + 783.3.

[0537] Step 3: A mixture 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, concentrated under reduced pressure, and the residue was purified by flash column chromatography (C18, eluent: 0-65% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford L37 (115 mg). m / z: [M+H] + 569.2.

[0538] Steps 4 & 5: Using the synthetic method of Linker-22, 4-(3-toluenesulfonyl-2-(toluenesulfonylmethyl)propionyl)benzoic acid and L37 were reacted to obtain Linker-26. m / z: [M+H] + 1161.2.

[0539] Synthesis of Linker-27

[0540] Step 1: To a solution of N,N'-diacetyldiaminomethane (20 g, 154 mmol) in anhydrous tetrahydrofuran (1 L) was added sodium hydroxide (60%, 8.61 g, 215 mmol). The reaction mixture was stirred at room temperature for 10 minutes. 3-Chloro-2-chloromethylpropene (20.2 g, 161 mmol) was then added dropwise. The reaction mixture was stirred at 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 afford L39 (12 g) as a yellow oil. m / z: [M+H] + 183.3.

[0541] Step 2: To a suspension of L39 (4 g, 22.0 mmol) in water (20 mL) was added sodium hydroxide (4.39 g, 110 mmol). The reaction was stirred at 110°C overnight. The reaction was cooled in an ice bath and solid sodium hydroxide (1.4 g) was added. The aqueous phase was extracted with dichloromethane, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to afford crude L40 (4.3 g), which was used directly in the next reaction.

[0542] Step 3: To a solution of L40 (2.15 g, 21.9 mmol) in dichloromethane (40 mL) was added HOBT (11.8 g, 87.6 mmol), EDCI (16.8 g, 87.6 mmol), diethylphosphinoacetic acid (14 g, 71.4 mmol), and DIPEA (14.2 g, 110 mmol) in an ice bath. The reaction mixture was slowly warmed to room temperature and stirred overnight. The mixture was then 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% to 60% acetonitrile in 0.05% trifluoroacetic acid in water) to afford L41 (3.5 g) as a colorless oil. m / z: [M+H] + 455.2.

[0543] Step 4: Ozone was introduced into a mixture of L41 (1 g, 2.2 mmol) in dichloromethane (20 mL) and methanol (5 mL) at -70°C and stirred for 10 minutes. Excess ozone was replaced by nitrogen gas, and dimethyl sulfide (409 mg, 6.6 mmol) was added. The reaction mixture was stirred and warmed to room temperature, then concentrated under reduced pressure to afford L42 (1 g) as a yellow oil. m / z: [M+H] + 457.2.

[0544] Step 5: To a solution of L42 (500 mg, 1.1 mmol) in methanol (5 mL) were added 3-(aminooxy)propionic acid (220 mg, 2.09 mmol) and sodium acetate (451 mg, 5.5 mmol). The reaction was stirred at room temperature overnight, quenched with water, and purified directly by flash column chromatography (C18, eluent: 0% to 35% acetonitrile in 0.05% trifluoroacetic acid in water) to afford L43 (560 mg) as a light yellow oil. m / z: [M+H] + 544.2.

[0545] Step 6: To a solution of L43 (97 mg, 0.18 mmol) in anhydrous tetrahydrofuran (5 mL) was added sodium hydroxide (60%, 36 mg, 0.9 mmol) under ice-bath conditions. The reaction mixture was stirred at 0°C for 30 minutes. Freshly prepared formaldehyde tetrahydrofuran solution (1.38 mL, 0.29 M) was added dropwise to the reaction mixture. The resulting reaction mixture was stirred at room temperature for 10 minutes. The reaction was quenched with water and then directly purified by flash column chromatography (C18, eluent: 0.05% trifluoroacetic acid in water, 1 minute) to afford Linker-27 (133 mg) as a yellow oil. m / z: [M+H] + 296.2.

[0546] Synthesis of Linker-32

[0547] Step 1: To a solution of L33 (1 g, 2.13 mmol) in dichloromethane (10 mL) was added TBSCl (390 mg, 2.56 mmol) and imidazole (190 mg, 2.77 mmol) in an ice bath. The reaction mixture was stirred at room temperature for 2 hours, quenched with water, 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. The residue was purified by flash column chromatography (ethyl acetate / petroleum ether = 1 / 3) to afford L45 (900 mg) as an off-white solid. m / z: [M+H] + 584.2.

[0548] Step 2: To a solution of L45 (120 mg, 0.21 mmol) in DMF (3 mL) was added linker-27 (124 mg, 0.21 mmol), DIPEA (54 mg, 0.42 mmol), and HATU (120 mg, 0.32 mmol) in an ice bath. The reaction mixture was stirred at room temperature for 2 days and then purified directly by flash column chromatography (C18, eluent: 0% to 75% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford L46 (10 mg) as an off-white solid. m / z: [M+Na] + 883.4.

[0549] Step 3: To a solution of L46 (10 mg, 0.012 mmol) in tetrahydrofuran (1 mL) was added triethylamine trihydrofluoride (10 mg, 0.06 mmol) dropwise under ice-cooling conditions. The reaction mixture was stirred at room temperature overnight and quenched with water. The aqueous phase was extracted with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to afford L47 (9 mg) as an off-white solid. m / z: [M+Na] + 769.2.

[0550] Step 4: To a solution of L47 (9 mg, 0.012 mmol) in dichloromethane (2 mL) was added dropwise thionyl chloride (3 mg, 0.024 mmol) under ice-bath conditions. The reaction mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure to afford Linker-32 (9 mg) as a yellow solid. m / z: [M+Na] + 787.2.

[0551] Synthesis of Linker-33

[0552] Step 1: To a solution of tert-butyl 5-formylsalicylate (1 g, 4.50 mmol) in acetonitrile (40 mL) was added acetyl bromide-α-D-glucose (1.85 g, 4.5 mmol) and silver oxide (3.13 g, 13.5 mmol). The reaction system was stirred at room temperature for 12 hours, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (methanol / dichloromethane = 0%-5%) to afford L48 (2.2 g) as a light yellow solid. m / z: [M+Na] + 575.2.

[0553] Step 2: A mixture 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 slowly adjusted to pH 6.0-6.5 with saturated aqueous sodium bicarbonate. The aqueous phase was extracted with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to afford L49 (1.5 g) as a light yellow solid. m / z: [M+Na] + 519.2.

[0554] Step 3: To a solution of L49 (1.5 g, 3.02 mmol) in tetrahydrofuran (5 mL) and ethanol (1 mL) was added portionwise sodium borohydride (110 mg, 3.02 mmol) in an ice bath. The reaction was stirred at room temperature for 3 hours. Acetone was added to quench the reaction, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (C18, 0%-50% acetonitrile in 0.1% trifluoroacetic acid in water) to afford L50 (1.3 g) as a white solid. m / z: [M+Na] + 521.2.

[0555] Step 4: To a solution of L50 (500 mg, 1 mmol) in dichloromethane (2 mL) was added thionyl chloride (240 mg, 2 mmol) under ice-bath conditions. The reaction mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure, and dichloromethane (5 mL) was added to the residue, followed by concentration under reduced pressure. This process was repeated twice to afford L51 (500 mg) as a white solid. m / z: [M+Na]+ 539.0.

[0556] 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 was stirred at room temperature for 0.5 h. The reaction solution was concentrated under reduced pressure and directly purified by flash column chromatography (C18, 0%-60% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to afford Linker-33 (80 mg) as a white solid. m / z: [M+Na] + 371.0.

[0557] Synthesis of Linker-34

[0558] Synthesis of L52: Using the synthesis method of Linker-27, replace 3-(aminooxy)propionic acid in step 5 with tert-butyl (2-(aminooxy)ethyl)carbamate to obtain L52. m / z: [M+Na] + 389.2.

[0559] Trifluoroacetic acid (1 mL) was added to a solution of L52 (1 g, 2.73 mmol) in dichloromethane (9 mL) under ice-cooling conditions. The reaction mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure. The residue was purified by flash column chromatography (C18, 0%-60% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford Linker-34 (400 mg) as a colorless oil. m / z: [M+H] + 267.2.

[0560] Synthesis of Linker-35

[0561] Step 1: To a solution of L53 (400 mg, 0.82 mmol) in dichloromethane (40 mL) was added TBSCl (160 mg, 1.07 mmol) and imidazole (150 mg, 2.13 mmol) in an ice bath. The reaction mixture was stirred at room temperature for 2 hours, quenched with methanol, and concentrated under reduced pressure at low temperature. The residue was purified by flash column chromatography (C18, 0%-95% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford L54 (470 mg) as a white solid. m / z: [M+H] + 600.2.

[0562] Step 2: To a solution of L54 (300 mg, 0.5 mmol) in DMF (2 mL) was added 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) under ice-cooling conditions. The reaction mixture was stirred at room temperature for 2 hours and then directly purified by flash column chromatography (C18, 0%-85% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford L55 (400 mg) as a white solid. m / z: [M+H] + 813.2.

[0563] Step 3: To a solution of L55 (390 mg, 0.31 mmol) in tetrahydrofuran (5 mL) was added triethylamine hydrofluoride (0.25 mL, 1.55 mmol) dropwise under ice-cooling conditions. The reaction mixture was stirred at room temperature for 2.5 hours and then directly purified by flash column chromatography (C18, 0%-49% acetonitrile in 0.05% trifluoroacetic acid in water) to afford L56 (210 mg) as a yellow solid. m / z: [M+H] + 699.2.

[0564] Step 4: To a solution of L56 (50 mg, 0.07 mmol) in dichloromethane (10 mL) was added dropwise thionyl chloride (26 mg, 0.22 mmol) under ice-bath conditions. The reaction mixture was stirred at room temperature for 3 hours and concentrated under reduced pressure to afford Linker-35 (50 mg) as a yellow solid. m / z: [M+Na] + 739.2.

[0565] Synthesis of Linker-38

[0566] Step 1: To a solution of L6 (500 mg, 1.1 mmol) in dichloromethane (10 mL) was added imidazole (90 mg, 1.32 mmol) and TBSCl (330 mg, 2.2 mmol) in an ice bath. The reaction mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure to remove the solvent. The residue was purified by flash column chromatography (C18, 90% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford L57 (600 mg) as a white solid. m / z: [M+H] + 570.2.

[0567] Step 2: To a solution of L57 (200 mg, 0.62 mmol) in DMF (8 mL) was added HATU (280 mg, 0.74 mmol), DIPEA (160 mg, 1.24 mmol), and L57 (390 mg, 0.68 mmol) in an ice bath. The reaction mixture was stirred at room temperature for 2 hours and then purified directly by flash column chromatography (C18, 95% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford L58 (500 mg) as a white solid. m / z: [M+Na] + 897.3.

[0568] Step 3: To a solution of L58 (350 mg, 0.4 mmol) in ethyl acetate (5 mL) was added palladium on carbon (10%, 355 mg). The reaction system was purged with hydrogen and stirred at room temperature under a hydrogen atmosphere for 2 hours. The reaction system was filtered through celite, and the filtrate was concentrated under reduced pressure to afford L59 (300 mg) as a white solid. m / z: [M+H] + 785.0.

[0569] Step 4: To a solution of L59 (230 mg, 0.29 mmol) in DMF (10 mL) was added 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-hexaoxanonadecan-19-amine (86 mg, 0.29 mmol) in an ice bath. The reaction mixture was stirred at room temperature for 2 hours and then purified directly by flash column chromatography (C18, 95% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford L60 (300 mg) as a colorless oil. m / z: [M+H] + 1062.5.

[0570] 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.

[0571] Synthesis of Linker-39

[0572] Step 1: To a solution of compound L45 (500 mg, 0.86 mmol) in methanol (15 mL) was added potassium carbonate (0.36 g, 2.58 mmol). The reaction system was stirred at room temperature for 1 hour, adjusted to pH 6-7 with acetic acid, and concentrated under reduced pressure at low temperature. The product was then purified directly by flash column chromatography (C18, eluent: 10%-90% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford L63 (330 mg) as a white solid. m / z: [M+H] + 416.3.

[0573] Step 2: To a solution of L63 (180 mg, 0.43 mmol) in DMF (5 mL) was added 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) under ice-cooling conditions. The reaction mixture was stirred at room temperature for 2 hours and then purified directly by flash column chromatography (C18, eluent: 0% to 85% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford L64 (270 mg) as a white solid. m / z: [M+Na] + 651.2.

[0574] Step 3: To a solution of L64 (200 mg, 0.32 mmol) in tetrahydrofuran (5 mL) was added triethylamine hydrogen fluoride (150 mg, 0.96 mmol) under ice-cooling conditions. The reaction mixture was stirred at room temperature for 2 hours and then purified directly by flash column chromatography (C18, eluent: 0% to 75% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford L65 (160 mg) as a white solid. m / z: [M+H] + 515.3.

[0575] Step 4: To a solution of L65 (30 mg, 0.058 mmol) in dichloromethane (3 mL) was added thionyl chloride (21 mg, 0.17 mmol) and a drop of DIPEA. The reaction was stirred at room temperature for 12 hours and concentrated under reduced pressure to afford Linker-39 (33 mg) as a light yellow oil. m / z: [M+H] + 579.0.

[0576] Synthesis of Linker-40

[0577] Step 1: To a solution of L35 (1.33 g, 2.34 mmol) and (S)-2-((tert-butoxycarbonyl)amino)pent-4-ynoic acid (500 mg, 2.34 mmol) in DMF (6 mL) were added HATU (1.16 g, 3.04 mmol) and DIPEA (756 mg, 5.85 mmol) in sequence. The reaction solution was stirred at room temperature for 5 hours, quenched with 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.

[0578] Step 2: Under nitrogen protection, sodium ascorbate (166 mg, 0.84 mmol), cuprous iodide (80 mg, 0.42 mmol) and DIPEA (54 mg, 0.42 mmol) were added to a DMF (2 mL) solution of L66 (320 mg, 0.42 mmol) and 19-azido-2,5,8,11,14,17-hexaoxa-n-nonadecane (148 mg, 0.46 mmol). The reaction solution was stirred at 60 ° C for 2 hours, cooled to room temperature, and quenched with water. The aqueous phase was extracted with ethyl acetate, and 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.

[0579] Step 3: To a solution of L67 (347 mg, 0.32 mmol) in dichloromethane (4 mL) was added trifluoroacetic acid (2 mL). The reaction mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The residue was purified by prep-HPLC (mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution from 10% to 60% mobile phase B over 25 minutes) to afford L68 (280 mg) as a light yellow solid. m / z: [M+H] + 872.4.

[0580] Step 4: To a solution of L68 (200 mg, 0.23 mmol) and 4-maleimidophenylacetic acid (53 mg, 0.23 mmol) in DMF (2 mL) was added HATU (105 mg, 0.28 mmol) and DIPEA (60 mg, 0.46 mmol) under ice-cooling conditions. The reaction mixture was stirred at 0°C for 2 hours and then directly purified by prep-HPLC (mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution from mobile phase B from 10% to 60% over 20 minutes) to afford L69 (300 mg) as a yellow solid. m / z: [1 / 2M+H]+ 543.2.

[0581] Step 5: To a solution of L69 (60 mg, 0.06 mmol) in dichloromethane (2 mL) was added thionyl chloride (26 mg, 0.22 mmol) under ice-bath conditions. The reaction mixture was slowly warmed to room temperature and stirred for 1 hour. The solution was concentrated under reduced pressure to afford Linker-40 (60 mg) as a yellow solid. m / z: [M+H] + 1103.4.

[0582] Synthesis of Linker-41 to Linker-44

[0583] Step 1: To a solution of CTC resin (2 g, 2.2 mmol) in dichloromethane (20 mL) were added Fmoc (1.03 g, 3.30 mmol) and N,N-diisopropylethylamine (2.13 g, 16.5 mmol). The reaction mixture was shaken for 2 hours and then washed four times with DMF and DCM, followed by two washes with methanol (10 minutes each). The resin was washed twice with a 20% diethylamine / DMF mixture (15 minutes each), four times with DMF and DCM, and then DMF (30 mL) was added. Fmoc (1.34 g, 4.32 mmol), HATU (1.67 g, 4.4 mmol), and DIPEA (1.4 g, 10.8 mmol) were then added. The reaction mixture was shaken for 2 hours and then washed four times with DMF and DCM. The resin was washed twice with a 20% diethylamine / DMF mixed solution (15 minutes each), and then washed four times with DMF and DCM, and then dried under reduced pressure. The above steps were repeated until the CTC resin-polysarcosine with the target i value was obtained.

[0584] Step 2: Add bromoacetic acid (2.45 g, 17.6 mmol) and N,N'-diisopropylcarbodiimide (3.6 g, 28.6 mmol) to a DMF (30 mL) solution of the CTC resin-polysarcosine obtained in Step 1. Shake the reaction mixture on a shaker for 1 hour and wash four times with DMF to obtain CTC resin-polysarcosine-2-bromoacetamide.

[0585] Step 3: To a solution of the CTC resin-polysarcosine-2-bromoacetamide obtained in Step 2 in DMF (20 mL) were added 2-azidoethylamine (280 mg, 3.3 mmol) and N,N'-diisopropylcarbodiimide (430 mg, 3.3 mmol). The reaction mixture was shaken for 2 hours and washed four times with DMF and DCM. The resin was then dissociated with a 1% TFA / DMF mixture (twice, 5 minutes each). The solution was then concentrated under reduced pressure. The residue was directly purified by prep-HPLC (mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution: mobile phase B from 10% to 35% over 20 minutes) to afford Linkers 41 to 44 as white solids.

[0586] Linker-41, i=6, m / z: [M+H] + 571.4.

[0587] Linker-42, i=12, m / z: [M+H] + 997.6.

[0588] Linker-43, i=18, m / z: [1 / 2M+H] + 712.4.

[0589] Linker-44, i=24, m / z: [1 / 2M+H] + 925.5.

[0590] Synthesis of Linker-47

[0591] Step 1: To a solution of L45 (150 mg, 0.26 mmol) and Boc-glycine (46 mg, 0.26 mmol) in DMF (3 mL) were added HATU (129 mg, 0.34 mmol) and DIPEA (67 mg, 0.52 mmol). The reaction mixture was stirred at room temperature for 3 hours and directly analyzed by prep-HPLC (mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution from 10% to 95% mobile phase B, elution time: 20 minutes). To the collected eluate was added 0.1% trifluoroacetic acid in water (20 mL), stirred at room temperature, and after LCMS monitoring, the reaction mixture was lyophilized to obtain L70 (95 mg) as a white solid. m / z: [M+Na] + 649.2.

[0592] Step 2: To a solution of L70 (95 mg, 0.15 mmol) in dichloromethane (2 mL) was added thionyl chloride (36 mg, 0.3 mmol) under ice-bath conditions. The reaction mixture was slowly warmed to room temperature and stirred for 1 hour. The mixture was concentrated under reduced pressure to afford Linker-47 (100 mg) as a white solid. m / z: [M+Na] + 667.2.

[0593] The above synthesis method is used to synthesize the linker shown in Table 1:

[0594] Table 1

[0595] Synthesis of tert-Butyl(chloromethyl)ethane-1,2-diyl bis(methylcarbamate)

[0596] Under ice bath conditions, pyridine (1.14 g, 14.4 mmol) and chloromethyl chloroformate (1.48 g, 11.5 mmol) were added to a solution of tert-butyl methyl (2-(methylamino)ethyl)carbamate (1.8 g, 9.56 mmol) in dichloromethane (20 mL). The reaction system was slowly warmed to room temperature and stirred for 12 hours. Water was added to quench the reaction, and the aqueous phase was extracted with dichloromethane. The organic phase was separated and concentrated under reduced pressure. The residue was purified by Flash column chromatography (petroleum ether / ethyl acetate = 1 / 3) to give tert-butyl (chloromethyl) ethane-1,2-diyl bis(methylcarbamate) (2.3 g).

[0597] Synthesis of 4-((tert-Butoxycarbonyl)(methyl)amino)butane(isobutylcarbonyl)anhydride

[0598] 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) under ice-cooling conditions. The reaction mixture was stirred at room temperature for 2 hours and concentrated under reduced pressure. The residue was dissolved in dichloromethane and filtered through a short silica gel column. The filtrate was concentrated under reduced pressure to give 4-((tert-butyloxycarbonyl)(methyl)amino)butane(isobutylcarbonyl)anhydride (110 mg) as a colorless oil. m / z: [M+Na] + 340.2.

[0599] Synthesis of tert-Butyl (S)-2-((((chloromethoxy)carbonyl)(2-(dimethylamino)ethyl)amino)methyl)pyrrolidine-1-carboxylate

[0600] Step 1: To a solution of (S)-tert-butyl 2-acetylpyrrolidine-1-carboxylate (15.8 g, 79.4 mmol) in dichloromethane (20 mL) was added N,N-dimethylethylenediamine (7 g, 79.4 mmol) and sodium triacetoxyborohydride (21 g, 99.3 mmol) in an ice bath. The reaction system was slowly warmed to room temperature and stirred for 3 hours. The reaction was quenched by water, and the aqueous phase was extracted with dichloromethane. The organic phases were combined and concentrated under reduced pressure. The residue was purified by flash column chromatography (dichloromethane / methanol = 1 / 8) to give (S)-tert-butyl 2-(((2-(dimethylamino)ethyl)amino)methyl)pyrrolidine-1-carboxylate (900 mg). m / z: [M+H] + 272.2.

[0601] Step 2: Under ice bath conditions, pyridine (180 mg, 2.22 mmol) and chloromethyl chloroformate (190 mg, 1.44 mmol) were added to a solution of the product obtained in step 1 (300 mg, 1.11 mmol) in dichloromethane (15 mL) in sequence. The reaction solution was slowly warmed to room temperature and stirred for 12 hours, and water (15 mL) was added to quench the reaction. The aqueous phase was extracted with dichloromethane, and the organic phases were combined and concentrated under reduced pressure. The residue was purified by Flash column chromatography (dichloromethane / methanol = 1 / 5) to give (S)-2-(((chloromethoxy)carbonyl)(2-(dimethylamino)ethyl)amino)methyl)pyrrolidine-1-carboxylic acid tert-butyl ester (110 mg). m / z: [M+H] + 364.2.

[0602] Synthesis of 4-((4-((tert-Butoxycarbonyl)amino)benzyl)mercapto)butane(isobutylcarbonyl)anhydride

[0603] Step 1: 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) was slowly added dropwise with an ice bath. After the addition, 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 reaction system. The reaction solution was stirred at room temperature for another 3 hours and then concentrated under reduced pressure. The residue was purified by flash column chromatography (C18, eluent: 0-60% acetonitrile in 0.1% aqueous trifluoroacetic acid) to yield 4-((4-((tert-butoxycarbonyl)amino)benzyl)oxy)butanoic acid (860 mg). m / z: [M+Na] + 348.2.

[0604] Step 2: To a solution of the product from Step 1 (350 mg, 1.08 mmol) and triethylamine (142 mg, 1.4 mmol) in dichloromethane (10 mL) was slowly added isobutyl chloroformate (134 mg, 0.98 mmol) under ice-cooling conditions. The reaction mixture was stirred at 0°C for 1 hour and then concentrated under reduced pressure. Dichloromethane (3 mL) was added to the residue, and the mixture was filtered through a short silica gel column. The filtrate was concentrated under reduced pressure to provide 4-((4-((tert-butyloxycarbonyl)amino)benzyl)mercapto)butane(isobutylcarbonyl)anhydride (500 mg) as a light yellow oil. m / z: [M+Na] + 448.2.

[0605] Synthesis of 4-((4-((tert-Butoxycarbonyl)amino)benzyl)dimercapto)butyric acid

[0606] Step 1: To a solution of thioacetic acid (5.3 g, 70 mmol) and triethylamine (8.85 g, 87.5 mmol) in toluene (75 mL) was slowly added dropwise a mixed solution of p-nitrobenzyl bromide (7.56 g, 35 mmol) in toluene (60 mL) and tetrahydrofuran (30 mL). After the addition was complete, the reaction solution was stirred at room temperature overnight. The mixture was concentrated under reduced pressure, and the residue was re-dissolved in a small amount of ethyl acetate. 10 volumes of n-heptane were added to precipitate a solid, 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.

[0607] Step 2: Add 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) to ethanol (50 mL) and water (15 mL). The reaction mixture was stirred at 90°C for 1.5 hours, filtered through celite, 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 in water) to afford S-(4-aminophenyl)thioacetate (1.3 g) as a yellow solid. m / z: [M+H] + 182.0.

[0608] Step 3: A solution of S-(4-aminophenyl)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 at room temperature overnight. The reaction solution was concentrated under reduced pressure, and the residue was purified by flash column chromatography (petroleum ether / ethyl acetate = 3 / 1) to give S-(4-((tert-butoxycarbonyl)amino)phenyl)thioacetate (840 mg) as a white solid. m / z: [M+Na] + 304.2.

[0609] Step 4: 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) was added lithium hydroxide monohydrate (94 mg, 3.9 mmol) under ice-cooling conditions. The reaction mixture was stirred at 0°C for 50 minutes and then concentrated under reduced pressure. The residue was purified by flash column chromatography (C18, eluent: 0-70% acetonitrile in 0.1% aqueous trifluoroacetic acid) to afford tert-butyl (4-(mercaptomethyl)phenyl)carbamate (430 mg) as a white solid. m / z: [M+Na] + 262.2.

[0610] Step 5: To a solution of 4-(pyridin-2-yldimercapto)butanoic acid (1 g, 4.38 mmol) in acetonitrile (10 mL) was slowly added a solution of tert-butyl (4-(mercaptomethyl)phenyl)carbamate (420 mg, 1.75 mmol) in acetonitrile (20 mL) over 30 minutes. The reaction mixture was stirred at room temperature for 2 hours and then purified directly by flash column chromatography (C18, eluent: 0-80% acetonitrile in 0.1% aqueous trifluoroacetic acid) to afford 4-((4-((tert-butoxycarbonyl)amino)benzyl)dimercapto)butanoic acid (480 mg) as a white solid. m / z: [M+Na] + 380.0.

[0611] Synthesis of 5-((4-(chloromethyl)phenoxy)methyl)-1-methyl-2-nitro-1H-imidazole

[0612] Step 1: 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) was stirred at 50° C. for 5 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by flash column chromatography (ethyl acetate / petroleum ether = 1 / 3) to give 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.

[0613] Step 2: To a mixed solution of the product obtained in step 1 (600 mg, 1.51 mmol) in tetrahydrofuran (2 mL) and methanol (2 mL) was added trifluoroacetic acid (1.5 mL). The reaction solution was stirred at room temperature overnight and then directly concentrated under reduced pressure. The residue was purified by flash column chromatography (C18, eluent: 0-50% acetonitrile in 10 mM aqueous ammonium bicarbonate) to give (4-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)phenyl)methanol (260 mg). m / z: [M+H] + 264.2.

[0614] Step 3: To a solution of the product obtained in Step 2 (20 mg, 0.076 mmol) in dichloromethane (5 mL) was added thionyl chloride (90 mg, 0.076 mmol) under ice-cooling conditions. The reaction mixture was stirred at 0°C for 1 hour and then concentrated under reduced pressure to yield 5-((4-(chloromethyl)phenoxy)methyl)-1-methyl-2-nitro-1H-imidazole (20 mg). m / z: [M+H] + 282.2.

[0615] Synthesis of (1-methyl-2-nitro-1H-imidazol-5-yl)methyl (4-nitrophenyl) carbonate

[0616] A solution of (1-methyl-2-nitro-1H-imidazol-5-yl)methanol (225 mg, 1.43 mmol), DIPEA (554 mg, 4.29 mmol), and di(p-nitrophenyl) carbonate (870 mg, 2.86 mmol) in DMF (2 mL) was stirred at room temperature for 2 hours. The reaction solution was directly purified by flash column chromatography (C18, 0%-65% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) 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.

[0617] Synthesis of 5-(Chloromethyl)-1,4-dimethyl-2-nitro-1H-imidazole

[0618] Step 1: Under nitrogen, to a solution of compound 1.1 (400 mg, 1.69 mmol) in 1,4-dioxane (5 mL) were added [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride dichloromethane complex (414 mg, 0.51 mmol) and cesium carbonate (1.10 g, 3.38 mmol). After addition, the reaction system was purged with nitrogen three times, and trimethylboroxine (0.97 mL, 3.38 mmol) was added. The reaction system was purged with nitrogen three times, sealed, and stirred at 100°C for 3 hours. The reaction mixture was then cooled to room temperature, filtered through celite, and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (C18, eluent: 0% to 23% acetonitrile in 0.05% aqueous trifluoroacetic acid) to afford (1,4-dimethyl-2-nitro-1H-imidazol-5-yl)methanol (80.7 mg) as a yellow solid. m / z:[M+H] + 172.2.

[0619] Step 2: To a solution of (1,4-dimethyl-2-nitro-1H-imidazol-5-yl)methanol (27.4 mg, 0.16 mmol) in tetrahydrofuran (2 mL) was added DIPEA (51.7 mg, 0.40 mmol) and then methylsulfonyl chloride (40.3 mg, 0.35 mmol) dropwise in an ice bath. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then 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 afford 5-(chloromethyl)-1,4-dimethyl-2-nitro-1H-imidazole (28.0 mg) as a yellow oil. m / z: [M+H] + 190.0.

[0620] Synthesis of (E)-4-((4-(chloromethyl)phenyl)diazenyl)-2-hydroxybenzoic acid

[0621] Step 1: Sodium nitrite (2.1 g, 30.5 mmol) and hydrogen chloride (6 g, 165 mmol) were added to a solution of (4-aminophenyl)methanol in water (100 mL). The reaction mixture was stirred at 0°C for 20 minutes. The reaction mixture was then added to a 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 mixture was stirred at 0°C for 1 hour. The pH was adjusted to 3-4 with hydrochloric acid (1 M). The aqueous phase was extracted with ethyl acetate, and the organic phases were combined and concentrated under reduced pressure. The residue was purified by flash column chromatography (C18, eluent: 0%-85% acetonitrile in 0.1% trifluoroacetic acid in water) to provide (E)-2-hydroxy-4-((4-(hydroxymethyl)phenyl)diazenyl)benzoic acid (500 mg) as a yellow solid. m / z:[M+H] + 273.2.

[0622] Step 2: To a solution of (E)-2-hydroxy-4-((4-(hydroxymethyl)phenyl)diazenyl)benzoic acid (35 mg, 0.13 mmol) in dichloromethane (5 mL) was added thionyl chloride (39 mg, 0.33 mmol) under ice-cooling conditions. The reaction mixture was slowly warmed to room temperature and stirred for 1 hour. The mixture was then concentrated under reduced pressure to afford (E)-4-((4-(chloromethyl)phenyl)diazenyl)-2-hydroxybenzoic acid (36 mg) as a yellow solid. m / z: [M+H] + 291.0.

[0623] Synthesis of N-(5-(chloromethyl)-2-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)phenyl)-2,5,8,11,14,17,20,23-oxahexacosa-26-amine

[0624] 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 hours. The mixture was then washed with saturated 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 afford 2-nitro-4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)phenol (2.89 g) as a yellow oil. m / z: [M+Na] + 276.1.

[0625] Step 2: Under nitrogen, palladium on carbon (10%, 364 mg) was added to a solution of the product from Step 1 (2.89 g, 11.4 mmol) in methanol (150 mL). The atmosphere was then replaced with hydrogen three times, and the reaction system was stirred at room temperature under a hydrogen atmosphere for 2 hours. The reaction mixture was then filtered through celite and concentrated under reduced pressure to yield 2-amino-4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)phenol (2.54 g) as a gray-black oil. m / z: [M+Na] + 246.2.

[0626] Step 3: To a solution of 3-methyl-2-nitroimidazole-4-methanol (1.0 g, 6.36 mmol), the product of 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) in an ice bath. The reaction was stirred at room temperature for 2 hours. The reaction solution was directly purified by flash column chromatography (C18, eluent: 0% to 25% acetonitrile in 0.1% trifluoroacetic acid in water) to afford 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.

[0627] Step 4: To a solution of the product from Step 3 (510 mg, 1.41 mmol) in dichloromethane (6 mL) was added trifluoroacetic acid (2 mL) dropwise under ice-cooling conditions. The reaction system was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure and purified by flash column chromatography (C18, eluent: 0% to 42% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford (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.

[0628] Step 5: To a solution of the product from Step 4 (280 mg, 1.01 mmol) in dichloromethane (10 mL) was added tert-butyldimethylsilyl trifluoromethanesulfonate (320 mg, 1.21 mmol) and imidazole (89.4 mg, 1.31 mmol) in an ice bath. The reaction system was stirred at room temperature for 10 minutes. The mixture was then washed with water, and 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 afford 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.

[0629] Steps 6 & 7: To a solution of 4,7,10,13,16,19,22,25-octaoxahexacosadecanoic acid (65.8 mg, 0.16 mmol) in DMF (3 mL) was added HATU (79.1 mg, 0.21 mmol) and DIPEA (41.4 mg, 0.32 mmol) on ice. The reaction was stirred at 0°C for 5 minutes. The product from Step 5 (60 mg, 0.15 mmol) was added to the reaction mixture, and the resulting mixture was slowly warmed to room temperature and stirred for 16 hours. The reaction solution was directly purified by flash column chromatography (C18, eluent: 0% to 64% acetonitrile in 0.05% trifluoroacetic acid in water). The collected solution was stirred at room temperature for 4.5 hours and then lyophilized to give N-(5-(hydroxymethyl)-2-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)phenyl)-2,5,8,11,14,17,20,23-oxahexacosa-26-amine (88.7 mg) as a yellow oil. m / z: [M+H] + 673.4.

[0630] Step 8: To a solution of the product from Step 7 (21.7 mg, 0.032 mmol) in dichloromethane (3 mL) was added dropwise thionyl chloride (7.6 mg, 0.064 mmol) in an ice bath. The reaction system was stirred at 0°C for 1 hour. The reaction solution was concentrated under reduced pressure at low temperature to yield N-(5-(chloromethyl)-2-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)phenyl)-2,5,8,11,14,17,20,23-oxahexacosa-26-amine (22.3 mg) as a yellow oil. m / z: [M+H] + 691.2.

[0631] Synthesis of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-azidotetrahydro-2H-pyran-3,4-diyl diacetate

[0632] Under nitrogen, ferric 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 the addition, the reaction system was purged with nitrogen three times and stirred at room temperature for 72 hours. The reaction mixture was washed sequentially with saturated 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% to 39% acetonitrile in 0.02% trifluoroacetic acid) to afford (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.

[0633] Synthesis 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 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

[0634] Step 1: Under nitrogen, triphenylphosphine (15.3 g, 58.4 mmol) was added to a solution of trehalose (10 g, 29.2 mmol) in DMF (100 mL). The reaction was stirred at room temperature for 15 minutes. N-bromosuccinimide (6.76 g, 38 mmol) was added to the solution in an ice bath and the reaction was stirred at 80°C for 8 hours. The reaction mixture was then cooled to room temperature, quenched with methanol, and concentrated under reduced pressure to remove the solvent. Water (100 mL) was added to dissolve the residue, and the aqueous phase was back-extracted with dichloromethane. The aqueous phase was concentrated under reduced pressure to give (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.

[0635] Step 2: To a pyridine (110 mL) 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) was added acetic anhydride (37 mL, 395 mmol) dropwise, and the reaction system was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (100 mL), washed sequentially with saturated aqueous sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give (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.

[0636] Step 3: 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) was added sodium azide (0.55 g, 8.45 mmol) and the reaction system was stirred at 55 °C overnight. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure. The residue was purified by flash column chromatography (C18, eluent: 0% to 57% acetonitrile in 0.05% trifluoroacetic acid aqueous solution) to afford (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.

[0637] 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), and the reaction system was stirred at room temperature for 20 hours. Ion exchange resin AMBERLITE IR-120(H) was then added to adjust the pH to neutral, the resin was removed by filtration, and the filtrate was concentrated under reduced pressure to yield (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.

[0638] Synthesis of 2-azido-N-((3R,4R,5R,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide

[0639] 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 celite, 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.

[0640] Step 2: 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) was added bromoacetic acid N-hydroxysuccinimide (3.32 g, 14.1 mmol) under ice-bath conditions. The reaction system was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The residue was slurried with petroleum ether / ethyl acetate = 10 / 1 (22 mL), the solid was removed by filtration, and the filtrate was concentrated under reduced pressure to give 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.

[0641] Step 3: 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) was added sodium azide (0.68 g, 10.5 mmol), and the reaction system was stirred at 20°C overnight. The reaction mixture was then diluted with ethyl acetate (20 mL), washed with a large amount of water, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined and concentrated under reduced pressure to give 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.

[0642] Step 4: To a 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) in methanol (15 mL) was added Dowex 50wx8 cation exchange resin (1 g). The reaction system was stirred at room temperature for 2 hours. Filtered through celite, the filter cake was rinsed with a small amount of methanol, and the combined filtrates were concentrated under reduced pressure. The residue was slurried with ethanol (20 mL), and the solid was collected and dried to give 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.

[0643] Synthesis of compounds:

[0644] Example 1: Synthesis of Compounds 1-1, 1-2, 1-3 and 1-4

[0645] Step 1: Compounds 1A (1.2 g, 2.49 mmol) and 1B (2.4 g, 2.74 mmol) were azeotropically concentrated with acetonitrile (10 mL) twice, then dissolved in acetonitrile (5 mL) and set aside. 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 No. 4 molecular sieves. The resulting mixture was stirred for 1 hour, and DDTT (0.61 g, 2.99 mmol) was added to the above reaction system. After stirring for another half hour, the molecular sieves were removed by filtration, and the filtrate was concentrated under reduced pressure to obtain compound 1C (1.3 g, crude product). m / z: [M+H] + 1289.2.

[0646] Step 2: Under nitrogen protection, a dichloromethane solution of DCA (0.6 M, 9 ml, 5.39 mmol) was added to a dichloromethane solution of compound 1C (1.0 g, 0.77 mmol) (10.0 mL) in an ice-water bath. The reaction system was stirred at room temperature for 0.5 hour. Triethylsilane (0.5 ml) and pyridine (1.0 mL) were added, and the mixture 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, pyridinium salt, crude) as a white solid. m / z: [M+H] + 987.0.

[0647] Step 3: Compound 1D (500 mg, 0.51 mmol) and pyridine (2 mL) were azeotropically concentrated three times, then dissolved in pyridine (2 mL) and slowly added dropwise to a pyridine solution of DPCP (685 mg, 2.55 mmol) at -40°C, followed by stirring at -20°C for 1 hour to obtain a reaction solution containing compound 1E, which was directly used in the next reaction.

[0648] Step 4: Water (459 mg, 25.5 mmol) and 3H-1,2-benzodisulfonol-3-one (102 mg, 0.61 mmol) were added directly to the reaction mixture of compound 1E prepared in step 3. After stirring at room temperature for 20 minutes, the reaction mixture 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.

[0649] Step 5: To a solution of compound 1F (180 mg, 0.18 mmol) in acetonitrile (2 mL) was added tert-butylamine (2 mL). The resulting mixture was stirred at room temperature for 0.5 hours and concentrated under reduced pressure. The residue was purified by flash column chromatography (C18, eluent: 0-35% acetonitrile in 10 mM aqueous ammonium bicarbonate). Compounds 1G-1 (130 mg) and 1G-2 (35 mg) were collected first and then collected as white solids. m / z: [M+H] + 948.0.

[0650] Step 6: Add ammonia water (3 mL) to a solution of compound 1G-1 (120 mg) in methanol (3 mL), stir at 50°C in a sealed tube for 5 hours, cool the reaction solution to room temperature, quench the reaction with acetic acid, and directly lyophilize to obtain compound 1-A.

[0651] Compound 1-A was purified by prep-HPLC to obtain the following compounds, all of which were ammonium salts

[0652] 1-1: white solid, 10.6 mg, m / z: [M+H] + 740.0, HPLC-RT (condition 2): 10.814 minutes, 1 H NMR (400MHz, D2O): δ7.70-8.35(m,3H),6.38(d,J=13.6Hz,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 (162MHz, D2O): δ54.72, 54.22, 53.46; 19 F NMR (376MHz, D2O): δ-201.09;

[0653] 1-2: white solid, 8.2 mg, m / z: [M+H] + 740.0, HPLC-RT (condition 2): 11.380 minutes, 1 H NMR (400MHz, D2O): δ8.08-8.35(m,3H),6.34-6.36(m,1H),6.07(d,J=7.6Hz,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 (162MHz, D2O): δ54.94, 52.83, 51.54; 19 F NMR (376MHz, D2O): δ-202.86;

[0654] 1-3: white solid, 8.2 mg, m / z: [M+H] + 740.0, HPLC-RT (condition 2): 10.370 minutes, 1 H NMR(400MHz,D2O): δ8.36(s,2H),8.12(s,1H),.61(s,1H),6.11(s,1H),5.79(s,1H),5.3 9(d,J=51.6Hz,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 (162MHz, DMSO-d6+D2O): δ55.759,51.401; 19 F NMR (376MHz, D2O): δ-202.92.

[0655] Compound 1-B was obtained by reacting compound 1G-2 using the synthetic method of compound 1-A. Compound 1-4 was purified by prep-HPLC to obtain the ammonium salt, a white solid, 5.2 mg, m / z: [M+H] + 740.0, HPLC-RT (condition 1): 11.650 min, 1H NMR (400MHz, D2O): δ8.51(s,1H),8.28(s,1H),8.18(s,1H),6.64(s,1H),6.45(d,J= 16.0Hz,1H),5.68-5.82(m,1H),5.26-5.43(m,2H),4.07-4.57(m,7H),3.41(s,3H); 31 P NMR (162MHz, D2O): δ54.64,52.83; 19 F NMR (376MHz, D2O): δ-201.55.

[0656] Single crystal diffraction experiment of compound 1-3

[0657] 1. Single crystal growth: Compound 1-3 (8 mg) was dissolved in a mixed solution of water, acetonitrile and tetrahydrofuran (1 / 8 / 1, 0.5 mL), slowly evaporated and crystallized at room temperature, and single crystals were collected for single crystal diffraction test.

[0658] 2. Test parameters are shown in Table 2 below:

[0659] Table 2

[0660] 3. Test results: The stereostructure of compound 1-3 is as follows. The absolute configuration of all chiral centers in the molecule is R configuration.

[0661] Example 2: Synthesis of Compound 1M and Compound A

[0662] Step 1: To a solution of compound 1H (2.5 g, 7.9 mmol) in DMF (12 mL) were added TBSCl (1.44 g, 10 mmol) and imidazole (1.62 g, 23.8 mmol) in sequence. The reaction mixture was stirred at room temperature for 4 hours, quenched with 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 obtain compound 1I (2.9 g, yield: 86%) as a white solid. m / z: [M+H] + 429.2.

[0663] 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 concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1) to afford compound 1J (3 g, yield: 65%) as a white solid. m / z: [M+H] + 675.2.

[0664] Step 3: Under nitrogen, 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 concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1 to 1 / 4) to afford compound 1L (2.7 g, 69% yield) as a white solid.

[0665] Step 4: A solution of compound 1L (2.7 g, 3.0 mmol) in 1,4-dioxane hydrochloride (40 mL, 2 M) was stirred at room temperature for 16 hours and then concentrated under reduced pressure. The residue was purified by flash column chromatography (C18, eluent: 0-35% acetonitrile in 10 mM aqueous ammonium bicarbonate) to obtain compound 1M (1.1 g, yield: 55%) as a light yellow solid. m / z: [M+H] + 662.1. 31 P NMR (162MHz, DMSO-d6+D2O): δ55.847.

[0666] Sodium iodide (3.6 mg, 0.024 mmol) was added to a solution of compound 1-3 (300 mg, 0.41 mmol) and benzyl chloride (52 mg, 0.41 mmol) in DMF (1 mL). The reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was directly purified by prep-HPLC (eluent: 10% to 55% acetonitrile in 0.1% aqueous ammonium bicarbonate) to obtain compound A (50 mg, yield: 15%) as a white solid. m / z: [M+H] + 830.0; 31 P NMR (162MHz, DMSO-d6+D2O): δ53.113,27.573.

[0667] Example 3: Synthesis of Compound 2

[0668] Under ice-cooling 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 added sequentially to a mixed solution of compound 1-3 (62 mg, 0.084 mmol) in anhydrous acetonitrile (1 mL) and dimethylacetamide (1 mL). The reaction solution was stirred at room temperature for 2 hours and then 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.

[0669] Example 4: Synthesis of Compound 3 and Compound 4

[0670] Step 1: To a solution of compound 1-3 (200 mg, 0.27 mmol) in DMF (5 mL) was added Linker-21 (180 mg, 0.27 mmol). The reaction mixture was stirred at room temperature for 2 hours and then directly purified by flash column chromatography (C18, eluent: 0-70% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford 3 (135 mg, yield: 40%) as a white solid. m / z: [1 / 2M+H] + 632.2.

[0671] Step 2: To a solution of compound 3 (80 mg, 0.063 mmol) in water (2 mL) was added a 1,4-dioxane hydrochloride solution (2 mL, 4 M). The reaction mixture was stirred at 35°C for 12 hours and then directly purified by flash column chromatography (C18, eluent: 0-50% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to afford compound 4 (50 mg, yield: 70%) as a white solid. m / z: [1 / 2M+H] + 562.2.

[0672] Example 5: Synthesis of Compound 5

[0673] Using the synthesis method of compound 3, compound 1-3 was reacted with iodomethyl pivalate to obtain compound 5. m / z: [M+H] + 853.9.

[0674] Example 6: Synthesis of Compound 6

[0675] Using the synthetic method of compound 2, compound 1-3 and 2-(chloromethyl)-5-nitrothiophene were reacted to obtain compound 6. m / z: [M+H] + 880.9.

[0676] Example 7: Synthesis of Compound 7

[0677] Using the synthetic method of compound 2, compound 1-3 and (E)-4-((4-(chloromethyl)phenyl)diazenyl)-2-hydroxybenzoic acid were reacted to obtain compound 7. m / z: [M+H] + 994.0.

[0678] Example 8: Synthesis of I-1

[0679] To a solution of compound 1-1 (10 mg, 0.01 mmol) in anhydrous DMF (2 mL) was added dropwise a solution of linker-1 (5.9 mg, 0.01 mmol) in anhydrous DMF (0.3 mL). The reaction mixture was stirred at 50°C for 3 hours. The reaction solution was then directly purified by prep-HPLC (mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 15% to 50% mobile phase B, elution time: 20 minutes) to afford 1-1 (3.6 mg) as a white solid. UPLC RT = 6.003 minutes; m / z: [M+H] + 1208.2.

[0680] Example 9: Synthesis of I-2

[0681] To a solution of compound 1-3 (10 mg, 0.01 mmol) in anhydrous DMF (2 mL) was added dropwise a solution of Linker-1 (5.9 mg, 0.01 mmol) in anhydrous DMF (0.3 mL). The reaction mixture was stirred at room temperature for 3 hours. The reaction solution was then directly purified by prep-HPLC (mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 15% to 65% mobile phase B, elution time: 25 minutes) to afford I-2 (3.6 mg) as a white solid. UPLC RT = 6.609 minutes; m / z: [M+H] + 1208.1.

[0682] Example 10: Synthesis of I-3

[0683] To a solution of compound 1-3 (15 mg, 0.02 mmol) in anhydrous DMF (2 mL) was added dropwise a solution of linker-2 (13.7 mg, 0.01 mmol) in anhydrous DMF (0.3 mL). The reaction mixture was stirred at room temperature for 3 hours. The reaction solution was then directly purified by prep-HPLC (mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 15% to 65% mobile phase B, elution time: 25 minutes) to afford 1-3 (8.9 mg) as a white solid. UPLC RT = 6.329 minutes; m / z: [M+H] + 1294.3. 31 P NMR (162MHz, DMSO-d6+D2O): δ54.318,27.733.

[0684] Example 11: Synthesis of I-4

[0685] To a solution of compound 1-1 (10 mg, 0.013 mmol) in anhydrous DMF (2 mL) was added linker-1 (17 mg, 0.029 mmol). The reaction was stirred at 50°C for 5 hours and then directly purified by prep-HPLC (mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 15% to 85% mobile phase B, elution time: 25 minutes) to afford I-4 (6.3 mg) as a white solid. UPLC RT = 6.893 minutes; m / z: [M+H] + 1677.8.

[0686] Example 12: Synthesis of I-5

[0687] To a solution of compound 1-3 (150 mg, 0.20 mmol) in anhydrous DMF (3.5 mL) was added linker-2 (340 mg, 0.50 mmol). The reaction was stirred at room temperature for 2 hours and then purified directly by flash column chromatography (C18, eluent: 0-65% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford I-5 (140 mg) as a white solid. UPLC RT = 6.577 min; m / z: [1 / 2M+H] + 924.8.

[0688] Example 13: Synthesis of I-6

[0689] Using the synthesis method of I-3, compound 1-1 and Linker-2 were reacted to give I-6 as a white solid. HPLC RT = 13.448 min; m / z: [1 / 2M+H] +647.8.

[0690] Example 14: Synthesis of I-7

[0691] Step 1: To a solution of compound 1-3 (140 mg, 0.19 mmol) in DMF (3 mL) were added 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.

[0692] Step 2: Trifluoroacetic acid (1 mL) was added dropwise to a solution of compound 4A (80 mg, 0.081 mmol) in dichloromethane (2 mL) under ice-cooling conditions. The reaction mixture 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 afford compound 4B (70 mg, 96% yield). m / z: [M+H] + 883.9.

[0693] Step 3: To a solution of compound 4B (40 mg, 0.045 mmol) in DMF (2 mL) were added linker-3 (43 mg, 58 mmol) and DIPEA (15 mg, 0.11 mmol) in sequence. The reaction mixture 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 afford compound I-7 (55 mg, 82% yield) as a white solid. HPLC RT = 14.510 min; m / z: [1 / 2M+H] + 741.8.

[0694] Example 15: Synthesis of I-8

[0695] 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 then directly purified by prep-HPLC (mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 10% to 45% mobile phase B, elution time: 20 minutes) to afford I-8 (3.2 mg) as a white solid. HPLC RT = 14.350 minutes; m / z: [M+H] + 919.1.

[0696] Example 16: Synthesis of I-9

[0697] 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 then directly purified by prep-HPLC (mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 5% to 65% mobile phase B over 20 minutes) to afford I-9 (3.2 mg) as a white solid. HPLC RT = 14.622 minutes; m / z: [M+H] + 1064.0.

[0698] Example 17: Synthesis of I-10

[0699] 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 then directly purified by flash column chromatography (C18, eluent: 0-70% acetonitrile in 10 mM aqueous ammonium bicarbonate) to obtain compound 5A (32 mg, yield: 52%). m / z: [M+H] + 927.0.

[0700] Steps 2 & 3: Using the synthetic method of steps 2 and 3 of III-8, compound 5A was reacted to obtain compound I-10 (prep-HPLC separation: mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 3% to 45% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 14.034 minutes; m / z: [M+H] + 1424.9.

[0701] Example 18: Synthesis of I-11

[0702] Using the synthetic method for compound I-8, compound 1-3 was reacted with 1-(2-iodoethyl)-1H-pyrrole-2,5-dione to give compound I-11 (prep-HPLC separation: mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 5% to 45% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 12.959 minutes; m / z: [M+H] + 863.1.

[0703] Example 19: Synthesis of I-12

[0704] Using the synthetic method for compound I-10, compound 1-3 was reacted with tert-butyl (17-iodo-3,6,9,12,15-pentaoxaheptadecanyl)carbamate to give compound I-12 (prep-HPLC: mobile phase A: 10 mmol / L aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 45% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 13.701 minutes; m / z: [M+H] + 1196.2.

[0705] Example 20: Synthesis of I-13

[0706] Steps 1 & 2: Using the synthetic method of 5B, 1-3 was reacted with tert-butyl (2-iodoethyl)carbamate to give compound 6B as a pale yellow solid. m / z: [1 / 2M+H] + 392.0.

[0707] Step 3: To a solution of compound 6B (25 mg, 0.032 mmol) in DMF (2 mL) was added Mc-Val-Cit-OH (18 mg, 0.038 mmol), HATU (20 mg, 0.038 mmol), and DIPEA (60 mg, 0.038 mmol) in an ice bath. The reaction mixture was stirred at room temperature for 1 hour and then directly purified by prep-HPLC (mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution from 5% to 45% mobile phase B, elution time: 20 minutes) to afford 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.

[0708] Example 21: Synthesis of I-14

[0709] Compound I-14 was obtained by reacting compound 1-3 with Linker-7 using the synthetic method of compound I-9 (prep-HPLC: mobile phase A: 10 mmol / L aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 5% to 50% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 13.968 minutes; m / z: [1 / 2M+H] + 650.7.

[0710] Example 22: Synthesis of I-15

[0711] Steps 1 & 2: Using the synthetic method for compound 4B, compound 1-3 was reacted with tert-butyl (2-(((chloromethoxy)carbonyl)oxy)ethyl)(methyl)carbamate to afford compound 7B as a pale yellow solid. m / z: [M+H] + 871.0.

[0712] Step 3: To a solution of compound 7B (10 mg, 0.011 mmol) in DMF (2 mL) were added linker-3 (8.1 mg, 0.11 mmol), DIPEA (2.8 mg, 0.022 mmol), and HOBT (2.2 mg, 0.017 mmol) in sequence. The reaction mixture was stirred at room temperature for 2 hours, concentrated under reduced pressure, and the residue was purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 55% mobile phase B over 20 minutes) to afford compound I-15 (5 mg, 28% yield) as an off-white solid. HPLC RT = 14.228 min; m / z: [1 / 2 M+H] + 735.2.

[0713] Example 23: Synthesis of I-16

[0714] Step 1: Compound 1-3 (50 mg, 0.068 mmol) was dissolved in dry pyridine (2 mL) and concentrated under reduced pressure. The above operation was repeated twice. Under ice bath conditions and nitrogen protection, pyridine (2 mL) and trimethylsilyl chloride (44 mg, 0.41 mmol) were added to the above 1-3. After the addition, the reaction solution was stirred at room temperature for 0.5 hours. Then, a solution of 4-((tert-butoxycarbonyl)(methyl)amino)butane(isobutylcarbonyl) anhydride (110 mg) in dry pyridine (0.5 mL) was added to the reaction solution and stirring was continued for 3 hours. The reaction solution was directly purified by flash column chromatography (C18, eluent: 0-60% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) to obtain compound 8A (30 mg, yield: 47%) as a white solid. m / z: [M+H] + 939.3.

[0715] Step 2: Using the synthetic method of 7B, react with 8A to obtain 8B as a white solid. m / z: [M+H] + 839.2.

[0716] Step 3: Using the synthetic method of step 3 of I-15, compound 8B was reacted to obtain compound I-16 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 15% to 70% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 13.067 minutes; m / z: [1 / 2M+H] + 719.3.

[0717] Example 24: Synthesis of I-17

[0718] Steps 1 & 2: Using the synthetic method for compound 4B, compound 1-3 was reacted with (S)-tert-butyl 2-((((chloromethoxy)carbonyl)(2-(dimethylamino)ethyl)amino)methyl)pyrrolidine-1-carboxylate to yield compound 9B as a pale yellow solid. m / z: [M+H] + 967.2.

[0719] Step 3: Using the synthetic method of compound I-15 in step 3, compound I-17 was obtained by reaction with 9B (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 2% to 65% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 13.891 minutes; m / z: [1 / 2M+H] + 783.0.

[0720] Example 25: Synthesis of I-18

[0721] Using the synthetic method of III-3, compound 1-3 and Linker-8 were reacted to obtain compound I-18 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 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.

[0722] Example 26: Synthesis of I-19

[0723] Steps 1 & 2: Using the synthetic method for compound 8B, compound 1-3 and 4-((4-((tert-butyloxycarbonyl)amino)benzyl)mercapto)butane(isobutylcarbonyl)anhydride were reacted to give compound 10B as a yellow solid. m / z: [M+H] + 947.0.

[0724] Step 3: Using the synthetic method of step 3 of I-15, compound 10B was reacted to obtain compound I-19 (prep-HPLC separation: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 5% to 70% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 13.762 minutes; m / z: [1 / 2M+H] + 773.2.

[0725] Example 27: Synthesis of I-20

[0726] A mixture 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, mobile phase B: acetonitrile; gradient elution from 5% to 70% mobile phase B over 20 minutes) to afford I-20 (3.73 mg, 30% yield) as a white solid. HPLC RT = 13.114 min; m / z: [1 / 2 M+H] + 698.6.

[0727] Example 28: Synthesis of I-21

[0728] Using the synthetic method of I-3, compound 1-3 and Linker-9 were reacted to give I-21 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 5% to 65% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 14.432 minutes; m / z: [M+H] + 1427.4.

[0729] Example 29: Synthesis of I-22

[0730] Steps 1 & 2: Using the synthesis method of compound 8B, compound 1-3 and linker-10 were reacted to obtain compound 11B as a light yellow solid. m / z: [1 / 2M+H] + 603.6.

[0731] Step 3: To a mixture of compound 11B (5.6 mg, 0.018 mmol) and succinimidyl 6-(maleimido)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, mobile phase B: acetonitrile; gradient elution from 5% to 70% mobile phase B, elution time: 20 minutes) to afford compound I-22 (1.35 mg, yield: 8%) as a white solid. HPLC RT = 13.496 minutes; m / z: [1 / 2M+H] + 700.2.

[0732] Example 30: Synthesis of I-23

[0733] Step 1: To a solution of 4-((4-((tert-butoxycarbonyl)amino)benzyl)dimercapto)butanoic acid (350 mg, 0.98 mmol) and triethylamine (141 mg, 1.4 mmol) in dichloromethane (10 mL) was added isobutyl chloroformate (134 mg, 0.98 mmol) under ice-cooling conditions. The reaction mixture was stirred at 0°C for 0.5 hours and then concentrated under reduced pressure. The residue was diluted with a small amount of dichloromethane and passed through a short silica gel layer. The resulting filtrate was used for later use. 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 the mixture was redissolved in dry pyridine (3 mL). Under nitrogen protection, trimethylsilyl chloride (91 mg, 0.84 mmol) was added to a pyridine solution of compound 1-3 in an ice bath. After the addition, the reaction solution was stirred at room temperature for 0.5 hours, and the dichloromethane filtrate was added. The reaction solution was 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) to obtain compound 12A (125 mg, yield: 86%) as a white solid. m / z: [1 / 2M+H] + 540.0.

[0734] Step 2: Trifluoroacetic acid (3 mL) was added to a solution of 12A (120 mg, 0.11 mmol) in dichloromethane (10 mL) under ice-cooling conditions. The reaction mixture was stirred at 0°C for 2 hours and concentrated under reduced pressure. The residue was purified by flash column chromatography (C18, eluent: 0-30% acetonitrile in 0.1% trifluoroacetic acid in water) to afford compound 12B (55.6 mg, 51% yield) as a white solid. m / z: [M+H] + 979.0.

[0735] Step 3: Using the synthetic method of I-20, 12B was reacted to give I-23 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 70% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 13.798 minutes; m / z: [1 / 2M+H] + 671.8.

[0736] Example 31: Synthesis of I-24

[0737] Step 1: To a solution of compound 1-3 (50 mg, 0.068 mmol) in DMF (3 mL) was added Linker-11 (52 mg, 0.068 mmol) under ice-cooling conditions. The reaction mixture was stirred at room temperature for 1.5 hours and then purified directly by flash column chromatography (C18, eluent: 0-75% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford compound 13A (38 mg, yield: 41%) as a light yellow solid. m / z: [1 / 2M+H] + 685.8.

[0738] Step 2: A mixture of compound 13A (20 mg, 0.015 mmol) in water (0.5 mL) and 1,4-dioxane hydrochloride (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, mobile phase B: acetonitrile; gradient elution from 5% to 55% mobile phase B over 20 minutes) to afford compound I-24 (5.76 mg, 31% yield) as a white solid. HPLC RT = 11.793 minutes; m / z: [1 / 2M+H] + 615.8.

[0739] Example 32: Synthesis of I-25

[0740] Using the synthetic method for compound I-20, compound 10B was reacted with Mc-Val-Ala-OH to give compound I-25 (Flash column chromatography: C18, eluent: 0-60% acetonitrile in 10 mM aqueous ammonium bicarbonate) as a white solid. HPLC RT = 13.447 min; m / z: [M+H] + 1310.0.

[0741] Example 33: Synthesis of I-26

[0742] Using the synthesis method for compound I-17, replacing Linker-3 in step 3 with Linker-4, compound I-26 was obtained (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 5% to 60% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 14.249 minutes; m / z: [M+H] + 1478.9.

[0743] Example 34: Synthesis of I-27

[0744] Using the synthetic method of I-3, compound 1-3 and Linker-13 were reacted to give I-27 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 3% to 50% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 13.656 minutes; m / z: [1 / 2M+H] + 825.3.

[0745] Example 35: Synthesis of I-28

[0746] Step 1: Using the synthesis method of I-3, compound 1-3 and linker-14 were reacted to obtain 14A as a white solid. m / z: [M+H] + 1569.0.

[0747] Step 2: To a solution of compound 14A (70 mg, 0.045 mmol) in DMF (2 mL) was added diethylamine (16 mg, 0.22 mmol). 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 afford compound 14B (55 mg, yield: 92%). m / z: [M+H] + 1347.0.

[0748] Step 3: Compound 14B (40 mg, 0.03 mmol) was stirred in water and a solution of hydrochloric acid-1,4-dioxane (2 mL, 4 M) at 20°C for 12 hours. The reaction solution was directly purified by flash column chromatography (C18, eluent: 0-70% acetonitrile in 10 mM aqueous ammonium bicarbonate) to obtain compound 14C (35 mg, yield: 98%). m / z: [M+H] + 1207.2.

[0749] Step 4: To a solution of compound 14C (18 mg, 0.015 mmol) in DMF (2 mL) was added succinimidyl 6-(maleimido)hexanoate (14 mg, 0.045 mmol) and 3 drops of DIPEA. The reaction was stirred at room temperature for 1.5 hours. The reaction solution was directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 2% to 50% mobile phase B over 20 minutes) to afford compound I-28 (3.1 mg, 14% yield) as a white solid. HPLC RT = 13.946 min; m / z: [M+H] + 1400.4.

[0750] Example 36: Synthesis of I-29

[0751] Using the synthesis method for compound I-17, replacing Linker-3 in step 3 with Linker-16, compound I-29 was obtained (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 3% to 65% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 6.378 minutes; m / z: [1 / 2M+H] + 917.0.

[0752] Example 37: Synthesis of I-30

[0753] Compound I-30 was obtained by reacting compound 10B with Linker-17 using the synthesis method of compound I-20 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 70% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 13.414 minutes; m / z: [1 / 2M+H] + 833.4.

[0754] Example 38: Synthesis of I-31

[0755] Step 1: Using the synthesis method of I-3, compound 1-3 and Linker-18 were reacted to give 15A as a white solid. m / z: [1 / 2M+H] + 645.2.

[0756] Step 2: To a solution of compound 15A (50 mg, 0.039 mmol) in DMF (2 mL) was added diethylamine (14.2 mg, 0.20 mmol). The reaction mixture was stirred at room temperature for 1 hour and then purified directly by flash column chromatography (C18, eluent: 0-60% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford compound 15B (55 mg, yield: 92%). m / z: [1 / 2M+H] + 534.6.

[0757] Step 3: To a solution of compound 15B (18 mg, 0.015 mmol) in DMF (2 mL) was added succinimidyl 6-(maleimido)hexanoate (13 mg, 0.042 mmol) and 3 drops of DIPEA. The reaction was stirred at room temperature for 1 hour. The reaction solution was directly purified by flash column chromatography (C18, eluent: 0-60% acetonitrile in 0.1% trifluoroacetic acid in water) to afford 15C (40 mg, 97% yield) as a white solid. m / z: [1 / 2M+H] + 631.2.

[0758] 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 hours. After cooling to room temperature, the reaction mixture was directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 2% to 60% mobile phase B, elution time: 20 minutes) to obtain compound I-31 (1.2 mg, yield: 9%) as a white solid. HPLC RT = 12.657 minutes; m / z: [M+H] + 1191.1.

[0759] Example 39: Synthesis of I-32

[0760] Step 1: Using the synthetic method of 8A, compound 1-3 and Linker-20 were reacted to obtain 16A as a white solid.

[0761] Step 2: To a solution of 16A (65 mg, 0.057 mmol) in DMF (2 mL) was added diethylamine (25 mg, 0.34 mmol). The reaction was stirred at room temperature for 2 hours and then purified directly by flash column chromatography (C18, eluent: 0-40% acetonitrile in 0.1% trifluoroacetic acid in water) to afford 16B (30 mg, yield: 57%) as a white solid. m / z: [M+H] + 928.2.

[0762] Step 3: 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 minutes under ice. Compound 16B (3.3 mg, 0.004 mmol) was added to the reaction mixture. The reaction mixture was stirred overnight at room temperature and then directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 70% mobile phase B over 20 minutes) to afford compound I-32 (1.8 mg, yield: 27%) as an off-white solid. HPLC RT = 13.021 minutes; m / z: [1 / 2M+H] + 869.3.

[0763] Example 40: Synthesis of I-33

[0764] Using the synthesis method of I-32, replacing Linker-19 in step 3 with Mc-Gly-Gly-Phe-OH, I-33 was obtained (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 80% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 10.141 minutes; m / z: [1 / 2M+H] + 691.8.

[0765] Example 41: Synthesis of I-34

[0766] To a solution of compound 4 (10 mg, 0.009 mmol) in DMF (2 mL) was added Linker-2 (7.3 mg, 0.011 mmol). The reaction mixture was stirred at room temperature for 1 hour and then directly analyzed by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 5% to 65% mobile phase B, elution time: 20 minutes) to afford compound I-34 (2 mg, yield: 13%) as a white solid. HPLC RT = 13.860 minutes; m / z: [1 / 2M+H] + 838.8.

[0767] Example 42: Synthesis of I-35

[0768] To a solution of compound 5 (20 mg, 0.023 mmol) in DMF (2 mL) was added Linker-2 (16 mg, 0.023 mmol). The reaction mixture was stirred at room temperature for 1 hour and then directly analyzed by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 5% to 50% mobile phase B, elution time: 20 minutes) to afford compound I-35 (12.3 mg, yield: 35%) as a white solid. HPLC RT = 16.147 minutes; m / z: [1 / 2M+H] + 704.8.

[0769] Example 43: Synthesis of I-36

[0770] To a solution of compound 2 (18 mg, 0.018 mmol) in DMF (1 mL) was added linker-2 (16 mg, 0.023 mmol). The reaction mixture was stirred at room temperature for 1 hour and then directly analyzed by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 10% to 30% mobile phase B, elution time: 20 minutes) to afford compound I-36 (7.8 mg, yield: 27%) as a white solid. HPLC RT = 15.783 minutes; m / z: [1 / 2M+H] + 770.4.

[0771] Example 44: Synthesis of I-37

[0772] Using the synthetic method of I-3, compound 1-3 and Linker-22 were reacted to give I-37 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 3% to 75% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 15.308 minutes; m / z: [M+H] + 1497.4.

[0773] Example 45: Synthesis of I-38

[0774] Using the synthetic method of I-3, compound 1-3 and Linker-24 were reacted to give I-38 (Flash column chromatography: C18, eluent: 0% to 48% acetonitrile in 10 mM aqueous ammonium bicarbonate) as a white solid. UPLC RT = 7.599 min; m / z: [M+H] + 1404.4. 31 P NMR (162MHz, DMSO-d6+D2O): δ52.848, 27.645.

[0775] Example 46: Synthesis of I-39

[0776] 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. The product was then directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 70% mobile phase B over 20 minutes) to afford compound I-39 (0.77 mg, 3% yield) as a white solid. UPLC RT = 4.985 minutes; m / z: [M+H] + 1236.1.

[0777] Example 47: Synthesis of I-40

[0778] Under ice-cooling conditions, DIPEA (2.3 mg, 0.02 mmol) was added 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). 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, mobile phase B: acetonitrile; gradient elution from 5% to 50% mobile phase B, elution time: 20 minutes) to afford compound I-40 (4.3 mg, yield: 19%) as a white solid. UPLC RT = 5.459 minutes; m / z: [1 / 2M+H] + 917.5.

[0779] Example 48: Synthesis of I-41

[0780] Using the synthetic method of I-3, compound 1-3 and Linker-25 were reacted to give I-41 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 75% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 7.650 minutes; m / z: [M+H] + 1583.3.

[0781] Example 49: Synthesis of I-42

[0782] Using the synthetic method of I-3, compound 1-3 and Linker-26 were reacted to give I-42 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 85% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 8.348 minutes; m / z: [1 / 2M+H] + 886.8.

[0783] Example 50: Synthesis of I-43

[0784] Sodium iodide (7.64 mg, 0.051 mmol) and 4-(chloromethyl)-1-methyl-2-nitro-1H-imidazole (3 mg, 0.017 mmol) were added to a solution of compound I-3 (22 mg, 0.017 mmol) in DMF (2 mL). The reaction mixture was stirred at room temperature for 2.5 hours and then directly purified using prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 5% to 60% mobile phase B, elution time: 20 minutes) to obtain compound I-43 (2.5 mg, yield: 10%) as a white solid. UPLC RT = 6.180 minutes; m / z: [1 / 2M+H] + 717.2.

[0785] Example 51: Synthesis of I-44

[0786] Using the synthetic method of I-43, compound I-3 was reacted with 5-(chloromethyl)-1-methyl-4-nitro-1H-imidazole to afford I-44 (Flash column chromatography: eluent: 0-55% acetonitrile in 0.1% trifluoroacetic acid in water) as a white solid. UPLC RT = 6.039 min; m / z: [1 / 2M+H] + 717.2.

[0787] Example 52: Synthesis of I-45

[0788] Using the synthesis method of I-36, compound 2 and linker-26 were reacted to give I-45 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 75% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 7.765 minutes; m / z: [1 / 2M + CH3CN + H] + 1050.4.

[0789] Example 53: Synthesis of I-46

[0790] Step 1: Compound 1-3 (90 mg, 0.12 mmol) and Linker-29 (94 mg, 0.13 mmol) in DMF (5 mL) were 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 afford 17A (40 mg, 20% yield) as an off-white solid. m / z: [1 / 2M+H] + 661.5.

[0791] Step 2: To a solution of 17A (40 mg, 0.03 mmol) in DMF (5 mL) was added diethylamine (22 mg, 0.3 mmol). The reaction was stirred at room temperature overnight and directly purified by flash column chromatography (C18, eluent: 0-45% acetonitrile in 0.05% trifluoroacetic acid in water) to afford 17B (25 mg, 52% yield) as a white solid. m / z: [1 / 2M+H] + 551.2.

[0792] Step 3: 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 on ice for 2 h. The reaction solution was directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 45% mobile phase B over 20 min) to afford compound I-46 (1.03 mg, 11% yield) as a white solid. UPLC RT = 5.702 min; m / z: [M+H] + 1378.5.

[0793] Example 54: Synthesis of I-47

[0794] Synthesis of 18B: Using the synthesis method of 17B, compound 1-3 and Linker-30 were reacted to obtain 18B as a white solid. m / z: [1 / 2M+H] + 582.5.

[0795] Using the synthesis method of I-46, compound 18B and Linker-31 were reacted to give I-47 (prep-HPLC: mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution from 5% to 45% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 6.211 minutes; m / z: [1 / 2M+H]+ 713.8.

[0796] Example 55: Synthesis of I-48

[0797] Using the synthesis method of I-46, compound 18B and Linker-27 were reacted to give I-48 (prep-HPLC: mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution from 5% to 45% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 6.021 minutes; m / z: [1 / 2M+H] + 720.7.

[0798] Example 56: Synthesis of I-49

[0799] Using the synthesis method of I-46, compound 18B and Linker-28 were reacted to give I-49 (prep-HPLC: mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution from 5% to 45% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 6.420 minutes; m / z: [1 / 2M+H] + 764.8.

[0800] Example 57: Synthesis of I-50

[0801] Sodium iodide (4 mg, 0.028 mmol) was added to a solution of compound 1-3 (10 mg, 0.014 mmol) and Linker-32 (9 mg, 0.013 mmol) in DMF (2 mL). The reaction system was stirred at room temperature for 2 days and then directly purified using prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 5% to 75% mobile phase B, elution time: 20 minutes) to obtain compound I-50 (3 mg, yield: 1 / 2) as a white solid. UPLC RT = 7.140 minutes; m / z: [1 / 2M+H] + 734.8. 31 P NMR (162MHz, DMSO-d6+D2O): δ52.902,27.667.

[0802] Example 58: Synthesis of I-51

[0803] 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% trifluoroacetic acid aqueous solution) to afford 19A (45 mg, 43% yield) as a light yellow solid. m / z: [1 / 2M+H] + 526.5.

[0804] Step 2: 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 on ice for 2 h. The reaction solution was directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate solution, mobile phase B: acetonitrile; gradient elution from 15% to 70% mobile phase B, elution time: 20 min) to give compound I-51 (22 mg, yield: 44%) as a white solid. UPLC RT = 4.785 min; m / z: [1 / 2 M+H] + 650.7.

[0805] Example 59: Synthesis of I-52

[0806] Using the synthesis method of I-50, compound 1-3 and Linker-35 were reacted to give I-52 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 75% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 14.703 minutes; m / z: [M+H] + 1420.9.

[0807] Example 60: Synthesis of I-53

[0808] Steps 1 & 2: Using the synthetic method for compound 4B, compound 1-3 and Linker-37 were reacted to give compound 20B as a white solid. m / z: [M+H] + 1265.5.

[0809] Step 3: To a solution of compound 20B (20 mg, 0.016 mmol) in DMF (1 mL) was added (1-methyl-2-nitro-1H-imidazol-5-yl)methyl(4-nitrophenyl)carbonate (6 mg, 0.018 mmol) and DIPEA (4 mg, 0.032 mmol). The reaction mixture was stirred at room temperature for 3 hours and then purified directly using prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 10% to 85% mobile phase B over 20 minutes) to afford compound I-53 (5 mg, 22% yield) as a white solid. UPLC RT = 7.240 min; m / z: [M+H] + 1448.5.

[0810] Example 61: Synthesis of I-54

[0811] Using the synthetic method of I-3, compound 1-3 and Linker-38 were reacted to give I-54 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 70% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 7.233 minutes; m / z: [M+H] + 1782.5.

[0812] Example 62: Synthesis of I-55

[0813] To a solution of I-3 (22 mg, 0.017 mmol) and sodium iodide (10.3 mg, 0.069 mmol) in DMF (2 mL) was added dropwise 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-oxahexacosa-26-amine (22.3 mg, 0.023 mmol) in acetonitrile (1 mL) under ice-cooling conditions. 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% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution from 5% to 55% mobile phase B over 20 minutes) to afford I-55 (6.25 mg, 12% yield) as a white solid. UPLC RT = 6.864 min; m / z: [1 / 2M+H] + 974.8.

[0814] Example 63: Synthesis of I-56

[0815] Using the synthesis method of I-46, replacing Linker-27 in step 3 with Linker-31, I-56 was obtained (C18, eluent: 0-40% acetonitrile in 10 mM aqueous ammonium bicarbonate) as a white solid. UPLC RT = 5.722 min; m / z: [1 / 2M+H] + 682.8.

[0816] Example 64: Synthesis of I-57

[0817] Using the synthetic method of I-43, I-56 was reacted with 5-(chloromethyl)-1-methyl-2-nitro-1H-imidazole to give I-57 (prep-HPLC: mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution from 5% to 85% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 6.160 minutes; m / z: [1 / 2M+H] + 752.8.

[0818] Example 65: Synthesis of I-58

[0819] Using the synthetic method of I-43, I-3 was reacted with 4-bromo-5-(chloromethyl)-1-methyl-2-nitro-1H-imidazole to give I-58 (prep-HPLC: mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution from 5% to 50% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 6.730 minutes; m / z: [1 / 2M+H] + 756.2.

[0820] Example 66: Synthesis of I-59

[0821] Using the synthetic method of I-3, compound 6 and Linker-2 were reacted to give I-59 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 70% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 7.007 minutes; m / z: [M+H] + 1435.3.

[0822] Example 67: Synthesis of I-60

[0823] Using the synthetic method of I-43, I-3 was reacted with 5-(chloromethyl)-1-methyl-2-nitro-1H-imidazole to give I-60 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 85% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 6.539 minutes; m / z: [M+H] + 1433.2.

[0824] Example 68: Synthesis of I-61

[0825] Using the synthetic method of I-43, I-3 was reacted with 5-(chloromethyl)-1,4-dimethyl-2-nitro-1H-imidazole to give I-61 (prep-HPLC: mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution from 5% to 55% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 6.448 minutes; m / z: [1 / 2M+H] + 724.2.

[0826] Example 69: Synthesis of I-62

[0827] Using the synthetic method of I-43, I-50 was reacted with 4-bromo-5-(chloromethyl)-1-methyl-2-nitro-1H-imidazole to give I-62 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 65% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 7.154 minutes; m / z: [M+H] + 1686.1.

[0828] Example 70: Synthesis of I-63

[0829] Using the synthetic method of I-43, I-50 was reacted with 5-(chloromethyl)-1-methyl-2-nitro-1H-imidazole to give I-63 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 65% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 6.919 minutes; m / z: [M+H] + 1607.5.

[0830] Example 71: Synthesis of I-64

[0831] Using the synthetic method of I-3, compound 7 and Linker-2 were reacted to give I-64 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 55% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 7.388 minutes; m / z: [M+H] + 1548.6.

[0832] Example 72: Synthesis of I-65

[0833] Using the synthetic method of I-3, compound 7 and Linker-39 were reacted to give I-65 (prep-HPLC: mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 80% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 9.125 minutes; m / z: [M+H] + 1490.5.

[0834] Example 73: Synthesis of I-66

[0835] 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) was added sodium iodide (4 mg, 0.03 mmol). The reaction mixture was stirred at 30°C for 2 days and then directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 10% to 65% mobile phase B, elution time: 20 minutes) to afford I-66 (14.7 mg, yield: 98%) as a white solid. HPLC RT = 14.541 minutes; m / z: [1 / 2M+H] + 903.8.

[0836] Example 74: Synthesis of I-67

[0837] Step 1: 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) was added sodium iodide (20 mg, 0.13 mmol). The reaction mixture was stirred at 30°C for 1 day and then directly purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 10% to 60% mobile phase B over 20 minutes) to afford 21A (65 mg, 34% yield) as a white solid. m / z: [1 / 2M+H] + 743.8.

[0838] Step 2: Under nitrogen, to a solution of 21A (15 mg, 0.01 mmol) and 37-azido-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-heptatriacontane (12 mg, 0.02 mmol) in DMF (1 mL) was added cuprous iodide (3.8 mg, 0.02 mmol) and DIPEA (4 mg, 0.03 mmol). The reaction was stirred at 60°C for 2 hours, cooled to room temperature, and quenched with water. The aqueous phase was extracted with ethyl acetate, and the combined organic phases were concentrated under reduced pressure. The residue was purified by prep-HPLC (mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 10% to 60% mobile phase B over 20 minutes) to afford compound I-67 (3.4 mg, 95% yield) as a white solid. HPLC RT = 17.565 min; m / z: [1 / 2M+H] + 1035.8.

[0839] Example 74: Synthesis of I-68

[0840] Using the synthetic method of I-67, 21A was reacted with 7,3-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-tetracosaheptatriacontane to give I-68 (mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 10% to 60% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 14.705 minutes; m / z: [1 / 2M+H] + 1300.8.

[0841] Example 75: Synthesis of I-69

[0842] Under nitrogen, 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% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution from 10% to 65% mobile phase B over 20 minutes) to afford compound I-69 (4.17 mg, 20% yield) as a white solid. UPLC RT = 5.785 min; m / z: [1 / 2M+H] + 1028.6.

[0843] Example 76: Synthesis of I-70

[0844] Using the synthesis method of I-69, 21A and Linker-42 were reacted to give I-70 (mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution from 10% to 65% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 13.507 minutes; m / z: [1 / 2M+H] + 1241.8.

[0845] Example 77: Synthesis of I-71

[0846] Compound I-71 was obtained by reacting 21A with Linker-43 using the synthesis method of I-69 (mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution from 10% to 65% mobile phase B, elution time: 20 minutes) as a white solid. UPLC RT = 5.478 minutes; m / z: [1 / 2M+H] + 1454.2.

[0847] Example 78: Synthesis of I-72

[0848] Compound I-72 was obtained by reacting 21A with Linker-44 using the synthetic method of I-69 (mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution from 10% to 65% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 13.407 minutes; m / z: [1 / 2M+H] + 1668.1.

[0849] Example 79: Synthesis of I-73

[0850] Compound I-73 was obtained by reacting compound 1-3 with Linker-47 using the synthesis method of I-7 (mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 10% to 45% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 15.289 minutes; m / z: [M+H] + 1549.9.

[0851] Example 80: Synthesis of I-74

[0852] Synthesis of compound 22A: Using the synthesis method of compound 1-7, steps 1 & 2, compound 1-3 and linker-48 were reacted to obtain 22A as a white solid. m / z: [M+H] + 1607.4.

[0853] Under ice-cooling conditions, PyBOP (8 mg, 14 μM) and DIPEA (3 mg, 19 μM) were added 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 was slowly warmed to room temperature and stirred 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 from 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.

[0854] Example 81: Synthesis of I-75

[0855] Compound I-75 was obtained by reacting compound 1-3 with Linker-49 using the synthesis method of I-74 (mobile phase A: 10 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 10% to 55% mobile phase B, elution time: 20 minutes) as a white solid. HPLC RT = 14.320 minutes; m / z: [1 / 2M+H] + 1292.8.

[0856] Example 82: Synthesis of I-76

[0857] Using the synthetic method of I-67, compound I-76 was obtained by reacting 21A with 2,3,4,6-tetra-O-acetyl-β-D-galactopyranose azidate (mobile phase A: 1 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 65% mobile phase B, elution time: 20 minutes). HPLC RT = 15.305 minutes; m / z: [1 / 2M+H] + 929.8.

[0858] Example 83: Synthesis of I-77

[0859] Using the synthesis method of I-67, compound I-77 was obtained by reacting 21A with 2,3,4,6-tetra-O-acetyl-β-D-glucopyranose (mobile phase A: 1 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 65% mobile phase B, elution time: 20 minutes). HPLC RT = 15.360 minutes; m / z: [1 / 2M+H] + 929.8.

[0860] Example 84: Synthesis of I-78

[0861] Using the synthetic method of I-67, compound I-78 was obtained by reacting 21A with 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-pyranose acyl azide (mobile phase A: 1 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 65% mobile phase B, elution time: 20 minutes). HPLC RT = 15.334 minutes; m / z: [1 / 2M+H] + 929.3.

[0862] Example 85: Synthesis of I-79

[0863] Using the synthetic method of I-67, compound I-79 was obtained by reacting 21A with (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-azidotetrahydro-2H-pyran-3,4-diyl diacetate (mobile phase A: 1 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 65% mobile phase B, elution time: 20 minutes). UPLC RT = 7.300 minutes; m / z: [1 / 2M+H] + 929.4.

[0864] Example 86: Synthesis of I-80

[0865] Using the synthetic method of I-67, compound I-80 was obtained by reacting 21A with 2,3,6,2',3',4',6'-heptadeca-O-acetyl-β-lactose azide (mobile phase A: 1 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 65% mobile phase B, elution time: 20 minutes). UPLC RT = 7.781 minutes; m / z: [1 / 2M+H] + 1073.7.

[0866] Example 87: Synthesis of I-81

[0867] Using the synthetic method of I-67, compound I-80 was obtained by reacting 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 (mobile phase A: 1 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 50% mobile phase B, elution time: 20 minutes). UPLC RT = 7.818 minutes; m / z: [1 / 2M+H] + 1073.8.

[0868] Example 88: Synthesis of I-82

[0869] Using the synthetic method of I-67, compound I-82 was obtained by reacting 21A with 2-azido-N-((3R,4R,5R,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (mobile phase A: 0.05% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution from 5% to 50% mobile phase B, elution time: 20 minutes). HPLC RT = 14.408 minutes; m / z: [1 / 2M+H] + 874.3.

[0870] Example 89: Synthesis of I-83

[0871] Using the synthetic method of I-67, compound I-83 was obtained by reacting 21A with 1-azido-1-deoxy-β-D-galactose (mobile phase A: 1 mM aqueous ammonium bicarbonate, mobile phase B: acetonitrile; gradient elution from 5% to 65% mobile phase B, elution time: 20 minutes). UPLC RT = 6.499 minutes; m / z: [1 / 2M+H] + 845.6.

[0872] Example 90: Synthesis of I-84

[0873] Using the synthetic method of I-67, compound I-84 was obtained by reacting 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 (mobile phase A: 0.05% trifluoroacetic acid in water, mobile phase B: acetonitrile; gradient elution from 5% to 50% mobile phase B, elution time: 20 minutes). HPLC RT = 13.832 minutes; m / z: [1 / 2M+H] + 926.8.

[0874] Example 91: Antibody Coupling Reaction

[0875] 1. General Method A for Antibody-Drug Conjugation Reaction (as shown in Formula 1):

[0876] Method 1: The antibody is ultrafiltered with phosphate buffer (50mM PBS, 2mM EDTA, pH 6.5) to adjust the protein concentration to 5-10mg / mL. A TCEP aqueous solution (6-12 equivalents per antibody molecule) is added and the reaction is carried out at room temperature for 60-120 minutes. After the reduction reaction is completed, the antibody concentration is diluted to approximately 5mg / mL using the above phosphate buffer. Then, 10-20 times the molar number of the antibody-containing compound (linker-payload) represented by Formula I is dissolved in an organic solvent (DMA or DMSO) at a total volume of 5%-20%. This is added to the reaction system and stirred at room temperature for 30-120 minutes. After the coupling reaction is completed, the solution is exchanged into MES buffer (25mM, pH 6.5) using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30kDa to obtain the antibody-immunostimulatory conjugate.

[0877] Method 2: Ultrafiltration exchange the antibody with phosphate buffer (50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate + 50 mM sodium chloride + 2 mM EDTA, pH 7.4 ± 0.2) to adjust the protein concentration to 10 ± 2 mg / mL. Add TCEP aqueous solution (2.2 equivalents per antibody molecule). Stir at 25 ± 2°C for at least 2 hours. Adjust the protein concentration to 5.0 ± 0.5 mg / mL with phosphate buffer. Add an appropriate amount of DMA dropwise to the reaction system while stirring. Mix thoroughly, then add a DMA solution of the compound represented by Formula I (Linker-payload) dropwise (5.5 equivalents per antibody molecule). Stir at 25 ± 2°C for at least 1 hour. Ultrafiltration exchange the antibody with exchange buffer (50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate + 6% trehalose, pH 6.5 + 0.1) to obtain the antibody-immunostimulatory conjugate.

[0878] Method 3: The antibody was diluted to a concentration of 5 mg / mL in phosphate buffer (25 mM PBS, 1 mM EDTA, pH 7.4). After vortexing, the antibody was reduced by adding 2 mg / mL TCEP aqueous solution (8 equivalents per antibody molecule). After vortexing, the antibody was reacted on a refrigerated thermomixer at 37°C for 2 hours. Ultrafiltration was performed four times through phosphate buffer. The ultrafiltered antibody was recovered and a dimethyl sulfoxide solution of the compound represented by Formula I (linker-payload) (4 equivalents per antibody molecule) was added. The dimethyl sulfoxide solution was supplemented to 10% of the total reaction volume. After vortexing, the antibody was reacted on a refrigerated thermomixer at 20°C for 2 hours. The linker-payload solution (4 equivalents per antibody molecule) was then added and reacted overnight at 20°C. The sample storage buffer was replaced with an ultrafiltration tube and the antibody-immunostimulatory conjugate was obtained by ultrafiltration 10 times through 30 mM His-HAc, pH 5.5.

[0879] Method 4: Antibody 5 or 8 was adjusted to a concentration of 3 mg / mL in phosphate buffer (50 mM PBS, 2 mM EDTA, pH 6.5). 20 equivalents (TCEP / antibody) of TCEP aqueous solution was added, mixed thoroughly, and allowed to react in a thermomixer at room temperature for 17 hours. Centrifuge in an ultrafiltration centrifuge tube (Millipore UFC803096) at 4300 rpm for 10 minutes, and ultrafiltration was repeated three times. The sample collected in the previous step was added to a centrifuge tube with 30 equivalents (DHAA / antibody) of DHAA oxidant, mixed thoroughly, and allowed to react in a thermomixer at room temperature for 2 hours. Ultrafiltration was repeated three times. The sample was cooled to approximately 4°C, and the compound (linker-payload) represented by Formula I (10 equivalents relative to one antibody molecule) was added. Mix thoroughly, and allow to react in a thermomixer at 4°C for 1 hour. Ultrafiltration was repeated three times to obtain the antibody-immunostimulatory conjugate.

[0880] 2. General Method B for Antibody-Drug Conjugation Reaction (as shown in Formula 1'):

[0881] The antibody-immunostimulatory conjugate obtained by General Method A was exchanged into phosphate buffered saline (50 mM PBS, 2 mM EDTA, pH 8.0) using an ultrafiltration tube (MWCO 30 kD, 4 mL, Millipore) and reacted overnight at 37°C. The antibody-immunostimulatory conjugate was then ultrafiltered 10 times using 30 mM His-HAc, pH 5.5, without dimethyl sulfoxide.

[0882] 3. General Method C for Antibody-Drug Conjugation Reaction (as shown in Formula 5 or 12):

[0883] The antibody was diluted to a concentration of 5 mg / mL in phosphate buffer (25 mM PBS, 1 mM EDTA, pH 7.4). After vortexing, 2 mg / mL TCEP aqueous solution (8 equivalents per antibody molecule) was added for antibody reduction. After vortexing, the solution was reacted on a refrigerated thermomixer at 37°C for 2 hours. Ultrafiltration was performed four times using 50 mM BBS, pH 8.0 buffer. The ultrafiltered antibody was recovered and a dimethyl sulfoxide solution of the compound represented by Formula I (Linker-payload) (6-8 equivalents per antibody molecule) was added. The dimethyl sulfoxide solution was supplemented to 10% of the total reaction volume. After vortexing, the solution was reacted on a refrigerated thermomixer at 20°C for 1 hour. The sample storage buffer was replaced with an ultrafiltration tube and the solution was ultrafiltered 10 times using 30 mM His-HAc, pH 5.5, to obtain the antibody-immunostimulatory conjugate.

[0884] IV. General Method D for Antibody-Drug Conjugation Reaction (as shown in Formula 13):

[0885] Antibody 12 in phosphate buffer (25 mM PBS, 1 mM EDTA, pH 7.4) and a dimethyl sulfoxide solution (10 mM, 6-8 equivalents per antibody molecule) of the compound represented by Formula I (Linker-payload) were reacted overnight at 37°C. The sample storage buffer was replaced with an ultrafiltration tube, and the antibody-immunostimulatory conjugate was obtained by ultrafiltration five times with 30 mM His-HAc, pH 5.5.

[0886] In the above general method, the antibody molecules used are anti-HER2 antibody 1: Trastuzumab, Roche; anti-HER2 antibody 2: Pertuzumab, Roche; anti-HER2 antibody 3: Trastuzumab-LALA (L234A / L235A), B801901, Shanghai Baiying Biotechnology Co., Ltd.; anti-HER2 antibody 4: Trastuzumab-AAG (L234A / L235A / P329G), MHDDD001, Shanghai Baiying Biotechnology Co., Ltd.; anti-HER2 antibody 5: Trastuzumab (HC-s239.5) (engineered Trastuzumab with 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), LPDDFD001, Shanghai Baiying Biotechnology Co., Ltd.; antibody 6: Anti-HEL human IgG1-Kappa Isotype control, B117901, Shanghai Bio-Technology Co., Ltd.; anti-EGFR antibody 7: Cetuximab, Merck; anti-5T4 antibody 8: huA1 (V H v2.0+V L v2.4) (can be expressed and purified using the sequence information table disclosed in US8044178B2 (SEQ ID NO: 54 and SEQ ID NO: 70, see Figures 9B and 9E)); Anti-HER2 Antibody 9: Trastuzumab (HC-s239.5, L234A / L235A / P329G), CZ7DUD001, Shanghai Bio-Technology Co., Ltd.; Antibody 10: Anti-HEL human IgG1 (L234A / L235A / P329G)-Kappa Isotype control, B422203, Shanghai Bio-Technology Co., Ltd.; Antibody 11: Enfortumab (Ha22-2) (can be expressed and purified according to the sequence information table disclosed in US10894090B2); Anti-EGFR Antibody 12: Prepared using Cetuximab using the method disclosed in Example 25 of CN115209921A; Anti-EGFR Antibody 13: Nimotuzumab, Bio-Technology Co., Ltd.

[0887] 5. Determination of the average drug attachment number (DAR) per antibody molecule in antibody-drug conjugates

[0888] The average number of drug linkages per antibody molecule in the antibody-drug conjugate can be determined by high performance liquid chromatography (HPLC) analysis using the following method.

[0889] 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.

[0890] Hydrophobic interaction chromatography can be used to determine the drug-antibody ratio (DAR) in antibody-drug conjugates. Unconjugated antibody-drugs are the least hydrophobic and elute first, while antibodies conjugated to eight drugs are the most hydrophobic and elute last. Peak area percentage represents the relative distribution of the ADC conjugated to a specific number of drugs. The weighted average DAR is calculated by combining peak area percentage and the number of conjugated drugs: SUM (peak area of ​​each component * corresponding DAR value) / total peak area.

[0891] 6. Analysis of size heterogeneity (SEC) of antibody-immunostimulatory conjugates

[0892] Method A: Column: Waters XBridge 7.8 x 300 mm, 3.5 μm; Mobile phase A: 100 mM PB + 200 mM 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 x 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.

[0893] Antibody-immunostimulatory conjugates were prepared using General Method A, as shown in Table 3 below:

[0894] Table 3

[0895] Antibody-immunostimulatory conjugates were prepared using General Method B, as shown in Table 4 below:

[0896] Table 4

[0897] Antibody-immunostimulatory conjugates were prepared using General Method C, as shown in Table 5 below:

[0898] Table 5

[0899] Antibody-immunostimulatory conjugates were prepared using General Method D, as shown in Table 6 below:

[0900] Table 6

[0901] Example 92: Synthesis of C-7

[0902] C-7 was prepared using Preparation Method 2 in General Method A using Antibody 1 and Compound 17 (WO2019129880A1), with DAR: 2.5 and SEC: 99.23%.

[0903] Biological Examples:

[0904] Experimental Example 1: Type I interferon activation level test

[0905] THP-1dual cells (purchased from Invivogen) were seeded at 100,000 cells / well in 96-well plates and induced with phorbol 12-myristate 13-acetate (PMA) at a final concentration of 30 ng / ml for 24 hours. After 24 hours, the supernatant was discarded, and the cells were rinsed twice with fresh culture medium. Test compounds were then added to the cells in a 3-fold serial dilution in 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) to a maximum final concentration of 10 μM and a minimum final concentration of 0.0015 μM. After the cells were returned to the incubator and incubated for 30 minutes, the supernatant was discarded, the cells were rinsed twice again with fresh culture medium, and fresh culture medium was added and returned to the incubator for further culture for 24 hours. After 24 hours, 10 μL of supernatant was taken from each well to a new 96-well plate, and 50 μL / well QUANTI-Luc reagent (purchased from Invivogen) was added, and the Luciferase reading was immediately read using TECAN M1000. The activation level of type I interferon is positively correlated with the detected fluorescence intensity. Graphpad Prism software was used to draw the dose response curve and analyze the EC of the test compound. 50 .

[0906] Experimental Example 2: Cytokine Detection in hPBMC System

[0907] Frozen human peripheral blood mononuclear cells (Allcells) were quickly thawed in a 37°C water bath and 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, and resuspend in culture medium to adjust the cell density to 4 × 10 6 / mL. Add 50 μL per well to a 96-well flat-bottom plate (Corning). Add 50 μL of culture medium to the culture system.

[0908] Prepare a 3x working concentration dilution of the test sample in culture medium and add 50 μL to the cell suspension, 150 μL in total. Initial concentration of the test sample was 0.67 μM, and the sample was serially diluted 3-fold. Supplement the blank control with 50 μL of culture medium and incubate at 37°C in a 5% CO2 incubator for 20 hours. Centrifuge at 500 × g for 5 minutes at room temperature, and collect the supernatant. TNFα concentrations were analyzed using HTRF (Cisbio) and Infinite M1000 PRO (TECAN).

[0909] The test results showed that the TNFα secretion values ​​of II-1, II-7, II-10 to 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 to II-61, II-67 to II-82 were all less than 500 pg / mL at a concentration of 500 nM.

[0910] Experimental Example 3: Cytokine Detection in the hPBMC and BT474 Co-culture System

[0911] Frozen human peripheral blood mononuclear cells (Allcells) were quickly thawed in a 37°C water bath and 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, and resuspend in culture medium to adjust the cell density to 4 × 10 6 / mL. Add 50 μL per well to a 96-well flat-bottom plate (Corning). Collect normal growth human breast cancer epithelial cells BT474 (Nanjing Kebai Biotechnology Co., Ltd.), resuspend in RPMI 1640 complete medium, and adjust the density to 4×10 5 / mL. Add 50 μL per well to a 96-well plate and mix well with hPBMCs.

[0912] Prepare a 3x working concentration dilution of the test sample in culture medium and add 50 μL to the cell suspension. A total of 150 μL of the system was prepared, with compounds 1-3 starting at 0.8 μM and then diluted 5-fold. Other test samples were diluted 5-fold and then diluted 0.1 μM. A blank control was supplemented with 50 μL of culture medium and incubated at 37°C in a 5% CO2 incubator for 20 hours. Centrifuge at 500 rpm for 5 minutes at room temperature, and collect the supernatant. TNF-α concentrations were analyzed using HTRF (Cisbio) and Infinite M1000 PRO (TECAN).

[0913] Test results showed that at equivalent concentrations, II-5 significantly induced TNF-α in the BT474 co-culture system, while its induction was significantly weakened in the hPBMC alone system. The induction of TNF-α by II-2 and II-5 in the BT474 co-culture system was significantly superior to that of compound 1-3, trastuzumab, and the combination of trastuzumab and 1-3.

[0914] 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, I The TNFα secretion values ​​of I-53, II-57~II-60, II-64, II-66, II-67, II-73, II-78, II-81 and II-82 at a concentration of 100nM are between 1500~9000pg / mL.

[0915] Experimental Example 4: Cytokine Detection in the hPBMC and MDA-MB-468 Co-culture System

[0916] Cryopreserved human peripheral blood mononuclear cells (Sai ​​Li Bio) were quickly thawed in a 37°C water bath and 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 (MEM NEAA), and 10 mM HEPES (all from Gibco). The cells were centrifuged at 400 × g for 5 minutes at room temperature, the supernatant discarded, and the cells were resuspended in culture medium to adjust the cell density to 4 × 106 / mL. 50 μL of the cells were added to a 96-well flat-bottom plate (Corning). Normally growing human breast cancer MDA-MB-468 cells (Nanjing Kebai Biotechnology Co., Ltd.) were collected and resuspended in RPMI 1640 complete medium to adjust the cell density to 4 × 106 / mL.5 / mL. Add 50 μL per well to a 96-well plate and mix well with hPBMCs.

[0917] Prepare a 3x working concentration dilution of the test sample in culture medium and add 50 μL to the cell suspension. A total of 150 μL of the sample solution was added, with an initial compound concentration of 500 nM, and then a 5-fold serial dilution was performed. A blank control was supplemented with 50 μL of culture medium and incubated for 20 hours at 37°C in a 5% CO2 incubator. The sample was centrifuged at 500 rpm for 5 minutes at room temperature, and the supernatant was collected. TNF-α concentrations were analyzed using HTRF (VKEY-BIO) and Infinite M1000 PRO (TECAN).

[0918] Experimental Example 5: Cell proliferation assay

[0919] On the first day, SK-BR-3 human breast cancer cells (Nanjing Kebai Biotechnology Co., Ltd.) in the logarithmic growth phase were obtained and trypsinized. Single-cell suspensions were prepared in RPMI 1640 (Gibco) medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cell counts were made and the cell density was adjusted. 100 μL of the suspension was plated per well in a 96-well plate, resulting in 2,500 cells per well. The plates were then incubated overnight in a 37°C, 5% CO2 incubator. On the second day, ADC was serially diluted fivefold using the aforementioned RPMI 1640 medium and added to the plates at a rate of 50 μL / well, achieving a maximum ADC concentration of 500 nM and a minimum ADC concentration of 0.00128 nM. A control group received culture medium. After a brief centrifugation, the plates were placed in a CO2 incubator and incubated for 120 hours. On the fifth day, the cell culture plates were removed and 50 μL of Cell Titer-Glo (Promega) detection reagent was added to each well. After incubation in the dark for ten minutes, luminescence was detected using a microplate reader (Tecan, Infinite M1000 Pro). Data were analyzed using Graphpad software, and a three-parameter equation was used to fit the curve and calculate the inhibitor IC. 50 value.

[0920] Experimental Example 6: Plasma stability experiment

[0921] The test samples were added to human and mouse plasma at a concentration of 0.05 mg / mL. 50 μL of the test sample solution was transferred to a new plate on days 0, 2, 4, 7, and 9. 1 μg / mL human Her2 / ErbB2 protein (acro, Cat# HE2-H5225) and 5 μg / mL anti-small molecule antibody (4222-03) were prepared in Coating Buffer. 30 μL were added to each well of a High Bind Microplate (SpectraPlate, Cat# 6007500) and incubated overnight at 4°C. Wash three times with wash buffer (90 μL per well). Add 60 μL of blocking buffer (5% BSA in PBS) to each well and incubate at 37°C for 1 hour. Then add 30 μL of sample or standard and incubate at room temperature for 2 hours. Wash three times with wash buffer (0.05% Tween-20 in PBS) (90 μL per well). Add 30 μL of detection antibody (Anti-Human IgG (Fab specific)-peroxidase: Sigma, Cat# A0293) and incubate at room temperature for 1 hour. Wash three times with wash buffer (90 μL per well). Add 30 μL of TMB solution (A+B) (Solarbio, Cat# PR1210-2*50 mL) and incubate at room temperature for 5 minutes in the dark. Add 30 μL of stop solution (2 M dilute sulfuric acid) and read the OD value at 450 nm. The results showed that the antibody-immunostimulatory conjugates shown in Formula II had good plasma stability, especially II-4, II-38, II-42, II-43, II-46, II-49, II-57, II-58, II-61, and II-66, whose active half-lives in human and rat plasma were greater than 4 days.

[0922] Experimental Example 7: PK concentration detection

[0923] 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 refrigerated overnight at 4°C. The next day, the plates were washed three times with washing buffer (PBS buffer containing 0.05% Tween-20) and blocked with blocking buffer (PBS buffer containing 5% BSA). The plates were incubated at 37°C for 1-2 hours and then washed three times with washing buffer. Diluted standards and test samples were added to the wells and incubated at room temperature for 2 hours. The plates were then washed three times with washing buffer. The detection antibody (Anti-Human IgG (Fab specific)-peroxidase: Sigma, Cat#A0293) diluted in PBS was then added and incubated at room temperature for 1 hour. Finally, the colorimetric solution was added, and the reaction was stopped with stop solution (2 M dilute sulfuric acid) at room temperature for 10 minutes. The OD values ​​were read at 450 nm.

[0924] Experimental Example 8: In vivo efficacy study of mouse colon cancer CT26-hHER2 subcutaneous transplant tumor model

[0925] Cell Culture: Mouse colon cancer CT26-hHER2 cells were maintained as monolayers in RPMI-1640 medium supplemented with 10% fetal bovine serum in a 37°C incubator with 5% CO₂. After treatment with trypsin-EDTA, cells were subcultured twice weekly. Cells in the exponential growth phase were harvested and counted for plating.

[0926] Experimental animals: BALB / c mice, 6-8 weeks old, 16-18 g, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0927] Six experimental groups were set up for 1-3, II-1, II-2, II-3, and II-4, as shown in Table 7 below:

[0928] Table 7

[0929] Two experimental groups were set for Trastuzumab, as shown in Table 8 below:

[0930] Table 8

[0931] Six experimental groups were set up for Trastuzumab, II-22, II-25, II-30, and II-37, as shown in Table 9 below:

[0932] Table 9

[0933] Twelve experimental groups were set up for Trastuzumab + I-3, II-4, II-6, II-38, II-43, II-42, II-44, II-49, II-53, II-57, and II-58, as shown in Table 10 below:

[0934] Table 10

[0935] Six experimental groups were set up for II-45, II-51, II-54, II-61, and II-62, as shown in Table 11 below:

[0936] Table 11

[0937] Six experimental groups were set up for II-46, II-55, II-64, II-65, and I-65, as shown in Table 12 below:

[0938] Table 12 Note: it: intratumoral injection, iv: intravenous injection, ip: intraperitoneal injection

[0939] Experimental method: CT26-hHER2 cell line (3.0×10 6 The tumor was inoculated subcutaneously on the right side of the back of the experimental mice. The inoculation volume for each mouse was 0.1 mL. The growth of the tumor was observed regularly. 3 The mice were randomly divided into groups according to tumor size and mouse weight at around 3 pm and given drugs according to the dosing plan. The mouse weight and tumor size were measured twice a week throughout the experiment.

[0940] Tumor size calculation formula: tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ).

[0941] The experimental results are shown in Tables 13 to 18:

[0942] Table 13

[0943] Table 14

[0944] Table 15

[0945] Table 16

[0946] Table 17

[0947] Table 18

[0948] The results showed that compared with compounds 1-3 and Trastuzumab, the compound of the present invention not only achieved systemic administration, but also showed better efficacy in the mouse colon cancer CT26-hHER2 subcutaneous transplant tumor model.

[0949] Experimental Example 9: In vivo efficacy study of mouse colon cancer CT26 subcutaneous transplant tumor model

[0950] Cell Culture: Mouse colon carcinoma CT26 cells were maintained as monolayers in RPMI-1640 medium supplemented with 10% fetal bovine serum in a 37°C incubator with 5% CO₂. After treatment with trypsin-EDTA, cells were subcultured twice weekly. Cells in the exponential growth phase were harvested and counted for plating.

[0951] Experimental animals: BALB / c mice, 6-8 weeks old, 16-18 g, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0952] II-42, II-58, I-3, I-38, I-43, I-44, I-53, I-58, I-59, I-61, and I-64 are set up as 12 experimental groups, as shown in Table 19 below:

[0953] Table 19 Intraperitoneal injection

[0954] Experimental method: CT26 cell line (3.0×10 6 The tumor was inoculated subcutaneously on the right side of the back of the experimental mice. The inoculation volume for each mouse was 0.1 mL. The growth of the tumor was observed regularly. 3 The mice were randomly divided into groups according to tumor size and mouse weight at around 3 pm and given drugs according to the dosing plan. The mouse weight and tumor size were measured twice a week throughout the experiment.

[0955] Tumor size calculation formula: tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ).

[0956] The experimental results are shown in Table 20:

[0957] Table 20

[0958] Experimental Example 10: In vivo efficacy study in a mouse melanoma B16F10-hHER2 subcutaneous transplant tumor model

[0959] Cell Culture: Mouse melanoma B16F10-hHER2 cells were maintained as monolayers in DMEM supplemented with 10% fetal bovine serum and 1 μg / mL puromycin at 37°C in a 5% CO2 incubator. Tumor cells were subcultured twice weekly. Cells in the exponential growth phase were harvested and counted for plating.

[0960] Experimental animals: C57 mice, 6-9 weeks old, 18-22 g, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0961] Four experimental groups were set for II-5, C-7, and Trastuzumab, as shown in Table 21 below:

[0962] Table 21 Note: it: intratumoral injection, ip: intraperitoneal injection

[0963] Experimental method: B16F10-hHER2 cell line (1.0×10 5 The tumor was inoculated subcutaneously on the right side of the back of the experimental mice. The inoculation volume for each mouse was 0.1 mL. The growth of the tumor was observed regularly. 3 The mice were randomly divided into groups according to tumor size and mouse weight at around 3 pm, and the drugs were administered on the 14th and 21st days according to the dosing plan. The mouse weight and tumor size were measured three times a week throughout the experiment.

[0964] Tumor size calculation formula: tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ).

[0965] The experimental results are shown in Table 22:

[0966] Table 22

[0967] The results showed that the efficacy of II-5 was significantly better than that of C-7 and Trastuzumab in the mouse melanoma B16F10-hHER2 subcutaneous transplant tumor model.

[0968] Experimental Example 11: In vivo efficacy study of human lung cancer cell NCI-H1373 subcutaneous transplant tumor model

[0969] Cell Culture: Human lung cancer NCI-H1373 cells were maintained as monolayers in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Tumor cells were subcultured twice weekly. Cells in the exponential growth phase were harvested and counted for plating.

[0970] Experimental animals: Balb / c nude mice, 6-8 weeks old, 20-25 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0971] Two experimental groups were set for II-63, as shown in Table 23 below:

[0972] Table 23 Note: iv: intravenous injection

[0973] Experimental method: NCI-H1373 cell line (5×10 6 The tumor was inoculated subcutaneously on the right side of the back of the experimental mice. The inoculation volume for each mouse was 0.1 mL. The growth of the tumor was observed regularly. 3 The mice were randomly divided into groups according to tumor size and mouse weight at around 3 pm and given drugs according to the dosing plan. The mouse weight and tumor size were measured twice a week throughout the experiment.

[0974] Tumor size calculation formula: tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ).

[0975] The experimental results are shown in Table 24:

[0976] Table 24

[0977] Experimental Example 12: In vivo efficacy study of human pancreatic cancer cell HPAC subcutaneous transplant tumor model

[0978] Cell Culture: Human pancreatic cancer HPAC cells were maintained as monolayers in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Tumor cells were subcultured twice weekly. Cells in the exponential growth phase were harvested and counted for plating.

[0979] Experimental animals: Balb / c nude mice, 6-8 weeks old, 20-25 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0980] Two experimental groups were set for II-63, as shown in Table 25 below:

[0981] Table 25 Note: iv: intravenous injection

[0982] Experimental method: HPAC cell line (5×10 6 The tumor was inoculated subcutaneously on the right side of the back of the experimental mice. The inoculation volume for each mouse was 0.1 mL. The growth of the tumor was observed regularly. 3The mice were randomly divided into groups according to tumor size and mouse weight at around 3 pm and given drugs according to the dosing plan. The mouse weight and tumor size were measured twice a week throughout the experiment.

[0983] Tumor size calculation formula: tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ).

[0984] The experimental results are shown in Table 26:

[0985] Table 26

[0986] Experimental Example 13: In vivo efficacy study in a subcutaneous transplanted human breast cancer MDA-MB-468 tumor model

[0987] Cell Culture: Human breast cancer MDA-MB-468 cells were maintained as monolayers in a culture medium containing 10% fetal bovine serum and 90% DMEM at 37°C in a constant-temperature incubator with 5% CO2. Tumor cells were subcultured twice weekly. Cells in the exponential growth phase were harvested and counted for plating.

[0988] Experimental animals: BALB / c-nude mice, 6-8 weeks old, 18-22 g, purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0989] Four experimental groups were set for II-48 and II-63, as shown in Table 27 below:

[0990] Table 27 Note: iv: intravenous injection

[0991] Experimental method: MDA-MB-468 cell line (2.5×10 6 The tumor was inoculated subcutaneously on the right side of the back of the experimental mice. The inoculation volume for each mouse was 0.2 mL. The growth of the tumor was observed regularly. 3 The mice were randomly divided into groups according to tumor size and mouse weight at around 3 pm and given drugs according to the dosing plan. The mouse weight and tumor size were measured twice a week throughout the experiment.

[0992] Tumor size calculation formula: tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ).

[0993] The experimental results are shown in Table 28:

[0994] Table 28

[0995] Experimental Example 14: In vivo efficacy study of B-hNECTIN4 in a subcutaneous transplanted MC38 colon cancer model

[0996] Cell Culture: Colon cancer B-hNECTIN4 MC38 cells (Beijing Biocytogen Pharmaceuticals Co., Ltd.) were maintained as monolayers in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Tumor cells were subcultured twice weekly. Cells in the exponential growth phase were harvested and counted for plating.

[0997] Experimental animals: C57BL / 6 mice, 6-8 weeks old, 17-21 g, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0998] Three experimental groups were set for II-83, as shown in Table 29 below:

[0999] Table 29 Note: iv: intravenous injection

[1000] Experimental method: B-hNECTIN4 MC38 cell line (5×10 5 The tumor was inoculated subcutaneously on the right side of the back of the experimental mice. The inoculation volume for each mouse was 0.1 mL. The growth of the tumor was observed regularly. 3 The mice were randomly divided into groups according to tumor size and mouse weight at around 3 pm and given drugs according to the dosing plan. The mouse weight and tumor size were measured twice a week throughout the experiment.

[1001] Tumor size calculation formula: tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ).

[1002] The experimental results are shown in Table 30:

[1003] Table 30

[1004] Experimental Example 15: In vivo efficacy study of mouse colon cancer CT26-hEGFR subcutaneous transplant tumor model

[1005] Cell Culture: Mouse colon cancer CT26-hEGFR cells were maintained as monolayers in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Tumor cells were subcultured twice weekly. Cells in the exponential growth phase were harvested and counted for plating.

[1006] Experimental animals: Balb / c mice, 6-8 weeks old, 18-22 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[1007] Four experimental groups were set up for II-47, II-85, and II-86, as shown in Table 31 below:

[1008] Table 31 Note: sc: subcutaneous injection

[1009] Experimental method: CT26-hEGFR cell line (5×10 6 The tumor was inoculated subcutaneously on the right side of the back of the experimental mice. The inoculation volume for each mouse was 0.1 mL. The growth of the tumor was observed regularly. 3 The mice were randomly divided into groups according to tumor size and mouse weight at around 3 pm and given drugs according to the dosing plan. The mouse weight and tumor size were measured twice a week throughout the experiment.

[1010] Tumor size calculation formula: tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ).

[1011] The experimental results are shown in Table 32:

[1012] Table 32

Claims

1. An antibody-immunostimulatory conjugate of formula II or a pharmaceutically acceptable salt thereof, Among them, Ab is an antibody; t is from 1 to 8; L is a linker having the following combination: -(L1) a -(Z) b -M-; L1 is connected to D, and 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 shown compound losing a hydrogen atom; the *-labeled P configuration is independently R, S or R / S; B1 and B2 are each independently Moreover, 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 optionally substituted at any position by 1 to 3 substituents selected from hydroxy, mercapto, dithiol, amino, C 1-6 alkylamino, C 1-6 alkylamino C 1-10 alkyl; R5 is H, -(L2) d -(Z) e -(maleimidyl) 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 each independently The c2 side of L1 is connected to D; the c2 side of L2 is connected to the sulfhydryl group in D; X is p is independently 1, 2 or 3; Q is a linking group, -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; said C 1-6 alkyl is unsubstituted or optionally substituted at any position by 1 C 1-6 alkylamino or C 1-6 alkylsulfonyl; R7 and R 7’ are each independently H or C 1-6 alkyl; R8 is phenyl or a 5- to 10-membered heteroaryl; said R8 is unsubstituted or optionally substituted 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, aldehyde, acetyl, methyl ester, amino-C 1-6 alkyl, hydroxy-C 1-6 alkyl and C 1- 6 alkylsulfonyl, and the substituents are substituted at any position; Each Z is independently selected from -(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; L3 is a linking key, -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 as -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 linker connected to Ab.

2. The antibody-immunostimulatory conjugate or a pharmaceutically acceptable salt thereof as shown in Formula II according to claim 1, wherein The antibody-immunostimulatory conjugate shown in Formula II satisfies one or more of the following conditions: (1) Ab is an anti-HER2 antibody, an anti-EGFR antibody or an anti-5T4 antibody; (2) t is any value from 2 to 8; (3) 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; (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- 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; the -(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; the -(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; (5) In M, the linker is a group formed by click chemical reaction connection, click-like chemical reaction connection, sulfhydryl connection, amino connection, oxime connection or hydrazone connection; (6) D is A group formed by the loss of a hydrogen atom from the indicated compound; For and / or For and / or 3. The antibody-immunostimulatory conjugate or a pharmaceutically acceptable salt thereof shown in Formula II as described in claim 2, wherein The antibody-immunostimulatory conjugate 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 - means non - existent, The c2 side is connected to D; (4)-(Z) b -is any one 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 -subcycloalkyl-(CH2) y -(c1), -C(O)-(CH2) x -subphenyl-(CH2) y -(c1), -C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -(c1), -C(O)-(CH2CH2O) n -(CH2) u -NHC(O)-(CH2) x -subphenyl-(CH2) y -(c1), -(A) v -(c1), -(A) v -C(O)-(CH2) x -(c1), -(A) v -C(O)-(CH2) x -subphenyl-(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 -subphenyl-(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 connecting head is The linker is connected to the rest of L through the c side; (6) R5 is H, -L2-(Z) e -(maleimidyl) or -L2-(Z) e -H.

4. The antibody-immunostimulatory conjugate or a pharmaceutically acceptable salt thereof as shown in Formula II according to claim 1, characterized in that, The antibody-immunostimulatory conjugate shown in Formula II satisfies one or more of the following conditions: (1)-(L1) a - is The c2 side is connected to D; (2) 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 Rs 10 ; or R A and the adjacent nitrogen atom form a five-membered heterocyclic group; The 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 alkoxy acyl is optionally substituted by one R8 at any position; (3) R6 is H or C 1-6 alkyl; said C 1-6 alkyl is unsubstituted or optionally substituted at any position by 1 dimethylamino or methylsulfonyl group; (4) B1 and B2 are each independently Moreover, at least one of B1 and B2 is 5. The antibody-immunostimulatory conjugate or a pharmaceutically acceptable salt thereof as shown in Formula II according to claim 1, wherein -(A) v - is each R A independently is hydrogen, methyl, ethyl, n-propyl, isopropyl, isobutyl, or R A forms a five-membered heterocyclic group with the adjacent nitrogen atom; v is 1, 2, 3 or 4; preferably, -(A) v - is 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- is connected to the mercapto group in D on the c2 side; e is 1; -(Z) e -H is C 1-6 alkyl Or, 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; Or, R5 is Q is a linking group, -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 -(maleimidyl); -L2- is The thiol group on the c2 side is linked to D; -(Z) e - is -(A) v -C(O)-(CH2) x -(c3); the c3 side is linked to the maleimide group; And / or, R8 is phenyl or a 5- or 6-membered heteroaryl group; the 5- or 6-membered heteroaryl group is preferably pyridyl, pyrimidinyl, pyrazinyl, pyrrolyl, thienyl, furyl, pyrazolyl, thiazolyl or oxazolyl; R8 is unsubstituted or optionally substituted by 1 to 3 substituents selected from hydroxy, 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.

7. The antibody-immunostimulatory conjugate of formula II or a pharmaceutically acceptable salt thereof as claimed in claim 1, wherein, D is the group formed by the loss of one hydrogen atom from an amino or mercapto group in the indicated compound; Or, D is The group formed by the loss of a hydrogen atom from the amino group in the indicated compound; R3 or R 3’ are each independently Or, D is The group formed by the sulfhydryl group in the shown compound losing a hydrogen atom; Or, D is The group formed by the sulfhydryl group in the shown compound losing a hydrogen atom.

8. The antibody-immunostimulatory conjugate or a pharmaceutically acceptable salt thereof as shown in Formula II according to claim 1, wherein, L is Or, D is any of the following structures: Or a pharmaceutically acceptable salt thereof.

9. The antibody-immunostimulatory conjugate or a pharmaceutically acceptable salt thereof as shown in Formula II according to claim 1, wherein, The antibody-immunostimulatory conjugate shown in Formula II is any antibody-immunostimulatory conjugate in Table A.

10. A cyclic dinucleotide compound shown in Formula I or a pharmaceutically acceptable salt thereof, D-LX (I) Among them, LX is a linker precursor having the following combination: -(L1) a -(Z) b -M'; L1 is connected to D; M' is a linker precursor; The definitions of L1, Z, D, a and b are as described in any one of claims 1-9.

11. The cyclic dinucleotide compound represented by 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 mercapto side chains of antibody amino acid residues; preferably ethynyl, vinyl, hydroxylamine group, R 11 is hydrogen or C 1-4 alkyl; Each R 12 and R 12’ are each 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 represented by Formula I or a pharmaceutically acceptable salt thereof according to claim 10, wherein LX is 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 represented by Formula I is Or a pharmaceutically acceptable salt thereof.

14. A compound represented by Formula D'-1 or D'-2, its stereoisomer or a pharmaceutically acceptable salt thereof; B1 and B2 are each independently Moreover, at least one of B1 and B2 is For and / or For and / or 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 group, -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; said C 1-6 alkyl is unsubstituted or optionally substituted at any position by 1 C 1-6 alkylamino or C 1-6 alkylsulfonyl; R7 and R 7’ are each independently H or C 1-6 alkyl; R8 is phenyl or a 5- to 10-membered heteroaryl; the 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 substituents are 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 represented by formula D'-1 or D'-2, its stereoisomer or its pharmaceutically acceptable salt as claimed in claim 14, characterized in that, R5 is -L2-(Z) e -H; -L2- is connected to the mercapto group in D on the c2 side; e is 1; -(Z) e -H is C 1-6 alkyl or, 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; Or, R5 is Q is a linking key, -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 a 5- or 6-membered heteroaryl group; the 5- or 6-membered heteroaryl group is preferably pyridyl, pyrimidinyl, pyrazinyl, pyrrolyl, thienyl, furyl, pyrazolyl, thiazolyl or oxazolyl; R8 is unsubstituted or optionally substituted by 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 the substituents of methylsulfonyl are substituted at any position.

16. The compound represented by formula D'-1 or D'-2, its stereoisomer or its pharmaceutically acceptable salt according to claim 15, characterized in that, R5 is connected to the mercapto group on the c2 side and in D.

17. The compound represented by formula D'-1 or D'-2, its stereoisomer or its pharmaceutically acceptable salt 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 substance K and a pharmaceutically acceptable excipient; said substance K is an antibody-immunostimulatory conjugate of formula II as described in any one of claims 1-9 or a pharmaceutically acceptable salt thereof, or a cyclic dinucleotide compound of formula I as described in any one of claims 10-13 or a pharmaceutically acceptable salt thereof, or a compound of formula D'-1 or D'-2 as described in any one of claims 14-17, its stereoisomer or a pharmaceutically acceptable salt.

19. Use of a substance K or the pharmaceutical composition according to claim 17 in the preparation of a medicament for treating and / or alleviating tumors, said substance K being an antibody-immunostimulatory conjugate of formula II as described in any one of claims 1-9 or a pharmaceutically acceptable salt thereof, or a cyclic dinucleotide compound of formula I as described in any one of claims 10-13 or a pharmaceutically acceptable salt thereof, or a compound of formula D'-1 or D'-2 as described in any one of claims 14-17, its stereoisomer or a pharmaceutically acceptable salt.

20. Use of a substance K or the pharmaceutical composition according to claim 17 in the preparation of a medicament for treating and / or alleviating tumors, said substance K being an antibody-immunostimulatory conjugate of formula II as described in any one of claims 1-9 or a pharmaceutically acceptable salt thereof, or a cyclic dinucleotide compound of formula I as described in any one of claims 10-13 or a pharmaceutically acceptable salt thereof, or a compound of formula D'-1 or D'-2 as described in any one of claims 14-17, its stereoisomer or a pharmaceutically acceptable salt, said substance K or pharmaceutical composition being used in combination with one or more other types of therapeutic agents.