Antibody-drug conjugate with connection system

Abstract

Provided are antibody-drug conjugates (ADCs) with linking systems, preparation methods therefor, and uses thereof. These linking systems have high linking stability and enhanced solubility, allowing for efficient conjugation of hydrophobic drugs and effective intracellular delivery of drugs.

Description

Antibody Drug Conjugates with Linker Systems

[0001] The present invention claims priority to patent application No. 2024101754438 filed with the State Intellectual Property Office of China on February 7, 2024, patent application No. 2024105358331 filed with the State Intellectual Property Office of China on April 30, 2024, patent application No. 2024107726728 filed with the State Intellectual Property Office of China on June 14, 2024, patent application No. 202411432986X filed with the State Intellectual Property Office of China on October 14, 2024, patent application No. 2024115803285 filed with the State Intellectual Property Office of China on November 6, 2024, and patent application No. 2025101207110 filed with the State Intellectual Property Office of China on January 24, 2025, all with the title “Antibody Drug Conjugate with Linker System”, the entire text of which is incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of medicinal chemistry, and in particular relates to an antibody-drug conjugate with a linker system. Background Art

[0003] Antibody-drug conjugates (ADCs) can selectively target and kill cancer cells while minimizing the effects on normal cells, ushering in a new era in cancer treatment. Several ADCs have been approved by the FDA for marketing, including Mylotarg, a CD33 antibody linked to calicheamicin; Adcetris, a CD30 antibody linked to MMAE, for the treatment of Hodgkin's lymphoma and undifferentiated large cell lymphoma; Kadcyla, a Her2 antibody linked to a maytansine derivative, for the treatment of HER2-positive breast cancer; DS8201, a Her2 antibody linked to a camptothecin derivative Dxd, for the treatment of HER2-positive breast cancer; and Sacituzumab govitecan, a drug targeting the TROP-2 antigen, for the treatment of triple-negative breast cancer.

[0004] According to the classic mechanism of action of ADC drugs, antibody-drug conjugates can specifically bind to cell surface proteins, and the resulting conjugate is internalized by the cells, thereby achieving targeted delivery of drug molecules to tumor cells. Therefore, the concentration of drug inside cells is directly related to the distribution density of target sites on the cell surface that can be specifically recognized by the antibody. However, the density of target sites on the molecular surface that can be recognized by the antibody is generally low, resulting in low drug concentrations inside the target cells. To address this problem, a commonly used approach is to increase the DAR value of the ADC, thereby increasing the amount of active drug that enters the cell interior and thus improving drug efficacy. However, according to a study by Hamblett et al. (Clin Cancer Res. 2004, 10, 7063), increasing the DAR value of the ADC often affects the ADC's pharmacokinetic properties in vivo. ADC molecules may aggregate, reducing plasma stability, and increasing the shedding of small molecule toxins in the blood, causing toxic side effects.

[0005] Since 2013, Daiichi Sankyo has filed several patent applications (CN201380053256.2, CN201910768778.X, CN201980061665.4, etc.), disclosing a series of antibody-drug conjugates with specific linker-toxin structures. Specifically, they have disclosed an ADC with the following typical structure, which exhibits excellent efficacy. However, the stability of this linker needs to be further improved to further expand the safety window of the ADC.

[0006] Therefore, it is still of great significance to develop a linker-toxin structure that can be stable in plasma and specifically released in tumor cells, and then develop new ADCs to improve the clinical safety window of antibody-drug conjugates. Summary of the Invention

[0007] The present invention provides a compound represented by formula (Ia) or (Ib), its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or prodrug compound:

[0008] M-Z1-Tr1-L1-D(Ia) or M-Z1-L2(Tr2)-D(Ib)

[0009] Wherein, M is a linker site with an antibody or antigen-binding fragment thereof; preferably, M comprises a sulfhydryl reactive group, an amino reactive group, a carboxyl reactive group, a proline residue reactive group, a tyrosine residue reactive group, a disulfide bond bridging group, etc.; for antibodies introduced with non-natural amino acids, it may also comprise a bioorthogonal reactive group; preferably, a sulfhydryl reactive group, such as a methylsulfone pyrimidine group, a methylsulfone pyridine group, or a maleimide group, wherein the pyrimidine group, pyridine group, or maleimide group may be optionally substituted with one or more substituents selected from the following: halogen, CN, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylene-OC 1-6 Alkyl, -O-(CH2CH2O) n1 -CH3; n1 is an integer selected from 1-36;

[0010] Z1 is a chemical bond or a divalent group or a trivalent group substituted by a hydrophilic group, such as -NH- or -(CH2) q CONH-; q is an integer selected from 0 to 6;

[0011] Tr1 is a bivalent trigger group, preferably a peptide residue or a modified peptide residue, wherein the peptide residue or the modified peptide residue comprises an optionally substituted natural or unnatural amino acid, L or D amino acid; selected from glycine-glycine-phenylalanine-glycine (GGFG), glutamic acid-valine-citrulline (EVC), valine-citrulline (VC), valine-alanine (VA), aspartic acid-valine-citrulline (DVC), glutamic acid-glycine-glycine-phenylalanine-glycine (EGGFG), aspartic acid-glycine-glycine-phenylalanine-glycine (DGGFG), lysine-glycine-glycine-phenylalanine-glycine (KGGFG);

[0012] L1 is the connecting part between Tr1 and the biologically active molecular structure fragment D; preferably a chemical bond, -NH-C 1-6 Alkyl-, -N(CH3)-C 1-6 alkyl-,

[0013] L1 is further preferably -NH-CH2- or

[0014] D is selected from biologically active molecular structural fragments.

[0015] L2 is a trivalent linking portion between Z1 or M, Tr2 and the biologically active molecular structure fragment D;

[0016] Tr2 is a monovalent trigger group, preferably a peptide residue, a β-glucuronide group or a β-galactoside group;

[0017] In formula (Ia) or formula (Ib), M or Z1 has at least one monovalent hydrophilic group Hp1 substituent; and / or Z1 has at least one divalent hydrophilic group Hp2 inserted or replaced, and the monovalent hydrophilic group or divalent hydrophilic group is connected to the other parts of formula (Ia) or formula (Ib) through any chemical bond or linking group:

[0018] The monovalent hydrophilic group Hp1 is selected from:

[0019] (1) a monovalent polyethylene glycol group, preferably: -(CH2CH2O)n1-T1, -(CH2CH2O(CH2)m1CONH)n1-T1 or a cyclic group containing a -(CH2CH2O)- fragment, wherein the cyclic group optionally contains 1-3 heteroatoms such as N;

[0020] (2) Monovalent polysarcosine: -(N(CH3)CH2CO)n2-T2;

[0021] (3) Monovalent polybetaine: -(CH2CH2N(CH3)(CH2COOH))n3-T3 (inner salt form: -(CH2CH2N + (CH3)(CH2COO - ))n3-T3) or -(CH2CH2N(CH3)(CH2SO3H))n3-T3;

[0022] (4) Groups containing carboxyl groups: -(CH2)m2-X-(CH2)m3-COOH;

[0023] (5) The combination of the above (1) to (4) with the following divalent hydrophilic groups -Hp2-Hp1 is also regarded as a monovalent hydrophilic group in the present invention;

[0024] The divalent hydrophilic group Hp2 is selected from the divalent group derived from the monovalent hydrophilic group after removing the end-capping group or the n4, n5 or n6 repeating units of -K(Hp1)-, preferably:

[0025] (1) A divalent polyethylene glycol group, preferably: -(CH2CH2O)n1-, -(CH2CH2O(CH2)m1CONH)n1-, -[K-(CH2CH2O)n1-T1]n4-, -[K-(CH2CH2O(CH2)m1CONH)n1-T1]n4- or a cyclic group containing a -(CH2CH2O)- fragment, wherein the cyclic group optionally contains 1-3 heteroatoms such as N, such as:

[0026] wherein n1′ and n1″ are any integers from 1 to 36, and q1 and q2 are integers of 1, 2 or 3;

[0027] (2) Divalent polysarcosine: -(N(CH3)CH2CO)n2- or -[K-(N(CH3)CH2CO)n2-T2]n5-;

[0028] (3) Divalent polybetaine: -(CH2CH2N(CH3)(CH2COOH))n3- or -[K-(CH2)m4N(CH3)2CH2COOH]n6-, or -(CH2CH2N(CH3)(CH2SO3H))n3- or -[K-(CH2)m4N(CH3)2CH2SO3H]n6-;

[0029] (4) any combination of (1) to (3) above;

[0030] in:

[0031] n1-n6 are integers from 1 to 36; m1-m5 are integers from 1 to 10;

[0032] X is O, S or NH;

[0033] K is any trivalent group, preferably a trivalent group derived from an amino acid;

[0034] T1 or T2 or T3 is selected from any end-capping group, preferably H, OH, C 1-6 Alkyl, carboxyl, -(CH2)m 5- CONH-(CH2)m4N(CH3)2CH2COOH, -(CH2)m4N(CH3)2CH2COOH, -C 1-6 Alkylene-COOH, -N(C 1-6 alkyl)2 or The capping group may be connected to the hydrophilic group via any linking group.

[0035] In a specific embodiment of the present invention, T1 or T2 or T3 is selected from a hydrophilic group comprising a monosaccharide, a disaccharide or an oligosaccharide, or a hydrophilic group comprising multiple (more than 2) carboxyl groups, multiple (more than 2) sulfonic acid groups or a chelating group.

[0036] In a specific embodiment of the present invention, T1 or T2 or T3 is selected from H, OH, methyl, carboxyl, -CH2CH2COOH, -N(CH2)2,

[0037] In a specific embodiment of the present invention, the monovalent hydrophilic group Hp1 is preferably:

[0038] -(CH2CH2O)n1-H, -(CH2CH2O)n1-CH3, -(CH2)m2-X-(CH2)m3-COOH;

[0039] Preferably, the divalent hydrophilic group Hp2 is selected from:

[0040] -(CH2CH2O) n1 -、

[0041] In a specific embodiment of the present invention, the formula (Ia) or formula (Ib) has the following structure:

[0042] M(Hp1)-Z1-Tr1-L1-D(Ia-1)

[0043] M-Z1-Hp2-Z2-Tr1-L1-D(Ia-2)

[0044] M-Z1(Hp1)-Z2-Tr1-L1-D(Ia-3)

[0045] M(Hp1)-Z1-Hp2-Z2-Tr1-L1-D(Ia-4)

[0046] M-Z1(Hp1)-Hp2-Z2-Tr1-L1-D(Ia-5)

[0047] M(Hp1)-Z1(Hp1)-Hp2-Z2-Tr1-L1-D(Ia-6)

[0048] M(Hp1)-Z1-L2(Tr2)-D(Ib-1)

[0049] M-Z1-Hp2-Z2-L2(Tr2)-D(Ib-2)

[0050] M-Z1(Hp1)-Z2-L2(Tr2)-D(Ib-3)

[0051] M(Hp1)-Z1-Hp2-Z2-L2(Tr2)-D(Ib-4)

[0052] M-Z1(Hp1)-Hp2-Z2-L2(Tr2)-D(Ib-5)

[0053] M(Hp1)-Z1(Hp1)-Hp2-Z2-L2(Tr2)-D(Ib-6);

[0054] Wherein Z2 is a chemical bond or a divalent group, and Z1 is a divalent group or a trivalent group substituted by Hp1.

[0055] In a specific embodiment of the present invention, M is selected from the following structures:

[0056] Lg does not exist or is a leaving group selected from halogen, sulfone, trifluoromethanesulfonyl, and methanesulfonyl;

[0057] Ring B is selected from a 5-14 membered heteroaryl ring and a 3-14 membered heterocyclic ring;

[0058] Each R b The same or different, independently selected from the following groups: halogen, cyano, oxo (=O), C 1-6 Alkyl, halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, 3-8 membered heterocyclic group, C 1-6 Alkyl-OC 1-6 Alkyl-, C 1-6 Alkyl-(5-6 membered) heteroaryl- or a monovalent hydrophilic group; r is an integer from 0 to 4;

[0059] L m1 Absent, or selected from unsubstituted or optionally substituted by one, two or more R m1 Substituted with the following groups: C 6-14 Aryl, 5-14 membered heteroaryl, 3-14 membered heterocyclic group; each R m1 the same or different, independently selected from H, halogen, cyano, C 1-6 Alkyl or HOOC-C 1-3 alkylene;

[0060] L m2 is selected from unsubstituted or optionally substituted with one, two or more R m2 Substituted with the following groups: -(CH2) s -(C=O)-, -C≡C-(CH2) t -(C=O)-; each R m2 the same or different, independently selected from H, halogen, cyano, C 1-6 Alkyl or -C 1-6 Alkylene-COOH, wherein the alkylene is optionally interrupted by one, two or more of the following groups: O, NH; s and t are the same or different and are independently selected from integers of 0 to 10;

[0061] According to some embodiments, ring B is selected from a 5-6 membered N-containing heteroaromatic ring, a 3-6 membered N-containing heterocyclic ring;

[0062] According to some embodiments, ring B is selected from a pyrimidine ring, a pyridine ring, a triazine ring (such as ),

[0063] According to some embodiments, each R b The same or different, independently selected from cyano, oxo (=O), methoxy, cyclopropyl, trifluoromethyl, or -O-(CH2CH2O) n1 -CH3; n1 is an integer selected from 1-36;

[0064] According to some embodiments, L m1 Absent, or selected from unsubstituted or optionally substituted by one, two or more R m1 Substituted from the following groups: phenyl, piperidinyl or piperazinyl;

[0065] According to some embodiments, L m1 Selected from

[0066] According to some embodiments, L m2 is selected from unsubstituted or optionally substituted with one, two or more R m2 Substituted with the following groups: -CH2-(C=O)-, -(CH2)2-(C=O)-, -(CH2)5-(C=O)-, -C≡C-(CH2)3-(C=O)-;

[0067] According to some embodiments, Formula M is as follows:

[0068] Wherein, t is an integer from 0 to 10, and Z is N or CR 22 , R 21 、R 22 and R 23 Each independently selected from H, halogen, cyano, C 1-6 Alkyl, halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, 3-8 membered heterocyclic group, C 1-6 Alkyl-OC 1-6 Alkylene or a monovalent hydrophilic group; provided that when Z is N, R 21 or R 23 Not at the same time H;

[0069] According to some embodiments, t is selected from 1, 2, 3, 4, 5 or 6;

[0070] According to some embodiments, Z is N or C-CN;

[0071] According to some embodiments, R 21 Selected from H, halogen, cyano, C 1-6 Alkoxy or C 1-6 Alkyl-OC 1-6 alkylene;

[0072] According to some embodiments, Z is N, R 21 Selected from halogen, cyano, C 1-3 Alkoxy or C 1-3 Alkyl-OC 1-3 alkylene;

[0073] According to some embodiments, Z is N, R 21 Selected from methoxy or CH3-O-CH2-;

[0074] According to some embodiments, Z is C-CN, R 21 Selected from H;

[0075] According to some embodiments, R 23 Selected from H.

[0076] According to some embodiments, M is selected from:

[0077] In a specific embodiment of the present invention, Z1-Hp2-Z2 has the following structure:

[0078] -Hp2-;

[0079] -NH-(CH2) m6 -HP2-;

[0080] -NH-(CH2) m6 -HP2-(CH2) m7 CO-;

[0081] -CO-(CH2) m6 -HP2-;

[0082] -CO-(CH2) m6 -HP2-(CH2) m7 CO-;

[0083] -CO-(CH2) m6 -HP2-(CH2) m7 O(CH2) m8 CO-;

[0084] Wherein m5 is an integer from 1 to 10.

[0085] Preferably, M-Z1-Hp2-Z2 has the following structure:

[0086] In a specific embodiment of the present invention, Z1(Hp1)-Z2 has the following structure:

[0087] N(-Z6-Hp1)-

[0088] -N(-Z6-Hp1)-(CH2)m9CO-

[0089] -N(-Z6-Hp1)-(CH2)m9O(CH2)m 10 CO-

[0090] Wherein Z3-Z7 is a chemical bond or a divalent group, preferably, Z3-Z6 is selected from C1-10 alkylene, -OC 1-10 Alkylene-, -OC 1-10 Alkylene-O-, -OC 1-10 Alkylene-CO-, -O-C1-10 alkylene-NH-, -NH-C 1-10 Alkylene-, -NH-C 1-10 Alkylene-O-, -NH-C 1-10 Alkylene-CO-, -C 1-10 Alkylene-CO- or -NH-C1-10 alkylene-NH-; m9 or m 10 An integer from 1 to 10.

[0091] Preferably, M-Z1(Hp1)-Z2 has the following structure:

[0092] In a specific embodiment of the present invention, Z1(Hp1)-Hp2-Z2 has the following structure:

[0093] -N(Hp1)-Hp2-

[0094] -N(Hp1)-Hp2-(CH2)m 11 CO-

[0095] -N(Hp1)-Hp2-(CH2)m 11 O(CH2)m 12 CO-;m 11 or m 12 An integer from 1 to 10.

[0096] Preferably, M-Z1(Hp1)-Hp2-Z2- has the following structure:

[0097] According to some embodiments, Tr1 is glycine-glycine-phenylalanine-glycine (GGFG), i.e.

[0098] Or valine-alanine (VA), that is:

[0099] In a specific embodiment of the present invention, the structure of L2 is:

[0100] Wherein X1 is O or NH;.

[0101] According to some embodiments, Tr2 is a valine-alanine (Val-Ala) or a β-glucuronide group, i.e.

[0102] In a specific embodiment of the present invention, the structure of M-Z1-Hp2-Z2-L2(Tr2)- is:

[0103] In a specific embodiment of the present invention, the compound represented by formula (Ia) or formula (Ib) is preferably:

[0104] wherein -Z1(Hp1)- is preferably:

[0105] N(-Z6-Hp1)-

[0106] -N(-Z6-Hp1)-(CH2)m9CO-

[0107] -N(-Z6-Hp1)-(CH2)m9O(CH2)m 10 CO-

[0108] Wherein Z3-Z7 is a chemical bond or a divalent group, preferably, Z3-Z6 is selected from C 1-10 Alkylene, -OC 1-10 Alkylene-, -OC 1-10 Alkylene-O-, -OC 1-10 Alkylene-CO-, -OC 1-10 Alkylene-NH-, -NH-C 1-10 Alkylene-, -NH-C 1-10 Alkylene-O-, -NH-C 1-10 Alkylene-CO-, -C 1-10Alkylene-CO- or -NH-C 1-10 Alkylene-NH-; t2, m9 or m 10 are each independently selected from an integer of 1-10;

[0109] Hp3 is H or Hp1, and the remaining groups have the definitions described herein.

[0110] In a specific embodiment of the present invention, the compound represented by formula (Ia) or formula (Ib) is preferably:

[0111] wherein t2, n7, m13 and m14 are each independently selected from an integer of 1-10.

[0112] In a specific embodiment of the present invention, the compound represented by formula (Ia) or formula (Ib) is preferably:

[0113] Among them, Z, R 21 、R 23 , n1, n2, Tr1, L1, T1, T2, m13, m14, and D have the definitions described in this document.

[0114] In a specific embodiment of the present invention, the compound represented by formula (Ia) or formula (Ib) is preferably:

[0115] Among them, Z, R 21 、R 23 , n1 have the definitions described in this article.

[0116] Preferably, n1 is selected from an integer of 2-12, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; more preferably, n1 is selected from an integer of 4-10.

[0117] In a specific embodiment of the present invention, the compound represented by formula (Ia) or formula (Ib) is preferably:

[0118] In a specific embodiment of the present invention, D is preferably:

[0119] In a specific embodiment of the present invention, the compound represented by formula (Ia) or formula (Ib) is further preferably 001-056 or 001'-003':

[0120] The present invention also provides an antibody-drug conjugate of formula (IIa) or (IIb) obtained from formula (Ia) or formula (Ib),

[0121] Ab-[M'-Z1-Tr1-L1-D] β (IIa) or Ab-[M'-Z1-L2(Tr2)-D] β (IIb)

[0122] Wherein, Ab is an antibody or an antigen-binding fragment thereof, M' is a fragment formed by coupling M with Ab, and β is an integer or decimal between 1 and 10. Z1, Tr1, L 1、 D, Tr2, and L2 have the definitions described herein.

[0123] According to some embodiments, Ab is an antibody or an antigen-binding fragment, wherein the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, (scFv)2, diabody and single-domain antibody (sdAb); and / or, the antibody is a murine antibody, a humanized antibody, a chimeric antibody, a bispecific antibody or a multispecific antibody.

[0124] According to some embodiments, Ab is an anti-HER 2 antibody or an antigen-binding fragment thereof;

[0125] Preferably, the heavy chain variable region (VH) of the antibody or antigen-binding fragment thereof may include the following CDRs or variants of these CDRs:

[0126] VHCDR1 is provided as SEQ ID NO. 1: GFNIKDTYIH

[0127] VHCDR2 is provided as SEQ ID NO. 2: RIYPTNGYTRYADSVKG

[0128] VHCDR3 is provided as SEQ ID NO. 3: WGGDGFYAMDY

[0129] The light chain variable region (VL) of the antibody or antigen-binding fragment thereof may include the following CDRs or variants of these CDRs:

[0130] VLCDR1 is provided as SEQ ID NO. 4: RASQDVNTAVA

[0131] VLCDR2 is provided as SEQ ID NO. 5: SASFLYS

[0132] VLCDR3 is provided as SEQ ID NO. 6: QQHYTTPPT

[0133] For example, Ab is trastuzumab or an antigen-binding fragment thereof, and the heavy chain variable region and light chain variable region combination are as shown in SEQ ID NO.7 and SEQ ID NO.8:

[0134] SEQ ID NO.7:

[0135] SEQ ID NO.8:

[0136] According to some embodiments, β is selected from an integer or decimal between 4 and 9 (e.g., 7, 7.71, 7.84, 7.92, 7.94, 7.97, 7.98, 7.99, 8, 8.02, 8.06, or 8.14).

[0137] According to some specific embodiments, the antibody-drug conjugate represented by formula (IIa) of the present invention has the following structure:

[0138] Among them, Ab, Z, R 21 、R 23 , n1 has the definition as described herein; preferably, n1 is selected from an integer of 2-12, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; more preferably, n1 is selected from an integer of 4-10.

[0139] The present invention also provides the following intermediate:

[0140] Among them, Z, R 21 、R 23 , n1, n2, Tr1, m13 and m14 are as defined herein, Y1 is selected from halogen, hydroxy, OSu or C 1-6 Alkoxy.

[0141] Preferably, Y1, Y3, and Y4 are the same or different and are independently selected from hydroxy, methoxy, ethoxy, isopropoxy, tert-butoxy, or OSu;

[0142] According to some embodiments, Z is N or C-CN;

[0143] According to some embodiments, R 21 Selected from H, halogen, cyano, C 1-6 Alkoxy or C 1-6 Alkyl-OC 1-6 alkylene;

[0144] According to some embodiments, Z is N, R 21 Selected from halogen, cyano, C 1-3 Alkoxy or C 1-3 Alkyl-OC 1-3 alkylene;

[0145] According to some embodiments, Z is N, R 21 Selected from methoxy or CH3-O-CH2-;

[0146] According to some embodiments, Z is C-CN, R 21 Selected from H;

[0147] According to some embodiments, R 23 Selected from H.

[0148] The present invention also provides a linker of formula (IIIa) or (IIIb):

[0149] M-Z1-Tr1-L1'(IIIa) or M-Z1-L2'(Tr2)(IIIb)

[0150] Wherein, L1' and L2' are the reaction forms of L1 and L2;

[0151] Preferably:

[0152] When L1 is a chemical bond, L1' is the reactive form of Tr1, for example, Tr1 is a peptide segment with a carboxyl group or active ester at the C-terminus;

[0153] When L1 is -NH-CH2-, then L1' is The wavy line shows the connection site with the peptide residue; for example,

[0154] When L1 is When L1' is a carbonic acid active ester of p-aminobenzyl alcohol, for example The wavy line indicates the site of attachment to the peptide residue.

[0155] Preferably, the linker structure is:

[0156] Among them, Z, R 21 、R 23 , n1 has the definition described herein, Y2 is selected from halogen, hydroxy, OSu or C 1-6 Alkoxy; preferably, Y2 is selected from hydroxy, methoxy, ethoxy, isopropoxy, tert-butoxy or OSu;

[0157] According to some embodiments, Z is N or C-CN;

[0158] According to some embodiments, R 21 Selected from H, halogen, cyano, C 1-6 Alkoxy or C 1-6 Alkyl-OC 1-6 alkylene;

[0159] According to some embodiments, Z is N, R 21 Selected from halogen, cyano, C 1-3 Alkoxy or C 1-3 Alkyl-OC 1-3 alkylene;

[0160] According to some embodiments, Z is N, R 21 Selected from methoxy or CH3-O-CH2-;

[0161] According to some embodiments, Z is C-CN, R 21 Selected from H;

[0162] According to some embodiments, R 23 Selected from H.

[0163] The present invention also provides a linker as shown below, which is used to obtain an antibody-drug conjugate formed by connecting an antibody to a drug via a linker.

[0164] Among them, Z, R 21 、R 23 , n1, n2, Tr1, L1, T1, T2, m13, and m14 have the definitions described herein, position 1 is connected to Ab, and position 2 is connected to D.

[0165] According to some embodiments, the linker is as follows:

[0166] Among them, position 1 is connected to Ab and position 2 is connected to D.

[0167] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the antibody-drug conjugate represented by formula (IIa) or (IIb).

[0168] According to an embodiment of the present invention, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0169] According to an embodiment of the present invention, the pharmaceutical composition may further contain one or more additional therapeutic agents.

[0170] The present invention also provides a method for treating tumor diseases, comprising administering to a patient a preventively or therapeutically effective amount of an antibody-drug conjugate represented by Formula (IIa) or (IIb), its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or prodrug compound thereof.

[0171] The present invention also provides a method for treating tumor diseases, comprising administering to a patient a preventive or therapeutically effective amount of the above-mentioned pharmaceutical composition.

[0172] The tumor disease is selected from breast cancer, gastric cancer, lung cancer, colorectal cancer, large intestine cancer, ovarian cancer, liver cancer, kidney cancer, esophageal cancer, cervical cancer, bladder cancer, pancreatic cancer, prostate cancer, nasopharyngeal cancer, melanoma or leukemia.

[0173] In some embodiments, the patient comprises a mammal, preferably a human.

[0174] The present invention also provides an antibody-drug conjugate of formula (IIa) or (IIb), its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or at least one of its prodrug compounds for treating tumor diseases, or a pharmaceutical composition thereof.

[0175] The present invention also provides the use of at least one of the antibody-drug conjugates represented by formula (IIa) or (IIb), its racemates, stereoisomers, tautomers, solvates, polymorphs, pharmaceutically acceptable salts, or prodrug compounds thereof, or the pharmaceutical compositions thereof, in the preparation of topoisomerase I inhibitors and / or in the preparation of drugs for preventing or treating diseases or conditions associated with topoisomerase I.

[0176] In some embodiments, the disease or condition is a tumor, which includes breast cancer, gastric cancer, lung cancer, colorectal cancer, large intestine cancer, ovarian cancer, liver cancer, kidney cancer, esophageal cancer, cervical cancer, bladder cancer, pancreatic cancer, prostate cancer, nasopharyngeal cancer, melanoma or leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0177] Figure 1 Schematic diagram of ADC-2 free toxin release in mouse, rat, monkey, and human plasma;

[0178] Figure 2 Schematic diagram of ADC-12 free toxin release in mouse, rat, monkey, and human plasma;

[0179] Figure 3 Schematic diagram of the release of free toxin from DS8201 in the plasma of mice, rats, monkeys, and humans;

[0180] Figure 4 Schematic diagram of drug efficacy evaluation in NCI-N87 tumor-bearing mice;

[0181] Figure 5 Schematic diagram of drug efficacy evaluation in JIMT-1 tumor-bearing mice;

[0182] Figure 6 Schematic diagram of drug efficacy evaluation in RT11284 tumor-bearing mice;

[0183] Figure 7 Schematic diagram of drug efficacy evaluation in HCT116 tumor-bearing mice;

[0184] Figure 8 Schematic diagram of drug efficacy evaluation in Capan-1 tumor-bearing mice;

[0185] Figure 9 Schematic diagram of the efficacy evaluation of NCI-N87-Enhertu resistant cell tumor-bearing mice;

[0186] Figure 10 Schematic diagram of drug efficacy evaluation in human gastric cancer LD1-0017-411335PDX tumor-bearing mice;

[0187] Figure 11 PK curve of ADC-2 in mice;

[0188] FIG12 Comparison of the in vivo tumor inhibition effects of ADC-2 and ADC-58 on the JIMT-1 transplant model;

[0189] FIG13 Comparison of the in vivo tumor inhibition effects of ADC-2 and ADC-58 on the NCI-N87 transplant model;

[0190] Figure 14 ADC-58 plasma stability test results

[0191] Figure 15 Comparison of in vivo tumor inhibition effects of ADC-2 and ADC-57 on 22RV1 transplant models

[0192] Definitions and Explanations of Terms

[0193] Unless otherwise indicated, the definitions of groups and terms in this specification and claims, including definitions used as examples, exemplary definitions, preferred definitions, definitions in tables, and definitions of specific compounds in the Examples, may be arbitrarily combined and coupled with one another. The group definitions and compound structures resulting from such combinations and couplings should be understood to be within the scope of this specification and / or claims.

[0194] As used herein, a "linker," "linker structure," "linker," or "linker unit" refers to a chemical structure fragment or bond that is connected to an antibody at one end and to a drug (drug compound) at the other end. Alternatively, the linker structure may be connected to another linker before being connected to the drug compound. The linker structure of the present invention can be synthesized by methods known in the art or using the methods described herein.

[0195] The "polyethylene glycol" in the present invention is a homogeneous compound containing 2-36 ethoxy units. The "polyethylene glycol" in the present invention includes homogeneous polyethers formed by directly linking ethoxy units, and also includes homogeneous compounds obtained by chemical reaction of compounds containing ethoxy units (e.g., NH2-CH2CH2O-CH2CH2O-CH2-COOH). That is, any number of ethoxy units in the "polyethylene glycol" can be separated or interrupted by optional chemical groups such as amide groups. Monovalent polyethylene glycol groups can be obtained based on homogeneous monofunctional polyethylene glycol derivatives (e.g., α-methoxy-ω-carboxyl oligoethylene glycol), and divalent polyethylene glycol groups can be obtained based on homogeneous bifunctional polyethylene glycol derivatives (e.g., α-amino-ω-carboxyl oligoethylene glycol).

[0196] The "divalent group" or "divalent linking group" described in the present invention is any divalent organic group, preferably selected from: -O-, -NH-, -CO-, -OC 1-6 Alkylene-, -N(R0)-C 1-6 Alkylene-, -C 1-6 Alkylene-O-, -C 1-6 Alkylene-N(R0)-, -CO-C 1-6 Alkylene-, -C 1-6 Alkylene-CO-, -OC 1-6 Alkylene-CO-, -C 1-6 Alkylene-CO-N(R0)-, -C 1-6 Alkylene-N(R0)-CO-, -CO-C 1-6 Alkylene-N(R0)-CO-C 1-6 Alkylene-, -CO-C 1-6 Alkylene-CO-N(R0)-C 1-6 Alkylene-, -C 1-6Alkylene-CO-N(R0)-C 1-6 Alkylene-, -C 1-6 Alkylene-N(R0)-CO-C 1-6 Alkylene-, -C 1-6 Alkylene-N(R0)-CO-C 1-6 Alkylene-COO-C 1-6 Alkylene-, -C 1-6 Alkylene-CO-N(R0)-C 1-6 Alkylene-COO-C 1-6 Alkylene-, -CO-C 1-6 Alkylene-N(R0)-CO-C 1-6 Alkylene-COO-C 1-6 Alkylene-, -CO-C 1-6 Alkylene-CO-N(R0)-C 1-6 Alkylene-COO-C 1-6 Alkylene-, etc.; R0 is selected from C 1-4 Alkyl groups, such as methyl; preferably, the alkyl groups are optionally interrupted by O atoms.

[0197] The "trivalent group" described in the present invention is any trivalent organic group, preferably a trivalent group obtained by substitution of the above-mentioned "divalent group", or preferably a trivalent group based on amino acid, benzene ring or heterocycle.

[0198] The "linking group" described in the present invention includes the above-mentioned "divalent group" or "trivalent group". When it connects two functional fragments (for example, the connection between the blocking group and the hydrophilic fragment, the connection between the antibody linker and the polypeptide fragment, etc.), it is a divalent group, and when it connects three functional fragments, it is a trivalent group (for example, the linker L2 between the hydrophilic fragment, the trigger group and the drug).

[0199] The "trigger group" described in the present invention is a group in a cleavable ADC linker that triggers drug release, such as a polypeptide fragment cleaved by cathepsin, or a fragment cleavable by β-glucuronidase and β-galactosidase. The trigger group is often coupled to the drug through a self-cleavable group, and the drug is released through a series of cascade reactions under the action of specific enzymes or other conditions.

[0200] The "antibody-drug conjugate" (ADC) described in the present invention refers to a targeting moiety connected to a biologically active drug through a stable linker unit.

[0201] The "bioactive molecules" mentioned in the present invention refer to cytotoxic drugs, which are chemical molecules that can strongly disrupt the normal growth of tumor cells.

[0202] Unless otherwise indicated, the numerical ranges recited in this specification and claims are equivalent to reciting at least each specific integer value therein. For example, the numerical range "1-36" is equivalent to reciting each integer value in the numerical range "1-36", namely 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, and 36. In addition, when certain numerical ranges are defined as "numbers", it should be understood that the two endpoints of the range, each integer within the range, and each decimal within the range are recited.

[0203] The term "integer from 0 to 10" means 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0204] The term "halogen" refers to fluorine, chlorine, bromine and iodine.

[0205] “C 1-10 "Alkyl" means straight-chain and branched alkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, "C 1-8 "Alkyl" means straight-chain and branched alkyl groups having 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms, "C 1-6 The term "alkyl" refers to straight-chain and branched alkyl groups having 1, 2, 3, 4, 5 or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or the like or isomers thereof.

[0206] “C 2-10 "Alkenyl" is understood to mean preferably a linear or branched monovalent hydrocarbon radical containing one or more double bonds and having 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, more preferably "C 2-8 Alkenyl". "C 2-10 "Alkenyl" is understood to mean preferably a linear or branched monovalent hydrocarbon radical containing one or more double bonds and having 2, 3, 4, 5, 6, 7 or 8 carbon atoms, e.g. 2-6 alkenyl), having 2 or 3 carbon atoms (i.e., C 2-3It is understood that when the alkenyl group contains more than one double bond, the double bonds may be separated from one another or conjugated. The alkenyl group is, for example, vinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, (E)-but-2-enyl, (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl, pent-4-enyl, (E)-pent-3-enyl, (Z)-pent-3-enyl, (E)-pent-2-enyl, (Z)-pent-2-enyl, (E)- Pent-1-enyl, (Z)-pent-1-enyl, hex-5-enyl, (E)-hex-4-enyl, (Z)-hex-4-enyl, (E)-hex-3-enyl, (Z)-hex-3-enyl, (E)-hex-2-enyl, (Z)-hex-2-enyl, (E)-hex-1-enyl, (Z)-hex-1-enyl, isopropenyl, 2-methylprop-2-enyl, 1-methylprop-2-enyl , 2-methylprop-1-enyl, (E)-1-methylprop-1-enyl, (Z)-1-methylprop-1-enyl, 3-methylbut-3-enyl, 2-methylbut-3-enyl, 1-methylbut-3-enyl, 3-methylbut-2-enyl, (E)-2-methylbut-2-enyl, (Z)-2-methylbut-2-enyl, (E)-1-methylbut-2-enyl, (Z)-1-methyl But-2-enyl, (E)-3-methylbut-1-enyl, (Z)-3-methylbut-1-enyl, (E)-2-methylbut-1-enyl, (Z)-2-methylbut-1-enyl, (E)-1-methylbut-1-enyl, (Z)-1-methylbut-1-enyl, 1,1-dimethylprop-2-enyl, 1-ethylprop-1-enyl, 1-propylvinyl, 1-isopropylvinyl.

[0207] The term "C 2-10 "Alkynyl" is understood to mean preferably a linear or branched monovalent hydrocarbon radical containing one or more triple bonds and having 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, e.g. having 2, 3, 4, 5, 6, 7 or 8 carbon atoms (i.e. "C 2-8 alkynyl”), having 2, 3, 4, 5, or 6 carbon atoms (i.e., “C 2-6 Alkynyl”), having 2 or 3 carbon atoms (“C 2-3The alkynyl group is, for example, ethynyl, prop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl, but-3-ynyl, pent-1-ynyl, pent-2-ynyl, pent-3-ynyl, pent-4-ynyl, hex-1-ynyl, hex-2-ynyl, hex-3-ynyl, hex-4-ynyl, hex-5-ynyl, 1-methylprop-2-ynyl, 2-methylbut-3-ynyl, 1-methylbut-3-ynyl, 1-methylbut-2-ynyl, 3-methylbut-1-ynyl, 1-ethylprop-2-ynyl, 3-methylpent-4-ynyl, 2-methylpent-4-ynyl, 1-methylpent-4-ynyl, In some embodiments, the alkynyl group is ethynyl, prop-1-ynyl or prop-2-ynyl.

[0208] The term "C 3-10 "Cycloalkyl" is understood to mean a saturated monovalent monocyclic, bicyclic (eg bridged, spiro) hydrocarbon ring or tricyclic alkane having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. 3-10 The cycloalkyl group may be a monocyclic hydrocarbon group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl, or a bicyclic hydrocarbon group such as borneol, indolyl, hexahydroindolyl, tetrahydronaphthyl, decahydronaphthyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.1]heptenyl, 6,6-dimethylbicyclo[3.1.1]heptyl, 2,6,6-trimethylbicyclo[3.1.1]heptyl, bicyclo[2.2.2]octyl, 2,7-diazaspiro[3,5]nonyl, 2,6-diazaspiro[3,4]octyl, or a tricyclic hydrocarbon group such as adamantyl.

[0209] Unless otherwise defined, the term "3-6 membered heterocyclyl" refers to a saturated or unsaturated non-aromatic ring or ring system, for example, a 4-, 5- or 6-membered monocyclic ring, and containing at least one, for example 1, 2, 3, 4, 5 or more heteroatoms selected from O, S and N, wherein N and S may also be optionally oxidized to various oxidation states to form nitrogen oxides, -S(O)- or -S(O)2- states. The heterocyclyl may include fused or bridged rings and spirocyclic rings. In particular, the heterocyclyl may include, but is not limited to, a 4-membered ring such as azetidinyl, oxetanyl; a 5-membered ring such as tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or a 6-membered ring such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl or trithianyl.

[0210] The term "C 6-14 "Aryl" is understood to mean preferably a monovalent aromatic or partially aromatic monocyclic, bicyclic or tricyclic hydrocarbon ring ("C 6-14 or a ring having 9 carbon atoms ("C9 aryl"), for example indanyl or indenyl, or a ring having 10 carbon atoms ("C 10 aryl) such as tetrahydronaphthyl, dihydronaphthyl or naphthyl, or a ring having 13 carbon atoms ("C 13 aryl), such as fluorenyl, or a ring having 14 carbon atoms ("C 14 aryl”), such as anthracenyl. When the C 6-20 When the aryl group is substituted, it may be monosubstituted or polysubstituted. Furthermore, there is no limitation on the position of substitution, and for example, substitution may be at the ortho, para or meta position.

[0211] The term "5-14 membered heteroaryl" is understood to include monovalent monocyclic, bicyclic or tricyclic aromatic ring systems having 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms, in particular 5 or 6 or 9 or 10 carbon atoms, and which contain 1 to 5, preferably 1 to 3, heteroatoms each independently selected from N, O and S and which, in each case, may additionally be benzofused. "Heteroaryl" also refers to a radical in which a heteroaromatic ring is fused to one or more aryl, alicyclic or heterocyclyl rings, wherein the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include 1-, 2-, 3-, 5-, 6-, 7-, or 8-indolizinyl, 1-, 3-, 4-, 5-, 6-, or 7-isoindolyl, 2-, 3-, 4-, 5-, 6-, or 7-indolyl, 2-, 3-, 4-, 5-, 6-, or 7-indazolyl, 2-, 4-, 5-, 6-, 7-, or 8-purinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, or 9-quinolizinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, 1-, 3-, 4-, 5-, 6-, 7-, or 8-isoquinolyl, 1-, 4-, 5-, 6-, 7- or 8-phthalazinyl, 2-, 3-, 4-, 5- or 6-naphthyridinyl, 2-, 3-, 5-, 6-, 7- or 8-quinazolinyl, 3-, 4-, 5-, 6-, 7- or 8-cinnolinyl, 2-, 4-, 6- or 7-pteridinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-4aHcarbazolyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-carbazolylcarbazolyl, 1-, 3-, 4-, 5-, 6-, 7-, 8- or 9-carbolinyl, 1-, 2-, 3- , 4-, 6-, 7-, 8-, 9- or 10-phenanthridinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-acridinyl, 1-, 2-, 4-, 5-, 6-, 7-, 8- or 9-piperidinyl, 2-, 3-, 4-, 5-, 6-, 8-, 9- or 10-phenanthrolinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8- or 9-phenazinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9- or 10-phenothiazinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9- or 10-phenanthrolinyl, , 3-, 4-, 5-, 6- or 1-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-benzoisoquinolyl, 2-, 3-, 4- or thieno[2,3-b]furanyl, 2-, 3-, 5-, 6-, 7-, 8-, 9-, 10- or 11-7H-pyrazino[2,3-c]carbazolyl, 2-, 3-, 5-, 6- or 7-2H-furo[3,2-b]pyranyl, 2-, 3-, 4-, 5-, 7- or 8-5H-pyrido[2,3-d]-o-oxazinyl, 1-, 3- or 5-1H-pyrazolo[4,3-d]-oxazolyl, 2-, 4- or 5-4H-imidazo[4,5-d]thiazolyl, 3-, 5- or 8-pyrazino[2,3-d]pyridazinyl, 2-, 3-, 5- or 6-imidazo[2,1-b]thiazolyl, 1-, 3-, 6-, 7-, 8- or 9-furo[3,4-c]cinnolinyl, 1-, 2-, 3-, 4-, 5-, 6-, 8-, 9-, 10 or 11-4H-pyrido[2,3-c]carbazolyl, 2-, 3-, 6- or 7-imidazo[1,2-b][1,2,4]triazinyl, 7-benzo[b]thiazolyl phenyl, 2-, 4-, 5-, 6-, or 7-benzoxazolyl, 2-, 4-, 5-, 6-, or 7-benzoimidazolyl, 2-, 4-, 4-, 5-, 6-, or 7-benzothiazolyl, 1-, 2-, 4-, 5-, 6-, 7-, 8-, or 9-benzoxapinyl, 2-, 4-, 5-, 6-, 7-, or 8-benzoxazinyl, 1-, 2-, 3-, 5-, 6-, 7-, 8-, 9-, 10-, or 11-4H-pyrrolo[1,2-b][2]benzazapinyl. Typical fused heteroaryl groups include, but are not limited to, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, 1-, 3-, 4-, 5-, 6-, 7-, or 8-isoquinolyl, 2-, 3-, 4-, 5-, 6-, or 7-indolyl, 2-, 3-, 4-, 5-, 6-, or 7-benzo[b]thienyl, 2-, 4-, 5-, 6-, or 7-benzoxazolyl, 2-, 4-, 5-, 6-, or 7-benzimidazolyl, and 2-, 4-, 5-, 6-, or 7-benzothiazolyl. When the 5-20 membered heteroaryl group is linked to other groups to form the compounds of the present invention, the carbon atoms on the 5-20 membered heteroaryl ring may be linked to the other groups, or heteroatoms on the 5-20 membered heteroaryl ring may be linked to the other groups. When the 5-20 membered heteroaryl group is substituted, it may be monosubstituted or polysubstituted. Furthermore, there is no limitation on the substitution site, for example, a hydrogen atom connected to a carbon atom on a heteroaryl ring may be substituted, or a hydrogen atom connected to a heteroatom on a heteroaryl ring may be substituted.

[0212] The term "spirocyclic" refers to a ring system in which two rings share one ring atom.

[0213] The term "fused ring" refers to a ring system in which two rings share two ring atoms.

[0214] The term "bridged ring" refers to a ring system in which two rings share three or more ring atoms.

[0215] Wavy lines intersecting chemical bonds Used to indicate the connection position of a group to other atoms or groups in the general structure.

[0216] Unless otherwise specified, a heterocyclic group, heteroaryl group, or heteroarylene group includes all possible isomeric forms thereof, such as positional isomers thereof. Thus, for some illustrative non-limiting examples, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-positions, etc. (if present) may include 1, 2, or more substituted or bonded forms thereof, including pyridin-2-yl, pyridin-2-ylene, pyridin-3-yl, pyridin-3-ylene, pyridin-4-ylene, and pyridin-4-ylene; thienyl or thienylene group includes thien-2-yl, thien-2-ylene, thien-3-ylene, and thien-3-ylene; pyrazol-1-yl, pyrazol-3-yl, pyrazol-4-yl, and pyrazol-5-yl.

[0217] The term "alkyloxy" refers to -O-(alkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include methoxy, ethoxy, propoxy, butoxy. Alkoxy groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkyloxy, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, or heterocycloalkyloxy.

[0218] The term "alkylamino" refers to -NH-(alkyl), wherein alkyl is as defined above. Non-limiting examples of alkylamino include methylamino, ethylamino, propylamino, isopropylamino, butylamino, and the like.

[0219] The term "(alkyl)2amino" refers to -N-(alkyl)2, wherein alkyl is as defined above. Non-limiting examples of (alkyl)2amino include dimethylamino, methylethylamino, diethylamino, dipropylamino, methylpropylamino, diisopropylamino, dibutylamino, and the like.

[0220] "Haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0221] The term "antibody" refers to an immunoglobulin-derived molecule that is capable of specifically binding to a target antigen through at least one antigen-binding site located in its variable region. When referring to the term "antibody," unless the context clearly indicates otherwise, it includes not only intact antibodies but also antigen-binding fragments that are capable of specifically binding to a target antigen. "Intact antibodies" are typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ). Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with heavy chains also containing a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of a single domain, CL. The constant domains are not directly involved in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding site. The distribution of amino acids in various regions or domains can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883. As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable region of each heavy chain and light chain contains three CDRs, designated CDR1, CDR2, and CDR3.The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. In addition, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). In the present invention, the CDR contained in the antibody of the present invention or its antigen-binding fragment can be determined according to various numbering systems known in the art. In certain embodiments, the CDR contained in the antibody of the present invention or its antigen-binding fragment is preferably determined by the Kabat numbering system. As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the antibody variable region except the CDR residues as defined above. The term "antibody" is not limited by any specific method for producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (for example, IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody. As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of an antibody can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies.Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, diabodies, single-domain antibodies, and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136. As used herein, the term "Fd" refers to an antibody fragment consisting of the VH and CH1 domains; the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL, and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; and the term "Fab' fragment" refers to the fragment obtained by reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and the Fd fragment of the heavy chain (consisting of the VH and CH1 domains). As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the entire binding site. As used herein, the term "Fc" means an antibody fragment formed by the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain bound via disulfide bonds. The Fc fragment of an antibody has a variety of different functions, but does not participate in the binding of antigens. The "effector functions" mediated by the Fc domain include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (such as B cell receptors); and B cell activation, etc. The Fc domain can include both a native Fc region and a variant Fc region. The native Fc region comprises an amino acid sequence that is consistent with the amino acid sequence of the Fc region found in nature, for example, the native sequence human Fc region includes a native sequence human IgG1 Fc region; a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and a native sequence human IgG4 Fc region, as well as naturally occurring variants thereof. The variant Fc region comprises an amino acid sequence that is different from the amino acid sequence of the native sequence Fc region due to at least one amino acid modification. In some embodiments, a variant Fc region may possess altered effector function (e.g., Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function) compared to a native Fc region.As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers are composed of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, but variants thereof may also be used. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments of the present invention, scFv may form a di-scFv, which refers to two or more single scFvs connected in series to form an antibody. In certain embodiments of the present invention, scFv may form a (scFv)2, which refers to two or more single scFvs connected in parallel to form an antibody. As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with the complementary domains of another chain and produce two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), and Poljak RJ et al., Structure 2: 1121-1123 (1994)). As used herein, the term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen bound by a full-length antibody. As used herein, the term "bispecific antibody" refers to an antibody that has binding specificity for two different antigens (or epitopes). The term "multispecific antibody" refers to an antibody that has binding specificity for at least two or more (e.g., three or four) different antigens (or epitopes). A bispecific antibody or multispecific antibody comprises a plurality of antigen-binding domains that have binding specificity for different antigens (or epitopes), thereby being able to bind to at least two different binding sites and / or target molecules. The individual antigen-binding domains comprised by a bispecific antibody or multispecific antibody can be independently selected from a full-length antibody (e.g., an IgG antibody) or an antigen-binding fragment thereof (e.g., an Fv fragment, a Fab fragment, a F(ab')2 fragment, or a scFv). In some cases, the individual antigen-binding domains are connected by a peptide linker. Each of the above-mentioned antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.Conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods) can be used to obtain antigen-binding fragments of antibodies (e.g., the above-mentioned antibody fragments) from given antibodies (e.g., antibodies provided by the present invention), and the antigen-binding fragments of antibodies can be specifically screened in the same manner as for complete antibodies. As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered, and its amino acid sequence has been modified to improve the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (e.g., variable region FR and / or constant region) are derived from human immunoglobulin (receptor antibody). In certain embodiments, the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (e.g., variable region FR and / or constant region) are derived from human immunoglobulin (receptor antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, etc. In the present application, the donor antibody can be a murine antibody with expected properties (e.g., antigen specificity, affinity, reactivity, etc.). To prepare a humanized antibody, the CDR region of the donor antibody can be inserted into a human framework sequence using methods known in the art. In some cases, the human framework sequence may include amino acid mutations replaced by corresponding non-human residues. In addition, the humanized antibody may also include residues that are not found in the initial donor antibody variable region (e.g., light chain variable region or heavy chain variable region) or human framework sequence to further improve or optimize the performance of the humanized antibody. As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain or / and heavy chain is derived from an antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain or / and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen. In certain embodiments, the term "chimeric antibody" may include antibodies in which the heavy chain variable region and light chain variable region of the antibody are derived from a first antibody, and the heavy chain constant region and light chain constant region of the antibody are derived from a second antibody.

[0222] In the present invention, the compounds referred to also include isotopically labeled compounds, which are the same as those shown in Formula I, but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of H, C, N, O, S, F, and Cl, such as 2 H. 3 H. 13C. 11 C. 14 C. 15 N. 18 O. 17 O. 32 P. 35 S. 18 F and 36 Cl. Compounds of the invention, prodrugs thereof, or pharmaceutically acceptable salts of said compounds or prodrugs containing the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the invention. Certain isotopically labeled compounds of the invention, for example, those incorporating radioactive isotopes (such as 3 H and 14 C) compounds can be used in drug and / or substrate tissue distribution assays. 3 H) and carbon 14 (i.e. 14 C) isotopes are particularly preferred due to their ease of preparation and detectability. 2 Substitution with hydrogen (H or D) may provide certain therapeutic advantages (e.g., increased in vivo half-life or reduced dosage requirements) derived from greater metabolic stability and may therefore be preferred in certain circumstances. The compounds of the present invention as claimed in the claims may be specifically limited to substitution with deuterium or tritium. Furthermore, the absence of separate listing of the term deuterium or tritium for hydrogen present in a substituent does not exclude deuterium or tritium, but rather may also include deuterium or tritium.

[0223] It will be appreciated by those skilled in the art that the compounds of formula (I) may exist in the form of various pharmaceutically acceptable salts. If these compounds have a basic center, they may form acid addition salts; if these compounds have an acidic center, they may form base addition salts; if these compounds contain both an acidic center (e.g., a carboxyl group) and a basic center (e.g., an amino group), they may also form internal salts.

[0224] The compounds of the present invention may exist in the form of solvates (e.g., hydrates), wherein the compounds of the present invention contain a polar solvent as a structural element of the crystal lattice of the compound, in particular water, methanol or ethanol. The amount of polar solvent, in particular water, may be present in a stoichiometric or non-stoichiometric ratio.

[0225] Depending on their molecular structure, the compounds of the present invention may be chiral and therefore may exist in various enantiomeric forms. Thus, these compounds may exist in racemic or optically active forms. The compounds of the present invention encompass isomers or mixtures thereof, racemates, in which each chiral carbon is in the R or S configuration. The compounds of the present invention or their intermediates can be separated into enantiomeric compounds by chemical or physical methods well known to those skilled in the art, or used in this form for synthesis. In the case of racemic amines, diastereomers are prepared from the mixture by reaction with an optically active resolving agent. Examples of suitable resolving agents are optically active acids, such as R and S forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, appropriate N-protected amino acids (e.g., N-benzoylproline or N-phenylsulfonylproline) or various optically active camphorsulfonic acids. Chromatographic enantiomer resolution can also be advantageously performed with the aid of optically active resolving agents (e.g., dinitrobenzoylphenylglycine, cellulose triacetate or other carbohydrate derivatives or chirally derivatized methacrylate polymers immobilized on silica gel). Suitable eluents for this purpose are aqueous or alcoholic solvent mixtures, e.g., hexane / isopropanol / acetonitrile.

[0226] The corresponding stable isomers can be separated according to known methods, for example by extraction, filtration or column chromatography.

[0227] The term "patient" refers to any animal including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses or primates, and most preferably humans.

[0228] The term "therapeutically effective amount" refers to that amount of an active compound or drug that will elicit the biological or medical response that a researcher, veterinarian, physician, or other clinician is seeking in a tissue, system, animal, individual, or human, and includes one or more of the following: (1) prevents disease, e.g., prevents a disease, disorder, or condition in an individual who is susceptible to the disease, disorder, or condition but who is not yet experiencing or developing the pathology or symptoms of the disease. (2) inhibits disease, e.g., inhibits the disease, disorder, or condition (i.e., prevents further development of the pathology and / or symptoms) in an individual who is experiencing or developing the pathology or symptoms of the disease, disorder, or condition. (3) alleviates disease, e.g., alleviates the disease, disorder, or condition (i.e., reverses the pathology and / or symptoms) in an individual who is experiencing or developing the pathology or symptoms of the disease, disorder, or condition.

[0229] Abbreviation Description DETAILED DESCRIPTION

[0230] The present disclosure is further described below with reference to the following embodiments, but these embodiments are not intended to limit the scope of the present disclosure.

[0231] Example

[0232] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). -6 The unit of ppm is given. NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), and tetramethylsilane (TMS) was used as the internal standard.

[0233] MS was determined using an Agilent 1260 / 1290 Infinity II liquid chromatography system (manufacturer: Agilent, MS model: 1260 / 1290 Infinity II) or a Shimadzu Prominence UFLC+LCMS-2020 (manufacturer: Shimadzu, MS model: Prominence UFLC+LCMS-2020).

[0234] High performance liquid chromatography (HPLC) analysis was performed using a Thermo UltiMate 3000 (manufacturer: Thermo, MS model: UltiMate 3000).

[0235] Chiral HPLC analysis was performed using a YMC K-PrepLAB100G high performance liquid chromatograph.

[0236] High performance liquid chromatography was performed using Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector and GX-281 Liquid Handler without pump, 4020 Syringe pump, 333-H3 Pump, 334-H3 Pump, 1741 UV Detector preparative chromatograph.

[0237] A Biotage Isolera One flash chromatograph was used for flash chromatography.

[0238] The thin layer chromatography silica gel plate used was Yantai Xinnuo GF254 silica gel plate. The specifications of the silica gel plate used in thin layer chromatography (TLC) were 0.15 mm to 0.2 mm, and the specifications used for thin layer chromatography separation and purification products were 0.4 mm to 0.5 mm.

[0239] Silica gel column chromatography generally uses Yantai Huanghai 200-300 mesh silica gel as the carrier.

[0240] Average kinase inhibition rate and IC 50 The values ​​were determined using a NovoStar microplate reader (BMG, Germany).

[0241] The known starting materials disclosed herein can be synthesized by methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Darui Chemicals, and other companies.

[0242] Unless otherwise specified in the examples, all reactions can be carried out under an argon atmosphere or a nitrogen atmosphere.

[0243] Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a capacity of about 1 L.

[0244] Hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.

[0245] The pressurized hydrogenation reaction uses a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0246] The hydrogenation reaction is usually carried out by evacuating the chamber and filling it with hydrogen, and the operation is repeated three times.

[0247] A CEM Discover-S 908860 microwave reactor was used for the microwave reaction.

[0248] Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0249] Unless otherwise specified in the examples, the reaction temperature is room temperature, 20°C to 30°C.

[0250] The reaction progress in the examples was monitored by thin layer chromatography (TLC). The developing solvent used in the reaction, the eluent system for column chromatography used to purify the compound, and the developing solvent system for thin layer chromatography included: A: dichloromethane / methanol system, B: n-hexane / ethyl acetate system. The volume ratio of the solvent was adjusted according to the polarity of the compound, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0251] Synthesis of intermediates

[0252] 1. 2,5-Dioxopyrrolidin-1-yl-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate Int-1

[0253] Step 1 5-Bromo-2-chloro-4-methoxypyrimidine Int-1b

[0254] 5-Bromo-2,4-dichloropyrimidine Int-1a (5.0 g, 22 mmol) was dissolved in methanol (60 mL), and a methanol solution of sodium methoxide (30%, 4.0 g, 22.0 mol) was added to the solution. The reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction, the reaction mixture was concentrated, extracted with ethyl acetate, and the organic phase was dried and concentrated to afford compound Int-1b (5.0 g, 100% yield).

[0255] MS m / z(ESI):223.1(M+1) + .

[0256] Step 2 5-Bromo-4-methoxy-2-(methylthio)pyrimidine Int-1c

[0257] To compound Int-1b (5.0 g, 22.0 mmol) in N,N-dimethylformamide (25 mL) was added sodium thiomethoxide (1.52 g, 22 mmol), and the reaction mixture was stirred at 40°C for 1 hour. After completion of the reaction, the reaction mixture was poured into water and extracted with ethyl acetate (50 mL x 3). The organic phases were combined, dried, and concentrated to obtain a crude product which was purified by silica gel column chromatography on System B to afford compound Int-1c (3.0 g, 60% yield).

[0258] MS m / z(ESI):235.0(M+1) + .

[0259] Step 3 6-(4-methoxy-2-(methylthio)pyrimidin-5-yl)hexyl-5-ynoic acid Int-1d

[0260] Compound Int-1c (3.0 g, 12.8 mmol) was dissolved in isopropanol (20 mL), and 5-hexynoic acid (1.43 g, 12.8 mmol), cuprous iodide (243 mg, 1.29 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (300 mg, 0.41 mmol), and sodium carbonate solution (5 M, 8 mL) were added. The reaction mixture was stirred at 80°C under a nitrogen atmosphere for 16 hours. After completion of the reaction, the reaction mixture was filtered, and the filtrate was extracted with ethyl acetate (50 mL x 3). The organic phases were combined, dried, and concentrated to obtain a crude product, which was purified by silica gel column chromatography on System B to afford compound Int-1d (3.0 g, 87% yield).

[0261] MS m / z(ESI):267.1(M+1) + .

[0262] Step 4: 6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoic acid Int-1e

[0263] Compound Int-1d (3.0 g, 11.2 mmol) was dissolved in a mixture of methanol and water (40 mL, V / V = 1:1), and potassium peroxodisulfate (11.6 g, 33.6 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction mixture was poured into water and extracted with ethyl acetate (50 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate to obtain a crude product, which was then purified by silica gel column chromatography (System B) to obtain compound Int-1e (2.5 g, 75% yield).

[0264] MS m / z(ESI):299.1(M+H) + .

[0265] Step 5: 2,5-dioxopyrrolidin-1-yl-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate Int-1

[0266] To a solution of compound Int-1e (700 mg, 2.3 mmol) and N-hydroxysuccinimide (396 mg, 3.45 mmol) in tetrahydrofuran (10 mL) was added N,N-diisopropylcarbodiimide (434 mg, 3.45 mmol), and the reaction was stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was directly concentrated to obtain a crude product which was purified by silica gel column chromatography using System B to afford compound Int-1 (600 mg, 60% yield).

[0267] MS m / z(ESI):395.5(M+1) + .

[0268] 2. 2,5-Dioxopyrrolidin-1-yl-6-(5-cyano-6-(methylsulfonyl)pyridin-3-yl)hexyl-5-ynoate Int-2

[0269] The first step 5-bromo-2-(methylthio)nicotinonitrile Int-2b

[0270] 5-Bromo-2-chloronicotinonitrile Int-2a (2 g, 9.2 mmol) was dissolved in ethylene glycol dimethyl ether (20 mL) and sodium thiomethoxide (640 mg, 9.2 mmol) was added under ice. The reaction was stirred at room temperature for 4 hours. After completion of the reaction, aqueous ammonium chloride was added to quench the reaction. The system was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, and the combined organic phases were concentrated. The resulting concentrate was purified by silica gel column chromatography on System B to afford compound Int-2b (1.5 g, 71% yield).

[0271] MS m / z(ESI):228.9,230.9(M+1) +.

[0272] Step 2 6-(5-cyano-6-(methylthio)pyridin-3-yl)hexyl-5-ynoic acid Int-2c

[0273] Compound Int-2b (1.5 g, 6.6 mmol) was dissolved in a mixed solvent of tetrahydrofuran (5 mL) and triethylamine (5 mL). 5-Hexynoic acid (1.5 g, 13.2 mmol), bistriphenylphosphine palladium dichloride (913 mg, 1.3 mmol), and cuprous iodide (133 mg, 0.7 mmol) were added. The reaction was stirred at 70°C for 2 hours. After completion of the reaction, the reaction solution was filtered and the mother liquor was directly mixed with the sample. The crude product was purified by silica gel column chromatography system B to obtain compound Int-2c (0.7 g, yield: 41%).

[0274] MS m / z(ESI):261.1(M+1) + .

[0275] Step 3 6-(5-cyano-6-(methylsulfonyl)pyridin-3-yl)hexyl-5-ynoic acid Int-2d

[0276] Compound Int-2c (0.7 g, 2.7 mmol) was dissolved in methanol (10 mL) and water (10 mL), and potassium peroxymonosulfonate (9.3 g, 27 mmol) was added. The reaction was stirred at room temperature for two hours. After the reaction was completed, the reaction was filtered, the mother liquor was poured into water, extracted three times with dichloromethane, dried over anhydrous sodium sulfate, and the combined organic phases were concentrated. The filtrate was collected to obtain the crude product Int-2d (815 mg), which was used directly in the next reaction without purification.

[0277] MS m / z(ESI):293.0(M+1) + .

[0278] Step 4: 2,5-dioxopyrrolidin-1-yl-6-(5-cyano-6-(methylsulfonyl)pyridin-3-yl)hexyl-5-ynoate Int-2

[0279] The crude compound Int-2d (815 mg) was dissolved in dichloromethane (10 mL), and N-hydroxysuccinimide (345 mg, 3.0 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.04 g, 5.4 mmol) were added and stirred at room temperature for 2 hours. After the reaction, the reaction solution was poured into water, extracted three times with dichloromethane, dried over anhydrous sodium sulfate, and the organic phases were combined and concentrated. The obtained concentrate was purified by silica gel column chromatography system B to obtain compound Int-2 (400 mg, yield: 37%). MS m / z (ESI): 390.0 (M+1) + .

[0280] 3. 2,5-Dioxopyrrolidin-1-yl-6-(4-(methoxymethyl)-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate Int-3

[0281] Step 1 5-Bromo-2-chloro-4-(methoxymethyl)pyrimidine Int-3b

[0282] 5-Bromo-2-chloropyrimidine Int-3a (10 g, 0.05 mol), silver nitrate (36 g, 0.2 mol), ammonium persulfate (57 g, 0.25 mol), and 2-methoxyacetic acid (5.4 g, 0.06 mol) were dissolved in acetonitrile (300 mL) and water (300 mL) and stirred at 60°C for 2 hours. The reaction solution was poured into water and extracted three times with ethyl acetate, washed once with saturated brine, and concentrated under reduced pressure. The crude product obtained was purified by silica gel column chromatography on System B to obtain compound Int-3b (1.2 g, yield: 13%).

[0283] MS m / z(ESI):236.9(M+1) + .

[0284] Step 2 6-(2-chloro-4-(methoxymethyl)pyrimidin-5-yl)hexyl-5-ynoic acid Int-3c

[0285] Compound Int-3b (1.2 g, 4.5 mmol) was dissolved in tetrahydrofuran (10 mL), and 5-hexynoic acid (0.76 g, 6.8 mmol), cuprous iodide (86 mg, 0.45 mmol), bistriphenylphosphine palladium dichloride (632 mg, 0.9 mmol), and triethylamine (1.4 g, 13.5 mmol) were added. The reaction solution was stirred at 60°C under a nitrogen atmosphere for 3 hours. After the reaction was completed, the reaction solution was filtered, the filtrate was separated into ethyl acetate and water, and the aqueous phase was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, dried, and concentrated to obtain a crude product which was purified by silica gel column chromatography system A to obtain compound Int-3c (600 mg, yield: 50%). MS m / z (ESI): 269.1 (M+1) + .

[0286] Step 3 6-(4-(methoxymethyl)-2-(methylthio)pyrimidin-5-yl)hexyl-5-ynoic acid Int-3d

[0287] Compound Int-3c (600 mg, 2.2 mmol) was dissolved in dimethyl sulfoxide (6 mL), and sodium thiomethoxide (154 mg, 2.2 mmol) and anhydrous magnesium sulfate (528 mg, 4.4 mmol) were added. The reaction solution was stirred at 50°C for 1 hour. After completion of the reaction, the reaction solution was poured into water and extracted with ethyl acetate (50 mL x 3). The combined organic phases were dried and concentrated to obtain the crude compound Int-3d (600 mg), which was directly used in the next reaction without purification.

[0288] MS m / z(ESI):281.1(M+1) + .

[0289] Step 4: 6-(4-(methoxymethyl)-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoic acid Int-3e

[0290] Compound Int-3d (600 mg) obtained in the previous step was dissolved in a mixed solvent of acetone and water (20 mL, V / V = 1:1), potassium peroxymonosulfate (7.6 g, 22 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was poured into water and extracted three times with ethyl acetate, washed once with saturated brine, and concentrated under reduced pressure. The crude product obtained was purified by silica gel column chromatography system A to obtain compound Int-3e (200 mg, yield: 30%).

[0291] MS m / z(ESI):313.0(M+1) + .

[0292] Step 5: 2,5-dioxopyrrolidin-1-yl-6-(4-(methoxymethyl)-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate Int-3

[0293] Compound Int-3e (200 mg, 0.64 mmol) and N-hydroxysuccinimide (110 mg, 0.96 mmol) were dissolved in dichloromethane (10 mL). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (246 mg, 1.28 mmol) was added, and the reaction was stirred at room temperature for 2 hours. After completion of the reaction, the reaction solution was directly concentrated to obtain a crude product, which was purified by silica gel column chromatography using System B to obtain compound Int-3 (100 mg, 38% yield).

[0294] MS m / z(ESI):410.1(M+1) + .

[0295] Example 1 N-(((S)-1-(((S)-1-([(S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino-3-methyl-1-oxobutan-2-yl)-1-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)-3,6,9,12,15,18-hexaoxaheneicosane-21-amide (001)

[0296] Step 1: (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl (amino)-3-methyl-1-oxobutan-2-yl)carbamate (001b)

[0297] N,N,N',N'-Tetramethylchloroformamidine hexafluorophosphate (333.8 mg, 1.19 mmol) was added to a solution of compound 001a (400.0 mg, 0.99 mmol), (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine-L-alanine (407.0 mg, 0.99 mmol), and N-methylimidazole (170.9 mg, 2.08 mmol) in N,N-dimethylformamide (18.4 mL), and stirred at 23°C for 3 hours. After the reaction, the reaction solution was added dropwise to saturated brine (50 mL), and a yellow solid precipitated. The solid was filtered, and the filter cake was dissolved with methanol and dichloromethane, washed twice with water. The organic phase was dried, filtered, and concentrated under reduced pressure to give compound 001b (560.0 mg, 71%).

[0298] MS m / z(ESI):796.2(M+1).

[0299] Step 2: (S)-2-amino-N-((S)-1-(((S,9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,2H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (001c)

[0300] Diethylamine (22.1 mg, 0.30 mmol) was added to a solution of compound 001b (80.0 mg, 0.10 mmol) in N,N-dimethylformamide (1 mL) at 0°C. The mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure to afford compound 001c (70.0 mg), which was used in the next step without purification.

[0301] MS m / z(ESI):574.2(M+1).

[0302] Step 3: Tert-butyl-28-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)-23-oxo-4,7,10,13,16,19-hexaoxa-22-azaoctacarbon-27-ynoate (001e)

[0303] N,N-Diisopropylethylamine (98.1 mg, 0.76 mmol) was added to a solution of compound Int-1 (100.0 mg, 0.25 mmol) and compound 001d (103.6 mg, 0.25 mmol) in N,N-dimethylformamide (5 mL) and stirred at room temperature for 2 hours. After the reaction was complete, ethyl acetate (50 mL) was added and the mixture was washed twice with water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford compound 001e (170.0 mg, 97%).

[0304] MS m / z(ESI):634.2(M-56+1).

[0305] Step 4: 28-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)-23-oxo-4,7,10,13,16,19-hexaoxa-22-azaoctacarbon-27-ynoic acid (001f)

[0306] Trifluoroacetic acid (132.2 mg, 1.16 mmol) was added to a solution of compound 001e (80.0 mg, 0.12 mmol) in dichloromethane (5 mL) at 0°C and stirred at room temperature for 1 hour. After completion of the reaction, the mixture was concentrated under reduced pressure to obtain compound 001f (80.0 mg), which was used directly in the next reaction without purification.

[0307] MS m / z(ESI):634.2(M+1).

[0308] Step 5: N-(((S)-1-(((S)-1-([(S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino-3-methyl-1-oxobutan-2-yl)-1-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)-3,6,9,12,15,18-hexaoxaheneicosane-21-amide (001)

[0309] Compound 001c (57.7 mg, 0.10 mmol) was added to a solution of compound 001f (70.1 mg, 0.11 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (57.3 mg, 0.15 mmol) and N,N-diisopropylethylamine (39.0 mg, 0.30 mmol) in N,N-dimethylformamide (3 mL), and stirred at room temperature for 2 hours. After completion of the reaction, the product was concentrated under reduced pressure, and the residue was purified by preparative high performance liquid chromatography (Waters MS-triggered Prep-LC with QDA detector, column: Xtimate C18, 21.2×250 mm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-min gradient, gradient ratio: acetonitrile phase 20%-45%, flow rate: 20 mL / min) to obtain compound 001 (17.3 mg, 14%).

[0310] MS m / z(ESI):595.5(M / 2+1).

[0311] 1 H NMR(400MHz,DMSO-d6)δ9.75(s,1H),8.80(s,1H),8.27(d,1H),7.97–7.89(m,3H),7.82(d,1H),7. 30(s,1H),6.51(s,1H),5.43(s,2H),5.26(s,2H),4.53(t,1H),4.25(dd,1H),4.07(s,3H),3.59(t, 2H),3.49–3.47(m,21H),3.39(s,4H),3.22–3.14(m,6H),2.99–2.96(m,2H),2.42–2.32(m,2H),2.2 4(t,2H),2.05–1.96(m,3H),1.90–1.83(m,2H),1.80–1.75(m,2H),1.39(d,3H),0.90–0.84(m,9H).

[0312] Example 2 N-((S)-1-((S)-1-((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-1-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-amide (002)

[0313] The first step is tert-butyl 34-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatetraaza-33-ynoate (002b)

[0314] Compound 002a (100 mg, 0.201 mmol) and compound Int-1 (87 mg, 0.221 mmol) were dissolved in N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (78 mg, 0.603 mol) was added dropwise to the reaction system at 0°C. The reaction solution was heated to 25°C and stirred for 2 hours. After the reaction was completed, the solvent was removed by rotary evaporation. Water (20 mL) was added to dissolve the mixture and extracted three times with dichloromethane (30 mL). The organic phase was washed with saturated brine (10 mL) and dried over anhydrous sodium sulfate. The product was dried by rotary evaporation to obtain compound 002b (142 mg, yield: 91%), which was used directly in the next step without purification. MS m / z (ESI): 722.3 (M+1-56).

[0315] Step 2: 34-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatetraaza-33-ynoic acid (002c)

[0316] Compound 002b (60 mg, 0.077 mmol) was dissolved in dichloromethane (1 mL). Trifluoroacetic acid (0.2 mL, 298 mg, 2.612 mmol) was then added at 0°C. The mixture was heated to 25°C and stirred for 1 hour. After completion of the reaction, the reaction solution was spin-dried to obtain compound 002c (50 mg), which was used directly in the next reaction without purification.

[0317] MS m / z(ESI):722.2(M+1).

[0318] Step 3: N-((S)-1-((S)-1-((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-1-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-amide (002)

[0319] Compound 002c (50 mg, 0.069 mmol), HATU (40 mg, 0.104 mmol), and N,N-diisopropylethylamine (27 mg, 0.208 mmol) were dissolved in N,N-dimethylformamide (1 mL). Compound 001c (40 mg, 0.070 mmol) was added at 0°C, and the reaction was stirred at 25°C for 2 hours. After completion of the reaction, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector, chromatographic column: Xtimate C18, 21.2*250 mm, 5 μm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20 min gradient, gradient ratio: acetonitrile phase 20%-45%, flow rate: 20 mL / min) to give compound 002 (22.6 mg, yield: 26%).

[0320] MS m / z(ESI):639.4(M / 2+1).

[0321] 1H NMR(400MHz,DMSO-d6)δ9.75(s,1H),8.80(s,1H),8.27(d,1H),7.97–7.89(m,3H),7.82(d,1H),7.31(s, 1H),6.50(s,1H),5.43(s,2H),5.26(s,2H),4.57–4.50(m,1H),4.27–4.24(m,1H),4.08(s,3H),3.60(t,2 H),3.50–3.48(m,30H),3.42–3.39(m,5H),3.22–3.14(m,4H),2.99–2.96(m,2H),2.47–2.35(m,2H),2.2 6–2.22(m,2H),2.06–1.95(m,3H),1.93–1.84(m,2H),1.82–1.74(m,2H),1.40(d,3H),0.90–0.84(m,9H).

[0322] Example 3 N-(((S)-1-(((S)-1-([(S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropyl-2-yl)amino-3-methyl-1-oxobutyl-2-yl)-1-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)-3,6,9,12,15,18,21,24,27,30-decaoxatriacontane-33-amide (003)

[0323] The first step is 40-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)-35-oxo-4,7,10,13,16,19,22,25,28,31-decaoxa-34-azatetraone-39-ynoic acid tert-butyl ester (003b)

[0324] Compound 003a (100 mg, 0.17 mmol) and compound Int-1 (67.5 mg, 0.17 mmol) were dissolved in N,N-dimethylformamide (5 mL) and N,N-diisopropylethylamine (66.1 mg, 0.51 mmol) was added. The mixture was stirred at room temperature for 2 hours. After the reaction, water and ethyl acetate were added for extraction, and the organic phase was washed twice with saturated sodium chloride. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column chromatography system A to obtain compound 003b (110 mg, 74.4%).

[0325] MS m / z(ESI):886.4(M+1).

[0326] Step 2: 40-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)-35-oxo-4,7,10,13,16,19,22,25,28,31-deca-34-azatetraone-39-ynoic acid (003c)

[0327] Compound 003b (100 mg, 0.17 mmol) was dissolved in dichloromethane (2 mL), cooled to 0°C, and trifluoroacetic acid (0.4 mL) was slowly added. The mixture was stirred at room temperature for 1 hour. After the reaction, the reaction solution was concentrated under reduced pressure to obtain crude compound 003c (90 mg), which was used directly in the next reaction without purification.

[0328] MS m / z(ESI):810.4(M+1).

[0329] Step 3

[0330] N-(((S)-1-(((S)-1-([(S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino-3-methyl-1-oxobutan-2-yl)-1-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)-3,6,9,12,15,18,21,24,27,30-decaoxatriacontane-33-amide (003)

[0331] Compound 003c (80 mg, 0.10 mmol) and compound 001c (56.7 mg, 0.10 mmol) were dissolved in N,N-dimethylformamide (2 mL) solution, and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (56.3 mg, 0.15 mmol) and N,N-diisopropylethylamine (38.2 mg, 0.30 mmol) were added, and the mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was filtered, and the crude product was purified by high performance liquid chromatography (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector, chromatographic column: Xtimate 5μm C18250×21.2mm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 21-minute gradient, gradient ratio: acetonitrile phase 20%-48%, flow rate: 20 mL / min) to obtain compound 003 (24.1 mg, 17.9%).

[0332] MS m / z(ESI):683.4(M / 2+1).

[0333] 1 H NMR(400MHz,DMSO-d6)δ9.78(s,1H),8.82(s,1H),8.29(d,1H),8.02–7.81(m,4H),7.33(s,1H),6. 53(s,1H),5.45(s,2H),5.28(s,2H),4.55(t,1H),4.31–4.22(m,1H),4.09(s,3H),3.61(t,2H),3. 51–3.50(m,34H),3.43–3.41(m,5H),3.24–3.18(m,5H),3.01–2.98(m,2H),2.70–2.68(m,1H),2.2 8–2.24(m,2H),2.09–1.96(m,4H),1.93–1.76(m,5H),1.41(d,3H),1.25(s,2H),0.92–0.86(m,9H).

[0334] Example 4 1-(6-(5-cyano-6-(methylsulfonyl)pyridin-3-yl)hexyl-5-ynamido)-N-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxaheneicosane-21-amide (004)

[0335] Referring to the synthetic route of Example 1, the starting material in the first step was replaced with Int-2. The crude product was purified by HPLC (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector, column: Xtimate C18, 21.2*250 mm, 5 μm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 20%-45%, flow rate: 20 mL / min) and lyophilized to afford Compound 004 (10.7 mg, 17%).

[0336] MS m / z(ESI):592.4(M+2 / 2).

[0337] 1 H NMR(400MHz,DMSO-d6)δ9.82(s,1H),9.04(d,1H),8.78(d,1H),8.33(d,1H),8.04–7.94(m,3H),7.88(d,1H),7.37 (s,1H),6.56(s,1H),5.49(s,2H),5.32(s,2H),4.62–4.56(m,1H),4.33–4.29(m,1H),3.67–3.64(m,2H),3.57–3. 53(m,20H),3.51(s,3H),3.49–3.45(m,4H),3.28–3.26(m,2H),3.24–3.21(m,2H),3.05–3.02(m,2H),2.48–2.39( m,2H),2.34–2.31(m,2H),2.08-2.03(m,2H)1.99-1.91(m,2H)1.86–1.82(m,2H),1.46(d,3H),0.97–0.89(m,9H).

[0338] Example 5 1-(6-(5-cyano-6-(methylsulfonyl)pyridin-3-yl)hexyl-5-ynamido)-N-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-amide (005)

[0339] Referring to the synthetic route of Example 2, the starting material in the first step was replaced with Int-2. The crude product was purified by HPLC (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector, column: Xtimate C18, 21.2*250 mm, 5 μm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 20%-46%, flow rate: 20 mL / min) and lyophilized to afford Compound 005 (10.7 mg, 13.7%).

[0340] MS m / z(ESI):1271.4(M+1).

[0341] 1 H NMR(400MHz,DMSO-d6)δ9.76(s,1H),8.98(d,1H),8.72(d,1H),8.27(d,1H),7.99–7.90(m,3H),7.83(d,1H),7. 31(s,1H),6.51(s,1H),5.44(s,2H),5.27(s,2H),4.55–4.52(m,1H),4.27–4.24(m,1H),3.61–3.58(m,2H),3.50 (s,28H),3.45(s,3H),3.42–3.39(m,4H),3.23–3.21(m,2H),3.18-3.16(m,2H)2.99–2.97(m,2H),2.42-2.33(m, 2H),2.29–2.25(m,2H),2.02–1.97(m,3H),1.91–1.84(m,2H),1.82-1.76(m,2H)1.40(d,3H),0.91–0.83(m,9H).

[0342] Example 6 1-(6-(5-cyano-6-(methylsulfonyl)pyridin-3-yl)hexyl-5-ynamido)-N-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27,30-decaoxatriacontane-33-amide (006)

[0343] Referring to the synthetic route of Example 3, the starting material in the first step was replaced with Int-2. After completion of the reaction, the reaction solution was directly purified by HPLC (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector, chromatographic column: Xtimate C18, 21.2*250 mm, 5 μm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 21% to 46%, flow rate: 20 mL / min) to obtain Compound 006 (11.2 mg, yield: 18%).

[0344] MS m / z(ESI):1360.5(M+1).

[0345] 1 H NMR (400MHz, CD3OD) δ8.85(d,1H),8.47(d,1H),7.97(d,1H),7.82(d,J=9.2Hz,1H),7.66(s,1H),5. 62(d,1H),5.42(d,1H),5.26(s,2H),4.72–4.58(m,2H),4.27(d,1H),3.77-3.74(m,2H),3.63-3.61 (m,38H)3.56-3.53(m,2H),3.39-3.33(m,4H),3.23-3.20(m,2H),3.08-3.04(m,2H),2.62–2.55(m, 4H),2.43-2.39(m,2H),2.21-2.12(m,3H),2.03-1.92(m,4H),1.58-1.56(m,3H),1.05-1.00(m,9H).

[0346] Example 7 N-(((S)-1-(((S)-1-([(S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino-3-methyl-1-oxobutan-2-yl)-1-(6-(4-(methoxymethyl)-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)-3,6,9,12,15,18-hexaoxaheneicosane-amide (007)

[0347] Referring to the synthetic route of Example 1, the starting material in the first step was replaced with Int-3. The crude product was purified by preparative HPLC (Waters MS-triggered Prep-LC with QDA detector, column: Xtimate C18, 21.2×250 mm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile 20%-45%, flow rate: 20 mL / min) to obtain Compound 007 (32.0 mg, 26%).

[0348] MS m / z(ESI):602.5(M / 2+1).

[0349] 1 H NMR(400MHz,DMSO-d6)δ9.75(s,1H),9.04(s,1H),8.27(d,1H),7.97–7.90(m,3H),7.82(d,1H),7 .30(s,1H),6.50(s,1H),5.43(s,2H),5.26(s,2H),4.70(s,2H),4.53(t,1H),4.25(dd,1H),3.59( t,2H),3.49–3.47(m,20H),3.41–3.38(m,8H),3.22–3.14(m,4H),2.97(t,2H),2.57(t,2H),2.46 –2.35(m,2H),2.27(t,2H),2.06–1.96(m,3H),1.91–1.78(m,4H),1.39(d,3H),0.90–0.84(m,9H).

[0350] Example 8 N-((S)-1-((S)-1-((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-1-(6-(4-(methoxymethyl)-2-(methylsulfonyl)pyrimidin-5-yl)hexadecyl)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-amide (008)

[0351] Refer to the synthetic route of Example 2, replacing the first raw material with Int-3. After the reaction, the reaction solution was filtered and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector, chromatographic column: Xtimate C18, 21.2*250mm, 5μm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 20%-45%, flow rate: 20 mL / min) to obtain compound 008 (18.1 mg, yield: 21%).

[0352] MS m / z(ESI):646.4(M / 2+1).

[0353] 1 H NMR (400MHz, DMSO) δ9.76(s,1H),9.05(s,1H),8.28(d,1H),7.97–7.91(m,3H),7.83(d,1H),7.31(s ,1H),6.51(s,1H),5.43(s,2H),5.26(s,2H),4.71(s,2H),4.57–4.50(m,1H),4.28–4.24(m,1H),3. 60(t,2H),3.50–3.48(m,28H),3.42–3.39(m,8H),3.23–3.14(m,4H),2.98(t,2H),2.58(t,2H),2.4 9–2.36(m,2H),2.28(t,2H),2.08–1.96(m,3H),1.93–1.79(m,4H),1.40(d,3H),0.91–0.85(m,9H).

[0354] Example 9 N-(((S)-1-(((S)-1-((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropyl-2-yl)amino-3-methyl-1-oxobutyl-2-yl)-1-(6-(4-(methoxymethyl)-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)-3,6,9,12,15,18,21,24,27,30-decaoxatriacontane-33-amide (009)

[0355] Referring to the synthetic route of Example 3, the starting material in the first step was replaced with Int-3. The crude product was purified by HPLC (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector, chromatographic column: Xtimate 5μm C18 250×21.2mm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 20%-48%, flow rate: 20 mL / min) to obtain Compound 009 (23.2 mg, 19.8%).

[0356] MS m / z(ESI):690.4(M / 2+1).

[0357] 1 H NMR(400MHz,DMSO-d6)δ9.78(s,1H),9.07(s,1H),8.29(d,1H),7.99–7.83(m,4H),7.33(s,1H),6.53(s,1 H),5.45(s,2H),5.29(s,2H),4.72(s,2H),4.57–4.53(m,1H),4.29–4.25(m,1H),3.61(t,2H),3.52–3.50( m,34H),3.43–3.42(m,7H),3.21–3.18(m,4H),3.01–2.98(m,2H),2.70–2.68(s,1H),2.62–2.56(m,3H),2. 36–2.27(m,3H),2.08–1.98(m,3H),1.91–1.82(m,4H),1.41(d,3H),1.27–1.24(m,2H),0.92–0.86(m,9H).

[0358] Example 10

[0359] N-(2-(3-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)ethyl)-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)-N-(2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-yl)hex-5-ynamide

[0360] Step 1: 2,5,8,11,14,17,20,23,26-nonoxyoctadecane-28-aldehyde 011b: Dissolve 011a (500.0 mg, 1.17 mmol) and Dess-Martin oxidant (742.3 mg, 1.75 mmol) in dichloromethane (10 mL) and stir at room temperature for 18 hours. After the reaction, add saturated sodium bicarbonate for washing. The organic phase is dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure to obtain a colorless oily liquid 011b (400.0 mg, 80%).

[0361] MS m / z(ESI):427.4(M+1).

[0362] Step 2 2,5,8,11,14,17,20,23,26,32-deca-29-aza-32-oxapentacarbonyl-35-acid tert-butyl ester 011c

[0363] 011b (100.0 mg, 0.23 mmol), tert-butyl 3-(2-aminoethoxy)propionate (39.9 mg, 0.21 mmol) and acetic acid (6.9 mg, 0.12 mmol) were dissolved in methanol (5 mL) and stirred at room temperature for 30 minutes. Sodium cyanoborohydride (29.5 mg, 0.47 mmol) was added, and the atmosphere was replaced with nitrogen three times. The mixture was stirred at room temperature for 18 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography system A to obtain colorless oily liquid 011c (35.0 mg, 25%).

[0364] MS m / z(ESI):600.4(M+1).

[0365] Step 3 29-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-2,5,8,11,14,17,20,23,26,32-decaoxa-29-aza-32-oxapentacarbon-35-oic acid tert-butyl ester 011d

[0366] 011c (35.0 mg, 0.06 mmol), 2,5-dioxopyrrolidin-1-yl-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate Int-1 (23.1 mg, 0.06 mmol), and N,N-diisopropylethylamine (22.6 mg, 0.18 mmol) were dissolved in N,N-dimethylformamide (2 mL) and heated to 50°C with stirring for 18 hours. After the reaction was complete, ethyl acetate and water were added for extraction. The organic phase was concentrated under reduced pressure, and the residue was purified by column chromatography using System A to obtain 011d (17.0 mg, 33%) as a colorless oil. MS m / z (ESI): 880.5 (M+1).

[0367] Step 4: 29-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-2,5,8,11,14,17,20,23,26,32-deca-29-aza-32-oxapentacarbonyl-35-oic acid 011e

[0368] 011d (17.0 mg, 0.02 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (44.1 mg, 0.39 mmol) was added at 0°C. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain crude product 011e (20.0 mg). The product was used directly in the next reaction without purification.

[0369] MS m / z(ESI):824.3(M+1).

[0370] Step 5: N-(2-(3-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)ethyl)-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)-N-(2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-yl)hex-5-ynamide

[0371] Compound 011e (20.0 mg, 0.020 mmol), compound 001c (11.5 mg, 0.02 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.4 mg, 0.03 mmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (7.8 mg, 0.06 mmol) was added. The mixture was stirred at room temperature for 18 hours. After the reaction, the reaction solution was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with QDA detector, column: Xtimate C18, 21.2×250 mm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20 min gradient, gradient ratio: acetonitrile phase 20%-45%, flow rate: 20 mL / min) to obtain compound 011 (3.4 mg, 12%).

[0372] MS m / z(ESI):690.6(M / 2+1).

[0373] 1 H NMR(400MHz,DMSO-d6)δ9.75(s,1H),8.81–8.79(m,1H),8.27(d,1H),7.95(d,1H),7.94–7.90(m,1H),7.82(d,1H),7.30(s,1H),6.5 0(s,1H),5.43(s,2H),5.26(s,2H),4.57–4.50(m,1H),4.28–4.23(m,1H),4.07(d,3H),3.61–3.54(m,2H),3.54–3.46(m,29H),3.46 –3.39(m,6H),3.38(s,3H),3.35–3.34(m,1H),3.30–3.26(m,2H),3.23(s,3H),3.18–3.12(m,2H),3.00–2.94(s,2H),2.56–2.52(m, 3H),2.48–2.43(m,2H),2.40–2.32(m,2H),2.06–1.94(m,4H),1.94–1.83(m,2H),1.81–1.76(m,2H),1.39(d,3H),0.90–0.84(m,9H).

[0374] Example 11

[0375] N-(4-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl)-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)-N-(2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-yl)hex-5-ynamide

[0376] The first step is 2,5,8,11,14,17,20,23,26-nonyloxy-29-azatricarbonyl-33-acid tert-butyl ester 012a

[0377] 011a (100.0 mg, 0.23 mmol), tert-butyl 4-aminobutyrate hydrochloride (41.3 mg, 0.21 mmol) and acetic acid (6.9 mg, 0.12 mmol) were dissolved in methanol (5 mL) and stirred at room temperature for 30 minutes. Sodium cyanoborohydride (29.5 mg, 0.47 mmol) was added, and the atmosphere was replaced with nitrogen three times. The mixture was stirred at room temperature for 18 hours. After the reaction, the reaction solution was purified by column chromatography system A to obtain compound 012a (77.0 mg, 58%).

[0378] MS m / z(ESI):570.4(M+1).

[0379] Step 2 29-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-2,5,8,11,14,17,20,23,26-nonoxa-29-azatricarbon-33-oic acid tert-butyl ester 012b

[0380] Compound 012a (77.0 mg, 0.14 mmol), 2,5-dioxopyrrolidin-1-yl-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate Int-1 (53.4 mg, 0.14 mmol), and N,N-diisopropylethylamine (52.4 mg, 0.41 mmol) were dissolved in N,N-dimethylformamide (5 mL) and heated to 50°C with stirring for 18 hours. After the reaction was complete, ethyl acetate and water were added for extraction. The organic phase was concentrated under reduced pressure, and the residue was purified by column chromatography using System A to obtain Compound 012b (60.0 mg, 52%). MS m / z (ESI): 850.4 (M+1).

[0381] Step 3 29-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-2,5,8,11,14,17,20,23,26-nonoxa-29-azatricarbon-33-oic acid 012c

[0382] 012b (60.0 mg, 0.07 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (161.0 mg, 1.41 mmol) was added at 0°C. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain crude compound 012c (65 mg), which was used directly in the next reaction without purification.

[0383] MS m / z(ESI):794.2(M+1).

[0384] Step 4: N-(4-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl)-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)-N-(2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-yl)hex-5-ynamide 012

[0385] Compound 012c (60.0 mg, 0.07 mmol), compound 001c (40.5 mg, 0.07 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (40.3 mg, 0.11 mmol) were dissolved in N,N-dimethylformamide (2 mL). N,N-diisopropylethylamine (27.4 mg, 0.21 mmol) was added and stirred at room temperature for 18 hours. After the reaction, the reaction solution was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with QDA detector, column: Xtimate C18, 21.2×250 mm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20 min gradient, gradient ratio: acetonitrile phase 20%-45%, flow rate: 20 mL / min) to obtain compound 012 (29.9 mg, 31%).

[0386] MS m / z(ESI):675.6(M / 2+1).

[0387] 1H NMR(400MHz,DMSO-d6)δ9.73(d,1H),8.79(s,1H),8.27(d,1H),7.95(d,1H),7.92–7.89(m,1H),7.81(d,1H),7.30(s,1H) ,6.50(s,1H),5.43(s,2H),5.26(s,2H),4.54–4.51(m,1H),4.26–4.18(m,1H),4.07(d,3H),3.51–3.47(m,32H),3.43–3. 40(m,4H),3.38(s,3H),3.23(s,3H),3.18–3.18(m,2H),3.00–2.93(m,2H),2.56–2.54(m,3H),2.46–2.43(m,2H),2.23–2 .12(m,2H),2.07–2.12(m,4H),1.92–1.83(m,2H),1.82–1.73(m,3H),1.68–1.64(m,1H),1.39(d,3H),0.90–0.84(m,9H).

[0388] Example 12

[0389] N-((5S,14S)-5-Benzyl-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-14-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)-1,4,7,10,13-pentaoxo-3,6,9,12-tetraazaoctan-18-yl)-2,5,8,11,14,17,20,23-octaoxehexacanoamide

[0390] Refer to the synthetic route of Example 13. The residue was purified by HPLC (Waters MS-triggered Prep-LC with QDA detector, column: Xtimate C18, 21.2 x 250 mm; mobile phase 1: water (containing 0.1% trifluoroacetic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile 20%-45%, flow rate: 20 mL / min) to afford compound 018 (5.8 mg, 15.8%).

[0391] MS m / z(ESI):762.5(M / 2+1) + .

[0392] 1 H NMR(400MHz,DMSO-d6)δ9.64(s,1H),8.79(s,1H),8.52–8.46(m,1H),8.24–8. 16(m,2H),8.04–8.02(m,2H),7.99–7.96(m,1H),7.90–7.86(m,1H),7.79–7.77 (m,1H),7.32(s,1H),7.27–7.25(m,3H),7.23–7.15(m,2H),6.51(s,1H),5.44 (s,2H),5.27(s,2H),4.60–4.54(m,1H),4.24–4.17(m,1H),4.07(s,3H),4.03– 3.99(m,2H),3.78–3.66(m,4H),3.59–3.55(m,2H),3.50–3.47(m,26H),3.25– 3.21(m,5H),3.19–3.14(m,4H),3.01–2.96(m,5H),2.88–2.82(m,2H),2.31–2. 27(m,4H),2.07–2.01(m,2H),1.90–1.86(m,2H),1.80–1.76(m,2H),1.72–1.57 (m,2H),1.54–1.41(m,2H),1.38–1.32(m,2H),1.27–1.23(m,2H),0.89(t,3H).

[0393] Example 13

[0394] N-((5S,14S)-5-Benzyl-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-14-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)-1,4,7,10,13-pentaoxo-3,6,9,12-tetraazaoctadec-18-yl)-2,5,8,11,14,17,20,23,26,29,35-dodecahydrotetratriacontamide

[0395] Step 1: (9H-fluoren-9-yl)methyl(2-((((S)-1-((2-([((((R)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino(019a)

[0396] 001 (100 mg, 0.248 mmol), (((9H-fluoren-9-yl)methoxy)carboxy)glycylglycylglycyl-L-phenylalanylglycine (138.46 mg, 0.248 mmol), and 1-methylimidazole (42.73 mg, 0.521 mmol) were added to DMF (5 mL) at room temperature, followed by N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (83.46 mg, 0.297 mmol). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The mixture was added to saturated aqueous sodium chloride (25 mL) and filtered, and the yellow solid was dissolved in DCM / CH3OH. The organic layer was washed twice with H2O and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to obtain the crude product 019a (132 mg, 56.4%).

[0397] MS m / z(ESI):943.6(M+1) + .

[0398] Step 2 (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((R)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-2-oxoethyl)-3-phenylpropanamide (019b)

[0399] To a solution of 019a (132 mg, 0.140 mmol) in DMF (2 mL) was added diethylamine (0.087 mL, 0.839 mmol) at 20° C. The mixture was then stirred at 20° C. for 1 h. The residue was purified by HPLC (TFA, H 2 O:MeCN=57%) to give the product 019b (76 mg, 75.31%).

[0400] MS m / z(ESI):721.6(M+1) + .

[0401] Step 3: Tert-butyl (S)-44-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecyloxy-39-azetidine-45-acid ester (019d)

[0402] To a solution of 019c (250 mg, 0.589 mmol) in DMF (4 ml) were added N,N-diisopropylethylamine (0.195 mL, 1.178 mmol), HATU (291.09 mg, 0.766 mmol) and undecyl glycol monomethyl ether propionic acid (346.66 mg, 0.589 mmol) at 25°C. The mixture was stirred under nitrogen at 25°C for 18 hours. After the reaction was complete, the mixture was partitioned between ethyl acetate (20 mL) and water (20 ml). The aqueous layer was extracted with ethyl acetate (20 mL*2), the combined organic layers were dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography system A to give the product 019d (467 mg, 79.69%).

[0403] MS m / z(ESI):995.4(M+1) + .

[0404] Step 4 (S)-44-amino-38-oxo-2,5,8,11,14,17,20,23,26,29,35-dodecyloxy-39-azatetracarboxylic acid tert-butyl ester 019e

[0405] To a solution of 019d (417 mg, 0.419 mmol) in DCM (2 mL) was added diethylamine (0.087 mL, 0.838 mmol) at 25° C. The mixture was then stirred at 25° C. for 1 hour. The solvent was removed under reduced pressure to give 019e (310 mg, 95.7%), which was used directly in the next reaction without purification.

[0406] MS m / z(ESI):773.6(M+1) + .

[0407] Step 5: Tert-butyl (S)-44-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecyloxy-39-azatetraazetidine-45-acid ester 019f

[0408] To a solution of 019e (310.0 mg, 0.401 mmol) in DMF (5 mL) was added N,N-diisopropylethylamine (0.133 mL, 0.802 mmol) and Int 1 (158.57 mg, 0.40 mmol) at 25° C. The mixture was then stirred at 25° C. for 2 hours. The residue was purified by reverse phase column to give the title product 019f (267 mg, 63.2%) as a colorless oil.

[0409] MS m / z(ESI):1052.6(M+1) + .

[0410] Step 6 (S)-4-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecyloxy-39-azatetraazapentadecaenoic acid (0.19 g)

[0411] 019f (110 mg, 0.104 mmol) was added to a mixed solution of DCM (1 mL) and TFA (0.2 mL) at 20°C. The mixture was then stirred at 20°C for 2 hours. The reaction solution was concentrated in vacuo to give the crude product 019g (104 mg, 99.8%). The product was used directly in the next reaction without purification.

[0412] MS m / z(ESI):996.6(M+1).

[0413] Step 7: N-((5S,14S)-5-benzyl-1-(((R)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-14-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)-1,4,7,10,13-pentaoxo-3,6,9,12-tetraazaoctadec-18-yl)-2,5,8,11,14,17,20,23,26,29,35-dodecanooxaoctadecane-38-amide (019)

[0414] To a solution of 019b (70 mg, 0.097 mmol) in DMF (1 mL) were added N,N-diisopropylethylamine (0.048 mL, 0.291 mmol), 019g (106.38 mg, 0.107 mmol) and HATU (55.32 mg, 0.145 mmol) at 0° C. The mixture was then stirred at 20° C. for 1 h. After the reaction, the reaction solution was directly purified by high performance liquid chromatography (GX-281Liquid Handler without pump, 4020Syringe pump, 333-H3Pump, 334-H3Pump, 1741U, chromatographic column: Bonnasil-BS C18, 20*250mm, 8um; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 20%-50%, flow rate: 20 mL / min) to obtain the title product 019 (16 mg, 0.009 mmol, 9.70%).

[0415] MS m / z(ESI):1700.7(M+1) + .

[0416] 1 H NMR(400MHz,DMSO-d6)δ9.64(s,1H),8.79(s,1H),8.49(d,1H),8.22–8.15(m,2H),8.04(d,2H),7.97(d,1H),7.87(d,1H),7.80-7.76 (m,1H),7.31(s,1H),7.29–7.24(m,4H),7.19(dd,1H),6.52(s,1H),5.44(s,2H),5.27(s,2H),4.57(dd,1H),4.26–4.16(m,1H),4.07( s,3H),4.04-3.88(m,3H),3.83–3.60(m,5H),3.60-3.54(m,3H),3.50–3.38(m,26H),3.33(s,7H),3.26–3.06(m,9H),2.99(d,5H),2. 85(dd,2H),2.54(d,2H),2.32-2.24(m,5H),2.04(s,2H),1.95–1.72(m,5H),1.71–1.44(m,3H),1.43–1.03(m,6H),0.92-0.84(m,3H).

[0417] Example 14

[0418] 6-(4-((2,5,8,11,14-pentaoxohexadecan-16-yl)oxy)-2-(methylsulfonyl)pyrimidin-5-yl)-N-((2S,5S)-1-((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-5-isopropyl-2-methyl-1,4,7-trioxy-10,13-dioxa-3,6-diazapentadecan-15-yl)hex-5-ynamide

[0419] Step 1: 6-(2,4-dichloropyrimidin-5-yl)hexanoic acid methyl ester (028b)

[0420] Compound 028a (6000 mg, 21.669 mmol), methyl 5-hexynoate (4100 mg, 32.504 mmol), triethylamine (6.0 mL, 43.339 mmol), cuprous iodide (825 mg, 4.334 mmol), and bistriphenylphosphine palladium dichloride (1686 mg, 2.167 mmol) were dissolved in THF (25 mL) and stirred at 70°C for 2 hours. After completion of the reaction, H2O (30 mL) was added to the reaction solution, and the mixture was extracted with EA (50 mL x 3). The organic phase was washed with brine and dried over anhydrous Na2SO4, concentrated under reduced pressure, and the mixture was passed through a column chromatography (PE:EA = 10 / 1) to afford compound 028b (3.6 g, 61% yield).

[0421] MS m / z(ESI):272.6(M+1).

[0422] Step 2: 6-(4-((2,5,8,11,14-pentaoxohexadecan-16-yl)oxy)-2-chloropyrimidin-5-yl)hexanoic acid methyl ester (028c)

[0423] Compound 028b (1100 mg, 4.028 mmol), 2,5,8,11,14-pentaoxa-16-hexadecanol (1016 mg, 4.028 mmol), and potassium carbonate (1113 mg, 8.056 mmol) were dissolved in DMF (6 mL) and stirred at 70°C under nitrogen for 18 hours. The reaction solution was purified by column chromatography system A to obtain compound 028c (600 mg, 30% yield).

[0424] MS m / z(ESI):489.0(M+1).

[0425] Step 3: 6-(4-((2,5,8,11,14-pentaoxohexadecan-16-yl)oxy)-2-(methylthio)pyrimidin-5-yl)hexanoic acid methyl ester (028d)

[0426] Compound 028c (400 mg, 0.818 mmol) and sodium thioethoxide (114 mg, 1.636 mmol) were dissolved in DMF (4 ml) and stirred at 25°C under nitrogen for 2 h. The reaction solution was purified by column chromatography system A to obtain compound 028d (200 mg, yield: 49%).

[0427] MS m / z(ESI):501.1(M+1).

[0428] Step 4: 6-(4-((2,5,8,11,14-pentaoxohexadecan-16-yl)oxy)-2-(methylthio)pyrimidin-5-yl)hexanoic acid (028e)

[0429] Compound 028d (300 mg, 0.599 mmol) and LiOH (50 mg, 1.198 mmol) were dissolved in THF (3 mL) / H2O (1 mL) and stirred at 25°C under nitrogen for 2 h. The reaction solution was purified by column chromatography system A to obtain compound 028e (270 mg, yield: 93%).

[0430] MS m / z(ESI):508.7(M+23) + .

[0431] Step 5: 6-(4-((2,5,8,11,14-pentaoxohexadecan-16-yl)oxy)-2-(methylsulfonyl)pyrimidin-5-yl)hexanoic acid (028f)

[0432] Compound 028e (250 mg, 0.514 mmol) was dissolved in THF (5 mL), and m-chloroperbenzoic acid (354 mg, 2.055 mmol) was slowly added at 0°C. The reaction solution was stirred at 0°C for 3 minutes. The reaction solution was purified by column chromatography system A to obtain compound 028f (245 mg, yield: 92%).

[0433] MS m / z(ESI):519.2(M+1) + .

[0434] Step 6: tert-Butyl 3-(2-(2-(6-(4-((2,5,8,11,14-pentaoxohexadecan-16-yl)oxy)-2-(methylsulfonyl)pyrimidin-5-yl)hexadecyl)ethoxy)propanoate (0.28 g)

[0435] Compound 028f (50 mg, 0.096 mmol), HATU (55 mg, 0.145 mmol), and N,N-diisopropylethylamine (37 mg, 0.289 mmol) were dissolved in N,N-dimethylformamide (1 mL). 3-(2-(2-aminoethoxy)ethoxy)propionic acid tert-butyl ester (25 mg, 0.106 mmol) was added at 0°C, and the reaction was stirred at 25°C for 18 hours. After the reaction, most of the solvent was removed by vortexing, water (10 mL) was added, and the mixture was extracted three times with dichloromethane (20 mL). The organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and then dried to give crude compound 028g (70 mg). The product was used directly in the next reaction without purification.

[0436] MS m / z(ESI):734.3(M+1) + .

[0437] Step 7: 3-(2-(2-(6-(4-((2,5,8,11,14-pentaoxohexadecan-16-yl)oxy)-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)ethoxy)propanoic acid (028h)

[0438] Compound 028g (70 mg, 0.095 mmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (0.2 mL, 298 mg, 2.612 mmol) was added at 0°C. The mixture was heated to 25°C and stirred for 1 hour. After the reaction, the reaction solution was dried to give crude compound 028h (60 mg).

[0439] MS m / z(ESI):678.2(M+1) + .

[0440] Step 8: 6-(4-((2,5,8,11,14-pentaoxohexadecan-16-yl)oxy)-2-(methylsulfonyl)pyrimidin-5-yl)-N-((2S,5S)-1-((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-5-isopropyl-2-methyl-1,4,7-trioxy-10,13-dioxa-3,6-diazapentadecan-15-yl)hex-5-ynamide (028)

[0441] 028h (60 mg, 0.089 mmol), HATU (50 mg, 0.133 mmol), and N,N-diisopropylethylamine (34 mg, 0.266 mmol) were dissolved in N,N-dimethylformamide (1 mL). Compound 001c (51 mg, 0.089 mmol) was added at 0°C. The reaction mixture was heated to 25°C and stirred for 1 hour. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector, chromatographic column: Xtimate C18, 21.2*250 mm, 5 um; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20 min gradient, gradient ratio: acetonitrile phase 20%-45%, flow rate: 20 mL / min) to give the title compound 028 (28.2 mg, yield: 26%).

[0442] MS m / z(ESI):1233.4(M+1) + .

[0443] 1 H NMR(400MHz,DMSO-d6)δ9.77(s,1H),8.81(s,1H),8.28(d,1H),7.98–7.82(m,4H),7.31(s,1H),6.51(s ,1H),5.44(s,2H),5.27(s,2H),4.63–4.61(m,2H),4.55–4.52(m,1H),4.28–4.24(m,1H),3.82–3.80(m ,2H),3.62–3.58(m,4H),3.53–3.48(m,16H),3.42–3.39(m,10H),3.23–3.15(m,8H),3.00–2.96(m,2H) ,2.26(t,2H),2.06–1.97(m,3H),1.91–1.84(m,2H),1.82–1.75(m,2H),1.40(d,3H),0.91–0.84(m,9H).

[0444] Example 15

[0445] 6-(4-((2,5,8,11,14,17,20-heptaoxetriacont-22-yl)oxy)-2-(methylsulfonyl)pyrimidin-5-yl)-N-((2S,5S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-5-isopropyl-2-methyl-1,4,7-trioxo-10,13-dioxa-3,6-diazapentadecan-15-yl)hexan-5-amide

[0446] Compound 029 was prepared according to the synthetic route described in Example 14. The final product was purified by HPLC (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector, column: Xtimate C18, 21.2*250 mm, 5 μm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 20%-45%, flow rate: 20 mL / min) to obtain Compound 029 (10.5 mg, yield: 17%).

[0447] MS m / z(ESI):661.4(M / 2+1) + .

[0448] 1 H NMR(400MHz,DMSO-d6)δ9.77(s,1H),8.81(s,1H),8.28(d,1H),7.99–7.82(m,4H),7.31(s,1H),6.51(s ,1H),5.44(s,2H),5.27(s,2H),4.66–4.60(m,2H),4.58–4.49(m,1H),4.31–4.23(m,1H),3.85–3.78(m ,2H),3.61-3.58(m,4H),3.52-3.48(m,22H),3.44–3.38(m,7H),3.23–3.15(m,7H),2.98(s,2H),2.68– 2.66(m,1H),2.47–2.31(m,4H),2.27-2.24(m,2H),2.10–1.75(m,8H),1.40(d,3H),0.91–0.84(m,9H).

[0449] Example 16

[0450] 6-(4-((2,5,8,11,14,17,20,23,26-nonaoxoctacosan-28-yl)oxy)-2-(methylsulfonyl)pyrimidin-5-yl)-N-((2S,5S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-5-isopropyl-2-methyl-1,4,7-trioxo-10,13-dioxo-3,6-diazapentadecan-15-yl)hexan-5-amide

[0451] Compound 030 was prepared according to the synthetic route described above, with reference to the synthetic method of Example 14. The final product was purified by HPLC (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector, column: Xtimate C18, 21.2*250 mm, 5 μm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 20%-45%, flow rate: 20 mL / min) to obtain the title compound 030 (22.7 mg, 25%).

[0452] MS m / z(ESI):1408.5(M+1) + .

[0453] 1 H NMR(400MHz,DMSO-d6)δ9.76(s,1H),8.81(s,1H),8.28(d,1H),7.98–7.81(m,4H),7.30(s,1H),5.43 (s,2H),5.26(s,2H),4.64–4.59(m,2H),4.56–4.49(m,1H),4.31–4.22(m,1H),3.83–3.78(m,2H),3.6 1-3.58(m,5H),3.52-3.50(m,4H),3.49–3.37(m,30H),3.27–3.14(m,8H),2.97(s,2H),2.53-2.50(m ,2H),2.48–2.33(m,4H),2.27-2.23(m,2H),2.07–1.74(m,8H),1.40-1.38(m,3H),0.90–0.84(m,9H).

[0454] Example 17

[0455] (S)-N-(2-(((R)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-2-(2-(2-(10-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-1,7-dioxo-4,10-diazacyclododec-4-yl)acetamido)-3-phenylpropanamide 040

[0456] The first step is tert-butyl 2-(1,7-dioxo-4,10-diazacyclododecan-4-yl)acetate 040b

[0457] Compound 040a (700 mg, 4.017 mmol), tert-butyl bromoacetate (705 mg, 3.616 mmol), and potassium carbonate (500 mg, 3.616 mmol) were dissolved in DMF (5 mL) and stirred at 25°C under nitrogen for 2 h. The reaction mixture was directly purified by reverse phase chromatography (MeCN / H2O = 1 / 1) to obtain the title compound 040b (280 mg, 24.2%).

[0458] MS m / z(ESI):288.9(M+1) + .

[0459] Step 2 tert-Butyl 2-(10-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynyl)-1,7-dioxo-4,10-diazacyclododecan-4-yl)acetate 040c

[0460] Compound 040b (150 mg, 0.520 mmol), Int1 (206 mg, 0.520 mmol), and DIEA (0.258 mL, 1.560 mmol) were dissolved in DMF (3 mL) and stirred at 25°C under N₂ for 18 h. The reaction mixture was directly purified by reverse phase chromatography (MeCN / H₂O = 1 / 1) to obtain the title compound 040c (20 mg, 6.8%).

[0461] MS m / z(ESI):569.2(M+1) + .

[0462] Step 3 2-(10-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynyl)-1,7-dioxy-4,10-diazacyclododecan-4-yl)acetic acid 040d

[0463] Compound 040c (20 mg, 0.0.35 mmol) was dissolved in a mixed solvent of DCM and TFA (1 mL, V / V = 1:1) and stirred at 25°C under nitrogen for 3 h. The reaction mixture was directly concentrated to give crude compound 040d (18 mg, 99.8%).

[0464] MS m / z(ESI):512.7(M+1) + .

[0465] Step 4: (S)-N-(2-(((R)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-2-(2-(2-(10-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-ynyl)-1,7-dioxo-4,10-diazacyclododecan-4-yl)acetamido)-3-phenylpropanamide 040

[0466] Compound 040d (18 mg, 0.035 mmol), 019b (25 mg, 0.035 mmol), HATU (20 mg, 0.053 mmol), and DIEA (0.017 mL, 0.105 mmol) were dissolved in DMF (2 mL) and stirred at 25°C for 1 h. After completion of the reaction, the reaction solution was directly purified by preparative HPLC (GX-281 Liquid Handler without pump, 4020 Syringe pump, 333-H3 Pump, 334-H3 Pump, 1741U, column: Bonnasil-BS C18, 20*250 mm, 8 μm; mobile phase 1: water (containing 0.1% TFA); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 20%-50%, flow rate: 20 mL / min) to obtain compound 040 (5 mg, 12%).

[0467] MS m / z(ESI):1215.3(M+1) + .

[0468] 1H NMR(400MHz,DMSO-d6)δ9.69(s,1H),8.90(s,1H),8.78(s,1H),8.52(s,1H),8.27–8.19(m,2H), 7.99–7.86(m,3H),7.31(s,1H),7.28–7.25(m,3H),7.20-7.17(m,1H),6.51(s,1H),5.43(s,2H), 5.27(s,2H),4.59(s,1H),4.19–4.00(m,7H),3.90–3.49(m,18H),3.25–2.93(m,8H),2.84-2.82( m,1H),2.67-2.66(m,1H),2.33-2.31(m,1H),2.11–1.72(m,8H),1.23(s,2H),0.90-0.85(m,3H).

[0469] Example 18

[0470] (S)-N-(2-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-2-oxoethyl)-2-(2-(2-(2-(16-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadec-7-yl)acetamido)-3-phenylpropanamide 041

[0471] Refer to the synthetic route of Example 17. The final product was purified by high-performance liquid chromatography (Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector, chromatographic column: Xtimate C18, 21.2*250 mm, 5 μm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; 20-minute gradient, gradient ratio: acetonitrile phase 15%-40%, flow rate: 20 mL / min) to obtain the title compound 041 (4.7 mg, yield: 4%). MS m / z (ESI): 653.0 (M / 2+1). + .

[0472] 1H NMR(400MHz,DMSO-d6)δ9.63(s,1H),8.80(s,1H),8.51–8.47(m,1H),8.22–8.18(m,1H),8.09–8.05(m,1H),7.99–7.95(m,2 H),7.91–7.87(m,1H),7.31(s,1H),7.27–7.22(m,4H),7.21–7.14(m,1H),6.51(s,1H),5.44(s,2H),5.27(s,2H),4.59–4.5 5(m,1H),4.07(s,3H),4.03–4.00(m,2H),3.78–3.70(m,3H),3.61–3.56(m,6H),3.56–3.47(s,22H),3.17–3.11(m,4H),3.1 1–3.07(m,4H),3.03–2.96(s,2H),2.88–2.82(m,2H),2.07–2.00(m,2H),1.91–1.84(m,2H),1.80–1.77(m,2H),0.89(t,3H).

[0473] Example 19

[0474] N-(2-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-2-oxoethyl)-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)-N-(2,5,8,11,14,17,20,23,26-nonaoxooctacosan-28-yl)hex-5-ynamide

[0475] Referring to the synthetic route of Example 20, the starting material in the first step was replaced with 042a. The final product was purified by HPLC (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge 5u C18 150 x 19 mm, 5 μm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 35%-45%, flow rate: 20 mL / min) to obtain compound 042 (16 mg, 38% yield).

[0476] MS m / z(ESI):1321.5(M+1)+ .

[0477] 1 H NMR(400MHz,DMSO-d6)δ9.41(d,1H),8.71(s,1H),8.02(d,1H),7.93(d,1H),7.84(d,1H),7.68–7.55(m,1H),7. 32(s,1H),6.16(s,1H),5.40(m,2H),5.22(s,2H),4.56(s,1H),4.30–4.20(m,1H),3.54–3.47(m,34H),3.47–3. 38(m,5H),3.33(s,3H),3.24(s,3H),3.14–3.10(m,2H),3.00–2.97(m,2H),2.55–2.52(m,2H),2.42–2.34(m,1H ),2.07–2.01(m,3H),1.94–1.85(m,2H),1.83–1.76(m,2H),1.44–1.38(m,3H),1.25(s,2H),0.92–0.87(m,9H).

[0478] Example 20

[0479] N-(3-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizinyl[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)-N-(2,5,8,11,14,17,20,23,26-nonaoxooctacosan-28-yl)hex-5-ynamide

[0480] The first step 29-((benzyloxy)carbonyl)-2,5,8,11,14,17,20,23,26-nonyloxy-29-azatriazetidine-32-acid benzyl ester 043b

[0481] β-Alanine benzyl ester p-toluenesulfonate 043a (300 mg, 0.85 mmol) was dissolved in acetonitrile (5 mL), and 28-bromo-2,5,8,11,14,17,20,23,26-nonaoxoctacosane (419.5 mg, 0.85 mmol), potassium carbonate (352 mg, 2.55 mmol), and potassium iodide (141 mg, 0.85 mmol) were added. The reaction was stirred at 80°C for 16 hours. After the reaction was completed, the temperature was cooled to room temperature, benzyl chloroformate (290 mg, 1.7 mmol) was added, and the mixture was stirred for 30 minutes. After the reaction was completed, water was added, and the system was extracted with ethyl acetate. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain the crude product. The crude product was purified by column chromatography using System B to obtain 043b (400 mg, 65% yield).

[0482] MS m / z(ESI):724.3(M+1) + .

[0483] Step 2 2,5,8,11,14,17,20,23,26-nonoxa-29-azatrimethylpentane-32-oic acid 043c

[0484] Compound 043b (400 mg, 0.55 mmol) was dissolved in methanol (10 mL) and Pd / C (200 mg) was added. The reaction was stirred at 25°C for 2 hours. After completion of the reaction, the solvent was evaporated to give the crude product 2,5,8,11,14,17,20,23,26-nonoxa-29-azatripentane-32-oic acid 043c (260 mg). The crude product was used directly in the next reaction without purification.

[0485] MS m / z(ESI):500.3(M+1) + .

[0486] Step 3 29-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-2,5,8,11,14,17,20,23,26-nonaoxa-29-azadotriacontane-32-oleic acid 043d

[0487] The crude product 2,5,8,11,14,17,20,23,26-nonoxa-29-azatrimethylpentane-32-oic acid 043c (130 mg, 0.26 mmol) was dissolved in DMF (2 mL), compound Int1 (103 mg, 0.26 mmol) and DIPEA (50 mg, 0.39 mmol) were added, and the reaction was stirred at room temperature for 1 hour. After the reaction, the reaction solution was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge 5u C18100 x 19 mm, 5 μm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 10 min gradient, gradient ratio: acetonitrile phase 27%-37%, flow rate: 20 mL / min) to obtain 29-(6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-2,5,8,11,14,17,20,23,26-nonaoxa-29-azadotriacontane-32-oleic acid 043d (71 mg, yield: 35%).

[0488] MS m / z(ESI):780.4(M+1) + .

[0489] Step 4: N-(3-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizinyl[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)-N-(2,5,8,11,14,17,20,23,26-nonaoxoctan-28-yl)hexane-5-acetamide 043

[0490] Compound 043d (25 mg, 0.032 mmol) was dissolved in N,N-dimethylformamide (1 mL), and 001c (25 mg, 0.044 mmol), HATU (18 mg, 0.048 mmol), and DIPEA (9 mg, 0.64 mmol) were added. The reaction was stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge 5u C18150 x 19 mm, 5 μm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 10-minute gradient, gradient ratio: acetonitrile phase 38%-48%, flow rate: 20 mL / min) to obtain compound 043 (8.7 mg, yield: 20%).

[0491] MS m / z(ESI):668.5(M / 2+1) + .

[0492] 1 H NMR(400MHz,DMSO-d6)δ9.75(d,1H),8.80(d,1H),8.29(dd,1H),8.11–7.98(m,1H),7.97–7.93(m,1H),7.82(d,1H) ,7.30(s,1H),6.50(s,1H),5.43(s,2H),5.26(s,2H),4.57–4.48(m,1H),4.34–4.18(m,1H),4.07(s,3H),3.51–3.4 8(m,30H),3.47–3.45(m,5H),3.43–3.41(m,3H),3.39(s,3H),3.23(s,3H),3.18–3.13(m,2H),3.01–2.94(m,2H),2 .06–1.95(m,4H),1.92–1.83(m,2H),1.82–1.74(m,2H),1.42–1.36(m,3H),1.28–1.21(m,2H),0.91–0.81(m,12H).

[0493] Example 21

[0494] N-(5-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentyl)-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)-N-(2,5,8,11,14,17,20,23,26-nonaoxooctacosan-28-yl)hex-5-ynamide

[0495] Referring to the synthetic route of Example 20, the starting material in the first step was replaced with 044a. The final product was purified using preparative HPLC using a Waters MS-triggered Prep-LC with an SQD2 detector (columns: Xbridge 5µm C18, 150 x 19mm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile 35% to 45%, flow rate: 20 mL / min) to afford compound 044 (20 mg, 35% yield).

[0496] MS m / z(ESI):1364.5(M+1) + .

[0497] 1 H NMR(400MHz,CD3OD)δ9.77(s,1H),8.80(s,1H),8.26(t,1H),8.02–7.93(m,1H),7.91–7.80(m,2H),7.31 (s,1H),6.52(s,1H),5.44(s,2H),5.27(s,2H),4.57–4.49(m,1H),4.27–4.20(m,1H),4.11–4.04(m,3H) ,3.53–3.46(m,32H),3.44–3.41(m,10H),3.25–3.23(m,3H),3.20–3.14(m,2H),3.01–2.95(m,2H),2.27 –2.14(m,2H),2.12–1.93(m,4H),1.91–1.74(m,4H),1.57–1.32(m,8H),1.24(s,1H),0.92–0.84(m,9H).

[0498] Example 22

[0499] N-(4-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl)-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)-N-(2,5,8,11,14,17,20,23,26,29,32,35,38-tridecahydrotetradec-40-yl)hex-5-ynamide

[0500] The first step 2,5,8,11,14,17,20,23,26,29,32,35,38-tridecanoatetradecane-40-yl-4-methylbenzenesulfonate 045b

[0501] 2,5,8,11,14,17,20,23,26,29,32,35,38-Tridecanoatetracontan-40-ol 045a (1 g, 1.7 mmol) was dissolved in dichloromethane (20 mL). p-Toluenesulfonyl chloride (0.65 g, 3.4 mmol), triethylamine (340 mg, 3.4 mmol), and 4-dimethylaminopyridine (1.04 g, 8.5 mmol) were added sequentially. The mixture was stirred at room temperature for 16 hours. After completion of the reaction, the solvent was dried and concentrated under reduced pressure to obtain the crude product, which was then purified on a silica gel column to afford compound 045b (1.2 g, 94% yield).

[0502] MS m / z(ESI):759.3(M+1) + .

[0503] Step 2 Benzyl 2,5,8,11,14,17,20,23,26,29,35,38-tridecahydro-41-nitropentahydro-45-pentadecanoate 045d

[0504] Benzyl 4-aminobutyrate 045c (112 mg, 0.58 mmol) was dissolved in acetonitrile (10 mL), and potassium carbonate (146 mg, 1.05 mmol) and 2,5,8,11,14,17,20,23,26,29,32,35,38-tridecyloxytetracarbon-40-yl-4-methylbenzenesulfonate 045b (400 mg, 0.53 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction, the reaction mixture was used directly in the next step without purification to obtain crude product 045d (411 mg).

[0505] MS m / z(ESI):780.4(M+1) + .

[0506] Step 3 Benzyl 41-((benzyloxy)carbonyl)-2,5,8,11,14,17,20,23,26,29,32,35,38-tridecahydro-41-nitropentadecane-45-ol 045e

[0507] Compound 045d (411 mg, 0.53 mmol) was dissolved in acetonitrile (10 mL), and benzyl chloroformate (108 mg, 0.64 mmol) was added. The mixture was stirred at room temperature for 1 hour. After the reaction, the solvent was dried and concentrated under reduced pressure to give the crude product, Compound 045e (180 mg, 37% yield).

[0508] MS m / z(ESI):931.4(M+18) + .

[0509] Step 4: 2,5,8,11,14,17,20,23,26,29,32,35,38-tridecahydro-41-nitropentahydro-45-carboxylic acid 045f

[0510] Compound 045e (180 mg, 0.2 mmol) was dissolved in methanol (10 mL), and palladium carbon (35 mg, 0.2 mmol) was added thereto. The reaction was stirred at room temperature under a hydrogen atmosphere for 1 hour. After the reaction was completed, compound 045f (120 mg, yield: 88%) was obtained by direct filtration.

[0511] MS m / z(ESI):690.4(M+1) + .

[0512] Step 5: 41-(6-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-2,5,8,11,14,17,20,23,26,29,32,35,38-tridecahydro-41-nitro-pentadecane-45-carboxylic acid 0.45g

[0513] Compound 045f (120 mg, 0.3 mmol) was dissolved in N,N-dimethylformamide (10 mL), and N,N-diisopropylethylamine (78 mg, 0.6 mmol) and 2,5-dioxopyrrolidin-1-yl 6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate Int-1 (230 mg, 0.33 mmol) were added. The reaction was stirred at room temperature for 1 hour. After completion of the reaction, the crude product was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge 5u C18150 x 19 mm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; gradient ratio: acetonitrile phase 30%-40%, flow rate: 20 mL / min) to give compound 045g (60 mg, yield: 20%).

[0514] MS m / z(ESI):970.3(M+1) + .

[0515] Step 6: N-(4-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutyl)-6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)-N-(2,5,8,11,14,17,20,23,26,29,32,35,38-tridecahydrotetradec-40-yl)hex-5-ynamide 045

[0516] Compound 045g (50 mg, 0.05 mmol) was dissolved in N,N-dimethylformamide (10 mL), and compound 001c (32.5 mg, 0.06 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (30 mg, 0.08 mmol), and N,N-diisopropylethylamine (14 mg, 0.1 mmol) were added thereto, and the mixture was stirred at room temperature for 2 hours. After the reaction, the solvent was dried to obtain a crude product, which was then purified by high-performance liquid chromatography (HPLC) (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge 5u C18 150 x 19 mm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; gradient ratio: acetonitrile 37% to 47%, flow rate: 20 mL / min) to obtain compound 045 (25.9 mg, yield: 33%). MS m / z (ESI): 763.8 (M / 2+1). + .

[0517] 1 H NMR (400MHz, CDCl3) δ8.97(s,1H),8.56(s,1H),8.02(s,1H),7.93–7.81(m,2H),7.61(s,1H),6.63(s ,1H),5.69(d,1H),5.30–5.23(m,1H),5.14–5.01(m,2H),4.84–4.76(m,1H),4.28(s,1H),4.17(s,3H) ,3.66–3.62(m,48H),3.39–3.37(m,4H),3.34–3.33(m,3H),3.11–2.94(m,5H),2.66–2.58(m,4H),2. 40–2.29(m,2H),2.14(s,4H),1.98–1.87(m,6H),1.70(s,1H),1.57–1.50(m,3H),1.08–0.94(m,12H).

[0518] Example 23

[0519] N-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-1-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-amide

[0520] Compound 057 was obtained by the same synthetic method as in Example 1.

[0521] MS m / z(ESI):624.3(M / 2+1) + .

[0522] 1H NMR(400MHz,CD3OD)δ8.95(s,1H),8.94(s,1H),7.96(d,1H),7.81(d,1H),7.65(s,1H),5.58(d,1H),5 .38(d,1H),5.33(t,1H),5.25(s,2H),4.64–4.57(m,2H),4.26–4.22(m,1H),3.73(t,3H),3.62–3.58(m ,30H),3.54–3.50(m,2H),3.36–3.33(m,2H),3.22–3.17(m,2H),3.09–3.01(m,2H),2.60–2.53(m,2H), 2.38(t,2H),2.21–2.10(m,2H),2.04–1.99(m,3H),1.98–1.88(m,4H),1.54(d,3H),1.04–0.97(m,9H).

[0523] Referring to the synthesis method of patent WO2020200880A1, the following compound 058 was synthesized:

[0524] Preparation of Antibody Drug Conjugates

[0525] Example A-1 Preparation of ADC-1

[0526] Trastuzumab (16 mg) was placed in a 50 mL centrifuge tube and 50 mM PBS, pH 7.97, was added to a final antibody concentration of 5 mg / mL. 0.2 M EDTA was then added to a final EDTA concentration of 2 mM. 15-fold molarity of TCEP was then added and the mixture was incubated at 37°C for 2 h with continuous mixing. Compound 001 (prepared in DMA) was added under ice at a final drug-to-antibody molar ratio of 15:1. DMA was supplemented to 10% of the total volume of the reaction solution, and the mixture was shaken and incubated at 22°C for 3 h, followed by 18 h at 4°C with continuous mixing. The reaction solution was purified using a Zeba desalting column, and the sample was concentrated using an Amicon buffer solution with a 30 mM His / HAc buffer, pH 5.5, to yield ADC-1 (14 mg). The average DAR value of the ADC was calculated using the HIC method, β = 8.

[0527] Referring to the method of Example A-1, Compound 001 was replaced with the compound in other examples of the present application to prepare the following antibody drug conjugate, whose structure and DAR value (β) are shown in the following table

[0528] The results showed that the cytotoxic drug-linker compounds of the present invention can be successfully coupled with antibodies to obtain antibody-drug conjugates.

[0529] Biological evaluation

[0530] Test Example 1 ADC Bioactivity Detection

[0531] 1. Test Purpose

[0532] The purpose of this experiment is to detect the inhibitory activity of ADC compounds on the proliferation of HER2-expressing NCI-N87 cells, SK-BR-3 cells and HER2-negative MDA-MB-468 cells in vitro. Luminescent Cell Viability Assay was used to detect cell proliferation. 50 The in vitro activity of the compound was evaluated.

[0533] 2. Test Method

[0534] (1) On the first day, tumor cells were plated in a 96-well plate, with 5000 cells / 100 μL culture medium in each well. 100 μL DPBS was inoculated into each empty well at the edge. The plates were incubated in a 37°C incubator overnight.

[0535] (2) On the second day, aspirate the old culture medium (50 μL / well) and add ADC at different concentrations. The starting concentration of ADC is 200 nM, and the ADC is diluted 5-fold to form 9 concentrations. The volume of drug addition is 50 μL / well.

[0536] (3) On the sixth day, thaw CellTiter-Glo Buffer and CellTiter-Glo Substrate at 4°C. Before use, aspirate 10 ml of Buffer and add it to the Substrate, mix well, and equilibrate to room temperature.

[0537] (4) On the seventh day, the 96-well plate was equilibrated at room temperature for 30 minutes, and 100 μL of Cell-Titer-Glo was added to each well. After shaking at room temperature in the dark for 5 minutes, the plate was incubated for 10 minutes. 100 μL of the liquid in each well was transferred to a white plate, and chemiluminescence was detected using a microplate reader.

[0538] 3. Data Analysis

[0539] The data were processed and analyzed using Microsoft Excel and Graphpad Prism 5 to test the inhibitory activity of the ADC compounds on the proliferation of NCI-N87 cells, SK-BR-3 cells, and MDA-MB-468 cells in vitro. The results are shown in Table 1 below.

[0540] Table 1

[0541] Conclusion: The antibody-drug conjugates targeting HER2 of the present invention have significant proliferation inhibitory activity against HER2-positive cells SK-BR-3 and NCI-N87; at the same time, they have weak proliferation inhibitory activity against HER2-negative cells MDA-MB-468 and have good selectivity.

[0542] Test Example 2 ADC plasma stability experiment

[0543] The mice used in this experiment were CD-1 mice, the rats used were SD rats, and the monkeys used were cynomolgus monkeys.

[0544] (1) Free toxin release test and results

[0545] DS8201 sample, ADC-2, ADC-12, and ADC-58 were added to the above-mentioned sterile mouse plasma, sterile rat plasma, sterile human plasma, and sterile monkey plasma, respectively, at a final concentration of 200 μg / mL and incubated in a 37°C cell culture incubator. The day of incubation was recorded as day 0, and samples were subsequently taken out on days 1, 4, 7, 14, and 21 for testing of free toxin content.

[0546] The free toxin release rates are shown in Figures 1 to 3 and 14 . The results indicate that ADC-2 and ADC-12 are quite stable in mouse, rat, human, and monkey plasma, with the maximum free toxin release rate not exceeding 0.2%, and are significantly better than the reference DS8201 and ADC-58.

[0547] (2)ADC DAR value test and results

[0548] DS8201 sample, ADC-2, ADC-12, and ADC-58 were added to the above-mentioned sterile human plasma at a final concentration of 200 μg / mL and incubated in a 37°C cell culture incubator. The day of incubation was recorded as day 0. Samples were then taken on days 1, 4, 7, 14, and 21 to detect changes in DAR values.

[0549] The experimental results of ADC DAR value changes are shown in Table 2. The results indicate that the change in DAR value of the conjugate formed by the small molecule linker of the present invention in human plasma is significantly smaller than that of DS8201 and ADC-58, showing better plasma stability, further confirming the stability of the small molecule linker of the present invention.

[0550] Table 2: ADC plasma stability (DAR value change)

[0551] Test Example 3: Efficacy Evaluation in NCI-N87 Tumor-Bearing Mice

[0552] 3.1 Purpose of the test

[0553] Balb / c nude mice were used as test animals to investigate whether tumor growth was inhibited, delayed or cured, and to evaluate the efficacy of the ADC in this application.

[0554] 3.2 Experimental steps

[0555] 3.2.1 Test drugs

[0556] Blank control / or vehicle control (Vehicle): PBS

[0557] Reference ADC (DS8201): 3 mg / kg

[0558] ADC-2: 3 mg / kg

[0559] ADC-12: 3 mg / kg

[0560] ADC-43: 1 mg / kg

[0561] 3.2.2 Preparation method: All preparations were diluted with PBS.

[0562] 3.2.3 Test methods

[0563] Mice were subcutaneously inoculated with NCI-N87 cells in the right flank. After 7 days of tumor growth, the animals were randomly divided into 5 groups (6 animals / group) (4 treatment groups + 1 blank control group). Drugs were administered via tail vein injection once. Tumor volume (diameter) and body weight were measured twice weekly for 4 weeks, and data were recorded. Data were analyzed using Excel 2023 statistical software: mean was calculated as avg; SD was calculated as STDEV; SEM was calculated as STDEV / SQRT; P values ​​for group differences were calculated using TTEST.

[0564] The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0565] The tumor inhibition efficacy of the compound was evaluated by TGI (%), and the tumor growth inhibition rate was calculated using the following formula:

[0566] TGI (%) = [1-(Ti-T0) / (Ci-C0)] × 100%, where Ti is the average tumor volume of a dosing group on a certain day, T0 is the average tumor volume of this dosing group at the start of dosing; Ci is the average tumor volume of the vehicle control group on a certain day (the same day as Ti), and C0 is the average tumor volume of the vehicle control group at the start of dosing.

[0567] 3.3 Experimental results and conclusions

[0568] A schematic diagram of the efficacy evaluation in NCI-N87 tumor-bearing mice is shown in Figure 4 , and the in vivo tumor inhibition effect (TGI) of the test drugs in the treatment group on the NCI-N87 transplant model is shown in Table 3 . The results show that the ADC molecules of this application can significantly reduce tumor volume and have a better tumor inhibition effect than the reference ADC (DS8201).

[0569] Table 3: In vivo tumor inhibition effect of ADC on NCI-N87 transplant model

[0570] Test Example 4: Efficacy Evaluation in JIMT-1 Tumor-Bearing Mice

[0571] 4.1 Test Purpose

[0572] SCID Beige mice were used as test animals to investigate whether tumor growth was inhibited, delayed or cured, and to evaluate the efficacy of the ADC in this application.

[0573] 4.2 Experimental steps

[0574] 4.2.1 Test Drugs

[0575] Blank control / or vehicle control (Vehicle): PBS

[0576] Reference ADC (DS8201): 1mg / kg

[0577] ADC-2: 1 mg / kg

[0578] ADC-5: 1 mg / kg

[0579] ADC-8: 1 mg / kg

[0580] ADC-11: 1 mg / kg

[0581] ADC-12: 1 mg / kg

[0582] 4.2.2 Preparation method: All preparations were diluted with PBS.

[0583] 4.2.3 Test methods

[0584] Mice were inoculated subcutaneously with JIMT-1 cells in the right flank. After 8 days of tumor growth, the animals were randomly divided into 7 groups (6 treatment groups + 1 blank control group). Drugs were administered via tail vein injection once. Tumor volume (diameter) and body weight were measured twice weekly for 4 weeks, and data were recorded. Data were analyzed using Excel 2023 statistical software: mean was calculated as avg; SD was calculated as STDEV; SEM was calculated as STDEV / SQRT; P values ​​for group differences were calculated using TTEST.

[0585] The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0586] The tumor inhibition efficacy of the compound was evaluated by TGI (%), and the tumor growth inhibition rate was calculated using the following formula:

[0587] TGI (%) = [1-(Ti-T0) / (Ci-C0)] × 100%, where Ti is the average tumor volume of a dosing group on a certain day, T0 is the average tumor volume of this dosing group at the start of dosing; Ci is the average tumor volume of the vehicle control group on a certain day (the same day as Ti), and C0 is the average tumor volume of the vehicle control group at the start of dosing.

[0588] 4.3 Experimental Results and Conclusions

[0589] The in vivo tumor suppression effects of the treatment groups in the JIMT-1 transplant model are shown in Table 4, and a schematic diagram of the efficacy evaluation in JIMT-1 tumor-bearing mice is shown in Figure 5. The results demonstrate that the ADC molecules of this application significantly reduced tumor volume and exhibited superior tumor suppression efficacy compared to the reference ADC (DS8201).

[0590] Table 4: In vivo tumor inhibition effect of ADC on JIMT-1 transplant model

[0591] Test Example 5: Efficacy Evaluation in RT11284 Tumor-Bearing Mice

[0592] 5.1 Test Purpose

[0593] BALB / c Nude mice were used as test animals to investigate whether tumor growth was inhibited, delayed or cured, and to evaluate the efficacy of the ADC in this application.

[0594] 5.2 Experimental steps

[0595] 5.2.1 Test Drugs

[0596] Blank control / or vehicle control (Vehicle): PBS

[0597] Reference ADC (DS8201): 3 mg / kg

[0598] Reference ADC (DS8201): 10mg / kg

[0599] ADC-2: 3 mg / kg

[0600] ADC-2: 10 mg / kg

[0601] ADC-12: 3 mg / kg

[0602] ADC-42: 3 mg / kg

[0603] ADC-43: 3 mg / kg

[0604] 5.2.2 Preparation method: dilute with PBS.

[0605] 5.2.3 Test method

[0606] Mice were inoculated subcutaneously with RT11284 cells in the right flank. After 10 days of tumor growth, the animals were randomly divided into 8 groups (7 treatment groups + 1 blank control group), 6 animals per group. Drugs were administered via tail vein injection once. Tumor volume (diameter) and body weight were measured twice weekly for 4 weeks, and data were recorded. Data were analyzed using Excel 2023 statistical software: mean was calculated as avg; SD was calculated as STDEV; SEM was calculated as STDEV / SQRT; P values ​​for group differences were calculated using TTEST.

[0607] The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0608] The tumor inhibition efficacy of the compound was evaluated by TGI (%), and the tumor growth inhibition rate was calculated using the following formula:

[0609] TGI (%) = [1-(Ti-T0) / (Ci-C0)] × 100%, where Ti is the average tumor volume of a dosing group on a certain day, T0 is the average tumor volume of this dosing group at the start of dosing; Ci is the average tumor volume of the vehicle control group on a certain day (the same day as Ti), and C0 is the average tumor volume of the vehicle control group at the start of dosing.

[0610] 5.3 Experimental Results and Conclusions

[0611] A schematic diagram of the efficacy evaluation in RT11284 tumor-bearing mice is shown in Figure 6 , and the in vivo tumor inhibition effects of the test drugs in the treatment group on the RT11284 xenograft model are shown in Table 5 . The results demonstrate that the ADC molecules of this application can significantly reduce tumor volume and exhibit superior tumor inhibition effects compared to the reference ADC (DS8201).

[0612] Table 5: In vivo tumor inhibition effect of ADC on RT11284 transplant model

[0613] Test Example 6: Efficacy Evaluation in HCT116 Tumor-Bearing Mice

[0614] 6.1 Test Purpose

[0615] BALB / c Nude mice were used as test animals to investigate whether tumor growth was inhibited, delayed or cured, and to evaluate the efficacy of the ADC in this application.

[0616] 6.2 Experimental steps

[0617] 6.2.1 Test Drugs

[0618] Blank control / or vehicle control (Vehicle): PBS

[0619] Reference ADC (DS8201): 3 mg / kg

[0620] Reference ADC (DS8201): 10mg / kg

[0621] ADC-43: 3 mg / kg

[0622] ADC-43: 10 mg / kg

[0623] ADC-2: 3 mg / kg

[0624] ADC-2: 10 mg / kg

[0625] 6.2.2 Preparation method: dilute with PBS.

[0626] 6.2.3 Test method

[0627] HCT116 cells were inoculated subcutaneously in the right flank of mice. After 14 days of tumor growth, the animals were randomly divided into 7 groups (6 treatment groups + 1 blank control group). Drugs were administered via tail vein injection once. Tumor volume (diameter) and body weight were measured twice weekly for 4 weeks, and data were recorded. Data were analyzed using Excel 2023 statistical software: mean was calculated as avg; SD was calculated as STDEV; SEM was calculated as STDEV / SQRT; P values ​​for group differences were calculated using TTEST.

[0628] The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0629] The tumor inhibition efficacy of the compound was evaluated by TGI (%), and the tumor growth inhibition rate was calculated using the following formula:

[0630] TGI (%) = [1-(Ti-T0) / (Ci-C0)] × 100%, where Ti is the average tumor volume of a dosing group on a certain day, T0 is the average tumor volume of this dosing group at the start of dosing; Ci is the average tumor volume of the vehicle control group on a certain day (the same day as Ti), and C0 is the average tumor volume of the vehicle control group at the start of dosing.

[0631] 6.3 Experimental Results and Conclusions

[0632] A schematic diagram of the efficacy evaluation in HCT116 tumor-bearing mice is shown in Figure 7 , and the in vivo tumor inhibitory effects of the test drugs in the treatment group on the HCT116 transplant model are shown in Table 6 . The results show that the ADC molecules of the present application can significantly reduce tumor volume and have a better tumor inhibitory effect than the reference ADC (DS8201).

[0633] Table 6: In vivo tumor inhibition effect of ADC on HCT116 transplant model

[0634] Test Example 7: Efficacy Evaluation in Capan-1 Tumor-Bearing Mice

[0635] 7.1 Test Purpose

[0636] SCID Beige mice were used as test animals to investigate whether tumor growth was inhibited, delayed or cured, and to evaluate the efficacy of the ADC in this application.

[0637] 7.2 Experimental Procedure

[0638] 7.2.1 Test Drugs

[0639] Blank control / or vehicle control (Vehicle): PBS

[0640] ADC (DS8201): 3 mg / kg

[0641] ADC (DS8201): 10 mg / kg

[0642] ADC-2: 3 mg / kg

[0643] ADC-2: 10 mg / kg

[0644] ADC-12: 3 mg / kg

[0645] ADC-12: 10 mg / kg

[0646] ADC-42: 3 mg / kg

[0647] ADC-43: 3 mg / kg

[0648] 7.2.2 Preparation method: dilute with PBS.

[0649] 7.2.3 Test method

[0650] Capan-1 cells were inoculated subcutaneously in the right flank of mice. After 8 days of tumor growth, the animals were randomly divided into 9 groups (8 treatment groups + 1 blank control group), with 6 animals per group. Drugs were administered via tail vein injection once. Tumor volume (diameter) and body weight were measured twice weekly for 4 weeks, and data were recorded. Data were analyzed using Excel 2023 statistical software: mean was calculated as avg; SD was calculated as STDEV; SEM was calculated as STDEV / SQRT; P values ​​for group differences were calculated using TTEST.

[0651] The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0652] The tumor inhibition efficacy of the compound was evaluated by TGI (%), and the tumor growth inhibition rate was calculated using the following formula:

[0653] TGI (%) = [1-(Ti-T0) / (Ci-C0)] × 100%, where Ti is the average tumor volume of a dosing group on a certain day, T0 is the average tumor volume of this dosing group at the start of dosing; Ci is the average tumor volume of the vehicle control group on a certain day (the same day as Ti), and C0 is the average tumor volume of the vehicle control group at the start of dosing.

[0654] 7.3 Experimental Results and Conclusions

[0655] A schematic diagram of drug efficacy evaluation in Capan-1 tumor-bearing mice is shown in Figure 8 , and the in vivo tumor inhibition effect (TGI) of the test drugs in the treatment group on the Capan-1 xenograft model is shown in Table 7 . The results demonstrate that the ADC molecules of this application can significantly reduce tumor volume and exhibit superior tumor inhibition compared to the reference ADC (DS8201).

[0656] Table 7: In vivo tumor inhibition effect of ADC on Capan-1 transplant model

[0657] Test Example 8: Efficacy evaluation of NCI-N87-Enhertu in mice bearing drug-resistant cells

[0658] 8.1 Test Purpose

[0659] NOD SCID mice were used as test animals to investigate whether tumor growth was inhibited, delayed or cured, and to evaluate the efficacy of the ADC in this application.

[0660] 8.2 Experimental Procedure

[0661] 8.2.1 Test Drugs

[0662] Blank control / or vehicle control (Vehicle): PBS

[0663] Reference ADC (DS8201): 3 mg / kg

[0664] Reference ADC (DS8201): 10mg / kg

[0665] ADC-2: 3 mg / kg

[0666] ADC-2: 10 mg / kg

[0667] ADC-43: 3 mg / kg

[0668] 8.2.2 Preparation method: dilute with PBS.

[0669] 8.2.3 Test method

[0670] Mice were subcutaneously inoculated with NCI-N87-Enhertu-resistant cells in the right flank. After 12 days of tumor growth, the animals were randomly divided into 6 groups (5 treatment groups + 1 blank control group). The drug was administered via tail vein injection once. Tumor volume (diameter) and body weight were measured twice weekly for four weeks, and the data were recorded. Data were statistically analyzed using Excel 2023 statistical software: mean was calculated as avg; SD was calculated as STDEV; SEM was calculated as STDEV / SQRT; P values ​​for group differences were calculated using TTEST.

[0671] The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0672] The tumor inhibition efficacy of the compound was evaluated by TGI (%), and the tumor growth inhibition rate was calculated using the following formula:

[0673] TGI (%) = [1-(Ti-T0) / (Ci-C0)] × 100%, where Ti is the average tumor volume of a dosing group on a certain day, T0 is the average tumor volume of this dosing group at the start of dosing; Ci is the average tumor volume of the vehicle control group on a certain day (the same day as Ti), and C0 is the average tumor volume of the vehicle control group at the start of dosing.

[0674] 8.3 Experimental Results and Conclusions

[0675] A schematic diagram of the efficacy evaluation in mice bearing NCI-N87-Enhertu-resistant cells is shown in Figure 9, and the in vivo tumor inhibition effect (TGI) of the test drugs in the treatment group on the NCI-N87-Enhertu-resistant cell transplant model is shown in Table 8. The results show that the ADC molecules of this application can significantly reduce tumor volume and have a better tumor inhibition effect than the reference ADC (DS8201).

[0676] Table 8: In vivo tumor inhibition effect of ADC on NCI-N87-Enhertu-resistant cell transplant model

[0677] Test Example 9: Efficacy Evaluation in Human Gastric Cancer LD1-0017-411335PDX Tumor-Bearing Mice

[0678] 9.1 Test Purpose

[0679] NU / NU mice were used as test animals to investigate whether tumor growth was inhibited, delayed or cured, and to evaluate the efficacy of the ADC in this application.

[0680] 9.2 Experimental Procedure

[0681] 9.2.1 Test Drugs

[0682] Blank control / or vehicle control (Vehicle): PBS

[0683] Reference ADC (DS8201): 10mg / kg

[0684] ADC-2: 10 mg / kg

[0685] 9.2.2 Preparation method: dilute with PBS.

[0686] 9.2.3 Test method

[0687] LD1-0017-411335 human gastric cancer tumor tissue was uniformly cut into tumor pieces approximately 3 mm × 3 mm × 3 mm (approximately 30-60 mg) and inoculated subcutaneously on the right flank of NU / NU mice. The mice were subsequently observed and their weight changes and tumor growth were monitored. When the average tumor volume of the grouped mice reached 140.81 mm 3 At 4 hr, the animals were randomly divided into 3 groups (2 treatment groups + 1 blank control group) according to tumor size, with 6 animals in each group. The day of grouping was designated as day 0, and the drugs were injected into the tail vein once.

[0688] Tumor volume and body weight were measured twice weekly for four weeks and the data were recorded. Data were analyzed using Excel 2023 statistical software: mean values ​​were calculated as avg; SD values ​​were calculated as STDEV; SEM values ​​were calculated as STDEV / SQRT; and P values ​​for intergroup differences were calculated using TTEST.

[0689] The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0690] The tumor inhibition efficacy of the compound was evaluated by TGI (%), and the tumor growth inhibition rate was calculated using the following formula:

[0691] TGI (%) = [1-(Ti-T0) / (Ci-C0)] × 100%, where Ti is the average tumor volume of a dosing group on a certain day, T0 is the average tumor volume of this dosing group at the start of dosing; Ci is the average tumor volume of the vehicle control group on a certain day (the same day as Ti), and C0 is the average tumor volume of the vehicle control group at the start of dosing.

[0692] 9.3 Experimental Results and Conclusions

[0693] A schematic diagram of the drug efficacy evaluation in mice bearing human gastric cancer LD1-0017-411335PDX tumors is shown in Figure 10 , and the in vivo tumor inhibition effect (TGI) of the test drugs in the treatment group on the human gastric cancer LD1-0017-411335PDX model is shown in Table 9 . The results demonstrate that the ADC-2 molecule of this application can significantly reduce tumor volume and has a superior tumor inhibition effect compared to the reference ADC.

[0694] Table 9: In vivo tumor inhibition effect of ADC on human gastric cancer LD1-0017-411335 PDX model

[0695] Test Example 10: Pharmacokinetic Evaluation in Mice

[0696] 10.1 Test Purpose

[0697] C57 mice were used as test animals to evaluate the in vivo pharmacokinetic properties of the ADC in this application.

[0698] 10.2 Experimental Procedures 10.2.1 Test Drugs

[0699] ADC-2: 10 mg / kg

[0700] 10.2.2 Preparation method: dilute with PBS.

[0701] 10.2.3 Test method

[0702] For each test drug, 15 C57 mice were administered a tail vein bolus injection of ADC (10 mg / kg) for approximately 1 minute ± 10 seconds, with a dosing volume of 5 mL / kg. Following dosing, blood was collected at 0.083 hours, 2 hours, 8 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours, 336 hours, 504 hours, and 672 hours, and serum was separated by centrifugation within 30-120 minutes. Free toxin molecules in blood samples were detected by conventional LC-MS, and total antibody and ADC concentrations in blood samples were determined by conventional ELISA.

[0703] The PK curve of ADC-2 in mice is shown in Figure 11, and the pharmacokinetic data are shown in Table 10. No free toxin small molecules were detected at any time point, indicating that the ADC of the present application has good stability in vivo and has good in vivo pharmacokinetic properties.

[0704] Table 10: Pharmacokinetic data of ADC-2 in mice

[0705] Test Example 11: Efficacy Evaluation in JIMT-1 Tumor-Bearing Mice

[0706] 11.1 Test Purpose

[0707] SCID Beige mice were used as test animals to investigate whether tumor growth was inhibited, delayed or cured, and to evaluate the efficacy of the ADC in this application.

[0708] 11.2 Experimental Procedures 11.2.1 Test Drugs

[0709] Blank control / or vehicle control (Vehicle): PBS

[0710] ADC-2: 1 mg / kg

[0711] ADC-58: 1 mg / kg

[0712] 11.2.2 Preparation method: dilute with PBS.

[0713] 11.2.3 Test method

[0714] JIMT-1 cells were inoculated subcutaneously in the right flank of mice. After 8 days of tumor growth, the animals were randomly divided into 3 groups (6 animals / group) (2 treatment groups + 1 blank control group). Drugs were administered via tail vein injection once. Tumor volume (diameter) and body weight were measured twice weekly for 4 weeks, and data were recorded. Data were analyzed using Excel 2023 statistical software: mean was calculated as avg; SD was calculated as STDEV; SEM was calculated as STDEV / SQRT; P values ​​for group differences were calculated using TTEST.

[0715] The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0716] The tumor inhibition efficacy of the compound was evaluated by TGI (%), and the tumor growth inhibition rate was calculated using the following formula:

[0717] TGI (%) = [1-(Ti-T0) / (Ci-C0)] × 100%, where Ti is the average tumor volume of a dosing group on a certain day, T0 is the average tumor volume of this dosing group at the start of dosing; Ci is the average tumor volume of the vehicle control group on a certain day (the same day as Ti), and C0 is the average tumor volume of the vehicle control group at the start of dosing.

[0718] 11.3 Experimental Results and Conclusions

[0719] The in vivo tumor inhibition effect of the treatment group on the JIMT-1 transplant model is shown in Table 11, and a schematic diagram of the efficacy evaluation in JIMT-1 tumor-bearing mice is shown in Figure 12. The results show that compared with the control, the ADC molecule of the present application can more significantly reduce the tumor volume.

[0720] Table 11: In vivo tumor inhibition effect of ADC on JIMT-1 transplant model

[0721] Test Example 12: Efficacy Evaluation in NCI-N87 Tumor-Bearing Mice

[0722] 12.1 Test Purpose

[0723] Balb / c nude mice were used as test animals to investigate whether tumor growth was inhibited, delayed or cured, and to evaluate the efficacy of the ADC in this application.

[0724] 12.2 Experimental Procedures 12.2.1 Test Drugs

[0725] Blank control / or vehicle control (Vehicle): PBS

[0726] ADC-2: 1 mg / kg

[0727] ADC-58: 1 mg / kg

[0728] 12.2.2 Preparation method: dilute with PBS.

[0729] 12.2.3 Test method

[0730] Mice were subcutaneously inoculated with NCI-N87 cells in the right flank. After 7 days of tumor growth, the animals were randomly divided into 3 groups (6 animals / group) (2 treatment groups + 1 blank control group). Drugs were administered via tail vein injection once. Tumor volume (diameter) and body weight were measured twice weekly for 4 weeks, and data were recorded. Data were analyzed using Excel 2023 statistical software: mean was calculated as avg; SD was calculated as STDEV; SEM was calculated as STDEV / SQRT; P values ​​for group differences were calculated using TTEST.

[0731] The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0732] The tumor inhibition efficacy of the compound was evaluated by TGI (%), and the tumor growth inhibition rate was calculated using the following formula:

[0733] TGI (%) = [1-(Ti-T0) / (Ci-C0)] × 100%, where Ti is the average tumor volume of a dosing group on a certain day, T0 is the average tumor volume of this dosing group at the start of dosing; Ci is the average tumor volume of the vehicle control group on a certain day (the same day as Ti), and C0 is the average tumor volume of the vehicle control group at the start of dosing.

[0734] 12.3 Experimental Results and Conclusions

[0735] A schematic diagram of the efficacy evaluation in NCI-N87 tumor-bearing mice is shown in Figure 13, and the in vivo tumor inhibition effect (TGI) of the test drugs in the treatment group on the NCI-N87 transplant model is shown in Table 12. The results show that compared with the control, the ADC molecules of the present application can more significantly reduce tumor volume.

[0736] Table 12: In vivo tumor inhibition effect of ADC on NCI-N87 transplant model

[0737] Test Example 13: Efficacy Evaluation in 22RV1 Tumor-Bearing Mice

[0738] 13.1 Test Purpose

[0739] Balb / c nude mice were used as test animals to investigate whether tumor growth was inhibited, delayed or cured, and to evaluate the efficacy of the ADC in this application.

[0740] 13.2 Experimental Procedures 13.2.1 Test Drugs

[0741] Blank control / or vehicle control (Vehicle): PBS

[0742] ADC-2: 5 mg / kg

[0743] ADC-57: 5 mg / kg

[0744] 13.2.2 Preparation method: dilute with PBS.

[0745] 13.2.3 Test method

[0746] Mice were inoculated subcutaneously with 22RV1 cells in the right flank. After 7 days of tumor growth, the animals were randomly divided into 3 groups (6 animals / group) (2 treatment groups + 1 blank control group). The drug was administered via tail vein injection once. Tumor volume (diameter) and body weight were measured twice weekly for 3 weeks, and the data were recorded. Data were analyzed using Excel 2023 statistical software: mean was calculated as avg; SD was calculated as STDEV; SEM was calculated as STDEV / SQRT; P values ​​for group differences were calculated using TTEST.

[0747] The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0748] The tumor inhibition efficacy of the compound was evaluated by TGI (%), and the tumor growth inhibition rate was calculated using the following formula:

[0749] TGI (%) = [1-(Ti-T0) / (Ci-C0)] × 100%, where Ti is the average tumor volume of a dosing group on a certain day, T0 is the average tumor volume of this dosing group at the start of dosing; Ci is the average tumor volume of the vehicle control group on a certain day (the same day as Ti), and C0 is the average tumor volume of the vehicle control group at the start of dosing.

[0750] 13.3 Experimental Results and Conclusions

[0751] A schematic diagram of the efficacy evaluation in 22RV1 tumor-bearing mice is shown in FIG15 , and the in vivo tumor inhibition effect (TGI) of the test drugs in the treatment group on the 22RV1 transplant model is shown in Table 13 . The results show that ADC-2 of the present application is more effective than ADC-57.

[0752] Table 13: In vivo tumor inhibition effect of ADC on 22RV1 transplant model

[0753] Test Example 14: Stability test of compound under antibody coupling conditions

[0754] 14.1 Test Purpose

[0755] The compounds were incubated in coupling buffer to evaluate their stability under coupling conditions and temperature.

[0756] 14.2 Test steps

[0757] Compounds 002 and 057 (2.1 mg, dissolved in 0.1 mL of DMA) were added to 3.2 mL of 50 mM PBS, pH 7.97, 0.1 mL of 0.1 M EDTA, and 0.33 mL of DMA. The mixture was incubated at 22°C for 6 hours, then at 4°C for 18 hours. Samples were taken at 1 hour, 2 hours, 3 hours, 6 hours, and 24 hours, and the purity of compounds 002 and 057 was determined by HPLC. The results are shown in Table 14. Compound 002 exhibited superior stability in buffer to 057, providing advantages in production process and quality control.

[0758] Table 14: Stability test results of compounds in coupling buffer

[0759] Test Example 15: ADC Thermal Stability Test

[0760] 15.1 Test Purpose

[0761] The thermal stability of ADCs coupled with different linker payloads was tested to evaluate the effects of different linker payloads on the thermal stability of ADCs.

[0762] 15.2 Test Compounds

[0763] ADC-2: Preparation buffer 20mM Histidine-HCl, 8% Sucrose, pH 5.5

[0764] ADC-57: Preparation buffer 20mM Histidine-HCl, 8% Sucrose, pH 5.5

[0765] 15.3 Test steps

[0766] Using Protein Thermal Shift Dye Kie TMThe thermal stability of proteins was detected using the Protein Thermal Shift Kit (Cat. No. 4461146). TM Dye was diluted 8-fold and the following 20 μl detection system was prepared: 8× dye 2.5 μl / well, Protein Thermal Shift TM Buffer (5 μl / well) and sample (12.5 μl / well) were added. Mix thoroughly, incubate at 4000 rpm for 1 minute, and centrifuge immediately. PCR was then performed using a real-time fluorescence quantitative PCR instrument (Thermo ABI7500) using Real-Time PCR Software v2.4. The results are shown in Table 15. These results demonstrate that ADC-2 exhibits superior thermal stability compared to ADC-57.

[0767] Table 15: ADC thermal stability test

[0768] The above is an exemplary description of the implementation methods of the technical solution of the present invention. It should be understood that the scope of protection of the present invention is not limited to the above implementation methods. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present invention shall be included in the scope of protection of the claims of this application.

Claims

1. A compound represented by formula (Ia) or (Ib), its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or prodrug thereof: M-Z1-Tr1-L1-D(Ia) or M-Z1-L2(Tr2)-D(Ib) Wherein, M is a linker site with an antibody or antigen-binding fragment thereof; preferably, M comprises a sulfhydryl reactive group, an amino reactive group, a carboxyl reactive group, a proline residue reactive group, a tyrosine residue reactive group, a disulfide bond bridging group, etc.; for antibodies introduced with non-natural amino acids, it may also comprise a bioorthogonal reactive group; preferably, a sulfhydryl reactive group, such as a methylsulfone pyrimidine group, a methylsulfone pyridine group, or a maleimide group, wherein the pyrimidine group, pyridine group, or maleimide group may be optionally substituted with one or more substituents selected from the following: halogen, CN, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylene-OC 1-6 Alkyl, -O-(CH2CH2O) n1 -CH3; n1 is an integer selected from 1-36; Z1 is a chemical bond, a divalent group or a trivalent group substituted by a hydrophilic group, such as NH or -(CH2) q CONH-; q is an integer selected from 0 to 6; Tr1 is a bivalent trigger group, preferably a peptide residue or a modified peptide residue, wherein the peptide residue or the modified peptide residue comprises an optionally substituted natural or unnatural amino acid, L or D amino acid; selected from glycine-glycine-phenylalanine-glycine (GGFG), glutamic acid-valine-citrulline (EVC), valine-citrulline (VC), valine-alanine (VA), aspartic acid-valine-citrulline (DVC), glutamic acid-glycine-glycine-phenylalanine-glycine (EGGFG), aspartic acid-glycine-glycine-phenylalanine-glycine (DGGFG), lysine-glycine-glycine-phenylalanine-glycine (KGGFG); L1 is the connecting part between Tr1 and the biologically active molecular structure fragment D; preferably a chemical bond, -NH-C 1-6 Alkyl-, -N(CH3)-C 1-6 alkyl-, L1 is further preferably -NH-CH2- or D is selected from the structural fragments of biologically active molecules; L2 is a trivalent linking portion between Z1 or M, Tr2 and the biologically active molecular structure fragment D; Tr2 is a monovalent trigger group, preferably a peptide residue, β-glucuronide or β-galactoside group; In formula (Ia) or formula (Ib), M or Z1 has at least one monovalent hydrophilic group Hp1 substituent; and / or Z1 has at least one divalent hydrophilic group Hp2 inserted or replaced, and the monovalent hydrophilic group or divalent hydrophilic group is connected to the other parts of formula (Ia) or formula (Ib) through any chemical bond or linking group: The monovalent hydrophilic group Hp1 is selected from: (1) a monovalent polyethylene glycol group, preferably: -(CH2CH2O)n1-T1, -(CH2CH2O(CH2)m1CONH)n1-T1 or a cyclic group containing a -(CH2CH2O)- fragment, wherein the cyclic group optionally contains 1-3 heteroatoms such as N; (2) Monovalent polysarcosine: -(N(CH3)CH2CO)n2-T2; (3) Monovalent polybetaine: -(CH2CH2N(CH3)(CH2COOH))n3-T3 or -(CH2CH2N(CH3)(CH2SO3H))n3-T3; (4) Groups containing carboxyl groups: -(CH2)m2-X-(CH2)m3-COOH; (5) The combination of the above (1) to (4) with the following divalent hydrophilic groups -Hp2-Hp1 is also regarded as a monovalent hydrophilic group in the present invention; The divalent hydrophilic group Hp2 is selected from the divalent group derived from the monovalent hydrophilic group after removing the end-capping group or the n4, n5 or n6 repeating units of -K(Hp1)-, preferably: (1) A divalent polyethylene glycol group, preferably: -(CH2CH2O)n1-, -(CH2CH2O(CH2)m1CONH)n1-, -[K-(CH2CH2O)n1-T1]n4-, -[K-(CH2CH2O(CH2)m1CONH)n1-T1]n4- or a cyclic group containing a -(CH2CH2O)- fragment, wherein the cyclic group optionally contains 1-3 heteroatoms such as N, such as: wherein n1′ and n1″ are any integers from 1 to 36, and q1 and q2 are integers from 1 to 10; (2) Divalent polysarcosine: -(N(CH3)CH2CO)n2- or -[K-(N(CH3)CH2CO)n2-T2]n5- (3) Divalent polybetaine: -(CH2CH2N(CH3)(CH2COOH))n3- or -[K-(CH2)m4N(CH3)2CH2COOH]n6-, or -(CH2CH2N(CH3)(CH2SO3H))n3- or -[K-(CH2)m4N(CH3)2CH2SO3H]n6-; (4) any combination of (1) to (3) above; in: n1-n6 are integers from 1 to 36; m1-m5 are integers from 1 to 10; X is O, S or NH; K is any trivalent group, preferably a trivalent group derived from an amino acid; T1 or T2 or T3 is selected from any end-capping group, preferably H, OH, C 1-6 Alkyl, C 1-6 Alkoxy, carboxyl, -(CH2)m 5- CONH-(CH2)m4N(CH3)2CH2COOH, -(CH2)m4N(CH3)2CH2COOH, -C 1-6 Alkylene-COOH, -N(C 1-6 Alkyl)2, or a hydrophilic group selected from a monosaccharide, a disaccharide or an oligosaccharide, or a hydrophilic group selected from a hydrophilic group comprising a plurality of (two or more) carboxyl groups, a plurality of (two or more) sulfonic acid groups or a chelate group; Preferably, T1 or T2 or T3 is selected from H, OH, methyl, carboxyl, -CH2CH2COOH, -N(CH2)2, T1, T2 or T3 may be linked to the hydrophilic group via any linking group.

2. The compound of formula (Ia) or (Ib) according to claim 1, its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or prodrug compound, wherein Hp1 is: -(CH2CH2O)n1-H、-(CH2CH2O)n1-CH3、 -(CH2)m2-X-(CH2)m3-COOH; Preferably, the divalent hydrophilic group Hp2 is selected from: -(CH2CH2O)n1-、 3. A compound represented by formula (Ia) or (Ib) according to claim 1 or 2, its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or prodrug compound, wherein the formula (Ia) or formula (Ib) has the following structure represented by (Ia-1)-(Ia-6) or (Ib-1)-(Ib-6): M(Hp1)-Z1-Tr1-L1-D(Ia-1) M-Z1-Hp2-Z2-Tr1-L1-D(Ia-2) M-Z1(Hp1)-Z2-Tr1-L1-D(Ia-3) M(Hp1)-Z1-Hp2-Z2-Tr1-L1-D(Ia-4) M-Z1(Hp1)-Hp2-Z2-Tr1-L1-D(Ia-5) M(Hp1)-Z1(Hp1)-Hp2-Z2-Tr1-L1-D(Ia-6) M(Hp1)-Z1-L2(Tr2)-D(Ib-1) M-Z1-Hp2-Z2-L2(Tr2)-D(Ib-2) M-Z1(Hp1)-Z2-L2(Tr2)-D(Ib-3) M(Hp1)-Z1-Hp2-Z2-L2(Tr2)-D(Ib-4) M-Z1(Hp1)-Hp2-Z2-L2(Tr2)-D(Ib-5) M(Hp1)-Z1(Hp1)-Hp2-Z2-L2(Tr2)-D(Ib-6); Wherein Z2 is a chemical bond or a divalent group, and Z1 is a divalent group or a trivalent group substituted by Hp1.

4. The compound of formula (Ia) or (Ib) according to any one of claims 1 to 3, its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or prodrug compound, wherein M is selected from the following structures: Lg does not exist or is a leaving group selected from halogen, sulfone, trifluoromethanesulfonyl, and methanesulfonyl; Ring B is selected from a 5-14 membered heteroaryl ring and a 3-14 membered heterocyclic ring; Each R b The same or different, independently selected from the following groups: halogen, cyano, oxo (=O), C 1-6 Alkyl, halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, 3-8 membered heterocyclic group, C 1-6 Alkyl-OC 1-6 Alkyl-, C 1-6 Alkyl-(5-6 membered) heteroaryl- or a monovalent hydrophilic group; r is an integer from 0 to 4; L m1 Not present, or selected from unsubstituted or optionally substituted by one, two or more R m1 Substituted with the following groups: C 6-14 Aryl, 5-14 membered heteroaryl, 3-14 membered heterocyclic group; each R m1 the same or different, independently selected from H, halogen, cyano, C 1-6 Alkyl or HOOC-C 1-3 alkylene; L m2 is selected from unsubstituted or optionally substituted with one, two or more R m2 Substituted with the following groups: -(CH2) s -(C=O)-, -C≡C-(CH2) t -(C=O)-; each R m2 the same or different, independently selected from H, halogen, cyano, C 1-6 Alkyl or -C 1-6 Alkylene-COOH, wherein the alkylene is optionally interrupted by one, two or more of the following groups: O, NH; s and t are the same or different and are independently selected from integers of 0 to 10; Preferably, ring B is selected from a 5-6 membered N-containing heteroaromatic ring, a 3-6 membered N-containing heterocyclic ring; Preferably, ring B is selected from pyrimidine ring, pyridine ring, triazine ring (such as )、 Preferably, each R b The same or different, independently selected from cyano, oxo (=O), methoxy, cyclopropyl, trifluoromethyl, Preferably, L m1 Not present, or selected from unsubstituted or optionally substituted by one, two or more R m1 Substituted from the following groups: phenyl, piperidinyl or piperazinyl; Preferably, L m1 Selected from Preferably, L m2 is selected from unsubstituted or optionally substituted with one, two or more R m2 Substituted with the following groups: -CH2-(C=O)-, -(CH2)2-(C=O)-, -(CH2)5-(C=O)-, -C≡C-(CH2)3-(C=O)-; Preferably, M is the following formula M-1: in, t is an integer from 0 to 10, and Z is N or CR 22 , R 21 、R 22 and R 23 Each independently selected from H, halogen, cyano, C 1-6 Alkyl, halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, 3-8 membered heterocyclic group, C 1-6 Alkyl-OC 1-6 Alkylene or monovalent hydrophilic group Hp1; provided that when Z is N, R 21 or R 23 Not at the same time H; Preferably, t is selected from 1, 2, 3, 4, 5 or 6; Preferably, Z is N or C-CN; Preferably, R 21 Selected from H, halogen, cyano, C 1-6 Alkoxy or C 1-6 Alkyl-OC 1-6 alkylene; Preferably, Z is N, R 21 Selected from halogen, cyano, C 1-3 Alkoxy or C 1-3 Alkyl-OC 1-3 alkylene; Preferably, Z is N, R 21 Selected from methoxy or CH3-O-CH2-; Preferably, Z is C-CN, R 21 Selected from H; Preferably, R 23 Selected from H; Preferably, M is selected from:

5. The compound of formula (Ia) or (Ib) according to any one of claims 1 to 4, its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or prodrug compound, wherein Z1-Hp2-Z2 has the following structure: -Hp2-; -NH-(CH2)m6-HP2-; -NH-(CH2)m6-HP2-(CH2)m7CO-; -CO-(CH2)m6-HP2-; -CO-(CH2)m6-HP2-(CH2)m7CO-; -CO-(CH2)m6-HP2-(CH2)m7O(CH2)m8CO- wherein m6, m7, and m8 are each independently selected from an integer of 0-10; Preferably, M-Z1-Hp2-Z2 has the following structure:

6. The compound of formula (Ia) or (Ib) according to any one of claims 1 to 5, its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or prodrug compound, wherein Z1(Hp1)-Z2 has the following structure: -N(-Z6-Hp1)- -N(-Z6-Hp1)-(CH2)m9CO- -N(-Z6-Hp1)-(CH2)m9O(CH2)m 10 CO- Wherein Z3-Z7 is a chemical bond or a divalent group, preferably, Z3-Z6 is selected from C 1-10 Alkylene, -OC 1-10 Alkylene-, -OC 1-10 Alkylene-O-, -OC 1-10 Alkylene-CO-, -OC 1-10 Alkylene-NH-, -NH-C 1-10 Alkylene-, -NH-C 1-10 Alkylene-O-, -NH-C 1-10 Alkylene-CO-, -C 1-10 Alkylene-CO- or -NH-C 1-10 Alkylene-NH-; m9 or m10 is an integer from 1 to 10; Preferably, M-Z1(Hp1)-Z2 has the following structure:

7. The compound of formula (Ia) or (Ib) according to any one of claims 1 to 6, its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or prodrug compound, wherein Z1(Hp1)-Hp2-Z2 has the following structure: -N(Hp1)-Hp2- -N(Hp1)-Hp2-(CH2)m 11 CO- -N(Hp1)-Hp2-(CH2)m 11 O(CH2)m 12 CO-;m 11 or m 12 An integer from 1 to 10; Preferably, M-Z1(Hp1)-Hp2-Z2- has the following structure:

8. The compound of formula (Ia) or (Ib) according to any one of claims 1 to 7, its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or prodrug compound, wherein Tr1 is glycine-glycine-phenylalanine-glycine (GGFG), that is: Or valine-alanine (VA), that is:

9. The compound of formula (Ia) or (Ib) according to any one of claims 1 to 7, its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or prodrug compound, wherein the structure of L2 is: Wherein X1 is O or NH; Preferably, Tr2 is a valine-alanine (Val-Ala) or β-glucuronide group, i.e. Preferably, the structure of M-Z1-Hp2-Z2-L2(Tr2)- is:

10. A compound of formula (Ia) or (Ib) according to any one of claims 1 to 9, its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or prodrug thereof, wherein the compound has the following structure: wherein -Z1(Hp1)- is preferably: -N(-Z6-Hp1)- -N(-Z6-Hp1)-(CH2)m9CO- -N(-Z6-Hp1)-(CH2)m9O(CH2)m 10 CO- Wherein Z3-Z7 is a chemical bond or a divalent group, preferably, Z3-Z6 is selected from C 1-10 Alkylene, -OC 1-10 Alkylene-, -OC 1-10 Alkylene-O-, -OC 1-10 Alkylene-CO-, -OC 1-10 Alkylene-NH-, -NH-C 1-10 Alkylene-, -NH-C 1-10 Alkylene-O-, -NH-C 1-10 Alkylene-CO-, -C 1-10 Alkylene-CO- or -NH-C 1-10 Alkylene-NH-; t2, m9 or m 10 are each independently selected from an integer of 1-10; Hp3 is H or Hp1, and the remaining groups have the meanings as defined in claims 1-9.

11. A compound of formula (Ia) or (Ib) according to any one of claims 1 to 10, its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or prodrug thereof, wherein the compound has the following structure: Among them, t2, n7, m 13 and m 14 are each independently selected from an integer of 1-10; the remaining groups have the meanings described in claims 1-10; the compound represented by formula (Ia) or formula (Ib) is further preferably: in, m 13 and m 14 Each independently selected from an integer of 1-10; the remaining groups have the meanings described in claims 1-10; The compound represented by formula (Ia) or formula (Ib) is further preferably: Among them, Z, R 21 、R 23 , n1 has the definition as described in any one of claims 1-10; Preferably, n1 is selected from an integer of 2-12, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; more preferably, n1 is selected from an integer of 4-10.

12. A compound of formula (Ia) or (Ib) according to any one of claims 1 to 11, a racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or a prodrug thereof, wherein the compound has the following structure:

13. The compound of formula (Ia) or (Ib) according to any one of claims 1 to 12, its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or prodrug thereof, wherein D is preferably:

14. The compound of formula (Ia) or (Ib) according to any one of claims 1 to 13, its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or prodrug thereof, wherein the compound of formula (Ia) or (Ib) has a 001-056 or 001'-003' structure:

15. An antibody drug conjugate of formula (IIa) or (IIb) obtained from the compound of formula (Ia) or (Ib) according to claims 1-14, its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or prodrug thereof: Ab-[M'-Z1-Tr1-L1-D] β (IIa) or Ab-[M'-Z1-L2(Tr2)-D] β (IIb) in, Ab is an antibody or an antigen-binding fragment thereof, M' is a fragment formed after coupling M with Ab, and β is an integer or decimal selected from 1-10; Preferably, Ab is an antibody or an antigen-binding fragment, wherein the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, (scFv)2, diabody and single-domain antibody (sdAb); and / or, the antibody is a murine antibody, a humanized antibody, a chimeric antibody, a bispecific antibody or a multispecific antibody; Preferably, Ab is an anti-HER2 antibody or an antigen-binding fragment thereof, for example, Ab is trastuzumab or an antigen-binding fragment thereof; Preferably, β is selected from an integer or decimal between 4 and 9 (e.g., 7, 7.71, 7.84, 7.92, 7.94, 7.97, 7.98, 7.99, 8, 8.02, 8.06, or 8.14); Preferably, the antibody drug conjugate represented by formula (IIa) has the following structure: Among them, Z, R 21 、R 23 , n1 has the definition as described in any one of claims 1-11.

16. A linker represented by formula (IIIa) or (IIIb): M-Z1-Tr1-L1'(IIIa) or M-Z1-L2'(Tr2)(IIIb) in, L1′ and L2′ are the reactive forms of L1 and L2; Preferably: When L1 is a chemical bond, L1' is the reactive form of Tr1, for example, Tr1 is a peptide segment with a carboxyl group or active ester at the C-terminus; When L1 is -NH-CH2-, then L1' is The wavy line shows the site of attachment to the peptide residue; When L1 is When L1' is a carbonic acid active ester of p-aminobenzyl alcohol, for example The wavy line shows the site of attachment to the peptide residue; Preferably, the linker is: Among them, Z, R 21 、R 23 , n1 has the definition as described in any one of claims 1 to 11, Y2 is selected from halogen, hydroxyl or C 1-6 Alkoxy; preferably, Y2 is selected from hydroxy, methoxy, ethoxy, isopropoxy, tert-butoxy.

17. The linker described below is used to obtain an antibody-drug conjugate formed by linking an antibody to a drug via a linker. in, Z, R 21 、R 23 , n1, n2, Tr1, L1, T1, T2, m13, and m14 have the definitions of any one of claims 1 to 11, position 1 is connected to Ab, and position 2 is connected to D; Preferably, the linker is selected from: Among them, position 1 is connected to Ab and position 2 is connected to D.

18. A pharmaceutical composition comprising a therapeutically effective amount of the antibody-drug conjugate of formula (IIa) or (IIb) according to claim 15; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients; Preferably, the pharmaceutical composition may further contain one or more additional therapeutic agents.

19. A method for treating a tumor disease, comprising administering to a patient a preventively or therapeutically effective amount of at least one of the antibody-drug conjugate of formula (IIa) or (IIb) of claim 15, its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or prodrug compound thereof.

20. A method for treating a tumor disease, comprising administering to a patient a preventively or therapeutically effective amount of the pharmaceutical composition of claim 18; Preferably, the tumor disease is selected from breast cancer, gastric cancer, lung cancer, colorectal cancer, large intestine cancer, ovarian cancer, liver cancer, kidney cancer, esophageal cancer, cervical cancer, bladder cancer, pancreatic cancer, prostate cancer, nasopharyngeal cancer, melanoma or leukemia; Preferably, the patient comprises a mammal, preferably a human.

21. An antibody-drug conjugate of formula (IIa) or (IIb) according to claim 15, or at least one of its racemates, stereoisomers, tautomers, solvates, polymorphs, pharmaceutically acceptable salts, or prodrug compounds, or a pharmaceutical composition thereof for treating tumor diseases.

22. Use of at least one of the antibody-drug conjugates of formula (IIa) or (IIb) according to claim 15, its racemates, stereoisomers, tautomers, solvates, polymorphs, pharmaceutically acceptable salts, or prodrug compounds thereof, or the pharmaceutical composition according to claim 18 in the preparation of a topoisomerase I inhibitor and / or in the preparation of a medicament for preventing or treating a disease or condition associated with topoisomerase I; Preferably, the disease or condition is a tumor, and the tumor includes breast cancer, gastric cancer, lung cancer, colorectal cancer, large intestine cancer, ovarian cancer, liver cancer, kidney cancer, esophageal cancer, cervical cancer, bladder cancer, pancreatic cancer, prostate cancer, nasopharyngeal cancer, melanoma or leukemia. 23.Intermediates: in, Z, R 21 、R 23 , n1, n2, Tr1, m13 and m14 have the definitions as described in any one of claims 1 to 11; Y1, Y3, Y4 are the same or different and are independently selected from halogen, hydroxyl, OSu or C 1-6 alkoxy; Preferably, Y1, Y3, and Y4 are the same or different and are independently selected from hydroxy, methoxy, ethoxy, isopropoxy, tert-butoxy, or OSu.

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