CN114053426B - Double-drug linked assembly unit and double-drug targeting connector-drug conjugate - Google Patents

Double-drug linked assembly unit and double-drug targeting connector-drug conjugate Download PDF

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CN114053426B
CN114053426B CN202110866925.4A CN202110866925A CN114053426B CN 114053426 B CN114053426 B CN 114053426B CN 202110866925 A CN202110866925 A CN 202110866925A CN 114053426 B CN114053426 B CN 114053426B
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drug
targeting linker
double
drug conjugate
dual
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CN114053426A (en
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黄金昆
吴成龙
冯超阳
鲁岳
耿嘉豪
李磊
徐鑫
刘俊强
张伟
陈方
谢德建
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Chengdu Scimount Pharmatech Co ltd
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Abstract

The invention discloses a double-drug linked assembly unit and a double-drug targeting connector-drug conjugate. Specifically provided is a double-drug linked assembly unit shown in a formula VI, or a stereoisomer thereof, or an optical isomer thereof. The double-drug linked assembly unit can be connected with a targeting linker to obtain the double-drug targeting linker-drug conjugate molecule shown in the formula I. The double-drug targeting linker-drug conjugate can target tumor cells, reduce toxic and side effects on normal cells, and effectively overcome drug resistance of drugs. With ADCs already on the market: compared with DS-8201a, the ADC provided by the invention has obviously improved inhibition effect on breast adenocarcinoma cells and gastric carcinoma cells. The double-drug linked assembly unit and the double-drug targeting connector-drug conjugate have wide application prospects in preparation of antitumor drugs for prevention and/or treatment.

Description

Double-drug linked assembly unit and double-drug targeting connector-drug conjugate
Technical Field
The invention belongs to the field of medicines, and particularly relates to a double-medicine linked assembly unit and a corresponding double-medicine targeting joint-medicine conjugate thereof.
Background
The Antibody-Drug Conjugate (ADC) can selectively deliver drugs to cancer cells and kill the cancer cells, but has less influence on normal cells, thus opening a new era of tumor treatment. As ADCs, there are a number of drugs available on the market that are FDA approved, such as Mylotarg with antibody CD33 linked to calicheamicin, adcetris with CD30 antibody linked to auristatin E for treatment of hodgkin lymphoma and undifferentiated large cell lymphoma patients, DS-8201 with Her2 antibody linked to camptothecin derivative Dxd for treatment of Her2 positive breast cancer patients, and Sacituzumab govitecan targeting TROP-2 antigen (also known as epithelial glycoprotein 1, egp-1).
Drugs contained in ADCs that have been recognized by the FDA to date are mainly targeted to DNA or tubulin. The marketed ADCs are respectively connected by antibodies and targeted DNA or tubulin drugs, no ADCs are marketed which connect the antibodies and the targeted DNA or tubulin drugs at the same time, and whether the targeted DNA or tubulin drugs are connected to the antibodies at the same time can kill tumors through two different antitumor action mechanisms or not can effectively play roles is not known.
As antitumor small molecule compounds, camptothecin derivatives such as SN-38, dxd, and Dx-8951, which are known as compounds that inhibit DNA topoisomerase I and exert antitumor effects, have been confirmed to have killing effects on various cancer cells both in vivo and in vitro, and have demonstrated strong antitumor effects. As compounds which inhibit tubulin and achieve antitumor effect, eribulin, MMAE, MMAF, maytansine and the like have proved to have killing effect on various cancer cells in vivo and in vitro and show strong antitumor effect. The anti-tumor drugs with two different mechanisms are connected to the same antibody, so that the anti-tumor activity can have a synergistic effect, and the anti-tumor activity can also have an antagonistic effect, so that the actual effect cannot be expected.
Therefore, there is a need to develop an ADC with a high-efficiency and safe multi-drug multi-target mechanism of action, which has very important significance in developing anti-tumor drugs with excellent anti-tumor effect and safety.
Disclosure of Invention
The invention aims to provide a double-drug linked assembly unit and a corresponding double-drug targeting connector-drug conjugate thereof.
The present invention provides a dual drug linked assembly unit of formula VI, or a stereoisomer thereof, or an optical isomer thereof:
wherein T is a tether group capable of linking to a targeting linker; the targeting linker is a substance capable of targeting the binding lesion;
U is a trifurcated linker moiety;
l1 and L2 are cleavable or non-cleavable linking groups;
d 1、D2 is a first drug unit and a second drug unit, respectively.
Further, the T is capable of reactive attachment to a thiol group on a targeting linker.
Further, the structure of the double-drug linked assembly unit is shown as a formula VI-1:
Wherein, K 1、K2 is independently selected from none, K aKbKc;Ka is phenyl, K b is selected from O, S or none, and K c is selected from 0-2 methylene;
n and p are each independently selected from integers of 0 to 10;
U is a substituted or unsubstituted aryl, heteroaryl, alkanyl, cycloalkyl or heterocycloalkyl; the substituent is selected from deuterium, halogen, cyano, hydroxy, C 1～6 alkyl or C 1～6 alkoxy;
X 1、X2 are each independently selected from Wherein a, b, C, d is independently selected from 0 or 1, K 3 is selected from C 1～4 alkylene which is unsubstituted, unsubstituted or substituted by R 1, K 4 is selected from C 1～4 alkylene which is unsubstituted, unsubstituted or substituted by R 2, K 5 is selected from C 1～4 alkylene which is unsubstituted, unsubstituted or substituted by R 3, K 6 is selected from C 1～4 alkylene which is unsubstituted, unsubstituted or substituted by R 4, r 1、R2、R3、R4 is independently selected from H, C 1～5 alkyl, substituted or unsubstituted benzyl, -L 1aNHCONH2、-L1aNHCOCR5NH2,L1a is 0-4 methylene, R 5 is C 1～3 alkyl; the substituent on the benzyl group is selected from deuterium, halogen, cyano, hydroxy, C 1～6 alkyl or C 1～6 alkoxy;
B 1,B2,C1,C2,E1,E2 are each independently selected from the group consisting of substituted or unsubstituted: l 2aCONHL3、L4OL5 CO or none; the substituents are each independently selected from/> C 1～5 alkyl; wherein each L 2a、L3、L4、L5、L6 is independently selected from 0 to 2 methylene groups;
d 1、D2 are each independently selected from cytotoxic drugs, drugs for the treatment of autoimmune diseases or anti-inflammatory drugs;
T is as described above.
Further, the structure of the double-drug linked assembly unit is shown in formula VII:
wherein n and p are each independently selected from integers of 0 to 10;
U is a substituted or unsubstituted aryl, heteroaryl, alkanyl, cycloalkyl or heterocycloalkyl; the substituent is selected from deuterium, halogen, cyano, hydroxy, C 1～6 alkyl or C 1～6 alkoxy;
X 1、X2 are each independently selected from Wherein a, b, c, d is independently selected from 0 or 1, R 1、R2、R3、R4 is independently selected from H, C 1～5 alkyl, substituted or unsubstituted benzyl and L 1aNHCONH2,L1a is 0-3 methylene; the substituent on the benzyl group is selected from deuterium, halogen, cyano, hydroxy, C 1～6 alkyl or C 1～6 alkoxy;
B 1,B2,C1,C2,E1,E2 are each independently selected from the group consisting of substituted or unsubstituted: l 2aCONHL3、L4OL5 CO or none; the substituents are each independently selected from/> C 1～5 alkyl; wherein each L 2a、L3、L4、L5、L6 is independently selected from 0 to 2 methylene groups;
d 1、D2 are each independently selected from cytotoxic drugs, drugs for the treatment of autoimmune diseases or anti-inflammatory drugs;
T is as described above.
Further, the structure of the double-drug linked assembly unit is shown as a formula VII-1:
Wherein, A 1、A2 is independently selected from 0 to 10 methylene, O, S, CO, NH, CONH, NHCO or none;
m,n,p,U,X1,X2,B1,B2,C1,C2,E1,E2,D1、D2 As described above.
Further, the structure of the double-drug linked assembly unit is shown as formula VIIIa or VIIIb:
Wherein ,A1、A2、m、n、p、X1、X2、B1、B2、C1、C2、E1、E2、D1、D2 is as described above;
M is hydrogen, deuterium, halogen, cyano, hydroxy, C 1～6 alkyl or C 1～6 alkoxy.
Further, the structure of the dual drug linked assembly unit is selected from one of the following structures:
Wherein D 1、D2 is as above, n and p are integers of 0-4; preferably, n=p.
Further, the structure of the double-drug linked assembly unit is one of the following structures:
Wherein D 1、D2 is as described above.
Further, each of said D 1、D2 is independently selected from a DNA-targeted drug unit or a tubulin-targeted drug unit; the DNA-targeting drug unit is preferably SN-38, dxd, dx-8951 or a derivative thereof, and/or the tubulin-targeting drug unit is preferably Eribulin, MMAE, MMAF, maytansine or a derivative thereof.
Further, the dual drug linked assembly unit is one of the following structures:
/>
/>
/>
/>
/>
/>
The invention also provides a double-drug targeting linker-drug conjugate molecule, or a stereoisomer or an optical isomer thereof, wherein the double-drug targeting linker-drug conjugate molecule is obtained by connecting a targeting linker and q double-drug linking assembly units; the targeting linker is a substance capable of targeting the binding lesion; the structure of the double-drug targeting linker-drug conjugate molecule is shown in formula I:
wherein Ab is a targeting linker, q is an integer from 1 to 20, T, U, L, L2, D 1、D2 are as described above.
Further, the targeting linker is an antibody, antibody fragment, protein or aptamer, preferably an antibody directed against a cell surface receptor and a tumor associated antigen;
And/or q is an integer of 1 to 8.
Further, the structure of the dual drug targeting linker-drug conjugate molecule is selected from one of the following structures:
/>
/>
/>
/>
/>
/>
/>
/>
The invention also provides a double-drug targeting linker-drug conjugate, or a stereoisomer or an optical isomer thereof, wherein the double-drug targeting linker-drug conjugate is obtained by connecting a targeting linker and the double-drug linking assembly unit; the targeting linker is a substance capable of targeting binding to a lesion, preferably an antibody, antibody fragment, protein or nucleic acid aptamer; the antibody is preferably an antibody against a cell surface receptor and a tumor-associated antigen.
Further, the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula IIa, and the DAR value of the double-drug targeting linker-drug conjugate is 6.00-7.00, preferably 6.41;
or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in a formula IIb, wherein the DAR value of the double-drug targeting linker-drug conjugate is 1.50-2.50, preferably 2.03;
Or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in a formula IIc, wherein the DAR value of the double-drug targeting linker-drug conjugate is 4.00-5.00, preferably 4.38;
or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in a formula IId, wherein the DAR value of the double-drug targeting linker-drug conjugate is 1.50-2.50, preferably 1.99;
or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in a formula IIe, wherein the DAR value of the double-drug targeting linker-drug conjugate is 3.00-4.00, preferably 3.35;
Or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in a formula IIf, wherein the DAR value of the double-drug targeting linker-drug conjugate is 2.00-3.00, preferably 2.31;
Or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in a formula IIg, wherein the DAR value of the double-drug targeting linker-drug conjugate is 3.00-4.00, preferably 3.47;
or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in a formula II SMP-52581, wherein the DAR value of the double-drug targeting linker-drug conjugate is 1.50-2.50, preferably 1.93;
Or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in a formula II SMP-96745, wherein the DAR value of the double-drug targeting linker-drug conjugate is 4.00-5.00, preferably 4.27;
Or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-39545, wherein the DAR value of the double-drug targeting linker-drug conjugate is 5.00-6.00, preferably 5.24;
or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in a formula II SMP-93954, wherein the DAR value of the double-drug targeting linker-drug conjugate is 4.00-5.00, preferably 4.32;
or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in a formula II SMP-11115, wherein the DAR value of the double-drug targeting linker-drug conjugate is 7.00-8.00, preferably 7.71;
or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-04404, wherein the DAR value of the double-drug targeting linker-drug conjugate is 4.00-5.00, preferably 4.67;
or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in a formula II SMP-37241, wherein the DAR value of the double-drug targeting linker-drug conjugate is 6.00-7.00, preferably 6.39;
Or the double-drug targeting joint-drug conjugate consists of two or more than two of double-drug targeting joint-drug conjugate molecules shown in a formula II SMP13432, wherein the DAR value of the double-drug targeting joint-drug conjugate is 4.00-5.00, preferably 4.26;
Or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-84984, wherein the DAR value of the double-drug targeting linker-drug conjugate is 5.00-6.00, preferably 5.69;
Or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in a formula II SMP-04140, wherein the DAR value of the double-drug targeting linker-drug conjugate is 2.00-3.00, preferably 2.47;
or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in a formula II SMP-76794, wherein the DAR value of the double-drug targeting linker-drug conjugate is 2.00-3.00, preferably 2.58;
or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-99767, wherein the DAR value of the double-drug targeting linker-drug conjugate is 6.50-7.50, preferably 7.14;
Or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-86145, wherein the DAR value of the double-drug targeting linker-drug conjugate is 6.00-7.00, preferably 6.45;
Or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-54418, wherein the DAR value of the double-drug targeting linker-drug conjugate is 7.00-8.00, preferably 7.26;
Or the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in a formula II SMP-44708, wherein the DAR value of the double-drug targeting linker-drug conjugate is 1.50-2.50, preferably 2.07.
The invention also provides the preparation method of the double-drug targeting linker-drug conjugate molecule or the double-drug targeting linker-drug conjugate, which comprises the following steps: coupling the targeting linker with a double-drug linked assembly unit, and purifying to obtain the compound; preferably, the purification means is dialysis.
The invention also provides a medicine for preventing and/or treating tumors, which is a preparation prepared by taking the double-medicine targeting linker-medicine conjugate molecule, or a stereoisomer or an optical isomer thereof, or the double-medicine targeting linker-medicine conjugate, or a stereoisomer or an optical isomer thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
The invention also provides the application of the double-drug targeting linker-drug conjugate molecule, or a stereoisomer thereof, or an optical isomer thereof, or the double-drug targeting linker-drug conjugate, or a stereoisomer thereof, or an optical isomer thereof in preparing medicaments for preventing and/or treating tumors.
Further, the tumor is selected from lung cancer, urinary tract cancer, large intestine cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, stomach cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell cancer, peritoneal cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulval cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.
Definition of terms used in connection with the present invention: unless otherwise indicated, the initial definitions provided for groups or terms herein apply to the groups or terms throughout the specification; for terms not specifically defined herein, the meanings that one skilled in the art can impart based on the disclosure and the context.
An "antibody" or "antibody unit" is within its scope and includes any moiety to which an antibody binds. This unit may bind, reactively associate, or complex with a receptor, antigen, or other receptor unit that the targeted cell population has. An antibody may be any protein or intra-protein molecule that can bind, complex or react with a portion of a cell population to be treated or biologically engineered.
Antibodies of the invention are capable of specifically binding to an antigen. The designed antigens include: tumor Associated Antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival modulators, cell proliferation modulators, molecules associated with tissue growth or differentiation (as known or predicted to be functional), lymphokines, cytokines, factors involved in regulation of the cell cycle, molecules involved in angiogenesis, and molecules associated with angiogenesis. The tumor-associated factor may be a cluster differentiation factor (e.g., CD protein). The antigen to which the antibodies of the invention bind may be one or a subset of the above classes, the other subset comprising other molecules/antigens having specific properties.
Antibodies for use in the dual drug linked assembly units and dual drug targeting linker-drug conjugates of the invention include, but are not limited to, antibodies directed against tumor-associated antigens of cell surface receptors. Tumor-associated antigens include, but are not limited to, the tumor-associated antigens listed below, including names and gene bank accession numbers. Antibodies target the corresponding tumor-associated antigen include all amino acid sequence variants and homologs that have at least 70%,80%,85%,90% or 95% homology to the sequences identified in the references, or that have biological properties and characteristics that are entirely identical to the tumor-associated antigen sequences in the references.
Tumor-associated antigens: BMPR1B (Genbank accession number: NM-001203), E16 (Genbank accession number: NM-003486), STEAP1 (Genbank accession number: NM-01449), 0772P (Genbank accession number: AF 361486), MPF (Genbank accession number: NM-005823), napi3B (Genbank accession number: NM-006424), sema 5B (Genbank accession number: AB 040878), PSCA hlg (Genbank accession number: AY 358628), ETBR (Genbank accession number: AY 275463), MSG783 (Genbank accession number: NM-017763), STEAP2 (Genbank accession number: AF 455138), trpM4 (Genbank accession number: NM-017636), CRI (Genbank accession number: gen-003203 or NM-003212), CD21 (Genbank GenM 26004), CD (Genbank Gen 2: gen-Brevican), CD 393 2 (Genbank accession number: DAP Brevican), NAP (Genbank-Brevican) and CD Brevican (Genbank A3932) are (Genbank-Brevican, NAP Brevican) and (Genbank-Brevican) respectively, fcRH1 (Genbank accession number: NP-443170.1), IRTA2 (Genbank accession number: NP-112571.1), TENB2 (Genbank accession number: AF 179274).
As used herein, "drug", "drug unit" or code "D" refers broadly to any compound having the desired biological activity and having reactive functional groups to prepare conjugates of the invention. Further, the drug includes a cytotoxic compound for cancer treatment, a protein or polypeptide having biological activity, including but not limited to camptothecin derivatives such as SN-38, dxd, dx-8951, compounds acting on tubulin such as Eribulin, MMAE, MMAF, maytansine, etc. (structure below).
According to intracellular drug release mechanisms, "linkers" or "antibody drug conjugate linkers" described herein can be divided into two classes: non-cleavable linkers and cleavable linkers.
For a bi-drug antibody drug conjugate containing a non-cleavable linker, the drug release mechanism is: after the conjugate is combined with the antigen and endocytosed by the cell, the antibody is subjected to enzymolysis in a lysosome to release a small molecule drug, and an active small molecule consisting of a linker and an antibody amino acid residue is formed.
Cleavable linkers cleave within the target cell and release the active agent (the small molecule drug itself), which can be divided into two main categories: chemically labile linkers and enzymatically labile linkers.
Chemically labile linkers can selectively cleave due to differences in plasma and cytoplasmic properties, including pH, glutathione concentration, and the like. Enzyme labile linkers, such as peptide linkers, can better control drug release. The peptide linker can be effectively cleaved by a protease in the lysosome, such as cathepsin, or plasmin. This peptide linkage is considered to be very stable in plasma because extracellular unfavorable PH values and serum protease inhibitors result in proteases that are normally inactive extracellular. In view of the high plasma stability and good intracellular cleavage selectivity and availability, enzyme labile linkers are widely used as cleavable linkers for antibody drug conjugates.
In the invention, "q" represents the number of double-drug linked assembly units coupled on one targeting linker in the double-drug targeting linker-drug conjugate molecule, and q is an integer.
However, the dual drug targeting linker-drug conjugates used in the art are typically mixtures of multiple dual drug targeting linker-drug conjugate molecules with different q values.
"DAR value" means the average number of double drug linked assembly units coupled to one targeting linker in a double drug targeting linker-drug conjugate, corresponding to the average of q values. The DAR value may not be an integer.
The invention provides a double-drug linked assembly unit, which can be connected with a targeting joint to obtain a corresponding double-drug targeting joint-drug conjugate, wherein the double-drug targeting joint-drug conjugate can target on tumor cells, reduce toxic and side effects on normal cells, and effectively overcome drug resistance of drugs. With ADCs already on the market: compared with DS-8201a, the ADC provided by the invention has obviously improved inhibition effect on breast adenocarcinoma cells and gastric carcinoma cells. The double-drug linked assembly unit and the double-drug targeting connector-drug conjugate have wide application prospects in preparation of antitumor drugs for prevention and/or treatment.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Detailed Description
The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.
Examples 1 to 22 are specific examples of the preparation of the double drug linked assembly unit of the present invention.
Synthesis of example 1, compound XIe:
Step 1: preparation of intermediate 4-2:
To a 15mL reaction flask was added raw material L-2 (300 mg), DCM (5 mL), and oxalyl chloride (0.3 mL) was slowly added dropwise at 0deg.C and the reaction was stirred at room temperature for 30 minutes. The DCM was removed by concentration under reduced pressure and the residue was taken up in DCM (20 mL); after 5-amino isophthalic acid (350 mg) was dissolved in DMF (2 mL), DIPEA (320 mg) was added thereto, and the above DCM solution was slowly added dropwise at 0℃to stir the reaction at room temperature for 2 hours. HPLC purification (H 2 O: ACN=10-90%) gave 4-2 as pure product, weighing 200mg. MS (EI) m/s:375.1 (M+H).
Step 2: preparation of intermediate 4-3:
to a 50mL reaction flask was added raw material 4-2 (53 mg), DMF (2.5 mL), HATU (54 mg), DIPEA (18 mg) and stirred at room temperature for 5 minutes. Raw material A (40 mg) was dissolved in DMF (1 mL) and then slowly added dropwise to the above reaction solution, followed by reaction at room temperature for 20 minutes. HPLC purification (H 2 O: ACN=10-90%) gave 4-3 pure, weighing 20mg. MS (EI) m/s:740.5 (M/2+H).
Step 3: preparation of the target product XIe:
To a 25mL reaction flask, raw material 4-3 (15 mg), DMF (1.5 mL), HATU (5.7 mg), DIPEA (2.5 mg) were added sequentially, and after stirring at room temperature for 1 minute, raw material C (11.5 mg) was added and reacted at room temperature for 20 minutes. HPLC purification (H 2 O: ACN=10-90%) to obtain XIe pure product, heavy 17.7mg.MS(EI)m/s：1299.3(M/2+H).1H NMR(400MHz,DMSO-d6)δ9.87(m,3H),8.29–8.13(m,5H),7.78(m,2H),7.57–7.44(m,5H),7.42–7.24(m,10H),6.81–6.66(m,3H),6.16(m,2H),5.41–5.31(m,4H),5.19(qm,1H),5.17–5.09(m,5H),5.07–5.00(m,3H),4.91(m,1H),4.49–3.68(m,28H),3.68–3.50(m,3H),3.44–3.36(m,1H),3.35–3.19(m,11H),3.18–3.01(m,4H),2.91(d,J＝7.8Hz,6H),2.68(m,1H),2.62–2.25(m,13H),2.25–1.46(m,40H),1.45–1.22(m,4H),1.18(m,3H),1.09(d,J＝7.0Hz,3H),1.02(d,J＝8.0Hz,3H),0.98–0.90(m,15H),0.90–0.77(m,15H).
Example 2 synthesis of compound XIc:
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step 1: preparation of the target product XIc:
To a25 mL reaction flask, raw materials 4-3 (15 mg), DMF (1.5 mL), HATU (5.7 mg), DIPEA (2.5 mg) were added sequentially, and after stirring at room temperature for 1 minute, raw material B (8.5 mg) was added and reacted at room temperature for 20 minutes. Purification by HPLC (H2O: ACN=10-90%) afforded the XIc pure product as 9.8mg. MS (EI) m/s:1016.5 (M/2+H).
Example 3 Synthesis of Compound SMP-52581:
Step 1: preparation of intermediate 4-2-B:
To a 50mL reaction flask was added raw material 4-2 (89 mg), DMF (2.5 mL), HATU (90 mg), DIPEA (30 mg) and stirred at room temperature for 5 minutes. Raw material B (50 mg) was dissolved in DMF (1 mL) and then slowly added dropwise to the above reaction solution, followed by reaction at room temperature for 20 minutes. HPLC purification (H 2 O: ACN=10-90%) gave pure 4-2-B, weighing 35mg. MS (EI) m/s:1197.2 (M+H).
Step 2: preparation of the target product SMP-52581:
To a 15mL reaction flask was added raw material 4-2-B (15 mg), DMF (1.5 mL), HATU (7.1 mg), DIPEA (3.2 mg) and stirred at room temperature for 5 minutes. After C material (14 mg) was dissolved in DMF (0.5 mL), it was slowly added dropwise to the above reaction solution, and reacted at room temperature for 20 minutes. Purification by HPLC (H 2 O: ACN=10-90%) gave pure product weighing 6.9mg. MS (EI) m/s:1157.5 (M+H).
Example 4 Synthesis of Compound SMP-96745:
step 1: preparation of the target product SPM-96745:
To a 50mL reaction flask was added raw material 4-2 (4.4 mg), DMF (1.0 mL), HATU (4.4 mg), DIPEA (2 mg) and stirred at room temperature for 1 min. Raw material B (20 mg) was added to the above reaction solution at once, and reacted at room temperature for 20 minutes. Purification by HPLC (H 2 O: ACN=10-90%) gave pure product weighing 10.5mg. MS (EI) m/s:1010.2 (M+H).
Example 5 synthesis of compound XIf:
Step 1: preparation of intermediate X-1:
Intermediate X (27 mg), DMF (1.5 mL), HATU (30 mg), DIPEA (31 mg) were added sequentially to a 25mL reaction flask and reacted at room temperature for 5 minutes, after which intermediate A (30 mg) was dissolved in DMF (0.5 mL) and clarified, and then slowly added dropwise to the above reaction mixture and reacted at room temperature for 30 minutes. Spot plate, intermediate a was fully reacted. Purification by HPLC (H 2 O: ACN=10-90%) afforded intermediate X-1 as pure product, weighing 18mg. MS (EI) m/s:723.4 (M/2+H).
Step 2: preparation of the target product XIf:
To a 15mL reaction flask was added raw material X-1 (15 mg), DMF (1.5 mL), HATU (3.9 mg), DIPEA (2.6 mg) and stirred at room temperature for 5 minutes. After C material (12 mg) was dissolved in DMF (0.5 mL), it was slowly added dropwise to the above reaction solution, and reacted at room temperature for 20 minutes. HPLC purification (H 2 O: ACN=10-90%) to obtain pure and heavy products 4.6mg.MS(EI)m/s：1281.7(M/2+H).1H NMR(400MHz,DMSO-d6)δ9.87(s,2H),8.45(d,J＝8.9Hz,1H),7.87(m,2H),7.75(m,2H),7.59–7.43(m,5H),7.43–7.23(m,10H),6.81–6.67(m,3H),6.16(m,2H),5.43–5.26(m,4H),5.23–5.09(m,6H),5.09–4.97(m,3H),4.96–4.87(m,1H),4.43–4.22(m,12H),4.22–4.09(m,4H),4.09–3.70(m,13H),3.64(t,J＝5.9Hz,2H),3.55(m,1H),3.43–3.35(m,1H),3.34–3.20(m,11H),3.19–3.00(m,4H),2.91(d,J＝7.8Hz,6H),2.68(m,1H),2.62–2.24(m,15H),2.22–1.44(m,42H),1.42–1.23(m,4H),1.18(m,3H),1.09(d,J＝7.0Hz,3H),1.02(d,J＝8.0Hz,3H),0.95(d,J＝7.6Hz,3H),0.92–0.78(m,27H).
Example 6 synthesis of compound XIb:
To a 15mL reaction flask was added raw material X-1 (15 mg), DMF (1.5 mL), HATU (3.9 mg), DIPEA (2.6 mg) and stirred at room temperature for 5 minutes. Raw material B (12 mg) was dissolved in DMF (0.5 mL) and then slowly added dropwise to the above reaction solution, followed by reaction at room temperature for 20 minutes. Purification by HPLC (H 2 O: ACN=10-90%) gave pure product weighing 5.6mg. MS (EI) m/s:1275.5 (M/2+H).
Example 7 Synthesis of Compound SMP-39545:
step 1: preparation of the target product SMP-39545:
To a 15mL reaction flask was added raw material X (2.3 mg), DMF (1.5 mL), HATU (2.3 mg), DIPEA (2 mg) and the reaction was stirred at room temperature for 1 min. Raw material A (15 mg) was dissolved in DMF (0.5 mL) and then slowly added dropwise to the above reaction solution, followed by reaction at room temperature for 20 minutes. HPLC purification (H 2 O: ACN=10-90%) gave pure SMP-39545, weighing 4.5mg. MS (EI) m/s:1272.5 (M/2+H).
Example 8 Synthesis of Compound SMP-93954:
step 1: preparation of intermediate X-2:
intermediate X (45 mg), DMF (1.5 mL), HATU (50 mg), DIPEA (22.7 mg) were added sequentially to a 25mL reaction flask and reacted at room temperature for 5 minutes, after which intermediate A (30 mg) was dissolved in DMF (0.5 mL) and clarified and then slowly added dropwise to the above reaction mixture and reacted at room temperature for 30 minutes. Dot plate, intermediate reacted completely. Purification by HPLC (H 2 O: ACN=10-90%) afforded intermediate X-2 as pure product, weighing 13mg. MS (EI) m/s:729.0 (M/2+H).
Step 2: preparation of the target product SMP-93954:
To a 15mL reaction flask was added raw material X-2 (15 mg), DMF (1.5 mL), HATU (3.9 mg), DIPEA (2.6 mg) and stirred at room temperature for 5 minutes. Raw material B (8.6 mg) was dissolved in DMF (0.5 mL) and then slowly added dropwise to the above reaction solution, followed by reaction at room temperature for 20 minutes. HPLC purification (H 2 O: ACN=10-90%) to obtain pure and heavy products 9.6mg.MS(EI)m/s：1141.0(M/2+H).1H NMR(400MHz,DMSO-d6)δ9.87(s,1H),8.45(d,J＝8.9Hz,1H),8.15–8.02(m,4H),7.88(m,2H),7.75(d,J＝8.9Hz,1H),7.54–7.46(m,2H),7.39–7.29(m,4H),7.29–7.18(m,6H),6.75(t,J＝7.4Hz,1H),6.70(s,2H),6.16(t,J＝4.8Hz,1H),5.87(s,1H),5.43–5.29(m,2H),5.24–5.17(m,2H),5.17–5.09(m,4H),5.09–4.99(m,4H),4.69(dd,J＝12.3,1.0Hz,1H),4.60–4.49(m,2H),4.42–4.35(m,2H),4.35–4.29(m,3H),4.29–4.21(m,4H),4.21–4.14(m,2H),4.13(m,2H),4.10–3.99(m,4H),3.99–3.86(m,3H),3.83–3.68(m,8H),3.64(m,3H),3.40–3.18(m,5H),3.18–3.01(m,2H),3.01–2.76(m,4H),2.61–2.26(m,13H),2.23(s,3H),2.20–2.09(m,3H),2.09–1.96(m,4H),1.96–1.80(m,7H),1.80–1.63(m,10H),1.63–1.45(m,9H),1.43–1.31(m,2H),1.02(d,J＝8.0Hz,3H),0.96(t,J＝8.1Hz,3H),0.85(t,J＝1.6Hz,3H),0.84(t,J＝1.6Hz,3H).
Example 9 Synthesis of Compound SMP-11115:
Step 1: preparation of the target product SMP-11115:
to a 15mL reaction flask was added raw material X (2.3 mg), DMF (1.5 mL), HATU (2.3 mg), DIPEA (2 mg) and the reaction was stirred at room temperature for 1 min. After the C material (25 mg) was dissolved in DMF (0.5 mL), it was slowly added dropwise to the above reaction solution, and reacted at room temperature for 20 minutes. HPLC purification (H 2 O: ACN=10-90%) gave pure SMP-11115, weighing 18mg. MS (EI) m/s:1287.5 (M/2+H).
Example 10 Synthesis of Compound SMP-04404:
step 1: preparation of the target product SMP-04404:
To a 15mL reaction flask was added, in order, raw material X (2.0 mg), DMF (1.5 mL), HATU (13 mg), DIPEA (5.3 mg) and the reaction was stirred at room temperature for 1 min. Raw material B (10 mg) was dissolved in DMF (0.5 mL) and then slowly added dropwise to the above reaction solution, followed by reaction at room temperature for 20 minutes. HPLC purification (H 2 O: ACN=10-90%) gave pure SMP-04404, weighing 3.4mg. MS (EI) m/s:1493 (M/2+H).
Example 11 Synthesis of Compound SMP-37241:
Step 1: preparation of intermediate Y:
To a 50mL reaction flask, H 2 O: THF=2:1=18 mL was added first, naHCO 3 (148 mg) and Y (200 mg) were added sequentially, and after stirring at room temperature until the solution was clear, L-3 (460 mg) was dissolved in DME (6 mL) and added to the reaction solution at once, and reacted at room temperature for 12 hours. Post-treatment: to the reaction mixture was added EA, meoh=30:1=25×3 (mL), and the mixture was extracted three times, and the organic phase was dried over anhydrous sodium sulfate and distilled under reduced pressure to obtain a crude product of L-3-1. Purification by HPLC (H 2 O: ACN=10-90%) afforded intermediate L-3-1 as pure product, weighing 45mg. MS (EI) m/s:327.1 (M+H)
Step 2: preparation of intermediate Y-1:
To a 25mL reaction flask, intermediate Y (51 mg), DMF (2.5 mL), HATU (60.4 mg), DIPEA (23 mg) were added sequentially, and after stirring at room temperature for 5 minutes, the intermediate C starting material (60 mg) was dissolved in DMF (1.0 mL) and was clarified, and then added dropwise to the above reaction mixture slowly, followed by reaction at room temperature for 30 minutes. Dot plate, intermediate reacted completely. HPLC purification (H 2 O: ACN=10-90%) of intermediate Y-1 pure product, heavy 40mg.MS(EI)m/s：1443.1(M+H).1H NMR(400MHz,DMSO-d6)δ9.87(s,1H),8.59(d,J＝8.9Hz,1H),7.76(m,2H),7.54–7.45(m,2H),7.34(m,2H),6.78–6.68(m,3H),6.16(t,J＝4.8Hz,1H),5.41–5.30(m,2H),5.19(m,1H),5.14(m,3H),5.04(m,2H),4.49–4.36(m,2H),4.36–4.29(m,3H),4.29–4.21(m,4H),4.21–4.13(m,2H),4.10–3.99(m,4H),3.96–3.86(m,2H),3.83–3.69(m,2H),3.64(m,3H),3.32(s,3H),3.27(m,2H),3.16–3.02(m,2H),2.71–2.57(m,2H),2.57–2.27(m,9H),2.21–2.14(m,2H),2.11–1.95(m,4H),1.93–1.45(m,24H),1.36(m,2H),1.02(d,J＝8.0Hz,3H),0.85(t,J＝1.6Hz,3H),0.84(t,J＝1.6Hz,3H).
Step3: preparation of the target product SMP-37241:
To a 25mL reaction flask, intermediate Y-1 (10 mg), DMF (1.5 mL), HATU (3.1 mg), DIPEA (1.3 mg) were added sequentially, and after stirring at room temperature for 1 minute, the intermediate B starting material (5.8 mg) was dissolved in DMF (0.5 mL) and was then clarified, and then added slowly dropwise to the above reaction mixture, followed by reaction at room temperature for 30 minutes. Dot plate, intermediate reacted completely. HPLC purification (H 2 O: ACN=10-90%) to obtain the target product SMP-37241 pure product, heavy 2.1mg.MS(EI)m/s：1133.5(M/2+H).1H NMR(400MHz,DMSO-d6)δ9.87(s,1H),8.60(d,J＝8.7Hz,1H),8.19–7.99(m,4H),7.90(d,J＝9.1Hz,1H),7.77(m,2H),7.54–7.46(m,2H),7.41–7.29(m,4H),7.30–7.17(m,6H),6.75(t,J＝7.4Hz,1H),6.70(s,2H),6.16(t,J＝4.8Hz,1H),5.87(s,1H),5.41–5.29(m,2H),5.24–5.17(m,2H),5.17–5.10(m,4H),5.09–5.00(m,4H),4.69(dd,J＝12.3,1.0Hz,1H),4.60–4.50(m,3H),4.42–4.36(m,2H),4.36–4.29(m,4H),4.29–4.21(m,4H),4.21–4.14(m,2H),4.13(m,2H),4.10–3.97(m,4H),3.97–3.86(m,2H),3.83–3.68(m,8H),3.64(m,3H),3.36–3.20(m,5H),3.16–3.01(m,2H),2.99–2.78(m,4H),2.61–2.44(m,6H),2.43–2.27(m,5H),2.26–2.10(m,6H),2.09–1.95(m,4H),1.95–1.44(m,27H),1.36(m,2H),1.02(d,J＝8.0Hz,3H),0.96(t,J＝8.1Hz,3H),0.85(t,J＝1.6Hz,3H),0.84(t,J＝1.6Hz,3H).
Example 12 Synthesis of Compound SMP-13432:
Step 1: preparation of the target product SMP-13432:
To a 25mL reaction flask, intermediate Y-1 (10 mg), DMF (1.5 mL), HATU (3.6 mg), DIPEA (1.2 mg) were added sequentially, and after stirring at room temperature for 1 minute, the intermediate A starting material (7.2 mg) was dissolved in DMF (0.5 mL) and was then clarified, and then added slowly dropwise to the above reaction mixture, followed by reaction at room temperature for 30 minutes. Dot plate, intermediate reacted completely. HPLC purification (H 2 O: ACN=10-90%) afforded the target product SMP-13432 as a pure product, weighing 2.6mg. MS (EI) m/s:1274.5 (M/2+H).
Example 13 Synthesis of Compound SMP-84984:
step 1: preparation of the target product SMP-84984:
To a 25mL reaction flask, intermediate Y (1.5 mg), DMF (1.5 mL), HATU (5.4 mg), DIPEA (2.4 mg) were added sequentially, and after stirring at room temperature for 1 minute, the intermediate B starting material (8 mg) was dissolved in DMF (0.5 mL) and was clarified, and then was slowly added dropwise to the above reaction mixture, followed by reaction at room temperature for 30 minutes. Dot plate, intermediate reacted completely. HPLC purification (H 2 O: ACN=10-90%) of intermediate SMP-84984, weighing 2.5mg. MS (EI) m/s:986.1 (M/2+H).
Example 14 synthesis of compound XIa:
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Step 1: preparation of intermediate Y-A:
Intermediate Y (26 mg), DMF (2.5 mL), HATU (31 mg), DIPEA (12 mg) were added sequentially to a 25mL reaction flask and reacted at room temperature for 5 minutes, after which intermediate A starting material (30 mg) was dissolved in DMF (1.0 mL) and clarified and then slowly added dropwise to the above reaction solution and reacted at room temperature for 30 minutes. Dot plate, intermediate reacted completely. Purification by HPLC (H 2 O: ACN=10-90%) afforded intermediate Y-A as pure product, weighing 20mg. MS (EI) m/s:1431.1
Step 2: preparation of target product XIa:
To a 25mL reaction flask, intermediate Y-A (10 mg), DMF (1.5 mL), HATU (2.5 mg), DIPEA (1.2 mg) were added sequentially, and after stirring at room temperature for 1 minute, the intermediate B starting material (5.8 mg) was dissolved in DMF (0.5 mL) and was then clarified, and then added slowly dropwise to the above reaction mixture, followed by reaction at room temperature for 30 minutes. Dot plate, intermediate reacted completely. Purification by HPLC (H 2 O: ACN=10-90%) afforded the target product XIa as pure product, weighing 2.5mg. MS (EI) m/s:1127.5 (M/2+H).
Example 15 Synthesis of Compound SMP-04140:
step 1: preparation of intermediate Z:
To a 50mL reaction flask, H 2 O: THF=2:1=36 mL was added first, naHCO 3 (1.05 g) and SM1 (500 mg) were added in this order, and after stirring at room temperature until the solution became clear, L-3 (1.9 g) was dissolved in DME (18 mL) and added to the reaction solution at once, and reacted at room temperature for 12 hours. Post-treatment: to the reaction mixture was added EA, meoh=30:1=35×3 (mL), and the mixture was extracted three times, and the organic phase was dried over anhydrous sodium sulfate and distilled under reduced pressure to obtain a crude product of Z. Purification by HPLC (H 2 O: ACN=10-90%) afforded intermediate Z as pure product, weighing 300mg. MS (EI) m/s:313.1 (M+H)
Step 2: preparation of intermediate Z-1:
Intermediate Z (22 mg), DMF (1.5 mL), HATU (26.6 mg), DIPEA (9.0 mg) were added sequentially to a 25mL reaction flask and reacted at room temperature for 5 minutes under stirring, then intermediate C starting material (20 mg) was dissolved in DMF (1.0 mL) and clarified, and then slowly added dropwise to the above reaction solution and reacted at room temperature for 30 minutes. Dot plate, intermediate reacted completely. Purification by HPLC (H 2 O: ACN=10-90%) afforded intermediate Z-1 as pure product, weighing 15mg. MS (EI) m/s:715 (M/2+H).
Step3: preparation of the target product SMP-04140:
To a 25mL reaction flask, intermediate Z-1 (10 mg), DMF (1.5 mL), HATU (4 mg), DIPEA (1.8 mg) were added sequentially, and after stirring at room temperature for 1 minute, the intermediate B starting material (5.8 mg) was dissolved in DMF (0.5 mL) and was then added dropwise slowly to the above reaction mixture after clarification, and reacted at room temperature for 30 minutes. Dot plate, intermediate reacted completely. HPLC purification (H 2 O: ACN=10-90%) afforded the target product SMP-04140 as a pure product, weighing 1.7mg. MS (EI) m/s:1126.5 (M/2+H).
Example 16 Synthesis of Compound SMP-76794:
step 1: preparation of the target product SMP-76794:
To a 25mL reaction flask, intermediate Z-1 (10 mg), DMF (1.5 mL), HATU (4 mg), DIPEA (1.8 mg) were added sequentially, and after stirring at room temperature for 1 minute, the intermediate A starting material (8.1 mg) was dissolved in DMF (0.5 mL) and was then added dropwise slowly to the above reaction mixture after clarification, and reacted at room temperature for 30 minutes. Dot plate, intermediate reacted completely. HPLC purification (H 2 O: ACN=10-90%) afforded the target product SMP-76794 as a pure product, weighing 1.8mg. MS (EI) m/s:1126.5 (M/2+H).
Example 17 Synthesis of Compound SMP-99767:
step 1: preparation of the target product SMP-99767:
To a 25mL reaction flask, intermediate Z (1.5 mg), DMF (1.5 mL), HATU (5.4 mg), DIPEA (2.5 mg) were added sequentially, and after stirring at room temperature for 1 minute, the intermediate B starting material (8 mg) was dissolved in DMF (1.0 mL) and was clarified, and then was slowly added dropwise to the above reaction mixture, followed by reaction at room temperature for 30 minutes. Dot plate, intermediate reacted completely. HPLC purification (H 2 O: ACN=10-90%) afforded the target product SMP-99767 as a pure product, weighing 3mg. MS (EI) m/s:979.3 (M/2+H).
Example 18 Synthesis of Compound SMP-86145:
step 1: preparation of intermediate Z-A:
Intermediate Z (21 mg), DMF (1.5 mL), HATU (25 mg), DIPEA (8.6 mg) were added sequentially to a 25mL reaction flask and reacted at room temperature for 5 minutes, then intermediate A starting material (25 mg) was dissolved in DMF (1.0 mL) and clarified, and then slowly added dropwise to the above reaction mixture and reacted at room temperature for 30 minutes. Dot plate, intermediate reacted completely. Purification by HPLC (H 2 O: ACN=10-90%) afforded intermediate Z-A as pure product, weighing 20mg. MS (EI) m/s:708 (M/2+H).
Step 2: preparation of the target product SMP-86145:
To a 25mL reaction flask, intermediate Z-A (10 mg), DMF (1.5 mL), HATU (4.0 mg), DIPEA (1.8 mg) were added sequentially, and after stirring at room temperature for 1 minute, intermediate B starting material (5.9 mg) was dissolved in DMF (0.5 mL) and clarified, and then added slowly dropwise to the above reaction mixture, followed by reaction at room temperature for 30 minutes. Dot plate, intermediate reacted completely. HPLC purification (H 2 O: ACN=10-90%) afforded the target product SMP-86145 as a pure product, weighing 1.8mg. MS (EI) m/s:1120 (M/2+H).
Example 19 Synthesis of Compound SMP-54418:
step 1: preparation of intermediate V-2:
To a 25mL reaction flask was added in order intermediate V-3 (1 g), DMF (20 mL), K2CO3 (1.16 g), bromoacetic acid (593 mg), nitrogen blanket, and reacted at room temperature for 12h. Post-treatment: after DMF was distilled under reduced pressure at 55 ℃ the remaining solid was dissolved in H2O (10 mL), ph=3 was adjusted at 0 ℃ and extracted three to five times with ethyl acetate and saturated brine, and the organic phase was dried over anhydrous sodium sulfate and distilled under reduced pressure to give crude V-2. Purification by HPLC (H 2 O: ACN=10-90%) afforded intermediate V-2 as pure product, weighing 400mg. MS (EI) m/s:340 (M+H).
Step 2: preparation of intermediate V-1:
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To a 25mL reaction flask was added intermediate V-2 (400 mg), TFA, nitrogen blanket, and reacted at room temperature for 20 minutes. The crude product of V-1 is obtained after distillation under reduced pressure at 55 ℃. Purification by HPLC (H 2 O: ACN=10-90%) afforded intermediate V-2 as pure product, weighing 140mg. MS (EI) m/s:240 (M+H). Step 3: preparation of intermediate V:
To a 15mL reaction flask was added raw material L-2 (123 mg), DCM (2 mL), and oxalyl chloride (0.1 mL) was slowly added dropwise at 0deg.C and the reaction was stirred at room temperature for 30 minutes. The DCM was removed by concentration under reduced pressure and the residue was taken up in DCM (20 mL); after dissolving V-1 (140 mg) in DCM (2 mL), DIPEA (140 mg) was added, and the above DCM solution was slowly added dropwise at 0deg.C and the reaction was stirred at room temperature for 2 hours. HPLC purification (H 2 O: ACN=10-90%) gave V pure, weighing 50mg. MS (EI) m/s:433 (M+H).
Step 4: preparation of intermediate 065-1:
Intermediate V (30 mg), DMF (1.5 mL), HATU (25 mg), DIPEA (8.6 mg) were added sequentially to a 25mL reaction flask and reacted at room temperature for 5 minutes, then intermediate A (21 mg) was dissolved in DMF (0.5 mL) and clarified, and then slowly added dropwise to the above reaction mixture and reacted at room temperature for 30 minutes. Spot plate, intermediate a was fully reacted. Purification by HPLC (H 2 O: ACN=10-90%) afforded intermediate 065-1 as pure product, weighing 8mg. MS (EI) m/s:769 (M/2+H).
Step 5: preparation of the target product SMP-54418:
To a 25mL reaction flask was added sequentially intermediate 065-1 (8 mg), DMF (1.5 mL), DIPEA (1.3 mg), HATU (2.9 mg) and then B (4.3 mg) was added in one portion and reacted at room temperature for 20 minutes. Dot plate, intermediate reacted completely. HPLC purification (H 2 O: ACN=10-90%) afforded the target product SMP-54418 as a pure product, weighing 7mg. MS (EI) m/s:1181 (M/2+H).
Example 20 Synthesis of Compound SMP-44708:
step 1: preparation of intermediate 066-1:
Intermediate V (15 mg), DMF (1.5 mL), HATU (16 mg), DIPEA (6.9 mg) were added sequentially to a 25mL reaction flask and reacted at room temperature for 5 minutes, then intermediate B (30 mg) was dissolved in DMF (0.5 mL) and clarified, and then slowly added dropwise to the above reaction mixture and reacted at room temperature for 30 minutes. Dot plate, intermediate B was fully reacted. Purification by HPLC (H 2 O: ACN=10-90%) afforded intermediate 066-1 as pure product, weighing 8.4mg. MS (EI) m/s:1255.5 (M+H).
Step 2: preparation of the target product SMP-44708:
To a 25mL reaction flask was added sequentially intermediate 066-1 (8.4 mg), DMF (1.5 mL), DIPEA (1.2 mg), HATU (3.0 mg) followed by one-time addition of starting material C (7.5 mg) for 20 minutes at room temperature. Dot plate, intermediate reacted completely. HPLC purification (H 2 O: ACN=10-90%) to obtain the target product SMP-44708 pure product, heavy 7mg.MS(EI)m/s：1185.5(M/2+H).1H NMR(400MHz,DMSO-d6)δ9.87(s,1H),8.15–8.02(m,4H),8.01–7.87(m,3H),7.75(d,J＝8.9Hz,1H),7.53–7.46(m,2H),7.38–7.29(m,4H),7.29–7.18(m,6H),7.12(dt,J＝8.8,1.0Hz,2H),6.89–6.84(m,2H),6.75(t,J＝7.4Hz,1H),6.70(s,2H),6.16(t,J＝4.8Hz,1H),5.87(s,1H),5.42–5.30(m,2H),5.24–5.17(m,2H),5.17–5.09(m,4H),5.09–4.99(m,4H),4.69(m,1H),4.61–4.51(m,2H),4.49(m,2H),4.43–3.98(m,18H),3.96–3.85(m,2H),3.83–3.70(m,8H),3.64(m,3H),3.36–3.19(m,5H),3.19–3.02(m,2H),3.02–2.79(m,6H),2.58–1.96(m,19H),1.96–1.43(m,28H),1.36(m,2H),1.02(d,J＝8.0Hz,3H),0.96(t,J＝8.1Hz,3H),0.85(t,J＝1.6Hz,3H),0.84(t,J＝1.6Hz,3H).
Example 21 synthesis of compound XId:
to a 25mL reaction flask was added intermediate 4-3 (12 mg) of example 1, DMF (1.5 mL), DIPEA (1.5 mg), HATU (3.5 mg) and then the D starting material (8.0 mg) was added in one portion and reacted at room temperature for 20 minutes. Dot plate, intermediate reacted completely. Purification by HPLC (H 2 O: ACN=10-90%) afforded the desired product XId as a pure product, weighing 7.3mg. MS (EI) m/s:1299.9 (M/2+H).
Example 22 synthesis of compound XIg:
To a 25mL reaction flask was added intermediate x-1 (8.0 mg) from example 5, DMF (1.5 mL), DIPEA (1.0 mg), HATU (3.0 mg) and then the D starting material (7.0 mg) was added in one portion and reacted at room temperature for 20 minutes. Dot plate, intermediate reacted completely. HPLC purification (H 2 O: ACN=10-90%) to obtain the target product XId pure product, heavy 6.7mg.MS(EI)m/s：1282.5(M/2+H).1H NMR(400MHz,DMSO-d6)δ9.87(m,2H),8.45(d,J＝8.9Hz,1H),7.87(m,2H),7.73(m,3H),7.57–7.46(m,5H),7.41–7.27(m,11H),7.27–7.15(m,5H),6.70(s,2H),6.16(m,2H),5.42–5.29(m,4H),5.11(m,4H),5.04(m,1H),4.95–4.86(m,1H),4.42–4.20(m,9H),4.18–4.08(m,4H),4.01–3.79(m,6H),3.64(t,J＝5.9Hz,2H),3.55(m,2H),3.44–3.36(m,2H),3.24(s,12H),3.18–3.01(m,4H),3.01–2.86(m,14H),2.65(m,2H),2.58–2.46(m,4H),2.40(s,4H),2.20–1.63(m,24H),1.62–1.44(m,6H),1.42–1.22(m,6H),1.18(m,3H),1.09(m,6H),0.95(d,J＝7.6Hz,6H),0.91–0.79(m,42H).
Example 23 is a specific preparation example of an antibody-drug conjugate (ADC) of the invention.
Example 23 preparation of ADC
The corresponding ADC was prepared using the double drug linked assembly units and antibodies prepared in examples 1 to 22, respectively, as raw materials. The general method is as follows:
Step 1: antibody reduction
Trastuzumab was formulated with PBS6.0/EDTA as a 10mg/mL solution (3.0 mL), to which 10mM tris (2-carboxyethyl) phosphine hydrochloride (TCEP) aqueous solution (0.0934 mL) and 1M dipotassium hydrogen phosphate aqueous solution (0.150 mL) were added to ensure the pH of the reaction solution was between 7.3 and 7.5, and after stirring for 1 minute, incubated at 37℃for 1 hour.
Step 2: antibody and drug conjugation
The temperature of the above solution was lowered to 10℃and DMSO solutions (0.0374 mL) containing 10mM each of the two-drug linked assembly units of examples 1 to 22 were added, followed by stirring for 1 minute and then standing for 2 hours.
Step 3: purification
Dialyzing the reaction solution obtained in the step 2 by using a10 KD semipermeable membrane, dialyzing at 25 ℃, dialyzing the solvent for 4L/time and dialyzing for 4 hours/time for 4 times, centrifuging and concentrating the obtained dialyzate to the concentration of about 5mg/mL of ADC by using a10 KD ultrafiltration centrifuge tube, and preserving at-20 ℃ to-30 ℃ for later use.
The prepared ADCs were designated as example 1-ADC to example 22-ADC, respectively, and the structures were as follows:
1. Example 14-ADC:
2. Example 6-ADC:
3. example 2-ADC:
4. example 21-ADC:
5. example 1-ADC:
6. Example 5-ADC:
7. Example 22-ADC:
8. example 3-ADC:
9. Example 4-ADC:
10. Example 7-ADC:
11. example 8-ADC:
12. example 9-ADC:
13. Example 10-ADC:
14. example 11-ADC:
15. example 12-ADC:
16. example 13-ADC:
17. example 15-ADC:
18. Example 16-ADC:
19. Example 17-ADC:
20. Example 18-ADC:
21. example 19-ADC:
22. example 20-ADC:
example 24 DAR value determination of the ADC of the present invention
Step 1: ADC reduction
Each ADC sample prepared in example 23 was diluted to 1mg/ml with Tris buffer at pH8.0, 50mmol/L, and then freshly prepared DTT stock solution (1 mol/L) was added to give final Dithiothreitol (DTT) concentration of 50mmol/L, and incubated at 37℃for 20-30 min for reduction.
Step 2: DAR value determination
And (3) injecting the sample obtained in the step (1) into an LC-MS (liquid Crystal-mass spectrometry) for detection, obtaining the HPLC peak areas of L+0, L+1, H+0, H+1, H+2, H+3 and H+4 after deconvolution treatment, and obtaining DAR values through calculation.
The DAR value here represents the average number of conjugated double drug linked assembly units on one antibody in the ADC sample to be tested.
The results are shown in Table 1 below.
DAR value measurement results of Table 1 ADC
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The beneficial effects of the present invention are demonstrated by the test examples below.
Test example 1: procedure for cell experiment
(1) Test method
The cells were plated in 96-well plates at a density of 3000 SK-BR-3 cells (human breast adenocarcinoma cells) per well and 1500N 87 cells (human gastric carcinoma cells) per well, respectively, and cultured overnight.
The next day, each ADC to be tested (i.e. example 1-ADC to example 22-ADC) and the positive control Trastuzumab-GGFG-Dxd (DS-8201 a, a known ADC) were formulated and diluted in a 1:5 ratio gradient to prepare 9 concentration gradient points with the final dosing concentration of 500nM as the highest concentration. Positive control CISPLATIN (cisplatin) was diluted in a 1:3 ratio in a gradient to give final drug concentration of 9 concentration gradient points with 100. Mu.M as the highest concentration.
Each prepared ADC to be tested and positive control were added to cells, and wells with medium alone were used as negative controls. Culturing for 144 hours after dosing, and then detecting the cell viability by a CTG (CELL TITER-GLO) method, and calculating the IC50 value of each tested drug on SK-BR-3 cells and N87 cells respectively. The results are shown in Table 2 below.
(2) Test results
TABLE 2 IC 50 values for each drug pair SK-BR-3 cells and N87 cells
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Compared with chemotherapeutic drug CISPLATIN (cisplatin), the ADC provided by the invention can obviously improve the inhibition effect on breast adenocarcinoma cells and gastric cancer cells. With ADCs already on the market: compared with DS-8201a, the ADC provided by the invention has obviously improved inhibition effect on breast adenocarcinoma cells and gastric carcinoma cells.
In summary, the invention provides a double-drug linked assembly unit, which can be connected with a targeting linker to obtain a corresponding double-drug targeting linker-drug conjugate, wherein the double-drug targeting linker-drug conjugate can target tumor cells, reduce toxic and side effects on normal cells, and effectively overcome drug resistance of drugs. With ADCs already on the market: compared with DS-8201a, the ADC provided by the invention has obviously improved inhibition effect on breast adenocarcinoma cells and gastric carcinoma cells. The double-drug linked assembly unit and the double-drug targeting connector-drug conjugate have wide application prospects in preparation of antitumor drugs for prevention and/or treatment.

Claims (21)

1. A dual drug linked assembly unit, or a stereoisomer thereof, or an optical isomer thereof, characterized in that: the structure of the double-drug linked assembly unit is selected from one of the following structures:
wherein, D 1、D2 is independently selected from cytotoxic drugs, drugs for treating autoimmune diseases or anti-inflammatory drugs, and n and p are integers of 0-4.
2. The dual drug linked assembly unit, or a stereoisomer thereof, or an optical isomer thereof, of claim 1, wherein: the n=p.
3. The dual drug linked assembly unit, or a stereoisomer thereof, or an optical isomer thereof, of claim 1, wherein: the structure of the double-drug linked assembly unit is one of the following structures:
Wherein D 1、D2 is as claimed in claim 1.
4. A dual drug linked assembly unit, or a stereoisomer thereof, or an optical isomer thereof, as claimed in any one of claims 1 to 3 wherein: the D 1、D2 is independently selected from a drug unit targeting DNA or a drug unit targeting tubulin.
5. The dual drug linked assembly unit, or a stereoisomer thereof, or an optical isomer thereof, of claim 4, wherein: the drug units targeting DNA are SN-38, dxd, dx-8951 or derivatives thereof, and/or the drug units targeting tubulin are Eribulin, MMAE, MMAF, maytansine or derivatives thereof.
6. The dual drug linked assembly unit, or a stereoisomer thereof, or an optical isomer thereof, of claim 1, wherein: the double-drug linked assembly unit is one of the following structures:
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7. a dual drug targeting linker-drug conjugate molecule, or a stereoisomer thereof, or an optical isomer thereof, characterized in that: the double-drug targeting linker-drug conjugate molecule is obtained by connecting a targeting linker and q double-drug linked assembly units according to any one of claims 1 to 6; the targeting linker is a substance capable of targeting the binding lesion; the structure of the dual drug targeting linker-drug conjugate molecule is selected from one of the following structures:
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Wherein Ab is a targeting linker, and q is an integer of 1 to 20.
8. The dual drug targeting linker-drug conjugate molecule, or stereoisomer thereof, or optical isomer thereof, of claim 7, wherein: the targeting linker is an antibody, antibody fragment, protein or nucleic acid aptamer;
And/or q is an integer of 1 to 8.
9. The dual drug targeting linker-drug conjugate molecule, or stereoisomer thereof, or optical isomer thereof, of claim 8, wherein: the antibodies are directed against cell surface receptors and tumor-associated antigens.
10. The dual drug targeting linker-drug conjugate molecule, or stereoisomer thereof, or optical isomer thereof, according to any one of claims 7 to 9, wherein: the structure of the dual drug targeting linker-drug conjugate molecule is selected from one of the following structures:
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11. a dual drug targeting linker-drug conjugate, or a stereoisomer thereof, or an optical isomer thereof, characterized in that: the double-drug targeting linker-drug conjugate is obtained by connecting a targeting linker and the double-drug linking assembly unit according to any one of claims 1 to 6; the targeting linker is a substance capable of targeting binding to a lesion site.
12. The dual drug targeting linker-drug conjugate, or stereoisomer thereof, or optical isomer thereof, of claim 11, wherein: the targeting linker is an antibody, antibody fragment, protein or nucleic acid aptamer.
13. The dual drug targeting linker-drug conjugate, or stereoisomer thereof, or optical isomer thereof, of claim 12, wherein: the antibodies are directed against cell surface receptors and tumor-associated antigens.
14. The dual drug targeting linker-drug conjugate, or stereoisomer thereof, or optical isomer thereof, of claim 11, wherein: the double-drug targeting linker-drug conjugate consists of two or more than two of the double-drug targeting linker-drug conjugate molecules shown in the formula IIa in claim 10, wherein the DAR value of the double-drug targeting linker-drug conjugate is 6.00-7.00;
or, the double-drug targeting linker-drug conjugate consists of two or more than two of the double-drug targeting linker-drug conjugate molecules shown in the formula IIb in claim 10, wherein the DAR value of the double-drug targeting linker-drug conjugate is 1.50-2.50;
or, the double-drug targeting linker-drug conjugate consists of two or more than two of the double-drug targeting linker-drug conjugate molecules shown in the formula IIc in claim 10, wherein the DAR value of the double-drug targeting linker-drug conjugate is 4.00-5.00;
or, the double-drug targeting linker-drug conjugate consists of two or more than two of the double-drug targeting linker-drug conjugate molecules shown in the formula IId in claim 10, wherein the DAR value of the double-drug targeting linker-drug conjugate is 1.50-2.50;
Or, the double-drug targeting linker-drug conjugate consists of two or more than two of the double-drug targeting linker-drug conjugate molecules shown in formula IIe in claim 10, wherein the DAR value of the double-drug targeting linker-drug conjugate is 3.00-4.00;
or, the double-drug targeting linker-drug conjugate consists of two or more than two of the double-drug targeting linker-drug conjugate molecules shown in the formula IIf in claim 10, wherein the DAR value of the double-drug targeting linker-drug conjugate is 2.00-3.00;
Or, the double-drug targeting linker-drug conjugate consists of two or more than two of the double-drug targeting linker-drug conjugate molecules shown in the formula IIg in claim 10, wherein the DAR value of the double-drug targeting linker-drug conjugate is 3.00-4.00;
Or, the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-52581 in claim 10, and the DAR value of the double-drug targeting linker-drug conjugate is 1.50-2.50;
or, the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-96745 in claim 10, and the DAR value of the double-drug targeting linker-drug conjugate is 4.00-5.00;
Or, the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-39545 in claim 10, and the DAR value of the double-drug targeting linker-drug conjugate is 5.00-6.00;
or, the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-93954 in claim 10, and the DAR value of the double-drug targeting linker-drug conjugate is 4.00-5.00;
Or, the double-drug targeting linker-drug conjugate consists of two or more than two of the double-drug targeting linker-drug conjugate molecules shown in the formula II SMP-11115 in claim 10, wherein the DAR value of the double-drug targeting linker-drug conjugate is 7.00-8.00;
or, the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-04404 in claim 10, and the DAR value of the double-drug targeting linker-drug conjugate is 4.00-5.00;
or, the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-37241 in claim 10, and the DAR value of the double-drug targeting linker-drug conjugate is 6.00-7.00;
Or, the double-drug targeting linker-drug conjugate consists of two or more than two of the double-drug targeting linker-drug conjugate molecules shown in the SMP13432 in the formula II in claim 10, wherein the DAR value of the double-drug targeting linker-drug conjugate is 4.00-5.00;
or, the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-84984 in claim 10, and the DAR value of the double-drug targeting linker-drug conjugate is 5.00-6.00;
Or, the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-04140 in claim 10, and the DAR value of the double-drug targeting linker-drug conjugate is 2.00-3.00;
Or, the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-76794 in claim 10, and the DAR value of the double-drug targeting linker-drug conjugate is 2.00-3.00;
Or, the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-99767 in claim 10, and the DAR value of the double-drug targeting linker-drug conjugate is 6.50-7.50;
Or, the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-86145 in claim 10, and the DAR value of the double-drug targeting linker-drug conjugate is 6.00-7.00;
Or, the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-54418 in claim 10, and the DAR value of the double-drug targeting linker-drug conjugate is 7.00-8.00;
Or, the double-drug targeting linker-drug conjugate consists of two or more than two of double-drug targeting linker-drug conjugate molecules shown in formula II SMP-44708 in claim 10, and the DAR value of the double-drug targeting linker-drug conjugate is 1.50-2.50.
15. The dual drug targeting linker-drug conjugate, or stereoisomer thereof, or optical isomer thereof, of claim 14, wherein: the double-drug targeting linker-drug conjugate consists of two or more than two of the double-drug targeting linker-drug conjugate molecules shown in the formula IIa in claim 10, wherein the DAR value of the double-drug targeting linker-drug conjugate is 6.41;
or, the dual-drug targeting linker-drug conjugate consists of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIb in claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 2.03;
Or, the dual-drug targeting linker-drug conjugate consists of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIc in claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.38;
Or, the dual-drug targeting linker-drug conjugate consists of two or more of the dual-drug targeting linker-drug conjugate molecules represented by formula IId in claim 10, the DAR value of the dual-drug targeting linker-drug conjugate is 1.99;
Or, the dual-drug targeting linker-drug conjugate consists of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIe of claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.35;
Or, the dual-drug targeting linker-drug conjugate consists of two or more of the dual-drug targeting linker-drug conjugate molecules of formula IIf in claim 10, the DAR value of the dual-drug targeting linker-drug conjugate being 2.31;
Or, the dual-drug targeting linker-drug conjugate consists of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula IIg of claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 3.47;
Or, the dual-drug targeting linker-drug conjugate consists of two or more than two of the dual-drug targeting linker-drug conjugate molecules shown in formula II SMP-52581 in claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 1.93;
Or, the dual-drug targeting linker-drug conjugate consists of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula II SMP-96745 in claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.27;
Or, the dual-drug targeting linker-drug conjugate consists of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula II SMP-39545 in claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 5.24;
Or, the dual-drug targeting linker-drug conjugate consists of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula II SMP-93954 in claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.32;
Or, the dual-drug targeting linker-drug conjugate consists of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula II SMP-11115 in claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 7.71;
Or, the dual-drug targeting linker-drug conjugate consists of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula II SMP-04404 of claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 4.67;
Or, the dual-drug targeting linker-drug conjugate consists of two or more than two of the dual-drug targeting linker-drug conjugate molecules shown in formula II SMP-37241 in claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 6.39;
Or, the dual-drug targeting linker-drug conjugate consists of two or more of the dual-drug targeting linker-drug conjugate molecules shown in the formula II SMP13432 in claim 10, wherein the DAR value of the dual-drug targeting linker-drug conjugate is 4.26;
or, the dual-drug targeting linker-drug conjugate consists of two or more than two of the dual-drug targeting linker-drug conjugate molecules shown in formula II SMP-84984 in claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 5.69;
Or, the dual-drug targeting linker-drug conjugate consists of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula II SMP-04140 in claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 2.47;
Or, the dual-drug targeting linker-drug conjugate consists of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula II SMP-76794 in claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 2.58;
Or, the dual-drug targeting linker-drug conjugate consists of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula II SMP-99767 in claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 7.14;
or, the dual-drug targeting linker-drug conjugate consists of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula II SMP-86145 in claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 6.45;
or, the dual-drug targeting linker-drug conjugate consists of two or more of the dual-drug targeting linker-drug conjugate molecules shown in formula II SMP-54418 in claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 7.26;
or, the dual-drug targeting linker-drug conjugate consists of two or more than two of the dual-drug targeting linker-drug conjugate molecules shown in formula II SMP-44708 in claim 10, and the DAR value of the dual-drug targeting linker-drug conjugate is 2.07.
16. The method of preparing a dual drug targeting linker-drug conjugate molecule according to any one of claims 7 to 10, or a dual drug targeting linker-drug conjugate according to any one of claims 11 to 15, characterized in that: the method comprises the following steps: coupling the targeting linker with the double-drug linked assembly unit, and purifying to obtain the final product.
17. The method according to claim 16, wherein: the purification mode is dialysis.
18. A medicament for preventing and/or treating a tumor, characterized in that: the preparation is prepared by taking the double-drug targeting linker-drug conjugate molecule, or a stereoisomer or an optical isomer thereof according to any one of claims 7 to 10, or the double-drug targeting linker-drug conjugate, or a stereoisomer or an optical isomer thereof according to any one of claims 11 to 15 as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
19. Use of a dual drug targeting linker-drug conjugate molecule according to any one of claims 7 to 10, or a stereoisomer thereof, or an optical isomer thereof, or a dual drug targeting linker-drug conjugate according to any one of claims 11 to 15, or a stereoisomer thereof, or an optical isomer thereof, for the preparation of a medicament for the prevention and/or treatment of a tumor.
20. Use according to claim 19, characterized in that: the tumor is selected from lung cancer, urethra cancer, carcinoma of large intestine, prostatic adenocarcinoma, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, colorectal cancer, uterine cancer, salivary gland cancer, renal cancer, vulval cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma or sarcoma.
21. Use according to claim 20, characterized in that: the colorectal cancer is colon cancer or rectal cancer.
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