wherein R is₁And R₂Each independently selected from C₁-C₃Alkyl or substituted alkyl, -H, -CF₃Aryl or substituted aryl; or R₁、R₂Together with the carbon atom to which it is attached, form cyclobutane, cyclopentane or cyclohexane; r₁、R₂Not hydrogen at the same time.

Preferably, R is₁Is hydrogen, R₂Is C₁-C₃Alkyl, -CF₃Aryl, heteroaryl, monofluoro substituted aryl or difluoride substituted aryl; or R₁And R₂Is C₁-C₃Alkyl, -CF₃Aryl, heteroaryl, monofluoro substituted aryl or difluoride substituted aryl; or R₁、R₂Together with the carbon atoms to which they are attached, constitute cyclobutane, cyclopentane or cyclohexane:

in the structural formula (a), R₂Independently is- (CH)₂)n¹-CH₃、-CF₃Aryl, heteroaryl, monofluoro substituted aryl or difluoride substituted aryl, wherein n¹0, 1 or 2;

in the structural formula (b), R₁And R₂Independently is- (CH)₂)n¹-CH₃、-CF₃Aryl, heteroaryl, monofluoro substituted aryl or difluoride substituted aryl, wherein n¹0, 1 or 2;

in the formula (c), R₁、R₂Together with the carbon atom to which it is attached, form cyclobutane, cyclopentane, cyclohexane, and m is 1, 2, or 3.

More preferably, R₁Is hydrogen, R₂Independently is- (CH)₂)n¹-CH₃、-CF₃Aryl, heteroaryl, monofluoro substituted aryl or difluoride substituted aryl, wherein n¹0, 1 or 2:

R₂the attached carbon has two configurations of R, S,

in the structural formula (a-1), R₂The carbon to which it is attached is in the R configuration,

in the structural formula (a-2), R₂The attached carbon is in the S configuration.

As a preferred example, the camptothecin compound or a pharmaceutically acceptable salt thereof comprises the following structure:

preferably, the camptothecin compound or a pharmaceutically acceptable salt thereof is an antitumor agent, and is used for solid tumors such as lung cancer, kidney cancer, urinary tract cancer, colon cancer, rectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, lung cancer, esophageal cancer and the like, and hematological tumors.

In another aspect of the invention, there is provided an antibody drug conjugate as shown in formula II, which exerts its pharmacological effect by releasing drug D upon reaching a target cell.

Wherein Ab is an antibody, antibody fragment or protein;

l is an optional linking unit;

d is selected from any one of the camptothecin compounds or pharmaceutically acceptable salts thereof, which is connected with L through a hydroxyl group in the molecule.

m is an integer from 1 to 20.

Preferably, the antibody drug conjugate comprises a linking unit L selected from the group consisting of-O-, -N (R) n₁-，-CH₂-，-CH(R)n₁-, amides, ester bonds, -S-, - (PEG) n₂-group of; n is₁Is selected from the integers 1-3, n₂Is selected from an integer of 1-20.

Another aspect of the invention includes a method of treating a patient in need thereof, comprising administering to the patient an antibody drug conjugate of any of the preceding claims, wherein the patient has a tumor, an autoimmune disease, or an infectious disease, and the antibody of the drug-ligand conjugate specifically binds to a target cell of the cancer, autoimmune disease.

Preferably, the antibody conjugated drug or the salt thereof is an antitumor drug or an anticancer drug, and is used for solid tumors such as lung cancer, kidney cancer, urinary tract cancer, colon cancer, rectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, esophageal cancer and the like, and blood tumors.

Drawings

Fig. 1 is a structural diagram of MTS tetrazolium salt and its formazan product.

Detailed Description

Abbreviations and Definitions

As used herein, the following terms and phrases are intended to have the following meanings unless otherwise indicated. When a brand name is used herein, the brand name includes the product formulation, general purpose drug, and active pharmaceutical ingredient of the brand name product, unless the context indicates otherwise.

The term "alkylene" refers to a divalent straight chain saturated hydrocarbon group having 1-20 carbon atoms, including groups of from 1 to 10 carbon atoms. Examples of alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2-CH2-), n-propylene, n-butylene, n-pentylene, and n-hexylene. Unless otherwise indicated, the term "aryl" refers to a polyunsaturated, generally aromatic, hydroxyl group, which can be a single ring or a fused or covalently linked plurality of rings (up to three rings). The term "heteroaryl" refers to an aryl (or ring) containing 1 to 5 heteroatoms selected from N, O or S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. The heteroaryl group may be attached to the rest of the molecule through a heteroatom. Non-limiting examples of aryl groups include: phenyl, naphthyl and biphenyl, while non-limiting examples of heteroaryl groups include: pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl (pyrimidinyl), triazinyl, quinolyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl (phthalazinyl), benzotriazinyl, purinyl, benzimidazolyl, benzpyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuranyl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridyl, thienopyrimidyl, pyridopyrimidyl, imidazopyridine, benzothiazolyl (benzothiazolyloxyl), benzofuranyl, benzothienyl, indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, and thienyl, and the like. When described as "substituted", the substituents of the above-described aromatic and heteroaromatic ring systems are selected from the following acceptable substituents.

Unless otherwise indicated herein, the substituents of the alkyl group may be a variety of groups selected from the group consisting of: -halogen, -OR ', -NR' R ', -SR', -SiR 'R' ", -OC (O) R ', -C (O) R', -CO₂R’、-CONR’R”、-OC(O)NR’R”、-NR”C(O)R’、-NR’-C(O)NR”R”’、-NR”C(O)₂R’、-NH-C(NH₂)＝NH、-NR’C(NH₂)＝NH、-NH-C(NH₂)＝NR’、 -S(O)R’、-S(O)₂R’、-S(O)₂NR’R”、-NR’S(O)₂R ", -CN and-NO₂The number of substituents is 0 to(2m '+ 1) wherein m' is the total number of carbon atoms in the group. R ', R ' and R ' each independently represent hydrogen, unsubstituted C_1-8Alkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted C_1-8Alkyl radical, C_1-8Alkoxy or C_1-8Thioalkoxy, or unsubstituted aryl-C_1-4An alkyl group. When R 'and R' are attached to the same nitrogen atom, they may form a 3-, 4-, 5-, 6-or 7-membered ring together with the nitrogen atom. For example, -NR' R "includes 1-pyrrolidinyl and 4-morpholinyl.

As used herein, a "derivative" of a compound refers to a substance that has a chemical structure similar to a compound but also contains at least one chemical group that is not present in the compound and/or lacks at least one chemical group that is present in the compound. The compounds to which the derivatives are compared are referred to as "parent" compounds. In general, a "derivative" can be produced from a parent compound in one or more chemical reaction steps.

L-ligands

The ligand unit is a targeting agent that specifically binds to the target moiety. The ligand is capable of specifically binding to a cellular component or to other target molecules of interest. The target moiety or target is typically on the cell surface. In some aspects, the ligand unit functions to deliver the drug unit to the particular target cell population with which the ligand unit interacts. Ligands include, but are not limited to, proteins, polypeptides and peptides, as well as non-proteins such as sugars. Suitable ligand units include, for example, antibodies, such as full-length (intact) antibodies and antigen-binding fragments thereof. In embodiments where the ligand unit is a non-antibody targeting agent, it may be a peptide or polypeptide, or a non-proteinaceous molecule. Examples of such targeting agents include interferons, lymphokines, hormones, growth factors and colony stimulating factors, vitamins, nutrient transport molecules, or any other cell binding molecule or substance. In some embodiments, the linker is covalently attached to the sulfur atom of the ligand. In some aspects, the sulfur atom is a sulfur atom of a cysteine residue, which forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine residue that has been introduced into a ligand unit, which forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine residue that has been introduced into a ligand unit (e.g., by site-directed mutagenesis or chemical reaction). In other aspects, the linker-bound sulfur atom is selected from cysteine residues that form interchain disulfide bonds of the antibody or additional cysteine residues that have been incorporated into ligand units (e.g., by site-directed mutagenesis or chemical reaction). In some embodiments, the numbering system is according to the EU index as in Kabat (Kabat E.A et al, (1991)) "protein Sequences of Immunological Interest" (Sequences of proteins of Immunological Interest), fifth edition, NIH publication 91-3242).

As used herein, "antibody" or "antibody unit" is within the scope of it, including any part of an antibody structure. This unit may bind, reactively associate, or complex with a receptor, antigen, or other receptor unit that is possessed by the targeted cell population. An antibody can be any protein or proteinaceous molecule that can bind, complex, or otherwise react with a portion of a cell population to be treated or biologically engineered.

The antibody constituting the antibody-drug conjugate of the present invention preferably retains its antigen-binding ability in its original wild state. Thus, the antibodies of the invention are capable of, preferably specifically, binding to an antigen. Antigens contemplated include, for example, Tumor Associated Antigens (TAA), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules associated with tissue growth and differentiation (e.g., known or predicted to be functional), lymphokines, cytokines, molecules involved in the regulation of cell circulation, molecules involved in angiogenesis, and molecules associated with angiogenesis (e.g., known or predicted to be functional). The tumor associated factor may be a cluster differentiation factor (e.g., a CD protein). As described in the present invention

Antibodies useful in antibody drug conjugates include, but are not limited to, antibodies directed against cell surface receptors and tumor associated antigens. Such tumor-associated antigens are well known in the art and can be prepared by antibody preparation methods and information well known in the art. In order to develop effective cellular level targets for cancer diagnosis and treatment, researchers have sought transmembrane or other tumor-associated polypeptides. These targets are capable of being specifically expressed on the surface of one or more cancer cells, while expressing little or no expression on the surface of one or more non-cancer cells. Typically, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells relative to the surface of non-cancer cells. The confirmation of such tumor-associated factors can greatly improve the specific targeting property of antibody-based cancer treatment.

Tumor associated antigens include, but are not limited to, tumor associated antigens (1) - (36) listed below. For convenience, antigen-related information well known in the art is labeled as follows, including name, other names, and GenBank accession numbers. Nucleic acid and protein sequences corresponding to tumor associated antigens can be found in public databases, such as Genbank. The corresponding tumor associated antigens targeted by the antibodies include all amino acid sequence variants and homologues, having at least 70%, 80%, 85%, 90%, or 95% homology with the sequences identified in the references, or having biological properties and characteristics that are fully identical to the tumor associated antigen sequences in the cited references.

The term "inhibit" or "inhibition of" refers to a reduction in a detectable amount, or a complete prevention.

The term "cancer" refers to a physiological condition or disease characterized by unregulated cell growth. "tumor" includes cancer cells.

The term "autoimmune disease" is a disease or disorder that results from targeting an individual's own tissue or protein.

The phrase "pharmaceutically acceptable salt" as used herein refers to a pharmaceutically acceptable organic or inorganic salt of a compound (e.g., a drug-linker or a ligand-linker-drug conjugate). The compounds may contain at least one amino or carboxyl group and may therefore form addition salts with corresponding acids or bases. Exemplary salts include, but are not limited to: sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, salicylate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, potassium salt, sodium salt and the like. In addition, pharmaceutically acceptable salts have more than one dotted atom in the structure. Examples where the plurality of charged atoms are part of a pharmaceutically acceptable salt can have multiple counter examples. For example, pharmaceutically acceptable salts have one or more charged atoms and/or one or more counter atoms.

According to the mechanism of drug release in cells, as used herein, a "linker" or a "linker of an antibody drug conjugate" can be divided into two categories: non-cleavable linkers and cleavable linkers.

For antibody drug conjugates containing a non-cleavable linker, the drug release mechanism is: after the conjugate is combined with antigen and endocytosed by cells, the antibody is enzymolyzed in lysosome to release active molecules consisting of small molecular drugs, linkers and antibody amino acid residues. The resulting structural change in the drug molecule does not reduce its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate into neighboring cells. Thus, such active drugs are unable to kill adjacent tumor cells that do not express the targeted antigen (antigen negative cells) (Ducry et al, 2010, Bioconjugate chem.21: 5-13).

Cleavable linkers, as the name implies, can cleave within the target cell and release the active drug (small molecule drug itself). Cleavable linkers can be divided into two main classes: chemically labile linkers and enzyme labile linkers.

Chemically labile linkers can be selectively cleaved due to differences in plasma and cytoplasmic properties. Such properties include pH, glutathione concentration, and the like.

The pH sensitive linker is often also referred to as an acid cleavable linker. Such a linker is relatively stable in the neutral environment of blood (pH7.3-7.5), but will be hydrolyzed in weakly acidic endosomes (pH5.0-6.5) and lysosomes (pH 4.5-5.0). The first generation of antibody drug conjugates mostly used such linkers as hydrazones, carbonates, acetals, ketals. Antibody drug conjugates based on such linkers typically have a short half-life (2-3 days) due to the limited plasma stability of the acid-cleaved linker. This short half-life limits to some extent the use of pH sensitive linkers in the next generation of antibody drug conjugates.

Linkers that are sensitive to glutathione are also known as disulfide linkers. Drug release is based on the difference between high intracellular glutathione concentrations (millimolar range) and relatively low glutathione concentrations in the blood (micromolar range). This is particularly true for tumor cells, where their low oxygen content leads to enhanced activity of the reductase and thus to higher glutathione concentrations. Disulfide bonds are thermodynamically stable and therefore have better stability in plasma.

Enzyme-labile linkers, such as peptide linkers, allow for better control of drug release. The peptide linker can be effectively cleaved by an endolytic protease, such as Cathepsin B or plasmin (increased levels of such enzymes in some tumor tissues). This peptide linkage is considered to be very stable in the plasma circulation, since proteases are generally inactive due to an undesirable extracellular pH and serum protease inhibitors. In view of higher plasma stability and good intracellular cleavage selectivity and effectiveness, enzyme-labile linkers are widely used as cleavable linkers for antibody drug conjugates. Typical enzyme-labile linkers include Val-Cit (vc), Phe-Lys, and the like.

The suicide linker is typically either chimeric between the cleavable linker and the active drug or is itself part of the cleavable linker. The mechanism of action of the suicide linker is: when the cleavable linker is cleaved under convenient conditions, the suicide linker is able to undergo spontaneous structural rearrangement, thereby releasing the active drug attached thereto. Common suicide linkers include para-aminobenzols (PAB) and beta-glucuronides (beta-Glucuronide), among others.

The invention will now be further illustrated by reference to specific examples, which are intended to be illustrative only and not to be limiting of the scope of the invention. Test methods without specific conditions noted in the following examples are generally performed according to conventional conditions or according to conditions recommended by the manufacturer. All percentages, ratios, or parts are by weight unless otherwise specified.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

EXAMPLE 1 Synthesis of Compound 2

Compound 1 (irinotecan mesylate, ex situ) (40mg,75.3mmol,1.0eq) and L-lactic acid (10mg,113.0 mmol,1.5eq) were dissolved in dry 5mL DMF and PyBop (58.8mg,113.0mmol,1.5eq) and DIEA (15.7 uL,113.0mmol,1.5eq) were added. After stirring at room temperature for 3 hours, the reaction was complete by TLC, quenched with water, extracted with dichloromethane (10 mL. times.3), the organic phases combined, dried over anhydrous sodium sulfate, filtered, the filtrate concentrated under reduced pressure, and the residue purified by column chromatography to give Compound 2(30.9mg, 81.1%). LC-MS: [ M + H ] +: 508.2. 1H NMR (400Mz, CDCl3/CD3OD) 0.91-0.94(3H, m),1.32-1.39(3H, m),1.71-1.83(2H, m),2.31(3H, s),2.78-3.02(2H, m),3.16-3.26(2H, m), 4.27-4.35(1H, m),4.81-4.92(1H, m),5.15-5.24(2H, m),5.49-5.76(2H, m),7.52(1H, d, J ═ 12.0Hz), 7.58(1H, s),7.75(1H, d, J ═ 12.0 Hz).

EXAMPLE 2 Synthesis of Compound 4

Compound 3: n-fluorenylmethoxycarbonyl-glycyl-glycine (10g,28.2mmol,1.0eq), lead tetraacetate (17.5g,55.3mmol,1.4eq), 200mL of dry tetrahydrofuran and 67mL of toluene were stirred uniformly, protected with nitrogen, and heated to 85 ℃ for reaction for 2.5 h. TLC monitoring, after the starting material had reacted, cooled to room temperature, filtered, the filtrate was concentrated under reduced pressure and the residue was purified by column chromatography to give compound 4(8.7g, 83.7%).

EXAMPLE 3 Synthesis of Compound 5

In a 25mL single-neck flask, compound 3(500mg, 1.4mmol, 1.0eq), p-toluenesulfonic acid monohydrate (26mg, 0.1mmol, 0.1eq) and 10mL THF were added, stirred well, then cooled to 0 deg.C, and then benzyl L-lactate (1.2g, 7.0mmol, 5eq) was slowly added, and after the addition was completed, the temperature was raised to room temperature for reaction. TLC, saturated NaHCO3 solution was added after the reaction was finished, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by reverse phase column to give compound 5(400mg, 60.3%). 1H NMR (400Mz, CDCl3):1.39(3H, d, J ═ 6.8Hz),3.78(2H, t, J ═ 4.0Hz),4.17-4.27(2H, m),4.42(2H, d, J ═ 4.0Hz), 4.72-4.85(2H, m),5.11-5.58(2H, m),5.43(1H, s),7.06(1H, t, J ═ 8.0Hz),7.25-7.33(6H, m),7.38(2H, t, J ═ 8.0Hz),7.57(2H, d, J ═ 8.0Hz),7.75(2H, d, J ═ 8.0 Hz).

EXAMPLE 4 Synthesis of Compound 6

In a 25mL single-neck flask, compound 5(400mg, 0.8mmol, 1.0eq) and 10mL of DMF were added, stirred well, then cooled to 0 deg.C, DBU (137mg, 0.9mmol, 1.1eq) was slowly added, and after addition, the temperature was raised to room temperature for reaction. TLC monitoring, after the reaction was finished, concentration was carried out to obtain crude compound 6 (550mg), which was directly used in the next step without purification.

EXAMPLE 5 Synthesis of Compound 7

A25 mL single vial was charged with Z-Gly-Gly-Phe-OH (372mg, 0.9mmol, 1.1eq), PyBOP (852mg, 1.6mmol, 2.0eq) and 3mL DMF, stirred at room temperature for 5 min, added with the crude compound 6 (550mg), reacted at room temperature, and monitored by HPLC. After the reaction was completed, water was added, extraction was performed with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by reverse phase column chromatography to obtain compound 7(326mg, 59.2%).

EXAMPLE 6 Synthesis of Compound 8

In a 25mL single-necked flask, compound 7(50mg, 1.0eq, 0.08mmol), 5% Pd/C (50mg), 3mL DMF was added and hydrogenated at room temperature. HPLC monitoring, after the reaction was completed, water was added and filtered, and the filtrate was concentrated to give crude compound 8(52mg), which was directly used in the next step without purification.

EXAMPLE 7 Synthesis of Compound 9

Compound 8(52mg), SMCC (23mg, 0.07mmol, 1.0eq), DIEA (22.2mg, 0.24mmol,2.5eq) and 3mLDMF were added to a 25mL single vial and reacted at room temperature, monitored by HPLC, purified for preparation and lyophilized to give compound 9(9.0mg, 18.1%). MS: [ M-H ] 655.1.

EXAMPLE 8 Synthesis of Compound 11

Compound 9(9.0mg, 0.014mmol, 1.0eq), irinotecan mesylate (6.6mg, 0.014mmol, 1.0eq), PyBOP (14.3mg, 0.028mmol,2.0eq), DIEA (6.2mg, 0.048mmol,3.5eq) and 0.5mL dmf were added to a 25mL single vial, reacted at room temperature, monitored by HPLC, purified, lyophilized to give compound 11(7.0mg, 48.3%). TOF: [ M + Na ] + 1096.42.

EXAMPLE 9 Synthesis of Compounds 12, 13

Compound 1 (irinotecan mesylate) (40mg,75.3mmol,1.0eq) and trifluoro lactic acid (16.3mg,113.0 mmol,1.5eq) were dissolved in dry 5mL DMF and PyBop (58.8mg,113.0mmol,1.5eq) and DIEA (15.7 uL,113.0mmol,1.5eq) were added. After stirring at room temperature for 3 hours, the reaction was complete by TLC, quenched with water, extracted with dichloromethane (10 mL. times.3), the organic phases combined, dried over anhydrous sodium sulfate, filtered, the filtrate concentrated under reduced pressure, and the residue purified by column chromatography to give compound 12(13.5mg, 32%). LC-MS: [ M + H ] +: 562.2. 1H NMR (400Mz, CDCl3/CD3OD) 0.91-0.95(3H, m),1.78-1.84(2H, m),2.34(3H, s),3.04-3.14(2H, m),3.27-3.32(2H, m),4.42-4.47(1H, m), 5.08-5.20(3H, m),5.41-5.58(2H, m),7.23-7.25(1H, m), 7.52-7.55(1H, m); compound 13(15.5mg, 36.7%). LC-MS: [ M + H ] +: 562.2. 1H NMR (400Mz, CDCl3/CD3OD) 0.90-1.00(3H, m),1.74-1.89(2H, m),2.34(3H, s),3.01-3.09(2H, m),3.32-3.38(2H, m),4.65-4.71(1H, m),4.89-4.96(1H, m), 5.17-5.30(2H, m),5.55-5.65(2H, m),7.53-7.61(2H, m).

EXAMPLE 10 Synthesis of Compound 14

Reacting trifluoro-lactic acid (3.5g,24.3mmol,1.0eq) with K₂CO₃(5.0g,36.5mmol,1.5eq) is dissolved in dry 35mL DMF, benzyl bromide (5.0g,29.2mmol,1.2eq) is added dropwise in an ice water bath, nitrogen is protected, the mixture is heated to room temperature after the addition is finished and reacts for 5 hours, the TLC detection reaction is complete, water is added for quenching, dichloromethane is used for extraction (100mL × 3), organic phases are combined and washed by saturated common salt water in sequence, anhydrous sodium sulfate is used for drying, filtration is carried out, filtrate is decompressed and concentrated, and residue is purified by column chromatography to obtain compound 14(3.14g, 55%). 1H NMR (400Mz, DMSO) 4.91-4.94(1H, m),5.25(2H, s),7.17-7.19(1H, d),7.39(5H, s);

EXAMPLE 11 Synthesis of Compound 15

In a 50mL single-neck flask were added compound 4(1.45g, 3.9mmol, 1.0eq), compound 14(1.84g, 7.8mmol, 2.0eq), Zn (OAc)₂(1.44g, 7.86mmol, 2.0eq) and 25ml of L mol under nitrogen protection, stirring uniformly, raising the temperature to 100 ℃ and reacting for 5.5 h. TLC monitored, product spot was evident, filtered, and the filtrate was concentrated to give a yellow oil (4.0g) and the crude product was purified by column chromatography to give compound 15(0.99g, 46%). 1H NMR (400Mz, CDCl3):3.68-3.83(2H, m),4.20-4.23(1H, m),4.49 (2H, d, J ═ 8.0Hz),4.73-4.78(1H, m),4.89-5.00(2H, m),5.19(1H, s),5.25(2H, s),7.11(1H, s,), 7.29-7.35(7H, m),7.43(2H, t, J ═ 8.0Hz),7.59(2H, d, J ═ 8.0Hz),7.79(2H, d, J ═ 8.0 Hz).

EXAMPLE 12 Synthesis of Compound 16

The compound 15(990mg, 1.8mmol, 1.0eq) and 10mL of DMF were added to a 25mL single-neck flask, stirred well, cooled to 0 deg.C, then DBU (335mg, 2.2mmol, 1.2eq) was added slowly under nitrogen protection, and after the addition was complete, the reaction was continued at 0 deg.C for 30 min. TLC monitoring, the raw materials are reacted completely, and the reaction solution is directly reacted in the next step.

EXAMPLE 13 Synthesis of Compound 17

Adding Z-Gly-Gly-Phe-OH (909mg, 2.2mmol, 1.2eq), PyBOP (1.4g, 2.7mmol, 1.5eq) and 10mL DMF (dimethyl formamide) into a 50mL single-neck bottle, dropwise adding DIEA under an ice water bath, continuing to react for 10min under the protection of nitrogen, slowly dropwise adding the reaction solution of the compound 16 into the reaction solution under the ice water bath, raising the temperature to room temperature after the addition is finished, reacting for 1.5h, monitoring by HPLC (high performance liquid chromatography), finishing the reaction, purifying, and freeze-drying to obtain the compound 17(0.91g, 71%).

EXAMPLE 14 Synthesis of Compound 18

In a 25mL single-necked flask, compound 17(85mg, 1.0eq, 0.12mmol), 5% Pd/C (85mg), 6mL DMF was added and hydrogenated at room temperature for 1 h. And (5) monitoring by HPLC, finishing the reaction of the raw materials, filtering the reaction solution, and directly feeding the filtrate to the next reaction.

EXAMPLE 15 Synthesis of Compound 19

Filtering the reaction solution of the compound 18 into a 25mL single-neck bottle, adding SMCC (80mg, 0.24mmol, 2.0eq), DIEA (62mg, 0.48mmol,4.0eq) and nitrogen protection in sequence under an ice-water bath, raising the temperature to room temperature for reaction for 1h after the addition is finished, monitoring by HPLC, preparing and purifying, and freeze-drying to obtain a compound 19(66mg, 78%), MS: [ M-H ] 709.2.

EXAMPLE 16 Synthesis of Compounds 20, 21

Dissolving compound 19(10mg,14umol,1.0eq), compound 1(9mg,21umol,1.5eq) and PyBop (14.6mg, 28mmol,2.0eq) in dry DMF (0.5mL), adding DIEA (5uL,28umol,2.0eq) under ice-water bath, reacting for 1h under nitrogen protection, raising the temperature after addition, monitoring by HPLC, completing the reaction of the starting compound 19, preparing the reaction solution directly by HPLC pure water to obtain compound 20(2.77mg, 17.5%), LC-MS: [ M + H ] +: 1128.0; compound 21(3.92mg, 24.8%), LC-MS: [ M + H ] +: 1128.0.

EXAMPLE 17 Synthesis of Compound 22

Filtering the reaction solution (0.15mmol,1.0eq) of the compound 18 into a 25mL single-neck bottle, adding MC (93mg, 0.3mmol, 2.0eq), DIEA (78mg, 0.6mmol,4.0eq) and nitrogen protection in sequence under an ice-water bath, raising the temperature to room temperature for reaction for 1h after the addition is finished, carrying out HPLC monitoring, preparing and purifying pure water, and freeze-drying to obtain a compound 22(90mg, 86%), MS: [ M-H ] 683.2.

EXAMPLE 18 Synthesis of Compounds 23, 24

Compound 22(15mg,21.9umol,1.0eq), compound 1(14.3mg,32.8umol,1.5eq) and PyBop (22.8mg,43.8mmol,2.0eq) were dissolved in dry DMF (0.8mL), DIEA (7.3uL,43.8umol,2.0eq) was added under ice-water bath, under nitrogen protection, then warmed to room temperature for 1h, monitored by HPLC, the starting compound 22 was reacted completely to give compound 23(6.01mg, 25%) respectively after direct HPLC pure water preparation of the reaction mixture, LC-MS: [ M + H ] +: 1102.0; compound 24(5.57mg, 23.2%), LC-MS: [ M + H ] +: 1102.0.

EXAMPLE 18 Synthesis of Compound 25

Mandelic acid (42mg, 0.09mmol, 1.1eq), irinotecan (35mg, 0.08mmol, 1.0eq), PyBOP (84mg, 0.16mmol,2.0eq), DIEA (36.4mg, 0.28mmol,3.5eq) and 1 mldff were added to a 5mL single vial and reacted at room temperature with HPLC monitoring, preparative purification and lyophilization afforded compound 25(15.0mg, 32.6%). 1H NMR (CDCl)₃，400Mz) 7.70(d，1H，J＝8.0Hz)，7.64(s，1H)，5.64-5.75(m，2H)，5.48-5.38(m，1H)，5.29-5.21 (m，1H)，5.19-5.11(m，1H)，3.37-3.11(m，2H)，2.55-2.38(m，4H)，2.32-2.15(m， 2H)，2.08-1.99(m，1H)，1.94-1.85(m，4H)，1.33-1.24(m，4H)，1.05(t，3H，J＝7.2Hz)； LC-MS：[M+H]548.4。

EXAMPLE 19 Synthesis of Compound 26

D-lactic acid (11.2mg, 0.08mmol, 1.1eq), irinotecan (30.0mg, 0.07mmol, 1.0eq), PyBOP (119.5mg, 0.14mmol,2.0eq), DIEA (31.2mg, 0.25mmol,3.5eq) and 1mL DMF were added to a 5mL single-neck flask, reacted at room temperature, HPLC monitored, purified, and lyophilized to give Compound 26(7.2m Mg，20.6％)。1H NMR(CDCl₃，400Mz) 7.75(d，1H，J＝10.4Hz)，7.70(s，1H)，5.75-5.63(m，2H)，5.46-5.38(m，1H)，5.30-5.16 (m，2H)，4.50-4.40(m，1H)，3.34-3.13(m，2H)，2.50-2.36(m，3H)，2.34-2.21(m， 1H)，2.05-2.02(s，1H)，1.96-1.84(m，2H)，1.35-1.23(m，3H)，1.06(t，3H，J＝4.0Hz)； LC-MS：[M+H]508.3。

EXAMPLE 20 Synthesis of Compound 27

In a 5mL single neck flask was added 2-methyl lactic acid (10.5mg, 0.10mmol, 1.1eq), irinotecan (40mg, 0.09mmol, 1.0eq), PyBOP (95.6mg, 0.18mmol,2.0eq), DIEA (41.4mg, 0.32mmol,3.5eq) and 1mL DMF, reacted at room temperature, HPLC monitored, purified, and lyophilized to give compound 27(10.0mg, 20.8%). 1H NMR (CDCl)₃，400Mz) 7.68(d，1H，J＝24Hz)，7.63(s，1H)，5.77-5.59(m，2H)，5.48-5.39(m，1H)，5.30-5.22 (m，1H)，5.19-5.11(m，1H)，3.33-3.10(m，2H)，2.24(s，3H)，1.71-1.63(m，2H)， 1.58-1.52(m，2H)，1.40-1.20(m，6H)，1.05(t，3H，J＝7.2Hz)；LC-MS：[M+H]522.2。

EXAMPLE 4 Synthesis of Compound 28

In a 5mL single neck flask were added R) - (-) -mandelic acid (15.2mg, 0.10mmol, 1.1eq), irinotecan (40mg, 0.09mmol, 1.0eq), PyBOP (95.6mg, 0.18mmol,2.0eq), DIEA (41.4mg, 0.32mmol,3.5eq) and 1mL dmf, reacted at room temperature, HPLC monitored, preparative purified, lyophilized to give compound 28(12.2mg, 23.3%). 1H NMR (CDCl)₃，400Mz)7.76(d，1H，J＝8.0Hz)，7.69(s，1H)，7.53-7.35(m，5H)，5.77-5.70(m，1H)， 5.65-5.55(m，1H)，5.34-5.20(m，4H)，3.32-3.31(m，2H)，2.47-2.40(m，3H)，2.30-2.27 (m，1H)，2.05-2.02(s，1H)，1.93-1.89(m，3H)，1.07(t，3H，J＝8.0Hz)；LC-MS：[M+H]570.2。

EXAMPLE 21 Synthesis of Compound 29

In a 5mL single vial was added 3, 5-difluoromandelic acid (23.7mg, 0.13mmol, 1.1eq), irinotecan (50mg, 0.11mmol, 1.0eq), PyBOP (119.5mg, 0.23mmol,2.0eq), DIEA (37.1mg, 0.29mmol,2.5eq) and 1mL DMF, reacted at room temperature, HPLC monitored, purified, and lyophilized to give compound 29(13.5mg, 19.4%). 1H NMR (DMSO, 400Mz)8.76(d, 1H, J ═ 8.4Hz), 7.81(d, 1H, J ═ 10.8Hz), 7.31(s, 1H), 7.27-7.07(m, 4H), 5.54-5.47(m, 1H), 5.43(s, 2H), 5.20-5.03(m, 4H), 3.20-3.09(m, 2H), 2.43-2.38(m, 3H), 2.17-2.09(m, 1H), 1.96-1.79(m, 3H), 0.88(t, 3H, J ═ 7.2 Hz); LC-MS: [ M + H ] 606.2.

EXAMPLE 22 Synthesis of Compound 30

In a 5mL single vial was added 3, 5-difluoromandelic acid (22.5mg, 0.13mmol, 1.1eq), irinotecan (50mg, 0.11mmol, 1.0eq), PyBOP (119.5mg, 0.23mmol,2.0eq), DIEA (37.1mg, 0.29mmol,2.5eq) and 1mL DMF, reacted at room temperature, HPLC monitored, purified, and lyophilized to give compound 30(8.2mg, 11.7%). 1H NM R (DMSO, 400Mz)8.60(d, 1H, J ═ 8.4Hz), 7.79(d, 1H, J ═ 11.2Hz), 7.31(s, 1H), 7.02-6.90 (m, 2H), 6.90-6.72(m, 1H), 6.05-5.93(m, 2H), 5.52-5.40(m, 2H), 5.19-5.09(m, 1H), 5.09-4.92(m, 2H), 2.98-2.85(m, 2H), 2.22-2.14(m, 3H), 1.94-1.83(m, 1H), 1.75-1.59 (m, 1H), 1.53-1.45(m, 2H), 0.66(t, 3H, J ═ 7.4 Hz); LC-MS: [ M + H ] 614.2.

EXAMPLE 23 Synthesis of Compound 31

S-2-hydroxybutyric acid (16.3mg, 0.16mmol, 1.1eq), irinotecan (68.0mg, 0.16mmol,1.0eq), HATU (59.4mg, 0.16mmol,1.0eq), DIEA (50.5mg, 0.39mmol,2.5eq) and 1mL DMF were added to a 5mL single vial and reacted at room temperature with HPLC monitoring, purified, lyophilized to give compound 31(16.3mg, 20.1%). 1H NMR (DMSO, 400Mz)8.36(d, 1H, J ═ 8.8Hz), 7.79(d, 1H, J ═ 11.2Hz), 7.31(s, 1H), 6.53(s, 1H), 5.60-5.52(m, 1H), 5.46-5.40(m, 3H), 5.24-5.17(m, 2H), 3.23-3.09(m, 2H), 2.45-2.38 (m, 3H), 2.28-2.08(m, 2H), 1.94-1.80(m, 2H), 1.79-1.66(m, 1H), 1.66-1.55(m, 1H), 1.05-0.84(m, 6H); LC-MS: [ M + H ] 522.3.

EXAMPLE 24 camptothecin drug cell Activity assay

The cytotoxic activity of camptothecin drugs was determined by the following experimental procedure: the camptothecin drugs were added to human tumor cell culture media expressed by A431, Fadu, Bxpc-3(EGFR positive expression cells) and U87-MG, SW620 (negative control cells), respectively, and cell viability was determined after 72 hours of cell culture. Cell-based in vitro experiments were used to determine cell viability, cytotoxicity and apoptosis induced by the camptothecin drugs of the invention.

The in vitro efficacy of camptothecin drugs was determined by cell proliferation assay. CellTiter

The aquousOneresolution Cell promotion Assay is commercially available (Promega Corp., Madison, Wis.). CellTiter

The AQueous One Solution Cell Proliferation Assay (a) is a reagent for measuring the number of living cells in Cell Proliferation and cytotoxicity experiments by colorimetry. The reagent contains a novel tetrazolium compound [3- (4, 5-dimethylthiozol-2-yl) -5- (3-carboxymethyloxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt; MTS]And an electron coupling agent (PES). PES has enhanced chemical stability which allows it to be mixed with MTS to form a stable solution. This convenient "single solution" model was in the first generation CellTiter

Improvement on the basis of AQueous Assay, CellTiter

The electron coupling agent PMS used in the AQueous Assay is provided separately from the MTS solution. MTS (Owen's reagent) is bioreduced by cells into a colored formazan product that can be directly dissolved in the culture medium (fig. 1). This conversion is most likely accomplished by the action of NADPH or NADH produced by dehydrogenases in metabolically active cells. When in detection, only a small amount of CellTiter is needed

The AQueous One Solution Reagent is directly added into the culture medium of a culture plate hole, incubated for 1-4 hours and then read the absorbance value of 490nm by a microplate reader.

The amount of formazan product detected at 490nm is directly proportional to the number of viable cells in culture. Since the formazan product of MTS is soluble in tissue culture medium, CellTiter

The AQueous One Solution Assay has fewer steps than the MTT or INT methods.

In the invention, A431, Fadu, Bxpc-3(EGFR positive expression cells), U87-MG and SW620 (negative control cells) are used as a research system for in vitro drug effect detection. In 96-well plates, plating was performed using appropriate cell density, and after 24 hours, camptothecin drug dosing was performed. After 24 hours, diluting camptothecin medicaments (1uM initial, 5 times dilution, 9 concentrations, adding a detection culture medium in the tenth column to be used as a blank control), adding the diluted camptothecin medicaments into corresponding cell holes, then shaking for 3min by a microplate shaker (model: MX100-4A), shaking at the speed of 550rpm/min, and putting the cells into a carbon dioxide incubator to incubate for 3 days after shaking. After 3 days 20ul MTS (Promega, G3581) was added to each well and reacted for 2 hours, and a microplate reader (Molecular Device, model: SpectraMAX190) was read at 490 nM. The proliferation inhibitory effect of camptothecin drugs on cells was evaluated by detecting the activity of dehydrogenase in the mitochondria.

SN38 is a classical highly active camptothecin drug and has been clinically demonstrated in IMMU-132 ADC. The inventor proves that the camptothecin derivative of the invention shows equivalent or higher cell activity than SN38 in representative tumor cells Fadu, BXPC-3, A431, U87-MG and SW620 through cell activity experiments.

EXAMPLE 25 preparation of ADC by coupling

And (3) replacing the antibody molecule C with the monomer rate of more than 95% after primary purification into a phosphate buffer solution with the concentration of 10mg/ml by using an ultrafiltration centrifugal tube. TCEP was added in an amount of 20 mol times the amount of the antibody molecules, and the reaction was carried out at room temperature for 4 hours to open the interchain disulfide bonds of the antibody. The resulting mixture was reacted at room temperature for 2 hours with the addition of 20 mol times of payload as many as the antibody. After the reaction is finished, the solution is changed into PBS by using an ultrafiltration centrifugal tube with the molecular weight cutoff of 30KDa, and the uncoupled payload is removed. The ADC samples after the exchange were filtered using a 0.22 micron sterile filter and ready for use. Compounds 11, 20, 21, 23 and 24 for conjugation were conjugated to antibody molecule C using the conjugation protocol described in example 25.

Compound numbering	ADC numbering obtained by coupling
		11	C-11
20	C-20
		21	C-21
23	C-23
		24	C-24

Example 26 ADC anti-tumor cell Activity assay

Similar to the method for testing the cell activity of camptothecin medicaments, A431, Fadu, Bxpc-3 (antigen positive expression cells) and SW620 (antigen negative control cells) are used as a research system for in-vitro drug effect detection. In 96-well plates, plating was performed using appropriate cell density, and 24 hours later, ADC drug dosing was performed. After 24 hours, the ADC drugs were diluted with the detection medium (1uM initial, 5-fold dilution, 9 concentrations, and detection medium added in the tenth column for blank control), added to the corresponding cell wells, and then shaken with a microplate shaker (model: MX100-4A) for 3min at a shaking speed of 550rpm/min, and then incubated in a carbon dioxide incubator for 3 days after shaking. After 3 days 20ul MTS (Promega, G3581) was added to each well and reacted for 2 hours, and a microplate reader (Molecular Device, model: SpectraMAX190) was read at 490 nM. The cell proliferation inhibitory effect of the ADC drug was evaluated by detecting the activity of dehydrogenase in the mitochondria.

Through the ADC cell activity test, the camptothecin medicament disclosed by the invention shows good anti-tumor activity in a plurality of antigen positive tumor cell lines after being coupled with an antibody through the connecting unit L, and has great clinical application value.

Example 27 ADC in vivo drug efficacy testing

In the invention, an A431 tumor-bearing mouse model is established to evaluate toxin ADCThe in vivo efficacy of the coupled drug is 3 × 10⁶The A431 cells are injected to the right side of BALB/C nude mice aged 4-6 weeks subcutaneously, when the average tumor size of the mice grows to 140-150 mm3, the mice are randomly grouped, 5 mice in each group are respectively administered with blank control (buffer solution blank) and antibody drug conjugate C-11 at 0,7,14 and 21 days, and the intravenous administration is carried out at a dose of 10 mg/kg. Tumor volume measurements are shown as mean tumor volume at the time of measurement ± SE, and changes in body weight of mice are recorded to observe preliminary in vivo toxicity of ADC drugs.

Through the in vivo efficacy experiment of the ADC mouse, the camptothecin medicament disclosed by the invention shows definite anti-tumor activity in a tumor-bearing mouse after being coupled with an antibody through the connecting unit L, and the average tumor body is obviously lower than that of a blank control. The body weight of the mice has no obvious change in the administration period, no mice in the group die, and the camptothecin medicament has good safety.

Claims

1. A camptothecin compound represented by formula I or a pharmaceutically acceptable salt thereof:

wherein R is₁And R₂Each independently selected from C₁-C₃Alkyl or substituted alkyl, -H, -CF₃Aryl, substituted aryl or heteroaryl; or R₁、R₂Together with the carbon atom to which it is attached, form cyclobutane, cyclopentane or cyclohexane; r₁、R₂Not hydrogen at the same time.

2. Camptothecin compounds according to claim 1Or a pharmaceutically acceptable salt thereof, characterized in that: r₁Is hydrogen, R₂Is C₁-C₃Alkyl, -CF₃Aryl, substituted aryl or heteroaryl; or R₁And R₂Is C₁-C₃Alkyl, -CF₃Aryl, heteroaryl or substituted aryl; or R₁、R₂Together with the carbon atoms to which they are attached, constitute cyclobutane, cyclopentane or cyclohexane:

in the structural formula (a), R₂Independently is- (CH)₂)n¹-CH₃、-CF₃Aryl, heteroaryl, substituted aryl, wherein n is¹0, 1 or 2;

in the structural formula (b), R₁And R₂Independently is- (CH)₂)n¹-CH₃、-CF₃Aryl, heteroaryl, substituted aryl, wherein n is¹0, 1 or 2;

in the formula (c), R₁、R₂Together with the carbon atom to which they are attached, to form cyclobutane, cyclopentane, cyclohexane, n²1, 2 or 3.

3. The camptothecin-based compound, or a pharmaceutically acceptable salt thereof, of claim 2 wherein: r₁Is hydrogen, R₂Independently is- (CH)₂)n¹-CH₃、-CF₃Aryl, heteroaryl or substituted aryl, wherein n is¹0, 1 or 2:

R₂the attached carbon has two configurations of R, S,

4. A camptothecin compound, or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 3 wherein: the aryl substituents are selected from halogen, alkyl, alkoxy, hydroxy, nitro, amino, hydroxy or cyano.

5. The camptothecin-based compound or pharmaceutically acceptable salt thereof of claim 1, wherein: selected from the following structures:

6. an antitumor agent comprising the camptothecin compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein: can be used for treating solid tumor or blood tumor such as lung cancer, renal cancer, urethral cancer, colon cancer, rectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, lung cancer or esophageal cancer.

7. An antibody drug conjugate of formula II, which exerts its pharmacological effect by releasing drug D upon reaching a target cell:

wherein Ab is an antibody, antibody fragment or protein;

l is an optional linker unit, one end linked to Ab and one end linked to drug D;

d is selected from the camptothecin compounds or pharmaceutically acceptable salts thereof according to any one of claims 1 to 6, which is connected with L through hydroxyl in the molecule;

m is an integer from 1 to 20.

8. The antibody drug conjugate of claim 7, wherein the antibody drug conjugate is a conjugate of the antibody drug and the antibody drugThe method comprises the following steps: the linking unit L structurally comprises a chemical bond selected from-O-, -N (R) n₁-，-CH₂-，-CH(R)n₁-, amides, ester bonds, -S-, - (PEG) n₂-group of; n is₁Is selected from the integers 1-3, n₂Is selected from an integer of 1-20.

9. The antibody drug conjugate of claim 7 or 8, wherein: the antibody of the drug-ligand conjugate specifically binds to target cells of cancer, autoimmune diseases.

10. A medicament comprising an antibody-conjugated drug according to any one of claims 7 to 9, wherein: can be used for treating solid tumor or blood tumor such as lung cancer, renal cancer, urethral cancer, colon cancer, rectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, hepatocarcinoma, bladder cancer, gastric cancer, esophageal cancer, etc.