Preparation and application of immune agonist antibody coupling medicine

Abstract

The invention relates to an immune agonist antibody coupling medicine and application thereof. Specifically, the invention relates to a compound represented by a formula (I) or an isotope labeled compound thereof, or an optical isomer, a geometric isomer, a tautomer or an isomer mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, and an application thereof in preparation of a drug for treating or preventing cancers, especially an antibody coupling drug. And P-L-D (I).

Description

Technical Field

[0001] This application relates to the field of biomedicine, and particularly to an immune agonist antibody conjugate and its use in cancer treatment. Background Art

[0002] Stimulator of interferon genes (STING), as a key signal transduction molecule involved in the innate immune response, is triggered by cytoplasmic DNA from pathogens and the host, and plays an important role in inducing the secretion of type I interferons and pro-inflammatory cytokines, defending against viral and intracellular bacterial infections, and regulating the generation of spontaneous anti-tumor immune responses in the body. The mechanism of action of immune agonists can include the following aspects: stimulating the activation and proliferation of immune cells, enhancing the defense response of the immune system. Inducing immune cells to produce more cytokines and chemokines, which can further activate immune cells and trigger an inflammatory response. Enhancing the function of NK cells, making them more effectively recognize and kill virus-infected cells or abnormal cells. Stimulating the immune system to produce antibodies, which can recognize and neutralize viruses, bacteria, and other pathogens to prevent them from infecting cells.

[0003] Immune agonist antibody conjugate (ISAC) is an antibody conjugate with immune-stimulating effects. It achieves systemic administration and tumor-targeted administration by conjugating an immune agonist with an antibody, overcoming the limitations of small molecule immune agonists in intratumoral or intravenous injection. The antibody part can recognize antigens on the surface of tumor cells and target the drug to tumor tissues; the immune agonist part can activate immune cells in the tumor microenvironment and enhance the anti-tumor immune response. The purpose of designing this drug is to simultaneously achieve targeted therapy and immune activation, thereby maximizing the therapeutic effect and reducing side effects.

[0004] The design and development of immune agonist antibody conjugates face many technical difficulties, such as the selection of antibodies, the payload of drugs, the conjugation method, and poor water solubility, etc. In response to these difficulties, researchers have been continuously exploring and optimizing to achieve the best therapeutic effect. Summary of the Invention

[0005] The content of the present invention is mainly to chemically bond a small molecule immune agonist with immune activation effects to a water-soluble linker, and then conjugate it with an antibody to form a novel drug with targeting effects.

[0006] In a first aspect, the present application prepares a compound of formula (I), or its isotope-labeled compound, or its optical isomer, geometric isomer, tautomer or mixture of isomers, or its pharmaceutically acceptable salt or solvate,

[0007] P-L-D(I)

[0008] Among them,

[0009] P has the following structure:

[0010]

[0011] m and n are each independently selected from any integer from 0 to 12;

[0012] X is selected from a bond, nitrogen, oxygen, sulfur, phosphorus, C1-C6 alkylene, imino, ester group, amide group, carbonate group, carbamate group, sulfone group, ketone group, urea group or phosphate group;

[0013] L has the following structure:

[0014]

[0015] n1 and n2 are each independently selected from any integer from 1 to 6;

[0016] R1 is selected from hydrophilic groups;

[0017] R2 and R3 are each independently selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, or R2 and R3 together with the carbon atom to which they are attached form a C3-C6 cycloalkyl, wherein one or more hydrogen atoms in the C1-C6 alkyl, C1-C6 haloalkyl or C3-C6 cycloalkyl are optionally substituted by halogen, hydroxyl, cyano, amino, C1-C3 alkyl or phenyl; and

[0018] D is an immune agonist molecule or other equivalent.

[0019] In one embodiment of the present invention, the hydrophilic group has at least three ether bonds and / or hydroxyl groups composed of oxygen-containing groups. Preferably, the hydrophilic group has any one of the following structures:

[0020]

[0021] In one embodiment of the present invention, R2 and R3 are each independently selected from H, F, Cl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or R2 and R3 together with the carbon atom to which they are attached form cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, wherein one or more hydrogen atoms in the methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl are optionally substituted by halogen, hydroxyl, cyano, amino, C1-C3 alkyl or phenyl.

[0022] In one embodiment of the present invention, the payload molecule is an immune agonist. Among them, the payload molecule is a cyclic dinucleotide, anthrone, aminobenzimidazole, 3-imidazole-pyridazine, benzothiophene, or triazole quinoxaline immune agonist molecule.

[0023] In a preferred embodiment of the present invention, the payload molecule is an aminobenzimidazole derivative having the following structure:

[0024]

[0025] For the sake of simplicity, the "compound of formula (I)" or "compound of the present application" mentioned hereinafter may also cover any isotopically labeled compound of the compound of formula (I), or its optical isomers, geometric isomers, tautomers or mixtures of isomers, or its pharmaceutically acceptable salts or solvates as the coupling precursor of the conjugate.

[0026] The term "optical isomers" means that when a compound has one or more chiral centers, each chiral center can exist in the R configuration or the S configuration, and the various isomers thus formed are optical isomers. Optical isomers include all diastereomers, enantiomers, meso forms, racemates or mixtures thereof. For example, optical isomers can be separated by a chiral chromatographic column or by chiral synthesis.

[0027] The term "geometric isomers" means that when a double bond exists in a compound, the compound can exist in cis isomers, trans isomers, E isomers and Z isomers. Geometric isomers include cis isomers, trans isomers, E isomers, Z isomers or mixtures thereof.

[0028] The term "tautomers" refers to isomers resulting from the rapid movement of a certain atom in a molecule between two positions. Those skilled in the art can understand that tautomers can be converted into each other and may coexist in a certain state in an equilibrium state.

[0029] Unless otherwise specified, when referring to the "compound of formula (I)" or "compound of the present invention" herein, it also covers isotopically labeled compounds obtained by replacing any atom in the compound with its isotopic atom. The present invention includes all pharmaceutically acceptable isotopically labeled compounds of the compound of formula (I), wherein one or more atoms are replaced by atoms having the same atomic number as the atoms usually found in nature but different atomic masses or mass numbers.

[0030] Examples of isotopes applicable to be included in the compounds of the present invention include isotopes of hydrogen, such as 2 H(D) and 3 H(T), isotopes of carbon, such as 11C, 13 C and 14 C, an isotope of chlorine, such as 37 Cl, an isotope of fluorine, such as 18 F, an isotope of iodine, such as 123 I and 125 I, an isotope of nitrogen, such as 13 N and 15 N, an isotope of oxygen, such as 15 O, 17 O and 18 O, and an isotope of sulfur, such as 35 S.

[0031] The isotopically labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by using appropriate isotopically labeled reagents in place of the previously used non-labeled reagents in a manner similar to the methods described in the examples and preparations appended hereto.

[0032] The compounds of formula (I) can exist in the form of pharmaceutically acceptable salts, for example, acid addition salts and / or base addition salts of the compounds of formula (I). Unless otherwise specified, the "pharmaceutically acceptable salts" used herein include acid addition salts or base addition salts that can occur in the compounds of formula (I).

[0033] Pharmaceutically acceptable salts of the compounds of formula (I) include their acid addition salts and base addition salts. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples thereof include, but are not limited to: acetates, adipates, aspartates, benzoates, benzenesulfonates, bicarbonates / carbonates, bisulfates / sulfates, borates, camphorsulfonates, citrates, cyclohexanesulfonates, ethanedisulfonates, formates, fumarates, glucoheptanoates, gluconates, glucuronates, hexafluorophosphates, 2-(4-hydroxybenzyl)benzoates, hydrochlorides / chlorides, hydrobromides / bromides, hydroiodides / iodides, 2-hydroxyethanesulfonates, lactates, malates, maleates, malonates, methanesulfonates, methylsulfates, naphthalenecarboxylates, 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, palmitates, phosphates / hydrogen phosphates / dihydrogen phosphates, pyroglutamates, glucosaccharinates, stearates, salicylates, tannates, tartrates, toluenesulfonates and trifluoroacetates. Suitable base addition salts are formed from bases which form non-toxic salts. Examples thereof include, but are not limited to: aluminum, arginine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases can also be formed, such as hemisulfates and hemicalcium salts. For a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection and Use by Stahl and Wermuth (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds described herein are known to those skilled in the art.

[0034] Certain compounds of the present invention may exist in non-solvated form as well as in solvated forms (including hydrated forms). In general, whether the compounds of formula (I) exist in solvated form or in unsolvated form, they are included within the scope of the present invention.

[0035] Certain compounds of the present invention may exist in different crystalline forms or amorphous forms, and regardless of the form, the compounds of formula (I) are included within the scope of the present invention.

[0036] To avoid ambiguity, definitions of the terms used herein are given below. Unless otherwise indicated, the meanings of the terms used herein are as follows.

[0037] The term "pharmaceutically acceptable" means that the corresponding compound, carrier or molecule is suitable for administration to humans. Preferably, the term means that it is certified by a regulatory agency such as CFDA (China), EMEA (Europe), FDA (USA) or any other national regulatory agency for use in mammals, preferably humans.

[0038] "Prodrug" refers to a derivative that is converted into a compound of the present invention in vivo under physiological conditions, such as by oxidation, reduction, hydrolysis, etc. catalyzed by enzymes, gastric acid, etc.

[0039] "Metabolite" refers to all molecules derived from any compound of the present invention in cells or organisms, preferably in humans.

[0040] As used herein, the term "substituted" means that one or more (preferably 1 to 5, more preferably 1 to 3) hydrogen atoms in a group are independently replaced by a corresponding number of substituents.

[0041] As used herein, the term "independently" means that when the number of substituents exceeds one, these substituents can be the same or different.

[0042] As used herein, the term "optionally" or "optionally" means that the event it describes can occur or not occur. For example, a group "optionally substituted" means that the group can be unsubstituted or substituted.

[0043] As used herein, the term "alkyl" refers to saturated aliphatic hydrocarbons, including straight-chain and branched-chain. In some embodiments, the alkyl group has 1-8, or 1-6, or 1-3 carbon atoms. For example, the term "C 1-8 alkyl" refers to a straight-chain or branched-chain group having 1-8 carbon atoms. The term "C 1-8 alkyl" includes the terms "C 1-6 alkyl", "C1-C3 alkyl", and "C1-C4 alkyl" in its definition. Examples of alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, isopentyl, neopentyl, (R)-2-methylbutyl, (S)-2-methylbutyl, 3-methylbutyl, 2,3-dimethylpropyl, 2,3-dimethylbutyl, hexyl, etc. The alkyl group can be optionally substituted with one or more (e.g., 1 to 5) suitable substituents.

[0044] As used herein, the term "haloalkyl" refers to an alkyl group having one or more halogen substituents (up to perhaloalkyl, i.e., each hydrogen atom of the alkyl group is replaced by a halogen atom). For example, the term "C1-C6 haloalkyl" refers to a C 1-6 alkyl group having one or more halogen substituents (up to perhaloalkyl, i.e., each hydrogen atom of the alkyl group is replaced by a halogen atom). Another example, the term "C1-C4 haloalkyl" refers to a C 1-4 alkyl group having one or more halogen substituents (up to perhaloalkyl, i.e., each hydrogen atom of the alkyl group is replaced by a halogen atom); the term "C 1-3"Halogenated alkyl" refers to a C 1-3 alkyl group having one or more halogen substituents (up to a perhalogenated alkyl group, i.e., each hydrogen atom of the alkyl group is replaced by a halogen atom); and the term "C 1-2 halogenated alkyl" refers to a C 1-2 alkyl group (i.e., methyl or ethyl) having one or more halogen substituents (up to a perhalogenated alkyl group, i.e., each hydrogen atom of the alkyl group is replaced by a halogen atom). As another example, the term "C1 halogenated alkyl" refers to a methyl group having 1, 2, or 3 halogen substituents. Examples of halogenated alkyl groups include: CF3, C2F5, CHF2, CH2F, CH2CF3, CH2Cl, etc.

[0045] As used herein, the term "C3-C6 cycloalkyl" refers to a cycloalkyl group having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0046] In this article, the number ranges related to the number of substituents, the number of carbon atoms, and the number of ring atoms represent the individual listing of all integers within that range, and the range is only used as a simplified notation. For example: "1-4 substituents" means 1, 2, 3, or 4 substituents; "3-8 ring atoms" means 3, 4, 5, 6, 7, or 8 ring atoms. Therefore, the number ranges related to the number of substituents, the number of carbon atoms, and the number of ring atoms also cover any of its sub-ranges, and each sub-range is also considered to be disclosed herein.

[0047] The compounds of the present application can be prepared in a variety of ways known to those skilled in the art of organic synthesis. Those skilled in the art can refer to the synthesis routes of specific compounds in the specific examples of the present application and appropriately adjust the reaction raw materials and reaction conditions to obtain the synthesis methods of other compounds.

[0048] In a second aspect, the present application provides a conjugate, which includes a compound of formula (I) according to the foregoing or an isotope-labeled compound thereof, or an optical isomer, geometric isomer, tautomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof as a conjugate precursor of the conjugate.

[0049] In one embodiment of the present invention, the conjugate includes a targeting moiety. As used herein, the term "targeting moiety" is a chemical moiety capable of binding to a biological entity, such as an antibody, a synthetic functionalized antibody, an enzyme, a protein or peptide, or any other targeting ligand. The targeting moiety can localize a therapeutic agent or a small molecule to a specific target site, such as a tumor or tissue. This can effectively increase the efficacy of the therapeutic agent at the target site while minimizing unwanted side effects on normal cells.

[0050] In one embodiment of the present invention, the targeting moiety may include but is not limited to an antibody or synthetically functionalized antibody specific for an antigen selected from the group consisting of HER2, EGFR, Claudin18.2, c-MET, Nectin-4, ROR1, GPNMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-α, mesothelin, ENPP3, guanylate cyclase C, SLC44A4, NaPi2b, CD70, mucin 1, STEAP1, connexin 4, 5T4, SLTRK6, SC-16, LIV-1, P-cadherin, PSMA, fibronectin extra domain B, endothelin receptor ETB, tenascin c, collagen IV, VEGFR2, periostin, CD30, CD79b, CD19, CD22, CD138, CD37, CD33, CD74, CD19, and CD98. In a preferred embodiment of the present invention, the targeting moiety may be trastuzumab or synthetically functionalized trastuzumab.

[0051] In one embodiment of the present invention, the targeting moiety may form free thiols (i.e., via -S-) by reduction of the interchain disulfide bonds for conjugation with the maleimide group of the compound of formula (I). In a preferred embodiment of the present invention, the DAR value of the antibody conjugate is any integer or decimal from 1 to 10, preferably 4 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any decimal therebetween; preferably, the DAR value is 4 to 8, more particularly 6.9, 7.45, etc.). As used herein, the term "DAR value" refers to the average number of toxin molecules conjugated to each monoclonal antibody.

[0052] In a third aspect, the present application provides a pharmaceutical composition comprising a compound of formula (I) or its isotopically labeled compound, or its optical isomer, geometric isomer, tautomer, or mixture of isomers, or its pharmaceutically acceptable salt, or its prodrug, or its metabolite, or the above conjugate; and a pharmaceutically acceptable carrier.

[0053] The pharmaceutically acceptable carrier may be an organic or inorganic inert carrier material. For example, suitable carriers include water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oil, polyalkylene glycol, petrolatum, mannitol, cellulose, cellulose derivatives, saccharin sodium, glucose, sucrose, magnesium carbonate, saline, glycerol, ethanol, etc. In addition, the pharmaceutical composition may further contain other pharmaceutical additives, such as flavoring agents, preservatives, stabilizers, emulsifying agents, buffering agents, diluents, binders, wetting agents, disintegrants, lubricants, glidants, etc.

[0054] The dosage form of the pharmaceutical composition of the present application can be a liquid dosage form, a solid dosage form or a semi-solid dosage form. The liquid dosage form can be a solution (including true solution and colloidal solution), an emulsion (including o / w type, w / o type and multiple emulsion), a suspension, an injection (including aqueous injection, powder injection and infusion), an eye drop, a nasal drop, a lotion, a liniment, etc.; the solid dosage form can be a tablet (including ordinary tablet, enteric-coated tablet, buccal tablet, dispersible tablet, chewable tablet, effervescent tablet, orally disintegrating tablet), a capsule (including hard capsule, soft capsule, enteric-coated capsule), a granule, a powder, a pill, a suppository, a film, a patch, an aerosol, a spray, etc.; the semi-solid dosage form can be an ointment, a gel, a paste, etc. The pharmaceutical composition of the present application can be made into an ordinary preparation, and can also be made into a sustained-release preparation, a controlled-release preparation, a targeted preparation and various particulate drug delivery systems.

[0055] In some embodiments, the dosage form of the pharmaceutical composition is selected from tablets, capsules, granules, powders, syrups, inhalants and injections.

[0056] Solid dosage forms for oral administration can include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert excipient (or carrier) (e.g., sodium citrate or dibasic calcium phosphate), which may further include: (a) fillers or extenders (e.g., starch, lactose, sucrose, glucose, mannitol and silicic acid); (b) binders (e.g., carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia); (c) humectants (e.g., glycerol); (d) disintegrants (e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain synthetic silicate esters, sodium carbonate); (e) solution retarders (e.g., paraffin); (f) absorption promoters (e.g., quaternary ammonium compounds); (g) wetting agents (e.g., cetyl alcohol and glyceryl monostearate); (h) adsorbents (e.g., kaolin and bentonite) and (i) lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate) or mixtures thereof.

[0057] Preparations suitable for parenteral administration, such as injections, can include aqueous and non-aqueous isotonic sterile solutions suitable for injection, as well as aqueous and non-aqueous sterile suspensions. The parenteral preparations provided herein are optionally contained in unit dose or multi-dose sealed containers (e.g., ampoules), and can be stored under lyophilized (freeze-dried) conditions that require only the addition of a sterile liquid carrier (e.g., water for injection) immediately prior to use. Examples of suitable diluents for reconstituting the pharmaceutical composition (e.g., prior to injection) include bacteriostatic water for injection, 5% aqueous glucose solution, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized water and combinations thereof.

[0058] The spray may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or a mixture of these substances. The spray may additionally contain conventional propellants, such as chlorofluorocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. The inhalant may contain an excipient such as lactose, or an aqueous solution containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or an oily solution administered as nasal drops or spray, or in the form of a gel.

[0059] The content of the compounds of the present application in their pharmaceutical compositions can be adjusted according to actual needs (such as dosage form, administration method, administration object, etc.), for example, 0.1-95% by weight, such as 1-95% by weight, 5-90% by weight, 10-80% by weight, etc.

[0060] Specifically, the pharmaceutical composition of the present application may particularly contain 0.001-10 g (such as 0.05 g, 0.1 g, 0.5 g, 1 g or 5 g, etc.) of the compound of the present application.

[0061] In a fourth aspect, the present application provides the use of a compound of formula (I) or its isotope-labeled compound, or its optical isomer, geometric isomer, tautomer or isomer mixture, or its pharmaceutically acceptable salt, or its prodrug, or its metabolite, or the above conjugate in the preparation of a drug for treating or preventing cancer in a subject in need thereof. The compound of formula (I) or its isotope-labeled compound, or its optical isomer, geometric isomer, tautomer or isomer mixture, or its pharmaceutically acceptable salt, or its prodrug, or its metabolite, or the above conjugate can be used to treat or prevent the said cancer in a subject in need thereof.

[0062] The term "subject" used in the present application refers to any human or non-human organism that can potentially benefit from treatment with a compound of formula (I). Exemplary subjects include humans or mammals of any age. Preferably, the subject is a human.

[0063] The term "treatment" as used herein includes treating a disease or symptom in a mammal, particularly a human, and includes: (a) inhibiting an infection, disease or symptom, i.e., curbing or delaying the development of an infection, disease or symptom; (b) alleviating an infection, disease or symptom, i.e., causing the subsidence of a disease or symptom, and / or (c) curing an infection, disease or symptom.

[0064] As used herein, the term "prevention" includes prophylactic therapies in mammals, particularly humans, aimed at reducing the likelihood of infection, disease, or symptom occurrence. Patients receiving prophylactic therapies can be selected based on factors such as an increased risk of infection or disease or symptoms compared to the general population. "Prevention" can include treating subjects who have not yet presented with infection or a clinical condition and preventing a second occurrence of the same or a similar infection or clinical condition.

[0065] In one embodiment of the present invention, the cancer can be various cancers known in the art, including but not limited to, for example, bone cancer, brain cancer, breast cancer, cervical cancer, laryngeal cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, spinal cancer, gastric cancer, uterine cancer, and / or lymphoma, etc. In a preferred embodiment of the present invention, the cancer can be a HER2-positive cancer. In a more preferred embodiment of the present invention, the HER2-positive cancer can be a cancer in which HER2 is overexpressed. In a further preferred embodiment of the present invention, the cancer can be breast cancer.

[0066] In a fifth aspect, the present application provides a method for treating or preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the above-mentioned drug or pharmaceutical composition of the present invention.

[0067] In some embodiments, the compounds of the present invention can be administered by oral, parenteral, intravenous, intramuscular, subcutaneous, nasal, buccal mucosa, ocular, pulmonary, respiratory, vaginal, rectal, intraperitoneal, intralesional, perilesional, and other routes.

[0068] "Therapeutically effective amount" means an amount of the compounds of the present application that is effective in treating cancer diseases or symptoms when administered alone or in combination.

[0069] The specific dosage administered will depend on the route of administration, the severity of the disease, the age and weight of the patient, and other factors typically considered by the attending physician in determining the individual regimen and dosage level most suitable for a particular patient. For example, the daily dosage of the compounds of the present application can particularly be 0.001 - 150 mg / kg body weight (such as 0.1 mg / kg body weight, 1 mg / kg body weight, 10 mg / kg body weight, or 100 mg / kg body weight, etc.).

[0070] The specific frequency of administration can be determined by those skilled in the relevant art, for example, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once every four weeks, twice a day, three times a day, etc.

[0071] Those skilled in the art can understand that the definitions and preferences described in one aspect of the present application are equally applicable to other aspects. Those skilled in the art can understand that the embodiments of various aspects of the present application can be combined in various ways without departing from the theme and idea of the present application, and these combinations are also within the scope of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. They are used together with the following specific embodiments to explain the present invention, but do not constitute a limitation to the present invention. In the accompanying drawings:

[0073] Figure 1 Dose-response curve of the binding activity to HER2 antigen when the final concentration range of naked anti-trastuzumab or antibody-immunostimulant conjugate drug is 0.011 - 11400 ng / mL

[0074] Figure 2 Dose-response curve of the affinity to SK-BR-3 cells when the final concentration range of naked anti-trastuzumab or antibody-immunostimulant conjugate drug is 0.01 - 20000 ng / mL

[0075] Figure 3 Dose-response curve of the affinity to human STING protein when the final concentration range of STING agonist or antibody-immunostimulant conjugate drug is 0.00038 - 10000 nM

[0076] Figure 4 Activation when co-culturing SK-BR-3 / THP1-Dual luciferase reporter gene cells and the final concentration range of STING agonist or antibody-immunostimulant conjugate drug is 0.03 - 200 nM

[0077] Figure 5 Activation when culturing THP1-Dual luciferase reporter gene cells alone and the final concentration range of STING agonist or antibody-immunostimulant conjugate drug is 0.03 - 200 nM

[0078] Figure 6 Cytotoxicity to SK-BR-3 cells when co-culturing SK-BR-3 cells and PBMC cells and the final concentration range of naked anti-trastuzumab or antibody-immunostimulant conjugate drug is 0.00256 - 100 nM DETAILED DESCRIPTION OF THE EMBODIMENTS

[0079] The compounds of formula (I) of the present application can be synthesized by various methods familiar to those skilled in the art of organic synthesis. The following specific examples give some synthetic methods of exemplary compounds of formula (I), which are well-known in the field of synthetic chemistry. Obviously, referring to the exemplary schemes in this patent, those skilled in the art can easily design other synthetic routes of compounds of formula (I) by appropriately adjusting the reactants, reaction conditions and protecting groups.

[0080] The present invention will be further illustrated below in conjunction with examples; however, these examples do not limit the scope of the present invention. Unless otherwise stated, all reactants used in each example are obtained commercially; the instruments and equipment used in synthetic experiments and product analysis and detection are all conventional instruments and equipment commonly used in organic synthesis.

[0081] The structure of the compound was determined by liquid chromatography-mass spectrometry (LCMS). The LCMS determination was performed using a Waters (ESI) mass spectrometer (model: Acquity H-Class liquid chromatography-mass spectrometry instrument). The HPLC determination was performed using an Agilent 1260 infinity II high-pressure liquid chromatography instrument. The preparative liquid phase was carried out using a Waters Prep-HPLC high-pressure liquid chromatography preparative chromatograph (Unitary C18 250×20mm chromatographic column). The normal-phase preparation was carried out using a Biotage Selekt chromatography purification system.

[0082] The abbreviations used herein have the definitions shown in Table 1 below.

[0083] Table 1

[0084]

[0085]

[0086]

[0087] Example 1 Preparation of Compound DL-1

[0088]

[0089] Step 1: Synthesis of 3-((tert-butoxycarbonyl)amino)-5-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-5-oxopentanoic acid

[0090] Compound 1 (50 mg, 0.202 mmol) and HATU (76 mg, 0.202 mmol) were added to N,N-dimethylformamide (3 mL), and the mixture was stirred at room temperature for 10 minutes. Then, N-methyl-D-glucamine (39 mg, 0.202 mmol) was added and stirred for 10 min. Finally, DIEA (104 mg, 0.808 mmol) was added and the mixture was stirred at room temperature for 1 hour. The reaction was considered complete when the amount of Compound 1 in the reaction solution was less than 3% as determined by LCMS. The solvent of the reaction solution was evaporated under reduced pressure, and the residue was purified by preparative liquid chromatography using acetonitrile / water as the mobile phase. Compound 2 (white solid, 70 mg, yield 81.4%) was obtained. MS: 425.45 [M+H] + 。

[0091] Step 2: Synthesis of 3-amino-5-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-5-oxopentanoic acid

[0092] Compound 2 (70 mg, 0.165 mmol) was added to dichloromethane (3 mL) and trifluoroacetic acid (1 mL), and the mixture was reacted at room temperature for 30 mins. The reaction was monitored by LCMS until the starting material Compound 2 was completely consumed. The solvent was removed by distillation under reduced pressure, and the residue was purified by preparative liquid chromatography using acetonitrile / water as the mobile phase. Compound 3 (white solid, 30 mg, yield 55.6%) was obtained. MS: 324.35 [M+H] + 。

[0093] Step 3: Synthesis of 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-33-(2-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-2-oxoethyl)-3,31-dioxo-7,10,13,16,19,22,25,28-octaoxa-4,32-diazapentacontane-35-carboxylic acid

[0094] Compound 4 (55 mg, 0.093 mmol) and HATU (42 mg, 0.112 mmol) were added to N,N-dimethylformamide (3 mL), and the mixture was stirred at room temperature for 10 minutes. Then, Compound 3 (30 mg, 0.093 mmol) was added and stirred for 10 min. Finally, DIEA (48 mg, 0.372 mmol) was added and the mixture was stirred at room temperature for 2 hours. The reaction was considered complete when the amount of Compound 4 in the reaction solution was less than 3% as determined by LCMS. The solvent of the reaction solution was evaporated under reduced pressure, and the residue was purified by preparative liquid chromatography using acetonitrile / water as the mobile phase. Compound 5 (white solid, 42 mg, yield 50.3%) was obtained. MS: 898.97 [M+H] + 。

[0095] Step 4: Synthesis of (E)-3-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-methyl-3-pyrazole-5-acetamido)-1H-benzo[d]imidazol-7-yl)oxy)propyl(tert-butoxycarbonyl)-L-alanine

[0096] Place Boc-L-alanine (2.5 mg, 0.013 mmol) and HATU (6 mg, 0.016 mmol) in a 25 mL single-necked flask, then add N,N-dimethylformamide (3 mL), stir at room temperature for 10 min, then add compound 6 (5 mg, 0.007 mmol), stir evenly, and then add DIEA (7 mg, 0.052 mmol). Stir at room temperature for 3 hours. When LCMS shows that the amount of compound 6 in the reaction solution is less than 3%, the reaction is considered complete. Evaporate the solvent of the reaction solution under reduced pressure, and purify the crude product by preparative liquid chromatography with acetonitrile / water. Compound 7 (white solid, 6 mg, yield 90.2%) is obtained. MS: 923.02 [M+H] +

[0097] Step 5: Synthesis of (E)-3-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-methyl-3-methyl-1H-pyrazole-5-acetamido)-1H-benzo[d]imidazol-7-yl)oxy)propyl L-alanine

[0098] Dissolve compound 7 (6 mg, 0.007 mmol) completely in dichloromethane (3 mL), then add HCl / 1,4-dioxane solution (0.5 mL, 4 M), stir at room temperature for 1 hour. When LCMS shows that the amount of compound 7 in the reaction solution is less than 3%, the reaction is considered complete. Evaporate the solvent of the reaction solution under reduced pressure to obtain crude compound 8 (7 mg, yield 130.8%). MS: 822.90 [M+H] +

[0099] Step 6: Synthesis of 3-((5-carbamoyl-1-((E)-4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-methyl-3-methyl-1H-pyrazole-5-acetamido)-1H-benzo[d]imidazol-7-yl)oxy)propyl (1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-33-(2-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-2-oxoethyl)-3,31-dioxo-7,10,16,19,22,25,28-octaoxa-4,32-diazapentacosa-35-yl)-L-alanine

[0100] Take compound 5 (4 mg, 0.0045 mmol) and HATU (2 mg, 0.0054 mmol) and place them in a 10 mL single-necked flask. Then add N,N-dimethylformamide (2 mL) and stir at room temperature for 10 min. After that, add compound 8 (3.7 mg, 0.0045 mmol). After stirring evenly, add DIEA (2.3 mg, 0.018 mmol) and stir at room temperature for 2 hours. When the amount of compound 8 in the reaction solution is less than 3% as determined by LCMS, the reaction is considered complete. Evaporate the solvent under reduced pressure from the reaction solution, and purify the crude product by preparative liquid chromatography with acetonitrile / water. Lyophilize to obtain compound DL-1 (white solid, 2 mg, yield 26.1%). MS: 1703.84 [M+H] +

[0101] Preparation of Compound DL-2 in Example 2

[0102]

[0103] Step 1: Synthesis of 14-((tert-butoxycarbonyl)amino)-12-oxo-2,5,8-trioxa-11-azapentadecanoic acid

[0104] Place compound 1 (113 mg, 0.461 mmol) and HATU (175 mg, 0.461 mmol) in a 25 mL single-necked flask. Then add N,N-dimethylformamide (3 mL) and stir at room temperature for 10 min. After that, add amino triglycol monomethyl ether (50 mg, 0.307 mmol). After stirring evenly, add DIEA (158 mg, 1.228 mmol) and stir at room temperature for 2 hours. When the amount of compound 1 in the reaction solution is less than 3% as determined by LCMS, the reaction is considered complete. Evaporate the solvent under reduced pressure from the reaction solution, and purify the crude product by preparative liquid chromatography with acetonitrile / water. Obtain compound 9 (white solid, 100 mg, yield 83.4%). MS: 393.45 [M+H] +

[0105] Step 2: Synthesis of 14-amino-12-oxo-2,5,8-trioxo-11-azahexadecanoic acid

[0106] Compound 9 (100 mg, 0.255 mmol) was added to dichloromethane (4 mL) and dissolved completely. Then, HCl / 1,4-dioxane solution (2 mL, 4 M) was added, and the reaction was stirred at room temperature for 0.5 h. The reaction was considered complete when the amount of compound 9 in the reaction solution was less than 3% as determined by LCMS. The solvent of the reaction solution was evaporated under reduced pressure to obtain crude compound 10 (72 mg, yield 97.3%). MS: 293.33 [M+H] +

[0107] Step 3: Synthesis of 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,31-dioxo-33-(12-oxo-25,8-trioxa-11-azatridecan-13-yl)-7,10,13,16,19,22,25,28-octaoxa-4,32-diazapentacosan-35-oic acid

[0108] Compound 4 (50 mg, 0.084 mmol) and HATU (38 mg, 0.101 mmol) were placed in a 25 mL single-necked flask. Then, N,N-dimethylformamide (4 mL) was added, and the mixture was stirred at room temperature for 10 min. Then, compound 10 (25 mg, 0.084 mmol) was added and stirred evenly, followed by the addition of DIEA (43 mg, 0.336 mmol). The reaction was stirred at room temperature for 2 h. The reaction was considered complete when the amount of compound 4 in the reaction solution was less than 3% as determined by LCMS. The solvent of the reaction solution was evaporated under reduced pressure, and the crude product was purified by preparative liquid chromatography using acetonitrile / water. Compound 11 (white solid, 46 mg, yield 63%) was obtained. MS: 867.96 [M+H] +

[0109] Step 4: Synthesis of 3-((5-carbamoyl-1-((E)-4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-methyl-3-methyl-1H-pyrazole-5-acetamido)-1H-benzo[d]imidazol-7-yl)oxypropyl(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,31-dioxo-33-an-35-yl)-L-alanine

[0110] Compound 11 (10 mg, 0.012 mmol) and HATU (5 mg, 0.012 mmol) were placed in a 25 mL single-necked flask, then N,N-dimethylformamide (3 mL) was added, and the mixture was stirred at room temperature for 10 min. Then compound 8 (3 mg, 0.004 mmol) was added. After stirring evenly, DIEA (2 mg, 0.016 mmol) was added, and the reaction was stirred at room temperature for 2 h. When the amount of compound 8 in the reaction solution was less than 3% as determined by LCMS, the reaction was considered complete. The solvent of the reaction solution was evaporated under reduced pressure, and the crude product was separated and purified by preparative liquid chromatography with acetonitrile / water. Compound DL-2 (white solid, 2 mg, yield 29.9%) was prepared. MS: 1671.84 [M+H] +

[0111] Preparation of Compound DL-3 in Example 3

[0112]

[0113] Step 1: Synthesis of (E)-3-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-methyl-3-methyl-1H-pyrazole-5-acetamido)-1H-benzo[d]imidazol-7-yl)oxy)propyl (tert-butoxycarbonyl)-L-valine

[0114] Boc-valine (3 mg, 0.013 mmol) and HATU (6 mg, 0.016 mmol) were placed in a 25 mL single-necked flask, then N,N-dimethylformamide (3 mL) was added, and the mixture was stirred at room temperature for 10 min. Then compound 8 (5 mg, 0.007 mmol) was added. After stirring evenly, DIEA (7 mg, 0.052 mmol) was added, and the reaction was stirred at room temperature for 2 h. When the amount of compound 8 in the reaction solution was less than 3% as determined by LCMS, the reaction was considered complete. The solvent of the reaction solution was evaporated under reduced pressure, and the crude product was separated and purified by preparative liquid chromatography with acetonitrile / water. Compound 12 (white solid, 6 mg, yield 90.2%) was prepared. MS: 951.07 [M+H] +

[0115] Step 2: Synthesis of (E)-3-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-methyl-3-methyl-1H-pyrazole-5-acetamido)-1H-benzo[d]imidazol-7-yl)oxy)propyl L-valine

[0116] Compound 12 (6 mg, 0.006 mmol) was added to dichloromethane (3 mL) and dissolved completely. Then trifluoroacetic acid (1 mL) was added, and the reaction was stirred at room temperature for 1 hour. When the amount of compound 12 in the reaction solution was less than 3% as determined by LCMS, the reaction was considered complete. The solvent of the reaction solution was evaporated under reduced pressure to obtain crude compound 13 (5 mg, yield 92.6%). MS: 850.95 [M+H] +

[0117] Step 3: Synthesis of 3 - ((5 - carbamoyl - 1 - ((E) - 4 - (5 - carbamoyl - 2 - (1 - ethyl - 3 - methyl - 1H - pyrazole - 5 - carboxamido) - 1H - benzimidazol - 1 - yl) but - 2 - en - 1 - yl) - 2 - (1 - methyl - 3 - methyl - 1H - pyrazole - 5 - acetamido) - 1H - benzimidazol - 7 - yl) oxypropyl (1 - (2,5 - dioxo - 2,5 - dihydro - 1H - pyrrol - 1 - yl) - 3,31 - dioxo - 33 - an - 35 - yl) - L - valine

[0118] Compound 11 (10 mg, 0.012 mmol) and HATU (5 mg, 0.012 mmol) were placed in a 25 - mL single - necked flask, and then N,N - dimethylformamide (3 mL) was added. The mixture was stirred at room temperature for 10 min. Then compound 13 (5 mg, 0.006 mmol) was added and stirred evenly, followed by the addition of DIEA (6 mg, 0.048 mmol). The reaction was stirred at room temperature for 2 hours. When the amount of compound 13 in the reaction solution was less than 3% as determined by LCMS, the reaction was considered complete. The solvent of the reaction solution was evaporated under reduced pressure, and the crude product was purified by preparative liquid chromatography using acetonitrile / water. After lyophilization, compound DL - 3 (white solid, 2.8 mg, yield 28%) was obtained. MS: 1699.90 [M+H] +

[0119] Preparation of Compound DL - 4 in Example 4

[0120]

[0121] Step 1: 2 - (3 - (tert - butoxy) - 3 - oxopropyl) - 34 - (2,5 - dioxo - 2,5 - dihydro - 1H - pyrrol - 1 - yl) - 4,32 - dioxo - 7,10,16,19,22,25,28 - octaoxy - 3,31 - diazacyclotrimethylcarboxylic acid

[0122] Compound 4 (33 mg, 0.055 mmol) and HATU (25 mg, 0.066 mmol) were placed in a 25 mL single-necked flask, then N,N-dimethylformamide (3 mL) was added, and the mixture was stirred at room temperature for 10 min. Then, compound 14 (11 mg, 0.055 mmol) and DIEA (28 mg, 0.22 mmol) were added, and the reaction was stirred at room temperature for 1 h. When the amount of compound 4 in the reaction solution was less than 3% as determined by LCMS, the reaction was considered complete. The solvent of the reaction solution was evaporated under reduced pressure, and the crude product was purified by preparative liquid chromatography with acetonitrile / water. Compound 15 (white solid, 31 mg, yield 72.3%) was obtained. MS: 778.86 [M+H] +

[0123] Step 2: Synthesis of tert-butyl 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-33-((2-(2-methoxyethoxy)ethoxyethyl)carbamoyl)-3,31-dioxo-7,10,13,19,22,25,28-octaoxa-4,32-diazapentatriacontane-36-carboxylate

[0124] Compound 15 (31 mg, 0.04 mmol) was placed in a 25 mL single-necked flask, HATU (18 mg, 0.048 mmol) and N,N-dimethylformamide (3 mL) were added, and the mixture was stirred at room temperature for 10 min. Then, amino triethylene glycol monomethyl ether (8 mg, 0.048 mmol) and DIEA (21 mg, 0.16 mmol) were added, and the reaction was carried out at room temperature for 3 h. The reaction of the starting compound 15 was monitored by LCMS until completion. After evaporation, the crude product was purified by preparative liquid chromatography with acetonitrile / water to obtain compound 16 (17 mg, yield 46.3%). MS: 924.06 [M+H] +

[0125] Step 3: Synthesis of 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-33-((2-(2-methoxyethoxy)ethoxyethyl)carbamoyl)-3,31-dioxo-7,10,16,19,22,25,28-octaoxa-4,32-diazapentatriacontane-36-carboxylic acid

[0126] Compound 16 (17 mg, 0.018 mmol) was completely dissolved in dichloromethane (3 mL), then trifluoroacetic acid (1 mL) was added, and the reaction was stirred at room temperature for 1 h. When the amount of compound 16 in the reaction solution was less than 3% as determined by LCMS, the reaction was considered complete. The solvent of the reaction solution was evaporated under reduced pressure, and the crude product was purified by preparative liquid chromatography with acetonitrile / water. Compound 17 (10 mg, yield 62.5%) was obtained. MS: 867.96 [M+H] +

[0127] Step 4: Synthesis of 3-((5-carbamoyl-1-((E)-4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-methyl-3-methyl-1H-pyrazole-5-acetamido)-1H-benzo[d]imidazol-7-yl)oxypropyl(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-33-(2-(2-(methoxyethoxy)ethoxyethyl)carbamoyl)-3,31-dioxo-7,10,13,16,12,22,25,28-octadecyloxy-4,3 2-diazahexacosan-36-yl)-L-alanine

[0128] Compound 17 (10 mg, 0.012 mmol) and HATU (5.2 mg, 0.014 mmol) were placed in a 25 mL single-necked flask, then N,N-dimethylformamide (3 mL) was added, and the mixture was stirred at room temperature for 10 min. Then compound 8 (9.5 mg, 0.012 mmol) and DIEA (6 mg, 0.046 mmol) were added, and the reaction was stirred at room temperature for 2 hours. The reaction was considered complete when the amount of compound 8 in the reaction solution was less than 3% as determined by LCMS. The solvent of the reaction solution was evaporated under reduced pressure, and the crude product was purified by preparative liquid chromatography using acetonitrile / water. The product was freeze-dried to obtain compound DL-4 (white solid, 1 mg, yield 5%). MS: 1671.84 [M+H] +

[0129] Example 5: Preparation of Compound DL-5

[0130]

[0131] Step 1: Synthesis of 3-((5-carbamoyl-1-((E)-4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-7-yl)oxy)propyl(((4-((S)-2-((S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrazole)ROL-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)-L-alanine ester

[0132] Compound 8 (2 mg, 0.0024 mmol) and compound 18 (14 mg, 0.019 mmol) were added to N,N-dimethylformamide (1 mL), and the mixture was stirred at room temperature for 10 minutes. Then DIEA (2 mg, 0.0155 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The reaction was considered complete when the amount of compound 18 in the reaction solution was less than 3% as determined by LCMS. The solvent of the reaction solution was evaporated under reduced pressure, and the crude product was purified by preparative liquid chromatography using acetonitrile / water. Compound DL-5 (white solid, 2 mg, 0.0014 mmol, yield 57.8%) was obtained. MS: 1420.56 [M+H] + .

[0133] Example 6: Preparation of ADC-1

[0134]

[0135] Under the condition of 37 °C, an aqueous solution of the prepared reducing agent TCEP (10 mM, 0.008 mL, 0.08 μmol) was added to an aqueous HEPES buffer solution of antibody Herceptin (0.05 M aqueous HEPES solution with pH = 7.0; 0.24 mL, 8.4 mg / mL, 0.014 μmol). Argon gas was filled, and the mixture was placed in a metal bath oscillator and reacted with shaking at 37 °C for 1.5 hours. After the reaction was stopped, the reaction solution was allowed to cool naturally to room temperature (25 °C) for subsequent reactions. An aqueous solution of compound STA-2 (0.142 mL, 4.0 mg / mL, 0.14 μmol) was added to the above 0.248 mL antibody reaction solution. Argon gas was filled, and the mixture was placed in a shaking mixer and reacted with shaking at 25 °C for 2.0 hours. The reaction was stopped, and an aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution. Argon gas was filled, and the mixture was placed in a shaking mixer and reacted with shaking at 25 °C for 0.3 hours. The reaction solution was ultrafiltered and exchanged with a 0.05 M PBS buffer solution with pH = 7.0 using an Amicon Ultra-15 30 kDa ultrafiltration tube to obtain a PBS buffer solution of the conjugate ADC-STA-2 (2.5 mg / mL, 0.7 mL), which was stored frozen at -80 °C. The average DAR value was calculated to be n = 6.0 by the reduction-reversed phase chromatography method.

[0136] Example 7: Preparation of ADC-2

[0137]

[0138] At 37 °C, an aqueous solution of the reducing agent TCEP (10 mM, 0.008 mL, 0.08 μmol) was added to an aqueous PBS buffer solution of the antibody Herceptin (0.05 M PBS aqueous solution at pH = 7.0; 0.175 mL, 11.47 mg / mL, 0.014 μmol). After purging with Ar gas, the mixture was placed in a metal bath shaker and reacted with shaking at 37 °C for 1.5 hours. After stopping the reaction, the reaction solution was allowed to cool naturally to room temperature (25 °C) for subsequent reactions. A 20% DMF / aqueous solution of compound STA-4 (0.113 mL, 4.0 mg / mL, 0.28 μmol) was added to the above 0.183 mL of the antibody reaction solution. After purging with Ar gas, the mixture was placed in a shaker and reacted with shaking at 25 °C for 2.0 hours. After stopping the reaction, an aqueous solution of compound NAC (0.005 mL, 100 mM, 0.5 μmol) was added to the reaction solution. After purging with Ar gas, the mixture was placed in a shaker and reacted with shaking at 25 °C for 0.3 hours. After stopping the reaction, the reaction solution was ultrafiltered and exchanged with a 0.05 M PBS buffer solution at pH = 7.0 using an Amicon Ultra-15 30 kD ultrafiltration tube to obtain a PBS buffer solution of the conjugate ADC-STA-4 (2.81 mg / mL, 0.42 mL), which was stored frozen at -80 °C. The average DAR value was calculated to be n = 5.4 by the reduction-reversed phase chromatography method.

[0139] Example 8: Preparation of ADC-3

[0140]

[0141] At 37 °C, an aqueous solution of the reducing agent TCEP (10 mM, 0.008 mL, 0.08 μmol) was added to an aqueous PBS buffer solution of the antibody Herceptin (0.05 M PBS aqueous solution at pH = 7.0; 0.175 mL, 11.47 mg / mL, 0.014 μmol). After purging with Ar gas, the mixture was placed in a metal bath shaker and reacted with shaking at 37 °C for 1.5 hours. After stopping the reaction, the reaction solution was allowed to cool naturally to room temperature (25 °C) for subsequent reactions. A 20% DMF / aqueous solution of compound STA-5 (0.113 mL, 4.0 mg / mL, 0.28 μmol) was added to the above 0.183 mL of the antibody reaction solution. After purging with Ar gas, the mixture was placed in a shaker and reacted with shaking at 25 °C for 2.0 hours. After stopping the reaction, an aqueous solution of compound NAC (0.005 mL, 100 mM, 0.5 μmol) was added to the reaction solution. After purging with Ar gas, the mixture was placed in a shaker and reacted with shaking at 25 °C for 0.3 hours. After stopping the reaction, the reaction solution was ultrafiltered and exchanged with a 0.05 M PBS buffer solution at pH = 7.0 using an Amicon Ultra-15 30 kD ultrafiltration tube to obtain an ADC-STA-5 conjugate in a PBS buffer solution (3.52 mg / mL, 0.42 mL), which was stored frozen at -80 °C. The average DAR value was calculated to be n = 5.3 by reduction-reversed phase chromatography method.

[0142] Example 9: Preparation of ADC-4

[0143]

[0144] At 37 °C, an aqueous solution of the reducing agent TCEP (10 mM, 0.008 mL, 0.08 μmol) was added to an aqueous PBS buffer solution of the antibody Herceptin (0.05 M PBS aqueous solution at pH = 7.0; 0.171 mL, 11.7 mg / mL, 0.014 μmol). After purging with Ar gas, the mixture was placed in a metal bath shaker and reacted with shaking at 37 °C for 1.5 hours. After the reaction was stopped, the reaction solution was allowed to cool naturally to room temperature (25 °C) for subsequent reactions. A 20% DMF / aqueous solution of compound STA-6 (0.149 mL, 3.0 mg / mL, 0.28 μmol) was added to the above 0.179 mL of the antibody reaction solution. After purging with Ar gas, the mixture was placed in a shaker and reacted with shaking at 25 °C for 2.0 hours. After the reaction was stopped, an aqueous solution of compound NAC (0.005 mL, 100 mM, 0.5 μmol) was added to the reaction solution. After purging with Ar gas, the mixture was placed in a shaker and reacted with shaking at 25 °C for 0.3 hours. After the reaction was stopped, the reaction solution was ultrafiltered and exchanged with a 0.05 M PBS buffer solution at pH = 7.0 using an Amicon Ultra-15 30 kD ultrafiltration tube to obtain an ADC-STA-6 conjugate in a PBS buffer solution (3.26 mg / mL, 0.49 mL), which was stored frozen at -80 °C. The average DAR value was calculated to be n = 4.7 by reduction-reversed phase chromatography method.

[0145] Example 10: Preparation of ADC-5

[0146]

[0147] At 37 °C, an aqueous solution of the reducing agent TCEP (10 mM, 0.002 mL, 0.02 μmol) was added to an aqueous HEPES buffer solution of the antibody Herceptin (0.05 M HEPES aqueous solution at pH = 7.0; 0.06 mL, 8.4 mg / mL, 0.0033 μmol). After purging with Ar gas, it was placed in a metal bath shaker and reacted with shaking at 37 °C for 1.5 hours. After stopping the reaction, the reaction solution was allowed to cool naturally to room temperature (25 °C) for subsequent reactions. A 50% DMF / aqueous solution of compound STA-3 (0.024 mL, 2.0 mg / mL, 0.033 μmol) was added to the above 0.062 mL of the antibody reaction solution. After purging with Ar gas, it was placed in a shaker and reacted with shaking at 25 °C for 2.0 hours. After stopping the reaction, an aqueous solution of compound NAC (0.002 mL, 100 mM, 0.2 μmol) was added to the reaction solution. After purging with Ar gas, it was placed in a shaker and reacted with shaking at 25 °C for 0.3 hours. After stopping the reaction, the reaction solution was ultrafiltered and exchanged with a 0.05 M PBS buffer solution at pH = 7.0 using an Amicon Ultra-15 30 kD ultrafiltration tube to obtain a PBS buffer solution of the conjugate ADC-STA-3 (2.1 mg / mL, 0.19 mL), which was stored frozen at -80 °C. The average DAR value was calculated to be n = 6.1 by reduction-reversed phase chromatography method.

[0148] Among them, the analysis and detection method of the ADC sample is as follows:

[0149] Analysis and test of DAR value

[0150] Prepare mobile phase A: 0.5‰ TFA acetonitrile solution, mobile phase B: 0.5‰ TFA aqueous solution. Take 20 μL of the sample to be tested (1 mg / mL), add 1 μL of DTT (10 mM), and react at room temperature for 30 min for reduction. Use a chromatographic column: Aglient PLRP-S 1000A 5 μm, 50 * 2.1 mm, and perform gradient elution (mobile phase A: 0 - 100%) at 70 °C.

[0151] Analysis and test of activity

[0152] We took ADC-1 and ADC-5 for activity analysis and testing, with trastuzumab as the control.

[0153] 1. Analysis of the binding ability of the conjugated drug to the HER2 antigen

[0154] The binding of the antibody-immunostimulant conjugated drug to the HER2 antigen was investigated by enzyme-linked immunosorbent assay (ELISA), using the EC of the naked anti-trastuzumab before conjugation and the antibody-immunostimulant conjugated drug after conjugation. 50The change in the ability to bind to the HER2 antigen was measured by the change in value. First, a certain amount of HER2 antigen was coated on the enzyme-linked immunosorbent assay (ELISA) plate overnight, and then a 4-fold serial dilution of naked trastuzumab or antibody-immunostimulant conjugate drug was added to the corresponding wells, with the final concentration ranging from 0.011 to 11400 ng / mL. After incubation at 37 °C for 1 hour, HRP-labeled goat anti-human IgG Fc antibody was added and incubated at room temperature for half an hour. Then, TMB chromogenic solution was added and incubated at room temperature for 15 minutes. The reaction was terminated by adding the stop solution, and the absorbance at 450 nm was measured using an ELISA reader. With the final concentration of the sample (ng / mL) as the abscissa and the corresponding absorbance value (OD 450 ) as the ordinate, a four-parameter curve was fitted using Graph Pad Prism 5 to calculate the EC 50 value. The results are shown in Figure 1 . The EC 50 values of naked trastuzumab, ADC-1, and ADC-5 were 18.55 ng / mL, 21.30 ng / mL, and 27.88 ng / mL, respectively, indicating that ADC-1 and ADC-5 have HER2 targeting, and the conjugation of the immunostimulant has little effect on the ability to bind to the HER2 antigen.

[0155] 2. Affinity analysis of conjugate drug with SK-BR-3 cells

[0156] The affinity of the antibody-immunostimulant conjugate drug for the HER2-positive tumor cell SK-BR-3 was investigated by flow cytometry. The change in the affinity before and after conjugation was measured by the change in the EC 50 values of naked trastuzumab before conjugation and the antibody-immunostimulant conjugate drug after conjugation. SK-BR-3 cells were cultured in McCoy's 5A medium containing 10% fetal bovine serum (FBS). Cells in the logarithmic growth phase were digested and collected, and then added to a 96-well U-bottom plate (200000 cells / well). The final concentration of the sample was 20000 ng / mL, 10000 ng / mL, 5000 ng / mL, 1000 ng / mL, 200 ng / mL, 40 ng / mL, 8 ng / mL, 1.6 ng / mL, 0.32 ng / mL, 0.06 ng / mL, 0.01 ng / mL, a total of 11 gradients. Cells and the test substance of the same volume were incubated together on ice for 1 hour, and then AF 488 Goat Anti-Human IgG(H+L)Antibody CrossAdsorbed was added and incubated at 4 °C in the dark for 40 min. Then, the fluorescence intensity was measured using a flow cytometer, and the detected fluorescence intensity was positively correlated with the concentration of the test substance bound. With the logarithm of the final concentration of the test sample (ng / mL) as the abscissa and the mean fluorescence intensity (MFI) as the ordinate, a four-parameter curve was fitted using Graph Pad Prism 5.01 to calculate the EC50 Values. The results are as follows Figure 2 shown. The ECs of naked anti-trastuzumab, ADC-1, and ADC-5 50 were 166.6 ng / mL, 139.0 ng / mL, and 240.1 ng / mL, respectively, indicating that at the cellular level, ADC-1 and ADC-5 have HER2 targeting properties, and the conjugation of the immune agonist has little effect on the affinity for SK-BR-3 cells.

[0157] 3. Analysis of the affinity of the conjugated drug for human STING protein

[0158] The affinity of the conjugated drug for human STING protein (Human STING WT BINDING KITS, Cisbio) was detected by competitive binding based on the principle of homogeneous time-resolved fluorescence (HTRF). The test substances were serially diluted 4-fold, with a total of 10 points, and 4 μL / well was added to a 384-well plate. The positive control was 2'3'-cGAMP of the same volume, and the blank control was DMSO of the same volume as the diluent; subsequently, a certain concentration of Human STING protein 6His tagged was added to the above wells, 4 μL / well, and centrifuged at 1000 rmp for 1 minute; incubated at 25 °C for 15 minutes; 8 μL of a mixture of 6His Tb Cryptate Antibody and STING WT ligand d2 reagent was added to the above wells, and centrifuged at 1000 rmp for 1 minute; incubated at 25 °C for 3 hours; the response values at 665 nm / 615 nm were read using a microplate reader, and the inhibition rate was calculated according to the following formula:

[0159] Relative Ratio (RR) = [(Ratio 665nm / 615nm - Ratio vehicle )],

[0160]

[0161] RR positive: The average RR of the positive control;

[0162] RR vehicle: The average RR of the blank control;

[0163] Using the logarithm of the final concentration (nM) of the test substance as the abscissa and the inhibition rate (%) as the ordinate, a four-parameter curve was fitted using Graphpad 8.0 to calculate the EC 50 value. The results are as follows Figure 3As shown, both ADC-1 and ADC-5 have inhibitory effects, and the inhibitory effects are significantly higher than those of the positive control 2'3'-cGAMP. The EC 50 values of 2'3'-cGAMP, ADC-1, and ADC-5 are 7.514 nM, 0.1236 nM, and 2.634 nM, respectively.

[0164] 4. Biological activity analysis of the conjugate drug (SK-BR-3 / THP1-Dual luciferase reporter gene cell co-culture)

[0165] The biological activity of the conjugate drug was detected by co-culturing SK-BR-3 cells (ATCC, HTB-30) and luciferase reporter gene cells THP1-Dual (InvivoGen, thpd-nfis). On the first day, 15,000 SK-BR-3 cells per well (50 μL / well) were seeded in a 96-well plate and incubated in an incubator at 37 °C and 5% CO2 for 6 hours. Then, 5 μL / well of the sample to be tested (starting from a final concentration of 200 nM, diluted 3-fold in 9 gradients) was added, and the plate was left to stand in the incubator for 20 minutes. Next, 30,000 THP1-Dual cells per well (50 μL / well) were added, and the plate was incubated in an incubator at 37 °C and 5% CO2 for 20 hours. On the second day, 20 μL / well of the above culture supernatant was added to a new plate, 50 μL / well of Quanti-luc Gold was added and mixed evenly, and the Luminesence value was immediately read using a multi-functional microplate reader (EnVision, PerkinElmer). With the logarithm of the final concentration (nM) of the sample to be tested as the abscissa and the Luminesence value as the ordinate, a four-parameter curve was fitted using Graph Pad Prism 5.01 to calculate the EC 50 value. The results are as Figure 4 shown. Both ADC-1 and ADC-5 can activate THP1-Dual cells, and the activation effect is more than 100 times that of the STING agonist. The EC 50 values of the STING agonist, ADC-1, and ADC-5 are 18 nM, 0.1204 nM, and 0.1620 nM, respectively.

[0166] 5. Biological activity analysis of the conjugate drug (THP1-Dual Luciferase reporter gene cell single culture)

[0167] The biological activity of the conjugated drug was detected by culturing the luciferase reporter gene cells THP1-Dual (Invivo Gen, thpd-nfis) alone. On the first day, 30,000 THP1-Dual cells were seeded in each well of a 96-well plate at a density of 50 μL / well, and 5 μL / well of the sample to be tested was added (starting from a final concentration of 200 nM, diluted 3-fold in a total of 9 gradients). The plate was then incubated in an incubator at 37 °C and 5% CO2 for 20 hours. On the second day, 20 μL / well of the above culture supernatant was added to a new plate, and 50 μL / well of Quanti-luc Gold was added and mixed well. The Luminesence value was immediately read using a multi-functional microplate reader (EnVision, Perkin Elmer). Using the logarithm of the final concentration (nM) of the sample to be tested as the abscissa and the Luminesence value as the ordinate, a four-parameter curve was fitted using Graph Pad Prism 5.01 to calculate the EC 50 value. The results are as shown in Figure 5 . ADC-1 and ADC-5 could not activate THP1-Dual cells, while the control STING agonist could activate THP1-Dual cells in a dose-dependent manner. This indicates that ADC-1 and ADC-5 cannot activate the STING pathway alone within the effective concentration range, and the activation of the STING pathway depends on the binding of target cells. The in vitro activity with target cells is more than 900 times that without target cells. The EC 50 of the STING agonist was 13.14 nM, while the EC 50 of ADC-1 and ADC-5 were both greater than 100 nM.

[0168] 6. Functional activity analysis of the conjugated drug (co-culture of SK-BR-3 cells and PBMC cells)

[0169] The functional activity of the conjugated drug was detected by co-culturing SK-BR-3 cells (Nanjing Kebai Biotechnology Co., Ltd., cell number CBP60413) and PBMC cells (Shanghai Rubai Biotechnology Co., Ltd., PBMNC050C). On the first day, 20,000 SK-BR-3 cells were seeded per well in a 96-well plate at a density of 50 μL / well, and incubated in an incubator at 37 °C and 5% CO2 for 6 hours. Then, 10 μL of the test sample per well (starting from a final concentration of 100 nM, serially diluted 5-fold for a total of 9 gradients) was added, and the plate was left in the incubator for 20 minutes. Next, 30,000 PBMC cells were added per well at a density of 50 μL / well, and the plate was incubated in an incubator at 37 °C and 5% CO2 for 40 hours. On the third day, the CellTiter-Glo reagent (Promega, catalog number G7571) and the culture plate were equilibrated at room temperature for 30 minutes. Then, 50 μL of CellTiter-Glo was added to each well, and the plate was shaken for 2 minutes. After that, the cell culture plate was equilibrated at room temperature for 10 minutes. The Luminescence value was read using a multimode microplate reader (BMG, Omega). With the logarithm of the final concentration (nM) of the test sample as the abscissa and the Luminescence value as the ordinate, a four-parameter curve was fitted using Graph Pad Prism 5.01 to calculate the EC 50 value and the maximum response value. The results are shown in Figure 6 . As shown, both ADC-1 and ADC-5 had significant killing effects on the target cells SK-BR-3. The killing rate at the highest concentration reached approximately 90%, which was approximately 30% higher than that of naked anti-trastuzumab. The IC 50 and the maximum killing rate are shown in Table 2:

[0170] Table 2 Co-culture of SK-BR-3 cells and PBMC cells

[0171] Trastuzumab ADC-1 ADC-5 <![CDATA[IC 50 (nM)]]> 0.01378 0.01842 0.01842 Maximum killing rate % 63 93 88

Claims

1. A compound of formula (I) or an isotopically labeled compound thereof, or an optical isomer, geometric isomer, tautomer or mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof, P-L-D(I) wherein, P has the following structure: m and n each independently selected from any integer from 0 to 12; X is selected from a bond, nitrogen, oxygen, sulfur, phosphorus, C1-C6 alkylene, imino, ester group, amide group, carbonate group, carbamate group, sulfone group, ketone group, urea group or phosphate group; L has the following structure: n1 and n2 each independently selected from any integer from 1 to 6; R1 is selected from hydrophilic groups; R2 and R3 are each independently selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, or R2 and R3 together with the carbon atom to which they are attached form a C3-C6 cycloalkyl, wherein one or more hydrogen atoms in the C1-C6 alkyl, C1-C6 haloalkyl or C3-C6 cycloalkyl are optionally substituted by halogen, hydroxyl, cyano, amino, C1-C3 alkyl or phenyl; and D is an immune agonist molecule or other equivalent.

2. The compound of formula (I) according to claim 1, wherein the hydrophilic group has at least three ether bonds and / or hydroxyl groups composed of oxygen-containing groups, preferably, the hydrophilic group has any one of the following structures:

3. The compound of formula (I) according to claim 1, wherein R2 and R3 are each independently selected from H, F, Cl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or R2 and R3 together with the carbon atom to which they are attached form cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, wherein one or more hydrogen atoms in the methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl are optionally substituted by halogen, hydroxyl, cyano, amino, C1-C3 alkyl or phenyl.

4. The compound of formula (I) according to claim 1, wherein the payload molecule is an immune agonist.

5. The compound of formula (I) according to claim 1, wherein the payload molecule is a cyclic dinucleotide, anthrone, aminobenzimidazole, 3-imidazolylpyridazine, benzothiophene, triazole quinoxaline immune agonist molecule.

6. The compound of formula (I) according to claim 1, wherein the payload molecule is an aminobenzimidazole derivative having the following structure:

7. A conjugate comprising the compound of formula (I) or an isotopically labeled compound thereof, or an optical isomer, geometric isomer, tautomer or mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 6 as a conjugate precursor of the conjugate.

8. The conjugate according to claim 7, wherein the conjugate further comprises a targeting moiety.

9. The conjugate according to claim 8, wherein the targeting moiety forms a free thiol group by reduction of an interchain disulfide bond for coupling with the maleimide group of the compound of formula (I).

10. The conjugate according to claim 8, having a DAR value which is any integer or decimal number from 1 to 10, preferably from 4 to 8.

11. The conjugate according to claim 8, wherein the targeting moiety is selected from an antibody, a synthetically functionalized antibody, a peptide, or other targeting ligands.

12. The conjugate according to claim 11, wherein the targeting moiety is an antibody or a synthetically functionalized antibody specific for an antigen overexpressed in cancer cells.

13. The conjugate according to claim 12, wherein the targeting moiety is an antibody or a synthetically functionalized antibody specific for an antigen selected from HER2, EGFR, Claudin18.2, c-MET, Nectin-4, ROR1, GPNMB, CD56, TACSTD2 (TROP2), CEACAM5, FRα, mesothelin, ENPP3, guanylate cyclase C, SLC44A4, NaPi2b, CD70, TF, B7-H3, B7-H4, GPC3, mucin 1, STEAP1, connexin 4, 5T4, SLTRK6, SC-16, LIV-1, P-cadherin, PSMA, fibronectin extra domain B, endothelin receptor ETB, tenascin c, collagen IV, VEGFR2, periostin, CD30, CD79b, CD19, CD20, CD22, CD138, CD37, CD33, CD74, and CD98.

14. The conjugate according to claim 13, wherein the targeting moiety is trastuzumab or a synthetically functionalized trastuzumab.

15. A pharmaceutical composition comprising a compound of formula (I) according to any one of claims 1 to 7 or an isotopically labeled compound thereof, or an optical isomer, geometric isomer, tautomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt thereof, or a conjugate according to any one of claims 8 to 14; and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition according to claim 15, wherein the dosage form is selected from tablets, capsules, granules, powders, syrups, inhalants, or injections.

17. Use of a compound of formula (I) according to any one of claims 1 to 7 or an isotopically labeled compound thereof, or an optical isomer, geometric isomer, tautomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt thereof, or a conjugate according to any one of claims 8 to 14 in the manufacture of a medicament for treating or preventing cancer in a subject in need thereof.

18. The use according to claim 17, wherein the cancer is HER2-positive cancer.

19. The use according to claim 18, wherein the HER2-positive cancer is a cancer in which HER2 is overexpressed.

20. The use according to claim 19, wherein the cancer is breast cancer.

21. The use according to claim 19, wherein the medicament is an antibody-drug conjugate.

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