CN117534725A - Linker, linker-drug conjugate, ligand-drug conjugate and uses thereof - Google Patents

Linker, linker-drug conjugate, ligand-drug conjugate and uses thereof Download PDF

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CN117534725A
CN117534725A CN202311489725.7A CN202311489725A CN117534725A CN 117534725 A CN117534725 A CN 117534725A CN 202311489725 A CN202311489725 A CN 202311489725A CN 117534725 A CN117534725 A CN 117534725A
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structural fragment
natural number
linker
antibody
ligand
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唐胜
王英召
狄维
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Beijing Haibu Pharmaceutical Technology Co ltd
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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Abstract

The invention relates to the technical field of biological medicines, in particular to a connector, a connector-drug conjugate, a ligand-drug conjugate and application thereof. The linker comprises a fragment of structure B,the linker carries two drugs with synergistic effects, thereby improving drug delivery efficiency, while being capable of optimizing stability and PK properties of ligand-drug conjugates.

Description

Linker, linker-drug conjugate, ligand-drug conjugate and uses thereof
Cross Reference to Related Applications
The present application claims priority from patent application No. 202211551221.9 entitled "linker, linker-drug conjugate, ligand-drug conjugate and uses thereof" filed on month 05 of 2021, national intellectual property agency of the people's republic of China, the entire contents of which are incorporated herein by reference.
Technical Field
The invention relates to the technical field of biological medicines, in particular to a connector, a connector-drug conjugate, a ligand-drug conjugate and application thereof.
Background
At present, the monoclonal antibody or the antibody fragment is connected with cytotoxin with biological activity through a stable chemical joint compound by the antibody-drug conjugate (antibody drug conjugate, ADC), so that the specificity of the antibody on the surface antigen of normal cells and tumor cells and the high efficiency of cytotoxic substances are fully utilized, and the defects of low curative effect, overlarge toxic and side effects of the former and the like are avoided. This means that the antibody-drug conjugate binds tumor cells more precisely and reduces the effect that would have on normal cells compared to conventional chemotherapeutic drugs. But only a few classes of drugs have proven to be sufficiently active as antibody drug conjugates, with suitable toxicity profiles and other pharmacological properties to warrant clinical development.
The design of chemical linkers for covalently binding antibodies to drugs to form ADCs also plays a role in the development of ADCs. For example, the linker should be stable in blood to limit damage to healthy tissue. The breakdown or decay of the ADC may release the cytotoxic drug prior to delivery of the ADC to the target site. Once the ADC reaches the target site, the ADC must efficiently release the cytotoxic drug in active form. The balance between plasma stability and effective drug release at the target cells, which may depend on linker design, remains to be explored. The linker technology affects ADC performance, specificity, and safety. There is a need for linkers for ADCs that can provide serum stability and increased solubility, allowing for efficient conjugation and intracellular delivery of hydrophobic drugs.
When the traditional linker is used, the phenomenon of instability and aggregation sedimentation of the ADC is caused, in addition, the phenomenon of high lipophilicity can lead to rapid metabolism elimination of metabolic organs such as liver and the like in vivo, and the PK property is deteriorated, so that the structure of the linker is necessary to explore, and the physicochemical and Pharmacokinetic (PK) properties of the ADC can be optimized while the high drug loading rate is ensured. Therefore, there is still a need for further development of ADC drugs with better therapeutic effects.
In view of this, the present invention has been made.
Disclosure of Invention
The present invention aims to provide linkers, linker-drug conjugates, ligand-drug conjugates and uses thereof. The linker increases drug loading, thereby increasing drug delivery efficiency, while being capable of optimizing stability and PK properties of ligand-drug conjugates.
The invention is realized in the following way:
in a first aspect, embodiments of the present invention provide a linker comprising a B structural fragment, wherein the B structural fragment is selected from the group consisting of:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6.
It should be noted that n represents the subscript n in the R and B structural fragments.
In a second aspect, embodiments of the present invention provide the use of a linker as described above in the preparation of a medicament comprising a linker-drug conjugate or a ligand-drug conjugate.
In a third aspect, embodiments of the present invention provide linker-drug conjugates of the linker having a structure represented by formula (II):
or a pharmaceutically acceptable salt thereof, wherein,
La is an extension unit capable of linking to the ligand unit;
the B structural fragment is selected from the following formulae:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6;
the Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, the methylene of the Lb structural fragment is connected with the methine of the B structural fragment, and the Lb structural fragment is connected with the Lc1 or Lc2 structural fragment through carbonyl;
lc1 and Lc2 structural segments are releasable assembly units, respectively;
d1 and D2 are each a drug unit.
In a fourth aspect, embodiments of the present invention provide a linker or a ligand-drug conjugate of the linker-drug conjugate having a structure represented by formula (V):
or a pharmaceutically acceptable salt thereof, wherein,
l is a ligand unit, wherein the subscript n is selected from any integer from 1 to 8, preferably from 4 to 8;
la is an extension unit;
the B structural fragment is selected from the following formulae:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6;
the Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, the methylene of the Lb structural fragment is connected with the methine of the B structural fragment, and the Lb structural fragment is connected with the Lc1 or Lc2 structural fragment through carbonyl;
lc1 and Lc2 are releasable assembled units, respectively;
d1 and D2 are each a drug unit.
In a fifth aspect, embodiments of the invention provide a method of preparing a ligand-drug conjugate comprising conjugating an antibody to a linker or said linker-drug conjugate.
In a sixth aspect, embodiments of the invention provide the use of a linker-drug conjugate or said ligand-drug conjugate in the manufacture of a medicament for the treatment of a disease in which a tumor antigen is overexpressed.
In a seventh aspect, embodiments of the invention provide the use of a linker-drug conjugate or said ligand-drug conjugate in the manufacture of a medicament for the treatment of a disease in which expression and/or activity of dnase topoisomerase I is abnormal;
preferably, the dnase topoisomerase I expression and/or activity is up-regulated.
In an eighth aspect, embodiments of the invention provide the use of a linker-drug conjugate or said ligand-drug conjugate as a dnase topoisomerase I inhibitor.
In a ninth aspect, embodiments of the invention provide the use of a linker-drug conjugate or said ligand-drug conjugate in the manufacture of a medicament, wherein said medicament is for cancer.
In a tenth aspect, embodiments of the invention provide a pharmaceutical composition comprising a linker-drug conjugate or said ligand-drug conjugate.
In an eleventh aspect, embodiments of the invention provide a kit comprising a linker-drug conjugate or said ligand-drug conjugate.
The invention has the following beneficial effects: the linker provided by the embodiment of the invention can carry two drugs with synergistic effect, so that the drug delivery efficiency is improved, and meanwhile, the stability and PK properties of the ligand-drug conjugate can be optimized.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate the embodiments of the present invention, and therefore should not be considered as limiting the scope, and other related drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the results of NCI-N87 cells in the cell proliferation inhibition test of Experimental example 1 of the present invention;
FIG. 2 is a graph showing the results of the test of SK-BR-3 cells in the cell proliferation inhibition test of Experimental example 1 of the present invention;
FIG. 3 shows the tumor inhibition effect of different drugs on human gastric cancer cell NCI-N87 model in the animal experiment of experimental example 2 of the invention;
FIG. 4 is a graph showing weight change of different drugs in animal experiments of experimental example 2 of the invention on human gastric cancer cell NCI-N87 tumor model mice;
FIG. 5 shows the tumor inhibition effect of different drugs on human breast cancer cells SK-BR-3 model in the animal experiment of experimental example 2;
FIG. 6 shows the weight change patterns of different drugs on mice with human breast cancer cells SK-BR-3 tumor model in the animal experiment of experimental example 2 of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In embodiments of the present invention, the term "antibody" is generally intended to be used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity (Milleretal (2003) journal of immunology 170:4854-4861). The natural form of an antibody is typically tetrameric and consists of two identical immunoglobulin chain pairs, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions (VL and VH) together are primarily responsible for binding to antigen. The light and heavy chain variable domains are composed of framework regions interrupted by three hypervariable regions (also known as "complementarity determining regions" or "CDRs"). The constant region is recognized by and interacts with the immune system. (see, e.g., janeway et al, 2001, immunol. Biology, 5 th edition, garland Publishing, new York). Antibodies can be of any type (e.g., igG, igE, igM, igD and IgA), class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2), or subclass. The antibodies may be derived from any suitable species. In some embodiments, the antibody is of human or murine origin. The antibody may be, for example, a human, humanized or chimeric antibody.
In embodiments of the invention, the term "monoclonal antibody" generally refers to an antibody obtained from a substantially homogeneous population of antibodies (i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts). Monoclonal antibodies are highly specific and are directed against a single antigenic site. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
In embodiments of the invention, an "intact antibody" is typically an antibody comprising an antigen binding variable region and light chain constant domain (CL) and heavy chain constant domains CH1, CH2, CH3 and CH 4. The constant domain may be a natural sequence constant domain (e.g., a human natural sequence constant domain) or an amino acid sequence variant thereof.
In embodiments of the invention, an "antibody fragment" generally comprises a portion of an intact antibody, including antigen-binding or variable regions thereof. Examples of antibody fragments include Fab, fab ', F (ab') 2 And Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single chain antibody molecules, scFv-Fc, multispecific antibody fragments formed from one or more antibody fragments, one or more fragments produced from a Fab expression library, or epitope-binding fragments of any of the foregoing that immunospecifically bind to a target antigen (e.g., a cancer cell antigen, a viral antigen, or a microbial antigen).
In embodiments of the present invention, the term "antigen" generally refers to an entity to which an antibody specifically binds.
In embodiments of the invention, the terms "specifically bind" and "specifically bind" refer to antibodies or antibody derivatives that will bind to their corresponding epitopes of a target antigen in a highly selective manner without binding to a variety of other antigens. Typically, the antibody or antibody derivative is present in an amount of at least about 1x10 -7 M, preferably 10 -8 M to 10 -9 M、10 -10 M、10 -11 M or 10 -12 M binds with an affinity that is at least twice greater than its affinity for a non-specific antigen other than the predetermined antigen or closely related antigen (e.g., BSA, casein).
In embodiments of the present invention, the term "inhibit" generally refers to a reduction in the amount detectable or a complete prevention.
In the present examples, the term "tumor" refers generally to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. In embodiments of the invention, the tumor may comprise a solid tumor and/or a hematological tumor. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are not mutually exclusive when referred to herein. In some embodiments, a tumor may refer to a body mass containing a majority of cancer cells, e.g., cells that display characteristics of any of the cancers described herein. Examples of tumors may include primary tumors of any of the above types of cancers or metastatic tumors at a second site derived from any of the above types of cancers.
In the present examples, the term "tumor antigen" generally includes the meaning known in the art, including any molecule expressed on (or associated with the development of) tumor cells, known or believed to have an effect on the tumorigenic properties of tumor cells. Many tumor antigens are known in the art. Whether a molecule is a tumor antigen can also be determined according to techniques and assays well known to those skilled in the art, such as clonogenic assays (clonogenic assays), transformation assays, in vitro or in vivo tumor formation assays, gel migration assays (gel migration assay), gene knockout analysis, and the like. The term "tumor antigen" may refer to a human transmembrane protein, i.e. a cell membrane protein anchored in the lipid bilayer of a cell. As used herein, a human transmembrane protein will typically comprise an "extracellular domain" that can bind a ligand, a lipophilic transmembrane domain, a conserved intracellular domain, such as a tyrosine kinase domain, and a carboxy-terminal signaling domain having several tyrosine residues that can be phosphorylated. Tumor antigens include molecules such as EGFR, HER2, HER3, HER4, epCAM, CEA, TRAIL, TRAIL receptor 1, TRAIL receptor 2, lymphotoxin beta receptor, CCR4, CD19, CD20, CD22, CD28, CD33, CD40, CD80, CSF-1R, CTLA-4, fibroblast Activation Protein (FAP), hepsin, melanoma-associated chondroitin sulfate proteoglycan (MCSP), prostate Specific Membrane Antigen (PSMA), VEGF receptor 1, VEGF receptor 2, IGF1-R, TSLP-R, TIE-1, TIE-2, TNF-alpha, TNF-like apoptosis weak inducer (TWEAK), or IL-1R.
In embodiments of the invention, "HER receptor" refers generally to receptor protein tyrosine kinases belonging to the HER receptor family, including EGFR, HER2, HER3 and HER4 receptors and other members of this family that will be identified in the future. HER receptors will typically comprise an extracellular domain that can bind HER ligands; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxy-terminal signal domain containing several tyrosine residues that can be phosphorylated. In some embodiments, the HER receptor is a native sequence human HER receptor.
The terms "ErbB1", "HER1", "epidermal growth factor receptor" and "EGFR" are used interchangeably herein to refer to, for example, carpenter et al, ann.rev.biochem.56:881-914 (1987), including naturally occurring mutant forms thereof (e.g., deleted mutant EGFR in Humphrey et al, PNAS (USA) 87:4207-4211 (1990)).
The terms "ErbB2" and "HER2" are used interchangeably herein and generally refer to, for example, semba et al, PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al, nature 319:230-234 (1986) (Genebank number X03363). The extracellular domain of "HER2" typically comprises 4 domains: domain I (about amino acid residues 1-195), domain II (about amino acid residues 196-319), domain III (about amino acid residues 320-488), and domain IV (about amino acid residues 489-630) (residue numbers excluding signal peptides). See Garrett et al, mol. Cell.11:495-505 (2003); cho et al, nature421:756-760 (2003); franklin et al, cancer Cell 5:317-328 (2004); or Plowman et al, proc.Natl.Acad.Sci.90:1746-1750 (1993).
In embodiments of the present invention, the terms "trastuzumab," "Pertuzumab," "intutmaab," "cetrimab," "panitumumab," "Necitumumab," "Matuzumab," and "Nimotuzumab" are used in accordance with their ordinary and customary meanings as understood in the art.
In embodiments of the present invention, the term "cytotoxic agent" or "cytotoxic agent" generally refers to a substance that has cytotoxic activity and causes cell destruction. The term is intended to include radioisotopes, chemotherapeutic agents, and toxins (e.g., small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin), including synthetic analogs and derivatives thereof. The term "cytotoxic activity" generally refers to the cell killing effect of intracellular metabolites of a drug (e.g., a camptothecin conjugate). Cytotoxic activity can be expressed as an IC50 value, which is the concentration per unit volume (mole or mass) of half the cell survival.
In embodiments of the present invention, the term "cytostatic agent" or "cytostatic agent" generally refers to a substance having cytostatic activity, including a substance that inhibits cell growth or proliferation. Cytostatics include inhibitors such as protein inhibitors, for example enzyme inhibitors. Cytostatic agents have cytostatic activity. The term "cytostatic activity" generally refers to the antiproliferative effect of intracellular metabolites of drugs (e.g., camptothecin conjugates).
In embodiments of the present invention, the term "camptothecin" refers generally to a pyrroloquinoline cytotoxic alkaloid consisting of a quinoline ring AB, a pyrrole ring C, a pyridone ring D, and an α -hydroxy lactone ring E, wherein the 20-position is in the S configuration, having the structure:
among them, the lactone ring (E ring), pyridone ring (D ring) and the hydroxyl group at C20 position are considered to be essential groups for CPT to exert topoisomerase I inhibitory effect to achieve antitumor effect.
In embodiments of the present invention, the term "10-difluoromethyl camptothecins" refers generally to the presence of a difluoromethyl-substituted camptothecin derivative (CN 201810497805) at the 10-position of camptothecins, which is incorporated by reference in its entirety into embodiments of the present invention.
In embodiments of the present invention, the terms "camptothecin," "irinotecan," "topotecan," "irinotecan," "belotecan," "lurotecan," "CKD-602," "gematecan," "kartinitecin," "BN-80915," "hydroxycamptothecin," "9-aminocamptothecin," "9-nitrocamptothecin," "7-ethyl-10-hydroxycamptothecin," "7-ethyl-10-difluoromethylcamptothecin," are used in accordance with their ordinary and customary meanings as understood in the art.
In the present examples, the term "pharmaceutically acceptable" component generally refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio.
In embodiments of the present invention, the term "solvate" generally refers to an association (assocI-Btion) or complex (complex) of one or more solvent molecules with a compound of an embodiment of the present invention. Non-limiting examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. The term "hydrate" generally refers to a complex in which the solvent molecule is water.
In embodiments of the present invention, the term "optical isomer" or "stereoisomer" generally refers to compounds having the same chemical composition but differing in the arrangement of atoms or groups in space, including any of the various stereoisomeric configurations that may exist, including geometric isomers. It is understood that substituents may be attached to the chiral center of a carbon atom. The term "chiral" refers to a molecule that has a non-overlapping characteristic with its mirror molecule pair, while the term "achiral" refers to a molecule that is capable of overlapping with its mirror molecule pair. Thus, the present invention includes enantiomers, diastereomers or racemates of the compounds. "enantiomers" are a pair of stereoisomers that do not mirror each other. A1:1 mixture of a pair of enantiomers is a "racemic" mixture. Where appropriate, the term is used to designate a racemic mixture. "diastereomers" are stereoisomers having at least two asymmetric atoms, but which are not mirror images of each other. Absolute stereochemistry was described according to the Cahn-lngo-PrelogR-S system. When the compounds are pure enantiomers, the stereochemistry of each chiral carbon may be designated as R or S. Resolution compounds of unknown absolute configuration can be designated (+) or (-) depending on their direction of rotation (right-hand or left-hand) of plane polarized light at the sodium D-line wavelength. Certain compounds described herein contain one or more asymmetric centers or axes, and thus can produce enantiomers, diastereomers, and other stereoisomeric forms, which may be designated as (R) -or (S) -, depending on the absolute stereochemistry.
In embodiments of the present invention, the term "tautomer" generally refers to structural isomers having different energies, which can be interconverted by a low energy barrier. For example, proton tautomers (also known as proton tautomers) include interconversions by proton transfer, such as keto-enol and imine-enamine isomerisation. Valence tautomers include interconversions by recombining some of the bond-forming electrons.
In embodiments of the present invention, the term "isotope" generally includes atoms having the same atomic number but different mass numbers. Examples of isotopes that can be incorporated into compounds of embodiments of the invention and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine, for example, 2 H、 3 H、 11 C、 13 C、 14 C、 15 N、 17 O、 18 O、 31 P、 32 P、 35 S、 18 F、 36 Cl、 123 i and 125 I. the disclosed subject matter of embodiments of the present invention may also include isotopically-labeled forms of the compounds described in embodiments of the present invention.
In embodiments of the present invention, the term "metabolite" generally refers to a product produced by metabolizing a specific compound or salt thereof in vivo. Metabolites of compounds may be identified using conventional techniques known in the art and their activity determined using assays as described in the examples of the invention. Such products may result from, for example, oxidation, hydroxylation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc. of the administered compound. Accordingly, embodiments of the present invention may also include metabolites of the compounds of embodiments of the present invention, including compounds produced by a method comprising contacting a compound of embodiments of the present invention with a mammal for a period of time sufficient to produce a metabolite thereof.
In embodiments of the invention, the term "prodrug" or "prodrug" generally refers to a compound that is a prodrug that undergoes a chemical conversion by a metabolic or chemical process upon administration to a subject, resulting in a compound of embodiments of the invention or a salt thereof. The content of prodrugs is well known in the art (see, e.g., berge et al (1977) "Pharmaceutical Salts", J.Pharm. Sci.66:1-19).
In the examples of the present invention, the term "pharmaceutically acceptable salt" refers to a salt capable of maintaining the biological effects and properties of the compounds of the examples of the present invention, which salt generally has no biological or other disadvantages. It includes pharmaceutically acceptable organic or inorganic salts, and exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, gluconate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1' -methylene-bis- (2-hydroxy-3-naphthoate) salts), alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may be referred to as comprising another molecule, such as an acetate ion, succinate ion, or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. In examples where the plurality of charged atoms are part of a pharmaceutically acceptable salt, the salt may have a plurality of counter ions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counter ions.
In embodiments of the present invention, the term "linker" or "linker unit" generally refers to a bifunctional moiety that links a drug (e.g., camptothecin) to a ligand unit in a drug-ligand conjugate. The linker units of the present examples have several components (e.g., in some embodiments, they have an extension unit of an alkaline unit, a branching unit that may or may not be present, a spacer unit, a releasable assembly unit).
In embodiments of the present invention, the term "PEG", "PEG unit" or "polyethylene glycol" is an organic moiety composed of repeating ethylene-oxy subunits and may be polydisperse, monodisperse, or discrete (i.e., having a discrete number of ethylene-oxy subunits). Polydisperse PEG is a heterogeneous mixture of size and molecular weight, whereas monodisperse PEG is generally purified from heterogeneous mixtures and thus has a single chain length and molecular weight. The PEG units of embodiments of the present invention may comprise one or more polyethylene glycol chains, each polyethylene glycol chain being composed of one or more ethyleneoxy subunits covalently linked to each other. Polyethylene glycol chains may be linked together, for example, in a linear, branched or star configuration. Typically, the terminal ethyleneoxy subunit of each polyethylene glycol chain that is not covalently linked to the remainder of the linker unit is modified with a PEG capping unit, typically an optionally substituted alkyl group such as-CH 3 、CH 2 CH 3 Or CH (CH) 2 CH 2 CO 2 H. In some embodiments, the PEG unit has 1 to 20-CH 2 CH 2 A single polyethylene glycol chain of O-subunits, covalently linked in tandem and terminated at one end by a PEG capping unit.
In embodiments of the present invention, the term "optionally substituted" means that the groups involved may be substituted or unsubstituted. When substituted, substituents of an "optionally substituted" group may include, but are not limited to, one or more substituents independently selected from the following groups, alone or in combination, or a specifically designated group of groups: alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, hydroxy, alkoxy, mercapto, cyano, halogen, carbonyl, thiocarbonyl, isocyanic acidGroups, thiocyanates, isothiocyanates, nitro groups, perhaloalkyl groups, and amino groups including mono-and di-substituted amino groups, and protected derivatives thereof. Non-limiting examples of optional substituents include halogen, -CN, =o, =n-OH, =n-OR, =n-R, -OR, -C (O) R, -C (O) OR, -OC (O) R, -OC (O) OR, -C (O) NHR, -C (O) NR 2 、-OC(O)NHR、-OC(O)NR 2 、-SR-、-S(O)R、-S(O) 2 R、-NHR、-N(R) 2 、-NHC(O)R、-NRC(O)R、-NHC(O)OR、-NRC(O)OR、S(O) 2 NHR、-S(O) 2 N(R) 2 、-NHS(O) 2 NR 2 、-NRS(O) 2 NR 2 、-NHS(O) 2 R、-NRS(O) 2 R、C 1-6 Alkyl, C 1-6 Alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C 1-6 Alkyl, and halogen substituted C 1-6 Alkoxy, wherein each R is independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C 1-6 Alkyl, and halogen substituted C 1-6 An alkoxy group. The position and number of such substituents are determined by the well known valence constraints of each group, e.g., =o is a suitable substituent for an alkyl group but not for an aryl group. The two substituents may be linked together to form a five, six or seven membered aromatic or non-aromatic carbocyclic or heterocyclic ring containing one to three heteroatoms, for example to form methylenedioxy or ethylenedioxy.
Where specific naming is concerned, substituents are generally placed before the groups being substituted, e.g. "C 1-3 Alkoxy C 3-8 Ring(s)
Alkyl C 1-6 Alkyl "means C 1-6 Alkyl, which is C 3-8 Cycloalkyl substitution, and C 3-8 Cycloalkyl radicals are again C 1-3 Alkoxy substitution, examples: the structural formula of the methoxycyclobutylmethyl is as follows:
in embodiments of the invention, the number of carbon atoms is generally by the prefix "C x -C y "OR" C x-y "means where x is the minimum number of carbon atoms in the substituent and y is the maximum number. For example, "C 1 -C 6 Alkyl "or" C 1-6 Alkyl "refers to an alkyl substituent containing 1 to 6 carbon atoms. Further by way of example, C 3 -C 6 Cycloalkyl or C 3-6 Cycloalkyl refers to saturated cycloalkyl groups containing 3 to 6 carbon ring atoms.
In embodiments of the present invention, the term "alkyl" generally refers to a straight, branched, or cyclic saturated substituent consisting of carbon and hydrogen. Non-limiting examples of alkyl groups include: methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl and tert-butyl), pentyl, isopentyl, hexyl, and the like. Optionally, alkyl groups may be optionally substituted on each carbon as defined in the claims. Typical substituents include, but are not limited to: fluorine, chlorine, OH, cyano, alkyl (optionally substituted), cycloalkyl, and the like.
In embodiments of the present invention, the term "cycloalkyl" generally refers to a saturated monocyclic hydrocarbon group. The monocyclic ring generally comprises 3 to 10 carbon atoms. Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, and the like.
In embodiments of the present invention, the term "heterocyclyl" or "heterocycle" generally refers to a non-aromatic heterocyclic group, bicyclic heterocyclic group, polycyclic heterocyclic group, etc., containing at least one heteroatom in the ring structure, and specifically may be, for example, 1 or more heteroatoms selected from O, S and N, optionally the same or different, in meaning.
The term "aryl" generally refers to a hydrocarbon monocyclic or bicyclic aromatic ring system, wherein such rings may be fused. If the rings are fused rings, one of the rings must be a fully unsaturated ring, and the fused rings may be fully saturated, partially unsaturated, or fully unsaturated rings. The term "fused" means that a second ring is present (i.e., linked or formed) with two adjacent atoms that are common to (i.e., share with) the first ring. The term "aryl" includes aromatic groups such as phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, 4- (pyridin-3-yl) phenyl, 2, 3-dihydro-1H-indenyl, and 1,2,3, 4-tetrahydronaphthyl.
The term "heteroaryl" generally refers to an aromatic group (e.g., pyrrolyl, pyridyl, pyrazolyl, indolyl, indazolyl, thienyl, furyl, benzofuranyl, oxazolyl, imidazolyl, tetrazolyl, triazinyl, pyrimidinyl, pyrazinyl, thiazolyl, purinyl, benzimidazolyl, quinolinyl, isoquinolinyl, benzothienyl, benzoxazolyl, 1H-benzo [ d ] [1,2,3] triazolyl, and the like) containing at least one heteroatom (e.g., oxygen, sulfur, nitrogen, or a combination thereof) in a 5-to 10-membered aromatic ring system. Heteroaromatic groups may consist of a single ring or a fused ring system. Typical mono-heteroaryl rings are 5-to 6-membered rings containing 1 to 3 heteroatoms independently selected from oxygen, sulfur and nitrogen, and typical fused heteroaryl ring systems are 9-to 10-membered ring systems containing 1 to 4 heteroatoms independently selected from oxygen, sulfur and nitrogen. The fused heteroaryl ring system may consist of two heteroaryl rings fused together, or of a heteroaryl group fused to an aryl group (e.g., phenyl).
In embodiments of the present invention, a solid wedge key is typically usedAnd wedge-shaped dotted bond->Representing the absolute configuration of a solid centre, using straight solid keys +.>And straight dotted bond->Representing the relative configuration of the stereo centers, using wavy lines +.>Representing a wedge solid key +.>And wedge-shaped dotted bond->Or by wave lines->Representing a straight solid keyAnd straight dotted bond->Unless otherwise indicated, all compounds present in the present invention are intended to include all possible optical isomers, such as single chiral compounds, or mixtures of various chiral compounds (i.e., racemates). Among all the compounds of the invention, each chiral carbon atom may optionally be in the R configuration or in the S configuration, or in a mixture of R and S configurations.
In embodiments of the present invention, the term "carrier" may include pharmaceutically acceptable carriers, excipients or stabilizers which are non-toxic to the cells or mammals at the dosages and concentrations employed. The physiologically acceptable carrier is typically an aqueous pH buffered solution. Non-limiting examples of physiologically acceptable carriers include buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid; a low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants such as tween (tm), polyethylene glycol (PEG), and pluronic stm. In certain embodiments, the pharmaceutically acceptable carrier is a non-naturally occurring pharmaceutically acceptable carrier.
In embodiments of the present invention, the term "abnormal" generally refers to deviations from a standard, such as a normal healthy subject or cell and/or a group of normal healthy subjects or cells. The term "aberrant expression" as used herein refers to an aberrant expression of a gene product (e.g., RNA, protein, polypeptide, or peptide) of a cell or subject as compared to a normal healthy cell or subject and/or a population of normal healthy cells or subjects. Such abnormal expression may be caused by gene amplification or by inhibition of gene expression. In some embodiments, "aberrant expression" in relation to dnase topoisomerase I refers to increased, decreased or inappropriate expression of dnase topoisomerase I. In particular embodiments, the term "abnormal activity" refers to deviations of dnase topoisomerase I from normal activity in a healthy cell or subject and/or a population of normal healthy cells or subjects.
In embodiments of the invention, the term "up-regulation of expression" generally refers to an increase in the expression of the mRNA level of a nucleic acid or an increase in the expression of the polypeptide level. The term may also relate to post-translational modifications required for increased polypeptide activity and/or function, e.g., addition of sugar moieties, phosphorylation, etc.
In the present examples, the term "inhibitor" generally refers to compounds/substances known in the art and which are involved in being able to prevent or reduce, in whole or in part, the physiological function (i.e. activity) of one or more specific proteins (e.g. topoisomerase I). Inhibitors are also known as "antagonists". In the context of the present invention, inhibitors of topoisomerase I may prevent or reduce or inhibit or inactivate the physiological activity of topoisomerase I.
In embodiments of the present invention, examples of the term "topoisomerase I inhibitor" include, but are not limited to, topotecan, gematecon (gimatecan), irinotecan, camptothecin and analogs thereof, 9-nitrocamptothecin, and macromolecular camptothecin conjugate PNU-166148 (compound A1 in WO 99/17804); 10-hydroxycamptothecin acetate; etoposide; idarubicin hydrochloride; irinotecan hydrochloride; teniposide; topotecan, topotecan hydrochloride; doxorubicin; epirubicin, epirubicin hydrochloride; 4' -epirubicin, mitoxantrone hydrochloride; daunorubicin, daunorubicin hydrochloride, valubicin, and dasatinib (BMS-354825).
In embodiments of the present invention, the term "treatment" or "treatment" generally refers to both therapeutic and prophylactic treatment, wherein the aim is to inhibit or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "treatment" may also mean an extended lifetime compared to the expected lifetime of an untreated patient. Those in need of treatment include those already with the disease or condition as well as those prone to the disease or condition. Within the scope of cancer, the term "treatment" may also include any or all of the following: killing tumor cells; inhibit the growth of tumor cells, cancer cells, or tumors; inhibit replication of tumor cells or cancer cells; reducing overall tumor burden or reducing the number of cancer cells; and ameliorating one or more symptoms associated with the disease.
The term "administration" or "administering" includes the route by which the compound is introduced into a subject to achieve its intended function. Non-limiting examples of routes of administration that can be used include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal), topical, oral, inhalation, rectal, and transdermal.
The term "contacting" generally means that two or more different types of substances are contacted together in any order, in any manner, and for any period of time. When applied to a cell, "contacting" means a method whereby a compound of an embodiment of the invention is delivered to a target cell or placed in direct proximity to a target cell, which delivery may be in vitro or in vivo.
In embodiments of the present invention, the term "effective amount" generally includes an amount effective to achieve the desired result within the necessary dosage and period of time. An effective amount of a compound may vary depending on factors such as the disease state, age, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. The dosage regimen may be adjusted to provide the optimal therapeutic response.
In embodiments of the present invention, the term "therapeutically effective amount" generally refers to an amount of a conjugate that is effective to treat a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of the conjugate may reduce the number of cancer cells; reducing tumor size; inhibit (i.e., delay to some extent, preferably terminate) infiltration of cancer cells into surrounding organs; inhibit (i.e., delay to some extent, preferably terminate) tumor metastasis; inhibit tumor growth to some extent; and/or to some extent, one or more symptoms associated with the cancer. To the extent that the drug can inhibit the growth of cancer cells and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer treatment, efficacy may be detected, for example, by assessing time to disease progression (TTP) and/or determining Response Rate (RR).
In embodiments of the invention, the term "subject" or "patient" refers to an animal, such as a mammal, including but not limited to primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, and the like. In certain embodiments, the subject is a human.
Connector
In a first aspect, embodiments of the present invention provide a linker comprising a B structural fragment, wherein the B structural fragment is selected from the group consisting of:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20; r is any natural number from 1 to 4; for example 1, 2, 3 and 4.Y is selected from any one of N, O and S; r is selected from any one of (CH 2) n, cycloalkyl and aryl; n is any natural number from 1 to 6, for example 1, 2, 3, 4, 5 and 6.
In certain embodiments, the B structural fragment is selected from the following formulae:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6.
In certain embodiments, the linker further comprises a B-Lb structural fragment, wherein the Lb structural fragment is selected from the following formulas:
wherein n is any natural number from 1 to 6, for example 1, 2, 3, 4, 5 and 6. The methylene group of the Lb structural fragment is linked to the methine group of the B structural fragment.
In certain embodiments, the linker further comprises a B-Lb-LC structural fragment, wherein the Lb structural fragment is selected from the following formulas:
where n is any natural number from 1 to 6, for example 1, 2, 3, 4, 5 and 6. The methine group of the B structural fragment is linked to the methylene group of the Lb structural fragment, which is linked to the Lc structural fragment by a carbonyl group, wherein Lc is a releasable assembly unit and Lc is capable of linking to a drug unit.
In certain embodiments, the Lc structural fragment is selected from the following formulae:
the Lc structural fragment is linked to the carbonyl group of the Lb structural fragment by an amine group, and Lc can be linked to the drug unit by a carbonyl group.
In certain embodiments, the linker has a structure represented by formula (I):
wherein,
the La structural fragment is an extension unit that is capable of linking to a ligand unit;
the structure of B is selected from the following formulas:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6; the B structural fragment is connected with the La structure through an amino group and is connected with the Lb structural fragment through a methine group;
the Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, and the methylene of the Lb structural fragment is connected with the methine of the B structural fragment;
the Lb structural fragment is linked to either the Lc1 or Lc2 structural fragment by a carbonyl group, wherein Lc1 and Lc2 are releasable assembled units respectively and both Lc1 and Lc2 are capable of linking to a drug unit.
Each of Lc1 and Lc2 is independently selected from any one of Lc.
In certain embodiments, the La structural fragment comprises a maleimide linker fragment.
In certain embodiments, the La structural fragment has the following structure:
which is linked to the B structural fragment via a carbonyl group and is capable ofCan be linked to the ligand unit via succinimide in position 3 and/or 4; and R' is selected from: optionally substituted C 1 -C 10 Alkyl, C 1 -C 10 Alkoxy, C 1 -C 10 Aminoalkyl and C 1 -C 10 Alkyl-aryl wherein the substituents are selected from: amino, halogen, nitro, hydroxy, acetyl, cyano, C 1 -C 10 Alkyl, C 1 -C 10 Alkoxy, C 1 -C 10 Aminoalkyl, C 1 -C 10 Haloalkyl, C 2 -C 10 Alkenyl, C 2 -C 10 Alkynyl, amido, C 3 -C 8 Cycloalkyl, C 3 -C 8 A heterocycloalkyl group.
In certain embodiments, R' is selected from: optionally substituted C1-C10 alkyl, C1-C10 alkoxy, C1-C10 aminoalkyl and C1-C10 alkyl-aryl, wherein the substituents are selected from the group consisting of: amino, halogen, hydroxy, C1-C10 alkyl, C1-C10 alkoxy, C1-C10 aminoalkyl, C1-C10 haloalkyl.
In certain embodiments, R' is selected from: C1-C10 alkyl, C1-C10 alkoxy, C1-C10 aminoalkyl and C1-C10 alkyl-aryl.
In certain embodiments, the La structural fragment is selected from the following formulae:
it is linked to the B structural fragment via a carbonyl group and can be linked to the ligand unit via the succinimide 3 and/or 4 position; wherein each subscript s is independently selected from any integer of from 1 to 10, preferably from 1 to 8, and more preferably from 1 to 5.
In certain embodiments, the La structure is selected from the following formulas:
it is linked to the B structural fragment via a carbonyl group and can be linked to the ligand unit via the succinimide 3 and/or 4 position; wherein subscript s is selected from any of 1 to 10The integer is preferably 1 to 8, more preferably 1 to 5.
In certain embodiments, the La structure is selected from the following formulas:
wherein the subscript s is selected from any integer from 1 to 10, preferably from 1 to 8, more preferably from 1 to 5, the La structural fragment is attached to the B structural fragment by a carbonyl group and is capable of being attached to the ligand unit via the 3-position of the succinimide.
In certain embodiments, the linker is selected from the following structures:
r' is selected from: optionally substituted C 1 -C 10 Alkyl, C 1 -C 10 Alkoxy, C 1 -C 10 Aminoalkyl and C 1 -C 10 Alkyl-aryl wherein the substituents are selected from: amino, halogen, nitro, hydroxy, acetyl, cyano, C 1 -C 10 Alkyl, C 1 -C 10 Alkoxy, C 1 -C 10 Aminoalkyl, C 1 -C 10 Haloalkyl, C 2 -C 10 Alkenyl, C 2 -C 10 Alkynyl, amido, C 3 -C 8 Cycloalkyl, C 3 -C 8 Heterocycloalkyl, q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6.
It should be noted that, n in the above structural formula and n in R are any natural number from 1 to 6.
In certain embodiments, the linker is selected from the following structures:
q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6, and subscript S is any natural number from 1 to 6.
In certain embodiments, the linker is selected from the following structures:
q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; n is any natural number from 1 to 6, and subscript S is any natural number from 1 to 6.
In another aspect, embodiments of the present invention provide the use of the foregoing linker in the preparation of a medicament comprising a linker-drug conjugate or a ligand-drug conjugate.
Linker-drug conjugates
In another aspect, the present invention provides a linker-drug conjugate comprising the foregoing linker, having a structure represented by formula (II):
or a pharmaceutically acceptable salt thereof, wherein,
la is an extension unit capable of linking to the ligand unit;
the B structural fragment is selected from the following formulae:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6;
the Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, the methylene of the Lb structural fragment is connected with the methine of the B structural fragment, and the Lb structural fragment is connected with the Lc1 or Lc2 structural fragment through carbonyl;
LcLc1 and Lc2 are releasable assembled units, respectively;
D1 and D2 are each a drug unit.
In certain embodiments, the linker-drug conjugate has a structure represented by formula (IIa):
or a pharmaceutically acceptable salt thereof, wherein,
la is an extension unit capable of linking to the ligand unit;
the B structural fragment is selected from the following formulae:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6;
the Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, the methylene of the Lb structural fragment is connected with the methine of the B structural fragment, and the Lb structural fragment is connected with the Lc1 or Lc2 structural fragment through carbonyl;
lc1 and Lc2 are releasable assembled units, L c1 and Lc2, respectively, each connected to the drug unit by a carbonyl group;
d1 and D2 are each a drug unit.
In certain embodiments, D1 and D2 are each independently selected from any one of a camptothecin topoisomerase inhibitor, a tubulin inhibitor, a DNA alkylating agent, a checkpoint kinase 1 inhibitor, a sting1 agonist, and a TLR7/8 agonist, and D1 and D2 cannot be both a single pharmaceutical unit.
In certain embodiments, the linker-drug conjugate has a structure represented by formula (III):
or a pharmaceutically acceptable salt thereof, wherein,
la is an extension unit capable of linking to the ligand unit;
the B structural fragment is selected from the following formulae:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S;r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6;
the Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, the methylene of the Lb structural fragment is connected with the methine of the B structural fragment, and the Lb structural fragment is connected with the L1 or Lc2c structural fragment through carbonyl;
lc1 and Lc2 are releasable assembled units, respectively;
represents camptothecin or a derivative thereof;
representing a cell cycle checkpoint kinase 1 inhibitor.
In certain embodiments, camptothecins and derivatives thereof include irinotecan, SN-38, and derivatives thereof.
In certain embodiments, the camptothecin and derivatives thereof are selected from the group consisting of: camptothecin, irinotecan, topotecan, irinotecan, belotecan, lurtoltecan, CKD-602, gem Ma Tikang, karenitecin, BN-80915, hydroxycamptothecin (HCPT), 9-aminocamptothecin, 9-nitrocamptothecin, 7-ethyl-10-hydroxycamptothecin, 7-ethyl-10-difluoromethylcamptothecin, and derivatives thereof;
Preferably, the camptothecin and its derivatives are selected from the group consisting of camptothecin, irinotecan, 7-ethyl-10-hydroxycamptothecin, 7-ethyl-10-difluoromethyl camptothecin, and solvates, hydrates, stereoisomers, tautomers, isotopic labels, metabolites, prodrugs, pharmaceutically acceptable salts thereof.
In certain embodiments, the tubulin inhibitors include DM1, DM4, MMAE, and MMAF;
DNA alkylating agents include melphalan;
cell cycle checkpoint kinase 1 inhibitors include AZD-7762, rabusertib and prexaservib.
In certain embodiments, the linker-drug conjugate is selected from the following structures:
ligand-drug conjugates
The embodiment of the invention provides a ligand-drug conjugate comprising the linker or the linker-drug conjugate, which has a structure shown in formula (V):or a pharmaceutically acceptable salt thereof, wherein L is a ligand unit, wherein the subscript n is selected from any integer from 1 to 8, preferably from 4 to 8;
la is an extension unit;
the B structural fragment is selected from the following formulae:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6;
the Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, the methylene of the Lb structural fragment is connected with the methine of the B structural fragment, and the Lb structural fragment is connected with the Lc1 or Lc2 structural fragment through carbonyl;
lc1 and Lc2 are releasable assembled units, respectively;
d1 and D2 are each a drug unit.
In certain embodiments, D1 and D2 are each independently selected from any one of a camptothecin topoisomerase inhibitor, a tubulin inhibitor, a DNA alkylating agent, a checkpoint kinase 1 inhibitor, a sting1 agonist, and a TLR7/8 agonist, and D1 and D2 cannot be both a single pharmaceutical unit.
In certain embodiments, it has a structure represented by formula (VI):
or a pharmaceutically acceptable salt thereof, wherein n is a natural number from 1 to 8;
la is an extension unit capable of linking to the ligand unit;
the B structural fragment is selected from the following formulae:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6;
The Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, the methylene of the Lb structural fragment is connected with the methine of the B structural fragment, and the Lb structural fragment is connected with the Lc1 or Lc2 structural fragment through carbonyl;
lc1 or Lc2 is a releasable assembly unit, respectively;
represents camptothecin or a derivative thereof; />
Representing a cell cycle checkpoint kinase 1 inhibitor.
In certain embodiments, the camptothecin and derivatives thereof are selected from the group consisting of: camptothecin, irinotecan, topotecan, irinotecan, belotecan, lurtoltecan, CKD-602, gem Ma Tikang, karenitecin, BN-80915, hydroxycamptothecin (HCPT), 9-aminocamptothecin, 9-nitrocamptothecin, 7-ethyl-10-hydroxycamptothecin, 7-ethyl-10-difluoromethylcamptothecin, and derivatives thereof;
preferably, the camptothecin and its derivatives are selected from the group consisting of camptothecin, irinotecan, 7-ethyl-10-hydroxycamptothecin, 7-ethyl-10-difluoromethyl camptothecin, and solvates, hydrates, stereoisomers, tautomers, isotopic labels, metabolites, prodrugs, pharmaceutically acceptable salts thereof.
In certain embodiments, the tubulin inhibitors include DM1, DM4, MMAE, and MMAF;
DNA alkylating agents include melphalan;
cell cycle checkpoint kinase 1 inhibitors include AZD-7762, rabusertib and prexaservib.
In certain embodiments, the ligand unit comprises an antibody;
preferably, the antibody comprises a monoclonal antibody, a polyclonal antibody, a dimer, a multimer, a multispecific antibody, an intact antibody, an antibody fragment, a human antibody, a humanized antibody, a chimeric antibody, or an antibody from another species;
preferably, the antibody fragment comprises: fab, fab ', F (ab') 2, fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies (e.g., sdabs), camelid VHH domains, or multispecific antibodies formed from antibody fragments;
preferably, the antibodies include modified or unmodified analogs and derivatives, and allow the antibodies to retain their antigen-binding immunospecificity.
In certain embodiments, the ligand-drug conjugate has a structure represented by the following structural formula:
or a pharmaceutically acceptable salt thereof, wherein,
ab represents an antibody, and subscript n is selected from any integer of 1 to 8, preferably 4 to 8;
la is an extension unit;
the B structural fragment is selected from the following formulae:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6;
the Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, the methylene of the Lb structural fragment is connected with the methine of the B structural fragment, and the Lb structural fragment is connected with the Lc1 or Lc2 structural fragment through carbonyl;
lc1 and Lc2 structural segments are releasable assembly units, respectively;
d1 and D2 are each a drug unit.
In certain embodiments, the ligand-drug conjugate has any one of the compounds described by the following structural formulas:
preferably, the ligand comprises a mAb or antigen-binding fragment thereof; preferably is
Or a pharmaceutically acceptable salt thereof, wherein, the mAb represents a monoclonal antibody, n is any natural number from 1 to 8;
most preferably:
wherein n is 1 to 8, preferably 4 to 8.
In certain embodiments, the ligand targets binding to a tumor antigen;
preferably, the ligand targets binding receptor tyrosine-protein kinase ERBB2 (HER 2) and/or Epidermal Growth Factor Receptor (EGFR) and/or TROP2 and/or FGFR2b;
preferably, the ligand comprises an anti-HER 2 antibody;
preferably, the anti-HER 2 antibody comprises trastuzumab (trastuzumab), pertuzumab (Pertuzumab) or itumomab (ineetamab);
Preferably, the ligand comprises an anti-EGFR antibody;
preferably, the anti-EGFR antibody comprises Cetuximab (Cetuximab), panitumumab (panitumumab), cetuximab (Necitumumab), matuzumab (Matuzumab), or Nimotuzumab (Nimotuzumab);
preferably, the anti-TROP 2 antibody comprises a golian Sha Tuozhu mab (Sacituzumab);
preferably, the anti-FGFR 2b antibody comprises Bemarituzumab.
The embodiments of the present invention also provide methods of preparing the ligand-drug conjugates described above, comprising conjugating an antibody to the linker described above or to the linker-drug conjugate described above.
Application of
The embodiment of the invention also provides the use of the linker-drug conjugate or ligand-drug conjugate in the preparation of a medicament for treating a disease that overexpresses a tumor antigen;
preferably, the tumor antigen comprises HER2 or EGFR.
The embodiment of the invention also provides the application of the connector-drug conjugate or the ligand-drug conjugate in preparing a medicament for treating diseases with abnormal expression and/or activity of DNase topoisomerase I;
preferably, the dnase topoisomerase I expression and/or activity is up-regulated.
The embodiments of the present invention also provide the use of the linker-drug conjugate or ligand-drug conjugate described above as a dnase topoisomerase I inhibitor.
The embodiment of the invention also provides application of the connector-drug conjugate or the ligand-drug conjugate in preparing medicines for cancers.
The ligand-drug conjugate of the embodiments of the invention can be used to inhibit proliferation of tumor cells or cancer cells, cause apoptosis of tumor or cancer cells, or treat cancer in a patient. Ligand-drug conjugates are accordingly used in a variety of settings to treat cancer. The ligand-drug conjugate is intended to be used to deliver a drug to a tumor cell or cancer cell. Without being bound by theory, in one embodiment, the ligand units of the ligand-drug conjugate will bind or conjugate with a cancer cell or tumor cell-associated antigen, and the ligand-drug conjugate is absorbed (internalized) within the tumor cell or cancer cell by receptor-mediated endocytosis or other internalization mechanisms. In some embodiments, the antigen is linked to a tumor cell or cancer cell, or may be an extracellular matrix protein associated with a tumor cell or cancer cell. Once in the cell, the drug is released inside the cell via activation of the activation unit. In an alternative embodiment, the free drug is released from the ligand-drug conjugate outside the tumor cell or cancer cell, and the free drug subsequently penetrates the cell. In one embodiment, the ligand unit binds to a tumor cell or a cancer cell. In another embodiment, the ligand unit binds to a tumor cell or cancer cell antigen on the surface of a tumor cell or cancer cell. In another embodiment, the ligand unit binds to a tumor cell or cancer cell antigen that is an extracellular matrix protein associated with the tumor cell or cancer cell.
Cancers intended to be treated with the ligand-drug conjugates of embodiments of the invention include, but are not limited to, cancers of the hematopoietic system such as, for example, lymphomas (hodgkin's lymphoma and non-hodgkin's lymphoma) as well as leukemias and solid tumors. Examples of cancers of the hematopoietic system include follicular lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, acute myelogenous leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and multiple myeloma. Examples of solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelioma, synovial carcinoma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, renal cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cancer, throat cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystic gland carcinoma, medullary carcinoma, bronchi cancer, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, bladder cancer, lung cancer, epithelial cancer, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniomama, ependymoma, pineal tumor, glioblastoma, auditory tumor, and neuroblastoma.
In certain embodiments, the cancer comprises a cancer in which HER2 expression and/or activity is up-regulated;
preferably, the cancer comprises a cancer in which EGFR expression and/or activity is up-regulated;
preferably, the cancer comprises non-small cell lung cancer, breast cancer, gastric cancer, colorectal cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, biliary tract cancer, paget's disease, pancreatic cancer, ovarian cancer, or uterine cancer sarcoma.
In certain embodiments, the present examples provide a pharmaceutical composition comprising a linker-drug conjugate as described above or a ligand-drug conjugate as described above, and a pharmaceutically acceptable carrier.
Embodiments of the present invention provide pharmaceutical compositions comprising a ligand-drug conjugate described herein and at least one pharmaceutically acceptable carrier. The pharmaceutical composition is in any form that allows the compound to be administered to a patient to treat a disorder associated with the expression of an antigen bound to a ligand unit. For example, the conjugate is in liquid or solid form. The preferred route of administration is parenteral. Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. In one embodiment, the composition is administered parenterally. In one embodiment, the conjugate is administered intravenously. Administration is by any convenient route, for example by infusion or bolus injection.
The pharmaceutical composition is formulated such that the ligand-drug conjugate is bioavailable upon administration of the composition to a patient. The compositions are sometimes in the form of one or more dosage units. The materials used in the preparation of the pharmaceutical composition are preferably non-toxic in the amounts used. It will be apparent to one of ordinary skill in the art that the optimal dosage of one or more active ingredients in a pharmaceutical composition will depend on a variety of factors. Relevant factors include, but are not limited to, the type of animal (e.g., human), the particular form of the compound, the mode of administration, and the composition employed. In some embodiments, the composition is in liquid form. In some of those embodiments, the liquid is for delivery by injection. In some embodiments, in addition to the ligand-drug conjugate, the composition for administration by injection comprises one or more excipients selected from the group consisting of: surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers and isotonic agents. In some embodiments, the liquid compositions, whether they are solutions, suspensions, or other similar forms, comprise one or more of the following: sterile diluents (e.g., water for injection), saline solutions (preferably physiological saline), ringer's solution, isotonic sodium chloride, fixed oils which can act as solvents or suspending media such as synthetic mono-or diglycerides, polyethylene glycol, glycerol, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine tetraacetic acid; buffers such as amino acids, acetates, citrates or phosphates; detergents, such as nonionic surfactants, polyols; and agents for modulating tonicity, such as sodium chloride or dextrose. Parenteral compositions are sometimes packaged in ampules, disposable syringes or multiple dose vials made of glass, plastic or other materials. Saline is an exemplary adjuvant. The injectable composition is preferably sterile.
Embodiments of the present invention provide a kit comprising the linker-drug conjugate or ligand-drug conjugate.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
This embodiment provides a method of synthesizing intermediate 9, comprising:
the synthesis was performed with reference to the following synthesis route:
in particular, the method comprises the steps of,
step 1: compound 1 (20 g,1 eq) was placed in a reaction flask, DMF (120 mL) was added, stirred at room temperature for dissolution, then compound 2 (1 eq), 2- (7-azabenzotriazol) -N, N' -tetramethylurea Hexafluorophosphate (HATU) (1.1 eq) was added sequentially, then the reaction solution was cooled in an ice bath, and N, N-Diisopropylethylamine (DIPEA) (2.3 eq) was slowly added dropwise to the system. After the addition, stirring at room temperature, detecting by using an LC-MS (liquid crystal-mass spectrometer), pouring the reaction solution into 600 mL water for quenching and crystallization after the reaction is finished, slowly stirring the system for 30 min, filtering, and washing a filter cake with water to obtain a white solid 3. The next step is directly carried out.
Step 2: compound 3 obtained in step 1 was added to 300 mL Dichloromethane (DCM), dissolved by stirring at room temperature, then 100 mL trifluoroacetic acid was added, and the reaction was stirred at room temperature for 30 hours, and the reaction was terminated by lc-MS detection. Adding 600 mL water into the reaction system, precipitating a large amount of solid, slowly stirring for 20 min, filtering, washing the filter cake with water and DCM sequentially, and drying to obtain white solid 4 (22.7 g)
Step 3: compound 4 (8 g,1 eq) was placed in a reaction flask, dissolved in DMF, then HATU (1.2 eq) was added, cooled in a water bath, DIPEA, t-butyl glycine were added in sequence, and the reaction was stirred at room temperature. LC-MS detection, pouring the reaction solution into 500 mL water after the reaction, extracting with Ethyl Acetate (EA) (250 mL ×2), mixing the organic phases, washing the organic phases with saturated saline for 1 time, concentrating under reduced pressure to obtain white solid 5,
step 4: compound 5 obtained in step 3 was added to 200 mL Dichloromethane (DCM), dissolved by stirring at room temperature, then 80 mL trifluoroacetic acid was added, and the reaction was stirred at room temperature for 20 hours, and the reaction was terminated by lc-MS detection. 600 mL water is added into the reaction system, stirred and crystallized for 1 h under ice bath cooling, filtered, and filter cake is washed by water and dried to obtain white solid 6 (8.5 g).
Step 5: placing compound 6 (8.5 g,1 eq) in a reaction bottle, adding DMF solvent (85 mL), then adding anhydrous copper acetate (0.38 eq), lead tetraacetate (1.15 eq) and anhydrous acetic acid (3 eq) in sequence under stirring, heating to internal temperature of 60-65 ℃ for reaction after the addition, LC-MS detecting, adding 500 mL water after the reaction is finished, extracting with dichloromethane (250 mL multiplied by 2), combining organic phases, concentrating under reduced pressure to obtain white solid 7 (12 g)
Step 6: placing the compound 7 (12 g) into a reaction bottle, adding 200mL of Tetrahydrofuran (THF), stirring and dissolving, then sequentially adding benzyl hydroxy acetate (4 eq), pyridinium p-toluenesulfonate (PPTS) (0.2 eq), heating to 40-45 ℃ in a water bath, stirring and reacting overnight, monitoring by LC-MS, concentrating the solvent to about 50mL after the reaction is finished, adding 400 mL of water, stirring for 20 min, precipitating yellow solid, filtering, heating the filter cake with 80 mL ethyl acetate (60-65 ℃) for dissolving, dropwise adding n-hexane (100 mL) after dissolving, naturally cooling after dropwise adding, crystallizing for 1h under an ice bath, and filtering to obtain white solid 8 (11 g).
Step 7: compound 8 was placed in a reaction flask, DMF (200 mL) was added and stirred for dissolution, then 8mL morpholine was added, stirred at room temperature overnight, monitored by LC-MS, and after the reaction was completed, concentrated to dryness under reduced pressure at 45 ℃ to give crude compound 9, which was used directly in the next step without purification.
Example 2
This embodiment provides a method of synthesizing intermediate 16 comprising:
the synthesis was performed with reference to the following synthesis route:
in particular, the method comprises the steps of,
step 1: compound 10 (1.0 eq) was placed in a reaction flask, DMF was added to dissolve, HATU (1.2 eq) was then added, cooled in a water bath, DIPEA (2.0 eq) and amino octaglyme (1.1 eq) were added in sequence and the reaction was stirred at room temperature. LC-MS detection, pouring the reaction solution into water after the reaction is finished, extracting by ethyl acetate, combining organic phases, washing by saturated sodium bicarbonate and saturated saline, concentrating under reduced pressure to obtain a crude product, and purifying by a column to obtain the compound 11.
Step 2: compound 11 (1 eq) was placed in a reaction flask, DCM (10V) was added for dissolution, TFA (3V) was then added, the reaction was carried out overnight at room temperature, monitored by LC-MS, after the reaction was completed, the reaction solution was poured into water, DCM was extracted, the organic phases were combined, washed with 3-5% brine and concentrated under reduced pressure to give colorless oil 12.
Step 3: compound 12 (1.0 eq) was placed in a reaction flask, DMF was added to dissolve, then DMTMM (1.4 eq), compound 9 (1.0 eq), DIPEA (1.0 eq) and a little water were added to catalyze the reaction at room temperature, LC-MS was monitored, after the reaction was completed, the reaction solution was poured into saturated brine, THF was extracted, the saturated brine was washed, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain gum 13, which was directly used for the next reaction without purification.
Step 4: compound 13 (1.0 eq) was placed in a reaction flask, DMF was added, heated to dissolve, cooled to room temperature, palladium on charcoal (3-5% w) was added and reduced by hydrogenation at normal pressure. After the reaction is finished, palladium-charcoal is removed by filtration, methyl tert-butyl ether (MTBE) with volume six times of that of DMF is added, solid is separated out, and the solid is filtered and dried to obtain the compound 14.
Step 5: compound 14 (1.0 eq) was placed in a reaction flask, DMF (10V), DIPEA (2.0 eq) was added and stirred at room temperature for dissolution, then the compound irinotecan (1.0 eq), HATU (1.2 eq) was added sequentially. After the addition, stirring at room temperature, detecting by using an LC-MS, pouring the reaction solution into water to quench after the reaction is finished, extracting by using DCM, washing an organic phase by using 1% hydrochloric acid and saturated saline, and concentrating under reduced pressure to obtain a compound 15. The next step is directly carried out.
Step 6: compound 15 (100 mg,1 eq) was placed in a reaction flask, DMF (5 mL), morpholine (0.3 mL) was added, stirring at room temperature was performed, LC-MS monitoring was performed, after the completion of the reaction, the solvent was evaporated to dryness, DMF (5 mL) water (1 mL) was added to dissolve, then Fmoc-glycine (1 eq), 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (DMTMM) (1.4 eq), DIPEA (1.1 eq) was added in sequence, stirring at room temperature was performed, LC-MS monitoring was performed, after the completion of the reaction, 50mL water was added, extraction with dichloromethane was performed, concentration was performed, and the crude product was purified by column chromatography to give Compound 16 (100 mg).
Example 3
This embodiment provides a method of synthesizing intermediate 19 comprising:
the synthesis was performed with reference to the following synthesis route:
in particular, the method comprises the steps of,
step 1: compound 14 (1.0 eq) was placed in a reaction flask and dissolved in DMF, then compound AZD7762 (1.0 eq), DIPEA (1.0 eq) and DMTMM (1.4 eq) were added in sequence and finally catalysed with a small amount of water. Room temperature reaction, LC-MS monitoring, water quenching after the reaction, DCM extraction, washing the organic phase with saturated saline, and concentrating under reduced pressure to obtain the compound 17 which is directly used in the next step
Step 2: the compound 17 is placed in a reaction bottle, DMF (5 mL), morpholine (0.3 mL) is added, stirring is carried out at room temperature, LC-MS monitoring is carried out, and after the reaction is finished, the solvent is evaporated to obtain a crude product of the compound 18, which is directly used in the next step.
Step 3: compound 18 was placed in a reaction flask, dissolved in DCM, ice-water bath temperature controlled at 0-5 ℃, followed by sequential addition of DIPEA (2 eq) and succinic anhydride (1.2 eq), monitored by LC-MS and after completion of the reaction the crude solvent was directly evaporated. MTBE was slurried to afford compound 19.
Example 4
This embodiment provides a method of synthesizing intermediate 24 comprising:
the synthesis was performed with reference to the following synthesis route:
in particular, the method comprises the steps of,
step 1: placing the compound 4 (1.0 eq) in a reaction bottle, adding DMF for dissolution, then sequentially adding the compound p-aminobenzyl alcohol (1.2 eq), DIPEA (2.0 eq) and HATU (1.3 eq), stirring at room temperature for reaction, monitoring by LC-MS, after the reaction is finished, dropwise adding the reaction solution into 1% alkene hydrochloride water with the volume being six times that of DMF, separating out solid, filtering, pulping MTBE to obtain the compound 20
Step 2: placing the compound 20 (1.0 eq) in a reaction bottle, adding ultra-dry THF for dissolution, adding TEA (2.0 eq), then dropwise adding a THF solution of the compound p-nitrophenyl chloroformate (1.5 eq), stirring at room temperature for reaction overnight, monitoring by LC-MS, adding saturated saline solution after the reaction is basically finished, separating liquid, extracting an aqueous layer by ethyl acetate, combining organic layers, washing the saturated saline solution, concentrating under reduced pressure to obtain a crude product, pulping the n-hexane to obtain 21
Step 3: placing the compound 21 (1.2 eq) in a reaction bottle, adding ultra-dry THF for dissolution, then sequentially adding TEA (2.0 eq) and the compound AZD7762 (1.0 eq), stirring at room temperature for reaction overnight, monitoring by LC-MS, and directly purifying by silica gel sample mixing column chromatography after the reaction is basically finished to obtain the compound 22
Step 4: placing the compound 22 (1.0 eq) in a reaction bottle, adding DMF, morpholine, stirring at room temperature, monitoring by LC-MS, evaporating the solvent after the reaction is finished, adding DMF for dissolving, then sequentially adding the compound 12 (1.1 eq), HATU (1.2 eq), DIPEA (2.0 eq), stirring at room temperature for reaction, monitoring by LC-MS, adding water after the reaction is finished, extracting with dichloromethane, concentrating, pulping with MTBE to obtain the compound 23
Step 5: placing the compound 23 into a reaction bottle, adding DMF (dimethyl formamide), morpholine, stirring at room temperature, monitoring by LC-MS (liquid Crystal-Mass Spectrometry), evaporating the solvent after the reaction is finished, adding DCM for dissolution, controlling the temperature of ice water bath to be 0-5 ℃, then sequentially adding DIPEA (1.5 eq) and succinic anhydride (1.2 eq), monitoring by LC-MS, and directly evaporating the crude solvent after the reaction is finished. MTBE was slurried to afford compound 24.
Example 5
This example provides a method of synthesizing a linker-drug conjugate (number 060101) comprising:
the synthesis was performed with reference to the following synthesis route:
In particular, the method comprises the steps of,
step 1: compound 26 (280 mg,1 eq) was placed in a reaction flask and dissolved in DMF, then compound 25 (1 eq), HATU (2 eq) and DIPEA (3.3 eq) were added in sequence, the reaction was stirred at RT, LC-MS monitoring was carried out, after the reaction was completed 10mL of saturated aqueous sodium bicarbonate solution, 50mL of water was added, then extracted with dichloromethane (50 mL. Times.2), the organic phases were combined, the organic phase was washed once with saturated brine and concentrated under reduced pressure to give crude compound 27 (1.2 g)
Step 2: compound 27 (1 eq) was placed in a reaction flask, 20mL of methylene chloride, 6mL of trifluoroacetic acid were added, the reaction was carried out at room temperature for about 3 hours, LC-MS monitoring was carried out, after the completion of the reaction, the solvent was evaporated to dryness under reduced pressure, THF (10 mL), water (2 mL) and potassium carbonate (2 eq) were added for dissolution, then fluorenylmethoxycarbonyl chloride (Fmoc-Cl) (1.1 eq) was added, the reaction was carried out at room temperature for 1 hour, LC-MS monitoring was carried out, the solution was evaporated to dryness under reduced pressure after the completion of the reaction, and the resultant was purified by column chromatography to give Compound 28 (590 mg).
Step 3: compound 16 (1.0 eq) was placed in a reaction flask, DMF, morpholine and stirring at room temperature were added, LC-MS was monitored, the solvent was evaporated after the reaction was completed, then DMF was added to dissolve, then compound 28 (1.0 eq), DIPEA (1.0 eq) and DMTMM (1.4 eq) were added sequentially, a little water was used to catalyze the reaction, stirring at room temperature was carried out overnight, LC-MS was monitored, water was added after the reaction was completed, then dichloromethane was used to extract, the organic phase was combined, washed once with saturated brine, concentrated under reduced pressure to obtain crude product, separation of liquid phase was prepared, DCM was extracted after acetonitrile was distilled under reduced pressure, and concentrated under reduced pressure to obtain compound 29.
Step 4: placing the compound 29 (1.0 eq) in a reaction bottle, adding DMF, morpholine, stirring at room temperature, monitoring by LC-MS, evaporating the solvent after the reaction is finished, then adding DMF for dissolving, then sequentially adding the compound 19 (1.0 eq), DIPEA (1.0 eq) and DMTMM (1.4 eq), catalyzing a little water, stirring at room temperature for reaction overnight, monitoring by LC-MS, concentrating under reduced pressure to remove DMF after the reaction is finished, pulping by acetone to obtain a crude product, preparing a liquid phase, purifying and freeze-drying to obtain linker+payload 060101.
Example 6
This example provides a method of synthesizing a linker-drug conjugate (number 060103) comprising:
the synthesis was performed with reference to the following synthesis route:
specifically, compound 29 (1.0 eq) is placed in a reaction bottle, DMF and morpholine are added, stirring is carried out at room temperature, LC-MS monitoring is carried out, the solvent is evaporated after the reaction is finished, then DMF is added for dissolution, then compound 24 (1.0 eq), DIPEA (1.0 eq) and DMTMM (1.4 eq) are sequentially added, a little water is used for catalysis, stirring reaction is carried out at room temperature overnight, LC-MS monitoring is carried out, after the reaction is finished, after DMF is removed by decompression concentration, MTBE is pulped to obtain a crude product, and liquid phase purification and freeze drying are carried out to obtain (linker+payload) 060103.
Example 7
The present embodiment provides a method for synthesizing an antibody-drug conjugate, comprising:
The synthesis was performed with reference to the following synthesis route:
in particular, the method comprises the steps of,
reduction of antibodies: the concentration of trastuzumab solution was adjusted to 10mg/mL with PBS buffer (pH 6.0), 1mL of antibody solution was taken, 60. Mu.L of TCEP solution (10 mM) was added thereto, the reaction solution was placed in a 37℃water bath for incubation for 1 hour, and the solution was taken out and cooled to room temperature for the next reaction.
Conjugation to antibodies: the linker-drug conjugate was dissolved in dimethylacetamide to adjust the concentration to 10mM, 0.1mL of the solution was diluted 10-fold with PBS buffer (pH 6.0), the solution was added to the reduced antibody solution, and the mixture was allowed to react at room temperature for 40 minutes with mixing, then 20. Mu.L of NAC solution (0.1M) was added to the reaction solution, and stirring was continued for 20 minutes to terminate the reaction.
Purification of drug-antibody conjugates: the reaction was passed through a zeba desalting centrifuge column (molecular weight cut-off 10 kDa) to remove small molecules, the resulting target product solution was concentrated by centrifugation using an Amico Ultra (10, 000MWCO,Millipore Co.) vessel, the buffer was replaced, and the product solution was adjusted to PBS (pH 7.2) at a concentration of 2mg/mL.
Evaluation of drug-antibody conjugate products: the product yield was 65%, and the average linkage of the antibody to 7.88 drug molecules per molecule was calculated by measuring the UV absorbance at 280nm and the UV absorbance at 370nm using the molar extinction coefficients of the antibody and drug at 280nm and 370nm, respectively.
Experimental example 1: cell proliferation inhibition assay
Cell culture
Cell lines Cell line type Growth characteristics Complete medium
NCI-N87 Gastric cancer cell Wall-attaching RPMI-1640+10％FBS
SK-BR-3 Breast cancer cells Wall-attaching RPMI-1640+10％FBS
Information on test compounds
Cell proliferation assay
a) All cell lines were cultured in complete medium at 37 ℃,5% CO 2;
b) Harvesting cells in the logarithmic growth phase, counting the cells by adopting a platelet counter, and detecting the cell activity by using a trypan blue exclusion method to ensure that the cell activity is more than 90%;
c) The cell density is adjusted by using a complete culture medium, and then the cells are inoculated in a 96-well cell culture plate, and 90 mu L of cells are inoculated in each well, wherein the total number of the cells is 3000;
d) Cells in 96-well plates were incubated at 37℃under 5% CO 2;
e) Preparing a 10-time drug solution, wherein the working concentration of test samples HB01, HB02 and HB03 is 20000ng/mL, the concentrations are 9, and the dilution is 3.16 times; test samples HB04, HB05 were at a working concentration of 10. Mu.M, 9 concentrations, 3.16-fold diluted, and then transferred into respective experimental wells of 10. Mu.L each of the serially diluted compounds to 96 well cell plates, with three duplicate wells per drug concentration;
f) Cells in the dosed 96-well plates were incubated at 37℃under 5% CO2 for 6 days, after which CTG analysis was performed;
g) The CTG reagent was thawed and the cell plates equilibrated to room temperature for 30 minutes.
h) An equal volume of CTG solution was added to each well.
i) Cells were lysed by shaking on an orbital shaker for 5 minutes.
j) The cell plates were left at room temperature for 20 minutes to stabilize the luminescence signal.
k) The luminescence value is read and the data is collected.
Data analysis:
the data were analyzed using GraphPad Prism 7.0 software, and non-linear S-curve regression was used to fit the data to yield the dose-response curve, and IC50 values were calculated therefrom.
Cell viability (%) = (Lum test drug-Lum broth control)/(Lum cell control-Lum broth control) ×100%.
The results are shown in tables 1, 2, fig. 1 and 2.
TABLE 1NCI-N87 cell test results
Sequence number Sample name IC50(ng/ml)
1 DS8201 30.53
2 Herceptin 46.41
3 Antibody-drug conjugate 060101 27.10
4 Antibody-drug conjugate 060103 31.84
TABLE 2SK-BR-3 cell test results
Sequence number Sample name IC50(ng/ml)
1 DS8201 17.21
2 Herceptin 29.85
3 Antibody-drug conjugate 060101 13.55
4 Antibody-drug conjugate 060103 28.19
According to Table 1 and FIG. 1, the antibody-drug conjugate provided by the embodiment of the invention has equivalent NCI-N87 tumor proliferation inhibition effect and is superior to Herceptin.
According to Table 2 and FIG. 2, the antibody-drug conjugates provided by the examples of the invention have the effect of inhibiting SK-BR-3 tumor proliferation.
Experimental example 2 animal experiment
(1) Human gastric cancer cell NCI-N87
Mice: female nude mice (species: mus Musculus, BALB/c nude) from 6-8 weeks were subjected to the experiment.
The process comprises the following steps: human gastric cancer strain NCI-N87 cells purchased from ATCC were suspended in physiological saline and inoculated subcutaneously on the right body side of female nude mice (day 0) 1X 10 7 Individual cells, the tumor volume reaches 80-120mm 3 Is randomly divided into 5 groups according to tumor volume, namely a control group, a Herceptin (Herceptin, trastuzumab trade name) group, a DS8201 (first commonly marketed under the trade name ENHERTU) group in Japan, a test sample antibody-drug conjugate 060101 group and an antibody-drug conjugate 060103 group, and is administered by tail vein injection 1 time with an injection amount of 10 mL/kg. The dosing doses of each group were: acetic acid buffer was administered to the blank group; the dosage of the herceptin group is 10mg/kg; DS8201 dose is 5mg/kg; the dosage of antibody-drug conjugate 060101 and antibody-drug conjugate 060103 was 0.5mg/kg. The administration was on day 7 post inoculation.
Tumor volume measurement calculation:
the major and minor diameters of the tumors were measured 2 times per week with calipers, and the tumor volume (mm) was calculated 3 )。
The calculation formula is as follows: tumor volume (mm) 3 ) =0.52×long diameter (mm) × [ short diameter (mm)] 2 。
The results are shown in fig. 3 and 4. Wherein, figure 3 shows the tumor inhibition effect of different drugs on human gastric cancer cell NCI-N87 model; FIG. 4 shows the weight change of mice with different drugs on the human gastric cancer cell NCI-N87 tumor model.
As can be seen from fig. 3 and 4, the human gastric cancer cell NCI-N87 model has the effect of inhibiting tumor proliferation compared with the solvent control group; DS8201, antibody-drug conjugate 060101 and antibody-drug conjugate 060103 have significantly better tumor-inhibiting effect than herceptin. The anti-tumor effect of the antibody-drug conjugate 060101 and the antibody-drug conjugate 060103 is stronger than that of DS8201, and the in-vivo tumor is basically cleared on the 10 th day (17 th day after inoculation) after the administration. The anti-tumor effect of the antibody-drug conjugate 060101 and the antibody-drug conjugate 060103 is equivalent.
During the test, the weight change of each group of mice is not significantly different. No significant toxicity was seen for the test samples.
The dosage of the antibody-drug conjugate 060101 and the antibody-drug conjugate 060103 is only 1/20 of that of Herceptin and 1/10 of that of DS8201, and no obvious weight reduction toxicity of mice is seen, so that the antibody-drug conjugate 060101 and the antibody-drug conjugate 060103 have obvious anti-tumor advantages.
(2) Human breast cancer SK-BR-3
Mice: female nude mice (species: mus Musculus, CB-17 SCID) from 6 to 9 weeks were subjected to the experiment.
Human breast cancer SK-BR-3 cells purchased from ATCC were suspended in physiological saline and inoculated subcutaneously on the right body side of female nude mice (day 0) 5X 10 6 Individual cells, the tumor volume reaches 80-120mm 3 Is randomly divided into 5 groups according to tumor volume, namely a control group, a Herceptin (Herceptin, trastuzumab trade name) group, a DS8201 (EnHERTU group of the first Co-market in Japan) group, and test sample antibody-drug conjugate 060101 group and antibody-drug conjugate 060103 group, which are administered by tail vein injection for 1 time with an injection amount of 10 mL/kg. The dosing doses of each group were: acetic acid buffer was administered to the blank group; the dosage of the herceptin group is 10mg/kg; DS8201 dose is 5mg/kg; the dosage of the antibody-drug conjugate 060101 and the antibody-drug conjugate 060103 of the sample to be tested is 0.5mg/kg. Dosing was on day 21 post inoculation.
Tumor volume measurement calculation:
measured 2 times per week with calipersThe major and minor diameters of the tumor were measured, and the tumor volume (mm 3 )。
The calculation formula is as follows: tumor volume (mm) 3 ) =0.52×long diameter (mm) × [ short diameter (mm)] 2 。
The results are shown in FIGS. 5 and 6. Wherein, figure 5 shows the tumor inhibition effect of different drugs on the human breast cancer cell SK-BR-3 model, and figure 6 shows the weight change of different drugs on the human breast cancer cell SK-BR-3 tumor model mouse.
As can be seen from fig. 5 and 6, each of the other doses has an effect of inhibiting tumor proliferation in comparison with the solvent control group for the human breast cancer SK-BR-3 model; DS8201, antibody-drug conjugate 060101 and antibody-drug conjugate 060103 have significantly better tumor-inhibiting effect than herceptin. The anti-tumor effect of the antibody-drug conjugate 060101 and the antibody-drug conjugate 060103 is stronger than that of DS8201, and the in-vivo tumor is basically cleared at the 21 st day (42 th day after inoculation) after the administration. The anti-tumor effect of the antibody-drug conjugate 060101 and the antibody-drug conjugate 060103 is equivalent.
During the test, the weight change of each group of mice is not significantly different. No significant toxicity was seen for the test samples.
The dosage of the antibody-drug conjugate 060101 and the antibody-drug conjugate 060103 is only 1/20 of that of Herceptin and 1/10 of that of DS8201, and no obvious weight reduction toxicity of mice is seen, so that the antibody-drug conjugate 060101 and the antibody-drug conjugate 060103 have obvious anti-tumor advantages.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (14)

1. A linker comprising a B structural fragment, wherein the B structural fragment is selected from the group consisting of:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6.
2. The linker of claim 1, wherein the linker comprises a B-structure fragment, wherein the B-structure fragment is selected from the group consisting of the following formulas:
Wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8;
preferably, the linker comprises a B-Lb structural fragment, wherein the Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, and the methylene of the Lb structural fragment is connected with the methine of the B structural fragment;
preferably, the linker comprises a B-Lb-LC structural fragment, wherein the Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, the methine group of the B structural fragment is linked to the methylene group of the Lb structural fragment, the Lb structural fragment is linked to the Lc structural fragment by a carbonyl group, wherein Lc is a releasable assembly unit and Lc is capable of linking to a drug unit;
preferably, the Lc structural fragment is selected from the following formulae:
the Lc structural fragment is linked to the carbonyl group of the Lb structural fragment by an amine group, and Lc can be linked to the drug unit by a carbonyl group;
preferably, the linker has a structure represented by formula (I):
wherein,
the La structural fragment is an extension unit that is capable of linking to a ligand unit;
the structure of B is selected from the following formulas:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6; the B structural fragment is connected with the La structure through an amino group and is connected with the Lb structural fragment through a methine group;
the Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, and the methylene of the Lb structural fragment is connected with the methine of the B structural fragment;
the Lb structural fragment is linked to the Lc1 or Lc2 structural fragment by a carbonyl group, wherein Lc1 and Lc2 are respectively releasable assembled units and Lc1 and Lc2 are capable of linking to a drug unit;
preferably, the La structural fragment comprises a maleamide-type linker fragment;
preferably, the La structural fragment has the following structure:
it is linked to the B structural fragment via a carbonyl group and can be linked to the ligand unit via the succinimide 3 and/or 4 position; and R' is selected from: optionally substituted C 1 -C 10 Alkyl, C 1 -C 10 Alkoxy, C 1 -C 10 Aminoalkyl and C 1 -C 10 Alkyl-aryl wherein the substituents are selected from: amino, halogen, nitro, hydroxy, acetyl, cyano, C 1 -C 10 Alkyl, C 1 -C 10 Alkoxy, C 1 -C 10 Aminoalkyl, C 1 -C 10 Haloalkyl, C 2 -C 10 Alkenyl, C 2 -C 10 Alkynyl, amido, C 3 -C 8 Cycloalkyl, C 3 -C 8 A heterocycloalkyl group;
preferably, the La structural fragment is selected from the following formulae:
it is linked to the B structural fragment via a carbonyl group and can be linked to the ligand unit via the succinimide 3 and/or 4 position; wherein each subscript s is independently selected from any integer of from 1 to 10, preferably from 1 to 8, more preferably from 1 to 5;
Preferably, the La structure is selected from the following formulae:
it is linked to the B structural fragment via a carbonyl group and can be linked to the ligand unit via the succinimide 3 and/or 4 position; wherein subscript s is selected from any integer of from 1 to 10, preferably from 1 to 8, more preferably from 1 to 5;
preferably, the La structure is selected from the following formulae:
wherein subscript s is selected from any integer from 1 to 10, preferably from 1 to 8, more preferably from 1 to 5, the la structural fragment is linked to the B structural fragment by a carbonyl group and is capable of being linked to the ligand unit via the 3-position of the succinimide;
further preferably, the linker is selected from the following structures:
r' is selected from: optionally substituted C 1 -C 10 Alkyl, C 1 -C 10 Alkoxy, C 1 -C 10 Aminoalkyl and C 1 -C 10 Alkyl-aryl wherein the substituents are selected from: amino, halogen, nitro, hydroxy, acetyl, cyano, C 1 -C 10 Alkyl, C 1 -C 10 Alkoxy, C 1 -C 10 Aminoalkyl, C 1 -C 10 Haloalkyl, C 2 -C 10 Alkenyl, C 2 -C 10 Alkynyl, amido, C 3 -C 8 Cycloalkyl, C 3 -C 8 Heterocycloalkyl, q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6;
more preferably, the linker is selected from the following structures:
q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6, and subscript S is any natural number from 1 to 6;
further preferably, the linker is selected from the following structures:
q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; n is any natural number from 1 to 6, and subscript S is any natural number from 1 to 6.
3. Use of a linker according to claim 1 or 2 for the preparation of a medicament, wherein the medicament comprises a linker-medicament conjugate or a ligand-medicament conjugate.
4. Linker-drug conjugate comprising the linker according to claim 1 or 2, characterized in that it has the structure represented by formula (II):
or a pharmaceutically acceptable salt thereof, wherein,
la is an extension unit capable of linking to the ligand unit;
the B structural fragment is selected from the following formulae:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6;
The Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, the methylene of the Lb structural fragment is connected with the methine of the B structural fragment, and the Lb structural fragment is connected with the Lc1 or Lc2 structural fragment through carbonyl;
lc1 and Lc2 are releasable assembled units, respectively;
d1 and D2 are each a drug unit.
5. The linker-drug conjugate according to claim 4, having a structure represented by formula (IIa):
or a pharmaceutically acceptable salt thereof, wherein,
la is an extension unit capable of linking to the ligand unit;
the B structural fragment is selected from the following formulae:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6;
the Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, the methylene of the Lb structural fragment is connected with the methine of the B structural fragment, and the Lb structural fragment is connected with the Lc1 or Lc2 structural fragment through carbonyl;
lc1 and Lc2 are releasable assembled units, respectively, each Lc1 and Lc2 being connected to the drug unit by a carbonyl group;
d1 and D2 are each a drug unit;
Preferably, D1 and D2 are each independently selected from any one of a camptothecin topoisomerase inhibitor, a tubulin inhibitor, a DNA alkylating agent, a cell cycle checkpoint kinase 1 inhibitor, a sting1 agonist, and a TLR7/8 agonist, and D1 and D2 cannot be both a single pharmaceutical unit;
more preferably, it has a structure represented by formula (III):
or pharmaceutically acceptable thereofA salt, wherein,
la is an extension unit capable of linking to the ligand unit;
the B structural fragment is selected from the following formulae:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6; />
The Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, the methylene of the Lb structural fragment is connected with the methine of the B structural fragment, and the Lb structural fragment is connected with the Lc1 or Lc2 structural fragment through carbonyl;
lc1 and Lc2 are releasable assembled units, respectively;
represents camptothecin or a derivative thereof;
representing a cell cycle checkpoint kinase 1 inhibitor;
further preferably, the camptothecine and derivatives thereof comprise irinotecan, SN-38 and derivatives thereof;
More preferably, the camptothecin and derivatives thereof are selected from the group consisting of: camptothecin, irinotecan, topotecan, irinotecan, belotecan, lurtoltecan, CKD-602, gem Ma Tikang, karenitecin, BN-80915, hydroxycamptothecin (HCPT), 9-aminocamptothecin, 9-nitrocamptothecin, 7-ethyl-10-hydroxycamptothecin, 7-ethyl-10-difluoromethylcamptothecin, and derivatives thereof;
preferably, the camptothecin and its derivatives are selected from the group consisting of camptothecin, irinotecan, 7-ethyl-10-hydroxycamptothecin, 7-ethyl-10-difluoromethyl camptothecin, and solvates, hydrates, stereoisomers, tautomers, isotopic labels, metabolites, prodrugs, pharmaceutically acceptable salts thereof;
preferably, tubulin inhibitors include DM1, DM4, MMAE and MMAF;
DNA alkylating agents include melphalan;
cell cycle checkpoint kinase 1 inhibitors including AZD-7762, rabusertib and prexaservib;
more preferably, the following structure is selected:
6. ligand-drug conjugate comprising the linker of claim 1 or 2 or the linker-drug conjugate of claim 4 or 5, characterized in that it has the structure of formula (V):
Or a pharmaceutically acceptable salt thereof, wherein,
l is a ligand unit, wherein the subscript n is selected from any integer from 1 to 8, preferably from 4 to 8;
la is an extension unit;
the B structural fragment is selected from the following formulae:
wherein q is selected from 1 to 20Any integer, preferably 3 to 10, most preferably 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6;
the Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, the methylene of the Lb structural fragment is connected with the methine of the B structural fragment, and the Lb structural fragment is connected with the Lc1 or Lc2 structural fragment through carbonyl;
lc1 and Lc2 are releasable assembled units, respectively;
d1 and D2 are each a drug unit.
7. The ligand-drug conjugate of claim 6, wherein D1 and D2 are each independently selected from any one of a camptothecin topoisomerase inhibitor, a tubulin inhibitor, a DNA alkylating agent, a cell cycle checkpoint kinase 1 inhibitor, a sting1 agonist, and a TLR7/8 agonist, and D1 and D2 cannot be both a single pharmaceutical unit;
preferably, it has a structure represented by formula (VI):
Or a pharmaceutically acceptable salt thereof, wherein n is a natural number from 1 to 8;
la is an extension unit capable of linking to the ligand unit;
the B structural fragment is selected from the following formulae:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n NaphtheneAny one of a group and an aromatic group; n is any natural number from 1 to 6;
the Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, the methylene of the Lb structural fragment is connected with the methine of the B structural fragment, and the Lb structural fragment is connected with the Lc1 or Lc2 structural fragment through carbonyl;
lc1 and Lc2 are releasable assembled units, respectively;
represents camptothecin or a derivative thereof; />
Representing a cell cycle checkpoint kinase 1 inhibitor;
preferably, the camptothecin and its derivatives are selected from: camptothecin, irinotecan, topotecan, irinotecan, belotecan, lurtoltecan, CKD-602, gem Ma Tikang, karenitecin, BN-80915, hydroxycamptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 7-ethyl-10-hydroxycamptothecin, 7-ethyl-10-difluoromethyl camptothecin, and derivatives thereof;
Preferably, the camptothecin and its derivatives are selected from the group consisting of camptothecin, irinotecan, 7-ethyl-10-hydroxycamptothecin, 7-ethyl-10-difluoromethyl camptothecin, and solvates, hydrates, stereoisomers, tautomers, isotopic labels, metabolites, prodrugs, pharmaceutically acceptable salts thereof;
preferably, tubulin inhibitors include DM1, DM4, MMAE and MMAF;
DNA alkylating agents include melphalan;
cell cycle checkpoint kinase 1 inhibitors including AZD-7762, rabusertib and prexaservib;
more preferably, the ligand unit comprises an antibody;
preferably, the antibody comprises a monoclonal antibody, a polyclonal antibody, a dimer, a multimer, a multispecific antibody, an intact antibody, an antibody fragment, a human antibody, a humanized antibody, a chimeric antibody, or an antibody from another species;
preferably, the antibody fragment comprises: fab, fab ', F (ab') 2, fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies, camelidae VHH domains, or multispecific antibodies formed from antibody fragments;
preferably, the antibodies include modified or unmodified analogs and derivatives, and allow the antibodies to retain their antigen-binding immunospecificity;
Further preferably, it has a structure represented by the following structural formula:
or a pharmaceutically acceptable salt thereof, wherein,
ab represents an antibody, and subscript n is selected from any integer of 1 to 8, preferably 4 to 8;
la is an extension unit;
the B structural fragment is selected from the following formulae:
wherein q is selected from any integer from 1 to 20, preferably from 3 to 10, most preferably from 5 to 8; r is any natural number from 1 to 4; y is selected from any one of N, O and S; r is selected from (CH) 2 ) n Any one of cycloalkyl and aryl; n is any natural number from 1 to 6;
the Lb structural fragment is selected from the following formulae:
wherein n is any natural number from 1 to 6, and the methylene and B structural pieces of the Lb structural fragmentThe methine connection of the segments, the Lb structural segment is connected with the Lc1 or Lc2 structural segment through carbonyl;
lc1 and Lc2 structural segments are releasable assembly units, respectively;
d1 and D2 are each a drug unit;
still more preferably, it has any one of the compounds described by the following structural formula:
preferably, the ligand comprises a mAb or antigen-binding fragment thereof; preferably is
Or a pharmaceutically acceptable salt thereof, wherein, the mAb represents a monoclonal antibody, n is any natural number from 1 to 8;
most preferably 4-8:
wherein n is 1 to 8, preferably 4 to 8;
Preferably, the ligand is targeted to bind a tumor antigen;
preferably, the ligand targets binding receptor tyrosine-protein kinase ERBB2 and/or epidermal growth factor receptor and/or TROP2 and/or FGFR2b;
preferably, the ligand comprises an anti-HER 2 antibody;
preferably, the anti-HER 2 antibody comprises trastuzumab, pertuzumab or itumomab;
preferably, the ligand comprises an anti-EGFR antibody;
preferably, the anti-EGFR antibody comprises cetuximab, panitumumab, cetuximab, matuzumab, or nituzumab;
preferably, the anti-TROP 2 antibody comprises a golian Sha Tuozhu mab;
preferably, the anti-FGFR 2b antibody comprises Bemarituzumab.
8. A method of preparing the ligand-drug conjugate of claim 6 or 7, comprising conjugating an antibody to the linker of claim 1 or 2 or the linker-drug conjugate of claim 4 or 5.
9. Use of the linker-drug conjugate of claim 4 or 5 or the ligand-drug conjugate of claim 6 or 7 in the manufacture of a medicament, wherein the medicament is for the treatment of a disease that overexpresses a tumor antigen;
Preferably, the tumor antigen comprises HER2 or EGFR.
10. Use of the linker-drug conjugate of claim 4 or 5 or the ligand-drug conjugate of claim 6 or 7 in the manufacture of a medicament, wherein the medicament is for the treatment of a disease with aberrant dnase topoisomerase I expression and/or activity;
preferably, the dnase topoisomerase I expression and/or activity is up-regulated.
11. Use of the linker-drug conjugate of claim 4 or 5 or the ligand-drug conjugate of claim 6 or 7 as dnase topoisomerase I inhibitor.
12. Use of the linker-drug conjugate of claim 4 or 5 or the ligand-drug conjugate of claim 6 or 7 in the manufacture of a medicament, wherein the medicament is for cancer;
preferably, the cancer comprises lymphoma, leukemia or solid tumors;
preferably, the cancer comprises a cancer in which HER2 expression and/or activity is up-regulated;
preferably, the cancer comprises a cancer in which EGFR expression and/or activity is up-regulated;
preferably, the cancer comprises non-small cell lung cancer, breast cancer, gastric cancer, colorectal cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, biliary tract cancer, paget's disease, pancreatic cancer, ovarian cancer, or uterine cancer sarcoma.
13. A pharmaceutical composition comprising a linker-drug conjugate according to claim 4 or 5 or a ligand-drug conjugate according to claim 6 or 7, and a pharmaceutically acceptable carrier.
14. A kit comprising the linker-drug conjugate of claim 4 or 5 or the ligand-drug conjugate of claim 6 or 7.
CN202311489725.7A 2022-12-05 2023-11-09 Linker, linker-drug conjugate, ligand-drug conjugate and uses thereof Pending CN117534725A (en)

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