Novel camptothecin derivatives and their conjugates

Abstract

The present disclosure provides novel camptothecin derivatives and conjugates thereof. The present disclosure further provides pharmaceutical compositions comprising the novel camptothecin derivatives or conjugates thereof, methods for producing the conjugates, and pharmaceutical uses of the novel camptothecin derivatives and conjugates thereof. In one aspect, the present disclosure provides a camptothecin derivative conjugate comprising at least one of the camptothecin derivatives disclosed herein, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form thereof; or a pharmaceutically acceptable salt of the camptothecin derivative conjugate of the present disclosure.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 387,737, filed December 16, 2022. The disclosure of the prior application is considered part of the disclosure of this application and is incorporated herein in its entirety.

[0002] Sequence Listing Reference The contents of the electronic sequence listing (SeqListing-BIONECURE-005.xml; size: 3.51 kilobytes; created on: December 14, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to the pharmaceutical field, specifically to camptothecin derivatives and conjugates thereof. [Background technology]

[0004] Today, cancer remains a leading cause of death worldwide, despite the development of many advanced diagnostic and therapeutic methods. A major obstacle to successful cancer treatment and prevention is the fact that many cancers remain unresponsive to current chemotherapy and immunotherapy interventions, and many patients experience recurrence or die even after aggressive treatment.

[0005] Camptothecin (CPT) (Figure 1) was originally isolated from the bark of Camptotheca acuminata, a tree native to the rocky slopes of northern China, and was isolated by Wall et al. [Wall ME, Wani MC, Cook CE, Palmer KH, McPhail AT, Sim GA. Plant antitumor agents. I. The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata (J Am Chem Soc 1966;88:3888-3890)]. CPT has been demonstrated to be effective against a wide spectrum of tumors. Its molecular target has been firmly established as DNA topoisomerase I (Topo I), which alters the topological state of duplex DNA by single-strand breaks and religation. [Chen AY,Liu LF.DNA topoisomerases: essential enzymes and lethal targets.Annu Rev Pharmacol Toxicol 1994;34:191-218;Hsiang YH,Hertzberg R,Hecht S,Liu LF.Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase IJ Biol Chem 1985;260:14873-14878. Hsiang YH, Liu LF. Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin. Cancer Res 1988;48:1722-1726].In vitro biochemical studies have revealed that CPT binds to the interface between topo I and DNA and specifically inhibits the religation step in the cleavage / religation reaction [Hsiang YH, Hertzberg R, Hecht S, Liu LF. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 1985;260:14873-14878. Svejstrup JQ, Christiansen K, Gromova II, Anderson AH, Westergaard O. New technique for uncoupling the cleavage and relegation reactions of eukaryotic topoisomerase I: the mode of action of camptothecin at a specific recognition site. J Mol Biol 1991;222:669-678].

[0006] CPT has attracted much attention due to its excellent antitumor activity against experimental animal tumor models, such as L1210 leukemia in mice and Walker 256 sarcoma in rats. However, its clinical development failed due to the major dose-limiting toxicities: reversible bone marrow suppression and hemorrhagic cystitis [Muggia FM, Creaven PJ, Hansen HH, Cohen MH, Selawry OS. Phase I clinical trial of weekly and yearly treatment with camptothecin (NSC-100880); correlation with preclinical studies. Cancer Chemother Res 1972;56:515-521. Schappi U, Fleischmann RW, Cooney DA. Toxicity of camptothecin (NSC-100880). Cancer Cemother Rep Part 1974;35:25-36]. Subsequent efforts to discover new camptothecin derivatives with greater anticancer activity and lower toxicity led to the discovery of a potent and safe camptothecin derivative, designated irinotecan or CPT-11 (Figure 1). CPT-11, one of the most prominent antineoplastic drugs widely used in clinical practice today, is a water-soluble prodrug that undergoes carboxylesterase-mediated hydrolysis to form SN-38, a potent Topo I inhibitor [Bencharit S, Morton CL, Howard-Williams EL, Danks MK, Potter PM, Redinbo MR. Structural insights into CPT-11 activation by mammalian carboxyesterases. Nat Struct Biol 2002;9:337-342.Andoh T, Ishii K, Suzuki Y, Ikegami Y, Kusunoki Y, Takemoto Y, et al. Characterization of mammalian mutant with a camptothecin resistant DNA topoisomerase I. Proc Natl Acad Sci USA 1987;84:5565-5569].

[0007] Antibody-drug conjugates (ADCs) combine the binding specificity of antibodies with the potency of drugs such as cytotoxic agents, anticancer drugs, and immunosuppressants. The use of ADCs enables targeted delivery of drugs that, when administered unconjugated, may cause unacceptable levels of toxicity to normal cells. The mechanism of ADCs is to recognize and bind to specific antigens through antibodies, triggering a series of reactions and then entering the cytoplasm through endocytosis, where the highly cytotoxic drug is released from the antibody after degradation by lysosomal enzymes and kills cancer cells. Compared to conventional chemotherapy, which indiscriminately damages both cancer cells and normal tissues, targeted drug delivery allows drugs to act directly on cancer cells, reducing damage to normal cells.

[0008] Recently, with the FDA approval of Trodelvy® and Enhertu®, two camptothecin derivatives, SN-38 and DXd, have been approved as ADC payloads. SN-38 is relatively less toxic, while the linker used by Trodelvy® is unstable. Interstitial lung disease (ILD) is a serious side effect associated with the DXd ADC. (Thomas M. Cardillo, Serengulam V. Govindan, Robert M. Sharkey, Preeti Trisal, and David M. Goldenberg, Clin Cancer Res;17(10);3157-3169. Yusuke Ogitani, Tetsuo Aida, Katsunobu Hagihara, Junko Yamaguchi, Chiaki Ishii, Naoya Harada, Masako Soma, Hiromi Okamoto, Masataka Oitate, Shingo Arakawa, Takehiro Hirai, Ryo Atsumi, Takashi Nakada, Ichiro Hayakawa, Yuki Abe, and Toshinori Agatsuma, Clin Cancer Res;22(20);5097-5108). There remains a great need for campothecin derivatives to be used as ADC payloads for use in the treatment of cancer and / or immune disorders or to prevent their recurrence. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Wall ME,Wani MC,Cook CE,Palmer KH,McPhail AT,Sim GA.Plant antitumor agents.I.The isolation and structure of camptothecin,a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata(J Am Chem Soc 1966;88:3888-3890) .

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[0010] The present disclosure provides novel camptothecin derivatives, conjugates thereof, and pharmaceutical compositions containing the novel camptothecin derivatives or conjugates thereof. The present disclosure also provides methods for producing the conjugates, as well as pharmaceutical uses of the novel camptothecin derivatives and conjugates thereof.

[0011] In one aspect, the present disclosure provides a compound represented by the following structural formula (A): [ka] and a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form thereof, wherein the CPT derivative is represented by: In the formula, X is -C(=O)-(CH2)n 1 -ON(R2)R3, -C(=O)-(CH2)n 1 -N(OR2)R3, -C(=O)-(CH2)n 1 -O-(CH2)n 1 -N(R2)R3, -C(=O)-(C(R4)(R5))n 1 -N(R2)R3, -S(=O)2-CH2-(CH2)n 1 -ON(R2)R3, -S(=O)2-CH2-(CH2)n 1 -N(OR2)R3, -S(=O)2-CH2-(CH2)n 1 -N(R2)R3, -C(=O)-O-(CH2)n 2 -ON(R2)R3, -C(=O)-O-(CH2)n 2 -N(OR2)R3, -C(=O)-O-(CH2)n 2 -N(R2)R3, -C(=O)-NH-(CH2)n 2 -ON(R2)R3, -C(=O)-NH-(CH2)n 2 -N(OR2)R3, -C(=O)-N(R2)-(CH2)n 2 -N(R2)R3, -C(=O)-NH-(CH2)n 2 -O-R3, -C(=O)-NH-(CH2)n 2 -S-R3, -C(=O)-O-(CH2)n 2 -O-R3, -C(=O)-S-(CH2)n 2 -O-R3, -C(=O)-S-(CH2)n 2 -S-R3, -C(=O)-O-(CH2)n 2 -S-R3, -C(=O)-(C(R4)(R5))n 1 -N(R2)-C(=O)-(C(R a )(R b ))n 3 -N(R c )-R3, -C(=O)-(C(R4)(R5))n 1 -N(R2)-C(=O)-(C(R a )(Rb ))n 3 -O-R3, -C(=O)-(C(R4)(R5))n 1 -N(R2)-C(=O)-(C(R a )(R b ))n 3 -Selected from S-R3; R1, R2, R3, and R c are each independently a hydrogen atom or C1-C6 alkyl; R4 and R5 each independently represent a hydrogen atom, a deuterium atom, a halogen atom, a halogenated alkyl atom, a deuterated alkyl atom, an alkoxyl atom, a hydroxyl atom, an amino atom, a nitro group, a cyano group, a hydroxyalkyl group, a heterocyclic C1-C6 alkyl group, or a 3- to 6-membered heterocyclic or cycloaryl ring; or R4 and R5 together with the carbon atom form a 3- to 6-membered cyclic ring; R a and R b each independently represents a hydrogen atom, a deuterium atom, a halogen atom, a halogenated alkyl atom, a deuterated alkyl atom, an alkoxyl atom, a hydroxyl atom, an amino group, a nitro group, a cyano group, a hydroxyalkyl group, a heterocyclic C1-C6 alkyl group, or a 3- to 6-membered heterocyclic ring or a cycloaryl ring; or R a and R b together with the carbon atoms to form a 3- to 6-membered cyclic ring; n 1 is 1, 2, 3, 4 or 5; n 2 is 2, 3, 4 or 5; n 3 is 1, 2, 3, 4 or 5.

[0012] In one aspect, the present disclosure provides a camptothecin derivative conjugate comprising at least one of a camptothecin derivative disclosed herein, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form thereof; or a pharmaceutically acceptable salt of a camptothecin derivative conjugate of the present disclosure.

[0013] In one aspect, the present disclosure provides a camptothecin derivative conjugate comprising: (1) at least one of a camptothecin derivative disclosed herein, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form thereof; (2) a linker; and / or (3) a targeting agent; or a pharmaceutically acceptable salt of a camptothecin derivative conjugate of the present disclosure.

[0014] In one aspect, the present disclosure provides a method for making a camptothecin derivative conjugate disclosed herein, or a pharmaceutically acceptable salt thereof, comprising reacting a targeting agent and / or a linker with a camptothecin derivative. In some embodiments, the camptothecin derivative used in the method for making a camptothecin derivative conjugate, or a pharmaceutically acceptable salt thereof, is a camptothecin derivative disclosed herein.

[0015] In one aspect, the present disclosure provides a pharmaceutical composition comprising a camptothecin derivative disclosed herein, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form thereof, a camptothecin derivative conjugate disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier, or excipient.

[0016] In one aspect, the present disclosure provides a method of treating or preventing a disease, comprising administering a therapeutically effective amount of a camptothecin derivative disclosed herein, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form thereof, a camptothecin derivative conjugate disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein to a subject in need thereof.

[0017] In one aspect, the present disclosure provides a compound of formula (I'): Ab-(LD) n (I') or a pharmaceutically acceptable salt thereof; wherein n is an integer from about 1 to about 12; Ab is an antibody or antigen-binding fragment thereof; D is a camptothecin derivative; L is a linker. Camptothecin derivatives include: [ka] Selected from; X is -C(=O)-(CH2)n 1 -ON(R2)-, -C(=O)-(CH2)n 1 -N(OR2)-, -C(=O)-(CH2)n 1 -O-(CH2)n1-N(R2)-, -C(=O)-(C(R4)(R5))n 1 -N(R2)-, -S(=O)2-CH2-(CH2)n 1 -ON(R2)-, -S(=O)2-CH2-(CH2)n 1 -N(OR2)-, -S(=O)2-CH2-(CH2)n 1 -N(R2)-, -C(=O)-O-(CH2)n 2 -ON(R2)-, -C(=O)-O-(CH2)n 2 -N(OR2)-, -C(=O)-O-(CH2)n 2 -N(R2)-, -C(=O)-NH-(CH2)n 2 -ON(R2)-, -C(=O)-NH-(CH2)n 2 -N(OR2)-, -C(=O)-N(R2)-(CH2)n 2 -N(R2)-, -C(=O)-NH-(CH2)n 2 -O-, -C(=O)-NH-(CH2)n 2 -S-, -C(=O)-O-(CH2)n 2 -O-, -C(=O)-S-(CH2)n 2 -O-, -C(=O)-S-(CH2)n 2 -S-, -C(=O)-O-(CH2)n2 -S-, -C(=O)-(C(R4)(R5))n 1 -N(R2)-C(=O)-(C(R a )(R b ))n 3 -N(R c )-, -C(=O)-(C(R4)(R5))n 1 -N(R2)-C(=O)-(C(R a )(R b ))n 3 -O-, -C(=O)-(C(R4)(R5))n 1 -N(R2)-C(=O)-(C(R a )(R b ))n 3 -Selected from S-; L is a member of the following formula (IIa and IIb): L 1 -L 2 -L 3 (IIa) L 1 -L 2 (IIb) is a linker represented by In the formula, L 1 is the linker moiety attached to the antibody, and L 1 comprises a reactive functional group selected from maleimide, bromoacetyl, iodoacetyl, thiol, amino, alkyl bromide, alkyl iodide, allenamide, carboxyl, and NHS ester; L 2 is a linker moiety comprising 2-4 AA peptides, optionally including a spacer, preferably a PEG spacer; L 3 is a linker moiety connected to the camptothecin derivative, and L 3 is the following: [ka] Contains one of the R1 is a hydrogen atom or C1-C6 alkyl; R2 is a hydrogen atom or C1-C6 alkyl; R3 is a hydrogen atom, C1-C6 alkyl, or -C(=O)-(CH2)n 3 -OH; R4 and R5 each independently represent a hydrogen atom, a deuterium atom, a halogen atom, a halogenated alkyl atom, a deuterated alkyl atom, an alkoxyl atom, a hydroxyl atom, an amino atom, a nitro group, a cyano group, a hydroxyalkyl group, a heterocyclic group, a C1-C6 alkyl group, or a 3- to 6-membered heterocyclic or cycloaryl ring; or R4 and R5 together with the carbon atom form a 3- to 6-membered cyclic ring; R a and R b each independently represents a hydrogen atom, a deuterium atom, a halogen, a halogenated alkyl, a deuterated alkyl, an alkoxy, a hydroxyl, an amino, a nitro, a cyano, a hydroxyalkyl, a heterocyclic, a C1-C6 alkyl, or a 3- to 6-membered heterocyclic or cycloaryl ring; or R a and R b together with the carbon atoms to form a 3- to 6-membered cyclic ring; R c is a hydrogen atom or C1-C6 alkyl; n 1 is 1, 2, 3, 4 or 5; n 2 is 2, 3, 4 or 5; n 3 is 1, 2, 3, 4 or 5; Y is -C(=O)-R6, -C(=O)-OR6, -C(=O)-NHR6, where R6 is C1-C6 alkyl; Z is NHR7, where R7 is C1-C6 alkyl, -CH2-CH2-OH, -CH2-CH2-OCH3, or -CH2-CH2-N(CH3)2 W is NH or O.

[0018] In one embodiment, L 1 is -C(=O)-(CH2) n1 - and -C(=O)-(CH2)n 1 -R8-[O-(CH2) n1 ] n1 -N(R2)-C(=O)-)-(CH2)n1 -, and R8 is alkylene-aryl-alkylene, aryl, 3- to 7-membered heterocyclyl, 3- to 7-membered cycloalkyl, heteroaryl, -alkylene-heteroaryl-alkylene-.

[0019] In one embodiment, L is [ka] [ka] is selected from.

[0020] In certain aspects of the present disclosure, L 2 is a dipeptide, tripeptide, or tetrapeptide containing naturally occurring and non-naturally occurring amino acids.

[0021] In certain aspects of the present disclosure, L 2 is selected from gly-gly, gly-gly-gly, phe-lys, val-ala, val-cit, gly-gly-phe-gly (GGFG) (SEQ ID NO: 1), val-cit-gly, val-gln-gly, val-glu-gly, phe-lys-gly, glu-val-ala, glu-val-cit, β-ala-gly-phe-gly (AGFG) (SEQ ID NO: 2), and gly-gly-phe-gly-gly (GGFGG) (SEQ ID NO: 3), wherein the amino acid sequence is in either orientation.

[0022] In one embodiment, the camptothecin derivative-linker is [ka] is selected from. In the formula, L is [ka] [ka] Selected from; z is an integer between 1 and 6; X 1 is a reactive functional group selected from maleimide, bromoacetyl, iodoacetyl, thiol, amino, alkyl bromide, alkyl iodide, allenamide, carboxyl, and NHS ester.

[0023] In one embodiment, the camptothecin derivative is [ka] is.

[0024] In one embodiment, the reactive functional group is [ka] is.

[0025] In one embodiment, -LX is [ka] [ka] Includes:

[0026] In one embodiment, the camptothecin derivative-linker is: ; [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] is selected from.

[0027] Another aspect of the disclosure is a pharmaceutical composition comprising an ADC compound of Formula I', a mixture of ADC compounds of Formula I', or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable diluent, carrier, or excipient.

[0028] Another embodiment provides a pharmaceutical combination comprising an ADC compound of Formula I' and a second compound having anti-cancer properties or other therapeutic effect.

[0029] Another embodiment is a method of killing or inhibiting the growth of a tumor or cancer cell, the method comprising treating the cell with an amount of an antibody-drug conjugate of Formula I', or a pharmaceutically acceptable salt or solvate thereof, effective to kill or inhibit the growth of the tumor or cancer cell.

[0030] Another aspect is a method of treating cancer comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition comprising an ADC of Formula I'.

[0031] In various embodiments, the present disclosure relates to antibody drug conjugates, wherein x is from about 1 to about 8. In various embodiments, x is from about 4 to about 7. In various embodiments, x is about 4. In various embodiments, x is about 6. In various embodiments, x is about 7.

[0032] In various embodiments, the present disclosure relates to derivatized camptothecin derivatives, L 1includes pyrroline-dione. In various embodiments of the present disclosure, the heterocyclyl ring is selected from saturated or unsaturated 4- to 6-membered nitrogen-containing heterocycles. Examples of saturated heterocyclic radicals include saturated 3- to 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms (e.g., pyrrolidinyl, imidazolidinyl, piperidino, piperazinyl); saturated 3- to 6-membered heteromonocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms (e.g., morpholinyl); and saturated 3- to 6-membered heteromonocyclic groups containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms (e.g., thiazolidinyl). Examples of unsaturated heterocyclic radicals, also known as "heteroaryl" radicals, include unsaturated 5- to 6-membered heteromonocyclyl groups containing 1 to 4 nitrogen atoms, such as pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl [e.g., 4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl]; unsaturated fused heterocyclic groups containing 1 to 5 nitrogen atoms, such as indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl. [e.g., tetrazolo[1,5-b]pyridazinyl]; unsaturated 5- to 6-membered heteromonocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as oxazolyl, isoxazolyl, and oxadiazolyl [e.g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, and 1,2,5-oxadiazolyl]; unsaturated fused heterocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as benzoxazolyl and benzoxadiazolyl; and unsaturated 5- to 6-membered heteromonocyclic groups containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, such as thiazolyl and thiadiazolyl [e.g., 1,2,4-thiadiazolyl]. In various embodiments of the present disclosure, cyclic alkyl rings, also known as cycloalkyl rings, are saturated cyclic alkyl groups derived by removing one hydrogen atom from a single carbon atom of a parent cycloalkane.Typical cycloalkyl groups include, but are not limited to, groups derived from cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, and the like.

[0033] In various embodiments, the ADC comprises a cleavable linker, L. In various embodiments, the ADC comprises an antibody conjugated to a camptothecin derivative through a linker that is sensitive to peptidase cathepsin. In various embodiments, the ADC comprises an antibody conjugated to a camptothecin derivative through a linker that is sensitive to peptidase cathepsin. In various embodiments, the ADC is an anti-Her2 antibody conjugated to a camptothecin derivative, where the camptothecin derivative is linked to the anti-Her2 antibody through a linker that is not acid-labile. In various embodiments, the ADC comprises an antibody conjugated to a camptothecin derivative through a linker that does not contain a disulfide bond. In various embodiments, the ADC comprises an antibody conjugated to a camptothecin derivative through a linker that provides stability in the circulation while allowing the drug to be released once inside a cell. Such linkers are contemplated to provide stability to the conjugated molecule prior to endocytosis, such as during circulation, to prevent premature linker degradation and release of the toxic drug, thereby minimizing the toxic effects of the drug.

[0034] In various embodiments, the compound of formula I′, wherein L and L 1 is a cysteine-reactive linker.

[0035] In various embodiments, the compounds of formulas I′ and II, wherein L and L 2 is a cleavable linker. In various embodiments, the number of bonds formed between the drug-linker and cysteine ​​residues on an antibody, such as an anti-Her2 antibody, is between 3 and 8. In various embodiments, the number of such bonds is at least 2, or alternatively at least 4 or 5. In various embodiments, the number of such bonds formed is 8 or less, or alternatively 7, 6, 5, or 4 or less. In various embodiments, each antibody, e.g., an anti-Her2 antibody, is conjugated, on average, to about 4 to 7 drug molecules through cysteines.

[0036] Another aspect of the present disclosure relates to pharmaceutical compositions of the cell-binding agent conjugate of Formula I' and a pharmaceutically acceptable carrier, excipient, or diluent thereof.

[0037] In various embodiments, the ADC constructs of the present disclosure comprise a targeting moiety, an Ab, such as an antibody, multispecific antibody, bispecific antibody, or antibody fragment that can bind to a tumor-associated antigen (TAA), a tissue-specific antigen, a cell surface molecule, an extracellular matrix protein or protease(s), or any post-translationally modified residue(s). In various embodiments, the ADC constructs of the present disclosure comprise a targeting moiety, an Ab, that exhibits binding affinity for diseased cells or tissues.

[0038] In various embodiments, the antibody, multispecific antibody, bispecific antibody, or antibody fragment is selected from the group consisting of tumor-associated calcium signaling factor 2 (also known as Trop-2), Her2, Her3, Her4, EGF, EGFR, CD2, CD3, CD5, CD7, CD13, CD19, CD20, CD21, CD23, CD30, CD33, CD34, CD38, CD46, CD55, CD59, CD69, CD70, CD71, CD97, CD117, CD123, CD127, CD134, CD137, CD138, CD146, CD147, CD152, CD154, CD174, CD195, CD200, CD205, CD212, CD223, CD227, CD25 3, CD272, CD274, CD276, CD278, CD279, CD309, CD319, CD326, CD340, DR6, Kv1.3, 5E10, MUC1, uPA, MAGE3, MUC16, KLK3, K-ras, mesothelin, p53, survivin, G250, PSMA, endoplasmin, BCMA, GPNMB, EphA2, EphB2, TMEFF2, integrin beta 6, 5T4, CA9, IGF-1R, Axl, B7H3, B7H4, CDH6, HAVCR1, STEAP-1, STEAP-2, UPK2, CLDN18.2, CLDN6, CLDN9, c-Met, MICA / B, LIV-1, ROR1, ADAM9, STn, Globo It is capable of binding to a TAA selected from the group consisting of H, SSEA-4, MG7-Ag, fucosyl GM-1, DLK-1, CEACAM5.

[0039] In various embodiments, the ADC comprises a TAA-binding Ab selected from the group consisting of a fully human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a Fab, a Fab', a Fab2, a Fab'2, an IgG, an IgM, an IgA, an IgE, a scFv, a dsFv, a dAb, a nanobody, a unibody, and a diabody. In various embodiments, the antibody is a chimeric antibody. In various embodiments, the antibody is a humanized monoclonal antibody. In various embodiments, the antibody is a fully human monoclonal antibody.

[0040] In another aspect, the disclosure provides a pharmaceutical composition comprising an isolated ADC construct in admixture with a pharmaceutically acceptable carrier.

[0041] In another aspect, the disclosure provides the use of an ADC construct for the preparation of a medicament for the treatment of cancer.

[0042] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of the present disclosure to a subject in need thereof.In one embodiment, the subject is a human subject.In various embodiments, the cancer is selected from pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, or rhabdomyosarcoma, or any cancer.

[0043] In various embodiments, the subject previously responded to treatment with an anti-cancer therapy but relapsed when the treatment was discontinued (hereinafter "recurrent cancer"). In various embodiments, the subject has a resistant or refractory cancer.

[0044] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical composition of the present disclosure in combination with a second-line therapy selected from the group consisting of cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, stem cell transplantation, cell therapy, such as CAR-T, CAR-NK, iPS-induced CAR-T or iPS-induced CAR-NK, and a vaccine, such as Bacillus Calmette-Guerin (BCG). In various embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of immunotherapy, including, but not limited to, treatment with depleting antibodies against specific tumor antigens; treatment with antibody-drug conjugates; agonistic, antagonistic, or blocking antibodies against costimulatory or co-inhibitory molecules (immune checkpoints), such as CTLA-4, PD-1, PD-L1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, Siglec7, Siglec8, Siglec9, Siglec15, and VISTA. treatment with a bispecific T cell-engaging antibody (BiTE®) such as blinatumomab; treatment involving administration of biological response modifiers such as IL-12, IL-21, GM-CSF, IFN-alpha, IFN-β, and IFN-γ; treatment with a therapeutic vaccine such as sipuleucel-T; treatment with a dendritic cell vaccine, or a tumor antigen peptide vaccine; treatment with chimeric antigen receptor (CAR)-T cells; treatment with CAR-NK cells; treatment with tumor-infiltrating lymphocytes (TILs); treatment with adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR transgenic); treatment with TALL-104 cells; and treatment with immunostimulants such as the Toll-like receptor (TLR) agonists CpG and imiquimod; and treatment with a vaccine such as BCG, which combination therapy optionally provides enhanced effector cell killing of tumor cells, i.e., a synergistic effect is achieved between the ADC construct and the immunotherapy when co-administered.

[0045] In another aspect, there are provided novel compounds described herein, as well as methods for making them. [Brief explanation of the drawings]

[0046] [Figure 1] The structures of CPT, CPT-11, SN-38, exatecan, and DXd are shown.

[0047] [Figure 2] 1 shows the conjugation procedure for mAb-reduced camptothecin derivatives.

[0048] [Figure 3] (A) Line graph showing the percent inhibition of SK-BR-3 breast cancer cells with increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-36309 in an in vitro cytotoxicity assay. (B) Line graph showing the percent inhibition of NCI-N87 gastric cancer cells with increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-36309 in an in vitro cytotoxicity assay. (C) Line graph showing the percent inhibition of MDA-MB-468 breast cancer cells with increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-36309 in an in vitro cytotoxicity assay. D shows a line graph depicting the percent inhibition of BxPC-3 pancreatic cancer cells using increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-36309 in an in vitro cytotoxicity assay. Cell viability is shown as a percentage of inhibition relative to the untreated control. A representative figure from three independent experiments is shown, and values ​​represent the mean ± SEM. The dotted circle represents Dxd, the solid square represents T-363, the dotted triangle represents Herceptin-Dxd, and the solid diamond represents Herceptin-36309 (ADC-25).

[0049] [Figure 4]Figure 1 shows a line graph depicting the percent inhibition of BT-474 breast cancer cells using increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-363-11 in an in vitro cytotoxicity assay. Cell viability is shown as the percentage of inhibition relative to untreated controls. A representative figure from three independent experiments is shown, and values ​​represent the mean ± SEM. Dashed circles represent Dxd, solid squares represent T-363, dotted triangles represent Herceptin-Dxd, and solid diamonds represent Herceptin-363-11 (ADC-4).

[0050] [Figure 5] Figure 1 shows a line graph depicting the percent inhibition of NCI-N87 gastric cancer cells using increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-363-11 in an in vitro cytotoxicity assay. Cell viability is shown as the percentage of inhibition relative to the untreated control. A representative figure from three independent experiments is shown, and values ​​represent the mean ± SEM. The dotted circle represents Dxd, the solid square represents T-363, the dotted triangle represents Herceptin-Dxd, and the solid diamond represents Herceptin-363-11.

[0051] [Figure 6] Figure 1 shows a line graph depicting the percent inhibition of SK-BR-3 breast cancer cells using increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-363-11 in an in vitro cytotoxicity assay. Cell viability is shown as the percentage of inhibition relative to untreated controls. A representative figure from three independent experiments is shown, and values ​​represent the mean ± SEM. Dashed circles represent Dxd, solid squares represent T-363, dotted triangles represent Herceptin-Dxd, and solid diamonds represent Herceptin-363-11.

[0052] [Figure 7]Figure 1 shows a line graph depicting the percent inhibition of BxPC-3 pancreatic cancer cells using increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-363-11 in an in vitro cytotoxicity assay. Cell viability is shown as the percentage of inhibition relative to the untreated control. A representative figure from three independent experiments is shown, and values ​​represent the mean ± SEM. Dashed circles represent Dxd, solid squares represent T-363, dotted triangles represent Herceptin-Dxd, and solid diamonds represent Herceptin-363-11.

[0053] [Figure 8] Figure 1 shows a line graph depicting the percent inhibition of NCI-H292 lung cancer cells using increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-363-11 in an in vitro cytotoxicity assay. Cell viability is shown as a percentage of inhibition relative to untreated controls. A representative figure from three independent experiments is shown, and values ​​represent the mean ± SEM. Dashed circles represent Dxd, solid squares represent T-363, dotted triangles represent Herceptin-Dxd, and solid diamonds represent Herceptin-363-11.

[0054] [Figure 9] Figure 1 shows a line graph depicting the percent inhibition of MDA-MB-468 breast cancer cells using increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-363-11 in an in vitro cytotoxicity assay. Cell viability is shown as the percentage of inhibition relative to untreated controls. A representative figure from three independent experiments is shown, and values ​​represent the mean ± SEM. Dashed circles represent Dxd, solid squares represent T-363, dotted triangles represent Herceptin-Dxd, and solid diamonds represent Herceptin-363-11.

[0055] [Figure 10](A) Percent inhibition of HER2-positive SK-BR-3 cells and HER2-negative MDA-MB-468 cells treated with the indicated concentrations of Herceptin-Dxd for 5 days, as well as the percent inhibition of MDA-MB-468 cells in SK-BR-3 and MDA-MB-468 cocultures treated with the indicated concentrations of Herceptin-Dxd for 5 days. (B) Percent inhibition of HER2-positive SK-BR-3 cells and HER2-negative MDA-MB-468 cells treated with the indicated concentrations of Herceptin-363-11 for 5 days, as well as the percent inhibition of MDA-MB-468 cells in SK-BR-3 and MDA-MB-468 cocultures treated with the indicated concentrations of Herceptin-363-11 for 5 days. Each point represents the mean and SD (n=3). Black columns represent SK-BR-3, white columns represent MDA-MB-468, and gray columns represent cocultures.

[0056] [Figure 11] The percentage of remaining total antibody and Herceptin-363-11 in human plasma at the indicated time points is shown. Solid circles represent total antibody, and dotted boxes represent ADC-4 (Herceptin-363-11).

[0057] [Figure 12] Figure 1 shows the mean tumor volume over time in female athymic mice bearing HCC-1569 HER2-positive subcutaneous xenografts treated with vehicle control or ADC-4. Five mice per group were intravenously administered when tumors reached a volume of approximately 150 mm3. A single dose of ADC-4 was administered at 1 or 5 mg / kg. Vehicle control mice were used as negative controls. Solid filled circles represent vehicle control, dotted open circles represent 1 mg / kg ADC-4, and solid open circles represent 5 mg / kg ADC-4.

[0058] [Figure 13](A) shows the mean tumor volume over time in female athymic mice bearing JIMT-1 HER2-positive subcutaneous xenografts treated with vehicle control or ADC-5. (B) shows the mean tumor volume over time in female athymic mice bearing NCI-N87 HER2-positive subcutaneous xenografts treated with vehicle control or ADC-5. Five mice per group were intravenously administered when tumors reached a volume of approximately 200 mm3. A single dose of ADC-5 was administered at 1 or 5 mg / kg. Vehicle control mice were used as negative controls. Solid filled circles represent vehicle control, dotted open circles represent 1 mg / kg ADC-5, and solid open circles represent 5 mg / kg ADC-5.

[0059] [Figure 14A] The mean tumor volume over time in female athymic mice bearing JIMT-1 HER2-positive subcutaneous xenografts treated with vehicle control or ADC-14 is shown. Five mice per group were administered intravenously when tumors reached a volume of approximately 150-200 mm3. A single dose of ADC-14 was administered at 1 or 5 mg / kg. Vehicle control mice were used as negative controls. Solid filled circles represent vehicle control, dotted open circles represent 1 mg / kg ADC-5, and solid open circles represent 5 mg / kg ADC-5. [Figure 14B] The mean tumor volume over time in female athymic mice bearing NCI-N87 HER2-positive subcutaneous xenografts treated with vehicle control or ADC-14 is shown. Five mice per group were administered intravenously when tumors reached a volume of approximately 150-200 mm3. A single dose of ADC-14 was administered at 1 or 5 mg / kg. Vehicle control mice were used as negative controls. Solid filled circles represent vehicle control, dotted open circles represent 1 mg / kg ADC-5, and solid open circles represent 5 mg / kg ADC-5. [Figure 14C]The mean tumor volume over time in female athymic mice bearing HCC-1569 HER2-positive subcutaneous xenografts treated with vehicle control or ADC-14 is shown. Five mice per group were administered intravenously when tumors reached a volume of approximately 150-200 mm3. A single dose of ADC-14 was administered at 1 or 5 mg / kg. Vehicle control mice were used as negative controls. Solid filled circles represent vehicle control, dotted open circles represent 1 mg / kg ADC-5, and solid open circles represent 5 mg / kg ADC-5.

[0060] [Figure 15] Figure 1 shows the average tumor volume in female athymic mice bearing NCI-N87 HER2-positive subcutaneous xenografts treated with vehicle control, reference ADC, or ADC-22. Five mice per group were intravenously administered when tumors reached a volume of approximately 200 mm3. A single dose of reference ADC or ADC-22 was administered at 5 mg / kg. Vehicle control mice were used as negative controls. Circles represent vehicle control, squares represent 5 mg / kg reference ADC, and triangles represent 5 mg / kg ADC-22.

[0061] [Figure 16] The figures show the mean tumor volumes in female athymic mice bearing NCI-N87 HER2-positive subcutaneous xenografts treated with vehicle control, reference ADC, or ADC-23. Five mice per group were administered intravenously when tumors reached a volume of approximately 150–200 mm3. A single dose of reference ADC or ADC-23 was administered at 2.5 mg / kg or 5 mg / kg. Vehicle control mice were used as negative controls. Circles represent vehicle control, inverted triangles represent 2.5 mg / kg reference ADC, diamonds represent 5 mg / kg reference ADC, squares represent 2.5 mg / kg ADC-23, and triangles represent 5 mg / kg ADC-23.

[0062] [Figure 17](A) Body weight over time in naive CD-1 mice treated with a blank control, PBS, or ADC-14 (160 mg / kg). Solid filled circles represent the blank control, solid open circles represent PBS, and dotted open circles represent ADC-14. Arrows pointing to the x-axis indicate the day of dosing. (B) Red blood cell, white blood cell, neutrophil, and lymphocyte counts in naive CD-1 mice treated with a blank control, PBS, or ADC-14 (160 mg / kg). Five mice were randomized to each group based on body weight. A single dose of ADC-14 at 160 mg / kg was administered on day 0. Body weights were measured on days 0, 3, 5, and 7 after dosing. Whole blood was collected via retro-orbital bleeding on days 3 and 7 for automated red blood cell, white blood cell, neutrophil, and lymphocyte measurements.

[0063] [Figure 18] (A) Body weight over time in naive CD-1 mice treated with a blank control, PBS, or ADC-19 (160 mg / kg). Solid filled circles represent the blank control, solid open circles represent PBS, and dotted open circles represent ADC-19. Arrows pointing to the x-axis indicate the day of dosing. (B) Red blood cell, white blood cell, neutrophil, and lymphocyte counts in naive CD-1 mice treated with a blank control, PBS, or ADC-19 (160 mg / kg). Five mice were randomized to each group based on body weight. A single dose of ADC-19 at 160 mg / kg was administered on day 0. Body weights were measured on days 0, 3, 5, and 7 after dosing. Whole blood was collected via retro-orbital bleeding on days 3 and 7 for automated red blood cell, white blood cell, neutrophil, and lymphocyte measurements.

[0064] [Figure 19](A) Body weight over time in naive CD-1 mice treated with a blank control, PBS, or ADC-20 (160 mg / kg). Solid filled circles represent the blank control, solid open circles represent PBS, and dotted open circles represent ADC-20. Arrows pointing to the x-axis indicate the day of dosing. (B) Red blood cell, white blood cell, neutrophil, and lymphocyte counts in naive CD-1 mice treated with a blank control, PBS, or ADC-20 (160 mg / kg). Five mice were randomized to each group based on body weight. A single dose of ADC-20 at 160 mg / kg was administered on day 0. Body weights were measured on days 0, 3, 5, and 7 after dosing. Whole blood was collected via retroorbital bleeding on days 3 and 7 for automated red blood cell, white blood cell, neutrophil, and lymphocyte measurements.

[0065] [Figure 20] (A) Body weight over time in naive CD-1 mice treated with a blank control, PBS, or ADC-21 (160 mg / kg). Solid filled circles represent the blank control, solid open circles represent PBS, and dotted open circles represent ADC-21. Arrows pointing to the x-axis indicate the day of dosing. (B) Red blood cell, white blood cell, neutrophil, and lymphocyte counts in naive CD-1 mice treated with a blank control, PBS, or ADC-21 (160 mg / kg). Five mice were randomized to each group based on body weight. A single dose of ADC-21 at 160 mg / kg was administered on day 0. Body weights were measured on days 0, 3, 5, and 7 after dosing. Whole blood was collected via retro-orbital bleeding on days 3 and 7 for automated red blood cell, white blood cell, neutrophil, and lymphocyte measurements.

[0066] [Figure 21](A) Body weight over time in naive CD-1 mice treated with a blank control, PBS, or ADC-22 (160 mg / kg). Solid filled circles represent the blank control, solid filled triangles represent PBS, and dotted open circles represent ADC-22. Arrows pointing to the x-axis indicate the day of dosing. (B) Red blood cell, white blood cell, neutrophil, and lymphocyte counts in naive CD-1 mice treated with a blank control, PBS, or ADC-22 (160 mg / kg). Five mice were randomized to each group based on body weight. A single dose of ADC-22 at 160 mg / kg was administered on day 0. Body weights were measured on days 0, 2, 4, and 7 after dosing. Whole blood was collected via retroorbital bleeding on days 3 and 7 for automated red blood cell, white blood cell, neutrophil, and lymphocyte measurements.

[0067] [Figure 22] (A) Body weight over time in naive CD-1 mice treated with a blank control, PBS, or ADC-23 (160 mg / kg). Solid filled circles represent the blank control, solid filled triangles represent PBS, and dotted open circles represent ADC-23. Arrows pointing to the x-axis indicate the day of dosing. (B) Red blood cell, white blood cell, neutrophil, and lymphocyte counts in naive CD-1 mice treated with a blank control, PBS, or ADC-23 (160 mg / kg). Five mice were randomized to each group based on body weight. A single dose of ADC-23 at 160 mg / kg was administered on day 0. Body weights were measured on days 0, 2, 4, and 7 after dosing. Whole blood was collected via retroorbital bleeding on days 3 and 7 for automated red blood cell, white blood cell, neutrophil, and lymphocyte measurements.

[0068] [Figure 23](A) Body weight over time in naive CD-1 mice treated with a blank control, PBS, or ADC-12 (160 mg / kg). Solid filled circles represent the blank control, solid filled triangles represent PBS, and dotted open circles represent ADC-12. Arrows pointing to the x-axis indicate the day of dosing. (B) Red blood cell, white blood cell, neutrophil, and lymphocyte counts in naive CD-1 mice treated with a blank control, PBS, or ADC-12 (160 mg / kg). Five mice were randomized to each group based on body weight. A single dose of ADC-12 at 160 mg / kg was administered on day 0. Body weights were measured on days 0, 2, and 5 after dosing. Whole blood was collected via retroorbital bleeding on days 2 and 5 for automated red blood cell, white blood cell, neutrophil, and lymphocyte measurements. DETAILED DESCRIPTION OF THE INVENTION

[0069] The present disclosure provides novel camptothecin derivatives and CPT derivative conjugates comprising at least one camptothecin derivative. In various embodiments, the disclosure provides CPT derivatives, CPT derivative conjugates, and methods relating to the use of such CPT derivatives or conjugates to treat diseases such as cancer. The targeting agent in the conjugate binds to, for example, a tumor-associated antigen (TAA) on cancer cells. In various embodiments, the targeting agent is conjugated to a novel camptothecin derivative and / or a linker, and the conjugate exerts a cytotoxic, cytostatic, or immunosuppressive effect on antigen-expressing cells to treat or prevent the recurrence of the antigen-expressing cancer or immune disorder. Importantly, the CPT derivative conjugates of the present disclosure have excellent drug / targeting agent ratios (e.g., DAR to ADC), improved solubility, enhanced CMC properties, and improved therapeutic efficacy, particularly against tumors with high antigen expression levels, while sparing normal tissues with low or no antigen expression levels. Furthermore, CPT derivative conjugates offer targeting to a broader patient population and to patients with refractory cancers or those who previously responded to treatment with anti-cancer therapy but experienced recurrence upon cessation of treatment (hereinafter "recurrent cancer").

[0070] definition Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms and plural terms shall include the singular term. Generally, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are commonly used and well known in the art. The methods and techniques of this disclosure, unless otherwise indicated, are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012), which is incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are commonly used and well known in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of subjects.

[0071] As used herein, the term "alkyl" refers to a fully saturated branched or unbranched hydrocarbon moiety having up to 20 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like.

[0072] As used herein, the terms "heterocyclyl," "heterocycloalkyl," or "heterocyclo" refer to a saturated or unsaturated non-aromatic ring or ring system containing at least one heteroatom selected from O, S, and N. The heterocyclyl can be attached at a heteroatom, a carbon atom, or both.

[0073] As used herein, the term "aryl" refers to an aromatic hydrocarbon group having 6 to 20 carbon atoms in the ring portion. Typically, aryl is a monocyclic, bicyclic, or tricyclic aryl having 6 to 20 carbon atoms. Furthermore, as used herein, the term "aryl" refers to an aromatic moiety that can be a single aromatic ring or multiple aromatic rings fused together. Non-limiting examples include phenyl, naphthyl, or tetrahydronaphthyl, each of which can be optionally substituted with 1 to 4 substituents, such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxy, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, thiol, alkyl-S-, aryl-S-nitro, cyano, carboxy, alkyl-OC(O)-, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonamido, phenyl, and heterocyclyl.

[0074] As used herein, "cyclic alkyl" or "cycloalkyl" refers to a saturated or unsaturated monocyclic, bicyclic, or tricyclic hydrocarbon group of 3 to 12 carbon atoms. Unless otherwise provided, cycloalkyl refers to a cyclic hydrocarbon moiety having 3 to 9 ring carbon atoms or 3 to 7 ring carbon atoms, each of which can be optionally substituted with one, two, three, or more substituents independently selected from the group consisting of alkyl, halo, oxo, hydroxy, alkoxy, alkyl-C(O)--, acylamino, carbamoyl, alkyl-NH-, (alkyl)N--, thiol, alkyl-S--, nitro, cyano, carboxy, alkyl-O--C(O)--, sulfonyl, sulfonamido, sulfamoyl, and heterocyclyl.

[0075] As used herein, unless otherwise specified, the term "optionally substituted" refers to a group that is unsubstituted or substituted with one or more, typically 1, 2, 3 or 4, suitable non-hydrogen substituents.

[0076] The point of attachment of a given moiety to the parent structure can be readily determined by one of ordinary skill in the art, and thus, while the point of attachment may not be explicitly indicated, it will be apparent to one of ordinary skill in the art based on general knowledge in the chemical arts.

[0077] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. In various embodiments, a "peptide," "polypeptide," and "protein" is a chain of amino acids whose alpha carbons are linked through peptide bonds. Thus, the terminal amino acid at one end of the chain (e.g., the amino terminus) has a free amino group, while the terminal amino acid at the other end of the chain (e.g., the carboxy terminus) has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated N-terminus) refers to the free □-amino group on the amino terminal amino acid of a peptide, or the □-amino group (imino group when involved in a peptide bond) of an amino acid at any other position within the peptide. Similarly, the term "carboxy terminus" refers to the free carboxyl group on the carboxy terminal amino acid of a peptide, or the carboxyl group of an amino acid at any other position within the peptide. Peptides also include essentially any polyamino acid, including, but not limited to, peptidomimetics such as amino acids linked by ether bonds as opposed to amide bonds.

[0078] Polypeptides of the present disclosure include polypeptides that have been modified in any way or for any reason, for example, to (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or alter other physicochemical or functional properties. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) may be made in a naturally occurring sequence (e.g., in a portion of a polypeptide outside the domain(s) that form intermolecular contacts). A "conservative amino acid substitution" refers to the replacement of an amino acid in a polypeptide with a functionally similar amino acid. A "non-conservative amino acid substitution" refers to the replacement of a member of one of these classes with a member of another class. According to various embodiments, the hydropathic index of amino acids may be considered when making such changes. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. They are isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0079] The importance of the hydropathic amino acid index in conferring interactive biological function to a protein is understood in the art (see, e.g., Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certain amino acids may be substituted with other amino acids having similar hydropathic indices or scores and still retain similar biological activity. When making changes based on the hydropathic index, various embodiments include substitution of amino acids whose hydropathic indices are within ±2, various embodiments include those within ±1, and various embodiments include those within ±0.5.

[0080] It is also understood in the art that substitutions of like amino acids can be made effectively on the basis of hydrophilicity, particularly when the biologically functional proteins or peptides so generated are intended for use in immunological embodiments as disclosed herein. In various embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

[0081] These amino acid residues have been assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartate (+3.0 +-.1); glutamate (+3.0 +-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 +-.1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). When making changes based on similar hydrophilicity values, various embodiments include substitution of amino acids with hydrophilicity values ​​within ±2, various embodiments include those within ±1, and various embodiments include those within ±0.5.

[0082] In some embodiments, the amino acids found in the linker are non-conservative amino acid substitutions. This class generally includes the corresponding D-amino acids, homo-amino acids, N-alkyl amino acids, beta-amino acids, and other non-naturally occurring amino acids. Non-conservative amino acid substitutions still fall within the descriptions specified above for equivalent amino acid substitutions (e.g., polar, non-polar, etc.). Examples of non-conservative amino acids are provided below.

[0083] Non-limiting examples of non-conservative amino acids of alanine are: D-alanine [Dala, (dA), a], N-acetyl-3-(3,4-dimethoxyphenyl)-D-alanine, N-Me-D-Ala-OH, N-Me-Ala-OH, H-β-Ala-β-naphthalene, L-(−)-2-amino-3-ureidopropionic acid, (R)-(+)-α-allylalanine, (S)-(−)-α-allylalanine, D-2-aminobutyric acid, L-2-aminobutyric acid, DL-2-aminobutyric acid, 2-aminoisobutyric acid, α-aminoisobutyric acid, (S)-(+)-2-allylalanine Amino-4-phenylbutyric acid ethyl ester, benzyl α-aminoisobutyrate, Abu-OH, Aib-OH, β-(9-anthryl)-Ala-OH, β-(3-benzothienyl)-Ala-OH, β-(3-benzothienyl)-D-Ala-OH, Cha-OH, Cha-OMe, β-(2-furyl)-Ala-OH, β-(2-furyl)-D-Ala-OH, β-iodo-Ala-OBzl, β-iodo-D-Ala-OBzl, 3-iodo-D-Ala-OMe, β-iodo-Ala-OMe, 1-Nal-OH, D-1-Nal-OH, 2-Na l-OH, D-2-Nal-OH, (R)-3-(2-naphthyl)-β-Ala-OH, (S)-3-(2-naphthyl)-β-Ala-OH, β-phenyl-Phe-OH, 3-(2-pyridyl)-Ala-OH, 3-(3-pyridyl)-Ala-OH, 3-(3-pyridyl)-D-Ala-OH, (S)-3-(3-pyridyl)-β-Ala-OH, 3-(4-pyridyl)-Ala-OH, 3-(4-pyridyl)-D-Ala-OH, β-(2-quinolyl)-Ala-OH, 3-(2-quinolyl)-DL-Ala-OH, 3-(3-quinolyl)-D L-Ala-OH, 3-(2-quinoxalyl)-DL-Ala-OH, β-(4-thiazolyl)-Ala-OH, β-(2-thienyl)-Ala-OH, β-(2-thienyl)-D-Ala-OH, β-(3-thienyl)-Ala-OH, β-(3-thienyl)-D-Ala-OH, 3-chloro-D-alanine methyl ester, N-[(4-chlorophenyl)sulfonyl]-β-alanine, 3-cyclohexyl-D-alanine, 3-cyclopentyl-DL-alanine, (-)-3-(3,4-dihydroxyphenyl)-2-methyl-L-alanine, 3,3-Diphenyl-D-alanine, 3,3-diphenyl-L-alanine, N-[(S)-(+)-1-(ethoxycarbonyl)-3-phenylpropyl]-L-alanine, N-[1-(S)-(+)-ethoxycarbonyl-3-phenylpropyl]-L-alanyl carboxyanhydride, N-(3-fluorobenzyl)alanine, N-(3-indolylacetyl)-L-alanine, methyl (RS)-2-(aminomethyl)-3-phenylpropionate, 3-(2-oxo-1,2-dihydro-4-quinolinyl)alanine , 3-(1-pyrazolyl)-L-alanine, 3-(2-pyridyl)-D-alanine, 3-(2-pyridyl)-L-alanine, 3-(3-pyridyl)-L-alanine, 3-(4-pyridyl)-D-alanine, 3-(4-pyridyl)-L-alanine, 3-(2-quinolyl)-DL-alanine, 3-(4-quinolyl)-DL-alanine, D-styrylalanine, L-styrylalanine, 3-(2-thienyl)-L-alanine, 3-(2-thienyl)-DL-alanine, 3-(2-thienyl)-DL-alanine, 3,3,3-Trifluoro-DL-alanine, N-methyl-L-alanine, 3-ureidopropionic acid, Aib-OH, Cha-OH, dehydro-Ala-OMe, dehydro-Ala-OH, D-2-Nal-OH, β-Ala-ONp, β-homoala-OH, β-D-homoala-OH, β-alanine, β-alanine ethyl ester, β-alanine methyl ester, (S)-diphenyl-β-homoala-OH, (R)-4-(4-pyridyl)-β-homoala-OH, (S)-4-(4-pyridyl)-β-homoala-OH, β-Ala-OH, (S)-diphenyl-β-homoala-OH, L-β-homoalanine , (R)-4-(3-pyridyl)-β-homoala-OH, α-methyl-α-naphthylalanine [Manap], N-methyl-cyclohexylalanine [Nmchexa], cyclohexylalanine [Chexa], N-methyl-cyclopentylalanine [Nmcpen], cyclopentylalanine [Cpen], N-methyl-α-naphthylalanine [Nmanap], α-naphthylalanine [Anap], N-methylalanine [Nmala], N-methylalanine [Dnmala], α-methyl-cyclohexylalanine [Mchexa], α-methyl-cyclopentylalanine [Mcpen]. Each possibility represents a separate embodiment.

[0084] Non-limiting examples of non-conservative amino acids of arginine are: homoarginine (hArg), N-methylarginine (NMeArg), citrulline, 2-amino-3-guanidinopropionic acid, N-iminoethyl-L-ornithine, Nω-monomethyl-L-arginine, Nω-nitro-L-arginine, D-arginine, 2-amino-3-ureidopropionic acid, Nω,ω-dimethyl-L-arginine, and NMeArg. Nitro-L-arginine, Nω-nitro-D-arginine, L-α-methylarginine [Marg], D-α-methylarginine [Dmarg], LN-methylarginine [Nmarg], D-N-methylarginine [Dnmarg], β-homoarg-OH, L-homoarginine, N-(3-guanidinopropyl)glycine [Narg], and D-arginine [Darg, (dR), r]. Each possibility represents a separate embodiment.

[0085] Non-limiting examples of non-conservative amino acids of asparagine are: L-α-methylasparagine [Masn], D-α-methylasparagine [Dmasn], LN-methylasparagine [Nmasn], DN-methylasparagine [Dnmasn], N-(carbamylmethyl)glycine [Nasn], and D-asparagine [Dasn, (dN), n]. Each possibility represents a separate embodiment.

[0086] Non-limiting examples of non-conservative amino acids of aspartic acid are: L-α-methylaspartate [Masp], D-α-methylaspartate [Dmasp], LN-methylaspartic acid [Nmasp], DN-methylaspartate [Dnmasp], N-(carboxymethyl)glycine [Nasp], and D-aspartic acid [Dasp, (dD), d]. Each possibility represents a separate embodiment.

[0087] Non-limiting examples of non-conservative amino acids of cysteine ​​are: L-cysteic acid, L-cysteine ​​sulfinic acid, D-ethionine, S-(2-thiazolyl)-L-cysteine, DL-homocysteine, L-homocysteine, L-homocystine, L-α-methylcysteine ​​[Mcys], D-α-methylcysteine ​​[Dmcys], LN-methylcysteine ​​[Nmcys], DN-methylcysteine ​​[Dnmcys], N-(thiomethyl)glycine [Ncys], and D-cysteine ​​[Dcys, (dC), c]. Each possibility represents a separate embodiment.

[0088] Non-limiting examples of non-conservative amino acids of glutamic acid are: γ-carboxy-DL-glutamic acid, 4-fluoro-DL-glutamic acid, β-glutamic acid, L-β-homoglutamic acid, L-α-methylglutamate [Mglu], D-α-methylglutamic acid [Dmglu], LN-methylglutamic acid [Nmglu], DN-methylglutamate [Dnmglu], N-(2-carboxyethyl)glycine [Nglu], and D-glutamic acid [Dglu], (dE), e]. Each possibility represents a separate embodiment.

[0089] Non-limiting examples of non-conservative amino acids of glutamine are: Cit-OH, D-citrulline, thio-L-citrulline, β-Gln-OH, L-β-homoglutamine, L-α-methylglutamine [Mgln], D-α-methylglutamine [Dmgln], LN-methylglutamine [Nmgln], DN-methylglutamine [Dnmgln], N-(2-carbamylethyl)glycine [Ngln], and D-glutamine [Dgln,(dQ),q]. Each possibility represents a separate embodiment.

[0090] Non-limiting examples of non-conservative amino acids of glycine are: tBu-Gly-OH, D-allylglycine, N-[bis(methylthio)methylene]glycine methyl ester, Chg-OH, D-Chg-OH, D-cyclopropylglycine, L-cyclopropylglycine, (R)-4-fluorophenylglycine, (S)-4-fluorophenylglycine, iminodiacetic acid, (2-indanyl)-Gly-OH, (±)-α -phosphonoglycine trimethyl ester, D-propargylglycine, propargyl-Gly-OH, (R)-2-thienylglycine, (S)-2-thienylglycine, (R)-3-thienylglycine, (S)-3-thienylglycine, 2-(4-trifluoromethyl-phenyl)-DL-glycine, (2S,3R,4S)-α-(carboxycyclopropyl)glycine, N-(chloroacetyl)glycine ethyl ester, (S )-(+)-2-Chlorophenylglycine methyl ester, N-(2-chlorophenyl)-N-(methylsulfonyl)glycine, D-α-cyclohexylglycine, L-α-cyclopropylglycine, di-tert-butyl-iminodicarboxylate, ethyl acetamidocyanoacetate, N-(2-fluorophenyl)-N-(methylsulfonyl)glycine, N-(4-fluorophenyl)-N-(methylsulfonyl)glycine, N-(2-furfurylideneacetyl)glycine methyl ester, N-(2-furoyl)glycine, N-(2-hydroxyethyl)iminodiacetic acid, N-(4-hydroxyphenyl)glycine, iminodiacetic acid, N-lauroylsarcosine sodium salt, L-α-neopentylglycine, N-(phosphonomethyl)glycine, D-propargylglycine, LC-propargylglycine, sarcosine, N,N-dimethylglycine, N,N-Dimethylglycine ethyl ester, D-Chg-OH, α-phosphonoglycine trimethyl ester, N-cyclobutylglycine [Ncbut], L-α-methylethylglycine [Metg], N-cycloheptylglycine [Nchep], L-α-methyl-i-butylglycine [Mtbug], N-methylglycine [Nmgly], LN-methyl-ethylglycine [Nmetg], L-ethylglycine [Etg], LN-methyl-t-butylglycine [Nmtbug], Lt-butylglycine [Tbug], N-cyclohexylglycine [Nchex], N-cyclodecylglycine [Ncd] ec], N-cyclododecylglycine [Ncdod], N-cyclooctylglycine [Ncoct], N-cyclopropylglycine [Ncpro], N-cycloundecylglycine [Ncund], N-(2-aminoethyl)glycine [Naeg], N-(N-(2,2-diphenylethyl)diphenylethyl)glycine [Nnbhm], N-(2,2-carbamylmethyl-glycine [Nbhm], N-(N-(3,3-diphenylpropyl)diphenylpropyl)glycine [Nnbhe], and N-(3,3-carbamylmethyl-glycine [Nbhe]. Each possibility represents a separate embodiment.

[0091] Non-limiting examples of non-conservative amino acids of histidine are: L-α-methylhistidine [Mhis], D-α-methylhistidine [Dmhis], LN-methylhistidine [Nmhis], DN-methylhistidine [Dnmhis], N-(imidazolylethyl)glycine [Nhis], and D-histidine [Dhis, (dH), h]. Each possibility represents a separate embodiment.

[0092] Non-limiting examples of non-conservative amino acids of isoleucine are: N-methyl-L-isoleucine [Nmile], N-(3-indolylacetyl)-L-isoleucine, allo-lle-OH, D-allo-isoleucine, L-β-homoisoleucine, L-α-methylisoleucine [Mile], D-α-methylisoleucine [Dmile], DN-methylisoleucine [Dnmile], N-(1-methylpropyl)glycine [Nile], and D-isoleucine [Dile, (dD),i]. Each possibility represents a separate embodiment.

[0093] A non-limiting example of a non-conservative amino acid of leucine is: D-leucine [Dleu, (dL), l]. Cycloleucine, DL-leucine, N-formyl-Leu-OH, D-tert-leucine, L-tert-leucine, DL-tert-leucine, L-tert-leucine methyl ester, 5,5,5-trifluoro-DL-leucine, D-β-Leu-OH, L-β-leucine, DL-β-leucine, L-β-homoleucine, DL-β-homoleucine, LN-methyl-leucine [Nmleu], DN-methyl-leucine [Dnmleu], L-α-methyl-leucine [Mleu], D-α-methyl-leucine [Dmleu], N-(2-methylpropyl)glycine [Nleu], D-leucine [Dleu, 1], D-norleucine, L-norleucine, DL-norleucine, LN-methylnorleucine [Nmnle], and L-norleucine [Nle]. Each possibility represents a separate embodiment.

[0094] Non-limiting examples of non-conservative amino acids of lysine are: DL-5-hydroxylysine, (5R)-5-hydroxy-L-lysine, β-Lys-OH, L-β-homolysine, L-α-methyl-lysine [Mlys], D-α-methyl-lysine [Dmlys], LN-methyl-lysine [Nmlys], DN-methyl-lysine [Dnmlys], N-(4-aminobutyl)glycine [Nlys], and D-lysine [Dlys,(dK),k]. Each possibility represents a separate embodiment.

[0095] Non-limiting examples of non-conservative amino acids of methionine are: L-β-homomethionine, DL-β-homomethionine, L-α-methylmethionine [Mmet], D-α-methylmethionine [Dmmet], LN-methylmethionine [Nmmet], DN-methylmethionine [Dnmmet], N-(2-methylthioethyl)glycine [Nmet], and D-methionine [Dmet, (dM), m]. Each possibility represents a separate embodiment.

[0096] Non-limiting examples of non-conservative amino acids of phenylalanine are: N-acetyl-2-fluoro-DL-phenylalanine, N-acetyl-4-fluoro-DL-phenylalanine, 4-amino-L-phenylalanine, 3-[3,4-bis(trifluoromethyl)phenyl]-L-alanine, Bpa-OH, D-Bpa-OH, 4-tert-butyl-Phe-OH, 4-tert-butyl-D-Phe-OH, 4-(amino)-L-phenylalanine, rac-β 2-Homophenylalanine, 2-methoxy-L-phenylalanine, (S)-4-methoxy-β-Phe-OH, 2-nitro-L-phenylalanine, pentafluoro-D-phenylalanine, pentafluoro-L-phenylalanine, Phe(4-Br)-OH, D-Phe(4-Br)-OH, Phe(2-CF3)-OH, D-Phe(2-CF3)-OH, Phe(3-CF3)-OH, D-Phe(3-CF3)-OH, Phe(4-CF3 )-OH, D-Phe(4-CF3)-OH, Phe(2-Cl)-OH, D-Phe(2-Cl)-OH, Phe(2,4-Cl2)-OH, D-Phe(2,4-Cl2)-OH, D-Phe(3-Cl)-OH, Phe (3,4-Cl2)-OH, Phe(4-Cl)-OH, D-Phe(4-Cl)-OH, Phe(2-CN)-OH, D-Phe(2-CN)-OH, D-Phe(3-CN)-OH, Phe(4-CN)-OH, D-Phe (4-CN)-OH, Phe(2-Me)-OH, D-Phe(2-Me)-OH, Phe(3-Me)-OH, D-Phe(3-Me)-OH, Phe(4-Me)-OH, Phe(4-NH2)-OH, Phe(4-NO 2)-OH, Phe(2-F)-OH, D-Phe(2-F)-OH, Phe(3-F)-OH, D-Phe(3-F)-OH, Phe(3,4-F2)-OH, D-Phe(3,4-F2)-OH, Phe(3,5-F2)- OH, Phe(4-F)-OH, D-Phe(4-F)-OH, Phe(4-I)-OH, D-3,4,5-trifluorophenylalanine, p-bromo-DL-phenylalanine, 4-bromo-L-phenylalanine, β-phenyl-D-phenylalanine, 4-chloro-L-phenylalanine, DL-2,3-difluorophenylalanine, DL-3,5-difluorophenylalanine, 3,4-dihydroxy-L-phenylalanine, 3-(3,4-dimethoxyphenyl)-L-alanine, N-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methoxy-L-phenylalanine, o-fluoro-DL-phenylalanine, m-fluoro-L-phenylalanine, m-fluoro-DL-phenylalanine, p-fluoro-L-phenylalanine, p-fluoro-DL-phenylalanine, 4-fluoro-D-phenylalanine, 2-fluoro-L-phenylalanine methyl ester, p-fluoro-DL-Phe-OMe, D-3-bromophenylalanine D-4-bromophenylalanine, L-β-(6-chloro-4-pyridinyl)alanine, D-3,5-difluorophenylalanine, L-3-fluorophenylalanine, L-4-fluorophenylalanine, L-β-(1H-5-indolyl)alanine, 2-nitro-L-phenylalanine, pentafluoro-L-phenylalanine, phe(3-br)-OH, Phe(4-Br)-OH, Phe(2-CF3)-OH, D-Phe(2-CF3)-OH, Phe(3-CF3)-OH, D-Phe(3-CF 3)-OH, Phe(4-CF3)-OH, D-Phe(4-CF3)-OH, Phe(2-Cl)-OH, D-Phe(2-Cl)-OH, Phe(2,4-Cl2)-OH, D-Phe(2,4-Cl2)-OH, Phe(3,4-Cl2)-OH, D-P he(3,4-Cl2)-OH, Phe(4-Cl)-OH, D-Phe(4-Cl)-OH, Phe(2-CN)-OH, D-Phe(2-CN)-OH, D-Phe(3-CN)-OH, Phe(4-CN)-OH, Phe(2-Me)-OH, Phe(3 -Me)-OH, D-Phe(3-Me)-OH, Phe(4-NO2)-OH, D-Phe(4-NO2)-OH, D-Phe(2-F)-OH, Phe(3-F)-OH, D-Phe(3-F)-OH, Phe(3,4-F2)-OH, Phe(3,5-F2)-OH, D-Phe(4-F)-OH, Phe(4-I)-OH, D-Phe(4-I)-OH, 4-(phosphonomethyl)-Phe-OH, L-4-trifluoromethylphenylalanine, 3,4,5-trifluoro-D-phenylalanine, L-3,4,5-Trifluorophenylalanine, 6-hydroxy-DL-DOPA, 4-(hydroxymethyl)-D-phenylalanine, N-(3-indolylacetyl)-L-phenylalanine, p-iodo-D-phenylalanine, 4-iodo-L-phenylalanine, α-methyl-D-phenylalanine, α-methyl-L-phenylalanine, α-methyl-DL-phenylalanine, α-methyl-DL-phenylalanine methyl ester, 4-nitro-D-phenylalanine, 4-nitro-L-phenylalanine, 4-nitro-DL-phenylalanine, (S)-(+)-4-nitrophenylalanine methyl ester, 2-(trifluoromethyl)-D-phenylalanine, 2-(trifluoromethyl)-L-phenylalanine, 3-(trifluoromethyl)-D-phenylalanine, 3-(trifluoromethyl)-L-phenylalanine, 4-(trifluoromethyl)-D-phenylalanine, 3,3',5-triiodo- L-Thyronine, (R)-4-Bromo-β-Phe-OH, N-Acetyl-DL-β-Phenylalanine, (S)-4-Bromo-β-Phe-OH, (R)-4-Chloro-β-Homophe-OH, (S)-4-Chloro-β-Homophe-OH, (R)-4-Chloro-β-Phe-OH, (S)-4-Chloro-β-Phe-OH, (S)-2-Cyano-β-Homophe-OH, (R)-4-Cyano-β-Homophe-OH, (S)-4-Cyano-β -Homophe-OH, (R)-3-cyano-β-Phe-OH, (R)-4-cyano-β-Phe-OH, (S)-4-cyano-β-Phe-OH, (R)-3,4-dimethoxy-β-Phe-OH, (S)-3,4-dimethoxy-β-Phe-OH, (R)-4-fluoro-β-Phe-OH, (S)-4-fluoro-β-Phe-OH, (S)-4-iodo-β-Homophe-OH, (S)-3-cyano-β-Homophe-OH, (S)-3,4-Difluoro-β-Homophe-OH, (R)-4-fluoro-β-Homophe-OH, (S)-β2-homophenylalanine, (R)-3-methoxy-β-Phe-OH, (S)-3-methoxy-β-Phe-OH, (R)-4-methoxy-β-Phe-OH, (S)-4-methyl-β-Homophe-OH, (R)-2-methyl-β-Phe-OH, (S)-2-methyl-β-Phe-OH, (R)-3-methyl-β-Phe -OH, (S)-3-methyl-β-Phe-OH, (R)-4-methyl-β-Phe-OH, (S)-4-methyl-β-Phe-OH, β-Phe-OH, D-β-Phe-OH, (S)-2-(trifluoromethyl)-β-Homophe-OH, (S)-2-(trifluoromethyl)-β-Homophe-OH, (S)-3-(trifluoromethyl)-β-Homophe-OH, (R)-4-(trifluoromethyl)-β-Homophe he-OH, (S)-2-(trifluoromethyl)-β-Phe-OH, (R)-3-(trifluoromethyl)-β-Phe-OH, (S)-3-(trifluoromethyl)-β-Phe-OH, (R)-4-(trifluoromethyl)-β-Phe-OH, (S)-4-(trifluoromethyl)-β-Phe-OH, β-Homophe-OH, D-β-Homophe-OH, (S)-2-methyl-β-Homophe-OH, (S)-3-methyl Chil-β-Homophe-OH, β-Phe-OH, β-D-Phe-OH, (S)-3-(trifluoromethyl)-β-Homophe-OH, L-β-homophenylalanine, DL-β-homophenylalanine, DL-β-phenylalanine, DL-homophenylalanine methyl ester, D-homophenylalanine, L-homophenylalanine, DL-homophenylalanine, D-homophenylalanine ethyl ester, (R)-β, 2-homophenylalanine, L-α-methyl-homophenylalanine [Mhphe], L-α-methylphenylalanine [Mphe], Da-methylphenylalanine [Dmphe], LN-methylhomophenylalanine [Nm phe], L-homophenylalanine [Hphe], LN-methylphenylalanine [Nmphe], DN-methylphenylalanine [Dnmphe], N-benzylglycine [Nphe], and D-phenylalanine [Dphe, (dF), f]. Each possibility represents a separate embodiment.

[0097] Non-limiting examples of non-conservative amino acids of proline are: homoproline (hPro), (4-hydroxy)pro(4HyP), (3-hydroxy)pro(3HyP), gamma-benzyl-proline, gamma-(2-fluoro-benzyl)-proline, gamma-(3-fluoro-benzyl)-proline, gamma-(4-fluoro-benzyl)-proline, gamma-(2-chloro-benzyl)-proline, gamma-(3-chloro-benzyl)-proline, gamma-(4-chloro-benzyl)-proline, gamma-(2-bromo-benzyl)-proline, gamma-(3-bromo-benzyl)-proline. gamma-(4-bromobenzyl)-proline, gamma-(2-methylbenzyl)-proline, gamma-(3-methylbenzyl)-proline, gamma-(4-methylbenzyl)-proline, gamma-(2-nitrobenzyl)-proline, gamma-(3-nitrobenzyl)-proline, gamma-(4-nitrobenzyl)-proline, gamma-(1-naphthalenylmethyl)-proline, gamma-(2-naphthalenylmethyl)-proline, gamma-(2,4-dichlorobenzyl)-proline, gamma-(3,4-dichlorobenzyl)-proline, gamma-(3,4-Difluoro-benzyl)-proline, gamma-(2-trifluoro-methyl-benzyl)-proline, gamma-(3-trifluoro-methyl-benzyl)-proline, gamma-(4-trifluoro-methyl-benzyl)-proline, gamma-(2-cyano-benzyl)-proline, gamma-(3-cyano-benzyl)-proline, gamma-(4-cyano-benzyl)-proline, gamma-(2-iodo-benzyl)-proline, gamma-(3-iodo-benzyl)-proline, gamma-(4-iodo-benzyl)-proline, gamma-(3-phenyl-allyl-benzyl)-proline, gamma-(3-phenyl-propyl-benzyl)-proline, gamma-(4-tert-butyl-benzyl)-proline gamma-(4-phenyl-2-benzoyl)-proline, gamma-benzhydryl-proline, gamma-(4-biphenyl-methyl)-proline, gamma-(4-thiazolyl-methyl)-proline, gamma-(3-benzothienyl-methyl)-proline, gamma-(2-thienyl-methyl)-proline, gamma-(3-thienyl-methyl)-proline, gamma-(2-furanyl-methyl)-proline, gamma-(2-pyridinyl-methyl)-proline, gamma-(3-pyridinyl-methyl)-proline, gamma-(4-pyridinyl-methyl)-proline, gamma-allyl-proline, gamma-propynyl-proline, alpha-modified proline residues, pipecolic acid, azetidine-3-carboxylic acid, L-beta-homoproline, L-beta 3 -homoproline, L-β-homohydroxyproline, hydroxyproline [Hyp], L-□-methylproline [Mpro], D-□-methylproline [Dmpro], LN-methylproline [Nmpro], DN-methylproline [Dnmpro], and D-proline [Dpro,(dP),p]. Each possibility represents a separate embodiment.

[0098] Non-limiting examples of non-conservative amino acids of serine are: (2R,3S)-3-phenylisoserine, D-cycloserine, L-isoserine, DL-isoserine, DL-3-phenylserine, L-β-homoserine, D-homoserine, D-homoserine, L-3-homoserine, L-homoserine, L-α-methylserine [Mser], D-α-methylserine [Dmser], LN-methylserine [Nmser], DN-methylserine [Dnmser], D-serine [Dser,(dS),s], N-(hydroxymethyl)glycine [Nser], and phosphoserine [pSer]. Each possibility represents a separate embodiment.

[0099] Non-limiting examples of non-conservative amino acids of threonine are: L-allo-threonine, D-thyroxine, L-β-homothreonine, L-α-methylthreonine [Mthr], D-α-methylthreonine [Dmthr], LN-methylthreonine [Nmthr], DN-methylthreonine [Dnmthr], D-threonine [Dthr, (dT), t], N-(1-hydroxyethyl)glycine [Nthr], and phosphothreonine [pThr]. Each possibility represents a separate embodiment.

[0100] Non-limiting examples of non-conservative amino acids of tryptophan are: 5-fluoro-L-tryptophan, 5-fluoro-DL-tryptophan, 5-hydroxy-L-tryptophan, 5-methoxy-DL-tryptophan, L-abrin, 5-methyl-DL-tryptophan, H-Tpi-OMe, β-Homotrp-OMe, L-β-homotryptophan, L-α-methyltryptophan [Mtrp], D-α-methyltryptophan [Dmtrp], LN-methyltryptophan [Nmtrp], DN-methyltryptophan [Dnmtrp], N-(3-indolylethyl)glycine [Nhtrp], D-tryptophan [Dtrp,(dW),w]. Each possibility represents a separate embodiment.

[0101] Non-limiting examples of non-conservative amino acids of tyrosine are: 3,5 diiodotyrosine (3,5-dITyr), 3,5-dibromotyrosine (3,5-dBTyr), homotyrosine, D-tyrosine, 3-amino-L-tyrosine, 3-amino-D-tyrosine, 3-iodo-L-tyrosine, 3-iodo-D-tyrosine, 3-methoxy-L-tyrosine, 3-methoxy-D-tyrosine, L-tyrosine Thyroxine, D-thyroxine, L-thyronine, D-thyronine, O-methyl-L-tyrosine, O-methyl-D-tyrosine, D-thyronine, O-ethyl-L-tyrosine, O-ethyl-D-tyrosine, 3,5,3'-triiodo-L-thyronine, 3,5,3'-triiodo-D-thyronine, 3,5-diiodo-L-thyronine, 3,5-diiodo-D-thyronine, D-meta-tyrosine, L-meta-tyrosine D-ortho-tyrosine, L-ortho-tyrosine, phenylalanine, substituted phenylalanines, N-nitrophenylalanine, p-nitrophenylalanine, 3-chloro-Dtyr-oh, Tyr(3,5-diI), 3-chloro-L-tyrosine, Tyr(3-NO2)-OH, Tyr(3,5-diI)-OH, N-Me-Tyr-OH, α-methyl-DL-tyrosine, 3-nitro-L -tyrosine, DL-o-tyrosine, β-Homotyr-OH, (R)-β-Tyr-OH, (S)-β-Tyr-OH, L-α-methyltyrosine [Mtyr], D-α-methyltyrosine [Dmtyr], LN-methyltyrosine [Nmtyr], DN-methyltyrosine [Dnmtyr], D-tyrosine [Dtyr, (dY), y], O-methyl-tyrosine, and phosphotyrosine [pTyr]. Each possibility represents a separate embodiment.

[0102] Non-limiting examples of non-conservative amino acids of valine are: 3-fluoro-DL-valine, 4,4,4,4',4',4'-hexafluoro-DL-valine, D-valine [Dval, (dV), v], N-Me-Val-OH [Nmval], N-Me-Val-OH, L-α-methylvaline [Mval], D-α-methylvaline [Dmval], (R)-(+)-α-methylvaline, (S)-(-)-α-methylvaline, and DN-methylvaline [Dnmval]. Each possibility represents a separate embodiment.

[0103] Other unnatural amino acids that can be substituted as non-conservative replacements include ornithine and its modifications: D-ornithine [Dorn], L-ornithine [Orn], DL-ornithine, L-α-methylornithine [Morn], D-α-methylornithine [Dmorn], LN-methylornithine [Nmorn], DN-methylornithine [Dnmorn], and N-(3-aminopropyl)glycine [Norn]. Each possibility represents a separate embodiment.

[0104] Alicyclic amino acids: L-2,4-diaminobutyric acid, L-2,3-diaminopropionic acid, N-Me-Aib-OH, (R)-2-(amino)-5-hexynoic acid, piperidine-2-carboxylic acid, aminonorbornyl-carboxylate [Norb], alpha-aminobutyric acid [Abu], aminocyclopropane-carboxylate [Cpro], (cis)-3-aminobicyclo[2.2.1]heptane-2-carboxylic acid, exo-cis-3-aminobicyclo[2.2.1]hept-5-ene-2-carboxylic acid, 1-amino- 1-Cyclobutanecarboxylic acid, cis-2-aminocycloheptanecarboxylic acid, 1-aminocyclohexanecarboxylic acid, cis-2-aminocyclohexanecarboxylic acid, trans-2-aminocyclohexanecarboxylic acid, cis-6-amino-3-cyclohexene-1-carboxylic acid, 2-(1-aminocyclohexyl)acetic acid, cis-2-amino-1-cyclooctanecarboxylic acid, cis-2-amino-3-cyclooctene-1-carboxylic acid, (1R,2S)-(-)-2-amino-1-cyclopentanecarboxylic acid, (1S,2R )-(+)-2-Amino-1-cyclopentanecarboxylic acid, cis-2-amino-1-cyclopentanecarboxylic acid, 2-(1-aminocyclopentyl)acetic acid, cis-2-amino-2-methylcyclohexanecarboxylic acid, cis-2-amino-2-methylcyclopentanecarboxylic acid, 3-amino-3-(4-nitrophenyl)propionic acid, 3-azetidinecarboxylic acid, amchc-oh, 1-aminocyclobutanecarboxylic acid, 1-(amino)cyclohexanecarboxylic acid, cis-2-(amino)-cyclohexanecarboxylic acid , trans-2-(amino)-cyclohexanecarboxylic acid, cis-4-(amino)cyclohexanecarboxylic acid, trans-4-(amino)cyclohexanecarboxylic acid, (±)-cis-2-(amino)-3-cyclohexene-1-carboxylic acid, (±)-cis-6-(amino)-3-cyclohexene-1-carboxylic acid, 2-(1-aminocyclohexyl)acetic acid, cis-[4-(amino)cyclohexyl]acetic acid, 1-(amino)cyclopentanecarboxylic acid, (±)-cis-2-(amino)cyclopentanecarboxylic acid, (1R,4S)-(+)-4-(amino)-2-cyclopentene-1-carboxylic acid, (±)-cis-2-(amino)-3-cyclopentene-1-carboxylic acid, 2-(1-aminocyclopentyl)acetic acid, 1-(amino)cyclopropanecarboxylic acid, 1-aminocyclopropanecarboxylate ethyl, 1,2-trans-achec-oh, 1-(amino)cyclobutanecarboxylic acid, 1-(amino)cyclohexanecarboxylic acid, cis-2-(amino)-cyclohexanecarboxylic acid, trans-2-(amino)cyclohexanecarboxylic acid, cis-4-(amino)cyclohexanecarboxylic acid, trans-4-(amino)cyclohexanecarboxylic acid, cis-[4-(amino)cyclohexyl]acetic acid, 1-(amino)cyclopentanecarboxylic acid, (1R,4S)-(+)- 4-(amino)-2-cyclopentene-1-carboxylic acid, (1S,4R)-(-)-4-(amino)-2-cyclopentene-1-carboxylic acid, 1-(amino)cyclopropanecarboxylic acid, trans-4-(aminomethyl)cyclohexanecarboxylic acid, β-Dab-OH, 3-amino-3-(3-bromophenyl)propionic acid, 3-aminobutanoic acid, cis-2-amino-3-cyclopentene-1-carboxylic acid, DL-3-aminoisobutyric acid, (R)-3-amino-2-phenylpropionic acid, (±)-3-(amino)-4-(4-biphenylyl)butyric acid, cis-3-(amino)cyclohexanecarboxylic acid, (1S,3R)-(+)-3-(amino)cyclopentanecarboxylic acid, (2R,3R)-3-(amino)-2-hydroxy-4-phenylbutyric acid, (2S,3R)-3-(amino)-2-hydroxy-4-phenylbutyric acid, 2-(aminomethyl)phenylacetic acid, (R)-3-(amino)-2-methylpropionic acid, (S)-3-(amino)-2-methylpropionic acid, (R)-3-(amino)-4-(2-naphthyl)butyric acid, (S)-3-(amino)-4-(2-naphthyl)butyric acid, (R)-3-(amino)-5-phenylpentanoic acid, (R)-3-(amino)-2-phenylpropionic acid, 3-(benzylamino)propionate ethyl, cis-3-(amino)cyclohexanecarboxylic acid, (S)-3-(amino)-5-hexenoic acid, (R)-3-(amino)-2-methylpropionate carboxylic acid, (S)-3-(amino)-2-methylpropionic acid, (R)-3-(amino)-4-(2-naphthyl)butyric acid, (S)-3-(amino)-4-(2-naphthyl)butyric acid, (R)-(-)-pyrrolidine-3-carboxylic acid, (S)-(+)-pyrrolidine-3-carboxylic acid, N-methyl-γ-aminobutyrate [Nmgabu], γ-aminobutyric acid [Gabu], N-methyl-α-amino-α-methylbutyrate [Nmaabu], α-amino-α-methylbutyrate [Aabu], N-methyl-α-aminoisobutyrate [Nmaib], α-aminoisobutyric acid [Aib], α-methyl-γ-aminobutyrate [Mgabu]. Each possibility represents a separate embodiment.

[0105] Phenylglycine and its modifications: Phg-OH, D-Phg-OH, 2-(piperazino)-2-(3,4-dimethoxyphenyl)acetic acid, 2-(piperazino)-2-(2-fluorophenyl)acetic acid, 2-(4-piperazino)-2-(3-fluorophenyl)acetic acid, 2-(4-piperazino)-2-(4-methoxyphenyl)acetic acid, 2-(4-piperazino)-2-(3-pyridyl)acetic acid, 2-(4-piperazino)-2-[4-(trifluoromethyl)phenyl]acetic acid, L-(+)-2-chlorophenylglycine, (±)-2-chlorophenylglycine, (±)-4-chlorophenylglycine, (R)-(-)-2-(2,5-dihydrophenyl)glycine, (R)-(-)-N-(3,5-dinitrobenzoyl)-α-phenylglycine, (S)-(+)-N-(3, 5-dinitrobenzoyl)-α-phenylglycine, 2,2-diphenylglycine, 2-fluoro-DL-α-phenylglycine, 4-fluoro-D-α-phenylglycine, 4-hydroxy-D-phenylglycine, 4-hydroxy-L-phenylglycine, 2-phenylglycine, D-(-)-α-phenylglycine, D-(-)-α-phenylglycine, DL-α-phenylglycine, L-(+)-α-phenylglycine, N-phenylglycine, (R)-(-)-2-phenylglycine methyl ester, (S)-(+)-2-phenylglycine methyl ester, 2-phenylglycinonitrile hydrochloride, α-phenylglycinonitrile, 3-(trifluoromethyl)-DL-phenylglycine, and 4-(trifluoromethyl)-L-phenylglycine. Each possibility represents a separate embodiment.

[0106] Penicillamine and its modifications: N-acetyl-D-penicillamine, D-penicillamine, L-penicillamine [Pen], DL-penicillamine, α-methylpenicillamine [Mpen], N-methylpenicillamine [Nmpen]. Each possibility represents a separate embodiment.

[0107] β-homopyrrolidine. Each possibility represents a separate embodiment.

[0108] Aromatic amino acids: 3-acetamidobenzoic acid, 4-acetamidobenzoic acid, 4-acetamido-2-methylbenzoic acid, N-acetylanthranilic acid, 3-aminobenzoic acid, 3-aminobenzoic acid hydrochloride, 4-aminobenzoic acid, 4-aminobenzoic acid, 4-aminobenzoic acid, 4-aminobenzoic acid, 4-aminobenzoic acid, 4-aminobenzoic acid, 4-aminobenzoic acid, 4-aminobenzoic acid, 2-aminobenzophenone-2'-carboxylic acid, 2-amino-4-bromobenzoic acid, 2-amino-5-bromobenzoic acid, 3-amino-2-bromobenzoic acid, 3-amino-4-bromobenzoic acid, 3-amino-5-bromobenzoic acid, 4-amino-3-bromobenzoic acid, 5-amino-2-bromobenzoic acid, 2- Amino-3-bromo-5-methylbenzoic acid, 2-amino-3-chlorobenzoic acid, 2-amino-4-chlorobenzoic acid, 2-amino-5-chlorobenzoic acid, 2-amino-5-chlorobenzoic acid, 2-amino-6-chlorobenzoic acid, 3-amino-2-chlorobenzoic acid, 3-amino-4-chlorobenzoic acid, 4-amino-2-chlorobenzoic acid, 4-amino-3-chlorobenzoic acid, 5-amino-2-chlorobenzoic acid, 5-amino-2-chlorobenzoic acid, 4-amino-5-chloro-2-methoxybenzoic acid, 2-amino-5-chloro-3-methylbenzoic acid, 3-amino-2,5-dichlorobenzoic acid, 4-amino-3,5-dichlorobenzoic acid, 2-amino-4,5-Dimethoxybenzoic acid, 4-(2-aminoethyl)benzoic acid hydrochloride, 2-amino-4-fluorobenzoic acid, 2-amino-5-fluorobenzoic acid, 2-amino-6-fluorobenzoic acid, 4-amino-2-fluorobenzoic acid, 2-amino-5-hydroxybenzoic acid, 3-amino-4-hydroxybenzoic acid, 4-amino-3-hydroxybenzoic acid, 2-amino-5-iodobenzoic acid, 5-aminoisophthalic acid, 2-amino-3-methoxybenzoic acid, 2-amino-4-methoxybenzoic acid, 2-amino-5-methoxybenzoic acid , 3-amino-2-methoxybenzoic acid, 3-amino-4-methoxybenzoic acid, 3-amino-5-methoxybenzoic acid, 4-amino-2-methoxybenzoic acid, 4-amino-3-methoxybenzoic acid, 5-amino-2-methoxybenzoic acid, 2-amino-3-methylbenzoic acid, 2-amino-5-methylbenzoic acid, 2-amino-6-methylbenzoic acid, 3-(aminomethyl)benzoic acid, 3-amino-2-methylbenzoic acid, 3-amino-4-methylbenzoic acid, 4-(aminomethyl)benzoic acid, 4-amino-2-methylbenzoic acid, 4-amino 3-amino-3-methylbenzoic acid, 5-amino-2-methylbenzoic acid, 3-amino-2-naphthoic acid, 6-amino-2-naphthoic acid, 2-amino-3-nitrobenzoic acid, 2-amino-5-nitrobenzoic acid, 2-amino-5-nitrobenzoic acid, 4-amino-3-nitrobenzoic acid, 5-amino-2-nitrobenzoic acid, 3-(4-aminophenyl)propionic acid, 3-aminophthalic acid, 4-aminophthalic acid, 3-aminosalicylic acid, 4-aminosalicylic acid, 5-aminosalicylic acid, 5-aminosalicylic acid, 2-aminoterephthalic acid, 2 -Amino-3,4,5,6-tetrafluorobenzoic acid, 4-amino-2,3,5,6-tetrafluorobenzoic acid, (R)-2-amino-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid, (S)-2-amino-1,2,3,4-tetrahydro-2-naphthalenecarboxylic acid, 2-amino-3-(trifluoromethyl)benzoic acid, 2-amino-3-(trifluoromethyl)benzoic acid, 3-amino-5-(trifluoromethyl)benzoic acid, 5-amino-2,4,6-triiodoisophthalic acid, 2-amino-3,4,5-Trimethoxybenzoic acid, 2-anilinophenylacetic acid, 2-Abz-OH, 3-Abz-OH, 4-Abz-OH, 2-(aminomethyl)benzoic acid, 3-(aminomethyl)benzoic acid, 4-(aminomethyl)benzoic acid, tert-butyl 2-aminobenzoate, tert-butyl 3-aminobenzoate, tert-butyl 4-aminobenzoate, 4-(butylamino)benzoic acid, 2,3-diaminobenzoic acid, 3,4-diaminobenzoic acid, 3,5-diaminobenzoic acid, 3,5-diaminobenzoic acid, 3,5-dichloroanthranilic acid, 4-(diethylamino)benzoic acid, 4,5-difluoroanthranilic acid, 4-(dimethylamino)benzoic acid, 4-(dimethylamino)benzoic acid N-amino)benzoic acid, 3,5-dimethylanthranilic acid, 5-fluoro-2-methoxybenzoic acid, 2-Abz-OH, 3-Abz-OH, 4-Abz-OH, 3-(aminomethyl)benzoic acid, 4-(aminomethyl)benzoic acid, 4-(2-hydrazino)benzoic acid, 3-hydroxyanthranilic acid, 3-hydroxyanthranilic acid, methyl 3-aminobenzoate, 3-(methylamino)benzoic acid, 4-(methylamino)benzoic acid, methyl 2-amino-4-chlorobenzoate, methyl 2-amino-4,5-dimethoxybenzoate, 4-nitroanthranilic acid, N-phenylanthranilic acid, N-phenylanthranilic acid, and sodium 4-aminosalicylate. Each possibility represents a separate embodiment.

[0109] Other amino acids: (S)-α-amino-γ-butyrolactone, DL-2-aminocaprylic acid, 7-aminocephalosporanic acid, 4-aminosilicic acid, (S)-(+)-α-aminocyclohexanepropionic acid, (R)-amino-(4-hydroxyphenyl)acetic acid methyl ester, 5-aminolevulinic acid, 4-amino-nicotinic acid, 3-aminophenylacetic acid, 4-aminophenylacetic acid, 2-amino-2-phenylbutyric acid, 4-(4-aminophenyl)butyric acid, 2-(4-aminophenylthio)acetic acid, DL-α-amino-2-thiopheneacetic acid, 5-aminovaleric acid, 8-benzyl (S)-2-aminooctanedioate, 4-(amino)-1-methylpyrrole-2-carboxylic acid, 4-(amino)tetrahydrothiopyran-4-carboxylic acid, (1R,3S,4S)-2-azabicyclo[2.2.1]Heptane-3-carboxylic acid, L-azetidine-2-carboxylic acid, azetidine-3-carboxylic acid, 4-(amino)piperidine-4-carboxylic acid, diaminoacetic acid, Inp-OH, (R)-Nip-OH, (S)-4-oxopiperidine-2-carboxylic acid, 2-(4-piperazino)-2-(4-fluorophenyl)acetic acid, 2-(4-piperazino)-2-phenylacetic acid, 4-piperidineacetaldehyde, 4-piperidylacetic acid, (-)-L-thioproline, Tle-OH, 3-piperidinecarboxylic acid, L -(+)-Canavanine, (±)-Carnitine, Chlorambucil, 2,6-Diaminopimelic Acid, Meso-2,3-Diaminosuccinic Acid, 4-(Dimethylamino)cinnamic Acid, 4-(Dimethylamino)phenylacetic Acid, (S)-N-Boc-Piperidine-3-carboxylate Ethyl, Piperazinoacetate Ethyl, 4-[2-(amino)ethyl]piperazin-1-ylacetic Acid, (R)-4-(Amino)-5-phenylpentanoic Acid, (S)-Azetidine-2-carboxylic Acid, Azetidine-3-carboxylic Acid, Guvacine, Inp-OH, (R)-Nip-OH, DL-Nip-OH, 4-phenyl-piperidine-4-carboxylic acid, 1-piperazineacetic acid, 4-piperidineacetic acid, (R)-piperidine-2-carboxylic acid, (S)-piperidine-2-carboxylic acid, (S)-1,2,3,4-tetrahydronorharman-3-carboxylic acid, Tic-OH, D-Tic-OH, iminodiacetic acid, indoline-2-carboxylic acid, DL-kynurenine, L-aziridine-2-carboxylate, methyl 4-aminobutyrate, (S)-2-piperazine carboxylic acid, 2-(1-piperazinyl)acetic acid, (R)-(-)-3-piperidinecarboxylic acid, 2-pyrrolidone-5-carboxylic acid, (R)-(+)-2-pyrrolidone-5-carboxylic acid, (R)-1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid, (S)-1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid, L-4-thiazolidinecarboxylic acid, (4R)-(-)-2-thioxo-4-thiazolidinecarboxylic acid, hydrazinoacetic acid, and 3,3',5-triiodo-L-thyronine. Each possibility represents a separate embodiment.

[0110] As used herein, the terms "polypeptide variant" and "polypeptide mutant" refer to a polypeptide comprising an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted relative to a parent polypeptide sequence. In various embodiments, the number of inserted, deleted, or substituted amino acid residues can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids in length. Variants of the present disclosure include fusion proteins.

[0111] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, e.g., conjugated to another chemical moiety, e.g., polyethylene glycol, albumin (e.g., human serum albumin), etc., phosphorylated, and glycosylated.

[0112] The term "tumor-associated antigen" (TAA) refers to a cell surface antigen that is selectively expressed by or overexpressed in cancer cells, e.g., compared to most normal cells. As used herein, the terms "TAA variant" and "TAA mutant" refer to a TAA that includes an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted relative to another TAA sequence.

[0113] The term "anti-TAA antagonist antibody" (interchangeably referred to as "anti-TAA antibody") refers to an antibody that can bind to a TAA and inhibit the biological activity of the TAA and / or downstream pathway(s) mediated by TAA signaling. As used herein, the term "antibody" refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as subtypes of these genes and the myriad immunoglobulin variable region genes. Light chains (LC) are classified as either kappa or lambda. Heavy chains (HC) are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. Antibodies can specifically bind to a particular antigen, e.g., a TAA.

[0114] As used herein, the terms "antigen-binding fragment" and "antigen-binding protein" refer to any protein that binds to a specific target antigen. "Antigen-binding fragment" includes, but is not limited to, antibodies and binding portions thereof, e.g., immunologically functional fragments. Exemplary antigen-binding fragments of antibodies are heavy chain CDR(s) and / or light chain CDR(s), or heavy chain variable region and / or light chain variable region.

[0115] Antibodies exist as intact immunoglobulins or as several well-characterized fragments. Such fragments include target antigen-binding Fab fragments, Fab' fragments, Fab2, F(ab)'2 fragments, single-chain Fv proteins ("scFv"), and disulfide-stabilized Fv proteins ("dsFv"). An scFv protein is a fusion protein in which an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region are joined by a linker, whereas in a dsFv, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. While various antibody fragments are defined in terms of the digestion of an intact antibody, those skilled in the art will appreciate that such fragments can be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, as used herein, the term antibody includes, for example, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single-chain Fvs (scFvs), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments exhibiting the desired biological activity, disulfide-linked Fvs (sdFvs), intrabodies, and epitope- or antigen-binding fragments of any of the above.

[0116] Bispecific antibodies or fragments can be in several configurations. For example, bispecific antibodies can resemble single antibodies (or antibody fragments) but have two different antigen-binding sites (variable regions). In various embodiments, bispecific antibodies of the present disclosure can have binding specificities for two different epitopes of one antigen or two separate antigens. In various embodiments, antibodies and fragments can also be heteroantibodies. Heteroantibodies are two or more antibodies, or antibody-binding fragments (e.g., Fabs), linked together, each antibody or fragment having a different specificity.

[0117] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies within the population are identical except for minor naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigen. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.

[0118] As used herein, the term "chimeric antibody" refers to an antibody having framework residues derived from one species, e.g., human, and CDRs (which generally confer antigen binding properties) derived from another species, e.g., a murine antibody, that specifically binds to a targeted antigen.

[0119] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs, and particularly CDR3, that are not encoded by human germline immunoglobulin sequences. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0120] As used herein, the term "humanized antibody" refers to an antibody comprising a humanized light chain immunoglobulin and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions with amino acids taken from the donor framework. A humanized or other monoclonal antibody may have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. In various embodiments, the framework region is constructed from human germline exon X. H , J H , V K , and J K Selected from an array. For example, V H The acceptor sequence for humanization of the FR of the domain is the genuine V H Exon V H 1-18 (Matsuda et al., Nature Genetics 3:88-94, 1993) or V H 1-2 (Shin et al., EMBO J. 10:3641-3645, 1991), and the hinge region (J H ) for Exon J H In another example, the germline V K Exon B3 (Cox et al., Eur. J. Immunol. 24:827-836, 1994) and J K Exxon J K -1 (Hieter et al., J. Biol. Chem. 257:1516-1522, 1982), but V L It can be chosen as an acceptor sequence for domain humanization.

[0121] Antigen-binding proteins, including antibodies, are at least 1 x 10 -6 M, or at least 1 × 10 -7 M, or at least 1 × 10 -8 M, or at least 1 × 10-9 M, or at least 1 × 10 -10 M, or at least 1 × 10 -11 Dissociation constant of M (K D An antigen-binding protein "specifically binds" to an antigen if it binds to the antigen with high binding affinity, as determined by its affinity (or corresponding Kb, defined below) value. An antigen-binding protein that specifically binds to a human antigen of interest may also bind to the same antigen of interest from other species with the same or different affinity. As used herein, "K D The term "antibody-antigen interaction" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0122] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in animals. A pharmaceutical composition contains a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. A "pharmacologically effective amount" refers to an amount of an agent effective to produce an intended pharmacological result. A "pharmaceutically acceptable carrier" refers to any of standard pharmaceutical carriers, vehicles, buffers, and excipients, such as phosphate-buffered saline, 5% aqueous dextrose, and emulsions such as oil / water or water / oil emulsions, as well as various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21st Ed. 2005, Mack Publishing Co., Easton. A "pharmaceutically acceptable salt" is a salt that can be formulated into a compound for pharmaceutical use, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0123] The terms "treat," "treating," and "treatment" refer to a method of alleviating or arresting at least one of a biological disorder and / or its attendant symptoms. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder, or condition. As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, attenuation of the extent of the disease, prevention or delay of disease spread (e.g., metastasis, e.g., to the lungs or lymph nodes), prevention or delay of disease recurrence, delay or slowing of disease progression, improvement of the disease state, and remission (partial or total). "Treatment" also encompasses alleviation of the pathological consequences of a proliferative disease. The methods of the present disclosure contemplate any one or more of these aspects of treatment.

[0124] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease, e.g., to improve, alleviate, relieve, and / or delay one or more of its symptoms. With respect to cancer or other unwanted cell proliferation, an effective amount includes an amount sufficient to (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, suppress, slow to some extent, and preferably stop cancer cell invasion into surrounding organs; (iv) inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor onset and / or recurrence; and / or (vii) alleviate to some extent one or more symptoms associated with cancer. An effective amount can be administered in one or more administrations.

[0125] The terms "patient," "individual," and "subject" may be used interchangeably and refer to a mammal, preferably a human or non-human primate, but also to domestic mammals (e.g., dogs or cats), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., horses, cows, pigs, sheep). The term "proliferative disease" includes neoplastic diseases (including benign or cancerous) and / or any metastasis. Proliferative diseases can include hyperproliferative conditions such as hyperplasia, fibrosis (particularly pulmonary, but also other types of fibrosis such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis, and smooth muscle proliferation within blood vessels, such as stenosis or restenosis after angioplasty. In some embodiments, the proliferative disease is cancer. In some embodiments, the proliferative disease is a non-cancerous disease. In some embodiments, the proliferative disease is a benign or malignant tumor.

[0126] A "linker" refers to a molecule that joins two other molecules, either by a covalent bond or through an ionic, van der Waals, or hydrogen bond. A linker may be cleavable or non-cleavable. A "cleavable linker" refers to a linker that can be degraded or otherwise cleaved to separate two components connected by the cleavable linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, etc. Cleavable linkers may also be cleaved by environmental triggers, such as, for example, changes in temperature, pH, salt concentration, etc.

[0127] In this application, the use of the singular includes the plural unless expressly stated otherwise. In this application, the use of "or" means "and / or" unless expressly stated otherwise. Furthermore, the use of the term "including," as well as other forms such as "includes" and "included," is not limiting. Also, terms such as "element" or "component" encompass both elements and components that contain one unit and elements and components that contain two or more subunits, unless expressly stated otherwise.

[0128] Camptothecin (CPT) derivatives In one aspect, the present disclosure provides novel camptothecin derivatives, pharmaceutically acceptable salts, stereoisomers, enantiomers, or deuterated forms thereof. CPT derivatives have the following structural formula (A): [Chemical formula] represented by wherein X is -C(=O)-(CH2)n 1 -O-N(R2)R3, -C(=O)-(CH2)n 1 -N(OR2)R3, -C(=O)-(CH2)n 1 -O-(CH2)n 1 -N(R2)R3, -C(=O)-(C(R4)(R5))n 1 -N(R2)R3, -S(=O)2-CH2-(CH2)n 1 -O-N(R2)R3, -S(=O)2-CH2-(CH2)n 1 -N(OR2)R3, -S(=O)2-CH2-(CH2)n 1 -N(R2)R3, -C(=O)-O-(CH2)n 2 -O-N(R2)R3, -C(=O)-O-(CH2)n 2 -N(OR2)R3, -C(=O)-O-(CH2)n 2 -N(R2)R3, -C(=O)-NH-(CH2)n 2 -O-N(R2)R3, -C(=O)-NH-(CH2)n 2 -N(OR2)R3, -C(=O)-N(R2)-(CH2)n 2 -N(R2)R3, -C(=O)-NH-(CH2)n 2 -O-R3, -C(=O)-NH-(CH2)n 2 -S-R3, -C(=O)-O-(CH2)n 2 -O-R3, -C(=O)-S-(CH2)n 2 -O-R3, -C(=O)-S-(CH2)n 2 -S-R3, -C(=O)-O-(CH2)n 2 -S-R3, -C(=O)-(C(R4)(R5))n 1 -N(R2)-C(=O)-(C(R a )(R b ))n[[ID=!5]] 3 -N(R c )-R3, -C(=O)-(C(R4)(R5))n 1 -N(R2)-C(=O)-(C(R It should be noted that there seems to be an incomplete or incorrect tag "!5" in the original text. This might need to be verified and corrected in the source material.a )(R b ))n 3 -O-R3, -C(=O)-(C(R4)(R5))n 1 -N(R2)-C(=O)-(C(R a )(R b ))n 3 -Selected from S-R3; R1, R2, R3, and R c are each independently a hydrogen atom or C1-C6 alkyl; R4 and R5 each independently represent a hydrogen atom, a deuterium atom, a halogen atom, a halogenated alkyl atom, a deuterated alkyl atom, an alkoxyl atom, a hydroxyl atom, an amino atom, a nitro group, a cyano group, a hydroxyalkyl group, a heterocyclic C1-C6 alkyl group, or a 3- to 6-membered heterocyclic or cycloaryl ring; or R4 and R5 together with the carbon atom form a 3- to 6-membered cyclic ring; R a and R b each independently represents a hydrogen atom, a deuterium atom, a halogen atom, a halogenated alkyl atom, a deuterated alkyl atom, an alkoxyl atom, a hydroxyl atom, an amino group, a nitro group, a cyano group, a hydroxyalkyl group, a heterocyclic C1-C6 alkyl group, or a 3- to 6-membered heterocyclic ring or a cycloaryl ring; or R a and R b together with the carbon atoms to form a 3- to 6-membered cyclic ring; n 1 is 1, 2, 3, 4 or 5; n 2 is 2, 3, 4 or 5; n 3 is 1, 2, 3, 4 or 5.

[0129] In some embodiments, the camptothecin derivative is represented by structural formula (A), wherein X is —C(═O)—(CH 2 ) n 1 -ON(R2)R3, -C(=O)-(CH2)n 1 -N(OR2)R3, -C(=O)-(CH2)n 1 -O-(CH2)n 1 -N(R2)R3, -C(=O)-(C(R4)(R5))n 1-N(R2)R3、-S(=O)2-CH2-(CH2)n 1 -ON(R2)R3、-S(=O)2-CH2-(CH2)n 1 -N(OR2)R3、-S(=O)2-CH2-(CH2)n 1 -N(R2)R3、-C(=O)-O-(CH2)n 2 -ON(R2)R3、-C(=O)-O-(CH2)n 2 -N(OR2)R3、-C(=O)-O-(CH2)n 2 -N(R2)R3、-C(=O)-NH-(CH2)n 2 -ON(R2)R3、-C(=O)-NH-(CH2)n 2 -N(OR2)R3、-C(=O)-N(R2)-(CH2)n 2 -N(R2)R3、-C(=O)-NH-(CH2)n 2 -O-R3、-C(=O)-NH-(CH2)n 2 -S-R3、-C(=O)-O-(CH2)n 2 -O-R3、-C(=O)-S-(CH2)n 2 -O-R3、-C(=O)-S-(CH2)n 2 -S-R3、-C(=O)-O-(CH2)n 2 -Selected from S-R3, Preferably、Xは、-C(=O)-(CH2)n 1 -ON(R2)R3、-C(=O)-(CH2)n 1 -O-(CH2)n 1 -N(R2)R3、-C(=O)-(C(R4)(R5))n 1 -N(R2)R3、-S(=O)2-CH2-(CH2)n 1 -ON(R2)R3、-S(=O)2-CH2-(CH2)n 1 -N(R2)R3、-C(=O)-O-(CH2)n 2 -ON(R2)R3、-C(=O)-O-(CH2)n 2 -N(R2)R3、-C(=O)-NH-(CH2)n 2 -ON(R2)R3、-C(=O)-N(R2)-(CH2)n 2 -N(R2)R3 is selected from; R1 is a hydrogen atom or a C1-C6 alkyl; R2 is a hydrogen atom; R3 is C1-C6 alkyl; R4 and R5 each independently represent a hydrogen atom, a deuterium atom, a halogen atom, hydroxyl, amino, nitro, or cyano.

[0130] In some embodiments, the camptothecin derivative is represented by structural formula (A), wherein X is —C(═O)—(CH 2 ) n 1 -ON(R2)R3, -C(=O)-(CH2)n 1 -N(OR2)R3, -C(=O)-(CH2)n 1 -O-(CH2)n 1 -N(R2)R3, -C(=O)-(C(R4)(R5))n 1 -N(R2)R3, -S(=O)2-CH2-(CH2)n 1 -ON(R2)R3, -S(=O)2-CH2-(CH2)n 1 -N(OR2)R3, -S(=O)2-CH2-(CH2)n 1 -N(R2)R3, -C(=O)-O-(CH2)n 2 -ON(R2)R3, -C(=O)-O-(CH2)n 2 -N(OR2)R3, -C(=O)-O-(CH2)n 2 -N(R2)R3, -C(=O)-NH-(CH2)n 2 -ON(R2)R3, -C(=O)-NH-(CH2)n 2 -N(OR2)R3, -C(=O)-N(R2)-(CH2)n 2 -N(R2)R3, -C(=O)-NH-(CH2)n 2 -O-R3, -C(=O)-NH-(CH2)n 2 -S-R3, -C(=O)-O-(CH2)n 2 -O-R3, -C(=O)-S-(CH2)n 2 -O-R3, -C(=O)-S-(CH2)n 2 -S-R3, -C(=O)-O-(CH2)n 2 -S-R3, preferably X is -C(=O)-(CH2)n 1-ON(R2)R3, -C(=O)-(CH2)n 1 -O-(CH2)n 1 -N(R2)R3, -C(=O)-(C(R4)(R5))n 1 -N(R2)R3, -S(=O)2-CH2-(CH2)n 1 -ON(R2)R3, -S(=O)2-CH2-(CH2)n 1 -N(R2)R3, -C(=O)-O-(CH2)n 2 -ON(R2)R3, -C(=O)-O-(CH2)n 2 -N(R2)R3, -C(=O)-NH-(CH2)n 2 -ON(R2)R3, -C(=O)-N(R2)-(CH2)n 2 - selected from N(R2)R3; R1 is a hydrogen atom or a C1-C6 alkyl; R2 is a hydrogen atom; R3 is C1-C6 alkyl; R4 and R5 each independently represent a hydrogen atom, a deuterium atom, a halogen atom, a halogenated alkyl atom, a deuterated alkyl atom, an alkoxyl atom, a hydroxyl atom, an amino atom, a nitro group, a cyano group, a hydroxyalkyl group, a heterocyclic C1-C6 alkyl group, or a 3- to 6-membered heterocyclic or cycloaryl ring; or R4 and R5 together with the carbon atom form a 3- to 6-membered cyclic ring;

[0131] In some embodiments, the camptothecin derivative is represented by structural formula (A), wherein X is —C(═O)—(CH 2 ) n 1 -ON(R2)R3, -C(=O)-(CH2)n 1 -N(OR2)R3, -C(=O)-(CH2)n 1 -O-(CH2)n 1 -N(R2)R3, -C(=O)-(C(R4)(R5))n 1 -N(R2)R3, -S(=O)2-CH2-(CH2)n 1 -ON(R2)R3, -S(=O)2-CH2-(CH2)n 1 -N(OR2)R3, -S(=O)2-CH2-(CH2)n 1-N(R2)R3, -C(=O)-O-(CH2)n 2 -ON(R2)R3, -C(=O)-O-(CH2)n 2 -N(OR2)R3, -C(=O)-O-(CH2)n 2 -N(R2)R3, -C(=O)-NH-(CH2)n 2 -ON(R2)R3, -C(=O)-NH-(CH2)n 2 -N(OR2)R3, -C(=O)-N(R2)-(CH2)n 2 -N(R2)R3, -C(=O)-NH-(CH2)n 2 -O-R3, -C(=O)-NH-(CH2)n 2 -S-R3, -C(=O)-O-(CH2)n 2 -O-R3, -C(=O)-S-(CH2)n 2 -O-R3, -C(=O)-S-(CH2)n 2 -S-R3, -C(=O)-O-(CH2)n 2 -S-R3, preferably X is -C(=O)-(CH2)n 1 -ON(R2)R3, -C(=O)-(CH2)n 1 -O-(CH2)n 1 -N(R2)R3, -C(=O)-(C(R4)(R5))n 1 -N(R2)R3, -S(=O)2-CH2-(CH2)n 1 -ON(R2)R3, -S(=O)2-CH2-(CH2)n 1 -N(R2)R3, -C(=O)-O-(CH2)n 2 -ON(R2)R3, -C(=O)-O-(CH2)n 2 -N(R2)R3, -C(=O)-NH-(CH2)n 2 -ON(R2)R3, -C(=O)-N(R2)-(CH2)n 2 - selected from N(R2)R3; R1 is a hydrogen atom or a C1-C6 alkyl; R2 and R3 are each a hydrogen atom; R4 and R5 each independently represent a hydrogen atom, a deuterium atom, a halogen atom, a halogenated alkyl atom, a deuterated alkyl atom, an alkoxyl atom, a hydroxyl atom, an amino atom, a nitro group, a cyano group, a hydroxyalkyl group, a heterocyclic C1-C6 alkyl group, or a 3- to 6-membered heterocyclic or cycloaryl ring; or R4 and R5 together with the carbon atom form a 3- to 6-membered cyclic ring;

[0132] In some embodiments, the camptothecin derivative is represented by structural formula (A), wherein X is —C(═O)—(C(R4)(R5))n 1 -N(R2)-C(=O)-(C(R a )(R b ))n 3 -N(R c )-R3, -C(=O)-(C(R4)(R5))n 1 -N(R2)-C(=O)-(C(R a )(R b ))n 3 -O-R3, -C(=O)-(C(R4)(R5))n 1 -N(R2)-C(=O)-(C(R a )(R b ))n 3 -S-R3, preferably X is -C(=O)-(C(R4)(R5))n 1 -N(R2)-C(=O)-(C(R a )(R b ))n 3 -N(R c )-R3, -C(=O)-(C(R4)(R5))n 1 -N(R2)-C(=O)-(C(R a )(R b ))n 3 - selected from O-R3; R1 is a hydrogen atom or a C1-C6 alkyl; R2, R3, and R c are each independently a hydrogen atom or a C1-C6 alkyl; R4 and R5 each independently represent a hydrogen atom, a deuterium atom, a halogen atom, a halogenated alkyl atom, a deuterated alkyl atom, an alkoxyl atom, a hydroxyl atom, an amino atom, a nitro group, a cyano group, a hydroxyalkyl group, a heterocyclic C1-C6 alkyl group, or a 3- to 6-membered heterocyclic or cycloaryl ring; or R4 and R5 together with the carbon atom form a 3- to 6-membered cyclic ring; R a and R b each independently represents a hydrogen atom, a deuterium atom, a halogen atom, a halogenated alkyl atom, a deuterated alkyl atom, an alkoxyl atom, a hydroxyl atom, an amino group, a nitro group, a cyano group, a hydroxyalkyl group, a heterocyclic C1-C6 alkyl group, or a 3- to 6-membered heterocyclic ring or a cycloaryl ring; or R a and R b together with the carbon atoms form a 3- to 6-membered cyclic ring.

[0133] In some embodiments, the camptothecin derivative is selected from any one of Table 1 below: [Table 1-1] [Table 1-2]

[0134] Camptothecin derivative conjugates In one aspect, the present disclosure provides camptothecin derivative conjugates, and pharmaceutically acceptable salts thereof, comprising at least one of the camptothecin derivatives disclosed herein, pharmaceutically acceptable salts, stereoisomers, enantiomers, and deuterated forms thereof.

[0135] In one aspect, the present disclosure provides camptothecin derivative conjugates, and pharmaceutically acceptable salts thereof, comprising (1) at least one of a camptothecin derivative disclosed herein, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form thereof, (2) a linker, and / or (3) a targeting agent.

[0136] In some embodiments, the linker is selected from at least one of the group consisting of cleavable and non-cleavable linkers, including, but not limited to, peptidase cathepsin-sensitive linkers, acid-sensitive linkers, glutathione-sensitive linkers, sulfatase-sensitive linkers, lysosomal protease-sensitive linkers, beta-glucuronide linkers, phosphatase-sensitive linkers, and pyrophosphatase-sensitive linkers.

[0137] In some embodiments, the linker attaches the targeting agent to one or more drug moieties (e.g., CPT derivatives) through a covalent bond(s). The linker is a bifunctional or polyfunctional moiety that can be used to link one or more drug moieties and a targeting agent to form a conjugate, e.g., a camptothecin derivative conjugate. The linker can be stable outside the cell, i.e., extracellularly, or can be cleavable by enzymatic activity, hydrolysis, or other metabolic conditions. CPT derivative conjugates can be conveniently prepared using a linker with a reactive functional group for attachment to the CPT derivative and the targeting agent. For example, a cysteine ​​thiol or amine, e.g., the N-terminus or an amino acid side chain, e.g., lysine, of the targeting agent can form a bond with the functional group of a linker reagent, a CPT derivative, or a CPT derivative-linker reagent.

[0138] The linker is preferably stable outside the target cell. Before being internalized into the cell, the CPT derivative conjugate is preferably stable and remains intact, i.e., the CPT derivative remains linked to the targeting agent. An effective linker (i) maintains the specific binding properties of the targeting agent; (ii) enables intracellular delivery of the conjugate or drug moiety; (iii) remains stable and intact, i.e., is not cleaved, until the conjugate is delivered to its target site; and (iv) maintains the cytotoxic, cell-killing, or cytostatic effect of the camptothecin derivative. The stability of the CPT derivative conjugate may be measured by standard analytical techniques, such as mass spectrometry, HPLC, and the separation / analysis technique LC / MS. The linker may be indestructible.

[0139] Covalent attachment of a targeting agent to a drug moiety, such as a CPT derivative, requires that the linker have two reactive functional groups. Bivalent linker reagents useful for attaching two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups, are known, and methods for obtaining such conjugates have been described (Hermanson, GT (1996) Bioconjugate Techniques; Academic Press: New York, p234-242).

[0140] In some embodiments, the linker can be substituted with a group that modulates solubility or reactivity. For example, a sulfonate substituent can increase the aqueous solubility of the reagent, and facilitate the coupling reaction between the linker reagent and the targeting agent or drug moiety, or between the targeting agent-linker and drug moiety, or between the drug moiety-linker and targeting agent, depending on the synthetic route used to prepare the CPT derivative conjugate.

[0141] In some embodiments, the linker has a reactive nucleophilic group that reacts with an electrophilic group present on a targeting agent, such as an antibody. Useful electrophilic groups on a targeting agent include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of the nucleophilic group on the linker can react with the electrophilic group on the targeting agent to form a covalent bond to the targeting agent. Useful nucleophilic groups on the linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. The electrophilic group on the targeting agent provides a convenient site for attachment to the linker.

[0142] Nucleophilic groups on targeting agents include, but are not limited to, (i) N-terminal amine groups, (ii) side-chain amine groups, such as lysine, (iii) side-chain thiol groups, such as cysteine, and (iv) sugar hydroxyl or amino groups when the targeting agent is glycosylated. Amine groups, thiol groups, and hydroxyl groups are nucleophilic and can react with electrophilic groups on linker moieties and linker reagents to form covalent bonds, including (i) active esters, such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl halides and benzyl halides, such as haloacetamides; and (iii) aldehydes, ketones, carboxyls, and maleimide groups. Certain targeting agents have reducible interchain disulfides, i.e., cysteine ​​bridges. Targeting agents can be made reactive for conjugation with linker reagents by treatment with a reducing agent, such as DTT (dithiothreitol). Each cysteine ​​bridge would thus theoretically form two reactive thiol nucleophilic species. Additional nucleophilic groups can be introduced into targeting agents by reacting lysines with 2-iminothiolane (Traut's reagent) to convert amines to thiols. Reactive thiol groups can be introduced into targeting agents, such as antibodies (or fragments thereof), by introducing one, two, three, four, or more cysteine ​​residues (e.g., preparing a mutant antibody containing one or more non-natural cysteine ​​amino acid residues). US2007 / 0092940 teaches engineering antibodies by introducing reactive cysteine ​​amino acids.

[0143] Nucleophilic groups on drug moieties, e.g., CPT derivatives, include, but are not limited to, the following: active esters, e.g., NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, e.g., haloacetamides; (iii) amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups, which can react with electrophilic groups on linker moieties and linker reagents, including aldehyde, ketone, carboxyl, and maleimide groups, to form covalent bonds.

[0144] The linker may be peptidic, comprising one or more amino acid units. Peptide linker reagents may be prepared by solid-phase or liquid-phase synthesis methods well known in the field of peptide chemistry (E. Schroder and K. Lubke, The Peptides, volume 1, pp. 76-136 (1965) Academic Press), including t-BOC chemistry (Geiser et al., "Automation of solid-phase peptide synthesis," in Macromolecular Sequencing and Synthesis, Alan R. Liss, Inc., 1988, pp. 199-218) and Fmoc / HBTU chemistry (Fields, G. and Noble, R. (1990) "Solid phase peptide synthesis utilizing 9-fluoroenylmethoxycarbonyl amino acids," Int. J. Peptide Protein Res. 35:161-214) using automated synthesizers, such as the Rainin Symphony peptide synthesizer (Protein Technologies, Inc., Tucson, AZ), or the Model 433 (Applied Biosystems, Foster City, OH). This includes operations on the Internet.

[0145] Exemplary amino acid linkers include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Exemplary dipeptides include valine-citrulline and alanine-phenylalanine. Exemplary tripeptides include glycine-valine-citrulline and glycine-glycine-glycine. Exemplary tetrapeptides include glycine-glycine-valine-citrulline and glycine-glycine-phenylalanine-citrulline. Amino acid residues comprising the amino acid linker component include naturally occurring amino acids as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. The amino acid linker component can be designed and optimized for selectivity for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0146] A targeting agent refers to any moiety that can bind to a specific target, including, but not limited to, an antibody or its antigen-binding fragment, a peptide, RNA, and a DNA molecule. In some embodiments, the CPT derivative conjugate of the present disclosure comprises a targeting agent selected from an antibody or its antigen-binding fragment, a peptide, RNA, and a DNA molecule.

[0147] In some embodiments, the targeting agent is capable of binding to a tumor-associated antigen (TAA), a tissue-specific antigen, a cell surface molecule, an extracellular matrix protein or protease(s), or any post-translationally modified residue(s). In various embodiments, the CPT derivative conjugates of the present disclosure comprise a targeting agent that exhibits binding affinity for diseased cells or tissues.

[0148] In some embodiments, the targeting agent is selected from the group consisting of Trop-2, Her2, Her3, Her4, EGF, EGFR, CD2, CD3, CD5, CD7, CD13, CD19, CD20, CD21, CD23, CD30, CD33, CD34, CD38, CD46, CD55, CD59, CD69, CD70, CD71, CD97, CD117, CD123, CD127, CD134, CD137, CD138, CD146, CD147, CD152, CD154, CD174, CD195, CD200, CD205, CD212, CD223, CD227, CD253, CD272, CD274, CD276, CD278, CD279, CD309, CD319, C The present invention is capable of binding to a tumor-associated antigen (TAA) selected from D326, CD340, DR6, Kv1.3, 5E10, MUC1, uPA, MAGE3, MUC16, KLK3, K-ras, mesothelin, p53, survivin, G250, PSMA, endoplasmin, BCMA, GPNMB, EphA2, EphB2, TMEFF2, integrin beta 6, 5T4, CA9, IGF-1R, Axl, B7H3, B7H4, CDH6, HAVCR1, STEAP-1, STEAP-2, UPK2, CLDN18, CLDN6, CLDN9, c-Met, MICA, LIV-1, ROR1, ADAM9, Stn, DLK-1, and CEACAM-5.

[0149] In preferred embodiments, the targeting agent is an antibody or binding fragment thereof, for example, an anti-Her2 antibody (eg, Herceptin) or a binding fragment thereof, or an anti-Trop-2 antibody or a binding fragment thereof.

[0150] In various embodiments, the CPT derivative conjugate comprises a TAA-binding antibody or antigen-binding fragment thereof selected from the group consisting of a fully human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, Fab, Fab', Fab2, Fab'2, IgG, IgM, IgA, IgE, scFv, dsFv, dAb, nanobody, unibody, and diabody. In various embodiments, the antibody is a chimeric antibody. In various embodiments, the antibody is a humanized monoclonal antibody. In various embodiments, the antibody is a fully human monoclonal antibody.

[0151] In various embodiments, the targeting agent is a multispecific antibody, such as a bispecific antibody.

[0152] In some embodiments, the camptothecin derivative conjugate has the following formula (I): Targeting Agents (LD) n (I) is represented by wherein n is an integer from 1 to 24; D represents at least one of the camptothecin derivatives disclosed herein, pharmaceutically acceptable salts, stereoisomers, enantiomers, and deuterated forms thereof; L is a group represented by the following formula (IIa and IIb): L 1 -L 2 -L 3 - (IIa) L 1 -L 2 - (IIb) a linker comprising a peptide portion of 2 to 8 amino acids represented by In the formula, L 1 is a linker moiety attached to the targeting agent, and L 1 comprises a reactive functional group selected from maleimide, bromoacetyl, iodoacetyl, thiol, amino, alkyl bromide, alkyl iodide, allenamide, carboxyl, and NHS ester; L 2is a linker moiety comprising a 2-8 amino acid peptide and, optionally, a spacer, preferably a PEG spacer; L 3 is a linker moiety connected to the camptothecin derivative, and L 3 is as follows: [ka] It includes at least one of the following:

[0153] In one embodiment, the reactive functional group is [ka] is.

[0154] In some embodiments, L 1 is -C(=O)-(CH2) n1 - and -C(=O)-(CH2)n 1 -R8-[O-(CH2) n1 ] n1 -N(R2)-C(=O)-)-(CH2) n1 -; R8 is alkylene-aryl-alkylene, aryl, 3- to 7-membered heterocyclyl, 3- to 7-membered cycloalkyl, heteroaryl, -alkylene-heteroaryl-alkylene-; n 1 is 1, 2, 3, 4 or 5.

[0155] In some embodiments, L is: [ka] [ka] is selected from one of In the formula, n=1 to 12, preferably 1 to 8.

[0156] In some embodiments, L 2is a dipeptide, tripeptide, or tetrapeptide containing naturally occurring and non-naturally occurring amino acids.

[0157] In some embodiments, L 2 is selected from gly-gly, gly-gly-gly, phe-lys, val-ala, val-cit, gly-gly-phe-gly (GGFG) (SEQ ID NO: 1), val-cit-gly, val-gln-gly, val-glu-gly, phe-lys-gly, glu-val-ala, glu-val-cit, β-ala-gly-phe-gly (AGFG) (SEQ ID NO: 2), and gly-gly-phe-gly-gly (GGFGG) (SEQ ID NO: 3), wherein the amino acid sequence is in either orientation.

[0158] In some embodiments, the camptothecin derivative conjugate has the following structure: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] Contains one of the following:

[0159] In some embodiments, the camptothecin derivative conjugate disclosed herein, or a pharmaceutically acceptable salt thereof, has a camptothecin derivative to targeting agent ratio of 4 to 12.

[0160] In some embodiments, a camptothecin derivative conjugate disclosed herein, or a pharmaceutically acceptable salt thereof, comprises any one of the following structures provided in Table 2: [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0161] According to some methods described herein, the CPT derivative conjugate or CPT derivative is internalized by targeted tumor cells or activated immune cells, where the CPT derivative conjugate or CPT derivative exerts a cytotoxic, cytostatic, or immunosuppressive effect on antigen-expressing cells to treat or prevent the recurrence of the antigen-expressing cancer or immune disorder. In certain embodiments, the CPT derivative conjugate or CPT derivative is not internalized, and the anti-target Ab is effective in depleting or inhibiting the target antigen-expressing cells by binding to the cell membrane. In certain embodiments, the CPT derivative conjugate or CPT derivative can target intracellular biomolecules (e.g., inflammatory agents) and accumulate in cells that secrete or bind the biomolecule or neighboring cells, where the therapeutic agent moiety exerts its effect (e.g., a cytotoxic, cytostatic, or immunosuppressive effect).

[0162] Importantly, the CPT derivative conjugates of the present disclosure have excellent drug / targeting agent ratios (e.g., DAR to ADC), improved solubility, enhanced CMC properties, and enhanced therapeutic efficacy against tumor cells with high antigen expression levels, while minimizing the impact on cells with low or no antigen expression levels, i.e., normal cells. Furthermore, the CPT derivative conjugates or CPT derivatives provide targeting for a broader patient population, including patients with refractory cancers or those who previously responded to treatment with anticancer therapy but experienced recurrence upon discontinuation of the therapy (hereinafter, "recurrent cancer").

[0163] In one aspect, the present disclosure provides a compound of formula (I'): Ab-(LD) n (I') or a pharmaceutically acceptable salt thereof; wherein n is an integer from about 1 to about 12; Ab is an antibody or antigen-binding fragment thereof; D is a camptothecin derivative; L is a bivalent linker. Camptothecin derivatives include:

Chem.

[0164] Thus, the drug moiety reagent has the following structure: [ka] [ka] [ka] [ka] Including, wherein R2 and Rc are each independently a hydrogen atom or a C1-C6 alkyl, and R3 and R4 are each independently a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, or a 3- to 6-membered heterocyclic or cycloaryl ring; or R3 and R4 together with the carbon atoms form a 3- to 6-membered cyclic ring, and Ra and R b are each independently a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, or a 3- to 6-membered heterocyclic or cycloaryl ring; or R a and R b together with the carbon atoms form a 3- to 6-membered group; n1 and n2 are each independently 0, 1, 2, 3, or 4.

[0165] Embodiments of the drug-linker reagent include: [ka] [ka] [ka] [ka] [ka] Contains, wherein R1 is H or C1-C6 alkyl and n is 0, 1, 2, 3, or 4; R2 and R3 are independently selected from hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, - an amino acid side chain selected from (CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl; R4 and R5 are each independently a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, or a 3- to 6-membered heterocyclic or cycloaryl ring; or R4 and R5 together with the carbon atoms form a 3- to 6-membered cyclic ring.

[0166] Preparation method The present disclosure provides a method of making a camptothecin derivative conjugate disclosed herein, or a pharmaceutically acceptable salt thereof, comprising reacting a targeting agent and / or a linker with a camptothecin derivative.

[0167] Pharmaceutical Composition The present disclosure provides pharmaceutical compositions comprising a camptothecin derivative disclosed herein, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form thereof, a camptothecin derivative conjugate disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier, or excipient.

[0168] Pharmaceutically acceptable excipients and carriers are well known and understood by those skilled in the art and are widely described (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack Publishing Company, 1990). Pharmaceutically acceptable carriers may be included for the purpose of modifying, maintaining, or preserving, for example, the pH value, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate or release rate, adsorption, or permeability of the composition. Such pharmaceutical compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the polypeptide.Suitable pharmaceutically acceptable carriers include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate, other organic acids); bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants; flavorings and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; and salt-forming counterions. (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., Pluronic®, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapar); stability enhancers (sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides (preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.

[0169] The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier can be water for injection, saline, or artificial cerebrospinal fluid, to which other ingredients common in compositions for parenteral administration may be added. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5, which may further contain sorbitol or a suitable substitute. In one embodiment of the present disclosure, a composition can be prepared for storage by mixing a selected composition having the desired purity with any formulation in the form of a lyophilized cake or aqueous solution (Remington's Pharmaceutical Sciences, supra). Additionally, therapeutic compositions can be formulated as lyophilizates using appropriate excipients, such as sucrose. The optimal pharmaceutical composition will be determined by one of skill in the art depending, for example, on the intended route of administration, delivery format, and desired dosage.

[0170] The pharmaceutical compositions of the present disclosure are typically suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any administration route characterized by physically disrupting a patient's tissue and administering the pharmaceutical composition through the tissue break, thus generally resulting in direct administration into the bloodstream, muscle, or internal organs. Thus, parenteral administration includes, but is not limited to, administering the pharmaceutical composition by injecting the composition, applying the composition through a surgical incision, applying the composition through a tissue-penetrating non-surgical wound, etc. In various embodiments, the pharmaceutical composition is formulated for parenteral administration via a route selected from, for example, subcutaneous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, intravenous injection, intraarterial injection, intrathecal injection, intraventricular injection, intraurethral injection, intracranial injection, intrasynovial injection, or via infusion.

[0171] When parenteral administration is intended, the therapeutic pharmaceutical composition may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired ADC in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the polypeptide is formulated as a sterile, isotonic solution and properly preserved. In various embodiments, pharmaceutical preparations suitable for injectable administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. In addition, suspensions of the active ingredient may be prepared as appropriate oily injection suspensions. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compound, allowing for the preparation of highly concentrated solutions. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as ampoules or multi-dose containers containing preservatives. Other useful parenteral formulations include those which contain the active ingredient in microcrystalline form or in a liposomal preparation. Formulations for parenteral administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

[0172] Any of the art-accepted methods for formulating and administering peptides, proteins, antibodies, and immunoconjugates may be suitably employed for administering the CPT derivative conjugates of the present disclosure.

[0173] Medicinal Use The present disclosure provides a method for treating or preventing a disease, the method comprising administering a therapeutically effective amount of a camptothecin derivative disclosed herein, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form thereof, a camptothecin derivative conjugate disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein to a subject in need of such treatment or prevention.

[0174] In some embodiments, the method of treating or preventing a disease further comprises administering a secondary therapy to the subject.

[0175] When a camptothecin derivative disclosed herein, a pharmaceutically acceptable salt thereof, stereoisomer, enantiomer, or deuterated form thereof, a camptothecin derivative conjugate disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein is administered in combination with a second-line therapy, the second-line therapy is selected from the group consisting of cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, stem cell transplantation, cell therapy such as CAR-T, CAR-NK, iPS-induced CAR-T, or iPS-induced CAR-NK, and a vaccine such as Bacillus Calmette-Guerin (BCG). In various embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of immunotherapy, including, but not limited to, treatment with depleting antibodies against specific tumor antigens; treatment with antibody-drug conjugates; agonistic, antagonistic, or blocking antibodies against costimulatory or co-inhibitory molecules (immune checkpoints), such as CTLA-4, PD-1, PD-L1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, Siglec7, Siglec8, Siglec9, Siglec15, and VISTA. treatment with a bispecific T cell-engaging antibody (BiTE®) such as blinatumomab; treatment involving administration of biological response modifiers such as IL-12, IL-21, GM-CSF, IFN-alpha, IFN-β, and IFN-γ; treatment with a therapeutic vaccine such as sipuleucel-T; treatment with a dendritic cell vaccine, or a tumor antigen peptide vaccine; treatment with chimeric antigen receptor (CAR)-T cells; treatment with CAR-NK cells; treatment with tumor-infiltrating lymphocytes (TILs); treatment with adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR transgenic); treatment with TALL-104 cells; and treatment with immunostimulants such as the Toll-like receptor (TLR) agonists CpG and imiquimod; and treatment with a vaccine such as BCG, which combination therapy optionally provides enhanced effector cell killing of tumor cells, i.e., a synergistic effect is achieved between the ADC construct and the immunotherapy when co-administered.

[0176] When a camptothecin derivative disclosed herein, a pharmaceutically acceptable salt thereof, stereoisomer, enantiomer, or deuterated form thereof, a camptothecin derivative conjugate disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein is administered with a second-line therapy, the administration may be simultaneous or sequential.

[0177] A list of exemplary chemotherapeutic agents includes, but is not limited to, daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, bendamustine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, tenox, thiazolinone ... These include cyclophosphide, cisplatin, carboplatin, oxaliplatin, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fluradabin, ifosfamide, hydroxyurea taxanes (such as paclitaxel and doxitaxel) and / or anthracycline antibiotics, as well as combinations of drugs such as, but not limited to, DA-EPOCH, CHOP, CVP or FOLFOX.

[0178] In some embodiments, the disease is selected from any one of the following: a proliferative disorder, an autoimmune disorder, a destructive bone disorder, an infectious disease, a viral disease, a fibrotic disease, a neurodegenerative disorder, pancreatitis, or a kidney disease.

[0179] In some embodiments, the proliferative disorder is cancer, preferably a cancer selected from the group consisting of pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, and rhabdomyosarcoma.

[0180] The present disclosure provides the use of a camptothecin derivative disclosed herein, a pharmaceutically acceptable salt thereof, a stereoisomer, an enantiomer, or a deuterated form thereof, a camptothecin derivative conjugate disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein in the preparation of a medicament in the treatment or prevention of disease.

[0181] The present disclosure provides a camptothecin derivative disclosed herein, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form thereof, a camptothecin derivative conjugate disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein, for use in the preparation of a medicament for the treatment or prevention of a disease.

[0182] In one aspect, the present disclosure relates to a method of treating a proliferative disease (e.g., cancer) in an individual, the method comprising administering to the individual a therapeutically effective amount of a camptothecin derivative disclosed herein, a pharmaceutically acceptable salt thereof, a stereoisomer, an enantiomer, or a deuterated form thereof, a camptothecin derivative conjugate disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein. In some embodiments, the cancer includes recurrent, resistant, or refractory cancer at surprisingly low doses.

[0183] Examples of tumor cell lines derived from human tumors and available for use in in vitro and in vivo testing include leukemia cell lines (e.g., CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPM1-8226, SR, P388 and P388 / ADR, H292, MV-4-11); non-small cell lung cancer cell lines (e.g., A549 / ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-H522, and and LXFL529); small cell lung cancer cell lines (e.g., DMS114 and SHP-77); colon cancer cell lines (e.g., COLO205, HCC-2998, HCT-116, HCT-15, HT29, KM12, SW-620, DLD-1, and KM20L2); central nervous system (CNS) cancer cell lines (e.g., SF-268, SF-295, SF-539, SNB-19, SNB-75, U251, SNB-78, and XF498); melanoma cell lines (e.g., LOX I, MVI, MALME-3M, M14, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62, RPMI-7951, and M19-MEL; ovarian cancer cell lines (e.g., IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, and SK-OV-3); kidney cancer cell lines (e.g., 786-0, A498, ACHN, CAKI-1, RXF393, SN1 2C, TK-10, UO-31, RXF-631 and SN12K1); prostate cancer cell lines (e.g., PC-3 and DU-145); breast cancer cell lines (e.g., MCF7, NCI / ADR-RES, MDA-MB-231 / ATCC, HS578T, MDA-MB-435, BT-549, T-47D and MDA-MB-468); and thyroid cancer cell lines (e.g., SK-N-SH).

[0184] In various embodiments, the methods described herein may be used in combination with other conventional anti-cancer therapeutic approaches for the treatment or prevention of proliferative disorders, including, but not limited to, chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, and stem cell transplantation. For example, such methods can be used for prior cancer prevention, for preventing cancer recurrence and metastasis after surgery, and also as an adjuvant to other conventional cancer therapies. The present disclosure recognizes that the effectiveness of conventional cancer therapies (e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced through the use of the fusion molecules described herein.

[0185] Target Antigens and Exemplary Antibodies Tumor antigens expressed on cell membranes are potential targets in immunotherapy, and ideal tumor antigens are not present on normal cells and are overexpressed on tumor cell surface.The CPT derivative conjugates used in the method of the present disclosure can include targeting agents specific to any tumor-associated antigens described in the art, such as antibodies, or antigen-binding antibody fragments, multispecific antibodies (e.g., bispecific antibodies), including any biosimilar, biogeneric, subsequent biological, or subsequent protein version of any TAA described in the art.TAA can be any peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate, or small organic molecule, or any combination thereof, that those skilled in the art want to induce immune response.

[0186] In various embodiments, the TAA, TAA variant, or TAA mutant contemplated for use in the combination methods of the present disclosure is selected from or derived from the list provided in Table 3. [Table 3-1] [Table 3-2]

[0187] In various embodiments, the TAA has an amino acid sequence that shares, e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% homology with any one of the sequences disclosed in Table 3.

[0188] In various embodiments, the CPT derivative conjugates of the present disclosure utilize targeting agents, such as antibodies or antigen-binding fragments thereof, that are polyclonal antibodies, monoclonal antibodies or antigen-binding fragments thereof, recombinant antibodies, diabodies, chimerized or chimeric antibodies or antigen-binding fragments thereof, humanized antibodies or antigen-binding fragments thereof, fully human antibodies or antigen-binding fragments thereof, CDR-grafted antibodies or antigen-binding fragments thereof, single-chain antibodies, Fv, Fd, Fab, Fab', or F(ab')2, and synthetic or semi-synthetic antibodies. The antibodies or antigen-binding fragments thereof can be monospecific (specifically binding to one antigen), bispecific (specifically binding to two antigens), or multispecific (specifically binding to more than two antigens).

[0189] In various embodiments, antibodies contemplated for use in the CPT derivative conjugates of the present disclosure include LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20, anti-CD20), rituximab (anti-CD20), obinituzumab (GA101, anti-CD20), lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS7 (anti-epithelial glycoprotein-1 (EGP-1, also known as TROP-2)), PAM4 or KC4 (both anti-mucins), MN-14 (anti-carcinoembryonic antigen (CEA, also known as CD66e or CEACAM5)), MN-15 or MN-3 (anti-CEACAM6), MU-9 (anti-colon specific antigen p), Immunoglobulin G (IMG)-1 (anti-IL-1), Immunoglobulin G ... 31 (anti-alpha-fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19), TAG-72 (e.g., CC49), Tn, J591, or HuJ591 (anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026 (anti-PSMA dimer), D2 / B (anti-PSMA), G250 (anti-carbonic anhydrase IX) MAb), L243 (anti-HLA-DR), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20); panitumumab (anti-EGFR); tositumomab (anti-CD20); PAM4 (also known as clivatuzumab, anti-mucin); trastuzumab (anti-ErbB2); and anti-CTLA4 antibodies, such as ipilimumab (Bristol-Myers Squibb) and tremelimumab (PFIZER).Such antibodies are known in the art (e.g., U.S. Patent Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730,300; 6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,230,085). Nos. 8,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491; 7,612,180; 7,642,239; and U.S. Patent Application Publication Nos. 20050271671; 20060193865; 20060210475; and 20070087001; see the Examples section of each. (The disclosures of which are incorporated herein by reference in their entirety.) Specific known antibodies that may be used include hPAM4 (U.S. Patent No. 7,282,567), hA20 (U.S. Patent No. 7,251,164), hA19 (U.S. Patent No. 7,109,304), hIMMU-31 (U.S. Patent No. 7,300,655), hLL1 (U.S. Patent No. 7,312,318), hLL2 (U.S. Patent No. 7,074,403), hMu-9 (U.S. Patent No. 7,387,773), and the like. ), hL243 (U.S. Patent No. 7,612,180), hMN-14 (U.S. Patent No. 6,676,924), hMN-15 (U.S. Patent No. 7,541,440), hR1 (U.S. Patent Application Serial No. 12 / 772,645), hRS7 (U.S. Patent No. 7,238,785), hMN-3 (U.S. Patent No. 7,541,440), AB-PG1-XG1-026 (U.S. Patent Application Serial No. 11 / 983,372, which has been deposited as ATCC PTA-4405 and PTA-4406), and D2 / B (WO2009 / 130575), the text of each listed patent or application being incorporated herein by reference.

[0190] In certain embodiments, the CPT derivative conjugates of the present disclosure comprise at least one antibody or fragment thereof that binds to Her2.

[0191] Bispecific antibodies or fragments can be of several configurations: for example, they can resemble a single antibody (or antibody fragment), but have two different antigen-binding sites (variable regions).

[0192] In various embodiments, bispecific antibodies of the present disclosure can have binding specificities for two different epitopes, at least one of which is a tumor-associated antigen. In various embodiments, antibodies and fragments can also be heteroantibodies. Heteroantibodies are two or more antibodies, or antibody-binding fragments (e.g., Fabs) linked together, each antibody or fragment having a different specificity. [Example]

[0193] The following examples are provided to more fully illustrate the present disclosure, but are not to be construed as limiting its scope.

[0194] Example 1. Synthesis of Compound 12 (363-11) [ka] Step 1. To a solution of sarcosine (0.9 g, 10 mmol) in THF (10 mL) and water (10 mL) was added K2CO3 (2.07 g, 15 mmol) and allyl chloroformate (1.45 g, 12 mmol). The mixture was stirred for 2-3 hours. 1N HCl solution was added to the mixture to acidify it to pH = 6. The resulting mixture was concentrated in vacuo to give a residue, which was dissolved in DCM / MeOH (v / v = 3:1, 20 mL). The mixture was filtered and evaporated to give crude compound 1.

[0195] [ka] Step 2. HATU (72 mg, 0.45 mmol) was added to a solution of exatecan mesylate (20 mg, 0.38 mmol) and compound 1 (130 mg, 0.75 mmol) in DMF (8 mL). DIPEA (130 μL, 0.75 mmol) was added to the mixture and stirred at room temperature for 1 h. The mixture was diluted with DCM and washed with aqueous sodium bicarbonate and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated by evaporation to give a residue, which was purified by flash chromatography on silica gel (eluent: MeOH / DCM = 0-10%) to give the desired compound 2 (180 mg, 81%).

[0196] [ka] Step 3. To a solution of compound 2 (180 mg, 0.30 mmol) in DCM (10 mL) was added Pd(PPh3)4 (17.6 mg, 0.015 mmol) and pyrrolidine (44 mg, 0.61 mmol). The mixture was stirred for 20 min. The reaction was quenched with 5 mL of brine, and the mixture was extracted with DCM (2 × 10 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by HPLC (eluent: ACN / HO = 5 to 80% for 25 min) to give the desired compound 3 (T-363) (40 mg, 26%) as a solid.

[0197] [ka] Step 4. HATU (34 mg, 0.089 mmol) was added to a solution of 3 (30 mg, 0.059 mmol) and N-Fmoc-glycine (35 mg, 0.118 mmol) in DMF (5 mL). DIPEA (15 mg, 0.118 mmol) was added to the mixture and stirred at room temperature for 1 h. The mixture was loaded onto an HPLC column for purification (eluent: ACN / HO = 5-80% for 25 min) to give the desired compound 4 (35 mg, 75%). LRMS: m / z = 786.3 [M+1] + . Molecular formula:C 44 H 40FN5O8; molecular weight: 785.81.

[0198] [ka] Step 5. To a solution of compound 4 (35 mg, 0.044 mmol) in DMF (5 mL), piperidine (1 mL) was added. The mixture was stirred for 10 min and evaporated to give a residue, which was purified by HPLC (eluent: ACN / HO = 5-80% for 25 min) to give the desired compound 5 (20 mg, 80%). LRMS: m / z = 564.2 [M+1] + . Molecular formula:C 29 H 30 FN5O6; molecular weight: 563.58.

[0199] [ka] Step 6. To a solution of 6-maleimidohexanoic acid (2.11 g, 10 mmol) in DMF (20 mL) was added tert-butyl glycinate (1.31 g, 10 mmol), HATU (4.56 g, 12 mmol), and DIPEA (3.51 mL, 20 mmol). The mixture was stirred for 2 h. The mixture was diluted with ethyl acetate (100 mL) and washed with NaHCO3 solution (2 × 10 mL), water (3 × 10 mL), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by silica gel chromatography (eluent: MeOH / DCM = 3-10%) to give the desired compound 6 (1.8 g, 56%).

[0200] [ka] Step 7. To a solution of compound 6 (1.8 g, 3.08 mmol) in DCM (10 mL) was added TFA (10 mL). The mixture was stirred for 2 hours. The volatiles were removed in vacuo, and the resulting residue was dissolved in 1 mL of ethyl acetate. Methyl tert-butyl ether was added to precipitate the product. The solid was filtered and washed with some methyl tert-butyl ether to give compound 7 (1 g, 68%) as a white solid.

[0201] [ka] Step 8. To a solution of compound 7 (1 g, 3.73 mmol) in DMF (10 mL) was added tert-butyl glycinate (0.5 g, 3.73 mmol), HATU (1.7 g, 4.47 mmol), and DIPEA (1.30 mL, 7.46 mmol). The mixture was stirred for 2 hours. The mixture was diluted with ethyl acetate (100 mL) and washed with NaHCO3 solution (2 × 10 mL), water (3 × 10 mL), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give crude compound 8.

[0202] [ka] Step 9. To a solution of crude compound 8 in DCM (10 mL) was added TFA (10 mL). The mixture was stirred for 2 hours. The volatiles were removed in vacuo, and the resulting residue was dissolved in 1 mL of ethyl acetate. Methyl tert-butyl ether was added to precipitate the product. The solid was filtered and washed with some methyl tert-butyl ether to give compound 9 (0.5 g, 42% from 7) as a white solid.

[0203] [ka] Step 10. To a solution of compound 9 (0.5 g, 1.53 mmol) in DMF (15 mL) was added HL-Phe-OtBu (0.4 g, 1.53 mmol), HATU (0.87 g, 2.30 mmol), and DIPEA (0.49 g, 3.84 mmol). The mixture was stirred for 2 h. The mixture was diluted with ethyl acetate (60 mL) and washed with NaHCO solution (2 × 50 mL), water (20 mL), and brine. The organic layer was dried over anhydrous NaSO, filtered, and evaporated to give a residue, which was purified by flash chromatography on silica gel (eluent: EtOAc / hexane = 5-50%) to give the title compound 10 (0.4 g, 49%).

[0204] [ka] Step 11. To a solution of compound 10 (0.4 g, 0.75 mmol) in DCM (20 mL) was added TFA (10 mL). The mixture was stirred for 3 hours. The volatiles were removed in vacuo, and the resulting residue was dissolved in 1 mL of DCM. Ether was added to precipitate the product. The solid was filtered and washed with ether to give compound 11 (0.15 g, 42%) as a white solid. LRMS: m / z=473.22 [M+1] + . Molecular formula:C 23 H 28 N4O7; molecular weight: 472.49.

[0205] [ka] Step 12. HATU (20 mg, 0.053 mmol) was added to a solution of 5 (20 mg, 0.035 mmol) and 11 (34 mg, 0.071 mmol) in DMF (3 mL). DIPEA (12 μL, 0.071 mmol) was added to the mixture and stirred at room temperature for 1 h. The mixture was loaded onto an HPLC column for purification (eluent: ACN / HO = 5-80% for 25 min) to give the title compound 12 (363-11) (12 mg, 34%). LRMS: m / z = 1018.4 [M+1] + . Chemical formula:C 52 H 56 FN9O 12 ;Molecular weight: 1018.05.

[0206] Example 2: Synthesis of Compound 17 [ka] Step 1. To a solution of exatecan methanesulfonate (90 mg, 0.17 mmol) in dichloromethane (10 mL) were added triethylamine (51.6 mg, 0.51 mmol) and di-tert-butyl 114 dicarbonate (55.6 mg, 0.255 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with DCM (15 mL), washed with water and brine, dried over anhydrous Na2SO4, and concentrated in vacuo to give crude compound 13 (189 mg) as a white solid, which was used in the next step without further purification. LRMS: m / z = 536.30 [M+H] + . Chemical formula:C 29 H 30 FN3O6; molecular weight: 535.57.

[0207] [ka] Step 2. N-acetylcysteine ​​of compound 13 (60 mg, 0.112 mmol) 1 ,N 1 A solution of 1,2-dimethylethane-1,2-diamine (1 mL) was stirred at 50° C. for 0.5 h. The reaction mixture was concentrated in vacuo to give crude compound 14 (75 mg, crude) as a yellow oil, which was used in the next step without further purification. LRMS: m / z=624.60 [M+H] + . Chemical formula:C 33 H 42 FN5O6; molecular weight: 623.73.

[0208] [ka] Step 3. To a solution of crude compound 14 (75 mg, 0.112 mmol) in pyridine (2 mL) was added propionic anhydride (29 mg, 0.224 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo to give a crude residue, which was diluted with ethyl acetate (30 mL), washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to give crude compound 15 (61.9 mg) as a yellow solid, which was used in the next step without further purification. LRMS: m / z=680.70 [M+H] + . Chemical formula:C 36 H 46 FN5O7; molecular weight: 679.79.

[0209] [ka] Step 4. To a solution of crude compound 15 (30 mg, 0.044 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (TFA, 215 mg, 1.88 mmol), and the resulting mixture was stirred at room temperature for 2 hours. TLC showed that the starting material had completely disappeared. The reaction mixture was concentrated in vacuo to give crude compound 16 (40 mg) as a yellow oil, which was used in the next step without further purification. LRMS: m / z=580.35 [M+H] + . Chemical formula:C 31 H 38 FN5O5; molecular weight: 579.67.

[0210] [ka] Step 5. To a solution of crude 16 (40 mg, 0.044 mmol) and DIPEA (17 mg, 0.132 mmol) in DMF (1.5 mL) was added 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate (25 mg, 0.033 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo at 28 °C to remove DMF. The residue was dissolved in EtOAc (20 mL) and washed with water and brine. The aqueous layer was extracted with a mixture of MeOH in DCM (v / v = 10%, 30 mL × 5), and the combined organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by preparative TLC (DCM / MeOH, 10 / 1) to give a crude material, which was further purified by preparative HPLC (eluent: ACN / HO = 5-80% over 25 min) to give the title compound 17 (1.6 mg, 4.1% yield) as a yellow solid. LRMS: m / z = 1179.1 [M+H] + . Chemical formula:C 60 H 76 FN 11 O 13 ;Molecular weight: 1178.33.

[0211] Example 3: Synthesis of Compound 21 [ka] Step 1. A solution of compound 13 (189 mg, 0.35 mmol) in 2-methoxyethan-1-amine (2 mL) was stirred at 50° C. for 0.5 hours. The reaction mixture was concentrated in vacuo to give crude compound 18 (230 mg) as a yellow solid, which was used in the next step without further purification. LRMS: m / z=611.3 [M+H] + . Chemical formula:C 32 H 39 FN4O7; molecular weight: 610.68.

[0212] [ka] Step 2. To a solution of compound 18 (230 mg, 0.17 mmol) in pyridine (1.5 mL) was added acetic anhydride (AcO, 45 mg, 0.44 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated at 28 °C. The residue was diluted with DCM (30 mL), washed with water and brine, dried over anhydrous NaSO, and concentrated in vacuo to give a crude material, which was purified via silica gel flash column chromatography (PE / EtOAc, 1 / 1 to 1 / 3, then DCM / MeOH = 50 / 1) to give the title compound 19 (96.3 mg, 86%) as a yellow solid. LRMS: m / z = 653.35 [M+H] + . Chemical formula:C 34 H 41 FN4O8; molecular weight: 652.72.

[0213] [ka] Step 3. To a solution of compound 19 (20 mg, 0.03 mmol) in DCM (2 mL) was added trifluoroacetic acid (TFA, 137 mg, 0.12 mmol), and the resulting mixture was stirred at room temperature for 4 hours. TLC showed that the starting material had completely disappeared. The mixture was concentrated in vacuo to give crude compound 20 (30 mg) as a yellow oil, which was used in the next step without further purification. LRMS: m / z=553.30 [M+H] + . Chemical formula:C 29 H 33 FN4O6; molecular weight: 552.60.

[0214] [ka] Step 4. To a solution of compound 20 (27.5 mg, 0.05 mmol) and DIPEA (195 mg, 1.5 mmol) in DMF (2 mL) was added 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-reidopentanamido)benzyl(4-nitrophenyl)carbonate (21 mg, 0.028 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo to remove DMF (29 °C). The crude was dissolved in half-brine (20 mL) and extracted with a mixture of methanol in DCM (10%, 15 mL × 6). The combined organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The crude was purified by preparative TLC (DCM / MeOH=10 / 1) to give the product, which was further purified by preparative HPLC (eluent: ACN / HO=5-80% over 25 min) to give the title compound 21 (4.0 mg, 11.5%) as a yellow solid. LRMS: m / z=1151.60 [M+H] + . Molecular formula:C 58 H 71 FN 10 O 14 ;Molecular weight: 1151.24.

[0215] Example 4: Synthesis of Compound 30 [ka] Step 1. To a solution of 2-hydroxybenzyl acetate (1.1 g, 6.62 mmol) in DMF (15 mL) was added imidazole (1.35 g, 19.86 mmol) and tert-butyldimethylsilyl chloride (TBSCl) (1.1 g, 7.28 mmol), and the resulting mixture was stirred at room temperature for 2 h. The reaction mixture was poured into water (110 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with saturated aqueous ammonium chloride solution, dried over anhydrous Na2SO4, and concentrated to give the crude product, which was purified via silica gel flash column chromatography (EtOAc / petroleum ether (PE) = 0% to 10%) to give compound 22 (1.75 g, 94%) as a colorless oil. 1H NMR (400 MHz, CDCl3): δ7.37-7.31 (m, 5H), 5.18 (s, 2H), 4.29 (s, 2H), 0.91 (s, 9H), 0.096 (s, 6H) ppm.

[0216] [ka] Step 2. A mixture of compound 22 (1.75 g, 6.24 mmol) and palladium on carbon (10%, 525 mg) in methanol (30 mL) was stirred overnight at room temperature under a hydrogen atmosphere. The catalyst was removed by filtration, and the filtrate was concentrated to give the crude product, which was purified by silica gel flash column chromatography (hexane / EtOAc = 3 / 1) to give the title compound 23 (197 mg, 16.5%) as a colorless oil. 1 H NMR (400 MHz, CDCl3): δ8.77 (brs, 1H), 4.23 (s, 2H), 0.93 (s, 9H), 0.14 (s, 6H) ppm.

[0217] [ka] Step 3. To a solution of exatecan mesylate (120 mg, 0.225 mmol) and compound 23 (128 mg, 0.675 mmol) in DMF (15 mL) was added DIPEA (175 mg, 1.35 mmol) and PyBOP (234 mg, 0.45 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (40 mL × 4). The combined organic layer was washed with brine, dried over anhydrous NaSO, and concentrated in vacuo to give crude compound 24 (349 mg, impure) as a pale yellow solid. LRMS: m / z = 608.20 [M+H] + . Chemical formula:C 32 H 38 FN3O6Si; molecular weight: 607.75.

[0218] [ka] Step 4. A solution of compound 24 (122 mg, 0.2 mmol) in 2-methoxyethan-1-amine (2 mL) was stirred at 50° C. for 1 hour. The reaction mixture was concentrated in vacuo to give crude compound 25 (145 mg) as a yellow oil, which was used in the next step without further purification. LRMS: m / z=683.30 [M+H] + . Chemical formula:C 35 H47FN4O7Si; molecular weight: 682.87.

[0219] [ka] Step 5. To a solution of crude compound 25 (145 mg, 0.2 mmol) in dichloromethane (11 mL) was added 4-dimethylaminopyridine (DMAP, 156 mg, 1.27 mmol) and tert-butyl (2-((chlorocarbonyl)(methyl)amino)ethyl)(methyl)carbamate (213 mg, 0.85 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo to give a crude residue, which was purified via silica gel flash column chromatography (PE / EtOAc = 1 / 1, then DCM / MeOH, 50 / 1 to 30 / 1) to give compound 26 (60 mg, 31.5%) as a yellow solid. LRMS: m / z = 897.45 [M+H] + . Chemical formula:C 45 H 65 FN6O 10 Si; molecular weight: 897.13.

[0220] [ka] Step 6. To a solution of compound 26 (48 mg, 0.054 mmol) in DCM (4 mL) was added tetrabutylammonium fluoride (TBAF, 1 M in tetrahydrofuran, 162 μL, 0.162 mmol) and acetic acid (13 mg, 0.216 mmol), and the resulting mixture was stirred at room temperature for 4 hours. TLC showed that the starting material had completely disappeared. The reaction mixture was used in the next step without further processing. LRMS: (ES+ ): m / z=783.3[M+H] + . Chemical formula:C 39 H 51 FN6O 10 ;Molecular weight: 782.37.

[0221] [ka] Step 7. To a solution of compound 28 in DCM (crude solution from the previous step, 0.054 mmol), trifluoroacetic acid (TFA, 0.1 mL) was added, and the resulting mixture was stirred at room temperature for 2 hours. TLC showed that the starting material had completely disappeared. The mixture was concentrated in vacuo to give crude compound 29 as a yellow oil, which was used in the next step without further purification. LRMS: m / z=683.25 [M+H] + . Chemical formula:C 34 H 43 FN6O8; molecular weight: 682.75.

[0222] [ka] Step 8. To a solution of crude compound 29 (200 mg) and DIPEA (156 mg, 1.21 mmol) in DMF (2 mL) was added 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate (32 mg, 0.043 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo at 28 °C to remove DMF to give the crude product, which was dissolved in DCM (20 mL), washed with water and brine, dried over anhydrous NaSO, and concentrated. The crude residue was purified by preparative TLC (DCM / MeOH = 10 / 1) to give an impure product, which was further purified by preparative HPLC (eluent: ACN / HO = 5-80% over 25 min) to give the title compound 30 (3.1 mg, 5.6% yield) as a yellow solid. LRMS: m / z = 1281.65 [M+H] + . Chemical formula:C63 H 81 FN 12 O 16 ;Molecular weight: 1281.41.

[0223] Example 5: Synthesis of Compound 43 [ka] Step 1. To a solution of Fmoc-Ala-OH (3.11 g, 10 mmol) in DMF (15 mL) was added H-Gly-OtBu (1.31 g, 10 mmol), HATU (5.7 g, 15 mmol), and DIPEA (2.58 g, 20 mmol). The mixture was stirred for 2 h. The mixture was diluted with DCM (200 mL) and washed with NaHCO3 solution (2 × 50 mL), water (20 mL × 2), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by flash chromatography on silica gel (eluent: EtOAc / hexane = 10-50%) to give compound 31 (3.1 g, 73%).

[0224] [ka] Step 2. To a solution of compound 31 (3.1 g, 7.3 mmol) in DCM (7.5 mL) was added TFA (7.5 mL). The mixture was stirred for 4 hours. The mixture was evaporated to give an oil, which was precipitated with ether. The solid was filtered and washed with some ether. The white solid was further dried under high vacuum to give compound 32 (2.2 g, 82%).

[0225] [ka] Step 3. To a solution of compound 32 (2.2 g, 6 mmol) in DMF (40 mL) was added Cu(OAc) (0.40 g, 2.2 mmol), HOAc (0.77 mL, 13.6 mmol), and Pb(OAc) (3.0 g, 6.8 mmol). The resulting mixture was heated to 60 °C for 15 min and then cooled to room temperature. The mixture was diluted with EtOAc (80 mL) and washed with water (3 × 50 mL) and brine. The mixture was dried over anhydrous NaSO, filtered, and evaporated to give a residue that was passed through a column (eluent: EtOAc / n-heptane = 10-75%) to give compound 33 (1.4 g, 61.3%). LRMS: m / z = 383.1 [M+1] + . Molecular formula:C 21 H 22 N2O5; molecular weight: 382.41.

[0226] [ka] Step 4. To a solution of compound 33 (1 g, 2.61 mmol) and benzyl glycolate (2.17 g, 13.1 mmol) in DCM (16 mL) was added 4 mL of TFA. The resulting mixture was stirred for 55 minutes. The mixture was evaporated to give a residue, which was dissolved in DCM. The mixture was washed with saturated NaHCO3 solution, water, and brine. The mixture was dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by silica gel chromatography (eluent: EtOAc / n-heptane = 30 to 100%) to give compound 34 (600 mg, 47%).

[0227] [ka] Step 5. To a solution of compound 34 (600 mg, 1.22 mmol) in 20 mL of DCM and 20 mL of ethanol was added Pd / C (10%, 60 mg). The mixture was stirred under a hydrogen atmosphere for 2 hours. The mixture was filtered through Celite, and the filtrate was evaporated to give crude compound 35 (400 mg, 81.7%).

[0228] [ka] Step 6. To a solution of compound 35 (50 mg, 0.125 mmol) and exatecan mesylate (50 mg, 0.094 mmol) in DMF (5 mL) was added HATU (54 mg, 0.141 mmol) and DIPEA (24 mg, 0.188 mmol). The mixture was stirred for 2 h. The mixture was diluted with DCM (60 mL) and washed with NaHCO3 solution (2 × 30 mL), water (20 mL × 2), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by flash chromatography on silica gel (eluent: MeOH / DCM = 5-10%) to give compound 36 (55 g, 72%).

[0229] [ka] Step 7. To a solution of compound 36 (55 mg, 0.067 mmol) in DMF (3 mL) was added piperidine (29 mg). The mixture was stirred for 1 hour and evaporated to give crude title compound 37, which was used in the next step without purification.

[0230] [ka] Step 8. To a solution of 2-azidoacetic acid (505 mg, 5 mmol) and H-Val-Ot-Bu (1.04 g, 5 mmol) in DMF (10 mL) was added HATU (2.47 g, 6.5 mmol) and DIPEA (0.97 g, 7.5 mmol). The mixture was stirred for 2 h. The mixture was diluted with EtOAc (60 mL) and washed with water (20 mL × 3), NaHCO3 solution (2 × 30 mL), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by flash chromatography on silica gel (eluent: EtOAc / hexane = 10 to 50%) to give compound 38 (950 mg, 74%).

[0231] [ka] Step 9. To a solution of compound 38 (0.95 g, 3.7 mmol) in 1,4-dioxane (8 mL) was added concentrated HCl (12 N, 4 mL). The mixture was stirred for 3 hours and evaporated to give crude compound 39 (0.69 g) as a pale yellow oil, which was used in the next step without purification.

[0232] [ka] Step 10. To a solution of crude compound 39 (17.4 mg, 0.0871 mmol) and 37 (crude, 0.067 mmol) in DMF (2 mL) was added HATU (38.2 mg, 0.1 mmol) and DIPEA (17.3 mg, 0.134 mmol). The mixture was stirred for 2 h. The mixture was diluted with DCM (60 mL) and washed with water (20 mL × 3), NaHCO3 solution (2 × 30 mL), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by flash chromatography on silica gel (eluent: MeOH / DCM = 0-10%) to give compound 40 (40 mg, 77%).

[0233] [ka] Step 11. N-acetylcysteine ​​of compound 40 (40 mg, 0.051 mmol) 1 ,N 1 A solution of 41-dimethylethane-1,2-diamine (5 mL) was stirred at 55° C. for 1 hour. The reaction mixture was concentrated in vacuo to give a yellow oil, which was dissolved in DCM (5 mL). To the mixture was added propionic anhydride (13.4 mg, 0.103 mmol) and DMAP (1.2 mg, 0.010 mmol). The mixture was stirred for 4 hours, then diluted with DCM (40 mL) and washed with brine. The organic layer was dried over Na2SO4, filtered, and evaporated to give a residue, which was purified by flash chromatography on silica gel (eluent: MeOH / DCM = 0-15%) to give compound 41 (20 mg, 42%).

[0234] [ka] Step 12. To a solution of 3-maleimidopropionic acid (170 mg, 1 mmol) and propargyl-PEG-2-amine (143 mg, 1 mmol) in DMF (5 mL) was added HATU (494 mg, 1.3 mmol) and DIPEA (194 mg, 1.5 mmol). The mixture was stirred for 30 min. The mixture was diluted with EtOAc (50 mL) and washed with water (20 mL × 3), NaHCO3 solution (2 × 30 mL), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by flash chromatography on silica gel (eluent: EtOAc / hexane = 10 to 50%) to give compound 42 (200 mg, 68%).

[0235] [ka] Step 13. Compounds 41 (20 mg, 0.021 mmol) and 42 (12.8 mg, 0.043 mmol) were dissolved in DMSO (1.4 mL) and water (0.34 mL). To this solution, cuprous bromide (9.3 mg, 0.065 mmol) and acetic acid (1 μL) were added. The reaction mixture was stirred for 1 h, then diluted with DCM (50 mL) and separated. The organic layer was washed twice with half-saturated aqueous sodium chloride solution (10 mL), dried over NaSO, filtered, and evaporated under vacuum to give a residue, which was purified by preparative HPLC (eluent: ACN / HO = 5-80% for 25 min) to give the title compound 43 (3 mg, 11%). LRMS: m / z = 1214.5 [M+1] + . Molecular formula:C 58 H 76 FN 13 O 15 ;Molecular weight: 1214.39.

[0236] Example 6: Synthesis of Compound 47 (36309) [ka] Step 1. Compound 44 (150 mg, 0.39 mmol) and 4-aminobenzyl alcohol (68 mg, 0.55 mmol) were dissolved in anhydrous DCM (9 ml) and anhydrous MeOH (3 ml). EEDQ (190 mg, 0.78 mmol) was added to the mixture, which was stirred at room temperature overnight. The reaction mixture was evaporated under vacuum to give a residue, which was purified by silica gel chromatography (eluent: MeOH / DCM = 3-10%) to give compound 45 (150 mg, 78.4%). LRMS: m / z = 487.2 (M+H). Chemical formula: C 25 H 34 N4O6; molecular weight: 486.56.

[0237] [ka] Step 2. To a mixture of compound 45 (150 mg, 0.3 mmol) in 4 mL of DMF, bis-p-nitrophenyl carbonate (0.19 g, 0.6 mmol) was added, and the reaction was continued at room temperature for 16 h. The reaction mixture was evaporated in vacuo to give a residue, which was purified by silica gel chromatography (eluent: MeOH / DCM = 3-10%) to give the title compound 46 (150 mg, 74.6%). LRMS: m / z = 652.2 (M+1); formula: C 32 H 37 N5O 10 ;Molecular weight: 651.66.

[0238] [ka] Step 3. To a solution of compound 3 (20 mg, 0.039 mmol) and compound 46 (51 mg, 0.079 mmol) in DMF (4 mL) was added HOAt (1 mg) and DIPEA (15.3 mg, 0.118 mmol). The resulting mixture was stirred at room temperature for 4 h. The reaction mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 47 (36309) (15 mg, 38% yield) as a yellow solid. LRMS: m / z = 1019.4 [M+H] + . Chemical formula:C 53 H59 FN8O 12 ;Molecular weight: 1019.08.

[0239] Example 7: Synthesis of Compound 53 [ka] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and Fmoc-Aib-OH (250 mg, 0.75 mmol) in DMF (8 mL) was added HATU (214 mg, 0.56 mmol) and DIPEA (164 μL, 0.94 mmol). The mixture was stirred for 1 h. The mixture was diluted with DCM (60 mL) and washed with water (20 mL × 3), NaHCO3 solution (2 × 30 mL), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give crude compound 48, which was used directly in the next step.

[0240] [ka] Step 2. To a solution of crude compound 48 (0.38 mmol) in DMF (5 mL) was added piperidine (1 mL). The mixture was stirred for 1 h and evaporated to give crude title compound 49, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give title compound 49 (120 mg, 62% yield) as a yellow solid. LRMS: m / z = 521.2 [M+H]+. Chemical formula: C 28 H 29 FN4O5; molecular weight: 520.55.

[0241] [ka] Step 3. To a solution of 49 (120 mg, 0.23 mmol) and 50 (121 mg, 0.28 mmol) in DMF (10 mL) was added HATU (132 mg, 0.35 mmol) and DIPEA (100 μL, 0.58 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 51 (150 mg, 69% yield) as a yellow solid. LRMS: m / z = 939.3 [M+H]+. Chemical formula: C 48 H 55 FN8O 11 ;Molecular weight:939.0

[0242] [ka] Step 4. To a solution of compound 51 (150 mg, 0.16 mmol) in DCM (1 mL) was added TFA (1 mL). The mixture was stirred for 15 minutes and then evaporated to give a residue which was dried under high vacuum to give crude 52. LRMS: m / z=839.3 [M+H] + . Chemical formula:C 43 H 47 FN8O9;Molecular weight:838.88

[0243] [ka] Step 5. To a solution of crude 52 (20 mg, 0.024 mmol) in DMF (5 mL) was added Mal-PEG2-NHS (12 mg, 0.029 mmol) and DIEA (10 μL, 0.06 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 53 (20 mg, 74% yield) as a solid. LRMS: m / z = 1049.4 [M+H] + . Chemical formula:C 57 H 65 FN 10 O 15 ;Molecular weight: 1049.18.

[0244] Example 8: Synthesis of Compound 54 [ka] To a solution of Step 1.52 (20 mg, 0.024 mol) and Mal-PEG4-acid (15 mg, 0.036 mmol) in DMF (10 mL) was added HATU (14 mg, 0.036 mmol) and DIPEA (11 μL, 0.063 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 54 (15 mg, 51% yield) as a solid. LRMS: m / z = 1237.3 [M+H] + . Chemical formula: C61H73FN10O17; Molecular weight: 1237.29.

[0245] Example 9: Synthesis of Compound 55 [ka] To a solution of Step 1.52 (20 mg, 0.024 mol) and Mal-PEG8-acid (17 mg, 0.029 mmol) in DMF (4 mL) was added HATU (14 mg, 0.036 mmol) and DIPEA (11 μL, 0.063 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 55 (20 mg, 61% yield) as a solid. LRMS: m / z = 1413.5 [M+H] + . Chemical formula: C69H89FN10O21; Molecular weight: 1413.50.

[0246] Example 10. Synthesis of Compound 58 [ka] To a solution of Step 1.11 (400 mg, 0.85 mmol) and L-alanine tert-butyl ester (111 mg, 0.85 mmol) in DMF (10 mL) was added HATU (38 mg, 1.0 mmol) and DIPEA (350 μL, 2.1 mmol). The mixture was stirred for 1 h. The mixture was diluted with ethyl acetate. The mixture was washed with saturated NaHCO solution and brine. The organic layer was dried over anhydrous NaSO, filtered, and evaporated to give crude compound 56.

[0247] [ka] To a solution of step 2.56 (0.496 g, 0.85 mmol) in DCM (10 mL) was added TFA (10 mL). The mixture was stirred for 2 hours. The volatiles were removed in vacuo, and the resulting residue was dissolved in 1 mL of DCM. Ether was added to precipitate the product. The solid was filtered, washed with ether, and dried under high vacuum to give compound 57 (0.30 g, 67%) as a white solid. LRMS: m / z=530.2 [M+1] + . Molecular formula:C 25 H 31 N5O8; molecular weight: 529.54.

[0248] [ka] Step 3. To a solution of 52 (20 mg, 0.038 mol) and 57 (40 mg, 0.077 mmol) in DMF (4 mL) was added HATU (22 mg, 0.057 mmol) and DIPEA (20 μL, 0.096 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 58 (3 mg, 7.7% yield) as a solid. LRMS: m / z = 1032.3 [M+H] + . Chemical formula:C 53 H 58 FN9O 12 ;Molecular weight: 1032.08.

[0249] Example 11. Synthesis of Compound 60 [ka] Step 1. To a solution of 52 (20 mg, 0.024 mol) and 57 (9.6 mg, 0.036 mmol) in DMF (4 mL) was added PyBOP (62 mg, 0.117 mmol) and DIPEA (12 μL, 0.069 mmol). The mixture was stirred for 4 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 60 (16 mg, 62% yield) as a solid. LRMS: m / z = 1089.3 [M+H] + . Chemical formula:C 54 H 57 FN 10 O 12 F; Molecular weight: 1089.16.

[0250] Example 12. Synthesis of Compound 65 [ka] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and 1-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)cyclopropanecarboxylic acid (250 mg, 0.75 mmol) in DMF (8 mL) was added HATU (214 mg, 0.56 mmol) and DIPEA (164 μL, 0.94 mmol). The mixture was stirred for 1 h. The mixture was diluted with DCM (60 mL) and washed with water (20 mL × 3), NaHCO3 solution (2 × 30 mL), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give crude compound 61, which was used directly in the next step.

[0251] [ka] Step 2. To a solution of crude compound 61 (0.38 mmol) in DMF (5 mL) was added piperidine (1 mL). The mixture was stirred for 1 h and evaporated to give crude title compound 62, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give title compound 62 (160 mg, 80% yield) as a solid. LRMS: m / z = 533.2 [M+H] + . Chemical formula:C 29 H 29 FN4O5; molecular weight: 532.56.

[0252] [ka] To a solution of step 3.62 (80 mg, 0.15 mmol) in THF (20 mL) was added DIPEA (158 μL, 0.90 mmol) and 2-(Fmoc-amino)acetyl chloride (189 mg, 0.60 mmol). The mixture was heated to 50 °C and stirred for 3 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 63 (50 mg, 41% yield) as a solid. LRMS: m / z = 812.2 [M+H] + . Chemical formula:C 46 H 42 FN5O8;Molecular weight:811.85

[0253] [ka] Step 4. To a solution of compound 63 (50 mg, 0.06 mmol) in DMF (5 mL) was added piperidine (1 mL). The mixture was stirred for 1 h and evaporated to give crude title compound 64, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give title compound 64 (20 mg, 55% yield) as a solid. LRMS: m / z = 590.3 [M+H] + . Chemical formula:C 31 H 32 FN5O6; molecular weight: 589.61

[0254] [ka] Step 5. To a solution of 64 (20 mg, 0.034 mol) and 11 (24 mg, 0.05 mmol) in DMF (4 mL) was added HATU (22 mg, 0.05 mmol) and DIPEA (15 μL, 0.085 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 65 (7 mg, 20% yield) as a solid. LRMS: m / z = 1044.3 [M+H] + . Chemical formula:C 54 H 58 FN9O 12 ;Molecular weight: 1044.09.

[0255] Example 13. Synthesis of Compound 68 [ka] Step 1. To a solution of 62 (80 mg, 0.15 mmol) and 50 (196 mg, 0.46 mmol) in THF (20 mL) was added HATU (170 mg, 0.46 mmol) and DIPEA (262 μL, 1.50 mmol). The mixture was heated to 50 °C and stirred for 3 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 63 (50 mg, 41% yield) as a solid. LRMS: m / z = 951.3 [M+H] + . Chemical formula:C 49 H 55 FN8O 11 ;Molecular weight:951.00

[0256] [ka] Step 2. To a solution of compound 66 (60 mg, 0.063 mmol) in DCM (5 mL) was added TFA (5 mL). The mixture was stirred for 15 minutes and then evaporated to give a residue which was dried under high vacuum to give crude 67. LRMS: m / z=851.3 [M+H] + . Chemical formula:C 44 H 47 FN8O9;Molecular weight:850.89

[0257] [ka] Step 3. To a solution of crude 67 (20 mg, 0.024 mmol) in DMF (5 mL) was added Mal-PEG2-NHS (16 mg, 0.047 mmol) and DIEA (10 μL, 0.06 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 69 (10 mg, 37% yield) as a solid. LRMS: m / z = 1161.4 [M+H] + . Chemical formula:C 58 H 65 FN 10 O 15 ;Molecular weight: 1161.19.

[0258] Example 14. Synthesis of Compound 70 [ka] Step 3. To a solution of crude 67 (20 mg, 0.024 mmol) in DMF (5 mL) was added Mal-PEG4-acid (20 mg, 0.047 mmol), HATU (14 mg, 0.036 mmol), and DIPEA (10 μL, 0.059 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 70 (9 mg, 31% yield) as a solid. LRMS: m / z = 1249.4 [M+H] + . Chemical formula:C 62 H 73 FN 10 O 17 ;Molecular weight: 1249.30.

[0259] Example 15. Synthesis of Compound 73 [ka] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and Fmoc-Ala-OH (245 mg, 0.75 mmol) in DMF (8 mL) was added HATU (172 mg, 0.45 mmol) and DIPEA (155 μL, 0.94 mmol). The mixture was stirred for 1 h. The mixture was diluted with DCM (60 mL) and washed with water (20 mL × 3), NaHCO3 solution (2 × 30 mL), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give a residue that was purified by flash chromatography to give compound 71 (150 mg, 54% yield). [ka]

[0260] Step 2. To a solution of compound 71 (150 mg, 0.20 mmol) in DMF (5 mL) was added piperidine (1 mL). The mixture was stirred for 1 h and evaporated to give crude title compound 72, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give title compound 72 (100 mg, 95% yield) as a solid. LRMS: m / z = 521.2 [M+H] + . Chemical formula:C 28 H 29 FN4O5; molecular weight: 520.55.

[0261] [ka] Step 3. To a solution of 72 (30 mg, 0.057 mmol) and 57 (61 mg, 0.115 mmol) in DMF (4 mL) was added HATU (33 mg, 0.086 mmol) and DIPEA (25 μL, 0.144 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 73 (15 mg, 25.4% yield) as a solid. LRMS: m / z = 1032.4 [M+H] + . Chemical formula:C 53 H 58 FN9O 12;Molecular weight: 1032.08.

[0262] Example 16. Synthesis of Compound 74 [ka] Compound 74 was synthesized according to the same protocol as compound 73. LRMS: m / z=1032.4 [M+H] + . Chemical formula:C 53 H 58 FN9O 12 ;Molecular weight: 1032.08.

[0263] Example 17. Synthesis of Compound 79 [ka] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and 2-{[(9H-fluoren-9-ylmethoxy)carbonyl](methyl)amino}-2-methylpropanoic acid (250 mg, 0.75 mmol) in DMF (8 mL) was added HATU (172 mg, 0.45 mmol) and DIPEA (160 μL, 0.94 mmol). The mixture was stirred for 1 h. The mixture was diluted with DCM (60 mL) and washed with water (20 mL × 3), NaHCO solution (2 × 30 mL), and brine. The organic layer was dried over anhydrous NaSO, filtered, and evaporated to give crude compound 75, which was used directly in the next step.

[0264] [ka] Step 2. To a solution of crude compound 75 (0.38 mmol) in DMF (5 mL) was added piperidine (1 mL). The mixture was stirred for 1 h and evaporated to give crude title compound 76, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give title compound 76 (200 mg, 99% yield) as a solid. LRMS: m / z = 534.2 [M+H] + . Chemical formula:C 29 H 31 FN4O5; molecular weight: 534.58.

[0265] [ka] To a solution of step 3.76 (100 mg, 0.18 mmol) in THF (20 mL) was added DIPEA (158 μL, 0.90 mmol) and 2-(Fmoc-amino)acetyl chloride (236 mg, 0.75 mmol). The mixture was heated to 50 °C and stirred for 3 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 77 (50 mg, 30% yield) as a solid. LRMS: m / z = 814.3 [M+H] + . Chemical formula:C 46 H 44 FN5O8;Molecular weight:813.87

[0266] [ka] Step 4. To a solution of compound 77 (50 mg, 0.06 mmol) in DMF (5 mL) was added piperidine (1 mL). The mixture was stirred for 1 h and evaporated to give crude title compound 78, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give title compound 77 (20 mg, 55% yield) as a solid. LRMS: m / z = 592.3 [M+H] + . Chemical formula:C 31 H 34 FN5O6; molecular weight: 591.63

[0267] [ka] Step 5. To a solution of 78 (20 mg, 0.034 mol) and 11 (32 mg, 0.068 mmol) in DMF (4 mL) was added HATU (20 mg, 0.051 mmol) and DIPEA (15 μL, 0.085 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 79 (3 mg, 8.5% yield) as a solid. LRMS: m / z = 1046.4 [M+H] + . Chemical formula:C 54 H 60 FN9O 12 ;Molecular weight: 1046.10.

[0268] Example 18. Synthesis of Compound 84 [ka] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and 1-(9H-fluoren-9-ylmethoxycarbonylamino)cyclopropanecarboxylic acid (183 mg, 0.56 mmol) in DMF (10 mL) was added HATU (214 mg, 0.56 mmol) and DIPEA (164 μL, 0.94 mmol). The mixture was stirred for 1 h. The mixture was diluted with DCM (60 mL) and washed with water (20 mL × 3), NaHCO3 solution (2 × 30 mL), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give crude compound 80, which was used directly in the next step.

[0269] [ka] Step 2. To a solution of crude compound 80 (0.38 mmol) in DMF (5 mL) was added piperidine (1 mL). The mixture was stirred for 1 h and evaporated to give crude title compound 81, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give title compound 81 (180 mg, 92% yield) as a yellow solid. LRMS: m / z = 519.2 [M+H] + . Chemical formula: C 28 H 27FN4O5; molecular weight: 518.54.

[0270] [ka] Step 3. To a solution of 81 (52 mg, 0.10 mmol) and 50 (52 mg, 0.12 mmol) in DMF (5 mL) was added HATU (57 mg, 0.15 mmol) and DIPEA (44 μL, 0.25 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 82 (70 mg, 75% yield) as a solid. LRMS: m / z = 937.3 [M+H] + . Chemical formula:C 48 H 53 FN8O 11 ;Molecular weight:936.98

[0271] [ka] Step 4. To a solution of compound 82 (70 mg, 0.075 mmol) in DCM (9 mL) was added TFA (3 mL). The mixture was stirred for 15 minutes and then evaporated to give a residue which was dried under high vacuum to give crude 83. LRMS: m / z=837.3 [M+H] + . Chemical formula:C 43 H 45 FN8O9;Molecular weight:836.86

[0272] [ka] Step 5. To a solution of crude 83 (15 mg, 0.018 mmol) in DMF (2 mL) was added Mal-PEG2-NHS (12 mg, 0.029 mmol) and DIEA (8 μL, 0.045 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 84 (15 mg, 73% yield) as a solid. LRMS: m / z = 1047.4 [M+H] + . Chemical formula:C 57 H63 FN 10 O 15 ;Molecular weight:1047.16

[0273] Example 19. Synthesis of Compound 89 [ka] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and Fmoc-D-Ala-OH (185 mg, 0.56 mmol) in DMF (10 mL) was added HATU (214 mg, 0.56 mmol) and DIPEA (164 μL, 0.94 mmol). The mixture was stirred for 1 h. The mixture was diluted with DCM (60 mL) and washed with water (20 mL × 3), NaHCO3 solution (2 × 30 mL), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give crude compound 85, which was used directly in the next step.

[0274] [ka] Step 2. To a solution of crude compound 85 (0.38 mmol) in DMF (5 mL) was added piperidine (0.5 mL). The mixture was stirred for 20 minutes and evaporated to give crude title compound 86, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 minutes) to give title compound 86 (180 mg, 92% yield) as a yellow solid. LRMS: m / z = 519.2 [M+H] + . Chemical formula: C 27 H 27 FN4O5; molecular weight: 518.54.

[0275] [ka] Step 3. To a solution of 86 (51 mg, 0.10 mmol) and 50 (52 mg, 0.12 mmol) in DMF (5 mL) was added HATU (57 mg, 0.15 mmol) and DIPEA (44 μL, 0.25 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 87 (70 mg, 75% yield) as a solid. LRMS: m / z = 925.3 [M+H] + . Chemical formula:C 47 H 53 FN8O 11 ;Molecular weight:924.97

[0276] [ka] Step 4. To a solution of compound 87 (70 mg, 0.075 mmol) in DCM (9 mL) was added TFA (3 mL). The mixture was stirred for 15 minutes and then evaporated to give a residue which was dried under high vacuum to give crude 88. LRMS: m / z=825.3 [M+H] + . Chemical formula:C 42 H 45 FN8O9; molecular weight: 824.85.

[0277] [ka] Step 5. To a solution of crude 88 (15 mg, 0.018 mmol) in DMF (2 mL) was added Mal-PEG2-NHS (12 mg, 0.028 mmol) and DIEA (8 μL, 0.045 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 89 (12 mg, 58% yield) as a solid. LRMS: m / z = 1135.4 [M+H] + . Chemical formula:C 56 H 63 FN 10 O 15 ;Molecular weight: 1135.15

[0278] Example 20. Synthesis of Compound 94 [ka] Step 1. To a solution of exatecan mesylate (150 mg, 0.28 mmol) and (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-cyclopropylacetic acid (100 mg, 0.28 mmol) in DMF (10 mL) was added HATU (161 mg, 0.42 mmol) and DIPEA (120 μL, 0.70 mmol). The mixture was stirred for 1 h. The mixture was diluted with DCM (60 mL) and washed with water (20 mL × 3), NaHCO solution (2 × 30 mL), and brine. The organic layer was dried over anhydrous NaSO, filtered, and evaporated to give crude compound 90, which was used directly in the next step.

[0279] [ka] Step 2. To a solution of crude compound 90 (0.28 mmol) in DMF (5 mL) was added piperidine (0.5 mL). The mixture was stirred for 20 min and evaporated to give crude title compound 91, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give title compound 91 (130 mg, 87% yield) as a yellow solid. LRMS: m / z = 533.2 [M+H] + . Chemical formula:C 29 H 29 FN4O5; molecular weight: 532.57.

[0280] [ka] Step 3. To a solution of 91 (53 mg, 0.10 mmol) and 50 (52 mg, 0.12 mmol) in DMF (5 mL) was added HATU (57 mg, 0.15 mmol) and DIPEA (44 μL, 0.25 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 92 (70 mg, 74% yield) as a solid. LRMS: m / z = 951.3 [M+H] + . Chemical formula:C49 H 55 FN8O 11 ;Molecular weight:951.00

[0281] [ka] Step 4. To a solution of compound 92 (70 mg, 0.075 mmol) in DCM (9 mL) was added TFA (3 mL). The mixture was stirred for 10 minutes and then evaporated to give a residue which was dried under high vacuum to give crude 93. LRMS: m / z=851.3 [M+H] + . Chemical formula:C 44 H 47 FN8O9; molecular weight: 850.89.

[0282] [ka] Step 5. To a solution of crude 93 (15 mg, 0.018 mmol) in DMF (2 mL) was added Mal-PEG2-NHS (12 mg, 0.028 mmol) and DIEA (8 μL, 0.045 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 94 (13 mg, 64% yield) as a solid. LRMS: m / z = 1161.4 [M+H] + . Chemical formula:C 58 H 65 FN 10 O 15 ;Molecular weight: 1161.19.

[0283] Example 21. Synthesis of Compound 95 [ka] Compound 95 was synthesized from (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2-cyclopropylacetic acid according to the same procedure as compound 94. LRMS: m / z=1161.4 [M+H] + . Chemical formula:C 58 H 65 FN 10 O15 ;Molecular weight: 1161.19.

[0284] Example 22. Synthesis of Compound 97 [ka] To a solution of Step 1.52 (50 mg, 0.060 mol) and Boc-N-amido-PEG4-acid (28 mg, 0.089 mmol) in DMF (6 mL) was added HATU (34 mg, 0.089 mmol) and DIPEA (26 μL, 0.15 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 96 (50 mg, 76% yield) as a solid. LRMS: m / z = 1098.4 [M+H] + . Chemical formula:C 55 H 68 FN9O 14 ;Molecular weight:1098.18

[0285] [ka] Step 2. To a solution of compound 96 (50 mg, 0.046 mmol) in DCM (9 mL) was added TFA (3 mL). The mixture was stirred for 10 min and then evaporated to give a residue that was dried under high vacuum to give the crude amine. To a solution of the crude amine (0.046 mmol) and 57 (18.3 mg, 0.068 mmol) in DMF (10 mL) was added PyBOP (118 mg, 0.228 mmol) and DIPEA (24 μL, 0.137 mmol). The mixture was stirred for 3 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 97 (35 mg, 62% yield) as a solid. LRMS: m / z = 1249.4 [M+H] + . Chemical formula:C 61 H 70 FN 11 O 15 S; molecular weight: 1249.34.

[0286] Example 23. Synthesis of Compound 100 [ka] To a solution of 3 (100 mg, 0.20 mmol) and 50 (100 mg, 0.24 mmol) in DMF (5 mL) was added HATU (98 mg, 0.26 mmol) and DIPEA (86 μL, 0.49 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 98 (130 mg, 71% yield) as a solid. LRMS: m / z = 925.3 [M+H] + . Chemical formula:C 49 H 55 FN8O 11 ;Molecular weight:925.00

[0287] [ka] Step 4. To a solution of compound 98 (130 mg, 0.14 mmol) in DCM (9 mL) was added TFA (3 mL). The mixture was stirred for 10 minutes and then evaporated to give a residue which was dried under high vacuum to give crude 99. LRMS: m / z=825.3 [M+H] + . Chemical formula:C 42 H 45 FN8O9; molecular weight: 824.85.

[0288] [ka] Step 5. To a solution of crude 99 (65 mg, 0.079 mmol) in DMF (2 mL) was added Mal-PEG2-acid (31 mg, 0.094 mmol), HATU (39 mg, 0.102 mmol), and DIEA (34 μL, 0.20 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 100 (52 mg, 58% yield) as a solid. LRMS: m / z = 1135.4 [M+H] + . Chemical formula:C 56 H 63 FN 10 O 15;Molecular weight: 1135.15.

[0289] Example 24. Synthesis of Compound 101 [ka] Step 5. To a solution of crude 100 (30 mg, 0.036 mmol) in DMF (2 mL) was added Mal-PEG8-acid (32 mg, 0.055 mmol), HATU (21 mg, 0.055 mmol), and DIEA (15 μL, 0.09 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 101 (20 mg, 40% yield) as a solid. LRMS: m / z = 1399.5 [M+H] + . Chemical formula:C 68 H 87 FN 10 O 21 ;Molecular weight: 1399.47. Example 25. Synthesis of Compound 102 [ka] Step 1. To a solution of exatecan mesylate (100 mg, 0.19 mmol) and cis-3-(Boc-amino)cyclobutanecarboxylic acid (49 mg, 0.23 mmol) in DMF (10 mL), HATU (107 mg, 0.28 mmol) and DIPEA (82 µL, 0.47 mmol) were added. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 114 (85 mg, 71% yield). LRMS: m / z = 633.2 [M+H] + . Chemical formula:C 34 H 37 FN4O7; molecular weight: 632.68.

[0290] [ka] Step 2. To a solution of compound 114 (85 mg, 0.134 mmol) in DCM (6 mL) was added TFA (3 mL). The mixture was stirred for 30 minutes and evaporated to give the title compound 102 (60 mg, 84% yield). LRMS: m / z = 533.2 [M+H] + . Chemical formula:C 29 H 29 FN4O5; molecular weight: 532.56.

[0291] Example 26. Synthesis of Compound 103 [ka] Compound 103 was synthesized from cis-4-(Boc-amino)cyclohexanecarboxylic acid according to the same procedure as compound 102. LRMS: m / z=561.2 [M+H] + . Chemical formula:C 31 H 33 FN4O5; molecular weight: 560.61.

[0292] Example 27. Synthesis of Compound 107 [ka] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and Fmoc-D-Ala-OH (245 mg, 0.75 mmol) in DMF (8 mL) was added HATU (172 mg, 0.45 mmol) and DIPEA (155 μL, 0.94 mmol). The mixture was stirred for 1 hour. The mixture was diluted with DCM (60 mL) and washed with water (20 mL × 3), NaHCO3 solution (2 × 30 mL), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give crude compound 104.

[0293] [ka] Step 2. To a solution of compound 104 (0.38 mmol) in DMF (5 mL) was added piperidine (1 mL). The mixture was stirred for 1 h and evaporated to give crude title compound 105, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give title compound 105 (100 mg, 77% yield) as a solid. LRMS: m / z = 521.2 [M+H] + . Chemical formula:C 28 H 29 FN4O5; molecular weight: 520.55.

[0294] [ka] Step 3. To a solution of compound 105 (120 mg, 0.23 mmol) and (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-cyclopropylacetic acid (93 mg, 0.28 mmol) in DMF (10 mL) was added HATU (131 mg, 0.35 mmol) and DIPEA (100 μL, 0.58 mmol). The mixture was stirred for 1 h. The mixture was diluted with DCM (60 mL) and washed with water (20 mL × 3), NaHCO3 solution (2 × 30 mL), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give crude compound 106, which was used directly in the next step.

[0295] [ka] Step 4. To a solution of crude compound 106 (0.23 mmol) in DMF (5 mL) was added piperidine (0.5 mL). The mixture was stirred for 20 minutes and evaporated to give crude title compound 107, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 minutes) to give title compound 107 (120 mg, 85% yield) as a yellow solid. LRMS: m / z = 618.2 [M+H] + . Chemical formula:C 33 H 36 FN5O6; molecular weight: 617.67.

[0296] Example 28. Synthesis of Compound 109 [ka] Step 1. To a solution of compound 3 (120 mg, 0.23 mmol) and (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-cyclopropylacetic acid (93 mg, 0.28 mmol) in DMF (10 mL) was added HATU (135 mg, 0.35 mmol) and DIPEA (100 μL, 0.58 mmol). The mixture was stirred for 1 h. The mixture was diluted with DCM (60 mL) and washed with water (20 mL × 3), NaHCO3 solution (2 × 30 mL), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give crude compound 108, which was used directly in the next step.

[0297] [ka] Step 2. To a solution of crude compound 108 (0.23 mmol) in DMF (5 mL) was added piperidine (0.5 mL). The mixture was stirred for 20 minutes and evaporated to give crude title compound 109, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 minutes) to give title compound 109 (100 mg, 70% yield) as a yellow solid. LRMS: m / z = 604.2 [M+H] + . Chemical formula:C 32 H 34 FN5O6; molecular weight: 603.64.

[0298] Example 29. Synthesis of Compound 111 [ka] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-2-cyclopropylacetic acid (159 mg, 0.45 mmol) in DMF (8 mL) was added HATU (215 mg, 0.56 mmol) and DIPEA (155 μL, 0.94 mmol). The mixture was stirred for 1 h. The mixture was diluted with DCM (60 mL) and washed with water (20 mL × 3), NaHCO3 solution (2 × 30 mL), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give crude compound 110, which was used directly in the next step.

[0299] [ka] Step 2. To a solution of compound 110 (0.38 mmol) in DMF (5 mL) was added piperidine (1 mL). The mixture was stirred for 1 h and evaporated to give crude title compound 111, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give title compound 111 (150 mg, 73% yield) as a solid. LRMS: m / z = 547.2 [M+H] + . Chemical formula:C 30 H 31 FN4O5; molecular weight: 546.59.

[0300] Example 30. Synthesis of Compound 113 [ka] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-2-cyclopropylacetic acid (160 mg, 0.45 mmol) in DMF (8 mL) was added HATU (215 mg, 0.56 mmol) and DIPEA (155 μL, 0.94 mmol). The mixture was stirred for 1 h. The mixture was diluted with DCM (60 mL) and washed with water (20 mL × 3), NaHCO3 solution (2 × 30 mL), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give crude compound 112, which was used directly in the next step.

[0301] [ka] Step 2. To a solution of compound 112 (0.38 mmol) in DMF (5 mL) was added piperidine (1 mL). The mixture was stirred for 1 h and evaporated to give crude title compound 113, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give title compound 113 (140 mg, 68% yield) as a solid. LRMS: m / z = 549.2 [M+H] + . Chemical formula:C 30 H 33 FN4O5; molecular weight: 548.60.

[0302] Example 31. Synthesis of Compound 116 [ka] Step 1. To a solution of compound 113 (25 mg, 0.046 mmol) and 22 (13 mg, 0.068 mmol) in DMF (1 mL) was added HATU (35 mg, 0.091 mmol) and DIPEA (24 μL, 0.137 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 115 (25 mg, 76% yield) as a solid. LRMS: m / z = 721.3 [M+H] + . Chemical formula:C 38 H49 FN4O7Si; molecular weight: 720.90.

[0303] [ka] Step 2. To a solution of compound 115 (25 mg, 0.035 mmol) in DCM (10 mL) was added TBAF (86 μL, 0.087 mmol) and acetic acid (173 μL, 0.173 mmol). The mixture was stirred overnight and evaporated to give a residue, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 116 (10 mg, 48% yield) as a solid. LRMS: m / z = 607.2 [M+H] + . Chemical formula:C 32 H 35 FN4O7; molecular weight: 606.64.

[0304] Example 32. Synthesis of Compound 118 [ka] Step 1. To a solution of compound 113 (100 mg, 0.18 mmol) and Fmoc-L-Ala-OH (74 mg, 0.24 mmol) in DMF (1 mL) was added HATU (104 mg, 0.27 mmol) and DIPEA (80 μL, 0.46 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 117 (120 mg, 78% yield) as a solid. LRMS: m / z = 842.3 [M+H]+. Chemical formula: C 48 H 48 FN5O8; molecular weight: 841.92.

[0305] [ka] Step 2. To a solution of compound 117 (0.38 mmol) in DMF (5 mL) was added piperidine (1 mL). The mixture was stirred for 1 h and evaporated to give crude title compound 118, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give title compound 118 (70 mg, 79% yield) as a solid. LRMS: m / z = 620.2 [M+H] + . Chemical formula:C 33 H 38 FN5O6; molecular weight: 619.68.

[0306] Example 33. Synthesis of Compound 124 [ka] Step 1. To a solution of 119 (250 mg, 0.45 mmol) in DMF (10 mL) was added HATU (255 mg, 0.67 mmol). The mixture was stirred for 30 minutes, and then 4-aminobenzyl alcohol (77 mg, 0.63 mmol) and DIPEA (195 μL, 1.12 mmol) were added. The mixture was stirred for 2 hours. The mixture was diluted with DCM (60 mL) and washed with water (20 mL × 3), NaHCO3 solution (2 × 30 mL), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give crude compound 120, which was used directly in the next step.

[0307] [ka] Step 2. To a solution of 120 (0.45 mmol) in DMF (10 mL) was added bis(4-nitrophenyl)carbonate (176 mg, 0.58 mmol) and DIPEA (155 μL, 0.90 mmol). The mixture was stirred for 3 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 121 (250 mg, 67% yield) as a solid. LRMS: m / z = 829.2 [M+H] + . Chemical formula:C 44 H 40 NO 11 ;Molecular weight:828.82.

[0308] [ka] Step 3. To a solution of 121 (108 mg, 0.13 mmol) in DMF (10 mL) was added 3 (51 mg, 0.1 mmol) and DIPEA (35 μL, 0.2 mmol). The mixture was stirred for 3 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 122 (80 mg, 67% yield) as a solid. LRMS: m / z = 1196.4 [M+H] + . Chemical formula:C 65 H 62 FN9O 13 ;Molecular weight: 1196.24.

[0309] [ka] Step 4. To a solution of compound 122 (80 mg, 0.067 mmol) in DMF (5 mL) was added piperidine (0.1 mL). The mixture was stirred for 20 minutes and evaporated to give crude title compound 123, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 minutes) to give title compound 123 (50 mg, 77% yield) as a solid. LRMS: m / z = 974.3 [M+H] + . Chemical formula:C 50 H 52 FN9O 11 ;Molecular weight: 974.00.

[0310] [ka] Step 5. To a solution of compound 123 (50 mg, 0.051 mmol) and Mal-PEG8-acid (40 mg, 0.067 mmol) in DMF (1 mL) was added HATU (29 mg, 0.077 mmol) and DIPEA (23 μL, 0.128 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 124 (60 mg, 75% yield) as a solid. LRMS: m / z = 1548.6 [M+H] + . Chemical formula:C 76 H 94 FN 11 O 23 ;Molecular weight: 1548.62.

[0311] Example 34. Synthesis of Compound 127 [ka] Step 1. To a solution of 121 (73 mg, 0.087 mmol) in DMF (10 mL) was added 113 (40 mg, 0.073 mmol) and DIPEA (51 μL, 0.29 mmol). The mixture was stirred for 3 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 125 (80 mg, 67% yield) as a solid. LRMS: m / z = 1238.4 [M+H] + . Chemical formula:C 68 H 68 FN9O 13 ;Molecular weight: 1238.31.

[0312] [ka] Step 2. To a solution of compound 125 (75 mg, 0.061 mmol) in DMF (5 mL) was added piperidine (0.1 mL). The mixture was stirred for 20 minutes and evaporated to give crude title compound 126, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 minutes) to give title compound 126 (50 mg, 81% yield) as a solid. LRMS: m / z = 1016.4 [M+H] + . Chemical formula:C53 H 58 FN9O 11 ;Molecular weight: 1016.08.

[0313] [ka] Step 3. To a solution of compound 126 (50 mg, 0.049 mmol) and Mal-PEG8-acid (38 mg, 0.064 mmol) in DMF (1 mL) was added HATU (28 mg, 0.074 mmol) and DIPEA (22 μL, 0.123 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 127 (43 mg, 55% yield) as a solid. LRMS: m / z = 1590.6 [M+H] + . Chemical formula:C 79 H 100 FN 11 O 23 ;Molecular weight: 1590.70.

[0314] Example 35. Synthesis of Compound 130 [ka] Step 1. To a solution of 107 (100 mg, 0.16 mmol) and 50 (85 mg, 0.19 mmol) in DMF (5 mL) was added HATU (92 mg, 0.24 mmol) and DIPEA (67 μL, 0.40 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 128 (120 mg, 72% yield) as a solid. LRMS: m / z = 1036.4 [M+H] + . Chemical formula:C 53 H 62 FN9O 12 ;Molecular weight:1036.11

[0315] [ka] Step 2. To a solution of compound 128 (120 mg, 0.12 mmol) in DCM (9 mL) was added TFA (3 mL). The mixture was stirred for 10 minutes and then evaporated to give a residue which was dried under high vacuum to give crude 129. LRMS: m / z=936.3 [M+H] + . Chemical formula:C 48 H 54 FN9O 10 ;Molecular weight: 935.99.

[0316] [ka] Step 3. To a solution of crude 129 (0.12 mmol) in DMF (2 mL) was added Mal-PEG8-acid (85 mg, 0.14 mmol), HATU (68 mg, 0.18 mmol), and DIPEA (50 μL, 0.30 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 130 (100 mg, 57% yield) as a solid. LRMS: m / z = 1510.6 [M+H] + . Chemical formula:C 74 H 96 FN 11 O 22 ;Molecular weight: 1510.61.

[0317] Example 36. Synthesis of Compound 132 [ka] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-cyclopropylacetic acid (190 mg, 0.56 mmol) in DMF (10 mL) was added HATU (215 mg, 0.56 mmol) and DIPEA (155 μL, 0.94 mmol). The mixture was stirred for 1 h. The mixture was diluted with DCM (60 mL) and washed with water (20 mL × 3), NaHCO3 solution (2 × 30 mL), and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give crude compound 131, which was used directly in the next step.

[0318] [ka] Step 2. To a solution of crude compound 131 (0.38 mmol) in DMF (5 mL) was added piperidine (0.5 mL). The mixture was stirred for 20 minutes and evaporated to give crude title compound 132, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 minutes) to give title compound 132 (180 mg, 92% yield) as a yellow solid. LRMS: m / z = 533.2 [M+H] + . Chemical formula:C 29 H 29 FN4O5; molecular weight: 532.57.

[0319] Example 37. Synthesis of Compound 135 [ka] Step 1. To a solution of 121 (60 mg, 0.72 mmol) in DMF (10 mL) was added 111 (33 mg, 0.60 mmol) and DIPEA (42 μL, 0.24 mmol). The mixture was stirred for 3 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 133 (50 mg, 67% yield) as a solid. LRMS: m / z = 1236.4 [M+H] + . Chemical formula:C 68 H 66 FN9O13 ;Molecular weight: 1236.30.

[0320] [ka] Step 2. To a solution of compound 133 (50 mg, 0.040 mmol) in DMF (5 mL) was added piperidine (0.1 mL). The mixture was stirred for 20 minutes and evaporated to give crude title compound 134, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 minutes) to give title compound 134 (35 mg, 85% yield) as a solid. LRMS: m / z = 1014.4 [M+H] + . Chemical formula:C 53 H 56 FN9O 11 ;Molecular weight: 1014.06.

[0321] [ka] Step 3. To a solution of compound 134 (35 mg, 0.035 mmol) and Mal-PEG8-acid (27 mg, 0.045 mmol) in DMF (5 mL) was added HATU (26 mg, 0.069 mmol) and DIPEA (18 μL, 0.10 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 135 (40 mg, 73% yield) as a solid. LRMS: m / z = 1588.6 [M+H] + . Chemical formula:C 79 H 98 FN 11 O 23 ;Molecular weight: 1588.68.

[0322] Example 38. Synthesis of Compound 137 [ka] Step 1. To a solution of compound 62 (25 mg, 0.046 mmol) and 22 (13 mg, 0.068 mmol) in DMF (1 mL) was added HATU (35 mg, 0.091 mmol) and DIPEA (24 μL, 0.137 mmol). The mixture was stirred for 1 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 136 (25 mg, 76% yield) as a solid. LRMS: m / z = 721.3 [M+H] + . Chemical formula:C 38 H 49 FN4O7Si; molecular weight: 720.90.

[0323] [ka] Step 2. To a solution of compound 136 (25 mg, 0.035 mmol) in DCM (10 mL) was added TBAF (86 μL, 0.087 mmol) and acetic acid (173 μL, 0.173 mmol). The mixture was stirred overnight and evaporated to give a residue, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 137 (10 mg, 48% yield) as a solid. LRMS: m / z = 607.2 [M+H] + . Chemical formula:C 32 H 35 FN4O7; molecular weight: 606.64.

[0324] Example 39. Synthesis of Compound 141 [ka] Step 1. To a solution of compounds 138 (55 mg, 0.14 mmol) and 113 (60 mg, 0.11 mmol) in DMF (6 mL) was added HATU (62 mg, 0.16 mmol) and DIPEA (47 μL, 0.27 mmol). The mixture was stirred for 5 h. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 139 (70 mg, 70% yield) as a solid. LRMS: m / z = 915.3 [M+H] + . Chemical formula:C 50H 51 FN6O 10 ;Molecular weight: 914.97.

[0325] [ka] Step 2. To a solution of compound 139 (70 mg, 0.077 mmol) in DMF (5 mL) was added piperidine (0.1 mL). The mixture was stirred for 20 minutes and evaporated to give crude title compound 140, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 minutes) to give title compound 140 (45 mg, 85% yield) as a solid. LRMS: m / z = 693.2 [M+H] + . Chemical formula:C 35 H 41 FN6O8; molecular weight: 692.73.

[0326] [ka] Step 3. To a solution of compound 11 (50 mg, 0.084 mmol) and 140 (45 mg, 0.065 mmol) in DMF (6 mL) was added HATU (49 mg, 0.13 mmol) and DIPEA (34 μL, 0.19 mmol). The mixture was stirred for 30 min. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 141 (50 mg, 61% yield) as a solid. LRMS: m / z = 1264.5 [M+H] + . Chemical formula:C 62 H 74 FN 11 O 17 ;Molecular weight: 1264.31.

[0327] Example 40. Synthesis of Compound 144 [ka] Step 1. To a solution of compounds 138 (43 mg, 0.12 mmol) and 62 (50 mg, 0.94 mmol) in DMF (6 mL) was added HATU (53 mg, 0.14 mmol) and DIPEA (39 μL, 0.23 mmol). The mixture was stirred for 30 min. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 142 (60 mg, 71% yield) as a solid. LRMS: m / z = 899.3 [M+H] + . Chemical formula:C 49 H 47 FN6O 10 ;Molecular weight: 898.93.

[0328] [ka] Step 2. To a solution of compound 142 (60 mg, 0.067 mmol) in DMF (5 mL) was added piperidine (0.1 mL). The mixture was stirred for 20 minutes and evaporated to give crude title compound 143, which was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 minutes) to give title compound 143 (40 mg, 89% yield) as a solid. LRMS: m / z = 677.2 [M+H] + . Chemical formula:C 34 H 37 FN6O8; molecular weight: 676.69.

[0329] [ka] Step 3. To a solution of compound 11 (42 mg, 0.071 mmol) and 143 (40 mg, 0.059 mmol) in DMF (8 mL) was added HATU (34 mg, 0.89 mmol) and DIPEA (25 μL, 0.15 mmol). The mixture was stirred for 30 min. The mixture was purified by preparative HPLC (eluent: ACN / HO = 25-80% for 25 min) to give the title compound 144 (20 mg, 27% yield) as a solid. LRMS: m / z = 1248.4 [M+H] + . Chemical formula:C 61 H 70 FN 11 O17 ;Molecular weight: 1248.27.

[0330] Example 41. Generation of ADC Some embodiments of the present disclosure relate to camptothecin derivatives linked to antibodies via cleavable linkers to generate antibody-drug conjugates. Table 2 provides a list of camptothecin derivatives. The conjugation procedure is illustrated in Figure 2. The cysteine ​​conjugation method used in the conjugation process produces more homogeneous ADCs compared to the lysine conjugation method. In these examples, anti-Her2 antibodies, Herceptin, or anti-Trop-2 antibodies were conjugated to camptothecin derivatives to form ADCs and evaluated for their ability to inhibit the growth of multiple cancer cell lines expressing different levels of Her2. Camptothecin derivatives stabilize the topoisomerase I complex after cleaving the DNA strand for replication, preventing DNA double helix break repair and thereby halting the replication process.

[0331] 41-1. Preparation example of ADC-1 [ka] Anti-Trop-2 antibody in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.004 μmol) was reduced by adding 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.0048 ml, 0.024 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an Amicon Ultra filter. A solution of camptothecin derivative-linker, compound 43, in DMA (10 mg / ml, 0.04 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to yield anti-Trop-2 ADC-1 (0.6 mg / ml, 0.2 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 4.5.

[0332] 41-2. Preparation example of ADC-2 [ka] Anti-Trop-2 antibody in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.01 μmol) was reduced by adding 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.012 ml, 0.08 μmol) solution. The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 21, in DMA (10 mg / ml, 0.12 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to yield anti-Trop-2 ADC-2 (4.6 mg / ml, 0.9 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 5.3.

[0333] 41-3. Preparation example of ADC-3 [ka] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.01 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.012 ml, 0.06 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 30, in DMA (10 mg / ml, 0.1 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-3 (2.15 mg / ml, 0.9 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 5.2.

[0334] 41-4. Preparation example of ADC-4 [ka] The anti-Her2 antibody Herceptin in PBS buffer (20 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.06 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.24 ml, 12 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 12, in DMA (10 mg / ml, 10 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to yield anti-Her2 ADC-4 (3.4 mg / ml, 5.1 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 6.6.

[0335] 41-5. Preparation example of ADC-5 [ka] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.6 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (4.2 ml, 21 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, Compound 100, in DMA (10 mg / ml, 12 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to yield anti-Her2 ADC-5 (6.3 mg / ml, 56.7 mg) in PBS (10 mM, pH 6) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.7.

[0336] 41-6. Preparation example of ADC-6 [ka] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (5 mg / ml, 0.04 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.2 ml, 1 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, Compound 101, in DMA (10 mg / ml, 0.8 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-6 (5.7 mg / ml, 2.9 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.9.

[0337] 41-7. Preparation example of ADC-7 [ka] The anti-Her2 antibody Herceptin in His buffer (20 mM, pH 6) containing 5 mM EDTA (3 mg / ml, 0.06 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.24 ml, 1.2 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 124, in DMA (10 mg / ml, 1.5 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to yield anti-Her2 ADC-7 (7.5 mg / ml, 4.5 mg) in His (20 mM, pH 6) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.5.

[0338] 41-4. Preparation example of ADC-8 [ka] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.2 ml, 1 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 65, in DMA (10 mg / ml, 0.8 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to yield anti-Her2 ADC-8 (4.3 mg / ml, 1.9 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.4.

[0339] 41-9. Preparation example of ADC-9 [ka] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (4 mg / ml, 0.03 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.13 ml, 0.75 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 68, in DMA (10 mg / ml, 0.6 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to yield anti-Her2 ADC-9 (9.1 mg / ml, 2.7 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion chromatography-high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography-HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 6.7.

[0340] 41-10. Preparation example of ADC-10 [ka] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.02 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.1 ml, 0.5 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 73, in DMA (10 mg / ml, 0.4 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-10 (3.0 mg / ml, 1.8 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.4.

[0341] 41-11. Preparation example of ADC-11 [ka] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.2 ml, 1 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 79, in DMA (10 mg / ml, 0.8 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-11 (5.7 mg / ml, 2.9 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.5.

[0342] 41-12. Preparation example of ADC-12 [ka] The anti-Her2 antibody Herceptin in His buffer (20 mM, pH 6) containing 5 mM EDTA (5 mg / ml, 0.83 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (2.5 ml, 12.5 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 135, in DMA (10 mg / ml, 16.6 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to yield anti-Her2 ADC-12 (11.1 mg / ml, 55.5 mg) in His (20 mM, pH 6) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.9.

[0343] 41-13. Preparation example of ADC-13 [ka] The anti-Her2 antibody Herceptin in His buffer (20 mM, pH 6) containing 5 mM EDTA (5 mg / ml, 0.8 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (2.7 ml, 16.6 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 127, in DMA (10 mg / ml, 20.7 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to yield anti-Her2 ADC-13 (11.3 mg / ml, 83.8 mg) in His (20 mM, pH 6) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.5.

[0344] 41-14. Preparation example of ADC-14 [ka] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.16 ml, 0.8 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 55, in DMA (10 mg / ml, 1 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to yield anti-Her2 ADC-14 (7.1 mg / ml, 4.3 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.3.

[0345] 41-15. Preparation example of ADC-15 [ka] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.16 ml, 0.8 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 54, in DMA (10 mg / ml, 1 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-15 (6.9 mg / ml, 3.4 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.4.

[0346] 41-16. Preparation example of ADC-16 [ka] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.2 ml, 1 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 58, in DMA (10 mg / ml, 0.8 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to yield anti-Her2 ADC-16 (5.7 mg / ml, 2.6 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.3.

[0347] 41-17. Preparation example of ADC-17 [ka] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.16 ml, 0.8 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 53, in DMA (10 mg / ml, 1 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to yield anti-Her2 ADC-17 (5.7 mg / ml, 2.8 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.5.

[0348] 41-18. Preparation example of ADC-18 [ka] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.16 ml, 0.8 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 60, in DMA (10 mg / ml, 1 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to yield anti-Her2 ADC-18 (9 mg / ml, 4.5 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.7.

[0349] 41-19. Preparation example of ADC-19 [ka] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.16 ml, 0.8 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 84, in DMA (10 mg / ml, 1 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-19 (5.5 mg / ml, 2.8 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.7.

[0350] 41-20. Preparation example of ADC-20 [ka] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.16 ml, 0.8 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 89, in DMA (10 mg / ml, 1 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to yield anti-Her2 ADC-20 (4.6 mg / ml, 2.7 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.7.

[0351] 41-21. Preparation example of ADC-21 [ka] The anti-Her2 antibody Herceptin in PBS buffer (20 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.16 ml, 0.8 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 94, in DMA (10 mg / ml, 1 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-21 (5.2 mg / ml, 3.1 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.6.

[0352] 41-22. Preparation example of ADC-22 [ka] The anti-Her2 antibody Herceptin in His buffer (20 mM, pH 6) containing 5 mM EDTA (3 mg / ml, 0.04 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.16 ml, 0.8 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 130, in DMA (10 mg / ml, 1 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to yield anti-Her2 ADC-22 (7.0 mg / ml, 3.5 mg) in His (20 mM, pH 6) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.8.

[0353] 41-23. Preparation example of ADC-23 [ka] The anti-Her2 antibody Herceptin in His buffer (20 mM, pH 6) containing 5 mM EDTA (5 mg / ml, 1 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (3 ml, 15 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 141, in DMA (10 mg / mM, 20 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to yield anti-Her2 ADC-23 (13.5 mg / ml, 108 mg) in His (20 mM, pH 6) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography-HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.9.

[0354] 41-24. Preparation example of ADC-24 [ka] The anti-Her2 antibody Herceptin in His buffer (10 mM, pH 6) containing 5 mM EDTA (5 mg / ml, 0.04 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.12 ml, 0.6 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 144, in DMA (10 mg / ml, 0.8 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to yield anti-Her2 ADC-24 (8.6 mg / ml, 4.3 mg) in His (20 mM, pH 6) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.3.

[0355] 41-25. Preparation example of ADC-25 [ka] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.02 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (0.06 ml, 0.3 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of camptothecin derivative-linker, compound 47, in DMA (10 mg / ml, 0.36 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-14 (3.3 mg / ml, 0.98 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography-HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 5.5.

[0356] 41-26. Preparation example of reference ADC (Herceptin-deruxtecan ADC) [ka] The anti-Her2 antibody Herceptin in His buffer (20 mM, pH 6) containing 5 mM EDTA (3 mg / ml, 0.48 μmol) was reduced by adding a solution of 5 mM TCEP (tris(2-carboxyethyl)phosphine) (1.92 ml, 9.6 μmol). The antibody / TCEP solution was incubated at 28°C for 2 hours. At the end of the reduction, the reaction solution was cooled to 25°C, and excess TCEP was removed by ultrafiltration using an amicon ultra filter. A solution of deruxtecan in DMA (10 mg / ml, 12 μmol) was then added to the reduced antibody solution. The reaction solution was incubated at 25°C for 2 hours, while the reduced antibody was conjugated to the toxin-linker via thiolmaleimide ligation. After conjugation was complete, the reaction mixture was desalted and concentrated by ultrafiltration using an Amicon Ultra filter to obtain the reference ADC (9.2 mg / mL, 35.4 mg) in His (20 mM, pH 6) buffer. The biochemical properties of the resulting ADC were characterized by size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine purity and aggregation levels, and by hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of drug molecules conjugated per antibody molecule is 7.7. A reference ADC with a DAR of 4.55 was prepared using the same procedure as the DAR7.7 ADC, except that the deruxtecan / antibody ratio used in the preparation was reduced to 10.

[0357] Example 42. In vitro cytotoxicity of camptothecin derivatives and ADCs in cancer cell lines expressing different Her2 expression levels The in vitro cytotoxicity of free camptothecin derivatives, Herceptin ADCs containing camptothecin derivatives, or anti-Trop-2 ADCs was tested in SK-BR-3 breast cancer cells, NCI-N87 gastric cancer cells, BT-474 breast cancer cells, NCI-H292 lung cancer cells, BxPC pancreatic cancer cells, and MDA-MB-468 breast cancer cells, along with the reference toxin DXd or the reference ADC (Herceptin-Doruxtecan ADC). Briefly, all cell lines were cultured in appropriate culture media at 37°C in a humidified incubator atmosphere with 5% CO2. Cells were plated in 96-well flat-bottom plates. Cell seeding numbers ranged from 3,000 cells / 100 μl / well to 6,000 cells / 100 μl / well. Cells were allowed to adhere overnight at 37°C in a humidified atmosphere with 5% CO2. Free camptothecin derivatives or Herceptin-ADC were prepared from stock solutions and diluted to the appropriate working concentrations 24 hours after cell seeding. Nine 10-fold serial dilutions were performed using culture medium. Final free camptothecin derivative concentrations ranged from 5,000 nM to 0.00005 nM, and Herceptin-ADC concentrations ranged from 1,000 nM to 0.00001 nM. Cells were incubated for 3 or 5 days. Cell Counting Kit-8 solution (Dojindo China Co., Ltd., Lot No. PL701) was added to the wells for 1 to 4 hours at 37°C, and absorbance at 450 nm was measured using a microplate reader (SpectraMax M5, Molecular Devices) and SoftMax Pro 5.4.1 software. Dose-response curves were generated, and IC50 values ​​were calculated using three-parameter curve fitting in GraphPad Prism 7.

[0358] Different camptothecin (CPT) derivatives and their in vitro cytotoxicity are listed in Table 4. ADCs and their in vitro cytotoxicity data are listed in Table 5.

[0359] Typically, Herceptin-36309 had a DAR of 5.5, Herceptin-363-11 had a DAR of 6.2, and Herceptin-DXd (reference ADC) had a DAR of 4.55 or 7.7. The ADCs were tested against BT-474 breast cancer cells, NCI-N87 gastric cancer cells, and SK-BR-3 breast cancer cells, which express high Her2, as well as BxPC-3 pancreatic cancer cells, NCI-H292 lung cancer cells, and MDA-MB-468 breast cancer cells, which express low levels / no Her2. In vitro cytotoxicity assays were performed. Free camptothecin derivatives were also tested in the same assays. Briefly, all cell lines were cultured in appropriate culture media at 37°C in a humidified incubator atmosphere of 5% CO2. Cells were plated in 96-well flat-bottom plates. Cell seeding numbers ranged from 500 cells / 100 μl / well to 6,000 cells / 100 μl / well. Cells were allowed to adhere overnight at 37°C in a humidified atmosphere of 5% CO2. ADCs or free camptothecin derivatives were prepared from stock solutions and diluted to the appropriate working concentrations 24 hours after cell seeding. Seven 10-fold serial dilutions were performed using culture medium. Final concentrations ranged from 10,000 nM to 0.001 nM. Cells were incubated with ADCs for 5 days. Cell Counting Kit-8 solution (Dojindo China Co., Ltd., Lot No. PL701) was added to the wells for 1 to 4 hours at 37°C, and absorbance at 450 nm was measured using a microplate reader (SpectraMax M5, Molecular Devices) and SoftMax Pro 5.4.1 software. Dose-response curves were generated and IC50s were calculated using three-parameter curve fitting in GraphPad Prism7.

[0360] Figures 3A-D show representative killing curves of free camptothecin derivatives (T-363, DXd) and ADCs containing 36309 or DXd in SK-BR-3, NCI-N87, MDA-MB-468, and BxPC3 cells.

[0361] Figures 4-9 show representative killing curves of free camptothecin derivatives (T-363, DXd) and ADCs containing 363-11 or DXd in BT-474 (Figure 4), NCI-N87 (Figure 5), SK-BR-3 (Figure 6), BxPC-3 (Figure 7), NCI-H292 (Figure 8), and MDA-MB-468 (Figure 9). Overall, ADCs containing various camptothecin derivatives demonstrate specific and potent in vitro killing activity in Her2-expressing cells.

[0362] Tables 6 and 7 summarize IC50 values ​​from in vitro cytotoxicity assays for camptothecin derivatives (T-363 and DXd) and representative ADCs. ADCs generated from the Val-Ala-PABC linker payload 36309 exhibited potent cytotoxicity in Her2-positive cells, but had a narrower therapeutic window compared to DXd, as indicated by some nonspecific killing of Herceptin-36309 in Her2-negative cells (Table 6). In addition, PABC is a hydrophobic moiety, which contributes to the aggregation issues observed with the high-DAR ADC of Herceptin-36309. Indeed, the results showed that Herceptin-363-11 and Herceptin-DXd induced potent cytotoxicity against highly Her2-expressing BT-474, SK-BR-3, and NCI-N87 tumor cells, with IC50 values ​​in the sub-nM range. The killing activity of Herceptin-363-11 was comparable to or better than that of Herceptin-DXd. In Her2-negative BxPC-3, MDA-MB-468, and NCI-H292 cells, the IC50s of both ADCs were above 100 nM. On the other hand, the free toxins T-363 and DXd were able to kill all three cell lines with similar IC50s (Table 7). These in vitro cytotoxicity results suggest a wide therapeutic window for the 363-11-containing ADCs. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 6] [Table 7]

[0363] Example 43. Bystander killing effect The ability of an ADC to kill adjacent antigen-negative cells surrounding antigen-positive cells is called the bystander effect. To confirm whether the camptothecin derivative-containing Herceptin ADC induces bystander killing, a flow cytometry-based cell killing assay was performed. Exponentially growing HER2-positive cells NCl-N87 and HER2-negative cells MDA-MB-468 were separately harvested, counted, and suspended in culture medium at an adjusted cell density. The two types of cells were then seeded in the same well of a 24-well cell culture plate at a 1:1 ratio and incubated overnight at 37°C. Herceptin-ADC was then added at a concentration of 10 nM per well. The cells were further incubated in the presence of the ADC at 37°C for 5 days. At the end of the incubation period, the cells were harvested, transferred to a round-bottom 96-well plate, rinsed with PBS, and resuspended in DPBS. Anti-Herceptin antibody was added and incubated for 60 minutes, followed by the addition of goat anti-human IgG H&L (FITC) antibody to detect HER2-positive cells. Flow cytometry analysis was performed using a BD Fortessa flow cytometer, and the number of HER2-positive and HER2-negative cells was analyzed using FlowJo software. The bystander killing effect was evaluated by the survival rate of antigen-negative cells (e.g., HER2-negative cells). This was calculated as the antigen-negative cells surviving after ADC treatment / the total number of antigen-negative cells used. In some experiments, the bystander killing effect of ADC was evaluated using the procedure described below.

[0364] To determine whether Herceptin-363-11 induces bystander killing, a transwell coculture cell killing assay was performed. Cell culture inserts were placed in a 24-well plate to create chambers. Her2-positive SK-BR-3 cells were seeded in the upper chamber, and Her2-negative MDA-MB-468 cells were placed in the lower chamber. The chambers were separated by a membrane, which restricts cell movement between the chambers but not the movement of soluble factors. Cells were incubated in the presence of ADC. The ADC concentrations were 1, 10, and 100 nM. After 5 days in culture, the remaining MDA-MB-468 cells in the lower chamber were analyzed by Cell Counting Kit-8 assay as described in Example 42. The percentage of growth inhibition was calculated by comparing with untreated samples. SK-BR-3 cells or MDA-MB-468 cells alone were also plated in a 24-well plate and treated with ADC for comparison. As shown in Figures 10A-10B, Herceptin-363-11 potently inhibited growth of Her2-positive SK-BR-3 cells but induced only minimal cell killing in Her2-negative MDA-MB-468 cells at concentrations above 10 nM. However, when both cells were cocultured in a transwell system, Herceptin-363-11 exhibited significant growth inhibitory effects on MDA-MB-468 cells. Because the ADC possesses Her2-specific cytotoxicity, the killing of MDA-MB-468 cells may have been caused by the released T-363, suggesting a bystander killing effect of Herceptin-363-11. Herceptin-DXd was included as a positive control. Herceptin-363-11 showed a similar bystander effect compared to Herceptin-DXd.

[0365] Relevant bystander killing efficacy data are shown in Table 8. [Table 8] It can be seen that ADCs including ADC-7, ADC-12, ADC-14, ADC-15, ADC-16, ADC-17, ADC-18, ADC-19, ADC-20, ADC-21, and ADC-23 exhibit strong bystander killing effects, whereas ADC-5, ADC-11, and ADC-22 exhibit moderate or no bystander killing effects.

[0366] Example 44. Plasma stability of Herceptin-363-11 To evaluate the in vitro plasma stability of the ADC, Herceptin-363-11 (0.1 mg / ml) was incubated in blank human plasma at 37°C for up to 96 hours. At each time point, samples were removed, and the remaining amounts of total antibody (naked + conjugated) and conjugated antibody (ADC) were measured using a quantitative sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, to measure total antibody, human Her2 protein was coated onto a microplate to capture Herceptin antibody. After removing unbound antibody, bound total Herceptin antibody was detected using a horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc-specific polyclonal antibody. For measurement of the Herceptin-363-11 ADC alone, a mouse anti-DXd antibody was added as a secondary antibody. The concentration of Herceptin-363-11 ADC was then detected using an HRP-conjugated goat anti-mouse IgG (H+L) antibody. Figure 11 shows the percentage of remaining total antibody and Herceptin-363-11 at different time points. The trend of Herceptin-363-11 was similar to that of total antibody. 79% of Herceptin-363-11 remained after 96 hours. These results indicate that Herceptin-363-11 is stable in human plasma.

[0367] Example 45. In Vivo Antitumor Activity The antitumor activity of Herceptin ADCs containing camptothecin derivatives (ADC-4, ADC-5, ADC-14, ADC-22, and ADC-23) was evaluated using mouse xenograft models of Her2-positive NCI-N87, JIMT-1, and HCC1569 cells. Five million NCI-N87, JIMT-1, or HCC1569 cells were harvested from culture flasks and subcutaneously implanted into the right flank of 6- to 7-week-old BALB / c nude mice (Shanghai SLAC Laboratory Animal Co., Ltd.) or NSG mice (Shanghai Model Organisms Center, Inc.). Tumors were approximately 150-200 mm. 3 When tumors reached a size of 100 μg / kg, five mice from each tumor model were randomly assigned to different groups. These groups included a Herceptin ADC group, a reference ADC group, and a vehicle control group, where the vehicle control was injected with the buffer used to dilute the ADC and served as a negative control. The ADC or vehicle control was administered intravenously via the lateral tail vein at the indicated doses. Tumor volume was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: 1 / 2 × L × W 2 ;Calculated using the formula where L = length and W = width.

[0368] The results are shown in Figures 12 to 16. Figures 12 to 16 show in vivo tumor volume changes after ADC administration for the JIMT-1, NCI-N87, and HCC-1569 models. It can be seen that all of the tested ADCs (ADC-4, ADC-5, ADC-14, ADC-22, and ADC-23) demonstrated potent antitumor activity in human xenograft tumor models.

[0369] Example 46. In vivo tolerability and safety Healthy adult female CD-1 (ICR) mice were used to determine the toxicity profile of camptothecin derivative ADCs. Mice were purchased from Charles River Laboratories (Beijing, China). After 1 week of adaptive feeding, mice were randomized based on body weight into groups (n = 5 per group) using a serpentine layout: blank control, negative control (PBS group), and 160 mg / kg treatment group (ADC group). For the negative and ADC treatment groups, PBS or ADCs diluted in PBS (ADC-12, ADC-14, ADC-19, ADC-20, ADC-21, ADC-22, and ADC-23) were intravenously injected via the tail vein. For the blank control group, no injection was performed. Whole blood was collected via retroorbital bleeding at various time points (e.g., 3 and 7 days after administration, or 2 and 5 days after administration) and transferred to EDTA-K2 tubes for further analysis. Complete blood counts were determined using an XN-1000V automated hematology analyzer (Sysmex Asia Pacific Pte Ltd., Japan). Nine hematological parameters were analyzed: red blood cells, hemoglobin, platelets, reticulocytes, white blood cells, lymphocytes, neutrophils, monocytes, eosinophils, and basophils. Mouse weights were also monitored throughout the experiment.

[0370] The results are shown in Figures 17A-B to 23A-B. Figures 17A-B to 23A-B show the in vivo tolerability and safety of various ADCs.

[0371] ADC-14, ADC-19, ADC-20, and ADC-21 showed slight decreases in body weight and WBC, neutrophil, and lymphocyte counts that rapidly recovered within 7 days, indicating good tolerability and safety even at significantly higher doses. ADC-22 and 23 showed good tolerability and safety, with no changes in body weight and WBC counts observed even at very high doses.

[0372] All of the articles and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the articles and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the articles and methods without departing from the spirit and scope of the present disclosure. All such modifications and equivalents, whether now existing or later developed, apparent to those skilled in the art are deemed to be within the spirit and scope of the present disclosure as defined by the appended claims. All patents, patent applications, and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All patents, patent applications, and publications are herein incorporated by reference in their entireties for all purposes to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The present disclosure illustratively described herein may suitably be practiced in the absence of any element or elements not specifically disclosed herein. Thus, while the present disclosure has been specifically disclosed by preferred embodiments and optional features, it is to be understood that modifications and variations of the concepts disclosed herein may be utilized by those skilled in the art, and that such modifications and variations are deemed to be within the scope of the present invention as defined by the appended claims.

Claims

1. The following structural formula (A): 【Chemistry 191】 a camptothecin (CPT) derivative represented by the formula: wherein X is -C(=O)-(CH ２ , ２ , ２ , ３ )(n １ -O-N(R ２ )R ３ , -C(=O)-(CH ２ )(n １ -N(OR ２ )R ３ , -C(=O)-(CH ２ )(n １ -O-(CH ２ )(n １ -N(R ２ )R ３ , -C(=O)-(C(R ４ )(R ５ ))(n １ -N(R ２ )R ３ , -S(=O ２ -CH ２ -(CH ２ )(n １ -O-N(R ２ )R ３ , -S(=O ２ -CH ２ -(CH ２ )(n​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ ２ ), R ３ , -C(=O)-N(R ２ )-(CH ２ )n ２ -N(R ２ )R ３ , -C(=O)-NH-(CH ２ )n ２ -O-R ３ , -C(=O)-NH-(CH ２ )n ２ -S-R ３ , -C(=O)-O-(CH ２ )n ２ -O-R ３ , -C(=O)-S-(CH ２ )n ２ -O-R ３ , -C(=O)-S-(CH ２ )n ２ -S-R ３ , -C(=O)-O-(CH ２ )n ２ -S-R ３ , -C(=O)-(C(R ４ )(R ５ )n １ -N(R ２ )-C(=O)-(C(R ａ )(R ｂ )n ３ -N(R ｃ )-R ３ , -C(=O)-(C(R ４ )(R ５ )n １ -N(R ２ )-C(=O)-(C(R ａ )(R ｂ )n ３ -O-R ３ , -C(=O)-(C(R ４ )(R ５ )n １ -N(R ２ )-C(=O)-(C(R ａ )(R ｂ )n ３ -S-R ３ selected from; R １ , R ２ , R ３ and R ｃ are each independently a hydrogen atom or C １ -C ６ is alkyl; R ４ and R ５ are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl halide, an alkyl deuterate, an alkoxy, a hydroxyl, an amino, a nitro, a cyano, a hydroxyalkyl, a heterocyclic C １ -C ６ represents an alkyl or a 3- to 6-membered heterocyclic or cycloaryl ring; or R ４ and R ５ together with the carbon atoms form a 3- to 6-membered cyclic ring; R ａ and R ｂ are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl halide, an alkyl deuterate, an alkoxy, a hydroxyl, an amino, a nitro, a cyano, a hydroxyalkyl, a heterocyclic C １ -C ６ represents an alkyl or a 3- to 6-membered heterocyclic or cycloaryl ring; or R ａ and R ｂ together with the carbon atoms form a 3- to 6-membered cyclic ring; n １ is 1, 2, 3, 4 or 5; n ２ is 2, 3, 4 or 5; n ３ is 1, 2, 3, 4 or 5, or a pharmaceutically acceptable salt, stereoisomer, enantiomer or deuterated form thereof.

2. Xが、-C(=O)-(CH ２ )n １ -O-N(R ２ )R ３ 、-C(=O)-(CH ２ )n １ -N(OR ２ )R ３ 、-C(=O)-(CH ２ )n １ -O-(CH ２ )n １ -N(R ２ )R ３ 、-C(=O)-(C(R ４ )(R ５ ))n １ -N(R ２ )R ３ 、-S(=O) ２ -CH ２ -(CH ２ )n １ -O-N(R ２ )R ３ 、-S(=O) ２ -CH ２ -(CH ２ )n １ -N(OR ２ )R ３ 、-S(=O) ２ -CH ２ -(CH ２ )n １ -N(R ２ )R ３ 、-C(=O)-O-(CH ２ )n ２ -O-N(R ２ )R ３ 、-C(=O)-O-(CH ２ )n ２ -N(OR ２ )R ３ 、-C(=O)-O-(CH ２ )n ２ -N(R ２ )R ３ 、-C(=O)-NH-(CH ２ )n ２ -O-N(R ２ )R ３ 、-C(=O)-NH-(CH ２ )n ２ -N(OR ２ ), R ３ , -C(=O)-N(R ２ )-(CH ２ )n ２ -N(R ２ )R ３ , -C(=O)-NH-(CH ２ )n ２ -O-R ３ , -C(=O)-NH-(CH ２ )n ２ -S-R ３ , -C(=O)-O-(CH ２ )n ２ -O-R ３ , -C(=O)-S-(CH ２ )n ２ -O-R ３ , -C(=O)-S-(CH ２ )n ２ -S-R ３ , -C(=O)-O-(CH ２ )n ２ -S-R ３ selected from Preferably, X is —C(═O)—(CH ２ ) n １ -O-N(R ２ ) R ３ , -C(=O)-(CH ２ ) n １ -O-(CH ２ ) n １ -N(R ２ ) R ３ , -C(=O)-(C(R ４ ) (R ５ ))n １ -N(R ２ ) R ３ , -S(=O) ２ -CH ２ - (CH ２ ) n １ -O-N(R ２ ) R ３ , -S(=O) ２ -CH ２ - (CH ２ ) n １ -N(R ２ ) R ３ , -C(=O)-O-(CH ２ ) n ２ -O-N(R ２ ) R ３ , -C(=O)-O-(CH ２ ) n ２ -N(R ２ ) R ３ , -C(=O)-NH-(CH ２ ) n ２ -O-N(R ２ ) R ３ , -C(=O)-N(R ２ )-(CH ２ ) n ２ -N(R ２ ) R ３ Selected from: R ２ is a hydrogen atom; R ３ But C １ -C ６ is alkyl; R ４ and R ５ 2. The camptothecin derivative, its pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form according to claim 1, wherein each independently represents a hydrogen atom, a deuterium atom, a halogen, hydroxyl, amino, nitro, or cyano.

3. where X is -C(=O)-(CH ２ )n １ -O-N(R ２ )R ３ 、-C(=O)-(CH ２ )n １ -N(OR ２ )R ３ 、-C(=O)-(CH ２ )n １ -O-(CH ２ )n １ -N(R ２ )R ３ 、-C(=O)-(C(R ４ )(R ５ )n １ -N(R ２ )R ３ 、-S(=O) ２ -CH ２ -(CH ２ )n １ -O-N(R ２ )R ３ 、-S(=O) ２ -CH ２ -(CH ２ )n １ -N(OR ２ )R ３ 、-S(=O) ２ -CH ２ -(CH ２ )n １ -N(R ２ )R ３ 、-C(=O)-O-(CH ２ )n ２ -O-N(R ２ )R ３ 、-C(=O)-O-(CH ２ )n ２ -N(OR ２ )R ３ 、-C(=O)-O-(CH ２ )n ２ -N(R ２ )R ３ 、-C(=O)-NH-(CH ２ )n ２ -O-N(R ２ )R ３ 、-C(=O)-NH-(CH ２ )n ２ -N(OR ２ ), R ３ , -C(=O)-N(R ２ )-(CH ２ )n ２ -N(R ２ )R ３ , -C(=O)-NH-(CH ２ )n ２ -O-R ３ , -C(=O)-NH-(CH ２ )n ２ -S-R ３ , -C(=O)-O-(CH ２ )n ２ -O-R ３ , -C(=O)-S-(CH ２ )n ２ -O-R ３ , -C(=O)-S-(CH ２ )n ２ -S-R ３ , -C(=O)-O-(CH ２ )n ２ -S-R ３ selected from Preferably, X is —C(═O)—(CH ２ ) n １ -O-N(R ２ ) R ３ , -C(=O)-(CH ２ ) n １ -O-(CH ２ ) n １ -N(R ２ ) R ３ , -C(=O)-(C(R ４ ) (R ５ ))n １ -N(R ２ ) R ３ , -S(=O) ２ -CH ２ - (CH ２ ) n １ -O-N(R ２ ) R ３ , -S(=O) ２ -CH ２ - (CH ２ ) n １ -N(R ２ ) R ３ , -C(=O)-O-(CH ２ ) n ２ -O-N(R ２ ) R ３ , -C(=O)-O-(CH ２ ) n ２ -N(R ２ ) R ３ , -C(=O)-NH-(CH ２ ) n ２ -O-N(R ２ ) R ３ , -C(=O)-N(R ２ )-(CH ２ ) n ２ -N(R ２ ) R ３ Selected from: R ２ is a hydrogen atom; R ３ But C １ -C ６ is alkyl; R ４ and R ５ are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl halide, an alkyl deuterate, an alkoxy, a hydroxyl, an amino, a nitro, a cyano, a hydroxyalkyl, a heterocyclic C １ -C ６ represents an alkyl or a 3- to 6-membered heterocyclic or cycloaryl ring; or R ４ and R ５ The camptothecin derivative according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form thereof, wherein, together with the carbon atom, forms a 3- to 6-membered cyclic ring.

4. Xが、-C(=O)-(CH ２ )n １ -O-N(R ２ )R ３ 、-C(=O)-(CH ２ )n １ -N(OR ２ )R ３ 、-C(=O)-(CH ２ )n １ -O-(CH ２ )n １ -N(R ２ )R ３ 、-C(=O)-(C(R ４ )(R ５ ))n １ -N(R ２ )R ３ 、-S(=O) ２ -CH ２ -(CH ２ )n １ -O-N(R ２ )R ３ 、-S(=O) ２ -CH ２ -(CH ２ )n １ -N(OR ２ )R ３ 、-S(=O) ２ -CH ２ -(CH ２ )n １ -N(R ２ )R ３ 、-C(=O)-O-(CH ２ )n ２ -O-N(R ２ )R ３ 、-C(=O)-O-(CH ２ )n ２ -N(OR ２ )R ３ 、-C(=O)-O-(CH ２ )n ２ -N(R ２ )R ３ 、-C(=O)-NH-(CH ２ )n ２ -O-N(R ２ )R ３ 、-C(=O)-NH-(CH ２ )n ２ -N(OR ２ ), R ３ , -C(=O)-N(R ２ )-(CH ２ )n ２ -N(R ２ )R ３ , -C(=O)-NH-(CH ２ )n ２ -O-R ３ , -C(=O)-NH-(CH ２ )n ２ -S-R ３ , -C(=O)-O-(CH ２ )n ２ -O-R ３ , -C(=O)-S-(CH ２ )n ２ -O-R ３ , -C(=O)-S-(CH ２ )n ２ -S-R ３ , -C(=O)-O-(CH ２ )n ２ -S-R ３ selected from Preferably, X is —C(═O)—(CH ２ ) n １ -O-N(R ２ ) R ３ , -C(=O)-(CH ２ ) n １ -O-(CH ２ ) n １ -N(R ２ ) R ３ , -C(=O)-(C(R ４ ) (R ５ ))n １ -N(R ２ ) R ３ , -S(=O) ２ -CH ２ - (CH ２ ) n １ -O-N(R ２ ) R ３ , -S(=O) ２ -CH ２ - (CH ２ ) n １ -N(R ２ ) R ３ , -C(=O)-O-(CH ２ ) n ２ -O-N(R ２ ) R ３ , -C(=O)-O-(CH ２ ) n ２ -N(R ２ ) R ３ , -C(=O)-NH-(CH ２ ) n ２ -O-N(R ２ ) R ３ , -C(=O)-N(R ２ )-(CH ２ ) n ２ -N(R ２ ) R ３ Selected from: R ２ and R ３ are each a hydrogen atom; R ４ and R ５ are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl halide, an alkyl deuterate, an alkoxy, a hydroxyl, an amino, a nitro, a cyano, a hydroxyalkyl, a heterocyclic C １ -C ６ represents an alkyl or a 3- to 6-membered heterocyclic or cycloaryl ring; or R ４ and R ５ The camptothecin derivative according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form thereof, wherein, together with the carbon atom, forms a 3- to 6-membered cyclic ring.

5. Xが、-C(=O)-(C(R ４ )(R ５ ))n １ -N(R ２ )-C(=O)-(C(R ａ )(R ｂ ))n ３ -N(R ｃ )-R ３ 、-C(=O)-(C(R ４ )(R ５ ))n １ -N(R ２ )-C(=O)-(C(R ａ )(R ｂ ))n ３ -O-R ３ 、-C(=O)-(C(R ４ )(R ５ ))n １ -N(R ２ )-C(=O)-(C(R ａ )(R ｂ ))n ３ -S-R ３ to be selected from Preferably, X is —C(═O)—(C(R ４ ) (R ５ ))n １ -N(R ２ )-C(=O)-(C(R ａ ) (R ｂ ))n ３ -N(R ｃ )-R ３ , -C(=O)-(C(R ４ ) (R ５ ))n １ -N(R ２ )-C(=O)-(C(R ａ ) (R ｂ ))n ３ -O-R ３ Selected from: R ２ , R ３ , and R ｃ are each independently a hydrogen atom or C １ -C ６ is alkyl; R ４ and R ５ are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl halide, an alkyl deuterate, an alkoxy, a hydroxyl, an amino, a nitro, a cyano, a hydroxyalkyl, a heterocyclic C １ -C ６ represents an alkyl or a 3- to 6-membered heterocyclic or cycloaryl ring; or R ４ and R ５ together with the carbon atoms form a 3- to 6-membered cyclic ring; R ａ and R ｂ are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl halide, an alkyl deuterate, an alkoxy, a hydroxyl, an amino, a nitro, a cyano, a hydroxyalkyl, a heterocyclic C １ -C ６ represents an alkyl or a 3- to 6-membered heterocyclic or cycloaryl ring; or R ａ and R ｂ The camptothecin derivative according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form thereof, wherein, together with the carbon atom, forms a 3- to 6-membered cyclic ring.

6. The camptothecin derivative is 【Chemistry 192-1】 【Chemistry 192-2】 6. The camptothecin derivative according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form thereof, selected from any one of the following:

7. A pharmaceutical composition comprising the camptothecin derivative according to any one of claims 1 to 6, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form thereof, and a pharmaceutically acceptable diluent, carrier, or excipient.

8. A camptothecin derivative conjugate comprising at least one of the camptothecin derivative according to any one of claims 1 to 6, a pharmaceutically acceptable salt, a stereoisomer, an enantiomer, or a deuterated form thereof; or a pharmaceutically acceptable salt thereof.

9. 10. The camptothecin derivative conjugate of claim 8, comprising: (1) at least one of the camptothecin derivative of any one of claims 1 to 6, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form thereof; (2) a linker; and (3) a targeting agent; or a pharmaceutically acceptable salt thereof.

10. 10. The camptothecin derivative conjugate or a pharmaceutically acceptable salt thereof according to claim 9, wherein the linker is selected from the group consisting of a cleavable linker and a non-cleavable linker, and the targeting agent is selected from an antibody or an antigen-binding fragment thereof, a peptide, an RNA, and a DNA molecule.

11. 11. The camptothecin derivative conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 9 to 10, wherein the linker is selected from the group consisting of a peptidase cathepsin-sensitive linker, an acid-sensitive linker, a glutathione-sensitive linker, a sulfatase-sensitive linker, a lysosomal protease-sensitive linker, a beta-glucuronide linker, a phosphatase-sensitive linker, and a pyrophosphatase-sensitive linker.

12. 12. The camptothecin derivative conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 9 to 11, wherein the targeting agent is capable of binding to a target selected from the group consisting of a tumor-associated antigen (TAA), a tissue-specific antigen, a cell surface molecule, an extracellular matrix protein or protease(s), and any post-translationally modified residue(s).

13. The target is selected from the group consisting of Trop-2, Her2, Her3, Her4, EGF, EGFR, CD2, CD3, CD5, CD7, CD13, CD19, CD20, CD21, CD23, CD30, CD33, CD34, CD38, CD46, CD55, CD59, CD69, CD70, CD71, CD97, CD117, CD123, CD127, CD134, CD137, CD138, CD146, CD147, CD152, CD154, CD174, CD195, CD200, CD205, CD212, CD22 3, CD227, CD253, CD272, CD274, CD276, CD278, CD279, CD309, CD319, CD326, CD340, DR6, Kv1. 3, 5E10, MUC1, uPA, MAGE3, MUC16, KLK3, K-ras, mesothelin, p53, survivin, G250, PSMA, endoplasmin, BCMA, GPNMB, EphA2, EphB2, TMEFF2, integrin beta 6, 5T4, CA9, IGF-1R, Axl, B7H3, B7H4, CDH6, HAVCR1, STEAP-1, STEAP-2, UPK2, CLDN18, CLDN6, CLDN9, c-Met, MICA, LIV-1, ROR1, ADAM9, Stn, DLK-1, and CEACAM-5.

14. 14. The camptothecin derivative conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 12 to 13, wherein the targeting agent is selected from the group consisting of an anti-Her2 antibody or a binding fragment thereof, and an anti-Trop-2 antibody or a binding fragment thereof.

15. The camptothecin derivative conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 9 to 14, wherein the targeting agent is a multispecific antibody.

16. The camptothecin derivative conjugate has the following formula (I): Targeted chemical - (L-D) ｎ (I) is represented by wherein n is an integer from 1 to 24; D represents at least one of the camptothecin derivatives according to any one of claims 1 to 6, pharmaceutically acceptable salts thereof, stereoisomers, enantiomers, and deuterated forms thereof; L is a group represented by the following formula (IIa and IIb): L １ -8 ２ -8 ３ - (A) L １ -8 ２ - (A) a linker comprising a peptide portion of 2 to 8 amino acids represented by In the formula, L １ is a linker moiety attached to the targeting agent, and L １ comprises a reactive functional group selected from maleimide, bromoacetyl, iodoacetyl, thiol, amino, alkyl bromide, alkyl iodide, allenamide, carboxyl, and NHS ester; L ２ is a linker moiety comprising a 2-8 amino acid peptide and optionally a spacer, preferably a PEG spacer; L ３ is a linker moiety connected to the camptothecin derivative, and L ３ but the following: 【Chemistry 193】 The camptothecin derivative conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 8 to 15, comprising at least one of:

17. Said L １ is -C(=O)-(CH ２ ) ｎ１ -, and -C(=O)-(CH ２ ) n １ -R ８ -[O-(CH ２ ) ｎ１ ] ｎ１ -N(R ２ )-C(=O)-)-(CH ２ ) ｎ１ - selected from; R ８ is alkylene-aryl-alkylene, aryl, 3- to 7-membered heterocyclyl, 3- to 7-membered cycloalkyl, heteroaryl, -alkylene-heteroaryl-alkylene-; n １ 17. The camptothecin derivative conjugate of claim 16, or a pharmaceutically acceptable salt thereof, wherein is 1, 2, 3, 4 or 5.

18. L is the following: 【Chemistry 194-1】 【Chemistry 194-2】 wherein n=1 to 12, preferably 1 to 8.

19. L ２ is a dipeptide, tripeptide, or tetrapeptide comprising a naturally occurring amino acid and a non-naturally occurring amino acid, or a pharmaceutically acceptable salt thereof.

20. L ２ is selected from gly-gly, gly-gly-gly, phe-lys, val-ala, val-cit, gly-gly-phe-gly (GGGFG) (SEQ ID NO: 1), val-cit-gly, val-gln-gly, val-glu-gly, phe-lys-gly, glu-val-ala, glu-val-cit, β-ala-gly-phe-gly (AGGFG) (SEQ ID NO: 2), and gly-gly-phe-gly-gly (GGGFGG) (SEQ ID NO: 3), wherein the amino acid sequence is in either direction.

21. The camptothecin derivative conjugate has the following structure: 【Chemistry 195-1】 【Chemistry 195-2】 【Chemistry 195-3】 【Chemistry 195-4】 【Chemistry 195-5】 【Chemistry 195-6】 【Chemistry 195-7】 【Chemistry 195-8】 【Chemistry 195-9】 【Chemistry 195-10】 【Chemistry 195-11】 【Chemistry 195-12】 【Chemistry 195-13】 The camptothecin derivative conjugate according to any one of claims 16 to 19, or a pharmaceutically acceptable salt thereof, comprising any one of:

22. 22. The camptothecin derivative conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 16 to 21, wherein the ratio of the camptothecin derivative to the targeting agent is from 4 to 12.

23. The following structure: 【Chemistry 196-1】 【Chemistry 196-2】 【Chemistry 196-3】 【Chemistry 196-4】 【Chemistry 196-5】 【Chemistry 196-6】 23. The camptothecin derivative conjugate of claim 22, or a pharmaceutically acceptable salt thereof, comprising any one of:

24. A pharmaceutical composition comprising the camptothecin derivative conjugate according to any one of claims 8 to 23 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier, or excipient.

25. A method for making the camptothecin derivative conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 8 to 23, comprising reacting a targeting agent and / or a linker with a camptothecin derivative.

26. A method for treating or preventing a disease, comprising administering to a subject in need of such treatment or prevention a therapeutically effective amount of the camptothecin derivative, or a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated form thereof, according to any one of claims 1 to 6.

27. A method for treating or preventing a disease, comprising administering to a subject in need of such treatment or prevention a therapeutically effective amount of the camptothecin derivative conjugate according to any one of claims 8 to 23, or a pharmaceutically acceptable salt thereof.

28. 28. The method of any one of claims 26 to 27, further comprising administering a secondary treatment to the subject.

29. 29. The method of any one of claims 26 to 28, wherein the disease is selected from the group consisting of a proliferative disorder, an autoimmune disorder, a destructive bone disorder, an infectious disease, a viral disease, a fibrotic disease, a neurodegenerative disorder, pancreatitis, and a kidney disease.

30. 30. The method of claim 29, wherein the proliferative disorder is a cancer selected from the group consisting of pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, and rhabdomyosarcoma.

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