Formulations of conjugates of tubulicin analogs and cell-binding molecules

Abstract

To provide a pharmaceutical composition of a conjugate of a tubulysin analog and a cell surface binding ligand for longer stability and storage as well as easier shipment.SOLUTION: Provided is a pharmaceutical composition that is a liquid composition prior to lyophilization, or in a formulated lyophilized solid, or the re-constituted formulation from the lyophilized solid, comprising the following compositions: a conjugate of a tubulysin analog to a cell-binding agent of Formula (I); a polyol selected from sucrose or trehalose; a surfactant selected from polysorbates; 1 or more preservatives; 1 or more amino acids; 1 or more antioxidants; one or more metal chelating agents; one or more buffer salts to adjust the pH of the formulation from pH 4.5 to 8.5; and one or more of isotonic agent for adjusting osmotic pressure between about 250 to 350 mOsm after reconstituted for administration to a patient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to formulations of conjugates of tubulicin analogs and cell surface receptor-binding molecules for targeted therapy. The invention also relates to the use of compositions comprising cell-binding molecule-tubulicin analog conjugates for the treatment of cancer, autoimmune diseases, and infections. [Background technology]

[0002] Targeted delivery of highly active cytotoxic drugs to specific disease sites in the human body via antibodies or other cell surface receptor conjugates has proven to be a very promising therapy, significantly expanding the therapeutic window for cytotoxic agents (Non-Patent Literature 1). In particular, since the US FDA approved Adcetris (brentuximab vedotin) in 2011 and Kadcyla (adtrastuzumab emtansine) in 2013, most large pharmaceutical and biotechnology companies have adopted the use of antibody-drug conjugates (ADCs) for cancer targeted therapy (Non-Patent Literature 2-4). To date, most ADCs in clinical evaluation utilize meitansinoids or auristatins, which are potent tubulin-interacting agents. A small number of clinical ADCs incorporate other potent effector molecules, such as the topoisomerase 1 inhibitor SN-38 or the DNA-interacting substances calicheamicin and pyrrolobenzodiazepines (Non-Patent Literature 5 and 6).

[0003] Several short-chain peptide compounds that have been found to possess biological activity have been isolated from natural sources. One of them, tubulicin (structure shown below), was initially isolated by Hofle and Reichenbach et al. (GBF Braunschweig) from a culture of the myxobacterium Archangim gephyra (Non-Patent Document 7 and Patent Document 1), and is a member of the anti-mitotic peptide group that inhibits the polymerization of tubulin in dividing cells, thereby inducing cell death. These anti-mitotic peptides are at the forefront of targeted therapy due to their superior potency compared to vinblastine, taxol, and epothyrons. Structurally, tetrapeptide tubulicins contain N-methylpipecolic acid (Mep) at the N-terminus, isoleucine (Ile) as the second residue, a unique thiazole-containing tubevaline (Tuv) as the third residue, and two possible γ-amino acids (tubu tyrosine (Tut) or tubu phenylalanine (Tup)) at the C-terminus. Recently, several tubulicins have been synthesized, but at doses necessary to achieve therapeutic effects, existing tubulicins exhibit significant general toxicity (>20% weight loss in animals), impairing their efficacy (Patent Document 2). We are interested in the field of cell surface-bound ligand conjugates, particularly the use of antibodies conjugated with tubulicin derivatives, as they generally have low toxicity and excellent therapeutic effects. Natural tubulicins exhibit IC50 at tens of picomoles for many cell lines. 50 While natural tubulicins are ideal payloads for ADCs due to their extremely potent efficacy within a certain range, it has been found that they are poorly metabolized in animal livers and are prone to causing severe hepatotoxicity. Tubulicin analogs, such as using 2-(dimethylamino)-2-methylpropionic acid instead of 1-methylpiperidine-2-carboxylic acid at the left end of the natural tubulicin structure, reduce the toxicity of the conjugate to the liver without compromising the efficacy of the antibody-conjugated compound.

[0004]

number

[0005] Antibody-drug conjugates and other protein-based molecules such as macrocomplex molecules are not suitable for oral administration because they are destroyed in the digestive system, and are generally administered parenterally, such as by intravenous or subcutaneous injection. Therefore, these protein-based drugs need to be administered in liquid form. However, ADCs and other macrocomplex protein molecules usually have poor stability in aqueous environments, which can shorten the drug's shelf life and significantly reduce its market value. Therefore, solid ADC pharmaceutical compositions can be re-formulated with a solvent immediately before administration to the patient, which would be more advantageous from the standpoint of stability, storage, and transport convenience. Solid protein-based pharmaceutical compositions include powders, lyophilized (or freeze-dried), spray-dried, spray-lyophilized, vacuum-dried, or supercritical fluid-dried components. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication WO98 / 13375 [Patent Document 2] U.S. Patent Application Publication No. 2010 / 0048490 [Non-patent literature]

[0007] [Non-Patent Document 1] Van den Mooter, T. et al Expert Opin Biol Ther. 2015, 15, 749-60 [Non-Patent Document 2] Chari, R. et al, Angew. Chem., Int. Ed. 2014, 53, 3796-3827 [Non-Patent Document 3] Sievers, EL et al. Annu Rev Med. 2013, 64, 15-29 [Non-Patent Document 4] Mehrling, T. Future Oncol, 2015, 11, 549 [Non-Patent Document 5] Anderl, J. et al, Methods Mol Biol. 2013;1045:51-70 [Non-Patent Document 6] Thomas, A., et al, Lancet Oncol. 2016 Jun;17(6):e254-e262 [Non-Patent Document 7] F. Sasse et al. J. Antibiot. 2000, 53, 879-885 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] This patent discloses a drug composition of a tubulicin analog and a cell surface-bound ligand that has longer-lasting stability and storage properties and is easier to transport, as well as the use of the conjugated composition for immediate reconstitution with water before administration to a patient for the treatment of cancer and immunological diseases. [Means for solving the problem]

[0009] Summary of the Invention A liquid composition for lyophilization, or a formulated lyophilized solid, or a reconstituted formulation from a lyophilized solid, having the following composition: a conjugate of a cell binder of formula (I) and a tubulicin analog, which may constitute 0.01 to 99 wt% as the main component in the formulation; 0.0% to 20.0% of one or more polyols; 0.0% to 2.0% of one or more surfactants; 0.0% to 5.0% of one or more preservatives; 0.0% to 30% of one or more amino acids; 0.0% to 5.0% of one or more antioxidants; 0.0% to 0.3% of one or more metal chelating agents; 0.0% to 30.0% of one or more buffer salts to adjust the pH of the formulation to pH 4.5 to 8.5; and 0.0% to 30.0% of one or more isotonic agents to adjust the osmotic pressure between approximately 250 to 350 mOsm after reconstitution for administration to a patient.

[0010] As an exemplary embodiment, a tubulysin conjugate having the formula (I), or a pharmaceutically acceptable salt, hydrate, or hydrate salt thereof; or a polymorphic crystal structure of these compounds; or an isotope, optical isomer, racemate, diastereomer, or enantiomer thereof:

Chemical formula

[0011] wherein, T is a target or a cell-binding molecule; L is a releasable linker;

Chemical formula

[0012] The content within the parentheses is a tubulysin analog, wherein R 1 , R 2 , R 3 , and R 4 are independently C1-C8 straight-chain or branched alkyl, alkyl alcohol; C2-C8 heteroalkyl, alkyl cycloalkyl, heterocycloalkyl, alkyl ether, alkyl carboxylate, alkylamine, alkyl ester, alkyl amide; C3-C8 aryl, Ar-alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkyl cycloalkyl, alkyl carbonyl; or two Rs, namely R 1 R 2 , R 3 R 4 , R 5 R 6 , or R 12 R 13 together form a 3- to 7-membered carbocycle, cycloalkyl, heterocycle, heterocycloalkyl, aromatic or heteroaromatic ring system; Y is N or C; further, R 1 , R 2 , R 3 , and R 4 may optionally not exist;.

[0013] In the formula, R 5 , R 6 , R 8 , and R 10 These are independently selected from H and linear or branched C1-C4 alkyl or C2-C4 heteroalkyl groups.

[0014] In the formula, R 7 H, R 14 , or -R 14 C(=O)X 1 R 15 ;-R 14 X 1 R 15 Selected from;X 1 is O, S, SS, NH, or NR 14 Selected from.

[0015] In the formula, R 9 H, -OH, -OR 14 -OC(=O)R 14 -OC(=O)NHR 14 -OC(=O)NR 14 R 15 -OC(=O)R 14 SSR 15 , OP(=O)(OR 14 ), or OR 14 OP(=O)(OR 15 )

[0016] In the formula, R 11 H, R 14 , -R 14 C(=O)R 16 , -R 14 C(=O)X 2 R 16 ,-R 14 X 2 R 16 ,-R 14 C(=O)X 2 And in the formula, X 2 -O-, -S-, -NH-, -NHS(O2), -N(R 14 )-, -OR 14 -, -SR 14 -, -S(=O)-R14 - or -NHR 14 - is.

[0017] In the formula, R 12 is H, R 14 , -O-, -S-, -N-, =N-, =NNH-, -OH, -SH, -NH2, =NH, =NNH2, -NH(R 14 ), -OR 14 , -C(O)O-, -C(O)OR 16 -, -COR 16 , -COOR 14 -, C(O)NH-, C(O)NH2, C(O)NHR 14 , -SR 14 , -S(=O)R 14 , -P(=O)(OR 16 ), -OP(=O)(OR 16 ), -CH2OP(=O)(OR 16 ), -SO2R 16 is.

[0018] In the formula, R 13 is a linear or branched alkyl, alkyl acid, alkyl amide, alkyl amine of C1 - C 10 ; or a heteroalkyl of C2 - C 10 ; or an Ar of C3 - C 10 . Ar refers to an aromatic or heteroaromatic group composed of one or more rings containing 4 to 10 carbon atoms, preferably 4 to 6 carbon atoms; The term heteroaromatic group refers to an aromatic group in which one or more carbon atoms are replaced by heteroatoms, preferably 1, 2, or 3 carbon atoms are replaced by O, N, Si, Se, P, or S, more preferably O, S, N; The term aryl or Ar independently replaces one or more H atoms with R 17 , F, Cl, Br, I, OR 16 , SR 16 , NR 16 R 17 , N=NR 16 , N=R 16 , NR 16 R 17 , NO2, SOR 16 R 17 , SO2R 16 , SO3R16 , OSO3R 16 , PR 16 R 17 , POR 16 R 17 , PO2R 16 R 17 , OP(O)(OR 17 )2, OCH2OP(O)(OR 17 )2, OC(O)OP(O)(OR 17 )2, PO(OR 16 )(OR 17 ), OP(O)(OR 17 )OP(O)(OR 17 )2, OC(O)R 17 , or OC(O)NHR 17 also refers to an aromatic group that can be replaced by

[0019] In the formula, R 14 and R 15 are independently H; a linear or branched alkyl of C1-C8; an alkenyl, alkynyl, heteroalkyl, heterocycle, carbocycle of C2-C8; an aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, alkylcarbonyl of C3-C8.

[0020] In the formula, when R 14 is divalent, R 14 is further connected to an additional functional group of 1-4 amino acid units, or (CH2CH2O) r (r is an integer in the range of 0-12), or a glycoside of C4-C 12 , a carboxylic acid of C1-C8.

[0021] In the formula, R 16 is H, OH, R 14 , or 1-4 amino acid units.

[0022] In the formula, R 17This includes H, C1-C8 linear or branched alkyl groups; C2-C8 alkenyl, alkynyl, heteroalkyl, heterocyclic; C3-C8 aryl, carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl, or C4-C 12 It is a glycoside or medicinal salt of [the substance].

[0023] In another embodiment, the effective tubulicin analog-binding molecule conjugate L has the formula --Ww-(Aa)r-Vv-, where --W-- is an extended unit; w is 0 or 1; each --Aa-- is independently an amino acid unit; r is independently an integer between 0 and 12; --V-- is a spacer unit; v is 0, 1, or 2; the extended unit W may independently contain a self-destructing spacer, a peptide unit, a hydrazone bond, a disulfide, or a thioether bond.

[0024] In another embodiment, cell surface binding molecule T is any type of cell-binding ligand currently known or to be known, such as peptides and non-peptides. Binding molecule T is an antibody; a single-chain antibody; an antibody fragment that binds to a target cell; a monoclonal antibody; a single-chain monoclonal antibody; or a monoclonal antibody fragment that binds to a target cell; a chimeric antibody; a chimeric antibody fragment that binds to a target cell; a domain antibody; a domain antibody cross-section that binds to a target cell; an antibody-mimicking adnectin; DARPins; lymphokines; hormones; vitamins; growth factors; colony-stimulating factors; or nutrient transport molecules; transferrin; a binding peptide, or protein, or a small molecule, polymer, dendrimer, liposome, nanoparticle, vesicle, or (viral) capsid bound to an antibody or albumin. Preferably, binding molecule T is a monoclonal antibody.

[0025] In yet another embodiment, compositions of formula (I) and its dependent formulas (II)-(VII) are used to treat cancer, autoimmune diseases, or infections in humans or animals. [Brief explanation of the drawing]

[0026] [Figure 1] The general synthesis of Tuv, a tubulicin analog, is shown. [Figure 2] This shows the synthesis of the tubulicin component. [Figure 3] This shows the synthesis of the tubulicin component. [Figure 4] The synthesis of tubulicin analog components is shown. [Figure 5] The synthesis of tubulicin analog components is shown. [Figure 6] The synthesis of tubulicin analog components is shown. [Figure 7] The synthesis of tubulicin analog components is shown. [Figure 8] The synthesis of tubulosin analog components, including conjugated ligatures, is shown. [Figure 9] This describes the synthesis of tubulicin analogues and their conjugates with antibodies. [Figure 10] The synthesis of the linked components is shown. [Figure 11] This describes the synthesis of tubulicin analogs, including conjugates, and their conjugates with antibodies. [Figure 12] This describes the synthesis of tubulicin analogs, including conjugates, and their conjugates with antibodies. [Figure 13] This describes the synthesis of tubulicin analogs, including conjugates, and their conjugates with antibodies. [Figure 14] This describes the synthesis of tubulicin analogs, including conjugates, and their conjugates with antibodies. [Figure 15] The synthesis of conjugates with tubulicin analogues containing the conjugate is shown, as well as with the conjugate component and conjugates with antibodies. [Figure 16] The synthesis of Tup and Tuv analog components is shown. [Figure 17] The synthesis of Tuv analog components is shown. [Figure 18] This describes the synthesis of tubulicin analogs, including conjugates, and their conjugates with antibodies. [Figure 19]This describes the synthesis of tubulicin analogs, including conjugates, and their conjugates with antibodies. [Figure 20] This describes the synthesis of tubulicin analogs, including conjugates, and their conjugates with antibodies. [Figure 21] This describes the synthesis of tubulicin analogs, including conjugates, and their conjugates with antibodies. [Figure 22] This describes the synthesis of tubulicin analogs, including conjugates, and their conjugates with antibodies. [Figure 23] The antitumor effects of conjugated compounds C-166a, C-719, C-720, and C-723 and T-DM1 were investigated by intravenous injection (6 mg / kg in a single injection for conjugated compounds C-166a, C-719, C-720, C-723, and T-DM1) into a human gastric tumor N87 cell model. The four conjugates tested here showed superior antitumor activity compared to T-DM1. Compounds C-166a, C-719, C-720, and C-723 resulted in no measurable tumors from day 22 to day 36, and all of them were able to inhibit tumor growth for more than 48 days. In contrast, a 6 mg / kg dose of T-DM1 failed to eliminate tumors and inhibited tumor growth for only 31 days. [Figure 24] This shows acute toxicity studies of ADC conjugates T-DM1, C-166a, C-719, C-720, and C-723 by observing changes in body weight (BW) of mice treated with doses of 75 mg / Kg (Figure 24-a) and 150 mg / Kg (Figure 24-b) for 12 days. The changes in body weight indicate that at both doses, the toxicity of conjugate C-723 was higher than that of T-DM1, conjugate C-720 was similar in toxicity to T-DM1 at the 75 mg / Kg dose but lower at the 150 mg / Kg dose, and at both test doses, the toxicity of conjugates C-166a and C-719 was significantly lower than that of T-DM1. [Figure 25]The images show the liver pathogens of mice treated with conjugates T-DM1, C-166a, C-719, C-720, and C-723 at a dose of 75 mg / kg, compared to PBS buffer on day 5. The images are magnified 40 times. As shown in the images, (1) the pathology of the T-DM1 group (Figure 25-(a)) and the T-DM1 75 mg / kg group shows hepatocyte swelling and multifocal necrosis. The lobular structure is indistinct. The central vena cava contains swollen hepatocytes, erythrocytes, and remaining red color. The size and degree of staining of the hepatocyte nuclei differ. The hepatocytes show blurred boundaries, increased volume, and eosinophil staining. The hepatocyte nuclei disappear. A clear proliferative phase is observed. (2) In the C-723 group (Figure 25-(b)), scattered unicellular necrosis and hydrodegeneration are the main pathological behaviors. In the swollen area, the hepatic lobular structure was lost, and the central vena cava was heavily congested with red blood cells. Hepatocytes were swollen, with indistinct boundaries and eosinophilic staining. The size and color of the nuclei varied. Mild proliferation was observed. (3) The pathology of the C-720 group (Figure 25-(c)) shows exudate in the central vein of the lobule, disordered arrangement of hepatocyte plates, and proliferation of hepatocytes. Occasionally, hypertrophy of Kupffer cells was observed. (4) In the C-719 (Figure 25-(e)) and C-166a (Figure 25-(f)) groups, the hepatic lobular structure was slightly disordered, the hepatic sinus was visible, and inflammatory cell infiltration was observed. Hypertrophy of Kupffer cells was rare, and hepatocytes were mildly swollen, with their ultrastructure similar to that of the control group PBS (Figure 25-(g)). [Figure 26] The protein dechaining temperatures (Tm) of compositions S7, S8, S9, S10, S11, and S12, measured using nanoDSF technology, are shown. The results indicate that component S10 is more stable than its counterpart, and the difference in thermal stability between components S7, S8, S9, S11, and S12 is small. [Figure 27] The tag onset times for compositions S7, S8, S9, S10, S11, and S12, measured using nanoDSF technology, are shown. The results indicate that component S10 is more stable than the other components, there is little difference in thermal stability between components S7, S8, S9, and S11, and S12 is slightly less stable than the other test components. [Figure 28]The protein dechaining temperatures (Tm) of compositions S12, S18, and S24, measured using nanoDSF technology, are shown. The results indicate that composition S24 is more stable than the other two components, while composition S12 is less stable than the other two components. [Figure 29] The tag onset times for compositions S12, S18, and S24, measured using nanoDSF technology, are shown. The results indicate that composition S24 is more stable than the other two components, while composition S12 is less stable than the other two components. [Modes for carrying out the invention]

[0027] definition

[0028] "Alkyl" refers to an aliphatic hydrocarbon group or monovalent group derived from an alkane by removing one or two hydrogen atoms from a carbon atom. It may be linear or branched, having C1-C8 (1-8 carbon atoms) in the chain. "Branched" refers to a linear alkyl group to which one or more lower C-number alkyl groups, such as methyl, ethyl, or propyl groups, are bonded. Specific examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl, and isooctyl. C1-C8 alkyl groups may be unsubstituted or substituted with one or more substituents (but not limited to the following substituents). Examples of the substituents include -C1-C8alkyl, -O-(C1~C8alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2, and -CN, where R' is independently selected from C1~C8alkyl and aryl.

[0029] "Halogen" refers to fluorine, chlorine, bromine, or iodine atoms, with fluorine and chlorine atoms being preferred.

[0030] A "heteroalkyl" refers to a C2-C8 alkyl group in which 1 to 4 carbon atoms are independently substituted with heteroatoms selected from the group consisting of O, S, and N.

[0031] A "carbocycle" refers to a saturated or unsaturated ring with 3 to 8 carbon atoms in a monocyclic system or 7 to 13 carbon atoms in a bicyclic system. Monocyclic carbocycles have 3 to 6, more typically 5 or 6, ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms and are arranged as bicyclic systems [4,5], [5,5], [5,6], or [6,6], or they have 9 to 10 ring atoms and are arranged as bicyclic systems [5,6] or [6,6]. Representative C3-C8 carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrielinyl, -cyclooctyl, and -cyclooctadienyl.

[0032] A C3-C8 carbocycle refers to a saturated or unsaturated non-aromatic carboncyclic compound with 3, 4, 5, 6, 7, or 8 members. The C3-C8 carbocycle may be unsubstituted or substituted with one or more substituents. These substituents include, but are not limited to, -C1-C8 alkyl, -O-(C1-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S(O)R', -S(O)2R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2, and -CN, where R' is independently selected from C1-C8 alkyl and aryl.

[0033] "Alkenyl" refers to an aliphatic hydrocarbon group that has 2 to 8 carbon atoms in its chain and contains a carbon-carbon double bond, and may be linear or branched. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbuto-2-enyl, n-pentenyl, hexylenyl, heptenyl, and octenyl.

[0034] "Alkynyl" refers to an aliphatic hydrocarbon group that has 2 to 8 carbon atoms in its chain and contains a carbon-carbon triple bond, and may be linear or branched. Examples of alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5-pentynyl, n-pentynyl, hexylinyl, heptynyl, and octynyl.

[0035] "Alkylene" refers to a saturated, linear, branched, or cyclic hydrocarbon group having 1 to 18 carbon atoms, having two monovalent centers derived from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkane. Typical alkylene groups include, but are not limited to, methylene (-CH2-), 1,2-ethyl (-CH2CH2-), 1,3-propyl (-CH2CH2CH2-), and 1,4-butyl (-CH2CH2CH2CH2-).

[0036] "Alkenylene" refers to an unsaturated, linear, branched, or cyclic hydrocarbon group having 2 to 18 carbon atoms, having two monovalent centers derived from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkene. Typical alkenylene groups include, but are not limited to, 1,2-ethylene (-CH=CH-).

[0037] "Alkynylene" refers to an unsaturated, linear, branched, or cyclic hydrocarbon group having 2 to 18 carbon atoms, having two monovalent centers derived from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkyne. Typical alkynylene groups include, but are not limited to, acetylene, propargyl, and 4-pentinyl.

[0038] "Aryl" or "Ar" refers to an aromatic or heteroaromatic group consisting of one or more rings containing 3 to 14 carbon atoms, preferably 6 to 10 carbon atoms. The term "heteroaromatic group" refers to an aromatic group in which some carbon atoms, preferably 1, 2, 3, or 4 carbon atoms, are replaced with O, N, Si, Se, P, or S, preferably O, S, and N. The terms aryl or Ar also refer to a case where one or more H atoms are independently replaced by -R', -halogen, -OR', or -SR', -NR'R'', -N=NR', -N=R', -NR'R'', -NO2, -S(O)R', -S(O)2R', -S(O)2OR', -OS(O)2OR', -PR'R'', -P(O)R'R'', -P(OR')(OR''), -P(O)(OR')(OR''), or -OP(O)(OR')(OR''). The R', R'' are independently H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, arylalkyl, carbonyl, or pharmaceutically salt.

[0039] A heterocycle is a ring system in which 1 to 4 ring carbon atoms are independently substituted with heteroatoms from the group O, N, S, Se, B, Si, and P. Preferred heteroatoms are O, N, and S. Heterocycles are described in The Handbook of Chemistry and Physics, 78th edition, CRC Press, Inc., 1997-1998, pp. 225-226. These are incorporated herein by reference. Preferred non-aromatic heterocycles include, but are not limited to, epoxy, azilidinyl, tyranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxylanyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, dioxolanyl, piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolidinyl, pyrazolinyl, thiazolidinyl, tetrahydropyranyl, dihydropyranyl, tetrahydropyridyl, dihydropyridyl, tetrahydropyrimidinyl, dihydrothiopyranyl, azepanyl, and condensation systems resulting from condensation with phenyl groups.

[0040] The term "heteroaryl" or aromatic heterocyclic refers to an aromatic hetero, monocyclic, bicyclic, or polycyclic ring with 3 to 14 members, preferably 5 to 10 members. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanil, benzofuranil, 1,2,4-thiadiazolyl, isothiazolyl, triazoyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, pyridyl-N-oxide, and condensation systems resulting from condensation with a phenyl group.

[0041] The terms "alkyl," "cycloalkyl," "alkenyl," "alkynyl," "aryl," "heteroaryl," and "heterocyclic" also refer to the corresponding "alkylene," "cycloalkylene," "alkenylene," "alkynylene," "arylene," "heteroarylene," and "heterocyclene" groups, which are formed by the removal of two hydrogen atoms.

[0042] "Arylalkyl" refers to carbon atoms, typically terminal or sp 3 This refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom is replaced by an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethane-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethane-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, and 2-naphthophenylethane-1-yl.

[0043] "Heteroarylalkyl" refers to carbon atoms, typically terminal or sp 3 Bonded to carbon atoms This refers to an acyclic alkyl group in which one of the hydrogen atoms is replaced by a heteroaryl group. Typical heteroarylalkyl groups include 2-benzimidazolylmethyl and 2-furylethyl.

[0044] Examples of "hydroxy protecting groups" include methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, t-butyldimethylsilyl ether, triphenylmethylsilyl ether, acetate esters, substituted acetate esters, pivaloates, benzoates, methanesulfonates, and p-toluenesulfonates.

[0045] A "leaving group" refers to a functional group that can be substituted by another functional group. Such leaving groups are well known in the art and include, for example, halides (e.g., chlorides, bromides, and iodides), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonate. Preferred leaving groups are selected from nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3'-sulfonate, acid anhydrides formed with themselves or other acid anhydrides (e.g., acetic anhydride, formic anhydride); or intermediates produced by condensation reagents for peptide coupling reactions or Mitsunobu reactions.

[0046] The following abbreviations may be used herein, with the definitions set forth below: Boc, tert-butoxycarbonyl; BroP, bromotrispirolidinophosphonium hexafluorophosphate; CDI, 1,1'-carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCE, 1,2-dichloroethane; DCM, dichloromethane; DEAD, diethylazodicarboxylate; DIAD, diisopropyl azodicarboxylate; DIBAL-H, diisobutylaluminum hydride DIPEA or DEA, diisopropylethylamine; DEPC, diethylphosphoroanidiate; DMA, N,N-dimethylacetamide; DEPC, diethylphosphocyanidate; DMA, N,N-dimethylacetamide; DMAP, 4-(N,N-dimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DTPA, diethylenetriaminepentaacetic acid; DTT, dithiothreitol; EDC, 1-(3-dimethylaminopropyl)-3-ethyl acetate Podiimide hydrochloride; ESI-MS, electrospray mass spectrometry; toluene, ethyl acetate; Fmoc, N-(9-fluorenyl methoxycarbonyl); HATU, O-(7-azabenzotriazol-1-yl)-N,N,N'-N'-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high-pressure liquid chromatography; NHS, N-hydroxysuccinimide; MeCN, acetonitrile; MeOH, methanol; MMP, 4-methylmorpho Phosphorus; PAB, p-aminobenzyl; PBS, phosphate-buffered saline (pH 7.0-7.5); Ph, phenyl; Phe, L-phenylalanine; PyBrop, bromo-tris-pyrrolidino-phosphonium hexafluorophosphate; PEG, polyethylene glycol; SEC, size exclusion chromatography; TCEP, tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Val, valine; TLC, thin-layer chromatography; UV, ultraviolet light.

[0047] "Amino acids" may be natural and / or non-natural amino acids, preferably α-amino acids. Natural amino acids are those encoded by the genetic code, such as alanine, arginine, These are asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan, and valine. Non-natural amino acids are derivatives of protein-forming amino acids. Examples include hydroxyproline, lanthionine, 2-aminoisobutyric acid, dehydroalanine, γ-aminobutyric acid (neurotransmitter), ornithine, citrulline, β-alanine (3-aminopropanoic acid), γ-carboxyglutamate, selenocysteine ​​(present in most eukaryotes but not directly encoded by DNA), pyrrolidine (found in some archaea and only one bacterium), N-formylmethionine (often the first amino acid in bacterial, mitochondrial, and chloroplast proteins), 5-hydroxytryptophan, L-dihydroxyphenylalanine, triiodothyronine, L-3,4-dihydroxyphenylalanine (DOPA), and O-phosphoserine. The term amino acid also includes amino acid analogs and mimics. Analogues are compounds that have the same common H2N(R)CHCO2H structure as natural amino acids, except that the R group is not found in natural amino acids. Examples of analogues include homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Preferably, amino acid mimetic compounds are compounds that have a different structure from the general chemical structure of α-amino acids but function similarly. The term "non-natural amino acid" is intended to represent the stereochemical form "D," while natural amino acids are in the "L" form. When 1 to 8 amino acids are used in this application, the amino acid sequence is preferably a protease cleavage recognition sequence.Many cleavage recognition sequences are known in this field, for example, Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244: 175 (1994); Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al. Meth. Enzymol. 244: 595 (1994); Smith et al. Meth. Enzymol. 244: 412 (1994); and Bouvier et al. Meth. Enzymol. 248: 614 (1995). See reference; its disclosure is incorporated herein by reference. In particular, sequences are selected from the group consisting of Val-Cit, Ala-Val, Ala-Ala, Val-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Cit-Cit, Val-Lys, Ala-Ala-Asn, Lys, Cit, Ser, and Glu.

[0048] A "glycoside" is a molecule in which a sugar group is linked to another group via a glycosidic bond through its anomeric carbon. Glycosides can be linked by O-(O-glycoside), N-(glycosylamine), S-(thioglycoside), or C-(C-glycoside) glycosidic bonds. The core empirical formula is C m (H2O) n(where m is different from n, and both m and n are < 36), and here glycosides include glucose (dextrose), fructose (levrose), allose, altrose, mannose, gross, iodose, galactose, talose, galactosamine, glucosamine, sialic acid, N-acetylglucosamine, sulfoquinovose (6-deoxy-6-sulfo-D-glucopyranose), ribose, arabinose, xylose, lyxose, sorbitol, mannitol, sucrose, lactose, maltose, trehalose, maltodextrin, raffinose, glucuronic acid (glucuronide), and stachyose. It may be a D-type or L-type, a pentatomic cyclic furanose type, a hexatomic cyclic pyranose type, or an acyclic type, an α-isomer (anomer carbon -OH below the plane of carbon atoms in the Haworth projection), or a β-isomer (anomer carbon -OH above the plane of carbon atoms in the Haworth projection). It is used herein as a monosaccharide, disaccharide, polyol, or oligosaccharide containing 3 to 6 sugar units.

[0049] As used herein, the term “antibody” refers to a full-length immunoglobulin molecule or a molecule comprising an immunologically active portion of a full-length immunoglobulin molecule, i.e., an antigen-binding site that immune-specifically binds to an antigen or part thereof of a key target, such targets including, but not limited to, cancer cells or cell populations that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein include any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (For example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclasses. Immunoglobulins are derived from any species. However, preferably, immunoglobulins are of human, mouse, or rabbit origin. Antibodies useful in the present invention are preferably monoclonal, and include, but are not limited to, polyclonal, monoclonal, bispecific, human, humanized, or chimeric antibodies, single-chain antibodies, Fv, Fab fragments, F(ab') fragments, F(ab')2 fragments, fragments produced by Fab expression libraries, anti-idiotype (anti-Id) antibodies, CDRs, and any of the above epitope-binding fragments that bind immunospecifically to cancer cell antigens, viral antigens, or microbial antigens.

[0050] Just as left and right hands are identical except that they are opposite each other along a single axis (simply changing direction does not make hands appear identical), enantiomers, also known as optical isomers, are two stereoisomers that are mirror images of each other but cannot be superimposed (they are not identical). A single chiral atom or similar structural feature within a compound can result in two non-superimal structures, each being a mirror image of the other. The presence of multiple chiral elements in a particular compound increases the number of possible geometric forms, but perfect mirror-image pairs may still exist. A pure enantiomeric compound refers to a sample with only one chirality within the detection limit. In a symmetric environment, enantiomers have identical chemical and physical properties except for their ability to rotate plane-polarized light (+ / -) by equal amounts in opposite directions (although polarization can be considered an asymmetric medium). For this reason, they are sometimes called optical isomers. A mixture of equal portions of an optically active isomer and its enantiomer is called a racemic mixture, and the net rotation of plane-polarized light is zero. This is because the positive rotation of each (+) form is precisely canceled out by the negative rotation of the (-) form. Members of an enantiomer often react differently with other enantiomers. Since many biomolecules are enantiomers, there can be significant differences in the effects of two enantiomers on living organisms. For example, in drugs, often only one of the drug's enantiomers is involved in the desired physiological effect, while the other enantiomer may be less active, inactive, or even harmful. This discovery allows for the development of drugs composed of only one enantiomer ("pure enantio") to enhance pharmacological effects and sometimes eliminate some side effects.

[0051] Isotopes are variations of a particular chemical element with different numbers of neutrons. All isotopes of a given element have the same number of protons in each atom. Each atomic number identifies a particular element, but not an isotope. Atoms of a given element can have a wide range of neutron numbers. The number of nucleons (both protons and neutrons) in the nucleus is the mass number of an atom, and each isotope of a particular element has a different mass number. For example, carbon-12, carbon-13, and carbon-14 are three isotopes of carbon with mass numbers 12, 13, and 14, respectively. The atomic number of carbon is 6. This is because all carbon atoms have 6 protons, so the neutron numbers of these isotopes are 6, 7, and 8, respectively. A hydrogen atom has protium ( 1 H), deuterium ( 2 H), and tritium ( 3 There are three isotopes of protium (H), with deuterium having twice the mass of protium and tritium having three times the mass of protium. Isotope substitution can be used to determine the mechanisms of chemical reactions or to investigate kinetic isotope effects. Isotope substitution can be used to investigate not only metabolic changes of substances in the body (e.g., by metabolic enzymes such as cytochrome P450 or glucuronosyltransferase enzymes), but also how the body is affected by specific xenobiotics / chemicals after administration through mechanisms of absorption and distribution, as well as the excretion pathways and effects of drug metabolites. This study is called pharmacokinetics (PK). Isotope substitution can be used to study the biochemical and physiological effects of drugs. Effects include those that appear in animals (including humans), microorganisms, or combinations of organisms (e.g., infections). This study is called pharmacodynamics (PD). Effects include those that appear in animals (including humans), microorganisms, or combinations of organisms (e.g., infections). Together, both influence the administration, benefits, and adverse effects of a drug. Isotopes contain either stable (non-radioactive) or unstable elements. Isotope substitution of drugs may result in therapeutic effects different from those of the original drug.

[0052] "Pharmacologically" or "pharmaceutically acceptable" means that the corresponding compound or compound composition, when administered appropriately to animals or humans, is not harmful, allergic, or otherwise adverse.

[0053] "Pharmacologically acceptable solvate" or "solvate" is disclosed as a compound of one or more solvent molecules. This refers to association with a compound. Examples of solvents that form pharmacologically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0054] Pharmaceutically acceptable auxiliary materials include all carriers, diluents, adjuvants, or molding agents, such as preservatives, antioxidants, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersing media, coatings, antibacterial agents, antifungal agents, isotonic agents, and absorption retarders. In the pharmaceutical field, adding these auxiliary materials to active drug components is a common practice. It can be said that adding auxiliary materials to drug components is appropriate unless the auxiliary material is incompatible with the drug-active component. Active auxiliary materials may be added to drug components to obtain favorable results.

[0055] In the present invention, "medicinal salt" refers to salt derivatives of the compound of the present invention. By appropriate modification, the compound of the present invention can be formed into a corresponding acid salt or alkali salt. Medicinal salts include commonly used non-toxic salts or quaternary ammonium compounds, which can be prepared with the compound of the present invention and a corresponding non-toxic inorganic or organic acid. For example, inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, and nitric acid, while organic acids include acetic acid, propioic acid, succinic acid, tartaric acid, citric acid, methanesulfonic acid, benzenesulfonic acid, glucuronic acid, glutamic acid, benzoic acid, salicylic acid, toluenesulfonic acid, oxalic acid, fumaric acid, and lactic acid, and these acids can be used in pharmaceutically acceptable salts. Other salts include ammonium salts such as trometamol, meglumine, and pyrroleethanol, and metal salts such as sodium, potassium, calcium, zinc, and magnesium.

[0056] In the present invention, pharmaceutical salts can be prepared from parent compounds containing acidic or basic residues by conventional chemical methods. Generally, these salts can be obtained by reacting the free acidic or free base form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of both. Preferred non-aqueous reaction solvents are generally ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. A list of suitable salts is given in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, PA, 1985, page 1418, which is incorporated by reference.

[0057] "Administering" or "administration" refers to any manner in which a pharmaceutical or other drug is transferred, delivered, introduced, or transported. Such manners include oral administration, topical contact, intravenous, intraperitoneal, intramuscular, lesional, nasal, subcutaneous, or intracavitary administration. Furthermore, the present invention intends to utilize devices or equipment for administering drugs. Such devices may utilize active or passive transport and may be slow-release or fast-release delivery devices.

[0058] "Therapeutic dose" is the amount effective in preventing or treating the pathological conditions referred to herein. This refers to the amount of compound / drug used.

[0059] The terms “patient” or “patient in need” refer to an animal or human being that is suffering from or is likely to suffer from any of the pathological conditions referred to herein. Preferably, the patient is human.

[0060] In the context of cancer, the term “treat” includes any or all of the following: inhibiting the growth of tumor cells or cancer cells, preventing the replication of tumor cells or cancer cells, reducing the overall burden of the tumor, and improving one or more symptoms associated with the disease.

[0061] In the context of autoimmune diseases, the term “treat” includes any or all of the following: preventing the replication of cells, but not limited to, that are associated with the pathogenesis of the autoimmune disease, that are capable of producing autoimmune antibodies; reducing the burden of autoimmune antibodies; and improving one or more symptoms of the autoimmune disease.

[0062] In the context of infectious diseases, the term “to treat” includes preventing the growth, proliferation, or replication of the pathogen causing the infection, and improving one or more symptoms of the infection, or all of these.

[0063] Examples of “mammals” or “animals” include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cattle, horses, dogs, cats, birds, and poultry.

[0064] Composition comprising a conjugate of a cell-binding ligand and a tubulicin analog.

[0065] As described above, the present invention provides a liquid composition for freeze-drying, or a reconstituted solution of a formulated freeze-dried powder, or a reconstituted formulation from a freeze-dried solid, the composition of which is as follows: a conjugate of a tubulicin analog of formula (I) as the main component in the formulation may constitute 0.01 to 99 wt%; 1 or more polyols in 0.0% to 20.0%; 1 or more surfactants in 0.0% to 2.0%; 1 or more preservatives in 0.0% to 5.0%; 1 or more amino acids in 0.0% to 30%; 1 or more antioxidants in 0.0% to 5.0%; 1 or more metal chelating agents in 0.0% to 0.3%; 1 or more buffer salts in 0.0% to 30.0% to adjust the pH of the formulation to pH 4.5 to 8.5; and 1 or more isotonic agents in 0.0% to 30.0% to adjust the osmotic pressure between approximately 250 to 350 mOsm after reconstitution for administration to a patient.

[0066] The tubulicin analog conjugates of formula (I) are the following, or their pharmaceutically acceptable salts, hydrates, or hydrated salts; or the polymorphic crystalline structures of these compounds; or their isotopes, optical isomers, racemates, diastereomers, or enantiomers; [ka]

[0067] In the formula, T is the target or cell-binding molecule; L is the releaseable conjugate; [ka] This is a linkage bond in which L is independently connected to the atom in parentheses; n is 1 to 20 and m is 1 to 10.

[0068] The terms in parentheses in formula (I) are valid tubulicin analogs, where R 1 , R 2 , R 3 , and R 4 These are independently C1-C8 linear or branched alkyls, alkyl alcohols; C2-C8 heteroalkyls, alkylcycloalkyls, heterocycloalkyls, alkyl ethers, alkyl carboxylates, alkylamines, alkyl esters, alkylamides; C3-C8 aryls, Ar-alkyls, heterocyclic, carbocyclic, cycloalkyls, heteroalkylcycloalkyls, alkylcarbonyls; or two Rs, i.e., R 1 R 2 , R 3 R 4 , R 5 R 6 , or R 12 R 13 Together, they form a 3- to 7-membered carbon ring, cycloalkyl, heterocyclic, heterocycloalkyl, aromatic, or heteroaromatic ring system; Y is N or C; further, R 1 , R 2 , R 3 , and R 4 It can exist independently and does not need to exist.

[0069] In the formula, R 5 , R 6 , R 8 , and R 10These are independently selected from H and linear or branched C1-C4 alkyl or C2-C4 heteroalkyl groups.

[0070] In the formula, R 7 H, R 14 , or -R 14 C(=O)X 1 R 15 ;-R 14 X 1 R 15 Selected from;X 1 is O, S, SS, NH, or NR 14 Selected from.

[0071] In the formula, R 9 H, -OH, -OR 14 -OC(=O)R 14 -OC(=O)NHR 14 -OC(=O)NR 14 R 15 -OC(=O)R 14 SSR 15 , OP(=O)(OR 14 ), or OR 14 OP(=O)(OR 15 )

[0072] In the formula, R 11 H, R 14 ,-R 14 C(=O)R 16 ,-R 14 C(=O)X 2 R 16 ,-R 14 X 2 R 16 ,-R 14 C(=O)X 2 And in the formula, X 2 -O-, -S-, -NH-, -NHS(O2), -N(R 14 )-, -OR 14 -, -SR 14 -, -S(=O)-R 14 -, or -NHR 14 - is

[0073] In the formula, R 12 H, R14 , -O-, -S-, -N-, =N-, =NNH-, -OH, -SH, -NH2, =NH, =NNH2, -NH(R 14 ), -OR 14 -C(O)O-, -C(O)OR 16 -, -COR 16 ,-COOR 14 -, C(O)NH-, C(O)NH2, C(O)NHR 14 , -SR 14 -S(=O)R 14 , -P(=O)(OR 16 )2, -OP(=O)(OR 16 )2, -CH2OP(=O)(OR 16 )2, -SO2R 16 That is the case.

[0074] In the formula, R 13 C1~C 10 Linear or branched alkyl, alkyl acid, alkylamide, alkylamine; or C2-C 10 heteroalkyl; or C3~C 10 Ar refers to an aromatic or heteroaromatic group composed of one or more rings containing 4 to 10 carbon atoms, preferably 4 to 6 carbon atoms; the term heteroaromatic group refers to an aromatic group in which one or more carbon atoms are replaced by heteroatoms, preferably 1, 2, or 3 carbon atoms replaced by O, N, Si, Se, P, or S, more preferably O, S, N; the terms aryl or Ar refer to an aromatic group in which one or more H atoms are independently R 17 F, Cl, Br, I, OR 16 , SR 16 , NR 16 R 17 N=NR 16 N=R 16 , NR 16 R 17 NO2, SOR 16 R 17 SO2R 16 SO3R 16 OSO3R 16 PR 16 R 17 , POR 16 R17 PO2R 16 R 17 , OP(O)(OR 17 )2, OCH2OP(O)(OR 17 )2, OC(O)OP(O)(OR 17 )2, PO(OR 16 )(OR 17 ), OP(O)(OR 17 )OP(O)(OR 17 )2, OC(O)R 17 , or OC(O)NHR 17 It also refers to aromatic groups that can be replaced by [a specific group].

[0075] In the formula, R 14 and R 15 These are independently H; C1-C8 linear or branched alkyl; C2-C8 alkenyl, alkynyl, heteroalkyl, heterocyclic, carbocyclic; C3-C8 aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, alkylcarbonyl;

[0076] In the formula, R 14 If it is divalent, R 14 This is further divided into 1-4 amino acid units, or (CH2CH2O) r (r is an integer in the range of 0 to 12.) or C4 to C 12 The glycoside is further connected to additional functional groups, which are carboxylic acids of C1-C8.

[0077] In the formula, R 16 H, OH, R 14 , or 1 to 4 amino acid units.

[0078] In the formula, R 17 This includes H, C1-C8 linear or branched alkyl groups; C2-C8 alkenyl, alkynyl, heteroalkyl, heterocyclic; C3-C8 aryl, carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl, or C4-C 12 It is a glycoside or medicinal salt of [the substance].

[0079] R 13 If the following is the base, R 1 R 3 They combine to form a 3- to 7-membered heterocycle or alkyl heterocycle: [ka]

[0080] In the formula, Z 1 H, CH2OP(O)(OR 18 )2, C(O)OP(O)(OR 18 )2, PO(OR 18 )2, C(O)R 18 , P(O)(OR 18 )OP(O)(OR 18 )2, C(O)NHR 18 , SO2(OR 18) , C5-C 12 It is a glycoside, C1-C4 alkyl, or alkylcarbonyl; R 18 R is H, C1-C8 alkyl or alkylcarbonyl; 19 H, OH, NH2, OSO2 (OR 18 ), XCH2OP(O)(OR 18 )2, XPO(OR 18 )2, XC(O)OP(O)(OR 18 )2, XC(O)R 18 XC(O)NHR 18 , C1-C4 alkyl, alkylcarbonyl, carboxylic acid, or pharmaceutically acceptable salt; X is O, S, or NH; Y 1 and Y 2 Independently, it is either N or CH; [ka] is a bond point of a carbon atom;

[0081] Suitable buffers for use in formulations include, but are not limited to, organic acid salts such as sodium, potassium, ammonium, or trihydroxyethylamino salts of citric acid, ascorbic acid, gluconic acid, carbonate, tartaric acid, succinic acid, acetic acid, or phthalates; and Tris, tromethamine hydrochloride, sulfate, or phosphate buffers. Furthermore, amino acid cationic components can also be used as buffers. Such amino acid components include, but are not limited to, arginine, glycine, glycylglycine, and histidine. Arginine buffers include arginine acetate, arginine chloride, arginine phosphate, arginine sulfate, arginine succinate, etc. In one embodiment, the arginine buffer is arginine acetate. Examples of histidine buffers include histidine chloride-arginine chloride, histidine acetate-arginine acetate, histidine phosphate-arginine phosphate, histidine sulfate-arginine sulfate, histidine succinate-arginine succinate, etc. The pH of the buffer formulation is 4.5 to 8.5, preferably about 4.5 to about 7.0, and more preferably about 5.0 to about 6.2. In some embodiments, the concentration of the organic acid salt in the buffer is about 10 mM to about 500 mM.

[0082] Polyols, optionally included in pharmaceutical formulations, are substances having multiple hydroxyl groups. Polyols can be used as stabilizing excipients and / or isotonic agents in both liquid and lyophilized formulations. Polyols can protect biopharmaceuticals from physical and chemical degradation pathways. Preferredly excluded cosolvents increase the effective surface tension of the solvent at the protein interface, thereby resulting in the most energetically favorable structural conformation having the smallest surface area. Polyols include sugars (reducing and non-reducing sugars), sugar alcohols, and sugar acids. Reducing sugars contain hemiacetal groups that can reduce metal ions or react covalently with lysine or other amino groups of proteins, while non-reducing sugars lack these properties of reducing sugars. Examples of reducing sugars include fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, and glucose. Non-reducing sugars include sucrose, trehalose, sorbose, melegitose, and raffinose. The sugar alcohol is selected from mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, sreitol, sorbitol, and glycerol. The sugar acid includes L-gluconates and their metal salts. The polyol in the liquid formulation or the prepared lyophilized solid may be 0.0% to 20% by weight. Preferably, a non-reducing sugar, sucrose, or trehalose is selected in the formulation at a concentration of about 0.1% to 15%, with trehalose being preferred over sucrose due to its solution stability.

[0083] The surfactants optionally included in the formulation are polysorbates (polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, etc.), poloxamers (e.g., poloxamer 188, poly(ethylene oxide)-poly(propylene oxide), poloxamer 407, or polyethylene-polypropylene glycol, etc.); triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamide Propyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmimidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methylcocoyl or sodium methyloleyl taurate; dodecyl betaine, dodecyldimethylamine oxide, cocamidopropyl betaine, and cocoamphoglycinate, the "MONAQUAT" (trademark) series (e.g., isostearylethylimonium ethosulfate); polyethyl glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., Plu Selected from (e.g., ronics, PF68). Preferred surfactants are polyoxyethylene sorbitan fatty acid esters such as polysorbate 20, 40, 60, or 80 (Tween 20, 40, 60, or 80). The concentration of the surfactant in the formulation is in the range of 0.0% to about 2.0% by weight. In certain embodiments, the concentration of the surfactant is about 0.01% to about 0.2%. In one embodiment, the surfactant concentration is about 0.02%.

[0084] The “preservatives” optionally included in a formulation are compounds that essentially reduce bacterial activity. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl group is a long-chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl, and benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The amount of preservative in a liquid formulation or prepared lyophilized powder may be 0.0% to 5.0% by weight. In one embodiment, the preservative as used herein is benzyl alcohol.

[0085] Suitable free amino acids for use as isotonic or osmotic regulators in bulk raw materials or formulations are selected from, but are not limited to, arginine, cystine, glycine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycylcyrrhamate, or aspartic acid. Basic amino acids, i.e., those containing arginine, lysine, and / or histidine, are preferred. If the composition contains histidine, it can function as both a buffer and a free amino acid; however, if a histidine buffer is used, it typically contains non-histidine free amino acids, such as a histidine buffer and lysine. The amino acids may be D- and / or L-, but are usually L-isomers. The amino acids may exist as any stable salt, such as a hydrochloride salt of arginine-HCl. The amount of amino acids in the liquid formulation or prepared lyophilized powder can be 0.0% to 30% by weight.

[0086] The antioxidant in the formulation may be optionally selected from methionine, glutathione, cysteine, cystine, or ascorbic acid, and the concentration in the liquid formulation may be up to approximately 5 mg / ml, or the concentration in the formulated lyophilized powder may be between 0.0 wt% and 5.0 wt%.

[0087] The formulation may optionally contain metal chelating agents such as EDTA or EGTA, with a maximum concentration of approximately 2 mM in the liquid formulation, or a concentration of 0.0% to 0.3% in the formulated lyophilized powder.

[0088] The final formulation can be adjusted to a preferred pH with a buffering agent (e.g., acids such as HCl, H2SO4, acetic acid, H3PO4, citric acid, or bases such as NaOH, KOH, NH4OH, ethanolamine, diethanolamine, or triethanolamine, sodium phosphate, potassium phosphate, trisodium citrate, or tromethamine). The formulation needs to be controlled to be "isotonic," meaning that the formulation has essentially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250-350 mOsm. Isotonicity can be measured, for example, using a vapor pressure or ice-type osmometer. The isotonic agent is selected from mannitol, sorbitol, sodium acetate, potassium chloride, sodium phosphate, potassium phosphate, trisodium citrate, or NaCl. Generally, both the buffer salt and the isotonic agent can account for up to 30% by weight in the formulation.

[0089] Other excipients that may be useful in either the liquid or lyophilized formulation of the patent application include, for example, fucose, cellobiose, maltotriose, melibiose, octulose, ribose, xylitol, arginine, histidine, glycine, alanine, methionine, glutamic acid, lysine, imidazole, glycylglycine, mannosylglycerate, and Tri. This includes ton X-100, Pluoronic F-127, cellulose, cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, dextran (10, 40 or 70 kD), polydextrose, maltodextrin, Ficol, gelatin, hydroxypropyl meta, sodium phosphate, potassium phosphate, ZnCl2, zinc, zinc oxide, sodium citrate, trisodium citrate, tromethamine, copper, fibronectin, heparin, human serum albumin, protamine, glycerin, glycerol, EDTA, metacresol, benzyl alcohol, phenol, polyhydric alcohol, or hydrogenated forms of carbohydrates having a carbonyl group reduced to a primary or secondary hydroxyl group.

[0090] In another embodiment, the conjugate of the tubulicin analog in the formulation is having the structure of formula (II), or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof; or a polymorphic crystalline structure of these compounds; or an isotope, optical isomer, racemate, diastereomer, or enantiomer thereof: [ka]

[0091] In the formula, T, L, n, m, Y, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 8 , R 10 , R 13 , R 14 , R 15 , R 16 , and R 17 This is the same as the definition of equation (I).

[0092] In the formula, R 7 H, R 14 , or -R 14 C(=O)X 1 R 15 ;-R 14 X1 R 15 Selected from;X 1 is O, S, SS, NH, or NR 14 Selected from.

[0093] In the formula, R 9 H, -OH, -OR 14 -OC(=O)R 14 -OC(=O)NHR 14 -OC(=O)NR 14 R 15 -OC(=O)R 14 SSR 15 , OP(=O)(OR 14 )2, or OR 14 OP(=O)(OR 15 )

[0094] In the formula, R 11 H, R 14 ,-R 14 C(=O)R 16 ,-R 14 C(=O)X 2 R 16 ,-R 14 X 2 R 16 ,-R 14 C(=O)X 2 And in the formula, X 2 -O-, -S-, -NH-, -NHS(O2), -N(R 14 )-, -OR 14 -, -SR 14 -, -S(=O)-R 14 -, or -NHR 14 - is

[0095] In the formula, R 12 H, R 14 , -O-, -S-, -N-, =N-, =NNH-, -OH, -SH, -NH2, =NH, =NNH2, -NH(R 14 ), -OR 14 -C(O)O-, -C(O)OR 16 -, -COR 16 ,-COOR 14 -, C(O)NH-, C(O)NH2, C(O)NHR 14, -SR 14 -S(=O)R 14 , -P(=O)(OR 16 )2, -OP(=O)(OR 16 )2, -CH2OP(=O)(OR 16 )2, -SO2R 16 That is the case.

[0096] The exemplary compounds in parentheses in formula (II) are preferably having the following structures, or pharmaceutically acceptable salts, hydrates, or hydrated salts thereof; or polymorphic crystalline structures of these compounds; or isotopes, optical isomers, racemates, diastereomers, or their equivalents. Nantiomer: [ka] TIFF0007853711000010.tif242170TIFF0007853711000011.tif254170TIFF000 7853711000012.tif246170TIFF0007853711000013.tif254170TIFF00078537110 00014.tif254170TIFF0007853711000015.tif229170TIFF0007853711000016.t if246170TIFF0007853711000017.tif254170TIFF0007853711000018.tif132170

[0097] In the formula, R 20 This is a linear or branched alkyl, heteroalkyl, or acyl (-C(O)R) of H;C1-C8. 17 ); C2-C8 linear or branched alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 linear or branched aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; carbonate (-C(O)OR 17 ), carbamate (-C(O)NR 17 R 18); or C1-C8 carboxylates, esters, ethers, or amides; or 1-8 amino acids; or formula (OCH2CH2) p Or (OCH2CH(CH3)) p Polyethylene oxy units (where p is an integer from 0 to approximately 1000); or R 20 It is absent, and oxygen forms ketones, or a combination of these; R 21 It is a linear or branched alkyl group consisting of H, C1-C8.

[0098] Z 3 and Z 3 These are independently H, OH, NH2, OR 17 NHR 17 COOH, COOR 17 , C(O)R 17 C(O)NHR 17 C(O)NHNHR 17 , C(O)NH2, R 18 , OCH2OP(O)(OR 18 )2, OC(O)OP(O)(OR 18 )2, OPO(OR 18 )2, NHPO(OR 18 )2, OP(O)(OR 18 )OP(O)(OR 18 )2, OC(O)R 18 , OC(O)NHR 18 , OSO2(OR 18 ), O-(C4-C 12 - Glycosides), C1-C8 linear, branched, or heteroalkyl groups. ;C2-C8 linear or branched alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl;C3-C8 linear or branched aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl;carbonate (-C(O)OR 17 ), carbamate (-C(O)NR 17 R 18 ) It is; R 17 and R 18These are independently H, C1-C8 linear or branched alkyl or heteroalkyl; C2-C8 linear or branched alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 linear or branched aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; carbonate (-C(O)OR 17 ), carbamate (-C(O)NR 17 R 18 ) is; R 19 H, OH, NH2, OSO2(OR 18 ), XCH2OP(O)(OR 18 )2, XPO(OR 18 )2, XC(O)OP(O)(OR 18 )2, XC(O)R 18 XC(O)NHR 18 , C1-C8 alkyl or carboxylate; C2-C8 alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl or alkylcarbonyl; or medicinal salt; X is O, S, NH, NHNH, NHR 17 , or CH2; R 7 This is the same definition as above;

[0099] During the ceremony, [ka] This is the part that connects with the linked structure L in equation (II).

[0100] In another embodiment, the conjugate of the tubulicin analog in the formulation is having the structure of formula (III), or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof; or a polymorphic crystalline structure of these compounds; or an isotope, optical isomer, racemate, diastereomer, or enantiomer thereof: [ka]

[0101] In the formula, T, L, m, Y, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , and n are the same as defined in equations (I) and (II).

[0102] In the formula, R 7 is, -R 14 -, or -R 14 C(=O)X 1 R 15 , or -R 14 X 1 R 15 - Selected from, R 14 and R 15 These are independently linear or branched C1-C8 alkyl, heteroalkyl; C2-C8 alkenyl, alkynyl; C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, alkylcarbonyl; X 1 is O, S, SS, NH, or NR 14 Selected from.

[0103] The exemplary compounds in parentheses in formula (III) are those having the following structures, or pharmaceutically acceptable salts, hydrates, or hydrated salts thereof; or polymorphic crystalline structures of these compounds; or isotopes, optical isomers, racemates, diastereomers, or enantiomeric compounds thereof. Omar is: [ka] TIFF0007853711000022.tif254170TIFF0007853711000023.tif254170TIFF000 7853711000024.tif254170TIFF0007853711000025.tif254170TIFF00078537110 00026.tif254170TIFF0007853711000027.tif254170TIFF0007853711000028.t if229170TIFF0007853711000029.tif238170TIFF0007853711000030.tif102170

[0104] In the formula, R 20 This is a linear or branched alkyl, heteroalkyl, or acyl (-C(O)R) of H;C1-C8. 17 ); C2-C8 linear or branched alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 linear or branched aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; carbonate (-C(O)OR 17 ), carbamate (-C(O)NR 17 R 18 ); or C1-C8 carboxylates, esters, ethers, or amides; or 1-8 amino acids; or formula (OCH2CH2) p Or (OCH2CH(CH3)) p Polyethylene oxy units (where p is an integer from 0 to approximately 1000); or R 20 It is either absent, oxygen forms ketones, or a combination of these.

[0105] R 21 and R 22 These are independently H, C1-C8 linear or branched alkyl groups.

[0106] Z 3 and Z 3 These are independently H, OH, NH2, OR 17 NHR 17 COOH, COOR17 , C(O)R 17 C(O)NHR 17 C(O)NHNHR 17 , C(O)NH2, R 18 , OCH2OP(O)(OR 18 )2, OC(O)OP(O)(OR 18 )2, OPO(OR 18 )2, NHPO(OR 18 )2, OP(O)(OR 18 )OP(O)(OR 18 )2, OC(O)R 18 , OC(O)NHR 18 , OSO2(OR 18 ), O-(C4-C 12 - Glycosides), C1-C8 linear, branched, or heteroalkyl groups. ;C2-C8 linear or branched alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl;C3-C8 linear or branched aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl;carbonate (-C(O)OR 17 ), carbamate (-C(O)NR 17 R 18 ) is R 17 and R 18 These are independently H, C1-C8 linear or branched alkyl or heteroalkyl; C2-C8 linear or branched alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 linear or branched aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, Heteroaryl; carbonate (-C(O)OR 17 ), carbamate (-C(O)NR 17 R 18 ) is R 19 H, OH, NH2, OSO2(OR 18 ), XCH2OP(O)(OR 18 )2, XPO(OR 18 )2, XC(O)OP(O)(OR 18 )2, XC(O)R 18 XC(O)NHR18 , C1-C8 alkyl or carboxylate; C2-C8 alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl or alkylcarbonyl; or medicinal salt. X, X 1 , and X 2 These are independently O, S, NH, NHNH, or CH2.

[0107] In another embodiment, the conjugate of the tubulicin analog in the formulation is a pharmaceutically acceptable salt, hydrate, or hydrated salt having the structure of formula (IV) thereof; or a polymorphic crystalline structure of these compounds; or an isotope, optical isomer, racemate, diastereomer, or enantiomer thereof: [ka]

[0108] In the formula, T, L, m, Y, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 10 , R 11 , R 12 , R 13 , and n are the same as defined in equation (II).

[0109] In the formula, R 9 These are independently H, -O-, and -OR 14 -OC(=O)R 14 -OC(=O)NHR 14 -OC(=O)NR 14 R 15 -OC(=O)R 14 SSR 15 , OP(=O)(OR 14 )O- and R 14 and R 15These are independently H, C1-C8 alkyl, heteroalkyl; C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl, or medicinal salts.

[0110] The exemplary compounds in parentheses in formula (IV) are those having the following structures, or pharmaceutically acceptable salts, hydrates, or hydrated salts thereof; or polymorphic crystalline structures of these compounds; or isotopes, optical isomers, racemates, diastereomers, or enantiomers thereof: [ka] TIFF0007853711000033.tif254170TIFF0007853711000034.tif246170TIFF0007853711 000035.tif246170TIFF0007853711000036.tif229170TIFF0007853711000037.tif23817 0TIFF0007853711000038.tif238170TIFF0007853711000039.tif254170TIFF0007853711 000040.tif254170TIFF0007853711000041.tif254170TIFF0007853711000042.tif39170

[0111] During the ceremony, [ka] , R 7 , R 20 , R 21 , R 22 , Z 2 , Z 3 , and X 2 This is the same definition as above.

[0112] In another embodiment, the conjugate of the tubulicin analog in the formulation is a pharmaceutically acceptable salt, hydrate, or hydrated salt having the structure of formula (V) thereof; or a polymorphic crystalline structure of these compounds; or an isotope, optical isomer, racemate, diastereomer, or enantiomer thereof: [ka]

[0113] In the formula, T, L, m, Y, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 12 , R 13 , and n are the same as defined in equation (II).

[0114] In the formula, R 11 is, -R 14 -, -R 14 C(=O)R 17 -, -R 14 C(=O)X 2 R 17 -, -R 14 C(=O)X 2 - and R 17 Independently, H, OH, C1-C8 alkyl; C2-C8 alkenyl, alkynyl, heteroalkyl; C3-C8 aryl, arylene, heterocyclic, carbocyclic, heterocycloalkyl; or amino acid or two amino acid units; X 2 -O-, -S-, -NH-, -NHS(O2), -NHS(O)-, -N(R 14 )-, -OR 14 -, -SR 14 -, -S(=O)-R 14 -, or -NHR 14 -is;R 14These include H, C1-C8 alkyl, heteroalkyl; C2-C8 alkenyl, alkynyl; C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, and alkylcarbonyl.

[0115] The exemplary compounds in parentheses in formula (V) are those having the following structures, or pharmaceutically acceptable salts, hydrates, or hydrated salts thereof; or the polymorphic crystalline structures of these compounds; or they These are isotopes, optical isomers, racemates, diastereomers, or enantiomers thereof: [ka] TIFF0007853711000046.tif254170TIFF0007853711000047.tif242170TIFF0007853711 000048.tif238170TIFF0007853711000049.tif246170TIFF0007853711000050.tif25417 0TIFF0007853711000051.tif238170TIFF0007853711000052.tif254170TIFF0007853711 000053.tif254170TIFF0007853711000054.tif254170TIFF0007853711000055.tif39170

[0116] During the ceremony, [ka] , R 7 , R 20 , R 21 , R 22 , Z 2 , Z 3 , and X 2 This is the same definition as described above.

[0117] In another embodiment, the conjugate of the tubulicin analog in the formulation is a pharmaceutically acceptable salt, hydrate, or hydrated salt having the structure of formula (VI) thereof; or a polymorphic crystalline structure of these compounds; or an isotope, optical isomer, racemate, diastereomer, or enantiomer thereof: [ka]

[0118] In the formula, T, L, m, Y, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 11 , R 13 , and n are the same as defined in equation (II).

[0119] In the formula, R 12 R is independent of R 14 , -O-, -S-, -NH-, =N-, =NNH-, -N(R 14 )-, -OR 14 -, -C(O)O-, -C(O)NH-, -C(O)NR 14 -, -SR 14 -S(=O)R 14 , -NHR 14 -, -CH2OP(=O)(OR 15 ), -P(=O)(OR 15 ), -OP(=O)(OR 15 )O-, -SO2R 14 And R 14 and R 15 These are independently C1-C8 alkyl, heteroalkyl; C2-C8 alkenyl, alkynyl; C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, and alkylcarbonyl.

[0120] The exemplary compounds in parentheses in formula (VI) are those having the following structures, or pharmaceutically acceptable salts, hydrates, or hydrated salts thereof; or the polymorphic crystalline structures of these compounds; or These are isotopes, optical isomers, racemates, diastereomers, or enantiomers thereof: [ka] TIFF0007853711000059.tif229170TIFF0007853711000060.tif111170

[0121] In the formula, R 7 , R 17 , R 20 , R 21 , Z 2 , Z 3 , and X 2 This is the same definition as described above.

[0122] In another embodiment, the conjugate of the tubulicin analog in the formulation is a pharmaceutically acceptable salt, hydrate, or hydrated salt having the structure of formula (VII) thereof; or a polymorphic crystalline structure of these compounds; or an isotope, optical isomer, racemate, diastereomer, or enantiomer thereof: [ka]

[0123] In the formula, T, L, n, m, Y, R 1 , R 1’ , R 2 , R 3 , R 4 , R 5 , R 6 , R 8 , R 10 , R 11 , and R 12 This is the same as the definition of equation (II);

[0124] R 13C1~C 10 The group is an alkyl, heteroalkyl, alkyl acid, alkylamide, alkylamine, or Ar; Ar is an aromatic or heteroaromatic group consisting of one or more rings containing 4 to 10 carbon atoms, preferably 4 to 6 carbon atoms; the term heteroaromatic group means that one or more carbon atoms on the aromatic group, preferably 1, 2, or 3, are O, N, S i, Se, P, or S, more preferably O, S, N are replaced; the terms aryl or Ar mean that one or more H atoms are independently R 18 F, Cl, Br, I, OR 16 , SR 16 , NR 16 R 18 N=NR 16 N=R 16 , NR 16 R 18 NO2, SOR 16 R 18 SO2R 16 SO3R 16 OSO3R 16 PR 16 R 18 , POR 16 R 18 PO2R 16 R 18 OPO3R 16 R 18 , or PO3R 16 R 18 This also refers to aromatic groups that are substituted by R, in the formula. 16 and R 18 These are independently H, C1-C8 alkyl; C2-C8 alkenyl, alkynyl, heteroalkyl; C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, alkylcarbonyl; or C4-C 12 A glycoside; or a pharmaceutically acceptable salt.

[0125] The exemplary compounds in parentheses in formula (VI) are those having the following structures, or pharmaceutically acceptable salts, hydrates, or hydrated salts thereof; or polymorphic crystalline structures of these compounds; or isotopes, optical isomers, racemates, diastereomers, or enantiomers thereof: [ka] TIFF0007853711000063.tif254170TIFF0007853711000064.tif254170TIFF0007853711 000065.tif244170TIFF0007853711000066.tif251170TIFF0007853711000067.tif25417 0TIFF0007853711000068.tif254170TIFF0007853711000069.tif254170TIFF0007853711 000070.tif254170TIFF0007853711000071.tif254170TIFF0007853711000072.tif72170

[0126] During the ceremony, [ka] , R 7 , R 20 , R 21 , Z 2 , Z 3 , and X 2 The definition is the same as above. X, X 1 , and X 3 These are independently O, S, NH, NHNH, NHR 17 , CH2, or not present; P 1 H, R 17 , P(O)(OH)2, P(O)(X 1 R 17 )2, CH2P(O)(OH)2, S(O2)(X 1 R 17 ), C6H12 O5 (glycoside), (CH2CH2O) p R 17 In the formula, p is selected from 0 to 100, and R 17 is defined above; furthermore, X 1 P 1 It is also acceptable for them to be absent (both H).

[0127] In another embodiment, the synthesis of tubulicin analogs of the present invention and conjugates thereof with cell surface receptor-binding molecules is shown in Figure 1-22, but the invention is not limited thereto.

[0128] In another embodiment, the releaseable conjugate (L) is selected from a chain of C, N, O, S, Si, and P atoms that covalently bond a cell surface-bound ligand (T) to an effective tubulicine analog. The conjugates are of varying lengths, for example, ranging from about 2 to about 100 atoms. The atoms used to form the conjugate can be combined in all chemically relevant ways, such as forming alkylenes, alkenylenes, and alkynylenes, ethers, polyoxyalkylenes, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, acyloxylamines, and hydroxamic acids. Furthermore, the atoms forming the releaseable conjugate (L) may be either saturated or unsaturated, or radicals, or cyclized with each other to form divalent cyclic structures in the conjugate, including cycloalkanes, cyclic ethers, cyclic amines, arylenes, heteroarylenes, and the like.

[0129] The term "releaseable conjugate" refers to a conjugate containing at least one bond that can break down under physiological conditions such as pH instability, acid instability, base instability, oxidative instability, metabolic instability, biochemical instability, or enzymatic instability. The state does not necessarily have to be a biological or metabolic process; instead, it may include standard chemical reactions such as hydrolysis or substitution reactions, for example, disulfide bond exchange reactions with endosomes having a pH lower than cytoplasmic pH, and / or intracellular thiols such as glutathione-rich glutamine molecules in malignant cells.

[0130] The releaseable conjugate L of the conjugate may have the formula --Ww-(Aa)r-Vv-, where --W-- is an extended unit; w is 0 or 1; each --Aa-- is independently an amino acid unit; r is independently an integer between 0 and 12; --V-- is a spacer unit; v is 0, 1, or 2.

[0131] If an extended unit (--W--) is present, it binds the target binding molecular unit (T) to the amino acid unit (--Aa--); if Aa is absent, it binds to V. The extended unit W may independently contain a self-destructing spacer, a peptide unit, a hydrazone bond, a disulfide bond, or a thioether bond. In this regard, the binding molecule (T) has a functional group that forms a bond with the functional group of the extended unit. Useful functional groups that exist naturally or chemically on the binding molecule include, but are not limited to, sulfhydryl (-SH), amino, hydroxyl, carbonyl, anomeric hydroxyl of carbohydrates, and carboxyl groups. Preferred functional groups are sulfhydryl, carboxyl, and amino. Sulfhydryl groups can be generated by reducing the intramolecular disulfide bond of the ligand. Alternatively, the sulfhydryl group can be generated by reacting the amino group of the lysine portion of the binding molecule with 2-iminothiolane (Trout's reagent), thiolactone, or another sulfhydryl generating reagent (for example, modifying T with a disulfide bond conjugate or a thiol ester by reduction or hydrolysis as described below).

[0132] An example of W connected to T has the following structure: [ka] TIFF0007853711000075.tif21170

[0133] In the formula, R 20 and R 21 These are independently -C1~C9 alkylene-, -C1~C7 carbocyclo-, -O-(C1~C8 alkyl)-, -arylene-, -C1~C9 alkylene-arylene-, -arylene, -C1~C9 alkylene-, -C1~C9 alkylene-(C1~C8 carbocyclo)-, -(C3~C7 carbocyclo)-C1~C9 alkylene-, -C3~C8 heterocyclo-, -C1~C 10 Alkylene-(C3~C8 heterocyclo)-,-(C3~C8 heterocyclo)-C1~C9 alkylene-,-(CH2CH2O) k -,-(CH(CH3)CH2O) k -, and -(CH2CH2O) k Selected from -CH2-; k is an integer in the range of 1 to 20; R' and R'' are independently H or CH3.

[0134] In another embodiment, the covalent conjugation of W and T as described above can be carried out by various chemical reactions.

[0135] Examples of amide bond formation: [ka]

[0136] In the formula, the extended unit includes a reaction site with E, which can form an amide bond with the primary or secondary amino group of the ligand. Examples of reactive E include, but are not limited to, hydroxysuccinimide esters (NHS, sulfo-NHS, etc.), 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl (including sulfotetrafluorophenyl) esters, acid anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

[0137] Examples of thiol ethers or disulfide bonds: [ka]

[0138] The extended unit contains a sulfhydryl reaction site and can form a thiol ether or disulfide bond with a thiol group generated by reduction of the intramolecular disulfide bond of ligand T or by chemical modification of the bound ligand T.

[0139] In another aspect of the present invention, the reactive group of the extended unit includes a reaction site that reacts with an aldehyde (-CHO) or ketone (-C(=O)R) that can chemically modify the binding molecule T. For example, the carbohydrate of the binding molecule T can be lightly oxidized using a reagent such as sodium periodate to generate an aldehyde or ketone (-C(=O)R) group; or the amine of the N-terminal amino acid of an antibody (or protein, peptide) can be reacted with pyridoxal 5'-phosphate (PLP) in buffer to introduce a ketone group (Scheck & Francis, ACS Chem. Biol. 2007, 2, 247-251). The resulting (-C=O) unit can be condensed with an extended unit containing functional groups such as hydrazides, oximes, primary or secondary amines, hydrazines, thiosemicarbazones, carboxylic acid hydrazines, and aryl hydrazides.

[0140] Examples of hydrazone, oxime, or imine bond conjugation: [ka]

[0141] In the formula, R 20 and R 21 As stated above, R 25 is an organic substituent of an amino acid.

[0142] Furthermore, the present invention also discloses that an extended unit (which may include a spacer unit V and / or an amino acid) can be attached to a binding molecule (T), and then, in a buffered aqueous solution, a tubulicin analog of the active ingredient is conjugated with the binding molecule-extended unit moiety. An example of two-step conjugation (R 16 The drugs related to this are omitted in the formula):

[0143] [ka]

[0144] In the formula, E includes, but is not limited to, hydroxysuccinimide esters (NHS, sulfo-NHS, etc.), 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl (including sulfotetrafluorophenyl) esters, acid anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. R' and R'' are independently H or CH3. 20 , R 16 , and Ar are defined in various embodiments of the present invention. 26 is independently H, F, or NO2. J is independently F, Cl, Br, I, tosylate (TsO), or mesylate (MsO), in the formula, [ka] is at least one tubulicin analog / drug [ka] It holds.

[0145] In another aspect of the present invention, the extended unit can first bind to a tubulicin analog, which is the active ingredient, and then be conjugated with the binding molecule (T) in a buffered aqueous solution at pH 3-10 (preferably pH 5-8.5) containing up to 50% organic cosolvent. Examples of these two-step conjugation:

[0146] [ka]

[0147] In the formula, E includes, but is not limited to, hydroxysuccinimide esters (NHS, sulfo-NHS, etc.), 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl (including sulfotetrafluorophenyl) esters, acid anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. R' and R'' are independently H or CH3. 16 , R 20 , and Ar are defined in various embodiments of the present invention. 26 It is independently H, F, or NO2. J is independent. The components are F, Cl, Br, I, tosylate (TsO), or methanesulfonate (MsO), in which, [ka] It contains at least one tubulicin analog.

[0148] The amino acid unit (--Aa--) links the extended unit and the spacer unit if a spacer unit is present, links the extended unit and the tubulicin analog unit if a spacer unit is not present, and links the binding molecule (T) and the tubulicin analog unit if neither an extended unit nor a spacer unit is present. --(Aa)r-- is a natural or unnatural amino acid, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, hepeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide unit, where r is an integer from 0 to 12. As used herein, the term amino acid generally refers to an aminoalkyl carboxylate, where the alkyl residue is optionally substituted with, for example, alkyl, acyl, hydroxyalkyl, sulfhydrylalkyl, aminoalkyl, carboxyalkyl, etc. The structures and peptides of natural and unnatural amino acids are described in the literature: G.C. Barrett and D.T. Elmore, “AminoAcid and Peptide”, Cambridge University Press, 2004. Furthermore, amino acids refer to β, γ, and long-chain amino acids containing methyl, benzyl, hydroxymethyl, thiomethyl, carboxyl, carboxymethyl, guanidinopropyl, etc. in their chains. More preferably, amino acids are selected from asparagine, aspartic acid, cysteine, glycine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, etc.

[0149] The amino acid units can be digested by one or more enzymes (including tumor-associated proteases) to release tubulicin analogs, and in one embodiment, they are protonated in vivo to release tubulicin analogs.

[0150] If a spacer unit (--V--) is present, and an amino acid unit is present, the spacer unit links the amino acid unit to the tubulicin analogue; or, if an amino acid unit is absent, the spacer unit links the extended unit to the tubulicin analogue. The spacer unit also links the tubulicin analogue to the binding molecule (T) if neither an amino acid unit nor an extended unit is present. Spacer conjugates may contain functional groups that substantially increase the solubility, biological transport, preferential renal clearance, uptake, absorption, biodistribution, and / or bioavailability of the conjugate. Spacer units generally exist in two types: self-destructive and non-self-destructive. After cleavage of the amino acid unit from the tubulicin analogue-conjugate-binding molecule conjugate or tubulicin analogue-conjugate compound, particularly after enzymatic cleavage, some or all of the spacer unit maintains its binding to the tubulicin analogue. The self-destructing unit comprises an aromatic compound electronically similar to the para-aminobenzylcarbamoyl (PAB) group, a 2-aminoimidazole-5-methanol derivative, a heterocyclic PAB analog, a β-glucuronide, and an o- or p-aminobenzyl acetal; or any of the following structures:

[0151] [ka]

[0152] In the formula, ( * Atoms labeled with ) are additional spacers or releaseable linkage units, mitotic inhibitors, and / or binding sites to cell adhesion molecules (T); X, Y, and Z 3 Independently, NH, O, or S; Z 2 is H, NH, O, or S; v is 0 or 1; Q is independently H, OH, C1-C6 alkyl, (OCH2CH2) n F, Cl, Br, I, OR 17 , SR 17 , NR 17 R 18 N=NR 17 N=R 17 , NR 17 R18 NO2, SOR 17 R 18 SO2R 17 SO3R 17 OSO3R 17 PR 17 R 18 , POR 17 R 18 PO2R 17 R 18 , OPO(OR 17 )(OR 18 ), or OCH2PO(OR 17 )(OR 18 ) and in the formula, R 17 and R 18 Each is independently selected from H, C1-C8 alkyl; C2-C8 alkenyl, alkynyl, heteroalkyl; C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl; or pharmaceutically acceptable cation salts;

[0153] Examples of non-self-destructing spacer connectors: [ka] TIFF0007853711000086.tif254170TIFF0007853711000087.tif248170TIFF0007853711000088.tif254170TIFF0007853711000089.tif136170 Or L- or D-, natural or non-natural peptides containing 1 to 20 identical or different amino acids.

[0154] In the formula, “*” and [ka] The atoms are additional spacers or releaseable linkers, tubulosine analogs, and / or attachment sites of the binding molecule; m is 1 to 10; n is 1 to 20; X2, X3, X4, X5, or X6 are independently NH;NHNH;N(R 12 );N(R 12 )N(R 12’ );O;S;C1-C6 alkyl;C2-C6 heteroalkyl, alkylcycloalkyl, heterocycloalkyl;C3-C8 aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl;CH2OR 12 CH2SR 12 CH2NHR 12 , or selected from 1 to 8 amino acids; in the formula, R 12 and R 12’ These are independently H; C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or esters, ethers, or amides having 1 to 8 carbon atoms; or formula (OCH2CH2) p Or (OCH2CH(CH3)) p Polyethylene These are lenoxy units (where p is an integer from 0 to approximately 1000), or combinations thereof.

[0155] The releaseable component of conjugate L, which is at least one bond of conjugate L, is at least one bond that can collapse under physiological conditions; including a pH-instable, acid-instable, base-instable, oxidative-instable, metabolic-instable, biochemical-instable, or enzymatically instable bond, and having one of the following structures: -(CR 15 R 16 ) m (Aa) r (CR 17 R 18 ) n (OCH2CH2) t -,-(CR 15 R 16 )m (CR 17 R 18 ) n (Aa) r (OCH2CH2) t -、-(Aa) r -(CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) t -、-(CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) r (Aa) t -、-(CR 15 R 16 ) m (CR 17 =CR 18 )(CR 19 R 20 ) n (Aa) t (OCH2CH2) r -、-(CR 15 R 16 ) m (NR 11 CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m (Aa) t (NR 21 CO)(CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m (OCO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m(OCNR 17 )(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m (CO)(Aa) t -(CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m (No. 21 CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m (OCO)(Aa) t (CR 19 R 20 ) n -(OCH2CH2) r -、-(CR 15 R 16 ) m (OCNR 17 )(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m (CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m -phenyl-(CO)(Aa) t (CR 17 R 18 ) n -、-(CR 15 R 16 ) m -フリル-(CO)(Aa) t (CR17 R 18 ) n -,-(CR 15 R 16 ) m -Oxazolyl-(CO)(Aa) t (CR 17 R 18 ) n -,-(CR 15 R 16 ) m -Thiazolyl-(CO)(Aa) t (CR 17 R 18 ) n -,-(CR 15 R 16 ) t -Chienil-(CO)(CR 17 R 18 ) n -,-(CR 15 R 16 ) t -Imidazolyl-(CO)(CR 17 R 18 ) n -,-(CR 15 R 16 ) t -Morphorino-(CO)(Aa) t (CR 17 R 18 ) n -,-(CR 15 R 16 ) t -Piperadino-(CO)(Aa) t (CR 17 R 18 ) n -,-(CR 15 R 16 ) t -N-methylpiperazine-(CO)(Aa) t (CR 17 R 18 ) n -,-(CR 15 R 16 ) m -(Aa) t Phenyl-,-(CR 15 R 16 ) m -(Aa) t Frill-,-(CR 15 R16 ) m -oxazolyl (Aa) t -,-(CR 15 R 16 ) m -thiazolyl (Aa) t -,-(CR 15 R 16 ) m -thienyl (Aa) t -,-(CR 15 R 16 ) m -imidazolyl (Aa) t -,-(CR 15 R 16 ) m -morpholino-(Aa) t -,-(CR 15 R 16 ) m -piperazino-(Aa) t -,-(CR 15 R 16 ) m -N-methylpiperazino-(Aa) t -,-K(CR 15 R 16 ) m (Aa) r (CR 17 R 18 ) n (OCH2CH2) t -,-K(CR 15 R 16 ) m (CR 17 R 18 ) n (Aa) r (OCH2CH2) t -,-K(Aa) r (CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) t -,-K(CR 15 R 16 ) m (CR 17 R<able> 18 ) n (OCH2CH2)<able) r (Aa)<able) t-、-K(CR 15 R 16 ) m (CR 17 =R 18 )(CR 19 R 20 ) n (Aa) t (OCH2CH2) r -、-K(CR 15 R 16 ) m (NR 11 CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-K(CR 15 R 16 ) m (Aa) t (NR 21 CO)(CR 19 R 20 ) n (OCH2CH2) r -、-K(CR 15 R 16 ) m (OCO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-K(CR 15 R 16 ) m (OCNR 17 )(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-K(CR 15 R 16 ) m (CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-K(CR 15 R 16 ) m (NR 21 CO)(Aa) t (CR 19 R 20 )n (OCH2CH2) r -, -K(CR 15 R 16 ) m (OCO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -, -K(CR 15 R 16 ) m (OCNR 17 )(Aa) t ( CR 19 R 20 ) n (OCH2CH2) r -, -K(CR 15 R 16 ) m (CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -, -K(CR 15 R 16 ) m -phenyl-(CO)(Aa) t (CR 17 R 18 ) n -, -K(CR 15 R 16 ) m -furyl-(CO)(Aa) t (CR 17 R 18 ) n -, -K(CR 15 R 16 ) m -oxazolyl-(CO)(Aa) t (CR 17 R 18 ) n -, -K(CR 15 R 16 ) m -thiazolyl-(CO)(Aa) t (CR 17 R 18 ) n -, -K(CR 15 R 16 ) t-Chienil-(CO)(CR 17 R 18 ) n -, -K(CR 15 R 16 ) t -Imidazolyl-(CO)(CR 17 R 18 ) n -, -K(CR 15 R 16 ) t -Morphorino-(CO)(Aa) t (CR 17 R 18 ) n -, -K(CR 15 R 16 ) t -Piperadino-(CO)(Aa) t (CR 17 R8) n -, -K(CR 15 R 16 ) t -N-methylpiperazine-(CO)(Aa) t (CR 17 R 18 ) n -, -K(CR 15 R 16 ) m -(Aa) t Phenyl-,-K(CR 15 R 16 ) m -(Aa) t Frill-, -K(CR 15 R 16 ) m -Oxazolyl (Aa) t -, -K(CR 15 R 16 ) m - Thiazolyl (Aa) t -, -K(CR 15 R 16 ) m -Chienil (Aa) t -, -K(CR 15 R 16 ) m -Imidazolyl (Aa) t -, -K(CR 15 R 16 ) m -Morphorino-(Aa)t -, -K(CR 15 R 16 ) m -Piperadino-(Aa) t G-, -K(CR 15 R 16 ) m -N-methylpiperazino -(Aa) t ;In the formula, m, Aa, m, n, R 13 , R 14 , and R 15 The definition is as above; t and r are independently between 0 and 100. ru;R 16 , R 17 , R 18 , R 19 , and R 20 These are independently H; halides; C1-C8 alkyl or heteroalkyl; C2-C8 aryl, alkenyl, alkynyl, ether, ester, amine, or amide; or one or more halides, CN, NR 12 R 12’ CF3, OR 12 aryl, heterocyclic ring, S(O)R 12 SO2R 12 -CO2H, -SO3H, -OR 12 , -CO2R 12 ,-CONR 12 , -PO2R 12 R 13 -PO3H, or P(O)R 12 R 12’ R 13’ Selected from C3-C8 aryls, which may be optionally substituted; K is NR 12 , -SS-, -C(=O)-, -C(=O)NH-, -C(=O)O-, -C=NH-O-, -C=N-NH-, -C(=O)NH-NH-, O, S, Se, B, Het(C3~C 12 It is a peptide containing a heterocyclic or heteroaromatic ring, or 1 to 20 identical or different amino acids.

[0156] The binding molecule (T) may be any currently known or discovered molecule that binds, complexes, or reacts with residues of a cell population that is to be therapeutically or otherwise biologically modified. The binding molecule unit acts to deliver a tubulicin analog to the specific target cell population to which the binding molecule (T) reacts.

[0157] Cell binding agents T include, but are not limited to, large molecular weight proteins such as full-length antibodies (polyclonal or monoclonal), dimers, multimers, multispecific antibodies (e.g., bispecific antibodies); single-chain antibodies; antibody fragments, such as Fab, Fab', F(ab')2, Fv [Parham, J. Immunol. 131, 2895-2902 (1983)], fragments obtained by Fab expression libraries, anti-idiotype (anti-Id) antibodies, CDRs, bispecific antibodies, trispecific antibodies, cancer cell antigens, viral antigens, microbial antigens, or specific antigens that can recognize, bind to, or express desirable biological activity. Any epitope-binding fragment of the foregoing that binds immunospecifically to proteins produced by the immune system; interferons (e.g., types I, II, III); peptides; lymphokines, e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, GM-CSF, or interferon-γ (IFN-γ); hormones, e.g., insulin, TRH (thyroid-stimulating hormone-releasing hormone), MSH (cytostimulating hormone), or steroid hormones such as androgens, estrogens, or melanocyte-stimulating hormone (MSH); growth factors and colony-stimulating factors, e.g., epidermal growth factor (EFG), granulocyte-macrophage colony - Stimulating factors (GM-CSF); transforming growth factors (TGF), e.g., TGFα, TGFβ; insulin and insulin-like growth factors (IGF-I, IGF-II) G-CSF, M-CSF, and GM-CSF [Burgess, Immunology Today, 5, 155-158 (1984)]; vaccine growth factor (VGF); fibroblasts Cell growth factors (FGF); small molecular weight proteins, polypeptides, peptides, and peptide hormones, e.g., bombesin, gastrin, and gastrin-releasing peptides; platelet-derived growth factor; interleukins and cytokines, e.g., interleukin-2 (IL-2), interleukin-6 (IL-6), leukemia inhibitors, granulocyte-macrophage colony-stimulating factor (GM-CSF); vitamins such as folic acid; apolipoproteins and glycoproteins, e.g., transferrin [O'Keefe et al, J. Bio. Chem. 260, 932-927 (1985)]; sugar-binding proteins or lipoproteins such as lectins; cellular nutrient transport molecules; and small molecule inhibitors, e.g., prostate-specific membrane antigen (PSMA) inhibitors, small molecule tyrosine kinase inhibitors (TKIs), non-peptides, or other cell-binding molecules or substances, e.g., bioactive polymers (Dhar, et al. al, Proc. Natl. Acad. Sci. 2008, 105, 17356-61, dendrimers (Lee, et al, Nat. Biotechnol. 2005, 23, 1517-26; Almutairi, et al; Proc. Natl. Acad. Sci. 2009, 106, 685-90), nanoparticles (Liong, et al, ACS Nano, 2008, 19, 1309-12; Medarova, et al, Nat. Med. 2007, 13, 372-7; Javier, et al, Bioconjugate Chem. 2008, 19, 1309-12), liposomes (Medinai, et al, Curr. Phar. Des. 2004, 10, 2981-9), viral capsids (Flenniken, et al, Viruses This includes Nanotechnol. 2009, 327, 71-93). Generally, when a suitable monoclonal antibody is available, a monoclonal antibody is preferred as a cell surface binder.

[0158] Preferably, T is selected from the group consisting of antibodies; single-chain antibodies; antibody fragments that bind to target cells; monoclonal antibodies; single-chain monoclonal antibodies; or monoclonal antibody fragments that bind to target cells; chimeric antibodies; chimeric antibody fragments that bind to target cells; domain antibodies; domain antibody cross-sections that bind to target cells; antibody-mimicking adnectin; DARPins; lymphokines; hormones; vitamins; growth factors; colony-stimulating factors; or nutrient transport molecules; transferrin; binding peptides, or proteins, or small molecules, polymers, dendrimers, liposomes, nanoparticles, vesicles, or (viral) capsids bound to antibodies or albumin.

[0159] More preferably, the cell binding agent / molecule T can target tumor cells, virus-infected cells, microbial-infected cells, parasitic-infected cells, autoimmune cells, activated cells, bone marrow cells, activated T cells, B cells, or melanocytes, or any of the following antigens or receptors: CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CD12w, CD13, CD14, CD15, CD16, CD16a, CD16b, CDw17, CD18, CD19, CD20, C D21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD32a, CD32b, CD33 , CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD49c, CD49d, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P 、CD63、CD64、CD65、CD65s、CD66、CD66a、CD66b、CD66c、CD66d、CD66e、CD66f、CD67、CD68、CD69、CD70、CD71、CD72、CD73、CD74、CD75、CD75s、CD76、CD77、CD78、CD79、CD79a、CD79b、CD80、CD81、CD82、CD83、CD84、CD85、CD85a、CD85b、CD85c、CD85d、CD85e、CD85f、CD85g、CD85g、CD85i、CD85j、CD85k、CD85m、CD86、CD87、CD88、CD89、CD90、CD91、CD92、CD93、CD94、CD95、CD96、CD97、CD98、CD99、CD100、CD101、CD102、CD103、CD104、CD105、CD106、CD107、CD107a、CD107b、CD108、CD109、CD110、CD111、CD112、CD113、CD114、CD115、CD116、CD117、CD118、CD119、CD120、CD120a、CD120b、CD121、CD121a、CD121b、CD122、CD123、CD123a、CD124、CD125、CD126、CD127、CD128、CD129、CD130、CD131、CD132、CD133、CD134、CD135、CD136、CD137、CD138、CD139、CD140、CD140a、CD140b、CD141、CD142、CD143、CD144、CD145、CDw145、CD146、CD147、CD148、CD149、CD150、CD151、CD152、CD153、CD154、CD155、CD156、CD156a、CD156b、CD156c、CD156d、CD157、CD158、CD158a、CD158b1、CD158b2、CD158c、CD158d、CD158e1、CD158e2、CD158f2、CD158g、CD158h、CD158i、CD158j、CD158k、CD159、CD159a、CD159b、CD159c、CD160、CD161、CD162、CD163、CD164、CD165、CD166、CD167、CD167a、CD167b、CD168、CD169、CD170、CD171、CD172、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179、CD179a、CD179b、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CDw186、CD187、CD188、CD189、CD190、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CD198、CD199、CDw198、CDw199、CD200、CD201、CD202、CD202(a、b)、CD203、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210、CDw210a、CDw210b、CD211、CD212、CD213、CD213a1、CD213a2、CD214、CD215、CD216、CD217、CD218、CD218a、CD218、CD21b9、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235、CD235a、CD235b、CD236、CD237、CD238、CD239、CD240、CD240ce、CD240d、CD241、CD242、CD243、CD244、CD245、CD246、CD247、CD248、CD249、CD250、CD251、CD252、CD253、CD254、CD255、CD256、CD257、CD258、CD259、CD260、CD261、CD262、CD263、CD264、CD265、CD266、CD267、CD268、CD269、CD270、CD271、CD272、CD273、CD274、CD275、CD276、CD277、CD278、CD279、CD281、CD282、CD283、CD284、CD285、CD286、CD287、CD288、CD289、CD290、CD291、CD292、CD293、CD294、CD295、CD296、CD297、CD298、CD299、CD300、CD300a、CD300b、CD300c、CD301、CD302、CD303、CD304、CD305、CD306、CD307、CD307a、CD307b、CD307c、CD307d、CD307eぁ , CD307f, CD308, CD309, CD310, CD311, CD312, CD313, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD 323, CD324, CD325, CD326, CD327, CD328, CD329, CD330, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD341, CD342, CD343, CD344, CD345, CD346, CD347, CD348, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD35 6, CD357, CD358, CD359, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, CD371, CD372, CD3 73, CD374, CD375, CD376, CD377, CD378, CD379, CD381, CD382, CD383, CD384, CD385, CD386, CD387, CD388, CD389, CRIPTO, CRIPTO, CR, CR1, CRGF, CRIPTO, CXCR5, LY64, TDGF1, 4-1BB, APO2, ASLG659, BMPR1B, 4-1BB, 5AC, 5T4 (trophoblast glycoprotein, TPBG, 5T4, Wnt activator 1 or WAIF1), adenocarcinoma antigen, AGS-5, AGS-22M6, activin receptor-like kinase 1, AFP, AKAP-4, ALK, α-integrated angiopoietin, α-vbeta6, aminopeptidase N, amyloid β, androgen receptor, angiopoietin 2, angiopoietin 3, annexin A1, anthrax toxin protective antigen, anti-transferrin receptor, Aoc3 (VAP-1), B7-H3, Bacillus anthracis anthrax, BAFF (B-cell activator), BCMA, B lymphoma cells, bcr-abl, bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, CarboniCAnhydrase 9), CALLA, CanAg, Canine Sirpus IL31, CarboniCAnhydraseIX、CardiaCMyosin、CCL11(CCChemokine 11)、CCR4(CCChemokine Receptor 4), CCR5, CD3E(ε), CEA (oncoemulsifying antigen), CEACAM3, CEACAM5 (oncoemulsifying antigen), CFD (factor D), Ch4D5, cholecystokinin 2 (CCK2R), CLDN18 (claudin-18), CLDN18.1 (claudin-18.1), CLDN18.2 (claudin-18.2), agglutinating factor A, cMet, CRIPTO, FCSF1R (colony-stimulating factor 1 receptor), CSF2 (colony-stimulating factor 2, granulocyte-macrophage colony (GMO-CSF), CSP4, CTLA4 (Cytotoxic T Lymphocyte-Associated Protein 4), CTAA16.88 Tumor Antigen, CXCR4, CXC Chemokine Receptor 4, Cyclic ADP Ribohydrolase, Cyclin B1, CYP1B1, Cytomegalovirus, Cytomegalovirus Glycoprotein B, Dabigatran, DLL3 (Delta-like Ligand 3), DLL4 (Delta-like Ligand 4), DPP4 (Dipeptidyl Peptidase 4), DR5 (Death Receptor 5), Escherichia coli Shiga toxin type 1, Escherichia coli Shiga toxin type 2, ED-B, EGFL7 (EGF-like domain-containing protein 7), EGFR, EGFRII, EGFRvIII, endoglin, endothelin B receptor, endotoxin, EpCAM (epithelial cell adhesion molecule), EphA2, epicyalin, ERBB2 (epidermal growth factor receptor 2), ERBB3, ERG (TMPRSS2ETS fusion gene), E. coli, ETV6-AML, FAP (fibroblast) activating protein (α), fibroblast surface antigen, FCGR1, α-fetoprotein, fibrin II, β-chain, fibronectin extradomain B, FOLR (folate receptor or), folate receptor body α, folate hydrolase, Fos-associated antigen 1F protein of respiratory syncytial virus, curled receptor, rock alcohol GM1, GD2 ganglioside, G-28 (cell surface antigen glycolipid), GD3 idiotype, GloboH, glypican 3, N-glycylneuraminic acid, GM3, GMCSF receptor α-chain, growth differentiation factor, GP100, GPNMB (transmembrane glycoprotein NMB), GUCY2C (guanate cyclase 2C, guanine cyclase) Ze C (GC-C), enteric guanylate cyclase, guanylate cyclase receptor, heat-stable enterotoxin receptor (hSTAR), heat shock protein, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1 (human epidermal growth factor receptor 1), HER2, HER2 / neu, HER3 (ERBB-3), IgG4, HGF / SF (hepatocyte growth factor / scattering factor), HHGFR, HIV-1, histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, human chorionic gonadotropin, HNGF, human scattering factor receptor kinase, HP VE6 / E7, Hsp90, hTERT, ICAM-1 (intercellular adhesion molecule 1), idiotype, IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-γ, influenza hemagglutinin, IgE, IgEFc region, IGHE, interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-17A, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-27 or IL-28), IL31RA, ILGF2 (insulin-like growth factor 2), integrins (α4, αIIbβ3, αvβ3, α4β7, α5β1, α6β4, α7β7, αllβ3, α5β5, αvβ5), gamma interferon-inducible protein, ITGA2, ITGB2, KIRD2, κIg, LCK, Le, regmine, Lewis-Y antigen, LFA-1 (lymphocyte function-related antigen 1, CD11a), LHRH, LINGO-1, lipoprotein acid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE -3, MAGEA1, MAGEA3, MAGE4, ​​MART1, MCP-1, MIF (macrophage migration inhibitor or glycosylation inhibitor (GIF)), MS4A1 (transmembrane 4-domain subfamily A member 1), MSLN (mesothelin), MUC1 (mucin 1, cell surface association (MUC1) or polymorphoemic mucin (PEM)), MUC1-KLH, MUC16 (CA125), MCP1 (monocyte proliferation protein 1), MelanA / MART1, ML-IAP, MPG, MS4A1 (transmembrane 4-domain subfamily A), MYCN, myelin-related Glycoprotein, myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen), nectin-4 (ASG-22ME), NGF, neuronal apoptosis regulatory protease 1, NOGO-A, Notch receptor, nucleoprotein, Neu tumor gene product, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized low-density lipoprotein), OY-TES1, P21, p53 non-mutant, P97, Page4, PAP, anti(NN-glycolylneuraminate), PAX3, PAX5, PCSK9, PDCD1 (PD-1, programmed cell death protein) Phytoprotein 1), PDGF-Rα (Platelet-derived growth factor receptor alpha), PDGFR-β, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, Platelet-derived growth factor receptor β, Sodium phosphate cotransporter, PMEL17, Polysialic acid, Protease 3 (PR1), Prostate cancer, PS (Phosphatidylserine), Prostate cancer cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, Rayby virus glycoprotein, RHD (Rh polypeptide 1 (RhPI)), Rh factor, RANKL, Rhoc, Ras variant, RGS5, ROBO4, Respiratory syncytial virus,RON, ROR1, sarcoma transition breakpoint, SART3, sclerostin, SLAMF7 (SLAM family member 7), SelectinP, SDC1 (syndecane 1), sLe(a), somatostatin C, SIP (sphingosine 1-phosphate), somatostatin, sperm protein 17, SSX2, STEAP1 (six transmembrane epithelial antigens of prostate 1), STEAP2, STn, TAG-72 (tumor-associated glycoprotein 72), Survivin, T cell receptor, T cell transmembrane protein, TEM1 (tumor endothelial marker 1), TENB2, tenascin C (TN-C), TGF-α, TGF-β (transforming growth factor β), TGF-β1, TGF-β2 (transforming growth factor β2), Tie (CD202b), Tie2, TIM-1 (CDX-014), Tn, TNF, TNF-α, TNFRSF8, TNFR, Cells expressing SF10B (tumor necrosis factor receptor superfamily member 10B), TNFRSF-13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblast glycoprotein), TRAIL-R1 (tumor necrotic cell apoptosis-inducing ligand receptor 1), TRAILR2 (death receptor 5 (DR5)), tumor-associated calcium signaling transducer 2, MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-1 (Trop1), TRP-2 (Trop2), tyrosinase, VCAM-1, VEGF, VEGF-A, VEGF-2, VEGFR-1, VEGFR2 or vimentin, WT1, XAGE1, or any insulin growth factor receptor, or any epidermal growth factor receptor.

[0160] In another specific embodiment, the cell-binding molecule may be a ligand or receptor agonist selected from: folate derivatives (binding to folate receptors, proteins overexpressed in ovarian cancer and other malignancies) (Low, PS et al 2008, Acc. Chem. Res. 41, 120-129); glutamate urea derivatives (binding to prostate-specific membrane antigens, surface markers of prostate cancer cells) (Hillier, SM et al, 2009, Cancer Res. 69, 6932-6940); somatostatin (also known as growth hormone inhibitor (GHIH), somatotropin release inhibitor (SRIF), or somatotropin release inhibitor hormone) and their derivatives, e.g., octreotide (sandostatin) and lanreotide (somatsurin) (especially for neuroendocrine tumors, GH-producing pituitary adenomas, paraganglionic tumors, dysfunctional pituitary adenomas, pheochromocytomas) (Ginj, M., et al, 2006, Proc. Natl. Acad. Sci. USA 103, 16436-16441); Somatostatin receptor subtypes (sst1, sst2, sst3, sst4, and sst5) (Reubi JC, Landolt, AM 1984 J. Clin. Endocrinol Metab 59:1148-51; Reubi JC, Landolt AM 1987 J Clin Endocrinol Metab 65:65-73; Moyse E, et al, J Clin Endocrinol Metab 61:98-103), gastrointestinal and pancreatic tumors (Reubi JC, et al, 1987 J Clin Endocrinol Metab 65:1127-34; Reubi, J. C, et al, 1990 Cancer Res 50:5969-77), pheochromocytoma (Epel-baum J, et al 1995 J Clin Endocrinol Metab 80:1837-44; Reubi JC (e.g., 1992 J Clin Endocrinol Metab 74:1082-9), neuroblastoma (Prevost G, 1996 Neuroendocrinology 63:188-197; Moertel, C. L, et al 1994 Am J Clin Path 102:752-756), thyroid nodules (Reubi, J. C, et al 1991 Lab Invest 64:567-573) small cells Cellular lung cancer (Sagman U, et al., 1990 Cancer 66:2129-2133), marrow adenoma, marrow budding tumor, and also nerve fibrosis (Reubi JC, et al., 1986 J Clin Endocrinol Metab 63:433-8; Reubi JC, et al., 1987). Cancer Res 47:5758-64; Fruhwald, M. C, et al 1999 Pediatr Res 45:697-708), Breast cancer (Reubi JC, et al 1990 Int J Cancer 46:416-20; Srkalovic G, et al 1990 J Clin Endocrinol Metab 70:661-669), Rhinoplasty (Reubi JC, et al 1992, Int J Cancer 50:895-900), Renal Cell Carcinoma (Reubi JC, et al 1992, Cancer Res 52:6074-6078), Mesenchymal Tumor (Reubi JC, et al 1996, Cancer Res 56:1922-31), Prostate (Reubi JC, et al 1995, J. Clin. Endocrinol Metab 80:2806-14; et al 1989, Prostate 14:191-208; Halmos G, et al J. Clin. Endo-crinol Metab 85:2564-71), ovary (Halmos, G, et al, 2000 J Clin Endocrinol Metab 85:3509-12; Reubi JC, et al 1991 Am J Pathol 138:1267-72), stomach (Reubi JC, et al 1999, Int J Cancer 81: 376-86; Miller, G. V, 1992 Br J Cancer 66:391-95), hepatocellular carcinoma (Kouroumalis E, et al 1998 Gut 42:442-7; Reubi JC, et al 1999 Gut 45:66-774) and nasopharyngeal carcinoma (Loh K. S, et al, 2002 Virchows Arch 441:444-8; Specific to carbonic anhydrase IX (marker for hypoxia and renal cell carcinoma) Specific aromatic sulfonamides (Neri, D., et al, Nat. Rev. Drug Discov. 2011, 10, 767-777); pituitary adenylate cyclase activation for pheochromocytoma and paraganglionic tumors. Peptides (PACAP) (PAC1); Vasoactive intestinal peptide (VIP / PACAP) and its receptor subtypes (VPAC1, VCAP2); α-melanocyte-stimulating hormone (α-MSH) receptor; Cholecystokinin (CCK) / gastrin receptor and its receptor subtypes (CCK1 (formerly CCK-A) and CCK2); Bombecin (Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-H is-Leu-Met-NH2) / gastrin-releasing peptide (GRP) ( BB1, GRP receptor subtypes (BB2, BB3, and BB4) (Ohlsson, B., et al, 1999, Scand. J. Gastroenterology 34 (12): 1224-9; Weber, HC, 2009, Cur. Opin. Endocri. Diab. Obesity 16(1): 66-71, Gonzalez N, et al, 2008, Cur. Opin. Endocri. Diab. Obesity 15(1), 58-64); Neurotensin receptor and its receptor subtypes (NTR1, NTR2, NTR3); Substance P receptor and its receptor subtypes (NK1 receptor in glial tumors, etc.), Hennig IM et al, 1995 Int. J. Cancer 61, 786-792); Neuropeptide Y (NPY) receptor and its receptor subtypes (Y1-Y6); R GD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), dimeric and multimeric cyclic RGD peptides (e.g. cRGDfV) (Laakkonen P, Vuorinen K. 2010, Integr Biol (Camb). 2(7-8): 326-337; Chen K, Chen X. 2011, Theranostics. 1:189-200; Garanger E, et al, Anti-Cancer Agents Med Chem. 7 (5): 552-558; Kerr, JS et al, Anticancer Research, 19(2A), 959-968; Thumshirn, G, et al, 2003 Chem. Eur. J. 9, 2717- 2725), TAASGVRSMH and LTLRWVGLMS (chondroitin sulfate proteoglycan NG2 receptor) and F3 peptide (a 31-amino acid peptide that binds to cell surface-expressed nucleolin receptors) (Zitzmann, S., 2002 Cancer Res., 62, 18, pp. 5139-5143; Temminga, K., 2005, Drug Resistance Updates, 8, 381-402; P. Laakkonen and K. Vuorinen, 2010 Integrative Biol, 2(7-8), 326-337; MA Burg, 1999 Cancer Res., 59(12)) Homing peptides including 2869-2874; K. Porkka, et al 2002, Proc. Nat. Acad. Sci. USA 99(11), 7444-9); cell-permeable peptides (CPPs) (Nakase I, et al, 2012, J. Control Release. 159(2), 181-188); peptide hormones, e.g., testosterone production. Similarly, luteinizing hormone-releasing hormone (LHRH) agonists and antagonists that act by targeting follicle-stimulating hormone (FSH) and luteinizing hormone (LH), as well as gonadotropin-releasing hormone (GnRH) agonists, such as buserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-NHEt), gonadrelin (Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), goserelin (Py r-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-AzGly-NH2), histrelin (Pyr-His-Trp-Ser-Tyr-D-His(N-benzyl)-Leu-Arg-Pro-NHEt), leuprolide (Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt), nafarelin (Pyr-His-Trp-Ser-Tyr-2Nal-Leu-Arg-Pro-Gly-NH2), triptorelin (Py r-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2), Nafarelin, Deslorelin, Abarelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu-isopropylLys-Pro-DAla-NH2), Cetrorelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-Cit- Leu-Arg-Pro-D-Ala-NH2), degarelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-4-aminoPhe(L-hydroorotyl)-D-4-aminoP he(carba-moyl)-Leu-isopropylLys-Pro-D-Ala-NH2), and Ganirelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-(N9, N10-diethyl)-homoArg-Leu-(N9, N10-diethyl)-homoArg-Pro-D-Ala-NH2)(Thundimadathil, J., J.Amino Acids, 2012, 967347, doi:10.1155 / 2012 / 967347; Boccon-Gibod, L.; et al, 2011, Therapeutic Advances in Urology 3 (3): 127-140; Debruyne, F., 2006, Future Oncology, 2(6), 677-696; Schally A. V; Nagy, A. 1999 Eur J Endocrinol 141:1-14; Koppan M, et al 1999 Prostate 38:151-158); For example, Toll-like receptors (TLRs), type C lectins, and Nodl recognize a wide range of biomacromolecules, from small molecules (imiquimod, guanidine, and adenosine analogs) to large and complex biomacromolecules such as lipopolysaccharides (LPS), nucleic acids (CpG DNA, poly(I;C)), and lipopeptides (Pam3CSK4) (Kasturi, SP, et al, 2011, Nature 470, 543-547; Lane, T., 2001, JR Soc. Med. 94, 316; Hotz, C., and Bourquin, C., 2012, Oncoimmunology 1, 227-228; Dudek, AZ, et al, 2007, Clin. Cancer Res. 13, 7119-7125). ike receptors (NLRs) (Fukata, M., et al, 2009, Semin. Immunol. 21, 242-253; Maisonneuve, C., et al, 2014, Proc. Natl. Acad. Sci. USA 111, 1-6; Botos, I., et al, 2011, Structure 19, 447-459; Means, TK, et al, 2000, Life Sci. 68, Pattern recognition receptors (PRRs) such as 241-258). Calcitonin receptors are 32-amino acid neuropeptides primarily involved in regulating calcium levels through their effects on osteoclasts and the kidneys. Body (Zaidi M, et al, 1990 Crit Rev Clin Lab Sci 28, 109-174; Gorn, AH, et al. 1995 J Clin Invest 95:2680-91); Integrins that generally play an important role in angiogenesis. The body and its receptor subtypes (α V β1, α V β3, α V β5, α V β6, α6β4, α7β1, α L β2, α IIb β3 (etc.) is expressed on the surface of various cells, particularly osteoclasts, endothelial cells, and tumor cells (Ruoslahti, E. et al, 1994 Cell 77, 477-8; Albelda, SM et al, 1990 Cancer Res.). (50, 6757-64). Short-chain peptides, GRGDSPK and cyclic (RGDfV)(L1), etc. Cyclic RGD pentapeptides and their derivatives [cyclo(-N(Me)R-GDfV), cyclo(R-Sar-DfV), cyclo(RG-N(Me)D-fV), cyclo(RGD-N(Me)fV), cyclo(RGDf-N(Me)V-)(silengitide)] showed high binding affinity to integrin receptors (Dechantsreiter, MA et al, 1999 J. Med.Chem. 42, 3033-40; Goodman SL et al, 2002 J. Med.Chem. 45, 1045-51).

[0161] Cell-binding molecules / ligands or cell receptor agonists can be Ig-based and non-Ig-based protein scaffold molecules. Ig-based scaffolds include, but are not limited to, nanobodies (derivatives of VHH (Calamidae Ig)) (Muyldermans S., 2013 Annu Rev Biochem. 82, 775-797); domain antibodies (dAb, derivatives of VH or VL domains) (Holt, L. J, et al, 2003, Trends Biotechnol. 21, 484-490); and bispecific T cell receptors. gager (BiTE, bispecific dimer) (Baeuerle, P. A, et al, 2009, Curr. Opin. Mol. Ther. 11, 22-30); dual affinity retargeting (DART, bispecific dimer) (Moore PA P, et al. 2011, Blood 117(17), 4542-4551);Tetravalent tandem antibody (TandAb The bispecific dimer can be selected from (Cochlovius, B, et al. 2000, Cancer Res. 60(16):4336-4341). The non-Ig scaffold is not limited, but anticarin (a derivative of lipocalin) (Skerra A. 2008, FEBS J., 275(11): 2677-2683; Beste G, et al. al, 1999 Proc. Nat. Acad. USA. 96(5):1898-1903; Skerra, A. 2000 Biochim Biophys Acta, 1482(1-2):337-350; Skerra, A. 2007, Curr Opin Biotechnol. 18(4):295-304; Skerra, A. 2008, FEBS J. 275(11):2677-2683); Adnectins (10FN3 (fibronectin)) (Koide, A, et al, 1998 J. Mol. Biol., 284(4):1141-1151; Batori V, 2002, Protein Eng. 15(12): 1015-1020; Tolcher, A. W, 2011, Clin. Cancer Res. 17(2):363-371; Hackel, B. J, 2010, Protein Eng. Des. Sel. 23(4):211-219); engineered ankyrin repeat proteins (DARPins) (derivatives of ankrin repeat (AR) proteins) (Boersma, YL, et al, 2011 Curr Opin Biotechnol. 22(6): 849-857), e.g., DARPinC9, DARPinEc4, and DARPinE69_LZ3_E01 (Winkler J, et al, 2009 Mol Cancer Ther. 8(9), 2674-2683; Patricia MK. M., et al, Clin Cancer Res. 2011;17(1):100-110; Boersma Y. L, et al, 2011 J. Biol. Chem. 286(48),41273-41285); Avimer (domain A / low-density lipoprotein (LDL) receptor) (Boersma Y. L, 2011 J. Biol. Chem. 286(48): 41273-41285; Silverman J, et al, 2005 Nat. Biotechnol., 23(12):1556-1561) You can choose.

[0162] Small molecules themselves, or small molecules coated or covalently bonded with proteins, nanoparticles, polymers, micelles, or lipids, can be bound to the cytotoxic agents of the present invention and used as cell binding agents. Examples of the structures of these small molecules are as follows: LB01 (folic acid), LB02 (PMSA ligand), LB03 (PMSA ligand), LB04 (PMSA ligand), LB05 (somatostatin), LB06 (somatostatin), LB07 (octreotide, growth), LB08 (lanreotide, somatostatin analog), LB09 (vapreotide (Sambar), somatostatin analog), LB10 (CAIX ligand), LB11 (CAIX ligand), LB12 (gastrin-releasing peptide receptor (GRPr), MBA), LB13 (luteinizing hormone-releasing hormone (LH-RH) ligand and GnRH), LB14 (luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand), LB15 (GnRH antagonist, abalelix), LB16 (cobalamin, vitamin LB17 (cobalamin, vitamin B12 analog), LB18 (for αvβ3 integrin receptor, cyclic RGD pentapeptide), LB19 (heterobivalent peptide ligand for VEGF receptor), LB20 (for neurotonin B), LB21 (glycoprotein for G protein-binding receptor), LB22 (TLR2 for Toll-like receptor), LB23 (for androgen receptor), LB24 (sirengitide / cyclo(-RGDfV-) for αv integrin receptor), LB23 (flucortisone), LB25 (rifabutin analog), LB26 (rifabutin analog), LB27 (rifabutin analog), LB28 (flucortisone), LB29 (dexamethasone), LB30 (fluticasone propionate), LB31 (diclobetamethasone), LB32 (acetonin LB33 (prednisone), LB34 (prednisolone), LB36 (prednisolone), betamethasone), LB37 (irinotecan analog), LB38 (crizotinib analog), LB39 (bortezomib analog), LB40 (carfilzomib analog), LB41 (carfilzomib analog), LB42 (leuprolide analog), LB43 (triptorelin analog), LB44 (clindamycin) The structures of LB45 (liraglutide analog), LB46 (semaglutide analog), LB47 (retamrin analog), LB48 (indigosine analog), LB49 (vinblastine analog), LB50 (lixisenatide analog), LB51 (ocitinib analog), LB52 (nucleoside analog), LB53 (erlotinib analog), and LB54 (lapatinib analog) are as follows:

[0163] [ka] TIFF0007853711000092.tif254170TIFF0007853711000093.tif236170TIFF0007853711000094.tif254170TIFF0007853711000095.tif238170 TIFF0007853711000096.tif245170TIFF0007853711000097.tif228170TIFF0007853711000098.tif254170TIFF0007853711000099.tif215170

[0164] During the ceremony [ka] X4 and Y1 are independently O, NH, NHNH, NR1, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N(R1), N(R1)C(O)N(R1), CH2, C(O)NHNHC(O), and C(O)NR1; X1 is H, CH2, OH, O, C(O), C(O)NH, C(O)N(R1), R1, NHR1, NR1, C(O)R1, or C(O)O; X5 is H, CH3, F, or Cl; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, N(R 1 R 1’ R 2 R 3 ) is; R 1 , R 1’ , R 2 , and R 3 It is defined by equation (I).

[0165] In the conjugation process, prior to conjugation with the tubulicin analogs of the present invention, the cell-binding molecule may be modified by linking to more specific peptides, proteins, drugs, or other functional molecules using heterobifunctional crosslinking agents, such as amine-nonselective (succinimidyl (NHS)-diaziline (SDA), NHS ester / azide), amine-sulfhydryl (NHS ester / maleimide, NHS ester / pyridyldithiol, NHS ester / haloacetyl), mercapto-carbohydrate (maleimide / hydrogenated compound, pyridyldithiol / hydrogenate), hydroxytolyl-sulfhydryl (isocyanate / maleimide), amine-DNA (NHS ester / posoralen), or amine-carboxyl (carbodiimide) linkages.

[0166] In SDA linkage modification, the NHS ester of the SDA conjugate reacts with the primary amino group of the binding molecular backbone in pH 6-9 buffer to form a stable amide bond upon NHS release. Subsequently, photoactivation of diazirine with long-wave ultraviolet light (330-370 nm) generates a reactive carbene intermediate that can react with the amino group of more specific peptides, proteins, or other functional molecules. The order of these two steps may differ, with the initial reaction between the amino group of the functional molecule and the SDA conjugate and the subsequent photoactivation reaction of the binding molecule with long-wave UV light (330-370 nm). The SDA crosslinked conjugate is cleavable (using an internal disulfide bond, as in the SDAD conjugate).

[0167] [ka]

[0168] In NHS ester / azide bond modification, the NHS ester of the conjugate reacts with the primary amino group of the binding molecular backbone in a buffer solution at pH 6-9 to form a stable amide. Next, the alkynyl group of a more specific peptide, protein, or other functional molecule reacts with the azide on the opposite side of the conjugate via azide-alkyne huisgen cycloaddition to form a 1,2,3-triazole bond (click chemistry). Alternatively, the NHS ester of the conjugate reacts with the primary amino group of the functional molecule in a buffer solution at pH 6-9 to form a stable amide. Next, the alkynyl group attached to the binding molecule reacts with the azide on the opposite side of the conjugate via 5-azide-alkyne huisgen cycloaddition to form a 1,2,3-triazole bond.

[0169] [ka]

[0170] In amine-sulfhydryl bond modification, the NHS ester of the conjugate reacts with the primary amino group of the binding molecule backbone in a buffer solution at pH 6-9 to form a stable amide bond. Then, a sulfhydryl group on a more specific peptide, protein, or other functional molecule reacts with maleimide, pyridyldithiol, or haloacetyl on the opposite side of the amine-sulfhydryl conjugate at pH 4.5-8.5 to form a thioether or disulfide bond. The bonding with the amine-sulfhydryl conjugate can occur in different orders. For example, the amino group of the functional molecule can first react with the conjugate to form an amide bond, followed by a reaction with the sulfhydryl group on the binding molecule. Alternatively, the sulfhydryl group of the functional molecule can react with the conjugate to first form a thioether or disulfide bond at pH 4.5-7, followed by a reaction with the amino group of the binding molecule at pH 6-9 to form an amide bond.

[0171] [ka]

[0172] In sulfhydryl-carbohydrate bond modification, the sulfhydryl group of the binding molecule first reacts with maleimide or pyridyldithiol on the conjugate to form a thioether or disulfide bond at pH 4.5-8, and then the carbonyl (aldehyde / ketone) group of the functional molecule reacts with the hydrazide to form a hydrazone bond. Alternatively, the sulfhydryl group of the functional molecule can first react with the conjugate to form a thioether or disulfide bond at pH 4.5-8, and then react with the carbohydrate, oxidized carbohydrate, or carbonyl (aldehyde / ketone) group on the binding molecule to form a hydrazone bond.

[0173] [ka]

[0174] In hydroxyl-sulfhydryl bond modification, the sulfhydryl group of the binding molecule first reacts with maleimide or pyridyldithiol on the conjugate to form a thioether or disulfide bond at pH 6-8, and then the hydroxyl group of the functional molecule reacts with the isocyanate of the conjugate to form a carbamate bond at pH 8-9. Alternatively, the sulfhydryl group of the functional molecule can first react with the conjugate to form a thioether or disulfide bond at pH 6-8, and then react with the hydroxyl group of the binding molecule to form a carbamate bond at pH 8-9.

[0175] [ka]

[0176] In yet another aspect of the present invention, the production of the antibodies used in the present invention includes in vivo or in vitro production processes or combinations thereof. Methods for preparing anti-receptor peptide polyclonal antibodies are well known, for example, as shown in U.S. Patent No. 4,493,795 (Nestor et al.). A typical method for preparing monoclonal antibodies is to fuse mouse spleen cells isolated from a specific antigen-immunized mouse with myeloma cells (Kohler, G; Milstein, C. 1975. Nature 256:495-497). Detailed procedures are described in antibodies—A Laboratory Manual, Harlow and Lane, eds., cold spring harbor laboratory press, new York (1988), and the contents of that document are incorporated herein by reference as forming part of this specification. In particular, monoclonal antibodies can be obtained by immunizing mice, rats, hamsters, or other mammals with the target antigen. Examples of target antigens include intact target cells, antigens isolated from target cells, whole viruses, weakened whole viruses, and viral proteins. Splenocytes and myeloma cells are fused using PEG6000. The hybridomas obtained after fusion are screened using their sensitivity to HAT (hypoxanthine-aminopterin-thymine). Hybridomas that produce monoclonal antibodies useful for carrying out the present invention are identified by inducing an immune response with or suppression of receptor activity of specific target cell receptors.

[0177] The monoclonal antibodies used in the present invention can be obtained by initiating the culture of monoclonal hybridoma cells in a nutrient medium containing hybridoma cells that secrete antibodies with appropriate antigen specificity. In this culture, it is necessary to maintain sufficient time and conditions for the hybridoma cells to secrete antibodies into the culture medium. After collecting the supernatant of the antibody-containing medium, the antibodies can be isolated by well-known techniques, such as protein A affinity chromatography; anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, and molecular sieve chromatography (particularly affinity chromatography and molecular sieve chromatography using antigen-crosslinked protein A); centrifugation, precipitation, or standard methods for purifying other proteins.

[0178] Culture media useful for preparing these compositions are well known in the art and commercially available, including synthetic media. An example of a synthetic medium is Dulbecco's Minimum Essential Medium (DMEM; Dulbecco et al., Virol. 8:396 (1959)) with 4.5 gm / L glucose, 0-20 mM glutamine, 0-20% fetal bovine serum, and ppm amounts of It consists of several heavy metals (e.g., Cu, Mn, Fe, or Zn) or / and heavy metals added in salt form, as well as an antifoaming agent (e.g., polyoxyethylene-polyoxypropylene block copolymer).

[0179] Furthermore, in addition to cell fusion technology, cell lines for antibody production can also be constructed by the following methods. For example, direct transformation of B lymphocytes with oncogenic DNA, or transfection with oncogenic viruses, such as Epstein-Barr virus (EBV, also known as human herpesvirus 4 (HHV-4)) or Kaposi's sarcoma-associated virus (KSHV) (see U.S. Patent Nos. 4341761;4399121;4427783;4444887;4451570;4466917;4472500;4491632;4493890 for details). Monoclonal antibodies can be prepared using anti-receptor peptides or peptides containing terminal carboxyl groups based on known methods (see Niman et al., Proc. Natl. Acad. for details). See Sci. USA, 80:4949-4953 (1983); Geysen et al. Proc. Natl. Acad. Sci. USA, 82:178-182 (1985); Lei et al. Biochemistry 34(20):6675-6688 (1995). Antireceptor polypeptides or polypeptide analogs can be used alone or conjugated to a crosslinked immunogenic carrier as immunogens for preparing antireceptor polypeptides of monoclonal antibodies.

[0180] There are many other well-known methods for producing monoclonal antibodies as binding molecules in the present invention. Among these, the method for producing fully human antibodies has attracted particular attention. Phage display technology allows for the acquisition of fully human antibodies that specifically bind to known antigens from a fully human antibody library through affinity selection. The literature contains detailed descriptions of phage display technology itself, vector construction, and library screening. For more information, see Dente et al. Gene. 148(1):7-13 (1994); Little et al. Biotechnol Adv. 12(3):539-55 (1994); Clackson et al. Nature 352:264-628 (1991); Huse et al. Science 246:1275-1281 (1989).

[0181] Monoclonal antibodies obtained from non-human species (e.g., mice) using hybridoma technology can be humanized to avoid human anti-mouse antibodies when administered to humans. Among the well-known methods for antibody humanization are the transplantation and remodeling of complementarity-determining regions. For details, see U.S. Patents 5,859,205 and 6,797,492; Liu et al., Immunol Rev. 222:9-27 (2008); Almagro et al., Front Biosci. 1; 13:1619-33 (2008); Lazar et al., Mol Immunol. 44(8):1986-98 (2007); Li et al., Proc. Natl. Acad. Sci. USA. 103(10):3557-62 (2006). The disclosures in the aforementioned literature are incorporated as references. Fully human antibodies can be prepared by antigen immunization against transgenic mice, rabbits, monkeys, and other mammals that possess most of the light and heavy chains of human immunoglobulins. Examples of such mice include Xenomouse (Abgenix, Inc.), HuMab-Mouse (Medarex / BMS), and VelociMouse (Regeneron). For details, see U.S. Patents 6,596,541, 6,207,418, 6,150,584, 6,111,166, 6,075,181, 5,922,545, 5,661,016, 5,545,806, 5,436,149, and 5,569,825. During human treatment, the immunogenicity produced in the human body by chimeric antibodies constructed by integrating mouse antibody variable region genes and human antibody constant region genes is far lower than that of mouse antibodies (Kipriyanov et al., Mol Biotechnol. 26:39-60 (2004); Houdebine, Curr Opin B iotechnol.13:625-9(2002)). The disclosures in the aforementioned literature are incorporated as references. Furthermore, antibody affinity and specificity can be improved by inducing specific mutagenesis in the antibody variable region (Brannigan et al., Nat Rev Mol Cell Biol.3:964-70(2002); Adams et al., J.Imm). unol Methods. 231:249-60 (1999). By partially replacing the constant region of the antibody, the cytotoxic effect can be enhanced by effectively promoting affinity with immunoeffector cells.

[0182] Immunospecific antibodies against malignant cell antigens can be obtained through commercial channels or several commonly used technical methods, such as chemical synthesis or recombinant expression techniques. Similarly, nucleotide sequences encoding immunospecific antibodies against malignant cell antigens can be obtained through commercial channels such as the GenBank database or other similar databases, publicly available literature, or routine cloning and sequencing.

[0183] Besides antibodies, polypeptides or proteins similarly interact with corresponding receptors or epitopes on the target cell surface by binding, blocking, attacking, or other means, acting as binding molecules. These peptides or proteins do not need to belong to the immunoglobulin family, as long as they can specifically bind to the epitope or its corresponding receptor. These polypeptides are also isolated using techniques similar to those for phage display antibodies (Szardenings, J Recept Signal Transduct Res. 2003;23(4):307-49). Peptide fragments obtained from random peptide libraries have applications similar to those of antibodies and antibody fragments. Polypeptide or protein molecules maintain their antigen-binding specificity by linking to several macromolecules or media via binding molecules. These macromolecules include, but are not limited to, albumin, polymers, liposomes, nanoparticles, or dendrimers.

[0184] Antibodies used for drug conjugation by the conjugates of the present invention for the treatment of cancer, autoimmune diseases, and infectious diseases include, but are not limited to, the following: 3F8 (anti-GD2 antibody), avagovomab (anti-CA-125 antibody), absiximab (anti-CD41 antibody (integrin α-IIb)), adalimumab (anti-TNF-α antibody), adalimumab (anti-EpCAM antibody, CD326), afelimomab (anti-TNF-α); aftuzumab (anti-CD20 antibody), and alacizumab pegol. pegol (anti-VEGFR2 antibody), ALD518 (anti-IL-6 antibody), alemtuzumab (also known as Campus, MabCampus, anti-CD52 antibody), artumomab (anti-CEA antibody), anatumomab (anti-tag-72 antibody), anlukinzumab (IMA-638, anti-IL-13 antibody), apolizumab (anti-HLA-DR antibody), alsitumomab (anti-CEA antibody), aselizumab (anti-L - Selectin (CD62L) antibody), Atlizumab (also known as Tocilizumab, Actemra, RoActemra, anti-IL-6 receptor antibody), Atorolimumab (anti-rhesus monkey factor antibody), Bapineozumab (anti-β-amyloid antibody), Basiliximab (Symlect, anti-CD25 (IL-2 receptor α chain) antibody), Bavituximab (Bavitu ximab (anti-phosphatidylserine antibody), bectumomab (also known as LymphoScan, anti-CD22 antibody), belimumab (also known as BENLYSTA, LymphoStat-B, anti-BAFF antibody), benralizumab (anti-CD125 antibody), vertilimumab (anti-CCL11 (eotaxin-1) antibody), becylesomab (also known as Scintimun, anti-CEA-related antigen antibody), bevacizumab (also known as Avastin, anti-VEGF antibody), bisilomab (also known as FibriScint, anti-fibrin IIβ chain antibody), vivacuzumab (anti-CD44v6 antibody), blinatumomab (also known as BiTE, anti-CD19 antibody), brentuximab (cAC10, anti-CD30 TNFRSF8 antibody), Briakinumab (anti-IL-12, IL-23 antibody), canakinumab (also known as Ilaris, anti-IL-1 antibody), cantuzumab (also known as C242, anti-CanAg antibody), capromab, catumakisomab (also known as removeb, anti-EpCAM, anti-CD3 antibody), CC49 (anti-TAG-72 antibody), cedelizumab (anti-CD4 antibody), certolizumab pegol (also known as CIMZIA, anti-TNF-α antibody), cetuximab (also known as elbitux, IMC-C225, anti-EGFR antibody), sitatuzumab (anti-EpCAM antibody), cyclostomab ( Cixutumumab (anti-IGF-1 antibody), clenoliximab (anti-CD4 antibody), clibatuzumab (anti-MUC1 antibody), conatumumab (anti-TRAIL-R2 antibody), CR6261 (anti-influenza A hemagglutinin antibody), dasetuzumab (anti-CD40 antibody), daclizumab (also known as Zenapax, anti-CD25C (IL-2 receptor α chain) antibody), daratumumab (anti-CD38 (cyclic ADP-ribose hydrolase)) Antibodies include: denosumab (also known as Prolia, anti-RANKL antibody), detumomab (anti-B-lymphoma cell antibody), dorlimomab, doruxizumab, ecromeximab (anti-GD3 ganglioside antibody), eculizumab (also known as Soliris, anti-C5 antibody), edovacomab (anti-endotoxin antibody), edrecolomab (also known as Panorex, MAb17-A1, anti-EpCAM antibody), efalizumab (also known as Raptiva, anti-LFA-1 (CD11a) antibody), and efangumab. umab (also known as Mycograb, anti-Hsp90 antibody), Elotuzumab (anti-SLAMF7 antibody), Elsilimomab (anti-IL-6 antibody), Enrimomab pegol (anti-ICAM-1 (CD54) antibody), Epitumomab (anti-epiciarin antibody), Epratuzumab (anti-CD22 antibody), Erlizumab (anti-ITGB2 (CD18) antibody), Ertumaxomab (also known as Rexomun, anti-HER2 / neu, CD3 antibody),Etalacizumab (also known as Abegrin, anti-integrin αvβ3), exibivirumab (anti-hepatitis B surface antigen antibody (HBs antibody)), fanolesomab (also known as NeutroSpec, anti-CD15 antibody), faralimomab antibody (anti-interferon receptor antibody), farletuzumab (anti-folate receptor 1 antibody), felvizumab (antibody against RSV), fezakinumab (anti-IL-22 antibody), figtum Figitumumab (anti-IGF-1 receptor antibody), Fontolizumab (anti-IFN-γ antibody), Foravirumab (anti-rabies virus glycoprotein antibody), Fresolimumab (anti-TGF-β antibody), Galiximab (anti-CD80 antibody), Gantenerumab (anti-β-amyloid antibody), Gavilimomab (anti-CD147 (basigin) antibody), Gemtuzumab (anti-CD33 antibody), Girentuximab (anti-carbonic anhydrase 9 antibody), Glembatumumab (also known as CR011, anti-GPNMB antibody), Golimumab (also known as Simponi, anti-TNF-α antibody), Gomiliximab (anti-CD23C (IgE receptor) antibody), Ibalizumab (anti-CD4 antibody), Ibritumomab (anti-CD20 antibody), Igovomab (also known as Indimacis-125, anti-CA-125 antibody), I Muciromab (also known as Myoscint, anti-cardiac myosin antibody), infliximab (also known as Remicade, anti-TNF-α antibody), intetumumab (anti-CD51 antibody), inolimomab (anti-CD25 (IL-2 receptor α chain) antibody), inotuzumab (anti-CD22 antibody), ipilimumab (anti-CD152 antibody), iratumumab (anti-CD30 (TNFRSF8) antibody), keriximab (anti-CD4 antibody),Rabetuzumab (also known as CEA-Cide, anti-CEA antibody) ), lebrikizumab (anti-IL-13 antibody), lemalesomab (anti-NCA-90 (granulocyte antigen) antibody), lerdelimumab (anti-TGFβ-2 antibody), lexatumumab (anti-TRAIL-R2 antibody), livivirumab (anti-hepatitis B surface antigen antibody), lintuzumab (anti-CD33 antibody), lucatumumab (anti-CD40 antibody), lumiliximab (anti-CD23 (IgE receptor) antibody), mapatumumab (anti-TRAIL-R1 antibody), maslimomab (Anti-T cell receptor antibody), Matuzumab (anti-EGFR antibody), Mepolizumab (also known as Bosatria, anti-IL-5 antibody), Metelimumab (anti-TGFβ-1 antibody), Miratuzumab (anti-CD74 antibody), Minretumomab (anti-TAG-72 antibody), Mitumomab (also known as BEC-2, anti-ganglioside antibody-GD3), Morolimmumab (anti-rhesus monkey factor antibody), Motavizumab (also known as Numax, anti-RSV antibody), Muromonab-CD3 (also known as Orthoclone OKT3 (anti-CD3 antibody), Nacolomab (anti-C242 antibody), Naptumomab (anti-5T4 antibody), Natalizumab (also known as Tysabri, anti-integrin α4 antibody), Nebacumab (anti-endotoxin antibody), Necitumumab (anti-EGFR antibody), Nerelimomab (anti-TNF antibody) -α antibody), nimotuzumab (also known as Theracim, Theraloc, anti-EGFR antibody), nofetumomab, ocrelizumab (anti-CD20 antibody), odulimomab (also known as Afolimomab, anti-LFA-1 (CD11a) antibody), ofatumumab (also known as Arzerra, anti-CD20 antibody), olalatumab (anti-PDGF-Rα antibody),Omalizumab (also known as Xolair, anti-IgEFc region antibody), oportuzumab (anti-EpCAM antibody), olegovomab (also known as OvaRex, anti-CA-125 antibody), otelixizumab (anti-CD3 antibody), pagibaximab (anti-LTA antibody), palivizumab (also known as Synagis, Abbosynagis, anti-RSV antibody), panimumab (also known as Vectibix, ABX-EGF, anti-EGFR antibody), pano Bakumab (Panobacumab) (anti-Pseudomonas aeruginosa antibody), Pascolizumab (anti-IL-4 antibody), Pemtumomab (also known as Theragyn, anti-MUC1 antibody), Pertuzumab (also known as Omnitarg, 2C4, anti-HER2 / neu antibody), Pexelizumab (anti-C5 antibody), Pintumomab (anti-adenocarcinoma antigen antibody), Priliximab (anti-CD4 antibody), Pritumumab (anti-vimentin antibody), P RO140 (anti-CCR5 antibody), racotumomab (also known as 1E10, anti-N-glycolylneuraminic acid (NeuGc,NGNA)-ganglioside (GM3) antibody), rafivirumab (anti-rabies virus glycoprotein antibody), ramucirumab (anti-VEGFR2 antibody), ranibizumab (also known as Lucentis, anti-VEGF-A antibody), laxibacumab (anti-anthrax toxin, protective antigen antibody), regavirumab (anti-CMV glycoprotein antibody) Protein B antibody), Reslizumab (anti-IL-5 antibody), Rilotumumab (anti-HGF antibody), Rituximab (also known as MabThera, Rituxanmab, anti-CD20 antibody), Robatumumab (anti-IGF-1 receptor antibody), Rontalizumab (anti-IFN-α antibody), Robelizumab (also known as LeukArrest, anti-CD11, CD18 antibody), Ruplizumab (also known as Antova,Anti-CD154 (CD40L) antibody), Satumomab (anti-TAG-7, (2 antibodies), sevilumab (anti-CMV antibody), cibrotuzumab (anti-FAP antibody), sifalimumab (anti-IFN-α antibody), siltuximab (anti-IL-6 antibody), ciprizumab (anti-CD2 antibody), (SMART) MI95 (anti-CD33 antibody), solanezumab ( Anti-β-amyloid antibody), sonepcizumab (anti-sphingosine-1-phosphate antibody), sontuzumab (anti-epiciarin antibody), stamulumab (anti-myostatin antibody), sulesomab (also known as LeukoScan, (anti-NCA-90 (granulocyte antigen) antibody)), tacatuzumab (anti-α-pheno Toprotein antibody), tadocizumab (anti-integrin αIIbβ3 antibody), talizumab (anti-IgE antibody), tanezumab (anti-NGF antibody), taplitumomab (anti-CD19 antibody), tefibazumab (also known as Aurexis, anti-clumping factor A antibody), terimomab, tenatumomab (Te natumomab (anti-tenascin C antibody), teneliximab (anti-CD40 antibody), teplizumab (anti-CD3 antibody), TGN1412 (anti-CD28 antibody) ), Ticilimumab (also known as Tremelimumab, anti-CTLA-4 antibody), Tigatuzumab (anti-TRAIL-R2 antibody), TNX-650 (anti-IL-13 antibody), Tocilizumab (Also known as Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor antibody), Toralizumab (anti-CD154 (CD40L) antibody), Tositumomab (anti-CD20 antibody), Trastuzumab (also known as Herceptin, anti-HER2 / neu antibody), Tremelimumab (anti-CTLA-4 antibody), Tucotuzumab cermoloukin)celmoleukin (anti-EpCAM antibody), tuvirumab (anti-hepatitis B antibody), urtoxazumab (anti-E. coli antibody), ustekinumab (also known as Stellara, anti-IL-12, IL-23 antibody), vapaliximab (anti-AOC3 (VAP-1) antibody), vedolizumab (anti-integrin α4β7 antibody), bertuzumab (anti-CD20 antibody), bepalimomab (anti-AOC3 (VAP-1) antibody), bicilizumab (also known as Nuvion, anti-CD3 antibody), vitaxin (anti-angiogenic integrin avb3 antibody), voloxiximab (anti-integrin Phosphorus α5β1), Botumumab (also known as HumaSPECT, antitumor antigen CTAA16.88 antibody), Saltumumab (also known as HuMax-EGFr, (anti-EGFR antibody)), Zanolimumab (also known as HuMax-CD4, anti-CD4 antibody), Diralimumab (anti-CD147 (basic immunoglobulin) antibody), Zolimomab (anti-CD5 antibody), Etanercept (registered trademark "Enbrel"), Alefacept (registered trademark "Amevive"), Abatacept (registered trademark "Orencia"), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (iron regulatory protein 2) antibody], 14G2a (Nat. Cancer (Anti-ganglioside GD2 antibody for melanoma and solid tumors from Inst.), J591 (Anti-PSMA antibody for treating prostate cancer from Weill Cornell Medical School), 225.28S [Anti-HMW-MAA (high molecular weight melanoma-associated antigen) antibody for melanoma, Sorin Radiofarmaci SRL (Milan, Italy)], COL-1 (Nat. Cancer Anti-CEACAM3 antibody for colorectal and gastric cancer (CGM1) from Inst., CYT-356 (registered trademark "Oncoltad" for prostate cancer), HNK20 (for RSV from Ora Vax Inc.), ImmuRAIT (from IMMUNOMEDICS) Lym-1 (anti-HLA-DR10 antibody, for tumors from Peregrine Pharm), MAK-195F (anti-TNF (tumor necrosis factor; TNFA, TNF-α; TNFSF2) antibody for sepsis and toxic shock from Abbott / Knoll), MEDI-500 (also known as T10B9, anti-CD3 antibody for graft-versus-host disease from MedImmune Inc, TRαβ (T cell receptor α / β)), RING SCAN (Neoprobe From Corp.: Anti-TAG72 (tumor-associated glycoprotein 72) antibody for breast cancer, colon cancer and colorectal cancer; Avicidin (anti-EPCAM (epithelial cell adhesion molecule) antibody); Anti-TACSTD1 (tumor-associated calcium signaling transducer 1) antibody; Anti-GA733-2 (gastrointestinal tumor-associated protein 2) antibody; Anti-EGP-2 (epithelial glycoprotein 2) antibody; Anti-KSA antibody; KS1 / 4 antigen; M4S; Tumor antigen 17-1A; CD326 for colon cancer, ovarian cancer, prostate cancer and non-Hodgkin lymphoma; LYMPHOCIDE (IMMUNOMEDICS, NJ); SmartID10 (Protein Design Labs); Oncolym (Techniclone Inc, CA); Allomune (BioTransplant, CA); Anti-VEGF antibody (Genentech, CA); CEAcide (Immunomedics, NJ); IMC-1C11 (ImClone Systems (NJ), and cetuximab (ImClone, NJ).

[0185] Other antibodies that function as binding ligands include, but are not limited to, antibodies against the following antigens: aminopeptidase N (CD13), annexin A1, B7-H3 (CD276, various cancers), CA125 (ovary), CA15-3 (carcinoma), CA19-9 (carcinoma), L6 (carcinoma), Lewis Y (carcinoma), Lewis X (carcinoma), alpha-fetoprotein (carcinoma), CA242 (colorectal), placental alkaline phosphatase (carcinoma), prostate-specific antigen (prostate), and prostatic acid. Phosphatase (prostate), epidermal growth factor (carcinoma), CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma), CD3ε (T-cell lymphoma, lung cancer, breast cancer, gastric cancer, ovarian cancer, autoimmune diseases, malignant ascites), CD19 (B-cell malignancies), CD20 (non-Hodgkin lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, systemic lupus erythematosus), CD30 (Hodgkin lymphoma), CD33 (leukemia, autoimmune diseases), CD38 (multiple myeloma), CD40 (lymphoma, multiple bone marrow CD51 (metastatic melanoma, leukemia (CLL)), CD52 (leukemia), CD56 (small cell lung cancer, ovarian cancer, Merkel cell carcinoma and humoral neoplasms, multiple myeloma), CD66e (cancer), CD70 (metastatic renal cell carcinoma and non-Hodgkin lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (cancer), mucin (carcinoma), CD221 (solid tumors), CD227 (breast cancer, ovarian cancer), CD262 (non-small cell lung cancer and other cancers), CD309 (ovarian cancer), CD326 ( Solid tumors), CEACAM3 (colorectal cancer, gastric cancer), CEACAM5 (carcinoembryonic antigen; CEA, CD66e) (breast cancer, colorectal cancer, and lung cancer), DLL4 (Δ-like-4), EGFR (epidermal growth factor receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD184, heme tumor, solid tumor), endoglin (CD105, solid tumor), EPCAM (epidermal cell adhesion molecule, bladder, head, neck, colon cancer, NHL prostate cancer, and ovarian cancer), ERBB2 (epidermal growth factor receptor 2;Lung cancer, breast cancer, prostate cancer), FCGR1 (autoimmune disease), FOLR (folate receptor, ovarian cancer), GD2 ganglioside (cancer), G-28G (cell surface antigen glycolipid, melanoma), GD3 idiotype (cancer), heat shock protein (cancer), HER1 (lung cancer, gastric cancer), HER2 (breast cancer, lung cancer, and ovarian cancer), HLA-DR10 (NHL), HLA-DRB (NHL, B-cell leukemia), human chorionic gonadotropin (carcinoma), IGF1R (insulin-like growth factor-1 receptor, solid tumor, hematological malignancy) ), IL-2 receptor (interleukin-2 receptor, T-cell leukemia and lymphoma), IL-6R (interleukin-6 receptor, multiple myeloma, RA, Castleman disease, IL-6 dependent tumors), integrins (αVβ3, α5β1, α6β4, αIIβ3, α5β5, αVβ5 for various cancers), MAGE-1 (carcinoma), MAGE-2 (carcinoma), MAGE-3 (carcinoma), MAGE-4 (carcinoma), anti-transferrin receptor (carcinoma), p97 (melanoma), MS4A1 (transmembrane 4-transferrin); Main family A member 1, non-Hodgkin B-cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast cancer, ovarian cancer, cervical cancer, bronchial and gastrointestinal cancer), MUC16 (CA125) (ovarian cancer), CEA (colon), gp100 (melanoma), MART1 (melanoma), MPG (melanoma), MS4A1 (transmembrane 4-domain family A member 1, small cell lung cancer, NHL), nucleolin, neurocarcinoma gene product (carcinoma), P21 (carcinoma), anti-(N-glucolneuraminic acid paratope (breast cancer, melanoma cancer), PLAP-like testicular alkaline phosphatase (ovarian cancer, testicular cancer), PSMA (prostate cancer), PSA (prostate), ROBO4, TAG72 (tumor-associated glycoprotein 72, leukemia (A ML), gastric cancer, colorectal cancer, ovarian cancer), T cell transmembrane protein (cancer), Tie (CD202b), TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, cancer), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, NHL, other cancers, RA and SLE), TPBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (TNF-related apoptosis ligand receptor 1, lymphoma, NHL, colorectal cancer, lung cancer), VCAM-1 (CD106, melanoma), VEGF, VEGF-A, VEGF-2 (CD309) (various cancers). Other tumor-associated antigens recognized by antibodies have already been reported (Gerber, et al, mAbs 1:3, 247-253 (2009);Novellino et al,cancer immunol immunother. 54 (3),187-207 (2005)Franke et al,cancer biother radiopharm. 2000,15,459-76). (Gerber, et al, mAbs 1:3, 247-253 (2009);Novellino et al, Cancer Immunol Immunother. 54(3),187-207 (2005). Franke, et al, Cancer Biother Radiopharm. 2000,See 15,459-76). Examples of these antigens that antibodies counteract include: many other differentiated clusters (CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11C, CD12w, CD14, CD15, CD16, CDw17, CD18, CD21, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD31, CD32 , CD34, CD35, CD36, CD37, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49C, CD53, CD54, CD55, CD58, CD59, CD61, CD62E, CD62L, CD62P, CD63, CD68, CD69, CD71, CD72, CD79, CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, CD91, CD95, CD96, CD100, CD103, CD105, CD106, CD109, CD117, CD120, C D127, CD133, CD134, CD135, CD138, CD141, CD142, CD143, CD144, CD147, CD151, CD152, CD154, CD156, CD158, CD163, CD166, CD168, CD184, CDw186, CD195, CD202(a, b), CD209, CD235a, CD271, CD303, CD304), Annexin A1, Nucleolin, Endoglin (CD105), ROBO4, Aminopeptidase N, Δ-like-4 (DLL4), VEGFR-2 (CD309), CXCR4 9CD184), Tie2, B7-H3, WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2 / neu, idiotype, MAGE A3, p53 non-mutant, NY-ESO-1, GD2, CEA, MelanA / MART1, Ras mutant, gp100, p53 mutant, Proteinase3(PR1), bcr-abl, tyrosinase, survivorbin, hTERT, sarcoma translocation breakpoint, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG(TMPRSS2ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl GM1, mesoserine, PSCA, MAGE A1, sLe(a), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonate anhydrous yeast Element IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Regmine, Tie2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, Fos-related antigen 1.

[0186] In another embodiment, the lyophilized liquid formulation or formulated lyophilized powder of the present invention has the following specific composition: a conjugate of formula (I), (II), (III), (IV), (V), (VI), or (VII) as the main component in the formulation may constitute 1% to 95% (wt), 0.0% to 15.0% polyol; 0.0% to 0.5% one or more surfactants; 0.0% to 10% one or more amino acids, 0.0% to 5% preservatives, and 0.0% to 10% buffer salts to adjust the pH value to pH 4.5 to 8.5.

[0187] In another specific embodiment, the lyophilized liquid formulation or the formulated lyophilized powder has the following specific composition: the conjugate of formula (I), (II), (III), (IV), (V), (VI), or (VII) as the main component in the formulation may constitute 10% to 85% (wt) of a polyol, for example, 0.0% to 10.0% selected from sucrose or trehalose dihydrate; 0.1% to 0.25% of one or more surfactants, for example selected from polysorbate 20 or polysorbate 80; 0.0% to 10% of one or more amino acids, for example selected from cystine, glycine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine, glutamic acid, or aspartic acid; and 0.5% to 5% of a preservative, for example selected from benzyl alcohol. For example, a 1% to 10% buffer salt, selected from sodium citrate or citric acid monohydrate, for adjusting the pH to pH 5 to 7.

[0188] In another specific embodiment, the lyophilized liquid formulation or the formulated lyophilized powder has the following specific composition: a conjugate of formula (I), (II), (III), (IV), (V), (VI), or (VII) as the main component in the formulation, which may constitute 15% to 85% of the formulation; 3% to 8% of polyols selected from, for example, sucrose or trehalose dihydrate; 0.1% to 0.25% of one or more surfactants selected from, for example, polysorbate 20 or polysorbate 80; 0.0% to 8.0% of one or more amino acids selected from, for example, arginine, glycine, histidine, ornithine, or alanine; 0.5% to 5% of preservatives selected from, for example, benzyl alcohol; 2.0% to 10.0% of buffer salts selected from, for example, sodium citrate or citric acid monohydrate, for adjusting the pH to pH 5 to 6.

[0189] In another specific embodiment, the conjugate of a tubulicin analog of the present invention with a cell-binding molecule is used to treat cancer according to the components and methods of the present invention. The cancers targeted are, but are not limited to, adrenocortical carcinoma, anal cancer, bladder cancer, brain tumors (adult, brainstem glioma, pediatric, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal and pineal tumors, visual tract and hypothalamic glioma), breast cancer, carcinoid tumors, gastrointestinal cancers, cancers of unknown primary origin, cervical cancers, colorectal cancers, endometrial cancers, esophageal cancers, extrahepatic bile duct cancers, Ewing family tumors (PNETs), extracranial malignant germ cell tumors, eye cancers, intraocular melanoma, gallbladder cancer, gastric cancer (stomach), germ cell tumors, extragonadal cancers, trophoblastic tumors of pregnancy, head and neck cancers, hypopharyngeal cancers, and islet cell carcinomas. , kidney cancer (renal cell carcinoma), laryngeal carcinoma, leukemia (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myeloid, pilocytic cell), lip and oral cancer, liver cancer, lung cancer (non-small cell, small cell), lymphoma (AIDS-related, central nervous system, cutaneous T cell, Hodgkin's disease, non-Hodgkin's disease, malignant mesothelioma, melanoma, Merkel cell carcinoma, metastatic squamous neck cancer of unknown primary origin, multiple myeloma and other plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloproliferative syndrome, nasopharyngeal cancer, neuroblastoma, oral cancer, pharyngeal cancer, osteosarcoma, ovarian cancer (epithelial, germ cell tumor, low-malignant potential tumor), pancreatic cancer (exocrine gland, This includes islet cell carcinoma, sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary cancer, plasmacytoma, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter (transitional cell), salivary gland cancer, Sézary syndrome, skin cancer, skin cancer (cutaneous T-cell lymphoma, Kaposi's sarcoma, melanoma), small intestine cancer, soft tissue sarcoma, gastric cancer, testicular cancer, thymoma (malignant), thyroid cancer, urethral cancer, uterine cancer (sarcoma), abnormal childhood cancers, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0190] In another specific embodiment, the conjugate of a tubulicin analog of the present invention with a cell-binding molecule is used to treat or prevent autoimmune diseases according to the components and methods of the present invention. These autoimmune diseases include, but are not limited to, autoimmune acid deficiency chronic active hepatitis, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-GMB / TBM nephritis, antiphospholipid syndrome, anti-synthetase syndrome, arthritis, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphocyte proliferation syndrome, and autoimmune hepatitis. Epidemic peripheral nervous system disorders, autoimmune pancreatitis, multiple autoimmune endocrine disorders types I, II, and III, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune uveitis, Barlow's disease / Barlow concentric sclerosis, Behçet's disease, Berger's disease, Bickerstaff's encephalitis, Blau syndrome, bullous pemphigoid, Castleman disease, Chagas disease, chronic fatigue-associated immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal osteomyelitis, chronic Lyme disease, chronic obstructive pulmonary disease, allergic Granulomatous vasculitis, bullous pemphigoid, celiac disease, Cogan syndrome, cold agglutinin disease, complement component C2 deficiency, cranial arteritis, Crest syndrome, Crohn's disease (idiopathic inflammatory bowel disease), Cushing's syndrome, cutaneous leukocytoclastic vasculitis, malignant atrophic papulosis, painful steatosis, herpetiform dermatitis, dermatomyositis, type 1 diabetes, diffuse scleroderma, myocardial infarction, lupus discoid, eczema, endometriosis, enthesitis-associated arthritis, eosinophilic fasciitis, acquired epidermolysis bullosa, erythema nodosum, idiopathic mixed cryoglobulinemia, Evans syndrome, progressive Fibrodysplasia ossificans, fibromyalgia, fibromyositis, alveolar fibrosis, gastritis, pemphigoid of the gastrointestinal tract, giant cell arteritis, nephroglonephritis, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, hemolytic anemia, allergic purpura, herpes zoster of pregnancy, hidradenitis suppurativa, Hughes' syndrome (antiphospholipid antibody syndrome), hypogammaglobulinemia, idiopathic inflammatory demyelinating disease, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (autoimmune thrombocytopenic purpura), IgA nephropathy (Berger's disease), inclusion body myositis,Inflammatory demyelinating polyneuropathy, interstitial cystitis, irritable bowel syndrome, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, mucocutaneous lymphadenopathy, Lambert-Eaton myasthenic syndrome, leukocytosis-destructive vasculitis, lichen planus, lichen sclerosing, linear IgA disease (LAD), Lou Gehrig's disease (amyotrophic lateral sclerosis), lupus-like hepatitis, lupus erythematosus, Blau syndrome, Meniere's disease, microscopic polyangiitis, Miller-Fischer syndrome Group, mixed connective tissue disease, scleroderma, Mucher-Jakob disease, Mackle-Wells syndrome, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's disease), neuromuscular dystrophy, ocular scarring pemphigoid, opsoclonus-myoclonus syndrome, Odo thyroiditis, relapsing rheumatoid arthritis, panda syndrome (child autoimmune neuropsychiatric disorder associated with streptococcal infection), tumor cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry-Romberg syndrome, Personesy-Georgia syndrome, squamous cell carcinoma, pemphigus, pemphigus vulgaris, pernicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red blood cell aplastic anemia, Rasmussen's encephalitis, Raynaud's disease, relapsing polychondritis, Writer Syndrome, restless leg syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis, schizophrenia, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, Sjögren's syndrome, spondyloarthritis, adhesive blood syndrome, Still's disease, Stiffman syndrome, subacute bacterial endocarditis, Suzak syndrome, acute febrile neutrophilodermatitis, Sydenham's chorea, sympathetic ophthalmitis, Takayasu's arteritis, temporal arteritis (giant cell arteritis), This includes inflammation, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis (idiopathic inflammatory bowel disease), undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, vasculitis, vitiligo, Wegener's granulomatosis, Wilson's syndrome, and Westcott-Aldrich syndrome.

[0191] In other specific embodiments, the conjugating molecules used to conjugate via the conjugates of the present invention for the treatment or prevention of autoimmune diseases are, but are not limited to, anti-elastin antibodies; Abys anti-epithelial cell antibodies; anti-basement membrane type IV collagen protein antibodies; antinuclear antibodies; anti-double-stranded DNA antibodies, anti-single-stranded DNA antibodies, anti-cardiolipin antibodies IgM, IgG; anti-celiac antibodies; anti-phospholipid antibodies IgK, IgG; anti-SM antibodies; anti-mitochondrial antibodies; thyroid antibodies; microsomal antibodies, T-cell antibodies; thyroglobulin antibodies, anti-scleroderma-70 antibodies (AntiSCL-70); This includes anti-Joe antibodies, anti-U1RNP antibodies, anti-La / SSB antibodies, anti-SSA antibodies, anti-SSB antibodies, anti-parietal cell antibodies, anti-histone antibodies, anti-RNP antibodies, C-ANCA, P-ANCA, anti-centromere antibodies, anti-fibrin antibodies, anti-GBM antibodies, anti-ganglioside antibodies, anti-desmosome glycoprotein 3 core antibodies (anti-Desmogein 3), anti-p62 antibodies, anti-sp100 antibodies, anti-mitochondrial (M2) antibodies, rheumatoid factor antibodies, anti-MCV antibodies, anti-topoisomerase antibodies, and anti-neutrophil cytoplasmic (cANCA) antibodies.

[0192] In one preferred embodiment, the binding molecule used in the conjugate of the present invention can bind to a receptor or receptor complex expressed by activated lymphocytes associated with autoimmune diseases. The receptor or receptor complex may be, for example, a member of the immunoglobulin gene superfamily (e.g., CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD28, CD30, CD33, CD37, CD38, CD56, CD70, CD79, CD90, CD125, CD147, CD152 / CTLA-4, PD-1, or ICOS), or the TNF receptor superfamily (e.g., CD27, CD40, CD95) Examples include / Fas, CD134 / OX40, CD137 / 4-1BB, INF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2 / TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3), integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins (type C, type S, or type I), or complement regulatory proteins.

[0193] In another specific embodiment, a useful cell-binding ligand having immunospecificity for a viral or microbial antigen is a humanized or human monoclonal antibody. The term “viral antigen” as used herein includes, but is not limited to, any viral peptides, polypeptide proteins (e.g., HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLVtax, herpes simplex virus glycoproteins (e.g., gB, gC, gD, and gE), and hepatitis B surface antigen) that can induce an immune response. The term “microbial antigen” as used herein includes, but is not limited to, any microbial peptides, polypeptides, proteins, sugars, polysaccharides, or lipid molecules (e.g., bacteria, fungi, pathogenic protozoa, yeast polypeptides (e.g., LPS and capsular polysaccharide 5 / 8)) that can induce an immune response. Examples of antibodies useful in treating viral or microbial infections include, but are not limited to, palivizumab (a humanized anti-respiratory syncytial virus monoclonal antibody used to treat RVS infection), PRO542 (a CD4 fusion antibody used to treat HIV infection), Ostavir (a human antibody used to treat hepatitis B virus), PROTVIR (a humanized IgG1 antibody used to treat cytomegalovirus), and anti-LPS antibodies.

[0194] The tubulicin analog-cell binding molecule conjugates of the present invention can be used to treat infectious diseases. These infectious diseases include, but are not limited to, Acinetobacter infection, actinomycosis, African sleeping sickness (African trypanosomiasis), and AIDS (acquired immunodeficiency virus). Syndrome), amebiasis, anaplasmosis, anthrax, bacterial tuberculosis infection, Argentine hemorrhagic fever, roundworm infection, aspergillosis, astrovirus infection, babesiosis, Bacillus cereus infection, bacterial pneumonia, bacterial vaginosis, Bacteroides infection, balantidiosis, Bailey's roundworm infection, BK virus infection, black sand hair, Blastosis hominis infection, Blastomyces, Bolivian hemorrhagic fever, Borrelia infection, botulism (and infant botulism), Brazilian hemorrhagic fever, brucellosis, Burkholderia infection, Buruli ulcer, infectious calicivirus ( Norovirus, sapovirus, Campylobacter infection, Candida infection (candidiasis, thrush), cat scratch disease, cellulitis, Chagas disease (trypanosomiasis), chancroid, chickenpox, chrysomeloidiasis, chrysomeloidiasis of pneumonia, cholereticus, chromocytic mycosis, liver fluke disease, Clostridium difficile infection, coccidioidomycosis, Colorado tick fever, common cold (acute viral nasopharyngitis, acute rhinitis), Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever, cryptococcus, cryptosporidiosis, cutaneous larval migration, Cyclospora infection Cysticercosis, cytomegalovirus infection, dengue fever, dinuclear amebiasis, diphtheria, diphyllobothrium tapeworm infection, dung beetle infection, Ebola hemorrhagic fever, hydatidosis, ehrlichiosis, pinworm infection, enterococcal infection, enterovirus infection, typhus, erythema infectiosum (fifth disease), acute childhood rash, hypertrophic trematode infection, unilateral trematode infection, fatal familial insomnia, filariasis, food poisoning caused by Clostridium perfringens, nonparasitic amebic infection, Fusobacterium infection, gas gangrene (Clostridium myonecrosis), diotrichumosis, Gerstmann-Sträussler-Schey Kunker syndrome, giardiasis, glanders, gnathostomiasis, gonorrhea, inguinal granuloma (Donovan's disease), group A streptococcal infection, group B streptococcal infection, Haemophilus influenzae infection, hand, foot, and mouth disease (HFMD), hantavirus pulmonary syndrome, Helicobacter pylori infection, hemolytic uremic syndrome, hemorrhagic fever with renal syndrome, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex virus, histoplasmosis, hookworm infection, human balkan virus infection, human ehrlichiosis Evans, human granulocytic anaplasmosis, human metapneumovirus infection, human monocytic erythematosus.Lychiosis, human papillomavirus infection, human parainfluenza virus infection, tapeworm infection, influenza, isosporiasis, Kawasaki disease, mononucleosis, Kim's bacillus infection, kuru, Lassa fever, Legionnaires' disease, Legionnaires' disease (Pontiac fever), leishmaniasis, leprosy, leptospirosis, listeriosis, Lyme disease (Lyme Borrelia), lymphophilia (elephantiasis), lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever, measles, meidiomyelitis (Whitmore's disease), meningitis, meningococcal disease, metago Nimus disease, microsporidia, molluscum contagiosum, mumps, typhus (endemic typhus), mycoplasma pneumonia, mycomas, myiasis, neonatal conjunctivitis (neonatal ophthalmitis), Creutzfeldt-Jakob disease (vCJD, nvCJD), nocardiosis, onchocerciasis (blindness-causing filariasis), paracoccidioidomycosis (South American blastomyces), lung paragonimiasis, pasteurellosis, head lice (head lice), body lice (body lice), pubic lice (pubic lice, crarbice), pelvic inflammatory disease, pertussis (wooping cough) cough), epidemic, pneumococcal infection, Pneumocystis pneumonia, pneumonia, polio, Prevotella infection, PAME, progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, rat bite fever, respiratory syncytial virus infection, rhinovirus infection, rickettsial infection, rickettsia, Rift Valley fever, Rocky Mountain spotted fever, rotavirus infection, rubella, salmonellosis, SARS (severe acute respiratory syndrome), scabies, schistosomiasis, sepsis, diarrhea (dysentery), herpes zoster, smallpox, sporotrichum, staphylococcal food poisoning, grapes This includes cocci infections, nematode infections, syphilis, tapeworm infections, tetanus (trismus), barber's sitch, tinea manuum, necrotosis, tinea pedis, onychomycosis, tinea versicolor, toxocariasis (ocular larval migration), toxocariasis (visceral larval migration), toxoplasmosis, trichinella, trichomoniasis, cryptobiosis (whipworm infection), pulmonary tuberculosis, tularemia, mycoplasma urea infection, Venezuelan encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, rhizobia minor, pseudotuberculosis infection, Yersiniasis, yellow fever, and zygomycosis.

[0195] The cell-binding molecule described in this application, preferably an antibody against a pathogenic strain, the pathogenic strain being, but not limited to, Acinetobacter baumannii, Actiomyces iss Laerii, Actinomyces odontolyticus, Propionibacterium propionicus, Trypanosoma brusey, HIV (Human Immunodeficiency Virus), Entamoeba histolytica, Anaplasma, Bacillus anthrax, Arcanobacterium haemolyticum, Junin virus, Roundworm, Aspergillus, Astroviridae, Babesia, Bacillus cereus, Multiple bacteria, Bacteroides, Colonic pouch ciliate, Ascaris baleyi, BK virus Russ, Piedraia hortae, Blastocystis hominis, Dermatitis spongiformis, Macpo virus, Borrelia, Clostridium botulinum, Sabia, Brucella, Burkholderia cepacia and other Burkholderia species, Mycobacterium ulcerans, Caliciviridae family, Campylobacter, Candida albicans and other Candida species, Bartonella henselae, Group A Streptococcus and Staphylococcus, Trypanosoma cruzi, Chancroid, Varicella zoster Herpes zoster virus (VZV), Chlamydia trachomatis, Chlamydia pneumoniae, Vibrio cholerae, Fonsecae pedrosoi, Hepatoparasitic fluke, Clostridium difficile, Coccidioides imitis, Coccidioides posadasi, Colorado tick fever virus, Rhinovirus, Coronavirus, Creutzfeldt-Jakob disease prion, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium, Cat hookworm, Coparasites, Cyclospora, Taenia solium, Cytomegalovirus, Dengue fever virus (DEN-1, D EN-2, DEN-3 and DEN-4) Flavivirus, Bikaryota amoeba, Corynebacterium diphtheriae, Diphyllobothrium, Dracunculus medinensis, Ebola virus, Echinococcus, Ehrlichia, Pinworm, Enterococcus, Enterovirus, Typhus rickettsia, Parvovirus B19, Human herpesvirus type 6, Human herpesvirus type 7, Hypertrophic fluke, Liver fluke and giant liver fluke, FFI prion, Filariophaeus superfamily, Clostridium perfringens, Fusobacterium, Clostridium perfringens, Other Clostridium species,Geotrichum candidiasis, GSS prion, Giardial amblia, Burkholderia bacillus, Gnathostoma nematode, Gnathostoma rhinosum, Neisseria gonorrhoeae, Granulomatous bacteria, Streptococcus pyogenes, Streptococcus agalactie, Haemophilus influenzae, Enterovirus, most Coxsackie A viruses, Enterovirus type 71, Sin Nombre virus, Helicobacter pylori, Escherichia coli O158:H7, Bunyaviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Herpes simplex virus type 1, Herpes simplex virus Rupesvirus type 2, Histoplasma capsulatum, Duodenal hookworm, American hookworm, Haemophilus influenzae, Boca human virus, Ehrlichia ewingii, Anaplasma phagocytophyllum, Human metapneumovirus, Ehrlichia schaffensis, Human papillomavirus, Human parainfluenza virus, Dwarf tapeworm, Miniature tapeworm, Epstein-Barr virus, Orthomyxoviridae, Isosporabelli, Kingella kingea, Klebsiella pneumoniae, Klebsiella otzena, Klebsiella rhinoscleromotis, Culpurium pteronyssinus, Lassa fever virus, Legionella pneumophila, Leishmania, Mycobacterium lepromatosis, Leptospira species, Listeria monocystis, Borrelia and other Borrelia species, Wuchereria bancrofti and Mycobacterium malayi, Lymphocytic choriomeningitis virus (LCMV), Plasmodium species (Pla Smodium genus), Marburg virus, measles virus, Bacillus pseudorhinoplasia (Burkholderia pseudomallei), Neisseria meningitidis, Tremora yokogawai, Microsporidia, Molluscum contagiosum virus (MCV), Mumps virus, Rickettsia cyfii, Mycoplasma pneumoniae, various bacteria (Actinomycetoma) and fungi (Mycoplasma), Diptera larvae of parasitic flies, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prion, Nocardia asteroides and other Nocardia species, Paragonimus rotundus, Blastomyces brasilis, Paragonimus and other Paragonimus genera,Pasteurella genus, head, White lice, body lice, Phthirus pubis, Bordetella pertussis, Plague bacillus, Streptococcus pneumoniae, Pneumocystis cysticercosis, poliovirus, Prevotella species, Naegleria amoeba, JC virus, Chlamydia psittaci, Coxiella barnettii, rabies virus, bead chain Escherichia coli and rat bite fever spirochete, respiratory respiratory syncytial virus, Rhinosporidium sebergii, rhinovirus, Rickettsia species, Rickettsia mites, Rift Valley fever virus, Rocky Mountain spotted fever Rickettsia, rotavirus, rubella virus, Salmonella species, Atypical pneumonia coronavirus, scabies mites, schistosomiasis, Shigella, varicella-zoster virus, smallpox or smallpox, Sporothrix schenkyi, Staphylococcus, Staphylococcus aureus, Streptococcus pyogenes, Strongyloides, syphilis spirochete, Tapeworm, Tetanus, tinea, Trichophyton tonsurans, tinea, Epidermophyton floccosum, Dermatophytes rubra and Trichophyton folliculitis, Dermatophytes rubra, Horthea wernecki, tinea, Malassezia, roundworms in dogs and cats, Toxoplasma, Trichinella, Trichomonas vaginalis, whipworm, Mycobacterium tuberculosis, Bacillus toulatus, Ureaplasma Ureaticum, Venezuelan encephalitis virus, Vibrio cholerae, Guanalitovirus, West Nile virus, Mycobacterium leukoderma, Mycobacter pseudotuberculosis, Yersinia enteritis, Yellow fever virus, Mucormycetes (Mucormycetes) and Mesh mold (Entomophthora disease), Pseudomonas aeruginosa, Campylobacter fetus (Vibrio), Aeromonas bacteria, Edwardsierella genus, Tarda, Plague bacillus, Shigella, Shigella, Shigella sonne, Salmonella typhi, Treponema pertenue, Treponema calateneum, Fensenburgdorferi, Borrelia burgdorferi, Leptospirosis hemorrhagic jaundice, Ni Pneumocystis carinii, bovine abortifacient, porcine abortifacient, Malta fever bacillus, Mycoplasma species, typhus lichen, rickettsia mites, Chlamydia species, pathogenic fungi (Aspergillus fumigatus, Candida albicans, Histoplasma maculatum); protozoa (Entamoeba histolytica, Trichomonas vaginalis, Trichomonas ensata, Trypanosoma gambiens, Rhodesia trypanosoma, Donovan's leishmania, Leishmania tropicalis, Leishmania brizola, Pneumocystis carinii pneumonia, Plasmodium vivax, Plasmodium falciparum, malignant malaria);or include helminths (such as Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematocephalus, and hookworms).

[0196] Other antibodies used in the present invention as cell-binding ligands for the treatment of viral diseases include, but are not limited to, antibodies against pathogenic viral antigens, and examples of such pathogenic viruses include, but are not limited to, Poxyiridae, Herpesviridae, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, Influenza virus, Parainfluenza virus, and Mumps virus. Measles, respiratory syncytial virus, rubella, arbovirus, rhabdovirus, arenaviridae, non-A / non-B hepatitis virus, rhinovirus, coronavirus, rotavirus, oncovirus [e.g., HBV (hepatocellular carcinoma), HPV (cervical cancer, anal cancer), Kaposi's sarcoma-associated herpesvirus (Kaposi's sarcoma), EB virus (nasopharyngeal cancer, Burkitt lymphoma, primary central nervous system lymphoma), MCPyV (Merkel cell carcinoma), SV40 (Simian virus 40), HCV (hepatocellular carcinoma), HTLV-I (adult T-cell leukemia) Lymphoma); Immune diseases caused by viruses: [e.g., human immunodeficiency virus (AIDS)], CNS viruses: [e.g., JCV (progressive multifocal leukoencephalopathy), MeV (subacute sclerosing panencephalitis), LCV (lymphocytic choriomeningitis), arboviral encephalitis, orthomyxovirus (presumably) (encephalitis lethargica), RV (rabies), bullous stomatitis, herpesvirus meningitis, Ramsay Hunt syndrome type II; poliovirus (poliomyelitis, post-polio syndrome), HTLV-I (tropical spastic paralysis)] ]; Cytomegalovirus (CMV retinitis, HSV (herpetic keratitis)); Cardiovascular virus [e.g., CBV (pericarditis, myocarditis)]; Respiratory system / acute nasopharyngitis / viral pneumonia: [EB virus (EBV infection / infectious mononucleosis), cytomegalovirus, SARS coronavirus (severe acute respiratory syndrome), orthomyxovirus family: influenza virus A / B / C (influenza / avian influenza), paramyxovirus: human parainfluenza virus (parainfluenza), R This includes viruses such as SV (human respiratory syncytial virus), hMPV; digestive system viruses [MuV (mumps), cytomegalovirus (CMV esophagitis); adenovirus (adenovirus infection); rotavirus, norovirus, astrovirus, coronavirus, HBV (hepatitis B virus), CBV, HAV (hepatitis A virus), HCV (hepatitis C virus), HDV (hepatitis D virus), HEV (hepatitis E virus), HGV (hepatitis G virus)]; and genitourinary system viruses [e.g., BK virus, MuV (mumps)].

[0197] In a further objective, the present invention also relates to pharmaceutical compositions for the treatment of cancer or autoimmune diseases, comprising the conjugate of the present invention together with a pharmaceutically acceptable carrier, diluent, or excipient. Methods for treating cancer, infections, and autoimmune diseases can be carried out in vitro, in vivo, or ex vivo. Examples of in vitro therapies include cell culture treatments to kill all cells except desirable variants that do not express a target antigen, or to kill variants that express an undesirable antigen. Examples of ex vivo therapies include treating hematopoietic stem cells (HSCs) prior to performing a transplant (HSCT) and returning them to the body of the same patient to kill diseased or malignant cells. For example, clinical ex vivo treatments to remove cancer cells or lymphocytes from bone marrow prior to autologous transplantation in the treatment of cancer and autoimmune diseases, or to remove T cells and other lymphocytes from allogeneic bone marrow or tissue prior to transplantation to prevent graft-versus-host disease, can be carried out as follows: After obtaining bone marrow cells from a patient or another individual, they are cultured at 37°C for 15 minutes to approximately 48 hours in serum-containing medium to which the conjugate of the present invention is added, so that the concentration range is 1 pM to 0.1 mM. The appropriate concentration conditions and culture time (=dose) can be easily determined by an experienced clinician. After the culture is complete, the bone marrow cells are washed with serum-containing medium and returned to the human body by known methods such as intravenous injection. If the patient is receiving other treatments (e.g., pharmacokinetic chemotherapy or total body irradiation) between the acquisition and reinfusion of bone marrow cells, the processed bone marrow cells are cryopreserved in liquid nitrogen using standard medical equipment.

[0198] Prescription and application

[0199] The freeze-dried compositions of the present invention can be prepared by methods generally known in the art to remove water from the composition. For example, the composition can be dehydrated by heating a sample to a suitable temperature for a suitable time. The composition can also be dehydrated at any suitable temperature under a reduced pressure atmosphere. The reduced pressure atmosphere can be any pressure lower than atmospheric pressure. The reduced pressure atmosphere can be heated to a temperature higher than room temperature, maintained at approximately room temperature, or cooled to a temperature lower than room temperature. For example, the components can be cooled to a temperature lower than room temperature under a reduced pressure atmosphere. Suitable temperatures include, but are not limited to, temperatures below room temperature or below 20°C, 15, 10, 5, 0, -5, -10, -15, -20, -25, -30, -40, -50, -60, or -65°C. When using a reduced pressure atmosphere, the reduced pressure atmosphere can be below atmospheric pressure, or below 100 Torr (mmHg), 50, 25, 10, 5, 4.58 (triple point of water), 4, 3, 2, 1, 0.5, 0.1, 0.05, or 0.01 Torr. The components can be cooled to below 0.01°C under reduced pressure of below 4.58 Torr (611 Pa or 0.006 atmospheres).

[0200] The compositions of the present invention can be prepared by methods generally known in the art. For example, the freeze-dried compositions of the present invention can be prepared by dissolving an excipient in a purified solution of a tubulicin analog conjugate, adjusting the mixture to a specific pH value such as pH 5.0, 5.5, or 6.0, adding water as needed to form a solution in which the concentrations of the conjugate and excipient are constant, then pouring the solution into a vial, cooling it to a temperature below 0°C, and freezing the mixture.

[0201] Generally, pre-lyophilized liquid formulations containing conjugated active ingredients at concentrations of 0.1 g / L to 300 g / L for delivery to patients without high levels of antibody aggregation may include one or more polyols (e.g., sugars), buffers having a pH of 4.5 to 7.5, surfactants (e.g., polysorbate 20 or 80), antioxidants (e.g., ascorbic acid and / or methionine), isotonic agents (e.g., mannitol, sorbitol or NaCl), chelating agents such as EDTA, metal complexes (e.g., Zn-protein complexes); biodegradable polymers such as polyesters; preservatives (e.g., benzyl alcohol); and / or free amino acids.

[0202] Suitable buffers for use in formulations include, but are not limited to, organic acid salts such as sodium, potassium, ammonium, or trihydroxyethylamino salts of citric acid, ascorbic acid, gluconic acid, carbonate, tartaric acid, succinic acid, acetic acid, or phthalates; and Tris, tromethamine hydrochloride, sulfate, or phosphate buffers. Furthermore, amino acid cationic components can also be used as buffers. Such amino acid components include, but are not limited to, arginine, glycine, glycylglycine, and histidine. Arginine buffers include arginine acetate, arginine chloride, arginine phosphate, arginine sulfate, arginine succinate, etc. In one embodiment, the arginine buffer is arginine acetate. Examples of histidine buffers include histidine chloride-arginine chloride, histidine acetate-arginine acetate, histidine phosphate-arginine phosphate, histidine sulfate-arginine sulfate, histidine succinate-arginine succinate, etc. The pH of the buffer formulation is 4.5 to 7.5, preferably about 4.5 to about 6.5, and more preferably about 5.0 to about 6.2. In some embodiments, the concentration of the organic acid salt in the buffer is about 10 mM to about 500 mM.

[0203] Polyols, optionally included in pharmaceutical formulations, are substances having multiple hydroxyl groups. Polyols can be used as stabilizing excipients and / or isotonic agents in both liquid and lyophilized formulations. Polyols can protect biopharmaceuticals from physical and chemical degradation pathways. Preferredly excluded cosolvents increase the effective surface tension of the solvent at the protein interface, thereby resulting in the most energetically favorable structural conformation having the smallest surface area. Polyols can also be used as fillers in lyophilized compositions to add bulk to the composition and, for example, to visualize the composition, particularly since lyophilized pellets are difficult to see. Polyols are useful to prevent the active ingredient (conjugate of tubulicine analogs) of the pharmaceutical composition from being blown away and / or to aid in freeze-protection of the composition. Polyols include sugars (reducing and non-reducing sugars), sugar alcohols, and sugar acids. "Reducing sugars" are those containing a hemiacetal group that can reduce metal ions or react covalently with lysine or other amino groups of proteins, while "non-reducing sugars" are those that lack these properties of reducing sugars. Examples of reducing sugars include fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, and glucose. Non-reducing sugars include sucrose, trehalose, sorbose, melegitose, and raffinose. Sugar alcohols are selected from mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, sreitol, sorbitol, and glycerol. Sugar acids include L-gluconates and their metal salts. The amount of polyol in the liquid formulation or the prepared lyophilized solid may be 0.0% to 20% by weight. Preferably, a non-reducing sugar, sucrose, or trehalose is selected in the formulation at a concentration of about 0.1% to 15%, and trehalose is preferred over sucrose due to its solution stability.

[0204] The surfactants optionally included in the formulation are polysorbates (polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, etc.); poloxamers (e.g., poloxamer 188, poly(ethylene oxide)-poly(propylene oxide), poloxamer 407, or polyethylene polypropylene) (Glycol, etc.), Triton; Sodium dodecyl sulfate (SDS), Sodium lauryl sulfate Sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostea The surfactants selected are: dilamidopropyl dimethylamine; sodium cocoyl methyl taurate or sodium methyl oleyl taurate; dodecyl betaine, dodecyl dimethylamine oxide, cocamidopropyl betaine, and cocoamphoglycinate; the "MONAQUAT™" series (e.g., isostearyl ethylimonium ethosulfate); polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.). Preferred surfactants are polyoxyethylene sorbitan fatty acid esters, e.g., polysorbate 20, 40, 60, or 80 (Tween 20, 40, 60, or 80). The concentration of the surfactant in the formulation is in the range of 0.0% to about 2.0% by weight. In certain embodiments, the concentration of the surfactant is about 0.01% to about 0.2%. In one embodiment, the surfactant concentration is about 0.02%.

[0205] Preservatives, which are optionally included in pharmaceutical formulations, are compounds that essentially reduce bacterial activity. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides, where the alkyl group is a long-chain compound), and benzethonium chloride. These types of preservatives include phenol, butyl and benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and aromatic alcohols such as m-cresol. The amount of preservative in the liquid formulation or the prepared lyophilized powder may be 0.0% to 5.0% by weight. In one embodiment, the preservative described herein is benzyl alcohol.

[0206] Suitable free amino acids in the formulation as bulk raw materials, isotonic agents, or osmotic pressure regulators are selected from, but are not limited to, arginine, cystine, glycine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine, glutamic acid, or aspartic acid. Inclusion of basic amino acids is preferred, i.e., arginine, lysine, and / or histidine. If the composition contains histidine, it may act as both a buffer and a free amino acid; however, when using a histidine buffer, it is common to include, for example, a histidine buffer and non-histidine free amino acids including lysine. Amino acids may exist in their D and / or L forms, but the L form is typical. Amino acids may exist as any suitable salt, such as the hydrochloride of arginine-HCl. The concentration of amino acids in the liquid formulation or prepared lyophilized powder can be 0.0% to 30% by weight.

[0207] The formulation may optionally contain methionine, glutathione, cysteine, cystine, or ascorbic acid as antioxidants at a concentration of up to approximately 5 mg / ml in the liquid formulation, or at a concentration of 0.0% to 5.0% by weight in the formulated lyophilized powder. The formulation may optionally contain metal chelating agents, such as EDTA or EGTA, at a concentration of up to approximately 2 mM in the liquid formulation, or at a concentration of 0.0% to 0.3% by weight in the formulated lyophilized powder.

[0208] The final formulation contains buffering agents (e.g., acids such as HCl, H2SO4, acetic acid, H3PO4, citric acid, or NaOH, KOH, NH4OH, ethanolamine, diethanolamine or triethanolamine, sodium phosphate, potassium phosphate, trisodium citrate). The pH can be adjusted to a preferred level with a base (such as thorium or tromethamine). The formulation needs to be controlled to be "isotonic," meaning that the formulation in question has essentially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250–350 mOsm. Isotonicity can be measured, for example, using a vapor pressure or ice-type osmometer. The isotonic agent is selected from mannitol, sorbitol, sodium acetate, potassium chloride, sodium phosphate, potassium phosphate, trisodium citrate, or NaCl. Generally, both buffer salts and isotonic agents can account for up to 30% by weight in the formulation. The pH of the reconstituted formulations and injectable compositions of the present invention can be any pH that provides the desired properties of the formulation or composition. Desired properties may include, for example, the stability of the tubulicin analog conjugate and improved filtration efficiency. In some embodiments, the pH of the reconstituted formulations and injectable components of the present invention may be about 3.5 to about 9.0, for example, about 5.0 to about 7.0. In certain embodiments, the pH of the reconstituted formulations and injectable components of the present invention may be 5.0±0.1, 5.1±0.1, 5.2±0.1, 5.3±0.1, 5.4±0.1, 5.5±0.1, 5.6±0.1, 5.7±0.1, 5.8±0.1, 5.9±0.1, 6.0±0.1, 6.1±0.1, 6.2±0.1, 6.3±0.1, 6.4±0.1, or 6.5±0.1.

[0209] In some embodiments, it may be advantageous to buffer the pH by including one or more buffers in the composition. In certain embodiments, the buffer may have a pKa of, for example, about 5.5, about 6.0, or about 6.5. Those skilled in the art can select a suitable buffer to be included in the composition of the present invention based on its pKa and other properties. Buffers are well known in the art. Therefore, the buffers described herein are not intended to constitute an exhaustive list, but rather to constitute only some examples of buffers that can be used in the formulations or compositions of the present invention. In certain embodiments, buffers include, but are not limited to, tris, trisHCl, potassium phosphate, sodium phosphate, sodium citrate, sodium ascorbate, combinations of sodium phosphate and potassium phosphate, tris / trisHCl, sodium bicarbonate, arginine phosphate, arginine hydrochloride, histidine hydrochloride, cacodylate, succinate, 2-(N-morpholino)ethanesulfonic acid (MES), maleate, bistris, phosphate, carbonate, and any pharmaceutically acceptable salts and / or combinations thereof.

[0210] Other excipients that may be useful in either the liquid or lyophilized formulation of the patent application include, for example, fucose, cellobiose, maltotriose, melibiose, octulose, ribose, xylitol, arginine, histidine, glycine, alanine, methionine, glutamic acid, lysine, imidazole, glycylglycine, mannosylglycerate, Triton X-100, and Pluoronic. F-127 includes cellulose, cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, dextran (10, 40 or 70 kD), polydextrose, maltodextrin, Ficol, gelatin, hydroxypropyl meta, sodium phosphate, potassium phosphate, ZnCl2, zinc, zinc oxide, sodium citrate, trisodium citrate, tromethamine, copper, fibronectin, heparin, human serum albumin, protamine, glycerin, glycerol, EDTA, metacresol, benzyl alcohol, phenol, polyhydric alcohol, or hydrogenated forms of carbohydrates having a carbonyl group reduced to a primary or secondary hydroxyl group.

[0211] Other intended excipients for use in the aqueous pharmaceutical composition of the patent application include, for example, flavoring agents, antibacterial agents, sweeteners, antioxidants, antistatic agents, lipids such as phospholipids or fatty acids, steroids such as cholesterol, protein excipients such as serum albumin (human serum albumin), recombinant human albumin, gelatin, and casein, and salt-forming counterions such as sodium. These and additional known pharmaceutical excipients and / or additives suitable for use in the formulation of the present invention are: For example, "The Handbook of Pharmaceutical Excipients, 4 th edition, Rowe et al., Eds., American Pharmaceuticals Association (2003); and Remington: The Science and Practice of Pharmacy, 21 thAs cited in edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005), this is well known in the art. Those skilled in the art will recognize that the selection of any one excipient may affect the selection of other excipients. For example, if a combination of excipients produces an undesirable effect, the selection of a particular excipient may eliminate the use of one or more additional excipients. Those skilled in the art may, if any, empirically determine the excipient and include it in the composition or formulation of the present invention.

[0212] Pharmaceutical containers or vessels are used to hold any of the pharmaceutical formulations of the conjugates of the patent application. The containers are vials, bottles, pre-filled syringes, pre-filled syringes, or auto-injector syringes. Liquid formulations can be freeze-dried or drum-dried in the form of cake or powder in borosilicate vials or soda-lime glass vials. Solid powders may also be prepared by efficient spray drying and then packed into vials or pharmaceutical containers for storage and distribution.

[0213] In further embodiments, the present invention provides a method for preparing a formulation, comprising the following steps: (a) lyophilizing a formulation comprising a conjugate, excipients, and a buffer system; and (b) reconstituting the lyophilized mixture from step (a) in a reconstitution medium so that the reconstituted formulation is stable. The formulation from step (a) may further comprise a stabilizer and one or more excipients selected from the group comprising the above-mentioned bulking agents, salts, surfactants, and preservatives. As the reconstitution medium, several diluted organic acids or water, i.e., sterile water or bacteriostatic water for injection (BWFI), can be used. The reconstitution medium can be selected in an amount of about 10 to about 250 mM from the group consisting of water, i.e., sterile water, bacteriostatic water for injection (BWFI), or acetic acid, propionic acid, succinic acid, sodium chloride, magnesium chloride, acidic solutions of sodium chloride, acidic solutions of magnesium chloride, and acidic solutions of arginine.

[0214] The liquid pharmaceutical formulations of the conjugates of this application need to exhibit various predefined properties. Stability is one of the main concerns for liquid pharmaceuticals, as proteins / antibodies tend to form soluble and insoluble aggregates during manufacturing and storage. Furthermore, various chemical reactions (deamination, oxidation, clipping, isomerization, etc.) can occur in solution, potentially leading to increased levels of degradation products and loss of biological activity. Preferably, the conjugates in liquid or lyophilized formulations need to exhibit a shelf life of more than 6 months at 25°C. More preferably, the conjugates, in either liquid or lyophilized formulations, need to exhibit a shelf life of more than 12 months at 25°C. The most preferred liquid formulations should exhibit a shelf life of approximately 24–36 months at 2–8°C, and the lyophilized formulations should exhibit a shelf life of up to approximately 60 months at 2–8°C. Both liquid and lyophilized formulations need to exhibit a shelf life of at least 2 years at -20°C or -70°C.

[0215] In some embodiments, the formulation is stable after freezing (e.g., -20°C or -70°C) and thawing, for example, after one, two, or three freeze-thaw cycles. Stability can be assessed by evaluating the drug / antibody (protein) ratio and aggregate formation (e.g., using ultraviolet, size exclusion chromatography, turbidity measurement, and visual inspection); assessing charge heterogeneity using electrofocusing such as cation exchange chromatography or image capillary electrophoresis; sequence analysis of amino or carboxyl group ends; mass spectrometry, or matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS), or HPLC-MS / MS; SDS-PAGE analysis for comparison of antibody degradation or integrity; peptide mapping (e.g., trypsin or lysine-carbon); and evaluation of the antibody's biological activity or antigen-binding function. The instability can be evaluated qualitatively and / or quantitatively by various methods, such as those described above. Instability may include one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), shear / hydrolysis / fracture (e.g., hinge fracture), succinimide formation, unpaired cysteine, N-terminal extension, C-terminal processing, and catabolic glycosylation.

[0216] The stable conjugate also needs to "maintain its biological activity" in the pharmaceutical formulation, for example, the biological activity of the conjugate at a specific point in time, e.g., 12 months, must be within approximately 20%, preferably within approximately 10% (within the assay error) of the biological activity determined by antigen-binding assays and / or in vitro cytotoxicity assays, etc., at the time the pharmaceutical formulation was prepared.

[0217] For in vivo clinical use, the conjugated formulation is provided as a solution or as a lyophilized solid that can be redissolved in sterile water for injection. In either case, the most common process for reconstituting the solid-form conjugated composition typically involves the following steps: recovering the solvent (such as water) from a first container and pouring it into a second container containing the solid-form conjugated composition; homogenizing the liquid in the second container to eliminate bubbles and / or dried aggregates; and removing the reconstituted conjugated composition from the second container for administration or for injection into a physiological buffer / salt solution held in an injection bag containing a sodium chloride injection for administration. Each of the above steps may require some object handling, including needles or spikes, and completion of defined processes. To ensure correct reconstitution and reduce misunderstandings among users, pharmaceutical companies provide users with "Instructions for Use" guiding them through the reconstitution process.

[0218] Depending on the operating steps and conjugated composition applied, the reconstitution step may result in long reconstitution times, the presence of trapped dry aggregates or gel regions that barely reach the solvent, the presence of trapped air bubbles or foams, either entirely or limited to the gas-liquid interface ring, and / or significant variations in reconstitution time. These problems would be unacceptable for the reconstitution of a conjugated composition. In most cases, this process involves a general solvent transfer step, and before final recovery, several stirring / swirling and settling steps are performed to wet the solid for homogenization and to observe rehydration until completely dissolved. There will be "do" or "don't" suggestions during this process. Furthermore, pharmaceutical companies may recommend user training, regardless of whether the user is a professional, patient, or relative, or may restrict represcription to a professional scope. For some lyophilized pharmaceutical ingredients, the complete reconstitution time can be as long as 30 minutes.

[0219] In some embodiments, the concentration of tubulicin analog conjugates in the reconstituted formulation is about 1 mg / ml to about 30 mg / ml, for example, about 5 mg / ml to about 25 mg / ml, before being transferred to an infusion bag containing physiological buffer or saline. In certain embodiments, the concentration of tubulicin analog conjugates in the reconstituted formulation is about 15 to 25 mg / ml before being transferred to an infusion bag containing physiological buffer or saline.

[0220] The appropriate protocol for conjugate administration is as follows: The conjugate is administered intravenously as a bolus daily, weekly, every two weeks, every three weeks, every four weeks, or monthly for 8 to 54 weeks. The bolus dose is dissolved in 50 to 1000 mL of physiological saline, to which human serum albumin may optionally be added (e.g., 0.5 to 1 mL of concentrated human serum albumin solution at 100 mg / mL). The drug dose is approximately 50 μg to 20 mg / kg body weight per week, administered intravenously (each injection ranging from 10 μg to 200 mg / kg). After 4 to 54 weeks of treatment, the patient may accept a second course of treatment. Detailed treatment methods, including pathways, excipients, diluents, dosages, and duration of treatment, can be determined by an experienced surgeon.

[0221] Examples of conditions that can be treated by selectively killing cell populations in vivo or ex vivo include any type of cancer, autoimmune diseases, transplant rejection, and infections (including those caused by viruses, bacteria, or parasites).

[0222] The amount of conjugate required to achieve the desired biological effect varies depending on many factors, including the chemical properties, efficacy, and bioavailability of the conjugate, the type of disease, the patient's race, the patient's medical condition, the route of administration, and all other factors that determine the required dosage, including the delivery and regimen used.

[0223] Generally speaking, the formulations of the present invention may be parenteral formulations dissolved in physiological buffer so as to contain the conjugate at a concentration of 0.1 to 10% w / v. Typical dose ranges are 1 μg / kg body weight to 0.1 g / kg body weight per day, week, two weeks, three weeks, or month, and preferred dose ranges are 0.01 mg / kg body weight to 20 mg / kg body weight per week, two weeks, three weeks, or month. Preferred drug doses may appropriately depend on variables such as the type and degree of disease or disability progression, the overall health status of the individual patient, the relative biological activity of the selected drug, the dosage form of the compound, the mode of administration (intravenous, intramuscular, or other), the pharmacokinetic characteristics of the drug in the selected mode of administration, and the rate of administration (single injection or continuous infusion) and the administration schedule (frequency of administration within a set period of time).

[0224] The conjugate of the present invention can also be administered in unit doses, where "unit dose" refers to a single dose administered to one patient. These units can be used in simple and convenient packaging and maintain physical and chemical stability as either the active conjugate itself or a pharmaceutically acceptable composition as described below. Therefore, a typical daily dose range is 0.01 to 100 mg / kg body weight. Generally, unit doses range from 1 to 3000 mg per day, week, two weeks, three weeks, or month. Preferably, the unit dose is 1 mg to 500 mg administered 1 to 4 times per month, and more preferably, 1 mg to 100 mg administered once every week, two weeks, or three weeks. The conjugate given herein can be prepared by adding one or more pharmaceutically acceptable excipients to a pharmaceutical composition. A unit dose of the drug can be administered orally as a tablet, simple capsule, or soft capsule; intranasally as a powder, nasal spray, or aerosol; or cutaneously as, for example, an ointment, cream, lotion, gel, spray, or skin patch. The composition can be conveniently administered in unit dose form by methods known in any pharmaceutical art, e.g., Remington: The Science and Practice of Pharmacy, 21 th It can be prepared by the method described in Lippincott Williams & Wilkins: Philadelphia, PA, 2005.

[0225] Preferred formulations include pharmaceutical compositions in which the compounds of the present invention are formulated for oral or parenteral administration. For oral administration, tablets, powders, capsules, etc., may contain one or more of the following components or other compounds having similar properties: binders such as microcrystalline cellulose or tragacanth gum; diluents such as starch or lactose; disintegrants such as starch or cellulose derivatives; lubricants such as magnesium stearate; flow promoters such as colloidal silica; sweeteners such as sucrose or saccharin; and flavoring agents such as peppermint or methyl salicylate. Capsules can take the form of hard capsules or soft capsules and are generally made from a mixture of gelatin optionally mixed with a plasticizer, similar to starch capsules. Furthermore, the unit dosage form may have a physical form such as sugar coating, shellac, or enteric coating. It may contain various other raw materials that change form. Other oral administration forms, such as syrups or elixirs, may contain sweeteners, preservatives, pigments, colorants, and flavorings. Furthermore, the active compound can be made into a rapidly dissolving administration form, a controlled-release administration form, or a sustained-release form through various processing and formulations, with the sustained-release form being preferably bimodal. Tablets preferably contain lactose, corn starch, magnesium silicate, croscarmellose sodium, polyvinylpyrrolidone, magnesium stearate, talc, and other combinations.

[0226] Liquid pharmaceuticals for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Liquid compositions may contain binders, buffers, preservatives, chelating agents, sweeteners, flavorings, and colorants. Non-aqueous solvents include alcohols, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Aqueous solvents include mixtures of alcohol and water, buffering media, and physiological saline. In particular, biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers can be used as excipients to control the release of active compounds. Intravenous media may include liquids and nutritional rehydration solutions, electrolyte rehydration solutions such as Ringer's dextrose base, etc. Other viable parenteral delivery systems for the active drugs of the present invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

[0227] Other dosage forms include inhalation formulations containing dry powder, aerosol, and droplets. Inhalation formulations may be, for example, aqueous solutions containing polyoxyethylene-9-lauryl ether, glycocholates, and deoxycholates, or oily solutions for administration in the form of nasal drops or gels applied into the nasal cavity. Formulations for buccal administration include, for example, lozenges or troches, which may contain flavoring agents such as sucrose or acacia, and other auxiliary materials such as glycocholates. Formulations for rectal administration are preferably provided as suppositories of a unit dose together with a solid carrier such as cocoa butter, and may contain salicylic acid. Formulations for topical application to the skin are preferably in the form of ointments, creams, lotions, pastes, gels, sprays, aerosols, or oils. Petrolatum, lanolin, polyethylene glycol, alcohol, or mixtures thereof can be used as drug carriers. Suitable formulations for transdermal administration can be provided as individual patches, which may be lipophilic emulsions or buffered aqueous solutions dissolved or dispersed in polymers or adhesives.

[0228] In certain embodiments, the conjugate of the present invention is administered concurrently with known or future therapeutic agents, such as chemotherapeutic agents, radiotherapy agents, immunotherapy agents, autoimmune disease agents, anti-infective agents, or other antibody-drug conjugates, thereby achieving a synergistic effect. In another specific embodiment, the synergistic drug or radiotherapy is administered or performed before or after the administration of the conjugate, in one embodiment, 1 hour, 12 hours, 1 day, 1 week, 2 weeks, 3 weeks, 1 month, or several months before or after the administration of the conjugate of the present invention.

[0229] In other embodiments, the synergistic drugs include, but are not limited to, the following:

[0230] 1) Chemotherapy agents: a) Alkylating agents: Nitrogen mustard: Chlorambucil, chlornafadin, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, mannomustine, mitobronitol, melphalan, mitractol, pipobromane, nobenbitin, phenesterine, prednimustine, thiotepa, trophosfamide, uracil mustard; CC-1065 and synthetic analogs of adzeresin, karzeresin and bizeresin; duocalmycin and its synthetic analogues KW-2189 and CBI-TMI are forms of benzodiazepine dimers or pyrrolobenzodiazepine (PBD) dimers, tomaimycin dimers, indolinobenzodiazepine dimers, imidazobenzothiadiazepine dimers, or oxazolidinobenzodiazepine dimers; nitrosourea compounds including carmustine, lomustine, chlorozotosine, fotemustine, nimustine, ranimustine; busulfan, treosulfan, imuprosulfan, and pipos Alkyl sulfonates containing ruphan; triazenes or dacarbazines; platinum-containing compounds including carboplatin, cisplatin, and oxaliplatin; aziridines, benzodopa, carboquan, metsuredopa, and uredopa; ethyleneimines, and methylamelamamines including altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramine, and trimethylolomelamine; b) Plant alkaloids: Vinca alkaloids including vincristine, vinblastine, vindesine, vinorelbine, and navelbine; taxoids including paclitaxel and docetaxel and their analogues; meitansinoids including DM1, DM2, DM3, DM4, DM5, DM6, DM7, meitansine, and anthamitosine and their analogues; cryptophycins such as cryptophycin 1 and cryptophycin 8; epotilons, eruterobin, discodermolds, bryostatins, drostatins, auristatins, tubulicins, cephalostatins; pancratistatin; sarcodictiin; spongstatin sugars; c) DNA topoisomerase inhibitors: 9-aminocamptothecin, camptothecin, cristinator, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoic acid (retinols), teniposide, topotecan, 9-nitrocamptothecin, or epipodophilins including RFS 2000; and mitomycins, and their analogues, etc. d) Antimetabolites: {[Antifolates: (e.g., DHFR inhibitors: methotrexate, trimethrexate, denopterin, pteropterin, aminopterin (4-aminopteroic acid), or other folate analogs); IMP dehydrogenase inhibitors (e.g., mycophenolic acid, thiazophrine, ribavirin, EICAR); ribonucleotide reductase inhibitors (e.g., hydroxyurea, deferoxamine)]; [Pyrimidine analogs: uracil analogs (e.g., Ancitabine, azacitidine, 6-azauridine, capecitabine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-fluorouracil, floxuridine, larcitrexed; cytosine analogs (e.g., cytarabine, cytosine arabinoside, fludarabine); purine analogs (e.g., azathioprine, fludarabine, mercaptopurine, thiamiprine, thioguanine); folic acid supplements such as folic acid; e) Hormone therapy agents: Receptor antagonists: [Anti-estrogens: (e.g., megestrol, raloxifene, tamoxifen); LHRH agonists: (including goserelin, leuprolide acetate); Antiandrogens: (e.g., bicalutamide, flutamide, carsterone, dromostanolone propionate, epithiostanol, goserelin, leuprolide, mepitiostane, nilutamide, testolactone, trilostane, and other similar androgens] (Dioxin inhibitors); Retinoids / Deltoid muscle: [e.g., Vitamin D3 analogs: (CB1093, EB1089, KH1060, cholecalciferol, ergocalciferol); Photodynamic therapy agents: (e.g., verteporfin, phthalocyanine, photosensitizer Pc4, demethoxyhypocrelin A); Cytokines: (e.g., interferon α, interferon γ, tumor necrosis factor (TNF), TNF domain-containing human proteins)], etc. f) Kinase inhibitors: BIBW2992 (anti-EGFR / Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080 (anti-VEGFR2), mbritinib, ponatinib, bafetinib, bosutinib, cabozantinib, bismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib sorafenib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, ispinesib, etc. g) Poly(ADP-ribose) polymerase (PARP) inhibitors such as olaparib, niraparib, iniparib, talazoparib, veliparib, CEP9722 (Cephalon), E7016 (Eisai), BGB-290 (Baygene), or 3-aminobenzamide; h) Endiyne antibiotics (e.g., calicheamicins, especially calicheamicin γ1, δ1, α1, or β1 (J. Med. Chem., 39(11), 2103-2117 (1996), Angew Chem) See Intl. Ed. Engl. 33:183-186 (1994); Dynemicins including dynemicin A and deoxydynemicin; selected from esperamicin, kedulcidin, C-1027, mazulopeptin, or neocardinostatin chromophores and related pigment protein enediin antibiotic chromophores; acrasinomycins, actinomycin, anthramycin, azaserin, bleomycins, cactinomycin, carabicin, carminomycin, cardinophilin; chromomycins, dactinomycin, daunorubicin Antibiotics such as detrubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, eribulin, esorubicin, idarubicin, marcelomycin, mitomycins, mycophenolic acid, nogaramycin, olibomycins, peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zolbicin; i) Others: Polyketides (acetogenins), especially bratacin and bratacinone; gemcitabine, epoxomicins (e.g., carfilzomib), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zyprestat, PLX4032, STA-9090, Stimuvax, allobectin-7, Zygeba, Provenge, Elboy, isoprenylation inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g., 1-methyl -4-phenylpyridine ion), cell cycle inhibitors (e.g., staurosporine), actinomycins (e.g., actinomycin D, dactinomycin), amanitins, bleomycins (e.g., bleomycin A2, bleomycin B2, peplomycin), anthracyclines (e.g., daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zolubicin), mitoxantrone, MDR inhibitors (e.g., verapamil), Ca 2+ATP inhibitors (e.g., thapsigargin), histone deacetylase inhibitors (including vorinostat, romidepsin, panobinostat, valproic acid, mosetinostat (MGCD0103), bellinostat, PCI-24781, entinostat, SB939, resminostat, gibinostat, AR-42, CUDC-101, sulforaphane, trichostatin A); thapsigargin, celecoxib, glitazones, epigallocatechin gallate, disulfiram, salinosporamide A, anti-adrenal drugs (selected from the group consisting of aminoglutethimide, mitotane, trilostampa); acegraton; aldofosphamide cricoside; aminolevulinic acid; amsacrine; arabinoside, bethrabusil; bisant Len; Edatrexate; Defofamine; Demecolsin; Diadicone; Elfornithine (DFMO), Elfomitin; Erliptinium acetate; Etocluside, Gallium nitrate, Gasitosine, Hydroxyurea; Ibandronate, Lentinan; Ronidamin; Mitoguazone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK®; Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadicone; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T2 toxin, Berkarin A, Loridine A, and Anguidin); Urethanes, siRNAs, Antisense drugs, and Nucleolytic enzymes;

[0231] 2) Anti-autoimmune drugs include, but are not limited to, cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticoids (e.g., amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, fluocortolone danazol, dexamethasone, triamcinolone acetonide, beclomethasone dipropionate), DHEA, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mofetil, mycophenolate, prednisone, sirolimus, and tacrolimus.

[0232] 3) Anti-infective drugs include, but are not limited to, the following: a) Aminoglycosides: Amikacin, Astromycin, Gentamycin (Netylmycin, Shisomycin, Isepamycin), Hygromycin B, Kanamycin (Amikacin, Arbekacin, Bekanamycin, Dibekacin, Tobramycin), Neomycin (Furamycin, Paromomycin, Ribostamycin), Netylmycin, Spectinomycin, Streptomycin, Tobramycin, Verdamicin; b) Amphenicols: Azidamphenicol, Chloramphenicol, Florphenicol, Thiamphenicol; c) Ansamycin derivatives: geldanamycin, harbimycin; d) Carbapenems: biapenem, doripenem, ertapenem, imipenem, cilastatin, meropenem, panipenem; e) Cephalosporins: Carbasephalm (loracalbef), cefacetril, cefaclor, cefradin, cefadroxil, cephalonium, cefaloridine, cephalothin or cephalothin, cephalexin, cephaloglysin, cephamandol, cefapillin, cefatoridine, cefazal, cefazedone, cefazolin, cefubperazone, cefcapene, cefdaroxime, cefepime, cefminox, cefoxitin, cefprodil, ceffloxazine, ceftezol, cefuroxime, cefixime, cefdinir, cefditoren, cefepime, ce Fetamet, cefmenoxime, cefozidime, cefonisid, cefoperazone, cefolanide, cefotaxime, cefotiam, cefozopran, cephalexin, cefpimisole, cefpyramide, cefpirome, cefpodoxime, cefprodil, cefquinome, cefsulodine, ceftazidime, cefteram, ceftibuten, cefthiolen, ceftizoxime, ceftobiprol, ceftriaxone, ceffuroxime, cefzonam, cephamycin (including cefoxitin, cefotetan, and cefmetazole), oxacepham (flomoxef, latamoxef); f) Glycopeptides: Bleomycin, vancomycin (including oritabancin and teravancin), teicoplanin (darbabancin), lamopranin; g) Glycylcyclines: e.g., tigecycline; h) β-lactamase inhibitors: Penam (sulbactam, tazobactam), Clavam (clavulanic acid); i) Lincosamides: clindamycin, lincomycin; j) Lipopeptides: Daptomycin, A54145, calcium-dependent antibiotics (CDA); k) Macrolides: Azithromycin, cesromycin, clarithromycin, dilithromycin, erythromycin, flurithromycin, josamycin, ketolides (telithromycin, cesromycin), midecamycin, myokamycin, oleandmycin, rifamycin (rifampicin, rifampin, rifabutin, rifapentin), rokitamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandmycin, telithromycin; l) Monobactams: Aztreonam, Tigemonam; m) Oxazolidinones: Linezolids; n) Penicillins: Amoxicillin, ampicillin, pivampicillin, hetacillin, bacampicillin, methampicillin, tarampicillin, azidocillin, azurocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethylpenicillin, clometocillin, procaine benzylpenicillin, carbenicillin (kalindacillin), cloxacillin, dicloxacillin, epicillin, flucloxacillin, mesilinum (pibmesilinum), mezurocillin, methicillin, nafcillin, oxacillin, penamecillin, penicillin, pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin, sulbenicillin, temocillin, ticalcillin; o) Polypeptides: bacitracin, colistin, polymyxin B; p) Quinolones: Alatrofloxacin, valofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, phloxin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, canotorobafloxacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q) Streptogramins: Pristinamycin, quinupristin / dalfopristin; r) Sulfonamides: mafenide, prontosil, sulfacetamide, sulfamethizol, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole (co-trimoxazole); s) Steroidal antibacterial agents: Selected from fusidic acid; t) Tetracyclines: doxycycline, chlortetracycline, chromocycline, demeclocycline, rimecycline, meclocycline, metacycline, minocycline, oxytetracycline, penimepicycline, lolitetracycline, tetracycline, glycylcycline (e.g., tigecycline); u) Other antibiotics: annonasin, arsphenamine, bactoprenol inhibitor (bacitracin), DADAL / AR inhibitor (cycloserine), dicthiostatin, discodermolide, eleuterobin, epothilon, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laurimalid, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitor (e.g., fosfomycin), nitrofurantoin, paclitaxel, platensimycin, pyrazinamide, quinupristin / dalfopristin, rifampicin (rifampin), tazobactamtinidazole, uvarcin;

[0233] 4) Antiviral drugs: a) Entry / fusion inhibitors: aplaviroc, maraviroc, bicriviroc, gp41 (enfuvirtide), PRO140, CD4 (ibalizumab); b) Integrase inhibitors: raltegravir, elvitegravir, globoidnan A; c) Maturation inhibitors: Bevirimat, Vivecon; d) Neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; e) Nucleosides and nucleotides: Abacavir, acyclovir, adefovir, amdoxovir, apricitabine, brivudine, cidofovir, klevudine, dexerbucitabine, didanosine (DDI), erbucitabine, emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU), and 3'-fluorosubstituted 2',3'-deoxynucleoside analogs (3'-fluoro-2',3'-dideoxythymidine (FLT) and 3'-fluoro-2',3'-dideoxyguanosine (FLG)). (including the group), homivirsen, ganciclovir, doxuridine, lamivudine (3TC), L-nucleosides (including the group consisting of β-L-thymidine and β-L-2'-deoxycytidine), penciclovir, lasivir, ribavirin, stampidine, stabidine set (d4T), taribavirin (viramidine), terbivudine, tenofovir, trifluridine, valacyclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f) Non-nucleosides: Amantadine, ateviridine, caplavirine, diallylpyrimidine (etravirine, rilpivirine), delaviridine, docosanol, emibilin, efavirenz, foscarnet (phosphorylformate), imiquimod, interferon α, roviride, rodenosine, methisazone, nevirapine, NOV-205, pegylated interferon α, podophyllotoxin, rifampicin, rimantadine, reciquimod (R-848), tromantadine; g) Protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, preconalil, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h) Other antiviral drugs: Abzyme, Arbidol, Caranolid A, Selagenin, Cyanobilin-N, Diallylpyrimidine, Epigallocatechin gallate (EGCG), Foscarnet, Griffiscin, Taribavirin (Pyramidine), Hydroxyurea, KP-1461, Miltefosin, Preconalil, Portmanto inhibitors, Ribavirin, Cericiclib.

[0234] 5) Radioisotopes used in radiotherapy. Examples of radioisotopes (radionuclides) are as follows: 3 H, 11 C, 14 C, 18 F, 32 P, 35 S, 64 Cu, 68 Ga, 86 Y, 99 Tc, 111 In, 123 I, 124 I, 125 I, 131 I, 133 Xe, 177 Lu, 211 At, or 213 Antibodies labeled with Bi (bicarbonate) radioisotopes are useful for receptor-targeted imaging experiments and can also be used in targeted therapies using antibody-radioisotope conjugates, etc. (Wu et al (2005) Nature Biotechnology 23(9):1137-46). Cell-binding molecules, such as antibodies, can be labeled with ligand reagents that bind to, chelate, or form complexes with radioisotope metals using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, New York, Pubs. (1991). Chelate ligands capable of complexing metal ions include DOTA, DOTP, DOTMA, DTPA, and TETA (Macrocyclics, Dallas, Tex. USA).

[0235] 6) Another cell-binding molecule-drug conjugate as a synergistic treatment. Preferred synergistic conjugates include tubulicin analogs, maytansinoid analogs, taxane analogs, CC-1065 analogs, daunorubicin and doxorubicin compounds, amanitaxin analogs, benzodiazepine dimers (e.g., pyrrolobenzodiazepine (PBD), tomaimycin, anthramycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinodibenzodiazepine dimers), calicheamicin and enediyne antibiotic compounds, actinomycin, azaserin, bleomycin, epirubicin, tamoxifen, idarubicin, drastatin, The company possesses cytotoxic agents such as auristatins (e.g., monomethyl auristatin E, MMAE, MMAF, auristatin PYE, auristatin TP, auristatin 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)), duocalmycin, geldanamycin, methotrexates, thiotepa, vindesines, vincristines, hemiasterines, nazmamides, microginines, radiosmins, topoisomerase I inhibitors, alterobactins, microclerodermines, theonellamides, esperamicins, PNU-159682, and their analogs and derivatives. It can be formed as a conjugate.

[0236] 7) Other immunotherapeutic agents: e.g., imiquimod, interferons (e.g., α, β), granulocyte colony-stimulating factors, cytokines, interleukins (IL-1 to IL-35), antibodies (e.g., trastuzumab, pertuzumab, bevacizumab, cetuximab, panitumumab, infliximab, adalimumab, basiliximab, d-omalizumab, PD-1 or PD-L1), protein-binding agents (e.g., Abraxane), antibodies conjugated with drugs selected from calicheamicin derivatives, May Tansine derivatives (DM1 and DM4), CC-1065, SN-38, exatecan, topotecan, topoisomerase I inhibitors, duocalmycin, PBD or IGN subgroove binders, potent taxol derivatives, doxorubicin, auristutuzumab antimitotics (e.g., trastuzumab-DM1, trastuzumab derquistecan (DS-8201a), inotuzumab ozogamicin, brentuximab vedotin, sacituzumab brolbotuzumab meltansine, AN-152) LMB2, TP-38, VB4-845, cantuzumab meltansine, AVE9633, SAR3419, CAT-8015 (anti-CD22), IMGN388, milbetuximab sorabtansine (IMGN853), enfortumab vedotin, milatuzuma (anti-CD70), anti-Her3-exetecan, anti-Trop2-exetecan, nnti-CD79b-MMAE, anti-Her2-MMAE, anti-trop2-MMAE, anti-Her2-MMAF, anti-trop2-MMAF, anti-CD22-calicheamicin derivative, anti-CD22-MMAE, anti-Her2-aulistatin derivative, anti-Muc1-aulistatin derivative, anti-cMet-aulistatin derivative, or anti-claudin-18,2-aulistatin derivative).

[0237] 8) A pharmaceutically acceptable salt, acid, or derivative of any of the above-mentioned drugs.

[0238] In other synergistic immunotherapies, checkpoint inhibitors, TCR (T cell receptor) T cells, or CAR (chimeric antigen receptor) T cells, or B cell receptor (BCR), natural killer (NK) cells, or cytotoxic antibody cells, or anti-CD3, CD4, CD8, CD16 (FcγRIII), CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD37, CD38, CD40, CD40L, CD45RA, CD45RO, CD56, CD57, CD57bright, CD70, CD79, CD79b, CD123, CD125, CD138, TNFβ, Fas ligand, MHC class I molecules (HLA-A, B, C), VEGF, or NKR-P1 antigen are preferably used in conjunction with the conjugates of this patent for synergistic therapy.

[0239] In yet another embodiment, a pharmaceutical composition comprising a therapeutically effective amount of a conjugate of formulas (I) to (IV), or any conjugate described throughout this patent, may be administered concurrently with other therapeutic agents such as chemotherapeutic agents, radiotherapy agents, immunotherapy agents, autoimmune deficiency agents, anti-infective agents, or other conjugates for the synergistic treatment or prevention of cancer, autoimmune diseases, or infections. The synergistic agent is preferably selected from one or more of the following drugs: abatacept, abemaciclib, abiraterone acetate, Abraxane, acetaminophen / hydrocodone, acalabrutinib, aducanumab, adalimumab, ADXS31-142, ADXS-HER2, afatinib dimalate, aldesleukin, alectinib, alemtuzumab, alitretinoin, adtrastuzumab emtansine, amphetamine / dextroamphetamine, anastrozole, aripiprazole, anthracycline, aripip Razole, atazanavir, atezolizumab, atorvastatin, averumab, axicaptagensiloleucel, axitinib, bellinostat, live BCG, bevacizumab, bexarotene, blinatumomab, bortezomib, bosutinib, brentukimab vedotin, brigatinib, budesonide, budesonide / formoterol, buprenorphine, cabazitaxel, cabozatinib, camatinib, capecitabine, carfilzomib, chimeric antigen receptor modified T (CAR-T) cells, celecoxib, ceritinib, cetuximab, tidamide, citric acid Rosporine, Cinacalcet, Crizotinib, Cobimetinib, Cosentyx, Crizotinib, CTL019, Dabigatran, Dabrafenib, Dacarbazine, Daclizumab, Dacomotinib, Dacomotinib, Daptomycin, Daratumumab, Darbepoetin alfa, Darunavir, Dasatinib, Deniroikin difutitox, Denosumab, Depacote, Dexlansoprazole, Dexmethylphenidate, Dexamethasone, Dignicap Cooling System, Dinutuximab, Doxycycline, Duloxetine, Dubellisib, Darumabumab, Ero Tuzumab, emtricibine / rilpivirine / tenofovir, disoproxil fumarate, emtricibine / tenofovir / efavirenz, enoxaparin, ensartinib, enzalutamide, epoetin alfa, erlotinib, esomeprazole, eszopiclone, etanercept, everolimus, exemestane, everolimus, exenatide ER, ezetimibe, ezetimibe / simvastatin, fenofibrate, filgrastim, fingolimod, fluticasone propionate, fluticasone / salmeterol, fluves Trant, Gaziba, Gefitinib, Glatiramer, Goserelin acetate, Icotinib, Imatinib, Ibritumomab tiucetan, Ibrutinib, Idelalisib, Ifosfamide, Infliximab, Imiquimod, ImmuCyst, ImmunoBCG, Iniparib, Insulin aspart, Insulin detemir, Insulin glargine, Insulin lispro, Interferon alpha, Interferon alpha-1b, Interferon alpha-2a, Interferon alpha-2b, Interferon beta, Interferon beta-1a, Interferon L-beta-1b, interferon-gamma-1a, lapatinib, ipilimumab, ipratropium bromide / salbutamol, ixazomib, Kanuma, lanreotide acetate, lenalidomide, lenariomid, lenvatinib mesylate, letrozole, levothyroxine, levothyroxine, lidocaine, linezolid, liraglutide, lisdexamfetamine, LN-144, loratinib, memantine, methylphenidate, metoprolol, mekinist, mericitabine / rilpivirine / tenofovir, modafinil, mometasone, Mycidac-C,Necitumumab, neratinib, nilotinib, niraparib, nivolumab, ofatumumab, obinutuzumab, olaparib, olmesartan, olmesartan / hydrochlorothiazide, omalizumab, omega-3 fatty acid ethyl ester, oncolin, oseltamivir, osimertinib, oxycodone, palbociclib, palivizumab, panitumumab, panobinostat, pazopa Nib, pembrolizumab, PD-1 antibody, PD-L1 antibody, pemetrexed, pertuzumab, pneumococcal conjugate vaccine, pomalidomide, pregabalin, ProscaVax, propranolol, quetiapine, rabeprazole, radium-223 chloride, raloxifene, raltegravir, ramucirumab, ranibizumab, regorafenib, ribociclib Rituximab, rivaroxaban, romidepsin, rosuvastatin, ruxolitinibrate, salbutamol, savolitinib, semaglutide, sevelamer, sildenafil, siltuximab, ciproisel-T, sitagliptin, sitagliptin / metformin, solifenacin, solanezumab, sonidegib, sorafenib, sunitinib, tacrolimus, tacrimus, tadalafil, tamoxifen, tafinlar, talimogenelaherparepvec, talazoparib, telaprevir, talazoparib, temozolomide, tensirolimus, tenofovir / emtricitabine, tenofovir disoproxil fumarate, testosterone gel, talidomide, TICE BCG, tiotropium bromide, tisagenlecleucel, toremifene, trametinib, trastuzumab, trabectin (ectenacidin 743), trametinib, tremelimumab, trifluridine / tipiracil, tretinoin, uro-BCG, ustekinumab, valsartan, veliparib, vandetanib, vemurafenib, venetoclax, vorinostat, ziv-aflibercept, and zostavax, as well as their analogues, derivatives, pharmaceutically acceptable salts, carriers, diluents or excipients, or combinations thereof.

[0240] For another purpose, the present invention also relates to a method for preparing the conjugate of the present invention. The conjugates and methods of the invention can be prepared by various methods well known to those skilled in the art. Tubulicin analogs used in the conjugates may also be synthesized, for example, by application or modification of the following methods, or by modifications understood by those skilled in the art. Appropriate modifications and substitutions are obvious to those skilled in the art, well known from the scientific literature, or readily available. In particular, such methods can be found in RCLarock, Comprehensive OrganiCTransformations, 2nd edition, Wiley-VCH Publishers, 1999.

[0241] In the reactions described below, reactive functional groups required for the final product, such as hydroxyl, amino, imino, thio, or carboxyl groups, may need to be protected to prevent them from participating in unwanted chemical reactions. Conventional protecting groups are used according to standard practice. For example, see PGWuts and TWGreene, Greene's Protective Groups in Organic Synthesis, Wi. See ley-Interscience; 4th edition (2006). Some reactions can be carried out in the presence of a base, acid, or suitable solvent. There are no particular limitations on the properties of the base, acid, and solvent used in these reactions; any base, acid, or solvent conventionally used in this type of reaction can be used, as long as it does not adversely affect the reaction. The reactions can be carried out over a wide temperature range. Typically, the reactions are carried out at -80°C to 150°C (more preferably room temperature to around 100°C). The time required for the reaction can be very long depending on many factors, especially the reaction temperature and the properties of the reagents. However, as long as the reactants are carried out under the preferred conditions described above, it is usually between 3 and 20 hours.

[0242] The reaction can be completed by conventional means. For example, the reaction product can be recovered by distilling off the solvent from the reaction mixture, or, if necessary, by distilling off the solvent from the reaction mixture, then pouring the residue into water, followed by extraction with a water-immiscible organic solvent and distillation off. Furthermore, if necessary, the product can be purified by recrystallization, reprecipitation, or various well-known chromatographic techniques, particularly silica gel column chromatography or preparative thin-layer chromatography. The synthesis of the tubulicin analogs and their conjugates of the present invention is shown in Figures 1 to 22.

[0243] Further examples of cell-binding molecules and potent tubulicin analogs have been shown, but are not limited by the description of the following examples in this specification. [Examples]

[0244] Experimental materials:

[0245] Mass spectra were acquired using a Bruker Esquire 3000 system. NMR spectra were recorded with a Bruker AVANCE 300 spectrometer. Chemical shifts were reported in ppm relative to TMS as an internal standard. Ultraviolet spectra were recorded with a Hitachi U1200 spectrophotometer. HPLC was performed using an Agilent 1100 HPLC system with a fraction collector and a tunable wavelength detector. Thin-layer chromatography was performed on Analtech GF silica gel TLC plates. Aminalic acid and its derivatives, as well as preloaded resins, are products of either Merck Chemicals International Co., Synthetech Co., Peptides International Inc., Chembridge International Co., or Sigma-Aldrich Co. Several conjugates, NHS ester / maleimide conjugates (AMAS, BMPS, GMBS, MBS, SMCC, EMCS, or Sulfo-EMCS, SMPB, SMPH, LC-SMCC, Sulfo-KMUS, SM(PEG)4, SM(PEG)6, SM(PEG)8, SM(PEG)12, SM(PEG)24); NHS ester / pyridyldithiol (SPDP, LC-SPDP or Sulfo-LC-SPDP, SMPT, Sulfo-LC-SMPT); NHS ester Tel / haloacetyl (SIA, SBAP, SIAB, or Sulfo-SIAB); NHS ester / diazirine (SDA, or Sulfo-SDA, LC-SDA, or Sulfo-LC-SDA, SDAD, or Sulfo-SDAD); maleimide / hydrazide (BMPH, EMCH, MPBH, KMUH); pyridyldithiol / hydrazide (PDPH); and isocyanate / maleimide (PMPI) were purchased from Thermo Fisher Scientific Co., and SPDB and SPP conjugates were prepared according to the reference (Cumber, A. et al, Bioconjugate Chem., 1992, 3, 397-401). T-DM1 and trastuzumab are from Genentec. All other chemicals or anhydrous solvents are from Sigma-Aldrich International or Aladdin Chemical (Shanghai) Ltd.

[0246] Example 1: Synthesis of di-tert-butyl-1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate [ka]

[0247] To a 150 ml DMF solution of hydrazine-1,2-dicarboxylate di-tert-butyl (8.01 g, 34.4 mmol), NaH (60% in oil, 2.76 g, 68.8 mmol) was added. After stirring at room temperature for 30 minutes, 2-bromoacetate-tert-butyl (14.01 g, 72.1 mmol) was added. The mixture was stirred overnight, quenched with methanol (3 ml), concentrated, diluted with RINKAN (100 ml) and water (100 ml), separated, and the aqueous layer was extracted with RINKAN (2 × 50 ml). The organic layers were combined, dried over MgSO4, filtered, evaporated, and purified by SiO2 column chromatography (RINKAN / hexane 1:5~1:3) to obtain the title compound as a colorless oil (12.98 g, 82% yield). MS ESI m / z;C 22 H 41N2O8[M+H] + Calculated value: 461.28, measured value: 461.40.

[0248] Example 2: Synthesis of 2,2'-(hydrazine-1,2-diyl)diacetic acid [ka]

[0249] To a 1,4-dioxane solution (40 ml) of 1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate di-tert-butyl (6.51 g, 14.14 mmol), HCl (12 M, 10 ml) was added. The mixture was stirred for 30 minutes, diluted with dioxane (20 ml) and toluene (40 ml), evaporated, and dried by co-evaporation with dioxane (20 ml) and toluene (40 ml) to obtain the title crude product (2.15 g, yield 103%, purity ~93%), which was used in the next step without further production. Used. MS ESI m / z;C4H9N2O4[M+H] + Calculated value: 149.05, measured value: 149.40.

[0250] Example 3. Synthesis of 2,2'-(1,2-bis((E)-3-bromoacryloyl)hydrazine-1,2-diyl)diacetic acid [ka]

[0251] (E)-3-bromoacryloyl bromide (5.01 g, 23.60 mmol) was added to a mixed solution of 2,2'-(hydrazine-1,2-diyl)diacetic acid (1.10 g, 7.43 mmol) in NaH2PO4 (0.1 M, 80 ml, pH 6.0) and THF (50 ml). The mixture was stirred for 6 hours, concentrated, and purified by elution with H2O / CH3CN (1:9) containing 3% formic acid by silica gel column chromatography to obtain the title compound (2.35 g, yield 77%, purity approximately 93%). MS ESI m / z;C 10H 11 Br2N2O6[M+H] + Calculated value: 412.89, measured value: 413.50.

[0252] Example 4; Synthesis of 2,2'-(1,2-bis((E)-3-bromoacryloyl)hydrazine-1,2-diyl)diacetyl chloride. [ka]

[0253] 2,2'-(1,2-bis((E)-3-bromoacryloyl)hydrazine-1,2-diyl)diacetic acid (210 mg, 0.509 mmol) was added to dichloroethane (15 ml), followed by (COCl)2 (505 mg, 4.01 mmol), and then 0.040 ml of DMF was added. After stirring at room temperature for 2 hours, the mixture was concentrated and dried by co-evaporation with dichloroethane (2 × 20 ml) and toluene (2 × 15 ml) to obtain the title crude product (unstable) (245 mg, yield 107%), which was used in the next step without further purification. MS ESI m / z;C 10 H9Br2Cl2N2O4[M+H] + Calculated values: 448.82, 450.82, 452.82, 454.82; Measured values: 448.60, 450.60, 452.60, 454.60.

[0254] Example 5: Synthesis of tert-butyl 2,8-dioxo-1,5-oxazocane-5-carboxylate [ka]

[0255] At 4°C, 3,3'-((tert-butoxycarbonyl)azandiyl)dipropanoic acid was added over 1 hour to a 1.0 M NaOH solution (300 mL) of 3,3'-azandiyldipropanoic acid (10.00 g, 62.08 mmol) of di-tert-butyl dicarbonate (22.10 g, 101.3 mmol) of THF solution (200 mL). After addition, the reaction mixture was stirred at 4°C for 2 hours. The reaction mixture was carefully acidified to pH 4 with 0.2 M H3PO4, concentrated under vacuum, extracted with CH2Cl2, dried with Na2SO4, evaporated, and prepared by flash SiO2 chromatography using AcOH / MeOH / CH2Cl2 (0.01:1:5) to obtain 3,3'-((tert-butoxycarbonyl)azandiyl)dipropanoic acid (13.62 g, yield 84%). ESI MS m / z;C 11 H 19 NO6[M+H] + Calculated value: 267.27, measured value: 262.40.

[0256] At 0°C, phosphorus pentoxide (8.70 g, 61.30 mmol) was added to a CH2Cl2 solution (500 ml) of 3,3'-((tert-butoxycarbonyl)azandiyl)dipropanoic acid (8.0 g, 30.6 mmol). The mixture was stirred at 0°C for 2 hours, then stirred at room temperature for 1 hour. The mixture was filtered through a short SiO2 column, and the column was rinsed with Â1 / CH2Cl2 (1:6). The filtrate was concentrated and ground with Â1 / Hexane to obtain the title compound (5.64 g, 74% yield). ESI MS m / z;C 11 H 17 NO5 [M+H] + Calculated value: 244.11, measured value: 244.30.

[0257] Example 6; Synthesis of 2,5-dioxopyrrolidine-1-ylpropiolate [ka]

[0258] Propiolic acid (5.00 g, 71.4 mmol), NHS (9.01 g, 78.3 mmol), and EDC (20.0 g, 104.1 mmol) in CH2Cl2 (150 ml) and DIPEA (5 ml, 28.7 mmol) were stirred overnight, evaporated, and purified by SiO2 column chromatography (SiO2 / hexane 1:4) to obtain the title compound as a colorless oil (9.30 g, yield 79%). 1 H NMR (500MHz, CDCl3) δ2.68(s,1H), 2.61(s,4H). MS ESI m / z;C7H5NaNO4[M+Na] + ;190.02, measured value 190.20.

[0259] Example 7: Synthesis of tert-butyl 2-propioloylhydrazine carboxylate [ka]

[0260] Propiolic acid (5.00 g, 71.4 mmol), tert-butylhydrazine carboxylate (9.45 g, 71.5 mmol), and EDC (20.0 g, 104.1 mmol) in CH2Cl2 (150 ml) and DIPEA (5 ml, 28.7 mmol) were stirred overnight, evaporated, and SiO2 column chromatography (SiO2 / hexane) was performed. The title compound was purified in a 1:5 ratio to obtain a colorless oil (7.92 g, yield 84%). 1 H NMR (500MHz, CDCl3) δ8.76 (m, 2H), 2.68 (s, 1H), 1.39 (s, 9H). MS ESI m / z;C5H 12 NaN2O2[M+Na] + Calculated value: 155.09, measured value: 155.26.

[0261] Example 8: Synthesis of propiorohydrazide HCl salt [ka]

[0262] At 4°C, tert-butyl 2-propioloylhydrazine carboxylate (4.01 g, 30.35 mmol) dissolved in 1,4-dioxane (12 mL) was treated with 4 mL of concentrated HCl. The reaction mixture was stirred for 30 minutes, diluted with dioxane (30 mL) and toluene (30 mL), and concentrated under vacuum. Using a mixture of methanol (5%-10%) and 1% formic acid in methylene chloride as the eluent, the crude mixture was purified with silica gel to obtain the title compound (2.11 g, yield 83%). ESI MS m / z;C3H5N2O[M+H] + Calculated value 85.03, measured value 85.30.

[0263] Example 9; Synthesis of Compound 2 [ka]

[0264] In a 10 L reactor at room temperature, 2,2-diethoxyacetonitrile (1.00 kg, 7.74 mol, 1.0 equivalent) was mixed with (NH4)2S (48% aqueous solution, 1.41 kg, 9.29 mol, 1.2 equivalents) in methanol (6.0 L). The internal temperature rose to 33°C and then returned to room temperature. After stirring overnight, the reaction mixture was concentrated under vacuum, and the residue was transferred to ethyl acetate (5 L) and washed with saturated NaHCO3 solution (4 × 1.0 L). The aqueous layer was separated into acetic acid. Back-extraction was performed with ethyl acetate (5 × 1.0 L). The organic layers were combined, washed with brine (3 L), dried over anhydrous sodium 2SO4, and concentrated. The resulting solid was collected by vacuum filtration and washed with petroleum ether. The filtrate was concentrated and ground over petroleum ether to obtain a white or pale yellow solid harvest. All harvests were combined to obtain 1.1 kg of the desired product (yield 87%). 1 H NMR (500MHz, CDCl3) δ7.81(d,J=71.1Hz,2H),5.03(s,1H),3.73(dq,J=9.4,7.1Hz,2H),3.64(dq,J=9.4,7.0Hz,2H),1.25(t,J=7.1Hz,6H).

[0265] Example 10: Synthesis of Compound 3 [ka]

[0266] Ethyl bromopyruvate (80% purity, 404 mL, 2.57 mol, 1.2 equivalents) was added within 30 minutes to a 5 L three-necked round-bottom flask equipped with a reflux concentrator and an additional funnel. Molecular sieve (3A, 500 g) and thioamide (350 g, 2.1) were then added to 3 L EtOH. It was added to a mixture of 4 mol (1.0 equivalent). During the addition, the internal temperature rose slightly. Next, the reaction mixture was heated under reflux and stirred for 30 minutes. After cooling to room temperature, the reaction mixture was filtered through Celite and the filter cake was washed with ethyl acetate. The filtrate was concentrated under vacuum. Two batches of the crude compound were combined and mixed with silica gel (1.5 kg), loaded onto a silica gel (10 kg pack) column, and eluted with ethyl acetate / petroleum ether (10-20%) to obtain thiazole carboxylate as a brown oily substance (509 g, 92% yield).

[0267] Example 11; Synthesis of Compound 4 [ka]

[0268] A 3.0 L acetone solution of acetal (300 g, 1.16 mol) was heated under reflux, and 250 mL of 4N HCl was added over 1.0 hour. TLC analysis showed that the raw materials were completely consumed. The reaction mixture was concentrated under reduced pressure, and the phases were separated. The organic phase was diluted with ethyl acetate (1.5 L), washed with saturated sodium bicarbonate solution (1.0 L), water (1.0 L), and brine (1.0 L), and dried over anhydrous Na₂SO₄. All aqueous phases were combined and extracted with ethyl acetate. The extracts were combined and dried over anhydrous Na₂SO₄. The organic solution was filtered and concentrated under reduced pressure. The crude product was ground with petroleum ether and diethyl ether (5:1), the resulting solid was collected by vacuum filtration, and washed with petroleum ether and ethyl acetate (10:1). The filtrate was concentrated and subjected to chromatography with 0-15% ethyl acetate / petroleum ether to obtain another batch of the desired compound. All were white to pale yellow. We collected the colored solids (40g, 43% yield). 1 H NMR (500MHz, CDCl3) δ10.08-10.06(m,1H),8.53-8.50(m,1H),4.49(q,J=7.1Hz,2H),1.44(t,J=7.1Hz,3H). MS ESI m / z;C7H8NO3S[M+H] + Calculated value: 186.01; Measured value: 186.01.

[0269] Example 12; Synthesis of Compound 6 [ka]

[0270] NaN3 (740 g, 11.4 mol) was dissolved in water (2.0 L), dichloromethane (2.0 L) was added, and the mixture was cooled to 0°C. Tf2O (700 mL, 4.10 mol, 1.8 equivalents) was added over 1.5 hours. After the addition was complete, the reaction mixture was stirred at 0°C for 3 hours. The organic phase was separated, and the aqueous phase was extracted with dichloromethane (2 × 500 mL). The combined organic phase was washed with saturated sodium bicarbonate solution (3 × 1.0 L). At room temperature, a dichloromethane solution of this trifuryl azide was added to a mixture of (L)-isoleucine (300 g, 2.28 mol, 1.0 equivalent), K2CO3 (472 g, 3.42 mol, 1.5 equivalent), and CuSO4·5H2O (5.7 g, 22.8 mmol, 0.01 equivalent) in water (3.0 L) and methanol (3.0 L). During the addition, the internal temperature rose slightly. The mixture was then stirred at room temperature for 16 hours. The organic solvent was removed under reduced pressure, the aqueous phase was acidified to pH 6-6.5 with concentrated HCl (approximately 280 mL), then diluted with phosphate buffer (0.25 M, pH 6.2, 6.0 L), washed with HCl (6 × 2.0 L) to remove by-product sulfonamide compounds. The solution was acidified to pH 3 with concentrated HCl (approximately 400 mL added) and extracted with HCl (4 × 2.0 L). The combined organic layers were washed with brine (2.0 L), dried over anhydrous Na2SO4, filtered, and concentrated to obtain compound 6 as a pale yellow oil (320 g, yield 89%). 1 H NMR(500MHz,CDCl3)δ12.01(s,1H),3.82(d,J=5.9Hz,1H),2.00(ddd,J=10.6,8.6,5.5Hz,1H) ,1.54(dqd,J=14.8,7.5,4.4Hz,1H),1.36-1.24(m,1H),1.08-0.99(m,3H),0.97-0.87(m,3H).

[0271] Example 13; Synthesis of Compound 10 [ka]

[0272] Under nitrogen at room temperature, (S)-2-methylpropane-2-sulfinamide (100 g, 0.825 mol, 1.0 equivalent) was dissolved in THF (1 L), to which Ti(OEt)4 (345 mL, 1.82 mol, 2.2 equivalents) and 3-methyl-2-butanone (81 mL, 0.825 mol, 1.0 equivalent) were added. The reaction mixture was refluxed for 16 hours, then cooled to room temperature, and ice was added. The mixture was poured into water (1 L). The mixture was filtered, and the filter cake was washed with dimethyl acetate. The organic layer was separated, dried over anhydrous Na2SO4, concentrated to obtain the residue, which was purified by vacuum distillation (15-20 torr, 95°C) to obtain compound 10 as a yellow oil (141 g, 90% yield). 1 H NMR (500MHz, CDCl3) δ2.54-2.44 (m, 1H), 2.25 (s, 3H), 1.17 (s, 9H), 1.06 (dd, J=6.9, 5.1Hz, 6H). MS ESI m / z;C9H 19 NaNOS[M+Na] + Calculated value: 212.12; Measured value: 212.11.

[0273] Example 14; Synthesis of Compound 11 [ka]

[0274] Under an N2 atmosphere, at -78°C, n-butyllithium (2.5M, 681mL, 1.70mol, 1.5 equivalents) was added to a 1L solution of diisopropylamine (264mL, 1.87mol, 1.65 equivalents) in anhydrous THF. The reaction mixture was warmed to 0°C for 30 minutes and then cooled to -78°C. Compound 10 (258g, 1.36mol, 1.2 equivalents) was added and washed with THF (50mL). After stirring the reaction mixture for 1 hour, ClTi(OiPr)3 (834g, 3.17mol, 2.8 equivalents) in THF (1.05L) was added dropwise. After stirring for 1 hour, compound 4 (210g, 1.13mol, 1.0 equivalent) dissolved in THF (500mL) was added dropwise over approximately 1 hour, and the resulting reaction mixture was stirred for 3 hours. TLC analysis showed that the reaction was complete. The reaction was quenched with a mixture of acetic acid and THF (v / v 1:1, 300 mL), then poured into brine (2 L), and extracted with dimethyl acetate (8 × 1 L). The organic phase was washed with water and brine, dried over anhydrous sodium 2 SO4, filtered, and concentrated. The residue was purified by column chromatography (DCM / dimethyl acetate / PE 2:1:2) to obtain compound 11 as a colorless oil (298 g, yield 74%). 1 H NMR(500 MHz, CDCl3)δ8.13(s,1H),6.63(d,J=8.2Hz,1H),5.20-5.11(m,1H),4.43(q,J=7.0Hz,2H),3.42- 3.28(m,2H),2.89(dt,J=13.1,6.5Hz,1H),1.42(t,J=7.1Hz,3H),1.33(s,9H),1.25-1.22(m,6H). MS ESI m / z;C 16 H 26 NaN2O4S2[M+Na] + Calculated value: 397.13, measured value: 397.11.

[0275] Example 15; Synthesis of Compound 12 [ka]

[0276] A solution of compound 11 (509 g, 1.35 mol, 1.0 equivalent) dissolved in THF (200 mL) was cooled to -78 °C, and Ti(OEt)4 (570 mL, 2.72 mol, 2. (0 equivalents) was slowly added. After the addition was complete, the mixture was stirred for 1 hour, and then NaBH Compound 4 (51.3 g, 1.36 mol, 1.0 equivalent) was added gradually over 90 minutes. The reaction mixture was stirred at -78°C for 3 hours. TLC analysis showed that the starting material was still present. EtOH (50 mL) was slowly added, and the reaction mixture was stirred for 1.5 hours. Then, the mixture was poured into brine (2 L; containing 250 mL of HOAc) and warmed to room temperature. After filtration through Celite, the organic phase was separated, washed with water and brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (siRNA / PE 1:1) to obtain compound 12 as a white solid (364 g, 71% yield). 1 H NMR(500MHz,CDCl3)δ8.10(s,1H),5.51(d,J=5.8Hz,1H),5.23-5.15(m,1H),4.41(q,J=7.0Hz,2H),3.48-3.40(m,1H),3.37(d,J=8.3Hz,1H),2 .29(t,J=13.0Hz,1H),1.95-1.87(m,1H),1.73-1.67(m,1H),1.40(t,J=7.1Hz,3H),1.29(s,9H),0.93(d,J=7.3Hz,3H),0.90(d,J=7.2Hz,3H). MS ESI m / z;C 16 H 28 NaN2O4S2[M+Na] + Calculated value: 399.15, measured value: 399.14.

[0277] Example 16; Synthesis of Compound 13 [ka]

[0278] At 0°C, 4N HCl in dioxane (590 mL) was slowly added to an ethanol solution (590 mL) of compound 12 (600 g, 1.60 mol, 1.0 equivalent). The reaction mixture was warmed to room temperature and stirred for 2.5 hours. A white precipitate formed, which was collected by filtration and washed with ethyl acetate. The filtrate was concentrated and ground with ethyl acetate. Two white solids were combined, weighing 446 g (90% yield).

[0279] Example 17; Synthesis of Compound 14 [ka]

[0280] Compound 10: Azide-Ile-OH (6,153 g, 0.97 mol, 2.0 equivalents) was dissolved in THF (1.5 L), cooled to 0°C, and NMM (214 mL, 1.94 mol, 4.0 equivalents) and chloroformate isobutyl (95 mL, 0.73 mol, 2.0 eq) were added sequentially. The reaction mixture was stirred at 0°C for 1.0 hour. Compound 13 (150 g, 0.49 mmol, 1.0 equivalent) was added in batches. After stirring at 0°C for 30 minutes, the reaction mixture was warmed to room temperature and stirred for 2 hours. The reaction was quenched by adding water at 0°C, and the resulting mixture was extracted three times with HCl. The combined organic layer was treated with 1N HCl, saturated NaHCO3, and lye. The solution was washed, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (0-30% ÃO / PE) to obtain a white solid (140 g, 70% yield). 1 H NMR(500MHz,CDCl3)δ8.14(s,1H),6.57(d,J=8.9Hz,1H),4.91(d,J=11.1Hz,1H),4.44(dd,J=13.2,6.3Hz,2H),4.08-3.95(m,2H),2 .21(dd,J=24.4,11.5Hz,2H),1.90-1.79(m,3H),1.42(t,J=6.6Hz,3H),1.37-1.27(m,2H),1.11(d,J=6.4Hz,3H),1.01-0.94(m,9H). MS ESI m / z;C 18 H30 N5O4S[M+H]+; Calculated value 412.19, Measured value 412.19.

[0281] Example 18; Synthesis of Compound 15 [ka]

[0282] Compound 11: To a CH2Cl2 solution (50 mL) of Compound 14 (436 g, 1.05 mol, 1.0 equivalent), imidazole (94 g, 1.37 mmol, 1.3 equivalents) was added, followed by chlorotriethylsilane (222 mL, 1.32 mol, 1.25 equivalents) at 0°C. The reaction mixture was warmed to room temperature over 1 hour and stirred for an additional time. Brine was added to the reaction mixture, the organic layer was separated, and the aqueous layer was extracted with RINKAN. The combined organic phases were dried, filtered, concentrated under reduced pressure, and purified by column chromatography with elution under a 15–35% RINKAN gradient in petroleum ether to obtain Compound 15 as a colorless oil (557.4 g, 95% yield). 1 H NMR(500MHz,CDCl3)δ8.12(s,1H),6.75(d,J=8.0Hz,1H),5.20-5.12(m,1H),4.44(q,J=7.0Hz,2H),4.06-3.97(m,1H),3.87(d,J=3.8Hz,1H),2.14(d ,J=3.8Hz,1H),2.01-1.91(m,3H),1.42(t,J=7.1Hz,3H),1.34-1.25(m,2H ),1.06(d,J=6.8Hz,3H),1.00-0.93(m,18H),0.88(dd,J=19.1,6.8Hz,6H). MS ESI m / z;C 24 H 44 N5O4SSi[M+H] + Calculated value: 526.28, measured value: 526.28.

[0283] Example 19; Synthesis of Compound 16 [ka]

[0284] At 0°C, sodium hydride (62.2 g, 1.55 mol, 2.0 equivalents, 60% mineral oil dispersion) was added to a THF solution (4 L) of compound 15 (408 g, 0.77 mol, 1.0 equivalent) and methyl iodide (145 mL, 2.32 mol, 3.0 equivalents). The mixture was stirred overnight at 0°C, and then poured into saturated ammonium chloride (5 L) cooled with ice water while stirring vigorously. The mixture was then extracted with toluene (3 × 500 mL), the organic layer was dried, filtered, concentrated, and purified by column chromatography with elution at a gradient of 15–35% toluene in petroleum ether to obtain compound 16 as a pale yellow oil (388 g, 93%). 1 H NMR(500MHz,CDCl3)δ8.09(s,1H),4.95(d,J=6.6Hz,1H),4.41(q,J=7.1Hz,2 H),3.56(d,J=9.5Hz,1H),2.98(s,3H),2.27-2.06(m,4H),1.83-1.70(m,2H), 1.41(t,J=7.2Hz,3H),1.29(ddd,J=8.9,6.8,1.6Hz,3H),1.01(d,J=6.6Hz,3H ),0.96(dt,J=8.0,2.9Hz,15H),0.92(d,J=6.6Hz,3H),0.90(d,J=6.7Hz,3H). MS ESI m / z;C 25 H 46 N5O4SSi[M+H] + Calculated value: 540.30, measured value: 540.30.

[0285] Example 20: Synthesis of Compound 17 [ka]

[0286] 0.1N HCl was continuously added dropwise to a methanol solution (15 mL) of compound 16 (1.01 g, 1.87 mmol) until a neutral pH was reached. After adding Pd / C (10 wt%, 583 mg), the mixture was stirred at room temperature under H2 (1 atm) for 16 hours. Next, Pd / C was removed by filtration, and the filter pad was washed with methanol. The filtrate was concentrated under reduced pressure, the residue was redissolved in RINKAN (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated to obtain compound 17 as a pale yellow oil (900 mg, yield 94%).

[0287] Example 21; Synthesis of Compound 22 [ka]

[0288] To a methanol solution (100 mL) of D-piperic acid (10.0 g, 77.4 mmol, 1.0 eq), formaldehyde (37% aqueous solution, 30.8 mL, 154.8 mmol, 2.0 equivalents) was added, followed by the addition of Pd / C (10% wt, 1.0 g). The reaction mixture was stirred overnight with H2 (1 atm), then filtered through Celite, and the filter pad was washed with methanol. The filtrate was concentrated under reduced pressure to obtain compound 22 as a white solid (10.0 g, 90% yield).

[0289] Example 22; Synthesis of Compound 23 [ka]

[0290] Pentafluorophenol (3.75 g, 20.4 mmol) and DCC (4.21 g, 20.4 mmol) were added to a 50 mL siRNA solution of DN-methylpipecolic acid (2.65 g, 18.5 mmol). The reaction mixture was stirred at room temperature for 16 hours and filtered through Celite. The filter pad was washed with 10 mL of siRNA. The filtrate was used immediately without further purification or concentration.

[0291] Example 23; Synthesis of Compound 28 [ka]

[0292] A mixture of 2-amino-2-methylpropionic acid (500 g, 4.85 mol, 1.0 equivalent), aqueous formaldehyde (37%, 1.0 L, 12.1 mol, 2.5 equivalents), and formic acid (1.0 L) was heated under reflux (80°C) for 3.0 hours, and then 6N HCl (850 mL) was added at room temperature. The reaction mixture was concentrated. The resulting solid was collected by filtration and washed three times with ethyl acetate (1.0 L). The solid was dissolved in water (1.5 L) and neutralized to pH 7.0 with 4N NaOH (approximately 1.0 L solution). The solution was concentrated and co-evaporated with ethanol (2.0 L) to remove residual water. MeOH (2.0 L) was added to the residue and washed with ethyl acetate, and the solid (NaCl) was filtered off. The filtrate was concentrated under reduced pressure to obtain 639.2 g of a white solid containing some NaCl, which was used without further processing.

[0293] Example 24; Synthesis of Compound 29 [ka]

[0294] Pentafluorophenol (163 g, 0.88 mol) and DIC (126 mL, 0.81 mol) were added to 1 L of HCl solution of 2-(dimethylamino)-2-methylpropionic acid (97 g, 0.74 mol). The reaction mixture was stirred at room temperature for 24 hours and then filtered through Celite. The filter pad was washed with 10 mL of ethyl acetate. The filtrate was used immediately without further purification or concentration.

[0295] Example 25; Synthesis of Compound 30 [ka]

[0296] Dried Pd / C (10 wt%, 300 mg) and azide compound 16 (3.33 g, 6.61 mmol) were added to pentafluorophenyl ester 23 in ethyl acetate. The reaction mixture was stirred under hydrogen for 27 hours, then filtered through a Celite plug, and the filter pad was washed with ethyl acetate. The combined organic fraction was concentrated and purified by column chromatography using a gradient of 0-5% methanol in ethyl acetate to obtain compound 30 (3.90 g, 86% yield). MS ESI m / z;C 32 H 59 N4O5SSi[M+H] + Calculated value: 639.39, measured value: 639.39.

[0297] Example 26; Synthesis of Compound 31 [ka]

[0298] Coupling product compound 30 (3.90 g, 6.1 mmol) was dissolved in AcOH / water / THF (v / v / v 3:1:1, 100 mL) and stirred at room temperature for 48 hours. Next, the reaction product was concentrated and purified by column chromatography (2:98~15:85 MeOH / siRNA) to obtain compound 31 (2.50 g, 72% yield in two steps). MS ESI m / z;C 26 H 45 N4O5S[M+H] + Calculated value: 525.30, measured value: 525.33.

[0299] Example 27; Synthesis of Compound 32 [ka]

[0300] Dioxane solution of compound 31 (2.50 g, 4.76 mmol, 1.0 equivalent) at 0°C. To (47.7 mL), an aqueous solution of LiOH (0.4 N, 47.7 mL, 19.1 mmol, 4.0 equivalents) was added. The reaction mixture was stirred at room temperature for 2 hours and then concentrated. The mixture was purified by column chromatography (100% CH2Cl2 to CH2Cl2 / MeOH / NH4OH 80:20:1) to obtain compound 32 as an amorphous solid (2.36 g, 99% yield). MS ESI m / z;C 24 H 41 N4O5S[M+H] + Calculated value: 497.27, measured value: 497.28.

[0301] Example 28; Synthesis of Compound 33 [ka]

[0302] At 0°C, acetic anhydride (2.25 mL, 24 mmol) was slowly added to a pyridine solution (50 mL) of compound 32 (2.36 g, 4.75 mmol). The reaction mixture was warmed to room temperature over 2 hours and stirred at room temperature for 24 hours. The reaction mixture was concentrated and then treated with dioxane / water (v / v 1:1, 10 mL) for 1 hour to destroy any anhydrides that may be formed. After concentration, the residue was purified by column chromatography (100% CH2Cl2 to CH2Cl2 / MeOH / NH4OH 50:50:1) to obtain compound 33 as an amorphous white solid (2.25 g, yield 88%). MS ESI m / z;C 26 H 43 N4O6S[M+H] + Calculated value: 539.28, measured value: 539.28.

[0303] Example 29; Synthesis of Compound 38 [ka]

[0304] Compound 16 (200 g, 0.37 mol) and dried Pd / C (10 wt%, 10 g) were added to a solution of pentafluorophenyl ester 29 with ethyl acetate. The reaction mixture was stirred under a hydrogen atmosphere (1 atm) for 27 hours, then filtered through a Celite plug, and the filter pad was washed with ethyl acetate. The combined organic fraction was concentrated and purified by column chromatography using a gradient of 0-5% methanol in ethyl acetate to obtain compound 38 (184 g, 79% yield). MS ESI m / z;C 31 H 58 N4O5SSi[M+H] + Calculated value: 627.39, measured value: 627.39.

[0305] Example 30; Synthesis of Compound 39 [ka]

[0306] Compound 38 (200 g, 0.32 mmol) was dissolved in AcOH / water / THF (v / v / v 3:1:1, 638 mL) and stirred at room temperature for 4 days. After concentrating the reaction mixture, toluene was added and the mixture was concentrated again. This process was repeated twice to obtain compound 39, which was used directly in the next step. MS ESI m / z;C 25 H 45 N4O5S[M+H] + Calculated value: 513.30, measured value: 513.30.

[0307] Example 31; Synthesis of Compound 40 [ka]

[0308] At 0°C, a solution of compound 39 (160 g, 0.319 mol, 1.0 equivalent) in MeOH (1.2 L) was mixed with an aqueous LiOH solution (0.4 N, 600 mL, 2.55 mol, 8.0 equivalents). The reaction mixture was stirred at room temperature for 2 hours and then concentrated. Compound 40 was obtained as an amorphous solid by column chromatography (80:20:1 CH2Cl2 / MeOH / NH4OH from pure CH2Cl2) (140 g, 91% yield in two steps). MS ESI m / z;C 23 H 40 N4O5S[M+H] + Calculated value: 485.27, measured value: 485.27.

[0309] Example 32; Synthesis of Compound 41 [ka]

[0310] Solutions of compound 27 (143 g, 0.30 mol, 1.0 equivalent) and DMAP (0.36 g, 2.95 mmol, 0.01 equivalent) in anhydrous THF (1.4 L) and anhydrous DMF (75 mL) were cooled to 0°C, and added to TEA (82.2 mL, 0.59 mmol, 2.0 equivalents) and acetic anhydride (56 mL, 0.59 mmol, 2.0 equivalents). The reaction mixture was warmed to room temperature, stirred for 24 hours, and then concentrated. Column chromatography (5-50% MeOH / ) was performed. Compound 41 was obtained as an amorphous solid by DCM (147 g, 95% yield). MS ESI m / z;C 25 H 44 N4O6S[M+H] + Calculated value: 527.28, measured value: 527.28.

[0311] Example 33; Synthesis of Compound 41a [ka]

[0312] Under an N2 atmosphere at room temperature, compound 41 (5.0 g, 9.5 mmol, 1.0 equivalent) was dissolved in anhydrous DCM (100 mL), to which EDC (4.6 g, 23.8 mmol, 2.5 equivalents) and pentafluorophenol (4.4 g, 23.8 mmol, 2.5 equivalents) were added. The mixture was stirred at room temperature for 2 hours, then diluted with DCM (100 mL), washed with water (2 × 200 mL) and brine (200 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by SiO2 column chromatography (50% Â / PE) to obtain compound 41 as a white solid (5.2 g, yield 79%). MS ESI m / z;C 31 H 42 F5N4O6S[M+H] + Calculated value: 693.27, Measured value: 693.27.

[0313] Example 34; Synthesis of Compound 95 [ka]

[0314] Triphenylphosphine (100 g, 381 mmol, 1.0 equivalent) and ethyl 2-bromopropionate (100 mL, 762 mmol, 2.0 equivalents) were added to a 500 mL round-bottom flask equipped with a magnetic stirring rod. The mixture was then heated to 50°C overnight under an N2 atmosphere. After the dissolution of the white solid (PPh3), a large amount of white solid was produced. The mixture was ground with petroleum ether / siRNA and filtered to obtain compound 95 as a white solid (135 g, 80% yield). MS ESI m / z;C 23 H 24 O2P[M-Br] + Calculated value: 363.15, measured value: 363.13.

[0315] Example 35; Synthesis of Compound 96 [ka]

[0316] While vigorously stirring, a 500 mL solution of compound 95 (135.42 g, 305.7 mmol) in dichloromethane was slowly added to a 450 mL solution of 10% NaOH. The organic solution immediately turned bright yellow. After 30 minutes, TLC analysis indicated that the reaction was complete. The layers were separated, and the aqueous layer was further extracted with CH2Cl2 (2 × 200 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated to obtain compound 96 as a yellow solid (104 g, 94% yield). MS ESI m / z;C 23 H 24 O2P[M+H] + Calculated value: 362.14, measured value: 363.13.

[0317] Example 36; Synthesis of Compound 98 [ka]

[0318] A mixture of Boc-L-Tyr-OMe (670 g, 2.27 mol, 1.0 equivalent), K2CO3 (358 g, 2.5 mol, 1.1 equivalent), and KI (38 g, 0.227 mol, 0.1 equivalent) in acetone (3 L) was slowly mixed with benzyl bromide (283 mL, 2.38 mol, 1.05 equivalent). The mixture was then refluxed overnight. Water (6 L) was added, and the reaction mixture was extracted with RINKAN (5 × 100 L). The combined organic layer was washed with brine (2 L), dried over anhydrous Na2SO4, filtered, concentrated, and purified by SiO2 column chromatography (4:1 hexane / RINKAN) to obtain chemical 98 as a white solid (795 g, 91% yield). 1 H NMR(500MHz,CDCl3)δ7.43(d,J=7.0Hz,2H),7.38(t,J=7.4Hz,2H),7.32(t,J=7.2Hz,1H),7.04(d,J=8.5Hz,2H),6 .91(d,J=8.6Hz,2H),5.04(s,2H),4.55(d,J=6.9Hz,1H),3.71(s,3H),3.03(qd,J=14.0,5.8Hz,2H),1.43(s,9H). ESI:m / z:C 22 H28 NO5 [M+H] + Calculated value: 386.19, measured value: 386.19.

[0319] Example 37; Synthesis of Compound 99 [ka]

[0320] At -78°C, DIBAL (1.0 M in hexane, 2.9 L, 2.9 equivalents) was added over 3 hours to a 1 L solution of anhydrous dichloromethane containing ester 98 (380 g, 987 mmol, 1.0 equivalent). After the addition was complete, the mixture was quenched with 3 L of ethanol. 1 N HCl was added dropwise until the pH reached 4. The resulting mixture was heated to 0°C. The layers were separated, and the aqueous layer was further extracted with SiO2 (3 × 3 L). The combined organic solution was washed with brine, dried over anhydrous Na2SO4, and concentrated. It was ground with PE / SiO2, filtered, and 99 was obtained. It was obtained as a white solid (263g, 75% yield). 1 H NMR(500MHz,CDCl3)δ9.65(s,1H),7.45(d,J=7.1Hz,2H),7.41(t,J=7.4Hz,2H),7.35(t,J=7.1Hz,1H),7.11(d,J= 8.6Hz,2H),6.95(d,J=8.6Hz,2H),5.07(s,2H),4.42(dd,J=12.4,6.1Hz,1H),3.09(d,J=6.2Hz,2H),1.46(s,9H). ESI:m / z:C 21 H 26 NO4 [M+H] + Calculated value: 356.18, measured value: 356.19.

[0321] Example 38; Synthesis of Compound 100 [ka]

[0322] At room temperature, lactone 96 (2.0 equivalents) in anhydrous dichloromethane (800 mL) was added to aldehyde 99 (81.4 g, 229 mmol, 1.0 equivalent) over 30 minutes. The mixture was stirred overnight at room temperature, then concentrated and purified by SiO2 column chromatography (6:1 petroleum ether / siRNA) to obtain white solid 100 (63.4 g, yield 63%). 1 H NMR(500MHz,CDCl3)δ7.45-7.41(m,2H),7.40-7.35(m,2H),7.33(d,J=7.2Hz,1H) ,7.10-7.06(m,2H),6.92-6.88(m,2H),6.50(dd,J=8.8,1.3Hz,1H),5.04(s,2H), 4.57(s,2H),4.18(q,J=7.1Hz,2H),2.86(d,J=8.5Hz,1H),2.72(dd,J=13.6,6.8H z,1H),1.71(d,J=1.4Hz,3H),1.41(d,J=2.2Hz,9H),1.28(td,J=7.5,5.1Hz,4H). MS ESI m / z;C 26 H 33 NaNO5[M+Na] + Calculated value: 462.24, measured value: 462.22.

[0323] Example 39; Synthesis of Compound 101 [ka]

[0324] In a hydrogenation bottle, compound 100 (30.2 g, 68.9 mmol) was dissolved in THF (100 mL) and methanol (300 mL), to which Pd / C (1.83 g, 10 wt%, 50% water) was added. The mixture was shaken overnight under H2 at 1 atm, filtered through Celite (filtration aid), and the filtrate was concentrated to obtain compound 101 as a colorless oil (25.0 g, theoretical yield). 1H NMR(500MHz,CDCl3)δ6.99(d,J=7.0Hz,2H),6.72(d,J=7.6Hz,2H),4.39(s,1H),4.18-4.04(m ,2H),3.82(s,1H),2.60(dd,J=37.2,20.9Hz,4H),1.95-1.81(m,1H),1.39(s,11H),1.24(dd,J =9.5,4.3Hz,3H),1.13(t,J=8.9Hz,3H). MS ESI m / z;C 19 H 31 NO5 [M+H] + ;352.20, measured value 352.19.

[0325] Example 40; Synthesis of Compound 102 [ka]

[0326] At room temperature, a 200 mL solution of compound 101 (5.96 g, 35.9 mmol, 1.0 equivalent) in anhydrous dichloromethane was mixed with Ac2O (3.2 mL, 33.9 mmol, 2.0 equivalents) and HNO3 (65%-68%, 3.5 mL, 50.79 mmol, 3.0 equivalents). The mixture was stirred at room temperature for 30 minutes, and TLC analysis indicated that the reaction was complete. The reaction solution was washed with water (3 × 200 mL), and the aqueous layer was back-extracted with dichloromethane (3 × 100 mL). The combined dichloromethane solution was washed with brine, dried over anhydrous Na2SO4, filtered, concentrated, and purified by SiO2 column chromatography (5:1 hexane / Â) to obtain compound 102 as a yellow solid (4.18 g, 72% yield). 1H NMR(500MHz,CDCl3)δ10.49(s,1H),7.89(s,1H),7.44(d,J=8.4Hz,1H),7.09(d,J=8.6Hz,1H),4.32(d,J=8.3Hz,1H),4.12(dd,J=14.0,7.0Hz,2H) ,3.80(s,1H),2.76(dd,J=13.0,6.8Hz,2H),2.59(s,1H),1.88(s,1H),1. 37(t,J=8.7Hz,9H),1.25(dd,J=13.5,6.9Hz,4H),1.16(t,J=8.0Hz,3H). MS ESI m / z;C 19 H 28 NaN2O7[M+Na] + Calculated value: 419.19, measured value: 419.17.

[0327] Example 41; Synthesis of Compound 103 [ka]

[0328] At room temperature, a solution of ester 102 (15.3 g, 38.6 mmol, 1.0 equivalent) in THF (100 mL) and methanol (100 mL) was mixed with an aqueous solution of LiOH·H2O (16.3 g, 389 mmol, 10.0 equivalent) (190 mL). The mixture was stirred at room temperature for 40 minutes, then diluted with water (400 mL), and 1N KHSO4 was added dropwise until the pH reached 3-4. After extraction with SiO2 (3 × 300 mL), the organic phase was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated to obtain 103 as a yellow solid (14.4 g, theoretical yield). 1 H NMR (500MHz, CDCl3). δ10.48(s,1H),7.98-7.88(m,1H),7.42(dd,J=18.4,8.2Hz,1H),7.14-7.03( m,1H),4.48(d,J=8.6Hz,1H),3.90(s,1H),2.82-2.53(m,3H),1.97-1.82(m,2 H),1.42-1.27(m,10H),1.21(d,J=6.7Hz,4H). MS ESI m / z;C 17 H23 N2O7[MH] - Calculated value: 367.16, measured value: 367.14.

[0329] Example 42; Synthesis of Compound 104 [ka]

[0330] In a hydrogenated bottle, a methanol solution (260 mL) of compound 103 (26.0 g, 70.6 mmol, 1.0 equivalent) was mixed with Pd / C (2.60 g, 10 wt%, 50% water). The mixture was shaken overnight under 1 atm of H2, then filtered through Celite (a filtration aid), and the filtrate was concentrated to obtain compound 104 as a green oil (24.0 g, yield >100%).

[0331] Example 43; Synthesis of Compound 106 [ka]

[0332] At room temperature, a mixture of tert-butyl 2-bromopropionate (255 g, 1.22 mol, 1.0 equivalent) and triphenylphosphine (320 g, 1.22 mol, 1.0 equivalent) in dry acetonitrile (1 L) was stirred for 18 hours. The acetonitrile was removed under reduced pressure, and the white precipitate was pulverized with toluene. Next, the toluene was decanted, and the white solid was dissolved in dichloromethane (1 L) and transferred to a separation funnel. When 10% NaOH (1 L) was added to the funnel, the organic layer immediately turned yellow after shaking. The organic layer was separated, and the aqueous layer was extracted once with dichloromethane (1 L). The dichloromethane layers were combined, washed once with brine (400 mL), dried over Na2SO4, filtered and concentrated to obtain ylide 106 as a yellow solid (280 g, 58%).

[0333] Example 44; Synthesis of compound 107. [ka]

[0334] Aldehyde 99 (450g, 1.27mol, 1.0 equivalent) was dissolved in dry dichloromethane (3L), and tert-butyl ester ylide 106 (546g, 1.40 mmol) was added. Add 1.1 equivalents of the compound, stir overnight at room temperature, and measure the completeness by TLC. Purification by column chromatography (10-50% EtOAC / hexane) yielded compound 107 as a white solid (444 g, yield 75%). ESI m / z;C 28 H 38 NO5 [M+H] + Calculated value: 468.27, measured value: 468.22.

[0335] Example 45; Synthesis of Compound 108 [ka]

[0336] Compound 107 (63 g, 0.13 mol) was dissolved in methanol (315 mL) and hydrogenated overnight at room temperature (1 atm, H2) using a Pd / C catalyst (10 wt%, 6.3 g). The catalyst was filtered off, and the filtrate was concentrated under reduced pressure to obtain compound 108 (45.8 g, 93% yield).

[0337] Example 46; Synthesis of Compound 109 [ka]

[0338] At room temperature, tert-butyl nitrite (1.06 kg, 10.3 mol, 10 equivalents) was added to a THF solution (4 L) of compound 108 (390 g, 1.03 mol, 1.0 equivalent), and the reaction mixture was stirred overnight. After removing the THF, the residue was purified by column chromatography (10-50% EtOAC / hexane) to obtain compound 109 as a pale yellow solid (314 g, yield 72%).

[0339] Example 47; Synthesis of Compound 110 [ka]

[0340] Under a nitrogen atmosphere, Pd / C (10 wt%, 16 g) was added to a 500 mL solution of 10⁹ (166 g, 0.392 mol, 1.0 equivalent) of HCl. The reaction flask was evacuated and purged three times with hydrogen. The reaction mixture was stirred at room temperature under hydrogen (1 atm) for 16 hours, then filtered through Celite and concentrated to obtain product 110 as a pale yellow foam (146 g, yield). 97%). 1 H NMR(400MHz,CDCl3)δ6.62(d,J=7.9Hz,1H),6.55(s,1H),6.43(d,J=7.3Hz,1H),4.39(dd,J=53.0,44.2Hz,1H), 3.77(s,4H),2.72-2.29(m,3H),1.83-1.58(m,1H),1.40(d,J=7.6Hz,18H),1.24(s,1H),1.06(t,J=5.7Hz,3H). MS ESI m / z;C 21 H 35 N2O5[M+H] + Calculated value: 394.25, measured value: 395.25.

[0341] Example 48; Synthesis of Compound 114 [ka]

[0342] At -78°C under a nitrogen atmosphere, n-butyllithium (2.5 M in hexane, 17.0 mL, 1.2 equivalents) was added over 30 minutes to a 200 mL solution of anhydrous THF containing (S)-4-isopropyloxazolidine-2-one (5.00 g, 38.7 mmol, 1.0 equivalent). The mixture was stirred at -78°C for 1 hour, and then propionyl chloride (4.0 mL, 42.58 mmol, 1.1 equivalents) was added dropwise. After stirring the mixture at -78°C for another 1 hour, TLC analysis indicated that the reaction was complete. Saturated ammonium chloride solution (250 mL) was added and extracted with siRNA (3 × 100 mL). The combined organic layers were washed with 1N NaOH solution (200 mL) and brine (300 mL), dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography (7:1 hexane / Â) to obtain compound 114 as a colorless oil (6.36 g, yield 89%). MS ESI m / z;C9H 16 NO3 [M+H] + Calculated value: 186.10, measured value: 186.10. 1 H NMR(400MHz,CDCl3)δ4.48-4.39(m,1H),4.27(t,J=8.7Hz,1H),4.21(dd,J=9.1,3.1Hz,1H),3.06-2 .82(m,2H),2.38(dtd,J=14.0,7.0,4.0Hz,1H),1.17(t,J=7.4Hz,3H),0.90(dd,J=17.0,7.0Hz,6H).

[0343] Example 49; Synthesis of Compound 115 [ka]

[0344] At 0°C and under an N2 atmosphere, (S)-4-isopropyl-3-propionyloxazolidine-2-one (2.00 g, 11.9 mmol, 1.1 equivalents) is dissolved in anhydrous dichloromethane (20 mL), to which DIPEA (2.3 mL, 12.9 mmol, 1.2 equivalents) and n-Bu are added. 2BOTf (1.0 M dichloromethane solution, 12.0 mL, 1.1 equivalents) was added. The mixture was stirred for 45 minutes, then cooled to -78°C, and compound 99 in dichloromethane (4.24 mL, 10.8 mmol, 1.0 equivalent) was added dropwise. The mixture was stirred at -78°C for 1 hour, then slowly warmed to room temperature. The mixture was stirred at room temperature overnight, and PBS (0.1 M, pH 7.0, 100 mL) was added. After phase separation, the aqueous phase was further extracted with dichloromethane (3 × 50 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude product was redissolved in methanol (100 mL) and treated with H₂O₂ (30% aqueous solution, 26 mL, 23 equivalents) at 0°C for 3 hours. Methanol was removed by rotary evaporation, and water (100 mL) was added. The resulting mixture was extracted with toluene (3 × 100 mL). The combined organic layers were washed with brine (300 mL), dried over anhydrous sodium 2SO4, filtered, concentrated, and purified by SiO2 column chromatography (3:1 hexane / toluene) to obtain compound 115 as a foamy solid (2.70 g, 49% yield). 1 H NMR(400MHz,CDCl3)δ7.52-7.26(m,5H),7.15(d,J=7.4Hz,2H),6.93(d,J=7.3 Hz,2H),5.05(s,2H),4.69(d,J=7.0Hz,1H),4.47(s,1H),4.36(t,J=7.8Hz,1H) ,4.17(d,J=8.5Hz,1H),3.93(d,J=7.1Hz,1H),3.85(s,2H),2.84(d,J=6.9Hz,2 H),2.31(s,1H),1.40-1.37(m,9H),1.31(s,3H),0.92(dd,J=13.4,6.6Hz,6H). MS ESI m / z;C 30 H 41 N2O7[M+H] + Calculated value: 541.28, measured value: 541.30.

[0345] Example 50; Synthesis of Compound 116 [ka]

[0346] A mixture of compound 115 (2.50 g, 4.63 mmol, 1.0 equivalent) and 1,1'-thiocarbonyldiimidazole (2.48 g, 13.89 mmol, 3.0 equivalents) in anhydrous THF (46 mL) was refluxed overnight. Water (100 mL) was added, and the resulting mixture was extracted with HCl (3 × 50 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na₂SO₄, filtered, concentrated, and purified by silica gel column chromatography (3:1 hexane / HCl) to obtain compound 116 as a yellow foam (2.33 g, 77% yield). 1 H NMR(400MHz,CDCl3)δ8.41(s,1H),7.67(s,1H),7.36(dt,J=16.0,6.9Hz,6H),7.09(s,1H),7.05(d, J=8.4Hz,2H),6.86(d,J=8.4Hz,2H),6.32(d,J=9.5Hz,1H),5.01(s,2H),4.56-4.43(m,2H),4.32(d dd,J=16.2,15.6,7.8Hz,3H),4.19(d,J=8.7Hz,1H),2.96(dd,J=14.6,4.4Hz,1H),2.49(dd,J=14.5 ,10.5Hz,1H),2.29(td,J=13.4,6.7Hz,1H),1.31(s,3H),1.29(s,9H),0.91(dd,J=13.9,6.9Hz,6H). MS ESI m / z;C 34 H 43 N4O7S[M+H] + Calculated value: 651.27, measured value: 651.39.

[0347] Example 51; Synthesis of Compound 117 [ka]

[0348] To a 30 mL solution of dry toluene containing compound 116 (1.90 g, 2.92 mmol, 1.0 equivalent), n-Bu3SnH (1.6 mL, 5.84 mmol, 2.0 equivalents) and azodiisobutyronitrile (0.05 g, 0.584 mmol, 0.1 equivalents) were added sequentially. The mixture was refluxed for 2.5 hours, then concentrated and purified by SiO2 column chromatography (5:1 hexane / siRNA) to obtain compound 117 as a white foam (1.21 g, 79% yield). 1 H NMR (400MHz, CDCl3). δ7.36(ddd,J=24.5,14.5,7.1Hz,5H),7.08(d,J=8.5Hz,2H),6.90(d,J=8.5Hz,2H),5.04(d,J=5 .1Hz,2H),4.48(d,J=4.2Hz,1H),4.33(t,J=8.4Hz,1H),4.22(d,J=9.7Hz,1H),4.15(d,J=8.8Hz, 1H),3.81(s,2H),2.73(dd,J=14.1,5.9Hz,1H),2.61(dd,J=14.0,7.2Hz,1H),2.29(dq,J=13.5, 6.8Hz,1H),2.11-2.00(m,1H),1.35(s,9H),1.20(d,J=6.9Hz,3H),0.89(dd,J=14.0,6.9Hz,6H). MS ESI m / z;C 30 H 41 N2O6[M+H] + Calculated value: 525.28, measured value: 525.37.

[0349] Example 52; Synthesis of Compound 118 [ka]

[0350] To a THF solution (30 mL) of compound 117 (1.20 g, 2.29 mmol, 1.0 equivalent), water (6 mL) and a 30% H2O2 solution (1.4 mL, 6.0 equivalents) were added. After stirring at 0°C for 3 hours, sodium bisulfite solution (1.5 M, 30 mL) was added to stop the reaction. After 30 minutes, 1N KHSO4 was added dropwise until the pH reached 4. Next, the reaction mixture was extracted with RINKAN (3 × 50 mL). The RINKAN solution was washed with brine, dried over anhydrous Na2SO4, filtered, concentrated, and purified by SiO2 column chromatography (3:1 hexane / RINKAN, including 1% HOAc) to obtain compound 118 as a white solid (0.78 g, yield 82%). 1 H NMR(400MHz,CDCl3)δ7.46-7.28(m,5H),7.07(d,J=7.7Hz,2H),6.91(d,J=7.8Hz,2H) ,4.52(d,J=8.5Hz,1H),3.87(d,J=41.8Hz,1H),2.82-2.43(m,3H),1.85(t,J=12.2Hz, 1H), 1.41 (s, 9H), 1.17 (d, J=6.9Hz, 3H). MS ESI m / z;C 24 H 32 NO5 [M+H] + Calculated value: 414.22, measured value: 414.21.

[0351] Example 53; Synthesis of Compound 119 [ka]

[0352] A mixture of compound 118 (0.77 g, 1.86 mmol, 1.0 equivalent) and Pd / C (10%, 0.25 g) in methanol (15 mL) was hydrogenated under 1 atm and H2 for 16 hours, and then filtered through Celite (filtration aid). The filtrate was concentrated to obtain compound 119 as a white solid (0.58 g, yield 96%). 1H NMR(400MHz,CDCl3)δ7.00(d,J=7.5Hz,2H),6.80(s,2H),4.51(d,J=9.0Hz,1H),3.88(s,1H) ,2.66(dd,J=65.6,22.6Hz,4H),1.88(t,J=12.2Hz,1H),1.42(s,9H),1.14(d,J=6.6Hz,3H). MS ESI m / z;C 17 H 26 NO5 [M+H] + Calculated value: 324.17, measured value: 324.16.

[0353] Example 54; Synthesis of Compound 120 [ka]

[0354] At 0°C, t-BuONO (0.63 mL, 5.28 mmol, 3.0 equivalents) was added to a THF solution (10 mL) of compound 119 (0.57 g, 1.76 mmol, 1.0 equivalent). The reaction mixture was stirred at 0°C for 1 hour, then stirred at room temperature for 1 hour. After adding water (50 mL), the reaction mixture was extracted with RINKAN (3 × 30 mL). The combined organic layer was washed with brine (100 mL), dried over anhydrous Na₂SO₄, filtered, concentrated, and purified by SiO₂ column chromatography (2:1 hexane / RINKAN, including 1% HOAc) to obtain compound 120 as a yellow solid (0.50 g, 77% yield). 1 H NMR(400MHz,DMSO)δ7.92(s,1H),7.47(d,J=8.3Hz,1H),7.05(d,J=8.5Hz,1H),3.73(s,1H),2.78(dd,J=13.6, 5.3Hz,1H),2.69-2.47(m,2H),1.87(t,J=11.9Hz,1H),1.47-1.37(m,1H),1.32(s,9H),1.17(d,J=7.2Hz,3H). MS ESI m / z;C 17 H 25 N2O7[M+H] + Calculated value: 369.15, measured value: 369.14.

[0355] Example 55; Synthesis of Compound 121 [ka]

[0356] A mixture of compound 120 (0.50 g, 1.36 mmol, 1.0 equivalent) and Pd / C (10 wt%, 0.02 g) in methanol (10 mL) was hydrogenated at room temperature for 1 hour (1 atm, H2), and then filtered through Celite (filtration aid). The filtrate was concentrated to obtain compound 121 as a white foam (0.43 g, yield 93%). MS ESI m / z;C 17 H 27 N2O5[M+H] + ;339.18, measured value 339.17. 1 H NMR(400MHz,MeOD)δ6.60(d,J=7.9Hz,2H),6.44(d,J=7.3Hz,1H),3.71(d,J=6.3Hz,1H),2 .62-2.37(m,3H),1.83(ddd,J=13.7,9.9,3.7Hz,1H),1.39(s,9H),1.13(d,J=7.1Hz,3H).

[0357] Example 56; Synthesis of Compound 124 [ka]

[0358] 4-aminobutyric acid (285 g, 2.76 mol) was added to a acetic acid solution (1 L) of maleic anhydride (268 g, 2.73 mol). After stirring at room temperature for 30 minutes, the reaction mixture was refluxed for 1.5 hours, cooled to room temperature, and evaporated under vacuum to obtain a residue. This residue was dissolved in EA, washed with water and brine, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was crystallized from RINKAN and PE to obtain a white solid (400 g, yield 80%). 1 H NMR (500MHz, CDCl3) δ6.71(s,2H),3.60(t,J=6.7Hz,2H),2.38(t,J=7.3Hz,2H),2.00-1.84(m,2H).

[0359] Example 57; Synthesis of Compound 125 [ka]

[0360] Compound 124 (400g, 2.18mol, 1.0 equivalent) was dissolved in CH2Cl2 (1.5L), and N-hydroxysuccinimide (276g, 2.40 mmol, 1.1 equivalent) was added. ) and DIC (303 g, 2.40 mol, 1.1 equivalents) were added at room temperature and stirred overnight. The reaction product was concentrated and purified by column chromatography (1:2 petroleum ether / RINKAN) to obtain NHS ester 125 as a white solid (382 g, yield 63%). 1 H NMR (500 MHz, CDCl3) δ6.74(s,2H),3.67(t,J=6.8Hz,2H),2.85(s,4H),2.68(t,J=7.5Hz,2H),2.13-2.03(m,2H).

[0361] Example 58; Synthesis of Compound 126 [ka]

[0362] While stirring at 0°C, NMM (85.3 mL, 984 mmol, 3.0 equivalents) was added to 600 mL of THF solution of 124 (60 g, 328 mmol, 1.3 equivalents), and then chloroformate isobutyl (44.6 mL, 426 mmol, 1.3 equivalents) was added dropwise. After stirring at 0°C for 2 hours, the resulting mixture was added dropwise to 400 mL of THF solution of 104 (102 g, 259 mmol, 1.0 equivalent) while maintaining the temperature at 0°C. After the addition was complete, the reaction mixture was stirred for a further 30 minutes, then quenched with water (300 mL), and extracted with RINKAN (3 x 300 mL). The combined organic layers were dried, filtered, concentrated, and purified by column chromatography using a 9–35% RINKAN / PE gradient to obtain compound 126 as a pale yellow solid (104 g, 73% yield). 1 H NMR(400MHz,CDCl3)δ8.86(s,1H),8.40(d,J=17.3Hz,1H),6.87(s,3H),6.70(s,2H),4.53-4.16(m,0H),3.79(s,1H),3.62(t,J=6 .1Hz,1H),2.63(s,1H),2.40(t,J=6.9Hz,1H),2.12-1.88(m,4H),1.84-1.64(m,1H),1.38(t,J=9.6Hz,6H),1.06(t,J=6.0Hz,3H).

[0363] Example 59; Synthesis of Compound 127 [ka]

[0364] Compound 126 (12.7 g, 22.7 mmol) dissolved in CH2Cl2 (20 mL) at 0°C was treated with TFA (40 mL), and the reaction mixture was warmed to room temperature and stirred for 3 hours. The mixture was concentrated and evaporated three times with toluene. The residue was pulverized with ether to obtain a pale yellow solid. 127 cells were collected (11.4g, theoretical yield).

[0365] Example 60; Synthesis of Compound 128 [ka]

[0366] To an HCl solution of carboxylic acid 33 (40 mg, 0.074 mmol, 1.0 equivalent), pentafluorophenol (27 mg, 0.148 mmol, 2.0 equivalents) and DCC (23 mg, 0.111 mmol, 1.5 equivalents) were added. The reaction mixture was stirred at room temperature for 16 hours, filtered through a Celite pad, and washed with HCl. The filtrate was concentrated and redissolved in DMA (6 mL), then compound 127 (56.6 mg, 0.13 mmol) and DIPEA (47.4 μL, 0.18 mmol) were added. The reaction mixture was stirred at room temperature for 24 hours, then concentrated and subjected to reverse-phase HPLC (C). 18 The compound 128 was purified by column chromatography (10-100% acetonitrile / water) to obtain a white solid (43 mg, 63% yield). ESI m / z;C 46 H 66 N7O 11 S[M+H] + Calculated value: 924.45, measured value: 924.45.

[0367] Example 61; Synthesis of Compound 132 [ka]

[0368] At 0°C, DIPEA (6.9 mL, 39.7 mmol, 2.5 equivalents) was added to a 100 mL DMF solution of compound 41a (11 g, 15.9 mmol, 1.0 equivalent) and compound 127 (12.3 g, 23.8 mmol, 1.5 equivalents). The reaction mixture was heated to room temperature and stirred for 1 hour. The mixture was concentrated under vacuum and purified using a silica gel column (100% DCM to 10% MeOH / DCM) to obtain compound 132 as an amorphous solid (10 g, 69% yield). MS ESI m / z;C 45 H 65 N7O 11 S[M+H] +;912.45, measured value 912.45.

[0369] Example 62; Synthesis of Compound 204 [ka]

[0370] Under an N2 atmosphere at -78°C, n-butyllithium (85.0 mL, 0.213 mol, 1.1 equivalents) was added to an anhydrous THF solution (1150 mL) of (R)-4-isopropyloxazolidine-2-one (203) (25.0 g, 0.194 mol, 1.0 equivalent), and the mixture was stirred at the same temperature for 1 hour to form a large amount of white solid. Next, propionyl chloride (20.0 mL, 0.232 mol, 1.2 equivalents) was added at -78°C, and the mixture was stirred at the same temperature for 1 hour. After monitoring the consumption of (S)-4-isopropyloxazolidine-2-one by TLC, the mixture was poured into a saturated ammonium chloride solution (1.2 L) and extracted with EA (700 mL, 350 mL x 2). The organic extract was washed with 1.0 N NaOH solution (1.0 L) and brine (1.0 L), dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, and purified by SiO2 column chromatography (PE:EA = 10:1) to obtain the title compound as a colorless oil (32.6 g, 90.8%). ESI m / z: C9H 17 NO3 [M+H] + Calculated value: 186.1, measured value: 186.1. 1 H NMR(400MHz,CDCl3)δ4.48-4.37(m,1H),4.27(t,J=8.7Hz,1H),4.21(dd,J=9.1,3.1Hz,1H) ,3.04-2.82(m,2H),2.45-2.30(m,1H),1.17(t,J=7.4Hz,3H),0.90(dd,J=17.1,7.0Hz,6H).

[0371] Example 63; Synthesis of compound 205. [ka]

[0372] Under an N2 atmosphere at -78°C, 200 mL of anhydrous DCM solution of (R)-4-isopropyl-3-propionyloxazolidine-2-one (18.4 g, 99.5 mmol, 1.1 equivalents) was mixed with Bu2BOTf (100 mL, 100 mmol, 1.1 equivalents in 1 M dichloromethane solution) and DIPEA (19 mL, 108.6 mmol, 1.2 equivalents), and the mixture was stirred at the same temperature for 45 minutes. At -78°C, 320 mL of dichloromethane solution of aldehyde 99 (32.2 g, 90.5 mmol, 1.0 equivalent) was added, and the mixture was stirred at the same temperature for 1 hour, after which the solution was slowly warmed to room temperature for 15 hours. The mixture was poured into 700 mL of potassium phosphate buffer (pH 7.0) and extracted with ethyl acetate. The organic extract was washed with brine, dried over anhydrous sodium sulfate, filtered, and then concentrated under vacuum. The residue was dissolved in methanol (730 mL), cooled to 0°C, and then 30% H2O2 aqueous solution (225 mL) was slowly added and stirred at the same temperature for 3 hours. After adding water (750 mL), the mixture was concentrated under vacuum to remove methanol. The resulting aqueous solution was then mixed with ethyl acetate (500 mL, Extraction was performed using 150 mL x 2, the organic extract was washed with 5% sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, and purified by SiO2 column chromatography (PE:EA=3:1) to obtain the title compound as a white foam (31.7 g, 64.8%). ESI m / z:C 30 H 41 N2O7[M+H] + : 541.3, measured value 541.3. 1 H NMR(400MHz,CDCl3)δ7.49-7.29(m,5H),7.17(t,J=10.7Hz,2H),6.93(d,J=7.0Hz,2H),5.06(s,2H),4.28(dd,J=44.4, 36.4Hz,3H),4.04-3.52(m,3H),3.11-2.73(m,2H),2.35(s,1H),1.41(t,J=16.3Hz,9H),0.91(dd,J=15.6,6.4Hz,5H).

[0373] Example 64; Synthesis of Compound 206 [ka]

[0374] To a 350 mL solution of compound 205 (28.3 g, 52.3 mmol, 1.0 equivalent) in anhydrous THF, 1,1-thiocarbonyldiimidazole (TCDI) (35.1 g, 157.0 mmol, 3.0 equivalents) was added, and the mixture was heated overnight under reflux. After monitoring the amount of raw materials consumed by TLC, the mixture was concentrated under vacuum and purified by SiO2 column chromatography (PE:EA=3:1) to obtain the title compound as a pale yellow foam (26.1 g, 76.8%). ESI m / z:C 34 H 43 N4O7S[M+H] + Calculated value: 651.3, Measured value: 651.3. 1 H NMR(400MHz,CDCl3)δ8.21(s,1H),7.43(d,J=11.8Hz,1H),7.42-7.28(m,5H),7.06(d,J =8.3Hz,2H),7.01(s,1H),6.80(d,J=8.3Hz,2H),6.17(dd,J=8.5,2.9Hz,1H),4.96(s,2 H),4.42-4.04(m,5H),2.83(dd,J=14.2,6.2Hz,1H),2.69(dd,J=14.2,7.1Hz,1H),2.32 (dd,J=6.8,4.2Hz,1H),1.37(s,9H),1.30(d,J=6.9Hz,3H),0.87(dd,J=9.9,7.0Hz,6H).

[0375] Example 65; Synthesis of Compound 207 [ka]

[0376] Under an N2 atmosphere, in a 350 mL solution of anhydrous toluene containing compound 206 (26.0 g, 40.0 mmol, 1.0 equivalent), n-Bu3SnH (21.5 mL, 80.0 mmol) 2.0 equivalents of 2,2'-azobis(2-methylpropionitrile) (AIBN) (0.066 g, 0.01 eq) were added. The mixture was heated under reflux for 1 hour. After monitoring the consumption of raw materials by TLC, the mixture was concentrated under vacuum and purified by SiO2 column chromatography (PE:EA=5:1) to obtain the title compound as a white foam (6.0 g, 37.3%). ESI m / z:C 30 H 41 N2O6[M+H] + Calculated value: 525.3, measured value: 525.3. 1 H NMR(400MHz,CDCl3)δ7.37(ddd,J=25.1,15.1,7.1Hz,5H),7.08(d,J=7.9Hz,2H),6.89(d, J=8.4Hz,2H),5.03(s,2H),4.61(d,J=8.4Hz,1H),4.40(s,1H),4.32-4.08(m,2H),3.91-3. 66(m,2H),2.83(d,J=8.4Hz,1H),2.60(t,J=10.1Hz,1H),2.33(s,1H),1.7 1(s,1H),1.41(s,9H),1.15(d,J=6.5Hz,3H),0.87(dd,J=17.0,7.0Hz,6H).

[0377] Example 66; Synthesis of Compound 208 [ka]

[0378] At 0°C, a solution of compound 207 (7.84 g, 15.0 mmol, 1.0 equivalent) in THF (90 mL) and water (30 mL) was mixed with a 30% H2O2 aqueous solution of LiOH·H2O (1.57 g, 37.5 mmol, 2.5 equivalents) (11.4 mL, 112.5 mmol, 7.5 equivalents). The mixture was stirred at the same temperature for 3 hours. At 0°C, a 1.5 M Na2SO3 solution (160 mL) was added, and the mixture was stirred at the same temperature for 30 minutes. Next, 1N KHSO4 was slowly added until the pH became 4. The reaction aqueous solution was extracted with EA (200 mL, 75 mL x 2), the organic extract was washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, and purified by SiO2 column chromatography (PE:EA = 2:1) to obtain the title compound as a white solid (6.18 g, 100%). ESI m / z:C 24 H 32 N1O5[M+H] + : Calculated value 414.2, Actual value 414.2. 1H NMR(400MHz,CDCl3)δ7.39(ddd,J=24.5,15.0,7.2Hz,5H),7.11(d,J=7.8Hz,2H),6.93(d,J=8.3Hz,2H),5.06(s,2H),4.44(t,J=8 .3Hz,1H),3.83(d,J=69.4Hz,1H),2.85-2.61(m,2H),2.61-2.40(m,1H),1.99-1.70(m,1H),1.39(d,J=26.1Hz,9H),1.19(s,3H).

[0379] Example 67; Synthesis of Compound 209 [ka]

[0380] In a hydrogenation flask, 0.6 g of Pd / C (10% Pd / C) was added to a 50 mL MeOH solution of compound 208 (6.18 g, 15.0 mmol, 1.0 equivalent). The mixture was shaken overnight under a hydrogen atmosphere of 1 atm, and then filtered. The filtrate was concentrated to obtain the title compound as a colorless oil (4.8 g, 99% yield). ESI m / z:C 17 H 26N1O5[M+H] + :324.2, measured value 324.2. 1 H NMR(400MHz,CDCl3)δ6.97(d,J=6.5Hz,2H),6.74(d,J=8.2Hz,2H),3.93-3.66(m,1H),2.58(tdd,J=19. 5,12.9,7.4Hz,3H),1.75(ddd,J=20.1,16.3,7.7Hz,1H),1.37(d,J=21.5Hz,9H),1.11(d,J=7.0Hz,3H).

[0381] Example 68; Synthesis of Compound 210 [ka]

[0382] At 0°C under an N2 atmosphere, t-BuONO (18.0 mL, 150 mmol, 10.0 equivalent) was slowly added to a 75 mL anhydrous THF solution of compound 209 (4.8 g, 15.0 mmol, 1.0 equivalent). The reaction mixture was stirred at the same temperature for 3 hours, and after monitoring the consumption of the raw materials by TLC, 1N KHSO4 was slowly added to the mixture until the pH reached 4. The resulting aqueous reaction solution was extracted with EA (150 mL, 75 mL x 2), the organic extract was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by SiO2 column chromatography (PE:EA = 3:1) to obtain the title compound as a yellow solid (3.6 g, 65.4%). ESI m / z:C 17 H 25 N2O7[M+H] + Calculated value: 369.2, measured value: 369.2. 1H NMR(400MHz,MeOD)δ7.93(d,J=2.0Hz,1H),7.48(dd,J=8.6,2.1Hz,1H),7.06(d,J=8.5Hz,1H),3.83-3.71(m,1H),2.82(dd,J=13.6,5 .0Hz,1H),2.66-2.41(m,2H),1.84(ddd,J=14.0,10.6,5.6Hz,1H),1.65-1.51(m,1H),1.28(d,J=24.9Hz,9H),1.15(d,J=7.0Hz,3H).

[0383] Example 69; Synthesis of Compound 211 [ka]

[0384] In a hydrogenation flask, 0.2 g of Pd / C (10% Pd / C) was added to a 20 mL MeOH solution of compound 210 (3.2 g, 7.74 mmol, 1.0 equivalent). The mixture was shaken for 3 hours under a 1 atm H2 atmosphere. After monitoring the consumption of raw materials by TLC, the mixture was filtered. The filtrate was concentrated to obtain the title compound as a white foam (2.3 g, yield 92.0%). ESI m / z:C 17 H 27 N2O5[M+H] + Calculated value: 339.2, Measured value: 339.2. 1 H NMR(400MHz,MeOD)δ6.61(d,J=8.0Hz,2H),6.45(d,J=6.3Hz,1H),3.72(d,J=7.3Hz,1H),2.68-2.34(m,3H),1.81-1.66(m,1H),1.56-1. 45(m,1H),1.36(d,J=29.0Hz,9H),1.08(d,J=6.9Hz,3H).

[0385] Example 70; Synthesis of Compound 390 [ka]

[0386] At -25°C, CCl4 (2.2 mL, 22.7 mmol, 9.0 equivalents) was added to a 10 mL acetonitrile solution of compound 102 (1.00 g, 2.52 mmol). After stirring for 10 minutes, diisopropylethylamine (0.88 mL, 5.04 mmol, 2.0 equivalents) and DMAP (0.03 g, 0.252 mmol, 0.1 equivalents) were added, followed by dibenzyl phosphite (0.84 mL, 3.78 mmol, 1.5 equivalents). The reaction mixture was allowed to reach room temperature over 1.5 hours or longer, and then quenched with KH2PO4 solution (0.5 M, 50 mL). The reaction mixture was extracted with RINKAN (3 × 50 mL). The combined organic extract was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was rinsed and purified by flash column chromatography (10-50% Â / PE) to obtain compound 390 as a colorless oil (1.60 g, 96% yield). MS ESI m / z;C 33 H 41 N2O 10 P[M+H] + Calculated value: 657, measured value: 657.

[0387] Example 71; Synthesis of Compound 391 [ka]

[0388] Pd / C (10 wt%, 160 mg) was added to a methanol solution (20 mL) of compound 390 (1.60 g, 2.43 mmol). The reaction mixture was stirred at room temperature under an H2 atmosphere (1 atm) for 3 hours, then filtered through Celite and concentrated under reduced pressure to obtain compound 391 as a white solid (1.00 g, 91% yield). MS ESI m / z;C 19 H 31 N2O8P[MH] - Calculated value: 447, measured value: 447.

[0389] Example 72; Synthesis of Compound 392 [ka]

[0390] A 10 mL ethanol solution of compound 391 (730 mg, 1.63 mmol) was treated overnight at room temperature with 1 N NaOH (16 mL, 16.3 mmol, 10 equivalents), and then concentrated under reduced pressure. The residue was dissolved in water (20 mL) and acidified to pH 6 with 1 N HCl. The aqueous solution was concentrated under reduced pressure, and the residue was pulverized with MeOH / Âi (80:20, 5 mL). Compound 392 was obtained as a white solid by filtration (0.68 g, 99% yield). MS ESI m / z;C 17 H 27 N2O8P[MH] - Calculated value: 417, measured value: 417.

[0391] Example 73; Synthesis of Compound 399 [ka]

[0392] 2-(2-aminoethoxy)ethanol (21.00 g, 200 mmol, 1.0 equivalent) and K2CO3 (83.00 g, 600 mmol, 3.0 equivalents) were mixed in acetonitrile (350 mL) with BnBr (57.0 mL, 480 mmol, 2.4 equivalents). The mixture was refluxed overnight. Water (1 L) was added and extracted with RINKAN (3 × 300 mL). The combined organic layer was washed with brine (1000 mL), dried over anhydrous Na2SO4, filtered, concentrated, and purified by SiO2 column chromatography (4:1 hexane / RINKAN) to obtain a colorless oil (50.97 g, yield 89.2%). MS ESI m / z;C 18 H 23 NO2Na[M+Na] + Calculated value: 309.17, measured value: 309.19.

[0393] Example 74; Synthesis of Compound 400 [ka]

[0394] Add sodium hydroxide solution (300 mL, 50%) to a DCM solution (560 mL) of 2-(2-(dibenzylamino)ethoxy)ethanol (47.17 g, 165.3 mmol, 1.0 equivalent), tert-butyl acrylate (72.0 mL, 495.9 mmol, 3.0 equivalents), and n-Bu4NI (6.10 g, 16.53 mmol, 0.1 equivalents)). The mixture was stirred overnight, the organic layer was separated, and the aqueous layer was extracted with toluene (3 × 100 mL). The organic layer was washed with water (3 × 300 mL) and brine (300 mL), dried over anhydrous sodium 2SO4, filtered, concentrated, and purified by SiO2 column chromatography (7:1 hexane / toluene) to obtain a colorless oil (61.08 g, yield 89.4%). ESI m / z;C 25 H 36 NO4 [M+H] + Calculated value: 414.2566, measured value: 414.2384.

[0395] Example 75; Synthesis of Compound 401 [ka]

[0396] In a hydrogenation bottle, tert-butyl 3-(2-(2-(2-(bis(benzylamino)ethoxy)ethoxy)propionate (20.00 g, 48.36 mmol, 1.0 equivalent) was dissolved in THF (30 mL) and MeOH (60 mL), to which Pd / C (2.00 g, 10 wt%, 50% wet) was added. The mixture was shaken overnight, filtered through Celite (filtration aid), and the filtrate was concentrated to obtain a colorless oil (10.58 g, yield 93.8%). MS ESI m / z;C 11 H 24 NO4 [M+H] + Calculated value: 234.1627, measured value: 234.1810.

[0397] Example 76; Synthesis of Compound 402 [ka]

[0398] At 0°C, compound 401 (0.23 g, 1 mmol) was added to a DCM solution (10 ml) of (E)-3-bromoacrylic acid (0.15 g, 1 mmol), DMAP (0.15 g, 1.2 mmol), and DCC (0.21 g, 1 mmol). The reaction mixture was warmed to room temperature and stirred overnight. The crude product was concentrated and purified by SiO2 column chromatography using an EA / DCM gradient to obtain the title product 402 (0.31 g, yield 85%). ESI MS m / z:C 14 H 25 BrNO5[M+H] + Calculated value: 366.08, measured value: 366.08.

[0399] Example 77; Synthesis of Compound 403 [ka]

[0400] At 0°C, compound 402 (0.31 g, 0.84 mmol) was dissolved in formic acid (4 mL), and then H2O (2 mL) was added. The reaction mixture was warmed to room temperature and stirred overnight. The crude product was concentrated and used in the next step without further purification. ESI MS m / z:C 10 H 17 BrNO5[M+H] + Calculated value: 310.02, measured value: 310.03.

[0401] Example 78; Synthesis of Compound 404 [ka]

[0402] Compound 303 (0.12 g, 0.39 mmol), NHS (0.067 g, 0.58 mmol), and EDCI (0.11 g, 0.58 mmol) were dissolved in DCM (10 mL), the mixture was stirred overnight at room temperature, concentrated, and purified by SiO2 column chromatography to obtain the title product 404 (0.13 g, 82% yield). ESI MS m / z:C 14 H 20 BrN2O7[M+H] + Calculated value: 407.04, Measured value: 407.04.

[0403] Example 79; Synthesis of Compound 426 [ka]

[0404] A 40 mL H2O solution of 4-aminobutyric acid (7.5 g, 75 mmol) and NaOH (6 g, 150 mmol) was cooled to 0°C, and a 32 mL THF solution of CbzCl (16.1 g, 95 mmol) was added dropwise. After 1 hour, the reaction mixture was warmed to room temperature and stirred for 3 hours. Under vacuum, the THF was removed, and the pH of the aqueous solution was adjusted to 1.5 by adding 6N HCl. The solution was extracted with ethyl acetate, the organic layer was washed with brine, dried, and concentrated to obtain compound 426 (16.4 g, 92% yield). MS ESI m / z;C 12 H 16 NO5 [M+H] + Calculated value: 238.10, measured value: 238.08.

[0405] Example 80; Synthesis of Compound 427 [ka]

[0406] To a 100 mL DCCM solution of 4-(((benzyloxy)carbonyl)amino)butyric acid (16.4 g, 69.2 mmol) and t-BuOH (15.4 g, 208 mmol), DMAP (0.8 g, 6.56 mmol) and DCC (17.1 g, 83 mmol) were added. After stirring overnight at room temperature, the rea...

Claims

1. A pharmaceutical composition having the following composition, which is a liquid composition before lyophilization, a formulated lyophilized solid, or a reconstituted formulation from a lyophilized solid: A conjugate of the cell binder of formula (VII) and a tubulicin analog in an amount of less than 0.01 to 96.99 wt%; 3% to 20.0% of a polyol selected from sucrose or trehalose; A surfactant selected from polysorbates in an amount of 0.01% to 2.0%; One or more preservatives in a concentration of 0.0% to 5.0%; One or more amino acids in a concentration of 0.0% to 30%; One or more antioxidants in a concentration of 0.0% to 5.0%; One or more metal chelating agents in an amount of 0.0% to 0.3%; One or more buffer salts in an amount of more than 0.0% to 30.0% to adjust the pH of the formulation to pH 5.8 to 8.5; After being reconstituted for administration to the patient, one or more isotonic agents are added in an amount of 0.0% to 30.0% to adjust the osmotic pressure between 250 and 350 mOsm; Includes, The conjugates of the tubulicin analogs of formula (VII) are those listed below, or their pharmaceutically acceptable salts, hydrates, or hydrated salts; or polymorphic crystals of these compounds; or their isotopes, optical isomers, racemates, diastereomers, or enantiomers: 【Chemistry 12】 In the formula, T is the target or cell-binding molecule; L is the releaseable conjugate; n is 1 to 20, and m is 1 to 10. T is an antibody; a single-chain antibody; an antibody fragment that binds to a target cell; a monoclonal antibody; a single-chain monoclonal antibody; or a monoclonal antibody fragment that binds to a target cell; a chimeric antibody; a chimeric antibody fragment that binds to a target cell; a domain antibody; a domain antibody cross-section that binds to a target cell; an antibody-mimicking adnectin; DARPins; lymphokines; hormones; vitamins; growth factors; colony-stimulating factors; or nutrient transport molecules; transferrin; a binding peptide, or protein, or a small molecule, polymer, dendrimer, liposome, nanoparticle, vesicle, or (viral) capsid bound to an antibody or albumin; The linked structure L has the formula --Ww-(Aa)r--, where --W-- is an extended unit; w is 1; each --Aa-- is independently an amino acid unit; r is independently an integer between 1 and 12; the extended unit W includes a non-self-destructing spacer; the W that binds to T has the following structure; 【Transformation 3】 In the formula, R ２０ Ha-C １ ~C ９ Alkylene-; The non-self-destructing spacer has the following structure: 【Transformation 5】 During the ceremony, `` ＊ The atoms of the '' are the attachment points for the additional spacers; The content within the parentheses of formula (VII) is a tubulysin analog. In the formula, R １ 、R ２ 、R ３ 、and R ４ are independently a straight-chain or branched alkyl, alkyl alcohol of C １ -C ８ ; a heteroalkyl, alkylcycloalkyl, heterocycloalkyl, alkyl ether, alkyl carboxylate, alkylamine, alkyl ester, alkyl amide of C ２ -C ８ ; an aryl, Ar-alkyl, heterocyclic ring, carbocyclic ring, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl of C ３ -C ８ ; or two Rs, namely R １ R ２ 、R ３ R ４ 、R ５ R ６ 、or R １２ R １３ together form a 3- to 7-membered carbocyclic ring, cycloalkyl, heterocyclic ring, heterocycloalkyl, aromatic or heteroaromatic ring system; Y is N or C; further, R １ 、R ２ 、R ３ and R ４ may independently not be present; R ５ , R ６ , R ８ , and R １０ These are H and linear or branched C independently. １ -C ４ Alkyl or C ２ -C ４ Selected from heteroalkyl groups; R ７ is H or R １４ Selected from; R ９ is -OC(=O)R １４ It is; R １１ R １４ , -R １４’ C(=O)R １６ , -R １４’ C(=O)X ２ R １６ And in the formula, X ２ is -O-, -NH-, -N(R １４ )-,-OR-R １４’ - or -NHR １４’ - is; R １２ is H or R １４ It is; R １３ teeth, 【Chemistry 19】 In the formula, X １ is either O or does not exist; X ２ is NH; P １ H is H. R １４ and R １５ H;C １ ~C ８ It is a linear or branched alkyl group; R １４’ is C １ ~C ８ It is a linear or branched alkylene; R １６ H, OH, R １４ , or 1 to 4 amino acid units.

2. The pharmaceutical composition according to claim 1, wherein the conjugate of the tubulicine analog of formula (VII) is a conjugate of a tubulicine analog having the structure represented by the following formula, or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof; or polymorphic crystals of these compounds; or isotopes, optical isomers, racemates, diastereomers, or enantiomers thereof: 【Chemistry 13】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 During the ceremony, R ２０ H; acyl(-C(O)R １７ ); or R ２０ It is absent, and oxygen forms ketones; R ２１ H, C １ ~C ８ It is a linear or branched alkyl group; X １ is either O or does not exist; X ２ is NH; P １ H is; R １７ and R １８ H and C are independent of each other. １ ~C ８ Linear, branched, or heteroalkyl groups; C ２ ~C ８ Linear or branched alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C ３ ~C ８ These are linear or branched aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; carbonate, carbamate; R ７ This is defined the same way as in claim 1; m = 1 to 10. 【Chemistry 14】 This is the part that connects to the connecting body L.

3. The pharmaceutical composition according to claim 1, wherein the conjugate of the tubulicin analog of formula (VII) has the following structure: 【Chemistry 15】 【change】 【change】 In the formula, mAb is the target or cell-binding molecule; n is between 1 and 20; and p is between 0 and 100.

4. The pharmaceutical composition according to claim 1, wherein the target or cell-binding molecule targets tumor cells, virus-infected cells, microbial-infected cells, parasitic-infected cells, autoimmune disease cells, activated tumor cells, bone marrow cells, activated T cells, affected B cells, or melanocytes, or cells expressing one or more of the following antigens or receptors: CD2, CD2R, CD3, CD3gd, CD3e, CD4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD12, CD12w, CD13, CD14, CD 15, CD15s, CD15u, CD16, CD16a, CD16b, CD17, CDw17, CD18, CD19, CD20, CD21 , CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, C D34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD44R, CD45, CD45RA, CD45RB, CD45RO, CD46, CD47, CD47R , CD48, CD49a, CD49b, CD49c, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, C D55, CD56, CD57, CD58, CD59, CD60, CD60a, CD60b, CD60c, CD61, CD62E, CD62 L, CD62P, CD63, CD64, CD65, CD65s, CD66, CD66a, CD66b, CD66c, CD66d, CD66 e, CD66f, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD74, CD75, CD75s , CD76, CD77, CD78, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CDw8 4, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CDw92, CD93, CD94, CD95 , CD96, CD97, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD104, CD105 , CD106, CD107, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113,CDw113、CD114、CD115、CD116、CD117、CD118、CD119、CDw119、CD120a、CD120b、CD121a、CD121b、CDw121b、CD122、CD123、CDw123、CD124、CD125、CDw125、CD126、CD127、CD128、CDw128、CD129、CD130、CD131、CDw131、CD132、CD133、CD134、CD135、CD136、CDw136、CD137、CDw137、CD138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD144、CD145、CDw145、CD146、CD147、CD148、CD149、CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CDw156c、CD157, CD158a、CD158b、CD159a、CD159b、CD159c、CD160、CD161、CD162、CD162R、CD163、CD164、CD165、CD166、CD167、CD167a、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CDw186、CD187、CD188、CD189、CD190、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CD198、CDw198、CD199、CDw199、CD200、CD200a、CD200b、CD201、CD202、CD202b、CD203、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210、CDw210、CD212、CD213a1、CD213a2、CDw217、CDw218a、CDw218b、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235ab、CD235b、CD236、CD236R、CD238、CD239、CD240、CD240CE, CD240D, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248 , CD249, CD252, CD253, CD254, CD256, CD257, CD258, CD261, CD262, CD263, C D265, CD266, CD267, CD268, CD269, CD271, CD273, CD274, CD275, CD276 (B7- H3), CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD289, CD292 , CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300c, CD30 0e, CD301, CD302, CD303, CD304, CD305, CD306, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CDw325, CD326, C Dw327, CDw328, CDw329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, C Dw338, CD339, CD340, CD341, CD342, CD343, CD344, CD345, CD346, CD347, CD 348, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD356, CD357, CD358 , CD359, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, C D369, CD370, CD371, CD372, CD373, CD374, CD375, CD376, CD377, CD378, CD37 9, CD381, CD382, CD383, CD384, CD385, CD386, CD387, CD388, CD389, CRIPTO, CRIPTO, CR, CR1, CRGF, CRIPTO, CXCR5, LY64, TDGF1, 4-1BB, APO2, ASLG659, BMPR1B, 4-1BB, 5AC, 5T4 (trophoblast glycoprotein, TPBG, 5T4, Wnt activator inhibitor 1 or WAIF1), adenocarcinoma antigen, AGS-5, AGS-22M6, activin receptor-like kinase 1, AFP, AKAP-4, ALK, α-integrin, αvβ6,Aminopeptidase N, amyloid β, androgen receptor, angiopoietin 2, angiopoietin 3, annexin A1, anthrax toxin protective antigen, anti-transferrin receptor, AOC3 (VAP-1), B7-H3, anthrax, BAFF (B-cell activator), BCMA, B-lymphoma cells, bcr-abl, bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, CanAg, canine IL31, carbonic anhydrase IX, cardiac myosin, CCL11 (C-C motif chemokine 11), CCR4 (CC chemokine receptor type 4, CD194), CCR5, CD3E (epsilon) CEA (carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen), CFD (factor D), Ch4D5, cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18), CLDN18.1 (Claudin-18.1), CLDN18.2 (Claudin-18.2), clamping factor A, cMet, CRIPTO, FCSF1R (colony-stimulating factor 1 receptor, CD115), CSF2 (colony-stimulating factor 2, granulocyte-macrophage colony-stimulating factor (GM-CSF)), CSP4, CTLA4 (cytotoxic T lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4, CXC chemokine receptor type 4, cADP ribose hydrolase, Cyclin B1, CYP1B1, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL3 (delta-like ligand 3), DLL4 (delta-like ligand 4), DPP4 (dipeptidyl peptidase 4), DR5 (death receptor 5), Escherichia coli Shiga toxin type 1, Escherichia coli Shiga toxin type 2, ED-B, EGFL7 (EGF-like domain containing protein 7), EGFR, EGFRII, EGFRvIII, endoglin, E Endoselin B receptor, endotoxin, EpCAM (epithelial cell adhesion molecule), EphA2, epicyalin, ERBB2 (epidermal growth factor receptor 2), ERBB3, ERG (TMPRSS2ETS fusion gene), Escherichia coli, ETV6-AML, FAP (fibroblast-activating protein α), fibroblast surface antigen, FCGR1, α-fetoprotein, fibrin II, β-chain, fibronectin external domain B, FOLR (folate receptor),Folate receptor α, folate hydrolase, RS virus Fos-related antigen 1F protein, Frizzled receptor, fucosyl GM1, GD2 ganglioside, G-28 (cell surface glycolipid antigen), GD3 idiotype, GloboH, glypican 3, N-glycolylneuraminic acid, GM3, GMCSF receptor α chain, growth differentiation factor, GP100, GPNMB (transmembrane protein NMB), GUCY2C (guanylate cyclase 2C, guanylate cyclase C (GC-C), enteric guanylate cyclase, guanylate cyclase-C) Receptors, heat-stable enterotoxin receptor (hSTAR), heat shock proteins, hemagglutinins, hepatitis B surface antigen, hepatitis B virus, HER1 (human epidermal growth factor receptor 1), HER2, HER2 / neu, HER3 (ERBB-3), IgG4, HGF / SF (stem cell growth factor / cell dispersal factor), HHGFR, HIV-1, histone complexes, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, human chorionic gonadotropin, HGGF, human cell scattering factor receptor kinase, HPV E6 / E7, Hsp90, hTERT, ICAM-1 (intercellular adhesion molecule 1), idiotype, IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-γ, influenza hemagglutinin, IgE, IgE Fc region, IGHE, interleukins (including IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-9, IL-10, IL-11, L-12, IL-13, IL-15, IL-17, IL-17A, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-27, or IL-28), IL-31RA, ILGF2 (insulin-like growth factor 2), integrins (α4, α, ＩＩＩｂ β ３ , αvβ3, α ４ β ７ α5β1, α6β4, α7β7, αIIβ3, α5β5, αvβ5), interferon-gamma-inducing protein, ITAGA2, ITGB2, KIR2D, Kappa Ig, LCK, Le, Regmine, Lewis-Y antigen, LFA-1 (Lymphocyte function-associated antigen 1, CD11a), LHRH, LINGO-1, Lipoteichoic acid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGEA1, MAGEA3, MAGE4, ​​MART1, MCP-1, MIF (Macrophage migration inhibitor or glycosylation inhibitor (GIF)), MS4A1 (Transmembrane 4-domain subfamily A member 1), MSLN (Mesothelin), MUC1 (M Tin 1, cell surface-associated (MUC1), or Polymorphic epithelial mucin (PEM), MUC1-KLH, MUC16 (CA125), MCP1 (monocyte chemotactic protein 1), MelanA / MART1, ML-IAP, MPG, MS4A1 (transmembrane 4-domain subfamily A), MYCN, myelin-related glycoprotein, myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen), Nectin-4 (ASG-22ME), NGF, neuronal apoptosis regulatory proteinase 1, NOGO-A, Notch receptor, nucleolin, Neu oncogene product, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized low-density lipoprotein), OY-TES1, P21, p53 non-mutant, P9 7, Page4, PAP, anti-(N-glycolylneuraminic acid) paratope, PAX3, PAX5, PCSK9, PDCD1 (PD-1, programmed cell death protein 1), PDGF-Rα (platelet-derived growth factor receptor α), PDGFR-β, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, platelet-derived growth factor receptor β, sodium phosphate cotransporter, PMEL17, polysialic acid, proteinase 3 (PR1), prostate cancer, PS (phosphatidylserine), prostate cancer cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI)), rhesus factor, RANKL, PhoC,Ras variant, RGS5, ROBO4, RS virus, RON, ROR1, sarcoma metastasis breakpoint, SART3, sclerostin, SLAMF7 (SLAM family member 7), selectin P, SDC1 (syndecane 1), sLe(a), somatomedin C, SIP (sphingosine-1-phosphate), somatostatin, sperm protein 17, SSX2, STEAP1 (six-transmembrane epithelium of prostate 1) Antigen), STEAP2, STn, TAG-72 (tumor-associated glycoprotein 72), Survivin, T cell receptor, T cell transmembrane protein, TEM1 (tumor epithelial marker 1), TENB2, Tenascin C (TN-C), TGF-α, TGF-β (transforming growth factor β), TGF-β1, TGF-β2 (transforming growth factor β2), Tie (CD202b), Tie2, TIM-1 (CDX-014), TN, TNF, TNF-α, TNFRSF8, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophotrophic membrane glycoprotein), TRAIL-R1 (tumor necrosis apoptosis-inducing ligand receptor 1), TRAILR2 (cell death receptor 5 (DR5)), tumor-associated calcium signaling Cells expressing Lanceducer 2, tumor-specific glycosylation of MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-1 (Trop1), TRP-2 (Trop2), tyrosinase, VCAM-1, VEGF, VEGF-A, VEGF-2, VEGFR-1, VEGFR2, or vimentin, WT1, XAGE1, or any insulin growth factor receptor, or any epidermal growth factor receptor.

5. The pharmaceutical composition according to claim 4, wherein the tumor cells are selected from lymphoma cells, myeloma cells, renal cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cell carcinoma cells, small cell lung cancer cells, non-small cell lung cancer cells, testicular cancer cells, or malignant cells.

6. The pharmaceutical composition according to claim 1 for the treatment or prevention of cancer, autoimmune diseases, or infectious diseases.

7. The pharmaceutical composition according to claim 1, which is held in liquid or lyophilized solid form in a vial, bottle, pre-filled syringe, or pre-filled auto-injector syringe.

8. The pharmaceutical composition according to claim 1, having cytotoxic activity in vitro, in vivo, or ex vivo.

9. The pharmaceutical composition according to claim 1, administered simultaneously with a chemotherapy agent, radiotherapy agent, immunotherapy agent, autoimmune disease agent, anti-infective agent, synergistic agent, or other conjugate for synergistically treating or preventing cancer, autoimmune disease, or infectious disease.

10. The synergistic agent is selected from one or more of the following drugs, in the pharmaceutical composition according to claim 9: (1) Chemotherapy agents selected from the following group: a) Alkylating agents selected from the group consisting of the following: Nitrogen mustard: Chlorambucil, Chlornafadin, Cyclophosphamide, Dacarbazine, Estramustine, Ifosfamide, Mechloretamine, Mechloretamine Oxide Hydrochloride, Mannomustine, Mitobronitol, Melphalan, Mitractol, Pipobroman, Nobenbitin, Fenesterine, Prednimustine, Thiotepa, Trophosphamide, Uracil mustard; CC-1065 and synthetic analogs of Adzelesin, Carzelesin and Bizelesin; Duocalmycin and its synthetic analogs KW-2189 and CBI-TMI; Benzodiazepine dimers or pyrrolobenzodiazepine (PBD) dimers, Tomimycin dimers, Indolinobenzodiazepine dimers Forms, imidazobenzothiadiazepine dimers, or oxazolidinobenzodiazepine dimers; nitrosourea compounds including carmustine, lomustine, chlorozotosine, fotemustine, nimustine, and ranimustine; alkyl sulfonates including busulfan, treosulfan, improsulfan, and biposulfan; triazenes or dacarbazines; platinum-containing compounds including carboplatin, cisplatin, and oxaliplatin; aziridines, benzodopa, carboquan, metsuredopa, and uredopa; ethyleneimines, and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramine, and trimethylolomelamine; b) Plant alkaloids selected from the following groups: vinca alkaloids including vincristine, vinblastine, vindesine, vinorelbine, and navelbine; taxoids including paclitaxel and docetaxel and their analogues; meitansinoids including DM1, DM2, DM3, DM4, DM5, DM6, DM7, meitansine, and anthamitosine and their analogues; cryptophycins including the groups cryptophycin 1 and cryptophycin 8; epothyrons, eruterobin, discodermolds, bryostatins, drostatins, auristatins, tubulicins, cephalostatins; pancratistatin; sarcodicuthiin; spongstatin; c) DNA topoisomerase inhibitors selected from the following: 9-aminocamptothecin, camptothecin, cristinator, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoic acid (retinols), teniposide, topotecan, 9-nitrocamptothecin, or epipodophilins including RFS 2000; and mitomycins, and their analogues; d) Antimetabolites: {[Antifolic acid: (DHFR inhibitors: including methotrexate, trimethrexate, denopterin, pteropterin, aminopterin (4-aminopteroic acid), or other folic acid analogs); IMP dehydrogenase inhibitors (including mycophenolic acid, thiazophrine, ribavirin, EICAR); Ribonucleotide reductase inhibitors (including hydroxyurea, deferoxamine)]; [Pyrimidine analogs: uraci Selected from the group consisting of: **Purine analogs (including ancitabine, azacitidine, 6-azauridine, capecitabine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-fluorouracil, floxuridine, and larcitrexed); cytosine analogs (including cytarabine, cytosine arabinoside, and fludarabine); purine analogs (including azathioprine, fludarabine, mercaptopurine, thiamiprine, and thioguanine); and folic acid supplements.** e) Hormone therapy agents: Receptor antagonists: [Anti-estrogens: (including megestrol, raloxifene, and tamoxifen); LHRH agonists: (including goserelin and leuprolide acetate); Antiandrogens: (bicalutamide, flutamide, carsterone, dromostanolone propionate, epithiostanol, goserelin, leuprolide, mepitiostane, nilutamide, testactone, trilostane, and other similar androgen inhibitors] Selected from: [Vitamin D3 analogues: (including CB1093, EB1089, KH1060, cholecalciferol, ergocalciferol); Photodynamic therapy agents: (including verteporfin, phthalocyanine, photosensitizer Pc4, demethoxyhypocrelin A); Cytokines: (including interferon α, interferon γ, tumor necrosis factor (TNF), TNF domain-containing human protein)]} f) Kinase inhibitors: Selected from the group consisting of BIBW2992 (anti-EGFR / Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080 (anti-VEGFR2), mbritinib, ponatinib, bafetinib, bosutinib, cabozantinib, bismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, and ispinesib; g) Poly(ADP-ribose) polymerase (PARP) inhibitors selected from the group consisting of olaparib, niraparib, iniparib, talazoparib, veliparib, CEP9722 (Cephalon), E7016 (Eisai), BGB-290 (Baygene), or 3-aminobenzamide; h) Endiyine antibiotics (selected from calicheamicin, calicheamicin γ1, δ1, α1, or β1; dynemycin including dynemycin A and deoxydynemycin; esperamicin, kedalcidin, C-1027, mazulopeptin, or neocardinostatin chromophore and related pigment protein enediine antibiotic chromophore), acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, cactinomycin, carabicin, carminomycin, cardinophilin; chromomycin, dactinomycin, dauno Antibiotics selected from rubicin, detrubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, eribulin, esorubicin, idarubicin, marcelomycin, mitomycins, mycophenolic acid, nogaramycin, olibomycins, peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zolbicin; i) Polyketides (acetogenins), bratacin and bratacinone; gemcitabine, epixomicins and carfilzomib, bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zyprestat, PLX4032, STA-9090, Stimavax, allobectin-7, Zygeba, Provenge, Elboy, isoprenylation inhibitors and lovastatin, dopaminergic neurotoxins and 1-methyl-4-phenyl Pyridine ions, cell cycle inhibitors (including staurosporine), actinomycins (including actinomycin D and dactinomycin), amanitins, bleomycins (including bleomycin A2, bleomycin B2, and peplomycin), anthracyclines (including daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, and zolubicin), mitoxantrone, MDR inhibitors, or verapamil, Ca ２＋ ATP inhibitors or thapsigargin, histone deacetylase inhibitors (including vorinostat, romidepsin, panobinostat, valproic acid, mosetinostat (MGCD0103), bellinostat, PCI-24781, entinostat, SB939, resminostat, gibinostat, AR-42, CUDC-101, sulforaphane, trichostatin A); thapsigargin, celecoxib, glitazones, epigallocatechin gallate, disulfiram, salinosporamide A, antiadrenal drugs (selected from the group consisting of aminoglutethimide, mitotane, and trilostampa); acegraton; aldofosphamide cricoside; aminolevulinic acid; amsacrine; arabinoside, best Lovesil; Bisanthren; Edatrexate; Defofamine; Demecolsin; Diadicone; Elfornithine (DFMO), Elfomitin; Erliptinium acetate; Etocluside, Gallium nitrate, Gasitosine, Hydroxyurea; Ibandronate, Lentinan; Ronidamin; Mitoguazone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadicone; 2,2',2''-Trichlorotriethylamine; Trichothecenes (including T2 toxin, Bercalin A, Loridine A, and Anguidin); Urethanes, siRNAs, Antisense drugs; 2) Anti-autoimmune disease drugs: cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticoids (including amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, fluocortron danazol, dexamethasone, triamcinolone acetonide, and beclomethasone dipropionate), DHEA, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mofetil, mycophenolic acid, prednisone, sirolimus, tacrolimus; 3) Anti-infective drugs: a) Aminoglycosides: Amikacin, Astromycin, Gentamycin (Netylmycin, Shisomycin, Isepamycin), Hygromycin B, Kanamycin (Amikacin, Arbekacin, Bekanamycin, Dibekacin, Tobramycin), Neomycin (Furamycin, Paromomycin, Ribostamycin), Netylmycin, Spectinomycin, Streptomycin, Tobramycin, Verdamicin; b) Amphenicols: Azidamphenicol, chloramphenicol, florphenicol, thiamphenicol; c) Ansamycins: geldanamycin, herbimycin; d) Carbapenems: biapenem, doripenem, ertapenem, imipenem, cilastatin, meropenem, panipenem; e) Cephalosporins: Carbasephalm (loracalbef), cefacetril, cefaclor, cefradin, cefadroxil, cephalonium, cefaloridine, cephalothin or cephalothin, cephalexin, cephaloglysin, cephamandol, cefapillin, cefatoridine, cefazal, cefazedon, cefazolin, cefuboperazone, cefcapene, cefdaroxime, cefepime, cefminox, cefoxitin, cefprodil, ceffloxazine, ceftezol, cefuroxime, cefixime, cefdinir, cefditoren, cefepime, ce Fetamet, cefmenoxime, cefozidime, cefonisid, cefoperazone, cefolanide, cefotaxime, cefotiam, cefozopran, cephalexin, cefpimisole, cefpyramide, cefpirome, cefpodoxime, cefprodil, cefquinome, cefsulodine, ceftazidime, cefteram, ceftibuten, cefthiolen, ceftizoxime, ceftobiprol, ceftriaxone, ceffuroxime, cefzonam, cephamycin (including cefoxitin, cefotetan, and cefmetazole), oxacepham (flomoxef, latamoxef); f) Glycopeptides: Bleomycin, vancomycin (including oritabancin and teravancin), teicoplanin (darbabancin), lamopranin; g) Glycylcyclines: Tigecycline; h) β-lactamase inhibitors: Penam (sulbactam, tazobactam), Clavam (clavulanic acid); i) Lincosamides: clindamycin, lincomycin; j) Lipopeptides: Daptomycin, A54145, calcium-dependent antibiotic (CDA); k) Macrolides: Azithromycin, cethromycin, clarithromycin, dilithromycin, erythromycin, flurithromycin, josamycin, ketolides (telithromycin, cethromycin), midecamycin, myokamycin, oleandmycin, rifamycin (rifampicin, rifampin, rifabutin, rifapentin), rokitamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandmycin, telithromycin; l) Monobactams: aztreonam, tigemonam; m) Oxazolidinones: linezolids; n) Penicillins: Amoxicillin, ampicillin, pivampicillin, hetacillin, bacampicillin, methanepicillin, tarampicillin, azidocillin, azurocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethylpenicillin, clometocillin, procaine benzylpenicillin, carbenicillin (kalindacillin), cloxacillin, dicloxacillin, epicillin, flucloxacillin, mesillinam (pibmesillinam), mezurocillin, methicillin, nafcillin, oxacillin, penamecillin, penicillin, pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin, sulbenicillin, temocillin, ticalcillin; o) Polypeptides: bacitracin, colistin, polymyxin B; p) Quinolones: Alatrofloxacin, valofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, floxin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, canotorobafloxacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q) Streptogramins: Pristinamycin, quinupristin / dalfopristin; r) Sulfonamides: Mafenide, prontosil, sulfacetamide, sulfamethizol, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole (co-trimoxazole); s) Steroidal antibacterial agents: Selected from fusidic acid; t) Tetracyclines: doxycycline, chlortetracycline, chromocycline, demeclocycline, rimecycline, meclocycline, metacycline, minocycline, oxytetracycline, penimepicycline, loritetracycline, tetracycline, glycylcycline (including tigecycline); u) Other antibiotics: annonasin, arsphenamine, bactoprenol inhibitor (bacitracin), DADAL / AR inhibitor (cycloserine), dicthiostatin, discodermolide, eleuterobin, epotilon, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laurimalid, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitor (fosfomycin), nitrofurantoin, paclitaxel, platensimycin, pyrazinamide, quinupristin / dalfopristin, rifampicin (rifampin), tazobactamtinidazole, uvarcin; (4) Antiviral drugs: a) Invasion / fusion inhibitors: aplaviroc, maraviroc, bicriviroc, gp41 (enfvirtide), PRO140, CD4 (ibalizumab); b) Integrase inhibitors: raltegravir, elvitegravir, globoidnan A; c) Maturation inhibitors: Bevirimat, Vivecon; d) Neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; e) Nucleosides and nucleotides: Abacavir, acyclovir, adefovir, amdoxovir, apricitabine, brivudine, cidofovir, klevudine, dexerbucitabine, didanosine (DDI), erbucitabine, emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU), 3'-fluorosubstituted 2',3'-deoxynucleoside analogs (3'-fluoro-2',3'-dideoxythymidine (FLT) and 3'-fluoro-2',3'- (Including the group consisting of dideoxyguanosine (FLG)), homivirsen, ganciclovir, idoxuridine, lamivudine (3TC), L-nucleosides (including the group consisting of β-L-thymidine and β-L-2'-deoxycytidine), penciclovir, lasivir, ribavirin, stampidine, stabidine set (d4T), taribavirin (viramidine), terbivudine, tenofovir, trifluridine, valacyclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f) Non-nucleosides: amantadine, ateviridine, caplavillin, diallylpyrimidine (etravirine, rilpivirine), delavillin, docosanol, emibirin, efavirenz, foscarnet (phosphorylformate), imiquimod, interferon α, roviride, rhodenosine, methisazone, nevirapine, NOV-205, pegylated interferon α, podophyllotoxin, rifampicin, rimantadine, reximod (R-848), tromantadine; g) Protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, preconalil, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h) Other antiviral drugs: Abzyme, Arbidol, Caranolid A, Selagenin, Cyanobilin-N, Diallylpyrimidine, Epigallocatechin gallate (EGCG), Foscarnet, Griffiscin, Taribavirin (Pyramidine), Hydroxyurea, KP-1461, Miltefosin, Preconalil, Portmanto inhibitors, Ribavirin, Cericiclib; (5) ３ H １１ C １４ C １８ F ３２ P ３５ S ６４ Cu ６８ Ga ８６ Y ９９ Tc １１１ In １２３ I １２４ I １２５ I １３１ I １３３ Xe １７７ Lu ２１１ At, or ２１３ a radioisotope for radiotherapy that can be selected from Bi (radionuclide); (6) Tubulicin analogs, meitansinoid analogs, taxanoid (taxane) analogs, CC-1065 analogs, daunorubicin and doxorubicin compounds, amatoxin analogs, benzodiazepine dimers (dimers of pyrrolobenzodiazepine (PBD), tomaimycin, anthramycin, indolinobenzodiazepine, imidazobenzothiadiazepine, or oxazolidinobenzodiazepine), calicheamicin and engine antibiotics, actinomycin, azaserin, bleomycin, epirubicin, tamoxifen, idarubicin, dorastatin, auristantin Other cell-binding molecule-drug conjugates containing cytotoxic agents analogs thereof, such as (monomethyl auristatin E, MMAE, MMEF, auristatin PYE, auristatin TP, auristatin 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)), duocalmycin, geldanamycin, methotrexate, thiotepa, vindesine, vincristine, hemiasterine, nazmamide, microginine, radiosmin, topoisomerase I inhibitors, arterobactin, microsclerodermine, theonellamide, esperamicin, PNU-159682, and their analogues; (7) Other immunotherapeutic agents selected from the following: imiquimod, interferon (α or β), granulocyte colony-stimulating factor, cytokines, interleukins (IL-1 to IL-35), antibodies (trastuzumab, pertuzumab, bevacizumab, cetuximab, panitumumab, infliximab, adalimumab, basiliximab, daclizumab, omalizumab, PD-1 or PD-L1), protein conjugates (Abraxane); antibodies conjugated with drugs selected from trastuzumab-DM1, trastuzumab derquistecan (DS-8201a), inotuzumab ozogamicin, brentuximab vedotin, sacituzumab govitecan, glenbatumumab vedotin, and lorbotuzumab meltan; AN-152LMB2, TP-38, VB4-845, Cantuzumab Meltansine, AVE9633, SAR3419, CAT-8015, IMGN388, Milbetuximab Sorabtansine (IMGN853), Enfortumab Vedotin, Miratuzumab Doxorubicin, SGN-75 (Anti-CD70), Anti-Her3-Exceltecan, Anti-Trop-2-Exceltecan, Anti-CD79b-MMAE, anti-Her2-MMAE, anti-trop2-MMAE, anti-Her2-MMAF, anti-trop2-MMAF, anti-CD22-calicheamicin derivative, anti-CD22-MMAE, anti-Her2-aulistantin derivative, anti-Muc1-aulistatin derivative, anti-cMet-aulistatin derivative, or anti-claudin-18,2-aulistatin derivative; (8) A pharmaceutically acceptable salt, acid, or derivative of any of the above drugs.

11. The synergistic agent is selected from one or more of the following drugs, according to claim 10: abatacept, abiraterone acetate, abraxane, acetaminophen / hydrocodone, acalabrutinib, aducanumab, adalimumab, ADXS31-142, ADXS-HER2, afatinib dimalate, aldesleukin, alectinib, alemtuzumab, alitretinoin, alpelisib, ad-trastuzumab emtansine, amphetamine / dextroamphetamine, analotinib, anastrozole, apalutamide, aripiprazole, anthracycline, aripiprazole, atazanavir, atezolizumab, atorvastatin, averumab, axicaptagen Siloleucel, axitinib, bellinostat, live BCG, bevacizumab, bexarotene, blinatumomab, bortezomib, bosutinib, brentukimab, vedotin, brigatinib, budesonide, budesonide / formoterol, buprenorphine, cabazitaxel, cabozatinib, camrelizumab, cammatinib, capecitabine, carfilzomib, chimeric antigen receptor modified T (CAR-T) cells, celecoxib, ceritinib, cetuximab, tidamide, cyclosporine, cinacalcet, crizotinib, cobimetinib, cosentyx, crizotinib, CTL019, dabigatran, dabrafenib, dacarbazine, dacrizumab, dacomitinib, dacomotinib, dap Tomycin, daratumumab, darbepoetin alfa, darolutamide, darunavir, dasatinib, denileukin difutitox, denosumab, depacote, dexlansoprazole, dexmethylphenidate, dexamethasone, Dignicap Cooling System, dinutuximab, doxycycline, duloxetine, duvelisib, darumabumab, elotuzumab, emtricibine / rilpivirine / tenofovir, disoproxil fumarate, emtricibine / tenofovir / efavirenz, enfortumab Vedotin-EJFV, Enoxaparin, Ensartinib, Entrectinib, Enzalutamide, Epoetin alfa, Erlotinib, Erdafitinib, Esomeprazole, Eszopiclone, Etanercept, Everolimus, Exemestane, Everolimus, Exenatide ER, Ezetimibe, Ezetimibe / Simvastatin, FAM-TrastuzumabDuraxtecan, fenofibrate, filgrastim, fingolimod, fluticasone propionate, fluticasone / salmeterol, fulvestrant, gaziva, gefitinib, glatiramer, goserelin acetate, icotinib, imatinib, ibritumomab tiusetane, ibrutinib, idelalisib, ifosfamide, infliximab, imiquimod, ImmuCyst, immunoBCG, iniparib, insulin aspar Insulin detemir, insulin glargine, insulin lispro, interferon alpha, interferon alpha-1b, interferon alpha-2a, interferon alpha-2b, interferon beta, interferon beta-1a, interferon beta-1b, interferon gamma-1a, lapatinib, ipilimumab, ipratropium bromide / salbutamol, ixazomib, kanuma, lanreotide acetate, lenali Domide, lenariomid, lenvatinib mesylate, letrozole, levothyroxine, levothyroxine, lidocaine, linezolid, liraglutide, lisdexamfetamine, LN-144, loratinib, memantine, methylphenidate, metoprolol, mekinist, mericitabine / rilpivirine / tenofovir, modafinil, mometasone, Mycidac-C, necitumumab, neratinib, nilotinib, niraparib, nivolumab, Ofatumumab, Obinutuzumab, Olaparib, Olmesartan, Olmesartan / Hydrochlorothiazide, Omalizumab, Omega-3 Fatty Acid Ethyl Ester, Oncolin, Oseltamivir, Osimertinib, Oxycodone, Palbociclib, Palivizumab, Panitumumab, Panobinostat, Pazopanib, Pembrolizumab, Pexidartinib Hydrochloride, PD-1 Antibody, PD-L1 Antibody, Pemetrexed, Pertuzumab, Pneumococcal Conjugate Vaccine, PolatuzumabVedotin, pomalidomide, pregabalin, ProscaVax, propranolol, quetiapine, rabeprazole, radium-223 chloride, raloxifene, raltegravir, ramucirumab, ranibizumab, relugolix, regorafenib, rituximab, rivaroxaban, romidepsin, rosuvastatin, ruxolitinibulinate, salbutamol, savolitinib, semaglutide, selinexor, sevelamer, sildenafil, siltuximab, siproisel-T (Sipuleu) sel-T), sitagliptin, sitagliptin / metformin, solifenacin, solanezumab, sonidegib, sorafenib, sunitinib, tacrolimus, tacrimus, tadalafil, tamoxifen, Tafinlar, talimogenella herparepvec, talazoparib, telaprevir, talazoparib, temozolomide, tensirolimus, tenofovir / emtricitabine, tenofovir disoproxil fumarate, testosterone gel, talidomide, TICE BCG, tiotropium bromide, tisagenlecleucel, toremifene, trametinib, trastuzumab, trastuzumab / hyaluronidase-oysk, trabectin (ectenacidin 743), trametinib, tremelimumab, trifluridine / tipiracil, tretinoin, tislerizumab, uro-BCG, ustekinumab, valsartan, veliparib, vandetanib, vemurafenib, venetoclax, vorinostat, zanubrutinib, ziv-aflibercept, and zostavax, as well as their analogues, derivatives, pharmaceutically acceptable salts, carriers, diluents or excipients, or combinations thereof.

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