Compounds and their complexes

Abstract

Targeted conjugates containing specific topoisomerase inhibitors, and compounds useful in their synthesis, are provided. [Solution] Equation 5: TIFF2023065346000124.tif32161 [In the formula, R L*prot is Q-Prot N (In the formula, Prot N is an amine protecting group), and Q is an amino acid residue selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp; or a specific dipeptide residue; or a specific tripeptide residue; or a specific tetrapeptide residue. A specific example is allyl ((S)-1-(((S)-1-((4-amino-5-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate.

Description

[Technical field]

[0001] The present invention relates to targeted complexes containing specific topoisomerase inhibitors, and compounds useful in their synthesis, as well as the delivered warheads. [Background technology]

[0002] Topoisomerase inhibitors Topoisomerase inhibitors are compounds that block the action of topoisomerases (topoisomerase I and II), a class of enzymes that control the alteration of DNA structure by catalyzing the cleavage and rejoining of the phosphodiester backbone of DNA strands during the normal cell cycle.

[0003] The following compounds: [ka] (racemate) is disclosed in EP 0 296 597 (Example 63) and in Physiol. 1999, 14:1311-1323 (as racemic compound 34), where its biological activity is discussed along with that of a number of related compounds.

[0004] Irinotecan and exatecan derivatives, as well as various topoisomerase inhibitors such as doxorubicin, have been included in antibody-drug conjugates. For example, Daiichi Sankyo has DS-8201a, which is in clinical trials: [ka] [wherein the antibody is Her2 (Non-Patent Document 2)]. This ADC is an exatecan derivative: [ka] is released.

[0005] Non-Patent Document 3 states: [ka] The present invention discloses a complex having the following structure: [ka] [The formula contains a PABC (para-aminobenzyloxycarbonyl) group] is linked via an amino group having the formula:

[0006] Immunomedics has sacituzumab govitecan (IMMU-132) currently undergoing clinical trials (Non-Patent Document 4). [ka] [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Sugimori, M., et al., J Med Chem, 1998, 41, 2308-2318 (DOI: 10.1021 / jm970765q) [Non-Patent Document 2] Takegawa,N.,et al.,Int J Cancer,2017,141,1682-1689(DOI:10.1002 / ijc.30870) [Non-Patent Document 3] Burke,PJ,et al.,Bioconjugate Chem.,2009,20,1242-1250 [Non-Patent Document 4] Cardillo, TM, et al.,Bioconjugate Chem,2015,26(5),919-931,DOI:10.1021:acs.bioconjchem.5b00223 Summary of the Invention [Means for solving the problem]

[0008] In a general aspect, the present invention provides the following topoisomerase inhibitor derivatives having a linker connected to the ligand unit: * , drug unit): [ka] The present invention also provides a conjugate comprising: * , and intermediates for their synthesis, as well as delivered warheads.

[0009] A first aspect of the present invention is a compound represented by formula I: [ka] [In the formula, R L is a linker connected to the Ligand unit, which is: (ia): [ka] (In the formula, Q: [ka] (In the formula, Q X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue; X is: [ka] (wherein a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, and wherein at least b1 or b2=0 (i.e., only one of b1 and b2 may be non-zero), and at least c1 or c2=0 (i.e., only one of c1 and c2 may be non-zero); G L is the ligand unit; (ib): [ka] (In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1); and salts and solvates thereof.

[0010] A second aspect of the present invention provides a method for preparing a compound of the first aspect of the present invention, the method comprising at least one of the method steps set out below.

[0011] In a third aspect, the present invention provides a compound of formula IV: L-(D L ) p (IV) where L is a ligand unit (i.e., a targeting agent) and D L is represented by formula III: [ka] (In the formula, R LL teeth, (ia'): [ka] (wherein Q and X are as defined in the first aspect, and G LL is a linker connecting the Ligand units; and (ib'): [ka] (In the formula, R L1 and R L2 is a linker connected to the Ligand unit selected from: p is an integer from 1 to 20. or a pharma- ceutically acceptable salt or solvate thereof.

[0012] Thus, the conjugate comprises at least one Drug unit (A) linked by a Linker unit (i.e., a Ligand unit to which one or more Drug-Linker units are attached). * ) covalently bound to the target moiety. The Ligand unit is described more fully below and is a targeting agent that binds to a targeting moiety. The Ligand unit may, for example, specifically bind to a cellular component (cell binding agent) or to other target molecules of interest. Thus, the present invention also provides methods for the treatment of, for example, various cancers and autoimmune diseases. These methods include the use of the conjugates, where the Ligand unit is a targeting agent that specifically binds to a target molecule. The Ligand unit may, for example, be a protein, polypeptide or peptide, such as an antibody, an antigen-binding fragment of an antibody, or other binding agent, such as an Fc fusion protein.

[0013] Drug loading is expressed as the number of drug units (p) per ligand unit (e.g., antibody). Drug loading can range from 1 to 20 drug units (D) per ligand unit (e.g., Ab or mAb). In the case of a composition, p represents the average drug loading of the conjugates in the composition, and p ranges from 1 to 20.

[0014] A fourth aspect of the invention provides the use of a conjugate of the third aspect of the invention in the manufacture of a medicament for treating a proliferative disease. The fourth aspect also provides a conjugate of the third aspect of the invention for use in treating a proliferative disease.

[0015] One of ordinary skill in the art can readily determine whether a candidate compound treats a proliferative condition for any particular cell type. For example, the Examples below describe assays that can be conveniently used to evaluate the activity offered by a particular compound.

[0016] Nakada, et al., Bioorg Med Chem Lett, 26 (2016), 1542-1545 (DOI: 10.1016 / j.bmcl.2016.02.020) describes a series of ADCs: [ka] discusses and concludes that the reduced cytotoxicity of ADCs (1) and (2) may be due to steric hindrance of the released drug moiety at the site of action of degradative enzymes in tumor cells. This document teaches the importance of spacing the peptide group from the bulky released drug moiety. In contrast, in the present invention, the peptide group is directly linked to the bulky released drug moiety.

[0017] A fifth aspect of the present invention provides compound A, either as a single enantiomer or in enantiomerically enriched form: [ka] It is.

[0018] A sixth aspect of the present invention relates to a compound of formula VI: [ka] [Q is as defined in the first aspect].

[0019] definition C 5~6 Arylene: The term "C 5~6 "Arylene," as used herein, pertains to a divalent moiety obtained by removing two hydrogen atoms from aromatic ring atoms of an aromatic compound.

[0020] In this context, prefixes (e.g., C 5~6 ) indicates the number of ring atoms or range of number of ring atoms, whether carbon atoms or heteroatoms.

[0021] The ring atoms may all be carbon atoms, such as in a "carbarylene group," in which case the group is called phenylene (C 6 ).

[0022] Alternatively, the ring atoms may contain one or more heteroatoms, as in "heteroarylene groups." Examples of heteroarylene groups include, but are not limited to, those derived from: N 1 : Pyrrole (azole) (C 5 ), pyridine (azine) (C 6 ); O 1 :Furan (Oxol) (C 5 ); S 1 : Thiophene (thiol) (C 5 ); N 1 O 1 : Oxazole (C 5 ), isoxazole (C 5 ), Isoxazine (C 6 ); N 2 O 1 : Oxadiazole (furazan) (C 5 ); N 3 O 1 : Oxatriazole (C 5 ); N 1 S 1 : Thiazole (C 5 ), isothiazole (C 5 ); N 2 : Imidazole (1,3-diazole) (C 5 ), pyrazole (1,2-diazole) (C 5 ), pyridazine (1,2-diazine) (C 6 ), pyrimidine (1,3-diazine) (C 6 ) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C 6 ); and N 3 : Triazole (C 5 ), triazine (C6 ).

[0023] C 1~4 Alkyl: As used herein, the term "C 1~4 "Alkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 4 carbon atoms, which may be aliphatic or alicyclic, and may be saturated or unsaturated (e.g., partially unsaturated or fully unsaturated). 1~n "Alkyl" refers to a monovalent moiety obtained by removal of a hydrogen atom from a carbon atom of a hydrocarbon compound having 1 to n carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g., partially unsaturated or fully unsaturated). Thus, the term "alkyl" includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.

[0024] An example of a saturated alkyl group is methyl (C 1 ), ethyl (C 2 ), Propyl (C 3 ), and butyl (C 4 ), but are not limited to these.

[0025] An example of a saturated straight-chain alkyl group is methyl (C 1 ), ethyl (C 2 ), n-propyl (C 3 ), and n-butyl (C 4 ), but are not limited to these.

[0026] An example of a saturated branched alkyl group is isopropyl (C 3 ), iso-butyl (C 4 ), sec-Butyl (C 4 ), and tert-butyl (C 4 ) are mentioned.

[0027] C 2~4 Alkenyl: As used herein, the term "C 2~4 "Alkenyl" refers to an alkyl group having one or more carbon-carbon double bonds.

[0028] Examples of unsaturated alkenyl groups include ethenyl (vinyl, -CH=CH 2 ), 1-propenyl (-CH=CH-CH 3 ), 2-propenyl (allyl, -CH-CH=CH 2 ), isopropenyl (1-methylvinyl, -C(CH 3 )=CH 2 ), and butenyl (C 4 ), but are not limited to these.

[0029] C 2~4 Alkynyl: As used herein, the term "C 2~4 "Alkynyl" refers to an alkyl group having one or more carbon-carbon triple bonds.

[0030] Examples of unsaturated alkynyl groups include ethynyl (-C≡CH) and 2-propynyl (propargyl, -CH 2 —C≡CH).

[0031] C 3~4 Cycloalkyl: As used herein, the term "C 3~4 "Cycloalkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, having from 3 to 7 carbon atoms, including from 3 to 7 ring atoms, and which is also an alkyl group, which is a cyclyl group.

[0032] Examples of cycloalkyl groups include, but are not limited to, those derived from: Saturated monocyclic hydrocarbon compounds: Cyclopropane(C 3 ) and cyclobutane (C 4 );and Unsaturated monocyclic hydrocarbon compounds: Cyclopropene (C 3 ) and cyclobutene (C 4 ).

[0033] Connecting indicator: Expression [ka] In the superscript sign C(=O) and NH indicates the group to which the atom is attached. For example, an NH group is shown attached to a carbonyl (not part of the depicted moiety) and a carbonyl is shown attached to an NH group (not part of the depicted moiety).

[0034] salt It may be convenient or desirable to prepare, purify, and / or handle corresponding salts of the active compounds, e.g., pharma- ceutically acceptable salts. Examples of pharma-ceutically acceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).

[0035] For example, the compound may be anionic or have a functional group that may be anionic (e.g., -COOH may be -COO - If so, salts can be made with suitable cations. Examples of suitable inorganic cations include Na + and K + Alkali metal ions such as Ca 2+ and Mg 2+ Alkaline earth cations such as Al +3 Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH 4 + ) and substituted ammonium ions (e.g., NH 3 R + , N.H. 2 R 2 + , N.H.R. 3 + , N.R. 4 +). Some suitable examples of substituted ammonium ions include: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as those derived from amino acids such as lysine and arginine. One common example of a quaternary ammonium ion is N(CH 3 ) 4 + It is.

[0036] The compound is cationic or has a functional group which may be cationic (e.g., -NH 2 is -NH 3 + If so, salts can be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

[0037] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, trifluoroacetic acid, and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

[0038] solvate It may be convenient or desirable to prepare, purify, and / or handle a corresponding solvate of the active compound. The term "solvate" is used herein in the conventional sense to denote a complex of solute (e.g., active compound, a salt of active compound) and solvent. When the solvent is water, the solvate may conveniently be referred to as a hydrate, e.g., a monohydrate, a dihydrate, a trihydrate, etc.

[0039] Isomers Certain compounds of the present invention may exist in one or more particular geometric isomers, optical isomers, enantiomers, diastereoisomers, epiisomers, atropisomers, stereoisomers, tautomers, conformational isomers, or anomeric isomeric forms, including, but not limited to, cis and trans forms; E and Z forms; c, t, and r forms; endo and exo forms; R, S, and meso forms; D and L forms; d and l forms; (+) and (-) forms; keto, enol, and enolate forms; syn and anti forms; synclinal and anticlinal forms; α and β forms; axial and equatorial forms; boat, chair, twist, envelope, and half-chair forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or isomeric forms).

[0040] The term "chiral" refers to a molecule that has the property of not being superimposable on its mirror image partner, while the term "achiral" refers to a molecule that is superimposable on its mirror image partner.

[0041] The term "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0042] "Diastereomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers can be separated by high resolution analytical techniques such as electrophoresis and chromatography.

[0043] "Enantiomers" refers to two stereoisomers of a compound which are non-superimposable mirror images of one another.

[0044] The definitions and conventions of stereochemistry used herein generally follow those of S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric or chiral centers and therefore may exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers, and atropisomers, as well as mixtures thereof, such as racemic mixtures, are part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the chiral centers of the molecule. The prefixes d and l or (+) and (-) are used to specify the direction of rotation of plane polarized light by the compound, with (-) or l meaning that the compound is levorotatory. Compounds preceded by (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer may be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, which is devoid of optical activity.

[0045] "Enantiomerically enriched form" refers to a sample of a chiral material in which the enantiomeric ratio is greater than 50:50 and less than 100:0.

[0046] It should be noted that, except as described below with respect to tautomeric forms, when the term "isomer" is used herein, structural (or constitutional) isomers (i.e., isomers that differ not only in the arrangement of the atoms in space, but also in the connections between the atoms) are specifically excluded. For example, the methoxy group, -OCH 3 The description of isomer is of its structural isomer, the hydroxymethyl group -CH 2 Similarly, a description of ortho-chlorophenyl is not to be considered a description of its structural isomer, meta-chlorophenyl. However, a description of a class of structures may well include structural isomeric forms that fall within that class (e.g., C 1~7 Alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

[0047] The above exclusion does not pertain to tautomeric forms, e.g., keto, enol, and enolate forms, such as the following tautomeric pairs: keto / enol (as illustrated below), imine / enamine, amide / iminoalcohol, amidine / amidine, nitroso / oxime, thioketone / enethiol, N-nitroso / hydroxyazo, and nitro / aci-nitro. [ka]

[0048] The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible across a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by reorganization of some of the bonding electrons.

[0049] It should be noted that the term "isomer" specifically includes compounds having one or more isotopic substitutions. For example, H can be in any isotopic form, including: 1 H, 2 H(D), and 3 H(T); C can be in any isotopic form, such as: 12 C. 13 C, and 14 C; O may be in any isotopic form, such isotopic forms include: 16 O and 18 O; etc.

[0050] Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, e.g., 2 H (deuterium, D), 3 H (tritium), 11 C. 13 C. 14 C. 15 N, 18 F, 31 P, 32 P, 35 S, 36 Cl, and 125The compounds of the present invention may be used in metabolic studies, kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including drug or substrate tissue distribution assays, or in radiation therapy of patients. Therapeutic compounds of the present invention labeled or substituted with deuterium may have improved DMPK (drug metabolism and pharmacokinetics) properties in relation to distribution, metabolism, and excretion (ADME). Substitution with heavier isotopes, such as deuterium, may provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements. 18F-labeled compounds may be useful in PET or SPECT studies. Isotopically labeled compounds of the present invention and their prodrugs can generally be prepared by replacing nonisotopically labeled reagents with readily available isotope-labeled reagents and carrying out the procedures disclosed in the schemes or examples and preparations described below. In addition, the substitution of heavier isotopes, particularly deuterium (i.e., 2H or D), may provide certain therapeutic advantages due to greater metabolic stability, such as extended in vivo half-life or reduced dosage requirements or improved therapeutic index. It is understood that deuterium in this context is considered a substituent. The concentration of such heavy isotopes, particularly deuterium, can be defined by the isotopic enrichment factor. In the compounds of the present invention, any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom.

[0051] Unless otherwise indicated, reference to a particular compound encompasses all such isomeric forms, including (fully or partially) racemic and other mixtures thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallization and chromatographic means) of such isomeric forms are either known in the art or readily obtained by application of known methods, either as taught herein or in a known manner.

[0052] Ligand Unit The ligand units can be of any type, including proteins, polypeptides, peptides, and non-peptide agents that specifically bind to a target molecule. In some embodiments, the ligand units can be proteins, polypeptides, or peptides. In some embodiments, the ligand units can be cyclic polypeptides. These ligand units can include antibodies, or fragments of antibodies that contain at least one target molecule-binding site, lymphokines, hormones, growth factors, or any other cell-binding molecule or substance that can specifically bind to a target.

[0053] The terms "specifically bind" and "specific binding" refer to the binding of an antibody or other protein, polypeptide, or peptide to a given molecule (e.g., an antigen). Typically, an antibody or other molecule binds to at least about 1×10 7 M -1 and binds to a given molecule with an affinity that is at least two-fold greater than its affinity for binding to a nonspecific molecule other than the given molecule or a closely related molecule (e.g., BSA, casein).

[0054] Examples of Ligand units include those agents described for use in WO 2007 / 085930, which is incorporated herein.

[0055] In some embodiments, the ligand unit is a cell binding agent that binds to an extracellular target in a cell. Such a cell binding agent can be a protein, a polypeptide, a peptide, or a non-peptide agent. In some embodiments, the cell binding agent can be a protein, a polypeptide, or a peptide. In some embodiments, the cell binding agent can be a cyclic polypeptide. The cell binding agent can also be an antibody or an antigen-binding fragment of an antibody. Thus, in one embodiment, the invention provides an antibody drug conjugate (ADC).

[0056] Cell-binding agents The cell-binding agent can be of any type, including peptides and non-peptides, and can include antibodies or antibody fragments, lymphokines, hormones, hormone mimetics, vitamins, growth factors, nutrient transport molecules, or any other cell-binding molecule or substance that contains at least one binding site.

[0057] peptide In one embodiment, the cell binding agent is a linear or cyclic peptide comprising 4-30, preferably 6-20, consecutive amino acid residues.

[0058] In one embodiment, the cell binding agent is integrin α ν β 6 This peptide is more α-binding than XYS. ν β 6 It may be selective for

[0059] In one embodiment, the cell binding agent comprises an A20FMDV-Cys polypeptide. A20FMDV-Cys has the sequence: NAVPNLRGDLQVLAQKVARTC. Alternatively, variants of the A20FMDV-Cys sequence in which one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues are replaced with another amino acid residue may be used. Additionally, the polypeptide may have the sequence NAVXXXXXXXXXXXXXXXRTC.

[0060] antibody In this specification, the term "antibody" is used in the broadest sense as long as they exhibit the desired biological activity, and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), multivalent antibodies, and antibody fragments (Miller et al (2003) Jour. of Immunology 170:4854-4861). The antibody may be a mouse antibody, a human antibody, a humanized antibody, a chimeric antibody, or an antibody from another species. An antibody is a protein produced by the immune system that can recognize and bind to a specific antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has multiple binding sites (also called epitopes) that are recognized by the CDRs of multiple antibodies. Antibodies that specifically bind to different epitopes each have a different structure. Thus, one antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen-binding site or a portion thereof that immunologically binds to a target antigen of interest, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. Immunoglobulins can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. Immunoglobulins can be from any species, including human, murine, or rabbit origin.

[0061] An "antibody fragment" includes a portion of a full-length antibody, typically the antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab') 2and scFv fragments; bispecific antibodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions), and epitope-binding fragments of any of the above that immunologically bind to cancer cell antigens, viral antigens, or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0062] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed to different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be made by the hybridoma method first described in Kohler et al (1975) Nature 256:495, or may be made by recombinant DNA methods (see U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol., 222:581-597, or can be isolated from transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008) Curr. Opinion 20(4):450-459).

[0063] As used herein, the monoclonal antibody specifically includes a chimeric antibody, a humanized antibody, and a human antibody.

[0064] Examples of cell binding agents include those described for use in WO 2007 / 085930, which is incorporated herein.

[0065] Tumor associated antigens and cognate antibodies for use in embodiments of the present invention are listed below and are described in further detail in WO 2017 / 186894, pages 14 to 86, which are incorporated herein by reference.

[0066] (1) BMPR1B (bone morphogenetic protein receptor type IB) (2) E16 (LAT1, SLC7A5) (3) STEAP1 (six-transmembrane epithelial antigen of the prostate) (4) 0772P (CA125, MUC16) (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin) (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute transporter family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b) (7) Sema5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM), and short cytoplasmic domain, (semaphorin) 5B) (8)PSCAhlg(2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene) (9) ETBR (endothelin type B receptor) (10) MSG783 (RNF124, hypothetical protein FLJ20315) (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, prostate six-transmembrane epithelial antigen 2, six-transmembrane prostate protein) (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation 5 channel, subfamily M, member 4) (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor) (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein Barr virus receptor) or Hs.73792) (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-related beta), B29) (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase anchor protein 1a), SPAP1B, SPAP1C) (17)HER2 (ErbB2) (18) NCA (CEACAM6) (19)MDP(DPEP1) (20)IL20R-α(IL20Ra, ZCYTOR7) (21) Brevican (BCAN, BEHAB) (22)EphB2R(DRT, ERK, Hek5, EPHT3, Tyro5) (23)ASLG659(B7h) (24) PSCA (Prostate Stem Cell Antigen Precursor) (25) GEDA (26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3) (27)CD22 (B cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814) (27a)CD22 (CD22 molecule) (28) CD79a (CD79A, CD79α), immunoglobulin-related α, B cell-specific protein that covalently interacts with Igβ (CD79B) to form a complex with IgM molecules on the surface and transmits signals involved in B cell differentiation), pI: 4.84, MW: 25028 TM: 2[P] gene chromosome: 19q13.2). (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor activated by the CXCL13 chemokine; this G protein-coupled receptor functions in lymphocyte migration and humoral defense and plays a role in HIV-2 infection and possibly the pathogenesis of AIDS, lymphoma, myeloma, and leukemia); 372aa, pI:8.54 MW:41959 TM:7[P] gene chromosome:11q23.3. (30) HLA-DOB (β subunit of MHC class II molecule (Ia antigen) that binds peptides and presents them to CD4+ T lymphocytes); 273aa pI: 6.56, MW: 30820. TM: [P] gene chromosome: 6p21.3) (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, which may be involved in synaptic transmission and neurogenesis, and its deficiency may contribute to the pathophysiology of idiopathic detrusor instability); 422aa), pI: 7.63, MW: 47206 TM: 1[P] gene chromosome: 17p13.3). (32)CD72 (B cell differentiation antigen CD72, Lyb-2); 359aa, pI: 8.66, MW: 40225, TM: 15[P] gene chromosome: 9p13.3). (33) LY64 (Lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family that regulates B cell activation and apoptosis, the loss of which is associated with increased disease activity in patients with systemic lupus erythematosus); 661aa, pI: 6.20, MW: 74147 TM: 1[P] gene chromosome: 5q12). (34) FcRH1 (Fc receptor-like protein 1, a putative receptor for immunoglobulin Fc domains, containing C2-type Ig-like domains and ITAM domains, which may play a role in B lymphocyte differentiation); 429aa, pI: 5.28, MW: 46925 TM: 1[P] gene chromosome: 1q21-1q22) (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immune receptor that may play a role in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in several B cell malignancies); 977aa, pI: 6.88, MW: 106468, TM: 1[P] gene chromosome: 1q21) (36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, related to the EGF / heregulin family of growth factors and follistatin; 374aa) (37)PSMA-FOLH1 (Folate hydrolase (prostate-specific membrane antigen) 1) (38) SST (somatostatin receptor; note that there are five subtypes) (38.1) SSTR2 (somatostatin receptor 2) (38.2) SSTR5 (somatostatin receptor 5) (38.3)SSTR1 (38.4)SSTR3 (38.5)SSTR4 AvB6-both subunits (39+40) (39) ITGAV (integrin, αV) (40) ITGB6 (integrin, β6) (41) CEACAM5 (Carcinoembryonic antigen-related cell adhesion molecule 5) (42) MET (met proto-oncogene; hepatocyte growth factor receptor) (43) MUC1 (Mucin 1, cell surface associated) (44) CA9 (Carbonic anhydrase IX) (45) EGFRvIII (epidermal growth factor receptor (EGFR), transcript variant 3) (46)CD33 (CD33 molecule) (47)CD19 (CD19 molecule) (48) IL2RA (Interleukin 2 receptor, α); NCBI reference sequence: NM_000417.2); (49)AXL (AXL receptor tyrosine kinase) (50)CD30-TNFRSF8 (Tumor necrosis factor receptor superfamily, member 8) (51) BCMA (B cell maturation antigen)-TNFRSF17 (Tumor necrosis factor receptor superfamily, member 17) (52)CT Ags-CTA (Cancer Testis Antigen) (53) CD174 (Lewis Y)-FUT3 (Fucosyltransferase 3 (galactoside 3(4)-L-fucosyltransferase, Lewis blood group) (54) CLEC14A (C-type lectin domain family 14, member A; Genbank accession number NM175060) (55) GRP78-HSPA5 (Heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa) (56)CD70 (CD70 molecule) L08096 (57) Stem cell specific antigens, such as: 5T4 (see entry (63) below) CD25 (see entry (48) above) CD32 LGR5 / GPR49 Prominin / CD133 (58)ASG-5 (59)ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase 3) (60)PRR4 (Proline-rich 4 (lacrimal gland)) (61)GCC-GUCY2C (Guanylate cyclase 2C (heat-stable enterotoxin receptor) (62) Liv-1-SLC39A6 (solute carrier family 39 (zinc transporter), member 6) (63)5T4, trophoblast glycoprotein, TPBG-TPBG (trophoblast glycoprotein) (64)CD56-NCMA1 (Neural Cell Adhesion Molecule 1) (65)CanAg (tumor-associated antigen CA242) (66)FOLR1 (Folate Receptor 1) (67) GPNMB (Glycoprotein (transmembrane) nmb) (68)TIM-1-HAVCR1 (Hepatitis A Virus Cellular Receptor 1) (69)RG-1 / Prostate Tumor Targeting Mindin-Mindin / RG-1 (70)B7-H4-VTCN1 (V-set domain-containing T cell activation inhibitor 1 (71)PTK7 (PTK7 Protein Tyrosine Kinase 7) (72)CD37 (CD37 molecule) (73)CD138-SDC1 (Syndecan 1) (74)CD74 (CD74 molecule, major histocompatibility complex, class II invariant chain) (75) Claudin-CL (Claudin) (76) EGFR (Epidermal Growth Factor Receptor) (77)Her3 (ErbB3)-ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian)) (78)RON-MST1R (Macrophage-stimulating receptor 1 (c-met-related tyrosine kinase)) (79)EPHA2 (EPH receptor A2) (80)CD20-MS4A1 (membrane-spanning 4-domains, subfamily A, member 1) (81) Tenascin-C-TNC (Tenascin-C) (82)FAP (Fibroblast Activation Protein, α) (83)DKK-1 (Dickkopf 1 homolog (Xenopus laevis) (84)CD52 (CD52 molecule) (85)CS1-SLAMF7 (SLAM family member 7) (86) Endoglin-ENG (Endoglin) (87) Annexin A1-ANXA1 (Annexin A1) (88)V-CAM(CD106)-VCAM1(vascular cell adhesion molecule 1)

[0067] Further tumor-associated antigens and cognate antibodies of interest are: (89) ASCT2 (ASC transporter 2, also known as SLC1A5) It is.

[0068] ASCT2 antibodies are described in WO 2018 / 089393, which is incorporated herein by reference.

[0069] The cell-binding agent can be labeled, either prior to incorporation into the complex or as part of the complex, for example to aid in detection or purification of the cell-binding agent. The label can be a biotin label. In another embodiment, the cell-binding agent can be labeled with a radioisotope.

[0070] Treatment method The complexes of the present invention can be used in therapeutic methods. Also provided are therapeutic methods, comprising administering a therapeutically effective amount of a complex of formula IV to a subject in need of treatment. The term "therapeutically effective amount" refers to an amount sufficient to show benefit to the patient. Such benefit may be at least an improvement in at least one symptom. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of what is being treated. Decisions regarding prescriptions for treatment, such as dosage, are within the responsibility of the medical practitioner and other physicians.

[0071] The conjugates can be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (administration of active agents, including, for example, drugs), surgery, and radiation therapy.

[0072] The pharmaceutical compositions according to and for use in accordance with the present invention may contain, in addition to the active ingredient, i.e. the complex of formula IV, pharma- ceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other materials will depend on the route of administration, which may be oral, or by injection, e.g., cutaneous, subcutaneous, or intravenous injection.

[0073] The pharmaceutical composition for oral administration may be in tablet, capsule, powder, or liquid form. Tablets may contain a solid carrier or adjuvant. Liquid pharmaceutical compositions generally contain a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline solution, dextrose, or other sugar solution, or glycols, such as ethylene glycol, propylene glycol, or polyethylene glycol, may be included. Capsules may contain a solid carrier, such as gelatin.

[0074] For intravenous, cutaneous or subcutaneous injection, or injection into the site of pain, the active ingredient is in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has appropriate pH, isotonicity and stability.Those skilled in the art are well able to prepare suitable solutions using isotonic vehicles such as, for example, sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc.Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as necessary.

[0075] The conjugates may be used to treat proliferative and autoimmune diseases. The term "proliferative disease" refers to unwanted or uncontrolled cell proliferation of excess or abnormal cells, such proliferation being undesirable, e.g., neoplastic or hyperplastic proliferation, in vitro or in vivo.

[0076] Examples of proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cell proliferations, including neoplasms and tumors (e.g., histiocytoma, glioma, astrocytoma, osteoma), cancer (e.g., lung cancer, small cell lung cancer, gastrointestinal cancer, intestinal cancer, colon cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemia, psoriasis, bone disease, fibroproliferative disorders (e.g., of connective tissue), and atherosclerosis. Other cancers of interest include, but are not limited to, hematological, such as leukemia; malignancies, and lymphomas, such as non-Hodgkin's lymphoma, and subtypes, such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin's lymphoma, AML, and other cancers of B- or T-cell origin. Any type of cell can be treated, including, but not limited to, lung, gastrointestinal (including, e.g., intestine, large intestine), breast (breast), ovarian, prostate, hepatic (liver), renal (kidney), bladder, pancreas, brain, and skin.

[0077] Examples of autoimmune diseases include rheumatoid arthritis, autoimmune demyelinating diseases (e.g., multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves' disease, glomerulonephritis, autoimmune liver disease, inflammatory bowel disease (e.g., Crohn's disease), anaphylaxis, allergic reactions, Sjogren's syndrome, type I diabetes, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, polyendocrine deficiency, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenal inflammation, thyroiditis, and the like. thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia areata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, digital sclerosis, and telangiectasia), male and female autoimmune infertility, ankylosing spondylitis, ulcerative colitis, mixed connective tissue disease, nodular multiple myelopathy, pulmonary tuberculosis ... Arteritis, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, antiphospholipid syndrome, farmer's lung, erythema multiforme, postcardiac surgery syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird fancier's lung, toxic epidermal necrolysis, Alport syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Samter's syndrome group, eczema, lymphomatoid granulomatosis, Behçet's disease, Caplan's syndrome, Kawasaki disease, dengue fever, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum, psoriasis, erythroblastosis fetalis, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuchs' cyclitis, IgA nephropathy, Henoch-Schönlein purpura, graft-versus-host disease, transplant rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenström's macroglobulinemia, Evans' syndrome,and autoimmune gonadal dysfunction.

[0078] In some embodiments, the autoimmune disease is a disorder of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), a disorder of Th1 lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjogren's syndrome, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft-versus-host disease), or a disorder of Th2 lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft-versus-host disease). In general, disorders involving dendritic cells include disorders of Th1 lymphocytes or Th2 lymphocytes. In some embodiments, the autoimmune disease is a T cell-mediated immune disorder.

[0079] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in "targeted therapy" as well as conventional chemotherapy agents.

[0080] Examples of chemotherapeutic agents include: erlotinib (TARCEVA®, Genentech / OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS number 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS number 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(II), CAS number 15663-27-1), carboplatin (CAS number 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ), trastuzumab (HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide, CAS number 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine, NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1 / 2, HPPD, and rapamycin.

[0081] Further examples of chemotherapeutic agents include: oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), Sutent (SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007 / 044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787 / ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™ (Cremophor-free), an albumin-modified nanoparticulate formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill), vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chlorambucil, AG1478, AG1571 (SU5271;Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa and cyclophosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelanin; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin). cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatins; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, calicheamicin gamma 1I, calicheamicin omega 11 (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, Mitomycins such as esorubicin, idarubicin, nemorubicin, marcelomycin, and mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, keramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; fludarabine Purine analogues such as purine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; amino Levulinic acid; Eniluracil; Amsacrine; Bestravcil; Bisantrene; Edatrexate; Defofamine; Demecolcine; Diaziquone; Elfornithine; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Maytansinoids such as maytansine and ansamitocins; Mitoguazone; Mitoxantrone; Mopidamol; Nitramin; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine;PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veraculin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; cisplatin and carboplatin. platinum analogs such as vincristine; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche); ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharma- ceutically acceptable salts, acids, and derivatives of any of the above.

[0082] The definition of "chemotherapeutic agent" includes: (i) anti-hormonal agents that act to regulate or inhibit the action of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®, tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyphene, ketoxifene, LY117018, onapristone, and FARESTON® (toremifene citrate); (ii) anti-hormonal agents that act to regulate or inhibit the action of hormones on tumors, such as, for example, 4(5)-imidazole, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane, Pfizer), formstane, fadrozole, RIVIS, and the like. (iii) antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, and troxacitabine (a 1,3-dioxolane nucleoside cytosine analogue); (iv) protein kinase inhibitors such as MEK inhibitors (WO 2007 / 044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, in particular oblimersen (GENASENSE®, Genta Inc.Also included are antisense oligonucleotides that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, e.g., PKC-α, Raf, and H-Ras, such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) gene therapy vaccines, e.g., vaccines such as ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitors, such as LURTOTECAN®; ABAREFIX® rmRH; (ix) angiogenesis inhibitors, such as bevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0083] Also included within the definition of "chemotherapeutic agent" are therapeutic antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech / Biogen Idec), pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody-drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

[0084] Humanized monoclonal antibodies with potential therapeutic potential as chemotherapeutic agents to be combined with the conjugates of the invention include: alemtuzumab, apolizumab, aselizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidofusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlotinib, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, , motivizumab, natalizumab, nimotuzumab, norobizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resivizumab, and rovelizumab. , ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, and visilizumab.

[0085] formulation While it is possible for the conjugate to be used (eg, administered) alone, it is often desirable to present it as a composition or formulation.

[0086] In one embodiment, the composition is a pharmaceutical composition (e.g., formulation, preparation, medicament) comprising a conjugate as described herein and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0087] In one embodiment, the composition is a pharmaceutical composition comprising at least one conjugate as described herein together with one or more other pharma- ceutically acceptable ingredients known to those skilled in the art, including, but not limited to, pharma- ceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents, and sweetening agents.

[0088] In one embodiment, the composition further comprises other active agents, for example, other therapeutic or prophylactic agents.

[0089] Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts, see, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

[0090] Another aspect of the present invention relates to a method for making a pharmaceutical composition, the method comprising the step of: 11 The method includes mixing a [C]-radiolabelled conjugate or conjugate-like compound as defined herein with one or more pharma- ceutically acceptable ingredients well known to those skilled in the art, such as carriers, diluents, excipients, etc. When formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of active compound.

[0091] The term "pharmacologically acceptable," as used herein, refers to compounds, ingredients, materials, compositions, dosage forms, etc., that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of a subject (e.g., a human) without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0092] Formulations can be prepared by any method known in the pharmaceutical field. Such methods include the step of bringing active compound into association with carriers that constitute one or more accessory ingredients. In general, formulations are prepared by bringing active compound into association with carriers (e.g., liquid carriers, finely divided solid carriers, etc.) uniformly and intimately, and then shaping the product if necessary.

[0093] The formulations may be prepared to provide fast or slow release; immediate, delayed, sustained, or extended release; or a combination thereof.

[0094] Suitable formulations for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). Such liquids may additionally contain other pharma- ceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant bodily fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of isotonic carriers suitable for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active ingredient in the liquid is about 1 ng / ml to about 10 μg / ml, e.g., about 10 ng / ml to about 1 μg / ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0095] Dosage Those skilled in the art will appreciate that appropriate dosages of the conjugates and compositions containing the conjugates may vary from patient to patient. Determining optimal dosages will generally involve balancing the level of therapeutic benefit against any risk or adverse side effects. The selected dosage level will depend on a variety of factors, including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds, and / or materials used in combination, the severity of the condition, and the patient's species, sex, age, weight, condition, general health, and previous medical history. The amount of the compound and the route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, but generally, the dosage will be selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial adverse or dangerous side effects.

[0096] Administration can be accomplished in one dose, which can be continuous or intermittent (e.g., divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those skilled in the art and will vary with the formulation used for treatment, the purpose of the treatment, the target cells being treated, and the subject being treated. Single or multiple administrations can be administered with the dose level and pattern selected by the attending physician, veterinarian, or clinician.

[0097] In general, suitable doses of active compound are in the range of about 100 ng to about 25 mg (more typically about 1 μg to about 10 mg) per kilogram of subject body weight per day. Where the active compound is a salt, ester, amide, prodrug, or the like, the amount administered is calculated based on the parent compound, so that the actual weight used will be increased proportionately.

[0098] The above dosages may apply to an effective amount of compound releasable upon cleavage of the conjugate or linker.

[0099] For prevention or treatment of disease, the appropriate dosage of the ADC of the invention will depend on the type of disease to be treated, the severity and course of the disease as defined above, whether the molecule is administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to the antibody, and the judgment of the attending physician. The molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 100 mg / kg or more of the molecule is a candidate initial dosage to be administered to the patient, whether, for example, in a single or multiple divided doses, or by continuous infusion. For repeated administration over several days or longer depending on the condition, treatment is sustained until a desired suppression of disease symptoms occurs. Other dosing regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0100] Drug Payload Drug loading (p) is the average number of drugs per ligand unit, which can be a cell binding agent (e.g., an antibody).

[0101] The average number of drugs per antibody when preparing ADCs from conjugation reactions can be characterized by conventional methods such as UV, reversed-phase HPLC, HIC, mass spectrometry, ELISA assays, and electrophoresis. The quantitative distribution of ADCs with respect to p can also be measured. ELISA can measure the average value of p in a particular preparation of ADCs (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11:843-852). However, the distribution of p(drug) values ​​cannot be distinguished due to the detection limits of antibody-antigen binding and ELISA. In addition, ELISA assays for detecting antibody-drug conjugates do not reveal where the drug moiety is attached to the antibody, e.g., to the heavy or light chain fragment, or to a particular amino acid residue. In some cases, separation, purification, and characterization of homogeneous ADCs with a particular value of p from ADCs with different drug loadings can be achieved by means such as reversed-phase HPLC or electrophoresis. Such techniques are also applicable to other types of conjugates.

[0102] For some antibody-drug conjugates, p may be limited by the number of binding sites on the antibody. For example, an antibody may have only one or more cysteine ​​thiol groups, or may have only one or more sufficiently reactive thiol groups to which a linker can be attached. Higher drug loading may cause aggregation, insolubility, toxicity, or loss of cell permeability for certain antibody-drug conjugates.

[0103] Typically, fewer than the theoretical maximum number of drug moieties are conjugated to the antibody during the conjugation reaction. An antibody may contain, for example, many lysine residues that do not react with a drug linker. Only the most reactive lysine groups can react with an amine-reactive linker reagent. Also, only the most reactive cysteine ​​thiol groups can react with a thiol-reactive linker reagent. In general, an antibody does not contain many, if any, free reactive cysteine ​​thiol groups that can be linked to a drug moiety. Most cysteine ​​thiol residues in the compound's antibody exist as disulfide bridges and must be reduced with a reducing agent (such as dithiothreitol (DTT) or TCEP) under partial or total reducing conditions. The loading capacity (drug / antibody ratio) of an ADC can be controlled in several different ways, including: (i) limiting the molar excess of drug linker relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reducing conditions of cysteine ​​thiol modification.

[0104] Certain antibodies have reducible interchain disulfides, i.e., cysteine ​​bridges. Antibodies can be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Thus, each cysteine ​​bridge theoretically becomes two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies by converting amines to thiols by reaction of lysine with 2-iminothiolane (Traut's Reagent). Reactive thiol groups can be introduced into antibodies (or fragments thereof) by engineering one, two, three, four, or more cysteine ​​residues (e.g., preparing a mutant antibody containing one or more non-natural cysteine ​​amino acid residues). U.S. Pat. No. 7,521,541 teaches antibody engineering by introduction of reactive cysteine ​​amino acids.

[0105] Cysteine ​​amino acids that are in the reactive sites of antibodies and do not form interchain or intermolecular disulfide bridges can be engineered (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729; U.S. Pat. No. 7,521,541; U.S. Pat. No. 7,723,485; WO 2009 / 052249). The engineered cysteine ​​thiols can react with drug linkers of the invention bearing thiol-reactive electrophilic groups (such as maleimides or α-haloamides) to form ADCs bearing cysteine ​​engineered antibodies. Thus, the placement of the drug unit can be designed, controlled, and known. Drug loading can be controlled because engineered cysteine ​​thiol groups typically react with drug linker reagents in high yield. An IgG antibody can be engineered to introduce a cysteine ​​amino acid by substitution at a single site in the heavy or light chain, resulting in a symmetric antibody with two new cysteines. Drug loadings approaching 2 can be achieved with near-homogeneity of the conjugated product ADC.

[0106] When two or more nucleophilic or electrophilic groups of an antibody react with a drug linker, the resulting product can be a mixture of ADC compounds with a distribution of drug units attached to the antibody (e.g., 1, 2, 3, etc.). Liquid chromatography methods such as polymeric reversed phase (PLRP) and hydrophobic interaction (HIC) can separate compounds from the mixture with drug loading values. Although preparations of ADCs with a single drug loading value (p) can be isolated, such ADCs with a single drug loading value can still be a heterogeneous mixture because multiple drug units can be attached to different sites on the antibody via the linker.

[0107] Thus, an antibody-drug conjugate composition of the invention may comprise a mixture of antibody-drug conjugates in which an antibody bears one or more drug moieties, and the drug moieties may be attached to the antibody at different amino acid residues.

[0108] In one embodiment, the average number of drugs per cell binding agent ranges from 1 to 20. In some embodiments, the range is selected from 1 to 10, 2 to 10, 2 to 8, 2 to 6, and 4 to 10.

[0109] In some embodiments, there is one drug per cell-binding agent.

[0110] General synthesis route Formula I[R L is of formula Ia] is a compound of formula 2: [ka] [In the formula, R L* is -QH] to obtain a compound of formula 3: [ka] or an activated form thereof.

[0111] Such reactions may be carried out under amide coupling conditions.

[0112] The compound of formula 2 may be represented by formula 4: [ka] [In the formula, R L*prot Q-Prot N (In the formula, Prot N is an amine protecting group).

[0113] Compounds of formula 4 can be prepared using the Friedlander reaction to afford compounds of formula 5: [ka] with compound A3.

[0114] The compound of formula 5 may be prepared according to formula 6: [ka] by removing the trifluoroacetamide protecting group.

[0115] The compound of formula 6 can be coupled with compound I7: L*prot It can be synthesized by -OH.

[0116] A compound of formula I, wherein R L is of formula Ia or Ib] from compound I11 to compound R L It can be synthesized by coupling the -OH or an activated form thereof.

[0117] Amine Protecting Groups Amine protecting groups are well known to those skilled in the art. Particular reference is made to Greene's Protecting Groups in Organic Synthesis, Fourth Edition, John Wiley & Sons, 2007 (ISBN 978-0-471-69754-1), pages 696-871 for disclosure of suitable protecting groups.

[0118] Further priorities The following preferences may apply to all aspects of the invention described above or may relate to a single aspect. The preferences may be combined together in any combination.

[0119] Q X In one embodiment, Q is an amino acid residue. The amino acid may be a natural amino acid or an unnatural amino acid.

[0120] In one embodiment, Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, where Cit is citrulline.

[0121] In one embodiment, Q comprises a dipeptide residue. The amino acids in the dipeptide may be any combination of natural and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. If the linker is a cathepsin-labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. In that case, the dipeptide is the recognition site for the cathepsin.

[0122] In one embodiment, Q is: NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-Lys- C=O , NH -Val-Cit- C=O , NH -Phe-Cit- C=O , NH -Leu-Cit- C=O , NH -Ile-Cit- C=O , NH -Phe-Arg- C=O , NH -Trp-Cit- C=O , and NH -Gly-Val- C=O Selected from; In the formula, Cit is citrulline.

[0123] Preferably, Q is: NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-Lys- C=O , and NH -Val-Cit- C=O is selected from.

[0124] Most preferably, Q is NH -Phe-Lys- C=O , NH -Val-Cit- C=O or NH -Val-Ala- C=O is selected from.

[0125] Other dipeptide combinations of interest include: NH -Gly-Gly- C=O , NH -Gly-Val- C=O , NH -Pro-Pro- C=O , and NH -Val-Glu- C=O Examples include:

[0126] Other dipeptide combinations may also be used, including those described in Dubowchik et al., Bioconjugate Chemistry, 2002, 13, 855-869, incorporated herein by reference.

[0127] In some embodiments, Q is a tripeptide residue. The amino acids in the tripeptide may be any combination of natural and non-natural amino acids. In some embodiments, the tripeptide comprises natural amino acids. If the linker is a cathepsin-labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. In that case, the tripeptide is the recognition site for the cathepsin. Particularly interesting tripeptide linkers are: NH -Glu-Val-Ala- C=O NH-Glu-Val-Cit- C=O NH -αGlu-Val-Ala- C=O NH -αGlu-Val-Cit- C=O It is.

[0128] In some embodiments, Q is a tetrapeptide residue. The amino acids in the tetrapeptide may be any combination of natural and non-natural amino acids. In some embodiments, the tetrapeptide comprises natural amino acids. If the linker is a cathepsin labile linker, the tetrapeptide is the site of action for cathepsin-mediated cleavage. In that case, the tetrapeptide is the recognition site for the cathepsin. Tetrapeptide linkers of particular interest are: NH -Gly-Gly-Phe-Gly C=O and NH -Gly-Phe-Gly-Gly C=O It is. In some embodiments, the tetrapeptide is: NH -Gly-Gly-Phe-Gly C=O It is.

[0129] In the above representation of peptide residues: NH - represents the N-terminus of the residue, - C=O represents the C-terminus of the residue. * It binds to the NH of

[0130] Glu is a glutamic acid residue, i.e.: [ka] Represents.

[0131] αGlu is a glutamic acid residue when bound via the α chain, i.e.: [ka] Represents.

[0132] In one embodiment, where appropriate, amino acid side chains are chemically protected. Side chain protecting groups may be as described above. Protected amino acid sequences are enzymatically cleavable. For example, dipeptide sequences containing Boc side chain protected Lys residues are cleavable by cathepsin.

[0133] Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog, and are discussed above.

[0134] G L G L teeth,

[0135] [Table 1]

[0136] [Table 2] [Wherein, Ar is C 5~6 represents an arylene group, e.g., phenylene; X is C 1~4 represents alkyl.

[0137] In some embodiments, G L is G L1-1 and G L1-2 In some of these embodiments, G L is G L1-1 It is.

[0138] G LL G LL teeth:

[0139] [Table 3] [Wherein, Ar is C 5~6 represents an arylene group, e.g., phenylene; X is C1~4 represents alkyl.

[0140] In some embodiments, G LL is G LL1-1 and G LL1-2 In some of these embodiments, G LL is G LL1-1 It is.

[0141] X X is: [ka] [In the formula, a=0 to 5, b1=0 to 16, b2=0 to 16, c=0 or 1, and d=0 to 5, and wherein at least b1 or b2=0, and at least c1 or c2=0].

[0142] a may be 0, 1, 2, 3, 4, or 5. In some embodiments, a is 0 to 3. In some of these embodiments, a is 0 or 1. In further embodiments, a is 0.

[0143] b1 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b1 is 0 to 12. In some of these embodiments, b1 is 0 to 8, and may be 0, 2, 3, 4, 5, or 8.

[0144] b2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b2 is 0 to 12. In some of these embodiments, b2 is 0 to 8, and may be 0, 2, 3, 4, 5, or 8. Only one of b1 and b2 may not be 0.

[0145] c1 may be 0 or 1. c2 may be 0 or 1. Only one of c1 and c2 may be non-zero.

[0146] d may be 0, 1, 2, 3, 4, or 5. In some embodiments, d is 0 to 3. In some of these embodiments, d is 1 or 2. In further embodiments, d is 2. In further embodiments, d is 5.

[0147] In some embodiments of X, a is 0, b1 is 0, c1 is 1, c2 is 0, d is 2, and b2 can be 0 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8.

[0148] In some embodiments of X, a is 1, b2 is 0, c1 is 0, c2 is 0, d is 0, and b1 can be 0 to 8. In some of these embodiments, b1 is 0, 2, 3, 4, 5, or 8.

[0149] In some embodiments of X, a is 0, b1 is 0, c1 is 0, c2 is 0, d is 1, and b2 can be 0 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8.

[0150] In some embodiments of X, b1 is 0, b2 is 0, c1 is 0, c2 is 0, and one of a and d is 0. The other of a and d is 1-5. In some of these embodiments, the other of a and d is 1. In others of these embodiments, the other of a and d is 5.

[0151] In some embodiments of X, a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 can be 0 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8.

[0152] In some embodiments, R L is of formula Ib. In some embodiments, R LL is of formula Ib'.

[0153] R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group.

[0154] In some embodiments, R L1 and R L2 are both H.

[0155] In some embodiments, R L1 is H and R L2 is methyl.

[0156] In some embodiments, R L1 and R L2 Both are methyl.

[0157] In some embodiments, R L1 and R L2 together with the carbon atom to which they are attached form a cyclopropylene group.

[0158] In some embodiments, R L1 and R L2 together with the carbon atom to which they are attached form a cyclobutylene group.

[0159] In group Ib, in some embodiments, e is 0. In other embodiments, e is 1 and the nitro group may be at any available position on the ring. In some of these embodiments, it is at the ortho position. In others of these embodiments, it is at the para position.

[0160] In some embodiments of the fifth aspect of the invention, the enantiomerically enriched form has an enantiomeric ratio of greater than 60:40, 70:30, 80:20, or 90:10. In further embodiments, the enantiomeric ratio is greater than 95:5, 97:3, or 99:1.

[0161] In some embodiments, R L teeth,

[0162] [Table 4]

[0163] [Table 5] In some embodiments, R LL is the above R L It is a group derived from the group.

[0164] In one embodiment of the first aspect of the invention, the compound of formula I is: [ka] It is.

[0165] Further priorities In some embodiments, the compound of formula I has formula I P : [ka] [In the formula, R LP is a linker connected to a cell binding agent, which is: (ia): [ka] (In the formula, Q P teeth: [ka] (In the formula, Q XP Q P is an amino acid residue, a dipeptide residue, or a tripeptide residue; X P teeth: [ka] (In the formula, aP=0 to 5, bP=0 to 16, cP=0 or 1, and dP=0 to 5); G L is the ligand unit; (ib): [ka] (In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1); and salts and solvates thereof.

[0166] aP may be 0, 1, 2, 3, 4, or 5. In some embodiments, aP is 0 to 3. In some of these embodiments, aP is 0 or 1. In further embodiments, aP is 0.

[0167] bP may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b is 0 to 12. In some of these embodiments, bP is 0 to 8, and may be 0, 2, 4, or 8.

[0168] cP may be 0 or 1.

[0169] dP may be 0, 1, 2, 3, 4, or 5. In some embodiments, dP is 0-3. In some of these embodiments, dP is 1 or 2. In further embodiments, dP is 2.

[0170] X P In some embodiments of the present invention, aP is 0, cP is 1, dP is 2, and bP can be from 0 to 8. In some of these embodiments, bP is 0, 4, or 8.

[0171] Q above for compounds of formula I X and preferred, where appropriate, Q XP may be applied to.

[0172] For compounds of formula I, L , R L1 , R L2 and e is preferably of formula I P This may also be applied to compounds of the above formula:

[0173] In some embodiments, the conjugate of formula IV is P : L-(D LP ) p (IV P ) where L is a ligand unit (i.e., a targeting agent) and D LP is represented by formula III P : [ka] (In the formula, R LLP teeth, (ia'): [ka] (In the formula, Q P and X P is as defined above, and G LL is a linker connecting the Ligand units; and (ib'): [ka] (In the formula, R L1 and R L2 is a linker connected to the Ligand unit selected from: p is an integer from 1 to 20. or a pharma- ceutically acceptable salt or solvate thereof.

[0174] In some embodiments, the compound of formula I has formula IP2 : [ka] [In the formula, R LP2 is a linker connected to a cell binding agent, which is: (ia): [ka] (In the formula, Q: [ka] (In the formula, Q X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue; X P2 teeth: [ka] (wherein aP2=0 to 5, b1P2=0 to 16, b2P2=0 to 16, cP2=0 or 1, dP2=0 to 5, and at least b1P2 or b2P2=0 (i.e., only one of b1 and b2 may not be 0); G L is the ligand unit; (ib): [ka] (In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1); and salts and solvates thereof.

[0175] aP2 may be 0, 1, 2, 3, 4, or 5. In some embodiments, aP2 is 0 to 3. In some of these embodiments, aP2 is 0 or 1. In further embodiments, aP2 is 0.

[0176] b1P2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b1P2 is 0 to 12. In some of these embodiments, b1P2 is 0 to 8, and may be 0, 2, 3, 4, 5, or 8.

[0177] b2P2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b2P2 is 0 to 12. In some of these embodiments, b2P2 is 0 to 8, and may be 0, 2, 3, 4, 5, or 8.

[0178] Only one of b1P2 and b2P2 may be non-zero.

[0179] cP2 may be 0 or 1.

[0180] dP2 may be 0, 1, 2, 3, 4, or 5. In some embodiments, dP2 is 0-3. In some of these embodiments, dP2 is 1 or 2. In further embodiments, dP2 is 2. In further embodiments, dP2 is 5.

[0181] X P2 In some embodiments, aP2 is 0, b1P2 is 0, cP2 is 1, dP2 is 2, and b2P2 can be 0 to 8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5, or 8.

[0182] X P2In some embodiments, aP2 is 1, b2P2 is 0, cP2 is 0, dP2 is 0, and b1P2 may be 0 to 8. In some of these embodiments, b1P2 is 0, 2, 3, 4, 5, or 8.

[0183] X P2 In some embodiments, aP2 is 0, b1P2 is 0, cP2 is 0, dP2 is 1, and b2P2 may be 0 to 8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5, or 8.

[0184] X P2 In some embodiments, b1P2 is 0, b2P2 is 0, cP2 is 0, and one of aP2 and dP2 is 0. The other of aP2 and d is 1-5. In some of these embodiments, the other of aP2 and d is 1. In others of these embodiments, the other of aP2 and dP2 is 5.

[0185] Q above for compounds of formula I X is preferably, where appropriate, of formula Ia P2 Q X may be applied to.

[0186] For compounds of formula I, L , R L1 , R L2 and e is preferably of formula I P2 This may also be applied to compounds of the above formula:

[0187] In some embodiments, the conjugate of formula IV is P2 : L-(D LP2 ) p (IV P2 ) where L is a ligand unit (i.e., a targeting agent) and D LP2 is represented by formula III P2 : [ka] (In the formula, R LLP2 teeth, (ia'): [ka] (Wherein, Q and X P2 is as defined above, and G LL is a linker connecting the Ligand units; and (ib'): [ka] (In the formula, R L1 and R L2 is a linker connected to the Ligand unit selected from: p is an integer from 1 to 20. or a pharma- ceutically acceptable salt or solvate thereof. [Brief description of the drawings]

[0188] [Figure 1] FIG. 1 shows a plot of the mean tumor growth in Example 15. EXAMPLES

[0189] General information Flash chromatography was performed using a Biotage® Isolera™. Fractions were checked for purity using thin layer chromatography (TLC). TLC was performed using Merck Kieselgel 60 F254 silica gel (fluorescent indicator on aluminum plates). TLC visualization was performed with UV light.

[0190] Extraction and chromatography solvents were purchased from VWR, UK and used without further purification.

[0191] Unless otherwise stated, all pure compounds were purchased from Sigma-Aldrich.

[0192] PEGylation reagents were obtained from Quanta biodesign US via Stratech UK.

[0193] LC / MS conditions Method A Positive mode electrospray mass spectrometry was performed using a Waters Aquity H-class SQD2.

[0194] The mobile phase used was solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). An initial composition of 5% B was held for 25 seconds, then increased from 5% B to 100% B over a period of 1 min 35 s'. The composition was held at 100% B for 50 seconds, then changed back to 5% B in 5 seconds and held there for 5 seconds. The total duration of the gradient run was 3.0 minutes. The flow rate was 0.8 mL / min. Detection was at 254 nm. Column: Waters Acquity UPLC® BEH Shield RP18 1.7 μm 2.1×50 mm at 50° C. equipped with a Waters Acquity UPLC® BEH Shield RP18 VanGuard precolumn, 130A, 1.7 μm, 2.1 mm×5 mm.

[0195] Method B HPLC (Waters Alliance 2695) was performed using a mobile phase of water (A) (0.1% formic acid) and acetonitrile (B) (0.1% formic acid).

[0196] The initial composition was 5% B held for 25 seconds, then increased from 5% B to 100% B over a period of 1 min 35 s'. The composition was held at 100% B for 50 seconds, then changed back to 5% B in 5 seconds and held there for 5 seconds. The total duration of the gradient run was 3.0 minutes. The flow rate was 0.8 mL / min. Wavelength detection range: 190-800 nm. Column: Waters Acquity UPLC® BEH Shield RP18 1.7 μm 2.1×50 mm at 50° C. equipped with a Waters Acquity UPLC® BEH Shield RP18 VanGuard precolumn, 130A, 1.7 μm, 2.1 mm×5 mm.

[0197] Method C HPLC (Waters Alliance 2695) was performed using a mobile phase of water (A) (0.1% formic acid) and acetonitrile (B) (0.1% formic acid).

[0198] The initial composition was 5% B held for 1 min, then increased from 5% B to 100% B over a period of 9 min. The composition was held at 100% B for 2 min, then changed back to 5% B in 0.10 min and held there for 3 min. The total duration of the gradient run was 15 min. Flow rate 0.6 mL / min. Wavelength detection range: 190-800 nm. Oven temperature: 50 °C. Column: ACE Excel 2 C18-AR, 2μ, 3.0 x 100 mm.

[0199] HPLC conditions Reversed-phase ultra-performance liquid chromatography (UFLC) was performed on a Shimadzu Prominence™ machine using a Phenomenex™ Gemini NX 5μC18 column (50°C) dimensions: 150×21.2 mm. The eluent used was solvent A (H 2 O containing 0.1% formic acid). 2 O) and solvent B (CH containing 0.1% formic acid) 3CN). All UFLC experiments were performed with the following gradient conditions: initial composition of 13% B increased to 30% B over a period of 3 min, then increased to 45% B over 8 min, increased to 100% again and held for 6 min, then decreased back to 13% in 2 min and held for 1 min. The total duration of the gradient run was 20.0 min. The flow rate was 20.0 mL / min and detection was at 254 and 223 nm.

[0200] NMR method Proton NMR chemical shift values ​​were measured on a Bruker AV400 at 400 MHz on the delta scale. The following abbreviations were used: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet; br, broad. Coupling constants are reported in Hz.

[0201] Synthesis of key intermediates [ka] a) N-(5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (I2) 5,6,7,8-Tetrahydronaphthalen-1-amine I1 (8.54 g, 58.0 mmol) was dissolved in dichloromethane (80 mL). Triethylamine (18 mL, 129 mmol) was added and the mixture was cooled to 0° C. Acetic anhydride (11.5 mL, 122 mmol) was added dropwise and upon complete addition the reaction mixture was allowed to warm to room temperature and stirred for 45 min, after which LCMS indicated the reaction was complete. The mixture was purified by CH 2 Cl 2 Dilute with H 2 O, saturated NaHCO 3 , washed with 10% citric acid, and the organic phase was washed with MgSO 4 The off-white solid was extracted with 1:3 Et 2 Trituration with O / isohexane gave I2 (10.8 g, 57.1 mmol, 98% yield) as a white solid which was used without further purification. LC / MS (Method A): Retention time 1.44 min (ES+) m / z 190 [M+H] +

[0202] b) N-(4-nitro-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (I3) N-(5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I2 (1.00 g, 5.2840 mmol) was added portionwise to sulfuric acid (15 mL, 281 mmol) at −5° C. Sodium nitrite (450 mg, 5.2945 mmol) was added portionwise to the reaction mixture and stirred at −5° C. for 30 min, after which LCMS showed no further reaction progress. The reaction mixture was poured onto ice with external cooling, and the aqueous mixture was diluted with CH 2 Cl 2 The organic phase was extracted with MgSO 4 Drying over RT and purification by Isolella (10-80% EtOAc in isohexane) afforded a mixture of N-(4-nitro-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I3 and N-(2-nitro-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (956 mg, 4.0811 mmol, 77% yield) as a white / yellow solid. LC / MS (Method A): Retention time 1.53 min (ES+) m / z 235 [M+H] +

[0203] c) N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (I4) N-(4-nitro-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide, I3 (1.01 g, 4.31 mmol), was dissolved in acetone (30 mL). Magnesium sulfate in water (3.9 mL, 5.9 mmol, 1.5 mol / L) was added and the mixture was cooled to 0° C. Potassium permanganate (2.07 g, 13.0 mmol) was added in small portions to the reaction mixture, the mixture was allowed to warm to room temperature and stirred for 50 min, when TLC showed the reaction was complete. The reaction mixture was filtered through Celite and the solid was removed with CHCl. 3 The resulting organic mixture was washed with H 2 HO, washed with brine, and MgSO 4Drying over RT and purification by Isolella (20-50% EtOAc in isohexane) gave a mixture of N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I4 and N-(2-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (709 mg, 2.86 mmol, 66%) as white / yellow solids. LC / MS (Method A): Retention time 1.44 min (ES+) m / z 190 [M+H] +

[0204] d) 8-Amino-5-nitro-3,4-dihydronaphthalen-1(2H)-one (I5) A mixture of N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I4 and N-(2-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (709 mg, 2.8561 mmol) and 6N hydrochloric acid (7 mL) was stirred at 80° C. for 2.5 hours, when LCMS showed the reaction was complete. The reaction mixture was cooled in an ice bath and 6N NaOH solution was added until the pH was basic. The aqueous mixture was purified by CH 2 Cl 2 The organic phase was extracted with MgSO 4 The mixture was dried over hexane and concentrated in vacuo. Isolera (0-50% EtOAc in isohexane) afforded 8-amino-5-nitro-3,4-dihydronaphthalen-1(2H)-one I5 (320 mg, 1.552 mmol, 54% yield) as a yellow / orange solid. LC / MS (Method A): Retention time 1.54 min (ES+) m / z 207 [M+H] +

[0205] e) 2,2,2-trifluoro-N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (I6) 8-Amino-5-nitro-3,4-dihydronaphthalen-1(2H)-one I5 (430 mg, 2.0854 mmol) was dissolved in dichloromethane (20 mL). Pyridine (340 μL, 4.20 mmol) was added and the mixture was cooled to 0° C. Trifluoroacetic anhydride (590 μL, 4.197 mmol) was added and stirred for 30 min, after which LCMS showed the reaction was complete. The mixture was purified by CH 2 Cl 2 Dilute with H 2 O and the organic phase was washed with MgSO 4 After drying over rt and concentration in vacuo, 2,2,2-trifluoro-N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I6 (630 mg, 2.0846 mmol, >99% yield) was obtained as a yellow solid, which was used without further purification. LC / MS (Method A): Retention time 1.86 min (ES+) m / z 301X [MH] -

[0206] f) N-(4-amino-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)-2,2,2-trifluoroacetamide (I7) Zinc (2.73 g, 41.7 mmol) was suspended in methanol (80 mL), formic acid (4 mL), and water (4 mL) and the mixture was cooled to 0° C. 2,2,2-trifluoro-N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I6 (568 mg, 2.0865 mmol) was added in small portions and the mixture was stirred at 0° C. for 30 min, when LCMS showed the reaction was complete. The reaction mixture was filtered and the filtrate was diluted with EtOAc and dissolved in saturated NaHCO 3 The organic phase was washed with MgSO 4 After drying over 100° C. and concentration in vacuo, N-(4-amino-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)-2,2,2-trifluoroacetamide I7 (568 mg, 2.0865 mmol, >99% yield) was obtained as a yellow solid, which was used without further purification. LC / MS (Method A): Retention time 1.65 min (ES+) m / z 273 [M+H] +

[0207] g) N-(4-acetamido-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)-2,2,2-trifluoroacetamide (I8) N-(8-amino-4-oxo-tetralin-5-yl)-2,2,2-trifluoro-acetamide I7 (568 mg, 2.0865 mmol) was dissolved in dichloromethane (20 mL). Triethylamine (580 μL, 4.16 mmol) was added followed by acetyl chloride (297 μL, 4.173 mmol) and the mixture was stirred for 30 min, when LCMS showed the reaction was complete. The reaction mixture was purified by CH 2 Cl 2 Dilute with H 2 O and the organic phase was washed with MgSO 4 After drying over 100° C. and concentration in vacuo, N-(8-acetamido-4-oxo-tetralin-5-yl)-2,2,2-trifluoro-acetamide I8 (655 mg, 2.084 mmol, >99% yield) was obtained as a yellow solid, which was used without further purification. LC / MS (Method A): Retention time 1.55 min (ES+) m / z 315 [M+H] +

[0208] h) N-(4-amino-5-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (I9) N-(8-acetamido-4-oxo-tetralin-5-yl)-2,2,2-trifluoro-acetamide I8 (2.77 g, 8.81 mmol) was dissolved in methanol (240 mL) and water (17 mL). Potassium carbonate (4.88 g, 35.3 mmol) was added and the mixture was stirred at 50° C. for 1.5 h, when LCMS showed the reaction was complete. The reaction mixture was cooled and concentrated in vacuo to give CH. 2 Cl 2 Dissolve in 10% MeOH in H 2 The organic phase was washed with MgSO 4 Dry on a sieve and perform isolator chromatography (CH 2 Cl 2Purification by elution with 2-15% MeOH in hexane afforded N-(8-amino-1-oxo-tetralin-5-yl)acetamide I9 (1.20 g, 5.50 mmol, 62.3% yield) as a yellow solid. LC / MS (Method A): Retention time 0.98 min (ES+) m / z 219 [M+H] +

[0209] [ka] i) (S)-N-(9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)acetamide (I10) N-(8-amino-1-oxo-tetralin-5-yl)acetamide I9 (641 mg, 2.94 mmol, 1.0 equiv), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione A3 (840 mg, 3.19 mmol, 1.1 equiv), and PPTS (740 mg, 2.95 mmol, 1.0 equiv) were dissolved in toluene (60 mL) and stirred at reflux for 3 h, when LCMS indicated that I9 was consumed. The reaction mixture was cooled and concentrated in vacuo. The resulting solid was triturated with acetonitrile followed by acetone to give I10 as a brown solid (1.26 g, 96%) with traces of TsOH contamination. LC / MS (Method A): Retention time 1.32 minutes (ES+) m / z447[M+H] +

[0210] j) (S)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (I11) (S)-N-(9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)acetamide (I10) (1.26 g, 2.83 mmol, 1.0 equiv.) was reacted with 1H 2 The mixture was stirred at 80° C. for 5 h, and LCMS showed that I10 was consumed. 2 Dilution with O and concentration in vacuo afforded (S)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione, I11 (1.51 g, 2.85 mmol, 90% by mass, 101% yield) as a red crystalline solid. LC / MS (Method A): Retention time 1.36 min (ES+) m / z 405 [M+H] +。

[0211] Alternative synthesis of I11 [ka]

[0212] IPC, Purity, and Assay Methods for this Synthesis

[0213] [Table 6]

[0214] a) 5-Bromo-8-nitro-tetralin-1-one (I13) A solution of potassium nitrate (1.15 eq, 13.83 g) dissolved in sulfuric acid (concentrated, 5.0 rel vol, 160 mL) was added under nitrogen to a solution of 5-bromotetralin-1-one (I12) (1.0 eq, 26.77 g) in sulfuric acid (concentrated, 5.0 rel vol, 160 mL) (addition time 4-12 h, maintaining temperature below 10 °C). Once the reaction was complete, the reaction mixture was transferred to a flask containing water (36 rel vol, 1.15 L), adjusting the transfer rate to keep the temperature below 10 °C. The resulting solid was filtered, washed three times with water (4.0 rel vol, 128 mL), and then dried at about 40 °C for 24 h. The dry cake was dissolved in a mixture of acetone (2.5 rel vol, 80 mL) and water (0.38 rel vol, 12.2 mL), heated to about 75 °C, and then cooled to about 20 °C. The resulting solid was removed by filtration. The solvent was exchanged to ethanol by distillation to reduce the volume of the solution to 2.0 relative volumes (64 mL). The solution was cooled to about 25° C. and the resulting solid was collected by filtration. The solid was washed with ethanol (1.0 relative volumes, 32 mL) and then dried under vacuum at 40° C. to give 5-bromo-8-nitro-tetralin-1-one, I13 (15.36 g, 40%) as a brown solid; rt 14.0 min.

[0215] Method 1: IPC, Purity, and Assay Method for Bromo-8-nitro-tetralin-1-one

[0216] [Table 7]

[0217] b) N-(8-nitro-1-oxo-tetralin-5-yl)acetamide (I14) A solution of bromo-8-nitro-tetralin-1-one (I13) (1.0 equiv., 18.0 g, 90.6% ww), acetamide (1.2 equiv., 4.72 g), tris(dibenzylideneacetone)dipalladium(0) (0.01 equiv., 0.61 g), and potassium phosphate (1.4 equiv., 19.8 g) in dioxane (15 rel. vol., 270 mL) under nitrogen was heated to about 70° C. Upon completion of the reaction, the solution was cooled to about 20° C., diluted with dioxane (5 rel. vol., 90.0 mL), and filtered. The solvent was exchanged into ethanol to reduce the volume to a total reaction volume of 3 rel. vol. (54.0 mL). The solution was cooled to about 20° C. and the resulting solid was collected by filtration and washed with MTBE (methyl tert-butyl ether) (1.0 rel. vol., 18.0 mL). The solid was dried under vacuum at 40° C. to give N-(8-nitro-1-oxo-tetralin-5-yl)acetamide I14 (10.0 g, 60.6%) as a dark yellow solid; room temperature 8.86 min.

[0218] c) N-(8-amino-1-oxo-tetralin-5-yl)acetamide (I15) Palladium hydroxide on carbon (20% w / w, 0.15 equiv, 5.25 g) was added to a solution of N-(8-nitro-1-oxo-tetralin-5-yl)acetamide (I14) (1.0 equiv, 32.6 g) in methanol (40 rel vol, 1250 mL). The reaction mixture was placed under hydrogen atmosphere at about 40 psi and about 40° C. for 8 hours. The hydrogen was removed, replaced with nitrogen and the catalyst was removed by filtering through cellulose and washing the cellulose with methanol (4.0 rel vol, 130 mL). The solution volume was reduced to 4.0 rel vol by distillation and then diluted with MTBE (4 rel vol, 130 mL). The resulting solid was collected by filtration, washed with MTBE (2 rel vol, 65 mL) and dried under vacuum at 40° C. to give N-(8-amino-1-oxo-tetralin-5-yl)acetamide I15 (21.1 g, 77.8%) as a grey-green solid; room temperature 5.44 min.

[0219] d) 5,8-diaminotetralin-1-one (I16) A solution of N-(8-amino-1-oxo-tetralin-5-yl)acetamide (I15) (1.0 eq, 10.0 g) in hydrochloric acid (5 M, 6.0 rel vol, 60 mL) was held at about 90° C. for 3 hours. The temperature was reduced to 25° C. and sodium hydroxide (2 M, 4.0 rel vol, 40 mL) was added until a pH of 10.0 was achieved, while maintaining the temperature at 25° C. The resulting solid was collected by filtration and washed with water (2.0 rel vol, 20 mL). The wet cake was dissolved in tetrahydrofuran (60 rel vol, 600 mL) and filtered. The solution was concentrated to 5.0 rel vol and heptane (20 rel vol, 200 mL) was added. The solution was concentrated to 10.0 rel vol and more heptane (20 rel vol, 200 mL) was added, then the volume was reduced again to 10.0 rel vol. The resulting solid was collected by filtration and washed with heptane (5.0 rel vol, 50 mL). The solid was dried under vacuum at 40° C. for 17 h to give 5,8-diaminotetralin-1-one (I16) (6.90 g, 82.7%) as a yellow solid; 1H NMR (400 MHz DMSO-d6) δ ppm 1.82 (m, 2H), 2.38 (t, J=2.0 Hz, 2H), 2.47 (t, J=2.0 Hz, 2H), 6.34 (d, J=2.0 Hz, 1H), 6.68 (d, J=2.0 Hz, 1H); RT 3.90

[0220] e) (S)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (I11) A solution of 5,8-diaminotetralin-1-one (I16) (1.0 equiv., 5.0 g), (4S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10-trione (A3) (1.06 equiv., 7.9 g), and pyridinium para-toluenesulfonate (1.0 equiv., 7.2 g) in toluene (50.0 rel. vol., 250 mL) was kept at 120° C. for 15 h. The volume of the solution was reduced to 2.0 rel. vol. and then diluted with acetonitrile (20 rel. vol., 100 mL) and water (20 rel. vol., 100 mL). The resulting slurry was filtered and the solid was washed with aqueous acetonitrile (1:1, 20 rel. vol., 100 mL). The solid was slurried in aqueous methanol (water:MeOH 3:1, 40 rel vol, 200 mL), filtered and washed with aqueous methanol (1:1, 20 rel vol, 100 mL). The solid was slurried in water at 50° C. (60 rel vol, 300 mL), filtered and washed with water (10 rel vol, 50 mL). The solid was slurried with aqueous acetonitrile (water:acetonitrile, 1:3, 40 rel vol, 200 mL) at 30° C., filtered, washed with aqueous acetonitrile (water:acetonitrile, 1:3, 5 rel vol, 50 mL) and then dried under vacuum at 40° C. to give (S)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (I11) as a white solid (5.0 g, 43.7%); room temperature 5.13.

[0221] Synthesis of I18 [ka] a) tert-Butyl (S)-(2-((2-((1-((2-((4-amino-5-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (I17) Boc-GGFG-OH (227 mg, 0.52 mmol) and EEDQ (157 mg, 0.634 mmol) were dissolved in CH 2 Cl 2 (25 mL) and the mixture was stirred for 15 min until the peptide was dissolved. Compound I16 (100 mg, 0.56747 mmol) was then added and the mixture was left stirring until complete. After 1 h the reaction appeared to be 90% complete by LVMC. The mixture thickened as the product crashed out. The mixture was left for an additional hour before being dried in vacuo. The crude was purified by ethanol with Et 2 The solid was filtered and then dissolved in CH.O (50 mL). 2 Cl 2 (50 mL) for further purification. The solid was filtered and dried to give the product I17 (273 mg, 0.459 mmol, 80.9% yield) as a grey solid. Analytical data: LCMS 3 min: ES + =1.46 min, m / z595.7[M+H] + .

[0222] b) (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-2-oxoethyl)-3-phenylpropanamide (I18) The aniline I17 (450 mg, 1.045 mmol), lactone A5 (280 mg, 1.064 mmol), and pyridinium p-toluenesulfonate (273 mg, 1.086 mmol) were solubilized in toluene (20 mL) and the mixture was heated to 150 °C (high reflux). MeOH (4 mL) was added to aid in solubilizing the mixture. After 7 h, the crude reaction was concentrated in vacuo to dryness. The crude product was purified by silica gel chromatography (CHCl 3 / MeOH, 100% to 65:35) to give the product I18 (259 mg, 0.359 mmol, 78.1 yield). Analytical data: LCMS 3 min: ES +=1.17 min, m / z722.8[M+H] + .

[0223] Alternative synthesis of I16 [ka] a) 5-Fluoro-8-nitro-tetralin-1-one (I20) 5-Fluorotetralin-1-one, I19 (4.7 g, 29 mmol) was solubilized in 1 / 2 volume of sulfuric acid (120 mL) in a three-necked round bottom flask. The mixture was stirred until all solids were dissolved and then cooled to 0-5 °C. In a dropping funnel, potassium nitrate (3 g, 29.6730 mmol) was dissolved in the remaining half of the sulfuric acid (120 mL) at 0-5 °C. It was slowly added to the SM mixture, ensuring that the solution remained cold (45 min). It was stirred at 0-5 °C until completion. After that, the reaction mixture was quenched with water (250 mL) and left stirring at 0-5 °C. The solid was filtered and washed with water (50 mL). The solid was dried in a vacuum oven at 50 °C for 2 h. The crude solid was dissolved in Et 2 The wet cake was slurried in O overnight, then cooled to 0 °C and filtered. 2 0 (50 mL) and left to dry in a vacuum oven at 50 °C to give the pure product I20 (5.5 g, 26 mmol, 92% yield) as a light pink fine powder. LCMS (Method B): ES + =1.55 min, m / z210.1[M+H] + .

[0224] b) 5-amino-8-nitro-tetralin-1-one (I21) Compound I20 (2.7 g, 13 mmol) was dissolved in CH 3 Solubilize in CN (2.5 mL) and HO 2 NH in O (8 mL, 40 mmol) 4 OH (21% by weight) was added to a sealed pressure tube and heated to 185 °C. Upon completion, the mixture was transferred to a round bottom flask and concentrated in vacuo. The crude was purified by silica gel column chromatography (CHCl 3 / MeOH; 100 to 99:1) to give the pure product I21 (1.1 g, 5.3 mmol, 41% yield) as a black solid. LCMS (Method B): ES + =1.34 min, m / z207.1[M+H] + .

[0225] c) 5,8-diaminotetralin-1-one (I16) Compound I21 (1.35 g, 6.55 mmol) was dissolved in methanol (20 mL), H 2 The mixture was dissolved in a mixture of 1 mL of HO (1 mL), and formic acid (1 mL). Zinc (8.5 g, 130 mmol) was added slowly, ensuring the temperature remained below 40° C. A little more formic acid / HO was added. 2 O (0.5 mL) was added to drive the reaction to completion. The reaction mixture was filtered, and the filtrate was diluted with EtOAc and CH 2 Cl 2 The crude product was purified by silica gel column chromatography (CHCl 3 / EtOAc; 100 to 7:3, then CHCl 3 Dry loading onto a 1:1 hexane / MeOH column (99:1 to 98:2) gave the pure product I16 (1.015 g, 5.760 mmol, 88.0% yield). LCMS (Method B): ES + =0.2 min, m / z not observed.

[0226] Example 1 [ka]

[0227] a) Allyl ((S)-3-methyl-1-oxo-1-(((S)-1-oxo-1-((5-oxo-4-(2,2,2-trifluoroacetamido)-5,6,7,8-tetrahydronaphthalen-1-yl)amino)propan-2-yl)amino)butan-2-yl)carbamate (A1) DCC (6.54 g, 31.7 mmol) and HOPO (3.36 g, 30.2 mmol) were reacted with alloc-Val-Ala-OH (9.09 g, 31.7 mmol) and I7 (7.85 g, 28.8 mmol) at 25 °C in CH 2 Cl 2 (300 mL). The resulting mixture was left stirring overnight. The white solid formed during the reaction was filtered off and diluted with cold CH 2 Cl 2 The filtrate was washed with water (150 mL) and brine (150 mL). The organic layer was washed with MgSO 4 It was dried over ice, filtered and evaporated. The crude product was purified by silica gel chromatography (Hex / EtOAc, 60:40). The isolated product A1 was contaminated with co-eluting DCU (21.1 g, 140% yield). LC / MS (Method B): ES + =1.81 min, m / z527.6[M+H] + .

[0228] b) Allyl ((S)-1-(((S)-1-((4-amino-5-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (A2) The protected aniline A1 (18 g, 34.19 mmol) was dissolved in MeOH and H 2 Solubilize in a 10:1 mixture of 1:2 O (165 mL) and 2 CO 3 (10 g, 72.36 mmol) was added. The mixture was stirred at 50° C. until completion. The mixture was concentrated in vacuo to near dryness and the residue was extracted with CH 2 Cl 2 Dissolved in H 2 After washing with HO and brine, 4 The mixture was dried over ice, filtered and evaporated. The crude product was purified by silica gel chromatography (CHCl 3 / MeOH, 100% to 7:3). The isolated product A2 was contaminated with a coeluting impurity (10.71 g, 73% yield). LC / MS (Method B): ES +=1.46 min, m / z431.7[M+H] + .

[0229] c) Allyl ((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methylbutan-2-yl)carbamate (A4) Aniline A2 (450 mg, 1.045 mmol), lactone A3 (280 mg, 1.064 mmol), and pyridinium p-toluenesulfonate (273 mg, 1.086 mmol) were solubilized in toluene (20 mL) and the mixture was heated to 130 °C (high reflux). Occasionally, a few drops of MeOH were added to help solubilize the mixture. After 7 h, the crude reaction was concentrated in vacuo to dryness. The crude product was purified by silica gel chromatography (CHCl 3 / MeOH, 100% to 95:5) to give the product A4 (360 mg, 52.3% yield). LC / MS (Method B): ES + =1.51 min, m / z658.8[M+H] + .

[0230] d) Allyl(S)-2-amino-N-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (A5) A4 (543 mg, 0.82 mmol) and PdP (Ph 3 ) 4 (89 mg, 0.08 mmol) of CH 2 Cl 2 To the solution in (15 mL) was added excess piperidine (642 μL). The mixture was stirred at room temperature for 20 min, at which point the reaction was complete (monitored by LC / MS). The reaction mixture was diluted with CH 2 Cl2 (25 mL) and the organic phase was diluted with H 2 The organic phase was washed with O (25 mL) and brine (25 mL). 4 Drying over rt, filtration, and removal of excess solvent by rotary evaporation under reduced pressure gave crude product A5, which was used directly in the next step. LC / MS (Method B): ES + =1.15 min, m / z574.6[M+H] + .

[0231] e) 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide)-N-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-amide (1) Pyridine (83 μL, 1.03 mmol) and Mal-dPEG 8 -OTFP (767 mg, 1.03 mmol) was dissolved in dry CH under argon atmosphere. 2 Cl 2 (50 mL). The reaction was stirred overnight and, since the reaction had not gone to completion, 0.5 equivalents of Mal-dPEG was added. 8 -OTFP was added to try to accelerate the reaction. 2 Cl 2 (25 mL) and the organic phase was diluted with H 2 HO (2 × 50 mL), washed with brine, then MgSO 4 The crude was dried over ice, filtered, and the solvent was removed by rotary evaporation under reduced pressure. 2 O / CH 3 The mixture was purified by elution with a gradient of CN+0.05% FA and lyophilized to give 1 (1.189 g, 31% yield over two steps). LC / MS (Method B): ES + =1.43 min, m / z1149.3[M+H]+ .LC / MS (Method C):ES + =5.37 min, m / z1149.4[M+H] + .

[0232] Example 2 [ka] 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (2) Mal-caproic acid (56 mg, 0.26 mmol) and EDCI.HCl (51 mg, 0.26 mmol) were added to the dry CH 2 Cl 2 (20 mL). The reaction was stirred overnight and as the reaction was not complete an additional 0.5 equivalents of Mal-caproic acid and EDCI.HCl were added. The reaction was stirred in CH 2 Cl 2 (25 mL) and the organic phase was diluted with H 2 HO (2 × 50 mL), brine, and then MgSO 4 The crude was purified by silica gel column chromatography (CHCl 3 / MeOH 95:5) to give 2 (31.6 mg, 20% yield over two steps). LC / MS (Method B): ES + =1.56 min, m / z767.8[M+H] + .LC / MS (Method C) 15 minutes: ES + =6.05 min, m / z767.8[M+H] + .

[0233] Example 3 [ka] (S)-2-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)acetamido)-N-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (3) Azide-dPEG 3 - Acid (77.5 mg, 0.31 mmol) and EDCI.HCl (60 mg, 0.31 mmol) were added to the dry CH 2 Cl 2 (20 mL) was added to the solution. The reaction was stirred overnight and, since the reaction had not gone to completion, an additional 0.5 equivalents of azido-dPEG was added. 3 -OH and EDCI.HCl were added. The reaction was cooled to 5°C. 2 Cl 2 (25 mL) and the organic phase was diluted with H 2 HO (2 × 50 mL), brine, and then MgSO 4 After drying over 100° C., filtration and removal of excess solvent by rotary evaporation under reduced pressure, the crude was purified by preparative HPLC and the fractions were lyophilized to give pure 3 (92.2 mg, 24.7% yield over two steps). LC / MS (Method B): ES + =1.69 min, m / z789.9[M+H] + .LC / MS (Method C):ES + =6.68 min, m / z790.0[M+H] + .

[0234] Example 4 [ka] N-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-4,7,10,13,16-pentaoxanonadec-18-ynamide (4) Propargyl-dPEG 5 - Acid (56 mg, 0.19 mmol) and EDCI.HCl (37 mg, 0.19 mmol) were added to crude A5 (assumed 0.19 mmol) in dry CH under argon atmosphere. 2 Cl 2 (10 mL) was added to the solution. The reaction was stirred overnight and, since the reaction had not gone to completion, an additional 0.5 equivalents of propargyl-dPEG was added. 5 -OH and EDCI.HCl were added. The reaction was cooled to 5°C. 2 Cl 2 (25 mL) and the organic phase was diluted with H 2 HO (2 × 50 mL), brine, and then MgSO 4 After drying over 100° C., filtration and removal of excess solvent by rotary evaporation under reduced pressure, the crude was purified by preparative HPLC and the fractions were lyophilized to give pure 4 (22 mg, 16.7% yield over two steps). LC / MS (Method B): ES + =1.54 min, m / z860.9[M+H] + .LCMS(Method C):ES + =5.57 minutes, m / z860.9[M+H] + .

[0235] Example 5 [ka] (S)-2-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetamide)-N-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (5) PM-acetate-OSu (64 mg, 0.19 mmol) was dissolved in dry CH under argon atmosphere. 2 Cl 2 (10 mL). The reaction was not progressing so DIPEA (51 μL, 0.28 mmol) was added. The reaction was stirred until complete. The mixture was diluted with CH 2 Cl 2 (25 mL) and the organic phase was diluted with H 2 HO (2 × 50 mL), washed with brine, then MgSO 4 After drying over 100° C., filtration and removal of excess solvent by rotary evaporation under reduced pressure, the crude was purified by preparative HPLC and the fractions were lyophilized to give pure 5 (2.5 mg, 1.6% yield over two steps). LC / MS (Method B): ES + =1.54 min, m / z787.7[M+H] + .LC / MS (Method C):ES + =5.61 min, m / z787.8[M+H] + .

[0236] Example 6 [ka] (R)-2-((3-nitropyridin-2-yl)disulfanayl)propyl((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbamate (6) (i) (2R)-2-[(3-nitro-2-pyridyl)disulfanyl]propan-1-ol, A6 (25 mg, 0.1015 mmol, 1.0 equiv.), was dissolved in dichloromethane (1 mL). Pyridine (8.5 μL, 0.11 mmol, 1.0 equiv.) was added followed by triphosgene (11 mg, 0.0370685 mmol, 0.33 equiv.) and the mixture was stirred under Ar for 45 min. LCMS (Et 2 NH quench) indicated the formation of the corresponding carbamate.

[0237] (ii) (S)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (I11) (43 mg, 0.09026 mmol, 1.0 equiv.) was dissolved in dichloromethane (2 mL), N,N-diisopropylethylamine (42 μL, 0.241 mmol, 2.7 equiv.), and pyridine (25 μL, 0.309 mmol, 3.4 equiv.). The reaction mixture from step (i) was added and the mixture was stirred for 30 min, at which point LCMS indicated the reaction was complete. The reaction mixture was concentrated in vacuo and purified by Isolella chromatography (CH 2 Cl 2 Purification by elution with 0-4% MeOH in hexane gave 6 (22 mg, 0.03256 mmol, 36% yield, QC=96.8%) as a yellow solid. LC / MS (Method B): rt=1.86 min, 676.6 [M+H] + .

[0238] Example 7 [ka] 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((2-(((S)-1-((2-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)hexanamide (7) Compound I18 (259 mg, 0.3588 mmol) was dissolved in CH 2 Cl 2 (25 mL). No starting material was dissolved so DMA (1 mL) was added. No improvement was observed so DIPEA (68 μL, 0.390 mmol) was added to dissolve all solids. Maleimidocaproic acid (69 mg, 0.358 mmol) was added and the mixture was left stirring at room temperature overnight at which point LCMS analysis revealed the reaction to be complete. The reaction mixture was quenched with MeOH (2 mL) and concentrated to dryness in vacuo. The crude product was purified by preparative HPLC followed by lyophilization to give compound 7 as an ochre solid (38.2 mg, 11% yield). Analytical data: LCMS 3 min: ES + =1.47 min, m / z916.2[M+H] + LCMS 15 min:ES + =5.46 min, m / z916.1[M+H] + .

[0239] Example 8 [ka] 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide)-N-(2-((2-(((S)-1-((2-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-amide (8) Compound I18 (70 mg, 0.096 mmol) was dissolved in CH 2 Cl 2 (5 mL). None of the starting material was dissolved so DMA (0.5 mL) was added. No improvement was observed so DIPEA (19 μL, 0.106 mmol) was added and all solids were dissolved. Mal-dPEG 8 -OH (63 mg, 0.106 mmol) and EDCI.HCl (19 mg, 0.099 mmol) were added and the mixture was left stirring at room temperature overnight, at which point LCMS analysis revealed the reaction to be complete. The reaction mixture was quenched with MeOH (2 mL) and concentrated to dryness in vacuo. The crude product was purified by preparative HPLC followed by lyophilization to give 8 as an ochre solid (30 mg, 24% yield). LCMS 3 min: ES + =1.44 min, m / z1297.6[M+H] + .

[0240] Alternative Synthesis of Example 9-1 [ka] (S)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione, I11 (371 mg, 0.779 mmol, 1.0 equiv.), was dissolved in dichloromethane (30 mL). N,N-Diisopropylethylamine (69 μL, 0.396 mmol, 0.51 equiv) and (2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoic acid (664 mg, 0.871 mmol, 1.1 equiv) in N,N-dimethylacetamide (10 mL) were added followed by EDCI.HCl (226 mg, 1.18 mmol, 1.5 equiv) and the mixture was stirred for 2 h, LCMS showed good conversion but the reaction had stopped. The reaction mixture was warmed to 30° C. and stirred for 30 min, LCMS showed no change, so CH 2 Cl 2 Remove in vacuo and add Et 2 O was added. The precipitated oil was collected and diluted with Et 2 The O was removed in vacuo and the precipitation process was repeated. The combined precipitates were purified by HPLC (10-60% B in A in 13 min) to give 1 (200 mg, 0.174 mmol, 98% purity, 22% yield) as a yellow residue after lyophilization. LC / MS (Method A): Retention time 1.44 min (ES + ) m / z 1149 [M+H] + 1H NMR (600MHz, chloroform-d) δ 8.81(s,1H),7.83(s,2H),7.48(s,1H),7.18(dd,J=18.7,7.5Hz,2H),6.69( s,2H),6.43(s,1H),5.68(d,J=16.1Hz,1H),5.27(d,J=16.1Hz,1H),5.03(d, J=18.4Hz,1H),4.90(d,J=18.4Hz,1H),4.75(p,J=7.2Hz,1H),4.32(dd,J=7. 4,5.8Hz,1H),4.05(s,1H),3.83(t,J=7.2Hz,3H),3.78-3.68(m,3H),3.68-3 .57(m,31H),3.53(t,J=5.1Hz,3H),3.40(q,J=5.3Hz,2H),3.06-2.91(m,3H) ,2.84(dt,J=16.3,6.2Hz,1H),2.63(ddd,J=14.8,8.5,4.2Hz,1H),2.57-2.4 4(m,4H),2.30(dq,J=13.4,6.7Hz,1H),2.10(p,J=6.4Hz,3H),1.91(ddt,J=1 6.8,14.3,7.2Hz,3H),1.54(d,J=7.1Hz,3H),1.02(dd,J=15.5,6.9Hz,10H).

[0241] Alternative Synthesis of Example 10-A2 [ka] Allyl ((S)-1-(((S)-1-((4-amino-5-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (A2) EDCI.HCl (7.71g, 31.2mmol) was dissolved in CH 2 Cl 2 (200 mL) and stirred for 15 min or until solubilized. I16 (5 g, 28.3 mmol) was then added and the resulting mixture was left stirring until the reaction was complete. The volatiles were removed under reduced pressure. The crude product was extracted with Et 22H 3 (50 mL) and the mixture was sonicated for 3 min. The solid was filtered, redissolved in CH2Cl2 (50 mL), sonicated for 3 min, and filtered again to give the pure product A2 as a grey solid (12.21 g, 79% yield). LC / MS (Method B): ES + =1.47 min, m / z431.5[M+H] + .

[0242] Example 11 [ka] a) (9H-fluoren-9-yl)methyl N2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oyl)-N5-((S)-1-(((S)-1-(((S)-9-ethyl-9- Hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-L-glutamate (A7) EDCI.HCl (0.10 mmol, 1.2 equiv) was added to a solution of A5 (0.087 mmol, 1.0 equiv) and Mal-PEG8-Glu-OH (0.10 mmol, 1.2 equiv) in DCM (5 mL) and the resulting mixture was stirred at room temperature overnight. The reaction mixture was evaporated to dryness and purified by column (8-12% MeOH / DCM) to leave the product as a white solid. Yield=80 mg (63%). LC / MS (Method B) RT 1.66 min m / z (1456.2) M+H.

[0243] b) N2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oyl)-N5-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-L-glutamine (9) 1-Methylpyrrolidine (200 μL) was added to a solution of A7 (0.06 mmol) in DMF (0.8 mL) and stirred at room temperature for 10 min. The solvent was removed under vacuum and the residue was purified by preparative HPLC (30% MeCN / water + 0.05% formic acid in 8.5 min). The fractions containing the product were lyophilized to give the product as an off-white solid. Yield = 23 mg (30%). LC / MS (Method B) RT 1.43 min m / z (1278.4) M+H.

[0244] Example 12 - Conjugation Herceptin-C239i antibody The Herceptin antibody was engineered so that a cysteine ​​was inserted between positions 239 and 240 and was generated according to the method described in Dimasi, N., et al., Molecular Pharmaceutics, 2017, 14, 1501-1516 (DOI:5 10.1021 / acs.molpharmaceut.6b00995).

[0245] ConjA A 50 mM solution of DL-dithiothreitol (DTT) in phosphate buffered saline (pH 7.4) (PBS) (150 molar equivalents / antibody, 40 micromolar, 800 μL) was added to a 10 mL solution of Herceptin-C239i antibody (40 mg, 267 nmoles) dissolved in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) for a final antibody concentration of 4.0 mg / mL. The reduction mixture was reacted on an orbital shaker with gentle shaking (60 rpm) for 4 hours 45 minutes at room temperature (or until complete reduction was observed by UHPLC). The reduced antibody was buffer exchanged into reoxidation buffer containing PBS and 1 mM EDTA via spin filter centrifugation to remove any excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 20 molar equivalents / antibody, 5.33 micromoles, 106.7 μL) in DMSO was added and the reoxidized mixture was allowed to react for 16 hours at room temperature with gentle (60 rpm) shaking at an antibody concentration of 4 mg / mL (or more DHAA was added and the reaction was left longer until complete reoxidation of cysteine ​​thiols to reform interchain cysteine ​​disulfides was observed by UHPLC). The reoxidized mixture was then sterile filtered and diluted with conjugation buffer containing PBS and 1 mM EDTA to a final antibody concentration of 3.6 mg / mL. Compound 1 was added as a DMSO solution (10 molar equivalents / antibody, 1.33 micromoles in 0.55 mL DMSO) to 5.0 mL of this reoxidized antibody solution (20 mg, 133 nanomoles) to a final DMSO concentration of 10% (v / v). The solution was mixed for 2 hours at room temperature, then conjugation was quenched by the addition of N-acetylcysteine ​​(6.67 micromolar, 67 μL at 100 mM) and then purified by spin filtration in PBS using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, sterile filtered, and analyzed.

[0246] UHPLC analysis of the ConjA reduced sample at 214 nm and 330 nm (specific for Compound 1) using a Thermo Scientific MAbPac 50 mm x 2.1 mm column on a Shimadzu Prominence system eluted with a gradient of water and acetonitrile showed uncomplexed light chain bound to a single molecule of Compound 1 and a mixture of uncomplexed heavy and heavy chains, corresponding to a drug per antibody ratio (DAR) of 1.89 molecules of Compound 1 per antibody.

[0247] UHPLC analysis of the ConjA sample at 280 nm using a Shimadzu Prominence system with a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6 x 150 mm column (with a 4 μm 3.0 x 20 mm guard column) eluted at 0.3 mL / min with sterile filtered SEC buffer containing 200 mM potassium phosphate (pH 6.95), 250 mM potassium chloride, and 10% isopropanol (v / v) shows 98% monomer purity. UHPLC SEC analysis gives a final concentration of 2.14 mg / mL ConjA in 6.5 mL, with a mass of ConjA obtained of 13.9 mg (70% yield).

[0248] ConjA * A 10 mM solution of tris(2-carboxyethyl)phosphine (TCEP) in phosphate buffered saline (pH 7.4) (PBS) (10 molar equivalents / antibody, 400 nmoles, 40 μL) was added to 2.4 mL of Tratuzumab antibody (6 mg, 40 nmoles) dissolved in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) for a final antibody concentration of 2.5 mg / mL. The reduction mixture was allowed to react on an orbital shaker with gentle agitation (60 rpm) for 16 hours at room temperature (or until complete reduction was observed by UHPLC). The reduced antibody was buffer exchanged (to remove any excess reducing agent) into complexation buffer containing PBS and 1 mM EDTA via spin filter centrifugation to a final antibody concentration of 2.0 mg / mL. Compound 1 was added as a DMSO solution (20 molar equivalents / antibody, 400 nmoles in 0.15 mL DMSO) to 1.35 mL of this reduced antibody solution (3 mg, 20 nmoles) for a final DMSO concentration of 10% (v / v). The solution was mixed at room temperature for 2 hours, then conjugation was quenched by the addition of N-acetylcysteine ​​(2 micromolar, 20 μL at 100 mM) and then purified by spin filter centrifugation using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, sterile filtered, and analyzed.

[0249] ConjA was measured at 214 nm and 330 nm (specific for compound 1) using a Thermo Scientific MAbPac 50 mm x 2.1 mm column on a Shimadzu Prominence system eluted with a gradient of water and acetonitrile. * UHPLC analysis of the reduced sample showed a mixture of unconjugated light chain bound to a single molecule of compound 1, light chain, unconjugated heavy chain bound to up to three molecules of compound 1, and heavy chain, corresponding to a drug per antibody ratio (DAR) of 7.89 molecules of compound 1 per antibody.

[0250] The Shimadzu Prominence system was used with a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6 × 150 mm column (with a 4 μm 3.0 × 20 mm guard column) and eluted with sterile filtered SEC buffer containing 200 mM potassium phosphate (pH 6.95), 250 mM potassium chloride, and 10% isopropanol (v / v) at 0.3 mL / min. ConjA was measured at 280 nm. * UHPLC analysis of the sample shows a monomer purity of 98.5%. UHPLC SEC analysis shows a final concentration of ConjA of 2.02 mg / mL in 1.25 mL. * The obtained ConjA * The mass is 2.5 mg (84% yield).

[0251] ConjB A 10 mM solution of tris(2-carboxyethyl)phosphine (TCEP) in phosphate buffered saline (pH 7.4) (PBS) (10 molar equivalents / antibody, 3.56 micromoles, 356 μL) was added to 11.1 mL of Tratuzumab antibody (53.4 mg, 356 nanomoles) in reduction buffer containing PBS (pH 7.4) and 1 mM ethylenediaminetetraacetic acid (EDTA) for a final antibody concentration of 4.84 mg / mL. The reduction mixture was allowed to react on an orbital shaker with gentle agitation (60 rpm) at 37° C. for 1 hour and 30 minutes (or until complete reduction was observed by UHPLC). Compound 2 was added as a DMSO solution (15 molar equivalents / antibody, 5.1 micromoles in 1.2 mL DMSO) to 10.5 mL of this reduced antibody solution (50.8 mg, 339 nanomoles) to a final DMSO concentration of 10% (v / v). The solution was mixed at room temperature for 1 hour and 30 minutes, then conjugation was quenched by the addition of N-acetylcysteine ​​(25.4 micromoles, 254 μL at 100 mM), and then purified on an AKTA™ Start FPLC using a GE Healthcare HiLoad™ 26 / 600 column packed with Superdex 200PG, eluted with PBS at 2.6 mL / min. The fractions corresponding to the ConjB monomer peak were pooled, concentrated, and buffer exchanged into 25 mM histidine 205 mM sucrose pH 6.0 buffer using a 15 mL Amicon Ultracell 50 KDa MWCO spin filter, sterile filtered, and analyzed.

[0252] UHPLC analysis of the ConjB reduced sample at 214 nm and 330 nm (specific for compound 2) using a Thermo Scientific MAbPac 50 mm x 2.1 mm column on a Shimadzu Prominence system eluted with a gradient of water and acetonitrile showed a mixture of uncomplexed light chain bound to a single molecule of compound 2, light chain, uncomplexed heavy chain bound to up to three molecules of compound 2, and heavy chain, corresponding to a drug per antibody ratio (DAR) of 7.93 molecules of compound 2 per antibody.

[0253] UHPLC analysis of the ConjB sample at 280 nm using a Shimadzu Prominence system with a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6 x 150 mm column (with a 4 μm 3.0 x 20 mm guard column) eluted at 0.3 mL / min with sterile filtered SEC buffer containing 200 mM potassium phosphate (pH 6.95), 250 mM potassium chloride, and 10% isopropanol (v / v) shows 98.9% monomer purity. UHPLC SEC analysis gives a final concentration of 2.4 mg / mL ConjB in 16 mL, with a mass of ConjB obtained of 38.4 mg (84% yield).

[0254] ConjC A 10 mM solution of tris(2-carboxyethyl)phosphine (TCEP) in phosphate buffered saline (pH 7.4) (PBS) (40 molar equivalents / antibody, 11.2 micromolar, 1.12 mL) was added to a 20 mL solution of Herceptin-C239i antibody (42 mg, 280 nanomolar) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) for a final antibody concentration of 2.1 mg / mL. The reduction mixture was allowed to react on an orbital shaker with gentle shaking (60 rpm) for 16 hours at room temperature (or until complete reduction was observed by UHPLC). The reduced antibody was buffer exchanged into reoxidation buffer containing PBS and 1 mM EDTA via spin filter centrifugation to remove any excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 30 molar equivalents / antibody, 7.0 micromoles, 141 μL) in DMSO was added to 22 mL of this reduced, buffer-exchanged antibody (35.2 mg, 235 nanomoles) at an antibody concentration of 1.6 mg / mL, and the reoxidation mixture was allowed to react for 2 hours and 30 minutes at room temperature with gentle (60 rpm) shaking (or additional DHAA was added and the reaction was left longer until complete reoxidation of cysteine ​​thiols to reform interchain cysteine ​​disulfides was observed by UHPLC). The reoxidation mixture was then sterile filtered. Compound 6 was added as a DMSO solution (20 molar equivalents / antibody, 2.3 micromoles in 1.36 mL DMSO) to 11.0 mL of this reoxidized antibody solution (17.6 mg, 117 nanomoles) (pH adjusted with 1.22 mL 1 M sodium bicarbonate) to a final DMSO concentration of 10% (v / v) and 10% (v / v) 1 M sodium bicarbonate. The solution was allowed to react for 2 hours at room temperature with gentle shaking. The conjugation was then quenched by the addition of N-acetylcysteine ​​(12 micromoles, 117 μL at 100 mM), which was then purified and buffer exchanged into 25 mM histidine 205 mM sucrose pH 6.0 buffer using a 15 mL Amicon Ultracell 50 KDa MWCO spin filter, sterile filtered and analyzed.

[0255] UHPLC analysis of an intact sample of ConjB at 214 nm and 330 nm (specific for compound 6) using a Shimadzu Prominence system with a Sepax Proteomix HIC Butyl-NP5 4.6 x 35 mm 5 μm column eluted with a gradient of 25 mM sodium phosphate, 1.5 M ammonium sulfate (pH 7.4 buffer), and 20% acetonitrile (v / v) in 25 mM sodium phosphate pH 7.4 buffer showed unconjugated and conjugated antibody bound to one or two molecules of compound 6, corresponding to a drug per antibody ratio (DAR) of 1.42 molecules of compound 6 per antibody.

[0256] UHPLC analysis of the ConjC sample at 280 nm using a Shimadzu Prominence system with a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6 x 150 mm column (with a 4 μm 3.0 x 20 mm guard column) and eluting with sterile filtered SEC buffer containing 200 mM potassium phosphate (pH 6.95), 250 mM potassium chloride, and 10% isopropanol (v / v) at 0.3 mL / min shows 98% monomer purity. UHPLC SEC analysis gives a final concentration of 1.06 mg / mL ConjC in 10.1 mL, with a mass of ConjC obtained of 10.7 mg (61% yield).

[0257] Example 13 - In vitro assays Solid test substances were dissolved in DMSO to a stock solution of 2 mM from which eight serial dilutions were made in a 1:10 ratio in DMSO and stored at -20°C until use.

[0258] Adherent NCI-N87 cells were washed with D-PBS and detached with trypsin-EDTA, then cell density and viability were measured in duplicate by trypan blue exclusion assay using an automated cell counter (LUNA-II™). Cell suspensions were diluted to 1 × 10 in growth medium (RPMI 1640 with Glutamax + 10% (v / v) HyClone™ fetal bovine serum). 5The cells were diluted to 100 cells / mL, vortexed, and dispensed at 2 mL per well into a sterile 3 mL polypropylene plate. The warhead dilutions were then dispensed at 10 μL / well into the appropriate wells and mixed by repeated pipetting. In control wells, 10 μL of DMSO was dispensed into 2 mL of cell suspension and mixed thoroughly. 100 μL of each sample was then removed and placed into two replicate wells of a sterile flat-bottom 96-well microplate and incubated at 37°C in CO 2 At the end of the incubation period (7 days), 20 μL / well was dispensed and incubated at 37°C, 5% CO 2 Cell viability was measured with the CellTiter 96™ aqueous One (MTS) assay, incubated for 4 hours at 4° C. Plates were then read on an EnVision™ Multi-label plate reader (Perkin Elmer) using absorbance at 490 nm.

[0259] The percentage of viable cells was calculated from the average absorbance of duplicate wells for each sample compared to the average absorbance in two control wells treated with DMSO only (100%). IC 50 was determined by fitting each data set to a sigmoidal dose-response curve with variable slope using a nonlinear curve-fitting algorithm in GraphPad Prism software (San Diego, Calif.).

[0260] All experiments in this report were performed and tested in three independent experiments, and data are reported as the mean of three independent replicates.

[0261] [Table 8]

[0262] Example 14 - ADC in vitro assay The concentration and viability of cells from subconfluent (80-90% confluency) T75 flasks were determined by trypan blue staining and counted using a LUNA-II™ automated cell counter. Cells were cultured at 2 × 10 5 / ml and dispensed into 96-well flat-bottom plates (50 μL / well).

[0263] A stock solution (1 ml) of antibody drug conjugate (ADC) (20 μg / ml) was made by diluting filter-sterilized ADC in cell culture medium. A set of eight 10-fold dilutions of the ADC stock solution was made in a 24-well plate by serially transferring 100 μL into 900 μL of cell culture medium. The ADC dilutions were dispensed (50 μL per well) into four replicate wells of a 96-well plate containing 50 μL of the previously seeded cell suspension. Control wells received 50 μL of cell culture medium. The 96-well plate containing cells and ADC was incubated at 4°C for 1 h at 4°C for 2 h at 4°C for 3 h at 4°C. The cells were incubated at 4°C for 1 h at 4°C for 3 h at 4°C for 1 h at 4°C. The cells were incubated at 4°C for 1 h at 4°C for 3 h at 4°C. The cells were then ... 2 The exposure time was incubated at 37° C. in a gassed incubator.

[0264] At the end of the incubation period, cell viability was measured by MTS assay. MTS (Promega) was dispensed into each well (20 μL per well) and incubated with CO 2 Incubated for 4 hours at 37°C in a gassed incubator. Well absorbance was measured at 490 nm. The percentage of viable cells was calculated from the average absorbance in the four ADC-treated wells compared to the average absorbance in the four control untreated wells (100%). IC 50 was determined from dose-response data using GraphPad Prism using a nonlinear curve-fitting algorithm: sigmoidal dose-response curve with variable slope;

[0265] ADC incubation times were 4 days for MDA-MB-468 and 7 days for NCI-N87. MDA-MB-468 and NCI-N87 were cultured in RPMI1640 with Glutamax + 10% (v / v) HyClone™ fetal bovine serum. NCI-N87 is a Her2-expressing cell line and MDA-MB-468 is a Her2-negative cell line.

[0266] [Table 9]

[0267] Example 15 - ADC in vivo assay Methods and Materials mouse Female severe combined immunodeficient mouse (Fox Chase SCID(TM), CB17 / Icr-Prkdc scid Mice (100-240 mm H2O, 100-280 mm L / IcoIcrCrl, Charles River) were 8 weeks old with a body weight (BW) range of 14.5-20.0 grams on day 1 of the study. Animals had free access to water (reverse osmosis, 1 ppm Cl) and were fed an NIH 31 Modified and Irradiated Lab Diet™ consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber ad libitum. Mice were housed in irradiated Enricho'cobs™ Laboratory Animal Bedding in static pressure microisolators at 20-22°C and 40-60% humidity on a 12-hour light cycle. CR Discovery Services specifically adheres to the recommendations of the Guide for Care and Use of Laboratory Animals regarding restraint, husbandry, surgery, specimen, and fluid regulation, as well as veterinary care. CR Discovery Services' animal care and use program is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which ensures compliance with accepted standards for the care and use of laboratory animals.

[0268] Tumor cell culture Human NCI-N87 gastric carcinoma lymphoma cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units / mL penicillin, 100 μg / mL streptomycin sulfate, and 25 μg / mL gentamicin. The cells were incubated at 37 °C for 2 h at 37 °C for 3 h at 37 °C. 2 and were grown in tissue culture flasks in a humidified incubator at 37°C in an atmosphere of 95% air.

[0269] In Vivo Implantation and Tumor Growth NCI-N87 cells used for implantation were harvested during logarithmic phase growth and resuspended in phosphate-buffered saline (PBS) containing 50% Matrigel™ (BD Biosciences). On the day of tumor implantation, each test mouse was injected with 1×10 7 Cells (0.1 mL cell suspension) were injected subcutaneously into the right flank, and tumor growth was monitored at 100–150 mm 3 After 12 days, designated as the first day of the study, the mice were divided into 14 groups according to the calculated tumor size, with 7 groups set for efficacy evaluation (n=10) and 7 groups set for sample collection (n=3), each ranging from 108 to 172 mm. 3 and individual tumor volumes ranging from 120 to 124 mm 3 The groups consisted of animals with a group mean tumor volume of 1000 x 1000 mm. Tumors were measured in two dimensions using calipers and volumes were calculated using the following formula: Tumor volume (mm 3 )=(w 2 ×l) / 2 where w=width and l=length in mm of the tumor. Tumor weight is calculated as 1 mg per mm of tumor volume. 3 It may be estimated by assuming that it is equivalent to

[0270] Therapeutic Agents ConjA * were stored protected from light at 4° C. Sterile PBS was used to dose the vehicle control group.

[0271] treatment On day 1 of the study, female SCID mice with established NCI-N87 xenografts were sorted into groups. Aliquots of the stock solution were diluted to the appropriate concentrations with PBS. Drugs were administered intravenously once via tail vein injection on day 1. The dose was 0.2 mL per 20 grams of body weight (10 mL / kg) and was adjusted to the body weight of each individual animal.

[0272] Mice in group 1 received the PBS vehicle and served as the control group. Group 2 received 4 mg / kg of ConjA * was administered.

[0273] Tumors were measured twice weekly using calipers and each animal was cultured until tumors reached a final volume of 800 mm 3 Animals were euthanized when they reached a final tumor volume or at the end of the study (day 68), whichever occurred first. Animals that completed the study were recorded as euthanized for tumor progression (TP) along with the date of euthanasia.

[0274] Criteria for reduction reaction Treatment efficacy can be determined from the incidence and magnitude of regression responses observed during the study. Treatment can result in partial (PR) or complete (CR) regression of tumors in animals. A PR response is when tumor volume is 50% or less than the volume on day 1 for three consecutive measurements during the study period and is less than or equal to 13.5 mm for at least one of those three measurements. 3 In the CR response, tumor volume was 13.5 mm3 on three consecutive occasions during the study. 3 The mean time to respond was less than 10 days. Animals were scored only once during the study for PR or CR events and scored as CR only if both PR and CR criteria were met. Animals with a CR response at the end of the study were further classified as tumor-free survivors (TFS). Animals were monitored for regression responses.

[0275] toxicity Animals were weighed daily on days 1-5 and then twice weekly until study completion. Mice were frequently observed for overt signs of any adverse, treatment-related (TR) side effects, and clinical signs were recorded if observed. Individual body weights were monitored according to protocol, and animals exhibiting weight loss of more than 30% on a single measurement or more than 25% on three consecutive measurements were euthanized as TR deaths. Group mean body weight loss was also monitored according to CR Discovery Services protocol. Acceptable toxicity was defined as less than 20% group mean body weight (BW) loss during the study, and 10% or less TR deaths.

[0276] result FIG. 1 shows a plot of mean tumor growth in:

[0277] [Table 10]

[0278] Mice in Group 1 were administered PBS vehicle intravenously qd × 1 and served as the control group. Median TTE in Group 1 was 24.8 days. All control tumors were 800 mm 3 The endpoint was reached.

[0279] Group 2 received 4 mg / kg of ConjA * was administered intravenously qd × 1. CR was observed in all 10 mice that were further classified as TFS at the end of the study.

[0280] In the treatment group, the body weight nadir was -9.5% on study day 50. No mortality due to TR was observed.

[0281] Description of the Invention 1. Formula I: [ka] [In the formula, R L is a linker connected to the Ligand unit, which is: (ia): [ka] (In the formula, Q: [ka] (In the formula, Q X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue; X is: [ka] (wherein a=0-5, b1=0-16, b2=0-16, c1=0 or 1, c2=0 or 1, d=0-5, and wherein at least b1 or b2=0, and at least c1 or c2=0); G L is the ligand unit; (ib): [ka] (In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1); and salts and solvates thereof.

[0282] 2.R L is of formula Ia.

[0283] 3. The compound according to statement 2, wherein Q is an amino acid residue.

[0284] 4. The compound according to statement 3, wherein Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, lie, Arg, and Trp.

[0285] 5. The compound according to statement 2, wherein Q is a dipeptide residue.

[0286] 6.Q: NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-Lys- C=O , NH -Val-Cit- C=O , NH -Phe-Cit- C=O , NH -Leu-Cit- C=O , NH -Ile-Cit- C=O , NH -Phe-Arg- C=O , NH -Trp-Cit- C=O , and NH -Gly-Val- C=O 6. The compound according to statement 5, selected from:

[0287] 7. Q is, NH -Phe-Lys- C=O , NH -Val-Cit- C=O , and NH -Val-Ala- C=O 7. The compound according to statement 6, selected from:

[0288] 8. The compound according to statement 2, wherein Q is a tripeptide residue.

[0289] 9.Q: NH -Glu-Val-Ala- C=O , NH -Glu-Val-Cit- C=O , NH -αGlu-Val-Ala- C=O , and NH -αGlu-Val-Cit- C=O 9. The compound of statement 8, selected from:

[0290] 10. The compound according to statement 2, wherein Q is a tetrapeptide residue.

[0291] 11.Q: NH -Gly-Gly-Phe-Gly C=O and NH -Gly-Phe-Gly-Gly C=O 11. The compound of statement 10, selected from:

[0292] 12.Q: NH -Gly-Gly-Phe-Gly C=O 12. The compound of claim 11,

[0293] 13. The compound according to any one of statements 2 to 12, wherein a is 0 to 3.

[0294] 14. The compound according to statement 13, wherein a is 0 or 1.

[0295] 15. The compound according to statement 13, wherein a is 0.

[0296] 16. The compound according to any one of statements 2-15, wherein b1 is 0-8.

[0297] 17. The compound according to statement 16, wherein b1 is 0.

[0298] 18. The compound according to statement 16, wherein b1 is 2.

[0299] 19. The compound according to statement 16, wherein b1 is 3.

[0300] 20. The compound according to statement 16, wherein b1 is 4.

[0301] 21. The compound according to statement 16, wherein b1 is 5.

[0302] 22. The compound according to statement 16, wherein b1 is 8.

[0303] 23. The compound according to any one of statements 2 to 15 and 17, wherein b2 is 0 to 8.

[0304] 24. The compound according to statement 23, wherein b2 is 0.

[0305] 25. The compound according to statement 23, wherein b2 is 2.

[0306] 26. The compound according to statement 23, wherein b2 is 3.

[0307] 27. The compound according to statement 23, wherein b2 is 4.

[0308] 28. The compound according to statement 23, wherein b2 is 5.

[0309] 29. The compound according to statement 23, wherein b2 is 8.

[0310] 30. The compound according to any one of statements 2 to 29, wherein c1 is 0.

[0311] 31. The compound according to any one of statements 2 to 29, wherein c1 is 1.

[0312] 32. The compound according to any one of statements 2 to 31, wherein c2 is 0.

[0313] 33. The compound according to any one of statements 2 to 30, wherein c2 is 1.

[0314] 34. The compound according to any one of statements 2 to 33, wherein d is 0 to 3.

[0315] 35. The compound according to statement 34, wherein d is 1 or 2.

[0316] 36. The compound according to statement 34, wherein d is 2.

[0317] 37. A compound according to any one of statements 2 to 33, wherein d is 5.

[0318] 38. A compound according to any one of statements 2 to 12, wherein a is 0, b1 is 0, c1 is 1, c2 is 0, d is 2, and b2 is 0 to 8.

[0319] 39. The compound according to statement 38, wherein b2 is 0, 2, 3, 4, 5, or 8.

[0320] 40. A compound according to any one of statements 2 to 12, wherein a is 1, b2 is 0, c1 is 0, c2 is 0, d is 0, and b1 is 0 to 8.

[0321] 41. The compound according to statement 40, wherein b1 is 0, 2, 3, 4, 5, or 8.

[0322] 42. A compound according to any one of statements 2 to 12, wherein a is 0, b1 is 0, c1 is 0, c2 is 0, d is 1, and b2 is 0 to 8.

[0323] 43. The compound according to statement 42, wherein b2 is 0, 2, 3, 4, 5, or 8.

[0324] 44. The compound according to any one of statements 2 to 12, wherein b1 is 0, b2 is 0, c1 is 0, c2 is 0, one of a and d is 0, and the other of a and d is 1 to 5.

[0325] 45. The compound according to statement 44, wherein the other of a and d is 1 or 5.

[0326] 46. ​​A compound according to any one of statements 2 to 12, wherein a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 is 0 to 8.

[0327] 47. The compound according to statement 46, wherein b1 is 0, 2, 3, 4, 5, or 8.

[0328] 48.G L but

[0329] [Table 11]

[0330] [Table 12] [Wherein, Ar is C 5~6 represents an arylene group, and X is C 1~4 A compound according to any one of statements 2 to 47, wherein R represents an alkyl group.

[0331] 49.G L But, G L1-1 and G L1-2 49. The compound according to statement 48, selected from:

[0332] 50.G L G L1-1 49. The compound according to statement 48,

[0333] 51.R L is of formula Ib.

[0334] 52.R L1 and R L2 and R are both H.

[0335] 53.R L1 is H and R L2 52. The compound according to statement 51, wherein is methyl.

[0336] 54.R L1 and R L2 and R are both methyl.

[0337] 55.R L1 and R L2 together with the carbon atom to which they are attached form a cyclopropylene group.

[0338] 56.R L1 and R L2 together with the carbon atom to which they are attached form a cyclobutylene group.

[0339] 57. The compound according to any one of statements 51-56, wherein e is 0.

[0340] 58. The compound according to any one of statements 51-56, wherein e is 1.

[0341] 59.Formula IV: L-(D L ) p (IV) where L is a ligand unit (i.e., a targeting agent) and D L is represented by formula III: [ka] (In the formula, R LL teeth, (ia'): [ka] (wherein Q and X are as defined in any one of statements 1 to 47; G LL is a linker connecting the Ligand units; and (ib'): [ka] (In the formula, R L1 and R L2 is a linker connected to the Ligand unit selected from: p is an integer from 1 to 20. or a pharma- ceutically acceptable salt or solvate thereof.

[0342] 60.G LL but,

[0343] [Table 13] [Wherein, Ar is C 5~6 represents an arylene group, and X is C 1~4 60. The conjugate of statement 59, wherein:

[0344] 61.G LL But, G LL1-1 and G LL1-2 61. The conjugate of statement 60, selected from:

[0345] 62.G LL G LL1-1 62. The complex of statement 61,

[0346] 63. The conjugate of any one of statements 59-62, wherein the ligand unit is a cell-binding agent.

[0347] 64. A conjugate according to any one of statements 59 to 62, in which the ligand unit is an antibody or an active fragment thereof.

[0348] 65. The conjugate according to statement 64, wherein the antibody or antibody fragment is an antibody or antibody fragment to a tumor-associated antigen.

[0349] 66. The conjugate according to statement 65, wherein the antibody or antibody fragment is an antibody that binds to one or more tumor-associated antigens or cell surface receptors selected from the following (1) to (89): (1)BMPR1B; (2) E16; (3) STEAP1; (4)0772P; (5) MPF; (6) Napi3b; (7) Sema 5b; (8)PSCA hlg; (9)ETBR; (10)MSG783; (11)STEAP2; (12)TrpM4; (13)CRIPTO; (14)CD21; (15)CD79b; (16)FcRH2; (17)HER2; (18)NCA; (19)MDP; (20)IL20R-α; (21)Brevican; (22)EphB2R; (23)ASLG659; (24)PSCA; (25)GEDA; (26)BAFF-R; (27)CD22; (28)CD79a; (29)CXCR5; (30)HLA-DOB; (31)P2X5; (32)CD72; (33)LY64; (34)FcRH1; (35)IRTA2; (36)TENB2; (37)PSMA-FOLH1; (38)SST; (38.1)SSTR2; (38.2)SSTR5; (38.3)SSTR1; (38.4)SSTR3; (38.5)SSTR4; (39)ITGAV; (40)ITGB6; (41)CEACAM5; (42)MET; (43)MUC1; (44)CA9; (45)EGFRvIII; (46)CD33; (47)CD19; (48)IL2RA; (49) AXL; (50)CD30-TNFRSF8; (51)BCMA-TNFRSF17; (52)CT Ags-CTA; (53)CD174(Lewis Y)-FUT3; (54)CLEC14A; (55)GRP78-HSPA5; (56)CD70; (57) Stem cell-specific antigen; (58)ASG-5; (59)ENPP3; (60)PRR4; (61)GCC-GUCY2C; (62)Liv-1-SLC39A6; (63)5T4; (64)CD56-NCMA1; (65)CanAg; (66)FOLR1; (67)GPNMB; (68)TIM-1-HAVCR1; (69)RG-1 / prostate tumor targeting Mindin-Mindin / RG-1; (70)B7-H4-VTCN1; (71)PTK7; (72)CD37; (73)CD138-SDC1; (74)CD74; (75) Claudin-CL; (76)EGFR; (77)Her3; (78)RON-MST1R; (79)EPHA2; (80)CD20-MS4A1; (81) Tenascin-C-TNC; (82)FAP; (83)DKK-1; (84)CD52; (85)CS1-SLAMF7; (86)Endoglin-ENG; (87) Annexin A1-ANXA1; (88)V-CAM(CD106)-VCAM1; (89)ASCT2(SLC1A5).

[0350] 67. A conjugate according to any one of statements 64 to 66, wherein the antibody or antibody fragment is a cysteine ​​engineered antibody.

[0351] 684. The conjugate of any one of statements 64 to 67, wherein the drug loading (p) of the drug (D) on the antibody (Ab) is an integer from 1 to about 10.

[0352] 69. The complex of statement 68, wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0353] 70. The mixture of conjugates of any one of statements 64 to 69, wherein the average drug loading per antibody in the mixture of antibody-drug conjugates is about 1 to about 10.

[0354] 71. A complex or mixture according to any one of statements 59 to 70 for use in therapy.

[0355] 72. A pharmaceutical composition comprising a complex or mixture according to any one of statements 59 to 70 and a pharma- ceutically acceptable diluent, carrier, or excipient.

[0356] 73. A complex or mixture according to any one of statements 59 to 70, or a pharmaceutical composition according to statement 72, for use in treating a proliferative disorder in a subject.

[0357] 74. The complex, mixture or pharmaceutical composition according to statement 73, wherein the disease is cancer.

[0358] 75. Use of a complex or mixture according to any one of statements 59 to 70, or a pharmaceutical composition according to statement 72, in a method of medical treatment.

[0359] 76. A method of medical treatment comprising administering to a patient a pharmaceutical composition according to statement 72.

[0360] 77. The method according to statement 76, wherein the medical treatment method is for the treatment of cancer.

[0361] 78. The method of statement 77, wherein the patient is administered a chemotherapeutic agent in combination with the conjugate.

[0362] 79. Use of a complex or mixture according to any one of statements 59 to 70 in a method for the manufacture of a medicament for the treatment of a proliferative disorder.

[0363] 80. A method for treating a mammal having a proliferative disease, the method comprising administering an effective amount of a complex or mixture described in any one of statements 59 to 70, or a pharmaceutical composition described in statement 72.

[0364] 81. Compound A as a single enantiomer or in enantiomerically enriched form: [ka] .

[0365] 82. Formula VI: [ka] wherein Q is as described in any one of statements 1, 3 and 12.

[0366] Description of the invention from the first priority use (P1) P1-1.Formula I: [ka] [In the formula, R Lis a linker connected to a cell binding agent, which is: (ia): [ka] (In the formula, Q: [ka] (In the formula, Q X is such that Q is an amino acid residue, a dipeptide residue, or a tripeptide residue; X is: [ka] (In the formula, a=0 to 5, b=0 to 16, c=0 or 1, and d=0 to 5); G L is the ligand unit; (ib): [ka] (In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1); and salts and solvates thereof.

[0367] P1-2.R L is of formula Ia.

[0368] P1-3. The compound according to statement P1-2, wherein Q is an amino acid residue.

[0369] P1-4. The compound according to statement P1-3, wherein Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, lie, Arg, and Trp.

[0370] P1-5. The compound according to statement P1-2, wherein Q is a dipeptide residue.

[0371] P1-6.Q: NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH Ala-Lys- C=O , NH -Val-Cit- C=O , NH -Phe-Cit- C=O , NH -Leu-Cit- C=O , NH -Ile-Cit- C=O , NH -Phe-Arg- C=O , NH -Trp-Cit- C=O , and NH -Gly-Val- C=O The compound according to statement P1-5, selected from:

[0372] P1-7.Q, NH -Phe-Lys- C=O , NH -Val-Cit- C=O , and NH -Val-Ala- C=O The conjugate according to statement P1-6, selected from:

[0373] P1-8. The compound according to statement P1-2, wherein Q is a tripeptide residue.

[0374] The compound according to any one of statements P1-2 to P1-8, wherein P1-9.a is 0 to 3.

[0375] The compound according to statement P1-9, wherein P1-10.a is 0 or 1.

[0376] The compound according to statement P1-9, wherein P1-11.a is 0.

[0377] The compound according to any one of statements P1-2 to P1-11, wherein P1-12.b is 0 to 8.

[0378] The compound according to statement P1-12, wherein P1-13.b is 0.

[0379] The compound according to statement P1-12, wherein P1-14.b is 4.

[0380] The compound according to statement P1-12, wherein P1-15.b is 8.

[0381] The compound according to any one of statements P1-2 to P1-15, wherein P1-16.c is 0.

[0382] The compound according to any one of statements P1-2 to P1-15, wherein P1-17.c is 1.

[0383] P1-18. The compound according to any one of statements P1-2 to P1-17, wherein d is 0 to 3.

[0384] The compound according to statement P1-18, wherein P1-19.d is 1 or 2.

[0385] The compound according to statement P1-19, wherein P1-20.d is 2.

[0386] P1-21. The compound according to any one of statements P1-2 to P1-8, wherein a is 0, c is 1, d is 2, and b is 0, 4 or 8.

[0387] P1-22.G L but

[0388] [Table 14]

[0389] [Table 15] [Wherein, Ar is C 5~6 represents an arylene group, and X is C 1~4 A compound according to any one of statements P1-2 to P1-21, selected from:

[0390] P1-23.G L But, G L1-1 and G L1-2 The compound according to statement P1-22, selected from:

[0391] P1-24.G L G L1-1 The compound according to statement P1-22,

[0392] P1-25.R L is of formula Ib.

[0393] P1-26.R L1 and R L2 The compound according to statement P1-25, wherein

[0394] P1-27.R L1 is H and R L2 The compound according to statement P1-25, wherein is methyl.

[0395] P1-28.R L1 and R L2 The compound according to statement P1-25, wherein both are methyl.

[0396] P1-29.R L1 and R L2 together with the carbon atom to which they are attached form a cyclopropylene group.

[0397] P1-30.R L1 and RL2 together with the carbon atom to which they are attached form a cyclobutylene group.

[0398] The compound according to any one of statements P1-25 to P1-30, wherein P1-31.e is 0.

[0399] The compound according to any one of statements P1-25 to P1-30, wherein P1-32.e is 1.

[0400] P1-33.Formula IV: L-(D L ) p (IV) where L is a ligand unit (i.e., a targeting agent) and D L is represented by formula III: [ka] (In the formula, R LL teeth, (ia'): [ka] (wherein Q and X are as defined in any one of the descriptions P1-1 to P1-21; G LL is a linker connecting the Ligand units; and (ib'): [ka] (In the formula, R L-1 and R L2 is a linker connected to the Ligand unit selected from: p is an integer from 1 to 20. or a pharma- ceutically acceptable salt or solvate thereof.

[0401] P1-34.G LL but,

[0402] [Table 16] [Wherein, Ar is C 5~6 represents an arylene group, and X is C 1~4 A conjugate according to statement P1-33, wherein R represents an alkyl group.

[0403] P1-35.G LL But, G LL1-1 and G LL1-2 The conjugate according to statement P1-34, selected from:

[0404] P1-36.G LL G LL1-1 A complex according to statement P1-35,

[0405] P1-37. A conjugate according to any one of statements P1-33 to P1-36, wherein the cell binding agent is an antibody or an active fragment thereof.

[0406] P1-38. The conjugate according to statement P1-37, wherein the antibody or antibody fragment is an antibody or antibody fragment to a tumor-associated antigen.

[0407] P1-39. The conjugate according to statement P1-38, wherein the antibody or antibody fragment is an antibody that binds to one or more tumor-associated antigens or cell surface receptors selected from the following (1) to (88): (1)BMPR1B; (2) E16; (3) STEAP1; (4)0772P; (5) MPF; (6) Napi3b; (7) Sema 5b; (8) PSCA hlg; (9)ETBR; (10) MSG783; (11)STEAP2; (12)TrpM4; (13) CRIPTO; (14)CD21; (15)CD79b; (16)FcRH2; (17)HER2; (18)NCA; (19)MDP; (20)IL20R-α; (21)Brevican; (22)EphB2R; (23)ASLG659; (24)PSCA; (25)GEDA; (26)BAFF-R; (27)CD22; (28)CD79a; (29)CXCR5; (30)HLA-DOB; (31)P2X5; (32)CD72; (33)LY64; (34)FcRH1; (35)IRTA2; (36)TENB2; (37)PSMA-FOLH1; (38)SST; (38.1)SSTR2; (38.2)SSTR5; (38.3)SSTR1; (38.4)SSTR3; (38.5)SSTR4; (39)ITGAV; (40)ITGB6; (41)CEACAM5; (42)MET; (43)MUC1; (44)CA9; (45)EGFRvIII; (46)CD33; (47)CD19; (48)IL2RA; (49)AXL; (50)CD30-TNFRSF8; (51)BCMA-TNFRSF17; (52)CTAgs-CTA; (53)CD174(Lewis Y)-FUT3; (54)CLEC14A; (55)GRP78-HSPA5; (56)CD70; (57) Stem cell-specific antigen; (58)ASG-5; (59)ENPP3; (60)PRR4; (61)GCC-GUCY2C; (62)Liv-1-SLC39A6; (63)5T4; (64)CD56-NCMA1; (65)CanAg; (66)FOLR1; (67)GPNMB; (68)TIM-1-HAVCR1; (69)RG-1 / prostate tumor targeting Mindin-Mindin / RG-1; (70)B7-H4-VTCN1; (71)PTK7; (72)CD37; (73)CD138-SDC1; (74)CD74; (75) Claudin-CL; (76)EGFR; (77)Her3; (78)RON-MST1R; (79)EPHA2; (80)CD20-MS4A1; (81) Tenascin-C-TNC; (82)FAP; (83)DKK-1; (84)CD52; (85)CS1-SLAMF7; (86)Endoglin-ENG; (87) Annexin A1-ANXA1; (88)V-CAM(CD106)-VCAM1; (89)ASCT2(SLC1A5).

[0408] P1-40. A conjugate according to any one of statements P1-37 to P1-39, wherein the antibody or antibody fragment is a cysteine ​​engineered antibody.

[0409] P1-41. The conjugate of any one of statements P1-37 to P1-40, wherein the drug loading (p) of the drug (D) relative to the antibody (Ab) is an integer from 1 to about 10.

[0410] A conjugate according to statement P1-41, wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0411] P1-43. A mixture of conjugates according to any one of statements P1-33 to P1-42, wherein the average drug loading per antibody in the mixture of antibody-drug conjugate compounds is from about 1 to about 10.

[0412] P1-44. A complex or mixture according to any one of statements P1-33 to P1-43 for use in therapy.

[0413] P1-45. A pharmaceutical composition comprising a complex or mixture according to any one of statements P1-33 to P1-43 and a pharma- ceutically acceptable diluent, carrier, or excipient.

[0414] P1-46. A complex or mixture according to any one of statements P1-33 to P1-43 or a pharmaceutical composition according to statement P1-45 for use in treating a proliferative disorder in a subject.

[0415] P1-47. The complex or mixture according to statement P1-46, wherein the disease is cancer.

[0416] P1-48. Use of a complex or mixture according to any one of statements P1-33 to P1-43 or a pharmaceutical composition according to statement P1-45 in a method of medical treatment.

[0417] P1-49. A method of medical treatment comprising administering to a patient a pharmaceutical composition according to statement P1-45.

[0418] P1-50. The method according to statement P1-49, wherein the medical treatment method is for the treatment of cancer.

[0419] P1-51. The method of statement P1-50, wherein the patient is administered a chemotherapeutic agent in combination with the conjugate.

[0420] P1-52. Use of a complex or mixture according to any one of statements P1-33 to P1-43 in a method for the manufacture of a medicament for the treatment of a proliferative disorder.

[0421] P1-53. A method for treating a mammal having a proliferative disease comprising administering an effective amount of a complex or mixture described in any one of statements P1-33 to P1-43, or a pharmaceutical composition described in statement P1-45.

[0422] P1-54. Compound A as a single enantiomer or in enantiomerically enriched form: [ka] .

[0423] Description of the invention from the second priority use (P2) P2-1.Formula I: [ka] [In the formula, R L is a linker connected to a cell binding agent, which is: (ia): [ka] (In the formula, Q: [ka] (In the formula, Q Xis such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue; X is: [ka] (wherein a=0-5, b1=0-16, b2=0-16, c=0 or 1, d=0-5, and at least b1 or b2=0); G L is the ligand unit; (ib): [ka] (In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1); and salts and solvates thereof.

[0424] P2-2.R L is of formula Ia.

[0425] P2-3. The compound according to statement P2-2, wherein Q is an amino acid residue.

[0426] P2-4. The compound according to statement P2-3, wherein Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, lie, Arg, and Trp.

[0427] P2-5. The compound according to statement P2-2, wherein Q is a dipeptide residue.

[0428] P2-6.Q: NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-Lys- C=O , NH -Val-Cit- C=O , NH -Phe-Cit- C=O , NH -Leu-Cit- C=O , NH -Ile-Cit- C=O , NH -Phe-Arg- C=O , NH -Trp-Cit- C=O , and NH -Gly-Val- C=O The compound according to statement P2-5, selected from:

[0429] P2-7.Q, NH -Phe-Lys- C=O , NH -Val-Cit- C=O , and NH -Val-Ala- C=O The compound according to statement P2-6, selected from:

[0430] P2-8. The compound according to statement P2-2, wherein Q is a tripeptide residue.

[0431] P2-9.Q: NH -Glu-Val-Ala- C=O , NH -Glu-Val-Cit- C=O , NH -αGlu-Val-Ala- C=O , and NH -αGlu-Val-Cit- C=O The compound according to statement P2-8, selected from:

[0432] P2-10. The compound according to statement P2-2, wherein Q is a tetrapeptide residue.

[0433] P2-11.Q: NH -Gly-Gly-Phe-Gly C=O and NH -Gly-Phe-Gly-Gly C=O The compound according to statement P2-10, selected from:

[0434] P2-12.Q: NH -Gly-Gly-Phe-Gly C=O The compound according to statement P2-11,

[0435] The compound according to any one of statements P2-2 to P2-12, wherein P2-13.a is 0 to 3.

[0436] The compound according to statement P2-13, wherein P2-14.a is 0 or 1.

[0437] The compound according to statement P2-13, wherein P2-15.a is 0.

[0438] The compound according to any one of statements P2-2 to P2-15, wherein P2-16.b1 is 0 to 8.

[0439] The compound according to statement P2-16, wherein P2-17.b1 is 0.

[0440] The compound according to statement P2-16, wherein P2-18.b1 is 2.

[0441] The compound according to statement P2-16, wherein P2-19.b1 is 3.

[0442] The compound according to statement P2-16, wherein P2-20.b1 is 4.

[0443] The compound according to statement P2-16, wherein P2-21.b1 is 5.

[0444] The compound according to statement P2-16, wherein P2-22.b1 is 8.

[0445] P2-23. The compound according to any one of statements P2-2 to P2-15 and P2-17, wherein b2 is 0-8.

[0446] The compound according to statement P2-23, wherein P2-24.b2 is 0.

[0447] The compound according to statement P2-23, wherein P2-25.b2 is 2.

[0448] The compound according to statement P2-23, wherein P2-26.b2 is 3.

[0449] The compound according to statement P2-23, wherein P2-27.b2 is 4.

[0450] The compound according to statement P2-23, wherein P2-28.b2 is 5.

[0451] The compound according to statement P2-23, wherein P2-29.b2 is 8.

[0452] The compound according to any one of statements P2-2 to P2-29, wherein P2-30.c is 0.

[0453] The compound according to any one of statements P2-2 to P2-29, wherein P2-31.c is 1.

[0454] The compound according to any one of statements P2-2 to P2-31, wherein P2-32.d is 0 to 3.

[0455] The compound according to statement P2-32, wherein P2-33.d is 1 or 2.

[0456] The compound according to statement P2-32, wherein P2-34.d is 2.

[0457] P2-35. The compound according to any one of statements P2-2 to P2-12, wherein a is 0, b1 is 0, c is 1, d is 2, and b2 is 0-8.

[0458] P2-36. The compound according to statement P2-35, wherein b2 is 0, 2, 3, 4, 5, or 8.

[0459] P2-37. The compound according to any one of statements P2-2 to P2-12, wherein a is 1, b2 is 0, c is 0, d is 0, and b1 is 0-8.

[0460] P2-38. The compound according to statement P2-37, wherein b1 is 0, 2, 3, 4, 5, or 8.

[0461] P2-39. The compound according to any one of statements P2-2 to P2-12, wherein a is 0, b1 is 0, c is 0, d is 1, and b2 is 0-8.

[0462] The compound according to statement P2-39, wherein P2-40.b2 is 0, 2, 3, 4, 5, or 8.

[0463] P2-41. The compound according to any one of statements P2-2 to P2-12, wherein b1 is 0, b2 is 0, c is 0, one of a and d is 0, and the other of a and d is 1-5.

[0464] P2-42. A compound according to statement P2-41, wherein the other of a and d is 1 or 5.

[0465] P2-43.G L but

[0466] [Table 17]

[0467] [Table 18] [Wherein, Ar is C 5~6 represents an arylene group, and X is C 1~4 A compound according to any one of statements P2-2 to P2-42, selected from:

[0468] P2-44.G L But, G L1-1 and G L1-2 The compound according to statement P2-43, selected from:

[0469] P2-45.G L G L1-1 The compound according to statement P2-43,

[0470] P2-46.R L is of formula Ib.

[0471] P2-47.R L1 and R L2 The compound according to statement P2-46, wherein

[0472] P2-48.R L1 is H and R L2 The compound according to statement P2-46, wherein is methyl.

[0473] P2-49.R L1 and R L2 The compound according to description P2-46, wherein both are methyl.

[0474] P2-50.R L1 and R L2 together with the carbon atom to which they are attached form a cyclopropylene group.

[0475] P2-51.R L1 and R L2 together with the carbon atom to which they are attached form a cyclobutylene group.

[0476] The compound according to any one of statements P2-46 to P2-51, wherein P2-52.e is 0.

[0477] The compound according to any one of statements P2-46 to P2-51, wherein P2-53.e is 1.

[0478] P2-54.Formula IV: L-(D L ) p (IV) where L is a ligand unit (i.e., a targeting agent) and D L is represented by formula III: [ka] (In the formula, R LL teeth, (ia'): [ka] (wherein Q and X are as defined in any one of the statements P2-1 to P2-42; G LL is a linker connecting the Ligand units; and (ib'): [ka] (In the formula, R L-1 and R L2 is a linker connected to the Ligand unit selected from: p is an integer from 1 to 20. or a pharma- ceutically acceptable salt or solvate thereof.

[0479] P2-55.G LL but,

[0480] [Table 19] [Wherein, Ar is C 5~6represents an arylene group, and X is C 1~4 The complex of statement P2-54, wherein R represents an alkyl group.

[0481] P2-56.G LL But, G LL1-1 and G LL1-2 The conjugate of statement P2-55, selected from:

[0482] P2-57.G LL G LL1-1 The complex according to statement P2-56,

[0483] P2-58. A conjugate according to any one of statements P2-54 to P2-57, wherein the ligand unit is an antibody or an active fragment thereof.

[0484] P2-59. The conjugate according to statement P2-58, wherein the antibody or antibody fragment is an antibody or antibody fragment to a tumor-associated antigen.

[0485] P2-60. The conjugate according to statement P2-59, wherein the antibody or antibody fragment is an antibody that binds to one or more tumor-associated antigens or cell surface receptors selected from the following (1) to (89): (1)BMPR1B; (2) E16; (3) STEAP1; (4)0772P; (5) MPF; (6) Napi3b; (7) Sema 5b; (8) PSCA hlg; (9)ETBR; (10) MSG783; (11)STEAP2; (12)TrpM4; (13) CRIPTO; (14)CD21; (15)CD79b; (16)FcRH2; (17)HER2; (18) NCA; (19)MDP; (20)IL20R-α; (21)Brevican; (22)EphB2R; (23)ASLG659; (24)PSCA; (25)GEDA; (26)BAFF-R; (27)CD22; (28)CD79a; (29)CXCR5; (30)HLA-DOB; (31)P2X5; (32)CD72; (33)LY64; (34)FcRH1; (35)IRTA2; (36)TENB2; (37)PSMA-FOLH1; (38)SST; (38.1)SSTR2; (38.2)SSTR5; (38.3)SSTR1; (38.4)SSTR3; (38.5)SSTR4; (39)ITGAV; (40)ITGB6; (41)CEACAM5; (42)METHOD; (43)MUC1; (44)CA9; (45)EGFRvIII; (46)CD33; (47)CD19; (48)IL2RA; (49)AXL; (50)CD30-TNFRSF8; (51)BCMA-TNFRSF17; (52)CTAgs-CTA; (53)CD174(Fluid Y)-FUT3; (54)CLEC14A; (55)GRP78-HSPA5; (56)CD70; (57) Stem cell-specific antigen; (58)ASG-5; (59)ENPP3; (60)PRR4; (61)GCC-GUCY2C; (62)Liv-1-SLC39A6; (63)5T4; (64)CD56-NCMA1; (65)CanAg; (66)FOLR1; (67)GPNMB; (68)TIM-1-HAVCR1; (69)RG-1 / prostate tumor targeting Mindin-Mindin / RG-1; (70)B7-H4-VTCN1; (71)PTK7; (72)CD37; (73)CD138-SDC1; (74)CD74; (75) Claudin-CL; (76)EGFR; (77)Her3; (78)RON-MST1R; (79)EPHA2; (80)CD20-MS4A1; (81) Tenascin-C-TNC; (82)FAP; (83)DKK-1; (84)CD52; (85)CS1-SLAMF7; (86)Endoglin-ENG; (87) Annexin A1-ANXA1; (88)V-CAM(CD106)-VCAM1; (89)ASCT2(SLC1A5).

[0486] P2-61. A conjugate according to any one of statements P2-58 to P2-60, wherein the antibody or antibody fragment is a cysteine ​​engineered antibody.

[0487] P2-62. The conjugate of any one of statements P2-58 to P2-61, wherein the drug loading (p) of the drug (D) relative to the antibody (Ab) is an integer from 1 to about 10.

[0488] A conjugate according to statement P2-62, wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0489] P2-64. A mixture of conjugates according to any one of statements P2-58 to P2-63, wherein the average drug loading per antibody in the mixture of antibody-drug conjugates is about 1 to about 10.

[0490] P2-65. A complex or mixture according to any one of statements P2-54 to P2-64 for use in therapy.

[0491] P2-66. A pharmaceutical composition comprising a complex or mixture according to any one of statements P2-54 to P2-64 and a pharma- ceutically acceptable diluent, carrier, or excipient.

[0492] P2-67. A complex or mixture according to any one of statements P2-54 to P2-64, or a pharmaceutical composition according to statement P2-66, for use in treating a proliferative disorder in a subject.

[0493] P2-68. The complex, mixture or pharmaceutical composition according to statement P2-67, wherein the disease is cancer.

[0494] P2-69. Use of a complex or mixture according to any one of statements P2-54 to P2-64 or a pharmaceutical composition according to statement P2-66 in a method of medical treatment.

[0495] P2-70. A method of medical treatment comprising administering to a patient a pharmaceutical composition according to statement P2-66.

[0496] P2-71. The method according to statement P2-70, wherein the medical treatment method is for the treatment of cancer.

[0497] P2-72. The method of statement P2-71, wherein the patient is administered a chemotherapeutic agent in combination with the conjugate.

[0498] P2-73. Use of a complex or mixture according to any one of statements P2-54 to P2-64 in a method for the manufacture of a medicament for the treatment of a proliferative disorder.

[0499] P2-74. A method for treating a mammal having a proliferative disease comprising administering an effective amount of a complex or mixture described in any one of statements P2-54 to P2-64, or a pharmaceutical composition described in statement P2-66.

[0500] P2-75. Compound A as a single enantiomer or in an enantiomerically enriched form: [ka] . [Sequence table] [ka] [ka]

Claims

1. Formula 5: 【Chemistry 1】 [In the formula, R Ｌ＊ｐｒｏｔ is Q-Prot Ｎ (In the formula, Prot Ｎ is an amine protecting group, and Q: (a) an amino acid residue selected from Phe, Lys, Val, Ala, Cit, Leu, lie, Arg, and Trp; or (b) ＮＨ -Phe-Lys- Ｃ＝Ｏ 、 ＮＨ -Val-Ala- Ｃ＝Ｏ 、 ＮＨ -Val-Ly- Ｃ＝Ｏ 、 ＮＨ -R-L93- Ｃ＝Ｏ 、 ＮＨ -Val-Cit- Ｃ＝Ｏ 、 ＮＨ t0.

5. Ｃ＝Ｏ , ＮＨ -Leu-C- Ｃ＝Ｏ 、 ＮＨ -Ile-EM- Ｃ＝Ｏ 、 ＮＨ t0.

5. Ｃ＝Ｏ , ＮＨ -Trp-Cit- Ｃ＝Ｏ , and ＮＨ -Gly-Val- Ｃ＝Ｏ or (c) ＮＨ -Glu-Val-Ala- Ｃ＝Ｏ 、 ＮＨ -Glu-Val-Cit- Ｃ＝Ｏ 、 ＮＨ -αGlu-Val-Ala- Ｃ＝Ｏ , and ＮＨ -αGlu-Val-Cit- Ｃ＝Ｏ or (d) ＮＨ -Gly-Gly-Phe-Gly Ｃ＝Ｏ and ＮＨ -Gly-Phe-Gly-Gly Ｃ＝Ｏ is a tetrapeptide residue selected from Where: ＮＨ - represents the N-terminus of the residue, Ｃ＝Ｏ represents the C-terminal residue. A compound having the formula:

2. Q, ＮＨ -Phe-Lys- Ｃ＝Ｏ , ＮＨ -Val-Cit- Ｃ＝Ｏ or ＮＨ -Val-Ala- Ｃ＝Ｏ 2. The compound of claim 1 ,

3. Q, ＮＨ -Val-Ala- Ｃ＝Ｏ 2. The compound of claim 1,

4. Allyl ((S)-1-(((S)-1-((4-amino-5-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate: 【Chemistry 2】 2. The compound of claim 1,

5. Formula 6: 【Chemistry 3】 [In the formula, R Ｌ＊ｐｒｏｔ is as defined in any one of claims 1 to 3. A compound having the formula:

6. Allyl ((S)-3-methyl-1-oxo-1-(((S)-1-oxo-1-((5-oxo-4-(2,2,2-trifluoroacetamido)-5,6,7,8-tetrahydronaphthalen-1-yl)amino)propan-2-yl)amino)butan-2-yl)carbamate 【Chemistry 4】 6. The compound of claim 5,

7. Formula 4: 【Chemistry 5】 [In the formula, R Ｌ＊ｐｒｏｔ is as defined in any one of claims 1 to 3. A compound having the formula: 【Request 8】 【Chemical 6】 8. The compound of claim 7, wherein

9. Formula VI: 【Chemistry 7】 [Q is (a) an amino acid residue selected from Phe, Lys, Val, Ala, Cit, Leu, lie, Arg, and Trp; or (b) ＮＨ -Phe-Lys- Ｃ＝Ｏ 、 ＮＨ -Val-Ala- Ｃ＝Ｏ 、 ＮＨ -V-Ly- Ｃ＝Ｏ 、 ＮＨ -R-L93- Ｃ＝Ｏ 、 ＮＨ -Val-Cit- Ｃ＝Ｏ 、 ＮＨ t0.

5. Ｃ＝Ｏ , ＮＨ -Leu-C- Ｃ＝Ｏ 、 ＮＨ -Ile-EM- Ｃ＝Ｏ 、 ＮＨ t0.

5. Ｃ＝Ｏ , ＮＨ -Trp-Cit- Ｃ＝Ｏ , and ＮＨ -Gly-Val- Ｃ＝Ｏ or (c) ＮＨ -Glu-Val-Ala- Ｃ＝Ｏ 、 ＮＨ -Glu-Val-Cit- Ｃ＝Ｏ 、 ＮＨ -αGlu-Val-Ala- Ｃ＝Ｏ , and ＮＨ -αGlu-Val-Cit- Ｃ＝Ｏ or (d) ＮＨ -Gly-Gly-Phe-Gly Ｃ＝Ｏ and ＮＨ -Gly-Phe-Gly-Gly Ｃ＝Ｏ is a tetrapeptide residue selected from Where: ＮＨ - represents the N-terminus of the residue, Ｃ＝Ｏ represents the C-terminus of the residue.

10. Q, ＮＨ -Phe-Lys- Ｃ＝Ｏ , ＮＨ -Val-Cit- Ｃ＝Ｏ or ＮＨ -Val-Ala- Ｃ＝Ｏ 10. The compound of claim 9,

11. Q, ＮＨ -Val-Ala- Ｃ＝Ｏ 10. The compound of claim 9,

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