Patent Publication Number: US-10329352-B2

Title: Pyrrolobenzodiazepines and targeted conjugates

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This is a continuation of U.S. patent application Ser. No. 15/182,649, filed on Jun. 15, 2016, which is a continuation of U.S. patent application Ser. No. 14/351,172, filed on Apr. 11, 2014, now U.S. Pat. No. 9,387,259, which is a U.S. national stage entry of International Patent Application No. PCT/US2012/059870, filed on Oct. 12, 2012, which claims priority to U.S. Provisional Patent Application No. 61/547,195, filed on Oct. 14, 2011, the entire contents of each of which are fully incorporated herein by reference. 
     The present invention relates to pyrrolobenzodiazepines (PBDs), in particular pyrrolobenzodiazepine dimers having a C2-C3 double bond and an aryl group at the C2 position in each monomer unit, and their inclusion in targeted conjugates. 
    
    
     BACKGROUND TO THE INVENTION 
     Some pyrrolobenzodiazepines (PBDs) have the ability to recognise and bond to specific sequences of DNA; the preferred sequence is PuGPu. The first PBD antitumour antibiotic, anthramycin, was discovered in 1965 (Leimgruber, et al.,  J. Am. Chem. Soc.,  87, 5793-5795 (1965); Leimgruber, et al.,  J. Am. Chem. Soc.,  87, 5791-5793 (1965)). Since then, a number of naturally occurring PBDs have been reported, and numerous synthetic routes have been developed to a variety of analogues (Thurston, et al.,  Chem. Rev.  1994, 433-465 (1994); Antonow, D. and Thurston, D. E.,  Chem. Rev.  2011 111 (4), 2815-2864). Family members include abbeymycin (Hochlowski, et al.,  J. Antibiotics,  40, 145-148 (1987)), chicamycin (Konishi, et al.,  J. Antibiotics,  37, 200-206 (1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al.,  Chem. Brit.,  26, 767-772 (1990); Bose, et al.,  Tetrahedron,  48, 751-758 (1992)), mazethramycin (Kuminoto, et al.,  J. Antibiotics,  33, 665-667 (1980)), neothramycins A and B (Takeuchi, et al.,  J. Antibiotics,  29, 93-96 (1976)), porothramycin (Tsunakawa, et al.,  J. Antibiotics,  41, 1366-1373 (1988)), prothracarcin (Shimizu, et al,  J. Antibiotics,  29, 2492-2503 (1982); Langley and Thurston,  J. Org. Chem.,  52, 91-97 (1987)), sibanomicin (DC-102)(Hara, et al.,  J. Antibiotics,  41, 702-704 (1988); Itoh, et al.,  J. Antibiotics,  41, 1281-1284 (1988)), sibiromycin (Leber, et al.,  J. Am. Chem. Soc.,  110, 2992-2993 (1988)) and tomamycin (Arima, et al.,  J. Antibiotics,  25, 437-444 (1972)). PBDs are of the general structure: 
     
       
         
         
             
             
         
       
     
     They differ in the number, type and position of substituents, in both their aromatic A rings and pyrrolo C rings, and in the degree of saturation of the C ring. In the B-ring there is either an imine (N═C), a carbinolamine(NH—CH(OH)), or a carbinolamine methyl ether (NH—CH(OMe)) at the N10-C11 position which is the electrophilic centre responsible for alkylating DNA. All of the known natural products have an (S)-configuration at the chiral C11a position which provides them with a right-handed twist when viewed from the C ring towards the A ring. This gives them the appropriate three-dimensional shape for isohelicity with the minor groove of B-form DNA, leading to a snug fit at the binding site (Kohn, In  Antibiotics III.  Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter,  Acc. Chem. Res.,  19, 230-237 (1986)). Their ability to form an adduct in the minor groove, enables them to interfere with DNA processing, hence their use as antitumour agents. 
     It has been previously disclosed that the biological activity of these molecules can be potentiated by joining two PBD units together through their C8/C′-hydroxyl functionalities via a flexible alkylene linker (Bose, D. S., et al.,  J. Am. Chem. Soc.,  114, 4939-4941 (1992); Thurston, D. E., et al.,  J. Org. Chem.,  61, 8141-8147 (1996)). The PBD dimers are thought to form sequence-selective DNA lesions such as the palindromic 5′-Pu-GATC-Py-3′ interstrand cross-link (Smellie, M., et al.,  Biochemistry,  42, 8232-8239 (2003); Martin, C., et al.,  Biochemistry,  44, 4135-4147) which is thought to be mainly responsible for their biological activity. One example of a PBD dimmer, SG2000 (SJG-136): 
     
       
         
         
             
             
         
       
     
     has recently entered Phase II clinical trials in the oncology area (Gregson, S., et al.,  J. Med. Chem.,  44, 737-748 (2001); Alley, M. C., et al.,  Cancer Research,  64, 6700-6706 (2004); Hartley, J. A., et al.,  Cancer Research,  64, 6693-6699 (2004)). 
     More recently, the present inventors have previously disclosed in WO 2005/085251, dimeric PBD compounds bearing C2 aryl substituents, such as SG2202 (ZC-207): 
                         
and in WO2006/111759, bisulphites of such PBD compounds, for example SG2285 (ZC-423):
 
     
       
         
         
             
             
         
       
     
     These compounds have been shown to be highly useful cytotoxic agents (Howard, P. W., et al.,  Bioorg. Med. Chem.  (2009), 19 (22), 6463-6466, doi: 10.1016/j.bmcl.2009.09.012). 
     Due to the manner in which these highly potent compounds act in cross-linking DNA, these molecules have been made symmetrically. This provides for straightforward synthesis, either by constructing the PBD moieties simultaneously having already formed the dimer linkage, or by reacting already constructed PBD moieties with the dimer linking group. 
     WO 2010/043880 discloses unsymmetrical dimeric PBD compound bearing aryl groups in the C2 position of each monomer, where one of these aryl groups bears a substituent designed to provide an anchor for linking the compound to another moiety. Co-pending International application PCT/US2011/032664, filed 15 Apr. 2011, discloses the inclusion of these PBD dimer compounds in targeted conjugates. 
     DISCLOSURE OF THE INVENTION 
     The present inventors have developed further unsymmetrical dimeric PBD compounds for inclusion in targeted conjugates, where the substituents on the C2 aryl group not bearing the anchor for linking the compound to another moiety are different to those previously described. These differing substituent groups may offer advantages in the preparation and use of the compounds, particularly in their biological properties and the synthesis of conjugates, and the biological properties of these conjugates. 
     The present invention comprises a compound with the formula I: 
                         
or a pharmaceutically acceptable salt or solvate thereof,
 
wherein:
 
     R 2  is of formula III: 
                         
where A is a C 5-7  aryl group, X is
 
                         
or NHR N , wherein R N  is selected from the group comprising H and C 1-4  alkyl and either
 
     (i) Q 1  is a single bond, and Q 2  is selected from a single bond and —Z—(CH 2 ) n —, where Z is selected from a single bond, O, S and NH and n is from 1 to 3; or 
     (ii) Q 1  is —CH═CH—, and Q 2  is a single bond; 
     R 12  is a C 5-10  aryl group, substituted by a group selected from OH, CO 2 H, CO 2 R ∘ , where R ∘  is selected from C 1-4  alkyl; 
     R 6  and R 9  are independently selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NRR′, nitro, Me 3 Sn and halo; 
     where R and R′ are independently selected from optionally substituted C 1-12  alkyl, C 3-20  heterocyclyl and C 5-20  aryl groups; 
     R 7  is selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NHRR′, nitro, Me 3 Sn and halo; either: 
     (a) R 10  is H, and R 11  is OH, OR A , where R A  is C 1-4  alkyl; or 
     (b) R 10  and R 11  form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or 
     (c) R 10  is H and R 11  is SO z M, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; 
     R″ is a C 3-12  alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NR N2  (where R N2  is H or C 1-4  alkyl), and/or aromatic rings, e.g. benzene or pyridine; 
     Y and Y′ are selected from O, S, or NH; 
     R 6′ , R 7′ , R 9′  are selected from the same groups as R 6 , R 7  and R 9  respectively and R 10′  and R 11′  are the same as R 10  and R 11 , wherein if R 11  and R 11′  are SO z M, M may represent a divalent pharmaceutically acceptable cation. 
     A second aspect of the present invention provides the use of a compound of the first aspect of the invention in the manufacture of a medicament for treating a proliferative disease. The second aspect also provides a compound of the first aspect of the invention for use in the treatment of a proliferative disease. 
     One of ordinary skill in the art is readily able to determine whether or not a candidate conjugate treats a proliferative condition for any particular cell type. For example, assays which may conveniently be used to assess the activity offered by a particular compound are described in the examples below. 
     A third aspect of the present invention comprises a compound of formula II: 
                         
or a pharmaceutically acceptable salt or solvate thereof,
 
wherein:
 
     R 2  is of formula III: 
                         
where A is a C 5-7  aryl group, X is
 
                         
or NHR N , wherein R N  is selected from the group comprising H and C 1-4  alkyl and either
 
     (i) Q 1  is a single bond, and Q 2  is selected from a single bond and —Z—(CH 2 ) n —, where Z is selected from a single bond, O, S and NH and n is from 1 to 3; or 
     (ii) Q 1  is —CH═CH—, and Q 2  is a single bond; 
     R 12  is a C 5-10  aryl group, substituted by a group selected from OH, CO 2 H, CO 2 R 602  , where R ∘  is selected from C 1-4  alkyl; 
     R 6  and R 9  are independently selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NRR′, nitro, Me 3 Sn and halo; 
     where R and R′ are independently selected from optionally substituted C 1-12  alkyl, C 3-20  heterocyclyl and C 5-20  aryl groups; 
     R 7  is selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NHRR′, nitro, Me 3 Sn and halo; either: 
     (a) R 10  is carbamate nitrogen protecting group, and R 11  is O-Prot ∘ , wherein Prot ∘  is an oxygen protecting group; or 
     (b) R 10  is a hemi-aminal nitrogen protecting group and R 11  is an oxo group; 
     R″ is a C 3-12  alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NR N2  (where R N2  is H or C 1-4  alkyl), and/or aromatic rings, e.g. benzene or pyridine; 
     Y and Y′ are selected from O, S, or NH; 
     R 6′ , R 7′ , R 9′  are selected from the same groups as R 6 , R 7  and R 9  respectively and R 10′  and R 11′  are the same as R 10  and R 11 . 
     A fourth aspect of the present invention comprises a method of making a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, from a compound of formula II, or a pharmaceutically acceptable salt or solvate thereof, by deprotection of the imine bond. 
     The unsymmetrical dimeric PBD compounds of the present invention are made by different strategies to those previously employed in making symmetrical dimeric PBD compounds. In particular, the present inventors have developed a method which involves adding each each C2 substituent to a symmetrical PBD dimer core in separate method steps. Accordingly, a fifth aspect of the present invention provides a method of making a compound of the first or third aspect of the invention, comprising at least one of the method steps set out below. 
     In a sixth aspect, the present invention relates to Conjugates comprising dimers of PBDs linked to a targeting agent, wherein the PBD dimer is of formula I, or a pharmaceutically acceptable salt or solvate thereof (supra). 
     In some embodiments, the Conjugates have the following formula IV:
 
L-(LU-D) p    (IV)
 
or a pharmaceutically acceptable salt or solvate thereof, wherein L is a Ligand unit (i.e., a targeting agent), LU is a Linker unit and D is a Drug unit that is a PBD dimer (see below). The subscript p is from 1 to 20. Accordingly, the Conjugates comprise a Ligand unit covalently linked to at least one Drug unit by a Linker unit. The Ligand unit, described more fully below, is a targeting agent that binds to a target moiety. The Ligand unit can, for example, specifically bind to a cell component (a Cell Binding Agent) or to other target molecules of interest. Accordingly, the present invention also provides methods for the treatment of, for example, various cancers and autoimmune disease. These methods encompass the use of the Conjugates wherein the Ligand unit is a targeting agent that specifically binds to a target molecule. The Ligand unit can be, for example, 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.
 
     In the conjugates of the present invention, the PBD dimer D is of formula I, or a pharmaceutically acceptable salt or solvate thereof, except that X is 
                         
wherein R N  is selected from the group comprising H and C 1-4  alkyl, and the asterix indicates the point of attachment to the remainder of the Drug unit and the wavy line indicates the point of attachment to the Linker Unit.
 
     The drug loading is represented by p, the number of drug molecules per Ligand unit (e.g., an antibody). Drug loading may range from 1 to 20 Drug units (D) per Ligand unit (e.g., Ab or mAb). For compositions, p represents the average drug loading of the Conjugates in the composition, and p ranges from 1 to 20. 
     In some embodiments, p is from about 1 to about 8 Drug units per Ligand unit. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is from about 2 to about 8 Drug units per Ligand unit. In some embodiments, p is from about 2 to about 6, 2 to about 5, or 2 to about 4 Drug units per Ligand unit. In some embodiments, p is about 2, about 4, about 6 or about 8 Drug units per Ligand unit. 
     The average number of Drugs units per Ligand unit in a preparation from a conjugation reaction may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC. The quantitative distribution of Conjugates in terms of p may also be determined. In some instances, separation, purification, and characterization of homogeneous Conjugates, where p is a certain value, from Conjugates with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis. 
     In a seventh aspect, the present invention relates to Linker-Drug compounds (i.e., Drug-Linkers) comprising dimers of PBDs (see above) linked to a linking unit. These Drug-linkers can be used as intermediates for the synthesis of Conjugates comprising dimers of PBDs linked to a targeting agent. 
     These Drug-Linkers have the following formula V:
 
LU-D   (V)
 
or a pharmaceutically acceptable salt or solvate thereof, wherein LU is a Linker unit and D is a Drug unit that is a PBD dimer.
 
     In the Drug-Linkers of the present invention, the PBD dimer D is of formula I, or a pharmaceutically acceptable salt or solvate thereof, except that X is is 
                         
wherein R N  is selected from the group comprising H and C 1-4  alkyl, and the asterix indicates the point of attachment to the remainder of the Drug unit and the wavy line indicates the point of attachment to the Linker Unit.
 
    
    
     
       FIGURES 
         FIG. 1  shows the effect on tumour volume of a conjugate of the present invention at two different doses; 
         FIG. 2  shows the effect on tumour volume of the same conjugate as in  FIG. 1  on a different tumour. 
     
    
    
     DEFINITIONS 
     Pharmaceutically Acceptable Cations 
     Examples of pharmaceutically acceptable monovalent and divalent cations are discussed in Berge, et al.,  J. Pharm. Sci.,  66, 1-19 (1977), which is incorporated herein by reference in its entirety and for all purposes. 
     The pharmaceutically acceptable cation may be inorganic or organic. 
     Examples of pharmaceutically acceptable monovalent inorganic cations include, but are not limited to, alkali metal ions such as Na +  and K + . Examples of pharmaceutically acceptable divalent inorganic cations include, but are not limited to, alkaline earth cations such as Ca 2+  and Mg 2+ . Examples of pharmaceutically acceptable organic cations include, but are not limited to, ammonium ion (i.e. NH 4   + ) and substituted ammonium ions (e.g. NH 3 R + , NH 2 R 2   + , NHR 3   + , NR 4   + ). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH 3 ) 4   + . 
     Substituents 
     The phrase “optionally substituted” as used herein, pertains to a parent group which may be unsubstituted or which may be substituted. 
     Unless otherwise specified, the term “substituted” as used herein, pertains to a parent group which bears one or more substituents. The term “substituent” is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, or if appropriate, fused to, a parent group. A wide variety of substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known. 
     Examples of substituents are described in more detail below. 
     C 1-12  alkyl: The term “C 1-12  alkyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). The term “C 1-4  alkyl” as used herein, pertains 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 which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). Similarly, the term “C 1-2 alkyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 2 carbon atoms, i.e. methyl or ethyl. 
     Thus, the term “alkyl” includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below. 
     Examples of saturated alkyl groups include, but are not limited to, methyl (C 1 ), ethyl (C 2 ), propyl (C 3 ), butyl (C 4 ), pentyl (C 5 ), hexyl (C 6 ) and heptyl (C 7 ). 
     Examples of saturated linear alkyl groups include, but are not limited to, methyl (C 1 ), ethyl (C 2 ), n-propyl (C 3 ), n-butyl (C 4 ), n-pentyl (amyl) (C 5 ), n-hexyl (C 6 ) and n-heptyl (C 7 ). 
     Examples of saturated branched alkyl groups include iso-propyl (C 3 ), iso-butyl (C 4 ), sec-butyl (C 4 ), tert-butyl (C 4 ), iso-pentyl (C 5 ), and neo-pentyl (C 5 ). 
     C 2-12  Alkenyl: The term “C 2-12  alkenyl” as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds. 
     Examples of unsaturated alkenyl groups include, but are not limited to, 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 ), butenyl (C 4 ), pentenyl (C 5 ), and hexenyl (C 6 ). 
     C 2-12  alkynyl: The term “C 2-12  alkynyl” as used herein, pertains to an alkyl group having one or more carbon-carbon triple bonds. 
     Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (—C≡CH) and 2-propynyl (propargyl, —CH 2 —C≡CH). 
     C 3-12  cycloalkyl: The term “C 3-12  cycloalkyl” as used herein, pertains to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms. 
     Examples of cycloalkyl groups include, but are not limited to, those derived from:
         saturated monocyclic hydrocarbon compounds:       

     cyclopropane (C 3 ), cyclobutane (C 4 ), cyclopentane (C 5 ), cyclohexane (C 6 ), cycloheptane (C 7 ), methylcyclopropane (C 4 ), dimethylcyclopropane (C 5 ), methylcyclobutane (C 5 ), dimethylcyclobutane (C 6 ), methylcyclopentane (C 6 ), dimethylcyclopentane (C 7 ) and methylcyclohexane (C 7 );
         unsaturated monocyclic hydrocarbon compounds:       

     cyclopropene (C 3 ), cyclobutene (C 4 ), cyclopentene (C 5 ), cyclohexene (C 6 ), methylcyclopropene (C 4 ), dimethylcyclopropene (C 5 ), methylcyclobutene (C 5 ), dimethylcyclobutene (C 6 ), methylcyclopentene (C 6 ), dimethylcyclopentene (C 7 ) and methylcyclohexene (C 7 ); and
         saturated polycyclic hydrocarbon compounds:       

     norcarane (C 7 ), norpinane (C 7 ), norbornane (C 7 ). 
     C 3-20  heterocyclyl: The term “C 3-20  heterocyclyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms. 
     In this context, the prefixes (e.g. C 3-20 , C 3-7 , C 5-6 , etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C 5-6 heterocyclyl”, as used herein, pertains to a heterocyclyl group having 5 or 6 ring atoms. 
     Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from: 
     N 1 : aziridine (C 3 ), azetidine (C 4 ), pyrrolidine (tetrahydropyrrole) (C 5 ), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C 5 ), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C 5 ), piperidine (C 6 ), dihydropyridine (C 6 ), tetrahydropyridine (C 6 ), azepine (C 7 ); 
     O 1 : oxirane (C 3 ), oxetane (C 4 ), oxolane (tetrahydrofuran) (C 5 ), oxole (dihydrofuran) (C 5 ), oxane (tetrahydropyran) (C 6 ), dihydropyran (C 6 ), pyran (C 6 ), oxepin (C 7 ); 
     S 1 : thiirane (C 3 ), thietane (C 4 ), thiolane (tetrahydrothiophene) (C 5 ), thiane (tetrahydrothiopyran) (C 6 ), thiepane (C 7 ); 
     O 2 : dioxolane (C 5 ), dioxane (C 6 ), and dioxepane (C 7 ); 
     O 3 : trioxane (C 6 ); 
     N 2 : imidazolidine (C 5 ), pyrazolidine (diazolidine) (C 5 ), imidazoline (C 5 ), pyrazoline (dihydropyrazole) (C 5 ), piperazine (C 6 ); 
     N 1 O 1 : tetrahydrooxazole (C 5 ), dihydrooxazole (C 5 ), tetrahydroisoxazole (C 5 ), dihydroisoxazole (C 5 ), morpholine (C 6 ), tetrahydrooxazine (C 6 ), dihydrooxazine (C 6 ), oxazine (C 6 ); 
     N 1 S 1 : thiazoline (C 5 ), thiazolidine (C 5 ), thiomorpholine (C 6 ); 
     N 2 O 1 : oxadiazine (C 6 ); 
     O 1 S 1 : oxathiole (C 5 ) and oxathiane (thioxane) (C 6 ); and, 
     N 1 O 1 S 1 : oxathiazine (C 6 ). 
     Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C 5 ), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C 6 ), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose. 
     C 5-20  aryl: The term “C 5-20  aryl”, as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 3 to 20 ring atoms. The term “C 5-7  aryl”, as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 5 to 7 ring atoms and the term “C 5-10  aryl”, as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 5 to 10 ring atoms. Preferably, each ring has from 5 to 7 ring atoms. 
     In this context, the prefixes (e.g. C 3-20 , C 5-7 , C 5-6 , C 5-10 , etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C 5-6  aryl” as used herein, pertains to an aryl group having 5 or 6 ring atoms. 
     The ring atoms may be all carbon atoms, as in “carboaryl groups”. 
     Examples of carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (C 6 ), naphthalene (C 10 ), azulene (C 10 ), anthracene (C 14 ), phenanthrene (C 14 ), naphthacene (C 18 ), and pyrene (C 16 ). 
     Examples of aryl groups which comprise fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-1H-indene) (C 9 ), indene (C 9 ), isoindene (C 9 ), tetraline (1,2,3,4-tetrahydronaphthalene (C 10 ), acenaphthene (C 12 ), fluorene (C 13 ), phenalene (C 13 ), acephenanthrene (C 15 ), and aceanthrene (C 16 ). 
     Alternatively, the ring atoms may include one or more heteroatoms, as in “heteroaryl groups”. Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from: 
     N 1 : pyrrole (azole) (C 5 ), pyridine (azine) (C 6 ); 
     O 1 : furan (oxole) (C 5 ); 
     S 1 : thiophene (thiole) (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 ); 
     N 3 : triazole (C 5 ), triazine (C 6 ); and, 
     N 4 : tetrazole (C 5 ). 
     Examples of heteroaryl which comprise fused rings, include, but are not limited to:
         C 9  (with 2 fused rings) derived from benzofuran (O 1 ), isobenzofuran (O 1 ), indole (N 1 ), isoindole (N 1 ), indolizine (N 1 ), indoline (N 1 ), isoindoline (N 1 ), purine (N 4 ) (e.g., adenine, guanine), benzimidazole (N 2 ), indazole (N 2 ), benzoxazole (N 1 O 1 ), benzisoxazole (N 1 O 1 ), benzodioxole (O 2 ), benzofurazan (N 2 O 1 ), benzotriazole (N 3 ), benzothiofuran (S 1 ), benzothiazole (N 1 S 1 ), benzothiadiazole (N 2 S);   C 10  (with 2 fused rings) derived from chromene (O 1 ), isochromene (O 1 ), chroman (O 1 ), isochroman (O 1 ), benzodioxan (O 2 ), quinoline (N 1 ), isoquinoline (N 1 ), quinolizine (N 1 ), benzoxazine (N 1 O 1 ), benzodiazine (N 2 ), pyridopyridine (N 2 ), quinoxaline (N 2 ), quinazoline (N 2 ), cinnoline (N 2 ), phthalazine (N 2 ), naphthyridine (N 2 ), pteridine (N 4 );   C 11  (with 2 fused rings) derived from benzodiazepine (N 2 );   C 13  (with 3 fused rings) derived from carbazole (N 1 ), dibenzofuran (O 1 ), dibenzothiophene (S 1 ), carboline (N 2 ), perimidine (N 2 ), pyridoindole (N 2 ); and,   C 14  (with 3 fused rings) derived from acridine (N 1 ), xanthene (O 1 ), thioxanthene (S 1 ), oxanthrene (O 2 ), phenoxathiin (O 1 S 1 ), phenazine (N 2 ), phenoxazine (N 1 O 1 ), phenothiazine (N 1 S 1 ), thianthrene (S 2 ), phenanthridine (N 1 ), phenanthroline (N 2 ), phenazine (N 2 ).       

     The above groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from themselves and the additional substituents listed below. 
     Halo: —F, —Cl, —Br, and —I. 
     Hydroxy: —OH. 
     Ether: —OR, wherein R is an ether substituent, for example, a C 1-7  alkyl group (also referred to as a C 1-7  alkoxy group, discussed below), a C 3-20  heterocyclyl group (also referred to as a C 3-20  heterocyclyloxy group), or a C 5-20  aryl group (also referred to as a C 5-20  aryloxy group), preferably a C 1-7 alkyl group. 
     Alkoxy: —OR, wherein R is an alkyl group, for example, a C 1-7  alkyl group. Examples of C 1-7  alkoxy groups include, but are not limited to, —OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr) (isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu) (isobutoxy), and —O(tBu) (tert-butoxy). 
     Acetal: —CH(OR 1 )(OR 2 ), wherein R 1  and R 2  are independently acetal substituents, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C 1-7  alkyl group, or, in the case of a “cyclic” acetal group, R 1  and R 2 , taken together with the two oxygen atoms to which they are attached, and the carbon atoms to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Examples of acetal groups include, but are not limited to, —CH(OMe) 2 , —CH(OEt) 2 , and —CH(OMe)(OEt). 
     Hemiacetal: —CH(OH)(OR 1 ), wherein R 1  is a hemiacetal substituent, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C 1-7  alkyl group. Examples of hemiacetal groups include, but are not limited to, —CH(OH)(OMe) and —CH(OH)(OEt). 
     Ketal: —CR(OR 1 )(OR 2 ), where R 1  and R 2  are as defined for acetals, and R is a ketal substituent other than hydrogen, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C 1-7  alkyl group. Examples ketal groups include, but are not limited to, —C(Me)(OMe) 2 , —C(Me)(OEt) 2 , —C(Me)(OMe)(OEt), —C(Et)(OMe) 2 , —C(Et)(OEt) 2 , and —C(Et)(OMe)(OEt). 
     Hemiketal: —CR(OH)(OR 1 ), where R 1  is as defined for hemiacetals, and R is a hemiketal substituent other than hydrogen, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C 1-7  alkyl group. Examples of hemiacetal groups include, but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe), —C(Me)(OH)(OEt), and —C(Et)(OH)(OEt). 
     Oxo (keto, -one): ═O. 
     Thione (thioketone): ═S. 
     Imino (imine): ═NR, wherein R is an imino substituent, for example, hydrogen, C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably hydrogen or a C 1-7  alkyl group. Examples of ester groups include, but are not limited to, ═NH, ═NMe, ═NEt, and ═NPh. 
     Formyl (carbaldehyde, carboxaldehyde): —C(═O )H. 
     Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, a C 1-7  alkyl group (also referred to as C 1-7 alkylacyl or C 1-7 alkanoyl), a C 3-20  heterocyclyl group (also referred to as C 3-20  heterocyclylacyl), or a C 5-20  aryl group (also referred to as C 5-20  arylacyl), preferably a C 1-7  alkyl group. Examples of acyl groups include, but are not limited to, —C(═O)CH 3  (acetyl), —C(═O)CH 2 CH 3  (propionyl), —C(═O)C(CH 3 ) 3  (t-butyryl), and —C(═O)Ph (benzoyl, phenone). 
     Carboxy (carboxylic acid): —C(═O)OH. Thiocarboxy (thiocarboxylic acid): —C(═S)SH. 
     Thiolocarboxy (thiolocarboxylic acid): —C(═O)SH. 
     Thionocarboxy (thionocarboxylic acid): —C(═S)OH. 
     Imidic acid: —C(═NH)OH. 
     Hydroxamic acid: —C(═NOH)OH. 
     Ester (carbon/late, carboxylic acid ester, oxycarbonyl): —C(═O)OR, wherein R is an ester substituent, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C 1-7  alkyl group. Examples of ester groups include, but are not limited to, —C(═O)OCH 3 , —C(═O)OCH 2 CH 3 , —C(═O)OC(CH 3 ) 3 , and —C(═O)OPh. 
     Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C 1-7  alkyl group. Examples of acyloxy groups include, but are not limited to, —OC(═O)CH 3  (acetoxy), —OC(═O)CH 2 CH 3 , —OC(═O)C(CH 3 ) 3 , —OC(═O)Ph, and —OC(═O)CH 2 Ph. 
     Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C 1-7  alkyl group. Examples of ester groups include, but are not limited to, —OC(═O)OCH 3 , —OC(═O)OCH 2 CH 3 , —OC(═O)OC(CH 3 ) 3 , and —OC(═O)OPh. 
     Amino: —NR 1 R 2 , wherein R 1  and R 2  are independently amino substituents, for example, hydrogen, a C 1-7  alkyl group (also referred to as C 1-7  alkylamino or di-C 1-7  alkylamino), a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably H or a C 1-7  alkyl group, or, in the case of a “cyclic” amino group, R 1  and R 2 , taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Amino groups may be primary (—NH 2 ), secondary (—NHR 1 ), or tertiary (—NHR 1 R 2 ), and in cationic form, may be quaternary (— + NR 1 R 2 R 3 ). Examples of amino groups include, but are not limited to, —NH 2 , —NHCH 3 , —NHC(CH 3 ) 2 , —N(CH 3 ) 2 , —N(CH 2 CH 3 ) 2 , and —NHPh. Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino. 
     Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR 1 R 2 , wherein R 1  and R 2  are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═O)NH 2 , —C(═O)NHCH 3 , —C(═O)N(CH 3 ) 2 , —C(═O)NHCH 2 CH 3 , and —C(═O)N(CH 2 CH 3 ) 2 , as well as amido groups in which R 1  and R 2 , together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl. 
     Thioamido (thiocarbamyl): —C(═S)NR 1 R 2 , wherein R 1  and R 2  are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═S)NH 2 , —C(═S)NHCH 3 , —C(═S)N(CH 3 ) 2 , and —C(═S)NHCH 2 CH 3 . 
     Acylamido (acylamino): —NR 1 C(═O)R 2 , wherein R 1  is an amide substituent, for example, hydrogen, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably hydrogen or a C 1-7  alkyl group, and R 2  is an acyl substituent, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20 aryl group, preferably hydrogen or a C 1-7  alkyl group. Examples of acylamide groups include, but are not limited to, —NHC(═O)CH 3  , —NHC(═O)CH 2 CH 3 , and —NHC(═O)Ph. R 1  and R 2  may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl: 
     
       
         
         
             
             
         
       
     
     Aminocarbonyloxy: —OC(═O)NR 1 R 2 , wherein R 1  and R 2  are independently amino substituents, as defined for amino groups. Examples of aminocarbonyloxy groups include, but are not limited to, —OC(═O)NH 2 , —OC(═O)NHMe, —OC(═O)NMe 2 , and —OC(═O)NEt 2 . 
     Ureido: —N(R 1 )CONR 2 R 3  wherein R 2  and R 3  are independently amino substituents, as defined for amino groups, and R 1  is a ureido substituent, for example, hydrogen, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably hydrogen or a C 1-7  alkyl group. Examples of ureido groups include, but are not limited to, —NHCONH 2 , —NHCONHMe, —NHCONHEt, —NHCONMe 2 , —NHCONEt 2 , —NMeCONH 2 , —NMeCONHMe, —NMeCONHEt, —NMeCONMe 2 , and —NMeCONEt 2 . 
     Guanidino: —NH—C(═NH)NH 2 . 
     Tetrazolyl: a five membered aromatic ring having four nitrogen atoms and one carbon atom, 
     
       
         
         
             
             
         
       
     
     Imino: ═NR, wherein R is an imino substituent, for example, for example, hydrogen, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably H or a C 1-7 alkyl group. Examples of imino groups include, but are not limited to, ═NH, ═NMe, and ═NEt. 
     Amidine (amidino): —C(═NR)NR 2 , wherein each R is an amidine substituent, for example, hydrogen, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably H or a C 1-7  alkyl group. Examples of amidine groups include, but are not limited to, —C(═NH)NH 2 , —C(═NH)NMe 2 , and —C(═NMe)NMe 2 . 
     Nitro: —NO 2 . 
     Nitroso: —NO. 
     Azido: —N 3 . 
     Cyano (nitrile, carbonitrile): —CN. 
     Isocyano: —NC. 
     Cyanato: —OCN. 
     Isocyanato: —NCO. 
     Thiocyano (thiocyanato): —SCN. 
     Isothiocyano (isothiocyanato): —NCS. 
     Sulfhydryl (thiol, mercapto): —SH. 
     Thioether (sulfide): —SR, wherein R is a thioether substituent, for example, a C 1-7  alkyl group (also referred to as a C 1-7 alkylthio group), a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C 1-7  alkyl group. Examples of C 1-7 alkylthio groups include, but are not limited to, —SCH 3  and —SCH 2 CH 3 . 
     Disulfide: —SS—R, wherein R is a disulfide substituent, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C 1-7  alkyl group (also referred to herein as C 1-7  alkyl disulfide). Examples of C 1-7  alkyl disulfide groups include, but are not limited to, —SSCH 3  and —SSCH 2 CH 3 . 
     Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfine substituent, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C 1-7  alkyl group. Examples of sulfine groups include, but are not limited to, —S(═O)CH 3  and —S(═O)CH 2 CH 3 . 
     Sulfone (sulfonyl): —S(═O) 2 R, wherein R is a sulfone substituent, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C, 1-7  alkyl group, including, for example, a fluorinated or perfluorinated C 1-7  alkyl group. Examples of sulfone groups include, but are not limited to, —S(═O) 2 CH 3  (methanesulfonyl, mesyl), —S(═O) 2 CF 3  (triflyl), —S(═O) 2 CH 2 CH 3  (esyl), —S(═O) 2 C 4 F 9  (nonaflyl), —S(═O) 2 CH 2 CF 3  (tresyl), —S(═O) 2 CH 2 CH 2 NH 2  (tauryl), —S(═O) 2 Ph (phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl), 4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl), 4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and 5-dimethylamino-naphthalen-1-ylsulfonate (dansyl). 
     Sulfinic acid (sulfino): —S(═O)OH, —SO 2 H. 
     Sulfonic acid (sulfo): —S(═O) 2 0H, —SO 3 H. 
     Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinate substituent, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C 1-7  alkyl group. Examples of sulfinate groups include, but are not limited to, —S(═O)OCH 3  (methoxysulfinyl; methyl sulfinate) and —S(═O)OCH 2 CH 3  (ethoxysulfinyl; ethyl sulfinate). 
     Sulfonate (sulfonic acid ester): —S(═O) 2 OR, wherein R is a sulfonate substituent, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C 1-7  alkyl group. Examples of sulfonate groups include, but are not limited to, —S(═O) 2 OCH 3  (methoxysulfonyl; methyl sulfonate) and —S(═O) 2 OCH 2 CH 3  (ethoxysulfonyl; ethyl sulfonate). Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C, 1-7  alkyl group. Examples of sulfinyloxy groups include, but are not limited to, —OS(═O)CH 3  and —OS(═O)CH 2 CH 3 . 
     Sulfonyloxy: —OS(═O) 2 R, wherein R is a sulfonyloxy substituent, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C 1-7  alkyl group. Examples of sulfonyloxy groups include, but are not limited to, —OS(═O) 2 CH 3  (mesylate) and —OS(═O) 2 CH 2 CH 3  (esylate). 
     Sulfate: —OS(═O) 2 OR; wherein R is a sulfate substituent, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C 1-7  alkyl group. Examples of sulfate groups include, but are not limited to, —OS(═O) 2 OCH 3  and —SO(═O) 2 OCH 2 CH 3 . 
     Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR 1 R 2 , wherein R 1  and R 2  are independently amino substituents, as defined for amino groups. Examples of sulfamyl groups include, but are not limited to, —S(═O)NH 2 , —S(═O)NH(CH 3 ), —S(═O)N(CH 3 ) 2 , —S(═O)NH(CH 2 CH 3 ), —S(═O)N(CH 2 CH 3 ) 2 , and —S(═O)NHPh. 
     Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide): —S(═O) 2 NR 1 R 2 , wherein R 1  and R 2  are independently amino substituents, as defined for amino groups. Examples of sulfonamido groups include, but are not limited to, —S(═O) 2 NH 2 , —S(═O) 2 NH(CH 3 ), —S(═O) 2 N(CH 3 ) 2 , —S(═O) 2 NH(CH 2 CH 3 ), —S(═O) 2 N(CH 2 CH 3 ) 2 , and —S(═O) 2 NHPh. 
     Sulfamino: —NR 1 S(═O) 2 OH, wherein R 1  is an amino substituent, as defined for amino groups. Examples of sulfamino groups include, but are not limited to, —NHS(═O) 2 OH and —N(CH 3 )S(═O) 2 OH. 
     Sulfonamino: —NR 1 S(═O) 2 R, wherein R 1  is an amino substituent, as defined for amino groups, and R is a sulfonamino substituent, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C 1-7  alkyl group. Examples of sulfonamino groups include, but are not limited to, —NHS(═O) 2 CH 3  and —N(CH 3 )S(═O) 2 C 6 H 5 . 
     Sulfinamino: —NR 1 S(═O)R, wherein R 1  is an amino substituent, as defined for amino groups, and R is a sulfinamino substituent, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C 1-7  alkyl group. Examples of sulfinamino groups include, but are not limited to, —NHS(═O)CH 3  and —N(CH 3 )S(═O)C 6 H 5 . 
     Phosphino (phosphine): —PR 2 , wherein R is a phosphino substituent, for example, —H, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably —H, a C 1-7  alkyl group, or a C 5-20  aryl group. Examples of phosphino groups include, but are not limited to, —PH 2 , —P(CH 3 ) 2 , —P(CH 2 CH 3 ) 2 , —P(t-Bu) 2 , and —P(Ph) 2 . 
     Phospho: —P(═O) 2 . 
     Phosphinyl (phosphine oxide): —P(═O)R 2 , wherein R is a phosphinyl substituent, for example, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably a C 1-7  alkyl group or a C 5-20  aryl group. Examples of phosphinyl groups include, but are not limited to, —P(═O)(CH 3 ) 2 , —P(═O)(CH 2 CH 3 ) 2 , —P(═O)(t-Bu) 2 , and —P(═O)(Ph) 2 . 
     Phosphonic acid (phosphono): —P(═O)(OH) 2 . 
     Phosphonate (phosphono ester): —P(═O)(OR) 2 , where R is a phosphonate substituent, for example, —H, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably —H, a C 1-7  alkyl group, or a C 5-20  aryl group. Examples of phosphonate groups include, but are not limited to, —P(═O)(OCH 3 ) 2 , —P(═O)(OCH 2 CH 3 ) 2 , —P(═O)(O-t-Bu) 2 , and —P(═O)(OPh) 2 . 
     Phosphoric acid (phosphonooxy): —OP(═O)(OH) 2 . 
     Phosphate (phosphonooxy ester): —OP(═O)(OR) 2 , where R is a phosphate substituent, for example, —H, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably —H, a C 1-7  alkyl group, or a C 5-20  aryl group. Examples of phosphate groups include, but are not limited to, —OP(═O)(OCH 3 ) 2 , —OP(═O)(OCH 2 CH 3 ) 2 , —OP(═O)(O-t-Bu) 2 , and —OP(═O)(OPh) 2 . 
     Phosphorous acid: —OP(OH) 2 . 
     Phosphite: —OP(OR) 2 , where R is a phosphite substituent, for example, —H, a C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably —H, a C 1-7  alkyl group, or a C 5-20  aryl group. Examples of phosphite groups include, but are not limited to, —OP(OCH 3 ) 2 , —OP(OCH 2 CH 3 ) 2 , —OP(O-t-Bu) 2 , and —OP(OPh) 2 . 
     Phosphoramidite: —OP(OR 1 )—NR 2   2 , where R 1  and R 2  are phosphoramidite substituents, for example, —H, a (optionally substituted) C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably —H, a C 1-7  alkyl group, or a C 5-20  aryl group. Examples of phosphoramidite groups include, but are not limited to, —OP(OCH 2 CH 3 )—N(CH 3 ) 2 , —OP(OCH 2 CH 3 )—N(i-Pr) 2 , and —OP(OCH 2 CH 2 CN)—N(i-Pr) 2 . 
     Phosphoramidate: —OP(═O)(OR 1 )—NR 2   2 , where R 1  and R 2  are phosphoramidate substituents, for example, —H, a (optionally substituted) C 1-7  alkyl group, a C 3-20  heterocyclyl group, or a C 5-20  aryl group, preferably —H, a C 1-7  alkyl group, or a C 5-20  aryl group. Examples of phosphoramidate groups include, but are not limited to, —OP(═O)(OCH 2 CH 3 )—N(CH 3 ) 2 , —OP(═O)(OCH 2 CH 3 )—N(i-Pr) 2 , and —OP(═O)(OCH 2 CH 2 CN)—N(i-Pr) 2 . 
     Alkylene 
     C 3-12  alkylene: The term “C 3-12  alkylene”, as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 3 to 12 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term “alkylene” includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below. 
     Examples of linear saturated C 3-12  alkylene groups include, but are not limited to, —(CH 2 ) n  —where n is an integer from 3 to 12, for example, —CH 2 CH 2 CH 2  —(propylene), —CH 2 CH 2 CH 2 CH 2  —(butylene), —CH 2 CH 2 CH 2 CH 2 CH 2  —(pentylene) and —CH 2 CH 2 CH 2 CH 2 —CH 2 CH 2 CH 2  —(heptylene). 
     Examples of branched saturated C 3-12  alkylene groups include, but are not limited to, —CH(CH 3 )CH 2 —, —CH(CH 3 )CH 2 CH 2 —, —CH(CH 3 )CH 2 CH 2 CH 2 —, —CH 2 CH(CH 3 )CH 2 —, —CH 2 CH(CH 3 )CH 2 CH 2 —, —CH(CH 2 CH 3 )—, —CH(CH 2 CH 3 )CH 2 —, and —CH 2 CH(CH 2 CH 3 )CH 2 —. 
     Examples of linear partially unsaturated C 3-12  alkylene groups (C 3-12  alkenylene, and alkynylene groups) include, but are not limited to, —CH═CH—CH 2 —, —CH 2 —CH═CH 2 —, —CH═CH—CH 2 —CH 2 —, —CH═CH—CH 2 —CH 2 —CH 2 —, —CH═CH—CH═CH—, —CH═CH—CH═CH—CH 2 —, —CH═CH—CH═CH—CH 2 —CH 2 —, —CH═CH—CH 2 —CH═CH—, —CH═CH—CH 2 —CH 2 —CH═CH—, and —CH 2 —C≡C—CH 2 —. 
     Examples of branched partially unsaturated C 3-12  alkylene groups (C 3-12  alkenylene and alkynylene groups) include, but are not limited to, —C(CH 3 )═CH—, —C(CH 3 )═CH—CH 2 —, —CH═CH—CH(CH 3 )— and —C≡C—CH(CH 3 )—. 
     Examples of alicyclic saturated C 3-12  alkylene groups (C 3-12  cycloalkylenes) include, but are not limited to, cyclopentylene (e.g. cyclopent-1,3-ylene), and cyclohexylene (e.g. cyclohex-1,4-ylene). 
     Examples of alicyclic partially unsaturated C 3-12  alkylene groups (C 3-12  cycloalkylenes) include, but are not limited to, cyclopentenylene (e.g. 4-cyclopenten-1,3-ylene), cyclohexenylene (e.g. 2-cyclohexen-1,4-ylene; 3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene). 
     Oxygen protecting group: the term “oxygen protecting group” refers to a moiety which masks a hydroxy group, and these are well known in the art. A large number of suitable groups are described on pages 23 to 200 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3 rd  Edition, John Wiley &amp; Sons, Inc., 1999, which is incorporated herein by reference in its entirety and for all purposes. Classes of particular interest include silyl ethers (e.g. TMS, TBDMS), substituted methyl ethers (e.g. THP) and esters (e.g. acetate). 
     Carbamate nitrogen protecting group: the term “carbamate nitrogen protecting group” pertains to a moiety which masks the nitrogen in the imine bond, and these are well known in the art. These groups have the following structure: 
                         
wherein R′ 10  is R as defined above. A large number of suitable groups are described on pages 503 to 549 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3 rd  Edition, John Wiley &amp; Sons, Inc., 1999, which is incorporated herein by reference in its entirety and for all purposes.
 
     Hemi-aminal nitrogen protecting group: the term “hemi-aminal nitrogen protecting group” pertains to a group having the following structure: 
                         
wherein R′ 10  is R as defined above. A large number of suitable groups are described on pages 633 to 647 as amide protecting groups of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3 rd  Edition, John Wiley &amp; Sons, Inc., 1999, which is incorporated herein by reference in its entirety and for all purposes.
 
     Conjugates 
     The present invention provides Conjugates comprising a PBD dimer connected to a Ligand unit via a Linker unit. In one embodiment, the Linker unit includes a Stretcher unit (A), a Specificity unit (L 1 ), and a Spacer unit (L 2 ). The Linker unit is connected at one end to the 
     Ligand unit (L) and at the other end to the PBD dimer compound (D). 
     In one aspect, such a Conjugate is shown below in formula IVa:
 
L-(A 1   a -L 1   s L 2   y -D) p    (IVa)
         or a pharmaceutically acceptable salt or solvate thereof, wherein:   L is the Ligand unit; and   -A 1   a -L 1   s -L 2   y - is a Linker unit (LU), wherein:   -A 1 - is a Stretcher unit,   a is 1 or 2,   -L 1 - is a Specificity unit,   s is an integer ranging from 0 to 12,   -L 2 - is a Spacer unit,   y is 0, 1 or 2;   -D is a PBD dimer; and   p is from 1 to 20.       

     In another aspect, such a Conjugate is shown below in formula IVb: 
     
       
         
         
             
             
         
       
     
     Also illustrated as:
 
L-(A 1   a -L 2   y (-L 1   s )-D) p    (IVb)
         or a pharmaceutically acceptable salt or solvate thereof, wherein:       

     L is the Ligand unit; and
         -A 1   a -L 1   s (L 2   y )- is a Linker unit (LU), wherein:   -A 1 - is a Stretcher unit linked to a Stretcher unit (L 2 ),   a is 1 or 2,   -L 1 - is a Specificity unit linked to a Stretcher unit (L 2 ),   s is an integer ranging from 0 to 12,   -L 2 - is a Spacer unit,   y is 0, 1 or 2;   -D is a PBD dimer; and   p is from 1 to 20.       

     Preferences 
     The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. The preferences may be combined together in any combination. 
     In one embodiment, the Conjugate has the formula:
 
L-(A 1   a -L 1   s -L 2   y -D) p  
 
L-(A 1   a -L s   1 -D) p ,
 
L-(A 1 -L 1 D) p  or
 
L-(A 1 -D) p  
         or a pharmaceutically acceptable salt or solvate thereof, wherein L, A 1 , a, L 1 , s, L 2 , D, y and p are as described above.       

     In one embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to the Drug unit (D), CBA is the Cell Binding Agent, L 1  is a Specificity unit, A 1  is a Stretcher unit connecting L 1  to the Cell Binding Agent, L 2  is a Spacer unit, which is a covalent bond, a self-immolative group or together with —OC(═O)— forms a self-immolative group, and L 2  is optional. —OC(═O)— may be considered as being part of L 1  or L 2 , as appropriate. 
           
         
       
    
     In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below:
 
CBA-A 1   a -L 1    s -L 2   y -*
         where the asterisk indicates the point of attachment to the Drug unit (D), CBA is the Cell Binding Agent, L 1  is a Specificity unit, A 1  is a Stretcher unit connecting L 1  to the Cell Binding Agent, L 2  is a Spacer unit which is a covalent bond or a self-immolative group, and a is 1 or 2, s is 0, 1 or 2, and y is 0 or 1 or 2.       

     In the embodiments illustrated above, L 1  can be a cleavable Specificity unit, and may be referred to as a “trigger” that when cleaved activates a self-immolative group (or self-immolative groups) L 2 , when a self-immolative group(s) is present. When the Specificity unit L 1  is cleaved, or the linkage (i.e., the covalent bond) between L 1  and L 2  is cleaved, the self-immolative group releases the Drug unit (D). 
     In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to the Drug (D), CBA is the Cell Binding Agent, L 1  is a Specificity unit connected to L 2 , A 1  is a Stretcher unit connecting L 2  to the Cell Binding Agent, L 2  is a self-immolative group, and a is 1 or 2, s is 1 or 2, and y is 1 or 2. 
           
         
       
    
     In the various embodiments discussed herein, the nature of L 1  and L 2  can vary widely. These groups are chosen on the basis of their characteristics, which may be dictated in part, by the conditions at the site to which the conjugate is delivered. Where the Specificity unit L 1  is cleavable, the structure and/or sequence of L 1  is selected such that it is cleaved by the action of enzymes present at the target site (e.g., the target cell). L 1  units that are cleavable by changes in pH (e.g. acid or base labile), temperature or upon irradiation (e.g. photolabile) may also be used. L 1  units that are cleavable under reducing or oxidising conditions may also find use in the Conjugates. 
     In some embodiments, L 1  may comprise one amino acid or a contiguous sequence of amino acids. The amino acid sequence may be the target substrate for an enzyme. 
     In one embodiment, L 1  is cleavable by the action of an enzyme. In one embodiment, the enzyme is an esterase or a peptidase. For example, L 1  may be cleaved by a lysosomal protease, such as a cathepsin. 
     In one embodiment, L 2  is present and together with —C(═O)O— forms a self-immolative group or self-immolative groups. In some embodiments, —C(═O)O— also is a self-immolative group. 
     In one embodiment, where L 1  is cleavable by the action of an enzyme and L 2  is present, the enzyme cleaves the bond between L 1  and L 2 , whereby the self-immolative group(s) release the Drug unit. 
     L 1  and L 2 , where present, may be connected by a bond selected from: 
     —C(═O)NH—, 
     —C(═O)O—,
         —NHC(═O)—,   —OC(═O)—,   —OC(═O)O—,   —NHC(═O)O—,   —OC(═O)NH—,   —NHC(═O)NH, and   —O— (a glycosidic bond).       

     An amino group of L 1  that connects to L 2  may be the N-terminus of an amino acid or may be derived from an amino group of an amino acid side chain, for example a lysine amino acid side chain. 
     A carboxyl group of L 1  that connects to L 2  may be the C-terminus of an amino acid or may be derived from a carboxyl group of an amino acid side chain, for example a glutamic acid amino acid side chain. 
     A hydroxy group of L 1  that connects to L 2  may be derived from a hydroxy group of an amino acid side chain, for example a serine amino acid side chain. 
     In one embodiment, —C(═O)O— and L 2  together form the group: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to the Drug unit, the wavy line indicates the point of attachment to the L 1 , Y is —N(H)—, —O—, —C(═O)N(H)— or —C(═O)O—, and n is 0 to 3. The phenylene ring is optionally substituted with one, two or three substituents as described herein. 
           
         
       
    
     In one embodiment, Y is NH. 
     In one embodiment, n is 0 or 1. Preferably, n is 0. 
     Where Y is NH and n is 0, the self-immolative group may be referred to as a p-aminobenzylcarbonyl linker (PABC). 
     The self-immolative group will allow for release of the Drug unit (i.e., the asymmetric PBD) when a remote site in the linker is activated, proceeding along the lines shown below (for n=0): 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the attachment to the Drug, L* is the activated form of the remaining portion of the linker and the released Drug unit is not shown. These groups have the advantage of separating the site of activation from the Drug. 
           
         
       
    
     In another embodiment, —C(═O)O— and L 2  together form a group selected from: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk, the wavy line, Y, and n are as defined above. Each phenylene ring is optionally substituted with one, two or three substituents as described herein. In one embodiment, the phenylene ring having the Y substituent is optionally substituted and the phenylene ring not having the Y substituent is unsubstituted. 
           
         
       
    
     In another embodiment, —C(═O)O— and L 2  together form a group selected from: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk, the wavy line, Y, and n are as defined above, E is O, S or NR, D is N, CH, or CR, and F is N, CH, or CR. 
           
         
       
    
     In one embodiment, D is N. 
     In one embodiment, D is CH. 
     In one embodiment, E is O or S.
         In one embodiment, F is CH.       

     In a preferred embodiment, the covalent bond between L 1  and L 2  is a cathepsin labile (e.g., cleavable) bond. 
     In one embodiment, L 1  comprises a dipeptide. The amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin. 
     In one embodiment, the group —X 1 —X 2 — in dipeptide, —NH—X 1 —X 2 —CO—, is selected from:
         -Phe-Lys-,   -Val-Ala-,   -Val-Lys-,   -Ala-Lys-,   -Val-Cit-,   -Phe-Cit-,   -Leu-Cit-,   -Ile-Cit-,   -Phe-Arg-, and   -Trp-Cit-;       

     where Cit is citrulline. In such a dipeptide, —NH— is the amino group of X 1 , and CO is the carbonyl group of X 2 . 
     Preferably, the group —X 1 —X 2 — in dipeptide, —NH—X 1 —X 2 —CO—, is selected from:
         -Phe-Lys-,   -Val-Ala-,   -Val-Lys-,   -Ala-Lys-, and   -Val-Cit-.       

     Most preferably, the group —X 1 —X 2 — in dipeptide, —NH—X 1 —X 2 —CO—, is -Phe-Lys-, Val-Cit or -Val-Ala-. 
     Other dipeptide combinations of interest include:
         -Gly-Gly-,   -Pro-Pro-, and   -Val-Glu-.       

     Other dipeptide combinations may be used, including those described by Dubowchik et al., which is incorporated herein by reference in its entirety and for all purposes. 
     In one embodiment, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed below. Protected amino acid sequences are cleavable by enzymes. For example, a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin. 
     Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog. Additional protecting group strategies are set out in Protective groups in Organic Synthesis, Greene and Wuts. 
     Possible side chain protecting groups are shown below for those amino acids having reactive side chain functionality:
         Arg: Z, Mtr, Tos;   Asn: Trt, Xan;   Asp: Bzl, t-Bu;   Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;   Glu: Bzl, t-Bu;   Gln: Trt, Xan;   His: Boc, Dnp, Tos, Trt;   Lys: Boc, Z-CI, Fmoc, Z;   Ser: Bzl, TBDMS, TBDPS;   Thr: Bz;   Trp: Boc;   Tyr: Bzl, Z, Z-Br.       

     In one embodiment, —X 2 — is connected indirectly to the Drug unit. In such an embodiment, the Spacer unit L 2  is present. 
     In one embodiment, —X 2 — is connected directly to the Drug unit. In such an embodiment, the Spacer unit L 2  is absent. 
     In one embodiment, the dipeptide is used in combination with a self-immolative group(s) (the Spacer unit). The self-immolative group(s) may be connected to —X 2 —. 
     Where a self-immolative group is present, —X 2 — is connected directly to the self-immolative group. In one embodiment, —X 2 — is connected to the group Y of the self-immolative group. Preferably the group —X 2 —CO— is connected to Y, where Y is NH. 
     In one embodiment, —X 1 — is connected directly to A 1 . Preferably the group NH—X 1 — (the amino terminus of X 1 ) is connected to A 1 . A 1  may comprise the functionality —CO— thereby to form an amide link with —X 1 —. 
     In one embodiment, L 1  and L 2  together with —OC(═O)— comprise the group —X 1 —X 2 -PABC-. The PABC group is connected directly to the Drug unit. In one example, the self-immolative group and the dipeptide together form the group -Phe-Lys-PABC-, which is illustrated below: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of L 1  or the point of attachment to A 1 . Preferably, the wavy line indicates the point of attachment to A 1 . 
           
         
       
    
     Alternatively, the self-immolative group and the dipeptide together form the group -Val-Ala-PABC-, which is illustrated below: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk and the wavy line are as defined above. 
           
         
       
    
     In another embodiment, L 1  and L 2  together with —OC(═O)— represent: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to the Drug unit, the wavy line indicates the point of attachment to A 1 , Y is a covalent bond or a functional group, and E is a group that is susceptible to cleavage thereby to activate a self-immolative group. 
           
         
       
    
     E is selected such that the group is susceptible to cleavage, e.g., by light or by the action of an enzyme. E may be —NO 2  or glucuronic acid (e.g., β-glucuronic acid). The former may be susceptible to the action of a nitroreductase, the latter to the action of a β-glucuronidase. 
     The group Y may be a covalent bond. 
     The group Y may be a functional group selected from:
         —C(═O)—   —NH—   —O—   —C(═O)NH—   —C(═O)O—,   —NHC(═O)—,   —OC(═O)—,   —OC(═O)O—,   —NHC(═O)O—,   —OC(═O)NH—,   —NHC(═O)NH—,   —NHC(═O)NH,   —C(═O)NHC(═O)—,   SO 2 , and   —S—.       

     The group Y is preferably —NH—, —CH 2 —, —O—, and —S—. 
     In some embodiments, L 1  and L 2  together with —OC(═O)— represent: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to the Drug unit, the wavy line indicates the point of attachment to A, Y is a covalent bond or a functional group and E is glucuronic acid (e.g., β-glucuronic acid). Y is preferably a functional group selected from —NH—. 
           
         
       
    
     In some embodiments, L 1  and L 2  together represent: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to the remainder of L 2  or the Drug unit, the wavy line indicates the point of attachment to A 1 , Y is a covalent bond or a functional group and E is glucuronic acid (e.g., β-glucuronic acid). Y is preferably a functional group selected from —NH—, —CH 2 —, —O—, and —S—. 
           
         
       
    
     In some further embodiments, Y is a functional group as set forth above, the functional group is linked to an amino acid, and the amino acid is linked to the Stretcher unit A 1 . In some embodiments, amino acid is β-alanine. In such an embodiment, the amino acid is equivalently considered part of the Stretcher unit. 
     The Specificity unit L 1  and the Ligand unit are indirectly connected via the Stretcher unit. 
     L 1  and A 1  may be connected by a bond selected from:
         —C(═O)NH—,   —C(═O)O—,   —NHC(═O)—,   —OC(═O)—,   —OC(═O)O—,   —NHC(═O)O—,   —OC(═O)NH—, and   —NHC(═O)NH—.       

     In one embodiment, the group A 1  is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 1 , L 2  or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5. 
           
         
       
    
     In one embodiment, the group A 1  is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 1 , L 2  or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5. 
           
         
       
    
     In one embodiment, the group A 1  is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 1 , L 2  or D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8. 
           
         
       
    
     In one embodiment, the group A 1  is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 1 , L 2  or D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8. 
           
         
       
    
     In one embodiment, the group A 1  is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 1 , L 2  or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5. 
           
         
       
    
     In one embodiment, the group A 1  is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 1 , L 2  or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5. 
           
         
       
    
     In one embodiment, the group A 1  is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 1 , L 2  or D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8. 
           
         
       
    
     In one embodiment, the group A 1  is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 1 , L 2  or D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8. 
           
         
       
    
     In one embodiment, the connection between the Ligand unit and A 1  is through a thiol residue of the Ligand unit and a maleimide group of A 1 . 
     In one embodiment, the connection between the Ligand unit and A 1  is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to the remaining portion of A 1 , L 1 , L 2  or D, and the wavy line indicates the point of attachment to the remaining portion of the Ligand unit. In this embodiment, the S atom is typically derived from the Ligand unit. 
           
         
       
    
     In each of the embodiments above, an alternative functionality may be used in place of the malemide-derived group shown below: 
     
       
         
         
             
             
         
       
         
         
           
             where the wavy line indicates the point of attachment to the Ligand unit as before, and the asterisk indicates the bond to the remaining portion of the A 1  group, or to L 1 , L 2  or D. 
           
         
       
    
     In one embodiment, the maleimide-derived group is replaced with the group: 
     
       
         
         
             
             
         
       
         
         
           
             where the wavy line indicates point of attachment to the Ligand unit, and the asterisk indicates the bond to the remaining portion of the A 1  group , or to L 1 , L 2  or D. 
           
         
       
    
     In one embodiment, the maleimide-derived group is replaced with a group, which optionally together with a Ligand unit (e.g., a Cell Binding Agent), is selected from:
         —C(═O)NH—,   —C(═O)O—,   —NHC(═O)—,   —OC(═O)—,   —OC(═O)O—,   —NHC(═O)O—,   —OC(═O)NH—,   —NHC(═O)NH—,   —NHC(═O)NH,   —C(═O)NHC(═O)—,   —S—,   —S—S—,   —CH 2 C(═O)—   —C(═O)CH 2 —,   ═N—NH—, and   —NH—N═.       

     Of these —C(═O)CH 2 — may be preferred especially when the carbonyl group is bound to —NH—. 
     In one embodiment, the maleimide-derived group is replaced with a group, which optionally together with the Ligand unit, is selected from: 
     
       
         
         
             
             
         
       
         
         
           
             where the wavy line indicates either the point of attachment to the Ligand unit or the bond to the remaining portion of the A 1  group, and the asterisk indicates the other of the point of attachment to the Ligand unit or the bond to the remaining portion of the A 1  group. 
           
         
       
    
     Other groups suitable for connecting L 1  to the Cell Binding Agent are described in WO 2005/082023. 
     In one embodiment, the Stretcher unit A 1  is present, the Specificity unit L 1  is present and Spacer unit L 2  is absent. Thus, L 1  and the Drug unit are directly connected via a bond. Equivalently in this embodiment, L 2  is a bond. 
     L 1  and D may be connected by a bond selected from:
         —C(═O)N&lt;,   —OC(═O)N&lt;, and   —NHC(═O)N&lt;,       

     where N&lt; is part of D. 
     In one embodiment, L 1  and D are preferably connected by a bond:
         —C(═O)N&lt;.       

     In one embodiment, L 1  comprises a dipeptide and one end of the dipeptide is linked to D. As described above, the amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin. 
     In one embodiment, the group —X 1 —X 2 — in dipeptide, —NH—X 1 —X 2 —CO—, is selected from:
         -Phe-Lys-,   -Val-Ala-,   -Val-Lys-,   -Ala-Lys-,   -Val-Cit-,   -Phe-Cit-,   -Leu-Cit-,   -Ile-Cit-,   -Phe-Arg-, and   -Trp-Cit-;       

     where Cit is citrulline. In such a dipeptide, —NH— is the amino group of X 1 , and CO is the carbonyl group of X 2 . 
     Preferably, the group —X 1 —X 2 — in dipeptide, —NH—X 1 —X 2 —CO—, is selected from:
         -Phe-Lys-,   -Val-Ala-,   -Val-Lys-,   -Ala-Lys-, and   -Val-Cit-.       

     Most preferably, the group —X 1 —X 2 — in dipeptide, —NH—X 1 —X 2 —CO—, is -Phe-Lys- or -Val-Ala-. 
     Other dipeptide combinations of interest include:
         -Gly-Gly-,   -Pro-Pro-, and   -Val-Glu-.       

     Other dipeptide combinations may be used, including those described above. 
     In one embodiment, L 1 -D is: 
     
       
         
         
             
             
         
       
         
         
           
             where —NH—X 1 —X 2 —CO is the dipeptide, —N&lt; is part of the Drug unit, the asterisk indicates the points of attachment to the remainder of the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of L 1  or the point of attachment to A 1 . Preferably, the wavy line indicates the point of attachment to A 1 . 
           
         
       
    
     In one embodiment, the dipeptide is valine-alanine and L 1 -D is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisks, —N&lt; and the wavy line are as defined above. 
           
         
       
    
     In one embodiment, the dipeptide is phenylalnine-lysine and L 1 -D is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisks, —N&lt; and the wavy line are as defined above. 
           
         
       
    
     In one embodiment, the dipeptide is valine-citrulline. 
     In one embodiment, the groups A 1 -L 1  are: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 2  or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5. 
           
         
       
    
     In one embodiment, the groups A 1 -L 1  are: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 2  or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5. 
           
         
       
    
     In one embodiment, the groups A 1 -L 1  are: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 2  or D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8. 
           
         
       
    
     In one embodiment, the groups A 1 -L 1  are: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 2  or D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 7, preferably 3 to 7, most preferably 3 or 7. 
           
         
       
    
     In one embodiment, the groups A 1 -L 1  are: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 2  or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5. 
           
         
       
    
     In one embodiment, the groups A 1 -L 1  are: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 2  or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5. 
           
         
       
    
     In one embodiment, the groups A 1 -L 1  are: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 2  or D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8. 
           
         
       
    
     In one embodiment, the groups A 1 -L 1  is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 2  or D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8. 
           
         
       
    
     In one embodiment, the groups L-A 1 -L 1  are: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 2  or D, S is a sulfur group of the Ligand unit, the wavy line indicates the point of attachment to the rest of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5. 
           
         
       
    
     In one embodiment, the group L-A 1 -L 1  are: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 2  or D, S is a sulfur group of the Ligand unit, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5. 
           
         
       
    
     In one embodiment, the groups L-A 1 -L 1  are: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 2  or D, S is a sulfur group of the Ligand unit, the wavy line indicates the point of attachment to the remainder of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8. 
           
         
       
    
     In one embodiment, the groups L-A 1 -L 1  are: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 2  or D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 7, preferably 4 to 8, most preferably 4 or 8. 
           
         
       
    
     In one embodiment, the groups L-A 1 -L 1  are: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 2  or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5. 
           
         
       
    
     In one embodiment, the groups L-A 1 -L 1  are: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 2  or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5. 
           
         
       
    
     In one embodiment, the groups L-A 1 -L 1  are: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 2  or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8. 
           
         
       
    
     In one embodiment, the groups L-A 1 -L 1  are: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 2  or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8. 
           
         
       
    
     In one embodiment, the Stretcher unit is an acetamide unit, having the formula: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to the remainder of the Stretcher unit, L 1  or D, and the wavy line indicates the point of attachment to the Ligand unit. 
           
         
       
    
     Linker-Drugs 
     In other embodiments, Linker-Drug compounds are provided for conjugation to a Ligand unit. In one embodiment, the Linker-Drug compounds are designed for connection to a Cell Binding Agent. 
     In one embodiment, the Drug Linker compound has the formula: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to the Drug unit (D, as defined above), G 1  is a Stretcher group (A 1 ) to form a connection to a Ligand unit, L 1  is a Specificity unit, L 2  (a Spacer unit) is a covalent bond or together with —OC(═O)— forms a self-immolative group(s). 
           
         
       
    
     In another embodiment, the Drug Linker compound has the formula:
 
G 1 -L 1 -L 2 -.
         where the asterisk indicates the point of attachment to the Drug unit (D), G 1  is a Stretcher unit (A 1 ) to form a connection to a Ligand unit, L 1  is a Specificity unit, L 2  (a Spacer unit) is a covalent bond or a self-immolative group(s).       

     L 1  and L 2  are as defined above. References to connection to A 1  can be construed here as referring to a connection to G 1 . 
     In one embodiment, where L 1  comprises an amino acid, the side chain of that amino acid may be protected. Any suitable protecting group may be used. In one embodiment, the side chain protecting groups are removable with other protecting groups in the compound, where present. In other embodiments, the protecting groups may be orthogonal to other protecting groups in the molecule, where present. 
     Suitable protecting groups for amino acid side chains include those groups described in the Novabiochem Catalog 2006/2007. Protecting groups for use in a cathepsin labile linker are also discussed in Dubowchik et al. 
     In certain embodiments of the invention, the group L 1  includes a Lys amino acid residue. The side chain of this amino acid may be protected with a Boc or Alloc protected group. A Boc protecting group is most preferred. 
     The functional group G 1  forms a connecting group upon reaction with a Ligand unit (e.g., a cell binding agent. 
     In one embodiment, the functional group G 1  is or comprises an amino, carboxylic acid, hydroxy, thiol, or maleimide group for reaction with an appropriate group on the Ligand unit. In a preferred embodiment, G 1  comprises a maleimide group. 
     In one embodiment, the group G 1  is an alkyl maleimide group. This group is suitable for reaction with thiol groups, particularly cysteine thiol groups, present in the cell binding agent, for example present in an antibody. 
     In one embodiment, the group G 1  is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 1 , L 2  or D, and n is 0 to 6. In one embodiment, n is 5. 
           
         
       
    
     In one embodiment, the group G 1  is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 1 , L 2  or D, and n is 0 to 6. In one embodiment, n is 5. 
           
         
       
    
     In one embodiment, the group G 1  is: 
     
       
         
         
             
             
         
       
     
     where the asterisk indicates the point of attachment to L 1 , L 2  or D, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably 4 or 8. 
     In one embodiment, the group G 1  is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 1 , L 2  or D, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4 or 8. 
           
         
       
    
     In one embodiment, the group G 1  is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 1 , L 2  or D, and n is 0 to 6. In one embodiment, n is 5. 
           
         
       
    
     In one embodiment, the group G 1  is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 1 , L 2  or D, and n is 0 to 6. In one embodiment, n is 5. 
           
         
       
    
     In one embodiment, the group G 1  is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 1 , L 2  or D, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably 4 or 8. 
           
         
       
    
     In one embodiment, the group G 1  is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 1 , L 2  or D, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4 or 8. 
           
         
       
    
     In each of the embodiments above, an alternative functionality may be used in place of the malemide group shown below: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the bond to the remaining portion of the G group. 
           
         
       
    
     In one embodiment, the maleimide-derived group is replaced with the group: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the bond to the remaining portion of the G group. 
           
         
       
    
     In one embodiment, the maleimide group is replaced with a group selected from:
         —C(═O)OH,   —OH,   —NH 2 ,   —SH,   —C(═O)CH 2 X, where X is Cl, Br or I,   —CHO,   —-NHNH 2      —C≡CH, and   —N 3  (azide).       

     Of these, —C(═O)CH 2 X may be preferred, especially when the carbonyl group is bound to —NH—. 
     In one embodiment, L 1  is present, and G 1  is —NH 2 , —NHMe, —COOH, —OH or —SH. 
     In one embodiment, where L 1  is present, G 1  is —NH 2  or —NHMe. Either group may be the N-terminal of an L 1  amino acid sequence. 
     In one embodiment, L 1  is present and G 1  is —NH 2 , and L 1  is an amino acid sequence —X 1 —X 2 —, as defined above. 
     In one embodiment, L 1  is present and G 1  is COOH. This group may be the C-terminal of an L 1  amino acid sequence. 
     In one embodiment, L 1  is present and G 1  is OH. 
     In one embodiment, L 1  is present and G 1  is SH. 
     The group G 1  may be convertable from one functional group to another. In one embodiment, L 1  is present and G 1  is —NH 2 . This group is convertable to another group G 1  comprising a maleimide group. For example, the group —NH 2  may be reacted with an acids or an activated acid (e.g., N-succinimide forms) of those G 1  groups comprising maleimide shown above. 
     The group G 1  may therefore be converted to a functional group that is more appropriate for reaction with a Ligand unit. 
     As noted above, in one embodiment, L 1  is present and G 1  is —NH 2 , —NHMe, —COOH, —OH or —SH. In a further embodiment, these groups are provided in a chemically protected form. 
     The chemically protected form is therefore a precursor to the linker that is provided with a functional group. 
     In one embodiment, G 1  is —NH 2  in a chemically protected form. The group may be protected with a carbamate protecting group. The carbamate protecting group may be selected from the group consisting of:
         Alloc, Fmoc, Boc, Troc, Teoc, Cbz and PNZ.   Preferably, where G 1  is —NH 2 , it is protected with an Alloc or Fmoc group.       

     In one embodiment, where G 1  is —NH 2 , it is protected with an Fmoc group. 
     In one embodiment, the protecting group is the same as the carbamate protecting group of the capping group. 
     In one embodiment, the protecting group is not the same as the carbamate protecting group of the capping group. In this embodiment, it is preferred that the protecting group is removable under conditions that do not remove the carbamate protecting group of the capping group. 
     The chemical protecting group may be removed to provide a functional group to form a connection to a Ligand unit. Optionally, this functional group may then be converted to another functional group as described above. 
     In one embodiment, the active group is an amine. This amine is preferably the N-terminal amine of a peptide, and may be the N-terminal amine of the preferred dipeptides of the invention. 
     The active group may be reacted to yield the functional group that is intended to form a connection to a Ligand unit. 
     In other embodiments, the Linker unit is a precursor to the Linker uit having an active group. In this embodiment, the Linker unit comprises the active group, which is protected by way of a protecting group. The protecting group may be removed to provide the Linker unit having an active group. 
     Where the active group is an amine, the protecting group may be an amine protecting group, such as those described in Green and Wuts. 
     The protecting group is preferably orthogonal to other protecting groups, where present, in the Linker unit. 
     In one embodiment, the protecting group is orthogonal to the capping group. Thus, the active group protecting group is removable whilst retaining the capping group. In other embodiments, the protecting group and the capping group is removable under the same conditions as those used to remove the capping group. 
     In one embodiment, the Linker unit is: 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of the Linker unit, as applicable or the point of attachment to G 1 . Preferably, the wavy line indicates the point of attachment to G 1 . 
           
         
       
    
     In one embodiment, the Linker unit is: 
                         
where the asterisk and the wavy line are as defined above.
 
     Other functional groups suitable for use in forming a connection between L 1  and the Cell Binding Agent are described in WO 2005/082023. 
     Ligand Unit 
     The Ligand Unit may be of any kind, and include a protein, polypeptide, peptide and a non-peptidic agent that specifically binds to a target molecule. In some embodiments, the Ligand unit may be a protein, polypeptide or peptide. In some embodiments, the Ligand unit may be a cyclic polypeptide. These Ligand units can include antibodies or a fragment of an antibody that contains 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. The ligand Unit is also referred to herein as a “binding agent” or “targeting agent”. 
     The terms “specifically binds” and “specific binding” refer to the binding of an antibody or other protein, polypeptide or peptide to a predetermined molecule (e.g., an antigen). 
     Typically, the antibody or other molecule binds with an affinity of at least about 1×10 7  M −1 , and binds to the predetermined molecule with an affinity that is at least two-fold greater than its affinity for binding to a non-specific molecule (e.g., BSA, casein) other than the predetermined molecule or a closely-related molecule. 
     Examples of Ligand units include those agents described for use in WO 2007/085930, which is incorporated by reference herein in its entirety and for all purposes. 
     In some embodiments, the Ligand unit is a Cell Binding Agent that binds to an extracellular target on a cell. Such a Cell Binding Agent can be a protein, polypeptide, peptide or a non-peptidic agent. In some embodiments, the Cell Binding Agent may be a protein, polypeptide or peptide. In some embodiments, the Cell Binding Agent may be a cyclic polypeptide. The Cell Binding Agent also may be antibody or an antigen-binding fragment of an antibody. Thus, in one embodiment, the present invention provides an antibody-drug conjugate (ADC). 
     In one embodiment the antibody is a monoclonal antibody; chimeric antibody; humanized antibody; fully human antibody; or a single chain antibody. One embodiment the antibody is a fragment of one of these antibodies having biological activity. Examples of such fragments include Fab, Fab′, F(ab′) 2  and Fv fragments. 
     The antibody may be a diabody, a domain antibody (DAB) or a single chain antibody. 
     In one embodiment, the antibody is a monoclonal antibody. 
     Antibodies for use in the present invention include those antibodies described in WO 2005/082023 which is incorporated by reference herein in its entirety and for all purposes. Particularly preferred are those antibodies for tumour-associated antigens. Examples of those antigens known in the art include, but are not limited to, those tumour-associated antigens set out in WO 2005/082023. See, for instance, pages 41-55. 
     In some embodiments, the conjugates are designed to target tumour cells via their cell surface antigens. The antigens may be cell surface antigens which are either over-expressed or expressed at abnormal times or cell types. Preferably, the target antigen is expressed only on proliferative cells (preferably tumour cells); however this is rarely observed in practice. As a result, target antigens are usually selected on the basis of differential expression between proliferative and healthy tissue. 
     Antibodies have been raised to target specific tumour related antigens including:
         Cripto, CD19, CD20, CD22, CD30, CD33, Glycoprotein NMB, CanAg, Her2 (ErbB2/Neu), CD56 (NCAM), CD70, CD79, CD138, PSCA, PSMA (prostate specific membrane antigen), BCMA, E-selectin, EphB2, Melanotransferin, Muc16 and TMEFF2. In any of the embodiments provided herein, the Ligand unit can be a monoclonal antibody that specifically binds to the Cripto antigen, CD19 antigen, CD20 antigen, CD22 antigen, CD30 antigen, CD33 antigen, Glycoprotein NMB, CanAg antigen, Her2 (ErbB2/Neu) antigen, CD56 (NCAM) antigen, CD70 antigen, CD79 antigen, CD138 antigen, PSCA, PSMA (prostate specific membrane antigen), BCMA, E-selectin, EphB2, Melanotransferin, Muc16 antigen or TMEFF2 antigen.       

     The Ligand unit is connected to the Linker unit. In one embodiment, the Ligand unit is connected to A, where present, of the Linker unit. 
     In one embodiment, the connection between the Ligand unit and the Linker unit is through a thioether bond. 
     In one embodiment, the connection between the Ligand unit and the Linker unit is through a disulfide bond. 
     In one embodiment, the connection between the Ligand unit and the Linker unit is through an amide bond. 
     In one embodiment, the connection between the Ligand unit and the Linker unit is through an ester bond. 
     In one embodiment, the connection between the Ligand unit and the Linker is formed between a thiol group of a cysteine residue of the Ligand unit and a maleimide group of the Linker unit. 
     The cysteine residues of the Ligand unit may be available for reaction with the functional group of the Linker unit to form a connection. In other embodiments, for example where the Ligand unit is an antibody, the thiol groups of the antibody may participate in interchain disulfide bonds. These interchain bonds may be converted to free thiol groups by e.g. treatment of the antibody with DTT prior to reaction with the functional group of the Linker unit. 
     In some embodiments, the cysteine residue is introduced into the heavy or light chain of an antibody. Positions for cysteine insertion by substitution in antibody heavy or light chains include those described in Published U.S. Application No. 2007-0092940 and International Patent Publication WO2008/070593, which are incorporated by reference herein in their entirety and for all purposes. 
     Methods of Treatment 
     The compounds or conjugates of the present invention may be used in a method of therapy. Also provided is a method of treatment, comprising administering to a subject in need of treatment a therapeutically-effective amount of a compound of formula I or conjugate thereof. The term “therapeutically effective amount” is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors. 
     A compound or conjugate may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs; surgery; and radiation therapy. 
     Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, i.e. a compound of formula I, or conjugate thereof, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser 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 precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous. 
     Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin. 
     For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer&#39;s Injection, Lactated Ringer&#39;s Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required. 
     The Compounds and Conjugates can be used to treat proliferative disease and autoimmune disease. The term “proliferative disease” pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo. 
     Examples of proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi&#39;s sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. Other cancers of interest include, but are not limited to, haematological; malignancies such as leukemias and lymphomas, such as non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers of B or T cell origin. 
     Examples of autoimmune disease include the following: rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves&#39; disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g., Crohn&#39;s disease), anaphylaxis, allergic reaction, Sjögren&#39;s syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener&#39;s granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt&#39;s syndrome, autoimmune uveitis, Addison&#39;s disease, adrenalitis, thyroiditis, Hashimoto&#39;s thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler&#39;s syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud&#39;s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, ankylosing spondolytis, ulcerative colitis, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture&#39;s syndrome, Chagas&#39; disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti-phospholipid syndrome, farmer&#39;s lung, erythema multiforme, post cardiotomy syndrome, Cushing&#39;s syndrome, autoimmune chronic active hepatitis, bird-fancier&#39;s lung, toxic epidermal necrolysis, Alport&#39;s syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu&#39;s arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter&#39;s syndrome, eczema, lymphomatoid granulomatosis, Behcet&#39;s disease, Caplan&#39;s syndrome, Kawasaki&#39;s disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman&#39;s syndrome, Felty&#39;s syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch&#39;s cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom&#39;s macroglobulemia, Evan&#39;s syndrome, and autoimmune gonadal failure. 
     In some embodiments, the autoimmune disease is a disorder of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture&#39;s syndrome, rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjogren&#39;s syndrome, Hashimoto&#39;s thyroiditis, Graves&#39; disease, primary biliary cirrhosis, Wegener&#39;s granulomatosis, tuberculosis, or graft versus host disease), or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn&#39;s syndrome, systemic sclerosis, or chronic graft versus host disease). Generally, disorders involving dendritic cells involve disorders of Th1-lymphocytes or Th2-lymphocytes. In some embodiments, the autoimmunie disorder is a T cell-mediated immunological disorder. 
     In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 10 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administerd ranges from about 0.05 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administerd ranges from about 0.1 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 4 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.05 to about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 2 mg/kg per dose. 
     Includes Other Forms 
     Unless otherwise specified, included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (—COOH) also includes the anionic (carbon/late) form (—COO − ), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (—N + HR 1 R 2 ), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (—O − ), a salt or solvate thereof, as well as conventional protected forms. 
     Salts 
     It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, et al.,  J. Pharm. Sci.,  66, 1-19 (1977). 
     For example, if the compound is anionic, or has a functional group which may be anionic (e.g. —COOH may be —COO − ), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na +  and K + , alkaline earth cations such as Ca 2+  and Mg 2+ , and other 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 + , NH 2 R 2   + , NHR 3   + , NR 4   + ). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH 3 ) 4   + . 
     If the compound is cationic, or has a functional group which may be cationic (e.g. —NH 2  may be —NH 3   + ), then a salt may 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, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous. 
     Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose. 
     Solvates 
     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 refer to a complex of solute (e.g. active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc. 
     Carbinolamines 
     The invention includes compounds where a solvent adds across the imine bond of the PBD moiety, which is illustrated below where the solvent is water or an alcohol (R A OH, where R A  is C 1-4  alkyl): 
     
       
         
         
             
             
         
       
     
     These forms can be called the carbinolamine and carbinolamine ether forms of the PBD. The balance of these equilibria depend on the conditions in which the compounds are found, as well as the nature of the moiety itself. 
     These particular compounds may be isolated in solid form, for example, by lyophilisation. 
     Isomers 
     Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric 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 I-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 halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”). 
     Note that, except as discussed below for tautomeric forms, specifically excluded from the term “isomers”, as used herein, are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, —OCH 3 , is not to be construed as a reference to its structural isomer, a hydroxymethyl group, —CH 2 OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling 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). 
     The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro. 
     
       
         
         
             
             
         
       
     
     Note that specifically included in the term “isomer” are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including  1 H,  2 H (D), and  3 H (T); C may be in any isotopic form, including  12 C,  13 C, and  14 C; O may be in any isotopic form, including  16 O and  18 O; and the like. 
     Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner. 
     General synthetic routes 
     The synthesis of PBD compounds is extensively discussed in the following references, which discussions are incorporated herein by reference in their entirety and for all purposes: 
     a) WO 00/12508 (pages 14 to 30); 
     b) WO 2005/023814 (pages 3 to 10); 
     c) WO 2004/043963 (pages 28 to 29); and 
     d) WO 2005/085251 (pages 30 to 39). 
     Synthesis Route 
     The compounds of the present invention, where R 10  and R 11  form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, can be synthesised from a compound of Formula 2: 
     
       
         
         
             
             
         
       
     
     where R 2 , R 6 , R 7 , R 9 , R 6′ , R 7′ , R9′, R 12 , X, X′ are R″ are as defined for compounds of formula I, Prot N  is a nitrogen protecting group for synthesis and Prot ∘  is a protected oxygen group for synthesis or an oxo group, by deprotecting the imine bond by standard methods. 
     The compound produced may be in its carbinolamine or carbinolamine ether form depending on the solvents used. For example if Prot N  is Troc and Prot ∘  is an oxygen protecting group for synthesis, then the deprotection is carried out using a Cd/Pb couple to yield the compound of formula (I). If Prot N  is SEM, or an analogous group, and Prot ∘  is an an oxo group, then the oxo group can be removed by reduction, which leads to a protected carbinolamine intermediate, which can then be treated to remove the SEM protecting group, followed by the elimination of water. The reduction of the compound of Formula 2 can be accomplished by, for example, superhydride or lithium tetraborohydride, whilst a suitable means for removing the SEM protecting group is treatment with silica gel. 
     Compounds of formula 2 can be synthesised from a compound of formula 3a: 
     
       
         
         
             
             
         
       
     
     where R 2 , R 6 , R 7 , R 9 , R 6′ , R 7′ , R 9′ , X, X′ and R″ are as defined for compounds of formula 2, by coupling an organometallic derivative comprising R 12 , such as an organoboron derivative. The organoboron derivative may be a boronate or boronic acid. 
     Compounds of formula 2 can be synthesised from a compound of formula 3b: 
     
       
         
         
             
             
         
       
     
     where R 12 , R 6 , R 7 , R 9 , R 6′ , R 7′ , R 9′ , X, X′ and R″ are as defined for compounds of formula 2, by coupling an organometallic derivative comprising R 2 , such as an organoboron derivative. The organoboron derivative may be a boronate or boronic acid. 
     Compounds of formulae 3a and 3b can be synthesised from a compound of formula 4: 
     
       
         
         
             
             
         
       
     
     where R 2 , R 6 , R 7 , R 9 , R 6′ , R 7′ , R 9′ , X, X′ and R″ are as defined for compounds of formula 2, by coupling about a single equivalent (e.g. 0.9 or 1 to 1.1 or 1.2) of an organometallic derivative, such as an organoboron derivative, comprising R 2  or R 12 . 
     The couplings described above are usually carried out in the presence of a palladium catalyst, for example Pd(PPh 3 ) 4 , Pd(OCOCH 3 ) 2 , PdCl 2 , Pd 2 (dba) 3 . The coupling may be carried out under standard conditions, or may also be carried out under microwave conditions. 
     The two coupling steps are usually carried out sequentially. They may be carried out with or without purification between the two steps. If no purification is carried out, then the two steps may be carried out in the same reaction vessel. Purification is usually required after the second coupling step. Purification of the compound from the undesired by-products may be carried out by column chromatography or ion-exchange separation. 
     The synthesis of compounds of formula 4 where Prot ∘  is an oxo group and Prot N  is SEM are described in detail in WO 00/12508, which is incorporated herein by reference in its entirety and for all purposes. In particular, reference is made to scheme 7 on page 24, where the above compound is designated as intermediate P. This method of synthesis is also described in WO 2004/043963, which is incorporated herein by reference in its entirety and for all purposes . Further reference is also made to the synthesis of compounds 8a and 8b in WO 2010/043880 (pages 36 to 45), which is incorporated herein by reference in its entirety and for all purposes. 
     The synthesis of compounds of formula 4 where Prot ∘  is a protected oxygen group for synthesis are described in WO 2005/085251, which synthesis is herein incorporated by reference. 
     Compounds of formula I where R 10  and R 10′  are H and R 11  and  R11′  are SO z M, can be synthesised from compounds of formula I where R 10  and R 11  form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, by the addition of the appropriate bisulphite salt or sulphinate salt, followed by an appropriate purification step. Further methods are described in GB 2 053 894, which is herein incorporated by reference. 
     In some embodiments of the invention, particularly where R 12  bears a substituent that is OH or CO 2 H, it may be desired in the above methods to add an organometallic derivative of R 12  where the substituent group is protected. For example, if R 12  bears CO 2 H, it may be preferred to join a compound where the carboxy is protected as an ester (e.g. C 1-4  alkyl ester) and then deprotect the carboxy group at a later stage in the synthesis. It may even be deprotected once part of the linker group for making a drug linker has been added. The OH substituent may be protected by phenol protecting groups as known in the art. 
     Nitrogen Protecting Groups for Synthesis 
     Nitrogen protecting groups for synthesis are well known in the art. In the present invention, the protecting groups of particular interest are carbamate nitrogen protecting groups and hemi-aminal nitrogen protecting groups. 
     Carbamate nitrogen protecting groups have the following structure: 
     
       
         
         
             
             
         
       
     
     wherein R′ 10  is R as defined above. A large number of suitable groups are described on pages 503 to 549 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3 rd  Edition, John Wiley &amp; Sons, Inc., 1999, which is incorporated herein by reference. 
     Particularly preferred protecting groups include Troc, Teoc, Fmoc, BOC, Doc, Hoc, TcBOC, 1-Adoc and 2-Adoc. 
     Other possible groups are nitrobenzyloxycarbonyl (e.g. 4- nitrobenzyloxycarbonyl) and 2-(phenylsulphonyl)ethoxycarbonyl. 
     Those protecting groups which can be removed with palladium catalysis are not preferred, e.g. Alloc. 
     Hemi-aminal nitrogen protecting groups have the following structure: 
                         
wherein R′ 10  is R as defined above. A large number of suitable groups are described on pages 633 to 647 as amide protecting groups of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3 rd  Edition, John Wiley &amp; Sons, Inc., 1999, which is incorporated herein by reference. The groups disclosed herein can be applied to compounds of the present invention. Such groups include, but are not limited to, SEM, MOM, MTM, MEM, BOM, nitro or methoxy substituted BOM, Cl 3 CCH 2 OCH 2 —.
 
     Protected Oxygen Group for Synthesis 
     Protected oxygen group for synthesis are well known in the art. A large number of suitable oxygen protecting groups are described on pages 23 to 200 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3 rd  Edition, John Wiley &amp; Sons, Inc., 1999, which is incorporated herein by reference in its entirety and for all purposes. 
     Classes of particular interest include silyl ethers, methyl ethers, alkyl ethers, benzyl ethers, esters, acetates, benzoates, carbonates, and sulfonates. 
     Preferred oxygen protecting groups include acetates, TBS and THP. 
     Synthesis of Drug Conjugates 
     Conjugates comprising PBD dimers as described herein can be prepared using the knowledge of the skilled artisan in combination with the teachings provided herein. For example, linkers are described in U.S. Pat. No. 6,214,345, U.S. Pat. No. 7,498,298 as well as WO 2009/0117531, each of which is incorporated herein by reference in their entirety and for all purposes. Other linkers can be prepared according to the references cited herein or as known to the skilled artisan. 
     Linker-Drug compounds can be prepared according to methods known in the art in combination with the teachings provided herein. For example, linkage of amine-based X substituents (of the PBD dimer Drug unit) to active groups of the Linker units can be performed according to methods generally described in U.S. Pat. Nos. 6,214,345 and 7,498,298; and WO 2009-0117531, or as otherwise known to the skilled artisan. Some examples are shown below. 
     Antibodies can be conjugated to Linker-Drug compounds as described in Doronina et al., Nature Biotechnology, 2003, 21, 778-784). Briefly, antibodies (4-5 mg/mL) in PBS containing 50 mM sodium borate at pH 7.4 are reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37° C. The progress of the reaction, which reduces interchain disulfides, is monitored by reaction with 5,5′-dithiobis(2-nitrobenzoic acid) and allowed to proceed until the desired level of thiols/mAb is achieved. The reduced antibody is then cooled to 0° C. and alkylated with 1.5 equivalents of maleimide drug-linker per antibody thiol. After 1 hour, the reaction is quenched by the addition of 5 equivalents of N-acetyl cysteine. Quenched drug-linker is removed by gel filtration over a PD-10 column. The ADC is then sterile-filtered through a 0.22 μm syringe filter. Protein concentration can be determined by spectral analysis at 280 nm and 329 nm, respectively, with correction for the contribution of drug absorbance at 280 nm. Size exclusion chromatography can be used to determine the extent of antibody aggregation, and RP-HPLC can be used to determine the levels of remaining NAC-quenched drug-linker. 
     Antibodies with introduced cysteine residues can be conjugated to Linker-Drug compounds as described in International Patent Publication WO2008/070593, which is incorporated by reference herein in its entirety and for all purposes or as follows. Antibodies containing an introduced cysteine residue in the heavy chain are fully reduced by adding 10 equivalents of TCEP and 1 mM EDTA and adjusting the pH to 7.4 with 1M Tris buffer (pH 9.0). 
     Following a 1 hour incubation at 37° C., the reaction is cooled to 22° C. and 30 equivalents of dehydroascorbic acid is added to selectively reoxidize the native disulfides, while leaving the introduced cysteine in the reduced state. The pH is adjusted to 6.5 with 1M Tris buffer (pH 3.7) and the reaction is allowed to proceed for 1 hour at 22° C. The pH of the solution is then raised again to 7.4 by addition of 1 M Tris buffer (pH 9.0). 3.5 equivalents of the PBD drug linker in DMSO is placed in a suitable container for dilution with propylene glycol prior to addition to the reaction. To maintain solubility of the PBD drug linker, the antibody itself is first diluted with propylene glycol to a final concentration of 33% (e.g., if the antibody solution was in a 60 mL reaction volume, 30 mL of propylene glycol was added). This same volume of propylene glycol (30 mL in this example) is added to the PBD drug linker as a diluent. After mixing, the solution of PBD drug linker in propylene glycol is added to the antibody solution to effect the conjugation; the final concentration of propylene glycol is 50%. The reaction is allowed to proceed for 30 minutes and then quenched by addition of 5 equivalents of N-acetyl cysteine. The ADC is purified by ultrafiltration through a 30 kD membrane. (Note that the concentration of propylene glycol used in the reaction can be reduced for any particular PBD, as its sole purpose is to maintain solubility of the drug linker in the aqueous media.) 
     For halo-acetamide-based Linker-Drug compounds, conjugation can be performed generally as follows. To a solution of reduced and reoxidized antibodies (having introduced cysteines in the heavy chain) in 10 mM Tris (pH 7.4), 50 mM NaCl, and 2 mM DTPA is added 0.5 volumes of propylene glycol. A 10 mM solution of acetamide-based Linker-Drug compound in dimethylacetamide is prepared immediately prior to conjugation. An equivalent amount of propylene glycol as added to the antibody solution is added to a 6-fold molar excess of the Linker-Drug compound. The dilute Linker-Drug solution is added to the antibody solution and the pH is adjusted to 8-8.5 using 1 M Tris (pH 9). The conjugation reaction is allowed to proceed for 45 minutes at 37° C. The conjugation is verified by reducing and denaturing reversed phase PLRP-S chromatography. Excess Linker-Drug compound is removed with Quadrasil MP resin and the buffer is exchanged into 10 mM Tris (pH 7.4), 50 mM NaCl, and 5% propylene glycol using a PD-10 desalting column. 
     Illustrative Synthesis Schemes for Drug Linkers 
     The following schemes are illustrative of routes for synthesising drug linkers—the PBD dimer is shown with specific substituents, and dimer links, but these may be varied within the scope of the present invention. 
                         
where Prot Sub refers to either the OH or CO 2 H phenyl substituent groups or their protected versions. The protection may be installed in light of the reactions carried out to introduce the linking unit, and may be removed when appropriate during the synthesis. In some embodiments, protection would be in place for step (i), but would be removed either before or after step (ii). In other embodiments, protection would be in place for step (i), but would be removed either after step (iii).
 
     The glucuronide linker intermediate S1 (reference: Jeffrey et al.,  Bioconjugate Chemistry,  2006, 17, 831-840) can be treated with diphosgene in dichlroromethane at −78° C. to afford the glucuronide chloroformate, which is then reacted with the PBD dimer S2 dissolved in CH 2 Cl 2  by dropwise addition. Warming the reaction to 0° C. over 2 hours followed by extraction will yield the compound S3. Treating a solution of S3 in an equal solvent mixture of MeOH, tetrahydrofuran, and water (cooled to 0° C.) with lithium hydroxide monohydrate for 4 hours, followed by reaction with glacial acetic acid will yield the compound S4. Adding maleimidocaproyl NHS ester to a solution of S4 in DMF, followed by diisopropylethylamine and stirring at room temperature under nitrogen for 2 hours will yield the desired drug linker S5. 
     This approach could also be used with PBD dimers containing aliphatic amines, such as benzylamine, e.g. S6: 
     
       
         
         
             
             
         
       
     
     The methods of Examples 2 and 3 could also be applied to a wide variety of the PBD dimers of the present invention in order to introduce peptidic linkers. 
     Further Preferences 
     The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. The preferences may be combined together in any combination. 
     In some embodiments, R6′, R 7′ , R 9′ , R 10′ , R 11′  and Y′ are preferably the same as R 6 , R 7 , R 9 , R 10 , R 11  and Y respectively. 
     Dimer Link 
     Y and Y′ are preferably O. 
     R″ is preferably a C 3-7  alkylene group with no substituents. More preferably R″ is a C 3 , C 5  or C 7  alkylene. Most preferably, R″ is a C 3  or C 5  alkylene. 
     R 6  to R 9    
     R 9  is preferably H. 
     R 6  is preferably selected from H, OH, OR, SH, NH 2 , nitro and halo, and is more preferably H or halo, and most preferably is H. 
     R 7  is preferably selected from H, OH, OR, SH, SR, NH 2 , NHR, NRR′, and halo, and more preferably independently selected from H, OH and OR, where R is preferably selected from optionally substituted C 1-7  alkyl, C 3-10  heterocyclyl and C 5-10  aryl groups. R may be more preferably a C 1-4  alkyl group, which may or may not be substituted. A substituent of interest is a C 5-6  aryl group (e.g. phenyl). Particularly preferred substituents at the 7-positions are OMe and OCH 2 Ph. Other substituents of particular interest are dimethylamino (i.e. —NMe 2 ); —(OC 2 H 4 ) q OMe, where q is from 0 to 2; nitrogen-containing C 6  heterocyclyls, including morpholino, piperidinyl and N-methyl-piperazinyl. 
     These preferences apply to R 9′ , R 6′  and R 7′  respectively. 
     R 2    
     A in R 2  may be phenyl group or a C 5-7  heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, A is preferably phenyl. In other embodiments, A is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl. 
     X is a group selected from NHR N , wherein R N  is selected from the group comprising H and C 1-4  alkyl, 
                         
In some embodiments, X may preferably be NHR N . X may more preferably be NHMe, NHEt, and NH 2 , and may even more preferably be: NH 2 .
 
     Q 2 -X may be on any of the available ring atoms of the C 5-7  aryl group, but is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably β or γ to the bond to the remainder of the compound. Therefore, where the C 5-7  aryl group (A) is phenyl, the substituent (Q 2 -X) is preferably in the meta- or para-positions, and more preferably is in the para-position. 
     In some embodiments, Q 1  is a single bond. In these embodiments, Q 2  is selected from a single bond and —Z—(CH 2 ) n —, where Z is selected from a single bond, O, S and NH and is from 1 to 3. In some of these embodiments, Q 2  is a single bond. In other embodiments, Q 2  is —Z—(CH 2 ) n —. In these embodiments, Z may be O or S and n may be 1 or n may be 2. In other of these embodiments, Z may be a single bond and n may be 1. 
     In other embodiments, Q 1  is —CH═CH—. 
     In some embodiments, R 2  may be —A—CH 2 —X and —A—X. In these embodiments, X may preferably be NH 2 . 
     R 12    
     R 12  may be a C 5-7  aryl group. A C 5-7  aryl group may be a phenyl group or a C 5-7  heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, R 12  is preferably phenyl. In other embodiments, R 12  is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl. 
     R12 may be a C 8-10  aryl, for example a quinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl group may be bound to the PBD core through any available ring position. For example, the quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4y1, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3-yl and quinolin-6-yl may be preferred. The isoquinolinyl may be isoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4y1, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl may be preferred. 
     R 12  bears a substituent selected from OH, CO 2 H, CO 2 R ∘ , where R ∘  is selected from C 1-4  alkyl. The substituent may be any position. 
     Where R 12  is C 5-7  aryl group, a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably β or γ to the bond to the remainder of the compound. Therefore, where the C 5-7  aryl group is phenyl, the substituent is preferably in the meta- or para- positions, and more preferably is in the para-position. 
     Where R 12  is a C 8-10  aryl group, for example quinolinyl or isoquinolinyl, it may bear any number of substituents at any position of the quinoline or isoquinoline rings. 
     R ∘  is preferably selected from C 1-2  alkyl, i.e. methyl and ethyl. 
     R 12  Groups 
     Particularly preferred substituted R 12  groups include, but are not limited to, 4-hydroxy-phenyl, 3-hydroxyphenyl, 4-carboxy-phenyl, 3-carboxy-phenyl, 4-methyloxycarbonyl-phenyl, 3-methyloxycarbonyl-phenyl, 4-ethyloxycarbonyl-phenyl and 4-ethyloxycarbonyl-phenyl. 
     M and z 
     It is preferred that M and M′ are monovalent pharmaceutically acceptable cations, and are more preferably Na + . 
     z is preferably 3. 
     Accordingly, compounds of the present invention include, for example, those of formula I, or a pharmaceutically acceptable salt or solvate thereof, wherein 
     (i) R 2  is of formula III: 
     
       
         
         
             
             
         
       
     
     where A is a phenyl group, X is NHR N , wherein R N  is selected from the group comprising H and C 1-4  saturated alkyl, Q 1  is a single bond, and the remainder of the substituents are as defined herein. 
     Compounds of the present invention include, for example, those of formula I, or a pharmaceutically acceptable salt or solvate thereof, wherein 
     (ii) R 2  is of formula III: 
                         
where A is a phenyl group, X is NHR N , wherein R N  is selected from the group comprising H and C 1-4  saturated alkyl, Q 1  is a single bond, Q 2  is selected from a single bond and —Z— (CH 2 ) n —, where Z is selected from a single bond and n is from 1 to 3; and the remainder of the substituents are as defined herein.
 
     (iii) Compounds of the present invention include, for example, those of formula I, or a pharmaceutically acceptable salt or solvate thereof, wherein R 12  is a phenyl group, substituted by a group selected from CO 2 H, CO 2 R ∘ , where R ∘  is selected from saturated C 1-4  alkyl; and the remainder of the substituents are as defined herein. 
     (iv) Compounds of the present invention include, for example, those of formula I, or a pharmaceutically acceptable salt or solvate thereof, wherein R 12  is a phenyl group, substituted by a group selected from CO 2 H, CO 2 R ∘ , where R ∘  is selected from methyl or ethyl; and the remainder of the substituents are as defined herein. 
     (v) Compounds of the present invention include, for example, those of formula I, or a pharmaceutically acceptable salt or solvate thereof, wherein 
     R 2  is of formula III: 
                         
where A is a phenyl group, X is NHR N , wherein R N  is selected from the group comprising H and C 1-4  saturated alkyl, Q 1  is a single bond, Q 2  is selected from a single bond and —Z— (CH 2 ) n —, where Z is selected from a single bond and n is from 1 to 3; R 12  is a phenyl group, substituted by a group selected from OH, CO 2 H, CO 2 R ∘ , where R ∘  is selected from C 1-4  saturated alkyl; and the remainder of the substituents are as defined herein.
 
     (vi) Compounds of the present invention include, for example, those of formula I, or a pharmaceutically acceptable salt or solvate thereof, wherein 
     R 2  is of formula III: 
                         
where A is a phenyl group, X is NHR N , wherein R N  is selected from the group comprising H and C 1-4  saturated alkyl, Q 1  is a single bond, Q 2  is selected from a single bond and —Z— (CH 2 ) n —, where Z is selected from a single bond and n is from 1 to 3; R 12  is a phenyl group, substituted by a group selected from CO 2 H, CO 2 R ∘ , where R ∘  is selected from methyl or ethyl; and the remainder of the substituents are as defined herein.
 
     Preferred compounds of the present invention include any of those described in (i) through (vi) wherein:
         (a) the substituent group on R 12  is in the meta- or para-position, and more preferably in the para- position,   (b) Y and Y′ are O,       

     (c) R″ is —(CH 2 )—(CH 2 )—(CH 2 )— or —(CH 2 )—(CH 2 )—(CH 2 )—(CH 2 )—(CH 2 )—, 
     (d) R 10  and R 11  form a nitrogen-carbon bond between the nitrogen and carbon atoms to which they are bound and R 10′  and R 11′  form a nitrogen-carbon bond between the nitrogen and carbon atoms to which they are bound, 
     (e) R 7  is methoxy or ethoxy and R 7′  is methoxy or ethoxy, or 
     (f) R 6 , R 9 , R 6′ , and R 9′  are hydrogen, or any combination of (a) through (f). 
     Particularly preferred compounds of the present invention are of formula Ia: 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt or solvate thereof, where 
     n is 1 or 3; 
     R 1a  is methyl or phenyl; 
     R 2a  is: 
                         
where R N  is selected from H and methyl;
 
     R 12a  is selected from: 
     
       
         
         
             
             
         
       
     
     Particularly preferred compounds include: 
                                           
or a pharmaceutically acceptable salt or solvate thereof.
 
     3 rd  Aspect 
     The preferences expressed above for the first aspect may apply to the compounds of this aspect, where appropriate. 
     When R 10  is carbamate nitrogen protecting group, it may preferably be Teoc, Fmoc and Troc, and may more preferably be Troc. 
     When R 11  is O-Prot ∘ , wherein Prot ∘  is an oxygen protecting group, Prot ∘  may preferably be TBS or THP, and may more preferably be TBS. 
     When R 10  is a hemi-aminal nitrogen protecting group, it may preferably be MOM, BOM or SEM, and may more preferably be SEM. 
     The preferences for compounds of formula I apply as appropriate to D in the sixth aspect of the invention. For example, in the sixth aspect, the PBD dimer is any of the compounds of formula I, or a pharmaceutically acceptable salt or solvate thereof, described herein expect that 
                         
is replaced with
 
                         
is replaced with
 
                         
and *—NHR N  is replaced with
 
                         
where the wavy line indicates the point of attachment to the Linker Unit.
 
     Accordingly, the Conjugates of the present invention include those having the following formula (IV)
 
L-(LU-D) p    (IV)
 
or a pharmaceutically acceptable salt or solvate thereof, wherein L is a Ligand unit (i.e., a targeting agent), LU is a Linker unit and the PBD dimer D is any of the compounds of formula I, or a pharmaceutically acceptable salt or solvate thereof, described herein expect that
 
                         
is replaced with
 
                         
is replaced with
 
                         
and *—NHR N  is replaced with
 
                         
where the wavy line indicates the point of attachment to the Linker Unit.
 
     (a) Conjugates of the present invention include, for example, those of the formula:
 
CBA-A 1 -L 1 -*
         where the asterisk indicates the point of attachment to the PBD dimer (D) or the Spacer unit, CBA is the Cell Binding Agent, L 1  is a Specificity unit that is cleavable by the action of an enzyme, and A 1  is a Stretcher unit connecting L 1  to the Cell Binding Agent.       

     (b) Conjugates of the present invention include, for example, those of the formula:
 
CBA-A 1 -*
         where the asterisk indicates the point of attachment to the PBD dimer (D), CBA is the Cell Binding Agent, L 1  and A 1  is a Stretcher unit connecting the Drug to the Cell Binding Agent.       

     (c) Conjugates of the present invention include, for example, those of the formula:
 
CBA-A 1 -L 1 -*
         where the asterisk indicates the point of attachment to the PBD dimer (D), CBA is the Cell Binding Agent, A 1  is a Stretcher unit connecting L 1  to the Cell Binding Agent and L 1  is a Specificity unit that is cleavable by the action of cathepsin, L 1  is a dipeptide, L 1  is a dipeptide that is cleavable by the action of cathepsin or L 1  is a dipeptide selected from -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, and -Val-Cit-.       

     Preferred conjugates of the present invention include any of those described in (a)-(c) wherein A 1 is 
     
       
         
         
             
             
         
       
         
         
           
             where the asterisk indicates the point of attachment to L 1  or D, the wavy line indicates the point of attachment to CBA, and n is 0 to 6 (preferably n is 5). 
           
         
       
    
     Particularly preferred conjugates of the present invention are of formula Ib, Ic, 1d, and 1e: 
                         
or a pharmaceutically acceptable salt or solvate thereof, where
 
     n is 1 or 3; 
     R 1a  is methyl; 
     R N  is H 
     R 12a  is selected from: 
     
       
         
         
             
             
         
       
     
     A 1  is a Stretcher unit; 
     L 1  is a dipeptide that is cleavable by the action of cathepsin; 
     Ab is an antibody; and 
     p is from 1 to 20. 
     In a particularly preferred embodiment of formulas Ib, Ic, Id, and le, or a pharmaceutically acceptable salt or solvate thereof, the connection between the antibody and the Linker Unit is formed between a thiol group of a cysteine residue of the antibody and a maleimide group of the Linker unit. 
     Particularly preferred conjugates include: 
                                           
or a pharmaceutically acceptable salt or solvate thereof, wherein
 
     n is 1 or 3. 
     A 1  is a Stretcher unit; 
     L 1  is a dipeptide that is cleavable by the action of cathepsin; 
     Ab is an antibody; and 
     p is from 1 to 20. 
     In a particularly preferred embodiment, for all of these preferred conjugates, the connection between the antibody and the Linker is formed between a thiol group of a cysteine residue of the antibody and a maleimide group of the Linker unit. 
     In a particularly preferred embodiment, for all of these preferred conjugates, the antibody is a monoclonal antibody that specifically binds to the Cripto antigen, CD19 antigen, CD20 antigen, CD22 antigen, CD30 antigen, CD33 antigen, Glycoprotein NMB, CanAg antigen, Her2 (ErbB2/Neu) antigen, CD56 (NCAM) antigen, CD70 antigen, CD79 antigen, CD138 antigen, PSCA, PSMA (prostate specific membrane antigen), BCMA, E-selectin, EphB2, Melanotransferin, Muc16 antigen or TMEFF2 antigen. 
     The preferences for compounds of formula I, or a pharmaceutically acceptable salt or solvate thereof, apply as appropriate to D in the seventh aspect of the invention. For example, in the seventh aspect, the PBD dimer is any of the compounds of formula I, or a pharmaceutically acceptable salt or solvate thereof, described herein expect that, expect that 
                         
is replaced with
 
                         
is replaced with
 
                         
and *-NHR N  is replaced with
 
                         
where the wavy line indicates the point of attachment to the Linker Unit.
 
     Particularly preferred Drug-Linkers of the present invention are of formula If, Ig, Ih and Ii: 
                         
or a pharmaceutically acceptable salt or solvate thereof, where
 
     n is 1 or 3; 
     R 1a  is methyl or phenyl; 
     R N  is H 
     R 12a  is selected from: 
     
       
         
         
             
             
         
       
     
     A 1  is a Stretcher unit;and 
     L 1  is a dipeptide that is cleavable by the action of cathepsin. 
     Particularly preferred drug-linkers include: 
                                           
or a pharmaceutically acceptable salt or solvate thereof, wherein
 
     n is 1 or 3. 
     A 1  is a Stretcher unit; and 
     L 1  is a dipeptide that is cleavable by the action of cathepsin. 
     EXAMPLES 
     General Experimental Methods for Example 1 
     Optical rotations were measured on an ADP 220 polarimeter (Bellingham Stanley Ltd.) and concentrations (c) are given in g/100 mL. Melting points were measured using a digital melting point apparatus (Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum 1000 FT IR Spectrometer.  1 H and  13 C NMR spectra were acquired at 300 K using a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively. Chemical shifts are reported relative to TMS (δ=0.0 ppm), and signals are designated as s (singlet), d (doublet), t (triplet), dt (double triplet), dd (doublet of doublets), ddd (double doublet of doublets) or m (multiplet), with coupling constants given in Hertz (Hz). Mass spectroscopy (MS) data were collected using a Waters Micromass ZQ instrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. Waters Micromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35; Extractor (V), 3.0; Source temperature (° C.), 100; Desolvation Temperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flow rate (L/h), 250. High-resolution mass spectroscopy (HRMS) data were recorded on a Waters Micromass QTOF Global in positive W-mode using metal-coated borosilicate glass tips to introduce the samples into the instrument. Thin Layer Chromatography (TLC) was performed on silica gel aluminium plates (Merck 60, F 254 ), and flash chromatography utilised silica gel (Merck 60, 230-400 mesh ASTM). Except for the HOBt (NovaBiochem) and solid-supported reagents (Argonaut), all other chemicals and solvents were purchased from Sigma-Aldrich and were used as supplied without further purification. Anhydrous solvents were prepared by distillation under a dry nitrogen atmosphere in the presence of an appropriate drying agent, and were stored over 4 Å molecular sieves or sodium wire. Petroleum ether refers to the fraction boiling at 40-60° C. 
     General LC/MS conditions: The HPLC (Waters Alliance 2695) was run using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%). Gradient: initial composition 5% B over 1.0 min then 5% B to 95% B within 3 min. The composition was held for 0.5 min at 95% B, and then returned to 5% B in 0.3 minutes. Total gradient run time equals 5 min. Flow rate 3.0 mL/min, 400 μL was split via a zero dead volume tee piece which passes into the mass spectrometer. Wavelength detection range: 220 to 400 nm. Function type: diode array (535 scans). Column: Phenomenex® Onyx Monolithic C18 50×4.60 mm 
     Example 1 
     
       
         
         
             
             
         
       
     
     (a) (S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethylsulfonyl-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentoxyoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c] [1,4]benzodiazepine-5,11(10H,11aH)-dione (2) 
     1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (1)(Compound 8b in WO 2010/043880) (2.8 g, 2.4 mmol, leq) was added to a mixture of sodium carbonate (388 mg, 3.66 mmol, 1.52 eq) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)aniline (509 mg, 2.32 mmol, 0.95 eq), in toluene/water/ethanol (20 mL/10 mL/10 mL). The reaction flask was flushed with argon and solid Pd(0)tetrakis triphenylphosphine (84 mg, 0.072 mmol, 0.03 eq) was added. The reaction was allowed to proceed for 2 hours at 26° C. with vigorous stirring under argon. The mixture was partitioned between ethyl acetate (200 mL) and water (100 mL). The organic phase was washed with water (100 mL), followed by brine (50 mL). The organic phase was dried over magnesium sulphate and the volatiles removed by rotoevaporation, followed by hard vacuum. The residue was purified by flash chromatography (gradient ethyl acetate/hexane, 30/70 up 100/0, v/v). The unsymmetrical amino triflate (2) was isolated in 46% yield (1.23 g). LC/MS rt 3.80 min m/z (1087.6) M+H. 932 mg (33%) of starting material and 400 mg (16%) of symmetrical 4-amino phenyl product were also obtained. 
     (b) (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]diazepin-2-yl trifluoromethanesulfonate(3) 
     The amino triflate (2) was dissolved in dry THF (15 mL) and cooled at -78° C. (1.2g, 1.1 mmol, 1 eq). A solution of super hydride in THF (1M, 3.3mL, 3.3 mmol, 3 eq) was injected slowly in the stirred reaction mixture. Reaction completion was observed after 15 minutes. The reaction mixture was quenched with water (10 mL) and later extracted with DCM (50 mL). The organics were washed with water (100 mL), then brine (50 mL). The organic phase was dried over magnesium sulphate and the volatiles removed by rotoevaporation, followed by hard vacuum. The crude carbinolamine (3)(1.10g) was not purified and used directly in the next step. LC/MS rt 2.68 min m/z (796) M+H for SEM deprotected imine (self-immolation under the acidic conditions of the LC/MS). 
     (c) (S)-2-(4-aminophenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methyloxycarbonylphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one (4) 
     The crude SEM protected carbinolamine triflate (3) obtained in the previous step (1.10 g, 1 mmol, 1 eg) was added to a mixture of sodium carbonate (341 mg, 3.2 mmol, 3.2 eq) and phenylboronic acid methyl ester (286 mg, 1.6 mmol, 1.6 eq), in toluene/water/methanol/THF (10 mL/5 mL/5 mL/5 mL). The reaction flask was flushed with argon and solid Pd(0)tetrakis triphenylphosphine (35 mg, 0.030 mmol, 0.03 eq) was added. The reaction was allowed to proceed overnight with vigorous stirring under argon. The mixture was partitioned between ethyl acetate (200 mL) and water (100 mL). The organic phase was washed with water (100 mL), followed by brine (50 mL). The organic phase was dried over magnesium sulphate and the volatiles removed by rotoevaporation, followed by hard vacuum. The residue was treated with DCM (50 mL), ethanol (140 mL), water (70 mL) and silica gel (100 g). The viscous mixture was allowed to stir at room temperature for 3 days. The mixture was filtered slowly through a sinter funnel and the silica residue washed with 90/10 chloroform/methanol v/v (500mL). The organic phase was washed with water (300 mL), brine (100 mL), dried (magnesium sulphate), filtered, and evaporated in vacuo to provide the crude material which was purified by flash chromatography (gradient methanol/chloroform, 0/100 up 4/96, v/v) to yield 200 mg (25%) of PBD dimer LC/MS rt 2.68 min m/z (782) M+H. 
     General Experimental Methods for Examples 2 to 3 
     All commercially available anhydrous solvents were used without further purification. Analytical thin layer chromatography was performed on silica gel 60 F254 aluminum sheets (EMD Chemicals, Gibbstown, N.J.). Radial chromatography was performed on Chromatotron apparatus (Harris Research, Palo Alto, Calif.). Analytical HPLC was performed on a Varian ProStar 210 solvent delivery system configured with a Varian ProStar 330 PDA detector. Samples were eluted over a C12 Phenomenex Synergi 2.0×150 mm, 4 μm, 80 A reverse-phase column. The acidic mobile phase consisted of acetonitrile and water both containing 0.1% formic acid. Compounds were eluted with a linear gradient of acidic acetonitrile from 5% at 1 min post injection, to 95% at 11 min, followed by isocratic 95% acetonitrile to 15 min (flow rate=1.0 mL/min). LC-MS was performed on a ZMD Micromass mass spectrometer interfaced to an HP Agilent 1100 HPLC instrument equipped with a C12 Phenomenex Synergi 2.0×150 mm, 4 μm, 80 Å reverse phase column. The acidic eluent consisted of a linear gradient of acetonitrile from 5% to 95% in 0.1% aqueous formic acid over 10 min, followed by isocratic 95% acetonitrile for 5 min (flow rate=0.4 mL/min). Preparative HPLC was carried out on a Varian ProStar 210 solvent delivery system configured with a Varian ProStar 330 PDA detector. Products were purified over a C12 Phenomenex Synergi 10.0×250 mm, 4 μm, 80 Å reverse phase column eluting with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The purification method consisted of the following gradient of solvent A to solvent B: 90:10 from 0 to 5 min; 90:10 to 10:90 from 5 min to 80 min; followed by isocratic 10:90 for 5 min. The flow rate was 4.6 mL/min with monitoring at 254 nm. 
     Example 2 
     
       
         
         
             
             
         
       
     
     (a) (S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanoic acid (5) 
     Maleimidocaproyl N-hydroxysuccinimide (1.619 g, 5.25 mmol, 1.05 eq.) and H-Val-Ala-OH (0.941 g, 5 mmol, 1 eq.) were placed in a 25 mL recovery flask with a stir bar and the flask was flushed with nitrogen. DMF (4.7 mL) was added and the resulting white slurry was stirred. DIPEA (0.87 mL, 5 mmol, 1 eq) was added and the mixture was allowed to stir at room temperature overnight. The mixture was cooled in an ice/water bath and 2M HCI (3 mL, 6 mmol) was added dropwise. The viscous mixture was transferred to a separatory funnel and the reaction vessel rinsed with sat. NaCl (7 mL), EtOAc (10 mL), sat NaCl (10 mL) and EtOAc (5 mL). After separation of the aqueous phase, it was extracted with additional EtOAc (2×15 mL). The combined organic extracts were washed with sat NaCl (4×15 mL), until the washings were pH ˜3.5. The organic extracts were dried over Na2SO4, filtered and concentrated under reduced pressure to give crude 5 as a white solid (2.172 g, 114% crude yield). Crude 5 was suspended in warm CH 2 Cl 2  (35 mL) and filtered to remove a fine white solid. The solids were rinsed with additional CH 2 Cl 2  (3 mL). Toluene (5 mL) was added and the mixture was cooled in an ice/water bath, which resulted in a thick slurry. The solids were collected by filtration, washed with a cold mixture of CH 2 Cl 2  (12 mL) and toluene (2 mL) and dried by pulling air through the sample overnight to give 5 as a white solid (1.327 g, 70% yield). TLC: Rf=0.26, 10% MeOH in CH 2 Cl 2 . 1H NMR (CDCl 3 ) 
     (ppm) 0.95 (d, J=17 Hz, 3H), 0.98 (d, J=17 Hz, 3H), 1.30 (m, 2H), 1.40 (d, J=17 Hz, 3H), 1.61 (m, 4H), 2.06 (m, 1H), 2.25 (dt, J=4, 19 Hz, 2H), 3.35 (s, 1H), 3.49 (t, J=17 Hz,2H), 4.20 (d, J=18 Hz, 1H), 4.38 (m, 1H), 6.80 (s, 2H). Analytical HPLC (0.1% formic acid): tR 9.05 min. LC-MS: t R  11.17 min, m/z (ES+) found 381.9 (M+H)+, m/z (ES−) found 379.9 (M−H)−. 
     (b) Methyl 4-((S)-8-((5-(((S)-2-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1 H-benzo[e]pyrrolo[1 ,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,11 a-dihydro-1H-benzo[e]pyrrolo[1 ,2-a][1,4]diazepin-2-yl)benzoate (6) 
     A 10 mL flask was charged with 5 (11 mg, 29 μmol), EEDQ (8.9 mg, 36 μmol), and 0.46 mL anhydrous CH 2 Cl 2 . Methanol (24 μL) was added to facilitate dissolution and the mixture was stirred under nitrogen for 15 min. Aniline 4 (18 mg, 24 μmol) was then added and the reaction mixture was stirred at room temperature for 4 hours, at which time LC-MS revealed conversion to product. The reaction was concentrated, dissolved in CH 2 Cl 2  (1 mL) and purified by radial chromatography on a 1 mm chromatotron plate eluted with CH 2 Cl 2 /CH 3 OH mixtures (100:0 to 90:10 CH 2 Cl 2 /CH 3 OH) to provide 6 (9.9 mg, 36%). Analytical HPLC: t R  12.10 min. LC-MS: t R  12.91 min, m/z (ES + ) found 1145.6 (M+H) + . 
     Example 3 
     
       
         
         
             
             
         
       
     
     9: R 1 ═H, R 2 ═CH 3    
     10: R 1 ═H, R 2 ═H 
     11: R 1 ═MC, R 2 ═H 
     Compound 7 was prepared in a similar fashion to compound 5 in Example 2(a) using allyl chloroformate in place of maleimidocaproyl N-hydroxysuccinimide and dichloromethane as the reaction solvent. 
     (a) Methyl 4-((S)-8-(3-(((S)-2-(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)benzoate (8) 
     To a 7 (52 mg, 0.192 mmol) in 5% methanol/dichloromethane (3 mL) was at 0° C. was added EEDQ (47 mg, 0.193 mmol) and the mixture was stirred for 15 minutes before addition of 4 (50 mg, 0.064 mmol). The reaction mixture was allowed to warm to an ambient temperature and was monitored by LC-MS. The mixture was aspirated onto a 1 mm radial chromatotron plate and eluted with 1 to 3% methanol/dichloromethane. Product containing fractions were combined and concentrated to give 43 mg (65%) of 8 as a yellow solid: MS (ES + ) m/z 1036.87 [M+H] + . 
     (b) Methyl 4-((S)-8-(3-(((S)-2-(4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzol[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)benzoate (9) 
     To a solution of 8 (43 mg) in anhydrous dichloromethane (3 mL) was added Ph 3 P (0.5 mg, 0.002 mmol), pyrollidine (7 μL, 0.082 mmol) and tetrakis palladium (1.1 mg, 0.001 mmol). After approximately 30 minutes, the reaction mixture was aspirated onto a 1 mm radial chromatotron plate and eluted with 5% and then 10% methanol in dichloromethane. The major band was collected and concentrated under reduced pressure to give 22 mg (56%) of 9: MS (ES + ) m/z 952.5 [M+H] + . 
     (c) 4-((S)-8-(3-(((S)-2-(4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1, 2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1 ,2-a][1, 4]diazepin-2-yl)benzoic acid (10) 
     To 9 (20 mg) in THF/CH 3 OH (2 mL) was added a lithium hydroxide solution (1 mL of a 0.1 M solution). The reaction mixture was stirred at an ambient temperature. At 5 hours, LC-MS revealed approximately a 30% conversion to desired product with significant decomposition. The reaction mixture was cooled to −80° C. for 16 hours. LC-MS showed a ˜1:1 mixture of 10 and 9. The reaction mixture was neutralized with 0.1N HCI (˜1 mL) and was concentrated to approximately 1 mL. DMSO (1 mL) and CH 3 CN (1 mL) were added, and the mixture was purified by preparatory reverse-phase HPLC. Product containing fractions were combined, frozen and lyophilized. This resulted in 1.7 mg (9%) of 10 as a yellow film: MS (ES + ) m/z 938 [M+H]+. 
     (d) 4-((S)-8-(3-(((S)-2-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1, 2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11 a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)benzoic acid (11) 
     To a mixture of 10 (1.7 mg, 1.8 μmol) in DMF (100 μL) was added DIPEA (1 μL, 5.75 μmol) and maleimidocaproyl-NHS ester (4.6 mg, 15 μmol). The reaction was monitored by LC-MS. After 1 hour, the reaction mixture was concentrated under reduced pressure, dissolved in 0.5 mL of DMSO, 0.5 mL of acetonitrile and 0.5 mL of water, and purified by preparative reverse-phase HPLC. The product containing fraction was frozen and lyophilized to give 0.2 mg (10%) of 11: MS (ES + ) m/z 1131.6 [M+H] + . 
     Example 4 
     
       
         
         
             
             
         
       
     
     (a) (S)-2-(4-aminophenyl-8-(3-(((S)-2-(4-hydroxyphenyl)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1, 2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzol[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione (13) 
     A flask was charged with aniline triflate 12 (compound 9, WO 2011/130613 A1) (520 mg, 490 μmol, 1 eq) dissolved in toluene (5.4 mL), ethanol (2.7 mL), and water (2.7 mL). To the stirred solution was added 4-hydroxyphenylboronic acid (88 mg, 640 μmol, 1.3 eq), sodium carbonate (83 mg, 780 μmol, 1.6 eq), and tetrakis(triphenylphosphine)palladium(0) (23 mg, 20 μmol, 0.04 eq), the reaction was stirred vigorously overnight at room temperature under nitrogen. After 22 hours the reaction had stalled. Additional tetrakis(triphenylphosphine)palladium(0) (100 mg, 87 μmol, 0.18 eq) and 4-hydroxyphenylboronic acid (88 mg, 640 μmol, 1.3 eq) were added and the reaction was stirred at 35° C. for an additional 24 hours, at which time LC/MS revealed conversion to product. The reaction was concentrated and then partitioned between ethyl acetate (100 mL) and water (100 mL). The aqueous layer was extracted two times with ethyl acetate (100 mL). The organic layer was then washed with water (100 mL), brine (100 mL), dried over sodium sulfate, and concentrated to dryness to provide crude SEM dilactam 13. The crude product was purified by flash chromatography, eluting with mixtures of hexanes:ethyl acetate (75:25 to 0:100), to provide pure product 13 (218 mg, 44%). LC-MS: t R  11.54 min, m/z (ES + ) found 1004.3 (M+H) + .  1 H NMR (CDCl 3 ) δ (ppm) 0.02 (s, 18H), 0.98 (m, 4H), 2.44 (m, 2H), 3.12 (m, 2H), 3.67 (m, 3H), 3.77 (m, 4H), 3.91 (m, 8H), 4.29 (t, J=5.9 Hz, 4H), 4.59 (dt, J=3.1, 10.2 Hz, 2H), 4.76 (dd, J=3.1, 10.2 Hz, 2H), 5.52 (d, J=10.2 Hz, 2H), 6.34 (bs, 1H), 6.66 (d, J=8.2 Hz, 2H), 6.83 (d, J=8.6 Hz, 2H), 7.22 (m, 4H), 7.27 (m, 6H), 7.39 (s, 2H). 
     (b) (S)-2-(4-aminophenyl-8-(3-(((S)-2-(4-hydroxyphenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1, 2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one (14) 
     A flame-dried flask was charged with SEM dilactam 13 (109 mg, 109 μmol, 1 eq) dissolved in anhydrous tetrahydrofuran (2.2 mL), and cooled to −78° C. Lithium triethylborohydride (0.33 mL of a 1 M solution in THF, 330 μmol, 3 eq) was added dropwise and the reaction was stirred under nitrogen for 2.5 hours, at which time LC revealed incomplete conversion to product. An additional 0.66 mL of reductant was added and the reaction was stirred for one more hour. The reaction was quenched through the addition of water (1 mL) and allowed to warm to room temperature, then diluted brine (25 mL) and extracted three times with dichloromethane (25 mL). The combined organics were washed with brine (25 mL), dried over sodium sulfate, and evaporated to dryness. The residue was dissolved in a mixture of dichloromethane (2.8 mL), ethanol (7.4 mL), and water (1.0 mL), and silica gel (2.7 g) was added. The resulting slurry was stirred at room temperature for 4 days. TLC analysis revealed conversion to imine dimer 14, at which time the slurry was filtered over a sintered glass funnel and the silica gel cake was washed with 10% methanol in chloroform until no further PBD absorbance was observed in the filtrate. Concentration of the filtrate provided crude imine dimer 14. The material was dissolved in minimal dichloromethane and purified by radial chromatography on a 1 mm chromatotron plate eluted with CH 2 Cl 2 /MeOH mixtures (100:0 to 80:20) to provide 14 (31 mg, 40%). LC-MS: t R  8.48 min, m/z (ES + ) found 712.2 (M+H) + . 
     Example 5 
     
       
         
         
             
             
         
       
     
     6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((4-((S)-8-(3-(((S)-2-(4-hydroxyphenyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1 ,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1, 2-a][1, 4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (15) 
     A flame-dried flask was charged with maleimidocaproyl-valine-alanine linker (Compound 36 of Example 13 in WO 2011/130613 A1) (11 mg, 29 μmol, 1.5 eq) dissolved in 0.8 mL of 5% methanol in anhydrous dichloromethane. The acid was pre-activated by addition of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (9 mg, 34 μmol, 1.8 eq), followed by stirring at room temperature under nitrogen for 15 minutes. The activated acid was then added to a flame-dried flask containing PBD dimer 14 (13 mg, 19 μmol, 1 eq). The reaction was stirred for 4 hours at room temperature under nitrogen, at which time LC-MS revealed conversion to product. The material was diluted in dichloromethane and purified by radial chromatography on a 1 mm chromatotron plate eluted with CH 2 Cl 2 /MeOH mixtures (100:0 to 80:20) to provide 15 (7.7 mg, 38%). LC-MS: m/z (ES + ) found 1075.5 (M+H) + . 
     Example 6 
     Preparation of PBD Dimer Conjugates 
     Antibodies with introduced cysteines: Antibodies to CD70 containing a cysteine residue at position 239 of the heavy chain were fully reduced by adding 10 equivalents of TCEP and 1 mM EDTA and adjusting the pH to 7.4 with 1M Tris buffer (pH 9.0). Following a 1 hour incubation at 37° C., the reaction was cooled to 22° C. and 30 equivalents of dehydroascorbic acid were added to selectively reoxidize the native disulfides, while leaving cysteine 239 in the reduced state. The pH was adjusted to 6.5 with 1M Tris buffer (pH 3.7) and the reaction was allowed to proceed for 1 hour at 22° C. The pH of the solution was then raised again to 7.4 by addition of 1 M Tris buffer (pH 9.0). 3.5 equivalents of the PBD drug linker in DMSO were placed in a suitable container for dilution with propylene glycol prior to addition to the reaction. To maintain solubility of the PBD drug linker, the antibody itself was first diluted with propylene glycol to a final concentration of 33% (e.g., if the antibody solution was in a 60 mL reaction volume, 30 mL of propylene glycol was added). This same volume of propylene glycol (30 mL in this example) was then added to the PBD drug linker as a diluent. After mixing, the solution of PBD drug linker in propylene glycol was added to the antibody solution to effect the conjugation; the final concentration of propylene glycol is 50%. The reaction was allowed to proceed for 30 minutes and then quenched by addition of 5 equivalents of N-acetyl cysteine. The ADC was then purified by ultrafiltration through a 30 kD membrane. (Note that the concentration of propylene glycol used in the reaction can be reduced for any particular PBD, as its sole purpose is to maintain solubility of the drug linker in the aqueous media.) 
     Example 7 
     Determination of Free Drug In Vitro Cytotoxicity 
     Cells as detailed below were collected and plated in 96 well black-sided plates at a density of 10,000 cells/well in 150 μL of medium. Serial dilutions of the test article (50 μL) were added, and incubation was carried out for 92 hours at 37° C. After addition of test compound, cultures were incubated to 96 hours at 37° C. Resazurin (0.25 mM, 50 μL, Sigma, St. Louis, Mo.) in medium was added and incubation was continued for 4 hours. The plates were read on a Fusion HT microplate reader (Packard, Meriden, Conn.) using an excitation wavelength of 525 nm and an emission wavelength of 590 nm. Data from all assays were reduced using GraphPad Prism Version 4 for Windows (GraphPad Software, San Diego, Calif.). The IC 50  concentrations compared to untreated control cells were determined using a 4 parameter curve fits. 
     The IC 50  (pM) values for compounds 4 and 14: 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 IC50 in pM following 48 hours treatment 
               
            
           
           
               
               
               
               
               
            
               
                 compound 
                 786-O 
                 Caki-1 
                 HL60 
                 HEL9217 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 4 
                 50 
                 20 
                 8 
                 8 
               
               
                 14 
                 200 
                 400 
                 30 
                 50 
               
               
                   
               
            
           
         
       
     
     Example 8 
     Determination of In Vitro Activity of Selected Conjugates 
     The in vitro cytotoxic activity of the selected antibody drug conjugates was assessed using a resazurin (Sigma, St. Louis, Mo., USA) reduction assay (reference: Doronina et al.,  Nature Biotechnology,  2003, 21, 778-784). The antibody drug conjugates were prepared as described above in Example 6. 
     For the 96-hour assay, cells cultured in log-phase growth were seeded for 24 hours in 96-well plates containing 150 μL RPMI 1640 supplemented with 20% FBS. Serial dilutions of ADC in cell culture media were prepared at 4× working concentration; 50 μL of each dilution was added to the 96-well plates. Following addition of ADC, the cells were incubated with test articles for 4 days at 37° C. Resazurin was then added to each well to achieve a 50 μM final concentration, and the plates were incubated for an additional 4 hours at 37° C. The plates were then read for the extent of dye reduction on a Fusion HT plate reader (Packard Instruments, Meridien, Conn., USA) with excitation and emission wavelengths of 530 and 590 nm, respectively. The IC 50  value, determined in triplicate, is defined here as the concentration that results in a 50% reduction in cell growth relative to untreated controls. 
     Referring to the tables below, the in vitro cytotoxicity of ADCs using the 96 hour assay is shown. The ADCs were tested against antigen positive and antigen negative cell lines. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 IC50 in pM following 96 hours treatment 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 antigen-negative 
               
               
                 ADC 
                 drugs/Ab 
                 786-O 
                 Caki-1 
                 cell line 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 h1F6ec-6 
                 1.8 
                 30 
                 0.1 
                 90,000 
               
               
                 h1F6ec-11 
                 1.8 
                 50 
                 30 
                 No effect 
               
               
                 h1F6ec-15 
                 2.0 
                 30 
                 13 
                 10,000 
               
               
                   
               
            
           
         
       
     
     Example 9 
     Determination of In Vivo Cytotoxicity of Selected Conjugates 
     All studies were conducted in accordance with the Animal Care and Use Committee in a facility that is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. ADC tolerability was first assessed to ensure that the conjugates were tolerated at the doses selected for the xenograft experiments. BALB/c mice were treated with escalating doses of ADC formulated in PBS with 0.5 M arginine and 0.01% Tween 20. Mice were monitored for weight loss and outward signs of morbidity following treatment; those that experienced greater than 20% weight loss or displayed signs of morbidity were euthanized. The antibody used was a CD70 antibody, humanized h1F6 (WO2006/113909), with a point mutation substituting cysteine for serine at position 239. Conjugation to the Drug Unit is through the introduced cysteine at position 239. An average of 2 drugs is loaded per antibody. 
     In vivo therapy experiments were conducted in xenograft models in mice bearing CD70+ renal cell carcinoma or non-Hodgkin lymphoma. Tumor fragments were implanted into nude mice. Mice were then randomized to study groups with each group averaging around 100 mm 3 . The ADCs were administered according to the schedule indicated. Tumor volume as a function of time was determined using the formula (L×W 2 )/2. Animals were euthanized when tumor volumes reached 1000 mm 3 . Mice showing durable regressions were terminated around day 100 post implant. 
       FIG. 1  shows the results of treatment studies using h1F6ec-compound 6 in CD70+ renal cell carcinoma (786-O), with single dose given IP. In the figure,   is untreated, ● is treatment with h1F6ec-6 at 0.03 mg/kg and ∘ is treatment with h1F6ec-6 at 0.1 mg/kg. 
       FIG. 2  show the results of treatment studies using h1F6ec-compound 6 in non-Hodgkin lymphoma (MHHPreB1), with dosing q7dx2. In the figure,   is untreated and ● is treatment with h1F6ec-6 at 0.1 mg/kg. 
     The results of a mouse tolerability experiment with h1F6ec-6 nominally loaded at 2 drugs/mAb demonstrated that a single dose of 1 mg/kg was well tolerated with no weight loss or signs of outward morbidity out to 30 days. Administration of a higher dose (2.5 mg/kg) resulted in weight loss. 
     The IC 50  (nM) values for ADCs with Compound 6: 
     
       
         
           
               
               
               
               
               
               
             
               
                   
               
               
                   
                 786-O 
                 Caki-1 
                 CD70 neg 
                 CD70 neg 
                 CD70 neg 
               
               
                   
                 cancer 
                 cancer 
                 cancer cell 
                 cancer cell 
                 cancer cell 
               
               
                 ADCs 
                 cell line 
                 cell line 
                 line 
                 line 
                 line 
               
               
                   
               
             
            
               
                 h1F6ec-6 
                 1 
                 0.5 
                 7491 
                 2074 
                 5327 
               
               
                 (1.8dr/Ab) 
               
               
                   
               
            
           
         
       
     
     The IC 50  (nM) values for ADCs with Compound 6 and Compound 11: 
     
       
         
           
               
               
               
               
               
               
             
               
                   
               
               
                   
                 786-O 
                 Caki-1 
                 CD70 neg 
                 CD70 neg 
                 CD70 neg 
               
               
                   
                 cancer 
                 cancer cell 
                 cancer cell 
                 cancer cell 
                 cancer cell 
               
               
                 ADCs 
                 cell line 
                 line 
                 line 
                 line 
                 line 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 h1F6ec-11 
                 4 
                 2 
                 No Effect 
                 7725 
                 Max 
               
               
                 (1.8dr/Ab) 
                   
                   
                   
                   
                 Inh = 50% 
               
               
                 h1F6ec-6 
                 2 
                 0.01 
                 7215 
                 1415 
                 Max 
               
               
                 (1.8dr/Ab) 
                   
                   
                   
                   
                 Inh = 45%